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.