Strategies for managing Asherman's syndrome and endometrial atrophy: Since the classical experimental models to the new bioengineering approach

Endometrial function is essential for embryo implantation and pregnancy, but managing endometrial thickness that is too thin to support pregnancy or an endometrium of compromised functionality due to intrauterine adhesions is an ongoing challenge in reproductive medicine. Here, we review current and emerging therapeutic and experimental options for endometrial regeneration with a focus on animal models used to study solutions for Asherman's syndrome and endometrial atrophy, which both involve a damaged endometrium. A review of existing literature was performed that confirmed the lack of consensus on endometrial therapeutic options, though promising new alternatives have emerged in recent years (platelet‐rich plasma, exosomes derived from stem cells, bioengineering‐based techniques, endometrial organoids, among others). In the future, basic research using established experimental models of endometrial pathologies (combined with new high‐tech solutions) and human clinical trials with large population sizes are needed to evaluate these emerging and new endometrial therapies.

be driven by the somatic stem cell population located in the niche of the basal layer of the human endometrium (Gargett et al., 2010;Cervelló et al., 2013). In rodents (mice and rats), the main animal model used in reproductive research, the reproductive cycle is called the estrous cycle and contains four phases (proestrus, estrus, metaestrus, and diestrus), occurring every 4 to 5 days. Unlikely humans and other primates, rodents do not menstruate (Goldman et al., 2007).
Endometrial anomalies impact fertility and can reduce the chance of pregnancy. Absolute uterine factor infertility results from the absence of a uterus or a nonfunctional uterus (Brännström et al., 2015) while less severe conditions, such as leiomyomas, adenomyosis, Müllerian duct anomalies, and endometrial alterations, impair reproductive outcomes but do not necessarily imply absolute infertility. Among the pathologies directly related to endometrial factor, some affect the endometrial lining, such as endometrial atrophy (EA), and others involve intrauterine adhesions (IUAs) or scar tissue formation, which in the most severe cases can completely obliterate the uterine cavity leading to Asherman's syndrome (AS) (Galliano et al., 2015). Women with EA have a thin endometrium, usually defined by an endometrial thickness, at the time of hCG administration (in an in vitro fertilization procedure), measured by ultrasonography of less than 6-8 mm. At the same time, women with AS syndrome have a severe degree of IUAs, accompanied by menstrual disturbances, infertility, recurrent pregnancy loss, and/or placental abnormalities (Conforti et al., 2013). In any case, this thin and/or fibrotic endometrium may impair implantation and lead to early pregnancy loss or diminish the probability of pregnancy (Mahajan & Sharma, 2016;Senturk & Erel, 2008). Therefore, regenerating the endometrial tissue in EA and AS patients, either to restore the endometrial integrity from fibrotic lesions or to thicken it, is a therapeutic option to allow for embryo implantation.
As detailed above, animal models have been developed to elucidate possible solutions and treatments for endometrial alterations. However, effective and standardized options are lacking. Stem cells, platelet-rich plasma, and bioengineering-derived methodologies may be useful in place of traditional surgical treatment methods and hormonal treatments, but further studies are needed to bring these techniques into clinical practice (García-Velasco et al., 2016). Different models are used to test and study mechanisms of action of these treatments, as well as to understand the pathogenesis of the endometrial variations before clinical translation to humans (Andersen et al., 2018), but a well-established uterine-damaged animal model is needed to test treatment options for endometrial regeneration. Mechanical damage using a needle (Alawadhi et al., 2014;Cervelló et al., 2015), a curette (Huberlant et al., 2014;Feng et al., 2020), or an electric scalpel  are proposed as ways to model uterine injury. Another option is to damage the endometrium by chemical methods, such as ethanol , which has gained wide acceptance over the years (Sun et al., 2019).
In this review, we summarize classical and emerging advances in experimental models, mainly rodents (mice and rats) of endometrial regeneration. The therapeutic alternatives for treating AS and EA, based on animal research, are shown in Table 1. These studies are grouped into two areas: stem cell therapies, growth factors, and other molecules, and emerging therapeutic alternatives (including platelet-rich plasma, tissue engineering, bioengineering solutions, and organoids). Different variables related to either endometrial regeneration evaluation or fertility restoration verification are also listed (Table 1). Among the methods discussed, bioengineering-derived techniques are the most promising in the management of an injured endometrium, as in AS and EA. To translate these techniques to clinical use, a wellestablished model of endometrial injury is essential. All the studies cited along with this study support the effectiveness of the different treatments to regenerate the endometrium in animal models (mainly rodents) of uterine damage. However, not all of them induce uterine damage using the same protocol.
Thus, this study is focused on the importance of animal models before translating novel therapies to human patients.

