【作者】 李晓；

【导师】 赵涵；

【作者基本信息】 山东大学 ， 妇产科学， 2023， 博士

【摘要】 研究背景妊娠期糖尿病(gestational diabetes mellitus,GDM)是指在妊娠期间发病或首次发现的糖代谢异常[1]。GDM能够明显增加不良妊娠结局的风险[2],为母亲和胎儿近远期健康带来诸多问题。如孕妇患妊娠期高血压疾病、子痫的风险增加,剖宫产率增加;胎儿容易出现巨大儿、死胎、早产、死产;新生儿容易出现低血糖、高胆红素血症、呼吸窘迫等。GDM母体远期易患2型糖尿病、代谢综合征、早发性心血管疾病等,子女成年后患上糖尿病和/或肥胖症、神经精神类疾病的风险显著增加[3,4]。GDM发病率因诊断标准、地域、种族的不同从1%至30%不等,而发展中国家的GDM发病率往往高于发达国家[3]。中国大陆GDM发病率高达15%,且逐年升高[5]。考虑到中国庞大的人口基数,这意味着中国具有世界上最多的GDM患者,将为社会的经济、医疗带来巨大负担。GDM的发病是一个遗传、环境因素多方面作用的复杂过程。目前已知的GDM的风险因素包括母亲高龄、超重或肥胖、2型糖尿病(type 2 diabetes,T2D)家族史、久坐的生活方式、西方饮食模式及其他社会心理因素等[3,6,7]。其中,遗传因素在GDM发病过程中的作用不容忽视。有糖尿病家族史、肥胖、多囊卵巢综合征(polycystic ovary syndrome,PCOS)患病史的孕妇,其GDM的发生率明显增高,这提示GDM和T2D、肥胖、PCOS可能具有相同的遗传易感基因。但是,目前系统地研究遗传因素与GDM之间的联系的报道十分有限,尤其是对中国GDM女性的遗传学背景研究更少,且受限于研究样本量,研究结果间缺乏可重复性。因此,亟需基于多地域的人群队列的大样本病例-对照研究,系统探究中国GDM女性的遗传结构,发现中国人群GDM易感基因位点及可能的发病机制,从而为中国女性GDM的遗传机制研究提供新的见解。菌群是与人体密切接触的环境因素之一,同时作为“人体的第二个基因组”,GDM女性的阴道、肠道、口腔的微生物组在妊娠期间均发生了显著改变,提示微生态失调可能参与疾病发生发展[8,9]。阴道微生物组是否在GDM的发展中起因果作用,目前尚不清楚。有研究提出,GDM人群存在的胰岛素抵抗、体重增加、较高的炎症水平等代谢特征,也可能导致其阴道微生物群落在妊娠期的生理性适应不同于健康女性[10]。即我们的关键科学问题是:在妊娠期间发现的GDM女性阴道微生物组成的改变是疾病本身的反映,还是在疾病发展前即存在,并可作为疾病的驱动因素或者风险预示因素。具备完整临床表征信息的大型前瞻性阴道微生物群落研究对于回答这个问题至关重要。针对上述科学问题,本研究基于多地域人群的大型前瞻性队列,综合了核基因组、线粒体基因组及阴道微生物组3个层面,旨在从遗传和微生态角度出发,更加全面地探究影响中国女性GDM发生发展的候选基因及微生物因素,以完善中国GDM女性的遗传结构及微生态特点,推动我国GDM的病因学研究,寻找GDM的分子遗传诊断及防治的新潜在靶点,以改善GDM患者母婴健康。第一章:妊娠期糖尿病患者遗传易感性分析研究目的GDM是最常见的妊娠并发症之一,遗传因素与GDM的发病过程密切相关。虽然基于全基因组关联分析(genome-wide association study,GWAS)的GDM遗传学研究取得了一定进展,但对中国女性GDM的遗传学背景的认知还相当有限。此外,受限于研究的样本量,各研究结果之间缺乏可重复性。基于芯片技术、连锁不平衡原理,GWAS研究将获得的基因型数据进一步填充,并基于此与疾病表型进行关联,因此,仅能分析常见变异位点对疾病的贡献,而无法直接通过测序数据评估罕见变异对疾病的可能影响,造成了复杂疾病遗传度的缺失。本研究旨在通过基于多地域人群队列的基因组学研究,从常见变异位点、低频变异位点、罕见变异位点多个层面,确定中国人群中GDM在遗传层面的风险因素和可能机制,从而为中国女性GDM的发病机制研究奠定基础。研究方法本研究是一项基于中国环境与遗传因素出生队列的巢式病例对照研究,主要选取从中国北部(北京)、中部(武汉)、西南(遵义)3个地区入组的妊娠期糖尿病患者及其同时期对照人群,获得其孕早期血液样本1947例进行全外显子测序(含定制位点)。经过样本和位点质控后,最终纳入702例GDM女性和859例对照女性进行遗传关联分析。我们同时对在gnomeAD数据库东亚人群中次等位基因频率(minor allele frequency,MAF)>1%且在研究人群中MAF>5%的常见变异位点、在gnomeAD数据库东亚人群中MAF>1%且在研究人群中MAF为1%-5%的低频变异位点及在gnomeAD数据库东亚人群中MAF<5‰的罕见变异位点进行单个位点的关联分析。通过基因负荷检验、SKAT检验、SKAT-O检验等分析,在基因水平上,确定位于同一个基因的多个罕见或低频变异位点的联合效应。对相关基因进行GO富集分析,探讨其可能机制。研究结果1、基于常见变异位点的遗传易感性分析:位于CEP89基因外显子区的rs73035551、位于MUC16基因外显子区的rs4992693,是新发现的、与GDM关联性最强的两个位点,均位于第19号染色体。rs73035551(g.33444588T>G)与较低的 GDM 发生风险显著相关(OR=0.37,95%CI=0.26-0.53,P=3.84×10-8)。此位点变异属于错义突变,导致转录本多肽链的天冬氨酸被丙氨酸取代。rs4992693(g.9012823T>C)也属于错义突变,导致转录本多肽链的组氨酸被亮氨酸取代,其对GDM亦存在保护效应(OR=0.35,95%CI=0.23-0.55,P=4.81×10-6)。2、常见变异位点中,另有4个位点与GDM的易感性关联达到中等显著程度,包括位于TMEM255B基因的rs7400029、位于MTNR1B基因上游的rs10830962、位于CACNA1D基因的 rs535481979、位于SLC1A2 基因的rs10836370(P=2.63×10-5,5.89×10-5,6.41×10-5,7.67×10-5)。既往研究报道位于MTNR1B基因的上游rs10830962位点与韩国、欧洲及中国南方女性的GDM易感性相关。3、基于罕见变异位点的遗传易感性分析,发现了 1个与GDM显著关联的新位点:位于 FBXL6基因 rs200985955(g.145579932C>T,OR=0.23,95%CI=0.14-0.37,PFisher=1.23× 10-9,PWald=2.11×10-9)。4、分析位于同一个基因的多个罕见或低频变异位点的联合效应,P2RX7基因、SCO2基因是最显著的GDM关联基因。5、基于常见变异位点的GO富集分析发现,胰岛素分泌及其相关调控通路显著富集;基于罕见变异位点的GO富集分析提示,(胆固醇、脂蛋白等)脂质运输、定位、合成分解等相关生物过程显著富集;基于低频位点的GO富集分析发现,差异基因显著定位于线粒体膜,参与线粒体氧化磷酸化过程。研究结论本研究发现了中国女性中GDM风险相关的2个新的常见遗传易感位点(位于CEP89基因、MUC16基因)和1个新的罕见遗传易感位点(FBXL6基因),基于罕见及低频位点的基因水平分析发现了 P2RX7、SCO2等多个基因与GDM的关联,还验证了MTNR1B基因rs10830962与中国女性GDM易感性的关联。