A cytological F1 RNAi screen for defects in Drosophila melanogaster female meiosis.

Genetic screens for recessive alleles induce mutations, make the mutated chromosomes homozygous, and then assay those homozygotes for the phenotype of interest. When screening for genes required for female meiosis, the phenotype of interest has typically been nondisjunction from chromosome segregation errors. As this requires that mutant females be viable and fertile, any mutants that are lethal or sterile when homozygous cannot be recovered by this approach. To overcome these limitations, we have screened the VALIUM22 collection of RNAi constructs that target germline-expressing genes in a vector optimized for germline expression by driving RNAi with GAL4 under control of a germline-specific promoter (nanos or mat-alpha4). This allowed us to test genes that would be lethal if knocked down in all cells, and by examining unfertilized metaphase-arrested mature oocytes, we could identify defects in sterile females. After screening >1,450 lines of the collection for two different defects (chromosome congression and the hypoxic sequestration of Mps1-GFP to ooplasmic filaments), we obtained multiple hits for both phenotypes, identified novel meiotic phenotypes for genes that had been previously characterized in other processes, and identified the first phenotypes to be associated with several previously uncharacterized genes.

A Cytological F1 RNAi Screen for Defects in Drosophila melanogaster Female Meiosis.

Genetic screens for recessive alleles induce mutations, make the mutated chromosomes homozygous, and then assay those homozygotes for the phenotype of interest. When screening for genes required for female meiosis, the phenotype of interest has typically been nondisjunction from chromosome segregation errors. As this requires that mutant females be viable and fertile, any mutants that are lethal or sterile when homozygous cannot be recovered by this approach. To overcome these limitations, our lab has screened the VALIUM22 collection produced by the Harvard TRiP Project, which contains RNAi constructs targeting genes known to be expressed in the germline in a vector optimized for germline expression. By driving RNAi with GAL4 under control of a germline-specific promoter (nanos or mat-alpha4), we can test genes that would be lethal if knocked down in all cells, and by examining unfertilized metaphase-arrested mature oocytes, we can identify defects associated with genes whose knockdown results in sterility or causes other errors besides nondisjunction. We screened this collection to identify genes that disrupt either of two phenotypes when knocked down: the ability of meiotic chromosomes to congress to a single mass at the end of prometaphase, and the sequestration of Mps1-GFP to ooplasmic filaments in response to hypoxia. After screening >1450 lines of the collection, we obtained multiple hits for both phenotypes, identified novel meiotic phenotypes for genes that had been previously characterized in other processes, and identified the first phenotypes to be associated with several previously uncharacterized genes.

Outcomes in Subsequent Pregnancies of Individuals With Opioid Use Disorder Treated in Multidisciplinary Clinic in Prior Pregnancy.

BACKGROUND: Untreated opioid misuse in pregnancy is associated with adverse outcomes. Limited information is available on maternal and perinatal outcomes in subsequent pregnancies for individuals initiated on medication for opioid use disorder (MOUD) in a prior pregnancy. OBJECTIVE: Evaluate maternal and neonatal outcomes in subsequent pregnancies for individuals initiated on MOUD in prior pregnancy. METHODS: Historical cohort study including individuals with opioid use disorder with ≥2 pregnancies between 2013 and 2020, received care in our colocated multidisciplinary clinic for >1 pregnancy, and delivered at our institution. Primary outcome was rate of preconception MOUD. Secondary outcomes included rate of neonatal opioid withdrawal syndrome requiring pharmacologic treatment and length of hospital stay. RESULTS: Forty-two individuals with opioid use disorder in their index pregnancies (n = 42) and 46 subsequent pregnancies were identified. Individuals were more likely to receive long-acting reversible contraception in subsequent pregnancies (35% vs 14%, P = 0.04). No differences in tobacco use, gestational age at initiation of prenatal care or delivery was noted. Individuals in their subsequent pregnancies were 6 times more likely to be on MOUD preconception (78% vs 36%; OR, 6.48; [95% CI, 2.52-16.64]) and 67% less likely to have positive illicit urine drug screen upon initiation of care (36% vs 64%; OR, 0.33; 95% [CI, 0.14-0.78]). Neonates had similar rates of neonatal abstinence withdrawal syndrome requiring pharmacological treatment, positive illicit toxicology results, and neonatal length of stay. CONCLUSIONS: Participation in multidisciplinary obstetric and opioid use disorder program increases rate of MOUD in subsequent pregnancy with decrease in illicit drug use.

Intrahepatic cholestasis of pregnancy: risk factors for severe disease.

