Luteinizing Hormone Receptor Research Articles

#50 : Effect of Dual vs. Single (hCG) Trigger in Maturation Rate of Oocytes in ART

Background and Aims: Due to its similar molecular structures and biological activities, human chorionic gonadotropin (hCG) is used as a substitute for luteinizing hormone (LH) in ART. The weaknesses of using hCG as a trigger are the possibility of ovarian hyperstimulation syndrome in high responders and lacked FSH activity which promotes LH receptor formation in luteinizing granulosa cells, nuclear maturation, and cumulus expansion. GnRH agonist (GnRHa) reduces the development of OHSS and induces a physiological surge similar to the natural menstrual cycle. Because of shorter amplitude and duration of GnRHa-induced LH surge, GnRHa-alone trigger results in a higher early miscarriage rate. Combining these two agents; dual trigger increases oocyte maturation with reduced OHSS. Here we present reports in our clinic to differentiate the dual vs. hCG trigger results. Method: We retrospectively analyzed the data from 68 patients who had undergone antagonist protocol from January 2021-January 2023. The final oocyte maturation was undertaken with a dual trigger with GnRHa (Buserelin 0.3 mg) combined with hCG (r-hCG 250 [Formula: see text]g) ([Formula: see text] = 33) or hCG alone ([Formula: see text] = 35). Results: The mean age of both groups was 34 years old. The AMH level of the dual group ranged from 0.06 – 7.17 ng/mL, and in hCG alone group ranged from 0.16 – 12.3 ng/mL. Women in both groups got similar amounts of follicles and MII oocytes; dual group (14.8 and 12.72 vs. 14.9 and 13). The retrieval rate of oocytes was identical in both groups (86.6% vs. 89.9%). However, the maturation rate of dual group was slightly lower than the hCG group (77% vs. 82%), but it was statistically insignificant (independent t-test p 0.99). Conclusion: We showed that dual trigger was not superior to hCG-alone trigger in terms of maturation rate of oocytes. Analysis of larger data is needed to show the real advantages.

Optimisation of hormonal treatment to improve follicular development in one-day-old mice ovaries cultured under in vitro condition.

Base medium containing knock-out serum replacement (KSR) has been found to support formation and maintenance of follicles in one-day-old mice ovaries, but has not been shown to properly support activation and growth of primordial follicles. The present study was conducted to tailor the hormonal content of base medium containing KSR to enhance development of primordial follicles in neonatal ovaries. One-day-old mice ovaries were initially cultured with base medium for four days, and then, different hormonal treatments were added to the culture media and the culture was proceeded for four additional days until day eight. Ovaries were collected for histological and molecular assessments on days four and eight. In experiment I, the main and interactive effects of FSH and testosterone were investigated and FSH promoted activation of primordial follicles and development of primary and preantral follicles, and upregulated genes of phosphoinositide 3-kinase (Pi3k ), KIT ligand (Kitl ), growth differentiation factor 9 (Gdf9 ) and follicle stimulating hormone receptor (Fshr ) (P Bmp15 ), Connexin-43 (Cx43 ) and luteinising hormone and choriogonadotropin receptor (Lhcgr ) (P P Lhcgr (P P >0.05). Supplementation of culture medium containing KSR with gonadotropins, particularly hMG, could improve follicular growth and expression of factors regulating follicular development. This study was a step forward in formulating an optimal medium for development of follicles in cultured one-day-old mice ovaries.

Molecular insights into the effects of tetrachlorobisphenol A on puberty initiation in Wistar rats

Tetrachlorobisphenol A (TCBPA) is the chlorinated derivative of bisphenol A (BPA). Several studies have found that BPA adversely affects the reproductive activity largely through binding to estrogen receptors and the critical period of BPA exposure advances the vaginal opening time in the female offspring via the kisspeptin/G protein-coupled receptor 54 (KGG) system. However, whether TCBPA can affect puberty initiation via KGG and the roles of estrogen receptors in this process remain unknown. Therefore, this study investigated the influence of TCBPA on the onset time of puberty in Wistar rats and the related molecular mechanisms by combing in vitro GT1–7 cells and molecular docking. In female Wistar rats, TCBPA at ≥100 mg/kg bw/day (49.2 μmol/L in rat body) markedly advanced vaginal opening time and increased serum levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and gonadotropin-releasing hormone (GnRH). It also increased the relative gene expression of LH receptor (LHR), GnRH1, and FSH receptor (FSHR) in hypothalamic-pituitary-gonadal (HPG) axis tissues. In GT1–7 cells, TCBPA increased genes and proteins associated with KGG pathway and activated the extracellular-regulated protein kinase 1/2 (Erk1/2) and phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt) pathways via G protein-coupled estrogen membrane receptor 1 (GPER1) and estrogen receptor alpha (ERα). Docking analyses supported its interactions with GPER1 and ERα, and treatment with specific inhibitors of ERα- and GPER1-modulated PI3K/Akt and Erk1/2 signaling suppressed its effects. Taken together, TCBPA-induced advancement of puberty initiation in Wistar rats thus results primarily from increased LH, GnRH, and FSH secretion together with GnRH1, FSHR, and LHR upregulation driven by ERα- and GPER1-modulated Erk1/2 and PI3K/Akt signaling. Our results provide new molecular insights into the reproductive toxicity of EDCs.

