The Relationship between The Expression of Sperm MicroRNA-149b and 34c and Sperm Quality in Men with Oligoasthenoteratozoospermia in Endometriosis

Male infertility is caused by many acquired, congenital, and idiopathic factors ( 1 , 2 ). Lifestyle parameters and environmental stressors, such as nutrition smoking, and alcohol consumption affect the function and dynamics of the reproductive system in males ( 3 ). Despite advancements in the field of human reproduction, the precise reason of infertility remains unknown in approximately 30% of males experiencing infertility, underscoring the importance of comprehending the specific molecular mechanisms that contribute to the pathogenesis of male infertility ( 4 ). Certain research has proposed the potential involvement of small non-coding RNA molecules, referred to as microRNAs, in male infertility. MicroRNAs are present in every eukaryotic cell, and it has been demonstrated that these molecules regulate diverse physiological processes by either up-regulating or down-regulating genes ( 5 ). Dysregulating these molecules is associated with the onset of various diseases, including viral infections, cancers, and neurodegenerative disorders ( 6 , 7 ). Several microRNAs have also been identified in samples of semen, the change in the level of expression of these microRNAs is related to normal parameters of sperm such as (reduction in number, low motility and abnormal morphology) ( 8 ). Bioinformatics analysis revealed that miR-34c-5p, miR-122, miR-149b-5p, miR-181a, miR374b, miR-509-5p , and miR-513a-5p have been found to be involved in spermatogenesis, cell proliferation, differentiation and target specific genes related to apoptosis are involved ( 9 ). miR-34 is a protected miRNA family that includes members such as miR-34a, miR-34b , and miR-34c , which play a role in regulating cell cycle, apoptosis, and cellular senescence. miR-34b and miR-34c show expression in the male gonads ( 9 ). The target genes of the miR-34 family include various cell cycle regulators. In human testes, the predicted target genes NOTCH1 and NOTCH2 of miR-34b and miR-34c are important regulators of germ cell survival and differentiation ( 10 ). miR-149b is as the miR-149a/b/c family member ( 11 ). In this family, miR-149b has greater prominence as it can be greatly expressed in the spermatozoon ( 12 ). A report has demonstrated that miR-34c and miR-149b play a crucial role in the initial cleavage division of mouse zygotes ( 13 ). It has also been found to be associated with clinical outcomes in patients undergoing intracytoplasmic sperm injection (ICSI) and in vitro fertilization (IVF) procedures ( 14 ). Research revealed that elevated levels of miR-34b and miR-34c in teratozoospermic and Asthenozoospermic sperm were not significantly associated with the rate of fertilization and high-quality embryos above 50%. However, these elevated levels were more likely to be correlated with higher rates of implantation, pregnancy, and live births ( 15 ). We investigated the correlation between the expression of miR-149b and miR-34c in sperm and sperm parameters in individuals with oligoasthenoteratozoospermia.

A molecular estimation of sperm miR-34c and miR-149b was conducted in two groups, each consisting of 30 individuals. Regarding the expression rate, a significant downregulation of miR-149b expression was observed in the OligoAsthenoTeratospermia group (0.24 ± 0.07) compared to the fertile group (1 ± 0.04, P=0.01). Also, significant down regulation of this miR34c was shown in OligoAsthenoTeratospermia patients (0.6 ± 0.02) as compared to the control or fertile men (1 ± 0.037, P=0.03, Fig .1A, B ). Comparison of the miR-149b and miR34c in OligoAsthenoTeratospermia and fertile groups. A. The evaluation of the miR-149b level expression in OligoAsthenoTeratospermia group by contrast to the fertile group. B. The evaluation of the miR34c level expression in OligoAsthenoTeratospermia group by contrast to the fertile group. *; P<0.05. The results from analyzing the semen in every parameter are displayed in Table 1. The mean of semen characteristics in the male partner were assessed in accordance with of the WHO 2010. The count of sperm in the group of OligoAsthenoTeratospermia was significantly different from the fertile group (13 ± 1.8 vs. 88.86 ± 10.7, P=0.004). A meaningful decrease was observed