Differences in prenatal diagnosis rate of congenital anomalies associated with singletons and multiple births: An observational study of more than 1.9 million births in Zhejiang Province, eastern China, during 2012-2018.

OBJECTIVE: To characterize differences in the prenatal detection of congenital anomalies (CAs) associated with singleton and multiple births. METHODS: This observational study covered all births registered in the CA surveillance system in Zhejiang Province of China during 2012-2018. Differences in the incidence and characteristics between singletons and multiple births with CAs were tested. Multivariate logistic regression models were performed to explore the associations of prenatal detection rate of CAs with multiple births. RESULTS: Totals of 49 872 singletons and 3324 multiple births with CAs were analyzed. The mean incidences of CA for single and multiple births were 27.12 and 54.42 per 1000 births, respectively. After adjustment for covariates, CAs associated with multiple births were less likely to be diagnosed prenatally (adjusted odds ratio [OR] 0.38, 95% confidence interval [CI] 0.34-0.43), as were congenital heart defects, congenital hydrocephalus, cleft lip with cleft palate, cleft lip without cleft palate, limb reduction defects, congenital diaphragmatic hernia, trisomy 21 syndrome, congenital malformation of the urinary system, and other chromosomal malformation, compared with singletons with CAs. CONCLUSION: Multiple birth is associated with a significantly higher risk of CA, but a significantly lower prenatal diagnosis rate. Therefore, the healthcare of women with multiple pregnancy and their fetuses should be strengthened.

Postoperative circulating tumor DNA testing based on tumor naïve strategy after liver metastasis surgery in colorectal cancer patients.

Objective: There is still a lack of highly sensitive methods for monitoring recurrence of colorectal cancer patients after liver metastasis surgery. The aim of this study was to evaluate the prognostic value of tumor-naive ctDNA detection after resection of colorectal liver metastases (CRLM). Methods: Patients with resectable CRLM were prospectively enrolled. Based on the tumor-naive strategy, NGS panels containing 15 colorectal cancer hotspot mutated genes were used to detect ctDNA 3-6 weeks after surgery. Results: A total of 67 patients were included in the study, and the positive rate of postoperative ctDNA was 77.6% (52/67). Patients with positive ctDNA had a significantly higher risk of recurrence after surgery (HR 3.596, 95% CI 1.479 to 8.744, P = 0.005), and a higher proportion relapsed within 3 months after surgery (46.7% vs 3.8%). The C-index of postoperative ctDNA in predicting recurrence was higher than that of CRS and postoperative CEA. The nomogram combining CRS and postoperative ctDNA can improve the accuracy of recurrence prediction. Conclusion: Tumor-naive ctDNA detection can detect molecular residual lesions in patients with colorectal cancer after liver metastasis, and its prognostic value is superior to conventional clinical factors.

Development of Multiplex Drop-Off Digital PCR Assays for Hotspot Mutation Detection of KRAS, NRAS, BRAF, and PIK3CA in the Plasma of Colorectal Cancer Patients.

The detection of mutations in KRAS, NRAS, BRAF, and PIK3CA has become essential in managing the treatment of metastatic colorectal cancer (CRC) with the approval of new targeted therapies. We developed novel multiplex drop-off digital PCR (MDO-dPCR) assays by combining amplitude-/ratio-based multiplexing with drop-off/double drop-off strategies that allow for the detection of at least the 69 most frequent hotspot mutations in all four genes with only three reactions. The analytical performance of the assays was assessed using synthetic oligonucleotides, which were further validated on plasma cell-free DNA samples from a large cohort of CRC patients and compared with next-generation sequencing data. The MDO-dPCR assays showed a high sensitivity with a limit of detection ranging from 0.084% to 0.182% in mutant allelic frequency. The screening of plasma cell-free DNAs from 106 CRC patients identified mutations in 42.45% of them, with a sensitivity of 95.24%, a specificity of 98.53%, and an accuracy of 96.98% for mutation detection, and a strong correlation of measured mutant allelic frequencies compared with next-generation sequencing results. The high sensitivity and comprehensive mutation coverage of the MDO-dPCR assays make them suitable for rapid and cost-effective detection of KRAS, NRAS, BRAF, and PIK3CA mutations in the plasma of CRC patients, and could be useful in early response assessment and longitudinal disease monitoring.