基因富集分析提示了胰岛素分泌及其调控、炎症、自身免疫、脂质代谢及线粒体代谢相关通路在GDM发病中的可能作用。第二章:妊娠期糖尿病患者的线粒体基因组变异分析研究目的妊娠期糖尿病是一种以胰岛素抵抗和胰岛β细胞功能紊乱为特征的代谢性疾病。线粒体作为细胞主要的供能细胞器,对维持细胞的代谢稳态至关重要。线粒体拥有独立于核基因组的遗传体系即线粒体DNA(mitochondrial DNA,mtDNA)。人类的mtDNA是一个含16569个碱基对的双链环状基因组,包含1个mtDNA转录、复制必需的非编码置换区(displacement loop region,D-loop)和37个基因。mtDNA由于缺乏组蛋白保护和有效的DNA修复系统,且位于靠近电子呼吸链的位置,更容易受到氧自由基的攻击,具有更高的突变率。研究表明,线粒体基因突变会引起线粒体基因的复制和转录的改变,并导致基因的表达以及mtDNA拷贝数的变化,由此造成电子传递链的改变、线粒体功能障碍,从而影响细胞内正常的能量代谢和ATP供应,可能导致多种疾病的发生和进展。既往在亚洲印度人群、沙特女性、新加坡女性的研究中,发现mtDNA突变与GDM发生相关。然而,所报道的研究中涉及的样本量相对较小,对中国汉族女性人群研究不足,而且很少有关于单倍群的分析。基于前述的多地域人群队列,本研究旨在通过线粒体基因组测序,比较两组的mtDNA变异及单倍群,探究mtDNA变异与GDM的相关性。研究方法基于前述多地域的GDM巢式病例对照研究,同时行线粒体DNA测序,最终纳入701例GDM女性和859例健康对照女性进行分析。通过Student’s t检验、卡方检验及logistic回归,比较两组的mtDNA变异在不同区域的分布、mtDNA蛋白编码区的非同义突变及单倍群划分,探究mtDNA变异与GDM的关联。研究结果1、发现了 6个与GDM相关的线粒体DNA变异,包含5个位于D-loop区域的mtDNA变异和1个位于COX1基因变异:D-loop区的m.73A>G、m.185G>A和m.16051A>G与GDM风险降低相关,而D-loop区的m.16092T>A、m.16291C>T与COX1基因的m.6228C>T与GDM风险增加相关。相较于正常妊娠女性,GDM女性的线粒体DNA的D-loop区携带更少的线粒体DNA变异。2、GDM组和对照组的线粒体单倍群的分布未发现明显差异。研究结论中国汉族女性中,线粒体全基因变异与妊娠期糖尿病的风险相关联,提示了在妊娠早期使用特定线粒体DNA变异测定可作为一种潜在的辅助手段,用于预测妊娠期糖尿病风险,为未来GDM防治亦提供了潜在靶点。第三章:妊娠期糖尿病患者的妊娠前阴道微生态群落组成分析研究目的既往研究发现,作为“人体的第二个基因组”,GDM女性的阴道、肠道、口腔的微生物组在妊娠期间均发生了显著改变,提示微生态异常可能也参与其发病,并提出将人体微生物群落的调控作为疾病预防和治疗的新策略。有研究提出,GDM人群存在的胰岛素抵抗、体重增加、较高的炎症水平等代谢特征,也可能导致其阴道微生物群落在妊娠期的生理性适应不同于健康女性。在妊娠期间发现的GDM女性阴道微生物组成的改变是疾病本身的反映,还是在疾病发展前即存在,并可作为疾病的驱动因素或者风险预示因素,目前尚无定论。此外,传统的研究着重关注了在妊娠期间检测到的GDM风险因素,越来越多的数据支持围产期、孕前危险因素在GDM发展中的重要作用。然而,迄今为止的关于GDM女性阴道微生物组的研究样本量相对较小,且仅针对妊娠期间的生殖道菌群进行研究。本研究旨在探究发展为GDM女性妊娠前的阴道微生物组成和群落状态类型,为评估微生物组调控作为GDM防治新策略的可能性提供新的证据。研究方法本章研究基于一个涵盖1万余名女性的生殖道微生态临床前瞻性队列,追踪其中通过辅助生殖助孕后获得单胎活产的女性,进行巢式病例对照设计,通过1:1倾向性评分匹配控制年龄、BMI、血压、糖脂代谢指标、糖尿病家族病史等重要混杂因素的影响,最终纳入196名GDM女性和196名健康足月产女性。考虑到GDM女性并发妊娠期高血压疾病(hypertensive disorders of pregnancy,HDP)的风险较高,将GDM女性进一步分层为单纯GDM组、合并HDP的GDM组进行分析。采集研究对象在妊娠前的阴道分泌物拭子,提取细菌总DNA,扩增细菌16S rRNA基因的V1-V2保守区,进行高通量测序。采用Usearch软件处理测序数据,分析阴道微生态群的α和β多样性、组成及结构,通过置换多元方差分析、主坐标分析比较组间菌群组成的差异,并根据样本内的最高丰度细菌对其阴道菌群进行阴道群落状态类型(community state types,CSTs)的划分。研究结果1、与对照组相比,GDM女性的妊娠前阴道菌群的α多样性丰富度增加,β多样性无显著差异。进一步分层发现,与对照组相比,单纯GDM组、合并HDP的GDM组α多样性、β多样性无显著差异。2、比较GDM组与对照组阴道菌的细菌丰度,在各个分类水平上均未发现差异细菌。比较单纯GDM组与对照组的妊娠前阴道菌群,未发现显著的差异菌。与对照组或单纯GDM组相比,合并HDP的GDM女性的妊娠前阴道微生物群表现为卷曲乳杆菌丰度的减少,奈瑟菌、丙酸杆菌、贪铜菌、无乳链球菌、棒状杆菌、罗伊氏乳酸杆菌、加氏乳杆菌等多种细菌的富集。3、总体人群中,由卷曲乳杆菌、惰性乳杆菌、加氏乳杆菌、詹氏乳杆菌4种乳杆菌主导的CSTs的比例分别为52.3%、37.2%、2.6%、1.5%,即93.6%女性的阴道优势菌是这4种常见的阴道乳杆菌。仅6.1%女性的阴道菌群由阴道加德纳菌、阿托波菌、普雷沃氏菌、链球菌、纤毛菌、双歧杆菌、脲原体或罗伊氏乳杆菌主导。4、GDM组与对照组的CSTs 比例无显著差异。将GDM组进一步分层,与对照组、单纯GDM组相比,合并HDP的GDM女性的以卷曲乳杆菌主导的CST比例呈现降低趋势,但不显著(49.5%vs.57.1%vs.38.1%),相反地,惰性乳杆菌主导CST比例则呈现增加趋势(38.3%vs.34.9%vs.47.6%)。研究结论本研究首次分析了中国妊娠期糖尿病女性的妊娠前阴道微生物组,同时控制了年龄、BMI、糖尿病家族史等重要混杂因素的影响,研究发现发展为GDM女性在妊娠前的阴道微生物群落变化不明显,仅表现为α多样性丰富度的增加。合并HDP的GDM女性的妊娠前阴道微生物群落表现为卷曲乳杆菌丰度的减少,奈瑟菌、丙酸杆菌、贪铜菌、无乳链球菌、棒状杆菌等多种细菌富集。这为阴道菌群与GDM及其并发症的关联补充了不同时间点的证据,从微生态角度增加了对GDM病因的理解。更多还原