| MATERIAL AND METHODS
The PubMed database and Google Scholar were searched to identify studies published through December 2020 assessing therapeutic options for endometrial pathologies. We used the following search terms: animal model, Asherman's syndrome, bioengineering, endometrial atrophy, endometrium, growth factors, hydrogel, microfluidics, murine model, organoids, platelet-rich plasma, scaffold, stem cells, and thin endometrium. Additional studies were found in the bibliographies of selected works. Only original articles in English were included. Studies from bovine, murine, ovine, and porcine models were reviewed as well as some human studies.

| Classical management of AS and EA
These classical techniques have been mainly described in humans.
Hysteroscopic adhesiolysis is the most common treatment for human AS (Khan & Goldberg, 2017;Roge et al., 1997). However, surgery is not always effective, and often (20% to 62.5%) the IUAs reappear (Hanstede et al., 2015). Thus, postoperative measures are frequently needed, such as the insertion of an intrauterine device, a Foley balloon, or hyaluronic acid treatment (Amer et al., 2005;Lin et al., 2013;March, 2011). But still, a systematic review from 2017 concluded that there is no clear evidence on the safety and effectiveness of antiadhesion treatment after hysteroscopy for improving the reproductive outcomes rates or for decreasing reappearance of IUAs (Bosteels et al., 2017).
T A B L E 1 Therapeutic alternatives for treating endometrial pathologies: animal model approaches.
Other classical approaches include the use of vasoactive substances to increase endometrial blood flood in AS and EA patients (Miwa et al., 2009;Ng et al., 2007). Some studies report higher implantation and clinical pregnancy rates after administration of lowdose aspirin in patients with EA (Urman et al., 2000;Weckstein et al., 1997) while other investigations reported any improvement (Check et al., 1998;Hsieh et al., 2000). In AS, an improvement in endometrial thickness has been reported after aspirin administration, but no change in reproductive prognosis was observed . Before that, another group postulated that aspirin restored endometrial blood flow, preventing relapse of adhesions after surgery (Chen et al., 2016). Sildenafil citrate has also been tested.
Several case studies show promising results using it in terms of endometrial thickness increase and reproductive outcomes in patients with EA (Sher & Fisch, 2000;2002;Takasaki et al., 2010) and AS (Zinger et al., 2006). However, a randomized clinical trial ( was observed. Pro-inflammatory cytokines, such as tumor necrosis factor (TNF-α) and interleukin (IL) 1β, were downregulated while basic fibroblast growth factor (bFGF) and IL6, both anti-inflammatory cytokines, were upregulated, promoting an immunotolerant environment . In 2018, (Gao et al., 2018)  involved in biological processes such as cell proliferation and migration, cytoprotection, angiogenesis, reducing fibrosis and apoptosis, ECM homeostasis, reducing inflammation, and immunosuppression (Baraniak & McDevitt, 2010;Gnecchi et al., 2008Gnecchi et al., , 2016. This suggests that the application of the biomolecules secreted by stem cells, called the secretome, including lipids, free nucleic acids, soluble proteins, and extracellular vesicles, could be sufficient to activate regeneration or restoration of a specific tissue rather than necessitating the transplantation of stem cells (Beer et al., 2017). Indeed, the secretome derived from MSCs has anti-inflammatory, antiapoptotic, antimicrobial, and angiogenic properties and promotes wound healing and tissue repair (Vizoso et al., 2017). This secretome-based approach has been tested in animal models of different human diseases such as liver (Driscoll & Patel, 2019) or cerebrovascular (Maki et al., 2018) diseases and clinical trials (Konala et al., 2016), rather than in the gynecological field. The few published works using the stem cell secretome for endometrial repair are mentioned further in the text, in the Hydrogels section.
Besides, growth factors and cytokines are secreted by human stem cells including those derived from bone marrow (Baberg et al., 2019;Oskowitz et al., 2011), umbilical cord , and adipose tissue (Chang et al., 2017;Mussano et al., 2017). Deeper investigation and identification of these paracrine factors will aid the development of noninvasive therapies that could replace stem cell therapy in gynecological pathologies. Our group has taken the first steps in this field, reporting that CD133 + BMDSCs injected in an AS murine model (Cervelló et al., 2015) activated Serpine 1, which promotes cell migration and is involved in decidualization (Lumbers et al., 2015), and Jun protooncogene, which promotes endometrial epithelial cell proliferation while decreasing the expression of cyclin D1, a regulator of the cell cycle, through a paracrine mechanism to aid endometrial regeneration. This creates an immunomodulatory environment in endometrial tissue that promotes regenerative processes (De Miguel-Gómez et al., 2020). Paracrine molecules can be also delivered from exosomes, nano-sized extracellular vesicles that release active paracrine molecules .
Exosomes were successfully used to promote endometrial regeneration and restore fertility rates in a murine model , implicating this delivery method as a promising treatment tool. Indeed, exosomes derived from adipose-derived MSCs may restore endometrium to normal morphology, decrease fibrosis, and increase the expression of proregenerative factors such as ITGβ3, LIF, and VEGF, thus supporting an improvement in implantation and pregnancy rate .

