Spatial and temporal changes in the expression of steroid hormone receptors in mouse model of endometriosis

Purpose Endometriosis is recognized as a steroid hormone-dependent disorder. However, controversies exist regarding the status of the steroid hormone receptor expression in endometriotic tissues. The purpose of this study was to determine the ontogeny of cellular changes in the expression of estrogen receptors (ERα, ERβ), G protein-coupled estrogen receptor 1 (GPER1), and progesterone receptors (PRs) in endometriosis using a mouse model.

We used the autologous uterine tissue transfer mouse model and studied the mRNA and protein expression of ERα, ERβ, GPER1, and PR in ectopic lesions at 2, 4, and 8 weeks of induction of endometriosis.

As compared to endometrium of controls, in the ectopic endometrium, ERα is reduced while ERβ was elevated in stromal cells; however, Gper1 and PR levels are reduced in both stromal and epithelial cells in a time-specific manner. There is a high inter-animal variation in the levels of these receptors in ectopic endometrium as compared to controls; the levels also varied by almost 100-fold within the same lesion resulting in “micro-heterogeneity.” The expression of all these receptors also deferred between two lesions from the same animal.

In the endometriotic tissue, there is extensive inter-animal and intra-lesion heterogeneity in the expression of ERα, ERβ, GPER1, and PR. These changes are not due to the influence of the peritoneal environment but appear to be tissue intrinsic. We propose that the variable outcomes in hormonal therapy for endometriosis could be possibly due to heterogeneity in the expression of steroid hormone receptors in the ectopic endometrium. Electronic supplementary material The online version of this article (10.1007/s10815-020-01725-6) contains supplementary material, which is available to authorized users.

Endometriosis, Steroid hormone receptors, GPER1, Estrogen receptor, Progesterone receptor, Mouse, Heterogeneity

Endometriosis is a chronic benign gynecological disorder that results from the growth of endometrial glands and stroma outside the uterine cavity. Globally, ~ 17% of women are estimated to suffer from endometriosis [1]. The symptoms of endometriosis include chronic pelvic pain, dysmenorrhea, dyspareunia, and peritoneal inflammation. Among the various possible therapeutic targets, the receptors of the ovarian steroids (estrogen and progesterone) have taken a central stage. This is because steroid hormones play a key role in maintaining endometrial physiology and are proposed to be involved in the pathogenesis of endometriosis [2–6]. Studies have shown that endometriosis is associated with the increased local synthesis of estrogen, and the response to progesterone is blunted [4, 5]. Based on these observations, anti-estrogens, estrogen receptor, progestins, and progesterone receptor modulators are evaluated for the therapeutic activity in endometriosis [2, 7, 8]. However, clinical observations have shown that nearly one-third of women with endometriosis do not respond to combined oral contraceptives and progestin-based therapies [7]. These therapies also have multiple side effects and often there is a recurrence of endometriosis upon cessation of therapy [2, 7, 8]. Presently, the reasons for the ineffectiveness of these therapies in a subset of women with endometriosis are unknown. Since estrogen and progesterone or their agonist/antagonist act via their receptors in the target tissue, it is possible that the expression of steroid hormone receptors may be altered in endometriosis. Estrogen acts on the target tissue via their receptors mainly the estrogen receptors (ERs) viz. estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ). In the normal endometrium, ERα is hormonally regulated and required for the proliferation of endometrial cells; ERβ is constitutively expressed and has anti-proliferative and inflammatory functions [9–12]. Studies have assessed the expression of both ERα and ERβ in endometriotic lesions; however, the results are inconsistent and often contradictory [13–19]. Few studies have shown lower expression of ERα in endometriotic tissues as compared to endometrium from women without endometriosis or the eutopic endometrium from the same women [6, 15, 16]; others have shown no difference or a higher expression of ERα in the ectopic endometrium [17, 18]. Similarly, some studies have reported higher levels of ERβ in endometriotic tissues [6, 15, 18]; other studies have failed to confirm these findings [14, 19]. Beyond the classical ERs, estrogen also mediates its biological effects through non-canonical mechanisms involving the G Protein-Coupled Estrogen Receptor 1 (GPER1) [20, 21]. In the endometrium, GPER1 is predominantly expressed in the epithelial cells and is thought to regulate cell proliferation [10, 20, 21]. Limited information is available regarding the expression of Gper1 in endometriosis. As compared to controls, expression of GPER1 is elevated in epithelial cells of both eutopic and ectopic endometrium; this increase is observed only in 50–60% of cases, and in the remaining cases, expression is lower than in controls [15, 22, 23]. However, two studies reported elevated expression of GPER1 in stromal cells of ectopic endometrium as compared to the endometrium of controls [15, 22]; other studies failed to detect any significant changes in the expression of GPER1 in stromal cells of women with endometriosis [24, 25]. Functionally, stimulation of GPER1 increases proliferation in endometriotic epithelial cells [26]; however, in the stromal cells, GPER1 agonist inhibits proliferation and induces apoptosis [27]. Thus, the involvement of GPER1 in the pathogenesis of endometriosis is unclear. Like the ERs, controversies exist regarding the expression profiles of progesterone receptors (PR) in endometriosis. In the normal endometrium, PR plays a key role in the attainment of receptivity for embryo implantation and decidualization [9, 28]. In the ectopic endometrium of women with endometriosis, the expression of PR is downregulated according to some studies [29, 30]; others have reported their levels to be unchanged or even increased [15, 31]. However, it is believed that the response to progesterone is generally reduced in endometriosis [30, 32–34]. Thus, it is evident that there is a significant discrepancy in the patterns of steroid hormone receptor expression profile in different studies. Such disparities across studies could be due to differences in patient selection criteria, stage, or type of endometriosis evaluated and method of detection [33, 34]. Further, it must also be noted that in the normal endometrium, changes in the expression of steroid hormone receptors during the cycle are cell type-specific [10, 11, 21, 28, 35]; however, in most studies of endometriosis, total changes in the expression of the steroid hormone receptors are measured, and cell type-specific changes (if any) are unknown. It is possible that there might be spatial changes in the expression of steroid hormone receptors in ectopic endometrium during the progression of endometriosis. However, cell type-specific changes (if any) during the time course of endometriosis development and disease progression have not been well documented. In the present study, we aimed to determine the spatial changes in the expression of steroid hormone receptors in the ectopic endometrium during the course of endometriosis. Since time-bound longitudinal studies are not possible in humans, we herein chose the autologous uterine tissue transfer mouse model that resembles many features of human endometriosis [36–38]. We examined the expression profiles of ERα, ERβ, GPER1, and PR to gain an insight into the ontogeny of steroid hormone receptor dysregulation in endometriosis.