Germline mutations in homologous recombination repair genes among Chinese pancreatic ductal adenocarcinoma patients detected using next-generation sequencing.

BACKGROUND: Genetic testing for pancreatic ductal adenocarcinoma (PDAC) patients is in constant development. However, the status of homologous recombination repair (HRR) genes in unselected Chinese PDAC has not been fully explored. This study aims to characterize the profile of germline mutations in HRR genes in Chinese PDAC patients. METHODS: A cohort of 256 PDAC patients were enrolled at Zhongshan Hospital Fudan University between 2019 and 2021. Germline DNA was analyzed by next-generation sequencing using a multigene panel of the 21 HRR genes. RESULTS: The germline pathogenic (P)/likely pathogenic (LP) variant rates were 7.0% (18/256) in unselected patients with pancreatic cancer. Among them, 1.6% (4/256) were identified as harboring BRCA2 variants, and 5.5% (14/256) patients carried non-BRCA variants. Variants were detected in eight non-BRCA genes, including ATM (4, 1.6%), PALB2 (4, 1.6%), ATR (1, 0.4%), BRIP1 (1, 0.4%), CHEK2 (1, 0.4%), MRE11 (1, 0.4%), PTEN (1, 0.4%), and STK11 (1, 0.4%). ATM, BRCA2, and PALB2 were the most prevalent variant genes. If only BRCA1/2 was tested, 5.5% of P/LP variants would have been lost. Further, we found that the landscape of P/LP HRR variants in various population cohorts was quite different. However, no significant difference was found in clinical characteristics between germline HRR P/LP carriers and non-carriers. In our study, one case carrying a germline PALB2 variant showed a long-time response to platinum-based chemotherapy and PARP inhibitor. CONCLUSION: This study comprehensively depicts the prevalence and characteristics of germline HRR mutations in unselected Chinese PDAC patients. Our findings show the clinical utility of a multigene panel may increase the detection of P/LP HRR carriers.

Epidemiology of birth defects in teenage pregnancies: Based on provincial surveillance system in eastern China.

Background: Healthcare for adolescents and birth defects (BD) prevention are highlighted public health issues. The epidemiology of birth defects in teenage pregnancies has not been studied extensively. Objectives: To investigate the prevalence trend and spectrum of BDs among teenage mothers. Methods: This observational study covered all births registered in the BD surveillance system in Zhejiang Province, China, during 2012-2018. The annual change in the prevalence of BDs among adolescent mothers was estimated. Crude relative ratios using the BD categories in teenage pregnancies were calculated and compared with those in women aged 25-29 years. Results: Overall, 54,571 BD cases among 1,910,977 births were included in this study, resulting in an overall prevalence of 234.64 to 409.07 per 10,000 births from 2012 to 2018 (P trend < 0.001) in total population. The prevalence of birth defects in teenage pregnancies increased from 247.19 to 387.73 per 10,000 births in 2012-2018 (P trend = 0.024). The risks of neural tube defects (relative risk [RR] = 3.15, 95% confidence interval [CI] 2.56, 3.87), gastroschisis (RR = 7.02, 95% CI 5.09, 9.69), and multiple birth defects (RR=1.27, 95% CI 1.07, 1.52) were higher in teenage pregnancies than those in women aged 25-29 years. Conclusions: We found a distinctive spectrum of BDs, with higher proportions of fatal or multiple anomalies in infants born to teenage mothers than in those born to adults aged 25-29 years. These results emphasize the importance of providing adolescents with better access to reproductive and prenatal care.

Role of Plasma methylated SEPT9 for Predicting Microvascular Invasion and Tumor Proliferation in Hepatocellular Carcinoma.

