COVID-19 mRNA vaccination status and concerns among pregnant women in Japan: a multicenter questionnaire survey.

BACKGROUND: mRNA vaccination is an effective, safe, and widespread strategy for protecting pregnant women against infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. However, information on factors such as perinatal outcomes, safety, and coverage of mRNA vaccinations among pregnant women is limited in Japan. Therefore, this study aimed to investigate the perinatal outcomes, coverage, adverse effects, and short-term safety of mRNA vaccination as well as vaccine hesitancy among pregnant women. METHODS: We conducted a multicenter online survey of postpartum women who delivered their offspring at 15 institutions around Tokyo from October 2021 to March 2022. Postpartum women were divided into vaccinated and unvaccinated groups. Perinatal outcomes, COVID-19 prevalence, and disease severity were compared between the two groups. Adverse reactions in the vaccinated group and the reasons for being unvaccinated were also investigated retrospectively. RESULTS: A total of 1,051 eligible postpartum women were included. Of these, 834 (79.4%) had received an mRNA vaccine, while 217 (20.6%) had not, mainly due to concerns about the effect of vaccination on the fetus. Vaccination did not increase the incidence of adverse perinatal outcomes, including fetal morphological abnormalities. The vaccinated group demonstrated low COVID-19 morbidity and severity. In the vaccinated group, the preterm birth rate, cesarean section rate, and COVID-19 incidence were 7.2%, 33.2%, and 3.3%, respectively, compared with the 13.7%, 42.2%, and 7.8% in the unvaccinated group, respectively. Almost no serious adverse reactions were associated with vaccination. CONCLUSIONS: mRNA vaccines did not demonstrate any adverse effects pertaining to short-term perinatal outcomes and might have prevented SARS-CoV-2 infection or reduced COVID-19 severity. Concerns regarding the safety of the vaccine in relation to the fetus and the mother were the main reasons that prevented pregnant women from being vaccinated. To resolve concerns, it is necessary to conduct further research to confirm not only the short-term safety but also the long-term safety of mRNA vaccines.

Prevalence of common aneuploidy in twin pregnancies.

The incidence of chromosomal abnormalities in twin pregnancies is not well-studied. In this retrospective study, we investigated the frequency of chromosomal abnormalities in twin pregnancies and compared the incidence of chromosomal abnormalities in dichorionic diamniotic (DD) and monochorionic diamniotic (MD) twins. We used data from 57 clinical facilities across Japan. Twin pregnancies of more than 12 weeks of gestation managed between January 2016 and December 2018 were included in the study. A total of 2899 and 1908 cases of DD and MD twins, respectively, were reported, and the incidence of chromosomal abnormalities in one or both fetuses was 0.9% (25/2899) and 0.2% (4/1908) in each group (p = 0.004). In this study, the most common chromosomal abnormality was trisomy 21 (51.7% [15/29]), followed by trisomy 18 (13.8% [4/29]) and trisomy 13 (6.9% [2/29]). The incidence of trisomy 21 in MD twins was lower than that in DD twins (0.05% vs. 0.5%, p = 0.007). Trisomy 21 was less common in MD twins, even when compared with the expected incidence in singletons (0.05% vs. 0.3%, RR 0.15 [95% CI 0.04-0.68]). The risk of chromosomal abnormality decreases in twin pregnancies, especially in MD twins.

Evaluation of the clinical performance of noninvasive prenatal testing at a Japanese laboratory.

