Unraveling the microRNAs, key players in folliculogenesis and ovarian diseases

Background Folliculogenesis is an intricate process that involves the development and maturation of ovarian follicles in females. During folliculogenesis, multiple factors including hormones, growth factors, and signaling pathways regulate the growth and maturation of follicles. In recent years, microRNA, short non-coding RNA molecules, has gained attention due to its roles in the physiology and pathophysiology of various diseases in humans. It is known to have an important part in ovarian health and illness and its functions extend to several cellular processes. Main body In this overview, we look at the importance of microRNAs in ovarian illnesses and how they function during follicle growth in the ovaries. Short RNA molecules (22 nucleotides) called microRNAs may influence several mRNA targets in different biological processes. The expression patterns of these small non-coding RNAs undergo dynamic changes during the several phases of follicular development; they play a function in post-transcriptional gene regulation. Follicle development, follicular atresia (regression of the follicles), and ovulation are all intricately regulated by the dynamic expression of distinct miRNAs throughout the various phases of folliculogenesis. The role of microRNAs (miRNAs), which are known to regulate gene expression, has recently come to light as crucial in the development and advancement of a number of ovarian diseases. Abnormalities of the human ovary, such as ovarian cancer, polycystic ovary syndrome (PCOS), and endometriosis, have prompted extensive research into the dysregulation of microRNAs. Endometriosis is associated with miRNAs that are known to have a role in processes such as invasion, cell growth, cell adhesion, angiogenesis, and epithelial-mesenchymal transition. The disturbance of target gene expression resulting from abnormal miRNA production is a potential factor contributing to cancer development. Some microRNAs (miRNAs) differ in expression levels between women with polycystic ovary syndrome and healthy controls, indicating that miRNAs may play a role in the development of PCOS. Conclusion Extensive research carried out over the last 20 years has illuminated the roles of microRNAs (miRNAs), demonstrating their critical importance in controlling gene expression and the cell cycle. Changes in the quantities of microRNAs (miRNAs) may affect the aggressiveness of cancer and contribute to a variety of gynecological disorders. It appears that microRNAs hold potential as diagnostic biomarkers and treatment potential for various ovarian diseases.

MicroRNAs, found in cells and bloodstreams, show significant variation in expression profiles between healthy individuals and those with various diseases [10].Researchers Lawrie et al. found microRNAs (miRNAs) in blood as a non-invasive way to diagnose DLBCL [10].Alterations in miRNA levels have been observed across various diseases, including cancer, inflammation, reproductive, metabolic, and cardiovascular disorders [11].Currently, microRNAs are being suggested as diagnosis and monitoring tools for cancers and other conditions [11].Dysregulation of miRNA expression has been extensively investigated in female reproductive system diseases [11][12][13][14][15][16].
The female reproductive system's core function is folliculogenesis, a crucial process that shapes ovarian dynamics [17].The process begins with primordial germ cells migrating towards the embryonic genital ridge, differentiation, and activation of primordial follicles [17].A significant reduction of these follicles forms the ovarian follicle reserve, which recruits growing follicles for further development [17].As follicles undergo apoptosis, only a minority become primary follicles, and the process continues with the transformation of primary follicles into pre-antral, antral, and Graafian follicles [17].The intricate orchestration of events is influenced by localized signals from both oocytes and somatic cells [17].
Follicular granulosa cells (FGCs) are crucial for supporting oocytes by releasing growth factors and growth hormones, and controlling oocyte development [18].Regulating follicular growth and FGC apoptosis is a crucial role for microRNAs [18].Understanding these processes is essential for advancing knowledge of ovarian development and related disorders [18].MicroRNAs are pivotal in overseeing a range of mammalian FGC functions like proliferation, differentiation, and cumulus expansion [18].Their significance in oocyte and fetal development in mammals is widely acknowledged [18].Nonetheless, the exact impacts and mechanisms of miRNA regulation, particularly regarding target genes and pathways, are not fully comprehended [19].Ovarian illnesses and disorders are greatly impacted by microR-NAs, a network of genes that regulate gene expression and are essential in the development and advancement of these conditions [19].
Ten percent of reproductive-age women suffer from endometriosis, a benign chronic illness [11].Finding endometrial glands and stroma outside of the uterus that are functioning and responsive to estrogen signaling is the hallmark of endometriosis [11].Endometriosis showcases miRNA signatures within lesions, which mediate processes like angiogenesis, inflammation, and cell proliferation, affecting the establishment and perpetuation of the condition [11].Both ovarian cancer and polycystic ovarian syndrome (PCOS) are endocrine illnesses that are affected by microRNAs [20].PCOS affects 8-13% of reproductive-aged women globally, while ovarian cancer risk affects 1 in 78 women during their lifetime [20].The altered miRNAs in PCOS disrupt hormonal balance, follicular development, and insulin resistance, while in ovarian cancer, it leads to oncogenic shifts and metastatic cell spread [21].In the battle against ovarian cancer, miR-NAs may act as indicators for diagnosis and as targets for treatment [22].
The purpose of this study is to provide a synopsis of the most current findings on microRNAs (miRNAs) and their impact on human follicular development and folliculogenesis.This review focuses on findings regarding miRNA roles in follicular development and ovarian diseases (PCOS, endometriosis, and ovarian cancer).

