A.-M. Andersson

Changes in Anti-Müllerian Hormone (AMH) throughout the Life Span: A Population-Based Study of 1027 Healthy Males from Birth (Cord Blood) to the Age of 69 Years

CONTEXTnAnti-Müllerian hormone (AMH), which is secreted by immature Sertoli cells, triggers the involution of the fetal Müllerian ducts. AMH is a testis-specific marker used for diagnosis in infants with ambiguous genitalia or bilateral cryptorchidism.nnnAIMnThe aim of the study was to describe the ontogeny of AMH secretion through life in healthy males.nnnSETTINGnThis was a population-based study of healthy volunteers.nnnPARTICIPANTSnPARTICIPANTS included 1027 healthy males from birth (cord blood) to 69 yr. A subgroup was followed up longitudinally through the infantile minipuberty [(in cord blood, and at 3 and 12 months), n=55] and another group through puberty [(biannual measurements), n=83].nnnMAIN OUTCOME MEASURESnSerum AMH was determined by a sensitive immunoassay. Serum testosterone, LH, and FSH were measured, and pubertal staging was performed in boys aged 6 to 20 yr (n=616).nnnRESULTSnSerum AMH was above the detection limit in all samples with a marked variation according to age and pubertal status. The median AMH level in cord blood was 148 pmol/liter and increased significantly to the highest observed levels at 3 months (P<0.0001). AMH declined at 12 months (P<0.0001) and remained at a relatively stable level throughout childhood until puberty, when AMH declined progressively with adults exhibiting 3-4% of infant levels.nnnCONCLUSIONnBased on this extensive data set, we found detectable AMH serum levels at all ages, with the highest measured levels during infancy. At the time of puberty, AMH concentrations declined and remained relatively stable throughout adulthood. The potential physiological role of AMH and clinical applicability of AMH measurements remain to be determined.

Hypothesis: exposure to endocrine-disrupting chemicals may interfere with timing of puberty

A recent decline in onset of puberty - especially among girls - has been observed, first in the US in the mid-1990s and now also in Europe. The development of breast tissue in girls occurs at a much younger age and the incidence of precocious puberty (PP) is increasing. Genetic factors and increasing prevalence of adiposity may contribute, but environmental factors are also likely to be involved. In particular, the widespread presence of endocrine-disrupting chemicals (EDCs) is suspected to contribute to the trend of earlier pubertal onset. The factors regulating the physiological onset of normal puberty are poorly understood. This hampers investigation of the possible role of environmental influences. There are many types of EDCs. One chemical may have more than one mode of action and the effects may depend on dose and duration of the exposure, as well as the developmental stage of the exposed individual. There may also be a wide range of genetic susceptibility to EDCs. Human exposure scenarios are complex and our knowledge about effects of mixtures of EDCs is limited. Importantly, the consequences of an exposure may not be apparent at the actual time of exposure, but may manifest later in life. Most known EDCs have oestrogenic and/or anti-androgenic actions and only few have androgenic or anti-oestrogenic effects. Thus, it appears plausible that they interfere with normal onset of puberty. The age at menarche has only declined by a few months whereas the age at breast development has declined by 1 year; thus, the time span from initiation of breast development to menarche has increased. This may indicate an oestrogen-like effect without concomitant central activation of the hypothalamic-pituitary axis. The effects may differ between boys and girls, as there are sex differences in age at onset of puberty, hormonal profiles and prevalence of precocius puberty.

Adverse trends in male reproductive health: we may have reached a crucial ‘tipping point’

Healthy men produce an enormous number of sperms, far more than necessary for conception. However, several studies suggest that semen samples where the concentration of sperms is below 40 mill/mL may be associated with longer time to pregnancy or even subfertility, and specimens where the concentration of sperms is below 15 mill/mL may carry a high risk of infertility. Historic data from the 1940s show that the bulk of young men at that time had sperm counts far above 40 mill/mL with averages higher than 100 mill/mL. However, recent surveillance studies of young men from the general populations of young men in Northern Europe show that semen quality is much poorer. In Denmark approximately 40 percent of the men have now sperm counts below 40 mill/mL. A simulation assuming that average sperm count had declined from 100 mill/mL in ‘old times’ to a current level close to 40 mill/mL indicated that the first decline in average sperm number of 20–40 mill/mL might not have had much effect on pregnancy rates, as the majority of men would still have had counts far above the threshold value. However, due to the assumed decline in semen quality, the sperm counts of the majority of 20 year old European men are now so low that we may be close to the crucial tipping point of 40 mill/mL spermatozoa. Consequently, we must face the possibility of more infertile couples and lower fertility rates in the future.

