The chorioallantoic membrane (CAM) is a useful model for the fluorescence diagnosis of experimentally induced endometriosis. In our experimental setup ...
Human Reproduction vol.15 no.3 pp.584–588, 2000
Fluorescence diagnosis of endometriosis on the chorioallantoic membrane using 5-aminolaevulinic acid
E.Malik1,4, A.Meyho¨fer-Malik1, Ch.Berg1, W.Bo¨hm2, K.Kunzi-Rapp3, K.Diedrich1 and A.Ru¨ck3 1Department
of Obstetrics and Gynaecology, Medical University Lu¨beck, Ratzeburger Allee 160, D-23538 Lu¨beck, 2Department of Obstetrics and Gynaecology, Medical University Ulm and 3Institut fu¨r Lasertechnologien in der Medizin und Messtechnik, Ulm, Germany 4To
whom correspondence should be addressed
The chorioallantoic membrane (CAM) is a useful model for the fluorescence diagnosis of experimentally induced endometriosis. In our experimental setup 75.7% of the histologically examined tissue preparations were viable and only 24.3% showed signs of necrosis on the CAM after various periods of incubation. Best results were obtained when grafting to the CAM was performed between days 7 and 9 and when implants were left on the CAM for 3–5 days (P ⬍ 0.05). We were able to demonstrate that 5-aminolaevulinic acid (ALA) is stored selectively in ectopic endometrium. The subsequent fluorescence of the endometrium shows a rapid increase that reaches a peak after 10–14 h which can be clearly differentiated from the weaker fluorescence of grafted normal peritoneum and fimbriae (P ⬍ 0.01). Key words: 5-aminolaevulinic acid/chorioallantoic membrane/ endometriosis/fluorescence diagnosis/photodynamic diagnosis
Introduction The intra-operative diagnosis of endometriosis by conventional laparoscopy is often difficult and inappropriate. Endometriosis has a wide range of manifestations, frequently non-pigmented and presenting as small vesicles, nodular lesions, or plaquetype implants. This pleiomorphic appearance is best explained by a recently introduced model (Leyendecker et al., 1998): pathological expression of the P450 aromatase leads to local production of extra-ovarian oestrogen which up-regulates the endometrial oxytocin mRNA and leads to uterine hyperperistalsis and increased transtubal seeding of endometrial tissue fragments. At the level of the endometriotic lesion, locally increased concentrations of oestrogen result in hyperproliferation and infiltrative endometriosis. Some authors suggest that proliferating and highly active endometrial implants often present as non-pigmented peritoneal changes (Vasquez et al., 1984; Jansen and Russell, 1986; Murphy et al., 1986; Redwine, 1987). Indeed serum concentrations of the inflammation-related human leukocyte antigens class I (sHLAI) and the intercellular adhesion molecule-1 (sICAM-1) have 584
been found to be higher in patients with non-pigmented peritoneal lesions (De Placido et al., 1998). The severity of the disease may be overlooked if only pigmented ‘powder burn’ lesions are biopsied. The diagnosis of endometriosis thus depends on the ability of the surgeon to identify unspecific non-pigmented peritoneal changes as being indicative of endometriosis, so that the diagnosis can then be confirmed by biopsy and histological examination. We therefore sought to visualize such non-pigmented peritoneal changes more accurately. To achieve this, we first conducted an in-vivo study evaluating the fluorescence diagnosis of endometriosis using 5-aminolaevulinic acid (ALA). The experiments were done using endometrial implants on the chorioallantoic membrane of fertilized chicken eggs. The results obtained have to be validated on patients in a subsequent pilot study. The enhanced visualization of certain cancerous and noncancerous tissues after treatment with a specific photosensitizer has been previously reported. The light-induced fluorescence after treatment with ALA is an experimental model used in the early diagnosis of cancerous disorders in urology (Kriegmair et al., 1993, 1994; Baumgartner et al., 1994) and pulmonology (Hung et al., 1991). In the field of dermatology, ALA is also used for photodynamic therapy (Peng et al., 1997). ALA is a precursor of protoporphyrin IX (Pp IX) in the haem pathway. It has no inherent photosensitizing activity. The rate-limiting step in the haem synthesis is the step of converting Pp IX to haem (Fehr et al., 1996). Exogenous ALA therefore induces an excess of Pp IX which accumulates in the cell and has a strong photosensitizing effect. It can therefore be used for fluorescence diagnosis by exposure to light at a given wavelength. In the field