In-vitro and in-vivo histochemical and thermal studies using a thermal balloon endometrial ablation system for varying treatment times

Jeremy Hawe1,3,4, Jason Abbott2, Graham Phillips2, Nafisa Wilkinson4, Sean Duffy4 and Ray Garry5,6

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 Women’s and Infant’s Health, King Edward Memorial Hospital, Perth, WA 6008, Australia

6 To whom correspondence should be addressed. e-mail: rgarry{at}obsgyn.uwa.edu.au


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusion
 Conflict of interest
 References
 
BACKGROUND: To assess the immediate zone of thermal necrosis (ZTN) using an enzyme histochemical staining technique and serosal temperatures for the Cavaterm endometrial balloon ablation system for different treatment times. METHODS: A thermal balloon ablation was performed initially post- (n = 6) and subsequently pre-hysterectomy (n = 15). Eight to 12 tissue blocks from each uterus were sectioned and stained using diaphorase respiratory enzyme techniques. Patients in the in-vivo group had temperature measurements taken from four serosal points, a myometrial gradient profile, the balloon surface and the endocervical canal. RESULTS: The serosal temperature sensors did not demonstrate any rise in temperature above 44.1°C. The mean temperature at the anterior wall, posterior wall, fundus and cornual areas was 37.1 (SD 1.3), 36.8 (SD 1.0), 37.4 (SD 1.8) and 36.7°C (SD 1.0), respectively. The immediate mean maximum ZTN was greatest for the 15-min treatment time (3.1 mm, SD 1.5) compared to the 10- and 7-min treatment times (3.0 mm, SD 1.4 and 2.2 mm, SD 0.7, respectively). The maximum ZTN recorded was 5.6 mm. No full thickness injuries were demonstrated either histochemically or suggested by the temperature studies. CONCLUSIONS: This study confirms that Cavaterm thermal balloon ablation produces a reproducible thermal injury without evidence of serosal heating. Results suggest that the treatment time could be reduced to 10-min with no detrimental effect on the clinical outcomes. This hypothesis is currently being evaluated by clinical trials.

Key words: diaphorase/endometrial ablation/serosal temperature/thermal balloon/zone of thermal necrosis


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusion
 Conflict of interest
 References
 
The CavatermTM thermal balloon ablation system (Wallsten Medical, Morges, Switzerland) was introduced clinically in May 1993 for the treatment of dysfunctional uterine bleeding. The first 36 patients were treated for 30 min, but since 1995 the treatment time has been reduced to 15 min. Published studies have shown it to be an effective technique achieving amenorrhoea rates of 22–68%, combined amenorrhoea and hypomenorrhoea rates of 56–82%, overall ‘success’ rates of 92–98% and high patient satisfaction rates (Friberg et al., 1998Go; Hawe et al., 1999Go, 2003Go; Friberg and Ahlgren, 2000Go; Pellicano et al., 2002Go).

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusion
 Conflict of interest
 References
 
Subjects
The local research and ethics committee approved the study. Patients undergoing hysterectomy for management of dysfunctional uterine bleeding or pelvic pain were eligible for inclusion in the study. Six patients for the in-vitro study and 15 patients for the in-vivo study were recruited. Prior to the study treatment, all the patients had a documented normal cervical smear, endometrial biopsy, combined with either outpatient or inpatient hysteroscopy or ultrasound scan to confirm a normal uterine cavity. Contra-indications included a uterine cavity length (internal os to fundus) <4 or >10 cm, or active infection.

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., 1999Go). 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 50–100 µl nitroblue tetrazolium incubating solution containing the {beta} 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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Figure 1. Diaphorase staining x2.5. Section taken from the mid body of the uterus. Diaphorase-positive (viable): blue/black; diaphorase-negative (non-viable): pink.

 


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Figure 2. Myometrial gradient curve.

 
Other treatment parameters such as heating power, element current, voltage and resistance and the intraballoon pressure were collected during the study, via additional analogue inputs.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusion
 Conflict of interest
 References
 
