Shedding Light on the Dark Matter, Noncoding RNAs in Human Reproduction

Abstract

Advances in human transcriptome have unveiled the crucial regulatory role of noncoding RNA (ncRNA) in most biological processes, including reproduction. Recent studies have elucidated some of the questions, highlighting the regulatory function of specific ncRNAs on concrete reproductive mechanisms. ncRNAs have been shown to be crucial for the maintenance of spermatogenesis, primordial germ cells (PGCs) survivals, folliculogenesis, oocyte maturation, and corpus luteum function. In addition, due to their unique expression and critical functions, they have been demonstrated to be associated with aspects of infertility such as premature ovarian failure (POF), recurrent implantation failure (RIF), polycystic ovarian syndrome (POCS), varicocele, sperm abnormalities, and testicular cancer. This chapter will discuss the current knowledge of the role of ncRNAs in spermatogenesis, and oogenesis and their potential utilization as a noninvasive diagnostic marker for reproductive disorders.

1. Introduction

Human reproduction is made of intricate but interconnected cellular and molecular processes through which a viable healthy offspring is born. Recent studies have suggested that the disruptive status of reproductive health has led to infertility-related problems. The World Health Organization (WHO) has recently prognosticated infertility to might be considered as a major health concern after cancer and cardiovascular disease [1]. Environmental and physiological disruptors render the reproductive process particularly susceptible to epigenetic alterations and modifications. Epigenetic changes have been indicated to play an important role in gametogenesis and reproductive-related disorders. Advanced technologies have proved ncRNA crucial in controlling reproductive functions. Mounting evidence indicates that ncRNAs in gametes are sensitive to environmentally derived epigenetic changes and can moderate the inheritance of paternally acquired disorders attributes [2]. The different types of ncRNAs, dynamic expression, and the complexity of the regulatory pathways have been described, such as the dark matter of the universe. However, recent studies have shed some light on modulatory functions of specific ncRNAs on concrete reproductive mechanisms. ncRNAs have been shown to participate in multiple levels of gene expression moderation in oogenesis and spermatogenesis such as gene imprinting, meiosis, cell proliferation, and differentiation [3]. ncRNAs have been reported to be crucial for the maintenance of spermatogenesis, primordial germ cells (PGCs) survivals, folliculogenesis, oocyte maturation, and corpus luteum functions [4]. In addition, due to their unique expression and critical functions, they have been demonstrated to be associate with infertility such as premature ovarian failure (POF), recurrent implantation failure (RIF), polycystic ovarian syndrome (POCS), varicocele, sperm abnormalities, and testicular cancer. However, there are many challenges to overcome to elucidate the actual effect of ncRNA aberrancy on reproductive biology and disease. This chapter aims to summarize the current knowledge of ncRNA-related reproductive processes. Here, we will also review recent discoveries linking ncRNAs to infertility-related disorders. An enhanced understanding of the role of ncRNAs can further advance the potential utilization of ncRNAs as a noninvasive diagnostic marker and therapeutic target for reproductive disorders, which will be covered in this chapter as well.

1.1 Overview of ncRNA

A decades-long discussion has been escalated with the surprising, challenging discovery of ncRNAs. Genome-wide sequencing technology and high throughput sequencing revealed that the majority (~93%) of the human genome is actually transcribed into RNA. However, only 2% of this RNA encodes protein [5, 6, 7]. Those RNA sequences that were previously described as gene transcriptional “noise” or junk DNA are now believed to play a very important role in human normal cells physiology and pathological processes [7]. The rapid progress and accessibility to the technology have allowed a deeper understanding of transcription on a wider scale and in different conditions. Next generation sequencing (NGS), in particular, offered the possibility to identify and classify noncoding RNAs [8, 9]. ncRNAs have been ranked based on their function, biogenesis, and size in two broad subfamilies: small ncRNA (sncRNA) with size <200 nucleotides (nts) long and include housekeeping ncRNAs, comprising small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), and other regulatory sncRNAs such as microRNA (miRNA), piwi-interacting RNAs (piRNAs), small endogenous interfering RNAs (endo-siRNAs), and transfer RNA (tRNA). Long ncRNA defines the second group with a size >200 nts in length [10, 11, 12].

1.2 Definition, biogenesis, and functions of noncoding RNA

It can be anticipated that the most well-characterized sncRNAs are miRNAs, endo-siRNA, and piRNA. All the three types act via an RNA interference (RNAi) key gene regulatory pathway [13]. Fundamental to this pathway is the generation of small noncoding transcripts to assemble the RNA-induced silencing complex (RISC) by Argonaute proteins to cleave target mRNA. Two subfamilies of Argonaute are known as the AGO and PIWI families. AGO protein binds to both miRNA and siRNA, whereas the PIWI subfamily is specific for piRNAs [13].

