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Introduction Sexual development in females and males are routinely measured according to the Tanner Stages. Sparse data exist on the timing of pubertal milestones in Pakistan. To fill this gap, the age of attainment of pubertal milestones and their relationship with nutritional status was explored among children and adolescents living in the rural district of Matiari, Pakistan. Methods Anthropometry, nutrition biomarkers and Tanner Stage were assessed among girls aged 9.0–14.9 years (n = 723) and boys aged 10.0–15.9 years (n = 662) who were free from known disease in the rural District of Matiari, Pakistan. Median age was calculated for all Tanner Stages and menarche. Multivariable linear regressions were undertaken to determine covariates associated with the timing (age) of pubertal milestones. Results Among participants living in this rural community, the median age of puberty onset for girls was 11.9 years (95%CI:10.9; 12.5) and boys was 12.3 years (95%CI:11.5; 12.9). Age at first menarche was 12.9 years (95%CI:12.1; 13.3). Undernutrition was widespread among adolescents in this community. Thirty-seven percent of females and 27.0% of males were stunted while 20.5% of females and 31.3% of males were thin. Only 8% (n = 58) of females and 12% (n = 78) of males were free from any nutrient deficiency with most adolescents having two or three nutrient deficiencies. Conclusions Undernutrition (stunting or thinness) was associated with relatively older ages for early puberty stages but not puberty completion. This may decrease the duration of the pubertal growth spurt and curtail potential catch-up growth that may occur during puberty. Efforts to decrease nutrient deficiencies, stunting and thinness beyond childhood should be made in rural Pakistan.

Objectives Staging sexual maturation is an integral component of adolescent research. The Pubertal Development Scale (PDS) is commonly used as a puberty self-assessment tool because it avoids the use of images. Among the youth living in rural Pakistan, we determined the accuracy of self-reported pubertal assessments using a modified PDS compared to the ‘gold standard’ of physically assessed Tanner stages by a physician. Methods The strength of agreement between self-assessed puberty using a modified PDS and the ‘gold’ standard of physician-assessed Tanner stages was reported using weighted kappa (κ w) for girls (n = 723) of 9.0–14.9 years of age or boys (n = 662) of 10.0–15.9 years of age living in the rural District of Matiari. Results Agreement between the gold standard and self-assessment for puberty was substantial, with a κ w of 0.73 (95% confidence interval [CI]: 0.67; 0.79) for girls and a κ w of 0.61 (95% CI: 0.55; 0.66) for boys. Substantial agreement was observed for both boys and girls classified as thinness but only for girls with a normal body mass index. Those who were classified as severely thin had moderate agreement. The prevalence of overestimation was 18.5% (95% CI: 15.9–21.5) for girls and 2.7% (95% CI: 1.7–4.3) for boys, while the prevalence of underestimation estimation was 8.0% (95% CI: 6.2–10.2) for girls and 29.0% (95% CI: 25.8–32.6) for boys. Conclusions Most girls and boys assessed their pubertal development with substantial agreement with physician assessment. Girls were better able to assess their puberty, but they were more likely to overestimate. Agreement for boys was also substantial, but they were more likely to underestimate their pubertal development. In this rural Pakistan population, the PDS seems to be a promising tool for self-assessed puberty.

Convergent evidence associates exposure to endocrine disrupting chemicals (EDCs) with major human diseases, even at regulation-compliant concentrations. This might be because humans are exposed to EDC mixtures, whereas chemical regulation is based on a risk assessment of individual compounds. Here, we developed a mixture-centered risk assessment strategy that integrates epidemiological and experimental evidence. We identified that exposure to an EDC mixture in early pregnancy is associated with language delay in offspring. At human-relevant concentrations, this mixture disrupted hormone-regulated and disease-relevant regulatory networks in human brain organoids and in the model organisms Xenopus leavis and Danio rerio , as well as behavioral responses. Reinterrogating epidemiological data, we found that up to 54% of the children had prenatal exposures above experimentally derived levels of concern, reaching, for the upper decile compared with the lowest decile of exposure, a 3.3 times higher risk of language delay.

Context Growth hormone (GH) is used to treat short children born small for gestational age (SGA); however, the effects of treatment on pubertal timing and adult height are rarely studied. Objective To evaluate adult height and peak height velocity in short GH-treated SGA children. Methods Prospective longitudinal multicenter study. Participants were short children born SGA treated with GH therapy (n = 102). Adult height was reported in 47 children. A reference cohort of Danish children was used. Main outcome measures were adult height, peak height velocity, age at peak height, and pubertal onset. Pubertal onset was converted to SD score (SDS) using Danish reference data. Results Gain in height SDS from start of treatment until adult height was significant in both girls (0.94 [0.75; 1.53] SDS, P = .02) and boys (1.57 [1.13; 2.15] SDS, P Conclusion GH treatment improved adult height. Peak height velocity was reduced, but age at peak height velocity did not differ compared with the reference cohort. SGA boys had an earlier pubertal onset compared with the reference cohort.

