Comparison of Heat Shock Protein 70 Expression in Response to Different Non-Ablative Lasers: An In Vitro Study.

Objective: The objective of our in vitro study was to compare the extent and timing of heat shock protein 70 (HSP70) expression following non-ablative fractional laser (NAFL) Er:Glass as well as Nd:YAG and alexandrite laser systems. Background: The role of HSP70 has been established in wound healing. Reports indicate that HSP70 expression increases after skin laser interventions. Methods: Human skin ex vivo explants were subjected to different fluences and pulse durations by using the three laser systems. Assessments were conducted on days 0, 1, 3, and 5 post-laser intervention. HSP70 expression was assessed by immunohistochemistry using anti-HSP70. Results: Er:Glass interventions caused the most significant increase in HSP70 expression whereas alexandrite interventions resulted in the least HSP70 expression, although significant compared with controls. HSP70 expression rose rapidly and dissipated quickly after Er:Glass and Nd:YAG interventions. Epidermal damage was most noted after Er:Glass interventions followed by the Nd:YAG and alexandrite systems. Conclusions: Non-ablative laser systems induce the expression of HSP70. Therefore, laser treatment may be considered 1-3 days before anticipated skin trauma to minimize scar formation provided the future validation of our data in an in vivo setting. The NAFL Er:Glass has the most potential for HSP70, yet it has also the most potential for complications.

Role of Laparoscopic Exploration Under Local Anesthesia in the Management of Hemodynamically Stable Patients with Penetrating Abdominal Injury.

BACKGROUND: In many trauma centers there is an ongoing controversy over the way of managing patients with penetrating abdominal injuries. This study was constructed to evaluate the role of diagnostic laparoscopy performed with local anesthesia for the management of penetrating abdominal injury. PATIENTS AND METHODS: Thirty hemodynamically stable patients with a penetrating stab wound in the anterior abdominal wall were included in this study after consent was obtained. Laparoscopic explorations were done with the patient having local anesthesia. If there was bleeding or intestinal content in the peritoneal cavity or visible injury to any abdominal organ, the procedure was converted to open exploration. If the peritoneal cavities showed no fluid and there was no injury to the abdominal viscera, the patient was followed up for the next 72 hours. Continuous variables were expressed as mean and standard deviation values. Categorical variables were expressed as frequencies and percentages. Student's t test was used to assess the statistical significance of the difference between the two study groups' means. Fisher's exact test was used to examine the relationship between categorical variables. A significance level of P < .05 was used in all tests. All statistical procedures were carried out using SPSS version 20 for Windows software (IBM, Armonk, NY). RESULTS: From the total of 30 patients who underwent laparoscopic exploration, 13 patients (43.3%) needed open exploration: 11 cases had intrabdominal organ injuries that needed laparotomy, 1 case had intraperitoneal blood collection where the only source of blood was the anterior abdominal wall wound, and 1 case had acute abdominal pain after 48 hours of negative laparoscopic exploration, in which an intestinal tear was found upon re-exploration. For the other 17 (56.7%) cases, 3 cases had no peritoneal penetration, whereas 14 cases had peritoneal penetration without any internal organ injuries, and these patients were followed up and discharged after 2-3 days. CONCLUSIONS: Laparoscopy performed with the patient having local anesthesia is an accurate diagnostic tool in the management of patients with an equivocal penetrating stab wound in the abdominal wall and can reduce the number of patients with negative open exploration.

Effects of advanced selection methods on sperm quality and ART outcome: a systematic review.

BACKGROUND: Current routine semen preparation techniques do not inclusively target all intrinsic sperm characteristics that may impact the fertilization potential. In order to address these characteristics, several methods have been recently developed and applied to sperm selection. The objective of this study was to systematically review the literature describing these advanced sperm selection methods focusing on their anticipated benefits on sperm quality and assisted reproductive technique (ART) outcome. METHODS: Systematic literature review was conducted by means of a Medline literature search. Sperm quality parameters assessed included: motility, morphology, viability, DNA integrity, apoptosis and maturity. ART outcomes assessed included: fertilization, embryo quality, pregnancy, abortion and live birth rates. RESULTS: A total of 44 studies were identified describing four advanced sperm selection methods based on: (i) surface charge (electrophoresis and zeta potential), (ii) apoptosis (magnetic cell sorting and glass wool), (iii) membrane maturity (hyaluronic acid binding) and (iv) ultramorphology (high magnification). Selection of high-quality sperm including improvements in DNA integrity, resulted from the application of these methods. Fertilization and pregnancy rates showed improvement following some of the advanced sperm selection techniques. CONCLUSIONS: While some of the advanced sperm selection methods are of value in specific clinical ART settings, others are in need of further evaluation. More clinical studies on safety and efficacy are needed before the implementation of advanced sperm selection methods could be universally recommended in ART.

