CCL20-CCR6 axis directs sperm-oocyte interaction and its dysregulation correlates/associates with male infertility‡.

The interaction of sperm with the oocyte is pivotal during the process of mammalian fertilization. The limited numbers of sperm that reach the fallopian tube as well as anatomic restrictions indicate that human sperm-oocyte encounter is not a matter of chance but a directed process. Chemotaxis is the proposed mechanism for re-orientating sperm toward the source of a chemoattractant and hence to the oocyte. Chemokines represent a superfamily of small (8-11 kDa), cytokine-like proteins that have been shown to mediate chemotaxis and tissue-specific homing of leukocytes through binding to specific chemokine receptors such as CCRs. Here we show that CCR6 is abundantly expressed on human sperms and in human testes. Furthermore, radioligand-binding experiments showed that CCL20 bound human sperm in a specific manner. Conversely, granulosa cells of the oocyte-surrounding cumulus complex as well as human oocytes represent an abundant source of the CCR6-specific ligand CCL20. In human ovaries, CCL20 shows a cycle-dependent expression pattern with peak expression in the preovulatory phase and CCL20 protein induces chemotactic responses of human sperm. Neutralization of CCL20 in ovarian follicular fluid significantly impairs sperm migratory responses. Conversely, analyses in infertile men with inflammatory conditions of the reproductive organs demonstrate a significant increase of CCL20/CCR6 expression in testis and ejaculate. Taken together, findings of the present study suggest that CCR6-CCL20 interaction may represent an important factor in directing sperm-oocyte interaction.

[Impact of lifestyle and environmental factors on male reproductive health]. / Einfluss von Lebensstil und Umweltfaktoren auf die reproduktive Gesundheit des Mannes.

The identification of potential environmental hazards may be clinically relevant in the diagnosis of male infertility. Knowledge about these factors will improve prevention of fertility disorders. Apart from drugs or factors related to lifestyle such as alcohol and tobacco smoke, various environmental and occupational agents, both chemical and physical, may impair male reproductive function. Reproductive toxicity may evolve at the hypothalamic-pituitary, testicular, or post-testicular level; endpoints comprise deterioration of spermatogenesis and sperm function as well as endocrine disorders and sexual dysfunction. With regard to the complex regulation of the male reproductive system, the available information concerning single exogenous factors and their mechanisms of action in humans is limited. This is also due to the fact that extrapolation of results obtained from experimental animal or in vitro studies remains difficult. Nevertheless, the assessment of relevant exposure to reproductive toxicants should be carefully evaluated during diagnostic procedures of andrological patients.

Experimental Escherichia coli epididymitis in rats: assessment of testicular involvement in a long-term follow-up.

The objective of this study was to investigate spermatogenesis and testicular inflammation in a rat model of unilateral Escherichia coli epididymitis in a long-term follow-up. Unilateral epididymitis was induced in 30 Sprague-Dawley rats by injecting E. coli into the right ductus deferens. Oral antimicrobial treatment with sparfloxacin (50 mg kg(-1) body weight/7 days) was administered in half of the animals 24 h after infection. Five treated and five untreated rats were killed at 2 weeks, 3 months and 6 months after infection. Spermatogenesis was investigated using a histological semi-quantitative score. The presence of inflammatory cells (B- and T lymphocytes, macrophages and granulocytes) in the testicular tissues was evaluated by immunohistochemistry. The testes were sterile at all times. Over the course of 6 months, spermatogenesis underwent significant incremental impairment on the inoculated side as compared to the contralateral side (P < 0.001). However, overall spermatogenesis scores were not significantly different between treated and untreated animals (P > 0.3 at each time point). Finally, loss of testicular architecture on the inoculated side was not associated with any cellular inflammatory response. Thus, adjuvant therapies need to be studied, and research is necessary on how to prevent deterioration of testicular function in bacterial epididymitis.

[Dermatological diseases and sexuality. How to proceed?]. / Dermatologische Erkrankungen und Sexualität. Wie geht man vor?

A variety of dermatological diseases is associated with male or female sexual dysfunction. Some sexual disorders are caused organically; erectile dysfunction in men with systemic sclerosis may be due to penile vascular alterations and corporal fibrosis. Other dermatoses such as psoriasis are associated with risk factors (metabolic syndrome) for sexual disorders and may therefore indirectly induce erectile dysfunction. However, the majority of sexual dysfunctions in dermatological patients is caused by reduced self confidence and sexual self esteem leading to affected partnership and sexuality. Dermatologists should be trained in basic sexual medicine and ask their patients for sexual problems. They may not treat sexual disorders, but they should be identified as sympathetic physicians for these problems and should be able to refer the patient to specialists in sexual medicine.

