2020 Article 2471

Malvezzi et al.
J Transl Med (2020) 18:311

https://doi.org/10.1186/s12967-020-02471-0 Journal of
Translational Medicine
REVIEW Open Access
Endometriosis: current challenges

in modeling a multifactorial disease of unknown
etiology
Helena Malvezzi1* , Eliana Blini Marengo2, Sérgio Podgaec1 and Carla de Azevedo Piccinato1*
Abstract
Endometriosis is a chronic inflammatory hormone-dependent condition associated with pelvic pain and infertility,
characterized by the growth of ectopic endometrium outside the uterus. Given its still unknown etiology, treatments
usually aim at diminishing pain and/or achieving pregnancy. Despite some progress in defining mode-of-action for
drug development, the lack of reliable animal models indicates that novel approaches are required. The difficulties
inherent to modeling endometriosis are related to its multifactorial nature, a condition that hinders the recreation of
its pathology and the identification of clinically relevant metrics to assess drug efficacy. In this review, we report and
comment endometriosis models and how they have led to new therapies. We envision a roadmap for endometriosis
research, integrating Artificial Intelligence, three-dimensional cultures and organ-on-chip models as ways to achieve
better understanding of physiopathological features and better tailored effective treatments.
Keywords: Endometriosis, Animal model, Drug efficacy, Organ-on-chip, Cell culture, Artificial intelligence
Background: setting and disease clinical symptoms and to detect the presence of ectopic
Endometriosis is a reproductive age-associated disease endometrial implants (so-called lesions) in the perito-
[1, 2] that has become the target of intense investigation, neal cavity and on pelvic organs [7]. Although imag-
as indicated by the increasing number of scientific papers ing methods such as transvaginal ultrasonography with
published. In the last 10 years, indeed, more than 75% bowel preparation and magnetic resonance are common
of endometriosis-related papers, appeared during that diagnostic tools, the gold standard diagnostic method
period according to Web of Science data. This surge of for endometriosis is still the histopathological analysis of
information directed to both laypersons and healthcare lesions collected during laparoscopic surgery [8]. The eti-
professionals improved the identification of symptoms, opathogenesis of endometriosis is not known. However,
augmented the odds of correct diagnosis as well as the there are theories on the origin of endometriotic lesions
awareness of available medical treatment [3]. in the peritoneal cavity. It is proposed by some investi-
Worldwide epidemiological studies show a mean prev- gators that stem cells originating the lesions are already
alence of 10% of endometriosis in the pre-menopausal there in the peritoneal cavity whereas others propose
population [4], with annual incidences in specific popu- that endometrium cells are seeded there by retrograde
lations varying from 0.112% [5] to 0.72% [6]. The dif- menstruation. The pathophysiology, however, is strongly
ficulties to reach the diagnosis comprise the need for influenced by other factors such as genetic predisposition
clinical and surgical expertise to evaluate correctly the and hormonal factors such as resistance to progesterone,
estrogen dependence; inflammation, angiogenesis, and
vascularization processes, oxidative stress, resistance to
*Correspondence: [email protected]; [email protected]
1
Hospital Israelita Albert Einstein, São Paulo, SP 05652‑900, Brazil apoptosis and immunological factors are also involved to
Full list of author information is available at the end of the article various degrees in lesion development (Fig. 1).
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Malvezzi et al. J Transl Med (2020) 18:311 Page 2 of 21
Fig. 1 Endometriosis characteristics. Legend: Endometriosis main characteristics and contributions to its knowledge—shown as puzzle pieces—
coming from studies using animal (blue pieces) or human-based models (orange pieces)
It is generally accepted that the lesions found in differ- The treatment for endometriosis usually aims at ame-
ent pelvic sites are the main cause of all clinical findings liorating symptoms, mainly pain, and/or achieving preg-
in endometriosis. However, many women have lesions nancy in infertile patients [14]. Early understanding of
while being asymptomatic, and such lesions are found the disease has come from experimental studies in ani-
during abdominal surgery done for reasons other than mals [15] while advances in the therapy of endometrio-
endometriosis. The prevalence of asymptomatic cases sis have come from observation of symptomatic patients
among studies is highly variable ranging from 6% [9] up during surgery [16], from research on patients’ data and
to 43.3% [10, 11]. The common clinical symptoms asso- from in vitro cell cultures [17].
ciated with endometriosis vary from mild to severe pain
and/or infertility. The pain observed in 30 to 80% of the The challenge of modeling endometriosis
cases can manifest itself as dysmenorrhea, acyclic pel- Currently, the main approaches to investigate endometri-
vic pain, deep dyspareunia, dysuria and dyschezia [12]. osis are human-based either in vivo or in vitro, using cells
Infertility is found in 30 to 40% of the cases [6]. Other or tissue samples. There are also experimental in vivo
symptoms are fatigue, diarrhea, constipation, bloating animal models [18]. The first type encompasses clinical
or nausea, mainly during menstrual periods, and pain- trials, patients’ observational and association studies.
related to mental health problems such as somatization Experimental in vitro studies include histopathological
or depression, heightened sensitivity and anxiety are also comparative experiments, as well as tissue fragments,
seen in endometriosis patients when compared to control cells and fluids obtained from resected lesions or aspira-
groups [13]. tion biopsies, in general called patient-derived tissue or
fluid. In vivo animal models are still required to test drug that the biological systems in all species are essentially
candidates that affect these processes and to support pre- a network of cellular and molecular mediators working
clinical trial testing. together for the organism’s survival.
The development of in vitro or in vivo models that rec- Rodents and non-human primates are the most com-
reate or exhibit the main characteristics of endometrio- mon animal-based models in endometriosis research
sis is a challenging task for several reasons. Firstly, it is with advantages and disadvantages. Rodents, such as rats,
not known how the disease starts and how it persists. mice, and hamsters have been used to investigate basic
Secondly, endometriosis comprises distinctive disease mechanisms and the development of new drugs. They
features such as ovarian endometrioma, and peritoneal are easy to handle and relatively inexpensive animals to
as well as deep infiltrative lesions. Lastly, endometrio- work with; however, endometriosis does not develop
sis has not a single pathophysiological process on which naturally in rodents, possibly because the endometrium
basis it could be modeled. Indeed, being a multifactorial is not shed during the estrous cycle. To work with this
and complex disease, endometriosis has also been associ- model, homologous or heterologous uterine tissue has to
ated to environmental [19], genetic [20], immunological be surgically introduced in the peritoneal cavity of these
factors [21] and hormonal changes (such as estrogen- animals [31].
dependency [22] and progesterone resistance [23]). Conversely, endometriosis develops spontaneously in
The difficulties to evaluate and model the onset of non-human primate models that have a natural men-
endometriosis stem because it is not clear when it actu- strual cycle, such as rhesus monkeys and baboons [32].
ally starts. It is accepted and reported that many women Similarly to women, the duration of the menstrual
had already had the disease for up to 12 years before the cycle in female Rhesus (Macaca mulatta), cynomolgus
appearance of symptoms [24]. The main mechanism put (Macaca fascicularis) and pigtailed macaques (Macaca
forward to explain the beginning of that disease is based nemestrina)) is around 28 days [33]. These animals also
on retrospective epidemiological studies [25]. They show have retrograde menstruation and their reproductive
that retrograde menstruation [16] is associated with anatomy (uterus, fallopian tubes and ovaries morphol-
endometrial implants attached to the peritoneal cavity, ogy) and endocrine influences are similar to humans
which would develop into endometrial lesions. How- [34]. Although non-human primates would be interest-
ever, almost all reproductive-age women (90%) have ret- ing models to study endometriosis, they are captivity-
rograde menstruation to the pelvic cavity, but only 10% sensitive animals and costly to maintain. In addition,
are actually affected by endometriosis. This suggests that spontaneous endometriosis develops at considerable
other associated factors are needed, besides retrograde low frequencies, which limits primates’ use for research
menstruation, for the onset and progression of the dis- purposes. Moreover, public opposition to non-human
ease in the peritoneal cavity [26]. primate research has been rising [35] and stricter rec-
Thus, the main current challenge to develop research ommendations exist for future work with non-human
models for the investigation of endometriosis goes primates are being designed. The latest report from the
beyond the creation of models that fully recapitulate the Scientific Committee on Health Environmental and
multitude of factors affecting this disease; that challenge Emerging Risks (SCHEER) on the need for non-human
requires the definition of measurable and clinically trans- primates in biomedical research, production and test-
latable effective endpoints. ing of products and devices, updated in 2017, proposes
Generally speaking, there is still a gap in the develop- 23 recommendations for future work with non-human
ment of adequate disease models to study essential bio- primates.
logical processes [27], as shown by the fact that between Some of those recommendations are requirements
50 and 70% of drugs that reach phase II and III in clini- already listed in the Europe Union Directive 2010/63,
cal trials fail to demonstrate effectiveness [28, 29]. Par- and have been merged into the committees’ series of rec-
ticularly, an ideal model to investigate endometriosis ommendations in order to amplify their importance and
should incorporate relevant disease characteristics, such to encourage full and rapid implementation, such as: to
as the same cellular pathways and the clinical behavior check, case by case, the need of using non-human pri-
observed in patients. mates; to provide solid harm-benefit assessment of using
Until now, the most common approach to this problem non-human primates; and to identify circumstances in
involved diverse animal models whose pathophysiologi- which to avoid using non-human primates [36].
cal processes are claimed to be somehow similar to the Other less representative models used in endometrio-
human ones [30]. This idea relies on the concept that the sis studies are chicken, rabbit, sheep and cow [37]. The
use of animal models facilitates the analysis of integrative studies using these models frequently focus on a particu-
and complex events that occur in vivo. Some may claim lar physiopathological aspect, which can be emulated.
Chickens have been used for disease mechanism studies, disease mechanisms, toxicity of compounds, teratogenic
as for instance, a chicken embryo chorioallantoic mem- effects, correct dosage and drugs delivery routes [49].
brane model was used to show the invasiveness potential Many studies using non-human primates were repeated
of epithelial and stromal endometriosis cells [40] and the many times to the point that ethical reasons emerged to
role of angiogenesis in lesions formation [38]. In turn, restrict the number of animals in investigation.
rabbits and cows have been used to model key points of An experiment in baboons with endometriosis,
endometriosis-associated infertility. The understanding revealed that, by reducing the expression of aromatase-
of reduced fertility with the increase of prostaglandins mRNA with Letrozole, an aromatase inhibitor, size and
was observed first in studies using rabbits [41]. In vitro volume of peritoneal lesions were diminished [50], sug-
oocyte maturation studies, using bovine oocytes and fol- gesting a correlation between estrogen restriction and
licular fluid collected from women with endometriosis, endometriosis lesion shrinking. In addition, progesterone
