Bioengineering strategies of the uterus towards improving current investigative models and female reproductive health

In this thesis, we intended to develop tissue-engineering approaches based on decellularized (DC) uterine tissues obtained from whole organs to improve several aspects within reproductive medicine. We hypothesized that that the decellularization of whole uteri from different species has not only the potential to, one day, create tissue-engineered, transplantable organs but that the DC endometrial fraction can also be processed further into thin sections, ECM hydrogels and coatings that can be used as a biocompatible tissue-specific substrate for cell and embryo culture. Moreover, we intended to corroborate if the differences in the cyclically and drastically changing endometrium are translated to these hydrogels and coatings, possibly affecting the development of embryo culture. In the first part of this thesis, we developed a perfusion-based protocol usable for large pig uteri. The effect of a prior freeze/thaw step was also assessed. For this, three frozen-thawed and three fresh uteri were subjected to a DC protocol we optimized for the pig uterus consisting of SDS and Triton X-100 cycles. Furthermore, the F/T step did not noticeably affect the ECM architecture in our experience. DC efficiency was tested by histology techniques and by DNA and protein quantification. Vascular corrosion cast, immunofluorescence (Collagen I & IV, elastin, laminin and fibronectin), scanning and transmission electron microscopy were performed to assess the effect on organ vasculature, ECM composition and its ultrastructure respectively. Finally, in vitro biocompatibility was tested by recellularizing endometrial sections using human endometrial Side Population stem cell lines. In the second part of the thesis, we created coatings and hydrogels from different DC endometrial tissues and compare in vitro embryo development between these substrata, with commercial coatings and with standard culture conditions. For this, DC uteri without ovarian stimulation and synchronous uteri with day 3 embryos were obtained. In total eight different embryo culture conditions were tested: on top of biological surface coatings and hydrogels made of non-synchronous, synchronous acellular endometrium and Matrigel and compared to standard culture. After that, hatching rates, morphometry and expression of three core pluripotency markers were analyzed and compared. Our results seem to demonstrate that synchronous endometrium coating would retain and release compounds that permit, in part, to mimic the endometrial environment under in vitro conditions.While DC organs could be used to create transplantable organs, endometrial ECM hydrogels and coatings have the potential to become a platform used in the culture of stem/progenitor cells and primary culture cells to better maintain their tissue-specific phenotype, improving in vitro models. They can also have in vivo applications, such as the treatment of ashermans syndrome and endometrial atrophy

Tissue-specific decellularized endometrial substratum mimicking different physiological conditions influences in vitro embryo development in a rabbit model

[EN] In the last decades, the decellularization (DC) of organs has become an established technique in the field of regenerative medicine to yield complex and vascularized bioscaffolds. Furthermore, it has been demonstrated in vitro that these decellularized scaffolds retain their native tissue-specificity. This is also the case when this tissue-specific extracellular matrix (ECM) is solubilized and used as hydrogels or coatings to create a biomimetic environment. In this study we investigated if this specificity not only remains when applied to distinct tissues but even more, that these differences can be distinguished within the same tissue at different stages of proliferation. To address this question, a sensitive in vitro animal model was used: rabbit embryos at the third day of development were cultured on coatings made from acellular endometrium that was non-proliferating (non-synchronous, NS) and proliferating (synchronous with the embryo, S) and their development was compared. For this, we obtained whole NS and S rabbit uteri and subjected them to an adapted decellularization protocol. The acellular endometrium was carefully separated by microdissection and converted into a pre-gel solution to be used as hydrogels and coatings for in vitro assays. First, the characteristics of these NS and S hydrogels were investigated by proteomic analysis, electron microscopy and gelling kinetics. When used as substrata for day 3 embryos culture, it became apparent that only the acellular ECM from synchronous endometrial coating achieved similar results to the gold standard culture protocols and conditions, possibly because of the slow release of growth factors present in the synchronous/proliferating endometrium. Statement of Significance It has been shown by in vitro culture of stem cells, progenitor cells and primary culture cells that decellularized tissues retain their specific functions and biochemical and structural compositions. The present work demonstrates that using a mild SDS and perfusion based decellularization (DC) protocol not only effectively decellularize whole rabbit uteri, adding to the growing field of reproductive tissue engineering, but more importantly that the differences in the proliferating endometrium are translated after DC. This implies that DC not only retains the interspecificity of tissues but also the intraspecificity of a developing hormonally stimulated tissue. For the first time, we demonstrate that the coating from decellularized synchronous endometrium acts as a biological support for in vitro embryo development, achieving comparable results with the current gold standard that only uses serum-containing media. (C) 2019 Acta Materialia Inc. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).This study was supported by the following entities: GRISO-LIA/2015/002 (HC); PI17/01039 (IC); Prometeo/2018/137 (IC); AGL2017-85162-C2-1-R; BES-2015-072429 (XGD); ACIF/2017/118 (SLM). The proteomics laboratory is a member of Proteored, PRB3 and is supported by grant PT17/0019, of the PE I + D + i 2013-2016, funded by ISCIII and ERDF.Campo, H.; Garcia-Dominguez, X.; López-Martínez, S.; Faus, A.; Vicente Antón, JS.; Marco-Jiménez, F.; Cervello, I. (2019). Tissue-specific decellularized endometrial substratum mimicking different physiological conditions influences in vitro embryo development in a rabbit model. Acta Biomaterialia. 89:126-138. https://doi.org/10.1016/j.actbio.2019.03.004S1261388

