RU2634263C1 - Prediction of development of common forms of infiltrative endometriosiss based on evaluation of cd15 expression in endometric biopsy materials - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention can be used to predict the development of common forms of infiltrative endometriosis. To do this, the result of immunohistochemical marker CD15 expression in endometrial biopsy materials is evaluated. Immunohistochemical examination is carried out on dewaxed sections 4-5 microns thick. CD15 expression is determined in epithelial cells of the endometrium, the results of the immunohistochemical reaction are evaluated in a semiquantitative manner in points. At that, the absence of immunostained cells (-) is 0 points; less than 5% of immunostained cells (+) is 0.5 points, less than 20% of immunostained cells (+) is 2 points; 20 to 40% of stained cells (++) is 4 points; more than 40% of stained cells (+++) is 6 points. When CD15 expression is evaluated from 2 to 6, common forms of infiltrative endometriosis are diagnosed.

EFFECT: prediction of the development of common forms of infiltrative endometriosis.

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Description

The invention relates to medicine, namely to gynecology, surgery and pathological anatomy, is intended for early diagnosis and prediction of common forms of infiltrative endometriosis.

The prevalence of endometriosis in the female population is currently 10-15% (Bulun SE, 2009, Guidice LC, 2010). In women of reproductive age, it is 5-15%, in postmenopausal women - from 3 to 5% (Modesitt SC, Tortolero-Luna G., Robinson JB, Gershenson DM, Wolf JK., 2002).

Deep infiltrative endometriosis, according to various authors, occurs in 20-50% of patients with genital endometriosis (Cornilhe F.J., 1990; Konickx P., 1994; Martin D., 1996).

Damage to the urinary system with endometriosis is rare and accounts for 1-2% of all cases of endometriosis (Donnez J, Spada F, Squifflet J, Nisolle M 2000; Vitagliano G, Villeta M, Castillo O, 2006). The bladder is most commonly affected (84%), the ureter is 10% affected, and the left ureter is affected more often than the right (Vercellini, P., Pisacreta, A., Pesole, A., Vincentini, S., Stellato, G. and Crosignani, E., 2000), kidneys - 4%, and the urethra is affected in 2% of cases. According to published data, the incidence rate has increased in recent years (0.3-12%).

To date, there is no theory or complex of theories that fully explain the pathogenesis of endometriosis. In the pathogenesis of urinary endometriosis, the authors support all the hypothetical mechanisms of the pathogenesis of endometriosis, including retrograde menstruation, metastatic (lymphatic and vascular) spread, as well as iatrogenic implantation of endometrial cells, coelomic metaplasia / induced mesenchymal differentiation of cells, and the development of endometrioid tissue.

Recently, researchers have been intensively studying endometrial stem cells and their role in the occurrence of ectopic endometriotic foci. (Isaac E. Sassona and Hugh S. Taylora, b Stem Cells and the Pathogenesis of Endometriosis Ann NY Acad Sci. 2008 Apr; 1127: 106-115.) Numerous experiments have shown that cells isolated from the endometrium can grow into endometrioid implants ( Seli E, et al. Pathogenesis of endometriosis. Obstet Gynecol Clinics North Am. 2003; 30:61. [PubMed]; TeLinde RW, Scott RB. Experimental endometriosis. Am J Obstet Gynecol. 1950; 60: 1173. [PubMed]; D'Hooghe TM, et al. Intrapelvic injection of menstrual endometrium causes endometriosis in baboons (Papio cynocephalus and Papio anubis) Am J Obstet Gynecol. 1995; 173: 134. [PubMed]; Ridley JH, Edwards IK. Experimental endometriosis in the human Am J Obstet Gynecol. 1958; 76: 790. [PubMed].

