FSH Release Research Articles

Metabolic hormonal alterations in functional hypothalamic amenorrhea and anovulation associated with Polycystic ovary syndrome

Background: Polycystic ovary syndrome (PCOS) is a multifaceted endocrine disorder affecting many women during their reproductive years. It is characterized by oligo/amenorrhea, anovulatory cycles, polycystic ovaries, and insulin resistance. This review explores the hormonal and metabolic alterations associated with PCOS, comparing them to functional hypothalamic amenorrhea (FHA). Key aspects include the abnormally high LH pulse frequency in PCOS, indicating hyperactive gonadotropin-releasing hormone (GnRH), and the role of hyperandrogenemia in exacerbating the condition by increasing LH pulse secretion from the pituitary gland. Additionally, the review examines the neuroendocrine basis for PCOS. Methods: The methodology involved analyzing neuroendocrine pathways and physical manifestations through PubMed, ScienceDirect, and Scopus databases. Findings indicate that PCOS is primarily characterized by androgen excess, ovulatory dysfunction, and disruption of the hypothalamic-pituitary-ovarian (HPO) axis. Hormonal dysregulation includes disturbances in GnRH, insulin, LH/FSH ratio, and androgens. GnRH stimulates LH and FSH release from the pituitary, regulating ovarian function, while Anti-Müllerian hormone (AMH) inhibits follicular development in PCOS. Conclusion: The review concludes by highlighting the hormonal alterations, including decreased frequency and amplitude of LH pulses, disruptions in GnRH, LH, and FSH. Genetic predispositions and disturbances in the LH/FSH ratio can lead to impaired follicle growth and polycystic ovaries. This comprehensive exploration underscores the importance of understanding the hormonal and neuroendocrine mechanisms underlying PCOS, contributing to better diagnosis and treatment strategies

The Impact of Administering Kisspeptin-10 Peripherally on FSH Release and Reproductive Performance in Estrus Synchronized Ewes

The Impact of Administering Kisspeptin-10 Peripherally on FSH Release and Reproductive Performance in Estrus Synchronized Ewes

#210 : The Comparison of Agonist Trigger and Dual Trigger in Our IVF Population - A Single Center Retrospective Study

Background: For years, human chorionic gonadotropin (hCG) has been the trigger of choice for oocyte maturation due to its molecular and biological similarity with LH, but its longer half-life compared to LH contributes to the ovarian hyperstimulation syndrome (OHSS) in hyper responders. Besides, hCG lacks FSH activity, which plays a role in the in vitro maturation of oocytes. Gonadotropin-releasing hormone (GnRH) agonists which can bring endogenous release of both FSH and LH and have a shorter mean duration of LH surge, and effectively reduce the incidence of OHSS in high responders. However, the substitution of agonist as trigger is associated with the risk of empty follicle syndrome, increased early pregnancy loss and decreased rates of ongoing pregnancy. The idea of a dual trigger where hCG component could serve as a rescue in case of poor response to GnRH-agonist, occurring in about 2.71% of ART population, and agonist bringing FSH & LH surge simultaneously leading to improved oocyte quality was the basis for present study. Aim & objective: To observe the difference between agonist trigger and dual trigger in various ovarian reserve groups. Materials & Methods: Retrospective cohort study in patients attending Eva fertility clinic & IVF center between 2021-2022. Total patients =157 agonist trigger =57 dual trigger = 100 Age: 24 years to 41 years Average BMI 26 AMH :2.0 ng-6.6 ng/ml Primary outcome: Clinical pregnancy rate & ongoing pregnancy rate Secondary outcome: Blastocyst formation rate Total M2 achieved. Result: Pregnancy’ in (AMH: <2 ng/mL)- no significant difference between the various groups in terms of distribution of Pregnancy ([Formula: see text]2= 0.209, p = 1.000). (Table 1) Pregnancy’ in (AMH: 2-5 ng/mL)- no significant difference ([Formula: see text]2= 0.023, p = 0.880). (Table 2) Pregnancy’ in (AMH: >5 ng/mL)- no significant difference ([Formula: see text]2= 0.286, p = 0.711). (Table 3) Limitation of the study- small sample size, lack of plain hCG trigger group to compare as an arm 3. Conclusion: Dual trigger treatment with GnRH agonist and hCG is not inferior in terms of live birth rate, blastocyst, & M2 oocytes compared to GnRH agonist & hCG trigger apart from avoiding the danger of OHSS.

