Luteinizing Hormone/chorionic Gonadotropin Receptor Research Articles

Effect Modification of LHCGR Gene Variant (rs2293275) on Clinico-Biochemical Profile, and Levels of Luteinizing Hormone in Polycystic Ovary Syndrome Patients.

Polycystic ovary syndrome (PCOS) is a common multifaceted endocrine disorder among reproductive-aged women. Deranged luteinizing hormone levels and associated downstream signaling cascade mediated by its receptor luteinizing hormone chorionic gonadotropin receptor (LHCGR) are pivotal in the etiopathogenesis of PCOS. Genetic variations in the LHCGR have been associated with PCOS risk. However, the results are mixed and inconclusive. We evaluated the association of the LHCGR rs2293275 polymorphic variant with PCOS risk and its association with clinico-biochemical features of PCOS. 120 confirmed PCOS cases and an equal number of age-matched controls were subjected to clinical, biochemical, and hormonal investigations. Genotyping for rs2293275 was performed using polymerase chain reaction-restriction fragment length polymorphism. Logistic regression models were used to calculate odds ratios (ORs) at 95% confidence intervals (95% CIs). In the current study, PCOS cases reported a lower number of menstrual cycles per year than respective controls. A significantly higher BMI, Ferriman Galway score, levels of serum testosterone, insulin, TSH, FSH, and fasting glucose were observed in cases than in controls (p < 0.01). Compared to GG carriers, we observed a higher risk of developing PCOS in the subjects who harbored GA (OR 10.4, p < 0.0001) or AA (OR 7.73, p = 0.02) genotype. The risk persisted in the dominant model (GA + AA) as well (OR 10.29, p = 0.01). On stratification, a higher risk of developing PCOS was observed in variant genotype carriers who had a family history of either type two diabetes mellitus (OR 117; p < 0.0001) or hirsutism (OR 79; p < 0.0001). We also found significantly elevated levels of serum LH levels in the subject harboring GA and AA genotypes when compared to GG carriers. In the present study, we report a significant association of the LHCGR rs2293275 variant with the PCOS risk.

RF33 | PSUN04 Luteinizing Hormone-Chorionic Gonadotrophin Receptor in Pheochromocytomas

Abstract Background Pheochromocytomas and paragangliomas (PPGL) are catecholamine-producing neuroendocrine tumors that arise from the adrenal medulla or ganglia and display the highest heritability rate among all human tumours. Genomic analyses allowed identification of molecular subgroups of PPGL which are organized into 2 main clusters. Cluster 1 contains SDHx- and VHL-mutated tumors which do not produce epinephrine while cluster 2 includes the epinephrine-secreting PPGL related to RET, NF1, TMEM127 and MAX mutations. PPGL must be early diagnosed and treated to prevent adrenergic crises which can be life-threatening. Detection of PPGL is particularly important during pregnancy since PPGL are associated with a high risk of either maternal or fetal complications in this context. Reciprocally, pregnancy can favor adrenergic crises in patients with previously undiagnosed or silent pheochromocytoma (PCC). These dangerous events can be triggered by tumor compression resulting from fetus growth, or labor and delivery. However, it is known that surges in plasma catecholamines may also occur during early gestation suggesting that pregnancy may also activate the secretory activity of PPGL through the involvement of non-mechanical factors, such as gestational hormones. Methods In the present study, we report a case of silent PCC revealed in a pregnant woman by life-threatening adrenergic myocarditis occurring at 31 weeks of gestation. Analysis of PPGL susceptibility genes showed the presence of a heterozygous germline RET variant of uncertain significance. The fact that the first symptoms of catecholamine excess had appeared during the first trimester of pregnancy led us to conduct in vitro studies to investigate the effects of estradiol and the pregnancy hormone chorionic gonadotropin (hCG) on epinephrine secretion by cultured cells derived from the resected patient's tumor. Expression of luteinizing hormone/chorionic gonadotropin receptor (LHCGR) was searched for in the tumor and an additional series of 12 PCC by RT-qPCR and immunohistochemistry. LHCGR expression was also analyzed in silico in the PPGL cohorts of the COMETE and The Cancer Genome Atlas (TCGA) databases. Results hCG stimulated epinephrine secretion by primary cultured PCC cells. The tumor expressed the LHCG receptor, which was colocalized with catecholamine-producing enzymes. A similar expression pattern of the LHCG receptor was also observed in 5 out of a series of 12 PCCs. Moreover, in silico studies revealed that PPGL display the highest expression levels of LHCGR mRNA among the 32 solid tumor types of TCGA cohort. Interestingly, expression of LHCGR was higher in cluster 2 than in cluster 1 PPGL. Conclusion We show that PCC can express functional LHCG receptor. Consequently, pregnancy may activate catecholamine production by previously silent pheochromocytoma as early as the first trimester of gestation especially in women with gene mutations that predispose to cluster 2 epinephrine-secreting PCC. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m., Monday, June 13, 2022 1:18 p.m. - 1:23 p.m.

