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

Ouni, Emna; Nedbal, Valerie; Pian, Marta Da; Cao, Hangbao; Haas, Kalina T.; Peaucelle, Alexis; Kerk, Olivier Van; Herinckx, Gaëtan; Marbaix, Étienne; Dolmans, Marie-Madeleine; Tuuri, Timo; Otala, Marjut; Amorim, Christiani Andrade; Vertommen, Didier

doi:10.1016/j.matbio.2022.03.005

Cited by 15 publications

(13 citation statements)

References 84 publications

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“…In contrast, post-menopausal ovaries primarily consist of populations such as endothelial cells, immune cells, lymphatic endothelial cells, perivascular cells, smooth muscle cells, and stromal cells ( Lengyel et al , 2022 ). Notably, these aging ovaries exhibit a characteristic inflammatory environment, which has been identified as one of the mechanisms contributing to follicle depletion ( Ansere et al , 2021 ; Lliberos et al , 2021 ; Ouni et al , 2022 ), suggesting that they are not conducive to supporting folliculogenesis. However, cells isolated from post-menopausal ovaries have demonstrated remarkable plasticity in vitro , being able to survive, proliferate, differentiate, secrete hormones, and synthesize extracellular matrix ( Asiabi et al , 2020 ; Maruhashi et al , 2020 ; Dadashzadeh et al , 2022 ).…”

Section: Discussionmentioning

confidence: 99%

Influence of ovarian stromal cells on human ovarian follicle growth in a 3D environment

Grubliauskaitė,

Vlieghe,

Moghassemi

et al. 2023

Human Reproduction Open

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STUDY QUESTION Do ovarian stromal cells (OSCs) influence the viability and growth of human pre-antral follicles in vitro? SUMMARY ANSWER A feeder layer of oSCs promotes the growth and transition of low developmental stage follicles to the primary/secondary stage while maintaining a high proportion of viable follicles. WHAT IS KNOWN ALREADY In the ovary, follicles rely on the support of ovarian cells, which secrete essential factors for their survival and development. This phenomenon has also been demonstrated in vitro through the 3D culture of isolated mouse primary and secondary follicles on a feeder layer of ovarian stromal cells. This co-culture notably enhances follicle survival and growth. STUDY DESIGN, SIZE, DURATION Pre-antral follicles were isolated from human frozen-thawed ovarian tissue (OT) biopsies and then encapsulated in 1% alginate scaffolds. These embedded pre-antral follicles were either placed directly on the OSCs feeder layer or at the bottom of a culture dish for a 7-day in vitro culture (control). The study compared follicle viability, growth and hormone production between the different groups. PARTICIPANTS/MATERIALS, SETTING, METHODS Primordial/intermediate and primary follicles were isolated from frozen-thawed OT of cancer patients (n = 6). Ovarian stromal cells were then isolated from OT of post-menopausal women and cultured as a feeder layer. Follicle diameter was measured on days 0 and 7 using an inverted microscope to assess their development based on the increase in diameter. Viability was evaluated by staining a subset of follicles (n = 87) with calcein AM and ethidium homodimer-I, followed by classification into healthy/minimally damaged and damaged/dead follicles using confocal fluorescence microscopy. Additionally, estradiol levels were measured using ELISA. MAIN RESULTS AND THE ROLE OF CHANCE A total of 382 human preantral follicles (370 primordial/intermediate and 12 primary) with a mean diameter of 40.8 ± 9.9 µm (mean±SD) were isolated, embedded in 1% alginate hydrogel, and placed either on a monolayer of oSCs or directly on the plastic. By Day 7, the preantral follicles showed a significant size increase under both culture conditions (p < 0.0001 for D0 vs D7). The mean diameter of follicles (quiescent and growing) cultured on the feeder layer was 80.6 ± 11.0 μm compared to 67.3 ± 7.2 μm without it (p = 0.07). During the 7-day in vitro culture, the viability of the follicles significantly decreased only in the group without an OSCs monolayer compared to the D0 viability (p < 0.05). Additionally, more follicles transitioned to a higher developmental stage in the presence of OSCs (D0 primordial/intermediate: 184, primary: 7 vs D7 primordial/intermediate: 51, primary/secondary: 93) compared to those cultured without OSCs (D0 primordial/intermediate: 186, primary: 5 vs D7 primordial/intermediate: 84, primary/secondary: 65; p < 0.001). Specifically, 66 and 44 follicles reached the secondary stage (75 < x < 200 μm) in the presence and absence of OSCs, respectively. Moreover, the estradiol level was significantly higher (p = 0.006) in the alginate beads containing primordial and growing follicles cultured on the OSCs (54.1 ± 14.2 pg/ml) compared to those cultured without oSCs (29.9 ± 4.0 pg/ml). LARGE SCALE DATA N/A LIMITATIONS, REASONS FOR CAUTION This study was conducted using a short-term culture, and none of the primordial/intermediate/primary follicles reached the antral stage. Further in vitro studies are required to investigate follicular developmental capacity, physiology and steroidogenesis in alginate scaffolds with human ovarian stromal cells. WIDER IMPLICATIONS OF THE FINDINGS Activating and growing human primordial/intermediate follicles to a secondary stage in in vitro short-term culture has posed a longstanding challenge. However, co-culturing with human ovarian stromal cells has shown the potential to overcome this limitation. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (FNRS-PDR Convention grant number T.0004.20 awarded to C.A.A., Ph.D. scholarship awarded to H.V.), Fondation Louvain (awarded to C.A.A.; Ph.D. scholarship awarded to S.M., as part of a legacy from Mr Frans Heyes, and Ph.D. scholarship awarded to A.D. as part of a legacy from Mrs. Ilse Schirmer), Foundation Against Cancer (grant 2018-042 awarded to A.C.), and the European Community Structural Funds and Lithuanian Research Council (Agreement registration No. D-19-0874). The authors have no conflicts of interest to declare.

