Evolution of biotechnological advances and regenerative therapies for endometrial disorders: a systematic review.

BACKGROUND: The establishment and maintenance of pregnancy depend on endometrial competence. Asherman syndrome (AS) and intrauterine adhesions (IUA), or endometrial atrophy (EA) and thin endometrium (TE), can either originate autonomously or arise as a result from conditions (i.e. endometritis or congenital hypoplasia), or medical interventions (e.g. surgeries, hormonal therapies, uterine curettage or radiotherapy). Affected patients may present an altered or inadequate endometrial lining that hinders embryo implantation and increases the risk of poor pregnancy outcomes and miscarriage. In humans, AS/IUA and EA/TE are mainly treated with surgeries or pharmacotherapy, however the reported efficacy of these therapeutic approaches remains unclear. Thus, novel regenerative techniques utilizing stem cells, growth factors, or tissue engineering have emerged to improve reproductive outcomes. OBJECTIVE AND RATIONALE: This review comprehensively summarizes the methodologies and outcomes of emerging biotechnologies (cellular, acellular, and bioengineering approaches) to treat human endometrial pathologies. Regenerative therapies derived from human tissues or blood which were studied in preclinical models (in vitro and in vivo) and clinical trials are discussed. SEARCH METHODS: A systematic search of full-text articles available in PubMed and Embase was conducted to identify original peer-reviewed studies published in English between January 2000 and September 2023. The search terms included: human, uterus, endometrium, Asherman syndrome, intrauterine adhesions, endometrial atrophy, thin endometrium, endometritis, congenital hypoplasia, curettage, radiotherapy, regenerative therapy, bioengineering, stem cells, vesicles, platelet-rich plasma, biomaterials, microfluidic, bioprinting, organoids, hydrogel, scaffold, sheet, miRNA, sildenafil, nitroglycerine, aspirin, growth hormone, progesterone, and estrogen. Preclinical and clinical studies on cellular, acellular, and bioengineering strategies to repair or regenerate the human endometrium were included. Additional studies were identified through manual searches. OUTCOMES: From a total of 4366 records identified, 164 studies (3.8%) were included for systematic review. Due to heterogeneity in the study design and measured outcome parameters in both preclinical and clinical studies, the findings were evaluated qualitatively and quantitatively without meta-analysis. Groups using stem cell-based treatments for endometrial pathologies commonly employed mesenchymal stem cells (MSCs) derived from the human bone marrow or umbilical cord. Alternatively, acellular therapies based on platelet-rich plasma (PRP) or extracellular vesicles are gaining popularity. These are accompanied by the emergence of bioengineering strategies based on extracellular matrix (ECM)-derived hydrogels or synthetic biosimilars that sustain local delivery of cells and growth factors, reporting promising results. Combined therapies that target multiple aspects of tissue repair and regeneration remain in preclinical testing but have shown translational value. This review highlights the myriad of therapeutic material sources, administration methods, and carriers that have been tested. WIDER IMPLICATIONS: Therapies that promote endometrial proliferation, vascular development, and tissue repair may help restore endometrial function and, ultimately, fertility. Based on the existing evidence, cost, accessibility, and availability of the therapies, we propose the development of triple-hit regenerative strategies, potentially combining high-yield MSCs (e.g. from bone marrow or umbilical cord) with acellular treatments (PRP), possibly integrated in ECM hydrogels. Advances in biotechnologies together with insights from preclinical models will pave the way for developing personalized treatment regimens for patients with infertility-causing endometrial disorders such as AS/IUA, EA/TE, and endometritis. REGISTRATION NUMBER: https://osf.io/th8yf/.

Addressing Key Questions in Organoid Models: Who, Where, How, and Why?

Organoids are three-dimensional cellular structures designed to recreate the biological characteristics of the body's native tissues and organs in vitro. There has been a recent surge in studies utilizing organoids due to their distinct advantages over traditional two-dimensional in vitro approaches. However, there is no consensus on how to define organoids. This literature review aims to clarify the concept of organoids and address the four fundamental questions pertaining to organoid models: (i) What constitutes organoids?-The cellular material. (ii) Where do organoids grow?-The extracellular scaffold. (iii) How are organoids maintained in vitro?-Via the culture media. (iv) Why are organoids suitable in vitro models?-They represent reproducible, stable, and scalable models for biological applications. Finally, this review provides an update on the organoid models employed within the female reproductive tract, underscoring their relevance in both basic biology and clinical applications.

