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Insulin-like factor 3 (INSL3) is an emerging testicular marker, yet larger studies elucidating the clinical role of INSL3 in patients with hypogonadism are lacking. The aim was to describe serum INSL3 concentrations analyzed by LC–MS/MS methodology in males with hypogonadotropic hypogonadism (HH) and Klinefelter syndrome (KS). This was a combined study from two tertiary centers in Denmark and France analyzing INSL3 concentrations by LC–MS/MS. In total, 103 patients with HH and 82 patients with KS were grouped into treated (HH: n = 96; KS: n = 71) or untreated (HH: n = 7; KS: n = 11). Treatment modalities included testosterone and hCG. Serum concentrations and standard deviation (SD) scores of INSL3, total testosterone, and LH according to age and treatment were evaluated. In both HH and KS, INSL3 concentrations were low. In HH, INSL3 was low regardless of treatment, except for some hCG-treated patients with normal concentrations. In untreated HH, testosterone was low, while normal to high in most testosterone- and hCG-treated patients. In untreated KS, INSL3 and testosterone concentrations were low to normal, while in testosterone-treated KS, serum INSL3 was low in most patients. INSL3 SD scores were significantly lower in untreated HH than in untreated KS (p = 0.01). The dichotomy between lower INSL3 and higher testosterone concentrations, particularly observed in hCG-treated patients with HH, confirms that INSL3 is a different marker of Leydig cell function than testosterone. However, the clinical application of INSL3 in males with hypogonadism remains unclear.

To investigate the psychiatric healthcare utilisation of refugees vis-a-vis their peers in Denmark during the ages 15–22. This paper utilises comprehensive full-population registry data from 1995 to 2016 to explore the psychiatric healthcare utilisation during the transition from childhood to adulthood for refugees (N = 13,027), a comparison group of children of labour migrants from Morocco, Pakistan, and Turkey (N = 13,413), and the majority population (N = 693,043) in Denmark. To test for population differences in types of admission for particular types of disorders, odds ratios for a first contact during ages 15–22 were calculated using logistic regression. For those with at least one diagnosis-specific hospital contact, differences in the amount and type of treatment were tested using negative binomial regression to estimate means ratios of days hospitalised, days in outpatient care, number of outpatient contacts, consultations with psychiatrists in private practice, and prescribed medicine purchases. Refugees and the comparison group were generally less likely than the majority population to have a first contact for most disorders (adjusted ORs 0.03–0.88), but not for schizophrenia for boys (adjusted ORs 0.92–2.13). Among those who did have a first contact, youths from the ethnic minority groups tended to have more or similar inpatient and emergency room contacts (MRs 0.89–2.10), hospitalisations of refugee girls being an exception (MR 0.46; CI [0.23–0.94]), but fewer outpatient contacts, consultations with psychiatrists in private practice, and prescribed medicine purchases (MRs 0.23–0.94). The results suggest that refugee and other ethnic minority groups may face barriers both to initial contact and to completing adequate treatment beyond the first contact.

BackgroundQuarterly intramuscular injections with long-acting testosterone undecanoate (TU) provide stable serum testosterone concentrations over time and are therefore preferred by many testosterone-deficient patients. However, the use of long-acting TU in elderly patients is limited due to lack of safety and feasibility studies.ObjectiveTo investigate long-acting TU pharmacokinetics and assess differences in treatment regimens and risk of adverse outcomes in younger versus elderly testosterone-deficient patients.Materials and methodsSingle-center longitudinal observational study. Patients who initiated long-acting TU treatment between 2005 and 2010 were included. Elderly patients were born before 1956 and younger patients between 1965 and 1985. TU dose was adjusted yearly through shortening or prolongation of time between injections. Treatment targets were as follows: (1) free testosterone between 0 and −1 SD from the age-adjusted mean, (2) no symptoms of testosterone deficiency, and (3) hematocrit within the normal range.ResultsThe study population consisted of 63 elderly and 63 younger patients. Median follow-up time during testosterone replacement was 12.1 years. Increasing intervals between TU injections were performed 44% more often in the elderly compared to younger patients and time between TU injections were prolonged 4% more in the elderly patients. The hematocrit, as well as the hematocrit for a given serum testosterone (hematocrit: testosterone ratio), increased with treatment time but did not differ between age groups. During follow-up, 40% of patients—both elderly and younger—experienced polycythemia. Risk of polycythemia did not differ with age.Discussion and conclusionAn increased number of adjustments of TU dose are necessary in elderly patients in order to reach treatment targets. TU treatment in elderly testosterone-deficient patients is not associated with an increased risk of polycythemia compared to younger patients if age-adjusted treatment targets are reached.

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

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.

Background Nephropathy is common in Fabry disease (FD). Prior studies of renal function during enzyme replacement therapy (ERT) have primarily used estimated glomerular filtration rate (eGFR). We studied the attrition of renal function in FD by measured GFR (mGFR) and urine protein excretion, and explored the influence of age. Methods This was a long-term observational study of a nationwide, family-screened cohort of FD patients. All Danish genetically verified FD patients on ERT, without end-stage renal disease at baseline and with three or more mGFR values were included. Results In all, 52 patients with consecutive mGFR values (n = 841) over median 7 years (range 1–13) were evaluated. Blood pressure remained normal and urine protein excretion was unchanged. Plasma globotriaosylceramide (Gb-3) levels normalized while plasma lyso-Gb-3 remained abnormal in 34% of patients. Baseline mGFR was 90 ± 3 mL/min/1.73 m2 and rate of renal function loss 0.9 ± 0.2 mL/min/1.73 m2/year. Baseline eGFR was 97 ± 5 mL/min/1.73 m2 and rate of renal function loss 0.8 ± 0.3 mL/min/1.73 m2/year. mGFR was age- adjusted to renal healthy non-FD subjects, giving a standard deviation score of −0.8 ± 0.2 with an annual slope of −0.03 ± 0.01 (P = 0.099), without differences between genders. Age grouping of age-adjusted data showed exaggerated renal function loss with age. Urine albumin–creatinine ratio (UACR) >300 mg/g was associated with faster renal function loss, independent of baseline mGFR, age and gender. Conclusions ERT-treated FD patients did not have a faster attrition of renal function than renal healthy non-FD subjects (background population). The rate of renal function loss with age was independent of gender and predicted by high UACR. We suggest cautious interpretation of non-age-adjusted FD renal data.

Background: The filaggrin gene (FLG) encodes an epidermal protein, filaggrin, which is important for normal skin barrier functions. We previously showed that FLG loss-of-function mutation carriers have a higher internal exposure to some non-persistent chemicals such as certain phthalates and parabens, suggesting increased trans-epidermal penetration. Several groups of non-persistent chemicals are suspected endocrine disrupters with potential to affect testicular function. Objectives: To investigate associations between exposure to non-persistent chemicals and testicular function in young Danish men with and without FLG mutations. Methods: We measured urinary concentrations of bisphenol A (BPA) and other simple phenols, parabens, and UV filters including benzophenones (BP-1, BP-3 and 4-HBP) in men genotyped for FLG R501X, 2282del4, and R2447X loss-of-function mutations; in total 65 mutation carriers and 130 non-carriers (controls) were included. Outcomes were markers of testicular function, assessed by serum reproductive hormones and semen quality. Results: We found that associations between urinary chemical concentrations and outcomes were different in cases and controls. Within the group of FLG mutation carriers, higher urinary concentrations of BPA, BP-1 and BP-3 were associated with higher testosterone and estradiol serum levels and lower FSH. Similar trends in hormone levels were observed for FLG mutation carriers with measurable levels of 4-HBP compared to those who had no detectable levels of urinary 4-HBP. Furthermore, those in the highest urinary BPA quartile had lower sperm motility than those in the lower quartiles. None of these associations were evident in the control group. In the control group, however, lower sperm motility and sperm concentration were observed in the men with detectable urinary 4-HBP compared to the men non-detectable urinary 4-HBP. We found no association between any parabens and outcomes, nor for the other measured phenols or UV filters. Conclusions: Associations between male reproductive health parameters and urinary levels of BPA and benzophenones such as BP-3, BP-1 and 4-HBP were observed in FLG mutation carriers but not in controls from the same study population. This difference between FLG mutation carriers and non-carriers is not explained solely by differences in exposure levels of the examined compounds as e.g. BPA and 4-HBP urinary levels did not differ between the two groups. We hypothesise that effects of exposure to these compounds may be modulated in FLG mutation carriers by either different levels of co-exposures or by route of uptake, with a higher fraction of the uptake by dermal uptake. Keywords: Endocrine disrupting chemicals, Bisphenol A, Benzophenone-3, Testicular function, Filaggrin gene

Purpose To examine whether family separation caused by prolonged waiting for family reunification is associated with the risk of mental disorders among refugee fathers. Method Based on full-population Danish registry data covering 1995–2015, we mapped arrival patterns among nuclear refugee family members resettled in Denmark (n = 76,776) and established a cohort of refugee fathers (n = 6176) who all arrived alone and later obtained family reunification with their wife and children. The fathers were followed for up to 24 years, from the day their residence permit was issued until their first psychiatric diagnosis, emigration, death, or study end, whichever came first. Using Cox proportional hazard regression, we estimated hazard ratios (HRs) of being diagnosed with a mental disorder (i) for the period while the fathers were still separated from their family and (ii) across varying lengths of family separation. Results The HR of any mental disorder was 2.10 (95% confidence interval (CI): 1.57–2.81) for fathers still separated from their family compared with those who had obtained family reunification. The HR increased with longer family separation. Compared with fathers separated for Conclusion Fathers waiting for their wives and children face an increased risk of mental disorders. Countries receiving refugees should be aware that delaying family reunification can lead to adverse mental health effects.

