Association between microRNA expression and risk of male idiopathic infertility in Iraq

SUMMARY

OBJECTIVE:

The World Health Organization defines infertility as the inability to get pregnant after 12 months of unprotected sexual activity. This study was conducted to estimate the levels of gene expression for two mature miRNAs (i.e., miR-122 and miR-34c-5p) to evaluate susceptibility to male infertility.

METHODS:

This study included 50 male patients with idiopathic infertility who were admitted to hospital from the period November 2021 to May 2022 and another group consisting of 50 apparently healthy individuals used as controls.

RESULTS:

miR-122 level was significantly highest in azoospermia and followed by oligospermia, 39.22 (31.88) versus 37.34 (20.45), respectively. In addition, there was a very significant difference in miR-34c-5p levels between the study groups (p<0.05).

CONCLUSION:

Two miRNAs, namely, miR-34c-5p and miR-122, can be used as predictive and diagnostic biomarkers for infertility.

Keywords
Male infertility; MicroRNAs; Gene expression; Azoospermia; Oligospermia

INTRODUCTION

Failure to become pregnant following a year of unprotected sexual intercourse is known as infertility. On average, it affects 15% of married couples. About 30% of instances involve men and 35% include women¹. Male infertility is not a single pathological condition but rather reflects several different pathological conditions, and the majority of men who are investigated for infertility cannot identify the cause because the causation of male infertility is obviously diverse and could be immunological, accidental, pathological, physiological, or even nutritional². Most cases appear as a lack of sperm in the ejaculate or a decline in sperm count without evident cause so considered idiopathic male infertility³.

Male infertility may be due to a defect in sperm morphology and their motility and concentration, as these causes can contribute to 40-50% of male infertility. On this basis, male infertility can be classified into azoospermia, oligozoospermia, asthenozoospermia, and teratozoospermia⁴.

MicroRNAs are short non-coding RNAs (i.e., less than 22 nucleotides) that control the amount of proteins by preventing the translation of mRNA and/or accelerating its degradation. The seed sequence of the microRNA, which is found from the second to the eighth nucleotide from the 5’-end of microRNAs, pairs with a complementary sequence in the target mRNA transcript, which is typically found in the 3’ untranslated region, to recognize the target mRNA⁵.

Spermatogenesis requires a highly organized gene expression. Organization of gene expression during spermatogenesis occurs at several levels such as transcriptional, post-transcriptional, and even epigenetic modification. Studies have identified around thousands of genes that are coding proteins responsible for the regulation of spermatogenesis⁶. Regulation of gene expression by miRNA is one of the most important levels of post-transcriptional regulation levels. miRNA expression varies at each stage of spermatogenesis, so there are specific types of miRNAs at each stage of sperm production and maturation and the aberration of miRNA is considered a molecular etiology of male infertility and dysregulated expression of miRNA can be inherited by progeny⁷.

Previous studies have discovered changed miRNAs in the seminal plasma, spermatozoa, and total semen of asthenozoospermia males⁸; therefore, miRNA emerged as a promising biomarker that can ravel out many molecular etiologies underlying cases of idiopathic infertility⁹. Defects in miRNA expression can explain many problems associated with sperm production, motility, and fertilization ability. Sertoli cells and Leydig cells, which provide the niche for spermatogonial stem cells (SSCs) and subsequently give nutritional and structural support for germ cells, are regulated in their functionality and development by miRNAs. As a result, miRNAs in somatic cells play crucial roles in spermatogenesis through the control of genes, which in turn encode proteins with significant functions in spermatogenesis⁷.

In recent years, several studies have focused on the role of miRNA in sperm maturation within the epididymis. These studies indicated that miRNA is a communication language between cells of epididymis epithelium and sperm, as transcription of sperm is repressed due to condensation of chromatin, and during spermatogenesis, miRNA plays an important role in the regulation of sperm activities after repressing of its genome and also a key role in early embryogenesis¹⁰.

CBL, a tumor suppressor gene called Casitas B-lineage lymphoma, is the miR-122-5p target gene. SSCs’ proliferation and DNA synthesis are enhanced and apoptosis is prevented when CBL is inhibited by miR-122-5p (SSCs)¹¹.

Deleted in azoospermia-like (dazl) gene is one of miR-34b and miR-34c’s likely targets¹². Members of the MiR-34/449 family perform important functions in spermatogenesis by controlling testicular functionality and spermatozoa maturation. Numerous features of idiopathic male infertility extending from oligozoospermia to non-obstructive azoospermia (NOA) are related to dysregulation of miR-34/449¹³.

