bluuebunny/crossref_metadata_2025 huggingface dataset:
Early Phenotypes in Girls at Risk for PCOS Replicate Metabolic and Reproductive Subtypes: An Unsupervised Clustering Analysis
Authors: Torchen Laura Christine , Sisk Ryan , Ratnam Sarayu , Dunaif Andrea Elizabeth
Published: May 2021

Both daughters of women with PCOS (PCOS-d) and overweight girls (OW-g) are proposed to be at increased risk of PCOS because they have peripubertal increases in testosterone (T) levels, a cardinal feature of PCOS. We are testing this hypothesis by performing longitudinal studies in these girls after menarche. In adult women with PCOS, we have recently identified reproductive and metabolic subtypes using unsupervised cluster analyses. These subtypes were associated with novel PCOS susceptibility genetic loci suggesting that the subtypes reflect biologically discrete entities. We performed similar analyses in our cohort of early postmenarchal PCOS-d and OW-g to test the hypothesis that these subtypes are present in girls at risk for PCOS.

Fifteen PCOS-d and 10 OW-g aged 11-16 years and with postmenarchal age less than 2 years were studied. Mothers of PCOS-d fulfilled NIH criteria for PCOS, mothers of OW-g were reproductively normal with no history of irregular menses or clinical hyperandrogenism. OW-g had a BMI above the 85th percentile for age. There was no BMI inclusion criterion for PCOS-d; four PCOS-d had a BMI above the 85th percentile. The girls were of comparable age, post-menarchal age and BMI z score. A fasting morning blood sample was drawn for T, SHBG, DHEAS, glucose and insulin. Leuprolide 10 mcg/kg SC was administered. LH and FSH levels were measured at baseline, 30 min, and 60 min following leuprolide.

Unsupervised hierarchical cluster analysis adjusted for age was performed on quantitative traits including BMI, T, fasting insulin, fasting glucose, DHEAS, SHBG, and LH and FSH. These are the same quantitative traits used for clustering in adult PCOS. The clustering revealed 2 distinct PCOS subtypes: a reproductive group (41%), characterized by higher SHBG levels, LH and FSH with relatively low BMI and insulin levels, and a metabolic group (41%), characterized by higher BMI and insulin levels and lower SHBG, LH, and FSH. Jaccard coefficients indicated cluster stability (0.70 reproductive, 0.69 metabolic). There was a significant difference in the distribution of the two subgroups in PCOS-d and OW-g: PCOS-d 60% reproductive, 13% metabolic, 27% indeterminate; OW-g 25% reproductive, 50% metabolic, 25% indeterminate (Chi Sq P=0.05).

We found that early postmenarchal PCOS-d and OW-g demonstrate reproductive and metabolic subtypes similar to those identified in adult women with PCOS. The majority of PCOS-d had the reproductive subtype. These findings suggest that this subtype, which is characterized by disordered gonadotropin secretion, is an early harbinger of PCOS. Longitudinal studies are ongoing to test this hypothesis.
Mechanisms Involved in Intrauterine Growth Restriction in Patients With Polycystic Ovary Syndrome: Primary and Secondary Prevention of Metabolic Syndrome and Cancer Risk in the Adult Life of Offspring
Authors: Mugnolo Domingo , Giraldo Erica , Lana Maria Perez , Campeni Susana Beatriz
Published: May 2021

Polycystic ovary syndrome (PCOS) is one of the most common hormonal disorders that affects between 5- 10% of women of reproductive age. It is currently considered a complex and multifactorial disease with metabolic, cardiovascular implications and represents per se an increased cancer risk. PATIENTS with PCOS routinely have menstrual disorders, hyperandrogenism, infertility and reproductive complications such as recurrent abortions, gestational diabetes, intrauterine growth restriction, pregnancy induced hypertension that give rise to underweight newborns and condition metabolic diseases to adult life and increased risk of cancer, especially breast and endometrial cancer. Insulin resistance and hyperandrogenism are the most important etiopathogenic factors in PCOS. On the other hand, subjects exposed to an adverse microenvironment in the intrauterine stage develop compensating responses to survive, a process called fetal programming. Prenatal exposure to androgens and/or insulin resistance may act as fetal programming factors and cause restriction of intrauterine growth, obesity and insulin resistance in offspring. Newborn may have an increased risk of metabolic syndrome, increased incidence of hypertensive, type 2 diabetes, heart disease and cerebrovascular disease. Prevention of these complications will be achieved if women with Polycystic Ovary Syndrome are treated appropriately throughout their lives, but especially before and during their pregnancy. Only in this way can the risk of them be reduced, representing a better quality and greater life expectancy.
A Re-Examination of the oCRH Stimulation Test for the Diagnosis of Ectopic ACTH Secretion
Authors: Newman Andrew Joseph , McGlotten Raven , Nieman Lynnette K
Published: May 2021