【Abstract】 BackgroundGestational diabetes mellitus(GDM)is characterized by hyperglycemia with onset or first recognition during pregnancy[1].GDM significantly increases the risk of adverse pregnancy outcomes and leads to many short-term and long-term health problems for both mother and offspring[2].For example,women with GDM have an increased risk of hypertensive disorders of pregnancy(HDP),eclampsia,and cesarean delivery.The fetus is prone to macrosomia,stillbirth,premature birth,and stillbirth.An increased risk of hypoglycemia,hyperbilirubinemia,and respiratory distress is observed in newborns of GDM-affected mothers.Women with a history of GDM are prone to metabolic disorders such as type 2 diabetes,metabolic syndrome,early-onset cardiovascular disease in the long term.And the risk of diabetes and/or obesity and neuropsychiatric diseases in offspring of GDM-affected mother is also significantly increased[3,4].The incidence of GDM varies from 1%to 30%depending on different diagnostic criteria,geography,and ethnicity.The incidence of GDM tends to be higher in developing countries than in developed countries[3].GDM complicates 15%of pregnancies in mainland China with an annual increasing incidence[5].Considering China’s huge population,this means that China has the largest number of GDM patients in the world,which will bring a huge burden to the social economy and health care.The pathogenesis of GDM is a complex process complicated by both genetic and environmental factors.Advanced maternal age,overweight or obesity,family history of type 2 diabetes(T2D),sedentary lifestyle,Western dietary patterns,and other psychosocial factors are well-known risk factors of GDM[3,6,7].Among them,genetic factors are indispensable to the pathogenesis of GDM.The incidence of GDM is significantly higher in pregnant women with a family history of T2D,obesity,or polycystic ovary syndrome(PCOS),suggesting that GDM may share susceptibility genes with T2D,obesity,and PCOS.However,the systemic studies reporting genetic risk factors for GDM are limited,especially in Chinese women.Additionally,there is a lack of reproducibility among different studies due to the limited sample size.Further studies are needed to characterize the genetic profiles of Chinese women with GDM from multiple perspectives.A large-scale cohort based on multi-geographic populations is of critical importance to identify susceptibility loci and underlying mechanisms in the pathogenesis of GDM,which will help provide new insights into the Chinese population-specific genetic basis of GDM.Microbiome is one of the environmental factors in close contact with the human body.As the "the second genome of human body",the microbiomes of the vagina,intestine,and oral cavity in women with GDM are significantly altered during pregnancy,suggesting that microecology dysbiosis may also be involved in its pathogenesis[8,9].Emerging researchers suggest manipulation of the microbiome as a novel strategy for disease prevention and treatment.Whether vaginal microbial alterations play a causal role in the development of GDM remains unclear.It has been proposed that metabolic features such as insulin resistance,weight gain,and higher levels of inflammation,which are overrepresented in GDM,may also lead to different physiological adaptations of vaginal microbiome during pregnancy in GDM-affected women from that in healthy