Exposure to bisphenol A in European women from 2007 to 2014 using human biomonitoring data - The European Joint Programme HBM4EU.

BACKGROUND: Bisphenol A (BPA; or 4,4'-isopropylidenediphenol) is an endocrine disrupting chemical. It was widely used in a variety of plastic-based manufactured products for several years. The European Food Safety Authority (EFSA) recently reduced the Tolerable Daily Intake (TDI) for BPA by 20,000 times due to concerns about immune-toxicity. OBJECTIVE: We used human biomonitoring (HBM) data to investigate the general level of BPA exposure from 2007 to 2014 of European women aged 18-73 years (n = 4,226) and its determinants. METHODS: Fifteen studies from 12 countries (Austria, Belgium, Denmark, France, Germany, Greece, Israel, Luxembourg, Slovenia, Spain, Sweden, and the United Kingdom) were included in the BPA Study protocol developed within the European Joint Programme HBM4EU. Seventy variables related to the BPA exposure were collected through a rigorous post-harmonization process. Linear mixed regression models were used to investigate the determinants of total urine BPA in the combined population. RESULTS: Total BPA was quantified in 85-100 % of women in 14 out of 15 contributing studies. Only the Austrian PBAT study (Western Europe), which had a limit of quantification 2.5 to 25-fold higher than the other studies (LOQ=2.5 µg/L), found total BPA in less than 5 % of the urine samples analyzed. The geometric mean (GM) of total urine BPA ranged from 0.77 to 2.47 µg/L among the contributing studies. The lowest GM of total BPA was observed in France (Western Europe) from the ELFE subset (GM=0.77 µg/L (0.98 µg/g creatinine), n = 1741), and the highest levels were found in Belgium (Western Europe) and Greece (Southern Europe), from DEMOCOPHES (GM=2.47 µg/L (2.26 µg/g creatinine), n = 129) and HELIX-RHEA (GM=2.47 µg/L (2.44 µg/g creatinine), n = 194) subsets, respectively. One hundred percent of women in 14 out of 15 data collections in this study exceeded the health-based human biomonitoring guidance value for the general population (HBM-GVGenPop) of 0.0115 µg total BPA/L urine derived from the updated EFSA's BPA TDI. Variables related to the measurement of total urine BPA and those related to the main socio-demographic characteristics (age, height, weight, education, smoking status) were collected in almost all studies, while several variables related to BPA exposure factors were not gathered in most of the original studies (consumption of beverages contained in plastic bottles, consumption of canned food or beverages, consumption of food in contact with plastic packaging, use of plastic film or plastic containers for food, having a plastic floor covering in the house, use of thermal paper…). No clear determinants of total urine BPA concentrations among European women were found. A broader range of data planned for collection in the original questionnaires of the contributing studies would have resulted in a more thorough investigation of the determinants of BPA exposure in European women. CONCLUSION: This study highlights the urgent need for action to further reduce exposure to BPA to protect the population, as is already the case in the European Union. The study also underscores the importance of pre-harmonizing HBM design and data for producing comparable data and interpretable results at a European-wide level, and to increase HBM uptake by regulatory agencies.

Serum Phthalate Concentrations and Biomarkers of Oxidative Stress in Adipose Tissue in a Spanish Adult Cohort.

The relationship between phthalates, a group of chemical pollutants classified as endocrine disruptors, and oxidative stress is not fully understood. The aim of the present hospital-based study was to explore the associations between circulating levels of 10 phthalate metabolites and 8 biomarkers of oxidative stress in adipose tissue. The study population (n = 143) was recruited in two hospitals in the province of Granada (Spain). Phthalate metabolite concentrations were analyzed by isotope diluted online-TurboFlow-LC-MS/MS in serum samples, while oxidative stress markers were measured by commercially available kits in adipose tissue collected during routine surgery. Statistical analyses were performed by MM estimators' robust linear regression and weighted quantile sum regression. Mainly, positive associations were observed of monomethyl phthalate (MMP), monoiso-butyl phthalate (MiBP), and mono-n-butyl phthalate (MnBP) (all low molecular weight phthalates) with glutathione peroxidase (GPx) and thiobarbituric acid reactive substances (TBARS), while an inverse association was found between monoiso-nonyl phthalate (MiNP) (high molecular weight phthalate) and the same biomarkers. WQS analyses showed significant effects of the phthalate mixture on GSH (ß = -30.089; p-value = 0.025) and GSSG levels (ß = -19.591; p-value = 0.030). Despite the limitations inherent to the cross-sectional design, our novel study underlines the potential influence of phthalate exposure on redox homeostasis, which warrants confirmation in further research.