Background Potentially harmful effects of persistent organic pollutants (POPs) such as polychlorinated biphenyls (PCBs) and dichlorodiphenyltrichloroethane (DDT) on prenatal development and the endocrine system have been controversially discussed. Methods Working with a German cohort of 324 pregnant women, we assessed POP levels and used robust linear regression models to determine potential associations between maternal POP concentrations and pre- and postnatal development in the children, as well as the thyroid hormone status of the mother and child. Results Maternal p,p′-dichlorodiphenyldichloroethylene (p,p′-DDE) and most measured PCBs positively correlated with postnatal weight gain. We detected no correlation between newborn birth weight and head circumference, respectively, and maternal PCB and p,p′-DDE serum levels, while body length at birth was negatively associated with the maternal serum concentration of PCB 183. Maternal p,p′-DDE and nearly all PCB serum levels showed a negative correlation with maternal free triiodothyronine (FT3). p,p′-DDE and PCB 74 and 118 were negatively associated with maternal thyroid-stimulating hormone levels. In addition, we identified significant associations between maternal POP levels and thyroid hormone parameters of the child. Conclusions These results indicate that POP exposure likely affects different aspects of pre- and postnatal development and impacts the thyroid hormone status of both mother and child. Impact Pregnant women in a German cohort display a substantial accumulation of POPs. Body mass index and age influence maternal serum POP levels. Maternal POP levels show correlations with the child’s length at birth and weight gain, and FT3 levels in the mother and child. Our data provide additional evidence for the potentially harmful influence of POPs. Our data indicate that POPs influence pre- and postnatal development.

Background Advances in three-dimensional culture technologies have led to progression in systems used to model the gonadal microenvironment in vitro. Despite demonstrating basic functionality, tissue organisation is often limited. We have previously detailed a three-dimensional culture model termed the three-layer gradient system to generate rat testicular organoids in vitro. Here we extend the model to human first-trimester embryonic gonadal tissue. Results Testicular cell suspensions reorganised into testis-like organoids with distinct seminiferous-like cords situated within an interstitial environment after 7 days. In contrast, tissue reorganisation failed to occur when mesonephros, which promotes testicular development in vivo, was included in the tissue digest. Organoids generated from dissociated female gonad cell suspensions formed loosely organised cords after 7 days. In addition to displaying testis-specific architecture, testis-like organoids demonstrated evidence of somatic cell differentiation. Within the 3-LGS, we observed the onset of AMH expression in the cytoplasm of SOX9-positive Sertoli cells within reorganised testicular cords. Leydig cell differentiation and onset of steroidogenic capacity was also revealed in the 3-LGS through the expression of key steroidogenic enzymes StAR and CYP17A1 within the interstitial compartment. While the 3-LGS generates a somatic cell environment capable of supporting germ cell survival in ovarian organoids germ cell loss was observed in testicular organoids. Conclusion The 3-LGS can be used to generate organised whole gonadal organoids within 7 days. The 3-LGS brings a new opportunity to explore gonadal organogenesis and contributes to the development of more complex in vitro models in the field of developmental and regenerative medicine.

In recent years, there has been an increased incidence in several of the most common reproductive disorders, including hypospadias and cryptorchidism in newborns, and testicular cancer and lower sperm quality in young adult males. In addition, the timing of puberty has also changed over time. Although the cause of these reproductive effects is a matter of intense debate, a link with the presence of ubiquitous compounds in the environment, or the exposure to specific groups of medications during foetal life, has been suggested. Results from epidemiological and experimental studies, as well as clinical observations in humans indicate that endocrine-disrupting chemicals may be associated with those disorders. In this review, we will summarize the results of epidemiological studies and experimental studies utilising human testicular cells or tissue. Due to increasing public interest and the recently published data, the main focus will be on the effects of prenatal exposure to mild analgesics.

Background A low oxygen supply to the fetus causes intrauterine growth restriction and can affect gonadal development of the offspring, having a potential impact on fertility. We investigated histology and gene expression in the postnatal rat ovary after fetal hypoxia induced by uterine artery ligation. Methods Sprague-Dawley rats underwent uterine artery ligation at day 19 of gestation. Offspring were sacrificed at 5, 20 and 40 days post-partum. Follicles were counted and classified in hematoxylin-eosin stained sections. Gene expression of 90 genes was analyzed by TaqMan® Low Density Array. Results A significantly lower number of total and primordial follicles was detected in 20 days post-partum intrauterine growth restricted animals. Follicle density was not different at 40 days post-partum, suggesting that compensatory mechanisms occurred during the pre-pubertal window. Uterine artery ligation modified the expression of 24 genes involved in different cellular functions, among which proliferation, apoptosis and metabolism. Conclusion Ovarian follicle pool was affected by fetal hypoxia in early life, but this effect did not persist in puberty. Genes involved in cellular processes were affected at all ages, potentially implying long-term genetic alterations. Further analyses are needed to elucidate later effects of fetal hypoxia on ovarian function and fertility. Electronic supplementary material The online version of this article (10.1186/s12958-019-0453-3) contains supplementary material, which is available to authorized users.

Type 1 diabetes mellitus (T1DM) is associated with impaired spermatogenesis and lower testosterone levels and epididymal weight. However, the underlying processes in the testis are unknown and remain to be elucidated. Therefore, the present study focused on the effects of T1DM on testicular function in a spontaneously diabetic rat model. BB/OKL rats after diabetes manifestation were divided into 3 groups: those without insulin treatment and insulin treatment for a duration of 2 and of 6 weeks. Anthropometrical data, circulating levels of gonadotrophins, testosterone, and inhibin B were measured. Intratesticular testosterone, oxidative stress, inflammation, and apoptosis were analyzed. Key enzymes of steroidogenesis were evaluated in the testis. Untreated diabetic rats had significantly lower serum follicle-stimulating hormone and luteinizing hormone levels. Serum and intratesticular testosterone levels significantly decreased in untreated diabetic rats compared to healthy controls. Key markers of Leydig cell function were significantly downregulated at the RNA level: insulin-like factor 3 (Insl3) by 53% (P = .006), Star by 51% (P = .004), Cyp11A1 by 80% (P = .003), 3Beta-Hsd2 by 61% (P = .005), and Pbr by 52% (P = .002). In the insulin-treated group, only Cyp11A1 and 3Beta-Hsd2 transcripts were significantly lower. Interestingly, the long-term insulin-treated group showed significant upregulation of most steroidogenic enzymes without affecting testosterone levels. Tumor necrosis factor α and apoptosis were significantly increased in the long-term insulin-treated rats. In conclusion T1DM, with a severe lack of insulin, has an adverse action on Leydig cell function. This is partially reversible with well-compensated blood glucose control. Long-term T1DM adversely affects Leydig cell function because of the process of inflammation and apoptosis.

