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An In Vitro Method to Study Male Reproductive Toxicology: A Review on the Bio-AlteR® Technology Allowing a “Physio-Toxicology” Approach

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DOI: 10.31038/JPPR.2022514

Introduction

Increasingly, toxicology studies are using toxicogenomics on lines of cultured cells to identify genes whose expression is altered by toxics. However, this approach is not feasible in testicular toxicity, because there is no line of germ cells to respond to this request because the differentiation of germ cells is dependent on interactions with the somatic cells of the testis [1,2]. During the golden age of French research, we have developed and validated two culture systems of germ cells together with Sertoli cells, in bicameral chambers, in a defined culture medium, which maintain the blood testis barrier [3-5] and enable studying the mitotic phase of spermatogenesis, the entire meiotic phase, and the first steps of spermiogenesis over a 4-week culture period in the 20 days old rat [5-7]. These in vitro systems should be a major methodological breakthrough in assessing toxicological potency of chemical compounds on a relatively long time period and enabling the study of many aspects of their mechanism of action while reducing greatly the number of animals needed. It must be noted that our systems of culture in bicameral chamber allow studying the effects of a toxic substance added to the basal compartment of the culture chamber, and thus mimics what could happen in vivo in the testis. Indeed, the cellular junctions, between sertoli cells and between germ cells and Sertoli cells, which are essential for spermatogenesis, are maintained or rebuilt in our systems of culture [3]. Therefore, before reaching the germ cells, in our system, the toxic substance must cross the barrier of Sertoli cells (the main component the blood-testis barrier). By contrast, in “conventional” cultures wells, a compound may be toxic to differentiated germ cells because it is placed directly in contact with these cells, whereas in vivo, it may not have access to the compartment of the seminiferous tubules where this population of germ cells is located.

In addition, the possibility of rather long term cultures of the cells will allow, most often, testing for the reversibility of the observed effects. Our cultures can be analyzed by cell physiology, cytology, biochemical and molecular biological approaches allowing the determination of the mechanisms responsible for the gonadal toxicity or carcinogenic effect of organic or mineral compounds and of nanoparticles.

In these models it is indeed possible to analyze:

  1. The alterations of the blood-testis barrier.
  2. Survival/death of somatic and/or germ cells, proliferation of Sertoli cells and spermatogonia (stem cells of spermatogenesis).
  3. The course of meiotic division (key stage of spermatogenesis during which genetic recombinations occur).
  4. Cytogenetic abnormalities of germ cells.
  5. The expression of specific genes in the germ cells or Sertoli cells (transcriptome).
  6. The peptide profiling of the culture supernatant/cultured cells (proteome).

Our co-cultures can also serve as an original tool for rapid screening test molecules for therapeutic purposes in order to improve a failing male fertility.

The use of a cell population exposed (treated) or not (control group) to the substance allows to get rid of much of the variability between animals that are encountered in testing in vivo and to optimize the power of the tests.

Scientific Validation of these Systems (Physiology)

Two important aspects of the results obtained using in vitro models are their reproducibility (see Figure 7 in Staub et al.) [5], and their relevance to the in vivo (physiological) situation (see below).

The germ cell-Sertoli cell coculture systems that we settled [8-10]; have been carefully validated from the physiological point of view, on many aspects, over the last decades. To our knowledge, there is no other system of culture of male germ cells and Sertoli cells which has been so carefully and extensively validated. Hence, the availability of a physiologically relevant in vitro systems allowing screening the mechanism of action of a large number of potentially toxic/active molecules, at low concentrations on a relatively long period of time, is of an outmost interest. We were the first [5] to show that the whole meiotic process could occur in vitro in a mammal (the rat). This was proven by cytological and cytometrical methods and by germ cell specific gene expression; subsequently we showed that the development of the meiotic step, in vitro, in the testis of pubertal rats was close to what happens in vivo when considering the changes in the cell populations of different ploidy, the gene expression of germ cells, the kinetics of differentiation of BrdU-labeled early or middle pachytene spermatocytes and the levels of apoptosis in the different cell populations, even if the rate of in vitro differentiation of BrdU-labeled spermatocytes slowed down when reaching the stage of middle pachytene spermatocytes [11]. Then, we also showed, there was no significant difference between the percentages of leptotene, zygotene, pachytene, and diplotene stages in 42-day-old rats and on day 16 of culture of seminiferous tubules from 23 day-old rats, indicating a similar development in vivo and in vitro [12].

We showed that FSH and testosterone have positive and somewhat overlapping effects on the meiotic divisions and the post-meiotic expression of a germ cell-specific gene, effects which cannot be related solely to their ability to reduce germinal cell apoptosis [13]. These results have been confirmed by others using KO mouse models [14-16]. We have shown that βNGF and its two receptors are similarly expressed in vivo and under our culture conditions and that both βNGF and TGFβ are able to regulate the second meiotic division of rat spermatocytes by blocking secondary spermatocytes in metaphase (metaphase II) [17,18]. In vertebrates, mature oocytes are arrested at metaphase II until fertilization, because of the presence of a cytostatic factor (CSF) in their cytoplasm. We have shown that Mos, Emi2, cyclin E and Cdk2, the four proteins of CSF are present in male rat meiotic cells. In co-cultures of pachytene spermatocytes with Sertoli cells, β-NGF increases the number of metaphases II, while enhancing Mos and Emi2 levels in middle to late pachytene spermatocytes, pachytene spermatocytes in division and secondary spermatocytes; these results suggest strongly that CSF is not restricted to the oocyte [6,7]. In addition, they reinforce the view that, βNGF by enhancing Mos in late spermatocytes, is one of the intra-testicular factors which adjusts the number of round spermatids that can be supported by Sertoli cells. Actually, the yield of meiosis in vivo is not four round spermatids from one pachytene spermatocyte, but only two round spermatids from one pachytene spermatocyte [19]. Thereafter, we have demonstrated that βNGF and TGFβ1 have a totally redundant effect on this step [6,7]. These results may offer an explanation to the study of Ingman and Robertson who did not observe any effect on male meiosis of the absence of TGFβ1 in SCID null mutant mice [20]. Indeed, male gametes synthesize TGFβ1 with greatest abundance detected in spermatocytes and early round spermatids i.e., precisely, the same germ cell types which synthesize β-NGF [18]. Therefore, it is more tempting to speculate that the absence of effects of TGFβ1 knock out reported by these authors was due to the redundant effect of β-NGF on that step. We have shown that meiotic progression of rat spermatocytes requires mitogen-activated protein kinases of Sertoli cells and close contacts between the germ cells and the Sertoli cells [21,22]. Now, connexin 43 is detected between spermatocytes and Sertoli cells in our cultures [21]. These results fit with a study [23] showing that replacement of connexin 43 by connexin 26 in transgenic mice impairs spermatogenesis leading to the absence of germ cells beyond primary spermatocytes.

Furthermore, we have shown that both meiotic divisions are blocked by pharmacological inhibitors of MPF [24], as could be expected [25]. On the mitotic step of spermatogenesis, we have got results consistent with a role of GDNF in inhibiting the S-phase entrance of a large subset of differentiated type A spermatogonia, together with an enhancing effect of the factor on a small population of undifferentiated (stem cells) spermatogonia [26]. Actually these studies fit quite well with the results of the in vivo studies [27-30]. Further, we have shown that Cx43 gap junctions between Sertoli cells participate in the control of Sertoli cell proliferation and that Cx43 gap junctions between Sertoli cells and spermatogonia are indirectly involved in germ cell number increase by controlling germ cell survival rather than germ cell proliferation [4]. Similarly, by using Sertoli cell conditional Cx43 knock-out mice recent findings have reported that Cx43 is essential for control of Sertoli cell proliferation and differentiation [22,31]. Recently, we showed that our seminiferous tubule culture model, in bicameral chambers, allowed the settlement of the blood-testis barrier (BTB) in 8-day-old male rat cultures and the differentiation of spermatogonia into round spermatids [32]. Taken together, such data support the view that our co-culture systems enable to highlight mechanisms pertinent to the physiological processes. Hence toxicological studies on toxicants, and/or their identified “active” metabolites, performed with these models are most relevant.

Scientific Validation of these Systems (Toxicology)

We compared cultures of normal, and irradiated by gamma rays germ cells. In spermatocytes for non-irradiated cultures, the comet assay revealed the presence of breaks of DNA, whose number decreased during the culture, resulting from the involvement of mechanisms of DNA repair associated with meiotic recombination. In irradiated cells, the development of DNA strand breaks was heavily modified. Thus, our model is able to detect genotoxic lesions and/or abnormal DNA repairing [33,34]. Numerous studies have shown the toxicity of heavy metals to spermatogenic cells. A growing body of studies is available regarding the reproductive effects of chromium (VI) in men and animals [35-37], but a detailed analysis of spermatogenesis is not available. Kawanishi et al. have demonstrated that chromium (VI) produce noxious ROS including superoxide anion, singlet oxygen, and hydroxyl radicals through the formation of chromium (V) intermediates [38]. In male mice exposed to chromium (V), the major finding reported was the alteration of permeability of the blood-testis barrier [37]. A significant reduction in semen quality is also observed in male welders occupationally exposed to chromium: decrease in sperm count, mobility and vitality, large number of morphologically abnormal spermatozoa, and these semen changes are dose dependent [36]. Low concentrations of Cadmium are found in food and water [39]. Cadmium loading of the environment is a result of human activities such as fossil fuel combustion, agriculture, and the manufacturing of Nickel-Cadmium batteries. High concentrations of Cadmium in male smokers’ seminal fluid and blood are associated with high oxidative stress and damage. The testis is extremely vulnerable to Cadmium as shown by in vivo and in vitro studies in several mammalian species [40-42]. Cadmium induces poor semen quality and carcinogenicity [43,44]. Although numerous studies describe the testicular damages induced by Cadmium, the mechanisms underlying its toxicity remain not completely understood. Chung and Cheng and Siu et al showed that the blood-testis barrier is particularly vulnerable to Cadmium which inhibits, dose-dependently, the assembly of Sertoli cells tight junction-permeability barrier [42,45]. It was hypothesized that activation of c-JNK signal transduction pathway by Cadmium could lead to general cellular apoptosis in the seminiferous epithelium [46]. Additionally hypothesized was the fact that Cadmium mimics the effects of androgens [47] and has potent estrogen-like activity [48].

We have studied, in our culture systems, the effects of concentrations of chromium or Cadmium similar to those which can be observed in the blood (“physio-toxicology”) on the spermatogenic process. [3,12,49]. The numbers of late spermatocytes and of round spermatids were decreased by chromium(VI) even at the lower concentration (1 µg/L). The percentage of synaptonemal complex abnormalities increased slightly with the time of culture and dramatically with chromium (VI) concentrations. This study shows that chromium (VI) is toxic for meiotic cells even at low concentrations, and its toxicity increases in a dose-dependent manner. The number of Sertoli cells did not appear to be affected by Cadmium. By contrast, spermatogonia and meiotic cells were decreased by 1 and 10 μg/L Cadmium in a time and dose dependent manner. Cadmium caused a time-and-dose-dependent increase of total abnormalities, of fragmented Synaptonemal Complexes and of asynapsis from concentration of 0.1 μg/L. Additionally, we observed a new Synaptonemal Complexe abnormality, the “motheaten” Synaptonemal Complexes. This abnormality is frequently associated with asynapsis and Synaptonemal Complexes widening which increased with both the Cadmium concentration and the duration of exposure. This abnormality suggests that Cadmium disrupts the structure and function of proteins involved in pairing and/or meiotic recombination. These resultsshow that Cadmium induces dose-and-time-dependent alterations of the meiotic process of spermatogenesis exvivo, and that the lowest metal concentration, which induces an adverse effect, may vary with the cell parameter studied. Our systems have been used to study the effects of low doses of Bisphenol A which is a plasticizer commonly found in many consumer and industrial products. Bisphenol A is an endocrine disruptor due to its structural similarity to estrogen and suspected to be harmful to spermatogenesis despite some controversy [50-53]. Our work has shown the deleterious effects of BPA (1 nM and 10 nM) by combining transcriptomic analysis and analysis of synaptonemal complexes by immunocytochemistry. BPA interferes with the course of meiosis by altering the synaptonemal complexes of spermatocytes. The transcriptomic analysis carried out on 120 genes involved in the first prophase of meiosis confirms the immunocytochemical observations because the transcription of 60 of these genes is modified [54].

Lilly Research Laboratories financed a pilot study to evaluate in our model the toxicity of 4 molecules known for their testicular toxicity in in vivo studies: 1,3-dinitrobenzene (at 6 µM or 60 µM), methoxyacetic acid (at 0.5 mM or 2.5 mM), Bisphenol A (at 5 µM or 50 µM) and lindane (at 5 µM or 30 µM). DNB is a nitroaromatic compound used in the production of polymers, pesticides and dyes. In vivo studies have shown that DNB induces severe effects including Sertoli cell vacuolation, spermatocyte depletion and multinucleated and misshapen spermatids [55,56]. MAA is the toxic metabolite of 2-methoxyethanol, a solvent used in printing inks, varnishes, and as a de-icing additive. The toxic effect of MAA on pachytene spermatocytes, has been described in vivo [57,58]. Although Leydig cells are a target of BPA [59], in vitro studies using primary Sertoli cells demonstrated direct targeting through disruption of cell-cell signaling [56-61]. Lindane is a pesticide used in both agriculture and parasiticidal treatment for lice. Lindane induces apoptosis in Sertoli cells as well as in spermatogonia and spermatocytes [62]. Bio-Alter®has made it possible to find the same effects as those described in vivo for these 4 compounds: Sertolian toxicity and disruption of spermatogenesis. In addition, we were able to hypothesize different mechanisms of action explaining their toxicity and also overcome the shortcomings of the European ReProTect project for 1,3-dinitrobenzene and methoxyacetic acid. It should be noted that this project (www.reprotect.eu) included a battery of 14 tests responsible for predicting the disruption of female and male fertility and embryonic development, but that the tests used were unable to predict the toxicity of 1,3-dinitrobenzene and of methoxyacetic acid on spermatogenesis [63]. The results show very close similarities between the results of the published in-vivo studies and the results obtained using the Bio-AlteR® technology. Our model can also respond to the need of some eco-toxycology studies. A large number of environmental factors can pollute air, water and food and may potentiate each other when present as a mixture [32,64,65]. Numerous studies suggest a decline in semen quality in some parts of the world [49,66-73]. This might occur in response to adverse environmental factors [74].

We then Tested Pesticides Selected on the Basis of Their Persistence in Water and Food

Carbendazim is a broad-spectrum benzimidazole fungicide used to prevent and eliminate fungal plant diseases [75]. Carbendazim has been reported to induce, in vivo, a number of testicular alterations such as atrophic seminiferous tubules [76,77], decreased germ cell numbers, sloughing of the seminiferous epithelium [77-80] and abnormal development of the acrosome [81]. Carbendazim has been also reported to induce dysfunction of the somatic Sertoli cells cell by inhibiting microtubule assembly in a colchicine-like manner [75,82-84]. However, the mechanism of action of carbendazim was not fully elucidated, especially at low doses, even if its status of endocrine disruptor has been raised [85,86]. We tested 3 low concentrations of carbendazim: 50 nM, 500 nM and 5 μM. It should be emphasized that 5 μM (IC50 for the colchicine-like effect) is 60 times lower than the serum concentration of rats treated with 25 mg of CBZ/kg weight/day for 48 days [87]. We have shown that Carbendazim induces an alteration of the blood-testicular barrier (decrease in Connexin 43 and its functionality) and proves to be an endocrine disruptor (androgen-like effect) at a concentration of 50 nM by regulating on the one hand the estrogen receptor messenger RNAs (Erα and ERß) and secondly by increasing the messenger RNAs of TP1 and TP2, two androgen-dependent genes specific to round spermatids [88]. An additional advantage of our culture systems is that they make it possible to test the effect of combinations of molecules at low concentrations for a period of several weeks.

As an example, we studied the cocktail (carbendazim 50 nM- iprodione 50 nM) because unlike carbendazim, iprodione, a dicarboximide fungicide, is known to have anti-androgenic effects by lowering circulating testosterone levels, inhibiting testicular testosterone production and delaying pubertal development in male rats [89]. Moreover, these two pesticides are present together in the environment following their use alone or in cocktails in marketed products. We have shown by transcriptomic and proteomic studies, in rat seminiferous tubule cultures, that the cocktail triggers effects greater than the sum of the cumulative effects of 50 nM CBZ and 50 nM IPR tested separately [90]. We also compared the effects of the cocktail (carbendazim 50 nM- iprodione 50 nM) and of each of these 2 fungicides at 50 nM in cultures of rat seminiferous tubules, on physiological parameters. Our results show that (i) the presence of iprodione with carbendazim (cocktail) cancels the effect of carbendazim on the increase in androgen-dependent TP1 and TP2 mRNAs and on the decrease in Erα and ERß mRNAs. Nevertheless, carbendazim alone or iprodione alone or the cocktail induces toxicity on spermatogenesis by decreasing the number of round spermatids (the direct precursors of spermatozoa). These results strongly suggest that at the concentrations used, the effects of iprodione and carbendazim do not solely depend on their respective anti-androgen and androgen-like effects but should involve several mechanisms of action [65].

A cocktail of two micropollutants (benzo[a]pyrene and atrazine at 1 µg/L each) was also tested. Atrazine has been one of the widely used agricultural pesticides all over the world. Atrazine concentrations varied from 0.2 to 14.7 μg/L in soil-water samples from La Côte Saint André (Isère, France) [91]. Studied reservoirs in Brazil showed the presence of atrazine at mean levels from 7.0 to 15.0 μg/L [92]. Although its use has been banned in France from 2003, and by the European Union from 2007, atrazine can be persistent in the soil and rivers. Atrazine is now recognized to display endocrine disrupting effects on the male reproductive system of mammals [93-95]. In rodents, in vivo studies have shown that exposure to atrazine delays puberty (Stoker et al., 2000)-[94], decreases testosterone and increases estradiol levels [95], alters meiosis [96], and reduces sperm counts [95,97]. Benzo[a]pyrene can be formed as a result of incomplete combustion from industrial processes, smoking tobacco, charring of grilled foods, and exhaust from diesel and gasoline engines. Inhalation and oral ingestion are two major routes of Benzo[a]pyrene exposure [98]. Values for oral exposure to Benzo[a]pyrene through drinking water range from 0.007 to 0.7 μg/L [99]. In vivo, Benzo[a]pyrene can affect the hormonal balance and gonadal tissue growth and development, induces apoptosis in male germ cells leading to decreased spermatozoa quality and quantity [100-104]. Inhibition of meiotic divisions of rat spermatocytes by Benzo[a]pyrene has been shown in an early in vitro study [104]. The effect of the mixture (benzo[a]pyrene and atrazine at 1 µg/L each) was investigated in our validated BioAlter® model, either during or after the establishment of the blood-testis barrier by using 8- or 20-22-day-old rats. Cultures were performed over a 3-week period. Our results showed that the mixture reduced the number of round spermatids by targeting the middle to late pachytene spermatocytes. These effects were observed in 8- and in 20-22-day-old rat seminiferous tubule cultures. However, the decrease of the number of round spermatids was faster and more marked in the 8-day- than in the 20-22-day-old rat seminiferous tubule cultures. Hence, our study emphasizes the possible influence of the age of an individual on the effect of (a) toxicant(s) on spermatogenesis [32].

We then tested the effects of Glyphosate alone, the most used herbicide in the world today [33]. Indeed, Glyphosate has been mixed with other chemicals to constitute glyphosate-based herbicides such as Roundup® which has been used in agricultural fields and home gardens [105]. Formulated glphosate and its degradation products accumulate in the environment [106]. Glyphosate has been detected in various waters: (i) from 2 to 430 μg/L (11.8 nM to 2.5 μM) in river water and stream water in the USA [107-110] (ii) from 0.1 to 2.5 μg/L i.e. 0.6 nM to 14.8 nM [111,112] in various waters in Europe. Higher levels (up to 165 μg/L i.e. 976 nM) were found in France [113] and Denmark [114]. Human beings may be exposed to glyphosate through food and drinking water [115]. Controversial in vivo studies exist on the effect of glyphosate alone on male reproductive system. Two studies have shown that glyphosate affects spermatogenesis (i) in mice by decreasing the number of spermatozoa and the plasma levels of testosterone [116], (ii) in rabbits resulting in a decline in ejaculate volume and sperm concentration [117]. By contrast, two groups have shown that glyphosate does not affect male reproduction in rat [118,119]. There are only very few in vitro studies on the effect of glyphosate alone on primary cultures of testicular cells. Seralini’s team showed that within 24-48 h glyphosate was essentially toxic to Sertoli cells [120]. The group of Meroni showed that glyphosate could alter the blood-testis barrier at a high concentration (100 μg/mL (588 μM) [121]. Toxic effects on sperm progressive motility but not on sperm DNA integrity were induced in human by glyphosate alone at 360 μg/L (2.1 μM), a concentration that greatly exceed environmental exposures in Europe [122]. We tested the effects of low concentrations of Glyphosate (50 nM, 500 nM, 5 µM or 50 µM) in our model. The observed decrease of Clusterin mRNAs by glyphosate at 50 nM or 500 nM suggested that glyphosate would target the integrity of Sertoli cells. Glyphosate targeted also young spermatocytes and middle to late pachytene spermatocytes resulting in a decrease of the numbers of round spermatids. This study underlines that the effect of a toxicant should be also studied at low doses and during the establishment of the blood-testis barrier [123]. More recently, we evaluated the effects of waters of different origins, hospital effluent waters and/or different drinking waters) on several parameters of the seminiferous epithelium. Concentrated culture medium was diluted with the waters to be tested (final concentrations of the tested waters were between 8 and 80%). The integrity of the blood-testis barrier was assessed by the trans-epithelial electric resistance (TEER). The levels of mRNAs specific of Sertoli cells, of cellular junctions, of each population of germ cells, of androgen receptor, of estrogen receptor α and of aromatase were also studied. We showed that some waters may have an impact on some parameters involved in the process and/or regulation of spermatogenesis, directly at the level of the seminiferous epithelium, including some endpoints related to possible endocrine disrupting effects [124]. Although our model allows reducing the number of rats by 20 to 30 as compared to in vivo studies; in agreement with the 3R rule (Reduction, Refinement and Replacement), it does not provide an answer to the cosmetics industry, which must no longer kill animals for toxicology studies.

In order to replace the use of animals in toxicology studies, we settled the culture of pre-pubertal domestic cat seminiferous tubules (veterinary wastes) in our model. We carried out a comparative study on the effects of 3 testicular toxicants, 1,3-dinitrobenzene at 60 µM, 2-methoxyacetic acid at 2.5 mM and carbendazim at 50 nM or 500 nM in cat or rat seminiferous tubule cultures for a period of 3 weeks. Sertoli cell or each germ cell populations as well as the levels of Sertoli cell or germ cell specific mRNAs were studied. The harmful effects of the 3 toxicants on pre-meiotic, meiotic and post-meiotic cell numbers and on Sertoli or germ cell specific mRNAs were clearly observed in the two species, even if there might be some small differences in the intensity of the effects on some of the studied parameters. Hence, the culture of prepubertal domestic cat seminiferous tubules in our validated BioAlter® model might be a solution to the requirements of the EU cosmetics directive and REACH legislation for male reproductive toxicology studies [125].

Conclusions

It should be underlined that most of the growth factors, cytokines, neurotrophins and steroid hormones produced within the testis, and necessary for spermatogenesis, are widely expressed in the organism and/or necessary for the regulation of other vital functions. For instance, (i) the glial cell-derived neurotrophic factor (GDNF) has been observed in the central nervous system [126] and peripheral organs including the kidneys, lungs, blood, and testes [127-129]; (ii) the receptor tyrosine kinase c-Kit and its ligand Stem Cell Factor (SCF) are involved in haemopoiesis, melanogenesis and spermatogenesis [130,131]. In this line, it has been reported that men with impaired semen parameters have an increased mortality rate suggesting semen quality may provide a fundamental biomarker of overall male health [132,133]. Taken together, these features suggest the possibility of using this in vitro bioassay as a “warning system”.

Acknowledgment

We wish to thank all our colleagues who participated in the realization of our presented studies