For the detection of endometrial stem cells and endometriotic foci, the authors used different markers. Oct-4 is a transcription factor and plays a large role in supporting cell pluripotency in embryonic and adult stem cells, germ cells and whole embryos at different stages of development. The authors also reported that this marker was detected in half of the studied endometrial samples. (Matthai, Horvath et al., 2006). Oct4 mRNA expression was found in all experimental endometrial samples in women in the follicular or luteal phase of the menstrual cycle (Bentz, Kenning et al., 2010). Expression of other stem cell markers, such as Bcl-2, C-kit (CD117) and CD34, have also been found in endometrial stem cells (Cho, Park et al., 2004). Musashi-1 (RNA-binding protein) expression was detected in endometrial samples, endometriotic foci, and endometrioid carcinoma (Gotte, Wolf et al., 2008). Musashi-1 expression was found in the epithelial and stromal cells of the proliferative endometrium. In endometrial cells, the number of Musashi-1 positive cells of the basal layer in the stromal component is 1.5 times and in the epithelial component 3 times higher than in the functional layer. In the endometrioid and cancer cells of the endometrium, high levels of Musashi-1 were also detected (Gotte, Wolf et al., 2008). Kato et al. Used two markers to differentiate between epithelial (CD13) and stromal (CD9) endometrial stem cells (Kato, Yoshimoto et al. , 2007).

Song et al. Studied the transcription of pluripotency factors (SOX2, NANOG (Nanog homeobox) and OCT4 in women of reproductive age with ovarian endometriosis and in women without this pathology. The results of the study showed that in the endometrium in women with ovarian endometriosis, these markers were significantly higher than in the endometrium in women without endometriosis.In addition, it was found that in the foci of endometriosis the expression of these markers significantly exceeded the values of eutopic endometrium (Song Y, Xiao L, Fu J, Huang W, Wang Q, ZhangX, Yang S. Increased expression of th e pluripotency markers sex-determining region Y-box 2 and Nanog homeobox in ovarian endometriosis. Reprod Biol Endocrinol, 2014; 12: 42). It has also been found that stem / bone marrow progenitor cells, when introduced into the endometrium, promote vascular remodeling (Pittatore G, Moggio A, Benedetto C, Bussolati B, Revelli A. Endometrial adult / progenitor stem cells: pathogenetic theory and new antiangiogenic approach for endometriosis therapy. Reprod Sci (2014) 21 (3): 296-304. doi: 10. 1177/1933719113503405) or can be transdifferentiated into endometrial cells (Du H, Taylor HS. Stem cells and reproduction. Curr Opin Obstet Gynecol (2010) 22 (3): 235-41. Doi: 10.1097 / GCO.0b013e328338c152; 8 Zhang WB, Cheng MJ, Huang YT, Jiang W, Cong Q, Zheng YF, et al. A study in vitro on differentiation of bone marrow mesenchymal stem cells into endometrial epithelial cells in mice. Eur J Obstet Gynecol Reprod Biol (2012) 160 (2) : 185-90. Doi: 10.1016 / j.ejogrb.2011.10.01.01). It was also revealed that regenerative endometrial cells isolated from menstrual blood can differentiate into various cell lines, such as cells synthesizing insulin (Li, Chen et al., 2010), osteoblasts (Ai, Mehrabani, 2010), odontoblasts (Ai, Tabatabaei et al. 2009), and neurons (Wolff, Gao et al., 2011 and Ai, Esfandiari et al., 2009a). Endometrial stem cells (EnSCs) have recently been used to replace dopaminergic neurons in a mouse model of Parkinson's disease (Wolff, Gao et al. 2011). Hida et al. Studied the cardiogenic potential of EnSCs (Hida, Nishiyama et al. 2008). The angiogenic potential of EnSC is also known, which determines the high regeneration of the endometrium (Esfandiari, Ai et al. 2007; Esfandiari, Khazaei et al. 2007; Esfandiari, Ai et al. 2008 and Ai, Esfandiari et al 2009a and 2009b). Antigenogenesis can play not only a key role in the process of endometrial regeneration, but can also participate in the formation and development of foci of endometriosis (Donnez, Smoes et al., 1998; Fujishita, Hasuo et al., 2000).