METHODS

Subject

This study included 50 male infertile patients with ages ranging from 20 to 43 years, who attended to the infertility and IVF center in Al-Sader City Hospital for the period from November 2021 to May 2022. Other (50) healthy subjects without any history of systemic disease were included as a control group. Any infertile man with reproductive system diseases, surgery history, diagnosed varicocele, patients having cryptorchidism and testicular tumors, or undergoing chemotherapy and radiotherapy were excluded from this study. Blood samples were taken; 3 mL of blood per case was drawn through vein puncture. The miR-34c-5p and miR-122 expression levels were then measured using real-time PCR after RNA had been extracted. After explaining the purpose and potential outcomes of the study to each study participant, their informed agreement was acquired.

Stem-loop RT-qPCR

The miRNA-122 and miRNA-34c-5p expressions in the serum of patient and control groups were measured by using the stem-loop RT-qPCR, and the housekeeping gene (GAPDH) was used as a reference. The “TRIzol^® reagent kit” was used to extract the total RNA from serum samples following the manufacturer’s guidelines. A Nanodrop spectrophotometer (THERMO, USA) was used to measure the purity and concentration of the extracted RNA by reading the absorbance at 260/280 nm. DNase I enzyme was used to treat the extracted RNA to eliminate any remaining genomic DNA in the eluted total RNA and done according to the instructions provided by Promega Company (USA). The Sanger Center miRNA database registry was used to choose the miRNA sequence, and the miRNA primer design tool was used to design the primers for miRNA-34c-5p and miRNA-122. The M-MLV Reverse Transcriptase kit was used to synthesize cDNA from the DNase-I treated RNA samples according to manufacturer’s instructions. The housekeeping gene (GAPDH) used in the normalization of the expression analysis of miRNA-34c-5p and miRNA-122 was quantified using a quantitative real-time PCR.

Statistical analysis

Data were either normally distributed or not normally distributed and recorded in a Microsoft Excel spreadsheet. Statistical analysis was carried out with the SPSS v 0.26 software (chi-square, independent sample t-test, Spearman, and Pearson correlation coefficient) after translating data into codes. The results were analyzed and assessed using appropriate statistical methods. When a p-value is less than 0.05, the significance level is considered.

RESULTS AND DISCUSSION

Demographic and clinical parameters

The infertile patients and controls had a similar age range. This study revealed that the majority of patients (50%) belonged to the age group of less than 30 years and 34% of the patients had the age group of 30-39 years (Table 1). The data showed that semen volume was not significant (p>0.05) when compared between oligospermia and azoospermia (Table 2).

Table 1.
Distribution of control and patients according to their age.

Age groups(years)	Controls N (%)	Patients N (%)
<30	24 (48%)	25 (50%)
30-39	18 (36%)	17 (34%)
≥40	8 (16%)	8 (16%)
Total	50 (100%)	50 (100%)

Table 2.
Seminal fluid volume in controls and infertile individuals (oligozoospermia and azoospermia).

Groups	No.	Volume (mL)
		Mean±SE	Minimum	Maximum
Oligospermia	25	2.31±0.11	1.3	3.2
Azoospermia	25	2.42±0.22	1.2	3.8
Controls	50	2.50±0.15	1.5	3.9

Previous research showed a rise in infertile males between the ages of 26 and 35 years (63.5%) because the youngest age groups are seeking more medical advice, are more focused on fathering children, and are also under more psychological stress due to family obligations. Sperm functions, ejaculation frequency, and semen quality were found to gradually decline with older age and begin to decline after 35 years¹⁴. This active age group may be highly susceptible to infertility due to economic difficulties, stress, and pollution¹⁵.

Semen volume in patients did not significantly alter, which is in agreement with the research by Majed et al.¹⁶, which found no significant increases in semen volume. The mean seminal fluid volume between the investigated groups of controls and infertile is larger than the World Health Organization (WHO)’s more stringent recommendations for how to determine a suitable seminal fluid volume, which is estimated to be at about 1.5 mL. The prostate gland and seminal vesicles and a small amount from the bulbourethral glands are mainly contributed to the volume of the ejaculate (WHO)¹.

Comparison of hsa-miR-122 and hsa-miR-34c-5p among patients and control groups

The levels of hsa-miR-122 and hsa-miR-34c-5p are compared between study groups (Table 3). miR-122 was significantly highest in azoospermia and followed by oligospermia, 39.22 (31.88) versus 37.34 (20.45), respectively, and the statistical significance level was high (p<0.05). In addition, there was a very significant difference in miR-34c-5p levels between study groups (p<0.05); the highest level was found in the oligospermia group followed by the azoospermia group, 36.64 (22.24) versus 22.31 (17.73), respectively (Table 3).