Identification of key miRNAs as regulatory biomarkers of gonadotropins leading to infertility in males

Introduction. Infertility is a highly fatal reproductive system disorder that affects the ability of a couple to reproduce. Over the past decades, a drastic uplift has been recorded in infertility cases among males ranging from 20 to 70 % indicating spermatogenesis impairment.Aim: to identify key microRNAs (miRNAs) as regulatory biomarkers of gonadotropins involved in dysregulation of fertility-related genes to propose potential therapeutic strategies that would combat the action of oncogenic miRNAs (oncomiRs).Materials and Methods. Interaction analysis was performed between miRNAs and fertility-related genes namely luteinizing hormone choriogonadotropin receptor (LHCGR), gonadotropin-releasing hormone receptor (GnRHR), follicle-stimulating hormone receptor (FSHR) and cystic fibrosis transmembrane conductance regulator (CFTR) to identify key miRNAs as regulatory biomarkers of gonadotropins leading to infertility in males.Results. A total of 10, 13, 31 and 18 strong and potential binding sites were predicted for miRNAs-LHCGR, miRNAs-GnRHR, miRNAs-FSHR, and miRNAs-CFTR respectively employing miRWalk (comprehensive genetic database including miRNA targets) followed by identification of 6, 18, 55 and 17 significant interactions through RNA22. Subsequently shortlisted miRNAs and messenger RNA (mRNA) regions were subjected to Vfold-Pipeline and RNAComposer individually for 3D structure prediction. Additionally molecular docking was carried out between miRNAs and mRNAs models that discovered potential and stable interactions elucidating miR-6880-FSHR(R2) as a highly stable complex with least binding affinity (-566.3) and high confidence score (0.999).Conclusion. Hence this study proposes key oncomiRs as a diagnostic biomarker and therapeutic target to bring about a promising treatment strategy against male factor infertility. However wet lab investigations are required for further validations of proposed study.

Genes of Hormone Receptors Affecting Egg Productivity and Reproductive Qualities of Chickens

The egg production of chickens is a complex polygenic type of inheritance and is controlled by many genes. The laying performance of chickens is the result of a complex process regulated by the hypothalamic-pituitary-gonadal system. This review summarizes the information on the effect of polymorphisms of follicle stimulating hormone (FSHR), luteinizing hormone (LHCGR), progesterone (PR) and prolactin (PRLR) receptor genes on the egg production and reproductive performance of chickens. The data presented show that the polymorphisms of these genes are promising for use in breeding programs to improve egg production and reproductive performance of chickens.

Significantly shortened telomere length and altered androgen receptor level in cumulus cells from women with polycystic ovary syndrome

ObjectiveThe aim of this study was to investigate the correlation between hormone receptor levels and telomere length (TL) in infertile women with and without polycystic ovary syndrome (PCOS). Materials and methodsThis prospective cohort study recruited a total of 431 cumulus oocyte complex (COC) from 88 infertile women between July 2012 and June 2014. The participants were divided into three groups: young age (<38 years, n = 42 and 227 COC), advanced age (≥38 years, n = 33 and 107 COC) and PCOS patients (n = 13 and 97 COC). Cumulus cells were collected from individual follicle during oocyte pick-up, and the mRNA levels of hormone receptors and TL were measured using real-time PCR. ResultsThe cumulus cells of PCOS patients demonstrated lower mRNA levels of LH receptor (75.57 ± 138.10 vs. 171.07 ± 317.68; p < 0.01) and androgen receptor (1.13 ± 1.52 vs. 4.08 ± 9.57; p < 0.01), as well as a shorter TL (2.39 ± 2.58 vs. 3.96 ± 4.72; p < 0.01) compared to those of the young age group. In the young age group, only androgen receptor mRNA level showed a significant association with TL (rho = 0.148, p = 0.026), while FSH receptor mRNA level was the only factor associated with TL (rho = 0.247, p = 0.015) in PCOS patients. For advanced-aged patients, no significant relationship was observed between hormone receptor mRNA levels and TL. Alternative splicing of androgen receptors was identified in some PCOS patients but not in young age controls. ConclusionThe findings suggest that the androgen receptor level and function may be altered in the cumulus cells of PCOS patients, leading to a shorter TL in cumulus cells in PCOS patients.

