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 Table of Contents  
Year : 2021  |  Volume : 5  |  Issue : 1  |  Page : 30-37

Granulosa cell biomarkers to predict oocyte and embryo quality in assisted reproductive technology

Medical Center for Human Reproduction of Beijing Chao-Yang Hospital, Beijing 100020, China

Date of Submission28-Aug-2020
Date of Decision23-Nov-2020
Date of Acceptance02-Mar-2020
Date of Web Publication16-Apr-2021

Correspondence Address:
Wen-Hui Zhou
Medical Center for Human Reproduction of Beijing Chao-Yang Hospital, 8 Gongren Tiyuchang Nanlu, Chaoyang District, Beijing 100020
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/2096-2924.313684

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With the development of human assisted reproductive technology (ART), an objective, accurate, and non-invasive method to assess the quality and viability of oocytes and embryos remains one of the most significant goals. Granulosa cells (GCs) play an essential role in oocyte development. GCs can differentiate into mural GCs (MGCs) and cumulus cells (CCs) under the influence of oocytes. MGCs promote the growth and development of follicles by secreting cytokines and steroid hormones. Simultaneously, CCs can form cumulus-oocyte complexes to communicate with oocytes through gap junctions and promote oocyte growth and maturation. Seeking suitable biomarkers in GCs provides a direction for the non-invasive assessment of oocyte and embryo abilities during ART procedures. To date, only a few studies have investigated potentially effective GC biomarkers during ART processes, such as the apoptosis of GCs, transcriptomic characteristics of GCs, quality and quantity of mitochondria in GCs, and telomere length of such cells. These are potential reference indices for screening high-quality oocytes and embryos. Independent studies on MGCs and CCs can provide more effective results. Although there is scope for optimization and improvement, the results have become increasingly accurate with the constant advances in technology. Due to the heterogeneity of the study population and technical limitations, clinical tests for GCs cannot be performed as part of routine tests, but their prospects are promising. This article reviews the biomarkers that have been studied in MGCs and CCs.

Keywords: Assisted Reproductive; Embryo Quality; Granulosa Cell; Oocyte Competence

How to cite this article:
Huang RH, Zhou WH. Granulosa cell biomarkers to predict oocyte and embryo quality in assisted reproductive technology. Reprod Dev Med 2021;5:30-7

How to cite this URL:
Huang RH, Zhou WH. Granulosa cell biomarkers to predict oocyte and embryo quality in assisted reproductive technology. Reprod Dev Med [serial online] 2021 [cited 2021 Jun 22];5:30-7. Available from: https://www.repdevmed.org/text.asp?2021/5/1/30/313684

  Introduction Top

Granulosa cells (GCs) play an important role in oocyte development. During the fetal period, primordial germ cells migrate to the future gonads, mitosis occurs, and oocytes are produced. Before birth, oocytes in the ovaries proliferate and grow into primary oocytes. After birth, the follicles of the ovaries are all primary oocytes that have undergone the first meiosis and stagnated during the prophase of division. The primordial follicle consists of a primary oocyte with a single layer of flat GCs surrounding it. As primordial follicles grow and differentiate, the GCs around the oocyte become cuboids[1] and form primary follicles, initiating GC proliferation in preparation for secreting hormones.[2] GCs then proliferate and form multilayer somatic cells surrounding the oocyte, forming secondary follicles. GCs then secrete fluid and accumulate between the cells, while the capillaries of the follicle membrane exude fluid, forming small and fluid-filled cavities within the follicles. These cavities coalesce to form early antral follicles.[3] Early in follicular development, GCs and oocytes deliver small molecules through gap junctions. As the follicle develops, GCs differentiate into mural GCs (MGCs) that connect the follicle wall and cumulus cells (CCs) that directly surround the oocyte. Despite the fact that both cell types differentiate from GCs, the functions of CCs and MGCs are different.[4] CCs form a corona radiata surrounding the oocyte during ovulation and when entering the oviduct. MGCs are closely associated with steroid production.[5] Follicle stimulating hormone (FSH) secreted by the pituitary gland stimulates further growth of secondary follicles, which later become preovulatory follicles. The follicular fluid consists of the exudate from the capillaries of the follicular membrane and the fluid secreted by GCs, containing nutrients and hormones that promote the continued development of the follicle. Follicle cells that stop growing and degenerate during development are called atretic follicles. Follicular atresia can occur at any stage of follicular development. GCs are closely associated with follicular atresia.[6] It can be seen that the growth and development of the follicle is the result of the interaction of oocytes surrounding GCs and theca cells,[7] with GCs playing a vital role.

