Pituitary LH Research Articles

P-577 A randomized trial to compare the live birth rate following the progestin-primed ovarian stimulation versus the antagonist protocol in women with anticipated high ovarian responses

Abstract Study question Is there difference in the live birth rate following the progestin-primed ovarian stimulation (PPOS) versus the antagonist protocol in women with anticipated high ovarian responses? Summary answer The live birth rates of the first frozen embryo transfer following PPOS and the antagonist protocol were comparable in women with anticipated high ovarian responses. What is known already Progestin when given orally inhibits the pituitary LH surge during ovarian stimulation when taken orally i.e. progestin-primed ovarian stimulation. Two randomized trials showed similar number of oocytes for PPOS and the antagonist protocol in oocyte donation cycles. However, there is still no randomized trial to compare the live birth rate between PPOS and the antagonist protocol. Study design, size, duration This is a randomized controlled trial of 784 infertile women conducted from June 2020 and October 2021. Participants/materials, setting, methods Infertile women aged <43 years undergoing the first IVF cycle in a tertiary fertility center and having antral follicle count >15 randomly assigned in a 1:1 ratio into two groups: PPOS group and the antagonist group. Medroxyprogesterone 10 mg daily was given from the start of ovarian stimulation until the day of ovulation trigger in the PPOS group. The primary outcome was the live birth rate of the first frozen embryo transfer. Main results and the role of chance 784 women were randomly assigned into two groups: PPOS group (n = 392) and antagonist group (n = 392). Embryo transfer was either cancelled or postponed in 62 (62/392, 15.8%) women in the PPOS group and 65 (65/392, 16.6%) in the antagonist group because of no transferable embryos or no frozen embryo transfer within 6 months following randomization. The two groups were similar in demographic characteristics and number of oocytes obtained/fertilized, the number of cleaving embryos, number of good quality embryos at day 3, number of blastocysts developed and number of embryo/blastocyst frozen. There was no statistically significant difference in the live birth rate of the first frozen embryo transfer cycle between the progestin-primed ovarian stimulation and antagonist groups based on both intention to treat [37.5.0% (147/392) versus 32.7% (128/392), RR 1.148 (95% CI = 0.949–1.390), P = 0.16] and per protocol analysis [44.5% (147/330) versus 39.1.% (128/327), RR 1.138 (95%CI = 0.950–1.364), P = 0.16]. Both groups showed comparable clinical pregnancy, ongoing pregnancy, miscarriage, multiple pregnancy, ectopic pregnancy and cumulative live birth rates. Limitations, reasons for caution Women with anticipated high ovarian responses were recruited and the results may not be extrapolated to the general population seeking IVF. This is not a blind study although the live birth rate as the primary outcome is objection. The duration of the study is 6 months since randomization. Wider implications of the findings In situations in which fresh embryo transfer is not required such as oocyte/embryo freezing for fertility preservation, oocyte donation or preimplantation genetic testing, the PPOS protocol may be preferred over the antagonist protocol. Trial registration number NCT04414761

Expression of genes related to gonadal development and construction of gonadal DNA methylation maps of Trachinotus blochii under hypoxia

Chronic hypoxia can affect the growth and metabolism of fish and potentially impact gonadal development through epigenetic regulation. Trachinotus blochii (Golden pompano) is widely cultured near the coast and is sensitive to low oxygen conditions. We found that hypoxia and reoxygenation processes acted on multiple targets on the HPG axis, leading to endocrine disorders. Changes in the expression of key genes in the brain (gnrh), pituitary (fsh and lh), ovaries (cyp19a1a, foxl2, and er), and testes (dmrt1, ar, sox9, and gsdf) were associated with significant decreases in estrogen and testosterone levels. Hypoxia and reoxygenation lead to changes in DNA methylation levels in the gonads. Hypoxia upregulated the expression of dnmt1, dnmt3a, dnmt3b, tet1, and tet2 in females and dnmt3a and dnmt3b in males, while reoxygenation down-regulated the expression of dnmt1, dnmt3a, dnmt3b, tet1, and tet2 in males. Whole genome methylation sequencing showed that the number of differentially methylated regions was highest on chromosome 10 (5192) and lowest on chromosome 24 (275). Differentially methylated genes in females and males, as well as between males and females, were enriched in the oxytocin signaling pathway, fatty acid metabolism pathway, and HIF-1a pathway. In summary, hypoxia and reoxygenation can induce endocrine disorders, affect the expression of HPG axis genes, change the methylation pattern and modification pattern of gonad DNA, and then have potential effects on gonad development.

