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Spermatozoan production is tightly controlled by the multistep process of spermatogenesis and spermiogenesis. Physiological and molecular disruption in spermatogenesis can lead to various reproductive disorders including male infertility. Male infertility is associated with various etiologies, but mechanism is not determined yet. MicroRNAs (miRNAs) are almost 22 nucleotides long, non-protein coding RNA that play an essential role in posttranscriptional regulations in various biological processes including spermatogenesis. The current review is aimed to summarize the recent literature on the role of miRNAs in male infertility and spermatogenesis and their potential in diagnosis, prognosis, and therapy of the disease. miRNAs have shown tremendous potential to be used as diagnostic and prognostic marker and therapeutic target in diseases related to male infertility. Experimental evidence reveals that aberrant expression of miRNAs affects different cell types and different stages of spermatogenesis, which ultimately leads to male infertility. To exploit the full potential of miRNAs, characterization of unidentified miRNAs is required to understand the miRNA-mediated regulatory mechanism related to male infertility.
*Address all correspondence to: mohsinmunawar37yahoo.com
1. Introduction
Infertility is the inability to conceive spontaneously in 1 year by a sexually active, non-contracepting couple. Primary infertility, the complete inability to conceive, ranges from 2 to 5%, while secondary infertility, indicating cessation of further fertility, globally has a prevalence rate of 20%. Approximately 15% (48.5 million) of couples cannot conceive worldwide [1].
Among infertile men, the etiology of 30−40% of cases remains unknown, so considered idiopathic male infertility [2]. The possible causes of male infertility are physiological anomalies, immunological factors, some genetic abnormalities [3], and some environmental toxicants, which can disturb the reproductive health of male by disturbing steroidogenesis, spermatogenesis, and histopathological structures [4]. Among all these possible causes of male infertility, several types of small non-coding RNAs, including microRNAs (miRNAs), are expressed in the male germ line and impair the mammalian spermatogenesis [5, 6].
Protein coding genes represent only 1.5%of the genome, which can be increased by up to 2% if untranslated regions (UTRs) are also included. The remaining 98% genome, the non-protein-coding region, has been considered a “black box” until the characterization of non-coding RNAs (ncRNAs) was done using novel nucleotide sequencing technologies. ncRNAs are present in cellular compartments, participating in multiple biological functions, including the male reproductive system, whereas some are identified in extracellular fluids, correctly named as circulating ncRNAs, where they can be detected in exosomes, bound on lipoprotein and free circulating molecules [7].
Ambros and his colleagues 1993 discovered the miRNA and then revealed the ncRNAs as gene regulators in eukaryotes [8, 9]. Lin-4 and let-7 in Caenorhabditis elegans were first discovered ncRNAs, later classified as miRNAs. Thus far, almost 38579 entries have been recorded, and among these, up to 1982 miRNAs are only from the human genome [10]. miRNAs code is either located in the intergenic region or introns of genes of the whole genome. In contrast, nearly half of total miRNAs are arranged nearby (within 50kb of another miRNA code) to form clusters. These clusters range from 2 to 46 miRNAs; C19MC is the major known cluster of miRNAs located on chromosome 19 in primates [11]. It is predicted that over 30% of mammalian and 60% of human genes are regulated by miRNAs [12, 13].
miRNAs are small RNA molecules, derived from the genome-encoded-precursor loop, consisting of almost 19–23 nucleotides. They are conserved in species and so regulate the expression of the mRNAs by recognizing the targeted mRNA through base-pair binding to the 3′UTR [14, 15].
MicroRNAs (prefix; “miR" for mature sequence whereas precursor hairpins are labeled as "mir") are named by using the number in sequential order, such as miR-1, miR-2. Despite the discovery of identical miRNAs in different organisms, the same number is assigned. Lettered suffix such as miR-1a and miR-1b is assigned to the sequences whose mature miRNAs sequences differ at one of two positions. Numbered suffix (e.g., miR-1-1 and miR-1-2) is used to label the identical miRNAs that have originated from distinct hairpin loci. The -3p and -5p suffixes refer to the arm from which the mature miRNA sequence originates. Detailed guidelines for naming the miRNAs can be seen in [16, 17].
miRNAs have a vital role as epigenetic regulators. The enzyme (Dicer protein) used for the biosynthesis of miRNA plays a very crucial role for the developmental processes [18]. During miRNAs biogenesis, following steps will be followed sequentially as elaborated in Figure 1:
Primarily, a long imperfect ~80 nt hairpin-like structure primary microRNA (pri-miRNA) is transcribed by RNA polymerase (RNA pol) II from intergenic non-coding region or intragenic region intron (untranslated region of protein-coding genes).
