Infertility diagnosis of patients. Key words: Infertility, male

is one of the reproductive-related disorders that there are several causes in
its development, and both genders can play a role in the disorder. Despite the
efforts made, in many cases, the cause remains idiopathic. In male infertility, epigenetic factors play an important role,
one of which is piRNAs that are considered as a class of non-coding RNAs and
play a crucial role in spermatogenesis. Therefore, these non-coding RNAs can
serve as a novel and promising approach to the diagnosis, treatment, and
prognosis of this disorder, although this still requires much research. Our
study is one of the first studies that reviewed the most recent investigations
performed on the potential role of piRNAs in male infertility and in human
population and it can help to better understand the etiology of this disorder
and diagnosis of patients.

words: Infertility, male infertility, epigenetic, piRNAs,
non-coding RNAs




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).


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, in primordial
germ cells (PGCs) occur, eliminating the epigenetic marks. Second, in male germ
cell nuclei occurs a reorganization and a condensation of its genome (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 most important ncRNAs are miRNA, siRNA and piRNA,
the differences of which are presented in the following table (10, 11) :



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) (12, 13). 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 (14).



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) (15). 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 (16). The AUB protein plays a similar role to PIWI and
forms the 5? end of piRNAs that associated with AGO3. This complex produces the
5? end of the antisense piRNAs by the cleavage of antisense piRNA precursors
and then these are loaded onto AUB (17). 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 (18).



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 (17). 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 1, the most important research performed on
male infertility and piRNAs is summarized:

The Moloney leukemia virus 10-like 1 (MOV10L1) gene is
a piRNA biogenesis- related gene that plays a role in the primary and secondary
processing (19). 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 (20). 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 (21, 22).

A recent study on
peripheral blood samples of 30 infertile men, showed
that rs10773767 and rs6982089 were two single nucleotide
polymorphisms (SNPs) in PIWIL1 and
PIWIL2 respectively. These polymorphisms were allele-specific
methylation-sensitive and suggests that DNA methylation changes in these genes
are associated with spermatogenesis disorders (23).

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 (24). These studies indicate that piRNAs may play a crucial role in male infertility.



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 (25). some studies have shown that sperm analysis cannot be
used accurately for diagnosis between fertile and infertile men (26). Therefore, identification of non-Invasive seminal
Biomarkers, can solve this problem. In 2015, Hong and colleagues identified 5
piRNAs by examining seminal plasma samples in infertile patients, which can be
used as diagnostic biomarkers for the detection of infertile men (These are
shown in Figure 2) (27). 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 and sperm concentration (28). Thus, these piRNAs may play an important role in the
fertilization process (Figure 3).



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
two disorders related to male infertility, namely, azoospermia and asthenozoospermia
(Figure 4) (27).