Over the last 50 years, there has been a steady decline in fertility rates in humans, which has occurred in parallel with an increasing incidence of obesity and metabolic disorders. The potential impact of these disorders and plausible mechanisms by which they negatively influence male reproduction are only partly understood and published data are often controversial. Obesity is one of the most important health challenges worldwide and is becoming more prevalent in children and adolescents. Obesity, the metabolic syndrome and related co-morbidities can lead to impaired male reproductive function, including adverse effects on spermatogenesis and steroidogenesis as illustrated by reduced sperm number and quality, decreased testosterone levels and elevated inflammatory markers. The incidence of diabetes mellitus type I is also dramatically increasing and may negatively impact spermatogenesis and testicular function, resulting in decreased serum testosterone and epididymal weight. In this review, we summarize and discuss the effects of metabolic diseases that typically develop during childhood and adolescence on later reproductive function and fertility. While impact on reproductive health is likely observed in both sexes, we have chosen to focus on the male in the current review. Specifically, we illustrate adverse effects of obesity, type 1 diabetes, the metabolic syndrome and insulin resistance on sperm function and testosterone metabolism. Identification of pathophysiological mechanisms during childhood may open up new avenues for early prevention and treatment resulting in better reproductive outcomes and improved fertility rates during adulthood.

Potentially harmful effects of persistent organic pollutants (POPs) such as polychlorinated biphenyls (PCBs) and dichlorodiphenyltrichloroethane (DDT) on prenatal development and the endocrine system have been controversially discussed.Working with a German cohort of 324 pregnant women, we assessed POP levels and used robust linear regression models to determine potential associations between maternal POP concentrations and pre- and postnatal development in the children, as well as the thyroid hormone status of the mother and child.Maternal p,p'-dichlorodiphenyldichloroethylene (p,p'-DDE) and most measured PCBs positively correlated with postnatal weight gain. We detected no correlation between newborn birth weight and head circumference, respectively, and maternal PCB and p,p'-DDE serum levels, while body length at birth was negatively associated with the maternal serum concentration of PCB 183. Maternal p,p'-DDE and nearly all PCB serum levels showed a negative correlation with maternal free triiodothyronine (FT3). p,p'-DDE and PCB 74 and 118 were negatively associated with maternal thyroid-stimulating hormone levels. In addition, we identified significant associations between maternal POP levels and thyroid hormone parameters of the child.These results indicate that POP exposure likely affects different aspects of pre- and postnatal development and impacts the thyroid hormone status of both mother and child.Pregnant women in a German cohort display a substantial accumulation of POPs. Body mass index and age influence maternal serum POP levels. Maternal POP levels show correlations with the child's length at birth and weight gain, and FT3 levels in the mother and child. Our data provide additional evidence for the potentially harmful influence of POPs. Our data indicate that POPs influence pre- and postnatal development.

Female reproductive dysfunction occurs in patients with pathological loss of adipose tissue, i.e. lipodystrophy (LD). However, mechanisms remain largely unclear and treatment effects of adipocyte-derived leptin have not been assessed in LD animals.In the current study, C57Bl/6 LD mice on a low-density lipoprotein receptor knockout background were treated with leptin or saline for 8 weeks and compared to non-LD controls.The number of pups born was 37% lower in breeding pairs consisting of LD female mice x non-LD male mice (n = 3.3) compared to LD male mice x non-LD female mice (n = 5.2) (p 0.05). Mean uterus weight was significantly lower in the saline-treated LD group (18.8 mg) compared to non-LD controls (52.9 mg; p 0.0001) and increased significantly upon leptin treatment (46.5 mg; p 0.001). The mean number of corpora lutea per ovary was significantly lower in saline-treated LD animals compared to non-LD controls (p 0.01) and was restored to non-LD control levels by leptin (p 0.05). Mechanistically, mRNA expression of ovarian follicle-stimulating hormone receptor (p 0.01) and estrogen receptor β (p 0.05), as well as of pituitary luteinizing hormone β subunit (p 0.001) and follicle-stimulating hormone β subunit (p 0.05), was significantly upregulated in LD mice compared to non-LD controls. In addition, mean time to vaginal opening as a marker of puberty onset was delayed by 12.5 days in LD mice (50.9 days) compared to non-LD controls (38.4 days; p 0.001).Female LD animals show impaired fertility which is restored by leptin. Future studies should assess leptin as a subfertility treatment in human leptin-deficiency disorders.

OBJECTIVE Obesity in females is often associated with metabolic complications and hyperandrogenism, but the sources of androgens are not completely understood. Therefore, this study investigated whether adipose tissue could be a source of androgens promoting hyperandrogenism development in obese female rats. METHODS Gene expression of steroidogenic enzymes and testosterone levels were determined in periovarian and inguinal adipose tissue and in the supernatant of cultured preadipocytes and adipocytes. The conversion of pregnenolone to androgens was analyzed by thin-layer chromatography. RESULTS Substantial amounts of testosterone in adipose tissue (25-153 ng/g tissue) and in the supernatant of adipocytes (0.33-0.69 ng/ten thousand cells]) were found. StAR and steroidogenic enzymes encoded by genes including Cyp11A1, Cyp17A1, Cyp19, Hsd3b2, Hsd17b3, and Srd5a2 were expressed in adipose tissue and cultured cells. Thin layer chromatography data revealed that preadipocytes and adipocytes were able to convert pregnenolone to testosterone. Higher levels for all steroidogenic enzymes were found in both depots of obese animals compared with lean animals, with significantly higher levels in inguinal tissue. CONCLUSIONS The whole steroidogenic machinery and capacity for testosterone biosynthesis were found in fat depots of female rats. These findings support the hypothesis that adipose tissue may contribute substantially to the hyperandrogenism in female obesity.

