Anogenital distance in a cohort of 169 infant boys with uni- or bilateral cryptorchidism including 18 boys with vanishing testes.

STUDY QUESTION: Do different boys with different types of cryptorchidism exhibit different anogenital distances (AGDs)? SUMMARY ANSWER: Length of AGD seemed to differ in different groups of patients with cryptorchidism. WHAT IS KNOWN ALREADY: AGD, which is used as an indicator of prenatal androgen action, tends to be shorter in boys with cryptorchidism compared to unaffected boys. Shorter AGDs have also been reported in boys with hypospadias, in men with poor semen quality, and in men with testicular cancer. STUDY DESIGN, SIZE, DURATION: A prospective descriptive cohort study was performed using data from consecutively selected boys with cryptorchidism (n = 169) operated in a single center over a period of 3 years (September 2019 to October 2022). PARTICIPANTS/MATERIALS, SETTING, METHODS: AGD was measured in 169 infant boys, at 3 to 26 months of age, during anesthesia with a vernier caliper measuring the distance from the anus to the base of the scrotum (AGDAS) and from the anus to the anterior base of the penis (AGDAP) in two body positions according to the methods by 'The Infant Development and the Environment Study' (TIDES) and 'Cambridge Baby Growth Study', resulting in four mean values per patient (TIDES AGDAS/AP and Cambridge AGDAS/AP). Normal values for AGD by age were set by our hospital Department of Growth and Reproduction based on a large cohort of healthy infant boys (n = 1940). Testicular biopsies were performed at orchidopexy as a clinical routine. The germ cell number (G/T) and type Ad spermatogonia number (AdS/T) per cross-sectional tubule of at least 100 and 250 tubules, respectively were measured and related to normal samples. Blood samples were obtained by venipuncture for measuring serum LH, FSH, and inhibin B. They were analyzed in our hospital Department of Growth and Reproduction where the normal reference was also established. Correlations between the four mean AGD measurements for each boy were evaluated by Spearman rank correlation analyses. The AGD measurement of every boy was transferred to the multiple of the median (MoM) of the normal AGD for age and named MoM AGD. MAIN RESULTS AND THE ROLE OF CHANCE: There were 104 boysoperated for unilateral, and 47 boys operated for bilateral, undescended testes, whereas 18 boys had vanished testis including one boy with bilateral vanished testes. Only 6% of cases with vanished testes had a MoM AGD higher than the normal median compared to 32% with undescended testes (P < 0.05). MoM AGD increased with the age at surgery for boys with vanished testis (Spearman r = 0.44), but not for boys with undescended testes (Spearman r = 0.14). Boys with bilateral cryptorchidism had longer AGDs and more often had hypogonadotropic hypogonadism than boys with unilateral cryptorchidism (P < 0.005) and (P < 0.000001). LIMITATIONS, REASONS FOR CAUTION: Although being the largest published material of AGD measurements of infant boys with cryptorchidism, one limitation of this study covers the quite small number of patients in the different groups, which may decrease the statistical power. Another limitation involves the sparse normal reference material on G/T and AdS/T. Finally, there are currently no longitudinal studies evaluating AGD from birth to adulthood and evaluating childhood AGD in relation to fertility outcome. Our study is hypothesis generating and therefore the interpretation of the results should be regarded as exploratory rather than reaching definite conclusions. WIDER IMPLICATIONS OF THE FINDINGS: The study findings are in agreement with literature as the total included group of boys with cryptorchidism exhibited shorter than normal AGDs. However, new insights were demonstrated. Boys with vanished testis had shorter AGDs compared to unaffected boys and to boys with undescended testes. This finding challenges the current concept of AGD being determined in 'the masculinization programming window' in Week 8 to 14 of gestation. Furthermore, boys with bilateral cryptorchidism had longer AGDs and more often had hypogonadotropic hypogonadism than boys with unilateral cryptorchidism, suggesting that the lack of fetal androgen in hypogonadotropic hypogonadism is not that significant. STUDY FUNDING/COMPETING INTEREST(S): No external funding was used and no competing interests are declared. TRIAL REGISTRATION NUMBER: The trial was not registered in an ICMJE-recognized trial registry.

Cohort profile: The Copenhagen Analgesic Study-The COPANA cohort.

