Wim Coucke

Cumulus cell gene expression is associated with oocyte developmental quality and influenced by patient and treatment characteristics

BACKGROUND Gene expression of cumulus cells (CC) could predict oocyte developmental quality. Knowledge of the genes involved in determining oocyte quality is scanty. The aim was to correlate clinical and biological characteristics during ovarian stimulation with the expression of 10 selected genes in CC. METHODS Sixty-three ICSI patients were stimulated with GnRH-agonist plus highly purified hMG (n = 35) or recombinant FSH (n = 28). Thirteen variables were analyzed: Age, BMI, duration of stimulation, serum concentrations of progesterone, 17beta-estradiol, FSH and LH on day of hCG, Ovarian Response, Oocyte Maturity, 2 pronuclei and three embryo morphology related variables: > or =7 cells, Low Fragmentation, Good Quality Embryos score. Expression of HAS2, VCAN, SDC4, ALCAM, GREM1, PTGS1, PTGS2, DUSP16, SPROUTY4 and RPS6KA2 was analyzed in pooled CC using quantitative PCR, and the relationship to the 13 variables was evaluated by multivariable analysis. RESULTS All 10 genes are expressed at oocyte retrieval, with PTGS1, SPROUTY4, DUSP16 and RPS6KA2 described in human ovary for the first time. The three variables that correlated most often with differential expression were Age, BMI and serum FSH level. Significant correlation was found with Oocyte Maturity (VCAN, P < 0.005), Low Fragmentation (RPS6KA2, P < 0.05), Embryos with > or =7 cells (ALCAM and GREM1, P < 0.05). The expression of the other genes was also correlated to oocyte developmental quality but to a less extent. SDC4, VCAN, GREM1, SPROUTY4 and RPS6KA2 showed gonadotrophin preparation-dependent expression and/or interactions (all P < 0.05). CONCLUSION The expression of ovulation related genes in CC is associated with patient and treatment characteristics, oocyte developmental potential and differs with the type of gonadotrophin used.

Cumulus cell gene expression predicts better cleavage-stage embryo or blastocyst development and pregnancy for ICSI patients

BACKGROUND Cumulus cell (CC) gene expression is suggested as a non-invasive analysis method to predict oocyte competence. There are, however, important between-patient differences in CC gene expression. These can be compensated when expression results are combined with patient and cycle characteristics using a multiple regression analysis model. METHODS From ICSI patients stimulated with GnRH antagonist and recombinant FSH (n= 25) or GnRH agonist and highly purified menotrophin (n= 20), CC were collected and oocytes were individually fertilized and cultured. CC were analyzed for the expression of Syndecan 4 (SDC4), Prostaglandin-endoperoxide synthase 2 (PTGS2), Versican (VCAN), Activated leukocyte cell adhesion molecule, Gremlin 1, transient receptor potential cation channel, subfamily M, member 7 (TRPM7), Calmodulin 2 and Inositol 1,4,5-trisphosphate 3-kinase A (ITPKA) using quantitative PCR. Results were analyzed in relation to the stimulation protocol. Within-patient variation in gene expression was related to oocyte maturity and developmental potential. Models predictive for normal embryo or blastocyst development and pregnancy in single embryo transfer cycles were developed. RESULTS Mature oocytes have higher PTGS2 and lower VCAN expression in their cumulus. All genes except VCAN had a positive correlation with good embryo or blastocyst morphology and were used to develop predictive models for embryo or blastocyst development (P< 0.01). Specific models were obtained for the two stimulation protocols. In both groups, better cleavage-stage embryo prediction relied on TRPM7 and ITPKA expression and pregnancy prediction relied on SDC4 and VCAN expression. In the current data set, the use of CC expression for pregnancy prediction resulted in a sensitivity of >70% and a specificity of >90%. CONCLUSIONS Multivariable models based on CC gene expression can be used to predict embryo development and pregnancy.

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.

Timing of Nuclear Maturation and Postovulatory Aging in Oocytes of In Vitro-Grown Mouse Follicles with or Without Oil Overlay

Meiotic maturation of the oocyte is a timed sequence of events induced by the ovulatory LH surge. In vitro maturation of oocytes is known to alter the meiotic time course. This study documented the timing of meiosis in oocytes grown in vitro for 12 days, from the preantral follicle stage onward, and the influence of an oil overlay. In the oil-free culture, the stability of the metaphase II spindle was further explored to determine the postovulatory aging events. After the maturation stimulus, in vitro-grown oocytes were collected at 2-h intervals spanning the period of meiosis (0-18 h) and at 3-h intervals during early postovulatory aging (18-27 h). Stage of maturation was assessed both morphologically and by detailed spindle analysis and chromosome alignment. Results revealed that oil overlay did not impair the competence of cultured oocytes to proceed to meiosis II, but delayed meiosis I progression. Oil overlay during culture causes a different hormonal exposure of the follicles by a differential segregation into the oil overlay. The use of a progesterone receptor antagonist, however, did not induce a delay in meiotic progression. Aging effects in oil-free cultured follicles were detected 5 h after the establishment of the metaphase II spindle, comparable to their in vivo grown counterparts. The predominant effect of aging was an interphase-like appearance of the cytoskeleton. So an optimal time window for fertilization after in vitro follicular growth was determined to be 16-21 h after maturation induction.

