Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-01-24
2004-06-01
Chin, Christopher L. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007100, C435S007200, C435S007210, C435S007230, C435S007400, C435S007500, C435S007920, C435S007940, C435S287200, C435S960000, C435S975000, C436S503000, C436S519000, C436S547000, C436S548000, C436S063000, C436S064000, C436S172000
Reexamination Certificate
active
06743595
ABSTRACT:
BACKGROUND OF THE INVENTION
(a) Field of the Invention
The invention relates to a method and a kit for the diagnosis of endometriosis using blood and endometrial leukocyte markers.
(b) Description of Prior Art
Endometriosis is one of the most common gynecological disorders, affecting up to 15% of women within reproductive age. It is closely associated with severe pelvic pain, dysmenorrhea, dyspareunia, infertility and several other symptoms such as intraperitoneal bleeding, back pain, constipation and/or diarrhea. It is a major threat to physical, psychological and social integrity of the patients.
Endometriosis is characterized by the implantation and growth of endometrial cells (which normally constitute the lining of the uterus) in extra-uterine sites such as the peritoneal cavity. Although the etiology and pathogenesis of endometriosis remain mainly unclear, the theory of retrograde menstruation is the most widely accepted to explain the presence of ectopic endometrial cells in the peritoneal cavity. However, this phenomenon occurs in most women and, thus, several other factors must be invoked to explain the implantation of endometrial cells and the subsequent development of endometriotic lesions. It is generally believed that initiation of endometriosis implies a complex cascade of events requiring several essential features. Retrogradely seeded endometrial cells must remain viable, be capable of adhering to the mesothelium and of proliferating. Local degradation of the extracellular matrix, as well as extensive vascularization, are also believed to play an essential role in promoting the invasion of the peritoneal cavity by endometrial cells. Furthermore, once implanted, ectopic endometrial cells must have the capacity to counteract the cytolytic action of the immune system. Indeed, this is supported by the observation of several immunological abnormalities in patients with endometriosis.
At present, direct visualization of the endometriotic lesions under surgical procedures (laparascopy or laparotomy) is the golden standard and the only reliable method available to diagnose endometriosis. However, this method is highly invasive (i.e. surgery under general anesthesia), costly (i.e. direct cost and indirect cost due to convalescence) and requires a well-trained surgeon who has the ability to identify endometriotic lesions with a variety of appearances. The type of lesions, their size and their localization will determine the stage of the disease (stage I minimal, stage II mild, stage III moderate, stage IV severe). However, there is still no clear consensus on how these parameters correlate with the stage of the disease and the prognostic of endometriosis. In addition, early or minimal endometriosis (which can involve microlesions) can be hardly diagnosed by surgical methods, as they are unlikely to be detected by direct visualization. Indeed, several studies have reported microscopic endometriotic lesions that were not detected laparoscopically. Because the diagnosis of endometriosis by surgical procedures is difficult, costly and invasive, in some cases, several physicians and patients tend to avoid it or at least seriously delay it. Hence, the length of time between the onset of symptoms and the diagnosis can be as long as 8 to 12 years. The possibility to diagnose endometriosis at an early stage would certainly improve the efficacy of the treatments, and reduce dramatically the number of years during which patients endure acute or chronic pain.
Imaging methods such as transvaginal ultrasound and magnetic resonance imaging have been designed for the diagnosis of endometriosis. However, these techniques can only be reliable for the detection of large (>1 cm diameter) endometriomas lesions detected among a very small proportion of patients with endometriosis. Moreover, the high cost of these techniques has limited their use for the diagnosis of endometriosis.
Serum proteins such as CA-125 and placental protein-14 have been proposed as diagnostic markers for endometriosis. Elevated levels of CA-125 have been observed in serum, menstrual effluent and peritoneal fluid of patients with endometriosis. However, these markers, when used alone, are of very limited value for a diagnosis test. Indeed, these markers are not suitable for screening or diagnostic purposes because they provide poor sensitivity. Furthermore, levels of CA-125 and placental protein-14 vary according to several factors such as the assay, the stage of the disease and the menstrual cycle. Finally these markers are known to be modulated by conditions other than endometriosis.
High concentrations of antibodies to endometrial antigens were found in the serum of patients with endometriosis, and thus were proposed as markers for a diagnostic test (International patent application publications WO 94/28021 and WO 92/18535). However, the levels of specificity and sensibility with these tests remain very low. In most cases, the antigens recognized by these antibodies are still poorly characterized or yet totally unknown.
In U.S. Pat. No. 5,478,725, low levels of &agr;v&bgr;3 integrin expression in endometrial samples during the secretory phase of the menstrual cycle is described as a predictor of endometriosis in infertile but not in fertile patients with endometriosis. This observation was associated with milder form of endometriosis (stages I and II) only and, thus, is not useful to detect advanced stages of the disease. Moreover, this method yielded a specificity of 91% but a very low sensitivity (38%).
Taking into account that a number immunological abnormalities have been reported in patients with endometriosis, it is conceivable that the proportion of leukocyte populations and/or their activation status may be modulated during the course of the disease and, thus, may provide some diagnostic value. Previous flow cytometric studies have shown that some T lymphocyte subpopulations (CD8+, CD45+/HLADR+, CD45+/CD3+/HLADR+or CD3+/CD25+) can be slightly modulated in the peritoneal fluid of subjects with endometriosis relative to normal controls (Oosterlyncck D. J., et al.,
Am J reprod. Immunol
., 31: 25-31, 1994; Becker J. L., et al.,
Am J Reprod. Immunol
., 34: 179-187, 1995; Wu M. Y., et al.,
Am. j. Reprod. Immunol
. 35: 510-516, 1996). However, these observations have limited value for the diagnosis of endometriosis because peritoneal fluid collection is an invasive, non-conventional procedure. Proportions of leukocyte populations have also been studied in peripheral blood and endometrium of patients with endometriosis. Wu et al., (supra) have reported a modest but significant decrease in the proportion of CD3+ T lymphocytes expressing either CD69 or CD25 activation marker in the blood of patients with advanced endometriosis but not in patients with mild stage of endometriosis or normal controls. This difference was observed in advanced cases of endometriosis only and it was too modest to be used as a diagnostic marker. In contrast, Oosterlynck et al., (Oosterlyncck D. J., et al.,
Am J reprod. Immunol
., 31: 25-31, 1994) and Ho et al. (Ho H. N., et al.,
Hum Reprod
., 97: 2528-2533, 1997) reported no significant difference in term of T lymphocyte subpopulations when comparing endometriosis subjects with normal controls. These inconsistent results may be explained by the very low number of samples tested in these studies.
Several studies have investigated whether leukocytes are also modulated in eutopic endometrium from patients with endometriosis. Results arising from these studies are contradictory, probably due to the fact that in most cases the methods used were only semi-quantitative and the number of samples tested were very low. For instance, by means of immunohistochemistry, Ota et al. (Ota H., et al.,
Am J Reprod. Immunol
., 35: 477-482, 1996) have reported that the number of CD3+, CD4+, or CD8+ T lymphocytes, cells bearing adhesion molecules (i.e. ICAM-1, LFA-1, CD2) or CD68+ cells were upregulated in the endometrium of patients with
Gagné Danièle
Gosselin Diane
Hugo Patrice
Miron Pierre
Chin Christopher L.
Crowell & Moring LLP
Gabel Gailene R.
Metriogene Biosciences Inc.
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