BACKGROUND: Development of the gonads during fetal life is complex and vital for adult reproductive health. Cell and animal studies have shown an alarming effect of mild analgesics on germ cells in both males and females. More than 50% of pregnant women use mild analgesics during pregnancy, which potentially could compromise the reproductive health of the next generation. OBJECTIVES: We present a research protocol designed to evaluate the effect of prenatal exposure to mild analgesics and endocrine-disrupting chemicals on gonadal function in the offspring. POPULATION: Healthy, singleton pregnant women and their partners. DESIGN: The COPANA cohort is a prospective, observational pregnancy and birth cohort. METHODS: Participants were enrolled during the first trimester of pregnancy. Information on the use of mild analgesics was collected retrospectively 3 months prior to pregnancy and prospectively every 2 weeks throughout the study. We collected extensive data on lifestyle and reproductive health. Biospecimens were collected in the first trimester (maternal and paternal urine- and blood samples), in the third trimester in conjunction with a study-specific ultrasound scan (maternal urine sample), and approximately 3 months post-partum during the infant minipuberty period (maternal and infant urine- and blood samples). A comprehensive evaluation of reproductive function in the infants during the minipuberty phase was performed, including an ultrasound scan of the testis or ovaries and uterus. PRELIMINARY RESULTS: In total, 685 pregnant women and their partners were included between March 2020 and January 2022. A total of 589 infants (287 males) and their parents completed the follow-up during the minipuberty phase (December 2020-November 2022). CONCLUSIONS: The Copenhagen Analgesic Study holds the potential to provide novel and comprehensive insights into the impact of early and late prenatal exposure to mild analgesics and other endocrine-disrupting chemicals on future reproductive function in the offspring.

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.

Correlation of anogenital distance from childhood to age 9 years-a prospective population-based birth cohort-the Odense Child Cohort.

STUDY QUESTION: Does anogenital distance (AGD) - distance from the anus to the genitals - correlate from infancy (3 months) to the age of 9 years in boys and girls? SUMMARY ANSWER: In boys, AGD correlated from infancy to 9 years of age, whereas in girls, correlations were weaker, especially between infancy and later childhood. WHAT IS KNOWN ALREADY: AGD is considered a marker for prenatal androgen action. In males, reduced AGD is associated with testicular cancer, infertility, and lower sperm count. In females, AGD is associated with endometriosis and polycystic ovary syndrome. STUDY DESIGN SIZE DURATION: In the Odense Child Cohort, a prospective population-based birth cohort, pregnant women were enrolled in early pregnancy. AGD and BMI were measured repeatedly in children at ages 3 and 18 months, as well as at 3, 5, 7, and 9 years. PARTICIPANTS/MATERIALS SETTING METHODS: AGD was measured from the anus to the scrotum (AGDas) and to the penis (AGDap) in 1022 boys, and to the posterior fourchette and the clitoris in 887 girls repeatedly between the age of 3 months to 9 years. In total, 7706 assessments were made. AGD was adjusted for body weight, and SD scores (the difference between individual AGD and the mean of AGD in the population divided by SD of AGD) were calculated for each child. Pearson correlation coefficient (r) of each measurement was performed to investigate whether individual AGD was stable during childhood. Short predictive values at 3 months (20th percentile) to 9 years were investigated using the AUC produced by the receiver operating characteristic curve. MAIN RESULTS AND THE ROLE OF CHANCE: In boys, AGD/body size-index SD score correlated significantly between infancy and 9 years, strongest for AGDas (r = 0.540 P > 0.001). In girls, weaker significant correlation coefficients were found between AGD at infancy and 9 years; higher correlation coefficients were found between AGD from 3 to 9 years (P > 0.001). Short AGDas in infancy predicted short AGDas in boys aged 9 years (AUC: 0.767, sensitivity 0.71, specificity 0.71). The predictive values of short infant AGDap, penile width (in boys), and AGD (in girls) concerning short outcomes at 9 years were low. LIMITATIONS REASONS FOR CAUTION: The AGD measurements are less precisely measurable in girls compared to boys, especially in infancy, resulting in less reproducible measurements. Additionally, because AGD is shorter in girls, the same absolute measurement error is relatively more significant, potentially contributing to greater variability and lower reproducibility in girls. This may contribute to the weaker correlations in girls compared to boys. WIDER IMPLICATIONS OF THE FINDINGS: In boys, AGDas, relative to body size, correlated from infancy to 9 years, suggesting that AGD in infancy can be considered a non-invasive marker of later reproductive health. Further follow-up studies are needed to evaluate long-term individual tracking of AGD as well as assessment of childhood AGD as early marker of adult reproductive health. STUDY FUNDING/COMPETING INTERESTS: This study was supported by Odense University Hospital, Denmark, the Region of Southern Denmark, the Municipality of Odense, Denmark, the University of Southern Denmark, Odense Patient data Exploratory Network (OPEN), Denmark, the Danish Research Council (4004-00352B_FSS), Novo Nordisk Foundation, Denmark (grant no. NNF19OC0058266 and NNF17OC0029404), Sygeforsikring Danmark (journalnr. 2021-0173), the Collaborative Foundation between Odense University Hospital and Rigshospitalet, and Helsefonden. There is no conflict of interest of any author that could be perceived as prejudicing the impartiality of the research reported. TRIAL REGISTRATION NUMBER: N/A.

