Y chromosome microdeletion is the most important genetic cause of impairment of spermatogenesis. Nevertheless, a significant proportion of patients with spermatogenic failure do not have this condition. This study investigated the expression level of AZF genes, DDX3Y (DBY), RBMY1, DAZ and TSPY in testicular tissues of 42 subjects with impaired spermatogenesis compared with 33 with normal spermatogenesis. Histopathological evaluation was performed in all subjects and tissues were classified according to Johnsen Score. Transcript amounts were determined by quantitative-competitive RT-PCR. Patients with complete Sertoli cell-only syndrome (SCOS) did not exhibit RBMY1, DAZ and TSPY gene expression, however, we detected very low expression of DDX3Y transcript. Tissue samples with focal SCOS showed significantly decreased expression of all genes (P < 0.001). Maturation arrest and hypospermatogenesis tissues expressed significantly low levels of DDX3Y testicular transcript (P < 0.001), while the mRNA levels of the other genes were similar to that in tissues from the normal spermatogenesis group. Negative or diminished gene expression of DDX3Y, RBMY1, DAZ and TSPY in tissues samples with SCOS or focal SCOS reflects the absence or the lower number of germ cells, respectively. The finding that the testicular transcript of DDX3Y is significantly decreased in patients with severe spermatogenenic failure, especially in those presenting maturation arrest, suggests an important role of DDX3Y during spermatogenesis.

BACKGROUNDThe pathophysiology of the testicular damage in varicocele has not been completely understood. Oxidative stress and related sperm DNA damage have been identified as significant causes of male infertility. The current study was designed to determine the extent of sperm nuclear DNA damage in patients with varicocele and to examine its relationship with oxidative stress.METHODSSemen samples from 55 patients with clinical varicocele and 25 normozoospermic donors were examined. Varicocele sperm samples were classified as normal or abnormal according to World Health Organization guidelines. Sperm DNA damage was evaluated by the sperm chromatin structure assay/flow cytometry and by the terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay. Levels of reactive oxygen species (ROS) and total antioxidant capacity were assessed by a chemiluminescence assay.RESULTSDNA fragmentation index (DFI) (percentage of sperm with denatured DNA) values and the percentage of TUNEL-positive cells were significantly greater in patients with varicocele, either with normal (DFI, 20.7 +/- 4.0; TUNEL positive, 26.1 +/- 3.2) or with abnormal (DFI, 35.5 +/- 9.0; TUNEL positive, 32.2 +/- 4.1) semen profile, compared with controls (DFI, 7.1 +/- 0.9; TUNEL positive, 14.2 +/- 1.2). Similarly, ROS levels were significantly higher (P < 0.01) in both groups of patients with varicocele.CONCLUSIONSThe presence of a varicocele is associated with high levels of DNA-damage spermatozoa even in the presence of normal semen profile. The results also indicate that oxidative damage is associated with sperm DNA damage in these patients.

OBJECTIVE:To investigate the expression of Fas protein on the surface of ejaculated spermatozoa of normozoospermic and nonnormozoospermic men.DESIGN:Prospective study.SETTING:University infertility clinic.PATIENT(S):Twenty-three volunteer normozoospermic men (controls) and 43 men undergoing infertility evaluation (cases).INTERVENTION(S):Analysis of ejaculated spermatozoa by indirect immunofluorescence of Fas protein by flow cytometry.MAIN OUTCOME MEASURE(S):Comparison of flow cytometric analysis of autofluorescence, control tests (secondary antibody and isotype control), and experimental tests (anti-Fas monoclonal antibody) in the spermatozoa of ejaculated samples.RESULT(S):No expression of Fas protein was found on the surface of ejaculated spermatozoa of controls and cases.CONCLUSION(S):The Fas molecules are not present in substantial amounts in the ejaculated spermatozoa of normozoospermic and nonnormozoospermic men. Therefore, our results do not support the "abortive apoptosis" theory.

We report a 12-7/12 year-old male with obesity, eunuchoid proportions, genetic stigmata of Turner's syndrome and mild developmental delay. We investigated whether cytogenetic alterations could be responsible for his phenotype. Conventional karyotype in 70 peripheral blood cells was 45,X(15%)/46,XYqh-(85%). Dual FISH on 1,000 nuclei revealed 8% of X0 cells (DXZ1 X-centromeric probe) and 92% of XY cells (DYZ3 Y-centromeric probe). We studied Y chromosome microdeletions by PCR. The patient showed a terminal Yq deletion from the 5I interval including the AZFb and AZFc regions. FSH, LH and testosterone (468 ng/dl) were within the normal range for his age. At Tanner IV pubertal development the spermiogram showed azoospermia and the testicular aspirate spermatic arrest. The present report suggests that Y chromosome deletions including AZFb and AZFc regions may cause spermatogenic arrest in early puberty.

