Time to pregnancy and life expectancy: a cohort study of 18 796 pregnant couples.

STUDY QUESTION: Is fecundity, measured as time to pregnancy (TTP), associated with mortality in parents? SUMMARY ANSWER: Prolonged TTP is associated with increased mortality in both mothers and fathers in a dose-response manner. WHAT IS KNOWN ALREADY: Several studies have linked both male and female fecundity to mortality. In women, infertility has been linked to several diseases, but studies suggest that the underlying conditions, rather than infertility, increase mortality. STUDY DESIGN, SIZE, DURATION: A prospective cohort study was carried out on 18 796 pregnant couples, in which the pregnant women attended prophylactic antenatal care between 1973 and 1987 at a primary and tertiary care unit. The couples were followed in Danish mortality registers from their child's birth date until death or until 2018. The follow-up period was up to 47 years, and there was complete follow-up until death, emigration or end of study. PARTICIPANTS/MATERIALS, SETTING, METHODS: At the first antenatal visit, the pregnant women were asked to report the time to the current pregnancy. Inclusion was restricted to the first pregnancy, and TTP was categorised into <12 months, ≥12 months, not planned, and not available. In sub-analyses, TTP ≥12 was further categorized into 12-35, 36-60, and >60 months. Information for parents was linked to several Danish nationwide health registries. Survival analysis was used to estimate the hazard ratios (HRs) with a 95% CI for survival and adjusted for age at the first attempt to become pregnant, year of birth, socioeconomic status, mother's smoking during pregnancy, and mother's BMI. MAIN RESULTS AND THE ROLE OF CHANCE: Mothers and fathers with TTP >60 months survived, respectively, 3.5 (95% CI: 2.6-4.3) and 2.7 (95% CI: 1.8-3.7) years shorter than parents with a TTP <12 months. The mortality was higher for fathers (HR: 1.21, 95% CI: 1.09-1.34) and mothers (HR: 1.29, 95% CI: 1.12-1.49) with TTP ≥12 months compared to parents with TTP <12 months. The risk of all-cause mortality during the study period increased in a dose-response manner with the highest adjusted HR of 1.98 (95% CI: 1.62-2.41) for fathers and 2.03 (95% CI: 1.56-2.63) for mothers with TTP >60 months. Prolonged TTP was associated with several different causes of death in both fathers and mothers, indicating that the underlying causes of the relation between fecundity and survival may be multi-factorial. LIMITATIONS, REASONS FOR CAUTION: A limitation is that fecundity is measured using a pregnancy-based approach. Thus, the cohort is conditioned on fertility success and excludes sterile couples, unsuccessful attempts and spontaneous abortions. The question used to measure TTP when the pregnant woman was interviewed at her first attended prophylactic antenatal care: 'From the time you wanted a pregnancy until it occurred, how much time passed?' could potentially have led to serious misclassification if the woman did not answer on time starting unprotected intercourse but on the start of wishing to have a child. WIDER IMPLICATIONS OF THE FINDINGS: We found that TTP is a strong marker of survival, contributing to the still-emerging evidence that fecundity in men and women reflects their health and survival potential. STUDY FUNDING/COMPETING INTEREST(S): The authors acknowledge an unrestricted grant from Ferring. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article, or the decision to submit it for publication. M.L.E. is an advisor to Ro, VSeat, Doveras, and Next. TRIAL REGISTRATION NUMBER: N/A.

Effect of 12-months testosterone replacement therapy on bone mineral density and markers of bone turnover in testicular cancer survivors - results from a randomized double-blind trial.

BACKGROUND: Testicular cancer survivors (TCS) are at risk of Leydig cell insufficiency, which is a condition characterized by elevated luteinising hormone (LH) in combination with low levels of testosterone. It has been suggested that this condition is associated with impaired metabolic profile and low bone mineral density (BMD). The primary aim of the randomized double-blind trial NCT02991209 was to evaluate metabolic profile after 12-months testosterone replacement therapy (TRT) in TCS with mild Leydig cell insufficiency. Here we present the secondary outcomes of changes in BMD and markers of bone turnover. METHODOLOGY: In total, 69 TCS with mild Leydig cell insufficiency were randomized 1:1 to 12 months TRT (n = 35) (Tostran, gel, 2%, applied transdermally, with a maximum daily dose of 40 mg) or placebo (n = 34). BMD and markers of bone turnover were evaluated at baseline, after 6- and 12-months TRT, and 3-months post-treatment. Linear mixed effects models were used to analyse changes in BMD, N-terminal propeptide of type 1 procollagen (P1NP) and C-terminal telopeptide of type I collagen (CTX). RESULTS: After 12 months treatment, TRT was not associated with a statistically significant difference in BMD compared to placebo; total body BMD: 0.01 g/cm2 (95% confidence interval (CI): -0.01 - 0.02), BMD of the lumbar spine: 0.01 g/cm2, (95% CI: -0.01-0.03), BMD of the left femoral neck: 0.00, (95% CI: -0.01-0.02). TRT was associated with a small but statistically significant increase in P1NP: 11.65 µg/L (95% CI: 3.96, 19.35), while there was no difference in CTX. CONCLUSION: 12 months of TRT did not change BMD, while there was as small and clinically irrelevant increase in P1NP compared to placebo in TCS with mild Leydig cell insufficiency. The findings need validation in a larger cohort.

Prenatal and early postnatal exposure to perfluoroalkyl substances and bone mineral content and density in the Odense child cohort.

INTRODUCTION: Exposure to perfluoroalkyl substances (PFAS) has been associated with lower bone mineral density (BMD) in animal and human studies, but prospective data from children are limited. OBJECTIVES: To determine associations between prenatal and early postnatal PFAS exposure and BMD at age 7 years. METHODS: In the Odense Child Cohort, Denmark, pregnant women were recruited in 2010-2012, and their children were invited for subsequent health examinations. At 12 weeks of gestation the pregnant women delivered a serum sample, and at age 18 months serum was obtained from the child to measure perfluorooctane sulfonic acid (PFOS), perfluorooctanoic acid (PFOA), perfluorohexane sulfonic acid (PFHxS), perfluorononanoic acid (PFNA) and perfluorodecanoic acid (PFDA) by LC-MS/MS. At age 7 years DXA scans were performed to measure bone mineral content (BMC) and BMD Z-score. PFAS in pregnancy (n = 924) and/or at age 18 months (n = 511) were regressed against DXA measurements, adjusted for maternal education, child height Z-score, sex (for BMC) and for postnatal exposure, additionally duration of total breastfeeding. We additionally performed structural equation models determining combined effects of pre-and postnatal PFAS exposures. RESULTS: Higher prenatal and early postnatal serum concentrations of all measured PFAS were associated with lower BMC and BMD Z-scores at age 7 years, all estimates were negative although not all significant. For each doubling of prenatal or 18-month exposure to PFDA, BMD Z-scores were lowered by -0.07 (95 % CI -0.10; -0.03) and -0.14 (-0.25; -0.03), respectively after adjustment. Pre- and postnatal PFAS were correlated, but structural equation models suggested that associations with BMD were stronger for 18-month than prenatal PFAS exposure. DISCUSSION: Bone density is established in childhood, and a reduction in BMD during early childhood may have long-term implication for peak bone mass and lifelong bone health. Future studies of the impact of PFAS exposure on fracture incidence will help elucidate the clinical relevance.

