Polygenic Scores for Adult Testosterone and SHBG Levels Are Associated With Reproductive Hormone Levels in Male Infants.

CONTEXT: The Hypothalamic-Pituitary-Gonadal (HPG) axis's transient activity in infancy, i.e, minipuberty, is considered crucial for male reproductive function. Historically, minipuberty has been considered a passive response triggered by the withdrawal of placental steroids at birth. However, given its potential link to adult reproductive function, we hypothesize that minipuberty is a partially genetically regulated process, suggesting a link between the genetic architecture of reproductive hormone concentrations across lifespan. OBJECTIVE: To investigate the association of UK Biobank Study-based Polygenic scores (PGS) of adult total Testosterone (T) and Sex hormone-binding globulin (SHBG) concentrations with trajectories of reproductive hormones concentrations in male infants. DESIGN: Prospective, longitudinal birth cohort (The COPENHAGEN Minipuberty Study, 2016-2018, ClinTrial: NCT02784184). Individual PGSs in male infants derived from published literature were calculated for total testosterone and SHBG. The associations with mean Standard Deviation Scores (SDS) of reproductive hormone concentrations in infancy were tested. SETTING: Population-based. PATIENTS OR OTHER PARTICIPANTS: Healthy, male, term, singleton newborns were followed with repeated clinical examinations including blood sampling during a one-year follow-up (n=109). MAIN OUTCOME MEASURES: Circulating reproductive hormone concentrations. RESULTS: T-PGSadult were significant associated with mean T-SDSinfancy, mean SHBG-SDSinfancy and mean LH-SDSinfancy (p=0.02, <0.001 and 0.03, with r2=0.05, 0.21 and 0.04, respectively). SHBG-PGSadult was significantly associated with mean SHBG-SDSinfancy (p<0.001, r2=0.18). T-PGSadult explained 5% and 21% of the phenotypic variation in infancy of mean T-SDSinfancy and SHBG-SDSinfancy, respectively. CONCLUSIONS: Our findings suggest that the genetic architecture underlying total testosterone and SHBG in adults also associates with hormone concentrations and their trajectories during infancy.

Urinary concentration of phthalates and bisphenol A during minipuberty is associated with reproductive hormone concentrations in infant boys.

BACKGROUND: The transient postnatal activation of the hypothalamic-pituitary-gonadal hormone axis is termed minipuberty and considered an important developmental period, which is highly sensitive to endocrine disruption. Here, we explore exposure-outcome associations during minipuberty between concentrations of potentially endocrine disrupting chemicals (EDCs) in urine of infant boys and their serum reproductive hormone concentrations. METHODS: In total, 36 boys participating in the COPENHAGEN Minipuberty Study had data available for both urine biomarkers of target endocrine disrupting chemicals and reproductive hormones in serum from samples collected on the same day. Serum concentrations of reproductive hormones were measured by immunoassays or by LC-MS/MS. Urinary concentrations of metabolites of 39 non-persisting chemicals, including phthalates and phenolic compounds, were measured by LC-MS/MS. Nineteen chemicals had concentrations above the limit of detection in ≥50% of children and were included in data analysis. Associations of urinary phthalate metabolite and phenol concentrations (in tertiles) with hormone outcomes (age- and sex-specific SD-scores) were analysed by linear regression. Primarily, we focused on the EU regulated phthalates; butylbenzyl phthalate (BBzP), di-iso-butyl phthalate (DiBP), di-n-butyl phthalate (DnBP), and di-(2-ethylhexyl) phthalate (DEHP) as well as bisphenol A (BPA). Urinary metabolites of DiBP, DnBP and DEHP were summed and expressed as ∑DiBPm, ∑DnBPm and ∑DEHPm. RESULTS: Compared to boys in the lowest ∑DnBPm tertile, urinary concentration of ∑DnBPm was associated with concurrent higher luteinizing hormone (LH) and anti-Müllerian hormone (AMH) SD-scores as well as lower testosterone/LH ratio in boys in the middle ∑DnBPm tertile (estimates (CI 95%) 0.79 (0.04; 1.54), 0.91 (0.13; 1.68), and -0.88 (-1.58;-0.19), respectively). Further, higher insulin-like peptide 3 (INSL3) SD-scores and lower DHEAS SD-score in boys in the highest ∑DnBPm tertile (0.91 (0.12; 1.70) and -0.85 (-1.51;-0.18), respectively) were observed. In addition, boys in the middle and highest ∑DEHPm tertile had higher LH (1.07 (0.35; 1.79) and 0.71 (-0.01; 1.43), respectively) and in the highest ∑DEHPm tertile also higher AMH (0.85 (0.10; 1.61)) concentration SD-scores, respectively. Boys in the highest BPA tertile had significantly higher AMH and lower DHEAS concentration compared to boys in the lowest BPA tertile (1.28 (0.54; 2.02) and -0.73 (-1.45; -0.01)), respectively. DISCUSSION: Our findings indicate that exposure to chemicals with known or suspected endocrine disrupting potential, especially the EU-regulated DnBP, DEHP and BPA, may modify male reproductive hormone concentrations in infant boys suggesting that minipuberty is a critical window sensitive to endocrine disruption.