Associations between urinary metabolites of di(2-ethylhexyl) phthalate and reproductive hormones in fertile men

Widely used man-made chemicals, including phthalates, can induce hormonal alterations through a variety of cellular and molecular mechanisms. A number of rodent and observational studies have consistently demonstrated the anti-androgenic effect of several phthalates. However, there are only limited data on the relationship between exposure to these chemicals and reproductive hormone levels in men. All men (n=425) were partners of pregnant women who participated in the Study for Future Families in five US cities and provided urine and serum samples on the same day. Eleven phthalate metabolites were measured in urine and serum samples were analysed for reproductive hormones, including follicle-stimulating hormone, luteinizing hormone, testosterone, inhibin B and oestradiol and sex hormone-binding globulin (SHBG). Pearson correlations and parametric tests were used for unadjusted analyses, and multiple linear regression analysis was performed controlling for appropriate covariates. We observed weak or no associations with urinary phthalates other than di(2-ethylhexyl) phthalate (DEHP). All measures of testosterone [total, calculated free testosterone and the free androgen index (FAI)] were inversely correlated with the urinary concentrations of four DEHP metabolites. After adjustment by appropriate covariates, there was no longer an association between urinary DEHP metabolite concentrations and total testosterone levels; however, FAI was significantly associated with the urinary concentrations of several DEHP metabolites. SHBG was positively related to the urinary concentrations of mono(2-ethylhexyl) phthalate, but not with other DEHP metabolites, an association that was attenuated after adjustment. Our results suggest that DEHP exposure of fertile men is associated with minor alterations of markers of free testosterone.

Association between perfluorinated compounds and time to pregnancy in a prospective cohort of Danish couples attempting to conceive

BACKGROUNDnPerfluorinated chemicals (PFCs) have been widely used and have emerged as important food contaminants. A recent study on pregnant women suggested that PFC exposure was associated with a longer time to pregnancy (TTP). We examined the association between serum concentrations of PFCs in females and TTP in 222 Danish first-time pregnancy planners during the years 1992-1995.nnnMETHODSnThe couples were enrolled in the study when discontinuing birth control and followed for six menstrual cycles or until a clinically recognized pregnancy occurred. Fecundability ratio (FR) was calculated using discrete-time survival models. In addition, odds ratio (OR) for TTP >6 cycles was calculated.nnnRESULTSnOR for TTP >6 cycles for those with PFC concentrations above the median were 0.96 [95% confidence interval (CI): 0.54-1.64] for perfluorooctane sulfonic acid (PFOS), the major PFC, compared with those below the median. FRs for those with PFOS concentrations above the median were 1.05 (95% CI: 0.74-1.48) compared with those below the median. Other PFCs showed the same lack of association with TTP. The results were not affected by adjustment for covariates. PFOS and perfluorooctanoic acid concentrations were similar to those observed in a previous Danish study.nnnCONCLUSIONSnThese findings suggest that exposure to PFCs affects TTP only to a small extent, if at all.

Habitual alcohol consumption associated with reduced semen quality and changes in reproductive hormones; a cross-sectional study among 1221 young Danish men.

Objective Study associations between three measures of alcohol consumption (recent, typical/habitual, binging), semen quality and serum reproductive hormones. Design Cross-sectional population based study. Setting and participants 1221 young Danish men, aged 18–28u2005years were recruited when they attended a compulsory medical examination to determine their fitness for military service from 2008 to 2012. Total alcohol consumption: (1) in the week preceding (habitual/typical) the visit (recent alcohol intake), (2) in a typical week and (3) frequency of ‘binge drinking’ (consuming more than 5 units/day)) in the past 30u2005days was estimated. Main outcome measures Semen quality (volume, sperm concentration, total sperm count, and percentages of motile and morphologically normal spermatozoa) and serum concentration of reproductive hormones (follicle-stimulating hormone, luteinising hormone, testosterone, sex hormone binding globulin, oestradiol, free testosterone and inhibin B). Results Sperm concentration, total sperm count and percentage of spermatozoa with normal morphology were negatively associated with increasing habitual alcohol intake. This association was observed in men reporting at least 5 units in a typical week but was most pronounced for men with a typical intake of more than 25 units/week. Men with a typical weekly intake above 40 units had a 33% (95% CI 11% to 59%) reduction in sperm concentration compared to men with an intake of 1–5 units/week. A significant increase in serum free testosterone with increasing alcohol consumption the week preceding the visit was found. Binging was not independently associated with semen quality. Conclusions Our study suggests that even modest habitual alcohol consumption of more than 5 units per week had adverse effects on semen quality although most pronounced associations were seen in men who consumed more than 25 units per week. Alcohol consumption was also linked to changes in testosterone and SHBG levels. Young men should be advised to avoid habitual alcohol intake.

