Gonadotropin-releasing Hormone Research Articles

9205 Copy Number Variation (CNV) in Self-Limited Delayed Puberty (SLDP)

Abstract Disclosure: Y. Al-Sayed: None. M. Ishida: None. C.L. Hall: None. S.R. Howard: None. SLDP is where the onset of puberty is more than 2-2.5 standard deviations later than the population mean age and is often familial with strong genetic determinants. The reproductive axis is regulated by gonadotropin-releasing hormone (GnRH), which plays a crucial role in initiating puberty and maintaining fertility through its pulsatile secretion. Disruption in GnRH neuron development or hypothalamic function can lead to DP. UK Biobank data has identified negative health outcomes associated with SLDP including early menopause/andropause and cognitive and psychosocial disabilities. Consequently, there has been extensive research to investigate genes affecting the hypothalamic-pituitary-gonadal (HPG) axis that might be implicated in the pathogenesis of DP by our group and others which has identified sequence variation contributing to the aetiology of SLDP. However, factors beyond nucleotide variations, such as epigenetic changes and CNVs can also lead to pubertal timing disorders. Moreover, CNVs are seen in multiple individuals (n=92) from the Deciphering Developmental Disorders study (https://www.deciphergenomics.org) with a phenotype including DP (HP:0000823). We performed whole genome sequencing on 49 probands with SLDP and subsequently, analysed the data for CNV. The data was initially called, annotated, filtered then partitioned for classification of CNVs using a command-line tool that implements the ACMG guidelines to evaluate the pathogenicity of germline duplications and deletions. Using an unbiased candidate gene approach, we identified several deletions that affected 60 known/predicted dosage sensitive genes and combines with functional enrichment analysis; This approach revealed potential molecular pathways and biological processes involved in the pathogenesis of SLDP, including histone acetylation, and neural plate development. To refine our findings, we applied additional filters, including known HPG axis genes, GWAS loci associated with pubertal timing, decipher CNV tracks, and DP gene panels, to narrow down large number of rare predicted damaging CNVs and prioritize those with potential relevance to DP. Our study highlights the potential role of CNVs in DP and expands our understanding of the molecular pathways and biological processes involved in this condition, shedding light on the complex mechanisms underlying DP. Presentation: 6/2/2024

7497 Association between Plasma Prolactin Levels and Menopausal Vasomotor Symptoms

Abstract Disclosure: F. Galbiati: None. J. Kang: None. D.J. Cote: None. J.E. Manson: None. U.B. Kaiser: None. Background: High prolactin (PRL) levels inhibit gonadotropin-releasing hormone (GnRH), causing hypogonadism. GnRH release is regulated by neurokinin B (NKB) and kisspeptin. NKB antagonists are used in treatment of menopausal hot flashes and vasomotor symptoms (VMS). In mice and humans with hyperprolactinemic anovulation, kisspeptin restored ovarian cyclicity and LH pulsatility. However, the interplay between kisspeptin and PRL is not fully understood. In two menopausal women with hyperprolactinemia and central hypogonadism, treatment with bromocriptine led to PRL normalization and new-onset VMS, suggesting that hyperprolactinemia inhibited VMS associated with hypogonadism. We aimed to investigate the association between PRL and menopausal VMS in women from the Nurses’ Health Study I and II (NHS, NHSII). We hypothesized that women with higher PRL levels would have a lower frequency/severity of menopausal VMS. Methods: We conducted a cohort study of 2618 females enrolled in NHS (n=1692) and NHSII (n=926) in whom blood samples were collected between 1989-99, were not pregnant or on hormonal therapy, and were either pre or post-menopausal as of blood draw. Subjects were free of breast, ovarian and other cancers, and provided complete data on VMS at menopause as well as in the past 4 weeks (assessed in 2008 in NHS and 2009, 2013, 2017 in NHSII). Plasma PRL was measured as part of 28 case-control studies of various health outcomes. We evaluated the presence and severity of VMS at menopause and 4 weeks prior to the questionnaire, as well as duration (in years) of VMS. We used multiple logistic regression, adjusting for covariates to estimate multivariable-adjusted relative risks (MVRRs) and 95% confidence intervals (CIs). Results: Women were aged 33-65 years at blood draw; range for years from blood draw to menopause was -10 to 10 (median=1.8) years; range for years from blood draw to report of VMS (both at menopause and current VMS) was 9.8 to 22 years. Mean PRL (SD; range) was 14.7ng/mL (10.1; 0.08-246). Women with the highest PRL levels were likely to be younger and less likely to be current smokers or obese. 77.4% of women reported VMS at menopause and 47.6% in recent 4 weeks; 52.3% reported moderate-to-severe VMS at menopause and 32.0% reported VMS in recent 4 weeks. Overall, PRL was not associated with duration of VMS or moderate-to-severe VMS at menopause or in the past 4 weeks; compared to the lowest quintile (≤8.0ng/mL), those in the highest PRL quintile (≥18.2ng/mL) had MVRR of 1.23(95%CI=0.94,1.60) for moderate-to-severe menopausal VMS and 1.03(95%CI=0.67,1.59) for recent VMS. Menopausal status did not affect the association between prolactin levels and VMS. Conclusion: PRL levels were not associated with occurrence, severity, or duration of menopausal VMS in a large cohort of women. Presentation: 6/1/2024

