Prenatal Androgen Research Articles

The association between the 2D:4D ratio and offspring sex at birth: A cross‐sectional study in Ghana

The 2D:4D ratio is the putative marker of prenatal androgen exposure. Low maternal 2D:4D ratio has been associated with high or male-biased secondary sex ratio. Hitherto, there has not been any study in Ghana regarding the maternal 2D:4D ratio and sex ratio at birth. This study sought to investigate this observation in a Ghanaian population. The study was cross-sectional from December 2020 to April 2021 involving 272 first-time mothers. The mean ± SD age of the mothers was 23.9 ± 3.67 years. The right (2D:4DR), the left (2D:4DL), the mean (M2D:4D) digit ratios and the right-left difference (Dr-l) of the mothers were measured using computer-assisted analysis. The mothers were stratified by their digit ratios, and the cohort sex ratio (CSR) for each stratum was then calculated as the proportion of sons. The mean ± SD of the 2D:4DR of mothers-with-daughters and mothers-with-sons were 0.941 ± 0.032 and 0.933 ± 0.037, respectively. The mean ± SD of the 2D:4DL of mothers-with-daughters was 0.934 ± 0.034, while that of mothers-with-sons was 0.931 ± 0.039. The offspring sex at birth and the CSR was not associated with either the mother's right, left, mean 2D:4D ratio or their difference (Dr-l). However, mothers-with-daughters showed rightward asymmetry in their 2D:4D ratios as the Dr-l significantly and positively deviated from zero (p < .010). The maternal 2D:4D ratio may not be associated with their offspring sex at birth among first-time mothers. This study adds to the limited data on studies regarding offspring sex at birth and the 2D:4D ratio in Ghana and Sub-Saharan Africa.

Chronic androgen excess in female mice does not impact luteinizing hormone pulse frequency or putative GABAergic inputs to GnRH neurons.

Polycystic ovary syndrome (PCOS) is associated with androgen excess and, frequently, hyperactive pulsatile luteinizing hormone (LH) secretion. Although the origins of PCOS are unclear, evidence from pre‐clinical models implicates androgen signalling in the brain in the development of PCOS pathophysiology. Chronic exposure of female mice to dihydrotestosterone (DHT) from 3 weeks of age drives both reproductive and metabolic impairments that are ameliorated by selective androgen receptor (AR) loss from the brain. This suggests centrally driven mechanisms in hyperandrogen‐mediated PCOS‐like pathophysiology that remain to be defined. Acute prenatal DHT exposure can also model the hyperandrogenism of PCOS, and this is accompanied by increased LH pulse frequency and increased GABAergic innervation of gonadotrophin‐releasing hormone (GnRH) neurons. We aimed to determine the impact of chronic exposure of female mice to DHT, which models the hyperandrogenism of PCOS, on pulsatile LH secretion and putative GABAergic input to GnRH neurons. To do this, GnRH‐green fluorescent protein (GFP) female mice received either DHT or blank capsules for 90 days from postnatal day 21 (n = 6 or 7 per group). Serial tail‐tip blood sampling was used to measure LH dynamics and perfusion‐fixed brains were collected and immunolabelled for vesicular GABA transporter (VGAT) to assess putative GABAergic terminals associated with GFP‐labelled GnRH neurons. As expected, chronic DHT resulted in acyclicity and significantly increased body weight. However, no differences in LH pulse frequency or the density of VGAT appositions to GnRH neurons were identified between ovary‐intact DHT‐treated females and controls. Chronic DHT exposure significantly increased the number of AR expressing cells in the hypothalamus, whereas oestrogen receptor α‐expressing neuron number was unchanged. Therefore, although chronic DHT exposure from 3 weeks of age increases AR expressing neurons in the brain, the GnRH neuronal network changes and hyperactive LH secretion associated with prenatal androgen excess are not evident. These findings suggest that unique central mechanisms are involved in the reproductive impairments driven by exposure to androgen excess at different developmental stages.

Prenatal Androgen Exposure and Traits of Autism Spectrum Disorder in the Offspring: Odense Child Cohort.