After in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI), the major criterion for embryo selection is the morphological appearance of the embryos, including cleavage rates, blastomere number, cytoplasmic appearance, and blastomere nuclear status;[8],[9] however, the morphological appearance of the oocytes and embryos cannot accurately predict embryo health. Up to 40% of embryos with normal morphology have abnormal chromosomes.[10] Therefore, more accurate tests are needed to assess embryo quality and predict pregnancy outcomes. As GCs play an important role in the development of oocytes, they can reflect the characteristics of the corresponding oocyte to a certain degree. Appropriate biochemical markers of GCs will help to optimize the selection of oocytes and embryos, leading to successful pregnancy.

  Biomarkers of Granulosa Cells Top

Apoptosis of human granulosa cells with respect to the assisted reproductive technology outcome

GC apoptosis induces follicular atresia. The apoptosis of GCs has been proposed to be a predictor of ovarian reserves in women undergoing assisted reproductive technology (ART) procedures. For example, apoptosis of GCs plays a critical role in sustaining the physiological functions of the ovary, including follicular growth and hormone synthesis.[11] Increased apoptosis of GCs from patients is closely related to low response,[12],[13] advanced age, and endometriosis.[14] Distinct biochemical and morphological changes were observed during GC apoptosis. Typical morphological changes include cytoplasmic and chromatin condensation, nuclear fragmentation, and apoptotic bodies.[15] Biochemically, apoptosis is characterized by the activation of caspases, which are highly specific proteases.[16] Several molecular mechanisms have been reported to control GC apoptosis during follicle selection. For example, apoptosis of GCs can be induced by heat stress,[17] and increased reactive oxygen species levels can be found.[18],[19] Interactions between anti-apoptotic factors, including inhibitors of apoptosis proteins and B-cell leukemia 2[20] and pro-apoptotic factors, such as P53,[21] determine the rate of GC apoptosis. Signaling pathways, such as p16/p19[22] and WNT2/β-catenin,[23] are reportedly involved in apoptosis. Recent studies have shown that certain microRNAs can also regulate GC apoptosis.[24],[25]

Some studies have separately studied the effects of apoptosis of MGCs and CCs and their relationship with infertility.

Apoptosis of mural granulosa cells

Apoptosis of MGCs may have a negative impact on fertilization rate and embryo quality, and it is closely related to IVF outcome and ovarian reserve.[26] However, another study showed that apoptosis of MGCs is not related to oocyte maturity and fertilization ability in ICSI patients.[27] Coincidentally, the study conducted by Jancar et al. also supports the fact that there were no differences in the characteristics of MGCs between the follicles with fertilized and non-fertilized oocytes in patients undergoing IVF.[28] These different results may be due to the adoption of different methods to observe the apoptosis of MGCs. Therefore, the use of apoptosis of MGCs as a predictor of oocyte and embryo quality remains controversial.

Apoptosis of cumulus cells

CCs are important for oocyte growth and the maintenance of meiotic arrest.[29] Apoptosis of CCs is closely related to pregnancy outcomes. The oocyte-cumulus complex (OCC) comprises an intimate relationship between a CC syncytium in conjunction with an oocyte involving large gap junctions.[30] Therefore, apoptosis of CCs may be more accurate than that of MGCs in predicting oocyte and embryo quality.[31] Apoptosis of CCs may influence the maturation of oocytes,[32] and increased apoptosis of CCs indicates low embryo quality. It has been hypothesized that a certain threshold of apoptotic CCs may have a negative impact on oocyte quality, but the threshold is still undefined, indicating that apoptosis of CCs is not a reliable quality marker of the corresponding oocyte.[33]

Preovulatory follicles contain MGCs and CCs. Most studies collected GCs through the follicular fluid. Some of the conflicting results are due to the fact that the researchers did not use the same test standard when observing apoptosis. Different methods of IVF and ICSI will also lead to a difference, as CCs surrounding oocytes do not have to be removed during IVF treatment. Therefore, evidence regarding the influence of apoptosis of CCs during IVF is still lacking. Moreover, some researchers have not distinguished MGCs from CCs. All of these factors could contribute toward conflicting results.

