Significance of FSHR and LHCGR gene polymorphisms on clinical outcomes in gonadotropin-releasing hormone antagonist protocol with freeze-all strategy: A case-control study.

Background: Follicle-stimulating hormone receptor (FSHR) and luteinizing hormone/choriogonadotropin receptor (LHCGR) are integral to ovarian function, facilitating follicle development and maturation through their respective hormonal interactions. The influence of receptor polymorphisms on the outcomes of freeze-all cycles remains unclear. Objective: This study investigates the impact of FSHR N680S and LHCGR N312S polymorphisms on clinical outcomes in freeze-all cycles. Materials and Methods: Women undergoing controlled ovarian stimulation for assisted reproductive technology participated in this study. They were administered a gonadotropin-releasing hormone antagonist protocol, with recombinant follicle-stimulating hormone (rFSH) dosages adjusted according to age, body mass index, antral follicle count, and individual hormonal responses. Additionally, human menopausal gonadotropin dosages were tailored based on the LHCGR N312S genetic variant. Results: Analysis revealed no significant differences in age, body mass index, antral follicle count, or marital status across the genotypes of FSHR N680S and LHCGR N312S. However, notable differences were observed in the rFSH dosage required daily and in total among the FSHR polymorphism genotypes. Genotypes of the LHCGR polymorphism correlated with fewer stimulation days. A significant interaction was observed between the 2 polymorphisms concerning total rFSH dosage. Conclusion: The presence of serine in the FSHR polymorphism was associated with higher rFSH dosage requirements. Both FSHR N680S and LHCGR N312S polymorphisms significantly influenced clinical pregnancy and live birth outcomes in freeze-all cycles, underscoring the potential of a pharmacogenomic approach to optimize hormone supplementation in controlled ovarian stimulation protocols during assisted reproductive technology treatments.

Extragonadal function of follicle-stimulating hormone: Evidence for a role in endothelial physiology and dysfunction.

AIMS: Follicle-stimulating hormone (FSH) plays a fundamental role in reproduction stimulating ovarian folliculogenesis, Sertoli cells function and spermatogenesis. However, the recent identification of FSH receptor (FSHR) also in extra-gonadal tissues has suggested that FSH activity may not be limited only to fertility regulation, with conflicting results on the possible role of FSH in endothelial cells. The aim of this study was to investigate FSH role on endothelial function in Human Umbilical Vein Endothelial Cells (HUVECs). RESULTS: Endothelial Nitric oxide synthase (eNOS) expression, eNOS phosphorylation and Nitric Oxide (NO) production resulted increased after the stimulation of HUVEC with recombinant human FSH (rhFSH) at 3.6x103 ng/ml, with increasing Calcium release from intracellular stores. Furthermore, IP3 production increased after rhFSH stimulation despite PTX treatment and NFAT1 was observed prevalently in nucleus. We observed a statistical difference between untreated cells and cells stimulated with 0.36x103 ng/ml and between cells stimulated with 0.36x103 ng/ml and cells stimulated with 1.8x103 ng/ml at 4 and 8 h by Wound healing assay, respectively. Furthermore, a higher cellular permeability was observed in stimulated cells, with atypical VE-cadherin distribution, as well as filamentous actin. CONCLUSIONS: Our findings suggest that FSH at high concentrations elicits a signalling that could compromise the endothelial membrane. Indeed, VE-cadherin anomalies may severely affect the endothelial barrier, resulting in an increased membrane permeability. Although NO is an important vasodilatation factor, probably an excessive production could impact on endothelial functionality, partially explaining the increased risk of cardiovascular diseases in menopausal women and men with hypogonadism.

FSH Therapy in Male Factor Infertility: Evidence and Factors Which Might Predict the Response.

