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Background: Sperm cryopreservation is presently used for conservation of male gametes in assisted reproduction technologies (ART). Despite the benefits of sperm banking, freeze-thawing process is injurious to sperm integrity due to induced oxidative stress by cold stress. Oxidative stress reduces sperm motility, viability and DNA integrity., Objective: To investigate the effect of alpha lipoic acid (ALA) on human sperm function during the freeze-thawing process., Materials and Methods: Thirty semen samples were collected and different concentrations (0, 0.05, 0.1, 0.2, 0.4, 0.8, and 8mM) of ALA were added to a sperm freeze medium and its effects on sperm motility, DNA damage, and lipid peroxidation of frozen-thawed spermatozoa were assessed., Results: The addition of 0.2 mM ALA to the sperm freeze medium resulted in significant improvement in percentage of sperm motility, less DNA damage and decreased lipid peroxidation during freeze-thawing process (p<0.05)., Conclusion: ALA improves the cryo-protective capacity of sperm freeze medium used for human semen by protecting the sperm from ROS attack induced by the freezing-thawing process. We suggest that sperm freeze medium supplemented with 0.2 mM ALA would be beneficial for the cryopreservation of male gametes in ART.

Background: Reproductive toxicity of lipopolysaccharide (LPS) on spermatozoa is well established., Objective: The aim of the present study was to show the potential benefits of alpha lipoic acid (ALA) as a strong antioxidant in alleviating the reproductive toxicity of LPS., Materials and Methods: Sperm cells and cumulus-oocyte complexes (COCs) were collected from healthy NMRI mice (body weights ranged from 25 to 35 g, 100 females and 200 males). Sperm cells were treated with varying doses of ALA (0.01, 0.02, and 0.04 mm) and 0.01 μg/mL of LPS for 4 h. The quality of spermatozoa (ROS production, DNA fragmentation, and spontaneous acrosome reaction), sperm fertilizability, and the consequent developmental competence of oocytes inseminated with ALA/LPS-treated spermatozoa were recorded., Results: The results showed that 0.04 mm of ALA abrogated LPS-reduced sperm motility, viability, ROS production, spontaneous acrosome reaction, fertilizability, and developmental competence. In addition, 0.04 mm ALA significantly reverted the negative effects of LPS on inner cell mass (ICM) cell counts, total cell number (TCM), and ratio between ICM and TCM., Discussion: Our data showed that ALA significantly could abrogate the negative effects of LPS on sperm quality and oocyte developmental competence. Therefore, ALA had the capacity for protecting sperm cells from LPS-induced damage and ensured fertilization and developmental competency., Conclusion: These in vitro findings suggested a therapeutic role for ALA in reducing the negative effects of LPS on spermatozoa and early embryonic development., (© 2019 American Society of Andrology and European Academy of Andrology.)

Background: To achieve sperm retrieval in azoospermic men, predicting the success rate seems to be necessary., Objectives: In the present study we aimed to assess expression of seven molecular markers [ESX1, DAZ, DAZL (pre-meiotic markers), ZMYND15, PRM1, TNP1, and SPEM1 (post-meiotic markers)] to predict the success of sperm retrieval., Materials and Methods: In this study, 63 azoospermic men [16 OA (obstructive azoospermia) and 47 NOA (nonobstructive azoospermia)] undergoing testicular tissue microdissection (micro-TESE) for intracytoplasmic sperm injection (ICSI). Expression levels of these target genes were determined by real-time reverse transcription polymerase chain reaction using the DDCt method, and efficacy of each gene was compared by receiver operating characteristic (ROC) curve analysis., Results: Expression of post-meiotic transcripts significantly decreases in NOA and its subgroups (SCOS: Sertoli cell only syndrome, MA: maturation arrest, and HS: hypospermatogenesis) with spermatogenic failure compared to normal spermatogenesis (OA), with an exception of ZMYND15 for the HS group. These findings suggest the differential expression of the post-meiotic ZMYND15 marker is in accordance with histological findings and can discriminate HS from SCOS and MA. Post-meiotic markers were significantly reduced in negative vs. positive sperm retrieval groups., Discussion and Conclusion: Among the seven markers, SPEM1 had the best positive prediction power (96%) and negative prediction power (85%) at a 0.086 cutoff with the area under the curve (AUC) of 0.91 for receiver operating characteristic 4 (ROC) to predict the micro-TESE outcome., (© 2018 American Society of Andrology and European Academy of Andrology.)

