Age-dependent inhibin B concentration in relation to FSH and semen sample qualities: a study in 2448 men.

Inhibin B is an important serum marker of spermatogenesis, whereas sensitivity and predicting power for the spermatogenic situation at several ages are under debate. We performed a retrospective analysis of data from 2448 men who attended our University-based male infertility clinic to evaluate inhibin B in relation to age and semen sample qualities in comparison with FSH. Moreover, the range of inhibin B in 82 nonobstructive azoospermic patients was correlated with the sperm retrieval in testicular sperm extraction procedures. Inhibin B correlated with FSH (Spearman rank correlation (R)=-0.50; P<0.00001). Inhibin B and inhibin B/FSH ratio (IFR) showed an inverse U-shaped dependence on age, whereas FSH showed a U-shaped dependence on age (optimum 20-40 years). However, in men with normal spermiograms inhibin B concentrations did not differ between age groups. Their levels of inhibin B amounted to 130.5, 54.5-247 ng/l (median, 10th-90th precentile), and of IFR to 38.3, 12.5-104.8 (median, 10th-90th percentile), which might be taken as the reference range. Using the 10th percentile of IFR, correct classification in normal or pathological semen groups was achieved in 99.1%. The percentage of aniline blue-negative spermatozoa, i.e. mature spermatozoa with protamines, did not correlate with FSH (P>0.05) but with inhibin B (R=0.15, P<0.001). The probability of retrieving testicular spermatozoa decreased with declining inhibin B: <20 ng/l sperm could never be found. Our results from a large group of men with a wide spectrum of semen qualities allow estimating reference values for inhibin B and IFR. Inhibin B and especially the IFR are more sensitive markers of male infertility than FSH alone.

Effects of post-density gradient swim-up on apoptosis signalling in human spermatozoa.

The inclusion of apoptotic spermatozoa during assisted reproductive techniques (ART) may be one reason for suboptimal success rates. The aim of our study was to evaluate the potential of routine semen preparation to eliminate spermatozoa with activated apoptosis signalling. Semen samples from 20 infertility patients scheduled for ART procedures were investigated. Following density gradient centrifugation (DGC) and swim-up, aliquots were taken from each sample to analyse motility, Caspase-3 activation (CP3) and integrity of the mitochondrial membrane potential (MMP) using flow cytometry. Aliquots from the neat semen served as controls. Semen samples of patients contained 53.8 +/- 17.7% spermatozoa with disrupted MMP and 51.8 +/- 14.9% with active CP3. Preparation by DGC and swim-up resulted in improvement of progressive motility (+43.5%) and reduction of spermatozoa with disrupted MMP (-34.3%) and activated CP3 (-25.7%, P < 0.01). Minimal reduction of spermatozoa with disrupted MMP and active CP3 was 6.0% and 0.7%, maximum reduction was 65.5% (disrupted MMP) and 49.3% (CP3). Semen samples of subfertile patients contain high levels of spermatozoa with activated apoptosis signalling. Although there was a reduction in the majority of the samples, profound interindividual differences in the separation effect demand further development of innovative molecular-based separation methods to deplete apoptotic spermatozoa.

Activity of nitric oxide synthase in mature and immature human spermatozoa.

Nitric oxide (NO) is known to be involved in multiple signal transduction pathways of male germ cells, including sperm capacitation. In somatic cells, NO production was found to be part of apoptosis signalling. The aim of our study was to further clarify the role of NO in spermatozoa by investigation of NO synthase activity with regard to sperm maturity and sperm apoptosis signalling. Semen specimens from 19 healthy donors were subjected to density gradient centrifugation to separate the predominantly mature and immature sperm fraction. NO synthase activity was evaluated using diaminofluoresceine-2-diacetate by FACS. Apoptosis signalling was monitored by flowcytometric analyses of caspase-3 (CP3) and integrity of the transmembrane mitochondrial potential (TMP). TUNEL assay was used to detect DNA fragmentations. Maturity of human spermatozoa was associated with increased NO synthase activity and inactivated apoptosis signalling (lower levels of disrupted TMP, active CP3 and DNA fragmentations, P < 0.05). Activation of apoptosis signalling was significantly negatively correlated to NO production, indicating a rather anti-apoptotic effect of NO. This might underline the recently proposed role of NO in physiological sperm signal transduction, e.g. during capacitation.

alpha1-antitrypsin prevents polymorphonuclear leucocyte-elastase effects on spermatozoa quality.