References

  1. Chen X, An H, Ao L, Sun L, Liu W, et al. (2011) The combined toxicity of dibutyl phthalate and benzo(a)pyrene on the reproductive system of male Sprague Dawley rats in vivo. Journal of Hazardous Materials 186: 835-841. [crossref]
  2. Archibong AE, Ramesh A, Niaz MS, Brooks CM, Roberson SI, et al. (2008) Effects of benzo(a)pyrene on intra-testicular function in F-344 rats. International Journal of Environmental Research and Public Health 5: 32-40. [crossref]
  3. Moffat I, Chepelev NL, Labib S, Bourdon-Lacombe J, Kuo B, et al. (2015) Comparison of toxicogenomics and traditional approaches to inform mode of action and points of departure in human health risk assessment of benzo[a]pyrene in drinking water. Critical Reviews in Toxicology 45: 1-43. [crossref]
  4. Boström CE, Gerde P, Hanberg A, Jernström B, Johansson C, et al. (2002) Cancer risk assessment, indicators, and guidelines for polycyclic aromatic hydrocarbons in the ambient air. Environmental Health Perspectives 110: 451-488. [crossref]
  5. Abarikwu SO, Adesiyan AC, Oyeloja TO, Oyeyemi MO, Farombi EO (2010) Changes in sperm characteristics and induction of oxidative stress in the testis and epididymis of experimental rats by a herbicide, atrazine. Archives of Environmental Contamination and Toxicology 58: 874-882. [crossref]
  6. Gely-Pernot A, Hao C, Becker E, Stuparevic I, Kervarrec C, et al. (2015) The epigenetic processes of meiosis in male mice are broadly affected by the widely used herbicide atrazine. BMC Genomics 16, 885. of Andrology 14: 584-590. [crossref]
  7. Victor-Costa AB, Bandeira SMC, Oliveira AG, Mahecha GAB, Oliveira CA (2010) Changes in testicular morphology and steroidogenesis in adult rats exposed to Atrazine. Reproductive Toxicology 29: 323-331. [crossref]
  8. Stoker TE, Laws SC, Guidici DL, Cooper RL (2000) The effect of atrazine on puberty in male wistar rats: an evaluation in the protocol for the assessment of pubertal development and thyroid function. Toxicological sciences : an official journal of the Society of Toxicology 58: 50-59. [crossref]
  9. Jin Y, Wang L, Fu Z (2013) Oral exposure to atrazine modulates hormone synthesis and the transcription of steroidogenic genes in male peripubertal mice. General and Comparative Endocrinology 184: 120-127. [crossref]
  10. Sousa, A.S., Duaví, W.C., Cavalcante, R.M., Milhome, M.A.L., Do Nascimento, R.F., 2016. Estimated Levels of Environmental Contamination and Health Risk Assessment for Herbicides and Insecticides in Surface Water of Ceará, Brazil. Bulletin of Environmental Contamination and Toxicology 96: 90-95. [crossref]
  11. Tasli S, Patty L, Boetti H, Ravanel P, Vachaud G (1996) Persistence and leaching of atrazine in corn culture in the Experimental Site of La Côte Saint André (Isère, France). Archives of Environmental Contamination and Toxicology 30: 203-212.
  12. Pisani C, Voisin S, Arafah K, Durand P, Perrard MH, et al. (2016) Ex vivo assessment of testicular toxicity induced by carbendazim and iprodione, alone or in a mixture. Altex 33: 393-413. [crossref]
  13. Blystone CR, Lambright CS, Furr J, Wilson VS, Gray LE (2007) Iprodione delays male rat pubertal development, reduces serum testosterone levels, and decreases ex vivo testicular testosterone production. Toxicology Letters 174: 74-81. [crossref]
  14. Carette D, Blondet A, Martin G, Montillet G, Janczarski S (2015) Endocrine Disrupting Effects of Noncytotoxic Doses of Carbendazim on the Pubertal Rat Seminiferous Epithelium: An Ex Vivo Study. Applied In Vitro Toxicology 1: 289-301.
  15. Rajeswary S, Mathew N, Akbarsha MA, Kalyanasundram M, Kumaran B (2007) Protective effect of vitamin E against carbendazim-induced testicular toxicity-histopathological evidences and reduced residue levels in testis and serum. Archives of Toxicology 81: 813-821. [crossref]
  16. Lu SY, JW L, Kuo M (2004) Endocrine-disrupting activity in carbendazim-induced reproductive and developmental toxicity in rats. J Toxicol Environ Health 67: 1501-1515. [crossref]
  17. Hsu YH, Chang CW, Chen MC, Yuan CY, Chen JH, et al. (2011) Carbendazim-induced androgen receptor expression antagonized by flutamide in male rats. Journal of Food and Drug Analysis 19: 418-428.
  18. Winder BS, Strandgaard CS, Miller MG (2001) The role of GTP binding and microtubule-associated proteins in the inhibition of microtubule assembly by carbendazim. Toxicological Sciences 59: 138-146. [crossref]
  19. NAKAI M, HESS RA, NETSU J, NASU T (1995) Deformation of the Rat Sertoli Cell by Oral Administration of Carbendazim (Methyl 2‐Benzimidazole Carbamate). Journal of Andrology 16: 410-416. [crossref]
  20. Correa LM, Miller MG (2001) Microtubule depolymerization in rat seminiferous epithelium is associated with diminished tyrosination of alpha-tubulin. Biology of reproduction 64: 1644-1652. [crossref]
  21. Nakai M, Toshimori K, Yoshinaga K, Nasu T, Hess RA (1998) Carbendazim-induced abnormal development of the acrosome during early phases of spermiogenesis in the rat testis. Cell and Tissue Research 294: 145-152[crossref]
  22. Yu G, Guo Q, Xie L, Liu Y, Wang X (2009) Effects of subchronic exposure to carbendazim on spermatogenesis and fertility in male rats. Toxicology and Industrial Health 25: 41-47. [crossref]
  23. Moffit JS, Bryant BH, Hall SJ, Boekelheide K (2007) Dose-dependent effects of sertoli cell toxicants 2,5-hexanedione, carbendazim, and mono-(2-ethylhexyl) phthalate in adult rat testis. Toxicologic pathology 35: 719-727. [crossref]
  24. Hess RA, Moore B, Forrer J, Linder RE, Abuel-Atta A (1991) The fungicide benomyl (methyl 1-(butylcarbamoyl)-2-benzimidazolecarbamate) causes testicular dysfunction by inducing the sloughing of germ cells and occlusion of efferent ductules. Fundamental and Applied Toxicology 17: 733-745. [crossref]
  25. Yu GC, Xie L, Liu Y, zhong, Wang X. fen (2009) Carbendazim affects testicular development and spermatogenic function in rats. Zhonghua nan ke xue = National journal of andrology 15: 505-510. [crossref]
  26. Carter SD, Hess RA, Laskey JW (1987) The fungicide methyl 2-benzimidazole carbamate causes infertility in male Sprague-Dawley rats. Biology of Reproduction 37: 709-717. [crossref]
  27. Lim J, Miller MG (1997) The role of the benomyl metabolite carbendazim in benomyl-induced testicular toxicity. Toxicology and Applied Pharmacology 142: 401-410. [crossref]
  28. Gabrielsen JS, Tanrikut C (2016) Chronic exposures and male fertility: the impacts of environment, diet, and drug use on spermatogenesis. Andrology 4: 648-661. [crossref]
  29. Van Waeleghe, K, De Clercq N, Vermeulen L, Schoonjans F, Comhaire F (1996) Deterioration of sperm quality in young healthy Belgian men. Human Reproduction 11: 325-329. [crossref]
  30. Sripada S, Fonseca S, Lee A, Harrild K, Giannaris D, et al. (2007) Trends in semen parameters in the northeast of Scotland. Journal of Andrology 28: 313-319. [crossref]
  31. Shine R, Peek J, Birdsall M (2008) Declining sperm quality in New Zealand over 20 years. New Zealand Medical Journal 121: 50-56. [crossref]
  32. Mukhopadhyay D, Varghese AC, Pal M, Banerjee SK, Bhattacharyya AK, et al. (2010) Semen quality and age-specific changes: A study between two decades on 3,729 male partners of couples with normal sperm count and attending an andrology laboratory for infertility-related problems in an Indian city. Fertility and Sterility 93: 2247-2254. [crossref]
  33. Lackner J, Schatzl G, Waldhör T, Resch K, Kratzik C, et al. (2005) Constant decline in sperm concentration in infertile males in an urban population: Experience over 18 years. Fertility and Sterility 84: 1657-1661. [crossref]
  34. Carlsen E, Giwercman A, Keiding N, Skakkebaek NE (1992) Evidence for decreasing quality of semen during past 50 years. BMJ (Clinical research ed) 305: 609-613. [crossref]
  35. Auger J, Kunstmann JM, Czyglik FC., Jouannet, P. (1995) Decline in semen quality among fertile men in paris during the past 20 years. New England Journal of Medicine 332: 281-285. [crossref]
  36. Adamopoulos DA, Pappa A, Nicopoulou S, Andreou E, Karamertzanis M, et al. (1996) Seminal volume and total sperm number trends in men attending subfertility clinics in the Greater Athens area during the period 1977-1993. Human Reproduction 11: 1936-1941. [crossref]
  37. Durand P, Martin G, Blondet A, Gilleron J, Carette D, et al. (2017) Effects of low doses of carbendazim or iprodione either separately or in mixture on the pubertal rat seminiferous epithelium: An ex vivo study. Toxicology in Vitro 45: 366-373. [crossref]
  38. Cotton J, Leroux F, Broudin S, Poirel M, Corman B, et al. (2016) Development and validation of a multiresidue method for the analysis of more than 500 pesticides and drugs in water based on on-line and liquid chromatography coupled to high resolution mass spectrometry. Water Research 104: 20-27. [crossref]
  39. Goldstein KM, Seyler DE, Durand P, Perrard MH, Baker TK (2016) Use of a rat ex-vivo testis culture method to assess toxicity of select known male reproductive toxicants. Reproductive Toxicology 60: 92-103. [crossref]
  40. Saradha B, Vaithinathan S, Mathur PP (2009) Lindane induces testicular apoptosis in adult Wistar rats through the involvement of Fas-FasL and mitochondria-dependent pathways. Toxicology 255: 131-139. [crossref]
  41. Li YJ, Song TB, Cai YY, Zhou JS, Song, et al. (2009) Bisphenol A exposure induces apoptosis and upregulation of fas/fasl and caspase-3 expression in the testes of mice. Toxicological Sciences 108: 427-436. [crossref]
  42. Li MWM, Mruk DD, Lee WM, Cheng CY (2010) Connexin 43 is critical to maintain the homeostasis of the blood-testis barrier via its effects on tight junction reassembly. Proceedings of the National Academy of Sciences of the United States of America 107: 17998-18003.
  43. Akingbemi BT, Sottas CM, Koulova AI, Klinefelter GR, Hardy MP (2004) Inhibition of Testicular Steroidogenesis by the Xenoestrogen Bisphenol a Is Associated with Reduced Pituitary Luteinizing Hormone Secretion and Decreased Steroidogenic Enzyme Gene Expression in Rat Leydig Cells. Endocrinology 145: 592-603. [crossref]
  44. Wade MG, Kawata A, Williams A, Yauk C (2008) Methoxyacetic acid-induced spermatocyte death is associated with histone hyperacetylation in rats. Biology of Reproduction 78: 822-831. [crossref]
  45. Chapin RE, Dutton SL, Ross MD, Sumrell BM, Lamb JC (1984) The Effects of Ethylene Glycol Monomethyl Ether on Testicular Histology in F344 Rats. Journal of Andrology 5: 369-380. [crossref]
  46. Hess RA, Linder RE, Strader LF, Perreault SD (1988) Acute Effects and Long‐Term Sequelae of 1,3‐Dinitrobenzene on Male Reproduction in the Rat II. Quantitative and Qualitative Histopathology of the Testis. Journal of Andrology 9: 327-342. [crossref]
  47. Elkin ND, Piner JA, Sharpe RM (2010) Toxicant-induced leakage of germ cell-specific proteins from seminiferous tubules in the rat: Relationship to blood-testis barrier integrity and prospects for biomonitoring. Toxicological Sciences 117: 439-448. [crossref]
  48. Ali S, Steinmetz G, Montillet G, Perrard MH, Loundou A, et al. (2014) Exposure to low-dose bisphenol a impairs meiosis in the rat seminiferous tubule culture model: A physiotoxicogenomic approach. PLoS ONE 9. [crossref]
  49. Tyl RW, Myers CB, Marr MC, Thomas BF, Keimowitz AR, et al. (2002) Three-generation reproductive toxicity study of dietary bisphenol A in CD Sprague-Dawley rats. Toxicological Sciences 68: 121-146. [crossref]
  50. Tohei A, Suda S, Taya K, Hashimoto T, Kogo H (2001) Bisphenol a inhibits testicular functions and increases luteinizing hormone secretion in adult male rats. Experimental Biology and Medicine 226: 216-221. [crossref]
  51. Meeker JD, Ehrlich S, Toth TL, Wright DL, Calafat AM, et al. (2010) Semen quality and sperm DNA damage in relation to urinary bisphenol A among men from an infertility clinic. Reproductive Toxicology 30: 532-539. [crossref]
  52. Cariati F, D’Uonno N, Borrillo F, Iervolino S, Galdiero G, et al. (2019) bisphenol a: An emerging threat to male fertility. Reproductive Biology and Endocrinology. [crossref]
  53. Geoffroy-Siraudin C, Perrard MH, Ghalamoun-Slaimi R, Ali S, Chaspoul F, et al. (2012) Ex-vivo assessment of chronic toxicity of low levels of cadmium on testicular meiotic cells. Toxicol Appl Pharmacol 262: 238-246. [crossref]
  54. Stoica A, Katzenellenbogen BS, Martin MB (2000) Activation of estrogen receptor-α by the heavy metal cadmium. Molecular Endocrinology 14: 545-553. [crossref]
  55. Martin MB, James Voeller H, Gelmann EP, Jianming L, Stoica EG (2002) Role of cadmium in the regulation of AR gene expression and activity. Endocrinology 143: 263-275. [crossref]
  56. Yu X, Hong S, Faustman EM (2008) Cadmium-induced activation of stress signaling pathways, disruption of ubiquitin-dependent protein degradation and apoptosis in primary rat sertoli cell-gonocyte cocultures. Toxicological Sciences 104: 385-396. [crossref]
  57. Chung NPY, Cheng CY (2001) Is cadmium chloride-induced inter-Sertoli tight junction permeability barrier disruption a suitable in vitro model to study the events of junction disassembly during spermatogenesis in the rat testis?. Endocrinology 142: 1878-1888. [crossref]
  58. Goyer RA, Liu J, Waalkes MP (2004) Cadmium and cancer of prostate and testis. Ln: BioMetals. Pg: 555-558.
  59. Benoff S, Hauser R, Marmar JL, Hurley IR, Napolitano B, et al. (2009) Cadmium concentrations in blood and seminal plasma: Correlations with sperm number and motility in three male populations (infertility patients, artificial insemination donors, and unselected volunteers). Molecular Medicine 15, 248-262. [crossref]
  60. Siu ER, Mruk DD, Porto CS, Cheng CY (2009) Cadmium-induced testicular injury. Toxicology and Applied Pharmacology. [crossref]
  61. Chia SE, Xu B, Ong CN, Tsakok FMH, Lee ST (1994) Effect of cadmium and cigarette smoking on human semen quality. International Journal of Fertility and Menopausal Studies 39: 292-298. [crossref]
  62. Benoff S, Auborn K, Marmar JL, Hurley IR (2008) Link between low-dose environmentally relevant cadmium exposures and asthenozoospermia in a rat model. Fertility and Sterility. [crossref]
  63. Järup L, Åkesson A (2009) Current status of cadmium as an environmental health problem. Toxicology and Applied Pharmacology. [crossref]
  64. Kawanishi S, Inoue S, Sano S (1986) Mechanism of DNA cleavage induced by sodium chromate (VI) in the presence of hydrogen peroxide. Journal of Biological Chemistry 261: 5952-5958. [crossref]
  65. De Lourdes Pereira M, Santos TM, Garcia E Costa F, Pedrosa De Jesus J (2004) Functional changes of mice Sertoli cells induced by Cr(V). Cell Biology and Toxicology 20: 285-291. [crossref]
  66. Danadevi K, Rozati R, Reddy PP, Grover P (2003) Semen quality of Indian welders occupationally exposed to nickel and chromium. Reproductive Toxicology 17: 451-456. [crossref]
  67. Aruldhas MM, Subramanian S, Sekar P, Vengatesh G, Chandrahasan G, et al. (2005) Chronic chromium exposure-induced changes in testicular histoarchitecture are associated with oxidative stress: Study in a non-human primate (Macaca radiata Geoffroy). Human Reproduction 20: 2801-2813. [crossref]
  68. Perrin J, Lussato D, De Méo M, Durand P, Grillo JM (2007) Evolution of DNA strand-breaks in cultured spermatocytes: The Comet Assay reveals differences in normal and γ-irradiated germ cells. Toxicology in Vitro 21: 81-89. [crossref]
  69. Benbrook CM (2016) Trends in glyphosate herbicide use in the United States and globally. Environmental Sciences Europe 28: 1-15. [crossref]
  70. Durand P, Blondet A, Martin G, Carette D, Pointis G, et al. (2020) Effects of a mixture of low doses of atrazine and benzo[a]pyrene on the rat seminiferous epithelium either during or after the establishment of the blood-testis barrier in the rat seminiferous tubule culture model. Toxicology In Vitro 62.
  71. Sridharan S, Simon L, Meling DD, Cyr DG, Gutstein DE (2007) Proliferation of Adult Sertoli Cells Following Conditional Knockout of the Gap Junctional Protein GJA1 (Connexin 43) in Mice1. Biology of Reproduction 76: 804-812. [crossref]
  72. Yomogida K, Yagura Y, Tadokoro Y, Nishimune Y (2003) Dramatic expansion of germinal stem cells by ectopically expressed human glial cell line-derived neurotrophic factor in mouse sertoli cells. Biology of Reproduction 69: 1303-1307.
  73. Tadokoro Y, Yomogida K, Ohta H, Tohda A, Nishimune Y (2002) Homeostatic regulation of germinal stem cell proliferation by the GDNF/FSH pathway. Mechanisms of Development 113: 29-39. [crossref]
  74. Meng X, Lindahl M, Hyvönen ME, Parvinen M, De Rooij DG, et al. (2000) Regulation of cell fate decision of undifferentiated spermatogonia by GDNF. Science 287: 1489-1493. [crossref]
  75. Creemers LB, Meng X, Den Ouden K, Van Pelt AMM, Izadyar F, et al. (2002) Transplantation of germ cells from glial cell line-derived neurotrophic factor-overexpressing mice to host testes depleted of endogenous spermatogenesis by fractionated irradiation. Biology of Reproduction 66: 1579-1584.
  76. Fouchécourt S, Godet M, Sabido O, Durand P (2006) Glial cell-line-derived neurotropic factor and its receptors are expressed by germinal and somatic cells of the rat testis. Journal of Endocrinology 190: 59-71. [crossref]
  77. Ortega S, Prieto I, Odajima J, Martín A, Dubus P, et al. (2003) Cyclin-dependent kinase 2 is essential for meiosis but not for mitotic cell division in mice. Nature Genetics 35: 25-31. [crossref]
  78. Godet M, Dametoy A, Mouradian S, Rudkin BB, Durand P (2004) Key Role for Cyclin-Dependent Kinases in the First and Second Meiotic Divisions of Rat Spermatocytes. Biology of Reproduction 70: 1147-1152. [crossref]
  79. Winterhager E, Pielensticker N, Freyer J, Ghanem A, Schrickel JW, et al. (2007) Replacement of connexin43 by connexin26 in transgenic mice leads to dysfunctional reproductive organs and slowed ventricular conduction in the heart. BMC Developmental Biology 7. [crossref]
  80. Brehm R, Zeiler M, Rüttinger C, Herde K, Kibschull M, et al. (2007) A sertoli cell-specific knockout of connexin43 prevents initiation of spermatogenesis. American Journal of Pathology 171: 19-31.
  81. Godet M, Sabido O, Gilleron J, Durand P (2008) Meiotic progression of rat spermatocytes requires mitogen-activated protein kinases of Sertoli cells and close contacts between the germ cells and the Sertoli cells. Developmental Biology 315: 173-188. [crossref]
  82. Ingman WV, Robertson SA (2007) Transforming growth factor-β1 null mutation causes infertility in male mice associated with testosterone deficiency and sexual dysfunction. Endocrinology 148: 4032-4043. [crossref]
  83. Yang Z, Wreford NG, De Kretser DM (1990) A quantitative study of spermatogenesis in the developing rat testis. Biology of Reproduction 43: 629-635. [crossref]
  84. Perrard MH, Vigier M, Damestoy A, Chapat C, Silandre D, et al. (2007) β-nerve growth factor participates in an auto/paracrine pathway of regulation of the meiotic differentiation of rat spermatocytes. Journal of Cellular Physiology 210: 51-62. [crossref]
  85. Damestoy, A., Perrard, M.H., Vigier, M., Sabido, O., Durand, P., 2005. Transforming growth factor beta-1 decreases the yield of the second meiotic division of rat pachytene spermatocytes in vitro. Reproductive Biology and Endocrinology 3. [crossref]
  86. Holdcraft RW, Braun RE (2004) Androgen receptor function is required in Sertoli cells for the terminal differentiation of haploid spermatids. Development 131: 459-467. [crossref]
  87. De Gendt K, Swinnen JV, Saunders PTK, Schoonjans L, Dewerchin M, et al. (2004) A Sertoli cell-selective knockout of the androgen receptor causes spermatogenic arrest in meiosis. Proceedings of the National Academy of Sciences of the United States of America 101: 1327-1332. [crossref]
  88. Abel M, Wootton A, Wilkins V, Huhtaniemi I, Knight P, et al. (2000) The Effect of a Null Mutation in the Follicle-Stimulating Hormone Receptor Gene on Mouse Reproduction. Endocrinology 141: 1795-1803. [crossref]
  89. Vigier M, Weiss M, Perrard MH, Godet M, Durand P (2004) The effects of FSH and of testosterone on the completion of meiosis and the very early steps of spermiogenesis of the rat: An in vitro study. Journal of Molecular Endocrinology 33: 729-742. [crossref]
  90. Geoffroy-Siraudin C, Perrard MH, Chaspoul F, Lanteaume A, Gallice P, et al. (2010) Validation of a rat seminiferous tubule culture model as a suitable system for studying toxicant impact on meiosis effect of hexavalent chromium. Toxicological Sciences 116: 286-296. [crossref]
  91. Perrard MH, Hue D, Staub C, Le Vern Y, Kerboeuf D, et al. (2003) Development of the meiotic step in testes of pubertal rats: Comparison between the in vivo situation and under in vitro conditions. Molecular Reproduction and Development 65: 86-95. [crossref]
  92. Weiss M, Vigier M, Hue D, Perrard-Sapori MH, Marret C, et al. (1997) Pre- and postmeiotic expression of male germ cell-specific genes throughout 2-week cocultures of rat germinal and Sertoli cells. Biology of Reproduction 57: 68-76. [crossref]
  93. Marret C, Durand P (2000) Culture of porcine spermatogonia: Effects of purification of the germ cells, extracellular matrix and fetal calf serum on their survival and multiplication. Reproduction Nutrition Development 40: 305-319.
  94. Hue D, Staub C, Perrard-Sapori MH, Weiss M, Nicolle JC, et al. (1998) Meiotic differentiation of germinal cells in three-week cultures of whole cell population from rat seminiferous tubules. Biology of reproduction 59: 379-387. [crossref]
  95. Perrard MH, Durand P (2009) Redundancy of the effect of TGFβ1 and β-NGF on the second meiotic division of rat spermatocytes. Microscopy Research and Technique 72: 596-602. [crossref]
  96. Perrard MH, Chassaing E, Montillet G, Sabido O, Durand P (2009) Cytostatic factor proteins are present in male meiotic cells and β-nerve growth factor increases Mos levels in rat late spermatocytes. PLoS ONE 4. [crossref]
  97. Staub C, Hue D, Nicolle JC, Perrard-Sapori MH, Segretain D (2000) The whole meiotic process can occur in vitro in untransformed rat spermatogenic cells. Experimental Cell Research 260, 85-95. [crossref]
  98. Gilleron J, Carette D, Durand P, Pointis G, Segretain D (2009) Connexin 43 a potential regulator of cell proliferation and apoptosis within the seminiferous epithelium. International Journal of Biochemistry and Cell Biology 41: 1381-1390. [crossref]
  99. Carette D, Perrard MH, Prisant N, Gilleron J, Pointis G, et al. (2013) Hexavalent chromium at low concentration alters Sertoli cell barrier and connexin 43 gap junction but not claudin-11 and N-cadherin in the rat seminiferous tubule culture model. Toxicology and Applied Pharmacology 268: 27-36. [crossref]
  100. Parvinen M (1982) Regulation of the seminiferous epithelium. Endocrine Reviews 3: 404-417. [crossref]
  101. Kierszenbaum AL (1994) Mammalian spermatogenesis in vivo and in vitro: A partnership of spermatogenic and somatic cell lineages. Endocrine Reviews 15: 116-134. [crossref]
  102. Jeng HA, Bocca SM (2013) Influence of Exposure to Benzo[a]pyrene on Mice Testicular Germ Cells during Spermatogenesis. Journal of Toxicology 2013: 1-9. [crossref]
  103. Ramesh A, Inyang F, Lunstra DD, Niaz MS, Kopsombut P (2008) Alteration of fertility endpoints in adult male F-344 rats by subchronic exposure to inhaled benzo(a)pyrene. Experimental and Toxicologic Pathology 60, 269-280. [crossref]
  104. Georgellis A, Toppari J, Veromaa T, Rydström J, Parvinen M (1990) Inhibition of meiotic divisions of rat spermatocytes in vitro by polycyclic aromatic hydrocarbons. Mutation Research – Fundamental and Molecular Mechanisms of Mutagenesis 231: 125-135. [crossref]
  105. Gillezeau C, Gerwen M van, Shaffer RM, Rana I, Zhang L, et al. (2019) The evidence of human exposure to glyphosate: a review | Environmental Health | Full Text. Environ health 18.
  106. Battaglin WA, Meyer MT, Kuivila KM, Dietze JE (2014) Glyphosate and its degradation product AMPA occur frequently and widely in U.S. soils, surface water, groundwater, and precipitation. Journal of the American Water Resources Association 50: 275-290.
  107. Battaglin WA, Rice KC, Focazio MJ, Salmons S, Barry RX (2009) The occurrence of glyphosate, atrazine, and other pesticides in vernal pools and adjacent streams in Washington, DC, Maryland, Iowa, and Wyoming, 2005-2006. Environmental Monitoring and Assessment 155: 281-307. [crossref]
  108. Battaglin WA, Kolpin DW, Scribner EA, Kuivila KM, Sandstrom MW (2005) Glyphosate, other herbicides, and transformation products in Midwestern streams, 2002. Journal of the American Water Resources Association 41: 323-332.
  109. Coupe RH, Kalkhoff SJ, Capel PD, Gregoire C (2012) Fate and transport of glyphosate and aminomethylphosphonic acid in surface waters of agricultural basins. Pest Management Science 68: 16-30. [crossref]
  110. Mahler BJ, Van Metre PC, Burley TE, Loftin KA, Meyer MT, et al. (2017) Similarities and differences in occurrence and temporal fluctuations in glyphosate and atrazine in small Midwestern streams (USA) during the 2013 growing season – News – Elsevier. Sci Total of Environ 579: 149-158. [crossref]
  111. Poiger T, Buerge IJ, Bächli A, Müller MD, Balmer ME (2017) Occurrence of the herbicide glyphosate and its metabolite AMPA in surface waters in Switzerland determined with on-line solid phase extraction LC-MS/MS. Environmental Science and Pollution Research 24: 1588-1596. [crossref]
  112. Skark C, Zullei-Seibert N, Schöttler U, Schlett C (1998) The occurrence of glyphosate in surface water. International Journal of Environmental Analytical Chemistry 70: 93-104.
  113. Villeneuve A, Larroud S, Humbert JF (2011) Herbicide Contamination of Freshwater Ecosystems: Impact on Microbial Communities. Pesticides – Formulations, Effects, Fate.
  114. Rosenbom AE, Olsen P, Plauborg F, Grant R, Juhler RK, et al. (2015) Pesticide leaching through sandy and loamy fields – Long-term lessons learnt from the Danish pesticide leaching assessment programme. Environmental Pollution 201: 75-90. [crossref]
  115. Bai SH, Ogbourne SM (2016) Glyphosate: environmental contamination, toxicity and potential risks to human health via food contamination. Environmental Science and Pollution Research 23: 18988-19001. [crossref]
  116. Pham TH, Derian L, Kervarrec C, Kernanec PY, Jegou B, et al. (2019) Perinatal exposure to glyphosate and a glyphosate-based herbicide affect spermatogenesis in mice. Toxicological Sciences 169: 260-271. [crossref]
  117. Yousef MI, Ibrahim HZ, Helmi S, Salem MH, Seehy MA, et al. (1995) Toxic effects of carbofuran and glyphosate on semen characteristics in rabbits. Journal of Environmental Science and Health Part B 30: 513-534. [crossref]
  118. Dai P, Hu P, Tang J, Li Y, Li C (2016) Effect of glyphosate on reproductive organs in male rat. Acta Histochemica 118: 519-526. [crossref]
  119. Johansson HKL, Schwartz CL, Nielsen LN, Boberg J, Vinggaard AM, et al. (2018) Exposure to a glyphosate-based herbicide formulation, but not glyphosate alone, has only minor effects on adult rat testis. Reproductive Toxicology 82: 25-31. [crossref]
  120. Clair É, Mesnage R, Travert C, Séralini GÉ (2012) A glyphosate-based herbicide induces necrosis and apoptosis in mature rat testicular cells in vitro, and testosterone decrease at lower levels. Toxicology in Vitro 26: 269-279. [crossref]
  121. Gorga A, Rindone GM, Centola CL, Sobarzo C, Pellizzari EH, et al. (2020) In vitro effects of glyphosate and Roundup on Sertoli cell physiology. Toxicology in Vitro 62. [crossref]
  122. Anifandis G, Katsanaki K, Lagodonti G, Messini C, Simopoulou, M., et al. (2018) The effect of glyphosate on human sperm motility and sperm DNA fragmentation. International Journal of Environmental Research and Public Health 15. [crossref]
  123. Blondet A, Martin G, Durand P, Perrrard MH (2022b) Low concentrations of glyphosate alone affect the pubertal male rat meiotic step: An in vitro study. Toxicology in Vitro 79. [crossref]
  124. Blondet A, Martin G, Paulic L, Perrard MH, Durand P (2021) An in vitro bioassay to assess the potential global toxicity of waters on spermatogenesis: a pilot study. Environmental Science and Pollution Research 28: 26606-26616. [crossref]
  125. Blondet A, Martin G, Durand P, Perrard MH (2022a) A comparative study of the effects of 3 testicular toxicants in cultures of seminiferous tubules of rats or of domestic cats (veterinary waste): an alternative method for reprotoxicology. Toxicology in Vitro. In press.
  126. Du Y, Dreyfus CF (2002) Oligodendrocytes as providers of growth factors. Journal of Neuroscience Research 68: 647-654. [crossref]
  127. Suter-Crazzolara C, Unsicker K (1994) GDNF is expressed in two forms in many tissues outside the CNS. NeuroReport 5: 2486-2488. [crossref]
  128. Suvanto P, Hiltunen JO, Arumäe U, Moshnyakov M, Sariola H, et al. (1996) Localization of glial cell line-derived neurotrophic factor (GDNF) mRNA in embryonic rat by in situ hybridization. European Journal of Neuroscience 8: 816-822. [crossref]
  129. Trupp M, Rydén M, Jörnvall H, Funakoshi H, Timmusk T, et al. (1995) Peripheral expression and biological activities of GDNF, a new neurotrophic factor for avian and mammalian peripheral neurons. Journal of Cell Biology 130: 137-148. [crossref]
  130. Ashman LK (1999) The biology of stem cell factor and its receptor C-kit. International Journal of Biochemistry and Cell Biology 31: 1037-1051. [crossref]
  131. Rossi P, Sette C, Dolci S, Geremia R (2000) Role of c-kit in mammalian spermatogenesis. Journal of Endocrinological Investigation 23: 609-615. [crossref]
  132. Eisenberg ML, Li S, Behr B, Cullen MR, Galusha D, et al. (2014) Semen quality, infertility and mortality in the USA. Human Reproduction 29: 1567-1574. [crossref]
  133. Jensen TK, Jacobsen R, Christensen K, Nielsen NC, Bostofte E (2009) Good semen quality and life expectancy: A cohort study of 43,277 men. American Journal of Epidemiology 170: 559-565. [crossref]
fig 3

Recurrent Frozen Shoulder – An autobiographical Case Report

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DOI: 10.31038/IJOT.2022514

Abstract

Frozen shoulder (FS) remains one of the most debated and ill-understood conditions causing painful shoulder. It is an extremely painful condition that can be treated in primary care facilities with a lengthy natural history resulting in resolution over 12-18 months. Codman, who coined the word “frozen shoulder” in 1934 had defined it as a painful condition of slow onset in the shoulder coupled with stiffness and sleeping discomfort on the affected side. He had described a pronounced decrease in forward elevation and outward rotation which are the disease’s hallmarks even today. The prevalence of frozen shoulder is estimated to affect 2-5% of the general population, from 10.8 to 30% of diabetic patients and much higher prevalence (27.2%) and incidence (10.9%) of hypothyroidism patients. FS has higher prevalence among men (70:30) than women. It usually affects the non-dominant shoulder although it can occur in either shoulder. Bilateral frozen shoulder occurs in around 14% of patients. Though the single most effective treatment is uncertain, response to a combination of conservative treatment results in gradual resolution of symptoms in 12-18 months.

Frozen shoulder once fully resolved rarely recurs among healthy population but not rare among the diabetes and Thyroid patients. Recurrence is known to occur usually in 5-7 years after the first episode. It’s unusual for frozen shoulder to recur on the affected side earlier. I present here an autobiographic case of rare recurrence of frozen shoulder. It is a rare case because it affected the non-dominant and same side (left shoulder) and after nearly 24 years in contradiction to recurrence in about 5-7 years known in the literature. Though the second and current episode is milder than the first with roughly one third pain and half restriction of range of movement and is being managed conservatively by physiotherapy without analgesics. Resistant cases that do not respond to conservative treatment for 6-9 months are offered surgical treatments called as 1) an arthroscopic capsular release (ACR) or 2) a manipulation under anaesthesia (MUA), both being equally effective. Manipulation process could result in unwarranted complications like fractures of humerus or rotator cuff tear, there for less preferred. Surgical procedures are out of the scope of present case report.

Keywords

Frozen shoulder, Adhesive capsulitis, Conservative treatment, Manipulation, Arthroscopic capsular release, AIIMS, Rehabilitation exercises

Introduction

Adhesive Capsulitis of the Shoulder (ACOS) commonly known as Frozen shoulder (FS) is a condition of uncertain aetiology characterized by significant restriction of both active and passive shoulder movements that occur in the absence of a known intrinsic shoulder disorder [1]. It was first described by Duplay in 1872 as ‘periarthritis’ scapulohumeral, later described as Frozen shoulder by Codman in 1934 [2], Adhesive capsulitis (Naviaser 1945) and Fibrotic capsulitis (Hsu 2011). International Society of Arthroscopy, Knee Surgery & Orthopaedic Sports Medicine (ISAKOS) prefers the term _ ‘frozen shoulder’_ and discourages adhesive capsulitis as there are no adhesions in the shoulder joint [3]. The ISAKOS Upper Limb Committee has classified a stiff shoulder into1. primary idiopathic stiff shoulder (frozen shoulder) and secondary stiff shoulder. Primary idiopathic stiff shoulder develops without any specific trauma or any underlying disease process, however a patient can have a condition that is known to have a link to stiffness (diabetes, thyroid disorders) but not necessarily known to cause stiffness. Secondary FS is shoulder stiffness following an underlying cause such as trauma, infection, or inflammatory disorder. The prevalence of frozen shoulder is estimated to affect 2-5% of the population, and affects men more than women. The peak incidence is observed between 40 and 60 years 20% of patients develop similar symptoms in the opposite shoulder in later years. Bilateral simultaneous involvement is reported in 14% of the patients.

Two conditions namely diabetes mellitus (DM) and thyroid dysfunction are associated with FS. The incidence of frozen shoulder in diabetic patients could vary from 10.8 to 30% [4,5] with a tendency of more severe symptoms and resistance to treatment. The prevalence of DM is ten folds in patients with frozen shoulder, and higher HbA1C and poorly controlled diabetes is associated with the development of FS [6]. Several studies have confirmed higher prevalence (27.2%) and incidence (10.9%) of hypothyroidism in patients with FS [7,8]. Other associated conditions with FS are smoking, cardiac disease, Parkinson’s disease, stroke, neck and cardiac surgery, hyperlipidaemia and Dupuytren’s contracture.

The diagnosis is most often by clinical examination and sometime supported by Xray, MRI or Scanning. Treatment options address reduction in pain, improved function, with high level of patient satisfaction. Minimum of 6 months of supervised conservative treatment should be attempted before surgical treatments are considered. Standard conservative therapy include i) Self-limited disease course: “supervised neglect” with analgesia ii) Non-steroidal anti-inflammatory medications (NSAIDS for 2-3 weeks, iii) Modalities – like Heat/Ice application iv) Oral corticosteroids to reduce night pain v) One or two Corticosteroid injections for pain relief – Glenohumeral/Intra-articular either Landmark-guided or Image-guided, Hydro dilatation and Suprascapular nerve blockade. The surgical procedures like Manipulation under anaesthesia (MUA) and b) Arthroscopic capsular release (ACR) are beyond the scope of this article. The modern evidence of frozen shoulder pathogenesis involves fibrosis and/or contracture of the tendons, joint capsule, and other soft tissues surrounding the glenohumeral joint specifically the rotator cuff interval. In severe cases, the RC interval is “obliterated,” and the coracohumeral ligament is “transformed into a tough contracted band,” like arthritis of soft tissues.

My Case Report

I started to have insidious onset of pain and stiffness in my left shoulder without any preceding traumatic, infective, or inflammatory event in mid May 2022. Pain is around the shoulder over deltoid muscle. Sleeping on my left side was a bit troublesome in the night. My left shoulder examination revealed restriction of both active and passive range of movements (ROM). The loss of external rotation with arm by the side of the chest and folding my left hand behind my back and raising hands overhead were the key symptoms. The range of movement is more than 100° in forward flexion, less than 30° in external rotation, and more than L5 vertebral level in internal rotation. The strength of the rotator cuff is relatively unaffected.

History of Significance

I am a known diabetic since August 1991, managing well on oral anti-diabetics, diet, and regular exercises. I had Left sided Frozen shoulder in 1998 (in the 8th year after diabetes diagnosis). I was treated conservatively at All India Institute of Medical Sciences (AIIMS) New Delhi. The treatment then included NSAIDS, Physiotherapy and Extracorporeal shock wave therapy. After about 3 months of intensive therapy and follow-up of another 12 months I had fully recovered with less than 5% restriction of the movements. I had some complications of Diabetes like Coronary artery disease undergoing By-pass surgery (CABG) in August 2005, Bilateral early cataract surgeries in 2010 and 2014, and Benign enlargement of the prostate. I am under annual check-ups for Diabetic retinopathy,Kidney function tests. My diabetes was fairly well managed until 2018. Since 2018 following relocation to Bengaluru, I am on Carbohydrates restricted diet, walking and muscle strengthening exercises for about 90 minutes per day for 6 days week and oral anti-diabetes drugs. This led to reduction of oral diabetes drugs requirements by one third of what I used to take in early 2018 and Hb1Ac has been around 6.5-7. The FS was almost forgotten.

Two events 1) A head injury due to a fall in the bathroom in August 2021 (Brain scan was normal) 2) the precautionary dose of Covid 19 vaccination on 25 January 2022 had disrupted my diet and exercise routine.

Current Clinical presentation

The current Frozen shoulder pain started insidiously around 15th of May 202 and in a week’s time range of movements of the shoulder got restricted. The pain this time in much less, not needing analgesics but with the restriction of movements (50% of what it was in the first episode). The first sign observed was of painful usual exercise of joining both hands behind the back around L5 level. I had to do this for left shoulder with passive pulling from right hand. Over last 4 weeks it is improved, but the first few attempts of all shoulder exercises in the morning are painful. The worst effected movement is folding (90°) at elbow keeping the shoulders and arms parallel to the ground and extending the left upper limb to touch the ground in supine position or stretching above head standing with my back to wall. Fortunately, there is no disturbance in the sleep. I intensified my shoulder exercises and in 4 weeks I see 25% improvement of ROM.

Physical Examination

Current examination findings include Painful and restricted active and passive ROM, especially of Elevation – forward flexion/abduction, Rotation – external/internal rotation. Difficult active and passive internal and external rotation with shoulder abducted 90° and tenderness of anterior and posterior capsule is felt. Has not affected my sleep.

Basic investigations on 26 May 2022, indicated a Fasting BS= 159 mg/Dl, Hb1Ac level of 7.0, Hb% of 12.2 g/Dl, B12= 150 mg/ml which were a matter of concern. Other biochemical parameters like serum calcium, creatinine, Iron, Vitamin D (25 Hydroxy) = 50 ng/ml and serum Cholesterol = 106 mg/Dl were well within acceptable limits. No Imaging is done this time.

My Management Approach in Current Episode

I am a 76 years aged male, diabetic for 32 years, FS diagnosis based on previous experience. The clinical stages are indistinguishable, ROM and pain are limited. Have been doing all activities as much as tolerated. I have not used either heat, or analgesic medications. I have been cautiously doing physiotherapy, including shoulder exercises as recommended in the first episode,listed later with illustrations, that were interrupted for about 3 months including stretching, strengthening and Pendulum stretch with weights, inwards and outwards rotations, and other Yoga asanas.

Prognosis

I intensified my shoulder exercises and in over last 4weeks ROM has improved,but the first few attampts of all shoulder exercises in the morning are painful.

Discussions

Two Episodes of My FS Episodes in Comparison with National Recommendations

Pathogenesis

A major role of inflammatory mediators (interleukins, cytokines, B- and T-lymphocytes, growth factors, matrix metalloproteinases, tumour necrosis factors and fibroblast activation markers) is postulated in FS characterized by intense inflammatory changes in capsule indicating a role of and disturbance in local collagen translation, which result in global fibroplasia. The capsule of the FS appears thick, congested, and inflamed, particularly around the rotator interval and anteroinferior capsule along with thickened coracohumeral ligament (CHL) and superior-middle-inferior glenohumeral ligaments to the naked eyes during surgery. This results in loss of flexion, abduction, and rotations. Microscopic tissue samples reveal dense collagen matrix and high population of fibroblasts and contractile myofibroblasts, a process like Dupuytren’s contracture, with the fibrotic process predominantly limited to anterior capsule.

Epidemiology

Exact cause is not known. Evolution of synovial inflammation to capsular fibrosis and a Combination/progression of inflammation and fibrosis like Dupuytren’s chronic inflammatory response with immunomodulated fibroblastic proliferation (Hand, JBJS, 2007). Contracture of the rotator interval, coracohumeral ligament, and anterior /inferior capsule is observed.

Literature review indicates that mostly men (60-70 %) are affected and Med Sport data indicated 67% (Housner, 2017). Majority (70-75%) of the affected are between 40-60 years old (peak age 50). My first episode was at the age of 46 year and the current at 76 years. While the first episode was well within expected age frame the second episode is well beyond known upper age limit.

Risk of recurrent in contralateral shoulder 15-20% usually within 5 years (range of 6 months to 7 years (Bridgman 1972, Reeves 1975, Shaffer 1992, Hand 2008) and Risk of recurrence in same shoulder essentially 0% (Codman 1934, Lippman 1944, Hsu 2011). My case is very rare as both these conditions are contradicted as the recurrence has occurred after 24 years and has affected same shoulder.