Follicle-stimulating hormone (FSH) administration is applied in the management of subjects affected by hypogonadotropic hypogonadism. Whilst this application is widely recognized and established alone or in combination with human chorionic gonadotropin (hCG), a similar strategy is empirically advocated in idiopathic male factor infertility (MFI). In this setting, FSH therapy has been used to increase sperm quantity, quality, and pregnancy rate when FSH plasma concentrations are below 8 IU/L and when the seminal tract is not obstructed. In the literature, several studies suggested that giving FSH to patients with idiopathic MFI increases sperm count and motility, raising the overall pregnancy rate. However, this efficacy seems to be limited, and about 10-18 men should be treated to achieve one pregnancy. Thus, several papers suggest the need to move from a replacement approach to an overstimulating approach in the management of FSH therapy in idiopathic MFI. To this aim, it is imperative to determine some pharmacologic markers of FSH efficacy. Furthermore, it should be useful in clinical practice to distinguish, before starting the treatment, among patients who might respond or not to FSH treatment. Indeed, previous studies suggest that infertile men who have normal levels of gonadotropins in plasma might not respond to FSH treatment and about 50% of patients might be defined as "non-responders". For these reasons, identifying predictive markers of FSH action in spermatogenesis and clinical markers of response to FSH treatment is a fascinating area of study that might lead to new developments with the aim of achieving personalization of the treatment of male infertility. From this perspective, seminal parameters (i.e., spermatid count), testicular cytology, genetic assessment, and miRNA or protein markers in the future might be used to create a tailored FSH therapy plan. The personalization of FSH treatment is mandatory to minimize side effects, to avoid lost time with ineffective treatments, and to improve the efficacy, predicting the most efficient dose and the duration of the treatment. This narrative review's objective is to discuss the role of the different putative factors which have been proposed to predict the response to FSH treatment in idiopathic infertile men.

Peroxiredoxin 4 deficiency induces accelerated ovarian aging through destroyed proteostasis in granulosa cells.

Ovarian aging, a complex and challenging concern within the realm of reproductive medicine, is associated with reduced fertility, menopausal symptoms and long-term health risks. Our previous investigation revealed a correlation between Peroxiredoxin 4 (PRDX4) and human ovarian aging. The purpose of this research was to substantiate the protective role of PRDX4 against ovarian aging and elucidate the underlying molecular mechanism in mice. In this study, a Prdx4-/- mouse model was established and it was observed that the deficiency of PRDX4 led to only an accelerated decline of ovarian function in comparison to wild-type (WT) mice. The impaired ovarian function observed in this study can be attributed to an imbalance in protein homeostasis, an exacerbation of endoplasmic reticulum stress (ER stress), and ultimately an increase in apoptosis of granulosa cells. Furthermore, our research reveals a noteworthy decline in the expression of Follicle-stimulating hormone receptor (FSHR) in aging Prdx4-/- mice, especially the functional trimer, due to impaired disulfide bond formation. Contrarily, the overexpression of PRDX4 facilitated the maintenance of protein homeostasis, mitigated ER stress, and consequently elevated E2 levels in a simulated KGN cell aging model. Additionally, the overexpression of PRDX4 restored the expression of the correct spatial conformation of FSHR, the functional trimer. In summary, our research reveals the significant contribution of PRDX4 in delaying ovarian aging, presenting a novel and promising therapeutic target for ovarian aging from the perspective of endoplasmic reticulum protein homeostasis.

Follicle-Stimulating Hormone Receptor (FSHR) Ser680Asn Genotype Does Not Affect the Follicular Fluid Hormonal Profile in Stimulated Cycles Using Different Gonadotropin Preparations for Ovulation Induction: A Pilot Study.

BACKGROUND: The existing literature lacks consensus on the effectiveness of utilizing polymorphisms to enhance outcomes in in vitro fertilization (IVF), particularly regarding ovulation induction protocols, oocyte and embryo quality, and pregnancy rates. Therefore, the present pilot study aims to assess whether the composition of different gonadotropin preparations affects the ovarian stimulation protocol concerning follicle-stimulating hormone receptor (FSHR) Ser680Asn genotypes (Ser/Ser, Ser/Asn, and Asn/Asn), in terms of ovulation induction parameters, including oocyte maturation rate, embryo quality, and pregnancy rate. METHODOLOGY: A total of 94 IVF patients underwent treatment using a GnRH antagonist protocol with four distinct gonadotropin preparations: HMG, HMG/hCG, rFSH, and rFSH/hCG. Follicular fluid (FF) samples were pooled for each patient for analysis. RESULTS: No statistical differences in the FF hormonal profile (progesterone, testosterone, androstenedione, estradiol, FSH, hCG) among the FSHR genotypes were reported either separately for each protocol or in combination for the four different preparations of gonadotropins. The maturation rate of MII oocytes and embryo quality did not differ among women carrying either Ser/Ser, Ser/Asn, or Asn/Asn genotype (p-value=0.475, and p-value=1.000, respectively). Moreover, no statistically significant correlation was revealed among Ser/Ser, Ser/Asn, and Asn/Asn carriers and pregnancy rate (p = 0.588). CONCLUSIONS: FF hormonal analysis of women undergoing IVF using different ovulation induction protocols and carrying either Ser/Ser, Ser/Asn, or Asn/Asn genotype revealed no significant correlations, in terms of maturation rate of MII oocytes, embryo quality, and pregnancy rate, indicating that the FSHR Ser680Asn genotype does not constitute a biomarker for a positive pregnancy outcome. Therefore, the existence of a different mechanism for the expression of FSHR Ser680Asn genotypes in the FF hormonal profile related to stimulated cycles is implied.

