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Developmental BiologyReproduction
Peer-reviewed journal article

Age-associated increased stiffness of the ovarian microenvironment impairs follicle development and oocyte quality and rapidly alters follicle gene expression

Sara PietroforteiD, Makenzie Plough, Farners AmargantiD

Abstract

In humans, aging triggers cellular and tissue deterioration, and the female reproductive system is the first to show signs of decline. Reproductive aging is associated with decreased ovarian reserve, decreased quality of the remaining oocytes, and decreased production of the ovarian hormones estrogen and progesterone. With aging, both mouse and human ovaries become pro-fibrotic and stiff. However, whether stiffness directly impairs ovarian function, folliculogenesis, and oocyte quality is unknown. To answer this question, we cultured mouse follicles in alginate gels that mimicked the stiffness of reproductively young and old ovaries. Follicles cultured in stiff hydrogels exhibited decreased survival and growth, decreased granulosa cell viability and estradiol synthesis, and decreased oocyte quality. We also observed a reduction in the number of granulosa cell-oocyte transzonal projections. RNA sequencing revealed early changes in the follicle transcriptome in response to stiffness. Follicles cultured in a stiff environment had lower expression of genes related to follicle development and greater expression of genes related to inflammation and extracellular matrix remodeling than follicles cultured in a soft environment. Altogether, our findings suggest that ovarian stiffness directly modulates folliculogenesis and contributes to the progressive decline in oocyte quantity and quality observed in women of advanced maternal age.

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Claude Opus 5.5 · xhigh effort · 6 Nov 2026 · from the full text of version 1 (10 Jun 2024) · CC BY-NC-ND 4.0

Hydrogels matching aged mouse ovary stiffness (5.85 kPa vs 0.84 kPa) cut follicle survival to 6.66% to 30%, reduce estradiol up to 533-fold, and alter 135 genes within 6 hours.

Aged ovaries stiffen through collagen deposition and hyaluronan loss, yet whether mechanical stiffness alone drives follicular decline was unclear. Encapsulating isolated pre-pubertal follicles in tuned 3D alginate hydrogels isolates biomechanical cues from endogenous aged matrix composition.

Key results

  1. ShownAlginate hydrogels at 2% weight/volume reproduce the elevated stiffness of reproductively old mouse ovaries without restricting nutrient diffusion.

    Piuma nanoindentation measured 0.5% alginate at 0.84 +/- 0.16 kPa and 2% alginate at 5.85 +/- 0.28 kPa (7-fold stiffer, matching 14-17 month mouse ovaries at 4.36 +/- 1.24 kPa vs 6-12 week ovaries at 1.98 +/- 0.42 kPa), while Alcian blue dye (1298.86 g/mol) equilibrated across both 0.5% and 2% beads within 3 minutes. · Nanoindentation; Diffusion test; A stiff microenvironment mimicking that of…

  2. ShownCulturing secondary follicles in 2% or 4% alginate arrests growth by day 6 and sharply reduces 12-day follicle survival across two mouse strains.

    On day 12, 100% of CB6F1 and over 80% of CD1 follicles survived in 0.5% alginate (reaching 247.6 +/- 13.76 um and 226.9 +/- 8.69 um by day 10), whereas only 6.66% of CB6F1 and 30% of CD1 follicles survived in 2% alginate (157.5 +/- 10.33 um and 160.8 +/- 4.03 um) and none survived in 4% alginate. · Follicle isolation, encapsulation, and growth; Follicle survival, growth, and granular pattern; A stiff microenvironment mimicking that of…

  3. ShownMicroenvironmental stiffness triggers peripheral granulosa cell death, depletes transzonal projections, reduces estradiol secretion, and lowers germinal vesicle oocyte quality.

