OBJECTIVE: We evaluated the effectiveness of sperm washing using the discontinuous two-layer density gradient centrifugation method to remove antisperm antibodies attached to the sperm surface. METHOD: We prospectively enrolled sixty-six men with unexplained infertility who were seeking evaluation. Each patient delivered one semen specimen for the study. We determined antisperm antibody levels using the direct immunobeads test. Specimens were classified into two groups according to the pre-washing levels of antibody-bound spermatozoa: group 1 (low antisperm antibodies levels, immunobeads test < 20%; n = 54), and group 2 (high antisperm antibodies levels, immunobeads test > 20%; n = 12). Sperm washing was carried out using the discontinuous two-layer colloidal density gradient centrifugation method. Pre- and post-wash levels of antisperm antibodies were compared in the groups. RESULTS: The pre- and post-wash percentage of spermatozoa with antisperm antibodies attached to their surface was 11% and 6.5% in group 1 (mean difference = 40%; p < 0.01), and 30% and 19.5% in group 2 (mean difference = 36.8%, p = 0.02), respectively. The effectiveness of density gradient centrifugation in removing antisperm antibodies was not different between the groups, but individual variations from - 52.3% to -3.9% were observed. CONCLUSIONS: Sperm washing by density gradient centrifugation is an overall effective method to remove antibodies bound to sperm membranes, regardless of the levels of antisperm antibodies in the neat semen. Due to an inter-individual variation in the effectiveness of the method, we recommend that each patient be tested before applying sperm washing by density gradient centrifugation in intrauterine insemination.

Objective: To study the resistance of human spermatozoa to cryoinjury in repeated cycles of thaw-refreezing by using the fast liquid nitrogen vapor method. Material and Methods: Semen specimens were obtained from sixteen normal and oligozoospermic individuals who required disposal at the sperm bank. Five of them had testicular cancer. Specimens were thawed and an aliquot was removed for analysis. The remaining specimens were refrozen without removing the cryomedia. Repeated freeze-thaw cycles were performed until no motile sperm were observed. Sperm motility, number of motile spermatozoa and viability were determined after thawing. Resistance to cryoinjury was compared between groups and also after each refreezing cycle within groups. Results: Motile spermatozoa were recovered after five and two refreeze-thawing cycles in normozoospermic and oligozoospermic specimens, respectively. There were no significant differences in the recovery of motile spermatozoa between thaws within each group of normal and oligozoospermic specimens, but percentage motility and total number of motile spermatozoa were significantly lower in the oligozoospermic one. Specimens from men with cancer were exposed to six refreeze-thawing cycles. Although recovery of motile spermatozoa was significantly impaired after each thawing, there were no significant differences in the recovery of motile sperm between thaws in cancer and non-cancer groups. Conclusions: Human spermatozoa resist repeated cryopreservation using the fast liquid nitrogen vapor method. Normozoospermic specimens withstand refreezing for an average two cycles longer than oligozoospermic ones. Specimens from cancer patients seem to resist repeated cryoinjury similarly to non-cancer counterparts. Resistance to repeated cryoinjury was related to the initial semen quality.