Natural History

Total duration may be 12 to 40 months; recovery is always sure and can be confidently expected but there is a Controversy over residual pain and/or loss of motion of 50% patients at 7 years with no functional limitation. My first episode had similar experience as I had recovered within 15 months fully, with a functional loss of less than 5% and absolutely no pain. The second episode now is insidious and much less painful and ROM. The different stages described below were evident and distinct in the first episode but not now.

Stage 1 (Painful or “Freezing”). Usually lasts up to 9 months from onset of symptoms, pain precedes the restriction in motion. Sharp pain at end range of motion and gradual loss of motion. The earliest to get affected and latest to return finding is usually loss of external rotation. May affect sleep if there is aggressive synovitis or angiogenesis. In my case in the first episode, my sleep was disturbed for nearly 2 months but not affected at all now.

Stage 2 (“Frozen”). 6 to 15 months from onset leading to loss of motion in all planes and throbbing pain worse with motion and usually disturbs sleep. MRI shows Capsuloligamentous fibrosis. My first episode had bothered me for about 6 months in this stage. The image inferences were like the ones described in the literature. In the current episode I have not gone for any imaging as the condition is evident and mild. I need to observe the long-term resolution.

Stage 3 (“thawing”). Lasts for 12 to 24 months from onset. It is gradual spontaneous improvement of shoulder mobility and function. Pain starts decreasing, range of motion improves, mild mobility deficits and pain may persist. Most patients report minimal to no disability. Pathologically Synovial involvement recedes. Poor function of reaching overhead (getting dressed, putting on deodorant), reaching behind back (putting on shirt/coat) and reaching out to the side (getting mail, using ATM). Patient finds it difficult to explain onset of pain to attributes to a trivial injury. My first episode had taken about 6 months in this stage. Pain had receded slowly over 6 months and after a total of about 15 months neither there was a residual pain or no was there any residual restriction of movements. In the current episode I need to observe this stage. Looking at the current progress in first 4 weeks, I strongly feel that the entire course of returning to normalcy may take around 6 months.

Treatment

By and large, conservative treatment of frozen shoulder is successful in up to 90% patients. Latest recommendation of the treatment is ‘use it or lose it’ by movement therapy [Jun 2022-11]. The age old conservative treatment incudes:

NSAIDs and Other Analgesics

NSAIDs remain one of the most common medical interventions, a short course of NSAIDs for 2-3 weeks minimises the intense pain of in treating frozen. In my first episode this was adopted for about 3 weeks. The current episode pain is tolerable and hence have not taken any analgesics.

Corticosteroids

Both oral steroid and local steroid injections are widely used. They are beneficial only in early stages to control inflammation and ensuing pain and may not be useful in late stages with established fibrosis without much inflammation. Oral steroids for improving pain, ROM and function when prescribed for ‘short term’ of up to 6 weeks in early stage is helpful. Systematic reviews and metanalysis have confirmed usefulness of steroid injections in improving pain and ROM in the short term, and moderate evidence in the midterm. Fortunately, neither in the first nor the current episodes this intervention was either advised or tried.

Hydro-dilatation (HD)

A single HD of the glenohumeral joint using saline, steroid, local anaesthetic agent is supposed to distend the capsule by breaking the ‘early intracapsular fibrosis’ helping in improving ROM In early/late frozen stage. However, more than one repeated HD after 2 weeks have no added effect over FS. There was no need of such an intervention in my case.

Calcitonin

Calcitonin is supposed to decrease the systemic inflammatory response and stimulate the release of endorphins, improving mRNA expression of fibrosis-related mole, but further research is required in this area to validate.

Extracorporeal Shock Wave Therapy (ECSWT)

ECSWT significantly improves the functional outcome and ROM without any adverse events. ECSWT was used for about 4 weeks twice a week in the first episode and was instrumental in remission of pain in 6 months as against expected time frame of 9-12 month, that was considered needed then.

Acupuncture

A few studies have reported reasonable relief in pain and improved forward flexion by using acupuncture in the treatment of FS.

Nerve Block

A single or multiple injections to block Suprascapular nerve in the treatment of FS have shown improved pain score and ROM Operative Management of Frozen Shoulder involves a) Manipulation under anaesthesia (MUA) and b) Arthroscopic capsular release (ACR). Both are equally effective, but ACR is ‘preferred’ surgical option for the treatment of refractory FS as it allows controlled and precise release of fibrosed capsule-ligament complex under vision, avoiding complications of MUA like as Humerus shaft fracture, rotator cuff tear, shoulder dislocation, labral tear, and nerve injury.

Rehabilitation

A Shoulder Range of Motions (Flexion, abduction, internal rotation, and external rotation) as detailed below was advised and done with the help of a physiotherapist of AIIMS for two weeks along with ECSWT and continued at home for another 4 months without ECSWT in my first episode of FS in 1998.

Pendulum Stretch

I was to relax my shoulders, stand and lean over slightly, allowing the affected arm to hang down. Swing the arm in a small circle, about a foot in diameter. Perform 11 revolutions in each direction, once a day. As my symptoms improved, increased the diameter of my swing, without forcing it. Slowly I increased the stretch by holding a light weight (1-5 kg) in the swinging arm even now [9] (Figure 1).

fig 1

Figure 1: Stretch by holding a light weight (1-5 kg) in the swinging arm

Towel/Rubber Band Stretch

I hold one end of a three-foot-long towel (of late a rubber stretch band) behind my back and grab the opposite end with your other hand. Held the towel in a horizontal position and used my good (right) arm to pull the affected arm upward to stretch it. I held the bottom of the towel with the affected arm and pull it toward the lower back with the unaffected arm for 11 times a day (Figure 2).

fig 2

Figure 2: Held the bottom of the towel with the affected arm and pull it toward the lower back with the unaffected arm for 11 times a day

Finger Walk

I faced a wall three-quarters of an arm’s length away. Reach out and touch the wall at waist level with the fingertips of the affected arm. With my elbow slightly bent, slowly walk my fingers up the wall, spider-like, until I had raised my arm as far as I was comfortable. Only my fingers worked and not my shoulder muscles. Slowly lower the arm (with the help of the good arm, if necessary) and repeat. Perform this exercise 11 times a day.

Cross-body Reach

While standing, I used my good arm to lift my affected arm at the elbow, and bring it up and across my body, exerting gentle pressure to stretch the shoulder and hold the stretch for 15 to 20 seconds. Repeating these 11 times per day.

Outward Rotation

Holding a rubber exercise band between my hands with my elbows at a 90-degree angle close to my sides, rotated the lower part of the affected arm outward two or three inches and hold for five seconds. Repeated these movements for 11 times, once a day (Figure 3).

fig 3

Figure 3: Repeated these movements for 11 times, once a day

Inward Rotation

Standing next to a closed door, hooked one end of a rubber exercise band around the doorknob. Holding the other end with the hand of the affected arm, and my elbow at a 90-degree angle, I pulled the band toward my body two or three inches and held for five seconds. Repeated this exercise for 11 times, daily at least 5 days a week. I had continued doing most of these exercises at least 3-4 days week since then. After relocating to Bengaluru in 2018 my apartment complex Gymnasium had equipment that provided for the above exercises – internal and external rotation exercises, that I did. In 2020 the gym was closed, and I continued to the similar exercising using the elastic stretch bands (Figure 4).

fig 4

Figure 4: Similar exercising using the elastic stretch bands

The international recommendations are for 10-15/20 movements each time. Keeping in Indian tradition of the odd number sanctity I advocate for 11 or 21 movements for each shoulder.

These exercises are field tested under the name of Pune Shoulder Rehabilitation Program (PSRP) in 2013. PSRP an exercise program involving exercises with low resistance, high repetition performed in sub impingement region for strengthening of scapular and rotator cuff muscles to normalize scapular muscle strength, normalize scapula humeral rhythm, pain relief, rotator cuff muscle strengthening, restoration of range of motion (ROM), restoration of function, and maintaining posture and core. The Pune study findings showed that over the 6th-week protocol, statistically significant improvements were found in pain reduction and shoulder range of motion, concluding that such exercise protocol is effective in increasing the range of motion and decreasing the pain in the shoulder caused by frozen shoulder [10].

Prevention: A few easy steps to help prevent FS are:

  • Stretching your shoulder and back muscles daily.
  • Stretching your tendons (by rotating hands and palms to stretch different tendons).
  • Practicing good ergonomics while sitting at a desk and using a computer.
  • Maintaining a healthy immune system.

Conclusion

These days, adhesive capsulitis is the precise descriptive jargon for the condition, but no better than “periarthritis. Frozen shoulder (FS) continues to be an extremely painful condition since it was first identified in 1934, that can be treated in primary care. It has a lengthy natural history resulting in resolution over 12-18 months. The shoulder is the only joint that often “freezes” like this and is a common biological puzzle. It’s hard to define precisely, diagnose accurately, or treat effectively. In fact, frozen shoulder treatment is one of the best examples of how musculoskeletal medicine is surprisingly primitive still. Of late there is a hypothesis that FS is more of Functional freezing than adhesive freezing. Three main ways that a functional limitation of shoulder ROM would probably work, are put forth as a) The brain can “shut down” a joint with neurological inhibition, b) because it has become sensitized or c) The muscles may have gotten rotten with trigger points.

Frozen shoulder once fully resolved does recur, though in small proportion among the diabetes and Hypothyroid patients. Usually, recurrence occurs in 5-7 years after the first episode and affects contralateral side. It’s extremely rare for frozen shoulder to recur on the same side affected earlier. Second episode is much milder, hardly disturbs sleep and can be managed with exercises only [11-13].

References

  1. Duplay S (1892) Archives Générales de Médecine.
  2. Codman E (1984) The Shoulder Rupture of the Supraspinatus Tendon and Other Lesions in or about the Subacromial Bursa. Medicine.
  3. Vivek Pandey et al. (2021) Clinical Guidelines in the Management of Frozen Shoulder. Journal List Indian J Orthop. 55: 2. [crossref]
  4. Jeffrey A, Housner. Adhesive Capsulitis of the Shoulder. Departments of Family Medicine, and Orthopaedic Surgery, University of Michigan.
  5. Future Sci OA (2020) Frozen shoulder: Overview of clinical presentation and review of the current evidence base for management strategies[crossref]
  6. J Zreik NH et al. (2016) Adhesive capsulitis of the shoulder and diabetes: A meta-analysis of prevalence. Muscles Ligaments Tendons. 6: 26-34. [crossref]
  7. Bridgman JF (1972) Periarthritis of the shoulder and diabetes mellitus, Annals of the Rheumatic Diseases. 31: 69-71. [crossref]
  8. Chan JH et al. (2017) The relationship between the incidence of adhesive capsulitis and haemoglobin A(1c). Journal of Shoulder and Elbow Surgery. 26: 1834-1837. [crossref]
  9. Schiefer M et al. (2017) Prevalence of hypothyroidism in patients with frozen shoulder, Journal of Shoulder and Elbow Surgery. 26: 49-55. [crossref]
  10. Cakir M et al. (2003) Musculoskeletal manifestations in patients with thyroid disease. Clinical Endocrinology – Oxford. 59: 162-167. [crossref]
  11. Shoulders stretching exercises for frozen shoulder.
  12. Seema Saini et al. (2022) Effectiveness of Pune shoulder rehab protocol on patients with frozen shoulder.
  13. Complete Guide to Frozen Shoulder.
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A Prospective, Randomized, Three Arm, Open Label, Parallel Group, Multicentric Study to Evaluate the Effectiveness of Supplementing MG-HT® in Reduction of Blood Pressure in Subjects with Stage 1 – Stage 2 Hypertension on any Antihypertensive Therapy

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DOI: 10.31038/JCRM.2022534

Abstract

Background: Worldwide, Hypertension (HTN) has emerged as the most highly prevalent modifiable risk factor for cardiovascular disease related morbidity and mortality, in terms of strongest evidence of causation and high prevalence for exposure. A preventive approach to control blood pressure (BP) may reduce these risks. Oral Magnesium (Mg) intake is inversely related with risk of HTN. Nutritional Magnesium has both direct and indirect impacts on regulation of BP through sodium (Na)-potassium (K) and intracellular Calcium (Ca) mediated Mg-driven Na-K and Ca pumps, impairment of which leads to vasoconstriction and HTN. Additionally, it increases endothelial nitric oxide, improves endothelial dysfunction, apart from inducing direct and indirect vasodilation. The efficacy of Mg, Beta-sitosterol, Pyridoxine, Niacinamide and L-carnitine, as individual ingredients in supporting alleviation of BP or associated conditions has been documented in literatures; however, no study has been done on the effectiveness of combination of these ingredients in HTN, specifically in Indian population. Despite improvement in primary therapeutics for HTN, there are reports of resistant hypertension in patients who are on more than three antihypertensives of different classes, and in many cases, achieving the goal BP becomes difficult in clinical practice. It has been suggested that addition of nutritional management for high blood pressure to the primary regimens can be a safe, sustainable, and cost-effective intervention, but their benefits are yet to be shown through appropriately designed studies. This study is aimed to evaluate the impact of MG-HT® in reduction of blood pressure when administered in conjunction with any antihypertensive therapy, such as, Calcium channel blocker, Angiotensin-converting enzyme (ACE) inhibitors, Thiazide diuretic, or Angiotensin II receptor blockers (ARB), in most cases, a combination of two drugs.

Objective: To evaluate the benefits of supplementation with MG-HT® administered twice daily with ongoing antihypertensive regimen versus MG-HT® administered once daily with ongoing antihypertensive drugs versus standard of care on the reduction of systolic blood pressure (SBP) and diastolic blood pressure (DBP) from baseline to study end at 90 days, in subjects with Stage 1 – Stage 2 Hypertension on any antihypertensive therapy, such as, Calcium channel blocker, ACE inhibitor, Thiazide diuretic, or ARB, in all cases a combination of two drugs.

Design, setting and participants: This is a Prospective, Randomized, Three-Arm, Open-Label, Parallel-Group, Multicentric Study involving 80 patients at 4 sites across India, who have Stage 1 – Stage 2 HTN and are on any existing antihypertensive therapy.

Intervention: Participants were randomized to existing antihypertensive therapy and MG-HT® once daily (Study arm 1), existing antihypertensive therapy and MG-HT® twice daily (Study arm 2), and existing antihypertensive therapy (Study arm 3) alone for 90 days, while all continued on the same dietary and activity advice.

Main outcomes and measures: The primary objective was to evaluate the benefits of additional supplementation with MG-HT® administered once daily with the existing antihypertensive therapy versus MG-HT® administered twice daily with the existing antihypertensive therapy versus standard of care with existing antihypertensive therapy alone in reduction of SBP and DBP from baseline to study end at 90 days from the beginning of the study. The secondary objectives were to evaluate the additional health benefits of MG-HT® in the study groups of subjects in terms of lipid profile from baseline to end of the study period at 90 days while they continued on existing dietary and activity advice and to analyze the relationship between the rise in serum magnesium levels to reduction in BP during the same period. Additionally, safety of MG-HT® in all the two study arms throughout the study period of 90 days was evaluated including the prevalence of subclinical magnesium deficiency at baseline in all enrolled subjects. The relationship between variations of serum magnesium levels (independent variable) and BP (dependent variable) was assessed by calculating the Odds Ratio (OR), using multivariate logistic regression analysis.

Results: The change in the Systolic Blood Pressure (SBP) in MG-HT® once daily arm from baseline to end of study was a reduction of 13.63 mmHg; in MG-HT® twice daily arm, there was a reduction of 7.87 mmHg, and in the comparator arm, and there was a reduction of 10.06 mmHg. The reduction in systolic blood pressure was higher in the arm receiving MG-HT® once daily as compared to twice daily and standard of care, respectively. The change in the Diastolic Blood Pressure (DBP) in MG-HT® once daily arm from baseline to end of study at 90 days was a reduction of 9.81 mmHg; in MG-HT® twice daily arm, there was a reduction of 6.16 mmHg and in the comparator arm, there was a reduction of 7.59 mmHg. The reduction in DBP was higher in the arm receiving MG-HT® once daily as compared to twice daily and the standard of care. However, the differences in SBP and DBP were not statistically significant (p-value >0.05). Subjects receiving MG-HT® once daily reported a greater reduction of BP in terms of returning to pre-hypertensive levels at the end of study when compared to MG-HT® twice daily and to subjects receiving standard of care. This difference was statistically significant (p-value >0.001).

The mean change of Mg levels from baseline to study end at 90 days was not statistically significant between study arms (p-value >0.05). The mean change of laboratory parameters from baseline to study end was not statistically significant between study arms (p-value >0.05).

The subjects who had lower magnesium levels at the baseline achieved normal serum mg levels at study end with an average value of 1.8 mg/dL. Subjects receiving MG-HT® once daily reported a greater reduction in SBP levels at study end with a reduction of 10 mmHg compared to 7 mmHg and 6 mmHg of MG-HT® twice daily arm and standard of care arm respectively. No change in DBP was seen. The SBP and DBP changes were not statistically significant between arms.

Conclusions: In this study, it was found that once-daily oral MG-HT® therapy added to standard antihypertensive regimens for 90 days reduced blood pressure in patients with stage 1 and 2 hypertension, improving clinical outcomes.

Keywords

Calcium channel blocker, Endothelial dysfunction, Hypertension, L-carnitine, Magnesium, MG-HT®, Niacinamide, Pyridoxine, Sterols

Introduction

Hypertension (HTN) has emerged as the most important risk factor for morbidity and mortality, worldwide [1]. It is the single largest contributor to the avoidable deaths and diseases in India [2]. It is also one of the major risk factors for noncommunicable diseases such as cardiovascular diseases, stroke, and renal diseases [3]. As per WHO currently, 35% of the world population is affected by HTN, and it might cross 50% by 2025 [4]. The prevalence of HTN in India is around 29.8%, with a higher prevalence seen in urban areas (33.8%), compared to rural areas (27.6%) [5].

In a latest Indian study, it is reported, that 25% of adults are hypertensive, with a substantial prevalence of 12% observed among the young adults aged between 18 to 25 years [6]. WHO ranks HTN as one of the prime causes of premature deaths globally. In India, 57% of all stroke deaths and 24% of all CHD are directly linked to HTN [7].

Despite availability of several comprehensive medical therapies, HTN remains a challenging clinical problem [8]. Majority of patients fail to achieve an optimal blood pressure control even with combination therapy with two or more drugs including a diuretic [9-13]. In India, a multidisciplinary consensus statement highlights that despite treatment, only about 9-20% of patients achieves goal target BP [10]. Another Indian study has reported that uncontrolled HTN may double the risks of cardiovascular events and stroke [11]. Number of drugs used to control uncontrolled hypertension matters, and any patient with uncontrolled hypertension may develop resistant hypertension, the prevalence of which is 20-30%, which would need addition of further medications for blood pressure control as an adjunct to conventional standard of care therapies [12].

Numerous studies have demonstrated that Mg supplementation may lower BP [14]. Mg may play a crucial role in BP regulation, by directly stimulating nitric oxide and prostacyclin formation, modulating endothelium-dependent and endothelium-independent vasodilation, reducing vascular tone and reactivity, and preventing vascular injury via its anti-inflammatory and antioxidant functions [14,15]. Mg also improves vascular smooth muscle tone and contractility by blocking the calcium channels and by inhibiting norepinephrine release [16,17]. An earlier study published in Japanese explored the relationship between Mg and HTN, demonstrating its relaxant effect on vascular smooth muscle cells through cationic regulation of intracellular Sodium (Na): Potassium (K) ratio and Calcium (Ca). This study also showed the benefits of Mg in hypertensive patients on antihypertensive medications, which required a lower dose in comparison to patients who were not on any antihypertensive medications [13].

Studies have shown that magnesium deficiency states may negatively influence functional and structural vascular changes in HTN. It is involved in the pathogenesis of HTN, endothelial dysfunction, dyslipidemia, and inflammation, contributing to arterial stiffness [14].

Pathophysiologically, several experimental models and cross-sectional and longitudinal population studies have reported an inverse correlation between Mg deficiency states and HTN [11-15]. On the other hand, several trials have reported inconsistent results with Mg supplementation on BP lowering effects with some showing a positive impact, while others showing none [15]. Various meta-analyses of Cohort studies and RCTs have confirmed the protective effects of Mg, establishing the association between dietary and supplemental Mg with HTN [16].

A systematic review by Rosanoff et al. (2021) of 49 clinical trials reported that oral Mg safely lowered BP in uncontrolled hypertensive patients on antihypertensive medications. However, patients with controlled hypertension or with normal blood pressure on oral Mg therapy did not show any BP lowering effect [21]. An interventional study by Banjamin et al. (2018) showed significant reduction in SBP and DBP by 8.9 mmHg and 5.8 mmHg respectively. Mg further significantly improved two hemodynamic parameters, reduced systemic vascular resistance index and left cardiac work index [18]. Several other studies have established that Mg increased the effectiveness of all antihypertensive drug classes due to its calcium channel blocker mimetic effect and circulating Na+K+ATPase suppressor activities that can reduce vascular tone [19-23].

Studies by Askarpou et al. (2019) and Craig et al. (2007) demonstrated that L-carnitine supplementation decreased SBP and DBP. It also increased brachial artery diameter by 2.3% [24,25].

Phytosterols (Beta-sitosterol) are the analogues of cholesterol, decrease the circulating cholesterol levels by competing with cholesterol for intestinal absorption. They decrease Low-Density Lipoprotein Cholesterol (LDL-c) by up to 10% and Triglycerides (TG), modulate the expression of lipid regulatory genes and de novo lipogenesis. They increase hepatic β-Hydroxy β-methylglutaryl-CoA (HMG-CoA) Reductase mRNA expression. Studies have shown that Beta-sitosterols lower risk of cardiovascular diseases [26,27]. Experimental studies with Beta-sitosterol showed that it improved nitric oxide levels and, hence, vascular function [28] that may support vasorelaxing effect of Mg.

Study by Zhang et al. (2021) demonstrated niacin-induced primary prevention of hypertension [29]. Bays et al. (2009) in a review reported significant BP lowering effects of Niacin in clinical trials involving hypertensive patients [30]. The role of niacin as an adjuvant therapy for reducing atherogenic lipoprotein levels in dyslipidemic patients has also been reported [31] including its efficacy in controlling high TG, supporting maintenance of High-Density Lipoprotein cholesterol (HDL-c) levels, and thus supporting management of lipid abnormalities associated with metabolic syndrome [32], which, in turn, is commonly associated with HTN.

Pyridoxal 5-phosphate regulates cellular calcium transport and thereby can be useful in controlling HTN (33). Aybak et al. (1995) concluded that pyridoxine administration significantly reduced SBP and DBP in hypertensive patients within 4 weeks [33, 34].

Since the current literature on the effectiveness of Mg, Beta-sitosterol, Pyridoxine, Niacinamide and L-carnitine in reduction of BP is predominantly from the western world and no combination study had been done even there, this study is envisaged to evaluate the benefits of proprietary nutraceutical formulation MG-HT® in reduction of BP when administered concomitantly with antihypertensive as a standard therapy in Indian population. Therefore, this study is the first study of its kind in India, which may contribute to better management of hypertensive patients, as a nutritional adjunct to the standard antihypertensive therapy, optimizing clinical outcomes. This could be more so useful in patients with uncontrolled HTN already on standard antihypertensive therapy, contributing to enhanced risks and resistance to treatment.

Materials and Methods

Study Design

This prospective, randomized, three arm, open label, parallel group, multicentric study investigated the clinical effectiveness of supplementing MG-HT® (Magnesium Bisglycinate 500 mg providing elemental Magnesium 70 mg, L-Carnitine L-Tartrate 500 mg, Niacinamide 8 mg, Pyridoxine Hydrochloride 1 mg, Beta-Sitosterol 20 mg, a proprietary nutraceutical formulation available in Indian market) in supporting reduction of BP in uncontrolled hypertensive patients, on standard antihypertensive therapy.

Site of the Study and Ethics

This is a multi-center, interventional study conducted at the 4 sites geographically distributed across India at Suraksha Polyclinic-Kolkata; Sanjeevani Hospital and Polyclinic-Mumbai; Diabetes Specialty Centre, Dwarka-New Delhi; and SRM Institute of Medical Sciences-Chennai. The study was approved by the Independent Ethics Committee (IEC) of each study center. It was performed in compliance with the ICH guidelines for Good Clinical Practice, ICMR guidelines, and declaration of Helsinki. Written informed consent for participation in the study was obtained from all participants. This study was registered in the Clinical Trials Registry of India (CTRI/2020/01/022864).

Study Population

80 Subjects of both sexes in age bracket 35-65 years with Stage 1 – Stage 2 hypertension as defined by the latest JNC 8 hypertension guidelines (Stage 1 HTN: SBP 140-159 mmHg, DBP 90-99 mmHg and Stage 2 HTN: SBP ≥160 mmHg, DBP ≥100 mmHg) and on any antihypertensive therapy for at least a month who have not shown any improvement in BP control or have mild elevation in BP but continue to fall under the same stage as at the time of diagnosis.

Inclusion Criteria

Subjects

  1. Able to provide signed informed consent
  2. Willing to adhere to protocol and study requirements during the entire study duration

Exclusion Criteria

Subjects

  1. With uncontrolled diabetes mellitus in the opinion of the investigator
  2. With history of myocardial infarction within the past 3 months of the start of the study, cardiac failure of class III and IV, Atrioventricular block II or III on ECG
  3. With chronic kidney disease or liver disorder
  4. With chronic terminal diseases, such as, malignancies, anemias and presence of serum electrolyte disturbances (Na, K, Cl), that might indicate an underlying secondary HTN
  5. On Mg supplements equal to or above the study dose of 70 mg, in which case a wash-out period of 7 days will be followed prior to enrolment
  6. With any other condition, which in the opinion of the investigator renders the patient unfit to participate
  7. Females, who are lactating, pregnant, or planning to conceive during the study period

Randomization

Computer generated random numbers were used to assign participants to the MG-HT® once daily with standard antihypertensive therapy or MG-HT® twice daily with standard antihypertensive therapy or standard of care groups in 1:1:1 allocation ratio.

Outcome Measures

The primary endpoint was the proportion of subjects with a reduction in SBP and DBP from baseline to study end after 90 days, mean change in SBP from baseline to study end after 90 days, and mean change in DBP from baseline to study end after 90 days.

The secondary endpoints were the mean change in lipid profile from baseline to study end after 90 days and mean change in serum Mg levels from baseline to study end after 90 days.

The safety endpoints were the solicited and unsolicited adverse events (AEs) in all the three arms and changes from baseline in the laboratory parameters of renal function tests and liver function tests.

The exploratory end point was the proportion of subjects with subclinical Mg deficiency at baseline.

BP Measurement Procedure: Seated BP of each enrolled subjects was measured using Diamond brand mercurial type BP instrument with standard U-tube manometer at each study visit after a rest period of 10 minutes. Subjects were made to sit comfortably with legs resting on the ground not crossed and with arm supported at the heart level. Each subject was advised to empty the bladder prior to BP recording. Cuff bladder was applied, encircling two-thirds of the subject’s arm circumference. The recordings were made on the right arm. The subject and the person taking the measurements were not allowed to speak during the procedure. The cuff was inflated to at least 30 mmHg above the point at which the radial pulse disappeared. The cuff was then deflated at a rate of 2 to 3 mmHg per second (or per pulse when the heart rate is slow). Deflation rates greater than 2 mmHg per second can cause the systolic pressure to appear lower and the diastolic pressure to appear higher. The first and last audible sounds were recorded as systolic and diastolic pressures, respectively. Measurements were given to the nearest 2 mmHg. An average of two readings was recorded. The BP was recorded by the investigator or a trained delegated study personnel. A calibrated sphygmomanometer was used at all sites. The same delegated personnel for each site recorded the BP at all visits. This maintained consistency and eliminated any interobserver bias.

Procedure to analyze MG-HT® batches allocated to each Intervention Group: The batch number and other details of MG-HT® product were recorded in the form of a list. Each batch allocated in the study had an assay undertaken at the manufacturing unit, and the Certificate of Analysis was available for record and corroboration. It was ascertained that the elemental Mg level in each batch was at least 70 mg.

Study Arms

  1. Study arm 1: Antihypertensive therapy and MG-HT® once daily (morning).
  2. Study arm 2: Antihypertensive therapy and MG-HT® twice daily.
  3. Comparator arm: Standard of care with antihypertensive therapy.
  4. Study product was supplied by Pharmed Limited, Bangalore, India.

Methodology

80 enrolled subjects ranging 35-65 years of age, diagnosed with Stage 1 – Stage 2 HTN and on any antihypertensive therapy for at least a month that have not shown any improvement in BP control or have mild elevation in BP but continue to fall under the same stage at the time of diagnosis were enrolled into the study. All enrolled subjects were screened according to the pre-defined inclusion and exclusion criteria, and subjects were randomized to either of the three study arms. Subjects randomized to study arm 1 received antihypertensive therapy and MG-HT® once daily (morning), subjects randomized to study arm 2 received antihypertensive therapy and MG-HT® twice daily, and subjects randomized to comparator arm received the investigator prescribed standard of care of antihypertensive therapy. There were three physical visits (V1-Screening, V3 and V6-Study end on Day 1, 30 and 90 respectively) and three telephonic follow-up visits (V2, V4 and V5 on Day 15, 45 and 60 respectively). All the subjects in each group were asked to continue with salt-restricted diet and usual activities, such as, daily walking as a part of standard of care before the screening.

After obtaining written informed consent from the participants in the prescribed format, detailed clinical history, and stage of HTN were recorded along with their relevant medical history and drugs used as antihypertensives or otherwise. Seated BP of all enrolled subjects were measured and recorded as an average of two readings. A fasting blood sample was collected from each subject for baseline laboratory investigations of HbA1c, Lipid Profile, Serum Mg, Renal Function Tests, Liver Function Tests, and an ECG was recorded at baseline on each subject.

Subjects were contacted telephonically at Day 15, Day 45 and Day 60 and enquired about their general well-being and compliance to study product consumption. Subjects were instructed to report to the study sites for the physical follow-up visits on Day 30 and Day 90. At all follow-up visits, a general examination was performed including BP recording, and subjects were enquired about any solicited or unsolicited AEs. Concomitant and rescue medications were reviewed as applicable in each case. Investigational Product (IP) accountability was also performed. At study end visit, a fasting blood sample was collected from each of the subjects for laboratory investigations, and an ECG was also recorded.

Statistical Analysis

To evaluate the overall effects of MG-HT® supplementation on BP, the mean changes of systolic and diastolic BP between treatment groups after treatment was compared by calculating mean differences and 95% confidence intervals (CIs). For comparison of normally distributed variables, One-Way ANOVA Test (or Kruskal-Wallis test for skewed data) to establish the differences between the groups was performed. The relationships between variations of serum Mg levels (independent variable) and BP (dependent variable) were assessed by calculating the Odds Ratio (OR) using multivariate logistic regression analysis. A 95% confidence interval (CI 95%) was considered, and p-value <0.05 defined the level of statistical significance. A sub-group analysis was performed to analyze the correlation between effects of MG-HT® and reduction in BP in various classes of antihypertensives prescribed. Associations between continuous variables were captured using Pearson’s Correlation Coefficient if the data followed normal distribution or Spearman’s Rank Correlation Coefficient if the data did not follow normal distribution. Safety analysis was performed on all enrolled subjects who have received at least one dose of the study product.

Results

A total number of 80 subjects satisfying inclusion and exclusion criteria were enrolled into the study and randomized to study arm 1, 2 and 3 or comparator arm. 28 subjects were enrolled in both the treatment arms of MG-HT® once daily and twice daily and 24 subjects were enrolled in the comparator arm. The lost to followup rate was considerably higher in the study owing to the unprecedented SARS-CoV-2 pandemic; a total of 28 subjects were lost to followup at visit 6, of which 12 were in MG-HT® once daily arm, 9 were in MG-HT® twice daily arm, and 7 were in the comparator arm. However, at each visit subjects were followed up telephonically, and the following is the subject disposition of all subjects who reported physically at site and followed up telephonically. The baseline characteristics of the study patients are shown in Table 1.

Table 1: Baseline characteristics of 80 subjects, the values representing the mean ± standard deviation (SD)

Parameters

 Arm-1 (n=28) Arm-2 (n=28)

Arm-3 (n=24)
Age (Y)

52.79 ± 8.359

 52.39 ± 8.5

48.08 ± 10.325
Height (cm)

162 ± 7.779

 163.87 ± 8.18

164.32 ± 8.706
Weight (Kg)

69.75 ± 10.429

 71.02 ± 11.262

72.63 ± 15.863
BMI (kg/m2)

26.62 ± 3.812

 26.43 ± 3.61

26.83 ± 5.252
History of duration of hypertension (months)

49.54 ± 68.692

 59.83 ± 61.198

37.71 ± 60.68
Blood Pressure (mmHg)

SBP:149.64 ± 7.395

DBP:90.39 ± 4.228

 SBP:147.55 ± 6.735

DBP:91.61 ± 3.9

SBP:148.56 ± 6.589

DBP:91.13 ± 4.785
Hypertension stage wise distribution (n)

Stage 1: 22

Stage 2: 6

 Stage 1: 24

Stage 2: 4

Stage 1: 20

Stage 2: 4
HbA1c (%)

6.46 ± 1.181

 6.83 ± 1.521

7.16 ± 1.807
Cholesterol Total (mg/dL)

168.22 ± 51.147

 178.96 ± 40.963

190.68 ± 42.956
HDL (mg/dL)

44.94 ± 9.427

 44.62 ± 10.993

44.31 ± 12.119
LDL (mg/dL)

96.78 ± 41.116

 106.07 ± 34.422

116 ± 35.68
VLDL (mg/dL)

27.05 ± 12.296

 27.34 ± 10.869

30.85 ± 12.887
Triglyceride (mg/dL)

145.96 ± 83.259

 150.94 ± 80.182

162.42 ± 90.644
Bilirubin Total (mg/dL)

0.64 ± 0.375

 0.61 ± 0.287

0.61 ± 0.502
Bilirubin Direct (mg/dL)

0.23 ± 0.12

 0.25 ± 0.136

0.21 ± 0.097
Bilirubin Indirect (mg/dL)

0.42 ± 0.3

 0.37 ± 0.189

0.4 ± 0.426
SGPT(ALT) (U/L)

27.16 ± 11.252

 25.91 ± 16.611

35.63 ± 25.901
SGOT (AST) (U/L)

28.2 ± 13.319

 23.94 ± 10.598

25.92 ± 11.204
Albumin (g/dL)

4.63 ± 0.506

 4.64 ± 0.462

4.55 ± 0.384
Alkaline Phosphatase (U/L)

92.64 ± 46.993

 85.59 ± 17.885

96.25 ± 26.122
Blood Urea Nitrogen (mg/dL)

11.42 ± 3.38

 10.43 ± 4.024

9.95 ± 2.703
Urea Serum (mg/dL)

23.93 ± 6.459

 22.61 ± 8.649

21.28 ± 5.785
Creatinine (mg/dL)

0.86 ± 0.228

 0.83 ± 0.293

0.78 ± 0.173
Magnesium (mg/dL)

2.02 ± 0.135

 1.9 ± 0.238

2.01 ± 0.226

Baseline values represent the mean ± standard deviation. BMI: Body mass index, HbA1c: Hemoglobin A1c, HDL: High-density lipoprotein, LDL: Low-density lipoprotein, VLDL: Very low-density lipoprotein, SGPT: Serum glutamic pyruvic transaminase, ALT: Alanine transaminase, SGOT: Serum glutamic oxaloacetic transaminase, AST: Aspartate transaminase

Blood Pressure Data

Systolic Blood Pressure

Under Intention-to-treat (ITT) analysis, the change in the SBP in study arm-1 from baseline to end of study was a reduction of 13.63 mmHg; in study arm-2, there was a reduction of 7.87 mmHg, and in the comparator arm, there was a reduction of 10.06 mmHg. The reduction in SBP was higher in the arm receiving MG-HT® once daily as compared to twice daily and standard of care. However, this difference was not statistically significant (p-value >0.05). The mean changes in SBP are shown in Table 2. It is also to be recognized at this point that there is high standard deviation noted in the data set, which is commensurate with other studies involving HTN.

