Parthenogenic activation of discarded in vitro matured, vitrified and rewarmed human oocytes for stem cell production

Abstract

OBJECTIVE: Oocyte parthenogenic activation (PA) is a potential source of embryonic stem cells. We performed PA on discarded human oocytes that were in vitro matured, vitrified and warmed, to assess the developmental potential of parthenotes for stem cell production. DESIGN: Prospective analysis of parthenogenically activated human oocytes. MATERIALS AND METHODS: Discarded oocytes were obtained from consenting patients. Immature oocytes were cultured in 5% Human Serum Albumin in Quinn's Cleavage Media for ∼20 hours to mature in vitro. All mature oocytes were vitrified and warmed using either the Medicult or Irvine Scientific systems (Medicult Vitrification Cooling and Warming kits of Medicult, Denmark; Irvine Scientific Vitrification Freeze and Warm kits of Irvine Scientific, Santa Ana, CA). Oocytes surviving ∼2 hours post-warming were used for PA. Oocytes were exposed to 5μM calcium ionomysin (CI) then puromycin dihydrochloride (puro, 10μg/ml) or 6-dimethylaminopurine (6-DMAP, 1mM). Activated oocytes were cultured in 10% Synthetic Serum Substitute in Quinn's Cleavage Media. Chi-square analysis was performed. RESULTS: See table.Table 1Vitrification SystemSurvival Rate (%)Activation MethodActivation Rate (%)Medicult48/50 (90)CI/puro16/24 (66.7)CI/6-DMAP10/24 (41.7)Irvine Scientific25/34 (73.5)CI/puro8/15 (53.3)CI/6-DMAP6/10 (60)Total73/84 (86.9)All40/73 (54.8) Open table in a new tab CONCLUSIONS: Oocyte PA has the potential to create HLA-matched stem cell lines. Using discarded oocytes that were matured in vitro, vitrified and warmed, we successfully demonstrated that such oocytes could undergo PA and develop to Day 3 (40/73, 54.8%) . Further studies to obtain Day 5 parthenotes for genetic and epigenetic analysis are ongoing. It is our hope to ultimately create diploid autologous stem cell lines for future experimentation and possible stem cell transfers.