Differential Gene Expressions of CALM1, PSMD6, and AK124742 Long Noncoding RNA in Cumulus Cells from Polycystic Ovary Syndrome Patients versus Normal Control Women.

Background: One of the well-known causes of subfertility is polycystic ovary syndrome (PCOS). Genetic components play a critical role in the etiology of PCOS. The recognition of differentially expressed genes in PCOS patients might provide a better understanding of the pathophysiology of this syndrome and paves the way for novel therapeutics. Gene expression profiles in cumulus cells (CCs) could be used as biological criteria for embryo competence and their analysis might lead to important molecular information about embryo quality. CALM1, PSMD6, and AK124742 are three well-known genes associated with embryo development. Therefore, the objective of this study was to compare the expression of CALM1, PSMD6, and AK124742 genes in the CCs of infertile PCOS patients with their expression in the CCs of the donor fertile group. Materials and Methods: CCs were collected from the follicular fluid of 33 patients with PCOS as the experimental group and 33 cumulus donor women who were referred to the infertility center for egg donation as the control group. CCs were frozen until genetic testing. The expression of CALM1, PSMD6, and AK124742 genes was detected by real-time polymerase chain reaction. Results: CALM1 and AK124742 gene expressions significantly increased (CALM1 P = 0.003) (AK124742 P = 0.000) and PSMD6 expression significantly decreased (P = 0.002) in the PCOS group compared to the cumulus donor (control) group. Conclusion: Therefore, our research findings suggest that the potential impact of Polycystic Ovary Syndrome (PCOS) on fertility could be attributed to modifications in the expression levels of genes that affect the reproductive.

The Effect of Endometrial Cell Culture on α3 and ß1 integrin Genes and Protein Expression in Type 2 Diabetic Rats at The Time of Implantation.

OBJECTIVE: Given the prevalence of fertility problems in couples and the defect in embryo implantation as well as the low success rate of assisted reproductive techniques, it is necessary to investigate the causes of this phenomenon. Type 2 diabetes mellitus (T2DM) is a metabolic disease with multiple effects on various organs as well as the endometrium. In this study, the effects of endometrial cell culture on the expression of α3 and ß1 integrin genes and protein in type 2 diabetic rats were investigated. MATERIALS AND METHODS: In this experimental study, 35 female rats were divided into five groups: control, sham, diabetic, Pioglitazone-treated and Metformin-treated groups. First, rats were maintained in diabetic condition for 4 weeks. Then, treatment was performed for the next four weeks. Four weeks after induction of diabetes, rats were sacrificed at the time of embryo implantation. The uterus was removed. Endometrial cells were isolated and cultured for 7 days. Immunocytochemistry staining was used to confirm endometrial cells. Expression of α3 and ß1 integrin genes was determined by real-time polymerase chain reaction (PCR) technique and the α3ß1 protein content measured using Western blot both before and after endometrial cell culture. RESULTS: The expression level of α3 integrin gene in the Pioglitazone-treated group compared with metformin-treated group was significantly decreased (P<0.001). The same result was observed in ß1 integrin gene expression (P=0.004). Also, the α3ß1 protein level increased in all diabetic groups, but its reduction was significantly greater in pioglitazonetreated group (P=0.004). CONCLUSION: T2DM altered the expression of α3 and ß1 integrin genes and related proteins, which endometrial cell culture regulated this disorder. According to these results, may be the endometrial cell culture can reduce the adverse effects of diabetes on α3 and ß1 integrin expression at the level of gene and protein, in endometrial cells.

Evaluation of vascular endothelial growth factor A and leukemia inhibitory factor expressions at the time of implantation in diabetic rats following treatment with Metformin and Pioglitazone.

