Infertility or carry for a woman who had


is described as the disability to conceive after 1 year of unprotected
intercourse, which it has a general prevalence of 9% (1). Primary
and secondary infertility is defined as childlessness and failure to conceive
or carry for a woman who had already had one or more children. Infertility
can occur in different ways in both genders (2). This
is a reproductive disease that can occur from many causes. Genetic, anatomical, immunological and endocrinological abnormalities
can lead to infertility (3). Male
factors contributing to infertility, included quality, motility,
sperm counts and
ejaculatory dysfunctions (3).

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Male Infertility


In 20% of
infertile couples, there is a defect in
male 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 cause
of male infertility remained unclear in approximately 25%
of cases that
is called as idiopathic infertility (5). Many studies have
examined the genetic causes of male infertility, but so far they have only been
able to identify about 15% of infertility cases (6). Consequently, there is
still a need for a better understanding of it, and we must consider other
approaches to understanding its causes. The epigenetic is one of these
promising approaches that can partly explain the causes of idiopathic cases. Therefore, the understanding of the epigenetic
basis of male infertility can be essential to appropriately
manage an infertile patients.


The role of the epigenetic
factors in male infertility


In fact, the
epigenetic modifications are alterations in phenotype caused by mechanisms that do not change the DNA sequence (7). These modifications
in sperm are
excluded for two reasons. First, the occurrence of the eliminating the
epigenetic marks in primordial germ cells (PGCs). Second, the occurrence of the
genomic condensation and reorganization in male germ cell nuclei (8). The most common of these
changes include
DNA methylation, Histones modifications, transition from canonical histones to
protamines 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 hot spot
regions (CpG islands) (10). Abnormalities in this
process can affect significant processes, including spermatogenesis, and may
cause male infertility (11, 12). Also, in the process of
replacing histones to protamines, numerous proteins are involved, including P1 and
P2. Mutations in proteins can lead to sperm abnormalities and infertility (13).  The non-coding RNAs (ncRNAs) are considered as
gene expression regulators involved in different cellular processes. The most
important ncRNAs are miRNA, siRNA, piRNA and lncRNA the differences of which
are presented in the following table (14, 15) :


The piRNAs as a non-coding RNA


In 2006, the first, a novel class of small noncoding RNA was
isolated from the mouse testis and Drosophila germ cells that were called piRNAs
(PIWI interacting RNAs) (16, 17). The length of the piRNA
is about 26-33 nucleotides which about 86% of them, there is a uracil
deflection at the 5′ end and play a crucial role in spermatogenesis (18). According to origin of
piRNAs, they can be divided into three classes: a-piRNAs originated from
transposons, b-piRNAs originated from mRNA, c-piRNAs originated from lncRNAs
(Long noncoding RNAs) (19).


Biogenesis of piRNA


The main distribution sites piRNA are the animal testes
spermatogonial cells and ovarian oocytes and in drosophila follicle cells (somatic
cells). There are two main pathways of the piRNA biogenesis: In germ cells, the
AUB dependent piRNA pathway (secondary piRNA processing) is active, while in somatic
cells, only pathway for producing piRNAs is the PIWI dependent pathway (primary
piRNA processing) (20). The primary antisense
transcripts of piRNA are preferably binds to PIWI protein. This complex is
called as piRISCs (piRNA-induced silencing complexes) which breaks the sense
transcript of transposons at positions 10 and 11 and generate the 5’ end of a
sense 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 (21). The AUB protein plays a
similar role to PIWI and forms the 5? end of piRNAs that associated with AGO3 (22). This complex has two
roles: On the one hand, it produces the 5? end of the antisense piRNAs by the
cleavage of antisense piRNA precursors and then these are loaded onto AUB, and
on the other hand, it produces secondary piRNAs (Figure 1). The HEN1 protein mediated
2??O-methylation of the 3? end of piRNA. Also, Mili and Miwi2
are two members of the mouse Piwi proteins that by processing of transposable
elements (TEs) produce piRNAs. This occurs in cytoplasmic granules called pi-bodies
and piP-bodies (23).


The role of piRNAs in
male infertility


The piRNAs can
play different roles in biological processes, including: Sex Determination, Gene Silencing,
Epigenetic Regulation and Cancer. Their most
important role is to protect the gametes genome from the transposon invasion
and is performed by PIWI-piRNA complexes with silencing their transcripts (22).
Consequently, piRNAs are usually used in the genome, but the aberrant
expression of each of the genes involved in biogenesis and function can lead to
modifications in the genome and different disorders. One of these disorders is
male infertility. In Figure 2, the most important
research 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 a role in the primary and secondary
processing (24). It can help to primary
piRNAs for binding to the PIWI protein. Some studies have confirmed that
several polymorphisms of this gene have a remarkable increase in infertile men (25). In human, the
association of four human PIWI proteins (HIWI, HILI, HIWI2 and PIWIL3) in male
fertility has been shown. In 2010 and 2017, investigations on Chinese and
Iranian populations with non-obstructive azoospermia revealed independently a
relationship between HIWI2 rs508485 (T>C) and non-obstructive azoospermia
and this variant can be considered as a risk factor for male infertility (26, 27).

