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Human cumulus cells transcriptome provides a poor predictive value for embryo transfer outcome

Open AccessPublished:January 19, 2023DOI:https://doi.org/10.1016/j.rbmo.2023.01.012

      Abstract

      Research Question

      Is cumulus cells transcriptome a good predictor of embryo developmental competence?.

      Design

      Cumulus cells transcriptome was analysed by RNAseq at >30*106 reads in samples grouped according to the developmental potential of their enclosed oocyte: not able to develop to blastocyst (Bl-), able to develop to blastocyst but failing to establish a pregnancy (P-) or able to develop to blastocyst and to establish pregnancy (P+).

      Results

      Cumulus cells trancriptome was largely independent of developmental potential, as using and FDR adjusted p value <0.05 only 10, 11 and 5 genes were differentially expressed (DEGs) for the comparisons P+ vs. P-, P+ vs. Bl- and P- vs. Bl- respectively, out of a total of 17469 genes expressed. Between DEGs, those showing little overlapping between samples of different groups were CHAC1, upregulated in P- and P+ groups compared to Bl-, and CENPE, CD93, PECAM1 and HSPA1B, showing the opposite expression pattern. Focussing on pregnancy potential, only EPN3 was consistently downregulated in P+ group compared to P- and Bl-.

      Conclusions

      Cumulus cells transcriptome is largely unrelated with pregnancy outcome following embryo transfer, although the expression level of a subset of genes in cumulus cells may indicate the ability to develop to the blastocyst stage.

      KEYWORDS

      INTRODUCTION

      The overall change in embryo transfer policy to transfer only one embryo at a time (Maheshwari et al., 2011) to prevent multiple pregnancies (Hazekamp et al., 2000) emphasizes the need for embryo selection to maximize the odds of attaining a successful pregnancy. Embryo morphology has been the most commonly used parameter to infer its prospective developmental ability (Gardner and Balaban, 2016). However, although morphological grading arguably constitutes the best available predictor of pregnancy success, clinical pregnancy rates remain around 35 % (European et al., 2020), suggesting than there is still room for improvement. Time-lapse screening of embryo development (Armstrong et al., 2019) can be considered as an evolution of conventional morphological criteria, as it allows to assess embryo morphology as a continuum, but there is no current consensus on its favorable impact on pregnancy rates (Chen et al., 2017). In this perspective, molecular proxies may serve as complementary methods to assist embryonic selection based on morphological parameters.
      Cumulus cells (CCs) are somatic cells closely connected to the oocyte that play essential metabolic and signaling functions during folliculogenesis and oocyte maturation (Robert, 2021). As oocyte competence is acquired through bidirectional signaling between the oocyte and the surrounding CCs (Richani et al., 2021), and these supporting cells are discarded prior to ICSI, they constitute an interesting biological material on which to perform molecular analyses to infer the developmental ability of their enclosed oocyte. To that aim, different studies have searched for possible transcriptional differences in cumulus cell samples associated to oocytes ultimately establishing pregnancy vs. those failing to succeed. Initial studies were conducted by quantitative PCR (qPCR) analysis for a select subset of genes, suggesting specific transcripts putatively associated to implantation potential (Anderson et al., 2009; Artini et al., 2017; Gebhardt et al., 2011; Iager et al., 2013; Kordus et al., 2019; Krieg et al., 2018; Wathlet et al., 2013; Wathlet et al., 2012; Wathlet et al., 2011). Subsequent studies employed microarray technology that allowed the analysis of a larger subset of genes (Assidi et al., 2015; Feuerstein et al., 2012; Hamel et al., 2010). Some of these transcripts have been validated in independent sample sets and even have been used to improve clinical outcomes (Van Vaerenbergh et al., 2021), but overall the cumulus cells transcripts putatively associated to pregnancy potential are highly divergent between studies performed by independent research groups, and other studies based on qPCR analysis of previously suggested genes (Burnik Papler et al., 2015b) or microarray (Burnik Papler et al., 2015a) have failed to replicate those results.
      In contrast to qPCR or microarrays, RNA sequencing (RNA-seq) provides an unbiased selection of transcripts and display a higher dynamic range, hence providing far more precise measurements of levels of transcripts (Wang et al., 2009). To date, only one study has investigated if the transcriptional levels of genes expressed by cumulus cells are associated with pregnancy prognosis by RNA-seq, finding no correlation between cumulus cells transcriptome and pregnancy outcome (Green et al., 2018). The objective of this study has been to employ RNA-seq technology to determine if cumulus cells transcription can be used as a proxy to enhance the already available predictive value of morphological evaluation. To that aim, cumulus cells were collected from individual oocytes and, once the developmental potential of each oocyte was known, the stored samples were allocated to one of three groups according to the oocyte´s developmental potential: 1) Oocytes not developing to blastocyst (Bl-), 2) oocytes developing to blastocyst but not establishing pregnancy (P-), or 3) oocytes ultimately establishing a pregnancy (P+).