BACKGROUND: Implantation requires intimate crosstalk between the embryo and uterus for a successful establishment of pregnancy. Type 2 diabetes mellitus may lead to implantation failure. The effect of diabetes and its therapeutic drugs on implantation is still largely unclear. OBJECTIVE: To assess the endometrial expression changes of vascular endothelial growth factor A (VEGFA) and leukemia inhibitory factor (LIF), at the time of implantation in diabetic rats following treatment with Metformin and Pioglitazone. MATERIALS AND METHODS: Twenty-eight 6-8-wk-old Wistar female rats weighing 200-250 gr were divided into four groups (n = 7/each). Type 2 diabetes was induced and Metformin and Pioglitazone were applied for 4 wk. The expression of VEGFA and LIF was measured by real-time reverse transcription-polymerase chain reaction and Western blot. RESULTS: The relative expression of VEGFA transcript was higher in the diabetic (p = 0.02) and Metformin-treated (p = 0.04) rats compared to the control group. Furthermore, the VEGFA transcript level significantly reduced in Pioglitazone-treated diabetic rats (p = 0.03). LIF expression was elevated in the Metformin- and the Pioglitazone-treated rats and reduced in the diabetic group in comparison with the control group. Compared to the diabetic rats, the expression of LIF was significantly elevated in the Metformin- (p = 0.01) and Pioglitazone-treated (p = 0.03) groups. CONCLUSION: The expressions of LIF and VEGFA were altered in diabetic rats during implantation which may be associated with diabetic-related infertility. Pioglitazone is able to restore the VEGFA and LIF expressions to their baseline levels more efficiently than Metformin.

Evaluation of Muc1 Gene Expression at The Time of Implantation in Diabetic Rat Models Treated with Insulin, Metformin and Pioglitazone in The Normal Cycle and Ovulation Induction Cycle.

BACKGROUND: Mucin-1(Muc1) is one of the first molecules in the endometrium that confronts implanting embryos. There is insufficient knowledge about the impacts of diabetes and drugs developed for diabetes treatment on expression of this molecule at the time of implantation. Therefore, this study aimed to investigate the impacts of diabetes and insulin, metformin and pioglitazone on Muc1 expression at the time of implantation. MATERIALS AND METHODS: This experimental study was conducted on a total of 63 female Wistar rats divided into 9 groups. To induce type 1diabetes, streptozotocin (STZ) and for induction of type 2 diabetes, nicotinamide (NA) and STZ were injected intraperitoneally. For superovulation, human menopausal gonadotropin (HMG) and human chorionic gonadotropin (HCG) were used. Insulin, metformin and pioglitazone were administered for two weeks. Finally, the endometrial expression of Muc1 was evaluated by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Muc1 expression was non-significantly increased in type 1 and type 2 diabetic groups compared to the control group (P=0.61 and 0.13, respectively); also, it increased in insulin-treated type 1 diabetic group compared to the control group (P=0.0001). Its expression was increased in insulin-treated type 1 diabetic group compared to untreated diabetic group (P=0.001). The expression level of Muc1 was significantly reduced in superovulated and insulintreated type 1 diabetic group compared to the insulin-treated type 1 diabetic group (P=0.001). CONCLUSION: One of the causes of fertility problems in diabetes, is changes in Muc1 expression during implantation. On the other hand, the use of insulin in these patients can even lead to overexpression of this gene and worsen the condition. However, these changes can be partially mitigated by assisted reproductive technology (ART) such as superovulation. Also, treatment with metformin and pioglitazone can restore Muc1 expression to near normal levels and has beneficial effects on implantation.

A Study on the Presence of Osteopontin and α3ß1 Integrin in the Endometrium of Diabetic Rats at the Time of Embryo Implantation.

BACKGROUND: Embryo implantation is a critical and multifactorial phenomenon which can be affected by any alteration in molecular micro construction of endometrium. The aim of the current study was to evaluate the effects of diabetes on osteopontin (OPN) and α3ß1 integrin proteins level at the time of endometrial receptivity. METHODS: Twenty-eight female rats were divided into control, diabetic, pioglitazone-treated and metformin-treated groups. Western blot was performed to determine the OPN and α3ß1 integrin proteins in rats' endometrium at the time of implantation. Data were analyzed by analysis of variance (ANOVA) and p<0.05 was considered statistically significant. RESULTS: OPN increased significantly in the diabetic group in comparison with control (p<0.001), metformin-treated (p=0.008) and pioglitazone-treated groups (p< 0.001). Furthermore, α3ß1 integrin protein level in diabetic group had a significant difference in comparison with that of the control (p<0.001), metformin-treated (p= 0.026) and pioglitazone-treated groups (p<0.001). CONCLUSION: OPN and α3ß1 integrin proteins are involved in embryo implantation and their changes in diabetic condition can affect fertility. Treatment with pioglitazone and metformin improved the level of OPN and α3ß1 integrin proteins while pioglitazone was more effective.

