microRNAs in the pathogenesis of non-obstructive azoospermia: the underlying mechanisms and therapeutic potentials

ABSTRACT

miRNAs are involved in different biological processes, including proliferation, differentiation, and apoptosis. Interestingly, 38% of the X chromosome-linked miRNAs are testis-specific and have crucial roles in regulating the renewal and cell cycle of spermatogonial stem cells. Previous studies demonstrated that abnormal expression of spermatogenesis-related miRNAs could lead to nonobstructive azoospermia (NOA). Moreover, differential miRNAs expression in seminal plasma of NOA patients has been reported compared to normozoospermic men. However, the role of miRNAs in NOA pathogenesis and the underlying mechanisms have not been comprehensively studied. Therefore, the aim of this review is to mechanistically describe the role of miRNAs in the pathogenesis of NOA and discuss the possibility of using the miRNAs as therapeutic targets.

Abbreviations: AMO: anti-miRNA antisense oligonucleotide; AZF: azoospermia factor region; CDK: cyclin-dependent kinase; DAZ: deleted in azoospermia; ESCs: embryonic stem cells; FSH: follicle-stimulating hormone; ICSI: intracytoplasmic sperm injection; JAK/STAT: Janus kinase/signal transducers and activators of transcription; miRNA: micro-RNA; MLH1: Human mutL homolog l; NF-κB: Nuclear factor-kappa B; NOA: nonobstructive azoospermia; OA: obstructive azoospermia; PGCs: primordial germ cells; PI3K/AKT: Phosphatidylinositol 3-kinase/protein kinase B; Rb: retinoblastoma tumor suppressor; ROS: Reactive Oxygen Species; SCOS: Sertoli cell-only syndrome; SIRT: sirtuin; SNPs: single nucleotide polymorphisms; SSCs: spermatogonial stem cells; TESE: testicular sperm extraction; TGF-β: transforming growth factor-beta.

KEYWORDS:

• Mirna
• azoospermia
• spermatogenesis
• sperm
• fertility

Introduction

Infertility affects 10–15% of couples globally, and about 50% of infertility cases are related to male factors (Hamada et al. 2011). One of the causes of male infertility is azoospermia that refers to the absence of sperm in semen and is categorized as obstructive and nonobstructive azoospermia (OA and NOA, respectively) (Ayhan et al. 2014). OA results from deficiencies in the vasal, epididymal, or ejaculatory duct, and the NOA can be caused by defective sperm production (Jarvi et al. 2015).

A testicular biopsy is used to determine different types of azoospermia and recover sperm for fertilization of eggs via intracytoplasmic sperm injection (ICSI) (Nistal et al. 2017). In 1999, a novel microsurgical method called microscopic testicular sperm extraction (Micro-TESE) was introduced for the recovery of sperm which is less invasive than the conventional testicular sperm extraction (TESE) biopsy (Schlegel 1999).

Azoospermia can arise from varicocele, cryptorchidism, deletion of deleted in azoospermia (DAZ) genes (DAZ1, DAZ2, DAZ3, and DAZ4), Yq microdeletion in the azoospermia factor region (AZFa, AZFb, and AZFc) (Totonchi et al. 2012; Kolon et al. 2014; Masterson and Ramasamy 2018), and pathogenic variants in different genes such as ADAD2, TERB1, SHOC1, MSH4, RAD21L1, BRDT, CHD5, MCM9, MLH3 and ZFX (Chen et al. 2020; Krausz et al. 2020). Furthermore, impairing key spermatogenic genes such as cAMP-response element modulator (CREM), testis-specific histone Hlt, heat shock protein (HSP), and Human mutL homolog l (MLH1) have been reported in azoospermia (Escalier 2001; Fattahi et al. 2017).

In addition to the protein-encoding genes, some noncoding RNA (ncRNAs) are responsible for posttranscriptional gene regulation throughout spermatogenesis (Robles et al. 2019; Daneshmandpour et al. 2020). ncRNAs are divided into long (> 200 nt) and small (< 200 nt) ncRNAs (lncRNAs and sncRNAs, respectively) based on their size (Zhang et al. 2019b). lncRNAs are dynamically expressed during spermatogenesis (Wichman et al. 2017). Interestingly, it has been demonstrated that the expression profile of lncRNAs in the testicular tissues of NOA patients is different from those with normal spermatogenesis (Bo et al. 2020). The three types of male germ cell sncRNAs include the Dicer-independent PIWI-interacting RNAs (piRNAs), Dicer-dependent microRNAs (miRNAs), and small interfering RNAs (siRNAs) (Meikar et al. 2011). Recent studies have shown that impaired piRNA biogenesis could cause germ cell death and infertility (Clark and Lau 2014). siRNAs, as another class of sncRNAs, are also expressed in male germ cells, especially SSCs (Song et al. 2011; Tan et al. 2014). Despite the dynamic expression of siRNAs during spermatogenesis, their function in spermatogenesis remains to be elucidated (Hilz et al. 2016).

