The effect of Phoenix dactylifera pollen on the expression of NRF2, SOD2, CAT, and GPX4 genes, and sperm parameters of fertile and infertile men: A controlled clinical trial

Abstract Background Oxidative stress is caused by the imbalance occurring between the creation and clearance of the reactive oxygen species (ROS), which is responsible for 30–40% of male infertility. The positive impact of phoenix dactylifera pollen (Date palm pollen, DPP) on the improvement of sperm parameters has been well documented in animal models. Objective For evaluating the effect(s) of DPP on sperm parameters, ROS levels, expression of antioxidant genes, and activity of antioxidant enzymes of infertile men. Materials and Methods In this controlled clinical trial, a total of 60 male case with infertility and 20 normospermic fertile men were recruited. Before and after the treatment with DPP, the case were administered 400 mg/kg of gelatinous capsules daily for 30 consecutive days and semen samples were taken. Quantitative real-time polymerase chain reaction was applied for the evaluation of the mRNA expression levels of Nuclear factor erythroid 2-related factor 2(NRF2), superoxide dismutase (SOD2), glutathione peroxidase 4(GPX4), and catalase (CAT) genes. Results The mRNA expression levels of NRF2, SOD2, GPX4, and CAT (p < 0.05 for all) and significantly increased after treatment with DPP. The increased expressions of all antioxidant genes and enzymes significantly correlated with improvement in semen parameters including count (p = 0.01), motility (p = 0.05), and morphology (p = 0.01) of sperm. A significant correlation between the alteration of SOD2 gene expression and SOD activity, GPX4 and GPX, and CAT were also observed (p = 0.05). Conclusion DPP can increase the expressions of NRF2, GPX4, SOD2, and CAT genes and also improve the semen quality in infertile men.


Introduction
As stated by the World Health Organization (WHO) guidelines, male infertility factors are responsible in roughly half of these couples (1). Moreover, male infertility has been proposed as one of the multifactorial disorders due to environmental, genetic, non-genetic factors, or a combination of these (2,3). Several factors including structural reproductive tract abnormalities, mutations in mitochondrial DNA, chromosomal disorders, endocrine disturbances, hypogonadism, erectile dysfunction, chronic illnesses, several medications, exposure to radiation, reproductive tract infections, and cryptorchidism have previously been proposed as causes of male infertility (4,5). However, the male infertility is still unknown in approximately 50% of the reported cases (4).
Accumulating evidence has shown oxidative stress (OS) as a major cause of the male infertility (6). OS has been reported to play an independent role as it has been considered as one of the conditions reflecting an imbalance between the production of oxygen-derived free radicals called ROS and the body's ability to the quenching of the ROS (7). In normal physiological conditions, ROS is generated at a low level in spermatozoa and plays a fundamental role in various biological mechanisms, namely capacitation, hyperactivation, and motility of sperms, acrosome reaction, and subsequent sperm-oocyte fusion and fertilization (8). Increased levels of ROS can pose a threat to sperms by causing peroxidation of sperm cellular membrane lipids, damage to sperm DNA integrity, decrease in sperm motility, oocyte-sperm fusion efficacy, and overall semen quality. Therefore, ROS plays a dual role as both deleterious and beneficial depending on their concentration in spermatozoa (9). Moreover, enzymatic antioxidants like SOD, CAT, and GPX, which are abundant in the midpiece, make up a great proportion of human sperms (10). Nuclear factor erythroid 2-related factor 2 (NRF2), as a key protein in antioxidant defense system, acts as nuclear transcriptional factor and regulates the expressions of GPX, SOD, and CAT genes (11). Altered expressions of NRF2, GPX4, SOD2, and CAT genes can be associated with increased OS, which could contribute to impaired fertilization capacity and male infertility (12).
Experts have employed herbal remedies for treating various diseases especially male infertility since ancient times (13). Because of various antioxidants constituents, Phoenix dactylifera [Date Palm Pollen (DPP)], a good source of hormones, minerals, enzymes, vitamins, proteins, and fatty acids, has been used as an herbal remedy in the treatment of male infertility but very few data are available about its molecular nature (14). Besides, its efficacy has been studied in animal models (15), but few studies have addressed the effectiveness of DPP on humans. In a very recent study, DPP administration was proven effective on testosterone and follicle-stimulating hormone (FSH) levels, as well as sperm motility; however, the subjects of this study were sub-fertile (16). In addition, although some studies have evaluated the effect of DPP on sperm parameters (17), no previous study has addressed the influence of DPP on male fertility at genomic level. Therefore, this research aimed at the determination of the impact(s) of DPP on the levels of mRNA expression of antioxidant genes (NRF2, GPX4, SOD2, and CAT) in infertile male. diagnosed with male infertility who were referred to the Om-e-Leila Fertility and Infertility Center, Om-e-Leila Hospital, Bandar Abbas, Iran, during 2016 (March)-17 ( June) (Figure 1). We excluded the case with these criteria from the research: history of genetic and systemic disorders, reproductive tract abnormality, testicular trauma, alcohol and substance abuse, and fertility medications at least for six months before participation. DPP powder 400 mg/kg in gelatinous capsules was used to treat the eligible subjects daily for 30 consecutive days and all of the case finished the study course. DPP powder was purchased from palm farmers in autumn and formed into capsules in the laboratory. Additionally, 20 fertile males who had fathered a child within the last 2 yr and their semen samples were recruited as the controls. Initially, the controls comprised of the same number of case as the case group, however, due to the inconvenience of sample collection for semen analysis, some case failed to cooperate and were excluded from the study. At the time of enrollment, demographic data were obtained from all case using a structured interview form.

