Martin Blomberg Jensen

Vitamin D receptor and vitamin D metabolizing enzymes are expressed in the human male reproductive tract

BACKGROUND The vitamin D receptor (VDR) is expressed in human testis, and vitamin D (VD) has been suggested to affect survival and function of mature spermatozoa. Indeed, VDR knockout mice and VD deficient rats show decreased sperm counts and low fertility. However, the cellular response to VD is complex, since it is not solely dependent on VDR expression, but also on cellular uptake of circulating VD and presence and activity of VD metabolizing enzymes. Expression of VD metabolizing enzymes has not previously been investigated in human testis and male reproductive tract. Therefore, we performed a comprehensive analysis of the expression of VDR, VD activating (CYP2R1, CYP27A1, CYP27B1) and inactivating (CYP24A1) enzymes in the testis, epididymis, seminal vesicle (SV), prostate and spermatozoa. METHODS Tissue samples were obtained after orchiectomy (testis n = 13; epididymis n = 7), prostatectomy (prostate n = 5 and SVs n = 3) and semen samples obtained after ejaculation (n = 13). mRNA was detected with RT-PCR and expression of proteins was determined by immunohistochemistry. RESULTS VDR and VD metabolizing enzymes were concomitantly expressed in round and elongated spermatids, vesicles within the caput epididymis, and glandular epithelium of cauda epididymis, SV and prostate. The expression pattern in ejaculated spermatozoa varied, although, concomitant expression of VDR, CYP2R1, CYP27B1 and CYP24A1 was observed in neck and midpiece in a subpopulation of mature spermatozoa. CONCLUSION On the basis of the marked expression of VDR and the VD metabolizing enzymes in human testis, ejaculatory tract and mature spermatozoa, we suggest that VD is important for spermatogenesis and maturation of human spermatozoa.

Human semen quality in the new millennium: a prospective cross-sectional population-based study of 4867 men

Objectives Considerable interest and controversy over a possible decline in semen quality during the 20th century raised concern that semen quality could have reached a critically low level where it might affect human reproduction. The authors therefore initiated a study to assess reproductive health in men from the general population and to monitor changes in semen quality over time. Design Cross-sectional study of men from the general Danish population. Inclusion criteria were place of residence in the Copenhagen area, and both the man and his mother being born and raised in Denmark. Men with severe or chronic diseases were not included. Setting Danish one-centre study. Participants 4867 men, median age 19 years, included from 1996 to 2010. Outcome measures Semen volume, sperm concentration, total sperm count, sperm motility and sperm morphology. Results Only 23% of participants had optimal sperm concentration and sperm morphology. Comparing with historic data of men attending a Copenhagen infertility clinic in the 1940s and men who recently became fathers, these two groups had significantly better semen quality than our study group from the general population. Over the 15 years, median sperm concentration increased from 43 to 48 million/ml (p=0.02) and total sperm count from 132 to 151 million (p=0.001). The median percentage of motile spermatozoa and abnormal spermatozoa were 68% and 93%, and did not change during the study period. Conclusions This large prospective study of semen quality among young men of the general population showed an increasing trend in sperm concentration and total sperm count. However, only one in four men had optimal semen quality. In addition, one in four will most likely face a prolonged waiting time to pregnancy if they in the future want to father a child and another 15% are at risk of the need of fertility treatment. Thus, reduced semen quality seems so frequent that it may impair the fertility rates and further increase the demand for assisted reproduction.

Vitamin D is positively associated with sperm motility and increases intracellular calcium in human spermatozoa