miRNA, a small noncoding RNA with a length of about 22 nucleotides, can hybridize with its complementary mRNA and suppress the translation of the mRNA (Hilz et al. 2016). miRNAs have crucial roles in controlling different biological processes, including proliferation, differentiation, and apoptosis (Shukla et al. 2011). It has been demonstrated that 38% of the miRNAs of the X chromosome are testis-specific (Song et al. 2009) and that X-linked miRNAs have a vital role in regulating SSCs renewal and their cell cycle (Guo et al. 2009). Therefore, deficiencies in the gene or expression of miRNAs may lead to spermatogenesis-associated male infertility (Chen et al. 2017). Moreover, differential miRNAs expression in seminal plasma of NOA patients has been compared to normozoospermic men (Finocchi et al. 2020). This review aims to describe the role of miRNAs in azoospermia and discusses the possibility of using the miRNAs as therapeutic targets for the treatment of azoospermia. In this review, we consider all miRNAs that have been reported to be involved in spermatogenesis and sperm function and shown to be associated with azoospermia as summarized in Table 1. We discuss possible mechanisms through which these miRNAs can be potentially involved in NOA development.

miRNAs involved in azoospermia

miR-449

miR-449 a, b, and c are highly expressed in cilia-containing tissues such as the male and female reproductive tracts (Song et al. 2014; Abu-Halima et al. 2014a; Pan et al. 2016; Yuan et al. 2019). miR-449 induces cell death, cell cycle arrest, and cell differentiation (Lizé et al. 2011). miR-449 expression has been observed during different stages of germ cell development and in various germ cells, including spermatogonia, spermatocytes, spermatids, and gonadal somatic cells (Leydig cells and Sertoli cells) (Bao et al. 2012; Hossain et al. 2012). The expression of this miRNA in germ cells at different stages of development, as well as testicular somatic cells, suggests its essential role in male fertility and spermatogenesis. In this respect, it has been reported that abnormal expression of miR-449 is related to infertility (Wu et al. 2014; Muñoz et al. 2015). Several studies have suggested that this miRNA is upregulated in the initiation of meiosis and is exclusively expressed in the spermatocytes and spermatids of adults (Comazzetto et al. 2014). It has been reported that the deletion of the miR-449a/b/c could lead to infertility due to abnormal spermatogenesis (Comazzetto et al. 2014). Moreover, downregulation of miR-449a in the testicular tissue of azoospermia patients with unsuccessful sperm retrieval and the downregulation of miR-449 in Sertoli cell-only syndrome (SCOS) have been observed; suggesting this miRNA may provide a biomarker for the diagnosis of male infertility (Abu-Halima et al. 2014b; Fang et al. 2019). P53 has an essential role in the meiotic process of spermatogenesis, and miR-449 may activate this signaling pathway (Kheir et al. 2011). The p53 signaling pathway is also involved in cell cycle arrest, DNA repair, spermatogenesis regulation, and maintenance of sperm DNA integrity (Raimondo et al. 2014, 2019). However, hyperactivity of p53 can lead to sperm apoptosis and can also negatively affect sperm morphology, motility, and viability (Cohen-Bacrie et al. 2009). The notch signaling pathway plays a key role in spermatid differentiation, epithelial function, sperm maturation, and germ cell meiosis. Notch1 and its ligand, Jagged 2, are found in Golgi complex vacuoles of primary spermatocytes. Interestingly, Notch1 has not been found in germ cells of NOA patients emphasizing the necessity of Notch signaling in spermatogenesis (Hayashi et al. 2001). It has been reported that miR-449 modulates this pathway through direct suppression of the Notch receptor and ligand (DLL1) (Lizé et al. 2011; Marcet et al. 2011). Blocking Notch signaling can yield morphologically defective spermatozoa and ultimately reduce fertility potential (Murta et al. 2014).

In addition to the above, miR-449 can also affect the E2F/Rb signaling pathway (Yan et al. 2012; Mao et al. 2016) during spermatogenesis (Bao et al. 2012; Rotgers et al. 2015). E2F/Rb is an essential signaling pathway that regulates the expression of the G1-S cell cycle transition genes controlling cellular development, proliferation, and apoptosis (Yan et al. 2012). Although the exact effects of miR-449 on spermatogenesis through this signaling pathway have yet to be elucidated, miR-449 plays a vital role in downregulating the E2F/Rb complex (Lizé et al. 2011). Downregulation of E2F/Rb pathway can influence spermatogenesis (Bao et al. 2012; Rotgers et al. 2015) as Rb plays an essential role in SSCs maintenance and maturation

miR-210

miR-210 is involved in various cellular processes, including angiogenesis, proliferation, apoptosis, cell cycle regulation, mitochondrial metabolism, protein modification, DNA damage repair, and spermatogenesis (Tang et al. 2016). It can be processed into miR-210-3p that incorporates into the RNA-induced silencing complex (RISC) as a guide-strand and miR-210-5p that is degraded as a passenger-strand (Bavelloni et al. 2017). The involvement of miR-210 in spermatogenesis and male infertility has previously been indicated (Tang et al. 2016; Xu et al. 2019, 2020). The expression of this miRNA in primordial germ cells (PGCs), spermatogonia, and spermatocytes imply a role in all stages of spermatogenesis (Bhin et al. 2015; Duan et al. 2016). The miR-210 negatively regulates spermatogenesis by targeting insulin-like growth factor 2 (IGF2) and nuclear receptor subfamily 1, group D, member 2 (NR1D2) expression in the transcriptional and translational phases (Duan et al. 2016; Tang et al. 2016). This miRNA could inhibit IGF2, one of the main factors involved in follicle-stimulating hormone (FSH) activity, which regulates Sertoli cell proliferation (Griffeth et al. 2014). Therefore, overexpression of miR-210 might result in the formation of structurally and functionally abnormal Sertoli cells and consequently impair spermatogenesis resolving as azoospermia. In this regard, elevated expression of miR-210 has been observed in patients with NOA (Tang et al. 2016). Additionally, miR-210 is involved in the regulation of transforming growth factor-beta (TGF-β) and nuclear factor kappa B (NF-κB) signaling pathways (Mizuno et al. 2009; Zhang et al. 2015), and it has been proved that these pathways are involved in spermatogenesis (Lilienbaum et al. 2000; Zhang et al. 2004). Studies have implicated that miR-210 inhibits activin/TGF-β signaling (Mizuno et al. 2009), and this signaling pathway controls Sertoli cell proliferation, germ cell maturation, and sperm functions (Nicholls et al. 2012; Sharkey et al. 2016). Moreover, NF-κB is one of the targets of miR-210 (Zhang et al. 2015). Its downregulation is associated with poor spermatogenesis and low sperm concentration (Ranganathan et al. 2002). Interestingly, miR-210 isolated from seminal fluid has been suggested as a male infertility biomarker (Xu et al. 2019).

