妊娠糖尿病的早期篩檢、預測及病理生理機轉： 來自基礎及臨床研究的新證據

Abstract

研究背景： 隨著生育年齡提高，愈來愈多婦女罹患妊娠糖尿病（gestational diabetes mellitus）。罹患妊娠糖尿病的孕婦，會增加媽媽及胎兒的周產期併發症及未來發生肥胖與糖尿病的風險，所以，了解妊娠糖尿病的病理生理機轉，進而發展預測與治療的方法，是臨床上很重要的問題。雖然治療妊娠糖尿病能改善許多周產期後果，但一些懷孕不良事件及長期後果的風險仍無法減輕。目前建議篩檢妊娠糖尿病的時間為孕期第24到28週，但文獻指出早在數週之前，罹患妊娠糖尿病的孕婦腹中的胎兒就已經加速成長。如果提早篩檢與介入，可能可以改善這群孕婦的懷孕結果及小孩的健康。本研究第一部分會測試早期篩檢與介入比起常規篩檢與介入能否改善懷孕後果。 在病理生理機轉方面，肥胖是導致妊娠糖尿病的危險因子且與胰島素阻抗相關，在過往文獻發現，human placental lactogen（CSH2）與growth hormone variant（GH2）與孕期胰島素阻抗以及β細胞增生有關，然而人類研究卻發現不一致的結果，因此應該還存在其他GDM病理生理機轉。Angiopoietin-like 4（ANGPTL4）是一種分泌醣蛋白，在非懷孕族群的研究中發現能促進胰島素分泌，同時造成胰島素阻抗，且與發炎反應有關。懷孕時，血中ANGPTL4會隨孕期增加，這些調節血糖的功能與懷孕時的變化與CSH2及GH2很類似，然而目前沒有文獻探討懷孕時ANGPTL4在血糖代謝與GDM病理生理機轉的角色。我們初步研究發現，肥胖孕婦胎盤ANGPTL4的表現，比正常體重的孕婦高，且孕期血中ANGPTL4濃度與胰島素阻抗有關。本研究第二部分會深入探討ANGPTL4在妊娠糖尿病的病理生理機轉所扮演的角色，我們的假說是：肥胖會使游離脂肪酸增加，刺激胎盤滋養細胞表現與分泌ANGPTL4。胎盤的ANGPTL4可能會引起內質網壓力與發炎反應，進一步影響CSH2與GH2的表現與分泌。此外，胎盤製造的ANGPTL4也會分泌到血液中，與CSH2及GH2一起影響孕婦全身的胰島素敏感性，最後導致妊娠糖尿病的發生。 另一方面，與妊娠糖尿病相關的懷孕後果中，大於胎齡（large for gestational age，LGA）是最常被選作主要目標，LGA且足月的胎兒會增加許多懷孕不良後果的風險，然而，在早產兒中，LGA與懷孕不良後果的關係仍不確定，本研究第三部分會完整評估足月兒與不同程度的早產兒，LGA與各種早產相關及懷孕常見併發症的關係。最後，台灣用來評估是否符合LGA的新生兒出生體重參考標準已經許久沒更新，本研究第四部分會分析台灣全國性新生兒出生體重，更新參考標準並製作生長曲線圖。 材料與方法： 第一部份，我們在臺大醫院及台大新竹分院執行開放式隨機分派試驗，預期收錄2054位懷孕婦女。這些婦女需生產單胞胎且從未被診斷為糖尿病，之後依隨機分配分成兩組，一組為早期篩檢，於孕期第18-20週執行75克糖水測試。另一組為常規篩檢，於孕期第24–28週執行糖水測試。若糖水測試時的血糖濃度，有任一個數值達標：空腹 ≥ 92 mg/dL、一小時 ≥ 180 mg/dL、二小時 ≥ 153 mg/dL，即診斷妊娠糖尿病。確診的孕婦會接受生活方式介入並執行自我血糖監控。如果4週內血糖沒有達標，會給予藥物治療。我們比較兩組主要結果的差異，包含剖腹產率、新生兒出生體重超過90百分位比率、新生兒低血糖率、臍帶血中C-胜肽超過90百分位比率、妊娠高血壓、子癲前症及生產創傷的綜合測量（目的1）。 第二部分，我們以之前收錄的孕婦世代進行人類研究，比較正常孕婦與肥胖孕婦胎盤的ANGPTL4、內質網壓力、發炎指標、CSH2與GH2的表現（目的2）；我們也檢測孕期的血液檢體，以了解不同孕期血中的ANGPTL4濃度與游離脂肪酸、CSH2、GH2濃度以及β細胞功能指標以及胰島素阻抗指標之間的關係（目的3），並釐清懷孕早期及中期是否能預測妊娠糖尿病的發生（目的4）。此外，我們利用3A-sub-E滋養細胞株，探討棕櫚酸會否刺激ANGPTL4表現（目的5），以及滋養細胞過度表現ANGPTL4是否會引起內質網壓力、發炎反應以及調控CSH2與GH2的表現與分泌（目的6）。最後，我們利用骨骼肌L6細胞株，探討外加ANGPTL4重組蛋白對於骨骼肌細胞胰島素敏感性與胰島素訊息傳遞的影響（目的7）。 第三部分，我們利用 2007–2018年台灣出生通報檔與健保資料庫連結，進行回溯性全國世代研究，納入24–42週單胞胎新生兒，並依其出生週數分為足月（37–42週）、晚期早產（34–36週）、中度早產（32–33週）、高度早產（28–31週）及極度早產（24–27週）。LGA依2013世界衛生組織生長曲線及台灣生長曲線定義。我們比較不同出生週數各組LGA與適當胎齡（appropriate for gestational age，AGA）新生兒之周產期後果（目的8）。 第四部分，我們分析2008–2017年台灣出生通報檔中，1,913,124名單胞胎新生兒的資料（目的9）。針對每一完整妊娠週數與性別，計算出生體重百分位，並採用非線性resistant smoothing技術，繪製平滑化的妊娠週數出生體重百分位曲線（目的10）。 結果： 第一部份之隨機分派試驗於期中分析後因無效性提前終止。意向性分析共納入967名受試者；主要結果在兩組間無顯著差異。與常規篩檢組相比，早期篩檢組的新生兒低血糖發生率顯著較低，而早期篩檢組中的妊娠糖尿病產婦之新生兒脂肪堆積顯著較高。兩組不良事件率相近。 在第二部分中，我們發現肥胖孕婦之胎盤ANGPTL4表現顯著高於正常體位對照組。棕櫚酸可誘導滋養層細胞表現ANGPTL4且表現量與棕櫚酸劑量成正比。於滋養層細胞過度表現ANGPTL4會引發內質網壓力，進而刺激GH2之表現與分泌，但不影響CSH2。在 L6 骨骼肌細胞中，分泌形式的ANGPTL4透過表皮生長因子受體（EGFR）/細胞外訊號調節激酶1/2（ERK1/2）路徑抑制胰島素介導的葡萄糖攝取。於孕婦世代中，第一孕期血漿ANGPTL4濃度可預測妊娠糖尿病發生，並與身體質量指數、血漿三酸甘油脂及GH2濃度呈正相關；而與第二孕期之胰島素敏感指數 ISI0,120呈負相關。 在1,602,638名台灣新生兒中，44,359名（2.8%）依世界衛生組織（WHO）標準被歸為LGA（第三部份）。與AGA相比，足月與晚期早產 LGA新生兒於初次剖腹產、產程遲滯、新生兒低血糖、生產創傷、缺氧缺血性腦病變、需光照治療的黃疸、呼吸窘迫、入住新生兒加護病房、新生兒敗血症與胎兒死亡之風險顯著升高；然而於中度、高度及極度早產組別，大多數上述風險並未增加。相反地，LGA會降低下列風險，包括在高度早產組別中的初次剖腹產、新生兒敗血症及肺支氣管發育不全，於中度與高度早產組別中的需光照治療的黃疸、呼吸窘迫及入住新生兒加護病房，以及在晚期、中度與高度早產組別中的早產兒視網膜病變。以台灣生長曲線標準重新分析結果一致。 