Semen treatment with progesterone and/or acetyl-L-carnitine does not improve sperm motility or membrane damage after cryopreservation-thawing

OBJECTIVE: To assess the effects of progesterone and acetyl-L-carnitine used before semen cryopreservation-thawing on sperm motility parameters and plasma membrane integrity. DESIGN: Prospective cohort study.Setting: Academic tertiary center. PATIENT(S): Subfertile men undergoing semen evaluation. INTERVENTION(S): Before cryopreservation, spermatozoa were incubated with water-soluble progesterone (1 and 10 microM), acetyl-L-carnitine (2.5, 5, 10, and 20 mM), or both (progesterone, 1 microM; and acetyl-L-carnitine, 5 mM). MAIN OUTCOME MEASURE(S): Postthaw change of motility parameters (computer-assisted measurements) and vitality-membrane integrity (examined with eosin-Y staining and annexin V-Cy3 binding assay). RESULT(S): There were no statistically significant differences between control samples and samples treated with progesterone and/or acetyl-L-carnitine for cryosurvival rate, motility parameters, or membrane integrity. The percentages of postthaw cells identified as live showed significantly different results with use of the eosin-Y staining and annexin V binding assay. CONCLUSION(S): Neither progesterone nor acetyl-L-carnitine seemed to prevent cryodamage assessed by motility changes or membrane integrity in human spermatozoa of subfertile men. Annexin V binding, a reflection of membrane translocation of phosphatidylserine, provided more distinct information about postfreezing membrane integrity changes than eosin-Y staining.     

Cryopreservation-thawing of fractionated human spermatozoa and plasma membrane translocation of phosphatidylserine

OBJECTIVE(S): [1] To evaluate sperm membrane damage during cryopreservation-thawing by the assessment of phosphatidylserine (PS) translocation and [2] to examine the relationship between reactive oxygen species (ROS) and cryopreservation-related alterations. DESIGN: Prospective cohort study. SETTING: University-based center. PATIENT(S): Men consulting for infertility and fertile donors (controls). INTERVENTION(S): Semen processing was performed by density gradient separation followed by cryopreservation and thawing. MAIN OUTCOME MEASURE(S): Membrane PS translocation was evaluated with annexin V binding, generation of ROS was detected by chemiluminescence, and motion parameters were assessed by computer analysis. RESULT(S): Annexin V binding was detected in the prefreeze fractions with high and low sperm motility. In the patient group, there were significantly higher postthaw levels of annexin V binding in both fractions when compared with prefreezing values. However, such induction of PS translocation was significantly higher in the fractions with high sperm motility. Significantly higher ROS levels were detected in prefreeze samples of the fractions with low sperm motility. CONCLUSION(S): In the population of men studied, [1] cryopreservation-thawing was associated with induction of membrane PS translocation; [2] postthaw ROS levels were lower than before freezing; and [3] neither annexin V binding results nor the generation of ROS were able to accurately predict sperm cryosurvival rates.     

Impact of sperm morphology on DNA damage caused by oxidative stress induced by beta-nicotinamide adenine dinucleotide phosphate

OBJECTIVE: To investigate the role of DNA damage induced by beta-nicotinamide adenine dinucleotide phosphate (NADPH) in human spermatozoa. DESIGN: Prospective controlled study. SETTING: Male infertility clinic at the Glickman Urological Institute, Cleveland Clinic Foundation, Cleveland, Ohio. PATIENT(S): Twenty-eight men undergoing infertility screening. INTERVENTION(S): Chemiluminescence assay and terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling (TUNEL) assay coupled flow cytometry after incubating mature and immature sperm separated by density gradient with 5 mM NADPH for 0, 3, and 24 hours. MAIN OUTCOME MEASURE(S): Reactive oxygen species (ROS) generation (10(6) counted photons per minute/10(6) sperm) and percentage of spermatozoa with fragmented DNA. RESULT(S): Immature sperm from teratozoospermic semen samples were characterized by a statistically significant presence of cytoplasmic residues in the mid-piece when compared with mature normozoospermic samples. Increased ROS production was observed in spermatozoa rich in cytoplasmic residues that showed a statistically significant positive correlation with sperm DNA damage in a time-dependent manner. CONCLUSION(S): Immature sperm contain high nicotinamide adenine dinucleotide phosphate (NADPH) in cytoplasmic droplets, but it has not yet been clear whether abnormal sperm morphology plays any role in sperm DNA damage induced by oxidative stress. Our data support the role of NAPDH in ROS-mediated sperm DNA damage and suggest that abnormal sperm morphology combined with elevated ROS production may serve as a useful indicator of potential damage to sperm DNA.     

