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

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

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

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

Paternal prescription medication before conception: A retrospective cohort study of all births in Denmark 1997-2017.

AIM: To study what medication fathers are being prescribed in the months preceding conception. METHODS: A retrospective cohort study of Danish national registries, comprising all births in Denmark 1997-2017 (1.3 million births). Time trends and absolute levels of paternal prescription medication in the 6 months prior to conception were assessed. While all medications were examined (N = 1335), we focused on the main medication groups, medications that have increased in use over time, and medications for which previous evidence exists of an effect on sperm quality. RESULTS: The average number of prescriptions increased over the study period (from 0.75 prescriptions to 0.82 per birth). Polypharmacy (three or more prescriptions) increased from less than 8% to 10% of fathers. The use of pain medication, proton-pump inhibitors, selective serotonin reuptake inhibitors and some inhalants have all increased markedly over the last 20 years. CONCLUSIONS: Potential harm to the offspring done by paternal medication may present an increasing problem. As paternal medication exposure is increasing, examination of generational effects, such as major birth defects, is necessary.

Bisphenol A Diglycidyl Ether (BADGE) and Bisphenol Analogs, but Not Bisphenol A (BPA), Activate the CatSper Ca2+ Channel in Human Sperm.

Aim: Evidence suggests that bisphenol A diglycidyl ether (BADGE), bisphenol A (BPA), and BPA analogs can interfere with human male fertility. However, the effect directly on human sperm function is not known. The CatSper Ca2+ channel in human sperm controls important sperm functions and is necessary for normal male fertility. Environmental chemicals have been shown to activate CatSper and thereby affect Ca2+ signaling in human sperm. BPA has previously been investigated for effects on Ca2+ signaling human sperm, whereas the effects of other BPA analogs are currently unknown. The aim of this study is thus to characterize the effect of BADGE, BPA, and the eight analogs BPG, BPAF, BPC, BPB, BPBP, BPE, BPF, BPS on Ca2+ signaling, and CatSper in human sperm. Methods: Direct effects of the bisphenols on Ca2+ signaling in human sperm cells were evaluated using a Ca2+ fluorimetric assay measuring changes in intracellular Ca2+. Effects via CatSper were assessed using the specific CatSper inhibitor RU1968. Effects on human sperm function was assessed using an image cytometry-based acrosome reaction assay and the modified Kremer's sperm-mucus penetration assay. Results: At 10 µM the bisphenols BPG, BPAF, BPC, BADGE, BPB, and BPBP induced Ca2+ signals in human sperm cells, whereas BPE, BPF, BPS, and BPA had no effect. The efficacy of the chemicals at 10 µM is BPG > BPAF > BPC > BADGE > BPB > BPBP. Dose-response relations of BPG, BPAF, BPC, BADGE, BPB, and BPBP yielded EC50-values in the nM-µM range. The induced Ca2+ signals were almost completely abolished using the CatSper inhibitor RU1968, indicating an effect of the bisphenols on CatSper. All bisphenols, except BPBP, were found to dose-dependently inhibit progesterone-induced Ca2+ signals, with BPG and BPAF displaying inhibition even in low µM doses. BPG and BPAF were shown to affect human sperm function in a progesterone-like manner. Conclusion: Our results show that the bisphenols BPG, BPAF, BPC, BADGE, BPB, and BPBP can affect Ca2+ signaling in human sperm cells through activation of CatSper. This could potentially disrupt human sperm function by interfering with normal CatSper-signaling and thus be a contributing factor in human infertility, either alone or in mixtures with other chemicals.

Bone mineral density is preserved in men with idiopathic infertility.

