The success of in-vitro fertilisation (IVF) depends mainly on the quality of gametes, specifically the DNA quality of sperm and the overall quality of the oocytes (1). Most research focuses on enhancing oocyte quality to improve IVF outcomes. Clinically, various control ovarian stimulation protocols are used, targeting different gonadotrophin receptors, and timely oocyte collection is performed, with or without intracytoplasmic sperm injection, aiming for optimum fertilisation and high success rates per cycle (2). However, the developmental potential of oocytes following ovarian stimulation varies amongst women, leading to inconclusive overall IVF outcomes. In addition to research on oocyte quality, the culture media and IVF laboratory protocols evolved. Efforts have been exerted to improve media substances with targeted concentration to minimise stress in cultured embryos and promote better progression, leading to the production of good-quality embryos (3, 4). Despite these strategies, the current worldwide IVF success rate remains at approximately 35%–45% (5–7). To address this issue, researchers are exploring the development of oocyte quality. Various factors have been established to contribute to a better quality of the oocytes – mainly the level of hormones, such as growth hormone (GH) with insulin-like growth factor-1 (IGF-1). A recent study reported that higher follicular fluid levels of GH and IGF1 appear to be associated with better oocyte competency. Otherwise, the same study also reported no association of levels of TSH, fT3, fT4, 25OHD, or antithyroid antibodies in follicular fluid for oocyte quality (8). These hormonal levels can be altered due to dietary habits and the women’s body mass index (BMI). In obese women, ovarian inflammation leads to the production of pro-inflammatory cytokines and activation of cell death mechanism, resulting in ovarian tissue damage, microenvironment alteration via the releasing of oxidation stress, and alteration of microbiome metabolism. Not surprisingly, all these factors negatively impact both meiotic and cytoplasmic oocyte maturation – leading to poor oocyte quality (9). In addition to that, hyperinsulinemia also altered the endogenous pathway of steroidogenesis, which may have contributed to the imbalance of the intrinsic pathway in folliculogenesis, leading to suboptimal oocyte development, thus affecting its quality (10).

At the molecular level, the study of oocyte competency aims to predict embryo outcomes more effectively through the use of various biomarkers and signalling pathways (11). A promising non-invasive method involves analysing the cumulus cells (CC), because they can reflect the competency of developing oocytes. Apart from offering mechanical protection, CC plays a crucial role in providing paracrine signalling, metabolism pathways and overall gene regulation that promote oocyte development and maturation competency (12, 13). The CC differentiation, proliferation and expansion will lead to a better oocytes’ developmental regulation – the signaling pathway influence the oocytes quality ( Figure 1 ). Therefore, promising evidence utilise cumulus-expressed genes as markers for assessing oocyte development competency, because they reflect the fundamental processes in the molecular environment (12–15). To date, several genes have been identified to coordinate the maturation of granulosa cells, leading to specific signalling pathways that contribute to CC expansion and promote oocyte development and maturation, primarily through growth differentiation factor 9 (GDF9) (16, 17). As established, GDF-9 is an oocyte-derived growth factor in the transforming growth factor β (TGF-β) superfamily. It plays a critical role in coordinating the expression of several genes that potentially contribute to oocyte and subsequent embryo quality (18, 19). These genes include Hyaluronan synthase 2 (HAS2), Prostaglandin-endoperoxide synthase 2 (PTGS2), Gremlin 1 (GREM1) and steroidogenic acute regulator protein (STAR) (20, 21). Distinctively, the available evidence strongly supports HAS2 and GREM1 as potential markers for CC in predicting oocyte competence compared with other genes. Additionally, oocyte nuclear maturation correlates with PTGS2, STAR, amphiregulin (AREG) and stearoyl-co-enzyme A desaturase 1 and 5 (SCD1 and SCD5), leading to a good CC expansion and development (22, 23). However, conflicting evidence reports that CC increases the expression of Pentraxin-related protein - PTX3, also known as TNF-inducible gene 14 protein (TSG-14), which is ultimately essential to correlate with oocyte maturation and influence its quality (24, 25). Regarding this matter, various genes and pathways are responsible for CC expansion, supporting oocyte development in animal and human models. However, the most established evidence suggests that specific GDF9 target genes, particularly HAS2, GREM1 and PTGS2, are closely correlated with the microenvironment in human oocytes (11, 12, 16, 23).

