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Introduction: Single intrauterine fetal death (sIUFD) in monochorionic diamniotic (MCDA) twin pregnancy may be associated with adverse clinical outcomes and possible metabolic changes in the surviving co-twin. Metabolomic profiling has not been undertaken before in these complex twin pregnancies. Methods: In this prospectively collected case-control study, three cross-cohort comparisons were made between sIUFD MCDA (n = 16), uncomplicated MCDA (n = 16, eight pairs), and uncomplicated singleton pregnancies (n = 8). To identify major sources of variation within the sIUFD MCDA cohort, a secondary comparison was conducted between spontaneous sIUFD (n = 8) and sIUFD in MCDA twins due to selective termination of a single abnormal fetus by radiofrequency ablation (RFA) (n = 8). Metabolomics analysis of placental tissue and umbilical cord plasma was performed using LC-MS profiling. The underlying metabolic networks and pathways were analyzed by web-based platforms. Associations and statistical correlations of all identified differential metabolites with neonatal birthweight and birth length were assessed by multivariable linear regression, adjusted for maternal age and gestation. Results: Across four comparisons, 131 and 111 differential metabolites were identified in placental tissue and cord plasma, respectively, with the highest variation seen between the spontaneous vs. single-induced IUFD in MCDA twins by RFA in the cord plasma. Conversely, the number of viable fetuses and the presence of sIUFD in MCDA twins had the highest impact on metabolite variation in placental tissue. Compounds correlated with fetal growth including placental acylcarnitines and gangliosides, along with specific amino acids (e.g., histidinyl-hydroxyproline), xenobiotics and biliverdin in cord plasma. Conclusion: sIUFD in MCDA twin pregnancy correlates with distinctive metabolic signatures, mostly in fatty acyls and complex lipids, in placental tissue and cord plasma of the surviving cotwin. Some

Objectives: To investigate metabolomic perturbations caused by twin-twin transfusion syndrome, metabolic changes associated with fetoscopic laser coagulation in both placental tissue and cord plasma, and to investigate differential metabolites pertinent to varying fetal outcomes, including hemodynamic status, birth weight, and cardiac function, of live-born babies. Methods: Placental tissue and cord plasma samples from normal term or uncomplicated preterm-born monochorionic twins and those complicated by twin-twin transfusion syndrome treated with or without fetoscopic laser coagulation were analyzed by high-performance liquid chromatography metabolomic profiling. Sixteen comparisons of different co-twin groups were performed. Partial least squares-discriminant analysis, metabolic pathway analysis, biomarker analysis, and Spearman's correlation analysis were conducted based on differential metabolites used to determine potential biomarkers in different comparisons and metabolites that are pertinent to neonatal birth weight and left ventricular ejection fraction. Results: These metabolomic investigations showed that the cord plasma metabolome has a better performance in discriminating fetuses among different hemodynamic groups than placental tissue. The metabolic alteration of twin-twin transfusion syndrome in these two types of samples centers on fatty acid and lipid metabolism. The fetoscopic laser coagulation procedure improves the metabolomic change brought by this syndrome, making the metabolomes of the treated group less distinguishable from those of the control and preterm birth groups. Certain compounds, especially lipids and lipid-like molecules, are noted to be potential biomarkers of this morbid disease and pertinent to neonatal birth weight and ejection fraction. Conclusions: Fetoscopic laser coagulation can ameliorate the metabolomic alteration caused by twin-twin transfusion syndrome in placental tissue and cord plasma, which are involved mainly in fatt

