In-vivo studies of targeted and localized cancer drug release from microporous poly-di-methyl-siloxane (PDMS) devices for the treatment of triple negative breast cancer.

Triple-negative breast cancer (TNBC) treatment is challenging and frequently characterized by an aggressive phenotype and low prognosis in comparison to other subtypes. This paper presents fabricated implantable drug-loaded microporous poly-di-methyl-siloxane (PDMS) devices for the delivery of targeted therapeutic agents [Luteinizing Hormone-Releasing Hormone conjugated paclitaxel (PTX-LHRH) and Luteinizing Hormone-Releasing Hormone conjugated prodigiosin (PG-LHRH)] for the treatment and possible prevention of triple-negative cancer recurrence. In vitro assessment using the Alamar blue assay demonstrated a significant reduction (p < 0.05) in percentage of cell growth in a time-dependent manner in the groups treated with PG, PG-LHRH, PTX, and PTX-LHRH. Subcutaneous triple-negative xenograft breast tumors were then induced in athymic female nude mice that were four weeks old. Two weeks later, the tumors were surgically but partially removed, and the device implanted. Mice were observed for tumor regrowth and organ toxicity. The animal study revealed that there was no tumor regrowth, six weeks post-treatment, when the LHRH targeted drugs (LHRH-PTX and LHRH-PGS) were used for the treatment. The possible cytotoxic effects of the released drugs on the liver, kidney, and lung are assessed using quantitative biochemical assay from blood samples of the treatment groups. Ex vivo histopathological results from organ tissues showed that the targeted cancer drugs released from the implantable drug-loaded device did not induce any adverse effect on the liver, kidneys, or lungs, based on the results of qualitative toxicity studies. The implications of the results are discussed for the targeted and localized treatment of triple negative breast cancer.

Gonadotropin-Releasing Hormone Receptor (GnRHR) and Hypogonadotropic Hypogonadism.

Human sexual and reproductive development is regulated by the hypothalamic-pituitary-gonadal (HPG) axis, which is primarily controlled by the gonadotropin-releasing hormone (GnRH) acting on its receptor (GnRHR). Dysregulation of the axis leads to conditions such as congenital hypogonadotropic hypogonadism (CHH) and delayed puberty. The pathophysiology of GnRHR makes it a potential target for treatments in several reproductive diseases and in congenital adrenal hyperplasia. GnRHR belongs to the G protein-coupled receptor family and its GnRH ligand, when bound, activates several complex and tissue-specific signaling pathways. In the pituitary gonadotrope cells, it triggers the G protein subunit dissociation and initiates a cascade of events that lead to the production and secretion of the luteinizing hormone (LH) and follicle-stimulating hormone (FSH) accompanied with the phospholipase C, inositol phosphate production, and protein kinase C activation. Pharmacologically, GnRHR can be modulated by synthetic analogues. Such analogues include the agonists, antagonists, and the pharmacoperones. The agonists stimulate the gonadotropin release and lead to receptor desensitization with prolonged use while the antagonists directly block the GnRHR and rapidly reduce the sex hormone production. Pharmacoperones include the most recent GnRHR therapeutic approaches that directly correct the misfolded GnRHRs, which are caused by genetic mutations and hold serious promise for CHH treatment. Understanding of the GnRHR's genomic and protein structure is crucial for the most appropriate assessing of the mutation impact. Such mutations in the GNRHR are linked to normosmic hypogonadotropic hypogonadism and lead to various clinical symptoms, including delayed puberty, infertility, and impaired sexual development. These mutations vary regarding their mode of inheritance and can be found in the homozygous, compound heterozygous, or in the digenic state. GnRHR expression extends beyond the pituitary gland, and is found in reproductive tissues such as ovaries, uterus, and prostate and non-reproductive tissues such as heart, muscles, liver and melanoma cells. This comprehensive review explores GnRHR's multifaceted role in human reproduction and its clinical implications for reproductive disorders.

Discovery of microglia gonadotropin­releasing hormone receptor and its potential role in polycystic ovarian syndrome.