showed damage to the meiotic spindle, probably caused antagonists (RU-486—mifepristone and ZK 98.299—
by elevated oxidative stress [39, 42]. onapristone) that block estrogen effects on endometrium
Even though some contribution to advancing the and cause endometrial atrophy by suppressing prolif-
knowledge of endometriosis physiopathology originated eration were shown to be effective at both the reduction
from animal research, none of the reported animal mod- of the lesions and control of clinical symptoms. These
els has yet led to successful novel therapies [43, 44]. In studies initially conducted in non-human primate mod-
contrast, patient tissue or fluid-derived in vitro models els reaffirmed the use of labeled drugs on endometriosis
as well as other human-based models, such as patient treatment by demonstrating how those therapies could
tissue and observation studies, have given substantial be effective at controlling clinical symptoms [51]. How-
knowledge to the development of possible therapy tar- ever, none of the recent studies carried on these primates
gets, which could be tested in human-based clinical tri- to test new drugs for endometriosis was found in the
als. In vitro models present several advantages over biomedical literature, possibly due to the prevalent cul-
animal models, namely the easy access to target cells ena- ture of avoiding publication of negative results [52, 53].
bling identification of critical cellular and molecular con- Therefore, there is the possibility that non-human pri-
tributors to the disease. Although not contemplating the mate models are failing to reveal novel treatment strate-
complete biological system, in vitro models enable high- gies, since there has not been real progress in drug (label
throughput screening for therapeutic compounds, with or off-label) management of endometriosis. The available
overall lower ethical issues and costs. Important findings treatments and/or surgery are often insufficient to elimi-
on the physiopathology of endometriosis have come from nate the disease and to prevent its recurrence.
classic in vitro cultures of endometriosis-derived stromal
[45] or epithelial cells [46]; more recently three-dimen- Contribution of rodent models to endometriosis research
sional (3D) cultures [47] also came into use with several Because rodent models can be used for disease studies
advantages. and can be genetically modified, they have been used to
investigate endometriosis pathophysiology by means of
Animal models several molecular techniques. The studies were focused
Contribution of studies with non‑human primates mainly on finding possible new therapeutic targets and/
to endometriosis research or on improving existing ones [54], even though extrapo-
Much of the knowledge of pathophysiological processes lating data between species is difficult.
such as tissue attachment [18], endometriosis-related Isogenic mouse strains allowed mechanistic and reg-
pain mediators [12], hormonal dependency, progesterone ulatory approaches to investigation without the inter-
resistance, angiogenesis, oxidative stress and inflamma- ference of the individual genetic variation existing in
tion has evolved from research on non-human primate humans. These models offered insights into the effects
models done in the past [48] and also from non-animal of ectopic endometrial tissue growth [55], in vitro fer-
research [25]. The main reason for conducting research tilization, embryo development and implantation, and
on human disease mechanisms in animal models is the oviduct transport [56]. Pain mechanisms [57], as well
simulation of the disease and, eventually, the translation as inflammation [58] and organ adhesion [59] were
of findings to humans. Although most of animal model also studied in mice. However, one of the major prob-
studies in general, including non-human primate models, lems of using rodents in endometriosis research is that
have been used to understand the mechanism of action only superficial lesions can be induced in these ani-
of approved drugs on endometriotic lesions and on the mals and those are the simplest and perhaps the least
treatment of other symptoms, they have also failed to clinically important types of lesion. No study to date
provide convincing translatable results related to the has been able to model deep endometriotic lesions and
perhaps this is one of the reasons for the lack of suc- Contribution of animal models to a breakthrough
cess of rodent models to generate applicable results to in endometriosis treatments: from disease experimental
human endometriosis in the areas of pathophysiology models to clinical trials
and therapeutics. While there is exponential growth in the number of pre-
Even though rodent models have provided informa- clinical endometriosis studies, the translation of findings
tion to endometriosis on inflammation [54], oxidative obtained by studies on animal models into clinical trials
stress [60] and animal reproduction [61] there are still has been poor. A recent search in PubMed using a sim-
gaps in knowledge. Immune modulators [62] and anti- ple searching strategy “endometriosis and experimen-
oxidant/oxidative stress compounds have also been tal model” retrieved 1230 published articles in February
studied in rodent models, but attempts to translate of 2019 compared to 469 in 2009, which amounts to a
the results to humans did not lead to affective endo- 150% increase. In fact, no novel therapeutic compounds
metriosis therapies. It became clear that a number of for endometriosis have been proposed and there is only
women treated in clinical trials with hormonal thera- a small number of published clinical studies on drug
pies translated from results obtained in animal mod- development for endometriosis. This is an indication that
els of the disease did not respond to these treatments the understanding of the disease and of its processes is
[63] needing surgical lesion excision to alleviate symp- still scarce [52, 69]. A fact that also reveals the failure of
toms. It is important to mention that the current avail- several attempts to translate experimental investigation
able hormonal therapies are not indicated for women results to the clinical sphere is evidenced by the number
trying to become pregnant because they interfere of collapsed phase II or III trials [28]. Again, most studies
with ovulation. Because alternative therapies such as producing negative results and/or with conflict of inter-
immune modulators have been shown to reduce lesion est are not published [52], a situation that leads to ana-
size in rodents [64] there was hope that they would lytical bias and affects the perception of the existing state
also be effective in women and might improve fertility. of research on endometriosis.
However, this hypothesis was not confirmed in women The high rate of potential drug therapies that fail at effi-
with endometriosis. cacy testing protocols in humans (Table 1) is notewor-
The rodent model requires the surgical induction of thy. Even when previous animal studies had shown that
endometriosis, usually done by transplanting endo- a treatment would be effective and safe, the subsequent
metrial tissue or cells into the animal’s abdominal failure rate in humans was higher than 80%. This situa-
cavity. This procedure leads to superficial endome- tion suggests that preclinical animal studies are either not
triotic lesions, a model that basically favors the study translatable at all or are not being accurately designed;
of inflammatory processes caused by the implanted therefore, studies of this type may be considered poor
lesions instead of those caused by endometriosis itself. predictors of therapeutic efficacy of novel drugs in
Recurrent studies in rodent models indicating puta- patients [70]. Although similar failure percentages are
tive therapeutic molecules, which eventually fail to be reported in drug discovery studies for other diseases, it
active in humans, are common in research on endome- brings into debate, in the case of endometriosis, the lack
triosis, but also on other human diseases. One particu- of reproducibility and translatability of animal studies to
lar example is the treatment with resveratrol. In the humans. The results stemming from animal research in
mouse endometriosis-induced model this natural phe- the field of drug development would be a contributing
nol showed promising results by reducing lesion size, factor to that observed failure when tested in human tri-
inhibiting angiogenesis and inflammation in several als, raising the question of the quality of the results [71,
overlapping studies [65–67]; however, when tested in 72].
affected women in a trial (ClinicalTrials.gov Identifier: We propose a thought-provoking illustration of that
NCT02475564) the results were disappointing. scenario in Table 2. We surveyed PubMed without deter-
Recently, a mouse model that mimics endometrial mining dates for published articles on endometriosis that
shedding similar to human menstruation was created. used animals as experimental models for testing anti-
It is claimed to provide a closer model to human repro- inflammatory drugs. With the intent of comparing the
ductive physiology. That model still requires more in- outcomes of animal studies, a search on clinicaltrials.gov
depth validation as it still presents limitations such as was performed for registered clinical trials of the same
variation in the endometrial response, and in the dura- drugs used as human medication. Clearly, the number
tion and quantity of the hormonal stimulus [54, 68]. of ongoing or complete clinical trials testing drugs pre-
viously evaluated in animal models for the same disease
is small. Out of 36 drugs which had been tested in ani-
mal studies of endometriosis, 32 were object of clinical
Table 1 Number and characteristics of registered Clinical Trials on endometriosis
Clinical Trials StudyTitle Phase Conditions Interventions Aim Comments Conclusion
Gov Identifier
NCT01968694 Effects of Intravenous Not applicable Endometriosis Drug: IV Lidocaine Reduce pain – No statistical
Lidocaine on Endome- Drug placebo: IV diphen- analysis
triosis Pain hydramine provided
Malvezzi et al. J Transl Med
NCT00902746 Efficacy and Safety, Long- 3 Dysmenorrhea Drug: NPC-01 (Nore- Reduce pain Single Group Assignment No statistical analysis
Term Study of NPC-01 thisterone and Ethinyl (no placebo) provided
to Treat Dysmenorrhea Estradiol)
Associated With Endo-
metriosis
(2020) 18:311
NCT02475564 Resveratrol for Pain Due to 4 Endometriosis Drug: Placebo Reduce pain Authors conclude that No difference in pain score,
NCT01620528 Endometriosis 3 Endometriosis Drug: Resveratrol Safety and effi- a longer follow-up, for CA-125 and prolactin
NCT01931670 A Clinical Study to 3 Endometriosis Drug: Placebo cacy + Reduce pain instance 6 months, may serum levels
NCT02143713 Evaluate the Safety and 3 Endometriosis Drug: Elagolix Safety and effi- impact the results The use of Elagolix reduced
NCT01760954 Efficacy of Elagolix in 3 Endometriosis Drug: Placebo cacy + Reduce pain Participants who com- pain and had adverse
Subjects With Moderate Drug: Elagolix Safety and effi- pleted the 6-month effects not yet analyzed
to Severe Endometriosis- Drug: Placebo cacy + Reduce pain treatment period in the by authors
Associated Pain (ELARIS Drug: Elagolix Safety and effi- pivotal Study M12-671 The use of Elagolix reduced
EM-I) Drug: Placebo cacy + Reduce pain (NCT01931670) could pain and had adverse
A Global Phase 3 Study to Drug: Elagolix enter this extension effects not yet analyzed
Evaluate the Safety and study by authors
Efficacy of Elagolix in Participants who com- The use of Elagolix reduced
Subjects With Moderate pleted the 6 month pain and had adverse
to Severe Endometriosis- treatment period in the effects not yet analyzed
Associated Pain (ELARIS pivotal study M12- by authors
EM-II) 665 (NCT01620528). The use of Elagolix reduced
Global Study to Evaluate The study consists of pain and had adverse
the Long-Term Safety 2 periods: a 6 month effects not yet analyzed
and Efficacy of Elagolix treatment period and a by authors
in Women With Moder- post treatment follow-
ate to Severe Endometri- up period of up to
osis-associated Pain 12 months.
Study to Evaluate the
Long-Term Safety and
Efficacy of Elagolix in
Adults With Moderate to
Severe Endometriosis-
Associated Pain
Page 6 of 21
Table 1 (continued)
Gov Identifier
NCT00973973 Efficacy and Safety Study 2 Endometriosis Drug: Placebo Evaluate effects on endo- All participants still The use of Elagolix reduced
of Elagolix in Women Drug: Elagolix metriosis related pelvic enrolled in the study pain and had adverse
With Endometriosis pain and its safety received 150 mg elago- effects not yet analyzed
lix once daily by authors