This study aimed at examining psychiatric morbidity in the perinatal period among refugees and family-reunified immigrants compared to Danish-born women, including predictors of psychiatric morbidity according to migration history. Inclusion criteria were women who had a residence permit in Denmark and gave birth to a live child between 1 April 1998 and 31 December 2014. The study included 7804 refugee women, 21,257 family-reunified women, and 245,865 Danish-born women. We estimated Odds Ratios (ORs) of having a first-time perinatal psychiatric episode (PPE) and specific risk for affective, psychotic, and neurotic disorders. Compared with Danish-born women, women family-reunified with immigrants had lower (aOR 0.37, 95% CI 0.22–0.64) and refugees had higher ORs of PPE (OR 1.46, 95% CI 1.22–1.76). In fully adjusted models, refugees no longer presented increased risk of PPE (OR 1.16, 95% CI 0.95–1.42) but showed higher ORs for psychotic (aOR 4.72, 95% CI 2.18–9.84) and neurotic disorders (aOR 1.31, 95% CI 1.01–1.72). Women family-reunified with refugees and to Nordic citizens had higher ORs of psychotic disorders. Among migrants, refugees had higher ORs of PPE. Results suggest that elevation in risk of PPE among refugees compared to Danish-born may be related to higher likelihood of poverty and single-parenting among refugees. Still, refugees appear to have increased risk for neurotic and psychotic disorders. In contrast, family-reunified to immigrants may have lower risk of PPE. Maternal health programs need to focus on promotion of mental health and tackle social risks that disproportionately affect immigrant women, particularly refugees.

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.

Background Refugees resettled in Western countries experience long periods of family separation due to waiting first for asylum, and then for family reunification. This study aimed to examine if family separation is associated with psychiatric disease among refugee fathers. Methods We traversed full-population Danish administrative data since 1986 to establish a cohort of refugee fathers from nuclear families in which the father was the first to arrive and resettled alone during 1995-2015, and where the mother and children were later family reunified. We followed these fathers for up to 24 years from the day their residence permit was issued until their first psychiatric diagnosis, emigration, death, or study end, whichever came first. Using Cox proportional hazard regression, we estimated the temporary and longer-term risks of psychiatric disorders for i) the total family separation period, and ii) the family separation decomposed into time waiting for, respectively, asylum and family reunification. Results 6176 fathers were included in the study. The HR assessing the temporary risk of psychiatric disorders was 2.23[95% CI: 1.72-2.89] for separated fathers compared with those who were already family reunified. As to the longer-term risk, compared with fathers separated for 0-9 months, the HR of psychiatric disorders was 1.54[1.08-2.21] after +9-12 months of separation, increasing to 1.84[1.29-2.62] after +24 months of separation. The decomposition analysis revealed that +6 months of waiting increased the risk of any psychiatric disorder by approximately 40% for both time waiting for asylum and time waiting for family reunification. Conclusions Strong evidence supports that fathers waiting for their wives and children face an increased risk of psychiatric disease. Countries receiving refugees should be aware that delayed family reunification can lead to adverse mental health effects. Key messages Family separation is associated with an increased risk of psychiatric disease among refugee fathers—both while the fathers are separated from their families and after family reunification. The risk of psychiatric disease is more than double among refugee fathers still separated from their family compared with those who have been family reunified.

Background Spermatogenesis depends on stimulation by follicle-stimulating hormone (FSH) which binds to FSH receptors (FSHR) on testicular Sertoli cells. Three FSH-related single nucleotide polymorphisms (SNPs); FSHB -211G>T (rs10835638), FSHR -29G>A (rs1394205) and FSHR 2039A>G (rs6166) affect FSH action, and have been suggested to affect testicular function, but the evidence is uncertain. Objective To describe the associations between the three SNPs and testicular function in a large and well-characterised cohort of men from the general population. Materials and methods A cross-sectional study of 2,020 Danish men unselected regarding testicular function. Outcome variables were semen parameters, reproductive hormones and testis size. Genotyping was done by competitive allele-specific quantitative PCR. Differences in genotype frequencies were tested by Chi-square test and associations between genotypes and outcomes were assessed by multivariate linear regressions. Results The SNPs affected serum FSH; carriers of the variant affecting FSH secretion (FSHB -211G>T) had lower FSH levels while carriers of variants affecting receptor expression (FSHR -29G>A) and receptor sensitivity (FSHR 2039A>G) had higher FSH levels. Carriers of FSHB -211G>T had lower calculated free-Testosterone/LH ratio. Although both FSHB -211G>T and FSHR 2039A>G were associated with smaller testis size, no clear association was detected in relation to any semen parameters, except a lower total number of morphologically normal spermatozoa in the heterozygous carriers of the FSHB -211G>T DISCUSSION AND CONCLUSION: The studied polymorphisms have only minor modulating influence on testis size and function in healthy men. We detected subtle effects of the three SNPs on FSH levels, but also effects of FSHB -211G>T on calculated free-Testosterone/LH ratio, compatible with altered Leydig cell function. Thus, the role of these FSH-related polymorphisms is complex and modest in men with normal testicular function, but the possible importance of FSH polymorphisms in men with impaired testicular function should be evaluated in future studies in more detail.

Context The lack of sensitive and robust analytical methods has hindered the reliable quantification of estrogen metabolites in subjects with low concentrations. Objective To establish sex-specific reference ranges for estrone (E1) and estradiol (E2) throughout life and to evaluate sex-differences using the state-of-the-art liquid chromatography tandem mass spectrometry (LC-MS/MS) method for quantification of E1, E2, and estriol (E3). Design LC-MS/MS method development and construction of estrogen reference ranges. Settings Population-based cross-sectional cohorts from the greater Copenhagen and Aarhus areas. Participants Healthy participants aged 3 months to 61 years (n = 1838). Results An isotope diluted LC-MS/MS method was developed and validated for measurements of serum E1, E2, and E3. Limits of detections (LODs) were 3 pmol/L (E1), 4 pmol/L (E2), and 12 pmol/L (E3), respectively. This sensitive method made it possible to differentiate between male and female concentration levels of E1 and E2 in children. In girls, E2 levels ranged from Conclusion Reference ranges of simultaneous quantification of E1 and E2 by this novel specific and highly sensitive LC-MS/MS method provide an invaluable tool in clinical practice and in future research studies.

Background The peptide hormone insulin-like factor 3 (INSL3) is a marker for Leydig cell function and the clinical use of serum INSL3 measurements has been suggested by several groups. Aim (1) To establish a reference range for liquid chromatography–tandem mass spectrometry (LC-MS/MS) of serum INSL3 in healthy boys and men; and (2) to compare the associations of serum INSL3 and testosterone (T) to pubertal stage, lifestyle factors, diurnal variation, body composition, and human chorionic gonadotropin (hCG) stimulation. Results In a reference range based on LC-MS/MS analysis of serum from 1073 boys and men, INSL3 increased from levels close to the detection limit (0.03 µg/L) in prepubertal boys to a maximum mean level of 1.3 µg/L (95% CI, 0.9-2.7) in young men (19-40 years of age) and decreased slightly in older men (0.1 µg/L per decade). Serum T, but not INSL3, was associated with body mass index or body fat percentage and with alcohol consumption. Smoking was positively associated with serum T, but negatively associated with INSL3. There were significant diurnal variations in both INSL3 and T in men (P Conclusion Measurement of serum INSL3 by LC-MS/MS has promise as a marker of testicular disorders.

Background Fabry disease (FD) is a lysosomal storage disorder resulting in systemic accumulation of globotriaosylceramide resulting in multi-organ dysfunction e.g. cerebral, cardiac, renal and audiologic complications. The audiologic involvement in FD has often been neglected; while not a lethal aspect of the disease, hearing loss can have a significantly negative impact on quality of life. Objectives To investigate baseline hearing status of the Danish Fabry cohort prior to treatment, compared to sex- and age-expected hearing levels and correlating hearing to renal and cerebral findings. Material and methods Retrospective study of baseline hearing status of the Danish Fabry cohort (n = 83, 9–72 years). Air conduction and speech discrimination scores were assessed at 6 frequencies between 0.25–8 kHz bilaterally. Data were collected between 2001–2014 and compiled in STATA using multilinear mixed modelling for statistical evaluation. Results Hearing thresholds at all frequencies deviated from the expected thresholds of an otologically normal cohort (p

Background: The prevalence of multimorbidity, defined by having two or more chronic diseases, is increasing in many Western countries. Simultaneously, the migrant population in Western countries has increased, making up a growing proportion of European populations. This study aims i) to determine the quantity and quality of multimorbidity patterns among refugees and family reunification immigrants from non-Western countries compared to Danish-born, and ii) to compare the mortality burden among those with multimorbidity in the two groups. Methods: Through the Danish Immigration Service, we conducted a historically prospective cohort study. We identified a total of 101,894 adult migrants who were sub-categorised into refugees and family reunification immigrants, and matched them to a Danish-born comparison group 1:6 on age and sex. Through the Danish National Patient Registry, we obtained information on all in- and outpatient data on hospitalised and ambulatory patients. To assess multimorbidity we used Charlson Comorbidity Index based on ICD-10 codes, together with ICD-10 diagnostic categories for psychiatric disease. We used Cox regression analysis to calculate risk of multimorbidity and risk of mortality in people with multimorbidity. Results: Overall refugees had higher risk of multimorbidity compared to Danish-born, while family reunification immigrants had a lower risk. When adjusting for civil status and mean income, the risk was lower for all migrant groups compared to Danish-born. Risk of mortality in people with multimorbidity, was lower for all migrant groups, compared to Danish-born. Conclusion: Refugees are an at-risk group for multimorbidity, however, mortality among those with multimorbidity is lower in all migrant groups compared to Danish-born.

