Relationship between potential sperm factors involved in oocyte activation and sperm DNA fragmentation with intra-cytoplasmic sperm injection clinical outcomes

Objective: The present study aimed to simultaneously evaluate the association between expression of three potential factors [post-acrosomal sheath WW domain-binding protein [PAWP], phospholipase Czeta [PLCzeta], and truncated form of the kit receptor [TR-KIT]] as candidates of oocyte activation with fertilization rate and early embryonic development

Materials and Methods: In this experimental study, semen samples were collected from 35 intra-cytoplasmic sperm injection [ICSI] candidates and analyzed according to World Health Organization criteria [2010]. Each sample was divided into two parts. The first part was processed for insemination by density-gradient centrifugation [DGC] and the second part was prepared for assessment of sperm morphology [Papanicolaou staining], DNA fragmentation [transferase dUTP nick end labeling [TUNEL]], and three Sperm-borne oocyte-activating factor [s] [SOAFs]-PLCzeta, PAWP, and TR-KIT

Results: Significant positive correlations existed between the percentages of PLCzeta, PAWP, and TR-KIT with fertilization rate. In addition, significant negative correlations existed between the percentage of DNA fragmentation with the percentages of PLCzeta and PAWP. We did not find a relationship between percentages of PLCzeta, PAWP, and TR-KIT with embryo quality and pregnancy rate [P>0.05]. There was a significant negative correlation between percentage of DNA fragmentation with fertilization and embryo quality

Conclusion: Oocyte activation was associated with the studied sperm factors [PAWP, PLCzeta, and TR-KIT]. These factors might hold the potential to be considered as diagnostic factors in the assessment of semen samples to evaluate their potential to induce oocyte activation. In addition, we observed a significant association between DNA fragmentation with fertilization, as well as embryo quality and expression of PAWP and PLCzeta, which indicated that men with high degrees of DNA fragmentation might require artificial oocyte activation. Whether such action should take place, and its cost and benefits should be evaluated in the future

Chances to have a boy after gender selection by pre-implantation genetic screening are reduced in couples with only girls and without a boy sired by the male partner

Background: Gender selection and family planning have their roots in human history. Despite great interest in these fields, very few scientific propositions exist which could explain why some family do not attain the desired sex. Therefore, the aim of this study was to evaluate whether sex of previous child or children could affect the outcomes of pre-implantation genetic screening [PGS]

Materials and Methods: This historical cohort study including 218 PGS cases referring to Isfahan Fertility and Infertility Center [IFIC]. Couples were grouped as those who their male child passed away or her husbands' has a son[s] from their previous marriage [n=70] and couples who just have daughter [n=148]. Male normal blastocysts were transferred for both groups. The outcomes of PGS including pregnancy, implantation and abortion rates, along with possible confounding factors were compared between the two groups

Results: Significant differences in pregnancy, implantation and abortion rates were observed between couples whose their male partner had/has one boy [n=70] compared to those who have just girl[s] [n=148] despite similar number and quality of male normal blastocyst transferred in the two groups. Confounding factors were also considered

Conclusion: The Y- bearing spermatozoa in male partners with no history of previous boy have lower ability to support a normal development to term, compared to male partners with previous history of boy requesting family balancing

Altered expression of KLC3 may affect semen parameters

Background: KLC3 protein as a member of the kinesin light-chain protein family plays an important role in spermatogenesis, during formation of mitochondrial sheath in the mid piece of the sperm tail

Objective:This study for the first time aims to compare the expression of the KLC3 gene between fertile and infertile individuals

Materials and Methods:Semen samples were collected from 19 fertile individuals who were selected from embryo-donor volunteers and 57 infertile individuals who had abnormal sperm parameters according to world health organization criteria. Sperm parameters using computer assisted sperm analysis and the quantitative KLC3-gene expression using the real-time PCR method were measured

Results: Our results revealed a significant correlations between sperm concentration with relative expression of KLC3 only in infertile groups [r=0.45, p=0.00]. A significant correlation was not found between KLC3 expression and sperm motility; however, the relative expression of KLC3 was significantly higher in asthenozoospermic compared to non-asthenozoospermic individuals