Prenatal and postnatal exposures to endocrine disrupting chemicals and timing of pubertal onset in girls and boys: a systematic review and meta-analysis.

BACKGROUND: Globally, the ages at pubertal onset for girls and boys have been decreasing during recent decades, partly attributed to excess body fat accumulation. However, a growing body of literature has recognized that endocrine disrupting chemicals (EDCs) may play an important role in this global trend, but the association has not yet been fully established. OBJECTIVE AND RATIONALE: EDCs can interfere with normal hormone function and metabolism and play a role in pubertal onset. We aimed to systematically identify and evaluate the current evidence on the timing of pubertal onset in girls and boys following prenatal or postnatal exposures to xenobiotic EDCs. SEARCH METHODS: Following PRISMA guidelines, we performed a systematic literature search of original peer-reviewed publications in the PubMed database through a block search approach using a combination of index MeSH and free text search terms. Publications were considered if they covered biomarkers of prenatal or postnatal exposures to xenobiotic EDCs (European Commission's list of category 1 EDCs) measured in maternal or child biospecimen and pubertal onset defined by the progression of the following milestones (and assessed in terms of the following measures): menarche (age), thelarche (Tanner staging) and pubarche (Tanner staging), in girls, and genital stage (Tanner staging), testicular volume (ml) and pubarche (Tanner staging), in boys. OUTCOMES: The literature search resulted in 703 references, of which we identified 52 publications fulfilling the eligibility criteria for the qualitative trend synthesis and 23 publications for the meta-analysis. The qualitative trend synthesis provided data on 103 combinations of associations between prenatal or postnatal exposure to EDC compounds groups and puberty outcomes and the meta-analysis enabled 18 summary risk estimates of meta-associations. WIDER IMPLICATIONS: Statistically significant associations in the qualitative trend synthesis suggested that postnatal exposure to phthalates may be associated with earlier thelarche and later pubarche. However, we did not find consistent evidence in the meta-analysis for associations between timing of pubertal onset in girls and boys and exposures to any of the studied xenobiotic EDCs. We were not able to identify specific pre- or postnatal windows of exposure as particularly critical and susceptible for effects of EDCs. Current evidence is subject to several methodological challenges and inconsistencies and evidence on specific exposure-outcome associations remains too scarce to firmly confirm EDC exposure as a risk factor for changes in age of pubertal onset in the general child population. To create a more uniform foundation for future comparison of evidence and to strengthen pooled studies, we recommend the use of more standardized approaches in the choice of statistical analyses, with exposure transformations, and in the definitions and assessments of puberty outcomes. The impact of mixtures of EDC exposures on the association also remains unestablished and would be valuable to elucidate for prenatal and postnatal windows of exposure. Future large, longitudinal epidemiological studies are needed to clarify the overall association.

Human sperm cells can form paracetamol metabolite AM404 that directly interferes with sperm calcium signalling and function through a CatSper-dependent mechanism.

STUDY QUESTION: Do paracetamol (N-acetyl-para-aminophenol (APAP) or acetaminophen) and/or its metabolites affect human sperm Ca2+-signalling and function? SUMMARY ANSWER: While APAP itself does not interact with Ca2+-signalling in human sperm, its metabolite N-arachidonoyl phenolamine (AM404), produced via fatty acid amide hydrolase (FAAH), interferes with human sperm Ca2+-signalling and function through a suggested CatSper channel-dependent action. WHAT IS KNOWN ALREADY: Studies have shown that adult men with high urinary levels of over-the-counter mild analgesic APAP have impaired sperm motility and increased time-to-pregnancy. STUDY DESIGN, SIZE, DURATION: This study consists of (i) an in vivo human pharmaceutical APAP exposure experiment to understand to what degree APAP reaches the sperm cells in the seminal fluid; (ii) in vitro calcium imaging and functional experiments in freshly donated human sperm cells to investigate CatSper channel-dependent activation by APAP and its metabolites; and (iii) experiments to understand the in situ capabilities of human sperm cells to form APAP metabolite AM404. PARTICIPANTS/MATERIALS, SETTING, METHODS: Three healthy young males participated in the in vivo human exposure experiment after prior consent. Human semen samples were provided by healthy young volunteer donors after prior consent on the day of the in vitro experiments. MAIN RESULTS AND THE ROLE OF CHANCE: Pharmaceutical APAP exposure reaches the seminal plasma in high micromolar concentrations and accumulates in the seminal plasma between 3 and 5 days of exposure (P-value 0.023). APAP and its primary metabolite 4-aminophenol (4AP) do not interact with human sperm Ca2+-signalling. Instead, the APAP metabolite AM404 produced via FAAH interferes with human sperm Ca2+-signalling through a CatSper-dependent action. Also, AM404 significantly increases sperm cell penetration into viscous mucous (P-value of 0.003). FAAH is functionally expressed in human sperm cells in the neck/midpiece region, as evidenced by immunohistochemical staining and the ability of human sperm cells to hydrolyse the fluorogenic FAAH substrate arachidonyl 7-amino, 4-methyl coumarin amide in an FAAH-dependent manner. Importantly, human sperm cells have the capacity to form AM404 in situ after exposure to 4AP (P-value 0.0402 compared to vehicle-treated sperm cells). LIMITATIONS, REASONS FOR CAUTION: The experiments were conducted largely in vitro. Future studies are needed to test whether APAP can disrupt human sperm function in vivo through the action of AM404. WIDER IMPLICATIONS OF THE FINDINGS: We hypothesize that these observations could, at least in part, be responsible for the negative association between male urinary APAP concentrations, sperm motility and time-to-pregnancy. STUDY FUNDING/COMPETING INTEREST(S): D.M.K. is funded by the Lundbeck Foundation, grant number R324-2019-1881, and the Svend Andersen Foundation. A.R. is funded by a BRIDGE-Translational Excellence Programme grant funded by the Novo Nordisk Foundation, grant agreement number: NNF18SA0034956. All authors declare no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Pubertal induction and transition to adult sex hormone replacement in patients with congenital pituitary or gonadal reproductive hormone deficiency: an Endo-ERN clinical practice guideline.

