Arielle Meisser

Biochemistry and molecular biology of trophoblast invasion.

Cytotrophoblastic cells (CTBs) from first‐trimester placenta form columns of invasive CTBs. This invasive behavior is due to the ability of CTBs to secrete matrix metalloproteinases (MMPs), since tissue inhibitor of MMPs (TIMP) inhibits their invasiveness in the extracellular space. Although CTBs behave like metastic cells, in vivo they are only transiently invasive (first trimester), and their invasion is normally limited only to the endometrium and to the proximal third of the myometrium. This temporal and spatial regulation of trophoblast invasion is believed to be mediated in an autocrine way by trophoblastic factors and in a paracrine way by uterine factors. Several types of regulators have been investigated: hormones, extracellular matrix glycoproteins, and cytokines or growth factors. This review is not intended to be an exhaustive catalogue of potential regulators of trophoblast invasion but is aimed at summarizing the most important categories of factors affecting trophoblast‐endometrium interactions.

Control of MMP-9 expression at the maternal–fetal interface

Cytotrophoblastic cells (CTB) from first trimester placenta form columns of invasive CTB. This invasive behaviour is due to the ability of CTB to secrete matrix metalloproteinases (MMPs) since tissue inhibitor of MMP (TIMP) inhibits their invasiveness. Although CTB behave like metastatic cells, in vivo they are only transiently invasive (first trimester) and their invasion is normally limited only to the endometrium and to the proximal third of the myometrium. This temporal and spatial regulation of trophoblast invasion is believed to be mediated in an autocrine way by trophoblastic factors and in a paracrine way by uterine factors. Several types of regulators have been investigated: hormones, extra-cellular matrix glycoproteins and cytokines or growth factors. This review is not intended to be an exhaustive catalogue of potential regulators of trophoblastic MMP-9 secretion but is aimed at summarising the most important signalling pathways involved in MMP-9 regulation.

Effects of decidua-conditioned medium and insulin-like growth factor binding protein-1 on trophoblastic matrix metalloproteinases and their inhibitors

The regulatory role of in vitro decidualized stromal cells (DESCM) and their main secretory product insulin-like growth factor binding protein-1 (IGFBP-1) was studied on the secretion of trophoblastic gelatinases and tissue inhibitor of metalloproteinase (TIMP-1). First trimester cytotrophoblastic cells (CTB) were obtained from abortions and cultured in vitro in presence or absence of DESCM or IGFBP-1. Secreted gelatinases were analysed in the culture supernatants by zymography and by measurements of the total gelatinolytic activity. TIMP-1, hCG, and fetal fibronectin (fFN) were measured by commercially available immunoassays. DESCM inhibited the total gelatinolytic activity of CTB but increased trophoblastic MMP-9, TIMP-1 and fFN. In contrast, IGFBP-1 increased the total gelatinolytic activity and TIMP-1, had no effect on MMP-2 , MMP-9 or fFN but inhibited hCG. It is concluded that a factor secreted by decidual cells inhibits the gelatinolytic property of trophoblast by increasing TIMP-1. Other decidual factors, as yet unidentified, increase MMP-2 and MMP-9 to an extent which does override the inhibitory effect of TIMP-1. Since in contrast to DESCM, IGFBP-1 increases the total gelatinolytic activity of CTB, it cannot be the primary active decidual factor regulating the proteolytic activity of CTB. The possibility of an integrin-mediated effect of IGFBP-1 on CTB is discussed.

Blood sampling as critical preanalytical determinant to use circulating MMP and TIMP as surrogate markers for pathological processes.

