Molecular and functional aspects of menstruation in the macaque

Abstract

Much of our understanding of the molecular control of menstruation arises from laboratory models that experimentally recapitulate some, but not all, aspects of uterine bleeding observed in women. These models include: in vitro culture of endometrial explants or isolated endometrial cells, transplantation of human endometrial tissue into immunodeficient mice and the induction of endometrial breakdown in appropriately pretreated mice. Each of these models has contributed to our understanding of molecular and cellular mechanisms of menstruation, but nonhuman primates, especially macaques, are the animal model of choice for evaluating therapies for menstrual disorders. In this chapter we review some basic aspects of menstruation, with special emphasis on the macaque model and its relevance to the clinical issues of irregular and heavy menstrual bleeding (HMB).

Fig. 1

Comparison of normal versus disordered and heavy menstrual bleeding (HMB) in rhesus macaques. Top panel: bleeding patterns of 8 animals with normal cycles. Bottom panel: bleeding patterns of 6 animals with abnormal cycles. Each row shows the days of bleeding of one animal followed for 110 days, aligned by the first day of bleeding. The lower panel shows the erratic bleeding patterns of monkeys with menstrual disorders including irregular bleeding and HMB

Fig. 2

Comparison of rhesus uterus in proliferative versus menstrual phase. Freshly removed uteri were cut in half along the fundal-cervical axis and photographed with macro lenses. a a dark line marks the endometrial-myometrial border and another line delineates the plane along which sections of the endometrium were cut. This specimen was taken on day 14 of the induced proliferative phase. Endo = endometrium. Myo = myometrium. b on day 2 of the menstrual cycle, bleeding is restricted to the upper third of the endometrium. The basalis and the lowest part of the functionalis does not bleed or slough. Scale bar = 1 cm; applies to both images

Fig. 3

Histology of the rhesus endometrium in the proliferative, secretory and menstrual phases. Histological sections of the endometrium were cut on a plane running from the luminal to the myometrial border. Straight versus tortuous glands are evident in (a versus b). Sections taken during the menstrual phase (c–e) show that menstrual breakdown is only evident in the functionalis (c–d) not the basalis (e). Scale bar for a–c = 1 mm; scale bar for d–e = 100 μm

Fig. 4

The rhesus macaque cervix. A cervix was freshly removed from a rhesus macaque at midcycle and cut longitudinally along the axis from the External os to Internal os. A projection of the cervical wall (colliculum) forces the cervical canal into a Z-shaped path. Scale bar = 1 cm

Fig. 5

mRNA expression of various MMPs in the rhesus menstrual cycle. Plots of northern hybridization data reveal two patterns of MMP mRNA expression. Top Row: Certain MMPs were strongly expressed during the menstrual phase and declined to minimal levels by days 5–6. Bottom Row: Other MMPs peaked similarly during menstruation but declined much more slowly

Fig. 6

Immunohistochemistry of MMP-2 during the proliferative phase. Endometrial sections were stained for MMP-2 on days (D) 0,1,2,3,4 and 14 after P withdrawal. The black line marks the myometrial border. Strong dark brown staining for MMP-2 protein was confined to the upper functionalis zone during the menstrual phase (D1-4). The staining became nondetectable by day 14. Scale bar = 1 mm; applies to all images

Fig. 7

Immunohistochemistry of hypoxia inducible factor in the macaque endometrium. Endometrial sections were stained for hypoxia inducible factor protein on days 0, 2,3, and 8 after P withdrawal. A mouse monoclonal antibody against HIF-1α at a concentration of 1:1000 (Novus Biologicals) was used. Nuclear HIF-1α staining was low on day 0, increased strikingly on days 1–3 of the cycle and then declined to undetectable by day 8. These increases occurred primarily in the glands and the small blood vessels of the upper functionalis zone, presumably in response to local hypoxia. Scale bar = 50 um; applies to all images

Fig. 8

Mean menstrual blood loss over 6 days in artificially cycled macaques. Menstruation peaked in all animals on day 3 after progesterone withdrawal. Short (21 days) menstrual cycles had no significant effect on total menstrual blood loss compared to normal length (28 days) cycles, but treatment with artificially amplified cycles (35 days) in which the secretory phase had elevated and prolonged exposure to progesterone resulted in increased bleeding (P < 0.01). Treatment with antifibrinolytics including tranexamic acid (TXA; 75 mg/kg/day) or ε-aminocaproic acid (EACA; 100 mg/kg/day) significantly reduced menstrual blood loss (P < 0.05). Values were compared statistically by Analysis of Variance and means were compared by Fisher’s protected LSD