An Endo-European Reference Network guideline initiative was launched including 16 clinicians experienced in endocrinology, pediatric and adult and 2 patient representatives. The guideline was endorsed by the European Society for Pediatric Endocrinology, the European Society for Endocrinology and the European Academy of Andrology. The aim was to create practice guidelines for clinical assessment and puberty induction in individuals with congenital pituitary or gonadal hormone deficiency. A systematic literature search was conducted, and the evidence was graded according to the Grading of Recommendations, Assessment, Development and Evaluation system. If the evidence was insufficient or lacking, then the conclusions were based on expert opinion. The guideline includes recommendations for puberty induction with oestrogen or testosterone. Publications on the induction of puberty with follicle-stimulation hormone and human chorionic gonadotrophin in hypogonadotropic hypogonadism are reviewed. Specific issues in individuals with Klinefelter syndrome or androgen insensitivity syndrome are considered. The expert panel recommends that pubertal induction or sex hormone replacement to sustain puberty should be cared for by a multidisciplinary team. Children with a known condition should be followed from the age of 8 years for girls and 9 years for boys. Puberty induction should be individualised but considered at 11 years in girls and 12 years in boys. Psychological aspects of puberty and fertility issues are especially important to address in individuals with sex development disorders or congenital pituitary deficiencies. The transition of these young adults highlights the importance of a multidisciplinary approach, to discuss both medical issues and social and psychological issues that arise in the context of these chronic conditions.

Testicular cancer survivors have shorter anogenital distance that is not increased by 1 year of testosterone replacement therapy.

STUDY QUESTION: Is anogenital distance (AGD) shorter in testicular cancer (TC) survivors than in men from the general population, and is AGD affected by testosterone replacement therapy in adulthood? SUMMARY ANSWER: AGD, measured as distance from anus to scrotum (AGDas), is shorter in TC survivors and does not change as a result of testosterone replacement therapy. WHAT IS KNOWN ALREADY: Animal studies have shown that AGD is a postnatal 'read-out' of foetal androgen action, and short AGD in male offspring is considered a sign of feminization caused by in utero disruption of the reproductive system. Likewise, measurement of AGD in human studies has suggested AGD to be part of the testicular dysgenesis syndrome hypothesis, which proposes that male reproductive disorders, such as hypospadias, cryptorchidism, some cases of impaired semen quality and TC, all share a common foetal origin. STUDY DESIGN, SIZE, DURATION: The aim was to assess AGD in men with a history of TC and controls, and furthermore to examine AGD during testosterone replacement therapy in adulthood. Study participants were TC survivors with a mild Leydig cell insufficiency who participated in a randomized double-blind study of testosterone replacement therapy versus placebo for 52 weeks (N = 69). Men from the general population were prospectively included from a study on testicular function as controls (N = 67). PARTICIPANTS/MATERIALS, SETTING, METHODS: We measured two variants of AGD; as our primary outcome the anoscrotal distance (AGDas) measured from the centre of the anus to the posterior base of the scrotum, and secondarily the anopenile distance (AGDap) measured from the anus to the cephalad insertion of the penis. Using multiple regression analysis, the mean difference in AGD between TC survivors and men from the general population was assessed, adjusted for height, BMI and examiner. Next, AGD was measured before and after 52 weeks of treatment with testosterone or placebo, and with covariance analysis differences between the two groups at follow-up was assessed after adjustment for baseline AGD, examiner, BMI and change in BMI during treatment. MAIN RESULTS AND THE ROLE OF CHANCE: TC survivors had a shorter AGDas (-0.84 cm, 95% CI: -1.31; -0.37) compared to men from the general population, and AGDas did not differ between the testosterone and placebo treated group at follow-up (0.11 cm, 95% CI: -0.22; 0.44). In contrast, AGDap was not shorter in TC survivors after adjustment (0.05 cm, 95% CI: -0.30; 0.39), and was 0.48 cm longer (95% CI: 0.13; 0.82) at follow-up in the testosterone treated compared to the placebo-treated group. LIMITATIONS, REASONS FOR CAUTION: A limitation of the study is that the number of included men was limited, and results need confirmation in a larger study. Furthermore, TC survivors were significantly older than controls. For the comparison of AGD in TC survivors and controls, it was not possible to conduct the examinations with the examiner being blinded to which group he was examining, and it cannot be excluded that this can cause a bias. WIDER IMPLICATIONS OF THE FINDINGS: The shorter AGDas in TC survivors compared to controls, which did not change upon adult testosterone replacement therapy, supports the hypothesis that reduced AGD is part of the testicular dysgenesis syndrome and may be a marker of disrupted foetal testicular development. By contrast, AGDap was not shorter in TC survivors and might be modestly sensitive to adult testosterone treatment, and thus inferior to AGDas as a constant postnatal marker of the foetal androgen environment. STUDY FUNDING/COMPETING INTEREST(S): Expenses were paid by the Department of Oncology, Copenhagen University Hospital, Rigshospitalet. Kiowa Kirin International covered expenses for Tostran and placebo. The Danish Cancer Society, The Danish Cancer Research Foundation, the Preben & Anna Simonsen Foundation, and Rigshospitalet have supported the study. L.P. was financed by the Research Fund of the Capital Region of Denmark. The authors have no competing interests. TRIAL REGISTRATION NUMBER: Part of the study is based on men participating in a randomized controlled trial registered at ClinicalTrials.gov, NCT02991209, 25 November 2016.

Prenatal exposure to maternal stressful life events and earlier age at menarche: the Raine Study.

