- Email: [email protected]
Susceptibility of uterine myomas and endometrium to cryosurgery using a carbon dioxide cryosurgical probe in vitro
Journal of Minimally Invasive Gynecology (2006) 13, 500 –504
Instruments and techniques
Susceptibility of uterine myomas and endometrium to cryosurgery using a carbon dioxide cryosurgical probe in vitro Julia F. Bodle, MD, MRCOG, Sean R.G. Duffy, MD, FRCOG, and David M. Binney, MMedSci From the Academic Department of Obstetrics and Gynecology, Saint James’s University Hospital, Leeds University, Leeds (Drs. Bodle and Duffy); and the Faculty of Health and Wellbeing, Collegiate Crescent, Sheffield Hallam University, Sheffield (Mr. Binney), UK. KEYWORDS: Cryosurgery; Uterine myoma; Endometrium; Carbon dioxide
Abstract STUDY OBJECTIVE: To determine the effect of a 9-mm diameter carbon dioxide cryoprobe, the Endocryo, on myomas and endometrial/myometrial tissue in vitro. DESIGN: Comparative laboratory study (Canadian Task Force classification II-2). SETTING: University laboratory. PATIENTS: Women with and without myomas, undergoing hysterectomy. INTERVENTION: A single 5-minute freeze followed by an active thaw was applied to uterine myomas and endometrial/myometrial tissue in vitro. MEASUREMENTS AND MAIN RESULTS: Endometrial/myometrial and uterine myoma temperature change was measured continuously during the cryosurgical procedure. Depth of cell death was measured using nicotinamide adenine dinucleotide diaphorase enzyme assay. There was no significant difference in temperature change and depth of cell death between myomas and endometrial/myometrial tissue in vitro. CONCLUSIONS: The Endocryo produces the same cryosurgical effect on both uterine myomas and endometrial/myometrial tissue in vitro, an important principal for future development of a clinically effective cryosurgical device for the treatment of menorrhagia in the presence of submucous myomas. © 2006 AAGL. All rights reserved.
Uterine myomas are the most common benign tumor affecting women; a prevalence of up to 65% has been reported in perimenopausal women.1 They are also the most common reason for hysterectomy.2 Myomas are described
Financial support was received from Integral Neurosciences, Andover, UK. Corresponding author: Dr. Sean R. G. Duffy, Level 9, Gledhow Wing, Saint James’s University Hospital, Leeds, LS9 7TF, UK. E-mail: [email protected] Submitted March 19, 2006. Accepted for publication June 19, 2006.
1553-4650/$ -see front matter © 2006 AAGL. All rights reserved. doi:10.1016/j.jmig.2006.06.012
by their location within the uterus as either submucous, intramural, or subserous. Submucous myomas have been shown to be associated with menorrhagia,3 with their removal resulting in improvement in symptoms.4,5 Hysteroscopically, the submucous myoma can be seen as a whitish smooth protuberance into the uterine cavity with a network of fragile blood vessels in the overlying endometrium. During menstruation, these blood vessels have been shown to bleed excessively, and this has been postulated as their relationship to menorrhagia.3 They are classified depending on the amount of the
Bodle et al
Cryosurgery of uterine myomas and endometrium
myoma that protrudes into the uterine cavity as type 0, type I, or type II. Submucous myomas can be removed in combination with endometrial resection using either electrosurgery6 or laser,7 with type 0 being more easily removed than type II.4 These procedures, however, are difficult to learn due to their technically demanding nature. A second-generation device for the surgical treatment of menorrhagia that is able to treat small submucous myomas during endometrial ablation would therefore be an advantage. Knowledge of the susceptibility of myomas to cryosurgical damage, compared with the endometrium, would enable development of a cryoablative device for this purpose. In vitro investigation of the effects of cryosurgical temperatures on myomas is limited largely to cell suspensions, with no investigation of the lethal temperature required for effective cryosurgery of whole myomas. Bishof et al. have looked at freezing parameters of myoma cells in suspension, investigating cooling rate, end temperature, hold time, and thawing rate. The most significant parameter was end temperature followed by hold time.8 They found a temperature lower than –30°C if held for 5 minutes consistently destroyed cells. They also looked at the effect of freezing rate on the mechanism of damage in uterine myoma tissue. Using microscopic assessment, they found rates of cooling less than 50°C/min lead to dehydration of the myoma cells whereas rates greater than 50°C/min resulted in the formation of intracellular ice.9 The Endocryo (Integral Neurosciences, Andover, UK), is a 9-mm diameter carbon dioxide cryoprobe with a tip temperature of – 45°C designed for endometrial cryoablation. It is capable of consistently producing 5-mm of endometrial/ myometrial cell death in vitro, the depth required to prevent endometrial regeneration.10 However its effect on uterine myomas is unknown. The temperatures produced during cryosurgery of uterine myomas were investigated and the corresponding depth of cell death measured. With this information, the effect of the Endocryo on uterine myomas can be determined and comparison made with endometrial cryoablation. Conclusions can then be drawn as to the feasibility of using cryosurgery for endometrial ablation in patients with small submucous myomas.