在第四部份中，我們納入出生通報檔資料庫中994,175名男嬰與918,949名女嬰進行分析。從2008至2017年，平均出生體重由3,098.10 g降至3,049.88 g（p < 0.0001）；低出生體重年發生率由5.59%升至6.98% （p < 0.0001）。同時觀察到平均妊娠週數微幅下降 （p < 0.001），外籍母親比例顯著下降（p < 0.0001）。本研究亦提供按妊娠週數分層之男、女單胞胎出生體重百分位。 結論： 比起現行標準做法，妊娠糖尿病普篩提前至18–20週對整體妊娠結果可能無明顯助益，但可能降低新生兒低血糖；透過早期篩檢確診之妊娠糖尿病產婦其新生兒具有較高新生兒脂肪堆積。其次，肥胖會誘導胎盤ANGPTL4表現，並透過引發內質網壓力及促進GH2分泌參與妊娠糖尿病之病理生理機轉；分泌形式的ANPGTL4可於骨骼肌誘發胰島素阻抗，並可作為早期預測妊娠糖尿病之生物標誌。此外，LGA 與足月及晚期早產新生兒之多項周產期併發症風險增加相關，但在更早週數之早產組別則無此現象，臨床應依妊娠週數調整LGA新生兒之處理策略。最後，本研究建立了台灣最新的全國性出生體重曲線，並確認 2008–2017年低出生體重率持續上升，建議進一步探討母體與環境因子對此趨勢的影響。

Background: The prevalence of gestational diabetes mellitus (GDM) has been increasing in parallel with the rising maternal age. Women with GDM are at increased risk of maternal and fetal perinatal complications, future development of maternal diabetes, and neonatal obesity. Therefore, elucidating the pathophysiology of GDM and advancing its prediction and treatment are of great clinical importance. Although treatment for GDM can improve many perinatal outcomes, risks of some adverse pregnancy events and long-term outcomes cannot be mitigated. Studies have pointed that women with GDM have excessive fetus growth weeks earlier than the current recommended screening period at 24-28 weeks of gestation. Therefore, earlier screening and intervention may improve pregnancy outcomes and the health of the offspring. In the first part of this study, we would test if early screening and intervention for GDM can improve pregnancy outcomes compared to standard screening and intervention. In the aspect of the pathophysiology of GDM, obesity is a risk factor for GDM and is associated with insulin resistance. Animal studies have reported that placental lactogen (CSH2) and growth hormone variant (GH2) were associated with insulin resistance and β-cell proliferation during pregnancy. However, human studies showed inconsistent results, which indicates that other pathophysiologies may exist in the disease process of GDM. Angiopoietin-like 4 (ANGPTL4) is a secretory glycoprotein. Researches in non-pregnant subjects have shown that ANGPTL4 could induce insulin secretion, while it also caused insulin resistance and inflammation. During pregnancy, the levels of plasma ANGPTL4 increased gradually. The function in glucose metabolism and changes during pregnancy of ANGPTL4 are similar to those of CSH2 and GH2. However, there was currently no literature regarding the role of ANGPTL4 in glucose metabolism during pregnancy and the pathophysiology of GDM. In our preliminary data, we found that placental ANGPTL4 expression was higher in obese pregnant women. In addition, serum ANGPTL4 levels at early pregnancy were positively associated with insulin resistance index. In the second part of this study, we would investigate the role of ANGPTL4 in the pathophysiology of GDM. We hypothesized that obesity results in an increase in plasma free fatty acid, which stimulates the expression and secretion of ANGPTL4 in placenta. ANGPTL4 