NCT00619866 An Efficacy and Safety 2 Endometriosis Drug: Placebo Safety and efficacyand to Only Elagolix at 8 week
Study of Elagolix (NBI- Drug: Elagolix see the effect, if any, on treatment had a statisti-
56418) in Women With bone mineral density. cal difference from the
Endometriosis placebo group. Adverse
effects and bone mineral
(2020) 18:311
density were not yet

analyzed by authors
NCT00797225 Efficacy and Safety Study 2 Endometriosis Drug:Leuprorelin Compare drugs safety Leuprorelin is an approved Elagolix treatment and
of Elagolix Versus Drug: Elagolix and beneficial effects of endometriosis therapy Leuprorelin had a statisti-
Placebo or Leuprorelin Drug: Placebo elagolix cal difference from the
Acetate in Endome- placebo group. Adverse
triosis effects and bone mineral
density were not yet
analyzed by authors
NCT01791413 Effect of Pre-operative 1 and 2 Endometriosis Drug: Placebo Ovarian reserve changes Ovarian endometrioma No statistical analysis
Depo Medroxypro- Drug: depot medroxypro- after preoperative provided
gesterone Acetate on gesterone acetate medication
Serum Anti-mullerian
Hormone Level After
Laparoscopic Ovarian
Cystectomy of Endome-
triomas
NCT01269125 GnRH-a and Pregnancy Not applicable Endometriosis Drug: Leuprolide Improve the oocyte qual- Measured clinical preg- No difference between
Rate in In Vitro Fertiliza- Infertility Procedure: IVF ity and the fertility nancy rate, embryo groups
tion (IVF) Cycles quality, fertilization rate,
follicular fluid’s TNF-a
concentration
Page 7 of 21
Table 1 (continued)
Gov Identifier
NCT01682642 The Influence of Adjuvant 4 Infertility Drug: Zoladex Impact of treatment prior Number of Metaphase II No statistical analysis
Medical Treatment of Endometriosis to IVF on pregnancy Cells, Pregnancy Rate,
Peritoneal Endometriosis rates Good Embryo Quality,
on the Outcome of IVF. A Number of Pro Nuclear
Prospective Randomized Cell (2PN), Number of

Analysis Cryopreserved Embryos
and Total Follicle Stimu-
lating Hormone (FSH)
Dose
NCT00474851 The Effect of Hormonal 2 Endometriosis Drug: Norethindrone Maintain skeletal health Bone Mineral Density, Total Body Bone Mineral
(2020) 18:311
Add-Back Therapy in acetate + estrogens and quality of life in Total Body Bone Mineral Content was higher on
Adolescents Treated Drug: norethindrone adolescents Content (BMC), the intervention group
With a GnRH Agonist acetate + placebo
for Endometriosis: A
Randomized Trial
NCT01791413 Effect of Pre-operative 1 and 2 Endometriosis Drug: depotmedroxypro- Evaluate ovarian reserve Percentage changes of No statistical analysis
Depo Medroxypro- gesteroneacetate Serum Anti-Mullerian
gesterone Acetate on Hormone (AMH) at
Serum Anti-mullerian 2-week and 3-month
Hormone Level After post surgery
Laparoscopic Ovarian
Cystectomy of Endome-
triomas
NCT01190475 BGS649 Monotherapy 2 Endometriosis Drug: BGS649 Assess the safety and Proportion of patients No statistical analysis
in Moderate to Severe Drug: Placebo tolerability who develop 2 or more
Endometriosis Patients follicles with diameter
16 mm or larger
NCT02203331 Bay98-7196, Dose Finding/ 2 Endometriosis Drug: Placebo Assess efficacy and safety Different Dose Combina- No difference between
POC Study Drug: Levonorgestrel tions groups
Drug: Anastrozole
Drug: Lupron/Leuprolide
acetate
NCT01294371 Observational Program Not applicable Genital Endometriosis Non-interventional, obser- Assess rates of admin- No statistical analysis
to Assess Routine Use vational study istration of add-back
of Add-back Therapy in therapy in patients with
Patients With Endo- endometriosis
metriosis in Russian
Federation, Planned
for 6-month Course of
Lucrin Depot® (Leu-
prorelin)
All information was taken from US National Library of Medicine, ClinicalTrials.gov without imposing dates or limits
Page 8 of 21
Table 2 All published research on endometriosis and anti-inflammatory drugs done in animal models was found by searching PubMed database
and the corresponding clinical trials until January 2019
Author, year Main drug Drug effect Animal model (n) Conclusion Clinical Trial using ClinicalTrial status New endometriosis
the same drug research advance
(ClinicalTrials.gov
Identifier)
Saltan et al. (2016) Viburnum opulus Antimicrobial, antioxi- Rat (30) Endometrioctic vol- The Efficacy of Vibur- Completed Endometriosis was an
[146] dant, hepatoprotec- ume reduced; Perito- num Opulus 3X in exclusion criteria
tive, hypogly-cemic, neal TNF-α, VEGF and the Treatment of Pri-
antinociceptive and IL-6 concentration mary Dysmenorrhea
anti-inflammatory reduced (NCT02467543)
(2020) 18:311
Bostanci et al. (2016) Aloe Vera Antioxidant Rat (24) Endometrioctic vol- 43 trials using Aloe – –
[127]) ume reduced; Perito- Vera, none using it to
neal fluid antioxidant treat endometriosis
levels raised
Zhou et al. (2012) [152] Salvia miltiorrhiza Anti-inflammatory, Rat (40) Reduced levels of 2 trials with Salvia – –
Bunge antioxidant CA-125, TNF-α and miltiorrhiza Bunge,
IL-18. Increased one for Polycystic
levels of Il-13 Ovary Syndrome and
the other for Pul-
monary conditions.
None for endome-
triosis
Neto et al. (2011) [142] Uncariatomentosa Anti-inflammatory, Rat (40) Contraception Phase II Clinical Unknown Not associated with
immunomodula- Trial of UncariaTo- endometriosis
tory, pro-apoptotic, mentosa (Cat´s
antioxidant and Claw) in Patients
contraceptive With Advanced
Solid Tumors
(NCT02045719)
Sun et al. (2011) [149] FubaoDangguiJiao Abortion prevention Rat (40) Endometrioctic vol- No clinicaltrials – –
and regulate men- ume reduced
struation
Neto et al. (2011) [143] Uncariatomentosa Anti-inflammatory, Rat (25) Endometrioctic vol- Phase II Clinical Unknown Not associated with
immunomodula- ume reduced Trial of UncariaTo- endometriosis
tory, pro-apoptotic, mentosa (Cat´s
antioxidant and Claw) in Patients
contraceptive With Advanced
Solid Tumors
(NCT02045719)
Page 9 of 21
Table 2 (continued)
(ClinicalTrials.gov
Identifier)
Qu et al. (2005) [145] Yiweining Anti-inflammatory Rat (50) Reduced serum levels Comparative study Completed Yiweining suppressed
of TNF-α, IL-6, and on the efficacy post-operational relapse