Methods Ethics approval was obtained for all parts of this study from the Saint James University Hospital ethics committee. Fully informed written consent was obtained from women who were undergoing hysterectomy for idiopathic menorrhagia and with and without myomas. Only those uteri that were considered of normal size and shape and assessed by hysterectomy, and that had no endometrial pathology were used for this study, excepting the presence of myomas for the myoma arm of the study. Eight experiments were carried out on single myomas dissected free from their respective uteri. Six experiments
501
Figure 1
Diagram of endometrial cryosurgery.
were undertaken on whole uteri. The Endocryo was used for all cryosurgical experiments. All experiments were carried out at room temperature, approximately 25°C, and used a single 5-minute freeze followed by an active thaw.
Cryoprobe procedure: endometrium After dilatation of the cervix to 9 mm, a single 5-minute freeze was applied with the tip of the cryosurgical device in one cornua. A multidepth thermocouple (Omega Engineering Ltd., Broughton Astley, Leicestershire, UK) was inserted perpendicular to the anterior uterine body until contact was made with the cryoprobe (Figure 1). Temperature change was measured at 1-, 3-, 5-, 7-, and 10-mm depth measured from the endometrial surface. Endometrial thinning agents were not used as the effect of the Endocryo on the endometrium has been shown previously to be the same as that on the myometrium.10 The total depth of cell death would therefore be the same regardless of the endometrial thickness. The exact position of the cryoprobe within the uterine cavity was not determined visually. Therefore, although the positioning demonstrated in Figure 1 is likely, it is also possible that mismatch between the cryoprobe and the endometrial cavity could affect tissue/cryoprobe contact and therefore temperature change and consequently depth of cell death.10 However positioning the multidepth thermocouple through the anterior wall of the uterus ensured apposition of the endometrium and cryoprobe, thereby overcoming this potential confounding.
Cryosurgical procedure: myomas After dissection of the myoma, a single 5-minute freeze was applied with the tip of the cryosurgical probe in direct contact with the surface of the myoma. Temperature change was measured at 1-, 3-, 5-, 7- and 10-mm depth measured from the myoma surface (Figure 2).
Assessment Temperature changes during myoma and endometrial cryosurgery were compared to determine whether the re-
502
Journal of Minimally Invasive Gynecology, Vol 13, No 6, November/December 2006 30
temperature (deg C)
20 10
10 mm 7 mm
0
5 mm 0
1
2
3
4
5
6
7
3 mm 1 mm
-10 -20 -30
Figure 2
time (min)
Diagram of myoma cryosurgery.
Figure 3 Typical freeze/thaw temperature pattern seen during cryosurgery.
Statistics Statistical analysis was carried out using SPSS software, version 11.5 (SPSS, Inc., Chicago, IL). Level of significance was set at p ⱕ.05. The unpaired t test or Mann Whitney U test was used depending on the normality of data sets analyzed. Where more than two variables were analyzed, two-way mixed ANOVA test was used.
Results Eight myomas ranging in weight from 0.7 g to 24.5 g (median 5.85 g) and six normal-sized uteri were used for experimentation. During cryosurgery, the ice ball was seen to grow; and immediately postcryosurgery, the cryolesion
was clearly demarcated by a blanched area approximating the size of the ice ball.
Temperature changes during cryosurgery Temperature change during cryosurgery of uterine myomas and endometrium/myometrium followed the typical pattern seen in cryosurgery at other sites.16 The greatest rate of fall during the freeze cycle occurred initially, with a flattening of the curve over time. During thawing, the initial rapid rise in temperature soon leveled off. This typical pattern is demonstrated in Figure 3. Mean tissue temperatures at 1-, 3-, 5-, 7-, and 10-mm perpendicular from the tissue surface during a single 5-minute freeze are shown in the Figure 4. The mean myoma temperature 5 mm from the cryoprobe after 5 minutes of freezing was 0.5°C (SD 4.27, 95% CI –2.92 to 3.92) and in the endometrial experiments was – 4.9°C (SD 1.39, 95% CI – 6.10 to –3.66). There was no significant difference between myoma and endometrial temperatures at 1-, 3-, 5-, 7-, and 10-mm during a single 5-minute freeze (p ⫽ .601).
