1 Imperial College School of Medicine at St Mary's, Norfolk Place, London W2 1PG, UK, 2 Department of Anatomy and Histology, Flinders University of South Australia, Adelaide, South Australia, 3 Sydney Centre for Reproductive Health Research, Department of Obstetrics and Gynaecology, University of Sydney, NSW and 4 Electron Microscope Department, University of Sydney, NSW, Australia
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Abstract |
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Key words: endometrium/Norplant/perfusion/progestogens/vaso-motion
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Introduction |
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Increased endometrial vascular fragility has been proposed as the mechanism by which superficial endometrial vessels break down, leading to breakthrough bleeding (Odlind and Fraser, 1990). Increased fragility of the superficial vasculature has been demonstrated at hysteroscopy in Norplant users (Hickey et al., 1996
), but the reason for the breakdown of endometrial vessels in vivo is unknown. Sex steroid hormones are known to influence uterine perfusion. Uterine blood flow is increased by oestradiol, and this effect is lost with the addition of progesterone (Fraser et al., 1987
; Hilliard et al., 1992
). Dysfunctional uterine bleeding is associated with characteristic changes in endometrial blood flow (Fraser et al., 1987
). Changes in the perfusion of superficial endometrial vessels may contribute to breakthrough bleeding, particularly if vessels are fragile. Alterations in tissue oxygenation and the release of local growth factors may stimulate angiogenesis, and contribute to the increase in endometrial microvascular density observed in Norplant users (Rogers et al., 1993
). In addition, changes in endometrial perfusion may alter the morphology and diameter of endometrial veins and capillaries, and may be associated with the dilated vessels observed in the superficial endometrium following progestogen exposure (Johannisson, 1990
; Hourihan et al., 1991
).
Endometrial perfusion cannot reliably be derived from measurements of total uterine perfusion, since the uterine artery also supplies the ipsilateral ovary, the Fallopian tube and the upper vagina as well as the myometrium (Bartelmez, 1933). External Doppler or ultrasonic measurements of uterine blood flow cannot be extrapolated to endometrial perfusion, since these two vascular beds have been shown to vary independently (Rogers and Gannon, 1981
). Once blood enters the uterus, it is distributed into myometrial and endometrial vascular beds, and this distribution is controlled by intermediate arterioles within the tissue. One method of estimating local tissue perfusion in the endometrium is laser-Doppler fluxmetry (Bungum et al., 1996
; Gannon et al., 1997
). This is a non-invasive technique for dynamic quantification of perfusion in microvascular beds, and is more sensitive temporally and spatially than other available methods to measure endometrial perfusion. This technique provides a relatively simple method of measuring localized endometrial perfusion that appears to be well tolerated.
The aim of this study was to measure endometrial perfusion in a small group of normal women during the secretory phase of the normal cycle, and again at 46 weeks after insertion of the low-dose levonorgestrel contraceptive implant system, Norplant. These findings were compared with those from a group of longer-term Norplant users, in order to assess changes in endometrial vascular perfusion over time. The relationship of endometrial vascular perfusion to menstrual bleeding patterns, systemic blood pressure and pulse rate and ovarian steroid hormones was explored. Endometrial vasomotion, indicating local regulation of perfusion, was also investigated.
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Materials and methods |
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Assessment of endometrial perfusion
Laser-Doppler fluxmetry uses the Doppler shift of a low energy laser beam to estimate microvascular perfusion. The laser beam is conveyed by a fibreoptic cable into tissue, where the light is scattered by both stationary and moving blood cells. When scattered by a moving red cell, the frequency of the light changes; the magnitude of this Doppler shift is proportional to the red blood cell (RBC) velocity, and the amount of reflected light is proportional to the volume (number) of RBC in the tissue. The RBC `flux' is calculated from an algorithm of the number and the mean velocity of cells in the measured volume of 12 mm3 below the fibreoptic probe (Bungum et al., 1996; Gannon et al., 1997
).
Endometrial perfusion was measured for 3 min at each of four to five sites on the endometrial surface of the middle of the uterus, on one occasion in the secretory phase of the menstrual cycle before Norplant insertion in six subjects and again, 46 weeks after insertion. The second measurement was timed to be as close to the time of the next menstrual period as was possible, and at a time normally associated with irregular bleeding patterns. This group was classed as early Norplant users. In the longer-term Norplant users, perfusion was measured in the same way on one occasion. Pulse rate and blood pressure were recorded at the time of each endometrial perfusion measurement.