Role of reactive oxygen species in the pathophysiology of human reproduction

OBJECTIVE: To summarize the role of reactive oxygen species (ROS) in the pathophysiology of human reproduction. DESIGN: Review of literature. SETTING: Fertility research center and obstetrics and gynecology department in a tertiary care facility. RESULT(S): ROS plays an essential role in the pathogenesis of many reproductive processes. In male-factor infertility. oxidative stress attacks the fluidity of the sperm plasma membrane and the integrity of DNA in the sperm nucleus. Reactive oxygen species induced DNA damage may accelerate the process of germ cell apoptosis, leading to the decline in sperm counts associated with male infertility. ROS mediated female fertility disorders share many pathogenic similarities with the ones on the male side. These similarities include a potential role in the pathophysiology of endometriosis and unexplained infertility. High follicular fluid ROS levels are associated with negative IVF outcomes, particularly in smokers. Moreover, oxidative stress may be responsible in hydrosalpingeal fluid mediated embryotoxicity as well as poor in vitro embryonic development. CONCLUSION(S): High levels of ROS are detrimental to the fertility potential both in natural and assisted conception states.     

Human sperm endothelial nitric oxide synthase expression: correlation with sperm motility

Objective: To characterize the pattern of endothelial nitric oxide synthase (eNOS) expression on human spermatozoa and to determine whether sperm eNOS expression correlates with sperm function.

Design: Prospective, observational study.

Setting: University infertility clinic.

Patient(s): Twelve nonazoospermic infertile men.

Intervention(s): Semen samples (n = 12) obtained from nonazoospermic infertile men were fractionated on discontinuous Percoll gradients. Endothelial nitric oxide synthase staining on spermatozoa was correlated with sperm motility in Percoll gradient–fractionated spermatozoa. Endothelial nitric oxide synthase protein was detected with the use of a previously characterized monoclonal antibody. Control slides were incubated with preabsorbed antibody or mouse immunoglobulin G.

Main Outcome Measure(s): Localization of eNOS on human spermatozoa and correlation between the pattern of sperm eNOS expression and sperm motility.

Result(s): Morphologically normal spermatozoa exhibited postacrosomal and equatorial eNOS immunostaining. However, abnormally shaped spermatozoa often exhibited aberrant staining (in the midpiece and/or head region). A significant negative correlation was observed between the percentage of sperm with aberrant eNOS immunostaining and the percentage of motile sperm (r = −.46).

Conclusion(s): The specific localization of eNOS to human spermatozoa suggests that nitric oxide may be involved in normal sperm physiology. However, aberrant patterns of sperm eNOS expression are associated with decreased sperm motility, possibly through the generation of excessive cytotoxic oxidants.     

Potential adverse effect of semen processing on human sperm deoxyribonucleic acid integrity.