Urinary Phthalates From 168 Girls and Boys Measured Twice a Year During a 5-Year Period: Associations With Adrenal Androgen Levels and Puberty

BACKGROUNDnLittle is known about the possible deleterious effects of phthalate exposure on endogenous sex steroid levels in children.nnnOBJECTIVEnOur objective was to investigate whether urinary phthalate metabolite levels are associated with circulating adrenal androgen levels and age at puberty.nnnMETHODSnThis was a longitudinal study of 168 healthy children (84 girls) examined every 6 months for 5 years. Serum levels of dehydroepiandrostenedione sulfate (DHEAS), Δ4-androstenedione, testosterone, and urinary morning excretion of 14 phthalate metabolites, corresponding to 7 different phthalate diesters were determined. A variation in urinary excretion of phthalates was evident in each child, which made a mean of repetitive samples more representative for long-term excretion than a single determination.nnnRESULTSnWe found that girls with excretion of monobutyl phthalate isomers (MBP) and di(2-ethylhexyl) phthalate metabolites above the geometric group mean (795 and 730 ng/kg, respectively) had lower levels of DHEAS and Δ4-androstenedione, although statistically significant only at 13 years of age. In boys, we found that excretion of monobenzyl phthalate above the geometric group mean (346 ng/kg) was associated with lower levels of DHEAS at 11 years of age but higher levels of testosterone at 13 years of age. The same trend was observed for MBP excretion, albeit not statistically significant. A lower age at pubarche was observed in boys with excretion of MBP above the geometric group mean (11.0 vs 12.3 years, P = 0.005).nnnCONCLUSIONnOur data indicate that exposure to dibutyl phthalate isomers (DBP) (in girls) and butylbenzyl phthalate (in boys) are negatively associated with adrenal androgen levels and in boys positively associated with testosterone level at 13 years of age. High exposure to DBP was associated with earlier age at pubarche in boys. In girls, no associations between phthalate exposure and age at pubertal milestones were observed.

Hormonal changes during GnRH analogue therapy in children with central precocious puberty.

Gonadotropin releasing hormone analogues (GnRHa) have been used for treatment of central precocious puberty (CPP) for more than 15 years. They are generally considered safe although data on potential long-term side effects are scarce. However, GnRHa therapy has profound effects on both the hypothalamopituitary-gonadal axis as well as on growth hormone (GH) secretion. Gonadal activity is increased in children with CPP; during GnRHa therapy secretion of gonadal hormones is suppressed as reflected by measurements of LH, FSH, and estradiol/testosterone. More recently, studies of levels of inhibin A and B as well as markers of androgen action such as SHBG and prostate specific antigen have demonstrated marked suppression of gonadal function possibly to infra-physiological levels. The possible long-term consequences of these observations have yet to be determined. Detailed analyses of the GH-IGF-I axis have revealed a decrease in levels of free, biologically active IGF-I during GnRHa treatment. These findings are in accord with the observed decrease in height velocity in children with CPP under treatment with GnRHa, and may also play a role in the relatively small gain in final height in most patients.

Compensated reduction in Leydig cell function is associated with lower semen quality variables: a study of 8182 European young men

STUDY QUESTIONnIs the Leydig cell function of young European men associated with semen quality?nnnSUMMARY ANSWERnCompensated reduction in Leydig cell function, defined as increased LH concentration combined with adequate testosterone production is associated with lower semen quality.nnnWHAT IS ALREADY KNOWNnSemen 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.nnnSTUDY DESIGN, SIZE AND DURATIONnA coordinated, cross-sectional population-based study of 8182 men undertaken in 1996-2010.nnnPARTICIPANTS, SETTING AND METHODnYoung 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.nnnMAIN RESULT AND ROLE OF CHANCEnSerum 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.nnnLIMITATIONS, REASON FOR CAUTIONnThe 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.nnnWIDER IMPLICATIONS OF THE FINDINGSnCompensated 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.nnnSTUDY FUNDING/COMPETING INTERESTSnSupport 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. Møller and wife Chastine McKinney Møllers 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.

Infant exposure to persistent organochlorine compounds is higher in Denmark than in Finland.

Significantly poorer male reproductive health has been documented in Danish compared to Finnish men, including a higher prevalence of cryptorchidism and hypospadias. Exposure to environmental pollutants with endocrine disrupting activities has been suggested as a possible contributing factor. In this study, we investigated whether there was a difference in milk and placenta concentrations of persistent organohalogen compounds between the two countries. Organohalogens were analyzed by high-resolution gas chromatography-high resolution mass spectrometry in human milk samples from Finland (n=65) and Denmark (n=65) and placentas (n=168 Danish, 112 Finnish), selected from a population-based cohort. p, p’-DDE was the dominant pollutant. β-HCH, HCB, END-I, dieldrin, OXC, c-HE, and p, p’-DDT were the other main organochlorines detected. Danish samples had significantly higher concentrations of p, p’-DDE, p, p’-DDT, β-HCH, HCB, dieldrin, c-HE, and OXC than Finnish. The organochlorine levels were higher in Danish samples, which suggests higher exposure of Danish infants.