Secondarily, pri-miRNA undergoes two steps enzymes (Drosha and Dicer from RNase III superfamily) catalyzed reaction.
Pri-miRNA (~80-nt hairpin structure) is excised to form an intermediate precursor (pre-) miRNA (~65–70 nt) processed by Nuclear Drosha-DGCR8(DiGeorge syndrome critical region 8), then transported to cytoplasm through export factor Exportin-5.
Pri-miRNA is processed by called Dicer-TRBP (trans-activating region binding protein), a second RNase III endonuclease enzyme, to remove the terminal loop of pre-miRNA resulting in ~20 bp miRNA: miRNA* duplex.
The Dicer-TRBP processed duplex is loaded onto Ago-2 (Argonaute) protein to form an miRNA induced silencing complex (miRISC). miRISC contains mature miRNA that regulates the gene expression, while another miRNA* will be released and degraded.
Finally activated region (miRNA) of miRISC binds with 3′ UTR of targeted mRNA, ultimately leading to translational repression, deadenylation, or degradation of targeted mRNA [19].
miRNAs play a vital role in the regulation of various biological phenomena including cellular differentiation, cell growth, programmed cell death, and embryonic development [20, 21, 22], whereas up- or downregulation may lead to serious abnormality. For instance, cardiovascular disease, cancer, diabetes, renal abnormality, schizophrenia, muscular dystrophy, and reproductive disorder especially related to sperm formation and its function have been linked with biosynthesis impairment, mutation, or improper regulation of miRNA [23, 24, 25, 26, 27, 28, 29]. miRNA is not solely responsible for the whole regulatory mechanism, but their activities are also dependent on other proteins or complexes depending upon the processor stage [30].
Mature human spermatozoa retain various miRNAs; moreover, variable expression of spermatozoal RNAs has been associated with male infertility [31]. miRNAs, present in primordial germ cells, spermatogonia, primary and secondary spermatocytes, spermatids, and mature spermatozoa, play an epigenetic role in the spermatogenesis and early development as well (Figure 2) [32].
Figure 2.
miRNAs expressed during different stages of spermatogenesis in mammals.
5. miRNA as potential biomarker in male infertility
In order to confirm the male infertility, various methods have been adopted, but the most recent way is the use of biomarker. Biomarkers can be used to diagnose the disease before the appearance of physiological symptoms. miRNA is known as the most reliable biomarker due to various known properties.
5.1 Properties of biomarker
A biomarker molecule should be stable, target-specific, and sensitive to detection, noninvasive, and from an easily accessible source. In the field of medical science, protein molecules, which are easily found in serum plasma, have been used as a biomarker for the detection of pathological conditions and their prognosis. However, as a biomarker, protein molecules have some limitations such as reduced diagnostic value due to low sensitivity and specificity of their detection. On the other hand, miRNAs present in the extracellular or intracellular environment appear much earlier and detected at minute concentration, thus allowing early diagnosis. Expressions of miRNAs are often biological-stage-specific [33, 34]. Moreover, miRNA is observed as the homogeneous population of molecules, but protein molecules may become heterogeneous due to different posttranslational modifications.
miRNAs are more stable than mRNA but lesser stable than DNA. Extracellular miRNAs exist in microvesicles, exosomes, and lipoproteins or form a complex with Ago-2 protein, so protected from endogenous RNAse activity, which suggests their reliable use as a noninvasive biomarker for spermatogenic impairment [35, 36, 37, 38]. Some miRNAs are stable at room temperature, sustain a repeated freeze-thaw cycle, and got stability under harsh conditions even found in degraded RNA preparations [39, 40, 41]. In a study, exosomal miRNAs were found to be stable at 4°C for 2 weeks, at −80°C for 2 months, and at −20°C for 5 year [42]. So their relative stability enhances their usage as biomarker molecules. miRNA (both extracellular and intracellular) can be isolated by using noninvasive procedures and detected with techniques in common routines such as RT-qPCR and miRNA microarray, which is more advantageous than conventional and invasive (e.g., prostate and testicular biopsy) diagnostic procedures [43, 44, 45, 46, 47]. Despite all these characteristics of miRNAs as a biomarker, the use of protein as a biomarker may not be completely replaced, but no doubt miRNAs can be labeled as a good candidate for complementary diagnosis tools.