BACKGROUND: Lower semen quality is associated with increased mortality and morbidity, which may include osteoporosis. OBJECTIVE: To assess whether infertile men have a lower bone mineral density (BMD) compared with fertile men at the time of fertility workup. METHODS: A total of 146 men from infertile couples with unexplained impaired semen quality, characterized by sperm concentration < 20 million/mL, progressive motility < 50% or < 12% morphologically normal spermatozoa. Men with infertility due to a genetic etiology or a condition that could cause testicular damage were excluded. A total of 271 men from couples with an ongoing naturally conceived pregnancy served as a control group. Lumbar, femoral, and total body BMD were measured by dual X-ray absorptiometry. RESULTS: Infertile men had similar BMD compared with fertile men (Beta coefficient (g/cm2 ) and 95% confidence interval for the difference between the two groups: -0.02 (-0.05; 0.01) for lumbar BMD, -0.02 (-0.05; 0.01) for femoral neck BMD, -0.01 (-0.04; 0.02) for total femur BMD, and -0.01 (-0.03; 0.01) for total body BMD). Semen parameters were not associated with BMD measurements. Furthermore, BMD did not differ between infertile men with the lowest semen quality vs. infertile men with better semen quality nor between infertile men with low testosterone vs. fertile men with normal testosterone levels. CONCLUSION: Bone mineral density is preserved in men with unexplained infertility at the time of fertility workup.

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

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

Semen quality of young men in Switzerland: a nationwide cross-sectional population-based study.

BACKGROUND: Sperm counts have been steadily decreasing over the past five decades with regional differences in the Western world. The reasons behind these trends are complex, but numerous insights indicate that environmental and lifestyle factors are important players. OBJECTIVE: To evaluate semen quality and male reproductive health in Switzerland. MATERIALS AND METHODS: A nationwide cross-sectional study was conducted on 2523 young men coming from all regions of Switzerland, recruited during military conscription. Semen volume, sperm concentration, motility, and morphology were analyzed. Anatomy of the genital area and testicular volume was recorded. Testicular cancer incidence rates in the general population were retrieved from Swiss regional registries. RESULTS: Median sperm concentration adjusted for period of sexual abstinence was 48 million/mL. Comparing with the 5th percentile of the WHO reference values for fertile men, 17% of men had sperm concentration below 15 million/mL, 25% had less than 40% motile spermatozoa, and 43% had less than 4% normal forms. Disparities in semen quality among geographic regions, urbanization rates, and linguistic areas were limited. A larger proportion of men with poor semen quality had been exposed in utero to maternal smoking. Furthermore, testicular cancer incidence rates in the Swiss general population increased significantly between 1980 and 2014. DISCUSSION: For the first time, a systematic sampling among young men has confirmed that semen quality is affected on a national level. The median sperm concentration measured is among the lowest observed in Europe. No specific geographical differences could be identified. Further studies are needed to determine to what extent the fertility of Swiss men is compromised and to evaluate the impact of environmental and lifestyle factors. CONCLUSION: A significant proportion of Swiss young men display suboptimal semen quality with only 38% having sperm concentration, motility, and morphology values that met WHO semen reference criteria.

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

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

An update on semen quality among young Finnish men and comparison with Danish data.

BACKGROUND: Finnish men used to have higher semen quality than Danish men. However, recent studies showed that semen quality in Finland has declined, but it has been relatively stable in Denmark. OBJECTIVE: This study aimed to compare new data on semen quality of the young Finnish men to that of Danish men. MATERIALS AND METHODS: In this cross-sectional study, 18- to 19-year-old men residing in Turku, Finland and Copenhagen, Denmark, were invited to participate in 2008-2011. Each man filled in a questionnaire, provided one semen sample and underwent andrological examination. Semen samples were analyzed according to WHO. Multiway ANOVA was used to adjust semen variables for duration of sexual abstinence and age (and time from ejaculation to the start of semen analysis for sperm motility). RESULTS: Altogether 287 Finnish men and 873 Danish men participated in the study. The adjusted median sperm concentrations were 49 and 47 million/mL for Finnish and Danish men, respectively (p = 0.48). The adjusted median total sperm counts were 148 million in Finland and 146 million in Denmark (p = 0.87). The adjusted median percentages of morphologically normal spermatozoa were 6.9% in Finland and 6.5% in Denmark, p = 0.27. Finnish men had higher adjusted median percentages of motile spermatozoa (A+B+C) than Danish men (80% vs. 69%, p < 0.001). The proportion of men who had low semen quality (sperm concentration, percentage of morphologically normal spermatozoa or percentage of progressively motile spermatozoa below WHO reference limits) was lower in Finland (25.4%) than in Denmark (34.6%), p = 0.004. DISCUSSION: Considerable percentage of men in both countries had low semen quality. The deteriorating semen quality in Finland may result in decreasing fecundity, which is a cause of concern. CONCLUSION: The formerly high semen quality in Finland has converged to the lower Danish levels. Our findings demonstrate the importance of continuing surveillance of semen quality.