miRNAs are short (21–22 nts) single-stranded ncRNAs that regulate gene expression posttranscriptionally via promoting mRNA translation silencing or degradation [4, 11]. miRNAs are initially derived from nuclear long primary transcripts that are then cleaved by nuclear RNA polymerase III protein complex containing Dorsha and DiGeotge critical region 8 (DGCR8) into 70 nt hairpin precursor miRNAs (pre-miRNAs). These small transcripts subsequently then leave the nucleus to the cytoplasm and are processed further by Dicer, another member of the nuclear protein complex, to form a mature double-stranded miRNA of 22 nt. The duplexes are then incorporated into a ribonuclear vesicle to assemble miRNA-RISC (miRISC) by Ago subfamily protein, where the complementary strand is degraded, and the mature miRNA form is retained. Mature miRNA can read and bind to the 3’end of their mRNA target by the first eight residues on the 5’end known as the “seed sequence,” leading to translation suppression and/or degradation of the target mRNA [14, 15, 16]. miRNAs embrace 1–5% of the genome and can typically modulate the expression of myriads of genes. Interestingly, it has also been reported that a single gene can be the target of multiple miRNAs, which, in fact, further complicates their regulatory pathways. Expression alteration of a single miRNA can lead to a profound impact on various cellular functions [11, 15, 17].

Similar to miRNAs, endogenous siRNA plays essential roles to posttranscriptionally regulating gene expression. It functions via binding to the 3’ UTR site of the target mRNA; however, unlike miRNA, it requires a full complementary sequence in order to silence mRNA translation or promote its decay [18]. siRNA exhibits the same size as miRNA, and both are processed from a hairpin double-strand precursors by Dicer-dependent mechanism [19]. piRNA are marginally longer (24-31 nt) than siRNA and miRNA. In contrast to miRNA and siRNA, piRNAs are processed from long single-stranded precursors by Dicer-independent mechanism. These transcripts are thought to be exported to the cytoplasm and subsequently processed into a primary piRNA [19, 20, 21]. In the cytoplasm, the secondary piRNA is generated when the primary piRNA loads onto the PIWI proteins and the homologous sequence. The later will preferentially bind to Piwi family proteins belonging to the Aubergine (Aub) superfamily, exciting the cleavage and generation of more piRNAs via the processing of antisense RNAs sequences [20]. Rounds of cleavage repeats produce several piRNAs in a process called a ping-pong cycle. The produced transcripts are then transported back to the nucleus by PIWI proteins to silence target genes. The Aub-piRNA, however, acts as a trigger to the ping-pong amplification cycle, by which, transposons are silenced and piRNAs are amplified.

The biogenesis of piRNA is elusive still and most of these data were from Drosophila which is believed similar to humans. piRNAs were ultimately found in germ cells and were reported to play a key role in germline specification, maintenance, and gametogenesis [11, 21]. A great deal of data is indicating a crucial regulatory function of piRNA in somatic cells via mRNA turnover, DNA rearrangement, transposon expression and silencing, translational modulation, and epigenetic programming. The dysregulation of piRNA has been shown to contribute to epigenetic modification-related disease stat [13, 20]. Recent studies demonstrated a differential expression of the PIWI-related proteins in the oocytes and surrounding granulosa cells (GC) in cases with diminished ovarian reserve (DOR). It’s interesting to note that PIWI mutant female mice seem to be unaffected [22], while the mutant male mice experience spermatogonia degeneration and altered spermatogenesis [11, 23]. Recent studies have indicated that the mouse genome encodes three PIWI family proteins named MIWI, MILI, and MIWI2. MIWI and MILI act as an RNA-slicing component crucial for retrotransposon inhibition and piRNA biogenesis. The loss of function of any of these three genes leads to spermatogenesis defects [24]. Tudor domain-containing (TDRD) protein has also been reported as essential for piRNA biosynthesis and repression of retrotransposon when complex with PIWI proteins [24, 25].

The biosynthesis of lncRNA in mammals has been preliminarily elucidated. It was indicated that many lncRNAs are transcribed by RNA polymerase II, followed by posttranscriptional 5′-cap addition [1, 26]. It’s fascinating to note that lnRNAs are generated through pathways similar to those for mRNAs, with comparable cleavage signals, intron/exon lengths, and histone-modification [27, 28]. According to study by Ma et al., in 2013, lnRNAs have been found to possess similar transcriptional activity [29]. However, other lncRNAs are processed from precursors into small transcripts of unknown function [28]. LncRNA can be classified based on its location within the genome into; 1. Long intergenic (LincRNA), which does not overlap protein-coding genes, 2. Sense intronic transcripts located within introns of a coding gene, however, do not overlap with any exons, 3. Sense intersecting transcripts from introns of protein-coding locus, 4. Antisense RNA, overlapping any exon of a protein-coding gene on the opposite strand, and 5. Spliced transcripts contain no open reading frame (ORF) and do not match any of the other categories [27, 28, 29]. The nucleus is the location of most lncRNAs, where they act as a scaffold to assist in chromatin structure modification or alternative splicing [11, 30]. However, it has recently been suggested that specific lncRNAs reside and function in the cytoplasm. They act to promote or inhibit mRNA decay, miRNA sponges, and regulate translation [11, 31, 36]. LncRNAs exhibit poor interspecies conservation, leading to uncertainty about their functionality. Possibly due to their low expression, leading to the proposed view of them being transcriptional noise [29]. Despite this, lncRNAs have been reported to interact with RNA, DNA, and proteins and can significantly impact biological processes such as protein localization, transcription, translation, splicing, imprinting, and reprogramming [11, 29]. It has been suggested to play an important role in infertility, reproduction-related disorders, and cancer disease progression [1, 29, 32]. The role of ncRNAs in regulating ovarian follicular development, spermatogenesis, as well as reproductive disease will be the next focus of this chapter.

