Human amniotic mesenchymal stem cells improve the follicular microenvironment to recover ovarian function in premature ovarian failure mice

Abstract

Background: Many adult women younger than 40 years old have premature ovarian failure (POF) and infertility. Previous studies confirmed that different tissue-derived stem cells could restore ovarian function and folliculogenesis in chemotherapy-induced POF mice. The aim of this study was to explore the therapeutic efficacy and underlying mechanisms of human amniotic mesenchymal stem cells (hAMSCs) transplantation for hydrogen peroxide-induced ovarian damage.

Methods: Bilateral ovaries of female mice were burned with 10% hydrogen peroxide to establish a POF model. After 24 h of treatment, hAMSCs and diethylstilbestrol were administered to POF mice by intraperitoneal injection and intragastric administration, respectively. After either 7 or 14 days, ovarian function was evaluated by the oestrus cycle, hormone levels, ovarian index, fertility rate, and ovarian morphology. The karyotype was identified in offspring by the G-banding technique. hAMSCs tracking, immunohistochemical staining, and real-time polymerase chain reaction (PCR) were used to assess the molecular mechanisms of injury and repair.

Results: The oestrus cycle was recovered after hAMSCs transplantation at 7 and 14 days. Oestrogen levels increased, while follicle-stimulating hormone levels decreased. The ovarian index, fertility rate, and population of follicles at different stages were significantly increased. The newborn mice had no obvious deformity and showed normal growth and development. The normal offspring mice were also fertile. The tracking of hAMSCs revealed that they colonized in the ovarian stroma. Immunohistochemical and PCR analyses indicated that changes in proteins and genes might affect mature follicle formation.

Conclusions: These results suggested that hAMSCs transplantation can improve injured ovarian tissue structure and function in oxidatively damaged POF mice. Furthermore, the mechanisms of hAMSCs are related to promoting follicular development, granulosa cell proliferation, and secretion function by improving the local microenvironment of the ovary.

Keywords: Diethylstilbestrol; Human amniotic mesenchymal stem cells; Hydrogen peroxide; Microenvironment; Ovary; Premature ovarian failure; Reproduction.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1

Morphology and molecular phenotype of hAMSCs. The cells morphology of passage 0 (a, × 40), 3 and 4 (b, c, × 100) hAMSCs. d Phenotypic analysis of the fourth-passage hAMSCs by flow cytometry

Fig. 2

The transplanted hAMSCs improve oestrus cycles in POF mice. Vaginal smears were obtained, and the oestrus cycles were evaluated by H&E staining. Representative photographs for proestrus (a), oestrus (b), metoestrus (c), and dioestrus (d) are shown (× 100). e The percentage of abnormal cyclicity was detected in mice at 7 and 14 days after cells transplantation (n = 6)

Fig. 3

Transplantation of hAMSCs improves serum oestrogen and FSH hormone levels in POF mice. Serum levels of oestrogen (a) and FSH (b) were detected at 7 and 14 days after cells transplantation. Data are presented as the mean ± S (x¯ ± s, n = 6), *P < 0.05

Fig. 4

The body weight and ovarian index of POF mice were obviously increased by transplanted hAMSCs. Body weight (a) and ovarian index (b) were detected at 7 and 14 days after cells transplantation (x¯ ± s, n = 6). *P < 0.05

Fig. 5

Transplanted hAMSCs improved the fertility rate in POF mice. a The different fertility rates are shown in each group, but the DES group showed infertility (n = 10). b The suckling mice of the hAMSC group were observed for 30 days. Karyotype analysis of the chromosomes of offspring mice in the normal group (c) and hAMSC group (d) were observed under oil immersion lenses (× 400)

Fig. 6

Transplantation of hAMSCs reduces ovarian injuries in POF mice. a The pathological changes of ovaries were evaluated by H&E staining in the four groups at 7 and 14 days after hAMSCs transplantation (× 40). Quantitation of follicle count from ovaries in mice of the four groups at 7 (b) and 14 (c) days after cell transplantation (n = 4). The black arrow shows granulosa cells, the red arrow shows the cumulus complex, and the green arrow shows mature follicles

Fig. 7

In vivo hAMSCs tracking. a, b PKH26-labelled hAMSCs showed red fluorescence (× 100). c The labelling rates of PKH26-labelled hAMSCs were detected by flow cytometry. Transplanted hAMSCs were observed at 7 (d, e) and 14 (f, g) days after cell transplantation in ovaries (× 100, n = 3). d and f show the bright field under a fluorescence microscope, and e and g show the dark field (× 100)

Fig. 8

The hAMSCs improved protein expression associated with follicular development in the ovarian microenvironment of POF mice. The brown particles of positive expression were observed on FSHR, VEGF, IGF-1, TNF-α, and IL-1β proteins by immunohistochemical staining at 7 days (a) and 14 days (c) after hAMSCs transplantation. At 7 (b) and 14 days (d) after cell transplantation, the mean integral optical density value (IOD) of FSHR-, VEGF-, IGF-1-, TNF-α-, and IL-1β-positive areas was determined in ovarian tissue by Image-Pro Plus (immunohistochemical staining, × 200). Data are presented as the mean ± S (x¯ ± s, n = 6), *P < 0.05

Fig. 9

The mRNA levels of FOXL2, OCT4, GDF-9, SCF, and LIF were regulated by hAMSCs in the ovarian tissues of POF mice by real-time PCR at 7 (a) and 14 (b) days after cells transplantation. Data are presented as the mean ± S (x¯ ± s, n = 6), *P < 0.05