12237 Monosynaptic Gabaergic Signaling From Suprachiasmatic Nucleus Neuromedin S Neurons To Preoptic Area Kisspeptin Neurons In Mice

Abstract Disclosure: W.I. Abdulmajeed: None. B.B. Jamieson: None. S.X. Thomas: None. Y. Rim: None. A.G. Novak: None. R.E. Campbell: None. R. Piet: None. In female rodents, projections from suprachiasmatic nucleus (SCN) neurons relay timing cues to the gonadotropin-releasing hormone (GnRH) neuronal network for the preovulatory surge. Kisspeptin (KISS1) neurons of the rostral periventricular area of the third ventricle (RP3V) are thought to integrate these cues with estradiol positive feedback to drive GnRH neuron activity, the gonadotropin surge and ovulation. We recently reported that SCN neuron projections release arginine vasopressin (AVP), thereby stimulating female RP3VKiss[1] neuron action potential firing in an estrous cycle stage-dependent manner. In the SCN, AVP is expressed in a subset of neuromedin S (NMS) neurons, a neuronal population essential for circadian rhythms. Moreover, a previous report indicates that exogenous NMS stimulates LH secretion in female rodents. SCNNMS neurons might, therefore, play a role in timing the preovulatory surge. However, whether SCNNMS neurons project to and regulate the activity of RP3VKISS[1] neurons is currently unknown. To address this question, we combined anterograde viral tract-tracing and channelrhodopsin-2 (ChR2)- assisted circuit mapping. Female mice expressing Cre recombinase in NMS cells (NMS-Cre) received stereotaxic injections in the SCN of adeno-associated viral vectors (AAVs) carrying the Cre-dependent sequence for mCherry. Fluorescence immunohistochemistry revealed that mCherry-expressing fibers come in close apposition with the great majority (>80%) of KISS1-immunoreactive neurons in the RP3V, suggesting a SCNNMS-to-RP3VKISS[1] neuron circuit. To interrogate this circuit, we next injected AAVs carrying Cre-dependent channelrhodopsin (ChR2) in the SCN of female NMS-Cre mice that also express the green fluorescent protein in KISS1 cells. Blue LED light (460-487 nm) activation of ChR2-expressing SCNNMS neuronal fibers in the RP3V evoked postsynaptic currents (PSCs) in most KISS1 neurons recorded in whole-cell voltage clamp (-60 mV) in brain slices. Evoked PSCs had short latencies and were blocked by bath application of 0.5 µM tetrodotoxin (TTX), indicating that they were generated by action potential-dependent release. Addition of 100 µM 4-Aminopyridine (4-AP, a potassium channel blocker) to TTX rescued these responses, indicating the existence of a monosynaptic SCNNMS-to-RP3VKISS[1] connection. In addition, evoked PSCs were suppressed by 5 µM gabazine, a GABAA receptor antagonist, but not by 20 µM NBQX and 50 µM D-AP5, AMPA- and NMDA-type glutamate receptor antagonists, respectively, revealing that SCNNMS projections release GABA onto RP3VKISS[1] neurons. Together, our findings are consistent with SCNNMS neurons extending monosynaptic GABAergic projections to RP3VKISS[1] neurons. Further studies are needed to determine the impact of these projections on RP3VKISS[1] neuron activity and the role they might play in the preovulatory surge. Presentation: 6/1/2024

8686 scRNA-seq of pituitary from letrozole-induced PCOS mouse model reveals abnormal prolactin secretion

Abstract Disclosure: G. De Robles: None. N. Ujagar: None. Z. Del Mundo: None. D. Nicholas: None. Polycystic ovary syndrome (PCOS) has a complex pathophysiology and is the most common endocrine disorder among reproductive-aged women. Ovarian hormonal dynamics are regulated via the secretion of the gonadotropin-releasing hormone (GnRH), which stimulates the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from the anterior pituitary gland. High serum levels of LH in PCOS patients indicate an increase in GnRH levels, but the mechanisms within the anterior pituitary at a cellular level are unclear. We performed single-cell mRNA sequencing (scRNA-seq) on the pituitaries of the letrozole-induced (LET) mouse model of PCOS to profile the functional genomics of endocrine cell populations relative to serum hormone levels. The population percentage of lactotropes significantly decreased; in contrast, two gonadotrope populations significantly increased in the LET mice compared to the placebo-treated mice. Consistent with these changes, an increasing trend of serum LH levels and a decreasing trend of serum prolactin (PRL) levels were observed in the LET mice, while no change occurred in serum growth hormone levels. A gene set enrichment analysis (GSEA) on the differentially expressed genes (DEGs) from the lactotropes revealed an association with negative regulation of protein kinase activity. These DEGs included Hspb1, Park7, Pcp4, and Ppia, which all share the function of mediating stress-induced apoptosis. A GSEA on the DEGs between the two gonadotrope clusters in the LET mice also confirmed a variation in biological pathways linked to ribosomal subunits, transcription regulation, and the regulation of steroid metabolic processes. Based on the scRNA-seq results of the anterior pituitaries of LET mice, alterations in the proportions of hormone-secreting cells may be a response to hyperandrogenism, in which functionally diverse gonadotropes could contribute to heterogeneity in reproductive phenotypes. Changes in the lactotrope population and PRL serum levels also support further research on the lactation hormone and its role in PCOS. PRL is involved in metabolic homeostasis and reproduction, and understanding its dysregulations may provide additional insight into the pathophysiology of PCOS. Presentation: 6/3/2024