| Growth factors and other molecules
Growth factors and other molecules have therapeutic effects both individually and in combination, though most studies are in vitro or animal models and the clinical translation to human treatment is undetermined.
Hepatocyte growth factor, an enhancer of in vitro proliferation and migration of human endometrial epithelial cells (Sugawara et al., 1997) and transforming growth factor β (TGF-β) isoforms promotes in vitro endometrial remodeling  along with platelet-derived growth factor (PDGF) isoforms, which stimulate proliferation and migration among cultured human endometrial stem cells for endometrial tissue repair and support endometrial tissue contraction and remodeling . Besides, epidermal growth factor (EGF), PDGF-BB, and basic fibroblast growth factor promote in vitro endometrial stromal and epithelial colony-forming units, an intrinsic characteristic of somatic stem cells (Gargett et al., 2008).
In vivo, stromal cell-derived factor 1 (SDF1α) improved stem cell engraftment in an AS mouse model receiving BMDSCs therapy (Ersoy et al., 2017). Later, the synergic effect of SDF1α and BMDSCs in a murine model of AS was also reported and endometrial regeneration levels after a single application of SDF1α were similar to those from stem cells alone (Yi et al., 2019). Similarly, BMDSC therapy improved endometrial thick-

| Emerging therapeutic alternatives
As discussed above, stem cell therapy is effective in inducing endometrial regeneration in animal models. However, stem cell therapy is costly, invasive, and painful, and therefore not an ideal intervention. Additionally, depending on the type of stem cells, other issues such as ethical and moral questions, risk of teratoma formation, and low retention of cells may arise . The use of single molecules has not been deeply explored, as we previously detailed, and most studies use them as enhancers of stem cell action. However, the lack of consensus and the weaknesses of the published studies have promoted the emergence of other therapeutic alternatives, such as platelet-rich plasma (PRP).

