FSH Receptor Expression Research Articles

Mechanism of negative modulation of FSH signaling by salt-inducible kinases in rat granulosa cells.

The optimal development of preovulatory follicles needs follicle-stimulating hormone (FSH). Recent findings revealed that salt-inducible kinases (SIKs) inhibit FSH actions in humans and rodents. This report seeks to increase our understanding of the molecular mechanisms controlled by SIKs that participate in the inhibition of FSH actions in primary rat granulosa cells (GCs). The results showed that FSH causes a transient induction of Sik1 mRNA. In contrast, SIK inhibition had no effects on FSH receptor expression. Next, we determined whether SIK inhibition enhances the effect of several sequential direct activators of the FSH signaling pathway. The findings revealed that SIK inhibition stimulates the induction of steroidogenic genes by forskolin, cAMP, protein kinase A (PKA), and cAMP-response element-binding protein (CREB). Strikingly, FSH stimulation of CREB and AKT phosphorylation was not affected by SIK inhibition. Therefore, we analyzed the expression and activation of putative CREB cofactors and demonstrated that GCs express CREB-regulated transcriptional coactivators (CRTC2) and that FSH treatment and SIK inhibition increase the nuclear expression of this factor. We concluded that SIKs target the FSH pathway by affecting factors located between cAMP/PKA and CREB and propose that SIKs control the activity of CRTC2 in ovarian GCs. The findings demonstrate for the first time that SIKs blunt the response of GCs to FSH, cAMP, PKA, and CREB, providing further evidence for a crucial role for SIKs in regulating ovarian function and female fertility.

Association between cumulus cells—mRNA levels of AMHR2 and FSHR with oocyte maturity

BackgroundThe gene expression of anti-Mullerian hormone receptor type 2 (AMHR2) and follicle stimulating hormone receptor (FSHR) in cumulus cells (CCs) isolated from mature and immature oocytes was studied as a possible non-invasive approach for determining oocyte maturity and quality. The CCs of 100 infertile women with different etiologic factors were subdivided into control (CCs from MII) and case (CCs from GV) group. Q-PCR was used to evaluate FSHR and AMHR2 mRNA expression levels in CCs from mature and immature oocytes.ResultsAMHR2 and FSHR genes are significantly overexpressed (4–6 fold) in CCs from immature relative to mature oocyte. The expression level of AMHR2 gene in terms of etiologic subgroups is significantly different (P value 0.000). FSHR mRNA expression levels in CCs show no significant difference regarding etiologic subgroups (P value 0.575).ConclusionIt seems that determining the expression level of AMHR2 and FSHR genes in CCs could help to understanding molecular mechanism of oocyte maturation process.

Effects of Different Energy Diets on FSHR mRNA Expression and DNA Methylation in Promoter Region of Duolang Sheep During Diestrus.

The aim of this study was to study the relationship between the methylation level of the promoter region of follicle-stimulating hormone receptor (FSHR) gene and the mRNA expression of Duolang sheep fed different energy diets. In this experiment, three groups of diets with different energy levels (dietary nutrient level reference (NY/T816 - 2004)) were designed to obtain medium energy level diets with metabolic energy of 10.88 MJ/d. The treatments with high and low energy levels increased and decreased by 15%, 12.51, and 9.25 MJ/d, respectively, on the basis of medium energy level. Total RNA and genomic DNA were extracted from the ovaries of Duolang sheep, and qPCR was performed. Bisulfite genomic sequencing PCR and sequence alignment were used to detect the relationship between the relative expression level of FSHR and methylation. The results showed that the expression of FSHR in high-energy group and medium-energy group was significantly higher than that in low-energy group (p < 0.01), and there was no significant difference between high-energy group and medium-energy group (p > 0.05). The methylation rate of the target fragment in the promoter region of FSHR gene was 41.67% in the high-energy group, 75.00% in the medium-energy group, and 83.33% in the low-energy group. This study revealed that different dietary energy levels had certain effects on DNA methylation level of FSHR gene and then affected gene expression, and the expression level was negatively correlated with methylation level.

P-666 Detecting gene variants correlated with MII oocytes developed during ovarian stimulation

