Infertility infertility, included quality, motility, sperm counts and

 Infertility is one of the reproductive-related disordersthat there are several causes in its development, and both genders can play arole in the disorder. Despite the efforts made, in many cases, the causeremains idiopathic.

In maleinfertility, epigenetic factors play an important role, one of which is piRNAs thatare considered as a class of non-coding RNAs and play a crucial role inspermatogenesis. Therefore, these non-coding RNAs can serve as a novel andpromising approach to the diagnosis, treatment, and prognosis of this disorder,although this still requires much research. Our study is one of the firststudies that reviewed the most recent investigations performed on the potentialrole of piRNAs in male infertility and in human population and it can help tobetter understand the etiology of this disorder and diagnosis of patients.Key words: Infertility, male infertility,epigenetic, piRNAs, non-coding RNAs IntroductionInfertilityInfertility is described as the disability to conceive after1 year of unprotected intercourse, which it has a general prevalence of 9% (1). Primaryand secondary infertility is defined as childlessness and failure to conceiveor carry for a woman who had already had one or more children. Infertilitycan occur in different ways in both genders (2). Thisis a reproductive disease that can occur from many causes. Genetic, anatomical, immunological and endocrinological abnormalitiescan lead to infertility (3).

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Malefactors contributing to infertility, included quality, motility,sperm counts andejaculatory dysfunctions (3).  Male Infertility In20% of infertile couples, there is a defect inmale fertility, and it can reach over 40% (4). The main causes of male infertility are varicocele (37%), semen disorders (10%), testicular insufficiency(9%), obstruction (6%), cryptorchidism (6%), and other abnormality(7%). Additionally, the causeof male infertility remained unclear in approximately 25%of cases thatis called as idiopathic infertility (5). Many studies haveexamined the genetic causes of male infertility, but so far they have only beenable to identify about 15% of infertility cases (6). Consequently, there isstill a need for a better understanding of it, and we must consider otherapproaches to understanding its causes.

The epigenetic is one of thesepromising approaches that can partly explain the causes of idiopathic cases. Therefore, the understanding of the epigeneticbasis of male infertility can be essential to appropriatelymanage an infertile patients.  The role of the epigeneticfactors in male infertility In fact, theepigenetic modifications are alterations in phenotype caused by mechanisms that do not change the DNA sequence (7). These modificationsin sperm areexcluded for two reasons. First, the occurrence of the eliminating theepigenetic marks in primordial germ cells (PGCs). Second, the occurrence of thegenomic condensation and reorganization in male germ cell nuclei (8).

The most common of thesechanges includeDNA methylation, Histones modifications, transition from canonical histones toprotamines and non-coding RNAs (ncRNAs) (9).  The event of adding a methyl group to the 5’cytosine pyrimidine ring, called as DNA methylation, commonly occurs in hotspot regions (CpG islands) (10). Abnormalities in thisprocess can affect significant processes, including spermatogenesis, and maycause male infertility (11, 12). Also, in the process ofreplacing histones to protamines, numerous proteins are involved, including P1and P2. Mutations in proteins can lead to sperm abnormalities and infertility (13).  The non-coding RNAs (ncRNAs) are considered asgene expression regulators involved in different cellular processes.

The mostimportant ncRNAs are miRNA, siRNA, piRNA and lncRNA the differences of whichare presented in the following table (14, 15) : The piRNAs as a non-coding RNA In 2006, the first, a novel class ofsmall noncoding RNA was isolated from the mouse testis and Drosophila germcells that were called piRNAs (PIWI interacting RNAs) (16, 17). The length of the piRNAis about 26-33 nucleotides which about 86% of them, there is a uracildeflection at the 5′ end and play a crucial role in spermatogenesis (18). According to origin ofpiRNAs, they can be divided into three classes: a-piRNAs originated from retrotransposons,b-piRNAs originated from mRNA, c-piRNAs originated from lncRNAs (Long noncodingRNAs) (19).The mostcommon piRNAs are derived from retrotransposons and are linked to AGO (Argonaute),PIWI (P-element Induced WImpy Testis in Drosophila) and AUB (Aubergine)proteins (20, 21)  The repression of transposon activity in germcells is main role of these small non-RNAs (22). Biogenesis of piRNA The main distribution sites piRNA arethe animal testes spermatogonial cells and ovarian oocytes and in drosophila folliclecells (somatic cells).