Differential leucocyte detection by flow cytometry improves the diagnosis of genital tract inflammation and identifies macrophages as proinflammatory cytokine-producing cells in human semen.

Genital tract inflammation is considered as a major cause of male infertility with leucocytospermia as widely used diagnostic marker. However, threshold of 10(6) leucocytes ml(-1) recommended by the WHO is a matter of debate. Moreover, leucocyte subpopulations and their impact cannot be identified by the routine peroxidase method (POM). Ejaculates of subfertile men (n = 47) were analysed by flow cytometry (FACS) using a bead-based method. Leucocytes were identified by CD18 and further divided into macrophages (HLA-Dr+/CD66abce-) and neutrophils (HLA-Dr-/CD66abce+). IL-1ß, TNF-α and IL-6 production was investigated in these subpopulations. It was found that CD18-positive cells correlated significantly with POM. However, only in samples with POM below 10(6) per millilitre, FACS detected significantly higher leucocyte numbers. Moreover, in 31% of these samples, FACS leucocyte detection reached threshold values greater than 1 × 10(6)  ml(-1) , fulfilling the criteria for diagnosis of leucocytospermia. Neutrophils were the predominating leucocyte population. Nevertheless, in 24% of samples, macrophages encountered more than 50% of leucocytes. Most interestingly, only macrophages produced significant amounts of IL-1ß, TNF-α and IL-6. It is concluded that FACS improves detection and functional differentiation of seminal leucocytes as one of the diagnostic hallmarks of male genital tract inflammation.

Validation of the German version of the 'Hypogonadism Related Symptom Scale' (HRS) in andrological patients with infertility, HIV infection and metabolic syndrome.

As commonly used self-reported screening instruments for male hypogonadism demonstrated lack of specificity, a Hypogonadism Related Symptom Scale (HRS) was developed in 2009 as a novel self-rating screening tool. As the questionnaire has not been validated, the purpose of our study was to perform a validation in patients presenting with different disorders (e.g. infertility, HIV infection or metabolic syndrome) and disease-related risk to develop hypogonadism. Two hundred and eighteen patients aged 19-71 years (40.1 ± 9.5) who completed the HRS and other common questionnaires [International Index Of Erectile Function (IIEF), National Institutes of Health Chronic Prostatitis Symptom Index (NIH-CPSI), Hospital Anxiety and Depression Scale (HADS), short form (SF)-12] were included. In all patients, blood levels of total testosterone, luteinizing hormone, follicle-stimulating hormone, oestradiol and sex hormone-binding globulin were determined and free testosterone was calculated. Cronbach's α for the scale was 0.896, split-half 0.871 for the 1st half and 0.807 for the 2nd half. Spearman-Brown coefficient was 0.767, and Guttman split-half coefficient was 0.759. Consistent correlations were found between HRS and IIEF5 (ρ = 0.57, P < 0.001), and HADS (ρ = -0.6, P < 0.001). In addition, HRS was significantly correlated with total testosterone (ρ = 0.135, P < 0.05), free testosterone (ρ = 0.148, P < 0.05) and oestradiol (ρ = -0.134, P < 0.05). Our validation study confirms the data from the initial development of the HRS questionnaire. Clinicians might have an additional advantage from the HRS when investigating males with suspected hypogonadism.

Male urogenital infections: impact of infection and inflammation on ejaculate parameters.

