RUNX2, GPX3 and PTX3 gene expression profiling in cumulus cells are reflective oocyte/embryo competence and potentially reliable predictors of embryo developmental competence in PCOS patients

Abstract

Background: Polycystic ovary syndrome (PCOS) is a common endocrine and metabolic disorder in women. The developmental competence of oocytes and embryos in PCOS patients is reduced to a certain extent (comparing to non-PCOS patients, the high quality embryo rate was decreased by 16% from the data of our centre) during the in vitro fertilization (IVF) process. Cross-talk between the oocyte and cumulus cells is critical for oocyte maturation and embryo competence. In this study, we have evaluated the transcription of specific genes in cumulus cells harvested from pre-ovulatory follicles of PCOS patients before IVF, according to individual oocyte nuclear maturity and developmental competence. Seven genes (RUNX2, PSAT1, ADAMTS9, CXCL1, CXCL2, CXCL3, and ITGB5) were targeted from our previous cDNA microarray data which isolated genes related to oocyte nuclear maturation in PCOS patients. Two additional genes which had been found to be associated with oocyte maturation or embryo quality in non-PCOS patients (GPX3 and PTX3) were also studied.

Methods: The mRNA expression levels of cumulus cells were detected by qRT- PCR.

Results: Consistent with our previous cDNA microarray data, with the exception of GPX3 and PTX3, the selected 7 genes were related to oocyte nuclear maturation in PCOS patients. Noticeably, the expression level of RUNX2 was lower in cumulus cells derived from oocytes that could develop into blastocysts than the level of expression from oocytes that could not. The PTX3 expression level was significantly lower in cumulus cells from oocytes with two normal pronuclei than that from oocytes that formed >2 pronuclei (MPN) after fertilization. GPX3 mRNA levels were decreased in cumulus cells isolated from oocytes that developed into blastocysts with high potential development competence.

Conclusions: Several cumulus cell genes were associated with oocyte maturation, fertilization and embryo quality in PCOS patients. RUNX2 and GPX3 are candidate genetic markers in the monitoring of embryo quality for PCOS patients, whereas PTX3 mainly played a role in fertilization process. Together with morphological evaluation, cumulus cells genes may serve as biomarkers of oocyte and embryo selection during the IVF process for PCOS patients and may advance our understanding of PCOS.

Figure 1

Distribution tree of COCs involved in the study. A total of 212 COCs were retrieved from the 15 PCOS patients. Except for 6 COCs which are atretic or with fractured zones pellucidae were discarded, there have 3 GV stage COCs, 30 MI stage COCs, and 173 MII stage COCs. On Day 1 (16–18 hours after insemination), 130 CCs separated from oocytes with normal fertilization (2PN) and 36 CCs separated from mature oocytes with abnormal fertilization (MPN) were classed into a “CC_2PN” and “CC_MPN” group, respectively. On Day 3, 54 of 127 embryos that developed from oocytes with normal fertilization (2PN) were transplanted or frozen, and their corresponding CCs were classified into six subgroups randomly. The other 73 embryos were cultured to the blastocyst stage. Cumulus cells from oocytes yielding blastulas after 5/6 days in vitro culture were classified as the “CC_B+” group (n = 37), and cumulus cells from oocytes that stopped developing at the embryo stage after Day 6 were divided into “CC_B-” groups (n = 36). COC, cumulus-oocyte-complex; CC_GV, cumulus cells from GV oocyte; CC_MI, cumulus cells from MI oocyte; CC_MII, cumulus cells from MII oocyte; CC_0PN, cumulus cells from oocyte which formed no pronuclei (0PN) after fertilization; CC_2PN, cumulus cells from oocyte which formed two pronuclei (2PN) after fertilization; CC_MPN, cumulus cells from oocyte which formed multi pronuclei (MPN) after fertilization; CC_WLGE, cumulus cells from oocyte yielding a weak or low grade embryo at Day3; CC_TQE, cumulus cells from oocyte yielding a top quality embryo at Day 3; CC_B-, cumulus cells from oocyte which stopped development at the embryo stage at Day 5/6; CC_B+, cumulus cells from oocyte yielding a blastocyst after 5/6 days of in vitro culture.

Figure 2

Transcripts levels of the target genes (RUNX2, PSAT1, ADAMTS9, CXCL1, CXCL2, CXCL3, ITGB5, GPX3 and PTX3) according to oocyte nuclear maturity in PCOS patients. mRNA expression of candidate genes (RUNX2, PSAT1, ADAMTS9, CXCL1, CXCL2, CXCL3, ITGB5, GPX3 and PTX3) in cumulus cells of PCOS patients, organized according to oocyte nuclear maturity stage (MI/GV stage versus MII stage). The signal intensity for each gene is shown on the y-axis in arbitrary units determined by qRT-PCR analysis with GAPDH as an endogeneous reference. * indicates a significant difference in gene expression between CC categories (** p < 0.01, * p < 0.05). The results are presented as the means ± SEM. CC_MI/GV: cumulus cells from oocyte at the MI stage or GV stage; CC_MII: cumulus cells from oocyte at the MII stage.

Figure 3

Transcripts levels of the target genes (RUNX2, PSAT1, ADAMTS9, CXCL1, CXCL2, CXCL3, ITGB5, GPX3 and PTX3) according to the fertilization process in PCOS patients. mRNA expression of candidate genes (RUNX2, PSAT1, ADAMTS9, CXCL1, CXCL2, CXCL3, ITGB5, GPX3 and PTX3) in cumulus cells of PCOS patients, organized according to pronuclei formation after fertilization (normal two pronuclei versus multi pronuclei). The signal intensity for each gene is shown on the y-axis in arbitrary units determined by qRT-PCR analysis with GAPDH as an endogeneous reference. * indicates a significant difference in gene expression between CC categories (** p < 0.01, * p < 0.05). The results are presented as the means ± SEM. CC_2PN: cumulus cells from oocyte which formed two normal pronuclei (2PN) after fertilization; CC_MPN, cumulus cells from oocyte which formed multi pronuclei (MPN) after fertilization.

Figure 4

Transcripts levels of the target genes (RUNX2, PSAT1, ADAMTS9, CXCL1, CXCL2, CXCL3, ITGB5, GPX3 and PTX3) according to embryo quality in PCOS patients. mRNA expression of candidate genes (RUNX2, PSAT1, ADAMTS9, CXCL1, CXCL2, CXCL3, ITGB5, GPX3 and PTX3) in cumulus cells of PCOS patients, organized according to oocyte development capability after normal fertilization. The signal intensity for each gene is shown on the y-axis in arbitrary units determined by qRT-PCR analysis with GAPDH as an endogeneous reference. * indicates a significant difference in gene expression between CC categories (** p < 0.01, * p < 0.05). The results were presented as the means ± SEM. CC_B+: cumulus cells from oocyte yielding blastocyst after 5–6 days of in vitro culture; CC_B-: cumulus cells from oocyte that had not developed into blastocyst at Day 5/6.