Freezer burn or learning curve? Prolonged time since blastocyst vitrification and impact on pregnancy outcomes

In this issue of Fertility and Sterility, Yan et al. (1Yan Y. Zhang Q. Yang L. Zhou W. Ni T. Yan J. Pregnancy and neonatal outcomes after long-term vitrification of blastocysts among 6,900 patients following their last live birth.Fertil Steril. 2023; 119: 36-44Abstract Full Text Full Text PDF Scopus (3) Google Scholar) presented a retrospective, observational, single-center cohort study to investigate the effect of time after blastocyst vitrification on pregnancy and neonatal outcomes. The 6900 women included had a live birth from a prior ovarian stimulation between January 2011 and January 2021 and underwent subsequent transfer of a vitrified-thawed blast from the same ovarian stimulation cohort <3–10.5 years after vitrification. Of these, 71 transfer cycles were canceled owing to blastocyst degeneration during thawing. Ultimately, 6,289 patients underwent their first vitrified-thawed blastocyst transfer after live birth and were analyzed into 5 storage duration groups: group 1 (1,874 transfers with storage duration <3 years), group 2 (2,672 transfers with storage duration 3–4 years), group 3 (1,329 transfers with storage duration 4–5 years), group 4 (569 transfers with storage duration 5–6 years), and group 5 (385 transfers with storage duration ≥6 years and ≤10.5 years). The mean storage duration for groups 1–5 was 30.1, 40.9, 52.6, 64.6, and 82.1 months, respectively. However, groups with embryos stored for 3–6 years did not illustrate significantly different biochemical, clinical pregnancy, live birth, miscarriage, or ectopic rates compared with embryos stored for <3 years. In both unadjusted and the multivariate logistic regression analyses controlling for maternal age at transfer, infertility diagnosis, the number of blastocysts transferred, and blastocyst quality at vitrification and transfer, vitrified blastocysts stored for ≥6 years were less likely to result in biochemical pregnancy 0.71 (95% CI, 0.56-0.90), clinical pregnancy 0.69 (95% CI, 0.54-0.87), or live birth 0.73 (95% CI, 0.58-0.93) when compared with embryos stored for <3 years. Neonatal outcomes including fetal sex, birth weight, and preterm delivery were statistically comparable between storage duration groups. This impressively large study of the first vitrified blastocyst embryo transfer after live birth adds to the body of literature assessing the influence of storage time on vitrified embryo survival and pregnancy and neonatal outcomes after the transfer. Previous studies have been limited by shorter cryostorage duration, the inclusion of cleavage stage embryos, and sample size (2Canosa S. Cimadomo D. Conforti A. Maggiulli R. Giancani A. Tallarita A. et al.The effect of extended cryo-storage following vitrification on embryo competence: a systematic review and meta-analysis.J Assist Reprod Genet. 2022; 39: 873-882Crossref PubMed Scopus (8) Google Scholar, 3Ueno S. Uchiyama K. Kuroda T. Yabuuchi A. Ezoe K. Okimura T. et al.Cryostorage duration does not affect pregnancy and neonatal outcomes: a retrospective single-centre cohort study of vitrified warmed blastocysts.Reprod Biomed Online. 2018; 36: 614-619Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 4Li J. Yin M. Wang B. Lin J. Chen Q. Wang N. et al.The effect of storage time after vitrification on pregnancy and neonatal outcomes among 24 698 patients following the first embryo transfer cycles.Hum Reprod. 2020; 35: 1675-1684Crossref PubMed Scopus (26) Google Scholar). A retrospective cohort study by Li et al. (4Li J. Yin M. Wang B. Lin J. Chen Q. Wang N. et al.The effect of storage time after vitrification on pregnancy and neonatal outcomes among 24 698 patients following the first embryo transfer cycles.Hum Reprod. 2020; 35: 1675-1684Crossref PubMed Scopus (26) Google Scholar) on 24,698 first vitrified embryo transfers after freeze-all cycles revealed that increasing the storage time of vitrified embryos negatively affected implantation rate, clinical pregnancy rate, and live birth rate. However, the findings of Li et al. (4Li J. Yin M. Wang B. Lin J. Chen Q. Wang N. et al.The effect of storage time after vitrification on pregnancy and neonatal outcomes among 24 698 patients following the first embryo transfer cycles.Hum Reprod. 2020; 35: 1675-1684Crossref PubMed Scopus (26) Google Scholar) have limited generalizability because >90% of embryos included in the study were vitrified cleavage stage embryos. Furthermore, the maternal age at oocyte retrieval was significantly higher with increasing storage time. Thus, the observed adverse pregnancy outcomes may be a result of increasing maternal age's effect on oocyte quality rather than prolonged cryopreservation. A meta-analysis by Canosa et al. (2Canosa S. Cimadomo D. Conforti A. Maggiulli R. Giancani A. Tallarita A. et al.The effect of extended cryo-storage following vitrification on embryo competence: a systematic review and meta-analysis.J Assist Reprod Genet. 2022; 39: 873-882Crossref PubMed Scopus (8) Google Scholar) assessed the putative impact of extended cryostorage on embryo competence, including both cleavage and blastocyst transfers. They found prolonged time since vitrification (>12 months) was associated with lower embryo survival with thawing, but showed no impact on pregnancy outcomes, even within subgroups of untested (N=13,596 embryos) and euploid vitrified blastocysts (N=2,712 embryos). Moreover, Canosa et al. compared the embryos stored for <12 months to those stored for ≥12 months, citing clinical relevance for the 12-month duration cut-off. Although most of the studies analyzed included embryos with <6 years of cryostorage, the study by Ueno et al. (4Li J. Yin M. Wang B. Lin J. Chen Q. Wang N. et al.The effect of storage time after vitrification on pregnancy and neonatal outcomes among 24 698 patients following the first embryo transfer cycles.Hum Reprod. 