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Article(id=1206995860018254169, tenantId=1146029695717560320, journalId=1189873630562394117, issueId=1206995859061952854, articleNumber=null, orderNo=null, doi=10.11855/j.issn.0577-7402.2022.12.1226, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1644163200000, receivedDateStr=2022-02-07, revisedDate=null, revisedDateStr=null, acceptedDate=1657814400000, acceptedDateStr=2022-07-15, onlineDate=1765699837926, onlineDateStr=2025-12-14, pubDate=1672156800000, pubDateStr=2022-12-28, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1765699837926, onlineIssueDateStr=2025-12-14, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1765699837926, creator=13701087609, updateTime=1765699837926, updator=13701087609, issue=Issue{id=1206995859061952854, tenantId=1146029695717560320, journalId=1189873630562394117, year='2022', volume='47', issue='12', pageStart='1169', pageEnd='1270', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=0, createTime=1765699837699, creator=13701087609, updateTime=1765700204449, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1206997397385859947, tenantId=1146029695717560320, journalId=1189873630562394117, issueId=1206995859061952854, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1206997397385859948, tenantId=1146029695717560320, journalId=1189873630562394117, issueId=1206995859061952854, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=1226, endPage=1231, ext={EN=ArticleExt(id=1206995860349604191, articleId=1206995860018254169, tenantId=1146029695717560320, journalId=1189873630562394117, language=EN, title=Correlation between mitochondrial copy number in IVF embryos and clinical outcome in patients with poor ovarian response, columnId=1190310109000602400, journalTitle=Medical Journal of Chinese People’s Liberation Army, columnName=Clinical Research, runingTitle=null, highlight=null, articleAbstract=

Objective To explore the role of mitochondrial copy number (MCN) of embryos in the outcome of embryo transfer in patients with poor ovarian response (POR) and the correlation with mother's age, embryonic karyotype and blastocyst grade. Methods A retrospective analysis was performed of 75 patients with normal ovarian reserve (NOR) and 74 patients with POR who underwent in vitro fertilization (IVF) and preimplantation genetic testing for aneuploidies (PGT-A) in the Sixth Medical Center of Chinese PLA General Hospital from September 2018 to October 2020. The blastocysts obtained after IVF were biopsied(NOR group: 205; POR group: 135), and 116 and 44 euploid blastocysts were obtained, respectively. The MCN of embryos in different clinical outcomes of each group were analyzed and compared, and the correlations of embryo MCN with maternal age, embryo karyotype and blastocyst grade were analyzed by multiple linear regression analysis. Results The mean MCN of biopsy blastocysts and euploid blastocysts were lower in POR group than those in NOR group with statistically significant difference (biopsy blastocysts: 267.4±174.0 vs. 319.0±264.8, P<0.05; euploid blastocysts: 216.0±95.7 vs. 305.4±273.2, P<0.01). Regardless POR group or NOR group, the MCN of early aborted embryos was higher than that of live embryos with statistically significant difference(POR group: 303.3±95.4 vs. 158.9±36.3, P<0.01; NOR group: 486.0±356.7 vs. 258.0±137.9, P<0.05). Multiple linear regression analysis showed the MCN of embryos in POR and NOR groups had no correlation with embryo karyotype and blastocyst grade, but was positively correlated with the mother's age (POR group: β=0.185, P=0.035; NOR group: β=0.215, P=0.002). Conclusion Higher MCN may be more likely to cause early miscarriage in POR patients, and the embryonic MCN was correlated with maternal age.

, correspAuthors=Wei Shang, authorNote=null, correspAuthorsNote=
*E-mail:
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目的 探讨胚胎的线粒体拷贝数(MCN)对卵巢低反应(POR)患者胚胎移植结局的影响及其与母亲年龄、胚胎核型和囊胚级别的相关性。方法 收集2018年9月—2020年10月于解放军总医院第六医学中心生殖中心经体外受精(IVF)治疗并行胚胎植入前非整倍体检测(PGT-A)的75例卵巢储备功能正常(NOR)患者和74例POR患者的临床资料进行回顾性分析。将体外受精培养后得到的囊胚(NOR组：205枚；POR组：135枚)进行活检，分别得到116枚和44枚整倍体囊胚。分析并比较两组患者不同临床结局胚胎的MCN，并采用多元线性回归分析胚胎MCN与母亲年龄、胚胎核型和囊胚级别的相关性。结果 POR组活检囊胚和整倍体囊胚的MCN明显少于NOR组，差异有统计学意义(活检囊胚：267.4±174.0 vs. 319.0±264.8，P<0.05；整倍体囊胚：216.0±95.7 vs. 305.4±273.2，P<0.01)。无论POR组还是NOR组，早期流产胚胎的MCN均多于活产胚胎，差异有统计学意义(POR组：303.3±95.4 vs. 158.9±36.3，P<0.01；NOR组：486.0±356.7 vs. 258.0±137.9，P<0.05)。多元线性回归分析显示，POR及NOR组胚胎的MCN与胚胎核型、囊胚级别无相关性，而与母亲年龄(POR组：β=0.185，P=0.035；NOR组：β=0.215，P=0.002)呈正相关。结论 较高的MCN可能更容易导致POR患者发生早期流产，且胚胎的MCN与母亲年龄相关。