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.

Benzophenones, bisphenols and other polychlorinated/phenolic substances in Danish infants and their parents - including longitudinal assessments before and after introduction to mixed diet.

BACKGROUND: Humans are widely exposed to chemicals with known or suspected endocrine disrupting effects. Among those are several benzophenones, bisphenols and other phenols commonly used in consumer products. OBJECTIVES: To provide human biomonitoring data from young families including infants and their parents as well as longitudinal data of infants exclusively breastfed versus on mixed diet. METHOD: Twenty-two benzophenones, bisphenols and other phenols, were measured in urine sample sets collected from more than 100 infants and their parents (the TRIO study) and in paired samples from 61 infants when exclusively breastfed and after introduction of mixed diet (the FOOD study). RESULTS: Twelve out of 22 substances were detectable in more than half of the urine samples from infants, mothers or fathers. Large variation in excreted levels of almost all the substances were observed. The TRIO study showed that infants had comparable or even significantly higher daily urinary excretion (DUE) of benzophenone, 4-hydroxy-benzophenone, bisphenol A, bisphenol S, triclosan and 2-phenylphenol than their parents. In the FOOD study, exclusively breastfed infants had higher or similar DUE of triclosan and benzophenones compared to when they received mixed diet. Urinary levels of triclosan and the benzophenones, BP-1 and BP-3 were significantly correlated between all trio members, indicating exposure from the same sources at home. For triclosan, BP-1 and BP-3, the within family variation was lower than between families in the TRIO study. Many substances were positively correlated both within infants and parents, indicating that some families were exposed to several of these substances concurrently. CONCLUSION: Participants in this study excreted relatively low chemical levels, however, simultaneous exposure to several chemicals with endocrine disrupting abilities is of concern due to the dose-additive effects of these substances in combination with other chemicals.

Dynamic Changes of Reproductive Hormones in Male Minipuberty: Temporal Dissociation of Leydig and Sertoli Cell Activity.

CONTEXT: The male hypothalamic-pituitary-gonadal (HPG) axis is transiently active during the first months of life with surging serum concentrations of reproductive hormones. This period, termed minipuberty, appears to be essential for priming testicular function. Despite the central role for male reproductive function, longitudinal data on HPG axis activation in infancy is sparse. OBJECTIVE: To explore the dynamics of HPG hormone activity in healthy male infants, to assess the association of HPG axis activity and testicular volume, and to establish reference curves for serum levels of reproductive hormones. DESIGN: Prospective, longitudinal birth cohort (the COPENHAGEN Minipuberty Study, 2016-2018, 1-year follow-up). SETTING: Population-based. PATIENTS OR OTHER PARTICIPANTS: Healthy, male, term, singleton newborns were followed from birth on with repeated clinical examinations including blood sampling during a 1-year follow-up. A total of 128 boys contributed to this study, while 119 participated in the postnatal follow-up. MAIN OUTCOME MEASURES: Serum reproductive hormone concentrations and testicular volume. RESULTS: Reproductive hormone concentrations showed marked dynamics during the first 6 months of age. Gonadotropins, total testosterone, and insulin-like factor 3 peaked at around 1 month of age. Inhibin B, anti-Müllerian hormone, and testicular volume peaked at around 4 to 5 months. Correlations largely recapitulated typical HPG axis pathways but also differed significantly from adult men. CONCLUSIONS: We demonstrate a temporal dissociation of Leydig and Sertoli cell activity during male minipuberty and provide reference curves for reproductive hormones.

Exposure to 15 phthalates and two substitutes (DEHTP and DINCH) assessed in trios of infants and their parents as well as longitudinally in infants exclusively breastfed and after the introduction of a mixed diet.