OBJECTIVE: Twin-twin transfusion syndrome (TTTS) causes perinatal mortality and morbidity in monochorionic twins. The early recognition of and interventional therapy for TTTS is associated with a more favorable overall prognosis. However, the prediction by the use of ultrasound in the first trimester has relatively poor sensitivity and specificity. This study aimed to identify metabolic biomarkers to aid in ultrasound screening of TTTS. METHODS: Maternal plasma was prospectively collected between 11 and 15 weeks of gestation in apparently uncomplicated monochorionic-diamniotic twin pregnancies. This cohort was divided into: (i) patients who were subsequently diagnosed with TTTS by using ultrasound; (ii) uncomplicated matched controls. Metabolome was profiled by using gas chromatography-mass spectrometry. RESULTS: The levels of fatty acids, organic acids, oxaloacetic acid, and beta-alanine were significantly lower in the TTTS maternal plasma at 11-15 weeks of gestation, and methionine and glycine were also higher (p < 0.05, FDR<0.12). Generally, in TTTS pregnancies, the metabolisms of amino acid, carbohydrate, cofactors, vitamins, and purine were "down-regulated"; whereas bile secretion and pyrimidine metabolism were "upregulated." CONCLUSIONS: The metabolomics scanning of early gestation maternal plasma may identify those pregnancies that subsequently develop TTTS; in particular, downregulated fatty acid levels may be biologically plausible to be implicated in the pathogenesis of TTTS.

Impaired invasion of extravillous trophoblasts and severe oxidative stress manifest the poor placentation in preeclampsia, which is life-threatening and more than a hypertensive disease of pregnancy. Previous studies have reported that G protein-coupled receptor kinases (GRKs) play a key role in initiating hypertension and hypertensive renal damage, yet little evidence so far suggests a link between GRKs and preeclampsia-related hypertension. Here, we demonstrate GRK2 expression is significantly downregulated (P < 0.0001) in preeclamptic placentae compared to normotensive controls. Knockdown or inhibition of GRK2 in placentae caused insufficient arterial remodeling and elevated trophoblast necroptosis in vivo. These further induced preeclampsia-like phenotype in mice: hypertension, proteinuria, and elevated pro-angiogenic cytokines. By human extra-villous invasive trophoblast cell line (HTR8/SVneo cells), we revealed the knockdown or inhibition of GRK2 triggered excessive death with typical necroptotic characteristics: nuclear envelope rupture and the activation of RIPK1, RIPK3, and MLKL. Necrostatin-1, an inhibitor of RIPK1, is able to restore the survival of trophoblasts. Together, our findings demonstrated that insufficient GRK2 activity compromises spiral artery remodeling and initiates necrotic events in placentae, thereby leading to preeclampsia. These findings advance our understanding of GRK2 in the pathogenesis of preeclampsia and could shed light on a potential treatment for preeclampsia.

AMP-activated protein kinase (AMPK) is an important regulator of glucose metabolism, and glucose transporter 3 (GLUT3) is an efficient glucose transporter in trophoblasts. Whether placental AMPK and GLUT3 respond accordingly to gestational diabetes mellitus (GDM) remains uncertain. Here, we explored the regulatory role of AMPK in the GLUT3-dependent uptake of glucose by placental trophoblasts and the viability of the cells. In this study, the level of glycolysis in normal and GDM-complicated placentas was assessed by LC-MS/MS. The trophoblast hyperglycemia model was induced by the incubation of HTR8/SVneo cells with a high glucose concentration. GDM animal models were generated with db/ + mice and C57BL/6J mice fed a high-fat diet, and AMPK was manipulated by the oral administration of metformin. The uptake of glucose by trophoblasts was assessed using 2-NBDG or 2-deoxy-D-[3H] glucose. The results showed that GDM is associated with impaired glycolysis, AMPK activity, GLUT3 expression in the plasma membrane (PM) and cell survival in the placenta. Hyperglycemia induced similar changes in trophoblasts, and these changes were rescued by AMPK activation. Both hyperglycemic db/ + and high-fat diet-induced GDM mice exhibited a compromised AMPK-GLUT3 axis and suppressed cell viability in the placenta as well as excessive fetal growth, and all of these effects were partially alleviated by metformin. Taken together, our findings support the notion that AMPK activation upregulates trophoblast glucose uptake by stimulating GLUT3 translocation, which is beneficial for viability. Thus, the modulation of glucose metabolism in trophoblasts by targeting AMPK might ameliorate the adverse intrauterine environment caused by GDM.

