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Podocalyxin is expressed on endothelial surface throughout the body and is likely released into the circulation during pregnancy in association with vessel remodeling. We have recently reported that serum podocalyxin is significantly increased in preeclampsia at disease presentation. In this study we investigated whether serum podocalyxin is altered prior to clinical presentation of preeclampsia. At 11-13 weeks of gestation, serum podocalyxin was significantly elevated in women who subsequently developed preeclampsia. Multi-factorial analysis suggests that combination of serum podocalyxin with the long isoform of HtrA3 and mean arterial pressure, may provide effective detection of late-onset preeclampsia at 11-13 weeks of pregnancy.

Background Preeclampsia is a pregnancy specific disorder affecting 3–5% of pregnancies worldwide. It is clinically divided into early-onset and late-onset subtypes. Placental factors are involved in the pathogenesis of preeclampsia. Growth differentiation factor 15 (GDF15), a protein of the transforming growth factor beta superfamily, is highly expressed in the placenta. However, it is unclear whether the circulating levels of GDF15 are altered in preeclampsia at the time of or prior to disease presentation. Methods Serum samples across three trimesters from 29 healthy pregnancies, third trimester sera from 34 women presenting with preeclampsia (early-onset n = 16, late-onset n = 18) and 66 gestation-age-matched controls, and sera at 11–13 weeks of pregnancy from women who later did (n = 36) or did not (n = 33) develop late-onset preeclampsia, were examined for GDF15 by ELISA. Results Serum GDF15 levels increased significantly with gestation in normal pregnancy. Serum GDF15 was significantly reduced in the third trimester in women presenting with preeclampsia compared to their gestation-age-matched controls. This reduction was apparent in both early-onset and late-onset subtypes, but it was more profound in late-onset cases. At 11–13 weeks of gestation, however, serum levels of GDF15 were similar between women who subsequently did and did not develop late-onset preeclampsia. Conclusion Serum GDF15 increased with gestation age, reaching the highest level in the third trimester. Serum GDF15 was significantly reduced in the third trimester in women presenting with preeclampsia, especially in late-onset cases. However, serum GDF15 was not altered in the first trimester in women destined to develop late-onset preeclampsia.

Introduction HtrA1 (high temperature requirement A1) is a serine protease of the HtrA family. We have previously shown that the placenta expresses the highest level of HtrA1 mRNA compared to other tissues in the human. Others have reported that placental HtrA1 is significantly up-regulated in preeclampsia (PE), a pregnancy-specific multi-systemic disorder associated with new onset hypertension and proteinuria. However, it is unclear how serum HtrA1 changes in a normal pregnancy and whether it is altered in PE pregnancies. Methods A sandwich ELISA highly specific to human HtrA1 and suitable for serum detection was developed and thoroughly validated. This assay was then applied to serum samples from different stages of normal pregnancy, as well as early-onset ( 34 weeks) PE pregnancies. Results Serum HtrA1 increased progressively with increasing gestation in normal pregnancies. However, this trend was perturbed in women with PE. Compared to respective gestation-age-matched normal pregnancies, HtrA1 serum levels were significantly increased in early-onset PE, but significantly reduced in late-onset PE. Discussion This is the first report to show a clear increase of HtrA1 in the maternal circulation during normal pregnancy, consistent with HtrA1 being highly expressed in the placenta. Importantly, this study identified that serum HtrA1 was altered differently in early-onset and late-onset PE pregnancies, highlighting the complex regulation of HtrA1 in the different subtypes. The significant increase of serum HtrA1 in early-onset PE suggests that it may be a potential biomarker for the diagnosis of early-onset PE at disease presentation.