Abstract Study question Is it possible to identify genetic factors influencing the number of MII oocytes produced in the course of ovarian stimulation? Summary answer Applying data exploration techniques made it possible to identify gene variants that markedly improved ability to predict the number of MII oocytes to be retrieved. What is known already There are well known clinical factors that correlate with the number of MII oocytes that can be retrieved as a result of ovarian stimulation. When deciding on an appropriate stimulation protocol and gonadotropin doses, clinicians mainly take into consideration patient’s age and selected markers of ovarian reserve. The choice of protocol and dosage itself influences the outcome of the stimulation. Certain genes have been shown to participate in the regulation of the expression of FSH, LG and HCG receptors although direct influence of genetic background on the number of MII oocytes still need to be elucidated. Study design, size, duration In the retrospective study, data collected between 05/08/2013 and 28/10/2020 was analyzed. The dataset consisted of 516 ovarian stimulations undergone by 264 patients as well as and 605 unique changes found in sequence data for 14 genomes (AMH, AMHR2, FSHB, FSHR, LHB, LHCGR, PRL, PRLR, AR, ESR1, ESR2, GDF9, BMP15, SOX3). Inclusion criteria set boundaries on women’s age (between 24 and 46 years) and Anti-Müllerian hormone results (AMH) (lower than 15). Participants/materials, setting, methods Neural Network model based on 7 statistically important clinical factors was trained to predict the number of MII oocytes and used as a benchmark for model including genetic factors. A model’s performance was evaluated with median of absolute error metric and validated on the test subset. Feature importance in the model was assessed using Shapley values. Important gene variants were selected using grouping algorithm Self Organizing Map and haplotype block generation algorithm Four Gamete Rule. Main results and the role of chance In the benchmark model, following clinical factors were included: AMH, antral follicle count on the first stimulation day (AFC), patient age, number of MII oocytes and denuded cumuluses obtained in the previous cycle (available for 37% of IVF processes), IVF protocol type and the presence of Polycystic ovary syndrome (PCOS). The trained model obtained median absolute error equal to 1,62. Out of all genetic data, 3 important variant combinations (haplotypes IV40-8, IV22-2 and a group of 6 variants labeled IV8-6) included in table 1 were identified. The addition of those 3 factors to the model resulted in a decrease in the median absolute error by 0.19 (12%). The average impact on the model output magnitude (mean SHAP) of genetic factors was greater than the impact of the chosen IVF protocol or the presence of PCOS. The presence of haplotypes IV40-8 and IV22-2 increased the expected number of MII oocytes, whereas the grater the number of variants grouped as IV8-6 found in the patient the lower the predicted number of MII oocytes. Additionally, if the patient’s previous cycle results were not given, the impact of genetic factors on the model magnitude was comparable to AFC on the first day of stimulation. Limitations, reasons for caution Considering the number of gene variants found (605) And the sample size of the study a broader study allowing for a better understanding of the influence of less frequently observed variants would be beneficial. Wider implications of the findings Research shows including gene factors in MII oocytes prediction could substantially increase the precision of MII oocyte predictions for patients and especially for patients undergoing ovarian stimulation for the first time. Knowing the expected MII oocytes count could help clinicians with deciding on gonadotropin dosing. Trial registration number Not applicable

Ovarian expression of follicle stimulating hormone and activin receptors genes in a prenatally-androgenized rat model of polycystic ovary syndrome in adulthood.

The expression of genes involved in basic pathways, such as folliculogenesis and steroidogenesis may be affected following prenatal androgen exposure. Besides, exposure to androgens during prenatal life plays a central role in developing polycystic ovary syndrome (PCOS) in females in later life. In the present study, we aimed to examine the expression of the follicle stimulating hormone receptor (FSHR) and activin receptor (actR) genes in ovarian granulosa cells (GCs) of a prenatally-androgenized rat model of PCOS in adulthood. In the adult rat model of PCOS and their controls (n = 8 in each group), different phases of the estrous cycle were determined by vaginal smear. Total RNA was extracted from the ovarian GCs using the TRIzol protocol, a reverse transcription kit was used for complementary DNA (cDNA) synthesis, and the expression of FSHR and actR genes was measured by SYBR-Green Real-Time PCR. GraphPad Prism was used for statistical analysis of data, and the t-Student'stestwasused to compare the results between the two groups. PCOS rats had longer and irregular estrous cycles compared to controls. The expression of FSHR and actR genes were significantly decreased in the rat model of PCOS compared to control rats. In PCOS rats, genes expression ratios for FSHR and actR were 0.91 ± 0.11 times (P = 0.008) and 0.42 ± 0.13 times (P = 0.048) less than controls, respectively. Reduced expression of the FSHR and actR genes in ovarian GCs may be one of the mechanisms mediating PCOS-related disorders, especially abnormal ovarian folliculogenesis and ovulation dysfunction, following exposure to androgens during fetal life.