Equipment
To measure endometrial perfusion, a TSI Laserflo BPM 403A laser-Doppler instrument (Vasamedics, St Paul, MN, USA) was used, operating from a laser diode at a wavelength of 780 ± 20 nm, delivering 2 mW at the probe tip. This was equipped with a 2 m long and 2.1 mm diameter flexible endoscope probe measuring at 90° to the probe axis (TSI PR-436). The probe was sterilized by immersion in surgical 2% glutaraldehyde (Aidal Plus, Whiteley Ind. Ltd, Mascot, NSW 2018, Australia) for 20 min and then rinsed in sterile water prior to each use. Indelible markings, 1 cm apart on the external white polymer casing of the probe allowed easy estimation of the extent of probe advancement through the cervix. The probe was carefully inserted into the uterus using sterile forceps for a distance of 5 cm through the cervix, until the tip of the probe was approximately at the middle of the uterus. Flow measurements from the instrument (averaging time 0.1 s) were charted on the inbuilt chart recorder (100 mm/min, sensitivityx4), and a MacLab/4eTM (AD Instruments Pty Ltd, Castle Hill, NSW, Australia) at 20 samples/s. Once 3 min of stable recording at any one site were made, the probe was moved by simultaneous rotation and advancement/withdrawal until four to five sites had been measured. Movement of the probe did not appear to consistently or adversely affect perfusion, based on inspection of the flux traces or temporal trends in the data. Measurements were made at the middle of the uterus, since no significant differences in perfusion along the length of the uterus were detected in preliminary studies. In these studies, we also determined that measurements of 3 min at five different sites allowed adequate sampling of both temporal and spatial variation in assessment of endometrial perfusion.
Flux calibration
All measurements were made using the same fibre optic probe. Values are reported as `units' (approximately equivalent to ml/min/100 mg tissue, as described by the manufacturer). The instrument and probe consistently recorded 25 TSI `units' (gain at x1) against a laser Doppler motility standard (Periflux PF-100; Perimed Inc., Sweden).
Vasomotion
Vasomotion was assessed by inspection of the number of cycles of endometrial flux/min (Gannon et al., 1997), using the display from the Maclab. Short-term cycles were classed as those with a well defined periodicity of >3/min and long-term cycles as (approximately) 1/min. Values obtained during movement of the probe or the subject were discarded.
Serum oestradiol and progesterone
Serum samples were obtained for measurement of oestradiol and progesterone at the time of measuring endometrial perfusion. Blood samples were allowed to clot, and centrifuged at 1600 g for 10 min within 2 h. Serum was stored at 20°C until analysis. Oestradiol and progesterone assays were performed using a chemiluminescent immunoassay (Immulite®, Diagnostic Products Corporation, Los Angeles, CA, USA). This assay has a reporting range of 73734 pmol/l for oestradiol and 0.6127 nmol/l for progesterone. The lower limits for detection were 44 pmol/l and 0.28 nmol/l respectively and the inter-assay variability was 10% for both steroid hormones. These assays are not known to interact with levonorgestrel. A progesterone concentration of >10 nmol/l was considered to be suggestive of luteal activity.
Vaginal bleeding patterns
Subjects prospectively recorded `bleeding' or `spotting' on a menstrual chart. Bleeding was defined as `any bloody vaginal discharge that required 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, 1988). The number of bleeding and spotting days in the 30 days prior to laser-Doppler measurements were recorded, and whether the subject was bleeding on the day that the measurement was made.
Statistical methods
The mean `flux', `volume', `velocity' and vasomotion cyclicity of red blood cell perfusion for each site were calculated, and used in subsequent analyses using the Statistics Package for Social Sciences (SPSS) program (Version 8; SPSS Inc., Chicago, IL, USA). Paired t-tests were used to assess changes in endometrial perfusion before and after insertion of Norplant in six subjects. Regression analysis and analysis of variance were used to assess changes in endometrial perfusion with time of exposure to Norplant, and the relationship of perfusion to sex steroid hormone concentrations, blood pressure and pulse rate using the SAS program JMP (Carey, NC, USA) using a Macintosh 6200/75. A probability (P) value of 0.05 was taken to indicate a statistically significant difference between samples. Data are presented as mean ± SEM unless otherwise reported.
