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.

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.

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.

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.

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.

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.

[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.

Lipid analysis of human spermatozoa and seminal plasma by MALDI-TOF mass spectrometry and NMR spectroscopy - effects of freezing and thawing.

In the present study, the applicability of proton NMR spectroscopy and matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) to the analysis of the lipid composition of human spermatozoa and seminal fluids as well as changes after cryopreservation of human spermatozoa was investigated. Whereas NMR spectra primarily indicated a high content of double bonds within the spermatozoa but no marked differences upon cryopreservation, MS detected intense peaks which could be assigned to phosphatidylcholines containing one docosahexaenoic and one palmitic or stearic acid residue (m/z=806 and 834). In contrast, the seminal plasma contained more saturated fatty acids and especially more sphingomyelin (SM). A freezing/thawing cycle markedly influences the lipid composition of spermatozoa. There was a diminution of phosphatidylcholines (16:0, 22:6 and 18:0, 22:6) and SM (16:0) and the appearance of lysophosphatidylcholines (16:0 and 18:0) and ceramide (16:0). These data demonstrate the release or activation of both phospholipase A(2) and sphingomyelinase in human spermatozoa due to the freezing/thawing cycle. These results were finally confirmed by experiments on the action of phospholipases on lipids containing docosahexaenoic acid.

Probability to retrieve testicular spermatozoa in azoospermic patients.

AIM: The degree of probability to retrieve spermatozoa from testicular tissue for intracytoplasmic sperm injection into oocytes is of interest for counselling of infertility patients. We investigated the relation of sperm retrieval to clinical data and histological pattern in testicular biopsies from azoospermic patients. METHODS: In 264 testicular biopsies from 142 azoospermic patients, the testicular tissue was shredded to separate the spermatozoa, histological semi-thin sections of which were then evaluated using Johnsen score. RESULTS: The retrieval of spermatozoa correlated significantly ( P < 0.001) with the testicular volume ( r = 0.49), the FSH concentration ( r = -0.66), the maximum score (r = 0.85) and the mean Johnsen score (r = 0.81). In the multivariate regression analysis the successful testicular sperm extraction showed the closest relationship to the maximum score. The testicular volume correlated significantly with the mean Johnsen score ( r = 0. 64, P < 0. 001), and the basal serum FSH concentration mainly with the maximum score ( r = - 0.77; P < 0.001). Patients with a history cryptorchidism showed a significantly lower Johnsen score compared to the patients who did not have any testicular disease in the past (3.7 +/- 2.4 vs. 5.9 +/- 2.5; P < 0. 01). CONCLUSION: In a limited range, the testicular volume and the FSH concentration in serum were related to the Johnsen score which correlated significantly with the sperm retrieval. The successful sperm retrieval can be expected in all azoospermic patients irrespective of the results of clinical examination. However, the probability of retrieval of spermatozoa decreased significantly in patients with a FSH level > 18 U/L, testicular volume < 5 mL, mean Johnsen score <5, and maximum Johnsen score <7.

Hidden effects of cryopreservation on quality of human spermatozoa.

The effects of cryopreservation on two characteristics of human spermatozoa were investigated: the early phases of disturbed plasma membrane function and the activity of enzymes in intact spermatozoa. The membrane function was detected by means of the calcium-dependent binding of fluorescein isothiocyanate (FITC)-conjugated Annexin V to sperm plasma membranes. Annexin V monitors the translocation of phosphatidylserine from the inner to the outer leaflet of the plasma membrane, which is one of the earliest features of membrane disintegration. For the second aim synthetic fluorogenic substrates for peptidases, proteinases, esterases, elastases and collagenases were applied. These substrates, CellProbe trade mark reagents consist of different peptide sequences, specific for the enzymes, and a fluorescein- or rhodamine 110-dye moiety. They enter the cells without previous membrane permeabilisation and exhibit fluorescence after cleavage depending on enzyme activity. The number of positive cells and the intensity of the fluorescence were determined by flow cytometric analysis comparing fresh spermatozoa with cryopreserved ones. Thirty-five semen samples collected from 35 donors were cryopreserved using the freezing medium TEST yolk buffer. All specimens showed normal spermiogram parameters. Twenty-five of the samples were used for detection of Annexin V-FITC binding and 10 semen samples for investigations of the intracellular enzymes. The Annexin V-assay applied two fluorescent dyes (Annexin V, AN and propidium iodide, PI) which led to three groups of spermatozoa (a) viable spermatozoa (AN V-negative and PI-negative), (b) dead spermatozoa (AN V-positive and PI-positive) and (c) cells with impaired but integer plasma membrane (AN V-positive and PI-negative). The percentage of vital Annexin V-negative spermatozoa (x +/- SEM) decreased significantly (p < 0.001) from fresh spermatozoa (51.6 +/- 3.1) to cryopreserved spermatozoa (26.6 +/- 2.2%) and was associated with their motility (57.9 +/- 1.9% motile fresh spermatozoa vs. 22.6 +/- 3.9% motile sperm after cryopreservation). Of the spermatozoa 28.2% were Annexin V-positive before and 44.4% after cryostorage even though they did not bind to PI. Thus, vital spermatozoa showed a disturbed membrane function indicating viability before as well as after cryostorage. Moreover, after cryopreservation the spermatozoal fluorescence increased applying substrates for butyryl esterase (p < 0.05), prolyl-aminopeptidase (p < 0.001) and val-lys-(VK)-cathepsin (p < 0.001). In contrast, the activities of fluorescein diacetate (FDA)- and FDA/sodium fluoride (NAF)-esterase (p < 0.05), ala-ala-pro-val-(AAPV)-elastase (p < 0.001), gly-pro-leu-gly-pro-(GPLGP)-collagenase (p < 0.05) and gly-gly-leu-(GGL)-subtilisin (p < 0.001) decreased after cryopreservation. The substrates for arg-gly-glut-ser-(RGES)-elastase, gly-phenyl-gly-ala-(GFGA)-collagenase and threo-pro-(TP)-cathepsin were not cleaved before as well as after cryostorage. In addition to the known effects of sperm cryopreservation our results showed two further alterations of human ejaculated spermatozoa: (a) disturbed plasma membrane function, which is not detectable by supravital staining and (b) a changed pattern of intracellular enzyme activities.

