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UDC 618.14-002:615.32
ENDOMETRIUM RECEPTION AND SOME IMMUNO-HISTOCHEMICAL PARAMETERS BY CHRONIC ENDOMETRITIS ON THE BACKGROUND OF TREATMENT BY THE EXTRACT OF ORTILIA SECUNDA
Altai State Medical University, Barnaul
O.A. Skoropatskaya, N.I. Fadeyeva, S.D. Yavorskaya, T.S. Taranina
Monocentric, prospective study in the group of female patients (p=20) with chronic endometritis, who received the extract of ortilia secunda at the stage of rehabilitation 2,5 mg/day during two menstrual periods. According to the therapy results there was stated a significant increase of estriol in the blood serum, activation of humoral antiviral immunity, restoration of endometrium structure and receptor apparatus of endometrium to sex steroids. The obtained results correspond to the results of the earlier experiment. Key words: ortilia secunda, chronic endometritis, phytotherapy, rehabilitation stage.
Chronic endometritis remains an actual problem of modern gynecology, as frequently, after etiotro-pic therapy, there remain disorders of microcirculation in the endometrium, a decrease in the sensitivity of reception to steroid hormones, inadequacy of local immunity [1]. Different methods of pre-gravid preparation by chronic endometritis are suggested, aimed at eliminating the above-listed disorders. Most often used means are nonspecific immunomodulators, hormone therapy, a number of preformed factors [2].
The anti-inflammatory, anticoagulant and immunomodulating properties of the dry extract of ortilia secunda have been experimentally proven [3]. There was also proved the antimicrobial activity of decoction of the ortilia secunda leaves against enterobacteria, pseudomonads, staphyllo-coccus, fungi of the genus Candida [4]. There had been previously conducted an experiment in 90 white outbred rats, which confirmed the efficacy of the ortilia secunda extract in the rapid restoration of the normal structure of the endometrium after experimental endometritis [5]. In a histolog-ical study of the endometrium, secretory changes were detected in rats which, for the purpose of treating experimental endometritis, received the ortilia extract. In the intact rats, such changes were not established. The data obtained suggested that the ortilia secunda extract possesses not only anti-inflammatory properties, but also contributes to the normalization of the endometrial receptor apparatus. The results obtained in the experiment were the reason for further research - the evaluation of the effectiveness of oral application of the ortilia secunda extract in patients with chronic endometritis.
Materials and methods
The research was conducted in accordance with the Helsinki Declaration of the World Association "Ethical principles of scientific medical research
with human participation", as amended in 2000 and the "Rules of Clinical Practice in the Russian Federation" approved by the Order of the Ministry of Health of the Russian Federation of June 19, 2003 No. 266.
The study included 20 patients aged 30-35 years, with echoscopically and histologically verified chronic endometritis. The inclusion criteria were: verified chronic endometritis, as an indication for pregravid preparation. The exclusion criteria were other organic diseases of the genitals, anovulations, subcompensated and decompen-sated somatic diseases; the patient's age over 35 years. All patients in the rehabilitation phase after etiotropic treatment of endometritis were assigned the ortilia extract at the dose of 2.5 mg/day during two menstrual cycles. The ortilia secunda extract used in the experiment was obtained on the basis of the laboratory for study and use of natural resources of the Research-and-development Center of Fundamental medicine of the Altai State Medical University by means of vacuum-pulse extraction. This extraction technique makes it possible to obtain highly purified and highly concentrated grass extracts [6].
In the blood serum of all patients, before and after the intake of the ortilia extract, on the 21st-25th day of the menstrual cycle, along with standard tests (clinical and biochemical blood analysis, urinalysis, bacterioscopy and PCR diagnostics of the cervical canal) there were determined estradiol, estriol, progesterone, DHEA, testosterone, 17-OHP and cortisol, there was performed the evaluation of the interferon status of blood (alpha and gamma-interferons) [7]. Pipel-biopsy of the endometrium was carried out on the 5th-7th day of the menstrual cycle with subsequent histo-logical and histochemical studies of the biopsy specimen. Immunohistochemical definition of the nature of inflammation was carried out according to the method of Ye.A. Mikhnina and co-authors
[8]. According to the proposed method, the number of cells of CD 56 +, CD 16+, HLA DR (II) + from 0 to 10 in the field of view is characteristic of healthy people. By the increase of only CD 56+ (above 10), autoimmune chronic endometritis is diagnosed.
