Nifedipine Prevents Apoptosis of Alcohol-Exposed First-Trimester Trophoblast Cells.

BACKGROUND: Maternal alcohol abuse leading to fetal alcohol spectrum disorder (FASD) includes fetal growth restriction (FGR). Ethanol (EtOH) induces apoptosis of human placental trophoblast cells, possibly disrupting placentation and contributing to FGR in FASD. EtOH facilitates apoptosis in several embryonic tissues, including human trophoblasts, by raising intracellular Ca2+ . We previously found that acute EtOH exposure increases trophoblast apoptosis due to signaling from both intracellular and extracellular Ca2+ . Therefore, nifedipine, a Ca2+ channel blocker that is commonly administered to treat preeclampsia and preterm labor, was evaluated for cytoprotective properties in trophoblast cells exposed to alcohol. METHODS: Human first-trimester chorionic villous explants and the human trophoblast cell line HTR-8/SVneo (HTR) were pretreated with 12.5 to 50 nM of the Ca2+ channel blocker nifedipine for 1 hour before exposure to 50 mM EtOH for an additional hour. Intracellular Ca2+ concentrations were monitored in real time by epifluorescence microscopy, using fluo-4-AM. Apoptosis was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), accumulation of cytoplasmic cytochrome c, and cleavage rates of caspase 3 and caspase 9. RESULTS: The increase in intracellular Ca2+ upon exposure to EtOH in both villous explants and HTR cells was completely blocked (p < 0.05) when pretreated with nifedipine, accompanied by inhibition of EtOH-induced release of cytochrome c, caspase activities, and TUNEL. CONCLUSIONS: This study indicates that nifedipine can interrupt the apoptotic pathway downstream of EtOH exposure and could provide a novel strategy for future interventions in women with fetuses at risk for FASD.

Trophoblast survival signaling during human placentation requires HSP70 activation of MMP2-mediated HBEGF shedding.

Survival of trophoblast cells in the low oxygen environment of human placentation requires metalloproteinase-mediated shedding of HBEGF and downstream signaling. A matrix metalloproteinase (MMP) antibody array and quantitative RT-PCR revealed upregulation of MMP2 post-transcriptionally in human first trimester HTR-8/SVneo trophoblast cells and placental villous explants exposed to 2% O2. Specific MMP inhibitors established the requirement for MMP2 in HBEGF shedding and upregulation. Because α-amanitin inhibited the upregulation of HBEGF, differentially expressed genes were identified by next-generation sequencing of RNA from trophoblast cells cultured at 2% O2 for 0, 1, 2 and 4 h. Nine genes, all containing HIF-response elements, were upregulated at 1 h, but only HSPA6 (HSP70B') remained elevated at 2-4 h. The HSP70 chaperone inhibitor VER 155008 blocked upregulation of both MMP2 and HBEGF at 2% O2, and increased apoptosis. However, both HBEGF upregulation and apoptosis were rescued by exogenous MMP2. Proximity ligation assays demonstrated interactions between HSP70 and MMP2, and between MMP2 and HBEGF, supporting the concept that MMP2-mediated shedding of HBEGF, initiated by HSP70, contributes to trophoblast survival at the low O2 concentrations encountered during the first trimester, and is essential for successful pregnancy outcomes. Trophoblast survival during human placentation, when oxygenation is minimal, required HSP70 activity, which mediated MMP2 accumulation and the transactivation of anti-apoptotic ERBB signaling by HBEGF shedding.

Enhancement of trophoblast differentiation and survival by low molecular weight heparin requires heparin-binding EGF-like growth factor.