| Platelet-rich plasma
PRP is a plasma fraction with a supra-physiologic platelet con-  al., 2016). These molecules can only be released after breaking the plasma membrane of platelets (i.e., using calcium chloride), a process called activation or degranulation (Fréchette et al., 2005).
Further, PRP can be easily obtained via centrifugation to create a gradient in which the lower part of the plasma fraction is enriched in platelets from a peripheral blood sample (Dohan Ehrenfest et al., 2018). This methodology is a minimally invasive procedure appropriate for autologous treatment in AS and EA patients (Pietrzak & Eppley, 2005).
In vitro experiments based on human endometrial cell processes such as cell migration or proliferation (Aghajanova et al., 2018;Wang et al., 2018) describe a positive effect of PRP on regeneration mechanisms. Overexpression of genes and proteins related to a healthy endometrium and regeneration processes, such as estrogen (ERα) and progesterone (PR) receptors (Marini et al., 2016), VEGF, and procollagen type I (Anitua et al., 2016), are also reported.
In vivo rodent models with injured endometrium also show promising results after intrauterine administration of autologous PRP, decreasing fibrosis and increasing expression of several markers of proliferation (Ki-67), angiogenesis (VEGF), and normal endometrial function (cytokeratin, homeobox A10 -HOXA10-) . Further, PRP administration improves endometrial morphology, reduces the degree of fibrosis, and produces a higher number of implantation sites and live-births . PRP was also described as a promotor of the regenerative action of BMDSCs . These authors suggested that PRP enhances stem cell differentiation through the nuclear factor κB pathway. They proposed that PRP regeneration activity was based on the activation of the NF-κB p50 subunit, which induces the upregulation of the anti-inflammatory cytokine IL-10, described to be involved in endometrial regeneration after injury (Xue et al., 2019). In addition, we recently corroborated that PRP promotes in vitro endometrial cell proliferation and migration and regenerates endometrial tissue after damage in a murine model. This effect is strengthened when blood is obtained from the umbilical cord, the most undifferentiated blood source .
These results suggest that the umbilical cord could be a good source of plasma for treating endometrial pathologies and other regenerative medicine applications, as demonstrated by other groups (Castellano et al., 2017;Ehrhart et al., 2018). PRP has also been tested in women with either thin endometrium Eftekhar et al., 2018;Kim, Shin et al., 2019;Molina et al., 2018;Nazari et al., 2016;Tandulwadkar et al., 2017) or AS (Javaheri et al., 2020;Zadehmodarres et al., 2017). However, not all these human works presented a robust study design. From the results obtained by those conducted as clinical trials Eftekhar et al., 2018;Javaheri et al., 2020;Tandulwadkar et al., 2017), it could be concluded that PRP is an effective therapeutic option for treating patients with thin endometrium in which the cause is not IUA (this was an exclusion criterion in the majority of existing studies).

| Tissue engineering solutions and bioengineering approaches
In 1988, tissue engineering was defined as the "application of engineering and life science basis toward the development of biological substitutes for improving, maintaining, or restoring tissue natural functions" (Skalak & Fox, 1988). Bioengineering is a fundamental pillar for tissue engineering based on the use of biomaterials to support tissue regeneration (Brien, 2011). Biomaterials used to model the human endometrium are typically collagen (Gentleman et al., 2003), proteoglycans (formed of glycosaminoglycans, such as heparin or keratin sulfates, covalently attached to a core protein) (Rnjak-kovacina et al., 2017), alginate (Nayak et al., 2020), and chitosan (Choi et al., 2016). They can be used alone or in combination with stem or fully differentiated cells, growth factors, or other biomolecules that work synergistically with the biomaterial (Brien, 2011).
Endometrial regeneration can occur in murine models using collagen scaffolds in combination with other approaches. Collagen and growth factors, such as bFGF (Sun et al., 2011) or VEGF (Lin et al., 2012), and stem cells, derived either from the bone marrow (Ding et al., 2014) or embryonic tissues (Song et al., 2015),