Synergistic activation of CatSper Ca2+ channels in human sperm by oviductal ligands and endocrine disrupting chemicals.

STUDY QUESTION: Does the chemosensory activation of CatSper Ca2+ channels in human sperm give rise to additive, sub-additive or even synergistic actions among agonists? SUMMARY ANSWER: We show that oviductal ligands and endocrine disrupting chemicals (EDCs) activate human CatSper highly synergistically. WHAT IS KNOWN ALREADY: In human sperm, the sperm-specific CatSper channel controls the intracellular Ca2+ concentration and, thereby, several crucial stages toward fertilization. CatSper is activated by oviductal ligands and structurally diverse EDCs. The chemicals mimic the action of the physiological ligands, which might interfere with the precisely coordinated sequence of events underlying fertilization. STUDY DESIGN, SIZE, DURATION: For both oviductal ligands and EDCs, we examined in quantitative terms whether stimulation of human sperm in vitro with mixtures results in additive, sub-additive or synergistic actions. PARTICIPANTS/MATERIALS, SETTING, METHODS: We studied activation of CatSper in sperm of healthy volunteers, using kinetic Ca2+ fluorimetry and patch-clamp recordings. The combined action of progesterone and prostaglandins and of the EDCs benzylidene camphor sulfonic acid (BCSA) and α-Zearalenol was evaluated by curve-shift analysis, curvilinear isobolographic analysis and the combination-index method. MAIN RESULTS AND THE ROLE OF CHANCE: Analysis of the action of progesterone/prostaglandin and BCSA/α-Zearalenol mixtures in human sperm by fluorimetry revealed that the oviductal ligands and EDCs both evoke Ca2+ influx via CatSper in a highly synergistic fashion. Patch-clamp recordings of CatSper currents in human sperm corroborated the synergistic ligand-activation of the channel. 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: These findings indicate that the fertilization process is orchestrated by multiple oviductal CatSper agonists that act in concert to control the behavior of sperm. Moreover, our results substantiate the concerns regarding the negative impact of EDCs on male reproductive health. So far, safety thresholds like the "No Observed Adverse Effect Level (NOAEL)" or "No Observed Effect Concentration (NOEC)" are set for individual EDCs. Our finding that EDCs act synergistically in human sperm challenges the validity of this procedure. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the German Research Foundation (SFB 645; CRU326), the Cells-in-Motion (CiM) Cluster of Excellence, Münster, (FF-2016-17), the 'Innovative Medical Research' of the University of Münster Medical School (BR121507), an EDMaRC research grant from the Kirsten and Freddy Johansen's Foundation, and the Innovation Fund Denmark (InnovationsFonden; 14-2013-4). The authors have no competing financial interests.

Quantification of the Leydig cell compartment in testicular biopsies and association with biochemical Leydig cell dysfunction in testicular cancer survivors.

A simple histological method to evaluate the Leydig cell compartment is lacking. We aimed to establish such a method and to investigate if Leydig cell hyperplasia of the biopsy contralateral to the tumour-bearing testicle in patients with testicular germ cell cancer is associated with biochemical signs of Leydig cell dysfunction after long-term follow-up. A case group of 50 long-term testicular germ cell cancer survivors without human chorionic gonadotropin elevation, 10 testicular germ cell cancer patients with elevated human chorionic gonadotropin and 10 controls without testicular malignancy were included. For each subject, 2-4 representative sections from their testicular biopsies were selected for analysis. Using the image processing program ImageJ (V.1.48, NIH), an area with a minimum of 50 tubules was selected and delineated (total selected area) and the total Leydig cell area was calculated by adding up every delineated Leydig cell group within the total selected area. Four different methods were tested for the ability to quantify the Leydig cell compartment. In the 50 testicular germ cell cancer survivors, associations between the area of the Leydig cell compartment and serum levels of testosterone and luteinising hormone were investigated using linear regression analysis. The Leydig cell compartment was best quantified by the total Leydig cell area/total selected area index, which was significantly larger in the human chorionic gonadotropin-positive patients than in controls (P = 0.00001). In the 50 human chorionic gonadotropin-negative testicular germ cell cancer survivors, increasing total Leydig cell area/total selected area was significantly associated with decreased levels of total testosterone and decreased total testosterone/luteinising hormone ratio after a median of 9-year follow-up. In conclusion, a new simple method, total Leydig cell area/total selected area, was established to estimate the Leydig cell compartment in testicular biopsies. The index identified Leydig cell hyperplasia in the contralateral biopsy in patients with testicular germ cell cancer, and it was associated with long-term biochemical Leydig cell dysfunction. Although in testicular germ cell cancer survivors, the clinical value is limited because the contralateral biopsies are not commonly available, we propose a closer andrological follow-up in any patient with an increased total Leydig cell area/total selected area index.