Abstract 106: Hypogonadism due to isolated luteinizing hormone receptor defect

Background: The human luteinizing hormone/chorionic gonadotropin receptor (LHCGR) belongs to G-protein coupled class A receptor with 12 Exons, 699 amino acids and molecular weight of 85-95KDa, the gene is located on chromosome 2p21. Mutations of LHCGR have been divided into two types based on the severity of the disease. Type 1 LCH (most severe form) causes male to female DSD, absence of sexual maturation at puberty, milder forms of resistance can present with micropenis and/or hypospadias or only infertility called type 2 LCH.Aims and Objectives: In this study we aim to describe a case with type 2 LCH and systematically review the literature of type 2 LCH cases with available genotype data.Results: Mr. AS presented from southern India born of third degree consanguineous marriage presented with micropenis at the age of 18 years. He had uneventful birth and was reared as a male. External genitalia showed Pubic hair stage 2, stretched penile length of 3.5 cm, bilateral testicular volume of 8 cc with no breast development. Karyotype of the patient was 46 XY and Hormonal evaluation (18 years) showed luteinizing hormone (LH) 19.92 mIU/ml, Follicular stimulating hormone (FSH) 7.62 mIU/ml, testosterone (T) 0.114 ng/ml, stimulated T 0.118 ng/ml, dihydrotestosterone (DHT) 0.199 ng/ml. Imaging showed small sized bilateral testes in the scrotum (right 2.5x1.6 cm, left 2.6x1.9 cm), with visible prostate gland, epididymis and no mullerian structures. Genetic analysis by next generation sequencing showed homozygous pathogenic mutation c.391T>C (p.Cys131Arg) in Exon 5 suggestive of leydig cell hypoplasia. He was treated with Inj testosterone and Inj Human chorionic gonadotropin (HCG) with which he had clinical improvement in terms of virilization and increase in testicular volume.Conclusion: Leydig cell hypoplasia is a rare cause of hypogonadism.

Effects of increasing intake of soybean oil on synthesis of testosterone in Leydig cells

BackgroundSoybean oil is a very common edible oil in daily life. With the changes in the dietary composition, the intake of soybean oil increased. However, the effects of dietary intake of soybean oil on testosterone production are still unclear.MethodsIn order to study the effects of increasing intake of soybean oil on the synthesis of testosterone in Leydig cells, we fed male C57BL/6 mice on the diet which added 20% soybean salad oil (SOY group). We detected the hormone levels by enzyme-linked immunosorbent assay (ELISA) kits and serum fatty acid composition by gas chromatography, and analyzed the expression of steroidogenic enzymes by Real-Time PCR or immunoblotting analysis.ResultsAfter the 16-week feeding period, serum linoleic acid (LA) and α-linolenic acid (ALA) significantly increased and serum palmitic acid (PA) significantly decreased in SOY group mice. Compared to the normal diet (ND group), increasing intake of soybean oil raised the luteinizing hormone (LH) levels and up-regulated luteinizing hormone/chorionic gonadotropin receptor (LHCGR), steroidogenic acute regulatory protein (StAR) and cytochrome P450 family 11 subfamily A member I (CYP11A1). Testosterone levels in SOY group were higher than that in the ND group, and significantly difference showed.ConclusionsIncreasing intake of soybean oil could raise the serum LA and ALA levels and decrease serum PA levels. This could activate the LH/LHCGR pathway and improve the function of steroid synthesis in Leydig cells, and finally lead to the elevated testosterone levels.

