Alternative Sample-Homogeneity Test for Quantitative and Qualitative Proficiency Testing Schemes.

Proficiency Testing (PT) External Quality Assessment (EQA) schemes are designed to ascertain the ability of individual laboratories to perform satisfactorily with respect to their peer laboratories or to limits imposed by external sources. Observed deviation of a laboratory result for a PT sample must be entirely attributed to the laboratory and not to the PT provider. To minimize the probability that deviations could be attributed to the PT provider, sample homogeneity should be assured. It is generally required that for quantitative parameters, the standard deviation among PT units should be calculated on the basis of duplicate measurements of at least 10 samples chosen at random, and the standard deviation among PT units should not exceed 0.3 times the standard deviation used to evaluate laboratories. Because this approach has important drawbacks, an alternative procedure is proposed by applying the theory of acceptance sampling to the assessment of sample heterogeneity for both quantitative and qualitative data and deriving acceptance limits on the basis of minimizing the probability of falsely evaluating laboratories. For obtaining acceptance limits for quantitative parameters, a distinction is made between laboratory evaluation using fixed limits on the one hand and laboratory evaluation using limits that are based on the variability of the reported results on the other hand. Sequential tests are proposed to evaluate sample heterogeneity by means of a comparison with the χ2 distribution. For qualitative parameters, acceptance-sampling plans are proposed that are based on minimizing the joint probability of rejecting batches that have a satisfactory amount of defective samples and accepting batches unnecessarily. The approach for quantitative parameters is applied on samples for a PT scheme of ethanol quantification and for qualitative parameters on the presence of monoblasts in a blood smear. It was found that five samples could already be enough to prove that the batch was homogeneous for quantitative parameters, although more than 20 samples were needed to prove homogeneity for qualitative parameters. This study describes a direct relation among the objective of an PT round, the criteria for evaluating the results, and the sample heterogeneity. When samples are effectively homogeneous, less measurements are needed than current practices require. A drawback of the proposed approach is that the number of samples to be tested is not known beforehand, and good knowledge of the analytical variability is crucial. The formulas to be applied are relatively simple. Despite the drawbacks, the proposed approach is generally applicable for both quantitative and qualitative data.

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

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.

Gene expression differences induced by equimolar low doses of LH or hCG in combination with FSH in cultured mouse antral follicles.

In a natural cycle, follicle growth is coordinated by FSH and LH. Follicle growth stimulation in Assisted Reproductive Technologies (ART) requires antral follicles to be exposed to both FSH and LH bioactivity, especially after GNRH analog pretreatment. The main aim was to detect possible differences in gene expression in granulosa cells after exposing the follicle during antral growth to LH or hCG, as LH and hCG are different molecules acting on the same receptor. Effects of five gonadotropin treatments were investigated for 16 genes using a mouse follicle culture model. Early (day 6) antral follicles were exposed to high recombinant FSH combined or not with equimolar concentrations of recombinant LH (rLH) or recombinant hCG (rhCG) and to highly purified human menopausal gonadotropin (HP-hMG) for 6 h, 12 h, or 3 days. Expression differences were tested for genes involved in steroidogenesis: Mvk, Lss, Cyp11a1, Hsd3b1, Cyp19a1, Nr4a1, and Timp1; final granulosa differentiation: Lhcgr, Oxtr, Pgr, Egfr, Hif1a, and Vegfa; and cytokines: Cxcl12, Cxcr4, and Sdc4. Lhcgr was present and upregulated by gonadotropins. Nr4a1, Cxcl12, and Cxcr4 showed a different expression pattern if LH bioactivity was added to high FSH in the first hours after exposure. However, no signs of premature luteinization were present even after a 3-day treatment as shown by Cyp19a1, Oxtr, Pgr, and Egfr and by estrogen and progesterone measurements. The downstream signaling by rhCG or rLH through the LHCGR was not different for this gene selection. Granulosa cells from follicles exposed to HP-hMG showed an enhanced expression level for several genes compared with recombinant gonadotropin exposure, possibly pointing to enhanced cellular activity.

New candidate genes to predict pregnancy outcome in single embryo transfer cycles when using cumulus cell gene expression.