We have recently developed a 3D culture system that allows the reorganization of rat primary testicular cells into organoids with a functional blood-testis barrier, as well as the establishment and maintenance of germ cells. The innovative aspect of our model, the three-layer gradient system (3-LGS), comprises cells combined with Matrigel placed between two layers of Matrigel without cells, which creates a gradient of cells and allows the reorganization of testicular cells into organized structures after 5-7 d in culture. This reorganization is not observed when testicular cells are suspended in only one layer of Matrigel, the methodology used in the majority of the protocols for generating organoids. The model can be applied to follow and quantify cell migration during testicular organoid formation, and to explore the role of growth factors and the toxic effects of drugs and environmental contaminants on germ cell maintenance and blood-testis barrier integrity. The 3-LGS is a robust and reproducible method that requires a small volume of Matrigel and a low number of cells (16 μl and 132,000 cells, respectively), enabling and facilitating high-throughput analysis of germ-to-somatic cell associations in vitro.

Finding robust culture conditions for in vitro maturation (IVM) of male germ cells is still a challenge. Recently, a testis organ culture method, using Knockout Serum Replacement (KSR), was suggested as a promising approach. However, the efficiency of that model is still not optimal. Hence, we have tried to establish the culture conditions in two laboratories, and to improve the reliability of the culture system to generate mature germ cells. Male mice at three days of age were sacrificed. Testes were cut into small pieces which were cultured atop agarose stands, using Minimum Essential Medium alpha supplemented with different supplements; melatonin, Glutamax, and different concentrations of KSR. The results showed that the duration of culture beyond 18 days had an impact on the number of differentiated germ cells. Supplementation with melatonin and Glutamax revealed a positive influence on the efficiency of male germ cell differentiation in vitro. Furthermore, the results confirmed that KSR had a positive effect on germ cell maturation and testosterone production, with a concentration of at least 10%. In conclusion, this study emphasizes the beneficial role of at least 10% KSR in the IVM of germ cells. Electronic supplementary material The online version of this article (doi:10.1007/s10439-017-1847-z) contains supplementary material, which is available to authorized users.

Prenatal events such as intrauterine growth restriction can affect gonadal development of the offspring and have an impact on reproductive health. To investigate the effects of intrauterine growth restriction induced by uterine artery ligation on the postnatal rat testis. Pregnant rats underwent uterine artery ligation at day 19 of gestation. Offspring were killed at 5, 20 and 40 days post-partum (dpp). At killing, one gonad was snap-frozen in liquid nitrogen and processed for RNA and steroid extraction. The other gonad was formalin-fixed for histology. Gene expression was analyzed by TaqMan Low-Density Array. Intratesticular testosterone, estradiol and serum gonadotrophins were measured. Thirty genes were dysregulated in intrauterine growth-restricted rats compared to controls, among which markers of Sertoli cell and Leydig cell function, cell metabolism and growth factors. Testis weights were significantly reduced at 5 and 20 dpp in intrauterine growth-restricted rats and caught-up by 40 dpp. Accordingly, Sertoli cell number was significantly lower in 5 dpp intrauterine growth-restricted rats. At 20 dpp, intratesticular testosterone was significantly increased in intrauterine growth-restricted rats, whereas serum gonadotrophins were unchanged. IUGR altered the gene expression in the rat testes up to peripubertal age and reduced testis size and Sertoli cell number in neonatal age. Multiple mechanisms encompassing genetic changes and steroid production by the testis may be involved in the catch-up growth phase that restored testis size by 40 dpp. Permanent consequences on organ function and gamete integrity cannot be excluded and deserve further investigations.

Background Some children born small for gestational age (SGA) experience supra-physiological insulin-like growth factor-I (IGF-I) concentrations during GH treatment. However, measurements of total IGF-I concentrations may not reflect the bioactive fraction of IGF-I which reaches the IGF-I receptor at target organs. We examined endogenous IGF-bioactivity using an IGF-I kinase receptor activation (KIRA) assay that measures the ability of IGF-I to activate the IGF-IR in vitro. Aim To compare responses of bioactive IGF and total IGF-I concentrations in short GH treated SGA children in the North European Small for Gestational Age Study (NESGAS). Material and method In NESGAS, short SGA children (n = 101, 61 males) received GH at 67 µg/kg/day for 1 year. IGF-I concentrations were measured by Immulite immunoassay and bioactive IGF by in-house KIRA assay. Results Bioactive IGF increased with age in healthy pre-pubertal children (n = 94). SGA children had low-normal bioactive IGF levels at baseline (-0.12 (1.8 SD), increasing significantly after one year of high-dose GH treatment to 1.1 (1.4) SD, P < 0.01. Following high-dose GH, 68% (n = 65) of SGA children had a total IGF-I concentration >2SD (mean IGF-I 2.8 SDS), whereas only 15% (n = 15) had levels of bioactive IGF slightly above normal reference values. At baseline, bioactive IGF (SDS) was significantly correlated to height (SDS) (r = 0.29, P = 0.005), in contrast to IGF-I (SDS) (r = 0.17, P = 0.10). IGF-I (SDS) was inversely correlated to delta height (SDS) after one year of high-dose GH treatment (r = -0.22, P = 0.02). Conclusion In contrast to total IGF-I concentrations, bioactive IGF stayed within the normal reference ranges for most SGA children during the first year of GH treatment.