STUDY QUESTION: Is there an association between prenatal exposure to stressful life events and age at menarche, and does childhood BMI mediate this association? SUMMARY ANSWER: Girls exposed to prenatal stress had a slightly earlier age at menarche, but this association did not show a dose-response effect and was not mediated by childhood offspring BMI. WHAT IS ALREADY KNOWN: Prenatal stress may impact on reproductive function in females including age at menarche, but human data are very limited. High childhood BMI is known to be associated with earlier age at menarche. Only one small study has measured the association between maternal stress and age at menarche and reported that childhood BMI mediated the association between maternal stress and earlier age at menarche. However, neither maternal stress nor age at menarche was prospectively recorded and the study was limited to 31 mother-daughter pairs. STUDY DESIGN, SIZE, DURATION: The Raine Study is a large prospective population-based pregnancy cohort study (n = 1414 mother-daughter pairs) continuously followed from prenatal life through to adolescence. In the present study, we examined the association between exposure to maternal stressful life events during early, late and total gestation and age at menarche in offspring using 753 mother-daughter pairs with complete case information. PARTICIPANTS/MATERIALS, SETTING, METHODS: Mothers prospectively reported stressful life events during pregnancy at 18 and 34 weeks using a standardized 10-point questionnaire. Exact date of menarche was assessed using a purpose-designed questionnaire at 8, 10, 14 and 17 years of age. Complete information on exposure, outcome and confounding variables was obtained from 753 mothers-daughter pairs. Multivariate linear regression complete case analysis was used to examine associations between maternal stressful life event exposure and age at menarche. Potential selection bias was evaluated using multiple imputations (50 datasets). The mediating effects of offspring childhood BMI (ages 5, 8, or 10 years) on these associations were measured in separate sub-analyses. MAIN RESULTS AND ROLE OF CHANCE: Most (580/753, 77%) daughters were exposed to at least one prenatal stressful life event. Exposure to maternal stressful life events during the entire pregnancy was associated with a non-linear earlier age at menarche. Exposure to one event and two or more psychological stressful events was associated with a 3.5 and 1.7-month earlier onset of puberty, respectively when compared to the reference group with no exposure maternal stressful life events. The estimates from multiple imputation with 50 datasets were comparable with complete case analysis confirming the existence of an underlying effect. No separate significant effects were observed for exposure during early or late gestation. The association between prenatal stressful events and age at menarche was not mediated by childhood BMI in the offspring. LIMITATIONS, REASONS FOR CAUTION: Stressful life events may have affected pregnant women in different ways and self-perceived maternal stress severity may have provided a more precise estimate of gestational psychological stress. The observed non-linear U-shape of the association between maternal psychological stress and age at menarche did not reflect a dose-response. This suggests that the first exposure to prenatal stress exerts a greater effect on fetal reproductive development. A potential mechanism is via dramatic initial activation of the hypothalamic-pituitary-adrenal (HPA) axis following the first stressful life event which is greater than that observed following subsequent exposure to two or more maternal stressful life events. Whilst we adjusted for a priori chosen confounders, we cannot exclude residual confounding or confounding by factors we did not include. Maternal age at menarche was not available so the effects of familial history/genetics could not be assessed. There was a large loss due to the number of girls with no information on date of menarche and missing confounder information implying risk of selection bias and multiple imputation analyses did not fully exclude this risk (similar direction but slightly weaker estimate magnitude). WIDER IMPLICATIONS OF THE FINDINGS: Menarche is a sentinel reproductive event and earlier age at menarche carries implications for psychological, social and reproductive health and for long-term risk of common non-communicable diseases. Understanding the factors regulating age at menarche has extensive health implications. This is the first population-based cohort study in humans to demonstrate that prenatal psychological stress might directly modify age at menarche. STUDY FUNDING/COMPETING INTEREST(S): Dr. Bräuner and Trine Koch's salaries were supported by Doctor Sofus Carl Emil Friis and spouse Olga Doris Friis foundation, The Danish Cancer Society (Kræftens Bekæmpelse, RP15468, R204-A12636, Denmark) and The Danish Health Foundation (Helsefonden, F-22181-23, Denmark). Martha Hickey was funded by NHMRC Practitioner Fellowships. The funding bodies played no role in the design, collection, analysis, or interpretation of data; in the writing of the manuscript; or in the decision to submit the manuscript for publication. Dr. Hart has received personal fees in his function as the Medical Director of Fertility Specialists of Western Australia and received educational sponsorship grants from MSD, Merck-Serono and from Ferring Pharmaceuticals. Dr Hart has also received personal fees from Shareholders in Western IVF outside the submitted work. TRIAL REGISTRATION NUMBER: NA.

The association between in utero exposure to maternal psychological stress and female reproductive function in adolescence: A prospective cohort study.

Background: Experimental studies suggest that prenatal stress affects reproductive function in female offspring, but human evidence is sparse and inconsistent. In this present study, we aim to investigate whether maternal psychological stress, quantified as stressful life events during pregnancy, affect reproductive function in the female offspring. Method: In a large population-based pregnancy cohort study (The Raine Study) continuously followed from prenatal life through to adolescence we examined the association between the number of maternal stressful life events in both early and late gestation and subsequent ovarian and uterine function in 228 female adolescent offspring. Mothers prospectively reported stressful life events during pregnancy at 18 and 34 weeks using a standardized 10-point questionnaire. Female offspring (n â€‹= â€‹228) age 14-16 years underwent gynecological examination including transabdominal abdominal ultrasound (TAUS) to measure uterine volume and ovarian AFC. Plasma samples on day 2-6 of the spontaneous menstrual cycle measured circulating AMH and inhibin B. Multivariate linear regression analysis was used to examine the associations between maternal stressful life events and reproductive function in female offspring. Adolescents taking hormonal contraception were excluded. Results: Most adolescents (145/228, 64%) were exposed to at least one stressful life event in early gestation and around half (125/228, 55%) were exposed to at least one in later gestation. Exposure to one or more maternal stressful life events in late gestation was associated with a greater uterine volume (ߠ​= â€‹0.13, 95% CI 0.04; 0.23) and higher ovarian AFC (ߠ​= â€‹0.19, 95% CI 0.02; 0.35) at age 14-16 years. No associations between maternal stressful events in late gestation and reproductive function were identified. No associations between stressful life events in early or late gestation and circulating AMH or Inhibin B were observed. Conclusion: Maternal psychological stress in late, but not early gestation was associated with a significantly greater uterine volume and ovarian antral follicle count (AFC) in adolescent offspring but did not affect ovarian production of antimullerian hormone (AMH) or Inhibin B. These findings suggest that female reproductive function is influenced by prenatal exposure to stress.

DIAGNOSIS OF ENDOCRINE DISEASE: Sex steroid action in adolescence: too much, too little; too early, too late.

ABSTRACT: This review aims to cover the subject of sex steroid action in adolescence. It will include situations with too little sex steroid action, as seen in for example, Turners syndrome and androgen insensitivity issues, too much sex steroid action as seen in adolescent PCOS, CAH and gynecomastia, too late sex steroid action as seen in constitutional delay of growth and puberty and too early sex steroid action as seen in precocious puberty. This review will cover the etiology, the signs and symptoms which the clinician should be attentive to, important differential diagnoses to know and be able to distinguish, long-term health and social consequences of these hormonal disorders and the course of action with regards to medical treatment in the pediatric endocrinological department and for the general practitioner. This review also covers situations with exogenous sex steroid application for therapeutic purposes in the adolescent and young adult. This includes gender-affirming therapy in the transgender child and hormone treatment of tall statured children. It gives some background information of the cause of treatment, the patient's motivation for medicating (or self-medicating), long-term consequences of exogenous sex steroid treatment and clinical outcome of this treatment.

In utero exposure to maternal stressful life events and risk of polycystic ovary syndrome in the offspring: The Raine Study.