#182 : Pharmacokinetics of Ovarian Stimulation for Projected Poor Responder Patients in IVF. A Randomised Trial Comparing a Corifollitropin Alfa Agonist Flare Protocol with a Corifollitropin Alfa Antagonist Protocol

Background & Aims: Approximately 10% of patients have a sub-optimal response to controlled ovarian stimulation. Many different protocols for superovulation have been developed for this group of patients but the optimal approach for “poor responder” patients is yet to be determined. Use of corifollitropin alfa (Elonva) in a flare (EF) protocol is a novel approach to ovarian stimulation for predicted poor responder patients. We hypothesised that the unique pharmacokinetic and pharmacodynamic profiles of corifollitropin alfa would maximise FSH exposure and follicular recruitment in the critical early follicular phase due to the initial suprathreshold release of bioactive FSH in the initial 2-3 days of exposure to Elonva, in combination with the ‘flare’ of endogenous FSH release. Method: Twenty-one women aged 31-41 were identified as predicted poor responder patients according to the Poseidon criteria. Each participant was randomised to treatment with Elonva/Synarel flare or Elonva/Orgalutran antagonist. Serum FSH and E2 were measured daily for five days following injection of Elonva. The study was approved by the Southeastern Sydney HREC. Results: Serum FSH concentrations were significantly higher at each time point in the EF group when compared with the antagonist group (p=0.022). Peak FSH two days after injection of Elonva, was also significantly higher 27 vs 11 IU/L. Serum E2 concentrations were also significantly higher in the EF group at each time point (p=0.039). On day seven of stimulation, E2 concentrations were 3000 vs 1850 pmol/L. Conclusion: The corifollitropin alfa agonist flare protocol produces significantly higher FSH and E2 concentrations than a standard corifollitropin alfa antagonist protocol for projected poor responder patients, with reduced patient burden for a group who may need multiple ovarian stimulation cycles to achieve a pregnancy. A larger RCT is required to study embryology and pregnancy outcomes, costs and patient burden.

Combined exposure of beta-cypermethrin and emamectin benzoate interferes with the HPO axis through oxidative stress, causing an imbalance of hormone homeostasis in female rats

The impact of pesticides on reproductive health has been increasingly recognized. β-cypermethrin (β-CYP) and emamectin benzoate (EMB) are commonly used with agricultural workers. There are few published studies on the effects of combined poisoning of these two pesticides on the reproductive system. This study investigated the toxic effects and mechanism of β-CYP and EMB on the reproductive system of female rats based on the hypothalamic-pituitary-ovarian (HPO) axis. The hypothalamic GnRH content tended to decrease, and Kiss-1 and GPR-54 mRNA and protein expression tended to increase in exposed rats. FSH content was elevated for the pituitary gland, and Kiss-1 and GPR-54 mRNA and protein expression were enhanced in all experimental groups compared with the control group. E2 content in rat ovaries and ERα mRNA and protein expression were reduced by β-CYP and EMB. Furthermore, there were interactive effects of β-CYP and EMB on FSH and E2 release, pituitary GPR-54 mRNA and protein, and ovarian ERα mRNA expression. To investigate causes of damage, oxidative damage indicators were tested and showed that exposure to β-CYP and EMB decreased GSH-Px and SOD activities in the HPO axis, increased MDA levels in the hypothalamus and ovary together with LDH activities in the HPO axis, with an interaction effect on GSH-Px and SOD activities in the hypothalamus and pituitary gland as well as on MDA in the ovary. The above results support the screening of sensitive molecular biomarkers and evaluation of the adverse effects of pesticide exposure in greenhouse operations on reproductive health.

FRI286 Stimulation Of Egr1 Expression Is Mediated By A Gαq/11-signaling Pathway In A Pattern Distinct From That Of MEK/ERK At High And Low GnRH Pulse Frequencies

Abstract Disclosure: G.A. Stamatiades: None. H.K. Kim: None. R.S. Carroll: None. U.B. Kaiser: None. Introduction: The pulsatile release of the hypothalamic decapeptide, GnRH, activates signal transduction cascades in pituitary gonadotropes to control the synthesis and secretion of LH and FSH, hormones critical for normal reproductive function and fertility. LH and FSH release are differentially regulated by varying GnRH pulse patterns, with LH secretion being preferentially stimulated at high (e.g., every 30 min) GnRH pulse frequencies, while FSH secretion is preferentially induced at low (e.g., every 2 hour) GnRH pulses. LH is a heterodimer comprised of a common α subunit and a distinct LHβ subunit, with Lhb gene (encoding LHβ) transcription being preferentially stimulated at the higher GnRH pulse frequencies. It is well established that Egr1 is a key inducer of Lhb expression upon pulsatile GnRH stimulation. In addition, it has been shown that MEKI/II inhibition abrogates the induction of Lhb mRNA levels at both high and low GnRH pulse frequencies. Hypothesis:Egr1 expression is mediated by a Gαq/11-signaling pathway that includes MEK/ERK at both high and low GnRH pulse frequencies. Methods: To determine whether a Gαq/11-mediated pathway regulates Egr1 expression upon GnRH stimulation, LβT2 cells transduced with lentiviruses encoding scrambled or Gnaq/11 shRNA were perifused and treated with pulsatile GnRH at either high (q30min) or low (q2h) pulse frequencies, or with medium only, for 20 hours. Following the same perifusion paradigm, ERK1/2 phosphorylation was also assessed. Results: LβT2 cells transduced with a scrambled control shRNA showed a GnRH pulse frequency-dependent pattern of induction of Egr1 expression, with significantly greater induction at high than at low GnRH pulse frequency, compared to cells perifused with media only. In addition, Gαq/11 depletion reduced Egr1 mRNA levels at both high and low GnRH pulse frequencies, but the frequency dependence was maintained. A GnRH pulse frequency-dependent pattern of induction of ERK1/2 phosphorylation was observed, with significantly greater induction at low than at high GnRH pulse frequency in control cells transduced with scrambled control shRNA, consistent with prior studies from our group. Following Gnaq/11 KD, the induction of ERK1/2 phosphorylation was reduced at both GnRH pulse frequencies, with loss of the pulse frequency dependence. Conclusion: Taken together, these findings suggest that a Gαq/11-signaling pathway involving Egr1 induces Lhb expression at both pulse frequencies. However, the link between MEK/ERK and Egr1 needs to be further investigated, given the differences in the GnRH pulse frequency dependent patterns of induction. Presentation: Friday, June 16, 2023