Background: Previous studies of the oCRH test for the differential diagnosis of ACTH-dependent Cushing’s syndrome gave imprecise point estimates of specificity because of small numbers (n<20) of patients with ectopic ACTH secretion (EAS). We examined a large EAS population to re-assess the test’s performance and whether benign tumors were associated with a positive response (i.e. Cushing’s disease (CD). Methods: We evaluated 103 EAS patients, including 58 females, with median age of 38 years (range 10 - 83); 99 had surgically-proven tumors and four had biochemical testing consistent with EAS. ACTH and cortisol were measured 5 and 0 minutes before and 15, 30 and 45 minutes after administration of oCRH, 1 ug/kg up to 100 ug, iv. Previous criteria were used to determine EAS responses: mean ACTH increase of <34% at 15 and 30 min or mean cortisol increase of <20% at 30 and 45 min. Mean responses and UFC (expressed as fold increase above upper limit of normal, ULN) were compared with t-tests; a p-value of < 0.05 was considered significant. Results: Tumor types included NET (pulmonary n=67; with 4 spindle type and 5 tumorlets, thymic n=9, pancreas n=7, appendix n=1, prostate n=1), SCLC (N=3), pheochromocytoma/paraganglioma (n=3/1), metastatic without known primary (n=3), teratoma with corticotroph elements (n=1) and occult (n=4). 23 patients had “CD” responses that were significantly higher than those of non-responders: 11 with ACTH (mean+SD: 83.7 + 49.8% vs 1.2 + 18.8%, p=00025), 15 with cortisol (mean+SD: 34.7 + 8.9% vs -1.7 + 14.2%, p< 0.00001). Three patients responded to both ACTH and cortisol. Among the ACTH (only) responders, 6 had cortisol increases of < 10% (range 0-2-8.5%); a similar discordant response was seen among cortisol (only) responders, of whom 7 had ACTH increases of <20% (range -18 - 13.4%). 70% of responders, including 3 with both ACTH and cortisol responses, had pulmonary NET (n=13), spindle cell NET (n=2) or tumorlets (n=4). Other responders had thymic NET (n=3), appendiceal NET n=1, teratoma (n=1) or were occult (n=2). The patient with corticotroph elements in a teratoma had an ACTH response only. UFC fold-increases above ULN were similar in responders and non-responders (20.20 + 26.9 xULN vs 31.4 + 38.4 xULN, p =0.21). Four of 10 patients with UFC cyclicity responded to CRH. Conclusions: Among patients with EAS the rate of false positive responses to CRH was similar to that of initial reports, 11–15%, and was associated with hormonal cyclicity and presence of a thymic carcinoid. The marked discordance of ACTH and cortisol responses may result from reduced sensitivity to ACTH, less biologically active ACTH, or autonomous cortisol release.
Sleep Characteristics Among Women With and Without PCOS
Authors: Oppermann Karen , Hemkemeier Stéfanie Zamboni Perozzo , Reichert Ana Victoria , Weber Lais , Rinaldi Laura
Published: May 2021

Clinical studies indicate that sleep disorders, including obstructive sleep apnea (OSA) and excessive daytime sleepiness, occur more frequently among women with PCOS compared to comparison groups without the syndrome. The presence of OSA in PCOS is associated with worsening of metabolic parameters. There is some evidence that obesity directly contributes to OSA among women with PCOS, although, it does not fully account for findings from community-and clinic-based studies. Questionnaires are used as screening for sleep disorders. The objective was to verify the quality of sleep, the prevalence of OSA and daytime sleepness among women with PCOS compared to control group. The sample size calculation was based on estimates bad quality of sleep among women with PCOS in 80% and among control women in 45% (1). The sample with 58 women, 29 each group, had a power of 80%, with a significance level of 0.05. This is a cross sectional study with 29 patients with PCOS and 31 controls from Gynecology Endocrinology Ambulatory of São Vicente de Paulo Hospital, Passo Fundo, RS, Brazil, who consulted between January 2017 and March 2020. Women with PCOS by Rotterdam criteria and controls were under 40 years old and no pregnant. Controls women had regular cycles, no history of PCOS or hirsutism and had normal ovaries on transvaginal ultrasound. Age, BMI, blood pressure (BP), waist circumference (WC) were measured. It was applied the validated questionnaires of Pittsburgh Sleep Quality Index, to classify in good and bad sleep quality; Epworth Sleepiness Scale for daytime sleepiness and Berlim Questionnaire for evaluate sleep apnea risk. The mean of age was 30.6 ± 5.9, PCOS 29.1± 6.7 versus 32.3±4.7, p=0.06. The group of PCOS women was heavier (BMI 32.4±6.1versus 28.0 ± 5.3, p=0.04) and presented higher WC (101.3±16.1 versus 91.6 ±14cm, p=0.03). The mean of BP was similar between the groups. The prevalence of bad sleep quality was 53.6% for women with PCOS and 63.1% for controls (p=0.29). The daytime sleepiness was present in 14,5% of the women with PCOS and 35.7% of controls (p=0,061) and the sleep apnea risk was 32.1% for women with PCOS and 21.4% for controls (p=0.27). The association of risk of OSA was verified with robust multivariate Poisson. The prevalence ratio (PR) of BMI ≥ 30 was 1.820 (CI 1.281-2.587) p<0.001, BMI ≥ 25 1.549 (IC 1.067- 2.250) p=0.02, adjusted for age, WC and PCOS diagnosis. In conclusion, there was no difference in prevalence of quality of sleep, OSA risk or daytime sleepiness between women with PCOS and controls. The risk of OSA was higher in obese women independently of age, abdominal circumference and PCOS diagnosis.

Reference: (1) Fernandez et al., Nature and Science of Sleep 2018; 10: 45–64.
ANGPTL3 Levels in Healthy and Mild Preeclamptic Pregnant Women
Authors: Garces Maria F , Franco - Vega Roberto , Maldonado - Acosta Luis M , Castro - Pinzón Andres , Eslava-Schmalbach Javier , Parada - Banos Arturo J , Angel–Müller Edith , Nogueiras Rubén , Dieguez Carlos , Castaño Justo , Ruiz–Parra Ariel I , Caminos Jorge Eduardo
Published: May 2021