MicroRNA biogenesis
Processing RNA polymerase II/III transcripts, either before or during transcription, initiates microRNA biogenesis [23].Two separate mechanisms, known as the canonical and non-canonical pathways, are involved in miRNA synthesis [24].
Beginning miRNA biogenesis in the canonical route is the production of the principal miRNA transcript (pri-miRNA) [24].In order to create the precursor miRNA, the microprocessor complex cleaves this pri-miRNA [24].Depending on Exportin5/RanGTP, the process of pre-miRNA export to the cytoplasm follows transcription in the nucleus [24].A mature miRNA duplex undergoes further processing in the cytoplasm; a miRNA-induced silencing complex (miRISC) is formed when one strand of the duplex is integrated with the Argonaute (AGO) proteins [24].
One of the non-canonical routes involves the microprocessor complex transferring short hairpin RNA (shRNA) to the cytoplasm via Exportin5/RanGTP after cleaving it [24].Extra processing happens via Dicer-independent AGO2-dependent cleavage [24].Although their nuclear and cytoplasmic transit is different, both Mirtrons and m7G-pre-miRNA need Dicer for cytoplasmic maturation.Mirtrons are exported by Exportin5/RanGTP, in contrast to m7G-pre-miRNA, which is exported through Exportin [24].
Regardless of the pathway, the end result is the formation of a functional miRISC complex [24].When miRISC attaches to target mRNAs, it will interfere with the eIF4F complex, leading to translational inhibition [24].The decapping complex is able to remove the m7G cap from the target mRNA because deadenylation begins with PAN2/3 and ends with the CCR4-NOT complex [24].At long last, the decapped messenger RNA is assisted in its 5′-3′ degradation by the exoribonuclease XRN1 [24].

Folliculogenesis: an overview
Folliculogenesis is the intricate process by which a woman's ovarian follicles grow and mature [25].The process starts with the recruitment of primordial follicles, which are then transformed into primary follicles after a sequence of growth and differentiation phases [25].Follicle development continues with the formation of secondary follicles, which are identified by the presence of an antrum, a hollow filled with fluid, and antral follicles [25].The final stage of folliculogenesis focuses on selecting a dominant follicle, responsible for further growth and eventually ovulation [25].Throughout folliculogenesis, numerous factors such as hormones, growth factors, and signaling pathways work together to coordinate the enlargement and maturation of the follicles [25].Particularly important are the granulosa cells that surround the oocyte, playing a vital role in supporting its maturation [25].The development of the oocyte depends on the nutrition and growth substances provided by these cells.The orchestration of folliculogenesis involves precise regulation and coordination, with intricate interactions among the oocyte, granulosa cells, and surrounding ovarian tissues [25].Any disruptions or abnormalities in this process can lead to various reproductive disorders and fertility issues [25].