OBJECTIVE: To examine the effect of standard Percoll density-gradient centrifugation on human sperm DNA denaturation. DESIGN: Prospective, observational study. SETTING: University-based infertility clinic. PATIENT(S): Twenty-five nonazoospermic men. INTERVENTION(S): Semen samples (n = 25) were obtained from consecutively seen nonazoospermic men presenting for infertility evaluation. Samples were processed by two-layer and four-layer Percoll density gradients. Sperm motility and sperm chromatin structure (evaluated by flow cytometry analysis of acridine orange-treated spermatozoa) were monitored before and after semen processing. Sperm chromatin integrity was expressed as the percentage of spermatozoa that demonstrated denatured DNA. MAIN OUTCOME MEASURE(S): Sperm motility and DNA integrity. RESULT(S): Mean sperm motility improved significantly after processing with two-layer and four-layer Percoll gradients compared with whole semen (54% and 57% motility versus 44% motility, respectively). In contrast, the percentage of sperm with denatured DNA increased after processing with two-layer and four-layer Percoll gradients compared with whole semen (34% and 32% versus 18%, respectively). CONCLUSION(S): Our data demonstrate that the improvement seen in sperm motility after Percoll processing is not associated with a similar improvement in sperm DNA integrity. These data suggest that we reexamine current sperm processing techniques to minimize sperm DNA damage and the potential transmission of genetic mutations in assisted reproductive cycles.     

Measuring apoptosis in human spermatozoa: a biological assay for semen quality?

OBJECTIVE: [1] To determine whether apoptosis can be measured in ejaculated spermatozoa by flow cytometry using the Annexin V assay, which measures expression of phosphatidylserine on the outer leaflet of the cell membrane, or the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP [deoxy-uridine triphosphate] nick end labeling) assay, which measures occurrence of DNA strand breaks and [2] to correlate the outcome with routine semen variables and the hypoosmotic swelling (HOS) test. DESIGN: Pilot study and clinical trial. SETTING: Large teaching hospital and fertility center. PATIENT(S): Men whose semen was studied for various reasons. MAIN OUTCOME MEASURE(S): Percentage of apoptotic spermatozoa by two different assays, percentage of necrotic spermatozoa, concentration and motility of spermatozoa, and outcome of the HOS test. RESULT(S): Apoptosis can be measured in spermatozoa by flow cytometry using the Annexin V assay and the TUNEL assay. Twenty percent of spermatozoa were apoptotic according to both assays. A significant inverse correlation was seen between phosphatidylserine expression (Annexin V assay) and sperm concentration (r = -0.389; P<.05) and motility (r = -0.289; P<.05). A highly significant inverse correlation was seen between DNA double-strand breaks (TUNEL assay) and sperm concentration (r = -0.629; P<.0001). CONCLUSION(S): Flow cytometry can easily and reliably detect phosphatidylserine expression on the outer leaflet of the cell membrane and DNA strand breaks, both of which are hallmarks of apoptosis. About 20% of ejaculated spermatozoa are apoptotic, and the concentration of spermatozoa is lower in men with more apoptotic spermatozoa.     

Detection of apoptotic markers in human ejaculated spermatozoa as new methods in human reproductive biology

Ejaculated spermatozoa, particularly in infertile men, have been shown to display numerous features that are typical of apoptosis in somatic cells including Fas expression, ROS production, activation of caspases, DNA fragmentation, reduction in mitochondrial membrane potential, plasma membrane translocation of phosphatidylserine and permeability. In this review we summarize the biological significance and the potential role of these markers in the exploration of men infertility.     

Correlations between two markers of sperm DNA integrity, DNA denaturation and DNA fragmentation, in fertile and infertile men

OBJECTIVE: To evaluate two different assays of human sperm DNA integrity, DNA denaturation (DD) and DNA fragmentation (DF), and to correlate these with standard semen parameters. DESIGN: Prospective, observational study. SETTING: University infertility clinic.Patient(s): Forty consecutive semen samples from 33 nonazoospermic men presenting for infertility evaluation and 7 fertile men presenting for vasectomy. Intervention(s): Assessment of sperm concentration, motility, morphology, DD and DF. MAIN OUTCOME MEASURE(S): Sperm DD and DF in fertile and infertile men. RESULT(S): The mean (+/-SE) rates of DD and DF were significantly higher in infertile subjects compared to fertile controls, respectively: 25.4 +/- 3.0 vs. 10.2 +/- 2.3 (P=.028) and 27.6 +/- 2.5 vs. 13.3 +/- 2.5% (P=.016). DF and DD correlated strongly (r = 0.71, P<.0001). Also, DD and DF correlated negatively with standard semen parameters (concentration, motility, and morphology), the strongest correlation being with sperm motility. CONCLUSION(S): The strong correlation between sperm DD and DF, and the higher levels of sperm DNA damage in infertile compared with fertile men, indicate that male infertility is associated with poor sperm DNA integrity. Although infertile men may father children with assisted conception, fertilization with DNA-damaged spermatozoa may increase the risk of genetic disease in the offspring.     

