Noninvasive single-cell-based prenatal genetic testing: A proof of concept clinical study.

OBJECTIVE: To clinically assess a cell-based noninvasive prenatal genetic test using sequence-based copy number analysis of single trophoblasts from maternal blood. METHODS: Blood was obtained from 401 (243 + 158) individuals (8-22 weeks) and shipped overnight. Red cells were lysed, and nucleated cells stained for cytokeratin (CK) and CD45 and enriched for positive CK staining. Automated scanning was used to identify and pick single CK+ /CD45- trophoblasts which were subjected to next-generation sequencing. RESULTS: Blood was obtained from 243 pregnancies scheduled for CVS or amniocentesis. Luna results were normal for 160 singletons while 15 cases were abnormal (14 aneuploidy and one monozygotic twin with Williams syndrome deletion). The deletion was confirmed in both fetuses. Placental mosaicism occurred in 7 of 236 (3.0%) Luna cases and in 3 of 188 (1.6%) CVS cases (total 4.6%). No scorable trophoblasts were recovered in 32 of 236 usable samples. Additionally, 158 low-risk pregnancies not undergoing CVS/amniocentesis showed normal results in 133 cases. Seven had aneuploidy results, and there were three likely pathogenic deletions/duplications, including one15q11-q13 deletion. CONCLUSION: Although the sample size is modest and statistically accurate measures of test performance are not possible, the Luna test detected aneuploidy and deletions/duplications based on concordance with CVS/amniocentesis.

Unindicated cervical cancer screening in adolescent females within a large healthcare system in the United States.

BACKGROUND: Current consensus recommendations are to not initiate cervical cancer screening for immunocompetent adolescent females before 21 years of age. This is in part because of the very low rate of 0.8 per 100,000 new cervical cancer cases diagnosed among women aged between 20 to 24 years. Timely human papillomavirus vaccination further decreases the incidence of cervical cancer to 4 cases per 100,000 persons by the age of 28 years. Screening before 21 years of age has demonstrated no clear benefit in cancer risk reduction or outcomes. In addition, unindicated screening among adolescents can lead to patient harm and increasing costs to the healthcare system. OBJECTIVE: It is important to assess the rates of overutilization of cervical cancer screening and to identify areas where improvements have occurred and where further opportunities exist. This study aimed to assess the trends over time and the practice and provider factors associated with unindicated cervical cancer screening tests in adolescent females within the largest healthcare system in the state. STUDY DESIGN: Cross-sectional data from patients aged 13 to 20 years who underwent cervical cancer screening between January 1, 2012, and December 31, 2018, across a large multihospital health system were reviewed. All cervical cancer screening results were included. The incidence rate of unindicated screening was analyzed over 6-month intervals using the Poisson regression analysis. RESULTS: The study included data from 118 providers and 794 women. Among the 900 screening results, most (90%) were unindicated: 87% with unindicated cytology testing alone and 14% with unindicated human papillomavirus testing. Screening tests were collected from patients aged 13 to 20 years, many of whom had multiple unindicated cytology tests, with 25 patients having ≥3 tests before the age of 21 years. Most results of cytology testing were negative for intraepithelial lesion or malignancy (77%). Moreover, 52 invasive diagnostic or therapeutic procedures (49 colposcopies and 3 conizations) were performed, of which 45 (87%) followed an unindicated screening test. Between 2012 and 2018, the incidence rate of unindicated cytology decreased by 33% (12.6 to 8.5 unindicated cytology per 1000 encounters). The incidence rate of unindicated screening was lower in the academic setting than in the community setting (incidence rate ratio, 0.43; P<.01). Even with decreases in the overall rates of unindicated screening throughout the study period, there were still 58 unindicated screening tests performed in the final year of this study. CONCLUSION: Despite substantial reductions in unindicated screening for women aged <21 years, there remained areas for improvement. Our data reflected practices of guideline nonadherence up to 7 years after the 2012 guideline. Now, with a new series of changes to the guidelines, which may be even more challenging for patients and providers, it is more important than ever to utilize evidence-based strategies to improve guideline dissemination and adherence.

Long-term outcomes of BRCA1/BRCA2 testing: risk reduction and surveillance.

BACKGROUND: For BRCA1/BRCA2 gene testing to benefit public health, mutation carriers must initiate appropriate risk management strategies. There has been little research examining the long-term use and prospective predictors of the full range of risk management behaviors among women who have undergone BRCA1/2 testing. We evaluated long-term uptake and predictors of risk-reducing mastectomy (RRM), risk-reducing bilateral salpingo-oophorectomy (RRBSO), chemoprevention, and cancer screening among women at a mean of 5.3 years after testing. METHODS: The study participants comprised 465 women who underwent BRCA1/2 testing. Prior to genetic counseling, we measured family/personal cancer history, sociodemographics, perceived risk, cancer-specific distress, and general distress. We contacted patients at a mean of 5.3 years after testing to measure use of RRM, RRBSO, chemoprevention, and breast and ovarian cancer screening. RESULTS: Among participants with intact breasts and/or ovaries at the time of testing, BRCA1/2 carriers were significantly more likely to obtain RRM (37%) and RRBSO (65%) compared with women who received uninformative (RRM, 6.8%; RRBSO, 13.3%) or negative (RRM, 0%; RRBSO, 1.9%) results. Among carriers, precounseling anxiety was associated with subsequent uptake of RRM. RRO was predicted by age. Carriers were also more likely have used breast cancer chemoprevention and have undergone magnetic resonance imaging screening. CONCLUSION: This prospective evaluation of the uptake and predictors of long-term management outcomes provides a clearer picture of decision making in this population. At a mean of 5.3 years after testing, more than 80% of carriers had obtained RRM, RRBSO, or both, suggesting that BRCA1/2 testing is likely to have a favorable effect on breast and ovarian cancer outcomes.

