Evaluation of atorvastatin efficacy and toxicity on spermatozoa, accessory glands and gonadal hormones of healthy men: a pilot prospective clinical trial.

BACKGROUND: Recommendations for cardiovascular disease prevention advocate lowering both cholesterol and low-density lipoprotein cholesterol systemic levels, notably by statin intake. However, statins are the subject of questions concerning their impact on male fertility. This study aimed to evaluate, by a prospective pilot assay, the efficacy and the toxicity of a decrease of cholesterol blood levels, induced by atorvastatin on semen quality and sexual hormone levels of healthy, normocholesterolaemic and normozoospermic men. METHODS: Atorvastatin (10 mg daily) was administrated orally during 5 months to 17 men with normal plasma lipid and standard semen parameters. Spermatozoa parameters, accessory gland markers, semen lipid levels and blood levels of gonadal hormones were assayed before statin intake, during the treatment, and 3 months after its withdrawal. RESULTS: Atorvastatin treatment significantly decreased circulating low-density lipoprotein cholesterol (LDL-C) and total cholesterol concentrations by 42% and 24% (p<0.0001) respectively, and reached the efficacy objective of the protocol. During atorvastatin therapy and/or 3 months after its withdrawal numerous semen parameters were significantly modified, such as total number of spermatozoa (-31%, p<0.05), vitality (-9.5%, p<0.05), total motility (+7.5%, p<0.05), morphology (head, neck and midpiece abnormalities, p<0.05), and the kinetics of acrosome reaction (p<0.05). Seminal concentrations of acid phosphatases (p<0.01), α-glucosidase (p<0.05) and L-carnitine (p<0.05) were also decreased during the therapy, indicating an alteration of prostatic and epididymal functions. Moreover, we measured at least one altered semen parameter in 35% of the subjects during atorvastatin treatment, and in 65% of the subjects after withdrawal, which led us to consider that atorvastatin is unsafe in the context of our study. CONCLUSIONS: Our results show for the first time that atorvastatin significantly affects the sperm parameters and the seminal fluid composition of healthy men.

Prostasomes as potential modulators of tyrosine phosphorylation in human spermatozoa.

Prostasomes, vesicles present in human semen, are known to play a role in male fertility. However, the mechanisms involved are still poorly understood. The present study looks at the direct influence of different concentrations of prostasomes on human sperm function in conditions supporting capacitation in vitro. Five million Percoll selected spermatozoa were incubated for 3 h at 37°C under an atmosphere of 5% CO2 in air, in 100 µl Biggers Whitten Whittingham's medium (BWW) containing polyvinyl alcohol (PVA, 1 mg/ml) and bovine serum albumin (BSA; 3 mg/ml) in the absence or presence of increasing concentrations of prostasomes (expressed in terms of their cholesterol content: 15; 30; 45 nmoles per 100 µL of incubation medium). After in vitro exposure of spermatozoa to prostasomes, our data indicate that i) tyrosine phosphorylation intensity of the 107 KDa protein band was dose dependently lower and ii) the percentage of viable and progressive motile spermatozoa was unchanged and the percentage of non-progressive motility decreased. In addition, the incubation of prostasomes with spermatozoa resulted in an enrichment of their lipid content. Our experiments suggest that adhesion of prostasomes to spermatozoa could be responsible for the decrease in Tyrosine phosphorylation and the alteration of the mean curvilinear velocity (VCL) and the average path velocity (VAP).

Activated caspases in thawed epididymal and testicular spermatozoa of patients with congenital bilateral absence of the vas deferens and intracytoplasmic sperm injection outcome.

OBJECTIVE: To analyze the expression of activated caspases and membrane permeability in thawed epididymal and testicular spermatozoa of patients with congenital bilateral absence of the vas deferens (CBAVD). DESIGN: Retrospective study. SETTING: Biology and medicine of reproduction in University hospital. PATIENT(S): Eight CBAVD patients. INTERVENTION(S): Staining of activated caspases and viability (propidium iodide, PI); intracytoplasmic sperm injection (ICSI). MAIN OUTCOME MEASURE(S): Proportion of viable (Casp-/PI-) or dead (Casp-/PI+) spermatozoa without activated caspases, viable (Casp+/PI-) or dead (Casp+/PI+) spermatozoa with activated caspases. ICSI results. RESULT(S): Higher percentage of dead (Casp+/PI+; 84.0% vs. 57.5%) and viable (Casp+/PI-; 12.0% vs. 0) spermatozoa with activated caspases were observed in testicular than in epididymal samples. No significant difference was observed between the percentage of total testicular and epididymal spermatozoa permeant for PI. The outcome of ICSI fertilization (67.5% vs. 57.4%), good morphology embryo at day 2 (75.9% vs. 61.3%), clinical pregnancy (26.7% vs. 15.4%), and implantation (15.6% vs 9.5%) rates were better when ICSI were performed with epididymal sperm samples. CONCLUSION(S): These results support the hypothesis of an abortive apoptotic process and demonstrate that combined staining of the activated caspases and membrane permeability provide complementary measurements for the evaluation of viable and functional spermatozoa to better understand ICSI outcomes with epididymal and testicular spermatozoa.