Implication of apoptosis in sperm cryoinjury.

Apoptosis is an ongoing physiological phenomenon that has been documented to play a role in male infertility, if deregulated. Caspase activation, externalization of phosphatidylserine, alteration of mitochondrial membrane potential and DNA fragmentation are markers of apoptosis found in ejaculated human spermatozoa. These markers appear in excess in subfertile men and functionally incompetent spermatozoa. Sperm cryopreservation is a widely used procedure in the context of assisted reproductive techniques. Cryopreservation and thawing is a procedure that inflicts irreversible injury on human spermatozoa. The damage is manifested by a decrease in recovery of viable spermatozoa with optimum fertilization potential. This review describes the implication of apoptosis as one of the possible mechanisms involved in sperm cryoinjury. Evidence shows significant increase in some apoptosis markers following cryopreservation and thawing. On the other hand, the increase in sperm DNA fragmentation following cryopreservation and thawing requires further investigation. Specific technical measures should be applied to minimize the induction of apoptosis in human spermatozoa during cryopreservation and thawing. These include standardization of freezing protocols and cryoprotectant use. Selection of non-apoptotic spermatozoa may also prove to be of benefit.

Increased sperm chromatin decondensation in selected nonapoptotic spermatozoa of patients with male infertility.

OBJECTIVE: To evaluate the sperm chromatin decondensation (SCD) rates of the annexin-negative (nonapoptotic) sperm fraction of patients with infertility using hamster intracytoplasmic sperm injection (H-ICSI). In healthy donors, the depletion of apoptotic sperm using annexin V-based magnetic-activated cell separation (MACS) enhances hamster oocyte sperm penetration but does not increase SCD rates following H-ICSI. DESIGN: A prospective-controlled study. SETTING: Male infertility clinic, European Academy of Andrology Center Leipzig. PATIENT(S): Twenty-one male infertility patients with subnormal spermiogram. INTERVENTION(S): Spermatozoa were separated by Annexin V-MACS. MAIN OUTCOME MEASURE(S): Apoptosis signaling (disruption of transmembrane mitochondrial potential, transmembrane mitochondrial potential [TMP], and activation of caspases-3 [CP3]) and SCD rates of human spermatozoa after hamster intracytoplasmic sperm injection. RESULT(S): Infertility patients showed high levels of sperm with active CP3 and disrupted TMP, which correlated negatively with SCD rates. Annexin V-MACS resulted in a significant enrichment of spermatozoa with inactive CP3 and intact TMP in the annexin-negative fraction. Similarly, annexin-negative sperm had the highest SCD rates following H-ICSI compared with controls and annexin-positive sperm. CONCLUSION(S): These results suggest that nonapoptotic spermatozoa prepared by annexin V-MACS display higher early fertilization potential following ICSI. The technique should be evaluated in a clinical setting for its impact on ICSI outcomes in patients diagnosed with infertility.

Association of sperm apoptosis and DNA ploidy with sperm chromatin quality in human spermatozoa.

OBJECTIVE: To examine the relationship among sperm apoptosis, sperm chromatin status, and DNA ploidy in different sperm fractions. DESIGN: Prospective study. SETTING: Reproductive research center in a tertiary care hospital. INTERVENTION(S): Sperm prepared by density gradient were evaluated for sperm count, motility, apoptosis, and sperm chromatin assessment. MAIN OUTCOME MEASURE(S): Sperm count, sperm motility, toluidine blue (TB) results, DNA fragmentation index (%DFI), high DNA stainability, DNA cytometry, and early and late apoptosis. RESULT(S): Sperm motility was related to late apoptotic and subhaploid apoptotic sperm (r = -0.56 and -0.53, respectively). The sperm %DFI showed significant correlation with late apoptotic and subhaploid sperm (r = 0.62 and 0.68). TB-stained sperm were significantly correlated with late apoptotic sperm (r = 0.51). Significantly higher proportions of haploid sperm and light blue TB-stained sperm were seen in mature compared with immature fractions. CONCLUSION(S): Even in semen samples with low %DFI, semen processing results in a lower incidence of nuclear immaturity and subhaploidy, but the incidence of late apoptotic sperm remains unchanged. Therefore, simultaneous evaluation of apoptosis and sperm chromatin status is important for processing sperm in assisted reproductive procedures.