High incidence of imperforate vagina in ADGRA3-deficient mice.

BACKGROUND: Ten percent of the female population suffers from congenital abnormalities of the vagina, uterus, or oviducts, with severe consequences for reproductive and psychological health. Yet, the underlying causes of most of these malformations remain largely unknown. ADGRA3 (GPR125) is involved in WNT signaling and planar cell polarity, mechanisms vital to female reproductive tract development. Although ADGRA3 is a well-established spermatogonial stem cell marker, its role within the female urogenital system remains unclear. RESULTS: In this study, we found Adgra3 to be expressed throughout the murine female urogenital system, with higher expression pre-puberty than after sexual maturation. We generated a global Adgra3-/- mouse line and observed imperforate vagina in 44% of Adgra3-/- females, resulting in distension of the reproductive tract and infertility. Ovarian morphology, plasma estradiol, ovarian Cyp19a1, and vaginal estrogen receptor α (Esr1) expression were unaffected. However, compared to controls, a significantly lower bone mineral density was found in Adgra3-/- mice. Whereas vaginal opening in mice is an estrogen-dependent process, 17ß-estradiol treatment failed to induce vaginal canalization in Adgra3-/- mice. Furthermore, a marked reduction in vaginal and ovarian progesterone receptor expression was observed concomitant with an upregulation of apoptotic regulators Bcl2, Bid, and Bmf in adult Adgra3-/- females with a closed vagina. CONCLUSIONS: Our collective results shed new insights into the complex mechanisms by which the adhesion receptor ADGRA3 regulates distal vaginal tissue remodeling during vaginal canalization via altered sex hormone responsiveness and balance in apoptotic regulators. This highlights the potential of ADGRA3 as a target in diagnostic screening and/or therapy for obstructive vaginal malformations in humans.

Time Patterns in Internal Human Exposure Data to Bisphenols, Phthalates, DINCH, Organophosphate Flame Retardants, Cadmium and Polyaromatic Hydrocarbons in Europe.

Human biomonitoring (HBM) data in Europe are often fragmented and collected in different EU countries and sampling periods. Exposure levels for children and adult women in Europe were evaluated over time. For the period 2000-2010, literature and aggregated data were collected in a harmonized way across studies. Between 2011-2012, biobanked samples from the DEMOCOPHES project were used. For 2014-2021, HBM data were generated within the HBM4EU Aligned Studies. Time patterns on internal exposure were evaluated visually and statistically using the 50th and 90th percentiles (P50/P90) for phthalates/DINCH and organophosphorus flame retardants (OPFRs) in children (5-12 years), and cadmium, bisphenols and polycyclic aromatic hydrocarbons (PAHs) in women (24-52 years). Restricted phthalate metabolites show decreasing patterns for children. Phthalate substitute, DINCH, shows a non-significant increasing pattern. For OPFRs, no trends were statistically significant. For women, BPA shows a clear decreasing pattern, while substitutes BPF and BPS show an increasing pattern coinciding with the BPA restrictions introduced. No clear patterns are observed for PAHs or cadmium. Although the causal relations were not studied as such, exposure levels to chemicals restricted at EU level visually decreased, while the levels for some of their substitutes increased. The results support policy efficacy monitoring and the policy-supportive role played by HBM.

Benzophenone-3: Comprehensive review of the toxicological and human evidence with meta-analysis of human biomonitoring studies.