Outcome of intracytoplasmic sperm injection for a couple in which the man is carrier of CFTR p.[R74W;V201M;D1270N] and p.P841R mutations and his spouse a heterozygous carrier of p.F508del mutation of the cystic fibrosis transmembrane conductance regulator gene.

OBJECTIVE: To document the phenotype associated with the p.[R74W;V201M;D1270N] and p.P841R mutations of cystic fibrosis transmembrane conductance regulator (CFTR) gene. DESIGN: Case report. SETTING: Biology and medicine of reproduction in a university hospital. PATIENT(S): A couple in which the man is carrier of the triple mutant p.[R74W;V201M;D1270N] allele in trans to p.P841R mutation and his spouse a heterozygous carrier for the severe p.F508del mutation of the CFTR gene, who became pregnant after intracytoplasmic sperm injection (ICSI) with twins. INTERVENTION(S): Genetic counseling; CFTR gene sequencing; ICSI; children's follow-up. MAIN OUTCOME MEASURE(S): First report of a male phenotype associated with the p.P841R mutation. RESULT(S): The triple mutant p.[R74W;V201M;D1270N] allele associated with the unknown p.P841R mutations were detected in this man with congenital bilateral absence of the vas deferens, which may presume p.P841R as a severe mutation. After genetic counseling, the couple preferred prenatal diagnosis after ICSI than preimplantation genetic diagnosis, which revealed that the boys were both carriers of p.[R74W;V201M;D1270N] and p.F508del mutations. They are now 4 years old and show normal growth without nutritional deficiency. CONCLUSION(S): This case report documents for the first time a male phenotype associated with the p.P841R mutation and underlines the difficulties in counseling a man with congenital bilateral absence of the vas deferens carrying uncommon mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene before ICSI.

Follicular growth and estradiol follow-up after subcutaneous xenografting of fresh and cryopreserved human ovarian tissue.

OBJECTIVE: To assess ovarian cortex surrounding benign ovarian cysts after cryopreservation and grafting to severe combined immunodeficient (SCID) mice. DESIGN: Animal study. SETTING: Academic research laboratories. PATIENT(S): Ovarian tissue obtained from 15 patients. INTERVENTION(S): Grafting of fresh and frozen/thawed ovarian tissue into the subcutaneous space of 22 SCID mice for 80 days. MAIN OUTCOME MEASURE(S): Histologic analysis before and after grafting. Serum E(2) measured before (after 37 days of grafting) and after FSH/LH supplementation (end of the study). RESULT(S): After grafting, follicular density had decreased for frozen/thawed tissue in all cases. The follicular distribution was modified in fresh tissue: Primordial follicles proportion was reduced (79% vs. 17%), whereas the primary and secondary ones were increased (21% vs. 57% and 0% vs. 23%, respectively). The same tendency was observed in frozen/thawed tissue. Significant E(2) secretion was obtained before and after FSH/LH supplementation in castrated mice, grafted with either fresh or frozen/thawed tissue. CONCLUSION(S): Fresh and cryopreserved ovarian cortex surrounding benign ovarian cysts grafted into the subcutaneous space of SCID mice is able to sustain ovarian tissue function, although follicular growth appears lower with frozen/thawed tissue.

Adenine nucleotide translocator promotes oxidative phosphorylation and mild uncoupling in mitochondria after dexamethasone treatment.

The composition of the mitochondrial inner membrane and uncoupling protein [such as adenine nucleotide translocator (ANT)] contents are the main factors involved in the energy-wasting proton leak. This leak is increased by glucocorticoid treatment under nonphosphorylating conditions. The aim of this study was to investigate mechanisms involved in glucocorticoid-induced proton leak and to evaluate the consequences in more physiological conditions (between states 4 and 3). Isolated liver mitochondria, obtained from dexamethasone-treated rats (1.5 mg.kg(-1).day(-1)), were studied by polarography, Western blotting, and high-performance thin-layer chromatography. We confirmed that dexamethasone treatment in rats induces a proton leak in state 4 that is associated with an increased ANT content, although without any change in membrane surface or lipid composition. Between states 4 and 3, dexamethasone stimulates ATP synthesis by increasing both the mitochondrial ANT and F1-F0 ATP synthase content. In conclusion, dexamethasone increases mitochondrial capacity to generate ATP by modifying ANT and ATP synthase. The side effect is an increased leak in nonphosphorylating conditions.