IL-8 of Yiweining and and dissemination of
Gestrinone for endometriosis III
post-operational
treatment of stage III
endometriosis
(2020) 18:311
Xiao et al. (2002) [151] Tripterygium Wilfordi- Anti-inflammatory, Rabbit (22) Endometriotic volume 6 clinical trials found – Its clinical use is limited
ipolyglycoside immune modulation, and antiendometrial on Pubmed. duo its severe toxicity
antiproliferative, and antibody reduced; Noneendometriosis-
proapoptotic serum FSH and LH related
levels decreased
Chen et al. (2010) 15-epi-lipoxin A4 Anti-inflammatory Mouse (45) Supression of lesion 14 trials, none using – –
[128]) growth for endometriosis
research
Machado et al. (2010) cyclooxygenase-2 Inhibitor of the Rat (20) Supression of lesion 682 trials, none using – –
[139] inhibitor enzyme responsible growth cyclooxygenase-2
for the production inhibitor to treat
of prostaglandins endometriosis
which are one of
the responsibles for
inflammation
Nenicu et al. (2017) 1-Telmisartan and 1-Anti-hypertensive; Mouse (42) Supression of lesion 282 and 47 trials, none – –
[141] 2-parecoxib 2-Inhibitor of the formation and using Telmisartan
(cyclooxygenase-2 enzyme responsible growth nor parecoxib to
inhibitor) for the production treat endometriosis
of prostaglandins respectively
which are one of
inflammation
Jana et al. (2012) [133]) Curcumin Anti-oxidant and anti- Mouse (48) Supression of lesion Curcumin Supplemen- Not yet recruting Curcumin was used
inflammatory growth tation for Gyneco- to trattubo ovarian
logical Diseases abcess, endometritis,
(NCT03016039) wound infection.
Matsuzaki et al. (2004) cyclooxygenase-2 Inhibitor of the Rat (74) Supression of lesion 682 trials, none using – –
[140] inhibitor enzyme responsible growth cyclooxygenase-2
for the production inhibitor to treat
of prostaglandins endometriosis
which are one of
inflammation
Page 10 of 21
Table 2 (continued)
(ClinicalTrials.gov
Identifier)
Laux-Biehlmann et al. 1-celecoxib (cyclooxy- 1-Inhibitor of the Mouse (number of 1-Decrease in percent- 1. 496 trials using – –
(2016) [138] genase-2 inhibitor); enzyme responsible animals was not age time in the celecoxib, none for
2-antinociceptive for the production described) standing position, endometriosis; 2- 55
(Nav1.8 blocker of prostaglandins associated with pain. trials for antinoci-
A-803467) which are one of ceptive none with
the responsibles endometriosis; 2-1
for inflammation; study using sodium
(2020) 18:311
2-induce analgesia channel blocker but