Fetal androgen exposure may be associated with autism spectrum disorder (ASD). We studied 1777 mother-child pairs in the prospective Odense Child Cohort. Prenatal androgen exposure was assessed by maternal 3rd trimester testosterone concentrations, maternal polycystic ovary syndrome (PCOS), and 3months offspring anogenital distance. ASD traits were assessed at age 3years with the ASD-symptom scale of the Child Behavior Checklist for ages 1½-5years. Maternal testosterone was positively associated with traits of ASD in boys (p < 0.05). Maternal PCOS was associated with increased offspring ASD traits (p = 0.046), but became non-significant after excluding parental psychiatric diagnosis. Offspring anogenital distance was not linked to ASD traits. Higher prevalence of ASD in boys could be linked to higher susceptibility to fetal androgen exposure.

Fully Masculinized 46,XX Individuals with Congenital Adrenal Hyperplasia: Perspective Regarding Sex of Rearing and Surgery.

Current guidelines for gender assignment for all 46,XX congenital adrenal hyperplasia (CAH) continue to be female. This decision is most challenging for individuals with a 46,XX karyotype born with (CAH) having severely masculin- ized genitalia (Prader 4 or 5). They may be at significant risk for quality of life (QoL) and psychological health. More outcome information currently exists for such individuals assigned male than female. Most available data for those raised females do not indicate the extent of masculinization at birth, so there are minimal outcome data to compare with those raised males. Gender dissatisfaction among those raised females may be related to the degree of prenatal androgen excess in the brain evidenced by external genital masculinization. Also, additional brain maturation after birth, especially during puberty, is impacted by postnatal androgen excess resulting from inadequate androgen sup- pression. The purpose of this perspective is to suggest that both female and male assignment be considered. Most who have been raised male at birth have positive adult outcomes. This consideration should occur after discussions with full disclosure to the parents. The lack of more outcome data highlights the need for further information. This perspective also suggests that surgery should be deferred whether assigned female or male at least until gender identity is apparent to preserve the potential for male sexual function and prevent irrevocable loss of sensitive erotic tissue. While the gender fluidity is recognized, it is important to consider potential subsequent need for gender reassignment and extent of masculinization, particularly at the time of gender determination.

Digit Ratio (2d:4d) in Patients with Antisocial Personality Disorder

Objective: The present study aimed to compare the 2nd finger to 4th finger ratio (2D:4D) of patients with antisocial personality disorder to the 2D:4D of a group of healthy subjects. Method: A total of thirty one male patients with antisocial personality disorder and the twenty-eight healthy controls were included in the present investigation. Finger (2D) and ring (4D) lengths and 2D:4D ratio of the subjects were determined. Results: In the present study, we found that patients with antisocial personality disorder had a significantly lower ratio of 2D:4D of both hands. Conclusion: In conclusion, we suggest that patients with antisocial personality seem to have a lower ratio of 2D:4D compared to healthy control subjects, leading us to think that higher prenatal gonadal androgens may be related to antisocial personality disorder.

Handgrip strength and 2D : 4D in women: homogeneous samples challenge the (apparent) gender paradox.

The length ratio between the second and the fourth digit (2D : 4D) is a retrospective, non-invasive biomarker for prenatal androgen exposure. It was found to be negatively correlated with handgrip strength (HGS) in men, but the evidence for women is mixed. Such studies in women call for increased detection sensitivity. The present study was designed to reduce potential confounding factors, especially age and ethnicity variation. We measured the digit ratios and HGS of 125 healthy women between 19 and 31 years of age from a remote region in Austria. 2D : 4D of both hands was significantly and negatively correlated with HGS (n = 125, right hand: r = –0.255, p = 0.002, left hand: r = –0.206, p = 0.011). Size, direction and significance of correlation coefficients remained stable when statistically controlling for age, body weight, body height, body mass index or hours of exercise per week. This yields theory-consistent evidence that HGS and 2D : 4D are clearly associated in women—when sufficiently reducing genetic variation (confounding 2D : 4D), the ontogenetic environment and age ranges (confounding HGS) in the study population. This finding implies similar organizing effects of prenatal androgens as in men, pointing to a more parsimonious developmental mechanism and a new look into its proximate and ultimate causes.

Evidence for Perinatal Steroid Influence on Human Sexual Orientation and Gendered Behavior.