Hypothalamic inflammation is a pathophysiological basis of polycystic ovarian syndrome (PCOS), while overactivated and/or excess M1 polarized microglia are considered to be the main reason for the occurrence of hypothalamic inflammation. Therefore, in vitro and in vivo experiments were performed to assess the relationships between microglia­mediated inflammatory reactions and endocrine functions in the PCOS hypothalamus. The expression of gonadotropin­releasing hormone (GnRH) receptor (GnRHR) was demonstrated in hypothalamic microglia, and it was found that low concentration, GnRH agonist, leuprolide acetate accelerated the expression of M2 polarization marker CD206, while high concentration leuprolide acetate increased the expression of M1 polarization marker CD86 in vitro. Furthermore, aerobic exercise not only reduced the levels of serum testosterone, luteinizing hormone and GnRH and the amount of overactivated microglia, but also increased the number of M2 microglia in the hypothalamus of letrozole­induced PCOS rats. In combination, these results not only demonstrated the expression of GnRHR in hypothalamic microglia, but also demonstrated that GnRH can induce microglial polarization, while aerobic exercise may improve the microglia­mediated inflammatory reaction by reducing the expression of GnRHR in the hypothalamic microglia of PCOS rats.

Role of gonadotropin-releasing hormone 2 and its receptor in human reproductive cancers.

Gonadotropin-releasing hormone (GnRH1) and its receptor (GnRHR1) drive reproduction by regulating gonadotropins. Another form, GnRH2, and its receptor (GnRHR2), also exist in mammals. In humans, GnRH2 and GnRHR2 genes are present, but coding errors in the GnRHR2 gene are predicted to hinder full-length protein production. Nonetheless, mounting evidence supports the presence of a functional GnRHR2 in humans. GnRH2 and its receptor have been identified throughout the body, including peripheral reproductive tissues like the ovary, uterus, breast, and prostate. In addition, GnRH2 and its receptor have been detected in a wide number of reproductive cancer cells in humans. Notably, GnRH2 analogues have potent anti-proliferative, pro-apoptotic, and/or anti-metastatic effects on various reproductive cancers, including endometrial, breast, placental, ovarian, and prostate. Thus, GnRH2 is an emerging target to treat human reproductive cancers.

GNRHR-related central hypogonadism with spontaneous recovery - case report.

BACKGROUND: Congenital hypogonadotropic hypogonadism (CHH) is a clinically and genetically heterogeneous disease characterized by absent or incomplete puberty and infertility. Clinical characteristics are secondary to insufficient gonadotropin secretion, caused by deficient gonadotropin-releasing hormone (GnRH) production, secretion, or action. Loss-of-function variants of the gonadotropin-releasing hormone receptor (GNRHR) are associated with CHH without anosmia. CHH was previously considered a permanent condition, but in the past two decades, cases of spontaneous recovery of CHH have been reported. The reversal of hypogonadism in CHH is currently unpredictable, and can happen unnoticed. CASE PRESENTATION: The male proband was diagnosed with CHH due to compound heterozygosity for two previously reported pathogenic missense variants in the GNRHR gene, NM_000406.2:c.416G > A (NP_000397.1:p.Arg139His) and c.785G > A (p.Arg262Gln) at 16 years of age. In addition to arrested partial puberty, he had a low testosterone level, gonadotropins in the range of early puberty, and a normal inhibin B level. A therapy with increasing doses of intramuscular testosterone undecanoate was received for 2.5 years, while there was no change in testicular volume. At the age of 19 years, testosterone supplementation was interrupted. During the next two years, he had spontaneous pubertal development to achieve a testicular volume of 20 mL, with normal adult levels of gonadotropins and testosterone. CONCLUSIONS: Genetic diagnostics can help discriminate congenital hypogonadotropic hypogonadism, deserving therapeutic intervention, from the self-limited constitutional delay of growth and puberty (CDGP). Patients with GNRHR associated hypogonadism can experience spontaneous recovery of the hypothalamic-pituitary-gonadal axis. Spontaneous testis enlargement in patients with central hypogonadism not taking gonadotropins or pulsatile GnRH therapy can indicate recovery of hypogonadism.

Pituitary gonadotroph-specific patterns of gene expression and hormone secretion.