The theory of retrograde menstruation and stem cells indicates a relationship between endometrium and endometrioid foci.

In our work, we performed a morphological study, analysis of the expression of immunohistochemical markers of stem cells in the endometrium in women with urinary tract endometriosis and in the endometrium of healthy women. These studies were carried out as part of scientific research carried out in the NCAC & them. Kulakova

An embodiment of the invention is predicting the development of common forms of infiltrative endometriosis by the expression intensity of the immunohistochemical marker CD15 in an endometrial biopsy.

The study was conducted on the clinical base of the 1 pathological department of the Federal State Budget Scientific Institution Scientific Center IN AND. Kulakova ”of the Ministry of Health of Russia and the Department of Obstetrics, Gynecology, Reproductology and Perinatology, GBOU VPO First MGMU im. THEM. Sechenov

The study included patients undergoing examination and treatment at the NIACAP named after V.I. Kulakova about infiltrative endometriosis with lesions of the urinary tract. The age of patients in both groups ranged from 23 to 42 years, the average age was 34 ± 1 year. Patients were divided into two groups. Group I consisted of patients with urinary tract infiltrative endometriosis. The second control group consisted of healthy women (surrogate mothers). The criterion for inclusion in group I was the presence of infiltrative endometriosis with the involvement of the urinary tract. Exclusion criteria were the absence of infiltrative endometriosis with lesions of the urinary tract and malignant tumors. The criterion for inclusion in group II was the absence of endometriosis and any gynecological and somatic diseases. Exclusion criteria were the presence of gynecological and somatic diseases.

Patients of groups I and II underwent a physical examination, all the necessary clinical laboratory and instrumental studies were carried out to clarify the presence of endometriosis and prepare for surgical treatment in patients from group I and to exclude any diseases in patients from group II.

For this study, endometrial scrapings were used, taken from patients with urinary tract endometriosis, and endometrium, taken from surrogate mothers.

The material for histological examination was fixed in buffered (phosphate) 10% neutral formalin, then the samples were processed according to the generally accepted standard method and enclosed in paraffin. For morphological studies, at least 10 step sections with a thickness of 4 μm were made from each block, after which hematoxylin and eosin staining was carried out according to the generally accepted method, then immunohistochemical reactions were set.

An immunohistochemical study was conducted in 20 patients - in 10 in each group.

Immunohistochemical studies were performed on dewaxed sections 4–5 µm thick according to generally accepted protocols (DAKO protocols).

By immunohistochemical method, stem markers CD15-monoclonal mouse antibody (clone MMA), 1:50, Cell Marque, Aldh1-rabbit monoclonal antibody (clone EP1933Y), 1: 400, GeneTex, SOX2 - rabbit monoclonal antibody (clone SP76), 1: 100, Cell Marque using monoclonal antibodies. Positive and negative control reactions were set as secondary antibodies and detection agents.

The results of the IHC reaction were evaluated by a semi-quantitative method in points according to the generally accepted method: the absence of immunostained cells (-) - 0 points; less than 5% of immunostained cells (+) - 0.5 points, less than 20% of immunostained cells (+) - 2 points; from 20 to 40% of stained cells (++) - 4 points; more than 40% of stained cells (+++) - 6 points.

The data obtained were statistically processed.

All received data was processed on a personal computer using the program Statistica for Windows v.8.0, StatSoft Inc. (USA). For each quantitative parameter, the following values were determined: mean value (M), standard deviation (δ), error of the mean (m), 95% confidence interval, median (Me), interquartile range (Q1-Q3), for qualitative data - frequency (% )

Before conducting a comparative analysis of quantitative data in the studied groups, the type of data distribution was determined (Kolmogorov-Smirnov test). In the normal form of data distribution, parametric statistics methods (Student's test) were used to assess differences in groups. In the absence of a normal distribution of data, non-parametric statistics methods were used - the Mann-Whitney test to compare data in groups.

