Gonadotropin-releasing Hormone Pulse Research Articles

Peripheral administration of a κ-opioid receptor agonist nalfurafine inactivates gonadotropin-releasing hormone pulse generator activity in goats

Neurons co-expressing kisspeptin, neurokinin B, and dynorphin A (KNDy neurons), located in the arcuate nucleus (ARC) of the hypothalamus, are indicated to be the gonadotropin-releasing hormone (GnRH) pulse generator. Dynorphin A is reported to suppress GnRH pulse generator activity. Nalfurafine is a selective agonist of the κ-opioid receptor (KOR), a receptor for dynorphin A, clinically used as an anti-pruritic drug. This study aimed to evaluate the effects of nalfurafine on GnRH pulse generator activity and luteinizing hormone (LH) pulses using female goats. Nalfurafine (0, 2, 4, 8, or 16 μg/head) was intravenously injected into ovariectomized Shiba goats. The multiple unit activity (MUA) in the ARC area was recorded, and plasma LH concentrations were measured 2 and 48 h before and after injection, respectively. The MUA volley interval during 0–2 h after injection was significantly increased in the nalfurafine 8 and 16 μg groups compared with the vehicle group. In 0–2 h after injection, the number of LH pulses was significantly decreased in the nalfurafine 8 and 16 μg groups, and the mean and baseline LH were significantly decreased in all nalfurafine-treated groups (2, 4, 8, and 16 μg) compared with the vehicle group. These results suggest that nalfurafine inhibits the activity of the GnRH pulse generator in the ARC, thus suppressing pulsatile LH secretion. Therefore, nalfurafine could be used as a reproductive inhibitor in mammals.

Efficacy of GnRH Pulses in Hypogonadism Secondary to Primary Empty Sella: Case Report.

This study aims to assess the effectiveness of pulsed gonadotropin-releasing hormone (GnRH) micropump replacement therapy in the treatment of hypogonadotropic hypogonadism (HH) caused by primary empty sella (PES).The efficacy of pulsed GnRH replacement therapy using the micropump was evaluated in a middle-aged male patient with HH who had experienced the loss of his only child. Relevant literature was also consulted to compare the differences between pulse GnRH treatment and conventional treatment in terms of the development of secondary sexual characteristics, sex hormone levels, sperm production rate, and sperm activity rate in male patient with HH.In this report, a 45-year-old male diagnosed with HH and PES presented with fatigue and decreased libido. The main characteristics included decreased follicle stimulating hormone (FSH) levels of 0.03 mIU/mL, luteinizing hormone (LH) levels of 0.02 mIU/mL, and testosterone (T) levels of 0.72 nmol/L. Magnetic resonance imaging (MRI) revealed an empty sella. Semen analysis showed a small number of normal sperm with reduced motility. During treatment with the micropump pulse GnRH, the patient experienced no side effects and showed improvements in fatigue, reduced libido, sexual urge, anxiety, and feelings of inferiority. LH, FSH, and T levels returned to normal, while sperm activity rate increased to 79.9%. Ultimately, the patient's spouse achieved a natural pregnancy.Pulsed gonadotropin delivery using the micropump demonstrates good efficacy and tolerability, and aligns more closely with the physiological rhythm of GnRH secretion in the human body.

P-708 melatonin alleviates circadian rhythm disruption-induced enhanced luteinizing hormone pulsatility and ovarian dysfunction in vivo and in vitro