Objectives: The purpose of this study was to evaluate whether a short exposure of oocytes to spermatozoa in human IVF-ET program in cases of non male factor infertility is better than those of the standard overnight incubation periods. In an attempt to evaluate if a prolonged exposure to spermatozoa could create detrimental conditions for sperm-oocyte fusion, the levels of lipid peroxidation (LPO) produced by the spermatozoa over time was also measured.Design: Prospective, randomized study involving 30 IVF cycles in which at least six oocytes were retrieved from each patient. All semen samples presented normal characteristics. Oocytes collected were randomly assigned to two study groups. Each patient oocytes were exposed to spermatozoa for either 3 h (study group) or the standard 16–18 h (control group) incubation period.Materials and Methods: Oocytes were inseminated in 100-μL drops of Human Tubal Fluid with 10% of Synthetic Serum Substitute (HTF/SSS) under oil with spermatozoa processed by the conventional swim up procedure. Fertilization was performed using 25 × 10 3 spermatozoa/100 μL. For both groups, corona cells were removed and oocytes examined for fertilization 16–18h following insemination. Cleavage was assessed after 48 and 72 h post-insemination at which time the embryos were transferred to the patient. Embryo quality was judged by the number of cells and morphology scoring. In order to assess the amount of LPO generated over time, spermatozoa processed for IVF were incubated under capacitating conditions and thiobarbituric acid reactive species (TBARS) were determined after 1, 3 and 18 h incubation.Results: No significant differences in fertilization rates could be found between the two groups (3h: 91%/18h: 93%). The proportion of better quality embryos (grades I–II) observed at 48 h (day 2) and 72 h (day 3) post-insemination was significantly higher (p<0.01) in the study group (short exposure) than in the controls (standard time). When evaluating the timing of embryo development on day 3, the study group generated a higher percentage (p<0.01) of 7 to 10 cell embryos when compared to control group (3h: 68%; 18h: 50%). A majority (87.5%) of the sperm preparations showed increased levels of TBARS between 1 and 3h incubation. Sixty percent of the normozoospermic patients have spermatozoa that showed increased concentrations of TBARS between 3 and 18 h incubation. The remaining 40% presented no changes in lipid peroxidation metabolites.Conclusions: A brief gamete coincubation during IVF, in cases apparently not involving a male factor offers a better outcome of embryonic development providing a good alternative to improve sperm-oocyte interaction. The beneficial effect may be associated with a decrease generation of peroxidized lipids and their breakdown products. FONDECYT 1990780. Objectives: The purpose of this study was to evaluate whether a short exposure of oocytes to spermatozoa in human IVF-ET program in cases of non male factor infertility is better than those of the standard overnight incubation periods. In an attempt to evaluate if a prolonged exposure to spermatozoa could create detrimental conditions for sperm-oocyte fusion, the levels of lipid peroxidation (LPO) produced by the spermatozoa over time was also measured. Design: Prospective, randomized study involving 30 IVF cycles in which at least six oocytes were retrieved from each patient. All semen samples presented normal characteristics. Oocytes collected were randomly assigned to two study groups. Each patient oocytes were exposed to spermatozoa for either 3 h (study group) or the standard 16–18 h (control group) incubation period. Materials and Methods: Oocytes were inseminated in 100-μL drops of Human Tubal Fluid with 10% of Synthetic Serum Substitute (HTF/SSS) under oil with spermatozoa processed by the conventional swim up procedure. Fertilization was performed using 25 × 10 3 spermatozoa/100 μL. For both groups, corona cells were removed and oocytes examined for fertilization 16–18h following insemination. Cleavage was assessed after 48 and 72 h post-insemination at which time the embryos were transferred to the patient. Embryo quality was judged by the number of cells and morphology scoring. In order to assess the amount of LPO generated over time, spermatozoa processed for IVF were incubated under capacitating conditions and thiobarbituric acid reactive species (TBARS) were determined after 1, 3 and 18 h incubation. Results: No significant differences in fertilization rates could be found between the two groups (3h: 91%/18h: 93%). The proportion of better quality embryos (grades I–II) observed at 48 h (day 2) and 72 h (day 3) post-insemination was significantly higher (p<0.01) in the study group (short exposure) than in the controls (standard time). When evaluating the timing of embryo development on day 3, the study group generated a higher percentage (p<0.01) of 7 to 10 cell embryos when compared to control group (3h: 68%; 18h: 50%). A majority (87.5%) of the sperm preparations showed increased levels of TBARS between 1 and 3h incubation. Sixty percent of the normozoospermic patients have spermatozoa that showed increased concentrations of TBARS between 3 and 18 h incubation. The remaining 40% presented no changes in lipid peroxidation metabolites. Conclusions: A brief gamete coincubation during IVF, in cases apparently not involving a male factor offers a better outcome of embryonic development providing a good alternative to improve sperm-oocyte interaction. The beneficial effect may be associated with a decrease generation of peroxidized lipids and their breakdown products. FONDECYT 1990780.