Animal ethics and animals This study was approved by the Institutional Animal Ethics Committee (IAEC) of the National Institute for Research in Reproductive Health (NIRRH), project number (05/12). Three- to four-month-old, regularly cycling C57BL/6 strain female mice were used for the study. Experimental tissue collection Endometriosis was surgically induced in the female mice as described previously with minor modifications [36]. In brief, 12-week-old female mice were anesthetized, one horn of the uterus was excised, and ~ 1 mm tissue piece was ligated onto the intestinal mesentery of the same animal. Mice were sacrificed after 2, 4, and 8 weeks of surgery either in estrus or diestrus stage as judged by the vaginal cytology [39]. To achieve this, surgery was performed in a cohort of animals and monitored for the stage of cycle on the day of sample collection; those animals in diestrus/estrus were used, and the rest of the animals continued till the next time point. Tissue from five animals in estrus and five in the diestrus stage were used for RNA extraction. For immunohistochemistry, tissues from diestrus stage animals (n = 5) were used. In a separate experiment, two fragments were separately ligated in 10 animals and lesions were collected at 8 weeks post-surgery and both the lesions were processed individually. As controls (n = 5), we used uterus from the stage and age-matched animals. On the day of sample collection, tissue attached to intestinal mesentery was excised upon laparotomy and either collected in TRIzol reagent (Invitrogen; CA, United States) for RNA extraction or fixed in 4% paraformaldehyde (Sigma Aldrich; Missouri, United States) for immunostaining. RNA extraction and complementary DNA synthesis Total RNA was extracted and reverse transcribed using random hexamers as described previously [40, 41]. Quantitative real-time polymerase chain reaction The messenger RNA (mRNA) levels for estrogen receptor 1 (Esr1), estrogen receptor 2 (Esr2), Gper1, and progesterone receptor (Pgr) were estimated by qPCR. The levels of 18s rRNA were used for normalization. qPCR reactions were done in duplicates using SYBR green chemistry (Bio-Rad, California, USA) in the iCycler Real-Time PCR System (Bio-Rad) as detailed previously [40, 41]. The primer sequences, their annealing temperatures, and expected product sizes are given in Supplementary Table 1. For each primer pair, the efficiency was estimated using 10-fold serial dilution of cDNA; the homogeneity of the PCR amplicons was verified by the melt curve method. Relative expression levels were calculated using the Pfaffl method as detailed previously [41]. Immunohistochemistry To study the spatial changes in levels of ERα, ERβ, GPER1, and PR, immunohistochemistry was performed as described previously [40, 41]. Briefly, 5-μm-thick sections were deparaffinized and antigens were retrieved using Tris-EDTA Buffer (pH 9). Blocking was done in 5% bovine serum albumin (MP Biomedicals; Maharashtra, India), and the sections were probed overnight with primary antibody diluted in phosphate buffer saline (PBS) at 4 °C. Negative controls were incubated with PBS instead of the primary antibody. The next day, slides were washed three times in PBS, and detection was done using the horse radish peroxidase (HRP) streptavidin method (ABC kit Santa Cruz Biotechnology; Texas, USA). All sections were counterstained with hematoxylin and mounted. Slides were viewed under a bright-field microscope (Olympus; Tokyo, Japan), and representative areas were photographed. The sources and the concentrations of primary antibodies used in the study are given in Supplementary Table 2. Quantification of immunostaining The intensity of the chromogen was quantified by the “Fiji” version of ImageJ software [42]. To detect the total amount of the receptors, at least three random sections were analyzed per lesion and the mean value was obtained to estimate the level of expression. To quantify cell-specific changes in immunostaining, five different areas of stroma and epithelia were selected randomly and quantified as above. To determine if there are any local changes in the expression of the steroid receptors within the tissue, ten random areas of three non-serial sections were selected and intensity was obtained as above. The intensity value of each area of each section of every lesion was plotted separately. Statistical analysis Data was calculated in terms of mean and standard deviation and statistically analyzed by Kruskal-Wallis nonparametric analysis of variance (ANOVA) test, followed by Dunn’s multiple comparison test in GraphPad Prism (Version 5, California). P value < 0.05 was accepted as statistically significant.

