1. The impact of culture conditions on early follicle recruitment and growth from human ovarian cortex biopsies in vitro 
Jana Liebenthron, M.Sc., Maria Köster, D.V.M., Christina Drengner, Jochen Reinsberg, Ph.D., Hans van der Ven, M.D., Markus Montag, Ph.D. 
Fertility and Sterility 
Volume 100, Issue 2, Pages e5 (August 2013)
DOI: /j.fertnstert
Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

2. Figure 1
Tissue processing. (A) Following preparation, an ovarian cortical tissue strip was cut into three pieces of similar size, allocated to subgroups (control [C], fluidic culture [FC], and conventional culture [CC]), and incubated in the respective culture system with serum-free medium for 6 days. (B) The complete trial setup with all investigations and an inset picture of the constructed FCe system. OTCM = ovarian tissue culture medium; PCR = polymerase chain reaction.
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3. Figure 2
Follicle counts. Histogram showing the mean ± SEM values of healthy fluorescent follicles in (A) 5 × 2.5 × 1 mm cortical pieces and (B) 4-μm histologic sections from 5 × 2.5 × 1 mm cortical pieces. The samples in each trial were taken from 10 different ovarian cortical biopsies at time of collection (day 0) and after 6 days of culture.
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4. Figure 3
Estradiol and progesterone production. The growth media supernatant from each cultured cortical piece was analyzed for (A and B) E2, (C and D) P, and (E and F) the E2/P ratio at day hours and at day 6. Each value was normalized to the respective follicle count. Data are presented as mean ± SEM values representing evaluations from 10 different ovarian cortical biopsies. Asterisks indicate significantly different results between treatments according to unpaired t test (P<.05).
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5. Figure 4
Gene expression profiles. Total RNA was extracted from 30 cortex fragments, three from each patient, before and after in vitro culture in a dynamic fluidic culture system or a static conventional culture system. Gene expression was assessed with the use of real-time polymerase chain reaction (PCR) followed by agarose gel electrophoresis of the PCR products (Supplemental Fig. 3, available online at The bars represent mean ± SEM values from the gene expression assays of early folliculogenesis genes: (A) Kit ligand, (B) growth differentiation factor 9, (C) bone morphogenetic factor 15, and (D) inhibin B. Asterisks indicate significantly different results from matched treatments according to unpaired t test (P<.05).
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6. Supplemental Figure 1
Evaluation of the viable follicle count and morphologic analysis before and after 6 days of culture. Viable follicles of digested ovarian cortex as viewed under fluorescence microscope (green) and with incident light (grey). (A, B) Example of primordial and primary healthy fluorescent follicles from uncultured cortex biopsies. (C, D) Example of healthy fluorescent follicles in different developmental stages (from primary to preantral stage) from biopsies cultured in the fluidic culture system. (E, F) Example of healthy fluorescent follicles in different developmental stages (from primary to preantral stage) from biopsies cultured in the static conventional culture system. Scale bars = 50 μm.
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7. Supplemental Figure 2
Histologic analysis after 6 days culture period. Histologic section, stained with haematoxylin and eosin, from a sample that was cultured in the dynamic fluidic culture system. All counted follicles show an intact healthy oocyte. Scale bar = 50 μm.
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8. Supplemental Figure 3
Agarose gel electrophoresis. DNA gel electrophoresis was performed for analytic evaluation of the polymerase chain reaction (PCR) products. Each lane represents a different gene: ribosomal protein L19 (RPL19) as housekeeping gene, Kit ligand (KIT-L), growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), and inhibin B (INHB). The bottom panel represents the negative control samples of each gene and excludes a contamination of the PCR mixes.
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Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions