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STUDY QUESTION Is prior testicular torsion associated with testicular function (semen quality and reproductive hormones) in young men from the general population? SUMMARY ANSWER In young men from the general population, no differences in semen parameters were observed in those who had experienced testicular torsion compared to controls and observations of higher FSH and lower inhibin B were subtle. WHAT IS KNOWN ALREADY Testicular function may be impaired after testicular torsion, but knowledge is sparse and based on studies with small sample sizes and no control group or a less than ideal control group. STUDY DESIGN, SIZE, DURATION A cross-sectional population-based study was carried out including 7876 young Danish men with unknown fertility potential, examined from 1996 to 2020. PARTICIPANTS/MATERIALS, SETTING, METHODS All men (median age 19.0 years) had a physical examination, provided a blood and semen sample, and filled in a questionnaire including information about prior testicular torsion, birth, lifestyle and current and previous diseases. Markers of testicular function, including testis volume, semen parameters and reproductive hormones, were compared between men operated for testicular torsion and controls, using multiple linear regression analyses. MAIN RESULTS AND THE ROLE OF CHANCE The average participation rate was 24% for the entire study period. In total, 57 men (0.72%) were previously operated for testicular torsion (median age at surgery 13.4 years) of which five had only one remaining testicle. Men with prior testicular torsion were more often born preterm (25% versus 9.5% among controls), and they had significantly higher FSH and lower inhibin B levels, and a lower inhibin B/FSH ratio than controls in crude and adjusted models. The association was mainly driven by the subgroup of men who had undergone unilateral orchiectomy. No differences in semen parameters were observed. LIMITATIONS, REASONS FOR CAUTION A limitation is the retrospective self-reported information on testicular torsion. Also, results should be interpreted with caution owing to the high uncertainty of the observed differences. WIDER IMPLICATIONS OF THE FINDINGS Overall, the results of our study are reassuring for men who have experienced testicular torsion, especially when treated with orchiopexy, for whom reproductive hormone alterations were subtle and without obvious clinical relevance. Our study found no differences in semen parameters, but follow-up studies are needed to assess any long-term consequences for fertility. STUDY FUNDING/COMPETING INTEREST(S) Financial support was received from the Danish Ministry of Health; the Danish Environmental Protection Agency; the Research fund of Rigshospitalet, Copenhagen University Hospital; the European Union (Contract numbers BMH4-CT96-0314, QLK4-CT-1999-01422, QLK4-CT-2002-00603, FP7/2007-2013, DEER Grant agreement no. 212844); A.P. Møller and wife Chastine Mckinney Møllers Foundation; Svend Andersens Foundation; the Research Fund of the Capital Region of Denmark; and ReproUnion (EU/Interreg). The authors have nothing to declare. TRIAL REGISTRATION NUMBER N/A. [ABSTRACT FROM AUTHOR]

Study Question: Are maternal urinary isoflavone (ISO) concentrations during pregnancy associated with anogenital distance (AGD) in infants at birth, and at 6 and 12 months of age?Summary Answer: Higher maternal urinary ISO concentrations during pregnancy were associated with longer AGD in infants of both sexes, and equol (EQU) and daidzein (DAD) were identified as the important ISO mixture components in the observed associations.What Is Known Already: Evidence of the association of prenatal exposure to ISO with offspring's AGD is mainly derived from animal studies, which used different study designs and had inconsistent results. Only one human study has been reported and it found null associations between maternal ISO exposure during pregnancy and AGD among boys at birth, with a small sample size and a wide range of exposure windows. No human study on girls was found.Study Design, Size, Duration: Prospective cohort study (Shanghai-Minhang Birth Cohort Study), with pregnant women recruited at 12-16 weeks of gestation in Shanghai, China between April and December 2012. One thousand two hundred and twenty-five live singletons were left in the cohort at delivery of which 480 mother-infant pairs had data on both maternal urinary ISO concentrations and at least one AGD measurement and were included in the present study. Anopenile distance (AGDAP) and anoscrotal distance (AGDAS) of boys and anoclitoral distance (AGDAC) and anofourchette distance (AGDAF) of girls were measured at birth and at 6 and 12 months of age.Participants/materials, Setting, Methods: Multiple linear regression models were used to examine the associations between maternal ISO concentrations and AGD. Bayesian kernel machine regression (BKMR) was implemented to examine both the overall effects of ISO mixture and the single effect of each ISO and identify important components of ISO mixture.Main Results and the Role Of Chance: A general profile of higher concentrations of maternal ISO associated with longer AGD in infants of both sexes was observed, when maternal education, parity, BMI before pregnancy (BMI, categorical variable), passive smoking during early pregnancy, age at delivery, gestational weeks and infant body size were adjusted for. Among boys, EQU was associated with increased AGDAS at birth and at 6 and 12 months, and DAD was associated with increased AGDAP at birth. Among girls, the associations of EQU and DAD with increased AGDAC and AGDAF at birth were found. When gestational weight gain and feeding patterns of infants in the first 6 months were additionally adjusted for, and maternal BMI was adjusted for as a continuous variable, more pronounced associations were observed, especially for associations of genistein (GEN), DAD and glycitein (GLY) with increased AGDAP and AGDAS at 6 months in boys. However, these associations were not always observed in the highest tertile group, and no consistent dose-response relationships were found. Similar results were observed in BKMR models, showing positive correlations of concentration of ISO mixture with increased AGDAS at both 6 and 12 months among boys, and increased AGDAC and AGDAF at birth among girls. Statistically significant increments of 4.96 mm (95% credible interval (CrI): 1.40, 8.52) and 1.07 mm (95% CrI: 0.02, 2.13) in AGDAS at 6 months among boys and AGDAC at birth among girls, respectively, were observed at the 75th percentile of ISO mixture, compared with 25th percentile. EQU and DAD were identified as the important components among ISO-AGD associations.Limitations, Reasons For Caution: First, due to the short half-lives of ISO, the accuracy of a single spot urine sample reflecting ISO exposure during pregnancy may be limited, and thus may cause non-differential misclassification. Second, despite the adjustments for several important covariates in the study, unmeasured and residual confounding factors may remain a concern. Third, false discovery due to multiple testing may remain. Finally, the reduced sample sizes attributed to the loss of follow-up and missing data of confounders may limit our ability to detect an association, if any existed.Wider Implications Of the Findings: Prenatal ISO exposure may affect the reproductive development of offspring. As ISO can be widely detected in pregnant women, especially in Eastern countries, more studies are warranted to provide evidence of the effects of prenatal ISO exposure on long-term reproductive outcomes.Study Funding/competing Interest(s): This work was supported by grants from the National Key Research and Development Program of China (2021YFC2701003), the National Natural Science Foundation of China (22076123), the Science and Technology Commission of Shanghai Municipality (21ZR1454700 and 20ZR1448000), the Shanghai Municipal Health Commission (20194Y0160) and Innovation-oriented Science and Technology Grant from NHC Key Laboratory of Reproduction Regulation (CX2022-04). The authors have no conflicts of interest to declare.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

Study Question: Should testicular sperm extraction (TESE) in non-mosaic 47,XXY Klinefelter syndrome (KS) patients be performed soon after puberty or could it be delayed until adulthood?Summary Answer: The difference in sperm retrieval rate (SRR) in TESE was not significant between the 'Young' (15-22 years old) cohort and the 'Adult' (23-43 years old) cohort of non-mosaic KS patients recruited prospectively in parallel.What Is Known Already: Several studies have tried to define predictive factors for TESE outcome in non-mosaic KS patients, with very heterogeneous results. Some authors have found that age was a pejorative factor and recommended performing TESE soon after puberty. To date, no predictive factors have been unanimously recognized to guide clinicians in deciding to perform TESE in azoospermic KS patients.Study Design, Size, Duration: Two cohorts (Young: 15-22 years old; Adult: 23-43 years old) were included prospectively in parallel. A total of 157 non-mosaic 47,XXY KS patients were included between 2010 and 2020 in the reproductive medicine department of the University Hospital of Lyon, France. However 31 patients gave up before TESE, four had cryptozoospermia and three did not have a valid hormone assessment; these were excluded from this study.Participants/materials, Setting, Methods: Data for 119 patients (61 Young and 58 Adult) were analyzed. All of these patients had clinical, hormonal and seminal evaluation before conventional TESE (c-TESE).Main Results and the Role Of Chance: The global SRR was 45.4%. SRRs were not significantly different between the two age groups: Young SRR=49.2%, Adult SRR = 41.4%; P = 0.393. Anti-Müllerian hormone (AMH) and inhibin B were significantly higher in the Young group (AMH: P = 0.001, Inhibin B: P < 0.001), and also higher in patients with a positive TESE than in those with a negative TESE (AMH: P = 0.001, Inhibin B: P = 0.036). The other factors did not differ between age groups or according to TESE outcome. AMH had a better predictive value than inhibin B. SRRs were significantly higher in the upper quartile of AMH plasma levels than in the lower quartile (or in cases with AMH plasma level below the quantification limit): 67.7% versus 28.9% in the whole population (P = 0.001), 60% versus 20% in the Young group (P = 0.025) and 71.4% versus 33.3% in the Adult group (P = 0.018).Limitations, Reasons For Caution: c-TESE was performed in the whole study; we cannot rule out the possibility of different results if microsurgical TESE had been performed. Because of the limited sensitivity of inhibin B and AMH assays, a large number of patients had values lower than the quantification limits, preventing the definition a threshold below which negative TESE can be predicted.Wider Implications Of the Findings: In contrast to some studies, age did not appear as a pejorative factor when comparing patients 15-22 and 23-44 years of age. Improved accuracy of inhibin B and AMH assays in the future might still allow discrimination of patients with persistent foci of spermatogenesis and guide clinician decision-making and patient information.Study Funding/competing Interest(s): The study was supported by a grant from the French Ministry of Health D50621 (Programme Hospitalier de Recherche Clinical Régional 2008). The authors have no conflicts of interest to disclose.Trial Registration Number: NCT01918280. [ABSTRACT FROM AUTHOR]

Study Question: Is histone H4 acetylation (H4ac) altered in the seminiferous tubules of patients affected by testicular tumours?Summary Answer: A considerable dysregulation of H4ac was detected in the cells of the seminiferous tubules adjacent to testicular tumours of different aetiology and prior to any treatment, while no comparable alterations were observed in patients with disrupted spermatogenesis.What Is Known Already: Altered H4ac levels have been associated with a variety of testicular pathological conditions. However, no information has been available regarding potential alterations in the spermatogenic cells adjacent to the neoplasia in testicular tumour patients.Study Design, Size, Duration: A retrospective analysis using testicular sections from 33 men aged between 21 and 74 years old was performed. Three study groups were defined and subjected to double-blind evaluation: a control group with normal spermatogenesis (n = 6), patients with testicular tumours (n = 18) and patients with spermatogenic impairments (n = 8). One additional sample with normal spermatogenesis was used as a technical internal control in all evaluations.Participants/materials, Setting, Methods: Immunohistochemistry against H4ac and, when needed, Placental-like alkaline phosphatase and CD117, was performed on testicular sections. The H4ac H-score, based on the percentage of detection and signal intensity, was used as the scoring method for statistical analyses. Protein expression data from the Human Protein Atlas were used to compare the expression levels of predicted secreted proteins from testicular tumours with those present in the normal tissue.Main Results and the Role Of Chance: We revealed, for the first time, a dramatic disruption of the spermatogenic H4ac pattern in unaffected seminiferous tubule cells from different testicular tumour patients prior to any antineoplastic treatment, as compared to controls (P < 0.05). Since no similar alterations were associated with spermatogenic impairments and the in silico analysis revealed proteins potentially secreted by the tumour to the testicular stroma, we propose a potential paracrine effect of the neoplasia as a mechanistic hypothesis for this dysregulation.Limitations, Reasons For Caution: Statistical analyses were not performed on the hypospermatogenesis and Leydig cell tumour groups due to limited availability of samples.Wider Implications Of the Findings: To the best of our knowledge, this is the first report showing an epigenetic alteration in cells from active seminiferous tubules adjacent to tumour cells in testicular tumour patients. Our results suggest that, despite presenting spermatogenic activity, the global epigenetic dysregulation found in the testicular tumour patients could lead to molecular alterations of the male germ cells. Since testicular tumours are normally diagnosed in men at reproductive age, H4ac alterations might have an impact when these testicular tumour patients express a desire for fatherhood.Study Funding/competing Interest(s): This work was supported by the European Union Marie Curie European Training Network actions and by grants to R.O. from the 'Ministerio de Economía y Competividad (Spain)' (fondos FEDER 'una manera de hacer Europa', PI13/00699, PI16/00346 and PI20/00936) and from EU-FP7-PEOPLE-2011-ITN289880. J.C. was supported by the Sara Borrell Postdoctoral Fellowship, Acción Estratégica en Salud, CD17/00109. J.C. is a Serra Húnter fellow (Universitat de Barcelona, Generalitat de Catalunya). F.B. has received grants from the Ministerio de Educación, Cultura y Deporte para la Formación de Profesorado Universitario (Spain) (FPU15/02306). A.d.l.I. is supported by a fellowship of the Ministerio de Economía, Industria y Competitividad (Spain) (PFIS, FI17/00224). M.J. is supported by the Government of Catalonia (Generalitat de Catalunya, pla estratègic de recerca i innovació en salut, PERIS 2016-2020, SLT002/16/00337). The authors have no conflicts of interest to declare.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Study Question: Is intake of sugar-sweetened beverages (SSB) or artificially sweetened beverages (ASB) associated with testicular function in young men?Summary Answer: Among young men unaware of their semen quality and reproductive hormone levels, intake of SSBs was associated with lower sperm concentration, lower total sperm count, and a lower ratio of serum inhibin-B/FSH.What Is Known Already: SSBs may adversely impact testicular function, but results are not consistent across studies. Moreover, the associations of ASB, energy-drinks or fruit juices with testicular function are unclear.Study Design, Size, Duration: Young healthy men and unselected for fertility status men enrolled in a cross-sectional study between 2008 and 2017.Participants/materials, Setting, Methods: A total of 2935 young (median age: 19 years) men enrolled in the study. Intake of SSBs, ASBs, fruit juices, and energy-drinks was assessed with a validated food frequency questionnaire. Testicular function was assessed through conventional semen quality parameters (semen volume, sperm concentration, total count, motility and morphology), testicular volume assessed with ultrasound, and serum reproductive hormone concentrations (total testosterone, free testosterone, E2, inhibin-B, LH, FSH, sex hormone-binding globulin) were measured.Main Results and the Role Of Chance: In multivariable-adjusted analyses, men in the highest category of SSB intake (median: 1.1 servings (∼220 ml)/day) had a 13.2 million/ml lower median sperm concentration (95% CI: -21.0, -5.5) than non-consumers. A similar pattern was observed with total sperm count (-28 million (95% CI: -48, -9)), serum inhibin-B (-12 pg/ml (95% CI: -21, -4)), and inhibin-B/FSH ratio (-9 (95% CI: -18, 0)). The adjusted median difference in sperm concentration and inhibin-B associated with increasing SSB intake by 1 serving (∼200ml)/day at the expense of water was -3.4 million sperm/ml (95% CI: -5.8, -1.0) and -7 pg/ml (95% CI: -11, -3), respectively.Limitations, Reasons For Caution: Inferring causality is limited owing to the cross-sectional design. We adjusted for a number of potential confounders but cannot exclude that unmeasured lifestyle and behavior associated with soft drink intake is associated with testicular function in these young men.Wider Implications Of the Findings: In the largest study to date, intake of SSBs was associated with lower sperm concentration, total sperm count, and serum inhibin-B/FSH ratio, consistent with a direct suppressive effect of SSB intake on testicular function among otherwise healthy men, potentially affecting fertility. However, the observed association between higher SSB intake and lower semen quality does not necessarily imply a decrease in fertility.Study Funding/competing Interest(s): Supported by research from the Danish Council for Strategic Research (2101-08-0058), Independent Research Fund Denmark (8020-00218B), European Union (212844), the Kirsten and Freddy Johansen's Foundation (95-103-72087), the Research Fund of the Capital Region of Denmark (A6176), and the NIH (P30DK046200). The authors report no conflict of interest.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

Study Question: How are germ cell numbers and initiation of folliculogenesis affected in fetal Turner syndrome (TS) ovaries?Summary Answer: Germ cell development was severely affected already in early second trimester pregnancies, including accelerated oogonia loss and impaired initiation of primordial follicle formation in TS ovaries, while the phenotype in TS mosaic ovaries was less severe.What Is Known Already: Females with TS are characterized by premature ovarian insufficiency (POI). This phenotype is proposed to be a consequence of germ cell loss during development, but the timing and mechanisms behind this are not characterized in detail. Only few studies have evaluated germ cell development in fetal TS and TS mosaic ovaries, and with a sparse number of specimens included per study.Study Design, Size, Duration: This study included a total of 102 formalin-fixed and paraffin-embedded fetal ovarian tissue specimens. Specimens included were from fetuses with 45,X (N = 42 aged gestational week (GW) 12-20, except one GW 40 sample), 45,X/46,XX (N = 7, aged GW 12-20), and from controls (N = 53, aged GW 12-42) from a biobank (ethics approval # H-2-2014-103).Participants/materials, Setting, Methods: The number of OCT4 positive germ cells/mm2, follicles (primordial and primary)/mm2 and cPARP positive cells/mm2 were quantified in fetal ovarian tissue from TS, TS mosaic and controls following morphological and immunohistochemical analysis.Main Results and the Role Of Chance: After adjusting for gestational age, the number of OCT4+ oogonia was significantly higher in control ovaries (N = 53) versus 45,X ovaries (N = 40, P < 0.001), as well as in control ovaries versus 45,X/46,XX mosaic ovaries (N = 7, P < 0.043). Accordingly, the numbers of follicles were significantly higher in control ovaries versus 45,X and 45,X/46,XX ovaries from GW 16-20 with a median range of 154 (N = 11) versus 0 (N = 24) versus 3 (N = 5) (P < 0.001 and P < 0.015, respectively). The number of follicles was also significantly higher in 45,X/46,XX mosaic ovaries from GW 16-20 compared with 45,X ovaries (P < 0.005). Additionally, the numbers of apoptotic cells determined as cPARP+ cells/mm2 were significantly higher in ovaries 45,X (n = 39) versus controls (n = 15, P = 0.001) from GW 12-20 after adjusting for GW.Limitations, Reasons For Caution: The analysis of OCT4+ cells/mm2, cPARP+ cells/mm2 and follicles (primordial and primary)/mm2 should be considered semi-quantitative as it was not possible to use quantification by stereology. The heterogeneous distribution of follicles in the ovarian cortex warrants a cautious interpretation of the exact quantitative numbers reported. Moreover, only one 45,X specimen and no 45,X/46,XX specimens aged above GW 20 were available for this study, which unfortunately made it impossible to assess whether the ovarian folliculogenesis was delayed or absent in the TS and TS mosaic specimens.Wider Implications Of the Findings: This human study provides insights about the timing of accelerated fetal germ cell loss in TS. Knowledge about the biological mechanism of POI in girls with TS is clinically useful when counseling patients about expected ovarian function and fertility preservation strategies.Study Funding/competing Interest(s): This work was supported by the International Center for Research and Research Training in Endocrine Disruption of Male Reproduction and Child Health (EDMaRC).Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

Study Question: Do selective serotonin reuptake inhibitor (SSRI) antidepressants affect the function of human sperm?Summary Answer: The SSRI antidepressant Sertraline (e.g. Zoloft) inhibits the sperm-specific Ca2+ channel CatSper and affects human sperm function in vitro.What Is Known Already: In human sperm, CatSper translates changes of the chemical microenvironment into changes of the intracellular Ca2+ concentration ([Ca2+]i) and swimming behavior. CatSper is promiscuously activated by oviductal ligands, but also by synthetic chemicals that might disturb the fertilization process. It is well known that SSRIs have off-target actions on Ca2+, Na+ and K+ channels in somatic cells. Whether SSRIs affect the activity of CatSper is, however, unknown.Study Design, Size, Duration: We studied the action of the seven drugs belonging to the most commonly prescribed class of antidepressants, SSRIs, on resting [Ca2+]i and Ca2+ influx via CatSper in human sperm. The SSRI Sertraline was selected for in-depth analysis of its action on steroid-, prostaglandin-, pH- and voltage-activation of human CatSper. Moreover, the action of Sertraline on sperm acrosomal exocytosis and penetration into viscous media was evaluated.Participants/materials, Setting, Methods: The activity of CatSper was investigated in sperm of healthy volunteers, using kinetic Ca2+ fluorimetry and patch-clamp recordings. Acrosomal exocytosis was investigated using Pisum sativum agglutinin and image cytometry. Sperm penetration in viscous media was evaluated using the Kremer test.Main Results and the Role Of Chance: Several SSRIs affected [Ca2+]i and attenuated ligand-induced Ca2+ influx via CatSper. In particular, the SSRI Sertraline almost completely suppressed Ca2+ influx via CatSper. Remarkably, the drug was about four-fold more potent to suppress prostaglandin- versus steroid-induced Ca2+ influx. Sertraline also suppressed alkaline- and voltage-activation of CatSper, indicating that the drug directly inhibits the channel. Finally, Sertraline impaired ligand-induced acrosome reaction and sperm penetration into viscous media.Limitations, Reasons For Caution: This is an in vitro study. Future studies have to assess the physiological relevance in vivo.Wider Implications Of the Findings: The off-target action of Sertraline on CatSper in human sperm might impair the fertilization process. In a research setting, Sertraline may be used to selectively inhibit prostaglandin-induced Ca2+ influx.Study Funding/competing Interest(s): This work was supported by the Swiss Centre for Applied Human Toxicology (SCAHT), the Département de l'Instruction Publique of the State of Geneva, the German Research Foundation (CRU326), the Interdisciplinary Center for Clinical Research, Münster (IZKF; Str/014/21), the Innovation Fund Denmark (grant numbers 14-2013-4) and the EDMaRC research grant from the Kirsten and Freddy Johansen's Foundation. The authors declare that no conflict of interest could be perceived as prejudicing the impartiality of the research reported.Trial Registration Number: NA. [ABSTRACT FROM AUTHOR]

Study Question: Are there temporal trends of sperm concentration (SC) and total sperm count (TSC) in Chinese healthy males from 1981 to 2019?Summary Answer: Our result indicated a temporal decrease in SC and TSC among 327 373 healthy Chinese men in the recent four decades.What Is Known Already: A review of 61 papers reported a temporal decline in SC and TSC from 1938 to 1990. This trend was later confirmed by a systematic review of 185 published papers from 1981 to 2013. However, the majority of the included individuals were from western countries. In China, whether SC and TSC have declined remains controversial.Study Design, Size, Duration: This systematic review of published articles used data extracted from Pubmed, Science Direct, Embase, China-National-Knowledge-Infrastructure (CNKI) and Wanfang Data to assess changes in SC and TSC in China from 1981 to 2019.Participants/materials, Setting, Methods: A total of 111 studies including 327 373 individuals who provided semen samples from 1981 to 2019 were extracted for the present analysis. Study selection and data extraction were performed by two independent researchers. The trends in SC and TSC were analysed using liner-regression and meta-regression before and after adjusting for potential covariates. Moreover, subgroups, categorised based on geographic region, fertility status or recruitment source, were also analysed.Main Results and the Role Of Chance: SC declined significantly (slope liner-regression = -0.748 million/ml/year; P = 0.005; slope meta-regression = -0.824 million/ml/year; P < 0.001) between 1981 and 2019 in China. Trends for TSC was similar to that for SC (slope liner-regression = -2.073 million/year; P = 0.032; slope meta-regression = -2.188 million/year; P = 0.003). In subgroup meta-regression analyses, males with definite fertility had continuous declines in SC (slope northern group=-2.268, P = 0.009; slope southern group=-1.014, P = 0.009) and TSC (slope northern group=-9.675, P = 0.010; slope southern group=-3.215, P = 0.042). However, in the unselected group, where fertility status was unknown, the obvious downward trend in SC was only seen in males from Northern regions (slope = -0.836, P = 0.003). Another subgroup analysis demonstrated that obvious decreases in SC (slope = -1.432, P < 0.001) and TSC (slope=-4.315, P = 0.001) were only seen in volunteer groups but not in pre-pregnancy examination groups and other recruitment groups. The results changed minimally in multiple sensitivity analyses.Limitations, Reasons For Caution: The validity of the meta-analysis results was limited mainly by the quality of the included studies. Additionally, our study spanned many decades and the recommended criteria for some semen parameter assessments have significantly changed, which may bring about some unavoidable bias. Moreover, the data remain insufficient especially in some provinces of China.Wider Implications Of the Findings: The present study is the first study to report significant decreases in SC and TSC in 327 373 healthy Chinese men between 1981 and 2019, indicating a serious reproductive health warning. Further studies on the causes of the declines are urgently needed.Study Funding/competing Interest(s): D.Z. is supported by the National Natural Science Funding of China, Natural Science Funding of Shaanxi Province, Science Funding of Health Department, Shaanxi Province, Fundamental Research Funds for the Central University and the Project of Independent Innovative Experiment for Postgraduates in Medicine in Xi'an Jiaotong University. The authors have no conflicts of interests to declare.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

Study Question: Does pituitary response to a GnRH stimulation test differ according to the different FSHB-211 G/T genotypes?Summary Answer: The promoter polymorphism FSHB-211 G > T affects the pituitary response to exogenous GnRH stimulation by reducing FSH and increasing LH outputs.What Is Known Already: The FSHB-211 G > T single nucleotide polymorphism (SNP) is known to affect pituitary FSH output by impairing the transcriptional activity of FSHB.Study Design, Size, Duration: This was a cross-sectional, retrospective study on 67 male subjects (mean age: 24.6 ± 10.3 years) undergoing a GnRH stimulation test for diagnostic purposes in cases of secondary hypogonadism.Participants/materials, Setting, Methods: A GnRH stimulation test was performed by administering an i.v. bolus of 100 µg of the GnRH-analogue gonadorelin acetate to all patients, with blood samples drawn from the cubital vein immediately prior to injection (T0) and 30 (T1) and 45 minutes (T2) after. Clinical and genetic data were retrieved from a computerized database. Linear longitudinal mixed-effect models were used to assess the effects of SNP genotype on FSH and LH levels over time via additive and recessive models.Main Results and the Role Of Chance: An overall marked increase in serum FSH and LH following administration i.v. of 100 µg of an LHRH-analogue was found (P < 0.0001 for linear trend, both models). Peak levels of LH were significantly higher in TT carriers than in GT and GG carriers (P = 0.012); no significant between-groups difference was found concerning stimulated FSH levels. In both the additive and recessive model, the main effect of T allele(s) did not reach statistical significance concerning FSH levels (P = 0.9502 and P = 0.8576, respectively), yet interaction effects over time demonstrated an attenuated response in T-allele carriers compared to the GG-allele carriers (P = 0.0219 and P = 0.0276). Main and interaction effects for LH were significant in both the additive (P = 0.0022 and P = 0.0013, respectively) and recessive model (P = 0.0025 and P = 0.0016, respectively).Limitations, Reasons For Caution: Given the retrospective nature of the study and the small number of TT carriers, results should be interpreted with caution.Wider Implications Of the Findings: The FSHB c.-211G>T polymorphism might result in an impaired response to endogenous, as well as exogenous, GnRH stimulation. This finding might contribute to the clinical phenotype of reduced testicular volume and sperm count for patients carrying one or two T alleles.Study Funding/competing Interest(s): Parts of the study were supported by the German Research Foundation (CRU326 Male Germ Cells). On behalf of all authors, the corresponding author states that there is no conflict of interest.Trial Registration Number: NA. [ABSTRACT FROM AUTHOR]

Study Question: Is testicular function associated within father-son pairs?Summary Answer: Familial resemblance in testis volume and serum markers of spermatogenesis was observed in father-son pairs.What Is Known Already: Studies suggest familial clustering of male subfertility and impaired spermatogenesis, but in men from the general population little is known about concordance in testicular function between fathers and sons.Study Design, Size, Duration: This cross-sectional study with simultaneous collection of data in fathers and sons included 72 pairs (144 fathers and sons), unselected regarding testicular function were included.Participants/materials, Setting, Methods: A subgroup of men from the background population and participating in a study on testicular function were asked permission to invite their fathers to participate in a similar setup. Fathers (median age of 53 years) and sons (median age of 19 years) participated in the same study setup including assessment of testis size, having a blood sample taken and analysed for serum levels of reproductive hormones (FSH, inhibin B, LH, testosterone, oestradiol, sex hormone-binding globulin (SHBG) and calculated free testosterone) and delivering a semen sample for assessment of traditional semen parameters. Mixed-effects models were fitted to estimate the familial resemblance as the proportion of variance in markers of testicular function due to shared factors for fathers and sons accounted for using random-effects. Variance components were calculated from both unadjusted and adjusted models.Main Results and the Role Of Chance: After adjustments, variance component analyses showed that familial resemblance between fathers and sons accounted for 48% (P < 0.001) of the variation in testicular volume, 32% (P = 0.009) of the variation in FSH, 31% (P = 0.009) of the variation in the inhibin B/FSH ratio, 33% (P = 0.007) and 45% (P < 0.001) of the variation in testosterone and free testosterone, respectively, and 31% (P = 0.009) of the variation in SHBG. None of the semen parameters were associated within father-son pairs.Limitations, Reasons For Caution: The present study may have lacked power to detect associations for semen quality, as large intra- and inter-individual variation occur in semen parameters.Wider Implications Of the Findings: In this study, testis volume, serum testosterone and serum markers of spermatogenesis including FSH were associated in fathers and sons, suggesting an impact of paternal genetics for testicular function in the son. However, the estimated familial resemblance for spermatogenesis markers highlights that other factors, such as maternal genetics and prenatal as well as adult exposures, are also of major importance for testicular function.Study Funding/competing Interest(s): The study has received funding from Danish Health Authority, Research Fund of the Capital Region of Denmark and Independent Research Fund Denmark (8020-00218B). None of the funders had any role in the study design, collection, analysis or interpretation of data, writing of the paper of publication decisions. The authors have nothing to disclose.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

Study Question: What are the consequences of ageing on human Leydig cell number and hormonal function?Summary Answer: Leydig cell number significantly decreases in parallel with INSL3 expression and Sertoli cell number in aged men, yet the in vitro Leydig cell androgenic potential does not appear to be compromised by advancing age.What Is Known Already: There is extensive evidence that ageing is accompanied by decline in serum testosterone levels, a general involution of testis morphology and reduced spermatogenic function. A few studies have previously addressed single features of the human aged testis phenotype one at a time, but mostly in tissue from patients with prostate cancer.Study Design, Size, Duration: This comprehensive study examined testis morphology, Leydig cell and Sertoli cell number, steroidogenic enzyme expression, INSL3 expression and androgen secretion by testicular fragments in vitro. The majority of these endpoints were concomitantly evaluated in the same individuals that all displayed complete spermatogenesis.Participants/materials, Setting, Methods: Testis biopsies were obtained from 15 heart beating organ donors (age range: 19-85 years) and 24 patients (age range: 19-45 years) with complete spermatogenesis. Leydig cells and Sertoli cells were counted following identification by immunohistochemical staining of specific cell markers. Gene expression analysis of INSL3 and steroidogenic enzymes was carried out by qRT-PCR. Secretion of 17-OH-progesterone, dehydroepiandrosterone, androstenedione and testosterone by in vitro cultured testis fragments was measured by LC-MS/MS. All endpoints were analysed in relation to age.Main Results and the Role Of Chance: Increasing age was negatively associated with Leydig cell number (R = -0.49; P < 0.01) and concomitantly with the Sertoli cell population size (R= -0.55; P < 0.001). A positive correlation (R = 0.57; P < 0.001) between Sertoli cell and Leydig cell numbers was detected at all ages, indicating that somatic cell attrition is a relevant cellular manifestation of human testis status during ageing. INSL3 mRNA expression (R= -0.52; P < 0.05) changed in parallel with Leydig cell number and age. Importantly, steroidogenic capacity of Leydig cells in cultured testis tissue fragments from young and old donors did not differ. Consistently, age did not influence the mRNA expression of steroidogenic enzymes. The described changes in Leydig cell phenotype with ageing are strengthened by the fact that the different age-related effects were mostly evaluated in tissue from the same men.Limitations, Reasons For Caution: In vitro androgen production analysis could not be correlated with in vivo hormone values of the organ donors. In addition, the number of samples was relatively small and there was scarce information about the concomitant presence of potential confounding variables.Wider Implications Of the Findings: This study provides a novel insight into the effects of ageing on human Leydig cell status. The correlation between Leydig cell number and Sertoli cell number at any age implies a connection between these two cell types, which may be of particular relevance in understanding male reproductive disorders in the elderly. However aged Leydig cells do not lose their in vitro ability to produce androgens. Our data have implications in the understanding of the physiological role and regulation of intratesticular sex steroid levels during the complex process of ageing in humans.Study Funding/competing Interest(s): This work was supported by grants from Prin 2010 and 2017. The authors have no conflicts of interest.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

Study Question: Are GnRH tests and serum inhibin B levels sufficiently discriminating to distinguish transient constitutional delay of growth and puberty (CDGP) from congenital hypogonadotropic hypogonadism (CHH) that affects reproductive health for life?Summary Answer: Both parameters lack the specificity to discriminate CDGP from CHH.What Is Known Already: GnRH tests and inhibin B levels have been proposed to differentiate CDGP from CHH. However, their diagnostic accuracies have been hampered by the small numbers of CHH included and enrichment of CHH patients with more severe forms.Study Design, Size, Duration: The aim of this study was to assess the diagnostic performance of GnRH tests and inhibin B measurements in a large cohort of CHH male patients with the whole reproductive spectrum. From 2008 to 2018, 232 males were assessed: 127 with CHH, 74 with CDGP and 31 healthy controls.Participants/materials, Setting, Methods: The participants were enrolled in two French academic referral centres. The following measurements were taken: testicular volume (TV), serum testosterone, inhibin B, LH and FSH, both at baseline and following the GnRH test.Main Results and the Role Of Chance: Among CHH patients, the LH response to the GnRH test was very variable and correlated with TV. Among CDGP patients, the LH peak was also variable and 47% of CHH patients had peak LH levels overlapping with the CDGP group. However, no patients with CDGP had an LH peak below 4.0 IU/l, while 53% CHH patients had LH peak below this threshold. Among CHH patients, inhibin B levels were also variable and correlated with TV and peak LH. Inhibin B was significantly lower in CHH patients than in CDGP patients but 50% of CHH values overlapped with CDGP values. Interestingly, all patients with CDGP had inhibin B levels above 35 pg/ml but 50% of CHH patients also had levels above this threshold.Limitations, Reasons For Caution: As CHH is very rare, an international study would be necessary to recruit a larger CHH cohort and consolidate the conclusion reached here.Wider Implications Of the Findings: Peak LH and basal inhibin B levels are variable in both CHH and CDGP with significant overlap. Both parameters lack specificity and sensitivity to efficiently discriminate CHH from CDGP. This reflects the varying degree of gonadotropin deficiency inherent to CHH. These two diagnostic procedures may misdiagnose partial forms of isolated (non-syndromic) CHH, allowing them to be erroneously considered as CDGP.Study Funding/competing Interest(s): This study was funded by Agence Française de Lutte contre le Dopage: Grant Hypoproteo AFLD-10 (to J.Y.); Agence Nationale de la Recherche (ANR): Grant ANR-09-GENO-017-01 (to J.Y.); European Cooperation in Science and Technology, COST Action BM1105; Programme Hospitalier de Recherche Clinique (PHRC), French Ministry of Health: PHRC-2009 HYPO-PROTEO (to J.Y.); and Programme Hospitalier de Recherche Clinique (PHRC) "Variété", French Ministry of Health, N° P081216/IDRCB 2009-A00892-55 (to P.C.). There are no competing interests.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

Study Question: What is the course of the LH/FSH ratio from infancy into adulthood in healthy individuals and in patients with Differences of Sex Development (DSD)?Summary Answer: The LH/FSH ratio had a marked overlap between the sexes after infancy and onwards throughout adulthood in healthy individuals and it was not a marker of hypogonadism in DSD patients.What Is Known Already: The LH/FSH ratio is a distinct marker of sex during minipuberty. No study has evaluated the LH/FSH ratio from infancy into adulthood.Study Design, Size, Duration: This was a combined study of prospective longitudinal and cross-sectional cohorts of healthy individuals totaling 6417 males and females aged 0-80 years. Retrospective data from a single, tertiary center on 125 patients with DSD was also included.Participants/materials, Setting, Methods: Based on the healthy males (n = 3144) and females (n = 3273) aged 0-80 years, reference ranges for LH, FSH and the LH/FSH ratio were established from infancy (after minipuberty) and onwards. LH, FSH, and the LH/FSH ratio in 125 patients with DSD not undergoing treatment were compared to the reference ranges. Included DSD diagnoses were: Klinefelter syndrome including mosaic variants (males: n = 14), Turner syndrome including mosaic variants without Y-chromosome material (females: n = 48), 45,X/46,XY mosaicism (males: n = 24 and females: n = 6), partial androgen insensitivity syndrome (males: n = 11), complete androgen insensitivity syndrome (females: n = 13) and anorchia (males: n = 9).Main Results and the Role Of Chance: An overlap was observed in the LH/FSH ratio reference curves between males and females. However, when comparing the sexes at specific time points, the LH/FSH ratio was significantly higher in healthy males during childhood and adulthood and significantly higher in healthy females during puberty. When compared with healthy participants, male patients with anorchia and 45,X/46,XY mosaicism had significantly lower ratios, while patients with androgen insensitivity, regardless of sex, had significantly higher ratios.Limitations, Reasons For Caution: The limitations of this study include that; (i) all healthy individuals were Caucasian, so conclusions may not apply to non-Caucasians; (ii) the calculated LH/FSH ratios were restricted to the specific analytical method used and may not be applicable to other laboratories; (iii) the samples from healthy individuals were stored for varying amounts of time up to 20 years which may affect the durability; and (iv) DSD diagnoses are heterogeneous thus making sturdy conclusions across diagnoses impossible.Wider Implications Of the Findings: In this study of combined cohorts of healthy participants, the largest normative ranges of LH, FSH, and the LH/FSH ratio to date were created. These reference ranges provide the opportunity for clinical as well as research use for all three markers. However, the previously rather undescribed LH/FSH ratio was not a distinct marker of sex after infancy nor a new marker of hypogonadism. Although there were significant differences between subgroups of DSD patients compared to healthy controls, the clinical significance of the LH/FSH ratio after infancy lacked. However, it can be speculated whether there are other areas of clinical application not investigated in this article, for example as a marker of fertility in select patient groups. As gonadotropin assays are readily available and gonadotropin measurements are part of regular workups, the LH/FSH ratio can easily be explored in further research without additional costs.Study Funding/competing Interest(s): M.L.L. was funded by the Absalon Foundation. Cohort 1 was funded by the European Commission, through the Biomed 2 Program (BMH4-CT96-0314), Environmental Reproductive Health (QLK4-CT1999-01422) and EXPORED (QLK4-2001-00269), by the Danish Council for Independent Research (9700833 and 9700909), and by the Svend Andersens Foundation. Cohort 2 was funded by the Danish Environmental Research Program (96.01.015.16.05). Cohort 3 was funded by Kirsten and Freddy Johansens Foundation.Trial Registration Number: NA.Date Of First Patient’s Enrolment: June 1990 (the launch of the department from which this project stems). [ABSTRACT FROM AUTHOR]

Study Question: Is WNT signalling functional in normal and/or neoplastic human male germ cells?Summary Answer: Regulated WNT signalling component synthesis in human testes indicates that WNT pathway function changes during normal spermatogenesis and is active in testicular germ cell tumours (TGCTs), and that WNT pathway blockade may restrict seminoma growth and migration.What Is Known Already: Regulated WNT signalling governs many developmental processes, including those affecting male fertility during early germ cell development at embryonic and adult (spermatogonial) ages in mice. In addition, although many cancers arise from WNT signalling alterations, the functional relevance and WNT pathway components in TGCT, including germ cell neoplasia in situ (GCNIS), are unknown.Study Design, Size, Duration: The cellular distribution of transcripts and proteins in WNT signalling pathways was assessed in fixed human testis sections with normal spermatogenesis, GCNIS and seminoma (2-16 individuals per condition). Short-term (1-7 h) ligand activation and long-term (1-5 days) functional outcomes were examined using the well-characterised seminoma cell line, TCam-2. Pathway inhibition used siRNA or chemical exposures over 5 days to assess survival and migration.Participants/materials, Setting, Methods: The cellular localisation of WNT signalling components was determined using in situ hybridisation and immunohistochemistry on Bouin's- and formalin-fixed human testis sections with complete spermatogenesis or germ cell neoplasia, and was also assessed in TCam-2 cells. Pathway function tests included exposure of TCam-2 cells to ligands, small molecules and siRNAs. Outcomes were measured by monitoring beta-catenin (CTNNB1) intracellular localisation, cell counting and gap closure measurements.Main Results and the Role Of Chance: Detection of nuclear-localised beta-catenin (CTNNB1), and key WNT signalling components (including WNT3A, AXIN2, TCF7L1 and TCF7L2) indicate dynamic and cell-specific pathway activity in the adult human testis. Their presence in germ cell neoplasia and functional analyses in TCam-2 cells indicate roles for active canonical WNT signalling in TGCT relating to viability and migration. All data were analysed to determine statistical significance.Large Scale Data: No large-scale datasets were generated in this study.Limitations, Reasons For Caution: As TGCTs are rare and morphologically heterogeneous, functional studies in primary cancer cells were not performed. Functional analysis was performed with the only well-characterised, widely accepted seminoma-derived cell line.Wider Implications Of the Findings: This study demonstrated the potential sites and involvement of the WNT pathway in human spermatogenesis, revealing similarities with murine testis that suggest the potential for functional conservation during normal spermatogenesis. Evidence that inhibition of canonical WNT signalling leads to loss of viability and migratory activity in seminoma cells suggests that potential treatments using small molecule or siRNA inhibitors may be suitable for patients with metastatic TGCTs.Study Funding and Competing Interest(s): This study was funded by National Health and Medical Research Council of Australia (Project ID 1011340 to K.L.L. and H.E.A., and Fellowship ID 1079646 to K.L.L.) and supported by the Victorian Government's Operational Infrastructure Support Program. None of the authors have any competing interests. [ABSTRACT FROM AUTHOR]

Study Question: Is there a difference in testicular function in early adulthood between men born with cryptorchidism and men born with normally descended testes?Summary Answer: In men from the general population, a history of cryptorchidism was associated with lower total testis volume and impaired semen quality as well as altered serum levels of reproductive hormones.What Is Known Already: The association between cryptorchidism and testicular function is well documented in studies based on sub-fertile or infertile men recruited from a clinical setting. However, the association has not previously been investigated in men from the general population, who were unselected regarding fertility status.Study Design, Size, Duration: This is a cross-sectional population-based study of 6376 young Danish men examined from 1996 to 2017.Participants/materials, Setting, Methods: This study is based on young men from the greater Copenhagen area, Denmark (median age of 19 years) who were unselected regarding fertility status and semen quality. The young men delivered a semen sample, had a blood sample drawn and underwent a physical examination including assessment of testis volume. Participants completed a questionnaire regarding cryptorchidism at birth, current lifestyle and their mother's pregnancy, after consulting their mother. The differences in markers of testicular function, including testis volume, semen parameters and reproductive hormones between men with and without a history of cryptorchidism were investigated with multiple linear regression analyses.Main Results and the Role Of Chance: The participation rate was 24% for the entire study period. Overall, a history of cryptorchidism was associated with reduced testicular function. In the adjusted models, a history of cryptorchidism was associated with a 3.5 ml lower total testis volume, determined by orchidometer (P < 0.001), 28% lower sperm concentration (95% CI: -37 to -20) and 26% lower inhibin B/FSH ratio (95% CI: -50 to -22) compared to men without a history of cryptorchidism, suggesting a reduced spermatogenetic capacity. Men with a history of cryptorchidism also had a slightly reduced Leydig cell function expressed as a 6% lower testosterone/LH ratio (95% CI: -12 to -0.7). The significant effect sizes and different markers of testicular function pointing in the same direction across the different models based on a large sample size support that the results are not chance findings.Limitations, Reasons For Caution: Information on cryptorchidism at birth and treatment modus was obtained by retrospective self-report, and each participant only delivered one semen sample.Wider Implications Of the Findings: The results suggest that men with a history of cryptorchidism could be at increased risk of experiencing fertility problems. However, among these men there is a wide variation in semen quality and further research is needed in order to identify the subgroup of boys born with cryptorchidism who are at the greatest risk of impaired semen quality when reaching adulthood.Study Funding/competing Interest(s): The study received financial support from the Research fund of Rigshospitalet, Copenhagen University Hospital; 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 founders 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. [ABSTRACT FROM AUTHOR]

Study Question: Does the X chromosome inactivation (XCI) of Klinefelter syndrome (KS)-derived human induced pluripotent stem cells (hiPSCs) correspond to female human pluripotent stem cells (hPSCs) and reflect the KS genotype?Summary Answer: Our results demonstrate for the first time that KS-derived hiPSCs show similar XCI behavior to female hPSCs in culture and show biological relevance to KS genotype-related clinical features.What Is Known Already: So far, assessment of XCI of KS-derived hiPSCs was based on H3K27me3 staining and X-inactive specific transcript gene expression disregarding the at least three XCI states (XaXi with XIST coating, XaXi lacking XIST coating, and XaXe (partially eroded XCI)) that female hPSCs display in culture.Study Design, Size, Duration: The study used hiPSC lines generated from two azoospermic patients with KS and included two healthy male (HM) and one healthy female donor.Participants/materials, Setting, Methods: In this study, we derived hiPSCs by reprograming fibroblasts with episomal plasmids and applying laminin 521 as culture substrate. hiPSCs were characterized by karyotyping, immunocytochemistry, immunohistochemistry, quantitative PCR, teratoma formation, and embryoid body differentiation. XCI and KS hiPSC relevance were assessed by whole genome transcriptomics analysis and immunocytochemistry plus FISH of KS, HM and female fibroblast, and their hiPSC derivatives.Main Results and the Role Of Chance: Applying whole genome transcriptomics analysis, we could identify differentially expressed genes (DEGs) between KS and HM donors with enrichment in gene ontology terms associated with fertility, cardiovascular development, ossification, and brain development, all associated with KS genotype-related clinical features. Furthermore, XCI analysis based on transcriptomics data, RNA FISH, and H3K27me3 staining revealed variable XCI states of KS hiPSCs similar to female hiPSCs, showing either normal (XaXi) or eroded (XaXe) XCI. KS hiPSCs with normal XCI showed nevertheless upregulated X-linked genes involved in nervous system development as well as synaptic transmission, supporting the potential use of KS-derived hiPSCs as an in vitro model for KS.Limitations, Reasons For Caution: Detailed clinical information for patients included in this study was not available. Although a correlation between DEGs and the KS genotype could be observed, the biological relevance of these cells has to be confirmed with further experiments. In addition, karyotype analysis for two hiPSC lines was performed at passage 12 but not repeated at a later passage. Nevertheless, since all XCI experiments for those lines were performed between passage 11 and 15 the authors expect no karyotypic changes for those experiments.Wider Implications Of the Findings: As KS patients have variable clinical phenotypes that are influenced by the grade of aneuploidy, mosaicism, origin of the X chromosome, and XCI 'escapee' genes, which vary not only among individuals but also among different tissues within the same individual, differentiated KS hiPSCs could be used for a better understanding of KS pathogenesis.Study Funding/competing Interest(s): This study was supported by grants from the Knut and Alice Wallenberg Foundation (2016.0121 and 2015.0096), Ming Wai Lau Centre for Reparative Medicine (2-343/2016), Ragnar Söderberg Foundation (M67/13), Swedish Research Council (2013-32485-100360-69), the Centre for Innovative Medicine (2-388/2016-40), Kronprinsessan Lovisas Förening För Barnasjukvård/Stiftelsen Axel Tielmans Minnesfond, Samariten Foundation, Jonasson Center at the Royal Institute of Technology, Sweden, and Initial Training Network Marie Curie Program 'Growsperm' (EU-FP7-PEOPLE-2013-ITN 603568). The authors declare no conflicts of interest. [ABSTRACT FROM AUTHOR]

