Pregnancy rates of day 4 and day 5 embryos after culture in an integrated time-lapse incubator

All IVF or ICSI cycles from January 2011 to April 2016 with a prolonged embryo culture until day 4 or 5 at a single university IVF center in Germany were retrospectively analyzed. Only cycles with culture in an integrated time-lapse incubator (EmbryoScope^©, Vitrolife A/S, Aarhus, Denmark), embryo transfer and documented HCG level were included. Frozen-thaw cycles were excluded.

Patients data included maternal age, causes for infertility, oocyte count, number of fertilized oocytes (pronuclear stages (PN)), day of transfer, stage and quality of transferred embryos, application of IVF laboratory techniques (assisted hatching, calcium ionophore or polar body biopsy), biochemical pregnancy rates, clinical pregnancy rates, presence of fetal heart beat at completed 6 weeks of gestation and the number of frozen PN stages and embryos.

The (long and ultralong) agonist as well as the antagonist protocol were applied and oocyte retrieval was performed 36 h after ovulation induction.

Out of n = 2207 IVF/ICSI cycles a day 4 or 5 transfer was performed in n = 804 cases. Only cycles with culture in an integrated time-lapse incubator (EmbryoScope^©), embryo transfer, documented HCG level and outcome until 6 completed weeks of gestation were included into the study (n = 599 (27.1%)). In 124 IVF/ICSI cycles the embryos were transferred on day 4, 475 transfers were performed on day 5. The decision to transfer the embryo(s) on day 4 instead of day 5 was mainly made for organizational reasons, for example when day 5 was a Sunday or public holiday. The German Embryo Protection Act allows a maximum of 3 embryos to be transferred and limits the number of embryos to be cultured. The transfer day was scheduled depending on the number of pronuclear stage oocytes (PNs) available on day 1. In case the number of available PNs exceeded the number of embryos to be transferred, prolonged culture until day 5 was scheduled with a maximum of 4–5 cultured embryos. Alternatively, day 4 transfer was scheduled in case of organizational reasons (n = 90/124 day 4 transfers) or rescheduled to day 4 when only 1–2 embryos were normally developed at day 3 (n = 34/124 day 4 transfers).

Cumulus–oocyte–complexes were isolated and rinsed in Phosphate Buffered Saline (SAGE 4012) supplemented with 0.05% Heparine (Ratiopharm) followed by washing in fertilization medium (Sydney IVF^©, Cook Medical, Bloomington, IN, USA) and incubation in fertilization medium covered under paraffine oil (Origio, Berlin) in humidified air at 6% CO₂ and reduced oxygen (5%) in a 4-well dish. Mature metaphase II (MII) oocytes were inseminated by IVF or ICSI. Prior to ICSI oocytes were denuded with hyaluronidase (SynVitro Hyadase^©, ORIGIO GmbH, Berlin). ICSI was performed at an inverted microscope (Nikon Ti-S^©, Tokio, Japan) equipped with a heated table and micromanipulators (Mikromanipulator MM89^©, Narishige, Tokyo, Japan). Spermatozoa were placed into polyvinylpyrrolidone solution (PVP Clinical Grade 10%, Origio, Berlin) in a 60 mm plastic dish (Nunc Microplate^©, Thermo Fisher Scientific Inc. Waltham, MA, USA). In the same dish, oocytes were placed into drops of gamete medium (Sydney IVF, Cook Medical, Bloomington, IN, USA) and were injected using appropriate micro-capillaries (Micro Injection Pipette^© K-MPIP 1035 and Holding Pipette^© K-HPIP 1035, Cook Medical, Bloomington, IN, USA). In case of previous fertilization failure or low fertilization rates oocyte activation by calcium-ionophore (Cult-active Ca-Ionophore^©, Gynemed Medizinprodukte GmbH & Co. KG, Lensahn, Germany) was applied according to the protocol described by Tesarik [24] with modifications according to Montag [25]. Fertilization was assessed 17–19 h after insemination by the presence of two pronuclei (PNs). Culture of embryos was performed in an EmbryoScope^© (Vitrolife) at 37 °C, 6.4% CO₂ and 5.0% O₂. According to the German Embryo Protection law and dependent on the previous cycles, patients age as well as oocyte quality a maximum of 4–5 embryos were cultured per patient. Supernumerary fertilized oocytes were frozen on day 1 at PN stage.

On day 3 the medium was changed by removing cleavage and adding blastocyst medium (Cook Medical, Bloomington, IN, USA). Assisted hatching procedure was performed in individual cases on day 3 as described by Germond [26] with a 1.48 μm diode laser. Embryo development was described according to the ESHRE Istanbul Consensus Conference [27], Feil [3] for day 4 embryos and blastocyst development was assessed as described by Gardner and Schoolcraft [28].

Two embryos with the best grading and morphokinetic development were selected for transfer. Occasionally, at request of the couple or in cases of certain maternal medical conditions, a single embryo was transferred.

Eight patients received three embryos due to implantation failure in past IVF attempts. Well-developed supernumerary embryos were cryopreserved on the transfer day.

On day 4 an embryo with complete compaction (morula) was defined as “ideal”, on day 5 blastocysts with stage 4 or higher and high grades for the inner cell mass (ICM) and trophectoderm (TE) (AA, AB and BA) quality were considered morphologically “ideal”. Embryos with developmental abnormalities (arrest for >24 h and direct cleavage) were considered as non-ideal albeit matching the above morphologic criteria on the transfer day.

Biochemical pregnancies were defined as serum HCG levels ≥10 IU/l on day 14 after oocyte retrieval. Clinical pregnancies were determined by ultrasonographic detection of an intrauterine gestational sac at 5 to 6 weeks of gestation and ongoing pregnancies were defined as presence of a fetal heart beat after completed 6 weeks of gestation. Implantation rates were calculated as described by the ICMART committee 2009 [29].

Data are presented as mean ± standard error of the mean or percentage. Statistical analysis was performed by exact χ²–test (Fisher-Yates-Test) and two-sample unpaired t-test for independent samples with SPSS 22.0. P-values <0.05 were considered significant.

Patient and cycle characteristics are shown in Table 1. Patient demographics and treatment characteristics including mean maternal age, number of oocytes retrieved, number of injected oocytes in ICSI cycles, number or fertilized oocytes and rate of cycles with special treatments (i.e. calcium ionophore, polar body biopsy or assisted hatching) were not significantly different between the day 4 and day 5 embryo transfer groups.

There was no significant difference between the biochemical (50.0% for day 4 and 53.1% for day 5), clinical (45.2% for day 4 versus 45.7% for day 5) and ongoing pregnancy rates (43.5% for day 4 vs 41.7% for day 5) between embryos transferred on day 4 or 5 (Table 2, Fig. 1a). Even after exclusion of single and triple embryo transfers, the pregnancy rates of both transfer days were comparable (Table 2, Fig. 1b). Additionally, no significant difference between the implantation rates after day 4 transfers and day 5 transfers could be observed (29.4% versus 33.0% with p = 0.310).

The rate of ideal embryos transferred did not differ significantly between both groups (41.6% on day 4 versus 44.1% on day 5, p = 0.508, Fig. 2a). However, the mean number of embryos transferred on day 4 was higher than on day 5 (1.92 vs 1.84, p = 0.023), but the lower number of transferred embryos on day 5 did not lead to significant changes in the rate of ongoing multiple pregnancies (Table 2).

The mean number of cryopreserved embryos was comparable between both groups (Table 1: 0.32 embryos/cycle on day 4 vs. 0.37 on day 5).