Non-Coding Nucleic Acid Sequences and Female Infertility

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BACKGROUND

Infertility is a disease of the reproductive system defined as the inability to achieve a clinical pregnancy after ≥12 months of regular unprotected sexual intercourse [1]. Up to 17.5% of couples experience infertility worldwide, which can negatively affect both individuals and society as a whole. Female factors account for approximately 37% of infertility cases [2].

In developed countries, the most common causes of female infertility are ovulatory disorders, occurring in 24.8% of cases of female infertility [1]. Ovulatory disorders include hypothalamic amenorrhea, polycystic ovary syndrome (PCOS), hyperprolactinemic anovulation, and thyroid dysfunction [3].

Globally, approximately 15% of couples of reproductive age—at least 48.5 million—encounter difficulties conceiving [3]. At present, this public health issue affects 20%–30% of women of reproductive age. Approximately 10%–15% of couples aged 20–45 years experience infertility [4–6]. According to the World Health Organization, infertility may affect nearly 80 million women worldwide [7].

Infertility can become a hopeless challenge for couples, leading to stress. Couples facing infertility are at higher risk for developing serious mental health conditions, such as anxiety and depression, than healthy couples [8, 9].

Female infertility is described as infertility primarily caused by female factors such as ovulatory dysfunction, diminished ovarian reserve, reproductive tract disorders, and chronic diseases [7]. Primary female infertility is diagnosed in women who have never given birth and secondary infertility in those who have previously had a live birth or miscarriage but are currently unable to achieve clinical pregnancy. In addition to physiological and age-related factors, conditions associated with the pathophysiology of the reproductive organs and other factors such as environmental exposures and lifestyle contribute to female fertility [10].

MOLECULAR AND BIOLOGICAL MECHANISMS OF INFERTILITY

Currently, various factors are associated with impaired oocyte maturation, including genetic causes and environmental influences [11]. However, recently, the condition of the immune system has been of greatest interest to researchers [12]. For example, in the peritoneal fluid of women with endometriosis, increased levels of tumor necrosis factor alpha (TNF-α), interleukin (IL)-6, and IL-8 have been observed [12]. In the uterine cavity, cytokines stimulate prostaglandin production by endometrial cells, leading to overexpression of other inflammatory cytokines. Moreover, these cytokines initiate inflammatory responses, promote angiogenesis, and participate in tissue injury and regeneration. Excessive levels of cytokines may impair follicular steroidogenesis and development, ovulation, and implantation, adversely affecting the possibility of achieving a natural pregnancy [12].

Furthermore, lipid peroxidation plays a crucial role in infertility-associated processes [2]. Physiological levels of reactive oxygen species are critical for essential regulatory functions in folliculogenesis, oocyte maturation, endometrial cycling, luteolysis, implantation, embryogenesis, and pregnancy maintenance via various signaling pathways.

Infertility is a heterogeneous condition, with genetic factors accounting for approximately 50% of all cases [10]. Next-generation sequencing, including whole-genome sequencing and whole-exome sequencing (WES), has accelerated the identification of potentially pathogenic variants (population polymorphisms) and private mutations [13].

In their study, Wang et al. [14] identified the genetic causes of oocyte maturation arrest (OMA), a disease that leads to female infertility and is a major cause of in vitro fertilization failure. OMA can occur at different stages of meiosis, and several genes involved in key processes, such as homologous recombination, spindle assembly, zona pellucida formation, and translational repression, have been implicated in its development [3].

Feng et al. (2016) conducted WES of five members of a four-generation family, three of whom experienced infertility due to oocyte meiotic arrest at meiosis I [15]. Then, Sanger sequencing of the candidate gene TUBB8 was performed on DNA from these individuals, additional relatives, and members of 23 other unrelated families affected by OMA. Consequently, seven mutations in the primate-specific TUBB8 gene were identified, which were responsible for meiosis I arrest in 7 of 24 families. TUBB8 expression is unique to oocytes and early embryos, wherein it represents nearly all β-tubulin expressions. These mutations impair chaperone-mediated folding and α/β-tubulin heterodimer assembly, disrupt microtubule behavior in cultured cells, alter microtubule organization in vivo, and lead to severe spindle assembly defects and maturation arrest when manifested in mouse and human oocytes. TUBB8 mutations induce dominant-negative effects that impair microtubule behavior, meiotic spindle assembly, and maturation in oocytes, resulting in female infertility [15].

