目的:与仅片段化相比,化学体外激活(cIVA)方案的作用是什么(Frag,也称为机械IVA)对基因表达的影响,
结论:尽管组织学评估显示cIVA与Frag相比显著增加了卵泡的存活和生长,与新鲜收集的卵巢组织相比,两种方案都刺激培养组织中广泛且几乎相同的转录组变化,包括能量代谢和炎症反应的显著变化。
背景:已对患有难治性早熟卵巢功能不全(POI)的患者进行了基于卵巢组织中磷酸酶和张力蛋白同源物(PTEN)-磷脂酰肌醇3-激酶(PI3K)途径的cIVA治疗,然后进行自体移植。然而,已经显示出与单纯的组织破碎相当的效果,质疑可能激活致癌反应的化学刺激的附加值。
■59例卵巢皮质活检来自接受选择性剖腹产(剖腹产)的妇女。将样品片段化以用于培养研究。在培养的最初24小时内,一半的片段暴露于bpV(HOpic)740Y-P(FragcIVA组),而另一半仅在培养基中培养(Frag组)。随后,两组均仅用培养基再培养6天.收集组织和培养基样品用于组织学,转录组,类固醇激素,和不同时间点的细胞因子/趋化因子分析。
方法:在培养7天之前和之后,通过计数和评分用苏木精和伊红染色的连续切片来评估对卵泡的影响。通过超高效液相色谱串联质谱法在不同时间点对类固醇进行定量来评估卵泡功能。通过多重分析测量细胞因子和趋化因子。在初始24小时培养后,通过组织的RNA测序(RNA-seq)测量转录组效应。通过定量PCR和免疫荧光在培养的卵巢组织以及KGN细胞(人卵巢颗粒样肿瘤细胞系)培养实验中验证了选定的差异表达基因(DEGs)。
结果:与Frag组相比,Frag+cIVA组表现出明显更高的卵泡存活率,次级卵泡数量增加,和更大的卵泡大小。此外,与Frag相比,Frag+cIVA组的组织产生的脱氢表雄酮较少。细胞因子测量显示两组在培养开始时都有强烈的炎症反应。RNA-seq数据显示Frag+cIVA和Frag组之间存在适度差异,只有164个DEG使用错误发现率(FDR)<0.1的宽松截止值识别。除了预期的PI3K蛋白激酶B(Akt)途径,cIVA还调节与缺氧相关的途径,细胞因子,和炎症。与新鲜收集的卵巢组织相比,基因表达在Frag+cIVA和Frag组中都受到显著影响,总共确定了3119和2900个DEG(FDR<0.001),分别。两组中最高富集的基因集包括几种已知调节卵泡生长的途径,例如哺乳动物雷帕霉素靶蛋白(mTOR)C1信号传导。与新鲜组织相比,两组中类固醇生成酶和经典颗粒细胞标记的编码基因的表达也发生了显着变化。有趣的是,我们在Frag和Frag+cIVA组中发现了与糖酵解及其上游调节因子相关的基因的深刻上调,cIVA治疗进一步促进了这些变化。细胞培养实验证实糖酵解相关基因是cIVA药物的直接靶标。总之,cIVA促进卵泡生长,正如预期的那样,但机制可能比单独的PI3K-Akt-mTOR更复杂,培养期后对卵泡功能和质量的影响仍然是一个悬而未决的问题。
方法:数据存储在GEO数据库中,登录号GSE234765。测序分析的代码可以在https://github.com/tialiv/IVA_project中找到。
结论:与已发布的IVA方案类似,我们研究的第一步是在体外培养模型中进行的,其中卵巢组织从下丘脑-垂体-卵巢轴的调节中分离.需要进一步的体内实验,例如在异种移植模型中,探索发现的影响的长期影响。从经历剖腹产的患者收集的组织可能与患有POI的患者的组织不可比。
结论:片段化和短(24小时)体外培养对卵巢组织基因表达的总体影响远远超过了cIVA的影响。然而,cIVA刺激了卵泡生长,这可能表明对可能稀释在大量RNA-seq中的特定细胞群体的影响。然而,我们使用细胞培养模型证实了cIVA对糖酵解的影响,表明对PI3K通路以外的细胞信号传导的影响。分裂和培养后炎症和糖酵解的深刻变化可能导致卵巢组织培养中的卵泡活化和丧失。以及在临床应用中,如通过卵巢组织自体移植保存生育力。
背景:这项研究由欧盟的“地平线2020”研究与创新计划(项目ERIN号。952516,FREIA编号825100),瑞典研究委员会VR(2020-02132),卡罗林斯卡学院的StratRegen资助,KI-中国奖学金理事会(CSC)计划和湖南省自然科学基金(2022JJ40782)。国际伊比利亚纳米技术实验室研究由欧盟的H2020项目Sinfonia(857253)和SbDToolBox(NORTE-01-0145-FEDER-000047)资助,北葡萄牙区域业务计划(NORTE2020)支持,根据葡萄牙2020年合伙协议,欧洲区域发展基金。没有竞争的利益被宣布。
OBJECTIVE: What is the effect of the chemical in vitro activation (cIVA) protocol compared with fragmentation only (Frag, also known as mechanical IVA) on gene expression, follicle activation and growth in human ovarian tissue in vitro?
