O-065 The effect of double vitrification on embryo reproductive potential and clinical outcome
Authors: Oraiopoulou C , Karagianni M , Toumpa O , Ioannidou D , Papadopoulou M , Christophoridis N , Papatheodorou A , Chatziparasidou A 
Published: June 2022

Study question

Do double-vitrified embryos have impaired implantation potential compared to embryos vitrified once?

Summary answer

Double-vitrified embryos demonstrated significantly lower percentages of positive β-HCG, clinical pregnancy (CPR) and live birth (LBR) compared to embryos vitrified once.

What is known already

Since vitrification became a core player in ART, many clinics adopted a freeze-all strategy, including vitrification of more than one embryo per vitrification device. Nowadays, the guidelines’ suggestion for single embryo transfers often results in a surplus of good quality embryos post warming which are re-vitrified to avoid embryo wastage. Thus, transfers of double-vitrified embryos have been performed worldwide, leading to successful pregnancies. It remains yet to be answered whether embryos after double vitrification exhibit the same reproductive performance as embryos vitrified once. By now, limited studies including various cryopreservation methods show relatively conflicting results.

Study design, size, duration

This retrospective cohort study was conducted in Embryolab Fertility Clinic, Greece. Assisted reproductive cycles included in the study were performed from January 2015 to December 2020. Both PGT cycles and cryopreserved oocyte cycles were excluded. The mean women age was 32.8 years old (±5.9) in the study group and 33 (±5.9) in the control group. There was no variation in the cryopreservation methods used, since all embryos were cryopreserved by the same vitrification protocol.

Participants/materials, setting, methods

For the present study, 233 embryo transfers of 334 double-vitrified embryos (309 blastocysts/25 cleavage embryos) and 172 transfers of 260 embryos vitrified once (246 blastocysts/14 cleavage) were analyzed to explore the impact of double vitrification on positive β-HCG, CPR, LBR and miscarriage rate. Moreover, the performance of embryos that were cultured for at least 24 hours before re-vitrification was compared to embryos warmed and re-vitrified on the same day, for the same parameters mentioned above.

Main results and the role of chance

All embryos survived vitrification (100% survival rate) in both study and control group. An alpha level of .05 was used for all statistical tests. Results showed that positive β-HCG percentage was significantly lower in the double vitrification group (45.1% for double -vitrified embryos -study group-, 57.0% for embryos vitrified once -control group-, p=.02), as well as CPR (28.3% in study group - 42.4% in control group, p=.003) and LBR (26.6% in study group - 40.4% in control group, p=.004). The miscarriage rate did not significantly differ between two groups (35% in study group - 25% in control group, p=.121). Moreover, positive β-HCG percentage was significantly higher in transfers with embryos that were cultured at least 24 hours before re-vitrification (group A: 48.9%), compared to embryos that were warmed and re-vitrified on the same day (group B: 30.6%, p=.02). Interestingly, although CPR (31% in group A, 18.4% in group B, p=.08), LBR (29.4% in group A, 16.3% in group B, p=.06) and miscarriage rate (35.6% in group A, 33% in group B, p=.87) were not significantly different between these groups, there was a tendency for rates to be higher in group A compared to group B.

Limitations, reasons for caution

This is a retrospective study, including transfers of embryos of different stage (day 2 – day 5). However, the control group was matched to the study group, in terms of embryo stage. The number of previous treatments per couple was not included in the study.

Wider implications of the findings

Although double-vitrified embryos can lead to successful pregnancies, our results showed an adverse effect of double vitrification on embryo reproductive potential. To avoid lower pregnancy rates, culture of embryos until day 5 and cryopreservation of one embryo per vitrification device should be considered.

Trial registration number

not applicabe