BACKGROUND: Benzophenone-3 (BP-3) and its major metabolite benzophenone-1 (BP-1) are widely used as UV filters in sunscreens and cosmetics to prevent sunburn and skin damage, or as stabilizers to prevent photodegradation in many commercial products. As a result, their presence is ubiquitous in the environment, wildlife and humans. Based on endocrine disruption concerns, international regulatory agencies are performing a closer evaluation. OBJECTIVE AND METHODS: This work aimed to comprehensively review the available human relevant evidence for safety issues in MEDLINE/PubMed in order to create a structured database of studies, as well as to conduct an integrative analysis as part of the Human Biomonitoring for Europe (HBM4EU) Initiative. RESULTS: A total of 1,635 titles and abstracts were screened and 254 references were evaluated and tabulated in detail, and classified in different categories: i) exposure sources and predictors; ii) human biomonitoring (HBM) exposure levels to perform a meta-analysis; iii) toxicokinetic data in both experimental animals and humans; iv) in vitro and in vivo rodent toxicity studies; and v) human data on effect biomarkers and health outcomes. Our integrative analysis showed that internal peak BP-3 concentrations achieved after a single whole-body application of a commercially available sunscreen (4% w/w) may overlap with concentrations eliciting endocrine disrupting effects in vitro, and with internal concentrations causing in vivo adverse female reproductive effects in rodents that were supported by still limited human data. The adverse effects in rodents included prolonged estrous cycle, altered uterine estrogen receptor gene expression, endometrium hyperplasia and altered proliferation and histology of the mammary gland, while human data indicated menstrual cycle hormonal alterations and increased risk of uterine fibroids and endometriosis. Among the modes of action reported (estrogenic, anti-androgenic, thyroid, etc.), BP-3 and especially BP-1 showed estrogenic activity at human-relevant concentrations, in agreement with the observed alterations in female reproductive endpoints. The meta-analysis of HBM studies identified a higher concern for North Americans, showing urinary BP-3 concentrations on average 10 and 20 times higher than European and Asian populations, respectively. DISCUSSION AND CONCLUSIONS: Our work supports that these benzophenones present endocrine disrupting properties, endorsing recent European regulatory efforts to limit human exposure. The reproducible and comprehensive database generated may constitute a point of departure in future risk assessments to support regulatory initiatives. Meanwhile, individuals should not refrain from sunscreen use. Commercially available formulations using inorganic UV filters that are practically not absorbed into systemic circulation may be recommended to susceptible populations.

Loss of Adgra3 causes obstructive azoospermia with high penetrance in male mice.

The adhesion receptor ADGRA3 (GPR125) is a known spermatogonial stem cell marker, but its impact on male reproduction and fertility has not been examined. Using a mouse model lacking Adgra3 (Adgra3-/- ), we show that 55% of the male mice are infertile from puberty despite having normal spermatogenesis and epididymal sperm count. Instead, male mice lacking Adgra3 exhibited decreased estrogen receptor alpha expression and transient dilation of the epididymis. Combined with an increased estradiol production, this indicates a post-pubertal hormonal imbalance and fluid retention. Dye injection revealed a blockage between the ejaculatory duct and the urethra, which is rare in mice suffering from infertility, thereby mimicking the etiologies of obstructive azoospermia found in human male infertility. To summarize, male reproductive tract development is dependent on ADGRA3 function that in concert with estrogen signaling may influence fluid handling during sperm maturation and storage.

Associations of circulating levels of phthalate metabolites with cytokines and acute phase reactants in a Spanish human cohort.

The associations between human phthalate exposure and the onset of chronic diseases with an immunological component (e.g., metabolic syndrome, cancer) remain unclear, partly due to the uncertainties in the underlying mechanisms. This study investigates cross-sectional associations of the concentrations of 10 phthalate metabolites with 19 cytokines and acute phase proteins in 213 serum samples of Spanish adults. The associations were explored by Spearman's correlation, multivariable linear regression, and weighted quantile sum regression analyses. In the multivariable analyses, levels of plasminogen activator inhibitor (PAI)-1 were positively associated with mono-n-butyl phthalate (fold-change per one IQR increase in phthalate levels, 95% Confidence Interval: 1.65, 1.45-1.88) and mono-iso-butyl phthalate (3.07, 2.39-3.95), mono-ethyl phthalate (2.05, 1.62-2.61), as well as categorized mono-iso-decyl and mono-benzyl phthalates. The same phthalates also were significantly associated with leptin, interleukin (IL)-18 and monocyte chemoattractant protein-1. Moreover, the proinflammatory markers IL-1ß, IL-17, IL-8, IL-6, IL-12, tumor necrosis factor, and lipopolysaccharide-binding protein showed positive and negative associations with, respectively, mono-(2-ethyl-hexyl) and mono-methyl phthalates. Finally, phthalate mixtures were positively associated with PAI-1, leptin, IL-18, IL-12, IL-8 and IL-1ß. Despite the cross-sectional design limitation, these associations point to relevant subclinical immuno-inflammatory actions of these pollutants, warranting confirmation in future studies.

Longitudinal evaluation of breast tissue in healthy infants: Prevalence and relation to reproductive hormones and growth factors.