Hybrid Endometrial-Derived Hydrogels: Human Organoid Culture Models and In Vivo Perspectives.

The endometrium plays a vital role in fertility, providing a receptive environment for embryo implantation and development. Understanding the endometrial physiology is essential for developing new strategies to improve reproductive healthcare. Human endometrial organoids (hEOs) are emerging as powerful models for translational research and personalized medicine. However, most hEOs are cultured in a 3D microenvironment that significantly differs from the human endometrium, limiting their applicability in bioengineering. This study presents a hybrid endometrial-derived hydrogel that combines the rigidity of PuraMatrix (PM) with the natural scaffold components and interactions of a porcine decellularized endometrial extracellular matrix (EndoECM) hydrogel. This hydrogel provides outstanding support for hEO culture, enhances hEO differentiation efficiency due to its biochemical similarity with the native tissue, exhibits superior in vivo stability, and demonstrates xenogeneic biocompatibility in mice over a 2-week period. Taken together, these attributes position this hybrid endometrial-derived hydrogel as a promising biomaterial for regenerative treatments in reproductive medicine.

Advances of xenogeneic ovarian extracellular matrix hydrogels for in vitro follicle development and oocyte maturation.

Research aimed at preserving female fertility is increasingly using bioengineering techniques to develop new platforms capable of supporting ovarian cell function in vitro and in vivo. Natural hydrogels (alginate, collagen, and fibrin) have been the most exploited approaches; however they are biologically inert and/or biochemically simple. Thus, establishing a suitable biomimetic hydrogel from decellularized ovarian cortex (OC) extracellular matrix (OvaECM) could provide a complex native biomaterial for follicle development and oocyte maturation. The objectives of this work were (i) to establish an optimal protocol to decellularize and solubilize bovine OC, (ii) to characterize the histological, molecular, ultrastructural, and proteomic properties of the resulting tissue and hydrogel, and (iii) to assess its biocompatibility and adequacy for murine in vitro follicle growth (IVFG). Sodium dodecyl sulfate was identified as the best detergent to develop bovine OvaECM hydrogels. Hydrogels added into standard media or used as plate coatings were employed for IVFG and oocyte maturation. Follicle growth, survival, hormone production, and oocyte maturation and developmental competence were evaluated. OvaECM hydrogel-supplemented media best supported follicle survival, expansion, and hormone production, while the coatings provided more mature and competent oocytes. Overall, the findings support the xenogeneic use of OvaECM hydrogels for future human female reproductive bioengineering.

Human umbilical cord platelet-rich plasma to treat endometrial pathologies: methodology, composition and pre-clinical models.

