Background

Single embryo transfer (SET) has been advocated as a strategy to reduce the frequency of multiple births after in vitro fertilization [1]. It has been established that the significantly lower pregnancy rate achieved after single cleavage stage embryo transfer as compared to a double embryo transfer can be compensated by pregnancies resulting from the first thawed cycle [2]. This finding underscores the important role of cryopreservation in enhancing the total reproductive potential of a single cycle [3]. The introduction of the vitrification technique as an efficient cryopreservation technique promises to further increase the impact of cryo-technology on the cycle specific pregnancy rates.

Fresh pregnancy rates can be optimized by performing blastocyst transfer as such approach yields higher live birth rates than single cleavage stage embryo transfer [4, 5]. However, a systematic blastocyst transfer policy holds the risk of cancelled embryo transfers, as extended embryo culture may be unsuccessful. Morphological scoring of embryo quality on day 3 may therefore offer a tool to identify patients that would potentially benefit from further embryo culture [6]. The threshold of four good embryos on the third day appeared to be a reassuring indication that the patient will undergo an embryo transfer [6].

The aim of present study is to compare two approaches, both aimed at a single blastocyst transfer. The outcome after elective blastocyst transfer was compared with the outcome of a blastocyst transfer or a cleavage stage transfer depending on whether the criteria of four good embryos on the third day were met. Moreover, the additional cryo-pregnancies and projected cryo-pregnancies through blastocyst vitrification have been evaluated.

There is an ongoing debate on the microbiological safety of cryopreservation since the report of a hepatitis B transmission from contaminated cryopreserved bone marrow and reports on potential contamination of reproductive cells in experimental conditions [7, 8]. Although no clinical reports of contamination have been reported for reproductive cells and tissues, research is ongoing aimed at eliminating such risks. In this study we evaluate the effectiveness of blastocyst vitrification performed with a high security vitrification system preventing all contamination risk during vitrification and storage.

Methods

Study design

A retrospective cohort study was performed. All fresh IVF or ICSI treatment cycles were performed in a 15 months period from March 2008 until June 2009. All cycles where the physicians' initial treatment plan envisaged eSET with either a fixed day 5 transfer or with a day 3 or day 5 transfer depending on the embryo quality on day 3 were included. Only the first treatment cycle was considered in case patients were undergoing multiple attempts.

Embryo culture, evaluation and selection day of transfer

Sperm preparation, IVF/ICSI procedures and embryo culture were carried out as described by Van Landuyt et al. [9]. Oocytes and embryos were cultured in sequential media and cultured at 37°C in an atmosphere of 6% CO2, 5% O2 and 89% N2. Fertilization was assessed 16-19 h after insemination or injection. On the morning of day 3 embryos were transferred from cleavage medium to blastocyst medium. Embryo quality was assessed daily was scored as described by Papanikolaou et al., [6] according to the following parameters: number of blastomeres, rate of fragmentation, multinucleation of the blastomeres and early compaction. Blastocyst quality on day 5/6 was assessed according to the criteria of Gardner and Schoolcraft [10].

Embryo cryopreservation procedures

For patients receiving transfer on day 3, supernumerary cleavage stage embryos were cryopreserved and thawed using standard slow freezing as described by Van den Abbeel et al., (2005). Blastocyst vitrification was performed using closed CBS-VIT High Security (HS) straws (Cryo Bio system) in combination with DMSO-EG-S as the cryoprotectants (Irvine ScientificR Freeze Kit). The vitrification procedure was carried out at room temperature (between 22 and 27°C). Blastocysts were vitrified one by one. The blastocyst was first incubated for 2 min in a 50 μl droplet of HEPES-buffered culture medium. Then, the blastocyst was brought in a 50 μl droplet of equilibration solution (ES) containing 7.5% (v/v) DMSO and 7.5% (v/v) ethylene glycol and incubated for 10 min. The blastocyst was then transferred consecutively into four 25-μl droplets with vitrification solution (VS) containing 15% (v/v) DMSO and 15% (v/v) ethylene glycol. The blastocyst was incubated for 5 sec in droplet 1 and 2 and for 10 sec in the 3rd droplet. The blastocyst was then transferred to the fourth droplet and immediately loaded onto the CBS-HS straw. The straw was heat sealed and plunged into liquid nitrogen. The total time needed to vitrify the blastocyst starting from VS droplet 1 to the loading of the straw and plunging into liquid nitrogen did not exceed 90 sec.