Performance of mutation pathogenicity prediction tools on missense variants associated with 46,XY differences of sex development

OBJECTIVES:Single nucleotide variants (SNVs) are the most common type of genetic variation among humans. High-throughput sequencing methods have recently characterized millions of SNVs in several thousand individuals from various populations, most of which are benign polymorphisms. Identifying rare disease-causing SNVs remains challenging, and often requires functional in vitro studies. Prioritizing the most likely pathogenic SNVs is of utmost importance, and several computational methods have been developed for this purpose. However, these methods are based on different assumptions, and often produce discordant results. The aim of the present study was to evaluate the performance of 11 widely used pathogenicity prediction tools, which are freely available for identifying known pathogenic SNVs: Fathmn, Mutation Assessor, Protein Analysis Through Evolutionary Relationships (Phanter), Sorting Intolerant From Tolerant (SIFT), Mutation Taster, Polymorphism Phenotyping v2 (Polyphen-2), Align Grantham Variation Grantham Deviation (Align-GVGD), CAAD, Provean, SNPs&GO, and MutPred.METHODS:We analyzed 40 functionally proven pathogenic SNVs in four different genes associated with differences in sex development (DSD): 17β-hydroxysteroid dehydrogenase 3 (HSD17B3), steroidogenic factor 1 (NR5A1), androgen receptor (AR), and luteinizing hormone/chorionic gonadotropin receptor (LHCGR). To evaluate the false discovery rate of each tool, we analyzed 36 frequent (MAF>0.01) benign SNVs found in the same four DSD genes. The quality of the predictions was analyzed using six parameters: accuracy, precision, negative predictive value (NPV), sensitivity, specificity, and Matthews correlation coefficient (MCC). Overall performance was assessed using a receiver operating characteristic (ROC) curve.RESULTS:Our study found that none of the tools were 100% precise in identifying pathogenic SNVs. The highest specificity, precision, and accuracy were observed for Mutation Assessor, MutPred, SNP, and GO. They also presented the best statistical results based on the ROC curve statistical analysis. Of the 11 tools evaluated, 6 (Mutation Assessor, Phanter, SIFT, Mutation Taster, Polyphen-2, and CAAD) exhibited sensitivity >0.90, but they exhibited lower specificity (0.42-0.67). Performance, based on MCC, ranged from poor (Fathmn=0.04) to reasonably good (MutPred=0.66).CONCLUSION:Computational algorithms are important tools for SNV analysis, but their correlation with functional studies not consistent. In the present analysis, the best performing tools (based on accuracy, precision, and specificity) were Mutation Assessor, MutPred, and SNPs&GO, which presented the best concordance with functional studies.

SUN-LB15 A Case of Familial Male-Limited Precocious Puberty With a Novel Mutation

Introduction:Familial Male-Limited Precocious Puberty (FMPP), also known as testotoxicosis, is a rare cause of precocious puberty in males. It is caused by a mutation in the luteinizing hormone/chorionic gonadotropin receptor (LHCGR) gene, resulting in the receptor to be constitutively activated. This results in precocious puberty in males with rapid skeletal maturation and compromised final adult height if not treated. A combination on anti-androgens and aromatase inhibitors are the mainstay of treatment.Case:A 16-month-old male presented to our clinic due to concerns of precocious puberty. Pubic hair was noted at 6 months of age along with rapid growth acceleration. There was no known exposure to testosterone and no family history of early puberty or subnormal adult height. Upon presentation, his height was over the 99th percentile while his target height is at the 50th percentile. Physical examination revealed prepubertal testes with pubic hair at Tanner stage II. A bone age was reported as 12-18 months at a chronologic age of 12 months. Initial lab evaluation revealed normal serum levels of 17 OHP, androstenedione and DHEA-S with a slightly elevated serum LH (0.7mIU/ml), and very elevated serum testosterone level (289ng/dl). Leuprolide and ACTH stimulation tests as well as MRI of the abdomen and pelvis were normal. Serum levels of AFP, β-hCG and IGF-1 were also normal. Bone age advanced to 6 years at a chronological age of 21 months and to 8 years by 26 months of age. At 26 months of age, his stretched penile length measured 11cm. LH receptor testing was obtained, which revealed a novel heterozygous missense mutation in the LHCGR gene (c.1733A>C; p.Asp.578Ala). The mutation was reported to be a variant of unknown significance though likely in the pathological end of the spectrum by variant analysis with SIFT and PolyPhen. He was started on treatment with anastrozole 1 mg daily and bicalutamide 25 mg daily at 2 and 9/12 years of age. Pubertal progression, linear growth and skeletal maturation slowed down with treatment. His bone age remained stable at 13 years from the age of 2 and 10/12 years to 4 years old. Pubertal stage and stretched penile length have also remained stable on treatment. He is tolerating treatment well.Conclusion:We report a novel mutation in the LHCGR gene causing FMPP in a 6-month-old male who is responding very well to a combination of bicalutamide and anastrazole with marked decrease in growth velocity, pubertal progression and skeletal advancement.