By the increase in only CD 16+ and HLA-DR (II) + (above 10), chronic endometritis with exacerbation or acute endometritis is diagnosed. By the increase in all indicators, chronic endometritis is diagnosed, without exacerbation. Immunohistochemical (IHC) study of hormonal receptors of the endome-trium (a-estrogen - ER and progesterone - PR) was carried out using monoclonal antibodies to a-ER (clone SP1, dilution 1: 200, Epitomics, USA), PR (clone YR85, dilution 1: 350, Epitomics, USA). The intensity of the IHC reaction of ER and PR was assessed by the method of the histological score "quickscore": Ax B, where A is the percentage of positively stained cells (counting no less than 1000 cells in 10 fields of vision) [9].
Statistical analysis of the obtained results was carried out with the help of the program for statistical processing Statistic 10. The values of p were considered significant values by less than 0.05. The Shapiro-Wilk criterion was used to estimate
The histochemical examination of the endo-metrium prior to therapy revealed an increase in CD20 +, CD138 +, natural killers (CD56 +, CD16 +) and lymphocytes expressing the activation marker of the histocompatibility complex of HLA-DR + II class, which is characteristic of chronic en-dometritis, without exacerbation [4]. A high level of CD56 + lymphocytes (from 25 to 60), detected in 30% of cases - a marker of pronounced autoimmune character of chronic endometritis [4].
After the course of therapy, there was a decrease in the frequency of detection in the endometrium of increased leukocyte activity: CD 20 + (100% and 15%, p = 0.0001); CD138 + (100% and 30%, p> 0.0002); CD 56+ (100% and 20% p> 0.0015); CD 16+ (100% and 20% p> 0.0015); HLA-DRII + (100%
the type of distribution of features in the case of small samples, in the case of large samples, the Kolmogorov-Smirnov test was used. The values of the interval values subject to the normal distribution are represented in the form M ± m, where M is the sample mean and the m - standard error of the mean. The values of qualitative characteristics are presented in the form of observed frequencies and percentages. In cases of normal distribution, as well as the equality of sample variances, Student's t-test was used to compare the samples.
Results and discussion
By the assessment of the hormonal profile of patients with chronic endometritis, it was established that the levels of all the hormones studied before and after treatment with the ortilia secunda extract were within the age limits. However, after the course of therapy, there was a significant increase in the average levels of estradiol, there was a trend towards an increase in the average level of estriol, progesterone and cortisol (Table 1), which can be regarded as an increase in ovarian activity under the influence of the ortilia seconda extract therapy.
and 30%, p> 0.0002) in combination with a decrease in the average level of activated leukocytes: CD 20+ (11.15 ± 1.2 and 3.0 ± 1.2 p = 0.0002 ); CD138 + (16.44 ± 3.4 and 1.55 ± 0.2, p> 0.0025; CD 56+ (26.88 ± 3.6 and 9.55 ± 0.32, p = 0.015); CD 16+ (17.3 ± 2.43 and 9.11 ± 0.18, p = 0.009), HLA-DRII + (19.55 ± 4.23, 9.88 ± 2.3, p = 0.006).