BACKGROUND The vitamin D receptor (VDR) is expressed in human spermatozoa, and VDR-knockout mice and vitamin D (VD) deficiency in rodents results in impaired fertility, low sperm counts and a low number of motile spermatozoa. We investigated the role of activated VD (1,25(OH)(2)D(3)) in human spermatozoa and whether VD serum levels are associated with semen quality. METHODS Cross-sectional association study of semen quality and VD serum level in 300 men from the general population, and in vitro studies on spermatozoa from 40 men to investigate the effects of VD on intracellular calcium, sperm motility and acrosome reaction. All men delivered samples for routine semen analysis and blood for measurements of follicle stimulating hormone, Inhibin B, 25-hydroxy-VD, albumin, alkaline phosphatase, calcium and parathyroid hormone (PTH). RESULTS In the association study, 44% were VD insufficient (<50 nM), and VD was inversely correlated with PTH (P < 0.0005). VD serum levels correlated positively with sperm motility and progressive motility (P < 0.05), and men with VD deficiency (<25 nM) had a lower proportion of motile (P = 0.027), progressive motile (P = 0.035) and morphologically normal spermatozoa (P = 0.044) compared with men with high VD levels (>75 nM). 1,25(OH)(2)D(3) increased intracellular calcium concentration in human spermatozoa through VDR-mediated calcium release from an intracellular calcium storage, increased sperm motility and induced the acrosome reaction in vitro. CONCLUSIONS 1,25(OH)(2)D(3) increased intracellular calcium concentration, sperm motility and induced the acrosome reaction in mature spermatozoa, and VD serum levels were positively associated with sperm motility, suggesting a role for VD in human sperm function.

Phthalate excretion pattern and testicular function: a study of 881 healthy Danish men.

Background: In animals, some phthalates impair male reproductive development and function. Epidemiological studies have reported inconsistent evidence of associations between phthalates and markers of human testicular function. Objectives: We aimed to provide estimates of the effects of phthalate exposure on reproductive hormone levels and semen quality in healthy men. Methods: A total of 881 men gave urine, serum, and semen samples. Serum levels of testosterone, estradiol (E2), sex hormone-binding globulin (SHBG), luteinizing hormone (LH), follicle-stimulating hormone (FSH), and inhibin-B; semen quality; and urinary concentrations of 14 phthalate metabolites, including metabolites of di(2-ethylhexyl) phthalate (DEHP) and diisononyl phthalate (DiNP), were assessed. The proportions of DEHP and DiNP excreted as their respective primary metabolites [mono(2-ethylhexyl) phthalate (MEHP) and mono-isononyl phthalate (MiNP)] were calculated and expressed as percentages (%MEHP and %MiNP, respectively). Results: The free androgen index was 15% lower [95% confidence interval (CI): –23, –8%] for men in the highest %MiNP quartile compared to the lowest quartile (p < 0.001) after adjusting for confounders, and 9% lower (95% CI: –16, –1%) in the highest %MEHP quartile (p = 0.02). %MEHP and %MiNP were negatively associated with the ratio of testosterone/LH and testosterone/FSH. %MEHP was negatively associated with total testosterone, free testosterone, and ratio of testosterone/E2. %MiNP was positively associated with SHBG. There was little evidence of associations between urinary phthalate metabolites or sums of phthalates with reproductive hormones or semen quality Conclusion: Our data suggest that both testosterone production and pituitary–hypothalamic feedback may be compromised in individuals excreting a high proportion of primary metabolites of long-chained phthalates relative to the proportion of secondary metabolites.

REGIONAL DIFFERENCES AND TEMPORAL TRENDS IN MALE REPRODUCTIVE HEALTH DISORDERS: SEMEN QUALITY MAY BE A SENSITIVE MARKER OF ENVIRONMENTAL EXPOSURES

The decline in semen quality has been the subject of an animated debate. A recent prospective study now irrefutably shows a decline in semen quality in men from Finland, a country that previously boasted good semen quality. Semen quality has, in some countries, reached a level where a considerable fraction of young men are at risk of fertility problems. Impaired semen quality, testicular cancer, cryptorchidism and hypospadias are risk factors for each other, and the testicular dysgenesis syndrome (TDS) has been put forward to explain the observations. This syndrome implies that the four disease entities share the same patho-physiological etiology caused by disturbed testicular development in early fetal life. It seems likely that the rapid rise in TDS-associated conditions can, at least partly, be explained by environmental factors. Animal studies provide strong evidence that manmade chemicals can disrupt the hormone dependent pathways responsible for fetal gonadal development, subsequently leading to TDS-like symptoms. In humans, fetal exposure to endocrine disrupting substances may play a role, although genetic factors are probably also involved. Recent studies indicate that exposure to endocrine disrupters also in adulthood may affect semen quality and reproductive hormones. Causal relationships are inherently difficult to establish in humans, and a clear connection between the disorders and specific toxicants has not been established. It seems likely that the cumulative effects of various low-dose exposures to endocrine disrupters in our environment are responsible for the adverse effects in the male reproductive system. Semen quality may be the most sensitive marker of adverse environmental exposures, and we suggest that standardized surveillance studies of semen quality are continued or initiated to monitor the combined effects of various preventive actions.