Insights into dysregulation of luteinizing hormone receptor (LHR) signaling in granulosa lutein (GL) cells from women with polycystic ovary syndrome (PCOS)

Insights into dysregulation of luteinizing hormone receptor (LHR) signaling in granulosa lutein (GL) cells from women with polycystic ovary syndrome (PCOS)

Luteinising hormone receptor signalling is reliant on formation of distinct multi-receptor complexes

Luteinising hormone receptor signalling is reliant on formation of distinct multi-receptor complexes

Betaine regulates steroidogenesis, endoplasmic reticulum stress response and Nrf2/HO-1 antioxidant pathways in mouse Leydig cells under hyperglycaemia condition

We studied the effects of betaine on steroidogenesis, endoplasmic reticulum stress and Nrf2 antioxidant pathways of mice Leydig cells under hyperglycaemia conditions. Leydig cells were grown in low and high glucose concentrations (5 mM and 30 mM) in the presence of 5 mM of betaine for 24 h. Gene expression was determined using a real-time PCR method. The protein levels were determined by Western blot analysis. The testosterone production was evaluated by the ELISA method. Cellular contents of reduced and oxidised glutathione were measured by colorimetric method. Hyperglycaemia caused impaired steroidogenesis and ERS in Leydig cells associated with the down-regulation of 3β-HSD, StAR, P450scc, LH receptor and increased expression of GRP78, CHOP, ATF6 and IRE1. Betaine could improve cell viability, attenuate the ERS, and restore testosterone production in Leydig cells under hyperglycaemia conditions. Betaine can protect Leydig cells against the adverse effects of hyperglycaemia by regulating steroidogenesis, antioxidants, and ERS.

Effects of Dietary Betaine on the Laying Performance, Antioxidant Capacity, and Uterus and Ovary Function of Laying Hens at the Late Stage of Production.

Betaine has been found to alleviate oxidative stress, inflammation, and apoptosis. However, whether dietary betaine can protect late-laying hens against these adverse effects is unknown. Here, 270 65-week-old Jinghong-1 laying hens were randomly divided into the Control, 0.1% Betaine, and 0.5% Betaine groups and fed a basal diet, 0.1%, and 0.5% betaine supplemented diet, respectively. The trial lasted for seven weeks. Birds that consumed 0.5% betaine laid more eggs with thicker eggshells. Accordingly, uterine reduced glutathione (GSH), glutathione peroxidase (GSH-PX), and ovarian superoxide dismutase (SOD) contents were increased. The uterine calcium ion content and the mRNA expression of ovalbumin, ovotransferrin, and carbonic anhydrase two were increased. Moreover, ovarian IL-1β, Caspase-1, Caspase-8, and Caspase-9 mRNA expressions were decreased; luteinising hormone receptor (LHR) and follicle-stimulating hormone receptor mRNA expressions were increased. Furthermore, dietary betaine decreased the ovaries' mRNA expression of DNA methyltransferase 1 (DNMT)1, DNMT3a, and DNMT3b. The methylation level at the promoter region of ovarian LHR decreased. These results indicated that dietary betaine consumption with a concentration of 0.5% could increase the laying rate and the eggshell thickness during the late-laying period. The underlying mechanism may include antioxidative, anti-apoptosis, and hormone-sensitivity-enhancing properties.

Effects of Calcium, Available Phosphorus and Microbial Phytase on Ovarian FSHR and LHR Expression in Laying Hens