On the day of transfer blastocysts were warmed one by one until one blastocyst was available for transfer. Blastocysts were warmed randomly, independent of the blastocyst stage or ICM/TE quality prior to vitrification. For warming, the Irvine ScientificR Thaw Kit was used. A Petri-dish containing two 25 μl droplets with thawing solution (TS, 1 M sucrose in Hepes buffered HTF medium supplemented with 20% DSS) was kept at 37°C. For warming the straw, the straw was transferred from the LN2 storage container to a transport dewar filled with liquid nitrogen. After cutting the straw and pulling the capillary from the straw, the gutter was placed in the first droplet with TS and the blastocyst was released from the gutter and kept at room temperature. The blastocyst was incubated for two times 1 min at room temperature in the two TS droplets. The blastocyst was then transferred to the first of two dilution solution droplets of 25 μl (DS; 0.5 M sucrose in Hepes buffered HTF medium supplemented with 20% DSS) and after that incubated for 2 min in a second DS droplet. Finally, the blastocyst was washed in 3 droplets (25 μl) of washing solution (Hepes buffered HTF medium supplemented with 20% DSS), each for 3 min. The blastocyst was transferred to a culture dish with blastocyst medium to assess its morphological survival. If the blastocyst was severely or completely damaged, a new one was warmed immediately. If the blastocyst was fully intact or showed moderate damage, expansion and re-expansion was assessed 1-2 hours later. If the morphological quality of the blastocyst was regressing or no signs of re-expansion were present, a second blastocyst was warmed until one blastocyst was suitable for transfer i.e. with at least moderate survival and expansion/re-expansion of the blastocyst

Statistical analysis

All data management and statistical analysis were performed using the Statistical Package for the Social Sciences version 17.0 (SPSS Inc. Chicago, OL, USA). Student't t-test were performed on continuous variables to determine differences in mean scores and standard deviation (SD). Categorical variables were analyzed using Chi square analysis. A significant level of 0.05 was accepted throughout.

Results

Patient demographics and stimulation characteristics (Table 1)

Table 1 Patient demographics and stimulation characteristics
Full size table

Overall 590 treatment cycles have been performed during the observation period with 341 (58%) in the fixed day 5 group and 247 (42%) in the day 3 or day 5 group. The two groups were comparable with regard to age, rank of trial, duration of stimulation and the total dose of gonadotrophins administered.

Fresh embryo transfer: embryology data (Table 2)

Table 2 Embryology data: fresh embryo transfer
Full size table

Overall, no differences were observed in the total number of cumulus-oocyte complexes, MII oocytes or 2PN oocytes. However, a significantly lower number of cumulus-oocyte complexes, MII oocytes and 2PN oocytes were available in the subgroup of patients with a transfer on day 3. Patients with the fixed day 5 approach had a lower subjects' fresh transfer rate (90.62% vs. 95.18%, p < 0.05) but the embryo fresh transfer rate and the embryo cryopreservation rate was similar. For patients in the day 3 or day 5 group, a higher embryo fresh transfer rate and a lower embryo cryopreservation rate were observed in patients with a transfer on day 3. The data reported separately for the day 3 and day 5 subgroups only included cycles with embryo transfer as cancelled cycles (n = 12) cannot be allocated to either one of the two subgroups.

Fresh embryo transfer: outcome (Table 3)

Table 3 Fresh embryo transfer outcome
Full size table

No differences were observed in terms of clinical pregnancy rate, ongoing pregnancy rate or live birth rate between the fixed day 5 and the day 3 or day 5 transfer policies. These outcome parameters also did not differ between the two subgroups within the day 3 or day 5 transfer policy. Multiple pregnancies were only observed in the fixed day 5 approach (1.29%) but did not statistically higher than in the other transfer approach group.

Frozen embryo transfer: embryology data (Table 4)

Table 4 Efficiency of cryopreservation and pregnancy potential of surviving embryos
Full size table

An overall embryo transfer rate of 69.9% was observed in the fixed blastocyst group which was not significantly different from the overall embryo transfer rate of 65.5% was observed for day 3 and day 5 embryos combined in the other group. The embryo transfer rate was statistically higher in the day 5 subgroup as compared to the day 3 subgroup (76.5% vs. 46.4%, p < 0.001). No differences were observed in the transfer specific or subject specific clinical pregnancy rates.

Frozen embryo transfer: outcome (Table 5)

Table 5 Frozen embryo transfer outcome
Full size table

Single embryo transfer was performed in 83% of all frozen blastocyst transfers in the fixed day 5 group. Single blastocyst transfers in the day 5 subgroup of the day 3 or day 5 group counted for 85.6% of the cycles which was significantly more than the 50% SET observed in the day 3 subgroup. The ongoing pregnancy rate per frozen embryo transfer in the fixed day 5 groups was 14.04%, which was not different from the overall ongoing pregnancy rate of the day 3 or day 5 group (14.63%). Multiple pregnancy rates were not different between the different approaches.