Aim To assess factors associated with the timing of puberty onset (Tanner Stage Breast 2/Genital 2) among adolescents living in an urban slum in Karachi, Pakistan. Methods Girls enrolled at 8-10 years (n = 1009) and boys 9-11 years (n = 863) were followed every 6 months from 2006 to 2010. Parametric survival analysis for interval-censored data was used to estimate median age at puberty onset and assess the association between exposures and timing of puberty onset. Results The overall median age at Tanner Stage Breast 2 (AAB2) was 10.1 years (95% CI: 10.1; 10.5), and the median age at Tanner Stage Genital 2 (AAG2) was 10.1 years (95% CI: 10.1; 10.6). Stunting delayed AAB2 1 year and AAG2 about 6 months when compared to non-stunted peers. In the multivariable model, after adjusting for age at enrolment, stunting, thinness and vitamin A deficiency (VAD) were significantly associated with delayed AAB2, while stunting and anaemia were significantly associated with delayed AAG2. Conclusion Among adolescents living in the Karachi slum, stunting and highly prevalent anaemia delayed AAG2, while stunting, thinness and VAD delayed AAG2. Parental and household factors were not significantly associated with the timing of puberty onset.

IntroductionAdolescence is a time of significant physical and emotional change, and there is emerging concern that adolescents living in low- and middle-income countries (LMIC) may face substantial challenges in relation to linear growth and mental health. Data on the global burden of stunting after 5 years of age are limited, but estimates suggest up to 50 per cent of all adolescents in some LMIC are stunted. Additionally, many LMIC lack robust mental health care delivery systems. Pakistan has one of the world’s largest populations of adolescents (10 to 19 years) at approximately 40 million. The Nash-wo-Numa study’s primary objective is to assess the prevalence and risk factors for stunting among early adolescents in rural Pakistan. The study also aims to determine the prevalence of poor mental health and identify factors associated with common mental health concerns during the childhood to adulthood transition.MethodsThis cross-sectional study will include girls (n= 738) 9.0 to 14.9 years of age and boys (n=687) 10.0 to 15.9 years of age who live in the rural district of Matiari, Pakistan. Participants will be assessed for anthropometrical measures, puberty development, nutritional biomarkers as well as symptoms of depression, anxiety and trauma using validated scales.Ethics and disseminationThe proposed study aims to complete the picture of child and adolescent health concerning linear growth and mental health by including puberty indicators. Ethics approval has been granted by the Ethics Review Committee at the Aga Khan University, Karachi, Pakistan, #5251-WCH-ERC-18 and Research Ethics Board at SickKids Hospital, Toronto, Canada, #:1000060684. Study results will be presented at relevant conferences and published in peer-reviewed journals.Trial registration numberNCT03647553; Pre-results.

Hypogonadotrophic hypogonadism (HH) is a rare disorder that manifests absent puberty and infertility. Genetic syndromes with hypogonadism, such as Klinefelter syndrome, are associated with an increased risk of neurodevelopmental disorders (NDDs). However, it is not clear whether patients with HH or transient delayed puberty in general, have an increased risk of NDDs. We performed a register-based study on a national cohort of 264 patients with HH and 7447 patients diagnosed with delayed puberty that was matched with 2640 and 74 470 controls, respectively. The outcome was defined as having any of the following NDD diagnoses: (i) autism spectrum disorder (ASD); (ii) attention deficit hyperactivity disorder (ADHD); or (iii) intellectual disability (ID). Additional sensitivity analyses were performed to control for different parental and birth variables, as well as diagnosed malformation syndromes and chromosomal anomalies (ie, Down's and Turner syndromes). Patients with HH had increased risk for being diagnosed with ASD (odds ratio [OR] = 5.7; 95% confidence interval [CI] = 2.6-12.6), ADHD (OR = 3.0; 95% CI = 1.8-5.1) and ID (OR = 18.0; 95% CI = 8.9-36.3) compared to controls. Patients with delayed puberty also had a significantly increased risk of being diagnosed with an NDD. These associations remained significant after adjustments. This is the first study to demonstrate a significant association between HH, delayed puberty and NDDs in a population-based cohort. Clinicians should be aware of the overlap between these disorders. Further studies should explore the mechanisms behind these associations.

Study question Do the organ culture conditions, previously defined for in vitro murine male germ cell differentiation, also result in differentiation of rat spermatogonia into post-meiotic germ cells exhibiting specific markers for haploid germ cells? Summary answer We demonstrated the differentiation of rat spermatogonia into post-meiotic cells in vitro, with emphasis on exhibiting, protein markers described for round spermatids. What is known already Full spermatogenesis in vitro from immature germ cells using an organ culture technique in mice was first reported 5 years ago. However, no studies reporting the differentiation of rat spermatogonia into post-meiotic germ cells exhibiting the characteristic protein expression profile or into functional sperm have been reported. Study design, samples/materials, methods Organ culture of testicular fragments of 5 days postpartum (dpp) neonatal rats was performed for up to 52 days. Evaluation of microscopic morphology, testosterone levels, mRNA and protein expression as measured by RT-qPCR and immunostaining were conducted to monitor germ cell differentiation in vitro. Potential effects of melatonin, Glutamax® medium, retinoic acid and the presence of epidydimal fat tissue on the spermatogenic process were evaluated. A minimum of three biological replicates were performed for all experiments presented in this study. One-way ANOVA, ANOVA on ranks and student's t-test were applied to perform the statistical analysis. Main results and the role of chance Male germ cells, present in testicular tissue pieces grown from 5 dpp rats, exhibited positive protein expression for Acrosin and Crem (cAMP (cyclic adenosine mono phosphate) response element modulator) after 52 days of culture in vitro. Intra-testicular testosterone production could be observed after 3 days of culture, while when epididymal fat tissue was added, spontaneous contractility of cultured seminiferous tubules could be observed after 21 days. However, no supportive effect of the supplementation with any factor or the co-culturing with epididymal fat tissue on germ cell differentiation in vitro or testosterone production was observed. Limitations, reasons for caution The human testis is very different in physiology from the rat testis, further investigations are still needed to optimize the organ culture system for future use in humans. Wider implications of the findings The successful differentiation of undifferentiated spermatogonia using the testis explant culture system might be employed in future to produce sperm from human spermatogonia as a clinical tool for fertility preservation in boys and men suffering infertility. Large scale data None. Study funding and competing interests This work was supported financially by the Frimurare Barnhuset in Stockholm, the Paediatric Research Foundation, Jeanssons Foundation, Sallskapet Barnavard in Stockholm, Swedish Research Council/Academy of Finland, Emil and Wera Cornells Foundation, Samariten Foundation, the Swedish Childhood Cancer Foundation as well as through the regional agreement on medical training and clinical research (ALF) between Stockholm County Council and Karolinska Institutet. All authors declare no conflicts of interests.

