The Protective Effects of Alpha Lipoic Acid on Human Sperm Function During Freezing-Thawing.

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.

Association between total globozoospermia and sperm chromatin defects.

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.

Cathepsin B inhibitor improves developmental competency and cryo-tolerance of in vitro ovine embryos.

BACKGROUND: Cathepsin B is a lysosomal cysteine protease involved in apoptosis and oocytes which have lower developmental competence show higher expression of Cathepsin B. Furthermore, expression of Cathepsin B show a decreasing trend from oocyte toward blastocyst stage. RESULTS: Present study assessed the effect of cathepsin B inhibitor, E-64, on developmental competency and cryo-survival of pre-implantation ovine IVF derived embryos. Cathepsin B inhibitor was added during day 3 to 8 of development. One µM E-64 was defined as the optimal concentration required for improving blastocyst rate. This concentration also reduced DNA fragmentation and BAX as apoptotic markers while increasing total cell number per blastocyst and improving anti-apoptotic marker, the BCL2. We further showed that addition of 1.0 µM of E-64 during day 3 to 8 of development improved re-expansion and hatching rates of blastocysts post vitrification. E-64 also reduced rate of DNA fragmentation and BAX expression and increased total cell number per blastocyst and BCL2 expression post vitrification. However, addition of E-64 post vitrification reduced the hatching rate. CONCLUSION: Therefore, it can be concluded that inhibition of cathepsin B in IVC, not only improves quality and quantity of blastocysts but also improves the cryo-survival of in vitro derived blastocysts.

Selection of physiological spermatozoa during intracytoplasmic sperm injection.

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.

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.

Long-term and efficient expression of human ß-globin gene in a hematopoietic cell line using a new site-specific integrating non-viral system.

Targeted integration of a therapeutic gene at specific loci in safe genomic regions by a non-viral vector can restore the function of the damaged gene. This approach also minimizes the potential genotoxic effects of transferred DNA. In this study, we have developed a non-viral vector that functions according to site-specific recombination (SSR). The vector contained a bacterial backbone and puromycin resistance gene (pur(r)), a ß-globin expressing cassette and an attB recombination site. We used phiC31 integrase to insert a copy of the vector into specific genomic locations of a human hematopoietic cell line. Site-specific integration of the vector with one or two copies in the transcriptionally active regions of the genome was confirmed. After genomic integration, we used Cre recombinase to remove the bacterial backbone and pur(r). This removal was verified by negative selection and genomic PCR screening. Following deletion of these sequences, the stable ß-chain expression was continued for several months in the absence of selective pressure. Consequently, this vector may potentially be a powerful tool for ex vivo correction of ß-globinopathies such as ß-thalassemia through successful genomic integration of a functional copy of the globin gene into the patient's target cells.

Glial cell line-derived neurotrophic factor in combination with insulin-like growth factor 1 and basic fibroblast growth factor promote in vitro culture of goat spermatogonial stem cells.

Growth factors are increasingly considered as important regulators of spermatogonial stem cells (SSCs). This study investigated the effects of various growth factors (GDNF, IGF1, bFGF, EGF and GFRalpha-1) on purification and colonization of undifferentiated goat SSCs under in vitro and in vivo conditions. Irrespective of the culture condition used, the first signs of developing colonies were observed from day 4 of culture onwards. The number of colonies developed in GDNF + IGF1 + bFGF culture condition was significantly higher than the other groups (p < 0.05). In contrast, the size of colonies developed in GDNF + EGF + LIF culture condition was significantly higher than the other groups (p < 0.05). Immunocytochemical stationing for specific biomarkers of somatic cells (vimentin, alpha-inhibin and α-SMA) and spermatogonial cells (PLZF, THY 1, VASA, alpha-1 integrin, bet-1 integrin and DBA) revealed that both cell types existed in developing colonies, irrespective of the culture condition used. Even though, the relative abundance of VASA, FGFR3, OCT4, PLZF, BCL6B and THY1 transcription factors in GDNF + IGF1 + bFGF treatment group was significantly higher than the other groups (p < 0.05). Additionally, goat SSCs developed in the latter culture condition could colonize within the seminiferous tubules of the germ-cell depleted recipient mice following xenotransplantation. Obtained results demonstrated that combination of GDNF with IGF1 and bFGF promote in vitro culture of goat SSCs while precludes uncontrolled proliferation of somatic cells.