SUN-035 Performance of Mutation Pathogenicity Prediction Tools on Missense Variants Associated with 46XY Disorders of Sex Development

INTRODUCTION: Single nucleotide variants (SNV) are the most common type of genetic variation among humans. The high-throughput sequencing methods increase the number of identified variants in the human genome but functional studies for disease-associated variants is laborious and time consuming. Different computational methods have been developed to predict SNP pathogenicity and phenotypic effect. They are based on different variant parameters, such as sequence homology, proteins structure and evolutionary conservation. OBJECTIVE/METHODS: To evaluate the performance of the 10 widely used pathogenicity prediction tools available in the internet in relation with functional studies: Fathmn, Mutation Acessor, Phanter and Sift sites (based on evolutionary information) and Mutation Taster, Polyphen-2, Align GVGD, CAAD, Provean and SNP-and-Go sites (based in a combination of protein structural, functional parameters and evolutionary information). We analyzed 40 described pathogenic mutation in 4 different genes associated with disorder of sex development (DSD): 17β-hydroxysteroid dehydrogenase (HSD17B3), Steroidogenic factor 1 (SF-1/NR5A1), Androgen receptor (AR) and Luteinizing hormone/chorionic gonadotropin receptor (LHCGR). All mutations are already published and functional studies showed loss of function proteins. To evaluate the false discovery rate of each tool, we analyzed 36 frequent (MAF >0.01) benign SNVs described in the same 4 DSD. The quality of the predictions was analyzed by five parameters: accuracy, precision, sensitivity, specificity and Matthews correlation coefficient (MCC). RESULTS: No method was in full accordance with the functional study. The best accuracy was observed in the Polyphen-2 and SNP-and-Go programs (0.81 and 0.82); the best precision was observed in Mutation Accessor and SNP-and-Go (0.96 and 0.84). The best specificity was observed for Mutation Accessor and SNP-and-GO (0.96 and 0.83). Five programs (Phanter, Sift, Mutation Taster, Polyphen-2 and CAAD) showed sensitivity > 0.80, but only SNP-and-GO program had specificity of 0.83. Performance ranged from poor (Align GVGD - MCC 0.13) to reasonably good (SNP-and-GO - MCC 0.63). CONCLUSION: Computational algorithms are important tools for SNV analysis but their correlation with functional studies is extremely variable. The overall best performing methods among the 10 prediction tools was SNP-and-GO, with accuracy reaching 0.82.

Sp1 regulates steroidogenic genes and LHCGR expression in primary human luteinized granulosa cells

Luteinizing hormone and human chorionic gonadotropin (hCG) bind to the luteinizing hormone/chorionic gonadotropin receptor (LHCGR). LHCGR is required to maintain corpus luteum function but the mechanisms involved in the regulation of LHCGR in human luteal cells remain incompletely understood. This study aimed to characterize the expression of LHCGR mRNA in primary human luteinized granulosa cells (hLGCs) obtained from patients undergoing in vitro fertilization and to correlate LHCGR expression with the response of hLGCs to hCG by assessing the expression of genes known to be markers of hCG actions. The results show that LHCGR expression is low in freshly isolated cells but recovers rapidly in culture and that hCG maintains LHCGR expression, suggesting a positive feedback loop. The activity of a LHCGR-LUC reporter increased in cells treated with hCG but not with follicle-stimulating hormone. Treatment with hCG also stimulated the expression of genes involved in steroidogenesis in a time-dependent manner. LHCGR promoter expression was found to be regulated by SP1, which we show is highly expressed in hLGCs. Moreover, SP1 inhibition prevented the stimulation of steroidogenic genes and the increase in LHCGR-LUC reporter activity by hCG. Finally, we provide evidence that a complex formed by SP1 and GATA4 may play a role in the maintenance of LHCGR expression. This report reveals the mechanisms involved in the regulation of the LHCGR and provides experimental data demonstrating that the proximal region of the LHCGR promoter is sufficient to drive the expression of this gene in primary hLGCs.

