Failure to become pregnant following a year of unprotected sexual intercourse is defined as infertility by the World Health Organization (WHO), which can affect approximately 15% of married couples. ¹ Half of the infertilities are assiociated with males, including anti-sperm immune response, ductal dysfunction, and spermatogenesis defect. ^{2– 4}

According to the routine laboratory studies, the semen fluid is analyzed based on three improtant factors consisting of sperm concentration, morphology and motility; regartding to these factors, the male infertility can be divided to Azoospermia (absence of spermatozoa in the semen fluid), Oligozoospermia (less than 15 million sperm cells per ml), Teratozoospermia (less than 4% morphologically normal sperm cells), and asthenozoospermia (less than 40% motile spermatozoa). ⁵

Infertile men can be dignosed through invasive and non-invasive approaches. Invasive diagnostic methods such as testicular and prostate biopsy, as well as fine-needle aspiration, are commonly used for assessing male infertility, but they may cause tissue impairment, emphasizing the need for non-invasive molecular investigations to provide valuable diagnostic insights. ^{6– 9} The evaluation of miRNA is considered as a non-invasive biomarker is gaining interest, as miRNA expression profiling in testicular tissue, sperm, and seminal plasma can complement histopathological diagnostics and aid in evaluating spermatogenic dysfunction. ^{10– 12}

MicroRNAs are known as 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 mRNA. ¹³

Regarding the important role of miRNAs in the cell, dysregulation of miRNAs has been observed in different types of diseases, in particular infertility. ¹⁴ For the first time, Ostermeier et al. identified miRNAs in the spermatozoa. ¹⁵ Until now, several investigations have been carried out to discover miRNAs with various expression patterns among infertile males as a novel and promising biomarker. ⁹ The aim of the present study was to evaluate the predictive value of miR-122-5p, miR-449a, miR-30b-5p, miR-34c-5p, and miR-34b-5p in assessing sperm retrieval success from testicular tissue in azoospermic infertile men.

According to the inclusion criteria for the present study‚ the age and body mass index of the patients were examined.

Regarding the demographic characteristics and semen analysis parameters for both the control and test groups, the mean age was 32.15 ± 4.23 years for the control group and 31.07 ± 3.72 years for the test group, with no statistically significant difference observed between groups (P = 0.32). Body mass index (BMI) measurements were similarly comparable between groups (control: 25.11 ± 2.72, test: 24.37 ± 2.72, P = 0.30), indicating demographic homogeneity.

In addition, relative expressions of selected miRNAs ( miR-34b-5p, miR-122-5p, miR-449a, miR-34c-5p, and miR-30b-5p) were assessed. The mean expression levels of miR-34b-5p, miR-449a, and miR-30b-5p were lower in the test group (1.17 ± 0.15, 1.15 ± 0.16, and 1.05 ± 0.15, respectively) compared to the control group (1.72 ± 0.50, 1.59 ± 0.39, and 1.37 ± 0.32, respectively). Conversely, miR-34c-5p showed a slightly higher expression in the case group (1.93 ± 0.56) than in controls (1.78 ± 0.77). miR-122-5p expression was almost identical between groups (test group: 1.12 ± 0.12, control group: 1.05 ± 0.09).

Furthermore, Cycle threshold (Ct) values, as well as ΔCt (Ct target gene - Ct reference gene), were calculated to quantify miRNA expression accurately. The Ct values for miR-34b-5p (25.17 vs. 27.28), miR-449a (23.70 vs. 25.39), miR-34c-5p (27.64 vs. 30.76), and miR-30b-5p (29.39 vs. 32.19) were consistently lower in the test group compared to the control group, indicating higher expression levels. However, miR-122-5p exhibited relatively similar Ct values between groups (test: 22.74, control: 22.26). ΔCt analysis further underscored these expression differences, particularly notable for miR-34b-5p, miR-449a, miR-34c-5p, and miR-30b-5p, suggesting their potential predictive significance in sperm recovery from testicular tissues in azoospermic infertile men.

microRNAs (miRNAs) have emerged as promising biomarkers and regulators in reproductive biology, playing critical roles in the regulation of gene expression at the post-transcriptional level. ¹⁶ This study specifically explored the predictive value of miR-122-5p, miR-449a, miR-30b-5p, miR-34c-5p, and miR-34b-5p concerning sperm recovery from testicular tissue in azoospermic infertile men, focusing on their differential expression patterns and potential mechanisms underlying these microRNAs’ involvement in fertility processes. Mentioned microRNAs play essential roles in spermatogenesis by regulating germ cell proliferation, differentiation, chromatin remodeling, and apoptosis through pathways such as p53, Notch, Wnt/β-catenin, and transition nuclear protein (TNP)-mediated chromatin condensation. Their dysregulation in azoospermia leads to impaired histone-protamine exchange, disrupted cell cycle progression, increased germ cell apoptosis, and defective sperm maturation, contributing to male infertility. ¹⁷

Our study revealed that miR-34b-5p, miR-449a, and miR-30b-5p exhibited decreased expression levels in the case group compared to the control one, while miR-34c-5p showed elevated expression in the case group; miR-122-5p expression remained virtually unchanged between groups. Reduced expressions of miR-34b, miR-449a, and miR-30b in azoospermic infertile men could be associated with disruptions in spermatogenic processes, as these miRNAs typically function in cell cycle regulation, apoptosis control, and differentiation pathways critical to sperm maturation. Specifically, miR-34b and miR-449a have been previously documented to regulate genes involved in germ cell proliferation, apoptosis, and motility; thus, their decreased expression likely indicates impaired spermatogenesis. Similar findings were reported by previous studies highlighting the downregulation of these miRNAs in infertile patients, further supporting their critical role in fertility and testicular function.