AIM: We aimed to evaluate the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of noninvasive prenatal testing (NIPT) in high-risk pregnant women. METHODS: Pregnant women who underwent GeneTech NIPT, the most commonly used NIPT in Japan, between January 2015 and March 2019, at Japan NIPT Consortium medical sites were recruited for this study. The exclusion criteria were as follows: pregnant women with missing survey items, multiple pregnancy/vanishing twins, chromosomal abnormalities in the fetus other than the NIPT target disease, and nonreportable NIPT results. Sensitivity and specificity were calculated from the obtained data, and maternal age-specific PPV and NPV were estimated. RESULTS: Of the 45 504 cases, 44 263 cases fulfilling the study criteria were included. The mean maternal age and gestational weeks at the time of procedure were 38.5 years and 13.1 weeks, respectively. Sensitivities were 99.78% (95% confidence interval [95% CI]: 98.78-99.96), 99.12% (95% CI: 96.83-99.76), and 100% (95% CI: 88.30-100) for trisomies 21, 18, and 13, respectively. Specificities were more than 99.9% for trisomies 21, 18, and 13, respectively. Maternal age-specific PPVs were more than 93%, 77%, and 43% at the age of 35 years for trisomies 21, 18, and 13, respectively. CONCLUSION: The GeneTech NIPT data showed high sensitivity and specificity in the detection of fetal trisomies 21, 18, and 13 in high-risk pregnant women, and maternal age-specific PPVs were obtained. These results could provide more accurate and improved information regarding NIPT for genetic counseling in Japan.

Isolation and characterization of fetal nucleated red blood cells from maternal blood as a target for single cell sequencing-based non-invasive genetic testing.

PURPOSE: Although non-invasive prenatal testing (NIPT) based on cell-free DNA (cfDNA) in maternal plasma has been prevailing worldwide, low levels of fetal DNA fraction may lead to false-negative results. Since fetal cells in maternal blood provide a pure source of fetal genomic DNA, we aimed to establish a workflow to isolate and sequence fetal nucleated red blood cells (fNRBCs) individually as a target for NIPT. METHODS: Using male-bearing pregnancy cases, we isolated fNRBCs individually from maternal blood by FACS, and obtained their genomic sequence data through PCR screening with a Y-chromosome marker and whole-genome amplification (WGA)-based whole-genome sequencing. RESULTS: The PCR and WGA efficiencies of fNRBC candidates were consistently lower than those of control cells. Sequencing data analyses revealed that although the majority of the fNRBC candidates were confirmed to be of fetal origin, many of the WGA-based genomic libraries from fNRBCs were considered to have been amplified from a portion of genomic DNA. CONCLUSIONS: We established a workflow to isolate and sequence fNRBCs individually. However, our results demonstrated that, to make cell-based NIPT targeting fNRBCs feasible, cell isolation procedures need to be further refined such that the nuclei of fNRBCs are kept intact.

Classification of factors involved in nonreportable results of noninvasive prenatal testing (NIPT) and prediction of success rate of second NIPT.

OBJECTIVE: To evaluate the reasons for nonreportable cell-free DNA (cfDNA) results in noninvasive prenatal testing (NIPT), we retrospectively studied maternal characteristics and other details associated with the results. METHODS: A multicenter retrospective cohort study in pregnant women undergoing NIPT by massively parallel sequencing (MPS) with failed cfDNA tests was performed between April 2013 and March 2017. The women's data and MPS results were analyzed in terms of maternal characteristics, test performance, fetal fraction (FF), z scores, anticoagulation therapy, and other details of the nonreportable cases. RESULTS: Overall, 110 (0.32%) of 34 626 pregnant women had nonreportable cfDNA test results after an initial blood sampling; 22 (20.0%) cases had a low FF (<4%), and 18 (16.4%) cases including those with a maternal malignancy, were found to have altered genomic profile. Approximately half of the cases with nonreportable results had borderline z score. Among the women with nonreportable results because of altered genomic profile, the success rate of retesting using a second blood sampling was relatively low (25.0%-33.3%). Thirteen (11.8%) of the women with nonreportable results had required hypodermic heparin injection. CONCLUSIONS: The classification of nonreportable results using cfDNA analysis is important to provide women with precise information and to reduce anxiety during pregnancy.

Fetal cell-free DNA fraction in maternal plasma for the prediction of hypertensive disorders of pregnancy.

