ASSESSMENT OF COMPLETE END TO END VAPOR PHASE NITROGEN SHIPPING AND STORAGE ON GAMETE AND BLASTOCYST QUALITY

BackgroundVapor phase Nitrogen storage allows for maximization of square footage and the application of automation to minimize specimen mishandling. The temperature variations that occur with vapor phase shipment and storage are largely unknown. Likewise, the impact of vapor phase compared with liquid phase storage on survival and viability remains a topic of study.ObjectiveTo assay the impact of shipment and storage of sperm, oocytes and blastocysts in vapor.Materials and MethodsFive blastocyst were shipped from one main laboratory (lab 1) in North America to both another domestic lab (lab 2) as well as an international lab location (lab 3) using a standard vapor shipping process. Matched controls were maintained in Liquid Nitrogen dewars. At lab 2 and lab 3 samples were reformatted using RFID tagged proprietary labware to ensure safe handling of embryos through subsequent shipment and storage, while providing a digital chain of custody. Environmental telemetry, including temperature at the embryo level, was started at this time and continued with once a minute measurement through return shipment to lab 1 and storage in a vapor phase TMRW platform using TMRW software, labware and robotics. Following at least 7-days of storage in the vapor tank, the blastocyst and their matched controls stored in liquid were thawed. To ensure a robust assessment, a previously described extended culture system was used. Data from the extended culture included: attachment to fibronectin coated plates and outgrowth area on outgrowth day (OGD) 2, 3, 4, and 5, the presence of epiblast cells, epiblast cell number and total cell number on OGD 5, and hCG concentrations in the spent media quantified by ELISA on OGD 3, 4, and 5. Additionally, sperm and oocytes were stored in both vapor and liquid phases and warmed to compare survival rates.ResultsThe initial study design called for 5 total lab locations. Coronavirus delayed the initiation of this study by 4 months. Approximately 4 days prior to reformatting of the samples, some locations had state and national lock down restrictions implemented. This limited the available personnel able to travel and necessitated a last-minute reassignment of schedules. Analysis of the available temperature data during shipment and storage in the TMRW platform is reassuring that the embryos did not experience a temperature excursion that would have compromised their integrity. The highest temperature recorded for lab 2 was -180.2OC with a mean of -190.4±0.5OC during shipment and -181.1±0.6OC during storage. Likewise, lab 3 had a high of -180.2OC with a mean of -193.5±0.6OC during shipment and -181.2±0.7OC during storage. Results from the extended culture assays have revealed no deleterious effect of shipment and storage in nitrogen vapor.ConclusionsShipping and storage in the vapor phase offers various logistical advantages. The recent Coronavirus pandemic resulted in interruptions in shipping and other logistics, demonstrating the need to validate the impact of vapor phase storage compare to liquid. The data collected during shipment and storage in the TMRW robotic platform provides clarity that gametes and embryos are not being damaged due to temperature excursions.Financial SupportThis project was supported by internal research funds of CCRM and TMRW Life Sciences, Inc. BackgroundVapor phase Nitrogen storage allows for maximization of square footage and the application of automation to minimize specimen mishandling. The temperature variations that occur with vapor phase shipment and storage are largely unknown. Likewise, the impact of vapor phase compared with liquid phase storage on survival and viability remains a topic of study. Vapor phase Nitrogen storage allows for maximization of square footage and the application of automation to minimize specimen mishandling. The temperature variations that occur with vapor phase shipment and storage are largely unknown. Likewise, the impact of vapor phase compared with liquid phase storage on survival and viability remains a topic of study. ObjectiveTo assay the impact of shipment and storage of sperm, oocytes and blastocysts in vapor. To assay the impact of shipment and storage of sperm, oocytes and blastocysts in vapor. Materials and MethodsFive blastocyst were shipped from one main laboratory (lab 1) in North America to both another domestic lab (lab 2) as well as an international lab location (lab 3) using a standard vapor shipping process. Matched controls were maintained in Liquid Nitrogen dewars. At lab 2 and lab 3 samples were reformatted using RFID tagged proprietary labware to ensure safe handling of embryos through subsequent shipment and storage, while providing a digital chain of custody. Environmental telemetry, including temperature at the embryo level, was started