Open Access Dissertation
Date of Award
D. Randall Armant
The placenta is vital for the short- and long-term health of the fetus, and significantly impacts the health of the mother. During the first and second trimesters of pregnancy, extravillous trophoblast (EVT) cells invade the uterus and remodel the maternal spiral arteries, which, if inadequate, leads to pregnancy complications, including early pregnancy loss (EPL), preeclampsia (PE), and intra-uterine growth restriction (IUGR). EVT migration into the uterine wall is dependent on growth factors and cytokines that signal between maternal and fetal tissues. The epidermal growth factor (EGF) signaling system plays a significant role in trophoblast function. Using immunocytochemistry (ICC), we evaluated EGF family growth factors in post-partum PE placentas (villi and basal plate), and maternal serum, comparing gestational age (GA)-matched adverse preterm pregnancies and uncomplicated term pregnancies. EGF, heparin-binding EGF-like growth factor (HBEGF), and transforming growth factor alpha (TGFA) were significantly decreased in placentas from PE compared to IUGR, preterm labor, and uncomplicated term placentas. It was uncertain whether reduced growth factor signaling contributed to disease, or was the result of late-stage pathology. However, EGF was significantly reduced in prenatal serum of pregnant patients with PE compared to GA-matched patients without PE, demonstrating that the EGF signaling system is disrupted before PE symptoms dictate delivery.
Currently, chorionic villous sampling is the only means by which intact placental cells can be obtained from an ongoing pregnancy. However, the earliest this invasive procedure can be preformed is 10 weeks GA, and it carries a risk of fetal loss. Trophoblast retrieval and isolation from the cervix (TRIC) can isolate intact trophoblast cells in a minimally invasive procedure from ongoing pregnancies as early as 5 weeks GA, using immunomagnetic isolation to target human leukocyte antigen-G, a protein present on trophoblast cells, but not on maternal cervical cells. ICC shows that the isolated cells are of the extravillous phenotype. The ability to isolate EVT cells from ongoing pregnancies not only provides clinicians with a tool to study the placenta in real time, but is also of benefit for prenatal genetic diagnosis (PGD). EVT protein expression was investigated, using TRIC with patient specimens obtained between 5-20 weeks GA. Using ICC, galectin 13 (LGALS13), galectin 14 (LGALS14), pregnancy-associated plasma protein-A (PAPPA), placental growth factor (PGF), endoglin (ENG), fms-like tyrosine kinase-1 (FLT1), and alpha-fetoprotein (AFP), proteins known to play a role in EVT function, were evaluated in EVT cells from patients that developed PE, IUGR, or EPL, and compared to pregnancies with uncomplicated term deliveries. Expression of LGALS14, PAPPA, and PGF significantly decreased in EVT cells from patients that developed IUGR, PE, or EPL, whereas expression of ENG, FLT1, and AFP were all significantly increased.
To evaluate the potential of TRIC for PGD, aneuploidy rates in EVT cells were studied in pregnancies with a diploid fetus, using fluorescent in situ hybridization (FISH). Diploid rates for chromosomes 13, 18, 21, and X/Y were 95.4 ± 2.3%, 93.4 ± 2.38%, 88.1± 1.96%, and 97.8± 0.57%, respectively. The percentage of EVT cells with a normal X/Y genotype in the cohort of EVT cells obtained from confirmed male fetuses was 96.9% ± 1.5%, confirming that TRIC isolates a homogenous population of EVT cells, free of maternal cells. Diploid rates for maternal cervical cells and fetal EVT cells were not significantly different. Interestingly, trophoblast yield by TRIC was unaffected by GA, maternal obesity, parity, or age, further highlighting the advantages of TRIC over other approaches for PGD. Finally, TRIC was used successfully for gender determination at 7 weeks GA in a case study of a pregnancy in which both parents were carriers of congenital adrenal hyperplasia (CAH). Early identification of male fetuses with TRIC PGD could eliminate the need for unnecessary prophylactic administration of high-dose dexamethasone prenatally to prevent the virilization of a female fetus.
Fritz, Rani, "Trophoblast Retrieval And Isolation From The Cervix (tric) For Non-Invasive Prenatal Genetic Diagnosis And Prediction Of Abnormal Pregnancy Outcome" (2015). Wayne State University Dissertations. 1310.