Authored by OA Melkozerova*
Abstract
Objective: To analyse the effect of the ultrasonic energy during MRI-guided high-intensity focused ultrasound ablation (HIFU) of uterine fibroids on molecular markers of endometrial receptivity in women of reproductive age.
Study design: A prospective comparative cohort study of 60 women of reproductive age was conducted. The main group consisted of 32 patients suffering from the symptomatic course of uterine fibroids who received treatment with HIFU ablation of uterine fibroids: 17 women with node localization at the front wall of the uterus and the reflected action of ultrasonic waves on the endometrium and 15 women with the back wall node localization and penetrating effect of the waves. The control group consisted of 28 healthy fertile women examined voluntarily. The endometrium obtained with pipelle-biopsia on days 20-22 of the cycle was examined by immunohistochemistry before and three months after the treatment. The results were processed by the method of variation statistics using the SPSS 22.0.
Results: A significant decrease in the stromal expression of CD95bright in the endometrium to the level comparable with control values was observed after HIFU ablation of uterine fibroids (from 70.22±9.77 c/s to 48.81±5.47 c/s; p<0.001; the control level – 47.80±2.13 c/s). The ratio and expression of steroid receptors, proliferation markers, p53-dependent apoptosis and itsblockers, regulators and markers of angiogenesis, LIF and LIF-R signalling molecules in thestroma and endometrial glands did not change significantly after treatment (p>0.05 in treatment dynamics). It was demonstrated that focused ultrasound causes an increase in the level of expression of estradiol alpha receptors in the endometrial glands (118.07±14.01 points vs.153.33±11.43 points; p=0.02) and a decrease in stromal p53 expression (30.80±21.61% vs.14.66±3.74%; p=0.007). No influence of the localization of fibroid node on the stromal expression of CD56bright+ was demonstrated. A significant increase in the stromal expression of CD34+ to the level statistically comparable with control values (from 26.00±1.63 c/s to 32.47±2.03 c/s; p=0.011; the control level - 37.99±1.23 c/s) was associated with ultrasound action.
Conclusion: This study did not reveal any significant negative effects of HIFU ablation of uterine fibroids on endometrial receptivity in women of reproductive age.
Keywords: Uterine fibroids; Magnetic resonance imaging guided high-intensity focused ultrasound ablation; Endometrial receptivity
Abbreviations: MRg: Magnetic Resonance Imaging Guided; HIFU: High-Intensity Focused Ultrasound
Introduction
Uterine fibroids are a clinically relevant problem due to their high incidence: about 25% of women older than 35 years have myomas and one-third of those patients have symptoms associated with fibroids [1-2]. A combination of uterine fibroids and infertility occurs in 1.2 - 2.4% of women, while the mechanisms of the influence of uterine fibroids on fertility today remain a subject of discussion [1-6].
The problem of choosing the therapeutic tactics of uterine fibroids in patients of reproductive age planning pregnancy is associated not only with a change in the functional state of the myometrium after treatment, but also with a corresponding change in the function of the endometrium, which plays a key role in the initiation of implantation and invasion of trophoblast [6,7].
In the presence of indications, myomectomy with careful layer- by-layer closure of the node bed is the first-line treatment method for patients with uterine fibroids planning a pregnancy [1,2,8- 10]. In this case, the risk of uterine rupture after laparoscopic myomectomy does not exceed 0.5 - 1% [11,12]. At the same time, when planning pregnancy for women of late reproductive age that have a certain decrease in ovarian reserve, the question of recovery time after myomectomy sometimes acquires fundamental importance. In addition, a uterine scar after myomectomy can consider an indication for abdominal delivery during subsequent pregnancy, which may increase perinatal risks in some way. In addition, there are certain risks of pregnancy with a scar on the uterus [2,12].
These circumstances dictate the feasibility of a thorough study of alternative methods of preparing patients with uterine fibroids for pregnancy. As an alternative, there are conservative regression methods for the treatment of uterine fibroids, including magnetic resonance imaging guided high- intensity focused ultrasound (HIFU), which allows accelerating pregravid preparation and to avoid surgical intervention with scar formation on the uterus [13,14].
The mechanisms of the influence of focused ultrasound rays passing through the endometrium during the procedure of HIFU ablation on the indicators of its receptivity are not fully understood, the results of individual studies are very contradictory. In this regard, it seems very relevant to study the functional activity of the endometrium after treatment using HIFU ablation of uterine fibroids.