Background Proprotein convertase 5/6 (PC6) is critical for endometrial epithelial receptivity and stromal cell decidualization for embryo implantation in women. We hypothesized that inhibiting PC6 could block implantation for contraception. The aim of this study was to prove this concept using human cell models and rabbits. Study Design A potential PC6 inhibitor, C1239-PEG-Poly R, was biochemically confirmed to be a potent PC6 inhibitor. The potential contraceptive action of the inhibitor was then tested in decidualization of primary human endometrial stromal cells in a human trophoblast spheroid attachment model and in vivo in rabbits. Results The PC6 inhibitor C1239-PEG-Poly R inhibited in a dose-dependent manner both decidualization and spheroid attachment. Vaginal delivery of 200 μL of the inhibitor at a final concentration of 5 mM to rabbits over a 3-day period starting 6 days after mating resulted in a 60% decrease in implantation and, hence, pregnancy. Conclusions This study presents proof of concept that PC6 inhibition has the potential to block embryo implantation, providing nonhormonal contraception for women.

Controlled invasion of extravillous trophoblast (EVT) is necessary for implantation and placentation. The serine protease HTRA3 is highly expressed in decidual cells in the late secretory phase of the menstrual cycle and throughout pregnancy. During the first trimester it is expressed in most trophoblast cell types, but not in the invading interstitial trophoblast. HTRA3 and its family members are down-regulated in a number of cancers and are proposed as tumour-suppressors. The current study investigated whether inhibiting HTRA3 in a first trimester trophoblast cell line expressing high levels of HTRA3 would alter invasion. HTR-8/SVneo (HTR-8, derived from first trimester placenta) and a number of choriocarcinoma cells (JEG-3, AC-1M88 and AC-1M32) were screened for HTRA3 expression. Only HTR-8 cells expressed high levels of HTRA3 mRNA, consistent with HTRA3 being down-regulated in cancer. Western blotting and immunofluorescence confirmed HTRA3 protein expression and localisation in HTR-8 cells. HTRA3 was detected in conditioned medium of HTR-8 cells, confirming its secretory nature. For functional studies, both long and short forms of recombinant human HTRA3, wild type and protease-inactive mutant (S 305 A) were produced using wheat-germ cell-free technology. Both have a similar molecular size, but the mutants have negligible protease activity. In addition, the mutants significantly inhibited the wild type protease activity, supporting their dominant-negative inhibition and utility as specific inhibitors of the wild type protein. Inhibition of HTRA3 by exogenous addition of HTRA3 mutant resulted in a significant increase in HTR-8 cell invasion. These results strongly support the hypothesis that HTRA3 is an inhibitor of trophoblast invasion during placental development.

Hemochorial placentation involves highly regulated interactions between fetal- and maternal-derived cells. HtrA3, a novel serine protease containing an insulin-like growth factor (IGF) binding domain, was previously shown to increase during early pregnancy in the mouse uterus, being dramatically upregulated post-implantation. The present study examined the regulation of HtrA3 gene in the mouse uterus from post-implantation to late gestation. Both mRNA and protein of HtrA3 were localized specifically in the maternal decidua. In contrast, HtrA3 expression was below detection in trophoblasts, including the giant cells that are in direct contact with the decidua. This pattern persisted from the early stages of placentation to near term. The level of decidual HtrA3 mRNA and its protein gradually decreased as the placenta matured. In the decidua, only the maternal decidual cells, but not blood vessels or uterine NK cells that are present in large numbers, were positive for HtrA3. The specific localization of a protease possessing an IGF-binding domain at the maternal-fetal interface suggests that HtrA3 plays a critical role in mediating maternal decidual remodelling and maintenance, likely in association with the IGF system, in placental development and function.