β-hCG promotes epithelial ovarian cancer metastasis through ERK/MMP2 signaling pathway

ABSTRACTEpithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy, with typically extensive intraperitoneal implantation leading to poor prognosis. Our previous study preliminarily demonstrated β-hCG can promote tumorigenesis in immortalized nontumorigenic ovarian epithelial cells. In this study, the roles and mechanisms of β-hCG in regulating EOC proliferation and metastasis were thoroughly explored. First, histologically, β-hCG was aberrantly overexpressed in human EOC metastatic tissues, and significantly correlated with FIGO stage, tumor size, differentiation, histologic grade and high grade serous ovarian carcinoma (HGSOC) (P < 0.05). However, serologically, β-hCG expression showed no significant difference between EOC and nonmalignant ovarian patients. Second, β-hCG was confirmed to have no significant effects on EOC proliferation in vitro and in vivo, while β-hCG upregulation was proven to promote migration and invasion ability in ES-2 and OVCAR-3 cells in vitro (P < 0.05), and β-hCG downregulation in SKOV3 cells had the opposite effect. Moreover, more invadopodia protrusions, mitochondria accumulations and cytoskeletal rearrangements were observed in β-hCG-overexpressing ES-2 cells, while β-hCG-depleted SKOV3 cells produced the opposite effect. Furthermore, β-hCG was confirmed to clearly facilitate intraperitoneal metastasis in nude mouse orthotopic ovarian xenograft models. Importantly, these effects of β-hCG were mediated by activation of the ERK/MMP2 signaling pathway, independently of luteinizing hormone/chorionic gonadotropin receptor (LHCGR) presence, and inhibition the pathway with the p-ERK1/2 inhibitor SCH772984 significantly impaired the tumor-promoting effects induced by β-hCG. Collectively, these data provide new insight into the roles and mechanisms of β-hCG in regulating EOC metastasis through ERK/MMP2 signaling pathway and may become a new target for therapeutic intervention.

Diseases caused by mutations in luteinizing hormone/chorionic gonadotropin receptor.

Accumulating evidence showed that the luteinizing hormone/chorionic gonadotropin receptor (LHCGR) is an essential regulator of sexual development and reproduction from zebrafish to human. Activating and inactivating mutations of LHCGR gene have been identified from patients of different phenotypes. Familial male-limited precocious puberty, Leydig cell hypoplasia, and empty follicle syndrome are caused by LHCGR mutations. More than 50 mutations have been reported from subjects of different ethnic backgrounds. Functional analyses of the mutant LHCGR revealed multiple defects, including cell surface expression, ligand binding, and signaling. The difference of the two native ligands and signaling pathway activated by LHCGR are illustrated. Potential therapeutic implications from the analyses of the naturally occurring LHCGR mutations, such as pharmacological chaperones, are highlighted.

MiR-122 Regulates LHR Expression in Rat Granulosa Cells by Targeting Insig1 mRNA

Luteinizing hormone/chorionic gonadotropin receptor (LHR) expression in the ovary is regulated by a messenger RNA (mRNA) binding protein, which specifically binds to the coding region of LHR mRNA. We have shown that miR-122, a short noncoding RNA, mediates LHR mRNA levels by modulating the expression of LHR mRNA-binding protein (LRBP) through the regulation of sterol regulatory element binding protein (SREBP) activation. The present results show that miR-122 regulates LRBP levels by increasing the processing of SREBP through the degradation of Insig1, the anchoring protein of SREBP. We present evidence showing that mRNA and protein levels of Insig1 undergo a time-dependent increase following the treatment of rat granulosa cells with follicle-stimulating hormone (FSH), which leads to a decrease in LRBP levels. Furthermore, overexpression of miR-122 using an adenoviral vector (AdmiR-122) abolished FSH-induced increases in Insig1 mRNA and protein. We further confirmed the role of Insig1 by showing that inhibition of Insig1 using a specific small interfering RNA prior to FSH treatment resulted in the abrogation of LHR upregulation. Silencing of Insig1 also reversed FSH-mediated decreases in SREBP and LRBP activation. These results show that decreased levels of miR-122 increase Insig1 and suppress SREBP processing in response to FSH stimulation of rat granulosa cells.

