International Journal of

 
1. Introduction
On average, it is estimated that infertility occurs in 10 to 15% of couples and 50% of infertility cases are due to male factor. The causes of 65-70% of male infertility cases is unknown and the correct mechanism has not been defined (1, 2). Genetic factors can be one of the causes of male infertility (3). But the role of miRNAs in the process of spermatogenesis and male infertility is very important. MiRNAs are non-coding RNAs, and play an important role in regulating gene expression (4, 5). MiRNAs regulate gene expression in two ways: by suppressing transcription and translation (RNAi) (6) or by activating transcription (RNAa) (7-9). The expression of miRNAs was shown in some types of male germ cells (10-14). The hsa-miR-449 was first detected in the fetal brain of mice(15, 16).
The hsa-miR-449 family has three members in mice and human, namely hsa-miR-449a, hsa-miR-449b, and hsa-miR-449c. These miRNAs have conserved sequence among different species and are located on the second intron of the Cdc20b gene. While the three hsa-miR-499 (miR-499 a, b, c) members have the same seed sequences, hsa-miR-449 members play the main role in the control of cell cycle and differentiation of epidermis (17-19).
In this regard, studies have shown that the hsa-miR449 family (hsa-miR-449a, hsa-miR449b, and hsa-miR-449c) and the hsa-miR-34 b, c (hsa-miR-34b-3p and hsa-miR-34c-5p) contain an identical seed sequence and have same sequence with the another miRNA, hsa-miR-34a-5p (17-21). About in addition, with respect to the role of hsa-miR-449 in spermatogenesis and male infertility, it was shown that the hsa-miR-449 family highly express in spermatocyte, spermatid, and adult testis. In one study was reported that, inactivation of both the hsa-miR34-b,c and hsa-miR-449 causes low sperm counts, motility   and high abnormal sperm morphology in animal models (22). CpG methylation of  mentioned genes is one possible reason for the their underexpression (20, 22), so that, under expression and high methylation of hsa-miR-449 (a, b, c) (as an important tumor-suppressor gene) have been shown in various cancers (23, 24). Besides, methylation of their promoter region has also been shown to be one of the mechanisms of expression reduction in adition to playing an important role in carcinogenesis of these microRNAs. Up to now, the patterns of hsa-miR-449 family expression and methylation has not been reported in different groups of infertile men and all the information in this regard has been based on animal models (25). Therefore, in  this study we  evaluated  the expression and methylation pattern of hsa-miR-449 family in infertile men.
 
2. Materials and Methods
2.1. Subject recruitment and sampling
In this case-control study, 74 infertile men with idiopathic asthenozoospermia (n = 14), teratozoospermia (n = 16), asthenoteratozoospermia (n = 28), and oligoasthenoteratozoospermia (n = 16) were collected during 2018-2019 from the ACECR Telemedicine Infertility Center Qazvin, IRAN, based on the WHO criteria. The condition of infertile men was as follows: a history of infertility for at least 1 yr with their wives having a normal gynecological evaluation. However, infertile men conditions such as cystic fibrosis, Klinefelter syndrome, varicocele, chemotherapy, AZF, and genes micro deletions were not included in this study. In addition, 30 fertile healthy men were recruited as the control group. A questionnaire was designed to evaluation of  the patients and controls’ information,  including medical history, occupational and environmental condition, smoking condition (an adult who has smoked 100 cigarettes in his lifetime and who currently smokes cigarettes)and reproduction status (Table I). Patients were advised not to have sexual abstinence for three days before sampling. After sampling, semen samples were stored at 37ºC for 30 min to complete the liquefaction. Then, based on  WHO criteria sperms concentration, motility, and morphology were evaluated (26).
 
