Study Design
This interventional, prospective, parallel, non-randomized, open-label controlled clinical trial was conducted on normo-ovulatory women who were referred to the Assisted Reproductive Unit of Orient Hospital, Damascus, Syrian Arab Republic, from December 2019 to August 2021. The Ethical Committee of Damascus University approved the study protocol, and a written informed consent was obtained from all participants. The clinical trial registration number is NCT04724343.
The trial was originally designed to be a randomized control trial (RCT). However, due to the medical supplies’ crisis in the Syrian Arab Republic, it was challenging to provide the GnRH antagonist (Cetrorelix, Cetrotide; Merck) during a certain period of the study duration, meanwhile supplying the GnRH agonist (Triptorelin, Decapeptyl; Ferring) was less troublesome. Thus, the randomization was broken, and more patients were recruited in the GnRH agonist arm.
Participants
In this study, a total of 83 normo-ovulatory women were allocated to take either the long GnRH agonist protocol (n=50) or the flexible GnRH antagonist protocol (n=33). Both the patients and the doctors were aware of the allocated arm. Patients who aged ≥ 40 years; or were diagnosed with polycystic ovary syndrome (PCOS), androgen-secreting tumors, Cushing’s syndrome, congenital adrenal hyperplasia, hyperprolactinemia, thyroid disorders, epilepsy, diabetes mellitus, cardiovascular diseases, liver diseases, kidney diseases, cancer; or had any conditions that might affect IVF outcomes like endometriosis, uterine fibroids, hydrosalpinx, adenomyosis, or autoimmune diseases were excluded. Women with three or more previous IVF failures, poor responders (Bologna criteria [51]), and those who were previously undergone unilateral oophorectomy were also excluded.
Controlled ovarian stimulation protocols:
Agonist Group (Long protocol):
The pituitary down-regulation in this group was carried out using 0.05-0.1 mg of Triptorelin acetate subcutaneously (SC) once daily from the mid-luteal phase (day 21) of the menstrual cycle until the ovulation triggering day. When the suppressive effect was obtained (Estradiol (E2) < 50 pg/ml, no cysts or follicles > 1 cm maximum diameter detected by ultrasound, endometrial thickness < 5 mm), ovarian stimulation was commenced with recombinant Follicle-Stimulating Hormone (r-FSH) and/or human Menopausal Gonadotropin (hMG), and the dose was adjusted according to the ovarian response, which was monitored by transvaginal ultrasound (Voluson TM E10, GE Healthcare Ultrasound, USA).
Antagonist Group (Conventional Flexible protocol):
The ovarian stimulation in this group was started with recombinant Follicle-Stimulating Hormone (r-FSH) and/or human Menopausal Gonadotropin (hMG) on the third day of the menstrual cycle, and the dose was adjusted according to the ovarian response, which was monitored by transvaginal ultrasound (Voluson TM E10, GE Healthcare Ultrasound, USA). The initiation of 0.25 mg of GnRH antagonist, Cetrorelix, took place after detecting a leading follicle diameter ≥ 14 mm and continued till the day of ovulation triggering.
Ovulation triggering and oocytes retrieval:
Ovulation was triggered by the administration of 10,000 IU of Human Chorionic Gonadotropin (hCG) when at least three follicles become more than 16-17 mm. After 35±2 hours of ovulation triggering, the oocytes were retrieved by transvaginal ultrasound-guided follicle aspiration.
IVF procedure and embryological outcomes assessment:
An Intra-Cytoplasmic Sperm Injection (ICSI) technique was used for insemination. The embryological outcomes were assessed by independent highly-trained embryologists. Each studied outcome was assessed by a single assessor for both groups to limit inter‑assessor variations. The same media and culturing methodology were used for both groups. The Thermo Scientific HERACELL 150i incubator (Thermo Fisher Scientific, USA) was used for COCs and oocytes cultures (humidified atmosphere at 37ºC, CO2 level at approximately 6%, and culture medium pH between 7.28-7.35), and the K-Systems G210 InviCell (K-Systems Kivex Biotec Ltd. Denmark) was used for Embryos cultures.
Oocyte’s denudation and maturation assessment:
Retrieved oocytes were first rinsed in G-MOPS TM Plus media (G-MOPS TM Plus, Vitrolife, Sweden) then maintained in G-IVF TM Plus culture (G-IVF TM Plus, VitroLife, Sweden) covered with paraffin oil (OVOIL, VitroLife, Sweden) before cumulus cell removal. The surrounding cumulus cells were removed within 2 hours after retrieval by the exposure to hyaluronidase (HYASE-10× in G-Mops TM Plus media, Vitrolife, Sweden) for several seconds before being transferred to G-MOPS TM Plus media where they were mechanically dissociated from the oocyte.
The denuded oocytes were classified according to their level of maturation using a Nikon SMZ1500 stereoscope. The number of Metaphase II Oocytes (MII; identified as oocytes with the extrusion of the first polar body), Metaphase I Oocytes (MI; identified as oocytes lack the presence of both the germinal vesicle and the polar body), Germinal Vesicle Oocytes (GV; identified as oocytes with Germinal Vesicle), and Atretic Oocytes (oocytes with signs of degeneration) were documented. The Maturation Rate was calculated by dividing the number of mature (MII) oocytes by the number of retrieved oocytes. In addition, the ovarian sensitivity index (OSI) was calculated by dividing the number of retrieved oocytes by the total dose of FSH used and multiplying the results by 1000 [52].
Oocytes morphological assessment:
Before being subjected to ICSI, MII oocytes from both groups were morphologically assessed using an inverted microscope Nikon Eclipse Ti2 (Nikon, Tokyo, Japan) under 400× magnification. The following dysmorphisms were studied:
- Cytoplasmic dysmorphisms: the presence of granulation, refractile bodies, smooth endoplasmic reticulum (SER) aggregations or vacuoles in the cytoplasm; or detecting dark cytoplasm.
- Extracytoplasmic dysmorphisms:
- Alterations in oocyte shape or size.
- Zona pellucida dysmorphisms: alterations in zona pellucida color, size, or thickness; the presence of a zona pellucida with a septum.
- Perivitelline space dysmorphisms: alterations in perivitelline space size or presence of perivitelline space fragments.
- Polar body dysmorphisms: alterations in polar body size, presence of polar body fragments, or presence of duplicated/triplicated polar body.
The oocytes were classified as normal oocytes, oocytes with cytoplasmic dysmorphisms, oocytes with extracytoplasmic dysmorphisms, and oocytes with both cytoplasmic and extracytoplasmic dysmorphisms. In addition, the oocytes were classified based on the quantity of the dysmorphisms observed.
Insemination and fertilization assessment:
Microinjections were performed at X400 magnification on a 37ºC heated stage inverted Nikon Eclipse Ti2 (Nikon, Tokyo, Japan). A Petri dish containing a microdroplet of ICSI TM media in the center (ICSI TM, VitroLife, Sweden) under paraffin oil (OVOIL, VitroLife, Sweden) was used for sperms selection and immobilization. On the same dish, a microdroplet of G-Gamete TM culture medium (G-Gamete TM, Vitrolife, Sweden) was used for placing the oocytes for microinjection. A single sperm was mechanically immobilized using the tip of the microinjection needle (Origio, USA) and then was aspirated inside the needle. The oocyte was held in place using a 35-degree angle holding micropipette (Origio, USA) with the polar body in the 6 or 12 o'clock position. Injection of a single spermatozoon within the oocyte cytoplasm was performed by using a micromanipulator (TransferMan® 4r, eppendorf, Germany). After ICSI, injected oocytes were cultured in G1‑Plus ™ medium (G1-Plus ™, VitroLife, Sweden). Fertilization was confirmed by the presence of two pronuclei and the extrusion of the second polar body approximately 16-18 h after ICSI. The Fertilization Rate was calculated by dividing the number of obtained zygotes (2PN) by the number of injected oocytes.
Embryos Grading, Cleavage rate, and high-quality embryos rate:
Embryos were morphologically evaluated using Nikon SMZ1500 stereoscope microscope (Nikon, Tokyo, Japan) and were graded based on ESHRE criteria (2011) [53]. According to these criteria, high-quality cleavage-stage embryos are defined as those with all of the following characteristics: 2-4 cells on day 2 or 6-8 cells on day 3, <10% fragmentation, symmetric blastomeres, and absence of multinucleation. Cleavage rate was calculated by dividing the number of cleavaged embryos by the number of obtained zygotes (2PN), while High-Quality Embryos Rate was calculated by dividing the number of high-quality embryos (Grade I) obtained by the total number of cleavaged embryos obtained.