Background: Methylated SEPT9 (mSEPT9) has a role in the occurrence and development of hepatocellular carcinoma (HCC). Here, we studied the significance of plasma mSEPT9 for predicting prognosis-associated pathological parameters in patients with HCC. Methods: We retrospectively analyzed data from 205 subjects, including 111 HCC patients, 53 patients with at-risk liver disease (ARD) and 41 healthy donors (HDs). Analysis of plasma mSEPT9 was performed using methylation-specific polymerase chain reaction. Levels of mSEPT9 among different groups were compared using a nonparametric Mann-Whitney U test or a one-way ANOVA test. Correlations between pretreatment plasma mSEPT9 and clinicopathological characteristics were analyzed using the Chi-square. Univariate and multivariate analyses were used to identify factors related to microvascular invasion (MVI). Performance of variables for MVI prediction was evaluated by receiver operating characteristics curve. Results: A specific increase of plasma mSEPT9 in HCC was found when compared with ARD and HDs (HCC vs ARD, P = 1.1 × 10-5 and HCC vs HDs, P = 3.7 × 10-10). Pretreatment plasma mSEPT9 was significantly correlated tumor number (P = .004), tumor size (P = 4.6 × 10-5), MVI (P = .002) and Barcelona Clinic Liver Cancer stage (P = .012). Levels of plasma mSEPT9 correlated significantly with Ki67 expression in tumor (r = 0.356, P = 1.3 × 10-4). Univariate and multivariate analyses showed that plasma mSEPT9 and serum protein induced by vitamin K absence or antagonist-II (PIVKA-II) were independent predictors for MVI. A combination of these 2 markers exhibited a larger areas under the curve (areas under the curve [AUC] = 0.72) than mSEPT9 or PIVKA alone (AUC = 0.67 and 0.65), especially in early-stage HCC. Conclusions: Plasma mSEPT9 is a promising noninvasive biomarker for predicting MVI and tumor proliferation in HCC. Integration plasma mSEPT9 detection into clinical settings might facilitate the patient management.

Trends in the prevalence, prenatal diagnosis, and outcomes of births with chromosomal abnormalities: a hospital-based study in Zhejiang Province, China during 2014-2020.

BACKGROUND: To investigate the prevalence and prenatal diagnosis rate of chromosomal abnormalities (CA) in Zhejiang Province, China. METHODS: We estimated the annual changes in the detected prevalence of CA and prenatal diagnosis rate among 681,590 births in Zhejiang Province, China, between 2014 and 2020. Data were derived from the provincial birth defects surveillance system, which represents 30% of annual births in Zhejiang Province. The effect of maternal age was also evaluated. RESULTS: The detected prevalence of sex chromosomal abnormalities (1.70-7.30 per 10,000 births, Ptrend < 0.001) and microdeletion and microduplication (0.30-6.81 per 10,000 births, Ptrend < 0.001) gradually increased, contributing to an upward trend in overall CA (12.09-39.22 per 10,000 births). The diagnosis rate before 22 gestational weeks constantly increased from 20.8 to 70.1% for trisomy 21 (Ptrend = 0.003). The prevalence rate ratio for maternal age of ≥ 35 years was higher than that for maternal age of 25-29 years for trisomy 21 (5.40, 95% confidence interval [CI] 4.59-6.35) and sex chromosomal abnormalities (3.28, 95% CI 2.48-4.33). CONCLUSIONS: The rising prevalence of CA in China may be attributable to the elevated maternal age and the innovation of prenatal diagnosis tools, Thus, studies should pay attention to the rare CA that were previously ignored, and select rational screening tools.

[Clinical Comparative Analysis of Early-Onset Preeclampsia Combined with HELLP Syndrome].