2.2. RNA extraction and qRT-PCR
After liqufication, we centrifuged the semen samples for 10 min at 500 g. Then 1 ml FSB (Merck, Germany) was added to sperms pellet and somatic cells removed.TRIZOL reagent was used for isolation of the total RNA from the sperms based on kit protocols (Invitrogen Life Technology Co., USA). hsa-miR-449-a (MI0001648), hsa-miR-449-b (MI0003673), and hsa-449-c (MI0003823) were studied in this research. hsa-miR30a-5p (MIMAT0000087) and hsa-miR100-5p (MIMAT 0000102) were used as internal controls. Rotor gene-Q real-time PCR system (Qiagene, Germany) was used to quantifing of the RNA expression. Total amount of master mix was 10 µl and  included 1 µl of reverse and forward primers (Exiqon, Denmark), 5 µl of Ampliqon real Q plus 2× master mix green (Ampliqone, Denmark), and 4 µl of diluted cDNA. For enzyme activation we incubated master mix for 15 min at 95ºC. Then reaction was runned in 40 cycle for 20 sec at 95ºC and 60 sec at 60^ºC. Ct values was used for evaluation of expression rate of studied miRNAs. hsa-miR30a-5p and hsa-miR100-5p were used as the endogenous controls. The 2^-△Ct method used for expression rate detection of target genes in comparision to internal controls.
 
2.3. DNA extraction and bisulfite modification
Phenol-chloroform method was used for DNA extraction. 2-5 µg of extracted DNA was bisulfited by using of EpiJET™ Bisulfite Conversion kit (Thermo Fisher Scientific, Inc).
 
2.4. MSPCR
The hsa-miR-449 methylation status was evaluated in all studied samples. For the targeted site, methylation-specific primers were designed. While the methylated primers were Forward: 5’-CGTTCGTTAATTTTTTCGTTTTTTGTCGC-3’) and Reverse: 5ꞌ-GTCAAAACCCGAATAAAATTCCCCGACG-3’, the unmethylated primers were Forward: 5’-TTGTTTGTTAATTTTTTTGTTTTTTGTTGT-3’ and Reverse: 5’- ATCAAAACCCAAATAAAATTCCCCAACA-3’. Methylation-specific PCR (MSP) was used to evaluation of methylation status of hsa-miR-449-abc promoter region. 1 µL of bisulfit converted DNA with methylated and unmethylated primers was amplifiedin in final 10 µL reaction mixture. "The PCR conditions for methylation status were: 95ºC for 15 min (Hot start), followed by 35 cycles at 95ºC for 20 sec (denaturation), 56.5ºC for 45 sec (annealing), and 72ºC for 45 sec (extension). PCR condition for unmethylated- primers was the same as the methylated condition except for the number of cycles and annealing temperature (60ºC)" (27).

 
2.5. Ethical considerations
This case-control study was approved by the Ethics Committee of Qazvin university of medical science with dedicated ID IR.QUMS.REC.1396.294. The recruited patients gave their informed written consent.
 
2.6. Statistical analysis
The GraphPad software (GraphPad PRISM V 5.04) was used for the data analysis. The analysis of variance test (ANOVA) was used for the evaluation of the miRNAs expression levels difference among the different studied groups. The frequency of promoter methylation pattern was evaluated by a nonparametric test (Kruskal-Wallis). The correlation between the miRNA expression rate, methylation with different sperm parameters was analyzed by Spearman's rank correlation. All P-values were two-tailed, with p < 0.05 considered as statistically significant.
 