Conclusion: Low expression of KLC3 may result in improper function of midpiece, which has important function in sperm motility. The results of this study show that aberrant expression of KLC3 might be associated with phenomena like oligozoospermia and asthenozoospermia. This article is extracted from student's thesis

Assessment of DPY19L2 deletion in familial and non-familial individuals with globozoospermia and DPY19L2 genotyping

Background: Globozoospermia is a rare syndrome with an incidence of less than 0.1% among infertile men. Researchers have recently identified a large deletion, about 200 kbp, encompassing the whole length of DPY19L2 or mutations in SPATA16 and PICK1 genes associated with globozoospermia. The aim of this study was to analyze the DPY19L2 gene deletion using polymerase chain reaction technique for the exons 1, 48, 11 and 22 as well as break point [BP] [a] in globozoospermic men

Materials and Methods: In this experimental study, genome samples were collected from 27 men with globozoospermia [cases] and 36 fertile individuals [controls], and genomic analysis was carried out on each sample

Results: Deletion of DPY19L2 gene accounted for 74% of individuals with globozoospermia. DPY19L2 gene deletion was considered as the molecular pathogenic factor for the onset of globozoospermia in infertile men. By quantitative real-time polymerase chain reaction [qPCR], we genotyped DPY19L2 deletion and identified carriers within the population

Conclusion: This technique may be considered as a method for family counseling and has the potential to be used as a pre-implantation genetic diagnosis, especially in ethnic community with high rate of consanguineous marriages

Zeta sperm selection improves pregnancy rate and alters sex ratio in male factor infertility patients: a double-blind, randomized clinical trial

Background: Selection of sperm for intracytoplasmic sperm injection [ICSI] is usually considered as the ultimate technique to alleviate male-factor infertility. In routine ICSI, selection is based on morphology and viability which does not necessarily preclude the chance injection of DNA-damaged or apoptotic sperm into the oocyte. Sperm with high negative surface electrical charge, named [Zeta potential], are mature and more likely to have intact chromatin. In addition, X-bearing spermatozoa carry more negative charge. Therefore, we aimed to compare the clinical outcomes of Zeta procedure with routine sperm selection in infertile men candidate for ICSI

Materials and Methods: From a total of 203 ICSI cycles studied, 101 cycles were allocated to density gradient centrifugation [DGC]/Zeta group and the remaining 102 were included in the DGC group in this prospective study. Clinical outcomes were com- pared between the two groups. The ratios of Xand Y bearing sperm were assessed by fluorescence in situ hybridization [FISH] and quantitative polymerase chain reaction [qPCR] methods in 17 independent semen samples

Results: In the present double-blind randomized clinical trial, a significant increase in top quality embryos and pregnancy rate were observed in DGC/Zeta group compared to DGC group. Moreover, sex ratio [XY/XX] at birth significantly was lower in the DGC/Zeta group compared to DGC group despite similar ratio of X/Y bearings sper- matozoa following Zeta selection

Conclusion: Zeta method not only improves the percentage of top embryo quality and pregnancy outcome but also alters the sex ratio compared to the conventional DGC method, despite no significant change in the ratio of Xand Ybearing sperm population

Evaluation of PLC[zeta] and PAWP expression in globozoospermic individuals

Objective: globozoospermia is a rare type of teratozoospermia with incidence of 0.1% among infertile individuals. Phospholipase C zeta [PLC[zeta]] and postacrosomal sheath WW domain binding protein [PAWP] are the main candidates in sperm taking responsibility for oocyte activation during fertilization. Therefore, we aimed to evaluate the expression of these two genes at RNA and protein levels in globozoospermic individuals and compare the results with fertile individuals

Materials and Methods: in this experimental study, semen samples of 21 infertile men with globozoospermia and 25 fertile men were collected. Expression of PLC[zeta] and PAWP at RNA and protein levels were assessed and compared between two groups by quantitative real time polymerase chain reaction [qPCR] and Western blot, respectively

Results: expression of both PLC[zeta] and PAWP were significantly reduced at RNA and protein levels in infertile men with globozoospermia compared to fertile men

