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Fritz, Rani; Feil, Heather; Jindal, Sangita K.; Buyuk, Erkan
ISSN: 0015-0282
CID: 4703122

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

Jain, Chandni V; Kadam, Leena; van Dijk, Marie; Kohan-Ghadr, Hamid-Reza; Kilburn, Brian A; Hartman, Craig; Mazzorana, Vicki; Visser, Allerdien; Hertz, Michael; Bolnick, Alan D; Fritz, Rani; Armant, D Randall; Drewlo, Sascha
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.
PMID: 27807286
ISSN: 1946-6242
CID: 3803622

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

Bolnick, Jay M; Kohan-Ghadr, Hamid-Reza; Fritz, Rani; Bolnick, Alan D; Kilburn, Brian A; Diamond, Michael P; Armant, D Randall; Drewlo, Sascha
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.
PMID: 27660926
ISSN: 2045-2322
CID: 3803612

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

Bolnick, Alan D; Fritz, Rani; Jain, Chandni; Kadam, Leena; Bolnick, Jay M; Kilburn, Brian A; Singh, Manvinder; Diamond, Michael P; Drewlo, Sascha; Armant, D Randall
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 β 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.
PMID: 26919977
ISSN: 1933-7205
CID: 3803602

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

Fritz, Rani; Kohan-Ghadr, Hamid Reza; Sacher, Alex; Bolnick, Alan D; Kilburn, Brian A; Bolnick, Jay M; Diamond, Michael P; Drewlo, Sascha; Armant, D Randall
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 β-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.
PMID: 26288006
ISSN: 1097-0223
CID: 3803592

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

Fritz, Rani; Kohan-Ghadr, Hamid-Reza; Bolnick, Jay M; Bolnick, Alan D; Kilburn, Brian A; Diamond, Michael P; Drewlo, Sascha; Armant, D Randall
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/METHODS:Case-control study. SETTING/METHODS:Academic medical center. PATIENT(S)/METHODS:Women with either early pregnancy loss (EPL, n = 10) or an uncomplicated term delivery (N = 10). INTERVENTION(S)/METHODS: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)/METHODS: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)/RESULTS: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)/CONCLUSIONS: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.
PMID: 26051097
ISSN: 1556-5653
CID: 3803582

Reduced expression of the epidermal growth factor signaling system in preeclampsia

Armant, D R; Fritz, R; Kilburn, B A; Kim, Y M; Nien, J K; Maihle, N J; Romero, R; Leach, R E
INTRODUCTION/BACKGROUND:The epidermal growth factor (EGF) signaling system regulates trophoblast differentiation, and its disruption could contribute to perinatal disease. We hypothesized that this pathway is altered in preeclampsia, a disorder associated with trophoblast apoptosis and failure to invade and remodel the uterine spiral arteries. METHODS:Six EGF family peptides and a truncated EGF receptor splice variant (p110/EGFR) were examined using immunohistochemistry in the trophoblast of placentas (N = 76) from women with preeclampsia, and compared to placentas from women of similar gestational age (GA) with preterm labor (PTL) or small for gestational age (SGA) fetuses, as well as normal term placentas. EGF, transforming growth factor-α (TGFA), and heparin-binding EGF-like growth factor (HBEGF) were evaluated using ELISA in maternal plasma from another 20 pregnancies with or without preeclampsia. Cell death was evaluated in the HTR-8/SVneo human cytotrophoblast cell line using TUNEL to evaluate the protective effects of EGF peptides. RESULTS:Trophoblast HBEGF, TGFA, and EGF were significantly reduced in preeclampsia compared to PTL and SGA, while p110/EGFR accumulated significantly on the surface of the chorionic villi (p < 0.05). Plasma EGF levels were significantly decreased in preeclamptic patients, compared to non-preeclamptic patients (p < 0.05). HBEGF, EGF, TGFA, epiregulin, and betacellulin each blocked cytotrophoblast cell death in vitro (p < 0.05). DISCUSSION/CONCLUSIONS:Three members of the EGF family are dysregulated in placentas with preeclampsia, whereas p110/EGFR, a potential EGF receptor antagonist, is overexpressed. These findings are consistent with the concept that disruption of the EGF signaling system contributes to aberrant trophoblast development associated with preeclampsia.
PMID: 25589361
ISSN: 1532-3102
CID: 4703172

Successful Laparoscopically Assisted Transcervical Suction Evacuation of Interstitial Pregnancy following Failed Methotrexate Injection in a Community Hospital Setting

Fritz, Rani B; Rosenblum, Neal; Gaither, Kecia; Sherman, Alonzo; McCalla, Alwyn
We report on a case of a patient with an early diagnosed cornual ectopic pregnancy following failed methotrexate treatment. The patient was subsequently taken to the operating room for a laparoscopic guided transcervical suction curettage of the cornual ectopic. The surgery was successful and the patient was followed up until her urine pregnancy test was negative. We conclude that in properly selected patients, cornual ectopic pregnancy may be treated with transcervical suction curettage.
PMID: 24649387
ISSN: 2090-6684
CID: 3803562

Cell signaling in trophoblast-uterine communication

Fritz, Rani; Jain, Chandni; Armant, D Randall
Intricate and precise communication between the blastocyst and the uterus orchestrates embryo implantation. However, many questions remain unanswered regarding the molecular complexities of implantation. On-time implantation requires a receptive uterus and a mature blastocyst with trophoblast cells capable of adhering to and invading the endometrium. Defects in uterine receptivity or embryo/uterine signaling can cause implantation failure or early pregnancy loss, whereas deficient trophoblast differentiation can generate placental abnormalities that produce adverse pregnancy outcomes. This review will discuss several examples of signaling pathways that regulate trophoblast and uterine development during this period. Leukemia inhibitory factor is involved in uterine priming for implantation. The epidermal growth factor signaling system contributes to trophoblast-uterine communication, as well as trophoblast adhesion and invasion. Indian hedgehog signaling synchronizes tissue compartments within the uterus, and WNT signaling mediates numerous interactions within the implantation site and developing placenta. The autocrine, paracrine and juxtacrine interactions mediated by these signaling pathways contribute significantly to the establishment of pregnancy, although there are many other known and yet to be discovered factors that synchronize the maternal and embryonic developmental programs.
PMID: 25023692
ISSN: 1696-3547
CID: 3803572

Defragmentation of low grade day 3 embryos resulted in sustained reduction in fragmentation, but did not improve compaction or blastulation rates

Keltz, Martin; Fritz, Rani; Gonzales, Eric; Ozensoy, Serhan; Skorupski, Josh; Stein, Daniel
In a prospective randomized fashion, this study evaluated embryo development in vitro after defragmentation versus assisted hatching alone of low grade day 3 embryos. Although a sustained decrease in day 5 fragmentation was observed in the defragmented group versus the assisted hatching only group, no difference in compaction rates or blastula formation rates were appreciated.
PMID: 20403590
ISSN: 1556-5653
CID: 3803552