Most of the current studies indicate that apoptosis of MGCs and CCs is a potential predictor of oocyte and embryo quality, and CCs are a more accurate predictor than MGCs. However, for patients undergoing IVF, the process of obtaining CCs still needs to be optimized. Considering the obtained results as well as the applied procedures in clinical practice, apoptosis of GCs is not a reliable marker.

Gene expression of granulosa cells

Transcriptomic analysis of CCs/MGCs has also been regarded as a potential noninvasive tool for assessing oocyte quality and viability to determine the reproductive potential of embryos.[34] Different gene expression in MGCs and CCs may correlate with morphological and physiological characteristics,[35] follicular competence, and fertility potential.[36] Compared to MGCs, oocytes communicate with the surrounding CCs through paracrine and gap-junction signaling.[37] Differences in the gene expression patterns of CCs may be more practical for use in the prediction of oocyte or embryo development and subsequent implantation. Furthermore, CCs may be collected while collecting MGCs from the follicular fluid. Therefore, many studies have attempted to analyze the genome-wide expression of genes in CCs instead of MGCs, and most patients analyzed in these studies were undergoing ICSI treatment.

By analyzing gene expression in CCs in early cleavage embryos using microarrays, it was found that numerous genes were differentially expressed and mainly involved in the cell cycle; angiogenesis; apoptosis; epidermal, fibroblast, and platelet-derived growth factor signaling; general vesicle transport; and chemokine and cytokine signaling.[38] There is no consistent standard to evaluate embryo quality, and different genes play different roles in the development of oocytes. Therefore, differential gene expression in CCs may change according to the observed time and the choice of the main outcome measures [Table 1].
Table 1: Gene expression as a predictor during ART

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Although high heterogeneity remains a problem in current studies, some variables related to oocyte development show a significant association with gene expression, such as oocyte maturity (VCAN, GJA1, and SERPINE2),[39] maturity of the nucleus (PTGS2), and oocyte fertilization ability (PRSS35).

The expression of some genes correlates with the morphological and physiological characteristics of the embryos. TRPM7 and ITPKA expression may predict better cleavage-stage embryos.[40] GREM1 and ALCAM are related to embryo cleavage speed. RPS6KA2 is related to the percentage of fragmentation of embryos.[41] PTGS2 cannot predict the morphology of the embryo on D5, but it is related to the ability of the embryo to develop to D5 or D6.[42] CCs with an increase in HAS2 and GREM1 expression are more likely to develop into high-quality embryos.[43]

The difference in gene expression is related to its function during pregnancy [Table 1]. Additional CC genes involved in homeostasis, signaling, and metabolism have been variably reported as upregulated or downregulated[44],[45] with respect to pregnancy or live births. Increased expression of oxidative stress-related genes in CCs, including inducible nitric oxide synthase, heme oxygenase-1, and those regulating the nuclear factor kappa B pathway, are associated with reduced oocyte fertilization by ICSI.[46] A significant downregulation of 11 genes involved in the PI3K/AKT pathway in CCs has been observed in oocytes that produced a positive pregnancy.[47]

A selected set of genes may be more accurate than a single gene. Compared to randomly selected metaphase II oocytes, those with the expression of HAS2 and a group of FSH receptors, including FSH receptor, VCAN, PR, ALCAM, and NRP1, were shown to have a high live birth rate.[48] Similarly, CC expression of 12 genes involved in glucose metabolism, transcription, gonadotropin regulation, and apoptosis appeared to have 78% accuracy in predicting pregnancy outcomes.[49]

Large differences in gene expression between patients have been found in many studies, which may be due to the application of different treatment protocols, such as the choice of ovarian stimulation methods. Compared with patients who had been stimulated with HP-hMG, increased and decreased SPROUTY4 and SDC4 expression, respectively, was detected in patients who had been treated with recombinant FSH.[23] The expression of VCAN, GREM1, and RPS6KA2 also varied considerably between the two treatment groups. Thus, CC gene expression analysis might also be a valuable tool for the further optimization of stimulation protocols.