Depth of cell death after cryosurgery Median depth of cell death after a single 5-minute freeze in vitro was 4.09 mm (range 1.51 to 5.48) in myomas. Mean depth of cell death in endometrium/myometrium was 4.55 mm (SD 0.71, 95% CI 4.22 to 4.87). All depths include
Temperature (deg C)
sponse of these two tissue types to the Endocryo in vitro is the same. Blocks from the myoma and uterine tissue in direct contact with the cryoprobe during cryosurgery were cut for assessment of depth of cell death. From the uteri, this constituted tissue from the lower part of the uterine body where apposition of the cryoprobe and endometrium was likely to be good. Ten-micron sections were stained for viability using nicotinamide adenine dinucleotide (NADH) diaphorase enzyme assay with nuclear fast red counter stain. First described by Nachlas in 1958,11 NADH staining has been used to assess endometrial damage after laser ablation12 and electrosurgery13 and after Thermachoice14 (Gynecare, Sommerville, NJ) and Cavaterm15 (Wallsten Medical SA, Morges, Switzerland) procedures. The standard operating procedure used has been specifically validated for the assessment of cryosurgical damage.10 Depth of cell death produced by the standard 5-minute freeze/thaw protocol in the myoma and endometrial experiments were compared to determine whether the response of these two tissue types to the Endocryo is the same. Correlating the temperature and depth of cell death data, the temperature at which cell death occurred during the myoma and endometrial experiments was determined. These data were compared to assess whether the effect of the Endocryo is the same on endometrium as on myomas.
20 15 10 5 0 -5 -10 -15 -20 -25 -30
Myoma Endometrium
1
3
5
7
10
Thermocouple depth (mm)
Figure 4 Temperatures after 5 minutes of freezing for myomas and endometrium.
Bodle et al
Cryosurgery of uterine myomas and endometrium
adjustment of 7.5% for shrinkage due to processing.17 There was no significant difference between depth of cell death in vitro in myomas and endometrium/myometrium (p ⫽ .439).
Temperature at which cell death occurred The mean temperature that produced cell death in the myomas was – 6.66°C (SD 6.58, 95% CI –10.84 to –2.48). This compares with a mean temperature producing cell death in endometrium/myometrium of – 8.74°C (SD 7.85, 95% CI –14.33 to – 4.39). There was no significant difference between lethal temperatures in vitro in myomas and uterine endometrium/myometrium (p ⫽ .406).
Discussion This is the first paper to look at the effects of low temperature on myoma tissue and compare them with those of the same cryosurgical device on endometrial tissue. The in vitro observation of uterine myomas during cryosurgery suggests that in terms of temperature change and cellular destruction their response is similar to that of endometrium and myometrium. Certainly there is no significant difference between the mean lethal temperature required to destroy myomas and that required by endometrium/myometrium. The mean depth of cell death produced in each tissue type by a standard protocol is also the same. Therefore, in theory, incorporating any submucous myomas within the cryolesion during cryoablation, provided the lethal temperature is reached, should result in destruction of those myomas. The first clinical investigation of endometrial cryoablation using a carbon dioxide probe published by Pittrof and colleagues in the early 1990s concluded it to be a promising development in the management of menorrhagia.18 Detailed laboratory experimentation of the Endocryo (the successor of Pittrof’s device) in the 1990s showed a single 2-minute freeze produced clinically effective endometrial/myometrial depth of cell death of 4.5 mm in vitro.10 However, application of this 2-minute and then a 5-minute freeze in vivo produced inadequate cell death.10 Further investigation using an in vitro uterine perfusion model determined that to consistently produce 4-mm depth of cell death in vivo with the Endocryo requires a freeze time exceeding 20 minutes.17 This is compounded by the need for two freeze/thaw cycles, one for the right side and one for the left side of the uterus. This does not compare favorably with other second-generation endometrial ablative devices. In addition, the effectiveness of the Endocryo has been shown to be significantly reduced when there is a gap of 1 mm or more between the probe and the endometrial surface.10 The same is likely to apply to the treatment of myomas. As submucous myomas by definition protrude into the uterine cavity there is likely to be variable contact between the probe and both the endometrium and the my-
503 oma. This is likely to reduce the cryosurgical effect on the treatment of both and when added to the long treatment time, is likely to render the Endocryo impractical for the treatment of women with menstrual disorders. However, a more powerful cryosurgical device, such as one of the currently marketed liquid nitrogen probes, which could overcome the problems of time and contact is likely to be useful for the treatment of women with menorrhagia and small submucous myomas. The evidence in this paper that the response of both tissues in vitro is the same is important for the development of these cryosurgical devices for this purpose.
