BACKGROUND: Testis volume, an indicator of the reproductive development during minipuberty, is commonly measured by Prader orchidometer, despite ultrasound being the gold standard. Data are lacking on the longitudinal relationship between these two measures and on the stability of boys\' relative testis size across infancy.
OBJECTIVE: To examine the relationship between ultrasound-based and orchidometer-based testis volume measurements and to assess the stability of relative testis size among individual boys in the study.
METHODS: The Infant Feeding and Early Development study is a longitudinal cohort of healthy infants recruited from hospitals in the Philadelphia area during 2010-2013. We measured testis size from birth to 28 weeks in 147 infants using Prader orchidometry (nine study visits) and ultrasound (five study visits). We modeled testis growth, extracted predicted volumes for each boy on each day of the study, and ranked these volumes from smallest to largest.
RESULTS: The average testis volume trajectory exhibited linear growth over the first 16 weeks followed by slower growth and then a plateau. Prader orchidometry overestimated testis size by almost 3-fold, compared to ultrasound. A range of ultrasound volumes corresponded to each bead size (e.g., bead size of 1 cm3 corresponded to an ultrasound-based volume between 0.11 and 0.87 cm3). Infants changed rankings of median of 22 positions (of 147) across the entire 6-month follow-up. Infants\' ranks near birth were highly correlated with their ranks at the end of the study.
CONCLUSIONS: Consistent with other studies, we found wide variability in testis size during infancy and that Prader orchidometry overestimates testis size. When compared to ultrasound, orchidometry only crudely estimates testis size in this age group. Ultrasound-based volumes generally showed stability in relative testis size across infancy.
CONCLUSIONS: Accurate measurement of testis size is difficult using orchidometry in infants. This highlights the need for ultrasound for accurate measurement, with a one-time measurement likely sufficient to determine relative testis size across the first 6 months of infancy.

UNASSIGNED: Atopic dermatitis (AD) is an inflammatory, pruritic, noncontagious, chronic relapsing skin disease. Skin barrier abnormalities, excessive T helper 2 activity, and immune dysregulation are held responsible. Androgens have a negative effect on the integrity of the epidermal skin barrier, while estrogen has a positive effect. We aimed to investigate whether hormones make a difference between healthy children and children with AD during minipuberty.
UNASSIGNED: A total of 96 infants (postnatal 4-13 weeks), 48 diagnosed with AD and 48 controls, were included. Each group consisted of 23 girls (47.9%) and 25 boys (52.1%). Anthropometric examinations and hormone measurements were compared.
UNASSIGNED: The two groups, having similar age, sex, body mass index, and weight-for-length standard deviation scores, were compared. Serum free thyroxine (FT4) levels were found to be lower and insulin-like growth factor binding protein-3 (IGFBP3) levels were found to be higher in children with AD (p < 0.001 and p = 0.038, respectively). In girls with AD, estradiol, FT4, and insulin-like growth factor-1 (IGF-1) levels were found to be lower, but thyroid-stimulating hormone (TSH) levels were found to be higher (p = 0.023, p < 0.001, p = 0.038, and p = 0.034, respectively). In boys with AD, the FT4 level was found to be lower (p = 0.023). Serum FT4 and TSH levels were within normal reference ranges in all comparisons.
UNASSIGNED: Especially in girls with AD, decreased estradiol and IGF-1 levels were observed compared to the controls during minipuberty. In the logistic regression model, decreased levels of serum estradiol, dehydroepiandrosterone sulfate, FT4, and IGF-1, and increased levels of IGFBP3 were associated with an increased likelihood of exhibiting atopic dermatitis.

To improve the mechanistic screening of reproductive toxicants in  chemical-risk assessment and drug development, we have developed a three-dimensional (3D) heterogenous testicular co-culture model from neonatal mice. Di-n-butyl phthalate (DBP), an environmental contaminant that can affect reproductive health negatively, was used as a model compound to illustrate the utility of the in vitro model. The cells were treated with DBP (1 nM to 100 µM) for 7 days. Automated high-content imaging confirmed the presence of cell-specific markers of Leydig cells (CYP11A1 +), Sertoli cells (SOX9 +), and germ cells (DAZL +). Steroidogenic activity of Leydig cells was demonstrated by analyzing testosterone levels in the culture medium. DBP induced a concentration-dependent reduction in testosterone levels and decreased the number of Leydig cells compared to vehicle control. The levels of steroidogenic regulator StAR and the steroidogenic enzyme CYP11A1 were decreased already at the lowest DBP concentration (1 nM), demonstrating upstream effects in the testosterone biosynthesis pathway. Furthermore, exposure to 10 nM DBP decreased the levels of the germ cell-specific RNA binding protein DAZL, central for the spermatogenesis. The 3D model also captured the development of the Sertoli cell junction proteins, N-cadherin and Zonula occludens protein 1 (ZO-1), critical for the blood-testis barrier. However, DBP exposure did not significantly alter the cadherin and ZO-1 levels. Altogether, this 3D in vitro system models testicular cellular signaling and function, making it a powerful tool for mechanistic screening of developmental testicular toxicity. This can open a new avenue for high throughput screening of chemically-induced reproductive toxicity during sensitive developmental phases.

BACKGROUND: 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.
OBJECTIVE: We present longitudinal measurements of fetal- and infant AGD. We evaluate the impact of testosterone and dihydrotestosterone at minipuberty on AGD and penile size.
METHODS: Secondary analyses of an observational, prospective pregnancy and birth cohort, COPANA (2020-2022).
METHODS: Copenhagen University Hospital - Rigshospitalet.
METHODS: 685 healthy, singleton pregnant women enrolled, 657 women attended 3rd trimester ultrasound, 589 infants completed follow-up.
METHODS: 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.