Identification of miRNAs involved in folliculogenesis
All stages of ovarian follicle development-from growth to regression (atresia) to ovulation-rely on microRNAs (miRNAs) [19].Figure 1 shows a summary of the folliculogenesis process and the miRNAs that play roles in each stage of follicular development.These tiny non-coding RNA molecules are involved in post-transcriptional gene regulation and undergo dynamic changes in their expression patterns during the various phases of follicular development in Fig. 1 [19].Multiple studies have revealed miRNA expression patterns during folliculogenesis (Table 1).
The following basic phases of follicle development are associated with specific microRNA (miRNA) expression profiles from primordial, to primary, pre-antral, tiny antral, and large antral follicles, continues to the preovulatory follicles, then early and late corpus luteum, and corpus albicans [26].Out of all the stages, let-7a, let-7b, miR-125b, and miR-21 had the highest levels of expression of microRNAs.While miR-199a-3p, miR-145, and miR-31 were all overexpressed during follicular development, their expression dropped dramatically during follicular-luteal transition [27].
Follicle development, also known as folliculogenesis, initiates with the breakdown of germ cell clusters and progresses with the formation of primordial follicles [27].A prior investigation discovered the expression of miR-143 within pre-granulosa cells through the application of in situ hybridization.This miRNA reportedly suppresses the expression of genes associated with the cell cycle and reduces the proliferation of pre-granulosa cells, hence inhibiting the formation of primordial follicles [50].More than ninety-nine percent of ovarian follicles undergo atresia degeneration during folliculogenesis [50].
Mature oocytes and follicular development (FD) take place in a woman's ovary [21].However, during these processes, a complex and natural phenomenon known as atresia occurs [21].The apoptosis of the granulosa cells (GCs) surrounding the oocytes is a hallmark of atresia, the spontaneous death of ovarian follicles [21].Ovulation occurs in less than one percent of mammalian ovarian follicles, whereas atresia affects more than ninety-nine percent [21].This phenomenon of atresia impacts follicular growth and development at all stages [21].A recent study found that follicular atresia and its development are influenced by certain miRNA clusters and families.Which miRNA cluster(s) linked to each stage of follicular development, however, remained undetermined [18].

Mechanisms of miRNAs in folliculogenesis regulation
Multiple variables, including Smads, ligand activation of type I receptors (also called activin receptor-like kinases, or ALKs), and members of the TGF-β superfamily, impact the complex process of follicle formation [27].The effect of microRNAs on these components is crucial to the control of follicle maturation [27].Research found that the TGF-β/Smad signaling pathway controls miRNA-224 expression [27].It has been discovered that elevated levels of miR-224, which target Smad4, enhance the proliferation of granulosa cells triggered by TGF-β [27].On the other hand, granulosa cell proliferation triggered by TGF-β1 is somewhat reduced when the endogenous miR-224 is suppressed [27].
Furthermore, miRNAs also influence ovulation indirectly [27].Research conducted by Hasuwa et al. on the effects of miR-200b and miR-429 on female mouse infertility found that blocking the production of luteinizing hormone (LH) was hindered by inactivating these miR-NAs [27].This finding indicated that these miRNAs indirectly contribute to ovulation by playing a role in the hypothalamus-pituitary-ovarian axis [27].
Researchers studying ovarian development have now recognized that follicular atresia, the process of degeneration and regression of ovarian follicles, involves granulosa cell (GC) apoptosis as a fundamental physical mechanism [51].This process initiates with the observation of pyknotic nuclei in GCs.Subsequently, the GC layer detaches, leading to basal membrane fragmentation [51].These changes eventually lead to the hypertrophy of thecal cells, disrupting thecal integration and thecal vessels [51].It is noteworthy that oocyte degeneration may happen at any stage of atresia [51].As a result of these findings, research on follicular atresia has shifted its focus toward investigating the molecular regulation of GC apoptosis [51].
Through altering the target genes' expression levels, miRNAs influence how follicular granulosa cells (FGCs) function [52].One such miRNA family, known as the miR-let-7 family, exhibits a high degree of sequence conservation across various animal species [52].There is a wide range of functions performed by members of the let-7 family, including regulating the differentiation and proliferation of cells, tissue development, and inhibition of tumor development [52].This miR-let-7 family demonstrates differential expression patterns during follicular atresia [52].MiR-let-7a, let-7b, let-7c, and let-7i gene expression levels were specifically observed to be lower in early and progressed stages of follicular atresia compared to healthy follicles [53,54].Premature ovarian failure (POF) is distinguished by lower levels of let-7c compared to healthy women, according to the research [54].This implies that let-7c likely helps in promoting normal follicular development.On the other hand, let-7g is highly expressed during atresia, unlike its family members, indicating a different role in follicles [54].
Blocking the expression of pri-miR-21 directly influences the expression of miR-21 in bovine oocytes and cumulus cells (CCs) [55].Enhancing the expression of miR-21 had a notable impact on decreasing apoptosis in cumulus cells (CCs) [55].Oocyte-secreted factors (OSFs) initiate the activation of the PI3K/Akt signaling pathway, resulting in increased miR-21 levels and the suppression of apoptosis in CCs [56].It has been demonstrated that oocytes and CCs withstand apoptosis more than other antral follicle components [56].

