Balkan Journal of Medical Genetics

EMBRYO QUALITY PREDICTIVE MODELS BASED ON CUMULUS CELLS GENE EXPRESSION
Devjak R, Burnik Papler T, Verdenik I, Fon Tacer K, Vrtačnik Bokal E
*Corresponding Author: Rok Devjak, M.D., Ph.D., Division of Medical Oncology, Institute of Oncology, Zaloška 2, 1000 Ljubljana, Slovenia. Tel: +386-1-5879-282; Fax: +386-1-5879-303. E-mail: rdevjak@onko-i.si
page: 5

MATERIALS AND METHODS

Patients and In Vitro Fertilization Treatment. In this study, 17 patients undergoing the classical IVF cycle at the Department of Obstetrics and Gynecology, University Medical Centre, Ljubljana, Slovenia, were included. The study was approved by the Republic of Slovenia National Medical Ethics Committee (http://www.kme-nmec. si/) and patients signed a written consent form prior to study inclusion. It included patients who were less than 35 years old and with body mass index (BMI) between 17 and 26 kg/m2. They attended the IVF program because of tubal factor infertility. The spermiograms of their partners were normal, according to the World Health Organization (WHO) criteria. As our previous study did not expose any differences in CC gene expression between patients who were treated with either GnRH agonists or antagonists in combination with recombinant follicle-stimulating hormone (rFSH) [10], we used both GnRH analogs in the present study. Ten patients were administered GnRH agonist buserelin acetate (Suprefact; Hoechst AG, Frankfurt/Main, Germany) starting from day 22 with a daily dose of 0.6 ml (600 pg) subcutaneously. When criteria for ovarian desensitization were fulfilled (eastradiol <0.05 nmol/L, follicles <5 mm in diameter), patients were subcutaneously administered 225 IU of gonadotropin folitropin α (Gonal F; Industria Farmaceutica Serono S.p.A, Bari, Italy). The other seven patients received 225 IU of gonadotropin folitropin α, subcutaneously administered on day 2. When the dominant follicle measured >14 mm in diameter, the GnRH antagonist cetrorelix acetate (Cetrotide; Asta Medica AG, Frankfurt, Germany) in a dose of 0.25 mg, was administered subcutaneously. Afterwards, all patients received 10,000 IU of the human chorionic gonadotropin (hCG) (Pregnyl; N.V. Organon, Oss, the Netherlands) when at least three follicles were >17 mm and serum oestradiol was >0.40 nmol/L per follicle; 34-36 hours later, ultrasound-guided transvaginal oocyte retrieval was performed. Cumulus Cells Collection and Oocyte Follow- Up. Oocytes were removed from the follicular fluid. Immediately after oocyte retrieval, a small sample of CC of each oocyte was removed using a needle and a glass denudation pipette (Swemed, Göteborg, Sweden). Oocytes were not denuded by this technique. Obtained CC samples were washed in phosphatebuffered saline (PBS), snap frozen in liquid nitrogen and stored at –80 °C in vials until RNA isolation. The oocytes were further inseminated (classical IVF) and cultivated individually. After 24 hours, oocyte fertilization status was assessed. Fertilized oocytes were further cultured to the blastocyst stage in the Universal IVF Medium followed by the BlastAssist System (M1 and M2; Origio, Målov, Denmark) for 5 days. On day 5, at most two embryos at the blastocyst or morula stage were transferred into the uterus. Supernumerary blastocysts were cryopreserved. Experimental Design. The models were built on the CC expression level values of five genes (AMHR2, LIF, SERPiNE2, VEGFC and FSHR) of two kinds of embryos: high quality embryos (n = 26), represented by morula and blastocyst stage embryos on day 5, and low quality embryos (n = 36), represented by embryos which arrested in development any time within 5 days of cultivation after fertilization. The decision trees probabilistic model was used to estimate sensitivity, specificity and area under the curve (AUC) of a proposed model. Quantitative Real Time Polymerase Chain Reaction Analysis. Quantitative real time PCR (qPCR) was used for CC gene expression using TaqMan Gene Expression pre designed assays (Applied Biosystems, Foster City, CA, USA). Peptidylprolyl isomerase B (PPIB) and 18s rRNA were added for normalization. Genomic DNA contamination was eliminated by DNAse treatment using DNAse I (F. Hoffmann-La Roche Ltd., Basel, Switzerland). cDNA for qPCR assays was prepared from 200 ng DNAsed RNA using SuperScript RT III (Invitrogen, Carlsbad, CA, USA) in a final volume of 20 μL. Following cDNA synthesis, RNAse-free water was added to increase the sample volume to 30 μL. Measurements were performed using a LightCycler 480 System (Roche Applied Science, Penzberg, Germany). Normalized mRNA levels were obtained by dividing the averaged, efficiency corrected values for mRNA expression by a normalization factor calculated from peptidylprolyl isomerase B (PPIB) and 18s rRNA values and are expressed in arbitrary units. The resulting values were log2 transformed (log2-fold change) for comparison with microarray data. Statistical Analysis and Decision Tree Model Construction. A Mann-Whitney U test and logistic regression model were performed with the Statistical Package for the Social Sciences (SPSS) version 17.0 (SPSS Inc., Chicago, IL, USA). For the decision tree model construction Orange® (www.orange.biolab.si) was used. Orange® is a data mining tool that works through preprogrammed widgets. Designing and testing decision trees is one of its functions.

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