Olivia J. Holland

Minor Histocompatibility Antigens Are Expressed in Syncytiotrophoblast and Trophoblast Debris: Implications for Maternal Alloreactivity to the Fetus

The fetal semi-allograft can induce expansion and tolerance of antigen-specific maternal T and B cells through paternally inherited major histocompatibility complex and minor histocompatibility antigens (mHAgs). The effects of these antigens have important consequences on the maternal immune system both during and long after pregnancy. Herein, we investigate the possibility that the placental syncytiotrophoblast and deported trophoblastic debris serve as sources of fetal mHAgs. We mapped the expression of four mHAgs (human mHAg 1, pumilio domain-containing protein KIAA0020, B-cell lymphoma 2-related protein A1, and ribosomal protein S4, Y linked) in the placenta. Each of these proteins was expressed in several placental cell types, including the syncytiotrophoblast. These antigens and two additional Y chromosome-encoded antigens [DEAD box polypeptide 3, Y linked (DDX3Y), and lysine demethylase5D] were also identified by RT-PCR in the placenta, purified trophoblast cells, and cord blood cells. Finally, we used a proteomic approach to investigate the presence of mHAgs in the syncytiotrophoblast and trophoblast debris shed from first-trimester placenta. By this method, four antigens (DDX3Y; ribosomal protein S4, Y linked; solute carrier 1A5; and signal sequence receptor 1) were found in the syncytiotrophoblast, and one antigen (DDX3Y) was found in shed trophoblast debris. The finding of mHAgs in the placenta and in trophoblast debris provides the first direct evidence that fetal antigens are present in debris shed from the human placenta. The data, thus, suggest a mechanism by which the maternal immune system is exposed to fetal alloantigens, possibly explaining the relationship between parity and graft-versus-host disease.

Review: Where is the maternofetal interface?

Ask where the maternofetal interface is and placental biologists will tell you, the syncytiotrophoblast and extravillous cytotrophoblasts. While correct, this is not full extent of the maternofetal interface. Trophoblast debris that is extruded into the maternal blood in all pregnancies expands the maternofetal interface to sites remote from the uterus. Trophoblast debris ranges from multinucleated syncytial nuclear aggregates to subcellular micro- and nano-vesicles. The origins of trophoblast debris are not clear. Some propose trophoblast debris is the end of the life-cycle of the trophoblast and that it results from an apoptosis-like cell death, but this is not universally accepted. Knowing whether trophoblast debris results from an apoptosis-like cell death is important because the nature of cell death that produced trophoblast debris will influence the maternal responses to it. Trophoblast debris is challenging to isolate from maternal blood making it difficult to study. However, by culturing placental explants in Netwells™ we can readily harvest trophoblast debris from beneath the Netwells™ which is very similar to debris that has been isolated from pregnant women. We have found that trophoblast debris from normal placentae shows markers of apoptosis and is phagocytosed by macrophages or endothelial cells, producing a tolerant phenotype in the phagocyte. Whereas, when we culture normal placental explants with factors such as antiphospholipid antibodies (a strong maternal risk factor for preeclampsia), or IL-6 (which is found at increased levels in the sera of preeclamptic women), the death process in the syncytiotrophoblast changes, such that the trophoblast debris becomes more necrotic. Phagocytosis of this necrotic debris leads to activation of endothelial cells. Trophoblast debris greatly expands the maternofetal interface and the nature of that debris is likely to strongly influence the responses of the maternal vascular and immune systems to the debris.

