Pre-Implantation Genetic Screening in Women Under the Age of 36 With Single-Embryo Transfer

Study design Between October 2004 and December 2006, ovulatory women undergoing infertility treatment were asked to participate in this RCT subject to the following inclusion criteria: maternal age below 36 years, need for ICSI and having motile sperm (sperm parameters: more than 1 x 106 sperm cells/ml) and both partners having a normal karyotype. Following written informed consent, eligible couples were randomly assigned to ICSI followed by blastocyst transfer either with or without PGS on the eight-cell stage embryo. Randomization was performed at the outpatient clinic, and each patient could be included only once. The consent form mentioned that patients allocated to PGS group would agree to donate embryos for research if these were chromosomally abnormal or unsuitable for transfer or cryopreservation. The study was approved by the Ethics Committee of the University Hospital. The patients were aware of the treatment allocation and were informed at the moment of transfer concerning the chromosomal abnormalities observed and about the morphological quality of the embryos.

Ovarian stimulation, oocyte retrieval and embryo culture Controlled ovarian hyperstimulation was carried out in an antagonist protocol using recombinant FSH combined with a GnRH antagonist (ganirelix, Orgalutran, NV Organon) (Kolibianakis et al., 2004) or an agonist protocol, using GnRH analogues for pituitary desensitisation (buserilin, Suprefact; Hoechst, Frankfurt, Germany), combined with human menopausal gonadotrophins (hMG)(Menopur, Ferring Pharmaceuticals A/S, Copenhagen, Denmark) or recombinant FSH (Puregon, NV Organon, Oss, The Netherlands)(Van de Velde et al., 1998). Final oocyte maturation was induced by the administration of 10,000 IU of HCG. Transvaginal ultrasound-guided oocyte retrieval was scheduled 36 h after HCG administration. ICSI was used to fertilize the oocytes. Sperm preparation, in vitro fertilization, intracytoplasmic sperm injection procedures and embryo culture were carried out as described by Van Landuyt et al. (2005).

Assessment of fertilization and embryo evaluation Fertilization was assessed as the number of 2PN zygotes per COC retrieved. Embryo evaluation was performed on days 2 and 3 by recording number of blastomeres and percentage of fragmentation. Grade A embryos were defined as embryos without anucleate fragments. Grade B embryos had blastomeres of equal or unequal sizes, with a maximum of 20 % of the volume of the embryo filled with anucleate fragments. In grade C embryos, anucleate fragments were present in 20-50 % of the volume of the embryo. Grade D embryos had more than 50% of the volume of the embryo filled with anucleate fragments. The embryos were evaluated daily until day 5. On day 5, the embryos were classified into arrested embryos showing no signs of compaction on day 5, compacting embryos (C1-C2), early blastocysts (Bl1-Bl2) and expanding blastocysts (Bl3-Bl7) according to the classification proposed by Gardner and Schoolcraft (1999). Embryos were considered for transfer at day 5 only from the compacting stage on. In both groups, supernumerary embryos were cryopreserved on day 5 or 6.

Embryo biopsy Embryos of grade A, B or C with at least five blastomeres were biopsied (Joris et al., 2003) in the morning of day 3 after ICSI. These selection criteria to decide whether an embryo was suitable for biopsy were the same as those used to decide whether an embryo was transferable on day 3 in the regular ICSI programme without PGD. Before biopsy of one blastomere, the blastomeres were checked for the presence of nuclei. If consequently no nuclear material was found during fixation, a second blastomere was removed. Embryo biopsy was carried out in HEPES-buffered medium under oil using laser technology (Fertilase) as was earlier described in detail (Staessen et al., 2004).

Spreading of the interphase nuclei and FISH procedure Using a mouth pipette, the individual blastomeres were first rinsed in medium and then transferred to a 1 µl droplet of 0.01 N HCl/ 0.1% Tween 20 solution (Coonen et al., 1994) on a Superfrost Plus glass slide (Kindler GmbH, Freiburg, Germany). A two-round FISH procedure was performed allowing us to detect the chromosomes X, Y, 13, 18, 21 (round 1) and 16, 22 (round 2) as was previously described in detail (Staessen et al., 2004).