Non-invasive prediction of endometriosis revisited; 3 biomarkers as Angiopoietin-2, Interleukin-1β and Vascular Endothelial Growth Factor

doi:10.4236/ojog.2013.37095

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Open Journal of Obstetrics and Gynecology, 2013, 3, 528-535 OJOG

http://dx.doi.org/10.4236/ojog.2013.37095 Published Online September 2013 (http://www.scirp.org/journal/ojog/)

Non-invasive prediction of endometriosis revisited;

3 biomarkers as Angiopoietin-2, Interleukin-1β and

Vascular Endothelial Growth Factor

Hoda Abdel Moety1*, Gihan I. Khalil1, Rania M. El Sharkawy1, Marwa K. El Ghandour1,

Fady S. Moiety2, Hesham A. F. Salem2

1Department of Chemical Pathology, Medical Research Institute, Alexandria University, Alexandria, Egypt

2Department of Obstetrics and Gynecology, Faculty of Medicine, Alexandria University, Alexandria, Egypt

Email: *[email protected]

Received 2 July 2013; revised 1 August 2013; accepted 9 August 2013

cense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ABSTRACT

Introduction: Endometriosis affects up to 1 every 5

women at their reproductive age, with variable and

complex symptomatology. Patients may be asympto-

matic but may have pain episodes or subfertility. Its

negative impact is on patients’ health and quality of

life. Objective: it was to investigate the serum and

peritoneal fluid (PF) concentrations of Angiopoietin-

2, Interleukin-1β, and Vascular Endothelial Growth

Factor, aiming to evaluate their diagnostic perform-

ance in endometriosis. Methods: Serum and perito-

neal fluid samples were taken from 112 women un-

dergoing laparoscopy for infertility, pelvic pain or

adnexal masses. 61 diagnosed with endometriosis and

51 controlled. Primary outcome was to estimate se-

rum and PF concentrations of Angio-2, IL-1β and

VEGF and secondarily correlate these concentrations

to disease stages thus assuming their diagnostic po-

tential. Results: Significant differences were found

between patients and control as regards serum and

PF concentration of all studied markers except serum

IL-1β. Serum Angio-2 and PF VEGF showed a sig-

nificantly higher level in more advanced stages of en-

dometriosis. PF VEGF showed a positively significant

correlation with the stage of the disease, spearman

coefficient t = 0.442 p = 0.014. PF concentrations of

Angio-2 and Serum VEGF did not show significant

pattern changes with stage-related levels. Diagnostic

potential of serum and PF concentrations of the 3

markers were assessed by the ROC curve. Angio-2

proved an excellent diagnostic ability for endometrio-

sis. PF and serum VEGF proved an equal diagnostic

performance, whereas, PF IL-1β was the least effi-

cient. Based on the results, we suggested preliminary

serum threshold values for these markers to be used

as diagnostic or follow-up landmarks with relatively

acceptable sensitivity, specificity, positive and nega-

tive predictive values. Conclusion: Non-invasive pre-

dictive biomarkers for endometriosis were Serum

Angio-2, IL-1β, and VEGF independently or in com-

bination with the estimated threshold values. Serum

Angio-2 merit is considered as a novel marker for en-

dometriosis due to its diagnostic power.

Keywords: Endometriosis; Angiopoietin-2; VEGF;

IL-1β

1. INTRODUCTION

Endometriosis affects up to 1 every 5 women at their

reproductive age group, with variable and often complex

symptomatology. Patients may be asymptomatic but may

have pain episodes, and/or subfertility [1]. Unlike its ae-

tiopathogenesis, its negative impact is on patients’ health

and quality of life is clear [2,3]. The stage of endome-

triosis can be morphologically classified by using the

revised American Fertility Society (rAFS) staging system

according to the localization and the size of nodes in the

peritoneum and ovaries, and the presence of adhesions in

the ovary and the fallopian tube [3]. This rAFS classifi-

cation includes the following stages: I = Minimal (1 - 5

points); II = Mild (6 - 15 points); III = Moderate (16 - 40

points); IV = Severe (<40 points) [4].

