Expression patterns of HENMT1 and PIWIL1 in human testis: implications for transposon expression.

This study aimed to define the expression patterns of HENMT1 and PIWI proteins in human testis and investigate their association with transposon expression, infertility sub-type or development of testicular germ cell tumours (TGCTs). Testis biopsies showing normal spermatogenesis were used to identify normal localisation patterns of HENMT1 and PIWIL1 by immunolocalisation and RT-PCR after laser microdissection. 222 testis biopsies representing normal spermatogenesis, hypospermatogenesis, spermatogenic arrests, Sertoli cell-only (SCO) tumours and TGCTs were analysed by RT-qPCR for expression of HENMT1/PIWIL1/PIWIL2/PIWIL3/PIWIL4 and LINE-1 Additionally, HENMT1-overexpressing TCam2 seminoma cell lines were analysed for the same parameters by RT-qPCR. We found that HENMT1 and PIWIL1 are coexpressed in pachytene spermatocytes and spermatids. Expression of HENMT1, PIWIL1 and PIWIL2 was mainly dependent on germ cell content but low levels of expression were also detected in some SCO samples. Levels of HENMT1, PIWIL1 and PIWIL2 expression were low in TGCT. Samples with HENMT1, PIWIL2 and PIWIL4 expression showed significantly (P < 0.05) lower transposon expression compared to samples without expression in the same histological group. HENMT1-overexpressing TCam2 cells showed lower LINE-1 expression than empty vector-transfected control lines. Our findings support that the transposon-regulating function of the piRNA pathway found in the mouse is conserved in adult human testis. HENMT1 and PIWI proteins are expressed in a germ-cell-specific manner and required for transposon control.

Current insights into the sulfatase pathway in human testis and cultured Sertoli cells.

Within the human testis, large amounts of sulfated steroid hormones are produced. As shown in breast tissue and placenta, these might not only be excretion intermediates, but re-activated in target cells by steroid sulfatase (STS). This process is called sulfatase pathway and may play a pivotal role in para- and/or intracrine regulation by creating a local supply for steroid hormones. This requires a facilitated transport via uptake carriers and efflux transporters as these hydrophilic molecules cannot pass the cell membrane. Moreover, blood-testis barrier formation in the testis requires a transport through Sertoli cells (SCs) to reach germ cells (GCs). Sertoli cells are therefore expected to play a key role as gate-keepers for sulfatase pathway in human seminiferous epithelium. We analyzed the mRNA and protein expression of uptake carriers and efflux transporters like organic anion-transporting polypeptides (OATP2B1, OATP3A1) and multidrug resistance-related proteins (MRP1, MRP4) in testicular tissue and cultured Sertoli cells (FS1, HSEC). Additionally, expression pattern of STS as well as sulfonating enzymes (SULTs) were assessed. OATP2B1, OATP3A1 and STS were detected in SCs as well as GCs, whereas MRP1 is only expressed in SCs, and SULT1E1 only in Leydig cells, respectively. By transcellular transport of [H3]DHEAS in HSEC, we showed a functional transport of sulfated steroids in vitro. Our data indicate that steroid synthesis via sulfatase pathway in Sertoli cells in vivo and in vitro is possible and may contribute to paracrine and intracrine regulation employing the local supply of sulfated and free steroid hormones inside seminiferous tubules.

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.

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.

Validation, Deployment, and Real-World Implementation of a Modular Toolbox for Alzheimer's Disease Detection and Dementia Risk Reduction: The AD-RIDDLE Project.

The Real-World Implementation, Deployment, and Validation of Early Detection Tools and Lifestyle Enhancement (AD-RIDDLE) project, recently launched with the support of the EU Innovative Health Initiative (IHI) public-private partnership and UK Research and Innovation (UKRI), aims to develop, test, and deploy a modular toolbox platform that can reduce existing barriers to the timely detection, and therapeutic approaches in Alzheimer's disease (AD), thus accelerating AD innovation. By focusing on health system and health worker practices, AD-RIDDLE seeks to improve and smooth AD management at and between each key step of the clinical pathway and across the disease continuum, from at-risk asymptomatic stages to early symptomatic ones. This includes innovation and improvement in AD awareness, risk reduction and prevention, detection, diagnosis, and intervention. The 24 partners in the AD-RIDDLE interdisciplinary consortium will develop and test the AD-RIDDLE toolbox platform and its components individually and in combination in six European countries. Expected results from this cross-sectoral research collaboration include tools for earlier detection and accurate diagnosis; validated, novel digital cognitive and blood-based biomarkers; and improved access to individualized preventative interventions (including multimodal interventions and symptomatic/disease-modifying therapies) across diverse populations, within the framework of precision medicine. Overall, AD-RIDDLE toolbox platform will advance management of AD, improving outcomes for patients and their families, and reducing costs.