Conclusion: this is the first study that simultaneously assessing the respective factors in a large population of globozoospermia, suggested that intra-cytoplasmic sperm injection [ICSI] along with artificial oocyte activation may rescue failed fertilization in routine ICSI

preliminary study: n-acetyl-l-cysteine improves semen quality following varicocelectomy

Background: Surgery is considered the primary treatment for male infertility from clinical varicocele. One of the main events associated with varicocele is excessive production of reactive oxygen species [ROS]. N-acetyl-L-cysteine [NAC], an antioxidant that scavenges free radicals, is considered a supplement to alleviate glutathione [GSH] depletion during oxidative stress. Despite beneficial effects of NAC in other pathological events, there is no report on the effect of NAC in individuals with varicocele. Therefore, the aim of this study is to evaluate the outcome of NAC on semen quality, protamine content, DNA damage, oxidative stress and fertility following varicocelectomy

Materials and Methods: This prospective clinical trial included 35 infertile men with varicocele randomly divided into control [n=20] and NAC [n=15] groups. We assessed semen parameters, protamine content [chromomycin A3 [CMA3]], DNA integrity [terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling [TUNEL]] and oxidative stress [2', 7'-dichlorodihydrofluorescein-diacetate [DCFH-DA]] before and three months after varicocelectomy

Results: Percentage of abnormal semen parameters, protamine deficiency, DNA fragmentation and oxidative stress were significantly decreased in both groups compared to before surgery. We calculated the percentage of improvement in these parameters compared to before surgery for each group, then compared the results between the groups. Only percentage of protamine deficiency and DNA fragmentation significantly differed between the NAC and control groups

Conclusion: The results of this study, for the first time, revealed that NAC improved chromatin integrity and pregnancy rate when administered as adjunct therapy post-varico-celectomy [Registeration Number: IRCT201508177223N5]

Selection of sperm based on hypo-osmotic swelling may improve ICSI outcome: a preliminary prospective clinical trial

The intra-cytoplasmic sperm injection [ICSI] technique selects sperm according to morphology and motility. However, these parameters cannot predict the chromatin integrity of sperm. Considering the detrimental effects of DNA-damaged sperm on reproductive outcomes, novel sperm selection procedures have been proposed to circumvent the possibility of inseminating DNA-damaged sperm. It has been shown that different potential hypo-osmotic swelling test [HOST] patterns possess the potential to differentiate between sperm that have intact or damaged chromatin. Therefore, for the first time, this preliminary study evaluates the role of HOST as a sperm selection procedure in a clinical setting. In this preliminary prospective clinical trial study, we divided infertile couples diagnosed with male infertility into two groups. In the treatment group [n=39], half of the oocytes were inseminated by sperm selected following density gradient centrifugation [DGC group]. The remaining oocytes from the treatment group were inseminated by sperm chosen according to HOST pattern [c, d or e] following DGC processing [HOST group]. In the control group [n=63], all oocytes were inseminated by sperm chosen after DGC. There was a significantly higher percentage of embryos that had good quality, implantation, and chemical pregnancy rates in the HOST group compared to the DGC group [p

Origin and role of DNA damage in varicocele

Varicocele is considered as one of the main etiologies of male infertility. Along with altered semen parameters, increased DNA fragmentation is believed to play an important role in varicocele-induced infertility. DNA damage may result from intra- or extra-testicular factors. Among these, apoptosis, abnormal chromatin packaging and oxidative stress are the most researched and are addressed in this review. Significant evidence suggests that varicoceles have a harmful effect on testicular function and a varicocelectomy not only prevents progressive decline in testicular function, but also reverses the damage. However, the degree to which varicocele repair improves pregnancy rates and the success of assisted reproductive technology [ART] remains controversial. Therefore, the role of varicocele repair on DNA fragmentation is also discussed

Semen parameters and chromatin packaging in microsurgical varicocelectomy patients