2020; 35: 1675-1684Crossref PubMed Scopus (26) Google Scholar) was the only one with embryos stored for >6 years. They compared the pregnancy and neonatal outcomes for embryos stored for 0–2, 2–12, vs. 13–97 months, finding no difference between the groups. However, only 1,181 embryos were frozen for 13–97 months, with likely a very small percentage of embryos cryopreserved for >6 years. Although the cryostorage time did not affect the live birth rate, the time from insemination to vitrification was negatively correlated with the live birth rate (4Li J. Yin M. Wang B. Lin J. Chen Q. Wang N. et al.The effect of storage time after vitrification on pregnancy and neonatal outcomes among 24 698 patients following the first embryo transfer cycles.Hum Reprod. 2020; 35: 1675-1684Crossref PubMed Scopus (26) Google Scholar). Yan et al. (1Yan Y. Zhang Q. Yang L. Zhou W. Ni T. Yan J. Pregnancy and neonatal outcomes after long-term vitrification of blastocysts among 6,900 patients following their last live birth.Fertil Steril. 2023; 119: 36-44Abstract Full Text Full Text PDF Scopus (3) Google Scholar) expanded on these works by only including blastocysts vitrified and thawed from a prior controlled ovarian hyperstimulation cycle that previously resulted in a live birth. The use of blastocysts derived from a solitary retrieval minimizes the oocyte’s quality heterogeneity which may be associated with different stimulations and more closely mimics clinical practice in which a prolonged gap in embryo transfer may occur after a successful transfer. Unfortunately, the investigators did not include the time from insemination to vitrification for embryos in their analysis, a variable that Ueno et al. found significantly affected the live birth rate. With the advent and refinement of cryopreservation techniques, vitrification has replaced the slow-freeze method in the past decades. In contrast to slow freezing, vitrification involves embryo exposure to increasing concentrations of permeating cryoprotectants, such as ethylene glycol or dimethyl sulfoxide; these permeating cryoprotectants diffuse through the cell membrane and form stabilizing hydrogen bonds, thereby preventing the formation of ice crystals within the cells. Thus, vitrification decreases the likelihood of cellular damage during thawing. As a result, vitrified/warmed embryos have repeatedly demonstrated superior embryo survival rates and clinical pregnancy rates when compared with slow-freeze/thawed embryos. Various systems and protocols have been developed and tried over time with variable mixtures and concentrations of cryoprotectants in closed vs. open systems (5Schiewe M.C. Anderson R.E. Vitrification: the pioneering past to current trends and perspectives of cryopreserving human embryos, gametes, and reproductive tissue.Journal of Biorepository Science for Applied Medicine. 2017; 5: 57-68Crossref Google Scholar). Thus, blastocyst vitrification techniques have been refined over time. Yan et al. (1Yan Y. Zhang Q. Yang L. Zhou W. Ni T. Yan J. Pregnancy and neonatal outcomes after long-term vitrification of blastocysts among 6,900 patients following their last live birth.Fertil Steril. 2023; 119: 36-44Abstract Full Text Full Text PDF Scopus (3) Google Scholar) included embryos vitrified in January 2011, at the inception of this novel technique, till January 2021, when vitrification became a routine procedure. The impact of time on vitrified embryo transfer success was only seen in the group with ≥6 years since cryopreservation. Thus, the differences in observed clinical outcomes may not have been due to cryopreservation duration, but rather variations in embryologists’ techniques, best practices, and cryoprotectant concentrations. Unfortunately, Yan et al. (1Yan Y. Zhang Q. Yang L. Zhou W. Ni T. Yan J. Pregnancy and neonatal outcomes after long-term vitrification of blastocysts among 6,900 patients following their last live birth.Fertil Steril. 2023; 119: 36-44Abstract Full Text Full Text PDF Scopus (3) Google Scholar) failed to control for both embryologist experience and alterations in vitrification media/technique which may have occurred during these 10 years. Despite these limitations, the research by Yan et al. (1Yan Y. Zhang Q. Yang L. Zhou W. Ni T. Yan J. Pregnancy and neonatal outcomes after long-term vitrification of blastocysts among 6,900 patients following their last live birth.Fertil Steril. 2023; 119: 36-44Abstract Full Text Full Text PDF Scopus (3) Google Scholar) is the largest study to date with the longest storage duration assessing the impact of storage time on pregnancy and neonatal outcomes after vitrified-thawed blastocyst transfer. Overall, the data are reassuring with up to 6 years of storage conferring minimal risk as evidenced by the lack of statistical difference in pregnancy outcomes between the groups with 3–6 years of storage compared with the embryos frozen for <3 years. Future studies controlling for vitrification media and embryologist experience as well as time from insemination to vitrification are needed to further characterize the lower implantation rates, clinical pregnancy rates, and live birth rates in patients undergoing vitrified-thawed blastocyst transfer after a storage duration of >6 years. Pregnancy and neonatal outcomes after long-term vitrification of blastocysts among 6,900 patients after their last live birthFertility and SterilityVol. 119Issue 1PreviewTo evaluate whether prolonged storage of vitrified blastocysts negatively impacts pregnancy and neonatal outcomes. Full-Text PDF