The aim of this study is to analyse the effect of MRg HIFU ablation of uterine fibroids on molecular markers of endometrial receptivity in women of reproductive age.
Material and Methods
A prospective comparative cohort study of 60 women of reproductive age was conducted. The main group consisted of 32 patients suffering from the symptomatic course of uterine fibroids who received treatment with HIFU ablation of uterine fibroids. The control group consisted of 28 healthy fertile women who were examined on a voluntary basis, and who did not have a history of miscarriage and had a history of normal vaginal delivery after the physiological pregnancy. Depending on the predominant localization of the uterine fibroids, the patients were divided into two subgroups: A - 17 women with localization of the dominant node at the front wall of the uterus (in this case the endometrium was impacted by reflected ultrasonic waves); B - 15 women with localization of the fibroids at the posterior wall of the uterus and direct impact of ultrasound energy upon the endometrium.
The procedure of HIFU ablation was performed on the ExAblate- 2000 (InSightec, Israel), combined into a single system with a 1.5 Tesla magnetic resonance imager (General Electric, USA). MR images were used for treatment localization, feedback control (beam guidance), real- time temperature mapping with the proton resonance frequency shift (PRFS) thermometry method, and post treatment verification of the ablated tissue.
The endometrial study was performed using the pipelle-biopsia method for LG7+ day, determined by the urinary test for ovulation, as the period of the alleged “implantation window” in both case groups.
For the Immunohistochemical study, a two-stage streptavidin- biotin-peroxidase method was used with antigen unmasking using standard sets of monoclonal and polyclonal antibodies from Bond RTU Primary, USA and DAKO, Denmark. Using the DakoCytomation imaging system, a Reaction was performed. The Super Sensitive Polymer-HPR Detection System (BioGenex, USA) bezbiotin- free detection system was used to visualize primary antibodies.
For Immunohistochemical studies used serial paraffin sections. For Immunohistochemical reactions, paraffin sections were processed according to the traditional method using murine monoclonal antibodies to estrogen receptors-α, progesterone receptors.
To analyse the results the H-score method of histological counting was used. To assess the expression of Ki-67, bcl-2, p53 antigens in the glands and stroma, proliferation and apoptosis indices were calculated - the ratio of the number of stained cell nuclei to the total number of nuclei in percent when counting at least 400 nuclei. Expression of CD56bright+ was evaluated by counting positive cells in the field of view at an increase of 400 when counting at least 10 fields of view. The expression of CD34+, VEGF-A and VEGFR-1 was determined in the epithelium, stroma of the endometrium and vascular endothelium. Activity was manifested in the form of staining of the membrane and cytoplasm of epithelial and endothelial cells. The expression of LIF, LIFR was determined on the cell membranes of the surface epithelium of the endometrial glands by counting the number of stained cells in the field of view at an increase of 400, while not less than 10 fields of view were studied.
Theory/Calculation
The inclusion criteria for the study of main group were as follows: women in reproductive age (18-45 years) with symptomatic uterine fibroids (pain, infertility, dysfunction of the pelvic organs); II-V types of nodes according to the FIGO classification (2011); sizes of nodes from 4 to 9cm; ovulatory cycle; informed voluntary consent to participate in the study signed by the patient. Criteria for not inclusion in the study: hyperplastic processes of the endometrium in combination with uterine myoma; the acute inflammatory process of the genitals; oncological diseases; severe extragenital pathology; pregnancy; taking hormone therapy less than 3 months before inclusion in the study; contraindications for the use of HIFU ablation.
Exclusion criteria from the study: the onset of pregnancy; severe complications during the treatment of fibroids; malignant and atypical changes according to histological examination of endometrium; refusal of the patient from further participation in the study. Statistical analysis of the results was carried out using SPSS 16.0, SPSS: IBM Company (USA) and Statistical 10.0, StatSoft (USA). Statistical data were specified as the mean ± standard deviation. Significance tests were performed according to normal distribution testing via t-test for independent and paired samples and Mann-Whitney-U and Wilcoxon testing for not normally distributed variables with a significance level of p<0.05. The required sample size was calculated using sealed envelope®. UK when taking an alpha level of 0.05 and a confidence level of 95% with a dropout of 10%.
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