Effects of pentoxifylline treatment before freezing on motility, viability and acrosome status of poor quality human spermatozoa cryopreserved by the liquid nitrogen vapor method

The objective of the present study was to investigate the effects of the direct addition of pentoxifylline (PF) to the ejaculates of men with poor sperm quality before freezing on post-thaw sperm motility, viability, acrosome integrity, and agonist-induced acrosome reaction. Semen specimens from 16 infertile men with impaired sperm count and motility (oligoasthenozoospermia) were divided into two equal aliquots: one received no treatment (control) while the other was incubated with 5 mM PF (treated). Both aliquots were cryopreserved by the liquid nitrogen vapor method. Motility was assessed according to WHO criteria. Acrosome integrity and spontaneous and calcium ionophore-induced acrosome reactions were assessed with fluorescein isothiocyanate-conjugated peanut agglutinin combined with a supra-vital dye (Hoechst-33258). Cryopreservation impaired sperm motility (percentage reduction: 87.4 (interquartile range, IQ: 70.3-92.9) vs 89.1 (IQ: 72.7-96.0%)), viability (25.9 (IQ: 22.2-29.7) vs 25.6 (IQ: 19.7-40.3%)) and acrosome integrity (18.9 (IQ: 5.4-38.9) vs 26.8 (IQ: 0.0-45.2%)) to the same extent in both treated and control aliquots. However, PF treatment before freezing improved the acrosome reaction to ionophore challenge test scores in cryopreserved spermatozoa (9.7 (IQ: 6.6-19.7) vs 4.8 (IQ: 0.5-6.8%); P = 0.002). These data show that pre-freeze treatment of poor quality human sperm with pentoxifylline did not improve post-thaw motility or viability nor did it prevent acrosomal loss during the freeze-thaw process. However, PF, as used, improved the ability of thawed spermatozoa to undergo the acrosome reaction in response to calcium ionophore. The present data indicate that treatment of poor quality human sperm with PF may enhance post-thaw sperm fertilizing ability.Universidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Laboratório de Reprodução HumanaCentro de Referência em Reprodução MasculinaUNIFESP, EPM, Laboratório de Reprodução HumanaSciEL

Effects of the technique of cryopreservation and dilution/centrifugation after thawing on the motility and vitality of spermatozoa of oligoasthenozoospermic men

OBJECTIVE: Comparing in human semen samples with low initial quality, the effects of 2 techniques of cryopreservation and dilution/centrifugation after thawing on the spermatic motility and vitality. MATERIALS AND METHODS: Semen samples from 15 oligo and/or asthenozoospermic individuals assisted in the infertility sector of a tertiary hospital were obtained through masturbation. The samples were divided into 2 portions of equal volume, and diluted (1:1; v/v) with the cryoprotector containing glycerol (Test yolk buffer). One portion was frozen through the technique of liquid nitrogen vapor with static phases (group I - GI), while the other was frozen through a programmable biological freezer with linear speed (Planer, Kryo 10, series III) (group II - GII). The following parameters were assessed before freezing and after thawing: percentage of spermatozoa with progressive motility (Prog%) and percentage of live spermatozoa (Vit%). After defrosting, Prog% was assessed before and after removal of cryoprotector diluent, in different time intervals (zero, 3 h, and 24 h). The statistical analysis has been accomplished by using the non-parametric tests of Wilcoxon and Friedman. RESULTS: There was significant reduction of Prog% and Vit% from before freezing to after defrosting in both groups, I and II (p < 0.001). Values of Prog% and Vit% were not statistically different between groups, after thawing. It has been observed a significant reduction in Prog% among portions frozen with the automated technique after dilution and centrifugation for removal of cryoprotector (p = 0.006). After cryoprotector removal, Prog% has been kept unaltered, in both groups, during the first 3 hours of incubation, although being superior in group I (p = 0,04). There was a significant decrease in Prog% after 24 hours of incubation, in both groups (p < 0,01). CONCLUSION: For human semen samples with low initial quality, freezing through vapor technique or through the automated technique showed to be equivalent in regarding recovery of live spermatozoa with progressive motility. The effects of dilution and centrifugation to remove the cryoprotector had a negative impact only in samples frozen through the automated technique. In both techniques, progressive motility is kept constant during the first 3 hours after thawing and removal of the cryoprotector, but is drastically diminished by the end of an incubation period of 24 hours.Universidade Federal de São Paulo (UNIFESP), Escola Paulista de Medicina (EPM) Division of UrologyUNIFESP, EPM, Division of UrologySciEL

Search and identification of spermatozoa and spermatids in the ejaculate of non-obstructive azoospermic patients

