{"paper_id":"2d26fa28-4f74-4266-8595-b9adc84da3f7","body_text":"Male infertility is caused by many acquired, congenital, and idiopathic factors ( 1 ,  2 ). Lifestyle parameters and\nenvironmental stressors, such as nutrition smoking, and\nalcohol consumption affect the function and dynamics of\nthe reproductive system in males ( 3 ). Despite advancements in the field of human reproduction, the precise\nreason of infertility remains unknown in approximately\n30% of males experiencing infertility, underscoring the\nimportance of comprehending the specific molecular\nmechanisms that contribute to the pathogenesis of male\ninfertility ( 4 ).\nCertain research has proposed the potential involvement of small non-coding RNA molecules, referred to as\nmicroRNAs, in male infertility. MicroRNAs are present\nin every eukaryotic cell, and it has been demonstrated that\nthese molecules regulate diverse physiological processes by either up-regulating or down-regulating genes ( 5 ).\nDysregulating these molecules is associated with the onset of various diseases, including viral infections, cancers,\nand neurodegenerative disorders ( 6 ,  7 ).\nSeveral microRNAs have also been identified in samples of semen, the change in the level of\nexpression of these microRNAs is related to normal parameters of sperm such as (reduction in\nnumber, low motility and abnormal morphology) ( 8 ). Bioinformatics analysis revealed\nthat  miR-34c-5p, miR-122, miR-149b-5p, miR-181a, miR374b, miR-509-5p , and\n miR-513a-5p  have been found to be involved in spermatogenesis, cell\nproliferation, differentiation and target specific genes related to apoptosis are involved\n( 9 ).\nmiR-34 is a protected miRNA family that includes members such as  miR-34a,\nmiR-34b , and  miR-34c , which play a role in regulating cell\ncycle, apoptosis, and cellular senescence. miR-34b and miR-34c show expression in the male\ngonads ( 9 ). The target genes of the miR-34 family include various cell cycle regulators. In\nhuman testes, the predicted target genes  NOTCH1  and  NOTCH2 \nof miR-34b and miR-34c are important regulators of germ cell survival and differentiation\n( 10 ).\nmiR-149b  is as the  miR-149a/b/c  family member ( 11 ). In\nthis family,  miR-149b  has greater prominence as it can be greatly expressed\nin the spermatozoon ( 12 ). A report has demonstrated that  miR-34c  and\n miR-149b  play a crucial role in the initial cleavage division of mouse\nzygotes ( 13 ). It has also been found to be associated with clinical outcomes in patients\nundergoing intracytoplasmic sperm injection (ICSI) and  in vitro \nfertilization (IVF) procedures ( 14 ). Research revealed that elevated levels of\n miR-34b  and  miR-34c  in teratozoospermic and\nAsthenozoospermic sperm were not significantly associated with the rate of fertilization and\nhigh-quality embryos above 50%. However, these elevated levels were more likely to be\ncorrelated with higher rates of implantation, pregnancy, and live births ( 15 ).\nWe investigated the correlation between the expression of  miR-149b  and\n miR-34c  in sperm and sperm parameters in individuals with\noligoasthenoteratozoospermia.\n\nThe experimental study received approval from the Ethics Committee of Qom Azad University\n(IR. IAU.QOM.REC.1401.087). The Written informed agreement was gained from all\nparticipants involved in the study. The case-control research involved 30\nOligoAsthenoTeratospermic men who were guided to the Infertility Research Center at the\nAcademic Center for Education, Culture, and Research (ACECR), located in Qom, Iran. The\nwhole of patients was recognized with OligoAsthenoTeratospermia based on semen analysis.\nTotal sperm number <(15×10<sup>6 </sup>per ejaculate), vol (mL), total\nmotility<(40%), and morphology <(4% abnormal forms). The inclusion criteria\nfor infertile men in this study were as follows: a history of infertility for at least 1\nyear, with their wives undergoing a normal gynecological evaluation. However, men with\nconditions such as cystic fibrosis, Klinefelter syndrome, varicocele, chemotherapy,\nazoospermia factors (AZF) abnormalities, and microdeletions in specific genes were\nexcluded from this study.