Pregnancy prediction in single embryo transfer cycles after ICSI using QPCR: validation in oocytes from the same cohort

Abstract

Cumulus cell (CC) gene expression is being explored as an additional method to morphological scoring to choose the embryo with the highest chance to pregnancy. In 47 ICSI patients with single embryo transfer (SET), from which individual CC samples had been stored, 12 genes using QPCR were retrospectively analyzed. The CC samples were at the same occasion also used to validate a previously obtained pregnancy prediction model comprising three genes (ephrin-B2 (EFNB2), calcium/calmodulin-dependent protein kinase ID, stanniocalcin 1). Latter validation yielded a correct pregnant/non-pregnant classification in 72% of the samples. Subsequently, 9 new genes were analyzed on the same samples and new prediction models were built. Out of the 12 genes analyzed a combination of the best predictive genes was obtained by stepwise multiple regression. One model retained EFNB2 in combination with glutathione S-transferase alpha 3 and 4, progesterone receptor and glutathione peroxidase 3, resulting in 93% correct predictions when 3 patient and treatment cycle characteristics were included into the model. This large patient group allowed to do an intra-patient analysis for 7 patients, an analysis mimicking the methodology that would ultimately be used in clinical routine. CC related to a SET that did not give pregnancy and CC related to their subsequent frozen/thawed embryos which ended in pregnancy were analyzed. The models obtained in the between-patient analysis were used to rank the oocytes within-patients for their chance to pregnancy and resulted in 86% of correct predictions. In conclusion, prediction models built on selected quantified transcripts in CC might help in the decision making process which is currently only based on subjective embryo morphology scoring. The validity of our current models for routine application still need prospective assessment in a larger and more diverse patient population allowing intra-patient analysis.

Figure 1. Overview of the samples used in this study.

This figure represents the distribution of the samples used for the different analyses performed in this study. The grey background fields delimit the samples that were used for the specific analyses which are marked on the left side of the field. SET: single embryo transfer; FRET: frozen embryo transfer cycle; rFSH: recombinant Follicle Stimulating Hormone.^a:.

Figure 2. t-test of normalized gene expression values of non-pregnant versus live birth related cumulus cell samples.

The graphs represent the differences in gene expression between the cumulus cell samples associated to an oocyte that after in vitro fertilization treatment resulted in a live birth (n = 19) or not (n = 28). Normalization was done to the mean of B2M and UBC. Only P-values <0.0042 were considered significant after Bonferroni correction. The total range of expressions found is depicted by the boxes and whiskers respectively representing the two inner and the two outer quartiles with centrally the median.

Figure 3. Receiver operating characteristic (ROC) curve of the 3 pregnancy models.

Multivariable models were built to predict the chance to pregnancy including the gene expression levels measured in cumulus cell samples associated to an oocyte that after in vitro fertilization treatment resulted in a live birth or not. For Model 3 patient and cycle characteristics were also included (from Table 3). Model 1 was limited to 3 genes and is composed of EFNB2, PGR and GSTA4. In Model 2 all genes were allowed into the model as long as they could improve the model. Five genes were retained for Model 2: EFNB2, PGR, GSTA4, GSTA3, GPX3. To try to improve Model 2, in Model 3 patient and cycle characteristics were allowed into the model if they could improve the P-value of the model: EFNB2, PGR, GSTA4, GSTA3, GPX3, age, Relative E2, and number of days of ovarian stimulation. The respective areas under the curve are 0.82, 0.93 and 0.95. Relative E2: E2 value measured on day of hCG over the number of cumulus oophorus complexes.