Diabetes mellitus increased integrins gene expression in rat endometrium at the time of embryo implantation.

BACKGROUND: Diabetes mellitus deeply changes the genes expression of integrin (Itg) subunits in several cells and tissues such as monocytes, arterial endothelium, kidney glomerular cells, retina. Furthermore, hyperglycemia could impress and reduce the rate of successful assisted as well as non-assisted pregnancy. Endometrium undergoes thorough changes in normal menstrual cycle and the question is: What happens in the endometrium under diabetic condition? OBJECTIVE: The aim of the current study was to investigate the endometrial gene expression of α3, α4, αv, Itg ß1 and ß3 subunits in diabetic rat models at the time of embryo implantation. MATERIALS AND METHODS: Twenty-eight rats were randomly divided into 4 groups: control group, diabetic group, pioglitazone-treated group, and metformin-treated group. Real-time PCR was performed to determine changes in the expression of Itg α3, α4, αv, ß1, and ß3 genes in rat's endometrium. RESULTS: The expression of all Itg subunits increased significantly in diabetic rats' endometrium compared with control group. Treatment with pioglitazone significantly reduced the level of Itg subunits gene expression compared with diabetic rats. While metformin had a different effect on α3 and α4 and elevated these two subunits gene expression. CONCLUSION: Diabetes mellitus significantly increased the expression of studied Itg subunits, therefore untreated diabetes could be potentially assumed as one of the preliminary elements in embryo implantation failure.

Impact of Metformin and Pioglitazone on Serum Level of Tumor Necrosis Factor-Alpha and Lipid Profiles during Implantation Window in Diabetic Rats.

BACKGROUND: The present study was designed to evaluate serum lipid profile and tumor necrosis factor-alpha (TNF-ɑ) level in diabetic rats at implantation time. Type 2 diabetes mellitus (T2DM) could affect various systems, including innate immune system and it causes chronic low-grade inflammation, increasing level of TNF-ɑ. Furthermore, T2DM is often accompanied by impaired lipid profile. Metformin and pioglitazone are used as the first and second lines of treatment for T2DM. MATERIALS AND METHODS: In this experimental study, 35 adult virgin female wistar rats, weighting 175-225 g, were randomly categorized into five groups: i. Control, ii. Sham, iii. Nicotinamide (NA)+streptozotocin (STZ) induced T2DM, iv. Diabetic+pioglitazone (20 mg/kg/day for 28 days oral administration), and v. Diabetic+metformin (100 mg/kg/day for 28 days oral administration). At the time of implantation, TNF-ɑ level in serum of rats was measured by ELISA kit. Glucose was measured using photometric method and lipid profiles were calculated by enzymatic methods. RESULTS: Level of TNF-ɑ in the diabetic group was significantly higher than other groups (P<0.001). In metformin treated group, TNF-ɑ serum level was also significantly higher than pioglitazone treated group (P<0.001). Fasting blood sugar (FBS) and lipid profiles were significantly higher in diabetic group. CONCLUSION: Metformin and pioglitazone have similar effects on glucose, lipid profiles and TNF-ɑ serum levels. Among these drugs, pioglitazone has more efficient influence on TNF-α serum level, in comparison with metformin.

Evaluation of Diabetes Effects on the Expression of Leukemia Inhibitory Factor and Vascular Endothelial Growth Factor A Genes and Proteins at the Time of Endometrial Receptivity after Superovulation in Rat Model.

BACKGROUND: Diabetes, a major metabolic disorder, seems to affect the fertility rates of women in various ways. Due to the uncertainty of the effects of diabetes along with superovulation treatment on the infertility, we investigate the effects of ovulation induction treatment as therapeutic approach on the expression of leukemia inhibitory factor (LIF) and vascular endothelial growth factor A (VEGFA) as two main factors which are involved in the implantation in the streptozotocin (STZ)-induced type 1 diabetic rats. MATERIALS AND METHODS: Type 1 diabetes was induced by injections of STZ in Wistar rats. The animals were kept in diabetic conditions for 4 weeks, while some were treated with insulin for treatment. After treatment, the ovulation was induced by human menopausal gonadotropin (hMG) and human chorionic gonadotropin (hCG). The rats were then sacrificed and the expression of LIF and VEGFA was checked by immunohistochemistry staining method, and the relative expression of LIF and VEGFA was measured by quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blotting methods. RESULTS: It was observed that diabetes and insulin treatment for diabetes altered the expression of Lif and VEGFA in both mRNA and protein levels. However, superovulation treatment seems to ameliorate this alternation for both factors. CONCLUSION: According to our results, diabetes and insulin therapy could alter the expression of Lif and VEGFA genes and proteins that are effective in endometrial receptivity and implantation process. It seems in diabetic cases, the effect of hCG and hMG therapy by itself could regulate the level of expression and presence of these two genes and proteins.

Evaluation of Osteopontin Gene Expression in Endometrium of Diabetic Rat Models Treated with Metformin and Pioglitazone.

BACKGROUND: Osteopontin (Opn) is one of the co-factors involved in cell adhesion and invasion during the implantation process. Several reports have shown Opn expression changes in diabetic condition in several tissues. In addition, an increased incidence of spontaneous abortion is reported in diabetic women. We, therefore, designed a study to evaluate the effects of diabetes on Opn expression at implantation time after treatment with metformin and pioglitazone. MATERIALS AND METHODS: In this interventional and experimental study, 28 rats were randomly divided into four groups, namely control, diabetic, pioglitazone-treated diabetic rats and metformin-treated diabetic rats. Streptozotocin (STZ) and nicotinamide (NA) were used to induce type 2 diabetes (T2D). During the implantation window, the endometrium was removed and the expression of Opn was analysed by reverse transcription quantitative polymerase chain reaction (RT-qPCR). RESULTS: Opn expression was significantly higher (30.70 fold-changes) in the diabetic group in comparison with the control group (P=0.04). Furthermore, the expression of Opn was significantly lower in the diabetic group treated with pioglitazone when compared with the diabetic group (P=0.04). CONCLUSION: According to the high Opn expression and the possibility of increased adhesion of endometrial epithelial cells, the invasion of blastocyst may be affected and thus reduced. As pioglitazone significantly reversed the upregulation of Opn in diabetic rats, it may be considered as a therapeutic compound for treating T2D.

A Survey on Main Semen Parameters in Natural Pregnancy and Intrauterine Insemination: Are There Any Significant Differences?

Intrauterine insemination (IUI) is a treatment of choice compared with other invasive and expensive techniques of assisted reproduction. Sperm quality is used to predict its outcome and success. Establishing threshold levels for sperm parameters is useful to avoid spending time and money to do other assisted reproductive techniques. This study was carried out to compare various semen parameters in a group of men eligible to participate in an IUI program with those of fertile men whose wives were pregnant at the time of the study. Two hundred and thirty-four semen samples were evaluated from subfertile men whose partners were candidates for IUI and 234 semen samples were evaluated from fertile men whose partners were pregnant less than 12 weeks. To assess the sensitivity and specificity of the main semen parameters, receiver operating characteristic (ROC) curves were used. Normal sperm morphology is more sensitive and specific compared with its progressive motility and concentration. No significant differences in various semen parameters of fertile men and those of the male partners of IUI candidates were observed. ROC analysis identified that sperm normal morphology using strict criteria may be a good indicator of fertility status in men. No significant difference in various semen parameters between the male partners of IUI candidates and the fertile men was seen. However, utilizing ROC curves, sperm morphology using strict criteria could be a good predictor of fertility.

The Study of Association Between Polymorphism of TNF-α Gene's Promoter Region and Recurrent Pregnancy Loss.

BACKGROUND: According to the literature review, polymorphisms of tumor necrosis factor alpha's (TNF-α) promoter region are probably the genetic risk factors of recurrent pregnancy loss. This study has investigated five single nucleotide polymorphisms in the TNF-α gene's promoter region to evaluate their relationship with recurrent pregnancy loss disorder. METHODS: Blood samples were taken from 65 women with recurrent pregnancy loss (Case group) and 65 healthy women with a history of successful pregnancy (Control group). Polymerase chain reaction with high resolution melting (PCR-HRM) analysis was done to determine the promoter region of -308G/A, -850T/C, -238G/A, -1031T/C and -863A/C TNF-α polymorphisms. The data were assessed using logistic regression models. P values less than 0.05 were considered statistically significant. RESULTS: Significant associations were found between recurrent pregnancy loss and -863C/A (p=0.000), -308G/A (p=0.045), and -238G/A (p=0.034) polymorphisms. TNF-α polymorphisms of -863C and -238G may be susceptible factors of recurrent pregnancy loss cases. The -308G polymorphism has an important role in maintaining pregnancy. CONCLUSION: The -863C/A and -238G/A TNF-α polymorphisms are possible genetic risk factors of recurrent pregnancy loss and might be its predictive markers.

PROTAMINE1 and PROTAMINE2 genes expression in the sperms of oligoasthenospermic individuals and intrauterine insemination candidates couples: Is there any significant differences?

BACKGROUND: Male infertility refers to a male's inability to cause pregnancy in a fertile female. It seems the large portion of this category of infertility, has roots in genetic factors. PROTAMINE family is one of the most important genes which are involved in male factor infertility. Hence, the aim of this study is to evaluate PROTAMINE1 and PROTAMINE2 (P1 and P2) genes expression in oligoasthenospermic individuals and intrauterine insemination (IUI) candidate couples' sperms. MATERIALS AND METHODS: Samples were gathered from the patients referred to the Isfahan Infertility Center of Shahid Beheshti, 80 semen samples were in IUI candidates groups and 16 semen samples were in oligoasthenospermia group was collected. The outcome of IUI procedure was followed up after 14 days. Through these samples, 16 couples achieved pregnancy (IUI+) and from the top of the list, 16 semen samples with negative ß-HCG were obtained (IUI-). After RNA extraction from sperms, PROTAMINE genes family expression was evaluated in our three groups by real time-reverse transcription polymerase chain reaction. RESULTS: Our study revealed that P1 gene expression has no significant differences between IUI-, IUI+, and oligoasthenospermia groups, whereas P2 gene expression showed significant differences between oligoasthenospermia with two IUI groups. Main sperm parameters have no significant differences between IUI groups. CONCLUSION: This study reveals P1 and P2 genes expression value have no significant differences between IUI- and IUI+. On the other hand, P2 gene expression value has significant differences between oligoasthenospermia with two IUI groups.

Study of association between promoter tumor necrosing factor alpha gene polymorphisms in -850T/C, -863 A/C, and endometriosis.

BACKGROUND: The purpose of this study was to determine whether variability in gene encoding for promoter of tumor necrosis factor participates to women differences in susceptibility to endometriosis. MATERIALS AND METHODS: The study involved 130 women; 65 endometriotic and 65 healthy control women. The blood samples were genotyped for -850 T/C and -863 C/A polymorphisms in TNF alpha gene promoter. Chi-square, odd ratio, and confidence interval 95% were used to evaluate genotypes and allele frequency differences between two groups. RESULTS: No significant differences in genotypes distribution of -850 T/C (P = 0.32) and 863 C/A (P = 0.34) polymorphisms were obtained between two groups. CONCLUSION: According to this study, these two polymorphisms have no risk or protective factor to develop endometriosis.

Evaluation of CD52 positive sperms in subfertile human semen samples: Is there any relationship with main semen parameters?

BACKGROUND: Sperm maturation and sperm membrane integration are the most important elements in male fertility. CD52 is one of the antigens. CD52 is a GPI (glycosylphosphatidylinositol) anchored that express on lymphocytes and epididymal cells. This antigen bind to sperm membrane during transition sperm from epididymal duct as well as its relationship with semenogelins in human seminal plasma. The aim of this study was to obtain any association between the percentage of CD52 positive sperms with main semen parameters such as percentage of motile sperms, percentage of sperm with normal morphology, and the presence of normal viscosity. MATERIALS AND METHODS: Semen samples from subfertile men were analyzed, the samples totally were 45 that divided according to their motility into three groups, first one, more than 40%, second one 10-40%, and the third one under 10% total motility. Fifteen samples in each group were evaluated by semen analysis according to WHO 2010 guidelines for infertility laboratory. Sperms were washed by Ham's F-10 and immunostaining with the monoclonal antibody CAMPATH-1G and then analyzed by flow cytometry. We compared each of the groups based on their motility and the data were analyzed by SPSS 20. RESULTS: Correlation between CD52 labeling and sperm motility was negatively significant, in the second group (r = -0.592, P = 0.020) and in the third group (r = -0.805, P = 0.00). CONCLUSION: Our results showed that the correlation between CD52 labeling and sperm motility was negatively significant, but we did not observe any relation with other semen parameters, such as sperm normal morphology, sperm concentration, and semen viscosity.

