This retrospective study was conducted to analyze the survival rate of cleavage stage embryos after vitrification/warming performed by CryoTouch and Cryotop methods, and the subsequent clinical outcomes in two infertility clinics located at Erfan Niayesh Hospital Tehran, Iran and Mehr Fertility Research Center, Rasht, Iran, from January 2018 to December 2020. The study protocol was done according to the Ethical Principles for Medical Research and approved by the Institutional Review Board of each center.
The details about patients’ basic characteristics, protocol of ovarian stimulation, ICSI procedure, embryo culture and evaluation methods, vitrification and warming protocols, FET protocol, and clinical outcomes were all obtained from registered documents at the mentioned infertility clinics. A total of 978 FET cycles involving embryos vitrified/warmed on day 3 of development were evaluated to enter the study based on the following inclusion/exclusion criteria:
The inclusion criteria were age range between 20 and 35 years old, body mass index (BMI) <30 kg/m2, serum level of follicle-stimulating hormone (FSH) < 10 mIU/ml on day 3 of menstrual cycle, having more than 8 cleavage stage embryos produced by ICSI procedure, and undergoing the first ICSI/FET cycle with gonadotropin-releasing hormone (GnRH) antagonist protocol for ovarian stimulation.
The exclusion criteria included the presence of anatomical uterine anomalies, space-occupying lesions, hormonal dysregulations, ovarian hyperstimulation syndrome (OHSS), inflammatory disorders, hydrosalpinx, endometriosis, autoantibodies, history of ectopic pregnancy, repeated implantation failure (RIF), miscarriage, and having less than 8 cleavage stage embryos produced by ICSI procedure.
Ovarian stimulation protocol
GnRH antagonist protocol was performed in all patients. Table 2 provides ovarian stimulation characteristics and final outcomes in patients undergoing an ovarian stimulation procedure. First, estradiol valerate (2 mg, PO, BID; Aburaihan Co., Tehran, Iran) was administered from day 21 of the natural cycle and continued until days 2–4 of the subsequent cycle. Then, follicular growth was stimulated by administration of recombinant FSH (150–225 IU, daily; Gonal F, Merck, Germany) from day 2 or 3 of the cycle. The prescribed dose was adjusted based on the follicular growth monitored using transvaginal ultrasonography. GnRH antagonist (Cetrotide, Merck, Germany) was administrated when the dominant follicles reached the size of 13–14 mm in diameter. GnRH antagonist administration was continued until the day of ovulation induction. Human chorionic gonadotropin (hCG, 10,000 IU; Choriomon, IBSA, Switzerland) was injected to induce oocyte maturation. Transvaginal ultrasound-guided ovum pick-up (OPU) was performed 36 h after hCG injection.
Cumulus cell-oocyte complex retrieval, oocyte denudation, and fertilization
All the used culture media were supplemented with 10 % (V/V) of human serum albumin (HSA) and were equilibrated for 8 h at 37 °C in 6% CO2 incubator before use. The retrieved cumulus cell-oocyte complexes (COCs) were washed in a handling medium (HTF w/HEPES, Fertilite®, Ravan Sazeh Co., Tehran, Iran ) and maintained in embryo culture medium (SingleCulture Medium, Fertilite®, Ravan Sazeh Co., Tehran, Iran ) for 2 h at 37 °C in 6% CO2 incubator prior to denudation. Oocyte denudation was conducted using both enzymatic (Hyaluronidase, Fertilite®, Ravan Sazeh Co., Tehran, Iran) and mechanical methods. ICSI procedure was performed on all matured oocytes (metaphase II stage) 3–4 h after OPU. Then, the injected oocytes were cultured in 30–50 µL of embryo culture medium (SingleCulture Medium, Fertilite®, Ravan Sazeh Co., Tehran, Iran) under mineral oil (RS Medical, Ravan Sazeh Co., Tehran, Iran) overlay. On day 3 of development, the resulting embryos were assessed morphologically and high-quality embryos were selected for vitrification.