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 such as umbilical cord blood and adult blood platelet-rich plasma (PRP) on the regeneration of endometrial damage. Design. Composition analysis, in vitro approaches and a pre-clinical murine model using plasma to promote endometrial regeneration. Setting. Hospital and university laboratories. Patients/Animals. Adult plasma from 4 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). All plasma samples contain molecules with a high potential for regeneration (SCF, PDGFBB, THBS1, VWF). 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 pro22 regenerative markers after applying umbilical cord plasma (Stat5a, Uba3, Thy1) in comparison to the other treatments (non-activated umbilical cord plasma, activated adult PRP and not treatment). Conclusion. 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

Stem cell paracrine actions in tissue regeneration and its potential therapeutic effect in human endometrium: a retrospective study.

Objective: Determining genetic and paracrine mechanisms behind endometrial regeneration in Asherman's Syndrome and Endometrial Atrophy (AS/EA) patients after autologous CD133+ bone marrow-derived stem cells (CD133+BMDSCs) transplantation. Design: Retrospective study using human endometrial biopsies and mouse models. Setting: Fundación-IVI, IIS-La Fe, Valencia, Spain. Samples: Endometrial biopsies collected before and after CD133+BMDSCs therapy, from 8 women with AS/EA (NCT02144987). And uterus from 5 mice, with only left horns receiving CD133+BMDSCs therapy. Methods: In human samples, hematoxylin and eosin (H&E) staining, RNA arrays, PCR validation and neutrophil elastase (NE) immunohistochemistry (IHQ). In mouse samples, PCR validation and protein immunoarrays

Identifying Fishes through DNA Barcodes and Microarrays

Background: International fish trade reached an import value of 62.8 billion Euro in 2006, of which 44.6% are covered by the European Union. Species identification is a key problem throughout the life cycle of fishes: from eggs and larvae to adults in fisheries research and control, as well as processed fish products in consumer protection. Methodology/Principal Findings: This study aims to evaluate the applicability of the three mitochondrial genes 16S rRNA (16S), cytochrome b (cyt b), and cytochrome oxidase subunit I (COI) for the identification of 50 European marine fish species by combining techniques of ‘‘DNA barcoding’’ and microarrays. In a DNA barcoding approach, neighbour Joining (NJ) phylogenetic trees of 369 16S, 212 cyt b, and 447 COI sequences indicated that cyt b and COI are suitable for unambiguous identification, whereas 16S failed to discriminate closely related flatfish and gurnard species. In course of probe design for DNA microarray development, each of the markers yielded a high number of potentially species-specific probes in silico, although many of them were rejected based on microarray hybridisation experiments. None of the markers provided probes to discriminate the sibling flatfish and gurnard species. However, since 16S-probes were less negatively influenced by the ‘‘position of label’’ effect and showed the lowest rejection rate and the highest mean signal intensity, 16S is more suitable for DNA microarray probe design than cty b and COI. The large portion of rejected COI-probes after hybridisation experiments (.90%) renders the DNA barcoding marker as rather unsuitable for this high-throughput technology. Conclusions/Significance: Based on these data, a DNA microarray containing 64 functional oligonucleotide probes for the identification of 30 out of the 50 fish species investigated was developed. It represents the next step towards an automated and easy-to-handle method to identify fish, ichthyoplankton, and fish products

The European Reference Genome Atlas: piloting a decentralised approach to equitable biodiversity genomics.

