1 Department of Obstetrics and Gynaecology, University of Glasgow, Glasgow Royal Infirmary, Glasgow, UK, 2 Department of Anatomy, University College, Cork, Ireland, 3 Department of Pathology, Glasgow Royal Infirmary, Castle Street, Glasgow and 4 Maternal, Fetal and Neonatal Physiology Group, Fetal Origins of Adult Disease Division, University of Southampton, Princess Anne Hospital, Coxford Road, Southampton, UK 5 To whom correspondence should be addressed at Department of Obstetrics and Gynaecology, University of Glasgow, Glasgow Royal Infirmary, 10 Alexandra Parade, Glasgow G31 2ER, UK. e-mail: gqta05{at}udcf.gla.ac.uk
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Abstract |
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Key words: breakthrough bleeding/endometrium/levonorgestrel intrauterine system/progestogen/vasculature
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Introduction |
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Both Norplant and the LNG-IUS have profound effects on endometrial structure that could in turn affect the function and stability of the endometrial vasculature (Skinner et al., 1999; Jones and Critchley, 2000
). The LNG-IUS delivers a high dose of LNG to the endometrium and induces a very rapid decidualization response in the endometrial stroma (Silverberg et al., 1986
; Telfer et al., 1997
; Critchley et al., 1998
). Ultrastructural examination has also demonstrated changes within the surface and glandular epithelium (Pakarinen et al., 1998
). Exposure to Norplant, although at a much lower effective dose to the endometrium, results in vascular changes that can be observed in tissue sections or by hysteroscopy (Rogers et al., 1993
; Hickey et al., 1998
). These changes include the presence of petechiae and ecchymoses in the superficial endometrium (Hickey et al., 1996, 1998; Hickey and Fraser, 2000b
). Mean superficial vascular diameter was greater in women using low dose LNG implants compared with those with menorrhagia (Hickey et al., 1998
).
In view of these in-vivo observations, we postulated that a common mechanism might underlie the pathogenesis of inter-menstrual bleeding irrespective of the dose or route of delivery of progestogen. We therefore sought to demonstrate the effect of short-term exposure to high-dose intrauterine LNG on endometrial vessels in a randomized, controlled study in women with menorrhagia scheduled for hysterectomy.
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Materials and methods |
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Specimen collection, processing and assessments
Following hysterectomy, the unfixed uterus was taken immediately to the pathology department where the pathologist cut a transverse mid-corpus slice (510 mm). This slice was then further divided into full thickness blocks from the anterior and posterior walls and lateral fornices. The blocks taken from the anterior and posterior wall were further divided for light microscopy. Tissue for wax embedding was fixed in 4% (v/v) buffered formaldehyde [Chemix (UK) Ltd, Standish, UK]. All specimens collected for research were examined by a consultant pathologist (C.J.R.S.) who was unaware of the group from which they were obtained. However as the effect of the LNG-IUS is so profound, the pathologist was not truly oblivious to the treatment. The specimens taken for routine examination were assessed independently by local pathologists and reports were obtained.
Immunocytochemistry
Serial sections (5 µm) were mounted on glass slides coated with 3-aminopropyltriethoxysilane (SigmaAldrich, Poole, UK). Vascular endothelium was identified in wax-embedded tissue sections (5 µm) using an antibody to CD31 (Clone JC 70A, dilution 1:500; Dako, Ely, UK). Smooth muscle cytoskeletal components, -smooth muscle actin (Clone 1A4, dilution 1:500; Sigma) and myosin (Clone HSM-V, dilution 1:150; Sigma) were localized (Kohnen et al., 2000
). Primary antibodies were diluted in 2% horse serum (Sigma) in phosphate-buffered saline pH 7.5. Antigen retrieval was used for detection of CD31 and myosin. Those sections were microwaved in 0.01 mol/l citrate buffer pH 6 for 8 and 45 min respectively (Kohnen et al., 2000
). Non-specific binding sites were blocked with 20% horse serum and 20% human serum in PBS for 30 min at room temperature. Endogenous peroxidase activity was blocked by immersing slides in 0.5% H2O2 in methanol. In the case of CD31, inactivation was carried out before microwaving. For myosin detection, blocking 0.5% H2O2 was used after addition of the secondary antibody. Antibody binding was detected using an avidinbiotin complex (ABC) peroxidase kit (Vector Laboratories, Peterborough, UK) and diaminobenzidine substrate tablets according to the manufacturers instructions (Sigma). Negative controls were used in which the primary antibody was replaced with a mouse monoclonal IgG1 to Aspergillus niger glucose oxidase (Dako). Nuclei were counterstained with Harris haematoxylin (BDH, Lutterworth UK).