in the OligoAsthenoTeratospermia group contrasted with the fertile group in terms of total motility (31.42 ± 9.60 vs. 70.18 ± 11.21, P=0.002), progressive motility (20.48 ± 8.57 vs. 44.54 ± 10.08, P=0.002), and abnormal morphology (98.02 ± 1.16 vs. 95.02 ± 3.16, P=0.002). Also, there was highly meaningful decrease in viability (75 ± 6.8 vs. 89 ± 4.1, P=0.001), in OligoAsthenoTeratospermia group than fertile group. Table 1 also reveals a meaningful increase in sperm DNA fragmentation in the OligoAsthenoTeratospermia samples (34.1 ± 2.8% vs. 9.14 ± 2.45%, P=0.003, Fig .2 ). Considering the results outcome, a significantly lower percentage of MMP (60.95 ± 7.03 vs. 75.95 ± 5.92, P=0.001, Fig .3 ), and a lower sperm capacity (7 ± 4.5 vs. 12.7 ± 4.4, P=0.002) was observed in the OligoAsthenoTeratospermia group compared to the fertile group. There was no significant difference in ejaculate volume between the two groups in this study (P=0.841). Comparison of sperm parameters of fertile, and OligoAsthenoTeratospermia groups All data are presented as mean ± SD. Statistically significant (P≤0.05) differences are detailed in bold. TM; Total motility, PM; Progressive motility, DFI; DNA fragmentation index, and MMP; Mitochondrial membrane potential. Sperm DNA fragmentation was assessed in two groups based on halo formation, using an Olympus CX21 light microscope (magnification: 100x, scale bar: 50 µm). A. Normal group and B. Oligoasthenoteratozoospermia group. The mitochondrial membrane potential of sperm was evaluated in different groups using rhodamine staining, employing the Olympus DP71 microscope from Japan 100x. A. Magnification: Normal group (scale bar: 50 µm) and B. Oligoasthenoteratozoospermia group (scale bar: 50 µm). Healthy sperm can be identified by the presence of a bright green middle piece, while damaged sperm typically lack this bright green middle piece. Table 2 displays the correlations between the examined miRNAs and seminal quality. In particular, miR-149b exhibited a negative correlation with sperm DNA fragmentation (r=-0.362, P=0.001) within the sample. While, a positive correlation between miR-149b and the count of sperm (r=0.515, P=0.004) and total motility (r=0.652, P=0.003), viability (r=0.078, P=0.001) and normal morphology (r=0.167, P=0.002). miR-149b correlated significantly and positively with sperm MMP (r=0.235, P=0.001), capacity (r=0.334, P=0.002). In this table, miR34c shows highly meaningful correlation with the count of sperm (r=0.468, P=0.003), total motility (r=0.568, P=0.001), viability (r=0.099, P=0.002), Normal morphology (r=0.218, P=0.001), capacity (r=0.522, P=0.001), MMP (r=0.537, P=0.001), and negatively with sperm DNA fragmentation (r=0.718, P=0.001). Furthermore, a strong correlation was observed between the two miRNAs and sperm quality. A significant decrease in the level of TAC was discovered in the seminal plasma of the OligoAsthenoTeratospermia group compared to the fertile group (1.82 ± 0.11 vs. 2.51 ± 0.13, P=0.001). Additionally, the level of MDA in the seminal plasma was significantly higher in the OligoAsthenoTeratospermia group (2.21 ± 0.01 vs. 1.77 ± 0.09, P=0.001) compared to the fertile group ( Fig .4 ). The evaluation of the sperm biochemical factors in OligoAsthenoTeratospermia group contrasted to the fertile group. TAC; Total antioxidant capacity, MDA; Malondialdehyde, and *; Significant difference between two groups. In the OligoAsthenoTeratospermia samples, we found a positive and significant correlation between the expression of miR-149b and sperm TAC (r=0.441, P=0.002). Additionally, a negative and significant correlation was observed between miR-149b expression and MDA levels (r=-0.201, P=0.003). The results of Pearson correlation test revealed a significant correlation between the expression of miR34c and both TAC (r=0.591, P=0.001) and MDA levels (r=-0.308, P=0.004) among the OligoAsthenoTeratospermia samples ( Table 2 ). Correlations between miR-149b, miR34c mRNA levels, sperm parameters, and biochemical factors The Pearson correlation coefficient (r) was used to measure the statistical correlation. A significance level of P<0.05 was considered significant. Statistically significant (P≤0.05) differences are detailed in bold. DFI; DNA fragmentation index, MMP; Mitochondrial membrane potential, TAC; Total antioxidant capacity, and MDA; Malondialdehyde.