Effective treatment could be achieved; however, di-

agnosis of the disease remains a problem, simply, be-

cause accurate diagnosis can only be attained via laparo-

scopic inspection, preferably with histopathologic con-

firmation of suspected lesions. This remains an invasive

technique, with possible complications and health care

*Corresponding author.

OPEN ACCESS

H. A. Moety et al. / Open Journal of Obstetrics and Gynecology 3 (2013) 528-535 529

costs [5]. However, ovarian endometriomas, and deep

infiltrating endometriotic nodules can be detected ade-

quately by vaginal ultrasound, but it does not rule out

peritoneal endometriosis or endometriosis-associated ad-

hesions [1]. This situation has led the researchers re-

cently to refocus on the possible mechanism by which

endometriosis occurs. The fact is that an altered immune

mechanism might be involved in the development of the

condition is now widely accepted, which explains why

some women are more prone than others to develop the

disease. The concentration of some immunological mar-

kers in the peritoneal fluid largely correlates with the

disease occurrence, progression, and symptoms [6].

There is substantial evidence that immunologic factors

play a role in the pathogenesis of endometriosis and en-

dometriosis-associated infertility. Decreased natural kil-

ler cell cytotoxicity leads to an increased likelihood of

implantation of endometriotic tissue and modulation of

growth and inflammatory behavior of ectopic endo-

metrial implants.

The alteration in cytokines level and other angiogenic

factors as Vascular Endothelial Growth Factor (VEGF)

may explain the adhesion, implantation and the progres-

sion of the transported fragment of endometrium in the

different site of endometriosis. Many cytokines have

been studied to prove the immunological theory of en-

dometriosis as Interleukin-1β (IL-1β). It has been gener-

ally accepted that the establishment of new biomarker as

angiopoietin-2 (Angio-2) played a key part in the pro-

gression of endometriosis, however this role still needs

further confirmation to suggest a relation to endometrio-

sis already proven for many pivotal angiogenesis stimu-

lators such as VEGF [7,8].

Setting a non-invasive biochemical diagnostic and

prognostic test for endometriosis with an acceptable level

of accuracy and sensitivity, would fundamentally change

those patients’ quality of life, save their costs and risks of

the more invasive tools, and eventually make it smoother

for health care providers to manage the condition with

ease.

The current study was designed to evaluate the serum

and peritoneal fluid concentrations of Angiopoietin-2,

Interleukin-1β, and Vascular Endothelial Growth Factor

in patients with endometriosis as well as controls, thus

adding some insight to the formulation of a non-invasive

test to screen, detect, or follow up patients with endome-

triosis.

The ultimate objective was to prove any of these mar-

kers independently or in combination as non-invasive

diagnostic biomarker in patients with endometriosis.

2. PATIENTS AND METHODS

Blood and (PF) samples were taken from 112 women

undergoing laparoscopy for infertility, pelvic pain, and/or

adnexal masses in their first half of the menstrual cycle.

Procedures were done at a specialized unit of Gyneco-

logic endoscopy at a University hospital.

Sixty one were histologically confirmed to have en-

dometriosis and 51 were controls. No cases were ex-

cluded. A full informed consent was taken from each

subject. Enrolment took place after formal approval of

the Ethical committee of the Faculty of Medicine, Alex-

andria University was obtained.

PF samples were collected by aspiration from the per-

itoneal cavity during laparoscopy.

Serum samples: Five ml of venous blood were asepti-

cally collected and the clear serum was stored at –70˚C

until analysis.

Measurement of IL-1β, VEGF, and Angio-2 in serum

and PF were done using a commercially available, en-

zyme-linked immunosorbent assays (ELISA) using the

reagents and protocol supplied with the ELISA kit (R&D

Systems, Minneapolis, Minnesota, United States) Frozen

serum and PF samples were thawed and then analyzed.