women.Therefore,we raise a critical question of whether the altered vaginal microbial composition in women with GDM during pregnancy is a reflection of the disease itself or is present prior to disease development and may serve as a driver or predictor of the disease.A large-scale,prospective,population-based study on vaginal microbiome with well-characterized clinical features is essential to answer this question.To address these scientific questions,this study aims to investigate the candidate predisposing genes and microbial factors that influence the development of GDM in Chinese women from a genetic and microecological perspective,based on a large prospective cohort of a multi-geographic population,integrating the nuclear genome,the mitochondrial genome and the vaginal microbiome.It could help develop in-depth knowledge of genetic background of GDM in Chinese populations,promote the etiology of GDM in China,find new potential targets for molecular genetic diagnosis and prevention of GDM,and achieve a better prognosis for both mothers and children.Part Ⅰ Genetic susceptibility analysis of patients with gestational diabetes mellitusObjectiveGDM is one of the most common pregnancy complications.Genetic factors are closely related to the pathogenesis of GDM.Despite efforts in genetic studies of GDM based on genome-wide association study(GWAS),the knowledge of the genetic background of GDM in Chinese women is still quite limited.In addition,there is a lack of reproducibility among the results of the studies due to relatively small sample sizes.Based on the principle of linkage disequilibrium,GWAS studies correlate array-based imputed genotype data with phenotypes.Therefore only the contribution of common variant loci can be analyzed in GWAS studies.Without direct sequencing data,the impact of rare variants on diseases cannot be fully evaluated,resulting in the lack of heritability of complex diseases.The aim of this study is to identify predisposing genes and possible underlying mechanisms of GDM in Chinese population through genomic studies based on a multi-geographic population cohort,at both common and rare/lowfrequency variant loci levels,thus laying the foundation for future studies of the pathogenesis of GDM in Chinese women.MethodsThis nested case-control study was based on a birth cohort studying on the effects of environmental and genetic factors in China,which included 1947 women from three regions in northern(Beijing),central(Wuhan),and southwestern(Zunyi)China.Blood samples from the study subjects were obtained during early pregnancy and then subjected to whole-exome sequencing(including additional customized SNP loci).After quality control of samples and variants,702 women with GDM and 859 controls were finally included in final analysis.We also performed analysis of individual loci for common variants,rare variants and low-frequency variant.Common variants were defined as loci with a minor allele frequency(MAF)>1%in the East Asian population of the gnomeAD database and a MAF>5%in the study population.Low-frequency variants were defined as loci with