Study Question: When should 'not so rare' Leydig cell tumors (LCTs) of the testis be suspected, diagnosed, and treated?Summary Answer: LCTs are more frequent than generally believed, are associated with male infertility, cryptorchidism and gynecomastia, and should be treated conservatively (in compliant patients) with active surveillance, which appears to be a safe alternative to surgical enucleation.What Is Known Already: Increasing referrals for testicular imaging have led to an increase in findings of LCTs. The features and natural history of these tumors remain largely unknown, as the available studies are small and heterogeneous. LCTs were previously treated aggressively and follow-up data are lacking.Study Design, Size, Duration: A case-cohort study of consecutive patients diagnosed with LCTs over a 10-year period was prospectively enrolled from 2009 to 2018 and compared to matched cohorts of patients with seminomas or no testicular lesions screened in the same timeframe.Participants/materials, Setting, Methods: Of the 9949 inpatients and outpatients referred for scrotal ultrasound, a total of 83 men with LCTs were included. Enrolled subjects underwent medical history and clinical examination and were asked to undergo routine blood tests, hormone investigations (FSH, LH, total testosterone, estradiol, inhibin B, sex hormone-binding globulin (SHBG), prolactin), and semen analysis. Patients who consented also underwent contrast-enhanced ultrasound, elastography, gadolinium-enhanced scrotal magnetic resonance imaging, and hCG stimulation test (5000 IU i.m.) with serum total testosterone and estradiol measured at 0, 24, 48, and 72 hours.Main Results and the Role Of Chance: In total, 83 patients diagnosed with LCTs were compared against 90 patients diagnosed with seminoma and 2683 patients without testicular lesions (NoL). LCTs were diagnosed by enucleation (48.2%), orchiectomy (13.3%), or clinical surveillance (38.5%). Testicular volume, sperm concentration, and morphology were lower (P = 0.001, P = 0.001, and P < 0.001, respectively) in patients with LCTs than in the NoL group. FSH, LH, and SHBG were higher and the testosterone/LH ratio was lower in LCTs than in the NoL group (P < 0.001). The LCT group showed higher SHBG (P = 0.018), lower sperm concentration (P = 0.029), and lower motility (P = 0.049) than the seminoma group. Risk factors for LCTs were cryptorchidism (χ2 = 28.27, P < 0.001), gynecomastia (χ2 = 54.22, P < 0.001), and low testicular volume (χ2 = 11.13, P = 0.001). Five cases were recurrences or bilateral lesions; none developed metastases during follow-up (median, 66 months).Limitations, Reasons For Caution: This study has some limitations. First, hCG and second-line diagnostic investigations were not available for all tumor patients. Second, ours is a referral center for infertility, thus a selection bias may have altered the baseline features of the LCT population. However, given that the comparison cohorts were also from the same center and had been managed with a similar protocol, we do not expect a significant effect.Wider Implications Of the Findings: LCTs are strongly associated with male infertility, cryptorchidism, and gynecomastia, supporting the hypothesis that testicular dysgenesis syndrome plays a role in their development. Patients with LCTs are at a greater risk of endocrine and spermatogenesis abnormalities even when the tumor is resected, and thus require long-term follow-up and prompt efforts to preserve fertility after diagnosis.LCTs have a good oncological prognosis when recognized early, as tissue-sparing enucleation is curative and should replace orchiectomy. Conservative surgery and, in compliant patients, active surveillance through clinical and radiological follow-up are safe options, but require monitoring of testicular failure and recurrence.Study Funding/competing Interest(s): The project was funded by the Ministry of University and Research Grant MIUR 2015ZTT5KB. There are no conflicts of interest.Trial Registration Number: ALCeP trial (ClinicalTrials.gov Identifier: NCT01206270). [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 (β = -0.31, 95% CI -0.58; -0.03), number of progressive motile sperm (β = -0.15, 95% CI -0.31; 0.00) and morning serum testosterone concentration (β = -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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Study Question: Does loss of DMRT1 in human fetal testis alter testicular development and result in testicular dysgenesis?Summary Answer: DMRT1 repression in human fetal testis alters the expression of key testicular and ovarian determining genes, and leads to focal testicular dysgenesis.What Is Known Already: Testicular dysgenesis syndrome (TDS) is associated with common testicular disorders in young men, but its etiology is unknown. DMRT1 has been shown to play a role in the regulation of sex differentiation in the vertebrate gonad. Downregulation of DMRT1 in male mice results in trans-differentiation of Sertoli cells into granulosa (FOXL2+) cells resulting in an ovarian gonadal phenotype.Study Design, Size, Duration: To determine the effect of DMRT1 repression on human fetal testes, we developed a novel system for genetic manipulation, which utilizes a Lentivral delivered miRNA during short-term in vitro culture (2 weeks). A long-term (4-6 weeks) ex vivo xenograft model was used to determine the subsequent effects of DMRT1 repression on testicular development and maintenance. We included first and second-trimester testis tissue (8-20 weeks gestation; n = 12) in the study.Participants/materials, Setting, Methods: Human fetal testes were cultured in vitro and exposed to either of two DMRT1 miRNAs (miR536, miR641), or to scrambled control miRNA, for 24 h. This was followed by a further 14 days of culture (n = 3-4), or xenografting (n = 5) into immunocompromised mice for 4-6 weeks. Tissues were analyzed by histology, immunohistochemistry, immunofluorescence and quantitative RT-PCR. Endpoints included histological evaluation of seminiferous cord integrity, mRNA expression of testicular, ovarian and germ cell genes, and assessment of cell number and protein expression for proliferation, apoptosis and pluripotency factors. Statistical analysis was performed using a linear mixed effect model.Main Results and the Role Of Chance: DMRT1 repression (miR536/miR641) resulted in a loss of DMRT1 protein expression in a sub-population of Sertoli cells of first trimester (8-11 weeks gestation) human fetal testis; however, this did not affect the completion of seminiferous cord formation or morphological appearance. In second-trimester testis (12-20 weeks gestation), DMRT1 repression (miR536/miR641) resulted in disruption of seminiferous cords with absence of DMRT1 protein expression in Sertoli (SOX9+) cells. No differences in proliferation (Ki67+) were observed and apoptotic cells (CC3+) were rare. Expression of the Sertoli cell associated gene, SOX8, was significantly reduced (miR536, 34% reduction, P = 0.031; miR641 36% reduction, P = 0.026), whilst SOX9 expression was unaffected. Changes in expression of AMH (miR536, 100% increase, P = 0.033), CYP26B1 (miR641, 38% reduction, P = 0.05) and PTGDS (miR642, 30% reduction, P = 0.0076) were also observed. Amongst granulosa cell associated genes, there was a significant downregulation in R-spondin 1 expression (miR536, 76% reduction, P < 0.0001; miR641, 49% reduction, P = 0.046); however, there were no changes in expression of the granulosa cell marker, FOXL2. Analysis of germ cell associated genes demonstrated a significant increase in the expression of the pluripotency gene OCT4 (miR536, 233%, P < 0.001). We used the xenograft system to investigate the longer-term effects of seminiferous cord disruption via DMRT1 repression. As was evident in vitro for second-trimester samples, DMRT1 repression resulted in focal testicular dysgenesis similar to that described in adults with TDS. These dysgenetic areas were devoid of germ cells, whilst expression of FOXL2 within the dysgenetic areas, indicated trans-differentiation from a male (Sertoli cell) to female (granulosa cell) phenotype.Limitations, Reasons For Caution: Human fetal testis tissue is a limited resource; however, we were able to demonstrate significant effects of DMRT1 repression on the expression of germ and somatic cell genes, in addition to the induction of focal testicular dysgenesis, using these limited samples. In vitro culture may not reflect all aspects of human fetal testis development and function; however, the concurrent use of the xenograft model which represents a more physiological system supports the validity of the in vitro findings.Wider Implications Of the Findings: Our findings have important implications for understanding the role of DMRT1 in human testis development and in the origin of testicular dysgenesis. In addition, we provide validation of a novel system that can be used to determine the effects of repression of genes that have been implicated in gonadal development and associated human reproductive disorders.Study Funding/competing Interest(s): This project was funded by a Wellcome Trust Intermediate Clinical Fellowship (Grant No. 098522) awarded to RTM. LBS was supported by MRC Programme Grant MR/N002970/1. RAA was supported by MRC Programme Grant G1100357/1. RMS was supported by MRC Programme Grant G33253. This work was undertaken in the MRC Centre for Reproductive Health which is funded by the MRC Centre grant MR/N022556/1. The funding bodies had no input into the conduct of the research or the production of this manuscript. The authors have declared no conflicts of interest. [ABSTRACT FROM AUTHOR]

Study Question: Is the anogenital distance (AGD) correlated to anthropometric, genital and sperm parameters in young adult men?Summary Answer: We observed that reduced AGD is strongly associated with altered semen parameters and reduced testicular volume.What Is Known Already: Abnormalities in the foetal development of the testis have been suggested as causative of common male reproductive disorders, such as cryptorchidism, hypospadias, reduced semen quality and testicular germ cell tumour, collectively defined as 'testicular dysgenesis syndrome'. In human epidemiological studies, alterations in AGD have been frequently associated with clinically relevant outcomes of reproductive health, suggesting AGD as a marker of foetal testicular development.Study Design, Size, Duration: This study was performed within the annual screening protocol to evaluate male reproductive health in the high schools of Padua and surroundings (Veneto Region, the North-East of Italy). Here we report the findings of 794 subjects who completed the study protocol between October 2016 and May 2017.Participants/materials, Setting, Methods: We evaluated 794 students aged 18-19 years recording the following parameters: height, weight, BMI, waist circumference, arm span, pubis-to-floor and crown-to-pubis length, penile length and circumference, testicular volumes, semen parameters and AGD (measured from the posterior base of the scrotum to the centre of the anus).Main Results and the Role Of Chance: Of the subjects, 49% had an abnormal arm span-height difference (>3 cm) and 63.4% had an altered ratio of crown-to-pubis/pubis-to-floor length (≤0.92). The rate of subjects with reduced testicular volume was 23%. Median sperm concentration was 51.0× 106/ml and total sperm count was 122.5 × 106. AGD showed a direct positive relation with testicular volume and penile length and circumference (R = 0.265, 0.176 and 0.095, respectively, all P < 0.05). No significant relation was observed between AGD and anthropometric parameters. Sperm concentration, total sperm count, progressive motility and normal morphology showed a significant and positive correlation with AGD (R = 0.205, 0.210, 0.216 and 0.117, respectively, all P < 0.05).Limitations, Reasons For Caution: Our cohort of young adults is not representative of the general population. Hormonal evaluation was missing.Wider Implications Of the Findings: Our findings show that AGD is associated with testicular volumes, penile measures and seminal parameters in young adult men. Because AGD is hormonally determined during foetal life, the reported high incidence of reduced semen quality and reduced testicular volume could be related to a reduced androgenic exposure in utero. AGD could represent a simple and useful method to evaluate testicular and penile development in adult men.Study Funding/competing Interest(s): The authors have no potential conflict of interest to declare. No external funding was obtained for this study.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

Study Question: When does germ cell loss and fibrosis occur in patients with Klinefelter syndrome (KS)?Summary Answer: In KS, germ cell loss is not observed in testicular tissue from fetuses in the second semester of pregnancy but present at a prepubertal age when the testicular architecture is still normal, while fibrosis is highly present at an adolescent age.What Is Known Already: Most KS patients are azoospermic at adult age because of a massive germ cell loss. However, the timing when this germ cell loss starts is not known. It is assumed that germ cell loss increases at puberty. Therefore, testicular sperm extraction (TESE) at an adolescent age has been suggested to increase the chances of sperm retrieval at onset of spermatogenesis. However, recent data indicate that testicular biopsies from peripubertal KS patients contain only a few germ cells.Study Design, Size, Duration: In this study, we give an update on fertility preservation in adolescent KS patients and evaluate whether fertility preservation would be beneficial at prepubertal age. The possibility of retrieving testicular spermatozoa by TESE was evaluated in adolescent and adult KS men. The presence of spermatogonia and the degree of fibrosis were also analysed in testicular biopsies from KS patients at different ages. The patients were divided into four age groups: foetal (n = 5), prepubertal (aged 4-7 years; n = 4), peripubertal (aged 12-16 years; n = 20) and adult (aged 18-41 years; n = 27) KS patients.Participants/materials, Setting, Methods: In peripubertal and adult KS patients, retrieval of spermatozoa was attempted by semen analysis after masturbation, vibrostimulation, electroejaculation or by TESE. MAGE-A4 immunohistochemistry was performed to evaluate the presence of germ cells in testicular biopsies from foetal, prepubertal, peripubertal and adult KS patients. Tissue morphology was evaluated by haematoxylin-periodic acid Schiff (H/PAS) staining.Main Results and the Role Of Chance: Testicular spermatozoa were collected by TESE in 48.1% of the adult KS patients, while spermatozoa were recovered after TESE in only one peripubertal patient (5.0%). Germ cells were detectable in testicular biopsies from 21% of adult men for whom no spermatozoa could be retrieved by TESE and in 31.5% of peripubertal KS boys. Very small numbers of spermatogonia (0.03-0.06 spermatogonia/tubule) were detected in three out of four (75%) prepubertal patients. At a foetal age, the number of germ cells was similar for KS and control samples. Increased signs of fibrosis were not present at foetal and prepubertal ages, but peripubertal and adult KS patients showed high levels of fibrosis.Large Scale Data: N/A.Limitations, Reasons For Caution: Only four prepubertal biopsies were included in this study, but they all showed a very low germ cell number. A high variability in the number of spermatogonia per mm2 was observed in the limited (n = 5) number of foetal biopsies. However, testicular biopsies from prepubertal and foetal Klinefelter patients are difficult to obtain.Wider Implications Of the Findings: Testicular tissue banking at a prepubertal age has been suggested as a potential method for fertility preservation in early diagnosed KS boys. However, our results show that a reduction in germ cell number has already taken place in childhood. Therefore, offering testicular tissue banking in young KS boys to prevent subsequent sterility might be a questionable strategy. However, this should be confirmed in a larger study population.Study Funding/competing Interest(s): This project was funded by the scientific Fund Willy Gepts from the UZ Brussel (D.V.S., J.D.S.), grants from the Vrije Universiteit Brussel (E.G.) and a Methusalem grant (K.S.). D.V.S is a post-doctoral fellow of the Fonds Wetenschappelijk Onderzoek (FWO; 12M2815N). No conflict of interest is declared. [ABSTRACT FROM AUTHOR]

Study Question: How are temporal trends in lifestyle factors, including exposure to maternal smoking in utero, associated to semen quality in young men from the general population?Summary Answer: Exposure to maternal smoking was associated with lower sperm counts but no overall increase in sperm counts was observed during the study period despite a decrease in this exposure.What Is Known Already: Meta-analyses suggest a continuous decline in semen quality but few studies have investigated temporal trends in unselected populations recruited and analysed with the same protocol over a long period and none have studied simultaneous trends in lifestyle factors.Study Design, Size, Duration: Cross-sectional population-based study including ~300 participants per year (total number = 6386) between 1996 and 2016.Participants/materials, Setting, Methods: The study is based on men from the Greater Copenhagen area, Denmark, with a median age of 19 years, and unselected with regard to fertility status and semen quality. The men delivered a semen sample, had a blood sample drawn and a physical examination performed and answered a comprehensive questionnaire, including information on lifestyle and the mother's pregnancy. Temporal trends in semen quality and lifestyle were illustrated graphically, and trends in semen parameters and the impact of prenatal and current lifestyle factors were explored in multiple regression analyses.Main Results and the Role Of Chance: Throughout the study period, 35% of the men had low semen quality. Overall, there were no persistent temporal trends in semen quality, testicular volume or levels of follicle-stimulating hormone over the 21 years studied. The men's alcohol intake was lowest between 2011 and 2016, whereas BMI, use of medication and smoking showed no clear temporal trends. Parental age increased, and exposure in utero to maternal smoking declined from 40% among men investigated in 1996-2000 to 18% among men investigated in 2011-2016. Exposure to maternal smoking was associated with lower sperm counts but no overall increase in sperm counts was observed despite the decrease in this exposure.Limitations, Reasons For Caution: Information of current and prenatal lifestyle was obtained by self-report, and the men delivered only one semen sample each.Wider Implications Of the Findings: The significant decline in in utero exposure to maternal smoking, which was not reflected in an overall improvement of semen quality at the population level, suggest that other unknown adverse factors may maintain the low semen quality among Danish men.Study Funding/competing Interest(s): The study has received financial support from the ReproUnion; the Research fund of Rigshospitalet, Copenhagen University Hospital; 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.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