OBJECTIVE: To relate the gene expression in cumulus cells surrounding an oocyte to the potential of the oocyte, as evaluated by the embryo morphology (days 3 and 5) and pregnancy obtained in single-embryo transfer cycles. DESIGN: Retrospective analysis of individual human cumulus complexes using quantitative real-time polymerase chain reaction for 11 genes. SETTING: University hospital IVF center. PATIENT(S): Thirty-three intracytoplasmic sperm injection patients, of which 16 were pregnant (4 biochemical and 12 live birth). INTERVENTION(S): Gene expression analysis in human cumulus complexes collected individually at pickup, allowing a correlation with the outcome of the corresponding oocyte. Multiparametric models were built for embryo morphology parameters and pregnancy prediction to find the most predictive genes. MAIN OUTCOME MEASURE(S): Gene expression profile of 99 cumulus complexes for 11 genes. RESULT(S): For embryo morphology prediction, TRPM7, ITPKA, STC2, CYP11A1, and HSD3B1 were often retained as informative. Models for pregnancy-biochemical or live birth-complemented or not with patient and cycle characteristics, always retained EFNB2 and CAMK1D together with STC1 or STC2. Positive and negative predictive values of the live birth models were >85%. CONCLUSION(S): EFNB2 and CAMK1D are promising genes that could help to choose the embryo to transfer with the highest chance of a pregnancy.

The cumulus cell gene expression profile of oocytes with different nuclear maturity and potential for blastocyst formation.

PURPOSE: Gene expression in human ART cumulus cell (CC) has been related to oocyte maturity and competence but requires further validation. Expression dynamics were investigated in CC of oocytes at different maturational stages and with different developmental competence in a standard in vivo mouse superovulation model. METHODS: Quantitative PCR analysis of Has2, Vcan, Sdc4, Alcam, Grem1, Ptgs1 and Ptgs2 in CC collected at regular time intervals from 0 to 24 h post hCG injection. RESULTS: Three expression patterns were observed each with strong regulation (4-230× differences). Immediately prior to ovulation CC of GVBD oocytes have 5× less Sdc4 and Ptgs1 and 5× more Ptgs2 when compared to the CC of freshly ovulated PB oocytes. When compared to the latter, the post-ovulatory aged PB oocytes had a 2× reduced blastocyst forming capacity and their CC expressed 2× more Sdc4 and 6× less Alcam. CONCLUSIONS: Morphologically identical cumulus oocyte complexes with different developmental competence can be differentiated by CC gene expression.

Differences in collagen expression in cumulus cells after exposure to highly purified menotropin or recombinant follicle-stimulating hormone in a mouse follicle culture model.

Extracellular matrix (ECM) formation by cumulus cells is an important process that determines fertilization and embryo quality. Several collagen types are present in the ovarian follicular ECM and are related to proliferation, steroidogenesis, and luteinization. In vitro mouse follicles can optimally grow and provide developmentally competent oocytes with 10 IU/L recombinant follicle-stimulating hormone (rFSH). As a model for superovulation, experiments with 100 IU/L rFSH or 100 IU/L highly purified menotropin (HP-hMG) exposure during antral growth were undertaken. Col4a1, Col4a2, and Col6a2 expression levels were analyzed at three time points during antral growth and at a 4-h interval up to 16 h after ovulation induction using quantitative PCR. The presence and induction of the collagen mRNA and protein were confirmed in cumulus from in vivo- and in vitro-grown follicles, and TGFBs 1 and 2 were assayed as potential regulators. The study revealed that exposure to 100 IU/L FSH, as in both superovulation conditions, significantly influenced the follicle morphology and slowed down nuclear maturation and mucification (P < 0.05). This coincided with an increased expression of the three collagens in the cumulus-oocyte complex at the end of antral growth and in the first hours following the ovulatory dose of human chorionic gonadotropin (P < 0.05). The increased expression might reflect a differentiation but is most likely due to a precocious luteinization of the cumulus. Growth in HP-hMG resulted in higher Tgfb1 mRNA and protein levels, fewer COCs with an increased collagen expression and with a more synchronous nuclear maturation. This suggests that the presence of luteinizing hormone activity tempered the effect of the elevated FSH dose.