in inflammatory pain not endometriosis
models related
Elmali et al. (2002) Caffeic acid phenethyl Anti-inflammatory Rat (30) Decrease in osxidative 1 clinical trial – –
[130]) ester stress parameters (NCT02744703) but
not endometriosis
related
Barretto et al. (2016) Acetylsalicylic acid Cytolytic and antineo- Rabbit (40) Partial supression of 1754 trails, none for – –
[125]) plastic lesion growth endometriosis treat-
ment
Abbas et al. (2013) β-Caryophyllene Anti-inflammatory Rat (27) Supression of lesion 1 trial (NCT03152578) – –
[123]) growth without but not endometrio-
interfering with sis related
fertility
Kizilay et al. (2017) 1- Curcumin; 2-Defer- 1-Anti-oxidant and Rat (30) Supression of lesion 58 trials using Defer- – –
[136] oxamine anti-inflammatory; growth oxamine, but none
2-Iron-chelating for endometriosis
treatment
Kiykac Altinbas et al. Montelukast Anti-inflammatory Rat (33) Supression of lesion 287 trails using – –
(2015) [135]) growth Montelukast (also
searched for Singu-
lair and MK 0476),
but none for endo-
metriosis research
Page 11 of 21
Table 2 (continued)
(ClinicalTrials.gov
Identifier)
Bayoglu Tekin et al. Resveratrol Anti-angiogenic, Rat (40) Reduction on Anti- Resveratrol for Pain Completed (phase 4) No difference between
(2015) [126]) antioxidant, antiangiogenic, antioxi- Due to Endome- groups regarding pain
inflammatory dant, anti-inflamma- triosis (ResvEndo) and serum CA125 and
tory parameters (NCT02475564) Prolactin Levels reduc-
tion after 42 days
of medication. 22
patients per group.
(2020) 18:311
Hull et al. (2005) [132] Nimesulide (COX-2 Anti-angiogenic, Mouse (30) No supression of 17 trails, none for – –
inhibitor) antioxidant, anti- lesion growth endometriosis treat-
inflammatory ment
Efstathiou et al. (2005) 1-Aspirin; 2-Celecoxib; Anti-angiogenic, Mouse (105) Celecoxib and Ibuprofen is only used – –
[129]) 3-Ibuprofen; 4-Indo- antioxidant, anti- indomethacin were in 2 clinical trials but
methacin; 5-Nap- inflammatory most efficacious as rescue medica-
roxen; 6-Sulindac; to supress lesion tion, not for testing
7-Rofecoxib growth (NCT02437175;
and aspirin had no NCT01942122)
effect
Takai et al. (2013) [150] Parthenolide Anti-cancer and anti- Mouse (30) Inhibited endome- 4 trials, none for endo- – –
inflammatory triosis like lesion metriosis propose
formation
Kurt et al. (2015) [137] Colchicine Anti-inflammatory Rat (16) Supression of lesion 127 trials, none to – –
formation and study endometriosis
growth
Güney et al. (2008) Melatonin Antioxidant and anti- Rat (25) Supression of lesion 441 trials using Mela- – –
[131] inflammatory formation and tonin, none using
growth it to treat endome-
triosis
Kilico et al. (2014) Dexketoprofen tro- Inhibitor of the Rat (60) Supression of lesion 18 trials using Dexke- – –
[134]) metamol (Cyclooxy- enzyme responsible formation and toprofen trometamol
genase-2 enzyme for the production growth (also searched as Enan-
inhibitor) of prostaglandins tyum), none to treat
which are one of endometriosis
inflammation
Rudzitis-Auth et al. Resveratrol Anti-angiogenic, Mouse (20) Suppression of new Resveratrol for Pain Completed (phase 4) No difference between
(2013) [65] antioxidant, anti- microvessels in Due to Endome- groups regarding pain
inflammatory lesions triosis (ResvEndo) and serum CA125 and
(NCT02475564) Prolactin Levels reduc-
tion after 42 days
of medication. 22
patients per group.
Page 12 of 21
Table 2 (continued)
(ClinicalTrials.gov
Identifier)
(2020) 18:311
Agostinis et al. 2015 1- N-acetyl cysteine; Antioxidant, anti- Mouse (16) Supression of lesion No endometriosis – –
[124]) 2-alpha-lipoic acid; inflammatory formation and related clinical trial
3- bromelain growth with neither drug
Soylu Karapinar et al. Dexpanthenol Antioxidant, anti- Rat (20) Supression of lesion 26 trials using Dexpan- – –
(2017) [148] inflammatory growth thenol (or Panthe-
nol and Bepanthen),
but nnone for endo-
metriosis treatment
Siqueira et al. (2011) Acetylsalicylic acid Cytolytic and antineo- Rabbit (40) Supression of lesion 1754 trails, none for – –
[147] plastic growth endometriosis treat-
ment
Olivares et al. (2011) Celecoxib (Cyclooxy- Anti-angiogenic, Mouse (48) Supression of lesion 1-Use of Rosiglitazone 1-Terminated; 2-With- –
[144] genase-2 enzyme antioxidant, anti- formation and in the Treatment drawn
inhibitor) and rosigli- inflammatory growth of Endometriosis
tazone (NCT00115661);
2-ffect of Rosiglita-
zone on Peritoneal
Cytokines in Women
With Endometriosis
(NCT00121953)
Page 13 of 21
trials not related to endometriosis, while only 4 were (or at all, as opposed to diminishing symptoms [76]. Accord-
are being) tested in clinical trials as treatments for endo- ing to Paolo Vercellini´s view, the treatment should not
metriosis, namely: Resveratrol (NCT02475564), Yiwein- target the lesions but the clinical signs, as researchers
ing [73], Curcumin (NCT03016039) and Rosiglitazone should aim at providing patients comfort and address
(NCT00115661; NCT00121953). Two of these four tri- their main complaints [74] instead of masking advances
als are finished by now, one is not yet recruiting, and one with endpoints of questionable use for the progression of
was interrupted (Table 2). new drugs discovery. Thus, it is still debatable what would
That small number of clinical trials shows that previ- be the most adequate approach to study endometriosis. If
ous animal testing for endometriosis-active drugs does endometriosis is seen as a chronic, yet incurable, but not
not translate in clinical trials to verify their effects on a life-threatening disease, then mitigation or the end of
patients. We searched the United States of America symptoms seem to be reasonable approaches to improve
National Library of Medicine—clinicaltrial.org website quality of life for the patients. At least, this would be a
and found 213 registered clinical trials relating to endo- goal until the etiology of endometriosis becomes clear
metriosis drug testing (last search was performed Janu- and, in consequence, research will seek novel approaches.
ary 2019), 105 were “Completed”, 36 were “Recruiting”
or “Enrolling by invitation”, 28 were “Not yet recruit- In vitro studies using patient‑derived tissue
ing”, “Active”, or with “Not recruiting status”, 20 were and fluids, and in vivo human‑based‑models
“Suspended”, “Terminated” or “Withdrawn” and 24 had Although experimental research on animals whose goal
“Unknown status”. was lesion regression failed to produce translatable data
For all completed trials, only 19 were published and, to humans, human-based research approaches to endo-
of these, 18 aimed at finding novel therapeutic activities metriosis (including clinical trials, observation and
for specific endometriosis symptoms. All the investigated association studies) are generating the most relevant
drugs had already been tested for other diseases and were publications in this field. Notably, the most cited original
approved by the FDA. Some of the tested drugs were and currently used article for understanding and classify-
specifically “labeled”, while others were “off-label” drugs ing endometriosis grades is the Revised American Society
used for endometriosis treatment (Table 1) [51]. Out of for Reproductive Medicine classification of endometriosis:
those 18 studies, seven aimed at pain reduction, four 1996. This article reports clinicians’ observations of dis-
were focused on fertility improvement, one study aimed ease behavior by visual and histopathological analysis of
at improving life quality of adolescents and nine studies biopsies [77]. In addition, when revising high-impact of
aimed at improving drug tolerance and safety (some tri- endometriosis-narrative reviews [78], the bibliography
als had multiples aims). Interestingly, none of these drugs cited by the reviewers focused mostly on in vitro patient-
undergoing new therapeutic testing originated from pre- derived tissue and/or fluids and on human-based studies,
clinical studies using animal models. the ones that were considered to advance our knowledge
Another important point that reveals the lack of com- of endometriosis. It is worth noticing that among most