could induce endoplasmic reticulum (ER) stress and inflammation, and regulate the expression and secretion of CSH2 and GH2 in placenta. Moreover, ANGPTL4 in placenta could be secreted into circulation to have an endocrine effect, which led to the development of insulin resistance and GDM during pregnancy, along with the effect of plasma CSH2 and GH2. On the other hand, among pregnancy outcomes associated with GDM, large for gestational age (LGA) is most commonly chosen as the primary outcome. Term infants with LGA carry an increased risk of various adverse pregnancy outcomes. However, the relationship between LGA and adverse pregnancy outcomes in preterm infants remains uncertain. Therefore, the third part of this study would comprehensively evaluate the association between LGA and prematurity-related and common pregnancy outcomes across term infants and infants with different degrees of prematurity. Lastly, the Taiwanese newborn birth weight reference standards used to determine LGA status have not been updated for many years. Hence, the fourth part of this study would analyze nationwide newborn birth weight data in Taiwan, update the reference standards, and produce new growth charts. Materials and methods: In the first part of this study, we aimed to conduct an open-label randomized controlled trial (RCT) in 2054 pregnant women, who delivered a singleton and who have not been diagnosed with overt diabetes mellitus at National Taiwan University Hospital and its HsinChu branch. These pregnant women were randomly assigned to two groups, an early screening group and a standard screening group. Subjects in the early screening group received a 75g 2-hour oral glucose tolerance test (OGTT) at 18-20 weeks of gestation and those in the standard screening group would receive an OGTT at 24-28 weeks of gestation. GDM was diagnosed if one of the plasma glucose levels at fasting, 1-hour, and 2-hour during an OGTT was equal or above 92 mg/dL, 180 mg/dL, or 153 mg/dL respectively. Subjects who were diagnosed with GDM received lifestyle intervention and self-monitoring of blood glucose. Pharmacological therapies were given when the target of glycemic control is not achieved within 4 weeks. We compared the primary outcome between the two groups, which was a composite measure of primary cesarean section, birth weight > 90th percentile, neonatal hypoglycemia, cord serum C-peptide > 90th percentile, gestational hypertension, preeclampsia, and birth trauma (specific aim 1). In the second part of this study, we used the samples collected previously in a pregnancy cohort. We compared the expression of ANGPTL4, ER stress markers, inflammatory markers, CSH2, and GH2 in the placenta of obese pregnant women and lean pregnant women (specific aim 2). We measured blood samples in the first and the second trimesters and analyze the relationship between serum ANGPTL4, free fatty acid, CSH2, GH2, β-cell function and insulin resistance