Context: The early activation of the hypothalamic-pituitary-gonadal axis during infancy can be used in the evaluation of infants suspected of disorders of sex development (DSD). However, few data exist on sex-specific reference ranges for these hormones during early life. Objective: To evaluate sex differences in reproductive hormone concentrations in serum from healthy infants to define sex-specific cutoff values and to apply these in infants with DSD. Design: A cross-sectional study. Setting: A tertiary center for pediatric endocrinology at the University Hospital of Copenhagen. Patients or Other Participants: Healthy infants (1840) and patients with DSD (27), aged 2 to 5 months. Main Outcome Measures: Serum concentrations of LH, FSH, testosterone (T), estradiol, sex hormone-binding globulin (SHBG), inhibin B, anti-Mullerian hormone (AMH), dehydroepiandrosterone (DHEA), DHEA sulfate (DHEAS), 17-hydroxyprogesterone (17-OHP), androstenedione, and LH/FSH ratio. Results: LH and FSH concentrations showed overlap between sexes, with LH being highest in boys and FSH being highest in girls. The LH/FSH ratio separated infant boys from girls with minimal overlap at a cutoff value of 0.32. Inhibin B and AMH concentrations were markedly higher in boys compared with girls, with minimal or no overlap. In infants with Klinefelter syndrome, 45,X/46,XY mosaicism and male phenotype, and Turner syndrome, the LH/FSH ratio matched the gender of rearing. However, infants with complete androgen insensitivity syndrome had LH/FSH ratios within the male range. Conclusions: Reference ranges for reproductive hormones and LH/FSH ratio during mini-puberty were established in this study. The classifiers that best separated sex in mini-puberty were AMH, LH/FSH ratio, and T. Use of the LH/FSH ratio may add valuable information in the workup of infants suspected of DSD. (J Clin Endocrinol Metab 103: 3028-3037, 2018)

** From Crossref via Jisc Publications Router. ** Licence for VoR version of this article starting on 18-05-2018: https://academic.oup.com/journals/pages/about_us/legal/notices Published in: Nephrology Dialysis Transplantation, Volume 33, Issue Supplement 1 (55th ERA-EDTA Congress, Copenhagen, Denmark, May 24th - 27th 2018, NDT Abstract Supplement 2018) Document number: SP400. DOI: https://doi.org/10.1093/ndt/gfy104.SP400

BACKGROUND: Anogenital distance (AGD) has been suggested to represent a phenotypic signature reflecting in utero androgen action. However, it is not known whether an individual's AGD at birth correlates to AGD later in life. We investigate correlations of AGD between 3 and 18 months and asses reproducibility of measurements.METHODS: We measured AGD from anus to scrotum (AGDas) and penis (AGDap) in 407 boys, and to posterior fourchette (AGDaf) and clitoris (AGDac) in 282 girls. Each measure was repeated three times at 3 and 18 months and some children were furthermore examined by two different examiners. We assessed age related changes and reproducibility of measurements.RESULTS: AGD increased between the two examinations and correlated within the child. A large proportion of the observed variation in AGD was due to true differences between the children (AGDas: 62%, AGDap: 40%, AGDaf: 30%, AGDac: 21%), and measurement error due to between- and within-examiner variation was low.CONCLUSIONS: Our study showed that measures of AGD within a child correlated during infancy, especially in boys and particularly for AGD measured as the distance between anus and scrotum. A planned cohort follow-up through childhood and puberty will reveal whether AGD represents a phenotypic signature throughout life.Pediatric Research accepted article preview online, 20 November 2017. doi:10.1038/pr.2017.287.

Background: Filaggrin is an epidermal protein that is important for normal skin barrier functions. Up to 10% of Europeans and Asians carry filaggrin gene (FLG) loss-of function mutations that appear to facilitate trans-epidermal penetration of certain chemicals. We previously showed that mutation carriers have higher internal exposure to certain phthalates, compared to controls, and hypothesized that they could have increased trans-epidermal penetration of other chemicals. Objectives: We investigated exposure to non-persistent chemicals in young Danish men with and without FLG mutations. Methods: Concentrations of eight simple phenols, six parabens and nine UV filters were analysed in urine from 65 FLG loss-of-function mutation carriers and 130 non-carriers (controls). Regression analyses, controlling for urinary dilution and confounders, were performed to estimate associations between FLG mutation status and chemical concentrations in urine. Results: FLG mutation carriers had 80% (13–180%) higher urinary concentrations of methyl paraben (MeP) and 91% (13–219%) higher concentrations of n-propyl paraben (n-PrP) than controls. For 13 compounds, levels were higher in FLG mutation carriers, although differences were only statistically significant for MeP and n-PrP. Combined statistical analysis of concentrations of all the 18 compounds that were detectable in >10% of subjects, suggested that concentrations were generally higher in mutation carriers (p=0.03). Conclusion: FLG loss-of-function mutation carriers have a higher internal exposure to some non-persistent chemicals, independently of atopic dermatitis. This may be due to increased trans-epidermal absorption and/or higher exposure, and mutation carriers may constitute a group susceptible to increased absorption of chemicals and topical medication. Keywords: Endocrine disrupting chemicals, Phenols, UV filters, Parabens, Filaggrin, Dermal exposure

In patients with Fabry disease (FD), left ventricular hypertrophy and arrhythmias are frequently observed and cardiac involvement is the leading cause of death. Long-term efficacy of enzyme replacement therapy (ERT) on cardiac involvement is unclear. We assessed and compared long-term progression of cardiac involvement according to ERT and non-ERT.We retrospectively assessed and compared long-term progression of cardiac involvement in adult patients with FD in the nationwide Danish cohort. We followed clinical signs, symptoms and findings by echocardiography, electrocardiography and Holter-monitoring.We included 66 patients; 47 patients (27 women) received ERT (ERT group) and 19 patients (15 women) did not (non-ERT group). The groups were followed for a median of 8 [0-12] years and 6 [0-13] years, respectively. Comparison between ERT and non-ERT receiving patients by left ventricular mass (echocardiographic assessment) and Sokolow-Lyon voltage- and Cornell product criteria (electrocardiographic assessment) revealed no significant differences. In the ERT group, we observed no change in left ventricular mass but a decrease in Sokolow-Lyon voltage- and Cornell product criteria from baseline to follow-up; 30 mm [15-53] vs. 25 mm [3-44], p 0.005 and 1710 mm·ms [480-3740] vs. 1520 mm·ms [550-5740], p .05, respectively. There were no changes within the non-ERT group. During follow-up, cardiac symptoms and use of cardiovascular procedures and -medication increased significantly in the ERT group, whereas no differences were observed within the non-ERT group.We raise concerns regarding the efficacy and benefit of ERT on cardiac involvement in Fabry disease and stress the need for further research.

Aim To compare HbA1c and fasting plasma glucose assessment, with the 2-h oral glucose tolerance test as reference, in screening for diabetes in people with turberculosis. Methods Individuals (N=268) with newly diagnosed smear-positive tuberculosis were screened for diabetes at a tertiary hospital in Lahore, Pakistan. Diabetes diagnosis was based on WHO criteria: thresholds were ≥ 48 mmol/mol (≥6.5%) for HbA1c and ≥7.0mmol/l for fasting plasma glucose. Results The proportion of participants diagnosed with diabetes was 4.9% (n =13) by oral glucose tolerance test, while 11.9% (n =32) and 14.6% (n =39) were diagnosed with diabetes using HbA1c and fasting plasma glucose criteria, respectively. The area under the receiver-operating characteristic curve was 0.79 (95% CI 0.64 to 0.94) for HbA1c and 0.61 (95% CI 0.50 to 0.73) for fasting plasma glucose, with a borderline significant difference between the two tests (P=0.07). Conclusions HbA1c and fasting plasma glucose performed equally in terms of diagnosing new diabetes cases in individuals with tuberculosis, but the proportion of participants falsely classified as positive was higher for fasting plasma glucose. This may be explained by acute blood glucose fluctuations when using fasting plasma glucose. HbA1c may be a more reliable test in individuals with transient hyperglycaemia. This article is protected by copyright. All rights reserved.