Elevated levels of polymorphonuclear leucocyte (PMN)-derived elastase, which is suggested as marker for inflammations in the male genital tract, correlate well with spermatozoa deterioration. PMN elastase caused a time- and concentration-dependent (up to a elastase concentration of 0.5 microg/mL) externalization of phosphatidylserine and intercalation of propidium iodide on human spermatozoa. There are apparently a limited number of target sites for elastase on spermatozoa surface, because the further enhancement of elastase amount did not fasten alterations in spermatozoa parameters. Analysis of flow cytometry data revealed that most spermatozoa were in a necrotic state after an exposure with elastase for 22 h. Some apoptotic cells were only detected at shorter incubation periods. Seminal plasma prevented in a concentration-dependent manner the PMN elastase-mediated loss of vitality of spermatozoa. We detected by blotting techniques large amounts of alpha(1)-antitrypsin in seminal plasma. This antiproteinase is known to inactivate elastase at inflammatory sites. Increasing concentrations of alpha(1)-antitrypsin prevented gradually spermatozoa deterioration induced by elastase. Thus, alpha(1)-antitrypsin contributes to an efficient protease/antiproteinase balance in seminal plasma. A disturbed balance will promote the development of chronic inflammations which can also be the reason for male infertility problems.

Interactions between apoptotic signal transduction and capacitation in human spermatozoa.

BACKGROUND: Capacitation of sperm is a prerequisite for successful fertilization, determined by hyperactivated motility, increased tyrosine phosphorylation (TyrP) and membrane changes. However, the exact molecular mechanism is not fully clarified. The calpain-calmodulin-system is essential for membrane fusion during capacitation. Recently, interactions with caspase (CP) activation, a main feature of apoptotic cells, were postulated. The objective of our study was to examine interactions between apoptosis signalling and the calpain-calmodulin-system during capacitation. METHODS: Semen samples from 20 healthy donors were incubated in human tubal fluid at 37 degrees C, 5% CO(2) for 3 h without additives (control), with 3% BSA (capacitation), 10 microM calpain-inhibitor III, 20 microM CP-1 inhibitor or 20 microM calmodulin-antagonist. Capacitation was monitored by computer assisted sperm motion analyzer, chlortetracycline (CTC)-assay and western blot (TyrP). Activation of caspases and integrity of transmembrane mitochondrial potential (TMP) were evaluated by flow cytometry. RESULTS: Capacitation, as measured by CTC assay, increased TyrP levels and hyperactivation, resulted in inactivation of CP-9, CP-3 and improved integrity of the TMP. Inhibition of calpain and CP-1 during capacitation reduced the capacitation-related parameters, but did not lead to apoptosis. Inhibition of calmodulin resulted in blocking of capacitation and stimulation of apoptosis. CONCLUSION: Interaction of the capacitation and apoptosis signalling systems seems to enable the capacitation process by prevention of apoptosis.

[Andrological testosterone replacement therapy]. / Andrologische Testosteronersatztherapie.

Male hypogonadism is characterised by decreased testicular function, especially testosterone deficiency. Its prevalence increases with age but may be acquired in all stages of life if it is not inborn. The lack of testosterone results in typical clinical symptoms. However, only about 10% of the affected men currently receive adequate treatment. A variety of testosterone preparations are available to meet the main needs of treatment, including intramuscular, transcutaneous, sublingual, and oral forms. In patients with prostate cancer, breast cancer, polycythemia, severe heart failure, obstruction of the lower urinary tract, or untreated sleep apnoea, testosterone treatment must be avoided. Therefore, clinical and laboratory screening are essential along with a long-term substitution.

Relationship between sperm apoptosis signalling and oocyte penetration capacity.

Human sperm have been documented to display apoptosis-like features such as externalization of phosphatidylserine (EPS), disruption of the transmembrane mitochondrial potential (MMP) and activation of caspases. Our aim was to evaluate possible association between activation of the apoptosis cascade in human sperm and its oocyte penetration capacity using the zona free hamster oocyte penetration assay (SPA). Semen specimens from 76 unselected donors were subjected to double density gradient centrifugation followed by incubation under capacitating conditions for 3 h and SPA. Apoptosis signalling was monitored by assessment of EPS, disruption of MMP and activation of caspase-3 by flow cytometry. Semen samples with subnormal SPA values (<20% penetrated oocytes) contained significantly higher amounts of spermatozoa with EPS, disrupted MMP and activated caspase-3 compared with those samples with normal SPA values (>20% penetrated oocytes, p < 0.01). All three apoptosis markers showed a significantly negative correlation with the percentage of penetrated oocytes (p < 0.01). Apoptosis-related signalling appears to have a negative association with sperm-oocyte penetration. The exclusion of sperm presenting with those apoptosis-related features during assisted reproduction may improve success rates of procedures such as intrauterine insemination and in vitro fertilization.