Pituitary gonadotrophs play a key role in reproductive functions by secreting luteinizing hormone (LH) and follicle-stimulating hormone (FSH). The LH secretory activity of gonadotroph is controlled by hypothalamic gonadotropin-releasing hormone (GnRH) via GnRH receptors and is accompanied by only minor effects on high basal Lhb gene expression. The secretory profiles of GnRH and LH are highly synchronized, with the latter reflecting a depletion of prestored LH in secretory vesicles by regulated exocytosis. In contrast, FSH is predominantly released by constitutive exocytosis, and secretory activity reflects the kinetics of Fshb gene expression controlled by GnRH, activin, and inhibin. Here is a review of recent data to improve the understanding of multiple patterns of gonadotroph gene expression and hormone secretion.

MicroRNA profiling predicts positive nodal status in papillary thyroid carcinoma in the preoperative setting.

BACKGROUND: The molecular characterization of thyroid nodules in cytological samples has so far been focused on discriminating between benign and malignant forms in a purely diagnostic setting. The evidence on the impact of molecular biomarkers to determine the risk of aggressiveness in cytologically "neoplastic" lesions is limited to genomic alterations (such as BRAF and TERT mutations). The aim of our study was to assess the preoperative role of microRNAs (miRNAs) in predicting the nodal status of patients with papillary thyroid cancer. METHODS: A pilot series of histological samples of papillary thyroid carcinoma with (6 cases) or without (6 cases) lymph node metastases, matched for other major clinical and pathological features, was analyzed for global miRNA expression in a screening phase. A set of miRNAs was then validated in a series of 63 consecutive cytological samples of papillary carcinomas: 48 pN-negative and 15 pN-positive at histology. RESULTS: Unsupervised cluster analysis segregated surgical pN-negative and pN-positive samples, except for 1 case. The 45 differentially expressed miRNAs in pN-positive versus pN-negative cases were predicted to regulate a wide range of cellular pathways, enriched for Wnt, gonadotropin-releasing hormone receptor, and cerulein/cholecystokinin receptor signaling. In agreement with their profiles in surgical samples, 4 miRNAs of the 10 selected for validation (miR-154-3p, miR-299-5p, miR-376a-3p, and miR-302E) had a significant differential expression in cytological samples of papillary carcinoma with lymph node metastases and predicted the positive nodal status with a relatively good performance. CONCLUSIONS: MiRNA profiling is a potential promising strategy to define papillary carcinoma aggressiveness in the preoperative setting.

Gonadotropin-releasing hormone receptor inhibits triple-negative breast cancer proliferation and metastasis.

BACKGROUND: Gonadotropin-releasing hormone receptor (GnRHR) is expressed in several malignant tumors and inhibits the proliferation and metastasis of cancer cells, but its role in triple-negative breast cancers (TNBCs) is unclear. This study investigated the biological effects of GnRHR and their influence on TNBC prognosis. METHODS: The GSE21653 database was used to obtain information about GnRHR expression and clinicopathological factors in patients with TNBC. GnRHR was activated in cultured MDA-MB-231 and MDA-MB-468 cells by leuprolide acetate and antagonized by elagolix sodium. Cell proliferation was assessed by the cell counting kit-8 and colony formation assays. Cell metastasis was detected by the wound healing assay and Transwell assay. Apoptosis and the cell cycle were investigated by flow cytometry. GnRHR protein expression was determined by western blotting. RESULTS: GnRHR mRNA expression was significantly higher in patients with TNBC than in hormone receptor+/human epidermal growth factor receptor (HER)2- and HER2+ patients with breast cancer. Patients with high GnRHR expression had significantly better disease-free survival than those with lower expression. Activated GnRHR significantly inhibited cell proliferation and metastasis, increased apoptosis, and enhanced GnRHR protein expression levels. CONCLUSION: GnRHR inhibits TNBC proliferation and metastasis, suggesting it could be targeted for TNBC treatment.

Quantitative proteomic analysis of GnRH agonist treated GBM cell line LN229 revealed regulatory proteins inhibiting cancer cell proliferation.