, correspAuthors=商微, authorNote=null, correspAuthorsNote=
商微，E-mail：
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孔娜娜，理学博士，主要从事卵巢功能减退方面的研究

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孔娜娜，理学博士，主要从事卵巢功能减退方面的研究

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孔娜娜，理学博士，主要从事卵巢功能减退方面的研究

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Fertil Steril, 2017, 107(1): 34-42.e3., articleTitle=Accurate quantitation of mitochondrial DNA reveals uniform levels in human blastocysts irrespective of ploidy, age, or implantation potential, refAbstract=null), Reference(id=1207064334941897492, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1206995860018254169, doi=null, pmid=null, pmcid=null, year=2018, volume=35, issue=5, pageStart=871, pageEnd=877, url=null, language=null, rfNumber=[24], rfOrder=26, authorNames=Klimczak AM, Pacheco LE, Lewis KE, journalName=J Assist Reprod Genet, refType=null, unstructuredReference=Klimczak AM, Pacheco LE, Lewis KE, et al. Embryonal mitochondrial DNA: relationship to embryo quality and transfer outcomes[J]. J Assist Reprod Genet, 2018, 35(5): 871-877., articleTitle=Embryonal mitochondrial DNA: relationship to embryo quality and transfer outcomes, refAbstract=null), Reference(id=1207064335029977877, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1206995860018254169, doi=null, pmid=null, pmcid=null, year=2021, volume=10, issue=1, pageStart=55, pageEnd=null, url=null, language=null, rfNumber=[25], rfOrder=27, authorNames=Busnelli A, Navarra A, Levi-Setti PE, journalName=Antioxidants (Basel), refType=null, unstructuredReference=Busnelli A, Navarra A, Levi-Setti PE. Qualitative and quantitative ovarian and peripheral blood mitochondrial DNA (mtDNA)alterations: mechanisms and implications for female fertility[J]. Antioxidants (Basel), 2021, 10(1): 55., articleTitle=Qualitative and quantitative ovarian and peripheral blood mitochondrial DNA (mtDNA)alterations: mechanisms and implications for female fertility, refAbstract=null), Reference(id=1207064335134835480, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1206995860018254169, doi=null, pmid=null, pmcid=null, year=2020, volume=35, issue=5, pageStart=1120, pageEnd=1129, url=null, language=null, rfNumber=[26], rfOrder=28, authorNames=Lan Y, Zhang S, Gong F, journalName=Hum Reprod, refType=null, unstructuredReference=Lan Y, Zhang S, Gong F, et al. The mitochondrial DNA copy number of cumulus granulosa cells may be related to the maturity of oocyte cytoplasm[J]. Hum Reprod, 2020, 35(5): 1120-1129., articleTitle=The mitochondrial DNA copy number of cumulus granulosa cells may be related to the maturity of oocyte cytoplasm, refAbstract=null), Reference(id=1207064335248081691, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1206995860018254169, doi=null, pmid=null, pmcid=null, year=2019, volume=111, issue=2, pageStart=205, pageEnd=211, url=null, language=null, rfNumber=[27], rfOrder=29, authorNames=Cecchino GN, Garcia-Velasco JA, journalName=Fertil Steril, refType=null, unstructuredReference=Cecchino GN, Garcia-Velasco JA. Mitochondrial DNA copy number as a predictor of embryo viability[J]. 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Comparison of clinical data and biopsy embryo information of the two groups of embryo transfer patients

, figureFileSmall=null, figureFileBig=null, tableContent=
项目POR组(n=74)NOR组(n=75)tP
年龄(岁，$\bar{x}±s$)38.2±4.436.8±4.4–1.8510.066
体重指数(kg/m2, $\bar{x}±s$)23.1±3.322.3±2.4–1.5640.125
流产次数($\bar{x}±s$)2.05±1.02.12±0.650.4490.648
活检囊胚数(个)135205––
整倍体囊胚数(个)44116––
整倍体囊胚率(%)32.656.6–<0.001
), ArticleFig(id=1207064331062166111, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1206995860018254169, language=CN, label=表1, caption=

两组胚胎移植患者临床资料与活检胚胎信息比较

, figureFileSmall=null, figureFileBig=null, tableContent=
项目POR组(n=74)NOR组(n=75)tP
年龄(岁，$\bar{x}±s$)38.2±4.436.8±4.4–1.8510.066
体重指数(kg/m2, $\bar{x}±s$)23.1±3.322.3±2.4–1.5640.125
流产次数($\bar{x}±s$)2.05±1.02.12±0.650.4490.648
活检囊胚数(个)135205––
整倍体囊胚数(个)44116––
整倍体囊胚率(%)32.656.6–<0.001
), ArticleFig(id=1207064331150246497, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1206995860018254169, language=EN, label=Tab. 2, caption=

Comparison of biopsy blastocysts MCN of the two groups of embryo transfer patients

