Relationship between phospholipase C-zeta, semen parameters, and chromatin status

ABSTRACT

The need for additional tests to complement basic sperm analysis in clinics is well appreciated. In this regard, a number of tests such as sperm DNA integrity test as a tool in diagnosis and treatment of infertility are suggested. But recent studies have focused on main sperm factors involved in oocyte activation such as phospholipase C-zeta (PLCζ) that initiate intracellular Ca²⁺ signaling and embryogenesis. Therefore, this study aimed to investigate the relationship between PLCζ, basic semen parameters, sperm DNA fragmentation (SDF), and protamine deficiency in men with normal (n=32) and abnormal (n=23) semen parameters. Unlike SDF and protamine deficiency, as negative factors related to fertility, the mean value of PLCζ as positive factor related to infertility was significantly lower in men with abnormal semen parameters compared to men with normal semen parameters. Significant correlations were also observed between sperm concentration, motility, and abnormal morphology with the percentage of PLCζ positive spermatozoa. In addition, logistic regression analysis revealed that sperm morphology is more predictive than sperm motility and concentration for PLCζ presence. In addition, a statistically significant negative relationship was observed between the percentage of PLCζ positive spermatozoa and SDF. These findings suggested during ICSI, selection of sperm based on morphology has a profound effect on its ability to induce oocyte activation based on the likelihood of PLCζ expression. Therefore, assessment of PLCζ as an index for fertilization potential of a semen sample in men with severe teratozoospermia may define individuals who are candidates for artificial oocyte activation (AOA) and may avoid failed fertilization post ICSI.

KEYWORDS:

• DNA fragmentation
• male infertility
• PLCζ
• protamine deficiency
• semen parameters

Introduction

An initial step in the evaluation of male factor infertility is based on non-invasive examination of gross sperm characteristics including concentration, motility, and morphology [Evgeni et al. 2015]. Semen analysis provides useful information regarding the status and function of testicles and epididymis, and helps the clinician with the management of infertility owing to the significant relationship established between semen parameters and conception rate in several studies [Baker and Sabanegh 2015; Hamada et al. 2012; WHO 2010]. However, the credibility of basic semen analysis to determine the underlying mechanisms of male factor infertility is limited as has been estimated. Indeed 15% of men with normal semen analysis profiles are infertile [Agarwal and Allamaneni 2005; Macanovic et al. 2015].

Moreover, numerous studies have shown overlap between the basic semen parameters of fertile and infertile men [Guzick et al. 2001; Lewis 2007]. Furthermore, important subtle aspects of sperm function (including DNA/protamine integrity, capacitation, acrosome reaction, sperm-oocyte interaction, and oocyte activation) are often obscured by mere evaluation of basic semen profile. Therefore, complementary sperm functional tests have been considered as indispensable in addition to basic semen analysis for diagnostic-therapeutic evaluation of male factor [Bieniek et al. 2016; Evgeni et al. 2015; Hebles et al. 2015; Speyer et al. 2015].

Sperm DNA fragmentation (SDF) among the well-documented factors is one of the indicators of probable male infertility. It has adverse impact on natural conception, and pregnancy outcomes following in vitro fertilization (IVF) and intra-cytoplasmic sperm injection (ICSI) [Khadem et al. 2014; Zini et al. 2008]. Generally, abnormal alterations occurring during sperm chromatin configuration or histone-to-protamine exchange can lead to SDF [Lewis et al. 2013]. Although Hamada et al. [2012] reported that SDF may be observed in 5–8% of men with normal semen parameters [Hamada et al. 2012], several studies have demonstrated a relationship between abnormal semen parameters and the percentage of sperm with DNA-damage [Moskovtsev et al. 2009; Zini et al. 2001]. In addition, numerous studies showed that protamine deficiency and DNA fragmentation in the bull sperm are also associated with reduced fertility [Ahmed et al. 2016; Dogan et al. 2015]. Therefore, assessment of SDF is an important attribute of semen quality in infertile men and provides useful information for diagnostic-therapeutic evaluation of male factor infertility [Bucar et al. 2015; Drobnis and Johnson 2015; Dada et al. 2011; Lewis et al. 2013; Majzoub et al. 2016; MalićVončina et al. 2016; Talebi et al. 2008].