Circulating (serum) miRNAs were firstly used as biomarkers for the detection of diffuse large B-cell lymphoma [48]. In another study, the downregulation of miR-34c, miR-122, and miR-181a was observed in the serum of patients suffering from oligoasthenospermia [49]. Wang and his colleagues reported a decreased level of various miRNAs in azoospermic patients when compared with control [44]. Many other such published literature suggested that miRNAs are suitable biomarkers for diagnostic purposes [50, 51].
In mammals, sperm deliver genome and epigenome to the oocyte, which includes protein factors, methylation of DNA, and non-coding RNAs [52]. During mammalian spermatogenesis, mRNAs undergo posttranscriptional and posttranslational regulation in differentiating germ cells [53], so the regulatory role of miRNAs during the process of spermatogenesis can be observed clearly.
Many miRNAs as families or individual candidates are found to be very crucial in many systems of humans including the reproductive system. The role of some miRNA's family and individual miRNA candidates is summarized.
5.2 miRNA families
Let-7 family is a large family encoded by 12 different loci [54]. A well-known regulatory role of the let-7 family is gamete differentiation and embryonic implantation in humans [55, 56]. Different members of this family were identified in the testis, spermatozoa, and seminal plasma with differential expression [57, 58, 59].
miR-30-5p family (five members miR30a/b/c/d/e-5p), encoded by six loci located on chromosomes 1, 6, and 8 in humans. All miRNAs are observed in male reproductive tissue cells [60]. These miRNAs were expressed differentially in spermatozoa and seminal plasma of asthenozoospermic, oligozoospermic, and normozoospermic infertile men [39].
miR-345p/449-5p family with five miRNAs (miR-34a-5p, miR-34c-5p, miR-449a-5p, miR-449b-5p miR-449c-5p) has shown a very important regulatory role in mitosis [61]. These are highly expressed in male reproductive cells but variably expressed in pathologically infertile men especially nonobstructive azoospermic and obstructive azoospermic patients [62]. Their role was also investigated in the process of fertilization, cleavage division, and pre-implantation. These miRNAs are highly expressed in the sperm, as miR-34c-5p is the most abundant human sperm miRNA [52].
MiR-303-3p/370-3p/372-3p and 520-3p family each member of family miR-302-3p regulates over 450 target genes [63]. Members of this family were seen to be differentially expressed in the testis having histopathological impairments and in spermatozoa of patients with asthenozoospermia [62].
miR-99-5P/100-5P family is one of the most ancient families with three members (miR-99a/b/c) located in distinct chromosomal regions (ch 21, 19, and 11 respectively) in the human genome [64]. The member of this family shows differential expression in the testis of NOA patients [57].
miR-888 family (miR-888/ Mir-890, miR-891a/b, and miR-892a/b) is located on the X chromosome of primates only which has specific expression in epididymal tissues [65, 66]. Computational functional analyses predicted that the targets of the miR-888 family were related to morphogenesis of epididymis and sperm maturation [67].
5.3 Individual miRNA
hsa-miR-10b-5p, located on chromosome no 2(1q31.1), plays a pivotal role in gametogenesis as it is highly expressed in the testes. The expression level of hsa-miR-10B-5P, among others, was upregulated in round spermatids of NOA patients [57].
hsa-miR-27b-3p, present on chromosome 9 (9q22.32), was differentially expressed in the mature spermatozoa of infertile men. The expression of miR-27 has correlated with the expression of the CRISP2 (cysteine-rich secretory protein 2) gene, the highly expressed gene in the testis [59]. CGN1-CRISP2 complex has an important role in the development of the sperm tail (sperm motility) and predicts the regulatory role of miR27b-3p in spermatozoa development and motility potential [58].
miR-320a, located on chromosome 8, is highly expressed miRNA in epididymis and spermatozoa so has an important role in multiple reproductive processes [68]. Mmu-miR-320a (hsa-miR-320a) was exclusively expressed in murine Sertoli cells (SCs). The induction of apoptosis elicited miRNA expression was observed in meiotic spermatocytes and haploid spermatids. Forced expression of exogenous miR-320a in SCs may cause oligospermia and defection in sperm mobility, thus compromising the male fertility [69].
miRNAs play a vital role in the whole spermatogenic process, and their dysregulation ultimately leads to spermatogenic impairments [70]. To determine the role of miRNA, in male reproductive system, various models (mice) have been established by using the Cre/Lox system, which enables conditional manipulation of target genes, including knockout, insertion, replacement, activation, or modification of gene expression [44]. To understand the exact function of concerned miRNA in vivo, target deletion is the key step; however, in the absence of one miRNA, usually someone other has a compensatory role.