3. Results
3.1. Expression of miRNAs in sperm samples of studied groups
About the expression rate, we saw significant downregulation of the hsa-miR-449a expression in infertile group (0.24 ± 0.2) in comparison to the control groups (0.98 ± 0.37) (p = 0.0001). Also, significant down-egulation of this miRNA was shown in oligoasthenoterathospermia (0.05 ± 0.02), asthenospermia (0.24 ± 0.11), terathospermia (0.28 ± 0.2), and asthenoterathospermia (0.3 ± 0.11) as compared to the control group (0.98 ± 0.37) (F = 7.1 p = 0.0001). MirRNA expression ratio had not significant difference  among the four infertile groups, (p = 0.21) (Figure 1A). The expression of hsa-miR-449b was downregulated significantly among the oligoasthenoterathospermia groups (0.022 ± 0.01) in comparison to the asthenospermia (0.04 ± 0.02), terathospermia (0.04 ± 0.032), asthenoterasthospermia (0.03 ± 0.02), and the control group (0.12 ± 0.09) (p = 0.0001, F = 2.9) (Figure 1B). However, in the case of hsa-miR-449c, oligoasthenoterathospermia patients showed significant downregulation of this miRNA (0.04 ± 0.03), also we observed this downregulation among the asthenospermia (0.15 ± 0.14), terathospermia (0.15 ± 0.14), and asthenoterasthospermia (0.1 ± 0.09) groups in  compared to the control group )0.37 ± 0.01) (F = 5.04, p = 0.001) (Figure 1C). The average of the expression of the three studied miRNAs in fertile men showed that hsa-miR-449a had the highest levels of expression in these individuals )0.98 ± 0.3), followed by hsa-miR-449c (0.37 ± 0.01) and hsa-miR-449b (0.12 ± 0.09) (Figure 2). As displayed in Figure 2, among the three studied miRNAs, hsa-miR-449b had the lowest expression ratio in infertile men especially in oligoasthenotaratospermic men (0.022 ± 0.01).
 
3.2. Investigating the relationship between miRNAs expressions and parameters of sperm
In all studied samples, we observed significant correlation between the expression of hsa-miR-449a and the  sperm progressive motility (r = 0.44, p = 0.0001), sperm count (r = 0.2, p = 0.03), and normal morphology (r = 0.46, p = 0.0001). The pearson test results showed, significant correlation between the expression of hsa-miR-449b miRNA and the spem progressive motility (r = 0.55, p = 0.0001), sperm count (r = 0.59, p = 0.0001), and normal morphology (r = 0.58, p = 0.0001). Also, there was significant correlation between the sperm count (r = 0.38, p = 0.0001), progressive motility (r = 0.22, p = 0.0322), and normal morphology (r = 0.32, p = 0.0001) with hsa-miR-449c experssion among all studied samples (Figure 3 A-I).
 
3.3. The methylation patterns of the promoter region of the hsa-miR-449a,b,c 
MSP results showed that, 60.8% and of the patients and 23.3% of the controls had methylated allele (p = 0.0001). The unmethylated allele was detected in all patients and controls. Oligoasthenoteratospermic (81.2%) and asthenothratospermic (61.2%) patients showed the highest frequency of methylation (Table II) (Figure 4 A, B). There was not significant correlation between hsa-hsa-miR-449abc promoter methylation with the liquefaction time (r = 0.022, p = 0.065) and viscosity (r = 0.969, p = 0.65). However, we observed a significant negative correlation between sperm count (r = -0.235, p = 0.003), progressive motility (r = -0.375, p = 0.0001), and normal morphology of sperms (r = -0.356, p = 0.0001) with  methylation pattern of hsa-miR-449abc promoter. Also, there was significant negative correlation between methylation of hsa-miR-449abc promoter region and hsa-miR-449a (r = -0.110, p = 0.02), hsa-miR-449b (r = -0.245, p = 0.01), and hsa-miR-449c (r = -0.348, p = 0.005) expression ratio (Table III). The results of the effect of smoking on methylation status of mir-449-abc promoter showed the high frequency of methylation in men who smoked (87.7%) in comparison to men who did not (75.2%, χ² = 4.2, p = 0.003).






