Keywords
CAM model - endometriosis - chorioallantoic membrane - 3D in vivo model - human endometriosis
tissue - human tissue
Schlüsselwörter
CAM-Modell - Endometriose - Chorioallantoismembran - 3D-in-vivo-Modell - humanes Endometriosegewebe
- humanes Gewebe
Introduction
Endometriosis
Women of reproductive age often suffer from endometriosis, which is defined by endometrial
tissue outside of the uterus. It affects around 10% of women worldwide and represents
one of the most common benign diseases in gynecology. Some of its effects are pain
and infertility and affect the women’s quality of life [1]. There are a few theories that try to explain the pathology of endometriosis but
the definite underlying cause is still unknown [2]
[3]. This makes its treatment and cure challenging. Endometriosis shows to be a tumor-like
disease as it has some characteristics that are defined as the hallmarks of cancer:
induction of angiogenesis, activation and invasion of metastasis, to name a few [4]
[5]. One of the disease’s characteristics is its dependance on high estrogen levels
[6]. The peritoneal fluid of patients with endometriosis consists of different hormones,
cells, and proangiogenic factors. Those and their signaling products seem to enhance
the spreading of new blood vessels, which lead to endometriotic implant survival [7]
[8]. Also, the endometrium has an angiogenic potential and both, endometrium and peritoneal
fluid influence lesion formation [9]
[10]. In patients, endometriosis is classified using the #Enzian classification that
categorizes endometriotic lesions into different groups, describing the depth of infiltration
and affected organs, allowing for a more precise assessment of the severity and spread
of the disease [11]
[12].
The chorioallantoic membrane model (CAM)
The chorioallantoic membrane (CAM) model, a 3D in vivo model using fertilized chicken
eggs, has emerged as a promising platform for translational research in endometriosis.
The CAM consists of three layers (ectoderm, mesoderm, and endoderm), with the mesoderm
being highly vascularized, creating an ideal environment for tissue engraftment [13]
[14]. The immunodeficient nature of the chicken embryo during early developmental stages
further supports the growth and invasion of heterologous tissue.
Endometriosis exhibits tumor-like features such as angiogenesis, local invasion, and
the formation of metastasis-like lesions, which can be effectively studied using the
CAM model. This innovative platform allows for real-time monitoring of engrafted endometriosis
tissue, providing a unique opportunity to analyze processes like inflammation, neuronal
interactions, and angiogenesis. The CAM model’s versatility also extends to drug testing,
offering a bridge between preclinical animal studies and clinical trials [15]
[16]
[17]
[18].
A key advantage of the CAM model is its alignment with the 3R principles – replacement,
refinement, and reduction of animal experiments [19]. By utilizing this model, researchers can minimize reliance on traditional animal
models, reducing ethical concerns while maintaining robust experimental outcomes.
Moreover, the CAM model’s ability to mimic the microenvironment of endometriotic lesions
enables detailed investigations into angiogenesis but also biomarker identification.
Aim
This study aims to investigate the role of angiogenesis and vascularization in endometriosis
using the CAM model in combination with immunohistochemistry and Laser Speckle Contrast
Analysis (LASCA) imaging. We hypothesize that endometriotic lesions induce increased
neovascularization, which can be visualized and quantified within the CAM model. Furthermore,
we propose that these angiogenic responses correlate with clinical parameters derived
from patient data, such as the #Enzian classification. The role of the chicken embryo’s
sex on perfusion was analyzed utilizing an in ovo sexing technique. By integrating
preclinical findings with patient-derived clinical information, this translational
approach aims to deepen our understanding of the vascular component of endometriosis
and support the development of targeted therapeutic strategies — all within the framework
of ethical and reductionist animal research.
Methods
Patient cohort and data
Human endometrium and endometriosis tissue was obtained from patients who underwent
laparoscopic endometriosis surgery at the EuroEndoCert certified Endometriosis Clinic
at the St. Marien Hospital Amberg between May 2023 and February 2024. Only patients
with intraoperatively and histologically confirmed endometriosis were ultimately included
in the study. Initial screening was based on a high clinical suspicion of endometriosis,
such as suggestive findings during ultrasound, a medical history consistent with endometriosis-related
symptoms, or a previously confirmed diagnosis during earlier surgery. Screening was
performed during visits to the endometriosis outpatient clinic. Exclusion criteria
included postmenopausal patients and patients without histologically confirmed endometriosis.
