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Corona cell RNA sequencing from individual oocytes revealed transcripts and pathways linked to euploid oocyte competence and live birth

Published:February 24, 2016DOI:https://doi.org/10.1016/j.rbmo.2016.02.002

      Abstract

      Corona cells surround the oocyte and maintain a close relationship through transzonal processes and gap junctions, and may be used to assess oocyte competence. In this study, the corona cell transcriptome of individual cumulus oocyte complexes (COCs) was investigated. Isolated corona cells were collected from COCs that developed into euploid blastocysts and were transferred in a subsequent frozen embryo transfer. Ten corona cell samples underwent RNA-sequencing to generate unique gene expression profiles. Live birth was compared with negative implantation after the transfer of a euploid blastocyst using bioinformatics and statistical analysis. Individual corona cell samples produced a mean of 21.2 million sequence reads, and 307 differentially expressed transcrpits (P < 0.05; fold change ≥2). Enriched pathway analysis showed Wnt signalling, mitogen-activated protein kinases signalling, focal adhesion and tricarboxylic acid cycle to be affected by implantation outcome. The Wnt/beta-catenin signalling pathway, including genes APC, AXIN and GSK3B, were independently validated by real-time quantitative reverse transcription. Individual, corona cell transcriptome was successfully generated using RNA-sequencing. Key genes and signalling pathways were identified in association with implantation outcome after the transfer of a euploid blastocyst in a frozen embryo transfer. These data could provide novel biomarkers for the non-invasive assessment of embryo viability.