Introduction: Breast tissue in infancy is a rather undescribed phenomenon. We aimed to describe the prevalence and progression of palpable breast tissue in healthy boys and girls aged 0-1 years and to evaluate clinical markers, individual serum hormone concentrations as well as combined hormone profiles as determinants of the persistence of breast tissue. Methods: In total, 233 term infants (119 boys, 114 girls) were included and followed from birth until 1 year of age in The COPENHAGEN Minipuberty Study (ClinicalTrials.gov #NTC02784184). Infants were followed up to six times with a clinical examination and serum sampling. Principal component analyses (PCAs) produced combined hormone profiles. Results: A total of 98% of all infants aged 0-1 year exhibited breast tissue at some point. 50% still had breast tissue present at 0.5-0.6 years in girls and 0.3-0.4 years in boys ('persistent'). At one year, more girls than boys had breast tissue present (p=0.010). Most clinical and hormonal markers did not differ in infants with/without persistent breast tissue. However, in those with persistent breast tissue, estradiol (first visit, girls, p=0.034), androstenedione, corticosterone, cortisol (first visit, boys, all p<0.050), length (first visit, boys, p=0.030), and testicular volume (0.3-0.4 years, p=0.040) were higher, while IGF-I (0.3-0.4, boys, p=0.033) was lower. In boys, a combined, PCA-derived hormone profile (first visit) was able to predict the persistence of breast tissue (area under the curve=83%) better than any single marker. Discussion: Palpable breast tissue in infancy is common in both sexes although it persists in significantly more girls than boys at one year of age. Data supports both the early origin of breast tissue (in utero- and early postnatal) as well as a role of endogenous hormone production in later development and maintenance.

A Biphasic Pattern of Reproductive Hormones in Healthy Female Infants: The COPENHAGEN Minipuberty Study.

CONTEXT: Minipuberty, a period of a transient activation of the hypothalamic-pituitary-gonadal (HPG) axis in both sexes, enables evaluation of gonadal function in infants suspected of hypogonadism. However, female minipuberty remains poorly elucidated. OBJECTIVE: We aimed to establish continuous reference ranges for the most commonly used reproductive hormones and to evaluate the dynamics of the HPG axis in females aged 0 to 1 year. DESIGN: The COPENHAGEN Minipuberty Study (ClinicalTrials.gov ID: NCT02784184), a longitudinal, prospective cohort study. SETTING: Healthy infants from Copenhagen. PATIENTS OR OTHER PARTICIPANTS: A total of 98 healthy, term female infants followed with 6 examinations including venipuncture during the first year of life. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Serum concentrations of LH, FSH, inhibin B, anti-Müllerian hormone (AMH), estrone (E1), estradiol (E2), and SHBG were quantified using highly sensitive methods in 266 serum samples. RESULTS: Reference ranges were established for LH, FSH, inhibin B, AMH, E1, E2, and SHBG. Two peaks were observed in normalized mean curves for all hormones. The first peaks were timed around postnatal days 15 to 27 followed by a general nadir for all hormones around days 58 to 92. The second peaks occurred around days 107 to 125 for inhibin B, AMH, E1, E2, and SHBG and days 164 to 165 for LH and FSH. CONCLUSIONS: We present age-related, continuous reference ranges of the most commonly used reproductive hormones and present novel data revealing a biphasic and prolonged female minipuberty. CLINICALTRIALS.GOV ID: NCT02784184.

The role of central serotonergic markers and estradiol changes in perinatal mental health.

OBJECTIVE: Women have an increased risk for mental distress and depressive symptoms in relation to pregnancy and birth. The serotonin transporter (SERT) may be involved in the emergence of depressive symptoms postpartum and during other sex-hormone transitions. It may be associated with cerebrospinal fluid (CSF) levels of the main serotonin metabolite 5-hydroxyindolacetic acid (5-HIAA). In 100 healthy pregnant women, who were scheduled to deliver by cesarean section (C-section), we evaluated 5-HIAA and estradiol contributions to mental distress 5 weeks postpartum. METHODS: Eighty-two women completed the study. CSF collected at C-section was analyzed for 5-HIAA, with high performance liquid chromatography. Serum estradiol concentrations were quantified by liquid chromatography tandem mass spectrometry before C-section and postpartum. Postpartum mental distress was evaluated with the Edinburgh Postnatal Depression Scale (EPDS). Associations between EPDS, 5-HIAA, and Δestradiol were evaluated in linear regression models adjusted for age, parity and SERT genotype. RESULTS: Higher levels of postpartum mental distress symptoms were negatively associated with a large decrease in estradiol concentrations (ßΔE2  = 0.73, p = 0.007) and, on a trend level, positively associated with high antepartum 5-HIAA levels (ß5-HIAA  = 0.002, p = 0.06). CONCLUSION: In a cohort of healthy pregnant women, postpartum mental distress was higher in women with high antepartum 5-HIAA (trend) and lower in women with a large perinatal estradiol decrease. We speculate that high antepartum 5-HIAA is a proxy of SERT levels, that carry over to the postpartum period and convey susceptibility to mental distress. In healthy women, the postpartum return to lower estradiol concentrations may promote mental well-being.