However, one healthy patient remained in the study to serve as a control, although
no clinical or histological lesion was confirmed, as healthy endometrium could be
extracted during an already planned hysterectomy. All patients signed an informed
consent. The study was approved by the Ethics Committee of the University of Regensburg
(23–3211–101, 22–2862–104), ensuring compliance with ethical standards for human tissue
research and patient data processing. Patient data were pseudonymized. Data processing
and analyses were conducted in accordance with the Declaration of Helsinki and the
General Data Protection Regulation of the European Union ([Fig. 1]
a, b).
Fig. 1
Methods – Overview. a, b Patient recruitment and tissue retrieval. c Assessment of tissue in the CAM model: sex determination of the chick, perfusion
measurements via LASCA, microscope images of engrafted tissue on inoculation and exoculation
day, respectively. d Immunohistochemical staining and histological analysis.
In ovo sex determination
The determination process is based on a patented in ovo sex determination method developed
by Prof. Dr. Einspanier (University of Leipzig) [20]. On embryonic developmental day (EDD) 11, the allantoic cavity was accessed by puncturing
the CAM, and allantoic fluid was collected ([Fig. 1]
c). Then the allantois laboratory kit was used, which works with a 96-well plate (NUNC
A/S, Roskilde, Denmark) coated with sheep anti-rabbit IgG (dilution 1 : 100 for E1S, Technische Universität München, Institute of Physiology, München, Germany). The
allantois samples were pipetted into the wells in duplicate. Also, two gender-specific
control fluids were used to assign the gender of the individual samples later. The
Elisa plates were evaluated by photometric determination at 450 nm wavelength in an
Elisa reader (Multiscan FC ThermoScientific) and subsequent evaluation via Microsoft
Excel was done. The device took advantage of the hormone level dependent color change
that occurs in the presence of the female sex hormone estrone sulfate to determine
the sex of the embryo. The calibration curve and final analysis was performed by
Prof. Dr. Einspanier and her group.
The endometriosis CAM Model
The CAM model was performed as previously described in the established protocol [21]. In brief, fertilized chicken eggs were incubated in a ProCon egg incubator (Grumbach,
Asslar, Germany) at 37.8 °C and 63% humidity. To access the CAM, the eggshells were
windowed to a size of approximately 1.5–2 cm and sealed with Leukosilk tape. Patient
tissue was sliced into approximately 3 × 3 mm pieces and engrafted onto the CAM. LASCA
measurements were conducted, and exoculation was performed ([Fig. 1]
c).
Angiogenesis measurements
Changes in blood flow in the CAM were measured as a surrogate for angiogenesis using
laser speckle contrast analysis (LASCA) with the PeriCAM perfusion speckle imager
(PSI) system, high-resolution (HR) model (PERIMED, Järfälla, Sweden), and Pimsoft
software version 1.5.4.8078 (64-bit) by Perimed, as previously described [21]
[22]
[23]. The LASCA technology enabled real-time visualization of tissue blood perfusion
through a small opening in the eggshell, providing insights into angiogenesis in chicken
eggs ([Fig. 1]
c). Measurements were performed on days 1, 2, and 3, with measurement day 1 conducted
after 2 days, measurement day 2 after 4 days, and measurement day 3 after 6 days following
tissue engraftment. For accurate results, LASCA was conducted during a low-movement
period of the embryo lasting at least 20 seconds, referred to as the time of interest
(TOI). A region of interest (ROI) was defined, including only the tissue surrounding
the CAM, with a 0.2 mm wide zone around the tissue samples to ensure precise readings.
