Dept of Obstetrics and Gynaecology, Monash University, Clayton, Victoria 3168, Australia
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Abstract |
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Key words: endometrium/immunohistochemistry/menorrhagia/proliferation index/vascular smooth muscle
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Introduction |
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Improved understanding of the structure and function of spiral arterioles may be an important step in understanding pathological conditions such as menorrhagia. Menorrhagia is excessive menstrual blood loss, >80 ml/cycle, and affects up to 15% of all women, increasing to 20% during the perimenopausal period (McKinley et al., 1992). These women suffer varying and often severe side effects, ranging from anaemia to disturbance of social or sexual functions and depression. Up to 50% of menorrhagia has no identifiable cause (Haynes et al., 1979
) and is termed idiopathic. Previous work has shown that women with menorrhagia lost blood at a greater rate than women with normal bleeding patterns (Haynes et al., 1979
), suggesting that while there may be a slight increase in length of menses in menorrhagic women, the primary problem is increased rate of bleeding. In the absence of a pathological cause for increased bleeding (fibroids, carcinoma, etc.) it seems likely that aberrations of the local mechanisms involved in menstruation may be involved in menorrhagia. The range of treatments available for menorrhagia (Fraser, 1992
) and the varied response to them by individual women suggests that several differing local mechanisms are involved in idiopathic menorrhagia.
Endothelin and transforming growth factor beta (TGF-ß) are two factors that may play an important role in the control and development of spiral arterioles. Both are found in the endometrium during the menstrual cycle (Salamonsen et al., 1992; Gold et al., 1994
; Polli et al., 1996
; Marsh et al., 1997
) and can be located around the spiral arterioles during the mid to late secretory stages. Endothelin is a potent vasoconstrictor and known vascular smooth muscle mitogen (Economos et al., 1992
; Salamonsen et al., 1992
; Giudice, 1994
; Marsh et al., 1997
), while TGF-ß inhibits VSMC proliferation and upregulates smooth muscle alpha actin (
SMA) in various cell types while also upregulating pre-proendothelin (Schwartz and Liaw, 1993
; Grainger et al., 1994
; Polli et al., 1996
). Their location in the endometrium around the spiral arterioles and their known effects on VSMC make them prime candidates for the control of spiral arteriole development.
We postulate that the development of spiral arterioles in menorrhagic women is altered so that the VSMC surrounding the arterioles are abnormal. The aims of this study were to: (i) quantify the menstrual blood loss (MBL) in women with and without menorrhagia to see if increased blood loss is due to increased length of bleeding period or to increased rate of blood loss; (ii) quantify VSMC proliferation in the endometrial arterioles of women across the menstrual cycle, and to compare VSMC proliferation levels in women with or without menorrhagia to observe if any differences occur that may relate to menorrhagia; and (iii) examine endothelin and TGF-ß1 expression in relation to VSMC proliferation.
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Materials and methods |
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Peripheral blood samples (5 ml) were taken for determination of follicle stimulating hormone (FSH) by immunoradiometric assay (FSH MAIA; Serono Diagnostics, Sydney, Australia) and steroid hormone concentrations by solid-phase radioimmunoassay (Diagnostic Products Corp., Los Angeles, CA, USA). Each woman answered a questionnaire to determine whether she suffered any perimenopausal symptoms. The primary definition of perimenopause was a FSH reading >9 IU/ml, and the woman reporting five or more perimenopausal symptoms. Menstrual irregularity in the 12 months prior to MBL evaluation was also taken into account. If FSH was elevated in a mid-cycle blood sample, with no evidence of perimenopausal symptoms, the woman was defined as being non-perimenopausal. The average age for the reproductive grouping was 43 (range 3447) years, while that of the perimenopausal groupings was 48 (range 4553) years. All experiments were performed with full institutional ethical approval.
Each subject was examined by microhysteroscopy, then biopsied using a Pipelle suction curette (Prodimed, 60530, Neuilly-en-Thelle, France). Where possible, this biopsy was taken from the functionalis of the endometrium; biopsies containing only basalis as subsequently confirmed by histology were excluded from the study. Biopsies were routinely fixed for 4 h in 10% buffered formalin, then paraffin embedded. Each biopsy was dated by an experienced histopathologist using the criteria established by Noyes et al. (Noyes et al., 1950) into six cycle stages: menstrual, early proliferative, mid proliferative, early-, mid- and late secretory (Table I
).
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Endothelin and TGF-ß1 single staining
Samples with the six highest and six lowest spiral arteriole VSMC proliferation indices were selected for immunolocalization of endothelin and TGF-ß1. Samples were selected in this manner as a pilot study to test the hypothesis that a difference existed between controls and women with menorrhagia. Slides were prepared and dewaxed as described previously and a 3% H2O2 peroxidase block applied (15 min). The primary antibody was then applied [anti human TGF-ß1, 1:50 in PBS with 1% BSA (polyclonal antibody; Dako, Carpentaria, CA, USA)/anti human ET-1, 1:2000 in PBS with 10% fetal calf serum (FCS) (polyclonal antibody; Cambridge Research Biochemicals, Norwich, UK)] at 37°C for 2 h. A biotinylated secondary antibody (goat anti-rabbit) was applied for 20 min. The horseradish peroxidasestreptavidin complex was then applied for 20 min, then aminoethyl carbazole red chromogen was applied (15 min, room temperature). After rinsing with distilled water, slides were mounted in Clearmount mounting medium. In all immunohistochemical studies, appropriate non-immune IgG were used as negative controls.
Scoring:
Blood loss data were analysed using Sigmaplot version 5.0 (SPSS, Chicago, IL, USA) software for statistical analysis. Quantification of SMA/PCNA staining was performed using a microscope linked via a colour video camera to a personal computer with the stereology software package Grid installed (Graffiti Data, Silkeborg, Denmark), with counts being performed from the computer screen by the operator. The number of proliferating VSMC/mm2 was counted from the whole section while the total VSMC/mm2 was estimated from sampling a minimum of 10% of the tissue in each section using a motorized stage to advance the slide at set intervals. A proliferation index was derived from the number of proliferating cells divided by the total number of VSMC and expressed as a percentage. Another analysis used the number of arterioles with VSMC proliferation/mm2. Non-parametric analyses using KruskalWallis and MannWhitney U-tests were performed at several levels using the statistical package Minitab (Minitab Inc., Pasadena, CA, USA).
Endothelin and TGF-ß1 staining was scored using a semi-quantitative scale where 0 = no staining and 3 = intense staining, using a stereo microscope linked to a colour monitor. The endometrium was divided into four compartments: glands/epithelium, stroma, VSMC and endothelial cells where each of these was identifiable. Each compartment was scored and analysed independently of the others.
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Results |
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Perimenopausal versus reproductive-aged women
When combined, perimenopausal women had significantly longer cycles than the reproductive-aged women (P <0.001). No significant differences were observed in MBL or days of MBL between the two groups.
Control versus menorrhagic women
When combined, menorrhagic women bled for significantly longer than controls (4.85 ± 0.25 versus 5.75 ± 0.35 days, P < 0.001). MBL in menorrhagic women was approximately 3.6-fold greater than that measured in controls (146.1 ± 17.5 versus 39.9 ± 3.4 ml respectively).
General immunostaining pattern for SMA and PCNA
Examples of PCNA staining were found in all cell types throughout the endometrium, while SMA staining marked the arteriole walls and some isolated vascular cells, probably pericytes (Figure 1a and b
). VSMC proliferation staining was heterogeneous. The predominant staining pattern was for vessels to have a mixture of proliferating and non-proliferating VSMC, with few vessels totally surrounded with positive or negatively stained cells (Figure 1a and b
). Similar numbers of spiral arterioles (arterioles that coil tightly in the stroma, with prominent VSMC layers and several vessel profiles close to each other) and straight arterioles (vessels with a VSMC coating other than spiral arterioles, usually a single vessel profile) were observed in control and menorrhagic tissues. Other
SMA staining was also seen in occasional cells scattered throughout the stroma. Throughout this study, no significant differences in staining patterns were found between perimenopausal and reproductive-age tissues, so these groups were combined to give control and menorrhagic groupings only.
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Endothelin staining levels were significantly reduced in the glandular/epithelium compartment of menorrhagic tissues when compared with controls (P < 0.05). No significant differences were found in endothelin staining in the other endometrial compartments (Figure 5). TGF-ß1 staining showed no significant differences between controls and menorrhagics (Figure 6
).
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Discussion |
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The adult vascular system is very stable, with low cell turnover unless injured or diseased. In comparison, the endometrium sheds and regenerates every 28 days, requiring constant regrowth and maturation of vessels. Straight arteriole VSMC proliferated at a similar rate across the cycle, with no significant differences between controls and menorrhagics until the late secretory stage. This suggests that these VSMC are proliferating enough to keep arteriole growth constant as the endometrium grows and is remodelled. VSMC are said to exist in two states, contractile and secretory (Mulvaney and Aalkjaer, 1990; Schwartz and Liaw, 1993
; Owens, 1995
). Secretory VSMC are thought to revert to a less-differentiated state to allow secretion of extracellular matrix proteins, and VSMC proliferation has been associated with this de-differentiated state in vivo (Aikawa et al., 1993
). It is possible that VSMC in the endometrium may be in a more de-differentiated state than in the rest of the body to allow for continuous growth and remodelling. Lower VSMC proliferation in straight vessels in menorrhagic women during the late secretory stage may contribute in some way to increased blood loss in menorrhagia, as not all blood loss is through the spiral arterioles.
Spiral arteriole proliferation in the control endometrium tissues follows a similar pattern to the total VSMC proliferation index. However, VSMC proliferation was significantly decreased in the spiral arterioles of menorrhagic women. Lower numbers of proliferating VSMC may reflect a change in VSMC phenotypes in menorrhagic women compared with controls. Evidence from previous studies suggests that SMC may exist in varying states between the two extremes of contractile and secretory (Aikawa et al., 1993; Owens, 1995
). It is possible that spiral arterioles require a variety of VSMC in varying phenotypic stages, to allow for greater control over coiling and constriction. The reduced VSMC proliferation levels in menorrhagic women may reflect an imbalance in VSMC phenotypes that ultimately weakens the spiral arterioles.
If decreased VSMC proliferation levels in spiral arterioles of menorrhagic women led to decreased VSMC numbers, the spiral arterioles might have thinner or weaker walls, leading to less control over menstruation and increased blood loss. However, this study found no significant difference in total spiral arteriole VSMC numbers between the two groups. PCNA is a proliferation marker that marks nuclei in the S phase of the cell cycle, when the DNA is replicated, but the cells have not yet divided. It may be that proliferation has been arrested in these cells, leading to larger VSMC. Spiral arteriole VSMC have been reported to be larger than normal VSMC (Ramsey, 1977), and cells with bulging nuclei may play a role in causing the arteriole to coil and twist, so that a lesser number of these cells could lead to a structurally incomplete spiral arteriole.
No significant difference was found in the number of spiral arterioles/mm2 observed in women with or without menorrhagia in this study. Spiral arteriole length, as measured by the number of loops or vessel profiles observed, did not differ significantly between control and menorrhagic women. These results correlate with previous work from this laboratory (Abberton et al., 1996), which showed no differences in spiral arteriole numbers between control and perimenopausal women, and with a study by Rees et al. (Rees et al., 1984
) which showed no difference in arteriole numbers between women with and without menorrhagia. This suggests that changes in spiral arteriole numbers do not play a significant part in idiopathic menorrhagia.
Menorrhagia occurs if one or more of the following situations arise: (i) increased or earlier breakdown of endometrial tissues and vessels at the start of menstruation; (ii) increased intensity of bleeding from the damaged blood vessels; or (iii) failure of the endometrial blood vessels to repair at the end of menstruation. MBL in menorrhagic women is 3- to 4-fold greater than in controls, demonstrating that increased intensity of bleeding is the major factor that results in the high blood losses that these women suffer. This agrees with the results of an earlier study (Haynes et al., 1979). Idiopathic menorrhagia may have more than one cause, with aberrations possible at several different stages of the menstrual process. A positive correlation between MBL and days of bleeding was shown to exist in both control and menorrhagic reproductive-aged groups, with menorrhagic women bleeding for approximately 24 h longer, suggesting that endometrial blood vessels may not be repairing properly. By contrast, no such relationship was seen in either of the perimenopausal groups, implying that different local mechanisms may be acting to cause the increased blood loss in perimenopausal women compared with reproductive-aged women.