STUDY QUESTION: Can human umbilical cord platelet-rich plasma (hUC-PRP) efficiently treat endometrial damage and restore fertility in a preclinical murine model? SUMMARY ANSWER: Local application of hUC-PRP promotes tissue regeneration and fertility restoration in a murine model of Asherman syndrome and endometrial atrophy (AS/EA). WHAT IS KNOWN ALREADY: AS/EA are well-described endometrial pathologies that cause infertility; however, there are currently no gold-standard treatments available. Recent reports have described the successful use of human platelet-rich plasma in reproductive medicine, and its regenerative potential is further enhanced using hUC-PRP, due to the ample growth factors and reduced pro-inflammatory cytokines in the latter. STUDY DESIGN SIZE DURATION: hUC-PRP (n = 3) was processed, characterized and delivered locally to endometrial damage in a murine model (n = 50). The hUC-PRP was either used alone or loaded into a decellularized porcine endometrium-derived extracellular matrix (EndoECM) hydrogel; endometrial regeneration, fertility outcomes and immunocompatibility were evaluated 2 weeks following treatment administration. PARTICIPANTS/MATERIALS SETTING METHODS: Umbilical cord blood was obtained from women in childbirth. Endometrial damage (mimicking AS/EA) was induced using ethanol in 8-week-old C57BL/6 mice, and treated with the most concentrated hUC-PRP sample 4 days later. Characterization of hUC-PRP and immunotolerance was carried out with multiplex technology, while uterine samples were analyzed by immunohistochemistry and quantitative PCR. The number of embryos and their morphology was determined visually. MAIN RESULTS AND THE ROLE OF CHANCE: Platelet density was enhanced 3-fold in hUC-PRP compared to that in hUC blood (P < 0.05). hUC-PRP was enriched with growth factors related to tissue regeneration (i.e. hepatocyte growth factor, platelet-derived growth factor-BB and epidermal growth factor), which were released constantly (in vitro) when hUC-PRP was loaded into EndoECM. Both treatments (hUC-PRP alone and hUC-PRP with EndoECM) were immunotolerated and caused significantly regeneration of the damaged endometrium, evidenced by increased endometrial area, neoangiogenesis, cell proliferation and gland density and lower collagen deposition with respect to non-treated uterine horns (P < 0.05). Additionally, we detected augmented gene expression of Akt1, VEGF and Ang, which are involved in regenerative and proliferation pathways. Finally, hUC-PRP treatment restored pregnancy rates in the mouse model. LARGE SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: This proof-of-concept pilot study was based on a murine model of endometrial damage and the use of EndoECM requires further validation prior to clinical implementation for women affected by AS/EA. WIDER IMPLICATIONS OF THE FINDINGS: The local administration of hUC-PRP has high impact and is immunotolerated in a murine model of AS/EA, as has been reported in other tissues, making it a promising candidate for heterologous treatment of these endometrial pathologies. STUDY FUNDING/COMPETING INTERESTS: This study was supported by the Ministerio de Ciencia, Innovación y Universidades; Conselleria de Innovación, Universidades, Ciencia y Sociedad Digital, Generalitat Valenciana; and Instituto de Salud Carlos III. The authors do not have any conflicts of interest to declare.

Human Umbilical Cord-Based Therapeutics: Stem Cells and Blood Derivatives for Female Reproductive Medicine.

There are several conditions that lead to female infertility, where traditional or conventional treatments have limited efficacy. In these challenging scenarios, stem cell (SC) therapies have been investigated as alternative treatment strategies. Human umbilical cord (hUC) mesenchymal stem cells (hUC-MSC), along with their secreted paracrine factors, extracts, and biomolecules, have emerged as promising therapeutic alternatives in regenerative medicine, due to their remarkable potential to promote anti-inflammatory and regenerative processes more efficiently than other autologous treatments. Similarly, hUC blood derivatives, such as platelet-rich plasma (PRP), or isolated plasma elements, such as growth factors, have also demonstrated potential. This literature review aims to summarize the recent therapeutic advances based on hUC-MSCs, hUC blood, and/or other plasma derivatives (e.g., extracellular vesicles, hUC-PRP, and growth factors) in the context of female reproductive medicine. We present an in-depth analysis of the principal molecules mediating tissue regeneration, compiling the application of these therapies in preclinical and clinical studies, within the context of the human reproductive tract. Despite the recent advances in bioengineering strategies that sustain delivery and amplify the scope of the therapeutic benefits, further clinical trials are required prior to the wide implementation of these alternative therapies in reproductive medicine.

Bioengineering trends in female reproduction: a systematic review.