OBJECTIVE: The purpose of this study is to compare the fetal fractions during non-invasive prenatal testing (NIPT) in singleton pregnancies according to gestational age and maternal characteristics to evaluate the utility of this parameter for the prediction of pregnancy complications including gestational diabetes mellitus (GDM) and hypertensive disorders of pregnancy (HDP). STUDY DESIGN: This study was a multicenter prospective cohort study. The present data were collected from women whose NIPT results were negative. The relationships between the fetal fractions and the gestational age, maternal weight and height, and incidences of miscarriage, preterm delivery, and pregnancy complications including GDM, HDP and placental abruption were assessed. RESULTS: A total of 5582 pregnant women with verified NIPT negative results were registered in the study. The demographic characteristics of the study populations were statistically analyzed, and the women with HDP tended to have a low fetal fraction in samples taken during early gestation. The area under the curve (AUC) in a receiver operating characteristic curve (ROC) analysis was 0.608 for women with HDP. CONCLUSION: A low fetal fraction on NIPT might be correlated with future HDP. However, predicting HDP during early pregnancy in women with a low fetal fraction might be difficult.

Hypertensive disorders of pregnancy: a strong risk factor for subsequent hypertension 5 years after delivery.

Hypertensive disorders of pregnancy are known to be a risk factor for future cardiovascular diseases. In contrast, there is a paucity of data on the not so distant future prognosis of hypertensive disorders of pregnancy. In the present study, we evaluated the incidence of the diseases causing cardiovascular problems (hypertension, diabetes mellitus, dyslipidemia and metabolic syndrome) 5 years after delivery in Japanese women with hypertensive disorders of pregnancy. We performed a double-cohort study and compared medical conditions between women with and without a history of hypertensive disorders of pregnancy. A total of 1513 women who participated in the cohort study were invited to undergo a medical checkup 5 years after the index delivery, of whom 829 responded. After excluding pregnant and lactating women at the time of examination, 25 women with hypertensive disorders of pregnancy and 746 control subjects were analyzed. The incidence of hypertension was significantly higher among women with hypertensive disorders of pregnancy than women who were normotensive during pregnancy (24.0 vs. 2.5%, P<0.001). They were also at an increased risk of subsequent hypertension 5 years after the index delivery, after adjusting for confounding factors such as age, body mass index, family history of hypertension and salt intake (odds ratio 7.1, 95% CI, 2.0-25.6, P<0.003). These is no significant difference in the incidence of diabetes mellitus, dyslipidemia and metabolic syndrome. In conclusion, hypertensive disorders of pregnancy are strong risk factors for subsequent hypertension only 5 years after delivery.

Current status of non-invasive prenatal testing in Japan.

AIM: The purpose of this study was to report the 3-year experience of a nationwide demonstration project to introduce non-invasive prenatal testing (NIPT) of maternal plasma for aneuploidy, and review the current status of NIPT in Japan. METHODS: Tests were conducted to detect aneuploidy in high-risk pregnant women, and adequate genetic counseling was provided. The clinical data, test results, and pregnancy outcomes were recorded. We discuss the problems of NIPT on the basis of published reports and meta-analyses. RESULTS: From April 2013 to March 2016, 30 613 tests were conducted at 55 medical sites participating in a multicenter clinical study. Among the 30 613 women tested, 554 were positive (1.81%) and 30 021 were negative (98.1%) for aneuploidy. Of the 289, 128, and 44 women who tested positive for trisomies 21, 18, and 13, respectively, and underwent definitive testing, 279 (96.5%), 106 (82.8%), and 28 (63.6%) were determined to have a true-positive result. For the 13 481 women with negative result and whose progress could be traced, two had a false-negative result (0.02%). The tests were performed on the condition that a standard level of genetic counseling be provided at hospitals. CONCLUSION: Here, we report on the 3-year nationwide experience with NIPT in Japan. It is important to establish a genetic counseling system to enable women to make informed decisions regarding prenatal testing. Moreover, a welfare system is warranted to support women who decide to give birth to and raise children with chromosomal diseases.

A survey on awareness of genetic counseling for non-invasive prenatal testing: the first year experience in Japan.

