Next Generation Sequencing-Based Fetal ABO Blood Group Prediction by Analysis of Cell-Free DNA from Maternal Plasma.

INTRODUCTION: ABO blood group incompatibility between a pregnant woman and her fetus as a cause of morbidity or mortality of the fetus or newborn remains an important, albeit rare, risk. When a pregnant woman has a high level of anti-A or anti-B IgG antibodies, the child may be at risk for hemolytic disease of the fetus and newborn (HDFN). Performing a direct prenatal determination of the fetal ABO blood group can provide valuable clinical information. OBJECTIVE: Here, we report a next generation sequencing (NGS)-based assay for predicting the prenatal ABO blood group. MATERIALS AND METHODS: A total of 26 plasma samples from 26 pregnant women were tested from gestational weeks 12 to 35. Of these samples, 20 were clinical samples and 6 were test samples. Extracted cell-free DNA was PCR-amplified using 2 primer sets followed by NGS. NGS data were analyzed by 2 different methods, FASTQ analysis and a grep search, to ensure robust results. The fetal ABO prediction was compared with the known serological infant ABO type, which was available for 19 samples. RESULTS: There was concordance for 19 of 19 predictable samples where the phenotype information was available and when the analysis was done by the 2 methods. For immunized pregnant women (n = 20), the risk of HDFN was predicted for 12 fetuses, and no risk was predicted for 7 fetuses; one result of the clinical samples was indeterminable. Cloning and sequencing revealed a novel variant harboring the same single nucleotide variations as ABO*O.01.24 with an additional c.220C>T substitution. An additional indeterminable result was found among the 6 test samples and was caused by maternal heterozygosity. The 2 indeterminable samples demonstrated limitations to the assay due to hybrid ABO genes or maternal heterozygosity. CONCLUSIONS: We pioneered an NGS-based fetal ABO prediction assay based on a cell-free DNA analysis from maternal plasma and demonstrated its application in a small number of samples. Based on the calculations of variant frequencies and ABO*O.01/ABO*O.02 heterozygote frequency, we estimate that we can assign a reliable fetal ABO type in approximately 95% of the forthcoming clinical samples of type O pregnant women. Despite the vast genetic variations underlying the ABO blood groups, many variants are rare, and prenatal ABO prediction is possible and adds valuable early information for the prevention of ABO HDFN.

Stable fetal hemodynamics measured by Doppler flow after initiation of anti-hypertensive treatment with methyldopa in pregnant women with diabetes.

AIM: To evaluate whether initiation of anti-hypertensive treatment with methyldopa affects fetal hemodynamics in women with pregestational diabetes. METHODS: Prospective study of unselected singleton pregnant women with diabetes (seven type 1 and two type 2 diabetes), normal blood pressure and kidney function at pregnancy booking. Methyldopa treatment was initiated at blood pressure >135/85 mmHg and/or urinary albumin excretion (UAE) >300 mg/g creatinine. Pulsatility indices (PI) of the uterine, umbilical, middle cerebral arteries before and 1 week after initiation of methyldopa treatment (250 mg three times daily) was performed and the cerebro-placental ratio (CPR) was calculated. RESULTS: Methyldopa treatment was initiated at median 249 (range 192-260) gestational days, mainly due to gestational hypertension (n = 7). Blood pressure declined from 142 (112-156)/92 (76-103) mmHg before to 129 (108-144)/82 (75-90) mmHg after initiation of methyldopa treatment (p = 0.11 and 0.04 for systolic and diastolic blood pressure, respectively). There were no significant changes in the umbilical artery PI (0.82 (0.72-1.40) versus 0.87 (0.64-0.95), p = 0.62) or CPR (1.94 (0.96-2.33) versus 1.78 (1.44-2.76), (p = 0.73). Gestational age was 265 (240-270) d. Apgar scores were normal. CONCLUSIONS: Stable Doppler flow velocity waveforms were documented after initiation of methyldopa treatment for pregnancy-induced hypertensive disorders in this cohort of pregnant women with pregestational diabetes.

[Selective screening for hepatitis B in pregnant women in Denmark]. / Selektiv hepatitis B-screening af gravide i Danmark.