3. STATISTICAL ANALYSIS

Data distribution was analyzed by using the Statistical

Packages for the Social Sciences (SPSS version 10.0,

Chicago, IL) and the Sigma-Stat software package (ver-

sion 3.5; SPSS, Chicago, IL). Normally distributed data

were presented as mean, standard deviation, whereas

skewed data were expressed as median and range. Pear-

son’s correlation was also used for evaluation of the lin-

ear relationship between different clinical variables. Pair-

wise comparisons between groups were performed using

the Wilcoxon-Mann-Whitney test. In order to determine

which serum marker could best represent patients with

endometriosis, sensitivity and specificity were assessed

with receiver operating characteristic ROC curves. The

area under the curve was approximately the percentage

correctly classified if the test was used as a diagnostic

tool. The same analyses were performed with the PF

measures and statistical significance was assessed using

two-tailed tests and an alpha level of p < 0.05.

4. RESULTS

A total of 112 women underwent laparoscopy for infer-

tility (n = 49), pelvic pain (n = 22), and/or adnexal

masses (n = 41). Sixty one were diagnosed with endome-

triosis and 51 were controls. No cases were excluded.

Out of the 61 patients with endometriosis, 25 (40.98%)

had early disease (stages I and II) and 36 (59.02%) had

late disease (stages III and IV).

The median age of the study group was 33.3 years,

median body mass index (BMI) was 27.0, and median

parity was 1.

No significant differences were found as regards age,

parity and BMI among the studied groups.

H. A. Moety et al. / Open Journal of Obstetrics and Gynecology 3 (2013) 528-535

530

OPEN ACCESS

5. CLINICAL DATA

Patients were presented with pain, discharge, and/or in-

fertility, those who presented with the latter were at ad-

vanced stages only (Table 1).

A significant difference was found between patients

and controls’ serum and PF concentration of all studied

markers except serum IL-1β (Table 2).

Meanwhile, significant difference was elicited between

early and late stages in terms of serum Angio-2 and PF

VEGF concentrations with significant increase in more

advanced stages (Z, p = 3.311 (0.042*), 2.188* 0.034

respectively), but the other studied markers with no spe-

cific pattern of stage-related level changes (Table 3).

Table 1. The relation between the serum and peritoneal fluid concentration of the 3 biomarkers and the stage of endometriosis among

women.

Pain Discharge Infertility

(pg/ml) Early Late Early Late Early Late

Serum Angio-2

Min - Max 3400 - 5320 3800 - 6240 3735 - 4300 3900 - 6240 - 3635 - 6530

Mean ± SD 4278.5 ± 618.6 4341.3 ± 692.1 5401.5 ± 399.5 4653.8 ± 1090.5 - 4302.5 ± 550.5

Median 4115.00 4230.00 4017.50 4237.50 - 4280.00

Z (p) 0.272 (0.786) 0.355 (0.533) -

PF Angio-2

Min - Max 2980 - 3990 3120 - 9000 2980 - 3990 3280 - 4840 - 3120 - 9500

Mean ± SD 3524.2 ± 372.4 4653.9 ± 2128.3 3575.0 ± 435.5 4060.0 ± 1103.1 - 4524.6 ± 1969.2

Median 3605.00 3805.00 3665.00 3842.50 - 4060.00

Z (p) 1.414 (0.157) 0.643 (0.800) -

Serum VEGF

Min - Max 600 - 3600 800 - 3750 650 - 1950 1210 - 3540 - 600 - 4020

Mean ± SD 1975.5 ± 971.2 2229.2 ± 961.3 1300.0 ± 919.2 2512.5 ± 1005.8 - 2060.1 ± 1128.8

Median 2008.00 2150.00 1300.00 2650.00 - 2054.00

Z (p) 0.571 (0.568) 0.165 (0.267) -

PF VEGF

Min - Max 650 - 3600 1100 - 14000 850 - 3600 2400 - 3900 - 1100 - 14000

Mean ± SD 2047.5 ± 1059.6 4233.1 ± 3967.8 2187.5 ± 1141.9 3150.0 ± 1060.7 - 4596.7 ± 4029.3