Average sperm count remains unchanged despite reduction in maternal smoking: results from a large cross-sectional study with annual investigations over 21 years.

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.

Maternal exposure to UV filters: associations with maternal thyroid hormones, IGF-I/IGFBP3 and birth outcomes.

BACKGROUND: Several chemical UV filters/absorbers ('UV filters' hereafter) have endocrine-disrupting properties in vitro and in vivo. Exposure to these chemicals, especially during prenatal development, is of concern. OBJECTIVES: To examine maternal exposure to UV filters, associations with maternal thyroid hormone, with growth factor concentrations as well as to birth outcomes. METHODS: Prospective study of 183 pregnant women with 2nd trimester serum and urine samples available. Maternal concentrations of the chemical UV filters benzophenone-1 (BP-1) and benzophenone-3 (BP-3) in urine and 4-hydroxy-benzophenone (4-HBP) in serum were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The relationships between 2nd trimester maternal concentrations of the three chemical UV filters and maternal serum concentrations of thyroid hormones and growth factors, as well as birth outcomes (weight, height, and head and abdominal circumferences) were examined. RESULTS: Positive associations between maternal serum concentrations of 4-HBP and triiodothyronine (T3), thyroxine (T4), insulin-like growth factor I (IGF-I) and its binding protein IGFBP3 were observed in mothers carrying male fetuses. Male infants of mothers in the middle 4-HBP exposure group had statistically significantly lower weight and shorter head and abdominal circumferences at birth compared to the low exposure group. CONCLUSIONS: Widespread exposure of pregnant women to chemical UV filters and the possible impact on maternal thyroid hormones and growth factors, and on fetal growth, calls for further studies on possible long-term consequences of the exposure to UV filters on fetal development and children's health.

EDC IMPACT: Chemical UV filters can affect human sperm function in a progesterone-like manner.

Human sperm cell function must be precisely regulated to achieve natural fertilization. Progesterone released by the cumulus cells surrounding the egg induces a Ca2+ influx into human sperm cells via the CatSper Ca2+-channel and thereby controls sperm function. Multiple chemical UV filters have been shown to induce a Ca2+ influx through CatSper, thus mimicking the effect of progesterone on Ca2+ signaling. We hypothesized that these UV filters could also mimic the effect of progesterone on sperm function. We examined 29 UV filters allowed in sunscreens in the US and/or EU for their ability to affect acrosome reaction, penetration, hyperactivation and viability in human sperm cells. We found that, similar to progesterone, the UV filters 4-MBC, 3-BC, Meradimate, Octisalate, BCSA, HMS and OD-PABA induced acrosome reaction and 3-BC increased sperm penetration into a viscous medium. The capacity of the UV filters to induce acrosome reaction and increase sperm penetration was positively associated with the ability of the UV filters to induce a Ca2+ influx. None of the UV filters induced significant changes in the proportion of hyperactivated cells. In conclusion, chemical UV filters that mimic the effect of progesterone on Ca2+ signaling in human sperm cells can similarly mimic the effect of progesterone on acrosome reaction and sperm penetration. Human exposure to these chemical UV filters may impair fertility by interfering with sperm function, e.g. through induction of premature acrosome reaction. Further studies are needed to confirm the results in vivo.

Presence of benzophenones commonly used as UV filters and absorbers in paired maternal and fetal samples.