Abstract Study question Is melatonin deficiency a reversible cause of abnormal pattern of luteinizing hormone (LH) pulsatility and ovarian dysfunction caused by circadian rhythm disruption (CRD)? Summary answer Melatonin could rescue the augmented LH pulsatility and reproductive abnormalities caused by CRD. What is known already Melatonin, mainly synthesized and released by pineal gland, engages in regulating circadian rhythms. When circadian rhythms are disrupted, the nocturnal productions of melatonin are suppressed. Evidence suggests that circadian rhythm disruption (CRD) is associated with reproductive dysfunctions, while melatonin plays a vital role for female reproduction as a free radical scavenger and a neuroendocrine regulator. The gonadotrophin-releasing hormone (GnRH) drives pulsatile luteinizing hormone (LH) secretion, which is also essential for fertility. Study design, size, duration We used 90 CBA/CaJ mice to establish the chronic jet-lag model in which mice were exposed to an 8 hr time shift every 3 days. After 8 weeks of jet-lag induction, some of the mice were given melatonin treatment for 4 weeks. Furthermore, we used a perfusion system in vitro to mimic the process of CRD-induce high-frequency GnRH pulses stimulation of LβT2 gonadotrope cells. Participants/materials, setting, methods Repetitive tail-tip blood collection was used to evaluate the pattern of LH pulsatility. Blood plasma samples were collected from 4 mice at 4-hour intervals to measure levels of melatonin. Follicular development was evaluated by ovarian histology. Reproductive hormones was detected by ELISA. Immunohistochemistry and western blot were used to detect the expression of melatonin receptor 1A (MT1). The LβT2 cells were exposed to pulsatile GnRH at high pulse frequencies to mimic augmented pulsatile GnRH stimulation. Main results and the role of chance CRD mice exhibited reproductive-endocrine dysfunction, including irregular estrous cycles, reduced number of corpus lutea (CL), and decreased levels of estradiol and progesterone. Additionally, CRD enhanced the frequency and amplitude of LH pulsatility and pituitary LH beta-subunit expression. Melatonin administration alleviated these CRD-induced reproductive abnormalities and reversed the augmented LH pulsatility. The expression of MT1 decreased in the anterior pituitaries of CRD mice but increased after melatonin treatment, indicating the essential role of MT1 in melatonin’s reversal of CRD-induced enhanced LH pulsatility. Limitations, reasons for caution Further research is needed to validate the applicability and safety of these findings in humans. Wider implications of the findings The results suggest that melatonin may be a beneficial option for patients with reproductive-endocrine abnormalities, particularly those with circadian rhythm disruption. Trial registration number 82101719

POMC neurons control fertility through differential signaling of MC4R in Kisspeptin neurons.

Inactivating mutations in the melanocortin 4 receptor ( MC4R ) gene cause monogenic obesity. Interestingly, female patients also display various degrees of reproductive disorders, in line with the subfertile phenotype of MC4RKO female mice. However, the cellular mechanisms by which MC4R regulates reproduction are unknown. Kiss1 neurons directly stimulate gonadotropin-releasing hormone (GnRH) release through two distinct populations; the Kiss1 ARH neurons, controlling GnRH pulses, and the sexually dimorphic Kiss1 AVPV/PeN neurons controlling the preovulatory LH surge. Here, we show that Mc4r expressed in Kiss1 neurons is required for fertility in females. In vivo , deletion of Mc4r from Kiss1 neurons in female mice replicates the reproductive impairments of MC4RKO mice without inducing obesity. Conversely, reinsertion of Mc4r in Kiss1 neurons of MC4R null mice restores estrous cyclicity and LH pulsatility without reducing their obese phenotype. In vitro , we dissect the specific action of MC4R on Kiss1 ARH vs Kiss1 AVPV/PeN neurons and show that MC4R activation excites Kiss1 ARH neurons through direct synaptic actions. In contrast, Kiss1 AVPV/PeN neurons are normally inhibited by MC4R activation except under elevated estradiol levels, thus facilitating the activation of Kiss1 AVPV/PeN neurons to induce the LH surge driving ovulation in females. Our findings demonstrate that POMC ARH neurons acting through MC4R, directly regulate reproductive function in females by stimulating the "pulse generator" activity of Kiss1 ARH neurons and restricting the activation of Kiss1 AVPV/PeN neurons to the time of the estradiol-dependent LH surge, and thus unveil a novel pathway of the metabolic regulation of fertility by the melanocortin system.

Raphe glucose-sensing serotonergic neurons stimulate KNDy neurons to enhance LH pulses via 5HT2CR: rat and goat studies

Dysfunction of central serotonergic neurons is known to cause depressive disorders in humans, who often show reproductive and/or glucose metabolism disorders. This study examined whether dorsal raphe (DR) serotonergic neurons sense high glucose availability to upregulate reproductive function via activating hypothalamic arcuate (ARC) kisspeptin neurons (= KNDy neurons), a dominant stimulator of gonadotropin-releasing hormone (GnRH)/gonadotropin pulses, using female rats and goats. RNA-seq and histological analysis revealed that stimulatory serotonin-2C receptor (5HT2CR) was mainly expressed in the KNDy neurons in female rats. The serotonergic reuptake inhibitor administration into the mediobasal hypothalamus (MBH), including the ARC, significantly blocked glucoprivic suppression of luteinizing hormone (LH) pulses and hyperglycemia induced by intravenous 2-deoxy-D-glucose (2DG) administration in female rats. A local infusion of glucose into the DR significantly increased in vivo serotonin release in the MBH and partly restored LH pulses and hyperglycemia in the 2DG-treated female rats. Furthermore, central administration of serotonin or a 5HT2CR agonist immediately evoked GnRH pulse generator activity, and central 5HT2CR antagonism blocked the serotonin-induced facilitation of GnRH pulse generator activity in ovariectomized goats. These results suggest that DR serotonergic neurons sense high glucose availability to reduce gluconeogenesis and upregulate reproductive function by activating GnRH/LH pulse generator activity in mammals.