Introduction: Throughout normal pregnancy, different metabolic and hormonal adaptations are presented, among others, significant modifications in the profile of lipids and lipoprotein metabolism. On the other hands, Angiopoietin-like protein 3 (ANGPTL3) are involved in the regulation of triglyceride metabolism in the fed state by inhibiting the enzyme lipoprotein lipase in oxidative tissues. Objective: Thus, the objective of this study was to determine the profile of serum ANGPTL3 levels during three periods of gestation and three months after delivery. Design, setting and Participants: Serum ANGPTL3 levels were analyzed by ELISA, throughout pregnancy in a case-control study nested within a longitudinal prospective cohort of healthy pregnant (n = 52) and mild preeclamptic women (n = 20), women in the third month postpartum (n = 20) and healthy non-pregnant women (n = 20). The results obtained were correlated with biochemical, hormonal, and anthropometric variables. Results: A significant reduction in ANGPTL3 levels was observed from the first to the third trimesters of pregnancy in healthy and preeclamptic pregnant women when compared with healthy non-pregnant and postpartum women (p<0.01). There were no significant differences in serum ANGPTL3 levels between normal and preeclamptic women. Serum ANGPTL3 levels were positively correlated with triglyceride, very-low-density lipoprotein cholesterol, and total cholesterol levels in healthy non-pregnant (p<0.05); whereas there were no significant correlations between ANGPTL3 with the same variables in healthy and preeclamptic pregnant women. Besides, there were no significant correlations between serum ANGPTL3 with body mass index, high-density lipoprotein cholesterol, glucose, insulin, leptin or HOMA-IR in the study groups described above. Conclusions: The results of the present study show for the first time that ANGPTL3 could be playing a fundamental role in the homeostasis of lipid metabolism throughout gestation. Thus, low levels of ANGPTL3 during pregnancy might favor the accumulation of lipid in oxidative tissues as a deposit of maternal energy source, while preserving glucose and amino acids for the fetus.
BMP6 Mediates BMP2-Increased Human Trophoblast Invasion
Authors: Deng Jianye , Li Yan
Published: May 2021

TGF-β superfamily proteins play divergent roles in regulating human extravillous trophoblast (EVT) invasion and their coordinated effects are essential for adequate placentation during pregnancy 1. Bone morphogenetic protein 2 (BMP2), which belongs to the BMP subfamily of TGF-β superfamily, has been shown to promote human EVT invasion and the acquisition of endothelial-like phenotype 2,3. It has been reported that BMP2 promotes EVT invasion by up-regulating Activin A, a growth factor which also belongs to TGF-β superfamily. However, whether BMP6 mediates the pro-invasive effect of BMP2 has yet to be determined. Herein, we firstly treated immortalized trophoblast cells (HTR8/SVneo) with recombinant BMP2 protein for 6 and 24 hrs, and our bulk-RNA sequencing results demonstrated significantly increased BMP6 mRNA levels after BMP2 treatment. Furthermore, we confirmed the up-regulatory effects of BMP2 on BMP6 mRNA and protein levels in both HTR8/SVneo and primary EVTs isolated from first-trimester villi. Notably, siRNA-mediated down-regulation of BMP6 significantly attenuated both basal and BMP2-induced cell invasion in HTR8/SVneo cells as measured by Matrigel-coated transwell invasion assay. In summary, our results firstly demonstrated the up-regulatory effect of BMP2 on BMP6 expression in human trophoblasts and identified the mediation role of BMP6 in BMP2-promoted EVT invasion, suggesting the interplay between BMP subfamily members during EVT invasion regulation. Our ongoing research focusing the underlying molecular mechanisms and signaling pathways could further benefit the advancement of diagnostic and therapeutic strategies for EVT invasion dysregulation-related pregnancy disorders, e.g., pre-eclampsia. Reference: (1) Li Yan et al., Trends Endocrinol Metab 2021 18: S1043-2760(20)30266-6. (2) Hong-Jin Zhao et al., FASEB J 2020;34(2):3151-3164. (3) Hong-Jin Zhao et al., Cell Death Dis 2018;9(2):174.
Effects of Dexamethasone on Offspring Survival and Intrauterine Growth Restriction
Authors: El Habbal Noura , Mulcahy Molly C , Redd JeAnna R , Bridges Dave
Published: May 2021

The placenta is the primary organ responsible for deactivating maternal glucocorticoids and reducing fetal exposure. Glucocorticoid use during pregnancy is a common treatment for asthma, allergies, and COVID-19. Several studies have reported adverse effects including intrauterine growth restriction as a result of glucocorticoid exposure, yet little is known about the mechanisms by which short and long-term maternal glucocorticoid exposures affect placental biology and fetal development. To better understand the role of glucocorticoids on placental and fetal outcomes, we used a mouse model exposed to the synthetic glucocorticoid, dexamethasone (Dex), prior to and throughout gestation. We conducted a randomized controlled trial in mice with a treatment arm of Dex exposure and water exposure as control. Virgin C57Bl/6J female mice were single-housed at 11 weeks of age, and Dex was introduced in the drinking water as a 1mg/kg/day dose. After one week of treatment, mice were bred with age-matched virgin males. Dam body composition, food, and water intake were monitored weekly. Maternal insulin sensitivity, pup survival rate, litter size, and pup birth weight at postnatal day (PND) 0.5 were also assessed. Dams treated with Dex lost significant lean mass after one week of treatment. Dex treatment did not appear to affect the dams’ ability to get pregnant, as both groups carried pups to term with similar lengths of gestation (p=0.838). Water and Dex-treated dams gained comparable weight during the first and second trimesters of pregnancy, however, the Dex group gained less lean mass than the water group during the third trimester. At PND0.5, Dex dams had fewer pups with a 40% reduction in litter size (p=0.01) and lighter pups with a 37% reduction in offspring weight (p<0.001), indicating substantial intrauterine growth restriction. All pups of Dex-treated dams died by PND1. Attempts to rescue pups of Dex-dams by cross-fostering with water-treated nursing dams or by feeding the pups 10% glucose at PND0.5 failed by PND1. These results demonstrate a novel finding regarding the chronic use of glucocorticoids before and during conception and pregnancy. The reduction in both pup weight and late-pregnancy maternal weight gain suggests potential growth restriction or placental insufficiency. Further molecular studies during multiple time points of gestation will help elucidate the mechanisms at play.
Effects of Peripartal Sertraline on Maternal Calcium Metabolism and Maternal Bone Health
Authors: Sheftel Celeste M , Sartori Luma C , Manuel Robbie S J , Fricke Hannah P , Charles Julia F , Hernandez Laura L
Published: May 2021

Lactation taxes mothers’ bone health, causing them to lose up to 10% of their bone mass for milk calcium. Selective serotonin reuptake inhibitor antidepressants (SSRIs) are commonly used during pregnancy and lactation but independently decrease bone mass. We previously demonstrated that both maternal and offspring bone densities were compromised by maternal treatment with the SSRI fluoxetine throughout pregnancy and lactation.

