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.

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.

The proper establishment and organogenesis of the placenta is crucial for intrauterine fetal growth and development. Endometrial invasion by the extravillous trophoblast cells, as well as formation of the syncytiotrophoblast (STB), are of vital importance for placental function. Trophoblast migration and invasion is often compared to tumor metastasis, which uses many of the same molecular mechanisms. However, unlike cancer cells, both initiation and the extent of trophoblast invasion are tightly regulated by feto-maternal cross-talk, which when perturbed, results in a wide range of abnormalities. Multiple factors control the trophoblast, including cytokines and hormones, which are subject to transcriptional regulatory networks. The relevance of epigenetics in transcriptional regulation of trophoblast differentiation and invasion, as well as in the onset of placenta-related pregnancy disorders, became recognized decades ago. Although, there has been tremendous progress in uncovering the molecular foundation of placental development, there is still much to be learned about the epigenetic machinery, and its role in trophoblast differentiation and invasion.This review will provide an overview of the epigenetic control of trophoblast differentiation and invasion. It will also highlight the major epigenetic mechanisms involved in pregnancy complications related to placental deficiencies.

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% O. 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% O 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% O, 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 O 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.

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 b 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.