parability between clinical trials and animal studies is the highly cited papers on endometriosis, there are two
fact that, while animal-based studies on experimental human-based studies. One of these deals with the delete-
drugs have lesion reduction as measured positive out- rious effect of continuous ovulatory cycles on endome-
come of the test, most clinical trials have as main out- triosis persistence [79] and the other revealed a mutation
come the reduction of disease symptoms (mainly pain in the ARID1Ain gene found in endometriosis-associated
and infertility, or fertility preservation). ovarian carcinomas [80]. Furthermore, a highly innova-
Moreover, the endpoint of most animal model studies tive target for the non-hormonal treatment of endome-
is based on lesion regression, as if it would be consid- triosis, P2X3, which is thought to be associated with
ered an important endpoint in endometriosis research. development and maintenance of chronic pelvic pain,
However, endometriosis clinical trials (Table 1), do not was also identified without using animal models [81].
concern and do not look for lesion regression when stud- Highlights of advances brought about from human-
ying a potential new drug. Curiously, lesion regression based research approaches comprise: large-scale
has never been chosen as an end-point of clinical stud- integrated genome-wide RNA sequencing [82]; endo-
ies. Clinical studies were aimed at finding ways to miti- metriosis peritoneal fluid a potent oxidative fluid and
gate symptoms, which seems to be the utmost point to modifier of miRNA expression profile in eutopic cells
be addressed for endometriosis [8, 74, 75]. An Interna- [83]; miRNA analysis associated with pelvic pain [84];
tional Consensus Workshop proposed a list of priorities modulation of pain [85], anxiety and depression [86];
for endometriosis research and under the “treatment and tissue-specific expression analysis of endometriosis tis-
outcome” section, lesion regression was not mentioned sue samples by laser capture microdissection (reviewed
in [87]); genetic associations between endometriosis and maintenance of endometriosis implants because NF-κB
obesity-related traits [88]. has the ability to regulate a large array of genes involved
Genomic wide association studies (GWAS) have in different processes of immune and inflammatory
emerged more than a decade ago [89] as an important responses, thus controlling genes related to cell prolifera-
tool to study complex genetic diseases. Particularly for tion, adhesion, apoptosis, and inflammation [97].
endometriosis, GWAS has helped to infer causality and These findings have emerged from in vitro observations
uncover pathogenic mechanisms. It may be helpful in and originated a whole new group of drugs for endome-
order to dissect all possible phenotypes of the disease, triosis treatment. One example are the progestins, a class
although this has not been broadly tested yet. To date, of drugs that inhibit inflammatory pathways, angiogen-
GWAS revealed more than 10 genomic regions associ- esis and oxidative stress in endometriotic cells. These and
ated with endometriosis, a condition that explains less other observations have opened up a new field, which
than < 4% of inheritance [82, 90]. Some of these studies may be critical for understanding mechanisms underly-
revealed, for instance, novel associations between endo- ing the development, progression, variability and symp-
metriosis (and some specific phenotypes) with the Wnt tomatology of endometriosis.
Family Member 4 (WNT4) locus [91] and the mitogen- In addition, a study with 2D cell cultures found that
activated protein kinase (MAPK)-related pathway [90]. inhibition of IL-1β in eutopic endometrial stromal cell
The gathered information has allowed new insights into cultures could potently alter decidualization in vitro,
the biological pathways that could be associated with the suggesting a way to improve endometrial receptivity and
pathology of endometriosis, besides being indicative of pregnancy success in women with endometriosis [98].
new potential therapeutic targets. Moreover, with the use of 2D culture of eutopic endome-
trial stromal cells, researchers found increased expres-
Contributions of in vitro models for endometriosis sion of a gene (H19 gene) in endometriosis. This gene is
research responsible for tumor cell invasion and migration, and is
Biopsies are important cell sources for in vitro culture. regulated by estrogen and progesterone. The dysfunction
In endometriosis research cells cultured on flat surfaces, of such a gene might act as an important factor for endo-
also called two-dimensional (2D) cellular models, have metrial stromal cell invasion and migration, contributing
generated hundreds of publications. Co-culture mod- to fibrous tissue formation [99].
els are cell cultures in which two (or more) different cell Basic research studies using cell cultures from endome-
types are cultured together, often separated by a fluid- triosis patients shed light on cellular behavior and pos-
permeable membrane. They are mostly used to assess the sibly new ways of managing cellular growth. Dienogest
effect of one cell type on another cell and also to verify was shown to downregulate the expression of CYP19A1
how cells interact [92]. These two previous models lack (aromatase gene), inflammatory and neuroangiogenesis
important features for cellular function feature such as in immortalized endometriotic epithelial cell lines [100],
a cellular physicochemical microenvironment, tissue- also in a spheroid cell culture system (3D system), dien-
specific architecture, and blood flow perfusion. In the ogest inhibited mRNA prostagladin synthases, protein
quest to create better models to observe cell to cell inter- expression, and the nuclear factor-κB activation, which
action, investigators have recently started to use three- could contribute to therapeutic effect on endometrio-
dimensional (3D) models (sometimes called spheroid or sis [101]. Recently, a double-blind phase 3 clinical trial
organoid cultures) [93]. These 3D models aim at provid- showed the benefits of using dienogest for endometriosis
ing better phenotype and gene expression than 2D cul- treatment, by reducing endometriosis-associated pelvic
ture systems, opening the possibility to test interactions pain [102], which could be also related to inhibition of
among different cells found in endometriotic lesions [94]. prostaglandin mRNA synthases.
By means of in vitro conducted experiments, research-
ers have found elevated levels of cytokine and inflamma- Roadmaps for endometriosis research:
tory mediators in the peritoneal cavity and phenotypic perspectives on new approaches of endometriosis
progesterone resistance, a condition seen in endome- The ultimate desirable goal of modeling endometriosis is
triosis patients [95]. Progesterone has anti-inflammatory to be able to develop a strong project, hypothesis-driven,
activity and the results show a link between progester- and based on solid science. We envision that with the
one-resistance and chronic inflammatory states. Sign- advance of high throughput genomics analysis, epig-
aling by progesterone in endometrial cells induces enomics, transcriptomics, proteomics and metabolomics
suppression of NF-κB, which stands for nuclear factor in cell biology, these analyses will be carried out in endo-
kappa-light-chain-enhancer of the family of B proteins metriosis-derived tissue samples. There will be a need
[96]. This suppression may lead to the establishment and for an integrated guideline of all “omics” to facilitate the
access to research data and to validate and increase their examination of cell shape changes under different condi-
reproducibility and robustness rates. This should also tions and the staining for surface and cytoplasmic mol-
facilitate both interpretation and functional use of such ecules by fluorescence-labeled antibodies.
data [103]. Importantly, it is expected that these data will In endometriosis, AI and text mining were put to prove
provide alternative strategies for diagnosis and target by unraveling endometriosis most important associ-
the treatment of symptoms. The high-dimensional gene ated genes that were already published, besides discov-