, figureFileSmall=null, figureFileBig=null, tableContent=
项目POR组(n=74)NOR组(n=75)tP
活检囊胚数(个)135205––
活检囊胚的MCN($\bar{x}±s$)267.4±174.0319.0±264.82.1650.031
整倍体囊胚数(个)44116––
整倍体囊胚的MCN($\bar{x}±s$)216.0±95.7305.4±273.2–3.0490.003
非整倍体囊胚数(个)9189––
非整倍体囊胚的MCN($\bar{x}±s$)292.2±196.4336.8±252.41.3110.191
), ArticleFig(id=1207064331284464228, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1206995860018254169, language=CN, label=表2, caption=

两组胚胎移植患者活检囊胚MCN比较

, figureFileSmall=null, figureFileBig=null, tableContent=
项目POR组(n=74)NOR组(n=75)tP
活检囊胚数(个)135205––
活检囊胚的MCN($\bar{x}±s$)267.4±174.0319.0±264.82.1650.031
整倍体囊胚数(个)44116––
整倍体囊胚的MCN($\bar{x}±s$)216.0±95.7305.4±273.2–3.0490.003
非整倍体囊胚数(个)9189––
非整倍体囊胚的MCN($\bar{x}±s$)292.2±196.4336.8±252.41.3110.191
), ArticleFig(id=1207064331376738922, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1206995860018254169, language=EN, label=Tab. 3, caption=

Comparison of MCN between the transferred embryos and the embryos with different pregnancy outcomes in the two groups of patients

, figureFileSmall=null, figureFileBig=null, tableContent=
项目POR组(n=74)NOR组(n=75)tP
移植囊胚数(个)2846––
移植囊胚MCN206.5±83.2291.1±227.12.2580.066
仅生化妊娠数(个)67––
仅生化妊娠囊胚MCN209.7±71.6222.3±92.90.2540.808
临床妊娠数(个)1331––
临床妊娠囊胚MCN192.2±82.6342.0±256.92.8640.051
活产数(个)1023––
活产囊胚MCN158.9±36.3(1)258.0±137.9(1)3.1170.038
早期流产数(个)35––
早期流产囊胚MCN303.3±95.4486.0±356.71.8860.480
未妊娠数(个)98––
未妊娠囊胚MCN225.1±87.3154.4±24.7(1)–2.1940.055
), ArticleFig(id=1207064331464819311, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1206995860018254169, language=CN, label=表3, caption=

两组患者移植胚胎及不同妊娠结局胚胎的MCN比较

, figureFileSmall=null, figureFileBig=null, tableContent=
项目POR组(n=74)NOR组(n=75)tP
移植囊胚数(个)2846––
移植囊胚MCN206.5±83.2291.1±227.12.2580.066
仅生化妊娠数(个)67––
仅生化妊娠囊胚MCN209.7±71.6222.3±92.90.2540.808
临床妊娠数(个)1331––
临床妊娠囊胚MCN192.2±82.6342.0±256.92.8640.051
活产数(个)1023––
活产囊胚MCN158.9±36.3(1)258.0±137.9(1)3.1170.038
早期流产数(个)35––
早期流产囊胚MCN303.3±95.4486.0±356.71.8860.480
未妊娠数(个)98––
未妊娠囊胚MCN225.1±87.3154.4±24.7(1)–2.1940.055
), ArticleFig(id=1207064331557094002, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1206995860018254169, language=EN, label=Tab. 4, caption=

Multiple linear regression analysis of embryonic MCN(logMCN) by age, karyotype and blastocyst grade in POR,NOR patients

, figureFileSmall=null, figureFileBig=null, tableContent=
变量非标准化系数标准化系数(β)tP
B标准误
POR患者
 常数2.2680.126 17.9400.000
 年龄0.0910.0430.1852.1280.035
 核型–0.0730.045–0.143–1.6300.105
 囊胚级别–0.0150.041–0.031–0.3660.715
NOR患者
 常数2.5230.122 20.6490.000
 年龄0.1200.0390.2153.0650.002
 核型–0.0470.039–0.084–1.2160.225
 囊胚级别0.0310.0390.0550.7920.430
), ArticleFig(id=1207064331640980087, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1206995860018254169, language=CN, label=表4, caption=

