Indocyanine Green and Near-Infrared Vision for Detection of Endometriosis (GRE-Endo Trial)
Phase: PHASE2
Conditions: Pelvic Endometriosis, Endometriosis Outside Pelvis
Sponsor: Catholic University of the Sacred Heart

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Polo per la Tutela e la Salute della Donna e della Vita Nascente 
Fondazione Policlinico A. Gemelli 
Università Cattolica del Sacro Cuore 
Roma 
 
Division of Gynecologic Oncology 
PRODCT:  
Indocyanine  Green  and  Near-infrared  fluorescence 
(NIRF) video endoscopic system 
STATE: 
Final Version n.1 
DATE: 29/01/2013 
TITOLE: 
 
 
 
PROTOCOL 
NUMBER: 
INDOCYANINE 
GREEN 
AND 
NEAR-
INFRARED  VISION  FOR  DETECTION  OF 
ENDOMETRIOSIS (GRE-ENDO TRIAL) 
 
736513. A 287 / C.E./2013 
 
 
 
Principal Investigator  
Professor Giovanni Scambia 
 
Co-investigators 
 
MDCRISTIANO ROSSITTO    cristiano.rossitto@libero.it 
MD VALERIO GALLOTTA valeriogallotta@hotmail.com 
MD SALVATORE GUELI ALLETTI gueliallettis@tiscali.it 
MD BARBARA COSTANTINI bacostantini@yahoo.it 
MD FRANCESCO COSENTINO cosentino.francesco@libero.it 
 
 
Confidential 
This protocol contains confidential information belonging to  Department of Oncology, Catholic University of Sacred Heart – Rome.  Except as may 
be otherwise agreed to in writing, by accepting or reviewing these materials, you agree to hold such information in confidence and not to disclose it to 
others (except where required by applicable law) nor use it for unauthorized purposes.  In the event of actual or suspected breach of this obligation, 
the principal investigator should be promptly notified. 

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SUMMARY 
 
a) BACKGROUND  
 
 
 
 
 
 
 
 
 
3 
• A- Endometriosis 
• B- Indocyanine Green 
 
b) DETAIL DESCRIPTION OF CLINICAL TESTING 
 
 
 
 
5 
 
a. Near-infrared fluorescence Imaging (NIRF) imaging as a supportive tool for localisation 
of deep infiltrating endometriosis (DIE) lesions during laparoscopic surgery 
 
b. Aim of the study 
 
c. Investigationpoints 
 
d. Study design and studypartecipants 
• Studypartecipants 
• Inclusioncriteria 
• Exclusioncriteria 
 
e. Procedure 
 
c) STUDY DURATION AND TIME PLAN 
a. Studyduration 
b. Planned start 
 
d) REFERENCES  
 
 
 
 
 
 
 
 
 
7 
 
 

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Background 
A. Endometriosis 
Endometriosis is a common gynecologic condition characterized by the presence of endometrial 
glands and stroma outside the uterine cavity.  Its prevalence in the general population among 
women undergoing tubal ligation is about 4%;1 however, the disease is much more common in 
women with pelvic pain and infertility, ranging from 40% to 60% and 20% to 30%, respectively.2 
Endometriosis is categorized into three different diseases: peritoneal, ovarian and deep 
endometriosis.3, 4  Peritoneal disease is defined by the presence of superficial peritoneal 
implants. Ovarian disease includes ovarian surface implants and ovarian endometriomas. Deep 
endometriosis, on the other hand, involves infiltrative lesions larger than 5 mm in depth affecting 
the rectovaginal septum, retro-cervical region, sigmoid, rectum, ureter or bladder.5   These 3 
endometriosis disease types are known to behave and affect women differently - it has been shown 
that compared to peritoneal and ovarian endometriosis, deep endometriosis is significantly 
correlated with pelvic pain and infertility.6 
In 2003, Abrao et al evaluated the histological patterns of endometriosis, observing these 
histological aspects of the disease:  stromal pattern, with the  presence of stroma morphologically 
similar to that of topical endometrium in any phase of the cycle, with no glandular epithelium; well-
differentiated glandular pattern, with presence of surface epithelium or epithelium forming 
glandular or cystic spaces (the morphology of the epithelial cells is indistinguishable from that of 
topical endometrium during the different phases of the menstrual cycle; undifferentiated glandular 
pattern, with presence of surface epithelium or epithelium forming glandular or cystic spaces (the 
epithelium is flattened or low cuboidal, with no correspondence with topical epithelium, resembling 
the mesothelium lining the peritoneum) and glandular pattern of mixed differentiation with presence 
of epithelium with a well-differentiated or undifferentiated pattern in the same biopsy. We 
concluded that the histological classification of endometriosis can predict the prognosis of the 
disease. 
B. Indocyanine Green 
In 1958, ICG (Indocyanine Green) was submitted for approval to the FDA for use in indicator-
dilution studies in humans. Absorption and fluorescence wave length of IGG lie in the near-infra-
red (nIR) spectrum. The fluorescence maximum varies with the solvent and lies around 830nm in 
plasma and 834nm in whole blood. The absorption maximum for blood and plasma is 805nm. 
Over the past several years, there has been an explosion of reports describing successful in vivo 
NIR fluorescence imaging. Many studies are qualitative; however, quantitative methods are 
beginning to emerge where dictated by necessity. Among the clinical applications for ICG 
fluorescence are: 
• Ophthalmic Angiography Studies  
• Hepatic Function Studies 
• Measurement of Cardiac Output 
• Sentinel Lymph Node Diagnosis  