BACKGROUND: Gonadotropin-releasing hormone (GnRH) receptor, a rhodopsin-like G-protein coupled receptor (GPCR) family member involved in GnRH signaling, is reported to be expressed in several tumors including glioblastoma multiforme (GBM), one of the most malignant and aggressive forms of primary brain tumors. However, the molecular targets associated with GnRH receptor are not well studied in GBM or in other cancers. The present study aims at investigating the effect of GnRH agonist (Gosarelin acetate) on cell proliferation and associated signaling pathways in GBM cell line, LN229. METHODS: LN229 cells were treated with different concentrations of GnRH agonist (10-10 M to 10-5 M) and the effect on cell proliferation was analyzed by cell count method. Further, total protein was extracted from control and GnRH agonist treated cells (with maximum reduction in cell proliferation) followed by trypsin digestion, labeling with iTRAQ reagents and LC-MS/MS analysis to identify differentially expressed proteins. Bioinformatic analysis was performed for annotation of proteins for the associated molecular function, altered pathways and network analysis using STRING database. RESULTS: The treatment with different concentrations of GnRH agonist showed a reduction in cell proliferation with a maximum reduction of 48.2% observed at 10-6 M. Quantitative proteomic analysis after GnRH agonist treatment (10-6 M) led to the identification of a total of 29 differentially expressed proteins with 1.3-fold change (23 upregulated, such as, kininogen-1 (KNG1), alpha-2-HS-glycoprotein (AHSG), alpha-fetoprotein (AFP), and 6 downregulated, such as integrator complex subunit 11 (CPSF3L), protein FRG1 (FRG1). Some of them are known [KNG1, AHSG, AFP] while others such as inter-alpha-trypsin inhibitor heavy chain H2 (ITIH2), ITIH4, and LIM domain-containing protein 1 (LIMD1) are novel to GnRH signaling pathway. Protein-protein interaction analysis showed a direct interaction of KNG1, a hub molecule, with GnRH, GnRH receptor, EGFR and other interactors including ITIH2, ITIH4 and AHSG. Overexpression of KNG1 after GnRH agonist treatment was validated using Western blot analysis, while a significant inhibition of EGFR was observed after GnRH agonist treatment. CONCLUSIONS: The study suggests a possible link of GnRH signaling with EGFR signaling pathways likely via KNG1. KNG1 inhibitors may be investigated independently or in combination with GnRH agonist for therapeutic applications.

Chronic Estrus Disrupts Uterine Gland Development and Homeostasis.

Female mice homozygous for an engineered Gnrhr E90K mutation have reduced gonadotropin-releasing hormone signaling, leading to infertility. Their ovaries have numerous antral follicles but no corpora lutea, indicating a block to ovulation. These mutants have high levels of circulating estradiol and low progesterone, indicating a state of persistent estrus. This mouse model provided a unique opportunity to examine the lack of cyclic levels of ovarian hormones on uterine gland biology. Although uterine gland development appeared similar to controls during prepubertal development, it was compromised during adolescence in the mutants. By age 20 weeks, uterine gland development was comparable to controls, but pathologies, including cribriform glandular structures, were observed. Induction of ovulations by periodic human chorionic gonadotropin treatment did not rescue postpubertal uterine gland development. Interestingly, progesterone receptor knockout mice, which lack progesterone signaling, also have defects in postpubertal uterine gland development. However, progesterone treatment did not rescue postpubertal uterine gland development. These studies indicate that chronically elevated levels of estradiol with low progesterone and therefore an absence of cyclic ovarian hormone secretion disrupts postpubertal uterine gland development and homeostasis.

Deletion of Gαq/11 or Gαs Proteins in Gonadotropes Differentially Affects Gonadotropin Production and Secretion in Mice.

Gonadotropin-releasing hormone (GnRH) regulates gonadal function via its stimulatory effects on gonadotropin production by pituitary gonadotrope cells. GnRH is released from the hypothalamus in pulses and GnRH pulse frequency differentially regulates follicle-stimulating hormone (FSH) and luteinizing hormone (LH) synthesis and secretion. The GnRH receptor (GnRHR) is a G protein-coupled receptor that canonically activates Gα q/11-dependent signaling on ligand binding. However, the receptor can also couple to Gα s and in vitro data suggest that toggling between different G proteins may contribute to GnRH pulse frequency decoding. For example, as we show here, knockdown of Gα s impairs GnRH-stimulated FSH synthesis at low- but not high-pulse frequency in a model gonadotrope-derived cell line. We next used a Cre-lox conditional knockout approach to interrogate the relative roles of Gα q/11 and Gα s proteins in gonadotrope function in mice. Gonadotrope-specific Gα q/11 knockouts exhibit hypogonadotropic hypogonadism and infertility, akin to the phenotypes seen in GnRH- or GnRHR-deficient mice. In contrast, under standard conditions, gonadotrope-specific Gα s knockouts produce gonadotropins at normal levels and are fertile. However, the LH surge amplitude is blunted in Gα s knockout females and postgonadectomy increases in FSH and LH are reduced both in males and females. These data suggest that GnRH may signal principally via Gα q/11 to stimulate gonadotropin production, but that Gα s plays important roles in gonadotrope function in vivo when GnRH secretion is enhanced.

