Objectives: Removal of a single blastomere for preimplantation genetic diagnosis (PGD) from day-3 embryo is a well established method. However, it is unknown whether the method of choice for zona pellucida (ZP) drilling for the biopsy procedure will influence further embryonic development. The most commonly applied technique for perforation of the ZP is zona drilling using acidified Tyrode's solution (AT), while only a few centers employ laser-assisted hatching (LAH). Because of the lack of studies comparing the two methods, many centers are reluctant to switch to the simpler and faster laser drilling. Thus, in the present study we assessed two zona drilling methods in terms of blastocyst development rates using sister embryos. Design: Prospective, randomized study. Sister embryos of 14 patients were randomly assigned to (1) AT zona drilling or to (2) laser zona drilling on day 3. After biopsy, subsequent embryo culture until the blastocyst stage (day 5) was performed. Materials and Methods: A total of 14 IVF-PGD cycles were included into the study that was performed between January and March of 2003. Patients underwent controlled ovarian hyperstimulation using recombinant FSH after pituitary down-regulation with long leuprolide acetate protocol. Embryos from the same patients (eligible for biopsy: >5 cells and <30% fragmentation) were randomly divided into two groups on day 3. In Group 1 embryos were submitted to Acidified Tyrode's zona drilling and in Group 2 embryos underwent laser-assisted hatching using a 1.48 μm diode laser (Octax). In both groups an opening of the zona of 15 to 20 micro-meter was formed followed by the removal of a single blastomere. Embryos were cultured sequentially in Sage cleavage and blastocyst medium until day 5. For statistical analysis One-way ANOVA, Kruskal-Wallis and chi-square tests were applied whenever appropriate. Results: The mean age (±S.D.) of the women in this study was 36.9 (±3.9). A total of 285 oocytes were recovered, and 175 oocytes fertilized normally (61%). There was an equal distribution of embryos in both groups based on cell stage, and percent fragmentation. Fourteen patients achieved pregnancy (50%) after transferring 30 blastocysts. The results are summarized in Table 1. Tabled 1 Conclusions: Blastocyst development rates (and blastocyst quality) were similarly high in both the AT group and in LAH group, indicating that laser hatching does not impair embryonic development to the blastocyst stage. Implantation rates were not compared because patients often had a combination of AT and LAH embryos used for transfer. Given that an equal number of LAH and AT embryos were transferred resulting in a 50% pregnancy rate, the data suggests that zona drilling by laser micro-beam does not produce additional risks for embryonic development beyond the blastocyst stage. Additionally, the results of the present study demonstrate that LAH is a suitable alternative to the use of acidified Tyrode's solution for zona drilling.

Objective: Historically, embryo transfers following embryo biopsy and preimplantation diagnosis (PGD) were performed on day 3 or 4. With the development of sequential media and blastocyst transfer, patients undergoing PGD now have the option of a day 4 or day 5 transfer. The objective of this study was to compare the pregnancy and implantation rates between patients who received a day 4 or day 5 embryo transfer following PGD for aneuploidy Design: Retrospective Analysis Materials/Methods: Embryo biopsy was performed in Ca2+/Mg2+ -free medium on day 3 and a diagnosis was determined using fluorescent in situ hybridization (FISH) for chromosomes X,Y,13,18 and 21. On the morning of day 4 all biopsied embryos were transferred into blastocyst culture medium. Patients who did not want their embryos cultured until day 5 had an embryo transfer on day 4. Embryos which were not transferred remained in culture until day 6. Results: Blastocyst transfer was offered to 50 patients post preimplantation genetic diagnosis. Twenty-two patients chose to have a day 5 transfer, while the remaining 40 requested to have a day 4 transfer. In the blastocyst transfer group 56% (13/23) of the patients had a positive pregnancy test of which 52% (12/23) are ongoing. The average age in this group was 38 and the implantation rate was 27% (16/59). Patients who received a day 4 embryo transfer had a 33.3%% (9/27) pregnancy rate of which 26% (7/27) are ongoing. The average age in this group was 39 and the implantation rate was 12% (8/66). Conclusions: The removal of a cell or cells from a day 3 embryo for the purpose of PGD does not preclude the subsequent in vitro development of that embryo to the blastocyst stage. The transfer of blastocysts instead of day 4 embryos post PGD may allow the embryo to better withstand the trauma of embryo transfer procedure. In this study blastocysts replaced to the uterus had a higher implantation rate than embryos transferred on day 4. Culture to the blastocyst stage may allow for selection of embryos for ET when multiple chromosomally normal embryos are available, or allow extra time for performing the genetic diagnosis or rechecking results. Many smaller IVF centers cannot justify the cost of equipment needed for PGD, though a micromanipulator is available. Cells removed from embryos can be shipped to a reference laboratory for analysis, allowing 48 hours for reporting results before embryo transfer.