In conclusion, the expression of a group of genes in CCs can effectively predict embryo development, implantation potential, and final pregnancy outcome. However, there are also many differences in the observation time points of the studies [Table 2], and the number of genes that needs to be screened is large. Because the selection of observation indicators is different, and the morphological score is a subjective judgment criterion, different studies have reached contradictory results, and it is not clear whether the differential expression of genes can predict the outcome of different stages of the embryo. The specific role of many genes in oocyte development remains presently unknown. Compared with morphological evaluation, the evaluation indexes of pregnancy outcome and live birth rate are more objective. According to the different predictors provided in different studies, it is of considerable importance to select a suitable gene or a group of genes to accurately predict pregnancy outcomes.
Table 2: Known functions of the studied genes

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Mitochondrial function in granulosa cells

MGCs are promising models for studying age-related ovarian dysfunction. The MGC micro-environment is similar to that of oocytes and is more conducive to analysis than that of CCs. Accumulating evidence shows that mitochondrial functions in MGCs are related to the fertility of women.[50],[51] It has been reported that the age-related decline in the mitochondrial oxidative phosphorylation function of MGCs elucidates ovary aging and reveals strategies to improve ART outcomes for aged women.[52]

OCC interactions mediate carbohydrate, lipid, and protein metabolism to ensure the proper energy balance required for meiosis and fertilization of the oocyte and to support early embryogenesis. Mitochondria in CCs are vital for oocyte development and competence, helping to metabolize glucose into pyruvate, which is then transported into the oocyte to produce ATP.[53] Serving as central agents of the energetic metabolic pathways, CC mitochondria directly participate in the establishment of oocyte competence during oogenesis.[54] The mitochondrial function of CCs is also closely related to fertility.[55]

However, acquiring an accurate number of mitochondria in the cells is challenging. The number, mass, and shape of mitochondria vary considerably depending on the cell type and physiological state.[56] The correct maintenance and expression of mtDNA are critical for cellular energy metabolism and mitochondrial biogenesis,[57] and mitochondrial function is reflected in mtDNA quality and quantity.[58] A suitable proxy measure for elucidating the function of mitochondria is mtDNA content. Thus, MGC and CC mitochondrial DNA may be a suitable predictor in ART procedures,[59] as the quantification of mGC and CC mtDNA is linked to embryo quality.[60] The mtDNA copy numbers in CCs seem to be closely related to the chance of embryo implantation during ART procedures.[61] Without freezing the embryos and postponing the transfer, this technique could be easily applied in clinical practice to select and transfer the embryos with the highest mtDNA content in CCs or MGCs during in vitro embryo development.

However, there are still some limitations. In addition to varying between cells, mtDNA content is affected by patient specificity, making it highly difficult to acquire a universal threshold value for all patients. Before its use in clinical practice, larger and prospective studies are required to confirm these preliminary results because of the small size of the population in most studies.

Telomeres in cumulus cells

Telomeres are specialized protein-DNA chromatin structures located at the ends of all eukaryotic chromosomes. Telomerase is an enzyme complex that binds to chromosome ends (telomeres). The maintenance of telomere length and integrity is regulated by the active telomerase complex, including the telomerase holoenzyme and its associated proteins.[62],[63] The telomerase activity varies in different cell types, such as germline cells, GCs, early embryos, stem cells, highly proliferative somatic cells, and many cancer cells.[64] It is linked to GC proliferation and differentiation status.[65] Telomere DNA length abnormalities may disturb folliculogenesis.[66] Both telomere length and telomerase activity in GCs are positively correlated with female infertility.[67],[68] In conclusion, telomere dysfunction is associated with GC function, which can also be evaluated by telomere loss, telomere deletion, and the formation of telomere doublets. A recent study also found that certain proteins could be markers of telomere function, such as cohesins SA1/SA2 and NAD-dependent deacetylase SIRT1.[69] The alteration of Tert, Terc, Trf2, and Pot1a gene expression may be associated with telomere shortening.[70] These could serve as new molecular signatures to assess oocyte quality, further explaining the dissatisfactory outcome of ART procedures.[71]

