Evaluation of Sperm Chromatin Integrity Using Aniline Blue and Toluidine Blue Staining in Infertile and Normozoospermic Men.

BACKGROUND: Male infertility is defined as a man lost his ability to fertilize a fertile female naturally. Diagnosis of male infertility cannot be made just according to basic semen analysis. It is necessity to have specific tests for evaluation of chromatin integrity. In this study, an attempt was made to evaluate the sperm chromatin quality in fertile men and infertile subgroup. METHODS: Among 1386 couples, 342 men were categorized into normospermia and 1044 were infertile and they were referred to Yazd Research and Clinical Center for infertility treatment. Standard semen analysis and sperm nuclear maturity tests including aniline blue (AB) and toluidine blue (TB) staining were done. Data were analyzed by SPSS software. The p≤0.05 was considered statistically significant. RESULTS: The mean value of TB staining was significantly higher in infertile group compared to normospermic group (p=0.005). Mean of sperm normal morphology was lower in idiopathic infertile men in comparison with normozoospermic men (p= 0.001). The highest negative correlation was obtained between sperm count and AB staining. Progressive motility was negatively correlated with AB and TB staining in both groups but there was no significant difference between AB staining and progressive motility in men normospermia group. CONCLUSION: Sperm chromatin staining using AB and TB showed a negative association between sperm chromatin condensation with sperm count, normal morphology and progressive motility. It seems that the AB and TB test may be useful for the assessment of male fertility potential.

Relationship between sperm protamine deficiency and apoptosis in couples with unexplained repeated spontaneous abortions.

BACKGROUND: Etiology of more than half of Recurrent Spontaneous Abortion. The etiology of more than 50 percent of Recurrent Spontaneous Abortions (RSA) cases has been remained unexplained. It is supposed that RSA may have "paternal effect" due to supply 50% of embryonic genomic content by male gamete. OBJECTIVE: The aim of present study was to evaluate the role of sperm apoptosis and protamine deficiency at same time in RSA cases. MATERIALS AND METHODS: Forty fertile (control) and 40 unfertile men with RSA (case) were enrolled in this case-control study. Semen analysis was performed in accordance with WHO criteria and sperm apoptosis and protamine deficiency were evaluated by cell apoptosis detection kit and chromomycin A3, respectively. RESULTS: Results showed significant different between normal morphology and total motility in two groups. Case group had higher percentage of spermatozoa with protamine deficiency and apoptosis compared to controls significantly. CONCLUSION: Our results showed that in cases of RSA, in addition to abnormal sperm parameters, we have a high percentage of spermatozoa with protamine deficiency and apoptosis and these two anomalies may consider as important causes of idiopathic recurrent abortions. It should be advised that sperm chromatin and DNA examinations are useful tools in the process of RSA treatments.

Sperm parameters, protamine deficiency, and apoptosis in total globozoospermia.

BACKGROUND: Globozoospermia is a severe form of teratozoospermia (incidence < 0.1%) in infertile men that is characterized by round headed sperm and acrosomeless in semen. OBJECTIVE: To compare the semen parameters, protamine deficiency, and apoptosis in ejaculated spermatozoa between globozoospermic and normozoospermic men. MATERIALS AND METHODS: Thirty six semen samples were divided into two groups including 15 infertile men with total globozoospermic (> 90% round-headed sperm) and 21 healthy donors with normal spermograms as controls. Semen analysis was performed according to World Health Organization criteria (2010). Sperm protamine deficiency was assessed using Chromomycin A3 (CMA3) staining and the rate of apoptotic spermatozoa was evaluated with TUNEL assay. RESULTS: Sperm concentration, motility, and normal morphology in globozoospermic men were significantly decreased compared with controls (p<0.05). The rate of CMA3-reacted spermatozoa (CMA3+) in globozoospermic men was higher than controls (65.93 ± 11.77 vs. 21.24 ± 7.37, respectively, p<0.0001). The rate of apoptotic spermatozoa (TUNEL positive) were significantly increased in globozoospermic cases with respect to the controls (17.60 ± 10.72 and 5.95 ± 3.02, respectively, p<0.0001). There was no significant correlation between sperm protamine deficiency and apoptosis in globozoospermic men. CONCLUSION: Globozoospermic samples contain a higher proportion of spermatozoa with abnormal chromatin packaging and DNA fragmentation than normozoospermic samples. Therefore, in addition to absence of acrosome in the spermatozoa of globozoospermic patients, the high percentage of spermatozoa with immature chromatin and apoptotic marker may be considered as the other etiologies of infertility in these patients.

