ObjectiveTo determine if sphingosine-1-phosphate (S1P) has any positive effect on the motilility, viability, or longevity of human sperm.DesignExperimental study of ejaculated sperm obtained from men in an in-vitro fertilization (IVF) program in which cellular viability was analyzed at specific intervals up to 40 hours.Materials and methodsEjaculated semen samples were examined after use in IVF for conventional insemination of oocytes. Samples from seven men exhibiting fertilization rates ranging from 25–100% and motility ranging from 41–97% 24–48 hours post insemination were selected for study. Each sample was divided for incubation with various concentrations of S1P (0 μM, 1 μM, 10 μM) in standard culture medium at 37°C under 5% CO2. Assessment of motility and forward progression were carried out by the same observer at several time intervals (0, 16, 24 and 40 hours). Cytological preparations of each sample were also made at the same times for assessing viability using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling of DNA (TUNEL assay). The data were plotted as time series and compared using Student´s t test.ResultsTreatment with 1 μM or 10 μM S1P had negligible effects on the decline in motility and forward progression of ejaculated sperm compared with controls. Over the course of 40 hours of observations, cellular viability fell to 0–20% in untreated samples but there was no significant beneficial effect on the survival curves with 1 μM and 10 μM S1P compared to control, although 100 μM S1P was found to be toxic. Moreover, results from the TUNEL assays confirmed morphological observations in showing no differences in cell death between groups over time.ConclusionsIn the present study, the effects of S1P on in vitro aging of sperm were tested because a natural compound promoting survival would be beneficial in clinically assisted conception and farm animal production. S1P is a sphingolipid involved in cellular homeostasis and promotes survival, and has been shown to protect oocytes from damage during heat shock or exposure to alkylating agents. However, it was found that neither physiological nor higher concentrations of S1P had any significant impact on three parameters of human sperm viability. ObjectiveTo determine if sphingosine-1-phosphate (S1P) has any positive effect on the motilility, viability, or longevity of human sperm. To determine if sphingosine-1-phosphate (S1P) has any positive effect on the motilility, viability, or longevity of human sperm. DesignExperimental study of ejaculated sperm obtained from men in an in-vitro fertilization (IVF) program in which cellular viability was analyzed at specific intervals up to 40 hours. Experimental study of ejaculated sperm obtained from men in an in-vitro fertilization (IVF) program in which cellular viability was analyzed at specific intervals up to 40 hours. Materials and methodsEjaculated semen samples were examined after use in IVF for conventional insemination of oocytes. Samples from seven men exhibiting fertilization rates ranging from 25–100% and motility ranging from 41–97% 24–48 hours post insemination were selected for study. Each sample was divided for incubation with various concentrations of S1P (0 μM, 1 μM, 10 μM) in standard culture medium at 37°C under 5% CO2. Assessment of motility and forward progression were carried out by the same observer at several time intervals (0, 16, 24 and 40 hours). Cytological preparations of each sample were also made at the same times for assessing viability using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling of DNA (TUNEL assay). The data were plotted as time series and compared using Student´s t test. Ejaculated semen samples were examined after use in IVF for conventional insemination of oocytes. Samples from seven men exhibiting fertilization rates ranging from 25–100% and motility ranging from 41–97% 24–48 hours post insemination were selected for study. Each sample was divided for incubation with various concentrations of S1P (0 μM, 1 μM, 10 μM) in standard culture medium at 37°C under 5% CO2. Assessment of motility and forward progression were carried out by the same observer at several time intervals (0, 16, 24 and 40 hours). Cytological preparations of each sample were also made at the same times for assessing viability using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling of DNA (TUNEL assay). The data were plotted as time series and compared using Student´s t test. ResultsTreatment with 1 μM or 10 μM S1P had negligible effects on the decline in