Future work This study made no attempt to investigate the in vivo effects of the Endocryo. Previous work on endometrial ablation has shown that the in vitro performance of the Endocryo is not sufficient to declare clinical effectiveness.10 Differences in perfusion may result in different effects of low temperature on myomas in vivo compared with endometrium. As the Endocryo is no longer a viable cryoablative device, future work should attempt to establish the roll of a cryoprobe with proven clinical effectiveness on the treatment of submucous myomas in women with menorrhagia.
Conclusion The Endocryo when applied to myoma tissue in vitro produces the same cryosurgical effect on both tissue types. In practical terms, however, it is unlikely that the Endocryo will become widely used for the treatment of submucous myomas hysteroscopically in conjunction with endometrial cryoablation, the limiting factors being the treatment time required and problems with contact.
References 1. Pokras R, Hufnagal VG. National Center for Health Statistics, Hysterectomies in the United States 1965–1984. Washington DC: Government Printing Office, 1987. 2. Vessy MP, Villard-Mackintosh L, McPherson K, et al. The epidaemiology of hysterectomy: findings of a large cohort study. Br J Obstet Gynaecol. 1992;99:402– 407. 3. Garry R. Hysteroscopic observations during menstruation. Middlesborough, UK: WEL Foundation CD-ROM, 1998. 4. Wamsteker K, Emanuel MH, de Kruif JH. Trans-cervical hysteroscopic resection of submucous fibroids for abnormal uterine bleeding: results regarding the degree of intramural extension. Obstet Gynecol. 1993;82:736 –740. 5. Feng ZC, Shi YP, Liu SP. Hysteroscopic resection of submucous fibroids: clinical analysis of 99 cases. Gynecological Endoscopy. 2002; 11:127–130. 6. DeCherney AH, Polan ML. Hysteroscopic management of intrauterine lesions and intractable uterine bleeding. Obstet Gynecol. 1983;61:392– 397.
504
Journal of Minimally Invasive Gynecology, Vol 13, No 6, November/December 2006
7. Baggish MS, Sze EH, Morgan P. Hysteroscopic treatment of symptomatic submucous myomata uteri with the Nd:YAG laser. J Gynecol Surg. 1989;6:89 –94. 8. Bishof J, Fahssi WM, Smith D, et al. A parametric study of freezing injury in ELT-3 uterine leiomyoma tumour cells. Hum Reprod. 2001; 16:340 –348. 9. Devireddy RV, Coad JE, Bischof JC. Microscopic and calorimetric assessment of freezing processes in uterine fibroid tumour tissue. Cryobiology. 2001;42:225–243. 10. Kremer C, Duffy S. In vitro studies of cryoablation of the endometrium. Am J Obstet Gynecol. 2000;183:22–27. 11. Nachlas MM, Walker DG, Seligman AM. A histological method for the demonstration of diphosphopyridine nucleotide diaphorase. Journal of Biophysics, Biochemistry and Cytology. 1958;4:29 –38. 12. Reid PCR. Nd:YAG Laser Ablation. Thesis/Dissertation. University of Manchester; 1989. 13. Duffy S, Reid PC, Smith JHF, et al. In vitro studies of uterine electrosurgery. Obstet Gynecol. 1991;78:213–220.
14. Shah AA, Stabinsky SA, Klusak T, et al. Measurement of serosal temperatures and depth of thermal injury generated by thermal balloon endometrial ablation in ex-vivo and in-vivo models. Fertil Steril. 1998;70:692– 697. 15. Hawe J, Abbot J, Phillips G, et al. In vitro and in vivo histochemical and thermal studies using a thermal balloon endometrial ablation system for varying treatment times. Hum Reprod. 2003;18:2603– 2607. 16. Rubinsky B. Thermodynamics and heat transfer in cryosurgery. In: Onik GM, Rubinsky B, Watson G, Albin RJ, eds. Percutaneous Prostate Cryoablation. St Louis, Mo.: Quality Medical Publishing, Inc; 1995:69 –77. 17. Bodle JF. Development of Cryosurgery for the Modern Surgical Management of Menorrhagia Using an in Vitro Perfusion Model. Thesis/ Dissertation. University of Leeds; 2004 (GENERIC). 18. Pittrof R, Majid F, Murray A. Transcervical endometrial cryoablation (ECA) for menorrhagia. Int J Gynaecol Obstet. 1994;47:135– 140.
