Attempted application of bioengineered/biosynthetic supporting matrices with phosphatidylinositol-trisphosphate-enhancing substances to organ culture of human primordial follicles

Abstract

Purpose: To improve human primordial follicle culture.

Methods: Thin or thick ovarian slices were cultured on alginate scaffolds or in PEG-fibrinogen hydrogels with or without bpV (pic), which prevents the conversion of phosphatidylinositol-trisphosphate (PIP3) to phosphatidylinositol-bisphosphate (PIP2) or 740Y-P which converts PIP2 to PIP3. Follicular growth was evaluated by follicular counts, Ki67 immunohistochemistry, and 17β-estradiol (E2) levels.

Results: BpV (pic) had a destructive effect on cultured follicles. Thawed-uncultured samples had more primordial follicles than samples cultured in basic medium and fewer developing follicles than samples cultured in PEG-fibrinogen hydrogels with 740Y-P. There were more atretic follicles in samples cultured on alginate scaffolds than in PEG-fibrinogen hydrogels, and in samples cultured in PEG-fibrinogen hydrogels with 740Y-P than in PEG-fibrinogen hydrogels with basic medium. Ki67 staining was higher in PEG-fibrinogen hydrogels than on alginate scaffolds. E2 levels were higher in thick than in thin slices.

Conclusions: PEG-fibrinogen hydrogels appear to have an advantage over alginate scaffolds for culturing human primordial follicles. Folliculogenesis is not increased in the presence of substances that enhance PIP3 production or with thin rather than thick sectioning.

Fig. 1

Photographs of the culture systems. a Molds for PEG-fibrinogen hydrogel formation. b Ovarian slice encapsulated in a PEG-fibrinogen hydrogel. c Human ovarian slice on alginate scaffold

Fig. 2

Graphs representing main and significant results from Table 1. I. Activation in culture by follicular counts. a Results are in percent of primordial follicles. Primordial follicle count is significantly higher in thawed-untreated samples than after culture with basic medium alone, regardless of the matrix (P < 0.05). b Results are in percent of developing follicles. Developing follicle count is higher in samples cultured with basic medium alone than in thawed-untreated samples, regardless of the matrix (NS). II. PEG-fibrinogen hydrogels versus alginate scaffolds. c Results are in percent of atretic follicles. Atretic follicle count is significantly higher on alginate scaffolds than atretic follicles in PEG-fibrinogen hydrogels, regardless of treatment (P < 0.04). d Results are in percents of developing follicles. Developing follicle count is higher in samples cultured in PEG-fibrinogen hydrogels than in thawed-untreated samples (NS). e Results are in percent of Ki67 positively stained follicles. The proportion of stained follicles is significantly higher on alginate scaffolds or in PEG-fibrinogen hydrogels with basic medium than in thawed-uncultured samples (P < 0.0001). III. Destructive effects of bpV (pic). Results are in percent of primordial follicles. f Primordial follicle count is significantly higher in thawed-untreated samples than in samples cultured with bpV (pic), regardless of the matrix (P < 0.05). (G) Developing follicles count is significantly higher in samples cultured in PEG-fibrinogen hydrogel with 740Y-P than in PEG-fibrinogen hydrogel with bpV (pic) (P < 0.05). h E₂ level is significantly higher in samples cultured with 740Y-P (P < 0.05) or with basic medium alone (P < 0.03) than with bpV (pic), regardless of the matrix. IV. Thick versus thin samples. Results are in pg/ml. Values represent mean±standard deviation. i E₂ level is significantly higher after culture of thick slices than thin slices (P < 0.0004), regardless of the matrix. j E₂ level is significantly higher after culture of thick slices than thin slices on alginate scaffolds (P < 0.04)

Fig. 3

Histological and Ki67 immunostaining of uncultured and cultured ovarian samples. a Thawed-uncultured ovarian section from a 22-year-old woman. Note the primordial follicles. Hematoxylin and Eosin staining, original magnification X400. b Section of ovarian sample with Ki67 immunohistochemical staining from a 13-year-old girl cultured in PEG-fibrinogen hydrogel for 1 week. Note the small secondary follicle, with the light red-brown staining indicating Ki67 expression in a portion of the granulosa cells (arrow). Original magnification X400. c Section of ovarian sample with Ki67 immunohistochemical staining from the same patient as in panel (A) cultured in PEG-fibrinogen hydrogel for 1 week. Note the primary and primordial follicles, with the red-brown staining indicating Ki67 expression in a portion of the granulosa cells (arrows). Original magnification X400. d Section of ovarian sample from a 10-year-old girl cultured on alginate scaffold with 740Y-P for 1 week. Note the two primordial-primary follicles (arrow) and the three atretic follicles (arrow heads). Hematoxylin and Eosin staining, original magnification X400. e Section of ovarian sample from the same patient as in panel (B) cultured on alginate scaffold for 1 week with bpV (pic). Note the atretic follicles (arrows). Hematoxylin and Eosin staining, original magnification X400