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

PURPOSE

Serum factors in patients with recurrent spontaneous abortions (RSA) inhibit mouse embryo development in vitro. Serum factors affecting DNA integrity 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 assay to assess DNA damage after exposure to patient sera and (b) to determine the effect of sera from gravidity 0 parity 0 patients to induce DNA apoptosis.

METHODS

Luteal phase sera were drawn 1 week after embryo transfer following assisted reproductive procedures. Frozen-thawed hamster zona intact oocytes at metaphase II were incubated in groups of eight in either control medium or medium supplemented with 50% patient serum for 1.5 h at 37 degrees C in 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 fragmentation of hamster oocyte DNA when compared with pregnant patient sera. This difference was also observed when adjusted for patient age. Sera of patients that had never been pregnant also resulted in oocyte DNA fragmentation.

CONCLUSIONS

The results suggested that sera from patients that did not conceive contained factors that did not support cell growth by causing DNA fragmentation and apoptosis. The level of the apoptotic factors varied from cycle to cycle. However, more studies are needed to determine if the sera factors actually reach the uterine environment to cause the undesirable effects.