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Section: Discussionmentioning

confidence: 99%

Influence of ovarian stromal cells on human ovarian follicle growth in a 3D environment

Grubliauskaitė,

Vlieghe,

Moghassemi

et al. 2023

Human Reproduction Open

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“…Follicle growth and cell–cell communication in follicles were evaluated by Ki67, connexin 43 (Cx43), and TZP staining on Days 4 and 7, by adapting the protocols from Ouni et al (2022) and Bus et al (2021) . Briefly, at the end of each culture period (4 or 7 days), the hydrogels were washed with PBS.…”

Section: Methodsmentioning

confidence: 99%

Mind the mechanical strength: tailoring a 3D matrix to encapsulate isolated human preantral follicles

Dadashzadeh

Moghassemi

Peaucelle

et al. 2023

Human Reproduction Open

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STUDY QUESTION Would a hydrogel with similar mechanical properties to the human ovarian cortex support preantral follicle development? SUMMARY ANSWER Yes, our tailored PEGylated fibrin hydrogel was shown to significantly improve follicle growth in vitro. WHAT IS KNOWN ALREADY One of the main challenges in developing an engineered ovary is to provide a 3D matrix that supports the follicle architecture and the interaction between granulosa cells and the oocyte as they are essential for folliculogenesis. Thanks to its biocompatibility and bioactivity, fibrin has been employed to fabricate a 3D matrix to encapsulate ovarian follicles. However, follicles lose their physical support within a few days owing to rapid fibrin degradation. Therefore, different strategies, including physical and chemical modifications, have been developed to enhance the stability of fibrin. STUDY DESIGN, SIZE, DURATION By developing a matrix made of a synthetic (polyethylene glycol: PEG) and natural polymer (fibrin), we aimed to overcome fibrin degradation by the chemical reaction of PEGylation and tailor a PEGylated fibrin hydrogel formulation with mechanical strength similar to the ovarian cortex in women of reproductive age. To this end, response surface methodology was employed to obtain a tailored formulation of PEGylated fibrin. This hydrogel was then tested to encapsulate and support isolated human preantral follicles in vitro. PARTICIPANTS/MATERIALS, SETTING, METHODS A PEGylated fibrin formulation was tailored using mathematical modeling software to mimic the mechanical properties of human ovarian tissue at reproductive age. Human preantral follicles were isolated from 11 patients of reproductive age and encapsulated in the tailored hydrogels, which were cultured in vitro for 4 or 7 days. Follicle survival and diameter were assessed on days 1 and 7. Furthermore, the follicles were subjected to confocal microscopy to evaluate their growth (Ki67 staining) on day 7 and analyze cell-cell communication (connexin 43 and transzonal projection staining) on day 4. MAIN RESULTS AND THE ROLE OF CHANCE In this study, mathematical modeling was applied to achieve the biomechanically tailored PEGylated fibrin formulation by targeting the specific goal of 3178 ± 245 Pascal, Young’s modulus of ovarian cortical tissue in reproductive-age women. Our results demonstrated that the PEGylated fibrin hydrogel consisting of 39.06 mg/ml of PEGylated fibrinogen and 50.36 IU/ml of thrombin was the optimum condition with the desirability of 97.5%. This tailored hydrogel yielded a high follicle survival rate (83%) after 7 days of in vitro culture and supported its development up to the secondary stage. Follicle growth was confirmed by the presence of Ki67-positive granulosa cells on day 7. Additionally, connexin 43 and Phalloidin staining indicated the retention of connections between granulosa cells and the oocyte. LARGE SCALE DATA Not applicable. LIMITATIONS, REASONS FOR CAUTION In this study, our tailored hydrogel was only tested in vitro, which is not the same as the physiological environment. It is crucial to conduct a study assessing the follicles following their encapsulation in the tailored hydrogel and transplantation, which will be the next step of our investigation. WIDER IMPLICATIONS OF THE FINDINGS The findings from this study introduced a suitable biomaterial similar to the ovarian cortex in reproductive-age women in terms of biomechanical properties for encapsulating human preantral follicles. This biomaterial allowed the radial growth of follicles and preserved their viability. Furthermore, PEGylation improved the stability of fibrin and the physical support of follicles. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by a grant from the Fondation Louvain (awarded to C.A.A.). The authors declare no competing interests. WHAT DOES THIS MEAN FOR PATIENTS? Survival rates of leukemia patients have been steadily increasing but, owing to cancer treatment, some survivors will face infertility at a very young age. One of the strategies under development to restore fertility in these patients is the transplantable ‘engineered ovary’, which should mimic the normal ovary as closely as possible. To assemble the engineered ovary, it is necessary to encase isolated preantral follicles (follicles at an early stage of growth that contain one immature egg) and ovarian cells in a 3D matrix. Developing this matrix is one of the main challenges in creating an engineered ovary, as it should support the follicle architecture as well as the interaction between granulosa cells (which produce estrogen, progesterone) and the oocyte (egg), as they are essential for follicle development. This study demonstrates that a matrix made of a synthetic (polyethylene glycol) and natural polymer (fibrin), with mechanical strength similar to that of the outer layer of the ovary in women of reproductive age, can successfully be used to encapsulate human preantral follicles. Indeed, we have shown that our 3D matrix yielded a high follicle survival rate and supported follicle growth to the secondary stage.

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“…This technique allows an enhanced image quality and does not rely on physical sectioning of the tissues. Combined with tissue clearing, light-sheet microscopy has been used to quantify follicle morphometrics in pig ovaries (Lin et al, 2017) and human ovaries that revealed marked architectural remodelling in certain ECM biomarkers at prepubertal, reproductive-age and menopausal stages (Ouni et al, 2022). Another powerful tool for studying follicle development in vivo is optical coherence tomography, which allows imaging of ovaries in situ and in real time with micron-scale imaging resolution (Wang et al, 2015;Watanabe et al, 2015).…”

Section: Quantitative Imaging Of Ovarian Dynamicsmentioning

confidence: 99%

Squeezing the eggs to grow: The mechanobiology of mammalian folliculogenesis

Biswas

Prabhakaran

et al. 2022

Front. Cell Dev. Biol.

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The formation of functional eggs (oocyte) in ovarian follicles is arguably one of the most important events in early mammalian development since the oocytes provide the bulk genetic and cytoplasmic materials for successful reproduction. While past studies have identified many genes that are critical to normal ovarian development and function, recent studies have highlighted the role of mechanical force in shaping folliculogenesis. In this review, we discuss the underlying mechanobiological principles and the force-generating cellular structures and extracellular matrix that control the various stages of follicle development. We also highlight emerging techniques that allow for the quantification of mechanical interactions and follicular dynamics during development, and propose new directions for future studies in the field. We hope this review will provide a timely and useful framework for future understanding of mechano-signalling pathways in reproductive biology and diseases.

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

Cited by 15 publications

References 84 publications

Influence of ovarian stromal cells on human ovarian follicle growth in a 3D environment

Influence of ovarian stromal cells on human ovarian follicle growth in a 3D environment

Mind the mechanical strength: tailoring a 3D matrix to encapsulate isolated human preantral follicles

Squeezing the eggs to grow: The mechanobiology of mammalian folliculogenesis

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