Endothelin-1 is a potent vasoconstrictor known to be present in the endometrium, as well as being a growth factor for both VSMC and endothelial cells (Economos et al., 1992; Giudice, 1994
). Our results have shown a decrease in the endothelin concentrations in the epithelium of menorrhagic tissues similar to that described in studies by Marsh et al. (Marsh et al., 1997
). The distance of the epithelium from the spiral arterioles would suggest a paracrine role in boosting concentrations of endothelin available to the spiral arterioles and their VSMC. If, as Marsh suggests, the metabolism of endothelin is increased and epithelial concentrations are reduced, this would lead to a decrease in the amount of endothelin available to components of the endometrium. Given that the production of endothelin in the VSMC and stroma is lowered in menorrhagic women (though not significantly), the further loss of endothelin from yet another source may affect spiral arterioles, through reduced VSMC proliferation and decreased vasoconstriction, leading to menorrhagia.
The role of prostaglandins in menorrhagia is well established, with anti-prostaglandins being used to treat idiopathic and intrauterine device-related menorrhagia (Van Eijkeren et al., 1989; Fraser, 1992
). The decrease in endothelin expression is similar to the relative decrease of the prostaglandin PGF2
in menorrhagic women (Hagenfeldt, 1987
). It has been suggested that endothelin-1 stimulates PGF2
release in endometrial cells (Cameron et al., 1991
) and that PGF2
and endothelin are both significantly reduced in the glandular epithelium of menorrhagic women. The decrease in both these vasoconstrictors, along with increased expression of PGE2
, a vasodilator, and PGI2 could affect the balance of vasoactive control, leading to menorrhagia.
TGF-ß inhibits VSMC proliferation, and maintains SMC in a contractile state (Schwartz and Liaw, 1993; Grainger et al., 1994
), and is also found throughout the endometrium. TGF-ß1 staining was similar to endothelin staining, being found in all endometrial compartments. No significant differences were seen in TGF-ß1 concentrations between control and menorrhagic tissues in any endometrial cell type in these samples. This suggests that TGF-ß1 concentrations at the mid/late secretory stages do not play a role in determining menorrhagia; however, this does not rule out a role earlier in the menstrual cycle. While immunolocalization is an appropriate method to use for the cellular localization of proteins such as endothelin and TGF-ß1, it may not be the best technique for quantifying endothelin and TGF-ß1 production. It is possible that a different technique, such as in-situ hybridization, would enable us to detect changes in the production of these factors that may relate to menorrhagia.
Every attempt was made to exclude samples with pathological causes for menorrhagia, and several samples from our study were removed after biopsy due to follow-up pathology reports showing excessive numbers of fibroids, large fibroids, adenomyosis or anovulatory cycles. Fibroids have a proven role in menorrhagia, with disturbances to the vascular system being probable causes of increased blood loss (Stewart and Nowak, 1996). Fibroids are also known to affect expression of growth factors such as vascular endothelial growth factor (VEGF), TGF-ß1 and basic fibroblast growth factor (bFGF) which may contribute to menorrhagia. As with many studies using human tissue, intersample variability was quite large, and may be masking some cyclic differences in VSMC proliferation in the straight arterioles. While it is possible that conditions such as mild adenomyosis and seedling fibroids may have been included in our study, the patient group used in this study met the current clinical definition for idiopathic menorrhagia.
In conclusion, we demonstrated a significant decrease in VSMC proliferation in the spiral arterioles of menorrhagic women in the mid/late secretory stages of the cycle compared with control tissues that may relate to menorrhagia. VSMC in straight arterioles were shown to proliferate at a similar rate across the cycle, and decrease significantly in the late secretory stage of the cycle in menorrhagic tissues. Endothelin concentrations were shown to be lower in menorrhagic tissues with low spiral arteriole VSMC proliferation rates, with a significant reduction being observed in the epithelium of menorrhagic women. No significant difference was observed in TGF-ß1 concentrations between the two groups. Further studies into the mechanisms which control growth and differentiation of endometrial VSMC are required to understand more fully the significance of these findings in relation to idiopathic menorrhagia.
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Notes |
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References |
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Submitted on June 18, 1998; accepted on December 16, 1998.