Luteinizing Hormone Pulse Amplitude Research Articles

Are menstrual disturbances associated with an energy availability threshold? A critical review of the evidence.

Exercising women have a high prevalence of menstrual disturbances. In 2003, it was suggested that disruption in luteinizing hormone (LH) pulsatility occurs below a threshold of energy availability (EA) of 30kcal/kg lean body mass (LBM)/day. This paper is a critical review of the evidence regarding the theory that disruptions to the reproductive axis and menstrual disturbances occur below the proposed threshold. Short-term laboratory studies demonstrated that 4-5 days of an EA below 30kcal/kg LBM/day, induced with or without exercise, decreased serum triiodothyronine and LH pulse frequency, and increased LH pulse amplitude in sedentary, regularly menstruating women. Fewer studies have investigated downstream ovarian effects after long-term exposure to low EA. The Sargent Camp Study was the first randomized trial that induced luteal phase defects, delayed menses, and anovulation by causing weight loss (-4± 0.3kg) with an abrupt increase in exercise volume for two menstrual cycles. The BioEnergetics Study was a randomized controlled trial that induced varying levels of energy deficits by manipulating energy intake and expenditure for three menstrual cycles. LH pulse frequency and triiodothyronine decreased, and 57% of women developed luteal phase defects, anovulation, and/or oligomenorrhea. An EA below 30kcal/kg fat free mass/day increased the chance of experiencing a menstrual disturbance by 50%. However, menstrual disturbances were observed above and below that EA threshold, and changes in LH pulse frequency predicted only luteal phase defects, not oligomenorrhea or anovulation. The proposed EA threshold is not a cut-off below which menstrual disturbances occur, but represents an increased risk of experiencing menstrual disturbances.

A high-fat eucaloric diet induces reprometabolic syndrome of obesity in normal weight women.

We examined the effects of 1 month of a eucaloric, high-fat (48% of calories) diet (HFD) on gonadotropin secretion in normal-weight women to interrogate the role of free fatty acids and insulin in mediating the relative hypogonadotropic hypogonadism of obesity. Eighteen eumenorrheic women (body mass index [BMI] 18-25 kg/m2) were studied in the early follicular phase of the menstrual cycle before and after exposure to an HFD with frequent blood sampling for luteinizing hormone (LH) and follicle-stimulating hormone (FSH), followed by an assessment of pituitary sensitivity to gonadotropin-releasing hormone (GnRH). Mass spectrometry-based plasma metabolomic analysis was also performed. Paired testing and time-series analysis were performed as appropriate. Mean endogenous LH (unstimulated) was significantly decreased after the HFD (4.3 ± 1.0 vs. 3.8 ± 1.0, P < 0.01); mean unstimulated FSH was not changed. Both LH (10.1 ± 1.0 vs. 7.2 ± 1.0, P < 0.01) and FSH (9.5 ± 1.0 vs. 8.8 ± 1.0, P < 0.01) responses to 75 ng/kg of GnRH were reduced after the HFD. Mean LH pulse amplitude and LH interpulse interval were unaffected by the dietary exposure. Eucaloric HFD exposure did not cause weight change. Plasma metabolomics confirmed adherence with elevation of fasting free fatty acids (especially long-chain mono-, poly-, and highly unsaturated fatty acids) by the last day of the HFD. One-month exposure to an HFD successfully induced key reproductive and metabolic features of reprometabolic syndrome in normal-weight women. These data suggest that dietary factors may underlie the gonadotrope compromise seen in obesity-related subfertility and therapeutic dietary interventions, independent of weight loss, may be possible.

PSIV-17 Postnatal Obesity Amplifies Gnrh-Induced Lh Release in Prenatal Testosterone-Treated Female Sheep

Abstract Polycystic ovary syndrome (PCOS) is the most common hormonal disorder in females of reproductive age. Hypersecretion of luteinizing hormone (LH), partly due to increased pituitary sensitivity to gonadotrophin-releasing hormone (GnRH), is a significant cause of infertility in women with PCOS. Moreover, obesity amplifies the severity of reproductive dysfunction in PCOS. In sheep, testosterone (T) excess during gestational days (GD) 30 to 90 results in GnRH-mediated LH hypersecretion and recapitulates the PCOS phenotype. This study investigated 1) if mid-gestation (GD 60 to 90) is the susceptibility window for programming pituitary hypersensitivity to GnRH, and 2) the contribution of postnatal obesity in modulating LH responsiveness to GnRH. Suffolk ewes received T propionate (T; 100 mg i.m.) or corn oil (C; vehicle) twice weekly from GD 60 to 90 (term = 147 d). At 5 mo of age, T lambs were assigned randomly to either a maintenance (100% of NRC requirements) or overfed diet (130% of NRC requirements), and Control lambs were fed the maintenance diet. During the anestrous period (late summer), ewes (n = 8/group; ~17 mo of age) received a GnRH bolus injection (2 ng/kg) intravenously every 1.5 h and blood samples were collected at 15-min intervals for a 6-h period. Despite animals being in seasonal anestrous, few endogenous LH pulses were evident during the 6 h-sampling period. To avoid confounding, only clear responses to GnRH challenge that occurred within 15 to 45 min after each GnRH injection were considered for further analyses. Mean number of exogenous GnRH-induced LH responses that met these criteria did not differ between groups and averaged 1.81 ± 0.27 pulses/animal. The parameters of these GnRH-induced responses were analyzed as one-way ANOVA with Tukey’s HSD post-hoc analysis. Mean LH pulse peak was greater (P &amp;lt; 0.05) in T overfed ewes (4.59 ± 0.49 ng/mL) compared with T maintenance (2.19 ± 0.61 ng/mL) and Control ewes (2.58 ± 0.53 ng/mL). Additionally, amplitude of LH pulses was greater (P &amp;lt; 0.05) in T overfed ewes (2.66 ± 0.39 ng/mL) compared with T maintenance (1.03 ± 0.48 ng/mL) and tended (P = 0.089) to be greater in T overfed compared with Control ewes (1.41 ± 0.42 ng/mL). LH pulse peak and amplitude in T maintenance did not differ from Control ewes. In conclusion, failure of GD 60 to 90 T treatment to increase GnRH responsiveness as opposed to our earlier finding of increased responsiveness in GD 30 to 90 T-treaded sheep indicates that the susceptibility window for programming pituitary hypersensitivity to GnRH lies between GD 30 to 60. Postnatal obesity may act as an independent activational signal in amplifying the pituitary sensitivity to GnRH in female sheep. This premise is supported by observations that obesity exacerbates pituitary-mediated LH hypersecretion in women with PCOS. Research support: NIH-NICHD (R01HD099096).