X chromosome inactivation in the human placenta is patchy and distinct from adult tissues.

In humans, one of the X chromosomes in genetic females is inactivated by a process called X chromosome inactivation (XCI). Variation in XCI across the placenta may contribute to observed sex differences and variability in pregnancy outcomes. However, XCI has predominantly been studied in human adult tissues. Here, we sequenced and analyzed DNA and RNA from two locations from 30 full-term pregnancies. Implementing an allele-specific approach to examine XCI, we report evidence that XCI in the human placenta is patchy, with large patches of either maternal or paternal X chromosomes inactivated. Further, using similar measurements, we show that this is in contrast to adult tissues, which generally exhibit mosaic X inactivation, where bulk samples exhibit both maternal and paternal X chromosome expression. Further, by comparing skewed samples in placenta and adult tissues, we identify genes that are uniquely inactivated or expressed in the placenta compared with adult tissues, highlighting the need for tissue-specific maps of XCI.

Erratum: X chromosome inactivation in the human placenta is patchy and distinct from adult tissues.

[This corrects the article DOI: 10.1016/j.xhgg.2022.100121.].

Seizing the Window of Opportunity Within 1 Year Postpartum: Early Cardiovascular Screening.

Background Our objective was to assess new chronic hypertension 6 to 12 months postpartum for those with hypertensive disorder of pregnancy (HDP) compared with normotensive participants. Methods and Results We performed a prospective cohort study of participants with singleton gestations and no known preexisting medical conditions who were diagnosed with HDP compared with normotensive women with no pregnancy complications (non-HDP). Participants underwent cardiovascular risk assessment 6 to 12 months after delivery. Primary outcome was onset of new chronic hypertension at 6 to 12 months postpartum. We also examined lipid values, metabolic syndrome, prediabetes, diabetes, and 30-year cardiovascular disease (CVD) risk. Multivariable logistic regression was performed to assess the association between HDP and odds of a postpartum diagnosis of chronic hypertension while adjusting for parity, body mass index, insurance, and family history of CVD. There were 58 participants in the HDP group and 51 participants in the non-HDP group. Baseline characteristics between groups were not statistically different. Participants in the HDP group had 4-fold adjusted odds of developing a new diagnosis of chronic hypertension 6 to 12 months after delivery, compared with those in the non-HDP group (adjusted odds ratio, 4.60 [95% CI, 1.65-12.81]), when adjusting for body mass index, parity, family history of CVD, and insurance. Of the HDP group, 58.6% (n=34) developed new chronic hypertension. Participants in the HDP group had increased estimated 30-year CVD risk and were more likely to have metabolic syndrome, a higher fasting blood glucose, and higher low-density lipoprotein cholesterol. Conclusions Participants without known underlying medical conditions who develop HDP have 4-fold increased odds of new diagnosis of chronic hypertension by 6 to 12 months postpartum as well as increased 30-year CVD risk scores. Implementation of multidisciplinary care models focused on CVD screening, patient education, and lifestyle interventions during the first year postpartum may serve as an effective primary prevention strategy for the development of CVD.

Insulin increases placental triglyceride as a potential mechanism for fetal adiposity in maternal obesity.

OBJECTIVE: Maternal obesity increases the incidence of excess adiposity in newborns, resulting in lifelong diabetes risk. Elevated intrauterine fetal adiposity has been attributed to maternal hyperglycemia; however, this hypothesis does not account for the increased adiposity seen in newborns of mothers with obesity who have euglycemia. We aimed to explore the placental response to maternal hyperinsulinemia and the effect of insulin-like growth factor 2 (IGF-2) in promoting fetal adiposity by increasing storage and availability of nutrients to the fetus. METHODS: We used placental villous explants and isolated trophoblasts from normal weight and obese women to assess the effect of insulin and IGF-2 on triglyceride content and insulin receptor signaling. Stable isotope tracer methods were used ex vivo to determine effect of hormone treatment on de novo lipogenesis (DNL), fatty acid uptake, fatty acid oxidation, and esterification in the placenta. RESULTS: Here we show that placentae from euglycemic women with normal weight and obesity both have abundant insulin receptor. Placental depth and triglyceride were greater in women with obesity compared with normal weight women. In syncytialized placental trophoblasts and villous explants, insulin and IGF-2 activate insulin receptor, induce expression of lipogenic transcription factor SREBP-1 (sterol regulatory element-binding protein 1), and stimulate triglyceride accumulation. We demonstrate elevated triglyceride is attributable to increased esterification of fatty acids, without contribution from DNL and without an acceleration of fatty acid uptake. CONCLUSIONS: Our work reveals that obesity-driven aberrations in maternal metabolism, such as hyperinsulinemia, alter placental metabolism in euglycemic conditions, and may explain the higher prevalence of excess adiposity in the newborns of obese women.

