Clinical and pathomorphological characteristics of patients with purulent inflammation of the uterine appendages

Pelvic inflammatory diseases continue to be a relevant gynecological problem, and chronic inflammation of appendages constitute the highest percentage in this group of diseases [1, 2]. Such factors as early age at first sexual intercourse, a large number of sexual partners, inadequate diagnosis and treatment of sexually transmitted infections contribute to the development of the disease. Chronic inflammation of appendages is often characterized by a recurrent course and sometimes it is complicated by the formation of abscesses, namely tubo-ovarian abscesses (TOA) [3, 4]. Both functional and morphological changes of tissues are detected in these cases. Unfortunately, adnexal structural changes are not always evaluated in a timely manner by clinicians; therefore, the choice of the wrong tactics for the management of patients leads to the delayed surgical treatment. All these can be caused by the use of non-informative diagnostic tests, the growth of autoimmune diseases, the impairment of the body’s immune response, the contradiction between the clinical picture of TOA and morphological changes in them.

Any inflammatory process and its outcome are known to be due to a complex immunopathological process, the launch of a cytokine cascade, and the balance of pro-inflammatory and anti-inflammatory cytokines [5]. A number of researchers conducted studies of cytokines in the peritoneal fluid in various gynecological diseases [6, 7].

Therefore, the study of the concentration of several cytokines both in serum and focus of inflammation is worth considering.

The aim of the research was to evaluate clinical and morphological characteristics, the expression of cytokines in serum and peritoneal fluid in patients with TOA.

Materials and methods

We conducted clinical and statistical, biochemical, immunological and morphological studies of 476 women. The main group consisted of 456 patients with TOA. The control group consisted of 20 healthy women who underwent laparoscopic sterilization with the removal of the fallopian tubes. The examination of the patients included taking a history, basic clinical laboratory examination, bacterioscopic studies (examination of vaginal smears), determining microbial DNA using polymerase chain reaction, instrumental (transvaginal and transabdominal ultrasound examination of pelvic organs), immunological, morphological and statistical research methods. The indicator of cell phagocytic protection was determined according to the formula of O.N. Buraya (1993); it was presented as the ratio of the sum of the number of rod-shaped, segmented neutrophils and blood monocytes to leukocytosis (the number of leukocytes) multiplied by 100.

There was a quantitative assessment of cytokine levels, such as interleukin‐1β (IL-1β), IL-4, IL-6, as well as tumor necrosis factor (TNF)-α, fibronectin in blood serum and peritoneal fluid. The levels were determined using the test system of JSC Vector-Best (Novosibirsk, Russia). Peritoneal fluid sampling was performed intraoperatively during the puncture of an adnexal abscess on the day of admission. In the control group, peritoneal fluid was collected during laparoscopic sterilization.

Patients with systemic autoimmune diseases, infectious genital and extragenital diseases, benign and malignant tumors, severe forms of extragenital pathology were not eligible for immunological examination.

All patients with TOA underwent surgical treatment followed by morphological examination of the surgical material (n=456). The operation consisted in unilateral removal of the appendages and was expanded to hysterectomy according to indications (n=62). In some cases, resection of the greater omentum (n=39) and appendectomy (n=24) were performed. For histological examination, the material was fixed in a 10% neutral formalin solution and then it was poured into paraffin; sections were prepared and subsequently stained with hematoxylin and eosin, picrofuxin, Mallory, fuchselin for elastic fibers. Periodic acid Schiff reaction with amylase control was performed; nerve fibers were impregnated with silver nitrate by Bilshovsky–Gross.

The morphometric study of the structural components of the surgical material was carried out according to G.G. Avtandilov (1990) using a digital video camera and Video TestMorpho software package, which makes it possible to take into account both dimensional and countable characteristics.

Statistical analysis

Statistical data processing was carried out with the use of a software package for Windows STATISTICA 10.0. The quantitative description of the variables was presented as arithmetic mean (M) and standard deviation (SD). Given the normal distribution of data, statistically significant differences between quantitative indicators were determined using the Student’s t-test. The critical significance level was assumed to be p<0.05.

Results and discussion

The average age of patients with TOA was 36.4 (1.2) years (from 18 to 54 years). Most women were of active reproductive age (20–40 years), 282/456 (61.8%). History of repeated hospitalizations due to chronic inflammation of appendages was noted in 297/456 (65.1%) women.

The medical history of patients revealed that almost a third of them took analgesics and antibiotics without doctor’s prescription or medical advice. Long-term outpatient treatment was prescribed to 93/456 (20.3%) patients by gynecologists and therapists due to fever. In these cases, doctors underestimated the seriousness of the situation which led to late hospitalization.

The main complaints of all patients were pelvic pains of varying severity, an increase in body temperature to 38–39.4°C, pathological discharge from the genital tract, general fatigue, malaise, loss of appetite.

On admission to the gynecological department, the condition of women was assessed as satisfactory in most cases; moderate disease was noted in 91/456 (19.9%) patients and severe in 74/456 (16.2%) women. The vaginal examination and ultrasound assessment confirmed the diagnosis of purulent adnexal inflammation in all cases.