Sample collection and isolation of spermatozoa from seminal fluid
Semen samples were obtained twice from cases, before and after the treatment period 30 consecutive days, and once from controls. Then, we collected the fresh semen samples in the sterile plastic containers by masturbation after three to five days of sexual abstinence. Then, we liquefied the samples at 37°C for 30 min and semen analysis (semen volume, pH, viscosity, sperm morphology, concentration, and motility) was immediately performed with the use of the Sperm Quality Analyzer IIC (SQA IIC, United Medical Systems Inc., Santa Ana, CA, USA) based on the WHO guidelines (18). In addition, in this randomized, single-blind, and comparative clinical trial, Semen analysis was performed by the blind technician. After purification of spermatozoa by Goodrich methods (19), BSA-free Ham's-F10 medium was used to wash the samples twice and then stored in RNALater solution (Qiagen: Germany) at a temperature of -80°C until RNA extraction. Sperms with smooth, rimmed, ovalshaped heads, 2.5-3.5 µm wide and 5-6 µm long, free of large vacuoles, an acrosome that covers 40-70% of the head, a midpiece of nearly the same length as the head but much slimmer, and un-coiled 45 µm-long tail thinner than the head and midpiece, and with no defects in the tail or head under light microscope are considered as having normal morphology.

Purification and measurement of free 8-Isoprostane
An affinity column (Cayman Chemical, Ann Arbor, MI, USA) available in the market was used to purify the free 8-Isoprostane in duplicate. For isolating of precipitates, all specimens were first centrifuged at 15000 g. Then, we used column buffer to dilute supernatant 1:5 and performed the next procedures based on the manufacturer's protocol. Concentration of 8-Isoprostane was assessed using 50 ml from specimens at a wave length of 405 nm by the enzyme-linked immunosorbent assay (ELISA) reader (STAT FAX 2100, USA).

RNA extraction and cDNA synthesis
NucleoSpin® RNA Midi kit (Macherey-Nagel, Düren, Germany) was used to extract the total RNA from the samples and then RNase-free DNase I (Thermo Scientific, USA), based on the manufacturer's manual, was used to treat the RNA yield. After that, agarose gel electrophoresis was employed for confirming the quality of the extracted RNA. Its quantity was confirmed using Nano Drop® ND-1000 Spectrophotometer (Thermo Scientific, USA). In accordance with the company's guidelines, we applied the Revert Aid TM First Strand cDNA Synthesis Kit (Fermentas, Canada) to reverse transcribe 20 ng of total RNA to cDNA.

Quantitative real-time polymerase chain reaction
Based on the company's instructions, we carried out duplicate real-time PCR with the gene-specific primers by means of SYBR® Premix Ex Taq TM II (Tli RNaseH Plus) kit (Takara Bio Inc., Shiga, Japan) produced by the Rotor-GeneTM 6000 system (Corbett Research: Australia). Thermal cycling consisted of a round of denaturation for 30 sec at 95°C, which was followed by 40 more 5-sec cycles at 95°C. Each primer pair was heated at their specific temperatures (shown in Table I) for 15 sec and another 20-sec round at 72°C. Using serial dilution of cDNA, standard curves were drawn and thus level of the genes' expression level was normalized based on the mean expression of β-actin (the housekeeping gene). No template negative control was used.

Ethical considerations
The Ethics and Human Rights Committee of Hormozgan University of Medical Sciences (under the ethics code: HEC-93-12-4) verified this research, and then the written informed consents were received from all participants after the purpose of study was explained based on the declaration of Helsinki.

Statistical analysis
According to the research design, SPSS 16.0 (SPSS Inc., Chicago, IL: USA) was employed for statistical analyses and finally we set p < 0.05 to be statistically significant. Then, we employed a paired t test for comparing the sperms' parameters before and after the treatment period. In addition, we applied student's t test to compare the sperm parameters between controls and case (after treatment). Analysis of the distribution of NRF2, GPX4, SOD2, and CAT genes expression levels with the use of the Kolmogorov-Smirnov test indicated the lack of normal distribution of the data. For this reason, we employed non-parametric statistical tests for comparing the data. The Wilcoxon two-sample test was applied to detect the significant differences found in the NRF2, GPX4, SOD2, and CAT genes expression levels before and after the treatment. To compare the expression levels of the genes between the treated and healthy control individuals, two-tailed Mann-Whitney U-test was used. Spearman's Rho correlation test and was also utilized to analyze the correlations between the NRF2, GPX4, SOD2, and CAT genes expression levels and the various sperm parameters and free 8-Isoprostane levels.

