Czy istnieje możliwość diagnostyki endometriozy z poziomu endometrium ? Is it possible to diagnose endometriosis at the level of endometrium ?

Maria Szubert1, Magdalena Ziętara2, Jacek Suzin1 Received: 25.03.2016 Accepted: 15.04.2016 Published: 29.04.2016 © Curr Gynecol Oncol 2016, 14 (1), p. 30–38 DOI: 10.15557/CGO.2016.0004 Czy istnieje możliwość diagnostyki endometriozy z poziomu endometrium? Is it possible to diagnose endometriosis at the level of endometrium?


E
ndometriosis is defined as the presence of a tissue similar to a glandular one and endometrial stroma outside the uterine cavity -mostly in the ovaries, uterine tubes, uterosacral ligaments, broad ligament, round ligament and Douglas cavity.The disease is also found in the large intestine, ureters, urinary bladder and rectovaginal septum (1) .Endometriosis is observed with 7-10% of women at a reproductive age.Roughly 60% of patients struggle against a pain syndrome, and ca.50% -infertility (2) .Moreover, as many as 20% of women with endometriosis is also diagnosed with irritable bowel syndrome, urinary bladder inflammation, fibromyalgia and migraine (1) .Researchers are unanimous in that endometriosis is promoted by conditions associated with hyperestrogenism, and the ectopic location of the endometrium induces an immunological reaction.Risk factors that may affect the development and progression of the disease involve: e.g.Caucasian race, age of 25-35, daily alcohol consumption of ca. 10 g, positive history towards the presence of endometriosis in the patient's mother, early menarche (below 11 years of age) and short menstrual cycles (under 25 days) with heavy bleeding (3) .What is more, the studies point to the fact that environmental factors -chronic exposition to formaldehyde, nickel or benzene, tobacco smoking, coffee drinking, consumption of food containing hormones, exposition to magnetic radiation from microwave ovens, contact with pesticides -as well contribute to the development of the disease (4)(5)(6) .Though endometriosis is a disease whose pathogenesis has already been sought after since the 19 th century and which is characterized by a high frequency, the mechanisms of its genesis remain unexplained.The surprising thing is that a constantly growing number of studies on pathogenesis, epidemiology and diagnostics fail to facilitate a better patogenezą, epidemiologią i diagnostyką nie przekłada się na lepsze poznanie endometriozy, a diagnoza, stawiana najczęściej przez sukcesywne wykluczanie innych jednostek chorobowych, pojawia się zbyt późno, już po znacznej progresji choroby (1,7) .Istnieją liczne teorie, w większości przestarzałe, które próbują tłumaczyć etiologię schorzenia, m.in.teoria wstecznej transplantacji Sampsona, teoria metaplazji nabłonka Waldeyera, teoria Halbana (szerzenia się endometriozy drogą krwiopochodną i limfopochodną) i teoria powstawania choroby z pozostałości przewodów Müllera, jednak żadna z nich nie tłumaczy dokładnie wszystkich aspektów patogenezy (8) .W ostatnich latach zwraca się uwagę na znaczenie zmian genetycznych (9) .Należy podkreślić ważną rolę diagnostyki w endometriozie.Według stanowiska Zespołu Ekspertów Polskiego Towarzystwa Ginekologicznego (PTG) na temat diagnostyki i leczenia endometriozy nieinwazyjnie chorobę rozpoznaje się na podstawie objawów klinicznych, palpacyjnego understanding of endometriosis.Still, the diagnosis, most often based on successive exclusion of other disease entities, is made too late, already after a major progression of the disease (1,7) .There exist numerous theories, most of them outdated, that strive to explain the etiology of the disease, i.a.Sampson's theory of retrograde transplantation, Waldeyer's theory of epithelial metaplasia, Halban's theory (of spreading of endometriosis in a blood-and lymph-derived manner) and a theory of disease formation from the remains of Müllerian ducts.However, none of these precisely explains all aspects of the pathogenesis (8) .In recent years researchers have paid a special attention to the significance of genetic changes (9) .The role of diagnostics in endometriosis should not be underestimated.In terms of diagnostics and non-invasive treatment of endometriosis, the Team of Experts at the Polish Gynaecological Society (Polskie Towarzystwo Ginekologiczne, PTG) claims that the disease is Tab. 1. Skala Biberoglu-Behrmana (11) (tłumaczenie własne) Tab. 1. Biberoglu-Behrman Score (11) badania ginekologicznego i badań obrazowych: ultrasonografii przez pochwowej i przezodbytniczej, a niekiedy rezonansu magnetycznego miednicy mniejszej (10) .Oceny narządu rodnego w badaniu dwuręcznym zestawionym można dokonać opisowo lub za pomocą skali Biberoglu-Behrmana, używanej jako narządzie obiektywizacji w większości badań i prób klinicznych (tab. 1) (11) .Zgodnie z wytycznymi European Society of Human Reproduction and Embryology (ESHRE) z 2005 roku, zmodyfikowanymi i opublikowanymi w roku 2014, badanie ultrasonograficzne miednicy mniejszej nie ma wartości diagnostycznej w odniesieniu do endometriozy otrzewnowej (poziom wiarygodności danych: A).Na podstawie systematycznego przeglądu literatury eksperci stwierdzili, że badanie ultrasonograficzne wykonywane głowicą przezpochwową lub przezodbytniczą może być narzędziem pomocnym w rozpoznawaniu torbieli endometrialnych recognized on the basis of clinic symptoms, palpable gynecologic tests and imaging tests: transvaginal and transrectal ultrasound, and sometimes magnetic resonance of the pelvis minor (10) .The evaluation of the reproductive organ in a bimanual examination can be made in a descriptive manner or by means of Biberoglu-Behrman score, used as a tool of objectification in most clinical tests and trials (Tab. 1) (11) .
deep infiltrating endometriosis of the urinary bladder or the rectum (12,13) .The above mentioned PTG claims that a significant diagnostic aspect is an image observed during an invasive operative intervention, confirmed by a histopathological test of endometrial specimens (10) .However, the indications for performing a diagnostic treatment should be carefully identified, as laparoscopy, though described as a minimally invasive procedure, involves a risk of complications (major and minor) assessed at 8.9%.
The indications for laparotomy are generally made in the case of advanced endometriosis -in patients who do not react to conservative treatment and in whose case the ultimate form of treatment leads to the removal of ovaries and the uterus (14,15) .Therefore, it currently becomes unquestionable to claim that only by means of an operation it is possible to diagnose endometriosis in a reliable way.
Often the patients are made to wait for years so that the cause of their discomfort can adequately be recognized (1) .Consequently, many researchers strive to diagnose endometriosis with the use of non-invasively available biological materials: plasma, serum, endometrium bioptates, and urine.However, these studies are limited in terms of small groups of patients, which translates into a low quality of obtained data (16,17) .