Study Question: Are the genetic variants FSHB -211 G>T (rs10835638), FSHR c.2039 A>G (Asn680Ser, rs6166) and FSHR -29 G>A (rs1394205) associated with serum FSH, LH and anti-Müllerian hormone (AMH) levels in reproductive age women, their menstrual cycle parameters and risk of infertility?Summary Answer: Only the FSHB -211 G>T variant was a major genetic determinant of serum gonadotropin levels in both, eumenorrheic healthy women and female infertility patients, and the T-allele carrier status was enriched among idiopathic infertility cases.What Is Known Already: There are accumulating data on common genetic variants modulating reproductive parameters and fertility potential. FSHB -211 G>T represents the strongest acknowledged genetic factor contributing to male circulating gonadotropins levels. Respective data in women are limited and the two previously published studies have reached conflicting results. In addition, previous studies have consistently associated FSHR c.2039 A>G (but not FSHR -29 G>A) with female serum FSH level.Study Design, Size, Duration: The study aimed to test robust and clinically meaningful genetic effects (if present) of the FSHB -211 G>T, FSHR c.2039 A>G and FSHR -29 G>A variants on female basal FSH, LH and AMH levels, and linked reproductive parameters. Genetic association testing was performed in two independent and clinically different study groups (i) eumenorrheic healthy women without known fertility problems (n = 169; 27.6 ± 6.1 years) and (ii) female partners of infertile couples (n = 186; 32.4 ± 4.7 years). The study groups were compared for allelic and genotypic distributions of the analysed variants.Participants/materials, Setting, Methods: All participants were recruited during the HAPPY PREGNANCY study (2013-2015) at the Women's Clinic, Tartu University Hospital, Estonia. Serum FSH, LH and AMH were measured in the follicular phase (Days 2-6) of the menstrual cycle. All three single nucleotide polymorphisms (SNPs) were genotyped by PCR and Taqman allelic discrimination assay. The effect of the analysed variants on hormonal measurements and menstrual cycle data was assessed using linear regression under additive and recessive models adjusted by age, BMI and smoking status. Results of the two subgroups were combined in a meta-analysis applying the fixed effects model. Restricted maximum likelihood analysis was applied to estimate the proportion of total phenotypic variance of analysed reproductive parameters, explainable by the tested genetic variants. In case-control analysis, genetic association with infertility status was tested using Fisher's exact test and logistic regression adjusted by age, BMI and smoking status.Main Results and the Role Of Chance: In both study groups, T-allele of the FSHB -211 G>T was associated with significantly higher serum levels of FSH and LH. Results of the meta-analysis (additive genetic model) remained significant after Bonferroni correction for multiple testing: FSH, T-allele effect 0.80 IU/L, P = 1.2 × 10-3; LH, 1.58 IU/L, P = 1.8×10-8. A more pronounced effect of T-allele of the FSHB -211 G>T on circulating LH was identified as a driving factor to increased LH/FSH ratio (meta-analysis, P = 4.7 × 10-3). In healthy women, the FSHB -211 G>T variant was estimated to explain 3.5 and 7.1% of the total variance of the measured serum FSH and LH levels, respectively. The corresponding numbers for the infertility patients were 1.6 and 10.5%. Women with idiopathic infertility compared to controls exhibited a doubled T-allele frequency (23.6 versus 12.4%; P = 8.9 × 10-3) and a >3-fold excess of TT homozygotes (5.6 versus 1.8%; P = 3.5 × 10-2). The only association of the FSHR c.2039 A>G was detected with serum FSH levels in eumenorrheic healthy women, explaining 3.9% of the total parameter variance (G-allele effect 0.56 IU/L, P = 4.6 × 10-3). In the study group of healthy reproductive age women, the highest serum FSH levels were detected among the FSHB -211 T-allele carriers with the FSHR c.2039 GG-genotype (median 7.7 IU/L). In contrast, the lowest hormone concentrations were measured for the women carrying the combination of the FSHB -211 GG- and the FSHR c.2039 AA-homozygosity (median 5.8 IU/L, P = 9.6 × 10-3). None of the analysed reproductive parameters was associated with the FSHR -29 G>A variant. In our study groups, the tested polymorphisms did not reach significant associations with serum AMH measurements, menstrual cycle length or age at menarche.Limitations, Reasons For Caution: Small sample size and the design involving two clinical groups with different reproductive histories may have limited the capacity to replicate the associations with the age at menarche and length of menstrual cycle, initially reported in large genome-wide association studies. Small sample size may have also affected the accuracy in estimating the contribution of the tested variants to the total phenotypic variance of measured gonadotropin concentrations. The group of eumenorrheic healthy women had its limitations as a control to estimate the true effect of analysed genetic variants on individual's fertility potential as the recruitment strategy had been targeted mostly towards younger women, who may not yet have planned to conceive a child by this age.Wider Implications Of the Findings: We propose that like in men, also in women the FSHB -211 G>T represents a key genetic modulator of circulating gonadotropin, leading to various possible downstream effects on reproductive physiology. This claim is strongly supported by the reports of genome-wide association studies on various female reproductive traits and diseases. In perspective, FSHB -211 G>T may have a diagnostic value in fertility clinics to detect female patients with genetically inherited elevated basal FSH and LH levels.Study Funding/competing Interest(s): The study was supported by Estonian Science Foundation Grant (ETF9030 for M.L.); Institutional Research Grant (IUT34-12 for M.L.) and European Union through the European Regional Development Fund (project HAPPY PREGNANCY, 3.2.0701.12-0047; for M.L. and K.R.). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the article. We have no competing interests to declare.Trail Registration Number: Not applicable. [ABSTRACT FROM AUTHOR]

Study Question: Does phthalate exposure from prescription drugs affect semen quality?Summary Answer: Exposure to phthalate-containing drugs is associated with poor semen quality.What Is Known Already: Phthalates and their metabolites have been shown to disrupt the hormone signalling in animal studies. One study has shown associations between medicinal phthalate exposure and poor semen quality, suggesting similar effects in humans.Study Design, Size, Duration: We included 18 515 males with poor semen quality (cases) and 31 063 males with normal semen quality (controls) registered in the Danish IVF Registry from 2006 to 2016.Participants/materials, Setting, Methods: Exposure to phthalate-containing drugs was assessed from the Danish Register of Medicinal Product Statistics. Outcome measures were obtained at the first contact with the fertility clinic, and categorized according to the International Classification of Diseases (ICD-10). The association between current use of phthalate-containing medications <90 days prior to semen sampling and reduced semen quality was analysed using unconditional logistic regression, adjusting for potential confounders.Main Results and the Role Of Chance: In total, 57 cases and 72 controls redeemed at least one prescription for a drug containing ortho-phthalates in the 90 days before their first semen sample, yielding an adjusted odds ratio (OR) of 1.30 (95% CI: 0.91-1.85) for poor semen quality when compared to males exposed to phthalate-free generic drugs. Similarly, 81 cases and 78 controls exposed to a drug containing polymers had increased odds of poor semen quality (OR = 1.71, 95% CI: 1.24-2.35). Current exposure to polymer containing products from alimentary tract and metabolism drugs was associated with the highest OR of 2.80 (95% CI: 1.63-4.84). Comparing males exposed to drugs containing ortho-phthalates or polymers with males unexposed to prescription drugs, we found adjusted ORs of 1.32 (95% CI: 0.93-1.87) and 1.73 (95% CI: 1.26-2.36), respectively. We saw no clear relationship between degree of exposure and odds of poor semen quality.Limitations, Reasons For Caution: The reliance on ICD-10 based register data restricted our ability to relate phthalate exposure to detailed semen parameters. Furthermore, due to imperfections in the registry, we could only include the first semen sample and could not follow semen quality over time.Wider Implications Of the Findings: Our results support the likely negative effect of phthalate exposure from medicinal drugs on semen quality. As exposures from medicinal products are readily avoidable, our findings may be of relevance to regulatory authorities.Study Funding/competing Interest(s): This work was supported by Odense University Hospital, Denmark (Grant number A1003). None of the authors declare conflict of interest. [ABSTRACT FROM AUTHOR]

Study Question: Is it possible, in an unbiased and clinical relevant way, to determine the number of viable acrosome-intact human spermatozoa in ejaculates and to use this as a measure of fertility chances?Summary Answer: Image cytometry enables easy and unbiased quantification of viable acrosome-intact spermatozoa and it correlates with semen quality and fertility status.What Is Known Already: The presence of the acrosome and its ability to respond to physiological inducers (e.g. progesterone) in the female reproductive tract at the appropriate time and place is required for fertilization. However, the available assays are labor intensive and therefore not used clinically.Study Design, Size, Duration: Washed semen samples and capacitated swim-up fractions from volunteers were used to develop the assay. Subsequently washed ejaculates from patients in fertility treatment (n = 156), proven fertile men (n = 54) and volunteers (n = 10) were assessed to evaluate the number of acrosome-intact spermatozoa in the ejaculate (acrosomal status) and compared to other semen parameters, fertility status, fertility treatments and pregnancy rates.Participants/materials, Setting, Methods: Image cytometry was used to assess the fluorescence intensity of Pisum sativum agglutinin and Propidium iodide.Main Results and the Role Of Chance: The assay was validated by inducing the acrosome reaction in swim-up-purified and capacitated spermatozoa with progesterone and ionomycin, and in repeated acrosomal status measurements of washed ejaculates a small coefficient of variation (3.7%) was observed. Men with poor semen quality had fewer viable acrosome-intact spermatozoa in the ejaculate (P = 0.0012; median 32.6% vs. 49.3%). A large proportion (44%) of normozoospermic men from infertile couples had less than the observed median fraction (46%) of viable acrosome-intact spermatozoa in the ejaculate. Furthermore, the total number of viable acrosome-intact spermatozoa was significantly lower among men with male factor infertility compared to fertile men (median 35 vs. 97 mill, P = 1 × 10-7). Men from couples going through one or more ICSI cycles had significant fewer viable acrosome-intact spermatozoa than men from couples who only underwent IUI (P = 0.002; 44.4% vs. 62.0%) and the fraction of viable acrosome-intact spermatozoa appeared better than classical semen parameters in classifying whether or not couples needed ICSI. A positive, although non-significant, tendency toward ongoing pregnancy with an increasing number of viable acrosome-intact spermatozoa was observed (P = 0.2).Large Scale Data: N/A.Limitations, Reasons For Caution: Even larger cohorts of infertile couples are needed to substantiate the clinical application of the assay in regard to estimation of fertility potential of an individual.Wider Implications Of the Findings: The presented assay makes it possible to measure the number of acrosome competent spermatozoa in an ejaculate in a standardized manner and hence may serve as a new biomarker for male fertility. Few spermatozoa in an ejaculate are acrosome competent and it might be a valuable measure when evaluating male reproductive function.Study Funding/competing Interest(s): This work was supported by grants from the Innovation Fund Denmark. M.G. and S.K. work at ChemoMetec, which produces the image cytometer used in the study, M.G. hold shares in the company. The other authors have no conflict of interest. [ABSTRACT FROM AUTHOR]

Study Question: Is the thrombophilia mutation factor V Leiden (FVL) associated with an increased total sperm count?Summary Answer: Carriers of FVL have a higher total sperm count than non-FVL-carriers, which could not be explained by genetic linkage or by observations in a FVL-mouse model.What Is Known Already: FVL has a high prevalence in Caucasians despite detrimental health effects. Carriers have been shown to have higher fecundity, which might partly explain this evolutionary paradox.Study Design, Size, Duration: We determined FVL status in two cohorts (Dutch, n = 627; Danish, n = 854) of consecutively included men without known causes for spermatogenic failure, and performed an individual patient data meta-analysis of these two cohorts together with one previously published (Dutch, n = 908) cohort. We explored possible biological underpinnings for the relation between sperm count and FVL, by use of a FVL-mouse model and investigations of genetic linkage.Participants/materials, Setting, Methods: Participants were male partners of subfertile couples (two Dutch cohorts) and young men from the general population (Danish cohort): FVL carrier rate was 4.0%, 4.6% and 7.3%, respectively. There were differences in smoking, abstinence time and age between the cohorts. We corrected for these in the primary analysis, which consisted of a mixed linear effects model, also incorporating unobjectified population differences. In public haplotype data from subjects of European descent, we explored linkage disequilibrium of FVL with all known single nucleotide polymorphisms in a 1.5 MB region around the F5 gene with an R2 cutoff of 0.8. We sequenced exons of four candidate genes hypothesized to be linked to FVL in a subgroup of FVL carriers with extreme sperm count values. The animal studies consisted of never mated 15-18-week-old C57BL/J6 mice heterozygous and homozygous for FVL and wild-type mice. We compared spermatogenesis parameters (normalized internal genitalia weights, epididymis sperm content and sperm motility) between FVL and wild-type mice.Main Results and the Role Of Chance: Human FVL carriers have a higher total sperm count than non-carriers, with an adjusted mean difference of 31 × 106 (95%CI 0.2-61.7; P = 0.048). Mice with the FVL mutation do not have increased spermatogenesis as compared to wildtype mice. None of the studied polymorphisms was in linkage disequilibrium, either in the public databases or in a subgroup of FVL carriers with extremely high sperm counts.Limitations, Reasons For Caution: The difference in total sperm count would benefit from confirmation in other cohorts. The finding of higher count in carriers was consistent however, with no heterogeneity between the cohorts. The lack of effect of murine FVL might suggest there is no direct causality. The exploratory efforts on genetic linkage do not rule out that the association is a reflection of FVL co-inheritance with a non-studied causative polymorphism.Wider Implications Of the Findings: A high sperm count in FVL-carrying males contributes to understanding the high prevalence of this otherwise disadvantageous mutation. The findings might provide directions for future research on male fertility.Study Funding/competing Interest(s): No conflicts of interest. Research was conducted with funding from the Netherlands Organisation for Scientific Research (NWO, VIDI innovative research grant 016.126.364 awarded to S. Middeldorp). The Danish cohort was supported by the Innovation Fund Denmark (InnovationsFonden, grant no. 14-2013-4), The Danish Ministry of Health and the Danish Environmental Protection Agency.Trial Registration Number: Not applicable. [ABSTRACT FROM AUTHOR]

Study Question: Does a short abstinence period of only 2 h yield spermatozoa with better motility characteristics than samples collected after 4-7 days?Summary Answer: Despite lower semen volume, sperm concentration, total sperm counts and total motile counts, higher percentages of motile spermatozoa with higher velocity and progressiveness were detected in samples obtained after 2 h.What Is Known Already: Most studies that have assessed the effect of abstinence periods on sperm motility parameters in men with a sperm concentration below 15 million/ml have detected a higher percentage of motile spermatozoa in samples obtained after short abstinence periods. Studies of men with sperm concentrations above 15 million/ml have reported significantly decreased motile sperm counts after 24 h of abstinence compared with longer abstinence periods.Study Design, Size, Duration: This study had a controlled repeated-measures design based on semen samples from 43 male partners, in couples attending for IVF treatment, who had a sperm concentration above 15 million/ml. Data were collected between June 2014 and December 2015 in the Fertility Unit of Aalborg University Hospital (Aalborg, Denmark).Participants/materials, Setting, Methods: Participants provided a semen sample after 4-7 days of abstinence followed by another sample after only 2 h. For both ejaculates, sperm concentration, total sperm counts, motility groups and detailed kinematic parameters were assessed and compared by using the Sperm Class Analyzer (SCA) computer-aided sperm analysis system before and after density gradient selection. The laboratory's local manual method (Makler chamber) was used for comparison.Main Results and the Role Of Chance: The second raw ejaculate demonstrated lower semen volume (P < 0.0001), sperm concentration (P = 0.003) and sperm counts in all motility sub-groups (P < 0.001) but higher percentages of spermatozoa with higher velocity (P < 0.01), progressiveness (P < 0.001) and hyperactivation (P < 0.001), compared with the first raw ejaculate.Limitations, Reasons For Caution: The first ejaculate in this study was also used for the IVF/ICSI treatments and therefore only patients with a semen volume ≥2 ml and concentration ≥15 million/ml were included. Further validation in large prospective randomized controlled trials, more purposely directed at normozoospermic males with partners having problems conceiving when there appears to be no female factor, is needed to confirm the potential advantage of using a second semen sample in improving fertilization and pregnancy rates in assisted reproduction.Wider Implications Of the Findings: Despite the significantly lower semen volume, sperm concentration and total sperm counts in all motility sub-groups, the significantly higher percentage of spermatozoa with better motility characteristics (velocity, progressiveness and hyperactivation) in the second ejaculate, may provide and allow for a simpler and more effective selection of higher quality spermatozoa. This could prove to be an advantage for ART procedures such as intracytoplasmic sperm injection where a large number of spermatozoa is not needed. It can also be speculated that pooling two consecutive ejaculates obtained after 4-7 days and after 2 h, could be an advantage for intrauterine insemination where a large number of motile spermatozoa are needed.Study Funding/competing Interest(s): This study was supported by internal grants from the Department of Health Science and Technology, Faculty of Medicine, Aalborg University (Aalborg, Denmark). The SCA® was provided by a grant from 'Ferring Pharmaceuticals' to Aalborg University Hospital (H.I.N). G.V.D.H. is an external senior scientific consultant to Microptic S/L (Barcelona, Spain). H.A. has provided scientific input and presentations for Microptic S/L (Barcelona, Spain) on several occasions. All other authors declare no conflict of interest.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

Study Question: What are the parameters of semen quality in Baltic men?Summary Answer: Combined parameters of sperm concentration, motility and morphology revealed that 11-15% of men had low semen quality, 37-50% intermediate and 38-52% high semen quality.What Is Known Already: Previous studies have revealed regional differences in semen parameters, and semen quality of Baltic men has been suggested to be better than that of other European men.Study Design, Size, Duration: This was a cross-sectional study of 1165 men aged 16-29 years from Estonia (N = 573), Latvia (N = 278) and Lithuania (N = 314) conducted in 2003-2004.Participants/materials Setting Methods: Men from the general population, median age 19.8 years, provided one semen sample each, had blood samples taken, had testis size determined, and provided information on lifestyle. Based on combined data of sperm concentration, sperm motility and morphology the cohort was classified into three categories: low, intermediate or high semen quality. Comparisons between groups (including subgroups of Estonian men of Russian versus Estonian ethnicity) were tested, adjusting for ejaculation abstinence and age.Main Results and the Role Of Chance: The median sperm concentration of the Estonian, Latvian and Lithuanian populations of Baltic men was 63 mill/ml. Low semen quality was detected in 11-15% of the men, intermediate in 37-50% and high in 38-52%. No crucial differences between national subgroups were detected, except that a higher percentage (9.6%) of the subgroup of Russian Estonians reported having had cryptorchidism compared to the other men (2.5-3.6%, P < 0.001). Smoking had an adverse impact on both sperm concentration and total sperm counts (P < 0.001).Limitations Reasons For Caution: The semen quality data were collected >10 years ago. Thus, a recent change in semen quality cannot be excluded. Owing to the study design, it is assumed, but unproven, that the men were representative of the general populations. Some men were very young (16 years), however, this was also the case for other European studies of similar populations. Assessment of sperm motility is associated with inter-observer variation, and no quality control was undertaken for sperm motility assessment to account for that. Thus, estimates of sperm motility should be interpreted with caution.Wider Implications Of the Findings: Analysis of the semen variables separately did not identify that a considerable percentage of Baltic men had low semen quality. The combined analysis, however, showed that more than one out of nine men had semen quality at a level indicating reduced fertility chances. We suggest that future studies of semen quality should be carried out reporting both results of single semen parameters and estimates that combine the most frequently assessed variables.Study Funding/competing Interest(s): The study was funded by the EU fifth framework project Number QLK4-1999-01422 'Envir.Repro.Health' extension to Baltic countries Number QLRT-2001-02911; Estonian Science Foundation, grant numbers 2991 and PUT181. There are no competing interests.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

Study Question: What are the relations among birthweight (BW), semen parameters and birth outcomes in a population-based sample?Summary Answer: BW is unrelated to semen parameters, which are in turn unrelated to birth outcomes.What Is Known Already: In clinical settings, there has been suggestion that semen parameters are related to BW when comparing fertile and infertile men; however, findings have been less clear in more general populations.Study Design, Size, Duration: Questionnaire data and semen samples were collected at baseline from 427 male participants of the population-based Longitudinal Investigation of Fertility and the Environment (LIFE) prospective cohort study from 2005 to 2009, who were followed prospectively to assess pregnancy outcomes among 226 singleton births.Participants/materials, Setting, Methods: Men of at least 18 years of age who were married or in a committed relationship and trying to conceive were eligible for participation; physician-diagnosed infertility was an exclusion criterion. Participants were recruited from two geographic areas and semen samples were analyzed for 34 quality parameters categorized as general, motility, morphology, sperm head and sperm chromatin structure using methods including computer-aided semen analysis integrated visual optical system and sperm chromatin structure assay. Linear and mixed models were used for statistical analysis of the relations between men's BW, semen parameters, and BW, gestational age at delivery, birth length, head circumference and ponderal index of singleton births.Main Results and the Role Of Chance: No association was observed between male BW and semen parameters or birth outcomes. Few associations were observed between semen parameters and birth outcomes, and the observed statistically significant associations were isolated and without a consistent pattern that would suggest an association between BW and birth outcomes.Limitations, Reasons For Caution: Men's BW was self-reported and may be subject to some imprecision. Semen analysis was performed the day after collection, an approach that impacts the assessment of motility and that may limit inference from our analyses of motility measures. In addition, inclusion criteria for selection into the cohort limits generalizability to generally healthy couples trying to conceive and without known subfertility.Wider Implications Of the Findings: Despite suggestions from prior studies of male in utero exposures impacting BW and male reproductive health, there appears to be little support for such relations in this generally healthy population.Study Funding/competing Interest(s): Supported by the Intramural Research Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development (N01-HD-3-3355, N01-HD-3-3356 and NOH-HD-3-3358). The authors report no competing interests, and a Memo of Understanding with the National Institute of Occupational Safety and Health (NIOSH) for semen analysis.Trial Registration Number: Not applicable. [ABSTRACT FROM AUTHOR]

Study Question: Is maternal use of mild analgesics in pregnancy associated with anogenital distance (AGD)-the distance from the anus to the genitals-in the offspring?Summary Answer: Maternal use of mild analgesics [especially simultaneous use of paracetamol and nonsteroidal anti-inflammatory drugs (NSAIDs)] during pregnancy was associated with a shorter AGD in boys whereas no effect was found in girls.What Is Known Already: Mild analgesics including paracetamol (acetaminophen) and NSAIDs (e.g. ibuprofen and acetyl salicylic acid) have endocrine disrupting properties and in utero exposure reduces AGD in male rats. In humans, maternal exposure has been associated with cryptorchidism and hypospadias in male offspring but no studies have examined AGD.Study Design, Size, Duration: A prospective birth cohort study. Between 2010 and 2012, 2500 pregnant women were recruited from the Odense Child Cohort. Children were examined 3 months after the expected date of birth.Participants/materials, Setting, Methods: Pregnant women were asked about use of medication including mild analgesics (paracetamol and NSAID) during pregnancy at recruitment (gestational age (GA) week 10-27) and at GA week 28. AGD and penile width were measured 3 months after expected date of birth by trained personnel. A total of 1027 women answered both questionnaires and their children were examined. Associations between prenatal exposure to mild analgesics and AGD and penile width were estimated using multivariable linear regression adjusting for age and weight-for-age SD score.Main Results and the Role Of Chance: A total of 40% of the women reported use of paracetamol and/or NSAIDs (4.4%) during the first 28 weeks of pregnancy. Exposure to analgesics during pregnancy was associated with a reduced AGD in boys, although statistically significant only for NSAIDs. The association was significant among 20 boys exposed to both paracetamol and NSAIDs (AGD -4.1 mm; CI 95%: -6.4; -1.7). Maternal intake of analgesics did not show any clear association with AGD in female offspring. No effect on penile width was found.Limitations Reasons For Caution: Only 27 boys and 18 girls were exposed to NSAIDs and most of them were also exposed to paracetamol. This makes it impossible to distinguish between exposures to NSAIDs alone and a potential mixture effect. Moreover, use of mild analgesics was self-reported up to 2 months after intake, which could have caused misclassification of exposure but is probably not associated with AGD as this was unknown to the women at time of reply to the questionnaire thereby underestimating the association. Confounding by indication may also explain our findings, as the condition for which the analgesic was taken may be associated with a reduction in AGD, rather than the use of the analgesic medication. This is the first study to report such an association in humans and further studies are needed to confirm our findings.Wider Implications Of the Findings: A negative association was observed between exposure to analgesics during pregnancy and AGD in boys, suggesting disruption of androgen action. The health implications of a shorter AGD are still uncertain, but in cross-sectional studies among adult men a shorter AGD is associated with poorer semen quality and lower testosterone. As 41% of the women used these painkillers the finding are of public health importance and pregnant women should be advised about the potentially harmful effects of painkiller use.Study Funding/competing Interests: The study was funded by the Danish Environmental Protection Agency by way of the Center on Endocrine Disruptors Danish Center for Hormone Disrupting Chemicals, the Danish Foundation for Scientific Innovation and Technology (09-067180), the Danish Research Council (4004-00352B_FSS), Novo Nordic Foundation (NNF15OC0017734), Ronald McDonald Children Foundation, K.A. Rohde's and wife's Foundation, Odense University Hospital and Region of Southern Denmark, Municipality of Odense, the Danish Council for Strategic Research, Program Commission on Health, Food and Welfare (2101-08-0058), Odense University Hospital Research Foundation and Odense Patient data Exploratory Network (OPEN). The authors declare they have no competing interests.Trial Registration Number: Not applicable. [ABSTRACT FROM AUTHOR]

Study Question: How knowledgeable are men about the medical, environmental and psychological factors that are associated with male infertility?Summary Answer: Men, across most demographic groups, have limited knowledge of the various factors that are associated with male infertility.What Is Known Already: Few surveys have focused on men's knowledge of their own fertility. Studies of both men and women have found that men are comparatively less knowledgeable about issues of fertility and reproductive health.Study Design, Size, Duration: A regionally representative sample of Canadian men completed a web-based survey of male fertility and reproductive health, over a 2-month period in 2015.Participants/materials, Setting, Methods: Men, aged 18-50 years, were recruited for the study. There were 701 male participants, with a mean age of 34.1 years. Each participant was asked to identify factors associated with male infertility; fertility knowledge was assessed through two open-ended questions and a comprehensive list of risk factors and attendant health issues.Main Results and the Role Of Chance: Men were only able to identify 51% of the risk factors and 45% of the health issues associated with male infertility. Men were most aware of the modifiable risk factors for infertility (e.g. sexually transmitted infections, smoking cigarettes), relative to their knowledge of fixed risk factors (e.g. delayed puberty, size of testicles) and the attendant health issues (e.g. cardiovascular disease, diabetes). The overall level of fertility knowledge did not vary by most demographic characteristics (e.g. age, education, employment, income), though men from ethnic minority groups displayed moderately greater awareness. Additionally, younger men, those with lower incomes and those who had no desire to have future biological children were more likely to identify themselves as unaware of associations with infertility in the open-ended questions. Self-reported knowledge was significantly associated with higher overall knowledge scores. More than half of the sample expressed an interest in obtaining information about male fertility and reproductive health, with the majority of these men indicating that medical professionals and online sources were their preferred methods for receiving information.Limitations, Reasons For Caution: Participants were self-selected and required to have Internet access in order to participate. This may affect the generalizability of results.Wider Implications Of the Findings: Previous studies of fertility knowledge have either omitted men from their samples or when men have been included, they were asked about general fertility or women's fertility. This is the first large-scale survey that focuses solely on men's knowledge of male fertility. Insight into the areas where men's knowledge may be lacking can inform strategies for disseminating fertility-related information and improving men's fertility awareness. Public health initiatives should tailor campaigns to educate men about the lesser known associations with male infertility, particularly those that are most prevalent and preventable through lifestyle modification.Study Funding/competing Interests: The study was funded by a grant from CIHR TE1-138296. No competing interests. [ABSTRACT FROM AUTHOR]

Study Question: Should fertility preservation be offered to children with Klinefelter syndrome (KS)?Summary Answer: Current evidence shows that fertility preservation should not be offered to adolescents with KS younger than 16 years because of lower retrieval rates for germ cells by testicular sperm extraction (TESE) compared with retrieval rates for adolescents and adults between 16 and 30 years.What Is Known Already: KS, the most common chromosomal disorder in men leading to non-obstructive azoospermia, is caused by the presence of at least one additional X chromosome. The onset of puberty in adolescents with KS leads to progressive degeneration of the testicular environment. The impact of the subsequent tissue degeneration on fertility potential of patients with KS is unknown, but in previous literature it has been suggested that fertility preservation should be started in adolescents as early as possible. However spermatozoa can be found by TESE in about 50% of adults with KS despite severe testicular degeneration. This review discusses the current evidence for fertility preservation in children and adolescents and possible prognostic markers for fertility treatment in KS.Study Design, Size, Duration: An extensive literature search was conducted, searching Pubmed, Embase, Cinahl and Web of Science from origin until April 2016 for 'Klinefelter syndrome' and 'fertility' and various synonyms. Titles and abstracts have been scanned manually by the authors for eligibility.Participants/materials, Setting, Methods: In total 76 studies were found to be eligible for inclusion in this review. Information from the papers was extracted separately by two authors.Main Results and the Role Of Chance: Various studies have shown that pre-pubertal children with KS already have a reduced number of germ cells despite a normal hormonal profile during childhood. The presence of spermatozoa in the ejaculate of adolescents with KS is extremely rare. Using TESE, the retrieval rates of spermatozoa for adolescents younger than 16 years old are much lower (0-20%) compared with those for adolescents and young adults between 16 and 30 years old (40-70%). Although spermatogonia can be found by TESE in about half of the peri-pubertal adolescents, there are currently no clinically functional techniques for their future use. Children and adolescents need to be informed that early fertility preservation before the age of 16 cannot guarantee fertility later in life and may even reduce the chances for offspring by removing functional immature germ cells which may possibly develop into spermatozoa after puberty. Furthermore, except for the age of patients with KS, there are no identified factors that can reliably be used as a predictive marker for fertility preservation.Limitations, Reasons For Caution: Most of the evidence presented in this review is based on studies including a small number of adolescents with KS. Therefore, the studies may have been underpowered to detect clinically significant differences for their various outcomes, especially for potential predictive factors for fertility preservation, such as hormone levels. Furthermore, the population of patients with KS diagnosed during childhood might be different from the adult population with KS where the diagnosis is based on infertility. Results based on comparisons between the two groups must be interpreted with caution.Wider Implications Of the Findings: Despite the limitations, this review summarizes the current evidence for managing fertility preservation in patients with KS to provide optimal health care.Study Funding/competing Interests: There was no funding for this study. S.F., Y.H., K.D., W.L.M.N., D.S., H.L.C.-v.d.G. and L.R. declare to have no conflicts of interests. D.D.M.B. reports grants from Merck Serono, grants from Ferring and grants from MSD, outside the submitted work. K.F. reports personal fees from MSD (commercial sponsor), personal fees from Ferring (commercial sponsor), grants from Merck-Serono (commercial sponsor), grants from Ferring (commercial sponsor) and grants from MSD (commercial sponsor), outside the submitted work. [ABSTRACT FROM AUTHOR]