BACKGROUND: PCOS is the most common endocrine disorder in reproductive age women. The origins of PCOS are unknown but experimental and limited human evidence suggests that greater prenatal exposure to androgens may predispose to PCOS. Experimental evidence suggests that maternal stressors may affect reproductive function in the offspring via changes in prenatal androgen exposure. In this present study, we aim to investigate whether maternal stressful life events during pregnancy are associated with polycystic ovary morphology (PCOM) or polycystic ovary syndrome (PCOS) in adolescent offspring. METHOD: In a large population-based pregnancy cohort study (The Raine Study) continuously followed from prenatal life through to adolescence we examined the association between maternal stressful life events during pregnancy in both early and late gestation, and subsequent circulating concentrations of ovarian and adrenal androgens, PCOM and PCOS in the normal menstrual cycle of offspring age 14-16 years. Maternal stressful life events were prospectively recorded during pregnancy at 18 and 34 weeks using a 10-point questionnaire. Female offspring (n = 223) completed a questionnaire about their menstrual cycles, underwent a clinical examination for hirsutism (Ferriman-Gallwey score) and transabdominal pelvic ultrasound examination to determine ovarian morphology according to standardized criteria for classification of PCOM. Plasma samples were obtained at day 2-6 of the normal menstrual cycle for measurement of androgens. PCOM was defined according to the international consensus definition, 2003 and the evidence-based guideline for the assessment and management of PCOS, 2018. PCOS was diagnosed according to Rotterdam criteria and National Institute of Health (NIH) criteria. Multivariate linear and logistic regression analyses were used to examine the associations between maternal stressful life event exposure and ovarian morphology (PCOM), circulating ovarian and adrenal androgens (clinical and biochemical hyperandrogenism (hirsutism)) and presence of PCOS. RESULTS: Of 223 recruited adolescent girls, 78 (35.9%) and 68 (31.3%) had PCOM by the 2003 and 2018 criteria respectively, while 66 (29.6%) and 37 (16.6%) had PCOS, using Rotterdam and NIH criteria, respectively. Most girls (141/223, 63.2%) were exposed to at least one stressful life event in early gestation and around half (121/223, 54.3%) were exposed to at least one stressful life event in late gestation. Maternal stressful life events in early gestation were associated with a statistically significant lower prevalence of PCOM when applying the 2003 criteria [adjusted odds ratio [aOR] and 95% confidence intervals (CI): 0.74 (95% CI: 0.55; 0.99)], and a similar association was detected when applying the 2018 PCOM criteria (aOR, 0.69, 95% CI: 0.50; 0.95)]. Maternal stressful life events in early gestation were also associated with lower circulating concentrations of testosterone (ß = -0.05, 95% CI: -0.09; -0.004) and androstenedione (ß = -0.05, 95% CI: -0.10; -0.002) in the offspring. No similar effects for PCOM or circulating androgens were detected in late gestation. No statistically significant associations between maternal stressful life events in early or late gestation with PCOS (neither Rotterdam nor NIH criteria) in adolescence were detected. The prospective collection of maternal stressful life events during both early and late gestation and direct measurement of PCOM, PCOS and circulating androgens in adolescence and key co-variates implies minimal possibility of recall, information bias and selection bias. CONCLUSION: Maternal exposure to stressful life events in early gestation is associated with significantly reduced circulating ovarian and adrenal androgen concentrations in adolescence (testosterone and androstenedione), and an indication of fewer cases of polycystic ovary morphology (PCOM) defined by the 2003 international consensus definition and by the 2018 international evidence-based guideline, but has no effect on polycystic ovary syndrome (PCOS), diagnosed using either Rotterdam or NIH criteria.

Maternal serum concentrations of bisphenol A and propyl paraben in early pregnancy are associated with male infant genital development.

STUDY QUESTION: Are maternal serum phthalate metabolite, phenol and paraben concentrations measured at 10-17 weeks of gestation associated with male infant genital developmental outcomes, specifically cryptorchidism, anogenital distance (AGD), penile length and testicular descent distance, at birth and postnatally? SUMMARY ANSWER: Maternal serum bisphenol A (BPA) concentration at 10-17 weeks of gestation was positively associated with congenital or postnatally acquired cryptorchidism, and n-propyl paraben (n-PrP) concentration was associated with shorter AGD from birth to 24 months of age. WHAT IS KNOWN ALREADY: Male reproductive disorders are increasing in prevalence, which may reflect environmental influences on foetal testicular development. Animal studies have implicated phthalates, BPA and parabens, to which humans are ubiquitously exposed. However, epidemiological studies have generated conflicting results and have often been limited by small sample size and/or measurement of chemical exposures outside the most relevant developmental window. STUDY DESIGN, SIZE, DURATION: Case-control study of cryptorchidism nested within a prospective cohort study (Cambridge Baby Growth Study), with recruitment of pregnant women at 10-17 postmenstrual weeks of gestation from a single UK maternity unit between 2001 and 2009 and 24 months of infant follow-up. Of 2229 recruited women, 1640 continued with the infancy study after delivery, of whom 330 mothers of 334 male infants (30 with congenital cryptorchidism, 25 with postnatally acquired cryptorchidism and 279 unmatched controls) were included in the present analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS: Maternal blood was collected at enrolment, and serum levels of 16 phthalate metabolites, 9 phenols (including BPA) and 6 parabens were measured using liquid chromatography/tandem mass spectrometry. Logistic regression was used to model the association of cryptorchidism with serum chemical concentrations, adjusting for putative confounders. Additionally, offspring AGD, penile length and testicular descent distance were assessed at 0, 3, 12, 18 and 24 months of age, and age-specific Z scores were calculated. Associations between serum chemical levels and these outcomes were tested using linear mixed models. MAIN RESULTS AND THE ROLE OF CHANCE: Maternal serum BPA concentration was associated with offspring all-type cryptorchidism both when considered as a continuous exposure (adjusted odds ratio per log10 µg/l: 2.90, 95% CI 1.31-6.43, P = 0.009) and as quartiles (phet = 0.002). Detection of n-PrP in maternal serum was associated with shorter AGD (by 0.242 standard deviations, 95% CI 0.051-0.433, P = 0.01) from birth to 24 months of age; this reduction was independent of body size and other putative confounders. We did not find any consistent associations with offspring outcomes for the other phenols, parabens, and phthalate metabolites measured. LIMITATIONS, REASONS FOR CAUTION: We cannot discount confounding by other demographic factors or endocrine-disrupting chemicals. There may have been misclassification of chemical exposure due to use of single serum measurements. The cohort was not fully representative of pregnant women in the UK, particularly in terms of smoking prevalence and maternal ethnicity. WIDER IMPLICATIONS OF THE FINDINGS: Our observational findings support experimental evidence that intrauterine exposure to BPA and n-PrP during early gestation may adversely affect male reproductive development. More evidence is required before specific public health recommendations can be made. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by a European Union Framework V programme, the World Cancer Research Fund International, the Medical Research Council (UK), Newlife the Charity for Disabled Children, the Mothercare Group Foundation, Mead Johnson Nutrition and the National Institute for Health Research Cambridge Comprehensive Biomedical Research Centre. Visiting Fellowship (J.M.): Regional Programme 'Jiménez de la Espada' for Research Mobility, Cooperation and Internationalization, Seneca Foundation-Science and Technology Agency for the Region of Murcia (No. 20136/EE/17). K.O. is supported by the Medical Research Council (UK) (Unit Programme number: MC_UU_12015/2). The authors declare no conflict of interest.

Influence of Nodal signalling on pluripotency factor expression, tumour cell proliferation and cisplatin-sensitivity in testicular germ cell tumours.