ABSTRACT: A global genome database of all of Earth’s species diversity could be a treasure trove of scientific discoveries. However, regardless of the major advances in genome sequencing technologies, only a tiny fraction of species have genomic information available. To contribute to a more complete planetary genomic database, scientists and institutions across the world have united under the Earth BioGenome Project (EBP), which plans to sequence and assemble high-quality reference genomes for all ∼1.5 million recognized eukaryotic species through a stepwise phased approach. As the initiative transitions into Phase II, where 150,000 species are to be sequenced in just four years, worldwide participation in the project will be fundamental to success. As the European node of the EBP, the European Reference Genome Atlas (ERGA) seeks to implement a new decentralised, accessible, equitable and inclusive model for producing high-quality reference genomes, which will inform EBP as it scales. To embark on this mission, ERGA launched a Pilot Project to establish a network across Europe to develop and test the first infrastructure of its kind for the coordinated and distributed reference genome production on 98 European eukaryotic species from sample providers across 33 European countries. Here we outline the process and challenges faced during the development of a pilot infrastructure for the production of reference genome resources, and explore the effectiveness of this approach in terms of high-quality reference genome production, considering also equity and inclusion. The outcomes and lessons learned during this pilot provide a solid foundation for ERGA while offering key learnings to other transnational and national genomic resource projects.info:eu-repo/semantics/publishedVersio

The European Reference Genome Atlas: piloting a decentralised approach to equitable biodiversity genomics

A genomic database of all Earth’s eukaryotic species could contribute to many scientific discoveries; however, only a tiny fraction of species have genomic information available. In 2018, scientists across the world united under the Earth BioGenome Project (EBP), aiming to produce a database of high-quality reference genomes containing all ~1.5 million recognized eukaryotic species. As the European node of the EBP, the European Reference Genome Atlas (ERGA) sought to implement a new decentralised, equitable and inclusive model for producing reference genomes. For this, ERGA launched a Pilot Project establishing the first distributed reference genome production infrastructure and testing it on 98 eukaryotic species from 33 European countries. Here we outline the infrastructure and explore its effectiveness for scaling high-quality reference genome production, whilst considering equity and inclusion. The outcomes and lessons learned provide a solid foundation for ERGA while offering key learnings to other transnational, national genomic resource projects and the EBP.info:eu-repo/semantics/publishedVersio

Three-Dimensionally Printed Agarose Micromold Supports Scaffold-Free Mouse Ex Vivo Follicle Growth, Ovulation, and Luteinization

Ex vivo follicle growth is an essential tool, enabling interrogation of folliculogenesis, ovulation, and luteinization. Though significant advancements have been made, existing follicle culture strategies can be technically challenging and laborious. In this study, we advanced the field through development of a custom agarose micromold, which enables scaffold-free follicle culture. We established an accessible and economical manufacturing method using 3D printing and silicone molding that generates biocompatible hydrogel molds without the risk of cytotoxicity from leachates. Each mold supports simultaneous culture of multiple multilayer secondary follicles in a single focal plane, allowing for constant timelapse monitoring and automated analysis. Mouse follicles cultured using this novel system exhibit significantly improved growth and ovulation outcomes with comparable survival, oocyte maturation, and hormone production profiles as established three-dimensional encapsulated in vitro follicle growth (eIVFG) systems. Additionally, follicles recapitulated aspects of in vivo ovulation physiology with respect to their architecture and spatial polarization, which has not been observed in eIVFG systems. This system offers simplicity, scalability, integration with morphokinetic analyses of follicle growth and ovulation, and compatibility with existing microphysiological platforms. This culture strategy has implications for fundamental follicle biology, fertility preservation strategies, reproductive toxicology, and contraceptive drug discovery

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

[EN] 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 degrees 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.This study was supported by Instituto de Salud Carlos III (PI17/01039-CP19/00149 [Irene Cervello]), Ministry of Economy, Industry and Competitiveness (AGL2017-85162-C2-1-R [Francisco Marco], BES-2015-072429 [Ximo Garcia]), Ministry of Science, Innovation and Universities (FPU18/06327 [Emilio Frances]) and Generalitat Valenciana (PROMETEO/2018/137 [Irene Cervello, Lucia de Miguel], ACIF/2017/118 [Sara Lopez]). The proteomics laboratory is a member of ProteoRed, PRB3, and is supported by grant PT17/0019 of the State Plan I+D+i 2013-2016, funded by Instituto de Salud Carlos III and European Regional Development Fund.Francés-Herrero, E.; De Miguel-Gómez, L.; López-Martínez, S.; Campo, H.; Garcia-Dominguez, X.; Diretto, G.; Faus, A.... (2021). Development of Decellularized Oviductal Hydrogels as a Support for Rabbit Embryo Culture. Reproductive Sciences. 28(6):1644-1658. https://doi.org/10.1007/s43032-020-00446-6S1644165828