STUDY QUESTION: Does low molecular weight heparin (LMWH) require heparin-binding epidermal growth factor (EGF)-like growth factor (HBEGF) signaling to induce extravillous trophoblast differentiation and decrease apoptosis during oxidative stress? SUMMARY ANSWER: LMWH increased HBEGF expression and secretion, and HBEGF signaling was required to stimulate trophoblast extravillous differentiation, increase invasion in vitro and reduce trophoblast apoptosis during oxidative stress. WHAT IS KNOWN ALREADY: Abnormal trophoblast differentiation and survival contribute to placental insufficiency syndromes, including preeclampsia and intrauterine growth restriction. Preeclampsia often manifests as a pro-thrombotic state, with unsuccessful transformation of the spiral arteries that reduces oxygen supply and can produce placental infarction. LMWH improves placental function by increasing blood flow. Recent data suggest that the actions of LMWH transcend its anti-coagulative properties, but the molecular mechanism is unknown. There is evidence that LMWH alters the expression of human HBEGF in trophoblast cells, which regulates human trophoblast pathophysiology. HBEGF, itself, is capable of increasing trophoblast survival and invasiveness. STUDY DESIGN, SIZE, DURATION: First-trimester placental explants and the HTR-8/SVneo cell line, established using extravillous trophoblast outgrowths from first-trimester villous explants, were treated in vitro with LMWH to examine the effects on HBEGF signaling and trophoblast function under normal physiological and pathological conditions. A highly specific antagonist of HBEGF and other inhibitors of HBEGF downstream signaling were used to determine the relationship between LMWH treatment and HBEGF. PARTICIPANTS/MATERIALS, SETTING, METHODS: Placental tissues (n = 5) were obtained with IRB approval and patient consent from first-trimester terminations. Placental explants and HTR-8/SVneo cells were cultured on plastic or Matrigel™ and treated with a therapeutic dose of LMWH (Enoxaparin; 10 IU/ml), with or without CRM197, pan Erb-B2 Receptor Tyrosine Kinase (ERBB) inhibitor, anti-ERBB1 or ERBB4 blocking antibodies, or pretreatment of cells with heparitinase I. Extravillous differentiation was assessed by immunocytochemistry to determine the relative levels of integrins α6ß4 and α1ß1. Trophoblast invasiveness was assessed in villous explants by measuring outgrowth from villous tips cultured on Matrigel, and by invasion assays with HTR-8/SVneo cells cultured on Matrigel-coated transwell insert. Placental explants and HTR-8/SVneo cells were exposed to oxidative stress in a hypoxia-reoxygenation (H-R) model, measuring cell death by TUNEL assay, caspase 3 cleavage, and BCL-2α expression. MAIN RESULTS AND THE ROLE OF CHANCE: LMWH induced extravillous differentiation, according to trophoblast invasion assays and integrin (α6ß4-α1ß1) switching. Treatment with LMWH rescued cytotrophoblasts and HTR-8/SVneo cells from apoptosis during exposure to reoxygenation injury, based on TUNEL, caspase 3 cleavage and BCL-2α expression. Experiments using CRM197, ERBB1 and ERBB4 blocking antibodies, pan-ERBB inhibitor and removal of cell surface heparin demonstrated that the effects of LMWH on trophoblast invasion and survival were dependent upon HBEGF signaling. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: The primary limitation of this study was the use of only in vitro experiments. Patient demographics from elective terminations were not available. WIDER IMPLICATIONS OF THE FINDINGS: These data provide new insights into the non-coagulation-related aspects of perinatal LMWH treatment in the management of placental insufficiency disorders. STUDY FUNDING/COMPETING INTEREST(S): This research was supported by grants from the National Institutes of Health (HD071408 and HL128628), the March of Dimes, and the W. K. Kellogg Foundation. There were no conflicts or competing interests.

Fetal genome profiling at 5 weeks of gestation after noninvasive isolation of trophoblast cells from the endocervical canal.

Single-gene mutations account for more than 6000 diseases, 10% of all pediatric hospital admissions, and 20% of infant deaths. Down syndrome and other aneuploidies occur in more than 0.2% of births worldwide and are on the rise because of advanced reproductive age. Birth defects of genetic origin can be diagnosed in utero after invasive extraction of fetal tissues. Noninvasive testing with circulating cell-free fetal DNA is limited by a low fetal DNA fraction. Both modalities are unavailable until the end of the first trimester. We have isolated intact trophoblast cells from Papanicolaou smears collected noninvasively at 5 to 19 weeks of gestation for next-generation sequencing of fetal DNA. Consecutive matched maternal, placental, and fetal samples (n = 20) were profiled by multiplex targeted DNA sequencing of 59 short tandem repeat and 94 single-nucleotide variant sites across all 24 chromosomes. The data revealed fetal DNA fractions of 85 to 99.9%, with 100% correct fetal haplotyping. This noninvasive platform has the potential to provide comprehensive fetal genomic profiling as early as 5 weeks of gestation.