Longitudinal evaluation of fetal and infant AGD in healthy children: association with penile size, testosterone and DHT.

CONTEXT: The anogenital distance (AGD) is considered a postnatal readout of early fetal androgen action. Little is known of prenatal AGD and how it correlates with AGD postnatally. OBJECTIVES: We present longitudinal measurements of fetal- and infant AGD. We evaluate the impact of testosterone and dihydrotestosterone at minipuberty on AGD and penile size. DESIGN: Secondary analyses of an observational, prospective pregnancy and birth cohort, COPANA (2020-2022). SETTING: Copenhagen University Hospital - Rigshospitalet. PARTICIPANTS: 685 healthy, singleton pregnant women enrolled, 657 women attended 3rd trimester ultrasound, 589 infants completed follow-up. MAIN OUTCOME MEASURES: 3rd trimester ultrasound (GW29-34): Fetal AGD. Minipuberty clinical examination (app. 3.5 months postpartum): infant AGD, penile width and stretched length (SPL), circulating testosterone and dihydrotestosterone (LC-MS/MS). RESULTS: AGD was available in 650/657 fetuses (310 boys) and 588/589 infants (287 boys). Boys had longer fetal and infant AGD compared to girls; fetal AGDas: mean (SD) 21.4 mm (±3.5), fetal AGDaf: 12.8 mm (±2.3), p < 0.001, infant AGDas: 32.0 mm (±5.6) and infant AGDaf: 15.8 (±3.3), p < 0.001. Fetal AGD correlated with infant AGD in boys and girls (Spearman's r = 0.275, p < 0.001 and r = 0.189, p = 0.001 respectively), but not with circulating testosterone or dihydrotestosterone at minipuberty. Penile size correlated positively with circulating androgen levels at minipuberty, i.e.: SPL vs testosterone: r = 0.235, p < 0.001. CONCLUSIONS: AGD is sexual dimorphic already in the 3rd trimester. Fetal and infant AGD correlates. AGD is associated with body size but not circulating androgen levels at minipuberty. These findings suggest that fetal and infant AGD, reflect androgen action during early fetal development.

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

CONTEXT: The hypothalamic-pituitary-gonadal 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 T and SHBG. The associations with mean SD 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 1-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 = .02, <.001 and .03, with r2 = 0.05, 0.21 and 0.04, respectively). SHBG-PGSadult was significantly associated with mean SHBG-SDSinfancy (P < .001, r2 = 0.18). T-PGSadult explained 5% and 21% of the phenotypic variation in infancy of mean T-SDSinfancy and SHBG-SDSinfancy, respectively. CONCLUSION: Our findings suggest that the genetic architecture underlying total T and SHBG in adults also associates with hormone concentrations and their trajectories during infancy.

Ovarian and Uterine Morphology in Minipuberty: Associations with Reproductive Hormones. A COPANA Study of 302 girls.

