1 Department of Gynecology, Université Catholique de Louvain, St Luc's Hospital, Brussels, and 2 Cell Biology Unit, Université Catholique de Louvain and Christian de Duve Institute of Cellular Pathology, Brussels, Belgium
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Abstract |
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Key words: cultured explants/experimental endometriosis/Ki-67/nude mice/VEGF
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Introduction |
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Autologous transplantation of uterine endometrium has previously been performed on rhesus monkeys, Macaca mulatta, on rabbits and on rats for elucidation of the pathophysiology of endometriosis in vivo (Jacobson, 1922; Katz and Szenes, 1926
; Te Linde and Scott, 1950
; Scott and Wharton, 1957
; Scott and Wharton, 1962
; Di Zerega et al., 1980
; Schenken and Asche, 1980; Jones, 1984
; Donnez et al., 1987
). Five studies of human endometrium transplanted into nude mice have previously been reported (Zamah et al., 1984
; Bergqvist et al., 1985
; Zaino et al., 1985
; Aoki et al., 1994
; Bruner et al., 1997
). Very recently, endometrial tissue was isolated into glands and stromal cells and the development of endometriosis after their injection into athymic mice was demonstrated (Tabibzadeh et al., 1999
).
Our aim was to evaluate the capacity of human endometrial explants, cultured for 24 h, to implant in nude mice, and the role of angiogenesis in the growth and proliferation of grafted tissue. For this purpose, endometrial tissue was cultured for 24 h in the presence of physiological concentrations of oestradiol and progesterone and deposited without sutures in the peritoneal cavity. Ki-67 and vascular endothelial growth factor (VEGF) immunohistochemical studies and a morphometric study of vascularization were carried out.
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Materials and methods |
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Fifteen nude 8-week-old female mice (Swiss nu/nu) were used. Three mice were housed per cage under laminar-flow HEPA filtered hoods in rooms maintained at 28°C with a 12-hour light/12-hour dark cycle. All housing materials, as well as food and water, were autoclaved prior to use. The mice were fed ad libitum with laboratory chow and acidified water. Handling was done under laminar-flow hoods.
Tissue sampling
During laparoscopy for tubal sterilization or infertility, specimens of human endometrium were obtained by endometrial biopsy from the fundus region of six women aged from 30 to 45 years. In two cases, biopsy was taken during menstruation, in two cases during the late proliferative phase, and in two cases during the late secretory phase. All the women had regular ovulatory cycles. The tissue samples were handled aseptically and cultured for 24 h as described in the next section, except for one part of each specimen, which was fixed in 10% formalin, dehydrated and embedded in paraffin. Sections of 6 µm thickness were stained with Gomori's trichrome for histological dating according to the criteria of Noyes (Noyes et al., 1950).
The use of human tissues for this study was approved by the Louvain Catholic University Ethical Institutional Board.
Tissue cultures
Endometrial biopsies were collected in ice-cold sterile phosphate-buffered saline, pH 7.4. Tissue preparations not exceeding 1 mm3 were cut with a sterile surgical blade and transferred on a membrane of Millicell-CM inserts (12 mm, Millipore, Brussels, Belgium) fitting to 4-well dishes (Nunc, Bornem, Belgium). They were cultured in Dulbecco's modified Eagle medium (Gibco Europe, Merelbeke, Belgium) without phenol red, supplemented with 1.5 mmol/l glutamine, 22 mmol/l glucose, 100 U/ml penicillin G, 100 µg/ml streptomycin, 250 ng/ml Fungizone® (Gibco) and buffered with 20 mmol/l HEPES (Marbaix et al., 1995; Kokorine et al., 1996
). Water-soluble complexes of 2-hydroxypropyl-ß-cyclodextrin with progesterone (100 nmol/l) and with 17ß-oestradiol (1 nmol/l) (SigmaAldrich, Bornem, Belgium) were added to the culture medium. Endometrial explants were transplanted into mice after 24 h of culture.
