| Sažetak | Endometrioza je česta kronična, složena i progresivna ginekološka bolest koja
najčešće zahvaća žene reproduktivne dobi. Definirana je nazočnošću i rastom ektopičnog
endometrija koji ima histološke značajke normalnog endometrija. Postavljanje dijagnoze
endometrioze delikatno je i složeno. Zlatni standard za postavljanje dijagnoze endometrioze
danas se smatra laparoskopija uz patohistološku potvrdu. Laparoskopija je invazivni
dijagnostički postupak, stoga je broj kliničkih studija usredotočen na otkrivanje neinvazivnih
dijagnostičkih postupaka koji bi omogućili postavljanje dijagnoze. Glavni cilj ovog
istraživanja bio je ispitati može li određivanje koncentracija vaskularnoga endotelnog
čimbenika rasta (VEGF), neuropilina 1 i aneksina A2 u serumu pridonijeti dijagnostici
endometrioze. U istraživanju je ispitana i dijagnostičku učinkovitost serumskih koncentracija
VEGF-a, neuropilina 1 i aneksina A2 u razlikovanju zdravih od oboljelih od endometrioze.
U istraživanje je bilo uključeno 200 ženskih osoba, raspona životne dobi od 18 do 39
godina. Serumske koncentracije VEGF-a, neuropiina 1 i aneksina A2 određene su u 120
bolesnica s endometriozom i 80 zdravih ispitanica. Serumske koncentracije sva tri analita
određene su ELISA metodom (od engl. enzyme-linked immunosorbent assay).
Bolesnice s endometriozom imale su povišene koncentracije VEGF-a, neuropilina 1 i
aneksina A2. Koncentracije VEGF-a i aneksina A2 bile su veće u bolesnica koje su imale viši
stadij endometrioze i veći broj lokacija zahvaćenih endometriozom. VEGF je pozitivno
korelirao s aneksinom A2. Kod bolesnica s pozitivnom obiteljskom anamnezom razdoblje od
pojave simptoma do dijagnosticiranja bolesti bilo je kraće. Koncentracije VEGF-a i aneksina
A2 bile su veće u bolesnica s endometriozom u kojih je prisutan simptom bola. Rezultati ROC
krivulja sva tri analita upućuju na to da neuropilin 1 (AUC = 0,97 kod granične vrijednosti >
11 μg/L ) i aneksin A2 (AUC = 0,99 kod granične vrijednosti > 16 μg/L) pokazuju izvrsne,
dok VEGF (AUC = 0,89 kod granične vrijednosti > 0,225 μg/L) pokazuje vrlo dobre
sposobnosti razlikovanja zdravih od oboljelih od endometrioze. Model koji uključuje
neuropilin 1 i aneksin A2 pokazao je jako dobru dijagnostičku učinkovitost u predviđanju
endometrioze (AUC 0,99 (95 % CI = 0,97 – 1,00)). Predloženim bi se modelom moglo
ispravno odrediti prisutnost endometrioze u 98 % bolesnica.
Bolje razumijevanje molekularnih mehanizama endometrioze koji uključuju VEGF,
neuropilin 1 i aneksin A2 moglo bi pomoći u dijagnozi i pronalaženju novih terapijskih meta
za endometriozu. Dijagnostičke karakteristike predloženog modela koji kombinira serumske
koncentracije VEGF-a, neuropilina 1 i aneksina A2 upućuju na to da bi se on mogao koristiti
kao vrijedan neinvazivan pristup u razlikovanju zdravih ispitanika od bolesnika s
endometriozom. |
| Sažetak (engleski) | Background: Endometriosis is a common chronic, complex, and progressive
gynecological disease which mainly affects women of reproductive age. It is defined by the
presence and growth of ectopic endometrial tissue which has the histological characteristics of
the normal endometrium. Researchers agree endometriosis is likely to be polygenic and
multifactorial, but the exact pathogenic mechanisms are still unclear. Each theory singularly
fails to account for all forms of endometriosis, thereby indicating multifactorial mechanisms.
Development can be divided into five basic processes: adhesion, invasion, recruiting,
angiogenesis, and proliferation. Genetics, biomolecular aberrations in the eutopic
endometrium, dysfunctional immune response, peritoneal environment may all ultimately be
involved. The most common symptoms are dysmenorrhea, dyspareunia, and lower back pain
that worsens during menses. Depending on the location of the implants, rectal pain and
painful defecation may also occur. The diagnosis of endometriosis should be considered
especially if a patient develops dysmenorrhea after years of pain-free menstrual cycles.
Infertility may also be the presenting complaint. Clinically, endometriosis should be
considered in any woman of reproductive age with pelvic pain or infertility. However, since
symptoms are often nonspecific, the disease may be misdiagnosed as other clinical conditions
characterized by chronic pelvic pain (irritable bowel syndrome, interstitial cystitis/painful
bladder syndrome, recurrent cystitisoveractive bladder), thus leading to inadequate treatment
and considerable diagnostic delay. The diagnosis of endometriosis is delicate and complex,
the ‘gold standard’ for a definitive diagnosis is laparoscopy with biopsy and histological
analysis. Laparoscopy is the invasive diagnostic procedure, therefore, huge number of clinical
studies have been focused on detection of non-invasive diagnostic procedures that would
facilitate the diagnosis.