Folliculogenesis, steroidogenesis, ovulation, and vitellogenesis are regulated by the effect of follicle stimulating hormone (FSH) and luteinizing hormone (LH) in the hypothalamus-pituitary-ovary axis and these hormones act via follicle stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR) in the ovary. Poultry ration and food additives are essential in the regulation of reproductive activity. Phytase is a supplement frequently added to laying hen diets to increase phosphorus (P) utilization. The aim of this study was to reveal the effects of a newly isolated microbial phytase together with different concentrations of calcium (Ca2+) and available phosphorus (AP) on ovarian FSHR and LHR expressions. For this purpose, 90 Lohmann LSL-White layers were first divided into three main diet groups (standard Ca2+ and AP, standard Ca2+ and low AP, low Ca2+ and AP) and then into three subgroups (no-phytase, commercial phytase, and microbial phytase). At the end of the experiment, all chickens were slaughtered and ovarian tissues were fixed in formalin. Routine avidin-biotin complex immunohistochemistry was performed using anti-FSHR and anti-LHR primary antibodies. Immunohistochemically, FSHR and LHR were expressed in granulosa/theca cells, oocytes, interstitial cells, and vitellus. While the expression intensity of the receptors increased in the microbial phytase-treated groups, the strongest expression was obtained in the granulosa/theca cells and oocytes in the standard Ca and low AP group. In conclusion, we suggest that the addition of newly isolated microbial phytase to diets of laying hens and feeding standard Ca and low AP may have positive effects on reproductive performance by increasing the FSHR and LHR expression in ovaries.

Luteinizing Hormone/Human Chorionic Gonadotropin Receptor N312S Single-Nucleotide Polymorphism and Its Impact on Clinical and Reproductive Outcomes in Assisted Reproductive Technology: A Prospective Cohort Study.

Objective The aim of this study was to determine the genotypic distribution of luteinizing hormone/human chorionic gonadotropin receptor (LHCGR) N312S single-nucleotide polymorphism (SNP) and to investigate its impact on clinical and reproductive outcomes in infertile Indian women undergoing assisted reproductive technology (ART). Study design and settings This was a prospective cohort study conducted at a tertiary care university hospital. Subjects and methods Infertile women aged between 21 and 40 years undergoing ART with an antagonist protocol were enrolled in this study. A 2-ml sample of peripheral venous blood was collected from each woman and genotyped for the LHCGR N312S SNP. Participants were divided into three groups based on their SNP: NN, NS, and SS. All subjects underwent controlled ovarian hyperstimulation (COH) through a gonadotropin-releasing hormone (GnRH) antagonist protocol and intracytoplasmic sperm injection (ICSI). Of the 140 women recruited based on selection criteria, 128 underwent embryo transfer. We compared the genotypic distribution of the LHCGR N312S SNP, baseline characteristics, clinical outcomes, and reproductive outcomes in ART among the three groups. Data were analyzed using IBM SPSS Statistics for Windows, Version 29 (Released 2022; IBM Corp., Armonk, New York, United States). The chi-square test and Fisher-Irwin test were employed to evaluate significant differences among the qualitative categorical variables. A p-value of less than 0.05 was considered statistically significant. Results Among the test subjects, 19.3% were homozygous for the LHCGR N312 SNP (NN group), 38.6% were heterozygous (NS group), and 42.1% were homozygous for the LHCGR S312 SNP (SS group). Baseline characteristics were similar among the three groups. In terms of ovarian reserve tests, significantly lower anti-Müllerian hormone (AMH) levels were observed in the SS group compared to the NS and NN groups (2.8 ± 2.1 vs. 3.2 ± 2.5 vs. 4.3 ± 3.3; p=0.03). No significant differences were observed in COH outcomes such as duration of stimulation, total gonadotropin requirement, oocyte yield, or the number of good-quality embryos among the three groups. The cumulative pregnancy rate (82.9% vs. 50.0% vs. 38.2%, p=0.0005), cumulative clinical pregnancy rate (78.8% vs. 44.7% vs. 34.5%, p = 0.0005), and cumulative live birth rate (50.0% vs. 20.2% vs. 20.0%, p=0.005) were significantly higher in the NN group than in the NS and SS groups. Conclusion The study's findings suggest that LHCGR N312 may help predict reproductive outcomes in ART, which may aid in providing better counseling to infertile couples. We need more studies on individualized/personalized COH using pharmacogenomics for follicle-stimulating hormone (FSH) and luteinizing hormone (LH) supplementation based on combined FSH and LH receptor SNP and to assess their effects on ART outcomes.

Luteinizing hormone stimulates ingression of mural granulosa cells within the mouse preovulatory follicle†.