Altered Biomarkers in Trophoblast Cells Obtained Noninvasively Prior to Clinical Manifestation of Perinatal Disease.

A contributing factor to poor placental perfusion, leading to intrauterine growth restriction and preeclampsia, is the failure of invading extravillous trophoblast (EVT) cells to remodel the maternal uterine arteries during the first and second trimesters of pregnancy. Noninvasive assessment of EVT cells in ongoing pregnancies is possible beginning three weeks after conception, using trophoblast retrieval and isolation from the cervix (TRIC). Seven proteins were semi-quantified by immunofluorescence microscopy in EVT cells obtained between gestational weeks 6 and 20 from pregnancies with normal outcomes (N = 29) and those with intrauterine growth restriction or preeclampsia (N = 12). Significant differences were measured in expression of PAPPA, FLT1, ENG, AFP, PGF, and LGALS14, but not LGALS13 or the lineage marker KRT7. These findings provide for the first time direct evidence of pathology-associated protein dysregulation in EVT cells during early placentation. The TRIC platform provides a novel approach to acquire molecular signatures of EVT cells that can be correlated with pregnancy outcome.

Reduced homeobox protein MSX1 in human endometrial tissue is linked to infertility.

STUDY QUESTION: Is protein expression of the muscle segment homeobox gene family member MSX1 altered in the human secretory endometrium by cell type, developmental stage or fertility? SUMMARY ANSWER: MSX1 protein levels, normally elevated in the secretory phase endometrium, were significantly reduced in endometrial biopsies obtained from women of infertile couples. WHAT IS KNOWN ALREADY: Molecular changes in the endometrium are important for fertility in both animals and humans. Msx1 is expressed in the preimplantation mouse uterus and regulates uterine receptivity for implantation. The MSX protein persists a short time, after its message has been down-regulated. Microarray analysis of the human endometrium reveals a similar pattern of MSX1 mRNA expression that peaks before the receptive period, with depressed expression at implantation. Targeted deletion of uterine Msx1 and Msx2 in mice prevents the loss of epithelial cell polarity during implantation and causes infertility. STUDY DESIGN, SIZE DURATION: MSX1 mRNA and cell type-specific levels of MSX1 protein were quantified from two retrospective cohorts during the human endometrial cycle. MSX1 protein expression patterns were compared between fertile and infertile couples. Selected samples were dual-labeled by immunofluorescence microscopy to localize E-cadherin and ß-catenin in epithelial cells. PARTICIPANTS/MATERIALS, SETTING METHODS: MSX1 mRNA was quantified by PCR in endometrium from hysterectomies (n = 14) determined by endometrial dating to be in the late-proliferative (cycle days 10-13), early-secretory (cycle days 14-19) or mid-secretory (cycle days 20-24) phase. MSX1 protein was localized using high-throughput, semi-quantitative immunohistochemistry with sectioned endometrial biopsy tissues from fertile (n = 89) and infertile (n = 89) couples. Image analysis measured stain intensity specifically within the luminal epithelium, glands and stroma during the early-, mid- and late- (cycle days 25-28) secretory phases. MAIN RESULTS AND THE ROLE OF CHANCE: MSX1 transcript increased 5-fold (P < 0.05) between the late-proliferative and early secretory phase and was then down-regulated (P < 0.05) prior to receptivity for implantation. In fertile patients, MSX1 protein displayed strong nuclear localization in the luminal epithelium and glands, while it was weakly expressed in nuclei of the stroma. MSX1 protein levels accumulated throughout the secretory phase in all endometrial cellular compartments. MSX1 protein decreased (P < 0.05) in the glands between mid- and late-secretory phases. However, infertile patients demonstrated a broad reduction (P < 0.001) of MSX1 accumulation in all cell types throughout the secretory phase that was most pronounced (∼3-fold) in stroma and glands. Infertility was associated with persistent co-localization of E-cadherin and ß-catenin in epithelial cell junctions in the mid- and late-secretory phases. LIMITATIONS, REASONS FOR CAUTION: Details of the infertility diagnoses and other patient demographic data were not available. Therefore, patients with uterine abnormalities (Mullerian) could not be distinguished from other sources of infertility. Antibody against human MSX2 is not available, limiting the study to MSX1. However, both RNAs in the human endometrium are similarly regulated. In mice, Msx1 and Msx2 are imperative for murine embryo implantation, with Msx2 compensating for genetic ablation of Msx1 through its up-regulation in a knockout model. WIDER IMPLICATIONS OF THE FINDINGS: This investigation establishes that the MSX1 homeobox protein accumulation is associated with the secretory phase in endometrium of fertile couples, and is widely disrupted in infertile patients. It is the first study to examine MSX1 protein localization in the human endometrium, and supported by genetic findings in mice, suggests that genes regulated by MSX1 are linked to the loss of epithelial cell polarity required for uterine receptivity during implantation. STUDY FUNDING/COMPETING INTERESTS: This research was supported by the NICHD National Cooperative Reproductive Medicine Network grant HD039005 (M.P.D.), NIH grants HD068524 (S.K.D.), HD071408 (D.R.A., M.P.D.), and HL128628 (S.D.), the Intramural Research Program of the NICHD, March of Dimes (S.K.D., S.D.) and JSPS KAKENHI grant 26112506 (Y.H.). There were no conflicts or competing interests.

