Effects of chemical in vitro activation versus fragmentation on human ovarian tissue and follicle growth in culture.

IF 8.3 Q1 OBSTETRICS & GYNECOLOGY Human reproduction open Pub Date : 2024-05-22 eCollection Date: 2024-01-01 DOI:10.1093/hropen/hoae028
Jie Hao, Tianyi Li, Manuel Heinzelmann, Elisabeth Moussaud-Lamodière, Filipa Lebre, Kaarel Krjutškov, Anastasios Damdimopoulos, Catarina Arnelo, Karin Pettersson, Ernesto Alfaro-Moreno, Cecilia Lindskog, Majorie van Duursen, Pauliina Damdimopoulou
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However, comparable effects with mere tissue fragmentation have been shown, questioning the added value of chemical stimulation that could potentially activate oncogenic responses.</p><p><strong>Study design size duration: </strong>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.</p><p><strong>Participants/materials setting methods: </strong>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.</p><p><strong>Main results and the role of chance: </strong>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.</p><p><strong>Large scale data: </strong>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.</p><p><strong>Limitations reasons for caution: </strong>Similar to the published IVA protocols, the first steps in our study were performed in an <i>in vitro</i> culture model where the ovarian tissue was isolated from the regulation of hypothalamic-pituitary-ovarian axis. Further <i>in vivo</i> 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.</p><p><strong>Wider implications of the findings: </strong>The general impact of fragmentation and short (24 h) <i>in vitro</i> 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.</p><p><strong>Study funding/competing interests: </strong>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.</p>","PeriodicalId":73264,"journal":{"name":"Human reproduction open","volume":null,"pages":null},"PeriodicalIF":8.3000,"publicationDate":"2024-05-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11128059/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Human reproduction open","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1093/hropen/hoae028","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2024/1/1 0:00:00","PubModel":"eCollection","JCR":"Q1","JCRName":"OBSTETRICS & GYNECOLOGY","Score":null,"Total":0}
引用次数: 0

Abstract

Study question: 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?

Summary answer: 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.

What is known already: 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.

Study design size duration: 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.

Participants/materials setting 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.

Main results and the role of chance: 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.

Large scale data: 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.

Limitations reasons for caution: 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.

Wider implications of the findings: 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.

Study funding/competing interests: 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.

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化学体外活化与破碎对培养中的人类卵巢组织和卵泡生长的影响。
研究问题化学体外活化(cIVA)方案与仅破碎(Frag,也称为机械体外活化)方案相比,对体外人类卵巢组织的基因表达、卵泡活化和生长有何影响?尽管组织学评估显示,与 Frag 相比,cIVA 能显著提高卵泡的存活率和生长率,但与新鲜采集的卵巢组织相比,两种方案都能刺激培养组织发生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明显变化:卵巢组织中磷酸酶和天丝蛋白同源物(PTEN)-磷脂酰肌醇 3-激酶(PI3K)通路的 cIVA 为基础,然后进行自身移植,这种治疗方法已用于难治性卵巢早衰(POI)患者,并获得了活产。然而,仅对组织进行破碎就能产生类似的效果,这就质疑了化学刺激的附加价值,因为化学刺激可能会激活致癌反应:59例卵巢皮质活检取自同意接受选择性剖腹产(C-section)的妇女。这些样本被切碎用于培养研究。一半的片段在培养的头 24 小时暴露于 bpV(HOpic)+740Y-P(Frag+cIVA 组),另一半只用培养基培养(Frag 组)。随后,两组都只用培养基再培养 6 天。在不同的时间点收集组织和培养基样本进行组织学、转录组学、类固醇激素和细胞因子/趋化因子分析:通过在 7 天培养前后对经苏木精和伊红染色的连续切片进行计数和评分,评估对卵泡的影响。在不同的时间点,通过超高效液相色谱串联质谱法对类固醇进行定量,从而评估卵泡功能。细胞因子和趋化因子通过多重检测法进行测定。在最初的 24 小时培养后,通过对组织进行 RNA 序列分析(RNA-seq)来测量转录组的影响。在卵巢组织培养和 KGN 细胞(人类卵巢肉芽肿样肿瘤细胞系)培养实验中,通过定量 PCR 和免疫荧光验证了部分差异表达基因(DEGs):与Frag组相比,Frag+cIVA组的卵泡存活率明显提高,次级卵泡数量增加,卵泡体积增大。此外,与 Frag 组相比,Frag+cIVA 组组织产生的脱氢表雄酮更少。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组与Frag组之间的差异不大,采用宽松的错误发现率(FDR)截断值,仅发现了164个DEGs:数据已存入 GEO 数据库,登录号为 GSE234765。测序分析代码见 https://github.com/tialiv/IVA_project.Limitations 注意事项:与已发表的 IVA 方案类似,我们研究的第一步也是在体外培养模型中进行的,即从下丘脑-垂体-卵巢轴调节中分离出卵巢组织。还需要进一步的体内实验,例如在异种移植模型中进行实验,以探索所发现的效应的长期影响。从剖腹产患者身上采集的组织可能无法与 POI 患者的组织相比较:破碎和短期(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刺激了卵泡的生长,这可能表明cIVA对特定细胞群产生了影响,而这些影响在大量RNA-seq中可能会被稀释。不过,我们利用细胞培养模型证实了 cIVA 对糖酵解的影响,这表明它对细胞信号的影响超出了 PI3K 通路。碎裂和培养后炎症和糖酵解的深刻变化可能会导致卵巢组织培养中的卵泡活化和丢失,以及临床应用中的卵泡活化和丢失,例如通过卵巢组织自身移植来保留生育能力:本研究得到了欧盟 "地平线2020 "研究与创新计划(ERIN项目编号:952516,FREIA项目编号:825100)、瑞典研究理事会VR(2020-02132)、卡罗林斯卡医学院StratRegen基金、国家留学基金委(CSC)项目和湖南省自然科学基金(2022JJ40782)的资助。 国际伊比利亚纳米技术实验室研究由欧盟 H2020 项目 Sinfonia (857253) 和 SbDToolBox (NORTE-01-0145-FEDER-000047)资助,由葡萄牙北部地区业务计划 (NORTE 2020)根据葡萄牙 2020 伙伴关系协议通过欧洲地区发展基金提供支持。未声明任何利益冲突。
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