Long-Term Advantages of Ovarian Reserve Maintenance and Follicle Development Using Adipose Tissue-Derived Stem Cells in Ovarian Tissue Transplantation

Abstract

(1) Background: Ovarian tissue transplantation with adipose tissue-derived stem cells (ASCs) has been shown to enhance graft vascularization and increase follicle survival after a short interval of 7 days. The aim of the present study was to investigate their long-term effects on primordial follicle pool maintenance and follicle development. (2) Methods: A total of 14 severe combined immunodeficient (SCID) mice were grafted with frozen-thawed human ovarian tissue with or without ASCs. Blood was taken monthly in order to quantify the anti-Müllerian hormone (AMH) and estradiol. After 6 months, all the grafts were retrieved and sent for histology and immunolabeling (AMH, AMH receptor II, estrogen receptors α and β, and c-kit/kit ligand). (3) Results: A significant upturn was observed in AMH and estradiol plasma levels 4 months after transplantation in both grafted groups. The primordial follicle pool was better preserved in the ASC group (41.86 ± 28.35) than in the standard transplantation group (9.65 ± 17.6, p < 0.05) compared to non-grafted controls (124.7 ± 140). (4) Conclusions: The use of ASCs prior to ovarian tissue transplantation yielded a larger primordial follicle pool and more physiological follicle distribution after long-term grafting. These findings suggested that ASC use might extend the ovarian tissue lifespan.

Keywords: AMH; ASCs; adipose tissue-derived stem cells; endocrine restoration; estradiol; folliculogenesis; long-term transplantation; ovarian tissue transplantation.

Figure 1

(A) Frozen-thawed ovarian tissue from 7 adult patients was distributed equally among the following groups: non-grafted controls, OT group (one-step transplantation to 7 SCID mice), and 2-step/ASCs+OT group (two-step transplantation to 7 SCID mice). The first step (ASCs loaded inside fibrin scaffolds) was carried out 2 weeks before the second step (OTT). Blood was taken every 4 weeks until euthanasia (after 24 weeks) and sample retrieval for histology and immunolabeling. (B) Human ovarian tissue retrieval from SCID mouse peritoneum after 6 months of transplantation in the 2-step/ASCs+OT group. Grafted ovarian tissue (1) with an antral follicle (2) and macroscopically visible vasculature (3) derived from mouse peritoneum (4). SCID: severe combined immunodeficient. ASCs: adipose tissue-derived stem cells. OT: ovarian tissue. OTT: ovarian tissue transplantation.

Figure 2

AMH (A) and estradiol (B) concentrations (pg/mL ± SEM) in mouse plasma ELISA assays were analyzed every 4 weeks by one-way analysis of variance (ANOVA) and Tukey’s post hoc test. Significant differences between time points are indicated as follows: * p < 0.05. No differences were detected between groups (OT vs. 2-step/ASCs+OT). AMH: anti-Müllerian hormone. ELISA: enzyme-linked immunosorbent assay. OT: ovarian tissue. ASCs: adipose tissue-derived stem cells.

Figure 3

H&E sections of 6-month grafted tissues showed antral and primordial follicles in the 2-step/ASCs+OT group (A,B) and antral and secondary follicles in the OT group (C). Scale bars: 1000 µm and 20 µm. (D) Follicle density (mean number of follicles/mm³) and (E) primordial follicle density (mean number of follicles/mm³) were compared between groups (non-grafted controls, OT, and 2-step/ASCs+OT) using two-way ANOVA and Tukey’s post hoc test. (F) Follicle survival rates (percentage, mean ± SEM) were compared between groups (non-grafted controls, OT, and 2-step/ASCs+OT) using one-way ANOVA and Tukey’s post hoc test. Significant differences between groups are indicated as follows: * p < 0.05; ** p < 0.01. H&E: hematoxylin and eosin. OT: ovarian tissue. ASCs: adipose tissue-derived stem cells.

Figure 4

Stacked percentages of each follicle stage (primordial, primary, secondary, and antral) were compared between groups (non-grafted controls, OT, and 2-step/ASCs+OT) with the one-way ANOVA and Tukey’s post hoc test. Differences between the grafted groups and non-grafted controls: ** p < 0.01, *** p < 0.0001. Differences between the OT and 2-step/ASC+OT groups: §§ p < 0.01). OT: ovarian tissue. ASCs: adipose tissue-derived stem cells. ns: not significant.

Figure 5

(A) AMH, (B) AMHRII, (C) ERα, and (D) ERβ staining concentrations (mean ± SEM) comparisons between groups (non-grafted controls, OT, and 2-step/ASCs+OT), and follicle stages (primordial, primary, secondary, and antral) were analyzed using Kruskal–Wallis and Fisher’s post hoc LSD tests. Significant differences between groups are indicated as follows: * p < 0.05; *** p < 0.001. AMH: anti-Müllerian hormone. AMHRII: anti-Müllerian hormone receptor type II. ER: estrogen receptor. OT: ovarian tissue. ASCs: adipose tissue-derived stem cells. Scale bars: 100 µm.