NK3R signalling in the posterodorsal medial amygdala is involved in stress-induced suppression of pulsatile LH secretion in female mice.

Psychosocial stress negatively impacts reproductive function by inhibiting pulsatile luteinizing hormone (LH) secretion. The posterodorsal medial amygdala (MePD) is responsible in part for processing stress and modulating the reproductive axis. Activation of the neurokinin 3 receptor (NK3R) suppresses the gonadotropin-releasing hormone (GnRH) pulse generator, under hypoestrogenic conditions, and NK3R activity in the amygdala has been documented to play a role in stress and anxiety. We investigate whether NK3R activation in the MePD is involved in mediating the inhibitory effect of psychosocial stress on LH pulsatility in ovariectomised female mice. First, we administered senktide, an NK3R agonist, into the MePD and monitored the effect on pulsatile LH secretion. We then delivered SB222200, a selective NK3R antagonist, intra-MePD in the presence of predator odour, 2,4,5-trimethylthiazole (TMT) and examined the effect on LH pulses. Senktide administration into the MePD dose-dependently suppresses pulsatile LH secretion. Moreover, NK3R signalling in the MePD mediates TMT-induced suppression of the GnRH pulse generator, which we verified using a mathematical model. The model verifies our experimental findings: (i) predator odour exposure inhibits LH pulses, (ii) activation of NK3R in the MePD inhibits LH pulses and (iii) NK3R antagonism in the MePD blocks stressor-induced inhibition of LH pulse frequency in the absence of ovarian steroids. These results demonstrate for the first time that NK3R neurons in the MePD mediate psychosocial stress-induced suppression of the GnRH pulse generator.

Long-term Recordings of Arcuate Nucleus Kisspeptin Neurons Across the Mouse Estrous Cycle.

The arcuate nucleus kisspeptin (ARNKISS) neurons represent the GnRH pulse generator that likely drives pulsatile gonadotropin secretion in all mammals. Using an improved GCaMP fiber photometry system enabling long-term continuous recordings, we aimed to establish a definitive profile of ARNKISS neuronal activity across the murine estrous cycle. As noted previously, a substantial reduction in the frequency of ARNKISS neuron synchronization events (SEs) occurs on late proestrus and extends into estrus. The SE amplitude remains constant throughout the cycle. During metestrus, we unexpectedly detected many multipeak SEs where many SEs occurred rapidly, within 160 seconds of each other. By applying a machine learning-based, k-means clustering analysis, we were further able to detect substantial within-stage variability in the patterns of pulse generator activity. Estrous cycle-dependent changes in SE activity occurred around the time of lights on and off. We also find that a mild stressor such as vaginal lavage reduces ARNKISS neuron SE frequency for up to 3 hours. These observations provide a comprehensive account of ARNKISS neuron activity across the estrous cycle, highlight a new pattern of multipeak SE activity, and introduce a new k-means clustering approach for analyzing ARNKISS neuron population behavior.

New methods to investigate the GnRH pulse generator.

The exact neural construct underlying the dynamic secretion of gonadotrophin-releasing hormone (GnRH) has only recently been identified despite the detection of multiunit electrical activity volleys associated with pulsatile luteinising hormone (LH) secretion four decades ago. Since the discovery of kisspeptin/neurokinin B/dynorphin neurons in the mammalian hypothalamus, there has been much research into the role of this neuronal network in controlling the oscillatory secretion of gonadotrophin hormones. In this review, we provide an update of the progressive application of cutting-edge techniques combined with mathematical modelling by the neuroendocrine community, which are transforming the functional investigation of the GnRH pulse generator. Understanding the nature and function of the GnRH pulse generator can greatly inform a wide range of clinical studies investigating infertility treatments.

Conditional Oprk1-dependent Kiss1 deletion in kisspeptin neurons caused estrogen-dependent LH pulse disruption and LH surge attenuation in female rats