      MATERIAL AND METHODS

      Sample collection

      Cumulus cells (CCs) were obtained from IVF treatments at IVF Spain Madrid between June 2018 and June 2020. All patients agreed to participate in the study, which was approved by the Ethical Committee from La Princesa University Hospital (Madrid). Inclusion criteria were the following: 1) absence of uterine abnormalities, 2) donor age ≤37 years, 3) recipient age ≤50 years, and 4) normal male sperm count (>2 millions spermatozoa/ml in ejaculated). Age, IMC and endometrial thickness were similar between experimental groups (Table 1). Only unfrozen oocytes and embryos obtained from donors and not submitted to preimplantation genetic testing were included in the analysis and only single embryo transfers were conducted.
      TABLE 1PATIENT CHARACTERISTICS.
      Bl-P-P+
      Age patient41.20±0.5241.28±0.6341.32±0.60
      BMI patient22.20±0.5521.78±0.5121.66±0.56
      Smoking patients2/251/255/25
      Endometrial thickness8.17±0.278.60±0.278.37±0.24
      Age donor23.92±0.8324.28±0.7324.84±0.75
      BMI donor23.06±0.6723.93±0.7422.97±0.86
      Smoking donors16/2515/2514/25
      Data are shown as mean±s.e.m., no significant differences were found between groups (ANOVA p>0.05, Fisher test for smoking status).
      BMI = body mass index
      Patients and donors were stimulated using FSH starting on the second menstruation day and ovulation was induced with GnRH analog or hCG when three or more follicles larger than 17 mm were present (Melo et al., 2009). Ovum pick-up (OPU) was performed 36 hours after induction by transvaginal ultrasound guidance. Endometrium stimulation treatment in recipients consisted in 6 mg/day of estradiol (Meriestra®) and embryo transfer was performed only if the endometrium measured 7-13 mm (Prapas et al., 1998). Luteal support started the OPU night with 400 mg of progesterone every 12 h until pregnancy test (Edwards et al., 1984).
      Cumulus-oocyte complexes (COCs) were retrieved following follicular aspiration, washed and individually cultured in G-IVF Plus Media (Vitrolife) (6.3% CO2, 5%O2, 89% N2 at 37°C). Denudation was performed individually two hours after COCs recovery, using hyaluronidase solution (Irvine). CCs detached from oocytes were collected from denudation medium, collected, pelleted at 1500 g for 10 min, snap frozen in liquid nitrogen and stored at -80°C until analysis. Oocytes were fertilized by Intracytoplasmic sperm injection (ICSI) using G-MOPS Plus (Vitrolife) and spermatozoa were allocated in 7 % Polyvinylpyrrolidone Solution (PVP) to slow their movement. Following ICSI, the presumptive zygotes were individually cultured in Continuous Single Culture SAGE 1-StepTM (Irvine) for 6 days, up to the blastocyst stage. Morphological score was used to select blastocysts for embryo transfer (Gardner et al., 2015). Only day 5 blastocyst with scores 4-5 were transferred (P- or P+ groups). Pregnancy was assessed at the fourth or fifth week post-fertilization by fetal heart rate detection by ultrasound echography. Once the embryo development was known, the previously stored cumulus cells were allocated into three groups according to the oocyte´s developmental potential: 1) Oocytes not developing to blastocyst (Bl-), 2) oocytes developing to blastocyst (scores 4-5 (Gardner et al., 2015)) but failing to establish pregnancy following embryo transfer (P-) or 3) oocytes developing to blastocyst able to establish pregnancy (P+).