Understanding the molecular mechanisms underlying the microenvironment of oocyte development competency is crucial to address the variability in IVF success rates amongst women. Extensive efforts have been made to identify key genes and signaling pathways involved in CC expansion and maturation. Notably, HAS2, a critical enzyme responsible for hyaluronan synthesis, has been associated with increased cumulus expansion, indicating its potential as a biomarker for oocyte quality (26). Similarly, GREM1, an antagonist of bone morphogenetic proteins, plays a vital role in modulating the transforming growth factor-beta (TGF-β) pathway, thereby influencing CC function and oocyte developmental competence (12, 27). Additionally, PTGS2, an enzyme involved in prostaglandin synthesis, has been implicated in regulating ovulation, CC expansion and embryo implantation. Unravelling the interplay between these genes and their regulatory pathways provides valuable insights into the complex molecular environment surrounding oocyte development and may hold the key to accurately predict IVF success (28). In recent years, advancements in molecular techniques have provided researchers with the ability to explore gene expression patterns in CC with great precision and sensitivity (14, 23). Through analysing the gene expression profile of CC, researchers aim to identify novel biomarkers that can reliably predict oocyte developmental competence, potentially revolutionising current IVF practices. Additionally, studying the relationship between CC gene expression and oocyte developmental potential offers a non-invasive approach to assess oocyte quality before fertilisation. This approach spares patients from unnecessary invasive procedures and minimises the risk of embryo wastage. Moreover, gaining insights into the molecular basis of oocyte competence may pave the way for the development of personalised IVF treatment strategies, tailored to each individual’s specific molecular profile.

Therefore, in this review, we have compiled published data to elucidate the role of gene expressions of Hyaluronan synthase 2 (HAS2), Gremlin 1 (GREM1) and Prostaglandin-endoperoxide synthase 2 (PTGS2) in CC and their association with human oocyte development competency, particularly in terms of fertilisation and embryo development capacity (11, 13, 25, 27). Additionally, we will explore the involvement of these genes in relevant pathways using integrated bioinformatic analysis. By examining the constellation of these gene expressions and their correlated importance, we aim to consolidate the evidence supporting CC as an excellent biomarker that reflects oocyte competency. This approach will provide a better understanding of oocyte development competency at the molecular level.

As established, the critical development of oocyte maturation occurs in the cumulus oocyte complex (37) during the follicular phase (38). Optimal expansion of CC is crucial to ensure better oocyte quality. Therefore, the oocyte developmental competency (ODC) can be assessed at the CC level based on gene expression (12, 13). Numerous studies have utilised GDF-9 derivatives such as Prostaglandin-endoperoxide synthase 2 (PTGS2), gremlin1 (GREM1), hyaluronic acid synthase 2 (HAS2) and pentraxin 3 (PTX3) as markers for ODC, as they reflect the immediate ODC during CC expansion (11, 18, 19). In this review, we found that GREM1 expression is an important marker for oocyte maturation rates (OMR) compared to both HAS2 and PTGS2 (32). Healthy oocytes tend to increase the expression of GREM1 in their CC, promoting folliculogenesis and enhancing spindle activities, ultimately leading to oocyte maturation (39, 40). The majority of the evidence aligns with these findings, consolidating the importance of GREM1 in OMR (12, 13, 17). Our review found that following rescue IVM, PTGS2 expression is reduced, indicating that proper IVM should not be preceded by hCG injection because it may interfere with oocyte maturation rates (34). Therefore, recent evidence recommends pre-IVM culture, as well as standard IVM procedures without hCG, to improve the microenvironment at the CC expansion level, aiming for a better OMR following IVM culture (41). Furthermore, our review revealed that GREM1 is also considered a predictor of good fertilisation rates (FR) and EDR (32). With optimum GREM1 expression following maturation, meiosis II (MII) oocytes can fertilise following insemination by sperm and subsequently undergo embryo development (42). The gathered evidence suggests that a reduction in GREM1 does affect the fertilisation rates and EDR, particularly in suboptimal cohorts, such as decrease ovarian reserved (DOR), or in cases of poor oocyte quality in women with endometriosis or polycystic ovarian syndrome (13, 43, 44).

Besides that, the expression of HAS2 in CC was associated with higher 2PN fertilisation (31). Most evidence postulates that an increase in the fold of HAS2 expression significantly correlates with better oocyte quality, leading to an increase in fertilisation (11, 16, 27, 32). Moreover, the unique expression of HAS2 is synergistically influenced by hCG or LH as maturation triggers (26). Therefore, standard IVM cycles without hCG will incorporate LH in the culture media to enhance HAS2 expression, aiming for better oocyte quality, fertilisation rates (FR) and EDR. Furthermore, our review agrees that the proportion of good quality of embryo (GQE) is influenced by all GDF-9 derivative gene expressions (32, 33). However, a higher fold of GREM1 expression is associated with an increase in the GQE cohort (32). In exploring the GDF-9 derivatives, most publications strongly support the importance of these three genes - HAS2, PTGS2 and GREM1 - in modulating embryo development (45, 46). Numerous pieces of evidence demonstrate that higher median expression of GDF-9 downstream predicts better quality embryo outcomes (11, 15, 19, 27, 40). The regulation of human follicles through GDF-9 has been well-established for decades. Any deviation or mutation in its molecular regulation can lead to possible subfertility, mainly due to a blockage of the early follicular phase, resulting in disruption of the meiosis process. Additionally, subsequent evidence suggests that GDF-9 also influences the later stages of oocyte development (47). Therefore, by dissecting the mechanisms of their regulation, a better understanding of the maturation of ongoing dominant follicles can be achieved. CC expansion is initiated by the secretion of GDF-9, with HAS2 playing a crucial role in promoting expansion through the production of hyaluronic acid. Subsequently, PTGS2 further promotes expansion by secreting prostaglandin E2. Both mechanisms are considered crucial for maturation and ovulation. Defects in these mechanisms, as observed in animal models, result in defects in ovulation, fertilisation, embryo quality and implantation (48). As for GREM1, it acts by inhibiting the signalling for BMP rather than GDF-9. During cumulus expansion, GREM1 promotes mural granulosa cell luteinisation, mostly during the oocyte maturation phase (22). Therefore, all three of these genes influence CC expansion, thus reflecting the ODC in the human reproductive field (49).