4. Discussion
In our project, significant downregulation of  the hsa-miR-449abc was seen in the sperm samples of infertile men. Briefly, miRNAs have the main role in incidence of male infertility through the gene expersion regulation (22). The miR-449 family have three members in mice and humans, hsa-miR-449a, hsa-miR-449b, and hsa-miR-449c. Three hsa-miR-499 (miR-499a, b, c) members are transcribed simultaneously and have the same seed sequence (17, 20).
The roles of hsa-miR-449 family in human reproduction, so far it has only been based on the results of animal models studeis (21). These studies report that hsa-miR-449 family is one of the most upregulated testicular miRNA and have major role in the initiation of  meiotic phase in the adult testes. Also, this group showed that “hsa-miR-449 is predominantly and exclusively expressed in spermatocytes and spermatids in the adult testes” (21). Liu and colleague showed a presence of hsa-miR-449 family in spermatozoa, but their absence in oocytes (28). In the line of above study, our results showed expression of these miRNAs was shown in sperm^,s s of the control (fertile) group, and hsa-miR-449a had the highest levels of expression. We also saw a significant downregulation of these miRNAs in the sperm samples of infertile patients, and hsa-miR-449b had the highest reduction. Another study reported  abnormal different sperm parameters in hsa-miR-34bc/449 knock-out mice with oligoteratoasthenospermia. They reported  that deficient mice had spermatozoa maturation stages problme, but hsa-miR-449 or hsa-miR-34 cluster Knockout (KO) mice showed no explained phenotype (22). This is because hsa-miR-449 and hsa-miR-34b/c have the same “seed sequence,” which maps between the second and seventh nucleotides; this core element is necessary for base pairing with target mRNAs, and can target the same set of mRNAs. On the other hand, hsa-miR-449 and hsa-miR-34b/c have the same expression profiles within testicular formation, and they are located to the precise identical spermatogenic cell types, such as spermatocytes and spermatids. In this regard, it is better in future studies that the expression pattern of hsa-miR-449 family and hsa-miR-34b/c be evaluated simultaneously. Interestingly, along with Comazzetto and colleague’s 2014 study, the highest downregulation of hsa-miR-449 family in oligoteratoasthenospermic men was observed, also we saw significant correlation between expression of this gene family and sperm progressive motility, count, and normal morphology (29).
Methylation is one of the mechanisms in expression regulation of miRNAs (30). Expression and methylation of the hsa-miR-449a,b,c as the tumor suppressor genes has been reported in different cancers such as prostate (31), hepatocellular carcinoma (32), and osteosarcoma (18). Also, the role of methylation in regulating the expression of miRNAs involved in spermatogenesis and infertility has also been demonstrated (33). To the best of our knowledge, there is no clinical data on the methylation status of hsa-miR-449a, b, c promoter in human sperm. In the study of hsa-miR-449family methylation, our results showed that, there was high frequency of methylation in infertile men (60.8%) specially in the oligoasthenoteratospermia patients. Interestingly, oligoasthenoteratospermia patients had the highest gene expression reduction. Also, in this research we observed a negative correlation between underexperssion and hypermethylation of hsa-miR-449a, b, c. This suggests that hypermethylation can be one of the downregulation mechanisms of these gene family. Further, we observed hypermethylation of promoter region among the 87.7% of smokers. Smoking and obesity have the major effect on the quantity of sperm miRNA (34). Recently, in the sperm samples of men exposed to lifestyle stress Dickson et al. reported under expression and hyper methylation of hsa-miR-449 and hsa-miR-34 (35). Acording to what was said, it is better to study the effect of other environmental factors on the rate of the hsa-miR-449a, b, c methylation.
 
5. Conclusion
Results of this research showed under-experssion and hypermethylation of the hsa-miR-449 a,b,c in sperm samples of infertile men.  According to the results of this study, it can be said that one of the possible causes of defective spermatogenesis in infertile people can be reduced expression and increased methylation of these miRNAs family.
 
Acknowledgments
We appreciate all of the participants in current study. We also thank Cellular and Molecular Research Center of Qazvin University of Medical Science for providing laboratory facilities.
 
Conflict of Interest
The authors declare that there is no conflict of interest.