MiRNA and steroidogenesis
miRNAs act as regulators of ovarian steroid hormones by aiming at hormone receptors and influencing hormone biosynthesis and release [57].A specific example is the regulation of estradiol (E2), which is crucial for ovarian follicle development and primarily controlled by the aromatase enzyme [57].A post-transcriptional mechanism that limits estradiol production in granulosa cells and downregulates aromatase expression was uncovered by Xu et al. as miR-378 [42].In contrast, miR-133b enhances the synthesis of ovarian estradiol by targeting Foxl2, a transcriptional regulator that represses the expression of StAR and CYP19A1 [58].By targeting Foxl2, miR-133b promotes the biosynthesis of estradiol, thereby facilitating the production of this hormone in the ovary [58].
Apart from controlling estradiol production, miRNAs also play a role in regulating the release of estradiol [59].In ovarian GCs, enhancement of estradiol happened by the release of miR-383 that inhibits RBMS1 [59].How this is accomplished by regulating RBMS1 mRNA stability, which in turn affects granulosa cell steroidogenesis by rendering c-Myc inactive [59].In addition, miR-378 and miR-423-5p target the CYP19A1 mRNA and are important in controlling estradiol production [59].They decrease the protein content and enzyme activity of CYP19A1, thereby exerting an inhibitory effect on estradiol synthesis.These regulatory mechanisms have been observed in newborn piglets [59].
Sirotkin et al. conducted a study that revealed several miRNAs' role in controlling reproductive functions [38].They identified a set of 36 miRNAs that inhibited the release of progesterone in granulosa cells [38].Conversely, they found that 16 miRNAs promoted progesterone release [38].Additionally, the research found that the following microRNAs were implicated in the inhibition of testosterone production in granulosa cells: mir-108, mir-122, let-7a, let-7b, let-7c, miR-16, miR-17-3p, miR-24, miR-25, and miR-26a [38].These findings emphasize the diverse regulatory effects of particular miRNAs on the release of reproductive hormones in the ovary [59].

Endometriosis
A benign inflammatory condition known as endometriosis affects 10% of reproductive-aged women.Hormonal, immunological, and genetic factors contribute to its etiology [51].Endometriosis is diagnosed when stroma and functioning endometrial glands are discovered in locations other than the uterus, such as the ovaries, pelvis, and rectovaginal septum [60].Current evidence has shown molecular defects in endometrial cells, with miRNA potentially acting as an endometriosis biomarker.Several dysregulated miRNAs have been directly linked to the disease's pathogenesis (Table 2) [6].

