Journal: Human Reproduction (Oxford, England)

Article Title: PCOS endometrium-derived epithelial organoids as a novel model to study endometrial dysfunction

doi: 10.1093/humrep/deaf113

Figure Lengend Snippet: Establishment and characterization of O-PCOS and O-Ctrl endometrium epithelial organoids (EEOs). ( a ) Hormone exposure protocol. EEOs were cultured for 8 days without hormones (baseline), or exposed to 10 nM β-estradiol (E2 exposure) for 6 days, or exposed to 10 nM E2 for 2 days, followed by 4-day exposure to a combination of 10 nM E2, 1 µM progesterone (P4), 0.25 mM cyclic adenosine monophosphate (cAMP), and 10 µM XAV-939 (EPCX exposure), all in the presence/absence of 100 nM dihydrotestosterone (DHT). ( b ) Representative brightfield images of overweight/obese PCOS (O-PCOS) or control (O-Ctrl) EEOs during 8-day culture without hormone exposure; scalebar = 200 µm. ( c ) Representative images of hematoxylin and eosin (H&E) staining and immunostaining for CDH1 (red) in EEOs and in endometrial (endom.) tissue as positive control. Blue indicates DAPI-stained nuclei, scalebar = 50 µm. ( d, e ) Volcano plots of differentially expressed genes (DEGs; adjusted P -value ( P .adj.)<0.05) in O-Ctrl EEOs after E2 (d) or EPCX exposure, (e) compared to non-exposed (baseline) Ctrl EEOs. Log2(FC), Log2(fold change). Green and blue dots indicate significantly up- and downregulated expression, respectively; gray dots denote non-significant changes. ( f ) Gene ontology (GO) enriched pathways ( P .adj.<0.05) in O-PCOS versus O-Ctrl EEOs at baseline. Green and blue bars represent up- and downregulated pathways, respectively. ( g ) Volcano plot of DEGs ( P .adj.<0.05) between O-PCOS and O-Ctrl EEOs after 8-day culture at baseline. Genes in bold were validated using RT-qPCR (see Fig. 1h ). ( h ) RT-qPCR gene expression analysis of OSMR, ICAM1 , and GPC6 in O-PCOS (purple) and O-Ctrl (blue) EEOs at baseline as relative to HPRT1 and GAPDH expression. ( i ) Representative images of immunostaining of OSMR (green) in O-EEOs of Ctrl and PCOS at baseline. Blue indicates DAPI-stained nuclei, scalebar = 100 µm. ( j ) Proportion of OSMR-positive cells in O-PCOS (purple) and O-Ctrl (blue) O-EEOs. N = 4 independent EEO cultures from different donors per group. EEOs from low passage ( P ≤ 5) were used. Student’s t -test or Mann–Whitney test; *= P < 0.05; ***= P <0.001. Data are presented as mean±SEM.

Article Snippet: For immunofluorescent staining of PR (1/300, Proteintech, USA, 25871-1-AP), and Oncostatin M Receptor (OSMR) (1/100, Proteintech, 10982-1-AP), slides were blocked for 1 h before primary and secondary antibody exposure using 5% donkey serum (Sigma-Aldrich) and 0.1% Tween 20 in Tris-buffered saline.

Techniques: Cell Culture, Control, Staining, Immunostaining, Positive Control, Expressing, Quantitative RT-PCR, Gene Expression, MANN-WHITNEY

Journal: Human Reproduction (Oxford, England)

Article Title: PCOS endometrium-derived epithelial organoids as a novel model to study endometrial dysfunction

doi: 10.1093/humrep/deaf113

Figure Lengend Snippet: L-PCOS endometrium epithelial organoids (EEOs) also show increased inflammatory gene expression. ( a ) Representative brightfield images of EEOs from lean PCOS (L-PCOS) and control (L-Ctrl) groups after 8 days of culture without hormones (baseline), or exposure to β-estradiol (E2), or exposure to E2, progesterone, cyclic adenosine monophosphate, and XAV-939 (EPCX). Scalebar = 200 µm. ( b ) Quantification of average EEO diameter of L-PCOS (purple) and L-Ctrl (blue) EEOs after 8 days of culture. Each symbol represents a different EEO sample, with the same samples shown across different hormone treatments. ( c ) RT-qPCR gene expression analysis of androgen receptor ( AR ) and ESR1 at baseline in L-PCOS (purple) and L-Ctrl (blue) EEOs as relative to GAPDH . ( d, e, f ) RT-qPCR gene expression analysis of OSMR, ICAM1 , and GPC6 at baseline (d), PRSS1 and PZP after E2 exposure (e), and leukemia inhibitory factor ( LIF ), progestagen-associated endometrial protein ( PAEP ), and PGR after EPCX exposure (f) as relative to GAPDH . L-PCOS (purple) and L-Ctrl (blue). ( g, i ) Representative images of immunostaining of PR at baseline and after E2-exposure (g); and of OSMR at baseline (i) in L-EEOs. Green indicates PR or OSMR protein, blue indicates DAPI-stained nuclei. Scalebar = 100 µm. ( h, j ) Proportion of PR- (h) and OSMR- (j) positive cells in L-PCOS (purple) and L-Ctrl (blue) EEOs. N = 3 (for size measurements), 4 (for RT-qPCR), or 3–4 (for immunostainings) independent EEO cultures from different donors per group. EEOs from low passage ( P ≤ 5) were used. Two-way ANOVA (b). Student’s t -test or Mann–Whitney test (c–f); *= P < 0.05; **= P < 0.01. Data are presented as mean±SEM.

Article Snippet: For immunofluorescent staining of PR (1/300, Proteintech, USA, 25871-1-AP), and Oncostatin M Receptor (OSMR) (1/100, Proteintech, 10982-1-AP), slides were blocked for 1 h before primary and secondary antibody exposure using 5% donkey serum (Sigma-Aldrich) and 0.1% Tween 20 in Tris-buffered saline.

Techniques: Gene Expression, Control, Quantitative RT-PCR, Immunostaining, Staining, MANN-WHITNEY