OBJECTIVE: To evaluate the impact of sperm defect severity and the type of azoospermia on the outcomes of intracytoplasmic sperm injection (ICSI). MATERIALS AND METHODS: This study included 313 ICSI cycles that were divided into two major groups according to the source of spermatozoa used for ICSI: 1) Ejaculated (group 1; n = 220) and 2) Testicular/Epididymal (group 2; n = 93). Group 1 was subdivided into four subgroups according to the results of the semen analysis: 1) single defect (oligo-[O] or astheno-[A] or teratozoospermia-[T], n = 41), 2) double defect (a combination of two single defects, n = 45), 3) triple defect (OAT, n = 48), and 4) control (no sperm defects; n = 86). Group 2 was subdivided according to the type of azoospermia: 1) obstructive (OA: n = 39) and 2) non-obstructive (NOA: n = 54). Fertilization (2PN), cleavage, embryo quality, clinical pregnancy and miscarriage rates were statistically compared using one-way ANOVA and Chi-square analyses. RESULTS: Significantly lower fertilization rates were obtained when either ejaculated sperm with triple defect or testicular sperm from NOA patients (63.4 + 25.9% and 52.2 + 29.3%, respectively) were used for ICSI as compared to other groups (~73%; P < 0.05). Epididymal and testicular spermatozoa from OA patients fertilized as well as normal or mild/moderate deficient ejaculated sperm. Cleavage, embryo quality, pregnancy and miscarriage rates did not differ statistically between ejaculated and obstructive azoospermia groups. However, fertilization, cleavage and pregnancy rates were significantly lower for NOA patients. CONCLUSION: Lower fertilization rates are achieved when ICSI is performed with sperm from men with oligoasthenoteratozoospermic and non-obstructive azoospermic, and embryo development and pregnancy rates are significantly lower when testicular spermatozoa from NOA men are used.

OBJECTIVE: The aim of this study was to analyze the influence of autoantibodies against spermatozoa present in the semen on the outcome of in vitro fertilization with intracytoplasmic sperm injection (ICSI). MATERIALS AND METHODS: We performed a retrospective analysis of clinical and laboratorial data from a six year-period ICSI cycles. Screening for the presence of ASA in the semen, by using the direct immunobeads test (IBT), was available for 351 cycles. According to the percentage of antibody-bound spermatozoa in the semen, we divided the cycles in four groups: I (n = 194): 0%-10% ASA; II (n = 107): 11%-20%; III (n = 33): 21%-50% and IV (n = 17): 51%-100% ASA. Additionally, a group of 349 ICSI cycles performed with ejaculated spermatozoa from oligo/asthenozoospermic men who had insufficient number of motile sperm available for ASA screening was included for comparison. ICSI outcomes were compared among groups and included fertilization rate (2 PN), cleavage rate, cleavage velocity, embryo quality, clinical pregnancy and miscarriage rates. Data were examined statistically, with an alpha level of 5% considered significant. RESULTS: Fertilization, cleavage rate and velocity, percentage of good quality embryos, as well as clinical pregnancy and miscarriage rates did not differ among different ASA levels groups. ICSI outcomes in men exhibiting different levels of autoimmunity against spermatozoa did not differ from those with severely abnormal seminal parameters. CONCLUSIONS: Our data indicate that intracytoplasmic sperm injection (ICSI) outcomes are not influenced by ASA levels on sperm.

OBJECTIVE: To assess the feasibility of refreezing human semen using the technique of liquid nitrogen vapor with static phases. MATERIALS AND METHODS: Twenty samples from 16 subjects who required disposal of their cryopreserved semen were thawed, corresponding to 6 cancer patients and 10 participants in the assisted reproduction (AR) program. Samples were refrozen using the technique of liquid nitrogen vapor with static phases, identical to the one used for the initial freezing, and thawed again after 72 hours. We assessed the concentration of motile spermatozoa, total and progressive percent motility and spermatic vitality, according to criteria of the World Health Organization (WHO), as well as spermatic morphology according to the strict Kruger criterion, after the first and after the second thawing. RESULTS: We observed a significant decrease in all the parameters evaluated between the first and the second thawing. Median values for the concentration of motile spermatozoa decreased from 2.0x10(6)/mL to 0.1x10(6)/mL (p < 0.01); total percent motility from 42% to 22.5% (p < 0.01); progressive percent motility from 34% to 9.5% (p < 0.01); vitality from 45% to 20% (p < 0.01); and morphology from 5% to 5% (p = 0.03). There was no significant difference in the spermatic parameters between the cancer and assisted reproduction groups, both after the first and after the second thawing. We observed that in 100% of cases there was retrieval of motile spermatozoa after the second thawing. CONCLUSIONS: Refreezing of human semen by the technique of liquid nitrogen vapor allows the retrieval of viable spermatozoa after thawing.