A physiological, rather than a superovulated, post-implantation environment can attenuate the compromising effect of assisted reproductive techniques on gene expression in developing mice embryos.

Assisted reproductive techniques (ARTs) may perturb the pre-/peri-conception microenvironments, which subsequently threaten the health of offspring. This study aimed to investigate the effects of superovulation, vitrification, in vitro culture, and embryo transfer on the expression of epigenetic modulators, imprinted genes, and pluripotency markers in expanded blastocysts and Day-9.5 (D9.5) concepti. Results revealed that 53.4% (8/15) and 86.7% (13/15) of genes in the fetus and placenta, respectively, have similar patterns of transcription in all D9.5 concepti, despite the perturbed mRNA expression observed at the blastocyst stage for each embryo-production technique. These observations indicate a counterbalancing of the abnormal expression pattern analyzed at the blastocyst stage during post-implantation development, particularly when the uterus of a naturally synchronized foster mother is employed. Superovulation resulted in the most abnormal expression patterns compared to other treatment groups, although these same blastocysts were able to develop in a synchronized uterus. Thus, superovulation creates a hormonal environment that negatively affected gene expression and impairs fetal growth more adversely during post-implantation development than other ART protocols, such as in vitro culture, vitrification, or embryo transfer-although each did contribute negatively to the implantation and development process. Together, these results may have implications for treating infertility in humans.

Effect of epigenetic modification with trichostatin A and S-adenosylhomocysteine on developmental competence and POU5F1-EGFP expression of interspecies cloned embryos in dog.

Adult canine fibroblasts stably transfected with either cytomegalovirus (CMV) or POU5F1 promoter-driven enhanced green fluorescent protein (EGFP) were used to investigate if pre-treatment of these donor cells with two epigenetic drugs [trichostatin A (TSA), or S-adenosylhomocysteine (SAH)] can improve the efficiency of interspecies somatic cell nuclear transfer (iSCNT). Fluorescence-activated cell sorting (FACS), analyses revealed that TSA, but not SAH, treatment of both transgenic and non-transgenic fibroblasts significantly increased acetylation levels compared with untreated relatives. The expression levels of Bcl2 and P53 were significantly affected in TSA-treated cells compared with untreated cells, whereas SAH treatment had no significant effect on cell apoptosis. Irrespective of epigenetic modification, dog/bovine iSCNT embryos had overall similar rates of cleavage and development to 8-16-cell and morula stages in non-transgenic groups. For transgenic reconstructed embryos, however, TSA and SAH could significantly improve development to 8-16-cell and morula stages compared with control. Even though, irrespective of cell transgenesis and epigenetic modification, none of the iSCNT embryos developed to the blastocyst stage. The iSCNT embryos carrying CMV-EGFP expressed EGFP at all developmental stages (2-cell, 4-cell, 8-16-cell, and morula) without mosaicism, while no POU5F1-EGFP signal was observed in any stage of developing iSCNT embryos irrespective of TSA/SAH epigenetic modifications. These results indicated that bovine oocytes partially remodel canine fibroblasts and that TSA and SAH have marginal beneficial effects on this process.

Effects of heat shock during the early stage of oocyte maturation on the meiotic progression, subsequent embryonic development and gene expression in ovine.