Luteinizing hormone (LH) induces ovulation by acting on its receptors in the mural granulosa cells that surround a mammalian oocyte in an ovarian follicle. However, much remains unknown about how activation of the LH receptor modifies the structure of the follicle such that the oocyte is released and the follicle remnants are transformed into the corpus luteum. The present study shows that the preovulatory surge of LH stimulates LH receptor-expressing granulosa cells, initially located almost entirely in the outer layers of the mural granulosa, to rapidly extend inwards, intercalating between other cells. The cellular ingression begins within 30 min of the peak of the LH surge, and the proportion of LH receptor-expressing cell bodies in the inner half of the mural granulosa layer increases until the time of ovulation, which occurs at about 10h after the LH peak. During this time, many of the initially flask-shaped cells appear to detach from the basal lamina, acquiring a rounder shape with multiple filipodia. Starting at about 4h after the LH peak, the mural granulosa layer at the apical surface of the follicle where ovulation will occur begins to thin, and the basolateral surface develops invaginations and constrictions. Our findings raise the question of whether LH stimulation of granulosa cell ingression may contribute to these changes in the follicular structure that enable ovulation.

Correlation between LHCGR and NR5A1 genes polymorphism and male infertility risk

IntroductionInfertility is one of the important phenomena in human reproduction. Genetic factors are the most important cause of male infertility. Here, we aimed to investigate the correlation between idiopathic male infertility and SNPs of the LHCGR (rs2293275) and NR5A1 (rs1057517779) genes in the Iranian-Azeri population. MethodsThis case-control study consisted of 100 males with infertility and 100 healthy males from the Iranian Azeri population. Genomic DNA isolation from whole blood samples and Tetra-primer amplification refractory mutation system-polymerase chain reaction (Tetra-ARMS-PCR) method was used for genotyping. The data analysis was performed by chi-square (χ2) and Fisher’s exact tests. ResultsGenotyping analysis for LHCGR (rs2293275) polymorphism indicated that the frequency of C in the case group was significantly higher than in the control group (P < .05). Moreover, genotyping analysis for NR5A1 (rs1057517779) polymorphism indicated that the frequencies of the A allele and heterozygote GA genotype in the case group were significantly higher than those in the control group (P < .05). ConclusionOur study demonstrated that the SNPs of LHCGR (rs2293275) and NR5A1 (rs1057517779) genes may play a critical role in male infertility in the Iranian Azeri population. However, further studies on other ethnic origins with larger sample sizes are essential for accessing more accurate results. Moreover, functional experiments might be needed to understand the role of these polymorphisms in the molecular pathways involved in male fertility.

Therapeutic dosing and targeting efficacy of Pt-Mal-LHRH towards triple negative breast cancer.

Pt-Mal-LHRH is a newly synthesized chemotherapeutic agent that was designed to selectively target the luteinizing hormone-releasing hormone (LHRH) receptor expressed by triple negative breast cancer (TNBC). The aim of this study was to evaluate the therapeutic dosing, tumor reduction efficacy, and selective distribution of Pt-Mal-LHRH in-vivo. LHRH tissue expression levels in-vivo were investigated using western blotting and LHRH was found to be increased in reproductive tissues (mammary, ovary, uterus). Further, Pt-Mal-LHRH was found to have increased TNBC tumor tissue platinum accumulation compared to carboplatin by inductively coupled plasma mass spectrometry analysis. The platinum family, compound carboplatin, was selected for comparison due to its similar chemical structure and molar equivalent doses were evaluated. Moreover, in-vivo distribution data indicated selective targeting of Pt-Mal-LHRH by enhanced reproductive tissue accumulation compared to carboplatin. Further, TNBC tumor growth was found to be significantly attenuated by Pt-Mal-LHRH compared to carboplatin in both the 4T1 and MDA-MB-231 tumor models. There was a significant reduction in tumor volume in the 4T1 tumor across Pt-Mal-LHRH doses (2.5-20 mg/kg/wk) and in the MDA-MB-231 tumor at the dose of 10 mg/kg/wk in models conducted by an independent contract testing laboratory. Our data indicates Pt-Mal-LHRH is a targeting chemotherapeutic agent towards the LHRH receptor and reduces TNBC tumor growth in-vivo. This study supports drug conjugation design models using the LHRH hormone for chemotherapeutic delivery as Pt-Mal-LHRH was found to be a more selective and efficacious than carboplatin. Further examination of Pt-Mal-LHRH is warranted for its clinical use in TNBCs, along with, other reproductive cancers overexpressing the LHRH receptor.

Phoenixin-14 as a novel direct regulator of porcine luteal cell functions†.