Protective effect of exogenous melatonin on testicular histopathology and histomorphometry of adult rats with domperidone-induced hyperprolactinemia

Hyperprolactinemia is a pathological condition resulting from increased prolactin that directly affects reproduction, as this condition inhibits the release of LH, FSH and gonadal steroidogenesis, bringing several negative clinical associations in reproduction. In contrast, melatonin (MEL) plays an important role in the regulation of steroidogenesis and modulates damages to the process of spermatogenesis. The objective was to analyze the protective effects of exogenous melatonin on the testis of hyperprolactinemic adult rats. Forty-eight male rats were used, divided into two treatment periods: 30 and 60 days, each treatment was subdivided into three groups: Control, Hyper (hyperprolactinemia), and Hyper+MEL (hyperprolactinemia and melatonin). Treatment with melatonin was 200 μg/100 g, subcutaneously. Induction of hyperprolactinemia was obtained with a dose of 4 mg/kg of domperidone, subcutaneously. The results of the histopathology demonstrated that the animals in the Hyper group presented degeneration of germ cells when compared to the control. In addition, the degenerations were presented in smaller quantities in the Hyper+MEL, in both treatment periods, evidencing the benefits of the melatonin in gonadal regeneration. The Hyper group of both treatment periods showed a decrease in tubular diameter, epithelium height, and tubular area, in addition to a decrease in Sertoli cells, when compared to the control and the Hyper+MEL group. In conclusion, the hyperprolactinemia can affect the germinal epithelium and testicular microstructure; the exogenous melatonin has a protective effect against hyperprolactinemia, reducing testicular damage.

EZH2 Gene Knockdown Inhibits Sheep Pituitary Cell Proliferation via Downregulating the AKT/ERK Signaling Pathway.

Pituitary gonadotropins perform essential functions in mammalian reproduction by stimulating gametogenesis and steroidogenesis in the ovaries and testicles. EZH2 is a histone methyltransferase that inhibits proliferation and aggravates apoptosis in stem cells subjected to pathological stimuli. However, the expression and molecular mechanisms of EZH2 in pituitary cells in vitro have not been extensively studied. In this study, the relative abundances of EZH2 mRNA (p < 0.01) and protein (p < 0.05) expression were larger in the pituitary cells of Hu sheep with relatively greater fecundity (GF) compared to those with lesser fecundity (LF). Loss-of-function examinations demonstrated that EZH2 gene knockdown led to an earlier induction of apoptosis in sheep pituitary cells (PCs). The relative abundance of CASP3, CASP9, and BAX was increased (p < 0.01), while BCL2's abundance was less decreased (p < 0.01) in PCs where there was EZH2 gene knockdown. Additionally, cell proliferation (p < 0.01) and viability (p < 0.01) were decreased in EZH2-knockdown sheep PCs, and the cell cycle was blocked compared to a negative control (NC). Notably, EZH2 gene knockdown led to reduced abundances of gonadotropin subunit gene transcripts (FSHβ, p < 0.05) and reduced FSH release (p < 0.01) from PCs. EZH2 gene knockdown led to reduced phosphorylation of AKT, ERK, and mTOR (p < 0.01). The results suggest that EZH2 regulates pituitary cell proliferation, apoptosis, and FSH secretion through modulation of the AKT/ERK signaling pathway, providing a foundation for further study of pituitary cell functions.

Low-Dose Estrogens as Neuroendocrine Modulators in Functional Hypothalamic Amenorrhea (FHA): The Putative Triggering of the Positive Feedback Mechanism(s).

