Lysophosphatidylcholine acyltransferase 1 protein is present in maternal blood in the third trimester and is upregulated by antenatal corticosteroids.

OBJECTIVES: Lysophosphatidylcholine acyltransferase 1 (LPCAT1) is involved in the production of fetal lung surfactant. We have shown that LPCAT1 mRNA is present in amniotic fluid and maternal plasma and that its quantity correlates with the amniotic fluid lamellar body count. The purpose of the present study was to assay maternal plasma for the LPCAT1 protein in term and preterm pregnancies; and to measure the impact of antenatal corticosteroids. METHODS: Maternal and newborn plasma samples were obtained from 7 women admitted to the hospital for induction of labor. Maternal plasma was also obtained before administration of corticosteroids and 24 h after the second dose of corticosteroids from 12 women with premature labor and premature rupture of membranes. After sample preparation, LPCAT1 protein levels were determined using sandwich ELISA. RESULTS: We discovered LPCAT1 protein in maternal plasma in measurable quantities after 32 weeks gestation. Further, there was a rise of maternal plasma LPCAT1 in response to the clinical administration of antenatal corticosteroids. CONCLUSIONS: Quantitation of maternal plasma LPCAT1 protein offers promise in the ongoing study of fetal lung maturation.

Long-Acting Reversible Contraception: Placement, Continuation, and Removal Rates at an Inner-City Academic Medical Center Clinic.

Long-Acting Reversible Contraception (LARCs) has the potential to decrease unintended pregnancies but only if women can easily access a requested method. Retrospective electronic chart review identified women desiring LARC placement over a one-year period ending 31 December 2016. Most of the 311 insertions were for family planning, with 220 new insertions and 60 replacements. Delays occurred in 38% (n = 118) of patients, averaged 5 ± 5 weeks, and 47% received interval contraception. Reasons included absence of qualified provider (n = 44, 37%), pending cultures (n = 31, 26%), and Mirena availability. Teenage LARC use favored Nexplanon whereas older women preferred Mirena (p < 0.01). Of the 11% choosing early LARC removal, a significant number were African Americans (p = 0.040) or teenagers (p = 0.048). Retention time varied by device type; most patients switched to other contraceptives. No patients experienced IUD expulsion. Understanding barriers, attempting to remedy them, and addressing the side effects associated with LARC use is of importance in this inner-city patient population in the United States.

Molecular mechanisms regulating lysophosphatidylcholine acyltransferase 1 (LPCAT1) in human pregnancy.

INTRODUCTION: Lysophosphatidylcholine Acyltransferase 1 (LPCAT1) is necessary for surfactant production in fetal lungs. Mechanisms responsible for its regulation during gestation remain to be elucidated. Our goal is to evaluate molecular mechanisms regulating LPCAT1 expression during gestation and after glucocorticoid administration. METHODS: Placentas throughout gestation were assayed for LPCAT1 protein levels. A placental cell line, HTR-8/SVneo (HTR), was used as a model to test the effects of placental oxygen tension found during pregnancy as well as the effects of dexamethasone used therapeutically in the clinic. RESULTS: LPCAT1 protein levels are maximal in late third trimester placental samples and are expressed strongly on the basal plate. LPCAT1 was maximally upregulated at 4% O2 (P < 0.01), corresponding to oxygen tension found in placenta at term. Mitochondrial nuclear retrograde regulator 1 (MNRR1), a bi-organellar (mitochondria and nucleus) regulator, transcriptionally activates LPCAT1. Antenatal corticosteroids (ACS) upregulate LPCAT1, at least in part, by an MNRR1-dependent pathway. HTR cells treated with 25 nM dexamethasone for 24 h exhibited a 2-fold increase in LPCAT1 levels compared to controls. In MNRR1 knockout cells, the response to ACS is significantly blunted. DISCUSSION: LPCAT1 appears to be induced by MNRR1. Hypoxia and corticosteroids increase LPCAT1 expression through an MNRR1 dependent pathway. LPCAT1 protein levels can be measured in maternal plasma and rise throughout gestation and in response to ACS.

Antisense techniques provide robust decrease in GnRH receptor expression with minimal cytotoxicity in GT1-7 cells.

