Biomarkers of human oocyte developmental competence expressed in cumulus cells before ICSI: a preliminary study

Abstract

Purpose: To identify reliable genomic biomarkers expressed in cumulus cells that accurately and non-invasively predict the oocyte developmental competence and reinforce the already used morphological criteria.

Methods: Eight consenting patients were selected for ovarian stimulation and ICSI procedures. Cumulus-oocyte complexes were transvaginally punctured and individually selected based on both good morphological criteria and high zona pellucida birefringence. Following ICSI, two 3-day embryos per patient were transferred. Pregnancy outcome was recorded and proven implantation was thereafter confirmed. Differential gene expression was assessed using two microarray platforms. Further real-time PCR validation, Ingenuity pathways analysis and intra-patient analysis were performed on 17 selected candidates.

Results: Seven genes were differentially (p ≤ 0.05) associated to successful pregnancy and implantation. These biomarkers could be used to predict the oocyte developmental competence.

Conclusions: These genomic markers are a powerful reinforcement of morphological approaches of oocyte selection. Their large-scale validation could increase pregnancy outcome and single embryo transfer efficiency.

Fig. 1

Summary of the experimental design used

Fig. 2

Schematic representation of the differentially expressed genes in CCs of ZGP oocytes compared to the ZGNP group according to both their fold change and the microarray platform

Fig. 3

Real-time PCR analysis of differentially expressed genes in individual CCs of ZGP group versus ZGNP. a, positive gene markers associated with pregnancy. b, negative markers associated with pregnancy failure. Candidates were ranked according to their p-values, which were determined following a T-test analysis achieved on normalized data at α = 0.05

Fig. 4

Bi-dimensional PCA representation of the six CCs samples to distinguish the positive embryo that led to successful pregnancy among the two transferred for each patient. For each patient and using the Euclidian distance and the origin (negative control), the nearer embryo is the false positive (FP, associated with pregnancy failure), while the farther is the true positive (TP, associated with successful pregnancy). If the distances have opposite signs, the embryo with positive Euclidian distance is automatically the true positive. (F1, F2), the two main PCA axes. (ZG), cumulus cells of oocytes with high zona score; (TP), true positive; (FP), false positive; (1, 2, 3), patient number

Fig. 5

Equation of the pregnancy prediction model. (P), probability of successful pregnancy

Fig. 6

Ingenuity pathways network generated from the QPCR validated candidates in human CCs prior to ICSI. Overexpressed genes (red) are RPL9 (ribosomal protein L9), UBQLN1 (ubiquilin 1), CALM1 (calmodulin 1), AR (androgen receptor), NRP1 (neuropilin 1), PKN2 (protein kinase N2), SYT11 (synaptotagmin XI), HIST1H4C (histone cluster 1, H4c), CALU (calumenin) and PSMD6 (proteasome (prosome, macropain) 26 S subunit, non-ATPase, 6). Underexpressed genes (green) are TOM1 (target of myb1 (chicken)) and CHGB (chromogranin B). Other genes: TMF1 (TATA element modulatory factor 1), CDC2L1 and CDC2L2 (cell division cycle 2-like 1 and 2), PXN (paxillin), CKAP4 (cytoskeleton-associated protein 4), HSPA13 (heat shock protein 70 kDa family, member 13), HERC3 (hect domain and RLD 3), Akt (protein kinase B), Hat (histone acetylase or acetyltransferase), PDK1 (pyruvate dehydrogenase kinase, isozyme 1), UBE3C (ubiquitin protein ligase E3C), and SELENBP1 (selenium binding protein 1)

Fig. 7

A custom-made IPA network made of selected list of microarray differentially expressed genes of human CCs between the ZGP and ZGNP groups. Overexpressed genes (red) are CALM1 (calmodulin 1), AR (androgen receptor), NRP1 (neuropilin 1), PKN2 (protein kinase N2), CALU (calumenin), and PLCB2 (phospholipase C, beta 2). Underexpressed genes (green) are TOM1 (target of myb1 (chicken)), GJA1 (gap junction protein, alpha 1, 43 kDa), PTX3 (pentraxin-related gene, rapidly induced by IL-1 beta), PGRMC1 (progesterone receptor membrane component 1), IL8 (interleukin 8), CASP9 (caspase 9, apoptosis-related cysteine peptidase), CAV1 (caveolin 1, caveolae protein, 22 kDa) and HSPD1 (heat shock 60 kDa protein 1 (chaperonin)). Other genes: hCG (human chorionic gonadotropin), FSH (follicle stimulating hormone), LH (luteinizing hormone), Akt (protein kinase B), AP1(JUN, enhancer-binding protein AP1), PDGF family (platelet-derived growth factor), VEGF family (vascular endothelial growth factor), PKC (protein kinase C), ERK1/2 and p38 MAPK (mitogen-activated protein kinase family), PI3K family (phosphoinositide-3-kinase), and HSP70 & 90 (heat shock protein 70 and 90 kDa family)