Subsequently, Feng et al. (2016) sequenced TUBB8 in oocytes arrested during maturation [16]. Seven heterozygous missense mutations and two homozygous mutations were determined. These mutations caused in vitro folding defects, varying degrees of microtubule disruption when expressed in cultured cells, and impaired meiotic spindle assembly in mouse oocytes at different extents. Some of the newly identified TUBB8 mutations led to phenotypic variability. For example, oocytes carrying one of the three missense mutations (i.e., I210V, T238M, and N348S) extruded the first polar body. Moreover, these oocytes could be fertilized, although the resulting embryos exhibited developmental arrest. The patient-derived oocytes were homozygous. Mutations that prevent the expression of a functional TUBB8 polypeptide contain identifiable spindles. Thus, Feng et al. expanded the phenotypic spectrum of oocyte dysfunction associated with TUBB8 mutations, emphasizing the independent nature of meiosis and human oocyte differentiation and broadening the group of genetic disorders classified as tubulinopathies [16].

Chen et al. revealed a homozygous mutation in PATL2 in a consanguineous family with OMA at the prophase I stage and biallelic PATL2 mutations in members of four additional families. Furthermore, they attributed the phenotypic variability in these patients to the extent of PATL2 dysfunction, with more severe impairment resulting in oocyte arrest at earlier stages [17].

A research team from the American College of Medical Genetics and Genomics hypothesized that genetic disorders predispose individuals to infertility and subsequent medical conditions. The exomes of 197 women with unexplained infertility were sequenced to detect pathogenic and potentially pathogenic gene variants. In four women (2%), pathogenic or potentially pathogenic variants in BRCA1 or BRCA2 were found, indicating a high risk of breast or ovarian cancer [18].

In a study, Huang (2014) described a form of infertility with autosomal recessive inheritance characterized by abnormal oocytes lacking the zona pellucida. A homozygous frameshift mutation in ZP1 was observed in six family members. In vitro studies showed that defective ZP1 and normal ZP3 proteins were distributed throughout the cell and were not expressed on the cell surface. This indicated that aberrant ZP1 causes ZP3 sequestration in the cytoplasm, preventing the formation of the zona pellucida around the oocyte [19].

These findings were confirmed using mouse models with the same mutations generated with the CRISPR/Cas9 gene-editing system, allowing to conclude that ZP mutations exhibit dosage effects that may cause female infertility in humans [20].

Further research focused on identifying candidate genes that may cause meiotic arrest in mice when ablated but have not yet been definitively associated with human infertility. Mutations in numerous genes may lead to OMA. One such candidate is poly(A)-binding protein cytoplasmic 1-like (PABPC1L), which encodes a protein that binds to the elongated poly(A) tail to stabilize polyadenylated messenger RNAs (mRNAs). Female mice deficient in Pabpc1l are infertile; their oocytes are dysmorphic, which fail to complete maturation due to impaired translational activation of maternal mRNAs [13].

Currently, noncoding nucleic acid sequences are gaining attention [21]. Study of microRNAs provides new opportunities for the diagnosis, treatment, and prevention of disease. Inhibited proliferation of granulosa cells (GCs) contributes to abnormal follicular maturation [3]. It has been shown that lnc-MAP3K13-7:1-dependent inhibition of DNA methyltransferase 1 regulates CDKN1A/p21 gene expression and suppresses cell proliferation [22].

Extracellular Nucleic Acids and Their Role in the Pathogenesis of Reproductive Organ Disorders.

MicroRNAs (miRNAs) are short noncoding RNA sequences approximately 18–25 nucleotides in length. Although they do not act as templates for protein synthesis, miRNAs are crucial in gene expression regulation, influencing nearly half of all protein synthesis processes. Additionally, they induce significant effects on various intracellular physiological functions, including cell growth, signal transduction, apoptosis, and pathologic processes [23].

MiRNAs can be detected within cells and in extracellular fluids such as serum, plasma, urine, follicular fluid, and cerebrospinal fluid. These molecules change in response to various cellular states and may be biomarkers for screening and predicting a wide range of diseases [24].

MiRNAs are present in various cells of the female reproductive system; thus, dysregulation of their activity may impair female reproductive potential [23]. They play a regulatory role in the uterine endometrium during the menstrual cycle. Moreover, increasing evidence demonstrates a key role of miRNAs in the proliferation and apoptosis of GCs, oocyte maturation, and oocyte apoptosis [25]. Table 1 presents the role of noncoding nucleic acid sequences in the development of infertility. It highlights the involvement of miR-324 and miR-155 [26]. Notably, miR-155 expression was found to be increases in the tissues of patients with PCOS. It has been shown that miR-155 enhances the proliferation, migration, and invasion of KGN-derived GCs [26, 27].