Associations of serum phthalate metabolites with thyroid hormones in GraMo cohort, Southern Spain.

The general population is continuously exposed to phthalates via various consumer products. Epidemiological research relating phthalate exposure to thyroid function during non-developmental periods is limited. This study aimed to investigate the associations between specific serum phthalate metabolites and indicators of thyroid function in adults. We measured 10 serum phthalate metabolites and thyroid hormones - total triiodothyronine (TT3), free thyroxine (FT4) and thyroid stimulating hormone (TSH) - in a subsample of 207 adults from the GraMo cohort. This subsample was made up of men and women (in equal proportions) of middle age (49 ± 17 years) and from Southern Spain (province of Granada). Data on age, sex, body mass index, residence area, tobacco use, alcohol consumption and attained education were obtained from a questionnaire. Phthalate metabolites were log-transformed and categorized into tertiles. Cross-sectional associations of each metabolite with thyroid hormones were analyzed using multivariable-adjusted linear regression models. The mixture effect of metabolite phthalates was assessed using weighted quantile sum regression. After multivariable-adjustment, the following phthalate metabolites were significantly associated with TT3 in a dose-response manner: MMP (ß = 0.90: 95% confidence interval 0.68,1.12), MEP (ß = 0.67: 0.44, 0.90), MiBP (ß = 0.49: 0.21, 0.77), MiDP (ß = 0.27: 0.03, 0.52), MBzP (ß = 0.51: 0.28, 0.73), MEHP (ß = -0.59: -0.82, -0.35) and MiNP (ß = -0.43: -0.71, -0.14), when comparing highest vs. lowest exposed. The sum of all metabolites was also linked to FT4 levels. No significant associations were observed for TSH except for MiNP. Although phthalate metabolites with different molecular weight showed opposite associations, overall metabolite concentrations seem to associate with increased TT3 and FT4 serum levels. The cross-sectional nature of this analysis limits causal inference.

Endocrine Disrupting Chemicals and Risk of Testicular Cancer: A Systematic Review and Meta-analysis.

The incidence of many hormone-dependent diseases, including testicular cancer, has sharply increased in all high-income countries during the 20th century. This is not fully explained by established risk factors. Concurrent, increasing exposure to antiandrogenic environmental endocrine disrupting chemicals (EDCs) in fetal life may partially explain this trend. This systematic review assessed available evidence regarding the association between environmental EDC exposure and risk of testicular cancer (seminomas and nonseminomas). Following PRISMA guidelines, a search of English peer-reviewed literature published prior to December 14, 2020 in the databases PubMed and Embase® was performed. Among the 279 identified records, 19 were eligible for quality assessment and 10 for further meta-analysis. The completeness of reporting was high across papers, but over 50% were considered subject to potential risk of bias. Mean age at diagnosis was 31.9 years. None considered effects of EDC multipollutant mixtures. The meta-analyses showed that maternal exposure to combined EDCs was associated with a higher risk of testicular cancer in male offspring [summary risk ratios: 2.16, (95% CI:1.78-2.62), 1.93 (95% CI:1.49-2.48), and 2.78 (95% CI:2.27-3.41) for all, seminoma, and nonseminoma, respectively]. Similarly, high maternal exposures to grouped organochlorines and organohalogens were associated with higher risk of seminoma and nonseminoma in the offspring. Summary estimates related to postnatal adult male EDC exposures were inconsistent. Maternal, but not postnatal adult male, EDC exposures were consistently associated with a higher risk of testicular cancer, particularly risk of nonseminomas. However, the quality of studies was mixed, and considering the fields complexity, more prospective studies of prenatal EDC multipollutant mixture exposures and testicular cancer are needed.

Novel functions of the luteinizing hormone/chorionic gonadotropin receptor in prostate cancer cells and patients.

