Follicle activation is a significant and immediate cause of follicle loss after ovarian tissue transplantation

Abstract

Purpose: Extensive follicle loss has been demonstrated in ovarian grafts post transplantation, reducing their productivity and lifespan. Several mechanisms for this loss have been proposed, and this study aims to clarify when and how the massive follicle loss associated with transplantation of ovarian tissue graft occurs. An understanding of the mechanisms of follicle loss will pinpoint potential new targets for optimization and improvement of this important fertility preservation technique.

Methods: Frozen-thawed marmoset (n = 15), bovine (n = 37), and human (n = 46) ovarian cortical tissue strips were transplanted subcutaneously into immunodeficient castrated male mice for 3 or 7 days. Histological (H&E, Masson's trichrome) analysis and immunostaining (Ki-67, GDF9, cleaved caspase-3) were conducted to assess transplantation-associated follicle dynamics, with untransplanted frozen-thawed tissue serving as a negative control.

Results: Evidence of extensive primordial follicle (PMF) activation and loss was observed already 3 days post transplantation in marmoset, bovine, and human tissue grafts, compared to frozen-thawed untransplanted controls (p < 0.001). No significant additional PMF loss was observed 7 days post transplantation. Recovered grafts of all species showed markedly higher rates of proliferative activity and progression from dormant to growing follicles (Ki-67 and GDF9 staining) as well as higher growing/primordial (GF/PMF) ratio (p < 0.02) and higher collagen levels compared with untransplanted controls.

Conclusions: This multi-species study demonstrates that follicle activation plays an important role in transplantation-induced follicle loss, and that it occurs within a very short time frame after grafting. These results underline the need to prevent this activation at the time of transplantation in order to retain the maximal possible follicle reserve and extend graft lifespan.

Keywords: Fertility preservation; Follicle activation; Ovarian tissue cryopreservation and transplantation.

Fig. 1

A Primordial follicle loss and activation following transplantation. Primordial (dark gray) and growing (light gray) follicle counts in frozen-thawed marmoset (A1), bovine (A2), and human (A3) ovarian tissue before (control) or after transplantation for 3 or 7 days s.c. in immunodeficient mice (counts per whole graft, mean ± SE, *p < 0.05 compared with untransplanted control). B Ratios of growing (GF) to primordial follicle (PMF) counts post transplantation. Ratios of GF/PMF in frozen-thawed marmoset (B1), bovine (B2), and human (B3) ovarian tissue before (untransplanted control, black) or 3 and 7 days after transplantation (white and stripes, respectively). Ratios in each time point significantly different from those of untransplanted control are marked by different letters (p < 0.05)

Fig. 2

Comparison of follicle density in human vs marmoset ovarian tissue grafts. PMF density in human ovarian tissue grafts (b) is substantially lower than in the equivalent size marmoset tissue (a). The result of this lack of density is increased variability between pieces of tissue

Fig. 3

Immunostaining demonstrates actively growing follicles in transplanted grafts. Histological analysis of marmoset (a), bovine (b), and human (c) frozen-thawed ovarian tissues before and 7 days post transplantation. Samples were stained with H&E (a i, vi; b i, vi; c i, v). Arrows pointing at PMF, with Masson’s trichrome for collagen (a ii, vii; b ii, vii; c ii, vi). Arrows pointing at collagen (blue), for Ki-67 (a iii, viii, b iii, viii; c iii, vii). Arrows pointing at Ki-67 expressing PMF, cleaved caspase-3 (a v, x; b v, x; c iv, viii). Arrows pointing at cleaved caspase-3 expressing PMF (a v; b v, x; c iv, viii), growing follicles (a v), and GDF9 (a iv, ix; b iv, ix). Arrows pointing at GDF9 expressing growing follicles. Magnification: ×40. Image analysis and quantification were performed for each antibody on four slides per group, and the mean counts per field ± SE are shown below the histological images (*p < 0.05, **p < 0.005, ***p < 0.0005 compared with untransplanted control average). Quantification is expressed as follows: for Ki-67 and caspase-3 expressed as cells in PMF/field, for GDF9 as GDF9 expressing follicles/field, and for collagen as pixels/field (using ImageJ software)

Fig. 3

Immunostaining demonstrates actively growing follicles in transplanted grafts. Histological analysis of marmoset (a), bovine (b), and human (c) frozen-thawed ovarian tissues before and 7 days post transplantation. Samples were stained with H&E (a i, vi; b i, vi; c i, v). Arrows pointing at PMF, with Masson’s trichrome for collagen (a ii, vii; b ii, vii; c ii, vi). Arrows pointing at collagen (blue), for Ki-67 (a iii, viii, b iii, viii; c iii, vii). Arrows pointing at Ki-67 expressing PMF, cleaved caspase-3 (a v, x; b v, x; c iv, viii). Arrows pointing at cleaved caspase-3 expressing PMF (a v; b v, x; c iv, viii), growing follicles (a v), and GDF9 (a iv, ix; b iv, ix). Arrows pointing at GDF9 expressing growing follicles. Magnification: ×40. Image analysis and quantification were performed for each antibody on four slides per group, and the mean counts per field ± SE are shown below the histological images (*p < 0.05, **p < 0.005, ***p < 0.0005 compared with untransplanted control average). Quantification is expressed as follows: for Ki-67 and caspase-3 expressed as cells in PMF/field, for GDF9 as GDF9 expressing follicles/field, and for collagen as pixels/field (using ImageJ software)

Fig. 4

The magnitude of follicle activation directly impacts PMF reserve in ovarian grafts. a Inducing activation in ovarian follicle grafts increases the number of growing follicles in the immediate short term at the expense of the PMF population, resulting in decreased graft lifespan. This may be suitable for immediate IVF retrieval but comes at the expense of the overall fertility potential and long-term functioning of the graft. b Limiting activation at the time of transplantation reduces PMF loss thereby extending graft lifespan. It is preferable to reduce the activation that occurs with transplantation in order to maximize the fertility outcomes of the procedure