The gonadotropin-releasing hormone (GnRH) pulse and surge are considered to be generated by arcuate kisspeptin/neurokinin B/dynorphin A (KNDy) neurons and anteroventral periventricular nucleus (AVPV) kisspeptin neurons, respectively, in female rodents. The majority of KNDy and AVPV kisspeptin neurons express κ-opioid receptors (KORs, encoded by Oprk1) in female rodents. Thus, this study aimed to investigate the effect of a conditional Oprk1-dependent Kiss1 deletion in kisspeptin neurons on the luteinizing hormone (LH) pulse/surge and fertility using Kiss1-floxed/Oprk1-Cre rats, in which Kiss1 was deleted in cells expressing or once expressed the Oprk1/Cre. The Kiss1-floxed/Oprk1-Cre female rats, with Kiss1 deleted in a majority of KNDy neurons, showed normal puberty while having a one-day longer estrous cycle and fewer pups than Kiss1-floxed controls. Notably, ovariectomized (OVX) Kiss1-floxed/Oprk1-Cre rats showed profound disruption of LH pulses in the presence of a diestrous level of estrogen but showed apparent LH pulses without estrogen treatment. Furthermore, Kiss1-floxed/Oprk1-Cre rats, with Kiss1 deleted in approximately half of AVPV kisspeptin neurons, showed a lower peak of the estrogen-induced LH surge than controls. These results suggest that arcuate and AVPV kisspeptin neurons expressing or having expressed Oprk1 have a role in maintaining normal GnRH pulse and surge generation, the normal length of the estrous cycle, and the normal offspring number in female rats.

Quantification of overnight urinary gonadotropin excretion predicts imminent puberty in girls: a semi-longitudinal study

PurposeWe explored the alternative of using overnight fold change in gonadotropin levels by comparing the last-night-voided (LNV) and first-morning-voided (FMV) urine concentrations of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) as a conceptual analogy to the invasive gonadotropin-releasing hormone (GnRH) stimulation test setting.MethodsWe investigated the nocturnal changes in the immunoreactivity levels of urinary gonadotropins between early and late prepubertal stages as well as between early and late pubertal stages in FMV and LNV urine samples from 30 girls, of whom those who were prepubertal were further investigated through follow-up visits within the 1-year period from the start of the study.ResultsROC analysis revealed that the FMV total U-LH and FMV U-FSH concentrations at or above 0.3 IU/L and 2.5 IU/L, respectively, were excellent predictors of forthcoming onset of puberty within 1 year (100% sensitivity, 100% specificity, AUC: 1.00, and n = 10, for both). FMV total U-LH concentration at or above 0.8 IU/L represented the cut-off for clinical signs of puberty. FMV/LNV total U-LH and FMV/LNV U-FSH ratios at or below 4.11 and 1.38, respectively, were also good predictors of the onset of clinical puberty within 1 year. An overnight increase (FMV/LNV ratio) in total U-LH concentrations and in the U-LH/U-FSH ratio at or below 1.2-fold in pubertal girls was associated with the postmenarcheal pubertal stage.ConclusionFMV total U-LH and U-FSH above 0.3 IU/L and 2.5 IU/L, respectively, can be used as cut-off values to predict the manifestation of the clinical signs of puberty within 1 year. FMV total U-LH concentrations 0.3–0.8 IU/L and 0.6 IU/L may represent the range and the threshold, respectively, that reflect the loosening of the central brake on the GnRH pulse generator. An overnight increase of 20% or less in total U-LH concentrations and in the U-LH/U-FSH ratio in an early pubertal girl may serve as an indicator of imminent menarche, a presumed timing of which can be unraveled by future longitudinal studies.

THU190 Deletion Of The Glucocorticoid Receptor From GABAergic Neurons Causes Early Puberty In Male And Female Mice

Abstract Disclosure: S.A. Pereira: None. L. Arvizu-Sanchez: None. A.N. Fontes: None. R.S. Carroll: None. V.M. Navarro: None. U.B. Kaiser: None. Background and Objectives: Glucocorticoid (GC) secretion induced by various types of stress can negatively impact reproduction through effects at several levels of the hypothalamic-pituitary-gonadal (HPG) axis, including inhibition of gonadotrophin-releasing hormone (GnRH) secretion and suppression of luteinizing hormone (LH) release. KNDy neurons, expressing kisspeptin, neurokinin B and dynorphin A, are the GnRH and LH pulse generator and are key regulators of puberty. These neurons are proposed to play an important role in mediating the effects of stress and GC on reproduction. While the precise mechanisms by which stress affects regulation of KNDy neurons remain to be fully characterized, it is known that KNDy neurons are subject to regulation by multiple neural networks, among which GABAergic neurons appear to play a determinant role. Importantly, a large proportion of GABAergic neurons express glucocorticoid receptors (GR). We therefore hypothesized that stress, through the action of GC on GR in GABAergic neurons, affects the activity of KNDy neurons to impact puberty timing. Methods and Results: We generated mice with conditional deletion of GR in GABAergic neurons using VgatCre mice crossed with GRlox/lox mice to generate VgatCre;GRlox/lox mice (GABAGRKO). The pubertal phenotypes of these GABAGRKO mice and their littermate controls (GRlox/lox) were characterized. Male GABAGRKO mice had earlier preputial separation compared to GRlox/lox littermate controls (GABAGRKO 29.8 ± 0.45 days vs. GRlox/lox 33.5 ± 0.57 days; p < 0.0001). Female GABAGRKO mice had a similar age of vaginal opening (GABAGRKO 25.7 ± 0.76 days vs GRlox/lox 24.5 ± 0.87 days; p = 0.2810) but had significantly earlier first estrus compared to their GRlox/lox littermate controls (GABAGRKO 32.4 ± 0.76 days vs GRlox/lox 37.9 ± 0.79 days; p < 0.0001). Male and female mice were euthanized at 30 days of age and their mediobasal hypothalamus (MBH) were collected for mRNA measurement by RT-qPCR of genes known to be related to pubertal development, including Kiss1, Tac2, Pdyn, Agrp and Pomc. Kiss1 and Pdyn expression were significantly higher in the MBH of GABAGRKO males compared to controls; in contrast, Pdyn expression was significantly lower in the MBH of GABAGRKO females and Kiss1 expression was unchanged compared to controls. Agrp expression was lower in the MBH of both GABAGRKO male and female mice compared to controls. No differences were observed in Tac2 and Pomc expression. Data sets are mean ± SEM. Statistical significance was considered at p < 0.05. Conclusions: The deletion of GR in GABAergic neurons results in early puberty onset, sexually dimorphic changes in Pdyn mRNA levels and downregulation of Agrp mRNA expression in male and female GABAGRKO mice. These findings suggest a role of glucocorticoids acting on GR in GABAergic neurons upstream of KNDy neurons to restrain the timing of their activation to initiate puberty. Presentation: Thursday, June 15, 2023

SAT375 Impact of Flutamide on Wake vs. Sleep LH Pulse Frequency in PCOS: An Interim Analysis

Abstract Disclosure: C.K. Matingou: None. S. Kim: None. M. Gilrain: None. C.M. Burt Solorzano: None. C.R. McCartney: None. Polycystic ovary syndrome (PCOS) is characterized by hyperandrogenism and ovulatory dysfunction. PCOS is also associated with luteinizing hormone (LH) excess, in part related to persistently high gonadotropin-releasing hormone (GnRH) pulse frequency. Furthermore, late pubertal girls with obesity and hyperandrogenemia did not exhibit sleep-associated decreases in LH (GnRH) pulse frequency when studied during the follicular phase, in contrast to non-hyperandrogenemic controls with or without obesity (JCEM 2014;99:2887-96). We therefore aimed to test two hypotheses: (1) sleep-associated reductions in LH pulse frequency are less pronounced in adult PCOS compared to late-follicular controls; and (2) sleep-associated reductions in LH pulse frequency are normalized in PCOS with 4 weeks of flutamide (androgen-receptor antagonist) pretreatment. To date, we have studied 5 women with PCOS (median age 30 y, BMI 42.6 kg/m2) and 5 ovulatory controls (median age 22 y, BMI 23.2 kg/m2). Subjects underwent two separate 16-hour frequent-blood-sampling studies to characterize pulsatile LH secretion, once after flutamide pretreatment (250 mg BID x 4 weeks) and once after placebo (PBO) pretreatment (BID for 4 weeks). In each study, 1500-2300 h and 2300-0700 h were designated the wake and sleep periods, respectively. The order of flutamide and PBO administration was randomized, and treatment allocation was switched after the first admission per a cross-over design. Controls were studied on cycle days 7-10, while those with PCOS were studied no earlier than cycle day 7. Serum progesterone concentrations were < 1.5 ng/dl for all admissions. With PBO pretreatment, participants with PCOS and controls exhibited the following: wake LH pulse frequency 1.19 (1.08-1.33) (median [interquartile range]) and 1.22 (0.97-1.27) pulses/h, respectively; sleep LH pulse frequency 0.93 (0.78-1.28) and 0.83 (0.78-0.99) pulses/h, respectively; and wake-to-sleep change in LH pulse frequency of -19.4 (-21.9 to +2.9) and -15.5 (-31.8 to -12.0) percent, respectively. For participants with PCOS, pretreatment with flutamide was associated with the following changes compared to PBO: wake LH pulse frequency +0.01 (-0.06 to +0.03) pulses/h; sleep LH pulse frequency +0.06 (+0.01 to +0.07) pulses/h; and wake-to-sleep LH pulse frequency change +5 (+7 to -12) percent. None of these differences were statistically significant by conservative nonparametric testing. We conclude that this interim analysis does not offer any indication that wake- and sleep-associated LH pulse frequencies are different between PCOS and late-follicular controls, nor does it suggest clear differences in wake-to-sleep changes in late-follicular LH pulse frequency between groups. Furthermore, this analysis does not seem to offer any indication that flutamide alone alters wake- and/or sleep-associated LH pulse frequencies in PCOS. Presentation Date: Saturday, June 17, 2023