289 Postnatal Obesity Amplifies the Effect of Prenatal Testosterone Excess in Reducing the Progesterone Negative Feedback Sensitivity in First-Generation Ewes

Abstract Polycystic ovary syndrome (PCOS) is the leading cause of anovulatory infertility in women of reproductive age. Women with PCOS exhibit reduced sensitivity of the GnRH neuronal system to the inhibitory effects of both progesterone and estradiol. Additionally, obesity amplifies the severity of the PCOS phenotype. In sheep, testosterone (T) excess during gestational days (GD) 30-90 and 60-90 reduces neuroendocrine sensitivity to the progesterone negative feedback and recapitulates the PCOS phenotype. This study investigated the contribution of postnatal obesity in amplifying or mitigating the effects of mid-gestational T excess in disrupting the responsiveness to progesterone negative feedback in sheep. Suffolk ewes received T propionate (T; 100 mg i.m.) or corn oil (CO; vehicle) twice weekly from GD 60-90 (term = 147 d). At 5 mo of age, F1 T lambs were assigned randomly to either a maintenance (100% of NRC requirements) or overfed diet (130% of NRC requirements) and control lambs were fed the maintenance diet. At 22 mo of age, F1 ewes (n = 8/group) were synchronized with two injections of PGF2α 11 d apart. After the second PGF2α administration, blood samples were collected daily for 14 d and progesterone concentrations were determined via radioimmunoassay. Ewes that were confirmed to be cycling and in the mid-luteal phase based on progesterone concentrations (progesterone ≥ 1 ng/ml on day 10 and ≥ 0.5 ng/ml on days 8-9 and 11-12) were used for LH pulse characterization. On day 10 after PGF2α synchronization (mid-luteal phase), blood samples were collected every 15 min for 6 h to characterize LH pulsatile secretion via radioimmunoassay. Data were analyzed as one-way ANOVA with Tukey's HSD post-hoc analysis. Frequency of LH pulses was greater (P &amp;lt; 0.05) in T maintenance (3.75 ± 0.41 pulses/6h) and T overfed ewes (3.50 ± 0.34 pulses/6h) compared with controls (2.00 ± 0.37 pulses/6 h). Luteinizing hormone pulse amplitude was greater (P &amp;lt; 0.05) in T overfed (2.03 ± 0.15 ng/mL) compared with T maintenance (1.44 ± 0.17 ng/mL), but not to controls (1.74 ± 0.21 ng/mL). Also, average LH concentration was greater (P &amp;lt; 0.05) in T overfed (2.03 ± 0.24 ng/mL), but not T maintenance (1.31 ± 0.35 ng/mL), compared with controls (0.93 ± 0.30 ng/mLl). In conclusion, these findings demonstrate the activational role of postnatal obesity in amplifying prenatal T-induced alterations in tonic LH secretion during progesterone negative feedback. The activational effect of postnatal obesity in increasing LH secretion and consequently thecal androgen production is likely a contributor to the severity of hyperandrogenic status in women with PCOS. Research support: NIH-NICHD (R01HD099096).