Prostate cancer (PCa) cells become castrate-resistant after initial tumor regression following castration-based lowering of testosterone (T). De-novo intra-tumoral steroid synthesis is a suggested biological mechanism of castration resistant PCa, but the regulators are unknown. Testicular T production is controlled by the luteinizing hormone/choriogonadotropin receptor (LHCGR). To elucidate the influence of LHCGR on PCa development the presence and effects of LHCGR in PCa and whether LHCGR in serum holds prognostic information in PCa patients is investigated. LHCGR expression was investigated by RT-PCR, WB, IHC, qPCR in PCa cell lines and prostatic tissue. Steroid production was measured in media from cell lines with LC-MS/MS and expression of steroidogenic enzymes with qPCR. Serum LHCGR (sLHCGR) was measured with ELISA in PCa patients (N = 157). Presence of LHCGR was established in prostatic tissue and PCa cell lines. Cell proliferation increased by 1.29-fold in LNCaP (P = 0.007) and 1.33-fold in PC-3 cells (P = 0.0007), when stimulated by luteinizing hormone. Choriogonadotropin stimulation decreased proliferation 0.93-fold in DU145 cells (P = 0.05), but none of the treatments altered steroid metabolite secretion. Low sLHCGR concentration was associated with a higher risk of biochemical failure after radical prostatectomy (HR = 3.05, P = 0.06) and castration resistance (HR = 6.92, P = 0.004) compared to high sLHCGR concentration. LHCGR is expressed in PCa and may exert a growth regulatory role in PCa derived cell lines. A potential prognostic role of sLHCGR for determining recurrence risk in PCa patients is found in this pilot study but needs verification in larger cohorts.

Ovarian reserve markers and endocrine profile during oral contraception: Is there a link between the degree of ovarian suppression and AMH?

The ovarian reserve markers anti-Müllerian hormone (AMH) and antral follicle count (AFC) are suppressed in varying degree during the use of combined oral contraceptives (COC). Further, long-term use of COC can mask a condition of premature ovarian insufficiency. A desirable clinical tool that could distinguish true low ovarian reserve markers from COC-induced low levels during use of COC is warranted. The aim of this multicenter study including 235 COC users was to assess whether low age-adjusted AMH levels during COC use were linked to concomitant low levels of LH, FSH, estradiol and androgens - as a potential future tool to differentiate between 'false', COC-induced low AMH levels vs. true low AMH. Study population and methods: In total, 235 COC users from the general population aged 19-40 years were included. AMH, AFC and a reproductive hormonal profile were measured during COC intake. Age-adjusted AMH levels (Z-scores) were calculated from a comparison group of 983 non-users of COC. Differences in hormonal profile were tested between women with low versus high age-adjusted AMH-quartiles based on non-parametric Wilcoxon rank sum tests. The outcomes of interest were levels of gonadotropins, estradiol and androgens according to the four the age-adjusted AMH quartiles to find out if women with low age-adjusted AMH levels had a stronger gonadotropin suppression compared with women with higher age-adjusted AMH levels. Mean age of COC users was 30.2 years (SD 3.8), median AMH 14 pmol/l (inter-quartile range (IQR) 8.7-23)), median AFC 16 (IQR 11-25). We found no significant differences across the age-adjusted AMH quartiles in either the levels of gonadotropins, estrogens or androgens, respectively. Thus, the degree of suppression of FSH, LH, androgens and estradiol are unlikely to be a useful tool to differentiate between false low and true low ovarian reserve markers during COC use. Presently, there seems to be no alternative to withdrawal of the COC and to re-test the ovarian reserve after 2-3 months. Trial registration Trial no. NCT02785809 (www.clinicaltrials.gov).

Male Gonadal Function after Allogeneic Hematopoietic Stem Cell Transplantation in Childhood: A Cross-Sectional, Population-Based Study.

Male gonadal dysfunction is a frequent late effect after pediatric hematopoietic stem cell transplantation (HSCT), but detailed insight into patterns of male gonadal function at long-term is limited by retrospective studies without semen sample data. In this study, we investigated the risk of azoospermia and testosterone deficiency, the diagnostic value of markers of spermatogenesis, and paternity at long-term follow-up after pediatric allogeneic HSCT. All male HSCT survivors age ≥18 years, transplanted in Denmark or Finland between 1980 and 2010, were invited to participate in this cross-sectional study. Examinations included a semen sample, measurements of reproductive hormones and testicular volume, and screening for chronic graft-versus-host disease (GVHD). Cumulative (pre-HSCT plus HSCT) treatment doses were calculated. Of 181 eligible patients, 98 participated, at a median 18 years (range, 8 to 35 years) after undergoing HSCT. Sperm was found in 30 patients, azoospermia in 42, and azoospermia during testosterone substitution in 24. A higher cumulative testicular irradiation dose was associated with increased risk of azoospermia and testosterone substitution (odds ratio [OR] per +1 Gy, 1.27; 95% confidence interval [CI], 1.14 to 1.46 [P < .001] and 1.21; 95% CI, 1.11 to 1.38 [P < .001], respectively). All patients treated with >12 Gy had azoospermia, and all but 1 patient treated with >16 Gy needed testosterone substitution. In patients treated with chemotherapy only (n = 23), a higher cumulative cyclophosphamide equivalent dose was associated with an increased risk of azoospermia (OR per +1 g/m2, 1.34; 95% CI, 1.01 to 2.15; P = .037). Prepubertal stage at HSCT was a risk factor for testosterone substitution (OR, 15.31; 95% CI, 2.39 to 315; P = .017), whereas chronic GVHD was unrelated to gonadal dysfunction. Inhibin B was the best surrogate marker of azoospermia (area under the curve, .91; 95% CI, .85 to .98; 90% sensitivity and 83% specificity) compared with follicle-stimulating hormone and testicular volume. Of 24 males who had attempted to conceive, 6 had fathered children. In conclusion, the risk of male gonadal dysfunction after pediatric HSCT is high and depends primarily on the cumulative testicular irradiation dose and pubertal stage at transplantation. Our findings support the need for fertility preservation before HSCT, as well as for prolonged follow-up of pediatric HSCT recipients into adulthood.