Honghua Xiaoyao tablet combined with estradiol improves ovarian function in D-galactose-induced aging mice by reducing apoptosis and affecting the release of reproductive hormones: an in vivo study.

Context: It is very necessary to delay ovarian aging and prevent age-related health problems. The active ingredient in Honghua Xiaoyao tablet (HHXYT) has the effects of anti-oxidation, anti-inflammation, immune regulation and so on. Objective: To explore the effect and mechanism of Honghua Xiaoyao tablet on aging model mice. Materials and methods: The aging model was established by intraperitoneal injection of D-galactose in model mice. The mice in the HHXYT-L,M,H group were given 0.3 g/kg, 0.6 g/kg and 1.2 g/kg Honghua Xiaoyao tablet suspension respectively, and the HHXYT-M + E2 group was given 0.6 g/kg HHXYT +0.13 mg/kg estradiol valerate for 30 days. In this study, ELISA, HE, Western blot, IH and TUNEL were used. Results: HHXYT + E2 can improve the gonadal index, estrous cycle of aging mice. In HHXYT-M + E2 group, the level of FSH and LH decreased, while E2 and AMH increased significantly. The number of growing follicles in HHXYT-M + E2 group increased, which was better than that of HHXYT alone. Western blot results showed that HHXYT-M + E2 group decreased the expression of Bax, cleaved-Parp, cleaved-Casp-3 and CytC molecules and increased the expression of Bcl-2 in ovarian tissue. FSHR expression decreased in model group and increased in HHXYT group. TUNEL staining showed that the number of apoptotic cells in HHXYT group was reduced, and the HHXYT-M + E2 group was the most significantly. Discussion and conclusion: HHXYT can improve the level of sex hormones and increase the number of growing follicles in aging mice. HHXYT-M + E2 group has the best effect, and its mechanism may be related to reducing ovarian granulosa cell apoptosis.

Puberty classifications in beef heifers are moderately to highly heritable and associated with candidate genes related to cyclicity and timing of puberty.

Introduction: Pubertal attainment is critical to reproductive longevity in heifers. Previously, four heifer pubertal classifications were identified according to attainment of blood plasma progesterone concentrations > 1 ng/ml: 1) Early; 2) Typical; 3) Start-Stop; and 4) Non-Cycling. Early and Typical heifers initiated and maintained cyclicity, Start-Stop started and then stopped cyclicity and Non-Cycling never initiated cyclicity. Start-Stop heifers segregated into Start-Stop-Discontinuous (SSD) or Start-Stop-Start (SSS), with SSD having similar phenotypes to Non-Cycling and SSS to Typical heifers. We hypothesized that these pubertal classifications are heritable, and loci associated with pubertal classifications could be identified by genome wide association studies (GWAS). Methods: Heifers (n = 532; 2017 - 2022) genotyped on the Illumina Bovine SNP50 v2 or GGP Bovine 100K SNP panels were used for variant component estimation and GWAS. Heritability was estimated using a univariate Bayesian animal model. Results: When considering pubertal classifications: Early, Typical, SSS, SSD, and Non-Cycling, pubertal class was moderately heritable (0.38 ± 0.08). However, when heifers who initiated and maintained cyclicity were compared to those that did not cycle (Early+Typical vs. SSD+Non-Cycling) heritability was greater (0.59 ± 0.19). A GWAS did not identify single nucleotide polymorphisms (SNPs) significantly associated with pubertal classifications, indicating puberty is a polygenic trait. A candidate gene approach was used, which fitted SNPs within or nearby a set of 71 candidate genes previously associated with puberty, PCOS, cyclicity, regulation of hormone secretion, signal transduction, and methylation. Eight genes/regions were associated with pubertal classifications, and twenty-two genes/regions were associated with whether puberty was attained during the trial. Additionally, whole genome sequencing (WGS) data on 33 heifers were aligned to the reference genome (ARS-UCD1.2) to identify variants in FSHR, a gene critical to pubertal attainment. Fisher's exact test determined if FSHR SNPs segregated by pubertal classification. Two FSHR SNPs that were not on the bovine SNP panel were selected for additional genotyping and analysis, and one was associated with pubertal classifications and whether they cycled during the trial. Discussion: In summary, these pubertal classifications are moderately to highly heritable and polygenic. Consequently, genomic tools to inform selection/management of replacement heifers would be useful if informed by SNPs associated with cyclicity and early pubertal attainment.