OBJECTIVE: Several phthalates have been restricted/banned due to their adverse endocrine disrupting properties. The use of other phthalates and substitutes has increased. Here we examine the current exposure to phthalates in family trios comprised of infants and their parents and in infants exclusive breastfed and following introduction to a mixed diet. METHODS: Metabolites of 15 phthalates and two substitutes, di(2-ethylhexyl)-teraphthalate (DEHTP) and diisononyl-cyclohexane-1,2-dicarboxylate (DINCH), were measured in urine samples collected from >100 infants and their parents and in paired urine samples collected from 67 infants, while they were exclusively breastfed and when they got mixed diet. RESULTS: Among infants and their parents, metabolites of nine out of 15 phthalates and both substitutes were detected in >74% of all samples. Estimated daily intake (DI) calculated as µg/kg/day, showed similar exposure levels among infants and their parents for several of the substances, and infants were more exposed to DEHTP than their mothers. Significantly higher estimated DIs were observed for some low-molecular phthalates in infants exclusively breastfed. In contrast, comparable estimated DIs were observed for many other phthalates and DEHTP regardless of feeding status. For most of the substances, the within-family variation, was lower than the between-family variation. Likewise, the within-infant variation on exclusively breast vs. mixed diet was lower than the between-infant variation. Independent of food status, some infants were concurrently exposed to almost all the measured phthalates and substitutes in higher amounts than others. CONCLUSION: Surprisingly, irrespective of diet status infants were exposed to several phthalates and substitutes some of which have been regulated for years. Exposure patterns and levels were similar in infants and their parents. Importantly, risk assessment based on new refined reference doses (RfD-AA) exceeded the safety level for anti-androgenic effects in a number of infants and parents, which is of concern.

Cohort profile: The COPENHAGEN Minipuberty Study-A longitudinal prospective cohort of healthy full-term infants and their parents.

BACKGROUND: The hypothalamic-pituitary-gonadal (HPG) axis governs sexual maturation and reproductive function in humans. In early postnatal life, it is transiently active during which circulating sex steroids reach adult levels. While this so-called minipuberty represents a universal phenomenon in infants of both sexes, its role for early maturation and growth remains incompletely understood. OBJECTIVES: To provide normative data on auxology as well as serum and urinary hormone levels in healthy, full-term infants throughout the first year of life and to investigate associations of postnatal HPG axis dynamics as well as hormonal, genetic and environmental exposures with early genital development and growth. POPULATION: Healthy, Danish, full-term, singleton newborns including their parents. DESIGN: Single-centre, prospective, observational longitudinal pregnancy and birth cohort. METHODS: Newborns were followed with six repeated clinical examinations during a one-year follow-up period. An umbilical cord blood sample was drawn at birth. At each visit, infants underwent a clinical examination focusing on auxology and genital development. Further, blood (serum, plasma, DNA) and urine samples were collected at each visit. Mothers and fathers underwent a clinical examination and provided blood samples prior to and after birth. A subset of parents provided urine samples and breast milk samples. Pregnancy and obstetrical outcomes, and detailed parental questionnaires were compiled. PRELIMINARY RESULTS: Between August 2016 and August 2018, 2481 women with singleton pregnancies were invited to participate of which 298, including their partners, were enrolled (12.0%). A total of 268 healthy, full-term newborns born appropriate for gestational age (AGA) were included at birth, 233 newborns participated in the postnatal follow-up period and 186 completed the one-year follow-up period (9.4% and 7.5%, respectively). CONCLUSION: The COPENHAGEN Minipuberty Study provides detailed, longitudinal data on early genital development and growth including hormonal and genetic profiles and environmental exposure in healthy infants including additional data in their parents.

Reproductive hormones during pubertal transition in girls with transient Thelarche.

CONTEXT: Transient thelarche (TT), that is, the appearance, regression and subsequent reappearance of breast buds, is a frequent phenomenon, but little is known about pubertal transition in these girls. OBJECTIVE: To describe pubertal progression, growth, genotypes, reproductive hormones and growth factors in girls with TT compared to those who do not present TT (non-TT). DESIGN: Retrospective analysis of a longitudinal population-based study. PATIENTS OR OTHER PARTICIPANTS: Girls (n = 508) of the Chilean Growth and Obesity cohort. MEASUREMENTS: Pubertal progression, reproductive hormones, follicle stimulating hormone (FSH) beta subunit/FSH receptor gene single nucleotide polymorphisms and growth. RESULTS: Thirty-seven girls (7.3%) were presented TT. These girls entered puberty by pubarche more frequently (51%) than girls with normal progression (non-TT; n = 471; 23%, P = .005). Girls with TT who were under 8 years old had lower androgens, anti-Müllerian hormone (AMH), luteinizing hormone (LH) and oestradiol (all P < .05) than older girls with TT. At the time of Tanner breast stage 2 (B2), girls with TT had higher androgens, LH, FSH, IGF1, LH, insulin and oestradiol (P < .01) than at the time of TT. TT girls were older at B2 (10.3 ± 1.1 vs. 9.2 ± 1.2 years, P < .001) and menarche (12.3 ± 0.8 vs. 12.0 ± 1.0 years, P = .040) than their counterparts (non-TT). No differences in anthropometric variables or FSHB/FSHR genotypes were detected. CONCLUSION: Transient thelarche is a frequent phenomenon that does not appear to be mediated by hypothalamic-pituitary-gonadal axis activation or by adiposity. Hormonal differences between earlier TT and later TT suggest that their mechanisms are different.

