Estrus Cycle Status Defined by Vaginal Cytology Does Not Correspond to Fluctuations of Circulating Estrogens in Female Mice

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Gender-oriented studies in shock, trauma, and/or sepsis require accurate monitoring of hormonal fluctuations as estrogens may influence various end points. Yet, monitoring is challenging in small laboratory animals: e.g., despite its subjectivity, vaginal smears are the major method for determination of estrus cycle phases in mice. Using female mice of different age, we aimed to (a) characterize general age-related changes in systemic estrogens and (b) examine the utility of determination of the estrus cycle by vaginal smears and/or impedance simultaneously comparing them with oscillation of systemic estrogens. In this study, 3-, 15-, and 20-month-old mice underwent vaginal smear and impedance examination each morning for 22 days. Ten hours after each morning checkup, feces were collected, and a second vaginal smear performed. Blood was collected on days 15 and 22. In 3-month-old females, estrus (by smears) was three times more frequent than in older mice, but mean concentrations of plasma and fecal estrogens never decreased with age. Collectively (not individually) plotted fecal estrogens values increased in the proestrus/estrus interphase (by smears) in 3-month-old mice only. Impedance typically peaked (4.5 Ω in 3-month-old mice) in the estrus phase, and only the prediction of estrus (highest area under the curve = 0.87 in 3-month-old) but not of other phases was possible. Regardless of age, individual cycle phase (by smears) never correlated with corresponding fecal estrogens, and estrus could not be predicted. In conclusion, while the fecal estrogens oscillation and frequency of estrus phase were affected by age, the systemic hormone release persisted. In mice, vaginal cytology did not reflect changes of systemic (fecal) estrogens, whereas impedance accurately identified estrus. The flaws and advantages of the examined monitoring methods should be considered in the design of future shock studies.

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