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.

Role of human papillomaviruses in persistent and glucocorticoid-resistant juvenile phimosis.

BACKGROUND: In adults, human papillomaviruses (HPV), lichen sclerosus et atrophicus (LSA) and phimosis are considered to be major risk factors for penile cancer. In boys, a possible association between phimosis, LSA and HPV has been suggested. OBJECTIVE: To investigate the role of HPV in the persistence of phimosis in children. PATIENTS AND METHODS: Out of a cohort of 420 boys presenting with foreskin problems, we prospectively sampled the preputial tissue of 82 patients during circumcision: 46 with steroid-naïve and 36 with steroid-resistant phimosis. All foreskins were assessed clinically and histopathologically with regard to appearance, inflammation, oedema, epithelial degeneration and fibrosis. The viral status of the foreskins was determined by immunohistochemistry and highly sensitive PCR, with subsequent subtyping by DNA hybridization (HPV types 6, 11, 16, 18, 31, 33, 35, 39, 42, 44, 45, 51-54, 56, 58, 59, 61, 62, 66-68, 70, 72, 73, 81-84, 90, 91). RESULTS: The foreskins appeared normal in 62 boys and suggestive of LSA in one single case. Small cracks or white scars were present in seven steroid-naïve and 12 steroid-resistant foreskins. LSA was diagnosed microscopically in two of the steroid-naïve and six of the steroid-pretreated group. No evidence of HPV was found in any of the juvenile foreskins. CONCLUSIONS: Our prospective study has provided evidence that HPV is not usually present in the foreskin of boys with persistent phimosis after their first year of life and that topical glucocorticoid treatment failure is not associated with HPV or any specific histopathological changes.

Validation of RiskCheck Bladder Cancer ©, version 5.0 for risk-adapted screening of bladder cancer.

INTRODUCTION: The aim of the study was to assess the strength of the online tool RiskCheck Bladder Cancer©, version 5.0 (RCBC) for early detection of bladder cancer (BC). MATERIALS AND METHODS: RCBC was evaluated retrospectively based on the data of 241 patients, of which 141 were suffering from BC. Statistical analysis was performed by descriptive statistics, nonparametric group comparison, classification tree analysis and ROC analysis. RESULTS: ROC analysis of the risk classification showed a sensitivity of 71.6%, a specificity of 56.5%, a positive predictive value of 67.8%, a negative predictive value of 52% and an accuracy of 63.5%. BC risk factors ranked by importance are time of smoking (p < 0.0001), gender (within the nonsmoking group: p < 0.009), occupational toxin exposure (within the group <35 years of smoking: p < 0.048) and amount of consumed cigarettes resulting in a 95% association with BC (within the group >35 years of smoking: p < 0.0001). CONCLUSIONS: The high predictive power of RCBC for the identification of asymptomatic patients living under risk could be demonstrated.

Prevention of recurrent urinary tract infections.

Urinary tract infections (UTI) are among the most frequent bacterial infections in the community and health care setting. Mostly young and, to some extent, postmenopausal women are affected by recurrent UTI (rUTI) defined as ≥3 UTI/year or ≥2 UTI/half year. In contrast, rUTI is rare in healthy men. On the other hand, rUTI are frequently found in female and male patients with complicating urological factors, e.g. urinary catheters, infection stones. Remediable predisposing factors in uncomplicated rUTI in women are rare. In complicated rUTI the success depends mainly on the possibility to eliminate or at leastimprove the complicating risk factors. Continuous antibiotic prophylaxis or postcoital prophylaxis, if there is close correlation with sexual intercourse, are most effective to prevent rUTI. Nitrofurantoin, trimethoprim (or cotrimoxazole), and fosfomycin trometamol are available as first-line drugs. Oral cephalosporins and quinolones should be restricted to specific indications. Antibiotic prophylaxis reduces the number of uropathogens in the gut and/or vaginal flora and reduces bacterial "fitness". Given the correct indication, the recurrence rate of rUTI can be reduced by about 90%. Due to possible adverse events and the concern of selecting resistant pathogens, according to the guidelines of the European Association of Urology antimicrobial prophylaxis should be considered only after counselling, behavioural modification and non-antimicrobial measures have been attempted. In postmenopausal patients vaginal substitution of oestriol should be started first. Oral or parenteral immunoprophylaxis is another option in patients with rUTI. Other possibilities with varying scientific evidence are prophylaxis with cranberry products, specific plant combinations or probiotics. The prophylaxis of catheter-associated UTI should employ strategies which result in a reduction of frequency and duration of catheter drainage of the urinary tract. The currently available catheter materials have only little influence on reducing catheter-associated rUTI.

