Journal: Molecular Human Reproduction

Article Title: A matrix-free 3D in vitro follicle culture system in mice exhibits enhanced oocyte meiotic and developmental competence compared to hydrogel encapsulation

doi: 10.1093/molehr/gaaf029

Figure Lengend Snippet: Study design. Secondary follicles (110–130 μm) were collected from 12- to 14-day-old mice and were cultured for 12 days in the AT, NA, ES, and PEG in vitro follicle culture systems. Endpoints are indicated in pink. On Day 12 of culture, follicle survival rates, antral rates, and alkaline phosphatase activity for theca cell localization were assessed. Moreover, immature COCs were denuded and collected for gene expression analysis. Following the ovulatory stimulus, on Day 13 of culture, COCs were either denuded to assess MII rates, oocyte diameters, and the gene expression profile of oocytes and cumulus cells, or they were in vitro fertilized to assess oocyte developmental competence (2-cell rates, total and hatched blastocyst rates, and blastomere count of blastocysts). Finally, hormone secretion levels were measured from spent media collected throughout the culture. AT, attachment; NA, non-attachment; ES, extracellular-matrix-derived soft hydrogel; PEG, Poly-Ethylene-Glycol; COCs, cumulus–oocyte complexes; MII, metaphase II; 2D, two-dimensional; 3D, three-dimensional. Created in BioRender. Anckaert, E. (2025) https://BioRender.com/ikgkpvm .

Article Snippet: To form PEG hydrogels, eight-arm Poly-Ethylene Glycol vinyl sulfone (PEG-VS, 40 kDa, >99% purity; JenKem Technology, Plano, TX, USA) was crosslinked with a 1:1 ratio of fast-degrading YKNR (GCYKNRGCYKNRCG) and slower-degrading YKNS (GCYKNSGCYKNSCG) trifunctional all-plasmin sensitive crosslinkers (>90% purity; GenScript, Piscataway, NJ, USA).

Techniques: Cell Culture, In Vitro, Activity Assay, Gene Expression, Derivative Assay

Journal: Molecular Human Reproduction

Article Title: A matrix-free 3D in vitro follicle culture system in mice exhibits enhanced oocyte meiotic and developmental competence compared to hydrogel encapsulation

doi: 10.1093/molehr/gaaf029

Figure Lengend Snippet: Growing follicles in different in vitro culture systems. Representative images of growing follicles from the early secondary to the antral follicle stage on Days 1, 4, 8, and 12 of culture in the AT, NA, ES, and PEG in vitro follicle culture systems. From Day 8 onwards, the adhesion and spreading of granulosa cells on the plate surface is apparent in the AT condition, whereas the three-dimensional morphology is maintained in the NA, ES, and PEG conditions. Images were obtained with an EVOS light microscope at 10× magnification; Scale bar, 400 µm. AT, attachment; NA, non-attachment; ES, extracellular-matrix-derived soft hydrogel; PEG, Poly-Ethylene-Glycol.

Article Snippet: To form PEG hydrogels, eight-arm Poly-Ethylene Glycol vinyl sulfone (PEG-VS, 40 kDa, >99% purity; JenKem Technology, Plano, TX, USA) was crosslinked with a 1:1 ratio of fast-degrading YKNR (GCYKNRGCYKNRCG) and slower-degrading YKNS (GCYKNSGCYKNSCG) trifunctional all-plasmin sensitive crosslinkers (>90% purity; GenScript, Piscataway, NJ, USA).

Techniques: In Vitro, Light Microscopy, Derivative Assay

Journal: Molecular Human Reproduction

Article Title: A matrix-free 3D in vitro follicle culture system in mice exhibits enhanced oocyte meiotic and developmental competence compared to hydrogel encapsulation

doi: 10.1093/molehr/gaaf029

Figure Lengend Snippet: Follicle development and oocyte maturation outcomes in different in vitro follicle culture systems. Comparison of the ( a ) follicle survival rates and ( b ) antral follicle rates on Day 12 of culture in the AT, NA, ES, and PEG conditions. ( c ) Recovered COC rates, ( d ) MII rates, and ( e ) diameters of MII oocytes in the AT, NA, and ES conditions on Day 13 of culture. Statistical differences were detected with a one-way ANOVA with Tukey’s post hoc test on arcsine transformed percentages for the survival, antral, COC, and MII rates and on log2 transformed values for the diameter. Data from five biological replicates are presented as mean ± SD, and P < 0.05 is considered significant; * P < 0.05; ** P < 0.01. ( f ) Representative images of denuded MII oocytes in the in IVO, AT, NA, and ES conditions. Images were obtained with an EVOS light microscope at 10× magnification; Scale bar, 400 µm. AT, attachment; NA, non-attachment; ES, extracellular-matrix-derived soft hydrogel; PEG, Poly-Ethylene-Glycol; COCs, cumulus–oocyte complexes; MII, metaphase II; IVO, in vivo control.

Article Snippet: To form PEG hydrogels, eight-arm Poly-Ethylene Glycol vinyl sulfone (PEG-VS, 40 kDa, >99% purity; JenKem Technology, Plano, TX, USA) was crosslinked with a 1:1 ratio of fast-degrading YKNR (GCYKNRGCYKNRCG) and slower-degrading YKNS (GCYKNSGCYKNSCG) trifunctional all-plasmin sensitive crosslinkers (>90% purity; GenScript, Piscataway, NJ, USA).

Techniques: In Vitro, Comparison, Transformation Assay, Light Microscopy, Derivative Assay, In Vivo, Control