Evaluation of sperm retrieval rate with bilateral testicular sperm extraction in infertile patients with azoospermia.

BACKGROUND: About 10% to 15% of infertile men have azoospermia, which could be obstructive or non-obstructive. Diagnostic biopsy from the testis and recently testicular sperm extraction (TESE) are the most precise investigations in these patients. Testicular biopsy can be done unilaterally or bilaterally. The worth of unilateral or bilateral testicular biopsy in men with azoospermia is controversial. OBJECTIVE: To evaluate the necessity of bilateral diagnostic biopsy from the testis in new era of diagnosis and treatment of male infertility. MATERIALS AND METHODS: In this retrospective study, we reviewed the results of testis biopsy in 419 azoospermic men, referred to Yazd Research and Clinical Center for Infertility from 2009-2013. Patients with known obstructive azoospermia were excluded from the study. RESULTS: In totally, 254 infertile men (60.6%) were underwent unilateral TESE, which in 175 patients (88.4%) sperm were extracted from their testes successfully. Bilateral testis biopsy was done in 165 patients (39.4%) which in 37 patients (22.4%), sperm were found in their testes tissues. CONCLUSION: Due to the low probability of positive bilateral TESE results especially when we can't found sperm in the first side, we recommend that physicians re-evaluate the risk and benefit of this procedure in era of newer and more precise technique of sperm retrieval like micro TESE.

Comparison of reactive oxygen species in neat and washed semen of infertile men.

BACKGROUND: Male are involved in near 50% of cases of infertility and reactive oxygen species (ROS) playing an important role in decreasing fertility potential. Accurate measurement of ROS seems to be important in evaluation of infertile male patients. OBJECTIVE: To compare ROS measurement in neat and washed semen samples of infertile men and define the best method for evaluation of ROS in these patients. MATERIALS AND METHODS: We measured the level of ROS in semen samples of thirty five non-azoospermic men with infertility. The semen samples were divided into two parts and the semen parameters and ROS levels in neat and washed samples were evaluated. We also evaluated the presence of pyospermia using peroxidase test. RESULTS: The differences regarding sperm count and quick motility were significant in neat and washed semen samples. The mean ROS level was significantly higher in neat samples compared with washed spermatozoa (7.50 RLU vs. 1.20 RLU respectively). Difference in ROS levels was more significant in patients with pyospermia compared to whom with no pyospermia (378.67 RLU vs. 9.48 RLU respectively). CONCLUSION: Our study confirmed that neat or unprocessed samples are better index of normal oxidative status of semen samples. Because we do not artificially add or remove factors that may play an important role in oxidative equilibrium status.

Sperm chromatin condensation, DNA integrity, and apoptosis in men with spinal cord injury.