BACKGROUND: Previous studies have demonstrated widespread exposure of humans to certain benzophenones commonly used as UV filters or UV absorbers; some of which have been demonstrated to have endocrine disrupting abilities. OBJECTIVES: To examine whether benzophenones present in pregnant women pass through the placental barrier to amniotic fluid and further to the fetal blood circulation. METHODS: A prospective study of 200 pregnant women with simultaneously collected paired samples of amniotic fluid and maternal serum and urine. In addition, unique samples of human fetal blood (n=4) obtained during cordocentesis: and cord blood (n=23) obtained at delivery, both with paired maternal samples of serum and urine collected simultaneously, were used. All biological samples were analyzed by TurboFlow-liquid chromatography - tandem mass spectrometry for seven different benzophenones. RESULTS: Benzophenone-1 (BP-1), benzophenone-3 (BP-3), 4-methyl-benzophenone (4-MBP), and 4-hydroxy-benzophenone (4-HBP) were all detectable in amniotic fluid and cord blood samples and except 4-HBP also in fetal blood; albeit at a low frequency. BP-1 and BP-3 were measured at ~10-times lower concentrations in fetal and cord blood compared to maternal serum and 1000-times lower concentration compared to maternal urine levels. Therefore BP-1 and BP-3 were only detectable in the fetal circulation in cases of high maternal exposure indicating some protection by the placental barrier. 4-MBP seems to pass into fetal and cord blood more freely with a median 1:3 ratio between cord blood and maternal serum levels. Only for BP-3, which the women seemed to be most exposed to, did the measured concentrations in maternal urine and serum correlate to concentrations measured in amniotic fluid. Thus, for BP-3, but not for the other tested benzophenones, maternal urinary levels seem to be a valid proxy for fetal exposure. CONCLUSIONS: Detectable levels of several of the investigated benzophenones in human amniotic fluid as well as in fetal and cord blood calls for further investigations of the toxicokinetic and potential endocrine disrupting properties of these compounds in order for better assessment of the risk to the developing fetus.

Is the FSHR 2039A>G variant associated with susceptibility to testicular germ cell cancer?

Testicular germ cell cancer (TGCC) is derived from germ cell neoplasia in situ (GCNIS), which arises due to niche disturbances affecting the Sertoli cells. It is believed that exogenous endocrine factors have a crucial role in governing neoplastic transformation but on a strong hereditary background. Follicle-stimulating hormone (FSH) is the major regulatory hormone of the Sertoli cells. FSH signalling-related single-nucleotide polymorphisms (SNPs) have previously been shown to affect FSH action in men at different levels. We aimed to investigate whether three FSH-related SNPs (FSHR 2039A>G, FSHR -29G>A and FSHB -211G>T) are associated with development of TGCC. A total of 752 Danish and German patients with TGCC from two tertiary andrological referral centres were included. Three control groups comprising 2020 men from the general population, 679 fertile men and 417 infertile men, were also included. Chi-squared test was performed to compare genotype- and allele frequencies. Kruskal-Wallis test was performed to compare age at diagnosis. Patients with TGCC had a higher frequency of the A-allele of FSHR 2039A>G compared to the group of fertile men with an AA-genotype frequency of 30.2% vs. 22.0%, respectively, p = 0.002. This variant is associated with higher FSH receptor activity. The distribution of the FSHR 2039A>G did not differ significantly between the patients with TGCC and the infertile or the general population. The frequency of the two other SNPs did not differ between patient with TGCC and any of the control groups. No differences were detected between genotypes and age distribution or histological subtype of the tumours. In conclusion, we observed that a genetic variant associated with FSHR activity may modulate the susceptibility to TGCC.

Possible involvement of the glucocorticoid receptor (NR3C1) and selected NR3C1 gene variants in regulation of human testicular function.