of gynaecology, ALA has previously been used as a diagnostic and therapeutic tool in tumour cell lines (Ishiwata et al., 1988; Rossi et al., 1996) and endometrium (Fehr et al., 1996; Steiner et al., 1996; Yang et al., 1996). A pilot study on the fluorescence diagnosis of endometriosis using ALA in patients has previously been published by our group (Malik et al., 1998) A simple and reliable in-vivo model of endometriosis was needed to show the selective uptake of the photosensitizer in ectopic endometrium and subsequent fluorescence in this tissue. We chose the chorioallantoic membrane (CAM) of the chicken embryo, a model that has been known since 1887 (Gerlach, 1887) and used extensively for the culture of all kinds of tissues and cell lines (Rubovits and Abrams, 1951; Hall, 1978; Kunzi-Rapp et al., 1992). Our experiments on the CAM should enable us to demonstrate (i) whether it is possible to attain selective fluorescence © European Society of Human Reproduction and Embryology
Fluorescence diagnosis of endometriosis
Table I. Histological scoring system for the viability of the endometrial implants on the chorioallantoic membrane (6–11 ⫽ viable; 0–5 ⫽ nonviable; complete necrobiosis ⫽ non-viable)
Glandular tissue Fibroblasts Necrobiosis Leukocytes
3 points
2 points
1 point
0 points
– Many None None
Intact Some Some Laminar
In dissociation In dissociation Intense Many
Dissociated Dissociated Complete Filled with
of ectopic endometrium using ALA and (ii) the time interval before maximum fluorescence of the endometrium is attained and hence the best one for fluorescence diagnosis. Materials and methods Endometrium, normal-looking peritoneum and fimbriae were grafted to the CAM of 107 chicken eggs. The endometrium was derived from hysterectomy specimens or endometrial biopsies obtained during the investigation of infertile patients. In hysterectomy patients the uterus was opened by means of a longitudinal cut and the endometrium retrieved from the cavity. The specimens were only used if pathological findings were excluded on histological examination. Tissue specimens were stored in sterile physiological saline solution until used. The time allowed to elapse between explantation of the specimen and grafting to the CAM was ⬍3 h. Specimens were minced to fragments of 2⫻2 mm using a scalpel in a sterile Petri dish. Tissue fragments that appeared to be necrotic were discarded. Fertilized chicken eggs were purchased from a breeding station (Zeh KG, Laichingen, Germany). They were stored blunt end up in an incubator at 60% humidity and 37°C until day 5 or 6 after fertilization. A circular window ~3 cm in diameter was then made in the sharp end of the shell using an electrical engraving tool and scissors. The window was then covered with a lid and incubation was continued until the egg was used. As the immune system of the embryo is known not to be sufficiently developed until day 16–17 of incubation, CAM culture could be performed to this point (Ausprunk et al., 1975). When grafting was performed between day 6 and 10, the window in the sharp end of the egg was widened and tissue fragments were placed on the CAM near the Y branch of a large blood vessel. Implants were left on the CAM for 3–7 days. Viable implants appeared white or pink while rejected tissue was found to be black within 1 or 2 days after grafting. The circulation of the CAM vessels could readily be evaluated. Implants were then explanted and stained with haematoxylin–eosin for histological evaluation of viability. All specimens were evaluated by an experienced gynaecological pathologist on the basis of the following criteria: (i) was there any glandular tissue in the preparation and was this tissue intact or in dissociation?; (ii) what was the quantity and the quality of fibroblasts in the preparation?; (iii) were there any signs of necrobiosis in the preparations? and (iv) was there any sign of an inflammatory reaction? The decision whether an implant was rated viable or non-viable was based on a scoring system shown in Table I. A maximum of 11 points could be achieved. Preparations with a score of 艌6 were rated viable. All preparations with a score ⬍6 were rated non-viable together with all preparations with complete necrobiosis. Following the experiments on the optimal time for grafting and explanting the specimen, we conducted additional experiments into the fluorescence activity induced by ALA (C5H10ClNO3; Merck, Darmstadt, Germany). Endometrium, normal peritoneum and fimbriae