Instruments and techniques
Susceptibility of uterine myomas and endometrium to cryosurgery using a carbon dioxide cryosurgical probe in vitro Julia F. Bodle, MD, MRCOG, Sean R.G. Duffy, MD, FRCOG, and David M. Binney, MMedSci From the Academic Department of Obstetrics and Gynecology, Saint James’s University Hospital, Leeds University, Leeds (Drs. Bodle and Duffy); and the Faculty of Health and Wellbeing, Collegiate Crescent, Sheffield Hallam University, Sheffield (Mr. Binney), UK. KEYWORDS: Cryosurgery; Uterine myoma; Endometrium; Carbon dioxide
Abstract STUDY OBJECTIVE: To determine the effect of a 9-mm diameter carbon dioxide cryoprobe, the Endocryo, on myomas and endometrial/myometrial tissue in vitro. DESIGN: Comparative laboratory study (Canadian Task Force classification II-2). SETTING: University laboratory. PATIENTS: Women with and without myomas, undergoing hysterectomy. INTERVENTION: A single 5-minute freeze followed by an active thaw was applied to uterine myomas and endometrial/myometrial tissue in vitro. MEASUREMENTS AND MAIN RESULTS: Endometrial/myometrial and uterine myoma temperature change was measured continuously during the cryosurgical procedure. Depth of cell death was measured using nicotinamide adenine dinucleotide diaphorase enzyme assay. There was no significant difference in temperature change and depth of cell death between myomas and endometrial/myometrial tissue in vitro. CONCLUSIONS: The Endocryo produces the same cryosurgical effect on both uterine myomas and endometrial/myometrial tissue in vitro, an important principal for future development of a clinically effective cryosurgical device for the treatment of menorrhagia in the presence of submucous myomas. © 2006 AAGL. All rights reserved.
Uterine myomas are the most common benign tumor affecting women; a prevalence of up to 65% has been reported in perimenopausal women.1 They are also the most common reason for hysterectomy.2 Myomas are described
Financial support was received from Integral Neurosciences, Andover, UK. Corresponding author: Dr. Sean R. G. Duffy, Level 9, Gledhow Wing, Saint James’s University Hospital, Leeds, LS9 7TF, UK. E-mail: [email protected] Submitted March 19, 2006. Accepted for publication June 19, 2006.
1553-4650/$ -see front matter © 2006 AAGL. All rights reserved. doi:10.1016/j.jmig.2006.06.012
by their location within the uterus as either submucous, intramural, or subserous. Submucous myomas have been shown to be associated with menorrhagia,3 with their removal resulting in improvement in symptoms.4,5 Hysteroscopically, the submucous myoma can be seen as a whitish smooth protuberance into the uterine cavity with a network of fragile blood vessels in the overlying endometrium. During menstruation, these blood vessels have been shown to bleed excessively, and this has been postulated as their relationship to menorrhagia.3 They are classified depending on the amount of the
Bodle et al
Cryosurgery of uterine myomas and endometrium
myoma that protrudes into the uterine cavity as type 0, type I, or type II. Submucous myomas can be removed in combination with endometrial resection using either electrosurgery6 or laser,7 with type 0 being more easily removed than type II.4 These procedures, however, are difficult to learn due to their technically demanding nature. A second-generation device for the surgical treatment of menorrhagia that is able to treat small submucous myomas during endometrial ablation would therefore be an advantage. Knowledge of the susceptibility of myomas to cryosurgical damage, compared with the endometrium, would enable development of a cryoablative device for this purpose. In vitro investigation of the effects of cryosurgical temperatures on myomas is limited largely to cell suspensions, with no investigation of the lethal temperature required for effective cryosurgery of whole myomas. Bishof et al. have looked at freezing parameters of myoma cells in suspension, investigating cooling rate, end temperature, hold time, and thawing rate. The most significant parameter was end temperature followed by hold time.8 They found a temperature lower than –30°C if held for 5 minutes consistently destroyed cells. They also looked at the effect of freezing rate on the mechanism of damage in uterine myoma tissue. Using microscopic assessment, they found rates of cooling less than 50°C/min lead to dehydration of the myoma cells whereas rates greater than 50°C/min resulted in the formation of intracellular ice.9 The Endocryo (Integral Neurosciences, Andover, UK), is a 9-mm diameter carbon dioxide cryoprobe with a tip temperature of – 45°C designed for endometrial cryoablation. It is capable of consistently producing 5-mm of endometrial/ myometrial cell death in vitro, the depth required to prevent endometrial regeneration.10 However its effect on uterine myomas is unknown. The temperatures produced during cryosurgery of uterine myomas were investigated and the corresponding depth of cell death measured. With this information, the effect of the Endocryo on uterine myomas can be determined and comparison made with endometrial cryoablation. Conclusions can then be drawn as to the feasibility of using cryosurgery for endometrial ablation in patients with small submucous myomas.