In laboratory animals, exposure to gonadal steroid hormones before and immediately after birth can exert permanent effects on many behaviors, particularly reproductive behaviors. The extent to which such effects occur in humans remains an open question, but several lines of evidence indicate that perinatal levels of both androgens and estrogens may affect adult human psychology and behavior, including sexual orientation and gender nonconformity. Some putative indicators of prenatal androgen exposure, including the ratio of the length of the index finger to that of the ring finger (2D:4D), have repeatedly indicated that lesbians, on average, were exposed to more prenatal androgens than straight women, suggesting that sufficient fetal androgen exposure predisposes a fetus to gynephilia (attraction to women) at maturity. The digit ratios of gay men do not differ from those of straight men, suggesting that prenatal androgen levels are not responsible for their androphilia (attraction to men). However, evidence that gay men who prefer an insertive anal sex role (ASR) have more masculine digit ratios than those preferring a receptive ASR suggests that early androgens influence some sexual preferences in men. Furthermore, digit ratios among gay men have been found to correlate with recalled childhood gender nonconformity (CGN). People with isolated gonadotropin-releasing hormone (GnRH) deficiency (IGD) offer further insight into the effects of perinatal gonadal steroid exposure. In people with IGD, gonadal hormone production is low or absent after the first trimester of gestation. However, because placental gonadotropins drive gonadal hormone secretion during the first trimester when genitalia sexually differentiate, individuals with IGD are unambiguously male or female at birth, consistent with their chromosomal and gonadal sex. Men with IGD report greater CGN, again suggesting that perinatal androgen exposure contributes to male-typical behavioral patterns in humans. Interestingly, women with IGD report less androphilia and more bisexuality than control women, suggesting that perinatal ovarian steroids in females typically augment androphilia in adulthood. Taken together, these findings indicate that the perinatal hormonal milieu influences human sexual orientation and gender conformity.

Prenatal androgenization causes expression changes of progesterone and androgen receptor mRNAs in the arcuate nucleus of female mice across development.

Prenatal exposure to excess androgens is associated with the development of polycystic ovary syndrome (PCOS). In prenatally androgenised (PNA) mice, a model of PCOS, progesterone receptor (PR) protein expression is reduced in arcuate nucleus (ARC) GABA neurons. This suggests a mechanism for PCOS-related impaired steroid hormone feedback and implicates androgen excess with respect to inducing transcriptional repression of the PR-encoding gene Pgr in the ARC. However, the androgen sensitivity of ARC neurons and the relative gene expression of PRs over development and following prenatal androgen exposure remain unknown. Here, we used a quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) of microdissected ARC to determine the relative androgen receptor (Ar) and progesterone receptor (Pgr) gene expression in PNA and control mice at five developmental timepoints. In a two-way analysis of variance, none of the genes examined showed expression changes with a statistically significant interaction between treatment and age, although PgrA showed a borderline interaction. For all genes, there was a statistically significant main effect of age on expression levels, reflecting a general increase in expression with increasing age, regardless of treatment. For PgrB and Ar, there was a statistically significant main effect of treatment, indicating a change in expression following PNA (increased for PgrB and decreased for Ar), regardless of age. For PgrA, there was a borderline main effect of treatment, suggesting a possible change in expression following PNA, regardless of age. PgrAB gene expression changes showed no significant main effect of treatment. We additionally examined androgen and progesterone responsiveness specifically in P60 ARC GABA neurons using RNAScope® (Advanced Cell Diagnostics, Inc.) in situ hybridization. This analysis revealed that Pgr and Ar were expressed in the majority of ARC GABA neurons in normal adult females. However, our RNAScope® analysis did not show significant changes in Pgr or Ar expression within ARC GABA neurons following PNA. Lastly, because GABA drive to gonadotropin-releasing hormone neurons is increased in PNA, we hypothesised that PNA mice would show increased expression of glutamic acid decarboxylase (GAD), the rate-limiting enzyme in GABA production. However, the RT-qPCR showed that the expression of GAD encoding genes (Gad1 and Gad2) was unchanged in adult PNA mice compared to controls. Our findings indicate that PNA treatment can impact Pgr and Ar mRNA expression in adulthood. This may reflect altered circulating steroid hormones in PNA mice or PNA-induced epigenetic changes in the regulation of Pgr and Ar gene expression in ARC neurons.

