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.
Department of Biomedical Science for Health, Universitá degli Studi di Milano, Via Macedonio Melloni 52, 20125 Milan, Italy.
Hum Reprod. 2018 Jun 1;33(6):1107-1116. doi: 10.1093/humrep/dey080.
Do adipose tissue-derived stem cells (ASCs) enhance vascularization and follicle survival in xenografted ovarian tissue using a two-step transplantation approach?
Higher rates of oxygenation and vascularization of ovarian tissue, as well as increased follicle survival rates, were detected in the early post-grafting period.
ASCs have multilineage differentiation potential, proangiogenic properties and enhance vascularization in a peritoneal grafting site. Some studies suggest that using ASCs may improve ovarian tissue quality by enhancing graft angiogenesis.
STUDY DESIGN, SIZE, DURATION: A total of 15 severe combined immunodeficient (SCID) mice were intraperitoneally grafted with frozen-thawed human ovarian tissue (OT) from five different patients. A peritoneal transplantation site had been previously prepared in a first step using either empty fibrin (Fi+OT group [n = 5]) or ASC-loaded fibrin (Fi/ASCs+OT group [n = 5]) for 14 days prior to grafting. Five mice underwent the standard one-step transplantation procedure and served as controls (OT group). Lithium phthalocyanine (LiPc) crystals were inserted into all grafted human ovarian tissue before transplantation. Levels of partial pressure of oxygen (pO2) in grafts were monitored in vivo by electron paramagnetic resonance (EPR) oximetry on Days 3 and 7. Samples for histology and immunohistochemistry (IHC) were collected after euthanizing the mice on Day 7 following EPR. One piece of ovarian tissue per patient was fixed for analysis to serve as non-grafted controls.
PARTICIPANTS/MATERIALS, SETTING, METHODS: Prospective experimental study conducted at the Gynecology Research Unit, Université Catholique de Louvain. All materials were used to perform pO2 measurements (EPR oximetry), histological (haematoxylin and eosin staining), immunohistochemistry (anti-mouse and human double CD34 and anti-human Ki-67) and TUNEL analyses.
A significant increase in pO2 was observed in all groups between Days 3 and 7 (P < 0.001). A significantly higher pO2 level was observed in the Fi/ASCs+OT group compared to the OT group on Day 7 (P = 0.028). Total CD34-positive vessel area on Day 7 was greater in the Fi/ASCs+OT group than in any other group (vs non-grafted group: P = 0.0014; vs OT group: P = 0.013; vs Fi+OT group: P = 0.018). Primordial follicle survival rates after grafting were higher in the Fi/ASCs+OT group than in the OT (P = 0.0059) or Fi+OT groups (P = 0.0307). TUNEL-positive follicle percentages after grafting were significantly lower in the Fi/ASCs+OT group than in any other grafted tissue (vs OT group: P = 0.045; vs Fi+OT group: P = 0.0268). Percentages of Ki-67-positive primordial follicles were significantly higher in all grafted groups compared to non-grafted tissue controls (P < 0.01).
As demonstrated by our results, the proposed two-step ovarian tissue transplantation procedure using ASCs enhances vascularization in the early post-grafting period, leading to increased follicle survival rates and decreased apoptosis. However, mechanisms involved in the proangiogenic behavior of ASCs remain to be elucidated.
Our results suggest that the proposed transplantation procedure with ASCs is a promising step towards potentially solving the problem of massive follicle loss after ovarian tissue grafting.
STUDY FUNDING/COMPETING INTEREST(S): This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (FNRS-PDR Convention T.0077.14, grant Télévie No. 7.6515.16 F to DDM and grant 5/4/150/5 awarded to MMD and CAA is research associate, FRS-FNRS), Fonds Spéciaux de Recherche, Fondation St Luc, and Foundation Against Cancer, and donations from the Ferrero family.
脂肪组织源性干细胞（ASCs）通过两步移植方法增强异种移植卵巢组织中的血管生成和卵泡存活吗？
在移植后早期，卵巢组织的氧合和血管化率更高，卵泡存活率也更高。
ASCs 具有多能分化潜能、促血管生成特性，并增强腹膜移植部位的血管生成。一些研究表明，使用 ASCs 可以通过增强移植物血管生成来改善卵巢组织质量。
研究设计、大小、持续时间：总共将 15 只严重联合免疫缺陷（SCID）小鼠的卵巢组织（OT）进行了冰冻解冻，来自 5 名不同患者的卵巢组织（OT）。在移植前，先用纤维蛋白（Fi+OT 组[ n = 5]）或 ASC 负载纤维蛋白（Fi/ASCs+OT 组[ n = 5]）预先进行了 14 天的腹膜移植部位制备，然后再进行移植。5 只小鼠进行了标准的一步移植程序作为对照（OT 组）。在移植前，将锂酞菁（LiPc）晶体插入所有移植的人卵巢组织中。在移植后第 3 天和第 7 天，通过电子顺磁共振（EPR）血氧饱和度测定法在体内监测移植物中的部分压力氧（pO2）水平。在 EPR 后第 7 天处死小鼠后收集用于组织学和免疫组织化学（IHC）的样本。每位患者固定一块卵巢组织作为非移植对照。
参与者/材料、设置、方法：在天主教鲁汶大学妇科研究单位进行的前瞻性实验研究。所有材料都用于进行 pO2 测量（EPR 血氧饱和度测定法）、组织学（苏木精和伊红染色）、免疫组织化学（抗小鼠和人双 CD34 和抗人 Ki-67）和 TUNEL 分析。
在第 3 天和第 7 天之间，所有组的 pO2 均显著增加（P < 0.001）。与 OT 组相比，Fi/ASCs+OT 组在第 7 天的 pO2 水平显著升高（P = 0.028）。第 7 天 Fi/ASCs+OT 组的总 CD34 阳性血管面积大于任何其他组（与非移植组比较：P = 0.0014；与 OT 组比较：P = 0.013；与 Fi+OT 组比较：P = 0.018）。与 OT 组（P = 0.0059）或 Fi+OT 组（P = 0.0307）相比，移植后原始卵泡存活率在 Fi/ASCs+OT 组更高。与任何其他移植组织相比，移植后 Fi/ASCs+OT 组的 TUNEL 阳性卵泡百分比显著降低（与 OT 组比较：P = 0.045；与 Fi+OT 组比较：P = 0.0268）。与非移植组织对照组相比，所有移植组的 Ki-67 阳性原始卵泡百分比均显著升高（P < 0.01）。
局限性/谨慎原因：正如我们的结果所示，使用 ASC 的两步卵巢组织移植程序增强了移植后早期的血管生成，导致卵泡存活率增加和凋亡减少。然而，ASCs 促血管生成行为的机制仍有待阐明。
我们的研究结果表明，使用 ASC 的拟议移植程序是解决卵巢组织移植后大量卵泡丢失问题的有前途的一步。
研究基金/利益冲突：这项研究得到了比利时国家科学研究基金会（FNRS-PDR 公约 T.0077.14、Télévie 第 7.6515.16 号赠款给 DDM 和 5/4/150/5 号赠款给 MMD 和 CAA，后者是研究助理，FRS-FNRS）、特别研究基金、圣卢克基金会和癌症基金会的资助，以及费雷罗家族的捐款。