Phoenixin is a neuropeptide with a well-established role in the central regulation of reproductive processes; however, knowledge regarding its role in the ovary is limited. One of the main active phoenixin isoforms is phoenixin-14, which acts through G protein-coupled receptor 173. Our research hypothesis was that phoenixin-14 is expressed in porcine corpus luteum and exerts luteotropic action by affecting the endocrine function of luteal cells through G protein-coupled receptor 173 and protein kinase signaling. Luteal cells were cultured to investigate the effect of phoenixin-14 (1-1000nM) on endocrine function. We showed that phoenixin-14 and G protein-coupled receptor 173 are produced locally in porcine corpus luteum and their levels change during the estrous cycle. We detected phoenixin-14 immunostaining in the cytoplasm and G protein-coupled receptor 173 in the cell membrane. Plasma phoenixin levels were highest during the early luteal phase. Interestingly, insulin, luteinizing hormone, progesterone, and prostaglandins decreased phoenixin-14 levels in luteal cells. Phoenixin-14 increased progesterone, estradiol, and prostaglandin E2 secretion, but decreased prostaglandin F2α, upregulated the expression of steroidogenic enzymes, and downregulated receptors for luteinizing hormone and prostaglandin. Also, phoenixin-14 increased the expression of G protein-coupled receptor 173 and the phosphorylation of extracellular signal-regulated kinase 1/2, protein kinase B, inhibited the phosphorylation of protein kinase A, and had mixed effect on AMP-activated protein kinase alpha and protein kinase C. G protein-coupled receptor 173 and extracellular signal-regulated kinase 1/2 mediated the effect of phoenixin-14 on endocrine function of luteal cells. Our results suggest that phoenixin is produced by porcine luteal cells and can be a new regulator of their function.

SAT649 Org43553, The First Low-molecular-weight Agonist Of The Human Luteinizing Hormone Receptor Induces Leanness And Energy Expenditure In Mice

Abstract Disclosure: D. Lizneva: None. A. Gumerova: None. K. Ievleva: None. O. Barak: None. F. Korkmaz: None. J. Gimenez Roig: None. T. Frolinger: None. G. Pevnev: None. F. Sultana: None. N. Kramskiy: None. S. Wizman: None. M. Orloff: None. A.R. Pallapati: None. S. Rojekar: None. V. Ryu: None. O. Moldavski: None. T. Yuen: None. M. Zaidi: None. We provide compelling evidence that LH is a pro–lean hormone, and that ORG43553, the first low-molecular-weight agonist of the luteinizing hormone receptor (LHCGR) induces leanness and promotes energy expenditure in mice. We detected Lhcgr transcripts in white adipose tissue (WAT) depots in male and female C57BL/6 mice (qPCR, IHC, RNAscope, Sanger sequencing), but not in Lhcgr-/- mice. Intraperitoneal AlexaFluor-488–labeled hCG bound to gonadal and subcutaneous fat pads in wild type mice, but not in Lhcgr-/- mice. Following i.v. 89Zr–LH injection, radioactivity was detected in mesenteric, inguinal and gonadal WAT. LH, hCG and ORG43553 rapidly induced ERK1/2 phosphorylation in differentiated 3T3.L1 cells. Female and male Lhcgr+/- mice on normal chow and high–fat diet, respectively, became obese, importantly, without changes in sex steroids. The gene–dosage effect of Lhcgr depletion suggests a dominant physiologic action of LH on body composition. 14–week–old male C57BL/6 mice were fed on high–fat diet and injected, i.p., with LH, hCG or vehicle, twice–a–week, for 6 weeks. Both LH and hCG markedly reduced body fat accrual (qNMR) but triggered a rise in serum testosterone. The hCG–induced lean phenotype persisted despite androgen receptor blockade by flutamide—confirming that the pro–lean actions of LH and hCG were largely independent of testosterone. Furthermore that LH failed to inhibit fat accrual in Lhcgr-/- mice testified to its action via the LHCGR. Injection of ORG43553 into male C57BL/6 mice for 9 weeks caused a significant reduction in fat mass (qNMR), with reduced WAT weight in fat depots. Remarkably, serum testosterone remained unchanged confirming that the anti–adiposity effect of ORG43553 was independent of testosterone. To study the mechanism of LHCGR activation on body fat, 3T3.L1 adipocytes were treated with hCG or ORG43553 for 12 hours. This resulted in reduced oil droplet accumulation. 3T3-L1–derived organoids treated with LH, hCG or ORG43553 also displayed a substantial reduction in the differentiated layer compared to vehicle–treated organoids. To study whether, in addition to reducing adipocyte differentiation, LHCGR agonism also enhanced thermogenesis, 3T3-L1 adipocytes were pretreated with ORG43553 for 1 hour before measuring oxygen consumption rate (OCR) on a Seahorse Xf96 Analyzer. ORG43553 increased OCR at baseline and upon oligomycin exposure, indicating mitochondria proton leak (thermogenesis). To confirm increased energy expenditure in vivo, mice were injected, s.c., with ORG43553 or vehicle. Oxygen consumption (VO2) and energy expenditure (EE) increased acutely in ORG43553–treated mice, with no change in locomotor activity. The findings suggest that ORG45335, which has been tested in humans for infertility, can potentially become a candidate drug for obesity. Presentation: Saturday, June 17, 2023

