1 Countess of Chester Hospital, Liverpool Road, Chester, UK, 2 University of New South Wales, Department of Endo-Gynaecology, Royal Hospital for Women, Randwick, New South Wales, Australia, 3 Academic Department of Gynaecological Surgery, James Cook University Hospital, Middlesbrough, UK, 4 St James University Hospital, Beckett Street, Leeds, UK, and 5 University of Western Australia, School of Womens and Infants Health, King Edward Memorial Hospital, Perth, WA 6008, Australia
6 To whom correspondence should be addressed. e-mail: rgarry{at}obsgyn.uwa.edu.au
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Abstract |
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Key words: diaphorase/endometrial ablation/serosal temperature/thermal balloon/zone of thermal necrosis
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Introduction |
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The first study on extirpated uteri was performed in 1992 demonstrating that endometrial destruction by hyperthermia using the Cavaterm system was a potential treatment for menorrhagia (Friberg et al., 1996). There were eight cases in total, five in-vitro cases and three in-vivo cases. The treatment time for the eight cases varied between 31 and 54 min. The maximum depth of destruction recorded was 8 mm with no significant increases in the uterine serosal temperatures recorded. In 1995 the treatment time was reduced to 15 min, but the histological studies were not repeated for the new treatment time.
The study will be presented in two phases. The in-vitro study was designed to test the validity of the histochemical method. The in-vivo study concentrated on the effect of the actual time differences for the treatment cycles.
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Materials and methods |
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Technique
For the in-vitro study, the hysterectomy specimens provided a source of freshly obtained uterine tissue, which could then be subjected to a thermal injury using the Cavaterm system. The Cavaterm thermal balloon ablation technique has been previously described (Hawe et al., 1999). Two cases at each treatment time (7, 10 and 15 min) were studied. For the in-vivo study, a standard Cavaterm procedure was performed after an endometrial curettage and placement of the thermal sensors. Four cases were performed for 7 min, six for 10 min and five for 15 min.
For the thermal studies, tissue temperature was measured using miniature negative temperature coefficient thermistors. The thermistors are housed individually or in groups of five. The sensors were calibrated on five points at regular intervals between 40 and 80°C. Post-calibration and precision tests were performed to ensure accuracy. A five-point sensor was used to monitor the balloon surface temperature and the other to measure the temperature gradient through the myometrial wall. The cervical temperature was assessed by using sensor number 5 on the five-point balloon sensor. The four single-point sensors were used to measure serosal temperatures at varying sites, including the cornual areas, isthmic region, anterior and posterior wall, and the fundus.
Tissue preparation and staining
Each case provided at least eight tissue blocks for assessment, two each from the lower uterine body, mid-body, upper body/fundus and the cornual areas. The zone of thermal necrosis (ZTN) was identifiable macroscopically. Sections were then cut, 1 cm square, to include the endometrium and the superficial myometrium. The sections were placed on a metal chuck in dry ice and fixed with optimal cutting temperature embedding matrix (Cell Path, Herts, UK) and snap-frozen in liquid nitrogen. The specimens were stored in a 80°C freezer until cut on a cryostat to thickness of 10 µm. Two sections underwent standard haematoxylin & eosin staining and four sections were mounted on slides for diaphorase staining (two sections per slide). The sections were allowed to reach room temperature and then covered with 50100 µl nitroblue tetrazolium incubating solution containing the
nicotinamide adenine dinucleotide (NAD). The sections were then placed in a humidity chamber and incubated at 37°C for 30 min. To make these sites of cell death more readily identifiable, the tissue is counter-stained with nuclear fast red, so that the areas of cell death stain pink (Figure 1)2. The depth of the zone of thermal injury was assessed by one author (J.H.) and checked by a further blinded pathologist (A.C.), using a graticule with an accuracy of ±0.1 mm.
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Results |
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The results of the diaphorase study to determine the mean maximum ZTN for the in-vitro and in-vivo study are shown in Table II. There is a trend to a deeper depth of thermal necrosis in the in-vitro study compared to the in-vivo study and with increasing treatment times. The depth of ablation appears to be tapered, with less destruction seen at the cornual and low-body sections. In the majority of cases the thermal injury was uniform, with very little difference noted between the minimum and maximum values (Figure 1). Viable tissue was identified in only one section at the lower body from a single 15-min case, which might be due to the section being taken from the upper cervix, which would have been untreated by the balloon. Viable tissue was identified in the majority of sections from the cornual areas. Here, the ablation effect was often tapered, with cell death identified at the uterine cavity end of the section and viable tissue deeper into the cornua.