Oocyte activation is initiated when a fertilizing sperm delivers sperm-borne oocyte activating factor(s) (SOAF) into the oocyte cytoplasm [Dozortsev et al. 1997; Kashir et al. 2015]. This factor(s) in the oocyte initiates a series of Ca²⁺ oscillations which could significantly influence fertilization, implantation, and embryo development [Ozil et al. 2006]. Previous studies have demonstrated that fertilization failure following ICSI could be mainly due to a sperm’s inability to induce oocyte activation [Sousa and Tesarik 1994]. A number of proteins have been proposed as SOAF candidates including PLCζ and PAWP. Despite extensive research and common functional similarities between the two molecules [Amdani et al. 2015; Machaty 2016], conclusions derived from a recent knockout animal model [Satouh et al. 2015], and data presented by Nomikos et al. [2014] regarding the inability of human PAWP to induce Ca²⁺ oscillation in mouse oocyte has questioned the role of PAWP as being the main candidate of SOAF.

PLCζ is expressed during early stages of spermiogenesis and is localized in the acrosomal, equatorial, and post-acrosomal regions of the sperm head and also the tail [Grasa et al. 2008; Heytens et al. 2009; Kashir et al. 2015; Nomikos 2015]. Microinjection of PLCζ cRNA or its recombinant protein into the mammalian oocyte initiates calcium oscillations and subsequent early embryonic development [Sanusi et al. 2015; Saunders et al. 2002]. In this regard, Kashir et al. [2013] and others [Aghajanpour et al. 2011; Ramadan et al. 2012; Yelumalai et al. 2015] demonstrated that the percentage of sperm exhibiting or expressing PLCζ was significantly lower in infertile men with failed oocyte activation compared to fertile men. From these studies PLCζ can be considered as a useful marker for evaluation of sperm to induce oocyte activation following ICSI.

The expression and localization of PLCζ takes place concomitant with late spermiogenic events such as histone-protamine remodeling involved in maintenance of sperm chromatin integrity. These events can simultaneously or independently affect fertility outcomes. Therefore this study aimed to evaluate PLCζ, SDF and histone/protamine exchange in individuals with normal and abnormal semen parameters.

Results

Comparison of conventional semen parameters

The mean age of men with normal and abnormal semen parameters were 33.18±1.31years and 31.01±1.7 years, respectively, with nonsignificant difference. The mean sperm concentration, sperm motility, and semen volume were significantly higher, and the percentage of abnormal sperm morphology was significantly lower in men with normal semen parameters than men with abnormal semen parameters (Table 1).

Table 1. Comparison of semen parameters between men with normal (n=32) and abnormal (n=23) semen parameters.

Parameters	Normal semen parameters	Abnormal semen parameters	P-value
Sperm concentration (10^6/ml)	74.84±5.21	26.82±4.62	0.001
Total sperm count (10^6/ejaculate)	350.72±30.78	104.54±22.52	0.001
Total sperm motility (%)	62.65±2.08	35.56±3.46	0.001
Progressive sperm motility (%)	39.77±1.98	14.41±2.42	0.001
Abnormal sperm morphology (%)	95.43±0.21	98.26±0.20	0.001
Semen volume (ml)	5.12±0.38	3.63±0.31	0.004

Data are presented as mean ± SEM and analyzed by independent-samples t-Test.

Comparison of PLCζ and sperm function tests

Percentages of SDF were 26.47%±3.2 and 14.82%±1.22 in men with abnormal and normal semen parameters, respectively. The mean of SDF was significantly higher in men with abnormal semen parameters compared to men with normal semen parameters (p≤0.001). Similarly, a significant difference (p<0.001) was observed in percentages of protamine deficient spermatozoa between men with abnormal semen parameters (41.13%± 2.56) and men with normal semen parameters (29.25% ± 1.53) (Figure 1). The mean percentage of PLCζ positive spermatozoa was also significantly (p≤0.001) lower in men with abnormal semen parameters (30.04% ± 3.82) compared to men with normal semen parameters (52.82% ± 4.68), respectively (Figure 1). Percentages of PLCζ positive spermatozoa in two semen samples with normal and abnormal semen parameters are shown in Figure 2A and B. In addition, fluorescence images of sperm stained for protamine deficiency and SDF are shown in Figure 2C and D.

Figure 1. Comparison of mean percentage of sperm PLCζ, SDF, and protamine deficiency between men with normal and abnormal semen parameters. The mean percentage of PLCζ positive spermatozoa was significantly lower, and percentage of SDF and protamine deficient spermatozoa were significantly higher in men with abnormal semen parameters compared to men with normal semen parameters, respectively. SDF: sperm DNA fragmentation; PLCζ: phospholipase C-zeta. # shows significant difference between the two groups at p<0.05.