Utility of magnetic cell separation as a molecular sperm preparation technique.

Assisted reproductive techniques (ARTs) have become the treatment of choice in many cases of infertility; however, the current success rates of these procedures remain suboptimal. Programmed cell death (apoptosis) most likely contributes to failed ART and to the decrease in sperm quality after cryopreservation. There is a likelihood that some sperm selected for ART will display features of apoptosis despite their normal appearance, which may be partially responsible for the low fertilization and implantation rates seen with ART. One of the features of apoptosis is the externalization of phosphatidylserine (PS) residues, which are normally present on the inner leaflet of the sperm plasma membrane. Colloidal superparamagnetic microbeads ( approximately 50 nm in diameter) conjugated with annexin V bind to PS and are used to separate dead and apoptotic spermatozoa by magnetic-activated cell sorting (MACS). Cells with externalized PS will bind to these microbeads, whereas nonapoptotic cells with intact membranes do not bind and could be used during ARTs. We have conducted a series of experiments to investigate whether the MACS technology could be used to improve ART outcomes. Our results clearly indicate that integrating MACS as a part of sperm preparation techniques will improve semen quality and cryosurvival rates by eliminating apoptotic sperm. Nonapoptotic spermatozoa prepared by MACS display higher quality in terms of routine sperm parameters and apoptosis markers. The higher sperm quality is represented by an increased oocyte penetration potential and cryosurvival rates. Thus, the selection of nonapoptotic spermatozoa by MACS should be considered to enhance ART success rates.

Evaluation of sperm recovery following annexin V magnetic-activated cell sorting separation.

Magnetic-activated cell sorting (MACS) using paramagnetic annexin V-conjugated microbeads eliminates spermatozoa with externalized phosphatidylserine, which is considered one of the features of apoptosis. The objective of this study was to evaluate sperm recovery following the use of MACS as a sperm preparation technique. Mature spermatozoa were separated and divided into two fractions: the first was prepared by density gradient centrifugation (DGC) and MACS, while the second was prepared by DGC only. Following MACS, the percentage of cells collected in the annexin-negative fraction was significantly higher than the annexin-positive fraction and the sperm recovery rate was 73.8 +/- 12.1%. In conclusion, the integration of MACS with DGC can be considered as an effective sperm preparation technique that does not lead to significant cell loss. Separating a distinctive population of non-apoptotic spermatozoa with intact membranes may optimize the outcome of assisted reproduction.

Oxidative stress in an assisted reproductive techniques setting.

OBJECTIVE: The manipulation of gametes and embryos in an in vitro environment when performing assisted reproductive techniques (ART) carries the risk of exposure of these cells to supraphysiological levels of reactive oxygen species (ROS). The main objective of this review is to provide ART personnel with all the necessary information regarding the development of oxidative stress in an ART setting, as well as the sources of ROS and the mechanisms of oxidative stress-induced damage during ART procedures. The impact of oxidative stress on ART outcome and the different strategies designed to prevent it are also discussed. DESIGN: Review of international scientific literature. A question-and-answer format was adopted in an attempt to convey comprehensive information in a simple yet focused manner. RESULT(S): The pO(2) to which gametes and the embryo are normally exposed in vivo is significantly lower than in vitro. This results in increased production of ROS. Increase in levels of ROS without a concomitant rise in antioxidant defenses leads to oxidative stress. Lipid, protein, and DNA damage have all been associated with oxidative stress. This may ultimately result in suboptimal ART success rates. CONCLUSION(S): Many modifiable conditions exist in an ART setting that may aid in reducing the toxic effects of ROS.

Role of total antioxidant capacity in the differential growth of human embryos in vitro.