Background: In patients with intestinal failure who are receiving home parenteral support (HPS), catheter-related bloodstream infections (CRBSIs) inflict health impairment and high costs.Objective: This study investigates the efficacy and safety of the antimicrobial catheter lock solution, taurolidine-citrate-heparin, compared with heparin 100 IE/mL on CRBSI occurrence.Design: Forty-one high-risk patients receiving HPS followed in a tertiary HPS unit were randomly assigned in a double-blinded, placebo-controlled trial. External, stratified randomization was performed according to age, sex, and prior CRBSI incidence. The prior CRBSI incidence in the study population was 2.4 episodes/1000 central venous catheter (CVC) days [95% Poisson confidence limits (CLs): 2.12, 2.71 episodes/1000 CVC days]. The maximum treatment period was 2 y or until occurrence of a CRBSI or right-censoring because of CVC removal. The exact permutation tests were used to calculate P values for the log-rank tests.Results: Twenty patients received the taurolidine-citrate-heparin lock and 21 received the heparin lock, with 9622 and 6956 treatment days, respectively. In the taurolidine-citrate-heparin arm, no CRBSIs occurred, whereas 7 CRBSIs occurred in the heparin arm, with an incidence of 1.0/1000 CVC days (95% Poisson CLs: 0.4, 2.07/1000 CVC days; P = 0.005). The CVC removal rates were 0.52/1000 CVC days (95% Poisson CLs: 0.17, 1.21/1000 CVC days) and 1.72/1000 CVC days (95% Poisson CLs: 0.89, 3.0/1000 CVC days) in the taurolidine-citrate-heparin and heparin arm, respectively, tending to prolong CVC survival in the taurolidine arm (P = 0.06). Costs per treatment year were lower in the taurolidine arm (€2348) than in the heparin arm (€6744) owing to fewer admission days related to treating CVC-related complications (P = 0.02).Conclusions: In patients with intestinal failure who are life dependent on HPS, the taurolidine-citrate-heparin catheter lock demonstrates a clinically substantial and cost-beneficial reduction of CRBSI occurrence in a high-risk population compared with heparin. This trial was registered at clinicaltrials.gov as NCT01948245.

Objective The aim of this study was to evaluate an individualised gonadotrophin starting dose regimen for women with anovulatory infertility. Study design We included 71 normogonadotrophic anovulatory infertile women in a prospective, observational study. All underwent one ovulation induction cycle in a flexible, low-dose step-up protocol. The gonadotrophin starting dose (75–150 IU/day) was individualised according to a nomogram incorporating menstrual cycle pattern (oligo- or amenorrhoea), BMI, and mean ovarian volume. The number of women who fulfilled the criteria for human chorionic gonadotrophin (hCG) administration (one follicle ≥17 mm or 2–3 follicles ≥15 mm) was assessed. Results Of the 50 women (70.4%) who fulfilled the hCG criteria and underwent intrauterine insemination, 34 (47.9%) achieved monofollicular growth and 16 (22.5%) developed 2–3 mature follicles. Seventeen (23.9%) cycles were converted to in vitro fertilisation (IVF) due to the development of >3 mature follicles, and one (1.4%) cycle was cancelled due to risk of ovarian hyperstimulation syndrome. Baseline total antral follicle count was found to be significantly associated with fulfillment of the hCG criteria (OR 0.96, 95% CI: 0.92–0.99, P = 0.01). Conclusions The nomogram-based dose regimen was not considered suitable for ovulation induction due to a tendency to overestimate the gonadotrophin starting dose. However, the model may serve as a mild IVF regimen, especially in women prone to excessive follicle growth.

Context: Detailed evaluation of pubertal progression in girls from longitudinal studies is sparse, and the phenomenon of transient thelarche (TT), defined as the appearance, regression, and subsequent reappearance of breast buds, in healthy girls remains undescribed. Objective: To describe TT in terms of pubertal progression, growth, genotypes, and reproductive hormones and to apply new puberty nomograms for breast stages, pubic hair, and menarche. Design: A prospective, longitudinal population-based study. Patients or Other Participants: Ninety-eight healthy Danish schoolchildren (Caucasian girls) followed longitudinally as part of the COPENHAGEN Puberty Study were included in the evaluation of TT. A total of 1466 girls from 2 cross-sectional studies were included in the creation of the puberty nomograms. Intervention(s): None. Main Outcome Measure(s): Pubertal progression, specifically thelarche, reproductive hormones, genotype, and growth. Results: Twelve of 98 (12%) girls experienced TT. A larger proportion of girls with TT entered puberty by the pubarche pathway (50%) compared with girls with normal progression (15.4%), P = 0.014. Girls with TT progressed through puberty normally when evaluated using puberty nomograms. Reproductive hormones and growth velocity were lower at the first (transient) thelarche than the second (permanent) thelarche. Conclusion: TT is a frequent phenomenon that appears to be a peripheral occurrence independent of central puberty. It does not appear to affect subsequent pubertal progression as evaluated by our new puberty nomograms.

Summary Background Children of refugees are often exposed to the consequences of parental post-traumatic stress disorder (PTSD), potentially leaving them vulnerable to intergenerational transmission of psychopathology. The present study aimed to determine whether parental PTSD is associated with childhood psychiatric morbidity among children of refugees. Methods This study is a two-generation nationwide cohort study using the Danish Immigration Services database. We followed up children younger than 18 years with at least one refugee parent until psychiatric contact, end of the study, their 18th birthday, emigration, or death. We excluded children if their parents were diagnosed only with psychiatric diagnoses other than PTSD or if they had received a psychiatric contact before parental PTSD diagnosis. Information on parental PTSD and offspring psychiatric morbidity was obtained from the Danish Psychiatric Central Research Register. We used Cox proportional hazards regression models to assess the risk of psychiatric contacts among children of refugees with PTSD compared with children of refugees with no psychiatric diagnosis. Findings Between Jan 1, 1995, and Dec 31, 2015, 102 010 refugees obtained residency permission in Denmark and 62 239 biological children of refugees were born in Denmark before Dec 31, 2015. 51 793 were eligible and included in the study (median follow-up 7·15 years [IQR 3·37–11·78]); of these, 1307 (2·5%) children had a psychiatric contact. 7486 (14·5%) children of refugees were exposed to parental PTSD. Parental PTSD significantly increased the risk of psychiatric contact in offspring (hazard ratio 1·49 [95% CI 1·17–1·89] for paternal PTSD, p=0·0011; 1·55 [1·20–2·01] for maternal PTSD, p=0·00084) after adjustment for sociodemographic variables. Interpretation Children of refugees exposed to parental PTSD are at increased risk of psychiatric morbidity. Targeted screening and interventions are necessary to prevent psychiatric morbidity and ensure access to adequate care. Funding Section for Immigrant Medicine, Department of Infectious Diseases, Copenhagen University Hospital Hvidovre.

STUDY QUESTION Is the Leydig cell function of young European men associated with semen quality? SUMMARY ANSWER Compensated reduction in Leydig cell function, defined as increased LH concentration combined with adequate testosterone production is associated with lower semen quality. WHAT IS ALREADY KNOWN Semen quality of young European men shows a heterogeneous pattern. Many have sperm counts below and in the lower WHO reference where there nevertheless is a significant risk of subfecundity. Little is known about differences in Leydig cell function between men with semen quality below and within the WHO reference range. STUDY DESIGN, SIZE AND DURATION A coordinated, cross-sectional population-based study of 8182 men undertaken in 1996-2010. PARTICIPANTS, SETTING AND METHOD Young men (median age 19.1 years) were investigated in centres in Denmark, Estonia, Finland, Germany Latvia, Lithuania, and Spain. The men originated from the general populations, all were young, almost all were unaware of their fecundity and each provided a semen and blood sample. Associations between semen parameters and serum levels of testosterone and luteinising hormone (LH), calculated free testosterone, and ratios between serum testosterone and LH were determined. MAIN RESULT AND ROLE OF CHANCE Serum testosterone levels were not associated with sperm concentrations, total sperm counts, or percentage of motile or morphologically normal spermatozoa. There was an inverse association between the semen parameters and serum LH levels, and accordingly a positive association to testosterone/LH ratio and calculated-free-testosterone/LH ratio. LIMITATIONS, REASON FOR CAUTION The size of the study mitigates the intra-individual variability concern. The distinction between different sub-categories of sperm motility and sperm morphology is subjective despite training. However, inter-observer variation would tend towards non-differential misclassification and would decrease the likelihood of detecting associations between reproductive hormone levels and semen variables, suggesting that the presented associations might in reality be even stronger than shown. Although we adjusted for confounders, we cannot of course exclude that our results can be skewed by selection bias or residual confounding. WIDER IMPLICATIONS OF THE FINDINGS Compensated reduction in Leydig cell function, defined as increased LH concentration combined with adequate testosterone production is associated with lower semen quality. This is apparent even within the WHO reference range of semen quality. It is unknown whether impaired Leydig cell function in young men may confer an increased risk of acquired testosterone deficiency later in life. STUDY FUNDING/COMPETING INTERESTS Support from The Research Fund of Rigshospitalet (grant no. R42-A1326) to N.J. made this study possible. The background studies of young men have been supported economically by several grants. ITALIC! Denmark: The European Union (contract numbers BMH4-CT96-0314, QLK4-CT-1999-01422, QLK4-CT-2002-00603 and most recently FP7/2007-2013, DEER Grant agreement no. 212844), The Danish Research Council (grants nos. 9700833 2107-05-0006), The Danish Agency for Science, Technology and Innovation (Grant no. 271070678), Rigshospitalet (Grant no. 961506336), The University of Copenhagen (Grant no. 211-0357/07-3012), The Danish Ministry of Health and the Danish Environmental Protection Agency, A.P. Moller and wife Chastine McKinney Mollers foundation, and Svend Andersens Foundation. ITALIC! Finland: European Union (contract numbers BMH4-CT96-0314, QLK4-CT-1999-01422, QLK4-CT- 2002-00603 and most recently FP7/2008-2012, DEER Grant agreement no. 212844), The Academy of Finland, Turku University Hospital Funds, Sigrid Juselius Foundation. ITALIC! Estonia, Latvia and Lithuania: European Union (QLRT-2001-02911), the Estonian Science Foundation, grant number 2991, Lithuanian Foundation for Research, Organon Agencies B.V. and the Danish Research Council, grant no. 9700833. ITALIC! Germany: European Union (contract numbers QLK4-CT-2002-00603). ITALIC! Spain: European Commission QLK4-1999-01422. M.F. received support from the Spanish Ministry of Science and Innovation (Program Ramon y Cajal). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. None of the authors have any competing interests to declare.

