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/.

Transcriptome analysis of adenomyosis eutopic endometrium reveals molecular mechanisms involved in adenomyosis-related implantation failure and pregnancy disorders.

BACKGROUND: Women with adenomyosis are characterized by having defective decidualization, impaired endometrial receptivity and/or embryo-maternal communication, and implantation failure. However, the molecular mechanisms underlying adenomyosis-related infertility remain unknown, mainly because of the restricted accessibility and the difficult preservation of endometrial tissue in vitro. We have recently shown that adenomyosis patient-derived endometrial organoids, maintain disease-specific features while differentiated into mid-secretory and gestational endometrial phase, overcoming these research barriers and providing a robust platform to study adenomyosis pathogenesis and the associated molecular dysregulation related to implantation and pregnancy disorders. For this reason, we aim to characterize the dysregulated mechanisms in the mid-secretory and gestational endometrium of patients with adenomyosis by RNA-sequencing. METHODS: Endometrial organoids were derived from endometrial biopsies collected in the proliferative phase of women with adenomyosis (ADENO) or healthy oocyte donors (CONTROL) (n = 15/group) and differentiated into mid-secretory (-SECorg) and gestational (-GESTorg) phases in vitro. Following RNA-sequencing, the significantly differentially expressed genes (DEGs) (FDR < 0.05) were identified and selected for subsequent functional enrichment analysis and QIAGEN Ingenuity Pathway Analysis (IPA). Statistical differences in gene expression were evaluated with the Student's t-test or Wilcoxon test. RESULTS: We identified 1,430 DEGs in ADENO-SECorg and 1,999 DEGs in ADENO-GESTorg. In ADENO-SECorg, upregulated genes included OLFM1, FXYD5, and RUNX2, which are involved in impaired endometrial receptivity and implantation failure, while downregulated genes included RRM2, SOSTDC1, and CHAC2 implicated in recurrent implantation failure. In ADENO-GESTorg, upregulated CXCL14 and CYP24A1 and downregulated PGR were related to pregnancy loss. IPA predicted a significant inhibition of ID1 signaling, histamine degradation, and activation of HMGB1 and Senescence pathways, which are related to implantation failure. Alternatively, IPA predicted an inhibition of D-myo-inositol biosynthesis and VEGF signaling, and upregulation of Rho pathway, which are related to pregnancy loss and preeclampsia. CONCLUSIONS: Identifying dysregulated molecular mechanisms in mid-secretory and gestational endometrium of adenomyosis women contributes to the understanding of adenomyosis-related implantation failure and/or pregnancy disorders revealing potential therapeutic targets. Following experimental validation of our transcriptomic and in silico findings, our differentiated adenomyosis patient-derived organoids have the potential to provide a reliable platform for drug discovery, development, and personalized drug screening for affected patients.

A biomechanical assessment of tissue-engineered polymer neo-uteri after orthotopic implantation.

OBJECTIVE: To assess the in vivo biomechanical maturation of tissue-engineered neo-uteri that have previously supported live births in a rabbit model. DESIGN: Nonclinical animal study. SETTING: University-based research laboratory. ANIMALS: Eighteen adult female rabbits. INTERVENTION: Biodegradable poly-DL-lactide-co-glycolide-coated polyglycolic acid scaffolds seeded with autologous uterine-derived endometrial and myometrial cells. Nonseeded scaffolds and seeded, tissue-engineered neo-uteri were implanted into one uterine horn of rabbits for 1, 3, or 6 months, excised, and biomechanically assessed in comparison to native uterine tissue. MAIN OUTCOME MEASURES: Tensile stress-relaxation testing, strain-to-failure testing, and viscoelastic modeling. RESULTS: By evaluating the biomechanical data with several viscoelastic models, it was revealed that tissue-engineered uteri were more mechanically robust than nonseeded scaffolds. For example, the 10% instantaneous stress of the tissue-engineered neo-uteri was 2.1 times higher than the nonseeded scaffolds at the 1-month time point, 1.6 times higher at the 3-month time point, and 1.5 times higher at the 6-month time point. Additionally, as the duration of implantation increased, the engineered constructs became more mechanically robust (e.g., 10% instantaneous stress of the tissue-engineered neo-uteri increased from 22 kPa at 1 month to 42 kPa at 6 months). Compared with native tissue values, tissue-engineered neo-uteri achieved or surpassed native tissue values by the 6-month time point. CONCLUSION: The present study evaluated the mechanical characteristics of novel tissue-engineered neo-uteri that have previously been reported to support live births in the rabbit model. We demonstrate that the biomechanics of these implants closely resemble those of native tissue, giving further credence to their development as a clinical solution to uterine factor infertility.