Studies have shown differences in the number of expressed microRNAs between women with normal and those with impaired reproductive function [27]. Additionally, dysregulation of microRNAs has been observed in various gynecologic malignancies and female disorders associated with infertility, such as PCOS, primary ovarian insufficiency, and endometriosis [26].

Moreover, microRNAs may be useful for determining the implantation window and improving outcomes of assisted reproductive technologies. Patients with PCOS exhibit lower miR-335-5p levels than those without PCOS. MiR-335-5p downregulates SGK3 expression and stimulates granulosa cell proliferation [28].

Upregulation of miR-9119 and miR-135a in the GCs of patients with PCOS may induce apoptosis [29]. In turn, miR-23a and miR-27a induce granulosa cell apoptosis in vitro by activating the SMAD5-mediated FasL–Fas signaling pathway [30].

Liu et al. reported that miR-146b-5p is involved in the development of PCOS in mice by γH2A phosphorylation suppression and Dab2ip/Ask1/p38-MAPK signaling pathway inactivation [20]. However, the role of miR-146b-5p in patients with PCOS remains unclear. Decreased miR-146b-5p expression has been observed in patients with PCOS [20].

The long noncoding RNA NEAT1 is retained in the nucleus, where it comprises a core structural component of subnuclear organelles called paraspeckles [30]. It may regulate transcription of numerous genes, including those involved in cancer progression. The antisense long noncoding RNA GNG12-AS1, located at the GNG12 gene locus, is considered a regulator of G protein-mediated signaling pathways. Studies have shown that GNG12-AS1 may influence signaling cascade activity such as the AKT/GSK-3β/β-catenin pathway, thereby affecting cell proliferation and migration [32]. It is believed to participate in G protein-coupled receptor-mediated signaling cascades and has been detected in extracellular exosomes. ZEB2-AS1 encodes a spliced long noncoding RNA that is a natural antisense transcript complementary to the 5’ untranslated region(5’-UTR) of the zinc finger E-box binding homeobox 2 (ZEB2) gene. This transcript regulates ZEB2 expression and may contribute to bladder cancer progression [31].

 

Table 1. Non-coding nucleic acid sequences associated with infertility

Type of sequence	Mechanism of action	Source
MicroRNA
miR-324	Regulate proliferation of KGN cells in PCOS.	[25]
miR-155	Enhance proliferation, migration, and invasion of granulosa cells.	[26, 27]
miR-335-5p	Decrease SGK3 expression and promote proliferation of granulosa cells.	[28]
miR-9119	May induce apoptosis in granulosa cells of patients with PCOS.	[29]
miR-23a	Induce apoptosis in granulosa cells in vitro via activation of the SMAD5-mediated FasL-Fas signaling pathway.	[30]
miR-27a
miR-146b-5p	Involved in PCOS pathogenesis in mice by suppressing γH2A phosphorylation and inactivating the Dab2ip/Ask1/p38-Mapk pathway.	[20]
Long noncoding nucleic acids
NEAT1	Produces long noncoding RNA (lncRNA), transcribed from the multiple endocrine neoplasia locus. This lncRNA is retained in the nucleus and is a structural component of paraspeckles. It may regulate transcription of numerous genes, including those involved in cancer progression.	[31, 32]
GNG12-AS1	Modulate the AKT/GSK-3β/β-catenin signaling cascade, crucial for cell division and migration. Localized in extracellular exosomes.
ZEB2-AS1	Produce a spliced long noncoding RNA as a natural antisense transcript corresponding to the 5’ UTR of zinc finger E-box binding homeobox 2 (ZEB2). This transcript regulates ZEB2 expression and contributes to bladder cancer progression.
LINC473	Participate in transcription, DNA matrix mediates decidualization of stromal cells through transcriptional regulation of PRL, IGFBP1, PGR, FOXO1, HOXA10, HOXA11, and WNT4.	[33]
STAT3	Functions as a transcription factor. Its expression markedly increases in endometrial stroma during decidualization, indicating its role in embryo implantation.
Circulating nucleic acids
let-7b	Expressed in granulosa and cumulus cells of mammalian and human ovaries; may predict blastocyst formation and growth.	[35]
miR-29a	Highly expressed in rat uterus during embryo implantation. Expression is regulated by blastocyst activation and uterine decidualization (regulatory role).
miR-30a	Overexpression of miR-30a in cultured human granulosa cells enhances BCL2A1, IER3, and cyclin D2 expressions by suppressing FOXL2.
miR-140	Functions as a tumor suppressor, downregulated in breast cancer via ERα-mediated signaling.
miR-320a	Reflects the quantity and quality of mature oocytes; its intrafollicular expression may vary with the quality of ovarian response in IVF patients.