The episodic pattern of gonadotropin-releasing hormone (GnRH) secretion from the hypothalamus is driven by an integrated network of cells termed the GnRH pulse generator. Cultured and immortalized GnRH neurons also produce a pulsatile pattern of GnRH secretions when grown in the absence of other cell types, suggesting the presence of an intrinsic oscillator mediating GnRH secretion. The mechanisms underlying such pulsatility comprise one of the most tantalizing problems in contemporary neuroendocrinology. In order to study the mechanism by which GnRH is produced in a pulsatile fashion, the autocrine effect of GnRH on GnRH-producing neurons must be eliminated. This may be performed by downregulating the expression of the GnRH receptor. Treatment with three 21-mer exogenous phosphorothioates and transient transfections with an inducible plasmid containing an antisense construct to the GnRH receptor gene decreased GnRH receptor expression further. This resulted in less cytotoxicity compared to inhibition of RNA or protein synthesis with actinomycin D, α-amanitin, puromycin, and cycloheximide. This study shows methods and optimized conditions established for the generation of a stable GT1-7 cell line containing an inducible construct allowing the downregulation of GnRH receptor expression. ABBREVIATIONS: ANOVA: analysis of the variance; DMEM: Dulbecco's modified Eagle's medium; GnRH: gonadotropin-releasing hormone; RXR: retinoid X receptor.

Maternal plasma LPCAT 1 mRNA correlates with lamellar body count.

Human lysophosphatidylcholine acyltransferase 1 (hLPCAT1) is a protein which helps produce surfactant in the fetal lung. We previously reported that levels of cell-free fetal mRNA for hLPCAT1 in amniotic fluid are correlated with lamellar body count (LBC) (r2=0.93). This short communication demonstrates that fetal hLPCAT1 mRNA is also present in maternal blood. Its quantity also correlates with amniotic fluid LBC (r2=0.81). Research in maternal plasma hLPCAT1 may assist in understanding fetal and placental maturational processes.

Amniotic fluid LPCAT1 mRNA correlates with the lamellar body count.

Lysophosphatidylcholine acyltransferase 1 (LPCAT1) is required in the biosynthesis of pulmonary surfactant. This short communication describes our assessment of LPCAT1 mRNA levels in human amniotic fluid. We found a direct correlation between LPCAT1 mRNA copies and the amniotic fluid lamellar body count (LBC). This finding corroborates an association between LPCAT1 and surfactant phospholipid biosynthesis in humans. It may provide a model for future research in perinatal medicine.

Ethics, Human Use, and the Department of Defense Serum Repository.

The Department of Defense Serum Repository (DoDSR) contains a growing archive of sera from service members collected to perform medical surveillance, clinical diagnosis, and epidemiologic studies to identify, prevent, and control diseases associated with military service. The specimens are a mandatory collection under DoD and U.S. regulations and do not include informed consent for uses beyond force health protection. Any use of the specimens for research requires deidentification of the samples and must be approved by Institutional Review Boards. However, as expansion of the DoDSR is contemplated, ethical considerations of sample collection, storage, and use must be carefully reconsidered. Other similar programs for research use of specimens collected for public health purpose are also undergoing similar reviews. It is recommended that at a minimum, service members are informed of the potential storage and use of their specimens and are allowed to opt out of additional use, or a broad informed consent is provided. The DoDSR provides a tremendous resource to the DoD and global health community, and to ensure its continued existence and improvement, the DoD must stay consistent with all principles of research ethics.

Validation of the performance of a comprehensive genotyping assay panel of single nucleotide polymorphisms in drug metabolism enzyme genes.

A class of genes, known as drug metabolism enzymes (DMEs) are responsible for the metabolism and transport of drugs and other xenobiotics. Variation in DME genes most likely accounts for a proportion of the variability in drug response in humans, and may contribute to complex diseases such as cancer (Nebert DW, Dieter MZ. Pharmacology 2000;61:124-135). To date, assessing the extent of this variation has proven difficult, especially because of sequence paralogy issues that cause difficulty when attempting to genotype polymorphisms in very closely-related gene families (Murphy MP. Pharmacogenomics 2000;1:115-123; Ingelman-Sundberg M. Drug Metab Rev 1999;31:449-459). We have developed and genotyped a panel of N=2,325 individual TaqMan genotyping assays for polymorphisms in >200 DME genes; many of the variants in the panel are single nucleotide polymorphisms (SNPs) that are of known or putative function (e.g., missense, nonsense or frameshift). Using these assays, we have examined genetic variation among several groups of populations, including: 1) the two SNP500 Cancer population panels (http://snp500cancer.nci.nih.gov; last accessed: 11 December 2007); and 2) the panel used in the International HapMap Project panel (www.hapmap.org; last accessed: 11 December 2007). We have developed a comprehensive validation strategy to ensure reproducibility and accuracy of the assays and estimated minor allele frequencies. Here, we present the results of these analyses, which strongly suggest that this panel of DME assays are of extremely high quality and produce robust, accurate, and reproducible results.