      Keywords

      Introduction

      Current estimates state that one in six couples in the USA will require the use of assisted reproductive techniques to successfully conceive a child (
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      Prevalence of infertility in the United States as estimated by the current duration approach and a traditional constructed approach.
      ). For many infertile couples, treatment will involve IVF and a subsequent embryo transfer. The oocyte is a major contributor to the success of an IVF cycle, with oocyte competence tightly linked to embryo viability. Numerous factors determine oocyte competence, including mitochondrial health, metabolomic processes, functional spindle assembly and successful meiotic division (
      • Keefe D.
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      Oocyte competency is the key to embryo potential.
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      A role of lipid metabolism during cumulus-oocyte complex maturation: impact of lipid modulators to improve embryo production.
      ). On the basis of current IVF protocols, the likelihood of a single aspirated oocyte developing to the blastocyst stage after fertilization, implanting and resulting in a live birth is about 5% (
      • Patrizio P.
      • Sakkas D.
      From oocyte to baby: a clinical evaluation of the biological efficiency of in vitro fertilization.
      ). Consequently, additional non-invasive methods to measure implantation outcome could represent a valuable asset to improve embryo selection and IVF outcomes.
      Follicular fluid is a potential non-invasive source of information indicating oocyte competence. The fluid is aspirated at the time of oocyte retrieval and discarded after oocyte isolation. Follicular fluid is contained within the follicular antrum surrounding the oocyte, and the composition of this fluid changes throughout folliculogenesis. Protein analysis of follicular fluid revealed diverse protein compositions containing mostly plasma proteins and proteins associated with lipid transport, complement pathways and blood coagulation (
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      Proteome mining of human follicular fluid reveals a crucial role of complement cascade and key biological pathways in women undergoing in vitro fertilization.
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      Peptide and protein profiles in serum and follicular fluid of women undergoing IVF.
      ). A recent study identified a subset of 75 follicular fluid proteins that were correlated with IVF outcome. Thirteen of these proteins are involved in acute response signalling, coagulation, prothrombin activation, complement system and growth hormone pathways, uniquely associated with IVF outcome (
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      Protein and steroid profiles in follicular fluid after ovarian hyperstimulation as potential biomarkers of IVF outcome.
      ). Metabolic analysis of follicular fluid has revealed metabolites that are correlated with oocyte maturity based on the depletion and appearance of specific amino acids: arginine, glutamate, glutamine, isoleucine and valine (
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      Amino acid turnover by human oocytes is influenced by gamete developmental competence, patient characteristics and gonadotrophin treatment.
      ). In addition to amino acids, carbohydrates and fatty acid ratios have been shown to correlate with developmental milestones, including embryogenesis, and form an overall predictive value of oocyte quality and developmental potential (
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      Metabolic profiling of human follicular fluid identifies potential biomarkers of oocyte developmental competence.
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      An investigation into the relationship between the metabolic profile of follicular fluid, oocyte developmental potential, and implantation outcome.
      ). Additional studies have linked oocyte quality with cytokines and growth factors in follicular fluid, including increased BMP2, interleukins (6, 8, 12 and 18), GDF-9, granulocyte-colony stimulating factor and amphiregulin (
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      Impact of follicular G-CSF quantification on subsequent embryo transfer decisions: a proof of concept study.
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      Comparison of follicular fluid amphiregulin and EGF concentrations in patients undergoing IVF with different stimulation protocols.
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      Follicular proinflammatory cytokines and chemokines as markers of IVF success.
      ,
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      • Nakagawa K.
      • Sugiyama R.
      • Isaka K.
      Bone morphogenetic protein 2 may be a good predictor of success in oocyte fertilization during assisted reproductive technology.
      ). Although follicular fluid molecular profiling seems to be promising, to date, no prospective studies using a panel of follicular fluid biomarkers to determine potential clinical value in identifying oocyte competence have been published.
      Another promising area of research into oocyte competence has been the molecular analysis of follicular cells. Corona cells surrounding the oocyte maintain a close relationship through transzonal processes and gap junctions, providing key nutrients and other factors essential for oocyte maturation and future developmental competence (
      • Anderson E.
      • Albertini D.F.
      Gap junctions between the oocyte and companion follicle cells in the mammalian ovary.
      ). Specific profiles of cumulus cell gene expression have been able to predict oocyte maturation, cumulus expansion and fertilization (
      • Anderson R.A.
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      • Bayne R.A.
      • Thong K.J.
      • De Sousa P.A.
      • Pickering S.
      Cumulus gene expression as a predictor of human oocyte fertilisation, embryo development and competence to establish a pregnancy.
      ,
      • Assou S.
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      • Pellestor F.
      • Klein B.
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      • Dechaud H.
      • De Vos J.
      • Hamamah S.
      The human cumulus – oocyte complex gene-expression profile.
      ,
      • Feuerstein P.
      • Puard V.
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      • Cadoret V.
      • Guerif F.
      • Houlgatte R.
      • Royere D.
      Genomic assessment of human cumulus cell marker genes as predictors of oocyte developmental competence: impact of various experimental factors.
      ,
      • McKenzie L.J.
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      • Carson S.A.
      • Kovanci E.
      • Cisneros P.
      • Buster J.E.
      • Amato P.
      • Matzuk M.M.
      Human cumulus granulosa cell gene expression: a predictor of fertilization and embryo selection in women undergoing IVF.
      ,
      • Ouandaogo Z.G.
      • Haouzi D.
      • Assou S.
      • Dechaud H.
      • Kadoch I.J.
      • De Vos J.
      • Hamamah S.
      Human cumulus cells molecular signature in relation to oocyte nuclear maturity stage.
      ,
      • Yerushalmi G.M.
      • Salmon-Divon M.
      • Yung Y.
      • Maman E.
      • Kedem A.
      • Ophir L.
      • Elemento O.
      • Coticchio G.
      • Dal Canto M.
      • Mignini Renzinu M.
      • Fadini R.
      • Hourvitz A.
      Characterization of the human cumulus cell transcriptome during final follicular maturation and ovulation.
      ). Corona cell gene expression is dynamic and can be affected by specific events, such as maternal ageing (
      • Al-Edani T.
      • Assou S.
      • Ferrieres A.
      • Bringer Deutsch S.
      • Gala A.
      • Lecellier C.H.
      • Ait-Ahmed O.
      • Hamamah S.
      Female aging alters expression of human cumulus cells genes that are essential for oocyte quality.
      ) and ovarian gonadotrophin treatments (
      • Assou S.
      • Haouzi D.
      • Dechaud H.
      • Gala A.
      • Ferrieres A.
      • Hamamah S.
      Comparative gene expression profiling in human cumulus cells according to ovarian gonadotropin treatments.
      ). One of the first studies to investigate oocyte competence identified several corona cells genes as biomarkers for assessing developmental potential, including BCL2L11, PCK1 and NFIB (
      • Assou S.
      • Haouzi D.
      • Mahmoud K.
      • Aouacheria A.
      • Guillemin Y.
      • Pantesco V.
      • Reme T.
      • Dechaud H.
      • De Vos J.
      • Hamamah S.
      A non-invasive test for assessing embryo potential by gene expression profiles of human cumulus cells: a proof of concept study.
      ). Follow-up investigations identified 45 corona cell genes of interest to be considered biomarkers of successful embryo development and pregnancy outcomes that were tested in a prospective study. The test group underwent a day-3 embryo transfer based on the 45 corona cell gene expression profiles, whereas the control group underwent a day 3 embryo transfer based on traditional embryo grading selection. Both implantation and ongoing pregnancy rates were significantly higher in the test group after embryos were chosen based upon their corona cell gene expression profile. Of the 267 corona cells samples studied, 27% had a corona cell gene expression profile that predicted positive pregnancy outcome, 42% that predicted negative pregnancy outcome and 13% that predicted early arrested development. Remarkably, no relationship between traditional morphological grading and corona cells gene expression was observed (
      • Assou S.
      • Haouzi D.
      • De Vos J.
      • Hamamah S.
      Human cumulus cells as biomarkers for embryo and pregnancy outcomes.
      ). Other retrospective studies have shown a significant correlation between the cumulus transcriptome and subsequent pregnancy outcomes where NRP1, UBQLN1, PSMD6, DPP8, HIST1H4C, CALM1, PTGS2, EFNB2 and CAMK1D gene expression were correlated with pregnancy and TOM1 with negative implantation (
      • Assidi M.
      • Montag M.
      • Van Der Ven K.
      • Sirard M.A.
      Biomarkers of human oocyte developmental competence expressed in cumulus cells before ICSI: a preliminary study.
      ,
      • Wathlet S.
      • Adriaenssens T.
      • Segers I.
      • Verheyen G.
      • Janssens R.
      • Coucke W.
      • Devroey P.
      • Smitz J.
      New candidate genes to predict pregnancy outcome in single embryo transfer cycles when using cumulus cell gene expression.
      ). Recently, the combinatorial expression of 12 corona cell genes involved in glucose metabolism, transcription, gonadotrophin regulation and apoptosis were used to predict pregnancy outcome for 55 patients with 78% accuracy (
      • Iager A.E.
      • Kocabas A.M.
      • Otu H.H.
      • Ruppel P.
      • Langerveld A.
      • Schnarr P.
      • Suarez M.
      • Jarrett J.C.
      • Conaghan J.
      • Rosa G.J.
      • Fernandez E.
      • Rawlins R.G.
      • Cibelli J.B.
      • Crosby J.A.
      Identification of a novel gene set in human cumulus cells predictive of an oocyte's pregnancy potential.
      ). Together, these studies indicate cumulus cells are a potential valuable source of information on oocyte competence. Inconsistency to date in the corona cells transcriptome data, however, may be due to differences in experimental design and methodology, including factors such as maternal age, ovarian stimulation protocols and various in-vitro laboratory protocols.
      In the present study, the individual corona cells transcriptome of chromosomally normal (euploid) embryos was investigated using RNA sequencing to identify novel gene transcription associated with implantation outcome. Results demonstrated that the canonical Wnt-signalling pathway and AXIN transcription are strong indicators of oocyte developmental competence and subsequent chromosomally normal live birth.