Endocrine patterns associated with ovarian development in female Pacific halibut (Hippoglossus stenolepis)

The Pacific halibut (Hippoglossus stenolepis) is a large migratory demersal flatfish species that occupies a top trophic role in the North Pacific Ocean and Bering Sea ecosystems, where it also supports various fisheries. As a first attempt to characterize the endocrine mechanisms driving sexual maturation in this important species, we collected pituitary, ovarian and blood samples from Pacific halibut females captured in the wild that were classified histologically into various female developmental stages. We conducted gene expression analyses of gonadotropin beta subunits in the pituitary and observed that mRNA expression levels of fshb gradually increased throughout vitellogenesis, remained elevated until before ovulation and declined after spawning. In contrast, the mRNA expression levels of lhb markedly increased during oocyte maturation and remained elevated until after spawning. Ovarian mRNA expression levels of the gonadotropin receptor genes fshr and lhr peaked during oocyte maturation and before spawning, respectively, immediately following the developmental stage at which pituitary fshb and lhb mRNA expression first reached maximum levels. The ovarian gene expression patterns of steroidogenic enzyme genes cyp19a1 and hsd20b2 paralleled those of fshr and lhr, respectively. Testosterone and 17β-estradiol (E2) plasma levels increased concomitantly with fshr and cyp19a1 mRNA expression levels, and vitellogenin plasma levels increased throughout vitellogenesis and reached maximum levels prior to spawning. These results are consistent with the notion that in female Pacific halibut, as in other teleosts, vitellogenesis and oocyte maturation and ovulation are likely under the control of pituitary gonadotropic hormones Fsh and Lh, respectively.

Function of secretoneurin in regulating the expression of reproduction-related genes in ovoviviparous black rockfish (Sebastes schlegelii).

Secretoneurin (SN), a conserved peptide derived from secretogranin-2 (scg2), also known as secretogranin II or chromogranin C, plays an important role in regulating gonadotropin in the pituitary, which affects the reproductive system. This study aimed to clarify the mode of action of scg2 in regulating gonad development and maturation and the expression of mating behavior-related genes. Two scg2 cDNAs were cloned from the ovoviviparity teleost black rockfish (Sebastes schlegelii). In situ hybridization detected positive scg2 mRNA signals in the telencephalon and hypothalamus, where sgnrh and kisspeptin neurons were reported to be located and potentially regulated by scg2. In vivo, intracerebral ventricular injections of synthetic black rockfish SNa affected brain cgnrh, sgnrh, kisspeptin1, pituitary lh and fsh and gonad steroidogenesis-related gene expression levels with sex dimorphism. In vitro, a similar effect was found in primary cultured brain and pituitary cells. Thus, SN could contribute to the regulation of gonadal development, as well as reproductive behaviors, including mating and parturition.

Effect of altrenogest on endogenous progesterone during early pregnancy in recipient mares

Altrenogest is commonly utilised for hormonal support of pregnancy, with undefined effects. We investigated effects of altrenogest supplementation on endogenous progesterone (P4) production in embryo recipient mares (RM). RM were managed onsite and selected on the basis of ‘best tone' on transfer day. Thirty RM received a 150mg depot altrenogest intramuscularly from transfer, at 7-day intervals, through to 42 days (ALT), a further 31 RM received no altrenogest (CON). Blood was collected at 7-day intervals, starting 7 days after transfer and P4 assayed by amplified enzyme-linked immunoassay. Data were analysed by total (tP4, ng/ml) and P4 corrected by number of corpora lutea (CL; cP4). Statistical comparisons of non-parametric data were performed by Kruskal-Wallis ANOVA and Friedman repeated measures; values presented as median and 95% confidence interval. Odds ratios (OR) were calculated to evaluate associations between ALT and secondary ovulation, and tP4 values of <3ng/ml. In ALT, tP4 was lower on days 21, 28 and 35 when compared to days 14 and 42 (p<0.01), and no difference was seen between days 14 and 42 (p=0.715). The same pattern was seen with cP4 lower on days 21, 28 and 35 compared to days 14 and 42 (p<0.01), and no difference between days 14 and 42 (p=0.273). A similar profile was seen in CON, with tP4 loweron days 21, 28 and 35 compared to days 14 and 42 (p<0.01) and no difference between days 14 and 42 (p=0.095). However, compared to day 14, cP4 was lower on days 21, 28, 35 (p<0.01), and also on day 42 (p=0.026). When compared to days 21, 28 and 35 there were no differences with day 42 (p>0.10). Secondary CL occurred in 17.24% CON and in 26.67% ALT (OR 1.55; 0.45-5.28, p=0.487). Total P4 was lower in ALT 4.8 (3.9-6.3), compared with CON 6.0 (5.4-7.3, p=0.04), and in ALT 4.7 (3.9-5.3), compared with CON 5.7 (5.1-7.4, p=0.02) at 21 and 28 days respectively. This held true for cP4 at 21 (ALT 3.7; 2.8-4.3 versus CON 4.7; 3.7-6.6, p=0.03) and 28 days (ALT 3.4; 2.5-4.3 versus CON 5.0; 4.0-5.8, p=0.005) where effect of multiple ovulations was nullified. In 40% of ALT, tP4 was <3.0 at ≥1 time-point compared to 3.2% in CON. The odds of a mare having a tP4 concentration <3.0 at any time-point was greater for ALT (OR 11.2; 1.37-91.83, p=0.024). Three ALT (10%) and no CON had tP4 ≤3.0 at 42 days (p=0.212). In conclusion, altrenogest led to suppressed endogenous P4 production with the greatest magnitude at 28 days of gestation; we hypothesize that this is mediated via negative feedback of altrenogest on pituitary LH release and effective reduction of its luteotrophic influence.