expression data presented by Linda Giudice’s group [104] ering new ones, building a genome wide gene network
represent the first steps toward that approach, by put- [107]. This network, coupled with other AI applications,
ting forward a molecular classification of endometrium formed a list of 5 top genes to be studied in endome-
candidate-genes and offering a correlation with disease triosis and perhaps used as biomarkers for the disease.
conditions and stage. Another work implemented machine learning AI to find
Besides inherent technical limitations such as dif- a relationship between endometriosis and other diseases,
ficulties to reach statistical power because of the large as for instance benign breast disease, cystitis and non-
number of observations required, GWAS has recently toxic goiter. Using a similarity matrix, the method evi-
allowed new insights into novel endometriosis-associated denced other diseases that women with endometriosis
biological and pathogenetic pathways and giving rise to had in common [108], showing that this would be a use-
new hypotheses to understand that disease. For instance, ful method for understanding how endometriosis works
a GWAS study suggested that the pathogenesis of and how it could be associated with other disease and
advanced endometriosis is likely to be distinct from less symptoms.
severe disease [82]. This hypothesis is possible due to the These techniques facilitate phenotype measure-
integration of molecular sequencing/expression results ments of individual cells and analyses of heterogeneous
with detailed clinical and demographic data. In fact, responses, providing in-depth insights into biological
GWAS seems to have the potential to cover the complete processes [109]. Convolutional neural network (CNN),
spectrum of disease-related effects [82]. In endometrio- multi-scale convolutional neural network (MSCNN) and
sis, no publication has yet explored the so-called phe- deep convolutional neural network (DCNN) are remark-
nomics (the set of phenotypes physical and biochemical able recent AI advances. Developing rapidly over the past
traits by the organism due to genetic and environmen- 6 years, those approaches are capable of fast “learning”
tal influences) approach, although this is a promising from captured images (by sensing pixel intensity values)
avenue [82]. Moreover, PhenoWAS, (Phenomics Wide or from input data. By teaching itself, these programs
Association Analysis) is an opposite way of looking at can automatically improve their analysis (machine learn-
data in comparison to GWAS classical approach, and ing) [110, 111]. In order to become the next-generation
might serve as a powerful way to investigate endometrio- tool for the diagnosis of human disease, CNN in all its
sis. PhenoWAS consists of investigating which diseases different forms, MSCNN and DCNN, are being tested
(many diseases at the same time) are associated with a and compared to diagnosis by clinicians, proving to be
given genetic variant [105]. This approach is only possible more accurate, sensitive and specific ways for the clas-
if a high-dimensional phenotypic dataset is available or sification of some diseases such as lung cancer [112]. A
very large and well phenotypically-characterized cohort potential contribution of such a system to clinical deci-
is used. sion making and therapeutic management of endo-
Years ago, it would have been impossible to foresee metriosis should help with earlier diagnosis. This may
the emergence of artificial intelligence (AI) models to contribute to identify disease phenotypes and how they
advance our knowledge on disease. However, text ana- relate to clinical symptoms, thus offering the possibility
lytics (text mining based on AI), high-throughput cell- of custom-tailored treatments according to phenotypes
based assays, automated microscopy-based high-content (based on symptoms, images or gene expression features)
screening and convolutional neural network can facili- and genotypes (based on DNA sequencing). In addi-
tate the way we look at data. Text mining is a process of tion, AI combined with bioinformatics can be useful for
adapting a massive amount of unstructured information retrieving relevant data on novel targets or markers for
into significant interpretable data for research analysis, endometriosis [113], as well as improving diagnosis, the
such as proteins, genes and other markers to eventually understanding of gene pathways and protein networks.
reach a set of findings relevant to endometriosis research Cell-culture has helped our understanding of gene
[106]. AI can be applied to high-throughput cell-based and protein expression [114], the roles of hormones in
assays’ data and to automated microscopy-based high- endometriosis [115] and also the possibility to recre-
content screenings. Both approaches allow the parallel ate early stages of endometriosis in a “Petri-dish” [116].
monitoring of multiple cell phenotypes, as well as the Three-dimensional models offer the opportunity to study
aspects of endometrial stromal and epithelial cells com- alternatives to in vivo testing of efficacy, toxicology and
munication and paracrine cross-talk between stromal safety of new drugs, and the real value of in vivo tests.
and epithelial cells [117]. While endometrial and endo- In vitro and in silico tests have been established and are
metriotic cells can also be cultivated with biopolymer under validation, particularly for efficacy studies, instead
and pre-fabricated scaffolds, hydrogels and cell sheets, of using naturally or induced animal models. Advances in
microfluidic devices like “organ-on-chips” are gaining in vitro modeling technologies, as for instance 3D blood
space as new research models for endometriosis [118]. vessels printing [122], are promising new platforms for
Organ-on-chips allows morphological and functional the understanding of angiogenesis and vasculogenesis
changes of cells to occur in a microfluidic device, with physiology.
the aim of reproducing enough cellular functions that
the model can be used to test therapeutic drugs and Conclusion
toxicity effects [119]. By creating an “endometrium-on- Finally, we envision personalized treatments that would
chip” to study the role of the perivascular stroma in the utilize complex decision algorithms (text mining, neural
human endometrium, this method could indirectly offer networks, etc.) capable of integrating genomics, tran-
information on in vivo mechanisms [120]. Moreover, the scriptomics, epigenomics, proteomics, microbiome,
breakthrough of cultivating the reproductive tract “on- exposures, behaviors, informatics and clinical/phono-
a-chip” [121] has offered hope to model cyclic hormonal typical data both cross-sectionally and throughout the
effects in hormone-dependent diseases, such as endome- patients’ lifetime. In our view, health regulatory agencies
triosis. Thus, endometriosis research can now count on should question the currently required preclinical data
several new approaches to improve our knowledge that from non-translatable animal models, and instead more
is, yet, rather limited. multicenter, randomized controlled trials should be per-
The lack of translation of results from animal to human formed to test different endometriosis treatment options
has been highly disappointing. Situations like this have against defined outcome measures, such as relief of pain
led many researchers to consider the use of animals to and/or infertility. This will bring back quality of life to
model human disease is a limited path concerning time, endometriosis-suffering women.
resources, and money. Perhaps a positive outcome of the
failure in translating research focused on therapy from
Abbreviations
animal experiments to human trials is the recognition CNN: Convolutional neural network; DCNN: Deep convolutional neural net-
of the limitations of using animals for experimental pur- work; GWAS: Genomic wide association studies; MSCNN: Multi-scale convolu-
poses [35]. tional neural network; SCHEER: Scientific Committee on Health Environmental
and Emerging Risks; 3D: Three-dimensional.
Final remarks Acknowledgments