POR、NOR患者的年龄、核型和囊胚级别对胚胎MCN (logMCN)的多元线性回归分析

, figureFileSmall=null, figureFileBig=null, tableContent=
变量非标准化系数标准化系数(β)tP
B标准误
POR患者
 常数2.2680.126 17.9400.000
 年龄0.0910.0430.1852.1280.035
 核型–0.0730.045–0.143–1.6300.105
 囊胚级别–0.0150.041–0.031–0.3660.715
NOR患者
 常数2.5230.122 20.6490.000
 年龄0.1200.0390.2153.0650.002
 核型–0.0470.039–0.084–1.2160.225
 囊胚级别0.0310.0390.0550.7920.430
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卵巢低反应患者IVF胚胎线粒体拷贝数与临床结局的相关性分析
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孔娜娜 1 , 王伟周 2 , 陈甫 1 , 刘斌 1 , 左海洋 1 , 沈可心 1, 3 , 商微 2, *
解放军医学杂志 | 临床研究 2022,47(12): 1226-1231
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解放军医学杂志 | 临床研究 2022, 47(12): 1226-1231
卵巢低反应患者IVF胚胎线粒体拷贝数与临床结局的相关性分析
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孔娜娜1, 王伟周2, 陈甫1, 刘斌1, 左海洋1, 沈可心1, 3, 商微2, *
作者信息
  • 1解放军总医院第六医学中心妇产科生殖中心，北京 100048
  • 2解放军总医院第七医学中心妇产医学部，北京 100700
  • 3华南理工大学医学院妇产科，广东广州 510006

    • 孔娜娜，理学博士，主要从事卵巢功能减退方面的研究

通讯作者:

商微，E-mail：
Correlation between mitochondrial copy number in IVF embryos and clinical outcome in patients with poor ovarian response
Na-Na Kong1, Wei-Zhou Wang2, Fu Chen1, Bin Liu1, Hai-Yang Zuo1, Ke-Xin Shen1, 3, Wei Shang2, *
Affiliations
  • 1Center for Assisted Reproductive Medicine, Department of Obstetrics and Gynecology, the Sixth Medical Center of Chinese PLA General Hospital, Beijing 100048, China
  • 2Department of Obstetrics and Gynecology, the Seventh Medical Center of Chinese PLA General Hospital, Beijing 100700, China
  • 3Department of Obstetrics and Gynecology, Medical College, South China University of Technology, Guangzhou, Guangdong 510006, China
出版时间: 2022-12-28 doi: 10.11855/j.issn.0577-7402.2022.12.1226
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摘要
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目的 探讨胚胎的线粒体拷贝数(MCN)对卵巢低反应(POR)患者胚胎移植结局的影响及其与母亲年龄、胚胎核型和囊胚级别的相关性。方法 收集2018年9月—2020年10月于解放军总医院第六医学中心生殖中心经体外受精(IVF)治疗并行胚胎植入前非整倍体检测(PGT-A)的75例卵巢储备功能正常(NOR)患者和74例POR患者的临床资料进行回顾性分析。将体外受精培养后得到的囊胚(NOR组：205枚；POR组：135枚)进行活检，分别得到116枚和44枚整倍体囊胚。分析并比较两组患者不同临床结局胚胎的MCN，并采用多元线性回归分析胚胎MCN与母亲年龄、胚胎核型和囊胚级别的相关性。结果 POR组活检囊胚和整倍体囊胚的MCN明显少于NOR组，差异有统计学意义(活检囊胚：267.4±174.0 vs. 319.0±264.8，P<0.05；整倍体囊胚：216.0±95.7 vs. 305.4±273.2，P<0.01)。无论POR组还是NOR组，早期流产胚胎的MCN均多于活产胚胎，差异有统计学意义(POR组：303.3±95.4 vs. 158.9±36.3，P<0.01；NOR组：486.0±356.7 vs. 258.0±137.9，P<0.05)。多元线性回归分析显示，POR及NOR组胚胎的MCN与胚胎核型、囊胚级别无相关性，而与母亲年龄(POR组：β=0.185，P=0.035；NOR组：β=0.215，P=0.002)呈正相关。结论 较高的MCN可能更容易导致POR患者发生早期流产，且胚胎的MCN与母亲年龄相关。

卵巢低反应  /  线粒体拷贝数  /  早期流产  /  体外受精

Objective To explore the role of mitochondrial copy number (MCN) of embryos in the outcome of embryo transfer in patients with poor ovarian response (POR) and the correlation with mother's age, embryonic karyotype and blastocyst grade. Methods A retrospective analysis was performed of 75 patients with normal ovarian reserve (NOR) and 74 patients with POR who underwent in vitro fertilization (IVF) and preimplantation genetic testing for aneuploidies (PGT-A) in the Sixth Medical Center of Chinese PLA General Hospital from September 2018 to October 2020. The blastocysts obtained after IVF were biopsied(NOR group: 205; POR group: 135), and 116 and 44 euploid blastocysts were obtained, respectively. The MCN of embryos in different clinical outcomes of each group were analyzed and compared, and the correlations of embryo MCN with maternal age, embryo karyotype and blastocyst grade were analyzed by multiple linear regression analysis. Results The mean MCN of biopsy blastocysts and euploid blastocysts were lower in POR group than those in NOR group with statistically significant difference (biopsy blastocysts: 267.4±174.0 vs. 319.0±264.8, P<0.05; euploid blastocysts: 216.0±95.7 vs. 305.4±273.2, P<0.01). Regardless POR group or NOR group, the MCN of early aborted embryos was higher than that of live embryos with statistically significant difference(POR group: 303.3±95.4 vs. 158.9±36.3, P<0.01; NOR group: 486.0±356.7 vs. 258.0±137.9, P<0.05). Multiple linear regression analysis showed the MCN of embryos in POR and NOR groups had no correlation with embryo karyotype and blastocyst grade, but was positively correlated with the mother's age (POR group: β=0.185, P=0.035; NOR group: β=0.215, P=0.002). Conclusion Higher MCN may be more likely to cause early miscarriage in POR patients, and the embryonic MCN was correlated with maternal age.