      Transcriptional analysis

      RNA extraction was performed on 5 samples per group and each sample was composed by cumulus cells obtained from 10 individual COCs. These samples were randomly selected between those collected once the oocyte´s developmental potential was known. The analysis in pooled samples allows to reduce the number of assessments required to analyze a large number of biological samples, although we acknowledge that contribution of each COC to the pool is not even, given that human COC are diverse in size. The rationale behind analyzing three experimental groups was to double-check for the correlation between transcriptional levels in cumulus cells and developmental competence in oocytes; for instance a “positive marker” for oocyte competence should be upregulated in P+ vs. P- but also between P- and Bl+. Total RNA was extracted using MagMAX mirVana Total Isolation Kit (Applied Biosystems) following the manufacturer´s instruction with minor modifications (Ramos-Ibeas et al., 2022). Briefly 200 µl of Lysis Binding Mix were added to the sample, followed by gently pipetting and 5 min incubation at room temperature. Then 20 µl of Binding Beads Mix were added and shaken gently during 5 min. Beads-mRNA complexes were washed once in each Wash Solution 1 and 2. Following the washing step, samples were treated with 50 µl of Turbo DNAse treatment, 50 µl of Rebinding Buffer and 100 µl of Isopropanol were added to the sample and mixed gently. Finally, following a double wash in Wash Solution 2, total RNA was eluted in 20 µl of Elution Buffer and stored at -80 ºC until analysis.
      RNA samples were quantified by Qubit RNA BR Assay (Thermo Fisher Scientific) and RNA integrity was estimated by using RNA 6000 Nano Bioanalyzer 2100 Assay (Agilent), with the minimum RIN value being 8.4. RNA-seq libraries were prepared with KAPA Stranded mRNA-seq Illumina Platforms Kit (Roche) following the manufacturer´s recommendations. Briefly, 50-100 ng of total RNA was used for the poly-A fraction enrichment with oligo-dT magnetic beads, following the mRNA fragmentation. The strand specificity was achieved during the second strand synthesis performed in the presence of dUTP instead of dTTP. The blunt-end double stranded cDNA was 3´adenylated and Illumina platform compatible adaptors with unique dual indexes and unique molecular identifiers (Integrated DNA Technologies) were ligated. The ligation product was enriched with 15 PCR cycles and the final library was validated on an Agilent 2100 Bioanalyzer with the DNA 7500 assay.
      The libraries were sequenced on HiSeq 4000 (Illumina) with a read length of 2×76bp+8bp+8bp using HiSeq 4000 SBS kit (Illumina) obtaining >30 M reads/sample. Image analysis, base calling and quality scoring of the run were processed using the manufacturer´s software Real Time Analysis (RTA 2.7.7). The number of raw reads (150 bp paired-end reads) ranged from 38 to 57 million per sample. Paired-end read fastq files were quality checked with FastQC (Andrews, 2010) and preprocessed with fastp (Chen et al., 2018). Resulting files were pseudoaligned and quantified using kallisto (Bray et al., 2016) against the reference transcriptome of Homo sapiens (cDNAs and ncRNAs from the genome assembly GCA_000001405.28). Differential expression was analysed by DESeq2 software (Love et al., 2014) obtaining raw p-value, FDR p adjusted value, and shrunken fold changes for all genes detected. A test on biological annotation enrichment was done with STRING (v11), but there were no enriched terms with the studied DEGs.
      RESULTS
      The expression of 17469 genes (including 15593 protein coding genes) was detected by RNA-seq in human cumulus cells. Sample clustering according to gene expression showed little divergence between the 15 CC samples analysed, and clusters were not grouped according to their developmental potential (Fig. 1). Principal Component Analysis (PCA) confirmed this observation, as the first two components explained only 37.4 % of the variance and no distinction between experimental groups was observed (Fig. 1).
      FIGURE 1:
      FIGURE 1Transcriptional analysis of cumulus cells surrounding oocytes of different developmental ability: unable to develop to blastocyst (Bl-), able to develop to blastocyst but not implanting (P-) or able to develop to blastocyst and implant (P+). A) Principal Component Analysis. B) Unsupervised hierarchical clustering.
      Using a raw p value <0.05, the analysis identified 920, 1354 and 1148 differentially expressed genes (DEGs) for the comparisons P+ vs. P-, P+ vs. Bl- and P- vs. Bl+, respectively (Suppl. Table). These DEGs were narrowed down to 10, 11 and 5 genes for the comparisons P+ vs. P-, P+ vs. Bl- and P- vs. Bl- respectively, when a FDR adjusted p value <0.05 was used (Fig. 2-4). Such adjusted p value takes into account the data overdisperssion inherently associated to RNA-seq data, yielding a more reliable result.
      FIGURE 2:
      FIGURE 2Differentially expressed genes (FDR padj <0.05) between cumulus cells surrounding oocytes unable to develop to blastocyst (Bl-) vs. able to develop to blastocyst but not establishing pregnancy following embryo transfer (P-). Dots on each box-plot indicate the value of each individual sample (5 samples/group).
      Box plots (Fig. 2-4) evidenced that the expression of some DEGs was similar in samples belonging to groups exhibiting different developmental ability, thereby precluding the establishment of a given threshold to blindly discriminate samples based on oocyte developmental potential. However, the expression levels of other DEGs showed little overlapping between groups and a consistent correlation with oocyte developmental potential when all three groups were considered. Between them, CHAC1 was upregulated in most samples obtained from oocytes able to reach the blastocyst stage (P- and P+ groups) compared to the samples obtained from oocytes unable to attain preimplantation development (Bl- group), and CENPE, CD93, PECAM1 and HSPA1B showed the opposite expression pattern (Fig. 2-3). Focussing on predictors of pregnancy potential (Fig. 3-4), only EPN3 showed a consistent pattern, being downregulated in P+ group compared to P- and Bl-. RMPR was upregulated in P+ vs. Bl-, but the association of RMPR expression and pregnancy outcome could be an artefact caused by the low abundance (0-40 counts/sample) of their transcripts.
      FIGURE 3:
      FIGURE 3Differentially expressed genes (FDR padj <0.05) between cumulus cells surrounding oocytes unable to develop to blastocyst (Bl-) vs. able to develop to blastocyst and establishing pregnancy following embryo transfer (P+). Dots on each box-plot indicate the value of each individual sample (5 samples/group).
      FIGURE 4:
      FIGURE 4Differentially expressed genes (FDR padj <0.05) between cumulus cells surrounding oocytes able to develop to blastocyst and establishing pregnancy (P+) or not (P-) following embryo transfer. Dots on each box-plot indicate the value of each individual sample (5 samples/group).