Table II. Histological outcome of 107 endometrial implants after various periods of incubation on the chorioallantoic membrane Day of grafting
Days of incubation
Viable (n)
Non-viable (n)
6 6 7 7 8 8 9 9 10 10 Total (%)
3–5 6–7 3–5 6–7 3–5 6–7 3–5 6–7 3–5 6–7
4 3 21 3 22 7 15 2 5 – 82 (76.6)
1 4 2 2 7 3 – – 6 – 25 (23.4)
were grafted to the CAM on day 7 of incubation and left for 3–5 days (Figure 1). ALA was used as a photosensitizer. Prior to each experiment, sterile ALA was freshly dissolved in phosphate-buffered saline. Vials containing the photosensitizer were covered with aluminium foil to prevent interaction with daylight. The pH was adjusted to 6.5 by adding sodium hydroxide. A final concentration of 25 mg/ml was used. Prior to the topical application, a silicone ring was placed around the graft on the CAM surface. 20 µl of the photosensitizer were then applied inside the ring using a micropipette. Fluorescence was assessed after 60 and 120 min and then every 120 min up to 36 h. After each measurement, the egg was covered with a lid and returned to the incubator. Fluorometric experiments were performed using a Zeiss Axiophot stereo-microscope (Zeiss, Oberkochen, Germany) coupled with a silicone-intensifying-target camera (SIT-camera 2400 Hamamatsu Photonics, Herrsching am Ammersee, Germany) able to detect even extremely weak fluorescence at low illumination (Schneckenburger et al., 1988). This camera was of special importance for the detection of the initial fluorescence prior to fluorescence diagnosis, as fastreacting photosensitizers tend to wear out quickly. ALA-mediated fluorescence was induced using a special filter-block BP436/LP470 (Zeiss) for illumination that allowed only blue light of 436 nm wavelength to pass. An additional red filter (⬎490 nm) was attached to the SIT-camera since only the red portion of fluorescence was to be detected. An additional image-intensifying system (video frame memory C 1901 Mark II; Hamamatsu) enhanced the signal background relationship by adding 64 single pictures. The fluorescence of the specimen was transformed into 16-bit gray scales. A gray scale was used to detect the relative level of fluorescence. Statistical analysis included Fisher’s exact test for the evaluation of the optimal time of implantation and explantation, and analysis of variance for the evaluation of the fluorescence measurements. Using receiver operator curve analysis (ROC), we defined the optimum cutoff-point for the diagnosis of endometriosis. Using this cut-off-point, we detailed the validity of our test by calculating sensitivity and specificity values.
Results The specimens were derived from a total of 18 patients (mean age 43.8 years). Of the 107 histologically assessed specimens, 82 (76.6%) were viable at the time of explantation. Twentyfive were non-viable (23.4%). Preparations with the best score of viability were obtained when grafting was performed between day 7 and 9 of incubation 585
E.Malik et al.
Figure 1. Endometrial implant after 4 days of incubation on the chorioallantoic membrane (CAM). Bar ⫽ 5 mm. Figure 3. Strong vascularization of the CAM adjacent to the endometrial implant. Bar ⫽ 1.4 mm.
Figure 2. Invasion of the endometrial implant by CAM vessels. Bar ⫽ 2.5 mm.
and when implants were allowed on the CAM for 3–5 days (Table II). When tissue was grafted onto the CAM on day 7 of incubation and kept on the CAM for 3–5 days, a significantly higher score of viability was obtained than was the case with grafting on days 6, 8 or 10 and culture for 3, 5 or 7 days respectively (P ⬍ 0.05). There was no statistically significant difference in all the other combinations. In 19 cases, erythrocytes with nuclear staining were observed in ectopic endometrium. Erythrocytes without nuclear staining were found in 13 cases. In order to detect the origin of the vessels, immunohistochemical staining of endothelium was performed in the cases where nuclear-stained erythrocytes were found. Monoclonal antibodies against factor VIII were used, but no vessels of human origin were detected. Figure 2 depicts the invasion of the graft by CAM vessels. Figure 3 shows the strong vascularization adjacent to the graft. We therefore conclude that vascularization (possibly neovascularization) by the CAM is one of the factors responsible for the survival of the graft. Exposure to light at a wavelength of 436 nm after topical ALA application revealed the selective uptake of the photosensitizer in ectopic endometrium. 586
Figure 4. Red portion of fluorescence of endometrium, peritoneum and fimbriae after topical 5-aminolaevulinic acid application.