Failure of phospholipid hydroperoxide glutathione peroxidase expression in oligoasthenozoospermia and mutations in the PHGPx gene.

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is a selenoprotein belonging to the family of glutathione peroxidases. PHGPx has long been considered a major antioxidant that, in cooperation with vitamin E, protects biomembranes. To determine the expression pattern of PHGPx mRNA in human, quantitative RT-polymerase chain reaction (PCR) analyses using RNA from different embryonal and adult tissues were performed. A predominant expression was found in testes. In spermatozoa, PHGPx was found to be localized in the mid-piece of spermatozoa. We studied the relationship between spermatozoa PHGPx expression, mutations in PHGPx gene and human oligoasthenozoospermia, a defect in which both the number and the motility of spermatozoa are significantly below normal. Spermatozoa specimens from 45 infertile males were analysed for fertility-related parameters according to World Health Organisation and were classified as suffering from oligoasthenozoospermia. Two patients (4.44%) showed no expression of PHGPx and in nine patients (20.00%), a reduced expression of the enzyme was observed. DNA sequences of various regions of the PHGPx gene (coding, 5'flanking region and intron 1) from these patients and 58 fertile volunteers were analysed for mutations by PCR amplification and direct sequencing. Sequence data revealed no cause/effect relationship for any of the variants. From these data it can be concluded that oligoasthenozoospermia is associated with a decrease in the level of expression of PHGPx in the spermatozoa of some infertile men (24.44%), but is not linked to mutations in PHGPx gene.

Destabilization of the acrosome results in release of phospholipase A2 from human spermatozoa and subsequent formation of lysophospholipids.

Phospholipase A(2) controls the phospholipid composition in spermatozoal membranes and is released from the acrosome of human spermatozoa. The extracellular phospholipase A(2) activity of human spermatozoa was determined by matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) mass spectrometry after destabilization of acrosome by the calcium-ionophore calcimycin. MALDI-TOF mass spectrometry allowed the monitoring of changes in both substrate and products of spermatozoal phospholipase A(2) (PLA(2)) without the use of labelled phospholipids. The spermatozoal PLA(2) was characterized as a secretory one (sPLA(2)). Secretory PLA(2) exhibited a high substrate specificity for 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (PDPC), the most abundant spermatozoal phospholipid. A time- and cell number-dependent formation of the lysophospholipid PDPC was observed following incubation of extracellular medium of calcimycin-treated spermatozoa (CTS) with PDPC. Antibodies against sPLA(2), specific inhibitors of sPLA(2) and Ca(2+)-chelators could inhibit its generation. An antibody against lysophospholipase enhanced the lysoproduct concentration in the extracellular medium of CTS containing sPLA(2) because further metabolization of these products was blocked. The results demonstrated that destabilization of the acrosome is able to induce a release of secretory phospholipase A(2) from human spermatozoa with subsequent generation of lysophosphocholine in the surrounding of spermatozoa.

[Scarring tinea profunda in the pubic area due to Trichophyton verrucosum]. / Vernarbende Tinea profunda des Mons pubis durch Trichophyton verrucosum.

A 22-year-old female veterinary student developed a severe inflammatory fungal infection of the pubic area 2 months after practical training in obstetrics at a cattle farm. Fungal culture revealed a slowly growing white dermatophyte, first differentiated as Trichophyton mentagrophytes. Because of the unusual location, molecular species differentiation was performed identifying Trichophyton verrucosum. Although the patient was treated with systemic antifungals, the infection resulted in scarring alopecia.

[Infertility in the Klinefelter syndrome]. / In-vitro-Fertilisation macht es in seltenen Fällen möglich. Vater mit Klinefelter-Syndrom.

The Klinefelter syndrome is characterized by one or more extra X-chromosomes, deficient androgens, increased gonadotropines, fibrosis and hyalinization of the seminiferous tubules, small testes, gynecomastia, disproportionately long legs, sparse facial hair, and diminished sexual activity. The incidence of Klinefelter syndrome in the general population is 0.1-0.2%, some 3% among infertile patients, and approximately 11% in patients with aspermia. In very rare cases, these patients may manifest focal residua of spermatogenesis. Employing the ICSI method (intracytoplasmic sperm injection into an oocyte), a patient may be helped to father a child. There is, however an increased risk of such a child being born with a chromosomal aberration.