A simple comparison of all three ncRNAs is included in Table 1.

	miRNA	piRNA	lncRNA
Length (nt)	~22	23–31	~200–800
Expression	All tissues	Germ cell line mainly	All tissues
Precursors	Transcripts with hairpins	Single strand transcripts	transcribed from intergenic regions and from introns of protein-coding genes
Drosha dependent	Yes	No	No
Dicer dependent	Yes	No	No
Argonaute proteins involved	AGO subfamily	PIWI subfamily	No
Function	Translational inhibition, mRNA degradation and storage	Transposable elements cleavage, Translational regulation, and DNA methylation	Cis or trans, splicing, translational, transcriptional and post-transcriptional regulation
Species specificity	Highly conserved across different species	Lack of conservation across the species	Less degree of sequence conservation across the species

Table 1.

Comparison of three the ncRNAs [33, 34, 35, 36].

2. ncRNA in reproduction

Germ cell replication and differentiation is a critical step during gametogenesis. Successful transcriptions of specific genes are fundamental for this process. However, the occurrence of any errors during this process can be the principal cause of infertility. [12, 18]. Studies indicate that ncRNAs are relevant from very early stages of germ cell development. They have been demonstrated to play a critical role in regulating transcriptional, posttranscriptional gene expression, and epigenetic-related mechanisms during spermatogenesis and oogenesis [18]. ncRNAs can intriguingly perform dimorphic distinct functions between males and females and have been suggested as a biomarker for oocyte and sperm quality. Here, we are focusing on miRNAs, piRNAs, and lncRNAs, as the most germline related classes of ncRNAs.

2.1 Roles of noncoding RNAs in sperm maturation and functions

Spermatogenesis is the process by which spermatogonial stem cells (SSCs) actively proliferate and differentiate into haploid male gametes. During sperm development, posttranscriptional regulation is particularly crucial at the later stages of their growth. ncRNAs have been shown to be critically important during this complex process at the transcriptional and posttranscriptional level. The process of spermatogenesis can be divided into three phases based on the relevance of ncRNAs: germ cell proliferation and spermatogonia formation, two rounds of meiosis to produce haploid round spermatid, and spermiogenesis (sperm maturation (Figure 1).

Figure 1.

Characterizations expression of miRNAs, piRNA, and lncRNAs during the different stages of spermatogenesis [12, 18, 37].

Hundreds of ncRNAs have recently been discovered throughout spermatogenesis. Of that, 7% of miRNAs were found to be selectively expressed in mouse and human immature and mature spermatozoa, as well as in the testis [1, 32, 38, 39], suggesting a crucial role of miRNA at almost every stage of sperm cell development. In mice germ cells, the inhibition of miRNA biogenesis components and therefore miRNA led to male infertility. A recent study by [40] further supported these findings. This study and two others indicated that miRNA processing key factors, in particular Dorsha and Dicer, depletion in Sertoli cells severely impaired testicular function, led to male infertility, oligoteratozoospermia, or azoospermia [40, 41, 42]. Moreover, the roles of several miRNAs have been described and pinpointed at different stages of spermatogenesis.

During the early stages (first phase of spermatogenesis), numerous miRNAs have been found to be highly expressed in mouse SSCs, such as miR-34c, miR-21, miR-146a miR-182, miR-183, and clusters of miR-17-92 and miRNA 290-295 [1, 14, 18]. The regulatory role of most of these miRNAs remains to be elucidated. miR-34c was particularly reported to promote SSCs differentiation and may affect their apoptosis [18, 43]. miR-21, miR-20, and miR-106 were found to play an important role in SSC self-renewal and differentiation via regulating homeostasis [18, 44]. Additionally, miR-10b has also been shown to act via targeting KLF4 to control SSC self-renewal, whereas miR-202-3p is involved in initiating spermatogonial meiosis [45, 46]. Niu and colleagues demonstrated a regulatory role of miR-204 in SSC proliferation via targeting Sirt1 [47]. Other miRNAs, such as miR-221/222, miR-146 act to maintain SSCs in an undifferentiated state [14, 18, 48].