The fluorescence of ectopic endometrium was measured in 81 preparations, each on an individual chicken egg for a total of 12 patients. Fluorescence of fimbriae was measured in 33 preparations from five patients and fluorescence of peritoneum in 21 preparations from four patients. The origin of the specimens was histologically confirmed in each case. The mean and standard deviation of the measured values were calculated. Figure 4 shows the intensity of fluorescence in these three tissues quantified in arbitrary units. Only the red portion of fluorescence was detected. A rapid increase in fluorescence was evident, reaching a maximum after 10–14 h when the fluorescence of the endometrium was twice as strong as that of the peritoneum and fimbriae and could be clearly differentiated (P ⬍ 0.01). At 8–10 h and 14–16 h after ALA application, the fluorescence of the endometrium was still significantly stronger than that of peritoneum but at a lower level (P ⬍ 0.05). At 16–18 h after ALA application, fluorescence of fimbriae reached a maximum and differed markedly from that of the peritoneum (P ⬍ 0.05). In summary, the fluorescence diagnosis of ectopic endometrium on the CAM is best achieved between 10 and 14 h after ALA
Fluorescence diagnosis of endometriosis
application. The optimal cut-off-point for the fluorescence was calculated using ROC analysis at a minimum sensitivity level of 0.8. At 10–14 h after ALA application, the optimal cut-off point was at 200 arbitrary fluorescence units. Using this cutoff value, the sensitivity and specificity values at 10, 12 and 14 h after ALA application were 0.9 and 0.6, 0.9 and 0.6, 0.8 and 0.7 respectively. Discussion On the basis of the results of the current investigation, CAM appears to be an appropriate model for fluorescence diagnosis in experimentally induced endometriosis. It is a cost-efficient and simple model that might replace animal experiments. In this respect, CAM culture contrasts sharply to the existing animal models of endometriosis. Endometrium was injected transperitoneally into rabbits (Manyak et al., 1990). Only four out of 10 investigated animals displayed endometriotic implants after this treatment; 20–23 weeks of incubation and subsequent laparotomy had to be performed in this investigation. In our model, 76.6% of the preparations were viable at histological examination and 23.4% showed signs of necrosis following various periods of incubation until grafting and various periods of incubation on the CAM. Experiments with regard to the best time for grafting and explanting various tissues when CAM culture is performed have previously been published (Knighton et al., 1977; Petruzelli et al., 1993; Kirchner et al., 1996). Using endometrium as a graft, our experiments did not differ significantly from these data. The pre-incubation period has to be individually chosen depending on the development of the CAM. This is mainly due to the varying conditions of temperature and humidity in which the eggs are stored prior to delivery. The topical application of ALA is simple, especially compared to i.v. injections in rats or rabbits. A rat model has been used to investigate the metabolism of ALA to Pp IX in experimentally induced endometriosis (Yang et al., 1996). This model compares with the cost-efficiency of the rabbit model used earlier (Manyak et al., 1990). Using our experimental model, we were able to show the selective uptake of ALA in ectopic endometrium. In various fields of medicine, numerous experiments have been conducted using ALA-induced fluorescence aiming in particular at photodynamic therapy. In the field of urology, most investigations have focused on bladder tumours (Kriegmair et al., 1994). Pigs (van Staveren et al., 1996) and rats (Kriegmair et al., 1995) were used as animal models. In gynaecology, ALA has been applied for the diagnosis and treatment of intrauterine endometrium. Maximum fluorescence of the endometrium was demonstrated after 2–4 h (Yang et al., 1996). Intravenous injections of ALA yielded higher rates of fluorescence than oral application. Uteri from hysterectomized patients were used to show that a maximum of fluorescence is reached after 4–8 h and that fluorescence of the endometrium is 48 times stronger than that of the underlying myometrium (Fehr et al., 1996). Our experiments demonstrate a rapid increase in fluorescence of the endometrium in contrast to peritoneum and fimbriae. A
maximum is reached after 10–14 h (P ⬍ 0.01), at which point the endometrium displays a fluorescence twice as strong as that of other tissues. Fimbriae, which are of special interest when ALA is applied via the transcervical, transuterine and transtubal routes, displayed up to 16 h the same fluorescence as the peritoneum. Only after 16–18 h was an increase in fluorescence in fimbriae noted, differing markedly from that of normal peritoneum (P ⬍ 0.05). At this time, fluorescence diagnosis should not be performed. However, histological examination of the fimbriae after fluorescence diagnosis revealed no signs of physical damage, therefore making impairment of the Fallopian tubes by fluorescence diagnosis unlikely. On the basis of these results, we can conclude that endometrium implanted to the CAM is a simple and efficient model for experimentally induced endometriosis. Selective uptake of ALA in ectopic endometrium was demonstrated and maximum fluorescence after topical application was evaluated in an in-vivo model. These results encouraged us to perform fluorescence diagnosis of endometriosis in patients. The results of our pilot study on the fluorescence diagnosis of endometriosis have previously been published (Malik et al., 1998, 1999). Acknowledgements We gratefully acknowledge the support of Takeda Pharma (Aachen, Germany). We also thank Karl Storz GmbH & Co. (Tuttlingen, Germany) for providing the equipment necessary for fluorescence diagnosis. Special thanks are due to Dr Moubayed for providing us with the histological scoring system for the viability of the implants on the chorioallantoic membrane.
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