LETROZOLE COMPARED WITH CLOMPIHENE CITRATE FOR POLYCYSTIC OVARIAN SYNDROME : A SYSTEMATIC REVIEW

According to the Consensus on the Management of PCOS, polycystic ovary syndrome, is an endocrine condition that produces oligo-anovulation, clinical and biochemical symptoms of hyperandrogenism, and distinctive ovarian morphological characteristics on ultrasonographic examination. PCOS is also associated with a higher risk of endometrial cancer. Both hyperandrogenism and anovulation are difficult conditions to manage. PCOS women often have higher GnRH pulsatility, which in turn causes increased pituitary LH secretion and an elevated LH/FSH ratio. Granulosa cells are responsible for the conversion of androgens to estrogen and the maturation of follicles, while LH is responsible for promoting androgen synthesis in theca cells. Intraovarian androgens produce atresia in later antral stages, although they do boost initial follicle recruitment by increasing preantral and early antral follicle growth. In contrast to CC on its own, letrozole was shown to increase the number of live births, and the overall level of evidence was moderate. There was insufficient evidence that there was a difference in the rate of live births between CC plus metformin and CC alone, and the overall certainty of the evidence was poor due to the risk of bias and imprecision. When contrasted with the use of CC by itself, the potential benefit of CC in combination with metformin was more apparent in women whose baseline blood insulin or HOMA-IR values were higher than average. In comparison to clomiphene citrate, letrozole was related with higher rates of ovulation, pregnancy, and successful delivery of a live baby. Despite the fact that the quality of the data is inconsistent, one study recommends letrozole over clomiphene citrate as an ovulation induction medicine for women who have infertility and PCOS.

Toward an optimal contraception dosing strategy.

Anovulation refers to a menstrual cycle characterized by the absence of ovulation. Exogenous hormones such as synthetic progesterone and estrogen have been used to attain this state to achieve contraception. However, large doses are associated with adverse effects such as increased risk for thrombosis and myocardial infarction. This study utilizes optimal control theory on a modified menstrual cycle model to determine the minimum total exogenous estrogen/progesterone dose, and timing of administration to induce anovulation. The mathematical model correctly predicts the mean daily levels of pituitary hormones LH and FSH, and ovarian hormones E2, P4, and Inh throughout a normal menstrual cycle and reflects the reduction in these hormone levels caused by exogenous estrogen and/or progesterone. Results show that it is possible to reduce the total dose by 92% in estrogen monotherapy, 43% in progesterone monotherapy, and that it is most effective to deliver the estrogen contraceptive in the mid follicular phase. Finally, we show that by combining estrogen and progesterone the dose can be lowered even more. These results may give clinicians insights into optimal formulations and schedule of therapy that can suppress ovulation.

OR08-1 PROP1 Is Not Essential For Pituitary Cells Terminal Differentiation

Abstract Introduction The pituitary gland controls several mechanisms as metabolism, growth, and reproduction, in response to hypothalamic stimuli. The adequate temporal/ spatial expression of transcription factors is mandatory for a normal pituitary development. PROP1 transcription factor is widely known as a key pituitary regulator. Pathogenic variants in the PROP1 gene are the main familial causes of hypopituitarism accounting for about 54% of the cases. Aim To characterize the expression of genes involved in pituitary cells determination and specification during pituitary high demand period such as sexual maturation compared to adulthood in the Ames mice. Methods Weight and naso-anal length were measured. Pituitary glands were collected from 5 wild type (WT) and 5 prop1 mutant (Mut) animals with 30 (P30), 40, (P40), and 60 (P60) days after birth. RT-qPCR was performed to analyze the pituitary transcription factors Sox2, Sox3, Hesx1, Otx2, and genes codifying the hormones GH, TSH, LH/ FSH, CGA, and PRL by SYBR® Green (QIAGEN, Valencia, CA). They were normalized by endogenous genes and performed in triplicate. The target genes relative quantification was performed using the mutant related to its age paired wild type as a calibrator and the results were expressed as fold change. Immunofluorescence of SOX2 (ab97959, Abcam, UK) (1: 1600) and pituitary hormones GH, LH, and FSH (1: 250) were done in pituitaries at P60 and its aged paired control, stained with propidium iodide (1: 1000) for nucleus and the secondary antibody Alexa FluorÒ488 (ab150077, Abcam, UK) (1: 1000). Results All mutant mice presented decreased weight and naso-anal length at the three analyzed periods. The expression of the pituitary stem/ progenitor cell marker Sox2 was increased at P30 and P60 and decreased at P40. Sox3 was increased at P30 and decreased at P40 and P60. Hesx1 was increased at P30 and P60 and decreased at P40. Otx2 was increased at all periods. At P30 the genes codifying pituitary hormones was decreased, except for Tsh and Fsh. At P40 it was observed increased expression of the genes codifying Gh, Lh, Fsh and Prl, while Tsh and Cga expression was reduced. At P60 all pituitary hormones codifying genes presented decreased expression. The immunofluorescence at P60 showed increased expression of SOX2, similar expression of FSH, and decreased expression of GH and LH related to their age paired WT. Conclusion Despite the absence of PROP1, Hesx1 is downregulated at P40. Pituitary cell subtype differentiation and hormonal production is observed at transcriptional and protein levels, showing that pituitary cells can differentiate and produce hormones in the absence of Prop1. The recovery of pituitary capacity to differentiate in mutant suggests the involvement of another factor in the pituitary cell differentiation pathway that could compensate the lack of Prop1. Presentation: Saturday, June 11, 2022 11:30 a.m. - 11:45 a.m.