The effect of many miRNAs during the later stages of spermatogenesis is well documented, at which they act via different targets and exhibit a regulatory role in meiosis and spermatocyte differentiation. miR-10a is a good example, demonstrating a critical modulatory role during human male germ cell meiosis via down-regulating RAD51. The overexpression of this miRNA may lead to complete meiosis quiescence and male sterility [1, 49]. In addition, the inhibition of miR-871 and 880 has been shown to result in meiosis stagnation [50]. Interestingly, miR-34b-5p and miR-34-c have been shown to be important during meiosis. The miR-34b-5p effect has been illustrated to act via targeting Cdk6, whereas miR-34-c acts via the notch signaling pathway. Both miRNAs have an additional role in pachytene spermatocytes and round spermatids associated with apoptosis events [1, 18, 51]. miR-449 cluster is another regulatory miRNA found to be involved in meiotic and postmeiotic events during the later stages of spermatogenesis. It has also been shown to be crucial in apoptotic events via BCL2 and AFT1 [18, 46].

piRNA is the prevalent ncRNA during spermatogenesis. It has been found to account for about 17% of the ncRNA in human testis tissue and is expressed highly in immature and mature sperms [32]. Recent data in mice has indicated a selective expression of piRNA during the pre-pachytene and pachytene stages. The pre-pachytene stage is rich in transposon sequences and mainly associated with prenatal and fetal male germ cell de novo DNA methylation. Pre-pachytene piRNA is co-expressed with MIWI2 and/or MILI. The complex of pachytene piRNA, MIWI, and the deadenylase CAF1 join to form the pi-RISC, by which it initiates the degradation of a huge number of mRNAs in elongating spermatids. The same complex plays a protecting function depending on the state of the complex and piRNA loading [14]. TDRD is believed to act as a scaffold to recruit PIWI proteins to form germline granules, a process essential for PIWI/piRNA function during spermatogenesis [14, 52].

Research on lncRNAs has just taken up a notch with reports on their expression profile and role in experimental models. Many lncRNAs were found to express at different stages of spermatogenesis and were reported most abundant in testicular tissues when compared to other tissue types [53]. However, only a few have been characterized to carry out important functions during germ cell development. Spga-lncRNAs1 & 2 have been described as spermatogonial specific and found essential for maintaining the stemness of spermatogonia. LncRNA033862 is another important regulator of SSC self-renewal via activating Gfra1gene. These lncRNAs are transcribed from the antisense strand of the GDNF alpha family receptors (Gfra1) gene. It was found to be highly expressed in spermatogonia and SSCs and can be dysregulated by GDNF (Gfra1 ligand) in mouse SSCs [18, 53]. The Dmr1-related gene is an intriguing lncRNA, unintentionally discovered by Zhong [54]. The group found an unexpected size sequence at the 3′ end while trying to clone the Dmrt1 gene. Further, investigation indicated that this part sequence was coded by the Dmr gene and located on chromosome 5, therefore forming a trans-splicing RNA isoform with Dmrt1 (located on chromosome 19). This disruption to the sequence resulted in the downregulation of the Dmrt protein and was later proposed to participate in regulating germ cell development via switching between mitosis and meiosis [37].

In the later stages of spermatogenesis, in human, Narcolepsy candidate-region 1 gene (NLCI-C) is a lncRNA found to express in the cytoplasm of spermatogonia and early spermatocytes. The inhibition of this lncRNA has been shown to promote apoptosis, whereas its overexpression stimulated cell growth. In agreement, the study by Nishant et al., [55] demonstrated that NLC1-C promoted the proliferation of spermatogonia and spermatocytes in maturation arrest patients [53, 55]. In rodents, HongrES2 lncRNA was reported to follow a pattern of expression in the cauda region of the epididymis that matches its proposed role as a mediator of sperm maturation. This lncRNA is processed in the nucleus to a 23 bp RNA known as mil-HongrES2. The overexpression of the processed transcripts resulted in a lower sperm capacitation [53, 56]. Moreover, lncRNA-HSVIII and lncRNA- T cam1 have been suggested to participate in the regulation of transcriptional expression of the spermatocyte-specific gene [57]. Testis-specific X-linked lncRNA is another crucial lncRNA located within the X-inactivation center. It is known to be highly expressed in the pachytene spermatocytes and has been proposed to regulate meiotic division [18, 53]. lncRNA-Tug1 is among the most conserved lncRNA between humans and mice. It resides on chromosome 11 in murine and was found to be widely expressed during different developmental stages in both humans and mice. Tug1^−/− males were presented with severe oligozoospermia with head and midpiece sperm morphological defects [53, 58]. Lnc32058, lnc98487, and lnc09522 have been identified to be involved in sperm motility-related regulatory processes [53]. Male important reproductive and fertility-related ncRNA are presented in Table 2.