Figure 2. Flow cytometry analysis of percentages of PLCζ positive spermatozoa in two semen samples with normal (A) and abnormal (B) semen parameters. Percentage of PLCζ positive spermatozoa was higher in individuals with normal parameters compared to abnormal parameters. Fluorescence images of spermatozoa stained with TUNEL (C) and CMA3 staining (D) for DNA fragmentation and protamine deficiency, respectively. Spermatozoa with green staining (TUNEL⁺) and bright yellow staining (CMA³⁺) were considered as protamine deficient and DNA fragmented, respectively. PLCζ: phospholipase C-zeta; CMA3: chromomycin A3; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labelling.

Correlation between percentage of PLCζ and sperm function tests with semen parameters

As depicted in Table 2, significant correlations were observed between sperm concentration with percentage of protamine deficient and PLCζ positive spermatozoa. In addition, significant correlations were observed between the percentage of abnormal sperm morphology with the percentages of SDF, protamine deficient, and PLCζ positive spermatozoa. Furthermore, statistically significant correlations were observed between the percentage of total sperm motility with the percentages of SDF, protamine deficient, and PLCζ positive spermatozoa.

Table 2. Correlation between percentage of spermatozoa presenting DNA fragmentation, protamine deficiency, and PLCζ with sperm concentration, sperm motility, and sperm abnormal morphology (n=55).

Parameters	Concentration (%)		Motility (%)	Abnormal morphology (%)
	R	0.476**	0.430**	-0.317*
PLCζ positive spermatozoa (%)	P	0.001	0.001	0.012
	R	-0.173	-0.629**	0.434**
DNA fragmented spermatozoa (%)	P	0.207	0.001	0.001
	R	-0.343*	-0.342*	0.475**
Protamine deficient spermatozoa (%)	P	0.011	0.011	0.001

PLCζ: phospholipase C-zeta. **Correlation is significant at the 0.01 level (2-tailed); *Correlation is significant at the 0.05 level (2-tailed).

The percentage of PLCζ positive spermatozoa shows significant correlation with sperm concentration, motility, and abnormal morphology. Therefore, a multiple regression analysis was carried out between the three parameters and the percentage of PLCζ positive sperm and the data showed that the sperm morphology was more predictive than the other two semen parameters for PLCζ (Table 3).

Table 3. Multiple regression analysis between sperm parameters and percentage of PLCζ positive spermatozoa.

	Adjusted OR (95% CI)	P-value
Sperm concentration	1 (0.97-1.08)	0.33
Sperm motility (%)	1 (0.95-1.11)	0.461
Abnormal sperm morphology (%)	0.25 (0.08-0.78)	0.018
PLCζ positive spermatozoa (%)
%≤50	0.4 (0.04-4.8)	0.49
%>51^ref	-	-

PLCζ: phospholipase C-zeta. Dependent variable in multinomial logistic regression with two categories; abnormal parameters were considered as reference category. OR: odd ratio; 95% CI: 95% confidence interval.

In addition, there was a statistically significant negative correlation between percentages of SDF and PLCζ positive spermatozoa (r=-0.3; p=0.026). But, no association was observed between the percentage of PLCζ (r=-0.05; p=0.702) and protamine deficient positive spermatozoa (Figure 3). Also a significant correlation was observed between percentages of SDF and protamine deficient positive spermatozoa (r = 0.3; p= 0.023).

Figure 3. Correlation between percentage of PLCζ with DNA fragmentation (r = -0.3; p=0.026) and protamine deficiency (r= -0.05; p=0.702) in total population (32 men with normal semen parameters and 23 men with abnormal semen parameters). PLCζ: phospholipase C-zeta.

