Elsevier

Contraception

Volume 70, Issue 4, October 2004, Pages 343-350
Contraception

Original research article
Effect of long-term progestin treatment on endometrial vasculature in normal cycling mice

https://doi.org/10.1016/j.contraception.2004.04.006Get rights and content

Abstract

The aim of this study was to develop a mouse model to investigate the effects of long-term progestin-only exposure on endometrial vascular structure. Normal cycling mice received Silastic implants containing either medroxyprogesterone acetate (MPA) or levonorgestrel (LNG) and were dissected after 1, 3 or 6 weeks. Endometrial vascular density increased significantly within 1 week of MPA (482 ± 40.2 vessels/mm2) or LNG (440 ± 26.5 vessels/mm2) treatment compared with normal cycling mice (293 ± 10.5 vessels/mm2). MPA increased stromal cell density within 1 week of treatment (13813 ± 1450 cells/mm2) compared with normal cycling mice (8256 ± 928 cells/mm2). However, although LNG significantly increased stromal cell density overall, the increase did not reach significance within the individual weeks examined. There was no significant change in the ratio of vascular to stromal cell density among treated and normal cycling mice. Epithelial cell height significantly decreased within 1 week of LNG (17.6 ± 1.3 μm) treatment compared with normal cycling mice (23.5 ± 1.3 μm); epithelial cell height also decreased within 1 week of MPA treatment (16.6 ± 2.1 μm), although this did not reach statistical significance. VEGF immunostaining increased significantly in luminal epithelium after MPA or LNG treatment, and in glandular epithelium after LNG treatment. These observations are similar to those reported in human endometrium, suggesting that this mouse model may facilitate further investigations into breakthrough bleeding due to long-term progestin use.

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.

References (19)

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