Follicle-stimulating hormone receptor expression in advanced atherosclerotic plaques.

Experimental evidence indicates that follicle-stimulating hormone (FSH), an essential hormone for reproduction, can act directly on endothelial cells inducing atherosclerosis activation and development. However, it remains unknown whether the FSH-receptor (FSHR) is expressed in human atherosclerosis plaques. To demonstrate the FSHR presence, we used immunohistochemical and immunoelectron microscopy involving a specific monoclonal antibody FSHR1A02 that recognizes an epitope present in the FSHR-ectodomain. In all 55 patients with atherosclerotic plaques located in carotid, coronary, femoral arteries, and iliac aneurysm, FSHR was selectively expressed in arterial endothelium covering atherosclerotic plaques and endothelia lining intraplaque neovessels. Lymphatic neovessels were negative for FSHR. M1-macrophages, foam cells, and giant multinucleated cells were also FSHR-positive. FSHR was not detected in normal internal thoracic artery. Immunoelectron microscopy performed in ApoEKO/hFSHRKI mice with atherosclerotic plaques, after injection in vivo with mouse anti-hFSHR monoclonal antibody FSHR1A02 coupled to colloidal gold, showed FSHR presence on the luminal surface of arterial endothelial cells covering atherosclerotic plaques. Therefore, FSHR can bind, internalize, and deliver into the plaque circulating ligands to FSHR-positive cells. In conclusion, we report FSHR expression in endothelial cells, M1-macrophages, M1-derived foam cells, giant multinucleated macrophages, and osteoclasts associated with human atherosclerotic plaques.

DENND1A desensitizes granulosa cells to FSH by arresting intracellular FSHR transportation.

Polycystic ovary syndrome (PCOS) is a complex disorder. Genome-wide association studies (GWAS) have identified several genes associated with this condition, including DENND1A. DENND1A encodes a clathrin-binding protein that functions as a guanine nucleotide exchange factor involved in vesicular transport. However, the specific role of DENND1A in reproductive hormone abnormalities and follicle development disorders in PCOS remain poorly understood. In this study, we investigated DENND1A expression in ovarian granulosa cells (GCs) from PCOS patients and its correlation with hormones. Our results revealed an upregulation of DENND1A expression in GCs from PCOS cases, which was positively correlated with testosterone levels. To further explore the functional implications of DENND1A, we generated a transgenic mouse model overexpressing Dennd1a (TG mice). These TG mice exhibited subfertility, irregular estrous cycles, and increased testosterone production following PMSG stimulation. Additionally, the TG mice displayed diminished responsiveness to FSH, characterized by smaller ovary size, less well-developed follicles, and abnormal expressions of FSH-priming genes. Mechanistically, we found that Dennd1a overexpression disrupted the intracellular trafficking of follicle stimulating hormone receptor (FSHR), promoting its internalization and inhibiting recycling. These findings shed light on the reproductive role of DENND1A and uncover the underlying mechanisms, thereby contributing valuable insights into the pathogenesis of PCOS and providing potential avenues for drug design in PCOS treatment.

Effects of Vaccination against Recombinant FSH or LH Receptor Subunits on Gonadal Development and Functioning Male Rats.

Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) play key roles in regulating testosterone secretion and spermatogenesis in male mammals, respectively, and they maintain the fertility of male animals by binding to their corresponding receptors. We designed and prepared a recombinant LH receptor (LHR) subunit vaccine and a recombinant FSH receptor (FSHR) subunit vaccine and used male Sprague Dawley (SD) rats as a model to examine their effects on testicular development, spermatogenesis, and testosterone secretion in prepubertal and pubertal mammals. Both vaccines (LHR-DTT and FSHR-DTT) significantly decreased the serum testosterone level in prepubertal rats (p < 0.05) but had no effect on the testosterone secretion in pubertal rats; both vaccines decreased the number of cell layers in the seminiferous tubules and reduced spermatogenesis in prepubertal and pubertal rats. Subunit vaccine FSHR-DTT decreased the sperm density in the epididymis in both prepubertal and pubertal rats (p < 0.01) and lowered testicular index and sperm motility in pubertal rats (p < 0.05), whereas LHR-DTT only reduced the sperm density in the epididymis in pubertal rats (p < 0.05). These results indicate that the FSHR subunit vaccine may be a promising approach for immunocastration, but it still needs improvements in effectiveness.

