The number of CAG and GGN triplet repeats in the Androgen Receptor gene exert combinatorial effect on hormonal and sperm parameters in young men.

Androgen receptor (AR) is a transcription factor that is activated upon binding to testosterone (T) and is implicated in regulating the expression of reproduction-related genes. The human AR gene (Xq11-12) spans 186,588 bp and eight exons. N-terminal transactivation domain of the encoded AR protein harbours two polymorphic stretches of identical amino acids, a polyglutamine tract (encoded by 8-37 CAG-repeats) and a polyglycine tract (encoded by 10-30 GGN-repeats). We set forward to analyse independent and combinatory effects of the length of these repetitive tracts on male reproductive hormones, testicular and sperm parameters in a population-based cohort of Baltic young men (n = 974; aged 20.1 ± 2.1 years). We designed an assay to amplify and detect simultaneously the variants of both polymorphic repeats. The study revealed that elongated AR CAG tract was associated with lower FSH (linear regression: p = 0.0002, effect per repeat -0.056 IU/L). As a novel finding, the carriers of GGN-stretch with ≥24 repeats showed a trend for decreased sperm concentration (p = 0.027). Although neither of the variants exhibited an isolated effect on circulating T, their allelic combinations modulated serum T levels, as well as sperm concentration. The lowest T was measured for men carrying the AR gene with long CAG (n ≥ 25) and short GGN (n ≤ 21) repeat tracts (mean 18.8 vs. 25.5-28.6 nmol/L for the other AR variants, p = 0.017). The lowest sperm concentration was detected among individuals with both elongated repetitive stretches (CAG, n ≥ 25 and GGN, n ≥ 24; mean 49.0 vs. 68.4-72.1 mill/mL for the other variants; p = 0.00059). The innovative study design enabled to clearly demonstrate a combinatory impact of CAG and GGN repeat lengths at male reproductive parameters. As AR regulates transcription of over 900 genes in many tissues and organs, the combinatory effects of these common repeat-length variants on male physiology in the wider context and across lifetime are still to be assessed.

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

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. 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.

'Carriers of variant luteinizing hormone (V-LH) among 1593 Baltic men have significantly higher serum LH'.

Luteinizing hormone (LH) is a pituitary heterodimeric glycoprotein essential in male and female reproduction. Its functional polymorphic variant (V-LH) is determined by two missense mutations (rs1800447, A/G, Trp8Arg; rs34349826, A/G, Ile15Thr) in the LH ß-subunit encoding gene (LHB; 19q13.3; 1111 bp; 3 exons). Among women, V-LH has been associated with higher circulating LH and reduced fertility, but the knowledge of its effect on male reproductive parameters has been inconclusive. The objective of this study was to assess the effect of V-LH on hormonal, seminal and testicular parameters in the Baltic young men cohort (n = 986; age: 20.1 ± 2.1 years) and Estonian idiopathic infertility patients (n = 607; 35.1 ± 5.9 years). V-LH was detected by genotyping of the underlying DNA polymorphisms using PCR-RFLP combined with resequencing of a random subset of subjects. Genetic associations were tested using linear regression under additive model and results were combined in meta-analysis. No significant difference was detected between young men and infertility patients for the V-LH allele frequency (11.0 vs. 9.3%, respectively). V-LH was associated with higher serum LH in both, the young men cohort (p = 0.022, allelic effect = 0.26 IU/L) and the idiopathic infertility group (p = 0.008, effect = 0.59 IU/L). In meta-analysis, the statistical significance was enhanced (p = 0.0007, resistant to Bonferroni correction for multiple testing; effect = 0.33 IU/L). The detected significant association of V-LH with increased serum LH remained unchanged after additional adjustment for the SNPs previously demonstrated to affect LH levels (FSHB -211G/T, FSHR Asn680Ser, FSHR -29A/G). Additionally, a suggestive trend for association with reduced testicular volume was observed among young men, and with lower serum FSH among infertility patients. The V-LH carrier status did not affect sperm parameters and other circulating reproductive hormones. For the first time, we show a conclusive contribution of V-LH to the natural variance in male serum LH levels. Its downstream clinical consequences are still to be learned.

Study in 1790 Baltic men: FSHR Asn680Ser polymorphism affects total testes volume.