BACKGROUND: Urogenital infections and inflammation may contribute significantly to ejaculate parameters essential for male infertility. METHODS: For this review, data were acquired by a systematic search of the medical literature of the last 5 years. RESULTS: We address the andrological relevance of male urogenital infections and inflammation on ejaculate parameters. The different classification systems of the WHO and NIH are illustrated. In most cases, a separation of the different areas of the urogenital tract, for example, of the prostate, epididymis and testicles, is not possible. The significance of bacteriospermia with common bacteria is discussed. Furthermore, HIV, ascending chlamydial, mycoplasmal and gonococcal infections are relevant. Especially, the relevance of sexually transmitted microorganisms seems to be underestimated. Leukocytospermia is not well defined in its biological significance. Seminal plasma elastase and the cytokine expression reveal better insights into the inflammatory response of the seminal pathways. Sperm antibodies and reactive oxygen species are not usable as indicators for infection and inflammation. Different aspects for an impairment of ejaculate quality have been demonstrated although a direct ascension of microorganisms to the prostate has not been confirmed. Probably, lesions of the epididymis may sustain an ongoing disturbance of sperm parameters. A potential negative influence of urogenital infections and inflammation on sperm function is under discussion. However, the severity of impairment differs according to the underlying infections and the involved compartments. CONCLUSIONS: Signs of infections and inflammation in the ejaculate of infertile men are common, and the relevance is often doubtful in spite of microbiological, spermatological and immunological facilities.

Prostatitis and andrological implications.

AIM: The prostatitis syndrome is a frequent disease affecting men in their reproductive age. The prostatitis syndrome is classified according to the National Institutes of Health (NIH) definition. Andrological implications of the prostatitis syndrome might encompass fertility issues, sexual dysfunctions and endocrinological alterations and influences. METHODS: A medline query using the terms prostatitis AND andrological implication, fertility, sexual dysfunction or endocrinology was performed. RESULTS: Acute bacterial prostatitis and andrological implications have not been adequately addressed. Patients with chronic bacterial prostatitis and chronic pelvic pain syndrome have been investigated evaluating sperm parameters. Some studies showed impaired sperm parameters. In chronic bacterial prostatitis, half of the patients reveal significant bacteriospermia with still debatable deleterious effects on sperm quality. Few interventional studies have addressed fertility issues in those patients. Anti-inflammatory treatment perhaps could have a positive impact on sperm parameters. Sexual dysfunction can be described by different components such as erectile, ejaculatory, orgasmic and sexual desire dysfunctions. Sexual dysfunction in chronic prostatitis adds to the number of positive symptom phenotypes and correlates therefore with increasing symptom scores in patients with chronic prostatitis syndromes. However, prospective interventional studies on the role of sexual dysfunctions are missing. Hormones have been found to modulate the inflammatory response via different receptors, particularly via estrogen receptor alpha. This evidence, however, is mainly limited to pre-clinical studies currently. CONCLUSION: Andrological implications are heterogenous and frequently described in patients with chronic prostatitis syndrome. Nonetheless, andrological factors have not been routinely addressed as primary variables in the different studies, which makes further research necessary.

Mutations at the mitochondrial DNA polymerase (POLG) locus associated with male infertility.

Human mitochondrial DNA polymerase, encoded by POLG, contains a polyglutamine tract encoded by a CAG microsatellite repeat. Analysis of POLG genotypes in different populations identified an association between absence of the common, ten-repeat allele and male infertility typified by a range of sperm quality defects but excluding azoospermia.

Influence of urogenital infections and inflammation on semen quality and male fertility.

BACKGROUND: Urogenital infections and inflammation are a significant etiologic factor in male infertility. METHODS: Data for this review were acquired by a systematic search of the medical literature. Relevant cross-references were also taken into account. RESULTS: We address infectious and inflammatory diseases of different compartments of the male genital tract and discuss their andrological sequelae. Chronic urethritis might be responsible for silent genital tract inflammation with negative impact on semen quality. In chronic pelvic pain syndrome, morphological abnormalities of spermatozoa and seminal plasma alterations are detectable. In the majority of men with epididymitis, a transient impairment of semen quality can be found during the acute infection. However, persistent detrimental effects are not uncommon, even after complete bacteriological cure. The relevance of chronic viral infections as an etiologic factor in male infertility is believed to be underestimated. Data concerning the impact of HIV infection on male fertility are of increasing interest as with the improvement in life expectancy, issues of sexuality and procreation gain importance. Moreover, effects of noninfectious systemic inflammation on the male reproductive tract have to be considered in patients with metabolic syndrome, a disorder of growing relevance worldwide. Finally, microbiological and related diagnostic findings in urine and semen samples are reviewed according to their relevance for male infertility. CONCLUSIONS: Available data provide sufficient evidence that in men with alterations of the ejaculate, urogenital infections and inflammation have to be considered.

Prognostic markers for competent human spermatozoa: fertilizing capacity and contribution to the embryo.

Management of male infertility largely depends on our understanding of cellular and molecular aspects of spermatogenesis as well as sperm function. Apart from standardized and comprehensive semen analysis, prognostic markers estimating the fertilizing capacity of either ejaculated spermatozoa or testicular spermatids are required. While there is general agreement that correct replacement of DNA-binding histones by protamines represents a prerequisite for achieving competent spermatozoa, especially in intracytoplasmic sperm injection (ICSI) where natural selection mechanisms are bypassed, the function of a variety of transcripts within the spermatozoa's cytoplasm and of remaining highly acetylated histones is still a matter of debate. Hence, this review brings the up-to-date research on mammalian spermatozoal chromatin composition into focus, which is discussed in conjunction with the paternal role on epigenetic reprogramming of the zygote following fertilization. As paternal transcripts have been demonstrated to be transmitted to the oocyte, it is now accepted that they represent more than solely remnants of previous transcriptional activity. Acetylation of histones, normaly a characteristic of transcriptional activity, was for a long time a miracle, as spermatozoa are transcriptionally inactive cells, but is now suggested to represent epigenetic marks that are transmitted to the oocyte and play an important role in the regulation of early gene expression in the developing embryo.

Immunodeviation towards a Th17 immune response associated with testicular damage in azoospermic men.

Infection and inflammation of the male reproductive tract are thought to be a primary aetiological factor of male infertility. Furthermore, several studies suggest that T lymphocytes are critically involved as regulator in the pathogenesis of male infertility under these conditions and are thought to induce autoimmune orchitis. In this context of autoimmunity the recently described T helper (Th) 17 subset has been suggested to play an essential role so that the aim of this study was to investigate the expression and characteristics of Th17 cells as well as the presence of Th17 inducing antigen presenting cells (APCs) in azoospermic testis with chronic inflammation (ATCI) compared with normal spermatogenesis. By stereological analysis, we detected base line expression of Th17 cells in Con. However, increased expression intensity and number of Th17 cells and their cytokines [interleukin (IL)-17A, IL-21, IL-22] and a decreased level of Foxp3(+) and interferon-γ(+) cells could be demonstrated in ATCI. Moreover, along with these data, increased numbers of Th17-inducing IL-23 producing CD11c(+) and CD68(+) APCs could be detected in ATCI. From these data, a picture emerges that Th17 cells orchestrated by IL-23 producing APCs are critically involved in chronic inflammation in ATCI.

Presence of histone H3 acetylated at lysine 9 in male germ cells and its distribution pattern in the genome of human spermatozoa.

During spermatogenesis, approximately 85% of histones are replaced by protamines. The remaining histones have been proposed to carry essential marks for the establishment of epigenetic information in the offspring. The aim of the present study was to analyse the expression pattern of histone H3 acetylated at lysine 9 (H3K9ac) during normal and impaired spermatogenesis and the binding pattern of H3K9ac to selected genes within ejaculates. Testicular biopsies, as well as semen samples, were used for immunohistochemistry. Chromatin immunoprecipitation was performed with ejaculated sperm chromatin. HeLa cells and prostate tissue served as controls. Binding of selected genes was evaluated by semiquantitative and real-time polymerase chain reaction. Immunohistochemistry of H3K9ac demonstrated positive signals in spermatogonia, spermatocytes, elongating spermatids and ejaculated spermatozoa of fertile and infertile men. H3K9ac was associated with gene promoters (CRAT, G6PD, MCF2L), exons (SOX2, GAPDH, STK11IP, FLNA, PLXNA3, SH3GLB2, CTSD) and intergenic regions (TH) in fertile men and revealed shifts of the distribution pattern in ejaculated spermatozoa of infertile men. In conclusion, H3K9ac is present in male germ cells and may play a role during the development of human spermatozoa. In addition, H3K9ac is associated with specific regions of the sperm genome defining an epigenetic code that may influence gene expression directly after fertilisation.

Validation of reference genes in human testis and ejaculate.