BACKGROUND: Testicular germ cell tumours (TGCTs) are characterised by an overall high cisplatin-sensitivity which has been linked to their continued expression of pluripotency factors. Recently, the Nodal signalling pathway has been implicated in the regulation of pluripotency factor expression in fetal germ cells, and the pathway could therefore also be involved in regulating expression of pluripotency factors in malignant germ cells, and hence cisplatin-sensitivity in TGCTs. METHODS: We used in vitro culture of the TGCT-derived cell line NTera2, ex vivo tissue culture of primary TGCT specimens and xenografting of NTera2 cells into nude mice in order to investigate the consequences of manipulating Nodal and Activin signalling on pluripotency factor expression, apoptosis, proliferation and cisplatin-sensitivity. RESULTS: The Nodal signalling factors were markedly expressed concomitantly with the pluripotency factor OCT4 in GCNIS cells, seminomas and embryonal carcinomas. Despite this, inhibition of Nodal and Activin signalling either alone or simultaneously did not affect proliferation or apoptosis in malignant germ cells in vitro or ex vivo. Interestingly, inhibition of Nodal signalling in vitro reduced the expression of pluripotency factors and Nodal pathway genes, while stimulation of the pathway increased their expression. However, cisplatin-sensitivity was not affected following pharmacological inhibition of Nodal/Activin signalling or siRNA-mediated knockdown of the obligate co-receptor CRIPTO in NTera2 cells in vitro or in a xenograft model. CONCLUSION: Our findings suggest that the Nodal signalling pathway may be involved in regulating pluripotency factor expression in malignant germ cells, but manipulation of the pathway does not appear to affect cisplatin-sensitivity or tumour cell proliferation.

Peptide hormone analysis in diagnosis and treatment of Differences of Sex Development: joint position paper of EU COST Action 'DSDnet' and European Reference Network on Rare Endocrine Conditions.

Differences of Sex Development (DSD) comprise a variety of congenital conditions characterized by atypical chromosomal, gonadal, or anatomical sex. Diagnosis and monitoring of treatment of patients suspected of DSD conditions include clinical examination, measurement of peptide and steroid hormones, and genetic analysis. This position paper on peptide hormone analyses in the diagnosis and control of patients with DSD was jointly prepared by specialists in the field of DSD and/or peptide hormone analysis from the European Cooperation in Science and Technology (COST) Action DSDnet (BM1303) and the European Reference Network on rare Endocrine Conditions (Endo-ERN). The goal of this position paper on peptide hormone analysis was to establish laboratory guidelines that may contribute to improve optimal diagnosis and treatment control of DSD. The essential peptide hormones used in the management of patients with DSD conditions are follicle-stimulating hormone, luteinising hormone, anti-Müllerian hormone, and Inhibin B. In this context, the following position statements have been proposed: serum and plasma are the preferred matrices; the peptide hormones can all be measured by immunoassay, while use of LC-MS/MS technology has yet to be implemented in a diagnostic setting; sex- and age-related reference values are mandatory in the evaluation of these hormones; and except for Inhibin B, external quality assurance programs are widely available.

Dysregulation of FGFR signalling by a selective inhibitor reduces germ cell survival in human fetal gonads of both sexes and alters the somatic niche in fetal testes.

STUDY QUESTION: Does experimental manipulation of fibroblast growth factor 9 (FGF9)-signalling in human fetal gonads alter sex-specific gonadal differentiation? SUMMARY ANSWER: Inhibition of FGFR signalling following SU5402 treatment impaired germ cell survival in both sexes and severely altered the developing somatic niche in testes, while stimulation of FGF9 signalling promoted Sertoli cell proliferation in testes and inhibited meiotic entry of germ cells in ovaries. WHAT IS KNOWN ALREADY: Sex-specific differentiation of bipotential gonads involves a complex signalling cascade that includes a combination of factors promoting either testicular or ovarian differentiation and inhibition of the opposing pathway. In mice, FGF9/FGFR2 signalling has been shown to promote testicular differentiation and antagonize the female developmental pathway through inhibition of WNT4. STUDY DESIGN, SIZE, DURATION: FGF signalling was manipulated in human fetal gonads in an established ex vivo culture model by treatments with recombinant FGF9 (25 ng/ml) and the tyrosine kinase inhibitor SU5402 (10 µM) that was used to inhibit FGFR signalling. Human fetal testis and ovary tissues were cultured for 14 days and effects on gonadal development and expression of cell lineage markers were determined. PARTICIPANTS/MATERIALS, SETTING, METHODS: Gonadal tissues from 44 male and 33 female embryos/fetuses from first trimester were used for ex vivo culture experiments. Tissues were analyzed by evaluation of histology and immunohistochemical analysis of markers for germ cells, somatic cells, proliferation and apoptosis. Culture media were collected throughout the experimental period and production of steroid hormone metabolites was analyzed in media from fetal testis cultures by liquid chromatography-tandem mass spectrometry (LC-MS/MS). MAIN RESULTS AND THE ROLE OF CHANCE: Treatment with SU5402 resulted in near complete loss of gonocytes (224 vs. 14 OCT4+ cells per mm2, P < 0.05) and oogonia (1456 vs. 28 OCT4+ cells per mm2, P < 0.001) in human fetal testes and ovaries, respectively. This was a result of both increased apoptosis and reduced proliferation in the germ cells. Addition of exogenous FGF9 to the culture media resulted in a reduced number of germ cells entering meiosis in fetal ovaries (102 vs. 60 γH2AX+ germ cells per mm2, P < 0.05), while in fetal testes FGF9 stimulation resulted in an increased number of Sertoli cells (2503 vs. 3872 SOX9+ cells per mm2, P < 0.05). In fetal testes, inhibition of FGFR signalling by SU5402 treatment altered seminiferous cord morphology and reduced the AMH expression as well as the number of SOX9-positive Sertoli cells (2503 vs. 1561 SOX9+ cells per mm2, P < 0.05). In interstitial cells, reduced expression of COUP-TFII and increased expression of CYP11A1 and CYP17A1 in fetal Leydig cells was observed, although there were no subsequent changes in steroidogenesis. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Ex vivo culture may not replicate all aspects of fetal gonadal development and function in vivo. Although the effects of FGF9 were studied in ex vivo culture experiments, there is no direct evidence that FGF9 acts in vivo during human fetal gonadogenesis. The FGFR inhibitor (SU5402) used in this study is not specific to FGFR2 but inhibits all FGF receptors and off-target effects on unrelated tyrosine kinases should be considered. WIDER IMPLICATIONS OF THE FINDINGS: The findings of this study suggest that dysregulation of FGFR-mediated signalling may affect both testicular and ovarian development, in particular impacting the fetal germ cell populations in both sexes. STUDY FUNDING/COMPETING INTEREST(S): This work was supported in part by an ESPE Research Fellowship, sponsored by Novo Nordisk A/S to A.JØ. Additional funding was obtained from the Erichsen Family Fund (A.JØ.), the Aase and Ejnar Danielsens Fund (A.JØ.), the Danish Government's support for the EDMaRC programme (A.JU.) and a Wellcome Trust Intermediate Clinical Fellowship (R.T.M., Grant no. 098522). The Medical Research Council (MRC) Centre for Reproductive Health (R.T.M.) is supported by an MRC Centre Grant (MR/N022556/1). The authors have no conflict of interest to disclose.

Voice break in boys-temporal relations with other pubertal milestones and likely causal effects of BMI.