,To compare numerical data (after checking the quantitative data for a normal distribution), Student t-test was used for 2 independent samples:

,

where M ₁ , M ₂ - arithmetic mean; σ ₁ and σ ₂ are standard deviations; N ₁ , N ₂ - sample sizes.

In case of discrepancy with the law of normal distribution or when comparing in small samples, the comparison of numerical indicators between the two groups was carried out using the Mann-Whitney criterion for unrelated populations:

where n ₁ is the number of units in the first sample; n ₂ is the number of units in the second sample; T _x is the larger of the two rank sums corresponding to the sample with n _x - units.

Differences were considered statistically significant at p <0.05 (95% significance level) and at p <0.01 (99% significance level).

When studying the results of immunohistochemical studies, the expression features of all 3 markers CD15, Aldh A1 and SOX2 in autologous endometrium in patients with endometriosis were found.

Aldh A1 was determined in the cytoplasm and in the nuclei of cells of the epithelial and stromal components of endometrioid tissue and autologous endometrium (group I). In the control group II, the expression of this marker was not detected. Despite the lack of expression in the control group, no statistically significant differences between the groups were found.

SOX2 expression was found in the nuclei of the cells of foci of endometriosis and in a small amount of autologous endometrium (group I). In the control group II, SOX2 expression was not detected. Expression is mainly detected in the epithelial components. In the stromal component, this marker in small quantities was determined only in the foci of endometriosis. In autologous endometrium, expression was evaluated at 1-2 points. No statistically significant difference was found between groups

Expression of CD15 was detected only in the epithelial cells of foci of endometriosis, autologous endometrium (group I) and was not detected in the control endometrium (group II). Expression in autologous endometrium was evaluated from 2 to 6 points. When comparing between groups revealed a significant statistical difference (p = 0,0174) (figure 1)

Thus, the statistically significant difference in the expression of CD15 between the groups allowed us to develop a method for predicting the development of common forms of infiltrative endometriosis by determining the expression of the CD15 marker in endometrial tissue samples obtained by pipelid biopsy or diagnostic curettage.

Clinical example 1

Patient S., 40 years old, complained of pain in the lower abdomen and lumbar region on the left, profuse painful menstruation.

From the anamnesis: 2011. At the place of residence, a diagnostic laparotomy was performed, a biopsy of the formation of the retrocervical region (histological conclusion - endometriosis of the RC region). Hormone therapy was prescribed - dipherilin 3.75 mg No. 6, then the therapy was continued by Jeanine on a contraceptive regimen. In 2013, stricture of the left ureter and hydronephrotic changes in the left kidney were revealed, and therefore, stenting of the left ureter was performed. When applying to the NCAGiP in 2014, infiltrative endometriosis of the lateral pelvic fiber was diagnosed with involvement of the left ureter. Hydronephrosis of the left kidney of the 1st Art. Adenomyosis II Art. Laparoscopy was performed - excision of endometrioid infiltrate, left ureterolysis, hysterectomy without appendages.

During histological examination, the endometrium corresponded to the proliferative phase.

Immunohistochemical study: Aldh A1 was determined in the cytoplasm and in the nuclei of cells of epithelial and stromal components. The expression of Aldh A1 in the epithelial and stromal components of the endometrium was evaluated at 6 points. SOX2 expression is found in the nuclei of cells. The expression intensity was estimated at 4 points in the epithelial component of the endometrium, and this marker was not found in the stromal component of expression. Expression of CD15 was detected only in the epithelial components of the endometrium and was evaluated at 6 points.

Clinical example 2

Patient I., 32 years old, complained of general weakness, pain in the lower abdomen during menstruation, lack of pregnancy, difficulty urinating during menstruation.

