Further insights into the impact of mouse follicle stage on graft outcome in an artificial ovary environment.
Are mouse preantral follicles differently affected by isolation, encapsulation and/or grafting procedures according to stage?



Isolated secondary follicles showed superior ability to survive and grow after transplantation, which was not related to a particular effect of the isolation and/or grafting procedure, but rather to their own ability to induce neoangiogenesis.



Isolated and encapsulated mouse preantral follicles can survive (6-27%) and grow (80-100%) in a fibrin matrix with a low concentration of fibrinogen and thrombin (F12.5/T1) after short-term transplantation.



An in vivo experimental model using 20 donor Naval Medical Research Institute (NMRI) mice (6-25 weeks of age) and 14 recipient severe combined immunodeficient (SCID) mice (11-39 weeks of age) was applied. Each NMRI mouse underwent mechanical disruption of both ovaries and isolation of primordial-primary and secondary follicles with ovarian stromal cells, in order to encapsulate them in an F12.5/T1 matrix. Twelve out of 40 fibrin clots were immediately fixed as controls (D0) (10 for histology and 2 for scanning electron microscopy [SEM]) and the others (n = 28) were grafted to the inner part of the peritoneum for 2 (16 fibrin clots) or 7 (12 fibrin clots) days (D2 and D7).



This study involved the participation of the Gynecology Research Unit (Universitè Catholique de Louvain) and the Physiological Sciences Department (University of Brasília). Specific techniques were used to analyze the follicle recovery rate (hematoxylin-eosin staining), vascularization (CD34) and follicle ultrastructure (transmission electron microscopy [TEM] and SEM).



After follicle isolation and encapsulation, a statistically higher percentage of normal follicles was observed in the secondary group (62%) than in the primordial-primary group (47%). Follicle recovery rates were 34% and 62% for primordial-primary and secondary follicles on D2, respectively, and 12% and 42% on D7, confirming that secondary follicles survive better than primordial-primary follicles after grafting. Concerning vascularization, both follicle stages exhibited similar vascularization to that seen in control mouse ovary on D7, but a significantly higher number of vessels and greater vessel surface area were detected in the secondary follicle group. Despite structural differences in fiber density between fibrin clots and ovarian tissue observed by SEM and TEM, preantral follicles appeared to be well encapsulated in the matrix, also showing a normal ultrastructure after grafting.



Not applicable.



As demonstrated by our results during the isolation procedure, we encapsulated a significantly higher number of round structures in the primordial-primary group than in the secondary group, which could partially explain the lower recovery rate of early-stage follicles in our previous study. However, it is not excluded that the physical and mechanical properties of the fibrin matrix may also play a role in follicle survival and growth, so further investigations are needed.



This research represents one more key step in the creation of the artificial ovary.



This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (FNRS) to C.A. Amorim as a research associate at FRS-FNRS and (grant 5/4/150/5 awarded to M.M. Dolmans), Fonds Spéciaux de Recherche, Fondation St Luc, Foundation Against Cancer, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES-Brazil) (grant #013/14 CAPES/WBI awarded to C.M. Lucci, with F. Paulini receiving a post-doctoral fellowship), and Wallonie-Bruxelles International, and donations from the Ferrero family. None of the authors have any competing interests to declare in relation to the topic.
Pôle de Recherche en Gynécologie, Institut de Recherche Expérimentale et Clinique, Université Catholique de Louvain, Avenue Mounier 52, bte. B1.52.02, 1200 Brussels, Belgium.
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MC
Chiti
Pôle de Recherche en Gynécologie, Institut de Recherche Expérimentale et Clinique, Université Catholique de Louvain, Avenue Mounier 52, bte. B1.52.02, 1200 Brussels, Belgium.|Gynecology Department, Cliniques Universitaires Saint-Luc, 1200, Brussels, Belgium.
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Dolmans
Physiological Sciences Department, Institute of Biological Sciences, University of Brasília, Brasília, DFBrazil.
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Lucci
Pôle de Recherche en Gynécologie, Institut de Recherche Expérimentale et Clinique, Université Catholique de Louvain, Avenue Mounier 52, bte. B1.52.02, 1200 Brussels, Belgium.
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Paulini
Society for Research into Infertility, 1200 Brussels, Belgium.
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Donnez
Pôle de Recherche en Gynécologie, Institut de Recherche Expérimentale et Clinique, Université Catholique de Louvain, Avenue Mounier 52, bte. B1.52.02, 1200 Brussels, Belgium.
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Amorim
D000818:Animals; D018929:Cell Culture Techniques; D018914:Cells, Immobilized / Q000166:cytology / Q000502:physiology / Q000637:transplantation*; D002828:Choristoma; D005260:Female; D005337:Fibrin / Q000737:chemistry; D005340:Fibrinogen / Q000737:chemistry; D006085:Graft Survival / Q000502:physiology*; D006801:Humans; D051379:Mice; D016513:Mice, SCID; D018919:Neovascularization, Physiologic; D006080:Ovarian Follicle / Q000166:cytology / Q000502:physiology / Q000637:transplantation*; D010537:Peritoneum; D017154:Stromal Cells / Q000166:cytology / Q000502:physiology / Q000637:transplantation; D013917:Thrombin / Q000737:chemistry; D014184:Transplantation, Homologous
D005337:Fibrin; D005340:Fibrinogen; D013917:Thrombin
artificial ovary; fibrin matrix; preantral follicles; ultrastructure; vascularization