Note: PCOS, polycystic ovary syndrome; IVF, in vitro fertilization.

 

LINC473 functions as an RNA scaffold and mediates stromal cell decidualization by regulating PRL, IGFBP1, PGR, FOXO1, HOXA10, HOXA11, and WNT4 transcription [31]. STAT3 is a transcription factor whose expression in the endometrial stroma markedly increases during decidualization owing to its critical role in embryo implantation [33]. Human endometrial epithelial cells provide a molecular environment for blastocyst attachment by regulating the expressions of integrins, cadherins, and other adhesion molecules, contributing to the formation of a receptive endometrium [34].

Another class of markers includes circulating microRNAs [34]. For example, let-7b is expressed in granulosa and cumulus cells of mammalian ovaries, including those of humans, and may be a predictor of blastocyst formation and development [35]. MiR-29a is highly expressed in the rat uterus during embryo implantation; its expression is regulated by blastocyst activation and uterine decidualization, indicating a regulatory role [34]. Moreover, miR-30a is crucial in oocyte maturation. Its overexpression in cultured human GCs promotes the expression of BCL2A1, IER3, and cyclin D2 by suppressing FOXL2 [35]. MiR-140 functions as a tumor suppressor and is downregulated in breast cancer by ERα signaling. MiR-320a reflects the quantity and quality of mature oocytes, and its intrafollicular expression may be modulated by the quality of ovarian response in patients undergoing in vitro fertilization [35, 36].

CONCLUSION

Some noncoding nucleic acid fragments may play a critical role in the development of female infertility. Investigating the functions of these fragments and microRNAs in the context of infertility may improve understanding of the molecular mechanisms underlying this condition. Further studies on the role of microRNAs in the development of infertility may lead to novel diagnostic and therapeutic approaches.

Additional information

Author contributions: M.M.A.: conceptualization, formal analysis, writing—review & editing, supervision; M.E.D.: methodology, validation, investigation, writing—original draft; S.L.V.: writing—review & editing, supervision. All authors approved the version of the manuscript to be published and agreed to be accountable for all aspects of the work, ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Ethics approval: Not applicable.

Funding sources: No funding.

Disclosure of interests: The authors have no relationships, activities, or interests for the last three years related to for-profit or not-for-profit third parties whose interests may be affected by the content of the article.

Statement of originality: No previously published material (text, images, or data) was used in this work.

Data availability statement: The editorial policy regarding data sharing does not apply to this work, as no new data was collected or created.

Generative AI: No generative artificial intelligence technologies were used to prepare this paper.

Provenance and peer review: This paper was submitted unsolicited and reviewed following the standard procedure. The review was conducted by two external reviewers, a member of the editorial board, and the journal’s scientific editor.

Дополнительная информация

Вклад авторов. М.М.А. — концептуализация, анализ, редактирование рукописи, общее руководство; М.Е.Д. — методология, валидация, исследование, создание черновика; С.Л.В. — редактирование рукописи, общее руководств. Все авторы одобрили рукопись (версию для публикации), а также согласились нести ответственность за все аспекты работы, гарантируя надлежащее рассмотрение и решение вопросов, связанных с точностью и добросовестностью любой её части.

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About the authors

Maxim A. Morozovsky

Siberian State Medical University

Email: morozovskiy0m@gmail.com
ORCID iD: 0009-0001-5841-0606

4 year student, Faculty of Medicine and Biology

Russian Federation, 2 Moskovsky trakt st, Tomsk, 634050

Liudmila V. Spirina

Siberian State Medical University; Cancer Research Institute — Tomsk National Research Medical Center

Email: spirinalvl@mail.ru
ORCID iD: 0000-0002-5269-736X
SPIN-code: 1336-8363

MD, Dr. Sci. (Medicine), Assistant Professor, Depart. of Biochemistry and Molecular Biology with a course in Clinical Laboratory Diagnostics, Leading research associate, Lab. of Tumor Biochemistry

Russian Federation, 2 Moskovsky trakt st, Tomsk, 634050; Tomsk

Evgeny D. Merkulov

Siberian State Medical University

Author for correspondence.
Email: evmerc@mail.ru
ORCID iD: 0000-0002-7082-9389

4 year student, Faculty of Medicine and Biology

Russian Federation, 2 Moskovsky trakt st, Tomsk, 634050

References

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