Complete Androgen Insensitivity Syndrome: From the Relevance of an Accurate Genetic Diagnosis to the Challenge of Clinical Management. A Case Report.

Introduction: Androgen insensitivity syndrome (AIS), an X-linked recessive disorder of sex development (DSD), is caused by variants of the androgen receptor (AR) gene, mapping in the long arm of the X chromosome, which cause a complete loss of function of the receptor. Case presentation: We report a patient diagnosed with complete AIS (CAIS) at birth due to swelling in the bilateral inguinal region. Transabdominal ultrasound revealed the absence of the uterus and ovaries and the presence of bilateral testes in the inguinal region. The karyotype was 46,XY. She underwent bilateral orchiectomy at 9 months and was given estrogen substitutive therapy at the age of 11 years. Genetic analysis of the AR gene variants was requested when, at the age of 20, the patient came to our observation. Methods: The genetic testing was performed by next-generation sequence (NGS) analysis. Results: The genetic analysis showed the presence of the c.2242T>A, p.(Phe748Ile) variant in the AR gene. To the best of our knowledge, this variant has not been published so far. Furthermore, the patient has a heterozygous c.317A>G, p.(Gln106Arg) variation of the gonadotropin-releasing hormone receptor (GNRHR) gene, a heterozygous c.2273G>A, p.Arg758His variation of the chromodomain helicase DNA binding protein 7 (CHD7) gene, and compound heterozygous c.875A>G, p.Tyr292Cys, and c.8023A>G, p.Ile2675Val variations of the Dynein Axonemal Heavy Chain 11 (DNAH11) gene. Conclusions: The case herein reported underlines the importance of an accurate genetic analysis that has to include karyotype and AR gene variant analysis. This is useful to confirm a clinical diagnosis and establish the proper management of patients with CAIS. Numerous variants of the AR gene have not yet been identified. Moreover, several pitfalls are still present in the management of these patients. More studies are needed to answer unresolved questions, and common protocols are required for the clinical follow-up of patients with CAIS.

Molecular basis for the evolved instability of a human G-protein coupled receptor.

Membrane proteins are prone to misfolding and degradation. This is particularly true for mammalian forms of the gonadotropin-releasing hormone receptor (GnRHR). Although they function at the plasma membrane, mammalian GnRHRs accumulate within the secretory pathway. Their apparent instability is believed to have evolved through selection for attenuated GnRHR activity. Nevertheless, the molecular basis of this adaptation remains unclear. We show that adaptation coincides with a C-terminal truncation that compromises the translocon-mediated membrane integration of its seventh transmembrane domain (TM7). We also identify a series of polar residues in mammalian GnRHRs that compromise the membrane integration of TM2 and TM6. Reverting a lipid-exposed polar residue in TM6 to an ancestral hydrophobic residue restores expression with no impact on function. Evolutionary trends suggest variations in the polarity of this residue track with reproductive phenotypes. Our findings suggest that the marginal energetics of cotranslational folding can be exploited to tune membrane protein fitness.

Effective accumulative temperature affects gonadal maturation by controlling expression of GnRH, GnRH receptor, serotonin receptor and APGWamide gene in Pacific abalone, Haliotis discus hannai during broodstock conditioning in hatcheries.