THU205 A Case Of Infant With Familial-Male Limited Precocious Puberty (FMPP) Inherited From Father Due To An Activating Mutation Of The Luteinizing Hormone/Chorionic Gonadotropin Receptor Gene

Abstract Disclosure: H. Yoo: None. J. Ha: None. M. Jung: None. Y. Choi: None. E. Yoo: None. We present an extremely rare case of FMPP with an activating mutation of the LHCGR gene in a Korean infant. A 16-month-old-boy was brought to the out-patient clinic with complaints of pubic hair, acne and rapid growth acceleration. His father had become aware of the symptoms 4 month before. His grandfather and father had a family history of precocious puberty and profound short stature (Height of grandfather and father: 148 cm and 158 cm respectively). At physical examination, his height was 83 cm (+1.2 SDS) and his weight was 14.3 kg (+2.8 SDS). The testicular volume was 2 mL and pubic hair was appropriate for Tanner stage 2. Stretched penile length was 7 cm (&amp;gt;2 SDS). He did not have any café au lait spots or hyperpigmentation. His bone age was significantly advanced to that of a 4 year old. Laboratory findings showed abnormally elevated serum testosterone (5.74 ng/mL) and slightly elevated α-fetoprotein (14.1 ng/ml; reference 0-10 ng/mL) with suppressed LH level (&amp;lt;0.07 mIU/mL). Serum levels of β-human chorionic gonadotropin, cortisol, 17-hydroxyprogesterone, androstenedione and dehydroepiandrosterone sulfate were within normal range. A gonadotropin releasing hormone (GnRH) stimulation test revealed a prepubertal response(peak LH 0.52). Brain magnetic resonance imaging study was reported to be normal. Ultrasound of the scrotum and adrenal gland were both normal. Due to the patient’s classic presentation and his family history of male-limited precocious puberty, a genetic analysis for LHCGR gene was performed. Molecular genetic analysis identified a pathogenic variant of LHCGR (c.1730 C&amp;gt;T, p.Thr577Ileu), which was previously reported in a FMPP patient. His father and paternal grand father also carry the same mutation. Anti-androgen treatment with bicalutamide (25mg/day) and aromatase inhibition with anastrozole (1mg/day) were initiated. After treatment, there was improvement in pubertal progression, accelerated growth velocity and rate of skeletal maturation without any specific side effects. Presentation: Thursday, June 15, 2023

OR08-01 Expression of Gonadal Differentiation Markers in Bilateral Macronodular Adrenocortical Disease

Abstract Disclosure: K. De Sousa: None. V. Pautasso: None. C. Duparc: None. S. Renouf: None. B. Ragazzon: None. F. Violon: None. L. Bouys: None. F. Bonnet-Serrano: None. M. Sibony: None. N. Driessens: None. S. Espiard: None. G. Raverot: None. J.Y. Bertherat: None. E. Louiset: None. H. Lefebvre: None. Context: Bilateral macronodular adrenocortical disease (BMAD) is a rare cause of primary Cushing syndrome, occurring mostly in women. ARMC5 and KDM1A mutations are responsible for distinct forms of BMAD. In particular, KDM1A mutations are associated with food-dependent Cushing syndrome. In addition, BMAD tissues can express illegitimate receptors, including LH receptor, that lead to aberrant control of cortisol secretion by various factors. Moreover, steroidogenic cells are able to abnormally produce bioactive factors, such as ACTH and the Leydig cell hormone INSL3. Since LH receptor, ACTH and INSL3 are known to be produced in gonads, we speculated on a gonadal-like differentiation of adrenocortical cells in BMAD tissues. Aim: The aim of our study was to investigate the presence of different gonadal markers in a series of BMAD tissues, and to search for secretion of adrenal and gonadal hormones by BMAD cells. Methodology: Gene expression of gonadal markers were evaluated by RNAseq. Immunohistochemical studies were performed on 41 BMAD and 9 normal adrenal samples to examine the presence of hormones (ACTH, INSL3, anti-Mullerian hormone [AMH]), the estrogen synthetizing enzyme aromatase, and transcription factors (GATA4, SOX9, FOXL2, MAGEA4) known to be expressed in testis or ovary. Perifusion experiments, coupled to immunoassays or liquid chromatography/mass spectrometry, were conducted to quantify hormone secretion by 2 adrenal explants. Results: BMAD tissues were collected from patients (60% women) without or with ARMC5 (32%) or KDM1A (13%) mutations. ARMC5-mutated tissues overexpressed FOXL2 and CYP19A1 (encoding aromatase) in comparison with other BMAD samples. In normal adrenals, cortisol-producing cells, located in the zona fasciculata, were immunonegative for INSL3, AMH, aromatase, GATA4 and SOX9, whereas FOXL2 and MAGEA4 immunostainings were detected in the cortex. In BMAH tissues, the presence of positive cells for ACTH (100% of BMAD), INSL3 (90%), AMH (50%), aromatase (77%), SOX9 (92%), GATA4 (97%), FOXL2 (100%) and MAGEA4 (100%) were observed in most of the samples regardless of the gender or mutational status. FOXL2 and MAGEA4 immunostainings were detected throughout BMAD tissues. Distribution of other gonadal markers was heterogenous in tissue sections with clusters of cells positive for ACTH, aromatase and GATA4 or ACTH and SOX9. Functional studies revealed pulsatile secretion of different glucocorticoids and INSL3 by BMAD explants. ACTH and hCG stimulated steroid production but were without effect on INSL3 release. Conclusion: These data show that steroidogenic cells in BMAD tissues exhibit a gonadal-type phenotype and abnormally secrete INSL3. They suggest that BMAD may result from a genetically driven defect occurring during early embryogenesis and affecting differentiation and development of adrenal tissues. Presentation: Friday, June 16, 2023