Follicle stimulating hormone promotes production of renin through its receptor in juxtaglomerular cells of kidney

BackgroundPost-menopausal hypertension has been attributed solely to declining estrogen levels. The purpose of the research is to elucidate the mechanism by which follicle stimulating hormone(FSH) increases renin production involved in the regulation of blood pressure.MethodsThe expression of follicle stimulating hormone receptors (FSHRs) in renal juxtaglomerular cells and a As4.1 juxtaglomerular mouse cell line was evaluated. We established a mouse model by ovariectomy (OVX). Ovariectomized mice were treated with gonadotropin-releasing hormone agonist (GnRHa) (OVX + GnRHa). Ovariectomized mice initially received physiological doses of estrogen and were then injected with recombinant FSH (OVX + E + FSH).ResultsWe found that FSHR was expressed in mouse renal juxtaglomerular cells labeled by renin antibody and in As4.1 cells. FSH promoted renin synthesis via Gsα-coupled FSHRs that activated protein kinase A, cyclic adenosine monophosphate(cAMP) response element-binding protein, extracellular signal-regulated kinase (Erk1/2), Protein kinase B(AKT), and c-Jun N-terminal kinase signaling pathways in As4.1 cells. We found increased serum FSH levels in the ovariectomized mouse with concurrent increases in renin, angiotensin II, heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial blood pressure (MAP). Additionally, increases in serum renin, angiotensin II, HR, SBP, DBP, and MAP were reduced by the additional injection of GnRHa. Exogenous FSH administration completely reversed decreases in renin, angiotensin II, HR, SBP, DBP, and MAP even in mice that received physiological doses of estrogen to maintain normal estradiol levels.ConclusionsElevated FSH stimulates renin production involving a mechanism that may be relevant to the expression of FSH receptors in renal juxtaglomerular cells.

Differential expression of FSHR and LHR genes and proteins during development of rabbit ovarian follicles.

The development of an ovarian follicle is a complex process at the cellular and molecular level that is mainly regulated by follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR). To elucidate the contribution of these receptors to ovarian follicle development, it is necessary to determine their expression profiles during this biological process. Therefore, this study aimed to investigate the relationship between ovarian development pattern and the differential ovarian expression pattern of FSHR and LHR genes as well as proteins at different developmental stages. Ovaries were collected from 30 New Zealand rabbits at day 0 (birth), week 2 (neonate), week 4 (cub), week 16 (maturity), and day 18 pregnancy. Ovarian histology, and gene as well as protein expression were determined using light microscopy, real-time PCR and western blotting, respectively. The results showed that the expression levels of FSHR mRNA and protein increased coincidently with age and the growth of ovarian follicles. The levels of LHR mRNA and protein remained low from the day of birth until week 4 and became significantly higher by week 16 coinciding with appearance of growing and antral follicles, which have a defined thecal layer. FSHR gene and protein expression decreased with pregnancy, whereas LHR increased, reaching a peak level during pregnancy. It can be concluded that changes in FSHR and LHR gene and protein expression could be related to the growth and development of follicles, indicating the regulatory role for these receptors in rabbit folliculogenesis.

Relative expression profile of Kisspeptin (Kiss1-Kiss1r) and gonadotrophin receptor in the ovarian follicular tissue and their association in the Buffalo (Bubalus bubalis)

In the past decade, kisspeptin research was primarily focussed on the regulation of GnRH release in hypothalamus. Present study was designed to explore the expression of extra-hypothalamic kisspeptinergic (Kiss1- Kiss1r) system in the follicular compartment of buffalo ovary. Buffalo genitalia (n=32) were collected immediately after exsanguinations and categorized into early luteal (EL), mid luteal (ML), follicular (FL) and acyclic (n=8 per group), based on the gross ovarian morphology. Ovarian follicular tissue samples were subjected to total RNA extraction, cDNA synthesis and qPCR amplification of Kiss1, Kiss1r, follicle stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR) along with an endogenous control (β-actin) gene. The expression of ovarian Kiss1 transcripts was abundant in the cyclic than acyclic stage. The fold change was significantly upregulated in ML (66.79 fold) followed by EL (28.64 fold) and FL (14.09 fold) stages against the acyclic stage (calibrator). Similarly, the Kiss1r expression was highest at ML (77.26 fold). The expression of FSHR was upregulated at FL (15.08 fold) stage in response to follicular activity and subsequently observed to be down regulated at EL (0.09 times) and ML (0.27 times). Further, the expression of Kiss1 was positively correlated with FSHR only at ML and FL. From this study, it could be concluded that Kiss1 and Kiss1r are expressed in the buffalo ovarian follicle and their expression is associated with the stage of estrous cycle.

Secoisolariciresinol Diglucoside Improves Ovarian Reserve in Aging Mouse by Inhibiting Oxidative Stress.