Expression in reproductive; tissues	miRNA	piRNA	lncRNAs	Function of target	Ref.
Spermatogonium	miR-10b; miR-202-3p; miR-34c, miR-21, miR-146a miR-182, miR-183, and clusters of miR-17-92 and miRNA 290–295.; miRNAs, such as miR-221/222, miR-146	TDRD	Spga-lncRNAs1 & 2, 033862,; Dmrt1,; NLCI-C	SSC self-renewal; Regulating germ cell development; Act as scaffold to recruit PIWI proteins.; Promote SSCs differentiation and self-renewal.; SSCs self-renewal.; Initiating spermatogonial meiosis; Act to maintain SSCs in an undifferentiated state.	[11, 18, 53]; [14, 52]; [18, 43]; [45, 59]; [14, 18, 48]
Spermatocytes	miR-469; miR-871 and miR-880, miR-34b-5p and miR-34-c		NLCI-C; Xist	Transition from pachytene spermatocyte to spermatid.; Regulate meiotic division.; Promoted the proliferation of spermatogonia and spermatocytes.; Regulate meiotic division.	[50]; [55]; [53] [18]
Spermatid	miR-122-a, miR-23b, miR-30c, miR—690.; miR-449, miR-34b-5p and miR-34-c			Cell adhesion and spermeation.; Regulate round spermatids associated with apoptosis events.	[1, 51, 18]
Sperm/Seminal plasma	miR-34c-5p, miR-122, miR-146b-5p, miR-181a, miR-509-5p, miR-513a-5p, miR-193b	piR-31,068, piR-31,098, piR-31,925, piR-43,773	Lnc32058, lnc98487, and lnc09522	Regulate sperm motility related processes.	[53]

Table 2.

Important ncRNA related to male reproduction and fertility.

Taken together, ncRNAs clearly, therefore, play a pivotal role during male reproduction. They are involved in SSCs differentiation, proliferation, and self-renewal. Moreover, they regulate the cell cycle, apoptosis, cell communications, and signaling pathways within the male reproductive system, and thus, ncRNAs can potentially be used as a critical biomarker for disease detection in the male reproductive tract. Although a number of database and analysis tools to support many of the potential discoveries of ncRNAs are now publicly available, functional annotations of different ncRNAs in spermatogenesis are in outset and need strong experimental evidence. The availability of the current technologies may allow us in the future to use ncRNA as a diagnostic or treatment target for male infertility.

2.2 Roles of noncoding RNAs in follicular development and oocyte maturation

The ovarian follicle is the principal unit of the ovary, encompassing the developing oocyte in a process called oogenesis. This is a highly precise, orchestrated process by which a small resting primordial follicle, containing the oocyte and the surrounding GCs, progressively develops into a maturing ovulatory follicle. It is a cyclic process controlled by both the follicle-stimulating hormone (FSH) and luteinizing hormone (LH). Every month, under the influence of FSH and other factors, only a few follicles are selected to grow to the preovulatory stage, after which, successive LH surges modulate the growth of only one follicle to become the dominant follicle (DF), which eventually ovulate. The rest of the growing subordinate follicles (SFs) will undergo atresia. The oocyte development and follicle maturation represent the most complex dramatic cellular proliferation, differentiation, and apoptotic pathways in females. It can only be achieved by bidirectional communication between the oocytes and GCs and is tightly regulated by a myriad network of interacting genes. Therefore, dysregulation of any of the key regulatory genes would negatively impact the fate of the developing follicle.

ncRNA posttranscriptional regulation has recently gained attention during oogenesis and ovarian functions (Figure 2). The core of publications focused on miRNA. The expression of miRNA has been reported to vary according to the cell type, stage, and functions [18, 60]. In mice knockout models, oocyte-specific Dicer removal caused spindle disruption and chromosome aggregation, and therefore meiosis stagnation [61]. The same experiment in GC resulted in accelerated use of follicle reserve, increased number of atretic follicles, and disruption of many follicular developmental key regulatory genes [18, 62, 63]. Several studies have indicated the key involvement of miRNAs in GC survival, proliferation, and differentiation, as well as steroidogenesis. miR-224, under the influence of transforming growth factor-beta1, has been shown to play a role in regulating GCs proliferation and function [1, 18]. Moreover, FSH was reported to modulate the expression of miR-29a and 30d in cultured GCs. Furthermore, the LH surge appeared to promote miR-21, miR-212, and miR132 expressions in GCs. The overexpression of miR-143 inhibited primordial follicle formation in 15.5 dpc murine ovaries [18].

Figure 2.

Characterizations expression of miRNAs, piRNA, and lncRNAs during the different stages of oogenesis [12, 18].

piRNAs have been found to be abundantly expressed in the ovaries. In humans, MILI was specifically localized to fetal oocytes, whereas MIWI and MILI were found in adult ovaries. piRNA has been described to be highly expressed in the oocytes of rhesus monkeys, bovine, as well as in humans [64]. However, their function is undescribed yet. PIWI proteins were localized to mouse oocytes, yet their loss did not affect their fertility. The PIWIL1 knockout model was interestingly able to generate mature fertilizable oocytes, nevertheless, arrested at the two-cell stage. The author indicated, as a result, the accumulation of transposons in the oocytes and zygotic gene activation failure [65].