      Materials and methods

      Corona cell collection

      After routine oocyte retrieval, cumulus cells were mechanically separated from each individual cumulus oocyte complex without disturbing the inner corona cells. Oocyte maturity was visually confirmed with the presence of a polar body and individual oocyte corona complexes were allowed to recover for 4 h in an incubator at 7% CO2, 5% O2 and 37°C. Corona cells were removed before intracytoplasmic sperm injection using a media solution containing 0.33 mg/ml hyaluronidase and rinsed through sequential 3 × 20 µL drops of phosphate buffered saline and bovine serum albumin solution before transfer into 10 µL of extraction buffer (PicoPure® RNA-isolation kit, Life Technologies, Carlsbad CA). All corona cells samples were individually snap-frozen in liquid nitrogen and stored at −80°C until later analysis.
      Corona cells were donated from individual cumulus oocyte complexes (n = 10), with patient consent. The study was approved by HCA – HealthONE Institutional Review Board on 5 November 2012 (reference number 231845-7). Female participants (n = 10) presented with normal ovarian reserve (anti-Müllerian hormone >1.0 ng/ml, FSH <10 mIU/ml, antral follicle count >6) and advanced maternal age (36–43 years) but no other infertility diagnosis, including male factor, to minimize potential inter-patient variables. Patients underwent routine IVF with morphologically mature oocytes (second metaphase stage) fertilized by intracytoplasmic sperm injection, and embryos were individually cultured to the blastocyst stage for a trophectoderm biopsy before vitrification. Culture conditions were identical, using established routine laboratory protocols. Only blastocysts graded 3BB or better were chosen for trophectoderm biopsy, based on the Gardner Schoolcraft grading system (
      • Gardner D.K.
      • Schoolcraft W.B.
      Towards Reproductive Certainty: Fertility and Genetics Beyond.
      ). Single-nucleotide polymorphism microarray-based comprehensive chromosome screening was carried out on biopsied trophectoderm cells. After the identification of a euploid blastocyst, patients were scheduled for a routine frozen embryo transfer (
      • Schoolcraft W.B.
      • Treff N.R.
      • Stevens J.M.
      • Ferry K.
      • Katz-Jaffe M.
      • Scott Jr., R.T.
      Live birth outcome with trophectoderm biopsy, blastocyst vitrification, and single-nucleotide polymorphism microarray-based comprehensive chromosome screening in infertile patients.
      ). Corona samples were subsequently grouped on the basis of implantation outcome (live birth n = 5 and negative implantation n = 5), with negative implantation defined as a complete lack of biochemical pregnancy. Both implantation groups had an equal number of day 5 and day 6 biopsied euploid blastocysts transferred.

      RNA sequencing

      Individual corona cell samples were isolated for RNA-sequencing using the PicoPure® RNA Isolation Kit (Life Technologies, Carlsbad CA) with modifications. Briefly, samples were lysed and bound to a silica-based filter, treated with RNase-free DNase I (Qiagen, Valencia CA) and washed several times before being recovered in 20 µL elution solution. Purified cDNA libraries were constructed from 50 ng total RNA, which was rRNA-depleted using the Ion Total RNA-Seq Kit v2, Whole Transcriptome Library Prep protocol (Life Technologies, Carlsbad CA). rRNA-depleted total RNA was fragmented using RNase III and purified by Agencourt Ampure XP micro-beads (Beckman Coulter Inc., Brea CA). The fragmented RNA was then hybridized and ligated before reverse transcription and addition of multiplexing barcode adaptor. The subsequent cDNA was then amplified by polymerase chain reaction and purified again. Barcoded libraries were equalized and pooled into an evenly represented final library consisting of 100 pM cDNA, which was then coupled onto templated capture beads and enriched using the Ion OneTouch 2 and OneTouch ES systems (Life Technologies, Carlsbad CA). Final libraries were prepped for loading on the ION PI v2 chip and then sequenced on the Ion Proton with a P1 200 v2 Sequencing kit (Life Technologies, Carlsbad CA).