Sex differences in early and term placenta are conserved in adult tissues.

BACKGROUND: Pregnancy complications vary based on the fetus's genetic sex, which may, in part, be modulated by the placenta. Furthermore, developmental differences early in life can have lifelong health outcomes. Yet, sex differences in gene expression within the placenta at different timepoints throughout pregnancy and comparisons to adult tissues remains poorly characterized. METHODS: Here, we collect and characterize sex differences in gene expression in term placentas (≥ 36.6 weeks; 23 male XY and 27 female XX). These are compared with sex differences in previously collected first trimester placenta samples and 42 non-reproductive adult tissues from GTEx. RESULTS: We identify 268 and 53 sex-differentially expressed genes in the uncomplicated late first trimester and term placentas, respectively. Of the 53 sex-differentially expressed genes observed in the term placentas, 31 are also sex-differentially expressed genes in the late first trimester placentas. Furthermore, sex differences in gene expression in term placentas are highly correlated with sex differences in the late first trimester placentas. We found that sex-differential gene expression in the term placenta is significantly correlated with sex differences in gene expression in 42 non-reproductive adult tissues (correlation coefficient ranged from 0.892 to 0.957), with the highest correlation in brain tissues. Sex differences in gene expression were largely driven by gene expression on the sex chromosomes. We further show that some gametologous genes (genes with functional copies on X and Y) will have different inferred sex differences if the X-linked gene expression in females is compared to the sum of the X-linked and Y-linked gene expression in males. CONCLUSIONS: We find that sex differences in gene expression are conserved in late first trimester and term placentas and that these sex differences are conserved in adult tissues. We demonstrate that there are sex differences associated with innate immune response in late first trimester placentas but there is no significant difference in gene expression of innate immune genes between sexes in healthy full-term placentas. Finally, sex differences are predominantly driven by expression from sex-linked genes.

Early sonographic detection of a succenturiate placenta after IVF in a 42-year-old woman with multiple comorbidities.

We present a case of a 42-year-old woman with a pregnancy resulting from in vitro fertilisation and a medical history including two spontaneous abortions, hypercoagulable state and other comorbidities. At 13 4/7 weeks' gestation, during research ultrasonography, the patient was noted to have an anterior succenturiate placental lobe. Following an episode of vaginal bleeding at 21 6/7 weeks, she was diagnosed with a low-lying posterior placental lobe. Velamentous cord insertion, placenta previa and vasa previa were excluded at that time. After elective induction for advanced maternal age at 39 0/7 weeks, arrest of labour and chorioamnionitis resulted in a primary low transverse caesarean section and delivery of a healthy girl at 39 3/7 weeks. Gross examination of the placenta showed an irregular, singleton placenta with an attached succenturiate lobe and a marginally inserting umbilical cord. Both lobes were connected by two vessels.

Time course analysis of RNA quality in placenta preserved by RNAlater or flash freezing.

PROBLEM: Preservation of biospecimen quality is critical to accurately and reliably assessing genes and proteins. We evaluated the effect of preparation method and storage duration on RNA quality in placenta and decidua. METHOD OF STUDY: Aliquots of nine placentas and decidua were placed in RNAlater® (RL) or flash frozen (FF) within 30 minutes of delivery. RNA was extracted immediately (baseline) and from matched samples stored at -80°C for 1 and 8-10 months. RNA Integrity Number (RIN) and housekeeping gene expression were quantified. RESULTS: At both time points, RL placenta had RIN and housekeeping gene Ct values similar to baseline. However, FF placenta had significantly lower RIN and higher Ct values at 1 and 8-10 months. In RL and FF decidua, RIN was unchanged from baseline. CONCLUSION: We found RNAlater more effectively and consistently preserved placenta, compared to flash freezing. However, for decidua, which is less dense than placenta, both modes yielded comparable RNA integrity over time.

Social network and census tract-level influences on substance use among emerging adult males: An activity spaces approach.

Social network and area level characteristics have been linked to substance use. We used snowball sampling to recruit 90 predominantly African American emerging adult men who provided typical locations visited (n=510). We used generalized estimating equations to examine social network and area level predictors of substance use. Lower social network quality was associated with days of marijuana use (B=-0.0037, p<0.0001) and problem alcohol use (B=-0.0050, p=0.0181). The influence of area characteristics on substance use differed between risky and non-risky spaces. Peer and area influences are important for substance use among men, and may differ for high and low risk places.