Ovarian reserve is a key factor in the reproductive function of the ovaries. Ovarian aging is characterized by a gradual decline in the quantity and quality of follicles. The underlying mechanism of ovarian aging is complex and age-related oxidative stress is considered one of the most likely factors. Secoisolariciresinol diglucoside (SDG) has been shown to have good scavenging ability against reactive oxygen species (ROS) which slowly accumulates in ovarian tissues. However, it is unknown whether SDG had beneficial effects on aging ovaries. In this study, we used 37-week-old female C57BL/6J mouse as a natural reproductive aging model to evaluate the role of SDG in ovarian aging. SDG (7 and 70 mg/kg) intragastric administration was performed in the mice daily. After 8 weeks, the effects of SDG on aging ovaries were evaluated by counting the number of follicles and the expression of follicle-stimulating hormone receptors (FSHR) in the ovary. The mechanism of SDG on the aging ovaries was further explored through ovarian metabolomics. It was found that SDG can effectively increase the number of growing follicles and increase the expression of the FSHR protein. The metabolomics results showed that the ovaries in the SDG intervention group achieved better uptake and transport of nutrients, including amino acids and glucose that are necessary for the development of oocytes. At the same time, the ovaries of the SDG intervention group showed that the drug reduced ROS generation. Additionally, we found that ovarian telomere length and ovarian mitochondrial DNA copy number that are highly susceptible to ROS damage and are also related to aging. The results showed that SDG can significantly increase mitochondrial DNA copy number and slow down the process of telomere shortening. These data indicate that SDG improves ovarian reserve by inhibiting oxidative stress.

Evaluating effect of acrylamide and ascorbic acid on oxidative stress and apoptosis in ovarian tissue of wistar rat

Acrylamide is one of the undesirable compounds created in food, which leads to oxidative stress. Under normal conditions of the body, there is a balance between the production and elimination of free radicals. Imbalance in this process causes oxidative stress. Ascorbic acid has antioxidant properties and can be used to prevent oxidative stress damage. In this study, we considered the effect of acrylamide as a substance that causes oxidative stress and ascorbic acid as an antioxidant. In this experiment, 28 rats were separated into 4 groups (n = 7). Mice were fed orally with acrylamide (10 mg/kg), ascorbic acid (200 mg/kg), both acrylamide and ascorbic acid, and water as AR, AA, AR&AA, and control groups, respectively. Blood and ovarian tissue samples were collected after the final feeding and anesthesia for hematological tests. Blood cells, anti-oxidation status and relative expression of BAX (Bcl-2 Associated X-protein), BCL-2 (B-cell lymphoma-2), Folliculogenesis Specific BHLH Transcription Factor (FIGLA), Follicle Stimulating Hormone Receptor (FSHR), and Klotho (KL) genes were assessed and compared between the study groups. Despite no change in the levels of other factors, white blood cells were significantly different between all groups. The total oxidant and antioxidant index had significant changes in the AR group compared to controls. Glutathione Peroxidase showed the least concentration in the AR group than others although this change was not significant. Gene expression of BAX, BCL-2, FIGLA, FSHR, and KL genes was not significant between the study groups (P > 0.05). The most ratio of BAX to BCL-2 belonged to the AR group compared to other groups. In conclusion, AR probably induces ovarian dysfunction by increasing the proportions of apoptosis-related genes, and the usage of antioxidants like AA can minimize its hazardous effects. However, more research is needed to uncover effective ways to limit AR exposure.

The Role of Inactivated NF-κB in Premature Ovarian Failure

Premature ovarian failure (POF) is defined as deployment of amenorrhea due to the cessation of ovarian function in a woman younger than 40 years old. The pathologic mechanism of POF is not yet well understood, although genetic aberrations, autoimmune damage, and environmental factors have been identified. The current study demonstrated that NF-κB inactivation is closely associated with the development of POF based on the data from literature and cyclophosphamide (Cytoxan)-induced POF mouse model. In the successfully established NF-κB-inactivated mouse model, the results showed the reduced expression of nuclear p65 and the increased expression of IκBα in ovarian granulosa cells; the reduced numbers of antral follicles; the reduction of Ki-67/proliferating cell nuclear antigen-labeled cell proliferation and enhanced Fas/FasL-dependent apoptosis in granulosa cells; the reduced level of E2 and anti-Müllerian hormone; the decreased expression of follicle-stimulating hormone receptor and cytochrome P450 family 19 subfamily A member 1 (CYP19A1) in granulosa cells, which was reversed in the context of blocking NF-κB signaling with BAY 11-7082; and the decreased expressions of glucose-regulated protein 78 (GRP78), activating transcription factor 6, protein kinase R-like endoplasmic reticulum kinase, and inositol-requiring enzyme 1 in granulosa cells. Dual-luciferase reporter assay demonstrated that p50 stimulated the transcription of GRP78, and NF-κB affected the expression of follicle-stimulating hormone receptor and promoted granulosa cell proliferation through GRP78-mediated endoplasmic reticulum stress. Taken together, these data indicate, for the first time, that the inactivation of NF-κB signaling plays an important role in POF.

