Proteome-wide and matrisome-specific atlas of the human ovary computes fertility biomarker candidates and open the way for precision oncofertility.

Our modern era is witnessing an increasing infertility rate worldwide. Although some of the causes can be attributed to our modern lifestyle (e.g., persistent organic pollutants, late pregnancy), our knowledge of the human ovarian tissue has remained limited and insufficient to reverse the infertility statistics. Indeed, all efforts have been focused on the endocrine and cellular function in support of the cell theory that dates back to the 18th century, while the human ovarian matrisome is still under-described. Hereby, we unveil the extracellular side of the story during different periods of the ovary life, demonstrating that follicle survival and development, and ultimately fertility, would not be possible without its involvement. We examined the human ovarian matrisome and described its remodeling from prepuberty until menopause, creating the first ovarian proteomic codex. Here, we confidently identified and quantified 98 matrisome proteins present in the three ovary groups. Among them, 26 were expressed differently among age groups, delineating a peculiar matrisomal fingerprint at each stage. Such proteins could be potential biomarkers phenotyping ovarian ECM at each age phase of female reproductive life. Beyond proteomics, our study presents a unique approach to understanding the data and depicting the spatiotemporal ECM-intracellular signaling networks and remodeling with age through imaging, advanced text-mining based on natural language processing technology, machine learning, and data sonification. Our findings provide essential context for healthy ovarian physiology, identifying and characterizing disease states, and recapitulating physiological tissues or development in vitro. This comprehensive proteomics analysis represents the ovarian proteomic codex and contributes to an improved understanding of the critical roles that ECM plays throughout the ovarian life span.

Ovarian extracellular matrix-based hydrogel for human ovarian follicle survival in vivo: A pilot work.

To successfully assemble a bio-engineered ovary, we need to create a three-dimensional matrix able to accommodate isolated follicles and cells. The goal of this study was to develop an extracellular matrix hydrogel (oECM) derived from decellularized bovine ovaries able to support, in combination with alginate, human ovarian follicle survival and growth in vitro. Two different hydrogels (oECM1, oECM2) were produced and compared in terms of decellularization efficiency (dsDNA), ECM preservation (collagen and glycosaminoglycan levels), ultrastructure, rigidity, and cytotoxicity. oECM2 showed significantly less dsDNA, greater retention of glycosaminoglycans and better rigidity than oECM1. Isolated human ovarian follicles were then encapsulated in four selected hydrogel combinations: (1) 100% oECM2, (2) 90% oECM2 + 10% alginate, (3) 75% oECM2 + 25% alginate, and (4) 100% alginate. After 1 week of in vitro culture, follicle recovery rate, viability, and growth were analyzed. On day 7 of in vitro culture, follicle recovery rates were 0%, 23%, 65%, 82% in groups 1-4, respectively, rising proportionally with increased alginate content. However, there was no difference in follicle viability or growth between groups 2 and 3 and controls (group 4). In conclusion, since pure alginate cannot be used to graft preantral follicles due to its poor revascularization and degradation after grafting, oECM2 hydrogel combined with alginate may provide a new and promising alternative to graft isolated human follicles in a bio-engineered ovary.

A blueprint of the topology and mechanics of the human ovary for next-generation bioengineering and diagnosis.

Although the first dissection of the human ovary dates back to the 17th century, the biophysical characteristics of the ovarian cell microenvironment are still poorly understood. However, this information is vital to deciphering cellular processes such as proliferation, morphology and differentiation, as well as pathologies like tumor progression, as demonstrated in other biological tissues. Here, we provide the first readout of human ovarian fiber morphology, interstitial and perifollicular fiber orientation, pore geometry, topography and surface roughness, and elastic and viscoelastic properties. By determining differences between healthy prepubertal, reproductive-age, and menopausal ovarian tissue, we unravel and elucidate a unique biophysical phenotype of reproductive-age tissue, bridging biophysics and female fertility. While these data enable to design of more biomimetic scaffolds for the tissue-engineered ovary, our analysis pipeline is applicable for the characterization of other organs in physiological or pathological states to reveal their biophysical markers or design their bioinspired analogs.

Divide-and-Conquer Matrisome Protein (DC-MaP) Strategy: An MS-Friendly Approach to Proteomic Matrisome Characterization.

