A mechanical origin for implantation defects in embryos from aged females - PubMed (original) (raw)
A mechanical origin for implantation defects in embryos from aged females
Kate E Cavanaugh et al. bioRxiv. 2025.
Abstract
Women over 35 experience a marked reduction in fertility. The origin of these fertility defects appears to reside in the implantation capacity of the embryo itself, but the mechanistic basis of this impairment is not well-understood. Here, we identify a core mechanical defect in embryos from aged mothers that impairs the process of implantation. Using mouse models, we find that reproductive aging yields increased contractility in the extra-embryonic trophectoderm, the outer epithelial tissue responsible for mediating uterine attachment and embryo implantation. This hypercontractile state elevates tissue surface tension and viscosity in the blastocyst, culminating in defective spreading during implantation. Enhanced contractility is necessary and sufficient for this age-related defect in implantation, and early embryo mechanics can be used to predict successful implantation for embryos from both young and aged mothers. Our work represents a potential foundation for improving embryo selection in Assisted Reproductive Technologies to resolve age-related defects in female fertility.
Keywords: Biophysics; Implantation; Morphogenesis; Reproductive Aging.
Figures
Fig. 1.. Embryos from aged mothers display low implantation.
a) Schematic of blastocyst structure with the Inner Cell Mass (ICM) (tan) sitting within the blastocyst and surrounded by the Trophectoderm tissue (gray). The ICM sits at the Polar Trophectoderm (pTE) region, with the opposite side being the Mural Trophectoderm (mTE) (left). Schematic of distinct developmental checkpoints spanning the 8-cell, morula, blastocyst, and implanting blastocyst stage. Species-specific differences occur at the site of implantation: the mouse embryo implants at the mural Trophectoderm, whereas the human embryo implants at the polar Trophectoderm (right). b) Schematic of Uterine Transfer workflow in Assisted Reproductive Technologies parsed from the HFEA. Embryos are transferred individually to the uterus. Embryos from Young or Aged females are transferred to either Young or Aged surrogate mothers (Y->Y n = 318,975 transferred embryos; A->A n = 34,255 transferred embryos; Y->A n = 482,730 transferred embryos). Violin plots show a reduction in age-related infertility that is rescued with embryos from younger women following transfer to older surrogates. p values from Mann-Whitney test. c) Schematic of NSET workflow to simulate Assisted Reproductive Technologies. Embryos from Young or Aged females are transferred to either Young or Aged surrogate mothers (N=4 for all transfer conditions, Y->Y n = 38 transferred embryos; A->A n = 35 transferred embryos; Y->A n = 36 transferred embryos). Violin plots show a reduction in age-related infertility that is rescued with developmentally younger embryos when transferred to older females. Two tailed p values from N-1 Chi-Square test. d) Diagram showing the experimental workflow for In Vitro Implantation Assays. E3.5 Blastocysts are denuded and transferred from KSOM-AA to IVC media that contained 650-FastAct to visualize the process of implantation on collagen-coated polyacrylamide gels. 24 hours post-implantation, the embryos are fixed (left). Views of the blastocyst in Z show mural Trophectoderm attachment, blastocyst cavity collapse, and spreading over the 24 hours post-implantation (middle). Mural Trophectoderm perimeter outlines show the evolution of mTE spreading over 24 hours. Perimeter outlines are color coded based on time post-implantation (right). Scale bars are 30μm and 50μm, as indicated. e) Representative images of Young and Aged implanting blastocysts over the 24 hours post-implantation. Blastocysts are initially non-adherent, after which they extend protrusions to mediate attachment, cavity collapse, and tissue spreading. Inlays display a single z-plane to visualize mural Trophectoderm protrusions at the substrate surface. Images of implanting blastocysts are displayed as a z-projection. Scale bars are 10 μm and 25 μm, as indicated. f) Graph of the evolution of the Trophectoderm’s spread area over 24 hours of implantation. Inlay shows a zoom of the first 4 hours of implantation, displaying an age-specific delay in attachment and spreading (Young N = 4, n = 26 implanting blastocysts; Aged N = 4, n = 31 implanting blastocysts). Lines and shading show mean and 95% Confidence Interval. g) Violin plot depicted total spread area post-implantation for both maternal conditions (Young N = 4, n = 26 implanting blastocysts; Aged N = 4, n = 31 implanting blastocysts). Maternally aged embryos show significant reductions in final spread area. p values from Mann-Whitney test.