In this study, we examined whether sertraline similarly affect maternal and offspring bone. Female C57BL/6 dams (n=23/group) were treated with sertraline (10 mg/kg/d) or a vehicle (DMSO) for 41 d (E0-lactation(L) 21). Pups were euthanized at weaning (L21). Dams were euthanized at peak lactation (L10, n=4/group), weaning (L21, n=9/group) or 3 mo post-weaning (n=10/group) for analysis.

Sertraline treatment did not affect dams’ weight gain during pregnancy or lactation and did not impact milk production/consumption, measured by weigh-suckle-weigh. Unlike fluoxetine, which increases circulating calcium, sertraline treatment reduced serum calcium concentration at L21 compared with control (p<0.05). Mammary calcium genes were not altered at L10, but at L21 we observed decreased mammary expression of the calcium transporters Orai1, Spca1, Spca2, and Serca2 (p<0.05), and a tendency for decreased Pthlh (a calcium-regulating peptide; p=0.08). In the intestinal duodenum, Calb1 (a cytosolic calcium trafficking protein) expression was increased at L10 and L21 (p<0.05), and the calcium transporters Trpv6, Pmca1 and Spca2 were increased by L21 (p<0.05). Sertraline treatment did not affect L21 calcium concentration in either tissue, although the mammary gland calcium was 3.5 times that of the duodenum.

Previously, we reported that circulating markers of bone formation (P1NP) or bone resorption (CTX) were unchanged in sertraline-treated dams at L21. This is unlike fluoxetine, which decreased P1NP without altering CTX. Furthermore, compared with control, sertraline treatment did not alter expression of bone resorption or formation genes (e.g. Trap, Rank/RankL, Alp, Runx2) in maternal femoral bone at L21 or 3 mo post-weaning.

Interestingly, in utero exposure to sertraline reduced litter size (5.4 vs 6.8 pups/dam, p<0.01) and increased pup mortality (20% vs 5% dead pups/litter, p<0.01) during the first 24 hours postpartum compared with controls, but did not affect length of gestation or live birth rate. Exposure to sertraline during lactation did not alter weight gain in the surviving offspring, nor did it alter circulating calcium in the pups at weaning, suggesting an adequate supply of calcium in the milk from the dam.

These data suggest that, compared with high-dose fluoxetine, low-dose peripartal sertraline has a milder impact on maternal bone and may reduce mammary calcium but could induce a failure to thrive in the offspring.
Aromatase Inhibitors in Erectile Dysfunction
Authors: Singh Santosh
Published: May 2021

Hyperestrogenism may cause erectile dysfunction(ED) by impeding normal penile development,increasing venous vascular permeability and leakage(via VEGF) and by inhibiting testosterone(T)production. Estrogen excess can impair spermatogenesis and may increase the risk of estrogen-sensitive cancers(viz.breast cancer)[1,2]Weekly and biweekly letrozole(2.5 mg),an aromatase inhibitor,has been reported to normalize serum T in males with obesity related hypogonadism and poor sperm quality,respectively.[3,4]A 40-year old insulin requiring,normotensive,obese(BMI-26.9),diabetic(12 years duration) was evaluated for ED.T was 187 ng/dl and estradiol(E2) was 69 pg/ml(normal-11-44 pg/ml) with normal LH and prolactin levels. There was normalization of T(increased to 487 ng/dl) with 2.5 mg letrozole every 3 weeks. Another patient,55 year old male, insulin requiring,hypertensive,obese(BMI-28.8),diabetic(21 years duration) had normalization of T(401 ng/dl) with baseline low T(224.4 ng/dl) and normal E2(33 pg/ml) with T-E2 ratio of less than 10,with weekly 2.5 mg letrozole. There was one kg weight gain and 0.2 ng/ml increase in PSA in two years. These cases highlight the significance of estimating both T and E2 in the evaluation of ED. Moreover,it also highlights the efficacy and safety of weekly and even every 3-week 2.5 mg letrozole therapy.

References: 1. .Schulster M,Bernie AM,Ramasamy R:The role of estradiol in male reproductive function.Asian Journal of Andrology,2016,18(3),435-440. 2. Goldfrank LR and Flomenbaum N:Goldfrank’s Toxicologic Emergencies,McGraw- Hill Professional,2006,p443. 3.Loves S,Ruinemans-Koerts,de Boer H:Letrozole once a week normalizes serum testosterone in obesity-related male hypogonadism. Eur J Endocrinol,2008,158,741-747.4. Singh SK:Aromatase inhibitors in male sex. Indian J Endocrinol Metab,2013,17,S259-261.
Aldosterone-Producing Adenoma: Does a Larger Tumor Secrete More Apparent Cortisol?
Authors: Xu Felicity , Yang Shumin , He Wenwen , Li Qifu
Published: May 2021