lncRNA-H19 was the first discovered functional lncRNA in the female reproductive tract. They were considered as a large functional RNA sequence with no coding proteins. H19 was described to be significantly expressed in endoderm and mesoderm-derived tissues. It has been shown to involve in the modulation of a co-expressing net of imprinting genes [66]. lncRNAs were recently found to play a role in regulating oocyte meiosis. It has been demonstrated that lncRNA Y00062 may be involved in NEK7 modulation, a known crucial gene for spindle assembly and mitosis. Moreover, lncRNA ENST00000502390 was reported to overexpress in poor-quality cumulus cells (CCs) and is associated with elongation of very long-chain fatty acid protein 5 (ELOVL5). ELOVL5 is known to involve in modulating oocyte maturation and ovulation and in the biosynthesis of highly unsaturated fatty acids, thus ENST00000502390 may participate in the same processes [67]. Thus, strengthening the evidence for the role of these ncRNAs in the oocyte maturation processes. Some examples of known female reproductive and fertility-related ncRNA are presented in Table 3.

Expression in reproductive tissues	miRNA	piRNA	lncRNAs	Function of target	Ref.
Folliculogenesis	miR-143		H19, Xist, AK124742, Amhr2		[68]
Granulosa cells	miR-133b, miR-98, miR-29a, miR-23a, miR-15a, miR-10a, miR-145, miR-151, miR-22-3p, miR-146a, miR-21, miR-106a, miR-24, miR-320a, miR-1275, miR-125a/b, miR-100, miR-224, miR-30d, miR—132, miR-212		H19, Let-7, Let -7 g, Neat1,ENST0000050239	Steroidogenesis, GC proliferation and apoptosis	[11]
Oocyte	miR-10a, miR-184, miR-100.; miR-20a, miR-15a, miR-602		lncRNA Y00062, ENST0000050239	spindle assembly and mitosis; Oocyte reprogramming, Repression nuclear receptors; regulation of oocyte specific genes expression.; Regulation of cell growth and division.	[67]; [69]

Table 3.

Important ncRNA related to female reproduction and fertility.

Overall, these data demonstrate the expression of the different ncRNAs within the various organs of the female reproductive tract, where they play an important role in modulating crucial cellular pathways for proper functioning. However, it is still a vague area of research and represents a great challenge in reproductive medicine. Upcoming studies, nevertheless, can provide a new insight into utilizing these ncRNAs as biomarkers and therapeutic targets for fertility disorders.

3. The role of noncoding RNAs in some reproductive disorders

The development of gametes is extremely complex, requiring both the sperm and egg to passage through several stages to eventually form the mature sperm in males, and the maturing follicle finally discharges the mature cumulus enveloped oocyte in females. This process is tightly controlled and the interruption of the sequence at any stage can impact one’s fertility. ncRNAs play a crucial part of all these processes.

3.1 The role of ncRNAs in male reproductive disorders

Male infertility is evident in about 40–50% of all infertility cases; however, the research into this topic is relatively spars. Molecular studies in the field of male fertility are important and can unveil the etiology of infertility. ncRNA dysregulation has been shown to implicate in male infertility such as prostatic cancer (PCa), varicocele, and sperm abnormalities.

3.1.1 Prostatic cancer (PCA)

PCa is one of the complex types of malignancies and is ranked as the second most lethal cancer type in men. A number of ncRNA transcripts have been identified and associated with PCa. These transcripts have been named as prostate cancer-associated ncRNA transcripts (PCATs). lncRNA PCAT-1, for example, has been found to be highly expressed in the cytoplasm and in low amount in the nucleus of PCa cells [10, 70]. In addition, the co-expression of lncRNA PCAT-1 with miR-34a and miR-3667-3 has been described as responsible for increased prostate tumor cell proliferation. This was achieved via upregulating the c-Myc protein, a crucial protein for cell cycle progression [10, 71]. Testis-specific transcript Y-linked 15 (TTTY15) lncRNA has been reported to be highly expressed in PCa patient samples when compared to the control group. The authors showed that TTTY15 acts as a sponge to let-7 miRNA to upregulate CDK6 and FN1 in those patients [10]. miR-648 has been identified as a novel diagnostic biomarker for PCa. The upregulation of miR-181a, miR-122, miR-181c, miR-193a, miR-193b, and miR-125a-b have been reported in PCa tissue samples, whereas miR-145, miR-125, and lect-7c were found dysregulated [72, 73, 74]. novel_ pir382289, novel_ pir158533, novel_ pir349843, and has_pir_002468 have been associated with PCa development [75].