TRIB3 regulates FSHR expression in human granulosa cells under high levels of free fatty acids

BackgroundGranulosa cells (GCs) in cumulus oophorus highly express follicle stimulating hormone receptor (FSHR), which is the most important mediator of both estradiol synthesis and oocyte maturation. Obese women have elevated free fatty acids (FFAs) levels in their follicular fluids and decreased FSHR expression in GCs, which is related to an altered protein kinase B/glycogen synthase kinase 3β (Akt/GSK3β) signaling pathway. Such FFA increases accompany 3-fold rises in pseudokinase 3 (TRIB3) expression and reduce the Akt phosphorylation status in both the human liver and in insulinoma cell lines. Therefore, in a high FFA environment, we determined if TRIB3 mediates regulation of FSHR via the Akt/GSK3β signaling pathway in human GCs.MethodsGCs from women undergoing in vitro fertilization were collected and designated as high and low FFAs cohorts based on their follicular fluid FFA content. GCs with low FFA levels and a human granulosa-like tumor (KGN) cell line were exposed to palmitic acid (PA), which is a dominate FFA follicular fluid constituent. The effects were assessed of this substitution on the Akt/GSK3β signaling pathway activity as well as the expressions of TRIB3 and FSHR at both the gene and protein levels by qPCR, Western blot and immunofluorescence staining analyses. Meanwhile, the individual effects of TRIB3 knockdown in KGN cells and p-AKT inhibitors were compared to determine the mechanisms of FFA-induced FSHR downregulation.ResultsThe average FSH dose consuming per oocyte (FSH dose/oocyte) was elevated and Top embryo quality ratio was decreased in women with high levels of FFAs in their follicular fluid. In these women, the GC TRIB3 and ATF4 protein expression levels were upregulated which was accompanied by FSHR downregulation. Such upregulation was confirmed based on corresponding increases in their gene expression levels. On the other hand, the levels of p-Akt decreased while p-GSK3β increased in the GCs. Moreover, TRIB3 knockdown reversed declines in FSHR expression and estradiol (E2) production in KGN cells treated with PA, which also resulted in increased p-Akt levels and declines in the p-GSK3β level. In contrast, treatment of TRIB3-knockdown cells with an inhibitor of p-Akt (Ser473) resulted in rises in the levels of both p-GSK3β as well as FSHR expression whereas E2 synthesis fell.ConclusionsDuring exposure to a high FFA content, TRIB3 can reduce FSHR expression through stimulation of the Akt/GSK3β pathway in human GCs. This response may contribute to inducing oocyte maturation.

The Effects of Zearalenone on the Localization and Expression of Reproductive Hormones in the Ovaries of Weaned Gilts.

This study aims to investigate the effects of zearalenone (ZEA) on the localizations and expressions of follicle stimulating hormone receptor (FSHR), luteinizing hormone receptor (LHR), gonadotropin releasing hormone (GnRH) and gonadotropin releasing hormone receptor (GnRHR) in the ovaries of weaned gilts. Twenty 42-day-old weaned gilts were randomly allocated into two groups, and treated with a control diet and a ZEA-contaminated diet (ZEA 1.04 mg/kg), respectively. After 7-day adjustment, gilts were fed individually for 35 days and euthanized for blood and ovarian samples collection before morning feeding on the 36th day. Serum hormones of E2, PRG, FSH, LH and GnRH were determined using radioimmunoassay kits. The ovaries were collected for relative mRNA and protein expression, and immunohistochemical analysis of FSHR, LHR, GnRH and GnRHR. The results revealed that ZEA exposure significantly increased the final vulva area (p < 0.05), significantly elevated the serum concentrations of estradiol, follicle stimulating hormone and GnRH (p < 0.05), and markedly up-regulated the mRNA and protein expressions of FSHR, LHR, GnRH and GnRHR (p < 0.05). Besides, the results of immunohistochemistry showed that the immunoreactive substances of ovarian FSHR, LHR, GnRH and GnRHR in the gilts fed the ZEA-contaminated diet were stronger than the gilts fed the control diet. Our findings indicated that dietary ZEA (1.04 mg/kg) could cause follicular proliferation by interfering with the localization and expression of FSHR, LHR, GnRH and GnRHR, and then affect the follicular development of weaned gilts.

Moxibustion improves ovarian function based on the regulation of the androgen balance.