BACKGROUND: Female minipuberty is characterized by complex dynamics of circulating reproductive hormones, while the relationship between ovarian and uterine morphology and reproductive hormones remains to be elucidated. AIM: To investigate the association between reproductive hormones and ovarian as well as uterine morphology by transabdominal ultrasound (TAUS) at minipuberty. METHODS: Secondary analysis from The Copenhagen Analgesic Study (COPANA) (ClinicalTrials.gov NCT04369222). Healthy infant girls (n=302, age 3.4 months (0.4) mean (±SD): mamma tissue diameter (mm), n=300. TAUS: uterine volume (n=230), endometrial thickness (n=255), ovarian volume, antral follicle count (total/2-4mm/≥5mm) (n=203).Blood samples (n=269/302=89%): AMH, FSH, LH, inhibin B (immunoassays), progesterone (PROG), androstenedione (Adione), testosterone (T), estrone (E1), estradiol (E2) (LC/MS-MS).Statistics: Pearson/Spearman´s correlation coefficient (parametric/non-parametric data). RESULTS: Total follicle count correlated positively with ovarian volume (r= 0.606, p<0.001), AMH (r=0.378, p<0.001), inhibin B (r=0.251, p<0.001), and negatively with FSH concentrations (r=-0.327, p<0.001). Larger follicles (≥5mm) correlated positively with AMH (r=0.264, p<0.001), inhibin B (0.230, p=0.002), E1 (r=0.209, p=0.004), E2 (r=0.269, p<0.001), PROG (r=0.160, p=0.031) and T (r=0.210, p=0.004) and negatively with FSH (r=-0.183, p=0.015). Circulating E1 and E2 levels correlated with the size of estrogen-responsive tissue sizes: E2 vs. uterine volume (r=0.134, p=0.054), E2 vs. endometrial thickness (r=0.155, p=0.020), E1 and E2 vs. mammary tissue diameter (r=0.213 and r=0.198, respectively, both p < 0.001). CONCLUSIONS: Correlations between reproductive hormones and the number of antral follicles suggest that negative feedback in the female HPG axis is established during minipuberty, with ovarian activity promoting uterine and glandular breast tissue growth We provide normative data of infant ovarian- and uterine morphology directly implementable to a clinical setting.

Serum Concentrations of Inhibin B in Healthy Females and Males Throughout Life.

OBJECTIVE: To describe the natural history of inhibin B throughout life according to sex, age, and pubertal development. METHODS: Based on serum samples from 2707 healthy controls aged 0 to 80 years, sex- and age-specific reference ranges of inhibin B concentrations were constructed. Concentrations were evaluated according to pubertal development and use of oral contraceptives (OCs). Also, measurements from 42 patients with Klinefelter syndrome were included. RESULTS: In both sexes, inhibin B concentrations were high during minipuberty, decreased in childhood, and increased significantly from Tanner stages B1 to B3 (peak: B4) in females and from G1 to G3 (peak: G3) in males. Despite variations in menstruating females, inhibin B concentrations remained relatively constant after puberty, until becoming unmeasurable at menopause. Despite a modest decrease, the inhibin B concentration in males remained relatively high from puberty onwards. At any age, males had highest concentrations. Inhibin B standard deviation (SD) scores were lower in OC-users (median SD score = -0.88) than in non-users (SD score = 0.35), p < 0.001. In patients with Klinefelter syndrome, inhibin B concentrations spanned the reference range until around 15 years of age, where they decreased to subnormal or unmeasurable levels. CONCLUSION: Valuable sex- and age-specific reference data for inhibin B concentrations were provided. In OC-users, decreased concentrations of inhibin B underlined the ovaries as the only place of inhibin B production. In patients with Klinefelter syndrome, the decline in inhibin B concentrations at puberty underlined the shift in regulation of inhibin B production at pubertal onset.

Sex- and age-specific reference intervals of 16 steroid metabolites quantified simultaneously by LC-MS/MS in sera from 2458 healthy subjects aged 0 to 77 years.

BACKGROUND: Reference intervals covering the whole life span for all the metabolites in the steroid hormone biosynthesis quantified by sensitive and robust analytical methods are sparse or not existing. OBJECTIVE: To develop a state-of-the-art LC-MS/MS method for simultaneous quantification of multiple steroid metabolites and to establish detailed sex- and age-specific reference intervals for 16 steroid metabolites. MATERIALS AND METHOD: An isotope diluted LC-MS/MS method was developed for simultaneous quantitation of 16 steroid hormones. Serum samples from cross-sectional cohorts of healthy infants, children, adolescents, and adults aged 0.17 months to 77 years (n = 2458) were analysed. RESULTS: With this novel, specific, and sensitive LC-MS/MS method, it was possible to quantify progesterone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, dehydroepiandrosterone sulfate, androstenedione, testosterone, dihydrotestosterone, 11-deoxycorticosterone, corticosterone, 11-deoxycortisol, cortisol, and cortisone in ≥90 % of the samples, while estrone sulfate, aldosterone and dehydroepiandrosterone were quantified in 77 %, 75 % and 60 % of the samples, respectively. 21-deoxycortisol was only detectable in 2.5 % of samples from healthy subjects. Sex- and age-dependent fluctuations observed in minipuberty, puberty and adulthood including the menopausal transition were modelled. This enabled us to establish valid reference intervals from birth to late adult life for both males and females. CONCLUSION: Detailed sex- and age-specific reference intervals of multiple, simultaneously quantified steroid metabolites by a novel and specific LC-MS/MS method provides a valuable tool for clinical practice and for future research.