Fig. 2.. Contractility is necessary and sufficient for maternal age-related implantation defects.
a) Schematic of contractile inhibitor pathways to regulate potential trophectoderm spreading behaviors. We hypothesized that RhoA activator CN03 would phenocopy aged embryos, while ROCK inhibitor Ripasudil would rescue aged spreading behaviors. b) Schematic of experimental workflow and the culture conditions for testing the necessity and sufficiency of contractility for maternal age-related implantation defects. Embryos are collected at E1.5, after which Young embryos are cultured in KSOM-AA with or without CN03 (Rho-based contractility activator), and Aged embryos are cultured in KSOM-AA with or without Ripasudil (Rho Kinase-based contractility inhibitor). Embryos are then denuded and transferred to IVC media for In Vitro Implantation assays and fixed 30 hours post-implantation. c) Representative images of Young (left) and Young-CN03 treated (right) embryos fixed and stained for actin. Violin plot shows a reduction of spread area in Young-CN03 treated embryos (Young N = 9, n = 43 implanting blastocysts; Young -CN03 N = 5, n = 25 implanting blastocysts). Scale bar is 50μm. p values from Mann-Whitney test. d) Representative images of Aged (left) and Aged-Ripasudil treated (right) embryos fixed and stained for actin. Violin plot shows a rescue of spread area in Aged-Ripasudil treated embryos (Aged N = 4, n = 27 implanting blastocysts; Aged-Ripasudil N = 3, n = 16 implanting blastocysts). Scale bar is 50μm. p values from Mann-Whitney test.
Fig. 3.. Advanced maternal age dampens blastocyst wetting.
a) Droplet spreading is a function of a liquid’s surface tension, adhesion between the liquid and the substrate, and viscosity within the liquid. These parameters collectively determine a droplet’s wetting behavior. When extrapolated to the blastocyst, key parameters dictating a tissue’s spreading behavior include surface tension, cell-cell adhesion, and cell-substrate adhesion. b) Schematic of an implanting blastocyst as a droplet with viscosity η whose spreading is driven by active traction forces Ta and opposed by surface tension γ. The inward-pointing traction forces on the substrate (-Ta) correspond to regions of cell pulling. c) Schematic of Micropipette Aspiration Assay for measuring surface tension of E4.5 Blastocysts (above). The glass pipette aspirates a portion of the mural Trophectoderm region. Violin plot of average surface tension measurements of E4.5 between the two maternal conditions (Young N = 3, n = 15 blastocysts; Aged N = 3, n = 15 blastocysts). p values from Mann-Whitney test. d) Representative images of implanted trophectoderm stained for 650-FastAct (black) along with calculated traction stresses (purple) for both maternal conditions. Scale bar is 50 μm. e) Plot with each dot representing a single embryo, analyzing Total Contractile Energy (pJ) as a function of the trophectoderm’s spread area (um2) with simple linear regression to show trends. Aged embryos show higher Contractile Energy per Area compared to younger maternal controls (Young N = 3, n = 18 implanted embryos, R2 = 0.81; Aged N = 3, n = 13 implanted embryos, R2 = 0.77). f) Contractile energy per Area as calculated by Traction Force Microscopy between both maternal conditions (Young n = 18 implanted embryos; Aged n = 13 implanted embryos). Maternally aged embryos show higher strain energy per area values across the 18 hours post-implantation. Bold lines represent the mean and are bounded by shaded SEM. g) Plot showing Maximal Traction T0 in both maternal conditions, as determined by fitting the physical model to the data, with higher maximal traction in the aged condition (Young n = 21 implanted embryos; Aged n = 31 implanted embryos). p values from Mann-Whitney test. h) Viscosity is higher in embryos from aged mothers, as determined by fitting the physical model to the data (Young n = 21 implanted embryos; Aged n = 31 implanted embryos). p values from Mann-Whitney test.