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.

Reference values for testicular volume, epididymal head size and peak systolic velocity of the testicular artery in adult males measured by ultrasonography.

PURPOSE: To obtain ultrasonography-based reference values for testicular volume, epididymal head size and peak systolic velocity (PSV) of the testicular artery in adult males of all ages. MATERIALS AND METHODS: Between 2009 and 2011, 306 Caucasian adult males (median age: 51 years; range: 18-88 years) without scrotal pathology underwent prospective scrotal ultrasonography. The testicular volume was calculated from the length (L), width (W), and height (H) using three formulas: a) 0.52 × L × W × H, b) 0.52 × L × W², and c) 0.71 × L × W × H. Thickness and height of the epididymal head and PSV of the testicular artery were measured. RESULTS: The median testicular volumes on the right (left) side were 13.9 (12.7) ml, 18.1 (16.5) ml, and 18.9 (17.3) ml for formula a), b), and c) respectively, and thus significantly different (p < 0.01 for all). The left testes were significantly smaller than the right testes (p < 0.01). The thickness and height of the right (left) epididymal head measured 7.5 (7.7) mm and 11.6 (11.3) mm, respectively. Median PSV of the right (left) testicular artery was 8.7 (8.6) cm/sec. No significant side-specific differences were documented with respect to epididymal size and PSV. CONCLUSION: It was possible to obtain virtually age-independent reference values for testicular volume, epididymal head size and PSV of the testicular artery in adults. With regard to testicular volumetry, it is essential to consider which formula has been used, since the calculated volumes differ significantly from formula to formula.

Mixed testicular atrophy related to atherosclerosis: first lessons from the ApoE(-/-)/ LDL receptor(-/-) double knockout mouse model.

Age-related testicular changes are associated with declining spermatogenesis and testosterone levels. A relationship to atherosclerosis has never been investigated systematically. The ApoE(-/-)/LDL receptor(-/-) double knockout mouse model, providing a remarkable homology to human atherosclerosis, is an ideal tool to investigate spermatogenetic alterations in this context. Testes (n = 10) from ApoE(-/-)/LDL receptor(-/-) double knockout mice at the age of 80 weeks were perfused in vivo with contrast agent, harvested and scanned with micro-CT at (4.9 µm³) voxel size. Testes (n = 8) of C57/BL mice at the same age served as controls. Testis volume (mm³) and total vascular volume fraction (mm³) were quantified using micro-CT. Serum testosterone levels were determined. Testicular histology and epididymal sections were analysed for tubular structure, spermatogenetic scores and sperm count. The expression of protamine 2 as a marker for elongated spermatids, inflammation markers (CD4, F4/80) and hypoxia inducible factor 1 alpha (HIF1 alpha) were investigated using immunohistochemistry. ApoE(-/-)/LDL receptor(-/-) double knockout mice exhibit diminished testis and vascular volume fraction with respect to that of controls (p < 0.001). These findings were associated with a reduction of testosterone levels (p < 0.001). Mixed atrophy was present in 41% of the seminiferous tubuli in ApoE(-/-)/LDL receptor(-/-) double knockout mice at the age of 80 weeks. Sperm counts from the epididymis demonstrated a significant decrease in ApoE(-/-)/LDL receptor(-/-) double knockout mice (p < 0.001). In addition, sperm specific protamine 2 expression was decreased in testicular tissue and epididymis of ApoE(-/-)/LDL receptor(-/-) double knockout mice compared with that of control mice. Peritubular inflammatory infiltration and the expression of the hypoxia related marker was observed. Mixed testicular atrophy in ApoE(-/-)/LDL receptor(-/-) double knockout mice is linked to reduced testis volume, vascular volume fraction and low testosterone serum levels, suggesting a direct relation between atherosclerosis and disturbed spermatogenesis.