BACKGROUND: To provide the optimal milieu for implantation and fetal development, the female reproductive system must orchestrate uterine dynamics with the appropriate hormones produced by the ovaries. Mature oocytes may be fertilized in the fallopian tubes, and the resulting zygote is transported toward the uterus, where it can implant and continue developing. The cervix acts as a physical barrier to protect the fetus throughout pregnancy, and the vagina acts as a birth canal (involving uterine and cervix mechanisms) and facilitates copulation. Fertility can be compromised by pathologies that affect any of these organs or processes, and therefore, being able to accurately model them or restore their function is of paramount importance in applied and translational research. However, innate differences in human and animal model reproductive tracts, and the static nature of 2D cell/tissue culture techniques, necessitate continued research and development of dynamic and more complex in vitro platforms, ex vivo approaches and in vivo therapies to study and support reproductive biology. To meet this need, bioengineering is propelling the research on female reproduction into a new dimension through a wide range of potential applications and preclinical models, and the burgeoning number and variety of studies makes for a rapidly changing state of the field. OBJECTIVE AND RATIONALE: This review aims to summarize the mounting evidence on bioengineering strategies, platforms and therapies currently available and under development in the context of female reproductive medicine, in order to further understand female reproductive biology and provide new options for fertility restoration. Specifically, techniques used in, or for, the uterus (endometrium and myometrium), ovary, fallopian tubes, cervix and vagina will be discussed. SEARCH METHODS: A systematic search of full-text articles available in PubMed and Embase databases was conducted to identify relevant studies published between January 2000 and September 2021. The search terms included: bioengineering, reproduction, artificial, biomaterial, microfluidic, bioprinting, organoid, hydrogel, scaffold, uterus, endometrium, ovary, fallopian tubes, oviduct, cervix, vagina, endometriosis, adenomyosis, uterine fibroids, chlamydia, Asherman's syndrome, intrauterine adhesions, uterine polyps, polycystic ovary syndrome and primary ovarian insufficiency. Additional studies were identified by manually searching the references of the selected articles and of complementary reviews. Eligibility criteria included original, rigorous and accessible peer-reviewed work, published in English, on female reproductive bioengineering techniques in preclinical (in vitro/in vivo/ex vivo) and/or clinical testing phases. OUTCOMES: Out of the 10 390 records identified, 312 studies were included for systematic review. Owing to inconsistencies in the study measurements and designs, the findings were assessed qualitatively rather than by meta-analysis. Hydrogels and scaffolds were commonly applied in various bioengineering-related studies of the female reproductive tract. Emerging technologies, such as organoids and bioprinting, offered personalized diagnoses and alternative treatment options, respectively. Promising microfluidic systems combining various bioengineering approaches have also shown translational value. WIDER IMPLICATIONS: The complexity of the molecular, endocrine and tissue-level interactions regulating female reproduction present challenges for bioengineering approaches to replace female reproductive organs. However, interdisciplinary work is providing valuable insight into the physicochemical properties necessary for reproductive biological processes to occur. Defining the landscape of reproductive bioengineering technologies currently available and under development for women can provide alternative models for toxicology/drug testing, ex vivo fertility options, clinical therapies and a basis for future organ regeneration studies.

Future Challenges and Opportunities of Extracellular Matrix Hydrogels in Female Reproductive Medicine.

Bioengineering and reproductive medicine have progressed shoulder to shoulder for several decades. A key point of overlap is the development and clinical translation of technologies to support reproductive health, e.g., scaffold-free constructs, polymeric scaffolds, bioprinting or microfluidics, and hydrogels. Hydrogels are the focus of intense study, and those that are derived from the extracellular matrix (ECM) of reproductive tissues and organs are emerging as promising new players given their results in pre-clinical models. This literature review addresses the recent advances in the use of organ-specific ECM hydrogels in reproductive medicine, considering the entire female reproductive tract. We discuss in-depth papers describing the development of ECM hydrogels, their use in in vitro models, and their in vivo application in preclinical studies. We also summarize the functions of hydrogels, including as grafts, carriers for cell transplantation, or drug depots, and present the potential and possible scope for use of ECM hydrogels in the near future based on recent scientific advances.

Establishment of Adenomyosis Organoids as a Preclinical Model to Study Infertility.

Adenomyosis is related to infertility and miscarriages, but so far there are no robust in vitro models that reproduce its pathological features to study the molecular mechanisms involved in this disease. Endometrial organoids are in vitro 3D models that recapitulate the native microenvironment and reproduce tissue characteristics that would allow the study of adenomyosis pathogenesis and related infertility disorders. In our study, human endometrial biopsies from adenomyosis (n = 6) and healthy women (n = 6) were recruited. Organoids were established and hormonally differentiated to recapitulate midsecretory and gestational endometrial phases. Physiological and pathological characteristics were evaluated by immunohistochemistry, immunofluorescence, qRT-PCR, and ELISA. Secretory and gestational organoids recapitulated in vivo glandular epithelial phenotype (pan-cytokeratin, Muc-1, PAS, Laminin, and Ki67) and secretory and gestational features (α-tubulin, SOX9, SPP1, PAEP, LIF, and 17ßHSD2 expression and SPP1 secretion). Adenomyosis organoids showed higher expression of TGF-ß2 and SMAD3 and increased gene expression of SPP1, PAEP, LIF, and 17ßHSD2 compared with control organoids. Our results demonstrate that organoids derived from endometria of adenomyosis patients and differentiated to secretory and gestational phases recapitulate native endometrial-tissue-specific features and disease-specific traits. Adenomyosis-derived organoids are a promising in vitro preclinical model to study impaired implantation and pregnancy disorders in adenomyosis and enable personalized drug screening.