markers (specific aim 3). We also clarified whether serum ANGPTL4 levels at first and second trimesters or the incremental levels could predict the incidence of GDM (specific aim 4). In addition, we used 3A-sub-E trophoblast cell line to test whether palmitic acid could induce ANGPTL4 expression (specific aim 5). We also explored the effect of ANGPTL4 overexpression of trophoblasts on ER stress, inflammation, and the expression and secretion of CSH2 and GH2 (specific aim 6). Using L6 skeletal muscle cell lines, we investigated the effect of recombinant ANGPTL4 treatment on insulin signaling and glucose uptake (specific aim 7). In the third part of this study, using the Birth Reporting Databases (BRD) (2007-2018) linked to Taiwan’s National Health Insurance Research Database, we conducted a retrospective nationwide cohort study of singleton neonates delivered between 24-42 weeks of gestation. Based on gestational age at delivery, the enrolled neonates were classified into term (37-42 weeks of gestation), late preterm (34-36 weeks of gestation), moderate preterm (32-33 weeks of gestation), very preterm (28-31 weeks of gestation), and extremely preterm (24-27 weeks of gestation). LGA was defined by the 2013 World Health Organization (WHO) growth standard and the Taiwan growth standard. Perinatal outcomes were compared between LGA and appropriate for gestational age (AGA) neonates across different gestational age groups (specific aim 8). In the fourth part of this study, we analyzed 1,913,124 singleton live births from the dataset of Taiwan BRD between 2008 and 2017 (specific aim 9). Birth weight percentiles were calculated for each completed gestational week and gender. A nonlinear, resistant smoothing technique was applied to develop smoothed birth weight-for-gestational-age percentile curves (specific aim 10). Results: The RCT in the first part was stopped early for futility after the interim analysis. Of the 967 women included in the intention-to-treat analysis, the primary outcome was not significantly different between the two groups. Neonatal hypoglycemia was significantly lower and neonatal adiposity in women with GDM was significantly higher in the early screening group compared with the standard screening group. Adverse event rates were similar between the two groups. In the second part, we found that ANGPTL4 expression in the placenta was higher in obese pregnant women than in lean controls. Palmitic acid significantly induced ANGPTL4 expression in trophoblast cells in a dose-response manner. ANGPTL4 overexpression in trophoblast cells resulted in ER stress, which stimulated the expression and secretion of GH2 but not CSH2. In L6 skeletal muscle cells, soluble ANGPTL4 suppressed insulin-mediated glucose uptake through the epidermal growth factor receptor (EGFR)/extracellular signal-regulated kinases 1/2 (ERK 1/2) pathways. In pregnant women, plasma ANGPTL4 concentrations in the first trimester predicted