Methods Ethics approval was obtained for all parts of this study from the Saint James University Hospital ethics committee. Fully informed written consent was obtained from women who were undergoing hysterectomy for idiopathic menorrhagia and with and without myomas. Only those uteri that were considered of normal size and shape and assessed by hysterectomy, and that had no endometrial pathology were used for this study, excepting the presence of myomas for the myoma arm of the study. Eight experiments were carried out on single myomas dissected free from their respective uteri. Six experiments
501
Figure 1
Diagram of endometrial cryosurgery.
were undertaken on whole uteri. The Endocryo was used for all cryosurgical experiments. All experiments were carried out at room temperature, approximately 25°C, and used a single 5-minute freeze followed by an active thaw.
Cryoprobe procedure: endometrium After dilatation of the cervix to 9 mm, a single 5-minute freeze was applied with the tip of the cryosurgical device in one cornua. A multidepth thermocouple (Omega Engineering Ltd., Broughton Astley, Leicestershire, UK) was inserted perpendicular to the anterior uterine body until contact was made with the cryoprobe (Figure 1). Temperature change was measured at 1-, 3-, 5-, 7-, and 10-mm depth measured from the endometrial surface. Endometrial thinning agents were not used as the effect of the Endocryo on the endometrium has been shown previously to be the same as that on the myometrium.10 The total depth of cell death would therefore be the same regardless of the endometrial thickness. The exact position of the cryoprobe within the uterine cavity was not determined visually. Therefore, although the positioning demonstrated in Figure 1 is likely, it is also possible that mismatch between the cryoprobe and the endometrial cavity could affect tissue/cryoprobe contact and therefore temperature change and consequently depth of cell death.10 However positioning the multidepth thermocouple through the anterior wall of the uterus ensured apposition of the endometrium and cryoprobe, thereby overcoming this potential confounding.
Cryosurgical procedure: myomas After dissection of the myoma, a single 5-minute freeze was applied with the tip of the cryosurgical probe in direct contact with the surface of the myoma. Temperature change was measured at 1-, 3-, 5-, 7- and 10-mm depth measured from the myoma surface (Figure 2).
Assessment Temperature changes during myoma and endometrial cryosurgery were compared to determine whether the re-
502
Journal of Minimally Invasive Gynecology, Vol 13, No 6, November/December 2006 30
temperature (deg C)
20 10
10 mm 7 mm
0
5 mm 0
1
2
3
4
5
6
7
3 mm 1 mm
-10 -20 -30
Figure 2
time (min)
Diagram of myoma cryosurgery.
Figure 3 Typical freeze/thaw temperature pattern seen during cryosurgery.
Statistics Statistical analysis was carried out using SPSS software, version 11.5 (SPSS, Inc., Chicago, IL). Level of significance was set at p ⱕ.05. The unpaired t test or Mann Whitney U test was used depending on the normality of data sets analyzed. Where more than two variables were analyzed, two-way mixed ANOVA test was used.
Results Eight myomas ranging in weight from 0.7 g to 24.5 g (median 5.85 g) and six normal-sized uteri were used for experimentation. During cryosurgery, the ice ball was seen to grow; and immediately postcryosurgery, the cryolesion
was clearly demarcated by a blanched area approximating the size of the ice ball.
Temperature changes during cryosurgery Temperature change during cryosurgery of uterine myomas and endometrium/myometrium followed the typical pattern seen in cryosurgery at other sites.16 The greatest rate of fall during the freeze cycle occurred initially, with a flattening of the curve over time. During thawing, the initial rapid rise in temperature soon leveled off. This typical pattern is demonstrated in Figure 3. Mean tissue temperatures at 1-, 3-, 5-, 7-, and 10-mm perpendicular from the tissue surface during a single 5-minute freeze are shown in the Figure 4. The mean myoma temperature 5 mm from the cryoprobe after 5 minutes of freezing was 0.5°C (SD 4.27, 95% CI –2.92 to 3.92) and in the endometrial experiments was – 4.9°C (SD 1.39, 95% CI – 6.10 to –3.66). There was no significant difference between myoma and endometrial temperatures at 1-, 3-, 5-, 7-, and 10-mm during a single 5-minute freeze (p ⫽ .601).