poor ovarian response  /  mitochondrial copy number  /  early miscarriage  /  in vitro fertilization
孔娜娜, 王伟周, 陈甫, 刘斌, 左海洋, 沈可心, 商微. 卵巢低反应患者IVF胚胎线粒体拷贝数与临床结局的相关性分析. 解放军医学杂志, 2022 , 47 (12) : 1226 -1231 . DOI: 10.11855/j.issn.0577-7402.2022.12.1226
Na-Na Kong, Wei-Zhou Wang, Fu Chen, Bin Liu, Hai-Yang Zuo, Ke-Xin Shen, Wei Shang. Correlation between mitochondrial copy number in IVF embryos and clinical outcome in patients with poor ovarian response[J]. Medical Journal of Chinese People’s Liberation Army, 2022 , 47 (12) : 1226 -1231 . DOI: 10.11855/j.issn.0577-7402.2022.12.1226
正文
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线粒体是细胞的能量来源，为细胞的生命活动提供能量，其基因组以线粒体DNA(mitochondrial DNA，mtDNA)的形式存在[1]。在人类细胞中，线粒体拷贝数(mitochondrial copy number，MCN)受到严格控制，以确保线粒体能产生适当水平的能量。卵母细胞中线粒体的质量和数量是成功受精及胚胎发育的重要指标，卵母细胞或胚胎中的mtDNA也被证实与卵巢功能不全、卵巢储备功能低下(diminished ovarian reserve，DOR)、子宫内膜异位症和女性年龄等不孕因素相关[2-5]。研究发现，MCN较低的囊胚更容易植入成功，MCN可作为胚胎植入的标志物[6]。卵巢低反应(poor ovarian response，POR)对促性腺激素刺激反应不良，常发生于DOR、高龄和既往对常规卵巢刺激反应不良的人群。胚胎植入前遗传学非整倍体检测(preimplantation genetic testing for aneuploidies，PGT-A)适用于POR患者以降低其流产率和分娩时间[7]，但目前胚胎MCN对POR患者移植后妊娠结局的影响尚未见报道。本研究回顾性分析卵巢储备功能正常(normal ovarian reserve，NOR)和POR患者行PGT-A的相关病例，探究MCN对其临床结局的影响，旨在预测胚胎的发育潜能，以期为其临床移植提供参考。
1 资料与方法
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1.1 研究对象
回顾性分析2018年9月－2020年10月于解放军总医院第六医学中心妇产科生殖中心行PGT-A的75例NOR患者[年龄(36.8±4.4)岁]和74例POR患者[年龄(38.2±4.4)岁]的临床资料。分别对NOR患者的205枚囊胚和POR患者的135枚囊胚进行活检。收集POR和NOR患者的年龄、体重指数、活检囊胚形态学评分、核型、移植情况、妊娠结局等信息。满足以下条件中的2项即可判断为POR：(1)高龄(≥40岁)或存在卵巢反应不良的其他危险因素；(2)前次体外受精(IVF)周期卵巢低反应，常规方案获卵≤3个；(3)卵巢储备下降，如早期窦卵泡数(antral follicle count，AFC)<5个或抗米勒管激素(anti-Müllerian hormone，AMH)<0.5 μg/L。NOR组的入组标准为AMH >2 μg/L，卵泡刺激素(FSH)<8 U/L，雌激素(E2)<60 ng/L，AFC>4个。所有纳入研究的女性均符合行PGT-A的指征，排除存在子宫内膜异位症、卵巢手术史、化疗或辐射暴露史、任何导致DOR的自身免疫性疾病或促性腺激素治疗、男女双方有任何一方染色体异常者。对于满足PGT-A指征的复发性流产、反复种植失败者，排除子宫解剖异常、母体疾病状态等情况。内膜厚度7~10 mm认为可以进行移植。此外，为避免严重男性因素的混杂因素，排除非梗阻性无精子症行睾丸取精的患者。本研究获解放军总医院第六医学中心伦理委员会批准(HZKY-PJ-2021-24)，患者胚胎在用于PGT-A前均已签署知情同意书。
1.2 方法
1.2.1 研究分组