      RNA-sequencing data and bioinformatic analysis

      Raw reads in FASTQ format were trimmed and filtered with FastQC such that only reads with Phred Q-scores over 20 and read lengths over 35 bp were retained. Duplicate reads were removed, thereby retaining unique reads with higher average base quality. Strand NGS v.2.1 (Strand Life Sciences Inc., Aurora CO) was used for subsequent RNA-Seq data analysis. The trimmed and filtered reads were aligned to NCBI RefSeq human reference genome (GRCh37/hg19) and human transcriptome. Transcript isoform assembly, abundance estimation and quantification were carried out with Strand NGS. Differential gene expression was carried out with DESeq v.3.0 normalization (
      • Anders S.
      • Huber W.
      Differential expression analysis for sequence count data.
      ) and fold change >2.0 (bioconductor.org). Strand NGS identified significantly differentially expressed up- and down-regulated genes using the Benjamini-Hochberg multiple test correction at P < 0.05. Gene annotations were provided by NCBI Entrez Gene database. Biological pathway analysis was carried out with Strand NGS based on WikiPathways Analysis, Reactome, GenMAPP and other pathway databases and the BioCyc pathway database. Significantly enriched pathways were identified with a threshold fold change >2.0.

      Quantitative real-time polymerase chain reaction validation

      Additional, independent corona cells samples (n = 10) were collected for quantitative real time polymerase chain reaction (qRT-PCR) to validate the RNA-sequencing data. The PicoPure® RNA Isolation Kit (Life Technologies, Carlsbad CA) was used for RNA isolation as previously described. Reverse transcription was carried out using the High Capacity Reverse Transcription cDNA Kit (Life Technologies, Carlsbad CA) to generate cDNA template for qRT-PCR.
      The ABI 7300 Real Time PCR System with the Power SYBR Green PCR Master Mix (Life Technologies, Carlsbad CA) was used to carry out qRT-PCR, in duplicate. After a 10-min incubation at 95°C, amplification was carried out for 40 cycles at 95°C for 15 s and 60°C for 1 min, then a dissociation stage for 15 s at 95°C, 1 min at 60°C, 15 s at 95°C, and 15 s at 60°C. Transcript quantification of APC, AXIN1, DUSP16, GSK3B, TNFRSF10A, and TXNIP, were calculated relative to the level of transcription of the housekeeping gene RPL19 in every sample. The polymerase chain reaction efficiency recorded R2 values ≥0.9, and the correlation coefficient was calculated to be greater than 0.99. Statistical analysis was carried out with REST-2009© software using bootstrap randomization techniques (Qiagen, Valencia CA). The standard error and the 95% confidence interval given by REST-2009© software are not derived from Ct values. They are the standard error and the confidence interval for the 50,000 iterations the programme runs to calculate the P-value; therefore no error bars are generated for graphical presentation. Gene expression fold differences with P < 0.05 were considered statistically significant.

      Results

      RNA-sequencing was successfully carried out for individual corona cell samples, with a mean number of sequence reads of 21.2 million per individual corona cell sample (range = 10.2 million to 29.8 million reads) to generate unique gene expression profiles in association with implantation outcome. After corona cell gene expression quantification with DESeq normalization, an unpaired t-test was carried out comparing live birth (n = 5) with negative implantation (n = 5), which identified 306 significantly differentially expressed genes (P < 0.05; fold change ≥1.5) between the two groups. Of these 307 differentially expressed corona cell genes, 67 were observed to have increased expression and 240 corona cell genes decreased expression in association with a live birth (Supplementary Figure S1a and S1b). A volcano plot was generated to visualize differences in transcript levels between live birth and negative implantation corona cells samples. Each dot on the volcano plot represents a transcript, red dots indicate transcripts that showed statistically significant differential expression between the two groups of corona cells samples, with P-value on the y-axis. The x-axis represents increased and decreased fold change reflecting differential gene expression observed between live birth and negative implantation (Figure 1). Upon further examination of these differentially expressed genes, 87% were observed to be protein-coding, with the remaining 13% being either pseudo or small regulatory RNAs. Several genes were identified in the RNA-sequencing data that displayed stable expression within each group, and differential expression between groups, including DUSP16, TXNIP and TNFRSF10A. These genes were targeted for validation using qRT-PCR using the additional independent corona cell samples. DUSP16 and TNFRSF10A displayed significantly increased transcription in association with negative implantation (P < 0.05) (Figure 2), and a trend towards increased expression was observed for TXNIP (Figure 2).
      Figure thumbnail rbmo1512-fig-0001
      Figure 1Volcano plot of transcripts from corona cells associated with live birth compared with negative implantation. Each dot represents a gene; red dots are significantly differentially expressed genes at P < 0.05 and fold change ≥2. Up-regulated genes are shown as positive values on the x-axis, and down-regulated genes are shown as negative values on the x-axis.
      Figure thumbnail rbmo1512-fig-0002
      Figure 2Fold change in negative implantation relative to live birth. Quantitative real-time polymerase chain reaction validation on additional individual corona cell samples displayed significantly increased differential expression of DUSP16 and TNSFRSF10A in negative implantation samples relative to live birth samples (P < 0.05), and a trend towards increased differential expression of TXNIP. P-values were calculated using REST-2009© software, hence no error bars are present.
      Gene ontology analysis revealed significantly enriched biological processes among the differentially expressed transcripts, including response to hormone stimulus, regulation of cellular protein metabolic processes, as well as transcription and transmembrane transport, in association with a live birth (P < 0.05; ≥ two-fold). Pathway analysis of the differentially expressed transcripts identified enriched downstream biochemical signalling pathways. Table 1 shows the enriched signalling pathways with increased differentially expressed transcripts in association with live birth that included Wnt signalling, mitogen-activated protein kinases signalling, focal adhesion and tricarboxylic acid cycle (TCA). In contrast, Table 2 shows the enriched signalling pathways identified with decreased differentially expressed transcripts in association with live birth, including degradation of beta-catenin, apoptosis, DNA damage response and the detoxification of reactive oxygen species.
      Table 1Enriched signalling pathways with increased differentially expressed transcripts in association with live birth.
      Enriched signalling pathways with increased expressionP-value
      Mitotic prometaphase0.00011
      Mitotic metaphase and anaphase0.00127
      Hedgehog signalling pathway0.00692
      Tricarboxylic acid cycle0.00789
      Glutamate removal from folates0.00789
      Methylglyoxal degradation VI0.00789
      Wnt signalling pathway and pluripotency0.00824
      Toll-like receptor signalling pathway0.00882
      Nucleosome assembly0.01291
      Signal transduction of S1P receptor0.01527
      Focal adhesion0.01570
      Histone modifications0.01607
      Activation of genes by ATF40.01651
      Table 2Enriched signalling pathways with decreased differentially expressed transcripts in association with live birth.
      Enriched signalling pathways with decreased expressionP-value
      Degradation of beta-catenin by the destruction complex0.000000367
      Metabolism of non-coding RNA0.000002327
      Metabolism of amino acids and derivatives0.000007400
      Mitogen-activated protein kinase signalling pathway0.000016274
      Mitotic prophase0.000024329
      Wnt signalling pathway and pluripotency0.000046015
      RNA polymerase II transcription0.000046015
      Apoptosis modulation and signalling0.000049197
      Cell cycle checkpoints0.000053863
      Synthesis of DNA0.000055260
      Metabolism of carbohydrates0.000074778
      Detoxification of reactive oxygen species0.000077281
      M-G1 transition0.000115366
      Examination of the two enriched pathways revealed Wnt signalling to be significantly associated with a live birth outcome, and the degradation of beta-catenin by the destruction complex pathway to be significantly associated with negative implantation. These pathways are linked as components of the larger intricate Wnt-canonical pathway. Further validation was carried out on additional independent corona cells samples that were collected and analysed individually using qRT-PCR for APC, AXIN1 and GSK3B gene transcription relative to RPL19. These three genes are part of the beta-catenin destruction pathway and showed consistent expression within each group and significantly increased expression in association with negative implantation compared with live birth, validating the RNA-sequencing data (P < 0.05) (Figure 3).
      Figure thumbnail rbmo1512-fig-0003
      Figure 3Fold change in negative implantation relative to live birth. WNT-canonical pathway genes AXIN1, GSK3B and APC were validated by quantitative real-time polymerase chain reaction on additional individual corona cell samples and showed significantly increased expression associated with negative implantation relative to live birth samples (P < 0.05). P-values were calculated using REST-2009© software, hence no error bars are present.