\nHealthy fertile males between the ages of 25 and 35,\nwho exhibited normal sperm parameters and had successfully fathered at least one healthy child within the past\nyear, were enrolled as normal fertile controls during the\nsame study period.\nSemen collection was done through masturbation after\na period of abstinence lasting 2 to 7 days and allowed to\nbecome liquid at room temperature for 30 minutes. All\nsamples of semen were initially analyzed through routine\nanalysis of semen following the guidelines provided by\nthe World Health Organization (WHO), and The seminal\nplasma was gained through centrifuging the samples of\nsemen at 40°C and 300 rpm for a duration of 5 minutes.\nSubsequently, the supernatant was carefully dismissed\nand reserved at -80°C prior to miRNA analysis. The\nnumber of sperms was evaluated by an improved Neubauer chamber after proper dilution, and Sperm motility\nwas evaluated using the computer-aided sperm analysis\n(CASA) system (LABOMED, SDC313B, Germany). The\nsperm appearance was evaluated by Papanicolaou staining ( 16 ), and a total of 100 sperm from different fields\nwere assessed to determine the presence of morphological\nabnormalities.\nThe SDFA kit (Cat#080910, Iran) has been applied\nfor DNA fragmentation before and following cryopreservation. The samples were diluted accordingly\nbased on their concentration in Ham’s F-10 medium\nand the agarose tube was positioned in a bath of water (90-100 C/5 minutes), followed by adding diluted\nsamples to the agarose tube. Then, pipetting of 50 µl\nof the agarose-sperm mixture was done into kits’ slides\nand the slides were located in the slide’s coverslip. After placing the slides on a flat plate and storing in the\nrefrigerator (4°C), the sperm microgel was appeared\nafter 5 minutes. The slides’ coverslips were removed at\nambient temperature and they were rapidly positioned\nhorizontally. The sperm agarose layer was added to lysing solution A and maintained in a dark place, followed\nby incubation in denaturation solution B after 7 minutes, and for 15 minutes. The slides were first washed\nin distilled water (DW) for a duration of 2 minutes.\nSubsequently, dehydration was performed by immersing the slides sequentially in 70, 90, and 100% ethanol\nfor 2 minutes each. Then, slides underwent incubation\nrespectively in marked solutions C, D, and E for 75\nSeconds, 3 minutes, and 2 minutes. The specimens\nwere rinsed using a gentle flow of water and examined\nunder an optical microscope (Olympus, Japan), about\n300 sperm cells were counted. The DNA fragmentation\nindex (DFI) represents the percentage of sperm exhibiting DNA breaks or fragmentation. The SDF frequency presents the fertility potential. Specimens with great\nfertility, fine fertility, and average to incomplete fertility exhibit SDF\\15%, 15%\\SDF\\30%, and SDF [30%],\nrespectively ( 17 ).\nThe viability of Sperm was assessed by Eosin-B and\nNigrosin marking (Merck, Germany). The defunct sperms\nwere stained red, whereas these living sperms were not stained. In every sperm specimen, a total of 200 sperm\nwere assessed, and the percentage of viable sperm was\ndetermined ( 18 ).\nThe sperm mitochondrial membrane potential (MMP)\nwas estimated considering Agnihotri et al.’s method ( 19 ).\nTo each tube containing the sperm suspension, 5 µl of\nrhodamine 123 dye (Sigma-Aldrich-62669-70-9, USA)\nat a final concentration of 1 mg/ml was added. The tubes\nwere then stored in the dark at a temperature of 25°C for\na duration of 25 minutes. The suspension was subjected\nto centrifugation at 300 rpm for 10 minutes, and the supernatant was separated. Afterward, phosphate-buffered\nsaline (PBS, 1 mL) solution was poured into the precipitate followed by centrifugation at 300 rpm for 10 minutes,\nand the supernatant was discarded. This step was repeated\ntwice 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\nthe suspension were placed on a slide, which was then\ncovered. 