本研究分别活检74例POR患者的135枚和75例NOR患者的205枚囊胚，得到44枚和116枚整倍体囊胚，其中用于移植的分别为28枚(POR组)和46枚(NOR组)。在探究与胚胎MCN相关的因素时，对母亲年龄、胚胎核型和囊胚级别进行了分析。基于母亲年龄为导致卵巢功能减退的重要因素之一且高龄会影响胚胎质量，本研究根据母亲年龄将活检胚胎分为<35岁与≥35岁两组(POR组：51例 vs. 84例；NOR组：97例 vs. 108例)。核型分为整倍体核型与非整倍体核型两组(POR组：44例 vs.91例；NOR组：116例 vs. 89例)。囊胚级别分为优质胚胎组与非优质胚胎组(POR组：68例 vs. 67例；NOR组：100例 vs. 105例)。根据Gardner标准对囊胚进行形态学评价，分组依据如下：优质胚胎组为囊胚腔的扩张程度在3期以上，内细胞团和滋养层细胞评分为AA/AB/BA/BB的囊胚；非优质胚胎为囊胚腔的扩张程度3期以上，内细胞团和滋养层细胞评分为AC/BC/CA/CB的囊胚。由于胚胎MCN的原始数据分布不符合正态性，故将数据进行转换处理[log(MCN)]。
1.2.2 IVF和胚胎培养
严格按照标准流程进行操作。采用细胞质内单精子注射(intracytoplasmic sperm injection，ICSI)的方式对第2次减数分裂中期的卵子(metaphase Ⅱ，MⅡ)与精子进行受精(D0)，受精后置于卵裂液中培养；胚胎培养48 h后(D3)转移至囊胚培养液中，继续培养48~72 h(D5-D6)。
1.2.3 胚胎活检
对D5或D6的囊胚进行滋养外胚层活检，具体操作如下：用激光破膜系统在内细胞团对侧的透明带上打孔，继续将囊胚培养4~6 h，使部分滋养层细胞形成疝。活检时，将内细胞团位于固定针一侧，活检针靠近形成疝的外滋养层细胞缓慢回吸，并进行激光打孔，直至活检的5~8个外滋养层细胞全部脱离囊胚。活检组织在配子缓冲液中清洗后装入含有2 μl单细胞裂解液的PCR管中，–20 ℃冷冻保存。
1.2.4 胚胎移植
移植胚胎数目按照《人类体外受精-胚胎移植实验室操作专家共识(2016)》[8]进行，结合胚胎形态学分级，选择正常核型的1枚囊胚进行移植。
1.2.5 全基因组扩增及测序
采用多重退火和环化扩增循环(multiple annealing and looping based amplification cycles，MALBAC)技术对活检组织进行单细胞全基因组扩增[9]。根据MALBAC试剂盒(YK001B，购自中国亿康基因公司)说明书，对活检的细胞进行全基因组扩增，再用HiSeq 2500系统(购自美国Illumina公司)进行二代测序(NGS)和数据分析。
1.2.6 MCN分析
计算特异性比对到线粒体基因组上的reads数目，并用比对到染色体基因组的reads数进行标准化，计算每个染色体基因组对应的线粒体数目。
1.2.7 指标定义
(1)整倍体囊胚率(%)=整倍体囊胚数/活检囊胚数×100%；(2)仅生化妊娠数，指人绒毛膜促性腺激素(human chorionic gonadotropin，HCG)阳性(即囊胚移植后12 d血HCG>30 U/ml) 但未见孕囊或胎心的移植数；(3)临床妊娠数，囊胚移植后28 d左右，阴道超声下可见孕囊或胎心的例数；(4)早期流产数，指妊娠12周前自然流产的移植数；(5)未妊娠数，指HCG阴性的移植数；(6)活产数，指有活产婴儿出生的移植数；(7)移植胚胎的临床结局，包括未妊娠、仅生化妊娠和临床妊娠的总和，其中临床妊娠的结局包含早、中、后期流产及活产。
1.2.8 指标分析
分别对POR组与NOR组未妊娠、仅生化妊娠和临床妊娠胚胎的MCN进行比较，分析两组间各临床结局胚胎MCN的差异，再对POR组与NOR组内早期流产和活产胚胎的MCN差异进行比较；分析母亲年龄、核型和囊胚级别与POR、NOR组囊胚MCN的相关性，再进一步探究母亲年龄与POR、NOR组囊胚MCN的相关性。
1.3 统计学处理
采用SPSS 17.0软件进行统计分析。计量资料以$\bar{x}±s$表示，POR和NOR组活检囊胚MCN的比较采用Welch t检验，POR和NOR组患者临床资料、移植胚胎和不同妊娠结局胚胎MCN的比较采用Student t检验；计数资料以例(%)表示，两组间比较采用χ2检验；将MCN数据进行转换处理为log(MCN)以获得数据的正态分布；采用多元线性回归分析探究年龄、核型和囊胚级别与胚胎MCN的相关性；采用Spearman检验分析年龄对胚胎MCN的影响。P<0.05为差异有统计学意义。
2 结果
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2.1 POR组与NOR组患者临床资料及活检胚胎信息的比较
两组患者的年龄、体重指数和流产次数差异无统计学意义。采用MALBAC-NGS对两组活检胚胎染色体拷贝数进行检测，结果显示，POR组患者135个囊胚中正常核型囊胚44个，整倍体囊胚率为32.6%；NOR组患者的205个囊胚中正常核型囊胚116个，整倍体囊胚率为56.6%，两组患者的整倍体囊胚率比较差异有统计学意义(P<0.01)(表1)。
2.2 POR组与NOR组活检囊胚MCN的比较
MALBAC-NGS检测不同核型囊胚MCN的结果显示，POR组活检胚胎的MCN明显少于NOR组(P=0.031)，且POR组整倍体囊胚的MCN明显少于NOR组(P=0.003)，而两组非整倍体囊胚的MCN差异无统计学意义(表2)。
