AMPK regulates homeostasis of invasion and viability in trophoblasts by redirecting glucose metabolism: Implications for pre-eclampsia.

Pre-eclampsia (PE) is deemed an ischemia-induced metabolic disorder of the placenta due to defective invasion of trophoblasts during placentation; thus, the driving role of metabolism in PE pathogenesis is largely ignored. Since trophoblasts undergo substantial glycolysis, this study aimed to investigate its function and regulatory mechanism by AMPK in PE development. Metabolomics analysis of PE placentas was performed by gas chromatography-mass spectrometry (GC-MS). Trophoblast-specific AMPKα1-deficient mouse placentas were generated to assess morphology. A mouse PE model was established by Reduced Uterine Perfusion Pressure, and placental AMPK was modulated by nanoparticle-delivered A769662. Trophoblast glucose uptake was measured by 2-NBDG and 2-deoxy-d-[3 H] glucose uptake assays. Cellular metabolism was investigated by the Seahorse assay and GC-MS.PE complicated trophoblasts are associated with AMPK hyperactivation due not to energy deficiency. Thereafter, AMPK activation during placentation exacerbated PE manifestations but alleviated cell death in the placenta. AMPK activation in trophoblasts contributed to GLUT3 translocation and subsequent glucose metabolism, which were redirected into gluconeogenesis, resulting in deposition of glycogen and accumulation of phosphoenolpyruvate; the latter enhanced viability but compromised trophoblast invasion. However, ablation of AMPK in the mouse placenta resulted in decreased glycogen deposition and structural malformation. These data reveal a novel homeostasis between invasiveness and viability in trophoblasts, which is mechanistically relevant for switching between the 'go' and 'grow' cellular programs.

Randomized, sham-controlled trial of acupuncture for post-cataract surgery dry eye disease.

PURPOSE: To determine the efficacy of acupuncture in patients with post-cataract surgery dry eye disease (DED). METHODS: Ninety participants with post-cataract surgery DED were randomly assigned (1:1) to receive true acupuncture (n = 44) or non-penetrating sham acupuncture (n = 46) twice weekly for 8 weeks. The primary outcome was the change from baseline to week 8 in the noninvasive tear film break-up time (TFBUT). Participants were followed until week 12. Secondary outcomes included Ocular Surface Disease Index (OSDI) score, tear meniscus height (TMH), corneal fluorescein staining (CFS) score, meiboscore and corrected distance visual acuity (CDVA). RESULTS: The estimated mean change from baseline in the noninvasive TFBUT was 1.52 for true acupuncture versus 0.77 for sham acupuncture (adjusted difference -0.75 [95% CI -1.39 to -0.12]; P = 0.02) at week 8 and 1.49 for true acupuncture versus 0.81 for sham acupuncture (adjusted difference -0.68 [95% CI, -1.29 to -0.07]; P = 0.029) at week 12. The mean change in the OSDI score was -20.13 for true acupuncture versus -13.44 for sham acupuncture (adjusted difference 6.69 [95% CI, 0.64 to 12.74]; P = 0.031) at week 8, but revealed no statistically between-group differences at week 12. Mean changes in TMH, CFS score, meiboscore and CDVA did not differ significantly between the two groups. CONCLUSIONS: Among patients with post-cataract surgery DED, acupuncture compared with sham acupuncture resulted in statistically significant benefit on tear film stability. TRIAL REGISTRATION: Chinese Clinical Trial Registry (ChiCTR1800020132).

Reactive Oxygen Species are Essential for Placental Angiogenesis During Early Gestation.

Background: Preeclampsia (PE) is associated with insufficient placental perfusion attributed to maldevelopment of the placental vasculature. Reactive oxygen species (ROS) are implicated in angiogenesis, but their regulatory effects and mechanisms in placental vascular development remain unclear. Methods: Placental oxidative stress was determined throughout gestation by measuring 4-hydroxynonenal (4HNE) and malondialdehyde (MDA). The antioxidant MitoQ was administered to pregnant mice from GDs 7.5 to 11.5; placental morphology and angiogenesis pathways were examined on GDs 11.5 and 18.5. Moreover, we established a mouse mFlt-1-induced PE model and assessed blood pressure, urine protein levels, and placental vascular development on GDs 11.5 and 18.5. Human umbilical vein endothelial cells (HUVECs) were treated with various H2O2 concentrations to evaluate cell viability, intracellular ROS levels, and tube formation capability. MitoQ, an AKT inhibitor and an ERK1/2 inhibitor were applied to validate the ROS-mediated mechanism regulating placental angiogenesis. Results: First-trimester placentas presented significantly higher MDA and 4HNE levels. MitoQ significantly reduced the blood vessel density and angiogenesis pathway activity in the placenta on GDs 11.5 and 18.5. Serum sFlt-1 levels were elevated, and we observed poor placental angiogenesis and PE-like symptoms in cases with mFlt-1 overexpression. Moderate H2O2 treatment promoted HUVEC proliferation and angiogenesis, whereas these improvements were abolished by MitoQ, AKT inhibitor, or ERK1/2 inhibitor treatment. Conclusions: Moderate ROS levels are essential for placental angiogenesis; diminishing ROS with potent antioxidants during placentation decreases placental angiogenesis and increases PE risk. Therefore, antioxidant therapy should be considered carefully for normal pregnant women during early gestation.