Functional hypothalamic amenorrhea (FHA) is a non-organic reversible chronic endocrine disorder characterized by an impaired pulsatile secretion of the gonadotropin-releasing hormone (GnRH) from the hypothalamus. This impaired secretion, triggered by psychosocial and metabolic stressors, leads to an abnormal pituitary production of gonadotropins. As LH and FSH release is defective, the ovarian function is steadily reduced, inducing a systemic hypoestrogenic condition characterized by amenorrhea, vaginal atrophy, mood changes and increased risk of osteoporosis and cardiovascular disease. Diagnosis of FHA is made excluding other possible causes for secondary amenorrhea, and it is based upon the findings of low serum gonadotropins and estradiol (E2) with evidence of precipitating factors (excessive exercise, low weight, stress). Treatments of women with FHA include weight gain through an appropriate diet and physical activity reduction, psychological support, and integrative approach up to estrogen replacement therapy. If no spontaneous ovarian function is restored, assisted reproductive technologies may be used when pregnancy is desired. Because subjects with FHA are hypoestrogenic, the use of low-dose estrogens has been proposed as a putative treatment to positively modulate the spontaneous restart of gonadotropin secretion, counteracting the blockade of the reproductive axis triggered by stress acting through the neuroendocrine pathways at the basis of positive feedback of estrogens. The mechanism through which low-dose estrogens acts is still unknown, but kisspeptin-secreting neurons may be involved.

134 Effect of circulating estradiol and progesterone concentrations on FSH release profile of beef heifers submitted to an estradiol/progesterone-based synchronization protocol

134 Effect of circulating estradiol and progesterone concentrations on FSH release profile of beef heifers submitted to an estradiol/progesterone-based synchronization protocol

Effect of Starvation on Female Reproductive Hormones of Female Albino Rats

Background: Starvation may alter energy metabolism and physiological activity of the gonads, with their cyclic production of sex hormones and this may affect reproduction. Aim: The aim of the study is to investigate the effects of intermittent and severe starvation on some female reproductive hormones in female albino Wistar rats. Methods: Thirty adult female albino rats weighing 160-180g were randomly subdivided into three groups-A, B and C. Group A serves as control and was fed with rat chow and water. Group B were subjected to intermittent starvation of food, each rat was fed 1g of feed and 2mls of water daily for 7 days. Group C was subjected to severe starvation of food, each rat fed with 1g of feed and 2mls of water daily for 14 days. The animals were sacrificed at the end experiment and blood samples were collected through cardiac punctures and stored in sterile plain containers. Serum FSH, LH, progesterone and estrogen were measured by enzyme-linked immunoassays for each group of rats. Results: The mean levels of FSH, LH, progesterone and estrogen were significantly lower in acutely and chronically starved rats (5.96± 0.32; 4.01 ± 0.29 vs 6.40 ± 0.64) when compared with the control (p &lt;0.005). The mean levels of all the assayed hormones in chronically starved rats (group C) were found to be significantly reduced when compared with the acutely starved rats (group B) (p &lt;0.005). This study indicated that starvation may result in decreased release of FSH, LH, estrogen and progesterone.

Male minipuberty involves the gonad-independent activation of preoptic nNOS neurons.

The maturation of the hypothalamic-pituitary-gonadal (HPG) axis is crucial for the establishment of reproductive function. In female mice, neuronal nitric oxide synthase (nNOS) activity appears to be key for the first postnatal activation of the neural network promoting the release of gonadotropin-releasing hormone (GnRH), i.e. minipuberty. However, in males, the profile of minipuberty as well as the role of nNOS-expressing neurons remain unexplored. nNOS-deficient and wild-type mice were studied during postnatal development. The expression of androgen (AR) and estrogen receptor alpha (ERα) as well as nNOS phosphorylation were evaluated by immunohistochemistry in nNOS neurons in the median preoptic nucleus (MePO), where most GnRH neuronal cell bodies reside, and the hormonal profile of nNOS-deficient male mice was assessed using previously established radioimmunoassay and ELISA methods. Gonadectomy and pharmacological manipulation of ERα were used to elucidate the mechanism of minipubertal nNOS activation and the maturation of the HPG axis. In male mice, minipubertal FSH release occurred at P23, preceding the LH surge at P30, when balanopreputial separation occurs. Progesterone and testosterone remained low during minipuberty, increasing around puberty, whereas estrogen levels were high throughout postnatal development. nNOS neurons showed a sharp increase in Ser1412 phosphorylation of nNOS at P23, a phenomenon that occurred even in the absence of the gonads. In male mice, nNOS neurons did not appear to express AR, but abundantly expressed ERα throughout postnatal development. Selective pharmacological blockade of ERα during the infantile period blunted Ser1412 phosphorylation of nNOS at P23. Our results show that the timing of minipuberty differs in male mice when compared to females, but as in the latter, nNOS activity in the preoptic region plays a role in this process. Additionally, akin to male non-human primates, the profile of minipuberty in male mice is shaped by sex-independent mechanisms, and possibly involves extragonadal estrogen sources.