Microscopy and stereology
Sections were examined by bright field microscopy (BX50; Olympus, London, UK) and digital images were captured with a 3-chip colour camera (JVC, London, UK) and the ImagePro Plus version 4 image analysis program (Media Cybernetics, Des Moines, IA, USA). Stereology was undertaken using a mixture of computer-assisted and manual methods. Microscope fields were selected in a tessellated pattern either laterally (for specimens with a smooth surface) or vertically (on specimens with an irregular surface where the former approach would have been problematic) beginning with a random starting point. In order to prevent bias, the entire height of the tissue was sampled. In the case of vascular surface density, a map image was produced by scanning the sections with a 35 mm film scanner (Coolscan III, Nikon, Kingston upon Thames, UK). Orthogonal sampling grids tailored in shape to each section were applied with an image analysis program (ImagePro). The location of the sampling points was chosen with respect to the grid at low magnification in order to prevent sampling bias. Analysis was then carried out at higher magnification. Volume fraction was measured on digital images by superimposing orthogonal grids using the image analysis program. A grid of 13x13 points was superimposed on the centre of each non-overlapping field. The outer surface of the endothelium was measured using cycloid grids oriented perpendicular to the vertical axis of the endometrium (Howard and Reid, 1998). Grid length was corrected for the proportion of the field occupied by tissue (Howard and Reid, 1998
). Vessel diameter was measured manually with a camera lucida attachment (Olympus, London, UK). It was necessary to examine the specimen using x20 and x40 objective lenses in order to measure the diameters of both large and small vessels accurately. The images were spatially calibrated using a 10 µm linear graticule (Graticules Ltd, Tonbridge, UK). The number of vessel lumen cross-sections per unit area was counted using the camera lucida and a x20 objective. A 13x13 orthogonal grid with an external guard zone was used to define the counting area and allow systematic accumulation of data. Vessels overlapping the basal and left lateral margins of the grid were excluded. Measurements were carried out on one section from each patient. Eleven to 20 fields were analysed per patient depending on the size of the tissue section. A total of 5548 point counts were made for the calculation of volume fraction. The total number of vessel lumen diameters measured was 2049. Preliminary analysis of the pooled data sets demonstrated that the data were not normally distributed. The median value of each data parameter was therefore obtained as an estimator for each patient. In addition the largest single vessel diameter was used as a non-arbitrary index for the presence of large vessels (Hourihan et al., 1986
). MannWhitney U-tests (Minitab v 13) were used to compare these sets of median or maximum values.
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Results |
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Discussion |
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It would appear after exposure to LNG-IUS that most of the vascular tree remains structurally intact after exposure to LNG-IUS; however, occasional larger diameter superficial thin-walled vessel segments develop. It is possible that these thin-walled vessels are more susceptible to breakdown during the course of events which lead to breakthrough bleeding. These findings are consistent with the previous findings of Hourihan et al. (1986) that revealed the presence of large vessels following oral progestogen exposure. Hysteroscopic observations of the endometrium after treatment with an LNG implant also support this conclusion (Hickey et al., 1998
). Hysteroscopy suggests that the large cross-sections observed in the present study are equivalent to the superficial lesions seen macroscopically within the endometrium. There may be common mechanisms which generate this type of lesion in women exposed to progestogen-only contraceptives and who suffer breakthrough bleeding.
Comparison of the median vessel diameter between the treated and control groups showed that these were almost identical. The normal size of most vessels also provides evidence that although there may be a specific lesion, there are no significant gross effects on vascular morphology. The observation that the arterioles also appeared well-formed reinforces the concept that the vascular tree is largely normal. This view is further supported by the fact that total vascular surface area is not altered. Despite this, there appeared to be an increase in vessel volume fraction and vessel cross-sectional number. These increases could explain the change in vascular volume density. However, the absence of a difference in vascular surface density is difficult to reconcile with an increase in these other parameters. It may be that the presence of the very large occasional vessel segments may contribute to the increase in volume fraction without producing gross changes in total surface area. Normally the observed increase in the number of luminal cross-sections would be expected to reflect change either in the length density (length per unit volume) or the branch pattern. The use of vertical sections in the present study, however, precluded the calculation of length density (Howard and Reid, 1998). In this study the entire endometrium was examined quantitatively, but qualitative observations suggest that changes may be more superficial. The quantitative effect may therefore be diluted by inclusion of the basal layer in the analysis.