Objective: To explore the clinical characteristics of early-onset preeclampsia (PE) combined with HELLP (hemolysis, elevated liver enzymes, and low platelets) syndrome, and to improve the capacity for early diagnosis and treatment. Methods: Pregnant women who received treatment at Women's Hospital, School of Medicine, Zhejiang University between March 2014 and October 2021 were retrospectively enrolled. There were two patient groups, the HELLP group consisting of 70 cases of early-onset PE combined with HELLP syndrome and the control group consisting of 140 cases of early-onset PE without HELLP syndrome. Patients in the two groups were matched by age. The general clinical data, characteristics of pathogenesis, and laboratory findings of the patients were collected and the perinatal outcomes of the two groups were compared and analyzed. Results: 1) There was no significant difference in gravidity, pre-delivery body mass index, years from the last delivery, and family history of diabetes mellitus and hypertension between the two groups. 2) The highest systolic blood pressure, highest diastolic blood pressure during the pregnancy, and the postpartum hospital length-of-stay ( P<0.001) in the HELLP group were higher than those in the control group. The gestational age at the time of the diagnosis of PE ( P=0.001) and the gestational age at delivery ( P<0.001) in the HELLP group were significantly earlier than those in the control group. The difference between the gestational age at the time of blood pressure elevation and that at the time of delivery ( P<0.001), and the gestational age difference between the diagnosis of early-onset PE and delivery ( P=0.027) were lower than those in the control group. The incidences of eclampsia in the HELLP group, pleural effusion, and ascites were higher than those of the control group. 3) Neonates in the HELLP group had a higher probability of being admitted to NICU and developing cyanotic/pale asphyxia ( P<0.001). 4) Before the termination of pregnancy, the HELLP group had higher levels of alanine aminotransferase, aspartate aminotransferase, total bilirubin, creatinine, urea, random glucose, lactate dehydrogenase, activated partial thromboplastin time, and the last 24-hour urine protein quantification than those of the control group (all P<0.05), while the platelet (PLT) counts were significantly lower than those of the control group ( P<0.001). 5) There was a significant correlation between PLT counts in the second trimester and the onset of HELLP syndrome ( P=0.006), with the area under the ROC curve reaching 0.746 (95% CI: 0.596-0.897). Conclusion: In comparison with early-onset PE patients without HELLP syndrome, patients with early-onset PE combined with HELLP syndrome are diagnosed for PE at an earlier gestational age, have higher blood pressure, are more prone to serious pregnancy complications, and have longer postpartum hospital length-of-stay and worse neonatal outcomes. Close monitoring of PLT counts of early-onset PE patients in the second trimester may help predict subsequent HELLP syndrome.

Validation of a highly sensitive Sanger sequencing in detecting EGFR mutations from circulating tumor DNA in patients with lung cancers.

BACKGROUND: The novel method, named blocker displacement amplification (BDA) Sanger, was applied to detect low variant allele frequency mutations in the circulating tumor DNA (ctDNA). This study aimed to evaluate the performance of the BDA Sanger method for the EGFR mutation detection in the ctDNA from lung cancer patients. METHODS: A total of 195 plasma samples of lung cancer patients were included. The EGFR mutation status in the ctDNA was detected by the BDA Sanger and Super-ARMS assays. Next-generation sequencing (NGS) was further used to verify the mutant of EGFR with inconsistencies. RESULTS: BDA Sanger assay was capable of detecting EGFR mutations with a 0.20% VAF from plasma samples. Among treatment-naive patients with paired tissue and plasma samples, the EGFR positive percent agreement (PPA) was 79% by BDA sanger. EGFR mutation was detected in 34.4% (67/195) ctDNA samples by the Super-ARMS and in 41.0% (80/195) ctDNA samples by the BDA Sanger assay. The overall concordance rate between the BDA Sanger and Super-ARMS assays was 82% (160/195). The BDA Sanger also enabled the detection of rare EGFR mutations, which were not discovered by the Super-ARMS. CONCLUSION: The results supported the validity and efficiency of the BDA Sanger method for EGFR detection in patients with lung cancer, indicating that BDA Sanger has a great potential for application in detecting mutations in the ctDNA.

Discrimination and structural validity evaluation of Zung self-rating depression scale for pregnant women in China.