A 550 perfusion units (PU) cutoff filter was applied to exclude areas with minimal
or no blood perfusion. LASCA-derived perfusion values are expressed in perfusion units
(PU), which are arbitrary units derived from optical speckle contrast analysis. As
the method provides relative, not absolute, measurements of blood flow, there is no
direct conversion to physiological units such as ml/min. Consequently, PU is the intrinsic
and appropriate unit for representing perfusion in LASCA-based assessments.
Immunohistochemistry
All excised human endometriosis and endometrium samples, along with surrounding CAM
tissue, were immediately fixed in 4% buffered formalin for 24 hours and subsequently
stored in sodium azide until paraffin embedding. The tissue was then cut into 6 µm
sections using a microtome. For histological and immunohistochemical analysis, sections
were stained with hematoxylin (Gill No. 3, Sigma-Aldrich, St. Louis, MO, USA) and
eosin (Chroma, Waldeck GmbH & Co. KG, Münster, Germany) (H&E staining) or subjected
to histochemical antibody staining. Endometrial glands were detected using an anti-cytokeratin
(CK) antibody (AE1/AE3 clone anti-mouse from DAKO, Santa Clara, USA), and endometrial
stromal cells were identified using a CD10 antibody (CD10–270-L-CE anti-mouse from
Leica Biosystems, Nußloch, Germany). For proliferation analysis, Ki67 staining was
performed with the ZytoChem Plus (HRP) Anti-Rabbit Kit (Biozol, Eching, Germany) using
the rabbit monoclonal Anti-Ki67 antibody [Sp6] (ab16667, Abcam, Cambridge, UK). For
the detection of apoptosis, cleaved Caspase-3 (Asp175) antibody #9661 from Cell Signaling
Technology, Inc. (Danvers, Massachusetts, USA) was used ([Fig. 1]
d). Slides were scanned with the Precipoint M8 and Precipoint Fritz scanner at various
magnifications, enabling standardized digital microscopy using Viewpoint Light (PreciPoint,
Munich, Germany).
Statistics
The statistical analysis was performed using International Business Machines Corporation
(IBM) SPSS Statistics, version 29.0.0.0 (241) (Armonk, New York, USA), and GraphPad
Prism 8, version 8.0.2 (263), by GraphPad Software Inc. (La Jolla, California, USA).
As sample sizes for each patient consisted of several CAM models, different statistical
tests were conducted depending on data distribution and scale level. For normally
distributed, paired data, paired t-tests were used. When normality could not be assumed,
the non-parametric Wilcoxon signed-rank test was applied instead. For comparing more
than two related samples, the Kruskal-Wallis test for related samples was used. In
cases of two independent groups with non-normally distributed data, the Mann-Whitney
U test was performed. All tests were two-sided, and p-values lower than 0.05 were
considered statistically significant. Furthermore, we evaluated and analyzed the collected
data from PeriCam using Microsoft Excel, version 2402, developed by Microsoft Corporation
(Redmond, Washington, USA) for chart creation, and GraphPad Prism for diagram generation.
Results
Description of patient cohort
Tissue samples were taken from different origins: pelvic wall, ovarian cyst, uterus,
peritoneum, mesosalpinx, or endometrium ([Fig. 1]
b, [Table 1]). A total number of 10 patients were included in the final analysis, of which nine
showed a histologically confirmed endometriosis. One healthy patient served as a control:
as surgery was performed due to dysmenorrhea and hypermenorrhea and included a hysterectomy,
endometrium tissue of this patient could be extracted. Patients were excluded if endometriosis
could not be confirmed histologically. CAM samples were excluded if the tissue failed
to adhere (n = 2) or if the movements of the chicks distorted the angiogenesis measurements,
resulting in falsely elevated values (n = 4).