7996 Neuroanatomic Characterization of Mouse Kisspeptin Neurons in the Bed Nucleus of the Stria Terminalis

Abstract Disclosure: S.T. Zdon: None. M.S. Silva: None. V.M. Navarro: None. Kisspeptin (Kiss1) neurons are essential for reproductive function as they are strong regulators of the hypothalamic-pituitary-gonadal axis (HPG). Mammalian Kiss1 neurons are mainly located in the hypothalamus, controlling gonadotropin-releasing hormone (GnRH) secretion, where the anteroventral periventricular nucleus (AVPV) population is morphologically and functionally sexually differentiated. Kiss1 neurons are also found in limbic areas, such as the bed nucleus of the stria terminalis (BNST). This nucleus is involved in the integration of the HPG axis with sex-dependent social behaviors and metabolic functions. However, it remains unclear whether these populations show sexually dimorphic features. The present study focused on the neuroanatomical characterization of Kiss1BNST neurons in mice on both sexes. A Kiss1cre;salsa6f mouse strain carrying the expression of tdTomato specifically in Kiss1 neurons was used to study the distribution of Kiss1BNST neurons. The BNST is divided into the anterior division of the BNST (aBNST), and a more posterior division called the principal component of the BNST (prBNST). Results indicate that male mice have significantly more Kiss1prBNST neurons than females (Male: 139.4 ± 23.32 vs. Female: 43.70 ± 5.33 neurons; **P < 0.01). This sex-biased distribution was absent in Kiss1aBNST neurons. Additionally, stereotaxically injecting a Cre-dependent recombinant adenovirus carrying the expression of mCherry reporter into the aBNST or prBNST in adult mice was performed to observe neuronal projections. We observed fibers from aBNST-injected Kiss1 neurons projecting widely to areas that include the AVPV, medial preoptic area (MPO), lateral preoptic area (LPO), and periventricular hypothalamic nucleus (PVH). Moreover, fibers and cell bodies from male prBNST-injected Kiss1 neurons projected to dorsal lateral BNST (dlBNST), juxta-periventricular lateral hypothalamic area (JPLH), and the posterolateral stria terminalis (STMPL). This study observed a sexual dimorphic Kiss1 neuronal population in the prBNST, with males having more Kiss1prBNST neurons present and prospective postsynaptic areas in the brain where these neurons project to, helping to aid future studies on characterizing the functional role of these neurons. Presentation: 6/1/2024

7269 Interim Analysis of Gonadotropin-Releasing Hormone Agonist (GnRHa) Treatment in Children with Central Precocious Puberty: Focus on FMV Urinary LH Levels and Clinical Outcomes

Abstract Disclosure: X. Luo: Advisory Board Member; Self; Takeda. L. Hou: Advisory Board Member; Self; Takeda. Y. Li: Advisory Board Member; Self; Takeda. Y. Sun: Advisory Board Member; Self; Takeda. X.K. Jin: Employee; Self; Takeda. Background: Central precocious puberty (CPP) refers to early activation of the hypothalamic–pituitary–gonadal (HPG) axis in children. There is an urgent need to understand the treatment pattern and clinical outcomes among children suffering from CPP. Studies have reported that first morning voided (FMV) urinary Luteinizing hormone (LH) and Follicle stimulating hormone (FSH) have substantial association with CPP tested in GnRH stimulation test and can be useful in diagnosis and management of CPP in children. Gonadotropin-releasing hormone agonists (GnRHa) such as Leuprolide acetate (LA) are the recommended treatment for children suffering from CPP. The current study aimed to provide the information on level of LH and FSH in FMV urine when CPP diagnosis at baseline and the present results may also revealed trend of FMV-LH and FMV-FSH change during LA intervention in children with CPP. Here we present interim results from this study. Methods: This is an open-label, multicentre, single arm, prospective study that enrolled 31 subjects with CPP. Individuals were administered LA depot 11.25 mg, subcutaneously every 12 weeks. The study comprises a screening period; a 6-month treatment period: 24 weeks; and a post-treatment follow-up period: 12 weeks. The FMV urinary LH and FSH were diagnosed by Clopper-Pearson method with a 95% confidence interval. Results: Due to the data collection time point for the interim analysis, only 8 female subjects reached the 12-week visit time point and had a 12-week visit for FMV urinary Gn testing. Therefore, for this interim analysis, only FMV Gn decline at week 12 of the 8 subjects who had a 12-week visit will be presented. At baseline, the mean value of FMV urinary LH and FSH is 0.58 IU/L and 2.27 IU/L respectively. The FMV Gn urinary LH was demonstrated a decrease in 87.5% (95% CI: 47.35,99.68) of the children after LA intervention. Additionally, urinary FSH value of FMV urinary Gn was found to decrease in 75.00% of the children (95% CI: 34.91,96.81) compared to baseline FSH value. Conclusion: The present study reveals a decreasing trend in FMV Gn urinary LH and FSH levels in CPP children treated with LA during interim analysis. LA was shown to be safe and effective in ameliorating the level of LH and FSH in children suffering from CPP. From the study, it can be concluded that urinary LH and FSH may act as a potential biomarker for diagnosis and management of CPP. Presentation: 6/3/2024