The purpose of this study is to summarize the results from a survey on awareness of genetic counseling for pregnant women who wish to receive non-invasive prenatal testing (NIPT) in Japan. As a component of a clinical study by the Japan NIPT Consortium, genetic counseling was conducted for women who wished to receive NIPT, and a questionnaire concerning both NIPT and genetic counseling was given twice: once after pre-test counseling and again when test results were reported. The responses of 7292 women were analyzed. They expressed high satisfaction with the genetic counseling system of the NIPT Consortium (94%). The number of respondents who indicated that genetic counseling is necessary for NIPT increased over time. Furthermore, they highly valued genetic counseling provided by skilled clinicians, such as clinical geneticists or genetic counselors. The vast majority (90%) responded that there was sufficient opportunity to consider the test ahead of time. Meanwhile, women who received positive test results had a poor opinion and expressed a low-degree satisfaction. We confirmed that the pre-test genetic counseling that we conducted creates an opportunity for pregnant women to sufficiently consider prenatal testing, promotes its understanding and has possibilities to effectively facilitate informed decision making after adequate consideration. A more careful and thorough approach is considered to be necessary for women who received positive test results.

Practicability of prenatal testing using lectin-based enrichment of fetal erythroblasts.

AIM: The aim of this study was to investigate the practicability and efficiency of lectin-based isolation of fetal erythroblasts for clinical use in non-invasive prenatal testing. METHODS: Peripheral blood samples were collected from 39 pregnant women. Leukocytes were removed with an anti-CD45 antibody after density gradient centrifugation. After blood cells were attached to slides by binding to a galactose-specific lectin and galactose-bound vinyl polymer, the slides were stained with May-Grünwald-Giemsa stain and cells were classified by automated image analysis based on their size and the nuclear area/cytoplasmic area ratio. In 14 samples from the women with male fetuses, fetal origin of the isolated erythroblasts was confirmed by detecting the Y chromosome using fluorescence in situ hybridization. In eight samples, single erythroblasts were collected by the laser capture microdissection technique for amplification of the sex-determining region Y gene to confirm fetal origin. RESULTS: Panning with an anti-CD45 antibody achieved stable removal of leukocytes without aggregation. In all samples, erythroblasts were successfully identified by automated image analysis (18-6000/10 mL of blood). The number of slides required to examine 10 mL of blood ranged from one to six, which was reasonable for clinical use. The Y chromosome was detected in 7.5-43.6% of erythroblasts by fluorescence in situ hybridization, and the sex-determining region Y gene was amplified in seven of eight samples. CONCLUSION: The combination of lectin-based erythroblast isolation and automated image analysis is a practical and efficient method for isolating fetal erythroblasts as a source of fetal genomes.

Fetal cell-free DNA fraction in maternal plasma is affected by fetal trisomy.

The purpose of this noninvasive prenatal testing (NIPT) study was to compare the fetal fraction of singleton gestations by gestational age, maternal characteristics and chromosome-specific aneuploidies as indicated by z-scores. This study was a multicenter prospective cohort study. Test data were collected from women who underwent NIPT by the massively parallel sequencing method. We used sequencing-based fetal fraction calculations in which we estimated fetal DNA fraction by simply counting the number of reads aligned within specific autosomal regions and applying a weighting scheme derived from a multivariate model. Relationships between fetal fractions and gestational age, maternal weight and height, and z-scores for chromosomes 21, 18 and 13 were assessed. A total of 7740 pregnant women enrolled in the study, of which 6993 met the study criteria. As expected, fetal fraction was inversely correlated with maternal weight (P<0.001). The median fetal fraction of samples with euploid result (n=6850) and trisomy 21 (n=70) were 13.7% and 13.6%, respectively. In contrast, the median fetal fraction values for samples with trisomies 18 (n=35) and 13 (n=9) were 11.0% and 8.0%, respectively. The fetal fraction of samples with trisomy 21 NIPT result is comparable to that of samples with euploid result. However, the fetal fractions of samples with trisomies 13 and 18 are significantly lower compared with that of euploid result. We conclude that it may make detecting these two trisomies more challenging.

Development of a fibrous DNA chip for cost-effective ß-thalassemia genotyping.