Double knockout of miR-34b/c and miR-449 in mice displayed severe disruption in spermatogenesis, dispensable fertilization, and pre-implantation development. Intracytoplasmic injection of miRNA-dKO sperm led to normal fertilization [52]. Deletion of miR34b/c and miR-449 in mice shows impaired spermatogenesis and spermatozoa maturation resulting in oligoasthenoteratozoospermia (OAT) [70]. Simultaneous inactivation of miR34b/c and miR449 in dKO mice leads to developmental defects and infertility [71, 72].
Salas-Huetos and his colleagues showed that 221 miRNAs were consistently detected in 10 healthy fertile men; among these most expressed, hsa-miR-191-5p was associated with sperm differentiation [73]. The first report on altered miRNA expression in patients with NOA showed that 154 miRNAs were differentially downregulated and 19 upregulated in NOA patients compared with fertile men [74]. Experimentally it is confirmed that specific genes are regulated by miRNAs involved in spermatogenesis. Decreased levels of estrogen-alpha (ERα) and a higher level of miR-100 and let-7b in oligospermia patients compared with fertile men indicated the regulatory role of miRNA over estrogen signaling [75, 76, 77]. Phosphatidylinositol-specific phospholipase C, X domain containing 3 (PLCXD3), expressed in spermatogenesis, was downregulated by miRNA34c-3p in severe oligozoospermic patients [78].
Conclusively, these results and some others (as shown in Table 1) confirm the regulatory role of miRNAs in male germ and somatic cells, and any change in their expression may lead to reproductive anomalies.
miRNA
Isolation of miRNA
Dysregulation
Cellular processes
Model organism
References
miR-19b and let-7a
Seminal plasma
Up-regulated
Failure in spermatogenesis
Human Oligozoospermia, non-obstructive and azoospermia
7. miRNAs: therapeutic advancements and infertility
In addition to the potential use of miRNAs as a biomarker, miRNAs have been considered therapeutic services against male infertility. miRNAs overexpression can be inhibited by anti-miR (laboratory designed molecule), or their downregulation might be supplemented with miRNAs mimics [89, 90]. A recent study showed that MRX34 (miRNA mimic of miR-34a) managed to suppress the tumor progression, and anti-miRs their inhibitory activity through binding to a miRNA, thus blocking it [91, 92, 93, 94].
In RNA-based therapeutics strategies, the challenge of degradation of the oligonucleotide by RNases in serum or cellular compartments is averted using two distinct approaches, either by altering oligonucleotide or through encapsulations of RNAs for protection (by adding phosphorothioate-like groups or developing delivery vehicles [95].
After several preclinical studies involving miRNA therapeutics, only a small number of miRNAs have moved into clinical development [96]. This is because of the many challenges, including;
Difficulty in identifying the best miRNA candidates or miRNA targets for each disease type and the absence of efficient delivery systems in specific tissues. As described below, several novel delivery systems are currently being developed to develop miRNA therapeutics using a systematic approach.
miRNA and its target identification by using bioinformatics tools, in silico interaction, and in vitro experimentation (cell culture, etc.)
Selection of miRNA chemistry such as 2’-methyl, locked nucleic acid (LNC), or phosphothionate.
Optimization of the delivery system by viral particle encapsulation or through liposomes.
In vivo testing by using a model organism so that target localization, the efficiency of delivery, and toxicity level can be reported.
Clinical trial for dose optimization, determination of the mode of drug delivery, and toxicity analysis.
Another challenge is to provide stability, accuracy in in vivo targeting, avoid toxicity and thwarting off-target complex to a therapeutic miRNA [96].
To achieve the efficient therapeutic functioning, correct in vivo dose concentration has not been determined yet [97].
miRNAs play a vital role in developing an organism and regulating many biological processes in the living organism. This chapter summarized the role of miRNAs in male infertility. Many miRNAs are reported as regulators of many functions, including developing reproductive organs, spermatogenesis, spermiogenesis, sperm mobility, and fertilization. Despite using miRNAs as biomarkers, miRNAs are also investigated as more reliable therapeutic agents for various diseases such as cancer, diabetes, and infertility.
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Written By
Mohsin Munawar, Irfana Liaqat and Shaukat Ali
Submitted: 24 May 2022Reviewed: 26 July 2022Published: 14 October 2022
Open access peer-reviewed chapter