Enhanced identification of endocrine disruptors through integration of science-based regulatory practices and innovative methodologies: The MERLON Project.

The prevalence of hormone-related health issues caused by exposure to endocrine disrupting chemicals (EDCs) is a significant, and increasing, societal challenge. Declining fertility rates together with rising incidence rates of reproductive disorders and other endocrine-related diseases underscores the urgency in taking more action. Addressing the growing threat of EDCs in our environment demands robust and reliable test methods to assess a broad variety of endpoints relevant for endocrine disruption. EDCs also require effective regulatory frameworks, especially as the current move towards greater reliance on non-animal methods in chemical testing puts to test the current paradigm for EDC identification, which requires that an adverse effect is observed in an intact organism. Although great advances have been made in the field of predictive toxicology, disruption to the endocrine system and subsequent adverse health effects may prove particularly difficult to predict without traditional animal models. The MERLON project seeks to expedite progress by integrating multispecies molecular research, new approach methodologies (NAMs), human clinical epidemiology, and systems biology to furnish mechanistic insights and explore ways forward for NAM-based identification of EDCs. The focus is on sexual development and function, from foetal sex differentiation of the reproductive system through mini-puberty and puberty to sexual maturity. The project aims are geared towards closing existing knowledge gaps in understanding the effects of EDCs on human health to ultimately support effective regulation of EDCs in the European Union and beyond.

AMH concentrations in infancy and mid-childhood predict ovarian activity in adolescence: A long-term longitudinal study of healthy girls.

Background: Anti-Müllerian hormone (AMH) is produced by granulosa cells in small growing ovarian follicles. In adult women, serum concentrations of AMH reflect the ovarian reserve of resting primordial follicles, and low AMH is associated with risk of early menopause. In contrast, patients with polycystic ovary syndrome (PCOS) have elevated AMH. The primary aim of this study was to evaluate the individual tracking of serum AMH concentrations, as well as whether AMH in early childhood reflects ovarian activity in adolescence. Methods: In this large longitudinal study of healthy girls were examined from infancy to adolescence (1997-2019) including physical examination, assessment of serum concentrations of reproductive hormones (in infancy, median age 0.3 yrs; mid-childhood, 7.2 yrs; puberty, 11.3 yrs; and adolescence, 15.9 yrs), transabdominal ultrasound (TAUS, puberty and adolescence) and magnetic resonance imaging (MRI, puberty) of the ovaries. Findings: Each girl maintained her relative AMH concentration (expressed as standard deviation (SD) scores) over time; mean variation of individual age adjusted AMH concentrations was 0.56 ± 0.31 SD.Serum concentrations of AMH in adolescence correlated with AMH in infancy and childhood; infancy: r = 0.347; mid-childhood: r = 0.637; puberty: r = 0.675, all p < 0.001.AMH correlated negatively with FSH concentrations in all age groups (infancy: r = -0.645, p < 0.001; mid-childhood: r = -0.222, p < 0.001; puberty: r = -0.354, p < 0.001; adolescence: n = 275, r = -0.175, p = 0.004).Serum AMH concentrations in mid-childhood correlated with the number of follicles in puberty (TAUS and MRI) as well as in adolescence (TAUS); e.g. total number of follicles: TAUS puberty (r = 0.607), MRI puberty (r = 0.379), TAUS adolescence (r = 0.414), all p < 0.001.AMH concentration in infancy as well as in mid-childhood predicted low AMH (<10 pmol/L) in adolescence; AMH infancy <7.5 pmol/L as predictor of low AMH in adolescence: sensitivity 0.71, specificity 0.70, AUC 0.759; AMH mid-childhood < 8.4 pmol/L as predictor of low AMH in adolescence: sensitivity 0.88, specificity 0.87, AUC 0.949.Girls with high serum AMH concentration in mid-childhood (AMH >30.0 pmol/L vs. other girls) had higher adolescent LH (median 4.53 vs. 3.29 U/L p = 0.041), LH/FSH ratio (1.00 vs 0.67, p = 0.019), testosterone (1.05 vs 0.81 nmol/L, p = 0.005), total number of follicles (23 vs. 19, p = 0.004), and higher prevalence of irregular cycles (10/15 = 67% vs. 28/113 = 25%, p = 0.002). Interpretation: The present findings suggest remarkably stable ovarian activity from small growing follicles in healthy girls, supporting AMH in early life as a useful clinical tool to predict future ovarian activity. Funding: The work was supported by The Center on Endocrine Disruptors (CeHoS) under The Danish Environmental Protection Agency and The Ministry of Environment and Food (grant number: MST-621-00 065), the EU (QLK4-CT1999-01422; QLK4-2001-00269), the Novo Nordisk Foundation and The Danish Ministry of Science Technology and Innovation (2107-05-0006). A.S.B. is funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) - 464240267. KM receives honoraria from Novo Nordisk A/S for teaching at the Danish annual postgraduate course of pituitary diseases.