at this time and continued with once a minute measurement through return shipment to lab 1 and storage in a vapor phase TMRW platform using TMRW software, labware and robotics. Following at least 7-days of storage in the vapor tank, the blastocyst and their matched controls stored in liquid were thawed. To ensure a robust assessment, a previously described extended culture system was used. Data from the extended culture included: attachment to fibronectin coated plates and outgrowth area on outgrowth day (OGD) 2, 3, 4, and 5, the presence of epiblast cells, epiblast cell number and total cell number on OGD 5, and hCG concentrations in the spent media quantified by ELISA on OGD 3, 4, and 5. Additionally, sperm and oocytes were stored in both vapor and liquid phases and warmed to compare survival rates. Five blastocyst were shipped from one main laboratory (lab 1) in North America to both another domestic lab (lab 2) as well as an international lab location (lab 3) using a standard vapor shipping process. Matched controls were maintained in Liquid Nitrogen dewars. At lab 2 and lab 3 samples were reformatted using RFID tagged proprietary labware to ensure safe handling of embryos through subsequent shipment and storage, while providing a digital chain of custody. Environmental telemetry, including temperature at the embryo level, was started at this time and continued with once a minute measurement through return shipment to lab 1 and storage in a vapor phase TMRW platform using TMRW software, labware and robotics. Following at least 7-days of storage in the vapor tank, the blastocyst and their matched controls stored in liquid were thawed. To ensure a robust assessment, a previously described extended culture system was used. Data from the extended culture included: attachment to fibronectin coated plates and outgrowth area on outgrowth day (OGD) 2, 3, 4, and 5, the presence of epiblast cells, epiblast cell number and total cell number on OGD 5, and hCG concentrations in the spent media quantified by ELISA on OGD 3, 4, and 5. Additionally, sperm and oocytes were stored in both vapor and liquid phases and warmed to compare survival rates. ResultsThe initial study design called for 5 total lab locations. Coronavirus delayed the initiation of this study by 4 months. Approximately 4 days prior to reformatting of the samples, some locations had state and national lock down restrictions implemented. This limited the available personnel able to travel and necessitated a last-minute reassignment of schedules. Analysis of the available temperature data during shipment and storage in the TMRW platform is reassuring that the embryos did not experience a temperature excursion that would have compromised their integrity. The highest temperature recorded for lab 2 was -180.2OC with a mean of -190.4±0.5OC during shipment and -181.1±0.6OC during storage. Likewise, lab 3 had a high of -180.2OC with a mean of -193.5±0.6OC during shipment and -181.2±0.7OC during storage. Results from the extended culture assays have revealed no deleterious effect of shipment and storage in nitrogen vapor. The initial study design called for 5 total lab locations. Coronavirus delayed the initiation of this study by 4 months. Approximately 4 days prior to reformatting of the samples, some locations had state and national lock down restrictions implemented. This limited the available personnel able to travel and necessitated a last-minute reassignment of schedules. Analysis of the available temperature data during shipment and storage in the TMRW platform is reassuring that the embryos did not experience a temperature excursion that would have compromised their integrity. The highest temperature recorded for lab 2 was -180.2OC with a mean of -190.4±0.5OC during shipment and -181.1±0.6OC during storage. Likewise, lab 3 had a high of -180.2OC with a mean of -193.5±0.6OC during shipment and -181.2±0.7OC during storage. Results from the extended culture assays have revealed no deleterious effect of shipment and storage in nitrogen vapor. ConclusionsShipping and storage in the vapor phase offers various logistical advantages. The recent Coronavirus pandemic resulted in interruptions in shipping and other logistics, demonstrating the need to validate the impact of vapor phase storage compare to liquid. The data collected during shipment and storage in the TMRW robotic platform provides clarity that gametes and embryos are not being damaged due to temperature excursions. Shipping and storage in the vapor phase offers various logistical advantages. The recent Coronavirus pandemic resulted in interruptions in shipping and other logistics, demonstrating the need to validate the impact of vapor phase storage compare to liquid. The data collected during shipment and storage in the TMRW robotic platform provides clarity that gametes and embryos are not being damaged due to temperature excursions.