Beta-actin (ACTB), glyceraldehyde-3-phosphate-dehydrogenase (GAPD), Heat Shock Protein 1, beta (HSPCB) and Adenosine Triphosphate subunit 5 beta (ATP5B) with distinct functional characteristics and expression patterns were investigated as suitable references for gene expression studies. We determined the expression stability of the four reference genes in ejaculates, cryopreserved as well as fixed and paraffin-embedded testicular tissue (from fertile and subfertile men) applying real-time qRT-PCR and statistical analysis. The mean gene expressions (mean Ct value) were compared for each gene between the fertile and subfertile donors by using the Wilcoxon-Mann-Whitney test. We did not observe significant statistical differences between variability of genes. To detect random effects, we used the two-way analysis of variance with a hierarchical model. The results show no significant statistical differences between proband and repetition within the probands. Taken together, we concluded that ACTB, GAPD, HSPCB and ATP5B have a variable expression within these samples, but this variability is not statistically significant. This finding demonstrated that all these genes could be appropriated for further studies on gene expression in ejaculate and testis tissue. Therefore, the selection of the suitable reference genes is highly specific for a particular experimental model and validation for each situation, on an individual basis, is a crucial requirement.

[Andrology]. / Andrologie.

Andrology deals with male infertility, erectile dysfunction, loss of libido, ejaculatory disorders, hypogonadism, delayed puberty, male contraception, gynecomastia and aspects of the aging male. New trends in reproductive medicine have influenced the evaluation of andrological patients in recent years. Even loss of ejaculated spermatozoa does not necessarily exclude paternity since testicular sperm extraction has been established in men with obstructive or non-obstructive azoospermia. The most important new aspect in andrology is the publication of the World Health Organization laboratory manual for semen analysis in 2010. Dramatic changes concerning sperm motility and morphology must now be considered for the interpretation of standard semen parameters.

[Andrological diagnostics prior to treatment by assisted reproduction]. / Andrologische Diagnostik vor einer reproduktionsmedizinischen Behandlung.

Infertility is defined as the inability of a couple to succeed in achieving a spontaneous pregnancy after 1 year. Male and female factors contribute to infertility with approximately 40% each. In the remaining cases factors that affect fertility can be found in both partners. The andrological work-up should be started simultaneously with the gynecological diagnostic procedure in order to identify and treat andrological factors related to infertility. Since the majority of intracytoplasmic sperm injection procedures are performed due to andrological infertility, andrological diagnostics can prevent a delay in assisted reproductive technology. The andrological work-up can be necessary before 12 months of unsuccessful conception if the female partner is older than 35 years or andrological factors are present that could impair male fertility.

Testicular immune cell populations and macrophage polarisation in adult male mice and the influence of altered activin A levels.

Detailed morphological characterization of testicular leukocytes in the adult CX3CR1 gfp/+ transgenic mouse identified two distinct CX3CR1 + mononuclear phagocyte (macrophage and dendritic cell) populations: stellate/dendriform cells opposed to the seminiferous tubules (peritubular), and polygonal cells associated with Leydig cells (interstitial). Using confocal microscopy combined with stereological enumeration of CX3CR1gfp/+ cells established that there were twice as many interstitial cells (68%) as peritubular cells (32%). Flow cytometric analyses of interstitial cells from mechanically-dissociated testes identified multiple mononuclear phagocyte subsets based on surface marker expression (CX3CR1, F4/80, CD11c). These cells comprised 80% of total intratesticular leukocytes, as identified by CD45 expression. The remaining leukocytes were CD3+ (T lymphocytes) and NK1.1+ (natural killer cells). Functional phenotype assessment using CD206 (an anti-inflammatory/M2 marker) and MHC class II (an activation marker) identified a potentially tolerogenic CD206+MHCII+ sub-population (12% of total CD45+ cells). Rare testicular subsets of CX3CR1 +CD11c+F4/80+ (4.3%) mononuclear phagocytes and CD3+NK1.1+ (3.1%) lymphocytes were also identified for the first time. In order to examine the potential for the immunoregulatory cytokine, activin A to modulate testicular immune cell populations, testes from adult mice with reduced activin A (Inhba+/-) or elevated activin A (Inha+/-) were assessed using flow cytometry. Although the proportion of F4/80+CD11b+ leukocytes (macrophages) was not affected, the frequency of CD206+MHCII+cells was significantly lower and CD206+MHCII- correspondingly higher in Inha+/- testes. This shift in expression of MHCII in CD206+ macrophages indicates that changes in circulating and/or local activin A influence resident macrophage activation and phenotype and, therefore, the immunological environment of the testis.

[Orchitis]. / Hodenentzündung.

