【作者】 罗平；

【导师】 梅冰；

【作者基本信息】 长江大学 ， 临床检验诊断学， 2023， 硕士

【摘要】 妊娠期糖尿病（gestational diabetes mellitus,GDM）是妊娠期发生的不同程度的糖耐量异常,不包括妊娠前已存在的糖尿病。GDM是最常见的妊娠并发症之一,可对孕妇及其胎儿产生较大的危害,除了导致剖宫产、难产、巨大儿、新生儿低血糖等围产期不良后果外,还会导致孕妇及其子代出现较多的远期并发症,如孕妇及其子代在将来更容易患上肥胖症、2型糖尿病（type 2 diabetes mellitus,T2DM）、代谢综合征和心血管疾病等,是我国乃至全球不容忽视的公共卫生问题。目前GDM的诊断需在孕24～28周行口服葡萄糖耐量试验（oral glucose tolerance test,OGTT）,待诊断后再进行干预治疗,此时高糖状态早已对孕妇和胎儿造成了影响。由于GDM的诊断尚缺乏早期诊断生物标志物,且临床用药的安全性尚不确定,所以在妊娠早期通过高危因素筛查出高危孕妇,进行早期临床管理,降低GDM的发病率,减轻GDM对母子的短期和长期影响尤为重要。GDM的病因和发病机制复杂,受到遗传和环境因素的共同影响,是在多基因遗传的基础上,由于妊娠时内分泌变化、慢性低度炎症状态、体重增加、饮食和运动习惯改变等作用,引起胰岛素相对和（或）绝对不足,出现以高血糖为特点的糖、脂肪、蛋白质、水及电解质紊乱的一组临床综合征。由于不同地区的人群遗传背景和生活环境的差异,不同地区的GDM孕妇表现出不同的代谢特征,并对妊娠结局产生不同的影响。研究本地区人群GDM的代谢特征,对GDM患者进行有针对性的预防和管理非常重要。虽然GDM的发病机制目前尚不明确,但普遍认为各种因素导致的胰岛素抵抗和胰岛β细胞功能障碍是GDM发病的主要机制。为了满足胎儿的生长需求,无论是正常糖耐量（normal glucose tolerance,NGT）的孕妇还是GDM孕妇,都会随着妊娠的进展而出现胰岛素的敏感性降低,以达到限制母体葡萄糖利用而满足胎儿能量供应的目的。NGT孕妇可通过增加胰腺β细胞分泌胰岛素来代偿,但GDM孕妇胰岛素分泌不足,当胰岛β细胞功能无法代偿胰岛素抵抗的增加时便出现了GDM。也就是说,良好的β细胞功能在妊娠期间维持葡萄糖稳态的机制中起着至关重要的作用。据报道,胰高血糖素样肽1受体（glucagon-like peptide-1 receptor,GLP-1R）基因多态性与胰岛β细胞功能有关,并影响胰岛素的分泌和T2DM的易感性。GDM与T2DM有相似的遗传背景和发病机制,故GLP-1R基因多态性可能也在GDM的发病机制中发挥重要作用,并影响GDM的易感性。研究GLP-1R遗传多态性与GDM的关系,有助于发现GDM的遗传易感基因,对GDM的病因学和机制研究有重要意义。目的本研究分析GDM在荆州地区的发病率及相关危险因素,并评估各因素的危险程度,描述荆州地区GDM孕妇的糖代谢特征,并评估其对妊娠结局的影响,探讨GLP-1R基因多态性与GDM易感性的关系,并分析其对糖代谢的影响,为GDM的预防、管理、病因和机制研究提供科学依据。方法在湖北省荆州市的两家医院（长江大学附属荆州医院和公安县妇幼保健院）招募研究对象1588人,组建“荆州母婴健康队列”。以研究对象的孕周为时间轴,分别于孕早期、孕中期和孕晚期在此前瞻性队列的基础上开展了三项巢式病例对照研究,对应本研究的三个部分。1.第一部分依据纳入和排除标准最终选取队列中GDM患者237人作为病例组（GDM组）,NGT孕妇1315人作为对照组（NGT组）。计算GDM的发病率,并收集研究对象的社会人口学资料、病史及孕早期临床特征数据和实验室检查结果,分析GDM的危险因素。先比较病例组和对照组的社会人口学变量、临床特征变量和实验室检查结果变量,筛选出两组有差异的变量,再将两组所有有差异的变量分别进行单因素logistic回归分析,最后将单因素logistic回归分析中与GDM有关联的变量全部纳入多因素logistic回归分析,探索本地区人群GDM的独立危险因素。2.第二部分采用1:1配对病例对照研究设计。选取第一部分所有GDM研究对象,并给每一位GDM对象在第一部分的NGT孕妇中按照匹配标准匹配一位对照,依据纳入和排除标准,最终得到病例组和对照组各200人。采用Sanger测序法对研究对象的GLP-1R基因的5个标签SNP进行分型。采用二元logistic回归模型分析GLP-1R基因多态性与GDM易感性的关系。所有对象于24～28周完成了75g OGTT试验,检测空腹血糖（fasting plasma glucose,FPG）、服葡萄糖后1小时血糖（1-hour plasma glucose,1h PG）、服葡萄糖后2小时血糖水平,并对其中109对孕妇检测了基于OGTT的空腹胰岛素（fasting insulin,FINS）、服葡萄糖后1小时胰岛素、服葡萄糖后2小时胰岛素浓度。通过一些测量和计算的指标来评估不同基因型个体的β细胞功能,并分析GLP-1R基因多态性对糖代谢的影响。3.第三部分选取第二部分中1:1配对的所有研究对象,去除无分娩信息的对象,最终得到病例组192人和对照组191人。利用第二部分的实验室检测数据,计算相关指标,分析研究对象的代谢特征。收集研究对象的分娩信息,采用逐步线性回归和二元logistic回归分析评估糖代谢指标与妊娠结局的关系。结果1.GDM的发病率和高危因素分析1.1 1552名孕妇中有237人发展为GDM,GDM发病率为15.27%。小于等于30岁孕妇人群的发病率是13.27%,31岁至34岁人群的发病率达16.39%,而大于等于35岁孕妇的发病率高达22.75%。正常体重的孕妇（BMI:18.5～23.9 kg/m²）GDM发病率为13.31%,体型偏瘦人群（BMI<18.5 kg/m²）GDM发病率为13.33%,超重人群（BMI:24～27.9 kg/m²）的GDM发病率为19.35%,肥胖人群（BMI≥28kg/m²）的GDM发病率为25%。1.2年龄超过35岁的孕妇患GDM的风险是年龄小于等于30岁孕妇的1.997倍;孕前体重超重和肥胖的孕妇患GDM的风险是正常体重孕妇的1.617倍和1.864倍;有内分泌疾病的孕妇患GDM的风险是无内分泌疾病孕妇的4.404倍。孕期体重每增长1kg,GDM风险就增加10.4%;早孕期舒张压每增高1mm Hg,GDM风险就增加5%;早孕期空腹血糖值每增加1mmol/L,GDM风险就增加246.5%,早孕期丙氨酸氨基转移酶值每增高1U/L,GDM风险就增加2.1%;孕妇配偶从事自由职业或其他职业是GDM的保护因素。2.GLP-1R基因多态性对GDM易感性和糖代谢的影响2.1在校正混杂因素后,GLP-1R的标签SNP rs6458093突变基因型AG+GG会增加GDM的风险,尤其在年龄小于35岁（P=0.024）和BMI大于等于24 kg/m²（P=0.041）的孕妇人群中更明显。2.2携带GLP-1R rs6458093基因型AG+GG的孕妇60分钟胰岛素分泌指数(insulinogenic index at 60 min,IGI₆₀)（P=0.032）和葡萄糖处置指数（disposition index,DI）（P=0.029）显著低于携带野生基因型AA的孕妇,而1h PG显著高于携带基因型AA的孕妇。