      Discussion

      In this study, RNA-sequencing technologies were used to generate an individual profile of corona cell gene expression in association with implantation outcome and successful live birth after the transfer of a euploid blastocyst. This is the first study to investigate corona cells gene expression in correlation with the success or failure of a frozen euploid blastocyst upon transfer, thereby controlling for embryonic chromosomes and an unstimulated uterine environment. As expected, embryo morphology is not always a good predictor of live birth and, although many of the embryos in this study had similar blastocyst grades, implantation success was not guaranteed, revealing the need for additional embryo selection methods (Table 3). The ongoing research surrounding corona cell gene expression biomarkers for non-invasive assessment of embryo competence could represent an additional tool for selection.
      Table 3Oocyte and embryo morphology with embryo transfer outcome.
      Number of cumulus oocyte complexesOocyte MII morphologyDay 3 cell number (grade)Blastocyst grade at biopsyEmbryo transfer outcome
      1Normal9 (4−)3BANegative
      2Normal8 (4-C)5BBNegative
      3Normal12 (4-C)5AANegative
      4Normal8 (4-)5BANegative
      5Normal9 (3+)4BBLive birth
      6Normal10 (4)5AALive birth
      7Normal8 (4)4BBLive birth
      8Normal8 (4-)5BBLive birth
      9Normal8 (3+)3BBNegative
      10Normal8 (4)4AALive birth
      MII, second metaphase.
      In the present study, RNA-sequencing was used to characterize the corona cells transcriptome, and has allowed us to investigate more genes than traditional microarray methods. To validate our approach, qRT-PCR validation was carried out on three genes identified by the RNA-sequencing data: DUSP16, TXNIP and TNFRSF10A. DUSP16 is responsible for encoding a mitogen-activated kinase phosphatase, a subfamily of the dual specificity phosphatase protein. DUSP16 gene expression has been shown to potentially regulate epidermal growth factor signalling, and is associated with oocyte quality (
      • Adriaenssens T.
      • Wathlet S.
      • Segers I.
      • Verheyen G.
      • De Vos A.
      • Van Der Elst J.
      • Coucke W.
      • Devroey P.
      • Smitz J.
      Cumulus cell gene expression is associated with oocyte developmental quality and influenced by patient and treatment characteristics.
      ). TXNIP is a protein coding gene. It is believed to act as an oxidative stress mediator, by limiting the bioavailability of thioredoxin. TXNIP is involved in the crosstalk between the oocyte and its somatic compartment in the preconception period, and has been implicated as a non-invasive, quality biomarker in the corona cells of bovine oocytes (
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      • Uzbekova S.
      In vitro maturation of oocytes alters gene expression and signaling pathways in bovine cumulus cells.
      ). TNFRSF10A is a member of the TNF-receptor superfamily. The TNFRSF10A receptor is activated by the tumour necrosis factor-related apoptosis inducing ligand (TNFSF10/TRAIL), which is responsible for inducing apoptosis and cell death. In this study, decreased TNFRSF10A expression levels were observed in relation to live birth. Overexpression of TNFRSF10A is indicative of disrupted cell regulation, which leads to increased apoptosis, and decreased developmental competence. Apoptosis is an essential pathway during folliculogenesis; however, a balance between cell death and division is crucial, with increased levels of apoptosis shown to be associated with impaired oocyte maturation, as well as fertilization and pregnancy outcome (
      • Host E.
      • Gabrielsen A.
      • Lindenberg S.
      • Smidt-Jensen S.
      Apoptosis in human cumulus cells in relation to zona pellucida thickness variation, maturation stage, and cleavage of the corresponding oocyte after intracytoplasmic sperm injection.
      ,
      • Lee K.S.
      • Joo B.S.
      • Na Y.J.
      • Yoon M.S.
      • Choi O.H.
      • Kim W.W.
      Cumulus cells apoptosis as an indicator to predict the quality of oocytes and the outcome of IVF-ET.
      ).
      Differential expression of transcripts in the Wnt-signalling pathway was associated with live birth in our study. This pathway is known to be functionally critical during pre-implantation and implantation (
      • Tepekoy F.
      • Akkoyunlu G.
      • Demir R.
      The role of Wnt signaling members in the uterus and embryo during pre-implantation and implantation.
      ). When Wnt binds to cell surface receptors, it turns off beta-catenin destruction which, in turn, stabilizes beta-catenin, allowing translocation into the nucleus (
      • Clevers H.
      Wnt/beta-catenin signaling in development and disease.
      ). Accumulation of beta-catenin within the nucleus activates genes by binding to transcriptional activators, including transcription factors belonging to the TCF/LEF family (
      • Clevers H.
      Wnt/beta-catenin signaling in development and disease.
      ), activating Wnt target gene transcription (
      • MacDonald B.T.
      • Tamai K.
      • He X.
      Wnt/beta-catenin signaling: components, mechanisms, and diseases.
      ) (Figure 4). Wnts are extracellular molecules that are secreted locally to exert control over a multitude of developmental processes, including cell-to-cell interactions, differentiation, cell-fate specification and regulation. Wnt signalling is vital for development, and is modulated by cytoplasmic, extracellular and nuclear regulations. Within the Wnt-signalling pathway, WNT4 regulates a number of genes involved in late follicular development, and is required for normal antral follicular development (
      • Boyer A.
      • Lapointe E.
      • Zheng X.
      • Cowan R.G.
      • Li H.
      • Quirk S.M.
      • Demayo F.J.
      • Richards J.S.
      • Boerboom D.
      WNT4 is required for normal ovarian follicle development and female fertility.
      ). In a mouse knock-out study, it was shown that inhibition of the Wnt4 gene led to a phenotype of compromised ovarian folliculogenesis and severely reduced fertility and premature ovarian failure (
      • Prunskaite-Hyyrylainen R.
      • Shan J.
      • Railo A.