Table 2: Mean changes in SBP & DBP at baseline, 1 month & 3 months, the values representing the mean ± standard deviation (SD)

Parameter (mmHg)

 Treatment Arms
Study Arm-1 Study Arm-2

Comparator Arm
Baseline SBP

149.64 ± 7.395

 147.55 ± 6.735

148.56 ± 6.589
End of 1-month SBP

135.5 ± 9.509

 137.73 ± 8.189

140.36 ± 6.801
End of 3 months SBP

133.75 ± 11.527

 138.68 ± 8.479

137.24 ± 8.864
Difference SBP

-13.63 ± 9.157

 -7.87 ± 7.341

-10.06 ± 9.666
Baseline DBP

90.39 ± 4.228

 91.61 ± 3.9

91.13 ± 4.785
End of 1-month DBP

81.79 ± 5.989

 84.93 ± 5.637

88.91 ± 8.549
End of 3 months DBP

80.75 ± 7.179

 84.89 ± 6.402

83 ± 6.275
Difference DBP

-9.81 ± 7.884

 -6.16 ± 8.14

-7.59 ± 6.472

Values represent the mean ± standard deviation

Diastolic Blood Pressure

The change in the DBP in study arm-1 from baseline to end of study was a reduction of 9.81 mmHg; in study arm-2, there was a reduction of 6.16 mmHg, and in the comparator arm, there was a reduction of 7.59 mmHg. The reduction in DBP was higher in the arm receiving MG-HT® once daily as compared to twice daily and standard of care. However, this difference was not statistically significant (p-value >0.05). The mean changes in DBP are shown in Table 2; although there was a high standard deviation, this compares well with the comparator arm.

Hypertension Stage Wise Distribution: Change from Baseline between Groups

In MG-HT® once daily arm, there were 22 subjects in stage 1 which reduced to 5 at study end. Similarly, there were 6 subjects in stage 2, and none at study end. Ten subjects became prehypertensive and 1 subject, normotensive; 12 subjects lost to follow-up before the end of the study. In MG-HT® twice daily arm, there were 24 subjects in stage 1 which reduced to 11 at study end. Similarly, there were 4 subjects in stage 2 and none at study end. Eight subjects became prehypertensive and none was normotensive; 9 subjects lost to follow-up before the end of the study. In standard of care arm, there were 20 subjects in stage 1 which reduced to 11 at study end. Similarly, there were 4 subjects in stage 2 and none at study end; 6 subjects became prehypertensive but none were normotensive; 7 subjects lost to follow-up before end of study. Subjects receiving MG-HT® once daily reported a greater reduction of BP in terms of returning to pre hypertensive levels at end of study when compared to MG-HT® twice daily and those receiving standard of care. This difference was statistically significant (p-value >0.001) despite a large standard deviation in the captured data set. The change in hypertension stage wise distribution is shown in Table 3.

Table 3: Hypertension stage wise distribution change from baseline between groups

Study Arm

 Stage Baseline

Visit 6
Antihypertensive therapy and MG-HT® once daily Stage 1

22

5
Stage 2

6

0
Pre-Hypertensive

0

10
Normal

0

1
Lost to follow-up

0

12

Total

 28

28
Antihypertensive therapy and MG-HT® twice daily Stage 1

24

11
Stage 2

4

0
Pre-Hypertensive

0

8
Normal

0

0
Lost to follow-up

0

9
Total

28

28
Standard of Care Stage 1

20

11
Stage 2

4

0
Pre-Hypertensive

0

6
Normal

0

0
Lost to follow-up

0

7
Total

24

24

Change in Magnesium (Mg) Levels from Baseline to Study End

The mean changes of Mg levels from baseline to study end were not statistically significant between study arms (p-value >0.05). The mean change in Mg levels are shown in Table 4.

Table 4: Mean changes in magnesium levels from baseline to study end, values represent the mean ± standard deviation

Antihypertensive therapy and MG-HT® once daily
Magnesium (mg/dL) Visit 1

(baseline)

 Visit 6

(study end)

 Difference in Mg levels (Visit 6 – Visit 1)
2.02 ± 0.135 2.05 ± 0.172 0.04 ± 0.159

Antihypertensive therapy and MG-HT® twice daily
Magnesium (mg/dL) Visit 1

(baseline)

 Visit 6

(study end)

 Difference in Mg levels (Visit 6 – Visit 1)
1.9 ± 0.238 2.07 ± 0.212 0.11 ± 0.125

Standard of Care with Antihypertensive therapy
Magnesium (mg/dL) Visit 1

(baseline)

 Visit 6

(study end)

 Difference in Mg levels (Visit 6 – Visit 1)
2.0 ± 0.226 2.06 ± 0.166 0.03 ± 0.179

Values represent the mean ± standard deviation

Change in Laboratory Parameters from Baseline to End of Study between Groups

The mean changes of laboratory parameters from baseline to study end were not statistically significant between study arms (p-value >0.05). The mean changes in laboratory parameters are shown in Table 5.

Table 5: Mean changes in laboratory parameters from baseline to study end, values represent the mean ± standard deviation

Parameter

 HbA1c (%) Visit 1

HbA1c (%) Visit 6
Arm-1

6.46 ± 1.181

6.42 ± 0.848
Arm-2

6.83 ± 1.521

6.35 ± 0.714
Arm-3

7.16 ± 1.807

6.61 ± 1.561

Parameter

 Cholesterol Total (mg/dL) Visit 1

Cholesterol Total (mg/dL) Visit 6
Arm-1

168.22 ± 51.147

168.46 ± 41.559
Arm-2

178.96 ± 40.963

178.81 ± 37.641
Arm-3

190.68 ± 42.956

179.45 ± 56.949

Parameter

 HDL (mg/dL) Visit 1

HDL (mg/dL) Visit 6
Arm-1

44.94 ± 9.427

42.73 ± 8.492
Arm-2

44.62 ± 10.993

45.97 ± 15.665
Arm-3

44.31 ± 12.119

43.33 ± 11.732

Parameter

 LDL (mg/dL) Visit 1

LDL (mg/dL) Visit 6
Arm-1

96.78 ± 41.116

94.69 ± 34.452
Arm-2

106.07 ± 34.422

100.28 ± 29.648
Arm-3

116 ± 35.68

99.88 ± 46.098

Parameter

 VLDL (mg/dL) Visit 1

VLDL (mg/dL) Visit 6
Arm-1

27.05 ± 12.296

31.04 ± 17.201
Arm-2

27.34 ± 10.869

29.16 ± 10.564
Arm-3

30.85 ± 12.887

35.74 ± 16.172

Parameter

 Triglyceride (mg/dL) Visit 1

Triglyceride (mg/dL) Visit 6
Arm-1

145.96 ± 83.259

161.33 ± 105.337
Arm-2

150.94 ± 80.182

178.27 ± 91.067
Arm-3

162.42 ± 90.644

196.94 ± 107.201

Parameter

 Bilirubin Total (mg/dL) Visit 1

Bilirubin Total (mg/dL) Visit 6
Arm-1

0.64 ± 0.375

0.77 ± 0.467
Arm-2

0.61 ± 0.287

0.65 ± 0.299
Arm-3

0.61 ± 0.502

0.62 ± 0.468

Parameter

 Bilirubin Direct (mg/dL) Visit 1

Bilirubin Direct (mg/dL) Visit 6
Arm-1

0.23 ± 0.12

0.29 ± 0.154
Arm-2

0.25 ± 0.136

0.26 ± 0.157
Arm-3

0.21 ± 0.097

0.21 ± 0.087

Parameter

 Bilirubin Indirect (mg/dL) Visit 1

Bilirubin Indirect (mg/dL) Visit 6
Arm-1

0.42 ± 0.3

0.48 ± 0.362
Arm-2

0.37 ± 0.189

0.38 ± 0.232
Arm-3

0.4 ± 0.426

0.4 ± 0.397

Parameter

 SGPT (ALT) (U/L)

 Visit 1

SGPT (ALT) (U/L)

Visit 6
Arm-1

27.16 ± 11.252

34.04 ± 19.129
Arm-2

25.91 ± 16.611

24.87 ± 12.862
Arm-3

35.63 ± 25.901

41.29 ± 32.789

Parameter

 SGOT (AST) (U/L)

 Visit 1

SGOT (AST) (U/L)

Visit 6
Arm-1

28.2 ± 13.319

34.89 ± 17.32
Arm-2

23.94 ± 10.598

25.94 ± 11.662
Arm-3

25.92 ± 11.204

29.19 ± 11.514

Parameter

 Albumin (g/dL) Visit 1

Albumin (g/dL) Visit 6
Arm-1

4.63 ± 0.506

4.37 ± 0.253
Arm-2

4.64 ± 0.462

4.67 ± 0.241
Arm-3

4.55 ± 0.384

4.47 ± 0.379

Parameter

 Alkaline Phosphatase (U/L) Visit 1

Alkaline Phosphatase (U/L) Visit 6
Arm-1

92.64 ± 46.993

102.19 ± 53.392
Arm-2

85.59 ± 17.885

82.74 ± 19.706
Arm-3

96.25 ± 26.122

88.71 ± 31.612

Parameter

 Blood Urea Nitrogen (mg/dL) Visit 1

Blood Urea Nitrogen (mg/dL) Visit 6
Arm-1

11.42 ± 3.38

11.44 ± 3.738
Arm-2

10.43 ± 4.024

10.47 ± 2.552
Arm-3

9.95 ± 2.703

10.82 ± 2.076

Parameter

 Serum Urea (mg/dL) Visit 1

Serum Urea (mg/dL) Visit 6
Arm-1

23.93 ± 6.459

25.11 ± 7.854
Arm-2

22.61 ± 8.649

21.9 ± 4.892
Arm-3

21.28 ± 5.785

23.15 ± 4.443

Parameter

 Creatinine (mg/dL) Visit 1

Creatinine (mg/dL) Visit 6
Arm-1

0.86 ± 0.228

0.89 ± 0.342
Arm-2

0.83 ± 0.293

0.83 ± 0.221
Arm-3

0.78 ± 0.173

0.82 ± 0.207

Values represent the mean ± standard deviation

Changes in Magnesium Levels in Magnesium Deficient Subjects from Baseline to End of 3 Months

There was a total of 9 subjects who had hypomagnesaemia at baseline with serum Mg levels below 1.7 mg/dL. Of these, 1 was in MG-HT® once daily arm, 6 were in MG-HT® twice daily arm out of which 2 subjects completed the visit 6 and remaining 4 were lost to follow-up before visit 6 and 2 were in the standard of care arm out of which, 1 subject completed the visit 6 and remaining 1 was lost to follow-up before visit 6. All subjects achieved normal serum Mg levels at study end with an average of 1.8 mg/dL. The mean change in Mg levels in hypomagnesaemic subjects are shown in Table 6, values representing the mean ± standard deviation.

Table 6: Mean changes in magnesium levels in subjects with Magnesium Deficiency from baseline to study end, values represent the mean ± standard deviation

Antihypertensive therapy and MG-HT® once daily

Visit 1

Visit 6
N

1

1
Magnesium (mg/dl)

1.7

1.8

Antihypertensive therapy and MG-HT® twice daily

Visit 1

Visit 6
N

6

2
Magnesium (mg/dl)

1.54 ± 0.15

1.8

Standard of Care with Antihypertensive therapy

Visit 1

Visit 6
N

2

1
Magnesium (mg/dl)

1.67 ± 0.042

1.8

Values represent the mean ± standard deviation

Change in SBP in Subjects with Hypomagnesaemia from Baseline to End of 3 Months

The changes in SBP in the 9 (1 subject in MG-HT® once daily arm, 6 subjects in MG-HT® twice daily arm and 2 subjects in Standard of Care arm) subjects who had hypomagnesaemia at baseline were not statistically significant between arms; out of these 9 subjects 4 in MG-HT® twice daily arm and 1 in Standard of Care arm lost to follow-up before study end visit. However, subjects receiving MG-HT® once daily reported a greater reduction in BP levels at study end with a reduction 10 mmHg compared to 7 mmHg and 6 mmHg of MG-HT® twice daily arm and standard of care arm respectively. The mean change in SBP in subjects with hypomagnesaemia is shown in Table 7.

Table 7: Mean changes in SBP in subjects with hypomagnesaemia from baseline to end of visit at 3 months, values represent the mean ± standard deviation

Antihypertensive therapy and MG-HT® once daily

Visit 1

Visit 6
N

1

1
SBP (mmHg)

150

140

Antihypertensive therapy and MG-HT® twice daily

Visit 1

Visit 6
N

6

2
SBP (mmHg)

144.5 ± 7.791

137.5 ± 3.536

Standard of Care with Antihypertensive therapy

Visit 1

Visit 6
N

2

1
SBP (mmHg)

146 ± 2.828

140

Values represent the mean ± standard deviation

Change in DBP in Subjects with Hypomagnesaemia from Baseline to End of 3 Months

The changes in DBP in the 9 subjects who had hypomagnesaemia at baseline were not statistically significant between arms. The mean changes in DBP in subjects with hypomagnesaemia are shown in Table 8.

Table 8: Mean changes in DBP in subjects with hypomagnesaemia from baseline to end of 3 months, values represent the mean ± standard deviation

Antihypertensive therapy and MG-HT® once daily

Visit 1

Visit 6
N

1

1
DBP (mmHg)

90

90

Antihypertensive therapy and MG-HT® twice daily
DBP (mmHg)

Visit 1

Visit 6
N

6

2
DBP (mmHg)

88.17 ± 4.491

90

Standard of Care with Antihypertensive therapy
DBP (mmHg)

Visit 1

Visit 6
N

2

1
DBP (mmHg)

94

90

Values represent the mean ± standard deviation

Relationship between Magnesium Levels and SBP and DBP between Arms (N=9)

As depicted in Figure 3, the correlation between changes in serum Mg levels and change in SBP was more marked in subjects receiving MG-HT® once daily compared to the other two arms. No correlation was seen between change in serum Mg levels and change in DBP in the treatment arms (Figure 3).

fig 1

Figure 1: Mean SBP change from baseline to study end

fig 2

Figure 2: Mean SBP change from baseline to study end

fig 3

Figure 3: Change in SBP, DBP and levels of magnesium in subjects with combined treatment arms vs. standard of care arm from baseline to end of 3 months

Change in SBP, DBP and Levels of Magnesium in Subjects with Combined Treatment Arms vs. Standard of Care Arm from Baseline to End of 3 Months

The pooled analysis of changes in SBP and DBP between control vs treatment (MGHT® once daily and twice daily combined) was not statistically significant. Mean changes are shown in Table 9.

Table 9: Mean change in SBP, DBP and levels of magnesium in subjects with combined treatment arms vs. standard of care arm from baseline to end of 3 months

SBP (mmHg) Visit 1

SBP (mmHg) Visit 6

N

 Mean ± SD N Mean ± SD

p-value
Treatment

56

 148.6 ± 7.087 35

136.43 ± 10.144

0.83
Standard of Care

24

 148.56 ± 6.589 17

137.24 ± 8.864
 

DBP (mmHg) Visit 1

DBP (mmHg) Visit 6
 

N

 Mean ± SD N Mean(SD)

p-value
Treatment

56

 91 ± 4.077 35

83 ± 6.987

0.982
Standard of Care

24

 91.13 ± 4.785 17

83 ± 6.275
 

Magnesium (mg/dL) Visit 1

Magnesium (mg/dL) Visit 6
 

N

 Mean ± SD N Mean ± SD

p-value
Treatment

55

 1.96 ± 0.199 35

2.06 ± 0.192

0.419
Standard of Care

24

 2.01 ± 0.226 17

2.06 ± 0.166

Change in Lab Parameters in Combined Treatment Arms vs. Standard of Care Arm from Baseline to End of 3 Months

The pooled analysis of change in laboratory parameters between control vs. treatment arms (MG-HT® once daily and twice daily combined) was not statistically significant. Mean changes are shown in Table 10.

Table 10: Mean change in lab parameters in combined treatment arms vs. standard of care arm from baseline to end of 3 months

 

 

HbA1c (%) Visit 1

 HbA1c (%) Visit 6

p-value
Treatment N

55

35

0.986
Mean ± SD

6.64 ± 1.359

6.38 ± 0.767
Standard of Care N

24

17
Mean ± SD

7.16 ± 1.807

6.61 ± 1.561
   

Cholesterol Total (mg/dl) Visit 1

 Cholesterol Total (mg/dl) Visit 6

p-value
Treatment N

55

35

0.391
Mean ± SD

173.49 ± 46.318

174.08 ± 39.236
Standard of Care N

24

17
Mean ± SD

190.68 ± 42.956

179.45 ± 56.949
   

HDL (mg/dl) Visit 1

 HDL (mg/dl) Visit 6

p-value
Treatment N

55

35

0.297
Mean ± SD

44.78 ± 10.132

44.49 ± 12.822
Standard of Care N

24

17
Mean ± SD

44.31 ± 12.119

43.33 ± 11.732
   

LDL (mg/dl) Visit 1

 LDL (mg/dl) Visit 6

p-value
Treatment N

55

35

0.606
Mean ± SD

101.34 ± 37.918

97.72 ± 31.575
Standard of Care N

24

17
Mean ± SD

116 ± 35.68

99.88 ± 46.098
   

VLDL (mg/dl) Visit 1

 VLDL (mg/dl) Visit 6

p-value
Treatment N

54

32

0.551
Mean ± SD

27.19 ± 11.523

30.1 ± 14.074
Standard of Care N

24

16
Mean ± SD

30.85 ± 12.887

35.74 ± 16.172
   

Triglyceride (mg/dl) Visit 1

 Triglyceride (mg/dl) Visit 6

p-value
Treatment N

55

35

0.888
Mean ± SD

148.41 ± 81.042

170.53 ± 96.742
Standard of Care N

24

17
Mean ± SD

162.42 ± 90.644

196.94 ± 107.201
   

Bilirubin Total (mg/dL) Visit 1

 Bilirubin Total (mg/dL) Visit 6

p-value
Treatment N

55

35

0.294
Mean ± SD

0.63 ± 0.332

0.7 ± 0.384
Standard of Care N

24

17
Mean ± SD

0.61 ± 0.502

0.62 ± 0.468
   

Bilirubin Direct (mg/dL) Visit 1

 Bilirubin Direct (mg/dL) Visit 6

p-value
Treatment N

55

35

0.256
Mean ± SD

0.24 ± 0.127

0.27 ± 0.154
Standard of Care N

24

17
Mean ± SD

0.21 ± 0.097

0.21 ± 0.087
   

Bilirubin Indirect (mg/dL) Visit 1

 Bilirubin Indirect (mg/dL) Visit 6

p-value
Treatment N

55

35

0.593
Mean ± SD

0.39 ± 0.251

0.43 ± 0.298
Standard of Care N

24

17
Mean ± SD

0.4 ± 0.426

0.4 ± 0.397
   

SGPT(ALT) (U/L) Visit 1

 SGPT (ALT) (U/L) Visit 6

p-value
Treatment N

55

35

0.661
Mean ± SD

26.55 ± 14.02

29.07 ± 16.447
Standard of Care N

24

17
Mean ± SD

35.63 ± 25.901

41.29 ± 32.789
   

SGOT (AST) (U/L) Visit 1

 SGOT (AST) (U/L) Visit 6

p-value
Treatment N

55

35

0.552
Mean ± SD

26.11 ± 12.14

30.03 ± 14.994
Standard of Care N

24

17
Mean ± SD

25.92 ± 11.204

29.19 ± 11.514
   

Albumin (g/dL) Visit 1

 Albumin (g/dL) Visit 6

p-value
Treatment N

55

35

0.509
Mean ± SD

4.63 ± 0.481

4.54 ± 0.286
Standard of Care N

24

17
Mean ± SD

4.55 ± 0.384

4.47 ± 0.379
   

Alkaline Phosphatase (U/L) Visit 1

 Alkaline Phosphatase (U/L) Visit 6

p-value
Treatment N

55

35

0.143
Mean ± SD

89.18 ± 35.649

91.63 ± 39.495
Standard of Care N

24

17
Mean ± SD

96.25 ± 26.122

88.71 ± 31.612
   

Blood Urea Nitrogen (mg/dL) Visit 1

 Blood Urea Nitrogen (mg/dL) Visit 6

p-value
Treatment N

55

35

0.795
Mean ± SD

10.93 ± 3.709

10.91 ± 3.138
Standard of Care N

24

17
Mean ± SD

9.95 ± 2.703

10.82 ± 2.076
   

Urea Serum (mg/dL) Visit 1

 Urea Serum (mg/dL) Visit 6

p-value
Treatment N

52

32

0.706
Mean ± SD

23.27 ± 7.587

23.41 ± 6.546
Standard of Care N

24

17
Mean ± SD

21.28 ± 5.785

23.15 ± 4.443
   

Creatinine (mg/dL) Visit 1

 Creatinine (mg/dL) Visit 6

p-value
Treatment N

55

35

0.931
Mean ± SD

0.84 ± 0.26

0.86 ± 0.28
Standard of Care N

24

17
Mean ± SD

0.78 ± 0.173

0.82 ± 0.207

Adverse Events

There were 8 adverse events in the study. The list of adverse events with the study arm distribution has been presented below. The causality assessment of all AEs reported were assessed to be unrelated to the study products. This means although sinus bradycardia and first-degree heart block had been reported to be associated with Mg administration, the incidence of such events are very low. None of the patients in study groups once or twice daily MG-HT® ever demonstrated any event of hypermagnesemia or critical hypomagnesemia, and thus it can be safely concluded that these adverse events did not result from any episode of hyper or hypomagnesemia, related to MG-HT® intervention. Additionally, the subjects had these problems even before the study. Therefore, these events were considered negligible, and all the adverse events were considered unrelated to the intervention.

The following AEs were reported, sinus bradycardia, first-degree heart block, insomnia, COVID-19, herpes zoster, weakness of leg and arm suspected due to Vitamin D deficiency, dyspepsia and constipation.

Discussion

The results of the present study show that MG-HT® once daily with continued antihypertensive regimen, MG-HT® twice daily with continued antihypertensive therapy and standard of care reduced BP in patients with stage 1 and 2 HTN. The mean change in SBP from baseline to end of study at 90 days was a reduction of 13.63 mmHg, 7.87 mmHg and 10.06 mmHg in MG-HT® once daily, MG-HT® twice daily and standard of care arm respectively. MG-HT® once daily arm showed higher SBP reduction as compared to MG-HT® twice daily arm and standard of care arm. However, this difference was not statistically significant (p-value > 0.05).

The mean change in DBP from baseline to end of study at 90 days was a reduction of 9.81 mmHg, 6.16 mmHg and 7.59 mmHg in MG-HT® once daily, MG-HT® twice daily and standard of care arm respectively. The reduction in DBP was higher in the arm receiving MG-HT® once daily as compared to MG-HT® twice daily and standard of care. However, this difference was not statistically significant (p-value > 0.05).

The pooled analysis of changes in SBP and DBP from baseline to end of 90 days between control vs. treatment (MG-HT® once daily and twice daily combined) was not statistically significant (p-value > 0.05).

These data showed wide variations in recorded BP, which could be due to two reasons, (1) wide variations in BP recordings in various other studies involving BP as a variable (2) a larger percentage of patients who were lost to follow-up due to prevalent COVID-19 situation in India. This wide variation led to a large standard deviation which might have contributed to lack of statistical significance of the findings in different data sets. The result also showed statistically non-significant improvement of lipid profile and serum Mg from baseline to study end between study arms (p-value > 0.05). Nine subjects who had hypomagnesaemia at baseline, achieved normal serum Mg levels at study end with an average value of 1.8 mg/dL. The mean change in SBP and DBP from baseline to end of study at 90 days in these 9 subjects with hypomagnesaemia was not statistically significant between study arms; however, subjects receiving MG-HT® once daily reported a greater reduction in SBP levels at the end of study with a reduction 10 mmHg compared to 7 mmHg and 6 mmHg for MG-HT® twice daily arm and standard of care arm respectively.

The change from baseline to study end in the stage wise distribution of number of subjects was statistically significant (p-value <0.001). There were 66 subjects (82.5%) in stage 1 which reduced to 27 subjects (33.8%) at study end. Similarly, there were 14 subjects (17.5%) in stage 2 and none at study end. A statistically significant high proportion of subjects, i.e., 24 subjects (30%) moved to prehypertensive levels with 15 subjects (18.8%) reporting prehypertensive levels of BP as early as, a month after treatment. One subject reported normal levels of BP at one month of treatment and maintained the same levels at the end of study.

In our study the causality assessment of all AEs reported were assessed to be unrelated to the study products. There were no serious adverse events reported in the present study.

Several studies have shown that inadequate intake of Mg may cause essential hypertension. Mg supplementation has been documented to decrease BP acting as Calcium antagonist on smooth muscle tone, leading to vasorelaxation, which appears to be the desired end result of all antihypertensive treatments and could be the final common physiological pathway for blood pressure regulation. This suggests an inverse correlation between dietary Mg and BP. Some studies have shown that Mg supplementation has been shown to decrease BP in normotensives; despite that, even now the clinical practice does not routinely recommend Mg as an active treatment for HTN. However, reduction of BP, albeit low, has been shown to be clinically significant in hypertensive patients, in that this can reduce the incidence of coronary heart disease, heart failure, and stroke or other complications, particularly in high-risk individuals. Thus, the reduction of BP as a result of Mg over and above that effected by standard of care can be of great and significant importance in management of HTN, more so when the clinical outcomes with standard of care are suboptimal or hypo-responsive, in addition to reduction of complications [35-38].

Some studies have established that Mg deficiency caused by lack of dietary or supplemental Mg intake leads to HTN. Due to the correlation between Mg and HTN, it has been suggested that supranutritional Mg intake may act as a mild antihypertensive agent. Although this antihypertensive effect of Mg is existent, it is also to be noted that these studies failed to demonstrate any significant association between serum Mg concentration and the risk of HTN. On the other hand, although most studies on Mg supplementation showed a reduction of 3-4 mmHg of SBP and 2-3 mmHg of DBP, one study reported significantly higher reduction of BP to the tune of 18.7 mmHg in SBP and 10.9 mmHg in DBP when the SBP was higher than 155 mmHg. This finding is significant and comparable to our study findings [21].

A landmark categorized systematic review of 49 clinical trials was published in 2021. In this, Rosanoff et al. categorized studies involving 4 categories; our study may belong to the second category which involved uncontrolled hypertensives, who were subjects using antihypertensive medications during and prior to the study but were still hypertensive at baseline. This study has demonstrated conclusively that uncontrolled hypertensive subjects respond to oral Mg therapy consistently and significantly lowered both SBP and DBP. This is in consonance with the findings in our study where once daily dose of Mg containing nutraceutical formulation added to the standard of care antihypertensive therapy leads to reduction of both SBP and DBP, which was resistant to treatment with standard of care therapy [21].

In our study, once daily MG-HT® added to standard antihypertensive regimen led to clinically significant BP reduction, in comparison to twice daily MG-HT®, contrary to general expectation that twice daily regimen will offer more Mg and thus will be more potent in terms of BP reduction. This can be explained from the findings of the categorized systematic review by Rosanoff et al. (2022) which showed that although all Mg doses ranging from 240-607 mg/day showed decreased BP in all uncontrolled hypertensives, it failed to reduce BP in controlled hypertensives or normotensive subjects. The Mg-replete patients showed no change in BP even at high Mg dose [21]. Another meta-analysis by Zhang et al. (2016) has demonstrated that the relationship between rise in Mg level and drop in BP is nonlinear and although there is a tendency of reduction of DBP by 2.26 mmHg for every 0.1 mmol/L increase in serum Mg level, depending on baseline Mg status. This relationship ceased to exist when the subjects were Mg replete, following which the BP response to Mg was invisible [15]. In another review by Houston, it has been stated that with Mg, patients with highest BP levels at entry had the largest reduction in BP. Additionally, in this review, the author quotes the findings of a randomized controlled study which shows that although Mg and potassium combination reduced BP to a significant extent, further addition of Mg failed to reduce BP further [23]. From these data, it is clear that just numerical enhancement of Mg dosage would not linearly reduce the BP, and there are several physiological factors that control the impact of Mg on BP, and Mg repletion annuls the impact of additional dose of Mg for further reduction of BP. This may explain why in our study a dose of 70 mg of Mg was effective in reducing BP, but a twice daily dose of Mg could not further enhance the BP reduction.

In our study intake of MG-HT® could reduce the nominal values of different laboratory parameters, but these lacked significances due mainly to the contracted sample size issue, and this is an important finding in that many of these parameters can reduce the CV risks, particularly in patients with HTN. Several studies have shown that oral Mg therapy could improve several cardiovascular health parameters, such as, serum and plasma Mg, endothelial function, fasting glucose and insulin resistance, triglycerides, and total cholesterol as well as high-density lipoproteins. Although adequately structured studies are needed to establish these parameters, it can be clearly stated that in uncontrolled hypertensives, addition of Mg in the management regimen would not only reduce the BP, would also reduce the hypertensive risks.

It has been shown that all forms of Mg, inorganic or organic are effective in reducing BP in uncontrolled hypertensive subjects. Additionally, some authors also have demonstrated that patients who are Mg replete respond poorly to Mg therapy in comparison to Mg deplete individuals. Several studies have also shown that many patients with baseline HTN, be it untreated or uncontrolled, respond to oral Mg therapy to demonstrate a BP lowering effect. Obviously, in relation to this, another prudent question may arise, particularly in case of hypertensive patients who are on standard of care therapy, like those who were randomized as subjects in this study, whether routine use of Mg should be considered in addition to antihypertensive therapy. Mg has vasorelaxant effect, leading to lowering down of both SBP and DBP. Therefore, patients who are already on antihypertensive therapy as standard of care at baseline may have additive effect on reduction of BP due to individual and independent action of both. This may lead to a clinical situation of hypotension with administration of Mg just in case the patient is normotensive at baseline with the standard of care therapy. One study by Hattori et al. (1998) has conclusively demonstrated that oral Mg does not demonstrate BP lowering effect on normotensive subjects, which is only demonstrable in hypertensive subjects [38]. Another systematic review in 2021 had also shown that a range of doses of Mg could not effect any change in normotensive subjects consistently [23].

Several studies including reviews and meta-analyses demonstrated minor adverse effects only among the participants, which were transient. Additionally, these adverse effects were reported in both experimental and control groups. This finding is identical to that in our study and therefore, it can be commented that the treatment offered by MG-HT® is also devoid of any considerable adverse effects. The effective dose in our study was 70 mg of elemental Mg from 500 mg of organic Mg salt. It has been stated in literature that the tolerable upper limit of Mg intake from non-food sources is 350 mg/day, and ICMR states, in Indian population, the RDA ranges from 370 to 440 mg (440 mg for males and pregnant women; 370 mg for nonpregnant females). Many studies reported mild gastrointestinal symptoms in this dosage range. It is also important to note that many studies which have supplemented substantially more than this range did not demonstrate any such adverse effects. It is well known from other literature that very high Mg intake can be dangerous to people despite not having renal or intestinal disease, but such concentrations are in the range of more than 5000 mg, about 70 times more than the strength used in our study product. This makes this product safe beyond any doubts or concerns, which has been demonstrated in the findings of this study, and this is presumably due to combination with other ingredients which have innate capability to reduce BP, in conjunction with oral Mg.

The evidence for a positive effect of Mg on high BP risk accentuates the importance of largely encouraging the intake of foods, such as, vegetables, nuts, whole cereals and legumes, restricting processed foods, which are deprived in Mg and lack other fundamental nutrients as well, in order to prevent high BP. In some cases when diet is not adequate to sustain a sufficient Mg status, Mg supplementation may be of advantage and has been shown to be well tolerated [35], particularly when the standard of care therapy fails to produce desired or optimum results in terms of BP in a hypertensive patient. This bears further importance in terms of risk reduction in vulnerable patient groups or in hypertensive patients who pose risk of complications related to uncontrolled HTN.

A pooled analysis of 7 RCTs showed that Mg supplementation significantly reduced SBP and DBP in type 2 diabetes mellitus patients [36]. A meta-analysis, based on evidence from 34 randomized, double-blind, placebo-controlled trials, showed a significant antihypertensive effect of Mg supplementation on both systolic and diastolic BP among normotensive or hypertensive adults. Findings from this meta-analysis suggested that oral Mg supplements can be recommended for the prevention of high BP or as adjunct to antihypertensive therapy [37] in patients where there are considerable risks of HTN or hypertensive complications. Our study also shows similar findings, and this is the first study of this kind in Indian population. Therefore, in the Indian population, the findings of this study can be translated into management practices in that all patients with Stage 1 and 2 uncontrolled HTN on any antihypertensive regimen can have clinically beneficial outcomes when a Mg containing supplement, MG-HT® is added to the regimen.

