Assessment of Daily Menstrual Blood Loss in Rhesus Macaques.

Abstract

Rhesus macaques exhibit 28-day menstrual cycles similar to those of women. Menstruation can be experimentally induced by progesterone (P) withdrawal in estradiol (E2) plus P primed macaques and women. However, little is known regarding daily menstrual blood loss (MBL) in hormone controlled subjects. To fill this gap, we tested the effect of artificially short and extended menstrual cycles on daily MBL in macaques. Our goal was to establish baseline daily MBL in macaques for future studies on novel therapies to suppress menses. Twelve ovariectomized rhesus macaques were used in this study. Animal protocols were reviewed and approved by the ONPRC Institutional Animal Care and Use Committee. The animals were treated with an E2-filled Silastic capsule that produced 100-130 pg E2 /ml in serum. After 14 days of E2 priming the animals received a P-filled capsule that produced 5-7 ng P/ml in serum. Removing the P capsule (leaving the E2 capsule in place) on day 28 induced menses (“normal length” cycles). Short 21-day cycles were created by treating animals with E2 for 14 days followed by E2 + P for 7 days. Extended 35 day cycles were induced by treating animals for 14 days with E2, followed by E2 plus one P implant for 7 days and then E2 plus two P implants for 14 days (producing 10-14 ng P/ml). Daily MBL was quantified by a modified alkaline haematin assay. Menstrual blood was collected daily by tampon. On light menstrual flow days Tampex Pearl "Lite" tampons (1.2 cm diameter), cut transversely at 2.5 cm in length, were placed in the vagina to collect menstrual blood. On heavy menstrual flow days Tampex Pearl "Regular" tampons, 3 cm in length, were used. The cut end of the tampons was sealed with Dow Corning Type A Silastic Medical Adhesive and the original tampon applicators were used to insert the tampons into the animals. The tampons were replaced twice each day on normal flow days and 3 times each day on heavy flow days. After collection the tampons were vacuum dried and mixed with 5% sodium hydroxide in a Stomacher Lab Blender. The resulting alkaline haematin extract was quantified spectrophotometrically at 564nm on Elisa plates. MBL was determined by comparing the alkaline haematin values with levels from similar extracts of whole blood standards (20-600 µl blood). Results were expressed as milliliters (ml) of blood lost. The alkaline haematin technique was highly reproducible with intraassay and interassay coefficient of variation estimates at 4.3 and 6.7, respectively (n=8 assays). MBL values were log transformed to correct for heterogeneity of variance and then subjected to ANOVA. The average total MBL during the menstrual cycle in 11 animals was 5.87 ± 0.6 ml (over 6 days). Menstruation peaked in all the animals on day 3 after P withdrawal. Short menstrual cycles had no significant effect on total MBL compared to normal-length cycles. However, treatment with artificially amplified secretory phases resulted in greater MBL (8.8 ± 2.2 ml/cycle). These animals had a 1 day delay of frank menses and then compensated with significantly heavier MBL on days 3, 4, and 5 after P withdrawal (P<0.05). We conclude that very accurate daily MBL measurements can be obtained from hormone controlled, artificially cycled rhesus macaques and that these animals can provide a useful animal model for evaluating factors that modulate menstrual bleeding. Supported by NIH grants HD18185 and RR000163. (poster)