The effect of androgens on follicular development and female reproduction has become an active research topic. Moxibustion is a Traditional Chinese Medicine therapy that has been reported to be able to prevent and treat numerous ovary-related problems. However, studies on the effect of moxibustion for diminished ovarian reserve (DOR) on androgen balance are still lacking. The present study aimed to assess the efficacy of moxibustion intervention prior to disease onset and at the early stage of disease in a rat model of DOR and explore the mechanisms of its effect on ovarian function. A total of 32 rats were randomly divided into four groups: Blank group, Model group (a drug-induced model of DOR), Moxibustion group 1 and Moxibustion group 2. Moxibustion was performed on the BL23 and RN4 acupoints of female rats daily for a total of 20 days (once a day, five times a week for a total of 4 weeks). The two moxibustion groups were established with different intervention times: One group was subjected to pre-disease intervention and the other group to early-disease intervention. The ovarian function was evaluated by detecting anti-Mullerian hormone (AMH), follicle-stimulating hormone (FSH), estradiol (E2), testosterone (T), dehydroepiandrosterone (DHEA), dihydrotestosterone (DHT) and androgen receptor (AR) levels in the serum or the ovary samples. To further investigate the downstream regulatory factors for AR after moxibustion treatment for pre-disease or early-disease intervention, FSH receptor (FSHR) and microRNA (miR)-125b expression in ovaries were also analyzed. The results indicated that AMH and DHT levels were reduced in the model group compared with those in the blank group, while FSH, T and DHEA levels were increased. AMH and DHT levels were increased in Moxibustion group 1 compared with those in the model group, while FSH, T and DHEA levels were reduced. There was no difference in E2 levels between Moxibustion group 1 and the model group. Compared with that in the model group, the AR content in the ovary was increased in Moxibustion group 1. There was no difference in FSHR mRNA in the ovaries between Moxibustion group 1 and the model group. miR-125b levels were significantly increased in Moxibustion group 1 as compared with those in the model group. Furthermore, AMH and DHT levels were increased in Moxibustion group 2 compared with those in the model group, while FSH, T and DHEA levels were reduced. E2 levels were significantly decreased in Moxibustion group 2 compared with those in the model group. The relative mRNA expression of AR, FSHR and miR-125b was decreased following establishment of the model. Compared with that in the model group, the AR content in the ovary was increased in Moxibustion group 2. In comparison with the blank and model groups, the FSHR content in the ovary of Moxibustion group 2 was significantly increased. miR-125b levels were not obviously altered in Moxibustion group 2 as compared with those in the model group. In addition, there was no significant difference in AMH, FSH, T and DHEA levels between the two moxibustion groups. E2 and DHT levels were higher in Moxibustion group 1 than in Moxibustion group 2. There was no difference in AR mRNA expression between the two moxibustion groups. FSHR mRNA levels were lower in Moxibustion group 1 than in Moxibustion group 2, while miR-125b mRNA levels were higher in Moxibustion group 1 than in Moxibustion group 2. In conclusion, the present study suggested that moxibustion intervention prior to disease onset and at the early disease stage was able to improve ovarian function via modulation of the AR-mediated stable equilibrium of androgens. However, the effects and mechanisms of moxibustion intervention for pre-disease and early-disease intervention of DOR appear to be different. The appropriate duration of treatment and the time-effect relationship require to be further studied.

Gonadotrophin stimulation reduces follicular fluid hormone concentrations and disrupts their quantitative association with cumulus cell mRNA

Research questionDo follicular fluid hormone concentrations and the mRNA expression of LHCG, FSH and androgen receptors, aromatase and anti-Müllerian hormone (AMH) in cumulus granulosa cells differ in naturally matured and stimulated follicles? DesignCross-sectional study involving 57 natural cycle IVF (NC-IVF) and 36 conventional gonadotrophin-stimulated IVF (cIVF) cycles performed between 2014 and 2016. cIVF was performed by ovarian stimulation with human menopausal gonadotrophin and gonadotrophin-releasing hormone antagonists. Hormone concentrations were determined in the follicular fluid of the leading follicle, and mRNA concentrations were quantified by reverse transcription polymerase chain reaction in RNA extracted from granulosa cells of the cumulus oophorus complex obtained from these fluids. ResultsFollicular fluid hormone concentrations were significantly lower in cIVF compared with NC-IVF follicles. Median concentrations were 0.50 and 14.5 mIU/ml for LH (P < 0.001), 16.1 and 46.5 nmol/l for testosterone (P < 0.001), 1270 and 2675 nmol/l for oestradiol (P < 0.001), and 12.3 and 28.9 pmol/l for AMH (P < 0.001), respectively. In cumulus granulosa cells, mRNA concentrations for LH receptor, FSH receptor, androgen receptor, aromatase and AMH did not differ between cIVF and NC-IVF follicles. Several hormone and mRNA concentrations were quantitatively associated in natural cycles such as follicular fluid AMH and cumulus granulosa cells AMH RNA (r2 = 0.107, P = 0.013), follicular fluid testosterone and cumulus granulosa cell AMH RNA (r2 = 0.158, P = 0.002), follicular fluid oestradiol and cumulus granulosa cell AMH RNA (r2 = 0.105, P = 0.014) and follicular fluid oestradiol and aromatase (r2 = 0.113, P = 0.011). In contrast, these associations were not observed in stimulated cycles. ConclusionThese findings indicate some effects of gonadotrophin stimulation on follicular physiology, which could be a cause for the previously suggested lower oocyte quality in cIVF compared with NC-IVF.

