A comparative assessment of RNF38 and P53 genes expression in the sperm samples obtained from males with normozoospermia and asthenospermia: A case-control study

Abstract Background Infertility is considered as a common problem appears in about 10-12% of couples in their reproductive ages. Ring finger protein 38 (RNF38) gene is a ubiquitin-protein ligase that can regulate Protein 53 (P53) and affect cellular motility. Objective Considering the role of P53 on cellular motility and RNF38 on the regulation of P53, the present study aimed to assess the difference between RNF38 and P53 genes expression in normozoospermic and asthenospermic samples as a diagnostic biomarker in males. Materials and Methods The present study was conducted among 21 asthenospermicsand 63 healthy individuals. First, the real-time polymerase chain reaction technique was applied to measure the expression level of the P53 and RNF38 genes extracted from sperm samples, and the glyceraldehyde-3phosphate dehydrogenase gene was selected as the reference gene. Results An increase and a decrease occurred in the level of P53 and RNF38 genes expressions in asthenospermic and normozoospermic samples, respectively. In addition, a significant difference was observed between increasing P53 gene expression (p < 0.001), reducing RNF38 one, and decreasing sperm motility (p < 0.001) in asthenospermic cells compared to that of normozoospermic ones. Conclusion Based on the results, an increase in the expression of the P53 gene and a decrease in the expression of the RNF38 gene had a significant relationship with asthenospermia in men. Therefore, it is expected that an effective step should be adopted to diagnose the asthenospermia expression pattern by using these results.


Introduction
Based on the world health organization report, about 10-12% of couples worldwide are suffering from infertility, half of which are related to males. Although there is no clear reason for male infertility in 30-40% of cases, the other 50% are related to sperm-related disorders (1)(2)(3) including asthenozoospermia or decreasing sperm motility in males medically (4) which appears because of disturbing genital system development and decreasing sperm motility and fertilization capacity by genetic disorders (5).
Sperm-specific RNAs are important biomarkers in the steps related to germ cells and the share of somatic cells. Thus, these RNAs can be considered valuable diagnostic markers for fertility, sperm viability, and motility, and can be used as a predictor for prognosis during in vitro fertilization (6)(7)(8).
The human Protein 53 (P53) gene is located in the short arm of chromosome 17 (17p139) (9) and encodes the P53 protein, which contains 393 amino acids (10,11). Numerous studies represented the importance of the function of P53 in apoptosis in preventing tumor growth. The P53 gene plays a role in many cellular processes such as metabolism, antioxidant response, and DNA repair (12,13). RNF38 is considered one of the P53-connected proteins (14,15). E3 disturbs the function of tumor suppressor genes or oncogene, which participates in cellular transduction and tumor progression (16,17). The main role of P53 is to provide signals since it can direct cells toward repairing or stopping growth, or even apoptosis by inducing the expression of a series of genes.
P53 is a central factor in cells, and RNF38 is a P53regulatory factor with inhibitory activity on RNF38.
Since no study has been done regarding the effect of P53 and RNF38 genes in asthenozoospermia, our purpose was whether P53 is involved in asthenozoospermia is an important process and whether its involvement is affected by RNF38. We sought to find out whether P53 affects motility through RNF38 or not. Thus, the expression of 2 genes was evaluated in the present study.
Additionally, the present study aimed to assay the level of DNA fragmentation by using a Sperm DNA fragmentation assay (SDFA) kit.
Assessing and recognizing this relationship can improve the efficiency of therapy, identifying new molecular biomarkers for the early diagnosis of asthenospermia in males, evaluating optimal therapeutic approaches, and diagnosing asthenozoospermic samples by assaying sperm in males.

Materials and Methods
In this case-control study, the count and motility of the sperm samples of the 84 participants, who were referred to Bu Ali Laboratory in Zanjan, Iran for 6 months from June-November 2020, were written on the day of sample preparation and the data were sorted from minimum motility to maximum one.

RNA extraction
RNA was extracted from collected samples using an RNA column extraction Kit (BioBasic Inc., America) and purified.

cDNA synthesis
Takara kit (TAKARA BIO INC., Japan) was used to synthesize cDNA from extracted RNA.
In the present study, the real-time polymerase chain reaction (PCR) technique was applied to assess the relationship between P53 and RNF38 genes expression in the sperm samples related to the males with normospermia and asthenospermia. Table I

Ethical considerations
The project was found to be under the ethical

Results
Based on the comparative assessment

SDFA results
Based on the results in SDF (Table III), an increase in RNF38 expression in poor cells was more than good, indicating a significant increase in expression (p = 0.01). In addition, an increase in P53 expression in poor cells was more than good, indicating an increase in expression, although it was insignificant (p = 0.81).

Discussion
Male infertility is considered the reason for 40-50% of infertilities (18,19), which is found in about 7% of males. The etiology of infertility in  and RNF38 genes expression in the cancer field, few studies were performed regarding the relationship between the expression of these genes and sperm motility and infertility.
On focusing on the expression of the RNF38 gene and the development of lung cancer, it was found that increasing the expression of RNF38 is significantly associated with metastasis to the lymph nodes in lung cancer, which can be considered as evidence of the effect of RNF38 expression in cellular motility (29).
The rate of RNF38 expression increased in liver carcinoma cells. An increase in RNF38 expression is related to cancer invasion and progression, as well as the inhibition of apoptosis in vivo and within the body (29,30). In addition, in line with the present study results, we examined P53 and its mechanism in prostate cancer and observed that prostate cancer cells have low P53 expression

Conclusion
The present study results represented a significant relationship between P53 and RNF38 gene expression and asthenospermia among males and changing their expression level in asthenospermic cells. Further, an increase in P53 expression in asthenospermic cells is significantly associated with decreased sperm motility. The present study could provide further research on the relationship between P53 and RNF38 gene expression alteration with motility in asthenospermia in males at a scale larger than both case and control groups.