Cloning of gonadotropin Gph-alpha, FSH-beta and LH-beta subunits and seasonal profiles of steroid hormones in wild-caught Nile perch, Lates niloticus

The Nile perch (np; Lates niloticus) is a freshwater teleost species with a potential for aquaculture in freshwater surroundings. However, wild-caught breeders have persistently failed to spawn spontaneously in captivity. Cloning of the gonadotropin subunits and analysing seasonal variation in reproductive hormone levels for a 1-year period were done to gain knowledge on the physiological basis underlying the reproductive biology of np. The β-follicle-stimulating hormone (FSH-β) and β-luteinizing hormone (LH-β) subunits and their common α-glycoprotein (Gph-α) subunit were cloned using 3′ and 5′ RACE-PCR. The nucleotide sequences of the npgph-α, npfsh-β, and nplh-β subunits were 664, 580 and 675 nucleotides in length, encoding peptides of 124, 120 and 148 amino acids, respectively. The deduced amino acid sequence of each mature subunit showed high similarity with its counterparts in other teleost. Sequence analysis showed that npFSH-β is more similar to higher vertebrate FSH-βs than to higher vertebrate LH-βs. Heterologous immunoassay was calibrated to analyse pituitary LH levels. While the LH immunoassay showed parallelism of npLH with that of tilapia (ta), no parallelism for FSH was found. Levels of pituitary LH were higher in females at gonadal stages of vitellogenic oocytes, mature secondary oocytes and mature tertiary oocytes with migrating nucleus than in pre-vitellogenic oocytes and early and late perinucleolus oocytes. Using competitive steroid ELISA, variations in the levels of the steroid hormones 11-ketotestosterone (11-KT) in males and E2 in females were characterized in relation to month and reproductive index of Nile perch. Our findings show that in females, gonadosomatic index and plasma E2 were highly correlated (R2 = 0.699, n = 172) and peaked from September to November while in males, the gonadosomatic index and plasma 11-KT peaked from October to November. In female fish, both steroid hormones were detected in the plasma but greatly varied in concentrations. E2 in particular, increased with the developmental stage of the gonads. The levels of steroid hormones, E2 and 11-KT in females and males respectively increased with fish size (total lengths) and suggest that females mature at a body length of 40–59 cm than their counter part males that mature at a total length of 60–70 cm. Taken together, we describe seasonal endocrine differences in wild-caught adult Nile perch which could potentially be exploited to manipulate the reproductive axis in cultured breeders.