P–612 Transdermal testosterone vs. Placebo (lubricant gel) pre-treatment in improving IVF outcomes in diminished ovarian reserve patients (POSEIDON group 3 and 4): a randomised controlled trial

Abstract Study question To compare the efficacy of transdermal testosterone with placebo (lubricant gel) in improving IVF outcomes using GnRH antagonist protocol in POSEIDON group 3 and 4 patients. Summary answer Patients receiving pre-treatment with testosterone gel had higher mean number of oocytes retrieved and grade A embryos as compared to the patients receiving lubricant gel. What is known already Diminished ovarian reserve (DOR) is associated with suboptimal ovarian response, higher cycle cancellation rate and lower clinical pregnancy rate following IVF cycles. Various treatment regimens have been devised for management of such patients and use of adjuvants in the form of oral or transdermal androgen is one of them. Androgens improves follicular response to gonadotropin stimulation as well as increase FSH receptor expression in granulosa cells, in turn leading to better oocyte yield and pregnancy rate. Aim was to compare the effect of transdermal testosterone gel with placebo gel on ART outcome in DOR patients (POSEIDON Group 3 and 4). Study design, size, duration A prospective, randomised controlled trial was carried out from 1st September 2019 to 31st October 2020 at a tertiary infertility centre in India. 50 patients fulfilling the criteria of Group 3 and Group 4 of POSEIDON classification were included in the study. Patients with endocrine disorders (thyroid, prolactin), endometrioma, history of surgery on the ovaries, sensitivity to testosterone gel, male factor infertility and deranged liver and renal function tests were excluded. Participants/materials, setting, methods Enrolled patients were randomised into two groups of 25 patients each, one group was pretreated (TTG group) with transdermal testosterone gel, 12.5 mg/day from day 6th of previous cycle to day 2nd of stimulation cycle while patients in other group took lubricant gel for the same duration before stimulation with GnRH antagonist fixed protocol followed by fresh Day 3 transfer. Main results and the role of chance The baseline characteristics of the two groups were comparable. The primary outcome measures were the number of oocytes retrieved and number of grade A embryos formed (according to Istanbul consensus). The secondary outcome measures were implantation rate, clinical pregnancy rate, miscarriage rate and ongoing pregnancy rate. The mean number of oocytes retrieved in TTG group was 5±1.02 which was significantly higher than placebo group–3.5±1.2, (p &amp;lt; 0.001). The mean number of Grade A embryos were also significantly higher (4.78±0.54 vs 3.00±0.23, p &amp;lt; 0.001) in TTG group. The TTG group had higher implantation rate (28% vs 20%, p = 0.49), clinical pregnancy rate (32% vs 18%, p = 0.41), ongoing pregnancy rate (32% vs 16%, p = 0.38) and lower miscarriage rate (0% vs 20%, p = 0.38), however, these differences were not statistically significant. Limitations, reasons for caution The study was done at a single centre with small sample size, replication with more subjects and in different centers is needed. Wider implications of the findings: Pre-treatment with testosterone gel in DOR patients improves ovarian response to stimulation and results in higher number of oocytes retrieved and good quality embryos resulting in improved clinical pregnancy rates. Transdermal testosterone is advantageous because of better bioavailability, easy application, patient friendly and less adverse effects. Trial registration number MCDH/2019/54

Dysregulation of anti-Mullerian hormone expression levels in mural granulosa cells of FMR1 premutation carriers

FMR1 premutation (55–200 CGG repeats) results in fragile X-associated primary ovarian insufficiency (FXPOI). We evaluated expression levels of folliculogenesis-related mediators, follicle-stimulating hormone (FSH) receptor and anti-Mullerian hormone (AMH), to gain insights into the mechanisms underlying the reduced ovarian function. Mural granulosa cells (MGCs) were collected from FMR1 premutation carriers and noncarriers undergoing IVF treatments. At baseline, MGCs of carriers demonstrated significantly higher mRNA expression levels of AMH (3.5 ± 2.2, n = 12 and 0.97 ± 0.5, n = 17, respectively; p = 0.0003) and FSH receptor (5.6 ± 2.8 and 2.7 ± 2.8, respectively; p = 0.02) and higher AMH protein expression on immunostaining. Accordingly, FMR1 premutation-transfected COV434 cells exhibited higher AMH protein expression than COV434 cells transfected with 20 CGG repeats. We conclude that FMR1 premutation may lead to dysregulation of AMH expression levels, probably due to a compensatory mechanism. Elucidating the pathophysiology of FXPOI may help in early detection of ovarian dysfunction and tailoring IVF treatments to FMR1 premutation carriers.

Leptin Modulates the mRNA Expression of Follicle Development Markers in Post-hatch Chicks in an Age-Dependent Manner.