FRI280 Partial Loss Of KNDy Neurons In Prenatally Androgen Treated Female Rats Alters The LH Secretion And Ovarian Morphology In A Model Of Polycystic Ovary Syndrome

Abstract Disclosure: N.S. Aquino: None. A. Campideli-Santana: None. L.M. Antunes: None. R. Araújo-Lopes: None. K.S. Silva: None. P.C. Henriques: None. D.D. Gusmão: None. M.P. Bernuci: None. R.E. Szawka: None. A.M. dos Reis: None. Polycystic Ovary Syndrome (PCOS) is a high-prevalent endocrine dysfunction, causing metabolic disorders and infertility. Women with PCOS show a disrupted hypothalamus-pituitary-gonadal (HPG) axis, which results in increased luteinizing hormone (LH) secretion, LH pulse frequency, and LH/follicle stimulating hormone (FSH) ratio. These changes are related to an impaired negative-feedback effect of ovarian steroids, estradiol and progesterone, on the gonadotrophin releasing-hormone (GnRH) neurons. Because the kisspeptin/neurokinin-B/dynorphin (KNDy) neurons of the arcuate (ARC) nucleus are responsible for mediating the ovarian steroid negative feedback, we investigated whether the ablation of KNDy neurons would change the LH secretion and ovarian morphology in a rat model of PCOS. Pregnant dams were treated with dihydrotestosterone (DHT, 3 mg/day; s.c) from days 16 to 19 of pregnancy. Three months after birth, the prenatally androgen-treated (PNA) offspring received intra-ARC stereotaxic injections of the neurokinin-3 receptor agonist conjugated with saporin (NK3-SAP) to induce the lesion of KNDy neurons and were submitted to serial blood sampling for LH measurement. PNA NK3-SAP rats had a moderate loss of ARC neurokinin-B-immunoreactive neurons (between 50-70%) compared with the Blank-SAP-injected PNA group. The PNA Blank-SAP rats displayed LH pulse frequency, pulse amplitude, and mean LH levels similar to control rats on diestrus. Remarkably, the partial loss of KNDy neurons increased the LH pulse amplitude and mean LH without changing the pulse frequency. Histological analysis was performed to investigate the outcome of the partial loss of KNDy neurons on the ovarian morphology in PNA rats. The numbers of primordial, primary, and secondary health follicles were decreased in PNA Blank-SAP compared with diestrus rats. Notably, the number of primordial follicles was partially restored in NK3-SAP rats. On the other hand, neither the number of atretic follicles nor the number of corpora lutea was changed in PNA Blank or NK3-SAP rats, indicating no change in the ovulatory function. Therefore, we provide evidence that KNDy neurons negatively modulate LH release and LH pulse amplitude in PNA rats. Moreover, the results suggest that KNDy neurons are involved in the reduction of the primordial follicle pool and ovarian reserve in PNA rats. Financial Support: Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), Brazilian National Council for Scientific and Technological Development (CNPq), British Society for Neuroendocrinology (BSN). Presentation: Friday, June 16, 2023

FRI279 Submaximal Loss Of KNDy Neurons Accelerates And Amplifies Pulsatile LH Secretion In Female Rats

Abstract Disclosure: A.C. Campideli-Santana: None. K.S. da Costa Silva: None. R. Araújo-Lopes: None. L.M. Antunes: None. R.E. Szawka: None. Kisspeptin is a powerful stimulator neuropeptide in the hypothalamus-pituitary-gonadal axis and is essential for fertility. Neurons in the arcuate nucleus (ARC) of the hypothalamus that coexpress kisspeptin, neurokinin B, and dynorphin (KNDy) play a crucial role in the control of luteinizing hormone (LH) secretion, but the neuroendocrine mechanisms involved in this regulation are not fully understood. Our previous study demonstrated that the moderate loss of KNDy neurons amplifies the LH surge induced by estradiol in ovariectomized rats (1). The current study aimed to perform a longitudinal evaluation of female rats in which KNDy neurons were ablated to determine the integrated role of this neuronal population in the control of pulsatile LH secretion. Adult female rats underwent a neurochemical ablation of KNDy neurons via intra-ARC stereotaxic injections of neurokinin-3 receptor agonist conjugated with saporin (NK3-SAP), while control animals received blank-saporin (Control). Estrous cyclicity was monitored over 21 days before and after the stereotaxic surgery. Three weeks after surgery, blood samples (10 µL) were withdrawn from the tail tip at 6-minute intervals for 180 minutes on the day of diestrus. Rats were then ovariectomized and the ovaries were processed for histological analysis. Whole blood LH levels were measured by ELISA and brains were immunohistochemically labeled for neurokinin B in the ARC. NK3-SAP rats bearing a submaximal loss of KNDy neurons (between 55-95%; n = 8) displayed irregular estrous cycles. The ovarian weight and the number of corpora lutea and atretic follicles were unaltered, whereas the number of healthy early antral follicles decreased in NK3-SAP rats. The frequency of LH pulses was increased and both pulse amplitude and mean LH levels were higher in NK3-SAP compared with Control rats. Thus, the submaximal loss of KNDy neurons affects the regularity of estrous cyclicity but does not preclude ovulation, while seems to stimulate the maturation of early antral follicles. Notably, KNDy neuron loss accelerates and amplifies LH pulsatile secretion during the rat estrous cycle. These results suggest a new role for KNDy neurons in restraining the frequency and amplitude of LH pulses in ovary-intact animals, with possible implications in the rate of recruitment of early antral follicles. Reference: Campideli-Santana et al., J Neuroendocrinol. 2022 Nov;34(11):e13204. Support: FAPEMIG, CNPq, CAPES, and Pro-reitoria de Pesquisa-UFMG. Presentation: Friday, June 16, 2023

SAT606 Comparison Of Psychosocial And Immune/Inflammatory Stress On Luteinizing Hormone Pulsatile Secretion