OBJECTIVE: The objective of this study was to examine the relationship of early human embryonic development parameters with day 1 culture media total antioxidant levels (day 1 TAC). DESIGN: Prospective study. SETTING: Patients undergoing assisted reproduction (ART) in a teaching hospital. PATIENT(S): Patients undergoing conventional IVF (n = 153; 167 cycles) and intracytoplasmic sperm injection (ICSI; n = 105; 116 cycles) were included. Both fertilization and early cultures were performed in human tubal fluid (HTF) with 5% serum substitute supplement. INTERVENTION(S): Levels of total antioxidants in the central well (sample) and the outer well (control) of each embryo culture dish were measured. MAIN OUTCOME MEASURE(S): Fertilization rate and embryo quality at days 3 and 5 were recorded for each cycle. Age, parity, and demographic features were also compared. RESULT(S): After controlling for all demographic and clinical variables, day 1 TAC was related to fertilization rates in both groups of patients. Day 1 TAC was significantly related to high (>7) day 3 cell number, low (<10%) fragmentation rate, and blastocyst development rate in both conventional IVF and ICSI cycles. Day 1 TAC was related to pregnancy in ICSI but not conventional IVF cycles. CONCLUSION(S): Day1 TAC appears to be an important biochemical marker for early embryonic growth. Decreased embryonic fragmentation, enhanced cleavage rate, and increased blastocyst development rate may be partially related to day 1 TAC in the culture media. Whether this relationship is a cause or effect needs further assessment.

Magnetic-activated cell sorting before cryopreservation preserves mitochondrial integrity in human spermatozoa.

Superparamagnetic annexin-V conjugated microbeads are able to eliminate spermatozoa with externalized phosphatidylserine, a membrane feature of apoptotic cells as well as spermatozoa with deteriorated plasma membrane. Our objective was to evaluate the effects of annexin-V Magnetic-Activated Cell Sorting (MACS) in cryopreservation-thawing protocols and on integrity of sperm mitochondrial transmembrane potential and mitochondrial integrity survival rate (MSR). Mature spermatozoa of 10 healthy donors were prepared by density gradient centrifugation and divided into 2 aliquots afterwards. The first one was subjected to annexin-V MACS followed by cryopreservation and thawing, while the second was cryopreserved-thawed without MACS to serve as control. Annexin-negative sperm separated by MACS showed significantly higher levels of intact mitochondria following cryopreservation-thawing (45.4+/-8.6%) compared to sperm that were not separated (15.8+/-4.6%, p<0.01). Separating a distinctive population of non-apoptotic spermatozoa with intact membranes may optimize cryopreservation-thawing outcome. MACS using annexin-V microbeads enhances the percentage of spermatozoa with intact transmembrane mitochondrial potential and mitochondrial integrity survival rates following cryopreservation.

Relationship between semen quality and tobacco chewing in men undergoing infertility evaluation.

OBJECTIVE: Male fertility is affected by a variety of lifestyle habits that include tobacco use. A large population of Indian men is addicted to tobacco chewing. The objective of our study was to assess the relationship between tobacco chewing in these Indian men--who were part of an infertile couple--and their sperm characteristics. DESIGN: Retrospective study. SETTING: Private infertility clinic. PATIENT(S): Six hundred thirty-eight male patients undergoing infertility evaluations were grouped according to the frequency of their tobacco chewing habit: mild (< 3 times/day, n = 177), moderate (3-6 times/day, n = 264), and severe (> 6 times/day, n = 197). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Sperm characteristics (concentration, motility, morphology, and viability). RESULT(S): Sperm concentration, percentage motility, morphology, and percentage viability were significantly higher in the mild group vs. the moderate group and in the moderate group vs. the severe group. The percentage of men with azoospermia rose with the level of addiction (1%, 3%, and 14%) as did the percentage of men with oligoasthenoteratozoospermia (2%, 8%, and 29%), although the differences were not statistically significant. CONCLUSION(S): In our study, use of chewing tobacco by a group of Indian men who were undergoing infertility evaluation was strongly associated with a decrease in sperm quality and to a lesser extent with oligoasthenozoospermia or azoospermia. Infertile men should be counseled about the adverse effects of tobacco chewing on sperm quality.

Advantage of combining magnetic cell separation with sperm preparation techniques.

The selection of vital, non-apoptotic spermatozoa is a prerequisite for achieving optimal conception rates in assisted reproductive techniques. Magnetic cell sorting using annexin-V microbeads can effectively separate apoptotic and non-apoptotic spermatozoa. The objective of the present study was to optimize the integration of magnetic cell sorting in standard sperm preparations and to correlate the effect of different sperm preparation procedures on apoptotic markers. Semen specimens collected from 15 healthy donors were prepared by either density gradient centrifugation or by one-step sperm wash technique separately and in combination with magnetic cell sorting. The preparation methods were evaluated by assessment of semen parameters (motility, viability and morphology) as well as markers of apoptosis (levels of active caspase-3, integrity of membrane mitochondrial potential and externalization of phosphatidylserine). The apoptotic markers were measured using fluorochrome dyes coupled with flow cytometry. The results showed that the combination of density gradient centrifugation and annexin-V magnetic cell sorting was superior to all other sperm preparation methods in terms of providing motile, viable and non-apoptotic spermatozoa. This study clearly shows the advantage of integrating magnetic cell sorting as a part of sperm preparation, which in turn may positively affect the success rates of assisted reproductive techniques.

