Treatment protocol and embryo quality assessment
In stimulation cycles, all patients were treated with agonist or antagonist protocol with the use of FSH or hMG as previously described12. The initial and ongoing dosage was adjusted according to the patient’s age, antral follicle count (AFC), BMI, and ovarian response. Ovarian response was monitored by means of transvaginal ultrasound measurements of follicular growth and serum E2 level every 1-3 days. When at least one follicle reached a mean diameter of 18 mm as determined by ultrasound, hCG was administrated. Oocyte retrieval was scheduled for 34-36 hours after hCG injection and was carried out under transvaginal ultrasound guidance.
Conventional IVF or ICSI was carried out depending on semen parameters and previous fertilization histories. In IVF cycles, cumulus-oocyte complexes were inseminated with approximate 1.5-3 X 105 progressively motile spermatozoa in fertilization culture medium (K-SIFM, Cook) for 4 h. Oocytes for ICSI were denuded 2h after ovum pickup, and sperm microinjection was performed 4 h after retrieval. Fertilization was checked about 17h post insemination/injection and was determined by the presence of two pronuclei (2PN).
All embryos were cultured under mineral oil in traditional incubators (C200, Labotect) at 37℃, 6% CO2, 5% O2. Cook IVF media (Cook Medical) was used for cleavage-stage embryos (K-SICM) and blastocysts (K-SIBM) culture in the form of microdroplet of 20 ul. On day 3, evaluation of embryo quality included the number of blastomere, the degrees of fragmentation and the uniformity of blastomeres. Cleavages were determined for fresh embryo transfer or blastocyst culture, and then were placed in blastocyst culture medium(K-SIBM). The quality of blastocysts were evaluated on Day 5 or Day 6 based on the Gardner and Schoolcraft grading system, and the score was dependent on blastocyst expansion, inner cell mass (ICM) development and trophectoderm (TE) appearance 13. Good quality embryos were blastocysts graded as AA, AB, BA and BB with expansion grade ≥3, while poor quality embryos were those defined as AC, CA, BC, CB and CC with expansion grade ≥3. In addition, Top quality embryo (TQE)were blastocysts graded as AA, AB and BA with expansion grade ≥4. Blastocysts were determined to be transferred on day 5 or vitrified for subsequent transfer. Blastocysts with poor morphological score (≤4CC) or low expansion grade (grades 1-2) were not considered for vitrification or transfer.
Vitrification and thawing
For vitrification, the Cryotop method was carried out as described by Kuwayama 14. Briefly, blastocysts were equilibrated for 3-5 min in equilibration solution (ES:7.5% dimethyl sulfoxide and 7.5% ethylene glycol), and were then placed into in vitrification solution (VS:15% dimethyl sulfoxide, 15% ethylene glycol, 10 mg/mL Ficoll-70, and 0.6 M sucrose). After 30-40 s in VS, embryos were transferred on the Cryotop strip and plunged into liquid nitrogen immediately. For thawing, blastocysts were directly immersed into thawing solution (TS) containing 1 M sucrose at 37℃ for 1 min, then was sequentially incubated in each of the following solutions for 3 minutes: 0.5 M sucrose, 0.25 M sucrose and sucrose-free TS. Then the embryos were placed in blastocyst culture medium (K-SIBM, Cook) and cultured in an incubator at 37℃ with 6% CO2 until transfer. Survival of thawed embryos were assessed under an inverted microscope depending on whether blastocysts showed a severely damaged cellular content or not.