Abstract Disclosure: J. Jang: None. M.J. Kreisman: None. K.M. Breen Church: None. Stress induces a physiological response in the body to return to the homeostatic state. It is recognized that many stress types impair reproduction, but the mechanisms conveying inhibition may be distinct and stressor-dependent. Our research aims to understand how stress inhibits the neuroendocrine mechanisms controlling the pulsatile release of luteinizing hormone (LH). This is especially important because pulsatile LH is pivotal to reproductive health in males and females. Immune/inflammatory stressors such as lipopolysaccharides (LPS) cause an inflammatory response, producing peripheral cytokines that can signal to the brain in distinct ways compared to the neural mechanisms employed in response to psychosocial stress. However, how LPS disrupts the neural mechanisms involved in gonadotropin pulsatility is still unknown. Here, we compared the alteration in LH pulses in response to psychosocial restraint stress or immune/inflammatory LPS stress and sought to understand stress-specific regulation of the upstream targets controlling LH synthesis and secretion. Our initial observations demonstrated, in ovariectomized (OVX) female mice, that restraint stress elicited suppression rapidly, within 30 minutes; yet, the response to LPS was delayed, occurring approximately 90 minutes from injection. Therefore, we investigated the prolonged response of the stressors on LH pulses, observing LH pulse profiles 90 minutes to 180 minutes after the initiation of stress. Restraint stress significantly suppressed mean LH, resulting from a suppression of pulse frequency. LPS injected intraperitoneal (IP, 1.5 ug/g) also significantly suppressed mean LH. In contrast to restraint stress, LPS induced a reduction in both frequency as well as LH pulse amplitude. We conducted two follow-up experiments to investigate the lowered LH pulse amplitude during immune/inflammatory stress. The first study assessed hypothalamic kisspeptin gene expression and pituitary LHβ and GnRH receptor expression, in LPS versus restraint animals, to test whether altered gene transcription in LPS animals resulted in lowered LH secretion. The results of this experiment are still pending. The second study tested the LH response to a single bolus of GnRH or kisspeptin. Interestingly, we found equivalent LH increases, to either agonist, in saline versus LPS-treated animals. Collectively, these data indicate that both restraint and immune/inflammatory stress inhibit LH pulse frequency. However, the endogenous LH pulse profiles suggest an additional stressor-specific mechanism that lowers pulsatile LH during immune/inflammatory stress via LPS. Identification of stress pathways that alter endocrine hormones is critical as it allows for the development of therapeutics and medications to treat endocrine disorders. Presentation: Saturday, June 17, 2023

The impact of undernutrition on KNDy (kisspeptin/neurokinin B/dynorphin) neurons in female lambs.

Undernutrition limits reproduction through inhibition of gonadotropin‐releasing hormone (GnRH)/luteinizing hormone (LH) secretion. Because KNDy neurons coexpress neuropeptides that play stimulatory (kisspeptin and neurokinin B [NKB]) and inhibitory (dynorphin) roles in pulsatile GnRH/LH release, we hypothesized that undernutrition would inhibit kisspeptin and NKB expression at the same time as increasing dynorphin expression. Fifteen ovariectomized lambs were either fed to maintain pre‐study body weight (controls) or feed‐restricted to lose 20% of pre‐study body weight (FR) over 13 weeks. Blood samples were collected and plasma from weeks 0 and 13 were assessed for LH by radioimmunoassay. At week 13, animals were killed, and brain tissue was processed for assessment of KNDy peptide mRNA or protein expression. Mean LH and LH pulse amplitude were lower in FR lambs compared to controls. We observed lower mRNA abundance for kisspeptin within KNDy neurons of FR lambs compared to controls with no significant change in mRNA for NKB or dynorphin. We also observed that FR lambs had fewer numbers of arcuate nucleus kisspeptin and NKB perikarya compared to controls. These findings support the idea that KNDy neurons are important for regulating reproduction during undernutrition in female sheep.

Kisspeptin Overcomes GnRH Neuronal Suppression Secondary to Hyperprolactinemia in Humans.

ContextHyperprolactinemia suppresses gonadotropin-releasing hormone (GnRH)-induced luteinizing hormone (LH) pulses. The hypothalamic neuropeptide kisspeptin potently stimulates the secretion of GnRH. The effects of exogenous kisspeptin administration on GnRH pulse generation in the setting of hyperprolactinemia have not previously been explored.ObjectiveThis work aimed to examine the effects of kisspeptin on GnRH secretion, as reflected by LH secretion, in women with hyperprolactinemia.MethodsWomen with hyperprolactinemia (n = 11) participated in two 12-hour visits. Before study visits, participants underwent washout of dopamine agonist and/or combined oral contraceptive. Frequent blood sampling was performed (1 sample was collected every 10 minutes). Visit 1 involved no intervention, to examine baseline LH pulsatility. During visit 2, kisspeptin 112–121 (0.24 nmol/kg) was administered every 1 hour, for 10 hours. At hour 11, one intravenous bolus of GnRH (75 ng/kg) was administered.ResultsRepetitive intravenous bolus kisspeptin administration increased the total number of LH pulses in the setting of hyperprolactinemia. The interpulse interval declined during the same time frames. LH pulse amplitude did not change, but the mean LH rose. In 6 participants with progesterone levels suggestive of an anovulatory state, mean LH and estradiol levels increased significantly at visit 2. In the entire cohort, follicle-stimulating hormone and prolactin levels did not change significantly across the 2 visits. A total of 73% of subjects exhibited an LH pulse within 30 minutes of first kisspeptin dose.ConclusionKisspeptin is capable of stimulating hypothalamic GnRH-induced LH pulses in the setting of hyperprolactinemia.