From the anamnesis: for the first time an endometrioid cyst of the right ovary was diagnosed in 2010. In 2012, in preparation for IVF, drugs, salpingo-ovariolysis, removal of endometrioid cysts of both ovaries were performed. After the operation, these complaints appeared. In 2014, RC endometriosis was diagnosed, and therefore was sent to the NCAGiP.

After a complete clinical, laboratory and instrumental examination, the diagnosis was made: a common form of OGE: RC endometriosis with germination and stenosis of the rectum, recto-sigmoid colon. Endometriosis KMC, pelvic scabies. Bladder endometriosis. Endometrioid cyst of the right ovary. Adhesive process of the abdominal cavity.

Hysteroscopy, diagnostic curettage of the endometrium was performed. Cystoscopy. Laparoscopy, resection of the right ovary, excision of infiltrative pelvic endometriosis, rectal shaving, disc-shaped resection of recto-sigmoid colon, excision of the endometriosis of the vesicoureteral fold, bladder resection, salpingo-ovariolysis.

During histological examination, the endometrium corresponded to the proliferative phase.

Expression of immunohistochemical markers in the endometrium: Aldh A1 expression was found only in the epithelial component of the endometrium and was evaluated at 2 points. SOX2 is also found only in the epithelial component and is rated at 2 points. Expression of CD15 in the epithelial component was evaluated at 4 points.

Clinical example 3

Patient E., 42 years old, came up with algomenorrhea, pain in the right ileal region, blood from the rectum in the first 2 days of menstruation, and mucus up to 10 days of menstruation, dyspareunia. These complaints have been disturbed for 3 years. After conducting clinical, laboratory and instrumental methods of research, the diagnosis was made: a common form of external genital endometriosis: retrocervical endometriosis with germination in the posterior vaginal fornix, rectum, endometriosis of the sacro-uterine ligaments and pelvic peritoneum, endometrioid ovarian cysts, infiltrative right endometriosis pelvis with signs of involvement in the process of the right obturator nerve, distal part of the right ureter, hydronephrotic transformer tion of the right kidney with stage III-hand hydroureter. Severe adhesions in the pelvic cavity. Uterine fibroids are small. With nephroscintigraphy, the absence of accumulative and excretory function of the right kidney was diagnosed.

Mid-midline laparotomy was performed. Right-sided nephrectomy. Appendectomy. Hysterectomy with right appendages, resection of the posterior vaginal fornix, resection of the left ovary. Low anterior resection of the rectum. Excision of endometrioid infiltrate of the right lateral wall of the pelvis. Neurolysis of the right obturator nerve. Establishment of transversostomy.

During histological examination, the endometrium was at the proliferation stage.

Expression of immunohistochemical markers in the endometrium was found only in the epithelial component. Expression of Aldh A1 was evaluated at 1 point, SOX2 at 1 point and CD15 at 2 points.

Clinical example 4

Patient A., 32 years old, was examined for entry into the surrogacy program. An examination of somatic and gynecological pathologies was not detected.

In the endometrial biopsy, expression of immunohistochemical markers CD15, Aldh A1, SOX2 was not found in the epithelial and stromal components.

Claims (1)

A method for predicting the development of common forms of infiltrative endometriosis based on evaluating the result of expression of the immunohistochemical marker CD15 in endometrial biopsies, characterized in that an immunohistochemical study is carried out on dewaxed sections 4-5 μm thick, the expression of CD15 is determined in epithelial cells of the endometrium, the results of the immunohistochemical reaction are evaluated by semi-quantitative method points: the absence of immunostained cells (-) - 0 points; less than 5% of immunostained cells (+) - 0.5 points, less than 20% of immunostained cells (+) - 2 points; from 20 to 40% of stained cells (++) - 4 points; more than 40% of stained cells (+++) - 6 points, and when assessing the expression of CD15 from 2 to 6, common forms of infiltrative endometriosis are diagnosed.

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