2.3 两组患者移植胚胎及不同妊娠结局胚胎MCN的比较
POR组共移植囊胚28枚，NOR组共移植囊胚46枚。POR组的活产囊胚MCN明显少于NOR组，差异有统计学意义(P=0.038)，而不同妊娠结局(仅生化妊娠、临床妊娠、早期流产囊胚)移植囊胚的MCN虽然均少于NOR组，但差异均无统计学意义(P>0.05)，且POR、NOR组内未妊娠、仅生化妊娠和临床妊娠胚胎的MCN差异也无统计学意义(P>0.05)。对获得临床妊娠的胚胎进一步分析，即比较活产和早期流产胚胎的MCN，结果显示，POR组及NOR组内的早期流产胚胎MCN均多于活产胚胎，差异有统计学意义(P<0.05)。此外，NOR组的早期流产胚胎MCN多于未妊娠胚胎MCN，差异有统计学意义(P<0.05)，而POR组的早期流产胚胎MCN虽然多于未妊娠胚胎MCN，但差异无统计学意义(P>0.05)(表3)。
2.4 POR、NOR患者的年龄、核型和囊胚级别对MCN的影响
多元线性回归分析结果显示，核型、囊胚级别对POR、NOR患者胚胎的MCN并无影响(P>0.05)，而年龄可明显影响POR、NOR患者胚胎的MCN，差异有统计学意义(P=0.035、P=0.002)(表4)。进一步采用Spearman分析探究年龄对POR、NOR患者胚胎MCN的影响，结果显示，年龄≥35岁的POR、NOR患者胚胎MCN均明显多于年龄<35岁的患者(POR：300.5±199.1 vs. 212.8±100.2；NOR：378.2±303.1 vs. 253.1±194.1)，且均呈正相关(P=0.004、P=0.001)。
3 讨论
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已有的研究认为，胚胎MCN含量与胚胎发育阶段相关，且母体年龄与线粒体DNA的增加呈正相关[3,6,10]，然而，目前尚未见在POR人群中探究胚胎MCN含量及与妊娠关系的报道。本研究对POR人群的胚胎MCN进行分析，探索其与NOR人群胚胎MCN的差异及与临床妊娠的关系，以期对临床筛选胚胎进行移植提供参考。
早期研究发现，PGT-A可降低POR患者的流产率，并减少非整倍体胚胎移植所损失的时间[7]。POR多发生于高龄DOR患者，DOR与卵母细胞的数量和质量下降有关，通常导致较差的临床结局。本研究对POR组[年龄(38.2±4.4)岁]的135枚囊胚和NOR组[年龄(36.8±4.4)岁]的205枚囊胚进行染色体拷贝数和MCN的分析；染色体拷贝数分析结果显示，POR组的整倍体囊胚率明显低于NOR组(32.6%vs. 56.6%)。已有的研究发现，DOR患者的非整倍体囊胚率较NOR患者偏高(66.0% vs. 51.7%)，与本研究中POR的非整倍体率67.4%相似[11]。此外，接受IVF/ICSI治疗的高龄DOR(年龄≥32岁)患者的早期流产物中含有较高的染色体畸变率[12]，提示POR患者非整倍体囊胚率较NOR患者高。早期研究认为，患有DOR和POR的年轻女性与年龄匹配的NOR患者比较，可表现出相近的囊胚率、非整倍体率和活产率[13]。本研究POR和NOR组患者平均年龄>35岁，高龄增加了DOR和非整倍体胚胎的风险，使DOR患者的胚胎形态学评分降低，非整倍体妊娠和流产增加[14-15]，故高龄可能增高了POR组囊胚的非整倍体率。
胚胎MCN来源于卵母细胞，并在受精至囊胚形成过程中保持不变。Boucret等[4]发现，与NOR患者相比，DOR患者卵母细胞中的MCN明显减少。最近的研究也表明，不论年龄大小，DOR患者的线粒体均表现出更严重的形态学改变，其中高龄DOR女性的线粒体超微结构异常最严重[16]。本研究发现，POR组的活检囊胚及整倍体囊胚的MCN均少于NOR组(P<0.05)，提示POR患者卵母细胞质量和胚胎质量明显降低。由于胚胎MCN代表了受精前卵母细胞的MCN，因此，在MⅡ期的卵母细胞需较多的MCN来支持受精和胚胎囊胚期前的发育。
为进一步探究囊胚MCN与妊娠结局的关系，本研究选取核型正常且胚胎形态学评级较好的囊胚用于移植，并分析了临床妊娠情况。POR组中的活产囊胚MCN更少，可能与移植囊胚和临床妊娠MCN较少有关，但目前并无关于POR和NOR患者活产囊胚MCN的相关研究，因此需要更多的数据进一步证实。本研究还发现，无论POR还是NOR组，早期流产胚胎的MCN均明显多于活产胚胎，而未妊娠、仅生化妊娠和临床妊娠胚胎的MCN之间差异无统计学意义，提示MCN似乎不能选择何种胚胎用于移植。早期的研究认为胚胎线粒体DNA可预测胚胎的发育潜能。如Fragouli等[6]发现，不能植入的整倍体囊胚MCN多于植入的整倍体囊胚，且研究者计算了胚胎mtDNA的阈值，超过这个阈值的胚胎移植则不能成功植入。Diez-Juan等[17]也证实了该结果，并用mtDNA评分来预测胚胎的植入潜能。然而，Fragouli等[6]和Diez-Juan等[17]的研究均未考虑到同一女性所有胚胎MCN的情况，MCN较多时是否更可能被植入。