Ovarian Grafts 10 Days after Xenotransplantation: Folliculogenesis and Recovery of Viable Oocytes

Abstract

Ovarian xenotransplantation is a promising alternative to preserve fertility of oncologic patients. However, several functional aspects of this procedure remained to be addressed. The aim of this study was evaluate the feasibility of xenotransplantation as a strategy to maintain bovine ovarian grafts and produce oocytes. Adult ovarian cortical pieces were xenotransplanted to the dorsal subcutaneous of female NOD-SCID mice (n = 62). Grafts were recovered ten days after xenotransplantation. Host and graft weights; folliculogenesis progression; blood perfusion, relative gene expression and number of macrophage and neutrophil of xenografts; in vitro developmental competence of graft-derived oocytes were evaluated. Folliculogenesis was supported in the grafts, as indicated by the presence of primordial, primary, secondary, antral, and atretic follicles. The xenografts showed a greater volumetric density of atretic follicles and higher hyperemia and number of host-derived macrophage and neutrophil (P<0.05), when compared to non-grafted fragments. There was a higher blood perfusion under the back skin in the transplantation sites of host animals than in control and non-grafted (P<0.01). BAX and PRDX1 genes were up-regulated, while BCL2, FSHR, IGF1R and IGF2R were down-regulated, when compared to the control (P<0.01). Twenty seven oocytes were successfully harvested from grafts, and some of these oocytes were able to give rise to blastocysts after in vitro fertilization. However, cleavage and blastocyst rates of xenograft derived oocytes were lower than in control (P<0.01). Despite showing some functional modifications, the ovarian xenografts were able to support folliculogenesis and produce functional oocytes.

Fig 1. Biometric data of the mice and grafts.

(a) There was no difference between host mice body weights (g) before and after xenotransplantation. However, (b) the graft weight (mg) 10 days after xenotransplantation was greater (P<0.01) than before.

Fig 2. Progression of folliculogenesis, morphometry and inflammatory cell numbers in xenografted and in control ovarian fragments.

(a) Primordial, (b) primary, (c) secondary, (d) antral and (e) atretic follicles were noted 10 days after xenotransplantation. (g) Volumetric density (%) of ovarian pieces before and after xenotransplantation showing a higher incidence of follicular atresia and hyperemia (f; P<0.01) after this procedure. (h, i) As demonstrated using (h) NAG and (i) MPO assays in xenografted ovarian wet tissue, higher concentration of host-derived macrophages and neutrophils were observed (P<0.01). Black arrows = healthy follicles; red arrows = atretic follicles; white arrows = blood vessels; black arrowhead = hyperemia; asterisks = oocytes. Bars in a, b, c and e = 80 μm and d and f = 150 μm.

Fig 3. Blood perfusion and gene expression pattern in ovarian xenografts.

(a, b) The back skin of xenotransplanted animals showed higher blood perfusion, when compared to non-grafted and control animals (P<0.01). (c) Furthermore, the analysis of gene expression, using real time PCR, showed that grafting entails in up-regulation (P<0.01) of genes related to apoptosis (BAX) and oxidative stress (PRDX1) whereas the genes related to follicle survival (BCL2) and progression of folliculogenesis (FSH-r, IGF1-r and IGF2-r) were down-regulated (P<0.01).

Fig 4

(a) Oocytes recovered from xenografts 10 days after transplantation. (b) Percentage of grade I, II and III oocytes derived from xenografts. (c) Cleavage and (d) blastocyst rates from control and xenograft groups. (e) Blastocysts (arrow), and early blastocyst (produced after in vitro fertilization of xenografted-derived oocytes. Bars in a = 200 μm and e = 800 μm.