7128 Efficacy and Safety of Leuprolide Acetate 3M Depot in Chinese Children with Central Precocious Puberty: Interim Analysis of Multicenter, Single-Arm, Prospective Study

Abstract Disclosure: X. Luo: Advisory Board Member; Self; Takeda. L. Hou: Advisory Board Member; Self; Takeda. Y. Li: Advisory Board Member; Self; Takeda. Y. Sun: Advisory Board Member; Self; Takeda. X.K. Jin: Employee; Self; Takeda. Background: Central precocious puberty (CPP) due to early activation of the hypothalamic-pituitary-gonadal axis leads to early puberty. CPP has a negative impact on adult height. Leuprolide acetate (LA), a gonadotropin-releasing hormone agonist (GnRHa) is the standard treatment for CPP. As CPP requires prolonged treatment, a 3-month (3M) depot formulation would increase patient compliance and reduce the injection burden. This interim analysis presents the effect of LA 3M depot on the gonadotropic axis and assesses safety in children with CPP. Methods: In this open-label, multicenter, single-arm, and prospective study, 31 children with CPP were included. Key inclusion criteria were the appearance of secondary sexual characteristics below the age of eight years in girls and nine years in boys with a confirmed diagnosis of CPP. Children treated with prior GnRHa and/or having underlying medical conditions were excluded. Eligible children were treated with LA 11.25 mg subcutaneously every 12 weeks. The primary endpoint of this interim analysis was the proportion of children achieving inhibition of peak LH defined as peak LH ≤ 3.0 IU/L in the GnRH stimulation test at weeks 24. The secondary endpoint was safety analysis in the children receiving at least one dose of LA 3M depot. Results: At baseline, 30 girls and one boy were enrolled. The median (95% CI) chronological age (CA) of the girls was 8.0 (7.0, 9.0) years and the boy was 10 years old. Girls had a median height of 136.95 (131.5, 139.7) cm, weight of 30 (27, 34) kg, and BMI of 16.4 (15.1, 17.9) kg/m2. Baseline bone (BA) age was 11 (10, 11) years, and BA/CA was 1.24 (1.2, 1.3). Boy’s characteristics at baseline differed in CA (10 years), height (144 cm), and BMI (17.4 kg/m2) from girls of this cohort. Due to the data collection time points for the interim analysis, 8 out of the 31 enrolled subjects had a visit at week 24, and 30 subjects reached the week 12 visit time point and had a visit at week 12. The percentage of children achieving inhibition of peak LH (≤ 3.0 IU/L) in the GnRH stimulation test at week 12 was 100 (88.4, 100) %. At week 24, all of them achieved inhibition of LH in the GnRH stimulation test 100 (63, 100) %. Decreases in BA/CA from baseline at weeks 12 and 24 are 93 (77.9, 99.2) % and 50 (15.7, 84.3) % respectively. No progression of the Tanner stage was observed in 96.7 (82.8, 99.9) % and 100 (63, 100) % at weeks 12 and 24. At the data cut-off, 4 (13.33%) patients experienced a total of 5 treatment-emergent adverse events (TEAEs) in the safety population of 30 subjects. No grade ≥3 AE or serious AE was reported. The incidence of injection site reaction was 3.33%. Conclusion: LA at 11.25mg/3M depot effectively suppresses the gonadotropic axis at weeks 12 and 24, normalizing growth and reversibly slowing the early onset of puberty in children with CPP. LA in a 3-month injecting frequency is a safe and convenient treatment option to improve patient compliance. Presentation: 6/3/2024

8208 Unexpected And Significant Rise In TSH In The Setting Of Relugolix Use

Abstract Disclosure: A.Y. Groysman: None. A. Poloju: None. P. Majety: None. T. Abraham: None. Background: Relugolix is a gonadotropin-releasing hormone antagonist used as androgen deprivation therapy for metastatic and nonmetastatic castration-resistant prostate cancer that was FDA-approved in December 2020. Common side effects include hot flashes, increased glucose and triglycerides, and decreased hemoglobin. We report a case of Relugolix interference with the absorption of levothyroxine, which has not been reported in the literature. Case: A 79-year-old white man with high-risk, Gleason 9 prostate cancer and Hashimoto’s hypothyroidism presented for evaluation of elevated thyroid stimulating hormone (TSH) level- 52.87 mIU/L (0.28-4.10 mIU/L). He did not have cold intolerance, constipation, fatigue, or weight gain. He was diagnosed with hypothyroidism in 1998 and was on a stable dose of levothyroxine 88mcg daily from 2016 until the time of this presentation. He is adherent and reports following a routine of having breakfast with coffee soon after taking levothyroxine for many years. In June 2022, his TSH was 4.48 mIU/L. In July 2022, he started taking Relugolix along with levothyroxine. Following this, his TSH levels increased to 30.08 mIU/L (FT4: 0.67 ng/dL), 35.43 mIU/L (FT4: 0.57 ng/dL), 52.87 mIU/L (FT4: 0.48 ng/dL) on 2/24/23, 3/3/23, and 3/14/23, respectively. No new medications were reported. His other medications were atorvastatin, tamsulosin, and vitamin D, which he took at night. He did not take calcium or biotin supplements. He had gradual weight gain (8 lbs) over the last 9 months. Levothyroxine dose was increased from 88 to 125 mcg daily and he was instructed to separate Relugolix from levothyroxine so he started taking Relugolix at bedtime. Repeat TSH was low at 0.12 mIU/L. The levothyroxine dose was eventually titrated back to 88 mcg daily and TSH was 5.2 (appropriate for age). Conclusion: Several medications are known to interfere with the absorption of levothyroxine. Our case suggests that Relugolix may be one of them. The significant rise in TSH and levothyroxine requirements months after starting Relugolix and the ability to return to the previous dose of levothyroxine only by separating Relugolix from levothyroxine strongly suggests that there is an interference between these medications that has not been reported. Presentation: 6/2/2024