From the bioinformatic analysis, GREM1 is clustered in cluster 1, whilst HAS2 and PTGS2 are separated in cluster 2. GREM1, along with its functional partner proteins, such as BMPR2, BMP2 and HFE2, is located in the extracellular space and cell surface, indicating its involvement in interactions with the cellular environment and adjacent cells. Communication is crucial for oocyte development. The cumulus-oocyte complex’s development is primarily driven by short-distance communication through extensive local cell-to-cell interactions, often involving members of the transforming growth factor-beta (TGF-β) family, such as inhibin, activin, anti-Müllerian hormone and growth and differentiation factor 9 (GDF9) (50). GREM1, being a downstream target protein of GDF9, plays a pivotal role in regulating the crosstalk between GDF9 signalling and BMP signalling pathways. It selectively inhibits the differential effect of the BMP signal whilst preserving the GDF9 signal. This unique regulation may promote luteinisation of ovarian granulosa cells whilst supporting CC expansion in the ovarian follicle (51). The proteins in cluster 1 also have roles in positive regulation of transcription from RNA polymerase II promoter and positive regulation of cell proliferation. GREM1, BMP4, BMP2 and BAMBI are associated with promoting cell proliferation, suggesting their potential roles in follicular development and CC expansion.

In contrast to GREM1, the proteins HAS2 and PTGS2 in cluster 2 are localised within the endoplasmic reticulum membrane. This cellular localisation suggests their involvement in cellular compartmentalisation and localisation processes within the oocyte. Both HAS2 and PTGS2 are integral components of key metabolic pathways. In particular, they play crucial roles in the arachidonic acid metabolism pathway, which is important for the production of bioactive lipid mediators, including prostaglandins. The prostaglandin biosynthetic process, enriched by HAS2 and PTGS2, is of particular significance because prostaglandins influence in vitro maturation of oocytes (52). PTGS2’s involvement in the cyclooxygenase pathway further emphasises its significance in prostaglandin synthesis, contributing to various reproductive processes. Moreover, the gene ontology terms associated with HAS2, such as positive regulation of cell migration, indicate its potential involvement in CC expansion, a critical process for successful oocyte maturation. Moreover, the enrichment of cellular response to interleukin-1 by HAS2 suggests its possible role in responding to inflammatory signals during oocyte development. Notably, exposure to high levels of IL-10 and IL-1β has been reported to decrease CC expression of GREM1, indicating that inflammation in the follicular fluid, particularly amongst obese women, could impact the cumulus oocyte complex and alter its microenvironment, significantly influencing GREM1 gene expression (44). Overall, the CC have demonstrated that GDF9 upregulates the expression of essential genes, including HAS2, PTGS2, GREM1 and STAR, whilst concurrently downregulating luteinising hormone receptor, a crucial factor for follicle development and cumulus expansion. The regulation of GDF9 and its downstream factors in CC presents a promising potential as an indicator of oocyte quality (12). Understanding these intricate molecular interactions offers valuable insights into oocyte development and maturation, which could lead to significant advancements in reproductive medicine and fertility treatments.

Our review summarizes the gene expression results of GDF-9 derivatives in CC, which can be reliable predictors in IVF, primarily in OMR, FR, EDR, and GQE. However, CC predictors are constellations of evidence rather than individuals, as different gene expressions represent different IVF parameters based on our review. Our analysis of the literature and bioinformatics provides evidence of a potential link between the expression levels of GREM1, PTGS2, and HAS2 in cumulus cells and the maturation of the oocyte and subsequent fertilization rates and embryo development. However, the link has yet to be consistently confirmed across all studies. Further research is necessary to verify the relevance of this connection. Our study has certain limitations, as the included papers did not correlate the outcomes with live birth rates. Hence, the molecular conclusions are limited to gene expression alone. Nonetheless, these candidate genes’ expression levels can be a valuable indicator correlated with IVF outcomes, predicting good embryo development and quality. These findings can help improve overall IVF outcomes.

AF: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing – original draft. ME: Conceptualization, Data curation, Formal analysis, Methodology, Software, Writing – original draft. NJ: Conceptualization, Formal analysis, Methodology, Resources, Writing – review & editing. MA: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing – review & editing. SS: Conceptualization, Data curation, Formal analysis, Methodology, Supervision, Writing – review & editing. AZ: Data curation, Formal analysis, Supervision, Writing – review & editing. NS: Data curation, Formal analysis, Methodology, Supervision, Validation, Writing – review & editing. AK: Project administration, Supervision, Writing – review & editing.