Kinetics of messenger RNA expression of steroid hormone receptors in ectopic endometrial lesions To assess the changes in expression of steroid hormone receptors, quantitative real-time PCR (qPCR) for steroid hormone receptor genes (Esr1, Esr2, Gper1, and Pgr) was performed on cDNA prepared from ectopic endometrial tissues at second, fourth, and eighth week post-surgery. Endometrium from animals in the same stage was used as control (n = 5 for each group both in estrus and diestrus) (Fig. 1). Estrogen receptor 1 and estrogen receptor 2 As compared to controls, irrespective of the stage of the estrous cycle, ectopic endometrial lesions had higher expression of Esr1 at all the time points tested but this increase was not statistically significant (Fig. 1a). Similarly, mRNA Levels of Esr2 were high at all the time points in ectopic lesions but this increase was statistically significant (P < 0.05) in ectopic lesions at the eighth week (Fig. 1b). Although the mean level of Esr1 was high, the increase was observed only in ~ 40% of animals while Esr2 was high in ~ 60% of animals; the levels were identical or lower than controls in other animals. G protein-coupled estrogen receptor 1 As compared to controls, Gper1 mRNA levels were low in endometriotic lesions at the second and fourth week which reached close to normal level in the eighth week (Fig. 1c). There was a high variation in the levels of Gper1 across biological replicates. This trend was observed in both the estrus and diestrus stage of animals. Progesterone receptor Mean levels of Pgr mRNA were identical between the endometrium of controls and endometriotic tissues at the second week. In endometriotic lesions at fourth week, Pgr mRNA levels were almost 10-fold lower than controls, while at the eighth week, the mean levels were higher than controls. The difference in the mRNA levels between endometriotic tissues and controls was statistically significant only in ectopic lesions at the fourth week (Fig. 1d). Irrespective of the time points or stage of the estrous cycle, there was high variation in the mRNA levels of all steroid hormone receptors across the biological replicates. This magnitude of variation was not observed among the controls. These variations also persisted when the levels of Gapdh were used instead of 18S rRNA for normalization (data not shown). Localization of steroid hormone receptors in ectopic endometrium Immunohistochemistry was performed to study localization and abundance of ERα, ERβ, GPER1, and PR proteins in endometriotic tissues at the second, fourth, and eighth week post-surgery in diestrus stage. For controls, endometrial tissues from diestrus stage animals were used (n = 5 for each group) (Fig. 2). Negative controls for each protein are shown in Supplementary Fig. 1. Estrogen receptor alpha ERα levels were low in endometriotic lesions at the second, fourth, and eighth week as compared to controls; however, this reduction was not statistically significant (Fig. 2a). In the epithelial cells, ERα levels were unchanged during the course of lesion development. In the stromal cells, the expression of ERα was reduced at all the time points; however, this reduction was significant only in the second week (Fig. 3a). Estrogen receptor beta The expression of ERβ significantly increased around 2–5-fold in the endometriotic tissues in the eighth week (Fig. 2b). In epithelial cells, the expression of ERβ was identical in controls and the endometriotic lesions. In stromal cells, levels of ERβ were higher by 1.5–3-fold in ectopic endometrium as compared to controls, but this increase was not statistically significant (Fig. 3b). G protein-coupled estrogen receptor In the endometrium of control mice, GPER1 staining was strong in the epithelium and weak in the stroma. As compared to control, the intensity of GPER1 was lower in the endometriotic tissues at all the time points tested (Fig. 2c). There was a ~ 5–10-fold reduction in the levels of GPER1 in endometriotic tissues both in epithelium and stroma at all the time points. However, a statistically significant reduction was noted only in the second week in the epithelial cells. In