the incidence of GDM, were positively associated with body mass index, plasma triglyceride, and plasma GH2 in the first trimester, and were negatively associated with insulin sensitivity index ISI0,120 in the second trimester. Among the 1,602,638 neonates in Taiwan, 44,359 were classified as LGA by the 2013 WHO growth standard (the third part). Compared to AGA neonates, LGA neonates in term and late preterm groups exhibited higher risks of primary cesarean section, prolonged labor, neonatal hypoglycemia, birth trauma, hypoxic ischemic encephalopathy, jaundice needing phototherapy, respiratory distress, neonatal intensive care unit (NICU) admission, newborn sepsis, and fetal death. However, most of these risks were not increased in moderate, very, and extremely preterm groups. Conversely, being LGA was associated with lower risks of primary cesarean section, newborn sepsis, and bronchopulmonary dysplasia in the very preterm group, and jaundice needing phototherapy, respiratory distress, and NICU admission in the moderate and very preterm groups, and retinopathy of prematurity in the late, moderate, and very preterm groups. These findings remained consistent when the Taiwan growth standard was applied. In the fourth part, we included 994,175 male neonates and 918,949 female neonates in the BRD cohort. During the study period, the annual mean birth weight decreased significantly from 3098.10g to 3049.88g (p < 0.0001). A significant increase in the annual rate of low birth weight (LBW) was observed, rising from 5.59% in 2008 to 6.98% in 2017 (p < 0.0001), coinciding with a slight and significant decrease in gestational age (p < 0.001) and a significantly declining proportion of foreign-born mothers (p < 0.0001) during the same period. In addition, birth weight percentiles by weeks of gestation in male and female singleton births were reported. Conclusion: Compared to standard practice, advancing universal GDM screening to 18-20 weeks of gestation may not improve pregnancy outcomes, except for a reduction in neonatal hypoglycemia. Newborns of women diagnosed with GDM through early screening had higher neonatal adiposity. Subsequently, placental ANGPTL4 is induced by obesity and is involved in the pathophysiology of GDM via the induction of ER stress and GH2 secretion. Soluble ANGPTL4 can lead to insulin resistance in skeletal muscle cells and is an early biomarker for predicting GDM. Additionally, being LGA is associated with increased risks of perinatal complications in term and late preterm neonates, but not in earlier preterm groups. These findings underscore the importance of tailoring management strategies for LGA neonates to consider different degrees of prematurity. Finally, this study developed an updated, nationwide birth weight chart for Taiwan. An increasing rate of LBW was observed from 2008 to 2017. Further investigation into maternal and environmental factors contributing to these trends is warranted.