We report reference ranges based on LC-MS/MS for testosterone (T), free testosterone (FT) and its precursors, i.e. 17-hydroxyprogesterone (17-OHP), dehydroepiandrosterone (DHEA), DHEA-sulfate (DHEAS) and androstenedione (Adione), in relation to different health markers and lifestyle factors. The study was based on 304 healthy men aged 30-61 years participating in a population-based cross-sectional study (Health2008). Examination program consisted of a clinical examination, completion of a self-administered questionnaire and blood sampling. Steroid metabolites were measured by a validated and sensitive LC-MS/MS method. Older age-groups were significantly associated with decreased concentrations of DHEA, DHEAS, Adione, and FT, while no significant associations with age were shown for 17-OHP or T. Participants with BMI≥30 kg/m(2) had lower age-related steroid metabolite z-scores compared to participants with BMI

Background Immigrants may face problems with accessing the Danish healthcare system due to, for example, lack of knowledge of how to navigate it, which may cause inappropriate healthcare-seeking. Danish municipalities provide a mandatory introduction and language programme for newly arrived immigrants, but no information on the healthcare system is offered. This study investigated what effects information about the Danish healthcare system may have on the hypothetical healthcare-seeking behaviour of newly arrived immigrants and their actual healthcare use. Methods A prospective intervention study of 1572 adult immigrants attending two language schools in Copenhagen was carried out. Two intervention groups received either a course or written information on the Danish healthcare system, respectively, while the control group received neither. Survey data included three case vignettes on healthcare-seeking behaviour (flu-like symptoms, chest pain and depression) and were linked to registry data on sociodemographic characteristics and healthcare use in the year to follow. Logistic regression and binomial regression analyses were performed. Results Appropriate hypothetical healthcare-seeking behaviour was reported by 61.8–78.8% depending on the vignette. Written information showed no effect on immigrants’ hypothetical healthcare-seeking behaviour, while the course showed a positive effect on hypothetical healthcare-seeking behaviour for flu-like symptoms (adjusted odds ratio [AOR] = 1.71, 95% confidence interval [CI] = 1.01–2.91, p-value = 0.0467), but not on chest pain or depression. The interventions did not affect immigrants’ actual healthcare use; all groups made lower use of health care services in the following year compared with the year where the study took place, except for the use of dental care which remained stable. Conclusions Information on the healthcare system embedded in the language school programme has the potential to facilitate immigrants’ access to healthcare. Yet, the results underscore the need for further refinement and development of educational interventions, as well as ensuring adequate utilisation of healthcare services by other means. Multi-dimensional and multi-sectional efforts are important for integration issues within healthcare in Europe. Trial registration Health-seeking behaviour among newly arrived immigrants in Denmark ISRCTN24905314, May 1, 2015 (Retrospectively registered).

Background: 'Remigration bias' is often referred to when explaining low mortality outcomes among migrants compared with local-born. The hypothesis suggests that severely ill migrants tend to return to their country of origin, but it has hitherto not been tested in a large-scale epidemiological study. Consequently, we studied whether migrants with severe disease were more likely to emigrate compared with migrants without severe disease. Methods: A historic prospective cohort study was conducted based on all adult refugees and family reunification immigrants (n = 114,331) who obtained residence permission in Denmark between 1 January 1993 and 31 December 2010. Migrants with severe disease were identified through the Danish National Patient Register. Emigrations during follow-up (1 January 1993 to 31 December 2011) were identified using the Danish Civil Registration System. Hazard ratios were calculated for emigration among migrants with different levels of disease severity, adjusting for sex, age and income. Results: Results showed progressively fewer emigrations with increasing disease severity. Migrants with low (HR = 0.92; 95% CI: 0.80-1.06), moderate (HR = 0.84; 95% CI: 0.67-1.06) and high (HR = 0.70; 95% CI: 0.55-0.90) disease severity had fewer emigrations to 'country of origin' compared with migrants without disease. For emigration to 'any country', results likewise showed fewer emigrations among migrants with low (HR = 0.86; 95% CI: 0.78-0.95), moderate (HR = 0.73; 95% CI: 0.62-0.87) and high (HR = 0.64; 95% CI: 0.53-0.76) disease severity. This tendency was also observed by nationality and migrant status. On the disease-specific level, no uniform tendencies in emigration patterns were identified. Conclusion: The results do not generally support the hypothesis of 'remigration bias' but rather suggest the opposite. Accordingly, remigration bias does not appear to explain lower mortality of migrants.

Individuals with newly diagnosed tuberculosis (TB) were screened for diabetes (DM) with fasting plasma glucose (FPG) in Pakistan. A significant decrease in FPG was observed when TB was treated. Of those with newly diagnosed DM, 46% and 62% no longer had hyperglycemia after 3 and 6 months, respectively. Individuals with known DM also showed a significant decrease in fasting plasma levels when treated for TB, but after 3 months none had normoglycemia, and after 6 months 9.2% were normoglycemic. Thus, TB-related DM may abate when the stress terminates, as is the case in gestational DM. However, because stress hyperglycemia may be associated with subsequent risk of developing DM, follow-up is recommended.

Context Results of animal models and cross-sectional cohort studies have suggested a beneficial role for vitamin D in male reproduction. Objective Determine the effect of vitamin D and calcium supplementation on semen quality in infertile men with serum 25-hydroxyvitamin-D (25OHD) levels ≤50 nmol/L. Design A single-center, triple-blinded, randomized clinical trial. Participants A total of 1427 infertile men were screened to include 330; 1002 men did not meet inclusion criteria and 95 did not wish to participate. Intervention The active group received cholecalciferol 300,000 IU initially, then 1400 IU cholecalciferol and 500 mg of calcium daily for 150 days; the other group received placebo. Results Serum concentrations of 25OHD and 1,25-dihydroxyvitamin D3 were significantly higher in men in the treatment group compared with the placebo group. Vitamin D supplementation was not associated with changes in semen parameters, although spontaneous pregnancies tended to be higher in couples in which the man was in the treatment group [7.3% vs 2.4%, Δ5.0% (-0.6%; 10.5%)]. Vitamin D treatment in a subgroup of oligozoospermic men increased the chance for a live birth compared with placebo [35.6% vs 18.3%, Δ17.3% (1.6%; 32.9%)]. Moreover, serum inhibin B levels were higher in men deficient in vitamin D who were randomly assigned to receive high-dose vitamin D [193 pg/mL vs 143 pg/mL, Δ49 pg/mL (8; 91 pg/mL)]; however, the increase in sperm concentration was not significantly higher than in the placebo group (P = 0.07). Conclusion High-dose vitamin D supplementation did not improve semen quality in vitamin D-insufficient infertile men. The positive impact of vitamin D supplementation on live birth rate and serum inhibin B in oligozoospermic and vitamin D-deficient men may be of clinical importance and warrant verification by others.

Clinical use of single serum gonadotropin measurements in children is limited by the pulsatile secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH). However, first morning voided (FMV) urine may integrate the fluctuating gonadotropin serum levels.We aimed to evaluate urinary and serum gonadotropin levels according to age, sex, and pubertal stage in healthy children and to assess the clinical use of FMV urinary gonadotropins in children with disordered puberty.Cross-sectional part of the COPENHAGEN Puberty Study and longitudinal study of patients.Population-based and outpatient clinic.Eight hundred forty-three healthy children from the COPENHAGEN Puberty Study and 25 girls evaluated for central precocious puberty (CPP).Clinical pubertal staging, including serum and urinary gonadotropin levels.Urinary gonadotropins increased with advancing age and pubertal development and were detectable in FMV urine before physical signs of puberty. FMV urinary LH correlated strongly with basal (r = 0.871, P0.001) and gonadotropin-releasing hormone (GnRH)-stimulated serum LH (r = 0.82, P0.001). Urinary LH was superior to urinary FSH in differentiating the pubertal stage. Receiver operating curve analysis revealed that a cut-off standard deviation (SD) score of 2 for urinary LH (IU/L) gave a sensitivity of 75% and a specificity of 92% in predicting a positive GnRH stimulation test (LHmax5 IU/L). Urinary concentrations of LH decreased after 3 months of GnRH treatment to levels below +2 SDs.Urinary gonadotropin levels increased before the onset of puberty and were elevated in girls with CPP. We suggest urinary LH as an alternative noninvasive method to improve diagnosing and therapeutic management of children with disordered puberty.