89 Does progesterone block the nerve growth factor–induced luteinizing hormone surge in llamas?

The central inhibitory effects of progesterone on gonadotrophin secretion have been well documented in several species, including camelids. Nerve growth factor (NGF) in seminal plasma triggers ovulation in camelids and is thought to act at the level of the hypothalamus. The objective of the study was to determine the effect of progesterone on NGF-induced LH release in llamas. In Experiment 1, llamas were assigned to a low, medium, or high progesterone group (n=4 per group). The low progesterone group consisted of non-mated (non-ovulatory) llamas, the medium progesterone group consisted of mated llamas (luteal phase; 3–4 weeks pregnant), and the high progesterone group consisted of non-mated llamas given a single intramuscular (IM) dose of progesterone (300mg IM, Progesterone BioRelease LA). A jugular catheter was placed, and the following day llamas were given an intravenous dose of 1mg of NGF isolated from seminal plasma. Blood samples were taken every 30min from 1h before to 5.5h after NGF treatment. In Experiment 2, the pituitary LH response to gonadotrophin-releasing hormone (GnRH) was compared between llamas treated with either Progesterone BioRelease LA or saline (n=4 per group). Sixteen hours later, llamas in both groups were given 50µg of gonadorelin (GnRH) IV, and blood samples were collected by jugular puncture at 0.5h before and 0, 1, 2, and 4h after GnRH. Blood samples were centrifuged, and plasma was stored frozen until radioimmunoassay for LH and progesterone. Data are presented as mean±s.e.m. Data were analysed by one-way ANOVA for single-point and repeated-measures, and independent or paired t-tests. In Experiment 1, plasma progesterone concentrations in the low, medium, and high progesterone groups were 0.6±0.3, 8.2±0.4, and 14.9±1.2ng mL−1, respectively, at the time of NGF treatment (P&amp;lt;0.05). Circulating concentrations of LH did not differ among progesterone groups (treatment, P=0.49; time, P&amp;lt;0.01; treatment×time interaction, P=0.65). In all groups, LH concentrations were elevated within 30min of NGF administration, reached a peak by 2h, and remained elevated beyond the sampling period. Comparison of samples collected during the pretreatment period (i.e. −60, −30, and 0min), however, revealed that plasma LH concentrations in the high progesterone group were half that of the low and medium progesterone groups (P&amp;lt;0.03). In Experiment 2, plasma progesterone concentrations in the progesterone- and saline-treated groups were 12.7±2.2 and 1.3±0.3ngmL−1, respectively (P&amp;lt;0.01). Despite the difference in circulating progesterone concentrations, the LH response to GnRH treatment was not different between groups (treatment group, P=0.43; time, P&amp;lt;0.01; treatment×time interaction, P=0.84). Results demonstrate a suppressive effect of progesterone on basal LH release in llamas, but no suppressive effect on NGF- or GnRH-induced surge release from the pituitary gland. Results are consistent with the hypothesis that the site of action of NGF is downstream of the hypothalamic site of action of progesterone. This research was supported by NSERC Canada.

Di-n-butyl phthalate diminishes testicular steroidogenesis by blocking the hypothalamic-pituitary-testicular axis: relationship with germ cell apoptosis in Japanese quail.

Although di-n-butyl phthalate (DBP) induces germ cell apoptosis, the underlying mechanism is not yet clear in quail. In this study, prepubertal quails were given a single dose of 500mg kg-1 DBP by gavage and were then killed 3, 6 and 24h after treatment. There was a significant reduction in intratesticular testosterone (ITT) concentrations and testicular steroidogenic enzyme mRNA expression and a significant increase in germ cell apoptosis in DBP-treated compared with control quails at all time points. Maximum apoptosis was detected 6h after treatment and the maximum reduction in testosterone concentrations was at 3h. To investigate whether DBP suppressed testicular steroidogenesis by affecting the hypothalamic-pituitary-testicular axis, we analysed pituitary LH subunit β (Lhb) mRNA expression and serum LH concentrations. At all time points, pituitary Lhb expression and serum LH concentrations were significantly decreased following DBP treatment. The present observations suggest the possibility that DBP blocked LH secretion from the hypothalamus and/or pituitary, thereby decreasing LH stimulation of Leydig cells and reducing ITT concentrations. DBP-induced decreases in ITT concentrations may cause changes to the physical structure of Sertoli cells, which, in turn, may induce germ cell apoptosis.

Control of follicular development during and after lactation in sows

Summary. Follicular development during early lactation in the sow is characterized by a large population of small-sized follicles and a small population of medium-sized follicles. As lactation progresses there is a gradual shift in number of follicles into medium- or large-sized categories and the percentage of follicles classified as atretic declines. Weaning at birth often leads to aberrant follicular development, apparently because the positive feedback response of LH to oestradiol does not occur during the first week post partum. Secretion of LH during lactation is primarily controlled by suckling intensity of the litter while FSH is controlled by a nonsteroidal ovarian factor, presumably inhibin. Suckling apparently limits secretion of GnRH and weaning leads to an increase of GnRH within the hypothalamus coincident with an increase of LH in the anterior pituitary and plasma and increased follicular growth. Follicular development during both lactation and post-weaning anoestrus can be stimulated by exogenous gonadotrophins and pulsatile administration of GnRH. Factors such as nutrition, season, boar exposure, litter size and split weaning affect follicular development during lactation and after weaning, probably because they affect secretion of LH.