The underlying mechanism for overexpression of this miRNA in patients with abnormal spermatogenesis such as azoospermia is not known, but it may be associated with hypoxia. It can be postulated that in azoospermia-related hypoxic diseases such as cryptorchidism and varicocele may be encouraged through the overexpression of miR-210 (Gat et al. 2010; Reyes et al. 2012).

miR-135a

miR-135a is highly expressed in the testis, and SSCs, playing an essential role in SSC maintenance by regulating the activity of forkhead box protein O1 (FoxO1). Downregulation of miR-135a and consequently abnormal activity of FoxO1 in cryptorchidism reduces the number of SSCs during early spermatogenesis when gonocytes are transitioning into spermatogonia. Therefore, it seems miR-135a works in favor of spermatogenesis and is required for SSCs function (Moritoki et al. 2014). In this regard, a significantly lower concentration of this miRNA has been found in the semen of patients with azoospermia in comparison to normozoospermic men (Tian et al. 2018). miR-135a is also involved in the regulation of Wnt and Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathways (Zhou et al. 2016; Mao et al. 2018) that is important for PGCs development, as well as germ cell proliferation and differentiation (Cheng et al. 2018). Accordingly, miR-135a could affect the Wnt and JAK/STAT signaling pathways modulating azoospermia.

miR-122a

miR-122a regulates spermatogenesis by affecting the TGF-β, Wnt/β-catenin, and Phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) signaling pathways (Wang et al. 2014; Sun et al. 2018). It is expressed in late-stage male germ cells, and it has been shown to negatively regulate the round spermatid expression of transition protein 2 (TNP2), which is involved in chromatin remodeling (Yu et al. 2005) and the histone protamine exchange (Martins and Krawetz 2007). It has been shown that miR-122a was downregulated in the sperm of patients with nonobstructive azoospermia and asthenozoospermia (Boissière et al. 2017). Downregulation of miR-122a that is involved in the antioxidant response (Qu and Zhang 2018) may increase oxidative stress that is well-known to negatively affect spermatogenesis (Aly et al. 2016).

miR-122a suppresses TGF-β signaling pathway (Sun et al. 2018), a pathway that is essential for normal testicular function and fertile sperm formation (Itman et al. 2011). Furthermore, this miRNA inhibits Wnt signaling (Wang et al. 2014), key signaling in the regulation of SSCs proliferation (Takase and Nusse 2016). PI3K/AKT pathway as another target of miR-122a (Yan et al. 2016) is also involved in self-renewal division of SSCs, Sertoli cells survival, and function (Lee et al. 2007; Sagare-Patil et al. 2013). Owing to the regulatory effects of miR-122a on the TGF-β, Wnt/β-catenin, and PI3K/AKT signaling pathways (Wang et al. 2014; Yan et al. 2016; Sun et al. 2018) and the involvement of these pathways in spermatogenesis (Lee et al. 2007; Itman et al. 2011; Sagare-Patil et al. 2013; Takase and Nusse 2016), abnormal levels of this miRNA may impair spermatogenesis, consistent with the development of azoospermia.

miR-181 c

The miR-181 family consists of four members, including miR-181a, miR-181b, miR-181 c, and miR-181d (Ji et al. 2011). Among these miRNAs, miR-181 c is abundant in testis and spermatozoa, and its role in male fecundity (Sendler et al. 2013) and fertility has previously been suggested (Cui et al. 2015). Moreover, miR-181 c is the most abundant miRNA in human spermatozoa and is upregulated in adult testis (Yan et al. 2007). In the testis, this miRNA plays a role in the differentiation of spermatogonia and spermatocytes via regulating the expression of SMAD family member 7 (Smad7) and round spermatid basic protein 1 (Rsbn1), as well as transcriptional regulation of haploid germ cells (Itman and Loveland 2008; Wang and Xu 2015).

A study conducted by (Noveski et al. 2016) showed that miR-181 c was downregulated in the testis of patients with hypospermatogenesis, while it was upregulated in patients with SCOS, AZFc deletion, or maturation arrest. In another study, it has been found that miR-181 c expression was higher in NOA patients with successful sperm retrieval compared to those with unsuccessful sperm retrieval. Therefore, it can be postulated that this miRNA is essential for spermatogenesis, and its downregulation can result in hypospermatogenesis or even lack of spermatogenesis (Fang et al. 2019).