Previous studies revealed that aldosterone-producing adenoma(APA) had a potential for excess co-secretion of cortisol. APA was considered to be composed of heterozygous cell types, including cortisol producing cells(expression of CYP11B1) and aldosterone-producing cells(expression of CYP11B2). The proportion of two cell types and the expression of two steroid synthases showed great heterogeneity in APAs, which may lead to differences in cortisol secretion. However, the hypothesis has not been tested. Therefore, we examined the correlation between tumor size and the ability to autonomously secrete cortisol of APA from the perspective of steroid hormones and immunohistochemistry. 165 patients with surgically proven APA were retrospectively studied. Patients with apparent excess cortisol co-secretion were excluded, which was confirmed by an overnight 1 mg dexamethasone suppression test(1mg-DST). Immunohistochemical staining was performed in 88 tumor specimens and CYP11B2-negative APAs were excluded. The expression of the steroidogenic enzymes was immunohistochemically analyzed, and semi-quantified according to the McCarty H-score system. All APA cases were classified into 2 groups according to the median(15mm) of maximum diameter detected by histological examination: the smaller(n=102) and larger group(n=63). 24am cortisol[78.7(47.7–121.5) VS. 54.6(33.6–81.4) nmol/l, p =0.002] and cortisol after 1mg-DST[28.8(19.1–40.9) VS. 24.1(16.8–30.7) nmol/l, p =0.024] were significantly higher in the larger APA group. The larger APA group had a higher H score of CYP11B1 (p=0.028) and a lower H score of CYP11B2 (p=0.036). Both 24pm cortisol (r =0.2424, p =0.0058)and cortisol after 1mg-DST(r =0.2476, p =0.042)were positively correlated with tumor diameter. The tumor diameter was positively correlated with the H score of CYP11B1 (p=0.0328) and inversely with the H score of CYP11B2(p=0.0285). We tentatively used a new ratio B1 index, calculated by H score of CYP11B1 divided by the sum of H score of CYP11B2 and CYP11B1, to represent the relative expression intensity of CYP11B1 in tumor specimens. B1 index had a stronger correlation with tumor diameter (r =0.3134,p=0.0041). Cortisol after 1mg-DST increased linearly with the tertiles of tumor diameter(p for trend<0.0001). These results suggested that APA without clinically overt cortisol secretion might have a higher proportion of cells expressing CYP11B1 to produce more cortisol with the increasing of tumor size, even developing to overt Cushing syndrome.
Measurement of Free Testosterone in Serum Using Equilibrium DialysisCoupled With ID-UHPLC-MS/MS: Comparison Between Equilibrium Devices
Authors: Zhou Hui , Ribera Ashley , Dabbs-Brown Amonae , Danilenko Uliana , Vesper Hubert W
Published: May 2021

Free testosterone (FT) has been used as a biomarker in clinical patient care and public health research to assess and manage patients with androgenic abnormalities. The latest Endocrine Society clinical practice guideline for testosterone therapy in men with hypogonadism recommends measuring FT for those with borderline and low total testosterone concentrations, or those who have conditions that change SHBG concentrations, such as some metabolic or hormonal diseases, certain medication use, or SHBG genetic polymorphisms. Measuring FT is technically challenging and shows high variability. The CDC clinical standardization program is developing a high throughput method using the gold-standard equilibrium dialysis (ED) procedure with isotope dilution ultra-high-performance liquid chromatography tandem mass spectrometry (ID-UHPLC-MS/MS).

A serum sample was dialyzed against a protein-free HEPES buffer (pH 7.4) at 37 °C until equilibrium. After isolating endogenous FT from protein-bound testosterone by ED, isotope-labeled internal standard (13C3-testosterone) was added to the dialysate for quantification. Certified pure primary reference material (National Measurement Institute M914) was used to prepare calibrators, enabling traceable quantitation and ensuring measurement trueness. FT was further isolated from the dialysate matrix using supported liquid extraction and a chromatographic separation from interfering compounds and quantitation by tandem MS. The dialysis step requires maintaining the endogenous free hormone equilibrium so that results in dialysate reflect FT concentrations in the blood without influence from dilution, temperature, or pH. The dialyzer system has a 1:1 sample-to-buffer volume and has been used in reference measurement procedures for free hormone measurements, serving as the standard for method performance comparison. Four commercially available devices designed for high throughput in a multiple well-plate format, requiring respective sample-to-buffer ratios, were evaluated for their recovery, speed, ease of automation by a liquid handling system, repeatability, and robustness. Preliminary results showed that a device with 1:1 sample-to-buffer volume had the most comparable results to those obtained from the standard dialyzer, with the mean bias less than 15%. The device with the highest sample-to-buffer ratio showed bias as high as 50%. These data suggest that controlling sample-to-buffer ratio is a critical step in ED FT method.
Recurring Painful Ectopic Gynecomastia in a Young Male - A Case Report
Authors: Pescador Antonio Ochoa , Ramos Celeste Ong
Published: May 2021

Painful lumps in the axillary area are relatively common and could normally be brought about by several etiologies, more commonly, lipomas, fibroadenoma, hidradenitis suppurativa, lymphoma, or breast cancer. However, recurring painful ectopic gynecomastia in the axillary area of a male patient is of rare occurrence with only few reports in the literature. Here, we report a case of a 25 year old male, who presented to our clinic due to recurring painful right axillary mass. He denied any prior history of trauma, infection, breast mass, or previous lymphadenopathy, decrease in libido nor erectile dysfunction. The mass appeared to be truly subcutaneous at the interface of skin between the superior axilla and the medial arm. Breast exam did not reveal any palpable masses nor abnormalities. Ultrasonography of the right axillary region revealed findings that may represent an accessory axillary breast tissue and histological evaluation revealed an accessory breast tissue with gynecomastia. For such cases, individual treatment requirements can range from simple reassurance to medical treatment or even surgery, all depending on the possible etiology. Due to the diversity of possible etiologies, performing a careful history and physical examination is imperative and the need for hormonal evaluation is warranted to be able to arrive at a certain diagnosis.
A Novel Mouse Model for Studying the Effects of Cyp17 Overexpression in a Temporal- and Spatial-Specific Manner
Authors: Secchi Christian , Belli Martina , Stupack Dwayne , Shimasaki Shunichi
Published: May 2021

