Successful vitrification of embryos ≤500µm without puncture or aspiration of the blastocoele

Abstract

To achieve good pregnancy rates following vitrification, embryos >300µm require micromanipulator-assisted puncture and blastocoele collapse. Without using a micromanipulator, Wilsher et al (Equine Veterinary Journal.2021;53:1227-1233) reported high pregnancy rates following vitrification of embryos 300-560µm using manual puncture but failed to achieve any pregnancies with non-punctured embryos. These manually punctured embryos were exposed to an equilibration medium (EM; HEPES culture medium + 7.5% DMSO + 7.5% ethylene glycol [EG]) for 6min. Extending the time to 8min in the non-puncture group did not improve results. The aim of this study was to determine if non-punctured embryos, exposed to considerably longer in EM (i.e.,15min) would, upon warming and transfer to recipients, result in acceptable pregnancy rates. Thirty, Grade 1 embryos were recovered on Day 7-8 post ovulation and divided into three groups based on size, G1 (≤300 µm; n = 8), G2 (>300 to ≤500µm; n=16) and G3 (>500µm; n=6). Median (range) embryo diameters for Groups 1 to 3 were 237.5 (180–280), 410 (310–500) and 575 (510–660) µm, respectively. Embryos were vitrified with a commercial human vitrification kit (Kitazato) utilising 2-step concentrations of DMSO and EG (7.5-15% v:v) in a HEPES culture medium containing gentamycin, trelahose and hydroxypropyl cellulose. At room temperature (RT) embryos were placed in EM for 15min prior toa vitrification solution for ≤90 sec, during which time they were loaded onto a Cryolock device, capped and plunged into LN2. Embryos were warmed in 1M sucrose solution of holding media (HM; H199 + 20% FBS + antibiotics) at 42°C for 1min before being transferred to HM + 0.5M sucrose at RT for 4min, then into Vigro Hold for a further 4min. Within 15min, embryos were transferred to recipient mares 6 days post ovulation. Mares were checked thrice for pregnancy and to ensure continuing embryonic development before administration of prostaglandin. Two mares were left pregnant until the heartbeat and expanding allantois stage in groups achieving pregnancies. G1 and G2 embryos achieved a higher pregnancy rate than G3 in which no embryos survived (87.5% [7/8] vs. 75% [12/16] vs. 0% [0/6], respectively; P<0.001). One early embryonic death (EED) occurred in G2 (350µm) between Days 12-14. The four embryos in G2 that did not result in a pregnancy were 330, 380, 425 and 500µm. This study shows that non-punctured embryos <500µm can survive vitrification and result in excellent pregnancy rates (19/24 [79%]) following a 15min exposure to equilibration medium containing 7.5%EG and 7.5%DMSO. Larger numbers of pregnancies are required to ensure that the longer exposure time to cryoprotectants does not increase the risk of EED. Nevertheless, this protocol negates the need to puncture embryos <500µm providing an easy and successful way to vitrify them.