In addition to the above, cancer cell studies have suggested that miR-181 c could also regulate PI3K/AKT and Hippo signaling pathways (Chen et al. 2015; Zhao et al. 2016). Interestingly, these pathways are involved in spermatogenesis, and their perturbation is associated with azoospermia (He et al. 2009). In this respect, it has been demonstrated that PI3K/AKT regulates differentiation, proliferation, and self-renewal division of SSCs, and also the survival of Sertoli cells (He et al. 2009). Hippo, which can be downregulated by miR-181 c, is also involved in testis development and normal spermatogenesis of sheep (Zhang et al. 2019a). This supports the view that perturbation of the signaling pathways involved in spermatogenesis by miR-181 c may contribute to azoospermia.

miR-192a

miR-192a is located on the human chromosome 11 (Sayadi et al. 2017), and studies have reported the role of this miRNA in spermatogenesis (Zhi et al. 2018). The expression of miR-192a has been examined in human testicular tissue as well as seminal fluid. It is similarly expressed in seminal fluid and testicular tissue, suggesting that miR-192a is mostly produced by germ cells in testes and secreted in the seminal plasma (Zhi et al. 2018). Subsequent investigations revealed that miR-192a could regulate the expression of genes that are associated with testicular venous hypertension, hypoxia, elevated oxidative stress, and elevated temperature in the spermatic vein (Zhi et al. 2018; Cannarella et al. 2020).

(Zhi et al. 2018) observed significantly higher expression of miR-192a in the seminal plasma and testicular tissue of NOA patients with unsuccessful sperm retrieval compared to normozoospermic men. In GC-2 cells (immortalized germ cells), transfection with this miRNA induced apoptosis increasing Caspase-3 providing a pathway to azoospermia. The involvement of an apoptotic mechanism is further supported by studies showing that miR-192a can directly affect p53 transcription and so act as an apoptotic factor (Sayadi et al. 2017) through p53 signaling that is involved in cell cycle arrest, apoptosis, sperm DNA integrity maintenance, and spermatogenesis (Beumer et al. 1998). Interestingly, elevated expression of p53 has been observed in NOA and OA males. Therefore, it can be postulated that the role of miR-192a in azoospermia is partly mediated by p53 (Rahbar et al. 2017). However, further studies are required to understand the exact role of miR-192a in azoospermia.

miR-188-3p

miR-188-3p is located on Xp11.23 (Pei et al. 2019) and is expressed in spermatocytes and spermatogonium. This miRNA has an important role in spermatogenesis and is involved in germ cell differentiation (Song et al. 2017). miR-188-3p is also involved in the apoptosis of damaged male germ cells via regulating p53 expression (Paul et al. 2007). miR-188-3p is downregulated in spermatogenic cells of NOA and OA patients due to hypo-acetylation of H3 and H4 in its promoter (Song et al. 2017). In confirming this mechanism, it has been shown that miR-188-3p binds to 3ʹUTR of the MLH1 gene and downregulates its expression in spermatogenic cells (Song et al. 2017). Furthermore, an association between the downregulation of miR-188-3p with elevated MLH1 expression has been found in azoospermic patients (Song et al. 2017). This miRNA can also negatively affect the PI3K/AKT signaling pathway (Yao et al. 2020). In the male reproductive system, the PI3K/AKT regulates the self-renewal division and differentiation of SSCs. This pathway is also essential for the survival of Sertoli cells and has a direct association with semen parameters such as motility (He et al. 2009; Sagare-Patil et al. 2013). Therefore, it can be hypothesized that in addition to the downregulation of miR-188-3p, its overexpression can also cause azoospermia via suppressing the PI3K/AKT signaling and subsequently inhibiting SSCs proliferation and differentiation; further studies are required to confirm this hypothesis.

miR-34 c

The miR-34 family consists of three miRNAs, including miR-34a, miR-34b, and miR-34 c (Achari et al. 2014). Previous studies have documented that miR-34 c is involved in spermatogenesis and enhancing germinal phenotypes, and its expression has been observed in the germ cells (Liang et al. 2012; Comazzetto et al. 2014). miR-34 c as a testis-specific miRNA is present in the spermatocytes and round spermatids and also is involved in the regulation of SSCs differentiation (Bouhallier et al. 2010). A study showed that the silencing of miR-34 c could increase the Bcl-2/Bcl-2-associated X protein (Bax) ratio and subsequently inhibit apoptosis of germ cells (Liang et al. 2012). Given the important role of miR-34 c in spermatogenesis, it might be involved in azoospermia (Finocchi et al. 2020). In this regard, it has been indicated that miR-34 c-5p was downregulated in seminal plasma and testicular tissue of SCOS, MA, and azoospermic patients (Abu-Halima et al. 2014a). Expression analysis of this miRNA in spermatozoa and testicular biopsies among infertile men suggested that miR-34 c could be used to discriminate patients with OA from NOA (Abu-Halima et al. 2014b).

miR-34 c can potentially be involved in the pathogenesis of azoospermia by affecting different signaling pathways such as Notch and TGF-β (Bouhallier et al. 2010). It has been reported that miR-34 c downregulates the Notch pathway, and as mentioned in previous sections, this signaling pathway is involved in spermatogenesis (Bouhallier et al. 2010). Studies have shown that TGF-β signaling can be activated by miR-34 c, and this signaling plays an important role in Sertoli cell proliferation, germ cell maturation, and the late stage of meiosis during spermatogenesis (Bouhallier et al. 2010). Furthermore, a study on colon cancer showed that miR-34 c could activate the JAK2/STAT3 pathway (Li et al. 2018), a pathway that is involved in the generation of germ cells and differentiation of SSCs (Zhang et al. 2020b). Since there is no evidence regarding the role of miR-34 c in azoospermia through the above-mentioned signaling pathways, more studies are required to clarify the mechanisms through which this miRNA can be involved in the pathogenesis of NOA and OA.