Fig. 4.. Compaction metrics predict implantation potential.
a) Schematic of developmental timing from the 4-cell stage through early blastocyst cavitation. At the 8-cell stage, contractility drives compaction and flattening of the embryo’s outer surface. Below are the quantifications of developmental tempo for the 4-cell, 8-cell, and cavitation stages. Maternally aged embryos display a significant reduction in the timing of the 8-cell stage (N=4, Young n = 95 embryos; Aged n = 119 embryos). p values from Mann-Whitney test. b) Schematic and representative images of Micropipette Aspiration Assays between early and late 8-cell stages, where the pipette aspirates the exposed apical region of the cell to calculate cortical surface tension. Analyses of cellular surface tensions show maternal age-associated increase in tension at the late 8-cell stage (Young N = 8, n = 44 Early 8-cell embryos, 26 Late 8-cell embryos; Aged N = 5, n = 24 Early 8-cell embryos, 19 Late 8-cell embryos). p values from Mann-Whitney test. Scale bar is 10μm. c) Histogram of compaction tempo between both maternal conditions (N=4, Young n = 95 embryos; Aged n = 119 embryos). Boxes highlight the expected implantation incompetent and implantation-competent windows with respect to compaction timing. d) Schematic of the indexing workflow for correlating compaction metrics with implantation potential. Embryos are stained for 650-FastAct and indexed at the 2-cell stage, imaged over the period of compaction, and then selected based on their developmental tempo to undergo In Vitro Implantation. Scale bar is 50μm. e) Representative images from timelapse imaging of embryos stained for 650-FastAct showing Normal (top) and Accelerated (bottom) compaction timing. Embryos in the Normal condition show significantly longer times to the first cell division into the 16-cell stage. Purple arrows denote cell divisions. Scale bar is 25μm. f) Representative images of embryos live-imaged at the 8-cell stages (black, 650-FastAct) undergoing compaction (left) and their representative fixed, implanted embryos (black, pMLC staining) (right). Scale bars are 30μm and 50μm, as indicated. g) Plot of compaction timing as a function of final Trophectoderm spread area 48 hours post-implantation for both maternal conditions (N=3, Young n = 56 implanted blastocysts; N=2, Aged n = 14 implanted blastocysts). Bold line represents simple linear regression and are bounded by 95% Confidence Interval (Young R2 = 0.40; Aged R2 = 0.55). h) Violin plots from (f) show spread area binned by their compaction timing of either >9hrs or <7.5hrs. Both Young and aged conditions show significant reductions in spread area if their compaction timing falls within the <7.5hrs compaction category (N = 3: Young n = 23 >9hrs, 29 <7.5hrs; N = 2: Aged n = 9 >9hrs, 5 <7.5hrs).
Fig. 5.. Summary Figure. Contractility shifts the proportion of implantation-competent embryos in the Young and Aged maternal condition.
a) Schematic of developmental trajectory in the embryos that are competent to implant. Compaction proceeds normally at ~ 9 h, followed by blastocyst formation and implantation to yield robust trophectoderm spreading. During wetting in these embryos, tissue-substrate adhesion forces dominate to produce robust trophectoderm spreading. The majority of Young embryos and minority of Aged embryos display this spreading behavior. Embryos that are incompetent to implant can be shifted to exhibit efficient wetting through lowering contractility. b) Schematic of developmental trajectory in the embryos that are incompetent to implant. Compaction is accelerated on average 2 hours faster due to aberrant contractility, that acts to increase surface tension at the blastocyst stage. Upon adhesion, tissue viscosity dominates to reduce wetting and slow trophectoderm spreading and implantation. The majority of Aged embryos and minority of Young embryos display this spreading deficiency. This spreading defect can be phenocopied in implantation-competent embryos by increasing contractile activity.
References
- Broekmans F. J., Soules M. R. & Fauser B. C. Ovarian Aging: Mechanisms and Clinical Consequences. Endocrine Reviews 30, 465–493 (2009). -PubMed
Publication types
Grants and funding
- R01 GM122902/GM/NIGMS NIH HHS/United States
- R01 GM148644/GM/NIGMS NIH HHS/United States
- R21 ES023297/ES/NIEHS NIH HHS/United States
- R35 GM118167/GM/NIGMS NIH HHS/United States
LinkOut - more resources
Full Text Sources