3.1.2 Varicocele (VC)

It is identified by an abnormal swelling of the pampiniform venous plexus within the scrotum. This condition can cause venous pressure and increased temperature, hypoxia-related oxidative stress, and apoptosis, leading to testicular dysfunction and impact sperm function and quality [10, 76]. Several studies indicated miRNA as an important player in the development of VC. A study by Ou and colleagues demonstrated that caspase-3 sperm cell apoptosis has been induced by miR-210-3P in patients with VC [77]. The same group further assessed the relationship between VC development and the expression profile of rno-miRNA-210, rno-miR-190a, rno-miR-6316, and rno-miR-135bb-5p. These findings provided evidence indicating the upregulation of these miRNA in VC patients’ samples [77]. Another study compared the expression of seminal miRNAs and oxidative stress-related apoptotic markers in VC patients. The group reported the downregulation of miR-122, miR-34c5, and miR-181a in infertile patients with VC [78].

The overexpression of lncRNA gadd7 (growth arrested DNA-damage inducible gene 7) has been associated with DNA damage and inhibition of cell growth. Increased expression of lncRNA gadd 7 has been studied in VC semen samples and was found to negatively impact sperm count [79].

3.1.3 Sperm abnormalities

Sperm abnormalities are a fundamental cause of male infertility such as obstructive and nonobstructive azoospermia, oligozoospermia, asthenozoospermia, and teratozoospermia [10]. miRNAs dysregulation has been linked to Sertoli cell only (SCO) syndrome, asthenozoospermia, germ cell arrest, and mixed atrophy (MA) [14]. Single-nucleotide polymorphisms have been localized in miRNA-binding sites of male fertility-associated genes, indicating a role of miRNAs in male infertility. In addition, Dorsha and Dicer SNPs have been shown to affect sperm quality, which further supports their role in male infertility.

Recent evidence demonstrated that multiple SNPs from human piwi genes are associated with increased risk of oligozoospermia or spermatogenic failure [80]. Moreover, spermatogenesis disruption can be due to PIWIL1/PIWIL2 allele-specific DNA methylation [81].

Differential expression of lncRNA in oligoasthenozoospermic and asthnozoospermic patients has been associated with spermatogenesis and sperm function. HOTAIR-lncRNA down-regulation has been shown to be related to decreased nuclear factor erythroid 22-related factor 2 (NRF2) gene expression. The expression levels of the NRF2 gene have been related to antioxidant gene expression and sperm quality. Thus, suggesting a protective function of HOTAIR against antioxidant sperm-damaging activity [14]. The level of expression of lncRNA32058, lnc98487, and lncRNA09522 have been reported to significantly impact sperm motility [82].

To date, thousands of ncRNAs have been identified to be associated with different male reproductive disorders. However, the current mechanisms and molecular pathways modulating these processes are still underway. Unveiling the link between the differential expression and functions of these ncRNAs and each male reproductive disease can identify needed biomarkers and support the development of new approaches to treat these conditions.

3.2 The role of ncRNAs in female reproductive disorders

ncRNAs have recently been explored as molecular markers for female reproductive-related disorders such as polycystic ovarian syndrome (PCOS), premature ovarian failure (POF), and repeated implantation failure (RIF).

3.2.1 Polycystic ovarian syndrome (PCOS)

PCOS is a common systematic endocrine disorder affecting women at their reproductive age. It is characterized by the dysregulation of androgens, which makes it a prevalent cause of anovulation. miRNA-mediated regulation of androgens has been explored recently in women with PCOS. The study results suggested an inverse relationship between the expression levels of serum miR-23a and serum testosterone [83]. Moreover, the overexpression of three miRNAs: miR-222, miR-30c, and miR-146a has been indicated in patients with PCOS when compared to controls [18]. Circulating miRNAs can be profiled in plasma, serum, and follicular fluid. A recent study demonstrated the upregulation of miR-32, miR-18b, miR-135a, miR34c, and miR-9 in the follicular fluid of PCOS patients. On the other hand, miR-320, and miR-132 were downregulated in the same group [69].

The role of lncRNA in the follicular fluid and somatic cells and the development of PCOS have recently been explored. For example, two GC and CC specific lncRNA, PWRN2 and HCG26 have been associated with PCOS. The overexpression of PWRN2 and its target gene ATP6V1G3 have been proposed to act via decreasing the follicular PH, and therefore oocyte dysplasia in these patients. HCG26 lncRNA has been shown to play a role in the regulation of cell proliferation and steroidogenesis. Therefore, alteration expression of this lncRNA in GC may contribute to the pathogenicity of this disorder.

3.2.2 Premature ovarian failure (POF)

POF is a multifactorial disorder characterized by hypoestrogenism and hypogonadism in women under the age of 40. Like PCOS, ncRNAs have been explored as epigenetic markers for the disease. The overexpression of circulating miRNAs, such as miR-126, miR-125b-2, miR-23a, miR-27a, miR146a, miR-139-3p and downregulation of miR-144, let-7c, and miR-22-3p, have been noted in patients with POF [69, 84].

Data on lncRNAs in POF is still quite limited and requires further research. The correlation between the expression profile of lncRNAs FMR4 and FMR6 and POF has quite recently been studied. The authors observed a significantly negative association between the expression level of FMR6 in GCs and the number of oocytes retrieved. Moreover, in mice, cyclophosphamide via upregulated lncRNA-Meg3-p53-p66Shc pathway has been proposed to induce ovarian injury and POF [68, 85].