      • Heinonen K.M.
      • Miinalainen I.
      • Yan W.
      • Shen B.
      • Perreault C.
      • Vainio S.J.
      Wnt4, a pleiotropic signal for controlling cell polarity, basement membrane integrity, and antimullerian hormone expression during oocyte maturation in the female follicle.
      ). Wnt signalling during embryogenesis participates in multiple processes, including cell differentiation, regulation and migration (
      • Logan C.Y.
      • Nusse R.
      The Wnt signaling pathway in development and disease.
      ), and loss of even a single Wnt gene can produce embryonic lethality (
      • Gilbert A.M.
      • Bursavich M.G.
      • Alon N.
      • Bhat B.M.
      • Bex F.J.
      • Cain M.
      • Coleburn V.
      • Gironda V.
      • Green P.
      • Hauze D.B.
      • Kharode Y.
      • Krishnamurthy G.
      • Kirisits M.
      • Lam H.S.
      • Liu Y.B.
      • Lombardi S.
      • Matteo J.
      • Murrills R.
      • Robinson J.A.
      • Selim S.
      • Sharp M.
      • Unwalla R.
      • Varadarajan U.
      • Zhao W.
      • Yaworsky P.J.
      Hit to lead studies on (hetero)arylpyrimidines – agonists of the canonical Wnt-beta-catenin cellular messaging system.
      ).
      Figure thumbnail rbmo1512-fig-0004
      Figure 4The Wnt-signalling pathway. Wnts are secreted extracellular signalling molecules that exert local control over diverse developmental processes including cell-fate specification, differentiation and regulation of cell-cell interactions. (A) As Wnt binds to cell surface receptors, it turns off beta-catenin destruction, which in turn stabilizes beta-catenin, allowing translocation into the nucleus and activation of Wnt target gene transcription. Wnt signalling was identified as an enriched pathway with increased differentially expressed transcripts in association with live birth (P < 0.05); (B) In the absence of Wnt signalling, increased expression of AXIN1, APC and GSK3 leads to the degradation of beta-catenin. The beta-catenin destruction complex was observed in association with negative implantation, and decreased differentially expressed transcripts (P < 0.05).
      In the absence of Wnt stimulus, cytoplasmic beta-catenin is targeted for proteolysis by a large multi-protein assembly called the beta-catenin destruction complex. Additionally, the relative levels of the destruction proteins AXIN, APC and GSK3 are critical for both homeostasis as well as responsiveness to Wnt (
      • Minde D.P.
      • Anvarian Z.
      • Rudiger S.G.
      • Maurice M.M.
      Messing up disorder: how do missense mutations in the tumor suppressor protein APC lead to cancer?.
      ). It has been suggested that Wnt signalling in cumulus cells may act by recruiting beta-catenin and promoting the formation of adherens junctions, and may be important for folliculogenesis (
      • Wang H.X.
      • Tekpetey F.R.
      • Kidder G.M.
      Identification of WNT/beta-CATENIN signaling pathway components in human cumulus cells.
      ).
      AXIN, APC and GSK3 within the beta-catenin destruction complex pathway were observed to have stable, increased expression in corona cell samples associated with negative implantation outcome. AXIN, which is expressed mostly within the cytoplasm, can function as a negative regulator of the Wnt-signalling pathway and is capable of inducing apoptosis. AXIN acts by recruiting beta-catenin into plasma membranes and promotes the formation of adherens junctions (
      • Wang H.X.
      • Tekpetey F.R.
      • Kidder G.M.
      Identification of WNT/beta-CATENIN signaling pathway components in human cumulus cells.
      ). AXIN functions also as a scaffold protein capable of shuttling between the nucleus and cytoplasm, allowing beta-catenin to be transported from the nucleus to the cytoplasm where it is degraded (
      • Wiechens N.
      • Heinle K.
      • Englmeier L.
      • Schohl A.
      • Fagotto F.
      Nucleo-cytoplasmic shuttling of Axin, a negative regulator of the Wnt-beta-catenin Pathway.
      ). The increase of AXIN observed in corona cell samples with negative implantation may be indicative of a degradation of beta-catenin, causing a reduction in Wnt-signalling, cellular proliferation, cellular migration and an increase in apoptosis.
      In this study, individual corona cell samples resulting in negative implantation displayed a significant increase in GSK3B activity, which may be indicative of beta-catenin instability and a reduction of nt signalling. GSK3B, a beta isoform of the GSK3 gene, is an evolutionarily conserved serine-threonine kinase that has been implicated in body pattern formation and energy metabolism and has been shown to interact with AXIN1 (
      • Mak B.C.
      • Takemaru K.
      • Kenerson H.L.
      • Moon R.T.
      • Yeung R.S.
      The tuberin-hamartin complex negatively regulates beta-catenin signaling activity.
      ,
      • Nakamura T.
      • Hamada F.
      • Ishidate T.
      • Anai K.
      • Kawahara K.
      • Toyoshima K.
      • Akiyama T.
      Axin, an inhibitor of the Wnt signalling pathway, interacts with beta-catenin, GSK-3beta and APC and reduces the beta-catenin level.
      ). The binding of Wnt by a seven trans-membrane domain receptor results in disheveled activation, down regulating GSK3 activity. This down-regulation of GSK3 contributes to the stabilization of beta-catenin, along with several other signalling actions. In the absence of a Wnt signal, GSK3 interacts with beta-catenin, AXIN and APC by phosphorylation of the associated proteins (
      • Eastman Q.
      • Grosschedl R.
      Regulation of LEF-1/TCF transcription factors by Wnt and other signals.
      ). This phosphorylation results in the Slimb/beta-TrCP-mediated ubiquination, as well as proteolytic degradation of beta-catenin (
      • Jiang J.
      • Struhl G.
      Regulation of the Hedgehog and Wingless signalling pathways by the F-box/WD40-repeat protein Slimb.
      ,
      • Maniatis T.
      A ubiquitin ligase complex essential for the NF-kappaB, Wnt/Wingless, and Hedgehog signaling pathways.
      ).
      APC is a tumour suppressor gene that acts as a negative regulator. APC controls the concentrations of nuclear beta-catenin. It has been suggested that APC may actively protect beta-catenin from dephosphorylation by PP2A (