Median 2375.00 3000.00 2150.00 3150.00 - 3085.00

Z (p) 1.687 (0.092) 0.240 (0.267) -

Serum IL-1β

Min - Max 33 - 254 56 - 260 65 - 201 198 - 215 - 33 - 254

Mean ± SD 138.9 ± 74.5 147.8 ± 65.6 143.5 ± 62.2 206.5 ± 12.0 - 142.2 ± 71.0

Median 122.00 142.00 154.00 206.50 - 132.50

Z (p) 0.381 (0.703) 0.165 (0.267) -

PF IL-1β

Min - Max 39 - 456 46 - 490 101 - 355 278 - 378 - 39 - 480

Mean ± SD 238.9 ± 126.2 289.3 ± 131.9 257.5 ± 114.2 328.0 ± 70.7 - 293.9 ± 147.4

Median 245.00 316.50 287.00 328.00 - 331.50

Z (p) 1.142 (0.253) 0.355 (0.533) -

Z: Z for Mann Whitney test. *Significant at p < 0.05.

H. A. Moety et al. / Open Journal of Obstetrics and Gynecology 3 (2013) 528-535 531

Table 2. The relation between endometriosis and serum and PF levels of the studied biomarkers.

Endometriosis

Cases n = 61 Control n = 51 Z (p)

Serum Angio II (pg/ml)

Min - Max 3400.00 - 6240.00 2566.00 - 5211.00

Mean ± SD 4307.83 ± 775.30 3765.30 ± 604.94

Median 4232.50 3642.50

2.794* (0.005)

PF Angio II (pg/ml)

Min - Max 2985.00 - 9500.00 2980.00 - 6169.00

Mean ± SD 4843.00 ± 1682.46 4211.83 ± 873.00

Median 4803.50 3700.00

3.622* (<0.001)

Serum VEGF (pg/ml)

Min - Max 600.00 - 4020.00 160.00 - 402.00

Mean ± SD 2158.37 ± 1026.87 286.83 ± 77.07

Median 2024.00 291.50

6.653* (<0.001)

PF VEGF (pg/ml)

Min - Max 650.00 - 14000.00 96.00 - 322.00

Mean ± SD 3174.67 ± 2857.60 214.30 ± 68.74

Median 2700.00 207.00

6.654* (<0.001)

Serum IL-1β (pg/ml)

Min - Max 33.00 - 260.00 25.00 - 240.00

Mean ± SD 142.73 ± 67.29 129.73 ± 66.24

Median 135.50 108.00

0.673 (0.501)

PF IL-1β (pg/ml)

Min - Max 39.00 - 490.00 35.00 - 302.00

Mean ± SD 277.63 ± 134.27 174.43 ± 84.04

Median 282.50 191.00

3.149* (0.002)

Z: Z for Mann Whitney test. *Statistically significant at p < 0.05.

In addition, a positive correlation of PF VEGF with

the stages of the disease was found, (spearman coeffi-

cient = 0.442*) (Table 4).

Non-invasive prediction of endometriosis and the di-

agnostic potential of the serum and PF concentrations of

the 3 markers were assessed by the Receiver-operating

characteristic (ROC) curves. Serum Angio-2 proved best

diagnostic performance of 100% with an outstanding

area under the curve AUC 1.000 followed by serum

VEGF AUC 0.920 and the PF AUC 0.810, then serum

IL-1β AUC 0.737 and PF IL-1β AUC 0.515 was the least

efficient.

Preliminary threshold values for the 3 studied markers

in serum were assumed to serve as diagnostic or fol-

low-up landmarks with relatively acceptable sensitivity,

specificity, positive and negative predictive values (Ta-

ble 5).

6. DISCUSSION

Our results confirmed that serum VEGF, Angio-2, and

IL-1β respectively have an excellent diagnostic ability to

screen cases with endometriosis with a good level of

sensitivity and specificity. This was not related to the

stage of the disease which makes these markers, prefera-

bly in combination, more suitable to construct a non-

surgical, screening system prototype rather than a staging

module. All samples were taken during the first half of

H. A. Moety et al. / Open Journal of Obstetrics and Gynecology 3 (2013) 528-535

532

Table 3. Comparison between the two stages of endometriosis according to serum and PF of the 3 biomarkers.