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Discussion |
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The aim of hysteroscopic endometrial ablation is to remove or destroy the endometrium, including its basal layer. Previous work has reported that in women pretreated with danazol, gland stumps are present to a mean depth of 3 mm below the endometrial/myometrial junction, with a maximal depth of 4.4 mm (Reid, 1989). It follows that for endometrial ablation to be effective there must be destruction of tissue to at least this depth.
In this study, macroscopic assessment of the uterus revealed a ZTN of between 4 and 8 mm for all cases, with the ZTN being greatest for the longer treatment times. In all cases the measured ZTN was less than that predicted macroscopically. Table II also demonstrates that the ZTN for all treatment times and areas sampled was greater for the in-vitro study compared to the in-vivo study. This finding was expected due to the loss of the heat-sink effect of the myometrial blood flow in the in-vitro cases. The in-vitro cases demonstrate the safety of ablation at all treatment times, as the serosal surface did not demonstrate any thermal injury even when the heat-sink effect has been removed. The ZTN did appear to vary depending on treatment times (Table II). Based on all the results it, would appear that a treatment time of 7 min would fail to achieve an adequate depth of ablation, whereas the difference between the 10- and 15-min in-vivo cases was not as great. The results obtained from this study are similar to other preclinical studies assessing second-generation endometrial ablation techniques (Donnez et al., 1996; Bustos-Lopez et al., 1998
; Hodgson et al., 1999
), and electrosurgical techniques of ablation and resection (Duffy et al., 1991
, 1992
).
An important consideration when assessing the depth of the ZTN is the shrinkage artifact secondary to the tissue preparation techniques. It is reported that the shrinkage associated with tissue processing was 10.3% in post-hysterectomy specimens, and shrinkage due to cessation of blood supply to the uterus and subsequent tissue processing was 25% (Duffy, 1993). Furthermore, the results from this study represent immediate cell death only. Previous work performed on perfused uteri in in-vitro work has shown that cell death continues after cryotherapy injury for up to 24 h (Kremer, 2001
). We found in 33 of the 70 in-vivo sections that the demarcation between viable and dead cells was not as obvious as expected, with a third zone [the transitional zone (TZ)] being identified. This appeared to be a stage between cells that showed no diaphorase activity at all, and those that showed normal activity. It is hypothesized that this area may represent cells that would go on to die with time. In a number of cases, when the ZTN and this TZ were combined the calculated depth was very similar to that seen macroscopically. Previous work has demonstrated that the initial destruction leading to a ZTN is followed by necrosis during the first month, and then a complex healing process in which fibrosis and scarring predominate, which are also thought to be important for the success of the procedure (Goldrath et al., 1981
; Reid et al., 1992
; Davis et al., 1998
; Colgan et al., 1999
; Tresserra et al., 1999
). The only way to assess the effect of time would be to perform the hysterectomy at different times post-ablative treatment, but this is not ethically possible.
One concern regarding endometrial ablation techniques is the risk of thermal spread through the uterine wall leading to thermal damage to surrounding tissues and there have been anecdotal reports of this. The uterus has thick muscular walls and rich blood supply, which provides an efficient heat-sink effect for thermal energy. The measured ZTN from all areas failed to show any evidence of full-thickness cell death. Duffy et al. demonstrated a reduction in measured temperature with an increasing distance from the active electrode secondary to uterine blood flow (Duffy et al., 1991). With the Cavaterm system, the high-pressure balloon tamponades only the superficial myometrial blood flow, allowing the normal heat-sink effect in deeper tissues. This effect was demonstrated by the myometrial gradient sensor measurements, which recorded lower temperatures with increasing myometrial depth (Figure 22). This confirms that, in a uterus of normal dimensions with a correctly placed catheter, there should be no risk of trans-mural thermal injury and that despite a high balloon pressure that the heat-sink effect of the myometrial blood supply is still effective. In addition, the maximal serosal surface temperature in this study was 44.1°C (mean 39.9°C). This temperature is still less than that which would be expected to cause irreversible cell damage. Serosal surface temperatures in this study (Table I) are in keeping with those previously reported.
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Conclusion |
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Conflict of interest |
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Acknowledgements |
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References |
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Submitted on February 24, 2003; accepted on August 22, 2003.