7115 Long-Term Exposure To Air Pollution And Precocious Puberty In Korea

Abstract Disclosure: J. Choi: None. J. Oh: None. R. Lee: None. E. Mun: None. K. Kim: None. E. Ha: None. H. Kim: None. Backgrounds and Aims The increasing prevalence of precocious puberty is emerging as a significant medical and social problem worldwide. However, limited research has investigated the relationship between long-term air pollution exposure and precocious puberty. We aimed to investigate the association between long-term air pollution exposure and first onset of precocious puberty in South Korea, nationwide. Methods: We investiagted a retrospective cohort using Korea National Health Insurance Database. Children born from 2007 to 2009 were followed-up from the age of six (2013 to 2015). We included boys aged ≤10 years and girls aged ≤ 9 years who visited hospitals for early pubertal development and were diagnosed with precocious puberty according to the ICD-10 (E228, E301,and E309) and gonadotropin-releasing hormone agonist (GnRHa) treatment. We conducted the association between long-term air pollution exposure and first onset of precocious puberty using a Cox proportional hazard model. We estimated the hazard ratios (HR) and 95% confidence intervals (CI) per 1 μg/m3 increase for fine particulate matter (PM2.5) and particulate matter (PM10) and per 1 ppb increase for sulfur dioxide (SO2), nitrogen dioxide (NO2), and ozone (O3). Results: A total of 1,205,784 childrens aged six years old were included during 2013­-2015. A positive association was found that the association between 48-month moving average PM2.5 (HR: 1.019, 95% CI: 1.012, 1.027), PM10 (HR: 1.009, 95% CI: 1.006, 1.013), SO2 (HR: 1.037, 95% CI: 1.018, 1.055), O3 (HR: 1.006, 95% CI: 1.001, 1.010) exposure and precocious puberty in girls. In contrast, no association was found in boys. Conclusions Our findings supported that long-term exposure to air pollution is associated with an increased risk of precocious puberty. The study provides valuable insights into the harmful effects of air pollution during childhood and adolescence, emphasizing that air pollution is a modifiable risk factor that can be managed and improved. Presentation: 6/1/2024

Evaluation of Synthetic GnRH-Analog Peforelin with Regard to Oocyte Differentiation and Follicular Development in C57BL/6J Mice.

In biomedical research, ovulation induction is a critical step in the reproductive biology of laboratory animals. This study evaluates the efficacy of peforelin, a synthetic gonadotropin-releasing hormone (GnRH) analog, in comparison to pregnant mare serum gonadotropin (PMSG, synonym: eCG), traditionally used for ovulation induction in mice. PMSG is derived from the serum of pregnant horses, and its production is becoming increasingly problematic due to animal welfare concerns and regulatory restrictions. The aim of this study was, therefore, to evaluate an ethically acceptable and less invasive alternative to PMSG. Female C57BL/6J mice, aged 3-4 weeks, were divided into two groups to receive either peforelin at three different concentrations or PMSG, followed by an injection of human chorionic gonadotropin (hCG) to induce ovulation. Key outcomes included the number and quality of oocytes collected, fertilization rates, ovary morphology, and follicular differentiation. Although the number of oocytes was significantly lower in the peforelin cohort, the fertilization rate was high. Ovarian morphology was not significantly altered compared to the PMSG cohort. This study showed that peforelin is suitable for superovulation in mice. These results suggest that peforelin could be an ethically acceptable alternative to PMSG stimulation for inducing superovulation in mice.

Effect of the Application of PGF2α Associated With Ovulation Induction in a Fixed-Time Superovulation Programme for Precocious Nellore Heifers.

