Study design and participants
A retrospective study of infertile women with normal ovarian reserve attending the Assisted Reproduction clinic, Shanghai First Maternity and Infant Hospital for IVF from January 2017 to December 2019 was undertaken, and each patient was followed for 18 months from the day of the ovarian stimulation. Ethical approval was not required for the retrospective analysis.
Women were included if they fulfilled the following inclusion criteria: (i) less than 40 years of age; (ii) having indications for IVF; (iii) regular menstrual cycles over the previous 3-month period (25 - 35 days in duration); (iv) antral follicle count (AFC) of more than 5 on menstrual cycle day 2 - 3, and basal serum FSH concentration of no more than 10 IU/L. Women were excluded if they had: (i) diagnosis of polycystic ovarian syndrome, (ii) an abnormal uterine cavity shown on hysterosalpingogram or hysteroscopy, (iii) moderate or severe endometriosis, (iv) use of donor eggs/sperm, (v) preimplantation genetic testing, (vi) rescue intracytoplasmic sperm injection (ICSI) or half ICSI, (vii) still having cryopreserved embryos but continuing to the next fresh IVF cycle.
Women were offered either progestin-primed ovarian stimulation protocol (PPOS group) or agonist long protocol (agonist group) at the discretion of the attending physicians or subject to the wishes of the couple.
Women started their IVF with ovarian stimulation using either PPOS or long agonist protocols. For the long agonist protocol, gonadotropin-releasing hormone analogue (GnRHa) (1.88mg Triptorelin acetate, Diphereline, Ipsen Pharma Biotech, France) was given for pituitary desensitisation from the mid-luteal phase in the previous cycle. On Day 2–3 of the menstrual cycle, they underwent transvaginal ultrasound examination and serum oestradiol measurement. Human menopausal gonadotrophin (hMG) (Lebaode, Lizhu, china) or recombinant FSH (Puregon, Organon, Dublin, Ireland or Gonal F, Merck Serono S.p.A, Modugno, Italy) was given at 150–225 IU per day based on the antral follicle count (AFC), age of women and previous ovarian response, according to the standard operation procedures of the centre. For the PPOS protocol, Medroxyprogesterone MPA (MPA, 10 mg/d, Shanghai Xinyi Pharmaceutical Co., China) was also given from day of the ovarian stimulation until the day of ovulation trigger. Ovarian response was monitored by serial transvaginal scanning with or without hormonal monitoring. Further dosage adjustments were based on the ovarian response at the discretion of the clinicians in charge.
When three leading follicles reached ≥18 mm in diameter, Ovidrel 250 microgram (Merck Serono S.p.A., Modugno, Italy) or triptorelin (0.1 mg; Decapeptyl, Ferring Pharmaceuticals, Netherlands) and hCG (2000 IU; Lizhu Pharmaceutical Trading Co., China) were given to trigger final maturation of oocytes. Oocyte retrieval was performed around 36 hours later.
Fertilization and embryo evaluation
Semen samples were prepared by the swim-up procedure. About 2 hours after oocyte retrieval, each oocyte was inseminated with approximately 20,000–30,000 motile spermatozoa. If the total number of motile sperm was <105 after washing or normal morphology was <1%, intracytoplasmic sperm injection (ICSI) was performed. Oocytes were decoronated and checked for the presence of two pronuclei to confirm fertilization. Embryos were graded on day 3 after retrieval as grade one to grade six according to the evenness of each blastomere and the percentage of fragmentation. Embryos of 6-8 cells and of grade one or two were regarded as top quality embryos. Some non-top-quality embryos were placed in extended culture until they reached the blastocyst stage.
Fresh embryo transfer
In the long agonist protocol, a maximum of two embryos was replaced on Day 3 after retrieval under transabdominal ultrasound guidance. Luteal phase support was given by vaginal or intramuscular progesterone at the discretion of the attending physicians. A pregnancy test was carried out 2 weeks after the transfer. All who had a positive pregnancy test had a transvaginal ultrasound scan 2 weeks after the positive pregnancy test (4 weeks after embryo transfer) to identify the presence of a gestation sac with a foetal heart signifying an ongoing pregnancy. All pregnant women were contacted or traced for the pregnancy outcomes after delivery or miscarriage.
