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ORIGINAL RESEARCH |
From the *State Key Laboratory of Provinces and Ministries, Institute of Ultrasound Engineering in Medicine, Biomedical Engineering Department, 
Department of Obstetrics and Gynecology, the First Affiliated Hospital, and 
Clinical Center for Tumor Therapy, the Second Affiliated Hospital, Chongqing University of Medical Sciences, Chongqing, China.
Address reprint requests to: Zhibiao Wang, MD, Institute of Ultrasound Engineering in Medicine, 1, Yixueyuan Road, Daping, Yuzhong Dist., Chongqing 400016, China; e-mail: wangzhibiao{at}netease.com.
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ABSTRACT |
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 ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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METHODS: A simple randomized phase 2 study was conducted in which a total of 76 patients (45 with squamous hyperplasia and 31 with lichen sclerosus) were treated with focused ultrasound therapy from 1999 to 2002. Before and after the treatment, the therapeutic responses were evaluated based on changes in clinical symptoms and signs. Pre- and posttreatment biopsy specimens were also assessed through the light and electron microscopic examinations. The positive expressions of CD34 and myelin basic protein (MBP) tests with the strepavidin-peroxidase immunohistochemistry method were used to evaluate the therapeutic response. Statistical analysis was performed using 
2 (McNemar 2) test and t test.
RESULTS: After the ultrasound treatment, clinical symptoms were dramatically improved with a total response rate of 94.74%. Three to 6 months later the skin of treated areas returned to normal appearance. In the 2-year follow-up, 49 of 76 cases (32 squamous hyperplasia and 17 lichen sclerosus) were cured, 23 (11 squamous hyperplasia and 12 lichen sclerosus) improved and 4 (2 squamous hyperplasia and 2 lichen sclerosus) persisted. The positive expressions of CD34 and MBP after treatment increased significantly at the treated region (P < .05). No major complications occurred.
CONCLUSION: Vulvar dystrophy could be effectively treated with focused ultrasound therapy. This approach appears to be a new promising treatment method, although further studies are still needed.
LEVEL OF EVIDENCE II-3:
Local approaches, such as hormone and herbal medications and laser and microwave exposures, are still the fundamental treatment options but have a high recurrence rate.5–8 The excision of a focal region or simple vulvectomy is effective for a period after the procedure, but the recurrence rate is very high (50%), and the vulvar scar shrinks to seriously affect the sexual life of the patient.9,10 Thus, it is imperative to develop new and effective treatment methods.
As a recently developed noninvasive technique, high-intensity focused ultrasound therpay has been used to treat benign and malignant tumors.11–14 An ultrasonic beam can be directed to the deep-seated target in the human body resulting in a temperature rising to above 70°C in less than 1 second, which leads to coagulative necrosis of the tissue at the focal region.15,16 As a result, well-delineated areas of protein denaturation, irreversible cell damage, and coagulative necrosis are produced, while the overlying and surrounding tissues are spared. Physical properties of an ultrasound beam enable it to be focused at a target region inside the body16; thus, a series of physical, chemical, and biochemical effects are produced to improve the nutrition and function of blood capillaries and nerve endings in the treated region.
Our preliminary study in an animal model demonstrated the feasibility of high-intensity focused ultrasound therapy for vulvar lesions. The purpose of this study was to investigate the feasibility and efficacy of focused ultrasound treatment of vulvar dystrophy in human beings.
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MATERIALS AND METHODS |
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ABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Between 1999 and 2002, 76 eligible female patients were selected from the First and Second Affiliated Hospitals of Chongqing University of Medical Sciences, the First People’s Hospital of Jiangbei District and Zibo Institute of Maternal and Child Health. Median age of the patients was 38.5 years and the mean course of their diseases was 5.8 years. Most patients in this group had previously received local treatment such as hormone and herbal medications and laser and microwave exposures 3 months before ultrasound treatment.
The Research Review Committee of the Institute of Ultrasound Engineering in Medicine approved this phase 2 clinical trial. Informed consents were obtained from all patients who decided to undergo the ultrasound therapy. The histologic patterns were classified according to ISSVD criteria,18,19 in which among the 76 patients, 31 had lichen sclerosis, and 45 had squamous hyperplasia (see Table 1).
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The focused ultrasound therapy equipment used in this study was developed and produced by Chongqing Haifu (HIFU) Technology Co. Ltd (Chongqing, China), which consists of the main system, power source, and water treatment system. The main system provides output electricity to the power source that transforms the electricity into an ultrasonic beam. The water treatment system is used to pump water to the head of the applicator. Frequencies of the transducer range from 5 to 8 MHz and the transducer is 12 mm in diameter. The applicator directly contacts the skin using water as the coupling medium.