This study evaluated the effect of prostaglandin F2α (PGF2α) associated with gonadotropin-releasing hormone (GnRH) for ovulation induction in precocious indicus heifers submitted to a fixed-time superovulation (SOV) programme. Precocious Nellore heifers (n = 35), aged 13 months, were subjected to the SOV protocol. On day 0 (D0), all animals received intravaginal insertion of a progesterone (P4) device along with intramuscular administration of 2 mg of oestradiol benzoate, plus 200 IU of follicle-stimulating hormone in decreasing doses, with 12-h intervals between D4 and D7, in addition to 150 μg of D-cloprostenol on D6 and device removal on D7. On D8, the donors received 10.5 μg of buserelin acetate and the treatment group received 300 μg of D-cloprostenol/PGF2α. Artificial insemination was performed 12 h and 24 h after GnRH administration using frozen semen. On D15 of the protocol (i.e., D7 after insemination), the embryos were collected and evaluated. All animals passed through the control and treatment groups. Results were evaluated by analysis of variance using an adjusted mixed-effects model (p < 0.05). There was no difference in the total number of embryos between the control and treatment groups (10.40 ± 1.52 vs. 9.60 ± 1.36; p = 0.63) or viable embryos (6.30 ± 1.22 vs. 4.30 ± 0.71). For precocious indicus heifers, treatment with PGF2α in association with GnRH did not affect embryo production in the fixed-time SOV protocol.

Sex-Dependent Changes in Gonadotropin-Releasing Hormone Neuron Voltage-Gated Potassium Currents in a Mouse Model of Temporal Lobe Epilepsy.

Temporal lobe epilepsy (TLE) is the most common focal epilepsy in adults, and people with TLE exhibit higher rates of reproductive endocrine dysfunction. Hypothalamic gonadotropin-releasing hormone (GnRH) neurons regulate reproductive function in mammals by regulating gonadotropin secretion from the anterior pituitary. Previous research demonstrated GnRH neuron hyperexcitability in both sexes in the intrahippocampal kainic acid (IHKA) mouse model of TLE. Fast-inactivating A-type (I A) and delayed rectifier K-type (I K) K+ currents play critical roles in modulating neuronal excitability, including in GnRH neurons. Here, we tested the hypothesis that GnRH neuron hyperexcitability is associated with reduced I A and I K conductances. At 2 months after IHKA or control saline injection, when IHKA mice exhibit chronic epilepsy, we recorded GnRH neuron excitability, I A, and I K using whole-cell patch-clamp electrophysiology. GnRH neurons from both IHKA male and diestrus female GnRH-GFP mice exhibited hyperexcitability compared with controls. In IHKA males, although maximum I A current density was increased, I K recovery from inactivation was significantly slower, consistent with a hyperexcitability phenotype. In IHKA females, however, both I A and I K were unchanged. Sex differences were not observed in I A or I K properties in controls, but IHKA mice exhibited sex effects in I A properties. These results indicate that although the emergent phenotype of increased GnRH neuron excitability is similar in IHKA males and diestrus females, the underlying mechanisms are distinct. This study thus highlights sex-specific changes in voltage-gated K+ currents in GnRH neurons in a mouse model of TLE and suggesting potential sex differences in GnRH neuron ion channel properties.

Genome sequencing projects reveal new insights into the mammalian Gonadotropin-releasing Hormone II system.

The type II gonadotropin-releasing hormone (GnRH-II) was first discovered in chicken (Gallus gallus) brain and then shown to be present in many vertebrates. Indeed, its structure is conserved unchanged throughout vertebrate evolution from teleost fish through to mammals suggesting a crucial function. Yet the functional significance has been largely unexplored. Studies in comparative endocrinology show that the GnRH-II system is differentially functional in mammalian species. Intact GnRH-II neuropeptide and receptor genes (GnRH2 and GnRH receptor 2 GnRHR2) occur in marmoset monkeys (Callithrix jacchus), musk shrews (Suncus murinus) and pigs (Sus scrofa). However, one or other or both of these genes are inactivated in other species, where mutations or remnants affecting GnRH2 neuropeptide and/or type II GnRHR exons are retained in conserved genomic loci. New data from DNA sequencing projects facilitate extensive analysis of species-specific variation in these genes. Here, we describe GnRH2 and GnRHR2 genes spanning a collection of 21 taxonomic orders, encompassing around 140 species from Primates, Scandentia, Eulipotyphla, Rodentia, Lagomorpha, Artiodactyla, Carnivora, Perissodactyls, Pholidota, Chiroptera, Afrotheria, Xenarthra and Marsupialia. Intact coding exons for both GnRH2 and GnRHR2 occur in monkeys, tree shrews, shrews, moles, hedgehogs, several rodents (degu, kangaroo-rat, pocket mouse), pig, pecarry and warthog, camels and alpaca, bears, Weddell seal, hyena, elephant, aardvark and marsupials. Inactivating mutations affecting GnRH2 and GnRHR2, some located at conserved sites within exons, occur in species of primates, most rodents, lagomorphs, bovidae, cetaceans, felidae, canidae and other carnivora, pangolins, most bats, armadillo, brushtail and echidna. A functional GnRH-II system appears retained within several taxonomic families of mammals, but intact retention does not extend to whole taxonomic orders. Defining how endogenous GnRH-II neuropeptide operates in different mammals may afford functional insight into its actions in the brain, especially as, unlike the type I GnRH system, it is expressed in the mid brain and not the hypothalamus.

Central δ/κ opioid receptor signaling pathways mediate chronic and/or acute suckling-induced LH suppression in rats during late lactation.