Review: Placental derived biomarkers of pregnancy disorders

Pregnancy is one of the greatest physiological challenges that a women can experience. The physiological adaptations that accompany pregnancy may increase the risk of developing a number of disorders that can lead to both acute and chronic physiological outcomes. In addition, fetal development may be impaired and, if the fetus survives, the child may be at an increased risk of disease throughout life. Pregnancy disorders are poorly predicted by traditional risk factors and maternal history alone. The identification of biomarkers that can predict incidence and severity of disease would allow for improved and targeted prophylactic therapies to prevent adverse maternal and fetal outcomes. Many of these pregnancy disorders, including preeclampsia, intrauterine growth restriction, gestational diabetes mellitus and preterm birth are known to be regulated at least in part by poor trophoblast invasion and/or dysregulated placental function. Cellular stress within the placenta increases the release of a number of factors into the maternal circulation. While many of these factors minimally impact maternal biology, others affect key physiological systems and contribute to disease. Importantly, these factors may be detected in physiological fluids and have predictive capacity making them ideal candidates as biomarkers of pregnancy disorders. This review will discuss what is known about these placental derived biomarkers of pregnancy disorders and highlight potential clinical opportunities for disease prediction and diagnosis.

Novel alleles in classical major histocompatibility complex class II loci of the brushtail possum (Trichosurus vulpecula).

We have investigated the diversity of class II major histocompatibility complex (MHC) loci in the brushtail possum (Trichosurus vulpecula), an important marsupial pest species in New Zealand. Immunocontraceptive vaccines, a method of fertility control that employs the immune system to attack reproductive cells or proteins, are currently being researched as a means of population control for the possum. Variation has been observed in the immune response of individual possums to immunocontraceptives. If this variability is under genetic control, it could compromise vaccine efficacy through preferential selection of animals that fail to mount a significant immune response and remain fertile. The MHC is an important immune region for antigen presentation and as such may influence the response to immunocontraceptives. We used known marsupial MHC sequences to design polymerase chain reaction primers to screen for possum MHC loci. Alpha and beta chains from two class II families, DA and DB, were found in possums throughout New Zealand. Forty new class II MHC alleles were identified in the possum, and the levels of variability in the MHC of this marsupial appear to be comparable to those of eutherian species. Preliminary population surveys showed evidence of clustering/variability in the distribution of MHC alleles in geographically separate locations. The extensive variation demonstrated in possums reinforces the need for further research to assess the risk that such MHC variation poses for long-term immunocontraceptive vaccine efficacy.

High variability in the MHC class II DA beta chain of the brushtail possum (Trichosurus vulpecula).

The diversity of class II major histocompatibility complex (MHC) loci was investigated in the brushtail possum, an important marsupial pest species in New Zealand. Immunocontraception, a form of fertility control that generates an autoimmune response, is being developed as a population control method for the possum. Because the immune response is partly under genetic control, an understanding of immunogenetics in possum will be crucial to the development of immunocontraceptive vaccines. MHC molecules are critical in the vertebrate immune response. Class II MHC molecules bind and present exogenously derived peptides to T lymphocytes and may be important in the presentation of immunocontraceptives. We used polymerase chain reaction primers designed to amplify the peptide binding region of possum class II MHC genes to isolate sequences from 49 animals. We have previously described 19 novel alleles from the DAB locus in the possum (Holland et al., Immunogenetics 60:449–460, 2008). Here, we report on another 11 novel alleles isolated from possum DAB, making this the most diverse marsupial locus described so far. This high level of diversity indicates that DAB is an important MHC locus in the possum and will need to be taken into account in the design of immunocontraceptive vaccines.

Review: Placental mitochondrial function and structure in gestational disorders

The aetiology of many gestational disorders is still unknown. However, insufficient trans-placental nutrient and oxygen transfer due to abnormal placentation is characteristic of several pathologies, and may alter the function of placental mitochondria. Mitochondria are multifunctional organelles that respond to a wide range of stimuli - such as physiological changes in cellular energy demands or various pathologies - by reshaping via fusion or fission, increasing/decreasing in number, altering oxidative phosphorylation, and signalling cellular functions such as apoptosis. Mitochondrial function is integral to tissue functions including energy production, metabolism, and regulation of various cellular responses including response to oxidative stress. This review details the functions of placental mitochondria and investigates mitochondrial function and structure in gestational disorders including preeclampsia, intrauterine growth restriction, diabetes mellitus, and obesity. Placental mitochondrial dysfunction may be critical in a range of gestational disorders which have important implications for maternal and fetal/offspring health.