2.3 GLP-1R rs3765467的突变杂合子基因型GA会降低GDM风险,且这种作用在年龄大于等于35岁的孕妇人群中更明显（P=0.037）,但未观察到该位点与糖代谢的关联。3.GDM的糖代谢特征及对妊娠结局的影响3.1 GDM组的空腹和餐后血糖指标都显著高于NGT组,而空腹和餐后胰岛素反应都显著低于NGT组。GDM组的稳态模型评估β细胞功能指数（homoeostasis model assessment ofβcell function,HOMA-β）、DI和松田胰岛素敏感指数(Matsuda insulin sensitivity index,ISI_Matsuda)均显著低于NGT组,但两组间稳态模型评估胰岛素抵抗指数无显著差异。3.2亚组分析显示单纯空腹血糖受损（impaired fasting glucose,IFG）亚组仅FINS/FPG显著低于NGT组,而单纯糖耐量受损（impaired glucose tolerance,IGT）亚组和空腹血糖和糖耐量均受损（impaired fasting and stimulated glucose,IFSG）亚组均存在空腹和餐后胰岛素反应不足。此外,IFSG亚组的葡萄糖指标最高而胰岛素指标最低,且ISI_Matsuda和HOMA-β显著低于NGT组。3.3 FPG值越高,新生儿出生体重越重。1h PG和FINS/FPG值越高,分娩孕周就越早。胰岛素曲线下面积、IGI60和DI与早产独立相关。IFG亚组剖宫产的风险是NGT组的2.319倍。FPG、FINS/FPG、葡萄糖曲线下面积、胰岛素曲线下面积与葡萄糖曲线下面积的比值和HOMA-β与巨大儿风险独立相关。结论1.本地区GDM的发病率是15.27%。年龄超过35岁、孕前超重或肥胖、孕期体重增长过快、早孕期血压过高、早孕期空腹血糖值偏高、早孕期丙氨酸氨基转移酶值偏高、患内分泌疾病是GDM的独立危险因素。孕妇配偶从事自由职业或其他职业是GDM的保护因素。2.GLP-1R标签SNP rs6458093基因多态性与GDM的遗传易感性有关,并影响β细胞功能和餐后糖代谢,突变基因型AG+GG是GDM的危险因素。GLP-1R标签SNP rs3765467基因多态性与GDM的遗传易感性有关,突变杂合子基因型GA是GDM的保护因素。3.导致荆州地区孕妇发生GDM的决定性因素是β细胞功能障碍而不是胰岛素抵抗。本研究人群GDM患者与NGT孕妇的妊娠结局未观察到显著性差异。IFG亚组和IGT、IFSG亚组可能有不同的发病机制,且IGT也许是IFSG的早期阶段。多项糖代谢参数（尤其是FPG）与不良妊娠结局有关,可作为妊娠期糖代谢监测指标。更多还原

【Abstract】 Gestational diabetes mellitus（GDM）is defined as“any degree of glucose intolerance during pregnancy,excluding overt diabetes in pregnancy”.GDM is one of the most common complications during pregnancy.GDM has become an important public health concern worldwide due to its adverse outcomes for both mothers and their offspring.Cesarean section,dystocia,macrosomia and neonatal hypoglycemia are the most common perinatal complications of GDM.In addition,women with a history of GDM and their offspring are more likely to become obese and develop type 2 diabetes mellitus（T2DM）,metabolic syndrome and cardiovascular disease in the long-term.Currently,the diagnosis of GDM depends on the oral glucose tolerance test（OGTT）at24～28 weeks of gestation when the pregnant women and the fetus have been affected by the hyperglycemic status.Due to the lack of early diagnostic biomarkers of GDM and the uncertain safety of clinical drugs,it is particularly important to reduce the incidence of GDM and the short-term and long-term effects on the mothers and their offspring by screening and managing high risk pregnant women through high risk factors in early pregnancy.The etiology and pathogenesis of GDM remain vague due to its complexity under the combined action of environmental and genetic factors.GDM is a group of clinical syndromes based on multigene inheritance characterized by hyperglycemia and disturbances of glucose,lipids,protein,water,and electrolytes caused by relative and/or absolute insufficiency of insulin due to hormonal changes,chronic low-grade inflammation,weight gain,and changes in eating and exercise habits during pregnancy.Due to the differences in genetic background and living environment,pregnant women with GDM in different regions show different metabolic characteristics and have different effects on pregnancy outcome.It is very important to explore the metabolic characteristics of GDM in the local population for targeted prevention and management.It