Direct investigation of the relationship between telomere dysfunction and maturation or preimplantation development in human oocytes or embryos has several limitations. Low quantities of DNA make it difficult to obtain access to oocytes and embryos. An effective method to simultaneously quantify telomere DNA and thoroughly diagnose aneuploidy in the same oocyte or embryo biopsy is also needed. The expression of TERT at the protein and messenger RNA (mRNA) levels is a critical component that determines telomerase activity.[72],[73] Detection of hTERT mRNA is an indicator of telomerase existence, and telomerase activity is determined using the telomeric repeat amplification protocol assay.[74] Electrochemiluminescence (ECL) is a highly sensitive detection technique. Using an ECL method, a new intracellular telomerase detection technique with high sensitivity and low cost is being devised.[75] Telomere function in GCs is important for the development of oocytes. Detecting telomere function is an excellent method to predict ART procedure outcomes.

  Discussion Top

As mentioned above, these methods have certain limitations. There is limited consensus on the transcriptomic characteristics of MGCs and CCs, and some studies have reached contradictory conclusions. First, there are differences in laboratory environments, outcome measures, study population sizes, population characteristics, and the techniques used. Second, the use of different ovarian stimulation protocols can affect transcriptomic characteristics. All of these factors create difficulties in the clinical application of gene transcriptomes. Third, most of the studies have used subjective morphological appearance as a surrogate endpoint. The main purpose to find markers related to ART is to indicate embryos with the highest probability of implantation leading to pregnancy and live birth. Moreover, better endpoints need to be considered. In actual clinical applications, despite the accuracy of the results, it is also necessary to consider the simplicity and feasibility of the operation, and a large number of prospective experimental studies are still required after the selection of genes with discriminatory value.

However, the limitations we described previously can be overcome by advancements in medical technology. For example, new sequencing techniques, such as RNA-Seq, are considered powerful tools for assessing transcript and gene expression levels. It would be worth studying whether other genes that can predict pregnancy outcomes and elucidate the function of these genes can be analyzed using new sequencing techniques. In addition to using the present experimental techniques, combining these methods may provide a more accurate prediction. Depending on the clinical circumstances, these methodologies may be appropriate as adjuncts to traditional approaches, such as morphological scores. Furthermore, current studies have shown that oocyte competence can be predicted more accurately using a set of genes. It is challenging to find a reliable group of genes to improve the accuracy of the test to predict embryo and oocyte competence.

Unfortunately, most reproductive centers face the challenge that the results from most studies were from a single institution and the sample size was not large. However, in the future, large-scale, multi-center studies should be able to address this limitation.

  Conclusion Top

The proliferation, differentiation, and function of GCs are highly correlated with oocyte capacity. Therefore, evaluating CCs or MGCs using their biomarkers is a potential measure to identify oocyte and embryo competence. Many studies have suggested promising biomarker candidates, including apoptosis rate, transcriptomics of CCs and MGCs, mitochondrial DNA copy number, and telomere length.

Although there are still some limitations that require more in-depth research, a bright future for such applications in clinical settings can be foreseen.

Financial support and sponsorship

This work was supported by the Beijing Natural Science Foundation (7202052), Beijing Municipal Administration of Hospitals Clinical Medicine Development of Special Funding Support (XMLX201825), National Natural Science Foundation of China (81471511), New Star Personnel Training Plan of Chao-Yang Hospital (to WHZ CYXX-2017-19).

Conflicts of interest

There are no conflicts of interest.

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  [Table 1], [Table 2]


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