The heparin-glutathione test: an alternative to the hypo-osmotic swelling test to select viable sperm for intracytoplasmic sperm injection

Objective: To evaluate the heparin-glutathione test (HEGLUT) for the selection of viable sperm for intracytoplasmic sperm injection (ICSI).

Design: A prospective study.

Setting: Department of Pediatrics, Obstetrics and Gynecology, University of Valencia and Instituto Valenciano de Infertilidad.

Patient(s): Semen samples from healthy donors and patients with infertility.

Intervention(s): Sperm samples were kept in culture for different periods in Ham’s F-10 medium supplemented or not supplemented with heparin, reduced glutathione (GSH), or a heparin-GSH mixture. Control and heparin-GSH–treated spermatozoa were injected into hamster oocytes. The HEGLUT and ICSI were performed.

Main Outcome Measure(s): Sperm nuclear decondensation, progressive and nonprogressive motility, and male pronucleus formation.

Result(s): The maximum proportion of sperm nuclear decondensation (28.7% ± 2.1% versus 2.6% ± 0.5% in the control group) was reached after 60 minutes of incubation in the presence of a heparin-GSH mixture. Differences in the percentages of progressive and nonprogressive motility among treatments and times of incubation, although statistically significant, were biologically negligible. No statistically significant differences were observed in the rate of sperm head decondensation (8.2% [4/49] versus 11.1% [6/54]) and male pronucleus formation (18.4% [9/49] versus 22.2% [12/54]) after the injection of control and treated spermatozoa into hamster oocytes.

Conclusion(s): The HEGLUT may offer an alternative to the hypo-osmotic swelling test for the selection of viable sperm for ICSI.     

Analysis of the functional and nuclear integrity of human spermatozoa.

The analysis of semen quality and sperm function has added a new dimension to the evaluation of male infertility. In the present investigation, some recent techniques were used to assess the nuclear, membrane, and acrosomal integrity of spermatozoa from semen of 15 subfertile males with low sperm count and motility. In the infertile semen, the live-to-dead ratio and percentage of spermatozoa showing swelling in hypoosmotic solution was lower than normal but correlated positively with the motility of spermatozoa (r = .85). Staining the spermatozoa with silver nitrate, using a modified technique developed in our laboratory, revealed a higher percentage of morphologically abnormal spermatozoa, in particular, with loss of acrosomal integrity in the subfertile males. Moreover, the percentage of green-fluorescing "fertile" spermatozoa, with intact double-stranded DNA (acridine orange fluorescence), was lower than normal, which correlated with the reduction in sperm nuclear DNA content. A low correlation was obtained between motility and the percentage abnormal spermatozoa (r = .44) and motility and percent green sperm (r = -.28), suggesting that both tests evaluate sperm functional properties independent of motility. The new parameters, assessed with recent techniques, indicate poor sperm fertilizability, which may therefore contribute to the low fertility of the cases studied.     

DNA strand breaks in human spermatozoa: a possible factor, to be considered in couples suffering from unexplained infertility.