Summary and projected outcome (Table 6)

Table 6 Summary and projected pregnancy rate compensating for the double counting of pregnancies in case a subject has more than one pregnancy
Full size table

The clinical and ongoing pregnancy rate per thawed embryo was not different between the two groups. Neither was the projected cumulative clinical and ongoing pregnancy rate different between the elective day 5 group (44.12%; 42.58%) versus the day 3 or day 5 group (44.04%; 39.84%).

Discussion

This study has shown that a single blastocyst transfer policy can be decided before the initiation of the in vitro fertilization (IVF) cycle. A day 3 or day 5 embryo transfer policy based on day 3 embryo evaluation does not increase cycle outcome. It merely divides the patient population based on their ovarian response and reduces the risks of a cancelled embryo transfer.

Randomized controlled trials have shown that a blastocyst transfer policy results in a significantly higher live birth rate [5, 11] However, the effect of a fresh blastocyst transfer policy on the cumulative pregnancy rate, including cryo-cycles has not been evaluated in these trials.

A recent prospective trial by Guerif et al., [12] did include embryo cryopreservation by slow freezing technique when comparing a single day 2 with a single blastocyst transfer policy. Although blastocyst transfer yielded a higher live birth rate in the fresh cycles, it appeared to result in the same cumulative pregnancy rate after adding cryo-cycles. The authors concluded that improvements in blastocyst cryopreservation were needed in order to claim superiority of the blastocyst transfer policy. The implementation of the vitrification technique may provide such effect, however it would also further increase cleavage stage cryopreservation outcome.

A meta analysis concludes that vitrification appears to be associated with a significantly higher postthawing survival rate than slow freezing [13]. The superiority of vitrification is also illustrated by comparing vitrification outcome in this study with previously published data on blastocyst slow-freezing at the same centre [14]. A reported embryo transfer rate ranging between 48.9% and 52.2% after slow freezing [14] has been substantially increased to rates varying between 69.9% and 76.5% after vitrification.

The question whether cumulative pregnancy chances are affected by a cleavage stage embryo transfer versus blastocyst culture and blastocyst transfer based on a day 3 embryo quality evaluation remained unanswered. This study showed that such a embryology based policy mainly divides the patient population based on their ovarian response. The inherent reduced reproductive potential is illustrated by the low transferrable embryo survival rate of 46.4% compared to 65.1% as previously published in elective day 3 population in the same centre [15]. An alternative interpretation of the dataset is that a day 5 transfer is not required for a large proportion of the patients as the day 3/day 5 subgroups comparison for pregnancy rates are comparable.

In this study we used an aseptic method for the vitrification of embryos. Although the transmission of infectious diseases through cryopreservation and storage of embryos has never been reported, such possibility exists under experimental conditions [7, 16]. Therefore, the application of hermetically sealed containers or secondary enclosure (for cryovials and open vitrification systems) is suggested [16]. The hermetically sealed device used in this study has the advantage that both vitrification and storage are perfomed under 'closed' conditions. Aseptical vitrification using open devices would require sterilization of liquid nitrogen and a secondary enclosure for storage [17].

For the vast majority of patients, the single embryo transfer policy was based on the Belgian law imposing a SET policy in women less than 36 years [18]. No strict SET policy was applied in the thawed embryo transfer cycles, with significantly more DET (double embryo transfer) in the frozen day 3 embryo transfers. The higher proportion of DET in the frozen day 3 cycles (50%) as compared to the frozen blastocyst cycles (14.6%) can be explained by a lower expected implantation rate and resulted in a similar ongoing pregnancy rate per transfer (15.38% vs. 14.43%). The p value for the subject specific clinical pregnancy rate comparing the day 3 and day 5 embryo transfer reaches 0.05, although no difference has been observed in the pregnancy rate per transfer. The high subject specific pregnancy rate in the day 5 group appears to be due to the significantly higher transferrable survival rate and the embryo transfers performed.

Although no differences were observed in patients' and treatment characteristics, the retrospective design remains a weakness. The allocation of patients to one or the other group is based on the view of the individual physician in the centre and the preferences of the patient. Establishing a prospective randomized controlled trial will generate anxiety amongst patients and clinicians. This retrospective data will be reassuring for centers wishing to investigate this subject further.