Background In Pakistan the prevalence of diabetes (DM) among adults is 6.9% and expected to double by 2040. DM may facilitate transmission and halter the elimination of tuberculosis (TB). We aimed to determine the prevalence of DM among TB patients in Pakistan and to investigate anthropometric biochemical, and haemodynamic associations between TB patients with and without DM. Methods We conducted a cross-sectional study at Gulab Devi Chest Hospital in Lahore, Punjab. A total of 3,027 newly diagnosed smear positive TB patients ≥ 25 years of age were screened for DM by HbA1c regardless of previous DM history. Results The prevalence of screen-detected DM and known DM among the TB participants was 13.5% and 26.1% respectively, resulting in a combined DM prevalence of 39.6%. Most participants were male (64.4%). Using bivariate analyses, participants with DM were significantly older (49.8 vs. 40.6 years) with higher haemoglobin (men, 12.1 vs. 11.8 g/dL, women 11.5 vs. 10.7 g/dL), body mass index (21.0 vs. 17.6 kg/m2) and waist-hip ratio (men, 0.87 vs. 0.81, women, 0.87 vs. 0.79) (all p

Perceived stress has been associated with decreased semen quality but the mechanisms have not been elucidated. It is not known whether cortisol, the major stress hormone in humans, can act directly via receptors in the testis, and whether variants in the gene encoding the glucocorticoid receptor (NR3C1) can possibly modulate the effect. To address these questions, we investigated the expression of the glucocorticoid receptor in human testicular tissue, including adult and fetal samples (n = 20) by immunohistochemical staining, and in silico analysis of publicly available datasets. In the adult testis NR3C1 protein was detected in peritubular cells, a subset of Leydig cells, Sertoli cells (weak), and spermatogonia, but not in spermatids. The NR3C1 expression pattern in fetal testis samples differed by a notably stronger reaction in Sertoli cells, lack of staining in gonocytes but the presence in a subset of pro-spermatogonia, and the almost absent reaction in nascent peritubular cells. In parallel, we explored the association between adult testicular function and three single nucleotide NR3C1 polymorphisms (BcII [rs41423247], 9β [rs6198], and Tth111I [rs10052957]) affecting glucocorticoid sensitivity. Testicular function was determined by semen analysis and reproductive hormone profiling in 893 men from the general population. The NR3C1 SNP BclI was associated with semen quality in an over-dominant manner with heterozygotes having better semen parameters compared to both homozygote constellations, and with sperm motility showing the strongest association. This association was supported by a higher inhibin B and inhibin B/FSH ratio, as well as a lower FSH in BclI heterozygotes. The SNPs 9β and Tth111I were not associated with semen parameters. Although the clinical impact of the findings is limited, the results substantiate a suggested link between stress and testicular function. Hence this investigation should be regarded as a discovery study generating hypotheses for future studies.

SummaryObjective To evaluate revised criteria for polycystic ovarian morphology (PCOM) in the diagnosis of polycystic ovary syndrome (PCOS) in anovulatory infertility. Design Prospective cohort study. Patients WHO Group II anovulatory infertile women (n = 75). Measurements Clinical, sonographic and endocrine parameters, including anti-Mullerian hormone (AMH). Results The Rotterdam criteria for PCOM (antral follicle count (AFC) ≥12 and/or ovarian volume >10 ml) were fulfilled in 93% of the women. The PCOM prevalence was 68% when increasing the threshold to AFC >20 and 76% according to an AMH-based threshold of >35 pmol/l. The most recently proposed AFC ≥ 25 threshold reduced the PCOM prevalence to 52% (n = 39), leaving 48% (n = 36) without features of PCOM. Comparing the 36 women with non-PCOM with the 39 women in the PCOM group according to AFC ≥ 25, 22% vs 59% (P = 0·001) had serum LH >10 IU/l, 11% vs 41% (P = 0·003) had an LH/FSH ratio >2 and 19% vs 41% (P = 0·04) had hirsutism and/or elevated total testosterone, free testosterone, and/or androstenedione. The non-PCOM group included significantly more women with secondary infertility. The median AMH in the non-PCOM group was 47 pmol/l, which was twofold lower than in the PCOM group but above the upper limit of normo-ovulatory women. Conclusions According to a revised threshold of 25 follicles, almost half the anovulatory infertile women do not have PCOM. The characteristics of these women may be compatible with hypothalamic anovulation, but according to AMH levels, the ovaries remain multifollicular. Perspectives A better distinction between hypothalamic amenorrhoea and PCOS could improve treatment strategies for anovulatory infertility.

Objectives The ‘healthy migrant effect’ (HME) hypothesis postulates that health selection has a positive effect on migrants' health outcomes, especially in the first years after migration. We examined the potential role of the HME by assessing the association between residence duration and disease occurrence. Methods We performed a historical prospective cohort study. We included migrants who obtained residence permits in Denmark between 1 January 1993 and 31 December 2010 (n = 114 331). Occurrence of severe conditions was identified through linkage to the Danish National Patient Register. Hazard Ratios (HRs) were modelled for disease incidence by residence duration since arrival (0–5 years; 0–10 years; 0–18 years) adjusting for age and sex. Results Compared with Danish-born individuals, refugees and family reunited immigrants had lower HRs of stroke and breast cancer within 5 years after arrival; however, HRs increased at longer follow-up. For example, HRs of stroke among refugees increased from 0.77 (95% CI: 0.66; 0.91) to 0.96 (95% CI: 0.88; 1.05). For ischaemic heart disease (IHD) and diabetes, refugees and family reunited migrants had higher HRs within 5 years after arrival, and most HRs had increased by end of follow-up. For example, HRs of IHD among family reunited migrants increased from 1.29 (95% CI: 1.17; 1.42) to 1.43 (95% CI: 1.39; 1.52). In contrast, HRs for TB and HIV/AIDS showed a consistent decrease over time. Conclusion Our analyses of the effect of duration of residence on disease occurrence among migrants imply that, when explaining migrants' advantageous health outcomes, the ruling theory of the HME should be used with caution, and other explanatory models should be included. Objectifs L'hypothese de “l'effet du migrant en bonne sante” (EMS) postule que la selection de la sante a un effet positif sur les resultats de sante des immigrants, en particulier dans les premieres annees apres la migration. Nous avons examine le role potentiel de l’EMS en evaluant l'association entre la duree de sejour et la survenue de la maladie. Methodes Nous avons realise une etude prospective de cohorte historique. Nous avons inclus les immigrants qui ont obtenu un permis de sejour au Danemark entre le 01 janvier 1993 et le 31 decembre 2010 (n = 114 331). La survenue de conditions severes a ete identifiee grâce aux liens avec le registre national danois des patients. Les rapports de risque (HR) ont ete modelises pour l'incidence de la maladie selon la duree de residence depuis l'arrivee (0 a 5 ans, 0 a 10 ans et 0 a 18 ans) et ajustes pour l’âge et le sexe. Resultats Comparativement aux personnes nees au Danemark, les refugies et les immigrants de regroupement familial avaient un HR inferieur d’AVC et de cancer du sein dans les 5 ans apres l'arrivee. Cependant, le HR augmentait sur un suivi plus long. Par exemple, le HR pour l’AVC chez les refugies passait de 0,77 (IC95%: 0,66–0,91) a 0,96 (IC95%: 0,88–1,05). Pour la maladie cardiaque ischemique (MCI) et le diabete, les refugies et les immigrants de regroupement familial avaient un HR plus eleve dans les 5 ans apres l'arrivee et la plupart des HR augmentaient a la fin du suivi. Par exemple, le HR pour la MCI chez les immigrants de regroupement familial a augmente de 1,29 (IC95%: 1,17–1,42) a 1,43 (IC95%: 1,39–1,52). En revanche, les HR pour la tuberculose et le VIH/SIDA ont montre une diminution constante au cours du temps. Conclusion Nos analyses de l'effet de la duree de residence sur la survenue de la maladie chez les immigrants impliquent que, pour expliquer les resultats de sante avantageux pour les immigrants, la theorie courante de l’EMS doit etre utilisee avec prudence et d'autres modeles explicatifs devraient etre inclus. Objetivos La hipotesis del “efecto del inmigrante sano” (EIS) postula que la seleccion de salud tiene un efecto positivo sobre los resultados de salud de los inmigrantes, especialmente en los primeros anos tras la migracion. Hemos examinado el papel potencial de la EIS evaluando la asociacion entre el tiempo de duracion de la residencia y la aparicion de enfermedad. Metodos Hemos realizado un estudio de cohortes historico y prospectivo. Hemos incluido inmigrantes que obtuvieron permisos de residencia en Dinamarca entre 01.01.1993 y 31.12.2010 (n = 114 331). Se identifico la aparicion de condiciones severas a traves del Registro Nacional de Pacientes de Dinamarca. Se modelaron los cocientes de riesgos instantaneos (HRs) para la incidencia de enfermedad segun la duracion de la residencia, desde la llegada 0–5 anos; 0–10 anos; 0–18 anos) ajustando para edad y sexo. Resultados Comparados con individuos nacidos en Dinamarca, los refugiados y los inmigrantes de familias reunificadas tenian un menor HR de accidente cerebrovascular isquemico (ACV) y cancer de mama dentro de los 5 anos posteriores a su llegada; sin embargo, los HRs aumentaban a mayor seguimiento. Por ejemplo, los HRs para ACV entre refugiados aumentaba del 0.77 (IC 95%:0.66; 0.91) al 0.96 (IC 95%:0.88; 1.05). Para la enfermedad cardiaca isquemica (ECI) y la diabetes, los refugiados y los inmigrantes de familia reunificadas tenian unos HRs mayores dentro de los 5 anos posteriores a la llegada, y la mayoria de los HRs habian aumentado al final del seguimiento. Por ejemplo, HRs de ECI los inmigrantes de familias reunificadas aumento del 1.29 (IC 95%:1.17; 1.42) al 1.43 (IC95%:1.39; 1.52). En con traste, los HRs para TB e VIH/SIDA mostraban una disminucion consistente a lo largo del tiempo. Conclusion Nuestro analisis sobre el efecto de la duracion de la residencia sobre la aparicion de enfermedad entre inmigrantes sugiere que, cuando se explican los resultados ventajosos para los inmigrantes, la teoria de descarte del EIS deberia utilizarse con precaucion, y deberian incluirse otros modelos explicativos.