BACKGROUND: The aim of this study was to assess the presence of DNA strand breaks and sperm cell morphology in men suffering from unexplained infertility, and to compare these results with normal fertile and oligozoospermic men. METHODS: One fresh sperm sample from proven fertile sperm donors (n=20) and from infertile men with oligozoospermia, (<20 x 10(6)/ml sperm cells) (n=74), and men suffering from unexplained infertility (>20 x 10(6)/ml sperm cells) (n=39) who delivered two sperm samples with 24 hours interval, were tested for the presence of DNA strand breaks in the spermatozoa by direct immunoperoxidase detection of digoxigenin-labeled genomic DNA. Correlations to other sperm parameters, sperm cell counts, motility, activation and Krüger's strict criteria were performed. RESULTS: DNA strand breaks in sperm cell nuclei were found significantly more often in sperm samples from men suffering from unexplained infertility compared to those from normal fertile men, and significantly more rarely compared with sperm samples from men with oligozoospermia. The percentages of normal spermatozoa (Krüger's strict criteria) were significantly lower in samples from men suffering from unexplained infertility compared to those of normal fertile men, but significantly higher compared to those of men with oligozoospermia. No difference was found between first and second day samples used for insemination, as regards DNA strand breaks, sperm cell morphology, total number of motile sperm cells, activation and motility degree. CONCLUSION: The present data suggest that a subgroup of men suffering from unexplained infertility have DNA strand breaks in their sperm cell DNA. This group might suffer from the same malfunction as many men with oligozoospermia, however, their apoptotic activated sites in the testis are different. Delivery of sperm samples with 24 hours interval does not affect any sperm cell counts, CASA, DNA strand breaks or morphology findings in sperm samples from men suffering from unexplained infertility.     

Eight-hydroxy-2'-deoxyguanosine in granulosa cells is correlated with the quality of oocytes and embryos in an in vitro fertilization-embryo transfer program

Objective: To determine the effects of oxidative stress on the quality of oocytes and embryos, 8-hydroxy-2′-deoxyguanosine (8-OHdG) in granulosa cells was quantitatively studied during an in vitro fertilization and embryo transfer (IVF-ET) program.

Design: Immunocytochemical staining of 8-OHdG in granulosa cells was quantitatively estimated using a charge-coupled device camera and analyzed using the National Institute of Health Image (NIH Image) freeware on a computer.

Setting: Obstetrics and gynecology department in a university hospital.

Patient(s): Ninety-six infertile couples undergoing IVF-ET treatment and intracytoplasmic sperm injection (IVF, N = 72; intracytoplasmic sperm injection, N = 24).

Intervention(s): Oocytes, granulosa cells, and follicular fluids were collected 35–36 hours after the administration of hCG.

Main Outcome Measure(s): 8-OHdG indices were obtained for mural [8-OHdG index (m)] and cumulus [8-OHdG index (c)] granulosa cells.

Result(s): A negative correlation between the fertilization rate and both 8-OHdG indices (c and m) was found. The rate of production of good embryos also showed a negative correlation with the 8-OHdG index (m) and the 8-OHdG index (c). Negative correlations between the 8-OHdG index (c) and E₂ levels in follicular fluid were observed. Endometriosis patients showed a higher 8-OHdG index (c) than did patients with other infertility causes, such as tubal, male factor, and unknown.

Conclusion(s): Oxidative stress in granulosa cells lowered fertilization rates and subsequently led to a decrease in the quality of embryos. The quality of oocytes for endometriosis patients was impaired by the presence of 8-OHdG. This might be one causative factor in infertility in endometriosis patients.     

Plasma membrane integrity of cryopreserved human sperm: an investigation of the results of the hypoosmotic swelling test, the water test, and eosin-y staining

Objective: [1] To examine the relationship between sperm membrane integrity and motion parameters before and after cryopreservation; [2] to determine the capacity of the membrane integrity tests to predict the outcome of cryopreservation in fertile and infertile men; and [3] to examine the degree of agreement between tail and head membrane integrity of testicular and ejaculated immotile sperm cryopreserved for intracytoplasmic sperm injection.

Design: Prospective study.

Setting: Academic tertiary care institution.