After routine disinfectant procedures, the patient was anesthetized with an intravenous injection of Disoprofol (Abbott Laboratories, Chicago, IL) and Fentanyl Citrate (Yichang Homanwell, Yichang, China) or under local anesthesia with Anestacon (ledocaine, lignostab, lidocaine; Xinan Pharmacedtics Co. Ltd. Chongqing, China). The applicator was used to scan the lesion with coupling of water. A linear scanning mode was utilized at a speed of 2–3 mm/s. Multiple linear scans crossing the lesion were performed until the whole target area was covered. The treatment area of each lesion included 5 mm of normal skin beyond the margin of each lesion. The treatment duration was from 15 to 60 minutes, which varied depending on the lesion sizes. After treatment, an ice bag was used to relieve the irritation if necessary.
Scoring criteria for symptoms and signs (eg, pruritus, skin elasticity, and color) of vulvar dystrophy were used according to a study by Cattaneo et al5 and other previous studies8,20,21; thus, the pathological classifications of vulvar dystrophy were made as follows: stage I, II and III referred respectively to early, fully developed and later lichen sclerosus and squamous hyperplasia (Table 2). Before and after the treatment, the therapeutic responses were evaluated based on the reduction of pruritus, changes of the skin elasticity and color, decreased percentage of the involved area, and changes in histological findings.
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All patients were followed up by the same gynecologist once per week for the first month, once per month for next six months, and then once per year for 2 years after the treatment, based on the scoring criteria of the clinical signs and symptoms.
The treatment responses were then classified as cured (no symptoms and/or lesions remained), improved (final scores lower than the initial ones), or persistent (scores remained the same) on the basis of patients’ responses to the therapy.
Before and 3 to 6 months after the treatment, biopsy specimens from the vulvar lesions were obtained for histopathological examination. Three methods were used to evaluate the changes before and after the treatment: light microscopic examination, electron microscopic examination, and standard immunohistochemical evaluation. The quantity of positive expressions of CD34 and myelin basic protein (MBP) were used to evaluate the therapeutic responses. CD34 to mark the epithelial cells of blood vessels and MBP to mark the oligodendrocytes and Schwann cells were tested by using the strepavidin-peroxidase immunohistochemistry method.
Measurements of the response rate of ultrasound therapy were analyzed with the 2 (McNemar 2) test. Because the positive expression of specific markers related to the count number of the epithelial cell of blood vessels, the oligodendrocytes and Schwann cell, a paired t test was used to analyze the change of CD34 and MBP before and after treatment. The level of significance (P value) was set at .05 for all tests.
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RESULTS |
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In up-to-2-year follow-up, 72 of 76 patients had recovered to various degrees with a total response rate of 94.74%. Among them, 49 (32 with squamous hyperplasia and 17 with lichen sclerosus) were cured and 23 (11 with squamous hyperplasia and 12 with lichen sclerosus) were improved. Four patients, 2 with squamous hyperplasia and 2 with lichen sclerosus, were persistent. There was no significant difference between the therapeutic response rate of lichen sclerosus and squamous hyperplasia (P > .05) (Table 3). A better effect was obtained in patients who had the disease for shorter time (P < .05; see Table 4).
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Four (5.26%) of the 76 patients had slight skin burns and a few blisters around the labia majora, which were cured after local anti-inflammatory therapy. Eight (10.52%) patients had a recurrence after focused ultrasound therapy, which was treated again with ultrasound therapy with good results and without any complications.
Vulvar biopsies of the involved regions before and 3 to 6 months after the therapy were obtained and analyzed. Pathological changes of the vulvar dystrophy before the treatment were characterized by a count reduction of the micro capillaries in the dermal layer. The pathological examination of the treated tissue samples showed that the squamous epithelium regained normal structure. Layers and the epithelial basal membrane recovered without epidermal hyperkeratosis or dermal edema, granular layer thickening, and lymphocyte infiltration. The fibroblasts also recovered their normal appearances (Fig. 2).
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Compared with those before treatment, the epithelial desmosome and pigment cells’ count in the cytoplasm increased dramatically, and the majority of melanin cells were seen in the basal layer. Epithelial cells aligned in order with distinct stratification. Rich free ribosomes were found in the basal layer cells and the mitochondrial count increased with normal shape. Melanin granule count and the hemidesmosome count increased as well. Desmosome structures in the echinocytes were well developed with normal nuclei. Rich microfilaments, mitochondrion, and endoplasmic reticulum were seen in the cytoplasm. Fibroblasts were seen in the dermis and the count of microvessels was increased. Lumens of vessels basically recovered. Nerve endings in the lower layer of the dermis increased, while infiltrative lymphocytes in the epithelium and dermis were obviously reduced and, in some cases, eliminated. (Fig. 3)
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The pathological specimens with CD34 stained microvessels and MBP-stained nerve fibers were examined by using a light microscope with 5x magnification. There were significant increases of the CD34 and MBP counts after treatment compared with those before ultrasound therapy (P < .01; Table 5 and Fig. 4).