Sperm genomic integrity has a significant effect on intracytoplasmic sperm injection (ICSI) outcomes, especially post-implantation. Spermatozoa selected based on motility and morphology do not guarantee the genomic integrity of spermatozoa. Nearly fifty percentage of spermatozoa in infertile men with normal morphology present different degrees of DNA fragmentation. However, capacitated or hyperactivated spermatozoa show lower degrees of DNA fragmentation. Therefore, selection of hyperactivated spermatozoa may improve ICSI outcome. Routinely, for ICSI, fast-moving spermatozoa with A or B motility pattern are mainly selected for injection. The result of this study shows that in processed semen samples, hyperactivated spermatozoa are mainly observed in B motility pattern while, in viscous medium like polyvinylpyrrolidone (PVP), hyperactivated spermatozoa are mainly present in spermatozoa with C pattern of motility (nonprogressive). Therefore, we propose spermatozoa with C motility pattern which contains the main population of physiological or hyperactivated spermatozoa should be selected for ICSI., (© 2016 Blackwell Verlag GmbH.)

To evaluate the predictive value of histone demethylase KDM3A to protamine 1 (PRM1) mRNA expression ratio as a reliable marker of sperm retrieval in men with obstructive and non-obstructive azoospermia (NOA). Fifty eight azoospermic men, including 44 with NOA and 14 with obstructive azoospermia (OA). Testis tissue samples were collected from azoospermic men who have been referred for testicular sperm extraction (TESE) and micro-TESE. Relative expression ratio of KDM3A to PRM1 was analyzed after selection of approved reference genes. Histological classification of testis biopsies was performed. Sperm retrieval following TESE and micro-TESE was evaluated. A sperm retrieval prediction sensitivity of 95% was established when the Cq of PRM1 became smaller than the Cq of both KDM3A and GAPDH genes. However, azoospermic men with down-regulated KDM3A and decreased expression of PRM1 mRNA showed very low success for sperm retrieval (<25%), even after micro-TESE surgery. The KDM3A to PRM1 mRNA expression ratio can be used as a reliable marker of successful testicular sperm extraction in men with obstructive and non-obstructive azoospermia with 95% sensitivity., (© 2016 American Society of Andrology and European Academy of Andrology.)

In individuals with varicocele, DNA is damaged due to high level of oxidative stress, and varicocelectomy can overcome this effect. Damaged DNA is less liable to DNA methylation, and antioxidant therapy appears to have the potential to reduce sperm oxidative stress and DNA damage and thereby maintain DNA methylation, while effect of varicocelectomy on DNA methylation patterns has remained unclear. In the light of these considerations, we aimed to examine the effect of varicocelectomy on sperm DNA methylation and functional characteristics. Fifty-two men with left-sided varicocele (grade II &III) were included. Sperm parameters, DNA fragmentation, protamine deficiency, oxidative stress and global DNA methylation were evaluated before and 3 months after surgery. Our data show that sperm concentration, percentages of spermatozoon with abnormal morphology, DNA fragmentation, protamine deficiency and oxidative stress significantly improved after surgery. Percentage of sperm motility, global DNA methylation and intensity of DNA methylation also improved after surgery, although the differences were not significant when compared with before surgery. Categorisation of individuals to subgroups revealed that improvement of DNA methylation appears to take place in oligozoospermic individuals, which are more severely affected by state of varicocele. However, this is a preliminary study, and further studies are required to solidify this conclusion., (© 2014 Blackwell Verlag GmbH.)

Assessment of sperm ubiquitination and DNA fragmentation as sperm functional markers are proposed to complement routine semen analysis. This study focuses on the evaluation of these markers in infertile men with varicocele or exposed to occupational background. The results were compared with normozoospermic men. Semen parameters in both groups were lower than those in the control group. Ubiquitination median, as a marker for functionality of the ubiquitin-proteasome system, was also lower in both groups. The ubiquitination median showed a significant positive correlation with motility in both groups, while it showed only a negative correlation with sperm morphology in the varicocele group. DNA fragmentation showed a significant correlation with semen parameters, in total varicocele and also total exposure groups. In conclusion, significant difference of sperm ubiquitination between normal and study groups further validates that sperm ubiquitination as a potential molecular marker for sperm evaluation in addition to routine semen analysis in clinical laboratories., (© 2013 Blackwell Verlag GmbH.)