Aim To construct new Danish growth charts for 0- to 20-year-olds and to compare them with Danish references from 1982 and with World Health Organization (WHO) standards for children aged 0–5 years from 2006, by applying similar inclusion and exclusion criteria. Methods Anthropometric data from three contemporary Danish population-based studies were combined. References for height were based on healthy Caucasian children born at term. A total of 12 671 height measurements (8055 in boys and 4616 in girls) were included. Reference charts were developed using the generalised additive models for location, scale and shape. Results From prepubertal ages, a secular increase in height was observed for both genders. The differences were most pronounced in puberty, and final heights were increased by 1.4 cm in boys and 2.9 cm in girls compared to 1982 references. In boys, but not girls an upward shift in body mass index (BMI) above median levels was found. Reference curves for height were superimposable with standard curves based on the selective WHO criteria. Danish children were longer/taller and heavier and they had larger head circumferences than those reported in the recent multiethnic WHO standards. Conclusion We recommend national implementation of these contemporary 2014 Danish references for anthropometric measurements.

BackgroundPubertal onset is usually defined by breast development in girls and testicular growth in boys. Pubarche is defined as the attainment of pubic hair and is considered as a sign of pubertal transition. Pubarche is preceded by a gradual increase in production of adrenal androgens, DHEA and Δ4-androstenedione (Adione), a process termed adrenarche.ObjectiveTo study the natural course of pubertal transition and the associations with adrenarche, body fat, and linear growth.Design and methodsA longitudinal study of 179 healthy children (89 girls) with higher socioeconomic background examined every 6 months for 5 years. Pubic hair stage, breast stage, genital stage, testicular volume (TV), height, weight, and four skinfolds were measured.ResultsIn girls, median age (25th and 75th percentiles) at thelarche (B2+) was 10.1 years (9.3–10.9). In boys, median age at attaining a TV >3 ml was 11.5 years (10.9–12.0). Median age at pubarche (PH2+) was 10.9 years (10.3–11.4) in girls and 11.6 years (10.8–12.4) in boys. Only 6.8% (4/59) of the girls and 24.6% (15/61) of the boys developed pubic hair as the first isolated sign of puberty. Serum DHEAS and Adione increased with age, although the increase in Adione was most pronounced in girls. No associations between early age at thelarche/testicular growth and increased body fat (BMI and sum of four skinfolds) were observed.ConclusionDanish children rarely experience pubarche as the first sign of puberty. No associations between age at pubertal onset and body composition were found. Circulating levels of Adione, but not DHEAS, increased with the onset of puberty, although with large interindividual variability.

Erratum in: Hum Reprod. 2014 Jul;29(7):1600. The authors would like to apologise for a typing error in Table V of this article. The confidence interval of the regression co-efficient for the effect of perfluoro-n-nonanoic acid (PFNA) on estradiol should be −0.075 (−0.130, −0.019) rather than the wrongly stated −0.075 (−0.013, −0.019). The authors would like to assure readers that this does not affect the other content or the conclusions of the article.; International audience; Is exposure to perfluorinated compounds (PFCs) associated with testicular function (reproductive hormone levels and semen quality) in healthy men? PFOS levels were significantly negatively associated with serum testosterone (total and calculated free), but not with any other reproductive hormones or semen quality. In animals, some PFCs have endocrine disrupting potential, but few studies have investigated PFCs in relation to human testicular function. Previously, we and others have observed a negative association between serum PFC levels and sperm morphology. The potential associations with reproductive hormones remain largely unresolved. A cross-sectional study of 247 men was conducted during 2008-2009. Healthy men from the general population, median age of 19 years, gave serum and semen samples. Serum samples were analysed for total testosterone (T), estradiol (E), sex hormone-binding globulin (SHBG), luteinizing hormone (LH), follicle-stimulating hormone (FSH) and inhibin-B and 14 PFCs, including perfluorooctanesulfonate (PFOS). Semen samples were analysed according to the WHO criteria. PFOS levels were negatively associated with testosterone (T), calculated free testosterone (FT), free androgen index (FAI) and ratios of T/LH, FAI/LH and FT/LH. Other PFCs were found at lower levels than PFOS and did not exhibit the same associations. PFC levels were not significantly associated with semen quality. PFOS levels in these samples collected in 20082009 were lower than in our previous study of men participating in 2003. Results were robust to adjustment for relevant confounders; however, the possibility of chance associations due to multiple testing or effects of uncontrolled confounding cannot be ruled out. Our previous findings of decreased sperm morphology in the most highly PFC exposed men were not replicated, possibly due to a lack of highly exposed individuals; however, a recent independent study also did corroborate such an inverse association. The negative association between serum PFOS and testosterone indicates that testosterone production may be compromised in individuals with high PFOS exposure. The authors received financial support from the European Commission (DEER, FP7-2007-212844), the Danish Agency for Science, Technology and Innovation (grant nos. 27107068 and 09-067180), Rigshospitalet (grant no. 961506336), the University of Copenhagen, the Danish Ministry of Health and the Danish Environmental Protection Agency (MST-621-00013), and Kirsten and Freddy Johansen Foundation (grant no. 95-103-72087). The funding organizations played no role in the design and conduct of the study, in collection, management, analysis and interpretation of the data; or in the presentation, review or approval of the manuscript. The authors declare that they have no competing financial interests.

One in four asylum applicants in Europe are children, and 23% of whom are unaccompanied and may be at increased risk of mental illness. This study contributes to the limited evidence base by comparing the incidence of psychiatric disorders among unaccompanied and accompanied refugee children. We linked a cohort of refugee children who obtained right of residency in Denmark between 01 January 1993 and 31 December 2010 to the Danish Psychiatric Central Register, and calculated incidence rates per 100,000 person years and incidence rate ratios of overall psychiatric disorder, psychotic disorders, affective disorders, and neurotic disorders for accompanied and unaccompanied minors using Poisson regression. We adjusted the analyses for sex, age at residency, and age at arrival (aIRR). Stratified analyses were conducted by nationality. Unaccompanied minors had significantly higher rates of any psychiatric disorder (aIRR: 1.38, 95% CI 1.14-1.68) and neurotic disorders (aIRR: 1.67, 95% CI 1.32-2.13) than accompanied minors. Among children from Afghanistan, unaccompanied minors had significantly higher rates of any psychiatric disorder (aIRR: 2.23, 95% CI 1.26-3.93) and neurotic disorders (aIRR: 3.50, 95% CI 1.72-7.11). Among children from Iraq, unaccompanied minors had higher rates of any psychiatric disorder (aIRR: 2.02, 95% CI 1.18-3.45), affective disorders (aIRR: 6.04, 95% CI 2.17-16.8), and neurotic disorders (aIRR: 3.04, 95% CI 1.62-5.70). Unaccompanied children were found to experience a higher incidence of any psychiatric disorder and neurotic disorders. Strategies are needed to address the specific mental health and social needs of unaccompanied minors.

Objective Gonadotropin-releasing hormone (GnRH) and human chorionic gonadotropin (hCG) stimulation tests may be used to evaluate the pituitary and testicular capacity. Our aim was to evaluate changes in follicular-stimulating hormone (FSH), luteinizing hormone (LH) and testosterone after GnRH and hCG stimulation in healthy men and assess the impact of six single nucleotide polymorphisms on the responses. Design GnRH and hCG stimulation tests were performed on 77 healthy men, 18–40 years (reference group) at a specialized andrology referral center at a university hospital. The potential influence of the tests was illustrated by results from 45 patients suspected of disordered hypothalamic–pituitary–gonadal axis. Methods Baseline, stimulated, relative and absolute changes in serum FSH and LH were determined by ultrasensitive TRIFMA, and testosterone was determined by LC–MS/MS. Results For the reference group, LH and FSH increased almost 400% and 40% during GnRH testing, stimulated levels varied from 4.4 to 58.8 U/L and 0.2 to 11.8 U/L and FSH decreased in nine men. Testosterone increased approximately 110% (range: 18.7–67.6 nmol/L) during hCG testing. None of the polymorphisms had any major impact on the test results. Results from GnRH and hCG tests in patients compared with the reference group showed that the stimulated level and absolute increase in LH showed superior identification of patients compared with the relative increase, and the absolute change in testosterone was superior in identifying men with Leydig cell insufficiency, compared with the relative increase. Conclusions We provide novel reference ranges for GnRH and hCG test in healthy men, which allows future diagnostic evaluation of hypothalamic–pituitary–gonadal disorders in men.

