Fig 1.
After hormonal synchronization, endometrial biopsies were sampled from young females (YF, n = 6) and old females (OF, n = 8) at Day 15 of the estrous cycle for experiment 1 (Exp. 1) and on YF (n = 5) and OF (n = 5) at Day 3 for experiment 2 (Exp. 2). For determining P4 levels, blood samples were collected on Days 2, 8, 14, and 22 (Exp. 1) and on Days 0 (estrus) and 3 (Exp. 2).
Fig 2.
Results of microarray data in experiment 1.
Transcriptomic analyses were performed using total RNA extracted from endometrial biopsies collected on Day 15 of the estrous cycle from old females (OF, n = 4) and young females (YF, n = 4). Statistical analyses were performed using the F-CROS method. (A) Volcano-plot of microarray data. Each circle represents a probe. The blue horizontal line represents the error threshold set at 5%. The vertical purple lines indicate the threshold used (|FC| ≥ 1.5). The green and red circles correspond to transcripts significantly over- or under-expressed in old females, respectively. (B) Table of the differentially expressed genes (DEG) between old and young females. FC = OF/ YF; f-value ≥ 0.975 or f-value ≤ 0.025.
Fig 3.
Canonical pathways associated with DEG identified in experiment 1.
Significant predicted canonical pathways associated with DEG were identified from endometrial biopsies collected on day 15 of the estrous cycle in old females (OF, n = 4) compared to young females (YF, n = 4). Canonical pathways are considered significant when -log (B-H p-value) ≥ 1.301 (vertical blue line). Ingenuity Pathway Analysis (IPA) defines a canonical pathway as inhibited or activated in OF if the Z-score is ≤ −2 or ≥ 2 respectively (vertical red lines). Only the canonical pathways that are predicted to be activated or inhibited are presented and ranked in descending order of Z-score.
Table 1.
Biological functions associated with DEG identified in experiment 1.
Fig 4.
Major networks associated with DEG identified in experiment 1.
Networks were generated from 859 differentially expressed genes (DEG) identified between old females (OF, n = 4) and young females (YF, n = 4) using IPA (Ingenuity Pathway analysis). The red node colour indicates up-regulated genes and the green color indicates down-regulated genes in OF versus YF endometrium. The colour intensity increases with the degree of fold change. Solid line indicates a direct interaction between nodes, and dashed line indicates an indirect relationship between nodes. (A) Network #1 – Cancer, Gastrointestinal Disease, Organismal Injury and Abnormalities. (B) Network #3 – Cancer, Organismal Injury and Abnormalities, Reproductive System Disease. (C) Network #15 – Cancer, Cellular Development, Cellular Movement.
Table 2.
Top 10 activated or inhibited upstream regulators, associated with DEG identified in experiment 1.
Table 3.
Response of endometrial cells to interferon tau (IFNT) in experiment 1.
Fig 5.
Response of endometrial explants to lipopolysaccharide (LPS) in experiment 2. Treat.: treatment. (B) Graphical representation of transcript levels for individual females after 24 hours incubation in the absence or presence of LPS. Mean + /- SEM.
Explants were derived from endometrial biopsies sampled on Day 3 of the estrous cycle. Explants were cultured in the absence or presence of 1 μg/mL LPS for 1 hour, 6 hours or 24 hours. Young females (YF), n = 5; old females (OF), n = 4. (A) Effect of LPS and ageing on transcripts level. Expression levels of candidate transcripts were quantified on total RNA using microfluidic RT-qPCR. Two-way repeated-measures ANOVA test using R software was performed to identify significant differences according to ageing and LPS treatment. Differences in transcript levels were considered statistically significant when P-value ≤ 0.05 or lower (two-tailed). Significant differences are indicated by a coloured box background. For the LPS treatment, fold changes « + LPS/ - LPS » are indicated. Only genes with significantly different expression levels for at least one condition are shown.
Fig 6.
Effect of LPS on cytokine concentrations in the culture medium of endometrial explant in experiment 2.
Explants were derived from endometrial biopsies sampled on Day 3 of the estrous cycle and were cultured in the absence or presence of 1 μg/mL LPS for 24 hours. Young females (YF), n = 5; old females (OF), n = 4. (A) Effect of LPS and ageing on cytokine concentration in the incubation medium. P-value was determined by an ANOVA test. Differences in cytokine levels were considered statistically significant when P-value ≤ 0.05 (two-tailed). Only cytokines with significantly different concentrations for at least one condition are shown. (B) Graphical representation of cytokine concentrations for individual females after 24 hours of incubation in the absence or presence of LPS. The cytokine concentration is expressed in picograms (pg) per milliliter of culture medium (mL), normalized to the weight of the explant tissue in milligrams (mg).