AMH and other markers of ovarian function in patients with Turner syndrome - a single center experience of transition from pediatric to gynecological follow up.

Turner syndrome (TS) is a chromosomal disorder that affects about 1 in 2500 female births and is characterized by the partial or complete absence of the second X chromosome. Depending on karyotype, TS is associated with primary ovarian insufficiency (POI). Approximately 50% of girls with a mosaic 45, X/46, XX karyotype may enter puberty spontaneously, but only 5-10% of women with TS achieve pregnancy without egg donation. In this review, we will evaluate the clinical use of markers of ovarian function in TS patients. Based on longitudinal studies of serum concentrations of reproductive hormones as well as ovarian morphology in healthy females and patients with TS, we will evaluate how they can be applied in a clinical setting. This is important when counseling patients and their families about future ovarian function essential for pubertal development and fertility. Furthermore, we will report on 20 years of experience of transition from pediatric to gynecological and adult endocrinological care in our center at Rigshospitalet, Copenhagen, Denmark.

Serum LH/FSH ratios in 87 infants with differences of sex development.

The ratio between luteinizing hormone (LH) and follicle-stimulating hormone (FSH) has previously been described as an excellent marker of sex in healthy infants. However, LH/FSH remains not fully described in patients with differences of sex development (DSD). The aim was therefore to describe LH/FSH in infants with DSD. This was a retrospective study of DSD patients, all aged 0-1.2 years. In total, 87 infants with DSD and at least one serum sample per infant were included. Longitudinal samples from single patients were included whenever possible. Serum LH/FSH ratios in these patients were plotted against recently published age-related and sex-dimorphic cutoffs. Overall, LH/FSH sometimes corresponded to assigned sex without any obvious pattern in terms of diagnoses. LH/FSH corresponded to the biological sex in all patients with Turner or Klinefelter syndrome. In patients with 46,XX or 46,XY DSD (except congenital adrenal hyperplasia (CAH)), the ratios did not correspond to the assigned sex in all cases and were interchangeably within the male and female range. In patients with CAH, the ratio corresponded to biological sex (based on sex chromosomes) in some cases but also ranged across the cutoffs. In the 15 patients with 45,X/46,XY mosaicism, the LH/FSH ratios corresponded to the assigned sex in all cases (12 were raised as males, 3 as females) and at all time points in cases with multiple sampling. While this study describes LH/FSH in infants with DSD, the exact clinical role of the ratio in the management of these patients remains to be further elucidated.

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.

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.

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.

Dynamic changes in LH/FSH ratios in infants with normal sex development.