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DISCUSSION |
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Up-to-2-year follow-up demonstrated that only transient congestion and edema occurred after ultrasound therapy without any tissue scaring, and skin in the treated region remained intact. The involved tissue structures recovered normal pigmentation. Melanin cells were seen in the basal layer. The count of the microvessels increased, and their lumens basically recovered. Nerve endings and fibroblast counts in the lower layer of the dermis also increased.
The capillary network of the dermal papilla supplies nutrition to the epithelium and mucosa as well as to the deeper dermal layer of the skin. Antibody CD34 is expressed in early lymphocytic hematopoietic stem cells and intraepithelial cells of the blood vessels, and it is mainly used to mark the intraepithelial cells of the blood vessels, which stains the cell membrane and cytoplasm yellow-brown. As the primary component of the structural protein of the myelin sheath, polyclonal antibody MBP marks basically the oligodendrocytes and Schwann cells of the nerve fibers, staining the cell membranes and cytoplasm yellow-brown. Examinations suggested that the count of the microvessels and nerve endings decreased due to the nutrient dysfunction before the ultrasound procedures. Thus, low positive expressions of CD34 and MBP were noted. The histopathological and ultrastructural results after ultrasound therapy supported the hypothesis that vulvar lesions were caused by degenerative changes due to microcirculation-dysfunction–induced insufficient cell energy supply.
It was found that ultrasonic exposure accelerated cell proliferation, and increased the synthesis of collagen protein and angiogenesis factors interleukin-8, basic fibroblast growth factor, and vascular endothelial growth factor,28 which suggested that ultrasound therapy could stimulate cell proliferation, protein synthesis, and vascularization. Recent studies also demonstrated that the interaction between an ultrasound beam and microbubbles in microvessels could enhance the permeability of cell membranes and lead to collapse of microvessels less than 7 µm in diameter and broaden the intraepithelial cell space.29,30
In ultrasound therapy, an ultrasound beam is directed to the dermal layer of the skin. This gives rise to thermal effects and cavitation causing damage to the microvessels and subsequently improves growth and reconstruction of the microcirculation. Due to the destruction of the endothelial cells, the permeability of the capillaries was evidently increased. As a result, there were transient congestion and edema after the therapy, but the epidermis in the treated region remained intact.
After ultrasound therapy, the structure of the epithelium, pigment metabolization and pigmentation recovered, and the positive expression of CD34 and MBP increased, indicating that a series of biological effects by ultrasound therapy had changed the function and nutritious state of microvessels and nerve endings. It was then clear that ultrasound treatment might accelerate tissue reconstruction and improve microcirculation and nutrition of the local tissues. Furthermore, it could reduce the stimulation of the nerve endings, and the pruritus of the vulva was palliated and even eliminated. Our preliminary clinical results suggested that the ultrasound therapy could not only cure the refractory vulvar pruritus but also result in recovery of the elasticity, shape, and appearance of the skin. Therefore, this technique is considered more effective than other treatments to control vulvar pruritus and to produce cutaneous lesion recovery.
There were 4 patients (5.26%) with squamous hyperplasia who had slight skin burn after ultrasound treatment, which may be caused by excessive ultrasound energy accumulation and an elevated temperature at the skin surface. The results suggested that local tissue would gradually change from a reversible lesion to an irreversible coagulative necrosis while the ultrasound exposure time was increased. Some measures, such as careful skin preparation and continuous water irrigation of the treated region during the procedure, are helpful to avoid skin burns. Postoperative local ice compression also helps lower the temperature and irritation of treated areas.
As a local therapy, focused ultrasound is noninvasive and nonionizing. It could be precisely targeted to the tissue of interest, without damaging the overlying tissue. This study has demonstrated the feasibility and efficacy of focused ultrasound therapy in patients with vulvar dystrophy. The preliminary results are very encouraging. Focused ultrasound therapy appears to be a promising method to treat vulvar dystrophy. Nevertheless, more studies are still necessary to evaluate its long-term effects.
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Footnotes |
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The authors thank Lian Wang, MD, Huaqin Wang, MD, and Youde Cao, MD, for their contributions to this study.
Received April 21, 2004. Received in revised form June 16, 2004. Accepted June 24, 2004.
doi:10.1097/01.AOG.0000139835.34168.b0
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