A prediction model to select PCOS patients suitable for IVM treatment based on anti-Müllerian hormone and antral follicle count

STUDY QUESTION Which baseline patient characteristics can help assisted reproductive technology practitioners to identify patients who are suitable for in-vitro maturation (IVM) treatment? SUMMARY ANSWER In patients with polycystic ovary syndrome (PCOS) who undergo oocyte IVM in a non-hCG-triggered system, circulating anti-Müllerian hormone (AMH), antral follicle count (AFC) and total testosterone are independently related to the number of immature oocytes and hold promise as outcome predictors to guide the patient selection process for IVM. WHAT IS ALREADY KNOWN Patient selection criteria for IVM treatment have been described in normo-ovulatory patients, although patients with PCOS constitute the major target population for IVM. With this study, we assessed the independent predictive value of clinical and endocrine parameters that are related to oocyte yield in patients with PCOS undergoing IVM. STUDY DESIGN, SIZE, DURATION Cohort study involving 124 consecutive patients with PCOS undergoing IVM whose data were prospectively collected. Enrolment took place between January 2010 and January 2012. Only data relating to the first IVM cycle of each patient were included. PARTICIPANTS/MATERIALS, SETTING, METHOD Patients with PCOS underwent oocyte retrieval for IVM after minimal gonadotrophin stimulation and no hCG trigger. Correlation coefficients were calculated to investigate which parameters are related to immature oocyte yield (patients age, BMI, baseline hormonal profile and AMH, AFC). The independence of predictive parameters was tested using multivariate linear regression analysis. Finally, multivariate receiver operating characteristic (ROC) analyses for cumulus oocyte complexes (COC) yield were performed to assess the efficiency of the prediction model to select suitable candidates for IVM. MAIN RESULTS AND THE ROLE OF CHANCE Using multivariate regression analysis, circulating baseline AMH, AFC and baseline total testosterone serum concentration were incorporated into a model to predict the number of COC retrieved in an IVM cycle, with unstandardized coefficients [95% confidence interval (CI)] of 0.03 (0.02-0.03) (P < 0.001), 0.012 (0.008-0.017) (P < 0.001) and 0.37 (0.18-0.57) (P < 0.001), respectively. Logistic regression analysis shows that a prediction model based on AMH and AFC, with unstandardized coefficients (95% CI) of 0.148 (0.03-0.25) (P < 0.001) and 0.034 (-0.003-0.07) (P = 0.025), respectively, is a useful patient selection tool to predict the probability to yield at least eight COCs for IVM in patients with PCOS. In this population, patients with at least eight COC available for IVM have a statistically higher number of embryos of good morphological quality (2.9 ± 2.3; 0.9 ± 0.9; P < 0.001) and cumulative ongoing pregnancy rate [30.4% (24 out of 79); 11% (5 out of 45); P = 0.01] when compared with patients with less than eight COC. ROC curve analysis showed that this prediction model has an area under the curve of 0.7864 (95% CI = 0.6997-0.8732) for the prediction of oocyte yield in IVM. LIMITATIONS, REASONS FOR CAUTION The proposed model has been constructed based on a genuine IVM system, i.e. no hCG trigger was given and none of the oocytes matured in vivo. However, other variables, such as needle type, aspiration technique and whether or not hCG-triggering is used, should be considered as confounding factors. The results of this study have to be confirmed using a second independent validation sample. WIDER IMPLICATIONS OF THE FINDINGS The proposed model could be applied to patients with PCOS after confirmation through a further validation study. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by a research grant by the Institute for the Promotion of Innovation by Science and Technology in Flanders, Project number IWT 070719.

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.

A new statistical method for evaluating long-term analytical performance of laboratories applied to an external quality assessment scheme for flow cytometry.

Abstract Background: The Belgian External Quality Assessment Scheme for Flow Cytometry evaluates the long-term analytical performance of participating laboratories by calculating a regression line between the target and reported values of each parameter for each laboratory during the past 3 years. This study aims to develop a method to find laboratories with aberrant variability or bias using robust techniques and to obtain robust estimates of the variability. Methods: A method is proposed to find outliers with respect to the individual regression line, followed by a step to find regression lines with excessive variability and finally a step to find regression lines with high bias. Results: The model was applied to the results obtained by 52 laboratories for CD4%. From the 1340 data points, 35 were determined to be regression outliers. The second step revealed one regression line with excessive variability; the third step detected three regression lines with exceeding bias. Conclusions: The methodology allows assessment of the long-term performance of laboratories, taking into account samples with different target values. Outliers in the first step indicate accidental mistakes, outliers in the second and third step point to high analytical variability or bias. Clin Chem Lab Med 2010;48:645–50.