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., (© 2012 Blackwell Verlag GmbH.)

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.

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., (© 2012 Blackwell Verlag GmbH.)

Heat-shock protein A2 (HSPA2) is a testis-specific member of the HSP70 family known to correlate with sperm maturity, function and fertility. The aim of this study was to compare expression of HSPA2 in fertile and infertile individuals using a recently marketed highly purified polyclonal antibody. Thus, after analysing sperm concentration, motility and morphology in semen sample of 49 individuals with male factor infertility and 47 fertile individuals according to World Health Organization guidelines, we evaluated HSPA2 by microscope florescence, flow cytometry, Western blot and RT-PCR. We observed higher percentage of sperm expressing HSPA2 in anterior and equatorial regions in fertile than infertile individuals by microscope fluorescence. Percentage of sperm expressing HSPA2 in two conditions (fixed permeabilised and capacitated) by flow cytometry showed that percentage of HSPA2-positive sperm in fixed permeabilised and also capacitated samples was significantly different between fertile and infertile individuals. Western blot analysis of HSPA2 in semen samples revealed high variation within the fertile and infertile population. The results of RT-PCR revealed higher expression of HSPA2 in the fertile compared to infertile individuals, but this difference was not significant. According to the results, we suggest that HSPA2 expression is heterogeneously expressed on different part of fixed permeabilised sperm and its expression is significantly higher in fertile compared to infertile individuals. The surface expression of this protein significantly increases following capacitation in both fertile and infertile individuals. HSPA2 expression significantly correlates with sperm concentration and morphology. Therefore, aberrant HSPA2 expression may play an important role in capacitation and fertilisation processes., (© 2012 Blackwell Verlag GmbH.)

Background: Intracytoplasmic sperm injection (ICSI) is the primary treatment for male infertility. However for this procedure, with the exception of visual morphological selection, there is no standardization for sperm selection. Recently, the hypo-osmotic swelling test (HOST) has been proposed to potentially select sperm with intact membranes. The aim of this study is to evaluate the ability of this technique to select functional sperm in terms of apoptosis and morphology, as well as nuclear integrity., Methods: A total of 20 semen samples were randomly collected from men who attended the Andrology Unit of the Isfahan Fertility and Infertility Center. Semen samples were washed and exposed to hypotonic conditions, before being fixed and simultaneously assessed for membrane integrity as well as abnormal morphology, DNA fragmentation and protamine deficiency by using Papanicolaou, TUNEL and CMA3 staining techniques, respectively. The remaining semen samples were washed with calcium buffer and stained by Annexin V, then exposed to hypotonic conditions before being assessed for early apoptosis along with membrane integrity., Results: HOST grade 'd', followed by grade 'c', showed the highest percentages of healthy sperm, whereas sperm of HOST grade 'g' in which anomalies in terms of apoptosis, abnormal head morphology or nuclear immaturity or membrane damage, were most frequently observed in the samples assessed., Conclusions: Integration of HOST into the sperm selection procedure may provide a valuable tool for selection of functional sperm required for ICSI. According to this study, insemination of HOST grade 'g' sperm should be avoided during 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., (© 2011 The Authors. International Journal of Andrology © 2011 European Academy of Andrology.)

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.

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.

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., (© 2010 The Authors. International Journal of Andrology © 2011 European Academy of Andrology.)

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.

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.

Purpose: To compare the efficiency of routine sperm selection method with HA-selection procedure for fertilization rate, embryo development, implantation and pregnancy rates as well as evaluating the relationship between HA-binding ability with sperm protamine deficiency and DNA fragmentation., Methods: Semen samples were obtained from the 50 couples undergoing ICSI. The percentage of fertilization rate, cleavage and quality of embryos compared between two procedures (routine sperm selection and HA-binding selection). The semen samples were assessed for DNA fragmentation and protamine deficiency by sperm chromatin dispersion (SCD) test and Chromomycin A3 (CMA3) staining, respectively., Results: A significant inverse correlation was observed between percentage of HA binding with protamine deficiency, DNA fragmentation and abnormal sperm morphology (P < 0.05). Furthermore, in current study, oocytes inseminated by HA sperm selection procedure had significantly higher fertilization rate (P < 0.05). While the pregnancy and implantation rates were insignificantly increased., Conclusion: The results suggest that normal sperm have higher chance to bind HA and therefore, HA sperm selection procedure may select sperm with normal protamine content and low DNA fragmentation, but to confirm the effect of HA sperm selection on the ICSI outcome requires further studies.