is also useful for understanding and explaining the mechanism of GDM.Although the pathogenesis of GDM is still not entirely clear,it is generally believed that insulin resistance and pancreaticβcells dysfunction are the main pathogenesis of GDM.To meet the energy needs of the growing fetus by limiting the mother’s consumption of glucose,insulin sensitivity decreases.Pregnant women with normal glucose tolerance（NGT）can compensate for increasing insulin resistance by increasing the secretion of insulin,while women with GDM have insufficient insulin secretion.GDM occurs when pancreaticβcell fail to compensate for increased insulin resistance.Genetic variants of glucagon-like peptide-1 receptor（GLP-1R）have been reported to be associated withβcell function and affect insulin secretion and susceptibility of T2DM,so they may also play an important role in the pathogenesis of GDM and affect susceptibility of GDM.Exploring the relationship between GLP-1R polymorphism and GDM is helpful to discover the genetic susceptibility genes of GDM,and has important significance for the etiology and mechanism of GDM.ObjectiveThe purpose of this study was to provide a scientific basis for the prevention,management,and interpretation of etiology and mechanism of GDM through analyzing the incidence and risk factors of GDM in Jingzhou and evaluating the risk degree of each factor,describing the metabolic characteristics of GDM and assessing their effects on perinatal outcomes,and exploring the relationship between GLP-1R polymorphism and susceptibility to GDM and valuing the influences of GLP-1R polymorphism on glucose metabolism.MethodsWe recruited 1588 subjects from Jingzhou Hospital Affiliated to Yangtze University and Gongan County Maternal and Child Health Care Hospital in Jingzhou,Hubei Province,and established the"Jingzhou maternal and child health cohort".Three nested case-control studies were conducted based on this prospective cohort with gestational age as the time axis.The three studies were conducted in the first trimester,the second trimester and the third trimester,corresponding to the three parts of this study.1.The first part included 237 pregnant women with GDM（case group）and 1315pregnant women with NGT（control group）selected from the cohort according to the inclusion and exclusion criteria.The incidence of GDM was calculated,and the risk factors of GDM were analyzed according to the socioldemographic data,medical history,clinical characteristics and laboratory results of the subjects.First,social demographic variables,clinical characteristics variables and laboratory test results variables were compared between the case group and the control group to screen out the variables with differences,and then all the variables with differences between the two groups were respectively carried out univariate logistic regression analysis.Finally,all variables associated with GDM in univariate logistic regression analysis were included in multivariate logistic regression analysis to explore independent risk factors for GDM of the local population.2.The second part was a 1:1 paired