Reformulation of PULSAR for Analysis of Pulsatile LH Secretion and a Revised Model of Estrogen-Negative Feedback in Mice.

The recent use of the tail-tip bleeding approach in mice has enabled researchers to generate detailed pulse and surge profiles of luteinizing hormone (LH) secretion in mice. However, the analysis of pulsatile LH secretion is piecemeal across the field with each laboratory using their own methodology. We have reformulated the once-popular PULSAR algorithm of Merriam and Wachter to operate on contemporary computer systems and provide downloadable and online pulse analysis platforms. As it is now possible to record the activity of the gonadotropin-releasing hormone pulse generator in freely behaving mice, we have been able to unambiguously define LH pulses in intact and gonadectomized male and female mice. These data sets were used to determine the appropriate PULSAR parameter sets for analyzing pulsatile LH secretion in the mouse. This was then used to establish an accurate model of estrogen negative feedback in the mouse. Intact and ovariectomized mice given Silastic capsules containing 1, 2, and 4 μg 17-β-estradiol/20 g body weight were tail-tip bled at 6-min intervals, and the resultant LH profiles were analyzed with PULSAR. Only the 4 μg 17-β-estradiol capsule treatment was found to return LH pulse amplitude and frequency to that of intact diestrous mice. Ultrasensitive mass spectrometry analysis showed that the 4 μg 17-β-estradiol capsule generated circulating estradiol levels equivalent to that of diestrous mice. It is hoped that the reformulation of PULSAR and generation of a realistic model of estrogen-negative feedback will provide a platform for the more uniform assessment of pulsatile hormone secretion in mice.

Adiposity Alters Follicle Dynamics in Women of Reproductive Age With Regular Menstrual Cycles

Obesity increases a woman’s likelihood of experiencing adverse reproductive health outcomes, including menstrual cycle irregularity, anovulation, and luteal phase dysfunction. Despite a heightened risk of reproductive dysfunction some women with obesity display ovulatory cyclicity. The degree to which adiposity affects menstrual cycle characteristics in this population is limited to endocrine assessments; evidence supports decreased luteinizing hormone pulse amplitude and reduced luteal phase progesterone. However, these endocrine disruptions have not been linked to alterations in follicular events. The objectives of the present analysis were to evaluate longitudinal changes in follicle size populations and growth kinetics of selectable follicles (6-9mm) and identify endocrine abnormalities associated with obesity in regular cycles. 14 non-obese women (BMI <30 kg/m2) and 15 obese women (BMI≥ 30 kg/m2) with regular cycles were evaluated by serial ovarian transvaginal ultrasonography and venipuncture every-other-day for one complete inter-ovulatory interval (IOI). The number and diameters of all follicles ≥2 mm at each visit were documented offline using the grid method. Growth profiles of individual follicles which grew to >7mm were assessed using the Identity Method. Diagnostic, morphologic, and endocrine features were compared across groups using parametric and non-parametric t-tests (i.e. cross-sectional features) and mixed models (i.e. longitudinal features). Non-obese and obese women with regular cycles showed similar IOI, follicular phase, and luteal phase lengths. The mean number of recruitment events, maximum dominant follicle diameter, and the growth rates of ovulatory follicles over time did not differ between groups, despite confirmation of compromised mean luteal progesterone production (8.23 ng/mL vs. 14.75 ng/mL, p=0.047) and decreased mean luteal FSH levels in women with obesity (2.33 mIU/mL vs. 5.83 mIU/mL, p=0.040). Over the IOI women with obesity showed an increased proportion of 2-5 mm follicles (β=5.3%; p<0.05) and a decreased proportion of 6-9mm follicles (β=-5.0%; p=0.05) versus non-obese women consistent with fewer follicles transitioning from the 2-5 mm pool to the selectable stage. This is the first comparison of follicle dynamics between non-obese and obese women with regular ovulatory cycles. These data suggest that a smaller pool of selectable follicles is present in women with obesity and may result in suboptimal follicle development luteal function. Future studies are needed to understand the impact of altered follicle populations and luteal hormone dynamics on endometrial receptivity and fertility/fecundity.