Table 2 miRNA expression profiles in endometriosis
according to the various types of lesions.The most often downregulated miRNA in endometriosis was miR-200b [52].An essential step in endometriosis, the epithelialmesenchymal transition (EMT) involves this microRNA [53,54].Other miRNAs were up-or downregulated in endometriotic lesions, and also involved in endometriotic lesion processes, angiogenesis, cell proliferation, adhesion, and invasion [53].
Endometriosis can cause diminished fertility and a shortened reproductive window through factors like disrupted folliculogenesis, poor oocyte quality, abnormal follicular development, and increased ROS levels [55].Endometriosis-related infertility is associated with changes in microRNAs that affect genes such as HOXA10, aromatase, progesterone receptors, matrix metalloproteinases (MMPs), and alphaV beta 3-integrin [56].Upregulation of miR-135a/b in endometriosis-affected women leads to repression of HOXA10, a transcription factor crucial for endometrial receptivity.This illustrates an early instance where dysregulated miRNA in endometriosis correlates with implantation failure [56].
One hallmark of endometriosis is progesterone resistance, which is aided by microRNAs such as the let-7 family, miR-29c, miR-125b, miR-135a/b, miR-194, and miR-196a [6].This resistance disrupts essential mechanisms like endometrial cell decidualization, affecting targets like FKBP4, PGR, and MMP26, thereby impairing fertility potential [6].As a result of its negative regulation of Ras oncogenes, the Let-7 family-the first human miRNA to be discovered-regulates cell differentiation and acts as a tumor suppressor.Endometriosis and several malignancies are associated with its downregulation [77].In severe endometriosis, polymorphisms at the KRAS gene's let-7 binding site increase, leading to higher KRAS mRNA and protein levels [42,78].Let-7 family is involved in estrogen biosynthesis and can be inhibited by aromatase inhibitors [79].
Enhancement of angiogenesis and anti-apoptotic process have been suggested as potential links between the pathophysiology and progression of endometriosis [80].During embryonic development in zebrafish, MiR-126 controls the response of endothelial cells to VEGF, leading to a decrease in vascular integrity and bleeding [79].It represses negative VEGF pathway regulators, potentially influencing vascular integrity and function [79].Mir-126 is also thought to affect the EGFL7 function which limits the endothelial cells' spatial distribution to control their migration [79].Endometriotic cyst stromal cells also showed disruption in the angiogenesis process through DNA hypermethylation in miR-503, which interacts with cyclin D1, BCL-2, Ras homology A, and VEFG-A, among others, and contributes to ECM contractility, angiogenesis, resistance to apoptosis, and proliferation [79].

Ovarian cancer
Dysregulation of miRNAs can disrupt their target genes' expression, thereby contributing to the onset of cancer development [81] (Table 3).Genetic abnormalities like chromosomal deletions, rearrangements, and mutations, together with epigenetic modifications, are among the pathways that might lead to this misexpression [81].Furthermore, abnormalities in transcription and posttranscription also play a major role in the development and advancement of ovarian cancer [81].MiRNA deregulation is influenced by factors like epigenetic changes, chromosome rearrangements, and genomic copy number alteration [82].
There is great promise for miRNAs as clinical indicators for the early detection of OC.If miRNA is to be used
Dysregulation of miRNAs in blood (exosomes) could improve early ovarian cancer diagnosis and prognosis.Both bodily fluids and tissue specimens contain miRNAs; however, tissue samples are only valuable after the early diagnosis of OC [109].Blood-based circulatory miRNA is less invasive for diagnosis but has low abundance [109].Prior to their clinical use, tissue-based miRNA and serum/plasma-based miRNA must be distinguished.To completely comprehend their roles, therapeutic potential, and usefulness as diagnostic or prognostic biomarkers in OC, additional investigation is required, despite the identification of numerous miRNAs with dysregulated patterns [109].

PCOS
Androgen excess and ovarian dysfunction define polycystic ovarian syndrome (PCOS), being the most prevalent endocrine disorder in reproductive-aged women globally [20].Diagnosis requires at least two criteria: chronic anovulation, hyperandrogenism, and polycystic ovaries, noting that other diagnoses mimicking PCOS features must be excluded [111].On a global scale, 8-13% of women of childbearing age have this illness, with an additional 70% going unidentified [112].Insulin resistance, excessive hair growth, difficulty conceiving, irregular ovulation, weight gain, high blood pressure, cancer, and depressive symptoms are some of the additional health conditions that may arise due to this disorder [112].Consequently, in order to minimize potential long-term health consequences, it is vital that women diagnosed with polycystic ovary syndrome (PCOS) get appropriate treatment measures as soon as possible.
New evidence suggests that specific microRNA expression levels differ between healthy persons and women with polycystic ovary syndrome [113,114] (Table 4).These observations suggest that miRNAs could potentially have significant involvement in the onset and progression of PCOS [113,114].Granulosa cells have been shown to have both elevated proliferation and apoptotic rates in relation to a large number of miRNAs that exhibit variable expression.These results may be rationally explained, even if they seem to be inconsistent at first.Potentially, the transformation of primordial follicles into primary follicles is responsible for the surge in primary follicles [115].Table 4 shows that aberrant miRNA expression may affect cell proliferation, apoptosis, steroidogenesis, folliculogenesis, glucose metabolism, and insulin sensitivity, all of which may play a role in the pathogenesis of polycystic ovary syndrome [115].Furthermore, circulating microRNAs may serve as potential biomarkers for distinguishing PCOS patients from healthy women [115].
As a modulator of the insulin-IGF-1, Wnt, and Akt signaling pathways, the klotho protein has recently emerged as a promising therapeutic target for polycystic ovary syndrome (PCOS).Researchers discovered that granulosa cell miR-129a-5p and miR-126-5p expression were substantially downregulated in PCOS patients and DHEA-induced PCOS animals [115].It is believed that aberrant folliculogenesis and metabolic problems in polycystic ovary syndrome (PCOS) are caused by granulosa cell death, and this discovery suggests that klotho plays a role in this process [115].Reducing klotho gene expression in PCOS GCs increased cell proliferation and mitigated insulin's anti-apoptotic effects [115].
In a comparative study of miRNAs, MiR-29a-5p, a recently discovered miRNA, has been found to be a superior diagnostic biomarker, demonstrating a significantly higher AUC value of 0.95, and is associated with metabolic disorders and cancers, involved in regulating cell growth, differentiation, and proliferation [132].Therefore, assessing the expression level of miR-29a-5p holds greater clinical significance compared to other miRNAs, making it a more valuable tool for diagnostic purposes [132].