Furthermore, Transposons are repetitive elements that use
the genome of a host cell to survive and amplification. For protecting of the
genomes of gametes from their invasion, PIWI-piRNA complexes target them to
silence of their transcripts. LINE-1 (L1) is one of the transposons studied
that by performing the examinations on patients with cryptorchidism revealed that
a consequence of alterations in the Piwi-pathway and derepression of
transposable elements in these patients is infertility (28). These studies indicate
that piRNAs may play a crucial role in male


The potential role of piRNAs as a diagnostic biomarker for
male infertility


According to the WHO, diagnosis of male infertility is based
on the semen parameters, which include the following: motility,
sperm concentration, seminal volume, pH and morphology (29). Some studies have shown
that sperm analysis cannot be used accurately for diagnosis between fertile and
infertile men (30). Therefore,
identification of non-Invasive seminal Biomarkers, can solve this problem. Cell
free RNA and non-coding RNAs can be important as non-invasive biomarkers in
controlling pregnancy and diagnosing reproductive-related disorders (31, 32).  In 2015, Hong and colleagues identified five
piRNAs by examining seminal plasma samples in infertile patients, which can be
used as diagnostic biomarkers for the detection of infertile men (33). Also, another study in patients
with idiopathic male infertility who experienced the first ICSI course, suggested
that there is a relationship between spermatozoa piRNA levels (piR-31704 and
piR-39888)  and sperm concentration (34). Thus, these piRNAs can
play an important role in the fertilization process.


The piRNAs and DNA methylation


DNA methylation, as one of the epigenetic markers, is
associated with many disorders. In germ cells, methylation is involved in the
silencing of TEs, genomic imprinting, and DNA compaction. Early studies have
shown that there is an abnormal methylation in men with low sperm quality (35). These changes in genes
involved in the processing of piRNAs can be associated with human spermatogenic
disorders. A recent study on peripheral blood samples of infertile men, showed that rs10773767 and rs6982089 were two single nucleotide polymorphisms (SNPs) in PIWIL1 and
PIWIL2, respectively, and these polymorphisms were allele-specific
methylation-sensitive (36). Thus, DNA methylation
changes in these genes are associated with spermatogenic disorders. Also, TDRD1
(a Tudor-domain-containing protein) which contributes to the MIWI function, some
of its variants may be associated with a risk of defects in spermatogenesis and
infertility (37, 38). Additionally, Considering
the relationship between the modified pattern of methylation of TEs and male
infertility (38, 39), these alterations may
be due to changed expression of the piRNAs. These results show that the study
of methylation patterns in the pathways of piRNAs processing can help us better
understand the etiology of male infertility.


The targeting of piRNAs as novel therapies


use of piRNAs is one of the therapeutic approaches that can be used in many
disorders. Based on the roles of piRNAs and PIWI proteins, there are two approaches
to change the expression of piRNAs: antibodies can be useful against PIWI
proteins at post-translational levels, while artificial piRNAs are a good
option for both transcriptional and post-translational approaches (Figure 3). The
anti-PIWI antibody prevents the formation of the piRISC complex, hence, the
piRNA expression can be changed. On the other hand, if the expression of a
piRNA is reduced in a disorder, the transposon levels may be increased, in this
case, the use of artificial piRNA is one of the approaches that can be used. Also,
in germ cells, transposons may alter the DNA methylation and inducing
methylation through artificial piRNAs could lead to gene silencing (40). Therefore, these are important in the assessment
of hereditary epigenetic alterations. However, these new approaches are in the
early stages and require more extensive research.



One of the benefits of understanding the epigenetic
abnormalities is that epigenetic modifications, unlike genetic mutations, can
be modified using specific drugs. Therefore, with a complete understanding of
these modifications, treatment for epigenetic-related diseases can be achieved.
The ncRNAs are the most common epigenetic regulators that their role has been
identified in many disorders. Among ncRNAs, piRNAs play an important role in
spermatogenesis and are candidates for further research on male infertility. The
studies presented in this review showed that investigating the role of piRNAs
in male infertility could be useful for multiple causes. First, determine a
non-invasive biomarker for early detection of male infertility. Second,
discover the causes of idiopathic male infertility. Also, piRNAs can be used to
diagnose different types of infertile patients. For example, piR-30198 is one
of piRNAs used for this purpose. This biomarker is able to distinguish between the
two disorders related to male infertility, namely, azoospermia and
asthenozoospermia (33).




authors appreciate the valuable contributions of the experts in the Research and Clinical Center for Infertility of Yazd,
especially in molecular and cytogenetic laboratories.


Financial support or sponsorship

There is no support in relation to this paper.



Conflict of interest statement

In the present study, all the authors declare to have no actual
or potential conflict of interest.


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