Objective: Little is known about the ratio between luteinizing hormone (LH) and follicle-stimulating hormone (FSH) during infancy. This study aimed to evaluate serum and urinary LH/FSH as a marker of sex with age-specific cutoffs in healthy infants. Design: A prospective, longitudinal cohort study of healthy infants aged 0-1.2 years. Methods: In total, 236 healthy infants (122 boys and 114 girls) from The COPENHAGEN Minipuberty Study (ClinicalTrials.gov ID: NCT02784184), with 567 serum and 603 urine samples, were included. Measures of diagnostic accuracy, including sensitivity and specificity, were used to assess the ability of LH/FSH to detect sex in healthy infants. Results: In both serum and urine, LH/FSH was highest in males with minimal overlap between the sexes. In contrast to isolated LH and FSH concentrations, LH/FSH ratios in both serum and urine were excellent markers of sex from 0 to 1.2 years with median sensitivities and specificities ranging from 93 to 100% with correspondingly narrow 95% CIs. Conclusions: Serum and urinary LH/FSH ratios are excellent discriminators of sex in healthy infants during the entire first year of life. The clinical role and application of the ratio remain to be elucidated.

Dynamic Changes in Serum IGF-I and Growth During Infancy: Associations to Body Fat, Target Height, and PAPPA2 Genotype.

CONTEXT: IGF-I is important for postnatal growth and may be of diagnostic value in infants suspected of pituitary disease; however, little is known about the impact of IGF-I and its determinants on infant growth. Importantly, detailed reference ranges for IGF-I and IGF binding protein-3 (IGFBP-3) concentrations during infancy are lacking. OBJECTIVE: To evaluate the rapid changes in weight and length as well as their determinants in healthy infants, and to establish age- and sex-specific reference curves for IGF-I and IGFBP-3 in children aged 0 to 1 years. DESIGN: Prospective longitudinal study. SETTING: Cohort study. PARTICIPANTS: A total of 233 healthy children (114 girls) with repeated blood samples during the first year of life. MAIN OUTCOME MEASURE(S): Serum concentrations of IGF-I and IGFBP-3, length velocity, weight velocity, and PAPPA2 (rs1325598) genotype. RESULTS: Individual trajectories of length and weight velocities were sex specific. We provide detailed reference curves based on longitudinal data for IGF-I and IGFBP-3 during infancy. In both girls and boys, IGF-I decreased during infancy, whereas IGFBP-3 remained stable. IGF-I and IGFBP-3, but not PAPPA2 genotype, were positively associated with weight gain, but not with longitudinal growth. When stratified by sex, the association between weight gain and IGF-I only remained significant in girls. CONCLUSIONS: Interestingly, we found a significant association between IGF-I and infant weight gain in girls, but not with longitudinal growth in the first year of life. Our findings highlight the role of IGF-I as an important anabolic hormone that is not limited to linear growth.

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.

Anogenital Distance in Healthy Infants: Method-, Age- and Sex-related Reference Ranges.

CONTEXT: The use of anogenital distance (AGD) in clinical and epidemiological settings is increasing; however, sex-specific reference data on AGD and data on longitudinal changes in AGD in children is scarce. OBJECTIVE: To create age-, sex-, and method-related reference ranges of AGD in healthy boys and girls aged 0-24 months, to assess the age-related changes in AGD and to evaluate the 2 predominantly used methods of AGD measurement. DESIGN: The International AGD consortium comprising 4 centers compiled data from 1 cross-sectional and 3 longitudinal cohort studies (clinicaltrials.gov [NCT02497209]). SETTING: All data were collected from population-based studies, recruiting from 4 maternity or obstetric centers (United States, Cambridge [United Kingdom], Odense, and Copenhagen [Denmark]). SUBJECTS: This study included a total of 3705 healthy, mainly Caucasian children aged 0-24 months on whom 7295 measurements were recorded. MAIN OUTCOME MEASURES: AGDAS (ano-scrotal), AGDAF (ano-fourchette), AGDAP (ano-penile), AGDAC (ano-clitoral), AGD body size indices (weight, body mass index [BMI], body surface area, and length), and intra- and interobserver biases. RESULTS: We created age-specific reference ranges by centers. We found that AGD increased from birth to 6 months of age and thereafter reached a plateau. Changes in AGD/BMI during the first year of life were minor (0-6% and 0-11% in boys and girls, respectively). CONCLUSIONS: Reference ranges for AGD can be used in future epidemiological research and may be utilized clinically to evaluate prenatal androgen action in differences-in-sex-development patients. The increase in AGD during the first year of life was age-related, while AGD/BMI was fairly stable. The TIDES and Cambridge methods were equally reproducible.