INTRODUCTION: Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy-associated hepatic disorder characterized by pruritus in the setting of elevated serum bile acids (BA). Risk factors for the disease include preexisting hepatobiliary disease, personal or family history of ICP, and advanced maternal age. Recent data suggests that patients with severe ICP (BA ≥100 µmol/L) have a higher risk of adverse pregnancy outcomes including stillbirth. MATERIAL AND METHODS: This was a retrospective cohort study of patients diagnosed with ICP between 2012 and 2019 at a tertiary referral center. ICP was defined as symptomatic pruritus combined with serum BA >10 µmol/L. Maternal characteristics and outcomes were abstracted from electronic medical records. Baseline characteristics were compared between patients with mild (BA <40 µmol/L), moderate (BA 40-99 µmol/L) and severe (BA ≥100 µmol/L) ICP. Obstetrics and neonatal outcomes for patients in each category were then analyzed. Shapiro-Wilk test was used to test for normality for continuous variables, and ANOVA, Kruskal-Wallis, Chi-squared or Fisher's exact tests were used as appropriate. A p-value <.05 was considered statistically significant. RESULTS: 438 patients were included in the analysis. Individuals with pregestational diabetes (p < .01), history of ICP (p < .01), prior cholecystectomy (p < .01), and tobacco use (p < .05) were more likely to have severe disease. When compared to individuals with moderate and mild disease, individuals with severe disease were more likely to be diagnosed earlier (29w1d vs 34w1d vs 34w1d, p < .05), have gestational diabetes (50% vs 6% vs 13%, p < .01), hypertensive disorders of pregnancy (42% vs 10% vs 15%, p = .02), and abnormal aspartate aminotransferase (91% vs 65% vs 27%, p < .01) and alanine aminotransferase levels (91% vs 60% vs 26%, p < .01). There were no differences in preterm labor, meconium-stained amniotic fluid, or neonatal respiratory distress syndrome and no stillbirths in this cohort. CONCLUSIONS: In patients with ICP, those with pregestational diabetes, history of ICP, prior cholecystectomy, and tobacco use are more likely to develop severe disease. Given the adverse outcomes associated with severe disease, serial BA measurements to monitor for development of severe disease may be warranted in this population.

Alteration of Membrane Cholesterol Content Plays a Key Role in Regulation of Cystic Fibrosis Transmembrane Conductance Regulator Channel Activity.

Altered cholesterol homeostasis in cystic fibrosis patients has been reported, although controversy remains. As a major membrane lipid component, cholesterol modulates the function of multiple ion channels by complicated mechanisms. However, whether cholesterol directly modulates cystic fibrosis transmembrane conductance regulator (CFTR) channel function remains unknown. To answer this question, we determined the effects of changing plasma membrane cholesterol levels on CFTR channel function utilizing polarized fischer rat thyroid (FRT) cells and primary human bronchial epithelial (HBE) cells. Treatment with methyl-ß-cyclodextrin (MßCD) significantly reduced total cholesterol content in FRT cells, which significantly decreased forskolin (FSK)-mediated activation of both wildtype (WT-) and P67L-CFTR. This effect was also seen in HBE cells expressing WT-CFTR. Cholesterol modification by cholesterol oxidase and cholesterol esterase also distinctly affected activation of CFTR by FSK. In addition, alteration of cholesterol increased the potency of VX-770, a clinically used potentiator of CFTR, when both WT- and P67L-CFTR channels were activated at low FSK concentrations; this likely reflects the apparent shift in the sensitivity of WT-CFTR to FSK after alteration of membrane cholesterol. These results demonstrate that changes in the plasma membrane cholesterol level significantly modulate CFTR channel function and consequently may affect sensitivity to clinical therapeutics in CF patients.

Extending the platinum-free interval: The impact of omitting 2nd line platinum chemotherapy in intermediate platinum-sensitive ovarian cancer.

OBJECTIVES: Patients with epithelial ovarian cancer (EOC) recurring between 6 and 12 months after primary platinum chemotherapy have worse prognosis than those recurring in >12 months. Artificially prolonging the platinum-free interval (PFI) with cytotoxic chemotherapy was tested in MITO-8 with poor outcomes. This study aimed to determine the impact of using non-platinum or targeted therapy in 2nd line treatment of EOC patients recurring 6-12 months after completion of primary platinum-based chemotherapy. METHODS: A multi-institutional retrospective review of 177 patients with recurrent EOC and PFI of 6-12 months following primary chemotherapy was performed comparing platinum versus non-platinum chemotherapy or targeted therapy for 2nd line treatment. PFI1 was defined as the date of last chemotherapy to date of recurrence. PFS2/3 were defined as start of 2nd or 3rd line chemotherapy to start of subsequent line. RESULTS: Of 177 patients, the majority of patients were Caucasian, had serous histology, and underwent primary cytoreductive surgery. Median PFI1 was 8.2 months (95% CI 8-9 months). Second line platinum was omitted in 28% of patients. Bevacizumab was used in 2nd line in 16% of patients; 19% received other targeted therapies. Median PFS2 for platinum chemotherapy was longer than non-platinum (7.1 vs 3 months, p = 0.0114). Median PFS2 was significantly longer for platinum vs. targeted therapy (7.1 vs. 3 months p = 0.0431). Median OS for platinum in 2nd line vs. no platinum was 43.6 vs. 37.6 months (p = 0.0174). CONCLUSIONS: Use of non-platinum chemotherapy and even targeted therapy to prolong PFI in patients with EOC recurring between 6 and 12 months leads to worse survival.

Trauma-induced delivery: A case report.

The term "birth trauma" is one that is well ingrained into the lexicon of medical providers. There is ample information of the types of injuries that are incurred during the birth process. However, there is no uniformed term for the process of an unforeseen act that leads to a precipitous birth. We would like to show a case report of such an act. The infant's injuries were sustained while in utero and the trauma induced a medical team to deliver the infant due to non-reassuring heart tones. Also, we would like to introduce the term of "trauma-induced delivery" into the medical literature as a way to describe similar types of deliveries that are influenced by factors related to physical forces applied to the mother's body, either intentional or unintentional.