Vitrification of immature mouse oocyte using step-wise protocol

Abstract

OBJECTIVE: To investigate if vitrification using step-wise protocol enhances post-thaw survival, fertilization and early embryonic development of immature mouse oocytes.DESIGN: Animal study.MATERIALS AND METHODS: The cumulus-enclosed oocytes (CEOs) were obtained from CD-1 female mice after injection of PMSG and were incubated for 18 hrs within IVM medium, which consisted of a commercial medium (SAGE-IVF) supplemented with 20% FBS and 75 mIU/mL FSH/LH. After denudation, the matured oocytes (with polar body extrusion) were cryopreserved using two vitrification protocols. In conventional vitrification, up to five oocytes were suspended in m-hTF–based equilibration medium (EM) containing 7.5% EG and 7.5% PROH for 5 min and transferred to vitrification medium (VM) (15% EG, 15% PROH, and 0.5 mol/L sucrose) for 45–60 sec at RT. In the stepwise protocol, the oocytes were placed on a 35 mm plastic culture dish filled with 170 μL of holding medium (HM) at RT. Then 10 μL mixture of 50% EG + 50% PROH was added and the dish was gently rotated for mixing and kept for 2 min, followed by a next-to-final (3rd) addition by the same manner. After exposure to VM for 45–60 sec, the oocytes were loaded onto a Cryoleaf and plunged into liquid nitrogen. For thawing, the Cryoleaf was directly inserted into a 37°C thawing medium (1.0 mol/L sucrose) for 1 min, and then, serially to 0.5 and 0.25 mol/L sucrose for 3 min each. After thawing, survived oocytes were inseminated by epididymal spermatozoa.Table 1Developmental competence of in vitro-matured mouse oocytes after vitrificationGroupsInitiated CEOsMatured oocyte (MR)Survived oocyte after vitrificationCleaved after insemination (FR)Blastocyst (BR)Non-vitrified control139104 (74.8%)-67 (64.4%)9 (13.4%)Conventional vitrification12990 (69.8%)85 (94.4%)28 (32.9%)a1 (3.6%)Step-wise protocol11292 (82.1%)85 (92.4%)53 (62.4%)1 (1.9%)MR: in vitro maturation rate, FR: fertilization rate (per matured), BR; blastocyst rate (per cleaved). a: P<0.05 when compared with non-vitrified control. Each group comprises 5 replicates. Open table in a new tab CONCLUSIONS: Step-wise protocol appears to be good vitrification method for in vitro-matured mouse oocytes. More progress is needed to enhance overall developmental potential of vitrified in vitro-matured oocytes. OBJECTIVE: To investigate if vitrification using step-wise protocol enhances post-thaw survival, fertilization and early embryonic development of immature mouse oocytes. DESIGN: Animal study. MATERIALS AND METHODS: The cumulus-enclosed oocytes (CEOs) were obtained from CD-1 female mice after injection of PMSG and were incubated for 18 hrs within IVM medium, which consisted of a commercial medium (SAGE-IVF) supplemented with 20% FBS and 75 mIU/mL FSH/LH. After denudation, the matured oocytes (with polar body extrusion) were cryopreserved using two vitrification protocols. In conventional vitrification, up to five oocytes were suspended in m-hTF–based equilibration medium (EM) containing 7.5% EG and 7.5% PROH for 5 min and transferred to vitrification medium (VM) (15% EG, 15% PROH, and 0.5 mol/L sucrose) for 45–60 sec at RT. In the stepwise protocol, the oocytes were placed on a 35 mm plastic culture dish filled with 170 μL of holding medium (HM) at RT. Then 10 μL mixture of 50% EG + 50% PROH was added and the dish was gently rotated for mixing and kept for 2 min, followed by a next-to-final (3rd) addition by the same manner. After exposure to VM for 45–60 sec, the oocytes were loaded onto a Cryoleaf and plunged into liquid nitrogen. For thawing, the Cryoleaf was directly inserted into a 37°C thawing medium (1.0 mol/L sucrose) for 1 min, and then, serially to 0.5 and 0.25 mol/L sucrose for 3 min each. After thawing, survived oocytes were inseminated by epididymal spermatozoa. MR: in vitro maturation rate, FR: fertilization rate (per matured), BR; blastocyst rate (per cleaved). a: P<0.05 when compared with non-vitrified control. Each group comprises 5 replicates. CONCLUSIONS: Step-wise protocol appears to be good vitrification method for in vitro-matured mouse oocytes. More progress is needed to enhance overall developmental potential of vitrified in vitro-matured oocytes.