Study Question: Is there an association between pubertal onset and subsequent reproductive health in young men?Summary Answer: Self-reported later onset of puberty was associated with reduced semen quality and altered serum levels of reproductive hormones among 1068 healthy, young Danish men.What Is Known Already: The long-term effects of variations in the onset of male puberty on subsequent reproduction remain largely unstudied.Study Design, Size, Duration: In a cross-sectional study, young healthy Danish men were approached when they attended a compulsory medical examination to determine their fitness for military service from 2008 to 2012.Participants/materials, Settings, Methods: A total of 1068 healthy, young Danish men (mean age 19 years) participated. They were asked to assess whether onset of penile and testicular growth, development of pubic hair and voice break occurred earlier, at the same time as or later than their peers. Their semen quality (semen volume, sperm concentration, total sperm count and percentages of motile and morphologically normal spermatozoa) and serum concentrations of sex hormones (LH, FSH, total testosterone, SHBG, inhibin B) and testicular size were determined.Main Results and the Role Of Chance: The response rate was 29%. Of the 1068 men who then participated, 652 answered the questions about penile growth and pubic hair development and were therefore included in the analysis. Self-reported later onset of puberty was associated with a 25% reduction in sperm concentration (95% CI -41%; -4%), a 40% reduction in total sperm count (-55%; -21%), a 1.6% age point reduction in morphological normal spermatozoa (-2.9; -0.3) and a 1.6 ml reduction in testicular size (-2.4 and -0.8 ml), after adjustment for confounders. Self-reported later onset of puberty was also associated with a 9% (3%; 15%) reduction in free testosterone and a 16% (2%; 31%) increase in FSH, after adjustment for confounders.Limitations, Reason For Caution: Our study was cross-sectional and reverse causality cannot be ruled out. In addition, we cannot rule out the possibility that the men with late puberty onset had not yet fully matured although most were in Tanner stage 5.Wider Implications Of the Findings: Approximately 15% of young Danish men have self-reported later onset of puberty than their peers. We found poorer testicular function in young men with a history of later pubertal development, suggesting that timing of pubertal onset may be a fundamental marker of male reproductive health. However, we cannot exclude the possibility that these men had not fully matured at the time of examination and therefore their semen quality may yet improve, which makes follow-up important.Study Funding/competing Interests: This work was supported by the Danish Council for Strategic Research, Program Commission on Health, Food and Welfare (project number 2101-08-0058), Rigshospitalet (grants 961506336 and R42-A1326), European Union, DEER (grant agreement no 212844), the Danish Ministry of Health and the Danish Environmental Protection Agency and Kirsten and Freddy Johansens Foundation (grant 95-103-72087). There are no competing interests. [ABSTRACT FROM AUTHOR]

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Study Question: Do variants of the genes encoding follicle stimulating hormone (FSH) beta subunit (B) and FSH receptor (R) impact circulating reproductive hormone levels and ovarian follicle maturation in healthy peripubertal girls?Summary Answer: FSHB and FSHR genetic variants exert, alone or their combination, distinct effects on reproductive hormone levels as well as ovarian follicle maturation in healthy peripubertal girls.What Is Known Already: FSHB and FSHR genetic variants impact reproductive hormone levels as well as associated pathologies in women. While FSHR c. 2039A>G is known to alter gonadotrophin levels in women, FSHR c.-29G>A has not yet been shown to exert effect and there are conflicting results concerning FSHB c.-211G>T.Study Design, Size, Duration: This population-based study included 633 girls recruited as part of two cohorts, the COPENHAGEN Puberty Study (2006-2014, a cross-sectional and ongoing longitudinal study) and the Copenhagen Mother-Child Cohort (1997-2002, including transabdominal ultrasound (TAUS) of the ovaries in a subset of 91 peripubertal girls).Participants/materials, Setting, Methods: Clinical examinations, including pubertal breast stage (Tanner's classification B1-B5) were performed. Circulating levels of FSH, luteinizing hormone (LH), estradiol, anti-Mullerian hormone (AMH) and inhibin-B were assessed by immunoassays. In a subset of the girls (n = 91), ovarian volume and the number/size of antral follicles were assessed by TAUS. Genotypes were determined by competitive PCR.Main Results and the Role Of Chance: FSHR c.2039A>G minor alleles were positively associated with serum FSH (β = 0.08, P = 0.004), LH (β = 0.06, P = 0.012) and estradiol (β = 0.06, P = 0.017) (adjusted for Tanner stages). In a combined model, FSHR c.-29G>A and FSHR c.2039A>G alleles were positively associated with FSH levels in early-pubertal girls (B2 + B3, n = 327, r = 0.1, P = 0.02) and in young adolescents (B4 + B5, n = 149, r = 0.2, P = 0.01). Serum AMH and inhibin B levels were not significantly influenced by the single nucleotide polymorphisms (SNPs). Single SNPs were not associated with follicles counts, however, a cumulative minor allele count (FSHB c.-211 G>T and FSHR c.-29G>A) was negatively associated with the number of large follicles (≥5 mm) (n = 91, P = 0.04) (adjusted for Tanner stages).Limitations, Reasons For Caution: Since we studied girls and young adolescents during pubertal transition, our study may not be fully comparable with previous studies on FSHB and FSHR variants in adult women. The group of young adolescents (Tanner B4 + B5) reflects the endocrine situation in adult women best, however, the group is not large enough to contribute substantially to the conflicting results concerning the influence of FSHB c.-211G>T in adult women. Furthermore, we have no information about the exact day of the menstrual cycle in the subgroup of girls with menarche.Wider Implications Of the Findings: The sex-specific interaction of FSHB and FSHR genetic variants and physiological as well as pathological conditions is being increasingly elucidated. The variant triplet set might serve as diagnostic and pharmacogenetic marker. For the first time, we show an additional effect of FSHR c.-29G>A on serum FSH levels in healthy girls. Moreover, morphological data suggest impaired FSH-induced maturation of ovarian follicles in minor allele carriers of FSHB c.-211G>T and FSHR c.-29G>A. This may explain previous findings of delayed pubertal onset in these girls.Study Funding/competing Interests: Funding was provided by the Danish Agency for Science, Technology and Innovation (09-067180), Danish Ministry of the Environment, CeHoS (MST-621-00065), Capital Region of Denmark (December 2011), Ministry of Higher Education and Science (DFF-1331-00113) and EDMaRC (Danish Ministry of Health). A.S.B. was funded from December 2015 by ReproUnion (EU Interreg Öresund-Kattegat-Skagerrak). The authors declare no conflict of interest. [ABSTRACT FROM AUTHOR]

Study Question: Does semen quality improve during early adulthood?Summary Answer: Semen variables change little during the third decade of life, however some improvement in sperm morphology and motility may occur.What Is Known Already: A suspicion of deteriorating semen quality has been raised in several studies. The longitudinal development of semen quality in early adulthood is insufficiently understood.Study Design, Size, Duration: A longitudinal follow-up of two cohorts of volunteer young adult Finnish men representing the general population was carried out. Cohorts A (discovery cohort, born 1979-1981, n = 336) and B (validation cohort, born 1983, n = 197) were followed up from the age of 19 years onward for 10 years.Participants/materials, Setting, Methods: Inclusion criteria included that both the men and their mothers were born in Finland. Semen analysis was performed in cohorts A and B at 2-4 year intervals over a period of 10 years. Semen volume, sperm concentration, total sperm count, motility, total motile count and morphology were the variables assessed in the analysis. A physical examination was carried out at each visit to detect any significant andrological abnormalities. The overall participation rate was 13.4%.Main Results and the Role Of Chance: During the follow-up, the percentage of sperm with normal morphology and the percentage of motile sperm increased significantly both in the discovery (A) (P < 0.001 at 19 versus 29 years for both) and validation (B) (P < 0.001 and P = 0.03 at 19 versus 29 years, respectively) cohort. Sperm concentration and total sperm count showed a significant increase with age only in cohort B (P = 0.03 at 21 versus 29 years, P = 0.009 at 19 versus 29 years, respectively).Limitations, Reasons For Caution: A limited number of men participated both in the first round and in the final fourth round (cohort A, n = 111 and cohort B, n = 90 men) and in all four rounds (cohort A, n = 61 and cohort B, n = 52).Wider Implications Of the Findings: Almost full spermatogenic capacity is reached by the age of 19 years. However, the improvement in sperm motility and morphology during early adulthood may slightly improve male fecundity.Study Funding/competing Interests: This study was supported by the European Commission (QLK4-CT-1999-01422, QLK4-CT-2001-00269, QLK4-2002-0063, FP7/2008-2012: DEER 212844), The Danish Medical Research Council (9700833, 9700909), Danish Agency for Science (Technology and Innovation 09-067180), the Svend Andersen's Foundation, Velux Foundation, and Novo Nordisk Foundation, the Turku University Hospital, Sigrid Jusélius Foundation and the Academy of Finland. There are no conflicts of interest. [ABSTRACT FROM AUTHOR]

Study Question: By investigating a birth cohort with a high ongoing participation rate to derive an unbiased population, what are the parameters and influences upon testicular function for a population not selected with regard to fertility?Summary Answer: While varicocele, cryptorchidism and obesity may impact on human testicular function, most common drug exposures and the presence of epididymal cysts appear to have no or minimal adverse impact.What Is Known Already: The majority of previous attempts to develop valid reference populations for spermatogenesis have relied on potentially biased sources such as recruits from infertility clinics, self-selected volunteer sperm donors for research or artificial insemination or once-fertile men seeking vasectomy. It is well known that studies requiring semen analysis have low recruitment rates which consequently question their validity. However, there has been some concern that a surprisingly high proportion of young men may have semen variables that do not meet all the WHO reference range criteria for fertile men, with some studies reporting that up to one half of participants have not meet the reference range for fertile men. Reported median sperm concentrations have ranged from 40 to 60 million sperm/ml.Study Design, Size and Duration: The Western Australian Pregnancy Cohort (Raine) was established in 1989. At 20-22 years of age, members of the cohort were contacted to attend for a general follow-up, with 753 participating out of the 913 contactable men. Of these, 423 men (56% of participants in the 20-22 years cohort study, 46% of contactable men) participated in a testicular function study. Of the 423 men, 404 had a testicular ultrasound, 365 provided at least one semen sample, 287 provided a second semen sample and 384 provided a blood sample.Participants/materials, Setting, Methods: Testicular ultrasound examinations were performed at King Edward Memorial Hospital, Subiaco, Perth, for testicular volume and presence of epididymal cysts and varicoceles. Semen samples were provided and analysed by standard semen assessment and a sperm chromatin structural assay (SCSA) at Fertility Specialists of Western Australia, Claremont, Perth. Serum blood samples were provided at the University of Western Australia, Crawley, Perth and were analysed for serum luteinizing hormone (LH), follicular stimulating hormone (FSH), inhibin B, testosterone, dihydrotestosterone (DHT), dehydroepiandrosterone (DHEA), estradiol, estrone and the primary metabolites of DHT: 5α-androstane-3α,17β-diol (3α-diol) and 5-α androstane-3-β-17-beta-diol (3β-diol). Serum steroids were measured by liquid chromatography, mass spectrometry and LH, FSH and inhibin B were measured by ELISA assays.Main Results and the Role Of Chance: Cryptorchidism was associated with a significant reduction in testicular (P = 0.047) and semen (P = 0.027) volume, sperm concentration (P = 0.007) and sperm output (P = 0.003). Varicocele was associated with smaller testis volume (P < 0.001), lower sperm concentration (P = 0.012) and total sperm output (P = 0.030) and lower serum inhibin B levels (P = 0.046). Smoking, alcohol intake, herniorrhaphy, an epididymal cyst, medication and illicit drugs were not associated with any significant semen variables, testicular volume or circulating reproductive hormones. BMI had a significantly negative correlation with semen volume (r = -0.12, P = 0.048), sperm output (r = -0.13, P = 0.02), serum LH (r = -0.16, P = 0.002), inhibin B (r = -0.16, P < 0.001), testosterone (r = -0.23, P < 0.001) and DHT (r = -0.22, P < 0.001) and a positive correlation with 3αD (r = 0.13, P = 0.041) and DHEA (r = 0.11, P = 0.03). Second semen samples compared with the first semen samples in the 287 participants who provided two samples, with no significant bias by Bland-Altman analysis. Testis volume was significantly correlated positively with sperm concentration (r = 0.25, P < 0.001) and sperm output (r = 0.29, P < 0.001) and inhibin B (r = 0.42, P < 0.001), and negatively correlated with serum LH (r = -0.24, P < 0.001) and FSH (r = -0.32, P < 0.001). SCSA was inversely correlated with sperm motility (r = -0.20, P < 0.001) and morphology (r = -0.16, P = 0.005). WHO semen reference criteria were all met by only 52 men (14.4%). Some criteria were not met at first analysis in 15-20% of men, including semen volume (<1.5 ml, 14.8%), total sperm output (<39 million, 18.9%), sperm concentration (<15 million/ml, 17.5%), progressive motility (<32%, 14.4%) and morphologically normal sperm (<4%, 26.4%), while all five WHO criteria were not met in four participants (1.1%).Limitations and Reasons For Caution: This was a large cohort study; however, potential for recruitment bias still exists. Men who did not participate in the testicular evaluation study (n = 282) did not differ from those who did (n = 423) with regard to age, weight, BMI, smoking or circulating reproductive hormones (LH, FSH, inhibin B, T, DHT, E2, E1, DHEA, 3α-diol, 3β-diol), but were significantly shorter (178 versus 180 cm, P = 0.008) and had lower alcohol consumption (P = 0.019) than those who did participate.Wider Implications Of the Findings: This study demonstrated the feasibility of establishing a birth cohort to provide a relatively unbiased insight into population-representative sperm output and function and of investigating its determinants from common exposures. While varicocele, cryptorchidism and obesity may impact on human testicular function, most common drug exposures and the presence of epididymal cysts appear to have little adverse impact, and this study suggests that discrepancies from the WHO reference ranges are expected, due to its derivation from non-population-representative fertile populations. [ABSTRACT FROM AUTHOR]

Study Question: What are the effects of experimentally manipulating meiosis signalling by addition of retinoic acid (RA) in cultured human fetal gonads?Summary Answer: RA-treatment accelerated meiotic entry in cultured fetal ovary samples, while addition of RA resulted in a dysgenetic gonadal phenotype in fetal testis cultures.What Is Known Already: One of the first manifestations of sex differentiation is the initiation of meiosis in fetal ovaries. In contrast, meiotic entry is actively prevented in the fetal testis at this developmental time-point. It has previously been shown that RA-treatment mediates initiation of meiosis in human fetal ovary ex vivo.Study Design, Size, Duration: This was a controlled ex vivo study of human fetal gonads treated with RA in 'hanging-drop' tissue cultures. The applied experimental set-up preserves germ cell-somatic niche interactions and the investigated outcomes included tissue integrity and morphology, cell proliferation and survival and the expression of markers of meiosis and sex differentiation.Participants/materials, Setting, Methods: Tissue from 24 first trimester human fetuses was included in this study, all from elective terminations at gestational week (GW) 7-12. Gonads were cultured for 2 weeks with and without addition of 1 µM RA. Samples were subsequently formalin-fixed and investigated by immunohistochemistry and cell counting. Proteins investigated and quantified included; octamer-binding transcription factor 4 (OCT4), transcription factor AP-2 gamma (AP2γ) (embryonic germ cell markers), SRY (sex determining region Y)-box 9 (SOX9), anti-Müllerian hormone (AMH) (immature Sertoli cell markers), COUP transcription factor 2 (COUP-TFII) (marker of interstitial cells), forkhead box L2 (FOXL2) (granulosa cell marker), H2A histone family, member X (γH2AX) (meiosis marker), doublesex and mab-3 related transcription factor 1 (DMRT1) (meiosis regulator), cleaved poly ADP ribose polymerase (PARP), cleaved Caspase 3 (apoptosis markers) and Ki-67 antigen (Ki-67) (proliferation marker). Also, proliferation was determined using a 5'-bromo-2'-deoxyuridine (BrdU) incorporation assay.Main Results and the Role Of Chance: A novel ex vivo 'hanging-drop' culture model for human fetal gonads was successfully established. Continued proliferation of cells without signs of increased apoptosis was observed after 2 weeks of culture. In cultured fetal ovaries treated with RA, an increased number of meiotic germ cells (P < 0.05) and DMRT1-positive oogonia initiating meiosis (P < 0.05) was observed, which is in agreement with a previous study. In fetal testes, RA-treatment resulted in a decreased number of gonocytes (P < 0.05), a reduced percentage of proliferating gonocytes (P < 0.05), altered expression pattern of the somatic cell markers AMH and COUP-TFII, as well as disrupted seminiferous cord structure and testis morphology.Limitations, Reasons For Caution: The number of samples included in this study was relatively small due to the limited availability of human fetal tissue.Wider Implications Of the Findings: The hanging-drop culture, similarly to other organ culture approaches, allows studies of germ cell-somatic niche interactions and determination of effects after manipulating specific signalling pathways. Our novel finding of disrupted fetal testis development after treatment with RA indicates that abnormal meiosis regulation can potentially cause gonadal dysgenesis. Further studies will elucidate the exact mechanisms and timing of observed effects.Study Funding/competing Interests: This work was supported in part by an ESPE Research Fellowship, sponsored by Novo Nordisk A/S to A.Jø. Additional funding for this project was obtained from The Research Council of the Capital Region of Denmark (E.R.-D.M.), The Research Fund at Rigshospitalet (A.Ju. and J.E.N.), Familien Erichssens Fund (A.Jø.), Dagmar Marshalls Fund (A.Jø.) and Aase & Ejnar Danielsens Fund (A.Jø.). The authors have no conflicts of interest. [ABSTRACT FROM AUTHOR]