Sperm premature chromatin condensation (PCC) has been considered as the second cause of failed fertilization post-intracytoplasmic sperm injection (post-ICSI). Cytoplasmic factors, including oocyte cytoplasmic immaturity have been suggested to induce PCC sperm. However, recent studies suggest that sperm chromatin anomaly might also lead to PCC sperm. During this study, human sperm from infertile patients with protamine deficiency or with adequate amount of protamine assessed by chromomycin A3 were injected into metaphase II mouse oocyte, treated with colcemid. Chromatin analysis was carried out on the injected oocyte. The results of this study show that contrary to the percentage of intact sperm, percentage of PCC sperm was significantly higher in oocytes injected with protamine deficient sperm (36.43 +/- 4.46) compared to oocytes injected with sperm with an adequate amount of protamine (11.99 +/- 3.54, P < 0.001). A significant correlation was also observed between percentage of PCC sperm and protamine deficiency (r = 0.46, P = 0.004). Therefore, it can be suggested that oocytes injected with protamine deficient sperm have a higher chance of forming PCC sperm and may result in failed fertilization post-ICSI.

During spermiogenesis, histones are replaced by protamines (P1 and P2), resulting in sperm chromatin condensation followed by a halt to gene expression in haploid spermatids and spermatozoa. As a consequence, protamine deficiency and aberrant P1/P2 ratio have a profound effect on both fertilization and embryo development. However, reports on the effect of the P1/P2 ratio on fertilization and embryo development after intracytoplasmic sperm injection (ICSI) are contradictory between human and animal studies. The question that still remains to be elucidated is which type of protamine deficiency is most common among protamine deficient samples. The present study has a direct bearing on this issue investigating the correlation of the P1/P2 ratio with protamine deficiency, fertilization, embryo quality and embryo development in ICSI patients. This study was carried out on 71 patients. Chromomycin A3 (CMA3) staining was used to determine protamine deficiency. Since this procedure does not indicate the type of protamine deficiency, the P1/P2 ratio was evaluated by nuclear protein extraction, acetic acid urea polyacrylamide gel electrophoresis and analysis of protein bands with software. Polyclonal anti-P1 and anti-P2 antibodies were used to confirm P1 and P2 presence. Results show a negative significant correlation of fertilization rate with protamine deficiency and P1/P2 ratio. No significant correlation was observed between protamine deficiency and P1/P2 ratio. Therefore, it can be concluded that altered P1/P2 ratio effects fertilization rate and embryo quality which subsequently may affect implantation and pregnancy outcome.

After aneuploidy, sperm premature chromatin condensation (PCC) is the next prevalent cause of fertilization failure. Therefore the aim of this study was to evaluate the effect of sperm protamine deficiency on sperm PCC formation post-ICSI. Chromatin analysis was carried out on failed fertilized oocytes post-ICSI and incidences of sperm PCC were evaluated and the results were compared with the extent of protamine deficiency assessed by chromomycin A3. The results show that incidence of sperm PCC was significantly different in failed fertilized oocytes injected from semen samples with greater or less than 30% CMA3 positivity (P = 0.04). However, except for fertilization rate (P < 0.001), the mean number of MII, MI and germinal vesicles oocytes and percentage normal sperm were not significantly different between the two groups (P > 0.05). A significant correlation was observed between sperm protamine deficiency with fertilization rate. Hence sperm protamine deficiency affects fertilization rate and possibly prones sperm to PCC post-ICSI.

The aim of this study was to evaluate the effect of sperm chromatin anomalies on fertilization outcome post-intracytoplasmic sperm injection (ICSI). Therefore, along with semen parameters, Chromomycin A3 (CMA3) staining for protamine deficiency, aniline blue staining for excessive histones, SDS for sperm chromatin stability and SDS + EDTA for the ability of sperm to undergo decondensation were carried out on 55 semen samples from patients referred to the Isfahan Fertility and Infertility Center for ICSI. The results showed that among the aforementioned tests and semen parameters only CMA3 showed a significant correlation with fertilization outcome post-ICSI. Patients were also grouped according to CMA3 level of <30% or >30% or fertilization rate of <50% or >50%. The results show that the mean percentage fertilization and mean percentage of CMA3 positivity is different in both groups, respectively. The area under receiver operator characteristics curve shows that CMA3 is a highly sensitive and specific test for prediction of fertilization outcome post-ICSI. In conclusion, that sperm protamine deficiency has profound effect on fertilization failure in ICSI.