Patient(s): Fertile donors and normozoospermic oligozoospermic, and asthenozoospermic subfertile men.

Intervention(s): Semen samples were cryopreserved and thawed for analysis.

Main Outcome Measure(s): Sperm membrane integrity and computer-assisted motion parameters.

Result(s): The hypoosmotic swelling test and water test had a significant and positive correlation in the fresh and cryopreserved ejaculates of all groups. The results of the hypoosmotic swelling test correlated positively with the percent motility in the fresh ejaculates of fertile and subfertile men. None of the membrane integrity tests correlated with the cryosurvival rate in any group. In the ejaculated and testicular samples with no postcryopreservation motility, the simultaneous assessment of hypoosmotic swelling test and eosin showed that of 33% sperm exhibiting coiling with the hypoosmotic swelling test, only 9% were eosin negative, whereas 24% were eosin positive.

Conclusion(s): [1] The water test may be a simpler replacement for the hypoosmotic swelling test; [2] none of the membrane integrity tests predicted sperm motility after cryopreservation; and [3] there was a high degree of disagreement between the hypoosmotic swelling test and eosin in the samples with no postcryopreservation motility.     

Inhibition of oocyte fertilization by assisted reproductive techniques and increased sperm DNA fragmentation in the presence of Candida albicans: a case report

The effects of Candida albicans on sperm parameters and the outcome of infertility treatment are unclear. This report describes a lack of fertilization after assisted reproductive techniques and increased sperm DNA fragmentation in an infertile patient with male accessory gland infection due to Candida albicans. He had normal sperm parameters and, therefore, underwent conventional IVF for a female factor of infertility. No spermatozoa or only one spermatozoon per oocyte were found attached to the zona pellucida of the six mature oocytes retrieved. A new semen sample was then requested from the patient to perform intracytoplasmic sperm injection (ICSI) on the same oocytes, but again no fertilization resulted. Candida albicans was detected in the medium where spermatozoa were co-incubated with oocytes and subsequently in the urethral swabs. It did not have any detrimental effect on sperm parameters soon after ejaculation or following separation of motile spermatozoa by swim-up technique. Fertilization failure after assisted reproduction treatment was associated with an increased percentage of motile spermatozoa having chromatin packaging abnormalities, externalization of phosphatidylserine and DNA fragmentation. In conclusion, Candida albicans did not affect sperm parameters, but increased sperm chromatin packaging damage and apoptosis that might have caused fertilization failure after assisted reproduction treatment in this couple.     

Cigarette smoke extract immobilizes human spermatozoa and induces sperm apoptosis

Cigarette smoking by the male partner adversely affects assisted reproductive techniques, suggesting that it may damage sperm chromatin/DNA and consequently embryo development. The effects of graded concentrations of research cigarettes smoke extract (CSE) on motility, mitochondrial membrane potential (MMP), chromatin integrity and apoptosis were evaluated in spermatozoa obtained from 13 healthy, non-smoking men with normal sperm parameters, by flow cytometry. CSE suppressed sperm motility in a concentration- and time-dependent manner and increased the number of spermatozoa with low MMP, the main source of energy for sperm motility. In addition, CSE had a detrimental effect on sperm chromatin condensation and apoptosis. Indeed, it increased the number of spermatozoa with phosphatidylserine externalization, an early apoptotic sign, and fragmented DNA, a late apoptotic sign, in a concentration- and time-dependent manner. These effects of CSE were of similar or even greater magnitude to those obtained following incubation with tumour necrosis factor-α, a cytokine known for its negative impact on sperm function, used as positive control. Since transmission of smoking-induced sperm DNA alterations has been found in pre-implantation embryos, and this may predispose offspring to a greater risk of malformations, cancer and genetic diseases, men seeking to father a child are recommended to give up smoking.     

