Oocyte comet assay of luteal phase sera from nonpregnant patients after assisted reproductive procedures

Purpose. Serum factors in patients with recurrent spontaneous abortions (RS A) inhibit mouse embryo development in vitro. Serum factors affecting DNA i ntegrity remain to be tested. The null hypothesis was that patient sera do not affect DNA integrity. The objectives were (a) to use the oocyte comet a ssay to assess DNA damage after exposure to patient sera and (b) to determi ne the effect of sera from gravidity 0 parity 0 patients to induce DNA apop tosis. Methods: Luteal phase sera were drawn 1 week after embryo transfer followin g assisted reproductive procedures. Frozen-thawed hamster zona intact oocyt es at metaphase II were incubated in groups of eight in either control medi um or medium supplemented with 50% patient serum for 1.5 h at 37 degreesC i n room air. The oocytes were fixed, stained in acridine orange, embedded in agarose, lysed, and alkaline electrophoresis performed. The intensities of the digitized fluorescent images were analyzed. Results: The sera of nonpregnant patients (64%) caused significant fragment ation of hamster oocyte DNA when compared with pregnant patient sera. This difference was also observed when adjusted for patient age. Sera of patient s that had never been pregnant also resulted in oocyte DNA fragmentation. Conclusions. The results suggested that sera from patients that did not con ceive contained factors that did not support cell growth by causing DNA fra gmentation and apoptosis. The level of the apoptotic factors varied from cy cle to cycle. However more studies are needed to determine if the sera fact ors actually reach the uterine environment to cause the undesirable effects .