In mammals, secretion of tonic (pulsatile) gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) is often suppressed during lactation. Suppression of GnRH/LH pulses in lactating dams is assumed to be caused by suckling stimuli and a chronic negative energy balance due to milk production. The present study aimed to investigate whether the central enkephalin-δ opioid receptor (DOR) signaling mediated the suppression of LH secretion by acute suckling stimuli and/or chronic negative energy balance due to milk production in rats during late lactation when dams were under a heavy energy demand. On postpartum day 16, the number of Penk (enkephalin mRNA)-expressing cells in the arcuate nucleus was significantly higher in lactating rats than in non-lactating control rats. Pulsatile LH secretion was suppressed in rats with chronic suckling or acute 1-h suckling stimuli 6 h after pup removal on day 16 of lactation. Central DOR antagonism significantly increased the mean LH concentrations and the baseline of LH pulses in rats with chronic suckling but not with acute suckling stimuli on day 16 of lactation. Besides, central κ opioid receptor (KOR) antagonism increased the amplitude of LH pulses in rats with the acute suckling stimuli on day 16 of lactation. These results suggest that central DOR signaling mediates the suppression of LH secretion caused by a negative energy balance in rats receiving chronic suckling during late lactation. On the other hand, central KOR signaling likely mediates acute suckling stimuli-induced suppression of LH secretion in rats during late lactation.

Potency evaluation of different GNRH analogues on ovulation induction and reproductive performance of doe rabbit.

Gonadotropin-releasing hormone (GnRH) -supplemented extenders have emerged as a welfare-orientated method to induce ovulation in the artificial insemination (AI) of rabbits. The main factor that limits the bioavailability of GnRH analogue on intravaginal administration is the proteolytic activity of enzymes present in rabbit seminal plasma. The use of GnRH analogues with higher biological potency would allow us to decrease their concentration in the seminal dose without compromising effectiveness. The current study was designed to assess the efficacy of various GnRH analogues concerning their ability to induce ovulation in rabbit AI. The base solution used for experimental extenders contained an aminopeptidase inhibitor. Four experimental groups were used, females from the Control group were induced to ovulate with an intramuscular administration of 1 μg of buserelin, while in the other three groups females received an intravaginal administration of 3.5 μg of buserelin (BUS), deslorelin (DES) or fertirelin (FER) within the seminal dose. Results showed that the ovulation frequency was similar in all groups studied. A concentration of 3.5 μg of the different GnRH analogues tested in this study showed similar potency in inducing ovulation in non-lactating females, yielding comparable results in terms of pregnancy rate at birth and prolificacy.

Progestin-primed ovarian stimulation

The use of progestins for the prevention of premature ovulation during ovarian stimulation is called progestin-primed ovarian stimulation (PPOS). Response to ovarian stimulation with PPOS and gonadotropin-releasing hormone (GnRH) analogs seem to be similar with the clinically insignificant differences being explained by endocrine characteristics as expected. PPOS has the advantage of oral administration and lower medication cost than GnRH analogs, but the mandatory cancellation of an otherwise possible fresh embryo transfer can render PPOS less cost-effective if a fresh transfer would have been possible. Oocytes collected in PPOS cycles have similar developmental potential, including blastocyst euploidy rates. Frozen embryo transfer outcomes of PPOS and GnRH analog cycles seem similar both in terms of ongoing pregnancy/live birth rates and obstetric and perinatal outcomes. While some studies reported lower cumulative live birth rates with PPOS, they suffer from methodological issues, including arbitrary definitions of cumulative live birth rates. PPOS has been used in all patient types with consistent results and seems a patient-friendly and cost-effective choice when a fresh embryo transfer is not intended.

Major regulatory factors for reproductive performances of female chickens

The reproductive performance of female chickens is critical for determining the efficiency of production and productivity and thus profitability. Studies have shown that the reproductive performance of female chickens is mainly regulated by the feed, hormones, genes, and light conditions. Herein, we review the major factors regulating female chicken reproductive performance and assess the reproductive organs and their functions. In the current review, we highlight how the interconnections of hormones, candidate genes, and photo-stimulation regulate female chicken reproductive hormones and thus regulate the reproductive organ performance. In this regard, the roles of main hormones [gonadotropinreleasing hormone (GnRH) and genes (GnRH-I)] in regulating sexual maturation and ovarian development and maintenance by influencing the survival and function of follicular granulosa cells were also reviewed. In addition, the current review also highlights how feeding female chickens with diets and artificial light-emitting diodes (LEDs) support the effective functioning of their reproductive capacity through the stimulation of sexual maturity at an appropriate age and regeneration of aged reproductive organs.

Effects of herbal medicine on ovulation in premature ovarian insufficiency patients during in vitro fertilization

Introduction: Premature ovarian insufficiency (POI) is a condition in which menopause occurs before the age of 40 and is known to be a relatively common disease that occurs in 1% of all women. The only way for patients diagnosed with POI to become pregnant is through oocyte donation, in which fertilized ovums are transplanted by receiving oocytes from donors.4 Can POI patients increase their chances of normal ovulation? POI patients were more likely to observe and collect follicles larger than the average 15 mm diameter when herbal medicine was administered. Methods: This study is a retrospective cohort study conducted on POI patients undergoing in vitro fertilization (IVF) who visited our hospital from 2020 to 2022. We would like to report the response rate to gonadotropin-releasing hormone (GnRH) during IVF in the hyperovulation induction low response group after herbal medicine treatment. Statistical analysis was performed using IBM’s SPSS Statistics to verify significance with a P-value&lt;0.05. Results: In recovering ovarian POI patient's ovarian function, herbal medicine treatment increased the response rate to GnRH (the number of oocytes collected) during IVF, follicle-stimulating hormone (FSH) and cortisol levels decreased, and estradiol (E2) and luteinizing hormone (LH) levels increased. Conclusions: It was confirmed that herbal medicine treatment can increase the probability of normal ovulation in patients with idiopathic POI with high cortisol levels. Until now, it has been known that ovarian function cannot be recovered once irreversibly decreased, but by increasing the probability of ovulation through herbal medicine treatment, it is significant that it may be possible to suppress ovarian aging or preserve fertility.