Treff等[18]的研究使用不同性别的同胞胚胎来评估mtDNA含量的预测价值，结果表明，MCN无法区分植入和未植入的胚胎，这与本研究中未妊娠和临床妊娠胚胎MCN无明显差异相似。本研究还发现，早期流产胚胎MCN明显多于活产胚胎，可能是早期流产胚胎线粒体DNA突变和功能障碍增加，即使MCN代偿性增加，仍无法维持线粒体的正常功能而引起早期流产。一项利用缺乏线粒体转录因子A的小鼠研究表明，卵母细胞和胚胎必须拥有一定阈值的MCN，以支持胚胎发育成活胎的能力[19]。另有研究发现，MCN多于阈值的胚胎实现持续妊娠的机会较少[3,20]。因此，笔者推测胚胎的MCN在一定范围内可能代表较高质量的卵母细胞和早期胚胎，有助于持续妊娠；少于或多于这个范围，可能均不利于胚胎的发育，临床结局也较差。因此，MCN与胚胎植入潜能之间的关系仍需进一步研究。
本研究多元线性回归分析结果显示，无论POR还是NOR患者，胚胎MCN与母亲年龄均呈正相关，而与核型、囊胚级别无关。多数研究表明，高龄女性囊胚中的mtDNA水平较高[6,20]。年龄相关的mtDNA不稳定可导致卵母细胞中mtDNA突变的积累和MCN的增加，而MCN代偿性的增加可能能够补偿线粒体的功能[21]。研究表明，与年轻的NOR患者相比，年轻的DOR患者卵母细胞质量似乎未受影响；相反，高龄DOR患者的临床妊娠率明显低于年轻的DOR患者，且流产率较高、优质胚胎率较低，提示高龄可明显影响DOR患者的卵母细胞质量[13,22]。目前的研究推测，年轻的DOR患者可能通过补偿性的MCN增加来弥补细胞能量供应的不足，而在高龄DOR患者中，线粒体的损伤无法有效地恢复，可能进一步导致更严重的不平衡性的代谢衰竭[16]，提示年龄可明显影响POR及NOR患者胚胎的MCN。
有研究使用NGS或qPCR方法检测胚胎mtDNA水平后再进行校正因子计算，认为囊胚mtDNA与胚胎植入能力、染色体核型无相关性[23]。一项回顾性研究探讨了MCN与囊胚质量之间的可能关系，发现虽然MCN可预测整个样本中质量非常差或非常好的胚胎，但在分析整倍体囊胚时却未发现存在统计学差异[24]，这与本研究中的囊胚级别与胚胎MCN无关相似。
综上所述，本研究证实较多的MCN可能更易导致POR与NOR患者发生早期流产，且年龄与胚胎MCN呈正相关。但本研究存在一定的局限性：受检测技术的限制，对于POR及NOR患者胚胎MCN的研究仅在行PGT-A的患者中进行筛选和检测，并非包括所有的POR及NOR人群；符合PGT-A指征的复发性流产和反复种植失败人群的子宫内膜因素也可能对胚胎妊娠结局有一定的影响，但本研究未进行探讨。目前，临床实践中使用MCN作为一种预测胚胎生存能力的指标仍然存在争议[25-27]。对MCN与胚胎植入潜能之间的关系仍需要大样本研究加以证实。
基金
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  • 国家重点研发计划(2018YFC1003003)
  • 解放军总医院第六医学中心院内创新培育基金(CXPY201807)
文献
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2022年第47卷第12期
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doi: 10.11855/j.issn.0577-7402.2022.12.1226
  • 接收时间：2022-02-07
  • 首发时间：2025-12-14
  • 出版时间：2022-12-28
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  • 收稿日期：2022-02-07
  • 录用日期：2022-07-15
基金
National Key Research and Development Program(2018YFC1003003)
国家重点研发计划(2018YFC1003003)
Innovation and Cultivation Fund of the Sixth Medical Center of PLA General Hospital(CXPY201807)
解放军总医院第六医学中心院内创新培育基金(CXPY201807)
作者信息
    1解放军总医院第六医学中心妇产科生殖中心，北京 100048
    2解放军总医院第七医学中心妇产医学部，北京 100700
    3华南理工大学医学院妇产科，广东广州 510006

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2种不同金属材料的力学参数
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Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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