Sperm caspases become more activated in infertility patients than in healthy donors during cryopreservation.

The programmed cell death (PCD, the apoptosis) is associated with the activation of the "death enzymes" caspases and very likely plays a role in the decrease of sperm vitality during cryopreservation. The activation of pan-caspases was examined in fresh and cryostored sperm by fluorescence microscopy applying a cell permeable and noncytotoxic carboxy-fluorescein labelled caspase inhibitor. Cryopreservation increased significantly the percentage of spermatozoa with activated pan-caspases (aCP) from 21 +/- 2.6 to 47 +/- 5.8 % (mean +/- SEM). Quantitative Western blot analyses confirmed the activation of caspase-1, -8, and -9 in detail on protein level after cryopreservation, whereas, the caspases of sperm of infertility patients showed a higher activation status than donors' sperm. This effect raised with increasing glycerol concentration from 7-14 %. The higher level in activation of caspases in cryostored spermatozoa of infertility patients may indicate that these cells have a lower cryotolerance and a higher susceptibility to caspase activation than does the sperm of donors.

Mature human spermatozoa do not transcribe novel RNA.

Mature spermatozoa contain subsets of mRNA that have been found in human testes before. Based on this finding it was hypothesized that the mRNAs of spermatozoa are transcribed during spermatogenesis. However, up to now there is no proof of the transcriptional inactivity of human sperm. To address this issue we performed in vitro labelling experiments with radio-labelled uridine triphosphate followed by analysis of cellular RNA. There was virtually no radioactive RNA detectable in the RNA purified from human spermatozoa proving the transcriptional inactivity of mature spermatozoa. The spermatozoal RNA obviously results from transcription during spermatogenesis and can be used for diagnostic purposes. These findings might have diagnostic and - possibly - therapeutic value in infertility patients as spermatozoal RNAs might complement the RNA pool of the oocyte after fertilization.

[Mitochondrial damage in human sperm caused by the antineoplastic agent betulinic acid]. / Mitochondriale Schädigung von Humanspermien durch das Onkotherapeutikum Betulinsäure.

BACKGROUND AND OBJECTIVE: Human spermatozoa post-ejaculation show all elements of intrinsic (via mitochondria) and extrinsic (via death receptors) programmed cell death or apoptosis. One experimental therapeutic agent for malignant melanoma is betulinic acid (BA), a cytotoxic agent which induces intrinsic apoptosis via direct effects on mitochondria. To assess the potential side effects of systemic BA, its effects on motility, mitochondrial transmembrane potential (mTMP) and the apoptotic enzymes caspase-9 and -3, were monitored in human ejaculated spermatozoa. PATIENTS AND METHODS: Semen samples from 33 healthy volunteers were examined after incubation with 60 microg/mL betulinic acid for 5 and 60 minutes. RESULTS: Treatment with betulinic acid caused an immediate disruption of mitochondrial transmembrane potential and activation of the "death enzymes" caspase-9 and -3. The loss of mitochondrial potential was accompanied by a significant decrease of spermatozoal motility. CONCLUSION: Our results suggest that inducers of the mitochondrial pathway of apoptosis used in the treatment of malignant melanoma damage the sensitive mitochondria of spermatozoa.

Cryopreservation and thawing is associated with varying extent of activation of apoptotic machinery in subsets of ejaculated human spermatozoa.

We investigated the impact of cryopreservation and thawing on levels of caspases-3, -8, and -9 activity, intact mitochondrial membrane potential (Deltapsim), and DNA fragmentation in human spermatozoa. Eleven pools of cryopreserved and eight pools of fresh semen samples were examined. Mature and immature fractions were separated on a two-layer density gradient (47% and 90%) and further subdivided based on the externalization of phosphatidylserine and its binding to annexin V-labeled superparamagnetic microbeads (ANMB). Levels of activated caspases were assessed using fluorescein-labeled inhibitors of caspases (FLICA), Deltapsim using a lipophilic cationic dye, and DNA fragmentation by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. Cryopreservation was significantly associated with activation of caspases-3, -8, and -9, as well as disruption of the mitochondrial membrane potential but no significant changes were observed in DNA fragmentation. In mature sperm, caspase activation was only detected in the ANMB+ fraction, whereas in immature sperm, both ANMB+ and ANMB- fractions showed activated caspase levels. In ANMB+ immature sperm, apoptosis seemed to be triggered by a surface ligand-receptor mechanism as well as by disruption of mitochondria, whereas in ANMB- immature sperm, apoptosis was induced by activation of caspase-9 following loss of intact Deltapsim. These results demonstrate that selection of annexin V-negative mature spermatozoa might be of clinical relevance for fertility preservation, as this sperm fraction shows no activated apoptosis during the cryopreservation process.