      • Su Y.
      • Fu C.
      • Ishikawa S.
      • Stella A.
      • Kojima M.
      • Shitoh K.
      • Schreiber E.M.
      • Day B.W.
      • Liu B.
      APC is essential for targeting phosphorylated beta-catenin to the SCFbeta-TrCP ubiquitin ligase.
      ). The APC protein is recruited and facilitates the phosphorylation of beta-catenin, becoming ubiquitin-conjugated, and subsequently destroyed by the 26S proteasome (
      • Clevers H.
      Wnt/beta-catenin signaling in development and disease.
      ). Interestingly, APC seems to compete with AXIN to bind to the same beta-catenin interaction interface (
      • Kimelman D.
      • Xu W.
      beta-catenin destruction complex: insights and questions from a structural perspective.
      ,
      • Xing Y.
      • Clements W.K.
      • Kimelman D.
      • Xu W.
      Crystal structure of a beta-catenin/axin complex suggests a mechanism for the beta-catenin destruction complex.
      ). This redundancy may represent a mechanism by which dramatic fluctuations in beta-catenin are protected, compensating for beta-catenin degradation (
      • Lee E.
      • Salic A.
      • Kruger R.
      • Heinrich R.
      • Kirschner M.W.
      The roles of APC and Axin derived from experimental and theoretical analysis of the Wnt pathway.
      ).
      Additional enriched corona cell signalling pathways in association with a live birth included the mitogen-activated protein kinase (MAPK) signalling pathway, which moderates multiple cellular processes including differentiation, growth and proliferation (
      • Ono K.
      • Han J.
      The p38 signal transduction pathway: activation and function.
      ). In oocytes, the MAPK signalling pathway is associated with maintaining a metaphase II arrest, although recently it has been discovered that when it is inhibited during the transition from meiosis I to meiosis II, meiosis I is accelerated, leading to an increase in aneuploidy in the metaphase II stage of development (
      • Nabti I.
      • Marangos P.
      • Bormann J.
      • Kudo N.R.
      • Carroll J.
      Dual-mode regulation of the APC/C by CDK1 and MAPK controls meiosis I progression and fidelity.
      ). MAPK signalling is mediated by FSH and epidermal growth factor, essential for cumulus expansion which is critical for oocyte advancement (
      • Dragovic R.A.
      • Ritter L.J.
      • Schulz S.J.
      • Amato F.
      • Thompson J.G.
      • Armstrong D.T.
      • Gilchrist R.B.
      Oocyte-secreted factor activation of SMAD 2/3 signaling enables initiation of mouse cumulus cell expansion.
      ). The focal adhesion pathway similarly showed increased expression in corona cells, resulting in live birth. This pathway is responsible for the transmission of mechanical force and regulatory signalling between extracellular matrices and cells (
      • Chen C.S.
      • Alonso J.L.
      • Ostuni E.
      • Whitesides G.M.
      • Ingber D.E.
      Cell shape provides global control of focal adhesion assembly.
      ). The focal adhesion pathway has been linked to oocyte maturation, thereby impacting oocyte developmental competence, and is partially involved in the communication between corona cells and the developing oocyte (
      • Ohtake J.
      • Sakurai M.
      • Hoshino Y.
      • Tanemura K.
      • Sato E.
      Expression of focal adhesion kinase in mouse cumulus-oocyte complexes, and effect of phosphorylation at Tyr397 on cumulus expansion.
      ). In corona cells, disruption of the MAPK/focal adhesion signalling pathways may affect critical processes involved in corona-oocyte communication, which may result in a less competent oocyte and embryo.
      Finally, the TCA cycle and respiratory electron transport pathway were observed to have increased corona cell gene expression in association with a live birth. These pathways are responsible for generating energy in eukaryotic cells in the form of adenosine triphosphate. Energy production can be glucose derived, or extracellular pyruvate may be metabolized by the mitochondria, producing most of the adenosine triphosphate found in the oocyte (
      • Dumollard R.
      • Ward Z.
      • Carroll J.
      • Duchen M.R.
      Regulation of redox metabolism in the mouse oocyte and embryo.
      ,
      • Dumollard R.
      • Carroll J.
      • Duchen M.R.
      • Campbell K.
      • Swann K.
      Mitochondrial function and redox state in mammalian embryos.
      ). In the mouse, the oocyte controls intercellular metabolomic cooperation for energy production through the TCA cycle between the corona cells and the developing oocyte. Sufficient energy production during development is crucial for oocyte competence (
      • Sugiura K.
      • Pendola F.L.
      • Eppig J.J.
      Oocyte control of metabolic cooperativity between oocytes and companion granulosa cells: energy metabolism.
      ).
      In conclusion, this study has demonstrated that RNA-sequencing technologies can successfully identify a unique, individual, corona cell transcriptome profile. The strength of this study is highlighted by the transfer of known euploid blastocysts, cultured and transferred under identical conditions, into an unstimulated uterine environment, specifically in relation to true negative implantation. Ongoing research to elucidate the biological and signalling pathways of corona cells associated with oocyte competence could contribute to advancements in embryo selection. A corona cell transcription assay developed for embryo viability, used in conjunction with advanced morphological algorithms and comprehensive chromosome screening, could result in overall improved IVF outcomes, and the routine use of single embryo transfers.

      Appendix. Supplementary material

      The following is the supplementary data to this article:

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      Biography

      Jason Parks leads the Embryology Team at the National Foundation for Fertility Research. He is currently pursuing his PhD in genetics through the University of Kent in the UK. Jason's research is focused on the window of implantation, the critical time when an embryo implants into the uterus and non-invasive assays for IVF.