STUDY QUESTION: How is timing of voice break related to other male pubertal milestones as well as to BMI? SUMMARY ANSWER: We provide a comprehensive temporal analysis of male pubertal milestones, including reproductive hormone dynamics, confirm voice break as a late milestone of male puberty and report a likely causal relationship between higher BMI and earlier age at voice break in men. WHAT IS KNOWN ALREADY: Voice break represents a late pubertal milestone and recalled age at voice break is frequently used in epidemiological studies as a measure of puberty. In contrast, clinical studies use mainly testicular enlargement and/or genital tanner stage as the marker of pubertal onset. However, neither correlation of pubertal milestones nor reproductive hormone dynamics have been assessed in detail previously. Further, although BMI and puberty timing are known to be closely linked, cause and effect between these traits are not known. STUDY DESIGN, SIZE, DURATION: The study included a population-based mixed cross-sectional and longitudinal cohort (2006-2014, COPENHAGEN Puberty Study) of 730 healthy Danish boys. Data for 55 871 male research participants from the 23andMe study were obtained, including genome-wide single nucleotide polymorphism data and age at voice break. PARTICIPANTS/MATERIALS, SETTING, METHODS: We performed a detailed evaluation of pubertal milestones and reproductive hormone levels (study population 1). A Mendelian randomization (MR) approach was used to determine the likely causal link between BMI and timing of voice break (study population 2). MAIN RESULTS AND THE ROLE OF CHANCE: Voice break occurred at mean age 13.6 (95% CI: 13.5-13.8) years. At voice break, mean (95% CI) testosterone levels, LH levels and bi-testicular volume were 10.9 (10.0-11.7) nmol/L, 2.4 (2.2-2.5) IU/L and 24 (23-25) mL, respectively. Voice break correlated moderately strongly with timing of male pubertal milestones, including testicular enlargement, gonadarche, pubarche, sweat odor, axillary hair growth and testosterone above limit of detection (r2 range: 0.43-0.61). Timing of all milestones was negatively associated with age-specific BMI (all P ≤ 0.001). MR analyses inferred likely causal effects of higher BMI on earlier voice break in males (-0.35 years/approximate SD, P < 0.001). LIMITATIONS, REASONS FOR CAUTION: Participation rate of the population-based cohort was 25%. Further, boys that were followed longitudinally were examined approximately every 6 months limiting the time resolution of pubertal milestones. Using adult BMI as exposure instead of prepubertal BMI in the MR analysis and the known inaccuracies of the testosterone immunoassay at low testosterone levels may be further limitations. WIDER IMPLICATIONS OF THE FINDINGS: We provide valuable normative data on the temporal relation of male pubertal milestones. Further, the likely causal relationship between BMI and puberty timing highlights the importance of preventing obesity in childhood. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by Danish Agency for Science, Technology and Innovation (09-067 180); Danish Ministry of the Environment, CeHoS (MST-621-00 065); Capital Region of Denmark (R129-A3966); Ministry of Higher Education and Science (DFF-1331-00 113); Innovation Fund Denmark (InnovationsFonden, 14-2013-4); The International Center for Research and Research Training in Endocrine Disrupting Effects of Male Reproduction and Child Health. B.H., F.R.D., J.R.B.P. and K.K.O. are supported by the Medical Research Council (MC_UU_12015/2). The 23andMe study is supported by the National Human Genome Research Institute of the National Institutes of Health (R44HG006981). Members of the 23andMe Research Team are employees of 23andMe, Inc. and hold stock or stock options in 23andMe. TRIAL REGISTRATION NUMBER: NCT01411527.

The association between in-utero exposure to stressful life events during pregnancy and male reproductive function in a cohort of 20-year-old offspring: The Raine Study.

STUDY QUESTION: Is exposure to gestational stress in the critical time window for the normal differentiation and growth of male reproductive tissue associated with male reproductive function in offspring in later life? SUMMARY ANSWER: Exposure to stressful life events (SLEs) in early, but not late gestation, are associated with reduced adult male reproductive function, consistent with the hypothesis that events during early prenatal life programme adult male reproductive function. WHAT IS ALREADY KNOWN: Animal studies suggest that gestational stress may impact on the reproductive function of male offspring, but human evidence is sparse. STUDY DESIGN, SIZE, DURATION: Using a prospective longitudinal cohort, we examined the association between number and type of maternal stressors during pregnancy in both early and late gestation and reproductive function in 643 male Generation 2 (offspring) at age 20 years. Mothers and their male Generation 2 (offspring) from The Raine Study participated. Mothers prospectively reported SLEs during pregnancy recorded at gestational weeks 18 and 34 using a standardized 10-point questionnaire. PARTICIPANTS/MATERIALS, SETTING, METHODS: The 643 male Generation 2 (offspring) underwent testicular ultrasound examination and semen analysis and provided serum for reproductive hormone analysis. Multivariate linear regression analysis was used to examine associations. MAIN RESULTS AND ROLE OF CHANCE: Of 643 recruited males, 407 (63%) were exposed to at least one SLE in early gestation. Fewer SLEs were reported in late gestation (n = 343, 53%). Maternal SLE exposure in early gestation was negatively associated with total sperm count (ß = -0.31, 95% CI -0.58; -0.03), number of progressive motile sperm (ß = -0.15, 95% CI -0.31; 0.00) and morning serum testosterone concentration (ß = -0.04, 95% CI -0.09; -0.00). No similar effects of maternal SLE exposure in late pregnancy were detected. The large sample size and an objective detailed direct assessment of adult male reproductive function with strict external quality control for sperm quality, as well as detailed prospectively collected information on prenatal SLEs in two distinct time windows of pregnancy reported by the women in early and late gestation along with other risk factors, imply minimal possibility of recall, information bias and selection bias. When assessing our results, we adjusted for a priori chosen confounders, but residual confounding or confounding by factors unbeknown to us cannot be ruled out. LIMITATIONS, REASONS FOR CAUTION: It is not possible to measure how SLEs impacted differently on the mother's experience or perception of stress. Resilience (coping) gradients may alter cortisol levels and thus modify the associations we observed and the mothers' own perception of stress severity may have provided a more precise estimate of her exposure. WIDER IMPLICATIONS OF THE FINDINGS: Our findings suggest that exposure to SLEs in early, but not late gestation, are associated with reduced adult male reproductive function. Improved support for women with exposure to SLEs during pregnancy, particularly during the first trimester, may improve the reproductive health of their male offspring in later life. Intervention studies of improved pregnancy support could provide more insight into this association and more information is needed about the potential specific epigenetic mechanisms underlying this association. STUDY FUNDING/COMPETING INTEREST(S): The male fertility sub-study was funded by NHMRC Grant 634 457. The core management of the Raine Study is funded by University of Western Australia, Curtin University, Telethon Kids Institute, Women and Infants Research Foundation, Edith Cowan University, Murdoch University, The University of Notre Dame Australia and Raine Medical Research foundation. Dr Bräuner's salary was supported by Læge Sofus Carl Emil Friis og Hustru Olga Doris Friis foundation in Denmark. All authors declare no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Anogenital distance is associated with semen quality but not reproductive hormones in 1106 young men from the general population.

