Optimal timing for blastomere biopsy of 8-cell embryos for preimplantation genetic diagnosis.

STUDY QUESTION: What is the optimal timing for blastomere biopsy during the 8-cell stage, at which embryos will have the best implantation potential? SUMMARY ANSWER: Fast-cleaving embryos that are biopsied during the last quarter (Q4) of the 8-cell stage and are less affected by the biopsy procedure, and their implantation potential is better than that of embryos biopsied earlier during the 8-cell stage (Q1-Q3). WHAT IS KNOWN ALREADY: Blastomer biopsy from cleavage-stage embryos is usually performed on the morning of Day 3 when the embryos are at the 6- to 8-cell stage and is still the preferred biopsy method for preimplantation genetic diagnosis (PGD) for monogentic disorders or chromosomal translocations. Human embryos usually remain at the 8-cell stage for a relatively long 'arrest phase' in which cells grow, duplicate their DNA and synthesize various proteins in preparation for the subsequent division. STUDY DESIGN, SIZE, DURATION: This is a retrospective cohort study. The study group (195 embryos) included all 8-cell stage embryos that underwent blastomere biopsy for PGD for monogenetic disorders and chromosomal translocations in our unit between 2012-2014 and cultured in the EmbryoScope until transfer. The control group (115 embryos) included all embryos that underwent intracytoplasmic sperm injection without a biopsy during the same period. PARTICIPANTS/MATERIALS, SETTING, METHODS: The 8-cell stage was divided into four quarters: the first 5 h post-t8 (Q1), 5-10 h post-t8 (Q2), 10-15 h post-t8 (Q3) and at 15-20 h post-t8 (Q4). Non-biopsied control embryos were divided into four equivalent quarters. Embryos were evaluated for timing of developmental events following biopsy including timing of first cleavge after biopsy, timing of comapction (tM) and start of blastulation (tSB). Timing of these events were compared between PGD and control embryos, as well as with 56 PGD implanted embryos with Known Implantation Data (PGD-KID-positive embryos). MAIN RESULTS AND THE ROLE OF CHANCE: Embryos that were biopsied during Q3 (10-15 h from entry into 8-cell stage) were delayed in all three subsequent developmental events, including first cleavage after biopsy, compaction and start of blastulation. In contrast, these events occurred exactly at the same time as in the control group, in embryos that were biopsied during Q1, Q2 or Q4 of the 8-cell stage. The results show also that embryos that were biopsied during Q1, Q2 or Q3 of the 8-cell stage demonstrated a significant delay from the biopsied implanted embryos already in t8 as well as in tM and tSB. However, embryos that were biopsied during Q4 demonstrated dynamics similar to those of the biopsied implanted embryos in t8 and tM, and a delay was noticed only in the last stage of tSB. LIMITATIONS, REASONS FOR CAUTION: This is a retrospective study that is limited to the timing of biopsy that is routinely performed in the IVF lab. A prospective study in which biopsy will be performed at a desired timing is needed in order to differ between the effect of biopsy itself and the cleavage rate of the embryo. WIDER IMPLICATIONS OF THE FINDINGS: Our findings showed that blastomere biopsy can be less harmful to further development if it is carried out during a critical period of embryonic growth, i.e during Q4 of the 8-cell stage. They also demonstrated the added value of time-lapse microscopy for determining the optimal timing for blastomere biopsy. STUDY FUNDING/COMPETING INTEREST(S): The study was funded by the routine budget of our IVF unit. TRIAL REGISTRATION NUMBER: N/A.

Human embryonic stem cells carrying an unbalanced translocation demonstrate impaired differentiation into trophoblasts: an in vitro model of human implantation failure.

Carriers of the balanced translocation t(11;22), the most common reciprocal translocation in humans, are at high risk of creating gametes with unbalanced translocation, leading to repeated miscarriages. Current research models for studying translocated embryos and the biological basis for their implantation failure are limited. The aim of this study was to elucidate whether human embryonic stem cells (hESCs) carrying the unbalanced chromosomal translocation t(11;22) can provide an explanation for repeated miscarriages of unbalanced translocated embryos. Fluorescent in situ hybridization and karyotype analysis were performed to analyze the t(11;22) in embryos during PGD and in the derived hESC line. The hESC line was characterized by RT-PCR and FACS analysis for pluripotent markers. Directed differentiation to trophoblasts was carried out by bone morphogenetic protein 4 (BMP4). Trophoblast development was analyzed by measuring ß-hCG secretion, by ß-hCG immunostaining and by gene expression of trophoblastic markers. We derived the first hESC line carrying unbalanced t(11;22), which showed the typical morphological and molecular characteristics of a hESC line. Control hESCs differentiated into trophoblasts secreted increasing levels of ß-hCG and concomitantly expressed the trophoblast genes, CDX2, TP63, KRT7, ERVW1, CGA, GCM1, KLF4 and PPARG. In contrast, differentiated translocated hESCs displayed reduced and delayed secretion of ß-hCG concomitant with impaired expression of the trophoblastic genes. The reduced activation of trophoblastic genes may be responsible for the impaired trophoblastic differentiation in t(11;22)-hESCs, associated with implantation failure in unbalanced t(11;22) embryos. Our t(11;22) hESCs are presented as a valuable human model for studying the mechanisms underlying implantation failure.

Nuclear membrane protein LAP2beta mediates transcriptional repression alone and together with its binding partner GCL (germ-cell-less).

LAP2beta is an integral membrane protein of the nuclear envelope involved in chromatin and nuclear architecture. Using the yeast two-hybrid system, we have cloned a novel LAP2beta-binding protein, mGCL, which contains a BTB/POZ domain and is the mouse homologue of the Drosophila germ-cell-less (GCL) protein. In Drosophila embryos, GCL was shown to be essential for germ cell formation and was localized to the nuclear envelope. Here, we show that, in mammalian cells, GCL is co-localized with LAP2beta to the nuclear envelope. Nuclear fractionation studies reveal that mGCL acts as a nuclear matrix component and not as an integral protein of the nuclear envelope. Recently, mGCL was found to interact with the DP3alpha component of the E2F transcription factor. This interaction reduced the transcriptional activity of the E2F-DP heterodimer, probably by anchoring the complex to the nuclear envelope. We demonstrate here that LAP2beta is also capable of reducing the transcriptional activity of the E2F-DP complex and that it is more potent than mGCL in doing so. Co-expression of both LAP2beta and mGCL with the E2F-DP complex resulted in a reduced transcriptional activity equal to that exerted by the pRb protein.

Expression analysis using DNA microarrays demonstrates that E2F-1 up-regulates expression of DNA replication genes including replication protein A2.

The transcription factor E2F-1 plays a pivotal role in the regulation of G1/S transition in higher eukaryotes cell cycle. We used a cell line containing an inducible E2F-1 and oligonucleotide microarray analysis to identify novel E2F target genes. We show that E2F-1 up-regulates the expression of a number of genes coding for components of the DNA replication machinery. Among them is the gene coding for the 32 Kd subunit of replication protein A (RPA2). Replication protein A is the most abundant single strand DNA binding complex and it is essential for DNA replication. We demonstrate that RPA2 is a novel E2F target gene whose expression can be directly regulated by E2F-1 via E2F binding sites in its promoter. In addition, expression of Topoisomerase IIalpha and subunit IV of DNA polymerase alpha is also up-regulated upon E2F-1 induction. Taken together, these results provide novel links between components of the DNA replication machinery and the cell growth regulatory pathway involving the Rb tumor suppressor and E2F.