Generation and use of recombinant gonadotropins in fish

Understanding the differential roles of the pituitary gonadotropins Fsh and Lh in gonad maturation is crucial for a successful manipulation of the reproductive process in fish, and requires species-specific tools and appropriate active hormones. With the increasing availability of fish cDNAs coding for gonadotropin subunits, the production of recombinant hormones in heterologous systems has gradually substituted the approach of isolating native hormones. These recombinant hormones can be continually produced without depending on the fish as starting material and no cross-contamination with other pituitary glycoproteins is assured. Recombinant gonadotropins should be produced in eukaryotic cells, which have glycosylation capacity, but this post-translational modification varies greatly depending on the cell system, influencing hormone activity and stability. The production of recombinant gonadotropin beta-subunits to be used as antigens for antibody production has allowed the development of immunoassays for quantification of gonadotropins in some fish species.The administration in vivo of dimeric homologous recombinant gonadotropins has been used in basic studies and as a biotechnological approach to induce gametogenesis. In addition, gene-based therapies using somatic transfer of the gonadotropin genes have been tested as an alternative for hormone delivery in vivo. In summary, the use of homologous hormonal treatments can open new strategies in aquaculture to solve reproductive problems or develop out-of-season breeding programs.

SAT-242 Isolated Adrenocorticotropic Hormone Deficiency Secondary to the PD-1 Inhibitor Pembrolizumab

Background: Immune checkpoint inhibitors (ICI’s) are now indicated in the treatment of several solid tumors and have improved the prognosis of patients with advanced malignancy. The expanded use of ICI’s has led to the rise of otherwise rare autoimmune sequelae. The overall incidence of ICI-induced autoimmune hypophysitis has increased to approximately 10%, but is only estimated to be 0.4% with PD-1 inhibitors specifically (1). Isolated ACTH deficiency from immune checkpoint inhibitors is rare, and very few cases secondary to the PD-1 inhibitor pembrolizumab have thus far been reported. Clinical Case: A 75-year old woman with history of Stage IV lung adenocarcinoma presented to oncology clinic in July 2019 with progressive weakness, weight loss, and confusion for several weeks. She was found to be hypotensive and dehydrated in the clinic and subsequently was admitted to the hospital. A comprehensive infectious work-up was non-contributory. History revealed that after treatment failure with carboplatin, she was treated with pembrolizumab from January 2017 to June 2019 with excellent response. Laboratory evaluation on admission demonstrated an undetectable AM cortisol level of < 1 ug/dL (n 5-25 ug/dL) with concomitant ACTH < 5pg/mL (Roche cobas, n 7.2-63 pg/mL), consistent with central adrenal insufficiency. Testing of the remainder of the pituitary axis, including TSH (0.83uU/mL, n 0.4-4.6 uU/mL), FSH (34.7 mIU/mL, n <150mIU/mL), LH (12.6mIU/mL, n <60 mIU/mL), and IGF-1 (33ng/mL, n 34-245ng/mL), all returned within normal limits. Further chart review verified that she had not been exposed to any form of glucocorticoids within the past 6 months. MRI brain with contrast demonstrated no obvious pituitary disease. The patient was started on 5mg of prednisone daily, with significant improvement in mental status, appetite, and blood pressure. She was discharged home on maintenance prednisone for adrenal insufficiency due to presumed isolated corticotroph destruction. Conclusions: Isolated ACTH deficiency is a very rare but potential consequence of pembrolizumab use. It can be especially difficult to diagnose in patients on chemotherapy who are at higher risk for dehydration and failure to thrive. Duration of pembrolizumab therapy should not preclude the diagnosis of isolated ACTH deficiency, as it can occur even as late as 2.5 years into therapy. Reference: (1) Chang, L., Barroso-Sousa, R., Tolaney S., Hodi F.S., Kaiser, U.B., Min, L. Endocrine Toxicity of Cancer Immunotherapy Targeting Immune Checkpoints, Endocr Rev. 2019;40;17–65.

SAT-268 Thyrotropin Secreting Pituitary Adenoma Initially Misdiagnosed as Primary Hyperthyroidism in a Taiwanese Man