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\n200 sperm were counted, focusing on identifying sperm\nwith natural MMP.\nTo evaluate sperm capacitation, Chlortetracycline\n(CTC) staining (Sigma, USA) was employed ( 20 ). One\nhundred sperms were assessed to determine the percentage of different CTC patterns for each specimen. Sperms\nhave been classified based on the succeeding acrosomal\nstaining patterns: i. Capacitated sperm: presence of Fluorescence-free (dark) band in the post-acrosomal region\nand ii. Incapacitated sperm: exhibiting Uniform bright\nfluorescence on the head.\nSeminal plasma was isolated and kept at -80°C until\nbiochemical factor analysis. All plasma specimens were\ntested for total antioxidant capacity (TAC) and malondialdehyde (MAD) using commercial kits (Zell Bio GmbH,\nWurttemberg and Germany).\nTo ensure the removal of non-gamete cells, an aliquot of the semen specimens was diluted\nusing PBS and subjected to osmotic shock. and then centrifugation was done at 13,000 rpm\nfor 15 minutes. The pellets underwent resuspension using cell lysis buffer [1 ml,\ndistilled H 2  O, 0.5% Triton X-100, and 0.1% sodium dodecyl sulfate (SDS)] and\nincubation for 60 minutes at 4°C. Confirmation of the absence of round cells was performed\nusing optical microscopy. Subsequently, the pellets were obtained by centrifuging the\nsamples at 13,000 rpm for 15 minutes and removing the supernatant. These pellets were then\nused for RNA extraction. The miRNeasy Mini Kit (Qiagen, Germany) was utilized for total\nRNA extraction from spermatozoa and the concentration of the extracted RNA was measured\nusing spectrophotometry, specifically the NanoDrop ND-2000 (Thermo Fisher Scientific,\nUSA). For cDNA synthesis, 10 ng of RNA was reverse-transcribed using the microRNA RT kit\n(Applied Biosystems, USA), following the provided instructions. The final reaction volume\nwas 15 μL. Digital polymerase chain reaction (ddPCR) was utilized to assess the expression\nof  miR-34c-5p  and  miR-149-5p  in spermatozoa. The\nreaction mixture consisted of 2×ddPCR Supermix for probes (11 μL, no dUTP) from Bio-Rad,\n1.5 μL of cDNA, and 1 μL of 20×TaqMan assay specific for each evaluated miRNA. The TaqMan\nassays used were hsa-miR-149b: 001608 (Thermo Fisher Scientific, USA) and hsamiR-34c:\n000428 (Applied Biosystems, USA). The droplet generator cartridge was loaded with the\nreaction mix and droplet generation oil (70 μL) was added to the cartridge wells. After\ntransferring the cartridge to the QX200 droplet generator and generating droplets, the\nresulting droplets (40 μL) were transferred to a ddPCR plate with 96 wells. The plate was\nthen covered with aluminum foil and sealed using the PX1 PCR plate sealer from Bio-Rad,\nUSA. The thermal cycling conditions consisted of an initial step of 95°C for 10 minutes,\nfollowed by 40 cycles of 94°C for 30 seconds and 60°C for 1 minute. Finally, an additional\nstep of 98°C for 10 minutes was performed to deactivate the enzyme. The plate was\nmaintained at 10°C for 4 hours to increase marked stabilization.\nThe findings are illustrated as the mean ± SD. To evaluate the difference in miRNA expression levels between\nthe two groups, a t test analysis was conducted. The correlation between miRNA expression rates and various\nsperm parameters was assessed using Pearson’s rank\ncorrelation. All P values were two-tailed, and a significance level of P<0.05 was considered statistically significant.\n\nA molecular estimation of sperm  miR-34c  and  miR-149b \nwas conducted in two groups, each consisting of 30 individuals. Regarding the expression\nrate, a significant downregulation of  miR-149b  expression was observed in\nthe OligoAsthenoTeratospermia group (0.24 ± 0.07) compared to the fertile group (1 ± 0.04,\nP=0.01). Also, significant down regulation of this miR34c was shown in\nOligoAsthenoTeratospermia patients (0.6 ± 0.02) as compared to the control or fertile men\n(1 ± 0.037, P=0.03,  Fig .1A, B ).