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.

Extracellular vesicles secreted by adenomyosis endometrial organoids contain miRNAs involved in embryo implantation and pregnancy.

RESEARCH QUESTION: Do extracellular vesicles secreted by the endometrium of women with adenomyosis contain miRNAs involved in adenomyosis-related infertility? DESIGN: A descriptive study using organoids from eutopic endometrium of women with adenomyosis (n = 4) generated and differentiated to secretory and gestational phases, in which miRNA cargo from extracellular vesicles secreted by these differentiated organoids in each phase was analysed by next-generation sequencing. miRNAs in secretory-extracellular vesicles and gestational-extracellular vesicles were selected based on the counts per million. miRNAs target genes in each phase were obtained from miRNet and gene ontology was used for enrichment analysis. RESULTS: miRNA sequencing identified 80 miRNAs in secretory-phase extracellular vesicles, including hsa-miR-21-5p, hsa-miR-24-3p, hsa-miR-26a-5p, hsa-miR-92a-3p, hsa-miR-92b-3p, hsa-miR-200c-3p and hsa-miR-423a-5p, related to adenomyosis pathogenesis and implantation failure. Further, 60 miRNAs were identified in gestational-phase extracellular vesicles, including hsa-miR-21-5p, hsa-miR-26a-5p, hsa-miR-30a-5p, hsa-miR-30c-5p, hsa-miR-222-3p and hsa-miR-423a-5p were associated with preeclampsia and miscarriage. Among the target genes of these miRNAs, PTEN, MDM4, PLAGL2 and CELF1, whose downregulation (P = 0.0003, P < 0.0001, P = 0.0002 and P = 0.0003, respectively) contributes to adenomyosis pathogenesis, and impaired early embryo development, leading to implantation failure and miscarriage, are highlihghted. Further, functional enrichment analyses of the target genes revealed their involvement in cell differentiation, proliferation, apoptosis, cell cycle regulation and response to extracellular stimuli. CONCLUSIONS: Eutopic endometrium in secretory and gestational phase from women with adenomyosis releases extracellular vesicles containing miRNAs involved in adenomyosis progression, impaired embryo implantation and pregnancy complications.

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.

Endometrial gene expression differences in women with coronavirus disease 2019.

OBJECTIVE: To study the potential effect of coronavirus disease (COVID-19) on the endometrial transcriptome of affected, symptomatic women for the detection of altered gene expression. DESIGN: Pilot study of the endometrial transcriptomes of women manifesting COVID-19 compared with those of women without COVID-19 undergoing hysteroscopic procedures for benign gynecologic disorders using RNA sequencing. SETTING: Hospital and university laboratories. PATIENT(S): Women with (n = 14) and without a COVID-19 (n = 10) diagnosis based on a nasopharyngeal swab analysis using quantitative reverse-transcription polymerase chain reaction. The endometrium of the patients with COVID-19 had previously been tested for severe acute respiratory syndrome coronavirus 2 infection, revealing the absence of the virus in this tissue. INTERVENTION(S): Endometrial biopsy sample collection. MAIN OUTCOMES MEASURE(S): Endometrial gene expression and functional analysis of symptomatic patients with COVID-19 vs. individuals without the infection. RESULT(S): The systemic disease COVID-19 altered endometrial gene expression in 75% of the women, with the patients exhibiting a preponderance of 163 up-regulated (e.g., UTS2, IFI6, IFIH1, and BNIP3) and 72 down-regulated genes (e.g., CPZ, CDH3, and IRF4) (false discovery rate<0.05). A total of 161 dysregulated functions (36 up-regulated and 125 down-regulated) were typically enriched in the endometria of the patients with COVID-19, including up-regulation in pathways involved in the development of immune responses to viruses and cytokine inflammation, reflecting elicitation of a COVID-19 response pathway. CONCLUSION(S): Coronavirus disease 2019 affects endometrial gene expression despite the absence of severe acute respiratory syndrome coronavirus 2 RNA in endometrial tissues.

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.

Decellularization Methods of Ovary in Tissue Engineering.

