Comparison of Different Cryopreservation Techniques: Higher Survival and Implantation Rate of Frozen–Thawed Mouse Pronuclear Embryos in the Presence of Beta‐Mercaptoethanol in Post‐Thaw Culture

Abstract

The objective of this study was to investigate the effects of beta-mercaptoethanol (β-ME) on post-thaw embryo developmental competence and implantation rate of mouse pronuclear (PN) embryos that were cryopreserved after slow freezing, solid surface vitrification (SSV) or open-pulled straw (OPS) vitrification methods. Mouse PN embryos were cryopreserved by using slow freezing, SSV and OPS methods. After cryopreservation, freeze-thawed PN embryos were cultured up to blastocyst stage in a defined medium supplemented without or with 50 μM β-ME. The blastocyst formation rate of embryos that were cryopreserved by slow freezing method (40.0%) or vitrified by OPS method (18.3%) were lower than those vitrified by SSV method (55.6%) and fresh embryos (61.9%) in the absence of 50 β-ME in the culture media (p < 0.05). The blastocyst formation rate of embryos that were cryopreserved by slow freezing method (53.1%) or by OPS method (41.9%) were lower than those vitrified by SSV method (79.5%) and that of fresh (85.7%) in the presence of β-ME in the culture media (p < 0.05). The embryos transfer results revealed that the implantation rate of blastocyst derived from mouse PN embryos vitrified by SSV method (31.9% vs 51.2%) was similar to that of the control (39.0% vs 52.5%), but higher than those cryopreserved by slow freezing (28.2% vs 52.0%) and by OPS method (0.0% vs 51.2%) (p < 0.05). In conclusion, supplementation of β-ME in an in vitro culture medium was shown to increase survival of embryo development and implantation rate of frozen-thawed mouse PN embryos after different cryopreservation protocols.

Comparison of the development of mouse embryos manipulatedwith different biopsy techniques

“…Similarly, the embryos were placed in biopsy medium for 30 min to loosen the bonds between the blastomeres to prevent compacting. In the study presented here, the rates of embryo development with the aspiration technique used in the eight-cell stage and the development rates in the study by Krzyminska et al (13) were found to be similar.…”

“…Bagis et al (13) obtained embryos from CB6F1 mice and cultured them in SAGE medium until the blastocyst stage. Their result for mean total cell number was 58.9.…”

“…CB6F1 (C57BL⁄6 × BALB⁄c) female mice were injected intraperitoneally with 10 IU of pregnant mare serum gonadotropin (Sigma PMSG). Superovulation protocol was completed by intraperitoneal administration of 7.5 IU of human chorionic gonadotropin (Organon hCG) 48 h after the injection, and female mice were mated with male CB6F1 (C57BL⁄6 × BALB⁄C) mice (13). The superovulated females were sacrificed 68-72 h after hCG administration.…”

Littermate Influence on Infant Growth in Mice: Comparison of SJL/J and ICR as Cotransferred Carrier Embryos

“…unknown damages caused by somatic cell nuclear transfer can reduce the number of healthy embryos [18], while cotransfer compensates for this by increasing the total number of healthy embryos to trigger and maintain pregnancy. Likewise, in the case of cryopreservation, some reports have described damage to freeze-thawed embryos [1,24], implying that cotransfer of carrier embryos could assist the implantation and subsequent development of freeze-thawed embryos.…”

Efficiency and Safety of Human Reproductive Cell/Tissue Vitrification

“…As it is valid for most of the rather new technologies, case reports on vitrification of zygotes were published in series (Jelinkova et al, 2002;Selman and El-Danasouri, 2002;Kumasako et al 2005). Even under aggravated conditions, such as the presence of an artificial gap in the zona pellucida after polar body biopsy, unsuspicious development to blastocyst stage and live births were reported for mice and humans (Isachenko et al, 2005a, Naether et al, 2008Macas et al, 2008Macas et al, , 2011Bagis et al, 2010;Vanderzvalmen et al 2012). Interestingly, re-vitrification of previously vitrified and warmed 2PN-stages turned out to be a feasible option Kumasako et al, 2009) although not always resulting in pregnancy and live birth (Hashimoto et al, 2007).…”