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Results |
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Serum oestradiol and progesterone
Serum oestradiol on the day of the perfusion measurement was not significantly affected by 46 weeks of Norplant exposure (Table I), and was not significantly different between the control group (280 ± 69 pmol/l) and the long-term Norplant group (288 ± 76 pmol/l). Serum progesterone concentrations indicating luteal activity (>10 nmol/l) were seen in all six control subjects. Serum progesterone decreased significantly (P < 0.05) after 46 weeks use of Norplant (Table I
), and was also significantly less (P < 0.05) in the long-term Norplant users (3.0 ± 1.3 nmol/l) than in the control group (32.2 ± 7.4 nmol/l).
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In the longer-term Norplant users, the mean number of bleeding and spotting days in the previous 30 day reference period was 8.08. Three of the 13 subjects were bleeding on the day of the procedure.
Endometrial perfusion before, and 46 weeks after, Norplant insertion
Endometrial perfusion was measured in the secretory phase of the control cycle (mean day 21 ± 0.9). The mean endometrial perfusion during the control cycle was 27.1 flux units (±3.3). Wide variations in endometrial perfusion were observed between and within subjects (see Figure 1 and Table I
).
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Long-term Norplant implantation
In the 13 longer-term Norplant users, the mean perfusion was 35.7 ± 7.2, which was no different from control values (27 ± 3.3, Figure 2). The time since insertion of Norplant had no significant effect on mean endometrial perfusion, although it tended to increase with time from its lowest values immediately after insertion. Neither velocity nor volume of endometrial perfusion was significantly related to time since Norplant insertion.
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Long-term vasomotion (1 cycle/min) in endometrial perfusion was observed in all but five of 126 measurements made in Norplant users, the pattern of the waveforms being similar to that seen in the control subjects. Short-term cyclicity was seen in two of 13 (15%) long-term Norplant users, compared to five of six (83%) all control subjects.
Correlations between variables
Neither the flux, velocity, volume nor vasomotion of endometrial RBC perfusion were related to subject age, BMI, blood pressure, pulse rate or cigarette smoking. There was no relationship between serum oestradiol and progesterone and these variables, or to recent or current endometrial bleeding.
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Discussion |
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Specific methods to assess endometrial perfusion have included the clearance of radioactive xenon gas from the uterine lumen (Fraser et al., 1987), and dissipation of locally applied heat to the endometrium (Akerlund et al., 1975
). The inert gas clearance techniques are laborious and time consuming, and cannot assess rapid changes in flow. The use of local heat may alter endometrial flow, thus invalidating the results of this method. The use of external ultrasonic Doppler to identify changes in endometrial artery pulsatility index relies on accurate discrimination of endometrial from intramyometrial vessels, which is unlikely to be accurate using external monitors.
Laser-Doppler flowmetry is a simple, continuous and dynamic method that can be used for prolonged time periods in the awake patient without causing undue discomfort or inducing local trauma. The fine laser-Doppler probe is only able to measure flow in the immediate area below the laser beam, and only to a depth of ~12 mm. When the endometrium is atrophic, the probe might be reaching a different vascular bed from that measured during the normal menstrual cycle.
This was a difficult study to recruit and execute, particularly since Norplant contraceptive implants were not generally available in Australia during the recruitment period. Multiple sites were measured within each subject in this study to assess the variation in endometrial perfusion as measured by this technique. Wide variations in perfusion were seen between patients given Norplant implants. Similarly, menstrual bleeding patterns in progestogen users show great variation, and it is not possible to predict which women will be troubled with frequent and prolonged bleeding before treatment. It is possible that this variation in endometrial vascular response to low-dose progestogens may reflect the clinical response of vascular breakdown.
Mean perfusion values during the secretory phase of the control cycle were similar to those previously reported (Fraser et al., 1987; Gannon et al., 1997
). The lack of a change in endometrial perfusion during early exposure to Norplant was an unexpected finding, since it corresponds with a time of increased bleeding and spotting and a reported increase in endometrial microvascular density (Rogers et al., 1993
). Laser-Doppler RBC flux values are derived from mean RBC velocity and the number (volume) of moving RBC, but it appears neither were significantly affected by the reported changes in endometrial vascular density and the regressed and atrophic glandular and stromal elements observed after exposure to Norplant (Rogers et al., 1993
).