Table 1
Patient characteristics (ID 1 to ID 10). Note: For the bivalent categories of the
#Enzian classification (such as B, O, T), only a single value (the highest value)
was recorded to simplify the analysis. n. s. = not specified.
|
ID
|
Tissue origin
|
#Enzian classification
|
Hormone therapy
|
Age at surgery
|
|
1
|
peritoneum, pelvic wall, ovary and ovarian cyst
|
P3 O2 T2 A1 B3 C0 FA
|
ethinyl estradiol 0.03 mg, levonorgestrel 0.15 mg
|
27
|
|
2
|
peritoneum, pelvic wall
|
P2 O3 T2 A0 B3 C1 FA
|
n. s.
|
24
|
|
3
|
peritoneum, pelvic wall, ovary, ovarian cyst and mesosalpinx
|
P3 O0 T2 A0 B2 C2 FA
|
no
|
38
|
|
4
|
ovary, ovarian cyst
|
P2 O3 T1 A1 B2 C1 FA
|
n. s.
|
31
|
|
5
|
peritoneum, pelvic wall
|
P3 O1 T0 A0 B1 C0 FA
|
chlormadinone acetate 2 mg
|
38
|
|
6
|
peritoneum, pelvic wall
|
P1 O0 T0 A1 B3 C1 FA
|
no
|
39
|
|
7
|
ovary, ovarian cyst
|
P0 O1 T0 A3 B2 C1
|
no
|
31
|
|
8
|
peritoneum, pelvic wall
|
P1 O0 T1 A0 B2 C0
|
dienogest 2 mg
|
25
|
|
9
|
Endometrium (healthy)
|
P0 O0 T0 A0 B0 C0
|
no
|
39
|
|
10
|
Endometrium (endometriosis)
|
P1 O1 T0 A0 B1 C0 FA
|
n. s.
|
24
|
Immunohistochemical staining
All tissue samples were routinely stained with H&E to enhance the visualization of
cellular structures. This staining served as a prescreening method for further immunohistochemical
analyses. Various markers were used to specifically identify tissue components, all
of which yielded positive results: the CD10 marker successfully identified stromal
cells, which were typically localized around the glands in endometrial tissue. Anti-CK
primarily stained the epithelial cells of endometrial glands, facilitating the differentiation
between epithelial and stromal regions. Ki67, a marker of cellular proliferation,
was used to assess cell growth activity. While Ki67 showed little to no proliferation
in endometrium, some proliferative activity was observed in endometriosis tissue before
and after engraftment on the CAM. Additionally, Caspase-3 staining showed some positive
results, revealing areas of necrosis within the endometriosis and endometrium tissue
after exoculation. The typical histological differences between endometriosis and
endometrium tissue were also seen in the tissue samples after exoculation: numerous
glands embedded within abundant stromal tissue for endometrium versus only sporadic
glands, with significantly less stromal tissue for endometriosis ([Fig. 2]).
Fig. 2
Immunohistochemical staining for human endometriosis and endometrium samples. Engrafted
tissue of endometriosis in the CAM model. a–f endometriotic tissue from patient ID 8 egg 7. g–l endometrium tissue from patient ID 10 egg 25 in the CAM model. a, g Overview image H&E staining. b, h H&E staining. c, i CD10 staining. d, j Caspase-3 staining. The tissue sections analyzed were from the same patient, respectively,
but not from the same egg, as the paraffin block was unfortunately completely depleted
before this staining method could be established. e, k Ki67 proliferation marker. f, l CK staining. 1 = stromal cells; 2 = gland; * = CAM.
Increase in angiogenesis over time for endometriosis patients
Depending on data distribution, paired t-tests for ID 1, 2, 5 and 10 or Wilcoxon tests
for ID 3, 4, 6 and 7 were applied (Supp. Table S1, Supplementary Material, Online). No tests were performed for ID 8 and 9 as the sample
sizes were too small. Across most endometriosis patients (75% of cases), an increase
in blood perfusion, hence angiogenesis, over time was observed from day 1 to day 3
([Fig. 3]
a–g1). For ID 1, a significant increase for PU was observed between day 1 and day 3 (p < 0.001).
ID 2 showed a significant increase between day 2 and day 3 (p = 0.016). In contrast,
no significant differences were detected for ID 3 across any of the measurement days.
For ID 4, a highly significant increase was observed between all measurement days
(p < 0.001 for all comparisons). ID 5 showed a significant increase between day 1
and day 2 (p = 0.015). ID 6 exhibited significant increases between day 1 and day
3 (p = 0.026) and between day 2 and day 3 (p = 0.008). Finally, ID 7 demonstrated
significant increases between day 1 and day 2 (p = 0.036) and between day 1 and day
3 (p = 0.025). ID 8 showed no visible consistent differences between all measurement
days.