BACKGROUND: Since the effectiveness of low-dose aspirin (LDA) in twin pregnancies is uncertain, we aimed to preliminarily assess whether LDA is beneficial in preventing preeclampsia in twin pregnancies. METHODS: This study is an observational study in two hospitals in China. Among 932 women, 277 in the First Affiliated Hospital of Chongqing Medical University were routinely treated with aspirin (100 mg daily) from 12 to 16 weeks to 35 weeks of gestational age, while 655 in Chongqing Health Center for Women and Children were not taking aspirin during pregnancy. We followed each subject and the individual details were recorded. RESULTS: LDA significantly reduced the risk of preeclampsia (RR 0.48; 95% CI 0.24-0.95) and preterm birth 34 weeks (RR 0.50; 95% CI 0.29-0.86) and showed possible benefits to lower the rate of SGA babies (RR 0.74; 95% CI 0.55-1.00). Moreover, the risk of postpartum hemorrhage was not increased by LDA (RR 0.89; 95% CI 0.35-2.26). CONCLUSIONS: Treatment with low-dose aspirin in twin pregnancies could offer some protection against adverse pregnancy outcomes in the absence of significantly increased risk of postpartum hemorrhage. TRIAL REGISTRATION: Chinese Clinical Trial Registry (ChiCTR); ChiCTR-OOC-16008203 , Retrospectively registered date: April 1st, 2016.

BACKGROUND: Gestational diabetes mellitus (GDM) is the most common metabolic disturbance during pregnancy and leads to an altered metabolic profile of human breast milk (HBM). The association between HBM metabolites and neonatal growth in GDM pregnancies has not been thoroughly investigated. AIMS: The primary aim was to quantify differences in the HBM metabolome between normal and GDM pregnancies. The secondary aim was to identify metabolites associated with neonatal growth during the first year postpartum. METHODS: In the present study, mothers intending to exclusively breastfeed (BF) and their newborns (mother-infant pairs) were recruited at delivery (n = 129 normal pregnancies and n = 98 GDM pregnancies). HBM samples (colostrum, transition milk, and mature milk) from mothers with normal pregnancies (n = 50) and GDM pregnancies (n = 50) were subjected to metabolomic profiling via liquid chromatography tandem mass spectrometry (LC-MS/MS). Receiver operating characteristic (ROC) analysis revealed the metabolomic fingerprints of GDM-associated mature HBM. Correlations between metabolites and neonatal body weight gain (BWG) were evaluated by Spearman correlation analysis. RESULTS: In total, 620 metabolites were identified in each HBM sample; 253 compounds had the same variation patterns, whereas 38 compounds had significantly different pattern transitions between the GDM and normal groups. Moreover, 12, 49 and 28 metabolites exhibited significant differences in the 3 milk types between the 2 groups. Twenty-two metabolites were confirmed by ROC analysis as metabolomic fingerprints in the mature BM of GDM patients. Ten compounds were significantly negatively correlated with neonatal growth, and only 2 unsaturated lipids (eicosatrienoic acid (FA 20:3) and lysophosphatidylcholine (LysoPC) (22:6)) were positively correlated with neonatal BWG. CONCLUSIONS: GDM is associated with alterations in the HBM metabolome. Only a small subset of compounds are associated with neonatal

Advanced maternal age (AMA) pregnancies are rapidly increasing and are associated with aberrant trophoblast cell function, poor placentation, and unfavorable pregnancy outcomes, presumably due to premature placental senescence. SIRT1 is an NAD+ -dependent deacetylase with well-known antiaging effects, but its connection with placental senescence is unreported. In this study, human term placentas and first-trimester villi were collected from AMA and normal pregnancies, and a mouse AMA model was established by cross breeding young and aged male and female C57 mice. SIRT1 expression and activity in HTR8/SVneo cells were genetically or pharmacologically manipulated. Trophoblast-specific Sirt1-knockout (KO) mouse placentas were generated by mating Elf5-Cre and Sirt1fl/fl mice. Trophoblast cell mobility was assessed with transwell invasion and wound-healing assays. SIRT1-binding proteins in HTR8/SVneo cells and human placental tissue were identified by mass spectrometry. We identified SIRT1 as the only differentially expressed sirtuin between AMA and normal placentas. It is downregulated in AMA placentas early in the placental life cycle and is barely impacted by paternal age. SIRT1 loss upregulates P53 acetylation and P21 expression and impairs trophoblast invasion and migration. Sirt1-KO mouse placentas exhibit senescence markers and morphological disruption, along with decreased fetal weight. In trophoblasts, SIRT1 interacts with vimentin, regulating its acetylation. In conclusion, SIRT1 promotes trophoblast epithelial-mesenchymal transition (EMT) to enhance invasiveness by modulating vimentin acetylation. AMA placentas are associated with premature senescence during placentation due to SIRT1 loss. Therefore, SIRT1 may be an antiaging therapeutic target for improving placental development and perinatal outcomes in AMA pregnancies.