CONCLUSIONS: Although histological assessment shows that cIVA significantly increases follicle survival and growth compared to Frag, both protocols stimulate extensive and nearly identical transcriptomic changes in cultured tissue compared to freshly collected ovarian tissue, including marked changes in energy metabolism and inflammatory responses.
BACKGROUND: Treatments based on cIVA of the phosphatase and tensin homolog (PTEN)-phosphatidylinositol 3-kinase (PI3K) pathway in ovarian tissue followed by auto-transplantation have been administered to patients with refractory premature ovarian insufficiency (POI) and resulted in live births. However, comparable effects with mere tissue fragmentation have been shown, questioning the added value of chemical stimulation that could potentially activate oncogenic responses.
UNASSIGNED: Fifty-nine ovarian cortical biopsies were obtained from consenting women undergoing elective caesarean section (C-section). The samples were fragmented for culture studies. Half of the fragments were exposed to bpV (HOpic)+740Y-P (Frag+cIVA group) during the first 24 h of culture, while the other half were cultured with medium only (Frag group). Subsequently, both groups were cultured with medium only for an additional 6 days. Tissue and media samples were collected for histological, transcriptomic, steroid hormone, and cytokine/chemokine analyses at various time points.
METHODS: Effects on follicles were evaluated by counting and scoring serial sections stained with hematoxylin and eosin before and after the 7-day culture. Follicle function was assessed by quantification of steroids by ultra-performance liquid chromatography tandem-mass spectrometry at different time points. Cytokines and chemokines were measured by multiplex assay. Transcriptomic effects were measured by RNA-sequencing (RNA-seq) of the tissue after the initial 24-h culture. Selected differentially expressed genes (DEGs) were validated by quantitative PCR and immunofluorescence in cultured ovarian tissue as well as in KGN cell (human ovarian granulosa-like tumor cell line) culture experiments.
RESULTS: Compared to the Frag group, the Frag+cIVA group exhibited a significantly higher follicle survival rate, increased numbers of secondary follicles, and larger follicle sizes. Additionally, the tissue in the Frag+cIVA group produced less dehydroepiandrosterone compared to Frag. Cytokine measurement showed a strong inflammatory response at the start of the culture in both groups. The RNA-seq data revealed modest differences between the Frag+cIVA and Frag groups, with only 164 DEGs identified using a relaxed cut-off of false discovery rate (FDR) <0.1. Apart from the expected PI3K-protein kinase B (Akt) pathway, cIVA also regulated pathways related to hypoxia, cytokines, and inflammation. In comparison to freshly collected ovarian tissue, gene expression in general was markedly affected in both the Frag+cIVA and Frag groups, with a total of 3119 and 2900 DEGs identified (FDR < 0.001), respectively. The top enriched gene sets in both groups included several pathways known to modulate follicle growth such as mammalian target of rapamycin (mTOR)C1 signaling. Significant changes compared to fresh tissue were also observed in the expression of genes encoding for steroidogenesis enzymes and classical granulosa cell markers in both groups. Intriguingly, we discovered a profound upregulation of genes related to glycolysis and its upstream regulator in both Frag and Frag+cIVA groups, and these changes were further boosted by the cIVA treatment. Cell culture experiments confirmed glycolysis-related genes as direct targets of the cIVA drugs. In conclusion, cIVA enhances follicle growth, as expected, but the mechanisms may be more complex than PI3K-Akt-mTOR alone, and the impact on function and quality of the follicles after the culture period remains an open question.
METHODS: Data were deposited in the GEO data base, accession number GSE234765. The code for sequencing analysis can be found in https://github.com/tialiv/IVA_project.
CONCLUSIONS: Similar to the published IVA protocols, the first steps in our study were performed in an in vitro culture model where the ovarian tissue was isolated from the regulation of hypothalamic-pituitary-ovarian axis. Further in vivo experiments will be needed, for example in xeno-transplantation models, to explore the long-term impacts of the discovered effects. The tissue collected from patients undergoing C-section may not be comparable to tissue of patients with POI.
CONCLUSIONS: The general impact of fragmentation and short (24 h) in vitro culture on gene expression in ovarian tissue far exceeded the effects of cIVA. Yet, follicle growth was stimulated by cIVA, which may suggest effects on specific cell populations that may be diluted in bulk RNA-seq. Nevertheless, we confirmed the impact of cIVA on glycolysis using a cell culture model, suggesting impacts on cellular signaling beyond the PI3K pathway. The profound changes in inflammation and glycolysis following fragmentation and culture could contribute to follicle activation and loss in ovarian tissue culture, as well as in clinical applications, such as fertility preservation by ovarian tissue auto-transplantation.
BACKGROUND: This study was funded by research grants from European Union\'s Horizon 2020 Research and Innovation Programme (Project ERIN No. 952516, FREIA No. 825100), Swedish Research Council VR (2020-02132), StratRegen funding from Karolinska Institutet, KI-China Scholarship Council (CSC) Programme and the Natural Science Foundation of Hunan (2022JJ40782). International Iberian Nanotechnology Laboratory Research was funded by the European Union\'s H2020 Project Sinfonia (857253) and SbDToolBox (NORTE-01-0145-FEDER-000047), supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund. No competing interests are declared.