The role of steroid hormones in the sexual differentiation of the human brain

Widespread sex differences in human brain structure and function have been reported. Research on animal models has demonstrated that sex differences in brain and behavior are induced by steroid hormones during specific, hormone sensitive, developmental periods. It was shown that typical male neural and behavioral characteristics develop under the influence of testosterone, mostly acting during perinatal development. By contrast, typical female neural and behavioral characteristics may actually develop under the influence of estradiol during a specific prepubertal period. This review provides an overview of our current knowledge on the role of steroid hormones in the sexual differentiation of the human brain. Both clinical and neuroimaging data obtained in patients with altered androgen levels/actions (i.e., congenital adrenal hyperplasia or complete androgen insensitivity syndrome [CAIS]), point to an important role of (prenatal) androgens in inducing typical male neural and psychosexual characteristics in humans. In contrast to rodents, there appears to be no obvious role for estrogens in masculinizing the human brain. Furthermore, data from CAIS also suggest a contribution of sex chromosome genes to the development of the human brain. The final part of this review is dedicated to a brief discussion of gender incongruence, also known as gender dysphoria, which has been associated with an altered or less pronounced sexual differentiation of the brain.

Temporal relationship between alterations in the gut microbiome and the development of polycystic ovary syndrome-like phenotypes in prenatally androgenized female mice.

It has been recently recognized that prenatal androgen exposure is involved in the development of polycystic ovary syndrome (PCOS) in adulthood. In addition, the gut microbiome in adult patients and rodents with PCOS differs from that of healthy individuals. Moreover, recent studies have suggested that the gut microbiome may play a causative role in the pathogenesis of PCOS. We wondered whether prenatal androgen exposure induces gut microbial dysbiosis early in life and is associated with the development of PCOS in later life. To test this hypothesis, we studied the development of PCOS-like phenotypes in prenatally androgenized (PNA) female mice and compared the gut microbiome of PNA and control offspring from 4 to 16weeks of age. PNA offspring showed a reproductive phenotype from 6weeks and a metabolic phenotype from 12weeks of age. The α-diversity of the gut microbiome of the PNA group was higher at 8weeks and lower at 12 and 16weeks of age, and the β-diversity differed from control at 8weeks. However, a significant difference in the composition of gut microbiome between the PNA and control groups was already apparent at 4weeks. Allobaculum and Roseburia were less abundant in PNA offspring, and may therefore be targets for future interventional studies. In conclusion, abnormalities in the gut microbiome appear as early as or even before PCOS-like phenotypes develop in PNA mice. Thus, the gut microbiome in early life is a potential target for the prevention of PCOS in later life.

Overexpression of Androgen Receptors Masculinizes 2D:4D Digit Ratios in Mice.

Studying the role of the prenatal endocrine environment in humans is challenging due to the ethical and practical considerations of measuring hormone levels of the developing fetus. Because it has been difficult to ascertain whether prenatal androgens contribute to the brain and behavior in humans as it does in non-human species, retrospective markers of prenatal androgens, such as the second-to-fourth finger digit ratio (2D:4D), are of interest to the studying of human behavioral endocrinology. To assess the validity of such markers, laboratory animals have been studied. Some strains of mice have been reported to show a sex difference in 2D:4D, and pharmacological and genetic manipulation of the androgen and estrogen receptors (AR and ER) has implicated a role for prenatal androgens in mediating this sex difference, although there have been conflicting reports. Here, we compared mice with global AR overexpression to mice with wildtype (WT) littermates and mice with neural-specific AR overexpression. We found a sex difference in the right hind paw, such that males had larger digit ratios than females. Regardless of sex, mice with global AR overexpression showed an increase in the right hind 2D:4D ratio compared with both WT and neural-specific AR overexpression mice. These results support a role for non-neural AR in the development of 2D:4D and suggest that increased sensitivity to androgens via increased AR is sufficient to increase the masculinization of digit ratios. Future directions for confirming the validity of 2D:4D as a marker for prenatal androgen exposure are discussed.