    By day 10, estradiol levels were 533-fold lower in CB6F1 and 35.1-fold lower in CD1 follicles in 2% versus 0.5% alginate; phalloidin confocal profiling at day 2 showed fewer transzonal projections per 10 um interface, and day-12 good-quality germinal vesicle oocytes fell from 73.5 +/- 4.91% (CB6F1) and 76.4 +/- 4.61% (CD1) in 0.5% to 7.9 +/- 3.96% and 31.1 +/- 6.87% in 2% alginate. · Estradiol measurement; Staining trans-zonal projections (TZPs); Age-associated ovarian stiffness impairs…; Microenvironment stiffness impairs oocyte…

  4. ShownFollicles mount an acute transcriptional response within 3 to 6 hours of stiff culture, upregulating inflammatory and mechanosensory genes while downregulating Yap1 targets.

    SMART-seq across 160 follicles at 3, 6, 12, and 24 hours identified 51 differentially expressed genes at 3 hours and 135 at 6 hours, with upregulation of inflammatory genes (Card9, Cfi, Ccl3, Nckap1l), downregulation of Yap1 targets (Ctgf, Ankrd1, Cyr61), and 91 extracellular matrix organization genes (including 34 collagen genes) enriched between 3 and 24 hours in 2% alginate. · RNA sequencing and data analysis; The follicle transcriptome quickly responds to a…; Pathways associated with inflammation and…; A stiff microenvironment increases expression of…

Methods, data and reproducibility

Isolated early secondary follicles (120-130 um) from day 12-13 CB6F1 and CD1 mice were encapsulated in 0.5%, 2%, or 4% w/v alginate beads, indented by Piuma nanoindenter, and assayed over 12 days for survival, diameter, calcein/ethidium homodimer-1 viability, ELISA estradiol, confocal phalloidin transzonal projections, germinal vesicle oocyte quality, and SMART-seq RNA-seq (3, 6, 12, and 24 hours).

  • Early secondary follicles from day 12-13 CB6F1 and CD1 mice in 0.5%, 2%, and 4% alginate · mouse · n = 30 follicles per condition (survival/growth); 160 follicles across 3, 6, 12, 24h
Methods & tools
Piuma nanoindenter (Optics 11), Fiji (ImageJ), SMART-seq mRNA kit, FastQC, STAR 2.7.0a, GenomicFeatures (SummarizeOverlap), DESeq2, ShinyGO 0.80, WebGestalt, GSEA 4.3.3 (MSigDB v2023.2.Mm), GraphPad Prism 9.4
Data & accessions
http://genome.ucsc.edu/http://www.ensembl.org/
Reagents & environment
STAR 2.7.0a, ShinyGO 0.80, GSEA Software 4.3.3 with Mouse MSigDB v2023.2.Mm (October 2023), and GraphPad Prism 9.4 specified; exact R, Bioconductor, and DESeq2 versions are not reported.
Assay conditions & compute
Illumina HiSeq 4000 sequencer generating ~90 million 50 bp single-end reads at the Northwestern University NUSeq Core.
Reproducibility gaps
Raw and processed SMART-seq RNA-seq datasets had not yet been deposited in GEO at preprint posting (marked as available upon request and to be uploaded upon acceptance in S24), and no custom analysis scripts are linked.
Limitations (3)
  • Follicles cultured in 2% alginate failed to reach the 180 um diameter threshold required for ex vivo in vitro maturation, precluding direct assessment of metaphase II spindle assembly or fertilization competence. · stated by the authors
  • Alginate hydrogels model bulk elastic modulus (0.84 to 5.85 kPa) using an inert polysaccharide without cell-adhesion ligands or fibrillar collagen architecture, so stiffness effects are not decoupled from integrin-mediated biochemical signaling present in aged ovaries. · noted in this summary
  • Bulk SMART-seq was performed on pooled intact follicles (5 follicles per replicate) only during the first 24 hours, which cannot separate oocyte from granulosa cell transcriptional changes or capture the day 4-10 degeneration trajectory. · noted in this summary

Who should read:Read this if you study ovarian aging, reproductive mechanobiology, or 3D hydrogel culture systems for mammalian folliculogenesis.

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