Depth of cell death after cryosurgery Median depth of cell death after a single 5-minute freeze in vitro was 4.09 mm (range 1.51 to 5.48) in myomas. Mean depth of cell death in endometrium/myometrium was 4.55 mm (SD 0.71, 95% CI 4.22 to 4.87). All depths include
Temperature (deg C)
sponse of these two tissue types to the Endocryo in vitro is the same. Blocks from the myoma and uterine tissue in direct contact with the cryoprobe during cryosurgery were cut for assessment of depth of cell death. From the uteri, this constituted tissue from the lower part of the uterine body where apposition of the cryoprobe and endometrium was likely to be good. Ten-micron sections were stained for viability using nicotinamide adenine dinucleotide (NADH) diaphorase enzyme assay with nuclear fast red counter stain. First described by Nachlas in 1958,11 NADH staining has been used to assess endometrial damage after laser ablation12 and electrosurgery13 and after Thermachoice14 (Gynecare, Sommerville, NJ) and Cavaterm15 (Wallsten Medical SA, Morges, Switzerland) procedures. The standard operating procedure used has been specifically validated for the assessment of cryosurgical damage.10 Depth of cell death produced by the standard 5-minute freeze/thaw protocol in the myoma and endometrial experiments were compared to determine whether the response of these two tissue types to the Endocryo is the same. Correlating the temperature and depth of cell death data, the temperature at which cell death occurred during the myoma and endometrial experiments was determined. These data were compared to assess whether the effect of the Endocryo is the same on endometrium as on myomas.
20 15 10 5 0 -5 -10 -15 -20 -25 -30
Myoma Endometrium
1
3
5
7
10
Thermocouple depth (mm)
Figure 4 Temperatures after 5 minutes of freezing for myomas and endometrium.
Bodle et al
Cryosurgery of uterine myomas and endometrium
adjustment of 7.5% for shrinkage due to processing.17 There was no significant difference between depth of cell death in vitro in myomas and endometrium/myometrium (p ⫽ .439).
Temperature at which cell death occurred The mean temperature that produced cell death in the myomas was – 6.66°C (SD 6.58, 95% CI –10.84 to –2.48). This compares with a mean temperature producing cell death in endometrium/myometrium of – 8.74°C (SD 7.85, 95% CI –14.33 to – 4.39). There was no significant difference between lethal temperatures in vitro in myomas and uterine endometrium/myometrium (p ⫽ .406).
Discussion This is the first paper to look at the effects of low temperature on myoma tissue and compare them with those of the same cryosurgical device on endometrial tissue. The in vitro observation of uterine myomas during cryosurgery suggests that in terms of temperature change and cellular destruction their response is similar to that of endometrium and myometrium. Certainly there is no significant difference between the mean lethal temperature required to destroy myomas and that required by endometrium/myometrium. The mean depth of cell death produced in each tissue type by a standard protocol is also the same. Therefore, in theory, incorporating any submucous myomas within the cryolesion during cryoablation, provided the lethal temperature is reached, should result in destruction of those myomas. The first clinical investigation of endometrial cryoablation using a carbon dioxide probe published by Pittrof and colleagues in the early 1990s concluded it to be a promising development in the management of menorrhagia.18 Detailed laboratory experimentation of the Endocryo (the successor of Pittrof’s device) in the 1990s showed a single 2-minute freeze produced clinically effective endometrial/myometrial depth of cell death of 4.5 mm in vitro.10 However, application of this 2-minute and then a 5-minute freeze in vivo produced inadequate cell death.10 Further investigation using an in vitro uterine perfusion model determined that to consistently produce 4-mm depth of cell death in vivo with the Endocryo requires a freeze time exceeding 20 minutes.17 This is compounded by the need for two freeze/thaw cycles, one for the right side and one for the left side of the uterus. This does not compare favorably with other second-generation endometrial ablative devices. In addition, the effectiveness of the Endocryo has been shown to be significantly reduced when there is a gap of 1 mm or more between the probe and the endometrial surface.10 The same is likely to apply to the treatment of myomas. As submucous myomas by definition protrude into the uterine cavity there is likely to be variable contact between the probe and both the endometrium and the my-
503 oma. This is likely to reduce the cryosurgical effect on the treatment of both and when added to the long treatment time, is likely to render the Endocryo impractical for the treatment of women with menstrual disorders. However, a more powerful cryosurgical device, such as one of the currently marketed liquid nitrogen probes, which could overcome the problems of time and contact is likely to be useful for the treatment of women with menorrhagia and small submucous myomas. The evidence in this paper that the response of both tissues in vitro is the same is important for the development of these cryosurgical devices for this purpose.