Operator experience and sample quality in genetic amniocentesis.

OBJECTIVE: We sought to relate the frequency of maternal cell contamination in amniotic fluid samples that were submitted to a single laboratory for cytogenetic analysis to the experience and training of the physician who performed the amniocentesis. STUDY DESIGN: We reviewed the database of a single cytogenetics laboratory to compare the number of amniocenteses that were performed annually per physician to the rate of maternal cell contamination in genetic amniocentesis samples. Only samples that resulted in a 46 XY male karyotype were studied so that maternal cell contamination could be identified as having occurred when the karyotype revealed > or = 1 cell with 2 X chromosomes. Samples were categorized as being submitted by a physician who submitted > or = 50 or more samples annually versus < 50 samples to this laboratory. The frequency of maternal cell contamination was compared with annual operator volume with 2 x 2 tables and analyzed by chi-squared testing. RESULTS: Between 2000 and 2004, the laboratory received 6332 mid-trimester amniotic fluid samples that generated a male karyotype result. Fourteen of 2081 samples (0.67%) that were submitted by physicians who submitted < 50 samples grew > or = 1 46 XX cells, compared with 8 of 4251 samples (0.19%; chi-squared, 9.47; degrees of freedom, 1; P = .0021). CONCLUSION: Maternal cell contamination occurs more frequently in genetic amniocentesis samples that are obtained by physicians who perform < 50 genetic amniocenteses annually.

Effects of DNA mass on multiple displacement whole genome amplification and genotyping performance.

BACKGROUND: Whole genome amplification (WGA) promises to eliminate practical molecular genetic analysis limitations associated with genomic DNA (gDNA) quantity. We evaluated the performance of multiple displacement amplification (MDA) WGA using gDNA extracted from lymphoblastoid cell lines (N = 27) with a range of starting gDNA input of 1-200 ng into the WGA reaction. Yield and composition analysis of whole genome amplified DNA (wgaDNA) was performed using three DNA quantification methods (OD, PicoGreen and RT-PCR). Two panels of N = 15 STR (using the AmpFlSTR Identifiler panel) and N = 49 SNP (TaqMan) genotyping assays were performed on each gDNA and wgaDNA sample in duplicate. gDNA and wgaDNA masses of 1, 4 and 20 ng were used in the SNP assays to evaluate the effects of DNA mass on SNP genotyping assay performance. A total of N = 6,880 STR and N = 56,448 SNP genotype attempts provided adequate power to detect differences in STR and SNP genotyping performance between gDNA and wgaDNA, and among wgaDNA produced from a range of gDNA templates inputs. RESULTS: The proportion of double-stranded wgaDNA and human-specific PCR amplifiable wgaDNA increased with increased gDNA input into the WGA reaction. Increased amounts of gDNA input into the WGA reaction improved wgaDNA genotyping performance. Genotype completion or genotype concordance rates of wgaDNA produced from all gDNA input levels were observed to be reduced compared to gDNA, although the reduction was not always statistically significant. Reduced wgaDNA genotyping performance was primarily due to the increased variance of allelic amplification, resulting in loss of heterozygosity or increased undetermined genotypes. MDA WGA produces wgaDNA from no template control samples; such samples exhibited substantial false-positive genotyping rates. CONCLUSION: The amount of gDNA input into the MDA WGA reaction is a critical determinant of genotyping performance of wgaDNA. At least 10 ng of lymphoblastoid gDNA input into MDA WGA is required to obtain wgaDNA TaqMan SNP assay genotyping performance equivalent to that of gDNA. Over 100 ng of lymphoblastoid gDNA input into MDA WGA is required to obtain optimal STR genotyping performance using the AmpFlSTR Identifiler panel from wgaDNA equivalent to that of gDNA.

Association of multiple DRD2 polymorphisms with anorexia nervosa.

To investigate whether the dopaminergic system plays a role in the etiology of anorexia nervosa (AN) via the dopamine D2 receptor, we investigated association and transmission disequilibrium at seven single-nucleotide polymorphisms (SNPs) spanning about 75 kbp of the gene DRD2. We studied 191 probands with a DSM-IV diagnosis of AN, 457 parents and affected relatives with a DSM-IV eating disorder diagnosis, and 98 unrelated, female, normal weight controls. The -141 C/- insertion/deletion (-141 Indel), previously shown to affect DRD2 transcription efficiency, and multiple exon seven polymorphisms, one of which has previously been shown to affect DRD2 transcript stability, exhibited statistically significant association with diagnosis in haplotype transmission disequilibrium and in haplotype case : control analyses. Significant linkage disequilibrium between the -141 Indel and two exon seven SNPs (939Y and 957Y) was observed over a distance of >50 kbp in the AN probands but not in the controls. Genetically transmitted variation in D2 dopamine receptor expression mediated by functional polymorphisms affecting transcription and translation efficiency may play a role in vulnerability to AN.