Follicle-stimulating hormone receptor (FSHR) contains two common linked polymorphisms, Thr307Ala (rs6165) and Asn680Ser (rs6166), shown to modulate ovarian function in women. The effect on male fertility and reproductive parameters has been inconclusive. We studied FSHR Asn680Ser polymorphism in a large study group (n = 1790) from the Baltic countries. The population-based Baltic male cohort (Estonians, Latvians, Lithuanians; n = 1052) and Estonian oligo-/azoospermic (sperm concentration <20 × 10(6) /mL) idiopathic infertile patients (n = 738) were genotyped for the FSHR Asn680Ser using PCR-RFLP. Genetic associations were tested using linear regression under additive model and results were combined in meta-analysis. No statistical difference was detected in allelic distribution of the FSHR Asn680Ser between the Baltic cohort and Estonian male infertility group. A consistent significant association was detected between the FSHR Ser680 allele and lower total testes volume in both, the Baltic cohort (p = 0.010, effect = -1.16 mL) and Estonian idiopathic infertility group (p = 0.007, effect = -1.77 mL). In meta-analysis, the statistical significance was enhanced (p = 0.000066, effect = -1.40 mL). Meta-analysis supported further associations with moderate effect between the FSHR Ser680 variant and higher serum FSH (p = 0.072), lower Inhibin B (p = 0.037) and total testosterone (p = 0.034). No statistically significant associations were identified with serum LH and estradiol, and sperm parameters. In conclusion, the study in 1790 Baltic men shows statistically highly significant association of the FSHR Asn680Ser with total testes volume and supportive association with serum reproductive hormone levels indicative to the functional effect of the alternative FSHR variants on male reproductive physiology.

Evaluation of male fertility potential by Toluidine Blue test for sperm chromatin structure assessment.

BACKGROUND: We have previously suggested that the Toluidine Blue (TB) test can be used for sperm chromatin structure assessment. In this study, we wished to evaluate the clinical applicability of the TB test in assessing male fertility potential using well-defined groups of fertile and infertile men. METHODS: Sixty-three fertile and 79 infertile men were tested. Infertility thresholds for the proportion of sperm with abnormal [TB dark cells (TBDCs)] and normal [TB light cells (TBLCs)] chromatin structure were set by the ROC curve analysis. RESULTS: Thresholds of 45% TBDC and 20% TBLC were highly predictive for infertility (specificity of the test: 92 and 90%, respectively), but they were poor predictors of the fertility (sensitivity of the test: 42 and 32%, respectively). Odds ratio for infertility was 7.5 [95% confidence interval (CI): 2.7-20.8] when the 45% TBDC threshold was used and 4.4 (95% CI: 1.7-11.6) when the 20% TBLC threshold was used. CONCLUSIONS: The TB test can be suggested for clinical use as a complementary test for standard semen analysis to diagnose male infertility.

The impact of androgen receptor polymorphism and parental ethnicity on semen quality in young men from Latvia.

Recent studies on young men from the general population have demonstrated geographic and ethnic differences in semen quality. The aim of this study was to investigate whether reported ethnic differences in semen quality might be associated with the maternally derived CAG and GGN polymorphisms in the androgen receptor gene or paternal ethnicity. In total 114 military conscripts from Latvia were included in the study. Information on maternal and parental ethnicity was collected by questionnaires. CAG and GGN repeats were analysed by direct sequencing of leukocyte DNA. Men with Latvian mothers (n = 83) had marginally shorter CAG repeat length (21.6 +/- 2.9) as compared with those with non-Latvian mothers (22.9 +/- 3.2, n = 31), not reaching statistical significance (p = 0.053). Sperm concentration did not differ significantly between these two groups (76 +/- 59 and 70 +/- 52, p = 0.9 respectively). In contrast, significantly higher sperm concentration and total sperm count were found in men with Latvian fathers (n = 77) as compared with men with non-Latvian fathers (n = 37) (80 +/- 61 vs. 62 +/- 48, p = 0.035, for sperm concentration and 225.7 +/- 209 vs. 158.4 +/- 134.4, p = 0.002, for total sperm count respectively). CAG repeat length did not correlate with any semen parameters in the whole population. However, GGN repeat length correlated with semen volume: men with GGN > 23 presented with higher semen volume (3.2 +/- 2.1) as compared with men with GGN = 23 (2.6 +/- 1.3, p = 0.04) or GGN < 23 (2.0 +/- 1.2, p = 0.006). We conclude that GGN repeat length has an impact on semen volume, whereas differences in sperm numbers are associated with the paternal ethnicity.

Seminal plasma albumin: origin and relation to the male reproductive parameters.

We wanted to investigate the origin of seminal plasma albumin and its relation to the male reproductive parameters. Semen samples from 916 men, under infertility assessment, were analysed according to guidelines of the World Health Organization. Seminal plasma constituents, i.e. albumin, markers of the epididymal (neutral alpha-glucosidase, NAG), prostatic (prostate-specific antigen, PSA, and zinc) and seminal vesicle function (fructose), as well as levels of reproductive hormones in plasma were measured. The sperm chromatin structure assay (SCSA) was applied on 267 of the 916 samples. A negative correlation was seen for seminal albumin and plasma follicle-stimulating hormone (r=-0.1, P=0.02) and a positive correlation for seminal albumin and serum inhibin B (r=0.2, P=0.004). Albumin exhibited positive correlations with the epididymal marker, NAG (r=0.5, P<0.001) and with the prostatic markers, PSA and zinc (r=0.1, P=0.001; r=0.2, P<0.001 respectively) as well as with age (r=0.2, P<0.001). A negative significant association was seen for seminal albumin and semen volume (beta=-0.60; 95% CI -0.80 to -0.30). The opposite trend was found regarding sperm concentration (beta=0.34; 95% CI 0.30-0.40), total sperm count (beta=0.30; 95% CI 0.20-0.40), and percentage morphologically normal spermatozoa (beta=0.70; 95% CI 0.10-1.0). No association was found between albumin and sperm motility, SCSA parameters, or fructose, the marker of seminal vesicles. Our results suggest testicular, epididymal and prostatic origin of seminal plasma albumin, in addition to the contribution from blood. This is the first study to demonstrate an association between seminal plasma albumin and sperm morphology. Further studies are needed to elucidate the role of seminal albumin in sperm morphology.

Sperm DNA integrity assessment in prediction of assisted reproduction technology outcome.

BACKGROUND: The sperm chromatin structure assay (SCSA) has been suggested as a predictor of fertility in vivo as well as in vitro. The available data however, have been based on limited numbers of treatments. We aimed to define the clinical role of SCSA in assisted reproduction. METHODS: A total of 998 cycles [387 intrauterine insemination (IUI), 388 IVF and 223 ICSI] from 637 couples were included. SCSA results were expressed as DNA fragmentation index (DFI) and high DNA stainable (HDS) cell fractions. Outcome parameters were biochemical pregnancy (BP), clinical pregnancy (CP) and delivery (D). RESULTS: For IUI, the odds ratios (ORs) for BP, CP and D were significantly lower for couples with DFI >30% as compared with those with DFI < or =30%. No statistical difference between the outcomes of ICSI versus IVF in the group with DFI < or =30% was seen. In the DFI >30% group, the results of ICSI were significantly better than those of IVF. CONCLUSIONS: DFI can be used as an independent predictor of fertility in couples undergoing IUI. As a result, we propose that all infertile men should be tested with SCSA as a supplement to the standard semen analysis. When DFI exceeds 30%, ICSI should be the method of choice.

Intra-individual variation in sperm chromatin structure assay parameters in men from infertile couples: clinical implications.

BACKGROUND: Sperm DNA integrity is an important factor in the prognosis of male fertility. In this study, we investigated intra-individual variation of sperm chromatin structure assay (SCSA) parameters in infertility patients undergoing assisted reproductive techniques (ARTs). METHODS: Retrospective study of 282 consecutive patients referred for ART [intrauterine insemination (IUI), IVF or ICSI] with repeated (between 2 and 5) SCSA measurements. RESULTS: Mean coefficient of variation (CV) of DNA Fragmentation Index (DFI) for repeated SCSA measurements was 29%. A high proportion [37%; 95% confidence interval (CI): 27%, 49%] of patients with DFI >30% in the first test had DFI <30% in the second test. Also, a considerable proportion (27%; 95% CI : 16%, 40%) of patients with 21-30% DFI values in the first test had DFI >30% in the second test. CONCLUSIONS: Intra-individual variability in DFI is significant, therefore repeated SCSA measurements are recommended. The biological mechanisms behind these variations remain to be elucidated.

Sperm chromatin structure and male fertility: biological and clinical aspects.

Sperm chromatin/DNA integrity is essential for the accurate transmission of paternal genetic information, and normal sperm chromatin structure is important for sperm fertilizing ability. The routine examination of semen, which includes sperm concentration, motility and morphology, does not identify defects in sperm chromatin structure. The origin of sperm DNA damage and a variety of methods for its assessment are described. Evaluation of sperm DNA damage appears to be a useful tool for assessing male fertility potential both in vivo and in vitro. The possible impact of sperm DNA defects on the offspring is also discussed.