PURPOSE: The applicability of the Zung self-rating depression scale (SDS) in pregnancy is unknown. We aimed to identify redundant items and evaluate the Zung SDS's structural validity. METHOD: Two samples of pregnant women were invited from two districts in Shanghai (Yangpu sample, n = 6468 and Huangpu sample, n = 402). The Yangpu sample was randomly split into YGroup1/2/3. Item's properties were evaluated via the item response theory in YGroup1. Exploratory and confirmatory factor analyses were correspondingly executed in YGroup2 and YGroup3. Those items with discrimination parameter (α) lower than 0.65 or factor loading smaller than 0.4 were deleted from the scale. The final structure was validated in the Huangpu sample. RESULTS: Items 4 (sleep), 7 (weight loss), 8 (constipation) and 9 (tachyarrhythmia) exhibited low discrimination power. Items 2 (diurnal variation), 5 (appetite), 10 (fatigue) and 19 (suicide idea) made a low contribution to all factors. A three-factor model was eventually constructed as cognitive (Items 14, 16, 17, 18 and 20), psychomotor (Items 6, 11 and 12) and affective (Items 1, 3, 13 and 15). CONCLUSION: The Zung SDS needs modification before applied to pregnant women in China. The items describing the overlap symptoms of the physical change in pregnancy and mood disorder should be deleted.

Predictive factors associated with disease recurrence in patients with severe intrahepatic cholestasis of pregnancy: a retrospective study of 118 cases.

PURPOSE: To compare the clinical characteristics of pregnant women and perinatal outcomes with or without recurrent severe intrahepatic cholestasis of pregnancy (sICP), and identify possible factors associated with disease recurrence. METHODS: A retrospective study of 164,603 deliveries was performed to identify pregnant women diagnosed with sICP in the previous pregnancy from January 2012 to December 2020. Eligible patients were divided into two subgroups according to the status of disease recurrence in the second pregnancy: recurrent severe ICP (r-sICP) and non-recurrent severe ICP (nr-sICP). Demographics, clinical characteristics, maternal and perinatal outcomes, and potential factors linked to disease recurrence were analyzed. RESULTS: Totally 118 patients were enrolled and respectively classified into the r-sICP group (n = 63) and the nr-sICP group (n = 55). The proportion of hepatitis B virus (HBV) infection (HBsAg+, HBeAg+, HBcAb+) and early-onset ICP (<28 weeks) in the r-sICP group in the previous pregnancy were higher than those in the nr-sICP group. In the second delivery, neonatal outcomes in the r-sICP group were worse than those in the nr-sICP group. Logistic regression analysis of predictive factors for disease recurrence in the second delivery revealed that the combination of HBV infection and early-onset ICP in the previous delivery had the steepest receiver-operating characteristic (ROC) curve value 0.720 (95%CI: 0.629-0.812). CONCLUSION: Patients with sICP displayed a higher recurrence rate in the second pregnancy. Being <28 weeks at the time of ICP diagnosis and having HBV infection in the previous delivery appear to be independent predictive factors for disease recurrence of sICP.

Role of blood mSEPT9 in evaluating tumor burden and disease monitoring in colorectal cancer patients.

PURPOSE: This study aimed to investigate the correlation between mSEPT9 and tumor burden as well as the role of mSEPT9 in monitoring colorectal cancer (CRC) patients. METHODS: A total of 309 patients were recruited and received mSEPT9 detection in this retrospective study. Clinicopathologic characteristics were collected, including age, gender, differentiation, gene mutation, stage, and tumor markers. The correlation between mSEPT9 and clinical tumor burden was analyzed. A relative mSEPT9 value was determined using the ΔΔCt method. RESULTS: The overall positivity rate of mSEPT9 was 39.8% in CRC patients. mSEPT9 status was significantly associated with disease status and tumor markers (CEA and CA19-9). The mSEPT9 positivity rates were 15.6%, 50.0%, 64.4%, and 70.0% for P0M0, P1M0, P0M1, and P1M1 patients, respectively (p < 0.001). Among 137 CRC patients who received mSEPT9 assay before surgery, the pre-operation mSEPT9 positivity rate increased significantly from stage I to stage IV (Stage I vs. II vs. III vs. IV 25% vs. 59.1% vs. 57.1% vs. 70.0%, respectively). Consecutive blood samples were obtained from 26 patients during therapy. The patients with increased mSEPT9 levels showed a higher progression rate. CONCLUSIONS: mSEPT9 was a biomarker reflecting tumor burden, and serial detections of mSEPT9 could be a promising strategy for disease monitoring in CRC patients.

Cross-platform comparison of next-generation sequencing and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for detecting KRAS/NRAS/BRAF/PIK3CA mutations in cfDNA from metastatic colorectal cancer patients.

