Can SCSA and TUNEL forecast apoptosis-related motility depletion in Asthenozoospermia?

This study is an attempt to determine the power of SCSA and TUNEL for the evaluation of apoptosis status and apoptosis-related motility depletion in Asthenozoospermia. Fifty-one semen samples from Asthenozoospermic and 20 samples from fertile men participated in this study. SCSA and TUNEL were applied for the assessment of DNA integrity by flow cytometry. Annexin V conjugated with FITC labelling and FLICA method were used for the assessment of externalisation of phosphatidylserine and spermatozoon with active Caspase 3 respectively. SCSA results were shown to have a significant correlation with EPS in live spermatozoon (r = .85, p value = .00) and spermatozoon with active Caspase 3 (r = .633, p value = .00). TUNEL result was revealed to have a nonsignificant positive correlation with them. Then, Asthenozoospermic individuals were divided into two groups, SCSA higher and SCSA lower than 27%. Results interestingly indicated that the two groups significantly differed from each other in terms of TUNEL, EPS in live spermatozoon, spermatozoon with active Caspase 3 and sperm vitality (p value = .00). Both SCSA and TUNEL were correlated with apoptosis-related motility depletion in Asthenozoospermia. However, SCSA might be more powerful than TUNEL and could provide reliable information about DNA, chromatin integrity and apoptosis status in Asthenozoospermia.

Effects of Tempol and Quercetin on Human Sperm Function after Cryopreservation.

　　BACKGROUND: Quercetin is a flavonoid with high reactive oxygen species (ROS) scavenging and ion chelating activity. It also enhances the activity of antioxidant enzymes and reduces enzymatic activity such as NADPH oxidase and NADH-dependent oxido-reductase. Tempol, as a superoxide dismutase mimetic agent, converts superoxide to less toxic hydrogen peroxide (H2O2), but cannot reduce highly toxic hydroxyl radicals in Fenton or Haber-Weiss reactions mediated with free iron or cupper. OBJECTIVE: The study was to compare the effect of Quercetin and Tempol in an optimized commercial cryo-protective media on ROS induced cryoinjury for the first time. MATERIALS AND METHODS: Following administration of these compounds during freezing process, sperm motility, viability, ROS production and DNA integrity were assessed before and after freezing/thawing process. RESULTS: Data showed that 10 µM Quercetin and 5 µM Tempol significantly improved sperm motility and viability, but they together had no additive effect. Supplementation with Quercetin alone or combination of Quercetin with Tempol decrease the ROS concentration, but the reduction was not significant for Tempol alone compared to control group. Quercetin and Tempol significantly decrease DNA fragmentation. CONCLUSION: The supplementation of Quercetin or Tempol, but not their combination improves the quality of cryopreserved human semen.

Relationship between fertilization rate and early apoptosis in sperm population of infertile individuals.

Integrity of the sperm membrane, of which phosphatidyl serine (PS) plays a central role, is essential for fertilization. The externalisation of PS (EPS) occurs during capacitation and the acrosome reaction. EPS, from the inner to the outer membrane, is considered as a sign of early apoptosis. Therefore, EPS may have a dual function in sperm. This study has evaluated the relationship between EPS and fertilization, embryo quality and pregnancy outcomes in couples who were candidates for ICSI. Semen samples were collected from 43 ICSI candidates and assessed according to World Health Organization guidelines for semen parameters. EPS was assessed by Annexin V and propidium iodide (PI) staining. Protamine deficiency was assessed by chromomycin A3 (CMA3) staining. A significant positive correlation was observed between the percentages of fertilization and annexin-positive PI-negative (An(+)PI(-)) sperm. There was a significant negative correlation between the percentages of protamine-deficient sperm with the percentage of fertilization. In addition, the percentage of An(+)PI(-) sperm in individuals with fertilization rates higher and lower than 50% significantly differed. The percentage of annexin-positive PI-positive (An(+)PI(+)) sperm in semen of the partners of pregnant women significantly differed from the partners of nonpregnant women. In conclusion, if An(+)PI(-) is a sign of capacitation and An(+)PI(+) is a sign of apoptosis, the results suggest that semen samples with a higher ability to undergo capacitation have a higher chance to result in successful fertilization post-ICSI. The presence of a high percentage of apoptotic sperm in the insemination sample before capacitation may reduce the chances of pregnancy.

Zeta potential vs apoptotic marker: which is more suitable for ICSI sperm selection?

