Biology subjects

Duncan, F.

Publications and source records attributed to Duncan, F..

10 recordsLinked to original sources

bioRxiv2026

Multimodal profiling establishes ovarian fibrosis as a measurable and targetable hallmark of human reproductive aging

ABSTRACT Ovarian aging underpins infertility, systemic morbidity, and mortality in women. Stromal fibrosis is implicated in ovarian aging but has not been defined in the human ovary in situ. We integrated shear wave elastography (SWE), extracellular matrix (ECM) neoepitope fingerprinting, and singlecell RNA sequencing of follicular fluid aspirates within the same individual to comprehensively profile ovarian fibrosis. In an age-stratified cohort (<33 and >37 years; N=32), age predicted ovarian stiffness, while mean stiffness was independently associated with reduced oocyte yield and follicular efficiency. ECM profiling revealed a dynamic fibrotic index favoring formation over degradation products. Single-cell transcriptomics demonstrated an age-associated fibroinflammatory stromal program enriched in pro-fibrotic pathways with stromal-immune crosstalk. In a larger validation cohort (25-45 years; N=100), age was similarly associated with ovarian stiffness. Thus, ovarian fibrosis is a quantifiable hallmark of human reproductive aging and represents a robust potential non-invasive biomarker and target for therapeutic intervention.

cell biology↗

bioRxiv2026

Biocompatible designated Resin-3D-printed polymers exhibit reproductive toxicity prevented by Parylene-C

Resin three-dimensional (3D) printing is an increasingly popular manufacturing and prototyping method used to create microphysiological systems (MPS), but resin cytotoxicity significantly hinders its adoption, especially when sensitive cell models are incorporated. The mammalian oocyte and early preimplantation embryo consist of cells that are highly sensitive to toxicants and thus represent stringent cell-based models for biocompatibility testing. We developed a Multi-Endpoint Oocyte Safety Assay (MEIOSA) to evaluate the biocompatibility of four ISO 10993 biocompatible BioMed resins (Clear, Durable, Elastic 50A, and Flex 80A). MEIOSA assesses the viability, morphology, meiotic stage, and meiotic spindle morphology of the oocyte after in vitro maturation (IVM). Oocytes were in vitro matured in plate inserts 3D printed with the four BioMed resins. Oocytes cultured in rigid resins (Clear and Durable) or elastomeric resins (Elastic 50A, and Flex 80A) exhibited impaired meiotic progression and complete oocyte degeneration, respectively, relative to controls cultured in polystyrene which matured normally. To determine whether such cytotoxicity could be prevented, we coated the resin inserts with a 5 {micro}m impermeable Parylene-C (PC) barrier. PC coating completely rescued the degeneration and meiotic maturation defect phenotypes for all resins. Remarkably, when the most cytotoxic material (Flex 80A) was coated with PC, the resulting eggs were fertilization-competent and produced embryos capable of normal preimplantation development via in vitro fertilization. Our findings demonstrate that standardized viability-based biocompatibility tests do not identify cytotoxic effects for all cell types and establish MEIOSA as a high sensitivity test model to robustly evaluate biomaterial biocompatibility. Furthermore, PC coating prevents the toxic effects of all resin-3D-printed materials tested, opening up a new toolbox to create MPS compatible with reproductive, and by extension, other sensitive cell cultures. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=197 HEIGHT=200 SRC="FIGDIR/small/730268v1_ufig1.gif" ALT="Figure 1"> View larger version (71K): org.highwire.dtl.DTLVardef@18fab44org.highwire.dtl.DTLVardef@1352e5forg.highwire.dtl.DTLVardef@77889forg.highwire.dtl.DTLVardef@1aabd89_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2026

Reproductive morph specialisation facilitated by a maternal sex-determining region in a fungus gnat (Bradysia coprophila)

Sexual reproduction is a ubiquitous process in eukaryotes, yet mechanisms of sex determination are strikingly diverse. One unique and understudied system is maternal genetic sex determination (mat-GSD), in which the genotype of the mother determines offspring sex. In some species, mothers further specialise into genetically determined reproductive morphs: gynogenic females that produce all-female broods, and androgenic females that produce all-male broods. Although this partition echoes the evolution of separate, dimorphic sexes, it remains unclear whether the morphs diverge in traits beyond offspring sex determination, and how this affects the evolutionary dynamics of the mating system. Here we address these questions in the dark-winged fungus gnat Bradysia coprophila, in which female morphs are determined by a large X-linked inversion. We show that maternal reproductive morphs diverge significantly in life history traits and gene expression profile, suggesting adaptive specialisation into their reproductive roles. We further evaluate potential drivers for specialisation and test two of them empirically, showing evidence in line with sex-specific maternal provisioning. By drawing explicit parallels with sex chromosome evolution and sexual dimorphism, our results extend core principles of reproductive specialisation to mat-GSD systems, and underscore the potential of unusual reproductive systems for extending fundamental evolutionary theory on how selection and genomic architecture interact to shape mating system evolution.

evolutionary biology↗

bioRxiv2026

The human ovary exhibits dynamic molecular remodeling in the decades post-menopause

The human ovary is among the first organs to show age-related functional decline, resulting in menopause. Beyond this transition, the postmenopausal ovary is often regarded as quiescent and remains poorly characterized. We analyzed the proteomes of healthy, non-pathological ovaries using mass spectrometry (data-independent acquisition) from 28 postmenopausal women (50-75 years old), stratified into three age groups (50-59, 60-69, [≥]70). We quantified 5,812 protein groups and observed progressive age-associated shifts, with 117 proteins significantly altered in the [≥]70 vs 50-59 age comparison. Multivariate analysis demonstrated clear separation between 50-59 and [≥]70-year-old age cohorts, with protein signatures shifting from RNA/gene-regulatory functions in younger ovaries to metabolic, trafficking, and innate immune/complement pathways in older ovaries. Across differential abundance, multivariate modelling, and covariate-adjusted linear modelling, we identified a convergent set of age-associated proteins that were integrated into the 26-protein Buck Postmenopausal Ovary Molecular Signature (BuckPOMS), a consensus molecular signature capturing progressive extracellular matrix remodeling, inflammatory signaling, and loss of structural and keratin-associated proteins with age. Representative BuckPOMS proteins, including the secreted matrisome proteins WNT4 and Fibromodulin (FMOD), were validated by immunohistochemistry. Pathway enrichment further identified an increase in inflammatory and matrisome pathways, and increased abundance of damage-associated secretory factors decades following menopause. These data fundamentally shift the notion of the postmenopausal ovary as an inert organ and instead demonstrate active and continuous molecular remodeling that has potential relevance to tissue signaling and implications for womens health.

biochemistry↗

bioRxiv2026

Proteogenomic Profiling Reveals a Distinct Endogenous p16INK4a-Associated Senescence Signature in the Human Ovary.

The tumor suppressor and cell cycle regulator, p16INK4a (p16), has been extensively linked to cellular senescence, and its accumulation can reflect endogenous senescence within ovarian tissue. However, gene and protein signatures associated with p16 have not been well defined in human tissue. We utilized immunohistochemical (IHC) staining for P16 to identify distinct positive (P16+) and negative (P16-) regions within the ovarian cortex and employed the GeoMx Digital Spatial Profiler for simultaneous proteomic and transcriptomic analyses on cortical tissue cores. Differential expression and translation between p16-positive and p16-negative cores identified genes and proteins that are cellular senescence related (e.g., CDKN1A, GADD45B, GADD45G, and MYC) or key regulators of the extracellular matrix (e.g., collagen I, ADAMTS4, and MMP11). Additionally, the transcriptomic signature identified here was significantly enriched for the spatially derived ovarian p16-associated signature, BuckSenOvary, but not for other senescence gene sets. Lastly, given the association between changes to the extracellular matrix in aged ovaries and ovarian cancer, we compared genes upregulated and downregulated in p16-positive regions relative to p16-negative regions against multiple ovarian cancer transcriptomic datasets. These findings provide new insight into the molecular landscape of naturally occurring ovarian senescence and its possible relationship to age-associated disease processes, including cancer development.

cell biology↗

bioRxiv2025

A complex phenotypic assay of mammalian oocyte maturation identifies compounds that block meiotic progression for non-hormonal contraceptive discovery

Despite the prevalent use and efficacy of hormonal contraception, it remains unsuitable for many due to the prevalence of perceived and actual side effects. Thus, there is a need for novel contraceptives for women worldwide, especially non-hormonal options. During ovulation, oocytes undergo a maturation process by which they progress through meiosis, a specialized cell division needed to reduce chromosome content. Given that meiotic progression is essential for generating a fertilizable egg but does not affect hormone production, it represents a promising biological mechanism to target for female non-hormonal contraception. In this study, we developed a phenotypic screening platform modeled after biopharmaceutical industry practices using mouse oocytes to screen for compounds that block meiotic progression. Oocytes were matured in vitro in the presence of selected compounds from a bioactive compound library, and confirmed hits were identified as those that potently and reproducibly blocked meiotic progression. In addition, we showed that the inhibitory effect of these compounds was persistent and reversible. Thus, our oocyte drug screening pipeline provides a powerful system to identify novel drug discovery candidates for further follow up for non-hormonal contraception development.