Assisted reproductive technology alters deoxyribonucleic acid methylation profiles in bloodspots of newborn infants.

OBJECTIVE: To evaluate the effect of infertility and intracytoplasmic sperm injection (ICSI) on DNA methylation of offspring. DESIGN: Microarray analysis of DNA methylation in archived neonatal bloodspots of in vitro fertilization (IVF)/ICSI-conceived children compared with controls born to fertile and infertile parents. SETTING: Academic research laboratory. PATIENT(S): Neonatal blood spots of 137 newborns conceived spontaneously, through intrauterine insemination (IUI), or through ICSI using fresh or cryopreserved (frozen) embryo transfer. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The Illumina Infinium HumanMethylation450k BeadChip assay determined genome-wide DNA methylation. Methylation differences between conception groups were detected using a Bioconductor package, ChAMP, in conjunction with Adjacent Site Clustering (A-clustering). RESULT(S): The methylation profiles of assisted reproductive technology and IUI newborns were dramatically different from those of naturally (in vivo) conceived newborns. Interestingly, the profiles of ICSI-frozen (FET) and IUI infants were strikingly similar, suggesting that cryopreservation may temper some of the epigenetic aberrations induced by IVF or ICSI. The DNA methylation changes associated with IVF/ICSI culture conditions and/or parental infertility were detected at metastable epialleles, suggesting a lasting impact on a child's epigenome. CONCLUSION(S): Both infertility and ICSI alter DNA methylation at specific genomic loci, an effect that is mitigated to some extent by FET. The impact of assisted reproductive technology and/or fertility status on metastable epialleles in humans was uncovered. This study provides an expanded set of loci for future investigations on IVF populations.

Trophoblast Retrieval and Isolation From the Cervix for Noninvasive, First Trimester, Fetal Gender Determination in a Carrier of Congenital Adrenal Hyperplasia.

Congenital adrenal hyperplasia (CAH) is an autosomal recessive defect in cortisol biosynthesis that elevates fetal androgen levels to cause genital ambiguity and external genital masculinization in newborn females. Introducing dexamethasone in utero by 7 weeks gestation precludes virilization of affected females. However, identification of a male fetus prior to week 7 could avert the necessity of steroid treatment in half of pregnancies at risk of CAH. We recently introduced trophoblast retrieval and isolation from the cervix (TRIC), an approach that noninvasively isolate homogeneous trophoblast cells from pregnant women as early as 5 weeks gestation, using a Papanicolaou test. Here, we have used TRIC to correctly identify male fetal DNA when both parents were carriers of the mutation that produces CAH and previously produced an affected child. Trophoblast cells (1400) obtained by TRIC were assessed using immunocytochemistry with an antibody against the trophoblast-specific ß subunit of human chorionic gonadotropin, which labeled 100% (17 of 17) of isolated cells, while none of the excluded maternal cervical cells were labeled. The isolated cells were examined by fluorescent in situ hybridization for chromosomes 18, X, and Y at a clinical cytogenetics laboratory, demonstrating 100% (18 of 18) of cells to be diploid 18/XY. Aliquots of DNA obtained from the isolated cells assayed for SRY and RNASEH genes by TaqMan assays confirmed a male fetus. This case study demonstrates the utility of TRIC to accurately identify fetal gender as a means of reducing the need for prophylactic administration of exogenous steroids in pregnancies at risk of CAH.