motility and forward progression of ejaculated sperm compared with controls. Over the course of 40 hours of observations, cellular viability fell to 0–20% in untreated samples but there was no significant beneficial effect on the survival curves with 1 μM and 10 μM S1P compared to control, although 100 μM S1P was found to be toxic. Moreover, results from the TUNEL assays confirmed morphological observations in showing no differences in cell death between groups over time. Treatment with 1 μM or 10 μM S1P had negligible effects on the decline in motility and forward progression of ejaculated sperm compared with controls. Over the course of 40 hours of observations, cellular viability fell to 0–20% in untreated samples but there was no significant beneficial effect on the survival curves with 1 μM and 10 μM S1P compared to control, although 100 μM S1P was found to be toxic. Moreover, results from the TUNEL assays confirmed morphological observations in showing no differences in cell death between groups over time. ConclusionsIn the present study, the effects of S1P on in vitro aging of sperm were tested because a natural compound promoting survival would be beneficial in clinically assisted conception and farm animal production. S1P is a sphingolipid involved in cellular homeostasis and promotes survival, and has been shown to protect oocytes from damage during heat shock or exposure to alkylating agents. However, it was found that neither physiological nor higher concentrations of S1P had any significant impact on three parameters of human sperm viability. In the present study, the effects of S1P on in vitro aging of sperm were tested because a natural compound promoting survival would be beneficial in clinically assisted conception and farm animal production. S1P is a sphingolipid involved in cellular homeostasis and promotes survival, and has been shown to protect oocytes from damage during heat shock or exposure to alkylating agents. However, it was found that neither physiological nor higher concentrations of S1P had any significant impact on three parameters of human sperm viability.

Objective: The role that individual multinucleated blastomeres (MNBs) play in further preembryo (PE) development is unclear, most reports complicated by an inconsistent presence and unclear origin of this particular finding. Here, we attempted to determine if the presence of MNBs, as a single variable, is associated with reduced developmental capacity. Design: Retrospective study. Materials and Methods: A total of 2016 preembryos from 200 patients were included in the analysis. One would expect to observe 2 cells on Day 1 (26 hrs after insemination or 24 hrs after ICSI), 4 cells on Day 2, and 8 cells on Day 3. Blastomere number and presence/absence of MNBs were evaluated daily. Preembryos were also analyzed for MNB size and distribution. Results: On Day 1, the frequency of MNBs in cleaved PE was low (9.5%). The highest frequency of MNBs was observed in 2-cell preembryos on Day 2 (34%), while a lower incidence was seen in greater cell stages on the same day (22%, 6%, and 12% for 3-, 4-, and 5-cells respectively). Interestingly, developmental rates did not appear to be compromised when MNBs were noted on Days 1 or 2; no differences in average cell number on Day 3 were observed between MNB and non-MNB groups. Growth rate on Day 2, however, did impact Day 3 findings. A difference in average cell number on Day 3 was associated with the cell stage on Day 2, significantly better quality preembryos developing from Day 2 PE with 4 and 5 cells as compared to 2 cells (7.5 vs. 5.4 cells on Day 3 for advanced and slower cleavage on Day 2, respectively). In addition, the average cell number on Day 3 in PE developing from 2-cell, with or without MNB on D1, was comparable to 4 and 5 cells on Day 2. PE persisting at the 2-and 3-cell stage on D2 (highest frequency of MNBs) developed primarily from slower developing conceptuses on D1 (90% from pronuclear stage). Four-cell PE on D2 developed equally from 2-cell (31%), sygamy (41%) and pronuclear (27%) stages on D1, regardless of MNB status. The size and distribution of MNB noted at various cell stages did not influence developmental outcome. Conclusion: The results suggest that the appearance of MNBs, used as a single variable, does not appear to influence subsequent development and confirms that MNBs are not consistently featured during sequential cell divisions. On the other hand, regular rate of development has the greatest positive influence on subsequent PE development in vitro.

