Εξέλιξη και βελτίωση της τεχνικής βιοψίας βλαστοκύστεων για μοριακές αναλύσεις και εκτίμηση της επιτυχίας εμφύτευσης και εγκυμοσύνης κατά την εξωσωματική γονιμοποίηση σε σχέση με το μοριακό υπόστρωμα

Abstract

IntroductionOne of the most difficult aspects in assisted reproductive technology (ART) is the selection of asuitable embryo for transfer to the patient’s uterus, in order to achieve implantation anddevelopment to term. This study was based on the hypothesis that preimplantation embryosmay have different gene expression profiles that characterize their ability to implant in theuterus and develop to a healthy baby at term.The main aim of this study was to investigate molecular markers associated with developmentalcompetence and successful implantation in ART. The primary aim of the study was to developand optimize a blastocyst biopsy method, suitable for application in clinical practice. Thesecondary aim of the study was to investigate the gene expression of beta Human ChorionicGonadotropin (CGβ) in blastocysts and correlate it with their morphology. Previously to thecurrent study, blastocyst biopsy was not implemented in clinical practice and no prior researchon the existence, quantification and standardization of transcripts of CGβ has been performedin blastocysts.MethodologyThe methodology for trophectoderm cell biopsy from blastocysts was developed and optimizedprimary to be a safe technique for the embryo and secondary to ensure biopsy of a sufficientnumber of cells, in order to allow the application of multiple molecular analyses. The blastocystbiopsy method involved three steps: A., opening of a hole in the zona pellucida using lowfrequency laser, B., blastocyst culture to allow trophectoderm cells to herniate from the holeand C., trophectoderm cell dissection of the blastocyst mass by laser ablation.The methodology for the investigation of CGβ gene expression in blastocysts, included RNAisolation, cDNA synthesis, amplification and quantification of CGβ transcripts. Because CGβ isencoded by a cluster of homologous genes (CGβ1, CGβ2, CGβ3, CGβ5, CGβ7, CGβ8),methodology was designed considering the homology between them into groups (A: CGβ1,CGβ2 and B: CGβ3, CGβ5, CGβ7, CGβ8). For group A, real time polymerase chain reaction (RealTime PCR, RT-PCR) was applied and then transcripts were identified using restriction enzymedigestion. For group B, nested polymerase chain reaction (Nested-PCR) was used incombination with polymerase chain reaction temperature decreasing hybridization (Touch-downPCR). Following amplification, the products were sequenced (DNA sequencing) for theiridentification.ResultsThe biopsy technique did not appear to impact on the blastocyst’s ability to reform a blastocoelecavity and continue to grow and hatch from the zona pellucida, as it was shown followingfurther in vitro culture. No blastocyst showed signs of morphological damage at the lightmicroscopic level. Blastocyst biopsy was applied in clinical practice in two steps: A., 49 couples undergoing IVF had a biopsy in 153 blastocysts. The implantation rate per blastocysttransferred was 34.3% and lead to 23 full-term pregnancies (46.9%) with 37 babies born. B.,24 couples undergoing IVF for PGD of monogenic diseases had biopsy in 144 blastocysts. Thediagnosis success rate was 93%, the implantation rate per blastocyst transferred was 40% andlead to 11 full-term pregnancies (50%) with 15 term newborns. Then, a randomized pilot studywas conducted with the aim to evaluate and compare the diagnosis and implantation successrates between patients undergoing blastomere biopsy and blastocyst transfer and those havingtrophectoderm biopsy and blastocyst transfer for the diagnosis of monogenic diseases. Theresults showed that the diagnosis success rate was superior in the blastocyst biopsy group,while implantation and pregnancy rates were not statistically significant between the twogroups.For the study of CGβ expression profiles 45 blastocysts were donated to research, of which 39generated trophectoderm cells cDNA libraries. RT-PCR revealed the presence of CGB3, CGB5,CGB7, CGB8 transcripts in 5 blastocysts. The transcripts CGB5, CGB7, CGB8 were expressed inone hatched and one hatching blastocysts (fair morphology on day 7 post insemination) and thetranscript CGβ3 was expressed in three hatched blastocysts (excellent morphology on day 5/6post insemination). The transcript CGβ1 was identified in one only blastocyst. Four blastocystswere biopsied in order to investigate whether CGβ expression can be detected at the minimallevel of few trophectoderm cells. No transcript was found in trophectoderm cell samples orbiopsied blastocyst proper.DiscussionIn recent years, many new technologies have been introduced in clinical practice of ART.Blastocyst biopsy since its first announcement in 2005, until today, has been adopted andintegrated into the application of preimplantation genetic diagnosis (Kokkali et al., 2005). Asblastocyst biopsy has the advantage of providing adequate number of cells for multipleanalyses, it has been lately used for the PGD for monogenic diseases in combination withhistocompatibility screening (HLA matching) or PGD for monogenic diseases screening forstructural or numerical chromosomal abnormalities. Besides its clinical application, blastocystbiopsy offers great opportunities for research, such as the study for the expression ofpreimplantation genetic profiles for the identification of the single most viable blastocyst amongthe cohort developing in vitro that will enable single blastocyst transfers without a concomitantreduction in pregnancy rates.In this study, we investigated whether the β HCG may be used as a predictive marker ofdevelopmental competence for human embryos. This study showed that CGβ gene expressionwas diverse and heterogeneous between blastocysts. Further studies need to be accomplishedto investigate this further.ConclusionsBlastocyst biopsy was developed and optimized to serve as powerful tool for diagnostics ofhuman diseases or to identify diagnostic markers of competence to develop to term for humanembryos.