Trophoblast retrieval and isolation from the cervix (TRIC) is unaffected by early gestational age or maternal obesity.

OBJECTIVE: The objective of this study is to evaluate whether trophoblast yield obtained by trophoblast retrieval and isolation from the cervix (TRIC) is affected by pregnancy outcome, gestational age (GA) at retrieval, maternal body mass index (BMI), parity, or maternal age. METHODS: TRIC was performed on 224 ongoing pregnancies between 5 and 20 weeks of GA. Trophoblast cells were isolated from cervical cells using anti-human leukocyte antigen-G antibody coupled to magnetic nanoparticles. Purity was assessed by the percentage of isolated cells that express ß-hCG. Patient records were monitored until delivery, and pregnancy outcomes were determined. Trophoblast yield was compared with GA at time of collection, maternal BMI, parity, maternal age, and outcome of pregnancy, using linear regression. RESULTS: There was no effect of GA, maternal BMI, parity, and maternal age on trophoblast yield. Trophoblast yield decreased significantly with early pregnancy loss compared with uncomplicated pregnancies that delivered at term. Trophoblast yield with preeclampsia or intrauterine growth restriction was decreased compared with healthy term outcomes; however, they did not reach statistical significance. CONCLUSIONS: If TRIC becomes available as a method for non-invasive prenatal testing, our data demonstrate that it is unaffected by BMI and is useful as early as 5 weeks of GA.

Noninvasive detection of trophoblast protein signatures linked to early pregnancy loss using trophoblast retrieval and isolation from the cervix (TRIC).

OBJECTIVE: To examine the expression pattern of biomarker proteins in extravillous trophoblast (EVT) cells obtained noninvasively by trophoblast retrieval and isolation from the cervix (TRIC) in patients with early pregnancy loss compared with control patients with uncomplicated term delivery. DESIGN: Case-control study. SETTING: Academic medical center. PATIENT(S): Women with either early pregnancy loss (EPL, n = 10) or an uncomplicated term delivery (N = 10). INTERVENTION(S): Endocervical specimens obtained from ongoing pregnancies at gestational ages of 5-10 weeks to generate an archive of EVT cells isolated by TRIC, with medical records examined to select specimens matched for gestational age at the time of endocervical sampling. MAIN OUTCOME MEASURE(S): Known serum biomarkers for adverse pregnancy outcome that are expressed by EVT cells were evaluated by semiquantitative immunocytochemistry, using antibodies against endoglin (ENG), FMS-like tyrosine kinase-1 (FLT-1), α-fetoprotein (AFP), pregnancy-associated plasma protein-A (PAPP-A), galectin-13 (LGALS13), galectin-14 (LGALS14), and placental growth factor (PGF). RESULT(S): The EVT purity was over 95% in all specimens, based on chorionic gonadotropin expression; however, the number of EVT cells obtained was significantly lower in women with EPL than the control group. There was a statistically significant elevation of AFP, ENG, and FLT-1, and statistically significant reduction of PAPP-A, LGALS14, and PGF in the EPL group compared with controls. CONCLUSION(S): In this pilot study, EVT cells isolated by TRIC early in gestation exhibited altered protein expression patterns before an EPL compared with uncomplicated term pregnancies.

Sildenafil stimulates human trophoblast invasion through nitric oxide and guanosine 3',5'-cyclic monophosphate signaling.

OBJECTIVE: To determine the effect of sildenafil, a phosphodiesterase type 5 inhibitor, on trophoblast invasiveness. DESIGN: Laboratory investigation. SETTING: Academic medical center. PATIENT(S): Placental tissues discarded after first-trimester terminations were obtained from patients with informed consent. INTERVENTION(S): A cell line, HTR-8/SVneo, established from first-trimester cytotrophoblast, and villous explants, was treated with or without sildenafil, guanosine 3',5'-cyclic monophosphate (cGMP) analog, cGMP inhibitor, or L-NAME (N(G)-nitro-L-arginine methyl ester hydrochloride) and cultured on fibronectin or Matrigel. Integrins α6ß4 and α1ß1 were detected by immunocytochemistry. MAIN OUTCOME MEASURE(S): Trophoblast outgrowth from villous tips, cytotrophoblast cell invasion, and integrin immunostaining were assessed in cytotrophoblast and explant cultures. RESULT(S): Integrin expression in trophoblast cells ex vivo switched from α6 to α1, and invasiveness increased, when exposed to sildenafil or cGMP agonist. Either cGMP antagonist or L-NAME blocked integrin switching and invasion induced by sildenafil. Elevation of nitric oxide pharmacologically induced invasion, but not when cGMP antagonist was present. CONCLUSION(S): Sildenafil altered trophoblast phenotype through a process dependent on nitric oxide availability and cGMP accumulation. In addition to its vasoactivity, sildenafil directly stimulates trophoblast extravillous differentiation, which would be favorable for implantation and reduce risk for adverse pregnancy outcomes.

