Synthetic hydrogel supports the function and regeneration of artificial ovarian tissue in mice

Abstract

Many prepubertal girls and young women suffer from premature ovarian insufficiency induced by chemotherapy given for treatment of cancer and autoimmune diseases. Autotransplantation of cryopreserved ovarian tissue could restore the lost ovarian endocrine function and fertility. Unfortunately, tissue ischemia, inconsistent graft quality and the risk of reintroducing malignant cells may stand in the way of the clinical translation of this approach. To address these risks and limitations, we engineered an artificial ovarian tissue from immature follicles using a synthetic hydrogel, poly(ethylene glycol) vinyl sulfone (PEG-VS), as a supportive matrix. Enzymatically isolated follicles from 6–7-day-old mice ovaries were encapsulated in 7% PEG-VS hydrogels modified with 0.5 mmol/l RGD and crosslinked with a trifunctional matrix metalloproteinase-sensitive peptide. PEG hydrogels with the encapsulated follicles were orthotopically implanted into ovariectomised mice to investigate whether PEG hydrogel supports folliculogenesis and steroidogenesis in vivo. After 30 days, grafts revealed multiple fully developed antral follicles and corpora lutea, which corresponded with regular ovulation cycles and follicle-stimulating hormone (FSH) levels. The elevated levels of FSH, caused by bilateral ovariectomy, were reversed by the implanted follicles and maintained at physiological levels for 60 days. Importantly, primordial and primary follicles still represented 60% of the follicular pool, demonstrating selective recruitment of primordial follicles into the growing pool. Functioning blood vessels in the grafts 30 and 60 days after implantation proved the capability of PEG hydrogels to undergo graft remodelling and revascularisation. Our results demonstrate that PEG hydrogels with encapsulated immature ovarian follicles successfully functioned as an artificial ovarian tissue for 60 days in vivo.

Bioengineering anin situovary (ISO) for fertility preservation

“…Follicle incubation time from isolation to transplant is a concern for cell therapy applications as it can directly impact viability 35 . Therefore, we adapted an existing enzymatic isolation protocol 36 to reduce the time needed to obtain a large pool of immature follicles for transplant. To enable identification of transplanted versus endogenous follicles, we isolated follicles from transgenic mice exhibiting ubiquitous GFP under the chicken β-actin (CAG) promoter.…”

A new possibility in fertility preservation: The artificial ovary

“…A small volume of ovarian cortical tissue is sufficient for cryopreservation because most follicles reside in ovarian cortex rather than the medulla. However, it is reasonable to remove the entire ovary prior to gonadotoxic cancer treatment because it provides a sufficiently large pool of follicles even after follicular loss during the processing (Dolmans et al, ; J. Kim, Perez et al, ; Paulini et al, ; Smith et al, ). Therefore, if patients have high risks of losing fertility, cryopreserving one entire ovary can result in more satisfactory results.…”

“…Inflammatory responses can be evaluated through CD45 and F4/80 immunostaining (Laronda et al, ; Vanacker, Dolmans, Luyckx, Donnez, & Amorim, ). Ovarian functional recovery in mice can be validated with lowered serum follicle stimulating hormone concentration, elevated estrogen level, increased inhibin level, and pregnancy (Carroll & Gosden, ; Kim, Perez, et al, ; Kim, Park, et al, ; Y. Y. Kim, H. Kim, Ku et al, ; Laronda et al, ; Laronda et al, ; Smith et al, ).…”

“…As securing oxygen supply via the blood vessels is crucial for a successful transplantation, the level of neoangiogenesis induced in different designs should also be comparatively analysed. New vessel formation in transplantation sites can be compared based on the number of vessels per area or the vessel surface area per total surface area (Kim, Perez et al, 2016;Laronda et al, 2017;Vanacker et al, 2012). Artificial ovaries transplanted with biological scaffold that resulted in pregnancies in animal studies promoted vessel formation either when vascular endothelial growth factors (VEGFs) were added to fibrin or when the gelatin scaffolds had reliable connectivity (Kniazeva et al, 2015;Laronda et al, 2017).…”

“…Ovarian functional recovery in mice can be validated with lowered serum follicle stimulating hormone concentration, elevated estrogen level, increased inhibin level, and pregnancy (Carroll & Gosden, 1993; Kim, Perez, et al, 2016;Kim, Park, et al, 2017;Laronda et al, 2015;Laronda et al, 2017;Smith et al, 2014).…”