Determination of vaginal cytology and MMP-9 expression during early pregnancy in mice

Precise pregnancy detection is critical for optimizing reproductive efficiency in animals. In mice, the conventional copulation plug method for pregnancy confirmation lacks reliability, particularly in inbred strains, with false-pregnancy rates exceeding 50 %. Here, to improve pregnancy detection efficiency in mice, we examined vaginal cell population changes using vaginal smear technique and analyzed MMP-9 expression in vaginal tract from day 0.5 to 6.5 of pregnancy. A total of forty-four female mice in the estrus stage were paired with sexually mature male mice for natural mating. The morning after mating was considered as embryonic day 0.5 (E0.5). Vaginal cytology was used to determine the vaginal cells of pregnant and non-pregnant mice from day 0.5 to 6.5. Furthermore, the protein and gene expression of MMP-9 were assessed through the utilization of immunohistochemistry and real-time PCR. Our findings reveal that pregnant mice display the higher number of leukocytes but the low number of nucleated/cornified epithelial cells in vagina compared to the non-pregnant counterparts. Furthermore, MMP-9 protein consistently presents in vaginal epithelial cells throughout the experimental period, with increased protein expression on day 4.5 of pregnancy followed by a decline on day 6.5 of pregnancy. This result is similar to MMP-9 gene expression, reaching the peak on day 5.5 of pregnancy. In conclusion, vaginal cytology proves effective for mouse pregnancy determination, while MMP-9 can serve as a potential biomarker for detecting pregnancy in mice.