Association of TP53 gene codon 72 polymorphism with endometriosis risk in Isfahan.

BACKGROUND: Endometriosis is a female health disorder that occurs when cells from the lining of the uterus grow in other areas of the body. The cause of endometriosis is unknown. OBJECTIVE: The purpose of this study was to investigate TP53 gene codon 72 polymorphism in women with endometriosis and compared it with healthy samples in Isfahan. MATERIALS AND METHODS: We undertook a case-control study to examine the possible association of the TP53 gene codon 72 polymorphism with the risk of endometriosis in Isfahan. Ninety whole blood specimens from normal people as controls and ninety endometriosis specimens were analyzed. p53 codon 72 genotypes were identified using allele-specific polymerase chain reaction. RESULTS: Frequency of genotype Arg/Arg (Arginine/Arginine) in the samples of endometriosis was 28.9% and in healthy samples 42.2%. Frequency of genotype Pro/Pro (Proline/Proline) in the samples of endometriosis was 15.6% and in healthy ones. Frequency of heterozygote's Arg/Pro was 55.6% in endometriosis samples and 54.45% in healthy ones 3.3%. By comparing statistical genotype Pro/Pro with two other genotypes in both groups there was a statistical meaningful difference between control group and endometriosis group. [p=0.009, CI=95%, OR=5.34 (1047-19.29)]. CONCLUSION: Recent research shows that genotype Pro/Pro codon72 exon4 TP53 gene may be one predisposing genetic factor for endometriosis in Isfahan.

Evaluation of Fas positive sperm and complement mediated lysis in subfertile individuals.

PURPOSE: Evaluation of Fas receptor on surface of sperm, as an apoptotic marker, using flow cytometry and confirming the results using an antibody-antigen complex through the classic complement pathway. MATERIALS AND METHODS: Semen samples were obtained from 10 fertile and 73 infertile individuals with diagnoses of male factor infertility. Expressions of Fas receptor and phosphatidyl serine on sperm were assessed by flow cytometry. Fas expression was further assessed by antibody-antigen complex through the complement pathway. Lysis was detected via PI (propidium iodide) staining. RESULTS: The mean Fas expression was considerably lower than previously reported values. No significant differences in the percentage of PI were detected before and after activation of the classic complement pathway. Annexin V positive samples showed low Fas expression. CONCLUSION: Our results have confirmed the presence of selected apoptotic markers such as Fas or phosphatidyl serine on ejaculate sperm, but suggest that Fas expression is low. Further studies are required to investigate the "abortive apoptosis" mechanism through Fas/Fas L.

Sperm MTT viability assay: a new method for evaluation of human sperm viability.

PURPOSE: MTT (3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay is commonly used as a cell proliferation assay. The objective of this study was to evaluate the capability of MTT assay to discriminate between viable and nonviable sperms and compare it sefficiency with E&N (eosin and nigrosin) and HOST (hypo-osmotic swelling test). METHODS: MTT assay was modified to obtain optimal result for assessment of sperm viability. After standardization of method, MTT, E&N, and HOST were carried out simultaneously on 57 semen samples from patients referring to Isfahan Fertility and Infertility Center. The correlation coefficient between these tests and sperm motility was calculated using the SPSS statistical program. Specificity and sensitivity of each test was also obtained. RESULTS: The optimal conditions for sperm MTT viability assay were 2 h after addition of sperm to MTT in HAM'S F10 + 25 mM HEPES + 10% HSA at 37 degrees C and pH 7.4-7.45. Inter- and intra-assay coefficients of variations were 9 and 7%, respectively. The sensitivity and specificity for sperm MTT viability assay, E&N, and HOST were 97,98, and 99%, and 100, 100, and 83% respectively. High significant correlations were obtained between sperm MTT viability assay, E&N, HOST and motility. CONCLUSIONS: Sperm MTT viability assay can be used as a diagnostic test for discrimination of viable sperms from sperm population.