However, this study has limitations hindering the generalization of the findings. Due to COVID-19, the number of patients lost to followup in each arm was large, totalling 28, which has contributed to large variation in data and has compromised the statistical significance. However even then, it could yield a very important finding of clinically significant numerical reduction of BP numbers with the study product. A large multicentric study in the same design may improve the different outcome parameters to a level of statistical significance which may impact the clinical management of HTN in future.

Conclusion

The findings of the present prospective, randomized, three-arm, open label, parallel group, multicentric study showed that adding oral MG-HT® to the existing antihypertensive regimens reduced BP in patients with stage 1 and 2 HTN. MG-HT® therapy holds potential as a way of safely achieving lower BP without increasing antihypertensive medications, specifically in persons where standard of care therapy fails to provide optimum BP control increasing risks of complications. Our findings suggested that oral MG-HT® supplement can be recommended for the prevention of HTN or as an adjuvant to antihypertensive therapy in patients with inadequate control, specifically in India. However, future large-scale, well-designed studies are warranted to provide more consistent evidence of MG-HT® supplementation benefits on BP among these patients.

Acknowledgments

The authors thank Dr. DB Anantha Narayana, Research Scholar, Bangalore, and Prof Roop Krishen Khar, Professor and Director, B.S. Anangpuria Institute of Pharmacy- Faridabad for their support in review and comments prior to publication.

Source of Funding

This study was funded by Pharmed Limited, Bangalore.

Availability of Data and Material

The data that support the findings of this study are available from the corresponding author upon reasonable request.

References

  1. Rajeev Gupta, Kiran Gaur, C Venkata S Ram (2019) Emerging trends in hypertension epidemiology in India. J Hum Hypertens 33: 575-587.
  2. Soumitra Ghosh, Manish Kumar (2019) Prevalence and associated risk factors of hypertension among persons aged 15-49 in India: a cross-sectional study. BMJ Open 9: e029714.
  3. Majid Jalalyazdi, Javad Ramezani, Azadeh Izadi-Moud, Fereshteh Madani-Sani, Shokufeh Shahlaei, et al. (2019) Effect of hibiscus sabdariffa on blood pressure in patients with stage 1 hypertension. J Adv Pharm Technol Res 10: 107-111.
  4. Hui-Fang Chiu, Kamesh Venkatakrishnan, Oksana Golovinskaia, Chin-Kun Wang (2021) Impact of Micronutrients on Hypertension: Evidence from Clinical Trials with a Special Focus on Meta-Analysis. Nutrients 13: 588.
  5. Senkadhirdasan Dakshinamurthy, Vartika Saxena, Ranjeeta Kumari, Anissa Atif Mirza, Minakshi Dhar (2020) Prevalence of hypertension and associated cardiometabolic risk factors in urban Rishikesh, Uttarakhand. J Family Med Prim Care 9: 2931-2939.
  6. Jonas Prenissl, Jennifer Manne-Goehler, Lindsay M Jaacks, Dorairaj Prabhakaran, Ashish Awasthi, et al. (2019) Hypertension screening, awareness, treatment, and control in India: A nationally representative cross-sectional study among individuals aged 15 to 49 years. PLoS Med 16: e1002801.
  7. Raghupathy Anchala, Nanda K Kannuri, Hira Pant, Hassan Khan, Oscar H Franco, et al. (2014) Hypertension in India: a systematic review and meta-analysis of prevalence, awareness, and control of hypertension. J Hypertens 32: 1170-1177.
  8. Carolina Guerrero-García, Alberto Francisco Rubio-Guerra (2018) Combination therapy in the treatment of hypertension. Drugs Context 7: 212531.
  9. Julian P Yaxley, Sam V Thambar (2015) Resistant hypertension: an approach to management in primary care. J Family Med Prim Care 4: 193-199.
  10. R Padmanabhan, Rana Gopal Singh, Govindan Unni, Bhupen Desai, Sanjeev Kumar Hiremath, et al. (2020) Multidisciplinary Consensus Document on the Management of Uncontrolled Hypertension in India. High Blood Pressure & Cardiovascular Prevention.
  11. Mayank Kapoor, Minakshi Dhar, Anissa Mirza, Vartika Saxena, Monika Pathania (2021) Factors responsible for Uncontrolled Hypertension in the Adults over 50 years of age: A pilot study from Northern India. Indian Heart Journal 73: 644-646.
  12. Rajkumar Bharatia, Manoj Chitale, Ganesh Narain Saxena, Raman Ganesh Kumar, Chikkalingaiah, et al. (2016) Management Practices in Indian Patients with Uncontrolled Hypertension 64: 14-21.
  13. Andrea Rosanoff (2005) Magnesium and hypertension. Clin Calcium 15: 255-260.
  14. Omid Asbaghi, Reza Hosseini, Behnoosh Boozari, Ehsan Ghaedi, Sara Kashkooli, et al. (2021) The Effects of Magnesium Supplementation on Blood Pressure and Obesity Measure among Type 2 Diabetes Patient: A Systematic Review and Meta-analysis of Randomized Controlled Trials. Biol Trace Elem Res 199: 413-424.
  15. Xi Zhang, Yufeng Li, Liana C Del Gobbo, Andrea Rosanoff, Jiawei Wang, et al. (2016) Effects of Magnesium Supplementation on Blood Pressure: A Meta-Analysis of Randomized Double-Blind Placebo-Controlled Trials. Hypertension 68: 324-333.
  16. Nikolina Banjanin, Goran Belojevic (2018) Changes of Blood Pressure and Hemodynamic Parameters after Oral Magnesium Supplementation in Patients with Essential Hypertension-An Intervention Study. Nutrients 10: 581.
  17. Ana Rosa Cunha, Jenifer D’El-Rei, Fernanda Medeiros, Bianca Umbelino, Wille Oigman, et al. (2017) Oral magnesium supplementation improves endothelial function and attenuates subclinical atherosclerosis in thiazide-treated hypertensive women. J Hypertens 35: 89-97.
  18. F Guerrero-Romero, M Rodríguez-Morán (2009) The effect of lowering blood pressure by magnesium supplementation in diabetic hypertensive adults with low serum magnesium levels: a randomized, double-blind, placebo-controlled clinical trial. J Hum Hypertens 23: 245-251.
  19. Ligia J Dominguez, Nicola Veronese, Mario Barbagallo (2021) Magnesium and Hypertension in Old Age. Nutrients 13: 139.
  20. Andrea Rosanoff, Rebecca B Costello, Guy H Johnson (2021) Effectively Prescribing Oral Magnesium Therapy for Hypertension: A Categorized Systematic Review of 49 Clinical Trials. Nutrients 13: 195.
  21. Nikolina Banjanin, Goran Belojevic (2018) Changes of Blood Pressure and Hemodynamic Parameters after Oral Magnesium Supplementation in Patients with Essential Hypertension-An Intervention Study. Nutrients 10: 581.
  22. Mark Houston (2011) The role of magnesium in hypertension and cardiovascular disease. J Clin Hypertens (Greenwich) 13: 843-847.
  23. Moein Askarpour, Amir Hadi, Azadeh Dehghani Kari Bozorg, Omid Sadeghi, Ali Sheikhi, et al. (2019) Effects of L-carnitine supplementation on blood pressure: a systematic review and meta-analysis of randomized controlled trials. J Hum Hypertens 33: 725-734.
  24. Craig J McMackin, Michael E Widlansky, Naomi M Hamburg, Alex L Huang, Susan Weller, et al. (2007) Effect of combined treatment with alpha-Lipoic acid and acetyl-L-carnitine on vascular function and blood pressure in patients with coronary artery disease. J Clin Hypertens (Greenwich) 9: 249-255.
  25. Chuanxun Yuan, Xueru Zhang, Xue Long, Jing Jin, Risheng Jin (2019) Effect of β-sitosterol self-microemulsion and β-sitosterol ester with linoleic acid on lipid-lowering in hyperlipidemic mice. Lipids Health Dis18: 157.
  26. Simin Feng, Zhuqing Dai, Anna B Liu, Jinbao Huang, Nihal Narsipur, et al. (2018) Intake of stigmasterol and β-sitosterol alters lipid metabolism and alleviates NAFLD in mice fed a high-fat western-style diet. Biochim Biophys Acta Mol Cell Biol Lipids 1863: 1274-1284.
  27. M K Radika, P Viswanathan, C V Anuradha (2013) Nitric oxide mediates the insulin sensitizing effects of β-sitosterol in high fat diet-fed rats. Nitric Oxide 32: 43-53.
  28. Zhuxian Zhang, Mengyi Liu, Chun Zhou, Panpan He, Yuanyuan Zhang, et al. (2021) Evaluation of Dietary Niacin and New-Onset Hypertension Among Chinese Adults. JAMA Netw Open 4: e2031669.
  29. H E Bays, D J Rader (2009) Does nicotinic acid (niacin) lower blood pressure? Int J Clin Pract 63: 151-159.
  30. William E Boden, Mandeep S Sidhu, Peter P Toth (2014) The therapeutic role of niacin in dyslipidemia management. J Cardiovasc Pharmacol Ther 19: 141-158.
  31. James McKenney (2003) Niacin for dyslipidemia: considerations in product selection. Am J Health Syst Pharm 60: 995-1005.
  32. Shyamala Dakshinamurti, Krishnamurti Dakshinamurti (2015) Antihypertensive and neuroprotective actions of pyridoxine and its derivatives. Can J Physiol Pharmacol 93: 1083-1090.
  33. M Aybak, A Sermet, M O Ayyildiz, A Z Karakilçik (1995) Effect of oral pyridoxine hydrochloride supplementation on arterial blood pressure in patients with essential hypertension. Arzneimittelforschung 45: 1271-1273.
  34. Ligia Dominguez, Nicola Veronese, Mario Barbagallo (2020) Magnesium and Hypertension in Old Age. Nutrients 13: 139.
  35. Omid Asbaghi, Reza Hosseini, Behnoosh Boozari, Ehsan Ghaedi, Sara Kashkooli, et al. (2021) The Effects of Magnesium Supplementation on Blood Pressure and Obesity Measure Among Type 2 Diabetes Patient: A Systematic Review and Meta-analysis of Randomized Controlled Trials. Biol Trace Elem Res 199: 413-424.
  36. Xi Zhang, Yufeng Li, Liana C Del Gobbo, Andrea Rosanoff, Jiawei Wang, et al. (2016) Effects of Magnesium Supplementation on Blood Pressure: A Meta-Analysis of Randomized Double-Blind Placebo-Controlled Trials. Hypertension 68: 324-333.
  37. L Kass, J Weekes, L Carpenter (2012) Effect of magnesium supplementation on blood pressure: a meta-analysis. Eur J Clin Nutr 66: 411-418.
  38. K Hattori 1, K Saito, H Sano, H Fukuzaki (1988) Intracellular magnesium deficiency and effect of oral magnesium on blood pressure and red cell sodium transport in diuretic-treated hypertensive patients. Jpn Circ J 52: 1249-1256.
fig 1a

Continuation of Temozolomide Chemotherapy in a Glioblastoma Patient After Resolution of COVID-19 Infection

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DOI: 10.31038/CST.2022732

Abstract

We present a case of a 33 years old patient with glioblastoma who was diagnosed with Coronavirus disease 2019 (COVID-19) while undergoing chemotherapy. The patient did not have any other medical comorbidities. Due to active infection and leucopenia, his chemotherapy with temozolomide was on hold for 1 month. Temozolomide was resumed after resolution of leucopenia, improvement of COVID symptoms and a negative COVID-PCR (polymerase chain reaction) test. Patient continued to do well after administration of subsequent temozolomide cycles. A repeat computed tomography (CT) chest 2 months post infection revealed resolution of consolidation and no new areas of consolidation. Temozolomide was safely administered in this patient without reactivation of COVID-19 infection. Patient did not have any thrombotic events.

Keywords

Glioblastoma, COVID-19 pneumonia, Temozolomide, Chemotherapy resumption

Case Report

We present a case of a previously healthy 33 years old patient who presented with severe headaches and was found to have a right parietal lobe heterogeneously enhancing intra-axial neoplasm on magnetic resonance imaging (MRI) of the brain. He underwent a complete resection of the enhancing tumor volume. Pathology revealed a WHO grade IV astrocytoma, isocitrate dehydrogenase enzyme (IDH wild-type), O6-methylguanine-DNA methyltransferase (MGMT) unmethylated, positive CDKN2A loss (cyclin-dependent kinase inhibitor). He was subsequently treated as per the Stupp protocol with concurrent temozolomide and radiation followed by maintenance temozolomide. He was non-compliant with tumor treating fields which he self-discontinued after few months of treatment.

After completion of 3 out of 6 temozolomide cycles, he was admitted for fever and upper abdominal pain. A COVID-19 polymerase chain reaction (PCR) test was positive. He acquired this infection in the pre-COVID-19 vaccine era. Laboratory tests revealed normal WBC count of 4.8 K/UL, normal absolute neutrophil count (3.5 K/MM3), grade 2 lymphopenia with low absolute lymphocyte count (0.7 K/MM3). Computed tomography (CT) of the chest revealed patchy peripheral bibasilar ground glass and consolidative opacities compatible with pulmonary infection, with viral etiology such as COVID-19 (Figure 1A). He did not have pulmonary symptoms including shortness of breath or cough. Fever and abdominal pain resolved after 2 weeks of supportive care. However, due to the active COVID-19 infection and leucopenia, temozolomide dosing was held. It was subsequently resumed (cycle 4) after resolution of clinical symptoms and signs of COVID, normalization of hematological parameters (resolution of lymphopenia) and a negative COVID-19 PCR test. He tolerated his chemotherapy well through the completion of 6 cycles of temozolomide. A repeat CT chest 2 months after the initial COVID-19 infection revealed resolution of the lung consolidation (Figure 1B). Patient did not have any thrombotic events.

fig 1a

Figure 1A: Non contrast computed tomography chest showing bibasilar ground glass and consolidative opacities

fig 1b

Figure 1B: resolution of bibasilar ground glass and consolidative opacities after 2 months

Discussion

COVID-19, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which was declared as a global pandemic in March 2020 [1].

Impact of anti-cancer therapies on the course of COVID-19, and risk factors affecting the COVID-19 outcome for patients with primary brain tumors in particular, have not yet been elucidated. The aggravated immune response to COVID infection mediated by endothelial cells cytokines can potentially change the permeability of the blood brain barrier (BBB) causing exposure of the cerebral tissue to viral proteins [2].

Reactivation of virus post cancer chemotherapy has been reported with hepatitis B [3,4]. Similar concern exists with resumption of chemotherapy after SARS-CoV-2 infection. The immunocompromised state of cancer patients makes them more vulnerable to infection and morbidity with COVID-19 [5-7]. This increased vulnerability is partially due to the chronic immunocompromised state which can be exacerbated by anti-cancer therapies. There are conflicting results on risk of mortality with cytotoxic chemotherapy in COVID-19 patients [8-12]. Poor outcomes of COVID-19 infection in patients with receipt of recent chemotherapy is likely related to other comorbidities (peri-COVID-19 lymphopenia, neutropenia, diabetes, hypertension, and cardiovascular diseases) [10,13]. American Society of Clinical Oncology guidelines suggest waiting for infected patients to become asymptomatic and register a negative COVID-19 test before resuming treatment [9,14]. It is crucial to avoid under-treatment and treatment delays.

A report from the Dutch Oncology COVID-19 Consortium included 30 primary brain tumor patients of which 60% were glioblastomas [15]. Most patients had received cancer treatment ≤90 days prior to COVID-19 diagnosis. There were no indications of a negative impact of systemic anti-cancer therapies on the outcome of COVID-19 [15]. The thoracic cancer international COVID-19 collaboration (TERAVOLT) registry is the first large dataset of patients with COVID-19 and thoracic malignancies, regardless of therapies administered. No statistically significant association was found between anti-cancer treatment with mortality rate of the patients with thoracic cancers who were infected with COVID-19 [16]. Safe resumption of chemotherapy after resolution of COVID symptoms (approximately a month after the positive PCR test) has been reported in a young breast cancer patient without significant comorbidities [17]. There was no re-activation of COVID-19 or pulmonary adverse events [17]. Similarly, chemotherapy was safely resumed (approximately a month after the positive PCR test) in a 60 years old lady with ovarian cancer who was infected with COVID-19 [18]. In both these cases chemotherapy was resumed after resolution of COVID symptoms and negative PCR test. A 29 years old gentleman with a poor risk germ cell tumor safely resumed cytotoxic chemotherapy despite positive SARS-CoV-2 nasopharyngeal swab (he did have resolution of COVID symptoms) [19]. Two patients from Poland had no complications with chemotherapy continuation after COVID infection [20]. One patient was a young gentleman with primary mediastinal B-cell lymphoma, other patient was 56 years old gentleman with sigmoid cancer with asymptomatic COVID infection when receiving chemotherapy [20]. A prospective multi-institutional study 39 cancer patients in China reported low risk of SARS-CoV-2 reactivation after chemotherapy administration [21]. This study involved patients with different cancer pathologies including a 41 years old patient with glioblastoma with no comorbidities [21]. A severe case of COVID pneumonia was reported due to viral re-activation after resumption of immunosuppressive therapy (lymphocyte- and antibody-depleting therapy) in a 55 years old lady with CD20+ B cell acute lymphoblastic leukemia as well as other comorbidities including diabetes and cardiovascular disease [22]. Chemotherapy in her case was resumed 2 months after the first positive COVID PCR test, she had complete resolution of COVID symptoms and a negative PCR test before initiation of chemotherapy [22]. Most of the above studies indicate safe resumption of chemotherapy after resolution of COVID infection in patients without significant comorbidities.

For brain tumor patients there is lack of reliable data on the optimal timing and safety of resumption of cytotoxic chemotherapy in patients infected with COVID-19. Malignant gliomas are aggressive tumors with poor survival rates. Delay or discontinuation of cytotoxic chemotherapy will result in tumor progression and neurologic adverse events. Safety of adjuvant chemotherapy with temozolomide for treating gliomas during covid-19 pandemic has been reported [23]. Nevertheless, risk and benefits of chemotherapy should be considered especially in systemic chemotherapy such as alkylating agents, moderate delay in systemic treatment is acceptable [24]. To the best of our knowledge, there has been no report of continuation of chemotherapy with temozolomide in patients with high grade glioma and symptomatic covid-19 infection. In the case reported here, successful resumption of standard temozolomide dosing was possible with resolution of the COVID-19 associated clinical symptoms and radiographic findings and normalization of the hematological parameters. This report may provide insight into timing of safe resumption of essential chemotherapy post COVID-19 infection in a high grade glioma patient without significant comorbidities.

References

  1. Guan WJ, Ni ZY, Hu Y, Liang WH, Ou CQ, et al. (2020) Clinical Characteristics of Coronavirus Disease 2019 in China. N Engl J Med 382: 1708-1720.
  2. Ljubimov VA, Ramesh A, Davani S, Danielpour M, Breunig JJ, et al. (2022) Neurosurgery at the crossroads of immunology and nanotechnology. New reality in the COVID-19 pandemic. Adv Drug Deliv Rev 181: 114033. [crossref]
  3. Yeo W, Chan PK, Zhong S, Ho WM, Steinberg JL, et al. (2000) Frequency of hepatitis B virus reactivation in cancer patients undergoing cytotoxic chemotherapy: a prospective study of 626 patients with identification of risk factors. J Med Virol 62: 299-307. [crossref]
  4. Lubel JS, Angus PW (2010) Hepatitis B reactivation in patients receiving cytotoxic chemotherapy: diagnosis and management. J Gastroenterol Hepatol 25: 864-871. [crossref]
  5. Zhang L, Zhu F, Xie L, Wang C, Wang J, et al. (2020) Clinical characteristics of COVID-19-infected cancer patients: a retrospective case study in three hospitals within Wuhan, China. Ann Oncol 31: 894-901. [crossref]
  6. Zhang H, Han H, He T, Labbe KE, Hernandez AV, et al. (2021) Clinical Characteristics and Outcomes of COVID-19-Infected Cancer Patients: A Systematic Review and Meta-Analysis. J Natl Cancer Inst 113: 371-380. [crossref]
  7. Liang W, Guan W, Chen R, Wang W, Li J, et al. (2020) Cancer patients in SARS-CoV-2 infection: a nationwide analysis in China. Lancet Oncol 21: 335-337. [crossref]
  8. Cioffi R, Sabetta G, Rabaiotti E, Bergamini A, Bocciolone L, et al. (2021) Impact of COVID-19 on medical treatment patterns in gynecologic oncology: a MITO group survey. Int J Gynecol Cancer 31: 1363-1368. [crossref]
  9. Lievre A, Turpin A, Ray-Coquard I, Le Malicot K, Thariat J, et al. (2020) Risk factors for Coronavirus Disease 2019 (COVID-19) severity and mortality among solid cancer patients and impact of the disease on anticancer treatment: A French nationwide cohort study (GCO-002 CACOVID-19). Eur J Cancer 141: 62-81. [crossref]
  10. Tian J, Yuan X, Xiao J, Zhong Q, Yang C, et al. (2020) Clinical characteristics and risk factors associated with COVID-19 disease severity in patients with cancer in Wuhan, China: a multicentre, retrospective, cohort study. Lancet Oncol 21: 893-903.
  11. Yang K, Sheng Y, Huang C, Jin Y, Xiong N, et al. (2020) Clinical characteristics, outcomes, and risk factors for mortality in patients with cancer and COVID-19 in Hubei, China: a multicentre, retrospective, cohort study. Lancet Oncol 21: 904-913.
  12. Lee LY, Cazier JB, Angelis V, Arnold R, Bisht V, et al. (2020) COVID-19 mortality in patients with cancer on chemotherapy or other anticancer treatments: a prospective cohort study. Lancet 395: 1919-1926. [crossref]
  13. Jee J, Foote MB, Lumish M, Stonestrom AJ, Wills B, et al. (2020) Chemotherapy and COVID-19 Outcomes in Patients With Cancer. J Clin Oncol 38: 3538-3546. [crossref]
  14. ASCO, Cancer Treatment & Supportive Care.
  15. De Joode K, Taal W, Snijders TJ, Hanse M, Koekkoek JAF, et al. (2022) Patients with primary brain tumors and COVID-19: A report from the Dutch Oncology COVID-19 Consortium. Neuro Oncol 24: 326-328. [crossref]
  16. Garassino MC, Whisenant JG, Huang LC, Trama A, Torri V, et al. (2020) COVID-19 in patients with thoracic malignancies (TERAVOLT): first results of an international, registry-based, cohort study. Lancet Oncol 21: 914-922. [crossref]
  17. Horiguchi J, Nakashoji A, Kawahara N, Matsui A, Kinoshita T (2021) Chemotherapy resumption in breast cancer patient after COVID-19. Surg Case Rep 7: 170. [crossref]
  18. Liontos M, Kaparelou M, Karofylakis E, Kavatha D, Mentis A, et al. (2020) Chemotherapy resumption in ovarian cancer patient diagnosed with COVID-19. Gynecol Oncol Rep 33: 100615. [crossref]
  19. Pedrazzoli P, Rondonotti D, Cattrini C, Secondino S, Ravanini P, et al. (2021) Metastatic Mediastinal Germ-Cell Tumor and Concurrent COVID-19: When Chemotherapy Is Not Deferrable. Oncologist 26: e347-e349. [crossref]
  20. Wozniak K, Sachs W, Boguradzki P, Basak GW, Stec R (2021) Chemotherapy During Active SARS-CoV2 Infection: A Case Report and Review of the Literature. Front Oncol 11: 662211. [crossref]
  21. Bi J, Ma H, Zhang D, Huang J, Yang D, et al. (2020) Does chemotherapy reactivate SARS-CoV-2 in cancer patients recovered from prior COVID-19 infection? Eur Respir J 56: 2002672. [crossref]
  22. Lancman G, Mascarenhas J, Bar-Natan M (2020) Severe COVID-19 virus reactivation following treatment for B cell acute lymphoblastic leukemia. J Hematol Oncol 13: 131.
  23. Pessina F, Navarria P, Bellu L, Clerici E, Politi LS, et al. (2020) Treatment of patients with glioma during the COVID-19 pandemic: what we learned and what we take home for the future. Neurosurg Focus 49: E10. [crossref]
  24. Weller M, Preusser M (2020) How we treat patients with brain tumour during the COVID-19 pandemic. ESMO Open 4: e000789. [crossref]
fig 3

A Study to Assess Outcome of Hip Fractures in Elderly Patients with Associated Hyponatraemia and Review of Literature

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DOI: 10.31038/IJOT.2022513

Abstract

Introduction: Hyponatremia is a clinical condition which can be summarized when serum sodium is less than 135 mMol/L, it is the most commonly encountered electrolyte imbalance in clinical practice. It is usually associated with poor clinical outcomes including falls, fractures, increased length of hospital stay, institutionalisation and mortality. Prevalence is known to increase in frail patient groups, such as elderly, hospitalised, peri-operative patients with a fracture. Elderly patients with fragility fractures (EPFF) have increased risk of hyponatremia as a result of degenerate physiology, multiple co-morbidities, polypharmacy, increased risk of dehydration due to hospitalisation and peri-operative fluid restriction, and homeostatic stress from fracture and subsequent surgical interventions.

Material and Method: We conducted a prospective interventional study in a tertiary care centre including 43 patients above the age of 60 years (Range 61 years to 90 years) (Mean age 71.23 years) admitted with fragility fracture around hip, which included inter-trochanteric fractures, sub-trochanteric fractures and neck of femur fractures, pubic ramus fracture, mode of trauma strictly being low energy injuries like trivial fall, patients with high energy trauma, and injuries like shaft of femur fracture, acetabular fracture, pelvic ring fractures were excluded from the study.

Results: Amongst 43 patients incidental hyponatraemia was seen in 36(83.72%), with mild (41.86%), moderate (27.90%) and severe (13.95%), incidence of diabetes mellites type 2 was seen in (74.41%), hypertension was (83.72%), chronic kidney disease upto stage 2 was seen in (6.9%) and (18.6%) were hypothyroid, there were (n=13) patients whose comorbidities were noticed after injury. Average duration of hospital stay and Barthel index (scale to measure activity of daily living) showed linear correlation with initial stage of hyponatraemia in patients, with length of hospital stay was average 24.75 days among the severe deficiency group and 15.25 days in normonatremic group, Barthel index was (90 to 100) among the normonatremic patients and less than 70 in patients with initial hyponatremia at 2 weeks from surgery.

Conclusion: With our study we can conclude that we have to look out for electrolyte disturbance, sodium being most common, incidence of fragility fractures with sodium disturbance, can be due to manifestation of osteoporosis, early diagnosis and management can help in preventing as well as in good outcome in such injuries.

Introduction

Hyponatremia is a clinical condition which can be summarized when serum sodium is less than 135 mMol/L, it is the most commonly encountered electrolyte imbalance in clinical practice [1,2]. It is usually associated with poor clinical outcomes including falls, fractures, increased length of hospital stay, institutionalisation and mortality [3]. Prevalence is known to increase in frail patient groups, such as elderly, hospitalised, peri-operative patients with a fracture. Elderly patients with fragility fractures (EPFF) have increased risk of hyponatremia as a result of degenerate physiology, multiple co-morbidities, polypharmacy, increased risk of dehydration due to hospitalisation and peri-operative fluid restriction, and homeostatic stress from fracture and subsequent surgical interventions [5]. They are also at higher risk of complications, making this group of special clinical importance. Hyponatremia itself may be responsible for the fracture. Reports of the prevalence of hyponatremia at admission in EPFF vary widely between 2.8%-26.5%, while 2.6-5.5% develop hyponatremia in the post-operative period. Hyponatremia occurs due to disruption of sodium and water homeostasis, normally maintained by complex multi-system physiological mechanisms. Consequently, there are numerous potential underlying causes of hyponatremia, spanning a broad spectrum of diseases, pharmacotherapy and pathophysiological variants each with different treatment requirements. When very acute or severe, hyponatremia may present with neurological symptoms which can result in serious complications e.g. hyponatraemic encephalopathy, non-cardiogenic pulmonary oedema, seizures, coma, death. However, 75-80% of cases of hyponatremia are mild and chronic (i.e. serum sodium 130-134 mMol/L, occurring over 24 hours) and typically devoid of obvious neurological symptoms [6]. As a result, chronic mild hyponatremia is frequently considered asymptomatic despite being strongly associated with major geriatric conditions and multi-organ pathological changes. These include abnormal gait patterns, falls, fractures, cognitive impairment, bone demineralisation, longer hospital stay, institutionalisation and increased mortality [7]. Despite this, older people may be at lower risk of hyponatraemic encephalopathy and subsequent complications of acute severe hyponatremia, where female gender, hypoxia and liver dysfunction are associated with poorer prognosis [8] whether hyponatremia is an independent predictor of patient outcomes or a marker of disease severity is controversial [9]. Nevertheless, it is very treatable, so its association with multiple poor clinical outcomes is important.

Materials and Methods

We carried out a prospective observational study of all adults aged 65 years or over admitted with a fragility fracture to a university teaching hospital from 7th January – 4th April 2021. Fragility fractures were defined as those occurring either without trauma or due to low energy trauma, equivalent to a fall from standing height or less than one metre. Anonymous baseline data (age, sex, fracture site and admission serum sodium) of all identified EPFF were recorded (Tables 1 and 2).

Table 1: Patient range

Age range

Number of patients

65-70

9
71-75

8
76-80

10
81-85

8
86-90

5
Above 90

3
Total

43

Table 2: Patients and Serum Na levels

Serum Na level at the time of admission

Number of patients Serum Na after administration of correction Serum Na at the time of discharge from hospital

Mean hospital stay from date of admission in days

135-145

7

 Nil given >135-140

15.25
131-135

18

 >136-140 >135-140

18.25
126-130

12

 >130 >135

23.55
121-125

6

 >130 >135

24.75

Inclusion Criteria

We included patients with age more than or equal to 65 years, patients who gave history of trivial trauma, like fall due to slipping of foot, who didn’t gave any history of direct trauma, patients with trauma around hip, so fractures included were inter-trochanteric, sub-trochanteric, pubic ramus fractures also we included neck of femur fractures, patients with medical comorbidities were included and their baseline investigations were done.

Exclusion Criteria

Adults with incapacity were excluded from recruitment and those associated with head injury or any other systemic trauma and with mode of trauma which included high velocity were also excluded. Patients with trauma which required prolonged bed rest were also excluded. Those with any history of metastatic illness were excluded. Participants were recruited from acute orthopaedic trauma wards and a geriatric assessment unit. Clinical data were collected daily until discharge, from patient interview, medical and nursing notes, observation and fluid balance charts and laboratory computer systems by a single investigator. Medication data were obtained by reconciling patient and/or carer histories, and primary and secondary care records. Also, clinical examination of volemic status was performed daily by the investigator. This involved measurement of skin turgor, capillary refill time, mouth moistness, axillary moistness, jugular venous distension, peripheral oedema and overall impression (signs selected based on previous research recommendations) [22]. Examination was carried out by one investigator to increase reliability of findings, maximise consistency and exclude effects of inter-observer variability. Cases of hyponatremia were defined as any serum sodium measurement, 135 mmol/L. An expert panel, consisting of two consultant geriatricians with special interest in hyponatremia and one consultant orthopaedic surgeon reviewed and determined aetiology of each case of hyponatremia. The panel did not examine patients themselves but instead retrospectively reviewed each case of hyponatremia relying on the detailed daily prospectively collected data and clinical examination findings provided by the investigator. This included all clinical information required to determine underlying cause(s) of hyponatremia – history, medications, detailed daily examination, fluid intake and output charts and laboratory results. Collectively, the expert panel used a diagnostic algorithm to determine underlying cause(s) of hyponatremia. The prevalence of hyponatremia upon admission and the incidence of cases developing in hospital were calculated. For incident cases, we recorded whether the hyponatremia was pre- or post-operative. The proportion of participants with known hyponatremia prior to their fracture was calculated by obtaining the last available serum sodium for each patient prior to their admission to hospital. The laboratory is the only public health service laboratory covering the study population so it was unlikely that patients had more recent investigations elsewhere. Prevalence of hyponatremia at discharge was calculated according to the last available serum sodium measurement prior to discharge (Figures 1 and 2).

fig 1

Figure 1: Barthel index of activities of daily living

fig 2

Figure 2: Interpretation of scoring on the Barthel index

Observation and Results

After Recruitment, participant information and data collection there were 167 patients with fragility fractures who presented to our hospital during that period, many of the patients had upper limb fracture mostly dominated by distal radius fracture, after applying our inclusion and exclusion criteria we included total 43 patients in our study. Those not recruited had incapacity to consent, declined participation, or agreed but were later excluded when the original diagnosis of fragility fracture was excluded. There were no statistically significant differences between participants and those who were eligible but declined participation. Amongst the patients we had 43 patients in all, with inter-trochanteric fractures in 10 patients, subtrochanteric fracture in 12 patients and neck of femur fracture in 18 patients and pubic ramus fractures in 3 patients, and among the associated co-morbidities diabetes mellitus type 2 was seen in (74.41%) patients and hypertension in (83.72%) chronic kidney disease upto stage 2 (6.91%) and hypothyroid was seen in (18.6%). Among all the Patients with associated comorbidities who were discovered at the time of admission, and so were not on any medication were total n=13(30.23%). Barthel index for daily activities was noted at the time of dmission, during hospital stay and time of discharge. We noticed we had 7 out of 43 patients who were normo-natremia, 18 patients had mild and 12 mad moderate and 6 had severe hyponatraemia, patients with severe hyponatraemia also had confusion and weakness during peri-traumatic period, and many scholarly articles also have published similar complaints, which was due to hyponatraemia. We noticed that patients with severe hyponatraemia at the time of presentation had mean hospital stay of 24.75 days and those with normo-natremia had mean hospital stay of 15.25 days, there was also similar observation in recovery of the patients with normo-natremia patients having a gradual and sustained increase in Barthel index, due to early mobilisation and shorter duration in trauma to surgery interval with mean od 4.3 days for surgery from trauma and 6.5 days for patients with deranged serum Na levels, for which correction was administered as per general medicine and nephrologist opinion, after serum Na correction there improvement in general condition as well as tolerance to physiotherapy post-surgery. Barthel index was (90 to 100) among the normonatremic patients and less than 70 in patients with initial hyponatremia at 2 weeks from surgery (Figures 3 and 4).

fig 3

Figure 3: Symptoms

fig 4

Figure 4: Chronic Hyponatremia

Discussion

We found that, compared to men with serum sodium ≥135 mmol/L men with serum sodium values <135 mmol/L had approximately a 3 fold increase in the risk of hip and incident morphometric spine fractures and a 2½ fold increase in the risk of prevalent morphometric spine fractures. Further, the strength of the relationship between hyponatremia and fractures was not substantially reduced after adjusting for multiple well established fracture risk factors, and in particular, for falls and bone mineral density [10].