Infliximab may reverse the toxic effects induced by tumor necrosis factor alpha in human spermatozoa: an in vitro model.

OBJECTIVE: To examine the toxic effects of tumor necrosis factor alpha (TNF-alpha) on ejaculated spermatozoa and evaluate the ability of infliximab to reverse these effects. DESIGN: Prospective controlled study. SETTING: Cleveland Clinic Foundation, Cleveland, Ohio. PATIENT(S): Thirty-one healthy sperm donors. INTERVENTION(S): Exposure of human spermatozoa to varying concentrations of TNF-alpha (100, 300, 400, 500 pg/mL, and 2.5 microg/mL) and infliximab (400 microg/mL). MAIN OUTCOME MEASURE(S): Sperm motility, functional integrity of plasma membrane, and DNA fragmentation. RESULT(S): Spermatozoa quality declined following incubation with TNF-alpha in a dose-dependent and time-dependent manner. Sperm motility and membrane integrity were higher in the samples incubated with TNF-alpha plus infliximab than in the samples treated with TNF-alpha only. These parameters improved significantly and were comparable with both controls and sperm incubated with infliximab alone. Similarly, the percentage of spermatozoa with DNA fragmentation improved significantly following incubation with TNF-alpha plus infliximab and again was comparable with both controls and sperm incubated with infliximab alone. CONCLUSION(S): Spermatozoa may be exposed to abnormal levels of TNF-alpha in the male reproductive tract or during their passage into the female reproductive tract (in cases of endometriosis). Exposing spermatozoa to pathological concentrations of TNF-alpha can result in significant loss of their functional and genomic integrity. Infliximab could potentially be used to help treat female infertility caused by endometriosis in those with elevated levels of TNF-alpha in their peritoneal fluid.

Novel association between sperm deformity index and oxidative stress-induced DNA damage in infertile male patients.

AIM: To investigate the impact of abnormal sperm morphology using the sperm deformity index (SDI) on reactive oxygen species (ROS) production and its correlation with sperm DNA damage. METHODS: Semen samples were collected from men undergoing infertility screening (n = 7) and healthy donors (n = 6). Mature spermatozoa were isolated and incubated with 5 mmol/L beta-nicotinamide adenine dinucleotide phosphate (NADPH) for up to 24 h to induce ROS. Sperm morphology was evaluated using strict Tygerberg's criteria and the SDI. ROS levels and DNA damage were assessed using chemiluminescence and terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labeling (TUNEL) assays, respectively. RESULTS: SDI values (median [interquartiles]) were higher in patients than donors (2 [1.8, 2.1] vs. 1.53 [1.52, 1.58], P = 0.008). Aliquots treated with NADPH showed higher ROS levels (1.22 [0.30, 1.87] vs. 0.39 [0.10, 0.57], P = 0.03) and higher incidence of DNA damage than those not treated (10 [4.69, 24.85] vs. 3.85 [2.58, 5.10], P = 0.008). Higher DNA damage was also seen following 24 h of incubation in patients compared to donors. SDI correlated with the percentage increase in sperm DNA damage following incubation for 24 h in samples treated with NADPH (r = 0.7, P = 0.008) and controls (r = 0.58, P = 0.04). CONCLUSION: SDI may be a useful tool in identifying potential infertile males with abnormal prevalence of oxidative stress (OS)-induced DNA damage. NADPH plays a role in ROS-mediated sperm DNA damage, which appears to be more evident in infertile patients with semen samples containing a high incidence of morphologically abnormal spermatozoa.

Effects of magnetic-activated cell sorting on sperm motility and cryosurvival rates.