Effects of electron-beam irradiation on whole genome amplification.

Electron-beam (E-beam) irradiation, currently being used to sterilize mail addressed to selected ZIP codes in the United States, has significant negative effects on the genomic integrity of DNA extracted from buccal-cell washes. We investigated the yield, composition, and genotyping performance of whole genome amplified DNA (wgaDNA) derived from 24 matched samples of E-beam-irradiated and nonirradiated genomic DNA (gDNA) as a model for the effects of degraded gDNA on the performance of whole genome amplification. gDNA was amplified using the Multiple Displacement Amplification method. Three methods of DNA quantification analysis were used to estimate the yield and composition of wgaDNA, and 65 short tandem repeat and single nucleotide polymorphism genotyping assays were used to evaluate the genotyping performance of irradiated and nonirradiated gDNA and wgaDNA. Compared with wgaDNA derived from nonirradiated gDNA, wgaDNA derived from irradiated gDNA exhibited a significantly reduced yield of wgaDNA and significantly reduced short tandem repeat and single nucleotide polymorphism genotyping completion and concordance rates (P < 0.0001). Increasing the amount of irradiated gDNA input into whole genome amplification improved genotyping performance of wgaDNA but not to the level of wgaDNA derived from nonirradiated gDNA. Multiple Displacement Amplification wgaDNA derived from E-beam-irradiated gDNA is not suitable for genotyping analysis.

Single nucleotide polymorphism genotyping by two colour melting curve analysis using the MGB Eclipse Probe System in challenging sequence environment.

Probe and primer design for single nucleotide polymorphism (SNP) detection can be very challenging for A-T DNA-rich targets, requiring long sequences with lower specificity and stability, while G-C-rich DNA targets present limited design options to lower GC-content sequences only. We have developed the MGB Eclipse Probe System, which is composed of the following elements: MGB Eclipse probes and primers, specially developed software for the design of probes and primers, a unique set of modified bases and a Microsoft Excel macro for automated genotyping, which ably solves, in large part, this challenge. Fluorogenic MGB Eclipse probes are modified oligonucleotides containing covalently attached duplex-stabilising dihydrocyclopyrroloindole tripeptide (DPI3), the MGB ligand (MGB is a trademark of Epoch Biosciences, Bothell, WA), which has the combined properties of allowing the use of short sequences and providing great mismatch discrimination. The MGB moiety prevents probe degradation during polymerase chain reaction (PCR), allowing the researcher to use real time data; alternatively, hybridisation can be accurately measured by a post-PCR two-colour melt curve analysis. Using MGB Eclipse probes and primers containing modified bases further enhances the analysis of difficult SNP targets. G- or C-rich sequences can be refractory to analysis due to Hoogsteen base pairing. Substitution of normal G with Epoch's modified G prevents Hoogsteen base pairing, allowing both superior PCR and probe-based analysis of GC-rich targets. The use of modified A and T bases allows better stabilisation by significantly increasing the Tm of the oligonucleotides. Modified A creates A-T base pairs that have a stability slightly lower than a G-C base pair, and modified T creates T-A base pairs that have a stability about 30 per cent higher than the unmodified base pair. Together, the modified bases permit the use of short probes, providing good mismatch discrimination and primers that allow PCR of refractory targets. The combination of MGB Eclipse probes and primers enriched with the MGB ligand and modified bases has allowed the analysis of refractory SNPs, where other methods have failed.

Specific roles of the obstetrician-gynecologist.

Obstetrician-gynecologists can play a major role in the prevention, identification, and treatment of substance abuse in women. Maintaining cognizance of abuse potential and consistently using office-screening techniques will identify patients who need assistance. Employing brief interventions at frequent office visits is an effective means of treating many of these patients. Those who do not respond to brief interventions may benefit from referral to an addictionologist. The US Supreme Court recently verified the Fourth Amendment rights of pregnant women by deciding that consent must be obtained to perform drug screening in pregnancy. Obstetrician-gynecologists will need to stay abreast of the changes that occur in their state laws as a result of this decision.