BACKGROUND: Vitamin D deficiency is a global public health issue in women and children and is associated with adverse impacts on child growth, such as rickets. However, prior studies have mainly focused on measuring vitamin D levels in singleton pregnant women and their offspring, and very limited studies have revealed the prevalence of vitamin D deficiency in twin pregnant women and their offspring. The aim of this study was to investigate vitamin D levels in twin-pregnant women and their neonates. We also explored the correlation of maternal vitamin D levels with neonatal outcomes and infant growth. METHODS: A prospective subcohort investigation was carried out among 72 dichorionic, diamniotic twin-pregnant mothers and their twin offspring from the Longitudinal Twin Study. Peripheral blood was collected from the mothers in the third trimester, and cord blood was collected from neonates at birth to identify 25[OH]D levels. Data on the characteristics of the mothers and neonates were collected. Infant growth data and food sensitivities were also collected. RESULTS: The average maternal 25[OH]D level was 31.78 ng/mL, with 19.4% being deficient and 20.8% insufficient, while the average neonatal 25[OH]D level was 15.37 ng/mL, with 99.3% being deficiency or insufficient. A positive correlation was found between maternal and neonatal 25[OH]D levels (beta-value: 0.43, 95% CI: 0.37, 0.49). Interestingly, the higher the maternal 25[OH]D level was, the smaller the cotwin birthweight discordance (beta-value: -2.67, 95% CI: - 5.11, - 0.23). In addition, the infants of mothers with vitamin D deficiency were more likely to be allergic to foods at 6 months than those of mothers with vitamin D sufficiency. CONCLUSIONS: Twin neonates were at high risk of vitamin D deficiency, although their mothers' vitamin D deficiency partially improved. Higher maternal vitamin D levels were associated with smaller discordance of cotwin birthweight. TRIAL REGISTRATION: Chinese Clinical Trial Regi

BACKGROUND: Gestational diabetes mellitus (GDM) is correlated with an increased risk of adverse perinatal outcomes for both the mother and offspring. Previous research has reported correlations between maternal dietary patterns and GDM, but such evidence for twin pregnancies is lacking. This study aimed to identify maternal dietary patterns in the second trimester and investigate their relationships with the risk of GDM among women who were pregnant with twins in China. METHODS: A longitudinal twin pregnancies birth cohort study of women who were pregnant with twins in China was conducted. Maternal dietary intake in the second trimester was recorded by using a food frequency questionnaire prior to the diagnosis of GDM among participants from the prospective twin pregnancies birth cohort in Chongqing City. GDM was diagnosed with a 75 g 2-h oral glucose tolerance test at 23-26 weeks of gestation. Dietary patterns were identified by principal components analysis, and the correlations between dietary pattern and GDM were examined using multivariable logistic regression analyses. RESULTS: Of the 324 participants, 101 (31.2%) were diagnosed with GDM. Four dietary patterns were identified: a vegetable-based pattern, a poultry-and-fruit-based pattern, a sweet-based pattern and a plant-protein-based pattern. Multivariate analysis showed that none of the dietary patterns were correlated with the risk of GDM among women who were pregnant with twins, but the sweet-based dietary pattern, which was associated with a higher GDM risk for quartile 4 versus quartile 1 (OR 2.69; 95% CI: 1.09, 6.66) among non-overweight women (prepregnancy BMI < 24.0). CONCLUSION: Dietary patterns were not correlated with later GDM risk among women who were pregnant with twins in western China, whereas a high intake of sweets was associated with a higher risk for GDM among women who were not overweight prior to pregnancy. TRIAL REGISTRATION: ChiCTR-OOC-16008203. Retrospectively registered on 1 April 20