miR-141

miR-141 belongs to the miR-200 superfamily and is a type of male germ cell stage-specific miRNA (Hayashi et al. 2008). The expression of miR141 has been detected in primordial germ cells as well as epididymal spermatozoa (Reza et al. 2019). It has been reported that miR-141 is involved in PGCs development and spermatogenesis (Landgraf et al. 2007; Buchold et al. 2010). A study demonstrated hypomethylation-mediated activation of miR-141 in patients with NOA and suggested an association between spermatogenesis and silencing of this miRNA (Wu et al. 2013). It has also been reported that the miR-141 was upregulated in the testicular tissue, seminal plasma, and sperm of NOA patients, and its overexpression was correlated with apoptosis in the germ cells (Wu et al. 2013). Therefore, it can be postulated that miR-141 is involved in the pathogenesis of the NOA by inducing germ cell apoptosis. Furthermore, miR-141 can regulate PI3K/AKT, NF-κB, and TGF-β signaling pathways in spermatogenesis (Wu et al. 2013; Qin et al. 2019). It has been demonstrated that miR-141 inhibits the expression of factors involved in TGF-β and PI3K/AKT signaling pathways (Wu et al. 2013; Qin et al. 2019) which both have important roles in self-renewal and differentiation of SSCs proliferation and survival of Sertoli cells as well as spermatogenesis (Lee et al. 2007; Wu et al. 2013). In ovarian cancer cells, the ability of miR-141 to induce NF-κB signaling was suggested (Van Jaarsveld et al. 2013), and that this signaling could enhance Sertoli cell and spermatocyte functions (Ranganathan et al. 2002; Teng et al. 2013).

miR-17-92

The miR-17-92 cluster consists of 6 miRNAs, including miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1, and miR-92a (Concepcion et al. 2012). miR-17-92 is expressed in the testis (particularly in Sertoli cells) and has a role in the development of testis cells as well as spermatogenesis (Hurtado et al. 2018). Moreover, a spermatogenic defect has been reported following the inactivation of this cluster (Tong et al. 2012; Xie et al. 2016). In mice, this cluster plays a role in the regulation of spermatogonial differentiation (Xie et al. 2016). It negatively regulates heat shock transcription factor 2 (HSF2) and inhibits apoptosis during spermatogenesis (Björk et al. 2010). The deficiency of the miR-17-92 cluster could increase apoptosis of germ cells and therefore cause testicular atrophy and reduced epididymal sperm production (Xie et al. 2016). Interestingly, it has been observed that the expression of this miRNA was significantly lower in the testis of NOA patients compared to control men, suggesting that the downregulation of the miR-17-92 cluster may induce apoptosis of germ cells and consequently pave the way for azoospermia (Lian et al. 2009). Furthermore, miR-17-92 can respectively activate and suppress both the PI3K/AKT and TGF-β signaling pathways (Mestdagh et al. 2010; Rao et al. 2012). The involvement of these signaling pathways in spermatogenesis has been well documented (He et al. 2009; Young et al. 2015), supporting the view that dysregulation of the cluster may cause abnormal function of these signaling pathways and subsequently abnormal spermatogenesis and azoospermia.

Lethal-7 family

Lethal-7 (let-7) miRNA family consists of nine miRNAs (let-7a to let-7i), and it has been shown that this family is the most abundant miRNA in the testes and sperm (Buchold et al. 2010). (Luo et al. 2015) reported the expression of eight members (a, b, c, d, e, f, g, and i) of the let-7 family in murine type B spermatogonia cells and primary spermatocytes. The let-7b expression has also been seen to be restricted to the germ cells in the seminiferous tubules of adult rats (Sangiao-Alvarellos et al. 2015). The widespread expression of the let-7 family members in the male reproductive tract and germ cells suggests a key role in spermatogenesis. In this respect, a study demonstrated that this family is involved in male PGCs differentiation through Lin-28 homolog A (Lin28)/let-7/Blimp1 signaling (Brieño-Enríquez et al. 2015). It has also been shown that the overexpression of the let-7 family in PGCs is required for male germline commitment (Hayashi et al. 2008). Moreover, it has been reported that let-7 c-5p and let-7i-5p are involved in spermatogenesis in mice through LIN28A (Tang et al. 2017). Let-7 g, another member of the let-7 family, has been shown to negatively regulate the germ cell pool in mice (Shinoda et al. 2013). The let-7 family possibly regulates spermatogonial differentiation in mice via targeting extracellular signal-regulated kinase 1/2 (Erk1/2) and PI3K signaling pathways (Shen et al. 2014). Moreover, it has been documented that retinoic acid could significantly upregulate six members of this family, including let-7a, b, c, d, e, and g (Tong et al. 2011). It has also been suggested that this family is involved in retinoic acid-induced spermatogonial differentiation (Tong et al. 2011). Fndc3a is essential for adhesion of spermatids and Sertoli cells and as one of the potential targets of let-7a. Its mutation could cause infertility (Obholz et al. 2006). (Ma et al. 2018) also reported that let-7 g-5p could inhibit porcine sperm apoptosis via targeting PMAIP1.

