Original research articleEffect of long-term progestin treatment on endometrial vasculature in normal cycling mice
Introduction
Progestin-based contraceptive methods, such as the injectable depot medroxyprogesterone acetate (MPA) and the levonorgestrel (LNG)-releasing implant and intrauterine systems, are known to be safe, effective and convenient and have gained worldwide popularity since they were introduced [1], [2]. However, these contraceptive methods are commonly associated with unpredictable and prolonged vaginal bleeding and spotting (breakthrough bleeding), particularly in the early months of use. These disturbances of endometrial bleeding, which are also experienced by a proportion of women using combined oral contraceptive and hormone replacement therapies [3], [4], are a major social and clinical problem and a common cause for women discontinuing use of these contraceptive methods [1], [5].
Progestin-only therapies cause differential changes among the various tissue components of the endometrium. A relative increase in vessel density was observed in the endometrium of women using the levonorgestrel implant Norplant in contrast to the apparent atrophy of other endometrial tissues [6], [7]. At higher doses than those delivered by Norplant, a reduction in vascular density has been observed [8]. Vessel structure and integrity are compromised by progestin use, with changes noted in the shape, amount of dilatation, components of the basement membrane and pericyte support (see [3], [9] and references therein). Together, the changes in vessel density with the associated decrease in the structural support have been hypothesized to contribute to vascular fragility and breakthrough bleeding [3]. Despite these observations, the mechanisms causing breakthrough bleeding remain unknown.
Varied levels of vascular endothelial growth factor (VEGF), a potent vascular permeability factor and mitogen for endothelial cells, have also been observed in the endometrium of progestin-only contraception users [10], [11], [12]. VEGF is known to have a pivotal role in endometrial angiogenesis, although the mechanisms by which VEGF acts to control blood vessel development at various stages of the menstrual cycle remain to be elucidated in full [13]. Whether VEGF plays a role in the endometrial vascular changes associated with long-term progestin use is unknown.
Research into the mechanisms causing breakthrough bleeding is limited for several reasons. Large human clinical trials are costly and invasive procedures are required to obtain the necessary samples. There is also a lack of animal models that are readily accessible and easy to manipulate. The aim of this research was to develop a mouse model of progestin-induced endometrial vascular changes. The reproductive cycle in mice differs significantly from humans; they do not menstruate and the endometrium lacks spiral arterioles. However, unlike normal menstrual bleeding, which has been hypothesized to arise predominantly from the spiral arterioles (based on intraocular endometrial explants in rhesus monkeys [14]), breakthrough bleeding is thought to arise from superficial vessels within the endometrium. In addition, spiral arteriole development is reduced after progestin exposure [15], [16]. We hypothesized that the changes observed in mouse endometrial microvasculature after long-term progestin-only treatment would be similar to those observed in the human endometrium, identifying the mouse as a potential model for fundamental research into the mechanisms causing breakthrough bleeding. The specific aims of this study were to quantify changes in endometrial vascular and stromal cell density, epithelial cell height, and VEGF production in response to long-term treatment with LNG or MPA.
Section snippets
Animals
Adult female mice (8–12 weeks, 25–30 g, C57BL/6J x CBA) were housed four per cage under controlled environmental conditions (20°C, 16:8-h light:dark cycle); food and water were provided ad libitum. While under Avertin anesthesia (25 mg/100 g body weight, 2,2,2-tribromoethanol; Aldrich Chemical Co., Milwaukee, WI, USA), a Silastic implant was inserted subcutaneously into the dorsal surface between the shoulders of each mouse; incisions were closed using a stainless steel wound clip (Becton
Capillary profile (CD31 immunostaining)
Endometrial capillary profiles from an untreated normal cycling mouse, or mice treated with MPA or LNG, are shown in Fig. 1. Although no morphological analysis of vessels was undertaken in this study, differences in vessel shape and size between normal cycling and progestin-treated mice were noted in some endometrial sections. Vascular density was significantly higher in mice treated with either MPA or LNG in comparison to untreated normal cycling females [F(2,35) = 52.8, p < 0.001]. Means
Discussion
Relative vascular density increased significantly in the mouse endometrium within 1 week of treatment with either MPA or LNG. This increase occurred concurrently with apparent regression of other endometrial tissues, as indicated by increasing stromal cell density (compacting of tissue) and decreasing epithelial cell height. These differential changes in various endometrial cellular compartments are in agreement with published observations of endometrium from women using progestin-based
Acknowledgements
The authors wish to thank Leonie Cann, Fiona Lederman and the staff of the Monash Medical Centre Animal House for technical help and assistance. The study was funded in part by a NH & MRC grant (143805) to Peter Rogers and a sponsored research agreement with SUGEN/Pharmacia.
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