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Figure: Chemical structure of IGC (left) and angiogram of the retina (right) 
The endoscopy system described in this document should enable the surgeon to visualize ICG 
fluorescence in laparoscopic surgery to reach the following goals: 
• Detection of ectopic endometrial lesions in the abdominal and pelvic space in the sense of 
deep infiltrating endometriosis 
• Detection of Sentinel Lymph Nodes during tumour resection of cervical or endometrial 
cancer. 
 
 

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2. Detail description of clinical testing:  
a) Near-infrared fluorescence Imaging (NIRF) imaging as a supportive tool for localisation of deep 
infiltrating endometriosis (DIE) lesions during laparoscopic surgery 
Endometriosis is a disease of the uterus in which tissue from the uterine cavity becomes implanted in the 
abdominal cavity and, rarely, metastasizes to organs at a distance from the uterus. Endometriosis tissue is 
biologically the same as basal endometrial tissue.  
Foci of endometriosis consist of glands, stromal cells, and smooth muscle; they are supplied by nerves 
(neurogenesis), lymphatic vessels, and blood vessels (angiogenesis). 
The diagnosis of endometrial lesions by simple laparoscopic visualization is difficult and often inaccurate.  
b) Aim of the Study 
Aim of the clinical testing is review the feasibility and potential to establish a new and more accurate method 
to visualize the peritoneal changes caused by endometriosis by ICG mediated fluorescence imaging. The 
results and scientific findings obtained within this study shall be used to design a follow-up controlled trial 
with a higher number of patients. 
c) Investigation Points 
• 
Question: Appearance of endometrial lesions and differentiation from surrounding tissue 
observed in the NIRF image in general 
• 
Hypothesis: 
• 
It is possible to increase the visual detection rate of peritoneal and deep endometriosis 
by means of ICG mediated fluorescence. 
• 
The estimation of extent and size of an endometriotic lesions can be aided by means of 
ICG mediated fluorescence prior to its excision. 
• 
Endometriosis of undifferentiated pattern may have different appearance by means of 
ICG mediated fluorescence than well differentiated and stromal disease 
 
d) Study Design and Study Participants 
• 
Study Participants:Prospective analysis of 25 patients undergoing laparoscopy for suspected 
endometriosis under white light illumination and ICG imaging diagnosis. 
• 
Inclusion criteria: 
• 
Suspected endometriosis with necessity for laparoscopic confirmation and resection 
• 
Regular menstrual cycles 
• 
Exclusion criteria: 
• 
Patients younger than 18 years and older than 50 years at time of operation 
• 
Subject has previous history of adverse reaction or allergy to ICG, iodine, shellfish or 
iodine dyes 
• 
Documented allergy to sulfur containing compounds 
• 
History of allergic reactions attributed to compounds of similar chemical or biologic 
composition to ICG 
• 
Subject has significant liver disease, cirrhosis or liver insufficiency with abnormal 
liver function tests (Total bilirubin increased by factor 1.5 than normal and/or serum 
glutamic oxaloacetic transaminase increased by factor 2 than normal) 
• 
Subject has uremia, serum creatinine (> 2.0 mg/dl) 
• 
Subject has severe coronary heart disease (instable angina pectoris) 
• 
Pregnant or breast-feeding women 