Perceived stress has been associated with decreased semen quality but the mechanisms have not been elucidated. It is not known whether cortisol, the major stress hormone in humans, can act directly via receptors in the testis, and whether variants in the gene encoding the glucocorticoid receptor (NR3C1) can possibly modulate the effect. To address these questions, we investigated the expression of the glucocorticoid receptor in human testicular tissue, including adult and fetal samples (n = 20) by immunohistochemical staining, and in silico analysis of publicly available datasets. In the adult testis NR3C1 protein was detected in peritubular cells, a subset of Leydig cells, Sertoli cells (weak), and spermatogonia, but not in spermatids. The NR3C1 expression pattern in fetal testis samples differed by a notably stronger reaction in Sertoli cells, lack of staining in gonocytes but the presence in a subset of pro-spermatogonia, and the almost absent reaction in nascent peritubular cells. In parallel, we explored the association between adult testicular function and three single nucleotide NR3C1 polymorphisms (BcII [rs41423247], 9ß [rs6198], and Tth111I [rs10052957]) affecting glucocorticoid sensitivity. Testicular function was determined by semen analysis and reproductive hormone profiling in 893 men from the general population. The NR3C1 SNP BclI was associated with semen quality in an over-dominant manner with heterozygotes having better semen parameters compared to both homozygote constellations, and with sperm motility showing the strongest association. This association was supported by a higher inhibin B and inhibin B/FSH ratio, as well as a lower FSH in BclI heterozygotes. The SNPs 9ß and Tth111I were not associated with semen parameters. Although the clinical impact of the findings is limited, the results substantiate a suggested link between stress and testicular function. Hence this investigation should be regarded as a discovery study generating hypotheses for future studies.

Assumed non-persistent environmental chemicals in human adipose tissue; matrix stability and correlation with levels measured in urine and serum.

The aim of this study was to (1) optimize a method for the measurement of parabens and phenols in adipose tissue, (2) evaluate the stability of chemical residues in adipose tissue samples, and (3) study correlations of these compounds in urine, serum, and adipose tissue. Samples were obtained from adults undergoing trauma surgery. Nine phenols and seven parabens were determined by isotope diluted TurboFlow-LC-MS/MS. The analytical method showed good accuracy and precision. Limits of detection (LOD) for parabens and phenols ranged from 0.05 to 1.83ng/g tissue. Good recovery rates were found, even when biological samples remained defrosted up to 24h. Benzophenone-3 (BP-3; range of values: 70% of adipose tissue samples, while bisphenol-A (BPA; 40% of adipose tissue samples. In general, levels were similar between adipose tissue and serum, while a correlation between adipose tissue and urine was only found for BP-3. In conclusion, adipose tissue samples in this study were found to contain environmental chemicals considered to be non-persistent, whose levels were weakly or not at all correlated with the urine burden. Therefore, adipose tissue may potentially provide additional information to that obtained from other biological matrices. Further investigations are warranted to explore whether adipose tissue might be a suitable matrix for assessment of the consequences for human health of mid/long-term exposure to these chemicals.

Chemical UV Filters Mimic the Effect of Progesterone on Ca2+ Signaling in Human Sperm Cells.

Progesterone released by cumulus cells surrounding the egg induces a Ca2+ influx into human sperm cells via the cationic channel of sperm (CatSper) Ca2+ channel and controls multiple Ca2+-dependent responses essential for fertilization. We hypothesized that chemical UV filters may mimic the physiological action of progesterone on CatSper, thus affecting Ca2+ signaling in human sperm cells. We examined 29 UV filters allowed in sunscreens in the United States and/or the European Union for their ability to induce Ca2+ signals in human sperm by applying measurements of the intracellular free Ca2+ concentration. We found that 13 UV filters induced a significant Ca2+ signal at 10 µM. Nine UV filters induced Ca2+ signals primarily by activating the CatSper channel. The UV filters 3-benzylidene camphor (3-BC) and benzylidene camphor sulfonic acid competitively inhibited progesterone-induced Ca2+ signals. Dose-response relations for the UV filters showed that the Ca2+ signal-inducing effects began in the nanomolar-micromolar range. Single-cell Ca2+ measurements showed a Ca2+ signal-inducing effect of the most potent UV filter, 3-BC, at 10 nM. Finally, we demonstrated that the 13 UV filters acted additively in low-dose mixtures to induce Ca2+ signals. In conclusion, 13 of 29 examined UV filters (44%) induced Ca2+ signals in human sperm. Nine UV filters primarily activated CatSper and thereby mimicked the effect of progesterone. The UV filters 3-BC and benzylidene camphor sulfonic acid competitively inhibited progesterone-induced Ca2+ signals. In vivo exposure studies are needed to investigate whether UV filter exposure affects human fertility.