OBJECTIVE: To search and to identify spermatozoa and spermatids, present in the ejaculate of non-obstructive azoospermic patients. MATERIALS AND METHODS: 27 patients, aged between 18 and 48 years, with initial diagnosis compatible with non-obstructive azoospermia, underwent up to 3 seminal samples, with assessment of macroscopic and microscopic parameters differentiated for each sample. In the first sample, 5 µL of semen were analyzed in a Horwell chamber in order to assess the presence or absence of spermatozoa. The procedure was repeated with 2 other aliquots. In the absence of spermatozoa, the entire sample was transferred to a conic tube and following centrifugation the sediment was freshly analyzed. The second seminal sample was collected only when no spermatozoa were found in the first sample and the research was performed in the same way. In cases where spermatozoa were not seen, the sample was centrifuged and the obtained sediment was stained by the panoptic method and observed under common light microscopy (1250X). The third seminal sample was collected only in cases when patients had not shown spermatozoa in the first and second seminal samples. RESULTS: 4/27 (14.8%) patients presented spermatozoa in the first seminal sample and 6/23 (26.1%), in the second seminal sample. No spermatozoa were seen in the third sample, however, 11/17 (64.7%) presented spermatids. CONCLUSION: In clinical situations where the initial diagnosis is non-obstructive azoospermia, one single routine seminal analysis is not enough to confirm this diagnosis and the analysis of the centrifuged sediment can have relevant clinical consequences. Among patients considered non-obstructive azoospermic, when duly assessed, 37% presented spermatozoa and 64.7%, spermatids.Universidade Federal de São Paulo (UNIFESP), Escola Paulista de Medicina (EPM) Division of UrologyUNIFESP, EPM, Division of UrologySciEL

Effects of the technique of cryopreservation and dilution/centrifugation after thawing on the motility and vitality of spermatozoa of oligoasthenozoospermic men

OBJECTIVE: Comparing in human semen samples with low initial quality, the effects of 2 techniques of cryopreservation and dilution/centrifugation after thawing on the spermatic motility and vitality. MATERIALS AND METHODS: Semen samples from 15 oligo and/or asthenozoospermic individuals assisted in the infertility sector of a tertiary hospital were obtained through masturbation. The samples were divided into 2 portions of equal volume, and diluted (1:1; v/v) with the cryoprotector containing glycerol (Test yolk buffer). One portion was frozen through the technique of liquid nitrogen vapor with static phases (group I - GI), while the other was frozen through a programmable biological freezer with linear speed (Planer, Kryo 10, series III) (group II - GII). The following parameters were assessed before freezing and after thawing: percentage of spermatozoa with progressive motility (Prog%) and percentage of live spermatozoa (Vit%). After defrosting, Prog% was assessed before and after removal of cryoprotector diluent, in different time intervals (zero, 3 h, and 24 h). The statistical analysis has been accomplished by using the non-parametric tests of Wilcoxon and Friedman. RESULTS: There was significant reduction of Prog% and Vit% from before freezing to after defrosting in both groups, I and II (p < 0.001). Values of Prog% and Vit% were not statistically different between groups, after thawing. It has been observed a significant reduction in Prog% among portions frozen with the automated technique after dilution and centrifugation for removal of cryoprotector (p = 0.006). After cryoprotector removal, Prog% has been kept unaltered, in both groups, during the first 3 hours of incubation, although being superior in group I (p = 0,04). There was a significant decrease in Prog% after 24 hours of incubation, in both groups (p < 0,01). CONCLUSION: For human semen samples with low initial quality, freezing through vapor technique or through the automated technique showed to be equivalent in regarding recovery of live spermatozoa with progressive motility. The effects of dilution and centrifugation to remove the cryoprotector had a negative impact only in samples frozen through the automated technique. In both techniques, progressive motility is kept constant during the first 3 hours after thawing and removal of the cryoprotector, but is drastically diminished by the end of an incubation period of 24 hours

Adolescents with varicocele have an impaired sperm-zona pellucida binding capacity

Universidade Federal de São Paulo, Div Urol, BR-04024002 São Paulo, BrazilUniversidade Federal de São Paulo, Human Reprod Sect, BR-04024002 São Paulo, BrazilEastern Virginia Med Sch, Jones Inst Reprod Med, Dept Obstet & Gynecol, Norfolk, VA USAUniversidade Federal de São Paulo, Div Urol, BR-04024002 São Paulo, BrazilUniversidade Federal de São Paulo, Human Reprod Sect, BR-04024002 São Paulo, BrazilWeb of Scienc