Evaluation of Serum Insulin-like Factor 3 Quantification by LC-MS/MS as a Biomarker of Leydig Cell Function.

BACKGROUND: The peptide hormone insulin-like factor 3 (INSL3) is a marker for Leydig cell function and the clinical use of serum INSL3 measurements has been suggested by several groups. AIM: (1) To establish a reference range for liquid chromatography-tandem mass spectrometry (LC-MS/MS) of serum INSL3 in healthy boys and men; and (2) to compare the associations of serum INSL3 and testosterone (T) to pubertal stage, lifestyle factors, diurnal variation, body composition, and human chorionic gonadotropin (hCG) stimulation. RESULTS: In a reference range based on LC-MS/MS analysis of serum from 1073 boys and men, INSL3 increased from levels close to the detection limit (0.03 µg/L) in prepubertal boys to a maximum mean level of 1.3 µg/L (95% CI, 0.9-2.7) in young men (19-40 years of age) and decreased slightly in older men (0.1 µg/L per decade). Serum T, but not INSL3, was associated with body mass index or body fat percentage and with alcohol consumption. Smoking was positively associated with serum T, but negatively associated with INSL3. There were significant diurnal variations in both INSL3 and T in men (P < 0.001), but serum INSL3 varied substantially less, compared with serum T (± 11% vs ± 26%). Mean serum INSL3 increased after hCG stimulation, but less than T (+ 17% vs + 53%). In both healthy men and in patients suspected of testicular failure, baseline serum INSL3 was more closely associated to the hCG-induced increase in serum T than baseline T itself. CONCLUSION: Measurement of serum INSL3 by LC-MS/MS has promise as a marker of testicular disorders.

Vitamin D and sex steroid production in men with normal or impaired Leydig cell function.

Production of testosterone is under tight control by human chorion gonadotropin (hCG) during fetal life and luteinizing hormone (LH) in adulthood. Several animal and human studies have linked vitamin D status with sex steroid production although it is not clear whether there exist a direct or indirect involvement in androgen production. Few studies have investigated this crosslink in young healthy men and putative direct or synergistic effect of activated vitamin D (1,25(OH)2D3) and LH/hCG on sex steroid production in vitro. Here, we present cross-sectional data from 300 young men and 41 hCG-stimulated men with impaired Leydig cell function combined with data from an ex vivo culture of human testicular tissue exposed to 1,25(OH)2D3 alone or in combination with hCG. Serum 25-OHD was positively associated with SHBG (ß:0.002; p = 0.023) and testosterone/estradiol-ratio (ß:0.001; p = 0.039), and inversely associated with free testosterone (%) (free testosterone/total testosterone) (ß:-0.002; p = 0.016) in young men. Vitamin D deficient men had higher total and free estradiol concentrations than men with higher vitamin D status (19% and 18%, respectively; p < 0.01). Interestingly, men with impaired Leydig cell function and vitamin D deficiency had a significantly lower hCG-mediated increase in total and free testosterone compared with vitamin D sufficient men (p < 0.05). Accordingly, testicular tissue exposed to 100 nM 1,25(OH)2D3 had a 15% higher testosterone release into the media compared with vehicle treated specimens (p = 0.030). In conclusion, vitamin D deficiency is associated with lower testosterone/estradiol ratio in young men and lower Leydig cell sensitivity after hCG-stimulation in men with impaired gonadal function. The significant effect of 1,25(OH)2D3 on testosterone production in a human testis model supports that the stimulatory effect at least in part may be direct. Larger placebo-controlled studies are needed to determine whether vitamin D supplementation can influence testosterone production.

Sex-specific Estrogen Levels and Reference Intervals from Infancy to Late Adulthood Determined by LC-MS/MS.

