P-241: Removal of bacterial contamination from developing blastocysts in extended culture via micromanipulation in PVP

Abstract

The presence of bacterial contamination in human embryos, resistant to antibiotics and sperm processing-purification, has been recently identified to occur after initiation of extended culture. Previous reports have identified Enterococcus faecalis, a Gram-positive bacteria that is resistant to many commonly used antibiotics, as the main cause of bacterial contamination. Removal of bacteria has been attempted by transferring the embryos via pipetting to fresh media every 24 hrs until the performance of ET. However, this approach represents a short-term solution because even minute amounts of the contaminated media may contain enough bacteria to colonize fresh media or affect the implantation and pregnancy In utero. Furthermore, another source for the bacteria may also be present in the zona pellucida (ZP). The objective of this study was to develop a method of containing or isolate the bacteria via micromanipulation in a medium of high viscosity before transferring the embryos to fresh media used for culture and ET. Case report. Bacterial contamination of culture media was detected on Day 4, following the transfer of embryos from cleavage media (Day 3) to blastocyst media for extended culture. Isolation of bacterial contamination was performed via micromanipulation of the embryos in PVP (7% (v/v) + 5 mg HSA/ml). The embryos were placed in a drop of PVP and a holding pipet was introduced next to the embryo. The selected embryo was moved to a separate area of the PVP drop and the holding pipet was raised over the plane of visibility in reference to the embryo at the 6 o’clock position. Negative pressure was applied to the holding pipet, which resulted in the formation of a vortex and a spinning motion on the embryo. The embryo was simultaneously moved in a zigzag route to aid in the removal and isolation of bacteria. The embryo was then placed in the opposite area of the PVP drop from where it was originally placed. The embryo was rinsed 3 times in blastocyst media and kept in culture until the time of ET or cryopreservation. The same treatment was applied to all embryos considered for ET or cryopreservation. All dishes where the embryos had contact with culture media or reagents were kept for examination on the next day. Most embryos were at the compact morula or early blastocyst stages when treated with PVP to isolate the bacteria. Culture drops in which the embryos resided before PVP treatment showed bacterial colonization at 24 hrs. The culture drops in which the embryos were cultured after PVP treatment were free of bacterial contamination at 24 hrs post-treatment. Embryos continued developing as expected for day 5 following PVP treatment. Two expanded blastocysts were transferred on day 5 resulting in an ongoing twin pregnancy. The remaining embryos were cryopreserved. Exposure and micromanipulation of embryos in PVP resulted in the isolation of bacteria as evident by the absence of colonization in fresh media 24 hrs after treatment. It is possible that the viscous properties of PVP serve to mechanically dislodge the bacteria from the ZP and also contain the bacteria and prevent re-colonization during the micromanipulation of the embryos. Treatment with PVP seems to be an adequate alternative to avoid transmitting bacteria once established in extended culture, and which can also represent risks for the establishment of implantation and pregnancy once the embryos are replaced in the uterus.