SAT601 The Role Of The Melanocortin System In Ovine Puberty

Abstract Disclosure: E.G. Aerts: None. M.J. Griesgraber: None. A. Thomson: None. M. Brown: None. A. Seman: None. S.L. Hardy: None. R.L. Goodman: None. C. Nestor: None. S.M. Hileman: None. The neural mechanisms that regulate the timing of puberty onset are not completely known. In several species, including sheep, puberty onset relies on an increase in gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) secretion as a result of decreased sensitivity to estradiol (E2) inhibition. Additionally, it is unclear how E2 acts in the hypothalamus to inhibit GnRH/LH secretion, as GnRH neurons do not contain estrogen receptor alpha (ERα). Neurons within the arcuate nucleus (ARC) of the hypothalamus that contain kisspeptin, neurokinin B, and dynorphin (KNDy neurons) are critical for the generation of GnRH pulses and contain ERα. Data from our laboratory suggests that KNDy neurons, while critical, may be responding to other inputs that time puberty onset. Proopiomelanocortin (POMC) and Agouti-related peptide (AgRP) neurons in the ARC have been shown to contain ERα and provide input to KNDy neurons, which contain the melanocortin 3 and 4 receptor (MC3R, MC4R). The goal of this work was to assess POMC- and AgRP-positive cell numbers, input onto KNDy neurons, and neuronal activity changes in a puberty-specific manner. Female sheep were ovariectomized and given subcutaneous E2 implants at 5 months (prepubertal, n=4), 8 months (peripubertal, n=5), and 10 months (postpubertal, n=5) of age. Two weeks after surgery, blood samples were collected via jugular venipuncture every 12 minutes for 4 hours and assessed for LH. The sheep were then sacrificed and hypothalamic tissue was perfused, collected, and prepared for staining. The tissue was assessed via immunohistochemistry for POMC, AgRP, kisspeptin, NKB, and c-Fos, a known marker of neuronal activity. Pulsatile LH secretion was, as expected, reduced at 5 months and highest at 10 months. ARC POMC cell numbers did not significantly change over development (p>0.10). The number of ARC POMC close contacts onto ARC kisspeptin neurons increased significantly with development (p<0.05), as well as the percentage of ARC POMC neurons expressing c-Fos (p<0.05). ARC AgRP cell numbers and the percentage of AgRP cells colocalized with c-Fos decreased significantly over time (p<0.05). The number of AgRP close contacts onto ARC NKB cells is currently being analyzed. The percentage of NKB cells colocalizing c-Fos throughout pubertal development also increased significantly (p<0.05). This data supports the hypothesis that the ARC melanocortin system impacts KNDy neurons to regulate GnRH/LH secretion throughout pubertal development. Presentation: Saturday, June 17, 2023

P-691 Does serum Kisspeptin levels discriminate between fertile and infertile women with polycystic ovary syndrome?