      DISCUSSION

      The search of molecular markers of oocyte competence in cumulus cells constitutes an enticing promise to improve the predictive value of conventional embryo selection. To raise the predictive value already available by embryo morphology assessment, routinely performed at a fixed time or by time-lapse equipment, such molecular markers must be able to distinguish between two cohorts of oocytes, both able to reach the blastocyst stage but differing in their ability to establish pregnancy. Unfortunately, RNA-seq analysis showed that cumulus cells transcriptome provides a poor predictive value of embryo transfer outcome. These results are in agreement with previous findings using qPCR (Burnik Papler et al., 2015b), microarray (Burnik Papler et al., 2015a) and RNA-seq (Green et al., 2018), and with the lack of coherence between the lists of transcripts putatively associated to oocyte competence provided by different articles employing qPCR or microarray techniques (Anderson et al., 2009; Artini et al., 2017; Assidi et al., 2015; Feuerstein et al., 2012; Gebhardt et al., 2011; Hamel et al., 2010; Iager et al., 2013; Kordus et al., 2019; Krieg et al., 2018; Wathlet et al., 2013; Wathlet et al., 2012; Wathlet et al., 2011). A plausible explanation for the lack of reliable markers of pregnancy potential in cumulus cells is that embryo transfer success depends on multiple variables and some of them, especially uterine receptivity (Craciunas et al., 2019), may outweigh the role of oocyte competence on prospective pregnancy outcome. In agreement, the search of other potential molecular markers of oocyte competence in cumulus cells, such as mitochondrial DNA abundance (Kumar et al., 2021, Liu et al., 2021, Martínez-Moro et al., 2022b), have also failed to predict post-transfer developmental potential.
      Another limitation to find usable markers of oocyte competence in cumulus cells is the heterogeneous nature of the samples, as follicle size, COC size or the stimulation protocol used (Borgbo et al., 2013) exert an effect on cumulus cell transcription that may mask potential predictors of oocyte quality. Our analyses was performed in pools of individual samples, an approach that focusses on identifying markers of oocyte competence usable on most patients, i.e. not affected by confounding individual co-variates. Sample pooling entails two limitations for the analysis: i) The contribution of each COC to the pool is not equal, as the number of cells/COC is variable between samples, and ii) the weight of individual factors of each patient or each specific COC on developmental outcome or cumulus cells transcription cannot be estimated. Morphological parameters of cumulus masses such as cell density or the degree of cell attachment have been correlated with oocyte developmental potential (Wang and Sun, 2006), and their effect on cumulus cell transcription may be obscured in pooled samples. However, if those morphological features of the COC are truly predictive of developmental outcomes -a topic requiring further investigation (Balaban et al. 1998)- they will be also associated to potential oocyte quality molecular markers. In this sense, “universal” rather than “feature specific” markers of oocyte competence would be required for clinical applications, as tailoring gene expression data to the myriad of potentially confounding patient or COC co-variates will not be a realistic option.
      In the search of a marker predicting pregnancy outcome, only EPN3 was overexpressed in most samples of P- and Bl- groups compared to P+. The role of EPN3 during cumulus cell development is unknown as Epn3 KO mice are fertile (Ko et al., 2010). However, as EPN3 seemingly mediates epithelial-to-mesenchymal transition (Spradling et al., 2001), a key process experienced by cumulus cells during follicle development (Mora et al., 2012), it could be hypothesized that the reduced expression of EPN3 in the most competent group (P+) may reflect an earlier stage of follicular development in the less competent (Bl- and P-) groups. On the other hand, it has been suggested that instead of applying a given threshold of expression of a single gene to predict the odds for embryo survival, multivariate models involving the expression of a subset of genes could be employed (Wathlet et al., 2011). Unfortunately, unsupervised clustering and principal component analysis were unable to deliver an applicable predictive value and, whereas models biased to few selected genes could cluster samples according to group in our data (SUPPLEMENTARY FIGURE 1), such cherry-picking data-tailored procedure would not be repeatable in independent data.