Sildenafil Prevents Apoptosis of Human First-Trimester Trophoblast Cells Exposed to Oxidative Stress: Possible Role for Nitric Oxide Activation of 3',5'-cyclic Guanosine Monophosphate Signaling.

Human first-trimester trophoblast cells proliferate at low O2, but survival is compromised by oxidative stress, leading to uteroplacental insufficiency. The vasoactive drug, sildenafil citrate (Viagra, Sigma, St Louis, Missouri), has proven useful in reducing adverse pregnancy outcomes. An important biological function of this pharmaceutical is its action as an inhibitor of cyclic guanosine monophosphate (cGMP) phosphodiesterase type 5 activity, which suggests that it could have beneficial effects on trophoblast survival. To investigate whether sildenafil can prevent trophoblast cell death, human first-trimester villous explants and the HTR-8/SVneo cytotrophoblast cell line were exposed to hypoxia and reoxygenation (H/R) to generate oxidative stress, which induces apoptosis. Apoptosis was optimally inhibited during H/R by 350 ng/mL sildenafil. Sildenafil-mediated survival was reversed by l-N(G)-nitro-l-arginine methyl ester hydrochloride or cGMP antagonist, indicating a dependence on both nitric oxide (NO) and cGMP. Indeed, either a cGMP agonist or an NO generator was cytoprotective independent of sildenafil. These findings suggest a novel intervention route for patients with recurrent pregnancy loss or obstetrical placental disorders.

Frequency of echogenic intracardiac focus by race/ethnicity in euploid fetuses.

OBJECTIVE: To determine the frequency of echogenic intracardiac focus (EIF) by race/ethnicity. METHODS: We performed a retrospective analysis from January 1996 through June 2003. We reviewed all initial sonograms from 14 to 23 weeks gestation in singleton pregnancies. Mothers on admission for delivery provided race/ethnicity. RESULTS: There were 8207 ultrasounds and deliveries that met study criteria. There were 4636 (56.5%) Caucasian, 2087 (25.4%) African-American, 1261 (15.4%) Hispanic and 223 (2.7 %) Asian subjects. There were 347 (4.2%) EIF detected. The frequency by race/ethnicity varied significantly (p < 0.0001). CONCLUSIONS: This large, population-based study showed that fetuses born to Asian mothers were significantly more likely to have an EIF. This racial difference should be taken into account when counseling patients about the potential for Down syndrome.

Use of the mitral valve-tricuspid valve distance as a marker of fetal endocardial cushion defects.

OBJECTIVE: The purpose of this study was to compare the mitral valve-tricuspid valve distance in second-trimester fetuses with normal cardiac anatomy versus those fetuses with endocardial cushion defects. STUDY DESIGN: We identified fetuses between 16 and 24 weeks of gestation. The distance between the insertions of the medial leaflets of the mitral and tricuspid valves were obtained. Linear regression curves were generated. RESULTS: The mean mitral valve-tricuspid valve distance for 86 fetuses with normal cardiac anatomy was 2.02 mm, compared with 0.37 mm in 13 fetuses with endocardial cushion defects ( P = .0001). Linear regression curve correlating mitral valve-tricuspid valve distance with gestational age showed a gradual slope (R 2 = 0.28; P < .0001). With a mitral valve-tricuspid valve distance < 5th percentile as a marker for the diagnosis of endocardial cushion defect gave a sensitivity of 69.2%, a specificity of 100%, a positive predictive value of 100%, a negative predictive value of 95.6%, and a false-positive rate of 0% ( P = .0001). CONCLUSION: The mitral valve-tricuspid valve distance is useful clinically in the detection of endocardial cushion defects in second-trimester fetuses.