DIAGNOSING ENDOMETRIOSIS AT THE LEVEL OF THE ENDOMETRIUM
A marker specific to endometriosis has not been identified yet.Scientists are currently searching for markers that in a specific combination would ensure maximum sensitivity and specificity of a non-invasive detection of endometriosis, even at early stages (18,19) .As neither serum nor plasma are a good diagnostic material in case of peritoneal endometriosis, the studies were shifted to the tissue of eutopic endometrium.This tissue is easily accessible due to its sampling without anesthesia, by means of endometrial biopsy.It was proven that changes in gene and protein expression specific to ectopic foci of the disease can also be found in the endometrium in the uterine cavity (20,21) .The assessment of these processes has recently fallen within the scope of numerous analyses and papers.Paraphrasing the researchers Liu and Lang, there arises a question if the pathogenic pathway looks as follows: initially changed endometrial cellsadhesion -invasion -angiogenesis -endometriosis foci in the peritoneal cavity.If so, such an ideal, minimally invasive diagnostic test should be based on markings made in endometrial bioptates (22) .This article focuses on analyzing four factors: interleukin 8 (IL-8), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and nerve growth factor (NGF) that could be the subject of diagnostic tests.Their significance in endometriosis was partly proven.In an analysis of a number of potential biomarkers for endometriosis, May et al. (23) distinguished the above mentioned as significant.The clinic, which the authors of this paper are Ich znaczenie w endometriozie zostało częściowo udowodnione.May i wsp. (23)podczas analizy szeregu potencjalnych biomarkerów dla endometriozy wyróżnili te wyżej wspomniane jako istotne.W klinice, w której pracują autorzy niniejszej pracy, prowadzony jest projekt badawczy dotyczący wykorzystania wymienionych czynników w diagnostyce endometriozy.

Interleukin 8 (IL-8)
IL-8 is a cytokine responsible for inducing neutrophil chemotaxis, it has highly angiogenic properties and stimulates the proliferation of various types of the body's cells (24) .It is beyond any doubt that it is one of the most specific cytokines connected with the pathogenesis of endometriosis (interleukin 6 is another specific cytokine of this kind) (25) .
There is evidence that in the endometrium of patients with endometriosis, the levels of mRNA and protein IL-8 are significantly elevated at early stages of hyperplasia and in late secretory phases, as compared to such levels in the mid-cycle.Additionally, some reports claim that IL-8 has an impact on the growing autocrine factors in the endometrium.What is more, there exists a tendency for a higher expression of IL-8 receptor in the endometrium in patients with endometriosis in comparison with a group of healthy women (24) .The observations made by Ulukus et al. prove that IL-8 and its receptors CXCR1 and CXCR2, present in the endometrium, have a significant impact on the pathogenesis of the disease (26) .As observed, IL-8 excites the adhesion of endometrial cells to fibronectin.This may influence the mechanism of a vicious circle of endometrial cell adhesion, cell growth and further secretion of this cytokine (24) .