Cleavage stage embryo quality evaluation
Cleavage stage embryo quality was assessed by recording morphological parameters according to the literature . The morphological parameters included the number and symmetry of blastomeres, degree of fragmentation (anucleate structures of blastomeric origin), presence of multinucleation, and presence of abnormalities in intracytoplasmic and extracytoplasmic compartments. High-quality cleavage stage embryos were defined as those with 4 blastomeres on day 2 or 6–8 blastomeres on day 3, having symmetric blastomeres, having less than 15% fragmentation, without multinucleation, without intracytoplasmic abnormalities such as vacuoles and inclusion bodies, and without extracytoplasmic abnormalities such as granularities in the perivitelline space and zona pellucida dysmorphism. Otherwise, the embryos were considered as low-quality embryos.
Vitrification and warming procedures
High-quality cleavage stage embryos were selected for the vitrification with two commercial methods and the procedure was performed based on the manufacturer’s protocol, CryoTouch method (RS Medical, Ravan Sazeh Co., Tehran, Iran) or Cryotop method (Kitazato BioPharma Co., Shizuoka, Japan). The method means the use of vitrification/warming media and vitrification devices from the same manufacturer. CryoTouch and Cryotop methods had similar protocols for vitrification and warming procedures with a little difference. Briefly, the equilibration was performed in equilibration solution for 7 min in CryoTouch method or 10 min in Cryotop method, both at room temperature. Afterwards, the embryos were placed in vitrification solution for 50–60 s in both protocols. Then, the embryos (maximum 3 in each vitrification device) were immediately aspirated with a minimum volume of the vitrification solution and placed onto the tip of CryoTouch® or Cryotop® vitrification devices. The loaded devices were immediately submerged vertically into liquid nitrogen, then placed in a goblet and stored in a liquid nitrogen tank.
In the morning of FET, the embryos were warmed in the corresponding commercial media based on the manufacturer’s protocol. Briefly, the vitrification device was quickly removed from liquid nitrogen and immersed in a warming solution (prewarmed at 37 °C). The embryos were detected and immediately transferred to another droplet of warming solution and incubated for 1 min. Subsequently, the embryos were transferred to the dilution solution and incubated for 3 min. In the next step, the embryos were transferred to the washing solution and maintained for 5 min. Ultimately, the embryos were placed in embryo culture medium and incubated at 37 °C in 6% CO2 until ET.
Embryo survival assay
The vitrified/warmed embryos were considered survived if they had 50 % or more viable blastomeres with no evidence of degenerated morphology.
Hormone replacement therapy (HRT) was performed for preparing the endometrium as a standard protocol. Estradiol valerate (6 mg/day, PO; Aburaihan Co., Tehran, Iran) was administered from days 2–3 of the menstrual cycle and continued up to 8 mg/day until the endometrial thickness reached 8 millimeters. Progesterone (400 mg, suppository, BID; Cyclogest, Actavis, England, UK) was initiated when the endometrial thickness was upper 8 mm. In the presence of a positive result for β-hCG test, the estradiol and progesterone administrations were continued until weeks 6 and 12 of gestation, respectively.
ET was conducted using an embryo transfer catheter (Guardia™ Access, Cook, USA) by an expert gynecologist under the guidance of ultrasound, based on the guideline provided by the American Society for Reproductive Medicine (ASRM). Single or double high-quality cleavage-stage vitrified/warmed embryos were selected for each ET cycle.
The embryo post-warming survival rate was calculated as the percentage of survived embryos, based on the mentioned definition, after the warming procedure.
The clinical pregnancy rate was calculated from the number of observed gestational sacs by ultrasonography per embryo transfer.
The implantation rate was calculated from the number of observed gestational sacs by ultrasonography per the number of transferred embryos.
The live birth rate was calculated from the number of live births per embryo transfer.
GraphPad Prism (GraphPad Software, USA) was used to analyze all the obtained data. Comparisons of the means were performed by Student’s t test. The P value < 0.05 was considered as the level of significance. Data are represented as mean ± standard deviation (SD).