Our study revealed a significant downregulation of miR34c and miR-149b in the sperm samples of infertile men. Furthermore, the expression levels of miR34c and miR-149b were found to be correlated with basic sperm parameters including count, motility, viability, and morphology, which is in line with studies suggesting an association between altered expression of miR-34c, miR149b , and sperm parameters ( 21 - 23 ). The levels of the two miRNAs studied in male factor infertility sperm have been the subject of limited research. It has been observed that men with oligozoospermia and asthenozoospermia have lower levels of sperm miR-34c compared to normozoospermic men ( 24 ). Additionally, men with idiopathic infertility have significantly lower levels of sperm miR-149b compared to men with normal semen parameters ( 25 ). miR34c is as the commonest sperm-borne miRNA In human models ( 26 ), and greatly conserved among various species, like mice, and pigs ( 13 , 26 ). Previous studies have demonstrated that miR-34c plays a regulatory role in the Notch signaling pathway ( 27 , 28 ). The Notch signaling pathway is a highly preserved system that consists of receptors, ligands, transcription factors, and downstream effectors ( 29 ). This pathway plays a crucial role in regulating various aspects of spermatogenesis, including the pace of spermatogenesis, proliferation, and differentiation of cells throughout the spermatogenic cycle ( 30 ). Dysregulated activation of the Notch signaling pathway has been associated with detrimental effects on spermatogenesis. It can disrupt the maintenance and differentiation factors of spermatogonial stem cells, leading to impaired spermatogenesis ( 31 ). The Notch 1 receptor and its ligand Jagged 2 have been found to be expressed in spermatocytes and spermatids in both human and rat testes ( 32 ). When the Notch signaling pathway is impeded in vivo , the expression patterns of Notch components in the testis are disrupted. This disruption leads to aberrations in male germ cell fate, a significant increase in germ cell apoptosis, particularly in the later stages of spermatogenesis, and an increase in spermatogenic maturation defects ( 33 ). The group of miR-149 consists of three members in both mice and humans, namely hsa-miR-149a, hsa-miR-149b , and hsamiR-149c . However, the understanding of the role of the hsa-miR-149 family in human reproduction is currently limited to findings from animal model studies ( 34 , 35 ). The findings from these studies indicate that the has-miR-149 family is highly regulated in the testes and is involved in the initiation of the meiotic phase in mature testes ( 35 ). Additionally, these studies demonstrate that hsa-miR-149 is predominantly and exclusively expressed in mature testis spermatocytes and spermatids ( 25 ). Emerging evidence indicates that microRNA-149 is directly regulated by the tumor suppressor p53. Under conditions of cellular stress, p53 is activated to protect against malignant transformation. It accomplishes this by activating DNA repair mechanisms to preserve the cell's integrity or by inducing apoptosis if the damage is irreparable, thereby eliminating the compromised cell ( 36 ). Of considerable interest in this study is the association between sperm miR34c and miR-149b and sperm DNA fragmentation. According to the relationship obtained, it can be concluded that the expression of miR-149 is positively influenced by p53, a crucial factor involved in the production of normal spermatogonial cells and the regulation of apoptosis ( 37 ). This suggests that miR-149 may play a role in promoting apoptosis during the regulation of spermatogenesis. Additionally, our observations reveal a correlation be tween low TAC level, and MDA level, and the expression of both miRNAs. Thus, the connection detected between sperm quality and stress oxidative factor can be through stress effects on the sperm miRNAs 149b and 34bc levels. Considering the relationship of miR-34 , and stress oxidative factors, the probable implication of such microRNAs on diseases leading to male infertility, like varicocele should be studied. Varicocele is linked to elevated testicular temperature, resulting in germ cell damage and temperature-dependent spermatogenic failure ( 21 ). Apoptosis, DNA damage oxidative stress (OS), and autophagy play a role in heat-related germ cell damage ( 38 ). Patients with impaired semen parameters and varicocele showed a significant reduction in miR34c levels than fertile men with normal testicular functionality and varicocele. In contrast, there exists a negative correlation between the levels of miR-34c and OS as well as apoptosis. Lower miR-34a levels are found in varicocele patients, suggesting that dysregulation of the miR-34 family is part of the pathophysiology of varicocele ( 26 ). Varicocele patients have decreased miR-34a expression levels and increased OS levels in their semen specimens, than healthy fertile controls ( 39 ). To understand the molecular mechanisms underlying the pathophysiology of varicocele, it is important to consider the anti-apoptotic effects of miR-34 . These microRNAs play a role in regulating apoptosis and may contribute to the modulation of cell survival pathways in the context of varicocele-induced molecular events ( 40 ). Also, miR-34/149 dysregulation is linked to increase germ cell apoptosis and OS in testis ( 21 ). According to the studies, the expression levels of miR-149 and miR-34c in sperm are associated with the quality of early embryonic development in conventional IVF treatment. While sperm-borne miR-149 is not essential for early embryonic development, it can serve as an additional biomarker. Notably, the decreased expression of miR-149 and miR-34c could potentially serve as an initial indicator of early embryonic development and offer valuable insights into the underlying biological factors in idiopathic infertile males. This study raised the possibility of stress-related miRNA changes of men’s sperms. Therefore, by conducting genetic assessments of sperm DNA, it becomes possible to evaluate the risk across generations. Additionally, future investigations may uncover the potential value of epigenetic testing of sperm miRNA, providing further insights and understanding.