Varicocelectomy is considered as standard treatment for male infertility for clinical varicocele. The aim of this study is to address the effects of varicocelectomy on semen parameters, chromatin packaging, and pregnancy outcome. This retrospective study was carried out between June 2006 and February 2011 on 145 infertile men with grade II or III varicocele. Microsurgical varicocelectomy was performed as part of patient management. Sperm count, motility, morphology, and chromatin packaging were assessed with a Makler counting chamber, light microscopy, Papanicoulaou and chromomycin A3 [CMA3] staining, respectively. In addition, we assessed spontaneous clinical pregnancy and miscarriage rates. The percentages of spontaneous cumulative pregnancies post-surgery were 33.1% [3 months], 42.06% [6 months], 46.2% [9 months], 48.9% [12 months], and 55.8% [after 12 months]. Percentages of spontaneous cumulative miscarriage post-surgery were 2.46% [3 months], 4.93% [6 months], 4.93% [9 months], 6.17% [12 months], and 6.17% [after 12 months]. Both sperm parameters improved and the percentage of sperm protamine deficiency decreased significantly after varicocelectomy. These results confirm that varicocelectomy improves sperm parameters and chromatin packaging, thereby improving the chance of pregnancy. Positive aspects of this study include the large number of patients studied, duration of follow up, one surgeon who performed all of the surgeries, and type of surgery [microsurgery]. The spontaneous pregnancy results also suggest that if pregnancy is not achieved within twelve months post-surgery, an alternative approach such as assisted reproductive technology [ART] treatment should be considered.

Flow cytometry: a novel approach for indirect assessment of protamine deficiency by CMA3 staining, taking into account the presence of M540 or apoptotic bodies

Chromomycin A3 [CMA3] staining, either by the slide method or fluorescence microscopy, is widely used for indirect assessment of protamine deficiency in a semen sample. Flow cytometry is the most suitable tool to improve assessment accuracy, both in terms of statistical analysis and for prevention of observer variation. This study provides a simple procedure to account for merocyanine 540 [M540] or apoptotic bodies, which result in underestimation of the percentage of CMA3 positivity, by using propidium iodide [PI] staining. Therefore, this study aims to evaluate the percentage of CMA3 by PI staining to exclude M540 bodies that prevent underestimation of CMA3 staining. This study is an experimental study. Semen samples collected from 104 infertile men who referred to the Andrology Unit of the Isfahan Fertility and Infertility Center were initially assessed according to World Health Organization [WHO] criteria. Samples were washed twice with Ham's. Each sample was divided into two portions, a control and the other processed for density gradient centrifugation [DGC]. Each portion was assessed for CMA3 staining by both the slide and flow cytometry methods. Coefficients of correlation and student t-test were carried out using the Statistical Package for the Social Studies [SPSS 11.5]. Detection of CMA3 staining was more appropriate with fluorescence detector 3 [FL-3] rather than fluorescence detector 2 [FL-2] in the evaluation of protamine deficiency to exclude M540 bodies. This study, for the first time, provides the basis for assessment of CMA3 staining for flow cytometry. However, since the maximum excitation for CMA3 is not covered by the 488 nm laser, we recommend further experimentation using a flow cytometer with optimal excitation

Etiology and evaluation of sperm chromatin anomalies

Evidence suggests that human sperm chromatin anomalies adversely affect reproductive outcomes and infertile men possess substantially amount of sperm with chromatin anomalies than fertile men. Routine semen analysis evaluates parameters such as sperm motility and morphology, but does not examine the nuclear DNA integrity of spermatozoa. It has been suggested that altered nuclear chromatin structure or damaged DNA in spermatozoa could modify the special cellular functions of human spermatozoa, and thereby affect the fertility potential. Intra-cytoplasmic sperm injection [ICSI] bypass the barriers to fertilization for such a sperm, then the effect of chromatin anomalies on the development remains a concern. Therefore, it is essential to develop and use accurate diagnostic tests, which may provide better prognostic capabilities than the standard sperm assessments. This review discusses our current understanding of the structure and organization of sperm DNA, the different procedures for assessment of sperm chromatin anomalies including comet assay, Chromomycin A3 [CMA3], sperm chromatin structure assay [SCSA], acridine orange test [AOT], terminal TdT-mediated dUTP-nick-end labelling [TUNEL] assay, aniline blue and sperm chromatin dispersion [SCD] test and the impact of chromatin anomalies on reproductive outcome