Bioengineered endometrial hydrogels with growth factors promote tissue regeneration and restore fertility in murine models.

Extracellular matrix (ECM) hydrogels obtained from decellularized tissues are promising biocompatible materials for tissue regeneration. These biomaterials may provide important options for endometrial pathologies such as Asherman's syndrome and endometrial atrophy, which lack effective therapies thus far. First, we performed a proteomic analysis of a decellularized endometrial porcine hydrogel (EndoECM) to describe the specific role of ECM proteins related to regenerative processes. Furthermore, we investigated the ability of a bioengineered system-EndoECM alone or supplemented with growth factors (GFs)-to repair the endometrium in a murine model of endometrial damage. For this model, the uterine horns of female C57BL/6 mice were first injected with 70% ethanol, then four days later, they were treated with: saline (negative control); biotin-labeled EndoECM; or biotin-labeled EndoECM plus platelet-derived GF, basic fibroblast GF, and insulin-like GF 1 (EndoECM+GF). Endometrial regeneration and fertility restoration were evaluated by assessing the number of glands, endometrial area, cell proliferation, neaoangiogenesis, reduction of collagen deposition, and fertility restoration. Interestingly, regenerative effects such as an increased number of endometrial glands, increased area, high cell proliferative index, development of new blood vessels, reduction of collagen deposition, and higher pregnancy rate occurred in mice treated with EndoECM+GF. Thus, a bioengineered system based on EndoECM hydrogel supplemented with GFs may be promising for the clinical treatment of endometrial conditions such as Asherman's syndrome and endometrial atrophy. STATEMENT OF SIGNIFICANCE: In the last years, the bioengineering field has developed new and promising approaches to regenerate tissues or replace damaged and diseased tissues. Bioengineered hydrogels offer an ideal option because these materials can be used not only as treatments but also as carriers of drugs and other therapeutics. The present work demonstrates for the first time how hydrogels derived from pig endometrium loaded with growth factors could treat uterine pathologies in a mouse model of endometrial damage. These findings provide scientific evidence about bioengineered hydrogels based on tissue-specific extracellular matrix offering new options to treat human infertility from endometrial causes such as Asherman's syndrome or endometrial atrophy.

Improved Models of Human Endometrial Organoids Based on Hydrogels from Decellularized Endometrium.

Organoids are three-dimensional (3D) multicellular tissue models that mimic their corresponding in vivo tissue. Successful efforts have derived organoids from primary tissues such as intestine, liver, and pancreas. For human uterine endometrium, the recent generation of 3D structures from primary endometrial cells is inspiring new studies of this important tissue using precise preclinical models. To improve on these 3D models, we decellularized pig endometrium containing tissue-specific extracellular matrix and generated a hydrogel (EndoECM). Next, we derived three lines of human endometrial organoids and cultured them in optimal and suboptimal culture expansion media with or without EndoECM (0.01 mg/mL) as a soluble additive. We characterized the resultant organoids to verify their epithelial origin, long-term chromosomal stability, and stemness properties. Lastly, we determined their proliferation potential under different culture conditions using proliferation rates and immunohistochemical methods. Our results demonstrate the importance of a bioactive environment for the maintenance and proliferation of human endometrial organoids.

Stem Cells and the Endometrium: From the Discovery of Adult Stem Cells to Pre-Clinical Models.