Due to the involvement of the let-7 family in the maintenance of germ cell pool (Shinoda et al. 2013) as well as spermatogenesis (Tong et al. 2011; Shen et al. 2014; Tang et al. 2017), it can be postulated that this family may be involved in azoospermia. In supporting this hypothesis, Mashizy and colleagues have reported that the expression levels of let-7b in sperm of azoospermic patients were about 18-fold higher than fertile males (Mashizy et al. 2019). Interestingly, studies demonstrated that hsa-let-7b-5p, hsa-let-7 c-5p and hsa-let-7i- 5p were upregulated in NOA patients (Lian et al. 2009; Zhuang et al. 2015). Furthermore, (Wu et al. 2012) found that levels of let-7a in the seminal plasma of NOA were significantly higher than fertile controls. In another study, higher expression levels of let-7e-5p and let-7b in pachytene spermatocytes as well as let-7 g-5p, let-7i-5p, and let-7 f-5p in round spermatids of NOA compared to OA patients have been observed (Yao et al. 2017).

Other miRNAs

Other miRNAs are involved in testicular functions and spermatogenesis, and their abnormal expression has been reported in azoospermia patients; however, their exact involvement in azoospermia and the underlying mechanisms remain to be clarified. Some of these miRNAs include miR-429, miR-7-1-3p, miR-299-5p, miR-372, miR-373, miR-510, miR-525-3p, miR-941, and miR-539-5p (Lian et al. 2009; Wu et al. 2013; Ji et al. 2016; Barceló et al. 2018; Tang et al. 2018). miR-429 and miR-7-1-3p were shown to be upregulated in the seminal plasma of patients with NOA. miR-429 could suppress spermatogenesis by downregulation of Nanos C2HC-type zinc finger 1 (NANOS1) (Wu et al. 2013). Lower levels of miR-372 and miR-373 were accompanied by an elevated rate of apoptosis in the testicular cells observed in NOA patients. However, these miRNAs can inhibit apoptosis and induce cell proliferation (Lian et al. 2009). Downregulation of miR-510 in testicular tissues of NOA patients has been reported. Interestingly, Peroxiredoxin 1 (PRDX1) is one of the target genes for miR-510, which is involved in ROS elimination (Ji et al. 2016), in which the role of oxidative stress in azoospermia has been well known (Aly et al. 2016). Association between miR-525-3p and sperm function has also been documented. In this respect, (Liu et al. 2012) have found that this miRNA was significantly lower in the seminal fluid of infertile men with abnormal semen. Moreover, (Zhou et al. 2019) found that miR-525-3p modulates the expression of Semenogelin 1 (SEMG1). These groups also indicated that low levels of this miRNA and high expression of SEMG1 in ejaculatory spermatozoa were associated with low progressively motile sperm and abnormal sperm morphology. Moreover, the lower levels of miR-539-5p in semen-derived exosomes of azoospermic patients compared with normozoospermic men have been shown (Barceló et al. 2018). More interestingly, (Barceló et al. 2018) demonstrated that a logistic model combining the expression values of exosomal miR-539-5p and miR-941 in the seminal fluid could predict sperm retrieval success in a testicular biopsy of azoospermic patients with high sensitivity and specificity (> 90%). In a recent study, it has been reported that miR-539-5p was upregulated in the testis tissues of NOA patients and this may be involved in the spermatogenesis failure (Zhang et al. 2020a).

Genetic variations of miRNA-related genes in azoospermia

Genetic variations exemplified by single nucleotide polymorphisms (SNPs) in miRNA-related genes can affect spermatogenesis and eventually lead to azoospermia (summarized in Table 2). One study indicates that rs5951785 polymorphism (A > G) near hsa-miR-506 and −507 genes could reduce the expression of these miRNAs and their binding ability to the target genes and therefore increase the risk of NOA by ~1.5 fold (Ji et al. 2016). One of the important targets of miR-506 in spermatogonial cells is Glioma-associated oncogene family zinc finger 3 (GLI3), which can inhibit cell proliferation and increases apoptosis (Persengiev et al. 1997). ADAM17 is involved in germ cell apoptosis, and its expression can be regulated by miR-507 (Moreno et al. 2011; Urriola-Muñoz et al. 2014). In contrast, rs1447393 polymorphism (C > G) near hsa-miR-510 could play a protective role and reduced the risk of NOA by half (Ji et al. 2016). PRDX1, a target of miR-510, is involved in the removal of ROS and hence inhibition of sperm DNA damage. Therefore, reducing the binding ability of the mutant-type miR-510 to its target can increase the expression of PRDX1 and ROS elimination and subsequently decrease the risk of azoospermia (Ji et al. 2016). However, (Vucic et al. 2014) reported a lack of association between rs895819 polymorphism in the miR-27a gene and NOA.