BACKGROUND: Examining tumor KRAS/NRAS/BRAF/PIK3CA status in metastatic colorectal cancer (mCRC) is essential for treatment selection and prognosis evaluation. Cell-free DNA (cfDNA) in plasma is a feasible source for tumor gene analysis. METHODS: In this study, we recruited mCRC patients and analyzed their KRAS/NRAS/BRAF/PIK3CA status in cfDNA using two platforms, next-generation sequencing (NGS) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF). The performance between the two platforms and the concordance rate between cfDNA and tissue were analyzed. The relationship between cfDNA-related variables and clinical variables was also assessed. Tumor mutations in cfDNA from patients receiving continuous treatments were monitored in the follow-ups. RESULTS: Next-generation sequencing and MALDI-TOF had similar specificity (100.0% vs. 99.3%) and negative predictive value (99.9% vs. 99.4%), whereas NGS had higher sensitivity (97.1% vs. 85.3% of MALDI-TOF) and positive predictive value (100% vs. 82.9% of MALDI-TOF). The overall concordance rate of NGS and MALDI-TOF was 98.6%. For the reportable types of mutations in both cfDNA and tissue, the concordance rate was 96.1%. Among 28 tissue-positive patients, the allele frequencies of tumor mutations in cfDNA were higher in patients with primary tumor burden (p = 0.0141). Both CEA and CA 19-9 were positively correlated with cfDNA concentration (r = 0.3278 and r = 0.3992). The allele frequencies of tumor mutations changed with disease progression. CONCLUSIONS: Next-generation sequencing showed slightly better performance in detecting cfDNA mutations and was more suitable for clinical practice. cfDNA-related variables reflected the tumor status and showed a promising potential in monitoring disease progression.

Chemotherapy-associated clonal hematopoiesis mutations should be taken seriously in plasma cell-free DNA KRAS/NRAS/BRAF genotyping for metastatic colorectal cancer.

BACKGROUND: Genotyping of plasma cell-free DNA (cfDNA) is an increasingly important method to assess the tumor mutation status in colorectal cancer (CRC) patients. Clonal hematopoiesis (CH) releases non-tumor somatic mutations into blood, causing false positive results in cfDNA-based tumor genotyping. It is still not clear if CH should be examined in all CRC patients undergoing cfDNA analysis. METHODS: We analyzed cfDNA KRAS, NRAS and BRAF genotypes in 236 metastatic CRC patients, who had matched tissue genotyping results, by next-generation sequencing using plasma cfDNA. The cfDNA-only mutations with allele frequencies (AFs) < 5% were highly suspicious for being CH-derived mutations. The origins of cfDNA mutations were confirmed by droplet digital polymerase chain reaction (ddPCR) using paired peripheral blood cells (PBCs) and CH-derived mutations were finally determined. One patient with a CH-derived mutation was followed up and the subpopulation of blood cells, in which CH was present, was investigated. RESULTS: Three CH-derived mutations, KRAS Q61H, KRAS G12D and KRAS G12V, were identified in the patient cohort. All three patients harboring corresponding CH-derived mutations had a prior chemotherapy history. The CH-derived KRAS G12V mutation in a patient was found only present in lymphocytes and persisting under treatment. For all cfDNA mutations, the CH-derived ones were clustered in the patients with < 5% mutation AF and prior chemotherapy. CONCLUSION: The prevalence of CH in CRC patients was limited, and prior chemotherapy was a contributing factor of CH. It is recommended for patients with < 5% mutation AF and prior chemotherapy to have genotyping analysis of their PBCs following plasma cfDNA genotyping.

Exposure to elevated per- and polyfluoroalkyl substances in early pregnancy is related to increased risk of gestational diabetes mellitus: A nested case-control study in Shanghai, China.