PURPOSE: Selection of appropriate sperm is considered as a decision making point in the ICSI procedure. Canonically, sperm selection is based on morphology and motility. Recent advances in this field, have shown that, this procedure can be assisted by further selection based on membrane surface charge (Zeta potential) and surface apoptotic marker (phosphatidylserine externalization) using magnetic activated cell sorter (MACS). Based on the literature, both these procedures improve quality of selected sperm population. Therefore, this study aims to compare the efficiency of these two procedures. METHODS: Semen samples were collected from 36 fertile and infertile (teratozoospermic and /or asthenozoospermic) individuals. Sperm DNA fragmentation, protamine deficiency and morphology were assessed by TUNEL, CMA3 and papanicolaou staining in unprocessed, MACS and Zeta processed samples. RESULTS: Although both MACS and Zeta were able to separate a higher percentage of sperm with normal morphology, and lower DNA fragmentation and protamine deficiency compared to unprocessed, MACS procedure could significantly isolate, a greater percentage of sperm with normal acrosome and protamine content compared to Zeta procedure. CONCLUSION: Both MACS and Zeta procedures improve the quality of the selected spermatozoa for ICSI. However, MACS procedure is more efficient in individuals with severe male factor infertility to select sperm with normal acrosome and protamine content but concern regarding transfer of MACS beads into the oocyte remains to be resolved.

Proper ubiquitination effect on the fertilisation outcome post-ICSI.

Ubiquitin is an 8.5-kDa protein that tags outlived proteins for degradation by the proteasome. It also marks defective spermatozoa during epididymal passage and has been proposed as a biomarker of sperm quality. This study evaluates the relationship between sperm ubiquitination, protamine deficiency, semen parameters and fertilisation rate in infertile individuals undergoing the intracytoplasmic sperm insemination (ICSI) procedure. Semen samples from 73 ICSI candidates were collected and analysed according to World Health Organization criteria. A portion of each sample was evaluated for sperm ubiquitination using the sperm ubiquitin tag immunoassay (SUTI) with flow cytometry, and protamine deficiency by chromomycin A3 (CMA3) staining. In addition, the relationship between the fertilisation rate and sperm ubiquitination was calculated in ICSI candidates. The intensity of ubiquitination showed a significant negative correlation with sperm concentration (r = -0.255, P = 0.032) and a positive correlation with fertilisation rate (r = 0.384, P = 0.013) post-ICSI. No correlation was observed between protamine deficiency and the percentage of ubiquitination or ubiquitination intensity. The results of this study suggest that sperm ubiquitination prior to capacitation may be considered as a marker of defective spermatozoon. Spermatozoa that undergo proper ubiquitination may have a higher chance for fertilisation, because they are made redundant by the ubiquitin-proteasome pathway in the epididymis compared to hypo-ubiquitinated spermatozoa.

New era in sperm selection for ICSI.

Spermatozoa contribute to approximately half of the genome of future progeny, and therefore, have a profound impact on embryo development post-fertilization. Sperm selection based on viability and normal morphology does not eliminate the chance for DNA damaged spermatozoa to be inseminated and may account for a considerable percentage of failed embryo development post-ICSI (Intra cytoplasmic sperm injection). Therefore, sperm selection based on functional sperm characteristics to preclude insemination of DNA damaged spermatozoa have paved the way for successful ICSI outcomes. In regard to this, different laboratories have introduced novel procedures to replace traditional or orthodox sperm selection methods. This review attempts to provide information on the scientific bases of each procedure, and pinpoint their advantages and disadvantages. In addition to data from our research, a systematic search on the literature, publications and presentations was carried out using such databases as PubMed and ISI-Web.

Density gradient centrifugation before or after magnetic-activated cell sorting: which technique is more useful for clinical sperm selection?

PURPOSE: Although, at present, the selection of sperm prior to ICSI is based on motility and morphology, undetectable anomalies, and more importantly damaged DNA are overlooked. In this regard, novel sperm selection procedures have gained much interest. For instance, sperm has been selected by Magnetic-Activated Cell Sorting (MACS) based on early apoptotic marker, the externalization of phosphatidylserine (EPS). Review of the literature has revealed that the efficiency of this technique has been mainly evaluated post Density Gradient Centrifugation (DGC). Therefore, there is a need to prove the efficiency of this technique independent of DGC. In addition, considering the fact that DGC induces EPS due to capacitation and acrosome reaction, therefore, the role of MACS before DGC(MACS-DGC) and MACS after DGC (DGC-MACS) should be assessed. METHODS: Semen samples from fifteen infertile men were divided into three separate fractions: control, DGC, and MACS. To carry out DGC-MACS, DGC samples were further divided into two fractions and MACS was carried on the second fractions. Similarly to carry out MACS-DGC, the MACS samples were further divided into two fractions and DGC was carried on the second fractions. Percentages of sperm with normal morphology, DNA fragmentation, protamine deficiency, EPS and caspase-3 activity were determined in each fraction. RESULTS: DGC is more efficient than MACS in separating intact sperm only in terms of normal morphology, DNA and chromatin integrity but not for active caspase. However, a combination of these procedures was more efficient than a single procedure to separate intact sperm for the aforementioned parameters. Comparison of the combined procedures showed only higher efficiency to separate active caspase in the MACS-DGC group. CONCLUSION: Based on these results, we propose MACS-DGC rather than DGC-MACS to be implemented in clinical settings.