Aim: The main aim of this study was to examine whether the determination of
vascular endothelial growth factor (VEGF), Neuropilin 1 and Annexin A2 concentrations in
serum may contribute to the diagnosis of endometriosis. The diagnostic accuracy of VEGF,
Neuropilin 1 and Annexin A2 in distinguishing between healthy subjects and patients with
endometriosis was also evaluated.
Participants and methods: A total of 200 women aged 18–39 years were included in
the results of this study. Preoperative serum samples were obtained after written informed
consent from 200 candidates for laparoscopic surgery due sterilisation and suspected
endometriosis based on ultrasound vaginal probe, clinical examination, infertility, medical
history. Our exclusion criteria for all groups were as follows: pregnancy before surgery or 6
months after surgery, active pelvic inflammatory disease, any anti-inflammatory or hormonal
or immunomodulatory medications use in the preceding 3 months, genital or any extragenital
malignant disease, polycystic ovaries, cysts, adenomyosis, fibroids, other defined causes of
infertility, use of oral contraceptives or gonadotropin-releasing hormone agonist (GnRH)
analogues, chronic or acute genital disease except endometriosis, history of any medical
problem (e.g., endocrine abnormalities, gastrointestinal, cardiovascular, and pulmonary
system diseases), any systemic diseases (e.g., chronic or acute inflammatory disease,
autoimmune disorders, hepatic or renal insufficiency). Candidates were divided into groups
(endometriosis and control). Endometriosis group was comprised of 120 patients with
endometriosis diagnosed by laparoscopy or laparotomy and confirmed by histopathological
examination. Finally, endometriosis group comprised 120 patients divided into 3 groups; the
first group (stage II endometriosis group) comprised 50 patients; while the second group
(stage III endometriosis group) comprised 62 patients and the third group comprised 8
patients diagnosed with stage IV endometriosis, respectively, according to rASRM
classification. Eighty healthy women who were undergoing laparoscopy because of
sterilization were the control group. Serum concentrations of VEGF, Neuropilin 1 and
Annexin A2 were determined in 120 patients with endometriosis and 80 healthy subjects.
Blood samples were taken from all women undergoing laparoscopy half an hour before the
induction of anesthesia, for the collection of serum. Five mililitres of venous blood were
aseptically collected and left to coagulate, after which samples were centrifuged for 15 min at
2000 g and the serum was separated in a sterile tube. One test tube for all subjects (for VEGF
and ANNXA2) was centrifuged after 30 minutes, and the NRP1 tube was centrifuged two
hours after were aseptically collected. Serum concentrations of all three analytes were assayed
by a standardised sandwich enzyme-linked immunosorbent assay (ELISA).
Results: Higher VEGF, Neuropilin 1 and Annexin A2 concentrations were found in
patients with endometriosis. Significant differences were detected between control (VEGF=
0,214 (0,201 – 0,230) μg/L, p<0.0001; NRP1 = 8.2 (7.3 – 9.4) μg/L, p<0.0001; ANXA2 =
12,4 (10,2 – 13,4) μg/L, p<0.0001) and endometriosis groups (VEGF= 0,254 (0,231 – 0,268)
μg/L, p<0.0001; NRP1 = 27,1 (25,6 – 28,1) μg/L, p<0.0001; ANXA2 = 29,1 (21,3 – 35,6)
μg/L, p<0.0001) in terms of VEGF, NRP1 and ANXA2 levels. VEGF and Annexin A2
concentrations showed a statistically significant difference depending on the severity of the
disease and a number of locations affected by endometriosis. There were no significant
correlations between VEGF anf Neuropilin 1 also between Neuropilin 1 and Annexin A2 in
patients with endometriosis. Annexin A2 were positively associated with VEGF (r=0,73;
p<0,001) in patients with endometriosis. Patients with a positive family history had a shorter
time period in diagnosing the disease. VEGF and Annexin A2 concentrations were higher in
patients with endometriosis in whom a symptom of pain was present. The results of the ROC
curves of all three analytes indicate that Neuropilin 1 (AUC = 0.97 at the cut-off value > 11
μg / L) and Annexin A2 (AUC = 0.99 at the cut-off value > 16 μg / L) showed excellent,
while VEGF (AUC = 0.89 at a cut-off value > 0.225 μg / L) showed very good ability to
distinguish between healthy and endometriosis patients. The model combining Neuropilin 1
and Annexin A2 demonstrated diagnostic performances (AUC (95%CI) = 0,96 (0,92 – 0,99)).
The proposed model correctly identifies 98 % of patients with endometriosis.
Conclusions: In complex diseases such as endometriosis, due to the complexity of the
pathophysiological background, it is unlikely that a single marker will reflect the changes and
predict the outcome of the disease. A diagnostically non-invasive marker of endometriosis is
needed to make a diagnosis, guide targeted treatment, and monitor activity and therapeutic
response at all stages of the disease. Since it cannot yet be concluded that all
pathophysiological processes in the development of endometriosis are known, markers need
constant reevaluation and retrospective studies to justify their use. Improved understanding of
the molecular mechanisms of endometriosis pathogenesis involving VEGF, Neuropilin 1 and
Annexin A2 could help identifying novel targets for diagnosis and therapeutic interventions in
endometriosis. Diagnostic characteristics of the proposed model, obtained by combining
serum concentrations of VEGF, Neuropilin 1 and Annexin A2 suggested potential value as a
noninvasive approach in endometriosis diagnosis. |