Puberty marks numerous physiological processes which are initiated by central activation of the hypothalamic–pituitary–gonadal axis, followed by development of secondary sexual characteristics. To a large extent, pubertal timing is heritable, but current knowledge of genetic polymorphisms only explains few months in the large inter-individual variation in the timing of puberty. We have analysed longitudinal genome-wide changes in DNA methylation in peripheral blood samples (n = 102) obtained from 51 healthy children before and after pubertal onset. We show that changes in single methylation sites are tightly associated with physiological pubertal transition and altered reproductive hormone levels. These methylation sites cluster in and around genes enriched for biological functions related to pubertal development. Importantly, we identified that methylation of the genomic region containing the promoter of TRIP6 was co-ordinately regulated as a function of pubertal development. In accordance, immunohistochemistry identified TRIP6 in adult, but not pre-pubertal, testicular Leydig cells and circulating TRIP6 levels doubled during puberty. Using elastic net prediction models, methylation patterns predicted pubertal development more accurately than chronological age. We demonstrate for the first time that pubertal attainment of secondary sexual characteristics is mirrored by changes in DNA methylation patterns in peripheral blood. Thus, modulations of the epigenome seem involved in regulation of the individual pubertal timing.

The pattern of testicular growth during puberty may provide important information about early testicular damage and reproductive potential in adulthood.To evaluate pubertal testicular growth in boys with congenital cryptorchidism and controls.Longitudinal case-control study.Andrological Research Center, University of Turku.Altogether, 119 boys participated: 51 cases with a history of congenital cryptorchidism and 65 controls fulfilled the inclusion criteria.None.Testicular volume by an orchidometer (mL) and ultrasound (mL), testicular length by a ruler (mm), and onset of pubertal testicular growth (y). Longitudinal testicular growth was analyzed with a nonlinear mixed-effect model.The mean age of the onset of pubertal testicular growth (age at the attainment of3 mL by orchidometer) was 11.7 and 11.8 years in cryptorchid cases and controls, respectively. The difference between cases and controls was not significant. Modeled postpubertal testicular size was smaller among bilaterally and unilaterally undescended testis than in controls. There was a high level of agreement between testicular sizes of 3 mL by orchidometer and 25 mm by ruler as cut-offs in definition of the onset of puberty. An orchidometer size of 3 mL and ruler length of 25 mm corresponded to 1.6 and 1.7 mL by ultrasound (with Lambert's formula), respectively.Testicular growth in puberty was impaired in congenitally cryptorchid boys. This suggests a poor perinatal development of the cryptorchid testis. The timing of the onset of pubertal testicular growth, however, did not differ which suggests an intact hypothalamic-pituitary axis.

Context: Epidemiological evidence on maternal and paternal heritability of the wide normal variation within pubertal timing is sparse. Objective: We aimed to estimate the impact of parental pubertal timing on the onset of puberty in boys and girls. Design: Annual pubertal examinations of healthy children in a longitudinal cohort study. Information on parental timing of puberty (earlier, comparable to, or later compared to peers) and menarche age was retrieved from questionnaires. Participants: A total of 672 girls and 846 boys. Main Outcome Measures: Age at onset of pubic hair (PH2+), breasts (B2+), and menarche in girls; and PH2+, genital stage (G2+), and testis >3 mL with orchidometer (Tvol3+) in boys. Results: In boys, pubertal onset was significantly associated with pubertal timing of both parents. PH2+ and Tvol3+ were earlier: −11.8 months (95% confidence interval, −16.8, −6.8)/−8.9 (−12.8, −4.9), and −9.5 (−13.9, −5.1)/−7.1 (−10.4, −3.7) if the father/mother, respectively, had early pubertal development compared to late. In girls, menarche was significantly associated with both parents' pubertal timing: −10.5 months (−15.9, −5.1)/−10.1 (−14.3, −6.0) if father/mother had early pubertal development compared to late. For the onset of PH2+ and B2+ in girls, estimates were −7.0 months (−12.6, −1.4) and −4.1 (−10.6, +2.4)/−6.7 (−11.0, −2.5), and −6.7 (−11.0, −2.0) for fathers/mothers, respectively. Maternal age of menarche was significantly associated with the onset of all pubertal milestones except PH2+ in girls. Conclusions: Maternal as well as paternal pubertal timing was a strong determinant of age at pubertal onset in both girls and boys. Age at breast and pubic hair development in girls, which has declined most during recent years, seemed to be least dependent on heritability.

Phthalates are a group of chemicals present in numerous consumer products. They have anti-androgenic properties in experimental studies and are suspected to be involved in human male reproductive health problems. A few studies have shown associations between phthalate exposure and changes in pubertal timing among girls, although controversies exist. We determined the concentration of 12 phthalate metabolites in first morning urine samples from 725 healthy Danish girls (aged 5.6-19.1 years) in relation to age, pubertal development (breast and pubic hair stage) and reproductive hormone levels (luteinizing hormone, oestradiol and testosterone). Furthermore, urinary phthalates were determined in 25 girls with precocious puberty (PP). In general, the youngest girls with less advanced pubertal development had the highest first morning urinary concentration of the monobutyl phthalate isoforms (∑MBP((i+n))), monobenzyl phthalate (MBzP), metabolites of di-(2-ethylhexyl) phthalate (∑DEHPm) and of di-iso-nonyl phthalate (∑DINPm). After stratification of the urinary phthalate excretion into quartiles, we found that the age at pubarche was increasing with increasing phthalate metabolite quartiles (except for MEP). This trend was statistically significant when all phthalate metabolites (except MEP) were summarized and expressed as quartiles. No association between phthalates and breast development was observed. In addition, there were no differences in urinary phthalate metabolite levels between girls with PP and controls. We demonstrated that delayed pubarche, but not thelarche, was associated with high phthalate excretion in urine samples from 725 healthy school girls, which may suggest anti-androgenic actions of phthalates in our study group of girls.

Vitamin D (VD) is important for male reproduction in mammals and the VD receptor (VDR) and VD-metabolizing enzymes are expressed in human spermatozoa. The VD-inactivating enzyme CYP24A1 titrates the cellular responsiveness to VD, is transcriptionally regulated by VD, and has a distinct expression at the sperm annulus. Here, we investigated if CYP24A1 expression serves as a marker for VD metabolism in spermatozoa, and whether CYP24A1 expression was associated with semen quality. We included 130 men (53 healthy young volunteers and 77 subfertile men) for semen analysis and immunocytochemical (ICC) detection of CYP24A1. Another 40 men (22 young, 18 subfertile) were tested for in vitro effects of 1,25(OH)(2)D(3) on intracellular calcium concentration ([Ca(2+)](i)) and sperm motility. Double ICC staining showed that CYP24A1 and VDR were either concomitantly expressed or absent in 80% of the spermatozoa from young men. The median number of CYP24A1-expressing spermatozoa was 1% in subfertile men and thus significantly (p 3% CYP24A1-positive spermatozoa distinguished young men from subfertile men with a sensitivity of 66.0%, a specificity of 77.9% and a positive predictive value of 98.3%. Functional studies revealed that 1,25(OH)(2)D(3) increased [Ca(2+)](i) and sperm motility in young healthy men, while 1,25(OH)(2)D(3) was unable to increase motility in subfertile patients. In conclusion, we suggest that CYP24A1 expression at the annulus may serve as a novel marker of semen quality and an objective proxy for sperm function.

BACKGROUND In adult women, the circulating level of anti-Mullerian hormone (AMH) is a novel marker of ovarian function, as it reflects the number of remaining ovarian follicles. Therefore, AMH has gained widespread attention in fertility clinics, and a low AMH is believed to predict impaired fertility and imminent menopause. However, the natural course of circulating AMH levels during female childhood and adolescence is not known. METHODS Serum levels of AMH and FSH were measured in girls participating in The COPENHAGEN Puberty Study. Longitudinal part: 85 healthy girls and adolescents were examined, and blood samples were drawn every 6 months for an average of 3 years: median (range) number of samples per girl was 6 (2-10), age at baseline was 9.2 (5.9-12.9) years. Cross-sectional part: 224 prepubertal girls (age 8.3, 5.6-11.7 years) were examined and each girl had one blood sample drawn. RESULTS The individual mean AMH levels in girls followed longitudinally ranged from 5 to 54 pmol/l (median 18 pmol/l). The mean intra-individual coefficient of variation of AMH was 22% (range 0-54%). Overall, each girl maintained her AMH level throughout childhood and adolescence although minor, but significant, changes occurred during pubertal transition. In prepubertal girls, AMH was negatively correlated with FSH (r = -0.31, P < 0.001). Twelve per cent (10/85) had mean AMH below a cut-off value of 8 pmol/l, indicating that the interpretation of low AMH as a marker of approaching menopause may not apply to pre- and peri-pubertal girls. CONCLUSIONS Circulating AMH exhibits only minor fluctuations during childhood and adolescence, and a random AMH measurement seems representative for a given girl. The negative AMH-FSH correlation in prepubertal girls supports the notion that AMH is a quantitative marker of ovarian follicles even in young girls.