Fig. 3
Angiogenesis measurements for endometriosis samples. a–g1: Representation of blood perfusion values (PU) on measurement days 1 to 3 for various
patients (ID 1 to ID 7). g2: Individual blood perfusion trends for 2 out of 10 eggs of ID 7 over days 1 to 3,
represented by colored points (red for egg 9, blue for egg 21). h: Summary of the average blood perfusion values (PU) for all measured eggs inoculated
with endometriosis tissue (n = 109, ID 1 to ID 8) on days 1 to 3. g3 and g4 (top row): LASCA images showing blood perfusion for egg 10 (red, Fig. g3) and egg 11 (blue, Fig. g4) on days 1 to 3. The perfusion images visualize regional differences in perfusion.
g3 and g4 (bottom row): The same images as the top row, with a PU filter of 550 nm applied to cancel out
areas with low to no perfusion. The black circle in the image represents the ROI.
Each bar represents the mean PU value with standard error of the mean (SEM). n = number
of eggs measured for each patient. * p < 0.05, ** p < 0.01 and *** p < 0.001. PU =
perfusion unit.
The overall average perfusion for all endometriosis IDs and all measured eggs (n = 109,
ID 1 to ID 8) over the three days increased ([Fig. 3]
h). The mean perfusion values increased progressively across the days, with an average
of 623.8 perfusion units (PU) on day 1, 644.2 PU on day 2, and 668.3 PU on day 3 (day
1 vs. day 2 p < 0.001, day 2 vs. day 3 p < 0.001, day 1 vs. day 3 p < 0.001).
When analyzed separately for each patient, the trends in perfusion values of the individual
eggs varied ([Fig. 3]
g2–4).
Increase in blood flow over time for endometrium of endometriosis patients
To investigate differences in blood perfusion of endometrium tissue, LASCA measurements
were performed on days 1 to 3 for a patient with and without endometriosis, respectively.
For endometrium of a healthy patient (ID 9) no visible consistent changes were observed
in perfusion levels in the CAM model over time. In contrast, a significant increase
in perfusion was observed for the endometrium sample of an endometriosis patient (ID 10):
between day 1 and day 2 (p = 0.004) ([Fig. 4], Supp. Table S1). The paired t-test was used to compare measurements across the three days for IDs
9 and 10.
Fig. 4
Perfusion measurements for endometrium samples: a PU values measured over three days of an endometrium sample of a healthy patient
(ID 9) without endometriosis. b PU values measured over three days in the endometrium of a patient (ID 10) with endometriosis,
classified with an #Enzian Score of P1 O1 T0 A0 B1 C0 FA. Each bar represents the
mean PU value with SEM. n = number of eggs measured for each patient. * p < 0.05,
** p < 0.01 and *** p < 0.001. PU = perfusion unit.
The role of the chicken embryo’s sex
To evaluate whether the sex of the chicken embryos influenced angiogenesis measurements,
sex determination for the eggs of ID 5, 6 and 7 was performed. The significance levels
of blood perfusion measurements for day 1, day 2, and day 3 were analyzed. Across
all three experimental runs, a total of n = 38 eggs were investigated, of which 16
were male and the remaining were female. The Mann-Whitney U-test revealed no significant
differences between sexes for blood perfusion measurements on day 1 (p = 0.866), day
2 (p = 0.651), and day 3 (p = 0.988).
The role of the #Enzian classification on angiogenesis in the CAM model
The analysis focused on the relationship between angiogenesis measurements via LASCA
over three days (day 1–3) and the #Enzian classification categories of the individual
patients (ID 1 to ID 8 and ID 10), particularly the categories A (vagina, rectocervical
and rectovaginal), O (ovary), P (peritoneum), and T (tubal/ ovarian), B (uterosacral
ligament, cardinal ligaments, and pelvic sidewall) and C (rectum) and FA (adenomyosis
uteri) ([Fig. 5]).