STUDY QUESTION: Is overweight associated with impaired sperm DNA integrity? SUM MARY ANSWER: High body mass index (BMI) is not associated with impaired sperm DNA integrity as assessed by the DNA Fragmentation Index (DFI). WHAT IS KNOWN ALREADY: Previous studies, based on fewer subjects and including mainly subfertile men, have shown conflicting results regarding the influence of overweight and obesity on sperm DNA integrity. STUDY DESIGN, SIZE, DURATION: This cross-sectional study was based on semen samples from 1503 men from the general population. PARTICIPANTS/MATERIALS, SETTING, METHODS: We included two cohorts (cohort A and B) of military recruits (n = 275, n = 304, respectively), one group (cohort C) of fertile men and men without known fertility problems (n = 724), and one group (cohort D) of men between 19 and 40 years without known fertility problems (n = 200). In all cohorts, data were available on BMI, DFI as measured by the sperm chromatin structure assay (SCSA), standard semen characteristics, and potential confounders (age, abstinence time, smoking habits). The subjects were categorized according to BMI into four groups: underweight (< 18.5 kg/m²), normal weight (18.5-24.9 kg/m²), overweight (25.0-29.9 kg/m²) and obese (≥30.0 kg/m²). Using a linear regression model, the inter-group differences in DFI were calculated. Furthermore with the normal-weight group as the reference, the odds ratios (ORs) for DFI ≥ 20% and DFI ≥ 30%, were calculated for the other groups. Calculations were made for the material as a whole and after exclusion of cohort C which included proven fertile men. MAIN RESULTS AND THE ROLE OF CHANCE: We found that normal-weight men had significantly higher DFI than overweight men, with a mean difference of 1.13% (95% CI: 1.05-1.22%); P = 0.001). Overweight men had a reduced risk of having DFI ≥ 20% and DFI ≥ 30%, compared with normal-weight men; adjusted odds ratio (OR) = 0.61 (95% CI: 0.42-0.88; P < 0.01) and adjusted OR = 0.48 (95% CI: 0.28-0.84; P < 0.01), respectively. When excluding cohort C, the statistical significance was lost. Regarding standard semen parameters, we found that obese men had a higher percentage of progressive motile spermatozoa than normal-weight men; mean difference 1.15% (95% CI: 1.02-1.30%, P < 0.05) but the significance was lost when excluding cohort C. All other standard semen parameters were unaffected by BMI. LIMITATIONS, REASONS FOR CAUTION: A main limitation might be the cross-sectional nature of the data. Furthermore our study included a significant proportion of men with proven fertility (75% of cohort C, n = 550), and could therefore be biased toward fertility. WIDER IMPLICATIONS OF THE FINDINGS: Our study indicates that overweight per se is not associated with a higher level of sperm DNA damage. STUDY FUNDING/COMPETING INTEREST(S): This research has been given grants from the following: EU 5th and 7th framework program (Inuendo and Clear projects, [Contracts no. QLK4-CT-2001 -00202 and FP7-ENV-2008-1-226217)]), the Swedish Research Council (Grants No. 2007-2590, 521-2004-6072 and 521-2002-3907); the Swedish Governmental Funding for Clinical Research, Skane county council's research and development foundation, MAS Funds, University Hospital MAS Foundation in Malmo, Crafoordska Fund, Ove Tulefjords Fund, Foundation for Urological Research, Fundacion Federico SA, and Gunnar Nilssons Cancer Fund. The authors declare that there are no conflicts of interest. [ABSTRACT FROM AUTHOR]

STUDY QUESTION What is the differentiation stage of human testicular interstitial cells, in particular Leydig cells (LC), within micronodules found in patients with infertility, testicular cancer and Klinefelter syndrome? SUMMARY ANSWER The Leydig- and peritubular-cell populations in testes with dysgenesis contain an increased proportion of undifferentiated cells when compared with control samples, as demonstrated by increased delta-like homolog 1 (DLK1) and decreased insulin-like peptide 3 (INSL3) expression. WHAT IS KNOWN ALREADY Normal LC function is essential for male development and reproduction. Signs of LC failure, including LC micronodules, are often observed in patients with reproductive disorders. STUDY DESIGN, SIZE, PARTICIPANTS In this retrospective study, a panel of markers and factors linked to the differentiation of LCs was investigated in 33 fetal and prepubertal human specimens and in 58 adult testis samples from patients with testicular germ cell tumours, including precursor carcinoma in situ (CIS), infertility or Klinefelter syndrome. PARTICIPANTS/MATERIALS, SETTING, METHODS The expression patterns of DLK1, INSL3, chicken ovalbumin upstream promoter transcription factor 2 (COUP-TFII), cytochrome P450, family 11, subfamily A, polypeptide 1 (CYP11A1) and smooth muscle actin (SMA) were investigated by immunohistochemistry and quantitative RT–PCR. The percentage of positive LCs was estimated and correlated to total LC numbers and serum levels of reproductive hormones. MAIN RESULTS AND THE ROLE OF CHANCE DLK1, INSL3 and COUP-TFII expression changed during normal development and was linked to different stages of LC differentiation: DLK1 was expressed in all fetal LCs, but only in spindle-shaped progenitor cells and in a small subset of polygonal LCs in the normal adult testis; INSL3 was expressed in a subset of fetal LCs, but in the majority of adult LCs; and COUP-TFII was expressed in peritubular and mesenchymal stroma cells at all ages, in fetal LCs early in gestation and in a subset of adult LCs. CYP11A1 was expressed in the majority of LCs regardless of age and pathology and was the best general LC marker examined here. SMA was weakly expressed in peritubular cells in the fetal and infantile testis, but strongly expressed in the adult testis. In pathological testes, the numbers of DLK1-positive interstitial cells were increased. The proportion of DLK1-positive LCs correlated with total LC numbers (R = 0.53; P < 0.001) and was higher in testis with enlargement of the peritubular layers (P < 0.01), which was also highly associated with DLK1 expression in the peritubular compartment (P < 0.001). INSL3 expression was absent in some, but not all LC micronodules, and in the majority of LCs, it was mutually exclusive of DLK1. LIMITATIONS, REASONS FOR CAUTION The number of samples was relatively small and no true normal adult controls were available. True stereology was not used for LC counting, instead LCs were counted in three fields of 0.5 µm2 surface for each sample. WIDER IMPLICATIONS OF THE FINDINGS The population of LCs, especially those clustered in large nodules, are heterogeneous and comprise cells at different stages of differentiation. The study demonstrated that the differentiation and function of LCs, and possibly also peritubular cells, are impaired in adult men with testicular pathologies including testis cancer and Klinefelter syndrome. STUDY FUNDING/COMPETING INTEREST(S) This work was funded by Rigshospitalet's research funds, the Danish Cancer Society and Kirsten and Freddy Johansen's foundation. The authors have no conflicts of interest. [ABSTRACT FROM PUBLISHER]

STUDY QUESTION Is there an association between alcohol intake and semen quality and serum reproductive hormones among healthy men from the USA and Europe? SUMMARY ANSWER Moderate alcohol intake is not adversely associated with semen quality in healthy men, whereas it was associated with higher serum testosterone levels. WHAT IS KNOWN ALREADY High alcohol intake has been associated with a wide range of diseases. However, few studies have examined the correlation between alcohol and reproductive function and most have been conducted in selected populations of infertile men or have a small sample size and the results have been contradictory. STUDY DESIGN, SIZE, DURATION A coordinated international cross-sectional study among 8344 healthy men. A total of 1872 fertile men aged 18–45 years (with pregnant partners) from four European cities and four US states, and 6472 young men (most with unknown fertility) aged 18–28 years from the general population in six European countries were recruited. PARTICIPANTS/MATERIALS, SETTING, METHODS The men were recruited using standardized protocols. A semen analysis was performed and men completed a questionnaire on health and lifestyle, including their intake of beer, wine and liquor during the week prior to their visit. Semen quality (semen volume, sperm concentration, percentage motile and morphologically normal sperm) and serum reproductive hormones (FSH, LH, testosterone, sex hormone-binding globulin, and inhibin B and free testosterone) were examined. MAIN RESULTS AND THE ROLE OF CHANCE The participation rate for our populations was 20–30%. We found no consistent association between any semen variable and alcohol consumption, which was low/moderate in this group (median weekly intake 8 units), either for total consumption or consumption by type of alcohol. However, we found a linear association between total alcohol consumption and total or free testosterone in both groups of men. Young and fertile men who consumed >20 units of alcohol per week had, respectively, 24.6 pmol/l (95% confidence interval 16.3–32.9) and 19.7 pmol/l (7.1–32.2) higher free testosterone than men with a weekly intake between 1 and 10 units. Alcohol intake was not significantly associated with serum inhibin B, FSH or LH levels in either group of men. The study is the largest of its kind and has sufficient power to detect changes in semen quality and reproductive hormones. LIMITATIONS, REASONS FOR CAUTION The participation rate was low, but higher than in most previous semen quality studies. In addition, the study was cross-sectional and the men were asked to recall their alcohol intake in the previous week, which was used as a marker of intake up to 3 months before. If consumption in that week differed from the typical weekly intake and the intake 3 months earlier, misclassification of exposure may have occurred. However, the men were unaware of their semen quality when they responded to the questions about alcohol intake. Furthermore, we cannot exclude that our findings are due to unmeasured confounders, including diet, exercise, stress, occupation and risk-taking behavior. WIDER IMPLICATIONS OF THE FINDINGS Our study suggests that moderate alcohol intake is not adversely associated with semen quality in healthy men, whereas it was associated with higher serum testosterone levels which may be due to a changed metabolism of testosterone in the liver. Healthy men may therefore be advised that occasional moderate alcohol intake may not harm their reproductive health; we cannot address the risk of high alcohol consumption of longer duration or binge drinking on semen quality and male reproductive hormones. STUDY FUNDING/COMPETING INTEREST(S) All funding sources were non-profitable and sponsors of this study played no role in ... [ABSTRACT FROM PUBLISHER]

STUDY QUESTION Are there quantitative alterations in the proteome of normozoospermic sperm samples that are able to complete IVF but whose female partner does not achieve pregnancy? SUMMARY ANSWER Normozoospermic sperm samples with different IVF outcomes (pregnancy versus no pregnancy) differed in the levels of at least 66 proteins. WHAT IS KNOWN ALREADY The analysis of the proteome of sperm samples with distinct fertilization capacity using low-throughput proteomic techniques resulted in the detection of a few differential proteins. Current high-throughput mass spectrometry approaches allow the identification and quantification of a substantially higher number of proteins. STUDY DESIGN, SIZE, DURATION This was a case–control study including 31 men with normozoospermic sperm and their partners who underwent IVF with successful fertilization recruited between 2007 and 2008. PARTICIPANTS/MATERIALS, SETTING, METHODS Normozoospermic sperm samples from 15 men whose female partners did not achieve pregnancy after IVF (no pregnancy) and 16 men from couples that did achieve pregnancy after IVF (pregnancy) were included in this study. To perform the differential proteomic experiments, 10 no pregnancy samples and 10 pregnancy samples were separately pooled and subsequently used for tandem mass tags (TMT) protein labelling, sodium dodecyl sulphate–polyacrylamide gel electrophoresis, liquid chromatography tandem mass spectrometry (LC-MS/MS) identification and peak intensity relative protein quantification. Bioinformatic analyses were performed using UniProt Knowledgebase, DAVID and Reactome. Individual samples (n = 5 no pregnancy samples; n = 6 pregnancy samples) and aliquots from the above TMT pools were used for western blotting. MAIN RESULTS AND THE ROLE OF CHANCE By using TMT labelling and LC-MS/MS, we have detected 31 proteins present at lower abundance (ratio no pregnancy/pregnancy < 0.67) and 35 at higher abundance (ratio no pregnancy/pregnancy > 1.5) in the no pregnancy group. Bioinformatic analyses showed that the proteins with differing abundance are involved in chromatin assembly and lipoprotein metabolism (P values < 0.05). In addition, the differential abundance of one of the proteins (SRSF protein kinase 1) was further validated by western blotting using independent samples (P value < 0.01). LIMITATIONS, REASONS FOR CAUTION For individual samples the amount of recovered sperm not used for IVF was low and in most of the cases insufficient for MS analysis, therefore pools of samples had to be used to this end. WIDER IMPLICATIONS OF THE FINDINGS Alterations in the proteins involved in chromatin assembly and metabolism may result in epigenetic errors during spermatogenesis, leading to inaccurate sperm epigenetic signatures, which could ultimately prevent embryonic development. These sperm proteins may thus possibly have clinical relevance. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the Spanish Ministry of Economy and Competitiveness (Ministerio de Economia y Competividad; FEDER BFU 2009–07118 and PI13/00699) and Fundación Salud 2000 SERONO13-015. There are no competing interests to declare. [ABSTRACT FROM AUTHOR]

STUDY QUESTION Can we diagnose intratubular germ cell neoplasia (IGCN) using the immunohistochemical markers placental-like alkaline phosphatase (PLAP) and OCT3/4 using a novel cell-processing method ‘AgarCytos’, applied to the remnants of testicular sperm extraction (TESE) specimens and what is the prevalence of a testicular germ cell (pre)malignancy in men with a non-obstructive azoospermia (NOA) undergoing TESE for fertility treatment? SUMMARY ANSWER IGCN can be successfully detected by immunohistochemical evaluation of AgarCytos, made of the remnants of TESE biopsies. The observed prevalence of a germ cell (pre)malignancy in this specific population was found to be 4.4%. WHAT IS KNOWN ALREADY Infertile men are at higher risk for testicular cancer than the general population. IGCN can be detected by immunohistochemistry using PLAP and OCT3/4 in standard testicular biopsies and, with less accuracy, in semen. STUDY DESIGN, SIZE, DURATION Between January 2011 and April 2012 a prospective cohort study was conducted at a Dutch tertiary care academic training hospital. All males with NOA (n = 182) undergoing a urological work-up followed by a diagnostic TESE for fertility treatment (n = 251) were included. PARTICIPANTS, SETTING, METHODS After cryopreservation of sperm, if present, an AgarCyto was made of the remnants of the TESE biopsies. Sections were stained with haematoxylin–eosin for pathological examination as well as PLAP and OCT3/4 for immunohistochemistry to detect IGCN. MAIN RESULTS AND THE ROLE OF CHANCE Eight men (4.4%) were diagnosed with a germ cell (pre)malignancy: six of them had seminoma, two without and four with concomitant IGCN, and two of them had IGCN only. Microscopic evaluation including immunohistochemical analysis of the AgarCytos diagnosed three (1.6%) more cases of a germ cell (pre)malignancy compared with scrotal ultrasound alone (one case of bilateral seminoma with concomitant IGCN and two cases of IGCN alone). No false-positive cytology results were found upon conventional histological evaluation. LIMITATIONS, REASONS FOR CAUTION The main limitation of this study is lack of a simultaneously taken standard testicular biopsy, to compare the results of our novel diagnostic method with. Nevertheless, in all but one of our cases orchidectomy followed and the diagnosis was confirmed by histology. In the remaining case repeat TESE showed similar results. WIDER IMPLICATIONS OF THE FINDINGS Simultaneous screening for IGCN is highly recommended to men with NOA undergoing TESE, because of the increased incidence of germ cell (pre)malignancies in this specific population. The principal advantage of our new method is that all available testicular tissue can be used for both sperm recovery and pathological evaluation, increasing the yield of spermatozoa as well as the chance to find (pre)malignant cells. In those cases where the disease is still in a premalignant stage, early diagnosis will allow for timely treatment and reduction of morbidity and mortality in this group of patients. STUDY FUNDING/COMPETING INTEREST(S) This study was (partially) funded by Merck Serono (the Netherlands). There are no conflicting interests to disclose. TRIAL REGISTRATION NUMBER N/A. [ABSTRACT FROM AUTHOR]