多囊卵巢综合征患者卵泡液的拉曼光谱分析及其对小鼠卵母细胞体外成熟的影响

Raman spectroscopy analysis of follicular fluid from patients with polycystic ovary syndrome and its effect on in vitro maturation of mouse oocytes

目的探究多囊卵巢综合征(polycystic ovary syndrome,PCOS)患者与非PCOS患者卵泡液中的差异性代谢物及其对小鼠卵母细胞体外成熟(in vitro maturation,IVM)和体外受精(in vitro fertilization,IVF)胚胎发育的影响。方法本研究的临床资料采取回顾性队列研究的研究方法获取;动物实验采取随机对照试验。收集2019年6月至2020年6月期间在上海集爱遗传与不育诊疗中心第一次接受IVF或卵胞质内单精子注射(intracytoplasmic sperm injection,ICSI)周期的女性患者的卵泡液。根据是否为PCOS患者,分为PCOS组(n=71)和非PCOS组(n=70),回顾性分析两组患者的临床资料,并利用深度学习的拉曼光谱分析技术检测两组患者卵泡液的代谢谱差异。后续进行实验性研究,在PCOS患者和非PCOS患者卵泡液中进行小鼠GV期卵母细胞IVM培养,收集两组成熟的鼠卵母细胞进行IVF,进一步探讨卵泡液中差异性代谢物对鼠卵成熟及胚胎发育的影响。结果PCOS组患者卵母细胞成熟率[82.19%(886/1078)]和受精第3天的有效胚胎率[51.30%(553/1078)]显著低于非PCOS组[85.85%(625/728),P=0.038;53.30%(388/728),P=0.042],两组患者的累积临床妊娠率和累积活产率差异均无统计学意义(均P>0.05)。PCOS患者和非PCOS患者卵泡液之间存在差异性拉曼代谢光谱。两组间特征性拉曼位移主要集中在600~1000 cm^(-1)、1168 cm^(-1)、1344 cm^(-1)、1440 cm^(-1)、1504 cm^(-1)、1632 cm^(-1)、1664 cm^(-1)。拉曼特征位移数据库显示,两组卵泡液样本的差异代谢物主要集中蛋白质、脂质、游离核酸、葡萄糖、胆固醇、类胡萝卜素和氨基酸等物质。利用两组卵泡液行鼠卵IVM,PCOS卵泡液组的鼠卵MⅡ率[49.04%(77/157)]较非PCOS组更低[65.07%(95/146),P=0.005],利用小鼠MⅡ卵进行IVF,PCOS卵泡液组卵裂率[46.75%(36/77)]显著低于非PCOS组[63.16%(60/95),P=0.031],而两组囊胚率差异无统计学意义(P>0.05)。结论PCOS患者卵泡液存在差异性拉曼代谢光谱,其差异性代谢物可能导致卵母细胞成熟障碍,并进一步影响IVF胚胎的发育,拉曼光谱为PCOS诊断和代谢组学的差异物分析提供了简便有效的方法。

Objective To explore the different metabolites in the follicular fluids(FFs)of polycystic ovary syndrome(PCOS)patients and non-PCOS patients and their effects on the maturation of mouse oocytes and the developmental potential of in vitro fertilization(IVF)embryos.Methods The clinical data were collected for the retrospective cohort study.Animal experiments were conducted in a randomized controlled trial.This study included PCOS(n=71)and non-PCOS(n=70)patients who underwent the first IVF or intracytoplasmic sperm injection(ICSI)cycle in Shanghai JIAI Genetics&IVF institute from June 2019 to June 2020.The patients'FFs were collected and the clinical data from these patients were analyzed.Raman spectroscopy analysis technology was used to detect differences in the metabolic spectra of FFs between the two groups.Mouse GV phase oocytes were placed in FFs from PCOS patients and non-PCOS patients for in vitro maturation(IVM)culture respectively,then the matured mouse oocytes were collected for IVF.The effects of differential metabolites in FFs on mouse oocyte maturation and embryonic development were further explored.The Raman spectrum was also applied to identify the differences of the IVM spent culture media.Results The MⅡrate[82.19%(886/1078)]and day 3 available embryo rate[51.30%(553/1078)]from PCOS group were significantly lower than those of the non-PCOS group[85.85%(625/728),P=0.038;53.30%(388/728),P=0.042].However,there were no significant differences between the two groups in the cumulative clinical pregnancy rate and the cumulative live birth rate(all P>0.05).Raman was capable of distinguishing PCOS from non-PCOS FFs.The characteristic Raman displacement difference between the two groups is mainly concentrated in the 600-1000 cm^(-1),as well as 1168 cm^(-1),1344 cm^(-1),1440 cm^(-1),1504 cm^(-1),1632 cm^(-1) and 1664 cm^(-1).The Raman characteristic shift database showed that the different metabolites of the two sets of FFs samples were mainly concentrated in protein,lipids,free nucleic acis,glucose,cholesterol,carotenoids,and amino acids.Mouse oocyte IVM results showed that the PCOS-FF group had a lower MⅡrate[49.04%(77/157)]than that of non-PCOS group[65.07%(95/146),P=0.005).IVF results showed the PCOS-FF group had a significantly lower cleavage rate[46.75%(36/77)]than that of non-PCOS group[63.16%(60/95),P=0.031],but there was no significant difference in the blastocyst rate between the two groups(P>0.05).Conclusion Differential metabolites detected by Raman spectrum in the PCOS FFs may cause defected maturation of the oocytes,leading to infertility,and Raman spectroscopy is an effective approach towards PCOS diagnosis and the identification of metabolomics differences.