Stages of e n dometriosis

Early Late Z (p)

Serum Angio-2 (pg/ml)

Min - Max 3400 - 5320 3800 - 6240

Mean ± SD 4285.6 ± 559.5 4341.3 ± 692.1

Median 4115.00 4550.00

3.311 (0.042*)

PF Angio-2 (pg/ml)

Min-max 2980 - 3990 3120 - 9500

Mean ± SD 3524.2 ± 372.4 4670.3 ± 2045.2

Median 3605.00 3842.50

1.566 (0.117)

Serum VEGF (pg/ml)

Min - Max 600 - 3750 800 - 4020

Mean ± SD 2111.2 ± 109.31 2229.2 ± 961.3

Median 2024 2150

0.233 (0.816)

PF VEGF (pg/ml)

Min - Max 650 - 1400 1100 - 3600

Mean ± SD 2047.5 ± 1059.6 3926.1 ± 3421.8

Median 2375.00 3005.0

2.188 (0.034*)

Serum IL-1β (pg/ml)

Min - Max 33 - 254 56 - 260

Mean ± SD 139.4 ± 65.3 147.8 ± 68.6

Median 132.50 142.00

0.381 (0.703)

PF IL-1β (pg/ml)

Min - Max 39 - 480 46 - 490

Mean ± SD 269.8 ± 131.1 289.3 ± 139.9

Median 273.00 316.50

0.381 (0.703)

*Statistically significant at p < 0.05.

Table 4. Correlation between serum and PF levels of Ang-2, VEGF and IL-1β with the stages of the disease.

Serum PF

Angio-2 VEGF IL-1B Angio-2 VEGF IL-1B

r 0.152 0.112 0.011 0.062

Angio-2

p 0.422 0.557 0.954 0.744

r 0.241 0.032

VEGF

p 0.199 0.865

IL-1β

rho 0.102 0.034 0.036 0.296 0.442* 0.034

Stages

Early & Late p 0.592 0.858 0.850 0.112 0.014 0.859

r: Pearson coefficient. rho: Spearman coefficient. *Statistically significant at p ≤ 0.05.

H. A. Moety et al. / Open Journal of Obstetrics and Gynecology 3 (2013) 528-535 533

Table 5. Diagonstic performance of the studied biomarkers with suggested threshold values.

Serum Ang-2 (pg/ml) Seru m VEGF (pg/ml) Serum IL-1β (pg/ml)

Threshold (cut of values) 3700 500 120

Sensitivity 100.00 90.00 60.00

Specificity 100.00 60.00 53.33

Positive predictive value (PPV) 100.00 69.23 56.25

Negative predictive value (NPV) 100.00 85.71 57.14

Accuracy 100.00 75.00 56.67

the menstrual cycle to neglect any effect of the sampling

time on the results. Bedaiwy et al. [5] made the sampling

time all through the menstrual cycle and found differ-

ences related to sampling time that they claimed not sig-

nificant, so we preferred to avoid those differences.

PF laparoscopic sampling; especially for VEGF, and

IL-1β can be more supportive in establishing diagnostic

tests in subtle or atypically-presenting cases, where evi-

dent endometriosis lesions would be hardly present to

assess histologically. We did not, however find any sig-

nificant correlation between symptoms such as pain with

the levels of PF studied markers including the cytokine;

IL-1β. This contradicts what Scholl et al. [9] stated about

an increased level of PF cytokine with the severe pain and

dysmenorrhea in endometriosis.

We were also able to estimate a preliminary cut-off

values, based on the number of patients studied, for the

serum markers in this cohort of patients, where Angio-2

showed the best sensitivity, and specificity followed by

VEGF and IL-1β respectively. There were relatively ac-

ceptable positive and negative predictive values, as well,

at the estimated cut-off levels. We assumed a promising

role for these makers that could be extended to similar

substances as diagnostic or follow-up landmarks in cases

destinated to develop endometriosis. Similarly, Mihalyi et

al. [10] concluded a diagnostic role of a panel of six

selected plasma biomarkers in the diagnosis of different

stages of endometriosis with good sensitivity and spe-

cificity. They studied, however, different biomarkers than

those in the present study, including some cytokines, and

obtained serum samples during the secretory phase or

during menstruation. Bedaiwy et al. [5] also assumed a

non- surgical diagnosis of endometriosis be possible by

measuring serum IL-6 and PF TNF-α.