Currently, the extracellular matrix (ECM) is considered a pivotal complex meshwork of macromolecules playing a plethora of biomolecular functions in health and disease beyond its commonly known mechanical role. Only by unraveling its composition can we leverage related tissue engineering and pharmacological efforts. Nevertheless, its unbiased proteomic identification still encounters some limitations mainly due to partial ECM enrichment by precipitation, sequential fractionation using unfriendly-mass spectrometry (MS) detergents, and resuspension with harsh reagents that need to be entirely removed prior to analysis. These methods can be technically challenging and labor-intensive, which affects the reproducibility of ECM identification and induces protein loss. Here, we present a simple new method applicable to tissue fragments of 10 mg and more. The technique has been validated on human ovarian tissue and involves a standardized procedure for sample processing with an MS-compatible detergent and combined centrifugation. This two-step protocol eliminates the need for laborious sample clarification and divides our samples into 2 fractions, soluble and insoluble, successively enriched with matrisome-associated (ECM-interacting) and core matrisome (structural ECM) proteins.

The Human Ovary and Future of Fertility Assessment in the Post-Genome Era.

Proteomics has opened up new avenues in the field of gynecology in the post-genome era, making it possible to meet patient needs more effectively and improve their care. This mini-review aims to reveal the scope of proteomic applications through an overview of the technique and its applications in assisted procreation. Some of the latest technologies in this field are described in order to better understand the perspectives of its clinical applications. Proteomics seems destined for a promising future in gynecology, more particularly in relation to the ovary. Nevertheless, we know that reproductive biology proteomics is still in its infancy and major technical and ethical challenges must first be overcome.

A Draft Map of the Human Ovarian Proteome for Tissue Engineering and Clinical Applications.

Fertility preservation research in women today is increasingly taking advantage of bioengineering techniques to develop new biomimetic materials and solutions to safeguard ovarian cell function and microenvironment in vitro, and in vivo,. However, available data on the human ovary are limited and fundamental differences between animal models and humans are hampering researchers in their quest for more extensive knowledge of human ovarian physiology and key reproductive proteins that need to be preserved. We therefore turned to multi-dimensional label-free mass spectrometry to analyze human ovarian cortex, as it is a high-throughput and conclusive technique providing information on the proteomic composition of complex tissues like the ovary. In-depth proteomic profiling through two-dimensional liquid chromatography-mass spectrometry, Western blotting, histological and immunohistochemical analyses, and data mining helped us to confidently identify 1508 proteins. Moreover, our method allowed us to chart the most complete representation so far of the ovarian matrisome, defined as the ensemble of extracellular matrix proteins and associated factors, including more than 80 proteins. In conclusion, this study will provide a better understanding of ovarian proteomics, with a detailed characterization of the ovarian follicle microenvironment, in order to enable bioengineers to create biomimetic scaffolds for transplantation and three-dimensional in vitro, culture. By publishing our proteomic data, we also hope to contribute to accelerating biomedical research into ovarian health and disease in general.

A novel fibrin-based artificial ovary prototype resembling human ovarian tissue in terms of architecture and rigidity.

PURPOSE: The aim of this study is to optimize fibrin matrix composition in order to mimic human ovarian tissue architecture for human ovarian follicle encapsulation and grafting. METHODS: Ultrastructure of fresh human ovarian cortex in age-related women (n = 3) and different fibrin formulations (F12.5/T1, F30/T50, F50/T50, F75/T75), rheology of fibrin matrices and histology of isolated and encapsulated human ovarian follicles in these matrices. RESULTS: Fresh human ovarian cortex showed a highly fibrous and structurally inhomogeneous architecture in three age-related patients, but the mean ± SD of fiber thickness (61.3 to 72.4 nm) was comparable between patients. When the fiber thickness of four different fibrin formulations was compared with human ovarian cortex, F50/T50 and F75/T75 showed similar fiber diameters to native tissue, while F12.5/T1 was significantly different (p value < 0.01). In addition, increased concentrations of fibrin exhibited enhanced storage modulus with F50/T50, resembling physiological ovarian rigidity. Excluding F12.5/T1 from further analysis, only three remaining fibrin matrices (F30/T50, F50/T50, F75/T75) were histologically investigated. For this, frozen-thawed fragments of human ovarian tissue collected from 22 patients were used to isolate ovarian follicles and encapsulate them in the three fibrin formulations. All three yielded similar follicle recovery and loss rates soon after encapsulation. Therefore, based on fiber thickness, porosity, and rigidity, we selected F50/T50 as the fibrin formulation that best mimics native tissue. CONCLUSIONS: Of all the different fibrin matrix concentrations tested, F50/T50 emerged as the combination of choice in terms of ultrastructure and rigidity, most closely resembling human ovarian cortex.