ß-thalassemia is one of the most common genetic disorders worldwide. Concerted efforts are being made to prevent the disease, as the medical and economic burden of thalassemia represents a major public health problem. The molecular diagnosis of the ß-globin mutations that cause the disease currently involves a combination of classic methodologies. A microarray-based assay for parallel one-shot detection of mutations has been developed, but the assay remains too expensive for routine application. We developed a cost-effective plastic fiber-based DNA chip for the fast and reliable detection of 25 types of ß-thalassemia mutations. Assay conditions were established and genotyping was successfully performed on a genomic sample from a ß-thalassemia patient. Our data show that this ß-thalassemia genotyping chip is an advantageous platform for mass genotyping because of its low cost, rapid results, and reliability.

Prediction of pregnancy-induced hypertension by a shift of blood pressure class according to the JSH 2009 guidelines.

Elevated blood pressure (BP) at early or mid pregnancy is a known risk factor for pregnancy-induced hypertension (PIH). However, the association between BP changes during the first half of pregnancy and subsequent PIH development is unknown. We used changes in maternal BP between 16 and 20 weeks of gestation to evaluate the risk of PIH. A total of 976 pregnant women with BP estimations recorded before 16 weeks and at 20 weeks of gestation participated in this study. BPs were classified by the Japanese Society of Hypertension 2009 Hypertension Treatment Guidelines (JSH 2009). There was a significant trend for future PIH in women whose JSH 2009 BP class increased between 16 and 20 weeks of gestation, and the risk of PIH was highest among women whose BP was Class IV Hypertension (systolic BP≥140 mm Hg and/or diastolic BP≥90 mm Hg). The risk of PIH increased in women whose BPs shifted from Classes I Optimal (systolic BP<120 mm Hg and diastolic BP<80 mm Hg) and II Normal (systolic BP 120-129 mm Hg and/or diastolic BP 80-84 mm Hg) before 16 weeks to Class III High-Normal (systolic BP 130-139 mm Hg and/or diastolic BP 85-89 mm Hg) at 20 weeks of gestation. These shifts in BP class were significantly correlated with the risk of PIH after adjustments for variables (P-value for trend <0.05). Within JSH 2009 Classes I, II and III, a shift in BP from a low to a high class between 16 and 20 weeks of gestation predicts the subsequent development of PIH.

Placenta accreta following uterine artery embolization.

OBJECTIVE: Uterine artery embolization (UAE) is becoming a common treatment for symptomatic leiomyoma. Several pregnancies following UAE have been reported. However, reports are still limited and the risk of complications remains unknown. CASE REPORT: A primigravida conceived after UAE for leiomyoma. She delivered spontaneously at 34 weeks plus 2 days after premature rupture of the membranes. The placenta was located on the interstitial leiomyoma. The patient required manual placental extraction owing to retained placenta and subsequently underwent emergency supracervical hysterectomy for severe postpartum hemorrhage. Placenta accreta was confirmed histologically. CONCLUSION: Placenta accreta may occur during pregnancy following UAE. When the implantation site is on the leiomyoma with a hyperechoic rim, there is a high risk of abnormal placental adherence.

Intra-Abdominal Bleeding during Pregnancy, Preterm Delivery, and Placental Polyp in a Long-Term Survivor of Neuroblastoma: A Case Report.

Background. There are few reports of pregnancies in long-term survivors of pelvic neuroblastoma. Case. A 30-year-old Japanese woman with a history of pelvic neuroblastoma in her childhood, which was treated with surgical resection, chemotherapy, and radiation. Her pregnancy continued with conservative management, but she delivered a 510 g female infant at 23 weeks of gestation due to sudden onset of labor pain. She also had a placental polyp and developed massive postpartum bleeding. Conclusion. Cancer treatment, especially radiation therapy, in childhood may cause adverse outcomes during pregnancy in long-term survivors of neuroblastoma.

[Case of external cephalic version under combined spinal-epidural anesthesia followed by vaginal delivery].

For a parturient with breech presentation, an external cephalic version is sometimes done to enable vaginal delivery. Usually external cephalic version has been done without anesthesiologist's management. However there have been several reports indicating a benefit of anesthesia for external cephalic version. We report successful case of external cephalic version under combined spinal-epidural anesthesia followed by vaginal delivery.

Endoscopic fetal urethrotomy for anterior urethral valves: a preliminary report.

