Recent advancements in cancer therapy have significantly improved survival rates, but fertility dysfunction after therapy is common due to negative side effects. Concerning women and cancer therapy, even low doses of radiation significantly reduce the number of primordial follicles [
1,
2]. In addition, the ovaries are particularly sensitive to alkylating cytotoxic drugs [
3], and chemotherapies have been associated with vascular damage and ovarian cortical fibrosis [
4,
5]. Hence, these gonadotoxic effects can lead to premature ovarian failure (POF), with accompanying early menopause and infertility. It is therefore important to consider quality-of-life after treatment, including fertility preservation [
6,
7]. Current fertility preservation methods for cancer patients include embryo/oocyte vitrification, ovarian transposition and ovarian cortex transplantation [
8‐
10]. Ovarian cortex transplantation is an effective method of fertility preservation and can potentially restore fertility in most female cancer survivors [
11].
However, fertility is particularly difficult to restore in young females with hematopoietic cancer types, such as leukemia, since ovarian cortex transplantation is related to a very high risk of re-introduction of malignant cells that may be spread in the ovarian tissue, particularly in connection with the microvascularity [
12,
13]. For these reasons, multiple groups investigated if isolated preantral follicles can be stimulated to growth in vitro with the aim to develop techniques to preserve fertility [
14‐
17]. Small, preantral follicles can be isolated from ovarian cortical tissue and these follicles do not include any blood cells and are thus free from any malignant cells. Follicles may therefore be considered safe to transplant back to the patient after cancer treatment. However, the follicles cannot survive and mature without an appropriate environment that supports follicular growth. Therefore, it has been proposed to use a biomaterial as supporting material that facilitates normal follicular maturation [
18‐
21]. Using ovarian scaffolds derived from fibrin- and/or alginate matrices, or three-dimensional (3D) printed structures of cross-linked gelatine, several groups successfully developed applications for rodents that supported folliculogenesis and the development of viable oocytes and births of healthy offspring [
22‐
26]. These reports serve as proof of concept that artificial ovarian tissue can support folliculogenesis in small mammals. However, the extraordinary follicular growth in larger mammals makes it more challenging, and current ovarian scaffolds are insufficient [
27]. Scaffolds derived from tissue-specific extracellular matrix (ECM) obtained by a concept known as decellularization received much attention in regenerative medicine and provided encouraging results for various organ/tissue reconstruction applications, including for uterine tissue [
28]. Tissue-specific ECM-derived 3D-scaffolds have shown to influence mitogenesis, chemotaxis and to induce constructive host tissue remodelling and differentiation of endogenous stem cells [
29,
30]. To our knowledge, there are only a few recent publications that explored decellularized tissues for ovarian bioengineering applications. For example, sliced bovine-, pig and human ovarian tissue have been decellularized and assessed for supporting structure for mixed primary ovarian cells [
31‐
34]. The potential of this application was exemplified by restoring the hormonal function and initiating puberty in ovariectomized mice [
31]. Furthermore, decellularized human skin was used in an attempt to improve graft vascularization and minimize the initial ischemic injury in two patients who underwent ovarian tissue transplantation [
35]. Even if a healthy baby was born from this procedure, the true benefit of the scaffold was not verified. However, extracellular-rich scaffolds with Matrigel-alginate proved more favourable compared to fibrin-alginate scaffolds [
36]. Collectively, these findings suggest that ECM scaffolds may be a good approach for ovarian tissue engineering. Various decellularization methods affect the recellularization ability, and consequently, the functionality of the constructed grafts. Sodium dodecyl sulfate (SDS) is commonly used as an effective decellularization reagent for many tissues and was mostly applied on ovarian tissue in earlier studies [
31,
32,
34]. However, this chemical seems to compromise recellularization efficiency in several tissues [
37,
38]. Moreover, using whole ovarian scaffolds instead of sliced structures may improve outcomes since it better mimics the natural 3D-structure and may allow a graft vascular anastomoses in future transplantation experiments that would reduce the initial ischemic injury. Therefore, the current study aimed to evaluate different decellularization protocols for whole mouse ovaries that later can be used as supporting structures for folliculogenesis in the rodent model, before moving towards studies in larger mammals.