THU452 Polyostotic Fibrous Dysplasia Complicated By Aneurysmal Bone Cyst In A Patient With McCune-Albright Syndrome

Abstract Disclosure: E. Milazzo: None. K.L. Hermayer: None. Introduction: McCune-Albright Syndrome (MAS) is a rare mosaic disorder with GNAS mutations, encoding the alpha-subunit of the Gs G-coupled protein receptor, activating it, and inappropriately producing cAMP. The hyperfunctioning endocrinopathies associated with MAS are due to signaling through the TSH, LH, FSH, GHRH, and ACTH receptors. MAS clinical presentation occurs with café-au-lait skin macules, precocious puberty, and fibrous dysplasia (FD) of the bone. FD is a benign tumor that leads to increased rates of bone fractures and if it causes facial deformities, can be complicated with orbital neuropathies. An aneurysmal bone cyst (ABC) is a blood-filled cyst that can be a primary or secondary lesion due to co-existing lesions. ABC is a rare complication of FD. We report a rare case of ABC in a MAS patient who presented with acute visual disturbance and enlargement of the forehead and was successfully treated with surgery. Clinical Case: A 20-year-old female diagnosed with MAS at the age of 2 who had precocious puberty by the 3rd year of life and developed polyostotic FD in the right arm, left femur, cranial, and skull bones with multiple long bone fractures by the age of 5-6 and required numerous orthopedic surgeries. She presented with the characteristic café-au-lait skin macules on the back and right arm. In 2018, a slow decline of left eye vision required left cranial decompression of the optic nerve, with pathology showing fibrous tissue compatible with FD. In 2021, she complained of acute right forehead enlargement, compressing her right eye with blurry vision. An MRI showed FD involving the facial and skull bones with a new expansible lesion in the right frontal bone with internal layering fluid, exerting a mild mass effect on the frontal lobe and extending superiorly to the orbit with inferior displacement of the globe. These findings were consistent with an ABC. Right frontal craniectomy and cranioplasty were performed in 12/2021, with pathology showing a fibrous pseudocyst wall with associated lymphoplasmacytic inflammation and evidence of prior hemorrhage with Ki-67 in the cyst wall at 15%. Lab work showed chronic alkaline phosphatase elevation at 567IU/L (42-106IU/L), normal calcium at 9.2mg/dl (8.4-10.3mg/dl), phosphate at 3.3mg/dl (3.3-5.1mg/dl), and PTH at 26pg/ml (15-65mg/dl), with Vitamin D insufficiency at 25.1ng/ml (30-100ng/ml). Endocrinology workup was negative for thyroid, adrenal, gonadotrophic, and GH dysfunction. 5 months post-surgery, the patient demonstrated complete recovery, and MRI showed no recurrence of the ABC. Conclusion: ABC is a benign solitary tumor generally arising from the metaphysis of long bones and rarely involves the cranium. ABC associated with FD has only been reported in a few cases. The treatment is surgical excision, with radiation and cryotherapy used in nonorbital cases. Presentation: Thursday, June 15, 2023