Application of Biomarkers in Obese Infertile Women: A Genetic Tool for a Personalized Treatment.

This study investigates links between CART and leptin gene expression, FSH receptor Asn680Ser polymorphism, and reproductive hormones in morbidly obese patients under 40 years old, facing infertility, and undergoing bariatric surgery. A total of 29 women were included in this study. A hormonal profile along with detection of CART and leptin gene expression was evaluated before and after bariatric surgery. Additionally, the presence or absence of Asn680Ser of the FSHR gene was studied. Following bariatric surgery, a mean reduction in BMI (16.03 kg/m2) was observed in all women. FSH levels preoperatively varied significantly among genotypes, with medians of 8.1, 9.5, and 10.3 for individuals without polymorphism, heterozygotes, and homozygotes, respectively (p = 0.0408). Post surgery, marginal differences in FSH levels were observed (5.8, 7.1, and 8.2, respectively) (p = 0.0356). E2 and LH levels exhibited no significant genotype-based differences pre and post surgery. Presurgical E2 levels were 29.6, 29.8, and 29.6, respectively (p = 0.91634), while postsurgical levels were 51.2, 47.8, and 47 (p = 0.7720). LH levels followed similar patterns. Our findings highlight bariatric surgery's positive impact on BMI reduction and its potential connection to genetic markers, hormones, and infertility. This suggests personalized treatments and offers a valuable genetic tool for better fertility outcomes in obese individuals.

The relationship between primary ovarian insufficiency and gene variations: a prospective case-control study.

Around 70 percent of cases of Primary Ovarian Insufficiency (POI) etiology remain unexplained. The aim of our study is to contribute to the etiology and genetic background of POI. A total of 37 POI patients and 30 women in the reproductive period were included in this prospective, case-control study between August 2020 and December 2021. The women were examined for 36 genes with next-generation sequencing (NGS) panel. Gene variations were detected in 59.5 percent of the patients in the case group. FSHR p.S680N (rs6166, c.2039 G>A) and FSHR p.A307T (rs6165, c.919 G>A) gene variants, which are most frequently located in exon 10 of the FSHR gene, were detected in both groups. Although it was not found that these gene variants were significantly different between the groups, it was also found that they were significantly different in POI patients under 30 years of age and in those with a family history of POI. Variations were detected in 12 genes in POI patients. Two gene variants (FGFR1 [c.386A>C, rs765615419] and KISS1 [c.58 G>A, rs12998]) were detected in both groups, and the remaining gene variants were detected only in POI patients. No differences were detected between the groups in terms of gene variations. However, the gene variations detected only in POI patients may play a role in the etiology of POI.

Association of FSHR gene polymorphisms with poor ovarian response in patients undergoing IVF: A systematic review and meta-analysis.

BACKGROUND: The results of studies on the association between polymorphisms in the FSHR gene and the risk of POR undergoing IVF have been inconsistent with each other, so we conducted a meta-analysis of all the available studies to explore the association between polymorphisms in the FSHR gene and the risk of POR. METHODS: Literature that met the inclusion criteria was collected by searching six electronic databases and basic data from included studies were extracted. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to assess the strength of association between follicle-stimulating hormone receptor (FSHR) gene polymorphism and poor ovarian response (POR) risk. Begg's and Egger's tests were used to determine whether there was publication bias, and sensitivity analysis and TSA analysis were used to verify the stability and reliability of the results. RESULTS: We included 24 articles, 22 of which explored rs6166, including 2,206 cases and 3,897 controls. 6 articles explored rs6165, including 444 cases and 875 controls. Under additive, heterozygote, and dominant models, rs6166 was significantly associated with POR (S vs. N: OR = 1.29, 95 % CI = 1.05-1.59, P = 0.017; NS vs. NN: OR = 1.33, 95 % CI = 1.02-1.74, P = 0.038; NS + SS vs. NN: OR = 1.38, 95 % CI = 1.04-1.84, P = 0.025). In ethnicity-based subgroup analyses, the additive, homozygote, heterozygote, and dominant models increased Asian POR risk. Among the five genetic models, rs6165 was significantly associated with POR (T vs. C: OR = 1.64, 95 % CI = 1.25-2.16, P = 0.000; TT vs. CC: OR = 2.76, 95 % CI = 1.43-5.32, P = 0.003; CT vs. CC: OR = 1.58, 95 % CI = 1.19-2.10, P = 0.001; TT vs. CC + CT: OR = 2.32, 95 % CI = 1.67-3.23, P = 0.000; CT + TT vs. CC: OR = 1.80, 95 % CI = 1.22-2.65, P = 0.003). In ethnicity-based subgroup analyses, all five genetic models increased the risk of POR in Caucasians. CONCLUSION: According to the current meta-analysis, the rs6166 S allele was significantly associated with an increased risk of POR, especially in Asian populations. The rs6165 T allele was significantly associated with an increased risk of POR, especially in Caucasian populations.

