Abstract

Ovarian tissue transplantation methods using cooled and cryopreserved samples have been attractive options for fertility preservation in animal models and humans. The aim of this study was to evaluate the impact of previous exposure to cooling, cryopreservation, and VEGF on the overall efficiency of equine ovarian tissue after heterotopic xenotransplantation in mice. The end points evaluated were follicular morphology and development, follicular and stromal cell densities, angiogenesis (i.e. the density of new and mature blood vessels), collagen types I and III fiber densities, and total fibrosis. Ovaries of adult mares were harvested after ovariectomy, and ovarian fragments were xenografted in the i.p. wall of BALB nude mice. Ten types of treatments involving different combinations of cooling, cryopreservation, xenografting procedures, and VEGF exposure were compared. The novel aspect of this study was the use of equine ovarian tissue xenotransplantation in mice, challenging the fragments with different combinations of treatments. The main findings were (i) cooling but not cryopreservation was effective in preserving the follicular morphology, (ii) a greater percentage of developing follicles but lower follicular and stromal cell densities were observed after ovarian tissue engraftment, (iii) exposure to VEGF increased new and mature vessels in cryopreserved-transplanted tissue, and (iv) an appropriate balance in the collagen types I and III fiber ratio in cooling-transplanted tissue was observed after exposure to VEGF. This study contributes to advancing knowledge in the preservation of ovarian tissue after cooling-cryopreservation and transplantation aiming to be applied to genetically superior/valuable horses, livestock, endangered animals, and, possibly, humans.Lay summaryDue to ethical limitations involving humans, the female horse (mare) has recently emerged as an alternative model for reproductive comparisons with women to optimize fertility restoration using ovarian tissue transplantation techniques. This study determined if ovarian tissue from donor mares (n = 3), exposed or not to vascular endothelial growth factor (VEGF) before transplantation, better survives for 7 days after transplantation into mouse hosts (n = 12). Tissues submitted to different combinations of cooling, freezing, and transplanting treatments, along with control groups, were evaluated using the parameters morphology, development, the density of immature eggs (follicles), the density of supportive (stromal) cells, collagen protein proportions, and density of blood vessels. Frozen-thawed treatments had lower percentages of normal follicles. Exposure to VEGF increased blood vessel densities in frozen tissue and favored adequate collagen levels in cooled-transplanted treatments. In conclusion, VEGF exposure seems to be beneficial for mare ovarian tissue transplantation and warrants further investigation.

Highlights

  • Cryopreservation and ovarian tissue transplantation (OTT) have been successfully used to restore fertility in animal models (Campbell et al 2014) and women affected by reproductive impairment

  • A total of 595 preantral follicles were analyzed in 6186 histological sections, with an overall mean number of 213.3 ± 11.5 sections evaluated per fragment

  • We investigated the overall effect of vascular endothelial growth factor (VEGF) by grouping the treatments in the absence or presence of VEGF, as well as the overall effect of the procedures regardless of the absence or presence of VEGF

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Summary

Introduction

Cryopreservation and ovarian tissue transplantation (OTT) have been successfully used to restore fertility in animal models (Campbell et al 2014) and women affected by reproductive impairment (for review, see Takae & Suzuki 2019). The OTT must use preferential animal models with 'dual-purpose and dual-benefit' that are able to improve clinical trials for human reproductive medicine (Langbeen et al 2016). In this regard, a comprehensive recent review article reported that the mare may be an excellent model for assisted reproductive technologies in women (for review, see Benammar et al 2021). The use of an equine model for studies focusing on preantral follicles has recently been revisited (for review, see Aguiar et al 2020, Gastal et al 2020)

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