Factors associated with birth outcomes from cryopreserved blastocysts: experience from 4,597 autologous transfers of 7,597 cryopreserved blastocysts  Kevin S. Richter, Ph.D., Daniella K. Ginsburg, B.A., Sharon K. Shipley, M.S., Josh Lim, M.S., Michael J. Tucker, Ph.D., James R. Graham, M.S., Michael J. Levy, M.D.  Fertility and Sterility  Volume 106, Issue 2, Pages 354-362.e2 (August 2016) DOI: 10.1016/j.fertnstert.2016.04.022 Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Figure 1 The percentages of live births per transfer cycle and live born children per transferred embryo according to patient age at the time of oocyte retrieval and embryo cryopreservation, with slow freeze (SF) illustrated in purple and vitrification (VIT) illustrated in green. Fertility and Sterility 2016 106, 354-362.e2DOI: (10.1016/j.fertnstert.2016.04.022) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Least squares means of live born children per transferred embryo and live births per transfer cycle, derived from GEE analysis accounting for repeated cycles by the same patients and adjusting for age at cryopreservation, cryopreservation day, percentage of intact cells and number of embryos transferred as appropriate. (A) According to cryopreservation day, after slow freezing. (B) According to cryopreservation day, after vitrification. (C) According to the percentage of intact cells, after slow freezing. (D) According to the percentage of intact cells, after vitrification. (E) According to the number of embryos transferred, after slow freezing. (F) According to the number of embryos transferred, after vitrification. Fertility and Sterility 2016 106, 354-362.e2DOI: (10.1016/j.fertnstert.2016.04.022) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Photographs illustrating representative examples of different percentages of intact cells in blastocysts after cryopreservation and thaw/warming. It must be noted that these photographic images are intended to illustrate representative examples corresponding to the indicated cell survival estimates following embryo cryopreservation and thawing/warming, to the best of our ability in this limited two-dimensional format. In practice, actual survival measures are determined based on thorough three-dimensional viewing and rotation, focusing carefully through numerous focal planes of each embryo. This process is to some extent unavoidably subjective, as it depends on the experience of the embryologist to estimate the percentage of cell loss through identification of such features as degenerative and necrotic foci and anuclear cellular fragments. Despite the subjectivity inherent in cell survival measurement, the clear and strong association between these cell survival estimates and birth outcomes documented in this study establishes the validity of this parameter as a valuable clinical tool in the assessment of blastocyst viability after cryopreservation. Fertility and Sterility 2016 106, 354-362.e2DOI: (10.1016/j.fertnstert.2016.04.022) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 1 The percentages of live born children per transferred embryo and live births per transfer cycle by treatment year. Fertility and Sterility 2016 106, 354-362.e2DOI: (10.1016/j.fertnstert.2016.04.022) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 2 The percentages of live born children per transferred embryo and live births per transfer cycle among blastocysts with delayed expansion and cryopreservation on day 7, according to the percentage of intact cells. Sample sizes of numbers of embryos and numbers of transfers are reported in parentheses. Fertility and Sterility 2016 106, 354-362.e2DOI: (10.1016/j.fertnstert.2016.04.022) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions