1 Department of Reproductive Science and Medicine, Imperial College School of Medicine at St Mary's, Norfolk Place W2 1PG, London, UK and 2 Electron Microscopy Unit and 3 Sydney Centre for Reproductive Health Research, Department of Obstetrics and Gynaecology, University of Sydney, NSW 2006, Australia
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Abstract |
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Key words: breakthrough bleeding/contraception/endometrium/progestogen/vascular fragility
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Introduction |
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BTB differs from normal menstrual bleeding in its pattern, duration, quantity and appearance (Odlind and Fraser, 1990). Normal menstrual bleeding is thought to arise primarily from the spiral arterioles, with endometrial capillaries making little contribution (Bartelmez, 1937
; Markee, 1940
). Breaks in capillaries and veins adjacent to the uterine lumen (Johanisson, 1990
) suggest that BTB arises primarily from these vessels and is related to increased capillary and venous fragility (Odlind and Fraser, 1990
; Hickey et al., 1996
). Vascular fragility cannot directly be assessed from endometrial biopsy specimens (Rogers et al., 1993
). Using out-patient hysteroscopy, vascular fragility in vivo in Norplant users was previously examined in a pilot study by the same authors (Hickey et al., 1996
), but has not been compared with normal cycling women. In this study, endometrial vascular fragility in Norplant users was compared with a group of women not exposed to exogenous sex steroids who complained of spontaneous menorrhagia. The relationship between vascular fragility and serum concentrations of oestradiol and progesterone was also investigated.
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Materials and methods |
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Twenty women referred to the hysteroscopy outpatient clinic complaining of spontaneous menorrhagia were used as a comparison group. These women complained of regular, heavy menstrual periods and fitted the clinical criteria of ovulatory dysfunctional uterine bleeding (DUB). This comparison group was chosen in order to compare the origins of abnormal bleeding. All were fully informed of the study and written consent for participation was obtained. Perimenopausal women, those with pre-existing intermenstrual or irregular bleeding, those with known uterine or pelvic pathology, those currently taking sex steroid hormones or regular non-steroidal anti-inflammatory drugs (NSAID) were excluded. When endometrial pathology was revealed at hysteroscopy or endometrial biopsy, subjects were excluded. These subjects kept a diary of all menstrual bleeding and spotting. Menstrual blood loss (MBL) was objectively measured using the alkaline haematin technique (Hallberg and Nilsson, 1964; Fraser et al., 1984
) in 12 women with DUB. Of these 12 women, 10 had MBL >60 ml (mean MBL 115 ± 27 ml; range 61172 ml) and two had MBL within the normal range (3540 ml) on at least one occasion. In the remaining eight women, MBL was not measured objectively.
Before insertion of Norplant, six serum samples were obtained for oestradiol and progesterone measurements; one sample was taken during the first and second weeks of the cycle, and two were taken during the third and fourth weeks. Following Norplant insertion, blood samples were taken on two occasions during the two weeks preceding the hysteroscopy. In the comparison group, blood samples were taken in the luteal phase of the menstrual cycle to confirm ovulation.
Blood was allowed to clot and then centrifuged at 200 g for 10 min within 2 h of sampling. The serum was removed and stored at -20°C until analysed. Oestradiol and progesterone assays were performed using a chemiluminescence immunoassay (Immulite®; Diagnostic Products Corp., Los Angeles, CA, USA). These assays had a reporting range of 73 to 734 pmol/l for oestradiol, and 0.6 to 127 nmol/l for progesterone. The lower limits for detection were 44 pmol/l and 0.28 nmol/l respectively, and the inter-assay variability in our laboratory was 10% for both steroid hormones. A progesterone concentration of >10 nmol/l in at least one sample was considered to be indicative of luteal activity.
Subjects prospectively recorded `bleeding' or `spotting' on a menstrual chart. Bleeding was defined as `any bloody vaginal discharge that requires the use of such protection as pads and tampons' and spotting as `any bloody vaginal discharge that is not large enough to require sanitary protection' (Belsey, 1991).
In all subjects, the presence of any bleeding or spotting on the day of the hysteroscopy was recorded, and the number of days of bleeding and spotting during the previous 30 days. In the comparison group, the date of the last normal menstrual period was prospectively recorded. The hysteroscopy technique and instruments used have been described previously in detail (Hickey et al., 1998).
There was no instrumentation of the cervical canal or uterine cavity before introduction of the hysteroscope. Superficial vascular fragility was assessed by controlled alteration of intrauterine pressure under direct hysteroscopic observation. This was achieved by slowly emptying the uterus of saline distension fluid by disconnecting the giving set, and allowing the remaining fluid in the cavity to drain out. As the cavity collapsed, any bleeding points were carefully observed. The term `vascular fragility' described the amount of bleeding arising from the superficial endometrial vessels on collapsing the uterine cavity. The volume of surface bleeding was assessed visually and scored numerically as: 1 = not present; 2 = present in slight quantity; 3 or 4 = present in moderate quantity; and 5 = present in large quantities. The occurrence and amount of bleeding was quantified by direct observation of bleeding vessels by two observers, the second of whom was blinded to the patient characteristics. The presence of subepithelial haemorrhages was recorded and the percentage of endometrial surface area covered by ecchymoses and petechiae was assessed using image analysis as described previously (Hickey et al., 1998).
The uterus was then slowly redistended with normal saline and the appearance of the superficial endometrial vessels as well as the extent and source of any bleeding were again recorded. The video-recording of the hysteroscopy was examined in detail after the procedure, and Polaroid photographs taken (Sony, Sydney, Australia).
Two hysteroscopies were performed in each Norplant user. For approximately one-third of the group, the initial hysteroscopy was performed at around 1 month of Norplant exposure, for a further one-third at 2 months, and for the remainder at 3 months. The second hysteroscopy was scheduled for 3 months later. One hysteroscopy was performed in each member of the comparison group. Hysteroscopy was performed in this group at various times through the menstrual cycle (mean day 17; range, day 5 to 30). None was performed during menstruation, but in 6/20 cases (30%) hysteroscopy was performed during the perimenstrual period (days 24 to 05 of a 28-day cycle).
Statistical analysis
Statistical analysis was performed using the SAS program JMP (Carey, Raleigh, NC, USA), on a Macintosh 6200/75 computer. Linear regression was used to assess the relationship between two continuous variables. Analysis of variance (ANOVA) was used to assess whether the relationship between these variables was significant. Ordinal logistic regression was used to analyse the relationship between an ordinal dependent variable (such as hysteroscopic appearance of the endometrial vessels) and a continuous independent variable (such as oestradiol and progesterone concentrations). A 2-test was used to assess whether the logistic model fit was significantly different from that of a fixed response rate across the whole sample. ANOVA was used to test whether there was a significant difference in means between two or more groups. When more than two groups were included in the ANOVA, the TukeyKramer post-hoc test was used to indicate which groups showed significant differences in means from which other groups. A probability value of < 0.05 was taken to indicate statistical significance.
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Results |
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The mean age of the 20 women in the menorrhagia comparison (DUB) group was 40 (range 2651) years, significantly older than the treatment group (F ratio = 63.9, P < 0.0001), and only one woman was nulliparous. The average weight and body mass index of the comparison group did not differ from the treatment group.
A total of 86 out-patient hysteroscopies was performed over a 1-year period; of these, 66 were in Norplant users (at between 2.6 and 41.5 weeks of exposure to the implants), and 20 were in DUB comparison subjects. The procedure was well tolerated in the majority of cases, but two women from the Norplant group declined a second hysteroscopy because of discomfort during the first procedure.
The mean number of bleeding and spotting days before Norplant insertion (five per 30 days) was increased to nine per 30 days after insertion. This did not change significantly over the 1-year follow-up period, but the number of spotting days tended to decrease with time of exposure.
The mean (± SD) number of bleeding or spotting days during the 30 days preceding the hysteroscopy was 8.5 ± 8.5 (range 0 to 30) days among Norplant users, and 5 ± 3 (range 4 to 10) days in the menorrhagia group.
The mechanical stress test to observe endometrial vascular fragility was performed in 56/66 (85%) of hysteroscopies in Norplant users, and 19/20 (95%) of menorrhagic subjects. The test was omitted when the subject was already bleeding heavily, thus obscuring a clear view of the endometrial vasculature, or for patient comfort in order to hasten the examination.
Superficial endometrial vascular fragility was significantly greater in Norplant users compared to women with menorrhagia (2 = 11.60, P = 0.02; Figure 1
). Vascular fragility was greater in those Norplant users who had experienced bleeding or spotting during the past 30 days, compared to those with amenorrhoea (
2 = 6.15, P = 0.01). Vascular fragility was also increased in those who were bleeding on the day of the hysteroscopy. Among 66 hysteroscopies carried out in Norplant users, 24 (36%) were performed during a bleeding episode. Of those women who presented for hysteroscopy during a bleeding episode, 21/24 (88%) bled heavily compared with 22/42 (52%) who were not bleeding on the day of the procedure (
2 = 12.18, P = 0.01).
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In the menorrhagia group, endometrial vascular fragility varied cyclically and was increased in the perimenstrual phase (days 24 to 05). Four of the six perimenstrual subjects bled heavily compared to only one of 14 subjects undergoing hysteroscopy during other phases of the menstrual cycle (2 = 12.83, P = 0.01; Figure 2
). If the perimenstrual group was excluded from this comparison of vascular fragility between the menorrhagic group and Norplant users, the difference between the groups was much more marked (
2 = 12.18, P = 0.0003). No significant differences in hysteroscopic appearance of vessels or in vascular fragility were observed between those subjects with measured menstrual blood loss >60 ml (10/20) and the remaining 10 subjects (
2 = 6.5, P = 0.16).
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No statistically significant change in mean serum oestradiol concentrations was seen in Norplant users. The mean concentration of oestradiol was 290 pmol/l before, and 180 pmol/l after, Norplant insertion. In the pre-insertion cycle, serum progesterone concentrations suggestive of ovulation (>10 nmol/l) were seen in all subjects but three. Following Norplant insertion, only two subjects showed serum progesterone concentrations of >10 nmol/l, and the mean serum progesterone concentration fell from a pre-treatment mean of 28.5 nmol/l to 2.1 nmol/l. No statistically significant associations were seen between vessel diameters or apparent fragility of superficial endometrial vessels and circulating concentrations of oestradiol or progesterone in Norplant users or in the menorrhagia group. There was no obvious relationship between subepithelial bleeding and circulating concentrations of oestradiol or progesterone.
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Discussion |
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The source of this vascular fragility is unknown. Reduced endometrial vessel integrity may arise from altered structure or function of endothelial cells, or intercellular adhesion of the surrounding and supporting tissues. During the normal menstrual cycle, migratory leukocytes (Clark and Daya, 1990; Clark, 1993
), and increased local activity of matrix metalloproteinases (MMP) (Hampton and Salamonsen, 1994
) are thought to precipitate endometrial vascular breakdown and menstruation. Vascular endothelial growth factor (VEGF) may mediate endothelial permeability, as well as promote vascular repair (Charnock-Jones et al., 1993
). Exposure to progestogen-only contraceptives is known to alter migratory leukocyte populations (Clark et al., 1996
), MMP expression (Skinner et al., 1999
; Vincent et al., 1999
) and VEGF activity (Lau et al., 1999
). The observation of increased vascular fragility in the menorrhagia group during the perimenstrual period suggests that vascular fragility may be a physiological event associated with vascular breakdown at this time. This requires further investigation in a larger population.
The observation of profuse subepithelial haemorrhages in Norplant users may be a further indication of vascular fragility. In other vascular beds, such as the skin, purpura may imply defective haemostasis (Rook et al., 1986). Normal capillary haemostasis depends upon numerous factors, including the integrity of the capillary endothelium, the basement membrane and pericytes, and the ability of platelets to plug defects (Rook et al., 1986
). Endometrial vascular basement membrane has been shown to be deficient in Norplant users, particularly during the early months of progestogen use when bleeding problems are most common (Hickey et al., 1999
). In addition, progestogen exposure is associated with reduced expression of Von Willebrand factor (vWF), crucial for platelet adhesion and plug formation (Marsh et al., 1995
).
Subepithelial haemorrhages were often observed without overt menstrual bleeding. In order for these haemorrhages to present as vaginal bleeding, a breach in the overlying epithelium must occur. Cytokeratin deficiency in the superficial epithelial architecture has been observed in Norplant users (Wonodiresko et al., 1997), and may contribute to epithelial fragility or deficient repair. Fragility or disruption of the epithelium may control whether endometrial vascular breakdown presents as frank vaginal bleeding or is contained beneath the epithelium. Oestrogens act to terminate BTB and are known to promote epithelial cell division (Gordon et al., 1995). The action of oestrogens may be to strengthen the epithelium and thus prevent overt bleeding.
Although mean oestradiol concentrations did not change following Norplant insertion in the present study, a number of reports have been made detailing variable oestradiol concentrations among Norplant users. Some earlier studies (Faundes et al., 1991, 1998
; Darney et al., 1996
) have suggested that oestradiol concentrations are decreased in these subjects, though others (Croxatto et al., 1988
) found no difference between mean oestradiol concentrations in Norplant and Copper-T intrauterine contraceptive device (IUCD) users. However, these studies have varied in the timing of pre- and post-implant oestradiol measurements and in the assay methods used.
Hysteroscopic inspection of the superficial endometrial vasculature revealed apparent differences in the source of bleeding between Norplant users and women with spontaneous menorrhagia (Hickey et al., 1999). These observations were consistent with the proposal that BTB arises from endometrial capillaries and veins (Hourihan et al., 1986
; Johanisson, 1990
). Normal menstrual blood loss is limited by spiral arteriole constriction, mediated by endometrial prostaglandins and endothelins (Cameron et al., 1991
). These mechanisms are likely to be less effective in limiting capillary and venous bleeding, and endometrial endothelin is also deficient in Norplant users (Marsh et al., 1995
).
In this study, bleeding from superficial endometrial vessels was provoked by the mechanical stress of gentle uterine distension and deflation at hysteroscopy. Since the uterus is mobile within the pelvis, shearing stress between endometrial surfaces may be sufficient to produce a mechanical stimulus capable of provoking bleeding when vessels are fragile.
This simple hysteroscopic mechanical stress test is not an ideal tool for the investigation of vascular fragility. The components, temperature and pressure of the distending medium may influence the microvascular circulation. The amount of saline used varied between subjects, and was related to the duration of the procedure. Pilot studies varying low uterine distension pressures, saline quantity and temperature did not appear greatly to alter endometrial vascular appearance at hysteroscopy, but these factors may have affected the endometrial vasculature in subtle ways. A more `physiological' environment may have been produced if an isotonic, body temperature distension medium had been used with minimal uterine distension pressures. In addition, the visual assessment of the source and quantity of bleeding was subjective.
A group of women with spontaneous menorrhagia and ovulatory DUB provided a comparison group. These subjects differed in age and parity from Norplant users, and these factors are of unknown significance. Alterations in local vasoactive control mechanisms in menorrhagia, such as endothelins and prostaglandins, may have affected the appearance and fragility of endometrial vessels quite differently in this population (Fraser et al., 1997). Hysteroscopy of normal volunteers or Norplant users before insertion of the implants would have provided a more legitimate control population. However, ethical concerns were raised about repeating hysteroscopies in these normal populations purely for research purposes.
In summary, this study has demonstrated that superficial endometrial vascular fragility appears to be greater in Norplant users than in a comparison group of women with spontaneous menorrhagia. These observations suggest that exposure to Norplant alters superficial vascular integrity. These changes may be associated with BTB.
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Acknowledgments |
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Notes |
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References |
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Submitted on June 28, 1999; accepted on March 22, 2000.