Orchitis can be acutely symptomatic or chronically asymptomatic. Among the acute forms is the rarer isolated orchitis, which is of viral origin in most cases as well as the more frequent secondary orchitis, which is usually the result of an ascending bacterial epididymitis. In addition, sterile forms of orchitis are also seen in patients with systemic autoimmune comorbidities. Chronic asymptomatic orchitis is the term used to describe cellular immune infiltrates in the testes, which are observed in approximately 25% of cases of azoospermia during testicular biopsy. The etiopathogenesis of these infiltrates is largely unknown with postinfection and primary pathogen-independent autoimmune reactions being discussed. Animal experimental models of orchitis may be helpful to investigate the immunological mechanisms involved as well as the therapeutic possibilities.

Activin A target genes are differentially expressed between normal and neoplastic adult human testes: clues to gonocyte fate choice.

BACKGROUND: Human testicular germ cell tumours (TGCT) arise from germ cell neoplasia in situ (GCNIS) cells that originate from foetal germ cell precursors. Activin A is central to normal foetal testis development, and its dysregulation may contribute to TGCT aetiology. OBJECTIVE: (i) To test whether the expression profiles of activin A targets in normal and neoplastic human testes indicates functional links with TGCT progression. (ii) To investigate whether activin A levels influence MMP activity in a neoplastic germ cell line. MATERIALS AND METHODS: (1) Bouin's fixed, paraffin-embedded human testes were utilized for PCR-based transcript analysis and immunohistochemistry. Samples (n = 5 per group) contained the following: (i) normal spermatogenesis, (ii) GCNIS or (iii) seminoma. CXCL12, CCL17, MMP2 and MMP9 were investigated. (2) The human seminoma-derived TCam-2 cell line was exposed to activin A (24 h), and target transcripts were measured by qRT-PCR (n = 4). ELISA (n = 4) and gelatin zymography (n = 3) showed changes in protein level and enzyme activity, respectively. RESULTS: (i) Cytoplasmic CXCL12 was detected in Sertoli and other somatic cells, including those surrounding seminoma cells. Anti-CCL17 labelled only the cytoplasm of Sertoli cells surrounding GCNIS, while anti-MMP2 and anti-MMP9 labelled germline and epithelial-like cells in normal and neoplastic testes. (ii) Exposing TCam-2 cells to activin A (50 ng/mL) elevated MMP2 and MMP9 transcripts (fourfold and 30-fold), while only MMP2 protein levels were significantly higher after activin A (5 ng/mL and 50 ng/mL) exposure. Importantly, gelatin zymography revealed activin A increased production of activated MMP2. DISCUSSION: Detection of CCL17 only in GCNIS tumours may reflect a change in Sertoli cell phenotype to a less mature state. Stimulation of MMP2 activity by activin A in TCam-2 cells suggests activin influences TGCT by modulating the tumour niche. CONCLUSION: This knowledge provides a basis for understanding how physiological changes that influence activin/TGF-ß superfamily signalling may alter germ cell fate.

Influence of moderate cycling on scrotal temperature.

Testicular temperature highly correlates with scrotal temperature. It has been postulated that cycling is associated with increased scrotal temperatures with time and consecutively with impaired semen quality. The aim of this study was to evaluate the influence of moderate cycling on scrotal temperature during highly standardized conditions in an experimental lab. A total of 25 volunteers without a history of infertility and normal andrological examination were included for scrotal temperature evaluation. Scrotal temperatures were measured every minute with a portable data recorder connected with two thermistor temperature sensors, which were attached on either side of the scrotum. A further thermistor sensor was attached on the central surface of the bicycle saddle. Ambient temperature in the study room was adjusted to 22 degrees C throughout the whole experiment. All volunteers started the experiment at the same daytime. Clothing of the volunteers consisted of standardized cotton wool trousers and shirts fitting to body size. After acclimatization to the study room in a sitting posture, each volunteer cycled on an exercise cycle for 60 min with a power of 25 Watt representing a speed of 25.45 km/h respectively. The saddle surface temperature reached in the median 35.59 degrees C after 60 min cycling. Median values of scrotal temperatures increased from 35.75 degrees C at the beginning to 35.82 degrees C after 60 min for the left side and from 35.50 to 35.59 degrees C for the right side. No correlation between cycling duration and scrotal temperatures could be found using multivariate anova for repeated measurements. However, scrotal temperatures during cycling were significantly lower (p < 0.001) compared with the last 10 min in sitting posture before starting cycling with a difference of 1.31 degrees C for the left and 1.46 degrees C for the right side. The present study suggests that moderate cycling under standardized conditions with a power of 25 Watt is not a major genital heat stress factor.