a MAF>1%in the East Asian population of the gnomeAD database and a MAF of 1%-5%in the study population.Rare variants were defined as with a MAF of<5‰ in the East Asian population of the gnomeAD database.The combined effect of multiple rare or low-frequency variants located in the same gene was determined by gene-level tests,namely burden test,SKAT test,and SKAT-O test.GO enrichment analysis was further performed on GDM-associated genes to explore the underlying mechanisms.Results1.Analysis based on common variant loci:rs73035551,located in the exonic region of CEP89,and rs4992693,located in the exonic region of MUC16,were the two newly identified loci with the strongest association with GDM,both on chromosome 19.rs73035551(g.33444588T>G)was significantly associated with a lower GDM risk(OR=0.37,95%95%CI=0.26-0.53,P=3.84×10-8).This variant was a missense mutation that results in the substitution of aspartate by alanine in polypeptide chain.rs4992693(g.9012823T>C)was also a missense mutation that resulted in the substitution of histidine by leucine in the polypeptide chain,which also had a protective effect against GDM(OR=0.35,95%CI=0.23-0.55,P=4.81×10-6).2.Among the common variants,another four loci were associated with GDM with moderate significance,including rs7400029 in TMEM255B,rs10830962 in the upstream of MTNR1B,rs535481979 in CACNA1D,and rs10836370 in SLC1A2(P=2.63×10-5,5.89×10-5,6.41×10-5,7.67×10-5,respectively).Previous studies have reported that rs10830962 located in the upstream MTNR1B is associated with GDM susceptibility in Korean,European and southern Chinese women.3.Among rare variant loci,one new locus was identified being significantly associated with GDM,namely rs200985955 in FBXL6(g.145579932C>T,OR=0.23,95%CI=0.14-0.37,PFisher=1.23×10-9,Pwald=2.11×10-9).4.Gene-level analysis of the combined effect of rare or low-frequency variants located in the same gene showed that P2RX7 and SCO2 were the most significant GDM-associated genes.5.GO enrichment analysis based on common variants revealed a potential role of insulin secretion and its related regulation in GDM development.GO enrichment analysis based on rare variants suggested involvement of biological processes related to lipid(cholesterol,lipoproteins,etc.)transport,localization,synthesis and catabolism in GDM.GO enrichment analysis based on low-frequency variants revealed that the roles of genes coding proteins on mitochondrion membrane and mitochondrial oxidative phosphorylation in GDM development.ConclusionIn this study,we identified two new common variants loci(in CEP89 and MUC16)and one new rare variant locus(in FBXL6)associated with GDM risk in Chinese women.Gene-level analysis revealed several genes including P2RX7 and SCO2 associated with GDM,and verified the association of polymorphisms in MTNR1B(rs10830962)with GDM susceptibility in Chinese women.Possible roles of insulin secretion and its regulation,inflammation,autoimmunity,lipid metabolism and mitochondrial metabolism were addressed in the pathogenesis of GDM.Part Ⅱ Mitochondrial DNA variation and risk of incident gestational