Cryopreservation and frozen embryo transfer (FET)
Surplus embryos of day 3 top quality embryos or good-morphology Day 5 or 6 blastocysts in the long agonist group and all the viable embryos/blastocysts in the PPOS group were cryopreserved using vitrification. Those who did not get pregnant in the stimulated IVF cycle and those who postponed embryo transfer would undergo frozen embryo transfer (FET) at least 2 months after the stimulated cycle if they had at least one frozen embryo.
Vitrification was performed with MediCult Vitrifification Cooling (Origio, Denmark) using ethylene glycol, propylene glycol, sucrose as cryoprotectant. Embryos were vitrified one by one at room temperature. For the warming procedure following vitrification, the straw was cut and the capillary was pulled from the straw out of the liquid nitrogen, and immediately warmed one by one using MediCult Vitrification Warming (Origio, Denmark). After warming, embryos were transferred to a culture dish for evaluation and further embryo development. Only embryos with more than 50% of blastomeres present after thawing were transferred in FET cycles.
FETs were carried out in natural cycles for ovulatory women and in clomiphene induced or hormonal cycles for anovulatory women. Up to two embryos or blastocysts were transferred in FET cycles.
The primary outcome measure was the cumulative live birth rate within 18 months from the first day of ovarian stimulation. LBR which was calculated by including the first live birth generated during the one complete IVF cycle including fresh and all subsequent FET cycles.
Secondary outcome measures included incidence of premature LH surge (LH ≥10 IU/l), fertilization rate, clinical pregnancy, ongoing pregnancy, live birth rate, miscarriage, multiple pregnancy, and implantation rates in both fresh and FET cycles. Number of cycle cancellations, number of oocytes retrieved, number of obtained oocytes, number of embryos available for transfer, number of cryopreserved embryos, number of FET cycles started, moderate and severe ovarian hyperstimulation syndrome (OHSS), time to ongoing pregnancy were also compared. A baby born alive after 22 weeks gestation was classified as a live birth. Clinical pregnancy was defined as the presence of at least one gestational sac on ultrasound at 6 weeks. Ongoing pregnancy was the presence of at least one foetus with heart pulsation on ultrasound beyond 10 weeks. Miscarriage rate was defined as the number of miscarriages before 22 weeks divided by the number of women with clinical pregnancy. Multiple pregnancy was a pregnancy with more than one gestational sac detected on ultrasound at 6 weeks. Fertilization rate was the percentage of zygotes with two visible pronuclei among inseminated oocytes. Implantation rate was calculated as the number of gestational sacs seen on scanning divided by the number of embryos replaced. Time to ongoing pregnancy leading to live birth as the time from day of ovarian stimulation to an ongoing pregnancy that led to a live birth.
We analyzed all cycles finished before 18 months after the first day of starting ovarian stimulation - whether cancelled, pregnant, or non - pregnant. To ensure validation of complete cycles, all enrolled subjects agreed to use all frozen embryos before proceeding with a new fresh IVF/ICSI cycle.
One sample of the Kolmogorov - Smirnov test was used to test the normal distribution of continuous variables. Continuous variables were given as mean ± SD if normally distributed, and as median (interquartile range) if not normally distributed. Statistical comparison was carried out by Student’s t-test, Mann - Whitney U-test for continuous variables and chi-square test for categorical variables, where appropriate.
Cox proportional hazard model was used to evaluate the relative prognostic significance of female age, BMI, the number of retrieved oocytes and the primary diagnosis of infertility in relation to CLBR.
Generalized estimated equation regression analyses(GEE) were made for the individual treatment groups in all FET cycles with transfer to evaluate the impact of independent variables on the total LBRs from FET (n = 919).
All pregnancies within 18 months from ovarian stimulation were analyzed, whether achieved by fresh or frozen IVF cycle. The Kaplan-Meier method was used to calculate the cumulative proportion of ongoing pregnancies leading to live births, and time to pregnancy was graphically depicted by cumulative incidence curves. The log-rank test was used to measure whether significant differences existed in the cumulative incidence curves. Patients who did not reach the primary outcome (live birth) including those achieved a continuing pregnancy that did not lead to live birth were censored. Statistical analysis was performed using the Statistical Program for Social Sciences (SPSS Inc., Version 24.0, Chicago, USA). The two-tailed value of P < 0.05 was considered statistically significant.