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.