The findings of this research indicate a reduced expression of miR-34c and miR-149b in sperm samples from infertile men. These results suggest that the decreased expression of these miRNA family members could potentially contribute to defective spermatogenesis, providing a possible explanation for infertility in affected individuals.

The experimental study received approval from the Ethics Committee of Qom Azad University (IR. IAU.QOM.REC.1401.087). The Written informed agreement was gained from all participants involved in the study. The case-control research involved 30 OligoAsthenoTeratospermic men who were guided to the Infertility Research Center at the Academic Center for Education, Culture, and Research (ACECR), located in Qom, Iran. The whole of patients was recognized with OligoAsthenoTeratospermia based on semen analysis. Total sperm number <(15×106 per ejaculate), vol (mL), total motility<(40%), and morphology <(4% abnormal forms). The inclusion criteria for infertile men in this study were as follows: a history of infertility for at least 1 year, with their wives undergoing a normal gynecological evaluation. However, men with conditions such as cystic fibrosis, Klinefelter syndrome, varicocele, chemotherapy, azoospermia factors (AZF) abnormalities, and microdeletions in specific genes were excluded from this study. Healthy fertile males between the ages of 25 and 35, who exhibited normal sperm parameters and had successfully fathered at least one healthy child within the past year, were enrolled as normal fertile controls during the same study period. Semen collection was done through masturbation after a period of abstinence lasting 2 to 7 days and allowed to become liquid at room temperature for 30 minutes. All samples of semen were initially analyzed through routine analysis of semen following the guidelines provided by the World Health Organization (WHO), and The seminal plasma was gained through centrifuging the samples of semen at 40°C and 300 rpm for a duration of 5 minutes. Subsequently, the supernatant was carefully dismissed and reserved at -80°C prior to miRNA analysis. The number of sperms was evaluated by an improved Neubauer chamber after proper dilution, and Sperm motility was evaluated using the computer-aided sperm analysis (CASA) system (LABOMED, SDC313B, Germany). The sperm appearance was evaluated by Papanicolaou staining ( 16 ), and a total of 100 sperm from different fields were assessed to determine the presence of morphological abnormalities. The SDFA kit (Cat#080910, Iran) has been applied for DNA fragmentation before and following cryopreservation. The samples were diluted accordingly based on their concentration in Ham’s F-10 medium and the agarose tube was positioned in a bath of water (90-100 C/5 minutes), followed by adding diluted samples to the agarose tube. Then, pipetting of 50 µl of the agarose-sperm mixture was done into kits’ slides and the slides were located in the slide’s coverslip. After placing the slides on a flat plate and storing in the refrigerator (4°C), the sperm microgel was appeared after 5 minutes. The slides’ coverslips were removed at ambient temperature and they were rapidly positioned horizontally. The sperm agarose layer was added to lysing solution A and maintained in a dark place, followed by incubation in denaturation solution B after 7 minutes, and for 15 minutes. The slides were first washed in distilled water (DW) for a duration of 2 minutes. Subsequently, dehydration was performed by immersing the slides sequentially in 70, 90, and 100% ethanol for 2 minutes each. Then, slides underwent incubation respectively in marked solutions C, D, and E for 75 Seconds, 3 minutes, and 2 minutes. The specimens were rinsed using a gentle flow of water and examined under an optical microscope (Olympus, Japan), about 300 sperm cells were counted. The DNA fragmentation index (DFI) represents the percentage of sperm exhibiting DNA breaks or fragmentation. The SDF frequency presents the fertility potential. Specimens with great fertility, fine fertility, and average to incomplete fertility exhibit SDF\15%, 15%\SDF\30%, and SDF [30%], respectively ( 17 ). The viability of Sperm was assessed by Eosin-B and Nigrosin marking (Merck, Germany). The defunct sperms were stained red, whereas these living sperms were not stained. In every sperm specimen, a total of 200 sperm were assessed, and the percentage of viable sperm was