Fig. 5
Angiogenesis measurements results for #Enzian categories. The blood perfusion from
days 1 to 3 (top to bottom) is shown for the following #Enzian classification categories:
A in panel a, O in panel b, P in panel c, and T in panel d. y-axis = PU; x-axis = extension of disease; box plots with mean (thick black line)
and standard deviation. *** p < 0.001, ** p < 0.01, * p < 0.05. n = 123.
The relationships were evaluated using Kruskal-Wallis tests for comparisons between
more than two groups. In category A (vagina and cervix), an inverse relationship was
observed, where higher #Enzian scores correlated with lower perfusion values on days
1 to 3. For example, lesions larger than 3 cm (A3) were associated with a lower perfusion
compared to cases without lesions (A0). The statistical significance of this relationship
was as follows: day 1: p < 0.001, day 2: p = 0.021 and day 3: p = 0.017. In category
O (ovary), no consistent effects on angiogenesis measurements were found. Significant
effects on angiogenesis measurements were identified in this category as follows:
day 1: p < 0.001 and day 2: p = 0.033. In contrast, higher #Enzian scores in category
P (peritoneum) significantly correlated with increased perfusion values on all three
days, indicating greater perfusion as lesion size increased (day 1: p < 0.001, day
2: p = 0.025, day 3: p = 0.020), demonstrating strong variation in perfusion across
the peritoneal extension categories. For category T (tube), the presence of adhesions
involving the pelvic sidewall and uterus (T2) was associated with significantly higher
perfusion values on day 1 to 3 compared to cases without adhesions (T0): day 1: p < 0.001,
day 2: p < 0.001 and day 3: p = 0.034. In category C, statistical significance was
observed on day 1 with p < 0.001, while on day 2 and 3, there was no significance.
Still, no clear pattern was seen. In category B, no statistical significance was detected
across all days. For category FA, significant results were found on day 1 (p = 0.010)
and day 3 (p = 0.040), showing no clear pattern. In summary, for #Enzian categories
B, C, O, and FA, the perfusion values fluctuated between the different classes, showing
neither a correlation nor an inverse trend compared to categories A (inverse correlation),
P, and T (positive correlation), respectively (Supp. Table S2, Supplementary Material, Online).
Discussion
This study is among the first to investigate human endometriosis tissue in the CAM
model – to our knowledge this has been performed by only four other groups. Liu et
al. focused on isolated endometrial stem cells from human endometriosis lesions in
the CAM model, measuring angiogenesis by evaluating the vascular area and branch count
[24]. Pluchino et al. investigated endometriosis tissue derived from human ovarian endometriomas
in the CAM model, applying local treatments with testosterone or anastrozole and measuring
lesion growth [25]. Wang et al. and Ria et al. also studied tissue from human ovarian endometriomas,
with Wang et al. treating lesions locally with estradiol or puerarin and assessing
angiogenesis by manually counting vessels and calculating an angiogenic index, while
Ria et al. analyzed angiogenesis using microscopic vessel counting after immunohistochemical
staining [26]
[27]. In contrast to these studies, our research analyzed tissue from a variety of endometriosis
lesions. Additionally, our method of measuring angiogenesis using LASCA provides the
advantage of dynamic, real-time measurements. This approach also allowed for multiple
assessments over the course of three separate days. In other prior endometriosis studies,
the CAM model has primarily been used to examine endometrial tissue, which was subsequently
classified as endometriosis tissue following engraftment onto the CAM [28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36].
Immunohistochemical staining
The analysis of human endometriosis tissue poses a challenge, as endometrial glands
are often only scarcely present compared to endometrium tissue and surrounded by scarring
tissue. Nevertheless, in the above study, the endometriosis tissue in the CAM model
demonstrated vitality, as evidenced by positive Ki67 staining, confirming cellular
activity, and only partly positive Caspase-3 staining, a marker for necrosis. We observed
more Ki67 positive cells in endometriosis tissue than in endometrium, although no
clear changes were seen after inoculation in the CAM model. This was also observed
by Nap et al., who found that Ki67 does not accurately reflect the ability to form
lesions in the CAM model [37]. The successful implantation of viable endometriosis tissue (glands and endometrial
stroma) onto the CAM was confirmed via CK and CD10 staining.