Embryos transfer and luteal phase support:
The Selected embryos were treated with EmbryoGlue® media (EmbryoGlue®, VitroLife, Sweden) before being transferred using a Sure-Pro Ultra catheter (Wallace, USA) under transvaginal ultrasound guidance on day 2-3 after insemination (cleavage stage embryos). Luteal phase support was achieved using vaginal micronized progesterone gel (Crinone ® 8%, Merck Serono). It was started from the day of oocyte retrieval and continued for 14 days when a pregnancy was carried out. If pregnancy was confirmed, progesterone administration was continued until the 12th week of pregnancy.
Embryo transfer was cancelled, and elective embryo cryopreservation was performed in cases that were highly suspected of developing life-threatening (critical) OHSS [54,55] or fulfill the criteria for OHSS hospitalization [56]. Cycle Cancellation Rate (CCR) was calculated by dividing the number of cycle cancellation cases by the total number of participants.
Follicular fluid collection and analysis:
Follicular fluid was aspirated from all follicles (>15) mm, and then it was centrifuged at 3000 g for 10 min at room temperature, and the supernatant was stored at −80 °C until assayed. Follicular fluid concentrations of AMH were assayed using an ELISA kit from Biorex diagnostics (United Kingdom). Follicular fluid concentrations of PlGF were assayed using an ELISA kit from DRG Instruments (Germany). The intra-assay and inter‑assay coefficients of variation for all assays were less than 5% and less than 10%, respectively.
Pregnancy assessment and follow up:
A serum pregnancy test (serum hCG) was performed 14 days after embryo transfer. All women with a positive test received a transvaginal ultrasound scan after one-two weeks (i.e., 3-4 weeks after embryo transfer) then followed up until week 12 of gestation. The following rates were calculated:
- Biochemical Pregnancy Rate (BPR): Biochemical pregnancy was defined as a positive serum beta-hCG pregnancy test after two weeks of embryo transfer [57]. BPR was calculated by dividing the number of women who were biochemically pregnant by the total number of participants (Per Woman) or the total number of women who had at least one embryo transferred (Per Embryo Transfer).
- Clinical Pregnancy Rate (CPR): Clinical pregnancy was defined as the presence of at least one gestational sac on ultrasound after 3-4 weeks of embryo transfer. In addition to intra-uterine pregnancy, it included a clinically documented ectopic pregnancy [57]. CPR was calculated by dividing the number of women who were clinically pregnant by the total number of participants (Per Woman) or the total number of women who had at least one embryo transferred (Per Embryo Transfer).
- Multiple Pregnancy Rate (MPR): MPR was calculated by dividing the number of pregnancies with two or more gestational sacs on ultrasound by the total number of participants (Per Woman) or the total number of women who had at least one embryo transferred (Per Embryo Transfer).
- Implantation Rate (IR): IR was calculated by dividing the number of gestational sacs observed by the number of embryos transferred.
- Ongoing Pregnancy Rate (OPR): Ongoing pregnancy was defined as a pregnancy that continued ≥ 12 weeks of gestation. OPR was calculated by dividing the number of ongoing pregnancies by the total number of participants (Per Woman) or the total number of women who had at least one embryo transferred (Per Embryo Transfer).
- Resolved Pregnancy of unknown location (RPUL) Rate: RPUL was defined as a pregnancy demise not visualized on transvaginal ultrasound with a resolution of serum β-hCG after expectant management or after uterine evacuation without chorionic villi on histology [58]. RPUL Rate was calculated by dividing the number of RPUL cases by the total number of participants (Per Woman) or the total number of women who had at least one embryo transferred (Per Embryo Transfer).
Statistical analysis
All statistical analyses were performed using a Statistical Package for the Social Sciences (SPSS) software version 24.0 (IBM Corp., Armonk, NY, USA). Continuous variables were expressed as mean ± standard deviation and categorical variables as counts with percentages. Between-group comparisons were performed using the independent t-test for normally distributed variables, the Mann–Whitney U test for non-normally distributed variables, and chi-square or Fisher’s exact test as appropriate for categorical variables. For testing all hypotheses, tests were two-tailed, and values less than 0.05 were considered statistically significant.