In vitro alachlor effects on reactive oxygen species generation, motility patterns and apoptosis markers in human spermatozoa.

Due to its extensive production and application, the toxicity of chloracetanilide herbicide alachlor[2-chloro-2',6'-diethyl-N-(methoxymethyl)-acetanilide] should be evaluated to establish minimum effects. In this study, we have examined the in vitro effects of alachlor on human sperm motion using a computer-assisted sperm analyser (CASA). An evaluation of both reactive oxygen species (ROS) and markers of apoptosis was also performed to investigate the mechanism by which alachlor modifies the sperm movement. After exposure up to 2 h to alachlor (0, 0.18, 0.37, 0.90 and 1.85 mM), the percentage of viable, motile spermatozoa and sperm velocities were concentration and/or time dependently decreased. The most sensitive parameters were progressive motility, mean average path velocity and mean straight velocity. Alachlor (1.85 mM) induced an increase in ROS production. A decrease of mitochondrial membrane potential (DeltaPsi(m)), an increase of both phosphatidylserine (PS) externalization and DNA fragmentation, which were concentration and/or time dependent, were also observed. It is possible that toxic effects of alachlor result in an oxidative stress which could act as a mediator of apoptosis. Alachlor could also contribute to some hypofertility cases.

Lipid remodeling of murine epididymosomes and spermatozoa during epididymal maturation.

We have isolated vesicular structures from mouse epididymal fluid, referred to as epididymosomes. Epididymosomes have a roughly spherical aspect and a bilayer membrane, and they are heterogeneous in size and content. They originate from the epididymal epithelium, notably from the caput region, and are emitted in the epididymal lumen by way of apocrine secretion. We characterized their membranous lipid profiles in caput and cauda epididymidal fluid samples and found that epididymosomes were particularly rich in sphingomyelin (SM) and arachidonic acid. The proportion of SM increased markedly during epididymal transit and represented half the total phospholipids in cauda epididymidal epididymosomes. The cholesterol:phospholipid ratio increased from 0.26 in the caput to 0.48 in the cauda epididymidis. Measures of epididymosomal membrane anisotropy revealed that epididymosomes became more rigid during epididymal transit, in agreement with their lipid composition. In addition, we have characterized the membrane lipid pattern of murine epididymal spermatozoa during their maturation. Here, we have shown that mouse epididymal spermatozoa were distinguished by high percentages of SM and polyunsaturated membranous fatty acids (PUFAs), principally represented by arachidonic, docosapentanoic, and docosahexanoic acids. Both SM and PUFA increased throughout the epididymal tract. In particular, we observed a threefold rise in the ratio of docosapentanoic acid. Epididymal spermatozoa had a constant cholesterol:phospholipid ratio (average, 0.30) during epididymal transit. These data suggest that in contrast with epididymosomes, spermatozoal membranes seem to become more fluid during epididymal maturation.

Human ovarian tissue from cortex surrounding benign cysts: a model to study ovarian tissue cryopreservation.

BACKGROUND: The scarcity of human ovarian tissue is a major problem in developing research on ovarian cryopreservation. We were interested in ovarian cortex surrounding benign ovarian cysts harvested during their requisite operations. METHODS: Ovarian tissue was collected from 25 women (mean age = 27.7 +/- 1.0 SEM) and frozen in serum-free cryoprotective medium. Histological and viability analysis were performed on fresh and frozen-thawed slices of tissue. RESULTS: Dermoid (n = 7), endometriosis (n = 13) and serous (n = 5) cysts were observed. Follicular densities (expressed per mm3) in ovarian cortex surrounding dermoid cysts were higher than in endometriosis and serous cysts for both histological (median of follicular densities: 13.04, 0.31 and 0.89 respectively) and viability analysis (2.93, 0.05 and 0.71 respectively). Freezing-thawing did not result in gross abnormality of follicle population either in number or morphology (80% of follicles preserved a normal pattern). However, a slight decrease of the density of living follicles (expressed per mm2) was reported. CONCLUSIONS: Ovarian cortex surrounding ovarian cysts, especially dermoid cysts, could be considered a source of ovarian tissue for future research. In our study, the cryopreservation procedure resulted in high follicular survival assessed by both histological and viability analysis. Nevertheless, further studies of in vivo and in vitro follicular maturation are needed to strengthen this model.

Electrophoretic characterization of the human sperm-specific enolase at different stages of maturation.