INTRODUCTION: The objective of this study was to evaluate the implementation of the present Danish selective screening strategy for possible hepatitis B infection in pregnant women who themselves or whose partners originate from medium- or high-endemic hepatitis B areas of the world. We also investigated whether children of hepatitis B carriers were treated with immunoglobulin and vaccination. MATERIALS AND METHODS: We did a retrospective study of 1,924 birth notification forms of women who had delivered between 1 January and 30 June 2000 in Frederiksborg County, Denmark. Women were selected who had foreign-sounding names. The hospital case notes were examined to determine the women's countries of origin and their hepatitis B screening status. We then contacted the Department of Clinical Immunology of each hospital and the Danish State Serum Institute to double-check the women's screening status. RESULTS: The study included 210 women, who made up nearly 11% of this cohort. Sixty-eight (32%) of the women had been tested according to the guidelines. One woman was a hepatitis B carrier. Her child had received immunoglobulin and vaccination within 24 hours of birth. CONCLUSION: This study indicates that a selective antenatal screening program for hepatitis B is difficult to implement in daily clinical practice. The fact that only one third of this well-defined risk group had been tested for hepatitis B emphasizes the need for a universal antenatal screening program.

Poor pregnancy outcome in women with type 2 diabetes.

OBJECTIVE: To evaluate the perinatal outcome and the frequency of maternal complications in pregnancies of women with type 2 diabetes during 1996-2001. RESEARCH DESIGN AND METHODS: Medical records of 61 consecutive singleton pregnancies in women with type 2 diabetes from 1996 to 2001 were studied. Pregnancy outcome was compared with that of pregnant women with type 1 diabetes during 1996-2000, the background population, and pregnant women with type 2 diabetes during 1980-1992 from the same department. RESULTS: The perinatal mortality in pregnancies complicated by type 2 diabetes (4/61, 6.6%) was increased four- and ninefold, respectively, and the rate of major congenital malformations (4/60, 6.7%) was more than doubled, although not statistically significant, compared with type 1 diabetic pregnancies and the background population. The glycemic control was similar or better in women with type 2 diabetes compared with women with type 1 diabetes. Multivariate logistic regression analysis in the pooled group of pregnancies with pregestational diabetes from 1996 to 2001 showed that high HbA(1c) at admission and type 2 diabetes were independently associated with a serious adverse fetal outcome (perinatal mortality and/or major congenital malformations). The perinatal mortality and the rate of major congenital malformations in type 2 diabetic pregnancies have increased during the last decade. CONCLUSIONS: The perinatal outcome of pregnancies in women with type 2 diabetes during 1996-2001 is poor. It is worse than the outcome of pregnancies in women with type 1 diabetes and the background population in the same period, as well as in women with type 2 diabetes studied during 1982-1990.

Insulin dose during glucocorticoid treatment for fetal lung maturation in diabetic pregnancy: test of an algorithm [correction of analgoritm].

OBJECTIVE: Poor glycemic control is often a serious clinical problem during glucocorticoid treatment for fetal lung maturation in pregnant women with diabetes. An algorithm for improved subcutaneous insulin treatment during glucocorticoid treatment in insulin-dependent diabetic women was developed and tested. STUDY DESIGN: The sample, divided into two cohorts, consisted of all insulin-dependent diabetic women (n=16) receiving glucocorticoid treatment (betamethasone 12 mg i.m., repeated after 24 h) from 1996 to 1999. In the first cohort the increments of insulin dose were based on the level of blood glucose obtained. Based on the first cohort an algorithm to determine increments of insulin dose was developed. In the second cohort (n = 8) the insulin dose was increased by up to 40%, according to the algorithm, starting immediately after glucocorticoid treatment; prior to a detectable increase in blood glucose. RESULTS: After betamethasone, the daily insulin dose for the following 5 days was increased by 6, 38, 36, 27 and 17% in the first cohort vs. 27, 45, 40, 31 and 11% in the second cohort. The algorithm was used in the second cohort. The median blood glucose was 6.7, 14.3, 12.3, 7.7 and 7.7 vs. 7.7, 8.2, 9.6, 7.0 and 7.4 mmol/l (p<0.05 for day 2 and 3) in the two cohorts, respectively. None developed ketoacidosis or severe hypoglycemia. CONCLUSION: An algorithm with an increasing insulin dose of up to 40% shortly after glucocorticoid treatment for fetal lung maturation in diabetic women prevents severe dysregulation of metabolic control.