Prenatal oestrogen-testosterone balance as a risk factor of migraine in adults

BackgroundMigraine is a common neurological disease with extremely debilitating, but fully reversible symptoms. Women suffer from migraine more often than men. It was assumed that fluctuation of oestrogen level during menstrual cycle is one of many factors responsible for more frequent migraine attacks. The second-to-fourth digit ratio (2D:4D) is considered as an indicator of prenatal sex steroids. Balance of prenatal androgens (testosterone) and oestrogen has been studied in numerous diseases that are affected by hormones. However, the relationship between migraine and the sex steroids balance in prenatal development is still unexplained.The aim of this paper is to provide an evidence of relationship between prenatal oestrogen and testosterone exposure following 2D:4D digit ratio, and migraine prevalence in adults.MethodsWe examined a group of 151 adults (33 males, 118 females) with migraine and a control group of 111 adults (45 males, 66 females). 2D:4D digit ratio of both hands was measured using sliding Vernier calliper.ResultsSignificant differences were found in the right hand. Female migraineurs had lower value of 2D:4D ratio than the control group and the right 2D:4D was lower than left 2D:4D (Δ2D:4D), suggesting prenatal testosterone dominance. The opposite relationship was observed in males. Male migraineurs had higher value of 2D:4D ratio and Δ2D:4D was greater than the control group, suggesting prenatal oestrogen dominance.ConclusionsOur results suggest that depending on sex, different proportion of prenatal sex steroids might be a risk factor of migraine in adults. Women with migraine were presumably exposed in prenatal life to higher testosterone levels relative to oestrogen, while men with migraine were probably exposed in prenatal life to higher levels of oestrogen relative to testosterone.

Understanding the Associations of Prenatal Androgen Exposure on Sleep Physiology, Circadian Proteins, Anthropometric Parameters, Hormonal Factors, Quality of Life, and Sex Among Healthy Young Adults: Protocol for an International, Multicenter Study.

BackgroundThe ratio of the second finger length to the fourth finger length (2D:4D ratio) is considered to be negatively correlated with prenatal androgen exposure (PAE) and positively correlated with prenatal estrogen. Coincidentally, various brain regions are sensitive to PAE, and their functions in adults may be influenced by the prenatal actions of sex hormones.ObjectiveThis study aims to assess the relationship between PAE (indicated by the 2D:4D ratio) and various physiological (sex hormone levels and sleep-wake parameters), psychological (mental health), and sexual parameters in healthy young adults.MethodsThis study consists of two phases. In phase 1, we will conduct a survey-based study and anthropometric assessments (including 2D:4D ratio and BMI) in healthy young adults. Using validated questionnaires, we will collect self-reported data on sleep quality, sexual function, sleep chronotype, anxiety, and depressive symptoms. In phase 2, a subsample of phase 1 will undergo polysomnography and physiological and genetic assessments. Sleep architecture data will be obtained using portable polysomnography. The levels of testosterone, estradiol, progesterone, luteinizing hormone, follicle-stimulating hormone, prolactin, melatonin, and circadian regulatory proteins (circadian locomotor output cycles kaput [CLOCK], timeless [TIM], and period [PER]) and the expression levels of some miRNAs will be measured using blood samples. The rest and activity cycle will be monitored using actigraphy for a 7-day period.ResultsIn Poland, 720 participants were recruited for phase 1. Among these, 140 completed anthropometric measurements. In addition, 25 participants joined and completed phase 2 data collection. Recruitment from other sites will follow.ConclusionsFindings from our study may help to better understand the plausible role of PAE in sleep physiology, mental health, and sexual quality of life in young adults.International Registered Report Identifier (IRRID)DERR1-10.2196/29199

Prenatal androgen excess impairs beta-cell function by decreased sirtuin 3 expression.

Prenatal androgen exposure induces metabolic disorders in female offspring. However, the long-term effect of maternal testosterone excess on glucose metabolism, especially on pancreatic beta-cell function, is rarely investigated. Our current study mainly focused on the effects of prenatal testosterone exposure on glucose metabolism and pancreatic beta- cell function in aged female offspring. By using maternal mice and their female offspring as animal models, we found that prenatal androgen treatment induced obesity and glucose intolerance in aged offspring. These influences were accompanied by decreased fasting serum insulin concentration, elevated serum triglyceride, and testosterone concentrations. Glucose stimulated insulin secretion in pancreatic beta cells of aged female offspring was also affected by prenatal testosterone exposure. We further confirmed that increased serum testosterone contributed to downregulation of sirtuin 3 expression, activated oxidative stress, and impaired pancreatic beta-cell function in aged female offspring. Moreover, over-expression of sirtuin 3 in islets isolated from female offspring treated with prenatal testosterone normalized the oxidative stress level, restored cyclic AMP, and ATP generation, which finally improved glucose-stimulated insulin secretion in beta cells. Taken together, these results demonstrated that prenatal testosterone exposure caused a metabolic disturbance in aged female offspring via suppression of sirtuin 3 expression and activation of oxidative stress in pancreatic beta cells.