Leutinizing hormone/choriogonadotropin receptor and follicle stimulating hormone receptor gene variants and risk of recurrent pregnancy loss: A case control study

We investigated the association of luteinizing hormone/chorionic gonadotropin receptor (LHCGR) and follicle stimulating hormone receptor (FSHR) polymorphisms with recurrent pregnancy loss (RPL). This was a retrospective case-control study involving 308 women with RPL and 310 control women. FSHR and LHCGR genotyping was done by allelic exclusion method. Lower minor allele frequency (MAF) of LHCGR rs7371084, and rs4597581, and FSHR rs6166, and higher MAF of rs1007541, and rs2055571 FSHR variants were seen in RPL cases. Significantly lower frequencies of heterozygous LHCGR rs2293275, rs7371084, and rs4597581, and homozygous FSHR rs6166, and higher frequency of homozygous FSHR rs2055571 carriers were seen in RPL cases, after controlling for key covariates. LHCGR rs7371084 and rs4597581 were negatively associated with RPL in non-obese subjects. On the other hand, FSHR rs1007541 and rs2055571 were positively, while FSHR rs6166 was negatively associated with RPL in obese subjects. Limited linkage disequilibrium was seen only among FSHR variants; higher frequency of GTAAA and AAGGA, and lower frequency of GTGGG FSHR haplotypes was seen in RPL cases; the frequencies of remaining FSHR haplotypes, and all LHCGR haplotypes were similar between cases and controls. This is the first study to demonstrate association of novel LHCGR (rs7371084 and rs4597581) and FSHR (rs6166, rs1007541 and rs2055571) SNPs with RPL, particularly according to obesity.

HCG: Biological Functions and Clinical Applications.

Human chorionic gonadotropin (hCG) is produced primarily by differentiated syncytiotrophoblasts, and represents a key embryonic signal that is essential for the maintenance of pregnancy. hCG can activate various signaling cascades including mothers against decapentaplegic homolog 2 (Smad2), protein kinase C (PKC), and/or protein kinase A (PKA) in several cells types by binding to luteinizing hormone/chorionic gonadotropin receptor (LHCGR) or potentially by direct/indirect interaction with transforming growth factor beta receptor (TGFβR). The molecule displays specialized roles in promoting angiogenesis in the uterine endothelium, maintaining myometrial quiescence, as well as fostering immunomodulation at the maternal-fetal interface. It is a member of the glycoprotein hormone family that includes luteinizing hormone (LH), thyroid-stimulating hormone (TSH), and follicle-stimulating hormone (FSH). The α-subunit of hCG displays homologies with TSH, LH, and FSH, whereas the β subunit is 80–85% homologous to LH. The hCG molecule is produced by a variety of organs, exists in various forms, exerts vital biological functions, and has various clinical roles ranging from diagnosis and monitoring of pregnancy and pregnancy-related disorders to cancer surveillance. This review presents a detailed examination of hCG and its various clinical applications.

Downregulation of LH and FSH receptors after hCG and eCG treatments in the porcine oviduct

The influence of induction of ovulation and superovulation with eCG and hCG on LH and FSH receptor levels in porcine oviducts on day 3 postcoitum was studied. In experiment I, gilts were assigned into cyclic (control; n = 5) and inseminated (n = 5) groups. In experiment II, there were 3 groups of animals: inseminated (n = 5), induced ovulation/inseminated (750 IU eCG, 500 IU hCG; n = 5) and superovulated/inseminated (1500 IU eCG, 1000 IU hCG; n = 5) gilts. Oviduct tissues were collected 3 d after insemination or PBS infusion. The messenger RNA (mRNA) expression of FSH receptor (FSHR) and luteinizing hormone/chorionic gonadotropin receptor (LH/CGR) was measured by real-time reverse transcription PCR and protein levels using Western blots. Localization of LH/CGR and FSHR-positive cells was studied by immunohistochemical staining. Insemination by itself did not influence mRNA and protein levels of LH/CGR. However, FSHR mRNA expression in the isthmus and ampulla of the oviduct was affected by insemination (P < 0.05). Similarly, insemination decreased FSHR protein level in the isthmus (P < 0.05). Stimulation with hCG and eCG did not affect LH/CGR and FSHR mRNA expression, either in the isthmus or in the ampulla. Nevertheless, superovulation decreased LH/CGR protein level in the oviductal ampulla (P < 0.05) in comparison with inseminated gilts. Similarly, protein levels of FSHR in the oviductal ampulla decreased after superovulation (P < 0.05). LH/CGR-positive cells were observed in the mucosa as well as in smooth muscle cells of both parts of the oviduct. Follicle-stimulating hormone receptor–positive cells were observed in smooth muscle cells and blood vessels of the isthmus. In the ampulla, FSHR-positive cells were observed in the smooth muscle as well as in the mucosa. Summarizing, the present study revealed for the first time that stimulation with eCG and hCG, especially in high doses, can change LH/CGR and FSHR levels in porcine oviducts. This may in turn alter many signaling pathways, eg, PGs or vascular endothelial growth factor synthesis, and consequently disturb the oviductal environment, with possible detrimental effects on fertilization and/or embryonic development.