cell biology↗

bioRxiv2025

A pre-menopausal single-cell atlas for ovarian drug discovery

Multi-tissue single-cell atlas efforts have transformed our understanding of cellular diversity across the human body and led to the creation of harmonized resources to advance research and therapeutic development. Ovarian biology, however, often remains underrepresented in these resources and is rarely analyzed with menopausal status as a biological variable. Here, we present the Menopause Cell Map (MenoMap), an integrated single-cell resource comprising more than 2 million cells from 13 healthy human tissues, including the ovary, from pre- and post-menopausal age donors. This harmonized atlas leverages curated samples from healthy female donors and enables transcriptomic comparisons across cell types, tissues, and organs while preserving menopausal status based on age as an interpretable variable. Using this resource, we show that menopause-associated gene expression changes are highly context dependent, with prominent remodeling in ovarian stromal, endothelial, immune, and reproductive cell populations. Within the ovary, post-menopausal remodeling rewired intercellular communication and shifted reproductive and steroidogenic programs toward collagen-integrin signaling, endothelial-to-mesenchymal transition, and senescence concentrated in the endothelial and stromal compartments. Cross-species comparison with young and aged mouse ovarian single-cell data showed these endothelial and stromal changes were conserved with age, most prominently in tumor necrosis factor-nuclear factor-kappa-beta signaling. Finally, we apply Human Protein Atlas-inspired specificity rules and fertility phenotype annotations to evaluate how menopausal age status affects tissue- and cell-type-specific gene classification and to prioritize ovary-enriched genes for downstream biological and translational investigation. Through this analysis, we find that the tissue specificity classification of several genes involved in reproductive-specific programs change in the pre- to post-menopausal transition, highlighting the need for age-aware and healthy donors in multi-tissue single-cell atlas efforts. Together, the MenoMap provides a human single-cell framework that leverages existing and standardized single-cell datasets curated for studying ovarian biology across reproductive aging, evaluating the influence of menopausal status on gene expression and tissue specificity, and nominating candidate genes for future investigation in reproductive biology, fertility, and target discovery.

bioinformatics↗

bioRxiv2024

The Effects of Heat Stress on the Ovary, Follicles and Oocytes: A Systematic Review

Climate change is driving significant environmental changes with profound implications for human health, including fertility. While the detrimental effects of heat on spermatogenesis are well-documented, the impact of elevated temperatures on ovaries and female fertility remains less explored. This review systematically examines the literature on heat stress (HS) effects on mammalian ovaries, follicles, and oocytes. Evidence from mammalian models indicates that HS significantly impairs ovarian function, disrupting hormone profiles, reducing ovarian size and weight, altering histology, decreasing granulosa cell viability, and compromising oocyte quality. Efforts to develop strategies and substances to mitigate these adverse effects are ongoing, but further research into the underlying mechanisms is urgently needed. Summary SentenceThis systematic review summarizes the evidence on the adverse effects of heat stress on the mammalian oocyte and ovary

developmental biology↗

bioRxiv2024

Multinucleated giant cells are hallmarks of ovarian aging with unique immune and degradation-associated molecular signatures

The ovary is one of the first organs to exhibit signs of aging, characterized by reduced tissue function, chronic inflammation, and fibrosis. Multinucleated giant cells (MNGCs), formed by macrophage fusion, typically occur in chronic immune pathologies, including infectious and non-infectious granulomas and the foreign body response1, but are also observed in the aging ovary2-4. The function and consequence of ovarian MNGCs remain unknown as their biological activity is highly context-dependent, and their large size has limited their isolation and analysis through technologies such as single-cell RNA sequencing. In this study, we define ovarian MNGCs through a deep analysis of their presence across age and species using advanced imaging technologies as well as their unique transcriptome using laser capture microdissection. MNGCs form complex interconnected networks that increase with age in both mouse and nonhuman primate ovaries. MNGCs are characterized by high Gpnmb expression, a putative marker of ovarian and non-ovarian MNGCs5,6. Pathway analysis highlighted functions in apoptotic cell clearance, lipid metabolism, proteolysis, immune processes, and increased oxidative phosphorylation and antioxidant activity. Thus, MNGCs have signatures related to degradative processes, immune function, and high metabolic activity. These processes were enriched in MNGCs compared to primary ovarian macrophages, suggesting discrete functionality. MNGCs express CD4 and colocalize with T-cells, which were enriched in regions of MNGCs, indicative of a close interaction between these immune cell types. These findings implicate MNGCs in modulation of the ovarian immune landscape during aging given their high penetrance and unique molecular signature that supports degradative and immune functions.

cell biology↗

bioRxiv2024

Systemic low-dose anti-fibrotic treatment attenuates ovarian aging in the mouse

The female reproductive system is one of the first to age in humans, resulting in infertility and endocrine disruptions. The aging ovary assumes a fibro-inflammatory milieu which negatively impacts gamete quantity and quality as well as ovulation. Here we tested whether the systemic delivery of anti-inflammatory (Etanercept) or anti-fibrotic (Pirfenidone) drugs attenuates ovarian aging in mice. We first evaluated the ability of these drugs to decrease the expression of fibro-inflammatory genes in primary ovarian stromal cells. Whereas Etanercept did not block Tnf expression in ovarian stromal cells, Pirfenidone significantly reduced Col1a1 expression. We then tested Pirfenidone in vivo where the drug was delivered systemically via mini-osmotic pumps for 6-weeks. Pirfenidone mitigated the age-dependent increase in ovarian fibrosis without impacting overall health parameters. Ovarian function was improved in Pirfenidone-treated mice as evidenced by increased follicle and corpora lutea number, AMH levels, and improved estrous cyclicity. Transcriptomic analysis revealed that Pirfenidone treatment resulted in an upregulation of reproductive function-related genes at 8.5 months and a downregulation of inflammatory genes at 12 months of age. These findings demonstrate that reducing the fibroinflammatory ovarian microenvironment improves ovarian function, thereby supporting modulating the ovarian environment as a therapeutic avenue to extend reproductive longevity.

developmental biology↗