Our results are consistent with previous studies that have reported on associations between hyponatremia and fractures. A case control study identified 513 cases of fractures (mainly hip and femoral neck) after a fall and reported an increased risk of fracture, by about 3 fold in men and women with hyponatremia (<135 mmol/L)[11], even after adjusting for medications and medical conditions known to confound that association between fracture and serum sodium. A second case control study reported on the prevalence of hyponatremia (<135 mmol/L) among 364 subjects presenting to the emergency department with fractures of the hip/pelvis and femur compared with the incidence of hyponatremia in 364 controls (subjects presenting to the emergency department with non-critical complaints)[12]. The incidence of hyponatremia in those with fractures was more than double that of controls. A recent cross sectional study by Arampatzis et al. reported that of 10,823 emergency department admissions among adults (≥50 years) there was an increased risk of osteoporotic fracture (OR= 1.46; 95%CI: 1.05 to 2.04) among individuals with diuretic-induced hyponatremia. A secondary data analysis of 1408 women participating in a study of chronic kidney disease determined that those with hyponatremia (again less than 135 mmol/L) had a 2 fold increase risk of fracture (based on self-report) even after adjusting for BMD [13,14].

Our analyses showed similar but generally stronger associations between hyponatremia and fracture risk compared to the only other prospective study of the issue; a secondary analysis performed within the Rotterdam Study that included 5208 men and women of which 399 (9%) had a hyponatremia (serum sodium <135 mmol/L)[15]. In the Rotterdam Study those with hyponatremia there was a 1.4 fold increase in nonvertebral fractures over 7.4 years of follow up and a 1.8 fold increase in prevalent but not incident vertebral fractures or hip fractures. As in our study, adjustment for factors such as disability index and falls did not substantially change the results. Of note, BMD was not associated with low serum sodium in the Rotterdam cohort [16,17].

There are several mechanisms by which low serum sodium might contribute to an increase risk of fracture. Hyponatremia, even when mild as in our study, might increase the risk of falls and fall related fractures by causing gait instability and attention deficits. One study reported that the threshold for gait deficits associated with hyponatremia was 134 mmol/L and 132 mmol/L for attention deficits. In our study we noted that 31% of men with hyponatremia reported falls in the past 12 months compared with 21% of men with serum sodium >135 mmol/L. However, adjusting for baseline fall history did not substantially change the relationship between hyponatremia and fracture.

There is growing evidence to suggest that unrecognized complications of hyponatremia include bone loss and osteoporosis, though the mechanisms by which this occurs is not clear. Cellular and animal data suggest that hyponatremia may have a direct effect on bone. Hyponatremia can directly stimulate osteoclast genesis and osteoclastic resorption without activation of signalling through osteoblasts [18,19]. Finally, it is possible that hyponatremia is a surrogate marker for other causes of fracture. In our study as well as in others, subjects with hyponatremia were older, in poorer health, more likely to report use of relevant medications (such as diuretics, SSRI’s) and have concomitant illnesses such as thyroid disease that increase the risk of fractures. However, in our analyses the associations between hyponatremia and fracture risk was not substantially altered by adjusting for many potential confounding factors including falls suggesting that hyponatremia might be associated with bone quality. Thus, assessment of BMD with DXA in mild hyponatraemic subjects may not represent the best available method to address microstructural skeletal alterations [20].

Our study had some limitations. Most importantly, this was not a randomized trial and as such it is not possible to definitively conclude that low serum sodium causes fractures, and that correcting serum sodium will reduce the risk of fractures. Another limitation of our study was the fact that serum sodium was measured only at baseline.

Our findings suggest that hyponatremia, one of the most common electrolyte abnormalities, is associated with up to a doubling in the risk of hip and morphometric spine fractures. The association we observed was strong despite the low prevalence of hyponatremia in our cohort and was not altered by adjusting for fall history or bone mineral density. Further studies are needed to determine if hyponatremia results in an increase in fractures, the mechanism by which this occurs, and whether treatment of hyponatremia reduces the incidence of fractures.

Conclusion

With our study we can conclude that we have to look out for electrolyte disturbance, sodium being most common, incidence of fragility fractures with sodium disturbance, can be due to manifestation of osteoporosis, early diagnosis and management can help in preventing as well as in good outcome in such injuries. From our study we can also draw a relationship between, hyponatraemia and osteoporosis and also weakness and associated confusion in severe hyponatraemia, though our study is limited with a exclusion of upper limb fragility fracture and also vertebral osteoporotic fractures, we can successfully draw a conclusion regarding early electrolyte restoration post trauma and also a scrutiny in suspected patients for early detection and prevention of such fractures.

References

  1. Gankam KF, Andres C, Sattar L, Melot C, Decaux G (2008). Mild hyponatremia and risk of fracture in the ambulatory elderly. 101: 583-588 [crossref]
  2. Tolouian R, Alhamad T, Farazmand M, Mulla ZD (2012). The correlation of hip fracture and hyponatremia in the elderly. J Nephrol 25: 789-793. [crossref]
  3. Sandhu HS, Gilles E, DeVita MV, Panagopoulos G, Michelis MF (2009). Hyponatremia associated with large-bone fracture in elderly patients. Int Urol Nephrol 41: 733-737.
  4. Arampatzis S, Gaetcke LM, Funk GC, et al. (2013). Diuretic-induced hyponatremia and osteoporotic fractures in patients admitted to the emergency department. Maturitas 75: 81-86. [crossref]
  5. Kinsella S, Moran S, Sullivan MO, Molloy MG, Eustace JA (2013). Hyponatremia independent of osteoporosis is associated with fracture occurrence. Clin J Am Soc Nephrol 5: 275-280. [crossref]
  6. Hoorn EJ, Rivadeneira F, van Meurs JB, et al. (2011). Mild hyponatremia as a risk factor for fractures: the Rotterdam Study. J Bone Miner Res 26: 1822-1828. [crossref]
  7. Blank JB, Cawthon PM, Carrion-Petersen ML, et al. (2005) Overview of recruitment for the osteoporotic fractures in men study (MrOS). Contemporary clinical trials.26: 557-568. [crossref]
  8. Orwoll E, Blank JB, Barrett-Connor E, et al. (2005) Design and baseline characteristics of the osteoporotic fractures in men (MrOS) study–a large observational study of the determinants of fracture in older men. Contemp Clin Trials 26: 569-58. [crossref]
  9. Cauley JA, Blackwell T, Zmuda JM, et al. (2012) Correlates of trabecular and cortical volumetric bone mineral density at the femoral neck and lumbar spine: the osteoporotic fractures in men study (MrOS). J Bone Miner Res 25: 1958-1971. [crossref]
  10. Genant HK, Wu CY, van Kuijk C, Nevitt MC (1993) Vertebral fracture assessment using a semiquantitative technique. J Bone Miner Res 8: 1137-1148. [crossref]
  11. Mackey DC, Lui LY, Cawthon PM, et al. (2007) High-trauma fractures and low bone mineral density in older women and men. JAMA 298: 2381-2388. [crossref]
  12. Pahor M, Chrischilles EA, Guralnik JM, Brown SL, Wallace RB, et al. (1994) Drug data coding and analysis in epidemiologic studies. Eur J Epidemiol 10: 405-411. [crossref]
  13. Bailey IL, Lovie JE (1976) New design principles for visual acuity letter charts. Am J Optom Physiol Opt 53: 740-745. [crossref]
  14. Levey AS, Stevens LA, Schmid CH, et al. (2009). A new equation to estimate glomerular filtration rate. Ann Intern Med 150: 604-612. [crossref]
  15. Renneboog B, Musch W, Vandemergel X, Manto MU, Decaux G (2006) Mild chronic hyponatremia is associated with falls, unsteadiness, and attention deficits. Am J Med 119: 7. [crossref]
  16. Barsony J, Sugimura Y, Verbalis JG (2011) Osteoclast response to low extracellular sodium and the mechanism of hyponatremia-induced bone loss. J Biol Chem 286: 10864-75. [crossref]
  17. Verbalis JG, Barsony J, Sugimura Y, et al. (2010) Hyponatremia-induced osteoporosis. J Bone Miner Res 25: 554-563. [crossref]
  18. Hoorn EJ, Liamis G, Zietse R, Zillikens MC (2011) Hyponatremia and bone: an emerging relationship. Nat Rev Endocrinol 8: 33-39. [crossref]
  19. Lindner G, Pfortmuller CA, Leichtle AB, Fiedler GM, Exadaktylos AK (2014) Age-related variety in electrolyte levels and prevalence of dysnatremias and dyskalemias in patients presenting to the emergency department. Gerontology 60: 420-423. [crossref]
  20. Adrogue HJ, Madias NE (2000) Hyponatremia. N Engl J Med 342: 1581-1589.
fig 3

Case Report of Surgical Treatment of Spiral Fracture of Medial Femoral Condyle by ORIF Using Lag Screws and Posterior Buttress Tibial Plate in Elderly Lady and Review of Literature

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DOI: 10.31038/IJOT.2022512

Abstract

Introduction: Fractures of medial condyle of femur are typically rare here we report a case of medial femoral condyle fracture with multiplanar displacement treated with cortico-cancellous screw for compression and posterior tibial plate used as an anti-glide plate/buttress for oblique/spiral configuration of fracture.

Case Presentation: A 78-year-old woman sustained trauma due to fall from stairs and was brought to our hospital with severe right knee pain and inability to bear weight and walk. Knee radiographs (Figure 1) revealed right femoral medial fracture in spiral configuration extending into intercondylar notch as well as breaking in posterior cortex. It is classified (AO classification 33-B2) Patient was initially stabilised and immobilised in splintage. All pre anaesthetic investigations performed and patient was planned for open reduction internal fixation. ORIF was done using two 6.5 mm partially threaded cancellous lag screws to compress the condyle and posteriorly extending oblique pattern of fracture compressed by proximal tibia posterior plate as anti-glide plate. The patient had uneventful post-operative recovery and recent follow up patient achieved a range of motion of zero to 120 degree and could walk without pain.

Conclusion: The locking compression plate for proximal tibia can be used solution for difficult femoral condyle fracture used in reversed position. However careful patient selection is important.

Introduction

Distal femur fractures account for 7% of all femur fractures if hip fractures are excluded then one third of femur fractures involve the distal portion [1], a bimodal age distribution exists with high incidence in young males due to high energy trauma such as motor vehicle or motorcycle accidents or fall from heights a second peak in elderly woman from minor falls there is ratio of 1:2 in men to women [1,2]. Femoral medial condyle fracture (AO classification 33-B2) is a considerably rare fracture. Limited literature is available to give clear view regarding treatment of this fracture configuration. Being metaphyseal region and intra-articular extension, anatomical reduction, and compression as well as stable fixation is a primary necessity to achieve better functional outcome [3]. However, in this case the spiral fracture pattern in distal femur metaphysic the fracture line itself has extended into two planes, Proximal fracture pattern was in coronal orientation and distal in sagittal plane. Hence compression could only have been achieved when screws are placed in the tangential plane to the plane of fracture line. Proximal end of the fracture being smaller in dimension to accommodate the lag screw. However, this compression in other plane could not be achieved through single plane orientation of the plate hence needs isolated another fixation modality. The plate must be precontoured to match posterior surface of distal femur to offer adequate compression till healing and combination construct was needed to be used. Considering the vertical line of fractures and the obliquity of the displacement, screw fixation compression can be done but to achieve stability however no current anatomical plates fit the femoral medial condyle [4,5]. We present a case of femoral medial condyle fracture treated with cancellous screws and posterior tibial plate used as anti-glide plate/buttress plate.

Case Presentation

A 78-year-old woman was brought to our hospital after sustaining trauma to her right knee due to fall from stairs. She was not suffering from chronic neurological illnesses. Parkinson’s disease or stroke or paralysis any signs of central nervous dysfunction or paralyses, all her vital signs were normal. On evaluation patient complained of pain and inability to move right lower limb, there was swelling and bruising over the knee, tenderness was elicited on palpation, distal neurology and vascularity were intact radiographs was done which demonstrated (Figure 1) a femoral medial condyle fracture of right side, the fracture was intra articular and simple oblique through the intercondylar notch (AO classification 33-B2) patient was admitted to our hospital for open reduction internal fixation to be performed following day after stabilization.

fig 1

Figure 1: radiograph done at time of presentation

Surgical Procedure

Patient was planned for open reduction and internal fixation, under all aseptic conditions, spinal and epidural anaesthesia, fixation patient was performed in supine position. Medial subvastus approach (Figure 2) was used, this approach is used for intra articular fractures of medial condyle of femur, medial Hoffa or in addition to lateral parapatellar approach in case of severe bicondylar articular fragmentation, a skin incision was started at the adductor tubercle and extends proximally just posterior to vastus medialis and the interval between vastus medialis and sartorius was identified, vastus medialis was elevated to expose the medial femoral condyle (Figure 2) the popliteal vascular bundle was protected which lies between adductor Magnus and intermuscular septum.

fig 2

Figure 2: intra-operative images

The fracture was initially compressed with cortico-cancellous screw in coronal plane (Figure 3) and a lag screw was passed to compress the fracture in sagittal plane and then posterior tibial plate was used upside down to fit the Bony surface, the plate was bent for achieving proper anatomical orientation and fixed with cortical and locking screws thus the plate was used as anti-glide/Buttress as per the configuration of the fracture, closure was done in layers. Patient had uneventful post-operative recovery, range of motion exercises were started on Day 1, the weight bearing protocol was toe touch gate for first 4 weeks, by 4th week partial weight bearing from 6 weeks half partial weight bearing and from eight weeks full weight bearing, at the latest follow up the patient had range of motion of zero to 120 degree without any pain, she could walk freely and joint surface restoration was maintained radiologically (Figure 4).

fig 3

Figure 3: Range of motion after 6 months of surgery

fig 4

Figure 4: follow up radiograph

Discussion

In elderly patients with osteoporosis a low energy trauma causes simple spiral or oblique fractures the shape of distal femur when viewed end on is a trapezoid with posterior part wide then the anterior part creating 25 degree of inclination on the middle surface and about 10 degree on the lateral surface the plate should lie flat on this lateral surface [6,7], a line that is drawn from anterior aspect of the lateral femoral condyle to the anterior aspect of the medial femoral condyle patellofemoral inclination slopes posteriorly approximately 10 degrees [8,9] this anatomical details are important when inserting any implant or plates, knowledge of the normal radiographic joint line angle helps to assess alignment during an operation the normal anatomical axis of femoral shaft relative to the knee or the anatomical lateral distal femoral angle(LDFA) is 80 to 84 degrees. Measured contralateral lateral distal femoral angle (LDFA) can be used for reference as assessment of coronal alignment, there is consistent pattern of mismatch at the proximal part of the 11-hole locking compression plate for distal femur that may cause valgus malalignment [10].

The quadriceps hamstring and the adductor muscle groups cause significant shortening and varus displacement especially when there are multiple fragments in the metaphysis, the gastrocnemius muscle originates from the posterior aspect of both femoral condyle and its unopposed action causes of flexion deformity of the distal fragment the typical deformity is of shortening with the proximal fragment displaced anteriorly piercing the quadriceps, sometimes the skin while the distal fragment is flexed in varus and rotated posteriorly.

In elderly patients with osteoporosis or Peri-prosthetic distal femoral fractures locking plate systems become valuable for solid fixation [11,12] in situations where there is limited bold stock in the distal fragment the basic principle in treating intra articular distal femoral fractures is based upon anatomical reduction of articular fragments under direct vision fixation is achieved by compressing the fragments with lag screws may be used when required.

However, there are no available anatomical plates that fit either the femoral medial condyle or for fracture fixation, except for the relatively short plate developed for digital femoral Osteotomy [13]. Past reports have shown possibility of screw fixation for the fracture. To date however no consensus exists regarding optimal implant due to very few cases [14]. We used 6.5 mm cancellous screw for compression of femoral condyles. 3.5 mm medial tibia locking plating in reverse mode is used for the same side as anti-glide plate to counteract the vertical and shearing forces. The plate fits the bone surface well after some level of bending. The clinical and radiological outcomes were acceptable the anatomical plate for distal femoral medial condyle should be considered for development as soon as possible.

Conclusion

In our case we used proximal tibial posterior plate upside down as a anti glide plate/Buttress for fixation of femoral medial condyle fracture although the plate needed bending to achieve congruence, it fit well and has yielded glued good clinical outcome the posterior tibial plate could become the method of choice for such fractures.

Funding

Nil

Conflict of interest

Nil

References

  1. Court-Brown CM, Caesar B (2006) Epidemiology of adult fractures: a review. Injury 37: 691-697. [crossref]
  2. Ehlinger M, Ducrot G, Adam P, Bonnomet F (2013) Distal femur fractures. Surgical techniques and a review of the literature, Orthop Traumatol Surg Res 99: 353-360. [crossref]
  3. Bel JC, Court C, Cogan A, Chantelot C, Pietu G, et al. (2014) Unicondylar fractures of the distal femur. Orthop Traumatol Surg Res 100: 873-877. [crossref]
  4. Agha RA, Borrelli MR, Farwana R, Koshy K, Fowler A, et al. (2018) For the SCARE Group, The SCARE 2018 statement: updating consensus surgical case report (SCARE) guidelines. Int J Surg 60: 132-136. [crossref]
  5. Gwathmey Jr. FW, Jones-Quaidoo SM, Kahler D, Hurwitz S, Cui Q (2010) Distal femoral fractures: current concepts. J Am Acad Orthop Surg 18: 597-607. [crossref]
  6. Murphy CG, Chrea B, Molloy AP, Nicholson P (2013) Small is challenging; distal femur fracture management in an elderly lady with achondroplastic dwarfism. BMJ Case Rep [crossref]
  7. Manfredini M, Gildone A, Ferrante R, Bernasconi S, Massari L (2001) Unicondylar femoral fractures: therapeutic strategy and long-term results. A review of 23 patients. Acta Orthop Belg 67: 132-138. [crossref]
  8. Dhillon MS, Mootha AK, Bali K, Prabhakar S, Dhatt SS, et al. (2012) Coronal fractures of the medial femoral condyle: a series of 6 cases and review of literature. Musculoskelet Surg 96 49-54. [crossref]
  9. Kiyono M, Noda T, Nagano H, Maehara T, Yamakawa Y, et al. (2019) Clinical outcomes of treatment with locking compression plates for distal femoral fractures in a retrospective cohort. J Orthop Surg Res 14: 384. [crossref]
  10. mcdonald TC, Lambert JJ, Hulick RM, Graves ML, Russell GV, et al. (2019) Treatment of distal femur fractures with the depuy-Synthes variable angle locking compression plate. J Orthop Trauma 33: 432-437.
  11. Gahlot N, Saini UC, Ss S, Aggarwal S. (2014) Triplane fracture of distal femur in an adult rare case study and review. Ortop Traumatol Rehabil 16: 523-30. [crossref]
  12. Neer CS, Grantham SA, Shelton ML (1967) Supracondylar fracture of the adult femur. A study of one hundred and ten cases. J Bone Joint Surg Am 49: 591-613. [crossref]
  13. Seinsheimer F (1980) Fractures of the distal femur. Clin Orthop Relat Res 153: 169-179.
  14. Müller ME, Allgöwer M, Schneider R, Willenegger H (1991) Manual of Internal Fixation. Springer, New York.
fig 4

Sauvé Kapandji Procedure to Salvage Wrist Function in a 2-month-old Distal End Radius and Ulna Fracture – A Case Report and Review of Literature

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DOI: 10.31038/IJOT.2022511

Abstract

The wrist joint pain and instability are frequently caused by distal radioulnar joint disorders. Most common aetiology is displaced fracture or malunion of distal radius and tears of the triangular fibrocartilage complex with DRUJ instability. A 65-year-old male patient presented to us with complains of pain and deformity of the right wrist of two months duration. Radiographs revealed a malunited distal end radius and a malunited distal ulna fracture. He underwent Sauvé kapandji procedure, in which his distal radius fracture was fixed with locking plate. Follow ups were done at periodic intervals and wrist physiotherapy was instituted. He had acceptable wrist motion at six weeks.

Keywords

Sauvé kapandji, Distal radius, Malunion

Introduction

The wrist joint also referred as the radio carpal joint is a condyloid synovial joint of the distal upper limb that connects and serves as transition point between the forearm and hand and distal radio ulnar articulation is a synovial pivot-type joint between two bones in forearm; the radius and ulna. The wrist joint performs very important functions in day to day life activities of a person, flexion and extension of wrist along with pronation and supination at distal radioulnar joint are fairly important for a person to perform daily activities, injury to wrist joint may result in digital radioulnar joint instability and can lead to deformity and degenerative changes this is often manifested with peanut ulnar side of wrist limited rotation of the forearm with loss of function.

In suave kapandji procedure arthrodesis of the distal radioulnar joint combined with creation of pseudo arthrosis of the digital ulnaArthrodesis of the distal radioulnar joint with creation of distal Nur pseudo arthrosis maintained the owner head in good position provided support for the ulnar corpus and allowed prono supination at the pseudoarthrosis [1]. The suave kapandji procedure is indicated for treatment of conditions that result in DRUJ pain or instability or both and that are refractory to non-surgical treatment, this procedure has been advocated as operation of choice for derangement of distal radial nerve joint in patients with high demand wrists and in particular for post traumatic problems of the distal radioulnar joint it is considered that retaining the head of ulna allows for more normal transmission of force through the rest very few publications have been reported regarding this procedure.

Case Report

A 65-year-old gentleman from a remote village with history of fall sustained injury to right wrist and right hip two months prior to presenting to us, had received conservative management in form of plaster for wrist joint and hip immobilisation, due to ongoing covid pandemic, he did not receive immediate surgical management. He presented with complaints of pain and deformity and restriction motion of the right wrist with pain over right hip and difficulty in standing and walking of two months duration. On clinical examination he had tenderness over distal radioulnar joint with mobility at fracture site and shortened radius with manus valgus deformity reduced the range of motion in all directions.

Radiographs revealed distal end radius communicated fracture with loss of radial height, radial inclination and distal ulna communicated fracture with displaced fragments.

Patient underwent open reduction internal fixation of distal end radius fracture with locking plate and augmentation with k wires to stabilise the fractured styloid fragment and prevent its rotation, comminuted fragments of ulnar remove from separate incision over ulna and distal ulnar head was fixed to radius with the cortico cancel screw this procedure restored the store wrist joint of the patient. a rigid plaster dressing was applied for 6 weeks, after 6 weeks k-wires were removed, and wrist physiotherapy was started at the end of six months he had acceptable range of painless range of motion at wrist with dorsiflexion of 70 degrees palmar flexion of 60 degrees supination 80 degrees pronation 60 degrees and wrist Mayo score of 80 which infers good result.

Discussion

Distal radius fractures (DRF) are very common in orthopaedic practice and are often accompanied by instability of the distal radio ulnar joint (DRUJ) The Sauvé-Kapandji procedure allows fusion at the joint thus decreasing ulnar instability as well as stabilizing the surrounding soft tissue support to the joint. Here the ulnar head is left intact and minimizes the potential for some of the complications that can follow its excision. The most commonly arising complication is the proximal stump instability. The Sauvé-Kapandji procedure is usually useful for treating various pathologic conditions that alter normal function of the DRUJ. The Sauvé-Kapandji procedure is designed to treat pain arising from the distal radio-ulnar joint by fusion, to correct the ulnar variance by recession of the ulnar head and maintain rotation of the forearm by creating a pseudarthrosis. There are strong biomechanical arguments for retaining the ulnar head, especially after trauma and ligamentous weakness of the radiocarpal joint. Conservation of the head maintains the triangular fibrocartilage complex to allow a more physiological transmission of forces from the hand to the forearm. It has been shown that approximately 20% of axial load is passed through the ulnar carpus and even minor derangements in this region can result in changes of load pattern. The ulnar head is also important in the mechanism of action of ECU, which further adds to stability.

Taleisnik [3] suggested that in cases involving subluxation or dislocation of the DRUJ, rupture of the interosseous membrane, which had contributed to static stability, could result in an excessively mobile distal ulna even after surgery. Because chronic derangement in most patients noting discomfort at the proximal ulnar stump was caused by DRUJ dislocation, Kapandji [4] suggested that leaving a short distal ulna fragment, fashioning the ulnar gap as far distally as possible, and creating a pseudarthrosis of approximately 10 mm would decrease the instability of the proximal ulnar stump. To obtain more stability of an excessively mobile proximal ulnar stump, Kapandji’s [4] recommendation should be followed combined with the tenodesis procedure, especially when the cause of DRUJ derangement is dislocation.

Deformities caused by different conditions at the DRUJ which can impair the normal function of the DRUJ can be treated by the Sauvé-Kapandji procedure [5]. A study done by Minami A et al has shown a combination of Sauvé-Kapandji procedure along with extensor carpi ulna tenodesis helps reduce the incidence of instability of the proximal stump [6]. Mohamed et al. [7] mentioned that patient with chronic post-traumatic derangement of DRUJ were treated by a modified Sauvé-Kapandji operation and the post-operative results were acceptable to the patients. Much has been written about problems of the ulnar stump associated with the Sauvé-Kapandji procedure. After the operation, the structures supporting the shaft of the ulna are the interosseous membrane (static), the tendons of ECU and FCU and the pronator quadratus muscle (dynamic). After injury these structures may be damaged, and rupture of the interosseous membrane may lead to a very mobile ulna. Most authors have described problems with pain and clicking of the ulnar stump, but this is usually only a minor inconvenience. Our patient was seldom troubled by symptoms of instability, most only experiencing minor, if any, discomfort. Various modifications have been described to decrease the incidence of this problem and good results have been reported. In our case we performed fixation of distal end radius fracture with locking plate augmented with K wires from styloid and listers tubercle and arthrodesis of distal radioulnar joint with creation of pseudo arthrodesis of digital ulna our patient had good wrist function (Figures 1-4).

fig 1

Figure 1: Pre-operative radiographic images

fig 2

Figure 2: Intra-operative images under c-arm

fig 3

Figure 3: Post-op images at 1 month interval

fig 4

Figure 4: Follow up radiographic images at 6 months

Conclusion

Our case highlights the importance of considering the patient needs and the type of fracture in choosing the management required, a blanket approach can be suitable only when fractures are typical, but such atypical fractures along with advanced age can be successfully managed with wrist joint salvaging procedure, our patient had good outcome with no residual pain.

Conflict of Interest

Nil

Funding

Nil

Ethical approval

Not required

Informed consent

Well informed consent taken after explanation of procedure

References

  1. Hironobu Inagaki, Ryogo Nakamura, Emiko Horii, Etsuhiro Nakao, Masahiro Tatebe (2006) Symptoms and Radiographic Findings in the Proximal and Distal Ulnar Stumps after the Sauvé-Kapandji Procedure for Treatment of Chronic Derangement of the Distal Radioulnar Joint. The Journal of Hand Surgery. 31: 780-784. [crossref]
  2. Lamey DM, Fernandez DL (1998) Result of the modified Sauvé-Kapandji procedure in the treatment of chronic posttraumatic derangement of the distal radioulnar joint. J Bone Joint Surg. 80A: 1758-1769. [crossref]
  3. Taleisnik J (1992) The Sauvé-Kapandji procedure. Clin Orthop. 275: 110-123. [crossref]
  4. Kapandji IA (1986) Opération de Kapandji-Sauvé. Techniques et indications dans les affections non rhumatismales. Ann Chir Main, 5:181-193.
  5. Aleisnik J (1992) The Sauve-Kapandji procedure. Clin Orthop Relat Res. 110-1023. [crossref]
  6. Minami A, Kato H, Iwasaki N (2000) Modification of the Sauvé-Kapandji procedure with extensor carpi ulnaris tenodesis. J Hand Surg Am. 25:1080-1084. [crossref]
  7. Mohamed MO (2016) Modified Sauve-Kapandji operation for treatment of chronic post-traumatic derangement of the distal radioulnar joint. Anti-Cancer Drugs. [crossref]
fig 13

Transparent Conductive Far-Infrared Radiative Film based on Polyvinyl Alcohol (PVA) with Carbon Fiber (CF) in Agriculture Greenhouse

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DOI: 10.31038/NAMS.2022513

Abstract

There are many types of transparent conductive films, the most common being made by depositing ITO (indium-Tin-oxide) on ultra-thin glass substrate by physical or chemical methods. In this study, PVA was used as basis material and CF as electrical conductive material, to mix both two materials and distribute the CF in the PVA solution, then by casting form transmittance electrical conductivity film. The newly developed film has an average Edge-to-Edge Resistance of 2069.58 Ω, light transmittance of 75.75% and has a heating capability of 23.38 W/m2 via far-infrared light (sample size is 200*200 mm and voltage in 220 V, through setting the facts, the Block Resistance reach 419.9208 Ω, heating capability of 115.26 W/m2 via far-infrared light, and the Light Transmittance reach 58.2790%,). The film is almost clearly transparent and is suitable for deployment as part of the retaining structure of agricultural greenhouses as it allows adequate sunlight penetration for the necessary photosynthesis of crops. This film is promising for the “solar greenhouse” industry and able to solve the long-term problems of agriculture in seasonable regions such as northern China during the winter. This film is a suitable energy efficient replacement to the current greenhouse facilities conventional electrical heaters to meet the temperature needs of crop growth.

Keywords

Transparent conductive film, Conductive film, Transparent film, PVA, Carbon fiber

Introduction

In recent years, researchers have developed a series of new transparent conductive films that can replace ITO, such as conductive polymers, carbon nanotubes, graphene and metal nanostructures [1]. The paper “Transparent Conductive Far-Infrared Radiative Film based on Cotton Pulp with Carbon Fiber in Agriculture Greenhouse” descried have developed a transparent conductive film by depositing conductive carbon fiber on cotton pulp substrate. The newly developed film has an average Edge-to-Edge Resistance of 545.65 Ω, light transmittance of 67.9% and has a heating capability of 88.70 W/m2 via far-infrared light. The film is almost clearly transparent and is suitable for deployment as part of the retaining structure of agricultural greenhouses as it allows adequate sunlight penetration for the necessary photosynthesis of crops. This film is promising for the “solar greenhouse” industry and is able to solve the long-term problems of agriculture in seasonable regions such as northern China during the winter. This film is a suitable energy efficient replacement to the current greenhouse facilities conventional electrical heaters to meet the temperature needs of crop growth [2].

This study research and develop a flexible transparent far-infrared radiation film made with CF as conductive material and PVA matrix respectively. The film is a kind of transparent conductive film, by flexible can be used as the retaining structure of agricultural greenhouses, by transparency can make the sun shining through, meet the needs of crop photosynthesis, by conductivity can make its radiate far infrared light, crop growth is the light of life, can meet the needs of agricultural greenhouse warming. The cheap raw materials and simple manufacturing process are suitable for the realization of industrial mass production, and can be used in the application of greenhouse heating and light supplement in facility agriculture, which is also the vitality of this study.

At present, the most widely used transparent conductive films are prepared on hard substrates such as glass and ceramics. Compared with the rigid transparent conductive film, the transparent conductive film prepared on the organic flexible substrate not only has the same photoelectric characteristics, but also has many unique advantages, such as: flexible, light weight, not easily broken, can be used to improve the efficiency of the industrial continuous production mode, easy to transport, etc. With the development of electronic devices towards lightening, flexible transparent conductive film is expected to become a replacement of rigid transparent conductive film.

From the perspective of the development pattern of modern facilities and horticulture in the world, most modern greenhouses which plastic film greenhouses are about 600,000 square kilometers, mainly distributed in Asia. Glass greenhouse about 40,000 square kilometers, mainly distributed in Europe and the United States. New cover material polycarbonate board (PC board) greenhouse in recent years, the development speed is very fast, at present about more than 10 thousand hectares, sporadic distribution in the world.

One important area, to apply flexible transparent conductive film, is the Protected Agriculture, the greenhouse, as a modern precision agriculture facility, has realized the artificial control of temperature, light, water, gas and fertilizer in the greenhouse to form an environment conducive to the growth of crops. In developed countries such as the United States and Israel, an intensive greenhouse industry has been formed. The greenhouse is a building with lighting covering material as the whole or part of the envelope structure material, which can be used for cultivating plants in winter or other seasons that are not suitable for the growth of open plants.

Greenhouse is a day lighting building, so light transmittance is one of the most basic indexes to evaluate the light transmittance of greenhouse. Transmittance is the percentage of the amount of light penetrating the greenhouse to the amount of light outside. The light transmittance of greenhouse is affected by the light transmittance of greenhouse cover material and the shadow rate of greenhouse skeleton, and the light transmittance of greenhouse is also changing with the different solar radiation Angle in different seasons. The light transmittance of greenhouse becomes the direct influence factor of crop growth and crop variety selection. In general, plastic greenhouse in 50%~60%, glass greenhouse transmittance in 60%~70%, solar greenhouse can reach more than 70% [3].

The transparent conductive film with both light transmittance and electrical conductivity makes it possible to integrate the enclosure structure and heating facilities of the agricultural greenhouse. According to the practical application experience of this kind of conductive film used for indoor heating, the resistance value of 900*600 mm conductive film is 96.8 Ω, the input power is 500 W (Voltage=220 V), and the surface temperature can reach 70~90°C. When the transparent conductive film in this experiment is used for heating in agricultural greenhouse, in order to avoid crops being roasted, the experience in the application process is that it is more appropriate to control the input power of the transparent conductive film at 200 W/m2 (Voltage=220 V) and the surface temperature of the film can reach 30°C.