There are two main pathways of the piRNA biogenesis: Ingerm cells, the AUB dependent piRNA pathway (secondary piRNA processing) isactive, while in somatic cells, only pathway for producing piRNAs is the PIWI dependentpathway (primary piRNA processing) (23). The primary antisensetranscripts of piRNA are preferably binds to PIWI protein. This complex iscalled as piRISCs (piRNA-induced silencing complexes) which breaks the sensetranscript of transposons at positions 10 and 11 and generate the 5′ end of asense Ago3-associated piRNA. In the secondary piRNA processing that is known as the Ping-Pong cycle,proteins of AUB and Argonaute 3 (AGO3) are involved (24). The AUB protein plays asimilar role to PIWI and forms the 5? end of piRNAs that associated with AGO3 (25).

This complex has tworoles: On the one hand, it produces the 5? end of the antisense piRNAs by thecleavage of antisense piRNA precursors and then these are loaded onto AUB, andon the other hand, it produces secondary piRNAs (Figure 1). The HEN1 protein mediated2??O-methylation of the 3? end of piRNA. Also, Mili and Miwi2are two members of the mouse Piwi proteins that by processing of transposableelements (TEs) produce piRNAs. This occurs in cytoplasmic granules called pi-bodiesand piP-bodies (26). The role of piRNAs inmale infertility The piRNAs canplay different roles in biological processes, including: Sex Determination, Gene Silencing,Epigenetic Regulation and Cancer. Their mostimportant role is to protect the gametes genome from the transposon invasionand is performed by PIWI-piRNA complexes with silencing their transcripts (25).

Consequently, piRNAs are usually used in the genome, but the aberrantexpression of each of the genes involved in biogenesis and function can lead tomodifications in the genome and different disorders. One of these disorders ismale infertility. In Figure 2, the most importantresearch performed on male infertility and piRNAs is summarized:The Moloney leukemia virus 10-like 1(MOV10L1) gene is a gene associated with the biogenesis of piRNA that plays arole in the primary and secondary processing (27). It can help to primarypiRNAs for binding to the PIWI protein.

Some studies have confirmed thatseveral polymorphisms of this gene have a remarkable increase in infertile men (28). In human, theassociation of four human PIWI proteins (HIWI, HILI, HIWI2 and PIWIL3) in malefertility has been shown. In 2010 and 2017, investigations on Chinese andIranian populations with non-obstructive azoospermia revealed independently arelationship between HIWI2 rs508485 (T>C) and non-obstructive azoospermiaand this variant can be considered as a risk factor for male infertility (29, 30).

Furthermore, Transposons are repetitiveelements that use the genome of a host cell to survive and amplification. Forprotecting of the genomes of gametes from their invasion, PIWI-piRNA complexestarget them to silence of their transcripts. LINE-1 (L1) is one of thetransposons studied that by performing the examinations on patients withcryptorchidism revealed that a consequence of alterations in the Piwi-pathwayand derepression of transposable elements in these patients is infertility (31). These studies indicatethat piRNAs may play a crucial role in maleinfertility. The potential role of piRNAs as a diagnosticbiomarker for male infertility According to the WHO, diagnosis of maleinfertility is based on the semen parameters, which include the following:motility,sperm concentration, seminal volume, pH and morphology (32).