Discussion

Analysis of semen parameters is the initial step in the evaluation of male factor infertility and over 80% of couples with male factor infertility are recognized with impaired spermatogenesis of unknown etiology [Flaherty et al. 1998; Mahutte and Arici 2003]. However, recently there is an increasing requirement for assessment of other sperm functional indices, especially genomic integrity in addition to semen analysis for diagnostic-therapeutic evaluation of male factor infertility [Drobnis and Johnson 2015; Dada et al. 2011; Lewis et al. 2013; MalićVončina et al. 2016; Majzoub et al. 2016; Talebi et al. 2008]. In couples with male infertility, ICSI can achieve a high fertilization rate as the first option offered. However, 3–5% of infertile couples face total failed fertilization following ICSI which has devastating emotional and financial effects [Esfandiari et al. 2005]. To overcome this inadequacy, artificial oocyte activation (AOA) can be implemented following ICSI [Ebner et al. 2015; Eldar-Geva et al. 2003 Montag et al. 2012; Nasr-Esfahani et al. 2008a; Nasr-Esfahani et al. 2010]. Recent studies unequivocally show that PLCζ is the main molecular mediator of oocyte activation [Kashir et al. 2015; Sanusi et al. 2015; Swann et al. 2006]. Therefore, in this study we assessed the relationship between PLCζ with semen parameters and chromatin status. The current study demonstrated that the percentage of PLCζ positive spermatozoa reached more than half of the sample and was significantly higher in men with normal semen parameters compared to their abnormal counterpart. In addition, the mean male ages were not different between the groups. This result is in agreement with a recent study by Yeste et al. [2016] demonstrating that “…male age is unlikely to cause problems in terms of the sperm’s fundamental ability to activate an oocyte”. Concomitant with literature, this data suggest that despite injection of sperm into the oocyte during ICSI, lack of oocyte activation may account for failed or low fertilization following ICSI in some ICSI cases [Bukulmez et al. 2000; Sunkara et al. 2011]. However, the effect of other confounding factors like SDF, protamine deficiency, and DNA methylation should not be ignored [Bahreinian et al. 2015; Simon et al. 2014; Tavalaee et al. 2009; Tavalaee et al. 2015].

Implementation of complementary sperm functional tests, in addition to its cost, is not an easy task. Therefore, in order to determine the infertile men that may benefit from the assessment of PLCζ, the relationship of this factor with semen parameters was evaluated. The present results revealed significant correlations between PLCζ with the three main semen parameters. The order of this relationship was higher with sperm concentration, then sperm motility, and finally sperm morphology. However, to see which of these parameters has a significant effect on the prediction of expression of PLCζ, we carried out multinomial logistic regression analysis using a cut off value of 50% PLCζ positive spermatozoa, based on the mean value for men with normal parameters. Despite a higher correlation between PLCζ with sperm concentration and motility, only sperm morphology appeared to have a significant predicative effect on the expression of PLCζ. These results further reiterate that proper selection of sperm based on morphology may have a profound effect on its ability to induce oocyte activation based on likelihood of PLCζ expression. This is consistent with previous literature referring to severe teratozoospermic samples, especially infertile men with globozoospermia, are suitable candidates of AOA [Mansour et al. 2009; Moaz et al. 2006; Nasr-Esfahani et al. 2008a]. Therefore, this data suggest that further studies are required in a larger population to define the cut-off point for expression of PLCζ in order to implement AOA.

Presence and location of PLCζ depends on proper expression of this gene at both the mRNA and protein levels. One of the factors affecting the transcription of this factor is the burden of oxidative stress faced during spermiogenesis [Park et al. 2015]. In order to evaluate the effect of oxidative stress on the expression of PLCζ, we analyzed the relationship between the expression of PLCζ and SDF as an indirect consequence of oxidative stress. The results revealed a significant correlation between these two parameters. In addition, the percentage of SDF was significantly higher in men with abnormal semen parameters compared to their normal counterpart. This indicated that sperm presenting a high degree of DNA damage or oxidative stress were less likely able to induce oocyte activation. These results are consistent with previous literature [Park et al. 2015;Yassine et al. 2015] and those indicating that the rate of fertilization is lower in sperm presenting a high degree of DNA fragmentation [Simon et al. 2014]. Despite this result, further studies are required to assess a direct relationship between oxidative stress and the expression of PLCζ. Perhaps, antioxidant therapy which has been shown to improve DNA integrity [Abad et al. 2013; Dattilo et al. 2014; Tunc and Tremellen 2009] may also have a positive effect on the percentage of sperm expressing PLCζ. The advantages of flow cytometry include that it is rapid and reproducible, and offers objectivity, accuracy, and statistical analysis power over microscopy. The sperm chromatin structure assay as a reliable test for assessment of DNA fragmentation in human and animal spermatozoa by flow cytometry is well established [Evenson et al 2016]. One of the limitations of our study was assessment of sperm DNA fragmentation by florescence microscope, since we had to perform several sperm functional tests simultaneously.

Unlike SDF, a significant correlation between the level of protamine deficiency and PLCζ positive spermatozoa was not obtained, despite that the mean value for protamine deficiency was different between the two groups. These data indicate that despite the role of protamine deficiency in progression spermatozoa to DNA fragmentation, it has no direct relation with the expression of PLCζ.