\nComparison of the  miR-149b  and  miR34c  in\nOligoAsthenoTeratospermia and fertile groups.  A.  The evaluation of the\nmiR-149b level expression in OligoAsthenoTeratospermia group by contrast to the\nfertile group.  B.  The evaluation of the miR34c level expression in\nOligoAsthenoTeratospermia group by contrast to the fertile group. *;\nP<0.05.\nThe 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\nwith of the WHO 2010. The count of sperm in the group\nof OligoAsthenoTeratospermia was significantly different from the fertile group (13 ± 1.8 vs. 88.86 ± 10.7,\nP=0.004). A meaningful decrease was observed in the\nOligoAsthenoTeratospermia group contrasted with the\nfertile group in terms of total motility (31.42 ± 9.60 vs.\n70.18 ± 11.21, P=0.002), progressive motility (20.48 ±\n8.57 vs. 44.54 ± 10.08, P=0.002), and abnormal morphology (98.02 ± 1.16 vs. 95.02 ± 3.16, P=0.002). Also,\nthere was highly meaningful decrease in viability (75\n± 6.8 vs. 89 ± 4.1, P=0.001), in OligoAsthenoTeratospermia group than fertile group. Table 1 also reveals\na meaningful increase in sperm DNA fragmentation in\nthe OligoAsthenoTeratospermia samples (34.1 ± 2.8%\nvs. 9.14 ± 2.45%, P=0.003,  Fig .2 ). Considering the results outcome, a significantly lower percentage of MMP\n(60.95 ± 7.03 vs. 75.95 ± 5.92, P=0.001,  Fig .3 ), and a\nlower sperm capacity (7 ± 4.5 vs. 12.7 ± 4.4, P=0.002)\nwas observed in the OligoAsthenoTeratospermia group\ncompared to the fertile group. There was no significant\ndifference in ejaculate volume between the two groups\nin this study (P=0.841).\nComparison of sperm parameters of fertile, and OligoAsthenoTeratospermia groups\nAll 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,\nand MMP; Mitochondrial membrane potential.\nSperm DNA fragmentation was assessed in two groups based on halo formation, using an Olympus CX21\nlight microscope (magnification: 100x, scale bar: 50 µm).  A.  Normal group\nand  B.  Oligoasthenoteratozoospermia group.\nThe mitochondrial membrane potential of sperm was evaluated in different groups using rhodamine\nstaining, employing the Olympus DP71 microscope from Japan 100x.  A. \nMagnification: Normal group (scale bar: 50 µm) and  B.\n Oligoasthenoteratozoospermia group (scale bar: 50 µm). Healthy sperm can be\nidentified by the presence of a bright green middle piece, while damaged sperm\ntypically lack this bright green middle piece.\nTable 2 displays the correlations between the examined miRNAs and seminal quality. In\nparticular,  miR-149b  exhibited a negative correlation with sperm DNA\nfragmentation (r=-0.362, P=0.001) within the sample. While, a positive correlation between\n miR-149b  and the count of sperm (r=0.515, P=0.004) and total motility\n(r=0.652, P=0.003), viability (r=0.078, P=0.001) and normal morphology (r=0.167, P=0.002).\nmiR-149b correlated significantly and positively with sperm MMP (r=0.235, P=0.001),\ncapacity (r=0.334, P=0.002). In this table,  miR34c  shows highly\nmeaningful correlation with the count of sperm (r=0.468, P=0.003), total motility\n(r=0.568, P=0.001), viability (r=0.099, P=0.002), Normal morphology (r=0.218, P=0.001),\ncapacity (r=0.522, P=0.001), MMP (r=0.537, P=0.001), and negatively with sperm DNA\nfragmentation (r=0.718, P=0.001). Furthermore, a strong correlation was observed between\nthe two miRNAs and sperm quality.\nA 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\nvs. 2.51 ± 0.13, P=0.001). Additionally, the level of MDA\nin the seminal plasma was significantly higher in the OligoAsthenoTeratospermia group (2.21 ± 0.01 vs. 1.77 ±\n0.09, P=0.001) compared to the fertile group ( Fig .4 ).