Association of infant feeding patterns with body fat mass and obesity in the first 2 years of life: a longitudinal twin birth cohort study.

Childhood obesity is a major public health problem with no effective intervention. We explored the influence of feeding patterns on infants' growth indices within the first 2 years in a twin birth cohort. Dietary intake at 12 months was recorded with a food frequency questionnaire, and dietary patterns were identified by principal component analysis. Milk feeding methods in first 6 months were categorised as breastfeeding or exclusive formula feeding. Correlations between feeding patterns and infants' growth indices were examined via generalised estimating equations. Two dietary patterns were identified and neither of which was related to growth indices. Breastfed infants had a higher body fat mass (BFM) percentage at 12 months, a higher body mass index (BMI) increment from birth to 6 months and a lower BMI increment from 6 to 12 months. Breastfed infants were likely positively correlated with BFM at 12 months; as complementary food was added, the effect of breastfeeding on growth gradually decreased.

Insufficient sleep during infancy is correlated with excessive weight gain in childhood: a longitudinal twin cohort study.

STUDY OBJECTIVES: To examine total sleep duration in infancy and the associations of insufficient sleep duration with later weight gain and the risk of overweight in a longitudinal twin cohort study. METHODS: The data for this study are from the Longitudinal Twin Study (LoTiS), a twin-pregnancy birth cohort study that was carried out in China (n = 186 pairs). The sleep data were collected at 6 months using the Brief Infant Sleep Questionnaire that was completed by parents with the assistance of a research assistant. Anthropometric data were obtained from the children's health clinic records at 6, 12, 18, and 24 months. RESULTS: There were no significant differences between infants with insufficient sleep and those with sufficient sleep in terms of height, weight, body mass index, incidence of overweight, and body fat mass, while infants with insufficient sleep duration were predisposed to gain excessive weight from 6 to 12 and 6 to 18 months of age (all P < .05). After adjusting for confounding variables, insufficient sleep duration was found to be correlated with excessive weight gain from 6 to 18 months of age (odds ratio: 3.47; 95% confidence interval, 1.23-9.78). The relationship was more pronounced in monozygotic twins than in dizygotic twins. CONCLUSIONS: Insufficient total sleep duration at the age of 6 months is correlated with the risk of excessive weight gain at 18 months of age in twins, particularly in monozygotic twins. CLINICAL TRIAL REGISTRATION: Registry: Chinese Clinical Trial Register; Name: Unraveling the complex interplay between genes and environment in specifying early life determinants of illness in infancy: a longitudinal prenatal study of Chinese Twins. URL: http://www.chictr.org.cn/showproj.aspx?proj=13839; Identifier: ChiCTR-OOC-16008203. CITATION: Yu J, Jin H, Wen L, et al. Insufficient sleep during infancy is correlated with excessive weight gain in childhood: a longitudinal twin cohort study. J Clin Sleep Med. 2021;17(11):2147-2154.

Advanced maternal age causes premature placental senescence and malformation via dysregulated α-Klotho expression in trophoblasts.

