FSH联合S1P干预小鼠卵巢玻璃化冻融过程对卵巢移植后生育功能的影响及相关机制

Emerging evidence has suggested that low oocyte quality post-cryopreservation ovary transplantation and abnormal oocyte increase are responsible for the low fertilization rate and low pregnancy rate during cryopreservation ovarian transplantation. Current research has focused on using Sphingosine 1-phosphase (S1P) to improve the quality of oocyte and cryopreserved ovarian activity. However, short bioavailable half-life of S1P remains the bottleneck for using it in ovary transplantation. Some studies have reported FSH can regulate the production of S1P, Our recent study has observed that FSH intervention (0.3 IU/ml) can improve mice fertility during cryopreserved ovary transplantation and S1P reduce the apoptosis. We speculated that FSH/S1P combinatorial intervention of ovarian vitrification cryopreservation on transplantation maintain high ratio of S1P/Cer in thawed ovary, which is conducive to the rapid reconstruction of ovarian blood supply and the maintenance of oocyte quality after transplantation. Therefore, in this study, we will use histology, molecular biology and the ovary transplantation related technology to study the effects and molecular mechanism of FSH/S1P combinatorial intervention of ovarian vitrification cryopreservation on transplantation associated reproductive functions in mice . Our study will provide the scientific rationale of studying the function and mechanism of FSH/S1P in the process of vitrified cryopreservation and ovarian transplantation, thus providing a new method for the clinical application of vitrified cryopreservation of ovary and transplantation .

最新研究表明玻璃化冻融卵巢移植后卵子质量低下及异常卵子增多是导致受精率和妊娠率低下的主要原因。1-磷酸鞘氨醇(S1P)提升卵母细胞质量及冻存卵巢活性已受到关注，而较短的生物半衰期成为其发挥移植后作用的瓶颈。文献报道FSH可调节S1P的生成，我们前期研究发现0.3 IU/mL FSH干预小鼠卵巢玻璃化冻融过程可提高移植后生育力，2μM S1P干预可提高正常卵泡比例，降低凋亡。我们推测卵巢冻融时适宜的FSH联合S1P干预会使解冻后卵巢维持较高的S1P/Cer比值，利于移植后卵巢血供的快速重建及卵子质量的维护。本研究将应用组织学、分子生物学及卵巢移植等方法探究卵巢玻璃化冻存过程中FSH联合S1P干预对冻存卵巢移植后生育力的保护效果及是否有调节S1P/Cer比值的相关机制，为揭示FSH和S1P在卵巢玻璃化冻存及移植过程中的作用及机制提供新的科学依据，为临床有效的卵巢玻璃化冻存及移植提供新的方法。

卵巢玻璃化冷冻及移植技术是青年癌症患者保存生育力的主要方法，但玻璃化冻融卵巢移植后卵子质量低下及异常卵子增多是导致受精率和妊娠率低下的主要原因。我们前期研究发现0.3 IU/mL FSH干预小鼠卵巢玻璃化冻融过程可提高移植后生育力，2μM S1P干预可提高正常卵泡比例，降低凋亡。但S1P较短的生物半衰期成为其发挥移植后作用的瓶颈。本项目应用组织学、分子生物学及卵巢移植等方法探索FSH联合S1P干预在卵巢玻璃化冻存移植过程中促卵泡存活及血管生成的作用及移植后对生育力的影响，探讨其对卵泡凋亡及血管形成相关信号分子的调控，以揭示FSH 联合S1P在玻璃化冻存及移植过程中对卵巢功能的保护作用机制。研究发现，0.3IU/ml FSH+2μM S1P联合干预冻存卵巢，可较好的保护冷冻卵巢的原始卵泡，提升PCNA的表达，提高Bcl-2/Bax 比值，抑制玻璃化冻存引起的卵巢细胞凋亡，促进卵泡发育和卵巢细胞增殖；FSH联合S1P干预能较好地保留移植后24h卵巢中的原始卵泡池，抵抗移植后缺血再灌注引起的凋亡损伤。移植后7天，闭锁卵泡比例显著降低，次级卵泡的比例较高，显示较好的卵泡质量。其作用通过增加Cx37和Cx43的表达促进移植卵巢中卵泡的存活，增加VEGF、CD31表达促进移植卵巢的血运重建，增加移植物中血管密度，抑制卵泡闭锁；FSH联合S1P干预可促进移植后卵巢E2和P4的分泌，维持其生殖内分泌功能；FSH联合S1P干预可上调卵巢中pSK-1和UGCG的表达，促进内源性S1P生成，降低Cer诱导的细胞凋亡。其作用可能是通过上调PI3K-AKT-mTOR信号通路及其下游蛋白HIF-1α、VEGFA的表达而实现。FSH 与 S1P 的联合干预可发挥更为有效的抗凋亡作用，有助于移植卵巢血供的快速重建及卵子质量的维护。研究为临床冻存卵巢自体移植后提高妊娠率提供新的手段及科学依据。