The Kisspeptin analogue C6 induces ovulation in jennies

Kisspeptins (KPs) are the most potent stimulating neurotransmitters of GnRH release, and consequently KP administration triggers LH and/or FSH release. In small ruminants, KP or its analogs induced an LH surge followed by ovulation in both cyclic and acyclic animals, while in the mare KP only increased LH plasma levels but failed to induce ovulation. This study in jennies compares the endocrinological effects, ovulatory and pregnancy rates of the KP analog C6 and the GnRH analog buserelin acetate. The ovarian activity of nine Amiata jennies was monitored daily by transrectal ultrasound for three complete estrous cycles. Jennies in estrus were assigned, to one of three treatment groups: 50 nmol of the KP analog C6 (injected twice, 24 h apart, C6 group); 0.4 mg buserelin acetate (injected once, Bu group); and 2 mL of saline (injected once, CTRL group). Blood samples were collected at Day-1 (−24 h) Day0 (h0, before treatment), h2, h4, h6, h8, h10, h24 (before second treatment with C6), h26, h28, h30, h32, h34, h48 and every 24 h until ovulation. Jennies were inseminated once at h24 with fresh extended semen from a donkey stallion. Pregnancy diagnoses were performed 14 days after ovulation. On days 5, 10, and 14 after ovulation, for every CL the cross-sectional area (CSA) and the vascularized area (VA) were recorded by color doppler ultrasound and measured. Significantly higher plasma LH levels were found after induction between the Bu and CTRL groups at h6 and h8 (P < 0.05), while tendentially higher differences were found between the Bu/C6 groups and CTRL at h10. Five/9, 4/9, and 2/9 jennies ovulated between 24 and 48 h after induction from the Bu, C6, and CTRL groups respectively, (P > 0.05). Correlations between corpora lutea CSA and VA with serum progesterone concentration were r = 0.31, P = 0.01, r = 0.38, P = 0.01, respectively. Pregnancy rates after artificial insemination did not differ among groups (CTRL: 6/9, 66.7%; C6: 7/9, 77.8%; Bu: 6/9, 66.7%; P > 0.05). Ovulation rates after C6 treatment were comparable to that of Bu, although not different from the CTRL. Pregnancy rates were comparable to the literature in terms of fresh extended donkey semen in every group. This study suggests that stimulation of the Kp system in jennies, in contrast to findings observed in mares, induces ovulation. Further studies using higher doses and/or more animals are needed to better characterize the efficacy of C6 in jennies.

Effect of administration of the C6 Kisspeptin analogue in jennies in estrus

Kisspeptin (KPs) are the most potent stimulating neurotransmitter of GnRH release from GnRH neurons. The administration of KP stimulates LH and/or FSH release. In small ruminants KPs analogues were able to induce an LH surge followed by ovulation in both cyclic and acyclic animals while in mares were able to lead to an increase of the LH/FSH plasma levels but not ovulation. Aim of this study was to compare the endocrinological effect, ovulatory and pregnancy rates effect of C6 KPs analogue in jennies. Ovarian activity of 9 Amiata jennies was monitored daily by transrectal ultrasound for 3 complete estrus cycles in which jennies in estrus were assigned, alternatively, to one of the 3 treatment groups: 50 nmol of the Kps analog C6 twice, 24 hours apart (C6 group); 0.4 mg buserelin acetate once (Bu group); 2 mL of saline once (CTRL group). Blood samples for [LH] were collected g-1 (-24h) g0 (h0, before treatment), h2, h4, h6, h8, h10, h24 (before second treatment with C6), h26, h28, h30, h32, h34, h48and every 24 hours until ovulation. Jennies were inseminated once at h24 with fresh extended semen of a donkey stallion. Pregnancy diagnoses were performed 14 days after ovulation. Significant higher plasma [LH] were found after induction between Bu and CTRL group at h6 and h8 (P<0.05), while tendentially higher differences were found between Bu/C6 groups and CTRL at h10. Five/9, 4/9 and 2/9 jennies of the Bu, C6 and CTRL groups, ovulated between 24 and 48 hours after induction, respectively (P>0.05). Pregnancy rates after artificial insemination were not different among groups (CTRL: 6/9, 66.7%; C6: 7/9, 77.8%; Bu: 6/9, 66.7%; P>0.05). In this study C6 showed to be able to tendentially and limited in time increase plasma [LH], compared to CTRL. Ovulation rates of C6 after induction were comparable to Bu even if not different to CTRL. Pregnancy rates were comparable to what reported in literature for donkey fresh extended semen for each group. Further studies using higher doses and/or more animals are needed.