In order to make the vegetables grow normally in the cold winter, maintain the high yield, the greenhouse ceiling is applied in modern agricultural planting, through the adoption of the transparent material, forming the local small climate, and creating the environment that is suitable for the growth of the crop. However, it is not necessarily possible to achieve the temperature of the vegetables, so the average greenhouse ceiling will install the heating equipment to ensure that the temperature in the shed can make the vegetables harvest.

As a transparent infrared radiation material, the transparent conductive film can be used for the maintenance of structural materials in agricultural greenhouses. On the one hand, it can store heat energy through sunlight，on the other hand, it can radiate far infrared through electric excitation to compensate the temperature of crops. The actual measurement of the internal light illumination of the agricultural greenhouse, target of LT > 70% is good. (According to the measurement of the agricultural greenhouses in use now, the actual light transmittance in the greenhouses is generally 70% in sunny weather.)

Plastic film has the property of heat preservation. After covering the film, the turbidity in the greenhouse will increase with the increase of the outside temperature and decrease with the decrease of the outside temperature. There are obvious seasonal changes and large diurnal temperature difference. The lower the temperature period, the greater the temperature difference. Generally, the daily temperature increase in the greenhouse in cold season can reach 3-6°C, and the temperature increase ability on cloudy days or at night is only 1-2°C. In the warm spring season, the temperature difference between the shed and the open field gradually increases, and the temperature increase can reach 6-15°C. When the outside temperature rises, the temperature inside the greenhouse increases relatively, up to more than 20°C. Therefore, there are high temperature and freezing hazards in the greenhouse, which need to be adjusted manually. In high temperature season, high temperature above 50°C can be produced in the shed. The whole shed is ventilated, and the shed is covered with straw curtain or built into “awning”, which is 1-2°C lower than the open air temperature. In winter, when the sun is clear, the minimum temperature at night is 1-3°C higher than the open field, and several branches are the same as the open field on cloudy days. Therefore, the main production season of greenhouses is spring, summer and autumn. Through heat preservation and ventilation and cooling, the temperature of the shed can be maintained at 15-30°C for growth [4].

In winter, the commonly used agricultural greenhouse warming measures are:

  1. One is to add a few warm fans in the shed, temporarily heating the place where the temperature is relatively low, but the need to pay attention to the high humidity in the shed, to avoid leakage caused by bad things.
  2. The second it is if the greenhouse is adjacent to have the condition that can use, if the supply hot gas such as brewery, bathhouse can try to be used abounding, saved cost already the repeat use that completed resource.
  3. The third is to cover the grass felt on the greenhouse, which is a relatively backward insulation method. What we need to pay attention to is to ventilate and receive abundant sunlight on time every day.

Plastic products have brought great convenience to people’s life, but discarded plastic garbage has caused unimaginable harm to the ecological environment. Plastic waste that is difficult to degrade causes the death of hundreds of thousands of Marine animals every year, and the generated micro-plastics are all over the earth, and even enter the bodies of animals and plants or other environments, posing a great threat to human health. In order to better prevent and control plastic pollution, it is urgent to develop a new generation of sustainable plastic alternative materials. PVA film in the natural environment can be degraded by microorganisms and non-toxic, its degradation products for carbon dioxide and water, is a new generation of environmentally friendly plastic film material, it completely solved the white pollution caused by plastic film material [5].

With the deepening of people’s environmental protection concept, the green packaging is getting higher and higher in the packaging field, and its varieties are also detected in endlessly. Water-soluble packaging film as an important type of green packaging materials, its good water solubility, barrier and environmental protection characteristics, more and more countries pay attention to the packaging field. Plants cannot grow everywhere. Their environment has to provide the right conditions for them to survive. Specifically, plants need water, air, sunlight, and suitable temperatures. In winter months, cold temperatures are often a limiting factor for plant growth [6]. Greenhouse heating is one of the most energy-consuming operational requirements during winter periods, having a significant impact on production cost [7]. The concept of heating greenhouses was first recorded in Korea in the 1400’s, as people in that cold country realized that they could add to the sun’s heat and open up more growing possibilities. Throughout the centuries the understanding of winter greenhouse technology improved, and farmers were able to precisely control the temperature, humidity, and chemical composition of the greenhouse atmosphere. Greenhouse heating systems eventually became automated, with digital controls and carefully regulated air circulation, and today’s global agricultural industry relies heavily on them [8]. Elizabeth Waddington et al, in ‘7 Innovative Ways To Heat Your Greenhouse In Winter’ said: As colder weather approaches, you’re probably wondering whether your greenhouse is up to the task. Will it fend off the frosts well enough to keep your crops growing all winter long? Whatever type of greenhouse you have, whether glass or plastic, you may need to think about heating it if you live in a colder climate zone. Where winter temperatures regularly drop well below freezing, some heating might be necessary to enable you to grow food year-round [9].

Greenhouse production in winter consumes a lot of energy. It has been pointed out that the ratio of fuel consumption produced by greenhouse to dry matter produced by greenhouse vegetables is 5:1 or 10:1, a lot of energy consumption, only 40% to 50% percent utilization. In Japan, it takes 5 L of oil to produce l0 kg of cucumbers, 50% to 60% times more energy than grain production. In the world agricultural production in a year, 35 percent of the energy consumption for greenhouse warming, greenhouse energy consumption costs accounted for 15% to 40% percent of the total greenhouse production costs [10].

PVA is a water-soluble polymer, characterized by good compactness, high crystallinity, strong adhesion, made of film flexible and smooth, oil resistance, solvent resistance, wear resistance, good gas permeability, and special treatment with water resistance, a wide range of uses. PVA is non-toxic, tasteless and harmless to human body. It has a good affinity with the natural environment. It does not accumulate and is pollution-free. PVA film is a kind of green and environment-friendly functional material with PVA as the main body and additives such as modifier, which can be completely degraded by microorganism in soil after special processing. It can degrade into carbon dioxide and water in a short time, and has the effect of improving the land. The biggest advantage of polyvinyl alcohol film is water solubility, the biggest disadvantage is poor water resistance. The reason for the poor water resistance is due to the hydrophilic hydroxyl group (-OH) in its molecules. The water resistance of PVA films can be improved if the hydroxyl group can be properly closed and connected with water resistant groups. PVA contains hydroxyl groups, which can take place in all typical reactions of polyols. Choosing appropriate polycondensation compounds, in the case of a small amount of addition, can appropriately interact with the hydroxyl groups in PVA, so that PVA forms a strong three-dimensional structure, which stabilizes the air tightness of PVA under wet conditions and improves the water resistance [11].

In this study, functional materials (CF) were added into the PVA membrane to improve the function and status of the PVA membrane, so that it has infrared radiation function, anti-static function, and electromagnetic shielding function, enrich the application field of the PVA membrane, and make the PVA membrane get a new life. In the industry, PVA films are generally manufactured with the use of a casting machine with roller press. For cost effectiveness, this study adapts the wet method is adapted for preparing the film. First, CF filament is mixed in diluted PVA and glycerin solution, then slowly blended for up to 2.5 hours to ensure the CF filament is dispersed evenly, and then casted in a mold and left for air drying up to 48 hours in ambient room temperate. The resulting product is a thin and flexible PVA-CF transparent conductive film. The process consists of manufacturing the transparent conductive film based on PVA-CF, and install current-carrying then install insulating film on double – sided for PVA-CF.

Agricultural greenhouses are mainly located in rural areas and suburbs, where electricity and other energy are in short supply. It is difficult for traditional power grids to reach these areas [12]. Therefore, self-made aluminum air battery is used as power supply in this study. The chemical reaction of al-air cells is similar to that of zinc-air cells. Al-air cells use high-purity Al (99.99% aluminum) as the negative electrode, oxygen as the positive electrode, and potassium hydroxide (KOH) or sodium hydroxide (NaOH) aqueous solution as the electrolyte. Aluminum absorbs oxygen from the air and is converted to aluminum oxide in a chemical reaction when the battery is discharged. The development of aluminum-air battery is very rapid, and its application in EV has achieved good results. It is a promising air battery [13]. The Schematic diagram of aluminum air battery see Figure 1.

fig 1

Figure 1: Schematic diagram of aluminum air battery

In agricultural greenhouse use, especially need to consider the requirements of environmental friendliness. In this study, K2SO4 is used as electrolyte (K2SO4 is a kind of high quality and efficient potassium fertilizer without chlorine). Aluminum-air batteries with K2SO4 electrolyte have a lower energy density than aluminum-air batteries with KOH or NaOH electrolyte, but they are environmentally friendly and can achieve long battery life. If the electrode material is directly immersed in the electrolyte structure (see the Figure 15, It has been running under load for 5 weeks), it can achieve 3 months of endurance, to meet the needs of a heating season.

Results and Discussion

The wet method process of fabricating the PVA-CF film are as follows (see Figure 2). CF filaments are mixed with diluted PVA. The mixture is after two steps of casting coating and drying (heat treatment), PVA film products can be obtained by stripping and coiling. The process is very simple and easy to operate, which is an efficient production mode. As below Figure 3 extrusion blown film process: obtain PVA raw materials, water, plasticizer and lubricant (pigment) and other materials need to be prepared, and then through the forced cycle mixing, the next step is the process of extrusion granulation, the following steps are melt extrusion blown film, modified treatment, coiling and obtain the finished product. This process is more complex than the previous method, and is much less operational and productive [14].

fig 2

Figure 2: PVA transparent conductive film manufacturing process

fig 3

Figure 3: The process of fabricate polyvinyl alcohol with carbon fiber transmittance conductive film

Industrial fabricating transmittance conductive film for PVA-CF, general using dissolute machine system to dissolute the PVA, using filtering machine system to filter out impurities in PVA solution, using dispersing system to disperse CF in PVA solution, and using Casting machine system to casting form PVA-CF film, this process shows as Figure 4.

fig 4

Figure 4: Four System industrial fabricating transmittance conductive for PVA-CF

Experiments are conducted using PVA as substrate material infused with CF filaments of lengths 3 mm, 6 mm, and 10 mm. The CF filaments provides conductive properties to the PVA solution mix, and when casted it results in a transmittance conductive film. An initial experiment for testing the suitable length of the CF used. Table 1 shows the ratio of the components used in the fabrication of the various film samples. The samples were fabricated with CF filament of 3 mm, 6 mm and 10 mm length were respectively cut and stir mixed with 30 g of PVA and 400 ml of water for two hours. The samples, which are shown in Figure 6 can then be examined for the dispersion uniformity of the CF in the PVA solution.

Table 1: Mixture ratio for fabrication of films with carbon fiber of various lengths

Materials

 CF-3 mm CF-6 mm

CF-10 mm
CF (g):PVA (g)

0.3:30

 0.3:30

0.3:30
Glycerin (mL)/(g)

15/18.95

 15/18.95

15/18.95
H2O (mL)

385

 385

385

Glycerin concentration is 1.26362 g/mL. Stir mixing time is 2 hours

The experimental results are shown in Figures 5 and 6.

fig 5

Figure 5: Different length carbon fiber dispersing in PVA solution (a) 3 mm, (b) 6 mm, (c) 10 mm

fig 6

Figure 6: Casted film with carbon fiber in PVA solution: (a) Casting mold. (b) Film with 3mm CF filament. (b) Film with 6mm CF filament. (d) Film with 10mm CF filament

Figure 5a shows the casting mold used for the test fabrication. From Figure 5b and 5c, it is observed that 3 mm CF filaments disperses well in the PVA, while the distribution of the 6 mm filaments is very uneven, and lumpy agglomerations have formed throughout the casted film. Meanwhile, Figure 5d shows that the 10 mm filaments are hardly dispersed in PVA solution and the fibers are entangled into groups, which is expected to interfere with the process of forming thin conductive films.

As the films are applied to agricultural greenhouse as heating films with high light transmissivity, the film needs to be thin with uniform conductivity. Overall, the experiment result shows that the carbon fiber filaments of lengths 6 mm and 10 mm do no disperse uniformly in the PVA solution and are not suitable for fabrication of thin transmittance film with uniform conductivity. In conclusion, the optimum CF length for film fabrication is 3 mm as it allows for the good dispersion of the CF in the PVA solution. Thus all other experiments were conducted using only 3 mm carbon fiber filaments. Table 2 shows the results of the 12 samples of pure PVA films and PVA-CF films tested for light transmissivity. It is noted that PVA film is superior to other films in glossiness and transparency, and when compared to common cellophane (PT) and PVC film, PVA film has reflectivity property and light transmissivity that is higher by 20% and 50% respectively.

Table 2: Properties of fabricated Pure-PVA with PVA-CF film

No.

 Pure-PVA CF-PVA
  LT (%) LT (%)

Edge-to-Edge Resistance (EER, Ω)

01

 91.02 77.80 3140
02 87.33 74.92

2140

03

 89.76 75.15 2220
04 89.12 80.18

1750

05

 88.83 74.92 2140
06 90.60 78.49

1410

07

 90.73 76.63 1950
08 88.43 79.87

3170

09

 90.73 69.13 1340
10 90.57 75.66

1840

11

 90.30 72.94 1900
12 88.73 71.30

2730

Mean

 89.68 75.75

2069.58

Sample size is 200 mm х 200 mm. Carbon fiber filament length is 3 mm, carbon fiber to PVA ratio is 1:100. LT denotes light transmissivity while EER denotes edge-to-edge resistance. Pure PVA film is nonconductive and has infinite edge to edge resistance. A sample calculation of using 220 V and Block Resistance of 419.9208 Ω gives heating capability of 115.26 W/m2 via far-infrared light.

In agricultural greenhouses, transmittance of sun light is essential for crop growth. Light transmissivity reflects the percentage of direct sun light from the environment that is passed through the greenhouse walls to the interior. As this percentage is affected by the incident angle of the solar radiation in different seasons, the necessary light transmissivity needed for various crops differs, and materials used for the outer structure of agricultural greenhouses vary depending on the crops. In general, the light transmissivity of plastic greenhouse is 50%~60%, whereas that of glass greenhouses is 60%~70% and solar greenhouse typically achieve a light transmissivity of more than 70% (Table 2).

When compared to pure PVA films (which have an average light transmissivity of 89.68%), the PVA-CF films (which have an average light transmissivity of 75.75%) have 15.53% lower light transmissivity. This is mainly due to the added CF blocking the light transmitted through the film. This value indicated that the film is suitable for solar greenhouses. For application of the film in agricultural greenhouses in regions with seasonal weather, electrical energy is also needed to be delivered to the film for heating purposes. The addition of carbon fiber filaments to PVA films makes the PVA-CF film electrically conductive. Table 2 shows that the average EER measured is 2,069.58 Ω, which is suitable for the conversion of electrical energy to heat. A discussion of the generated heat will be presented as a later section. Table 4 in Supporting Information shows the effects of various CF to PVA ratio on the Edge-to-Edge Resistance (Ω) or Block Resistance (Ω) and light transmissivity (%) properties of the PVA-CF film. The films are fabricated in sets of 5 different carbon fiber to PVA ratios: 0.1:30 (0.33%), 0.2:30 (0.66%), 0.3:30 (1.0%), 0.6:30 (2.0%) and 1.0:30 (3.33%) (which translates to carbon fiber percentage of 0.33%, 0.66%, 2.0%, and 3.3% respectively). The effect of solution volume on the achievable thickness of the final molded film is also presented in the table.

From the first set of results in Table 4, it is observed that a low carbon fiber to PVA ratio of 0.1:30 (0.33%) produces film samples with average edge-to-edge resistance of 554.5 kΩ and average achievable light transmissivity of 75.825%. While the light transmissivity is optimum, the low range of conductivity is not suitable for infrared application in the agricultural greenhouse settings.

Table 4: Distribution of carbon fibers of different content in polyvinyl alcohol film formation

Sample No.

 CF (g):PVA (g)

(CF (g)/PVA(g) х%)

 Solution Quantity in every mold (mL) Measured TH (mm) Edge-to-Edge Resistance (Ω) Block Resistance (Ω) LT (%)

Material Resistivity ρ (Ω•mm)

80.0

 0.6020 945,000.00 945,000.00 84.89 568,890.00
0.1:30 100 0.7525 756,000.00 756,000.00 80.08

568,890.00
Sample-01

0.33%

 120 0.9030 604,800.00 604,800.00 73.63

546,134.40

140

 1.0535 483,840.00 483,840.00 70.70

509,725.44
Mean

110

 0.82775 697,41000 697,410.00 77.33

548,409.96
80.0 0.6020 1,260.00 1,260.00 75.08 758.52
0.2:30 100 0.7525 1,008.00 1,008.00 72.22

758.52
Sample-02

0.66%

 120 0.9030 806.40 806.40 69.29 728.18
140 1.0535 645.12 645.12 67.07

679.63
Mean 110 0.8278 929.88 929.88 70.92

731.21
80.0 0.6020 560.87 560.87 70.07 337.64
0.3:30 100 0.7530 448.69 448.69 60.72

325.64
Sample-03

1.00%

 120 0.9030 358.95 358.96 59.83 324.14
140 1.0540 287.16 287.17 55.08

302.53
Mean 110 0.8280 413.92 413.92 61.425

322.49
80.0 0.6020 430.25 430.25 62.36 259.01
0.6:30 100 0.7530 344.20 344.20 59.98

259.01
Sample-04

2.00%

 120 0.9030 275.36 275.36 58.74 248.65
140 1.0540 220.28 220.29 56.98

232.07
Mean 110 0.8280 317.52 317.52 59.52 249.69

80.0

 0.6020 380.56 380.56 51.04

229.09
Sample-05

1:30

 100 0.7530 304.44 304.45 45.25 229.09
3.33% 120 0.9030 243.55 243.56 28.22

219.93

140

 1.0540 194.84 194.85 23.08

205.27
Mean

110

 0.8280 280.85 280.85 36.89

220.85

TH denotes thickness of the film. LT denotes light transmissivity under LED lightning at average 5,395.5 Lux. Edge-to-Edge Resistance (Ω) is comparable to Block Resistance (Ω) as the samples are square shaped. Specimen size is 200 × 200 mm and carbon fiber length is 3 mm.

When the samples are fabricated using high carbon fiber to PVA ratio of 1:30 (3.33%), the extreme opposite effects are observed. The edge-to-edge resistance average value is 280.85 Ω and average achievable light transmissivity is only 36.89%. While the resistance is suitable for infrared application, the light transmissivity is too low. In general, for infrared applications, an edge-to-edge resistance below 1 kΩ is suitable, while for agricultural greenhouse applications, a light transmissivity value of approximately 60% is comparable to plastic and glass greenhouses. For the results shown in Table 4, is can be seen that film samples with carbon fiber to PVA ratios of 0.2:30 (0.66%), 0.3:30 (1.0%) and 0.6:30 (2.0%) have edge-to-edge resistances suitable for infrared applications.

Figure 7 shows the effects of film thickness on the block resistance, which directly affects the usability of the film in infrared applications. As shown in Figure 8, block resistance decreases with increased thickness. While this indicates that thicker films have good electrical properties for infrared applications, it also translates to an inverse effect of poor light transmissivity as previously discussed. Thus, a balance between the two inversely related properties needs to be made when deciding on film thickness.

fig 7

Figure 7: The relationship on the sample for Block Resistance (Ω) vs. Thickness (mm): (a) Sample-01: CF:PVA of 0.1:30 (0.33%), (b) Sample-02, CF:PVA of 0.2:30 (0.66%), (c) Sample-03, CF:PVA of 0.3:30 (1%), (d) Sample-04, CF:PVA of 0.6:30 (2%), and (e) Sample-05, CF:PVA of 1:30 (3.33%).

fig 8

Figure 8: The relationship on the sample for Light Transmissivity vs. Thickness with equation for CF-PVA: (a) Sample-01: CF:PVA of 0.1:30 (0.33%), (b) Sample-02, CF:PVA of 0.2:30 (0.66%), (c) Sample-03, CF:PVA of 0.3:30 (1%), (d) Sample-04, CF:PVA of 0.6:30 (2%), and (e) Sample-05, CF:PVA of 1:30 (3.33%).

From the various graphs shown in Figure 7, it is observed that samples with a CF:PVA ratio of higher than 0.3:30 (1.0%) have suitable edge-to-edge resistances across all film thickness range, while samples with a CF:PVA ratio of 0.2:30 (0.66%) and above are only suitable when thickness is greater than 7.5 mm, and samples with a CF:PVA ration of 1:30 (0.33%) have extremely high edge-to-edge resistances and are not suitable across all film thickness range. Figure 8 shows the relationship between light transmissivity vs. film thickness, which directly affects the suitability of the film as building material for agricultural greenhouses. As shown in Figure 8, light transmissivity reduces with increase in film thickness. In other words, the transmittance of the film is inversely proportional to the thickness of the film. To achieve suitable light transmissivity, thinner films are necessary.

The various graphs in Figure 8 shows that samples with a CF:PVA ratio of lower than 0.3:30 (1.0%) have light transmissivity comparable to a solar greenhouse across all film thickness range, while samples with a CF:PVA ratio of 0.2:30 (0.66%) are able to accomplish light transmissivity of solar and glass greenhouses as the thickness varies from a low of 0.6 mm to a high of 1.05 mm. Whereas samples with a CF:PVA ratios of 0.3:30 (1%) and higher have wide ranging light transmissivity from 70% down to 23%. Designing films at these CF:PVA ratios would require a close monitoring of the achievable light transmissivity at difference desired film thickness. To determine a film with acceptable light transmissivity for use as building material for agricultural greenhouse, while having good edge-to-edge resistance for infrared applications requires further study of the relationship between the two. Figure 7 shows the relationship between light transmissivity and block resistance for the various samples at different CF:PVA ratios with increasing film thickness.

Figure 9 shows the relationship of Light Transmissivity vs. Block Resistance for the various samples. As shown, light transmissivity increases with block resistance. Higher block resistance is a result of lower CF content, which increases the film’s transparency as the ratio of the PVA material, which has high light transmissivity property, increases.

fig 9

Figure 9: The relationship on the sample for Light Transmissivity vs. Block Resistance with Equations for CF-PVA. (a) Sample-01, CF:PVA of 0.1:30, (b) Sample-02, CF:PVA of 0.2:30, (c) Sample-03, CF:PVA of 0.3:30, (d) Sample-04, CF:PVA of 0.6:30, (e) Sample-05, CF:PVA of 1.0:30.

Figure 10 shows the relationship of Material Resistivity vs. Light Transmissivity. In general, material resistivity increases with the increase of light transmittance. This is in agreement with the results shown in Figure 11, as material resistivity is a function of carbon percentage content of fiber content which blocks light transmission through the PVA based films.

fig 10

Figure 10: The relationship on the sample for Material Resistivity vs. Light Transmissivity with Equations. (a) Sample-01, CF:PVA of 0.1:30, (b) Sample-02, CF:PVA of 0.2:30, (c) Sample-03, CF:PVA of 0.3:30, (d) Sample-04, CF:PVA of 0.6:30, (e) Sample-05, CF:PVA of 1.0:30.
Note: R2 is the percentage of the dependent variable variation that a linear model explains.
R2 = variance explained by the model / total variance. Larger R2 typically means the regression model has a better fit to the data.

fig 11

Figure 11: The process of fabricating PVA-CF transmittance conductive film

Figures 7-10 also presents the various design equations obtained through regression analysis, which allows us to match corresponding film thickness and required CF:PVA ratios to desired Light Transmissivity and Resistance values. As the Light Transmissivity and Resistance of the film is highly dependent on the film thickness, which in turn is affected by the CF:PVA ratios, a set of equation is required for each specific CF:PVA ratio. One example of the design equations are as follows for the CF:PVA of 0.3:30 (1%) film sample:

formulas

Where BR is the Block Resistance in Ω, TH is the film thickness in mm, LT is light transmissivity in %, and MR is material resistivity in Ω.mm.

Eqns. (1) and 2 allows for the calculation of Light Transmissivity and Block Resistance given the film thickness, whereas Eqns. (3) and 4 relate Light Transmissivity to Block Resistance and Material Resistivity to Light Transmissivity. As the thickness of a film can be controlled in an industrial manufacturing setting, the equations can be used to determine the achievable block resistance and light transmissivity. Table 4 shows the sample calculated values using this set of equations.

From Table 3, it is observed that while the Light Transmissivity calculated using Eqns. (2) and (3) differs by 3.763%. This is due to the less-than-ideal R2 value in the linear regression when obtaining Eqn. (3). After we are done with all the general testing, certain specs are chosen to design the final film used for the agricultural greenhouse. As have obtained the condition on PVA with CF:

0.33% < CF/PVA < 3.33% (5)

CF/PVA < 0.33% which the conductivity is too low, the film cannot meet need of infrared (the Edge-to-Edge Resistance is low for radiation far-infrared on greenhouse), and the CF/PVA > 3.33% which the transmissivity is too low, the film cannot meet need of the transmissivity (the transmissivity is low for crop growth needs on greenhouse).

Table 3: Sample calculations for Sample-03 CF:PVA=0.3:30 (1%) at LT=0.8 mm

Relationship

 Equation R2

Calculate Value
BR vs. TH

 (Eqn. 1)

 1

BRTH,03=419.9208 Ω
LT vs. TH

1

LTTH,03= 58.2790%
LT vs. BR

0.9878

LTBR,03=62.0420%
MR vs. LT

0.9885

MRLT03=311.5902

BR denotes Block Resistance, TH denotes film Thickness, LT denotes Light Transmissivity, and MR denotes Material Resistivity. R2 is an indicator of the fitting degree of the equation to the data set when performing linear regression. A value of 1 indicates a fit of high reliability.

In the experiment shown using transparent conductive film in agricultural greenhouse, installed transparent conductive at 2600 W (72 W/m2), when the air average temperature in one month 15.03°C to 3.8°C, warming the greenhouse with PVA-CF light transparent conductive film on 12 hours every, the average temperature in the greenhouse keep 12.33°C to 10.06°C. Illustrates the PVA-CF light transparent conductive film be applied for warming greenhouse that it is can be use (see the Table 5 in Supporting Information).

Table 5: The data of Test warming temperature in greenhouse at morning 10:00 and night 10:00

Date (11/2011)

 In outside greenhouse Air Temperature Test warming Temperature in greenhouse
  The Highest The Lowest Am 10:00

Pm 10:00

01

 15°C 6°C 12°C 8°C
02 16°C 9°C 12°C

9°C

03

 20°C 9°C 15°C 10°C
04 22°C 11°C 16°C

11°C

05

 21°C 14°C 16°C 16°C
06 19°C 12°C 16°C

12°C

07

 14°C -3°C 12°C 8°C
08 5°C -1°C 10°C

8°C

09

 10°C 2°C 8°C 8°C
10 12°C 3°C 10°C

8°C

11

 11°C 2°C 9°C 8°C
12 13°C 2°C 10°C

8°C

13

 17°C 3°C 12°C 12°C
14 17°C 4°C 12°C

12°C

15

 15°C 4°C 12°C 12°C
16 15°C 5°C 13°C

12°C

17

 18°C 7°C 14°C 12°C
18 20°C 6°C 16°C

14°C

19

 18°C 8°C 16°C 14°C
20 18°C 10°C 16°C

12°C

21

 11°C 0°C 12°C 12°C
22 3°C -3°C 10°C

8°C

23

 9°C -1°C 7°C 8°C
24 13°C -2°C 12°C

8°C

25

 14°C -1°C 12°C 8°C
26 12°C -1°C 12°C

8°C

27

 13°C 4°C 12°C 8°C
28 14°C 5°C 12°C

10°C

29

 16°C 2°C 14°C 10°C
30 6°C -2°C 10°C

8°C

Mean

 15.03°C 3.8°C 12.33°C

10.06°C

Note: warming time night 10:00 to next morning 10:00 every day

Experimental Section

Experimental Setup

PVA-CF films can be manufactured using either solution salivation method (wet method) or extrusion blown film method (dry method). In this study, the wet method is used. In the industry, PVA films are generally manufactured with the use of a casting machine with roller press. For the initial experimental work on determining the feasibility and properties of the PVA-CF films, a wet method is adapted for preparing the films (see Figure 11). First, CF filament is mixed in diluted PVA and glycerin solution. The addition of glycerin ensures that the resulting film is flexible and not rigid. The mixture is then slowly blended for up to 2.5 hours to ensure the CF filament is dispersed evenly. The mixture is then casted in a mold and left for air drying up to 48 hours in ambient room temperate. The resulting product is a thin and flexible PVA-CF transparent conductive film. Note that this method is only suitable for fabricating small size films.

For the application of the developed film for agricultural greenhouse, two custom made specialized fabrication machines are constructed to produce the film in larger sizes. The first machine is used for fabrication of the basic flexible conductive PVA-CF films, while the second machine is then used to apply metal copper foil strips for electric current delivery to the film and to laminate the film to provide electrical insulation and ensure the durability of the film. The two fabrication machines are respectively shown in Figures 12 and 13.

fig 12

Figure 12: Fabrication machine for basic flexible conductive PVA-CF. 1: Mixer drum for mixing and blending of PVA, CF and glycerin. 2 & 4: pump. 3: Vacuum cavitation cylinder. 5: Horizontal slit funnel. 6: Heated conveyor steel belt. 7: PVA-CF film. 8: Winding machine.

fig 13

Figure 13: Modified food packaging machine for application of metal copper foil strips and protection laminate. Blue line indicates the feed path of the film. 1: current-carrying, 2: PVA-CF film roller, 3: Top laminate. 4: Glue applicator roller box. 5: Guide roller. 6: Forced convection oven. 7: Button laminate. 8: Hot drum, 9: Winding machine.

As shown in Figure 12, to produce the basic flexible conductive PVA-CF films, the mixture of CF filament in diluted PVA and glycerin solution is first blended in a mixer drum for 2.5 hours. The mixture is then pumped into a vacuum cavitation cylinder that removes all air bubbles from the mixture solution before the mixture is pumped squirted through a horizontal slit funnel onto a 10-meter length conveyor steel belt that is heated at 80°C. The mixture which is spread over the metal hot plate is then then left to air dry as it is slowly transferred along the heated steel belt for up to 5 minutes before being winded into a roll.

PVA-CF films are fabricated by infusing CF filaments in PVA substrate. CF is electrically conductive, and the mixture of the two material allows for the casting of an electrically conductive film.

For the application of the metal copper foil strips and protection laminate, a modified food packaging machine was used (see Figure 13). First, the current carrying metal copper foil strips are aligned and rolled onto the edges of the roll of conductive PVA-CF film. The top laminate is then applied onto the aligned copper foil strips and PVA-CF film to form a 2-layer film. Next, the 2-layer film is fed through a glue applicator roller box and then sent to a forced convection oven for drying. Finally, the bottom laminate is applied to the 2-layer film and sent through two hot drums for the final pressing to obtain the finished laminated film. The result is a durable PVA-CF film that can be applied directly in agricultural greenhouse setting.

For rural application of the film in agricultural greenhouses, aluminum air batteries are fabricated and applied for testing purposes. I have made an aluminum air batter which compose in 200 single aluminum air batteries with 12 V and 20 mA on PVA-CF transparent conductive film for 7 weeks now up till 01/Jan/2022: capacity = 20 mA x 24 hour x 7 days x 7 weeks = 235.2 Ah, see Figure 14. This battery be made at Carbon Rod as anode and aluminum sheet as cathode in size Ø18 mm by 200 mm. A single batter is 0.6 V and 0.2 mA, to drive a 1000 mm by 500 mm PVA-CF transparent conductive film (Edge-to-Edge is 480 Ω).

fig 14

Figure 14: Aluminum-air batteries with K2SO4 electrolyte (a) compose in 200 single aluminum air batteries with 12V and 20 mA for PVA-CF transparent conductive film, (b) To parallel connect 10 single battery and series connect 20 battery groups and all in one.

Experimental Method

Prior to the actual fabrication of the films for testing, an initial experiment to determine a suitable CF filament length is performed. 0.3 g of CF filaments of 3 mm, 6 mm and 10 mm length are respectively cut and mixed with 30 g of PVA, 15 ml of glycerin and 385 ml of distilled water. The solution is blended for two hours and the left to settle for 2 hours for the air bubbles disperse. The uniformity of the CF filament dispersion in the mixture is then observed. Next, 3 samples were casted using the 3 mixtures and the CF filament dispersion is further observed in the produced films.

Next, using the fabrication machines, various 200 mm by 200 mm samples using 3 mm CF filaments at CF to PVA ratio of 1:100 are fabricated. The samples are tested for light transmissivity using LH – 206 Optical Transmittance Meter made in Tianjing China.

To determine if the films are suitable for both heating and building usage in agricultural greenhouses, various film samples are fabricated at different CF to PVA ratios of 0.1:30 (0.33%), 0.2:30 (0.66%), 0.3:30 (1.0%), 0.6:30 (2.0%) and 1.0:30 (3.33%). The thickness, edge-to-edge resistance, block resistance, light transmissivity and material resistivity are observed.

Finally, to determine the effectiveness of the fabricated conductive film in agricultural greenhouse, films were fabricated at CF to PVA ratio of 0.6:30 (2.0%). The film has the following properties: edge-to-edge resistance of 480 Ω, power rating of 100.83 W at 220 V, and dimension of 1000 mm х 500 mm (see Figure 15a). A custom 9 m х 4 m х 4 m agricultural greenhouse is used as the test bed of the films. A total of 26 sheets of the fabricated film is applied to the side walls of the greenhouse (see Figure 15b) and the temperature difference is observed.

fig 15

Figure 15: The PVA-CF Light Transparent conductive film be installed on greenhouse wall. (a) PVA-CF transparent conductive film. (b) Installation of 26 sheets of PVA-CF to the custom built agricultural greenhouse.

Supporting Information

Please find below in Tables 4 and 5.

Conclusion

The newly developed film has an average Edge-to-Edge Resistance of 2069.58 Ω (Table 4), light transmittance of 75.75% (Table 4) and has a heating capability of 23.38 W/m2 (2202/2069.58 Ω) via far-infrared light (sample size is 200*200 mm and voltage in 220 V), which is far from meeting the temperature requirements of crop growth. To solve this problem, one is to increase the content of carbon fiber, sacrifice the transmittance, increase the power of the film, to improve the infrared radiation capacity; Second, increase the heating time to increase the accumulation of the overall infrared radiation.

From Table 3 shows, through setting the facts, the Block Resistance reach 419.9208 Ω, heating capability of 115.26 W/m2 (220 V/419.9208 Ω) via far-infrared light, and the light transmissivity reach 58.2790%, It can basically meet the requirements of agricultural greenhouse lighting rate and suitable crop growth.

