The inclusion criteria for participating in the study were women age 18–40 years, body mass index (BMI, calculated as weight in kilograms divided by height in meters squared) < 30 kg/m and high risk for OHSS (having more than 20 follicles > 12mm on day of trigger administration).
Patients with a history of diabetes, hyperprolactinemia, asthma, hypertension, hemorrhagic patients, and history of heart disease were excluded. Also, patient dissatisfaction with continued treatment was considered as an exclusion criteria.
At the begging of the study, women underwent complete evaluation such as clinical history, physical exam, laboratory test, transvaginal sonography, and profile hormone. If they met the inclusion criteria, entered the study.
Ovarian stimulation protocols
The participants underwent GnRH antagonist protocol ovarian stimulation for preparing IVF.At first, all patients investigated with Transvaginal ultrasound on the second day of menstrual cycle. If the endometrial thickness was less than 5 mm and the ovaries were quiet, ovarian stimulation would be started.
Stimulation protocol treatment was initiated on the third day of menses with the daily use of recombinant human follicle-stimulating hormone (FSH, CinnaGen, Tehran, Iran) 150–225 IU. The dose of gonadotropin individually adjusted according to reserve tests of ovary, BMI, age and response in prior cycles.
Once the leading follicle reached a size of 13 mm, co-treatment with GnRH antagonist 0.25 mg/day Cetrotide (Merck Serono, Mississauga, Canada) and highly purified human menopausal gonadotropin (Menotropin 75 IU, Darou Pakhsh Pharmaceutical Co,Tehran ,Iran) were commenced. Follicle growth and hormone levels were serially monitored by ultrasound .The dose of the drug was changed based on response ovary. All ultrasound exams were performed by a one researcher using a Phillips affinity 70 device at the Milad infertility center.
When the dominant follicles reached an average diameter of 18–20 mm (the morning of the trigger day) the investigators recruited the patients for the study. After the participants provided a written informed consent, based on the physician's preference (36.9%), 118 patients in GnRH-a group, (15.3%) 49 patients in hCG group and finally (47.8%) 153 patients in dual group (GnRH-a and hCG) were assigned .In hCG group, patients were triggered for final follicular maturation with hCG (PDpreg 5000 IU;pooyesh Darou, Tehran,Iran), in the dual trigger group, patients were triggered with GnRHagonist (Decapeptyl 0.2 mg, Ferring, Germany) and low dose hCG 1500IU and in GnRHagonist group, used GnRHagonist (Decapeptyl 0.2 mg ) alone to trigger. oocyte retrieval was performed via transvaginal US–guided needle (K-OPSD-1730-A-L; Cook Australia Pty Ltd., Brisbane, Australia) puncture 36 hours after injection of trigger .
Follicular fluids were observed under a microscope and then Cumulus-oocyte complexes (COCs) were washed in Cleave medium (Origio-Denmark) at 37 ° C with 26% CO and 5% o2, two hours after collection of oocytes, the oocytes were denuded from their cumulus cell and were evaluated for their maturity under Polarized Light Microscopy (PLM)
Based on Oocyte morphological such as oocyte shape, size, characteristic of polar body, perivitelline space, zona pellucida, granulation and cytoplasmic parameters like refractile bodies, vacuoles, smooth endoplasmic reticulum, Oocytes are divided into three categories, M I,MII,GV. The best quality oocyte selected for intracytoplasmic sperm injection (ICSI). On the same day, semen samples were collected and examined to evaluate sperm parameters including concentration, morphology, and motility, according to the World Health Organization guidelines (WHO laboratory manual, 2010) (20). The semen was prepared with Density gradient centrifugation and injection into oocyte were done 2 hours after CCs removal. After16-18 h, normal fertilization was confirmed with the presence of two pronuclei (2PN) and the extrusion of the second polar body. The zygotes were placed on the tip of the Cryotop (Kitazato Corporation, Tokyo, Japan) then they were plunged in liquid nitrogen.
Other results are recorded, including the absence of pronuclear, presence of one pronuclear, and degeneration.
Embryo quality was assessed on the third day of fertilization and evaluated according to Cummins et al. standard as grade 1 (excellent embryos with 8 blastomeres with cell regularity and size equality without necrosis and fragmentation, grade 2 (embryos 1–20% fragmentation), grade 3 (21–50% fragmentation) and grade 4 ( fragmentation greater than 50%).(21)
The primary outcome measure was the number of oocytes derived in every group and the number of M I, M II, and GV oocytes. The secondary outcome measure was the rate of fertilization and rate of embryos grade 1, 2, 3. Fertilization rate was calculated the ratio of oocyte injected divided by the number of embryos fertilized.
OHSS classification is based on the Humaidan et al. criteria (22). Mild OHSS was determined by the existence of pelvic discomfort, abdominal distension, and the presence of ascites in the Douglas pouch and enlarged ovaries in ultrasonography. Moderate OHSS was stated in the presence of pelvic discomfort, abdominal distension, ultrasonic evidence of fluid in Douglas pouch and around the uterus (major pelvis), Ovarian enlargement, and hemoconcentration (hematocrit > 45%).
Severe OHSS was defined in the presence of both objective criteria (fluid collection in the pelvic pouch and around intestinal loops, hematocrit > 45 percent, white blood cells > 15,000, urine outputs < 600 mL per 24 hours) and subjective criteria (pelvic discomfort, abdominal distension, severe dyspnea, and enlarged ovaries).