Are survival and implantation potential of cryopreserved zygotes affected by time of freezing and duration of storage?

Abstract

OBJECTIVE: There is no consensus on optimum time for freezing pronucleate stage embryos (2PN's). Additionally, little is known regarding any possible adverse effects of long-term storage on implantation potential. Given the dynamic events that occur during syngamy, time-related sensitivities to freezing may exist and may differ for IVF and ICSI 2PN's. The present study was designed to test the hypotheses that: 1) survival and quality of frozen 2PN's is affected by time interval between insemination (IVF) or ICSI and freezing; and 2) that shorter storage time improves 2PN survival and implantation potential. DESIGN: Retrospective cohort study at our academic center. MATERIALS AND METHODS: Autologous cycles were included that resulted in a 2PN freeze-all from 1/1998 through 5/2007 with at least one subsequent cycle in which some of these frozen 2PN's were thawed and transferred on day 3 (CET; n=282; mean age at retrieval: 34.2±4.1y). 2PN's were frozen between 15-25 h post-IVF or ICSI, using Leibo's standard slow freezing and thaw protocol. Since ICSI results in earlier PN formation, cycles were stratified and analysed by type (IVF, n=174; ICSI, n=108). Linear regression analyses were performed to assess the association between time of freezing (i.e. time interval between IVF/ICSI and freeze) and 2PN survival, implantation rate and live-birth rate. Interaction between time to freeze and storage was also investigated. Wald p-values are two-sided. RESULTS: No significant associations were identified for either IVF or ICSI cycles between time of freezing or any of the outcome variables (overall %survival: 86.0±17.4, mean±SD, implantation rate: 20.2%, live birth rate: 39.7%). Similarly, there were no associations between duration of storage and any of these outcome variables. There was also no interaction between time of freezing and duration of storage (range from 0.05 to 6.27 y) for either IVF or ICSI. The only significant observation was an inverse relation between storage time and the number of transferred embryos having at least 6-cells (p=0.03). CONCLUSIONS: Our data show that time of freezing for IVF or ICSI zygotes does not affect survival, implantation or live birth rates. Furthermore, storage of zygotes (at least up to 6.27y) does not affect clinical outcome even when time interval between IVF/ICSI and freeze was considered. Our observation that quality of IVF embryos decreased with increasing years of storage warrants further investigation.