Advanced maternal age (AMA) pregnancy is associated with higher risks of adverse perinatal outcomes, which may result from premature senescence of the placenta. α-Klotho is a well-known antiaging protein; however, its expression and effect on the placenta in AMA pregnancies have not yet been fully elucidated. The expression patterns of α-Klotho in mouse and human placentas from AMA pregnancies were determined by Western blotting and immunohistochemistry (IHC) staining. α-Klotho expression in JAR cells was manipulated to investigate its role in trophoblastic senescence, and transwell assays were performed to assess trophoblast invasion. The downstream genes regulated by α-Klotho in JAR cells were first screened by mRNA sequencing in α-Klotho-knockdown and control JAR cells and then validated. α-Klotho-deficient mice were generated by injecting klotho-interfering adenovirus (Ad-Klotho) via the tail vein on GD8.5. Ablation of α-Klotho resulted in not only a senescent phenotype and loss of invasiveness in JAR cells but also a reduction in the transcription of cell adhesion molecule (CAM) genes. Overexpression of α-Klotho significantly improved invasion but did not alter the expression of senescence biomarkers. α-Klotho-deficient mice exhibited placental malformation and, consequently, lower placental and fetal weights. In conclusion, AMA results in reduced α-Klotho expression in placental trophoblasts, therefore leading to premature senescence and loss of invasion (possibly through the downregulation of CAMs), both of which ultimately result in placental malformation and adverse perinatal outcomes.

Gestational diabetes mellitus-associated changes in the breast milk metabolome alters the neonatal growth trajectory.

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 body weight (BW). TRIAL REGISTRATION: ChiCTR-ROC-17011508. Prospectively registered on 26 May 2017 (http://www.chictr.org.cn/listbycreater.aspx).

The Potent Antioxidant MitoQ Protects Against Preeclampsia During Late Gestation but Increases the Risk of Preeclampsia When Administered in Early Pregnancy.

Aims: Although preeclampsia (PE) has been attributed to excessive oxidative stress (OS) in the placenta, mild antioxidants failed to prevent PE in clinical trials. As mitochondria are a major source of OS, this study assessed the potential of a potent mitochondria-targeting antioxidant MitoQ in the prevention of PE. Results: Placentas from women with PE and from reduced uterine perfusion pressure (RUPP) mice demonstrated significantly higher OS, along with increased mitochondrial damage and compromised glutathione peroxidase (GPx) activities. MitoQ administration during late gestation alleviated RUPP-induced PE; whereas early-pregnancy MitoQ treatment not only exacerbated blood pressure, fetal growth restriction, and proteinuria but also reduced the labyrinth/spongiotrophoblast ratio and blood sinuses in the labyrinth. Invasion (Matrigel transwell) and migration (wound healing assay) of trophoblasts were greatly improved by 1 µM hydrogen peroxide (H2O2), but this improvement was abolished by MitoQ or MitoTempo. Mild OS enhanced the expression of miR-29b-3p, which regulates five genes involved in viability and mobility, in HTR8-S/Vneo cells. Innovation and Conclusions: Although the potent mitochondrial-targeting antioxidant MitoQ protects against hypertension and kidney damage induced by RUPP in mice when administered in late gestation, it exacerbates the PE-like phenotype when given in early gestation by interfering with placenta formation because mild OS is required to stimulate trophoblast proliferation, invasion, and migration. Eliminating trophoblastic OS during early pregnancy may lead to compromised placentation and a risk of diseases of placental origin. Therefore, antioxidant therapy for pregnant women should be carefully considered.

Gestational Diabetes Mellitus Changes the Metabolomes of Human Colostrum, Transition Milk and Mature Milk.

BACKGROUND Gestational diabetes mellitus (GDM) is a pregnancy complication that is diagnosed by the novel onset of abnormal glucose intolerance. Our study aimed to investigate the changes in human breast milk metabolome over the first month of lactation and how GDM affects milk metabolome. MATERIAL AND METHODS Colostrum, transition milk, and mature milk samples from women with normal uncomplicated pregnancies (n=94) and women with GDM-complicated pregnancies (n=90) were subjected to metabolomic profiling by the use of gas chromatography-mass spectrometry (GC-MS). RESULTS For the uncomplicated pregnancies, there were 59 metabolites that significantly differed among colostrum, transition milk, and mature milk samples, while 58 metabolites differed in colostrum, transition milk, and mature milk samples from the GDM pregnancies. There were 28 metabolites that were found to be significantly different between women with normal pregnancies and women with GDM pregnancies among colostrum, transition milk, and mature milk samples. CONCLUSIONS The metabolic profile of human milk is dynamic throughout the first months of lactation. High levels of amino acids in colostrum and high levels of saturated fatty acids and unsaturated fatty acids in mature milk, which may be critical for neonatal development in the first month of life, were features of both normal and GDM pregnancies.

Hematopoietic repopulating ability of CD34⁺ progenitor cells ex vivo expanded with different cytokine combinations.