Core fucosylation regulates the ovarian response via FSH receptor during follicular development.

INTRODUCTION: Ovarian low response to follicle-stimulating hormone (FSH) causes infertility featuring hypergonadotropic hypogonadism, ovarian failure, and/or defective ovarian response. OBJECTIVES: N-glycosylation is essential for FSH receptor (FSHR). Core fucosylation catalyzed by fucosyltransferase 8 (FUT8) is the most common N-glycosylation. Core fucosylation level changes between individuals and plays important roles in multiple physiological and pathological conditions. This study aims to elucidate the significance of FUT8 to modulate FSHR function in female fertility. METHODS: Samples from patients classified as poor ovary responders (PORs) were detected with lectin blot and real-time PCR. Fut8 gene knockout (Fut8-/-) mice and FUT8-knockdown human granulosa cell line (KGN-KD) were established and in vitro fertilization (IVF) assay, western blot, molecular interaction, immunofluorescence and immunoprecipitation were applied. RESULTS: Core fucosylation is indispensable for oocyte and follicular development. FSHR is a highly core-fucosylated glycoprotein. Loss of core fucosylation suppressed binding of FSHR to FSH, and attenuated FSHR downstream signaling in granulosa cells. Transcriptomic analysis revealed the downregulation of several transcripts crucial for oocyte meiotic progression and preimplantation development in Fut8-/- mice and in POR patients. Furthermore, loss of FUT8 inhibited the interaction between granulosa cells and oocytes, reduced transzonal projection (TZP) formation and caused poor developmental competence of oocytes after fertilization in vitro. While L-fucose administration increased the core fucosylation of FSHR, and its sensitivity to FSH. CONCLUSION: This study first reveals a significant presence of core fucosylation in female fertility control. Decreased fucosylation on FSHR reduces the interaction of FSH-FSHR and subsequent signaling, which is a feature of the POR patients. Our results suggest that core fucosylation controls oocyte and follicular development via the FSH/FSHR pathway and is essential for female fertility in mammals.

Study on the Mechanism of Guanyuan Mingmen Sequential Acupuncture Activating FSHR/cAMP/PKA Pathway to Promote Granulosa Cell Proliferation in POI Model Rats / 广州中医药大学学报