OBJECTIVE: To evaluate the effect of magnetic-activated cell sorting in cryopreservation-thawing protocols on sperm motility and cryosurvival rate. DESIGN: Prospective-controlled study. SETTING: Andrology department at a university-based medical institution. PATIENT(S): Ten healthy volunteer sperm donors. INTERVENTION(S): Sperm populations were separated using annexin-V magnetic-activated cell sorting before and after the cryopreservation-thawing process. MAIN OUTCOME MEASURE(S): Sperm motility and cryosurvival rate. RESULT(S): Annexin-negative sperm separated by magnetic-activated cell sorting had statistically significantly higher motility following cryopreservation-thawing than sperm that were not separated. Similarly, annexin-negative spermatozoa also had higher cryosurvival rate than sperm cryopreserved without magnetic-activated cell sorting and sperm that were annexin-positive. CONCLUSION(S): Superparamagnetic annexin V-conjugated microbeads can separate spermatozoa with externalized phosphatidylserine, which is considered one of the early features of late apoptosis. The separation of a distinctive population of nonapoptotic spermatozoa with intact membranes may optimize the cryopreservation-thawing outcome. Magnetic-activated cell sorting using annexin-V microbeads enhances sperm motility and cryosurvival rates following cryopreservation.

Phenotypic characterization of the immune and mast cell infiltrates in the human testis shows normal and abnormal spermatogenesis.

OBJECTIVE: To assess the types, distributions, and numbers of immune cell infiltrates in the testes of men with azoospermia. DESIGN: Prospective study. SETTING: University research and clinical institutes. PATIENT(S): Thirty-one men with azoospermia showing normal spermatogenesis (n = 10), germ cell maturation arrest (GA, n = 12), and Sertoli cell-only syndrome (SCO, n = 9). INTERVENTION(S): Testicular tissue biopsies. Sections were stained with routine (hematoxyline and eosin), special (Masson Trichrome, Gordon, Periodic Acid Schiff, Aldehyde Fuchsin, and Orcein stains), and immunoperoxidase stains (using monoclonal antibodies for B and T cells and CD68 macrophages). MAIN OUTCOME MEASURE(S): Serum levels of T, FSH, and LH in addition to histopathological analysis. RESULT(S): Hormonal profiles were unremarkable in all patients. Marked deposition of the reticular and collagen fibers was seen more in abnormal than normal spermatogenesis. The immune (B and T lymphocytes and CD68 macrophages) and mast cells were found in the interstitium, tubular walls, and lumens of all the testes analyzed. The differential counts of these cells (B and T lymphocytes, CD68 macrophages, and mast cells, respectively) were higher in SCO (1.66 +/- 0.46, 9.14 +/- 1.30, 2.26 +/- 1.68, 3.35 +/- 0.23) and GA (2.03 +/- 0.48, 4.70 +/- 1.00, 2.61 +/- 0.70, and 4.18 +/- 0.13) when compared with those in normal spermatogenesis (1.22 +/- 0.19, 5.41 +/- 0.58, 1.55 +/- 0.33, and 2.26 +/- 0.13). Increased cellular counts were not statistically significant for T and B cells and macrophages. However, these differences were statistically significant for mast cells. CONCLUSION(S): Abnormal spermatogenesis is associated with increased numbers of the immune and mast cells. Our findings may reflect an exaggerated immune response in these cases.

Caspase activation in human spermatozoa in response to physiological and pathological stimuli.

OBJECTIVE: To investigate caspase activation in response to a variety of pathological and physiological stimuli in light of the fact that current research offers no clear consensus about caspase activation pathways in spermatozoa. DESIGN: A prospective, controlled study. SETTING: Male infertility clinic, Glickman Urological Institute, Cleveland Clinic Foundation, Cleveland, Ohio. PATIENT(S): Fifteen healthy volunteers. INTERVENTION(S): Spermatozoa were exposed to [1] Fibroblast-associated (Fas) death receptor activation, [2] mitochondrial apoptosis induction using betulinic acid, [3] oxidative stress, and [4] prolonged incubation up to 3 hours without any external stimuli. MAIN OUTCOME MEASURE(S): Active caspases-1, -3, -8, and -9 were examined in human spermatozoa by flow cytometry using carboxyfluorescein derivatives. RESULT(S): Inducing Fas antibody did not result in any caspase activation. Conversely, betulinic acid significantly triggered caspase-9 and -3 activation. The application of oxidative stress and prolonged incubation (3 hours) failed to result in caspase activation. CONCLUSION(S): These results suggest that Fas has no functional relevance in mediating caspase activation in human ejaculated spermatozoa. Although spermatozoal mitochondria are highly susceptible to specific agonists of apoptosis such as betulinic acid via caspase activation, oxidative stress-induced apoptosis appears to be caspase independent.