Background: Cyp17 plays a key role in theca cells (TCs) to produce androgens, which, in turn, are converted to estrogens in granulosa cells. Intrinsic alterations in ovarian steroidogenesis contribute to excessive ovarian androgen production that characterizes polycystic ovary disease (PCOS)1,2. Hyperandrogenism has been associated with higher levels of Cyp17 in TCs, and correlate with increased numbers of antral follicles3. While androgen excess is one of the hallmark features of PCOS, its putative role in the follicular development and function remains poorly known. Most efforts have used androgen administration or Cyp19 blockade approach to study how androgens prolong folliculogenesis4. Although some insights have been made, it is not clear if these models accurately address the cascade of effects that follow ovarian hyperandrogenism. Aim: Here, we aim to study the specific effects of hyperandrogenemia on ovarian morphology, follicle function and fertility with a new transgenic (TG) mouse model expressing elevated Cyp17 levels exclusively in TCs. Methods: We generated a breeding line of triple TG mice using a combination of the Tet-dependent expression system and the Cre/LoxP gene control system. Specifically, we used Cyp17 promoter-iCre mice crossed with trans-activator mice (R26-STOP-rtTA-IRES-EGFP transgene, Jackson Lab) and with a responder mouse carrying the TRE-Cyp17 transgene. Cyp17 promoter-iCre mice were used to ensure rtTA/EGFP is expressed specifically in TCs of secondary follicles. After the DNA segment between the two LoxP sites is excised by Cyp17iCre specifically in TCs, the R26-STOP-rtTA gene remains activated in all daughter TCs. Only upon treatment with Doxycycline (DOX) can suppression be relieved and active transcription of TRE-Cyp17 be induced in a dose-dependent manner. Results: Cyp17 mRNA expression levels in TCs of TG mice treated with 20, 100 or 200 mg/Kg DOX compared with corresponding untreated control mice showed a modulation in a dose-dependent manner (P=0.01 ANOVA). Confocal and RNAscope analysis validated (i) the effective combination of the Cyp17iCre/rtTA expression system visualizing the rtTA/EGFP specifically expressed in ovarian TCs and (ii) the DOX-induced increase of Cyp17 expression compared with the WT mice. DOX treated TG females were acyclic, being mostly arrested in diestrus. Analysis of estrous cycle stages revealed that treated TG females spent significantly more time in diestrus than control females (P=0.007, ANOVA). Conclusions: Our new in vivo model is the first that analyzes androgen impact independent of any extraovarian source of androgen, complementing current clinical efforts to study the occurrences of TCs elevated androgen levels in normal and PCOS women. 1 Rosenfield, R. L. et al. Endocr Rev (2016)2 Azziz, R. et al. Nat Rev Dis Primers (2016)3 Comim, F. V., et al. Hum Reprod (2013)4 Stener-Victorin, E. et al. Endocr Rev (2020)
Analysis of BMP15-Induced Transcriptome in Human Granulosa Cells for the Identification of Novel Candidate Genes for Primary Ovarian Insufficiency
Authors: Rossetti Raffaella , Fornili Marco , Moleri Silvia , Ferrari Ilaria , Gentilini Davide , Paffoni Alessio , Somigliana Edgardo , Biganzoli Elia , Marozzi Anna , Brancati Francesco , Moretti Costanzo G , Giuliani Cristina , Persani Luca
Published: May 2021

Primary Ovarian Insufficiency (POI) is a female fertility disorder which affects 1% of women before 40 years of age and manifests with amenorrhea, elevation of serum gonadotrophins and low estrogens. POI has a strong genetic component with incomplete penetrance. Several candidate genes have been described so far, however, its etiopathogenesis is mostly unknown. In order to discover the POI-related causative mechanisms, microarray transcriptome analysis in human granulosa cells (hGCs) stimulated with recombinant human BMP15 (rhBMP15) and next generation sequencing analysis (NGS) on the identified differentially expressed genes in a selected group of patients with POI were conducted on NGS Illumina platform. In the present study, we obtained 19 differentially expressed genes upon rhBMP15 stimulation in hGCs. Results: showed that all identified genes were upregulated and associated to pluripotency, inhibition of apoptosis, cell proliferation, BMP signaling and apoptosis. Moreover, we identified nine POI patients bearing six rare variants in 5 of the BMP15-induced genes (SAMD11, SMAD6, ID1, USP35, GPCR137C). The BMP15-induced transcriptome analysis in hGCs contributed the understanding of BMP15 role as transcriptional regulator, through the activation of transcriptional repressors, by inducing pathways inhibiting the ovarian follicle maturation, thus possibly maintaining an undifferentiated state of hGCs. These findings lead to the identification of novel candidate genes for POI.
Genetic Sex Effects of Polycystic Ovary Syndrome Reveal Distinct Metabolic Etiology
Authors: Actkins Ky’Era , Edwards Digna R Velez , Aldrich Melinda , Davis Lea K
Published: May 2021

