Methods

This was a retrospective cohort study evaluating freeze-only cycles performed in a private IVF centre in Sao Paulo, Brazil. All procedures in this study are part of the routine care in the assisted reproductive centre and written informed consent was obtained from all patients before treatment, consenting to the treatment procedures and to the use of their data in scientific publications with no patient identification. This study is based on databank of anonymized data and according to Brazilian legislation it was exempt from approval by the Institutional Review Board and specific Informed Consent is not applicable.
The database included all IVF cycles performed between 2011 and 2019 at Monteleone Assisted Reproduction Center, São Paulo – Brazil, that were potentially eligible for this study. The inclusion criteria were cycles of patients in which all embryos were cryopreserved (freeze-only cycles) and no fresh embryo transfer were placed. From 5156 cycles performed in the period of study, we selected 2725 freeze-only cycles. From those, we excluded cycles using donated oocytes, testicle sperm, embryo biopsy, cycles with less than three embryos cryopreserved and more than 2 embryos transferred in the frozen-thawed ET. Missing data were not a reason for case exclusion, and all cycle analysed had all essential data (associated to inclusion or exclusion criteria) and the most of other additional information. Thus, missing data did not compromised the analysis. The final number of 500 freeze-only cycles with elective Frozen-Thawed Embryo Transfer (eFET) of one or two embryos was analysed. The cycles were split into two study groups where the elective Double Embryo Transfer group (eDET group) was composed by 291 cycles in which two embryos were placed in the first eFET and had least one surplus embryo cryopreserved and the elective Single Embryo Transfer group (eSET group) with 209 cycles in which patients underwent a eSET in their first eFET and had least one surplus embryo cryopreserved. For those who underwent eSET and did not become pregnant, a second frozen-thawed SET was performed for 60 patients (Figure 1).

IVF protocol

Patients underwent ovarian stimulation and oocyte pickup according to routine medical criteria. Briefly, pituitary blockage was obtained with a GnRH antagonist (Cetrotide, Merck). Ovarian stimulation was accomplished using recombinant FSH (rFSH, Gonal-F, Merck) at 150 IU/day as the starting dose for women up to 35 years of age and 225 IU/day for women older than 35 years and the dose was adjusted according to the ovarian response. Follicular maturation was triggered when at least two follicles reached a diameter of 18 mm by using a GnRH agonist (Gonapeptyl, Ferring). Oocyte retrieval was performed after 35 to 36 h by transvaginal ultrasound-guided aspiration. All oocytes were fertilized by ICSI (22) according to routine procedures and embryos were cultured using standard methods in a triple gas incubator (90% N2, 5% O2 and 6% CO2) at 37°C until vitrification.
All good quality embryos were vitrified on D3 or D5 using the Vitrification Freeze kit (Irvine Scientific) with a Cryotip device (Irvine Scientific), following the manufacturer’s instructions. For warming, a Vitrification Thaw kit (Irvine Scientific) was used. Embryos were evaluated by morphological criteria on day 3 (D3) and/or day 5 (D5). The embryos on D3 were considered good quality when they presented 8 to 10 symmetric blastomeres, no multinucleation and a maximum fragmentation level of 20% (23). Blastocysts on D5 were considered good quality when they were expanded, inner cell mass grade 3 or 4 and the trophectoderm was classified as A or B (24).
For frozen-thawed embryo transfers, endometrial preparation was conducted with 100 µg of oestradiol valerate (Estradot, Novartis) for 14 days plus 800 mg of vaginal micronized progesterone (Utrogestan, Farmoquimica) beginning 5 days before the transfer. Embryos were warmed and evaluated for survival and morphology and a higher quality blastocyst was preferentially transferred when available. Clinical pregnancy was defined by the presence of a gestational sac with heartbeat at 2 weeks after biochemical confirmation of pregnancy with serum beta-hCG measurement.

Data collection and statistical analysis

Data were obtained from the clinical report forms and tabulated for this study. The primary endpoint was the ongoing pregnancy defined by the presence of a gestational sac with heartbeat and the ongoing pregnancy rate (PR) was calculated as the number of patients presenting an ongoing PR divided by the number of patients with embryos transferred. Additionally, for the calculation of the cumulative ongoing PR considering the 2nd SET for patients who did not become pregnant in the 1st SET (eSET-SET group), we used a formula previously described by Luke and colleagues (2015). The cumulative ongoing PR was equal to [ongoing PR for the 1st SET + the ongoing PR for the 2ndSET * (1 - the ongoing PR for the 1st SET)]. This calculation assumes no contraindication during cycle 1 for continuing into cycle 2. The implantation rate (IR) was calculated as the number of gestational sacs divided by the number of embryos transferred and miscarriage rate was defined as number of miscarriage divided by the number of patients with gestational sac.
Data analysis was performed using SPSS V.21 (IBM SPSS Software, USA). Normality distribution tests were performed and patient demographic data were evaluated using descriptive statistics, including the means and frequencies. As data were normally distributed, parametric tests to compare means (Student’s t test) were used to continuous variables. Pearson’s chi-squared test was used to compare frequencies as appropriated. Regression analysis was used to evaluate the associations between variables, and multivariate models included possible confounders. We considered p-values ≤0.05 to be statistically significant.