Prolonged maturation reduces cleavage and blastocyst rates after ICSI

Abstract

Most equine assisted reproduction laboratories use overnight room-temperature holding of immature oocytes, followed by in vitro maturation (IVM) for ∼30 hours. The period of maturation may vary according to laboratory workload and shipment schedules. Prolonged IVM (36 h) has been reported to increase blastocyst rates, and in vivo-matured oocytes are typically cultured until 40 to 46 h after gonadotrophin administration before ICSI is performed. There is no information on whether prolonged IVM (48 h) of immature oocytes supports acceptable blastocyst production. We compared maturation, cleavage and blastocyst rates for oocytes held overnight before being cultured for 30 or 48 h before ICSI. We noted that after IVM, some oocytes appeared mature (intact plasmalemma, heterogenous cytoplasm) but lacked a visible polar body, and we assessed whether these oocytes could produce blastocysts. Oocytes from slaughterhouse-derived ovaries were held at room temperature overnight (17 to 20 h) then randomly divided into two groups (n = 115 each): T30, 30 hours IVM culture; and T48, 48 hours IVM culture. After IVM, oocytes were divided into those with a visible polar body (MII) and those that appeared mature but without a polar body (PB-). Frozen-thawed semen from a single stallion was used for ICSI.The injected oocytes were cultured in vitro using a sequential mix of human embryo culture medium, DMEM/F-12, and FBS. Differences between groups were analyzed by Fisher's exact test. There was no difference (P > 0.1) between T30 and T48 in percentage of MII oocytes (37.4% and 32.3%, respectively), percentage of PB- oocytes (8.7% and 15.7%, respectively) or total percentage of oocytes with intact cytoplasm (MII+PB-; 46.1% and 47.8%, respectively). Within maturation duration, there were no significant differences in cleavage or blastocyst rate between MII and PB- oocytes (e.g. for T30, 27.9% and 20% blastocysts, respectively), so data for MII and PB- were combined. The cleavage rate was significantly higher for T30 than T48 (69.8% vs. 41.8%, p = 0.004). Similarly, the blastocyst rate per injected oocyte (26.4% vs. 5.5%, p = 0.003) and the blastocyst rate per cleaved oocyte (38% vs. 13%; p = 0.045) were significantly higher for T30 than T48. We conclude that increasing the duration of IVM to 48 hours does not increase the number of mature oocytes obtained, and indeed significantly decreases oocyte developmental competence. However, we were able to obtain blastocysts after 48 hours IVM, which has not been reported previously. After 30 h IVM, oocytes with a mature morphology but without a visible polar body may be used effectively for ICSI.