the stromal cells, levels of GPER1 were significantly lower at the second and eighth week as compared to controls (Fig. 3c). Progesterone receptor In the ectopic endometrium, the intensity of PR staining was low when compared to controls. Quantitatively, the expression of PR was almost 5–10-fold low in endometriotic tissues at all the time points (Fig. 2d). In epithelium, expression of PR was similar to controls in the ectopic endometrium at second week which declined significantly at the fourth and eighth week while in the stroma, and the expression of PR was low at all the time points but statistically significant only at the fourth week post-surgery (Fig. 3d). Micro-heterogeneity in the expression of steroid hormone receptors in ectopic endometrial lesion During the immunostaining quantification in Fig. 2, we observed that the expression of steroid hormone receptors was highly heterogeneous across the sections. We quantified the intensity of immunostaining in 10 random areas of non-serial sections of endometriotic tissues obtained at the eighth week post-surgery and represented the data individually (Fig. 4). In the controls, the expression of ERα, ERβ, GPER1, and PR was uniform across the tissues with minimal inter-sample variability. However, there was high variability in the intensity of staining in different areas of the same sections obtained from endometriotic tissue. For both ERα and ERβ, the expression in some areas of the endometriotic tissue sections was more than controls; in other areas of the same sections, the expression was lower than controls (Fig. 4a, b). Similarly, the expression of GPER1 and PR was also heterogeneous across the sections obtained from ectopic tissues (Fig. 4c, d). The differences in the expression of these receptors were almost of 100 orders in magnitude within the same lesion. These differences were seen in all three biological replicates. This heterogeneity is specific to steroid hormone receptors as the expression of both cytokeratin (epithelial marker) and vimentin (stromal marker) was found to be uniformed across the entire section of the endometriotic lesion (Supplementary Fig. 2). Tissue autonomous regulation of steroid hormone receptors in ectopic endometrial lesion To test if the changes and heterogeneity in the expression of steroid hormone receptors observed in endometriotic tissues is due to alteration in the peritoneal environment or is independent lesions, two pieces of endometrial tissue were ligated separately and the comparisons were made in the paired tissue obtained from the same animal (Figs. 5 and 6). Estrogen receptors In 6/7 animals, discordance was observed in the levels of Esr1 between the two lesions obtained from the same animal. There was a 5–10-fold difference in the expression of Esr1 between the two endometriotic tissues (Fig. 5a). The protein product of Esr1 (ERα) was also discordant between the two lesions obtained from the same animal (n = 3); the differences were in the range of 1.5–5-fold (Fig. 6a). The expression of Esr2 was discordant between the two lesions in all the seven mice; in 3/7 mice, there were more than 10-fold differences in the expression of Esr2 (Fig. 5b). At the protein level, ERβ expression was also found to be highly discordant between the two lesions obtained from the same animal. This discordance was observed in all three biological replicates (Fig. 6b). The expression of Gper1 was discordant between the two lesions in 6/7 mice; the difference was more than 5-fold (Fig. 5c). GPER1 protein expression was also discordant between the two lesions with differences in the range of 1.5- to 10-fold in all three biological replicates (Fig. 6c). Progesterone receptor In 5/7 animals, the expression of Pgr was discordant between two lesions from the same animal; in 2/7 animals, expression was similar in both the lesions. In three animals, the difference was almost 10-fold while in the other two animals, the difference was more than 100-fold (Fig. 5d). Protein levels of PR were also found to be discordant between the two lesions obtained from the same animal (n = 3); variation was in the range of 1.5–3-fold (Fig. 6d).

In the present study, we demonstrate a dysregulation in the expression of steroid hormone receptors (ERα, ERβ, Gper1, and PR) in cell type and time-specific manner during the course of endometriosis development in mice. Within the endometriotic lesions, this dysregulation is not uniform and there is extensive micro-heterogeneity in the expression of steroid hormone receptors. The dysregulation in the expression of ERα, ERβ, Gper1, and PR is not due to changes in the peritoneal environment but appears to be independently occurring in the ectopic lesions. Steroid hormones mainly estrogen and progesterone are crucial for endometrial functions and also play a role in the pathogenesis of endometriosis [12, 13, 28–35, 43–45]. Studies have shown that the systemic levels of estrogen are not significantly different in women with and without endometriosis, but estrogen and ERs are essential for the induction of endometriosis [12, 37, 43–45]. However, controversies exist regarding the expression profiles of estrogen receptors in endometriosis. Few studies have shown the increased expression of both ERα and ERβ in the ectopic endometrium of women with endometriosis while others have shown the reduced expression of both ERα and ERβ in ectopic endometrium [16, 33, 34]. In the case of non-canonical estrogen receptors, most of the studies have reported increased expression of GPER1 [15, 22, 26, 27] and lower levels of PR in the endometrium of women with endometriosis [29, 30]. However, there is high variability in the results of different studies and presently, there is no census on the status of these receptors in endometriotic tissues. It is suggested that differences in study design, patient selection criteria, stage of cycle, and type of endometriosis could be the possible reasons for such discrepant findings. To address these issues, we chose to determine if steroid hormone receptors are altered in experimental endometriosis where the confounding variables are minimized. We used the autologous uterine transplantation mouse model and profiled the mRNA and cognate protein levels of steroid hormone receptors during the course of lesion development. The results show that the mRNA and protein levels of ERα, ERβ, GPER1, and PR are altered in the ectopic tissues; these changes are not significant due to high inter-animal variability. Irrespective of the stage of the estrous cycle, some animals show higher than normal expression while others show reduced expression in the ectopic lesions as compared to controls. Similar inter-individual variability in the expression of ERα, ERβ, GPER1, and PR is reported in human endometriosis [15, 17, 30, 33]. Beyond the inter-individual variability, immunostaining of the endometriotic tissues revealed that the expression of these receptors changes spatially during the course of lesion development. ERα expression is unchanged in epithelial cells; it is reduced in stromal cells during the early stages of endometriosis that reverts near to normal at later stages. In contrast, ERβ expression is increased only in the stromal cells from early time points while the epithelial expression is maintained. The expression of GPER1 and PR is reduced in endometriosis; this reduction is observed in both epithelial and stromal cells at most of the time points. However, this difference is not statistically significant at all the time points as there is high heterogeneity across the biological replicates. These observations imply that the alterations occurring in the expression of steroid hormone receptors in the endometriotic tissues are not only cell type-specific but also highly heterogeneous in the population. Since these alterations are independent of age, stage of the estrous cycle, or time scale of endometriosis development, it appears that inter-individual heterogeneity in the expression of steroid hormone receptors is an intrinsic characteristic of the disease. In addition to high inter-sample and cell type-specific variability, quantitative analysis of different regions of the same tissue revealed that the expression of ERα, ERβ, GPER1, and PR was very high in certain areas of the endometrial lesions while it was almost absent in other areas. We termed this intra-lesion variability as micro-heterogeneity and quantified it across the biological replicates. The results revealed that irrespective of cell type, the expression of all these proteins varies as much as 100-fold between adjacent areas of the same tissue in all the endometriotic lesions examined but not in the normal endometrium. This micro-heterogeneity is specific to steroid hormone receptors as the expression of cytokeratin and vimentin were uniform in the same section (Supplementary Fig. 2). A similar micro-heterogeneity in the expression of PR has also been reported in human endometriotic lesions [30]. These results imply that along with time- and cell type-specific alteration, micro-heterogeneity in the expression of steroid hormone receptors is an inherent characteristic of endometriosis. It is known that as compared to normal women, the peritoneal environment is altered in women with endometriosis [46, 47]. Few studies have suggested that alterations in the peritoneal environment such as inflammation may be a cause of differential expression of steroid hormone receptors in endometriotic tissues as compared to normal endometrium [48, 49]. To test if the peritoneal environment is the cause of altered steroid hormone receptors in the endometrium, we ligated two fragments of uterine tissue juxtaposed to each other and studied them separately after 8 weeks of induction of endometriosis. The hypothesis was that if the peritoneal environment is the cause of alteration of steroid hormone receptors, both the lesions must show similar changes in the pattern of expression as compared to normal endometrium. The results revealed that the levels of the ERα, ERβ, GPER1, and PR varied as much by 100 orders of magnitude between paired lesions taken from the same animal. These observations imply that the alteration in steroid hormone receptors in ectopic endometrium is not due to a systemic effect like inflammation in peritoneum, but it appears to be independently regulated in ectopic lesion. The non-uniform alterations in the expression of steroid hormone receptors in different ectopic lesions from the same animal prompt us to suggest that one has to exert caution while interpreting the molecular phenotypes of ectopic lesions in women with endometriosis, and more than one lesion needs to be tested before drawing major conclusions. What is the cause of such heterogeneity in endometriotic tissues is difficult to ascertain. Cell type and time-dependent changes in DNA methylation of the steroid hormone receptor genes has been reported in women with endometriosis [50]. Abnormal proteolysis of steroid hormone receptor chaperons has also been observed in the ectopic endometrium of women with endometriosis [51, 52]. Whether these could result in the observed heterogeneity in the expression of ERα, ERβ, GPER1, and PR needs to be investigated. The high variability in the spatial and temporal expression of steroid hormone receptors implies that the sensitivity of the endometriotic tissue of exogenous steroid hormones may not be uniform. Indeed, several systematic reviews including the Cochrane studies have observed that despite high-quality study design, there is inconsistency in the outcomes of steroidal therapies between different studies [8, 53, 54]. A recent study has also shown that the differential response to progestin therapy in women with endometriosis directly correlates with the levels of PR in the lesions [30]. Thus, it is plausible that temporal, spatial, and lesion-specific differences in the expression of steroid hormone receptors coupled with lesion- specific micro-heterogeneity may alter the effectiveness of steroid hormone analogs resulting in variable outcomes of combined oral contraceptives and progestin-based therapies in women with endometriosis.