Decellularized scaffolds
Scaffolds made from ECM after decellularization (removal of all cellular components of a biological scaffold while retaining the ECM structure) of tissues or whole organs have also been evaluated.
Scaffolds are a relatively new concept first applied in the assisted reproduction field in 2014 in a rat uterus (Santoso et al., 2014). A longitudinal segment of a rat uterus was decellularized using two different methods, sodium dodecyl sulfate (SDS) and high hydrostatic pressure, and both supported regular pregnancies. Later, other groups reseeded the decellularized scaffolds before replanting them in animal models. Successful endometrial regeneration was noted in rat models using decellularized uterine scaffolds, obtained using SDS or Triton-X 100, grafted into an injured uterus after reseeding with rat primary uterine cells (Miyazaki & Maruyama, 2014) and mesenchymal BMDSCs (Hellström et al., 2016). Both studies reported comparable reproductive outcomes in the groups with cell-seeded scaffold transplant and control groups. Maruyama's group also described the importance of scaffold orientation and reported that if the uterine patches were reverse oriented (luminal part in the outside while the serosal side remained in the inside, or lumen), the regenerated uterine tissue was aberrant (Miki et al., 2019).
Not only single fragments but also the decellularization of the whole uterus has been achieved in different animal models. The decellularization of an entire rat uterus using several protocols, of which sodium deoxycholate was the best for preserving ECM, was reported (Hellström et al., 2014). Similar results were also reported in a later study using a whole sheep uterus. In this study, rings from a bioengineered uterus were recellularized using sheep fetal BMDSCs and the decellularized uterus fragments maintained the reseeded cells in vitro for 2 weeks (Tiemann et al., 2020). The same recellularizing procedure using human endometrial stem cells was also applied to decellularized scaffolds obtained from a whole porcine uterus, which successfully maintained ECM and vascular network integrity (Campo et al., 2017). Further, an ovine acellular uterus scaffold was harvested in rats and became recellularized with endometrial tissue and vascular cells (Daryabari et al., 2019). Finally, decellularized endometrial scaffolds recellularized again with endometrial cells, both obtained from human samples, responded to 28day hormone treatment in vitro, complete with the secretion of decidual markers .

Hydrogels
Another bioengineering approach is the use of hydrogels, which are three-dimensional hydrophilic polymer networks (Hoffman, 2002) derived from hyaluronic acid Kim, Park et al., 2019) or different poloxamers Zhang et al., 2020), among other biomaterials. To enhance the synergies of this novel approach and other classical therapies, artificial hydrogels have been applied in several murine models together with other factors or cells. These combinations have been performed using estradiol embedded in an aloe-poloxamer (Yao et al., 2020) and a heparin-poloxamer hydrogel  for restoring endometrium in rat models of intrauterine adhesions. Both studies reported improvement in the endometrial morphology status and a reduction in fibrosis, as well as the overexpression of cell proliferation and endometrial regeneration factors. Hydrogels in combination with the chemokine SDF1α restored the endometrium and improved endometrial thickness, fibrotic area, number of glands, and embryo implantation rate (Wenbo et al., 2020). Different types of cells or their derivatives, such as endometrial stromal cells (Kim, Park et al., 2019), BMDSCs , and the stem cell secretome  are reported to improve therapeutic effects when combined with hyaluronic acid or poloxamer-based hydrogels in murine models of endometrial damage.
Hydrogels could be directly related to decellularized scaffolds because they can also derive from decellularized ECM (Saldin et al., 2016).