BACKGROUND The aim of this study was to assess the correlation between selective growth restriction (sGR) and co-twin utero-placental perfusion discordance by using three-dimensional power Doppler (3DPD). MATERIAL AND METHODS We prospectively recruited 60 sGR and 64 normal monochorionic-diamniotic (MCDA) twin pregnancies. Vascularization index (VI), flow index (FI), and vascularization flow index (VFI) were assessed by 3DPD, while umbilical artery pulsatility index (UA-PI), middle cerebral artery peak systolic velocity (MCA-PSV), pulsatility index (MCA-PI), and cerebroplacental ratio (CPR) were assessed by conventional Doppler imaging. RESULTS In sGR co-twins, the VI, FI, VFI, MCA-PI, and CPR were significantly lower, while the UA-PI and MCA-PSV were significantly greater, in the smaller fetuses compared with the larger fetuses; significant differences were also observed in the VI, FI, VFI, CPR, and UA-PI in normal co-twins. Compared with the appropriately grown twins, the discordances of the VI, FI, VFI, UA-PI, MCA-PI, and CPR were increased in the sGR cohort. The discordances of the VI, FI, VFI, UA-PI, MCA-PI, and CPR were associated with birthweight discordance, and the FI discordance and CPR discordance were independently associated with sGR. The combination of the FI and CPR discordance showed a higher predictive accuracy for sGR, with an area under the ROC curve of 0.813, and a sensitivity and specificity of 68.33% and 85.94%, respectively. CONCLUSIONS MCDA twin pregnancies with birthweight discordance presented utero-placental perfusion deterioration assessed by 3DPD prior to sGR diagnosis. Co-twin utero-placental perfusion discordance was significantly correlated with growth discordance, and this correlation was more predictive of sGR when 3DPD was combined with conventional Doppler imaging.

Monocarboxylate transporter 8 (MCT8) is a well-established thyroid hormone (TH) transporter. In humans, MCT8 mutations result in changes in circulating TH concentrations and X-linked severe global neurodevelopmental delay. MCT8 is expressed in the human placenta throughout gestation, with increased expression in trophoblast cells from growth-restricted pregnancies. We postulate that MCT8 plays an important role in placental development and transplacental TH transport. We investigated the effect of altering MCT8 expression in human trophoblast in vitro and in a Mct8 knockout mouse model. Silencing of endogenous MCT8 reduced T3 uptake into human extravillous trophoblast-like cells (SGHPL-4; 40%, P<0.05) and primary cytotrophoblast (15%, P<0.05). MCT8 over-expression transiently increased T3 uptake (SGHPL-4: 30%, P<0.05; cytotrophoblast: 15%, P<0.05). Silencing MCT8 did not significantly affect SGHPL-4 invasion, but with MCT8 over-expression T3 treatment promoted invasion compared with no T3 (3.3-fold; P<0.05). Furthermore, MCT8 silencing increased cytotrophoblast viability (similar to 20%, P<0.05) and MCT8 over-expression reduced cytotrophoblast viability independently of T3 (similar to 20%, P<0.05). In vivo, Mct8 knockout reduced fetal: placental weight ratios compared with wild-type controls at gestational day 18 (25%, P<0.05) but absolute fetal and placental weights were not significantly different. The volume fraction of the labyrinthine zone of the placenta, which facilitates maternal-fetal exchange, was reduced in Mct8 knockout placentae (10%, P<0.05). However, there was no effect on mouse placental cell proliferation in vivo. We conclude that MCT8 makes a significant contribution to T3 uptake into human trophoblast cells and has a role in modulating human trophoblast cell invasion and viability. In mice, Mct8 knockout has subtle effects upon fetoplacental growth and does not significantly affect placental cell viability probably due t