BACKGROUND: Long-chain per- and polyfluoroalkyl substances (PFASs) and their short-chain alternatives have been produced and used extensively in China. However, it is unclear whether these compounds contribute to the risk of gestational diabetes mellitus (GDM) in women residing in contaminated areas. OBJECTIVE: The study was performed to explore the association between PFASs varying in chain length and the risk of developing GDM. METHOD: A nested case-control study was conducted in a prospective cohort of 2,460 pregnant women between July 1, 2017, and January 31, 2019 in Shanghai, China. Twelve PFASs of interest were measured using ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF MS) in the sera of pregnant women at 16-20 weeks. GDM was diagnosed by an oral glucose tolerance test administered over 24-28 gestational weeks. The cases and controls were matched by maternal age. The relationship between maternal serum PFAS level and GDM risk was determined by conditional logistic and linear regression analyses. RESULTS: A total of 165 GDM cases and 330 controls were enrolled in the study cohort. The frequencies of detection of PFHpA, PFDS, and PFOSA were all ≤80%. Hence, they were excluded from any further risk analysis. Perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) were detected at relatively high serum levels (medians 6.57 ng/mL and 8.07 ng/mL, respectively). The serum levels of perfluorobutanesulfonic acid (PFBS) and perfluorododecanoic acid (PFDoA) were significantly higher in the GDM group than they were in the control group (P = 0.02 and P < 0.01, respectively) according to a nonparametric Wilcoxon rank sum test. A quartile analysis showed that the odds ratio of GDM would significantly increase at the highest PFBS and PFDoA levels. In the core model, the adjusted ORs were 2.02 (95% CI = 1.04-3.79) and 13.00 (95% CI = 4.74-24.59), respectively, after adjusting for maternal age, sampling time, parity and body mass index [BMI]). CONCLUSION: Elevated maternal serum PFBS and PFDoA levels in early pregnancy may be associated with a substantially higher GDM risk.

Model-Driven Engineering of N-Linked Glycosylation in Chinese Hamster Ovary Cells.

Chinese hamster ovary (CHO) cells are used for industrial production of protein-based therapeutics (i.e., "biologics"). Here we describe a method for combining systems-level kinetic models with a synthetic biology platform for multigene overexpression to rationally perturb N-linked glycosylation. Specifically, we sought to increase galactose incorporation on a secreted Immunoglobulin G (IgG) protein. We rationally design, build, and test a total of 23 transgenic cell pools that express single or three-gene glycoengineering cassettes comprising a total of 100 kilobases of engineered DNA sequence. Through iterative engineering and model refinement, we rationally increase the fraction of bigalactosylated glycans five-fold from 11.9% to 61.9% and simultaneously decrease the glycan heterogeneity on the secreted IgG. Our approach allows for rapid hypothesis testing and identification of synergistic behavior from genetic perturbations by bridging systems and synthetic biology.

Insights into the loss of protein sialylation in an fc-fusion protein-producing CHO cell bioprocess.

Sialylation affects circulating half-life, charge distribution, and other biochemical properties of therapeutic glycoproteins. Loss of protein sialylation during glycoprotein-producing bioprocesses could lead to a low final protein sialylation level and bring negative effects on subsequent clinical efficacy. In this work, an Fc-fusion protein-producing Chinese hamster ovary cell fed-batch culture process was studied and insights into the loss of protein sialylation during the Fc-fusion protein production phase (days 5 to 13) were presented. The results showed that the decreased total sialic acid content was 13.84 µg/mg during the production phase, which accounted for 24% of the total sialic acid content on day 5. The lost sialic acids were predominantly from α 2-3 sialylation on N- and O-glycans. Through cell-free incubation and kinetics studies, it was found that the decreased sialic acid content caused by extracellular sialic acid degradation and incomplete glycan biosynthesis were 7.79 µg/mg and 6.05 µg/mg, respectively. The two processes had a nearly equal contribution to the loss of final product sialylation. Detailed characterizations revealed that decreases in sialic acid content were due either to extracellular sialic acid degradation via hydrolysis of α 2-3 sialic acids probably by released cytosolic sialidase or to a lack of galactosylated glycan availability for sialylation during late-stage glycosylation. Our work provides a better understanding of losses in protein sialylation during glycoprotein manufacturing.

The value of ultrasound in predicting isolated inter-twin discordance and adverse perinatal outcomes.

