Cell-free therapy with the secretome of adipose tissue-derived stem cells in rats' frozen-thawed ovarian grafts

Abstract

The use of secretome may be a new strand of cell therapy, which is equal to or even superior to the injection of live cells, called cell-free therapy. In ovarian transplantation, this approach may be a therapeutic possibility for the ovarian graft in hypoxia. We designed the present study to evaluate whether the cell-free therapy with the secretome of adipose tissue-derived stem cells (ASCs) in rat frozen-thawed ovarian grafts could protect a graft against ischemic injury. A single dose of rat ASCs secretome or vehicle was injected into the bilateral frozen-thawed ovaries of 18 adult female rats immediately after an autologous transplant. Nine animals were used to control the cryopreservation protocol and were evaluated before and after the cryopreservation process. Daily vaginal smears were performed for estrous cycle evaluation until euthanasia on postoperative day 30. Follicle viability by trypan blue, graft morphology by HE, and apoptosis by TUNEL and cleaved-caspase-3 were assessed. No differences were found with respect to estrous cycle resumption and follicle viability (p > 0.05). However, compared with the vehicle-treated grafts, the morphology of the secretome-treated grafts was impaired, showing reduced follicular population and increased apoptosis (p < 0.05). ASC secretome impaired the rat frozen-thawed ovarian graft from ischemic injury. However, more studies are needed to evaluate the factors involved and the possibility of applying the secretome in scaffolds to optimize its use.

Keywords: Fertility preservation; Ovarian transplantation; Rat; Secretome; Stem cells.

Fig. 1

Assessment of follicular viability by trypan blue staining. Photomicrograph of viable ovarian follicles stained with white (a) and of nonviable follicles stained with blue (b). Blue arrow: trypan blue crystals. Red arrow: blood cells. × 200. Percentages (c) and mean number ± standard deviation (d) of viable ovarian follicles in fresh and thawed tissue after isolation and trypan blue staining. p > 0.05, paired t test

Fig. 2

Photomicrographs of fresh and thawed ovarian tissue by HE staining and immunohistochemistry for apoptosis by TUNEL. Morphology is similar between groups. a Quantification of viable ovarian follicles in a 500-μm² area, data are shown as the mean ± SD. Immunohistochemistry was performed on the ovarian follicles; dark brown-stained cells are considered positive (red arrow). b Results are expressed as a percentage of the positive area (arbitrary unity/mm²). Non-significant differences between groups in both analyses. p > 0.05, paired t test. HE × 100. TUNEL × 400. F follicles, CL corpora lutea, Fi fibrosis, V vessel

Fig. 3

Quantitative PCR of apoptotic genes profile in fresh (control group) and thawed (group 1) ovarian tissue. a Upregulated genes. b Downregulated genes. Data were analyzed using Web Basis Data Analysis ;at https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=2&cad=rja&uact=8&ved=2ahUKEwi_mPepoKTeAhUFTZAKHa9LA2IQFjABegQICRAB&url=https%3A%2F%2Fdataanalysis.sabiosciences.com%2Fpcr%2Farrayanalysis.php%3Fwuid%3D8fa7191b-bb49-409d-b896-8ce28966b04e%26logindata%3D%26customerdata%3D%26customeremail%3D%26platform%3DcustomArray%26format%3DX&usg=AOvVaw23-l6Q74gr7qsQXNr95B-L

Fig. 4

Photomicrography of cryopreserved ovarian grafts treated with vehicle or ASC secretome 30 days after an autologous avascular transplant. Treatment with secretome induced atrophy of the graft, with predominance of fibrosis and few viable follicles (HE). Immunohistochemistry for apoptosis (TUNEL and cleaved-caspase-3) was performed on the ovarian follicles, and dark brown-stained cells were considered positive (red arrows). The results are expressed as a percentage of the positive area (arbitrary unity/mm²). Apoptosis increased in TUNEL assay in grafts treated with the secretome (a) and remained similar in caspase (b). *p < 0.05, unpaired t test. HE × 50. Immunohistochemistry × 400. ASC adipose tissue-derived stem cells, CL corpora lutea, CA corpora albicans, FI fibrosis