Background:TSH (Thyrotropin) secreting pituitary adenoma (TSHoma) account for less than 1% of all causes of hyperthyroidism and 1% of all functioning pituitary tumors. Definite diagnosis and treatment of TSHoma are clinical challenges in practice. Here we report laboratory data, imaging findings, endocrine dynamic test, and treatment outcomes in a 50-year-old Taiwanese man with pituitary plurihormonal adenoma secreting TSH and LH.Clinical case:The patient was initially diagnosed as goiter with primary hyperthyroidism and DM while medical check-up by primary care physician in 2014. He had no significant hyperthyroidism symptoms and signs except goiter and mild palpitation. He received propylthiouracil and Metformin. Two years later, he visited to Endocrinologist’s clinic for poor glycemic control. Central hyperthyroidism was diagnosed due to measurable TSH level in the presence of increased serum thyroid hormone level. Moreover sella MRI revealed left sided pituitary lesion. He was referred to Taipei Veteran General Hospital for further management. There was no family history of thyroid disease. Physical examination was not remarkable except diffuse grade 3 goiter and tachycardia (HR 100~115 bpm). Follow up laboratory data showed TSH 4.89; range 0.4~4.0 uIU/ml, free T4: 3.05; range 0.9~1.8 ng/dl, T4: 16.02; range 4.50~12.50 μg/dl, T3: 249; range 58~159 ng/dl, free T3: 8.0; range 2.3~4.3 pg/ml. Two times of TRH stimulation test showed blunted TSH response. Normal limit of thyroid autoantibodies level were found. Thyroid sonography revealed heterogenous echogenicity with increased size and vascularity of both lobes. I-131 uptake was homogenous uptake (94%). Other pituitary hormones level were within normal limit except mild elevation of testosterone 12.69 ng/ml. Sella MRI with contrast showed macroadenoma (size 10x10x7.6 mm) at left pituitary gland. Taken together, he was diagnosed as central hyperthyroidism related to left sided pituitary macroadenoma. Surgery was performed after one year of definite diagnosis due to personal reason. TSH level returned to normal ranges (0.799 uIU/ml) in 1st post operative day. Histologically, the pituitary mass was compatible with plurihormonal adenoma and immunohistochemistry showed positivity for TSH (4+) and LH (3+). Post operative condition was well. Antithyroid agent was discontinued after operation. His blood glucose became well controlled after operation.Clinical lessons:A biochemical hallmark of TSHoma is an escape of TSH from the feedback loop that is detectable TSH levels in the presence of increased serum thyroid hormone level. Diagnosis of TSHoma was frequently unrecognized and thus much delayed despite its relatively straightforward. Physician should keep in mind that the importance interpretation of simple laboratory tests to avoid delay diagnosis and unnecessary treatments.

SAT-LB79 Biotin Depleting Procedure Does Not Impact the Reliability of TSH, LH and FSH Immunoassays

Background: Biotin interference has become a major problem with some laboratory immunoassays, leading to erroneous tests results and potentially harmful clinical consequences. There is therefore a clinical need to easily identify the interference and to overcome its effect to avoid harmful consequences for patients. The aim of this study was to demonstrate the VeraPrep BiotinTM procedure, a biotin depleting device, does not impact the immunoreactivity of TSH, LH and FSH immunoassays.Materials and methods: Nine samples (4 from women and 5 from men) were tested with two-sites electrochemiluminescent immunoassays (Cobas e602, Roche diagnostics) for the measurement of three pituitary hormones, TSH, LH and FSH. The samples were first measured without any treatment (baseline) and then retested after treatment with VeraPrep Biotin™ procedure. Percent (%) difference was calculated between post‐treatment and baseline to determine if the biotin depleting device impact the immunoassays immunoreactivity.Results: The median baseline concentrations for TSH, LH and FSH were 2.3 mIU/L (standard deviation (SD): 1.5), 4.9 IU/L (19.0) and 5.3 IU/L (17.5), respectively. The median concentrations after treatment with the VeraPrep Biotin™ procedure for TSH, LH and FSH were 2.3 mIU/L (1.5), 4.9 IU/L (18.9) and 4.9 IU/L (16.5), respectively, and were not statistically different from baseline (P = 0.976). The mean recoveries after treatment were 99.5%, 98.6% and 95.4% for TSH, LH and FSH, respectively.Conclusions: The concentrations of the TSH, LH and FSH assays were not impacted by the pretreatment with the VeraPrep Biotin™ procedure, confirming its ability to help clinical laboratories to overcome biotin interference.

REGOLAZIONE DELLA FERTILITÀ: ARTE E NEUROSCIENZE

The term ‘fertility’ (from Latin language getting abundant harvests) leads back to several fields of the sciences; however, when it is linked to the physiology of reproduction it became, along with nutrition, as a fundamental behavior for the living species. The ‘mystery’ of human reproduction has been an attractive issue and extensively represented in figurative arts (paintings, sculptures, ect.) throughout the centuries and different cultures, being itself an ‘art’. Many scientists have been so fascinated by the art to have modified their personal and scientific lives. Art and sciences, in particular the neurosciences, share their origin during human history, being both the expression of human abilities to formulate an abstract thought to understand and describe the complexity of the world around us. For instance, paintings as the Cabanel’s Birth of Venus as well as the Primavera by Botticelli summarize several aspects of the sexual desire and reproduction; however, the neurosciences provide a more complex and artistic picture of the control of fertility. From the first descriptions, by Geoffrey Harris and Luciano Martini, of the control of the hormonal cascade governing reproductive functions by the hypothalamus, an important region of our brain, it came to the identification of the GnRH, the master regulator of hormonal reproductive axis. GnRH is produced by specific hypothalamic neurons and it is released in the blood vessels directed toward the pituitary gland in a pulsatile manner, finely tuned for rhythm and intensity, to regulate the secretion of pituitary gonadotropins LH and FSH. Released gonadotropin will exert trophic and regulatory effects on gonadal functions and on the production of sex hormones (testosterone and estrogens). A number of neuroendocrine signals control the development and the activity of GnRH neurons and whose functional alterations are responsible of several reproductive system diseases. According to the data so far available, it is fair to say that the development of such complex mechanisms, under the brain control, endowed with the extraordinary ability to create new individuals could be a real artwork.