\nThe evaluation of the sperm biochemical factors in OligoAsthenoTeratospermia group contrasted to the fertile group. TAC; Total antioxidant capacity, MDA; Malondialdehyde, and *; Significant difference\nbetween two groups.\nIn the OligoAsthenoTeratospermia samples, we found a positive and significant correlation\nbetween the expression of  miR-149b  and sperm TAC (r=0.441, P=0.002).\nAdditionally, a negative and significant correlation was observed between\n miR-149b  expression and MDA levels (r=-0.201, P=0.003). The results of\nPearson correlation test revealed a significant correlation between the expression of\nmiR34c and both TAC (r=0.591, P=0.001) and MDA levels (r=-0.308, P=0.004) among the\nOligoAsthenoTeratospermia samples ( Table 2 ).\nCorrelations between miR-149b, miR34c mRNA levels, sperm parameters, and biochemical factors\nThe Pearson correlation coefficient (r) was used to measure the statistical correlation. A\nsignificance level of P<0.05 was considered significant. Statistically significant (P≤0.05)\ndifferences are detailed in bold. DFI; DNA fragmentation index, MMP; Mitochondrial membrane potential, TAC; Total antioxidant capacity, and MDA; Malondialdehyde.\n\nOur study revealed a significant downregulation of  miR34c  and\n miR-149b  in the sperm samples of infertile men. Furthermore, the\nexpression levels of  miR34c  and  miR-149b  were found to be\ncorrelated with basic sperm parameters including count, motility, viability, and morphology,\nwhich is in line with studies suggesting an association between altered expression of\n miR-34c, miR149b , and sperm parameters ( 21 - 23 ).\nThe levels of the two miRNAs studied in male factor infertility sperm have been the subject\nof limited research. It has been observed that men with oligozoospermia and\nasthenozoospermia have lower levels of sperm  miR-34c  compared to\nnormozoospermic men ( 24 ). Additionally, men with idiopathic infertility have significantly\nlower levels of sperm  miR-149b  compared to men with normal semen parameters\n( 25 ).\nmiR34c  is as the commonest sperm-borne miRNA In human models ( 26 ), and\ngreatly conserved among various species, like mice, and pigs ( 13 ,  26 ). Previous studies have\ndemonstrated that  miR-34c  plays a regulatory role in the Notch signaling\npathway ( 27 ,  28 ). The Notch signaling pathway is a highly preserved system that consists of\nreceptors, ligands, transcription factors, and downstream effectors ( 29 ). This pathway plays\na crucial role in regulating various aspects of spermatogenesis, including the pace of\nspermatogenesis, proliferation, and differentiation of cells throughout the spermatogenic\ncycle ( 30 ). Dysregulated activation of the Notch signaling pathway has been associated with\ndetrimental effects on spermatogenesis. It can disrupt the maintenance and differentiation\nfactors of spermatogonial stem cells, leading to impaired spermatogenesis ( 31 ).\nThe Notch 1 receptor and its ligand Jagged 2 have been found to be expressed in\nspermatocytes and spermatids in both human and rat testes ( 32 ). When the Notch signaling\npathway is impeded  in vivo , the expression patterns of Notch components in\nthe testis are disrupted. This disruption leads to aberrations in male germ cell fate, a\nsignificant increase in germ cell apoptosis, particularly in the later stages of\nspermatogenesis, and an increase in spermatogenic maturation defects ( 33 ). The group of\nmiR-149 consists of three members in both mice and humans, namely  hsa-miR-149a,\nhsa-miR-149b , and  hsamiR-149c . However, the understanding of the\nrole of the hsa-miR-149 family in human reproduction is currently limited to findings from\nanimal model studies ( 34 ,  35 ).\nThe findings from these studies indicate that the  has-miR-149  family is\nhighly regulated in the testes and is involved in the initiation of the meiotic phase in\nmature testes ( 35 ). Additionally, these studies demonstrate that\n hsa-miR-149  is predominantly and exclusively expressed in mature testis\nspermatocytes and spermatids ( 25 ). Emerging evidence indicates that\n microRNA-149  is directly regulated by the tumor suppressor p53. Under\nconditions of cellular stress, p53 is activated to protect against malignant transformation.