Biochemical markers of sperm function: male fertility and sperm selection for ICSI

The expression of a 70 kDa chaperone protein, HspA2 (formerly called CK-M), has been identified in mature human spermatozoa. The central role of HspA2 has been established, as the expression level of this protein is related to sperm cellular maturity, DNA integrity, chromatin maturity, chromosomal aneuploidy frequency and sperm function, including fertilizing potential. The spermiogenetic events of cytoplasmic extrusion and remodelling of the plasma membrane, which facilitate the formation of zona pellucida binding site(s) in human spermatozoa, are related. Finally, the presence of the hyaluronic acid (HA) receptor on the plasma membrane of mature sperm coupled with the HA-coated slide sperm-binding assay, facilitates the testing of infertile men and the selection of single mature spermatozoa for ICSI. Because mature spermatozoa have no residual cytoplasm, the HA-bound sperm fraction is also enriched in spermatozoa that are normal by the Kruger strict morphology method.     

Absence of block to polyspermy at the human oolemma

Objective: To study the fusiogenic ability of human spermatozoa and oocytes.

Design: Retrospective study of 3,027 oocytes included in a program of subzonal insemination (SUZI).

Setting: Assisted fertilization program in an academic research environment.

Patient(s): Couples with characterized male factor infertility or previous unexplained IVF failures.

Intervention(s): Subzonal insemination (SUZI) was performed after study of the sperm pathology. The number of microinjected spermatozoa was controlled.

Main Outcome Measure(s): Fertilization and polyspermia rates were analyzed according to the number of microinjected spermatozoa and to the indication of SUZI.

Result(s): The fertilization rate increased linearly between one and three microinjected spermatozoa. Above this number, the rate plateaued around 25%. Polyspermia was correlated with the number of microinjected spermatozoa (r = 0.97). The fusiogenic ability of motile sperm cells was dependent on the semen characteristics and the sperm pathology. Observed diploid and polyspermia rates did not differ from the calculated probability of microinjecting only one or at least one fertilizing spermatozoon into the perivitelline space, respectively.

Conclusion(s): Data support the hypothesis that a physiologic block at the human oolemma is absent. The post-SUZI fertilization rate also can be explained by the probability of finding one fertilizing spermatozoon among those that were microinjected and by the limited number of sperm heads allowed to decondense in the ooplasm.     

Combined effect of GSTT1 and GSTM1 polymorphisms on human male infertility in north Indian population

Genes of different pathways regulate spermatogenesis, and complexity of spermatogenic process indicates that polymorphisms or mutations in these genes could cause male infertility. Detoxification pathway is involved in the regulation of spermatogenesis by reducing oxidative stress and contributes to the maintenance of global methylation in concert with other pathways. Glutathione S-transferases (GSTs) belong to the family of phase II antioxidant enzymes involved in the cellular detoxification of various physiological substances. Glutathione S-transferases act as an antioxidant and protect spermatozoa from oxidative stress. Increase in the levels of reactive oxygen species (ROS) along with reduced activity of GSTs may result in sperm membrane damage and DNA fragmentation. A case-control study was done to elucidate the role of deletion polymorphism of GSTT1 and GSTM1 genes from GSTs family on idiopathic human male infertility. The study comprises 2 groups: 113 nonobstructive azoospermia patients and 91 healthy fertile controls. Genomic DNA was analyzed by polymerase chain reaction for GSTT1 and GSTM1 genes. The study showed statistically significant protective association of GSTT1 null genotype with human male infertility (odds ratio [OR]: 0.3, 95% confidence interval [CI] 0.143-0.9966, P = .048) but not with GSTM1 null genotype (OR: 0.66, 95% CI 0.3653-1.2234, P = .19). Also, combination of null genotypes of GSTM1 and GSTT1 confers protective effect (OR: 0.28, CI 0.0801-0.948; P = .04). Probably, individuals bearing GSTM1 and GSTT1 (-/-) genotypes may have protective effect by gene-gene interaction mechanism. In summary, our study underscores the significance of combined effect of GSTT1 and GSTM1 null genotypes in modulating the risk of male infertility.