Gonadotropin response to insulin and lipid infusion reproduces the reprometabolic syndrome of obesity in eumenorrheic lean women: a randomized crossover trial

To study the reprometabolic syndrome in normal-weight, eumenorrheic women by infusing a combination of insulin and lipid. Women with obesity have been shown to have reduced gonadotropins and impaired luteinizing hormone (LH) and follicle-stimulating hormone (FSH) response to gonadotropin-releasing hormone (GnRH). Randomized crossover. Academic medical center. Fifteen women, median age 32 (interquartile ranged [IQR] 26, 36) years and body mass index 21.9 (IQR 20.2, 22.9) kg/m2 were recruited. Early follicular phase, 6-hour infusions of insulin (20-40 mU/m2 per minute) and lipid (Intralipid)-insulin/lipid infusion; or saline infusion (controls). The first 4 hours of each study assessed endogenous gonadotropins; at 4 hours, GnRH (75 ng/kg) bolus was administered and sampling continued until 6 hours. Linear mixed model analysis was used to determine differences between insulin/lipid and saline influence on endogenous LH pulse amplitude (primary outcome), mean FSH, and area under the curve (AUC) response to GnRH (secondary outcomes). Twelve women completed both intended studies and an additional 3 women completed only 1 of the 2 studies. LH pulse amplitude, mean FSH, and both AUC responses to GnRH were reduced by insulin/lipid, mean FSH and AUC for LH were at or near statistical significance. LH response to GnRH was significantly reduced when 1 participant with very high LH and antimullerian hormone levels was excluded. Acute infusion of insulin/lipid to eumenorrheic, normal-weight women recapitulated the reprometabolic syndrome of obesity. These findings imply that specific circulating factors in obese women contribute to their subfertility and thus may be amenable to discovery and treatment. NCT02653092.

Effect of calfhood nutrition on metabolic hormones, gonadotropins, and estradiol concentrations and on reproductive organ development in beef heifer calves.

This study examined the effect of plane of nutrition on the endocrinological regulation of the hypothalamic-pituitary-ovarian (HPO) axis in beef heifer calves during a critical sexual developmental window early in calf hood. Forty Holstein-Friesian × Angus heifers (mean age 19 d, SEM = 0.63) were assigned to a high (HI; ADG 1.2 kg) or moderate (MOD; ADG 0.50 kg) nutritional level from 3 to 21 wk of life. Intake was recorded using an electronic calf feeding system, BW was recorded weekly, and blood samples were collected on the week of age 5, 10, 15, and 20 for metabolite, reproductive, and metabolic hormone determination. At 19 wk of age, on sequential days, an 8-h window bleed was carried out for luteinizing hormone (LH), follicle-stimulating hormone (FSH), and estradiol analysis. To characterize anterior pituitary gland function, an intravenous GnRH challenge was conducted (19 wk of age). Blood was collected via a jugular catheter every 15 min for 135 min for the analysis of LH, FSH, and estradiol. Calves were subsequently euthanized at 21 wk of age; the anterior pituitary, metabolic organs, and reproductive tract were weighed, and ovarian surface follicular numbers and oocytes recovered were recorded. Mean ADG was 1.18 and 0.50 kg for HI and MOD, respectively, resulting in a 76.6-kg difference in BW (P < 0.001). Blood insulin, glucose, and IGF-1 concentrations were greater (P < 0.001) for HI compared with MOD. There was a diet × time interaction for leptin (P < 0.01); concentrations were greater in HI compared with MOD at 20 wk of age with no difference between treatments before this. Dietary treatment did not alter the concentrations of adiponectin or anti-mullerian hormone. There was a diet × time interaction for FSH, whereby MOD had greater concentrations than HI at 10, 15, and 20, but not at 5 wk of age. Over the duration of an 8-h window bleed (19 wk of age), serum concentrations of LH, LH pulse frequency, and LH pulse amplitude were unaffected by treatment, whereas FSH (0.23 vs. 0.43 ng/mL) and estradiol (0.53 vs. 0.38 ng/mL) concentrations were less than and greater, respectively, for HI than MOD (P < 0.05). Likewise, following a GnRH challenge, the area under the curve analysis revealed greater (P < 0.01) estradiol and lesser (P < 0.01) FSH concentrations in calves on the HI relative to MOD diet, whereas concentrations of LH were unaffected (P = 0.26) between treatments. Ovarian surface follicle numbers were greater (P < 0.05) in HI compared with MOD. Total reproductive tract, uterus, and ovarian tissue expressed relative to BW were greater (P < 0.05) for HI compared with MOD. In conclusion, enhanced nutrition in early calfhood advances the ontogeny development of the HPO axis.

Tachykinin Signaling Is Required for Induction of the Preovulatory Luteinizing Hormone Surge and Normal Luteinizing Hormone Pulses

Tachykinins (neurokinin A [NKA], neurokinin B [NKB], and substance P [SP]) are important components of the neuroendocrine control of reproduction by direct stimulation of Kiss1 neurons to control GnRH pulsatility, which is essential for reproduction. Despite this role of tachykinins in successful reproduction, knockout (KO) mice for Tac1 (NKA/SP) and Tac2 (NKB) genes are fertile, resembling the phenotype of human patients bearing NKB signaling mutations, who often reverse their hypogonadal phenotype. This suggests the existence of compensatory mechanisms among the different tachykinin ligand-receptor systems to maintain reproduction in the absence of one of them. In order to test this hypothesis, we generated complete tachykinin-deficient mice (Tac1/Tac2KO). Male mice displayed delayed puberty onset and decreased luteinizing hormone (LH) pulsatility (frequency and amplitude of LH pulses) but preserved fertility. However, females did not show signs of puberty onset (first estrus) within 45 days after vaginal opening, they displayed a low frequency (but normal amplitude) of LH pulses, and 80% of them remained infertile. Further evaluation identified a complete absence of the preovulatory LH surge in Tac1/Tac2KO females as well as in wild-type females treated with NKB or SP receptor antagonists. These data confirmed a fundamental role of tachykinins in the timing of puberty onset and LH pulsatility and uncovered a role of tachykinin signaling in facilitation of the preovulatory LH surge. Overall, these findings indicate that tachykinin signaling plays a dominant role in the control of ovulation, with potential implications as a pathogenic mechanism and a therapeutic target to improve reproductive outcomes in women with ovulation impairments.