Abstract Study question Does serum Kisspeptin (KP) levels discriminate between fertile and infertile women with polycystic ovary syndrome (PCOS)? Summary answer Serum KP levels can be used as a potential discriminatory biomarker for infertility in women with PCOS. What is known already PCOS is the most common cause of anovulatory infertility and not fully elucidated pathology. A persistent rapid gonadotropin-releasing hormone (GnRH) pulse frequency in patients with PCOS exist throughout ovulatory cycle. KP is a neuropeptide that increases GnRH pulsatile release during ovulation. Therefore, KP may have a key role as a central regulator of fertility in PCOS patients. Although, recent studies showed that the KP concentration is higher in PCOS patients, the evidence is limited as to whether serum KP levels determine infertility in PCOS. Study design, size, duration This was a single center prospective cohort study conducted at Goztepe Prof. Dr. Suleyman Yalcin City Hospital Affiliated to Istanbul Medeniyet University, Istanbul, Turkey between January 2023 to February 2023. Our study enrolled 30 fertile and 30 infertile women with PCOS, who were aged between 18 and 45 years. PCOS was defined according to the criteria of the Rotterdam ESHRE- ASRM sponsored consensus group (2004). Participants/materials, setting, methods Venous blood samples from the participants were obtained after fasting and between 8 a.m. and 10 a.m. on days 2-5 of the menstrual cycle. Samples were thawed, and a human KISS1 (Kisspeptin 1) ELISA kit (Elabscience, USA,lot no:E-EL-H5618) was applied to measure KP serum concentrations. Patient characteristics and KP concentrations were compared among the two groups. Statistical analysis was performed by Mann-Whitney, Spearman correlations, and linear regression analysis. A p-value <0.05 was considered significant. Main results and the role of chance The mean age of the patients was 27.57±5.39 years in the fertile PCOS group and 26.80± 4.85 years in the infertile PCOS group (p = 0.63). There was no significant difference among the groups in terms of body mass index (BMI), duration of infertility, serum FSH, LH, LH-to-FSH ratio, E2, antimullerian hormone (AMH), TSH, prolactin, 17OHP, total testosterone, SHBG, HbA1c, HOMA-IR, neutrophil–lymphocyte ratio levels and antral follicle count (AFC). DHEA-S was significantly higher in the infertile PCOS group (306.73±94.869 mcg/ dL), compared to the fertile PCOS group (258.08±84.29 mcg/ dL, p = 0.037). The mean KP level was significantly higher in the infertile PCOS group (444.80±136.61 ng/ mL), compared to the fertile PCOS group (333.02±131.10 ng/ mL, p = 0.001). The KP level was positively correlated with AFC, AMH, and total testosterone levels (R = 0.495, R = 0.548, R = 0.362, p < 0.05, respectively) in the infertile PCOS group. Furthermore, ROC analysis showed that the optimal cut-point value was found to be 285.59. When this cut-off value of serum KP levels was taken, the sensitivity was 0.96 and the specificity was 0.50. Linear regression analysis revealed that AMH was positively associated with KP levels (p = 0.022). Limitations, reasons for caution Due to the limited number of women with available samples, we were not able to analyze KP serum levels according to specific PCOS phenotypes Wider implications of the findings Serum KP levels are higher in infertile women with PCOS compared to fertile women with PCOS. Infertility caused by PCOS can be predicted by serum KP levels. In the future identification, KP in PCOS may be applied to develop potential therapeutic agents. Trial registration number Not applicable

KNDy neurones and GnRH/LH pulse generation: Current understanding and future aspects.

Uncovering the central mechanism underlying mammalian reproduction is warranted to develop new therapeutic approaches for reproductive disorders in humans and domestic animals. The present study focused on the role of arcuate kisspeptin neurones (also known as KNDy neurones) as an intrinsic gonadotropin-releasing hormone (GnRH) pulse generator, which plays a fundamental role in mammalian reproduction via the stimulation of pituitary gonadotropin synthesis and release and thereby in gametogenesis and steroidogenesis in the gonads of mammals. We also discuss the mechanism that inhibits pulsatile GnRH/gonadotropin release under a negative energy balance, considering that reproductive disorders often occur during malnutrition in humans and livestock.

Dynamics of pulsatile activities of arcuate kisspeptin neurons in aging female mice.

Reproductive senescence is broadly observed across mammalian females, including humans, eventually leading to a loss of fertility. The pulsatile secretion of gonadotropin-releasing hormone (GnRH), which is essential for gonad function, is primarily controlled by kisspeptin neurons in the hypothalamic arcuate nucleus (ARCkiss), the pulse generator of GnRH. The pulsatility of GnRH release, as assessed by the amount of circulating gonadotropin, is markedly reduced in aged animals, suggesting that the malfunctions of ARCkiss may be responsible for reproductive aging and menopause-related disorders. However, the activity dynamics of ARCkiss during the natural transition to reproductive senescence remain unclear. Herein, we introduce chronic in vivo Ca2+ imaging of ARCkiss in female mice by fiber photometry to monitor the synchronous episodes of ARCkiss (SEskiss), a known hallmark of GnRH pulse generator activity, from the fully reproductive to acyclic phase over 1 year. During the reproductive phase, we find that not only the frequency, but also the intensities and waveforms of individual SEskiss, vary depending on the stage of the estrus cycle. During the transition to reproductive senescence, the integrity of SEskiss patterns, including the frequency and waveforms, remains mostly unchanged, whereas the intensities tend to decline. These data illuminate the temporal dynamics of ARCkiss activities in aging female mice. More generally, our findings demonstrate the utility of fiber-photometry-based chronic imaging of neuroendocrine regulators in the brain to characterize aging-associated malfunction.

Clomiphene citrate therapy for testosterone deficiency: a proposed clinical care pathway.