Purpose: To consider the relationship between different sperm nuclear maturity tests and in vitro fertilization (IVF) rate, in order to select the most sensitive, specific, and independent factor(s) for prediction of in vitro fertilization., Methods: Infertile couples (101) were randomly selected from IVF candidates referred to Isfahan Fertility and Infertility center. Semen samples were collected on the day of oocyte recovery. Following routine semen analysis, major portion of the semen was prepared for routine IVF insemination and the remaining was used for following sperm nuclear maturity tests: chromomycin A3 (CMA3), aniline blue, sodium dodecyl sulphate (SDS) test, and acridine orange test with or without heat shock (87 degrees C, 5 min). Sperms (200) were evaluated for each test. The results were recorded and analyzed for their correlation to fertilization rate, using correlation coefficient, logistic regression analysis, student t-test, and receiver operating characteristics (ROC) curve., Results: Among these tests, aniline blue and CMA3, and semen parameters, sperm morphology, and sperm motility showed a significant correlation with fertilization rate. Using logistic regression analysis, sperm morphology and CMA3 were the only independent factors related to in vitro fertilization. ROC curves showed that among above tests, CMA3 is the most specific and sensitive for sperm nuclear maturity., Conclusion: Among CMA3, aniline blue, SDS test, and acridine orange, CMA3 was the most sensitive and specific test that can be used along with routine semen analysis for more precise prediction of fertilization rate.

Purpose: Our purpose was to investigate the association between percentage chromomycin A3 (CMA3) positivity of spermatozoa with some sperm parameters and in vitro fertilization rate., Methods: Spermatozoa were collected from 139 men, washed in PBS, fixed in methanol/glacial acetic acid (3:1), and then spread on slides. CMA3 positivity is expressed as the percentage in 200 spermatozoa., Results: Percentage of CMA3 positivity showed not only a negative correlation with fertilization rate but also a significant difference between fertilizing and nonfertilizing patients. Moreover, percentage of CMA3-positive spermatozoa showed a negative correlation with count and percentage motility and a positive correlation with percentage of abnormal morphology. Percentage of CMA3 positivity also had a positive correlation with some abnormalities of head such as amorphous and macrocephaly. Ultrastructural study showed chromatin unpackaging in high CMA3-positive semen samples in comparison with low CMA3-positive semen samples., Conclusion: There is a close relationship among fertilization rate, sperm parameters, and CMA3 positivity and CMA3 could be considered as a useful tool for evaluation of male fertility prior to infertility treatment.

Summary: Globozoospermia is a severe sperm morphological anomaly leading to primary infertility and low fertilisation following intracytoplasmic sperm injection (ICSI). This phenotype is observed in less than 0.1% of infertile men and is determined by small, round‐headed spermatozoa with absence of an acrosomal cap, acrosome protease and also cytoskeletal proteins. Failure of oocyte activation is considered as the main cause of fertilisation failure in these individuals post‐ICSI. Therefore, artificial oocyte activation (AOA) along with ICSI is commonly implemented. However, based on previous report, fertilisation rate remains low despite implementation of ICSI‐AOA. Therefore, other mechanisms like sperm chromatin packaging and DNA fragmentation may account for low fertilisation and development post‐ICSI‐AOA. Therefore, this study aims to assess and compare the degree of sperm protamine deficiency and DNA fragmentation in large population of infertile men with total globozoospermia (30 globozoospermic men presenting with 100% round‐headed spermatozoa) with 22 fertile individuals using chromomycin A3 and TUNEL assay respectively. Results clearly show that mean of sperm concentration and percentage of sperm motility were significantly lower, while percentage of sperm abnormal morphology, protamine‐deficient and DNA‐fragmented spermatozoa were significantly higher in infertile men with globozoospermia compared to fertile men. Therefore, increased sperm DNA damage in globozoospermia is likely related to defective DNA compaction and antioxidant therapy before ICSI‐AOA could be recommended as an appropriate option before ICSI‐AOA. [ABSTRACT FROM AUTHOR]