High total acrosin activity in varicocele individuals.

Varicocele is a common cause of male infertility and reports indicate that varicocelectomy has a beneficial effect on male fertility. The aims of this study were to evaluate and compare the total acrosin activity along with DNA integrity in semen samples obtained from 70 varicocele individuals with male factors infertility presenting grades II and III varicocele before and after the surgery and 30 fertile individuals without any clinical presentation of varicocele. Total acrosin activity, protamine deficiency, DNA fragmentation, and semen parameters including sperm concentration, motility and sperm morphology were assessed by spectrophotometery, CMA3 staining, terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay and WHO criteria, respectively. Total acrosin activity (P = 0.03), percentage CMA3 positivity (P = 0.00) and TUNEL-positive spermatozoa (P = 0.04) were higher in the varicocele individuals before the surgery compared with the fertile individuals; yet, all the aforementioned criteria decreased significantly after surgery (P < 0.05). The results of this study reveal that DNA fragmentation and protamine deficiency, as negative parameters in fertility, improve post-surgery; however, total acrosin activity as a positive parameter in fertility is higher in the varicocele individuals compared with fertile and decreases to a value close to the fertile control post-surgery. High levels of total acrosin activity in varicocele individuals need more research in future.

Evaluation of ubiquitin and annexin V in sperm population selected based on density gradient centrifugation and zeta potential (DGC-Zeta).

PURPOSE: Sperm that bypass natural apoptosis and the ubiquitin-proteasome system may find their way into semen. In order to avoid the insemination of such sperm during an intracytoplasmic sperm injection (ICSI) treatment, novel sperm selection procedures such as the Zeta procedure have been implemented. Therefore, the aim of this study was to evaluate extent of ubiquitination and external phosphatidylserine (EPS) in sperm populations selected by combines density gradient centrifugation (DGC) and Zeta electric potential in comparison to DGC and neat semen samples. METHODS: Semen samples were collected from 51 infertile men and divided into control, DGC and DGC-Zeta groups. Semen analysis was carried out according to World Health Organization criteria. The percentages of protamine deficiency, DNA fragmentation, EPS and ubiquitinated sperm were assessed by chromomycin A3 (CMA3), TUNEL, Annexin V, and immunostaining, respectively. RESULTS: Sperm selected by the DGC-Zeta procedure presented a lower percentage of sperm with protamine deficiency, abnormal morphology and DNA fragmentation while the percentage of annexin V and ubiquitin-positive sperm increased. CONCLUSION: The results of this study reveal that, DGC-Zeta improves the quality of the selected spermatozoa for ICSI and increases ubiquitination and EPS rates. We propose these alterations are part of the normal physiological process of capacitation.

Quantitative expression of phospholipase C zeta, as an index to assess fertilization potential of a semen sample.

BACKGROUND: Failed fertilization post-ICSI has been mainly attributed to the sperm's inability to induce oocyte activation. Phospholipase C zeta (PLCζ) is considered to be one of the factors for the induction of oocyte activation. The aim of this study was to quantitatively assess the expression of PLCζ in globozoospermic men or those with previously low or failed fertilization in comparison with fertile men or those with high fertilization potential. In addition, the relationship between expression of PLCζ and that of other sperm markers was evaluated. METHODS: Real-time PCR was carried out to evaluate relative expression of PLCζ mRNA. Chromatin maturity and acrosin activity were assessed by CMA3 staining and a colorimetric method. RESULTS: The expression of PLCζ was significantly lower in globozoospermic men (P< 0.01, n= 8) or individuals with previously low or failed fertilization (P< 0.01, n= 36) in comparison to fertile men (n= 24). In addition, a significant difference was observed between globozoospermic (P< 0.01) and individuals with previously low or failed fertilization (P= 0.003) in comparison to high fertilization individuals (n= 17). Expression of PLCζ was not correlated with either chromatin maturity or acrosin activity. However, a significant correlation was observed between the percentage of fertilization and relative expression of PLCζ (r= 0.4, P< 0.01). CONCLUSION: In this study, for the first time, we have shown that assessment of relative expression of PLCζ may provide a useful marker for the ability of sperm to induce oocyte activation after ICSI.

