O-2: Effects of Oocyte Cryopreservation on a Specific Oolemma Bound Protein, CD9: A Potential Functional Marker After Cryopreservation

Abstract

Background: Oocyte cryopreservation affects the zona pellucida and oolemma which may account for reduced fertilization in the mouse and human. These negative effects may be overridden by micro fertilization techniques. Although fertilization may be achieved, embryo development is often compromised. After cryopreservation, little is known about the integrity of critical oolemma proteins required for normal fertilization and embryo development. CD9, a plasma membrane protein, is known to be important in sperm-egg fusion during fertilization. It has also been hypothesized that CD9 may be involved in oocyte signaling along with embryonic implantation and placentation. However, the effect of cryopreservation on CD9 has not been evaluated. Objective: To compare the relative quantity of CD9 in frozen vs. fresh oocytes Materials and Methods: IRB approval was granted through Stanford University. B6C3F1 mouse MII oocytes were harvested after 5 IU PMSG and hCG stimulation. Oocytes were either slow frozen using the standard Testart/LaSalle method, stored, thawed then underwent reverse transcriptase (RT) or directly had RT performed. All oocytes were pooled in pairs for RT. mRNA expression of CD9 in both viable thawed and fresh oocytes were determined by comparative quantitative real time PCR (QPCR). A t-test was performed for statistical analysis. Results: 40 fresh oocytes and 38 slow frozen viable oocytes were compared using QPCR. Frozen oocyte mRNA expression levels of CD 9 was reduced by 73 fold (p<0.0001) compared to fresh oocytes (Figure I). Conclusions: This is the first report showing specific adverse membrane effects after oocyte cryopreservation. The relative decrease in CD9 may account for poorer outcomes reported after oocyte cryopreservation, even after achieving normal fertilization via micromanipulation techniques. CD9 may potentially be used as a molecular marker for evaluating successful oocyte cryopreservation and/or as a quantitative measure for evaluating different cryopreservation protocols in the future.