The presence of a sperm-specific enolase isoform (ENO-S) in human ejaculated spermatozoa was previously demonstrated. The objective of this study was to characterize this ENO-S in spermatozoa at different steps of maturation. Sperm ENO-S was characterized in testicular, epididymal, and ejaculated spermatozoa to determine whether any change occurred in the isoform patterns of this enzyme during epididymal maturation. In testicular sperm, ENO-S was present under 2 main bands named S1 and S3. In epididymal sperm, S1 and S3 bands and a prominent additional S2 band, with the same electrophoretic properties as the S isoform of ejaculated sperm, were visualized. In the testicular extracts obtained from testes in which no spermatozoa were visualized by histologic analysis, none of the 3 ENO-S bands was found. ENO-S exists as different isoforms (electrophoretic variants) in the different stages of sperm maturation. Passage through the epididymis seems to play a major role in the maturational process of this sperm-specific enolase.

Annexin V binding to plasma membrane predicts the quality of human cryopreserved spermatozoa.

Cryopreservation-induced stress may result in membrane injury with consequent decrease of sperm motility and fertilizing capacity. This study has investigated the relationship between human spermatozoa tolerance to cryopreservation and the loss of plasma membrane asymmetry, especially translocation of phosphatidylserine (PS) from the inner to the outer leaflet. The prospective study was performed on semen samples from 31 men. Conventional characteristics of 20 semen were analysed, before and after cryopreservation as well as externalization of PS assessed by annexin V-staining in combination with the propidium iodide which stains dead cells. The fertilizing capacity was evaluated by a zona free hamster egg penetration test in 11 freeze/thaw spermatozoa samples. The percentages of vital annexin V-negative and annexin V-positive spermatozoa in post-thaw samples were significantly lower than in pre-freeze ones (10 +/- 3 vs. 25 +/- 5% and 22 +/- 3 vs. 34 +/- 4% respectively), while the percentages of dead spermatozoa annexin V-negative and annexin V-positive had increased (42 +/- 4 vs. 23 +/- 4% and 23 +/- 4 vs. 16 +/- 2% respectively). The percentages of progressive and total motile spermatozoa were significantly correlated with the percentage of vital annexin V-negative spermatozoa before as well as after cryopreservation. Furthermore, recovery of motile spermatozoa after freeze/thaw was strongly correlated (p < 0.002) with the proportion of vital annexin V-negative spermatozoa in fresh semen. The percentage of penetration of zona-free hamster eggs was correlated (p < 0.02) with the percentage of live annexin V-negative cryopreserved spermatozoa capacitated for 3 h. These findings provide evidence that annexinV-binding staining in combination with PI brings additional information to predict the outcome of cryopreserved ejaculated sperm and may be used as a novel and reliable marker to study the freeze/thaw process.

Enolase isoforms activities in spermatozoa from men with normospermia and abnormospermia.

Total enolase as well as enolase-alphaalpha. (ENO-alphaalpha, ubiquitous) and enolase-S (ENO-S, sperm-specific) activities were measured in total and Percoll-selected sperm from 30 normospermic fertile men and 20 abnormospermic infertile patients. The total enolase activity was significantly higher in total sperm from patients with abnormospermia compared with normospermic patients (11.1 +/- 1.9 vs. 4.8 +/- 0.5 mlU/10(7) sperm P < .05). ENO-alphaalpha activity was significantly higher in total sperm from abnormospermic men than from normospermic men (P < .05). ENO-alphaalpha activity in Percoll-selected sperm was significantly lower compared with total sperm in both group of patients; however, for the same sperm fraction ENO-alphaalpha activity did not differ between normospermic and abnormospermic men. ENO-alphaalpha activity was related to the cell contamination ratio and to the percentage of spermatozoa with abnormal morphology. Furthermore, ENO-alphaalpha was positively correlated with the percentage of immature sperm showing an excess of residual cytoplasm. ENO-S activity was significantly higher in total sperm from normospermic patients than from abnormospermic patients (P < .05). ENO-S activity in Percoll-selected sperm was not significantly different compared with total sperm in both group of patients. However, this activity was significantly lower in Percoll-selected sperm from abnormospermic men compared with normospermic men (P < .05). ENO-S activity was not related to the cell contamination ratio but was significantly correlated with the percentage of spermatozoa with normal morphology. The 2 enolase isoforms seem to reflect 2 opposite aspects of sperm cells quality: ENO-alphaalpha is associated with abnormal spermatozoa, immature spermatozoa, or both; and ENO-S is associated with normal spermatozoa. As an additional index to distinguish normal from abnormal semen, the ENO-S:ENO-alphaalpha ratio was evaluated for total and Percoll-selected sperm in both groups. This ratio seems to be a new, valuable marker of the global sperm quality in a given semen sample, and may represent a predictive index of sperm fertilizing potential.