diabetes mellitusObjectiveGestational diabetes mellitus(GDM)is a metabolic dysbiosis characterized by insulin resistance and islet β-cell dysfunction.Mitochondria,as the main energysupplying organelle of the cell,are essential for maintaining the metabolic homeostasis of the cell.Mitochondria possess an independent genome system,namely mitochondrial DNA(mtDNA).Human mtDNA is a double-stranded circular genome of 16,569 base pairs.It contains a displacement loop region(D-loop),which is critical for mtDNA transcription and replication,and 37 genes.Due to lack of protection from histone and an effective DNA repair system,and its location near electron respiratory chain,mtDNA is more susceptible to oxygen radicals’ attack.Therefore,a higher mutation rate can be observed in mtDNA.It is shown that mutations of mtDNA may lead to alterations of replication,transcription and expression of mitochondrial genes,together with copy number changes in mtDNA.It will further result in changes in the electron transport chain and mitochondrial dysfunction,thus hindering normal intracellular energy metabolism and ATP supply.The mitochondrial dysfunction is generally associated with the development and progression of various diseases.Previous studies in Asian Indian populations,Saudi women,and Singaporean women have found that mtDNA mutations are associated with the development of GDM.However,evidence about mtDNA variants and the risk of incident GDM is insufficient in Han Chinese women.Few analyses were performed on haplogroups and the sample size of the reported studies has been relatively small.Based on the aforementioned multi-geographic population cohort,this nested case-control study aims to investigate the association of mtDNA variants with GDM by comparing the mtDNA variants and haplotypes between the two groups through mitochondrial genome sequencing.MethodsBased on the aforementioned multi-geographic nested case-control study,blood samples of the study subjects were obtained and then sequenced for mitochondrial DNA.701 women with GDM and 859 healthy control women were included in final analysis.The distribution of mtDNA variants in different regions,nonsynonymous mutations in the coding region of mtDNA,and haplogroup assignment were compared between the two groups by Student’s t-test,chi-square test and logistic regression to investigate the link between mtDNA variants and GDM.Results1.Six mtDNA variants associated with GDM were identified,including five mtDNA variants located in the D-loop region and one in COX1 gene.m.73A>G,m.185G>A and m.16051A>G in the D-loop region were associated with a reduced risk of GDM,while m.16092T>A and m.16291C>T in the D-loop region and m.6228C>T in COX1 gene were associated with an increased risk of GDM.Compared with normal pregnant women,GDM-affected women carry fewer mtDNA variants in the D-loop region of mitochondrial DNA.2.No significant difference in the distribution of haplogroups was found between the GDM and control groups.ConclusionThis study reveals an association between mtDNA variations and GDM in Chinese Han population,highlighting