Zaditen (Ketotifen), as mast cell blocker, improves sperm quality, chromatin integrity and pregnancy rate after varicocelectomy.

Surgery is considered the most common choice for the treatment of male infertility with clinical varicocele. Increased numbers of mast cells (MCs) have been associated with different types of infertility, including varicocele. Despite there being different reports on improved fertility following administration of MC blockers, there is no report on the effect of a MC blocker on varicocele or after varicocelectomy. Therefore, the aim of this study was to evaluate the effect of Zaditen on semen quality, protamine content, DNA damage and fertility post-surgery. The study included 103 infertile men who were referred to Isfahan Fertility and Infertility Center for varicocelectomy. Varicocele individuals were randomly divided into control (52) and treatment groups (51). Semen parameters, WBC/mL, protamine content (chromomycin A3 staining) and DNA integrity (sperm chromatin dispersion test) were assessed before and 3 months after surgery. Comparison of the aforesaid parameters between the two groups revealed significant improvement in the treatment group compared with the control group, with the exception of DNA integrity. In addition, the cumulative pregnancy significantly improved by 9 months post-surgery in the treatment group (41.17%) compared with the control group (21.15%). The results of this study, for the first time, reveal that MC blockers such as Zaditen improve semen parameters, chromatin integrity and pregnancy rates when administered as adjunct therapy post-varicocelectomy.

Evaluation of zeta and HA-binding methods for selection of spermatozoa with normal morphology, protamine content and DNA integrity.

Sperm selection parameters based on morphology and motility for ICSI might not be relevant to chromatin integrity. Thus sperm selection based on sperm characteristics has been suggested. Therefore, the aim of this study was to evaluate the efficiency of the zeta and hyaluronic acid (HA) sperm selection procedures with neat semen, for recovering spermatozoa with normal morphology, protamine content and DNA integrity in infertile men. Semen samples from 77 infertile couples were assessed during this study. Semen analysis was carried out according to World Health Organization criteria. Protamine content, DNA integrity and sperm morphology were assessed by chromomycin A3, sperm chromatin dispersion and Papanicolaou staining respectively. The results show that both HA and zeta methods were efficient to recover spermatozoa with normal morphology and protamine content. In terms of the latter parameters, there was no superiority between the two procedures. However, in terms of DNA integrity, the zeta method was more efficient compared with the control and HA procedure and no significant difference was observed between HA and the controls. Therefore, the zeta method appears to be a suitable procedure to recover spermatozoa with normal DNA integrity.

Selection of sperm based on combined density gradient and Zeta method may improve ICSI outcome.

BACKGROUND: Reducing the percentage of sperm anomalies in insemination samples remains a goal to be achieved in the intra-cytoplasmic sperm insemination (ICSI) procedure. The aim of this study was to evaluate the efficiency of density gradient centrifugation (DGC) and Zeta methods to recover sperm with intact chromatin, and to evaluate whether the combined DGC/Zeta procedure improved ICSI outcome. METHODS: In Experiment 1, DGC and Zeta methods were carried out on 60 unprocessed semen samples. The samples were then assessed by chromomycin A3 staining, acridine orange test, terminal deoxynucleotidyl transferase dUTP nick end labeling assay and the sperm chromatin dispersion test for protamine deficiency and DNA fragmentation. In Experiment 2, sibling oocytes from 30 ICSI candidates were divided into two groups; one group was inseminated with sperm processed by DGC and the second with sperm processed by DGC/Zeta. The outcomes of 30 ICSI cycles were compared between the two groups and also with 34 ICSI candidates whose oocytes were inseminated by DGC-processed sperm. RESULTS: Both procedures were efficient for the recovery of sperm with normal protamine content and low DNA fragmentation. However, the Zeta method yielded a greater number of sperm with less DNA fragmentation. Fertilization and pregnancy rates were improved following the combined DGC/Zeta procedure. Compared with DGC alone, the pregnancy rate appeared improved but this was not statistically significant (P = 0.091). CONCLUSIONS: Combining DGC and Zeta procedures improves the quality of semen samples which may increase fertilization rates and possibly pregnancy rates.