A potential role for the adrenal gland in autism

Androgens have been implicated in autism pathophysiology as recently, prenatal exposure to elevated androgens has been proposed as risk factor. However, published data on postnatal sex hormone levels in autistic children are controversial and the source of prenatal androgen exposure in autism remains unknown. Therefore, this study investigated postnatal sex hormone levels and dehydroepiandrosterone (DHEA) to shed light on a potential role for the adrenal gland in autism pathophysiology. A case-control study investigating estradiol (E2), DHEA, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels was conducted with 31 Saudi males with autism and 28 healthy, age-matched boys plasma. Moreover, correlation analysis with measured hormones and previously measured total testosterone (TT) and free testosterone (FT) in the same group of autism was conducted. DHEA was significantly higher (p < 0.05) in the autism group compared to controls. DHEA positively correlated with previously measured TT (r = + 0.79, p < 0.001) and FT (r = + 0.72, p < 0.001) levels in the same autism group. FSH levels were also significantly higher in the autism group than in the control group (p < 0.01). To the best of our knowledge, this is the first study to report a strong positive correlation between TT, FT and DHEA, suggesting an adrenal source for elevated androgen levels.

Maternal polycystic ovary syndrome and attention deficit hyperactivity disorder in offspring at 3years of age: Odense Child Cohort.

Previous data suggested a link between maternal polycystic ovary syndrome (PCOS) and offspring attention deficit hyperactivity disorder (ADHD), which could be mediated by higher prenatal androgen exposure. The study was part of the prospective Odense Child Cohort and included 1776 pregnant women, 165 (9%) with PCOS and 1607 (91%) controls. ADHD symptoms at 3years of age were defined using the parent-reported questionnaire Child Behavior Checklist/1.5-5 (scores >90th centile of Danish national standard). Maternal blood samples were collected in the third trimester measuring total testosterone by mass spectrometry, sex hormone-binding globulin, and calculated free testosterone. Offspring anogenital distance was measured at 3months of age. Regression models were performed with presence of ADHD symptoms as the dependent variable and adjusted for maternal age, body mass index, parity, smoking status, educational level, and parental psychiatric diagnoses. ADHD symptoms were present in 105/937 (11%) boys and 72/839 (9%) girls. In boys, maternal PCOS was positively associated with ADHD symptoms (unadjusted odds ratio [OR] 1.91, 95% CI 1.07-3.43, p=0.03, adjusted OR 2.20, 95% CI 1.20-4.02, p=0.01), whereas maternal PCOS was not associated with ADHD symptoms in girls. Maternal total testosterone, free testosterone, and offspring anogenital distance were not associated with higher risk of ADHD symptoms in the offspring. Higher risk of ADHD in boys born of mothers with PCOS were not associated with maternal third-trimester testosterone levels or offspring anogenital distance.

Prenatal Androgen Treatment Does Not Alter the Firing Activity of Hypothalamic Arcuate Kisspeptin Neurons in Female Mice

Neuroendocrine control of reproduction is disrupted in many individuals with polycystic ovary syndrome (PCOS), who present with increased luteinizing hormone (LH), and presumably gonadotropin-releasing hormone (GnRH), release frequency, and high androgen levels. Prenatal androgenization (PNA) recapitulates these phenotypes in primates and rodents. Female offspring of mice injected with dihydrotestosterone (DHT) on gestational days 16–18 exhibit disrupted estrous cyclicity, increased LH and testosterone, and increased GnRH neuron firing rate as adults. PNA also alters the developmental trajectory of GnRH neuron firing rates, markedly blunting the prepubertal peak in firing that occurs in three-week (3wk)-old controls. GnRH neurons do not express detectable androgen receptors and are thus probably not the direct target of DHT. Rather, PNA likely alters GnRH neuronal activity by modulating upstream neurons, such as hypothalamic arcuate neurons co-expressing kisspeptin, neurokinin B (gene Tac2), and dynorphin, also known as KNDy neurons. We hypothesized PNA treatment changes firing rates of KNDy neurons in a similar age-dependent manner as GnRH neurons. We conducted targeted extracellular recordings (0.5–2 h) of Tac2-identified KNDy neurons from control and PNA mice at 3wks of age and in adulthood. About half of neurons were quiescent (<0.005 Hz). Long-term firing rates of active cells varied, suggestive of episodic activity, but were not different among groups. Short-term burst firing was also similar. We thus reject the hypothesis that PNA alters the firing rate of KNDy neurons. This does not preclude altered neurosecretory output of KNDy neurons, involvement of other neuronal populations, or in vivo networks as critical drivers of altered GnRH firing rates in PNA mice.