The Effect of Moringa Oleifera Leaf Water Extract on the Number of Ovarian Follicles and the Enzyme Levels of Superoxide Dismutase of Rattus Norvegicus Rats Exposed to Depot Medroxyprogesterone Acetate (DMPA)

Depot Medroxyprogesterone Acetate (DMPA) contraceptives result in reduced release of FSH and LH inhibiting ovarian follicular development and preventing ovulation. Prolonged hypoestrogen causes oxidative stress due to increased ROS. SOD enzymes prevent the formation of ROS. Moringa is a plant that contains high antioxidants and minerals which are cofactors for enzymes that protect against the effects of free radicals by neutralizing them before they damage cells and cause disease. The aims of this study were to determine the effect of Moringa oleifera leaf water extract on the number of ovarian follicles and levels of the enzyme superoxide dismutase in Rattus norvegicus rats exposed to Depo Medroxy Progesterone Acetate. The study was carried out at the Bukittingi Veterinary Laboratory and the Biomedical Laboratory, Faculty of Medicine, Andalas University, Padang. The study began in April - May 2021. The type of study was experimental with a Post Test Only Control Group Design with the number of sample of 30 female rats. The data were analyzed using the Shapiro Wilks normality test. After the parametric test is fulfilled, the hypothesis test is continued using one way ANOVA. The results of the analysis showed that there was a significant difference between the group k- mean (3.40) K+ mean (1.00) in primary follicles and mean k-(3.80) and K+(1.20). In secondary follicles and mean k- (4.20) the mean k+(1.40) in tertiary follicles and mean k-(2.60) mean k+(1.00) in Degraaf follicles. The significance value for all types of follicles with ANOVA test with p value &lt;0.05. The dose of 300 grams / kg / body weight per day is the dose that is most able to increase the number of ovarian follicles and SOD enzyme levels in the ovaries with a comparison rate close to the negative control group treatment value. The conclusion of the study was that administration of Moringa leaf water extract could increase the number of ovarian follicles and levels of the ovarian SOD enzyme in rats exposed to DMPA. Keywords: DMPA, Ovarian Follicle, SOD, Moringa leaf water extract.

OVULATION INDUCTION AND SUCCESSFUL LIVE DELIVERY IN A 46.5-YEAR-OLD WOMAN IN OVERT MENOPAUSE BY RESTORING FOLLICULAR SENSITIVITY TO FOLLICLE STIMULATING HORMONE (FSH)

To try to up-regulate FSH receptors on granulosa-theca cells in a woman of very advanced reproductive age in overt menopause whose FSH receptors had been internalized related to elevated serum FSH levels, by inhibiting release of FSH from the pituitary by negative feedback of estrogen. The overall objective was to deliver a live healthy baby. Ethinyl estradiol (EE) 20 micrograms was given to a woman age 46.5 who had amenorrhea for 9 months and who was in overt menopause, as evidenced by failure to have menses following 10mg medroxyprogesterone acetate x 13 days. In addition, the patient had a serum estradiol (E2) of <15 pg/mL, a serum FSH of 117 mIU/mL, and a serum anti-Mullerian level of <0.09 ng/mL. She was treated with EE to lower serum FSH levels rather than E2 because the former does not contribute to the total serum E2 level, and thus allows the treating physician to detect when a follicle has been recruited by rising serum E2 levels. A rising E2 would dictate the addition of pelvic sonography to help determine when a mature dominant follicle was achieved (18mm average diameter with serum E2 ≥200pg/mL). If a mature follicle was achieved, 1mg granulocyte colony stimulating factor (G-CSF) would be given the day before an injection of 10,000 units human chorionic gonadotropin (hCG) to help the oocyte release. At age 42 she had been treated for infertility and was found to have the luteinized unruptured follicle syndrome. She failed to release the oocyte with either hCG or leuprolide acetate but was successful with hCG when G-CSF was added (and delivered a healthy baby despite a day 3 serum FSH of 47 mIU/mL). Progesterone vaginal suppositories 200mg twice daily was supplemented in the luteal phase. After 44 days of EE she was able to attain a mature follicle (E2 273 pg/mL, follicular size 18.8mm average diameter). She released the oocyte, conceived and successfully delivered a full-term healthy baby. A previous case was reported in Human Reproduction in 2000 of a 45-year-old in overt menopause. Successful reversal of menopause was achieved, and she delivered a live baby utilizing this same technique of restoration of FSH sensitivity by using EE without any exogenous FSH stimulation. Precedents are important for patient-physician decisions regarding treatment options. This case shows a live delivery is possible even in a 46.5-year-old menopausal woman using her own oocytes.

