Xenograft model of heterotopic transplantation of human ovarian cortical tissue and its clinical relevance

Abstract

In brief: Xenografts of human ovarian cortical tissue provide a tractable model of heterotopic autotransplantation that is used for fertility preservation in patients undergoing ablative chemo/radiotherapy. This study describes the behavior of hundreds of xenografts to establish a framework for the clinical function of ovarian cortex following autotransplantation over short- and long-term intervals.

Abstract: More than 200 live births have been achieved using autotransplantation of cryopreserved ovarian cortical fragments, yet challenges remain to be addressed. Ischemia of grafted tissue undermines viability and longevity, typically requiring transplantation of multiple cortical pieces; and the dynamics of recruitment within a graft and the influence of parameters like size and patient age at the time of cryopreservation are not well-defined. Here, we describe results from a series of experiments in which we xenografted frozen/thawed human ovarian tissue (n = 440) from 28 girls and women (age range 32 weeks gestational age to 46 years, median 24.3 ± 4.6). Xenografts were recovered across a broad range of intervals (1-52 weeks post-transplantation) and examined histologically to quantify follicle density and distribution. The number of antral follicles in xenografted cortical fragments correlated positively with the total follicle number and was significantly reduced with increased patient age. Within xenografts, follicles were distributed in focal clusters, similar to the native ovary, but the presence of a leading antral follicle coincided with increased proliferation of surrounding follicles. These results underscore the importance of transplanting ovarian tissue with a high density of follicles and elucidate a potential paracrine influence of leading antral follicles on neighboring follicles of earlier stages. This temporal framework for interpreting the kinetics of follicle growth/mobilization may be useful in setting expectations and guiding the parameters of clinical autotransplantation.

Figure 1

Primordial follicle retention and antral follicle output in short- and long-term ovarian cortical xenografts. (A) Schematic of the experimental design for xenotransplantation of ovarian cortical fragments from patients and organ donors. (B) A fibrin clot containing a graft harvested at 4 weeks post-transplantation (Patient 19, Table 1). (C and D) Density (C) and percentage (D) of primordial follicles retained in xenografts upon recovery between 1- and 2-, 3- and 4-, 8- and 11-, and 14- and 22-week intervals. (E, F, G, and H) The density of primordial follicles retained in xenografts at 1- to 2- (E), 3- to 4- (F), 8- to 11- (G), and 14- 22-week intervals (H) related to patient/donor age in years at the time of tissue cryopreservation. Bars in (C and D) represent median percentages of follicle sub-types, with each dot representing one replicate and error bars indicating the 95% CI.

Figure 2

Dynamics of antral follicle growth in xenografts. (A) The number of xenografts at 8 weeks contains one or more antral follicles. (B) Relationship of the number of antral follicles to the number of total follicles in xenografts containing one or more antral follicles. (C) Number of antral follicles in xenografts relative to age in years of donor/patient at the time of tissue cryopreservation. (D) Antral follicle number (y-axis) in ovarian cortical fragments of varying sizes (x-axis); all 17 replicates are from the same patient (DOV 4, Table 1). (E and F) Mouse was xenografted with tissue from a 6-year-old donor (Patient 12, Table 1) and monitored by MRI. (E) Coronal and (F) sagittal section of the xenografts bearing mouse. (G) The mouse was sacrificed at 16 weeks post-transplantation, and grafts were harvested for histological analysis, H&E staining. Inset in G is enlarged in the associated box. Scale bar = 500 μm. Black arrows identify primordial follicles.

Figure 3

Distribution of follicles within the graft and between replicates from different patients. (A, B, and C) Graphs show the distribution of total (A) or primordial (B and C) follicles across slides encompassing the total xenograft; representative examples are shown for five grafts from four patients (Table 1) in (A) and across four recovered xenografts after 8 (B) and 14 (C) weeks from the same patient (DOV 4, Table 1). (D) The primordial to growing follicle ratio in grafts where no antral follicles were detected compared to grafts with one or more antral follicles. (E) The primordial to growing follicle ratio was measured in three different time points: 4, 8, and 14 weeks. Bars in D and E represent median ratios, with each dot representing one replicate and error bars indicating the 95% CI.

Figure 4

Assessment of cell proliferation in consecutive temporal windows of follicle development. (A) Schematic of the experimental approach for sequential labeling with thymidine analogs; red and green dots indicate times of EdU and CldU injections, respectively. (B) Labeling detected proliferative cells in growing follicles of all stages (primary to antral). (C, D, E, F, G, and H) Quantification of proliferative cells within the GC and activated stroma compartment of follicles. (C, D, E, and F) Follicles from xenografts containing leading follicles showed a significant increase in GC and TS proliferation when compared to follicles in grafts without leading antral follicles, observed at secondary (D) (up to four layers of GCs), preantral (E) (five layers or more of GCs), and antral stages (F), as measured by incorporation of EdU, CldU, or both. (G and H) ST compartment proliferation decrease with increased size approaching ovulatory competence. (B, C and G) Scale bars in (B and C) are 50 μm; in (G), scale bars are 500 μm and 100 μm in the insets.

Figure 5

Reproductive hormones/steroids in host serum reflect follicle activity in xenografts. (A) E₂ was measured in the serum of animals bearing xenografts for short (2–8 weeks) and long (11–22 weeks) intervals. (B) Serum human E₂, mFSH, and mLH were measured in a subset of mice bearing xenografts for short- and long-term intervals. (C, D, E, and F) AMH (C and E) and testosterone (D and F) were measured in the serum of animals bearing xenografts for short- and long-term intervals; levels of AMH (E) and testosterone (F) relative to E₂ are shown for samples that had detectable levels. (G) H&E micrograph from neonatal (32 weeks gestational age) ovarian tissue that was xenografted for 52 weeks; magnified stroke boxes show primordial follicles. Bars in (A) represent median E₂ levels (pg/mL) in the serum, with each dot representing one replicate and error bars indicating the 95% CI. Bars in (C) represent median AMH levels (ng/mL) in the serum, with each dot representing one replicate and error bars indicating the 95% CI. Bars in (D) represent median testosterone levels (ng/dL) in the serum, with each dot representing one replicate and error bars indicating the 95% CI.