The External Genitalia Score (EGS): A European Multicenter Validation Study.

CONTEXT: Standardized description of external genitalia is needed in the assessment of children with atypical genitalia. OBJECTIVES: To validate the External Genitalia Score (EGS), to present reference values for preterm and term babies up to 24 months and correlate obtained scores with anogenital distances (AGDs). DESIGN, SETTING: A European multicenter (n = 8) validation study was conducted from July 2016 to July 2018. PATIENTS AND METHODS: EGS is based on the external masculinization score but uses a gradual scale from female to male (range, 0-12) and terminology appropriate for both sexes. The reliability of EGS and AGDs was determined by the interclass correlation coefficient (ICC). Cross-sectional data were obtained in 686 term babies (0-24 months) and 181 preterm babies, and 111 babies with atypical genitalia. RESULTS: The ICC of EGS in typical and atypical genitalia is excellent and good, respectively. Median EGS (10th to 90th centile) in males < 28 weeks gestation is 10 (8.6-11.5); in males 28-32 weeks 11.5 (9.2-12); in males 33-36 weeks 11.5 (10.5-12) and in full-term males 12 (10.5-12). In all female babies, EGS is 0 (0-0). The mean (SD) lower/upper AGD ratio (AGDl/u) is 0.45 (0.1), with significant difference between AGDl/u in males 0.49 (0.1) and females 0.39 (0.1) and intermediate values in differences of sex development (DSDs) 0.43 (0.1). The AGDl/u correlates with EGS in males with typical genitalia and in atypical genitalia. CONCLUSIONS: EGS is a reliable and valid tool to describe external genitalia in premature and term babies up to 24 months. EGS correlates with AGDl/u in males. It facilitates standardized assessment, clinical decision-making and multicenter research.

FSHB -211 G>T Polymorphism as Predictor for TESE Success in Patients With Unexplained Azoospermia.

CONTEXT: Testicular sperm extraction (TESE) followed by assisted reproductive techniques often remains the only therapeutic option for men with azoospermia due to spermatogenic failure. Reproductive parameters, such as gonadotropin levels and testicular volume or histopathology, contribute to the prediction of sperm retrieval rate (SRR) in TESE. However, there is an eminent lack of noninvasive predictive factors for TESE outcome. OBJECTIVE: To clarify the impact of three common genetic variants affecting FSH and its cognate receptor on testicular histopathology patterns and SRR in TESE. DESIGN: We evaluated the association of the single-nucleotide polymorphisms (SNP) FSHB -211G>T (rs10835638), FSHR -29G>A (rs1394205), and FSHR c.2039A>G (rs6166) with testicular histopathology and SRR in patients with azoospermia. SETTING: Tertiary referral center for andrology. PATIENTS OR OTHER PARTICIPANTS: Men (n = 1075) with azoospermia who underwent TESE (grouped by clinical pathologies). INTERVENTION(S): All participants underwent TESE. MAIN OUTCOME MEASURE(S): Testicular histopathology, SRR, and reproductive hormone levels. RESULTS: FSHB -211G>T was significantly associated with reduced chances of sperm retrieval in patients with unexplained azoospermia. Indicating an additional mechanism, the association of the SNP with SSR could not be solely attributed to decreased FSH levels. CONCLUSION: A common genetic factor was significantly associated with SRR in TESE. In perspective, a calculator or score including the noninvasive parameters FSH level, testicular volume, and FSHB haplotype should be considered to estimate the chances for sperm retrieval in men with azoospermia.

Deletion in the uridine diphosphate glucuronyltransferase 2B17 gene is associated with delayed pubarche in healthy boys.