Heat shock may affect different aspects of oocyte maturation and its subsequent development to the blastocyst stage. A series of in vitro experiments was performed to determine whether physiologically heat shock (41°C) disrupts the progression of the ovine oocytes through meiosis, activation and blastocyst formation. Cumulus-oocyte complexes (COCs) were aspirated from 2-6-mm follicles and cultured at 38.5°C (control) or 41°C (heat shock) for the first 12 h of maturation. The oocytes were incubated at 38.5°C during the last 10 h of maturation and 8 days after activation. Results showed that most of the oocytes matured under heat-shock conditions remained at the germinal vesicle breakdown (GVBD) stage and they showed an aberrant chromatin configuration. After heat shock, oocyte diameter and time spent for zona pellucida dissolution increased (P < 0.05). The heat-shocked group had a higher percentage of oocytes with incomplete migration of cortical granules (P < 0.05). The heat-shock condition decreased (P < 0.05) cleavage rates (56.19 versus 89.28%) and morula formation (26.85 versus 37.81%). However, there was no significant difference in blastocyst formation and percentage of hatched blastocysts. At 12 h, heat shock had an adverse effect on embryo quality and reduced inner cell mass number (P < 0.05). Quantitative gene expression analysis showed greater transcripts (P < 0.05) for Na/K-ATPase mRNA in heat-shocked oocytes. To sum up, heat shock has disruptive effects on ovine oocyte maturation and can impair cellular and molecular factors that are important for embryo development.

Effect of varicocelectomy on sperm functional characteristics and DNA methylation.

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.

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.

Sperm ubiquitination and DNA fragmentation in men with occupational exposure and varicocele.

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.

Optimization protocol for storage of goldfish (Carassius auratus) embryos in chilled state.

A series of five experiments were conducted to explore suitable conditions for storing of goldfish embryos in a chilled state. The factors studied were embryo stage, storage temperature, physiological saline solutions and goldfish artificial coelomic fluid (GFACF) medium, antibiotics (penicillin and streptomycin), antioxidants (vitamin E, vitamin C), buffer (Hepes, Tris) and BSA (bovine serum albumin). First, goldfish embryos at eight developmental stages were incubated in aerated and dechlorinated tap water at 0 °C for 24 h. Result shows that early developmental stages were most sensitive to chilling. Heartbeat-stage goldfish embryos were chilled at 0, 4 or 8 °C for up to 72 h in water, and chilled storage was possible only for up to 18, 24 and 48 h at 0, 4 and 8 °C, respectively, without a decrease in viability. Chilling of goldfish embryos at 8 °C in GFACF medium and Dettlaff's solution instead of water and other physiological saline solutions prolonged their viability (p < 0.01). Nevertheless, viability of chilled embryos in GFACF medium was slightly, but non-significantly, higher than in Dettlaff's solution. Supplementation of the GFACF medium with antibiotics, Hepes or BSA increased the viability of chilled embryos, but the tested vitamin E analogue Trolox, vitamin C or Tris concentration had no effect on embryo viability. The outcome of this series of experiments shows that heartbeat-stage goldfish embryos could be chilled for 60 h in GFACF supplemented with 25 mm Hepes, 100 U/ml penicillin, 10 µg/l streptomycin and 1 g/l BSA in such a way that embryonic development does not proceed, and viability is not lost.

Assisted oocyte activation overcomes fertilization failure in globozoospermic patients regardless of the DPY19L2 status.

STUDY QUESTION: Does DPY19L2 status influence intracytoplasmic sperm injection (ICSI) outcomes with or without assisted oocyte activation (AOA)? SUMMARY ANSWER: DPY19L2 mutations have no major impact on ICSI outcomes in globozoospermic patients. WHAT IS KNOWN ALREADY: Globozoospermia is a rare and severe teratozoospermia characterized by round-headed spermatozoa lacking an acrosome. Recently, it has been shown that DPY19L2 mutations can be found in a vast majority of, but not all, globozoospermic patients (66.7%). These patients suffer from primary infertility due to a sperm-related oocyte activation deficiency secondary to the absence of an acrosome that can be overcome by the application of AOA. STUDY DESIGN, SIZE, DURATION: Cohort study, retrospective, 34 patients, 83 cycles. MATERIALS, SETTING, METHODS: Clinical and biologic data were collected from 29 patients mutated for DPY19L2 and 5 non-mutated patients. In total, 35 ICSI cycles using AOA and 48 conventional ICSI cycles were included in the analysis. Patients were divided into groups according to whether or not they were mutated for DPY19L2 and whether or not they received AOA. MAIN RESULTS AND THE ROLE OF CHANCE: Regardless of the presence of a DPY19L2 mutation, the fertilization rates with AOA are restored to normal when compared with conventional ICSI in our cohort of globozoospermic patients. Also, when performing ICSI plus AOA, both mutated and non-mutated cases have similar positive hCG rates, ongoing pregnancy rates and live birth rates per transfer. On the contrary, the fertilization rate in globozoospermic patients using conventional ICSI is correlated with the presence of a DPY19L2 mutation, with slightly better, although still very low, fertilization rates in patients carrying a DPY19L2 mutation. Nevertheless, when performing conventional ICSI, both mutated and non-mutated cases have similar very low positive hCG rates, ongoing pregnancy rates and live birth rates per transfer. LIMITATIONS: A limitation of this study is the low number of included non-mutated cases. WIDER IMPLICATIONS OF THE FINDINGS: We propose a pathway for the clinical management of globozoospermic patients depending on the phenotype that includes several diagnostic and therapeutic steps. STUDY FUNDING/COMPETING INTEREST(S): None.