VEGF is a heparin-binding glycoprotein with potent

angiogenic, endothelial cell-specific mitogenic and vas-

cular permeability activities.

Studies have demonstrated that VEGF is involved in

both the etiology and maintenance of peritoneal endome-

triosis [11]. Moreover, in normal endometrium the ex-

pression of VEGF is potentiated by a variety of cytokines,

especially Interleukin-1β (IL-1β) [12].

In this study we confirmed a good diagnostic per-

formance of VEGF both in serum and PF.

We found significant differences of PF VEGF and se-

rum Angio-2 levels related to the stage of endometriosis,

this was in accordance with Meresman et al. [13] who

reported same results stating that VEGF-A levels in the

serum and PF of patients with severe endometriosis

(stages III-IV) were significantly higher than in those

with minimal endometriosis owing to the modulation of

this marker by the disease itself.

Angio-2 is a cytokine that can amplify the effects of

other angiogenic factors, mainly VEGF but it can not

independently activate angiogenesis. Angio-2 is the en-

dogenous antagonist of Angiopoietin-1, binding the same

receptor. Angio-1 has been demonstrated in endometrial

and periendothelial cells and in glandular and stromal

cells of endometriotic lesions.

Zhang et al. [14] suggested overexpression of Angio-2

gene responsible for development and/or progression of

endometriosis.

Data on Angio-1 and Angio-2 remain too scarce to

clarify any role or mechanism in the aetiopathogenesis of

endometriosis. We assumed a role for serum Angio-2 in

non-invasive prediction of endometriosis based on our

results which demonstrated an out-standing diagnostic

ability for serum Angio-2 of 100% with (AUC 1.000),

however It was much less efficient for PF Angio-2 (AUC

= 0.937), this results were in accordance with Di Carlo et

al. [15] who stated that patients with endometriosis had

higher levels of angiogenic factors including Angio-2

compared with normal controls. Meanwhile, those an-

giogenic factors’ concentrations in serum and PF, as re-

ported by Bourlev et al. [16] normalize within a week

after surgical removal of endometriotic lesions in women

with advanced endometriosis.

IL-1β, a proinflammaory cytokine, was produced main-

ly by monocytes macrophages, and dendritic cells. In

health, circulating human blood monocytes or bone mar-

row aspirate do not constitutively express IL-1



, Stimu-

lants such as the complement component C5a, hypoxia,

and adherence to surfaces or clotting of blood induce the

synthesis of large amounts of IL-1 mRNA in monocytic

H. A. Moety et al. / Open Journal of Obstetrics and Gynecology 3 (2013) 528-535

534

cells [17-19].

A predictive performance of IL-1



was also suggested

in the light of this work. Based on our analysis, the PF

IL-1



was significantly higher in patients than controls

wit—h better diagnostic potential compared to serum IL-



. This could be assumed to the fact that, being a proin-

flammatory cytokine, locally released in the vicinity of

active endometriotic lesions. Menstrual pain scores with

endometriosis were assessed in relation to PF cytokine

concentration in a study by Scholl et al. [9] where a sig-

nificant difference was found according to the pain score.

Cytokine profiles were also tackled in a study by Kalu et

al. [20] where an elevated level of PF interleukins but

not serum confirmed a role in the pathogenesis of endo-

metriosis.

Hou et al. [21] suggested a possible clinical strategy

for the treatment of endometriosis by neutralization of

IL-1



, thus counteracting its effect on endometrial stro-

mal cells.

Serum Angio-2 and VEGF markers with the estimated

peritoneal fluid levels would be an addition to the diag-

nostic potential in endometriosis.

7. CONCLUSION

Non-invasive predictive biomarkers for endometriosis as

Serum Angio-2, VEGF and IL-1β independently or in

combination were estimated with threshold values. Se-

rum angio-2 merit is considered as a novel marker for

endometriosis due to its diagnostic power.

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