the potential of screening for specific mtDNA variants during early pregnancy as a means of predicting GDM risk,which can also be a promising target for the prevention and therapy.Part Ⅲ Pregestational vaginal microbiome compositions and risk of incident gestational diabetes mellitus:a nested casecontrol studyObjectivePrevious studies have found that the vaginal,intestinal,and oral microbiomes,"the second genome of human body",are significantly altered during pregnancy in women with GDM,suggesting that microecology may also be involved in their pathogenesis.Emerging researchers suggest manipulation of the microbiome as a novel strategy for disease prevention and treatment.However,it has been proposed that metabolic features such as insulin resistance,weight gain,and higher levels of inflammation present in the GDM may also lead to physiological adaptations in their vaginal microbiome during pregnancy that are different from those of healthy women.It is contentious whether the altered vaginal microbial composition found in women with GDM during pregnancy is a reflection of the disease itself or is present prior to disease development and may serve as a driver or risk predictor of the disease.In addition,traditional studies have focused on risk factors for GDM detected during pregnancy,and emerging evidence supports the vital role of perinatal,pregestational risk factors in the development of GDM.However,studies to date on the vaginal microbiome of GDM-affected women have included relatively small samples and have only examined the reproductive tract flora during pregnancy.The study aims to investigate the pregestational vaginal microbial composition and vaginal community state types in women developing GDM and to provide new evidence for evaluating the possibility of microbiome modulation as a new strategy for GDM prevention and treatment.MethodsBased on a prospective clinical cohort of more than 10,000 women with reproductive tract microbiome sequencing,women who received assisted reproductive therapy followed by singleton births were eligible for this nested case-control design.We further perform 1:1 propensity score matching(PSM)to control the important confounding factors such as age,BMI,blood pressure,indicators of glucolipid metabolism and family history of diabetes.After matching,196 women with a subsequent development of GDM and 196 healthy women with term birth were finally included.Considering that GDM-affected women are at a higher risk for comorbid hypertensive disorders of pregnancy(HDP),women with GDM were further stratified into a GDM-only group(GDM-only)and a GDM group with combined HDP(GDMHDP)in further analysis.Vaginal discharge swabs from the subjects before pregnancy were collected.Total bacterial DNA was extracted,and the V1-V2 regions of 16S rRNA gene were amplified for high-throughput sequencing.The sequencing data were processed using Usearch software.The a and β diversity,composition and structure of the vaginal microbiota were analyzed.The differences in compositions of the vaginal microbiomes between groups were tested by