There was considerable variation in fluxmetry measurements from site to site within subjects; this variation was not affected by Norplant exposure, implying that heterogeneity of perfusion was not affected by Norplant. During normal menstruation, reduced local perfusion or vascular stasis was thought to precede bleeding (Markee, 1950), although mean perfusion as measured by laser-Doppler is not reduced at this time (Gannon et al., 1997
), perhaps because the spatial resolution of the laser-Doppler technique makes it difficult to detect the focal nature of menstrual perfusion and bleeding. There is increasing evidence that breakthrough bleeding is focal in nature, and may arise primarily from small vessels on the endometrial surface rather than from spiral arterioles (Olind and Fraser, 1990; Hickey et al., 1996
).
It is likely the region supplied by a single spiral arteriole is slightly larger than that sampled by the laser Doppler probe, assuming that the laser Doppler probe used in this study samples from a volume of a sphere of ~1 mm diameter (Johansson et al., 1991; Mayrovitz, 1992
), and that individual human spiral arterioles supply separate territories (arterial fields) of 49 mm2 at the lumenal surface (Bartelmez, 1933
). However, it was calculated that if the vasculature of the endometrial functionalis is arranged as an array of similar sized contiguous hexagonal vascular territories each centred on a spiral arteriole (Bartelmez, 1933
), the probability that the randomly placed probe was measuring from the flow territory of more than one spiral arteriole ranged from 64 to 46% for vascular territory sizes of 49 mm2 of endometrial surface respectively. Thus, it seems unlikely that laser Doppler technique would detect the perfusion of individual spiral arterioles in the endometrium, yet it is still capable of estimating the heterogeneity of endometrial perfusion. As a corollary, it follows that any effect of Norplant on endometrial perfusion is most likely at the level of individual spiral arterioles, rather than across the endometrial vasculature as a whole. On the other hand, mean perfusion across the entire endometrium (as measured by laser Doppler fluxmetry) can be significantly altered by simple routine procedures such as tubal ligation (Verco et al., 1998
). Clearly, the relationship between endometrial structure and its perfusion is a complex one, which cannot be easily predicted from histological or hormonal studies.
Alterations in focal endometrial perfusion may lead to changes in the endometrial vasculature via the release of local vasoactive factors. In the retina, loss of autoregulation of blood flow leads to increased retinal flow and increased tissue oxygenation. This, in turn provokes a locally regulated reduction in flow and tissue hypoxia. Hypoxia provokes the release of proteolytic, vasoactive and angiogenic substances and the proliferation of fragile vessels (Shweiki et al., 1992). If a similar process occurred in the endometrium, this may lead to focal vascular fragility and the breakthrough bleeding commonly reported after Norplant.
The marked changes in vasomotion observed in Norplant users may reflect a marked alteration in the local regulation of the endometrial microvasculature. Vasomotion describes the spontaneous and rhythmic dilation and constriction of microvessels. The oscillations range from 2 to 11 cycles/min and appear more frequently in areas with increased flow. Endometrial blood flow displays short-term cyclicity at 58 cycles/min due to vasomotion and long-term cyclicity at ~1 cycle/min, similar to that seen in other human tissues (Gannon et al., 1997). In this study, the marked decrease in short-term cyclicity in Norplant users may reflect the redistribution of endometrial blood flow through an increased number of small endometrial vessels, due to an increase in vascular density (Rogers et al., 1993
). Vasomotion is thought to represent the alternate opening and closing of contiguous vascular territories, and is a mechanism for distributing local blood flow whilst maintaining peripheral resistance and central blood pressure. If this phenomenon is lost during Norplant use, blood flow may be simultaneously spread through an increased number of capillaries, leading to a decreased perfusion in individual microvessels whilst maintaining total perfusion to the tissue overall. This loss of autoregulation may be associated with changes in the morphology and fragility of individual elements of the microvasculature of the endometrium.
In summary, this study demonstrates the use of laser-Doppler flowmetry in the measurement of endometrial perfusion in a small group of women exposed to the long-acting low-dose levonorgestrel contraceptive implant, Norplant. Endometrial perfusion was not changed during the early months of Norplant use, a time when irregular bleeding and spotting are common, and was no different from controls after longer periods of Norplant exposure. No direct relationships between endometrial perfusion and ovarian steroid hormones were demonstrated. Short-term endometrial vasomotion was largely abolished during Norplant exposure. This may reflect changes in the autoregulation of endometrial blood flow, which may contribute to breakthrough bleeding. Further studies are needed to ascertain whether other contraceptive steroids alter endometrial perfusion and microvascular regulation in this way, and this information may shed further light on the possible mechanisms of breakthrough bleeding with these preparations.
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Notes |
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References |
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Submitted on June 21, 1999; accepted on February 1, 2000.