(79) Embryology at IVF in the Common Marmoset

Rebecca A. Holton1; Joshua Brennan1; James Pickel1; Bhuchitra Singh1; Nia Bryan2; Xiaoqin Wang2; James Segars1 

1. Department of Gynecology and Obstetrics, Johns Hopkins University School of Medicine, Baltimore, MD, USA;
2. Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, USA 


Abstract Text: The marmoset has emerged as a model species for genetic studies of complex neurodevelopmental conditions because of their brain development, behavior and communication, and compact size. In vitro fertilization (IVF) has been used to generate wild type and transgenic marmoset embryos, but inefficiency of IVF in the marmoset compared to other primates poses challenges for genetic manipulation. To address this limitation, we analyzed the association between variables of age, number of cycles, and progesterone levels with the IVF outcomes of number of oocytes collected, fertilization rate, and embryonic cleavage rates. Serum progesterone levels were measured after Estrumate (1.0 mg/animal) using ELISA, prior to ovarian stimulation with FSH. Typically, FSH (25 IU) was administered every day, beginning on the day following Estrumate. Oocytes were collected in Porcine Oocyte Media (POM) containing Heparin and matured overnight in hormone supplemented POM at 38 degrees C and 5% CO2. Mature eggs were fertilized with 2 µL sperm (approximately 500 million motile sperm per mL) and cultured in FBS fortified cleavage media (SAGE). Embryos were scored according to the stage of development. Data were analyzed using linear regression, T-test or Chi-square and a p< 0.05 was used to assign significance. An analysis of data collected over a two-year period indicate that progesterone levels 24 hours after Estrumate administration, the number of days of ovarian stimulation during an IVF cycle, and the number of oocyte retrievals an animal had undergone all influenced IVF cycle outcomes. Specifically, there was a positive correlation between progesterone level post Estrumate reset (typically <10ng/mL) and number of cycles a marmoset had undergone (R squared=0.3752, p=0.0152, n=15), with higher progesterone levels with each subsequent IVF cycle. Higher serum progesterone levels at cycle start were negatively correlated with the likelihood of fertilized eggs progressing to the cleavage stage of embryo development (R squared=0.3519, p=0.0154, n=16). Additionally, a shorter duration of ovarian stimulation (ranging 7 to 14 days of medication) correlated with an increase percentage of fertilized eggs (R squared=0.2065, p=0.0025, n=42). We observed a trend that a shorter hormonal stimulation cycle length was associated with a higher percentage of embryos reaching the cleavage stage (R squared=0.0519, p=0.0519, n=42). These findings suggest that shorter IVF cycles and a progesterone level < 10 ng/mL at cycle start were correlated with an increased likelihood of a cleavage stage embryo development. This work was supported by U01DA056556.