MiRNA-592 is downregulated and may target LHCGR in polycystic ovary syndrome patients

The polycystic ovary syndrome (PCOS) is an endocrine disorder mainly associated with infertility. Abnormal regulation of relevant genes is required for follicular development in PCOS. In the current study, the expression of serum miRNAs of PCOS patients was explored using miRNA array followed by qRT-PCR assays. The circulating level of miR-592 was significantly down-regulated in PCOS patients in comparison with healthy controls. Furthermore, we found that miR-592 was inversely correlated with the level of luteinizing hormone/chorionic gonadotropin receptor (LHCGR). Computational analysis predicted that miR-592 interacts with the LHCGR mRNA via binding to a site located in the 3′UTR region. Using a luciferase-based reporter assay we found that miR-592 directly targeted the LHCGR. In KGN cell line, miR-592 overexpression inhibited cell viability and the transition of phase G1 to phase S. Knocking down of LHCGR inhibited cell viability and cell cycle progression in KGN cells, and LHCGR co-transfection reversed the inhibitory effect of miR-592. These results shed new light on the diagnosis and treatment of PCOS syndrome.

Genetic Models for the Study of Luteinizing Hormone Receptor Function.

The luteinizing hormone/chorionic gonadotropin receptor (LHCGR) is essential for fertility in men and women. LHCGR binds luteinizing hormone (LH) as well as the highly homologous chorionic gonadotropin. Signaling from LHCGR is required for steroidogenesis and gametogenesis in males and females and for sexual differentiation in the male. The importance of LHCGR in reproductive physiology is underscored by the large number of naturally occurring inactivating and activating mutations in the receptor that result in reproductive disorders. Consequently, several genetically modified mouse models have been developed for the study of LHCGR function. They include targeted deletion of LH and LHCGR that mimic inactivating mutations in hormone and receptor, expression of a constitutively active mutant in LHCGR that mimics activating mutations associated with familial male-limited precocious puberty and transgenic models of LH and hCG overexpression. This review summarizes the salient findings from these models and their utility in understanding the physiological and pathological consequences of loss and gain of function in LHCGR signaling.

Human chorionic gonadotropin stimulates spheroid attachment on fallopian tube epithelial cells through the mitogen-activated protein kinase pathway and down-regulation of olfactomedin-1

To study the effect of human chorionic gonadotropin (hCG) on olfactomedin-1 (Olfm1) expression and spheroid attachment in human fallopian tube epithelial cells in vitro. Experimental study. Reproductive biology laboratory. Healthy nonpregnant women. No patient interventions. Luteinizing hormone/chorionic gonadotropin receptor (LHCGR) and Olfm1 expression in fallopian tube epithelium cell line (OE-E6/E7 cells). OE-E6/E7 cells treated with hCG, U0126 extracellular signal-regulated kinase (ERK) inhibitor, or XAV939 Wnt/β-catenin inhibitor were analyzed by Western blotting, real-time polymerase chain reaction, and in vitro spheroid attachment assay. Human chorionic gonadotropin increased spheroid attachment on OE-E6/E7 cells through down-regulation of Olfm1 and activation of Wnt and mitogen-activated protein kinase (MAPK) signaling pathways. U0126 down-regulated both MAPK and Wnt/β-catenin signaling pathways and up-regulated Olfm1 expression. XAV939 down-regulated only the Wnt/β-catenin signaling pathway but up-regulated Olfm1 expression. Human chorionic gonadotropin activated both ERK and Wnt/β-catenin signaling pathways and enhanced spheroid attachment on fallopian tube epithelial cells through down-regulation of Olfm1 expression.

Association of luteinizing hormone chorionic gonadotropin receptor gene polymorphism (rs2293275) with polycystic ovarian syndrome.