GnRH antagonist protocol with hCG triggering ameliorates fertilization defect caused by failure of cumulus cell pentraxin-3 expression in unilateral endometriomas.

The aim of the study was to determine the expression pattern of long pentraxin 3 (PTX3) mRNA in cumulus cells (CCs) isolated from metaphase II oocytes of women with unilateral endometrioma undergoing controlled ovarian stimulation using a gonadotropin-releasing hormone antagonist (GnRHa) protocol. A total of 60 CC samples, 30 from the affected ovary and 30 from the contralateral ovary, were collected from 12 patients with unilateral endometrioma who underwent flexible GnRHa protocol with recombinant human chorionic gonadotropin (rhCG) trigger. Thirty CC samples collected from the left ovary of 12 women with male factor infertility were used as external controls. Long PTX3 mRNA expression in each group was analyzed by real-time polymerase chain reaction (RT-PCR). Relative gene expression (fold-change) was calculated according to the 2-ΔΔCt equation. Fertilization rates after intracytoplasmic sperm injection (ICSI) were recorded for each group. CC-PTX3 mRNA expression in the unilateral endometrioma group was significantly lower than the mRNA expression of the disease-free ovary (0.90±0.01 vs. 0.25±0.02, p<0.01). CC-PTX3 mRNA expression of MII oocytes of the disease-free ovary was found to be similar to the control group (1.02±0.03 vs. 0.90±0.01, p=0.107). A significant 3.6-fold downregulation was observed in the CC-PTX3 mRNA expression of the endometrioma group compared to the CC-PTX3 mRNA expression in the contralateral ovary. CC-PTX3 mRNA expression in the endometrioma group was downregulated 4.08-fold compared to the CC-PTX3 mRNA expression of the control group (1.02±0.03 vs. 0.25±0.02, p<0.001). The cumulus morphologies of the endometrioma group with low CC-PTX3 expression and the groups with normal CC-PTX3 levels were similar. Fertilization rates of the endometrioma group were similar to the contralateral ovary and control groups. Unilateral endometrioma reduces CC-PTX3 expression but does not affect disease-free ovaries. The GnRHa protocol improved the fertilization rates, suggesting that failed CC-PTX3 expression is an in vivo pathology.

N-acetylcysteine and zinc sulphate abate di-2-ethylhexyl phthalate-mediated reproductive dysfunction in rats: Focus on oxidative and sex hormone receptors mechanisms

Objective: To investigate the potential of N-acetylcysteine (NAC) and zinc sulphate (ZnSO4) in mitigating reproductive dysfunction caused by di-2-ethylhexyl phthalate (DEHP) in rats and to understand the underlying mechanisms, specifically oxidative stress and sex hormone receptor activity. Methods: Thirty-five male Wistar rats were randomly divided into five equal groups (n=7 per group). Group 1 was administered 0.5 mL of distilled water and served as the control group. Group 2 was given only DEHP (750 mg/kg/day), while group 3, 4 and 5 were given DEHP (750 mg/kg/day) plus NAC (100 mg/kg/day), DEHP (750 mg/kg/day) plus ZnSO4 (0.5 mg/kg/day), and DEHP (750 mg/kg/day) plus NAC (100 mg/kg/day) as well as ZnSO4 (0.5 mg/kg/day), respectively. All treatments lasted for 21 days. Samples were obtained after the rats were sacrificed, and hormones levels in the serum and markers of oxidative stress in the testicles were analyzed using the enzyme-linked immunosorbent assay. The amount of androgen receptors in the testicles was determined by immunohistochemistry, and the susceptibility of testosterone and DEHP to bind to androgen receptor and 5α-reductase was determined by molecular docking studies. Results: DEHP decreased reproductive hormones, testicular antioxidant enzymes, increased malondialdehyde levels, and negatively impacted histology of the pituitary and testes. NAC or ZnSO4 treatment showed a marked improvement in testicular antioxidant status and hormone levels, as well as a positive effect on the histology of the pituitary and testes. The combination of both treatments appeared to be more effective. The affinity of DEHP to bind to androgen receptors may lead to disruption of androgen receptor signaling, which can further result in dysfunction of hormones related to androgen. However, NAC is more likely to form stronger binding interactions with follicle stimulating hormone and luteinizing hormone receptors, as well as gonadotropin-releasing hormone receptors, when compared to DEHP. Conclusions: The possibility that NAC and ZnSO4 could downregulate DEHP-induced sex hormone changes is suggested by their potential to reduce toxicity.