Table 1. Role of miRNAs involved in azoospermia

miRNAs	Role in spermatogenesis	Mechanisms	Down/up-regulation in NOA and its outcome	Ref.
miR-449	Regulation of germ cell meiosis Induction of spermatids differentiation Induction of sperm maturation	Modulation of Notch signaling
	Regulation of meiotic process of spermatogenesis Induction of SSCs maintenance Induction of Sertoli cell maturation	Induction of p53 signalingDownregulation of E2F/Rb signaling pathway	Downregulated in NOA Spermatogenesis arrest	(Abu-Halima et al. 2014a)
miR-210	Regulation of Sertoli cell proliferation Regulation of germ cell maturation	Modulation of Activin/TGF-β and NF-κB signaling pathways	Upregulated in NOA Formation of Sertoli cells with abnormal structure and function Impaired spermatogenesis	(Tang et al. 2016)
miR-135a	Induction of SSCs maintenance, proliferation, and differentiation	Modulation of Wnt/β-catenin and JAK/STAT signaling pathways	Downregulated in NOA Reduced SSCs numbers	(Tian et al. 2018)
miR-122a	Regulation of Sertoli cell proliferation Regulation of germ cell maturation	Modulation of TGF-β signalingModulation of Wnt/β-catenin	Downregulated in NOA Oxidative stress during spermatogenesis Spermatogenesis impairment	(Boissière et al. 2017)
	Regulation of SSCs proliferation Regulation of self-renewal division of SSCs Regulation of Sertoli cells’ survival and function.	Modulation of PI3K/AKT signaling pathways
miR-181 c	Induction of SSCs differentiation, proliferation, and self-renewal division Induction of Sertoli cells survival	Induction of PI3K/AKT signaling	Upregulated in NOA Spermatogenesis arrest Reduced SSCs numbers	(Fang et al. 2019)
	Induction of PGCs development Induction of spermatogonia proliferation and differentiation	Induction of Wnt/β-catenin
miR-192a	Regulation of meiotic process of spermatogenesis	Induction of p53 signaling	Upregulated in NOA Reduced SSCs numbers Increased germ cells apoptosis	(Zhi et al. 2018)
miR-188-3p	Regulation of germ cells differentiation Regulation of SSCs differentiation and proliferation Induction of damaged male germ cells apoptosis	Modulation of PI3K/AKT signaling pathway	Downregulated in NOA Increased apoptosis of SSCs	(Song et al. 2017)
miR-34 c	Regulation of SSCs differentiation	Modulation of Notch signaling	Downregulated in NOA Reduced SSCs numbers Increased germ cells apoptosis	(Abu-Halima et al. 2014a)
	Induction of Sertoli cell proliferation Induction of germ cells maturation Induction of meiosis during the spermatogenesis	Activation of TGF-β signaling
	Induction of germ cell generation Induction of SSCs differentiation	Activation of JAK2/STAT3 signaling
miR-141	Regulation of PGCs development Regulation of SSCs self-renewal and differentiation Regulation of Sertoli cells proliferation and survival	Modulation of PI3K/AKT and TGF-β signaling pathways	Upregulated in NOA Increased germ cells apoptosis	(Wu et al. 2013)
	Induction of Sertoli cell and spermatocyte functions	Activation of the NF-κB signaling pathway
miR-17-92	Regulation of spermatogonial differentiation	Activation of PI3K/AKT	Downregulated in NOA Testicular atrophy Reduced epididymal sperm production Increased apoptosis of germ cells	(Lian et al. 2009)
	Inhibition of apoptosis during spermatogenesis	Modulation of HSF2
	Regulation of SSCs differentiation and proliferation	Modulation of TGF-β signaling pathway
Let-7 family	Regulation of male germline commitment	Modulation of Lin-28 homolog A (Lin28)/let-7/Blimp1 signaling	Upregulation of let-7a, b, c, and i in NOA Spermatogenesis impairment Increased apoptosis of germ cells	(Lian et al. 2009; Wu et al. 2012; Zhuang et al. 2015; Mashizy et al. 2019)
	Regulation of spermatogonial differentiation	Modulation of Erk1/2 and PI3K signaling pathways
	Inhibition of sperm apoptosis	Downregulation of PMAIP1

NOA, non-obstructive azoospermia; SSCs, spermatogonial stem cells; E2F/Rb, E2F transcription factor/retinoblastoma protein; NF-κB, nuclear factor kappa B; JAK/STAT, Janus kinase/signal transducers and activators of transcription; PI3K/AKT, Phosphatidylinositol 3-kinase/protein kinase B; PGCs, primordial germ cells; HSF2, Heat Shock Transcription Factor 2; Erk1/2, extracellular signal-regulated kinase 1/2; PMAIP1, Phorbol-12-Myristate-13-Acetate-Induced Protein 1

Table 2. The genetic variations of miRNAs and their biogenesis/function-related genes in azoospermia

Variation(S)	Affected gene(s)	Effects	Association with azoospermia	Reference(s)
rs5951785	hsa-miR-506 and −507	Downregulating expression of the miRNAs Reducing the binding ability of the miRNAs	Increase of the NOA risk about 1.5 fold	(Ji et al. 2016)
rs1447393	hsa-miR-510	Reducing the binding ability of the miRNA	Reduction of the NOA risk by half	(Ji et al. 2016)
rs895819	miR-27a	Not determined	No association with NOA	(Vucic et al. 2014)
rs197388	Gem-associated protein 3 (GEMIN3) gene	Disruption in the miRNAs-related regulation of chromatoid body functions	Positive association with idiopathic azoospermia	(Ay et al. 2017)
rs1057035 and rs3742330	DICER1	Disruption in miRNA biogenesis and maturation	Positive association with azoospermia	(Moghbelinejad et al. 2018)

NOA, non-obstructive azoospermia

There are also studies showing the association between azoospermia and polymorphism of genes that are involved in biogenesis and functions of miRNAs. For example, (Ay et al. 2017) reported that the AA genotype of rs197388 in the Gem-associated protein 3 (GEMIN3) gene might be a cause of idiopathic azoospermia. A complex of GEMIN3, Pumilio RNA binding family member 2 (PUM2), NANOS1 proteins with miRNAs can regulate protein translation within the chromatoid body of human germ cells; this process is necessary for spermatogenesis. GEMIN3 gene may alter the regulatory function of miRNA in the chromatoid body (Ay et al. 2017). In another study, (Moghbelinejad et al. 2018) evaluated the association of azoospermia with rs12323635, rs1057035, rs13078, and rs3742330 polymorphisms in the DICER1 gene and rs10719, rs642321, and rs2291102 polymorphisms in the drosha ribonuclease III (DROSHA) gene. They found that the CC genotype of rs1057035 polymorphism (T > C) in the DICER1 gene could be related to azoospermia. This gene encodes an RNase III enzyme that plays an important role in miRNA biogenesis and maturation (Park et al. 2011). DICER1 is also necessary for germ cell differentiation and sperm survival during spermatogenesis (Papaioannou et al. 2009). (Fu et al. 2016) also indicated a positive association between the AA genotype of rs3742330 polymorphism in the DICER1 gene and azoospermia.