Evidence for the positive impact of ISO 9001 and ISO 15189 quality systems on laboratory performance – evaluation of immunohaematology external quality assessment results during 19 years in Austria

Abstract Background ISO 9001 and ISO 15189 have been established as continuative models for quality systems beyond national laws, mandatory standards and guidelines of expert associations regarding analytical and organisational performance of medical laboratories and transfusion services. Although widely used, their impact on laboratory performance has not been investigated. Methods We retrospectively analysed the results of 167 laboratories in 59 distributions of the Austrian red cell immunohaematology external quality assessment (EQA) scheme in the years 1999–2017. The performance for each parameter and trends of individual participants were compared with respect to certification or accreditation status of participants’ quality systems and to laboratory type. Results Considering more than 52,000 EQA results, the absence or presence of a laboratory quality management system showed different error rates. Laboratories with ISO 9001 or ISO 15189 certification/accreditation had 0.7% incorrect results, while this rate was doubled without such quality systems (1.4%, p=0.0002). Statistically significant error reductions were seen upon ISO 9001/ISO 15189 implementation (1.3% before vs. 0.7% after; p=0.0468). Transfusion services had fewer errors (0.9%) compared to hospital and independent laboratories (both 1.2%). Conclusions Implementation and maintenance of quality systems according to ISO 9001 or ISO 15189 as well as laboratory specialisation result in better analytical performance as can be seen in immunohaematology EQA results. The conclusion is that these results apply to other laboratory tests and perhaps to other areas of health care.

A new approach to define acceptance limits for hematology in external quality assessment schemes

Abstract: Background: A study performed in 2007 comparing the evaluation procedures used in European external quality assessment schemes (EQAS) for hemoglobin and leukocyte concentrations showed that acceptance criteria vary widely. For this reason, the Hematology working group from the European Organisation for External Quality Assurance Providers in Laboratory Medicine (EQALM) decided to perform a statistical study with the aim of establishing appropriate acceptance limits (AL) allowing harmonization between the evaluation procedures of European EQAS organizers. Methods: Eight EQAS organizers from seven European countries provided their hematology survey results from 2010 to 2012 for red blood cells (RBC), hemoglobin, hematocrit, mean corpuscular volume (MCV), white blood cells (WBC), platelets and reticulocytes. More than 440,000 data were collected. The relation between the absolute value of the relative differences between reported EQA results and their corresponding assigned value (U-scores) was modeled by means of an adaptation of Thompson’s “characteristic function”. Quantile regression was used to investigate the percentiles of the U-scores for each target concentration range. For deriving AL, focus was mainly on the upper percentiles (90th, 95th and 99th). Results: For RBC, hemoglobin, hematocrit and MCV, no relation was found between the U-scores and the target concentrations for any of the percentiles. For WBC, platelets and reticulocytes, a relation with the target concentrations was found and concentration-dependent ALs were determined. Conclusions: The approach enabled to determine state of the art-based ALs, that were concentration-dependent when necessary and usable by various EQA providers. It could also easily be applied to other domains.

Peer groups splitting in Croatian EQA scheme: a trade-off between homogeneity and sample size number

Abstract Background: Laboratory evaluation through external quality assessment (EQA) schemes is often performed as ‘peer group’ comparison under the assumption that matrix effects influence the comparisons between results of different methods, for analytes where no commutable materials with reference value assignment are available. With EQA schemes that are not large but have many available instruments and reagent options for same analyte, homogenous peer groups must be created with adequate number of results to enable satisfactory statistical evaluation. We proposed a multivariate analysis of variance (MANOVA)-based test to evaluate heterogeneity of peer groups within the Croatian EQA biochemistry scheme and identify groups where further splitting might improve laboratory evaluation. Methods: EQA biochemistry results were divided according to instruments used per analyte and the MANOVA test was used to verify statistically significant differences between subgroups. The number of samples was determined by sample size calculation ensuring a power of 90% and allowing the false flagging rate to increase not more than 5%. When statistically significant differences between subgroups were found, clear improvement of laboratory evaluation was assessed before splitting groups. Results: After evaluating 29 peer groups, we found strong evidence for further splitting of six groups. Overall improvement of 6% reported results were observed, with the percentage being as high as 27.4% for one particular method. Conclusions: Defining maximal allowable differences between subgroups based on flagging rate change, followed by sample size planning and MANOVA, identifies heterogeneous peer groups where further splitting improves laboratory evaluation and enables continuous monitoring for peer group heterogeneity within EQA schemes.