Progesterone and Luteinizing hormone secretion patterns in early pregnant gilts.

We studied luteinizing hormone (LH) pulsatility and episodic progesterone release of the corpus luteum (CL) on Day 11 and Day 21 in inseminated gilts and aimed to establish a relationship between these two hormones. Blood was collected at 15-min intervals for 12hr on Days 11, 16 and 21 from a vena cava caudalis catheter. At euthanasia, eight gilts were pregnant and six gilts were not pregnant. Progesterone parameters (basal, mean, pulse frequency and pulse amplitude) did not differ between pregnant and non-pregnant gilts on Day 11, LH pulse frequency and amplitude tended to differ (p=.07 and p=.079). In pregnant gilts, basal and mean progesterone, progesterone pulse amplitude and frequency declined significantly from Day 11 to Day 21 (p<.05). A significant decline was also seen in the LH pulse amplitude from Day 11 to Day 21 (p<.05). None of the LH pulses was followed by a progesterone pulse within 1hr on Day 21. On Day 11 and Day 21 appeared a synchronicity in the LH pulse pattern, as there were two or three LH pulses in 12hr and these LH pulses appeared in the same time window. We conclude that on Day 11 and Day 21 of pregnancy in gilts, progesterone pulses do not follow an LH pulse within one hour. Further we demonstrated that the successful or not successful formation of a CL of pregnancy is independent of progesterone release on Day 11 after insemination. We confirmed the decline of progesterone from Day 11 to Day 21 in the vena cava caudalis and could demonstrate that this decline is partly due to lower progesterone pulse amplitude and frequency and that the decline occurs simultaneously with a decline in LH pulse amplitude.

Aromatase Inhibition Ameliorates Decreased LH Output Found in Obese Women

In obese ovulatory women, serum luteinizing Hormone (LH) and follicle stimulating hormone (FSH) are lowered compared with normal weight women. This relative hypogonadotropic hypogonadism represents a potential etiology for overall decreased fertility in obesity. The objective was to determine if administration of an aromatase inhibitor (AI) to ovulating obese women would normalize LH and FSH by interrupting estradiol negative feedback. Letrozole (2.5–5 mg) was given daily to 22 women, 12 obese and 10 normal weight, for 7 days. On the last day of administration, 8 h of blood sampling was done every 10 min before and after a bolus of GnRH at 4 h. We obtained data from 21 ovulatory women (10 normal weight and 11 obese) who had undergone a similar protocol of frequent blood sampling but no aromatase inhibitors (AI) treatment. Serum LH and FSH levels and pulse characteristics were measured. Treatment with AI only significantly affected obese women. Further, in women with obesity, LH secretion, prior to the GnRH bolus, was significantly higher in AI treated compared with non-treated (p = 0.011). AI treatment doubled LH pulse amplitude in obese women (p = 0.004). In response to aromatase inhibition, LH secretion in ovulatory women with obesity is increased and similar to levels found in untreated normal weight women. The increase in LH pulse amplitude indicates that the AI effect is mediated at the level of the pituitary. Our results suggest that the hypogonadotropic phenotype of simple obesity is subject to modulation by interruption of estradiol negative feedback.

Role of progesterone concentrations during early follicular development in beef cattle: I. Characteristics of LH secretion and oocyte quality

Objective was to investigate the effect of different progesterone (P4) concentrations during early follicular development on luteinizing hormone (LH) secretion and oocyte characteristics in beef cows. Primiparous cows (n = 24) were estrous pre-synchronized and follicular ablation was performed (d 0) 6 days following the time of ovulation. At the time of follicular ablation, cows were assigned to either: 1) high P4 treatment - HiP4; a new CIDR was inserted on d 0 to supplement P4 from the existing corpus luteum [CL], or 2) low P4 treatment - LoP4; a previously-used CIDR and two doses of PGF 8 to 12 h apart were given on d 0. Concentrations of P4 were greater (P < 0.01) in the cows of the HiP4 than LoP4 group on d 1.5, 2.5, and 3.5. Peripheral concentrations of E2 were greater (P < 0.05) in the cows of the LoP4 than HiP4 group on d 2.5 and 3.5. Frequency of LH pulses was greater (P < 0.05) in the LoP4 than HiP4 group on d 2.5, but mean LH concentration and pulse amplitude did not differ between treatments. Number of follicles aspirated per cow, total oocytes recovered, recovery rate, percentage of oocytes graded 1 to 3, oocyte diameter, percentage BCB+ oocytes, and relative abundance of oocyte mRNA for FST did not differ (P > 0.10) between treatments. In conclusion, lower P4 concentrations during early follicular development resulted in increased LH pulse frequency and E2 concentrations, but did not affect characteristics of oocyte developmental competence.

