Infertility infertility, included quality, motility, sperm counts and


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


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 retrotransposons,
b-piRNAs originated from mRNA, c-piRNAs originated from lncRNAs (Long noncoding
RNAs) (19).
The most
common 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 germ
cells is main role of these small non-RNAs (22).


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) (23). 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 (24). The AUB protein plays a
similar role to PIWI and forms the 5? end of piRNAs that associated with AGO3 (25). 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 (26).


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 (25).
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 (27). 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 (28). 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 (29, 30).

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 (31). 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:
sperm concentration, seminal volume, pH and morphology (32). Some studies have shown
that sperm analysis cannot be used accurately for diagnosis between fertile and
infertile men (33). 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 (34, 35).  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 (36). 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 (37). 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 (38). 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,
that rs10773767 and rs6982089 were two
single nucleotide polymorphisms (SNPs) in PIWIL1 and PIWIL2, respectively, and these
polymorphisms were allele-specific methylation-sensitive (39). 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 (40, 41). Additionally, Considering
the relationship between the modified pattern of methylation of TEs and male
infertility (41, 42), 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

The ncRNAs, as one of the new therapies, can be used in many disorders. It
has been made many improvement in the field of miRNAs clinical applications, such
as the use of Miravirsen (an oligonucleotide with 15-nucleotide) in the
treatment of HIV infections (43) and miRNAs replacement and inhibition techniques
in the treatment of many cancers (44, 45). Also, 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) (46). 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 the 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 (47). 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 (36).




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


Financial support or sponsorship

There is no support in relation to this



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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