Faculty Bibliography

Study question: What do RNA seq-profiles tell us about gene activity in preimplantation embryos known to be normal vs. trisomic for chromosomes 21, 18, 15 and 22. Summary answer: RNA-Seq profiles are karyotype-dependent and appear to correlate with viability. X and Y transcription is surprisingly active at the preimplantation stage. What is known already: Embryonic karyotypic abnormalities are the most common cause of implantation failure and miscarriage. Some such embryos can progress to viability as evidenced by the birth of children with Trisomy 21, 18 and 15. An abnormal karyotype dictates developmental aberrations, and for these differences to affect phenotype, variations in gene expression must occur in the developing embryo. We set out to determine if such changes could be identified in embryos as early as the pre-implantation stage. Study design, size, duration: We used a cohort of 19 pre-implantation embryos (day 5 and 6 blastocysts); three being normal, five trisomy 15, two trisomy 22, three trisomy 21 and three trisomy 18, and three of unknown karyotype. Participants/materials, setting, methods: After written consent, analysis was performed on high quality embryos that previously underwent trophectoderm biopsy with array comparative genomic hybridization (aCGH) or next generation sequencing (NGS) for 24-chromosome aneuploidy screening and vitrified. Blastocysts were lysed immediately after thawing, and cDNA was synthesized and amplified, followed by RNA-Seq library preparation for deep Illuminabased sequencing. Sex and chromosomal aneuploidy were used as parameters for comparative analysis using a bioinformatics pipeline using a sensitivity of 0.05. Main results and the role of chance: Principal Component analysis (PCA) revealed that normal embryos clustered in proximity to trisomies 21 and 18, while 15 and 22 clustered separately. This suggested that early gene expression may correlate with viability. PCA did not distinguish between male and female embryos. Differential gene expression was calculated using DESEQ2, an R package that estimates the variance-mean dependence and tests for differential expression using a model using the negative binomial distribution. A comparison of sex-specific gene expression showed that the top differentially expressed genes were on the sex chromosomes, including various Y-linked transcription factors, helicases, and ribosomal proteins, as well as a testis-specific regulatory transcript, and >200 X-chromosome genes. Trisomy 21 embryos were the closest to normal embryos, with only 3 genes on chromosome 21 expressed more highly in the trisomic embryos. In contrast, trisomy 22 embryos (non viable), had 684 differentially expressed genes, 32 of which on chromosome 22. Trisomy 18 embryos had only one highly expressed significant gene, and it is located on chromosome 18. Trisomy 15 embryos had 829 differentially expressed genes, of which 83 were in chromosome 15. Our results suggest that the less viable trisomies have bigger gene expression differences, even at this very early stage. Limitations, reasons for caution: While the karyotypes were analyzed by stringent methods, embryos may have had elements of mosaicism or chromosomal structural abnormalities. The genes identified by RNA-Seq need to be validated using orthogonal methods. Wider implications of the findings: We have expanded the knowledge of the transcriptome of the human pre-implantation embryo as they relate to aneuploidy and sex. This information can now be used to further our understanding of early embryonic development and stem cell biology, and to identify biomarkers for non-invasive preimplantation genomic screening

BACKGROUND: Preimplantation genetic screening (PGS) affords couples the knowledge of embryo ploidy status prior to embryo transfer (ET). Many patients arrive at donor egg (DE) after multiple failed autologous in vitro fertilization (IVF) cycles, of which many may be due to aneuploidy. Our goal was to assess if knowledge of ploidy status decreases disposition time to DE and, ultimately, live birth (LB). OBJECTIVE: To determine if patient knowledge of embryo ploidy status through use of PGS using array comparative genomic hybridization (aCGH) changes disposition time DE enrollment at a large university-based fertility center. MATERIALSAND METHODS: Patients who enrolled in the DE program between 2011 and 2014 at the NYU Fertility Center that had a prior in vitro fertilization (IVF) cycle were identified. The number of IVF egg retrievals (ER) and ET with and without PGS performed before enrolling in DE were collected. The primary outcome was time in months from initial consultation visit to first DE transfer and to live birth. If the patient had a prior LB, the consultation visit was the first visit after the LB to discuss continued childbearing. Unpaired t-tests and chi-square were used for analysis with p< 0.05 defined as significance. RESULTS: A total of 110 patients had both IVF and DE cycles at NYUFC. There were 9 patients that underwent day 3 embryo biopsy and PGS with aCGH and 19 patients that had previously undergone trophectoderm biopsy and PGS with aCGH. Of these patients that did PGS, only 7/28 (25%) made at least one euploid embryo. Use of PGS did not decrease the number of IVF cycles, disposition time to DE, or time to DE LB. Prior parity and pregnancy rates were similar in both groups. CONCLUSIONS: One might expect that knowledge of embryo aneuploidy would affect disposition time to DE. This small retrospective cohort study shows no difference in disposition time to DE. However, given that trophectoderm biopsy with aCGH is a relatively new technology, it may be too early to assess the true impact on knowledge of ploidy status on disposition to DE (Figure Presented)

OBJECTIVE: To determine the relationship between blastocyst growth parameters and birth weight. DESIGN: Cohort study. SETTING: University-affiliated fertility center. PATIENT(S): In vitro patients who delivered a singleton after a single-blastocyst transfer. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Birth weight adjusted for gestational age at delivery and gender, with adjusted birth weight examined for association with blastocyst scores and grades. RESULT(S): After standard in vitro fertilization (IVF) and thawed embryo transfers, greater birth weight was associated with a higher inner cell mass grade. The grade of the trophectoderm and stage of the blastocyst did not relate to weight. CONCLUSION(S): Embryonic growth as early as day 5 can predict the progress of fetal development as measured by birth weight.