case-control study.All subjects with GDM in the first part were selected into the case group and each GDM subject was matched to one control in the control group of the first part according to the matching criteria.Finally,200 pregnant women with GDM and 200 pregnant women with NGT were included according to the inclusion and exclusion criteria and genotyped for five tag SNPs of GLP-1R using Sanger sequencing.Binary logistic regression was used to evaluate the relationship between GLP-1R polymorphisms and GDM risk.All subjects underwent 75g OGTT at 24～28 weeks to measure fasting plasma glucose（FPG）,1-hour plasma glucose（1h PG）and 2-hour plasma glucose levels,and 109 pairs of pregnant women were tested for OGTT-based fasting insulin（FINS）,1-hour insulin and 2-hour insulin concentrations.Several measured and calculated indices were used to evaluate theβcell function of the individuals with different genotypes and analyze the effect of GLP-1R gene polymorphisms on glucose metabolism.3.The third part included 192 subjects with GDM and 191 subjects with NGT selected from all the subjects in the second part excluding the subjects without delivery data.Several indices were calculated to describe the metabolic characteristics of the subjects based on the concentrations of glucose and insulin in the second part.The relationship between glucose metabolism parameters and pregnancy outcomes was evaluated using stepwise linear regression and binary logistic regression.Results1.The incidence and risk factors of GDM1.1 Among the 1552 subjects,237 subjects developed GDM.The incidence of GDM was 15.27%.The incidence of GDM is 13.27%in pregnant women under the age of 30years old,16.39%in those between 31 and 34 years old,and 22.75%in those who are at least 35 years old.The incidence of GDM was 13.31%in subjects with normal weight（BMI:18.5～23.9 kg/m²）,13.33%in lean women（BMI<18.5 kg/m²）,and 19.35%in overweight women（BMI:24～27.9 kg/m²）,and 25.00%in obese people（BMI≥28 kg/m²）.1.2 The risk of GDM in pregnant women older than 35 years was 1.997 times higher than that in pregnant women younger than 30 years.The risk of GDM was 1.617 times and 1.864 times higher in overweight and obese pregnant women than in normal weight pregnant women.The risk of GDM in pregnant women with endocrine diseases was 4.404times higher than that in pregnant women without endocrine diseases.The risk of GDM increased by 10.4%for every extra kilogram of weight gained.For every 1mm Hg increase of diastolic blood pressure during early pregnancy,the risk of GDM increased by 5%.The risk of GDM increased by 246.5%for every 1 mmol/L increase of fasting blood glucose and 2.1%for every 1 U/L increase of alanine aminotransferase in early pregnancy.Freelance work or other occupations of a pregnant woman’s spouse was a protective factor for GDM.2.The effects of GLP-1R polymorphisms on GDM susceptibility and glucose metabolism2.1 Mutant genotype AG+GG of GLP-1R tag SNP rs6458093 increased GDM risk（P=0.049）,especially among subjects younger than 35 years（P=0.024）and with BMI no less than 24 kg/m²（P=0.041）after adjusting