Acknowledgements

The authors would like to thank the Faculty of Engineering, Built Environment and Information Technology (FoEBEIT), SEGi University for supporting the research work. The authors would also like to thank Xiaofei Wang from Beijing Biyan Special Materials Co., LTD for her help and support in fabricating the carbon fiber-cotton pulp conductive film under industry manufacturing conditions. The authors would also like to thank Shuting Yu and Mingqing Lu from Shangdong Hengtian Special Materials Co., LTD for their help and support in building the equipment for the transparent conductive process. The authors would also like to thank the Yuquanwa National Demonstration Center of Efficient Agriculture for providing the use of the agricultural greenhouse site for the practical testing of the transparent conductive film. Tzer Hwai Gilber Thio would like to acknowledge the support of research fund from SEGi University (SEGiIRF/2018-11/FoEBE-18/81).

References

  1. Hecht DS, Hu LB, Irvin G (2011) Emerging Transparent Electrodes Based on Thin Films of Carbon Nanotubes, Carphene and Metallie Nanostructures. Mater. 23:1482-1513. [crossref]
  2. Beiting Wang, Tzer Hwai Gilbert Thio, Hock Siong Chong. Transparent Conductive Far-Infrared Radiative Film based on Cotton Pulp with Carbon Fiber in Agriculture Greenhouse.
  3. Greenhouses for modern agricultural facilities.
  4. Obtainedfrom:https://baike.baidu.com/item/%E6%B8%A9%E5%AE%A4%E5%A4%A7%E6%A3%9A/9128827.
  5. agricultural grennhouse,
  6. Research status and application of polyvinyl alcohol films. 2014.
  7. Svenja Lohner (2020) Heating with the Greenhouse Effect. Science Buddies.
  8. De Pascale S, Maggio A (2004) Sustainable protected cultivation at Mediterranean climate, perspectives and challenges. Acta Horticulturae 691: 29-42.
  9. How to Heat a Green house in the Winter.
  10. Elizabeth Waddington (2020) 7 Innovative Ways To Heat Your Greenhouse In Winter.
  11. Fang Jiare (2022). Greenhouse heating, greenhouse agriculture.
  12. PVA film
  13. Advantages and development prospects of water-soluble pva.
  14. Li Fei Xiao, Gensheng Song Weisheng.(2016). UV resistance of polyvinyl alcohol composite film. Packaging Engineering. 21
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DOI: 10.31038/NRFSJ.2022514

Abstract

A total of 377 respondents from three countries (France, Germany, UK), selected pizza as a product of interest from a set of 30 different food products, and immediately participated in an approximately 15 minute experiment executed on the internet, the study one in the either French, German or UK English, depending upon the country.. Each respondent evaluated an individualized set of 60 different vignettes about pizza, the vignettes constructed according to a single basic experimental design, that design ‘permuted’ to create different combinations. The elements or messages within the vignette came from answers to four questions, each question addressing a different topic (Question A = product feature; Question B = consumption features; Question C = emotion benefits; Question D = tag lines and sales/restaurants appropriate for each country). Each respondent rated each vignette on a 9-point rating (1=does not crave. 9=craves the product as described). The initial analysis generated an additive model for each respondent, showing the contribution of each element to overall craveability. The three countries showed differences but without a clear underlying pattern. Clustering the respondents by the pattern of their individual coefficients generated clearer differences. Clustering the respondents first into two segments, then three segments, then four segments, five segments, and finally six segments revealed two general behaviors. The first behavior, emerging from Question B (venue) and Question C (emotion) can be labelled ORDERLY. No major surprises occurred for four, five and six segments. Most of the learning occurred for two or three segments. The second behavior emerged from Question A (food feature) can be labelled as DISORDERLY. For two or three segments, certain elements were important, other elements were not. When 4-6 segments were extracted, previously unimportant elements now became important. The disruptive emergence of elements with more segments of respondents (mind-sets) based on product features confirm the fact that in most countries, products like pizza will most easily be differentiated on the basis of flavor, allowing the marketer to identify new, promising products to introduce. Using venue and emotion will be less successful because it is likely that no previously poor-performer is likely to become a strong performer when more segments are uncovered.

Introduction – Mind Genomics and the Experimental Analysis of Preferences

Mind Genomics is a newly emerging branch of psychology focusing on the decision-making process for the world of the everyday. Mind Genomics works with the situations facing people every day. The goal of Mind Genomics is to explore, the everyday, how we make decisions, and how people differ from each other in the way they make the decision. Mind Genomics does not alter the world of the respondent to identify key factors driving decisions, although it could. Rather, Mind Genomics works with statements about issues and situations of everyday life, looking for the patterns without disturbing the situation. In contrast, experimental psychologists often put people into artificial situations, seeking to isolate phenomena of interest, and manipulate those phenomena as permitted by the artificial situation.

Mind Genomics traces its heritage to three different disciplines:

  1. Psychophysics, an early branch of experimental psychology, with the goal to understand how we perceive the outside world and how that physical world is transformed into of perception [1]. A typical psychophysical study of the traditional type concerns the sweetness of a soda versus how much sugar is in the soda. Psychophysics looks for the relation between physical ‘intensities’ and subjective ‘intensities’.
  2. Consumer research and opinion polling, which focus on what people do in their daily, relevant world [2]. Mind Genomics also focuses on the presumably more serious worlds of society, education, the law, medicine, and so forth, aspects dealing with society.
  3. The world of statistics, especially experimental design [3]. Mind Genomics sets ups simple-to-execute experiments, with either real stimuli, or with stimuli described as terms. These stimuli comprise systematically combined variables, often simply messages on a variety of topics.

In the actual implementation of Mind Genomics, the approach becomes a template. The researcher identifies the topic, creates a set of questions which ‘tell a story’, creates answers to the questions in the form of short, declarative statements, and then combines these answers into vignettes according to an underlying plan, the aforementioned experimental design. The researcher presents the respondent with these vignettes in a randomized order, and obtains a rating from the respondent. Each respondent evaluates a different, and unique set of vignettes, rather than re-evaluating the vignettes presented to another respondent.

The respondent, confronted with the systematic variation, cannot simply select a strategy and remain with it because the vignettes, the combinations keep changing. By presenting the respondent with these systematically created combinations of messages as the test stimuli, and by instructing the respondent to rate the combination, the researcher forces the respondent into the type of situation the respondent typically faces, viz., a set of compound stimuli of varying composition, where the respondent must abstract the relevant information without any hint of what is expected, what is ‘correct’, and so forth. In other words, the test situation mirrors everyday life.

This approach, combining the test stimuli, presenting them to a respondent, measuring the response, and deducing how the different features of the stimuli drive the response, constitutes the ‘secret sauce’ to Mind Genomics. The experimental design enables the researcher to pinpoint the drivers of response to the vignettes, and the magnitude of those drivers, using statistics appropriate for experimental design.

The Mind Genomics approach has been templated, and made available to the public for one specific, easy to use experimental design, the so-called 4 x 4 (Topic, four questions, answers to each question). The website is www.BimiLeap.com

Mind-sets as a Key Output from Mind Genomics

Beyond the experimentation, which allows the researcher to understand everyday behavior, albeit in controlled conditions through messages, emerges the second key output of Mind Genomics, namely different ways of looking at the same test stimulus. We are accustomed to the compound and complex nature of the external world. We also recognize at an intuitive level that people respondent to different parts of the world to which they are exposed. The power of Mind Genomics is that it specifies these different ways of looking at the world (mind-sets), finding out what is important to various groups (mind-sets), and the nature of these mind-sets (viz., WHO has the mind-sets, do the mind-sets change, etc.).

From many studies published using Mind Genomics in a variety of topics, ranging from health to law to food, to stores, to beauty, and so forth, clearly different groups of mind-sets emerge. The mind-sets emerge based upon the statistical method of clustering [4]. Clustering is an easy-to-implement approach for data emerging from Mind Genomics. Each respondent generates a model, an equation. The equation is expressed as: Rating = k0 + k1 (A1) + k2 (A2)…kn (An). The number of coefficients is a function of the number of elements or messages tested. Often the rating is transformed to a binary value (0/100) because managers understand binary (no/yes) more easily than actual rating values on a 9-point scale. The equation, whose parameters are estimated for each respondent, thus becomes the foundation for a database.

The statistical analysis underlying the discovery of the mind-sets is primarily ‘objective,’ viz., without reference to the content. Only the selection of the number of mind-sets and the naming of the mind-sets require the researcher to offer an input. The goal is to extract as few clusters or mind-sets from the data (parsimony), while at the same time ensuring that the mind-sets make sense.

In virtually every topic explored with Mind Genomics, with one exception (murder from the legal point of view) [5]; the abovementioned approach generates often, two or three, occasionally four different clusters (viz., mind-sets of respondents). These clusters appear to be meaningful, viz., they ‘tell a story’, and the stories are usually coherent, although not necessary ‘crisp.’ They ability to generate different groups of respondents is remarkable because 2-3 different groups emerge again and again. The effort does not product perfect clusters, a production which that requires an artist ‘sculpting the data’. The clusters are certainly not perfect, however, often incorporating elements or messages which seem not to belong.

Mind Genomics, specifically the search for mind-sets to understand the experience of the everyday, has started to address other issues such as how to uncover small groups of individuals in the populations, rather than large basic groups. In one of these studies, conducted in 2010, the focus was to discover a group of respondents who would be positive to the then-novel idea of health insurance for animals. The Trupanion Corporation approached the author with the request to investigate the world of pet owners, seeking a group that would be positive to animal health insurance. The analytic approach was kept simple. The strategy was to work with a large group of pet owners as respondents, and carry out the clustering beyond three and four clusters, to six clusters. That that point, database would be increasingly segmented, until a clear group of respondents emerged whose coefficients suggest strong interest in and acceptance animal insurance. The validity of the approach was demonstrated by the significantly growth of sales (100%), call center conversion rate (40%), web sales (25%, field sales (50%) [6].

A similar type of issue emerged two years later, in 2012, with the issue of what makes a product ‘taste great’. The focus of this second issue was to discover, if possible, a group of respondents to whom ‘texture’ rather than taste/flavor, was the most important. Once again the strategy was to extract an increasing number of clusters or mind-sets until a cluster emerged which showed the highest coefficients for the elements describing texture. The high coefficients, specifically much higher than those for appearance, aroma, and test, was assumed to represent texture-oriented respondents [7].

Applying Mind Genomics to Study Pizza

The importance of pizza to the world of eating cannot be overestimated. Every town, village, and of course every city can boast of at least one, often two and sometimes more outlets which sell pizza to consumers whether selling complete pizzas, selling slices (e.g. along with a beverage), or simply sell pizza as one of their products. The academic literature on pizza is large, because it is so popular, providing a substrate which can accommodate different ingredients as ‘toppings,’ these ingredients often driven by cultural norms [8-11].

Pizza provides an idea topic to study food preferences using Mind Genomics. The product is simple, easy to describe, has evolved in a number of directions, ranging from the cheese to the non-cheese ingredients. The changes in the product can be described in words, making it possible to study pizza through the text-based description of the product.

In light of the increasing competitiveness in the food world, a competition in 2003-2004 which now seems slow, the McCormick Company of Hunt Valley, Maryland, USA invested in a set of studies about the response to products, with the information about the product (features, venue, emotion, outlets and tag lines) embedded in short, easy-to-read vignettes, assembled according to an underlying experiment design [12]. Each respondent evaluated a unique set of 60 vignettes, comprising 2-4 elements, one from each type of information about the product (e.g. one product feature, one outline, one emotion, etc.). This strategy produced a great deal of information about the reactions of respondents to the different vignettes (viz. combination of elements).

The approach was called the It! study, which has been extensively discussed in previous papers [13,14]. The It! approach allowed the respondent to select a topic of interest from a ‘wall of products’, so that respondent was interested in the product to begin with the studies that we will address here come from the second-generation of the Crave It! studies, which we called Eurocrave. The studies were done with the same products, and mostly but not all the same elements across three countries (France, Germany, UK).

The data reported here come from the set of It! studies called Eurocrave, run in 2002 in France, Germany and the UK. The Eurocrave studies comprised 30 studies, the same 30 studies in each of the three countries, with the same raw material (elements), except for store outlet. Store outlet was particularized to the country.

The respondent was invited by a local field service. There were 30 studies available for the respondent, who could only choose one. As soon as 130 respondents selected a study, and completed that study, the study temporary ‘disappeared’ from the of available studies from which one could choose. Figure 1 thus shows the 19 available studies in Germany, 11 studies having been completed for Germany. The respondent was sent to the study chosen. The study had to be complete in order to count against the quote of 120 respondent [15].

FIG 1

Figure 1: The ‘wall’ of available studies for the German Eurocrave project

Moving to specifics, Table 1 shows the four questions for pizza, and the nine answers or elements for each question. Combinations of these text elements will become the stimuli that will be evaluated by the respondents. These combinations are known as vignettes. A hallmark of Mind Genomics is the effort made to have the elements present word pictures, not just simple phrases. That is, the elements or answers should paint an evocative picture involving pizza. The four questions provide a narrative of one’s experience with pizza.

Table 1: The 36 elements for pizza, shown in four groups. The respondent only saw the text, not the group, viz., not the organizing principle

table 1 (1)

table 1 (2)

The Mind Genomics process executed in the early years of Mind Genomics (1996-2006), used a large experimental design, known as the 4 x 9, four questions or aspects, each with nine different elements. The 4 x 9 design involves 36 different elements, shown for pizza, in English. Table 1 shows these elements. As noted above, the elements present the outlets (Elements E28-E33) were particularized for each country, comprising the relevant outlets for those countries. The data from Question (E27-E36) will be used in the preliminary analysis in this study, but not discussed in the results, because of the difficulty of comparing across countries.

It is important to note that the Mind Genomics effort is not a single, final study which proposes to quantify the way people think about a product, in this case pizza. Rather, the Mind Genomics effort is simply an experiment, one experiment in a series when so desired, with the experiment showing concretely the importance of the element as a driver of attitude.

Once the elements have been chosen and polished, the next step is to combine these elements into small combinations, as noted above, the so-called vignettes. The rationale for vignettes are that we typically don’t see sequences of single ideas, one after another, to which we must react. Rather, we see combinations of pieces of information, often combinations which appear to be haphazard, or at least we fail in the short time allotted to uncover the pattern below the combinations. Yet, again and again we emerge unscathed from what seems to be hard to discuss, namely haphazard combinations. We react, and often don’t even realized the nature of our thinking.

The Mind Genomics was designed eliminate two biases. The first bias is that one-at-a-time presentation of messages is simply not the way we work. We are gluttons for information, of all types, mixed in ways.

The second bias is that we unconsciously adjust our thinking to accommodate the nature of the stimulus. We do not judge all stimuli the same way. For example, brand names are judged differently than product performance. The criteria we used to judge brand names versus product performance are different. The rationale, perceptive individual will adjust her or his criteria depending upon what particularly is being evaluated. For example, were we to read only the individual elements in Table 1, rating one element at a time, it might be easy to adopt different criteria, depending upon the specific element. If the respondent were answer elements from the set of E1-E9, the respondent might adopt the criterion of ‘do i like the flavor’. In contrast when it comes to the third set of elements (E19-E27), the criterion would not be ‘liking’, but rather fun to eat in a situation. The change in criterion might be quick, virtually unconscious, but sufficient to create a situation where the assessments might not be commensurate because the respondent uses different criteria, such criteria a function of the specific nature of the single message presented. What we measure, as a result, may seem like it should be valid because the respondent does the operation, but the validity might be a chimera, a false result.

Each respondent in the study is exposed to these elements, but in the form of vignettes. The vignettes are combinations of elements, as specified by an underlying experimental design. The design specifies combinations of 2-4 elements. Each vignette, the aforementioned combination, comprises at most one element from each group, but often the vignette is lacking one or two elements.

The specific structure of the vignettes, defined as the underlying experimental design, comes to 60 different combinations. Each respondent tests a different permutation of the basic design. That is, the mathematical structure of the 60 vignettes remains the same, but what changes is the specific set of 60 combinations. This strategy, working with a fixed design that is permuted, ends up having three key benefits:

1. Ability to Work with the Rating Scale, to Simplify it for the Manager

As noted above, the ratings, assigned on a 9-point scale (1=do not crave at all…. 9=crave extremely) are converted to a simpler binary scale (ratings 1=6 converted to 0, ratings 7-9 converted to 100). It will be these binary transformed ratings that will be used as the dependent variables in the individual-level and group models. After the transformation, a vanishingly small random number added to each transformed value (value < 10-5), in order to ensure that the binary transformed rating always shows some variation across the 60 vignettes evaluated by a single respondent.

2. Create a Model (viz., Equation) for Each Individual Showing How the Elements ‘Drive’ the Rating

Each respondent tests just the correct combinations to allow statisticians to estimate the contribution of each of the 36 elements to the rating. This is called a permuted design [12]. The ability to do the statistical modeling down to the level of a single respondent is very important. One does not need to balance the sample, and go through other ‘gyrations’ to ensure that the study is balanced The typical equation is expressed as: The typical equation is written as: (Binary Transformed) Rating = k0 + k1 (E01) + k2 (E02)…kn(E36).

3. No Requirement that the Researcher ‘Know’ the Correct Region to Test

The typical experiment covers so little ground that the typical experiments ends up validating the ingoing hypothesis of the researcher, rather than exploring and learning. In effect, the research focuses on a microscopically small volume of the possible design space. In contrast, with Mind Genomics, each respondent provides unique data, not provided by the other respondents. Each respondent provides a separately oriented snapshot of the mind of the respondent. The result is coverage of a lot of the possible combinations, albeit a coverage achieved with the aid of the many respondents. A good metaphor for the approach is the MRI used in medicine, which takes snapshots of the same tissue from different angles, and then reconstitutes a single snapshot of the tissue through computer analysis.

Coefficients for Product Elements (Total vs. Countries vs. Mind-sets)

Our first focus will be on the coefficients achieved by E01-E09, the nine elements presenting information about the actual product itself, viz., the ingredients and the form. To summarize the analytic strategy, All data presented in Table 2 come from abstracting the coefficients for the individual models, these models having been created from the data for one respondent across the 60 vignettes, the 36 elements, and the rating scale transformed to a binary scale (0/100; ratings of 1-6 → 0; ratings 7-9 → 100).

Table 2: The coefficients for the nine description-based elements for pizza (E01-E09), and the additive constant. The numbers in the body of the table are the coefficients from the OLS (ordinary least-squares) models or equations relating the presence/absence of the elements to the transformed overall rating. The table only shows element coefficients of +10 or higher.

table 2 (1)

table 2 (2)

table 2 (3)

table 2 (4)

table 2 (5)

Mind Genomics studies ‘throw off’ a great deal of data. For most studies, it has become increasingly clear that the best way to discern patterns is to estimate all the coefficients, but then only focus on the coefficients which are strong. The large positive coefficients are the key elements which ‘drive’ the response. The small positive and many negative coefficients are harder to interpret, often adding noisy to what would otherwise be an easy naming.

Table 2 shows the coefficients for E01-E09, for six blocks of results. Recall that elements E01-E09 are features of the pizza. For the individual models, only coefficients of +10 or higher are shown. These coefficients are statistically significant, approaching a t-statistic of 2.0. The additive constant is shown for every key subgroup, however,

Block 1 = Total panel, France, Germany, United Kingdom

The data in Block 1 come from the coefficients for the total panel, without any further processing. The acceptance of pizza ranges from a low of 31 (Germany) to a high of 40 (France). What emerges is the modest acceptance of pizza without any elements, as shown by the modest-sized additive constant. It will be the elements which generate the acceptable.

It should not come as a surprise that when we deal with foods, the strong performing elements are those which present information about the product itself, the constituents, viz., and the ingredients. These elements are ‘concrete’, painting a word picture. For most food and beverage products, these are the elements which excite the consumer, even when the elements are embedded in a vignette with other information.

For the total panel, only two elements even reach the coefficient value 10: Soft and gooey slices of pizza with cheese and Pizza with a filled or twisted crust. It is important to keep in mind that these two elements deal with form, and do not invoke unusual flavors.

Blocks 2-6 (Clustering to Generate Mind-sets, viz., Segments of Respondents with Similar Patterns of Coefficients for Elements E01-E09

The initial analysis to generate the coefficients was done on the complete data-set for each respondent, viz., all 60 vignettes for the data base, and all 36 elements as independent variables. Thus each respondent generated a totally separate model, made possible by the previously discussed individual-level design. For the analyses in Blocks 2-6 m only the coefficients for E01-E09 were used in order to create the clusters, viz., and the mind-set. The method used was k-means clustering [16], with the measure of distance between pairs of respondents computed on the basis of the expression (Distance = 1-Pearson Correlation). It is important to emphasize that the clustering is done without any preconceived bias. The clustering algorithm is strictly an algorithm with no need to know the ‘meaning’ of the variables that it is using in its mathematical computations.

Block 2 (Two Mind-sets)

The simplest clustering using k-means clustering, ended up dividing the respondents into two groups, the basic likers (Mind-Set 22), and the feature likers (Mind-Set 21). Mind-Set 22 is larger, likes but does not love pizza (additive constant 55), and is indifferent to the features of the pizza. That is, no element emerge with coefficients over +10. In contrast, the smaller group Mind-Set 21), constituting approximately 1/3 of the respondents, shows a no general desire for pizza in the absence of elements (additive constant 5), but with a strong desire for the features of traditional pizza, Four examples of the exaggerated response by Mind-Set 21 to features (coefficient > 20) are:,

Soft and gooey slices of pizza with cheese

Pizza with a thick crust and a rich topping

Pizza with a filled or twisted crust

Pizza with a lot of tomato sauce, ham, and cheese

Block 3 (Three Mind-sets)

Mind-Set 32 is interested in pizza in general (additive constant 62), but does not find any of the product features very interest. In contrast, Mind-Sets 31 and 33 show low basic interest in pizza (additive constant 9 and 5), but strong interest in specific features of the product. Mind-Set 31 appears to want the more traditional features of pizza, whereas Mind-Set 33 appears to want to more ‘unusual features.’ It is important to keep in mind that these differences are not imposed on the data, but emerge from the patterns across people and countries.

Block 4 (Four Mind-sets), Block 5 (Five Mind-sets), and Block 6 (Six Mind-sets)

These three additional clusters, again based only on the coefficients for E01-E9, show similar patterns.

a. One of the mind-set clusters shows a high additive constant, meaning that the respondents like all of the pizza ideas. This cluster, however, shows no elements describing the pizza which score very well. This cluster with the high additive constant is the non-discriminating, acceptor, abbreviated NDA.

b. The remaining mind-sets show different, unpredictable patterns, with low additive constant, but with some specific product elements presenting a very high coefficient.

c. An element can perform poorly with total panel, and with two or three mind-sets, viz., demonstrate a low positive coefficient, or a negative coefficient, but with more mind-sets extracted this poorly performing element, one that might be overlooked, might suddenly become promising. An example is: Pizza with fish and seafood.

d. Table 2 suggests that the dynamics of the system are complicated. An element may be irrelevant until a sufficient number of mind-sets or clusters are allowed, at which point the element shows its strength, and for one mind-set the element generates a high coefficient, whereas for the other mind-sets this previously ‘minor’ element remains minor.

e. The key word here is unpredictability. The lesson is the possibility of identifying mind-sets, albeit as an exercise in data analysis, and the practical use of the clusters for formulating marketing strategy. What is disappointing is the lack of specific, repeated patterns emerging from the clustering, patterns that could for the basis of a culinary psychology of pizza.

The Deeper Dynamics of Elements as Drivers of Mind-sets

It may well be that a different way is needed to understand the way elements perform. Rather than looking at the coefficient of the element in different arrays of mind-sets (viz., 2 vs. 3 vs. 4 vs. 5 vs. 6), an alternative way looks at the variability generated by the element vs. error, and seeing how much real variability the generates with increasing number of mind-sets. This second way computes the F ratio for each element, with the F ratio being a measure of signal to noise. Higher F ratios mean that the element really ‘drives’ the segmentation. Lower F ratios mean that the element does not ‘drive’ the segmentation.

For this analysis, we look at the three sets of elements (Question A – product features; Question B – Consumption Occasions; Question C – Emotional benefits and outcomes.). We do not look at Question D because the elements for the ‘purchase location’ (E28 – E33) changed by country.

The analyses are done separately question, following the approach below. We describe the approach for one of the three sets of elements. The same approach is applied separately to the other two sets of elements.

  1. For each question, collect all the sets of nine coefficients across all the respondents. Each respondent generates a row of nine numbers, one cell for each element. This step generated three data sets, based on elements E01-E09; E10-E18, and E19-E27, respectively,
  2. For each question separately, cluster the respondents, using the appropriate set of elements and their coefficients., Use k-means clustering as one before, with the distance between pairs of respondents defined as (1-Pearson R computed on the nine corresponding elements for the two respondents). Do the clustering five times, extracting first two clusters, then separately three clusters, then separately four clusters, then separately five clusters, and finally separately six clusters. The term ‘separately’ is repeated to emphasize the fact that the clustering starts anew each time.
  3. Estimate the F ratio for each of the nine elements. The magnitude of the F ratios is a measure of the degree to which the element ‘drives the segmentation.
  4. Keep in mind that the data set is balanced in terms of respondents. Thus, we have removed the subject effect, and dealing only with the degree to which the element drives the segmentation.

When we follow this protocol, computing the F ratio for each element when the clustering goes sequentially from two to six mind-sets, we uncover the deeper structure, viz. a structure which may truly underlie the different groups. Table 3 shows the F ratios for each of the 27 elements, computed for the five sequential clustering. Each clustering generates a set of 27 F ratios.

Table 3: F ratios for elements emerging from the clustering. High F ratios mean that the element is a strong driver of the classification. All F ratios of 10 or higher are shown in in a shaded ell

table 3

Keeping in mind that the F ratio is a measure of signal to noise, we can sort Table 3 five times, each time identifying the two (or more) strongest performers. The structure below begins to emerge, suggesting that there are two strong groups; traditional pizza and pizza with fish and seafood. There may be more, but these are the repeating themes.

Two mind-sets

Pizza with a thick crust and a rich topping

Pizza with a crust that doubles as breadstick

Three mind-sets

Pizza with a thick crust and a rich topping

Pizza with fish and seafood

Four mind-sets

Pizza with fish and seafood

Pizza with a thick crust and a rich topping

Five mind-sets

Pizza with fish and seafood

Vegetarian pizza with vegetables and cheese

Six mind-sets

Pizza with fish and seafood

Vegetarian pizza with vegetables and cheese

The dynamics of segmentation suggest that it is both the nature of the topic whose representatives are being segmented and the number of available segments which make a difference. An element may be less important than another when there are two or three segments, and so the less important element simply ‘goes along’. When it comes to more segment, e.g. four or five or six, this hitherto minor, unimportant element becomes important, and becomes the center of a new mindset. This finding may seem a bit awkward, but it is testimony to the fact that it is not only people differences in which are working, but the channels in which those differences are allowed to emerge. Reduce the opportunity and the segmentation waiting to flower is simply suppressed, not allowed to show itself.

The variability in preferences underlying the segmentation is clear greater for product (Question A), and then for emotion/benefit (Question C), and finally for consumption situation (Question B). One possible conclusion is that the potential segmentation is greatest for the actual product, something which manifests itself in the market today, where companies offering pizza offer different variations of them. The other groups offer the possibility of segmentation, but the F ratios are smaller. There are differences across mind-sets for a specific element, but there are few surprising re-emergences of elements as driers of segmentation when new mind-sets are opened up. The practical implication is that for marketing, most of the efforts, where possible, will be attempts to present new features of the pizza product itself.

Discussion and Conclusion

The world of foodservice is forever seeking to discover both what people like, and what they will like. There is the notion of habituation, that a stimulus which is presented again and again will lose its ability to excite [17,18]. People get bored with the same food, although the dynamics of such boredom, and habituation are yet to be worked out. One can see food trends come and go, with trend spotters looking for the latest and greatest, the cuisines, and of course the star items that will excite everyone. At the same time, observations in restaurants, especially diners with their massive choice of foods, suggest that people often stick with the same foods, no matter how big the choice. Indeed, the notion of paradox of choice has been raised to describe the observation that the larger the choice, the more conservative people become, often reverting to their favorite [19].

When we narrow our focus from the world of foods to the world of pizza we turn from a world of products people may or may not like to a world of product variations of what we might be to be a basically acceptable product. Indeed, for the most part people assume that pizza is a universally loved product, perhaps one of the most beloved in the world.

The data from this study suggest that the world of pizza comprises a universe until itself. The data suggest that there is strong differentiation among the different aspects of pizza, the strong differentiation appearing in issues involving the ingredients and flavors, less so in issues involving emotion and consumption situation.

The Pizza Study in the Context of Mind Genomics and the It! Studies

When the study was run two decades, in 2002, the ingoing assumption was that we would naturally uncover country to country differences in what people liked in pizza, especially the flavors of the pizza. It was intuitively obvious that we would not find a simple linkage between country and preference for pizza flavor. That would be too easy, too deceptive, even though one of thinks of countries in terms of ‘general preferences’, based upon the cuisines they offer. With pizza, the ingoing assumption was that we would find groups of respondents with clearly different mind-sets, different tastes as reflected in their responses to the different elements describing pizza [20], and then use these patterns of preferences to create new foods.

The structure of the worked-up data, after an interval of two decades, presents a working model of how thinking underlying Mind Genomics has evolved. The original efforts in Mind Genomics, represented most faithfully in the parallel studies known as the It! research, appeared to stop at the remarkable discovery that across products and countries, three different ‘canonical’ mind-sets would emerge, especially for foods and beverages. These mind-sets were the Traditionalists who wanted things the way they had always been; the Experientials ls who responded strongly to the description of emotions and situations; and finally the Elaborates who respondent most strongly the fancified description of the product features [13]. At the time of the initial analyses, the recurring pattern across foods sufficed excite. The patterns seemed repetitive, and worthy of report.

Over the years, however, as the data from the It! studies led to practical applications, the applications themselves opened up new issues, such as discovering relevant but numerically small groups of what would be important respondent groups. The solutions, clustering the data to smaller and smaller groups, began to provide business answers, such as the discovery of key groups. At the same, the dynamics suggested that such groups might be found in flavor, but not necessarily in packaging, and so forth. It was the demand for understanding the data in a deeper fashion which has led to the reanalysis of data, a reanalysis eminently possible because of the tight, comprehensive, balanced structure of the underlying permuted experimental design. This paper may be seen as a continuation of the early effort, using the same data, but with the experience and insight of two decades, along with the realization that these It! studies were stepping forth onto a new continent, with new horizons. This paper is a progress report, appearing two decades later.

Acknowledgment

The author acknowledges the efforts and inputs of his colleagues of two decades ago, Pieter Aarts of Belgium, and Klaus Paulus of Germany and the contribution of the late Hollis Ashman of the Understanding and Insight Group. The studies reported here were done under the aegis of It! Ventures, Inc., and are published with permission of It! Ventures, Inc.

References

  1. Luce RD, Krumhansl CL (1988) Measurement, scaling, and psychophysics. Stevens’ handbook of experimental psychology 1: 3-74.
  2. Peter JP, Olson JC, Grunert KG (1999) Consumer behaviour and marketing strategy (pp. 329-348). London, UK, McGraw-hill.
  3. Mead R (1990) The design of Experiments: Statistical Principles for Practical Applications. Cambridge University Press.
  4. Rokach L, Maimon O (2005) Clustering methods. In Data Mining and Knowledge Discovery Handbook (pp. 321-352). Springer, Boston, MA.
  5. Moskowitz HR, Papajorgji P, Wren J (2020) Mind Genomics and the Law, Chapter 5, Arson & Murder. Lambert Publications, Germany.
  6. Rubin H, Craig Wallace, Darry Rawlings (Trupanion senior management) Personal communication to Howard Moskowitz and Stephen Onufrey, June 2010).
  7. Moskowitz HR (2014) Mind Genomics and Texture: The experimental science of everyday life. In: Food Texture design and optimization (ed.Dar, Y.L. and Light, J.M) Helstosky C (2008) Pizza: a global history. Reaktion books.
  8. Jaitly M (2004) Symposium 4: Food Flavors: Ethnic and International Taste Preferences: Current Trends in the Foodservice Sector in the Indian Subcontinent. Journal of Food science 69: SNQ191-SNQ192.
  9. Kumar R (2015) A comparative study between Pizza Hut and Domino’s Pizza. International Journal of Marketing and Technology 5: 89-123.
  10. Lestari AD, Baktiono A, Wulandari A (2020) The effect of market segmentation strategy on purchasing decisions of pizza in Surabaya. Quantitative Economics and Management Studies 1: 1-8.
  11. Gofman A, Moskowitz H (2010) Isomorphic permuted experimental designs and their application in conjoint analysis. Journal of sensory studies 25: 127-145.
  12. Moskowitz MR., Ashman H, Minkus-McKenna D, Rabino S, Beckley JH (2006) Data basing the shopper’s mind: approaches to a ‘Mind Genomics’. Journal of Database Marketing & Customer Strategy Management 13: 144-155.
  13. Moskowitz HR, Gofman A (2007) Selling Blue Elephants: How to Make Great Products that People Want before They Even Know They Want them. Pearson Education.
  14. Aarts P, Paulus K, Beckley J, Moskowitz HR (2002) September. Food craveability and business implications: The 2002 EuroCrave™ database. In ESOMAR Congress, Barcelona, Spain.
  15. Likas A, Vlassis N, Verbeek JJ 2(003) The global k-means clustering algorithm. Pattern Recognition 36: 451-461.
  16. Fishbach A, Ratner RK, & Zhang Y (2011) Inherently loyal or easily bored?: Nonconscious activation of consistency versus variety seeking behavior. Journal of Consumer Psychology 21: 38-48.
  17. Zeithammer R, Thomadsen R (2013) Vertical differentiation with variety-seeking consumers. Management Science 59: 390-401.
  18. Schwartz B (2004) January. The Paradox of Choice: Why More is Less. New York: Ecco.
  19. Gere A, Moskowitz H (2021) Assigning people to empirically uncovered mind-sets: a new horizon to understand the minds and behaviors of people. Consumer‑based new product development for the food industry. Royal Society of Chemistry 132-149.