OBJECTIVES: To evaluate the effect of cord injury on (1) sperm parameters and (2) DNA chromatin status. DESIGN: Case-control study. SETTING: Data were collected from men referred to Research and Clinical Center for Infertility, Yazd, Iran. PARTICIPANTS: Thirty infertile men with the presence of any level of spinal cord injury (SCI) were compared with 30 healthy donors with definite fertility and normal sperm parameters. INTERVENTIONS: Not applicable. OUTCOME MEASURES: Sperm chromatin integrity was assessed using aniline blue (AB), chromomycin A3 (CMA3), toluidine blue (TB), and acridine orange (AO) assays. The rate of apoptotic spermatozoa was evaluated with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) staining. RESULTS: Sperm concentration, motility, and morphology in men with SCI were significantly decreased compared with control group (P < 0.05). In addition, with regard to cytochemical staining and TUNEL test, the rate of reacted spermatozoa was increased significantly in SCI group when compared with the controls (P < 0.05). The majority of AB, TB, AO, and CMA3-reacted spermatozoa were higher than the "cut-off" value in men with SCI, as were the number of apoptotic spermatozoa stained with TUNEL. CONCLUSION: Results showed that SCI disturbs sperm parameters, nuclear maturity, and DNA integrity of spermatozoa. Therefore, the production of spermatozoa with less condensed chromatin and more apoptotic rate increases after cord injury and this may be one possible cause of infertility following SCI.

Seminal bacterial contaminations: Probable factor in unexplained recurrent pregnancy loss.

BACKGROUND: It is estimated that about 50% of causes of recurrent pregnancy loss (RPL) cases remain unknown. Sperm factors are suggested to have probable role in cases with RPL. OBJECTIVE: The goal was to determine the possible relationship between semen bacterial contaminations with unexplained RPL. Also, the correlation between number of bacterial colony and sperm chromatin condensation was examined. MATERIALS AND METHODS: This study consisted of 30 fertile men (group A) and 30 infertile (group B) men with unknown RPL. Semen collection and analysis were done according to WHO manuals. Sperm count and motility were evaluated by Makler chamber. Eosin-Nigrosin and Papanicolaou staining methods were applied for viability and morphology assessment, respectively. The semen samples from both groups were cultured for aerobic bacteria. Aniline blue (AB) and toluidine blue (TB) staining methods were applied for evaluating sperm chromatin condensation. RESULTS: The numbers of colonies were significantly higher in group B when compared to group A. Also, S. aureus and E. coli contaminations showed significant differences between two groups. Both AB+ and TB+ sperm cells showed significant increase in group B compared to group A. There was a significant negative correlation between colony number and progressive motility (p=0.01), and sperm viability (p=0.007). In addition, positive correlations were found between colony number and AB(+) (p=0.001) and TB+ (p=0.004) as well. CONCLUSION: Bacterial contaminations in semen of men from RPL couples had significantly higher levels when compared to fertile controls. Presence of microorganisms in semen may be correlated with irregular sperm parameters and quality.

The frequency of Yq microdeletion in azoospermic and oligospermic Iranian infertile men.

BACKGROUND: About 15% of couples have infertility problems which 40% of them are related to the male factors. Genetic factors are candidate for about 10% of male infertility conditions. Among these, AZFa, AZFb, AZFc and AZFd regions on the Yq are considered most important for spermatogenesis. Microdeletions of these regions are thought to be involved in some cases of azoospermic or oligospermic infertile men. OBJECTIVE: We studied the prevalence of AZF microdeletions among Iranian infertile men with non-obstructive azoospermia and oligospermia. MATERIALS AND METHODS: A total of 50 Iranian azoospermic and oligospermic infertile men were selected for case group and 50 men with normal spermogram as control group. The molecular study of Y chromosome microdeletions was done by multiplex polymerase chain reaction (M-PCR) method by using of 13 sequence tagged site (STS) markers from AZF region. RESULTS: Four (8%) patients showed Y chromosome microdeletions among case group, deletion in AZFc region was the most frequent (80%) followed by AZFb (20%), in AZFa and AZFd region we did not detect any deletions. No deletion was detected in control group; the ratio of Y chromosome microdeletion in azoospermic men was higher than this ratio in oligospermic men [19% (3/16) among azoospermic men and 3% (1/34) among oligospermics]. Serum FSH level in men with microdeletions was higher than this level in men with no deletions (p=0.034). CONCLUSION: Because of relatively high prevalence of microdeletions on the long arm of Y chromosome among Iranian azoospermic and oligospermic patients, screening of this microdeletion may be advised to infertile men particularly azoospermic and oligospermic men before using assisted reproductive treatments.