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• 
Subject is actively participating in another drug, biologic and/or device protocol 
• 
The presence of medical conditions contraindicating general anesthesia or standard 
surgical approaches 
• 
Subject has any medical condition, which in the judgment of the Investigator and/or 
designee makes the subject a poor candidate for the investigational procedure 
e) Procedure: 
• 
Patients are recruited within the clinical routine after indication for laparoscopy under 
consideration of inclusion and exclusion criteria. After obtaining the patient’s consent the patient 
is included into the study. Withdrawal of the patient from the study is possible at any time 
without resulting disadvantages or drawbacks in the patient’s treatment. 
• 
During the procedure, the sites is initially subjected to visual inspection using direct laparoscope 
visualization under white light conditions.  All areas suspected of peritoneal endometriosis are 
classified as white, black and red lesions and recorded together with their anatomic location in 
the surgical record for the purposes of subsequent extirpation.  All areas suspected of deep 
infiltrating endometriosis are recorded together with their anatomic location in the surgical 
record for the purposes of subsequent extirpation (retrocervical, vaginal, rectosigmoid, bladder 
lesions) 
• 
After initial visual inspection the patient is administered with 0.05 – 0.25 mg /(kg BW) ICG 
intravenously. The ICG imaging mode of the Olympus ICG Imaging System is activated and 
suspect areas in ICG imaging mode are recorded with their corresponding appearance in white 
light mode.  
• 
Areas that are conspicuous in ICG imaging mode shall be differentiated into white, red, and 
black lesions and inconspicuous peritoneum.  
• 
If areas suspect in either white light or ICG imaging mode were present, specific sample 
excisions will be taken from these areas.  
• 
In addition control biopsy specimens from inconspicuous peritoneum will be taken. The control 
biopsy specimens will be taken at least 2 cm away from any conspicuous structures of the 
peritoneum. 
f) Study Duration and Time Plan 
• 
Study Duration: 
6 Month (data acquisition phase) 
• 
Planned Start: 
T.B.D. 
 
 

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REFERENCES 
 
1. Eskenazi B, Warner ML. Epidemiology of endometriosis. ObstetGynecolClin North Am. 1997 
Jun;24(2):235-58. 
2. Ajossa S, Mais V, Guerriero S, Paoletti AM, Caffiero A, Murgia C, Melis GB..The prevalence 
of 
endometriosis 
in 
premenopausal 
women 
undergoing 
gynecological 
surgery. 
ClinExpObstetGynecol. 1994;21(3):195-7. 
3. Koninckx  PR,  Martin  DC.  Deep  endometriosis:  a  consequence  of  infiltration  or  retraction  or 
possibly adenomyosisexterna? FertilSteril. 1992 Nov;58(5):924-8 
4. Nisolle M, Donnez J. Peritoneal endometriosis, ovarian endometriosis, and adenomyotic nodules 
of the rectovaginal septum are three different entities. FertilSteril. 1997 Oct;68(4):585-96. 
5. Cornillie FJ, Oosterlynck D, Lauweryns JM, Koninckx PR..Deeply infiltrating pelvic 
endometriosis: histology and clinical significance. FertilSteril. 1990 Jun;53(6):978-83. 
6. Abrao  MS,  Gonçalves  MO,  Dias  JA  Jr,  Podgaec  S,  Chamie  LP,  Blasbalg  R.  Comparison 
between clinical examination, transvaginalsonography and magnetic resonance imaging for the 
diagnosis of deep endometriosis. HumReprod. 2007 Dec;22(12):3092-7. Epub 2007 Oct 18. 
7. Abrao,  M.  S.,  Neme,  R.  M.,  Carvalho,  F.  M.,  Aldrighi,  J.  M.  and  Pinotti,  J.  A.  (2003) 
Histological  classification  of  endometriosis  as  a  predictor  of  response  to  treatment.  Int  J 
Gynaecol Obstet, 82, 31-40.