      The identification of DEGs between COCs differing in their ability to reach the blastocyst stage does not provide an additional predictive value of pregnancy potential over conventional embryo selection, but it may uncover interesting clues to understand the mechanisms underpinning oocyte developmental competence. Previous studies have reported that the expression of different genes in cumulus cells can predict pre-hatching developmental potential, assessed by embryo morphology (Adriaenssens et al., 2010; Cillo et al., 2007; Huang et al., 2013; Liu et al., 2018; McKenzie et al., 2004; Zhang et al., 2005), time-lapse parameters (Parks et al., 2016) or development to blastocysts (Feuerstein et al., 2007; Scarica et al., 2019). Unfortunately, these studies show very poor correlation between them and could not be replicated by others employing microarray (Feuerstein et al., 2012) or RNA-seq ((Green et al., 2018) and our analysis). Studies in animal models, particularly in bovine, have uncovered a small, yet larger than in humans, set of transcripts associated to development to blastocysts (Bunel et al., 2015, Martinez-Moro et al., 2022a). The easier detection of oocyte competence markers in animals models may obey three fundamental differences compared to studies conducted in humans samples: 1) animal samples are obtained from a more homogeneous and fertile population, which potentially reduces the confounding effects of co-variates, 2) the interference of male factor is eliminated by using semen from a single male to fertilize all the oocytes of the study, and 3) in contrast to humans, animal oocytes are obtained from immature follicles and matured in vitro, thereby constituting a more divergent population in terms of oocyte competence where differences in molecular markers are expected to be more evident.
      A series of genes were identified as potentially associated to oocyte competence to attain preimplantation development in our analysis, but these results must be taken carefully, as no validation in an independent set of individual samples was conducted. Between them, CHAC1 was positively associated with developmental competence. CHAC1 (ChaC glutathione specific gama-glutamylcyclotransferase 1, previously known as BOTCH) is a protein involved in glutathione cleavage and a negative regulator of Notch signalling (Chi et al., 2012), a pathway involved in pre-granulosa cell recruitment (Vanorny et al., 2014). The upregulation of CHAC1 –i.e., Notch inhibition- may indicate a more advanced developmental stage of cumulus cell in oocytes able to develop to blastocyst, when cell recruitment is no longer needed. Also in agreement with a positive association with developmental competence, CHAC1 was found to be downregulated in cumulus cells from cows suffering metritis, a condition exerting detrimental effects on follicle development (Piersanti et al., 2019). Other genes, including CENPE, CD93, PECAM1 and HSPA1B were less abundant in cumulus cells surrounding oocytes able to develop to blastocyst. CENPE encodes for centromere specific protein E and its higher abundance in less competent COCs could be linked to a higher rate of cell replication probably associated to immature stages of follicle development, as cell replication is largely absent in fully matured oocytes. CD93 is a phagocytic receptor and its upregulation in COCs unable to develop to blastocysts could indicate a pro-phagocytosis state in less competent COCs, as granulosa cells have been recently suggested to eliminate apoptotic oocytes by phagocytosis (Yefimova et al., 2020). Platelet endothelial cell adhesion molecule-1 (PECAM-1) inhibits apoptosis by activating Akt and eNOS (Fleming et al., 2005; Limaye et al., 2005), and it has been reported to be downregulated in primordial follicle following chemotherapy with cyclophosphamide (Titus et al., 2021). Its upregulation in COCs unable to develop to blastocyst may again reflect a lesser degree of follicular development compared to more competent COCs, as at later stages of follicular development Akt can be activated by high oestradiol levels (Quirk et al., 2006) and thereby may not require from PECAM-1-mediated apoptosis inhibition. A previous study reported that PECAM1 protein levels in follicular fluid were not associated to pregnancy outcome (Benifla et al., 2001), which agrees with the lack of differences between P- and P+ groups in our study. Finally, heat shock protein family A (hsp70) member 1A (HSPA1A) belongs to the Hsp70 protein family, being one of the major proteins induced by heat and other stress stimuli (Huang et al., 2001) and therefore, its upregulation in COCs unable to develop to blastocyst may be associated to prior exposure to stressful conditions.
      In conclusion, transcriptome analysis of human cumulus cells obtained from COCs exhibiting diverse developmental competence revealed genes potentially associated with oocyte competence to reach the blastocyst stage, but was unable to identify useful markers to predict pregnancy potential.