Anterior urethral valves are a rare congenital anomaly associated with distal urethral obstruction, which can result in a poor prognosis. We report on the endoscopic creation of a fetal urethrotomy for obstructive uropathy resulting from anterior urethral valves. A 33-year-old woman was evaluated at 17 weeks gestation due to fetal megacystis. The diagnosis of anterior urethral valves was confirmed by the characteristic sonographic feature of a dilated membranous penile urethra. Oligohydramnios with normal-appearing kidneys and favorable urinary electrolytes led to fetal intervention. Ablation on the ventral site of the fetal penis for a cutaneous urethrotomy was performed using a YAG laser under a 1-mm fetoscope at 19 weeks gestation. Urine was drained from the incision and the dilated penis and the distended bladder shrunk with an increase in amniotic fluid. However, the fetus died unexpectedly on postoperative day 3, and chorioamnionitis was suspected as the etiology. While the outcome was unfavorable, our preliminary experience shows that fetal urethrotomy for obstructive uropathy can be achieved in utero using an endoscopic laser approach. Further experience will be required to evaluate the therapeutic value of this new procedure in the management of fetal anterior urethral valves.

Selection of hematopoietic stem cells with a combination of galactose-bound vinyl polymer and soybean agglutinin, a galactose-specific lectin.

BACKGROUND: Selection of hematopoietic stem cells can be used to prevent graft-versus-host disease (GVHD) after allograft transplantation. The purpose of the study was to examine a novel cell separation system comprising a galactose-bound vinyl polymer (Gal-VP) and soybean agglutinin (SBA), a galactose-specific lectin. STUDY DESIGN AND METHODS: A vinyl polymer (VP) containing alpha-1,6- and beta-1,4-linked galactose terminals was used to facilitate cell separation. A VP containing an alpha-1,4-linked glucose terminal (alpha-1,4-Glu-VP) was also synthesized as a control for alpha-1,6- and beta-1,4-Gal-VP. Peripheral blood samples were collected from healthy volunteers and umbilical cord blood cells were collected after normal labor. RESULTS: The sugar-VP was adsorbed on the surface of various materials. In the presence of SBA, T lymphocytes bound to beta-1,4-Gal-VP-coated microbeads, but not to alpha-1,4-Glu-VP-coated microbeads. When peripheral or cord blood cells were cultured on alpha-1,6-Gal-VP-coated plates, most red blood cells, lymphocytes, granulocytes, and monocytes adhered to the plate in the presence of 300 mg per mL SBA, whereas few CD34+ cells attached, even with 800 mg per mL SBA. CONCLUSION: SBA binds selectively to blood cells by recognizing cell-surface sugars, which are dependent on the extent of cellular differentiation. Therefore, the combination of alpha-1,6-Gal-VP and SBA might be useful for separation of blood cells according to their stage of differentiation and lineage.

Development of noninvasive fetal DNA diagnosis from nucleated erythrocytes circulating in maternal blood.

BACKGROUND: A considerable effort is being spent in developing noninvasive prenatal DNA diagnostic procedures. We recently reported that nucleated erythrocytes (NRBCs) can be enriched from maternal blood by a galactose-specific lectin method. In the present study, to prove that fetal NRBCs are definitely present in maternal blood and are a good source for fetal genetic diagnosis, we evaluated methods for lectin enrichment and subsequent fluorescence in situ hybridization (FISH) analysis through fetal gender determination. METHODS: Peripheral blood samples were collected from pregnant women (median 15, range: 10-18 weeks). From the blood samples, NRBCs were enriched based on galactose-specific lectin method. After detecting them by their morphology, NRBCs are separated and taken in a new glass slide by micromanipulator. We analyzed fetal gender using X and Y-chromosome-specific FISH probes. The results were compared with fetal gender analysis using Y-chromosomal sequences in maternal plasma. RESULTS: The fetal gender analyses by FISH in 20 pregnant women were all in accordance with the results from maternal plasma analyses. It is confirmed that fetal NRBCs were present in maternal blood and that 30.4% of NRBCs in maternal blood were fetal in origin. CONCLUSION: We have successfully carried out a noninvasive prenatal DNA diagnosis of fetal gender by using galactose-specific lectin method and subsequent FISH analysis.