Adult stem cells (ASCs) were long suspected to exist in the endometrium. Indeed, several types of endometrial ASCs were identified in rodents and humans through diverse isolation and characterization techniques. Putative stromal and epithelial stem cell niches were identified in murine models using label-retention techniques. In humans, functional methods (clonogenicity, long-term culture, and multi-lineage differentiation assays) and stem cell markers (CD146, SUSD2/W5C5, LGR5, NTPDase2, SSEA-1, or N-cadherin) facilitated the identification of three main types of endogenous endometrial ASCs: stromal, epithelial progenitor, and endothelial stem cells. Further, exogenous populations of stem cells derived from bone marrow may act as key effectors of the endometrial ASC niche. These findings are promoting the development of stem cell therapies for endometrial pathologies, with an evolution towards paracrine approaches. At the same time, promising therapeutic alternatives based on bioengineering have been proposed.

Development of Decellularized Oviductal Hydrogels as a Support for Rabbit Embryo Culture.

The oviducts (fallopian tubes in mammals) function as the site of fertilization and provide necessary support for early embryonic development, mainly via embryonic exposure to the tubal microenvironment. The main objective of this study was to create an oviduct-specific extracellular matrix (oviECM) hydrogel rich in bioactive components that mimics the native environment, thus optimizing the developmental trajectories of cultured embryos. Rabbit oviducts were decellularized through SDS treatment and enzymatic digestion, and the acellular tissue was converted into oviductal pre-gel extracellular matrix (ECM) solutions. Incubation of these solutions at 37 °C resulted in stable hydrogels with a fibrous structure based on scanning electron microscopy. Histological staining, DNA quantification and colorimetric assays confirmed that the decellularized tissue and hydrogels contained no cellular or nuclear components but retained important components of the ECM, e.g. hyaluronic acid, glycoproteins and collagens. To evaluate the ability of oviECM hydrogels to maintain early embryonic development, two-cell rabbit embryos were cultured on oviECM-coated surfaces and compared to those cultured with standard techniques. Embryo development was similar in both conditions, with 95.9% and 98% of the embryos reaching the late morula/early blastocyst stage by 48 h under standard culture and oviECM conditions, respectively. Metabolomic analysis of culture media in the presence or absence of embryos, however, revealed that the oviECM coating may include signalling molecules and release compounds beneficial to embryo metabolism.

Comparison of different sources of platelet-rich plasma as treatment option for infertility-causing endometrial pathologies.

OBJECTIVE: To study the effect of human plasma from different sources, namely, umbilical cord blood and adult blood platelet-rich plasma (PRP), on the regeneration of endometrial damage. DESIGN: Composition analysis, in vitro approaches, and a preclinical murine model using plasma to promote endometrial regeneration. SETTING: Hospital and university laboratories. PATIENT(S)/ANIMAL(S): Adult plasma from four Asherman syndrome/endometrial atrophy patients and one fertile woman, commercial umbilical cord plasma, and uterine-damaged NOD/SCID mice model were used. INTERVENTION(S): Endometrial stromal cells from primary culture and an endometrial stem cell line were cultured in vitro, and uterine-damaged NOD/SCID mice were treated with plasma samples from several origins. MAIN OUTCOME MEASURE(S): To investigate the possible beneficial effects of PRP from Asherman syndrome/endometrial atrophy patients. To test if plasma from human umbilical cord blood had a stronger effect than adult PRP in endometrial regeneration. To demonstrate if PRP from Asherman syndrome/endometrial atrophy patients was as effective as PRP from a healthy woman and could therefore be used for autologous treatment. RESULT(S): All plasma samples contained molecules with a high potential for regeneration (stem cell factor, platelet-derived growth factor BB, thrombospondin-1, von Willebrand factor). Furthermore, the highest increase in in vitro proliferation and migration rate was found when endometrial stromal cells were treated with umbilical cord plasma; adult PRP also revealed a significant increment. In the mouse model, a higher expression of Ki67 and Hoxa10 in the endometrium was detected after applying adult PRP, and the proteomic analysis revealed a specific protein expression profile depending on the treatment. The damaged uterine tissue showed more proregenerative markers after applying umbilical cord plasma (Stat5a, Uba3, Thy1) compared with the other treatments (nonactivated umbilical cord plasma, activated adult PRP, and no treatment). CONCLUSION(S): Human PRP possesses regeneration properties usable for endometrial pathologies. Besides that, these regenerative effects seem to be more apparent when the source of obtaining is umbilical cord blood.