Divergent expression patterns of pituitary gonadotropin subunit and GnRH receptor genes to continuous GnRH in vitro and in vivo

Continuous, as opposed to pulsatile, delivery of hypothalamic gonadotropin-releasing hormone (GnRH) leads to a marked decrease in secretion of pituitary gonadotropins LH and FSH and impairment of reproductive function. Here we studied the expression profile of gonadotropin subunit and GnRH receptor genes in rat pituitary in vitro and in vivo to clarify their expression profiles in the absence and continuous presence of GnRH. Culturing of pituitary cells in GnRH-free conditions downregulated Fshb, Cga, and Gnrhr expression, whereas continuous treatment with GnRH agonists upregulated Cga expression progressively and Gnrhr and Fshb expression transiently, accompanied by a prolonged blockade of Fshb but not Gnrhr expression. In contrast, Lhb expression was relatively insensitive to loss of endogenous GnRH and continuous treatment with GnRH, probably reflecting the status of Egr1 and Nr5a1 expression. Similar patterns of responses were observed in vivo after administration of a GnRH agonist. However, continuous treatment with GnRH stimulated LH secretion in vitro and in vivo, leading to decrease in LH cell content despite high basal Lhb expression. These data suggest that blockade of Fshb expression and depletion of the LH secretory pool are two major factors accounting for weakening of the gonadotroph secretory function during continuous GnRH treatment.

Gnrh1-Induced Responses Are Indirect in Female Medaka Fsh Cells, Generated Through Cellular Networks.

Reproductive function in vertebrates is stimulated by GnRH that controls the synthesis and release of the two pituitary gonadotropins, FSH and LH. FSH and LH, which regulate different stages of gonadal development, are produced by two different cell types in the fish pituitary. This is in contrast to the situation in mammals and birds, and it enables investigation of their differential regulation. In the present study, we used fluorescence in situ hybridization to show that Lh cells in adult female medaka express Gnrh receptors, whereas Fsh cells do not. This result was confirmed by patch-clamp recordings and by cytosolic Ca2+ measurements on dispersed pituitary cells, where Lh cells, but not Fsh cells, responded to Gnrh1 by biphasic alteration in action-potential frequencies and cytosolic Ca2+ levels. In contrast, both Fsh and Lh cells are able to respond to Gnrh1 in brain-pituitary tissue slices both electrically and by elevating the cytosolic Ca2+ levels. Using Ca2+ uncaging in combination with patch-clamp recordings and cytosolic Ca2+ measurements, we show that Fsh and Lh cells form homotypic and heterotypic networks in the pituitary. Taken together, these results show that the effects of Gnrh1 on Fsh release in adult female medaka are indirect and probably mediated via Lh cells.

SRXN1 Is Necessary for Resolution of GnRH-Induced Oxidative Stress and Induction of Gonadotropin Gene Expression.

A defining characteristic of the hypothalamus-pituitary-gonad reproductive endocrine axis is the episodic secretion of the pituitary gonadotropin hormones LH and FSH by the anterior pituitary gonadotropes. Hormone secretion is dictated by pulsatile stimulation, with GnRH released by hypothalamic neurons that bind and activate the G protein-coupled GnRH receptor expressed by gonadotropes. Hormone secretion and synthesis of gonadotropins are influenced by the amplitude and frequency of GnRH stimulation; variation in either affects the proportion of LH and FSH secreted and the differential regulation of hormone subunit gene expression. Therefore, proper decoding of GnRH signals is essential for appropriate gonadotropin synthesis and secretion. The GnRH receptor robustly activates downstream signaling cascades to facilitate exocytosis and stimulate gene expression and protein synthesis. It is necessary to rapidly quench signaling to preserve sensitivity and adaptability to changing pulse patterns. Reactive oxygen species (ROS) generated by receptor-activated oxidases fulfill the role of rapid signaling intermediates that facilitate robust and transient signaling. However, excess ROS can be detrimental and, unchecked, can confuse signal interpretation. We demonstrate that sulfiredoxin (SRXN1), an ATP-dependent reductase, is essential for normal responses to GnRH receptor signaling and plays a central role in resolution of ROS induced by GnRH stimulation. SRXN1 expression is mitogen-activated protein kinase dependent, and knockdown reduces Lhb and Fshb glycoprotein hormone subunit mRNA and promoter activity. Loss of SRXN1 leads to increased basal and GnRH-stimulated ROS levels. We conclude that SRXN1 is essential for normal responses to GnRH stimulation and plays an important role in ROS management.