Objective To observe the therapeutic effects and mechanisms of Guanyuan Mingmen Sequential Acupuncture on rats with premature ovarian insufficiency(POI)model.Methods Female SD rats were divided into the blank group,the model group,the protein kinase A(PKA)inhibitor(H89)+acupuncture group,and the acupuncture group,with 12 rats in each group.Except for the blank group,the POI model was prepared by gavage with Tripterygium Glycosides Tablets in the other three groups of rats.After the model was successfully established,the blank group and the model group were bundled once a day;in the acupuncture group,Guanyuan(RN4)point was taken during the intermotility period,and in the pre-motility period,Mingmen(DU4)point was taken;in the H89+acupuncture group,the intervention was performed in accordance with the acupuncture protocol of the acupuncture group,and H89 was injected intraperitoneally for 30 minutes prior to each acupuncture session.Continuous intervention was performed for 20 days.Samples were taken from each group of rats in the first estrus period and in proestrus period after intervention.Enzyme-linked immunosorbent assay(ELISA)was used to measure the levels of follicle stimulating hormone(FSH)and estradiol(E2)during the estrous phase,Western Blot was used to measure the protein expressions of follicle stimulating hormone receptor(FSHR)and aromatase P450(P450arom)during the estrous phase,and the activity of granulocytes during the estrous phase and the proestrus phase were measured using the cell-counting kit 8(CCK-8)method.The immunohistochemistry method was used to detect the protein expression of pre-motility proliferating cell nuclear antigen(PCNA).Results(1)Compared with the blank group,the serum FSH level of the model group and H89+acupuncture group was significantly increased(P＜0.01),and the E2 level was significantly decreased(P＜0.001);there was no difference between the FSH level of the H89+acupuncture group and that of the model group(P＞0.05),and the E2 level of the H89+acupuncture group was lower than that of the model group(P＜0.05);the FSH level of the acupuncture group was lower than that of the model group and that of the H89+acupuncture group(P＜0.05),had no difference with the blank group(P＞0.05),E2 level was significantly higher than the model group and H89+ acupuncture group(P＜0.01),still being lower than the blank group(P＜0.05).(2)The protein expressions of FSHR and P450arom in the model group and H89 + acupuncture group was lower than those in the blank group;the protein expression of FSHR in the H89 + acupuncture group was not different from that in the model group(P＞0.05),while the protein expression level of P450arom was lower than that in the model group(P＜0.05);the protein expression levels of FSHR and P450arom in the acupuncture group were higher than those in the model group and H89 + acupuncture group,but still lower than those in the blank group(P＜0.05).(3)Both GCs activity and average optical density value of PCNA in the model group and H89+acupuncture group were lower than the blank group(P＜0.05);both GCs activity and average optical density value of PCNA in the H89+acupuncture group were lower than the model group(P＜0.05);the activity of GCs and average optical density value of PCNA of the acupuncture group were significantly higher than that of the model group and H89+acupuncture group(P＜0.05 or P＜0.01).Conclusion Guanyuan Mingmen Sequential Acupuncture can regulate sex hormone levels,increase GCs activity and promote GCs cell proliferation by up-regulating protein expressions of follicle stimulating hormone receptor(FSHR)/cyclic adenosine monophosphate(cAMP)/protein kinase A(PKA)pathway FSHR,P450arom,thus improving POI.

The amelioration of ovarian dysfunction by hesperidin in malathion-treated mice through the overexpression of PCNA and FSHR proteins.

Objective: Malathion (MAL), a pesticide used for decades, is a highly toxic substance. Several studies have documented the negative effects of such agents on reproductive organ physiology, but the precise mechanism of action in the induction of ovarian dysfunction remains unclear. Therefore, the purpose of this research was to examine the effects of the antioxidant hesperidin (HES) on ovarian damage and toxicity caused by malathion. Materials and methods: In this experiment, forty adult female bulb/c mice weighing 27-30 g were categorized into four groups, namely hesperidin (20 mg/kg, i.p.), malathion (3 mg/kg, i.p.), malathion + hesperidin, and control groups. Following a period of 35 consecutive days of treatment, mice were euthanized, and their ovarian tissues were gathered for the purposes of histopathological analysis by H&E staining, immunohistochemical assessment via proliferating cell nuclear antigen (PCNA) and follicle-stimulating hormone receptor (FSHR) immunostaining, and biochemical evaluation via measuring the levels of malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), tumor necrosis factor-alpha (TNF-α), and interleukin-1 beta (IL-1ß). In addition, serum samples were collected from the blood of mice to perform hormonal analyses, especially 17ß-estradiol (E2), progesterone (P4), luteinizing hormone (LH), and follicle-stimulating hormone (FSH). Results: The results demonstrated that MAL exposure resulted in the development of abnormalities in the architecture and structure of ovaries. Also, the treatment of mice with MAL led to declined follicular counts at all three stages, namely, primary, secondary, and tertiary, reduced serum levels of sex hormones, decreased immunoreactivity of FSHR and PCNA, and diminished activity of CAT and SOD enzymes. In contrast, it caused an increase in MDA, IL-1ß, and TNF-α, as well as the count of atretic follicles. Nonetheless, it was observed that HES exhibited the ability to ameliorate the deleterious impacts of malathion across all the aforementioned parameters. Conclusion: Treatment with HES via upregulating the protein expression of PCNA and FSHR and activating antioxidant defense was able to ameliorate the adverse effects of MAL on ovarian tissues.

Paeoniflorin Alleviates Cisplatin-Induced Diminished Ovarian Reserve by Restoring the Function of Ovarian Granulosa Cells via Activating FSHR/cAMP/PKA/CREB Signaling Pathway.