For ICSI, semen samples are processed and to a certain degree different populations of sperm are separated from each other. In this regard, Kashir et al. [2011] demonstrated that density-gradient washing could separate spermatozoa with high PLCζ content. Therefore, proper sperm processing and novel sperm selection procedures may pave the way to reduce failed or low fertilization post ICSI [Bucar et al. 2015, Kashir et al. 2011, Nasr-Esfahani et al. 2008b; Nasr-Esfahani et al. 2012; Nasr-Esfahani and Tavalaee 2013].

Overall the results of this study suggest that PLCζ which is required for oocyte activation is not detectable in all spermatozoa and this may account for failed fertilization post ICSI in some oocytes. This effect is substantiated in teratozoospermic individuals and raises more concern. Additional measures are required when carrying ICSI for these couples. To overcome this inadequacy, AOA can be implemented but there is also an increasing requirement to avoid unnecessary AOA as the long term effects of these procedures need to be evaluated, despite several studies emphasizing safety of this procedure [Deemeh et al. 2015; D’haeseleer et al. 2014; Vanden Meerschaut et al. 2014]. Therefore, certain criteria are required to select the correct candidate for AOA. For example, successful ICSI has been reported for globozoospermia with normal ICSI or ICSI along with AOA. Success rate was substantially higher when AOA was implemented along with ICSI [Chansel-Debordeaux et al. 2015; Deemeh et al. 2015]. Indeed, in this study and also in previous studies [Kashir et al. 2013; Lee et al. 2014] some of normozoospermic individuals or fertile men presented low percentage of sperm with positive PLCζ. Therefore, the results of the present study suggest that assessment of PLCζ, especially in cases of severe teratozoospermic individual or in couples with previous repeated low fertilization rate, prior to ICSI may pave the way to avoid unnecessary AOA. In addition, implementation of this test along with semen analysis in the future may help to avoid low or failed fertilization in first cycle ICSI-candidates but to reach this conclusion further multi-central studies are required.

Materials and methods

This study was approved by the review board of the Royan Institute and Isfahan Fertility and Infertility Center (IFIC). Written informed consent to share the results of semen analyses for research purposes was obtained from all couples.

Sample selection

Semen samples were collected from men based on their semen analysis outcome according to WHO [2010] guidelines from December 2015 to April 2016. Samples with sperm concentration ≥15million per ml, percentage of sperm total motility higher than 40% and/or percentage of abnormal morphology lower than 96% were defined for individuals with normal semen parameters (n=32). While semen sample analysis under these thresholds were considered as “abnormal” (n=23). Individuals with leukocytospermia, varicocele, fever approximately 90 d prior to the seminal analysis, excessive alcohol and drug use, urogenital infections, Klinefelter’s syndrome, and cancer were excluded in this study. In addition, semen samples with greater than one million white blood cells (WBC) or other cell types were also excluded from our study.

Semen analysis

Ejaculated semen was obtained by masturbation into a sterile plastic container after 3–5 d of abstinence and was allowed to liquefy at room temperature. Briefly, sperm concentration, sperm motility, and sperm morphology were evaluated using a sperm chamber (Sperm meter; Sperm Processor, Aurangabad, India), Computer Assisted Semen Analysis (CASA, Video Test, Ltd: version Sperm 2.1, Russia), and Papanicolaou staining, respectively. After mixing the raw sample, semen was applied to the sperm chamber and 5 fields of ten squares were evaluated from 2 drops in each sample. Percentage of sperm motility was determined by calculating the mean value from the two drops. For assessment of sperm morphology, at least 200 spermatozoa per sample were examined at x100 magnification. The remaining of each semen sample was used for the other tests.

