Embryo culture and transfer

The zygote was rinsed with fresh G1 culture medium (Vitrolife; Gothenburg, Sweden), and then transferred to G1 medium droplets one by one. Fertilization was observed one day after embryo culture under a microscope at a magnification of ×400. Embryos containing double pronuclei and two polar bodies in the perivitelline space were identified as normally fertilized embryos, while zygotes with a single pronucleus, three or more pronuclei, or without a pronucleus were defined as abnormal fertilization or infertilization. Embryo development was observed 2 and 3 days post-culture and embryos were graded as following. Cleavage-stage embryos were graded with a 1 to 4 scoring system, based on speed of embryo development, fragmentation, cell symmetry, and number of blastomere, and Grade 1 and Grade 2 cleavage-stage embryos were identified as “top quality” embryos, while Grade 1 to 3 cleavage-stage embryos were defined as transferrable/frozen embryos. Two embryos with the highest scores were transferred to the embryo glue, and the remaining transferable embryos were transferred to G2 blastocyst culture medium (Vitrolife; Gothenburg, Sweden) for a further incubation until blastocyst formation. After embryo culture for 5 to 6 days, if blastocysts were produced, top-quality blastocysts were screened using a 1 to 6 scoring system based on the degree of expansion and hatching status of each blastocyst, with Grade 3 to 6 blastocysts graded according to the inner cell mass (ICM) and trophectoderm (TE), and cryopreserved by vitrification.

Embryo transfer was performed 72 h after egg retrieval, guided by transvaginal B-mode ultrasonography. For women aged less than 35 years, 2 embryos were transferred at the first cycle and 3 embryos were transferred at the second and higher cycle, while 3 embryos were transferred for those at ages of over 35 years, as recommended by the Ministry of Health, People’s Republic of China. Following embryo transfer, the women were stayed in bed for 0.5 h. At the day of egg retrieval, progesterone was administered by intramuscular injection at a daily dose of 60 to 100 mg for luteal phase support.

Urine hCG assay was performed 14 days or serum hCG levels were measured 14 to 16 days after embryo transfer to identify pregnancy. Luteal phase support was continued to 30 days in women positive for pregnancy testing, and the detection of gestational sac and fetal heartbeat by B-mode ultrasonography was considered successful clinical pregnancy. All pregnant women were followed up, and 6 to 7 weeks of gestation was defined as clinical pregnancy, and 10 to 12 weeks of gestation as ongoing pregnancy.