Aberrant miRNA-mRNA regulatory network in polycystic ovary syndrome is associated with markers of insulin sensitivity and inflammation.

BackgroundPolycystic ovary syndrome (PCOS) is a common multifactorial metabolic and endocrine disorder in women of reproductive age. Increasingly, evidence indicates that the microRNA (miRNA)-mRNA axis contributes to the development of PCOS.MethodsTo investigate the molecular mechanisms through which miRNA-mRNA expression affects hyperandrogenism, ovarian tissue samples from prenatally androgenized (PNA) mice were subjected to miRNA-seq and RNA-seq analysis. Analyses were performed to identify differentially expressed microRNAs (DEmiRs) and differentially expressed genes (DEGs). In combination with our previous data obtained from clinical samples, we have performed an integrated miRNA-mRNA analysis of PNA mice and granulosa cells (GCs) from patients with PCOS. The changes in expression were validated by RT-qPCR in more mouse models and clinical samples.ResultsIn total, 3,432 genes and 16 miRNAs were differentially expressed in PNA mice compared with the control mice. We investigated the regulation pattern of miRNAs-mRNAs and observed a total of 12 miRNA-mRNA pairs involved in negative regulation. Functional analysis concentrated on insulin resistance, the T cell receptor signaling pathway, and other inflammation-related pathways. Verification of these results by RT-qPCR showed that the expression of miR-106-5p and miR-155-5p in clinical GCs was consistent with that in PNA mice. After predicting the target genes of miR-106-5p and miR-155-5p and performing negative regulation analysis, six target genes were obtained in GCs. The integration analysis showed that the network of miR-106-5p/miR-155-5p targets was mostly concentrated in pathway related to insulin resistance and inflammation. In addition, the upregulation of the inflammatory genes Il18/IL18 and Socs3/SOCS3 was validated in the PNA mice and GCs from patients compared with the appropriate control sample. The in vitro experiments showed that the regulatory relationship observed may be related to the direct stimulation of DHT.ConclusionsOur results showed that the miRNA-mRNA regulatory network in PCOS was associated with markers of insulin sensitivity and inflammation. Our study provides a new genetic basis and novel insight regarding the pathogenesis of PCOS.

P–632 Examination of temporal changes in phenotype and gut microbiome during the process of growth in polycystic ovary syndrome (PCOS) model induced by prenatal androgen exposure