miRNAs as therapeutic targets for azoospermia

As described in the previous sections, various miRNAs are directly and indirectly involved in the pathogenesis of azoospermia. Studies have shown that abnormal expression of these miRNAs could negatively affect spermatogenesis yielding NOA. Therefore, targeting these miRNAs to manipulate the miRNA-related signaling in favor of spermatogenesis can be considered as a potential therapeutic approach. Although there is no study regarding azoospermia prevention and treatment using miRNAs, there are several strategies to use these small noncoding RNA molecules as therapeutic targets. In general, there are two approaches, miRNA inhibition and replacement, depending on whether the miRNA is upregulated or downregulated in the disease (Krützfeldt 2016). Different methods can be suggested to inhibit miRNAs that are abnormally upregulated, including anti-miRNA antisense oligonucleotide (AMO), miRNA sponge, and miRNA masking (Liu et al. 2008). In the AMO method, antagomir as a single-stranded molecule is used, which is complementary to the target miRNA. However, due to the susceptibility of antagomir to endonuclease, stabilization by chemical modification such as 2ʹ-O-methylation, phosphorothioate backbones, cholesterol moiety at the 3ʹ-UTR, locked nucleic acid (LNA), and peptide nucleic acid (PNA) (Fabani et al. 2010; Garzon et al. 2010) is required. For example, miR-210, which is also upregulated in azoospermia patients, was successfully inhibited in leukemic cells using PNA modified anti-miR-210 (Manicardi et al. 2012). A miR-mask is like an AMO, but instead of directly binding to the miRNA, it binds to the miRNA binding sites of the target mRNA. Thus the miR-mask inhibits miRNA access to the target mRNA and reduces its activity (Lee et al. 2012). Recently, a new approach has also been introduced for miRNA downregulation called miRNA sponge, a transcript with complementary sequence to a target miRNA (Ebert and Sharp 2010). The encoded RNA of the sex-determining region Y (SRY) is a typical example of a circular RNA with a sponge function (Hansen et al. 2013).

For microRNA replacement, the miRNA can be transferred into the cells using vectors and aptamers (Christopher et al. 2016). Different viral (e.g., adenoviruses, retroviruses, and lentiviruses) and non-viral (peptides, dendrimers, lipid-based nanocarriers, and polymeric) vectors can be applied for the miRNA delivery; it should be noted that using viral vectors has a risk of toxicity and immune response (Geisler and Fechner 2016; Bai et al. 2019). Aptamers can also be used for the delivery of miRNAs to the target cells (Wilner et al. 2012). In a study, (Russo et al. 2018) delivered aptamers-miR-34 c to non-small-cell lung cancer cells and affected cell proliferation; interestingly miR-34 c is also downregulated in azoospermia patients. Moreover, miRNA mimics can be applied for the induction of miRNA-related pathways in cases with abnormally downregulated miRNA (Walayat et al. 2018). In this regard, miR-188-3p mimics were transferred into colorectal cancer cell lines by lentiviral vectors and could produce a stable miR-188-3p overexpressing cell line (Pichler et al. 2017). As shown above in miR-188-3p of the section titled “miRNAs involved in azoospermia”, miR-188-3p is reduced in azoospermia patients (Song et al. 2017). This collection of strategies and techniques for miRNA silencing and induction in target cells can also be investigated for the treatment of miRNA-related abnormalities in spermatogenesis.

Conclusion

Given the over-and under-expression of different miRNAs that are involved in spermatogenesis in NOA patients and the importance of the miRNAs-targeting pathways in successful spermatogenesis, it can be postulated that miRNAs are one of the important players in spermatogenesis failure and NOA development. Since one type of miRNA can affect several target signaling pathways and subsequently change the cell fate, miRNA-related abnormal spermatogenesis cannot be treated by targeting the downstream signaling pathways. Therefore, the miRNA itself should be considered as an upstream target for the treatment of NOA. In this regard, different efficient techniques have been introduced to manipulate miRNAs expression or activities in the cells (miRNAs as therapeutic targets for azoospermia), and it can be hypothesized that these methods can be considered for the prevention of NOA development or treatment of NOA patients. Nevertheless, studies have yet to be conducted on the treatment of NOA using miRNA-targeting strategies.

Authors’ contributions

Study design and critical revision: AF and RD; Critical revisions: MN and NZ; Clinical advisement and study design: YA; Literature review: YRR, HO, NN, ZBA; Manuscript drafting: YRR, RZ, and SN.

Acknowledgments

We thank the academic staff of the Department of Reproductive Biology, Tabriz University of Medical Sciences, for their valuable advice. This study has been extracted from the thesis registered in Stem Cell Research Center, Tabriz University of Medical Sciences (Thesis No. IR.TBZMED.REC.1398.662). ‎

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was financially supported by Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran (approval ID: IR.TBZMED.REC.1398.662)