PFOS (perfluorooctanesulfonate) in serum is negatively associated with testosterone levels, but not with semen quality, in healthy men

STUDY QUESTION Is exposure to perfluorinated compounds (PFCs) associated with testicular function (reproductive hormone levels and semen quality) in healthy men? SUMMARY ANSWER PFOS levels were significantly negatively associated with serum testosterone (total and calculated free), but not with any other reproductive hormones or semen quality. WHAT IS KNOWN ALREADY 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. STUDY DESIGN, SIZE, DURATION A cross-sectional study of 247 men was conducted during 2008-2009. PARTICIPANTS/MATERIALS, SETTING, METHODS 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. MAIN RESULTS AND THE ROLE OF CHANCE 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 2008-2009 were lower than in our previous study of men participating in 2003. LIMITATIONS, REASONS FOR CAUTION 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. WIDER IMPLICATIONS OF THE FINDINGS 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. STUDY FUNDING/COMPETING INTEREST(S) 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.

High dietary intake of saturated fat is associated with reduced semen quality among 701 young Danish men from the general population

BACKGROUND Saturated fat intake has been associated with both cardiovascular disease and cancer risk, and a newly published study found an association between saturated fat intake and a lower sperm concentration in infertile men. OBJECTIVE The objective was to examine the association between dietary fat intake and semen quality among 701 young Danish men from the general population. DESIGN In this cross-sectional study, men were recruited when they were examined to determine their fitness for military service from 2008 to 2010. They delivered a semen sample, underwent a physical examination, and answered a questionnaire comprising a quantitative food-frequency questionnaire to assess food and nutrient intakes. Multiple linear regression analyses were performed with semen variables as outcomes and dietary fat intakes as exposure variables, adjusted for confounders. RESULTS A lower sperm concentration and total sperm count in men with a high intake of saturated fat was found. A significant dose-response association was found, and men in the highest quartile of saturated fat intake had a 38% (95% CI: 0.1%, 61%) lower sperm concentration and a 41% (95% CI: 4%, 64%) lower total sperm count than did men in the lowest quartile. No association between semen quality and intake of other types of fat was found. CONCLUSIONS Our findings are of potentially great public interest, because changes in diet over the past decades may be part of the explanation for the recently reported high frequency of subnormal human sperm counts. A reduction in saturated fat intake may be beneficial for both general and reproductive health.

Vitamin D and male reproduction

Vitamin D is a versatile signalling molecule with a well-established role in the regulation of calcium homeostasis and bone health. The spectrum of vitamin D target organs has expanded and the reproductive role of vitamin D is highlighted by expression of the vitamin D receptor (VDR) and enzymes that metabolize vitamin D in testis, male reproductive tract and human spermatozoa. The expression levels of VDR and CYP24A1 in human spermatozoa serve as positive predictive markers of semen quality, and VDR mediates a nongenomic increase in intracellular calcium concentration that induces sperm motility. Interestingly, functional animal models show that vitamin D is important for estrogen signalling and sperm motility, while cross-sectional studies support the positive association between serum 25-hydroxyvitamin D level and sperm motility in both fertile and infertile men. Expression of VDR and enzymes that metabolize vitamin D in fetal testis indicates a yet unknown role during development, which may be extrapolated from invasive testicular germ cell tumours where 1α,25-dihydroxyvitamin D induces a mesodermal differentiation of the pluripotent testicular cancer cells. Taken together, vitamin D signalling has a positive effect on semen quality, increases estrogen responsiveness and differentiates germ cell tumours. Future studies are needed to determine when 1α,25-dihydroxyvitamin D acts in a paracrine manner and whether systemic changes, which are subject to pharmacological modulation, could influence male reproductive function.Vitamin D is a versatile signalling molecule with a well-established role in the regulation of calcium homeostasis and bone health. The spectrum of vitamin D target organs has expanded and the reproductive role of vitamin D is highlighted by expression of the vitamin D receptor (VDR) and enzymes that metabolize vitamin D in testis, male reproductive tract and human spermatozoa. The expression levels of VDR and CYP24A1 in human spermatozoa serve as positive predictive markers of semen quality, and VDR mediates a nongenomic increase in intracellular calcium concentration that induces sperm motility. Interestingly, functional animal models show that vitamin D is important for estrogen signalling and sperm motility, while cross-sectional studies support the positive association between serum 25-hydroxyvitamin D level and sperm motility in both fertile and infertile men. Expression of VDR and enzymes that metabolize vitamin D in fetal testis indicates a yet unknown role during development, which may be extrapolated from invasive testicular germ cell tumours where 1α,25-dihydroxyvitamin D induces a mesodermal differentiation of the pluripotent testicular cancer cells. Taken together, vitamin D signalling has a positive effect on semen quality, increases estrogen responsiveness and differentiates germ cell tumours. Future studies are needed to determine when 1α,25-dihydroxyvitamin D acts in a paracrine manner and whether systemic changes, which are subject to pharmacological modulation, could influence male reproductive function.