Table 3.
Comparison of the protein expression for miRNAs in the study groups.

miRNA	Control n = 50	Azoospermia n = 25	oligospermia n = 25	P1	P2	P3
miR-122
Median (IQR)	1 (0.11)	39.22 (31.88)	37.34 (20.45)	p<0.05 HS	p<0.05 HS	p<0.05 HS
Range	0.28—1.77	4.69—99.55	2.32—95.46
miR-34c-5p
Median (IQR)	1 (0.08)	22.31 (17.73)	36.64 (22.24)	p<0.05 HS	p<0.05 HS	p<0.05 HS
Range	0.16—1.12	3.66—97.80	1.03—99.14

According to a recent study by Fang et al.¹⁷, patients with NOA who had unsuccessful sperm retrievals had lower expression of miR-34c-5p in their testicular tissue than those who had successful retrievals. Also, the expression profile of miR-34c-5p was similar between patient-specific testicular tissue and seminal plasma samples.

In NOA patients’ seminal fluid compared with controls, the expression of all miRNAs examined, namely, miR-34c-5p, miR-34b-3p, miR-122-5p, and miR-509-5p, was significantly decreased (p<0.001)¹⁸. Seminal plasma exosomes showed differential expression of miR-34c-5p and miR-122-5p in normozoospermic and azoospermic individuals¹⁹. Patients with NOA had significantly lower levels of the miRNAs (hsa-miR-202-3p and hsa-miR-34c-5p) in their seminal plasma compared with healthy fertile individuals (p<0.05)²⁰. Recent research by Liu et al.²¹ revealed 173 miRNAs that differed in their expression between spermatids and spermatocytes in human azoospermic patients. Among those, miR-34b-5p and miR-34c-5p significantly decreased in round spermatids as compared with spermatocytes, and it has been suggested that these miRNAs are essential for spermatocyte meiosis.

Mokánszki et al.²² demonstrated that infertile males (asthenozoospermic and oligozoospermia groups) had significantly lower expression levels of three microRNAs (i.e., miR-122, miR-34b, and miR-15b) in their seminal plasma and spermatozoa than normal men. Significant differences between asthenozoospermic and oligozoospermia ejaculates were found in the relative expression level of these miRNAs in spermatozoa (p<0.05). In the comparison of microarray and qRT-PCR analyses, we found that miR-122 expression was downregulated, whereas miR-141 expression was upregulated in the oligoasthenozoospermia group. Moreover, miR-141 expression was upregulated and miR-122 expression was downregulated in the asthenozoospermic comparison of microarray and qRT-PCR analyses²³. Abu-Halima et al.²⁴ showed that hsa-miR-429 was significantly upregulated, while three miRNAs (i.e., hsa-miR-122, hsa-miR-34c-5p, and hsa-miR-34b) were significantly downregulated. When compared with control males, the expression levels of the downregulated miRNAs in the spermatozoa of subfertile men ranged from 3.84-fold for hsa-miR-122, 2.67-fold for hsa-miR-34b, and 2.56 for hsa-miR-34c-5p, up to 7.73-fold for hsa-miR-34b.

As miRNAs are found in the seminal plasma, extracellular vesicles, sperm cells, testis, and epididymis, they may affect spermatogenesis and embryogenesis by altering the target gene’s expression. Clearly, such disturbances have the potential to lead to a variety of infertility conditions²⁵.

CONCLUSIONS

There was a very significant difference in miR-34c-5p and miR-122 levels between study groups. The prevalence of infertility has significantly increased worldwide. Therefore, it is advised that the health institution undertake a prescreen for the genes of these miRNAs, which may serve as a prognostic and diagnosed marker of patients with infertility.

ETHICAL APPROVAL

This study obtained ethical approval from the University of Al-Qadisiyah, and written consent was taken from all participants in the research (patient group and control group).

ACKNOWLEDGMENTS

I am grateful to the Medical College of Al-Qadisiyah University and the Infertility and IVF Center in Al-Sader City Hospital for fulfilling all my study needs.

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• Funding: The source of funding for this work was personal finance.

Publication Dates

• Publication in this collection
  18 Sept 2023
• Date of issue
  2023

History

• Received
  22 Mar 2023
• Accepted
  25 June 2023