Infertility piRNAs that are considered as a class of

Infertility 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.

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

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Infertility 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 (17). 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
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 2, 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 (Figure 3) (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 (piR-31704 and
piR-39888)  and sperm concentration (28). Thus, these piRNAs may
play an important role in the fertilization process.






















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 (27).