The changes in peripheral blood are considered to be one of the objective diagnostic criteria for any inflammatory process. The most characteristic signs were anemia, leukocytosis (neutrophil shift), toxic granularity of leukocytes. Monocytes were absent in 8/456 (91.75%) patients, and every tenth patient (47/456 (10.3%)) had lymphocytopenia below 3% of cells.

The number of leukocytes in women with TOA in the peripheral blood ranged from 2.9–16.9×109/L, on average it was 10.88 (0.97)×109/L, neutrophil shift reached 15%. According to the formula of O.N. Buraya, the indicator of cell phagocytic protection was 680.9 (35.6), which was 1.6 times lower than in healthy women (1182.6 (64.3)). This was indicative of phagocyte insufficiency in patients with inflammatory diseases. All patients with TOA showed an increase in the level of ESR, on average it was 35.57 (8.2) mm/h; the level of C-reactive protein increased as well, on average it was 76.3 (9.2) mg/L. Biochemical blood analysis showed a change in protein fractions even with normal total protein content, hypoalbuminemia, a decrease in the protein (albumin-globulin) coefficient to 0.67 in severe cases. The degree of dysproteinemia depended on the severity of the inflammatory process. The inflammatory process is usually accompanied by hypercoagulation. In our studies, the average level of fibrinogen in patients with TOA was 4.96 (0.52) g/L versus 2.43 (0.4) g/L in patients of the control group; there was an increase of fibrinogen level to 16.6 g/L in severe cases.

The examination of vaginal microcenosis revealed candidiasis (85/456 (18.6%)), bacterial vaginosis (79/456 (17.3%)), trichomoniasis (18/456 (3.9%)), gonococcal infection (7/456 (1.5%)), chlamydia infection (28/456 (6.1%)) in the patients of the main group. The inflammatory smear was identified in 86.8% (396/456): a large number of white blood cells and coccoid bacteria were predominant.

Any inflammatory process is known to be caused by the launch of a cytokine cascade. According to literature data, inflammatory processes are characterized by hyperproduction of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, IL-8) and relative insufficiency of synthesis of anti-inflammatory cytokines (IL-4 and IL-10). Since almost all cells of the body have receptors for IL-1, this cytokine activates them very quickly for the participation in a local inflammatory reaction. Our studies showed that the average content of IL-1ß in the blood of healthy women was 14.22 (1.33) pg/ml, and it was almost 27 times higher in patients with TOA (Table). In the peritoneal fluid, the level of IL-1ß in patients with TOA was also significantly increased, which may be a criterion for the severity of the inflammatory process.

According to our data, the concentration of TNF-α was increased in all the biological media which were studied. A number of experimental and clinical studies have shown that TNF-α is capable of both inhibiting the production of erythropoietin and activating hepcidin, which can cause anemia of chronic inflammation [8]. Anemia was detected in 146/456 (32.0%) patients with TOA; therefore, in case of detection of anemia and absence of a severe pain syndrome in patients, it is necessary for the general practitioner to suggest the possible presence of an inflammatory process, in particular, chronic inflammation of appendages.

IL-1 and TNF-α are considered to be the first-wave cytokines that cause a number of clinical effects such as fever and leukocytosis; they also facilitate the secretion of IL-6 in macrophages. IL-6 then triggers the secretion of acute-phase proteins, primarily C-reactive protein and fibrinogen [9, 10]. In our study we confirmed an increase in all the above-mentioned molecules both in the blood serum and in the focus of purulent inflammation.

The analysis of IL-4 content showed its decrease both in blood serum (by 1.47 times) and in exudate (by 1.77 times) in comparison with the control group (Table).

The level of fibronectin was noticed to be increased in serum and peritoneal fluid in patients of the main group. The concentration of fibronectin in the exudate of patients with TOA was more than four times higher than the same indicator in patients of the control group. The sources of tissue and plasma fibronectin are neutrophils, macrophages, platelets, fibroblasts; it lies on the surface of these cells in the form of a mesh network. Vascular endothelium, monocytes, and tissue basophils can also contain fibronectin. As a local opsonin, plasma fibronectin largely determines the phagocytic function of the reticuloendothelial system in the protection against infection. Fibronectin plays a significant role in the purification of the blood from collagen degradation products [11]. We believe that an increase in the serum level of fibronectin is a typical reaction of the body to inflammation.

All patients underwent surgical treatment.

On a macroscopic level, TOA was a single conglomerate of irregular shape up to 15-20 cm in diameter of a purple-grey color. The external surface of the peritoneum was pale and hyperemic with fibrinous deposits in the form of threads and flakes of fibrin. In all cases, the inflammatory infiltrate included the uterus, a strand of the greater omentum, as well as the loops of the intestine and the wall of the bladder which were covered with fibrin in most patients. Macroscopic manifestations of the disease were confirmed by microscopic studies. The histological study of the TOA wall revealed a diffuse inflammatory infiltrate which consisted of monocyte-macrophage elements and lymphocytes. In some areas of the infiltrate there were focal infiltrates with predominant leukocytes; that is, there were peculiar microabsesses which suggested a periodic exacerbation of the process and supported the release of pro-inflammatory cytokines. Muscular layers of the tube wall underwent a significant change and were replaced by connective tissue. According to the literature, fibronectin plays an important role in the formation of the extracellular connective tissue matrix, as it has a marked affinity for collagen and fibrin. In our opinion, a high concentration of fibronectin in the TOA zone leads to the increased formation of connective tissue in the focus of inflammation and changes the histological structure of the fallopian tube and ovary. Fragments of this glycoprotein are also one of the chemoattractants for macrophages migrating to the focus of inflammation [11]. All the above-mentioned factors, as well as the presence of bacterial biofilms, seem to constantly support the source of inflammation.