3.2.3 Ovarian cancer

Errors during the developmental process and the dysregulation of molecular pathways can give rise to different types of cancer. Several miRNAs have been associated with the manifestation of ovarian cancers and others appear to act as cancer suppressors [84]. LIN28 protein is specifically expressed in ESCs and known as a principal modulator of human embryonic stem cells (hESC) pluripotency. It has been reported to function as an oncogene to promote tumor progression. Zhong and colleagues demonstrated that the action of this protein may be suppressed in hESCs and cancer cells by the expression of four miRNAs, miR-9, miR-125, miR-30, and let-7 [54].

HOTAIR lncRNA has been shown to be highly expressed in endothelial ovarian tissues and serous ovarian cancers [86]. Another lncRNA, Xist, is a transcript of an inactive X chromosome and has been found to express in very small amounts in ovarian cancer cell lines [87]. Furthermore, H19 lncRNA is one of the first lncRNAs to express in epithelial ovarian cancers. MEG3 lncRNA has been reported to suppress tumor cell development and progression in various types of cancers, including ovarian cancer [86, 88].

It is indeed well documented that ncRNAs play an important role in female reproductive disease-related conditions. However, there is still a challenge in determining their specific regulatory contribution in relation to the disease state. Further research and studies are required to understand the exact mechanisms of their involvement and how they can be targeted for therapeutic interventions. Further, studies are needed to further provide evidence and expand our database for the diagnostic and therapeutic targets for female-related-reproductive disorders.

4. Potential applicability of noncoding RNAs as a diagnostic noninvasive biomarker and as a predictor of oocyte/embryo quality and reproductive disorders

Growing evidence indicates a crucial role of ncRNAs in infertility and reproductive health-related disorders. The detection of these ncRNAs can be easily isolated from various human biological fluids such as serum, plasma, seminal plasma, and follicular fluids, which makes them a reliable tool for infertility-related care.

At present, the quality of retrieved oocytes and developing embryos during the assisted reproductive program can only be assessed morphologically or via an invasive aggressive embryo biopsy. Thus, improving techniques to isolate and evaluate ncRNAs in spent culture medium can provide an interesting noninvasive tool to assess those embryos. In addition, in vivo or in vitro aging of postovulatory oocyte is a challenging scenario. Often, those oocytes are presented with chromosomal abnormalities, polyspermy, lower fertilization, and abnormal embryo development [17, 89]. It has been reported that these abnormalities are due to various cellular and molecular changes occurring within the aging oocytes. These changes include intracytoplasmic adenosine triphosphate (ATP) and antioxidant GSH reduction, increased reactive oxygen species (ROS), morphological and organelle alterations, reduction in expression of antiapoptotic factor Bcl-2, Ca²⁺ dysregulation, and increased apoptosis [17, 90]. Consequently, oxidative stress on these oocytes leads to the trigger of a cascade of events that results in postovulatory oocytes aging. ncRNAs have been shown to be involved in controlling these processes and therefore, understanding the regulatory mechanism can aid the prevention and maybe the development of a supplementation to protect these oocytes in vitro. Interestingly, the use of hCG and vitamin C supplementation in the culturing medium has been shown to alter the expression profile of miRNAs in murine GCs and oocytes during in vitro growth [84, 91]. In addition, current data provides some understanding regarding the involvement of ncRNAs in hESCs, which literally can comprehend some human reproductive entity development, and therefore some cancer manifestation mysteries. Thus, enhance and support reproductive-related cancer treatment protocols.

Early diagnosis can aid the management and treatment of various reproductive disorders. Yet, the profiling of ncRNAs from the culturing medium and biological fluids is still challenging. The small amount of ncRNAs in these samples creates some technical difficulties, including material expression variations, handling procedures may introduce contamination or cause RNA degradation, and the accuracy of the technology used for isolation, all of which can result in diversity in the reported profiles. For example, high-throughput sequencing analysis provides large data of short sequence reads, which often contain contaminant sequences in addition to the other primer sequencing. Therefore, the development of highly advanced and complex algorithms is needed to clear out any inaccurate reads and produce more robust data. In addition, the intricate ncRNA networks that are responsible for regulating reproductive health can make it extremely difficult to understand how they function. The amount of complexity involved in these molecular pathways can be quite challenging to decipher. In fact, it raises questions about wither it is the actual cause or part of the targeted pathway associated with the pathological condition. It has, however, been clearly shown that ncRNAs may provide a great potential for its use as a biomarker for the diagnosis and treatment of reproductive disorders.

5. Conclusion

Growing evidence indicates a crucial role of ncRNAs in epigenetic modulation and regulation of infertility-related disorders. Recent advancements in molecular testing and informatics approaches are well established and have eased ncRNA research. Discoveries of more novel species on ncRNAs are ongoing and can contribute to unveiling the “dark matter” of the genome, to further knowledge of ours in infertility-related disorders.