permutational multivariate analysis of variance(PERMANOVA)and visualized by principal co-ordinates analysis(PCoA).To simplify analysis and stratify populations,vaginal profiles were assigned to community state types(CSTs)based on the most abundant taxon.Results1.Compared to the control group,the pregestational vaginal microbiome in women with GDM had an increased a diversity(richness).No significant difference inβ diversity of pregestational vaginal microbiome was shown between groups.After further stratification of the GDM group,no significant difference in α diversity and βdiversity in the GDM-only and the GDM-HDP groups was observed,compared with the control group.2.When comparing the bacterial abundance of vaginal bacteria in the GDM group with the control group,no differential bacteria was found at each taxonomic level.No significant differential bacteria was found between the GDM-only group and the control group.However,the pregestational vaginal microbiome of women in GDM-HDP group presented a depleted abundance of Lactobacillus crispatus and an enrichment of various bacteria such as Neisseria,Propionibacterium,Cupriavidus,Streptococcus agalactiae,Corynebacterium,Lactobacillus reuteri,and Lactobacillus gasseri compared to that of the control or GDM-only groups.3.The vaginal community state types(CSTS)dominated by Lactobacillus crispatus,Lactobacillus iners,Lactobacillus gasseri,and Lactobacillus jensenii accounted for 52.3%,37.2%,2.6%,and 1.5%of total populations,respectively.That is to say that 93.6%of women had four common Lactobacillus species as the dominant vaginal bacteria.Only 6.1%of women presented vaginal microbiomes dominated by Gardnerella vaginalis,Atopobium,Prevotella,Streptococcus,Sneathia,Bifidobacterium,Ureaplasma or Lactobacillus reuteri.4.There was no significant difference in the proportion of CSTs between the GDM group and the control group.Compared to the control and GDM-only groups,the proportion of CSTs dominated by Lactobacillus crispatus in GDM-HDP group showed a decreasing trend(49.5%in control vs.57.1%in GDM-only vs.38.1%in GDM-HDP)while the proportion of CSTs dominated by Lactobacillus iners showed an increasing trend(38.3%in control vs.34.9%in GDM-only vs.47.6%in GDM-HDP).ConclusionThis study analyzed the pregestational vaginal microbiome of Chinese women with GDM for the first time.By PSM,the study controlled the influence of important confounding factors such as age,BMI,family history of diabetes.We found limited alterations in the pregestational vaginal microbiome of women who developed GDM,which is only manifested as an increase in the richness.Of note,the pre-gestational vaginal microbiome of GDM women with HDP presented a decreased abundance of Lactobacillus crispatus,and a higher abundance of various bacteria such as Neisseria,Propionibacterium,Cupriavidus,Streptococcus agalactiae,Corynebacterium.This study complements the knowledge of the association of vaginal microbiome with GDM and its complications at different time points and improves the understanding of the etiology of GDM from a microecological perspective.更多还原