Water temperature is a crucial environmental factor that influences reproductive function of abalone. Broodstock conditioning exposed to effective accumulative temperature (EAT) is a common practice in abalone hatcheries. To understand the molecular mechanism underlying the regulation of gonadal maturation and reproduction of Haliotis discus hannai exposed to EAT and induced spawning period, changes in expression of neuroendocrine genes encoding two gonadotropin releasing hormone (Hdh-GnRH, GnRH-like peptide), GnRH receptor (HdhGnRH-R), serotonin receptor (5-HTHdh) and Hdh-APGWamide in neural ganglia and gonadal tissues were examined. Gonadosomatic index (GSI) was significantly increased with increasing EAT °C-days. Expression levels of Hdh-GnRH, GnRH-like peptide, HdhGnRH-R, 5-HTHdh and Hdh-APGWamide mRNA were significantly increased with increasing EAT °C-days in ganglion (where the gene synthesized) and gonadal tissues. The significant increase in mRNA expression of each examined gene started from EAT 500 to 750°C-days, reached an initial peak at 1000°C-days, suggesting gonadal maturation started from the onset of EAT and slowly continued until 750°C-days, then at 1000°C-days reached to initial peak developmental period. The maturation reached to spawning state at 1000°C-days and peaked at 1500°C-days. Hdh-GnRH showed significantly higher mRNA expression in pleuropedal ganglion and branchial ganglion, whereas GnRH like peptide showed higher expression in cerebral ganglion, and HdhGnRH-R, 5-HTHdh and Hdh-APGWamide showed higher expression in pleuropedal ganglion. All genes were expressed higher at higher EAT °C-days. During induced spawning period, higher mRNA expression of examined genes was observed at the time of spawning; however, a sharp decrease occurred after spawning, suggesting that these genes are involved in spawning activities. Taken together, these results indicate that an increase of EAT °C-days can increase expression of neuroendocrine genes and enhance gonadal maturation. Besides all these genes are involved in the process of spawning induction, and increase of GSI has a positive correlation with the increase of gene expression.

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.

[Clinical and molecular genetic features of cases of isolated hypogonadotropic hypogonadism, associated with defects in GNRHR genes].

Congenital hypogonadotropic hypogonadism (CHH) is a rare disorder characterised by lack of pubertal development and infertility, due to deficient production, secretion or action of gonadotropin-releasing hormone (GnRH). Clinically, there are variants of CHH with hypo-/anosmia (Kalman syndrome) and normosmic hypogonadotropic hypogonadism. Given a  growing list of gene mutations accounting for CHH, the application of next generation sequencing (NGS) comprises an excellent molecular diagnostic approach because it enables the simultaneous evaluation of many genes. Biallelic mutations in GNRHR gene lead to the development of hypogonadotropic hypogonadism with normosmia. In this paper, we describe 16 patients with proven GnRH resistance and estimate the frequency of pathogenic variants in the GNRHR gene in the Russian population.

Association between GnRH Receptor Polymorphisms and Luteinizing Hormone Levels for Low Ovarian Reserve Infertile Women.

The choice of ovarian stimulation protocols in assisted reproduction technology (ART) cycles for low ovarian reserve patients is challenging. Our previous report indicated that the gonadotrophin-releasing (GnRH) agonist (GnRHa) protocol is better than the GnRH antagonist (GnRHant) protocol for young age poor responders. Here, we recruited 269 patients with anti-Müllerian hormone (AMH) < 1.2 ng/mL undergoing their first ART cycles for this nested case-control study. We investigated the genetic variants of the relevant genes, including follicular stimulating hormone receptor (FSHR; rs6166), AMH (rs10407022), GnRH (rs6185), and GnRH receptor (GnRHR; rs3756159) in patients <35 years (n = 86) and patients ≥35 years of age (n = 183). Only the genotype of GnRHR (rs3756159) is distributed differently in young (CC 39.5%, CT/TT 60.5%) versus advanced (CC 24.0%, CT/TT 76.0%) age groups (recessive model, p = 0.0091). Furthermore, the baseline luteinizing hormone (LH) levels (3.60 (2.45 to 5.40) vs. 4.40 (2.91 to 6.48)) are different between CC and CT/TT genotype of GnRHR (rs3756159). In conclusion, the genetic variants of GnRHR (rs3756159) could modulate the release of LH in the pituitary gland and might then affect the outcome of ovarian stimulation by GnRHant or GnRHa protocols for patients with low AMH levels.

Gonadotropin-releasing hormone receptor pathway affects the function of human EBV-transformed B lymphocytes in an age-independent way.