The aim of this study was to determine the predictive value of sperm penetration into the perivitelline space of human cadaveric oocytes on in vitro fertilization outcome. Forty-two patients with tubal infertility undergoing ovarian stimulation with gonadotropin for in vitro fertilization and embryo transfer participated in the study. The number of spermatozoa bound to the human zona pellucida, the percentage of cadaveric oocytes with one or more spermatozoa in the perivitelline space, and the in vitro fertilization outcome were evaluated. Spermatozoa from 37 of 42 patients were able to penetrate the perivitelline space of cadaveric oocytes as well as to fertilize human oocytes in vitro. In three individuals, no penetration of the perivitelline space of cadaveric oocytes was observed and no in vitro fertilization was detected. Only two patients were able to fertilize the couple's oocytes without penetration of the cadaveric oocytes. Based on these results the specificity and the sensitivity of the assay to predict in vitro fertilization was 100% and 94.1%, respectively. Accordingly, these results suggest that sperm-zona penetration is a useful bioassay to predict male fertility potential in IVF outcome.

The pregnancy rate reduction is mainly attributed to a low fertilization rate due to decreased sperm count and/or motility or a spermatozoa with morphological abnormalities. The aim of the present investigation was to determinate whether the percentage of apoptotic cells (programmed cell death) is related to the percentage of motile cell. 32 men participating in the IVF in our institute. The DNA fragmentation was analyzed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method (TUNEL). According to the seminal samples motility two groups of patients, Group I with seminal motility greater than or equal to 50% and Group II with seminal motility < 50%. A significant differece was observed between apoptotic cells after swim-up and at 24 hours of incubation. Our data suggest that higher percent of apoptotic spermatozoa is associated with seminal motility.

We studied the variability of traditional semen analysis parameters as well as sperm motion characteristics analyzed by computer-assisted-semen analyzer in semen and after 2 hours of incubation in capacitation conditions. Twenty men with probed fertility provided three semen samples and 60 men provide one. The higher variability was observed with seminal sperm concentration, the lower variation was observed for percent motility and percent of normal morphology. Ten Oligospermic donors (12,5%) were observed in our population without other semen parameters abnormalities. Their motion characteristics were comparable to cells analyzed from normospermic men. A lower variability of sperm motion characteristics was observed during sperm capacitation compared to sperm movement in seminal plasma; suggesting that sperm motion characteristics during capacitation may be a usefull method additional to semen analysis to evaluate male fertility potential.

The hypo-osmotic swelling (HOS) test has been proposed as a useful assay for evaluation of the functional competence of the human sperm membranes. To assess this further, the HOS-test was evaluated in 187 semen samples collected from fertile men and from male patients consulting for infertility. These samples were classified as normal, oligo-, astheno- or oligoasthenozoospermic on the basis of their standard semen variables. The percentage of total sperm tail swelling and of sperm exhibiting different tail swelling patterns was recorded. In the fertile men and in the group of patients with normal semen variables, significantly more (P less than 0.001) HOS-reactive sperm were observed after hypo-osmotic treatment in comparison with those groups exhibiting abnormal semen parameters. Swelling of the sperm in a hypo-osmotic medium was highly correlated with both progressive motility (r = 0.62, P less than 0.001) and sperm viability (r = 0.65, P less than 0.001). A weak positive correlation was also observed between sperm swelling and sperm morphological features (r = 0.31, P less than 0.005) and between sperm swelling and sperm concentration (r = 0.31, P less than 0.005). No significant correlation was observed between sperm swelling and in-vitro sperm fertilizing capacity as assessed by the zona-free hamster oocyte penetration assay. However the majority of the semen samples (87.3%) showing a normal penetration rate (greater than or equal to 10%) also exhibited a 60% (or higher) reaction in the HOS-test.(ABSTRACT TRUNCATED AT 250 WORDS)