Electronic data base systems support the evaluation of male infertility factors, example cryptorchidism.

BACKGROUND: A new data base system was applied to analyse our patient group with two aims: (a) to analyze the effects of former cryptorchidism on the fertility of OUR infertility patients in comparison with the data of the literature and (b) to evaluate this system in a clinical study. MATERIALS AND METHODS: Using the electronic data base Winsperm 2000, 1,648 infertility patients, 79 patients with testicular cancer and 201 healthy semen donors were examined. RESULTS: A history of cryptorchidism, treated at 6.8 +/- 3.3 years of life, was found in 10.1% of our infertility patient group. The routine spermiogram parameters, as well as basal hormone concentration of FSH, LH and testosterone, differed significantly from those of the healthy semen donor group. Comparison between patients with former unilateral and bilateral cryptorchidism differed significantly only in total sperm count. 27.7% of patients with a history of unilateral and 5.4% of patients with a history of bilateral cryptorchidism showed a sperm concentration within the normal range (p < 0.01). Azoospermia was detected in 13.1% of patients with unilateral cryptorchidism and in 29.7% of patients with former bilateral cryptorchidism (p < 0.05). The patients responding to our conception questionnaire realised a total conception rate of 46.1% in the 'non-cryptorchidism group' and of 20.6% in the 'cryptorchidism-group' (p < 0.05), whereas the conception rates did not differ between former unilateral and bilateral cryptorchidism (p > 0.05). Sixteen (20.3%) of the 79 patients with testicular neoplasm were previously treated for cryptorchidism. CONCLUSION: The results of our patient group underline the significance of former cryptorchidism for infertility and testicular neoplasm. The new data base system facilitates rapid data retrieval and examination.

[Molecular genetic mutation analysis of the PTPN11 gene in the multiple lentigines (LEOPARD) syndrome]. / Molekulargenetischer Mutationsnachweis im PTPN11-Gen beim Multiple-Lentigines- (LEOPARD-)Syndrom.

BACKGROUND AND OBJECTIVE: LEOPARD syndrome (MIM #151100) is a rare autosomal dominant condition with characteristic skin anomalies, facial dysmorphism, hypertelorism, cardiac anomalies, and occasional conductive hearing loss. Mutations in the PTPN11 gene are described as the causal gene defect for the clinical features of Noonan syndrome (MIM #163950), but also for LEOPARD syndrome. For confirmation of the clinical diagnosis of multiple lentigines syndrome, the molecular genetic mutation analysis in the PTPN11 gene could be helpful. PATIENTS/METHODS: We report on a family with LEOPARD syndrome in which the mutation analysis in the father and his daughter in the PTPN11 gene was carried out us:ng PCR, DHPLC, and automated sequencing. RESULTS: We could identify both father and daughter as carriers of the mutation Y279C in the PTPN11 gene, which is known as a disease-related mutation. CONCLUSIONS: The allelic affinity to Noonan syndrome could thus be further supported.

Leptin and leptin receptor in human seminal plasma and in human spermatozoa.

Leptin, a 167 amino acid peptide, is known to influence the gonads via direct and indirect effects. Recent studies provide contradictory proposition about the peripheral impact of leptin in the male gonads. Thus, we examined leptin and its receptors in human seminal plasma and in human ejaculated spermatozoa by Western blot technique and fluorescence microscopy. In seminal plasma we found a free leptin band (16 kDa) by an anti-leptin polyclonal antibody. Incubation of seminal plasma with recombinant leptin caused a statistically significant increase in the amount of free leptin (p < 0.01) and supports this finding. Furthermore, a soluble leptin receptor (145 kDa) was found in human seminal plasma in the same specimen. We also detected a 145-kDa leptin receptor isoform in ejaculated spermatozoa as a possible target of leptin action in the male genital tract, which was localized at the tail of spermatozoa by immunofluorescence microscopy only. This receptor was significantly associated with the intactness of sperm plasma membranes. Spermatozoa with deteriorated membranes contained 49.2 +/- 6.9% leptin receptor signal intensity compared with spermatozoa having intact membranes (p < 0.01). This finding is difficult to interpret and may be caused by a leakage of OB-R due to loss of membrane integrity. In conclusion, these data provide further hints for a peripheral role of leptin in the male genital tract, possibly, by an interaction between leptin and spermatozoa via sperm leptin receptors.