IVF/ICSI protocols of controlled ovarian stimulation (COS)
Ultra-long protocol: to receive the first injection of long-acting GnRHa (Triptorelin Acetate®; Ipsen, France; Leuprorelin Acetate®; Lizhu, China; Goserelin Acetate®; AstraZeneca, UK) with a dose of 3.75 mg or 3.6mg subcutaneously on day 2 or 3 of the menstrual cycle. The anteroposterior diameter of uterus was measured 28 days after each injection. If the diameter was more than 70 mm, another injection of long acting GnRHa was repeated until the third injection. 28 days after the last dose, standard downregulation was evaluated by transvaginal ultrasonography(TVS) and serum hormone examination. Eligible downregulation was defined as endometrium thickness ≤5mm, serum estradiol ≤50pg/mL, LH ≤5IU/L, diameters of follicles <8mm in bilateral ovary and no functional cysts.
Long protocol: a daily dose of triptorelin acetate (0.05–0.1mg) for pituitary downregulation lasted for 14 days. If downregulation criteria were achieved, ovarian stimulation started.
Short protocol: a daily dose (0.05–0.1 mg) of GnRHa (triptorelin acetate®; Ipsen, France) was given on day 2-4 of the menstrual cycle until HCG trigger. After 1-2 days of GnRHa, gonadotropin was used for ovarian stimulation and lasted for about 8-12 days.
Antagonist protocol: a daily dose (0.25mg) of GnRH-ant injection (Orgalutran®, MSD, Netherlands) was used following fixed protocol (started daily on the fifth or sixth day of goandotropin till trigger day) or flexible protocol (started when primary oocyte diameter≥14 mm or the primary oocyte diameter≥12mm and the level of estrogen ≥300 pg/ml).
Ovarian stimulation
150–300 IU gonadotropin (Gonal F®, Merck Serono, Switzerland; Puregon®, MRK, China; Lishenbao®, Lizhu, China) daily was administered for COS according to age, body mass index (BMI) and ovarian reserve. The adjustment of gonadotropin and addition of recombinant LH (Luveris®, Merck Serono, Germany) were decided by follicular development. Routinely, 8000-10000 IU of urinary human chorionic gonadotropin(HCG) (hCG®; Livzon, China) was used intramuscularly for triggering when at least two follicles measured ≥18mm. If high risk of ovarian hyperstimulation syndrome (OHSS) existed, 2000-4000 IU of HCG combining with 0.2mg GnRHa was administrated. Oocyte retrieval was performed 34h–36h later. The choice of IVF or ICSI depended on sperm quality. Cancellation of fresh ET and whole embryo frozen was carried out in the conditions of high risk of OHSS, hydrosalpinx or unsynchronized endometrium status. All embryos were cultured for 3 or 5 days with two high-quality cleavage-stage embryos on day 3 or one blastocyst on day 5 transferring into the uterus under the guidance of abdominal ultrasound. The high-quality embryos were defined as 2PN-derived embryos with 7-10 cells and scores ≥ 3 on day 3 or ≥ 4BC on day 5(12). Oral dydrogesterone tablet (Duphaston®, Abbott, Netherlands) 10 mg twice daily and vaginal progesterone gel (Crinone gel®, Merck Serono, Switzerland) 90 mg once daily or oral dydrogesterone tablet (Duphaston®, Abbott, Netherlands) 20 mg twice daily and vaginal progesterone soft capsules (Utrogestan®, Besins, Belgium) 200 mg once daily were administered as luteal phase support.
GnRHa pretreatment before FET: GnRHa ptreatment was performed in patients with severer adenomyosis before FET for≥1 months with 3.75 mg or 3.6mg of GnRHa per month. The uterine anteroposterior diameter was measured 28 days after each injection. If it was more than 70 mm, injection of the same dose of GnRHa was repeated until the third injection. Hormone replacement cycles would be administered 4 weeks after the last dose of GnRHa.
Protocols of endometrial preparation in FET cycles
Oral estradiol valerate (Progynova®; Bayer, Germany) was administrated in a dose-escalating method, 4mg/day for the first 5 days and subsequently 6mg/day for the second 5 days. According to the assessment of endometrial thickness and serum hormone levels of E2, 8mg/day for another 3-4 days was continued or not. When endometrial thickness ≥7 mm, progesterone addition was started and the detailed protocol was same as those in fresh ET cycles. 5 days after progesterone addition one frozen-thawed blastocyst was transferred into the uterus. Other protocols for endometrial preparation in FET cycles had been described in previous study(13).