CONTEXT: The lack of sensitive and robust analytical methods has hindered the reliable quantification of estrogen metabolites in subjects with low concentrations. OBJECTIVE: To establish sex-specific reference ranges for estrone (E1) and estradiol (E2) throughout life and to evaluate sex-differences using the state-of-the-art liquid chromatography tandem mass spectrometry (LC-MS/MS) method for quantification of E1, E2, and estriol (E3). DESIGN: LC-MS/MS method development and construction of estrogen reference ranges. SETTINGS: Population-based cross-sectional cohorts from the greater Copenhagen and Aarhus areas. PARTICIPANTS: Healthy participants aged 3 months to 61 years (n = 1838). RESULTS: An isotope diluted LC-MS/MS method was developed and validated for measurements of serum E1, E2, and E3. Limits of detections (LODs) were 3 pmol/L (E1), 4 pmol/L (E2), and 12 pmol/L (E3), respectively. This sensitive method made it possible to differentiate between male and female concentration levels of E1 and E2 in children. In girls, E2 levels ranged from

Nodal Signaling Regulates Germ Cell Development and Establishment of Seminiferous Cords in the Human Fetal Testis.

Disruption of human fetal testis development is widely accepted to underlie testicular germ cell cancer (TGCC) origin and additional disorders within testicular dysgenesis syndrome (TDS). However, the mechanisms for the development of testicular dysgenesis in humans are unclear. We used ex vivo culture and xenograft approaches to investigate the importance of Nodal and Activin signaling in human fetal testis development. Inhibition of Nodal, and to some extent Activin, signaling disrupted seminiferous cord formation, abolished AMH expression, reduced androgen secretion, and decreased gonocyte numbers. Subsequent xenografting of testicular tissue rescued the disruptive effects on seminiferous cords and somatic cells but not germ cell effects. Stimulation of Nodal signaling increased the number of germ cells expressing pluripotency factors, and these persisted after xenografting. Our findings suggest a key role for Nodal signaling in the regulation of gonocyte differentiation and early human testis development with implications for the understanding of TGCC and TDS origin.

The Possible Impact of Antenatal Exposure to Ubiquitous Phthalates Upon Male Reproductive Function at 20 Years of Age.

Phthalates are ubiquitous environmental endocrine-disrupting chemicals suspected to interfere with developmental androgen action leading to adverse effects on male reproductive function. Prenatal exposure studies in rodents show cryptorchidism, hypospadias and reduced testicular volume (TV), testosterone and anogenital distance in males. It is postulated that there is a developmental window in utero when phthalate exposure has the most potent adverse effects. Some human studies show associations between prenatal phthalate exposure and reduced calculated "free" serum testosterone in infant boys and shorter anogenital distance. However, there are no data available yet which link antenatal exposure to long-term effects in men. We aimed to correlate antenatal phthalate exposure with adult TV, semen parameters and serum reproductive hormone concentrations. 913 men from the Western Australian (Raine) Pregnancy Cohort were contacted aged 20-22 years. 423 (56%) agreed to participate; 404 underwent testicular ultrasound examination; 365 provided semen samples, and reproductive hormones were measured in 384. Maternal antenatal serum phthalate metabolite measurements were available for 185 and 111 men, who provided serum and semen, respectively. Maternal serum collected at 18 and 34 weeks gestation, stored at -80°C, was pooled and analyzed for 32 phthalate metabolites by liquid chromatography-tandem mass spectrometry. TV was calculated, semen analysis performed by WHO approved methods, and serum concentrations of gonadotrophins, inhibin B, and testosterone measured. Eleven phthalate metabolites were detected. Primary and secondary metabolites of di-(2-ethyl-hexyl) phthalate (DEHP) and di-iso-nonyl phthalate (DiNP) were positively correlated. After correction for adult height, BMI, presence of a varicocele and exposure to maternal smoking mono-iso-nonyl phthalate (MiNP) (r = -0.22) and sums of DEHP and DiNP metabolites (r = -0.24) and the sum of the metabolites of the high molecular weight phthalates (r = -0.21) were negatively correlated with TV (all p < 0.05). After adjustment for BMI adult serum total testosterone was positively associated with exposure to the following antenatal serum phthalate metabolites: mono-(2-ethylhexyl) phthalate (r = 0.26), MiNP (r = 0.18), the sum of metabolites for DEHP (r = 0.21) and DiNP (r = 0.18), and the sum of high molecular phthalates (r = 0.20) (p = 0.0005 to p = 0.02). Given sample size, storage duration and confounding through postnatal exposures, further studies are required.