Vascular endothelial growth factor (VEGF)
VEGF is one of the most important angiogenic factors.
It is responsible for the course of physiological angiogenesis and the one that occurs in pathological conditions.During a menstrual cycle angiogenesis facilitates the regeneration of the endometrium and formation of the corpus luteum, whereas at the early stage of pregnancy it is crucial in embryogenesis (27) .Scientists suggest that VEGF plays a significant role in the process of angiogenesis in the development of endometriosis.It is also believed that the ectopic glandular endothelium in patients with endometriosis has a huge potential for forming new vessels.This is due to a high level of VEGF in a late secretory phase of the cycle in comparison with healthy women (28) .VEGF binds with one of the receptors for tyrosine kinase -Flt (fms-like tyrosine kinase) and KDR (kinase domain receptor) that are found in endothelial cells.Their VEGF-induced activation leads to growing cell proliferation, cell membrane permeability and the release of proteolytic enzymes engaged in the process of angiogenesis.

Czynnik wzrostu nerwów (NGF)
NGF to główny biomarker odpowiedzialny za promowanie wzrostu włókien nerwowych, a zarazem czynnik inicjujący i podtrzymujący ból w różnych chorobach (36) .Niewątpliwie ma też znaczący wpływ na żeński układ rozrodczy.Udowodniono, że czynnik wzrostu nerwów wpływa na wiele procesów zachodzących w jajniku: dojrzewanie pęcherzyka, sekrecję steroidów, owulację i utrzymanie ciąży przez a significant expression of mRNA for VEGF in the cells of eutopic endometrium, confirm that in a group of women with endometriosis, this expression is significantly higher (30) .Still, Cho et al. (31) prove that in the peritoneal fluid the expression of VEGF and its soluble receptor (sFlt-1) is significantly higher in patients with endometriosis, as compared to the control group.The expression of VEGF and a soluble receptor sFlt-1 in the peritoneal tissue was elevated as well, whereas in the endometrial tissue sFlt-1 expression appeared to be significantly lower in patients with endometriosis.
As the above studies prove, the pathogenesis of endometriosis is affected not only by an abnormal expression of angiogenic factors.The development of the disease may also involve a disturbed expression of anti-angiogenic factors in the peritoneal fluid and in the endometrium.

Platelet-derived growth factor (PDGF)
PDGF is a well-known factor responsible for inducing proliferation of many types of cells (32) .It is synthesized not only by blood platelets but also by endothelial cells, fibroblasts and smooth muscle cells.PDGF, whose expression takes place in the normal endometrium, is a mitogenic factor for endometrial cells.Thanks to this, these cells are activated for proliferation (28) .Immunohistochemical studies confirm that PDGF is present in the uterine mucous membrane, whereas the staining intensity of its receptor, PDGFR-β, grows in the secretory phase of the cycle.Moreover, in vitro studies prove that in endometriosis PDGF plays a significant role in the stromal cells proliferation.It led to the hypothesis that the process of angiogenesis in endometriosis does not solely depend on VEGF.It may also be triggered by a mutual interaction between all three growth factors (the third being the fibroblast growth factor -FGF) (33) .Moreover, PDGF is described as a key factor for the pathogenesis of endometriosis, being a product of secreting macrophages in the phase of endometrial cell proliferation (34) .The concept of PDGF expression in the endometrium of women with endometriosis in the first phase of the cycle was for the first time presented by the authors of this paper.It is worth mentioning that in the eutopic endometrium of the patients, PDGF is practically untraceable (35) .
this factor in the uterus is regulated by progesterone.NGF takes part in the pathogenesis of breast cancer and epithelial ovarian cancer.Moreover, it is assumed to affect the development of polycystic ovary syndrome and endometriosis (37) .The main symptom of the described condition, namely pain, accompanies ca.60% of women with endometriosis.NGF plays here an important role in the formation and sustention of the disease.This is particularly crucial in the case of the most symptomatic form of the diseasedeep infiltrating endometriosis affecting lymph nodes and nerve fibers (38) .What is more, deep changes in endometriosis have very rich innervation and a stronger NGF expression, as compared to superficial changes.It should be emphasized that among deep infiltrating changes these are rectal changes that are characterized by the strongest innervation (39) .

SUMMARY
Endometriosis is observed in 7-10% of women at a reproductive age.Roughly half of these patients struggle with two main symptoms: pain syndrome and infertility.In many cases the diagnosis is made too late, although the changes begin already in early years of life.Not until women develop chronic pelvic pains that intensify during a cycle do they turn for the doctor's help due to infertility, dysmenorrhea, dyspareunia and tenderness in the pelvic area.This is undoubtedly influenced by the lack of non-invasive diagnostic methods, whereas delays in diagnostics often lead to significant consequences, i.e. disease progression.Searching for a marker in the endometrium is difficult as the conducted studies are not uniform in the case of different groups of patients and phases of cycles, where researchers analyze the concentration of angiogenic factors.Therefore, the main challenge is currently to detect the most responsible, minimally invasive method of diagnosing endometriosis, which shall bring great benefits both to the doctors as well as patients.According to current reports, such a diagnostic test -as a gold standard -should be based on markings performed in endometrial bioptates.