      Data availability

      The data underlying this article are available within it.
      Acknowledgements
      The authors want to acknowledge the slaughterhouse “Transformación Ganadera de Leganés S.A.” for providing bovine ovaries to conduct experiments.
      Author´s roles
      Human samples were collected by AMM, EPR & JGB. Transcriptome analysis was performed by AMM, LGB, ILT and PBA. LGB performed bioinformatics analysis. PBA designed experiments and managed funding. AMM, LGB and PBA wrote the manuscript, being supervised by all authors.
      Funding information
      This work was funded by the projects IND2017/BIO-7748 from Madrid Region Government and AGL2017-84908-R and PID2020-117501RB-I00 by the Spanish Ministry of Economy, Industry and Competitiveness (MINECO). AMM was funded by project IND2017/BIO-7748 and ILT by a FPI fellowship by MINECO.
      Conflict of interest
      The authors declare no conflict of interest.
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      Data Availability

      • Data will be made available on request.

      Appendix. Supplementary materials

      Biography

      Alvaro Martínez-Moro is a PhD candidate at CSIC-INIA. He holds a degree in Biology and MScs in Human Reproduction, Advanced Biotechnology, Genetics and Health Law and Biomedicine. He is embryologist at IVF-Spain Madrid (former Procreatec) and he is currently investigating molecular markers of oocyte competence and kinetics of embryo development.
      Key Message
      Molecular analysis in cumulus cells may be useful to predict embryo developmental competence and help in embryo selection. To that aim, mtDNA content in cumulus cells has been suggested as a good proxy, but herein we report that it provides a poor predictive value in both human and bovine samples.