Lipoamino acid-modified GnRH analogs with receptor-mediated antiproliferative activity in prostate and ovarian cancer cells

Gonadotropin-releasing hormone (GnRH) analogs (e.g., triptorelin) are developed to treat hormone-dependent reproductive cancers. However, these analogs lack a significant direct antitumor activity to make them suitable for hormone-refractory reproductive cancers. In this study, we examined different biological properties of lipid-modified GnRH analogs, with/without D-amino acid substitution at position 6 in prostate and ovarian cancer cells. We revealed that the improved metabolic stability due to lipid-modification and D-amino acid substitution played a pivotal role in enhancing GnRH receptor-mediated direct antiproliferative activity up to 4.5-fold higher than triptorelin. Furthermore, a comparable FSH release and higher LH release activity in pituitary cells than triptorelin was observed, indicating an improved specificity and/or binding affinity to GnRH receptors. We confirmed the important role of sex steroids in the antitumor activity of the lipopeptides, which were contrasting in prostate and ovarian cancer cells. The superior activity of these GnRH analogs over commercial peptides renders promises for developing new GnRH receptor ligands to treat hormone-dependent and -refractory cancers, as well as emerging new targeting moieties for the delivery of anticancer agents in GnRH receptor-overexpressing cancers.

Progesterone Positive Feedback on Pulsatile LH Secretion and FSH Release May Be Blunted in Estradiol-Pretreated Women With PCOS

In women pretreated with estradiol (E2), exogenous progesterone (P4) acutely augments LH and FSH release (P4 positive feedback). Women with PCOS exhibit impaired P4 negative feedback on LH pulse frequency, but it remains unclear whether such women exhibit impaired P4 positive feedback on LH/FSH release. We sought to explore the latter notion as an a priori secondary hypothesis in a study primarily designed to assess whether P4 acutely suppresses LH pulse frequency. We studied 12 women with PCOS and 12 normally-cycling, non-hyperandrogenic controls. After 3 days of transdermal E2 pretreatment (0.2 mg/day), subjects were admitted to the Clinical Research Unit (CRU) for a 24-hour frequent blood sampling protocol starting at 2000 h. (CRU admissions occurred no earlier than cycle day 7 in PCOS and between days 7 and 11 inclusive in controls.) At 0600 h, subjects received either 100 mg oral micronized P4 or placebo (PBO). In a subsequent menstrual cycle, subjects underwent an identical CRU protocol except that P4 was exchanged for PBO or vice versa. LH secretion was analyzed using Autodecon, a deconvolution program that provides estimates of LH pulse frequency, pulsatile LH secretion (amount of LH secreted as pulses), and basal (non-pulsatile) LH secretion. Results were analyzed using 2-period crossover design analysis of covariance. In both groups, neither LH pulse frequency nor basal LH secretion changed significantly with P4 (compared to changes with PBO). Mean LH increased with P4 in both groups—3.1-fold (95% CI, 2.4–4.0) in controls and 2.7-fold (95% CI, 2.1–3.5) in PCOS; in both groups, P4-related changes were significantly greater than PBO-related changes (Bonferroni-corrected p=0.012 and 0.010, respectively). In controls, pulsatile LH secretion increased 3.5-fold (95% CI, 2.3–5.2) with P4—significantly more than with PBO (p=0.029); while in PCOS, a 2.6-fold (95% CI, 1.8–3.9) increase with P4 was not significantly different from changes with PBO (p=0.911). In controls, mean FSH increased 2.0-fold (95% CI, 1.7–2.3) with P4—significantly more than with PBO (p=0.004); but in PCOS, a 1.5-fold (95% CI, 1.3–1.8) increase was not significantly different from changes with PBO (p=0.072). Despite the above, between-group (PCOS vs. controls) differences in P4-induced changes in pulsatile LH secretion and mean FSH were not formally (statistically) demonstrable. Between-group differences representing potential confounders included age (median 25.5 vs. 19.0 y; p=0.029), body mass index (29.9 vs. 21.8 kg/m2; p=0.006), and cycle day of CRU admissions (day 45.0 vs. 10.4 for P4 admissions; 30.0 vs. 10.0 for PBO admissions). In summary, these data suggest that P4-induced increases in pulsatile LH secretion and mean FSH may be blunted in PCOS compared to controls, which could contribute to ovulatory dysfunction in PCOS. However, our results do not confirm this possibility, and further study is needed.

Obese Women Exhibit Reduced Inhibin B and Estradiol SecretionFollowing Pulsatile Intravenous FSH Administration