for confounders.2.2 Compared with subjects with wild genotype AA,subjects with genotype AG+GG of GLP-1R tag SNP rs6458093 also showed significantly lower insulinogenic index at 60min(IGI₆₀)（P=0.032）and disposition index（DI）（P=0.029）,as well as significantly higher 1-hour plasma glucose levels（P=0.045）.2.3 The mutant heterozygous genotype GA of GLP-1R tag SNP rs3765467 decreased GDM risk among subjects older than 35 years（P=0.037）but showed no association with insulin secretion and glucose homeostasis.3.Metabolic characteristics of GDM and the effects on pregnancy outcomes3.1 Compared with the NGT group,the GDM group showed significantly higher fasting and postprandial glucose parameters but significantly lower fasting and postprandial insulin responses.Meanwhile,the GDM group had significantly lower homoeostasis model assessment ofβcell function（HOMA-β）,DI and Matsuda insulin sensitivity index(ISI_Matsuda)but comparable homoeostasis model assessment of insulin resistance.3.2 The impaired fasting glucose（IFG）subgroup showed significantly lower FINS/FPG only,while the impaired glucose tolerance（IGT）and impaired fasting and stimulated glucose（IFSG）subgroups showed deficiency in both fasting and postprandial insulin response.The IFSG subgroup had the highest glucose parameters and the lowest insulin parameters,as well as significantly lower ISI_Matsuda and HOMA-βthan the NGT group.3.3 The higher the level of FPG,the heavier the birth weight.The higher the 1h PG and FINS/FPG values,the earlier the gestational age of delivery.The area under the insulin curve（AUC-INS）,IGI₆₀ and DI were related to premature delivery risk after adjusting for confounders.The IFG subgroup of GDM was 2.319 times more likely to be subjected to cesarean section than the NGT group.FPG,FINS/FPG,area under the glucose curve（AUC-GLU）,AUC-INS/AUC-GLU and HOMA-βwere related to macrosomia risk.Conclusion1.The incidence of GDM in Jingzhou was 15.27%.Advanced age（over 35 years old）,overweight or obese before pregnancy,rapid weight gain during pregnancy,high blood pressure,FPG or ALT in early pregnancy and endocrine diseases were independent risk factors for GDM.Freelance work or other occupations of a pregnant woman’s spouse was a protective factor for GDM.2.Tag SNP rs6458093 of GLP-1R was associated with increased GDM risk and affectedβcell function and postprandial glucose metabolism,and mutant genotype AG+GG of rs6458093 might be a risk factor for GDM.Tag SNP rs3765467 of GLP-1R was associated with decreased GDM risk,and mutant heterozygote genotype GA might be a protective factor for GDM.3.βcell dysfunction rather than insulin resistance determined the occurrence of GDM in Jingzhou pregnant women.Women with GDM had a similar risk of adverse perinatal outcomes to women with NGT in this study.The IFG group might have a different pathogenesis from the IGT and IFSG groups and that IGT might be a transitional stage of IFSG.Several glucose metabolism parameters（especially FPG）were associated with adverse perinatal outcomes and could be used as indicators to monitor glucose metabolism during pregnancy.更多还原