The ovaries or female gonads are situated in the ovarian fossa of the abdominal cavity. These are paired, almond-shaped organs measuring about 3.5 cm long and 1.5 cm thick and exist out of a central medullary zone and a peripheral cortex that are enclosed in a fibrous capsule called the tunica albuginea. The ovaries serve 2 main functions, the first one being the production of female gametes called oocytes (oogenesis). Interestingly, the number of primary oocytes that reside in the ovary is determined at birth. About 400 oocyte-containing follicles successfully go through all the developmental stages from this limited pool during folliculogenesis throughout the female reproductive life. In this process, primordial follicles grow and advance until forming a mature or Graafian follicle; during ovulation, secondary oocytes are released and the remaining follicular wall collapses and forms the highly vascularized corpus luteum or luteal gland. This ovarian cycle is regulated by several hormones secreted from the adenohypophysis and lasts about 28 days. During this cycle, the ovaries also serve as endocrine glands and produce female sex hormones such as estrogens and progesterone (steroidogenesis), influencing the growth and development of tissues sensitive to these hormones such as the endometrium. Hence, the endometrial cycle goes synchronized with the ovarian cycle.

Decellularization Methods of Uterus in Tissue Engineering.

A new field of investigation which aims to design tissues and organs similar to their native origin has been developed recently, named as regenerative medicine (tissue engineering and bio-engineering). Uterus is the main organ for regeneration and contributes in the fertility. At an ultimate level, the uterus plays a role in embryo implantation, sperm migration and fetal nutrition. Uterine congenital anomalies, attained uterine lesions and immune system disorders may affect such uterine functions preventing successful pregnancy. Due to following reasons, it is essential to consider regenerative medicine as a new approach for the treatment of uterine dysfunctions to overcome the failures that cannot be treated with clinical medication.

Decellularization Methods of Vagina and Cervix in Tissue Engineering.

The vagina is a fibromuscular elastic tubular tract that connects the cervix with the outer genitals and has an important function discharging uterine secretions, sexual intercourse and acts as the passage for the full-term fetus. Currently, a new field of investigation which aims to design tissues and organs similar to their native origin has been developed recently and was named regenerative medicine (tissue engineering and bioengineering). Malformations in cervix tissue represent a hard challenge for medicine. Experts in bioengineering have tried to reconstruct vaginas or cervix with the aim to achieve cervicovaginal disorders, most of them with congenital cause. However, only few research groups have launched themselves upon the decellularization. The aim of this chapter is investigating the decellularization methods for cervix and vaginal tissues.

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.

5-aza-2'-deoxycitidine inhibits cell proliferation, extracellular matrix formation and Wnt/ß-catenin pathway in human uterine leiomyomas.

BACKGROUND: Uterine leiomyoma is a benign tumor with unclear pathogenesis and inaccurate treatment. This tumor exhibits altered DNA methylation related to disease progression. DNMT inhibitors as 5-aza-2'-deoxycytidine (5-aza-CdR), have been suggested to treat tumors in which DNA methylation is altered. We aimed to evaluate whether DNA methylation reversion with 5-aza-CdR reduces cell proliferation and extracellular matrix (ECM) formation in uterine leiomyoma cells to provide a potential treatment option. METHODS: Prospective study using uterine leiomyoma and adjacent myometrium tissues and human uterine leiomyoma primary (HULP) cells (n = 16). In tissues, gene expression was analyzed by qRT-PCR and DNMT activity by ELISA. Effects of 5-aza-CdR treatment on HULP cells were assessed by CellTiter, western blot, and qRT-PCR. RESULTS: DNMT1 gene expression was higher in uterine leiomyoma vs myometrium. Similarly, DNMT activity was greater in uterine leiomyoma and HULP cells (6.5 vs 3.8 OD/h/mg; 211.3 vs 63.7 OD/h/mg, respectively). After 5-aza-CdR treatment on HULP cells, cell viability was reduced, significantly so at 10 µM (85.3%). Treatment with 10 µM 5-aza-CdR on HULP cells significantly decreased expression of proliferation marker PCNA (FC = 0.695) and of ECM proteins (COLLAGEN I FC = 0.654; PAI-1, FC = 0.654; FIBRONECTIN FC = 0.733). 5-aza-CdR treatment also decreased expression of Wnt/ß-catenin pathway final targets, including WISP1 protein expression (10 µM, FC = 0.699), c-MYC gene expression (2 µM, FC = 0.745 and 10 µM, FC = 0.728), and MMP7 gene expression (5 µM, FC = 0.520 and 10 µM, FC = 0.577). CONCLUSIONS: 5-aza-CdR treatment inhibits cell proliferation, ECM formation, and Wnt/ß-catenin signaling pathway targets in HULP cells, suggesting that DNA methylation inhibition is a viable therapeutic target in uterine leiomyoma.

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.