\nIt accomplishes this by activating DNA repair mechanisms to preserve the cell's integrity or\nby inducing apoptosis if the damage is irreparable, thereby eliminating the compromised cell\n( 36 ).\nOf considerable interest in this study is the association between sperm\n miR34c  and  miR-149b  and sperm DNA fragmentation.\nAccording to the relationship obtained, it can be concluded that the expression of\n miR-149  is positively influenced by p53, a crucial factor involved in the\nproduction of normal spermatogonial cells and the regulation of apoptosis ( 37 ).\nThis suggests that miR-149 may play a role in promoting apoptosis during the regulation of\nspermatogenesis. Additionally, our observations reveal a correlation be tween low TAC level,\nand MDA level, and the expression of both miRNAs. Thus, the connection detected between\nsperm quality and stress oxidative factor can be through stress effects on the sperm\n miRNAs 149b  and  34bc  levels. Considering the\nrelationship of  miR-34 , and stress oxidative factors, the probable\nimplication of such microRNAs on diseases leading to male infertility, like varicocele\nshould be studied.\nVaricocele is linked to elevated testicular temperature, resulting in germ cell damage and\ntemperature-dependent spermatogenic failure ( 21 ). Apoptosis, DNA damage oxidative stress\n(OS), and autophagy play a role in heat-related germ cell damage ( 38 ). Patients with\nimpaired semen parameters and varicocele showed a significant reduction in\n miR34c  levels than fertile men with normal testicular functionality and\nvaricocele. In contrast, there exists a negative correlation between the levels of\n miR-34c  and OS as well as apoptosis. Lower  miR-34a \nlevels are found in varicocele patients, suggesting that dysregulation of the\n miR-34  family is part of the pathophysiology of varicocele ( 26 ).\nVaricocele patients have decreased  miR-34a  expression levels and increased\nOS levels in their semen specimens, than healthy fertile controls ( 39 ). To understand the\nmolecular mechanisms underlying the pathophysiology of varicocele, it is important to\nconsider the anti-apoptotic effects of  miR-34 .\nThese microRNAs play a role in regulating apoptosis and may contribute to the modulation of\ncell survival pathways in the context of varicocele-induced molecular events ( 40 ). Also,\n miR-34/149  dysregulation is linked to increase germ cell apoptosis and OS\nin testis ( 21 ). According to the studies, the expression levels of  miR-149 \nand  miR-34c  in sperm are associated with the quality of early embryonic\ndevelopment in conventional IVF treatment. While sperm-borne  miR-149  is not\nessential for early embryonic development, it can serve as an additional biomarker. Notably,\nthe decreased expression of  miR-149  and  miR-34c  could\npotentially serve as an initial indicator of early embryonic development and offer valuable\ninsights into the underlying biological factors in idiopathic infertile males.\nThis study raised the possibility of stress-related miRNA changes of men’s sperms. Therefore, by conducting\ngenetic assessments of sperm DNA, it becomes possible\nto evaluate the risk across generations. Additionally, future investigations may uncover the potential value of\nepigenetic testing of sperm miRNA, providing further insights and understanding.\n\nThe findings of this research indicate a reduced expression of  miR-34c  and\n miR-149b  in sperm samples from infertile men. These results suggest that\nthe decreased expression of these miRNA family members could potentially contribute to\ndefective spermatogenesis, providing a possible explanation for infertility in affected\nindividuals.","source_license":"CC0","license_restricted":false}