In total 21 hysterectomies were performed, six in the in-vitro study (two cases for each treatment time) and 15 in the in-vivo study (four, six and five at 7, 10 and 15 min respectively). Nineteen cases were performed by a laparoscopic approach, one vaginal and one abdominal. The mean balloon length was 5.6 cm (SD 1.1), with mean balloon volume of 5.4 ml (SD 2.9). The mean pressure for the cases studied was 207.2 mmHg (SD 2.9). Initially during the heating phase the power output from the unit is high (mean 70.1 W, SD 3.9), but this stabilizes (mean 34.4 W, SD 4.8) after 90–120 s, once the intra-balloon fluid has reached treatment temperature. The temperature of the fluid within the balloon is constantly monitored by the central processing unit; the mean temperature for all the cases was 76.2°C (SD 2.1). In contrast, the maximum stabilized temperatures at the balloon surface and the length of time spent at this temperature are shown in Table I. The mean maximum stabilized balloon surface temperature for all cases was 67.7°C (SD 3.2). The mean peak recorded temperature using the distal fifth sensor of the five-point balloon thermistor was 43.9°C (SD 4.7, range 35.7–62.1°C), which was felt to represent the temperature in the upper cervical canal.


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Table I. Results from the balloon five-point sensor, serosal sensors and the endo-cervical canal temperature
 
Results from the serosal surface sensors are shown in Table I. There were no significant rises in serosal temperatures. The highest recorded temperature was 44.1°C at the fundus. The maximum mean temperature for all the serosal surface sensors was 37.4°C. All measured cases (nine) demonstrated a gradual decrease in temperature through the myometrial wall as predicted. Each sensor records the temperature at that area for the treatment time. A typical curve is seen in Figure 22.

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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Table II. ZTN as assessed by diaphorase staining technique (Max = maximum depth of ablation for that section)
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusion
 Conflict of interest
 References
 
Cell death is difficult to identify and quantify accurately using standard histochemical techniques. Histologically, areas of coagulative necrosis may suggest cell death. In this study, all specimens demonstrated crush artifact in the residual endometrium and coagulative necrosis in the endometrium and the superficial myometrium. Enzyme histochemistry offers reproducibility and easier demarcation of viable and non-viable tissue. Histochemical staining identifies sudden cell death by a lack of cellular activity. In this study, the respiratory enzyme NAD diaphorase was examined. The original histochemical method was described by Nachlas et al. (1958Go), and was modified by Scarpelli et al. (1958Go) and Young (1971Go). Using this technique, sites of diaphorase activity appear as deposits of blue–black metal formazan, whereas sites without diaphorase activity (areas of cell death) are unstained. 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). This histochemical technique has been used in previous work to assess cell death in the endometrium and myometrium following insult with the Nd:YAG laser (Reid, 1989Go), electrosurgery (Duffy et al., 1991Go, 1992Go) and second-generation ablation techniques (Shah et al., 1998Go).

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, 1989Go). 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., 1996Go; Bustos-Lopez et al., 1998Go; Hodgson et al., 1999Go), and electrosurgical techniques of ablation and resection (Duffy et al., 1991Go, 1992Go).

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, 1993Go). 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, 2001Go). 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., 1981Go; Reid et al., 1992Go; Davis et al., 1998Go; Colgan et al., 1999Go; Tresserra et al., 1999Go). 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., 1991Go). 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.


    Conclusion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusion
 Conflict of interest
 References
 
This study demonstrates that when using the Cavaterm system in uteri of normal dimensions with correct catheter placement there is no evidence of trans-mural thermal spread and that histochemical evidence of cell death is restricted to the superficial myometrium. The results obtained also suggest that there is little difference between the measured ZTN for 10 and 15 min. Immediate histological evaluation of depth of endometrial destruction cannot accurately assess the clinical efficacy of a technique. Variables such as sectioning technique, length of time from treatment to freezing, tissue freezing technique and the effect of time on cell death cannot be assessed by this study. However, the available results suggest that a clinical study comparing the treatment effect of a 10- and 15-min treatment time is justified.


    Conflict of interest
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusion
 Conflict of interest
 References
 
This study was supported by a grant from Wallsten Medical, Morges, Switzerland, the manufacturers of the Cavaterm ablation system.


    Acknowledgements
 
We would like to thank Consultant Pathologist, Dr A. Caslin for his blinded assessment of the ZTN, Pars Henrickson, Wallsten Medical for his expertise for the thermal studies and Ms Jane Ramsdale, St James’s University Hospital, Leeds for her help with tissue sectioning and preparation prior to staining.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusion
 Conflict of interest
 References
 
Bustos-Lopez, H.H., Baggish, M., Valle, R.F., Vallillo-Ortega, F., Ibarra, V. and Nava, G. (1998) Assessment of the safety of intrauterine instillation of heated saline for endometrial ablation. Fertil. Steril., 69, 155–160.[CrossRef][ISI][Medline]

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Submitted on February 24, 2003; accepted on August 22, 2003.





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