Abstract Study question From when do abnormality in gut microbiome and phenotypes of PCOS appear during the process of growth? Summary answer Reproductive phenotypes of PCOS appear from 6 weeks and metabolic phenotypes from 12 weeks onward. Alteration in gut microbiome appears as early as 4 weeks. What is known already The etiology of PCOS remains largely unknown, however PCOS is considered as a complex multigenic disorder with strong epigenetic and environmental influence. Previous studies have suggested that fetal over-exposure to androgens could be the main factor of the development of PCOS after birth. On the other hands, recent studies on both human and PCOS rodent models have demonstrated the association between PCOS and alteration of gut microbiome in adulthood. Furthermore, it was recently reported that gut microbiome in obese adolescent with PCOS is different from obese adolescent without PCOS. Study design, size, duration A rodent PCOS model induced by prenatal dehydroepiandrosterone (DHT) exposure was applied to this study. Phenotypes and gut microbiome were compared between PCOS model mice (n = 12/group) and control mice (n = 10/group) at each stage of growth; 4 weeks (prepuberty), 6 weeks (puberty), 8 weeks (adolescent), 12 weeks (young adult), and 16 weeks (adult). The determinants for PCOS phenotypes are onset of puberty, estrous cycle, morphology of ovaries, serum testosterone level, body weight, and insulin resistance. Participants/materials, setting, methods Pregnant dams were subcutaneously injected on days of 16, 17, and 18 of gestation with either sesame oil for control groups or sesame oil containing 250µg of DHT for prenatal DHT groups. The evaluation of PCOS phenotypes and gut microbiome in female offspring were performed at each stage of growth. For examination of gut microbiota, next generation sequencing and bioinformatics analysis of 16S rRNA genes were performed on DNA extracted from mouse fecal samples. Main results and the role of chance Prenatal DHT mice exhibited delayed puberty onset, disrupted estrous cycle, and significantly increased testosterone levels from 6 weeks onward. Significantly increased atretic antral follicles were observed in prenatal DHT mice at 6, 12, and 16 weeks. Prenatal DHT mice showed significantly decreased body weight at 4, 6, 8 weeks and increased body weight from 12 weeks onward. As for gut microbiome, alpha-diversity was significantly different between control and prenatal DHT mice from 8 weeks onward and beta-diversity was significantly different at 6 and 8 weeks. Altered composition of gut microbiota was observed as early as 4 weeks. At phylum level, Firmicutes are significantly increased in prenatal DHT mice at 4 and 8 weeks and decreased at 16 weeks. Actinobacteria phylum showed significant decrease at 6 and 8 weeks in prenatal DHT mice. At genus level, relative abundance of several bacterial taxa significantly differed between control and prenatal DHT mice; some taxa, such as Allobaculum, Adlercreutzia, Bilophila, Clostridium, Gemella, Gemmiger, Roseburia, Ruminococcus, Staphylococcus, and Sutterella, exhibited constant increase or decrease in prenatal DHT mice during the process of growth. Interestingly, Roseburia was never detected in prenatal DHT mice, while approximately half of control mice harbored Roseburia at 12 and 16 weeks. Limitations, reasons for caution It is not clearly determined whether alteration in gut microbiome is cause or result of PCOS development, although the changes in gut microbiome seemed to precede the appearance of typical PCOS phenotypes in the present study. Mouse model does not completely recapitulate human PCOS. Wider implications of the findings: Our findings suggest that prenatal androgen exposure causes alteration of gut microbiome from pre-puberty onward, even before PCOS phenotypes become apparent. Intervention for girls at risk of PCOS with pre/pro-biotics may prevent them from developing PCOS in future. Trial registration number Not applicable

Prenatal Androgen Exposure Alters KNDy Neurons and Their Afferent Network in a Model of Polycystic Ovarian Syndrome.

Polycystic ovarian syndrome (PCOS), the most common endocrinopathy affecting women worldwide, is characterized by elevated luteinizing hormone (LH) pulse frequency due to the impaired suppression of gonadotrophin-releasing hormone (GnRH) release by steroid hormone negative feedback. Although neurons that co-express kisspeptin, neurokinin B, and dynorphin (KNDy cells) were recently defined as the GnRH/LH pulse generator, little is understood about their role in the pathogenesis of PCOS. We used a prenatal androgen-treated (PNA) mouse model of PCOS to determine whether changes in KNDy neurons or their afferent network underlie altered negative feedback. First, we identified elevated androgen receptor gene expression in KNDy cells of PNA mice, whereas progesterone receptor and dynorphin gene expression was significantly reduced, suggesting elevated androgens in PCOS disrupt progesterone negative feedback via direct actions upon KNDy cells. Second, we discovered GABAergic and glutamatergic synaptic input to KNDy neurons was reduced in PNA mice. Retrograde monosynaptic tract-tracing revealed a dramatic reduction in input originates from sexually dimorphic afferents in the preoptic area, anteroventral periventricular nucleus, anterior hypothalamic area and lateral hypothalamus. These results reveal 2 sites of neuronal alterations potentially responsible for defects in negative feedback in PCOS: changes in gene expression within KNDy neurons, and changes in synaptic inputs from steroid hormone-responsive hypothalamic regions. How each of these changes contribute to the neuroendocrine phenotype seen in in PCOS, and the role of specific sets of upstream KNDy afferents in the process, remains to be determined.