Polycystic ovaries and irregular menstruation/anovulation are important diagnostic criteria along with hyperandrogenism as per the Androgen Excess Society-2006 criteria for polycystic ovarian syndrome (PCOS). In the etiopathogenesis of PCOS, one of the candidate genes causing ovarian failure is the luteinizing hormone (LH) chorionic gonadotropin hormone receptor (LHCGR). Our aim was to study the association of LHCGR polymorphism (rs2293275) with PCOS in our study population. Genetic case-control study from multiple gynecological centers from Hyderabad, a cosmopolitan city in South India. The study involved 204 women with PCOS and 204 healthy, sex-, and age-matched controls. Anthropometric and biochemical profiles were taken in a well-designed pro forma. Isolation of deoxyribonucleic acid (DNA) and genotype analysis were done for the entire study population using the polymerase chain reaction-restriction fragment length polymorphism method followed by 12% polyacrylamide gel electrophoresis. In this study, we have demonstrated an association between LHCGR (rs2293275) polymorphism and PCOS. The frequency of the G allele was 0.60 in PCOS and 0.49 in controls (odds ratio [OR] 1.531, confidence interval [CI] 1.16-2.01, and p-value=0.0026), which indicates that the G allele is associated with PCOS in our population. The GG genotype conferred a significant risk of developing PCOS (OR 3.36, CI 1.96-5.75, and p-value<0.0001). We found a significant association of the GG allele with body-mass index, waist to hip ratio, insulin resistance, LH, and LH/follicle-stimulating hormone (FSH) ratio in PCOS when compared with controls. The AA allele showed high basal FSH levels. This study suggests that LHCGR (rs2293275) polymorphism is associated with PCOS and could be used as a relevant molecular marker to identify women with the risk of developing PCOS in our population and may provide an understanding about the etiology of PCOS.

MicroRNA miR-513a-3p acts as a co-regulator of luteinizing hormone/chorionic gonadotropin receptor gene expression in human granulosa cells

The luteinizing hormone/chorionic gonadotropin receptor (LHCGR) is essential for normal male and female reproductive processes. The spatial and temporal LHCGR gene expression is controlled by a complex system of regulatory mechanisms which are crucial for normal physiological function, especially during the female cycle. In this study, we aimed to elucidate whether microRNAs are involved in this network and play a role in regulating LHCGR expression. Computational analysis predicted that miR-513a-3p interacts with the LHCGR mRNA via three binding sites located in the 3′UTR region, enabling a synergistic action. Moreover, using a luciferase-based reporter assay we found that miR-513a-3p targets the LHCGR, resulting in a significant down-regulation of its expression. In human primary granulosa cell cultures we detected a dynamic, inversely associated expression pattern of miR-513a-3p and the LHCGR. In addition, transfection with miR-513a-3p or its specific inhibitor led to a down- or up-regulation at the LHCGR mRNA level, respectively. An increased amount of miR-513a-3p resulted in the down-regulation of the LHCGR mRNA, reflected by the attenuation of cAMP synthesis after hormonal stimulation. In conclusion, these data demonstrate that miR-513a-3p is involved in the control of the LHCGR expression by an inversely regulated mechanism at the post-transcriptional level and show for the first time that this kind of post-transcriptional process contributes to the multifaceted system of the human LHCGR regulation.

Neonatal exposure to 17α-ethynyl estradiol affects ovarian gene expression and disrupts reproductive cycles in female rats

Neonatal exposure to synthetic estrogen causes delayed reproductive dysfunction in female rats. Exposure to 17α-ethynyl estradiol (EE, low: 20 and high: 2000μg/kg) induced an abnormal estrous cycle during PND171–190 in low-dose and PND126–145 in high-dose group. At PND90 within normal estrous cycle, high-dose animals showed lack of LH surge and low of ovarian hormones in serum level. Gene expression analysis demonstrated that level of mRNA encoding luteinizing hormone/chorionic gonadotropin receptor (LHCGR) was higher in EE-treated ovaries than in control ovaries, and LHCGR protein colocalized with apoptosis-related proteins in the interstitial area of the ovary. At PND1, ovarian LHCGR mRNA levels were higher in EE-treated rats than in control rats, and direct induction of LHCGR expression by EE was observed in vitro. Our results indicate that neonatal exposure to EE induces irregular LHCGR expression in the immature ovary, which may influence the occurrence of delayed reproductive dysfunction in adult animals.