While researchers working with rodent and other animal We would like to thank Dra. Ises de Almeida Abrahamsohn for English revision
and for critically reading the manuscript. We would like to acknowledge Isis
models of endometriosis investigate reduction of lesion Mozetic for technical support on drawing the figures. We would like to thank
size and of cell proliferation, apoptosis parameters, or Marcia Triunfol, Lindsay Marshall and Brinda Poojary for their comments and
protein and gene expression data, from the point of view suggestions to the manuscript.
of achieving or advancing therapy for human endome- Authors’ contributions
triosis these models have provided scanty results. Analy- HM made substantial contributions to the conception, design of the work;
sis of available reported publications shows that animal acquisition, analysis, interpretation of data; drafted the work and revised it. HM
also approves the submitted version and is accountable for her own contribu-
models hardly produce robust data to provide candidate tions and had ensure that questions related to the accuracy or integrity of any
drugs for clinical trials. Thus, it is questionable whether part of the work are appropriately investigated, resolved, and documented in
these models should constitute the main strategy for the literature. EBM substantially revised the work and approves the submitted
version and is accountable for her own contributions and had ensure that
understanding endometriosis pathophysiology and for questions related to the accuracy or integrity of any part of the work are
the development of new therapies. appropriately investigated, resolved, and documented in the literature. SP
Hardly new compounds will emerge for the treatment have made substantially contribution to the design of the work and substan-
tively revised it. SP approves the submitted version and is accountable for
of endometriosis based on therapeutic interventions in his own contributions and had ensure that questions related to the accuracy
animal models. It is important to note that although the or integrity of any part of the work are appropriately investigated, resolved,
use of animals presents handling and physiological dis- and documented in the literature. CAP made substantial contributions to the
conception, design of the work, interpretation of data and revised it. CAP also
advantages, regulatory agencies (i.e., FDA United States approves the submitted version and is accountable for her own contributions
and ANVISA Brazil) still require small and large animals and had ensure that questions related to the accuracy or integrity of any part
for new drug testing, particularly for efficacy, toxicology of the work are appropriately investigated, resolved, and documented in the
literature. All authors read and approved the final manuscript.
and safety analysis. A topic of discussion by the scien-
tific community, industries and regulatory agencies are
Funding 13. de Fonseca M, Aragao LC, Sessa FV, de Resende JA, Crispi CP, Crispi CP.
This work was supported by the Human International Society. HM is sup- Interrelationships among endometriosis-related pain symptoms and
ported by São Paulo Research Foundation (FAPESP-Fundação de 464 Amparo their effects on health-related quality of life: a sectional observational
à Pesquisa do Estado de São Paulo; Grant Number 2018/11042-0). CAP has study. Obstet Gynecol Sci. 2018;61(5):605–14.
a visiting Scientist institutional Grant from Sociedade Beneficiente Israelita 14. Farquhar C. Endometriosis. BMJ. 2007;334(7587):249–53.
Brasileira Albert Einstein (UNIEMP). 15. Colette S, Donnez J. Animal models in endometriosis experimental
research| Modèles animaux dans la recherche expérimentale sur
Availability of data and materials l’endométriose. Gynecol Obstet Fertil. 2012;40(9):494–6.
All data generated or analysed during this study are included in this published 16. Sampson JA. Metastatic or embolic endometriosis, due to the men-
article [and its supplementary information files]. strual dissemination of endometrial tissue into the venous circulation.
Am J Philol. 1927;3:2.
Ethics approval and consent to participate 17. Brueggmann D, Templeman C, Starzinski-Powitz A, Rao NP, Gayther
Not applicable. SA, Lawrenson K. Novel three-dimensional in vitro models of ovarian
endometriosis. J Ovarian Res. 2014;7:1.
Consent for publication 18. Greaves E, Critchley HOD, Horne AW, Saunders PTK. Relevant human tis-
Not applicable. sue resources and laboratory models for use in endometriosis research.
Acta Obstet Gynecol Scand. 2017;96(6):644–58.
Competing interests 19. Bellelis P, Podgaec S, Abrão MS. Environmental factors and endometrio-
The authors declare that they have no competing interests. sis. Rev Assoc Med Bras. 2011;57(4):448–52.
20. Kobayashi H, Imanaka S, Nakamura H, Tsuji A. Understanding the role of
Author details epigenomic, genomic and genetic alterations in the development of
1
Hospital Israelita Albert Einstein, São Paulo, SP 05652‑900, Brazil. 2 Instituto endometriosis (review). Mol Med Rep. 2014;9(5):1483–505.
Butanta- EstabilidadeBiotech Quality Control, São Paulo, SP 05503‑900, Brazil. 21. Augoulea A, Alexandrou A, Creatsa M, Vrachnis N, Lambrinoudaki I.
Pathogenesis of endometriosis: the role of genetics, inflammation and
Received: 1 May 2020 Accepted: 28 July 2020 oxidative stress. Arch Gynecol Obstet. 2012;286(1):99–103.
22. Piccinato CA, Malvezzi H, Gibson DA, Saunders PTK. SULFATION
PATHWAYS: contribution of intracrine oestrogens to the aetiology of
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Malvezzi et al.

J Transl Med (2020) 18:311

REVIEW Open Access

Endometriosis: current challenges

lix once daily by authors

density were not yet

Prospective Randomized Cell (2PN), Number of

of TNF-α, IL-6, and on the efficacy post-operational relapse

2-induce analgesia channel blocker but

Final remarks Acknowledgments

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