Study design and patients
This study was reviewed and approved by the Ethics Committee at The Seventh Medical Center of PLA General Hospital (Research License 2021-31). All embryos were obtained from donors at the Center of Reproductive Medicine in The Seventh Medical Center of PLA General Hospital. Recruitment took place from April 2005 to February 2015.
Written informed consent was provided before donating their surplus frozen embryos for research. The donors were financially compensated for the effort, time and inconvenience related to the donation process. A total of 162 embryos donated from 52 couples with normal karyotypes were used in this study. All of the women with normal ovaries, uterus, and regular menstrual cycles had normal basal FSH levels (< 10 mIU/mL on day 3 of the menstrual cycle). We performed preimplantation genetic screening (PGS) for chromosome aneuploidy analysis in embryos derived from in vivo (IVF group) and in vitro matured human (IVM group) oocytes.
Controlled ovarian hyperstimulation (COH) in IVF group
For patients with COH cycle, the treatment was initiated from a baseline transvaginal ultrasound scan on day 3 of the menstrual cycle to ensure that there were more than seven small antral follicles in both ovaries. Ovary stimulation was carried out with exogenous gonadotropins after a desensitization protocol with GnRH analogues according to a long or a short protocol, depending on the patient’s previous history of gonadotropin response and other factors. An ultrasound scan was performed on day 7–9 and subsequently until one follicle reached 18 mm and two reached 16 mm in diameter, and then 10,000 IU human chorionic gonadotropin (hCG) was administered. Oocyte retrieval was performed approximately 36 h after hCG administration.
Follicle retrieval and in-vitro maturation (IVM)
IVM is routinely performed in our center for patients who had normal ovaries with > 7 antral follicles in both ovaries. Briefly, the treatment was initiated from a baseline transvaginal ultrasound scan on day 3 of the menstrual cycle to ensure that there were more than seven small antral follicles in both ovaries. On day 7–9 ultrasound scans were repeated. When the dominant follicle reached 12–14 mm in diameter and/or the endometrial thickness was ≥ 6 mm, hCG (10,000 IU) was administered and oocyte retrieval was performed 36 h later.
The oocyte retrieval was performed using transvaginal ultrasound-guided aspiration was performed with a 17-gauge double-lumen needle (Cook, Eight Mile Plains, Queensland, Australia) for aspiration of the leading follicles. A 19-gauge single-lumen needle (Cook) for the small follicles. For aspiration, a portable pump was connected to the needle with a pressure of < 100 mm Hg for leading follicles and < 40 mm Hg for small follicles. After assessing the nuclear maturity of the retrieved oocytes under the dissecting microscope as the commonly used method [7]. The collected matured oocytes were inseminated 2 or 3 h later by intracytoplasmic sperm injection (ICSI), while the collected immature oocytes were cultured in IVM medium.
The immature oocytes at Metaphase-I (MI) and GV stage were cultured in a 1 mL maturation medium containing 30% serum of the patient’s own (inactivated at 56° for 30 min) with 75 mIU/mL human FSH ((Gonal-F; Merck Serono, Switzerland), 75 mIU/ml human menopausal gonadotrophin (hMG, Lizhu Pharmacy) and 10 ng/ml recombinant human epidermal growth factor (Invitrogen, Carlsbad, CA, USA) to induce final oocyte maturation at 37 °C in 5% CO2, 5% O2 and 90% N2 with high humidity for 24 or 48 h. Then the oocytes matured in vitro were also inseminated by ICSI [8].
Embryo culture
All embryos were fertilized using ICSI. The zygotes were cultured in individual 20-µL droplets of G1-PLUS medium (Vitrolife, Gothenburg, Sweden) overlaid with 2.5-mL mineral oil (Vitrolife, Gothenburg, Sweden) in a 30-mm Falcon culture dish and incubated at 37 °C in an atmosphere containing 5% O2 and 6.0% CO2. On day 3 or day 4 oocyte retrieval, the embryos were vitrification for the further genetic screen.
Whole-genome amplification and DNA sequencing
After thawing, whole embryo (WE) samples were transferred into RNase- and DNase-free PCR tubes containing 5 μL cell lysis buffer (Yikon Genomics, China), and frozen immediately in liquid nitrogen stored at − 80 °C until further processing.
DNA for whole-genome amplification was amplified with the multiple annealing and looping based amplification cycles (MALBAC) technique and library generation (Cat No. YK001B, Yikon Genomics) as previously described [9, 10]. Amplification products were sequenced on an Illumina HiSeq 2500 platform (Illumina, San Diego, CA, USA) with approximately two million sequencing reads per sample. The read numbers were counted along the whole genome with a bin size of 1 Mb and normalized based on GC content and a reference dataset. The number of reading counts served as the index of ploidy: a 50% increase indicates an increase in the number of chromosomes from 2 to 3, whereas a 50% decrease indicates a reduction in the number of chromosomes from 2 to 1 [11, 12].
Statistical analysis
Continuous data were reported based on mean ± standard deviations. Data analysis was performed using SPSS (version 20). The frequency between the groups were assessed using Fisher’s exact test. Differences were considered significant at P < 0.05.