miR-34b contributes to azoospermia by disrupting critical molecular pathways involved in spermatogenesis, particularly those regulating cell cycle control, apoptosis, and germ cell differentiation. As a key target of p53, miR-34b typically facilitates spermatogonial cell cycle arrest and apoptosis by inhibiting cyclin-dependent kinases (CDK4, CDK6) and transcription factors like E2F3, which are essential for germ cell proliferation. When miR-34b expression is altered—either reduced or dysregulated—it leads to defective germ cell division, excessive apoptosis, and impaired differentiation, ultimately resulting in the absence of mature sperm in the testes and contributing to male infertility. ^{18, 19} Paoli et al. demonstrated that in non-obstructive azoospermia (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). ²⁰ Moreover, miR-449a is a key regulator of spermatogenesis and cell cycle control, primarily by targeting genes involved in apoptosis, differentiation, and motility, such as CDK6, Notch1, and Bcl-2. Mechanistically, it suppresses the Notch and E2F signaling pathways, leading to enhanced cell cycle arrest and apoptosis in germ cells, which is crucial for maintaining proper sperm maturation and testicular homeostasis. ^{21, 22} Recent research by Abu-Halima et al. revealed that five miRNAs ( miR-34b, miR-34b, miR-34c-5p, miR-449a, and miR-449b) down regulated among azoospermia men. ²³ According to miR-30b, it can lead to azoospermia by modulating CDK6, Notch1, Wnt/β-catenin, and TGF-β pathways, where its decreased expression leads to impaired germ cell differentiation, excessive apoptosis, and defective sperm maturation, ultimately disrupting spermatogenesis. ²⁴ Furthermore, Abu-Halima showed that miR-23a, miR-23b, miR-30b, miR-27a, and miR-100 showed an underexpression in semen samples of men with infertility as compared with fertile control men. ²⁵

miR-34c is specifically expressed in germ cells and plays a crucial role in spermatogenesis by regulating germ cell differentiation through the downregulation of genes that are normally expressed at very low levels in these cells. ^{26– 28} In azoospermia, miR-34b and miR-34c downregulate E2F-pRb, TGIF2 and NOTCH2, targets of miR-34c and miR-34b that are necessary for germ-cell differentiation and subsistence during spermatogenesis. ¹⁷ Contrary to the commonly reported downregulation of miR-34c in infertile males, particularly in azoospermic individuals, our case group showed upregulated expression of this miRNA. ^{29– 31} This increased expression might represent a compensatory mechanism activated under stress conditions, potentially aiming to restore spermatogenesis or mitigate cellular damage by regulating apoptosis or cell cycle checkpoints. Interestingly, contradictory findings regarding miR-34c have been documented previously, with some studies reporting downregulation linked to infertility while others found elevated expressions in specific pathological states, suggesting context-dependent behavior influenced by genetic or environmental factors. Therefore, further investigation into the precise biological context and molecular targets of miR-34c is essential.

In infertile men, miR-122 is downregulated, leading to reduced expression of transition protein 2 (TNP2) by targeting the untranslated region of its mRNA, which is specifically produced in round spermatids, thereby disrupting spermatogenesis, testicular development, and sperm maturation. Additionally, miR-122 influences spermatogenesis by suppressing TNP2 expression, affecting sperm differentiation, and promoting apoptosis through the repression of the anti-apoptotic gene Bcl-w, ultimately activating the intrinsic apoptotic pathway. ^{32– 34} Analysis of miR-122-5p expression in our study population demonstrated negligible differences between azoospermic and control groups, indicating that this miRNA is unlikely to play a direct or specific role in the molecular mechanisms underlying azoospermia. This result aligns with studies suggesting miR-122-5p is primarily involved in hepatic metabolism rather than reproductive processes, given its predominant expression in liver tissue and its well-documented roles in lipid homeostasis and hepatic function. ³⁴ The results of the present study are consistent with prior observations reported in other studies on male infertility, in which miR-122-5p levels also remained unaltered. This consistency lends further support to the notion that miR-122-5p is unlikely to serve as a reliable predictive biomarker for successful sperm retrieval from testicular tissue in individuals with azoospermia. In contrast, Khadhim and colleagues documented markedly increased expression levels of miR-122 in azoospermic patients (p < 0.05). ³¹ On the other hand, Abu-Halima et al. showed that mir-122 was downregulated in infertile men. ³⁵ The discrepancy in miR-122-5p expression across studies may be due to differences in patient selection criteria, sample sizes, methodologies for RNA extraction and quantification, or variations in underlying genetic and environmental factors affecting spermatogenesis in different populations.

In conclusion, the expression patterns of miR-34b-5p, miR-449a, miR-30b-5p, and miR-34c-5p highlight their potential utility as biomarkers in predicting sperm recovery outcomes in azoospermic infertility, whereas miR-122-5p appears less informative in this context. Further comprehensive research with larger sample sizes and mechanistic studies are warranted to fully elucidate their clinical applicability.

The study protocol was approved by the Ethics Committee of the University of Science and Art in October 2022 with the No: IR.ACECR.JDM.REC.1401.096. All of the participants signed an informed consent before entering the study.

The ethical principles of the Declaration of Helsinki were applied with respect to the confidentiality and veracity of the data collected during the course of the study, which are faithfully presented. Personal identity data and patient privacy were protected. Authorship contributions and transparency in conflicts of interest were reported. ³⁶