Transplantation
The mice were anaesthetized with an intraperitoneal injection of 0.07 ml of Imalgène 500 (Rhône Merieux, Brussells, Belgium) and 0.16 ml of Rompun 2% (diluted 100x, Bayer, Brussels, Belgium). Laparotomy was performed on the ventral midline just caudal to the umbilicus in order to place the cultured explants in a peritoneal pouch created by a 7/0 nylon continuous suture so that they could be easily located during the second laparotomy. As the explants were not sutured to the peritoneal layer, no damage was caused to this layer. The entire procedure was aseptically performed and took about 15 min. Four mice were grafted with explants of menstrual endometrium, four with proliferative, and seven with late secretory premenstrual endometrium. The 15 animals received 0.03 mg of oestradiol benzoate (Schering, Brussels, Belgium) intramuscularly on the day of tissue transplantation and on day 14.
Transplants
The mice were killed on day 21 and the grafts were surgically removed. After extirpation, tissues were fixed in 10% formalin and embedded in paraffin. The whole sample was cut into serial sections, which were stained either with Gomori's trichrome for histological evaluation or with specific antibodies for immunohistochemical studies. The proliferation index and the vascularization of the grafts were evaluated.
Measurement of proliferative activity
The proliferative status of endometriotic cells was measured by evaluating the percentage of Ki-67 positive-staining nuclei (proliferation index) (Nisolle et al., 1997). Ki-67 labelling was performed with immunoperoxidase using the peroxidase-antiperoxidase (PAP) complex. Tissue sections of 6 µm thickness were mounted on Superfrost Plus slides and stained according to the immunocytochemical assay described by Immunotech (Clone MIB1, Immunotech, Marseille, France). This monoclonal antibody does not cross-react with rodent Ki-67 (data not shown). Labelling was developed with a diaminobenzidene hydrogen peroxide substrate until nuclear staining was detectable. The resultant staining was evaluated by determining the distribution of staining within each tissue component. The number of labelled glandular and stromal nuclei per 500 cells was counted using the 40x objective of a light microscope (Zeiss, Oberkochen, Germany).
Immunohistochemical study of VEGF
VEGF labelling was performed by immunoperoxidase techniques using the peroxidase-antiperoxidase (PAP) complex. Immunohistochemical staining was carried out with the (1/100 dilution) polyclonal rabbit anti-VEGF IgG antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) (Smith, 1996; Donnez et al., 1998
). Slides were lightly counterstained in haematoxylin. Positive and negative controls were included in the series.
The VEGF H-score was calculated as follows: H-score = Pi, where i is the intensity: from 0 (negative cells) to 3 (high staining intensity); and P is the percentage of stained cells for each given i; P = 1 (<15% positive-staining cells), P = 2 (1550% positive-staining cells), P = 3 (5085% positive-staining cells), P = 4 (>85% positive-staining cells), and P = 5 (100% positive-staining cells).
Morphometric study of the stromal and interface layer (fatty and muscular) vascularization
The endometriotic lesions and the interface layer were analysed field by field using the 40x objective of the Axioskop light microscope and the CCD camera. Histological structures of interest such as the stroma, the interface layer (muscular or fatty) and the capillaries were drawn by moving a cursor, as previously described (Nisolle et al., 1993).
The image analysis program allowed us to evaluate the capillary relative surface area at the level of the stroma and the interface layer.
The 2 test, the median test, and one-way analysis of variance were used for statistical analysis (Siegel, 1956
).
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Results |
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Typical endometrial glands and stroma were observed in 13 grafts (87 %) at the fatty tissuemuscular layer interface (Figure 1). Proliferative glands were found in the 13 cases, even when the endometrial biopsy was taken during the late secretory phase. Indeed, the seven mice grafted with secretory endometrial biopsies revealed, in all cases, endometrial glands characteristic of the proliferative phase (epithelial height > 10 µm, mitoses, no signs of secretory activity). Interestingly, in areas where the stroma was highly developed, the epithelium was very high but where the stroma was scanty, the glandular epithelium was flattened (Figure 2
). The lesions exhibited a distinctive glandular epithelium surrounded by an intact basement membrane and adjacent stromal cell layer. An extensive vascular network (Figure 3
) was observed at the interface between the graft and the surrounding tissue.