Obesity has recently been linked with reduced fertility, and the mechanisms underpinning this effect are currently unknown. The adipokine leptin is dysregulated in obesity and affects reproductive tracts; therefore, we investigated the dose-dependent effects of leptin on Leydig cell function and spermatogenesis. Eight-week-old leptin-deficient obese (ob/ob) male mice were treated with subphysiological (0.1- or 0.5-mg/kg body weight [BW]/d) or physiological (3.0-mg/kg BW/d) doses of leptin or saline for 12 weeks (chronic treatment) or 72 hours (acute treatment). We then evaluated male reproductive function markers. Mean testis weight increased significantly in the 0.1- and 3.0-mg/kg BW/d groups compared with saline controls (both P < .05). Intratesticular testosterone levels relative to testis weight significantly increased in the 0.5-mg/kg BW/d group compared with saline controls (P < .05). FSH levels increased in a dose-dependent manner with leptin treatment, whereas LH levels did not change. Leptin treatment significantly up-regulated both mRNA and protein expression of the steroidogenic enzyme cytochrome P450 17A1. Spermatogenesis improved in leptin-treated animals. Significantly more seminiferous tubules were observed in stages I-VIII (P < .01), and there were fewer abnormal seminiferous tubule structures (P < .01). Acute treatment with physiological leptin doses partially improved male reproductive markers without changing BW. Administration of subphysiological to physiological doses of leptin improves Leydig cell function and spermatogenesis.

BACKGROUND: Genetic susceptibility to insulin resistance is associated with lower adiposity in adults. Insulin resistance, and therefore adiposity, may alter sensitivity to GH. We aimed to determine the relationship between adiposity, genetic susceptibility to insulin resistance or insulin secretion, and response to GH treatment in short children born small for gestational age (SGA). METHODS: In 89 short prepubertal SGA children (age, 6.2 ± 1.6 y; 55 boys) treated with GH for 1 year in a multicenter study, body fat percentage was estimated at baseline and 1 year using dual-energy x-ray absorptiometry. The main outcome measures were treatment-related changes in height, IGF-1 standard deviation score, insulin sensitivity, insulin secretion, and disposition index. Combined multiallele gene scores based on single nucleotide polymorphisms with known associations with lower insulin sensitivity (gene scores for insulin resistance [GS-InRes]) and insulin secretion (gene scores for insulin secretion [GS-InSec]) were analyzed for their relationships with adiposity. RESULTS: Mean percentage body fat at baseline was low compared to normative data (P = .045) and decreased even further on GH treatment (baseline vs 1-year z-scores, -0.26 ± 1.2 vs -1.23 ± 1.54; P < .0001). Baseline percentage body fat was positively associated with IGF-1 responses (p-trends = .042), first-year height gains (B [95% confidence interval], 0.61 cm/y [0.28,0.95]; P < .0001), insulin secretion at baseline (p-trends = .020) and 1 year (p-trends = .004), and disposition index at 1 year (p-trends = .024). GS-InRes was inversely associated with body mass index (-0.13 SD score per allele [-0.26, -0.01]; P = .040), body fat (-0.49% per allele [-0.97, -0.007]; P = .047), and limb fat (-0.81% per allele [-1.62, 0.00]; P = .049) at baseline. During GH treatment, GS-InRes was related to a lesser decline in trunk fat (0.38% per allele [0.16, 0.59]; P = .001) and a higher trunk-limb fat ratio at 1 year (0.04 per allele [0.01, 0.08]; P = .008). GS-InSec was positively associated with truncal fat (0.36% per allele [0.09, 0.63]; P = .009). CONCLUSIONS: Adiposity in SGA children has favorable effects on GH sensitivity and glucose metabolism. The associations with multiallele scores support a causal role of insulin resistance in linking lesser body fat to reduced sensitivity to exogenous GH.

In the past, explant tissue-culture methodologies have been used to grow gonads and study their development. Results from in vitro cultures of human gonads showed limited progress toward gonadal cell differentiation and were focused mainly on germ-cell differentiation. Thus, detailed studies focusing on human first-trimester gonadal tissue functionality in vitro are still missing. In this study we investigated the endocrine function of human first-trimester gonads in vitro. We included 27 female and 28 male gonadal samples, derived from a total of 55 cases, at postconceptional ages of 4.5 to 10.5 weeks. Tissues were cultured using an explant tissue-culture system for 14 days. Assays for testosterone (liquid chromatography-tandem mass spectrometry), anti-Mullerian hormone (AMH; ELISA), and inhibin B (ELISA) were performed using media collected after 7 and 14 days of culture. We demonstrated sex- and age-dependent secretion profiles of testosterone, AMH, and inhibin B in the culture media, which resemble the pattern of hormone production in human gonads in vivo, from the few available studies at the same age range. Our study shows that explant tissue-culture conditions are robust for culture of human first-trimester gonadal somatic cells. Thus, it can be used to study human gonadal development and related diseases as well as the effect of potentially hormone-disturbing substances in human gonads during development. However, detailed molecular studies are needed for better understanding of the mechanistic control of the endocrine function of human first-trimester gonads.