determined ( 18 ). The sperm mitochondrial membrane potential (MMP) was estimated considering Agnihotri et al.’s method ( 19 ). To each tube containing the sperm suspension, 5 µl of rhodamine 123 dye (Sigma-Aldrich-62669-70-9, USA) at a final concentration of 1 mg/ml was added. The tubes were then stored in the dark at a temperature of 25°C for a duration of 25 minutes. The suspension was subjected to centrifugation at 300 rpm for 10 minutes, and the supernatant was separated. Afterward, phosphate-buffered saline (PBS, 1 mL) solution was poured into the precipitate followed by centrifugation at 300 rpm for 10 minutes, and the supernatant was discarded. This step was repeated twice to ensure thorough cleaning of the sperm. Subsequently, the precipitate was mixed with 1 ml of PBS solution in the final step. After pipetting, a few microliters of the suspension were placed on a slide, which was then covered. The slide was examined using a fluorescent microscope (Olympus, DP71, Japan) equipped with a suitable filter, camera, and a magnification of 1000x. A total of 200 sperm were counted, focusing on identifying sperm with natural MMP. To evaluate sperm capacitation, Chlortetracycline (CTC) staining (Sigma, USA) was employed ( 20 ). One hundred sperms were assessed to determine the percentage of different CTC patterns for each specimen. Sperms have been classified based on the succeeding acrosomal staining patterns: i. Capacitated sperm: presence of Fluorescence-free (dark) band in the post-acrosomal region and ii. Incapacitated sperm: exhibiting Uniform bright fluorescence on the head. Seminal plasma was isolated and kept at -80°C until biochemical factor analysis. All plasma specimens were tested for total antioxidant capacity (TAC) and malondialdehyde (MAD) using commercial kits (Zell Bio GmbH, Wurttemberg and Germany). To ensure the removal of non-gamete cells, an aliquot of the semen specimens was diluted using PBS and subjected to osmotic shock. and then centrifugation was done at 13,000 rpm for 15 minutes. The pellets underwent resuspension using cell lysis buffer [1 ml, distilled H 2 O, 0.5% Triton X-100, and 0.1% sodium dodecyl sulfate (SDS)] and incubation for 60 minutes at 4°C. Confirmation of the absence of round cells was performed using optical microscopy. Subsequently, the pellets were obtained by centrifuging the samples at 13,000 rpm for 15 minutes and removing the supernatant. These pellets were then used for RNA extraction. The miRNeasy Mini Kit (Qiagen, Germany) was utilized for total RNA extraction from spermatozoa and the concentration of the extracted RNA was measured using spectrophotometry, specifically the NanoDrop ND-2000 (Thermo Fisher Scientific, USA). For cDNA synthesis, 10 ng of RNA was reverse-transcribed using the microRNA RT kit (Applied Biosystems, USA), following the provided instructions. The final reaction volume was 15 μL. Digital polymerase chain reaction (ddPCR) was utilized to assess the expression of miR-34c-5p and miR-149-5p in spermatozoa. The reaction mixture consisted of 2×ddPCR Supermix for probes (11 μL, no dUTP) from Bio-Rad, 1.5 μL of cDNA, and 1 μL of 20×TaqMan assay specific for each evaluated miRNA. The TaqMan assays used were hsa-miR-149b: 001608 (Thermo Fisher Scientific, USA) and hsamiR-34c: 000428 (Applied Biosystems, USA). The droplet generator cartridge was loaded with the reaction mix and droplet generation oil (70 μL) was added to the cartridge wells. After transferring the cartridge to the QX200 droplet generator and generating droplets, the resulting droplets (40 μL) were transferred to a ddPCR plate with 96 wells. The plate was then covered with aluminum foil and sealed using the PX1 PCR plate sealer from Bio-Rad, USA. The thermal cycling conditions consisted of an initial step of 95°C for 10 minutes, followed by 40 cycles of 94°C for 30 seconds and 60°C for 1 minute. Finally, an additional step of 98°C for 10 minutes was performed to deactivate the enzyme. The plate was maintained at 10°C for 4 hours to increase marked stabilization. The findings are illustrated as the mean ± SD. To evaluate the difference in miRNA expression levels between the two groups, a t test analysis was conducted. The correlation between miRNA expression rates and various sperm parameters was assessed using Pearson’s rank correlation. All P values were two-tailed, and a significance level of P<0.05 was considered statistically significant.