OBJECTIVE: Only a few genetic loci are known to be associated with male pubertal events. The ability of excreting testosterone (T) and other steroids in the urine depends on sulfation and glucuronidation. One of several essential glucuronidases is encoded by the UGT2B17 gene. In a preliminary report, we found that homozygous deletion of UGT2B17 in boys was associated with lower urinary excretion of T. We hypothesized that boys with a lower glucuronidation capacity may have altered androgen action and excretion affecting pubarche, as this represents a T-dependent event. DESIGN, PARTICIPANTS AND MEASURES: 668 healthy boys (cross-sectional) aged 6.1-21.9 years (COPENHAGEN puberty study conducted from 2005 to 2006) were included. 65 of the boys where followed longitudinally every 6 months. Participants were genotyped for UGT2B17 copy number variation (CNV). Clinical pubertal staging including orchidometry, anthropometry and serum reproductive hormone levels. RESULTS: 59 of the 668 boys (8.8%) presented with a homozygous deletion of UGT2B17 (del/del). These boys experienced pubarche at a mean age of 12.73 years (12.00-13.46) vs 12.40 years (12.11-12.68) in boys heterozygous for deletion of UGT2B17 (del/ins) vs 12.06 years (11.79-12.33) in boys with the wild-type genotype (ins/ins) (P = 0.029, corrected for BMI z-score). The effect accounted for 0.34 years delay per allele (95% CI: 0.03-0.64). A comparable trend was observed for onset of testicular enlargement >3 mL but did not reach significance. CONCLUSION: CNV of UGT2B17 is a factor contributing to the timing of male pubarche.

The AMH genotype (rs10407022 T>G) is associated with circulating AMH levels in boys, but not in girls.

OBJECTIVE: Fetal anti-Müllerian hormone (AMH) is responsible for normal male sexual differentiation, and circulating AMH is used as a marker of testicular tissue in newborns with disorders of sex development. Little is known about the mechanism of action in postnatal life. A recent genome wide association study (GWAS) reported genetic variation of AMH affecting AMH levels in young men. This study investigated the effect of genetic variation of AMH and AMH type II receptor (AMHR2) (AMHrs10407022 T>G and AMHR2rs11170547 C>T) on circulating reproductive hormone levels and pubertal onset in boys and girls. DESIGN AND METHODS: This study is a combined longitudinal and cross-sectional study in healthy Danish boys and girls from the general population. We included 658 boys aged 5.8-19.8 years and 320 girls aged 5.6-16.5 years. The main outcome measures were genotyping of AMH and AMHR2, pubertal staging and serum levels of reproductive hormones. RESULTS: AMHrs10407022T>G was associated with higher serum levels of AMH in prepubertal boys (TT: 575 pmol/L vs TG: 633 pmol/L vs GG: 837 pmol/L, P = 0.002) and adolescents (TT: 44 pmol/L vs TG: 58 pmol/L vs GG: 79 pmol/L, P < 0.001). Adolescent boys carrying the genetic variation also had lower levels of LH (TT: 3.0 IU/L vs TG: 2.8 IU/L vs GG: 1.8 IU/L, P = 0.012). Hormone levels in girls and pubertal onset in either sex did not seem to be profoundly affected by the genotypes. CONCLUSION: Our findings support recent GWAS results in young adults and expand our understanding of genetic variation affecting AMH levels even in boys prior to the pubertal decline of circulating AMH.

Polymorphisms in JMJD1C are associated with pubertal onset in boys and reproductive function in men.

JMJD1C, a member of the Jumonji-domain containing histone demethylases protein family, has been associated with levels of sex-hormone binding globulin (SHBG) and testosterone in men, and knock-out rodent models show age-dependent infertility. The objective of this study was to investigate whether single nucleotide polymorphisms (SNPs) nearby JMJD1C are associated with pubertal onset in boys and with male reproduction. 671 peri-pubertal boys, 1,027 young men, 315 fertile men, and 252 infertile men were genotyped for two JMJD1C SNPs (rs7910927 and rs10822184). rs7910927 and rs10822184 showed high linkage. Boys with the rs7910927 TT genotype entered puberty 3.6 months earlier than their peers (p = 2.5 × 10-2). In young men, the number of T alleles was associated with decreased levels of SHBG, follicle-stimulating hormone (FSH), testosterone, and testosterone x luteinizing hormone, as well as increased levels of Inhibin B, Inhibin B/FSH ratio, and testis size. No significant associations with semen parameters were observed and the genotype distribution was comparable among fertile and infertile men. In conclusion, genetic variation in the vicinity of JMJD1C had a surprisingly large impact on the age at pubertal onset in boys as well as levels of reproductive hormones and testis size in men, emphasizing the relationship between JMJD1C and reproductive functions.