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.

Regional pulmonary blood flow in the lung of the chicken.

It is known that alterations in respiratory gases in birds can cause a nonhomogenous redistribution of pulmonary blood flow between the 2 separate gas-exchanging regions of the avian lung, the paleopulmo (PALEO) and neopulmo (NEO); however, the effect of alterations in respired gas content on the distribution of pulmonary blood flow in birds, such as the chicken, that possess a highly developed NEO is not known. This study used a colorimetric microsphere method to determine the effects of hypoxia and hypercapnia on the relative distribution of pulmonary blood flow in anesthetized chickens (Gallus domesticus) during control (normoxic) and experimental (hypoxic or hypercapnic) conditions, where the relative regional distribution of blood flow in the lung is expressed as the ratio NEO/PALEO. Administration of a hypoxic gas mixture (16.0% O(2)) produced a 13.4% increase in NEO/PALEO, and, administration of a hypercapnic gas mixture (5.0% CO(2)) resulted in a 27.8% increase in NEO/PALEO. Our results are consistent with a mechanism in which the regional redistribution of pulmonary blood flow is mediated by local intrapulmonary factors.

Apoptotic gene expression in potentially fertile and subfertile men.

Human sperm contain similar amounts of protamine-1 (P1) and protamine-2 (P2). Although an aberrant protamine ratio have been observed in subfertile men, functional evidence is provided by protamine knockout mice exhibiting male infertility. As sperm DNA integrity is known to be linked with DNA fragmentation and apoptosis, we investigated whether the DNA fragmentation factor 40 (DFF40) ratio or caspase (Casp4, Casp6) and tumor necrosis factor superfamily member 10 (TNFSF10) ratio together with the P1/P2 ratio represents a reliable biomarker to discriminate between fertile and subfertile men. Real-time quantitative RT-PCR was used for amplification of P1, P2 and DFF40 in 49 testicular biopsies. Casp4, Casp6 and TNFSF10 have been selected from a PCR apoptosis array and were further investigated in another group of testicular biopsies (22 subfertile men versus 11 potentially fertile men). Using Spearman's rank correlation coefficient analysis, we did not find a correlation between DFF40 and P1, P2, P1/P2, score, fertilization rate and age. In addition, logistic regression analysis demonstrated no statistically significant effect of the analyzed variables on pregnancy. A two-way analysis of variance with repeated measures of relative expression of Casp4, Casp6 and TNFSF10 versus P1 or P2 in potentially fertile men and subfertile patients demonstrated statistically significant differences between both groups, all tested gene combinations and the interaction between two genes and both groups in all cases analyzed. Furthermore, significant differences in the expression of Casp4 and TNFSF10 between the groups of potentially fertile and subfertile men could be demonstrated. In addition, the means of differences of selected gene combinations revealed that the protamine to apoptotic gene ratio is statistically different between both groups. Our data suggest that Casp4, Casp 6 and TNFSF10 are differentially expressed in potentially fertile and subfertile men and represent useful biomarkers for predicting male fertility in combination with P1 and P2.

Color Doppler ultrasound imaging in varicoceles: is the venous diameter sufficient for predicting clinical and subclinical varicocele?

PURPOSE: Investigating the diagnostic value of color Doppler ultrasound for defining the varicocele grade according to WHO criteria. METHODS: A total of 217 men (129 with clinical varicocele and 88 without clinical varicocele) were investigated by physical examination and color Doppler ultrasound and categorized according to WHO varicocele criteria (0, subclinical, I, II, and III). Diameter and reflux of the largest vein in the pampiniform plexus were measured bilaterally with the patient in the supine position in rest and during the Valsalva maneuver. To assess the possibility of differentiating varicocele grade by venous diameter, optimal cut-point values were determined by receiver-operator characteristic (ROC) analysis. RESULTS: With increased varicocele grade, a larger vein diameter was more significant in rest and during Valsalva (in all cases P < 0.05), except between grade I and grade II. Retrograde peak flow velocities were similar in every group (in all cases P > 0.1). Only grade III varicoceles demonstrated significantly increased peak flow values compared with all other grades (P < 0.001). There were no side-related differences when comparing identical varicocele grades (in all cases P > 0.1). Venous diameters above 2.45 mm in rest (sensitivity 84%, specificity 81%) or 2.95 mm during Valsalva (sensitivity 84%, specificity 84%) predicted the presence of a clinical varicocele. CONCLUSIONS: Our findings support the hypothesis that clinical varicoceles can be predicted with high accuracy based only on the diameter of testicular veins using cut-point values of >2.45 mm in rest or >2.95 mm during Valsalva maneuver in the supine position.