Assessment of PLCζ by flow cytometry

Evaluation of PLCζ was performed according to modified protocols by Grasa et al. [2008]. All the procedures were carried out at room temperature unless otherwise stated. Briefly, semen samples were washed by centrifugation (300g, 5 min) in phosphate buffered saline (PBS; Merck, Darmstadt, Germany) and the pellets were fixed in 4% paraformaldehyde (Sigma, Saint Louis, MO, USA) for 30 min at room temperature. Then, sperm pellets were washed twice with PBS for 5 min at 300g. For permeabilization, spermatozoa were treated with 0.5% Triton X-100 (Merck) for 30 min at room temperature, followed by washing twice with PBS. Sperm pellets were then incubated with a solution of 3% bovine serum albumin (BSA; Sigma) in PBS for 1 h to block non-specific binding sites. Then, affinity-purified anti-human polyclonal antibody (PLCζ (1:500); Covalab, Villeurbanne, France) prepared in PBS containing 1% BSA were applied overnight at 4°C. Samples were then washed twice with PBS and incubated with goat anti-rabbit IgG secondary antibody FITC conjugated (1:100; Sigma) for 60 min at 37°C. Ultimately, the samples were washed twice with PBS followed by incubation with propidium iodide (PI: 1 μg/ml; Sigma) for sperm DNA staining. The samples were analyzed by a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA) equipped with a 488 nm argon-ion laser for excitation, at a flow rate of <300 cells/s. Then, gating of the sperm population was used to select sperm population from clumps and debris in the forward light scatter/side light scatter (FSC/SSC) dot plot. At least 10,000 sperm was counted in each sample. Green fluorescence (FITC) was detected in the fluorescence detector 1 (FL-1) with a 530/30 nm band pass filter, and red fluorescence (PI) was measured in the fluorescence detector 2 (FL-2) with a 585/42 nm band pass filter.

Assessment of SDF using TUNEL assay

SDF was assessed using a detection kit (Apoptosis Detection System Fluorescein; Promega, Mannheim, Germany). All the procedures were carried out at room temperature. Briefly, semen samples were washed twice in PBS pH 7.4. A droplet of prepared sperm suspension was smeared on glass microscopic slides, air-dried, and fixed by 4% paraformaldehyde for 25 min. Then, the slides were washed with PBS, treated with 0.2% Triton X-100 for 5 min and washed again with PBS. TUNEL-staining procedure was carried out according to the manufacturer’s instructions. For each sample, at least 200 sperm cells were randomly assessed using an epifluorescent microscope (Olympus: BX51, Tokyo, Japan) equipped with the appropriate filters (460–470 nm) at x100 magnification. The percentage of green fluorescing (TUNEL-positive) sperm cells was calculated as for SDF rate [Kheirollahi-Kouhestani et al. 2009].

Indirect assessment of protamine deficiency by chromomycin A3 (CMA3) staining

All the procedures were carried out at room temperature unless otherwise stated. Washed samples were fixed in Carnoy’s solution (methanol: glacial acetic acid (3:1); Merck) at 4°C for 5 min. Smears were prepared and each slide was treated for 20 min with 100 µl of CMA3 (Sigma) solution (0.25 mg/ml in McIlvaine buffer (7 ml citric acid; Sigma) 0.1 M, 32.9 ml Na₂HPO₄.7H₂O 0.2 M (Merck), pH 7.0, containing 10 mM MgCl₂ (Sigma)). Then, the slides were washed two or three times with PBS and mounted with buffered glycerol (Merck) and were evaluated using a fluorescent microscope (Olympus: BX51) with the appropriate filters (460–470 nm) [Nasr-Esfahani et al. 2004]. CMA3-positive (or protamine deficient) sperm cells were defined as having a light yellow stain, whereas CMA3-negative sperm (or sperm with a normal amount of protamine) were defined as having a dark yellow stain. On each slide, at least 200 sperm were evaluated.

Statistical analysis

Microsoft Excel and Package for the Social Studies (SPSS Science, Chicago, IL, USA) was used to analyze data. Skewness value was used to assess normal distribution. We used independent – samples t test to compare the mean value between different groups. Two-tailed Pearson correlation test was used to assess correlations between parameters. Multinomial logistic regression was used to investigate whether semen parameters had a significant effect on prediction of PLCζ expression. Results were expressed as mean ± SEM and differences with values of p < 0.05 were considered as statistically significant.

Declarations of interest

This study was supported by the Royan Institute. None of the authors have any conflicts of interest to disclose and all authors support submission of this manuscript to this journal.

Acknowledgments

This study was supported by Royan Institute and we would like to express our gratitude to staff of Isfahan Fertility and Infertility for their full support. We would like to thank Dr. Saber Khazaei for his guidance in data analysis of this manuscript.

Additional information

Notes on contributors

Mohammad H. Nasr-Esfahani

Conception, design, data analysis, interpretation, manuscript writing, and final approval of manuscript: MHN-E; Conception, design, collection and/or assembly of data, data analysis, interpretation, manuscript writing, and final approval of manuscript: MT. Flow cytometric analysis: AK-E.