PURPOSE: To investigate the value of ultrasound approaching delivery to predict isolated inter-twin discordance and adverse perinatal outcomes. METHODS: We retrospectively included twin pregnancies with sonography approaching delivery in ten maternal-foetal medicine centres in China from 2013 to 2014. Estimated foetal weight (EFW) and inter-twin EFW disparity (EFWD) were calculated based on biometry parameters. Percentage errors between EFW and actual birthweight or between EFWD and actual inter-twin disparity were calculated. ROC curves and multiple logistic regression were applied to evaluate the ability of EFWD to predict inter-twin disparity ≥ 25%, stillbirth, asphyxia and admission to a neonatal intensive unit (NICU). Chorionicity-stratified analysis was further performed. RESULTS: Two hundred sixty-six monochorionic and 760 dichorionic twin pregnancies were analysed. The percentage errors in foetal weight estimations were 7-13%, whereas percentage errors in the estimation of inter-twin disparity were nearly 100%. Among eight formulas, Hadlock1 performed best, with a detectable rate of 65% and a false positive rate of 5% when predicting inter-twin disparity ≥ 25%. EFWD ≥ 22% was strongly associated with stillbirth (OR = 4.17, 95% CI 1.40-12.40) and NICU admission (OR = 3.48, 95% CI 2.03-5.97) after adjustment for gestational age, parity and abnormal umbilical systolic/diastolic ratio. Ultrasound had better predictive ability in monochorionic twins. CONCLUSION: The predictive value of ultrasound for isolated inter-twin discordance and adverse perinatal outcomes was limited, which was possibly due to the magnifying of systematic errors in the disparity estimation compared with weight estimation. Despite this, abnormal biometry was an independent contributor for the poor prognosis of neonates.

Elucidating the effects of pH shift on IgG1 monoclonal antibody acidic charge variant levels in Chinese hamster ovary cell cultures.

Charge variants, especially acidic charge variants, in recombinant monoclonal antibodies are critical quality attributes, which can affect antibodies' properties in vitro and in vivo. Meanwhile, charge variants are cumulative effects of various post-translational modifications and chemical degradations on antibody. In this work, to investigate the effect of lowering culture pH in the stationary phase on acidic charge variant contents in fed-batch cultures and its mechanism, cell culture experiments in 2-L bioreactors were firstly performed to explore the changes in the charge distribution under the pH downshift condition using weak cation exchange chromatography. It is found that acidic charge variant contents were significantly decreased by pH downshift. Then, to reveal the mechanism by which the content of acidic charge variants is reduced under pH downshift condition, the variation of post-translational modifications and chemical degradations under the pH downshift condition was explored. Meanwhile, the structure of the acidic charge variants was characterized. Several analysis experiments including size exclusion chromatography, capillary electrophoresis-sodium dodecyl sulfate under non-reducing conditions, tryptic peptide map, and reduced antibody mass were applied in this study. The results show that the mechanism by which the content of acidic charge variants is reduced is that the contents of disulfide bond reduction, galactosylation, and asparagine deamination of the HC-N388 in the Fc domain were reduced by pH downshift.

Galactose supplementation enhance sialylation of recombinant Fc-fusion protein in CHO cell: an insight into the role of galactosylation in sialylation.

Sialic acid levels of therapeutic glycoprotein play an important role in plasma half-life. An undesirable decrease of sialic acid content was observed when we increased Fc-fusion protein productivity fourfold in a GS-CHO cell line by bioprocess optimization. We investigated the potential mechanism for the sialic acid content reduction. We found that limited nucleotide sugar precursor and the extracellular sialidase were not responsible for the reduction of the sialic acid content after titer improvement. Oligosaccharide analysis revealed that the lack of protein galactosylation was the potential cause for the reduction of sialic acid content. Thus we validated this notion by evaluated galactose supplementation in 2 L bioreactors. Cell culture performance was not impacted by addition of up to 40 mM galactose except for the glucose consumption rate. Addition of 20 mM galactose to the bioreactor resulted in the increase of 44 % for total sialic acid content and 20.3 % for sialylated glycans. These data were further validated when the process was run on 200 L scaled bioreactor. These data together show that the galactosylation plays an apparent role in sialylation in our current system.