Sperm-mediated oocyte activation critically depends upon appropriate expression and assembly of sperm-borne oocyte-activating factors (SOAFs) during spermiogenesis. Several factors have been considered as candidate for SOAFs over the recent years. However, little is known about the expression profile of these candidates and their potential contribution to the clinical outcomes of intra-cytoplasmic sperm injection ( ICSI), particularly in globozoospermia. This study investigated the expression profile of PLCζ, PAWP, and TR- KIT and clinical outcomes of ICSI in 12 men with total globozoospermia and compared with 12 fertile individuals. Levels of PLCζ, PAWP, and TR- KIT mRNA in the spermatozoa of fertile men were significantly higher than the corresponding values of the globozoospermic subjects. Interestingly, at protein level, expressions of these factors in the cases assessed were low in globozoospermic individuals. Fertilization rates following artificial oocyte activation ( AOA) in the majority of globozoospermic couples were higher than the expected 30% cut-off value reported for individuals with failed or low fertilization rate. Clinical outcomes of ICSI- AOA were dependent on inter-individual variation in globozoospermic couples. None of the SOAFs assessed could provide a greater prediction value with respect to fertilization rate in globozoospermic couples which underwent ICSI- AOA. High fertilization (56.06%) and pregnancy (41.7%) rates accomplished in this study following ICSI- AOA indicated that expression profiles of PLCζ, PAWP, and TR- KIT were low in globozoospermic individuals, and ICSI combined with artificial oocyte activation could mimic physiological calcium changes which occur during fertilization. [ABSTRACT FROM AUTHOR]

Different methods have been used to evaluate the beneficial effect of varicocelectomy; these include semen parameters and pregnancy rate. Because of high biological variability of semen parameters, sperm functional tests have been considered as an efficient end point in assessment of fertility. Therefore, the aim of this study was to evaluate the effect of varicocelectomy on semen parameters and sperm protamine deficiency in 192 patients. The results of the present study show that all the three semen parameters and percentage of sperms with normal protamine content have improved post-surgery. The cumulative pregnancy rate was 34.6%. Comparing the results of the semen parameters and protamine content between patients whose partner became pregnant to those who did not benefit from varicocelectomy before and 6 months after surgery, show that patients may benefit from varicocelectomy that had higher initial semen density and better sperm morphology prior to surgery. Detailed analyses of sperm morphology, along with aforementioned results reveal that the factors which account for pregnancy difference are: (i) improvement in early events of spermatogenesis, possibly during spermatocytogensis and reduction division; and (ii) late spermiogenesis events. Thus, it can be suggested that patients with low initial sperm count may benefit more from assisted reproductive techniques or varicocelectomy followed by assisted reproduction. [ABSTRACT FROM AUTHOR]

Introduction: Cryopreservation causes physical and chemical damages and it can effect on sperm vitality, motility and lipid composition of sperm membrane. Increase of imbalance between production of reactive oxygen species (ROS) and semen antioxidant levels may responsible for this damage. Tempol considered as antioxidant with ability to penetrate membrane and reduce stress oxidative, especially hydrogen peroxide. Therefore aim of this study for the first time was to evaluate effect of Tempol during human sperm cryopreservation on sperm parameters and ROS level. Materials and Methods: Semen sample from 42 normozoospermic men were collected and divided two groups. In control group, semen samples were cryopreserved accordingly vitrolife protocol. In test group, Tempol (5uM) was added in sperm freeze solution. Motility, viability and stress oxidative assessed with computer-aided sperm analysis (CASA), eosin-nigrosin staining, and H2DCFDA (2'7'-dichlorodihydrofluorescein diacetate) probe respectively. Data was analyzed with SPSS 18.5 software Results: Sperm motility and viability were significantly (p<0.05) higher in the test group (33.61±2.6, 45.63±1.95) compared to control group (33.94±2.52, 23.10±2.17) after freeze and thawed sample. In addition in Tempol group, stress oxidative insignificantly lower than control group (38.29±4.6 vs. 45.99±4.4 p>0.05). Conclusion: Tempol may preserve sperm motility and vitality after cryopreservation via inhibition of sperm membrane peroxidation. Tempol with anti-oxidant activity like super oxide dismutase function may decrease the level of ROS and hydrogen peroxide generation than control group. [ABSTRACT FROM AUTHOR]