Accelerated Episodic Luteinizing Hormone Release Accompanies Blunted Progesterone Regulation in PCOS-like Female Rhesus Monkeys (Macaca Mulatta) Exposed to Testosterone during Early-to-Mid Gestation

Background/Aims: Ovarian theca cell hyperandrogenism in women with polycystic ovary syndrome (PCOS) is compounded by androgen receptor-mediated impairment of estradiol and progesterone negative feedback regulation of episodic luteinizing hormone (LH) release. The resultant LH hypersecretion, likely the product of accelerated episodic release of gonadotropin-releasing hormone (GnRH) from the median eminence of the hypothalamus, hyperstimulates ovarian theca cell steroidogenesis, enabling testosterone (T) and androstenedione excess. Prenatally androgenized (PA) female monkeys exposed to fetal male levels of T during early-to-mid gestation, when adult, demonstrate PCOS-like traits, including high T and LH levels. This study tests the hypothesis that progesterone resistance-associated acceleration in episodic LH release contributes to PA monkey LH excess. Methods: A total of 4 PA and 3 regularly cycling, healthy control adult female rhesus monkeys of comparable age and body mass index underwent (1) a 10 h, frequent intravenous sampling assessment for LH episodic release, immediately followed by (2) IV infusion of exogenous GnRH to quantify continuing pituitary LH responsiveness, and subsequently (3) an SC injection of a progesterone receptor antagonist, mifepristone, to examine LH responses to blockade of progesterone-mediated action. Results: Compared to controls, the relatively hyperandrogenic PA females exhibited ~100% increase (p = 0.037) in LH pulse frequency, positive correlation of LH pulse amplitude (p = 0.017) with androstenedione, ~100% greater increase (p = 0.034) in acute (0–10 min) LH responses to exogenous GnRH, and an absence (p = 0.008) of modest LH elevation following acute progesterone receptor blockade suggestive of diminished progesterone negative feedback. Conclusion: Such dysregulation of LH release in PCOS-like monkeys implicates impaired feedback control of episodic release of hypothalamic GnRH reminiscent of PCOS neuroendocrinopathy.

Sex bias of the birth litter affects surge but not tonic LH secretion in gilts.

The physiology and behavior of gilts that develop in a male-biased litter can differ from gilts that develop in a female-biased litter. We hypothesized that gilts from male-biased litters will have a delayed and attenuated luteinizing hormone (LH) surge, and reduced LH pulse frequency and amplitude compared to gilts from female-biased litters. Gilts were selected at birth from male-biased (>60% males n = 10) or female-biased (>60% females n = 9) litters. From 18 wk of age, detection of puberty using daily boar contact began and their subsequent estrous periods were synchronized with oral progestogen (altrenogest). On day 3 after altrenogest withdrawal, blood samples were obtained from 6 gilts per sex bias group at 10 min intervals from 0900 to 2100 h to determine LH pulse amplitude and frequency. From 0900 on day 4, all 19 gilts were sampled every 4 h until the end of estrus to characterize LH surge dynamics. There were no differences between groups in LH pulse characteristics. Compared to gilts from female-biased litters, the LH surge in gilts from male-biased litters was delayed [56.00 ± 3.32 h vs. 43.11 ± 3.76 h (mean ± standard error of the mean (SEM)), P < 0.05], the duration was decreased [29.78 ± 2.12 h vs. 37.71 ± 1.19 h (mean ± SEM), P < 0.05] and the total secretion as measured by area under the curve was decreased (91.42 ± 9.52 ng/mL vs. 120.28 ± 9.48 ng/mL, P < 0.05). Our results indicate that a male-biased uterine environment has different effects on the tonic secretion of LH than the LH surge, with only some elements of the LH surge being affected.

Progesterone administration does not acutely alter LH pulse secretion in the mid-follicular phase in women.

It remains unclear how rapidly progesterone suppresses luteinizing hormone (LH) pulse frequency in women. Previous studies suggested that progesterone markedly increases LH pulse amplitude but does not slow LH pulse frequency within 10 h in estradiol‐pretreated women studied during the late follicular phase. However, this experimental paradigm may be a model of preovulatory physiology, and progesterone may have different effects at other times of the cycle. We studied regularly cycling, nonobese women without hyperandrogenism to assess the acute effect of progesterone during the midfollicular phase and in the absence of estradiol pretreatment. The study involved two admissions in separate cycles (cycle days 5–9). For each admission, either oral micronized progesterone (100 mg) or placebo was administered at 0900 h in a randomized, double‐blind fashion. Frequent blood sampling was performed between 0900 and 1900 h to define 10‐h LH pulsatility. Treatment crossover (placebo exchanged for progesterone and vice versa) occurred in a subsequent cycle. After an interim futility analysis, the study was halted after 7 women completed study. Mean progesterone concentrations after placebo and progesterone administration were 0.5 ± 0.1 (mean ± SD) and 6.7 ± 1.6 ng/mL, respectively. Compared to placebo, progesterone was not associated with a significant difference in 10‐h LH pulse frequency (0.79 ± 0.35 vs. 0.77 ± 0.28 pulses/h, P = 1.0) or amplitude (3.6 ± 2.8 vs. 4.3 ± 2.8 IU/L, P = 0.30). This study suggests that LH pulse frequency is not rapidly influenced by progesterone administration during the midfollicular phase.