Cumulus expansion is impaired with advanced reproductive age due to loss of matrix integrity and reduced hyaluronan

Abstract Reproductive aging is associated with ovulatory defects. Age‐related ovarian fibrosis partially contributes to this phenotype as short‐term treatment with anti‐fibrotic compounds improves ovulation in reproductively old mice. However, age‐dependent changes that are intrinsic to the follicle may also be relevant. In this study, we used a mouse model to demonstrate that reproductive aging is associated with impaired cumulus expansion which is accompanied by altered morphokinetic behavior of cumulus cells as assessed by time‐lapse microscopy. The extracellular matrix integrity of expanded cumulus–oocyte complexes is compromised with advanced age as evidenced by increased penetration of fluorescent nanoparticles in a particle exclusion assay and larger open spaces on scanning electron microscopy. Reduced hyaluronan (HA) levels, decreased expression of genes encoding HA‐associated proteins (e.g., Ptx3 and Tnfaip6), and increased expression of inflammatory genes and matrix metalloproteinases underlie this loss of matrix integrity. Importantly, HA levels are decreased with age in follicular fluid of women, indicative of conserved reproductive aging mechanisms. These findings provide novel mechanistic insights into how defects in cumulus expansion contribute to age‐related infertility and may serve as a target to extend reproductive longevity.


Figure S1. Assessment of cumulus expansion.
A) COC area was measured before and after IVM using FIJI (scale bars -400 mm) B) Cumulus cell layer thickness was measured in 4 quadrants (at 3, 6, 9, 12 o'clock positions) using FIJI and averaged to obtain mean cumulus cell layer thickness.C) Representative images of subjective cumulus expansion scoring.The image brightness/contrast was adjusted for the ease of visualization.p=0.17

Figure S2 .Figure S3 .Figure S4 .
Figure S2.Cumulus expansion is impaired in COCs from reproductively old (14-17 months) CB6F1 mice during IVM.A) Average cumulus cell layer thickness was decreased in COCs from reproductively old mice pre-and post-expansion.B) The change in average cumulus cell layer thickness (post-expansion thickness -pre-expansion thickness) was significantly less in COCs from reproductively old mice COCs during IVM (testing the difference between differences).Data are represented as mean ± SD.Experiments were repeated 5 times with the comparison of all COCs from one young and one old mouse per experiment (5-29 COCs per mouse).Two-sided Student's ttest or Mann-Whitney U test were used to compare continuous variables depending on normality.P<0.05 was considered statistically significant.

Figure S5 .
Figure S5.Cumulus expansion is not altered during IVM in COCs isolated from mid-reproductive age CD1 mice.A) Fewer COCs were obtained from mice at mid-reproductive age.B) Oocyte maturation stages, C) Chromosome alignment on metaphase II spindles, D) COC subjective expansion scores, E) COC area pre-and post-expansion, F) The change in COC area, G) Average cumulus cell layer thickness, and H) The change in average cumulus cell layer thickness were not significantly different between reproductively young and mid-age mice after IVM of COCs in a conventional incubator.Data are represented as mean ± SD.Experiments were repeated 3 times with all COCs from 2 young and 2 old mice pooled per experiment (10-25 COCs per group).Two-sided Student's t-test or Mann-Whitney U test were used to compare continuous variables depending on normality.Chi-square test was used to compare categorical variables.P<0.05 was considered statistically significant.GVBD-germinal vesicle breakdown, MII-metaphase of meiosis II

Figure S6 .
Figure S6.HA staining of expanded mouse COCs using the HABP assay.A)The loss of fluorescent signal after hyaluronidase treatment validated the specificity of the HABP assay.B) HA levels were higher in the corona radiata (i.e., in cumulus cell layer immediately surrounding the oocyte; arrows).C) Plasma membrane blebbing was observed on cumulus cells at high magnification.D) HA staining was detected on the zona pellucida (arrowhead), in the perivitelline space (short arrow) and at the plasma membrane (long arrow).Scale bars -40 mm.

Figure S8 .
Figure S8.List of genes analyzed in the customized RT 2 Profiler PCR Hyaluronan Network array and corresponding category key.The genes were categorized into 7 groups.The fifth column displays 5 housekeeping genes and 3 controls of the array experiment.RTCreverse transcriptase control; PPC -Positive PCR control; GDC -genomic DNA control.

Figure S9 .
Figure S9.Whole ovaries and enriched stromal fractions from reproductively young and old mice demonstrate similar differential expression of 9 genes analyzed in COCs.Whole ovaries and stromal compartments enriched from pooled ovaries of 4 reproductively young and 4 old mice were used once to perform the same customized RT 2 Profiler PCR Hyaluronan Network array reported in Figure 5. Nine out of 88 transcripts exhibited age-related differential patterns of gene expression that were similar to those observed in COCs.

Figure S10 .
Figure S10.HA levels (A) and weighted average molecular mass of HA (B) in follicular fluid of women undergoing infertility treatment displayed as a scatterplot for each individual patient.Arrows in A point to 2 circles represented by 2 patients each -these patients had the same age and very similar HA levels.

Figure S12 .
Figure S12.HA polydispersity in follicular fluid of women undergoing infertility treatment is not different between younger and older women (n=10 for <34 yo and n=9 for >39 yo group).Each box and whiskers plot represents an individual patient.Each colored dot represents the size of an individual HA molecule for the patient.The box extends from the 25th to 75th percentiles, and the whiskers go from the minimum to the maximum value.Data were analyzed under a Box-Cox transformation Ynew = (Y^(-.4242)-1)/(-0.4242)using a nested t-test to account for the correlation between measures on the same patient.

age young mid-age young mid-age young mid-age
). Two-sided Student's t-test or Mann-Whitney U test were used to compare continuous variables depending on normality.Chi-square test was used to compare categorical variables.P<0.05 was considered statistically significant.GVBD-germinal vesicle breakdown, MII-metaphase of meiosis II