The mammalian uterus is normally not receptive to embryo implantation except during the very limited ‘window of implantation’. To identify genes that may be responsible for this phenomenon the technique of RNA differential display (DD-PCR) was applied to implantation and inter-implantation sites on day 4.5 of pregnancy in the mouse, the time at which the blastocyst becomes attached to the endometrium. Three of these genes were identified as splicing factor SC35, calbindin-D9k and monoclonal non-specific suppressor factorβ (MNSFβ). Expression of SC35 mRNA, which is responsible for removal of introns from pre-mRNA, is much higher in implantation than in interimplantation sites during pregnancy. Expression of alternatively spliced mRNAs for SC35 is differentially regulated by early pregnancy and steroid hormones. By contrast, calbindin-D9k, a regulator of calcium, is upregulated by progesterone and its mRNA increases in the uterus during early pregnancy compared with during the cycle, although it is significantly lower in implantation sites than in interimplantation sites on days 4.5–5.5 of pregnancy, but subsequently becomes barely detectable in both sites. The mRNA for calbindin-D9k is predominantly in endometrial luminal epithelium. MNSFβ, a cytokine involved in regulation of the immune system, showed lower expression at implantation sites than interimplantation sites on day 4.5 of pregnancy, when embryos first attach to the uterus and initiate implantation, and on day 5.5 when implantation has advanced. Immunohistochemically, the protein was localized to endometrial stromal cells in the non-pregnant uterus, but disappeared as decidualization progressed. The precise roles of these three proteins in the process of embryo implantation remains to be determined. Homologues of the proteins may contribute to the development of the ‘window of implantation’ in the human and hence be appropriate targets for new post-coital contraceptives or may be manipulated to improve fertility.

Accurate quantitation of mRNA levels of a number of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in very small samples such as human biopsy material has not been generally possible. This paper describes the development, validation and application of a quantitative RT-PCR (Q-RT-PCR) assay that allows the detection and quantitation of mRNAs encoding genes of three MMPs (MMP-1, MMP-2, MMP-3), three TIMPs (TIMP-1, TIMP-2, TIMP-3) and GAPDH simultaneously from small amounts of RNA (4 microg). A multispecific competitor which shares the same primer-binding sequences as the cellular mRNA of all seven genes, but yields different sized PCR products, was constructed by adding primers specific for the MMPs and TIMPs to a core molecule (mutated GAPDH) by sequential PCR and cloning, and its multispecificity was experimentally validated. Application of the technique to measurement of transcriptional levels of MMPs and TIMPs in cultured human endometrial stromal cells provided support to the hypothesis that progesterone withdrawal alters the ratio of MMPs to TIMPs in favor of MMPs. This Q-RT-PCR method is a relatively simple, highly specific and nonradioactive procedure and is widely applicable.

Understanding the mechanisms of disease responsible for the syndrome of preeclampsia as well as early risk assessment is still a major challenge. The concentrations of circulating proteins in maternal blood such as placental growth factor, soluble vascular endothelial growth factor receptor-1 and soluble endoglin are altered weeks before the onset of clinical symptoms of the syndrome. Recently, other proteins in maternal serum, such as activin A, inhibin A, PAPP-A, and PP13 have been suggested to be of value in first trimester risk assessment. Since preeclampsia is a syndrome, it seems unlikely that a single test will predict all forms of preeclampsia. This realization has led to the formulation of a new conceptual framework suggesting that a combination of markers (biochemical and/or biophysical) may be required to conduct comprehensive risk assessment for the syndrome.

The pathogenic origin of preeclampsia is defective placental development (placentation) during early pregnancy. Owing to the lack of reliable early detection biomarkers, preeclampsia is not diagnosed until later in pregnancy and delivery remains the sole effective therapy. HtrA3 is a recently cloned gene with high expression during placentation in the mouse, rhesus monkey and human. We established that in women, placental HtrA3 protein was maximally produced in the 1st trimester, then dramatically down-regulated, especially in the syncytiotrophoblasts. HtrA3 was secreted into the maternal circulation with a serum profile reflecting placental production. Oxygen tension, which is dynamic during placentation, regulated HtrA3; hypoxia enhanced, while hypoxia-normoxia transition decreased, HtrA3 protein production and secretion specifically in syncytiotrophoblast, reflecting placental HtrA3 production. Importantly, maternal serum HtrA3 levels around the time of placental oxygen switch (∼13-14 weeks) significantly differed between women who subsequently experienced normal or preeclamptic pregnancies, being much higher in women destined to develop preeclampsia. This delay in HtrA3 down-regulation, likely reflects prolonged placental hypoxic exposure due to abnormalities in vessel remodeling (the root cause of preeclampsia). We thus provide experimental evidence and a molecular rationale suggesting abnormal maternal serum HtrA3 levels during early pregnancy are associated with development of preeclampsia.