Some studies have shownthat sperm analysis cannot be used accurately for diagnosis between fertile andinfertile men (33). Therefore,identification of non-Invasive seminal Biomarkers, can solve this problem. Cellfree RNA and non-coding RNAs can be important as non-invasive biomarkers incontrolling pregnancy and diagnosing reproductive-related disorders (34, 35).  In 2015, Hong and colleagues identified fivepiRNAs by examining seminal plasma samples in infertile patients, which can beused as diagnostic biomarkers for the detection of infertile men (36). Also, another study in patientswith idiopathic male infertility who experienced the first ICSI course, suggestedthat there is a relationship between spermatozoa piRNA levels (piR-31704 andpiR-39888)  and sperm concentration (37).

Thus, these piRNAs canplay an important role in the fertilization process.  The piRNAs and DNA methylation DNA methylation, as one of theepigenetic markers, is associated with many disorders. In germ cells,methylation is involved in the silencing of TEs, genomic imprinting, and DNAcompaction. Early studies have shown that there is an abnormal methylation inmen with low sperm quality (38).

These changes in genesinvolved in the processing of piRNAs can be associated with human spermatogenicdisorders. A recent study on peripheral blood samples of infertile men,showedthat rs10773767 and rs6982089 were twosingle nucleotide polymorphisms (SNPs) in PIWIL1 and PIWIL2, respectively, and thesepolymorphisms were allele-specific methylation-sensitive (39). Thus, DNA methylationchanges in these genes are associated with spermatogenic disorders.

Also, TDRD1(a Tudor-domain-containing protein) which contributes to the MIWI function, someof its variants may be associated with a risk of defects in spermatogenesis andinfertility (40, 41). Additionally, Consideringthe relationship between the modified pattern of methylation of TEs and maleinfertility (41, 42), these alterations maybe due to changed expression of the piRNAs. These results show that the studyof methylation patterns in the pathways of piRNAs processing can help us betterunderstand the etiology of male infertility. The targeting of piRNAs as noveltherapiesThe ncRNAs, as one of the new therapies, can be used in many disorders. Ithas been made many improvement in the field of miRNAs clinical applications, suchas the use of Miravirsen (an oligonucleotide with 15-nucleotide) in thetreatment of HIV infections (43) and miRNAs replacement and inhibition techniquesin the treatment of many cancers (44, 45). Also, based on the roles of piRNAs and PIWIproteins, there are two approaches to change the expression of piRNAs: antibodiescan be useful against PIWI proteins at post-translational levels, while artificialpiRNAs are a good option for both transcriptional and post-translationalapproaches (Figure 3) (46).

The anti-PIWI antibody prevents theformation of the piRISC complex, hence, the piRNA expression can be changed. Onthe other hand, if the expression of a piRNA is reduced in the disorder, thetransposon levels may be increased, in this case, the use of artificial piRNAis one of the approaches that can be used. Also, in germ cells, transposons mayalter the DNA methylation and inducing methylation through artificial piRNAscould lead to gene silencing (47). Therefore, these are important in the assessmentof hereditary epigenetic alterations. However, these new approaches are in theearly stages and require more extensive research.  One of the benefits of understandingthe epigenetic abnormalities is that epigenetic modifications, unlike genetic mutations,can be modified using specific drugs.

Therefore, with a complete understandingof these modifications, treatment for epigenetic-related diseases can beachieved. The ncRNAs are the most common epigenetic regulators that their role hasbeen identified in many disorders. Among ncRNAs, piRNAs play an important rolein spermatogenesis and are candidates for further research on male infertility.

The studies presented in this review showed that investigating the role ofpiRNAs in male infertility could be useful for multiple causes. First, determinea non-invasive biomarker for early detection of male infertility. Second,discover the causes of idiopathic male infertility. Also, piRNAs can be used todiagnose different types of infertile patients.

For example, piR-30198 is oneof piRNAs used for this purpose. This biomarker is able to distinguish between thetwo disorders related to male infertility, namely, azoospermia andasthenozoospermia (36).  AcknowledgementsThe authors appreciate the valuable contributions of theexperts in the Research and Clinical Center forInfertility of Yazd, especially in molecular and cytogeneticlaboratories. Financial support or sponsorshipThere is no support in relation to thispaper.  Conflict of interest statementIn the present study, all the authorsdeclare to have no actual or potential conflict of interest.