Chronic undernutrition is characterized by long-term exposure to food of insufficient quality and inadequate quantity, including restricted intake of energy, protein, fat, micronutrients, essential amino acids, vitamins and minerals. Physiologically, in a state of chronic food insufficiency, the human body conserves energy by prioritizing essential metabolic processes resulting in impaired linear growth and delayed reproductive maturation. Consequently, height can theoretically be considered a measure of an individual’s cumulative health and nutrition. Therefore, a deviation from the ‘normal’ height relative to one’s age represents a deviation from one’s optimal growth and, potentially, the presence of other issues. Similarly, the delayed onset of puberty is another common physiological response to food insufficiency, often accompanying impaired linear growth. Chronic undernutrition can arise from chronic disease, congenital abnormalities and insufficient food intake. In this review, we will explore the hypothesis of CUG during adolescence, given the relationship between impaired linear growth and the delayed onset of puberty in children suffering from chronic undernutrition due to a lack of sufficient quality and quantity of food.

Masculinization of the external genitalia in humans is dependent on formation of 5α-dihydrotestosterone (DHT) through both the canonical androgenic pathway and an alternative (backdoor) pathway. The fetal testes are essential for canonical androgen production but little is known about the synthesis of backdoor androgens despite their known critical role in masculinization. In this study, we have measured plasma and tissue levels of endogenous steroids in second trimester human male fetuses using multi-dimensional and high-resolution mass-spectrometry. Results show that androsterone is the principal backdoor androgen in the fetal circulation and that DHT is undetectable (

Chronic diseases are known to cause premature aging and frailty. Data about telomere length and telomere length-regulating proteins after pediatric KTx are scarce. Leukocyte telomere length and gene expression level of eight telomere-binding proteins were analyzed in 20 KTx recipients, eight childhood NBL survivors, and nine healthy controls. The influence of key clinical parameters on telomere length and on regulators of telomere length was evaluated. The telomere length in the KTx recipients tended to be shorter (0.53 AU) than in the healthy controls (0.64 AU) but longer than in the NBL survivors (0.38 AU). There was no significant difference in telomere length between the NBL survivors and the KTx recipients (P = .110). The gene expression level of telomere length-preserving protein RPA1 was significantly higher in the KTx recipients than among the NBL survivors or healthy controls, while the expression of TRF2 and the tumor suppressor gene p16 was significantly higher in the KTX recipients when compared to the controls. TRF2 and TIN2 correlated significantly with hsCRP; additionally, TRF2 showed significant correlation with plasma creatinine and eGFR. KTx recipients have near to normal telomere length, but they have significantly higher gene expression levels of telomere regulatory proteins compared with healthy controls, suggesting activation of mechanisms preserving telomere length among KTx recipients. Our results suggest that declined graft function and consequent inflammatory response may have influence on telomerase activity.

Obesity in females is often associated with metabolic complications and hyperandrogenism, but the sources of androgens are not completely understood. Therefore, this study investigated whether adipose tissue could be a source of androgens promoting hyperandrogenism development in obese female rats.Gene expression of steroidogenic enzymes and testosterone levels were determined in periovarian and inguinal adipose tissue and in the supernatant of cultured preadipocytes and adipocytes. The conversion of pregnenolone to androgens was analyzed by thin-layer chromatography.Substantial amounts of testosterone in adipose tissue (25-153 ng/g tissue) and in the supernatant of adipocytes (0.33-0.69 ng/ten thousand cells]) were found. StAR and steroidogenic enzymes encoded by genes including Cyp11A1, Cyp17A1, Cyp19, Hsd3b2, Hsd17b3, and Srd5a2 were expressed in adipose tissue and cultured cells. Thin layer chromatography data revealed that preadipocytes and adipocytes were able to convert pregnenolone to testosterone. Higher levels for all steroidogenic enzymes were found in both depots of obese animals compared with lean animals, with significantly higher levels in inguinal tissue.The whole steroidogenic machinery and capacity for testosterone biosynthesis were found in fat depots of female rats. These findings support the hypothesis that adipose tissue may contribute substantially to the hyperandrogenism in female obesity.

Human embryonic stem (hES) cells represent an important tool to study early cell development. The previously described use of human recombinant laminin (LN) 521 represented a step forward in generating clinically safe culture conditions. To test the short-term effect of LN521 on cultured hES cells, five male hES cell lines were cultured on human foreskin fibroblasts (hFFs), Matrigel, LN521, and LN121 and characterized by qPCR, immunofluorescence analysis, as well as their potential for three-germ layer differentiation. Variations in gene expression related to pluripotency, stemness, and testicular cells at different passages and culture conditions were evaluated by qPCR. All cell lines expressed pluripotency markers at protein and RNA level and were able to differentiate into cell types of the three germ layers after being cultured on LN521 for nine passages. Reduction in variation of pluripotency marker expression could be observed after culturing the cells on LN521 for nine passages. hES cells cultured on LN521 exhibited less differentiation, faster cell growth, and attachment when compared to hES cells cultured on LN121 or Matrigel. Our results indicate a positive effect of LN521 in stabilizing pluripotency gene expression and might be the first step towards more controllable and robust culture conditions for hES cells.