Microfluidics
Microfluidic technology has emerged as a method to model reproductive organs in vitro and to assist in evaluating therapeutic solutions for endometrial pathologies . A microfluidic device included a coculture of primary human stromal and endothelial cells in which the hormonal changes occurring during the menstrual cycle were simulated (Gnecco et al., 2017); this approach allowed the study of the implication of the vascular endothelium during the decidualization process (Gnecco et al., 2017). A microfluidic platform termed EVATAR, containing reproductive tract tissues and peripheral organs mimicking a 28-day human menstrual cycle was also described . These technologies will promote the in vitro study of new therapeutic options not only for the endometrium but also for the rest of the reproductive organs.

| Endometrial organoids
In the last years, organoids, defined as genetically stable in vitro-cultured 3D structures that encompass key features of in vivo organs (Schutgens & Clevers, 2020), have emerged as an alternative to conventional in vitro cell culture systems. These 3D biological structures have been revealed as key models for several diseases, drug screenings, testing, and benchmarking for novel therapeutic approaches, as well as a potential tool of personalized medicine (Clevers, 2016). Thus, for endometrial management, not only in AS/EA patients but also in endometriosis, organoids could be a promising instrument either to better understand the pathogenesis of AS/EA or screen incoming untested therapies. This novel approach could complement or even reduce the studies performed in animal models before the clinical translation to humans.
In the last decade, organoids have been derived from different human tissues such as the liver (Huch et al., 2015) or prostate (Karthaus et al., 2014). More recently, several groups have obtained them from human endometrial tissue. These organoids exhibited the characteristics of uterine glands in vivo, expressing specific epithelial, such as epithelial cell adhesion molecule (EPCAM), and secretory, such as mucin-1, markers.
These organ-like structures also responded to hormonal stimulation (estrogen and progesterone) by the overexpression of ERα and PR or the secretion of the progesterone-associated endometrial protein (PAEP) that DE MIGUEL-GÓMEZ ET AL.
| 535 reveals decidualization, among other features (Turco et al., 2017). Similarly, in 2018, another group isolated organoids not only from the human endometrium but also from murine samples (Boretto et al., 2017). They reported the endometrial epithelium-like phenotype by expression of Ecadherin, ERα, and cytokeratin, by mucin-1 secretion, and by the response to ovarian hormones. This group also published organoids directly derived from human patients, opening then the door to disease modeling and personalized medicine for endometrial-associated pathologies (Boretto et al., 2019).
After the revision of all works cited along with this review and despite those describing human studies, we want to remark the importance of basic science and standard animal models in the study of novel treatments for specific endometrial disorders (AS/EA) before the clinical translation. The generally smaller size of the animal models together with the bigger litter size, short generation times, and more availability of tissue (endometrium in this case) for molecular studies are the main advantages of using animals prior testing in humans (Carter, 2020). Besides, while promising therapies and study platforms are emerging, they still need to be further explored.

| CONCLUSION
Classical management of AS and EA is lacking effectiveness, so new approaches have emerged for endometrial regeneration to increase fertility options when this tissue is damaged (Figure 1 Lastly, the emergence of new research tools, like organoids, can also change and improve the current methods to study AS and EA and to screen different therapies. F I G U R E 1 Overview of the research and preclinical process in the management of endometrial pathologies. Patients with damaged endometrium (Asherman's syndrome (AS) or Endometrial atrophy (EA)) still lack completely effective treatments. The animal models are essential in the pursuit of therapy for these patients. In those animals (mainly mice and rats), mechanical (needles, electrical scalpels) and chemical (ethanol) methods can generate endometrial damage. Over the years, stem cell therapy has been revealed as the most promising option, however other approaches such as the use of the stem cells' secretome, platelet-rich plasma (PRP), and bioengineering techniques (hydrogels, collagen scaffolds, microfluidics), or even the combination of all of them, have emerged as good alternatives in the field. Moreover, the in vitro systems are also very useful but have many limitations for being able to extrapolate results. The development of endometrial organoids, which simulate more closely an in vivo tissue or organ, are emerging as promising research tools. Figure