STUDY QUESTION: Is anogenital distance (AGD) associated with semen quality and reproductive hormones in men from the general population? SUMMARY ANSWER: Short AGD measured from the anus to the base of scrotum (AGDAS) was associated with reduced sperm counts and morphology but not with sperm motility or reproductive hormones. WHAT IS KNOWN ALREADY: AGD is longer in males than in females. In rodents, AGD is a well-established and sensitive marker of disruption during the masculinization programming window in utero and it has been suggested to be so in humans as well. Therefore, the average AGD would be expected to be shorter in men with poor semen quality, which some studies have confirmed while others have not. STUDY DESIGN, SIZE, DURATION: This cross-sectional population-based study was of 1106 men included between 2012 and 2016. PARTICIPANTS/MATERIALS, SETTING, METHODS: Men from the general Danish population (median age 19 years), unselected with regard to fertility status and semen quality, delivered a semen sample, had a blood sample drawn, which was analyzed for concentrations of reproductive hormones, and answered a comprehensive questionnaire. They also had a physical examination performed including determination of AGD measured as the distance between anus and scrotum (AGDAS) and penis (AGDAP). Odds ratios (OR) and 95% CI were estimated for a man having abnormal semen parameters according to the World Health Organization's reference values or a low/high concentration of reproductive hormones (defined as the lowest or highest 10%) depending on AGD. AGD was categorized in four strata: ≤10th percentile, 10th-30th percentile, 30th-50th percentile and >50th percentile. MAIN RESULTS AND THE ROLE OF CHANCE: Men with the 10% shortest AGDAS had a more than doubled risk (OR: 2.19, 95% CI: 1.40-3.42) of being in the subfertile range for either sperm concentration (<15 million/mL) or sperm morphology (<4%) compared to men with AGDAS above the median (reference). Men in the 10th-30th percentile also had an increased OR of 1.48 (95% CI: 1.06-2.08) but not men in the 30th-50th percentile (OR: 1.14, 95% CI: 0.81-1.62). AGDAP was only weakly related to semen quality. AGD was not associated with testicular volume or any of the reproductive hormones. LIMITATIONS, REASONS FOR CAUTION: Limitations include the potential non-differential misclassification of reproductive outcomes based on a single semen and blood sample and some between-examiner differences in AGD measurements which introduces noise and may result in an underestimation of observed associations. WIDER IMPLICATIONS OF THE FINDINGS: Our study of men from the general population confirmed associations between AGD and semen quality, supporting the hypothesis that AGD in humans could be a marker of fetal testicular development. This suggests that the low semen quality in Danish men may partly be explained by prenatal factors. STUDY FUNDING/COMPETING INTEREST(S): The study has received financial support from the ReproUnion (L.P.); the Research fund of Rigshospitalet, Copenhagen University Hospital (N.J.); Grants R01ES016863-04 and R01ES016863-02S4; National Institute of Environmental Health Sciences (NIEHS) and National Institute of Environmental Health Sciences grant (P30ES023515) (S.S.); the European Union (Contract numbers BMH4-CT96-0314, QLK4-CT-1999-01422, QLK4-CT-2002-00603, FP7/2007-2013, DEER Grant agreement no. 212844); the Danish Ministry of Health; the Danish Environmental Protection Agency; A.P. Møller and wife Chastine McKinney Møllers foundation; and Svend Andersens Foundation. None of the funders had any role in the study design, collection, analysis or interpretation of data, writing of the paper or publication decisions. The authors have nothing to declare. TRIAL REGISTRATION NUMBER: N/A.

Decrease in semen quality and Leydig cell function in infertile men: a longitudinal study.

STUDY QUESTION: Are infertile men with reduced semen quality at risk of a further decrease in testicular function? SUMMARY ANSWER: Infertile men with severely reduced semen quality risk further deterioration of semen quality 15 years after treatment for infertility, and a lower baseline sperm concentration was associated with a more pronounced increase in LH and decrease in testosterone/LH ratio at follow-up. WHAT IS KNOWN ALREADY: Male factors account for up to 50% of human infertility. The most common finding is spermatogenic failure (SgF) yet the life course of semen quality and testosterone production in such men has not been described. STUDY DESIGN, SIZE, DURATION: A follow-up study of men with SgF was performed 15 years after the initial infertility assessment between January 1995 and December 2000. PARTICIPANTS/MATERIALS, SETTING, METHODS: Hospital records were used to identify potential participants in the study. A total of 137 men with primary male infertility due to SgF and 70 controls with good semen quality from couples with female factor infertility who attended a tertiary referral centre were included: the participation rate was 31% and 26%, respectively. The men provided semen samples and underwent a physical examination. Blood samples were taken to measure levels of reproductive hormones (FSH, LH, testosterone, sex hormone-binding globulin, estradiol and inhibin B). Current results were compared with results from the initial assessments. MAIN RESULTS AND THE ROLE OF CHANCE: At the time of follow up the SgF men had significantly lower Leydig cell capacity than the control group as well as much lower semen quality. For the SgF men, between baseline sampling and follow up, the median sperm concentration decreased from 1.9 to 0.6 mill/ml and total sperm count from 7.7 to 2.0 million (P = 0.019 and 0.012, respectively), and 10% developed azoospermia. Calculated free testosterone (cFT), but not total testosterone (tT) decreased in the SgF group by ~0.6% (95% CI 0.1-1.2%) per year. In the SgF group, LH increased by 1.6% (CI 0.9-2.3%) annually, and consequently tT/LH and cFT/LH ratios had decreased by 1.3% (CI 0.5-2.1) and 2.1% (CI 1.2-3.0%), respectively. The increase in LH and the decreases in tT/LH and cFT/LH ratios were more pronounced in men with lower baseline sperm concentrations. LIMITATIONS, REASONS FOR CAUTION: We consider the case group as representative of infertile men not in need of testosterone treatment at baseline investigation, but do not have information on those that chose not to participate in the follow-up study. There were alterations in some hormone analysis methods during the follow-up period that may introduce uncertainty in interpretation of long-term changes in hormone levels despite rigorous quality control. The validity of the control group suffers from a lack of hormone values at baseline. Also, at follow-up, for practical reasons only one semen sample could be obtained, which makes the effect estimate more uncertain and there is a risk of non-differential misclassification. WIDER IMPLICATIONS OF THE FINDINGS: Without being able to predict individual outcomes, it is prudent to consider sperm cryopreservation or advise not to postpone fertility treatment when men present with infertility due to impaired semen quality. Whether partly compensated Leydig cell insufficiency in men with SgF will eventually develop into overt testosterone deficiency cannot be determined from our study. STUDY FUNDING/COMPETING INTEREST(s): Aase and Einar Danielsen (Grant no. 10-001053), Nordic Research Committee (Grant no. 5109), The Kirsten and Freddie Johansen Fund, and Rigshospitalet's Research Fund (grant no. R24-A812). There are no competing interests.