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There were no significant differences in the glandular and stromal proliferation indexes of the transplants when they were evaluated according to the phase of the endometrial biopsy. Indeed, when the endometrial biopsy was taken during the menstrual phase, the median proliferation index of the endometriotic lesion was 2 and 0.7% in the glandular epithelium and stroma respectively. When the endometrial biopsy was taken during the secretory phase, the proliferation index was 4.9 and 0.2% in the glandular epithelium and stroma respectively.
Angiogenesis
VEGF positive cells were observed in both the glandular epithelium and stroma of the 13 grafts (Figure 4). The mean VEGF H score was significantly (P < 0.0001) higher in the glandular cells than in the stromal cells (9.3 ± 1.3 and 2 ± 1.5 respectively).
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Discussion |
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After 24 h of culture, explants of human endometrium (menstrual, proliferative and late secretory) were transplanted into the nude mouse intraperitoneally, into a peritoneal pouch, in an attempt to observe the process of implantation and angiogenesis and the biological behaviour of such tissues in an immune-deficient animal. As the endometrial tissue was placed in a peritoneal pouch, there was no mesothelial disruption at the site of implantation. Our study demonstrated that an intact peritoneum does not prevent endometrial implantation. This contradicts the hypothesis of Van der Linden, who suggested that mesothelial disruption was required to allow endometrial implantation (Van der Linden et al., 1996).
Our study confirms the ability of endometrial tissue to establish ectopic lesions after a period of in-vitro culture, with 87% of mice receiving endometrial tissue maintained in a continuous oestradiol and progesterone environment developing viable endometrial implants. Indeed, in our study, of the 15 grafted mice, only two exhibited no endometriotic tissue at all, which could be explained by the inferior quality of the endometrial biopsy which was haemorrhagic and atrophic.
In the present study, we were thus able to demonstrate that short-term (24 h) culture of endometrial tissue did not compromise the ability of human tissue to establish viable ectopic lesions in nude mice.
Recently, Bruner et al. tested whether steroid treatment of endometrial tissue in vitro differentially affects the ability of human tissue to establish ectopic lesions in the nude mouse (Bruner et al., 1997). Although treatment of endometrial tissue in vitro with oestradiol resulted in the establishment of ectopic lesions in 100% or 90% of recipient mice, on the contrary, significantly fewer animals developed ectopic lesions in the absence of oestradiol treatment of endometrial tissue in vitro. In contrast to the positive effect of oestradiol on the establishment of experimental endometriosis, progesterone treatment of endometrial tissues blocked lesion formation in recipient mice, regardless of the steroid treatment of the animal.
Our data, however, are very different. Firstly, in the study by Bruner et al., only proliferative endometrium samples were obtained. In our series, implantation of endometrial tissue occurred regardless of the phase (proliferative or secretory) of the cycle in which sampling was performed. Secondly, treatment of endometrial tissue with oestradiol and progesterone did not block lesion formation in recipient mice. Indeed, when the explants were cultured with oestradiol and progesterone, lesions were observed in 87% of cases in our series, whereas in the Bruner et al. study, no mice developed endometriotic-like lesions, even if the animals were supplemented with oestrogens.
We thus disagree with one of the conclusions of Bruner's study, which suggested that suppressing metalloproteinase secretion in vitro with progesterone treatment inhibits the formation of ectopic lesions in this experimental model. In our model, treatment of cultured tissue with progesterone did not inhibit implantation. The cellular mechanisms by which oestrogen and progesterone may affect the establishment of the disease, including a specific role for MMPs (Bruner et al., 1997), remain speculative.