Leptin is involved in regulating reproductive function in chickens, and the development of the leptin system is initiated during the early embryonic stage; however, whether leptin has a specific role in regulating the ovarian development in early post-hatch days is still not fully understood. This study investigated the expression of ovarian functional markers in growing juvenile chickens, along with the effects of leptin on gene expression in the hypothalamus–pituitary–gonadal (HPG) axis on specific ovarian-remodeling days. Leptin receptor (LEPR), follicle-stimulating hormone receptor (FSHR), and the mRNA expression of aromatase (CYP19A1) tended to increase with age in the ovaries of growing chicks. In the ovaries of 7-day-old chicks, intraperitoneally injected leptin significantly increased the mRNA expressions of LEPR, FSHR, and CYP19A1, and this resulted in the increased serum estradiol levels. However, leptin had no effect on hypothalamic LEPR, gonadotropin-releasing hormone 1 (GnRH1), or gonadotropin-inhibitory hormone (GnIH) mRNAs; however, in the pituitary gland, leptin significantly increased the mRNA expression of luteinizing hormone beta subunit (LHB) but had no effect on the mRNA expression of follicle-stimulating hormone beta subunit (FSHB). In 28-day-old chicks, hypothalamic and pituitary mRNAs were unaffected by leptin administration, except hypothalamic LEPR mRNA that was upregulated by a high dose of leptin. In the ovary, leptin dose-dependently decreased the mRNA expression of LEPR; low doses of leptin significantly increased the mRNA expression of FSHR, whereas high doses significantly decreased this expression; leptin did not affect the mRNA expression of CYP19A1; and high leptin doses significantly reduced the serum estradiol levels. Collectively, the results of this study show that leptin modulates ovarian development and folliculogenesis marker genes by primarily acting on ovaries on the specific ovarian-remodeling days in post-hatch chicks, which may alter folliculogenesis and ovarian development toward puberty in chicken.

Rescue of Cell Surface Expression and Signaling of Mutant Follicle-Stimulating Hormone Receptors.

Mutations in G protein-coupled receptors (GPCRs) underlie numerous diseases. Many cause receptor misfolding and failure to reach the cell surface. Pharmacological chaperones are cell-permeant small molecules that engage nascent mutant GPCRs in the endoplasmic reticulum, stabilizing folding and "rescuing" cell surface expression. We previously demonstrated rescue of cell surface expression of luteinizing hormone receptor mutants by an allosteric agonist. Here we demonstrate that a similar approach can be employed to rescue mutant follicle-stimulating hormone receptors (FSHRs) with poor cell surface expression using a small-molecule FSHR agonist, CAN1404. Seventeen FSHR mutations described in patients with reproductive dysfunction were expressed in HEK 293T cells, and cell surface expression was determined by enzyme-linked immunosorbent assay of epitope-tagged FSHRs before/after treatment with CAN1404. Cell surface expression was severely reduced to ≤18% of wild-type (WT) for 11, modestly reduced to 66% to 84% of WT for 4, and not reduced for 2. Of the 11 with severely reduced cell surface expression, restoration to ≥57% of WT levels was achieved for 6 by treatment with 1 µM CAN1404 for 24 h, and a corresponding increase in FSH-induced signaling was observed for 4 of these, indicating restored functionality. Therefore, CAN1404 acts as a pharmacological chaperone and can rescue cell surface expression and function of certain mutant FSHRs with severely reduced cell surface expression. These findings aid in advancing the understanding of the effects of genetic mutations on GPCR function and provide a proof of therapeutic principle for FSHR pharmacological chaperones.

Protective Effect of Hydroalcoholic Extract of Orange Peel on PCNA and FSH-R Gene Expression in Histological Damage and Oxidative Stress Due to Ovarian Torsion in Adult Rats

Objectives: The aim of this study was to evaluate the protective effect of the hydroalcoholic extract of orange peel on proliferating cell nuclear antigen (PCNA) and follicle-stimulating hormone receptor (FSH-R) gene expression in histological injuries and acid stress caused by ovarian torsion in adult rats. Materials and Methods: In this experimental study, 32 adult female rats were randomly divided into 4 groups. In group 1 (Sham), the abdominal wall was cut without applying torsion and in group 2, ovarian torsion was performed for 2 hours, followed by detorsion for 2 weeks. The hydro-alcoholic extract of orange peel was added to their diet for two weeks in group 3, followed by ovarian torsion for 2 hours and detorsion for 2 hours. Group 4 received the orange peel extract for two weeks and after then ovarian resection for the evaluation of histological damage and blood sampling to examine the serum level of antioxidant enzymes, as well as the expression of PCNA and FSH-R genes in the ovarian tissue. Results: Histological changes in the ovary tissue of rats showed that torsion and detorsion have destructive effects on the ovarian tissue, and torsion/detorsion led to a reduction in the expression of PCNA and FSH-R (P &lt; 0.05). Based on biochemical and hormonal results, the ovarian torsion resulted in an imbalance in the oxidative stress markers and hormone profile of rats. Finally, the administration of the hydroalcoholic extract of orange peel due to its high antioxidant properties improves these effects. Conclusions: In general, administering an appropriate dose of the hydroalcoholic extract of orange peel for two consecutive weeks in the diet had a protective effect on the ovarian tissue at the risk of torsion/detorsion.