Conclusion and future direction
MicroRNA, a subset of small RNAs, accounts for various biological pathways involved in folliculogenesis and related diseases.One way it works is by blocking the translation of certain messenger RNAs.A number of biological processes, including angiogenesis, cell adhesion, invasion, apoptosis, and proliferation, have been shown to rely on microRNAs (miRNAs).Atresia and follicular development are both thought to be impacted by certain miRNA clusters and families.In the early phases of follicular atresia, relative to healthy follicles, the expression of this family of miR-lets is decreased, indicating differential expression during follicular atresia.Through their targeting of hormone receptors and their influence on hormone production and release, microRNAs (miRNAs) regulate ovarian steroid hormones.
Over the last 20 years, scientists have learned a great deal about miRNAs and their roles in gene expression and cell cycle control.Alterations in miRNA expression levels can impact cancer aggressivity and contribute to various gynecological disorders such as PCOS and endometriosis, impacting various molecular processes.To conclude, It appears that microRNAs potential as a diagnostic biomarker and enable more effective treatment potential as the future therapeutic targets for the diseases.Further exploration of functional studies on miRNA and its role in targeting specific mRNA will be needed with several notes.
Recent studies regarding microRNAs in ovarian disorders are still conducted on a relatively small scale.The limited sample sizes compromise the generalizability of findings, hindering the ability to extrapolate results to the broader population of individuals with ovarian disorders.Selection bias and insufficient statistical power further challenge the reliability of these studies, potentially leading to overlooked associations and biased conclusions.Confounding variables, publication bias, and technical variability in laboratory methodologies add layers of complexity, requiring researchers to approach findings with a critical lens.Recruitment bias may happen in this case and may affect the external validity of the findings and limit the applicability to different patient groups.Several contradictory findings in the recent studies can be due to various factors.There are several potential sources of discrepancies, including study design, patient diversity, and microRNA measurement techniques.Additionally, the limited exploration of miRNA networks and interactions among multiple miRNAs, temporal variability, and validation challenges emphasize the need for more comprehensive and well-powered research.
To address these limitations, future research endeavors should prioritize larger sample sizes, diverse participant cohorts, and standardized methodologies.Collaborative efforts within the scientific community can facilitate the validation of findings across independent cohorts, improving the robustness and reliability of identified miRNA associations in ovarian disorders compared to independent cohorts alone.Last but not least, creating useful apps for the detection and treatment of ovarian diseases will depend on resolving these obstacles.

Fig. 1
Fig. 1 microRNA regulates the folliculogenesis.Blue text represents the upregulated miRNA and red text represents the downregulated miRNA arranged key steps in the follicular development process.This includes the development of primordial follicles from pre-granulosal cells, and progression through the stages of primary, secondary, and antral follicle development, with some follicles experiencing atresia.The pre-ovulatory follicle, influenced by luteinizing hormone (LH) and follicle-stimulating hormone (FSH), undergoes ovulation and transitions into the luteal stage

Table 1
MicroRNA expression profile in folliculogenesis

Table 4
MiRNAs expression profiles in PCOS