Selenium supplementation induces mitochondrial biogenesis in trophoblasts

INTRODUCTIONnPlacental oxidative stress has been implicated in pregnancy complications and previous work has shown that selenium can protect trophoblast mitochondria from oxidative stress. This report examines mitochondrial function and content in trophoblasts supplemented with selenium.nnnMETHODSnSwan-71, JEG-3 and BeWo cells and placental tissue were incubated with sodium selenite or selenomethionine. Mitochondrial function was examined in a respirometer. Mitochondrial content was determined using RT-PCR. The levels of the mitochondrial biogenesis markers selenoprotein H, PGC-1α and NRF-1 was examined by western blotting.nnnRESULTSnMitochondrial respiration was significantly enhanced post selenium supplementation in cells and tissues. Selenium supplementation increased mitochondrial content and up-regulated mitochondrial biogenesis mediators in cells.nnnDISCUSSIONnThese results emphasise the importance of selenium in mitochondrial regeneration in trophoblasts.

IFPA Meeting 2011 workshop report III: Placental immunology; epigenetic and microRNA-dependent gene regulation; comparative placentation; trophoblast differentiation; stem cells

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialised topics. At IFPA meeting 2011 there were twelve themed workshops, five of which are summarized in this report. These workshops related to various aspects of placental biology: 1) immunology; 2) epigenetics; 3) comparative placentation; 4) trophoblast differentiation; 5) stem cells.

Identification of novel major histocompatibility complex class I sequences in a marsupial, the brushtail possum (Trichosurus vulpecula)

The major histocompatibility complex (MHC) is an essential part of the vertebrate immune response. MHC genes may be classified as classical, non-classical or non-functional pseudogenes. We have investigated the diversity of class I MHC genes in the brushtail possum, a marsupial native to Australia and an introduced pest in New Zealand. The MHC of marsupials is poorly characterised compared to eutherian mammal species. Comparisons between marsupials and eutherians may enhance understanding of the evolution and functions of this important genetic region. We found a high level of diversity in possum class I MHC genes. Twenty novel sequences were identified using polymerase chain reaction (PCR) primers designed from existing marsupial class I MHC genes. Eleven of these sequences shared a high level of homology with the only previously identified possum MHC class I gene TrvuUB and appear to be alleles at a single locus. Another seven sequences are also similar to TrvuUB but have frame-shift mutations or stop codons early in their sequence, suggesting they are non-functional alleles of a pseudogene locus. The remaining sequences are highly divergent from other possum sequences and clusters with American marsupials in phylogenetic analysis, indicating they may have changed little since the separation of Australian and American marsupials.

Detection of Fetal Sex, Aneuploidy and a Microdeletion from Single Placental Syncytial Nuclear Aggregates

Objectives: A key problem in prenatal screening using extra-embryonic cells is the feasibility of extracting usable DNA from a small number of cells. Syncytial nuclear aggregates (SNAs) are multinucleated structures shed from the placenta. This study assesses the potential of SNAs as a source of fetal DNA for the detection of genetic abnormalities. Methods: SNAs were collected in vitro. Whole-genome amplification was used to amplify DNA from single SNAs, and DNA quality and quantity was assessed by spectrophotometry and PCR. Confocal microscopy was used to count nuclei within SNAs, determine metabolic activity and investigate DNA damage. Fetal sex and chromosomal/genetic abnormalities were investigated with array-comparative genomic hybridization (aCGH). Results: DNA was amplified from 81% of the individual SNAs. A mean of 61 ± 43 nuclei were found per SNA. DNA strand breaks were found in 76% of the SNAs. Seventy-five percent of SNAs yielded whole-genome-amplified DNA of sufficient quality for aCGH after storage and shipping. Individual SNAs from the same pregnancy reliably gave the same chromosomal profile, and fetal sex and trisomies could be detected. A microdeletion was detected in one pregnancy. Conclusion: SNAs could provide a source of extra-embryonic DNA for the prenatal screening/diagnosis of fetal sex and chromosomal and sub-chromosomal genetic abnormalities.