Immunohistochemical (IHC) study of hormonal receptors of the endometrium (a-estrogen - ER and progesterone - PR) in dynamics allowed us to establish that upon completion of therapy with the orthilia secunda extract in all patients, the average level of estrogen receptivity of the endometri-um increased significantly, as in stromal cells (22,22 ± 2.3 and 44.88 ± 2.6 p = 0.00003) and in the glands (20.22 ± 4.2 and 41 ± 2.2, p = 0.00003). In addition,
Table 1
The dynamics of the average levels of certain hormones in the blood, taken on the 21st-23rd day of the menstrual cycle
in patients with chronic endometritis
Level of hormones before treatment Level of hormones after treatment P Reference values on the 21st-23rd day of the menstrual cycle
Estradiol, pg / ml 152,78±7,34 209,22±10,43 P = =0,004 48,3 -211
Cortisol, |jg / dL 2,79±0,53 3,80±0,26 P =0,25 6,2-19,9
Estriol, nmol / l 0,8±0,35 1,26±0,31 P = =0,004 0 - 1,4
Progesterone, nmol / l 5,47±2,62 7,01± 1,89 P =0,17 0,5-9,4
17-OHP, nmol / l 5,96 ± 0,32 5,67±0,33 P =0,98 1,0 - 11,5
ACTH, pg / ml 38,44±22,8 37,66±21,29 P =0,94 0 - 46
DHEA, |jg / dL 295,5± 26,3 302,2±24,8 P =0,95 98,8-340
Total testosterone, nmol / l 0,32±0,03 0,36±0,02 P =0,99 0,45 - 3,75
the mean progesterone receptivity of the endome-trium in the stromal cells (32.55 ± 4.3 and 48.77 ± 4.3, p = 0.008) and in the glands (32.66 ± 2.2 and 48.44 ± 2.2, p = 0.02) significantly increased.
By the evaluation of the level of alpha and gamma interferons in heparinized blood on 21st-23rd day of the cycle, the initial insufficiency of the first degree of alpha interferon was detected in 20% of cases, whereas at the completion of therapy, the frequency of its detection increased to 40% of cases (p = 0.08 ), which was accompanied by a decrease in the average level of the studied indices in the blood (849.55 ± 120 and 646 ± 120 U/ml, p = 0.06).
Lack of gamma interferon of the first degree was initially determined in 90% of cases, which may be associated with insufficient antiviral immunity [1]. Upon the completion of therapy, insufficiency was detected only in 10% of cases (p = 0.0001), there was a general tendency to the increase of the average level of gamma interferons (104.44 ± 6 and 193.55 ± 4 U / ml, p = 0.0001), which can be regarded as the restoration of antiviral and antiproliferative activity [1,2].
By the histological examination of the endo-metrial pile-biopsy specimens, on the 5th-7th day of the cycle, inflammatory infiltrates were detected in all cases: diffuse infiltrates in 40% and focal infiltrates in 60% of cases. Upon completion of therapy, all patients showed an improvement in the his-tological pattern in terms of a decrease in the frequency of leukocyte infiltration, represented only by the diffuse form (100% and 20%, p = 0.00001). Focal fibrosis, revealed initially in 50% of cases, was preserved after the therapy only in 20% of cases, in combination with diffuse leukocyte infiltrate. In each second patient, after the completion of the course of therapy, histological markers of chronic endometritis is not established.
Thus, a two-month course of chronic endo-metritis therapy with the ortilia secunda extract at the dose of 2.5 mg/day significantly increased the average level of estradiol in the blood serum and the average level of estrogen and progesterone receptors in the endometrium; activated humoral antiviral immunity by reducing the frequency of gamma-interferon deficiency and increasing the average level of gamma interferons in the blood; increased the frequency of the normal value of activated leukocytes CD 20+, CD138 +, CD 56+, CD 16+, HLA-DRII +; eliminated focal leukocyte infiltrates and reduced diffuse leukocyte infiltrates while maintaining focal fibrosis.
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Contacts
Corresponding author: Skoropatskaya Olga Alek-
seyevna, post-graduate student od the Department
of Obstetrics and Gynecologyof Altai State Medical
University, Barnaul. 656038, Barnaul, ul. Molode-
zhnaya, 9.
Tel.: 8-906-942-9138.
Email: [email protected]