Paeoniflorin (PAE) is the main active compound of Radix Paeoniae Rubra (a valuable traditional Chinese medicine and a dietary supplement) and exerts beneficial effects on female reproductive function. However, the actions of PAE on diminished ovarian reserve (DOR, a very common ovarian function disorder) are still unclear. Herein, our study investigated the effect and potential mechanism of PAE on DOR by using cisplatin-induced DOR mice and functional impairment of estradiol (E2) synthesis of ovarian granulosa-like KGN cells. Our data show that PAE improved the estrous cycle, ovarian index, and serum hormones levels, including E2, and the number of antral follicles and corpora lutea in DOR mice. Further mechanism results reveal that PAE promoted aromatase expression (the key rate-limiting enzyme for E2 synthesis) and upregulated the FSHR/cAMP/PKA/CREB signaling pathway in the ovaries. Subsequently, PAE improved the levels of E2 and aromatase and activated the FSHR/cAMP/PKA/CREB signaling pathway in KGN cells, while these improving actions were inhibited by the siRNA-FSHR and FSHR antagonist treatments. In sum, PAE restored the function of E2 synthesis in ovarian granulosa cells to improve DOR by activating the FSHR/cAMP/PKA/CREB signaling pathway, which exhibited a new clue for the development of effective therapeutic agents for the treatment of DOR.

Combined Multiplexed Phage Display, High-Throughput Sequencing, and Functional Assays as a Platform for Identifying Modulatory VHHs Targeting the FSHR.

Developing modulatory antibodies against G protein-coupled receptors is challenging. In this study, we targeted the follicle-stimulating hormone receptor (FSHR), a significant regulator of reproduction, with variable domains of heavy chain-only antibodies (VHHs). We built two immune VHH libraries and submitted them to multiplexed phage display approaches. We used next-generation sequencing to identify 34 clusters of specifically enriched sequences that were functionally assessed in a primary screen based on a cAMP response element (CRE)-dependent reporter gene assay. In this assay, 23 VHHs displayed negative or positive modulation of FSH-induced responses, suggesting a high success rate of the multiplexed strategy. We then focused on the largest cluster identified (i.e., PRC1) that displayed positive modulation of FSH action. We demonstrated that PRC1 specifically binds to the human FSHR and human FSHR/FSH complex while potentiating FSH-induced cAMP production and Gs recruitment. We conclude that the improved selection strategy reported here is effective for rapidly identifying functionally active VHHs and could be adapted to target other challenging membrane receptors. This study also led to the identification of PRC1, the first potential positive modulator VHH reported for the human FSHR.

Membrane estrogen receptor and follicle-stimulating hormone receptor.

Follicle-stimulating hormone (FSH) and estrogens are fundamental to support reproductive functions. Beside the well-known FSH membrane receptor (FSHR), a G protein-coupled estrogen receptor (GPER) has been found, over the last two decades, in several tissues. It may trigger rapid, non-genomic responses of estradiol, activating proliferative and survival stimuli. The two receptors were co-characterized in the ovary, where they modulate different intracellular signaling cascades, according to the expression level and developmental stage of ovarian follicles. Moreover, they may physically interact to form heteromeric assemblies, suggestive of a new mode of action to regulate FSH-specific signals, and likely determining the follicular fate between atresia and dominance. The knowledge of FSH and estrogen membrane receptors provides a new, deeper level of comprehension of human reproduction.

Polymorphisms in FSHR modulating susceptibility to polycystic ovary syndrome: an updated meta-analysis.

BACKGROUND: Two polymorphisms, rs6165 and rs6166 located in the intracellular domain of FSHR has been reported to affect folliculogenesis, steroidogenesis and oocyte maturation. Several studies have highlighted the role of FSHR polymorphisms in PCOS but the findings are conflicting. A meta-analysis was carried out to decipher the emerging perspectives. METHODOLOGY: A comprehensive literature search was made using PubMed, PCOSkb, and Google Scholar. New Ottawa Scale has been utilized to evaluate the quality of each article. To evaluate the strength of association under different genetic models of rs6165 and rs6166 polymorphisms, odds ratio with a 95% confidence interval (CI) was calculated. RESULTS: A total of 20 articles were selected for the present study. In pooled analysis and after the stratification by ethnicity, polymorphism rs6165 remains unrelated to the onset of PCOS. Besides, rs6166 exhibits significant protection in the Indian population under recessive, additive, and allele models (OR = 0.7, CI: 0.54-0.9, p = 0.006, OR = 0.65, CI: 0.48-0.89, p = 0.006, OR = 0.82, CI: 0.7-0.95, p = 0.01, respectively) and low to moderate risk in the Caucasian population under allele model (OR = 1.17, CI: 1.04-1.32, p = 0.01). CONCLUSION: This meta-analysis suggests that GG genotype of rs6166 provides protection against PCOS, in a population-specific manner.