In our series, lesions exhibited a distinctive glandular epithelium surrounded by an intact basement membrane and adjacent stromal cell layer of variable thickness and organization. An active vascular network was observed in the implant and the surrounding tissue. Although it seemed that most blood vessels arose in the abdominal fat pad with fewer in the interphase layer, sufficient angiogenesis occurred in the latter for an active transplantation process to take place. Preservation of specific endometrial stroma was necessary for the maintenance of the normal morphological features of the glandular epithelium, suggesting the importance of the interaction between the stromal and epithelial compartments in cell growth and regulation (Cunha et al., 1985; Osteen et al., 1994
). This interaction was recently reported by in-vitro studies (purified cultures of epithelial and stromal endometrial cells), which confirmed that there is paracrine regulation of induction expression of matrix metalloproteinase-1 and that interleukin-1
, secreted by epithelial cells, plays a key role in this induction (Singer et al., 1997
).
Mitotic activity was observed in both components of the endometriotic-like foci. The presence of this mitotic activity was confirmed by the study of proliferative activity using the Ki-67 antibody (anti-human monoclonal antibody). Our study proved mitotic activity in grafts even if the biopsy was taken during the late secretory phase when mitoses are absent in the epithelium. Moreover, the use of anti-human monoclonal antibody not cross-reacting with rodent Ki-67 demonstrates the survival of human tissue in nude mice. It also proves that the endometriotic-like foci result from transplantation and not from metaplasia induction. Moreover, a significantly higher glandular proliferation index was observed in tissue transplants when compared to the 24-h cultured explant, proving the viability and independence of the grafted endometrial tissue in nude mice. This contrasts with the findings of Zamah et al., who observed necrosis not only in unsupplemented mice, but also in oestradiol-supplemented mice (Zamah et al., 1984).
In this study, we demonstrated for the first time that transplanted endometrium is able to undergo histological change when placed into nude mice, whatever the original phase of the cycle. Indeed, all the transplants demonstrated characteristics of the proliferative phase without secretory signs even if the endometrial tissue was obtained during the secretory phase. This suggests that after implantation, the tissue has its own activity. The centre of the implant was never necrotic, proving that the tissue had undergone secretory transformation with a disappearance of the secretory signs, and had started proliferative activity.
Survival of the grafted endometrial tissue was only possible if active angiogenesis allowed the development of a vascular network. The establishment of a new blood supply is essential for the survival of the endometrial implant and the development of endometriosis. In all cases, numerous blood vessels were observed in the stromal component of the endometriotic lesion. These transplants had capillaries at the interface of attachment to fat or the serosa. VEGF has been demonstrated in human endometrium and in ectopic endometrium (Donnez et al., 1998) and may be important in both physiological and pathological angiogenesis (Schifren et al., 1996
; McLaren et al., 1996
; Donnez et al., 1998
).
The high VEGF content observed in these grafts may provoke an increase in the subperitoneal vascular network and facilitate implantation and viability (Donnez et al., 1998). Moreover, VEGF, in addition to being angiogenic, causes increased permeability of the capillary bed, and similarly to the mechanisms involved in pathological angiogenesis, this could suggest that in freshly implanted endometriotic lesions, the higher expression of VEGF might explain a higher permeability of the capillary bed (Folkman and Shing, 1992
). This may lead to a leakage of fibrin products into the extracellular space, which will increase the recruitment of macrophages and their secretion of tumour necrosis factor-
, which, in addition to its angiogenic activity, promotes adhesion of human endometrial stromal cells to peritoneal mesothelial cells in vitro in a dose-dependent fashion (Zhang et al., 1993
).
In conclusion, focusing on the first stages of endometrial tissue implantation, we were able to demonstrate that even when not fixed to the mesothelial layer, endometrial tissue could attach to several surfaces, questioning the hypothesis of Van der Linden et al., who suggested that an intact peritoneum could prevent adhesion and implantation. The fact of culturing endometrium for 24 h in the presence of oestradiol and progesterone did not interfere with the implantation process. We also demonstrated that whatever the phase of the cycle at which biopsy was taken, the transplanted endometrium underwent histological changes (proliferative) when placed into nude mice. Indeed, strong proliferative and angiogenic activity was demonstrated in the implanted tissue, allowing it to become vascularized and remain viable.
Further studies are needed to prove the primordial role of angiogenesis, e.g. by blocking the production of VEGF, in order to see if it will prevent the development of endometriosis.
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Notes |
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References |
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Submitted on July 8, 1999; accepted on November 29, 1999.