Enrichment of non-apoptotic human spermatozoa after cryopreservation by immunomagnetic cell sorting.

Cryopreservation increases the rate of apoptotic spermatozoa with decreased capability to fertilise oocytes. In order to optimise the fertilisation rates, especially in assisted reproduction the use of apoptotic sperms should be avoided. Early events of apoptosis in cryopreserved spermatozoa are not detectable by conventional methods. However, the surface of apoptotic spermatozoa is characterised by externalisation of phosphatidylserine (PS), which has a high affinity to Annexin V. Therefore, colloid paramagnetic Annexin-V-conjugated microbeads (AN-MB) were tested for their ability to eliminate apoptotic spermatozoa from a total of 40 fresh and in TEST yolk buffer cryopreserved semen samples which were provided by 15 healthy volunteers. By passing through a magnetic field (MiniMACS, Miltenyi Biotec) the sperm suspensions were divided into 2 sperm fractions depending on bound magnetic Annexin V-microbeads (AN-MB) to spermatozoa. As additional markers of apoptosis CD95 (Fas, APO-1) on the sperm surface and activated caspases in the cytosol were detected in both fractions. Supplementary investigations comprised eosin-supravital staining and computer assisted sperm motion analysis. The separation was supervised by flow cytometric analysis of spermatozoa labelled with FITC-conjugated anti Annexin V-antibodies. Analyses of the magnetic inactive sperm fraction (AN-MB-negative) showed CD95 on 0.6 +/- 0.3% (X +/- SEM) of spermatozoa and only 3.2 +/- 0.5% were stainable with eosin, whereas, 40.6 +/- 6.7% of the remaining cells in the column appeared to be CD95 positive and 99.8 +/- 0.1% stainable with eosin after cryopreservation. Indeed the overall amount of CD95 positive spermatozoa did not significantly increase after cryopreservation (2.5 +/- 0.5% vs. 4.3 +/- 1.2%; p > 0.05). Activated caspases were found in 21.8 +/- 2.6% of the spermatozoa in fresh and in 47.7 +/- 5.8% of cryopreserved semen samples (p < 0.01). The separation procedure of the cryopreserved spermatozoa reduced significantly the quantity of those containing activated caspases to 9.3 +/- 2.2% within the AN-MB-negative fraction. In contrast 89.1 +/- 2.3% of AN-MB-positive sperms showed activation of these proteolytic enzymes. Flow cytometric analyses using FITC-conjugated anti Annexin V-antibodies for monitoring of AN-MB-binding to spermatozoa showed 5.2 +/- 1.0% labelled spermatozoa in the AN-MB negative fraction and 72.6 +/- 2.7% labelled spermatozoa in the AN-MB positive one. There was no significant influence of the separation column and the magnetic field on the sperm functions. The passage through the column led to a sperm loss of 0.8 +/- 1.2%. Conclusion: The binding of paramagnetic Annexin V-conjugated microbeads is an excellent method to eliminate spermatozoa at early apoptotic stages from cryopreserved semen samples. A deleterious influence of the separation column and the magnetic field on the spermatozoa was not observed.

Semen samples with teratozoospermia show a lower percentage of spermatozoa with detectable adhesion molecules (AM).

Adhesion molecules (AM) are cell surface proteins which interact with ligands and mediate cell-cell bindings. Possibly, the AM are involved in the spermatozoal adhesion to oocytes. Human spermatozoa showed several adhesion molecules on their surface: the alpha chains of the beta 1-integrins VLA alpha 4 (CDw 49d), VLA alpha 5 (CDw 49e) and VLA alpha 6 (CDw 49f), the beta-chain of beta 2-integrins (CD 18) and the matrix proteins laminin and fibronectin. Semen samples with severe teratozoospermia were characterized by a significantly lower percentage (P < 0.01) of spermatozoa with VLA alpha 4, VLA alpha 5 and laminin. VLA alpha 5 (the alpha-chain of the fibronectin receptor) showed the most significant difference between the "normal" and teratozoospermic semen sample groups. This phenomenon could contribute to the explanation of a lower fertilizing capacity of teratozoospermic semen samples.

[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.

[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.

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.

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.

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.

[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.