Objectives: Testicular sperm extraction (TESE) for intracytoplasmic sperm injection (ICSI) is an effective treatment for men with non-obstructive azoospermia. Overall, approximately 35% of these men have no spermatozoa found in their testicular tissue. The success of TESE cannot be predicted based on preoperative hormonal profiles (FSH, testosterone), nor based on testicular volume. Even a prior diagnostic testis biopsy does not absolutely predict the success of TESE, due to the heterogeneous nature of sperm maturation within seminiferous tubules of the testicle. Preliminary data in recent literature suggest a correlation between microdeletions involving the AZFb of the Y-chromosome and the absence of spermatozoa with TESE (Brandell et al., Human Reprod., 1998). We hereby report our expanded experience of the relationship between microdeletions involving AZFb and TESE results in men with non-obstructive azoospermia.Design: A retrospective analysis of azoospermic men who underwent TESE from 1996 to 2000 was performed. The outcomes of men with AZFb microdeletion of the Y-chromosome were analyzed.Materials and Methods: All TESE were performed under local or general anesthesia by one surgeon. TESE were performed with either multiple biopsies or with a single large incision with multiple samples of the seminiferous tubules under microscopic vision (microdissection TESE). The procedures were performed in conjunction with planned in-vitro fertilization cycles with ICSI for the female partner. Y-chromosomal microdeletion was tested in all patients using DNA extracted from peripheral leukocytes with a series of 35 sequence-tagged sites on Yq using polymerase chain reaction.Results: Among 181 patients who undergone TESE, 10 (6%) were found to have microdeletion involving the AZFb regions and 5 of them (3%) have microdeletion in AZFb region only. For men with non-obstructive azoospermia and no detectable deletions of the AZFb region, 114/171 (67%) men had sperm retrieved with TESE, whereas none of the 11 patients with microdeletions involving the AZFb region had sperm found with TESE (p<0.001). The average duration of TESE in patients with deletions involving the AZFb region was 3.5 hours.Conclusions: Genetic studies may provide important prognostic information in the management of patients with severe male infertility, as illustrated by our observation that the presence of an AZFb deletion is a significantly adverse prognostic finding for TESE. When counseling men with AZFb deletions, clinicians should apprise them of these results before attempting TESE-ICSI. Objectives: Testicular sperm extraction (TESE) for intracytoplasmic sperm injection (ICSI) is an effective treatment for men with non-obstructive azoospermia. Overall, approximately 35% of these men have no spermatozoa found in their testicular tissue. The success of TESE cannot be predicted based on preoperative hormonal profiles (FSH, testosterone), nor based on testicular volume. Even a prior diagnostic testis biopsy does not absolutely predict the success of TESE, due to the heterogeneous nature of sperm maturation within seminiferous tubules of the testicle. Preliminary data in recent literature suggest a correlation between microdeletions involving the AZFb of the Y-chromosome and the absence of spermatozoa with TESE (Brandell et al., Human Reprod., 1998). We hereby report our expanded experience of the relationship between microdeletions involving AZFb and TESE results in men with non-obstructive azoospermia. Design: A retrospective analysis of azoospermic men who underwent TESE from 1996 to 2000 was performed. The outcomes of men with AZFb microdeletion of the Y-chromosome were analyzed. Materials and Methods: All TESE were performed under local or general anesthesia by one surgeon. TESE were performed with either multiple biopsies or with a single large incision with multiple samples of the seminiferous tubules under microscopic vision (microdissection TESE). The procedures were performed in conjunction with planned in-vitro fertilization cycles with ICSI for the female partner. Y-chromosomal microdeletion was tested in all patients using DNA extracted from peripheral leukocytes with a series of 35 sequence-tagged sites on Yq using polymerase chain reaction. Results: Among 181 patients who undergone TESE, 10 (6%) were found to have microdeletion involving the AZFb regions and 5 of them (3%) have microdeletion in AZFb region only. For men with non-obstructive azoospermia and no detectable deletions of the AZFb region, 114/171 (67%) men had sperm retrieved with TESE, whereas none of the 11 patients with microdeletions involving the AZFb region had sperm found with TESE (p<0.001). The average duration of TESE in patients with deletions involving the AZFb region was 3.5 hours. Conclusions: Genetic studies may provide important prognostic information in the management of patients with severe male infertility, as illustrated by our observation that the presence of an AZFb deletion is a significantly adverse prognostic finding for TESE. When counseling men with AZFb deletions, clinicians should apprise them of these results before attempting TESE-ICSI.