Angiogenesis measurements
A key observation was the increase in blood perfusion in endometriosis tissue throughout
the CAM experiments, indicative of active angiogenesis. When analyzed individually,
75% of all patients showed an increase in blood perfusion, underscoring significant
interindividual differences. These variations could be influenced by factors such
as patient age, disease severity, the age of the endometriotic lesions, phase of the
menstrual cycle, different tissue origin or differences in proangiogenic factors within
the individual tissues. However, no definitive explanation for these differences could
be identified, highlighting the need for further investigation and consideration of
the above-mentioned factors in future experiments. The comparison between healthy
and endometriotic endometrium revealed significant differences. While healthy endometrium
showed no visible consistent changes in perfusion, the diseased endometrium exhibited
a notable increase. However, these observations are limited by the small sample size
and should be interpreted with caution. On the contrary, Gescher et al. observed no
differences in vessel density index analysis or vascular epithelial growth factor
(VEGF) mRNA expression between the endometrium of endometriosis patients and that
of non-endometriosis patients when incubated on the CAM for varying durations. This
suggests that factors other than the eutopic endometrium may contribute to the angiogenesis
of endometriosis lesions [30].
The role of the chicken embryo’s sex
Another focus of the study was the potential influence of the chicken embryo’s sex
on blood perfusion, as endometriosis is highly hormone-dependent and influenced by
differences in estrogen receptors. Female chicken embryos exhibit higher estrone sulfate
levels, leading to the hypothesis that blood perfusion might be gender dependent.
However, the results showed no significant differences between male and female embryos,
suggesting that perfusion in the CAM model is not primarily hormone-dependent and
that the sex of the chicken embryos does not affect the degree of blood perfusion.
Hence, a gender stratification does not seem mandatory for future angiogenesis investigations
in the endometriosis CAM model. Still, in ovo sexing could be relevant for analyzing
endometriosis tissue growth in the CAM model, as Pluchino et al. observed gender-specific
differences in the growth of endometriosis lesions [25].
The role of the #Enzian classification on angiogenesis in the CAM model
Noteworthy was the analysis of perfusion across different categories of the #Enzian
classification. A positive correlation was seen in perfusion and the #Enzian categories
P (peritoneum) and T (tube): higher classifications in both categories resulted in
higher perfusion values for all measurement days, indicating robust angiogenesis.
The higher the extent of endometriosis at the anatomical sites of the peritoneum or
the peritubarian region, the higher the angiogenesis. Paradoxically, category A, when
correlated with the angiogenesis measurements, demonstrated an inverse effect. The
lower the extent of endometriosis in the rectovaginal space and adjacent areas, the
higher the induced angiogenesis in the CAM model. This effect might be possible due
to specific tissue characteristics or local factors. Categories O (ovary), C (rectum),
and FA (adenomyosis uteri) showed inconsistent results without clear trends, while
category B (uterine ligaments) displayed no significant differences. These findings
should be interpreted cautiously and warrant further investigation as not all subgroups
of the #Enzian classification (such as A2, B0 and C3) were represented. The observed
correlation between the clinical extent of endometriosis and perfusion changes in
the CAM model can be explained by the presence of proangiogenic factors. In categories
P and T, the larger volume of affected tissue likely is accompanied by higher local
levels of VEGF and other angiogenic mediators, promoting increased perfusion. In contrast,
the inverse relationship observed in category A may result from specific tissue properties,
which could suppress angiogenesis. The lack of clear trends in other categories might
be due to variability in tissue composition or local microenvironmental factors.
