Alginate encapsulation supports the growth and differentiation of human primordial follicles within ovarian cortical tissue

Abstract

Purpose: In vitro follicle growth (IVFG) is an investigational fertility preservation technique in which immature follicles are grown in culture to produce mature eggs that can ultimately be fertilized. Although progress has been made in growing primate primary and secondary follicles in vitro, it has been a relatively greater challenge to isolate and culture primordial follicles. The purpose of this study was to develop methods to grow human primordial follicles in vitro using alginate hydrogels.

Methods: We obtained human ovarian tissue for research purposes through the National Physicians Cooperative from nationwide sites and used it to test two methods for culturing primordial follicles. First, primordial follicles were isolated from the ovarian cortex and encapsulated in alginate hydrogels. Second, 1 mm × 1 mm pieces of 500 μm-thick human ovarian cortex containing primordial follicles were encapsulated in alginate hydrogels, and survival and follicle development within the tissue was assessed for up to 6 weeks.

Results: We found that human ovarian tissue could be kept at 4 °C for up to 24 h while still maintaining follicle viability. Primordial follicles isolated from ovarian tissue did not survive culture. However, encapsulation and culture of ovarian cortical pieces supported the survival, differentiation, and growth of primordial and primary follicles. Within several weeks of culture, many of the ovarian tissue pieces had formed a defined surface epithelium and contained growing preantral and antral follicles.

Conclusions: The early stages of in vitro human follicle development require the support of the native ovarian cortex.

Fig. 1

The effects of ovarian transport at 4 °C for up to 24 h on tissue and follicle health. a) The percent of TUNEL-positive cells in fresh and transported tissue is plotted for each participant. b–e) H&E images of ovarian tissue, from participants N and S, that were fixed immediately (fresh) or fixed post-transport (transport). Representative TUNEL-staining for each sample is shown in the insets (TUNEL-positive cells are green, scale bar, 50 μm)

Fig. 2

Preantral follicles remained viable and functional when transported at 4 °C for up to 24 h. a) The number of secondary follicles are presented for the age-matched cohort of participants for fresh and transport tissue analysis. b) The follicle diameters upon collection (day 0) and upon termination (day 19–40) of culture are presented for fresh (F) and transported (T) tissue. c) Representative images of secondary follicles isolated (day 0) and tracked on day 7, 14, 21 and 28 of culture appear to grow similarly whether isolated from fresh (participant BB) or transported (participant V) tissue (scale bar, 100 μm). b) Follicular function is determined by the percentage of follicles that formed antrums in culture following isolation from fresh (F) or transport (T) tissue is shown. e) Normal follicular structure and cytoskeleton morphology is demonstrated within a cultured follicle isolated from transported tissue through staining with F-actin (red) and Germ cell-specific Y-box-binding protein (MSY2; green; scale bar, 10 μm)

Fig. 3

Heterogeneous human ovarian cortex contributed to inconsistent primordial follicle isolation and culture protocols. a–d) Masson’s Trichrome stain of ovarian cortical tissue highlights the variable collagen density (blue) surrounding primordial follicles between individual participants (A, participant H; B, participant FF; C, participant F; D, participant C; scale bar, 50 μm). e) Survival of primordial follicles following isolation protocols was poor as indicated by loss of integrity between the somatic cells and oocytes. Arrows highlight denuded oocytes. f) Follicles that remained intact following isolation were encapsulated in 0.5-2 % alginate (scale bar, 100 μm), but the granulosa cells dissociated from the oocytes by 3 days of culture (inset; scale bar, 25 μm)

Fig. 4

Primordial follicles were reliably identified using only light microscopy within 500 μm sections of ovarian cortex. a) Transmitted light image of a thin section of ovarian cortex containing primordial follicles, which are visible as areas of clearing within the tissue (inset, arrowheads). b) H&E staining confirmed primordial follicles within the tissue (scale bar, 100 μm). Primordial follicles are characterized by an oocyte surrounded by an incomplete layer of squamous granulosa cells (inset, arrowheads). c) Follicles were counted within ovarian cortical tissue sections at 100 μm increments. There were between 2.96 and 59.15 follicles per mm². A line indicates the median follicle number (18.89 per mm²)

Fig. 5

The majority of ovarian cortical tissue survives long-term culture encapsulated in alginate hydrogels. Tissue survival was assessed by quantifying the number of pieces of ovarian cortex that contained a) live cells (hematoxylin-positive nuclear material) and b) dead cells (hematoxylin-negative). Representative images of stromal tissue are shown (scale bar, 100 μm). c) The percent survival of ovarian cortex was calculated as the number of tissue pieces that contained live cells over the total number of tissue pieces analyzed for the particular culture time point. Data from the entire 6-week culture period is plotted

Fig. 6

Healthy ovarian tissue undergoes epithelial surface remodeling during culture. a) Processing of ovarian tissue into 1 mm × 1 mm pieces of 500 μm-thick cortex for encapsulation disrupts the edges of the tissue (arrows; participant O). b) This disruption likely stimulates a wound healing response, which causes the encapsulated tissue to differentiate to form an epithelial-like surface (arrows; participant R). Tissue pieces with clear epithelial differentiation typically contain healthy stroma and follicles (arrowheads). Representative images are shown (scale bar, 50 μm)

Fig. 7

Alginate-encapsulated culture of ovarian cortex maintains and supports follicles for at least 4 weeks. Representative cortical strip pieces fixed on day 0 (a, participant R; b, participant M) or following 2 weeks (c, participant S; d, participant, R), 3 weeks (e, participant R), 4 weeks (f, participant P) or 6 weeks (g, participant R; h, participant S) of culture are shown (scale bar, 100 μm). Arrows highlight the differentiated surface epithelium edge of the cortical tissue in 2, 3 and 4-week cultures. Evidence of general tissue degeneration was common at 6 weeks. The arrowhead points to a disorganized secondary follicle. This degeneration corresponded to the absence of the defined epithelial surface that was present earlier in culture

Fig. 8

Alginate encapsulation and culture of human ovarian cortex supports folliculogenesis. Representative images of a primordial (a, participant M, day 0) and a secondary follicle (b, participant T, 2 weeks) within the cultured ovarian tissue are shown. An arrow and arrowhead highlight the squamous granulosa cells and multiple granulosa layers, respectively. c) An antral follicle was identified within a piece of ovarian cortex that was cultured for 6 weeks (participant R). The cumulus-oocyte-complex is highlighted by an asterisks and the antral cavity is apparent as the white fluid-filled space (scale bars, 50 μm). d) The distribution of follicle classes at each culture time point is plotted for individual participants