Several testosterone (T) therapy options are currently available, including oral, intranasal, transdermal (gel, patches), intramuscular, and subcutaneous agents. The practice guidelines of some professional societies, including the American Urological Association and the European Association of Urology but not all international societies,1,2 suggest treating with aromatase inhibitors or selective estrogen receptor modulators for men with infertility. However, selective estrogen receptor modulators (ie, clomiphene citrate [CC]) may be a reasonable alternative for men experiencing low T with infertility or a wish to maintain their fertility.3 In clinical practice, CC—a nonsteroidal triphenylethylene derivative distantly related to diethylstilbestrol and a selective estrogen receptor modulator—is also used as an effective treatment for men with low T to improve T levels and T deficiency symptoms. CC is an oral agent that acts centrally. CC is a mixture of enclomiphene (62%), which is a trans isomer with pure antiestrogen effect, and zuclomiphene (38%), which is a cis isomer that has a mixed action with a predominant estrogen receptor agonistic property. It selectively binds to estrogen receptors primarily in the hypothalamus to produce a partial estrogen agonist effect resulting in an estrogenic negative feedback inhibition, thus increasing the hypothalamic gonadotropin-releasing hormone pulse frequency and, as a result, elevating luteinizing hormone (LH) secretion to stimulate the gonads to increase sexual hormones—for men, the Leydig cells to raise T levels.4 In vitro data have also suggested that CC has a pituitary site of action where it causes an increase in the gonadotropin response to gonadotropin-releasing hormone.5

The enigma of the gonadotropin-releasing hormone pulse frequency governing individual secretion of luteinizing hormone and follicle-stimulating hormone

Luteinizing hormone and follicle-stimulating hormone are the two gonadotropic pituitary hormones stimulated by one hypothalamic gonadotropin-releasing hormone (GnRH) in a pulsatile way. Under several experimental conditions, it appears that a low pulse frequency promotes follicle-stimulating hormone secretion, pointing to an elegant mechanism by which, under governance of one stimulating hormone, the responses of two separate hormones can be individualized. Several experimental and fundamental studies have indicated the underlying mechanisms at the level of gene expression and post receptor events. In this article, an additional explanation is hypothetically put forward on the basis of dynamic and kinetic differences between both hormones in response to GnRH, with a key role of their difference in serum half-life combined with some GnRH-related desensitization features. Although experimentally demonstrated, under clinical conditions its effect remains obscure, likely because of overwhelming hormonal gonadal feedback.

Investigation of irisin's role in pubertal onset physiology in female rats

ObjectiveThe timing of pubertal development is closely related to metabolic status and energy reserves. It is thought that irisin, which is involved in the regulation of energy metabolism and is shown to be present in the hypothalamo-pituitary-gonadal (HPG) axis, may play a role in this process. In our study, we aimed to investigate the effect of irisin administration on pubertal development and HPG axis in rats. Design-methods36 female rats were included in the study were divided into 3 groups: 100 ng/kg/day irisin treatment group (irisin-100), 50 ng/kg/day irisin treatment group (irisin-50), and control group. On the 38th day, serum samples were taken to determine levels of luteinizing hormone (LH) and follicle stimulating hormone (FSH), estradiol and irisin. Brain hypothalamus samples were taken to determine levels of pulsatile gonadotropin-releasing hormone (GnRH), kisspeptin, neurokinin-B, dynorphin (Dyn), and makorin ring finger protein-3 (MKRN3). ResultsVaginal opening and estrus were seen firstly in the irisin-100 group. At the end of the study, the highest rate of vaginal patency was found in the irisin-100 group. Hypothalamic protein expression levels of GnRH, NKB and Kiss1 in homogenates; serum FSH, LH, and estradiol levels were the highest in the irisin-100 group, followed by the irisin-50 and control groups, respectively. Ovarian sizes were significantly greater in the irisin-100 group compared to the other groups. The hypothalamic protein expression levels of MKRN3 and Dyn were the lowest in the irisin-100 group. ConclusionsIn this experimental study, irisin triggered the onset of puberty in a dose-dependent manner. Irisin administration caused the excitatory system to dominate in the hypothalamic GnRH pulse generator.

Hypothalamic kisspeptin neurons as potential mediators of estradiol negative and positive feedback

Gonadal steroid feedback regulates the brain’s patterned secretion of gonadotropin-releasing hormone (GnRH). Negative feedback, which occurs in males and during the majority of the female cycle, modulates the amplitude and frequency of GnRH pulses. Positive feedback occurs in females when high estradiol induces a surge pattern of GnRH release. These two forms of feedback and their corresponding patterns of GnRH secretion are thought to be mediated by kisspeptin-expressing neurons in two hypothalamic areas: the arcuate nucleus and the anteroventral periventricular area. In this review, we present evidence for this theory and remaining questions to be addressed.