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.

Protamine-1 represents a sperm specific gene transcript: a study in Callithrix jacchus and Bos taurus.

Spermatozoa are transcriptionally inactive cells, but contain acetylated histones, normally a characteristic of transcriptionally active cells. Acetylgroups are thought to represent epigenetic marks that are transmitted to the oocyte and are involved in starting gene expression in the zygote and in regulating gene expression during early embryogenesis. We performed reverse transcription polymerase chain reaction (RT-PCR) in the common marmoset monkey (Callithrix jacchus) and in bovine spermatozoa, oocytes, zygotes, two- and four-cell embryos to evaluate the presence of specific transcripts known to play a role during fertilisation and early embryo development, namely protamine-1 (PRM1), protamine-2 (PRM2), histone H1 (H1), histone H3 (H3), histone H4 (H4), cAMP-responsive element modulator (CREM), DNA methyltransferase-1 (DNMT1), TATA box-binding protein (TBP). All transcripts tested were present in spermatozoa of the common marmoset, while bull spermatozoa lack PRM2. Marmoset oocytes exhibited transcripts for H1, H3, H4 and TBP, whereas bovine oocytes revealed H1, H3, H4, CREM, DNMT and TBP mRNAs. In zygotes, we amplified H1, H4, TBP (marmoset) and PRM1, H1, H3, H4, CREM, DNMT1 and TBP (bovine). Two-cell embryos showed PCR products for H1, H3 and TBP in the marmoset. In the bovine, all transcripts could be observed except PRM2. In four-cell embryos, PCR signals were obtained for PRM1, H1, H3, H4 and TBP in the marmoset. In the bovine, all transcripts were detected except PRM2. Our data suggest that, in both C. jacchus and Bos taurus, PRM1 transcripts are delivered by the spermatozoon to the oocyte.

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.

The European Academy of Andrology (EAA) ultrasound study on healthy, fertile men: Scrotal ultrasound reference ranges and associations with clinical, seminal, and biochemical characteristics.

BACKGROUND: Scrotal color Doppler ultrasound (CDUS) still suffers from lack of standardization. Hence, the European Academy of Andrology (EAA) has promoted a multicenter study to assess the CDUS characteristics of healthy fertile men (HFM) to obtain normative parameters. OBJECTIVES: To report and discuss the scrotal organs CDUS reference ranges and characteristics in HFM and their associations with clinical, seminal, and biochemical parameters. METHODS: A cohort of 248 HFM (35.3 ± 5.9years) was studied, evaluating, on the same day, clinical, biochemical, seminal, and scrotal CDUS following Standard Operating Procedures. RESULTS: The CDUS reference range and characteristics of the scrotal organs of HFM are reported here. CDUS showed a higher accuracy than physical examination in detecting scrotal abnormalities. Prader orchidometer (PO)- and US-measured testicular volume (TV) were closely related. The US-assessed TV with the ellipsoid formula showed the best correlation with the PO-TV. The mean TV of HFM was ~ 17 ml. The lowest reference limit for right and left testis was 12 and 11 ml, thresholds defining testicular hypotrophy. The highest reference limit for epididymal head, tail, and vas deferens was 12, 6, and 4.5 mm, respectively. Mean TV was associated positively with sperm concentration and total count and negatively with gonadotropins levels and pulse pressure. Subjects with testicular inhomogeneity or calcifications showed lower sperm vitality and concentration, respectively, than the rest of the sample. Sperm normal morphology and progressive motility were positively associated with epididymal head size/vascularization and vas deferens size, respectively. Increased epididymis and vas deferens sizes were associated with MAR test positivity. Decreased epididymal tail homogeneity/vascularization were positively associated with waistline, which was negatively associated with intratesticular vascularization. CDUS varicocele was detected in 37.2% of men and was not associated with seminal or hormonal parameters. Scrotal CDUS parameters were not associated with time to pregnancy, number of children, history of miscarriage. CONCLUSIONS: The present findings will help in better understanding male infertility pathophysiology, improving its management.