According to our data, the volume density of connective tissue in the TOA wall was 51.2 (3.7)%, and the content of muscle tissue was 6.09 (0.05)%, which was 8.5 times less than the same indicator in the control group. These signs are the objective evidence of loss of tubal and ovarian function in chronic purulent adnexal inflammation. The thickness of the walls of small arteries significantly increased due to the deposition of periodic acid-Schiff -positive substances in them and proliferation of connective tissue fibers. The walls were 12.5 (4.3) microns thick in the patients of the main group versus 6.6 (0.5) microns thick in the control group. When some of the tissue structures associated with such vessels were increased and their walls were thickened, the vessels were unable to provide normal tissue exchange. In our opinion, this is one of the mechanisms for maintaining the inflammatory process.

The impregnation of nerve fibers of the fallopian tubes in the patients of the control group revealed separate thin nerve fibers which were slightly twisted. The nerve fibers of the patients in the main group were characterized by uneven thickness, bulbous thickening, fragmentation and disintegration, which could cause a decrease in pain syndrome and non-apparent clinical picture in a number of cases. All these factors might result in delayed medical treatment and late diagnosis.

It is obvious that normalization of the properties of damaged receptors, conductors, synapses and neurons during the inflammatory process is an important condition for restoring integrity of the tissue. Damage to the peripheral nervous system, defragmentation and breakdown of nerve fibers were accompanied by an irregular growth of connective tissue, which was confirmed by our morphological studies.

The cytokine levels which were obtained in the focus of purulent inflammation are consistent with the results of histological studies. In addition, the study of the exudate showed a marked change in protein metabolism. The content of total protein and globulin fraction in purulent exudate was almost the same as in blood serum, which is indicative of increased vascular permeability (Table). Plasma proteins leave the blood vessels mainly through the dilated interendothelial cell gaps in the venules. The decrease in albumin in the exudate of patients with TOA confirms the hypothesis that it is consumed in large quantities as a plastic material in the area of inflammation and is involved in the formation of a granulation inflammatory tissue. M.F. Mazurik (1984) proposed using the prognostic coefficient of the course of the wound process as the ratio of the total blood plasma protein to the protein of the wound discharge. Normally it is equal to 1.2–1.3 and it decreases if the patient’s general condition gets worse. When calculating our own data (the ratio of total plasma protein to exudate protein), we found out that this indicator in peritoneal fluid in healthy women was 2.45 (0.14), and it was 1.08 (0.07) in purulent inflammation, that is, 2.26 times lower.

It is noteworthy that besides surgical treatment, all patients underwent complex antibacterial (carbapenems, cephalosporins, semi-synthetic penicillins and other antibiotics), detoxification, nonspecific anti-inflammatory therapy, as well as physiotherapy in the postoperative period. All the women were discharged home in a satisfactory condition.

Thus, this study confirmed that processes of alteration, exudation and proliferation occur simultaneously in chronic inflammation. It can develop as a consequence of acute inflammation, it can run asymptomatically and result in late diagnosis and treatment [12, 13]. The results of our study suggest that the local inflammatory response, as a protective one, is not quite effective in cases of TOA, since the relationship between damage, inflammation, regeneration and fibrosis is distorted. The effect of proinflammatory cytokines IL-1, TNF-α leads to the activation of fibroblasts, smooth myocytes and the endothelium in the focus of inflammation. Their levels in the blood serum and peritoneal fluid are significant criteria for determining the severity of the clinical course of the disease. The analysis reflects the depth of pathological destructive changes in the focus of inflammation and the proposed tests may help to look through the “open window into the mesenchyme” [14].

Conclusion

Cytokines play an important role in the development of inflammation, and their diagnostic value increases when they are determined directly in the focus of inflammation. The obtained results indicate that patients with TOA showed an increase in the production of pro-inflammatory cytokines, along with a decrease in the production of anti-inflammatory ones. Such changes can eventually lead to the development of complications and loss of function of the appendages.

Activation of cells, increased production of pro-inflammatory cytokines (IL-1, IL-6, TNF-α, etc.) are necessary in the initial phases of inflammation. However, they become problematic if the degree of activation ceases to be adequate, when the initial protective mechanism develops into a pathological one, and that was confirmed by our morphological studies.

Moreover, the study of these indicators justifies the possibility of choosing pathogenetic therapy, namely, timely surgical treatment, which can lead to normalization of intercellular relationships of the immune system.