Future work This study made no attempt to investigate the in vivo effects of the Endocryo. Previous work on endometrial ablation has shown that the in vitro performance of the Endocryo is not sufficient to declare clinical effectiveness.10 Differences in perfusion may result in different effects of low temperature on myomas in vivo compared with endometrium. As the Endocryo is no longer a viable cryoablative device, future work should attempt to establish the roll of a cryoprobe with proven clinical effectiveness on the treatment of submucous myomas in women with menorrhagia.
Conclusion The Endocryo when applied to myoma tissue in vitro produces the same cryosurgical effect on both tissue types. In practical terms, however, it is unlikely that the Endocryo will become widely used for the treatment of submucous myomas hysteroscopically in conjunction with endometrial cryoablation, the limiting factors being the treatment time required and problems with contact.
References 1. Pokras R, Hufnagal VG. National Center for Health Statistics, Hysterectomies in the United States 1965–1984. Washington DC: Government Printing Office, 1987. 2. Vessy MP, Villard-Mackintosh L, McPherson K, et al. The epidaemiology of hysterectomy: findings of a large cohort study. Br J Obstet Gynaecol. 1992;99:402– 407. 3. Garry R. Hysteroscopic observations during menstruation. Middlesborough, UK: WEL Foundation CD-ROM, 1998. 4. Wamsteker K, Emanuel MH, de Kruif JH. Trans-cervical hysteroscopic resection of submucous fibroids for abnormal uterine bleeding: results regarding the degree of intramural extension. Obstet Gynecol. 1993;82:736 –740. 5. Feng ZC, Shi YP, Liu SP. Hysteroscopic resection of submucous fibroids: clinical analysis of 99 cases. Gynecological Endoscopy. 2002; 11:127–130. 6. DeCherney AH, Polan ML. Hysteroscopic management of intrauterine lesions and intractable uterine bleeding. Obstet Gynecol. 1983;61:392– 397.
504
Journal of Minimally Invasive Gynecology, Vol 13, No 6, November/December 2006
7. Baggish MS, Sze EH, Morgan P. Hysteroscopic treatment of symptomatic submucous myomata uteri with the Nd:YAG laser. J Gynecol Surg. 1989;6:89 –94. 8. Bishof J, Fahssi WM, Smith D, et al. A parametric study of freezing injury in ELT-3 uterine leiomyoma tumour cells. Hum Reprod. 2001; 16:340 –348. 9. Devireddy RV, Coad JE, Bischof JC. Microscopic and calorimetric assessment of freezing processes in uterine fibroid tumour tissue. Cryobiology. 2001;42:225–243. 10. Kremer C, Duffy S. In vitro studies of cryoablation of the endometrium. Am J Obstet Gynecol. 2000;183:22–27. 11. Nachlas MM, Walker DG, Seligman AM. A histological method for the demonstration of diphosphopyridine nucleotide diaphorase. Journal of Biophysics, Biochemistry and Cytology. 1958;4:29 –38. 12. Reid PCR. Nd:YAG Laser Ablation. Thesis/Dissertation. University of Manchester; 1989. 13. Duffy S, Reid PC, Smith JHF, et al. In vitro studies of uterine electrosurgery. Obstet Gynecol. 1991;78:213–220.
14. Shah AA, Stabinsky SA, Klusak T, et al. Measurement of serosal temperatures and depth of thermal injury generated by thermal balloon endometrial ablation in ex-vivo and in-vivo models. Fertil Steril. 1998;70:692– 697. 15. Hawe J, Abbot J, Phillips G, et al. In vitro and in vivo histochemical and thermal studies using a thermal balloon endometrial ablation system for varying treatment times. Hum Reprod. 2003;18:2603– 2607. 16. Rubinsky B. Thermodynamics and heat transfer in cryosurgery. In: Onik GM, Rubinsky B, Watson G, Albin RJ, eds. Percutaneous Prostate Cryoablation. St Louis, Mo.: Quality Medical Publishing, Inc; 1995:69 –77. 17. Bodle JF. Development of Cryosurgery for the Modern Surgical Management of Menorrhagia Using an in Vitro Perfusion Model. Thesis/ Dissertation. University of Leeds; 2004 (GENERIC). 18. Pittrof R, Majid F, Murray A. Transcervical endometrial cryoablation (ECA) for menorrhagia. Int J Gynaecol Obstet. 1994;47:135– 140.