From the result of the present study, we conclude that there is high spatial, temporal, and inter-lesion variability in the expression of steroid hormone receptors in the ectopic endometrium. Such heterogeneity in the expression of steroid hormone receptors in ectopic lesions provides a plausible explanation for the failure of first-line medical therapies in a subset of women with endometriosis. Further, studies will be needed to understand the cause of steroid hormone receptor dysregulation for developing rational management strategies for endometriosis. Electronic supplementary material Acknowledgments We express our gratitude to Dr. Stacy Colaco and Mr. Abhishek Tiwari for help during the preparation of the manuscript. We thank Dr. Uddhav Chaudhari and the staff of the Animal House (NIRRH) for their help during surgeries and animal maintenance. Funding information The manuscript bears the NIRRH ID: RA/756/03–2019. DM lab is funded by grants from ICMR, Govt of India. The study was funded by grants from the Department of Biotechnology (DBT); (BT/OR6587/MED/30/886/2012) and Department of Science and Technology (SR/SO/HS-0277/2012) Govt of India to DM. AM is a recipient of the junior and senior research fellowship from the University Grants Commission (UGC), Govt of India. MG was the recipient of the ICMR postdoctoral fellowship (6thBatch). NS is a recipient of the senior research fellowship from ICMR. Compliance with ethical standards This study was approved by the Institutional Animal Ethics Committee (IAEC) of the National Institute for Research in Reproductive Health (NIRRH), project number (05/12). Conflict of interest The authors declare that there is no conflict of interest. Footnotes Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Anuradha Mishra and Mosami Galvankar contributed equally to this work.

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