Introduction: Maternal obesity is an independent risk factor for reduced reproductive fitness. Decreased secretion of FSH in women with obesity is well documented but poorly understood. Furthermore, obese women secrete less protein and steroid hormones from their ovaries. In mice, prior studies have demonstrated that pulsatile release of FSH enhances ovarian function and fertility. Hypothesis: We hypothesize that insufficient FSH pulsatility, as seen in women with obesity, results in inadequate folliculogenesis and reduced ovarian steroid production. We attempt to correct pulsatile FSH secretion in obese women by administering exogenous FSH to compensate for the suppressed circulating ovarian hormones. Our primary outcome is the change in peak inhibin B between pre- and post-treatment. We present results from our interim analysis. Methods: Reproductive aged, regularly menstruating, normal weight (NW) (BMI 18.5-24.9) and obese (OB) (BMI >30) women were recruited for a 26hr study during the early follicular phase. Frequent blood sampling (q10min) for 10h was performed to obtain baseline hormone levels. At 10h, 3 mg of cetrorelix, a gonadotropin hormone antagonist, was given followed by a secondary dose (0.25mg) 6h later. At this time, hourly IV recombinant (r)FSH (30IU) was initiated and frequent blood sampling continued for 10h. LH, FSH, estradiol (E2) were measured by immunoassay (Advia Centaur XP, Siemens). Inhibin B was measured using an ELISA kit (Ansh labs). Differences between groups were modeled by linear regression, adjusted for age and cycle day (continuous). The relationship between change in peak inhibin B and change in peak E2 was estimated in a linear regression. Results: A total of 36 participants (19 NW and 17 OB) were included in our interim analysis. There were no differences in age, cycle day of study, race, and waist/hip ratio. Inhibin B and E2 rises following the intervention were statistically significant within each group. Peak Inhibin B and E2 levels following intervention were lower in obese women compared to normal weight (133.4 vs 202.5 pg/mL and 85.8 vs 126.4 pg/mL, respectively). The difference in pre and post peak inhibin B levels trended lower in the obese group (-40.1 (95%CI: -86.2, 6.1, p=0.087). No difference was seen in maximal E2 response. There was no relationship between inhibin B and E2 response [0.08 (95%CI -0.26, 0.42), p=0.634]. Conclusions: These early results suggest obese women may have a lower response to pulsatile rFSH as compared to normal weight counterparts even with intravenous administration. We speculate this may be due to decreased uptake of rFSH in obese patients or a sign of ovarian dysfunction in obese women. Additional subjects are recruited to detect these differences.

145 Comparison of single to multiple injections of follicle-stimulating hormone before ovum pickup in Holstein heifers: Oocyte recovery and embryo production

The demand for invitro-produced embryos from heifers with high genetic merit has increased over time. Synchronization and stimulation of follicular growth before ovum pickup (OPU) has been used to improve oocyte quality and, consequently, embryo production. Multiple injections involve extra labour and stress for both personnel and cattle. The release of FSH can be prolonged by using 0.5% hyaluronan (HA) as a diluent, allowing a decrease in the number of injections. The objective of this study was to compare oocyte recovery and embryo production between single or multiple injections of FSH before OPU of Holstein heifers. During April and May 2020, 20 Holstein heifers (8 to 15 mo old) from Ruann Dairy (Riverdale, CA) were randomly divided and submitted to two different treatments (crossover design). Gonadotrophin-releasing hormone (GnRH; Fertagyl®, Merck, 129µg, IM) was given to synchronize the follicular wave emergence. Treatment 1×FSH consisted of a single intramuscular (IM) injection of 100mg of FSH (Folltropin®, Vetoquinol) 36h after GnRH. The FSH consisted of a 2.5-mL injection of 400mg of FSH diluted in 10mL of 0.5% HA. OPU was performed 48 to 50h after FSH. Treatment 5×FSH consisted of 100mg of FSH divided into 5 equal IM injections (10-14h intervals) 36h after GnRH. The FSH consisted of 5×1-mL injections of 400mg of FSH in 20mL of saline. OPU was performed 18 to 20h after the last FSH injection. All donors received both treatments at a 14-day interval and the recovered oocytes were fertilized with the same sexed female-sorted semen in both rounds. OPU, oocyte classification, IVM, IVF, and culture (IVC) were performed as described by Demetrio et al. (2020 Anim. Reprod. 17, e20200053). All oocytes went into IVM, except for degenerated oocytes. The number of 4-cell (or more) embryos on Day 3 of IVC divided by the number of oocytes in IVC after IVF is defined as the cleavage rate. The number of blastocysts (early to hatched) on Day 7 of IVP divided by the number of oocytes in IVC after IVF is defined as the blastocyst rate. Poisson-normal (count data) and Logistic-normal (proportion data) models were used to analyse the data. Treatment, donor (random effect), and sire were included in the models. The results are summarized in Table 1. There were no differences between the two treatments on the number of oocytes recovered per OPU (total and grade 1 and 2), percentage of grade 1 and 2 oocytes, cleavage rate, blastocyst rate and number of embryos (total and grade 1). Oocyte recovery and embryo production are highly donor dependent. Stimulation of the follicular growth before OPU with one single injection of FSH diluted in 0.5% HA 36h after GnRH can be efficiently used for IVP in Holstein heifers, without decreasing the number of oocytes recovered and/or embryos produced with the advantage of reducing labour and stress of handling cattle. Table 1. Number and quality of oocytes and cleavage and blastocyst rates Treatment OPU Oocytes per donor Grade 1 and 2 oocytes (%) Cleavage rate (%) Blastocyst rate (%) Total embryos per OPU Grade 1 embryos per OPU 1×FSH 20 17.0 45.7 84 39.8 6.2 3.8 5×FSH 20 19.9 46.5 82 35.6 6.3 4.0