Angiogenesis in the CAM model
Endometriosis induced angiogenesis in the CAM model might be explained by the tissue
accompanying human angiogenic factors such as VEGF or angiopoietin 2 found in the
microenvironment of the tissue. Nap et al. demonstrated that human anti-VEGF effectively
inhibited neovascularization in the CAM model, but only when angiogenesis was stimulated
by the engraftment of endometrial tissue. They hypothesized that this effect was driven
by the presence of accompanying human VEGF. In contrast, no impact on vessel growth
was observed when the human anti-VEGF antibody was applied to control CAMs without
human tissue [34]. It has also been suggested that tissue engraftment onto the CAM initially places
the tissue in a hypoxic state. Since hypoxia is a key driver of angiogenic factor
expression, this condition stimulates the tissue to produce higher levels of angiogenic
factors. This, in turn, promotes increased neovascularization and facilitates the
integration of the engrafted tissue, enabling it to connect with the chick’s vascular
system [31].
Angiogenesis plays a crucial role in endometriosis and is driven by proangiogenic
factors. These mechanisms explain the increase in perfusion and support the hypothesis
that endometriosis and tumors share similar angiogenic processes as angiogenesis has
been identified as a hallmark of cancer [7]
[8]
[38]. The CAM model is a versatile 3D-in-vivo-model, widely acknowledged for its ability
to assess tissue growth, metastasis, invasion, and angiogenesis, as well as to test
potential drugs and substances [39]. Its unique structure and accessibility make it particularly suited for studying
angiogenesis dynamics [38]. Notably, the CAM model facilitates precise evaluations of angiogenesis and can
be used to test the effects of angiogenesis-modulating substances, a method that has
also been employed by others [40].
Limitations and strengths of the study
Although angiogenesis was evaluated functionally via perfusion measurements, the molecular
mechanisms driving neovascularization – such as VEGF expression or hypoxia-induced
pathways – were not directly quantified in this study. No molecular assays were performed
to directly assess their involvement in the CAM model. Future studies should integrate
molecular analyses to better elucidate the mechanistic basis of angiogenesis in endometriosis.
A major strength of this study is the application of real-time, dynamic perfusion
measurement using LASCA over multiple time points, which offers a functional perspective
on angiogenesis in viable human endometriosis tissue. Furthermore, the comparison
across a range of lesion localizations using the #Enzian classification adds clinical
relevance. However, the limited sample size and the exclusion of direct molecular
profiling represent methodological constraints. Additionally, variability in tissue
origin and quality may introduce heterogeneity that is difficult to control. A key
limitation of this study is the absence of data on the menstrual cycle phase of the
included patients. To enhance experimental standardization in future research, it
is recommended to include only patients in a defined phase of the menstrual cycle
– such as the proliferative phase – and to exclude those using hormonal contraceptives,
as their use may represent a potential confounding factor. Another limitation is that,
for the bivalent categories of the #Enzian classification (such as B, O, T), only
a single value was recorded, which may reduce the resolution of subgroup analyses.
Conclusion
This study demonstrates that higher #Enzian scores correlate with increased blood
perfusion in categories P and T, while an inverse relationship was observed for category
A. The gender of the chicken embryo had no impact on outcomes, but blood flow increased
over time in 75% of endometriosis cases, contrasting with stable perfusion in healthy
tissue. This study is limited by its small sample size, which restricts the generalizability
of the findings. Furthermore, the lack of long-term follow-up data prevents conclusions
regarding sustained angiogenic activity or tissue viability beyond the observation
window. The absence of molecular analyses also limits mechanistic insight into the
angiogenic processes observed. An additional limitation is that for bivalent categories
of the #Enzian classification, only a single value per case was recorded, which may
reduce accuracy in reflecting multifocal lesion distribution. These limitations underscore
the need for further studies with larger cohorts, and the inclusion of molecular endpoints.
By successfully analyzing angiogenesis in human endometriotic tissue using the CAM
model, this work lays the foundation for future studies with larger sample sizes to
confirm these findings, explore further clinical correlations, and extend investigations,
for example, with angiogenesis-modulating substances.
Supplementary Material
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Supplementary Table S1: Angiogenesis measurements results for all IDs. D1–3 = Measurement Day 1–3, σ = standard
error, PU = perfusion units.
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Supplementary Table S2: Angiogenesis measurements results for #Enzian classification categories. D1–3 =
Measurement Day 1–3, σ = standard error, PU = perfusion units.