Ex-vivo expansion technologies were developed for application of hematopoietic stem cells (HSCs) derived from cord blood (CB). The cytokine combination was essential to expand HSCs ex vivo and maintain the function of expanded HSCs. However the optimal cytokine combination was not determined. In this study, two combinations of cytokines were applied in ex-vivo expansion of HSCs to investigate the effect on the hematopoietic repopulating ability of expanded HSCs. CB CD34(+) cells were expanded with SCF + TPO + FL (STF) or SCF + TPO + FL + IL-3 + IL-6 (STF36) for 7 days and got 30.3 ± 6.4 and 39.8 ± 7.3 folds of total cells, respectively. The cells cultured by both STF and STF36 could engraft and repopulate in non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice effectively; however, the STF group achieved higher level of engraftment. These result demonstrated that the cytokine combination of STF36 favored the expansion of cells, while the cytokine combination of STF facilitated the engraftment and multi-lineage repopulating in vivo. These findings may have important implications for the cell therapy.

[Hematopoietic reconstitution of fresh and cultured cord blood CD34+ cells in NOD/SCID mice].

AIM: To study the hematopoietic reconstitution ability of fresh and cultured cord blood CD34(+) cells by sublethally irradiated NOD/SCID mice. METHODS: Mononuclear cells (MNC) were separated from cord blood and then cultured with stem cell factor (SCF)+ flt3 ligand (FL)+ thrombopoietin (TPO)+ interleukin-3 (IL-3)+ IL-6 for 14 d. CD34(+) cells were isolated from fresh or cultured MNC using miniMACS magnetic separation system. Then 4x10(5) CD34(+) cells together with 5x10(6) CD34(-) cells were injected into NOD/SCID mice. After transplantation, the dynamics of hematopoieitc recovery in the mice were measured. After 6 weeks, bone marrow (BM) and spleen samples were obtained from mice for detecting human cells. RESULTS: The number of total cells increased 1.78-fold after 14 d of culture. All recipients received fresh and cultured CD34(+) cells survived. Human cells, human lineage cells and human ALU sequence could be detected in BM and spleen. The peripheral blood counts of all transplanted mice recovered. The engraftment levels of cultured CD34(+) cells were similar to those of fresh CD34(+)cells but the percentage of human lineage cells was higher than that of fresh CD34(+) cells. CONCLUSION: CD34(+) cells cultured for 14 d still preserved the hematopoietic reconstitution ability and showed better multilineage reconstitution ability than fresh CD34(+) cells.

[Hematopoietic repopulating ability of human CD34+ cells and CD34- cells in NOD/SCID mice].

The hematopoietic repopulating ability of fresh and cultured CD34+ cells and CD34- cells derived from cord blood were compared by nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mouse model. Fresh CD34+ cells and CD34- cells were isolated from fresh cord blood. Cultured CD34+ cells and CD34- cells were separated from cultured mononuclear cells (MNC). We transplanted these cells into sublethally irradiated NOD/SCID mice via the tail vein and sacrificed surviving mice after 6 weeks. The peripheral blood, spleen and bone marrow from each mouse were harvested for flow cytometry, colony-forming cells and human Alu sequences analyses. The proportions of CD45+ cells and human multilineage hematopoietic cells in NOD/SCID mice received CD34+ cells were close to that in the mice received both CD34+ cells and CD34- cells, while it was significantly higher than that in the mice received CD34- cells. Six weeks after transplantation, all the mice injected with cultured CD34- cells dead. The survival rate of mice injected with cultured CD34+ cells was 66.7%. All of the mice injected with both cultured CD34- and CD34+ cells survived. Moreover, CD45+ cells could be detected in all surviving mice, and human CD34, CD3, CD19, CD33 and CD71 antigen also could be detected on these CD45+ cells. The results showed that both fresh and cultured CD34+ cells had the capability of engraftment and hematopoiesis reconstitution, but CD34- cells hadn't the ability. However, CD34- cells had assistant effect on the hematopoietic repopulating ability of CD34+ cells.

Hematopoietic reconstitution of CD34+ cells grown in static and stirred culture systems in NOD/SCID mice.

The hematopoietic reconstitution of cord blood (CB) CD34(+)cells grown in static and stirred system was studied. Static cultures were better than stirred cultures for cell expansion. Engraftment of stirred-culture hematopoietic stem cells (HSCs) was higher than static-culture HSCs. Stirred-culture HSCs had better multilineage reconstitution ability and colony-forming ability than static-culture HSCs. Static cultures thus favor the expansion of HSCs and stirred cultures are more effective in preserving functional HSCs.