Females with polycystic ovary syndrome (PCOS) have an increased risk of developing metabolic disorders such as insulin resistance, obesity, and type 2 diabetes (T2D). High-risk groups or individuals with a family history are more likely to have a greater genetic susceptibility to these diseases, which drastically increases their risk of developing other chronic health conditions. In this study, we systematically evaluated the bidirectional genetic burden of PCOS and its comorbidities among females and males. First, we analyzed the pleiotropic effects of the PCOS polygenic risk score (PRS), a measurement of genetic liability to PCOS, across 1,857 medical conditions recorded in the Vanderbilt University Medical Center electronic health record. We conducted a phenome-wide association study (PheWAS) adjusted for median age, sex, and genetic ancestry. In the European sex-combined model (n = 72,824), we observed that PCOS PRS was significantly (Bonferroni corrected p < 7.86e-06) associated with T2D (OR = 1.11, p = 8.75e-08) and hypertension (OR = 1.06, p = 1.13e-07) in addition to polycystic ovaries (OR = 1.11, p = 1.91e-07). In the sex-stratified model, we found that males (n = 32,022) with a higher PRS for PCOS were more likely to develop cardiovascular diseases (CVD) compared to females (n = 40,802) who had higher odds of developing T2D. Although we were underpowered to detect any phenome-wide significant effects in our African descent sample (n=15,283), uterine leiomyoma (OR = 1.24, p = 3.79e-03), osteoarthritis (OR = 1.21, p = 3.94e-03), and benign neoplasm of uterus (OR = 1.24, p = 4.00e-03) were the top three nominal significant results (p < 0.05) in females (n = 9,418). To understand the genetic relationships observed in the PheWAS, we used LD score regression to determine the genetic correlation between the phenotypes. We found that PCOS was positively correlated with T2D (rg = 31%), systolic blood pressure (rg = 12%), and pulse pressure (rg = 15%). However, we found no significant associations between the genetic risk of CVD and PCOS diagnosis in the European or African descent samples. Our findings show that the genetic architecture of PCOS has distinct metabolic sex differences, but the genetic risk of those comorbidities is not predictive of a PCOS diagnosis. This suggests that other drivers are contributing to the endocrine and cardiovascular comorbid signatures that underlie PCOS.
Gonadotropin Dependent Neuregulin1 Signaling Regulates Luteal Cell Survival
Authors: Jones Jennifer , Banerjee Saswati , Thompson Winston E , Chowdhury Indrajit
Published: May 2021

The formation of a functional corpus luteum (CL) is an absolute requirement for reproductive success and is induced by the mid-cycle surge of luteinizing hormone (LH). The CL is a transient ovarian endocrine structure that maintains pregnancy in primate during the first trimester and in rodents during the entire pregnancy by producing steroid hormone progesterone (P4). CL growth and differentiation are tightly regulated by both survival and cell death signals, including endocrine (LH), intra-ovarian regulators, and cell-cell interactions. Neuregulin-1 (NRG1) is a member of the epidermal growth factor-like factor family that mediates it’s effect through the erythroblastoma (ErbB) family. However, the detailed mechanisms associated with the interplay of NRG1 and its receptors in CL function is not known. Therefore, we examined the role and action of NRG1 and its receptors in the gonadotropin signaling pathway that impacts CL functions. Immunocolocalization of NRG1 and ErbB2/3 in pregnant rat CL on day 14 and 21 suggest that both NRG1 and ErbB2/3 are differentially expressed in CL. Moreover, both NRG1 and ErbB2/3 are highly expressed in rat CL on day 14 compared to day 21. Furthermore, in vitro studies revealed that rat luteal cells (LCs) treated with exogenous tumor necrosis factor-α (TNFα, an inflammatory cytokine) promoted apoptosis in LCs in a dose and time-dependent manner. However, the effects of TNFα was attenuated in presence of exogenous NRG1. Under these experimental conditions, immunoblot analysis indicated that exogenous TNFα treatment in the presence of NRG1 inhibits apoptosis through increased levels of the anti-apoptotic proteins Bcl2 and Bclxl, and activation of ErbB2-ErbB3-PI3K-Akt signaling pathway. Collectively, these studies provide new insights on the NRG1-mediated anti-apoptotic mechanism in LCs through ErbB3-ErbB2-PI3K-Akt→Bcl/Bcl-xL pathway and may have important clinical implications.

Acknowledgements: This study was supported in part by National Institutes of Health Grants 1 SC1 GM130544-01A1, 1SC3GM113751 and G12RR03034. This research was conducted in a facility constructed with support from the Research Facilities Improvement Grant C06RR018386 from the National Institutes of Health National Center for Research Resources.
Molecular Background of Estrogen-Dependent and -Independent Gene Expressions in Endometriotic Lesions
Authors: Izawa Masao , Azuma Yukihiro , Taniguchi Fuminori , Harada Tasuku
Published: May 2021

Background: Endometriosis is an estrogen-dependent disease, and the role of estrogen is obvious because the symptoms associated with endometriosis often disappear after menopause, and GnRH agonists or progestin relieve the pelvic lesions and endometriosis-associated pain. However, there are limitations to these treatments that target the estrogen reduction in endometriotic lesions. As a possible background, we hypothesized a role of the local environment with high estrogen depending on aromatase upregulation in endometriotic lesions. Objective: To test our hypothesis, we re-evaluated the expression profile of estrogen receptor (ER), and then searched for the estrogen-dependent gene expressions in endometriotic cells. Finally, we approached the epigenetic background of gene expressions in endometriotic cells.