Nuclear transfer technique affects mRNA abundance, developmental competence and cell fate of the reconstituted sheep oocytes.

The effect of technical steps of somatic cell nuclear transfer (SCNT) on different aspects of cloned embryo development was investigated in sheep. In vitro-matured oocytes were enucleated in the presence or absence of zona and reconstituted by three different SCNT techniques: conventional zona-intact (ZI-NT), standard zona-free (ZF-NT) and intracytoplasmic nuclear injection (ICI-NT). Stepwise alterations in nuclear remodeling events and in mRNA abundances, throughput and efficiency of cloned embryo development and cell allocation of the resulted blastocysts were assessed. Early signs of nuclear remodeling were observed as soon as 2 h post-reconstitution (hpr) for fusion-based methods of nuclear transfer (ZI-NT and ZF-NT) but were not observable until 4 hpr with the ICI-NT method. The relative mRNA abundances of HSP90AA1 (HSP90), NPM2 and ATPase genes were not affected by i) presence or absence of zona, ii) oocyte enucleation method and iii) nuclear transfer method. After reconstitution, however, the relative mRNA contents of POU5F1 (OCT4) with the ZI-NT and ZF-NT methods and of PAPOLA (PAP) with ZF-NT were significantly lower than those for the ICI-NT method. Zona removal doubled the throughput of cloned blastocyst development for the ZF-NT technique compared with ZI-NT and ICI-NT. Cleavage rate was not affected by the SCNT protocol, whereas blastocyst yield rate in ICI-NT technique (17.0±1.0%) was significantly (P<0.05; ANOVA) higher than in ZF-NT (7.1±1.5%) but not in the ZI-NT group (11.2±3.3%). Despite the similarities in total cell number, SCNT protocol changed the distribution of cells in the blastocysts, as ZF-NT-cloned blastocysts had significantly smaller inner cell mass than ZI-NT. These results indicate that technical aspects of cloning may result in the variety of cloning phenotypes.

Kit ligand and glial-derived neurotrophic factor as alternative supplements for activation and development of ovine preantral follicles in vitro.

In vitro growth of preantral follicles has the potential to produce considerable numbers of competent oocytes for use in medicine, agriculture, and even wildlife conservation. The critical regulatory role of growth factors and hormones in the development of preantral follicles has been established. This study investigated the effect of glial-derived neurotropic factor (GDNF) and kit ligand (KL) on the in vitro development of ovine preantral follicles. Results indicated that both GDNF and KL significantly improved activation of primordial follicles, similar to co-addition of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), which are commonly used for in vitro follicular development. Importantly, GDNF had a more profound effect on follicle health, development, and differentiation compared with KL alone. Furthermore, the combination of GDNF and KL in the presence of EGF and bFGF had a positive, synergic effect on health, development, and differentiation of preantral follicles, as determined by histological and hormonal assessments. The results of this study may provide a foundation for further studies that will unravel the molecular mechanisms of follicular development to further improve the current status of in vitro preantral follicle culture.

Evaluation of HSPA2 in fertile and infertile individuals.

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.

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.

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.