Immune system function changes during aging, but the molecular mechanisms of this phenomenon are not fully understood. The present study identified pathways that are associated with age-associated changes in human B lymphocytes. Initial in silico analysis of 1355 genes involved in aging revealed the strongest association (p = 4.36E-21) with the gonadotropin-releasing hormone receptor (GnRHR) pathway. Extended analysis of 2736 aging-related genes using updated databases confirmed such association (p = 2.41E-16). Genes involved in both aging and the GnRHR pathway were significantly involved in lymphocyte B and T activation and aging-related phenotypes, including hyperinsulinemia and diabetes, arthritis, cerebrovascular disease, and cancers. We, therefore, examined non-tumorigenic Epstein-Barr virus (EBV)-transformed B-lymphocyte cell lines that originated from 12 young subjects (20-31 years old) and 10 centenarians (100-102 years old). Gonadotropin-releasing hormone I (GnRH-I) and GnRHR levels did not depend on the age of the cell donors. Inhibition of the GnRHR pathway age-independently decreased cell proliferation (p < 0.001) and increased apoptosis (p < 0.001). However, the decrease in immunoglobulin G synthesis (p < 0.01) was twice as high in centenarian cells than in young cells. In conclusion, the GnRHR pathway regulated essential properties of B lymphocytes. However, upon EBV transformation, memory class-switched B cells became the dominant cell subpopulation. Therefore, the observed effects of GnRHR inhibition were attributable to this subpopulation.

Genetics of hypogonadotropic Hypogonadism-Human and mouse genes, inheritance, oligogenicity, and genetic counseling.

Hypogonadotropic hypogonadism, which may be normosmic (nHH) or anosmic/hyposmic, known as Kallmann syndrome (KS), is due to gonadotropin-releasing hormone deficiency, which results in absent puberty and infertility. Investigation of the genetic basis of nHH/KS over the past 35 years has yielded a substantial increase in our understanding, as variants in 44 genes in OMIM account for ~50% of cases. The first genes for KS (ANOS1) and nHH (GNRHR) were followed by the discovery that FGFR1 variants may cause either nHH or KS. Associated anomalies include midline facial defects, neurologic deficits, cardiac anomalies, and renal agenesis, among others. Mouse models for all but one gene (ANOS1) generally support findings in humans. About half of the known genes implicated in nHH/KS are inherited as autosomal dominant and half are autosomal recessive, whereas only 7% are X-linked recessive. Digenic and oligogenic inheritance has been reported in 2-20% of patients, most commonly with variants in genes that may result in either nHH or KS inherited in an autosomal dominant fashion. In vitro analyses have only been conducted for both gene variants in eight cases and for one gene variant in 20 cases. Rigorous confirmation that two gene variants in the same individual cause the nHH/KS phenotype is lacking for most. Clinical diagnosis is probably best accomplished by targeted next generation sequencing of the known candidate genes with confirmation by Sanger sequencing. Elucidation of the genetic basis of nHH/KS has resulted in an enhanced understanding of this disorder, as well as normal puberty, which makes genetic diagnosis clinically relevant.

Proliferating primary pituitary cells as a model for studying regulation of gonadotrope chromatin and gene expression.

The chromatin organization of the gonadotropin gene promoters in the pituitary gonadotropes plays a major role in determining how these gene are activated, but is difficult to study because of the low numbers of these cells in the pituitary gland. Here, we set out to create a cell model to study gonadotropin chromatin, and found that by optimizing cell culture conditions, we can maintain stable proliferating cultures of primary non-transformed gonadotrope cells over weeks to months. Although expression of the gonadotropin genes drops very low, these cells are enriched in gonadotrope markers and respond to GnRH. Furthermore, >85% of the cells contained Lhb and/or Fshb mature transcripts; though these were virtually restricted to the nuclei. The gonadotropes were harvested initially due to expression of dTOMATO, following activation of Cre recombinase by the Gnrhr promoter. Over 6 mo in culture, a similar proportion of the recombined DNA was maintained (i.e. cells derived from the original gonadotropes or having acquired Gnrhr-promoter activity), together with cells of a distinct origin. The cells are enriched with markers of proliferating pituitary and stem cells, including Sox2, suggesting that multipotent precursor cells might have proliferated and differentiated into gonadotrope-like cells. These cell cultures offer a new and versatile methodology for research in gonadotrope differentiation and function, and can provide enough primary cells for chromatin immunoprecipitation and epigenetic analysis, while our initial studies also indicate a possible regulatory mechanism that might be involved in the nuclear export of gonadotropin gene mRNAs.