Patients: Institutional Review Boards approved this project. We obtained the informed consent from all patients. The chocolate cyst lining in ovaries of patients with endometriosis was the source of endometriotic tissue. As the control, the eutopic endometrial tissues were obtained from uteri of premenopausal women who had uterine leiomyoma. Methods: Stromal cells were prepared from endometriotic and endometrial tissues. Gene expression was evaluated using RT-PCR. Specific primer sets of unique 5’-UTR exons/exon 2 in ESR1 and specific primer sets of unique 5’-UTR exons/exon1 in ESR2 were used for the analysis of promoter usage. Primer sets of exon 7 and exon 8 in ESR2 were used to evaluate the expression of ERβisoform. Using SERM(PPT and DPN), ER-dependent gene expression was estimated. The potential function of hypomethylated gene sequence as an active enhancer was evaluated by ChIP analysis and eRNA expression. Results: 1) Relative expression of ERα mRNA in endometriotic cells was estimated to be one tenth of that in endometrial cells. 2) Relative expression of ERβ1 mRNA was 40-fold higher than that in endometrial cells, which is almost at a comparable level of the ERα. 3) In addition to ERβ1 mRNA, a splice variant ERβ2 was expressed at a comparable level of the ERβ1. 4) Top ten genes, up- or down-regulated in response to SERM, were extracted in endometriotic cells. 5) TGFA expression was upregulated at a comparable level in response to PPT and DPN. 6) A stretch of hypomethylated sequence, which includes an ERE at 50kb upstream from the TSS, was suggested as active enhancer. 7) ESR1 and ESR2showed a marginal response to SERM. 8) GATA6 and CYP19 were highly expressed in endometriotic cells, and hypomethylated sequences in these genes were suggested as active enhancer. Conclusion: In the hope of overcoming the limitations of endocrine treatments in endometriosis, we examined ER-dependent and -independent gene expressions using endometriotic cells. The results suggest one aspect of gene expression in endometriosis lesions.
The Effects of Leptin and Irisin on Steroidogenic Enzyme Gene Expression in Human Ovarian Granulosa Cells - Initial Studies
Authors: Avtanski Dimiter Bogdanov , Ziskovich Karina , Singer Tomer , Yeshua Ariel , Cantor Tal , Poretsky Leonid
Published: May 2021

Fertility and energy metabolism are closely associated, and the cytokines produced by the adipose and muscle tissue play a role in this association. Leptin, predominantly produced by the white adipose tissue, and irisin, produced by the brown adipose and skeletal muscle tissues, are cytokines that are important in balancing energy metabolism. This study aimed to investigate the effects of leptin and irisin on steroidogenic enzyme gene expression in human ovarian granulosa cells in vitro. Granulosa cells were retrieved and isolated from ovarian follicular fluid during in vitro fertilization (IVF) procedures. Cells were placed in primary in vitro cultures and treated with increasing concentrations of leptin (25, 50, 100, 200, and 400 ng/ml) or irisin (125, 250, 500, 1,000, and 2,000 ng/ml) for 24, 48, and 72 hours. mRNA expression levels of CYP11A1, CYP19A1, CYP21A2, HSD3B1, and HSD17B3 were measured by qRT-PCR analysis. Leptin treatment of granulosa cells resulted in significant upregulation of CYP21A2 mRNA levels, while irisin significantly downregulated mRNA levels of CYP11A1, CYP19A1, and HSD3B1. Taken together, these early experiments demonstrate that leptin and irisin may affect steroid hormone production in the ovary by targeting the gene expression of key steroidogenic enzymes. Additional experiments are in progress.
A Genetically Defined Male Counterpart of Polycystic Ovary Syndrome: Evidence for Ovarian-Independent Pathogenesis
Authors: Zhu Jia , Wittemans Laura Brigitte Leen , Lindgren Cecilia , Hirschhorn Joel N , Chan Yee-Ming
Published: May 2021

Background: Polycystic ovary syndrome (PCOS) is a heterogeneous condition that affects 6-10% of women of reproductive age. PCOS is often characterized by a triad of ovulatory dysfunction, hyperandrogenism, and cardiometabolic dysfunction. Both ovarian-related and ovarian-independent factors have been implicated in the pathogenesis of PCOS, but it remains to be determined which are the inciting events and which are the secondary consequences. Studies of male relatives of women with PCOS have proposed a male counterpart of PCOS, which suggests that PCOS is not always a primary disorder of female reproduction, but rather can be, at least in part, a condition of cardiometabolic dysregulation and hyperandrogenism, with ovarian dysfunction as a secondary consequence.

Methods: To investigate a genetically defined male counterpart of PCOS, we optimized a polygenic risk score (PRS) algorithm for predicting PCOS based on 206,851 unrelated women of European ancestry in the UK Biobank, then used this algorithm to calculate PCOS PRS for 176,360 men in the UK Biobank. We used logistic regression to calculate odds ratios for dichotomous outcomes by comparing men with high and low PRS (testing a variety of percentile cutoffs) and ANCOVA to compare continuous outcomes across deciles of PRS. All analyses were adjusted for age, age2, assessment center, genotyping array, and the first 10 principal genetic components to account for ancestry.

Results: Men who carried a high PCOS PRS (top 20%) had a 17% increased risk of obesity defined as BMI ≥30 kg/m2 (OR 1.17, 95% confidence interval [CI] 1.14-1.20, p=1.3x10-30), 15% increased risk of type 2 diabetes mellitus (OR 1.15, 95% CI 1.09-1.20, p=5.3x10-8), 5% increased risk of coronary artery disease (OR 1.05, 95% CI 1.01-1.09, p=0.03), and 5% increased risk for androgenic alopecia (OR 1.05, 95% CI 1.01-1.08, p=0.01). BMI, hemoglobin A1c, triglycerides, and the free androgen index all increased across deciles of the PRS, while HDL and SHBG decreased across PRS deciles (p all <0.001). The relationship between the PCOS PRS and coronary artery disease, HDL, and triglycerides appeared to be mediated by BMI. In contrast, the associations between the PCOS PRS and type 2 diabetes mellitus and hemoglobin A1c remained significant after adjusting for BMI, suggesting independent mechanisms of pathogenesis.

Conclusions: By demonstrating associations between PCOS genetic risk factors and cardiometabolic dysfunction and androgenic conditions in men, we have shown that these genetic risk factors can act independently of ovarian function. Thus, at least in some cases, the reproductive dysfunction of PCOS in women may arise secondarily from disruption of biological pathways common to both men and women. Future dissection of these biological pathways will further inform efforts to identify pathological mechanisms underlying PCOS.