Managing patients unable to produce sex steroids using gonadotropins to mimic minipuberty in hypogonadotropic hypogonadism, or sex steroids in patients with Klinefelter or Turner syndrome, is promising. There is a need to pursue research in this area, with large prospective cohorts and long-term data before these treatments can be routinely considered.

OBJECTIVE: The aim of this study was to investigate the feasibility of different gonadotropin assays for determining total and intact luteinizing hormone (LH), and follicle-stimulating hormone (FSH) immunoreactivity in urine (U-LH-ir and U-FSH-ir, respectively) during early infancy.
METHODS: Morning urine samples were obtained from 31 infants, aged between 0 and 6 months, to study the age-related course of urinary gonadotropins. Additionally, we investigated bi-hourly urine samples of a 5-day-old male neonate for 24 h to observe the course of urinary gonadotropins during a daily cycle. We employed different immunofluorometric assays for measuring total and intact U-LH-ir, and U-FSH-ir.
RESULTS: In neonates up to 21 days of age, the U-LH-ir levels measured by the regular LH assay (also detecting hCG) were significantly higher than those determined by the total (specific) LH assays (p = .004). U-FSH-ir was higher in girls than boys during both the first and the next 5 months (p = .02 and p < .001, respectively), whereas total U-LH-ir was higher in boys until 6 months of age (p < .001). Total U-LH-ir/U-FSH-ir ratio was significantly higher in boys than girls across the first half-year (p < .001).
CONCLUSIONS: The assessment of total U-LH-ir and U-FSH-ir, and their respective ratio constitutes a noninvasive, practical and scalable tool to investigate sex-specific changes during early infancy, with the ratio being significantly higher in boys than girls. Only highly specific LH assays detecting beta-subunit and its core fragment in addition to intact LH should be used for determining U-LH-ir in the neonatal period to avoid potential cross-reactivity with hCG of placental origin.

In humans, the first 1000 days of life are pivotal for brain and organism development. Shortly after birth, gonadotropin-releasing hormone (GnRH) neurons in the hypothalamus are activated, a phenomenon known as minipuberty. This phenomenon, observed in all mammals studied, influences the postnatal development of the hypothalamic-pituitary-gonadal (HPG) axis and reproductive function. This review will put into perspective the results of recent studies showing that the impact of minipuberty extends beyond reproductive function, influencing sensory and cognitive maturation. Studies in mice have revealed the role of nitric oxide (NO) in regulating minipuberty amplitude, with NO deficiency linked to cognitive and olfactory deficits. Additionally, findings indicate that cognitive and sensory defects in adulthood in a mouse model of Down syndrome are associated with an age-dependent decline of GnRH production, whose origin can be traced back to minipuberty, and point to the potential therapeutic role of pulsatile GnRH administration in cognitive disorders. Furthermore, this review delves into the repercussions of COVID-19 on GnRH production, emphasizing potential consequences for neurodevelopment and cognitive function in infected individuals. Notably, GnRH neurons appear susceptible to SARS-CoV-2 infection, raising concerns about potential long-term effects on brain development and function. In conclusion, the intricate interplay between GnRH neurons, GnRH release, and the activity of various extrahypothalamic brain circuits reveals an unexpected role for these neuroendocrine neurons in the development and maintenance of sensory and cognitive functions, supplementing their established function in reproduction. Therapeutic interventions targeting the HPG axis, such as inhaled NO therapy in infancy and pulsatile GnRH administration in adults, emerge as promising approaches for addressing neurodevelopmental cognitive disorders and pathological aging.

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.
OBJECTIVE: 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.
METHODS: Healthy, singleton pregnant women and their partners.
METHODS: 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.
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.

BACKGROUND: 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.
METHODS: 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.
METHODS: Population-based.
METHODS: Healthy, male, term, singleton newborns were followed with repeated clinical examinations including blood sampling during a 1-year follow-up (n = 109).
METHODS: 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.
CONCLUSIONS: Our findings suggest that the genetic architecture underlying total T and SHBG in adults also associates with hormone concentrations and their trajectories during infancy.

The postnatal activation of the hypothalamic-pituitary-gonadal (HPG) axis is usually known as “minipuberty”. There are still open questions about its biological function and significance depending on sex, gestational age (GA) and birth weight (BW) with few available longitudinal data.
A single-centre, longitudinal study to quantify urinary follicle stimulating hormone (uFSH), luteinizing hormone (uLH) and testosterone (uTs) in male neonates. Neonates were enrolled and stratified into three subgroups: full-term boys appropriate for GA (FT AGA); FT boys with BW ≤3rd centile [FT small for gestational age (SGA)]; and preterm (PT) boys ≤33 weeks of GA. Urinary hormones were correlated to simultaneous auxological parameters, linear growth and external genitalia at scheduled time-points.
Forty-six boys were recruited, with subgroup sizes FT AGA n=23, FT SGA n=11 and PT n=12. PT boys display a pulsatile pattern of urinary gonadotropins (uGns) with higher levels of uLH and a gradual increase of uTs. Testicular descent started from 29-32 weeks with the peak of uTs. During the first 12-months post-term age (PTA), FT AGA boys displayed a better linear growth (p<0.05). PT showed higher uGns levels until 3-months PTA. PT babies had higher uLH levels than FT AGA, with a peak at 7 and 30 days, during the first 90 days of life (p<0.001) and higher uTs levels. Correlation analysis between penile growth of all neonates and uTs was significant (p=0.04) but not within subgroups.
This study investigated postnatal HPG axis activation in term and PT infants. Minipuberty may involve an early window of opportunity to evaluate the functionality of the HPG axis. Further studies with a long-term follow-up are needed with a special focus on possible consequences of GA and BW.