Numerous studies have reported on testicular toxicity of phthalates in different experimental paradigms and showed that Leydig cells (LCs) were one of the main targets of phthalate actions. Adverse effects of phthalates on LCs steroidogenesis have been attributed to their metabolites, monophthalates. This study focuses on investigation whether LCs responsiveness to monophthalates action is associated with their potential to produce androgens. We found that of 3 monophthalates investigated [ie, mono-2-ethylhexyl phthalate (MEHP), mono-n-butyl phthalate, and mono-n-benzyl phthalate] only MEHP caused biological effects on the mouse LCs function. This monophthalate stimulated basal steroidogenesis associated with upregulation of StAR protein expression with no effect on hCG-stimulated androgen production by LCs from CBA/Lac and C57BL/6j mouse genotypes were observed. Further, MEHP attenuated ATP production and increased superoxide generation by both phenotypes of mouse LCs that indicated on mitochondrial dysfunction induced by the monophthalate. All together, our data indicate that MEHP-mediated stimulation of steroidogenesis and perturbation in mitochondrial function are not associated with the capacity of the LCs to synthesize androgens. We suggest that this effect of MEHP observed in LCs of rodent origin needs to be taken into consideration in analysis of earlier start of puberty in boys and may highlight a possible influence of phthalates on reproductive health in males.

Children are not just small adults. A number of anatomical and physiological features are different in children than in adults and biology at a young age differs from that of later ages. This has impo

Convergent evidence associates endocrine disrupting chemicals (EDCs) with major, increasingly-prevalent human disorders. Regulation requires elucidation of EDC-triggered molecular events causally linked to adverse health outcomes, but two factors limit their identification. First, experiments frequently use individual chemicals, whereas real life entails simultaneous exposure to multiple EDCs. Second, population-based and experimental studies are seldom integrated. This drawback was exacerbated until recently by lack of physiopathologically meaningful human experimental systems that link epidemiological data with results from model organisms.We developed a novel approach, integrating epidemiological with experimental evidence. Starting from 1,874 mother-child pairs we identified mixtures of chemicals, measured during early pregnancy, associated with language delay or low-birth weight in offspring. These mixtures were then tested on multiple complementary in vitro and in vivo models. We demonstrate that each EDC mixture, at levels found in pregnant women, disrupts hormone-regulated and disease-relevant gene regulatory networks at both the cellular and organismal scale.

Objective The effect of a common polymorphism in the Growth Hormone (GH) receptor (d3-GHR) gene on growth, metabolism and body composition was examined in short children born small for gestational age (SGA) on GH treatment. Design In 96 prepubertal, short SGA children treated with high-dose GH (67 μg/kg/day) in the NESGAS study, insulin sensitivity (IS), insulin secretion and disposition index (DI) were determined during the first year of treatment. Body composition was analysed by DXA. The d3-GHR locus was determined by simple multiplex PCR. Results At baseline, children in the d3-GHR group (d3/fl (n = 37), d3/d3 (n = 7)) had significantly lower IS (median (25–75 percentile)) (223.3% (154.4–304.8)) vs. (269.7% (185.1–356.7)) (p = 0.03) and higher concentrations of glucose (mean (SD)) (4.4 mmol/L (0.6) vs. 4.2 mmol/L (0.7)) (p = 0.03), C-peptide (232.1 pmol/L (168.8–304.1) vs. 185.1 pmol/L (137.7–253.9)) (p = 0.04) and insulin (19.2 pmol/L (11.8–32.2)) vs. (13.7 pmol/L (9.3–20.8)) (p = 0.04) compared to children homozygous for the full length allele (fl/fl-GHR (n = 52)). There were no differences in DI or insulin secretion. Postnatal, spontaneous growth was significantly greater in the d3-GHR group compared to the fl/fl-GHR group (p = 0.02). There were no significant differences in growth response, body composition or metabolism after one year of GH therapy. Conclusion Short SGA children carrying the d3-GHR polymorphism had increased spontaneous growth, lower IS and a compensatory increase in glucose, C-peptide and insulin before GH therapy compared to children homozygous for the full-length allele.

Dramatic increase of gender dysphoria in youth In the past decade there has been a dramatic increase in the number of young people with gender dysphoria seeking help for gender-confirming medical interventions. From a situation of no more than a few patients annually, there were almost 200 referrals of gender dysphoria to the Astrid Lindgren Children's Hospital in 2016. This child and adolescent psychiatric unit has the whole country as a catchment area for patients16 years. Gender-confirming medical interventions are regulated by a special law that sets a minimum age for legal and surgical gender reassignment to 18 years. The law, which is under revision, does not prevent medical investigations, hormonal therapy, and some surgical procedures before the age of 18. Gender dysphoria is defined as a persistent desire to live and be accepted as the opposite sex, usually accompanied by a perceived inconsistency with the sex assigned at birth and a desire to change the body in accordance with the perceived sex. The cause is unknown despite attempts of etiological mapping including genetic analyses, hormonal studies and modern brain imaging techniques. Repeated studies have shown that only a minority (about 20 %) of prepubertal children with gender dysphoria will have a persistent desire for later gender-confirming medical interventions, while the majority of those whose gender dysphoria is reinforced during puberty will later meet the diagnostic criteria for gender dysphoria (DSM-5) and transsexualism (ICD-10) (so called persisters). Persisters can be offered treatment with puberty stopping hormones to avoid the development of undesirable secondary sexual characteristics. Gender-confirming medical interventions are the only recommended treatment for gender dysphoria, and early treatment facilitates the ability to successfully pass in the desired sex, which is associated with a significantly better prognosis.