Similar conclusions about the effects of exposure to levonorgestrel on vascular development and maturation were drawn with Norplant (Rogers et al., 2000). These authors classified the degree of vessel maturity by grading the amount of circumferential smooth muscle actin staining. Women treated with Norplant who also suffered breakthrough bleeding had an increased percentage of vessel cross-sections not circumscribed with
-actin. This finding suggested that exposure to levonorgestrel can modulate vascular development or maturation.
It seems likely that these vascular abnormalities may not persist when the tissue becomes atrophic, and this is probably associated with the resolution of breakthrough bleeding. It would be difficult, if not impossible, to obtain full thickness tissue specimens from women successfully treated with the LNG-IUS over a protracted period. We can therefore only speculate that as localized breakdown and shedding ensues and atrophy begins, the superficial part of the endometrium may be replaced by a tissue without superficial lesions.
The irregular appearance of the endometrial surface after treatment with LNG-IUS is not specific to intrauterine delivery of levonorgestrel per se. Polypoidal structures are known to develop after treatment with tamoxifen and often manifest with breakthrough bleeding (Hann et al., 2001), which suggests that endometrial surface irregularity or micro-polyps may contribute directly to bleeding patterns observed after exposure to the LNG-IUS. This view is supported by the hysteroscopic observations of Brechin et al. (2000
) who reported the presence of polyps in three women after long-term exposure to the LNG-IUS. It is noteworthy that the bleeding problems resolved after surgical removal of these polyps.
The presence of irregularities and micropolyps may be caused by changes within the epithelium and stroma (Figure 5). Thinning of the superficial and glandular epithelium noted here and previously observed in an ultrastructural study (Gu et al., 1995) may result in mechanical weakness. Weakening of the epithelium might in turn result in loss of glandular structural integrity causing the openings of the glands to widen. A driving force behind these changes might be the expansion of the underlying stroma resulting from localized and patchy decidualization (Gu et al., 1995
; Kohnen et al., 1998
). Localized shedding of superficial tissue could further exacerbate the development of irregularity.
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As there is a lack of nuclear receptors for estrogen and progesterone within the endometrial vasculature (Kohnen et al., 2000), steroid responsive genes expressed in the surrounding stromal cells may be of importance in the vascular response to levonorgestrel. It is not clear at the present time whether such genes would be involved in stromal decidualization, vascular growth, vascular wall maturation or in events that could lead to the breakdown of tissue. Previous studies have shown that the total volume of menstrual blood loss is reduced after exposure to LNG-IUS despite an increase in inter-menstrual bleeding (Andersson and Rybo, 1990
; Irvine et al., 1998
). This increase in inter-menstrual bleeding presents a significant constraint to compliance. However, counselling that informs the patient of the likely side-effects has been shown to be effective as an interim measure to improve continuation of treatment (Cameron, 2001
). In this group of women who sought hysterectomy as a treatment for menorrhagia, the response to the LNG-IUS may have been somewhat different from women using the device for contraception or from those who do not experience breakthrough bleeding. Regardless of the reason for treatment, in the longer term it may be possible to devise strategies which modulate tissue growth and vessel development prior to insertion of the LNG-IUS. These strategies could possibly minimize the changes in vascular morphology that are seen in the first few months after the initiation of treatment.
In conclusion, morphological examination of the endometrial vascular system after short-term exposure to the LNG-IUS revealed changes within the superficial endometrium that may explain the observed high incidence of breakthrough bleeding. The irregularity of the endometrial surface before the onset of treatment-induced atrophy may also be linked to bleeding patterns. Similar changes reported after exposure to other progestogens or tamoxifen suggest a common underlying effect in treatment-induced inter-menstrual bleeding. These data suggest that future approaches should consider either pre-treatment to make the endometrium atrophic or unresponsive, or the concomitant administration of other agents (such as antiprogestogens or inhibitors of metalloproteinase activity) to limit these adverse effects of progestogen on the endometrial blood vessels.
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Acknowledgements |
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Submitted on August 1, 2002; accepted on October 4, 2002