Results
Demographic information of the participants are demonstrated in Table II and also change information the sperm count, semen volume and other parameters of case significantly enhanced after treatment with DPP. Comparison of the mean concentration of free 8-isoprostane and its significant reduction after capsule treatment was one of the most significant results of this study (p < 0.05) (Table III). Next, we assessed the possible correlation between the 8-Isoprostane concentration and seminal parameters. The common frequency of abnormal parameters of the participants in this study is also mentioned (Table  IV).
Comparative evaluation of mRNA expression of NRF2, GPX4, SOD2 and CAT genes before and after DPP treatment and in healthy individuals using quantitative real-time PCR and evaluation of the ratio of these genes at the mRNA level using the 2 -ΔΔct method significantly increased showed the expression of these genes. The mRNA expression levels of NRF2, GPX4, and CAT genes in healthy control was significantly higher than the case after the treatment (p = 0.04, p < 0.05, and p = 0.03) (Figure 2) However, the differences between the mRNA expression levels of SOD2 gene in healthy controls and case after treatment with DPP were not statistically significant (p = 0.16).
Examination of seminal enzymatic activity results of GPX4, SOD2 and CAT genes in in case and healthy controls showed that the mean activity of seminal GPX4, SOD2 and CAT enzymes were higher in controls compared with case, in addition Our findings showed that the mean activity of GPX4, SOD2, and CAT enzymes were higher in case after the treatment compared with case before the treatment (Table V).
The associations between fold changes of NRF2, GPX4, SOD2, and CAT genes expressions with sperm parameters in this article indicated that increase in the NRF2, GPX4, SOD2, and CAT genes expressions after treatment with DPP is significantly associated with increased the sperms' count, motility, and volume, and improved morphology of sperm (Table VI).
Spearman's correlation analysis showed SOD2 mRNA expression was significantly correlated with count, motility, and morphology of sperm, moreover, we found a relationship between the GPX4 expression at mRNA level and sperm count, sperm motility, morphology of sperm, pH of semen fluid, and seminal plasma free 8-Isoprostane level. The CAT expression at mRNA level was significantly related to the count of sperm, motility, and morphology of sperm and seminal plasma free 8-Isoprostane level (Table VII). We also detected a positive correlation between the NRF2 mRNA expression levels and sperm count, sperm motility, appearance and morphology of sperm, and pH of semen fluid.

Discussion
As mentioned earlier, excessive levels of ROS can negatively affect the process of spermatogenesis and impair the ability of spermatozoa to fertilize ova (9). Antioxidants and antioxidant enzymes in the male reproductive tracts are the main antioxidant defense systems that protect spermatozoa from oxidative damage caused by ROS (6,9). Therefore, antioxidant therapy could be beneficial in increasing the scavenging capacity of seminal plasma and improving the semen quality in infertile males.
In traditional medicine, DPP has an extensive utilization as one of the folk remedies to treat the male infertility (14). This study was designed for investigating the mRNA expression levels of the antioxidant genes NRF2, GPX4, SOD2, and CAT in infertile males before and after treatment with DPP and healthy controls. We also investigated the impact of DPP on the sperm parameters of infertile man. We found that taking DPP in infertile males significantly improves the semen quality including sperm count, semen volume, morphology, and motility of sperm (p < 0.05) (Table IV). In accordance with our data, Al-Snafi reported that the sperms' motility and count enhanced in the infertile males who were given treatment with DPP (20). Similarly, evaluated the effects of DPP on the sperm parameters of infertile man; sperm motility, count, morphology, as well as forwardprogressive motility significantly increased with DDP (21). Al-Dujaily also reported a significant improvement in the sperms' motility and count after the addition of DPP extract to sperm activation medium (22). In a study on adult male Sprague-Dawley rats, it was found that DPP can promote the count, motility, morphology, and DNA quality of sperms. Moreover, it increased the weight of testis and epididymis (23). Adaay  reported an association between severe abnormalities in spermatozoa and depletion of GPX4 in spermatocytes (29). Furthermore, it has been shown that GPX4 is involved in condensation of chromatin and protection of sperm DNA against oxidation, a necessary process in the maintenance of sperm quality (30). It has been reported that mitochondrial One limitation of the current study was that the treatment was given to the infertile subjects for a duration of one month due to their lack of cooperation and limited resources for the study; however, the minimum time required for a complete cycle of spermatogenesis is 86 days. Therefore, future studies should consider a three-month treatment period for better results.

Conclusion
We could present further documents for supporting this hypothesis: altered expression of antioxidant genes NRF2, GPX4, SOD2, and CAT at mRNA levels are correlated to the greater risks of males' infertility. We also conclude that administering DPP in infertile males enhances the levels of expression of NRF2, GPX4, SOD2, and CAT genes and improves the semen quality including sperm count, semen volume, and morphology and motility of sperm.