Freezing of cleavage stage human embryos negatively impacts metabolism compared to cryopreservation by vitrification

Abstract

Slow freezing of human embryos conceived through IVF has been widely adopted for over two decades. However, in animal models slow freezing induces significant cellular trauma, including altered metabolism, which can compromise subsequent embryo development. In contrast, the process of ultra rapid vitrification has been shown to impart minimal cellular stress on gametes and embryos, and has now replaced slow freezing as the preferred method of cryopreservation in animal models. The aim of this work was to determine whether slow freezing and vitrification had different effects on human cleavage stage embryo metabolism and development. Laboratory study. Day 3 human embryos were donated for research with consent. Cleavage stage embryos underwent cryopreservation by slow freezing using 1,2-propanediol as the cryoprotectant. Alternatively, embryos underwent ultra rapid vitrification using the Cryoloop using ethylene glycol and 1, 2-propanediol as the cryoprotectants. Upon thawing or warming, embryos were incubated in 1 μl of G2 for 3h for metabolic analysis, and then cultured individually in 10 μl of G2 for 48h. Development to blastocyst and blastocyst quality were scored. The uptake of pyruvate on day 3 following slow freezing was 11.4 ± 0.9 pmol/embryo/h, compared to 19.1 ± 1.1 for embryos that had been vitrified (P<0.01). Blastocyst development and quality were enhanced in the vitrified group. Following warming, cryosurvived embryos (93.5%) were cultured for 24 h in medium G2 and then transferred. A mean of 2.4 embryos were transferred in 7 patients, resulting in a pregnancy rate of 57%. Tabled 1Table.Slow freezingVitrificationCryosurvival (%)98/110 (89.0)103/109 (94.4)Embryos with 100% blastomere survival (%)50/98 (51.0)80/103 (77.6)∗P<0.01.Blastocyst formation (%)49/98 (50.0)62/103 (60.1)Blasts.≥ 3 AA (%)21/49 (42.8)32/62 (51.6)Hatching blastocyst (%)10/49 (20.4)19/62 (30.6)∗ P<0.01. Open table in a new tab This study has shown for the first time that human embryo metabolism is significantly depressed following slow freezing. In contrast, embryos that were vitrified had a metabolism activity almost twice that of the frozen embryos. Subsequent embryo development appeared to be elevated following vitrification. These data support the hypothesis that vitrification is associated with less cellular trauma than slow freezing and should be considered as the primary method of human embryo cryopreservation.