Analysis of meiosis regulators in human gonads: a sexually dimorphic spatio-temporal expression pattern suggests involvement of DMRT1 in meiotic entry

The mitosis-meiosis switch is a key event in the differentiation of germ cells. In humans, meiosis is initiated in fetal ovaries, whereas in testes meiotic entry is inhibited until puberty. The purpose of this study was to examine the expression pattern of meiosis regulators in human gonads and to investigate a possible role of DMRT1 in the regulation of meiotic entry. The expression pattern of DMRT1, STRA8, SCP3, DMC1, NANOS3, CYP26B1 and NANOS2 was investigated by RT-PCR and immunohistochemistry in a series of human testis samples from fetal life to adulthood, and in fetal ovaries. DMRT1 was expressed in testes throughout development but with marked spatio-temporal changes. At the early fetal period of 8-20 gestational weeks (GW) and at infantile mini-puberty, DMRT1 was predominantly expressed in Sertoli cells, whereas at later stages of gestation (22-40 GW), during childhood and in post-pubertal testes, DMRT1 was most abundant in spermatogonia, except in the A-dark type. In fetal ovaries, DMRT1 was detected in oogonia and oocytes until 20 GW, but was completely down-regulated following meiotic entry. STRA8, SCP3 and DMC1 were expressed mainly in oocytes and spermatogonia in accordance with their role in initiation and progression of meiosis. The putative meiosis inhibitors, CYP26B1 and NANOS2, were primarily expressed in Leydig cells and spermatocytes, respectively. In conclusion, the expression pattern of the investigated meiotic regulators is largely conserved in the human gonads compared with rodents, but with some minor differences, such as a stable expression of CYP26B1 in human fetal ovaries. The sexually dimorphic expression pattern of DMRT1 indicates a similar role in the mitosis-meiosis switch in human gonads as previously demonstrated in mice. The biological importance of the changes in expression of DMRT1 in Sertoli cells remains to be established, but it is consistent with DMRT1 reinforcing the inhibition of meiosis in the testis.

Vitamin D metabolism, sex hormones and male reproductive function

: The spectrum of vitamin D (VD)-mediated effects has expanded in recent years, and VD is now recognized as a versatile signaling molecule rather than being solely a regulator of bone health and calcium homeostasis. One of the recently identified target areas of VD is male reproductive function. The VD receptor (VDR) and the VD metabolizing enzyme expression studies documented the presence of this system in the testes, mature spermatozoa, and ejaculatory tract, suggesting that both systemic and local VD metabolism may influence male reproductive function. However, it is still debated which cell is the main VD target in the testis and to what extent VD is important for sex hormone production and function of spermatozoa. This review summarizes descriptive studies on testicular VD metabolism and spatial distribution of VDR and the VD metabolizing enzymes in the mammalian testes and discusses mechanistic and association studies conducted in animals and humans. The reviewed evidence suggests some effects of VD on estrogen and testosterone biosynthesis and implicates involvement of both systemic and local VD metabolism in the regulation of male fertility potential.