Dear Sir, It has been suggested that circulating matrix metalloproteinases (MMP) and tissue inhibitors of matrix metalloproteinases (TIMP) may be useful diagnostic and prognostic indicators in malignant and nonmalignant diseases. Numerous reports seem to confirm that assumption although it is unknown whether increased MMP concentration are based on the initial step of tissue damage or the following repairing process. There are also contrasting results when similar patients were studied, i.e., increased plasma MMP-9 concentrations but unchanged serum concentrations were found in patients with colon carcinoma. We believe that in addition to the analytical and clinical reasons to explain these differences between various studies, more attention should be given to the preanalytical conditions of sampling. In this respect, we read with great interest a recent article published in this journal. Gianelli et al. found no difference in serum concentrations of MMP-9 and TIMP-1 between healthy women and patients with breast cancer, but decreased MMP-9 and increased TIMP-1 after surgery in comparison with initial concentrations. The authors concluded that these changes might serve as prognostic indices in the follow-up of breast cancer patients. It should be remembered that, however, that using serum material as samples for the determination of those analytes, the authors had rather inadequately considered the impact of blood collection as an important preanalytical determinant of MMP and TIMP results. There is rising evidence that blood sampling and handling markedly determine the concentrations of circulating MMP and TIMP. Thus, to interpret that data correctly and to avoid wrong expectations in future studies using these markers, we want to draw the attention of interested clinicians to these facts that were particularly discussed in analytical journals. Although the pitfalls in the measurement of circulating growth factors and cytokines for diagnostic purposes have become generally accepted, similar evidences for MMP and TIMP have been widely ignored. Results of own experiments (summarized in Fig. 1) demonstrate the differences of MMP-9 and TIMP-1 in serum and plasma as the analytes come into question. Briefly, for MMP-9 measurements, blood samples from 12 healthy volunteers were collected in Vacutainer tubes (Becton Dickinson, Franklin Lakes, NJ) containing a clot activator for serum preparations or lithium heparinate, dipotassium EDTA or sodium citrate for plasma preparations. Corresponding prepared plastic tubes (Monovettes; Sarstedt, N€urtingen, Germany) were used for blood sampling for TIMP-1 measurements. For the MMP-9 experiments, the tubes were either centrifuged (1,600g, 15 min) immediately (considered as t0) or after 0.5 or 2 hr storage at room temperature and after 24 hr storage at 4 C, respectively. Within 5 days of sampling while the samples were stored at 4 C MMP-9 measurements were carried out using our own enzyme immunoassay described previously. Briefly, samples (and standards) were incubated overnight at room temperature in microplate wells coated with our own polyclonal sheep antihuman MMP-9 raised against MMP-9 purified from U937 cell supernatant. After incubation and washing steps, sheep anti-MMP-9 (The Binding Site, Birmingham, UK) was added for 2 hr at room temperature on a rotating platform. After washing, plates were reincubated for 30 min at 20 C with peroxydase-labeled rabbit anti-sheep antibodies (DakoCytomation, Glostrup, Denmark) before adding the substrate 1,2-phenylenediamine for 10 min and measuring the absorbance at 492 nm. For the TIMP-1 experiments, the samples were prepared within 30 min after venipuncture and stored at 280 C until assayed using a commercially assay for TIMP-1 measurements (Biotrak; Amersham, Little Chalfont, UK). Statistical analyses were carried out by ANOVA using the Fisher test. Values (p < 0.05) were considered as statistically significant. Figure 1a shows that MMP-9 concentration depends on the time between venipuncture and centrifugation as well as on the type of sample preparation. The MMP-9 concentration in serum was about 7–10 times higher than in plasma. Similar results were shown for TIMP-1 although the effect of time between blood sampling and centrifugation was not investigated in detail. The TIMP-1 concentrations were about 5–7 times higher in serum than in plasma (Fig. 1b). Because platelets and leukocytes contain high concentrations of MMP-9 and TIMP-1, the observed differences between plasma and serum are attributed to the different release of these analytes from blood cells during platelet activation or sampling process. Thus, it can be assumed that serum MMP-9 and TIMP-1 as measured by Giannelli et al. are possible misleading markers, because they obviously reflect a high nonspecific background by the release from blood cells and are not in direct relationship to cancer. In addition, Giannelli et al. did not describe the time between venipuncture and centrifugation and whether they used tubes available commercially with or

Involvement of trophoblast in embryo implantation: regulation by paracrine factors.

In order to investigate the regulatory role of only one endometrial cell type on trophoblastic invasion, we explored the effects of culture medium conditioned by in vitro decidualised stromal cells (DCM) and of insulin-like growth factor binding protein-1 (IGFBP-1, the main secretory product of decidual cells) on the trophoblastic secretion of gelatinases and tissue inhibitor of metalloproteinases (TIMP-1). First trimester cytotrophoblastic cells (CTB) were obtained from abortions and cultured in vitro in presence or absence of DCM or IGFBP-1. Secreted gelatinases were analysed in the culture supernatants by zymography and by measurements of the total gelatinolytic activity. Tissue inhibitor of metalloproteinases (TIMP-1) was measured by a commercially available immunoassay. DCM inhibited the total gelatinolytic activity of CTB but increased trophoblastic MMP-9 and TIMP-1. In contrast, IGFBP-1 increased the total gelatinolytic activity and TIMP-1 and had no effect on MMP-2 and MMP-9. We conclude that a factor secreted by decidual cells (possibly TGFbeta) inhibits the total gelatinolytic activity of trophoblast by increasing TIMP-1 but other factors, as yet unidentified, increase MMP-2 and MMP-9 to an extent which does not shift the equilibrium between the gelatinases and TIMP-1 in favour of the gelatinases. In contrast to DCM, IGFBP-1 increases the total gelatinolytic activity probably by stimulating another gelatinase (stromelysin-1?) as MMP-2 and MMP 9 are unchanged by IGFBP-1. The possibility of an integrin mediated effect of IGFBP-1 on CTB is discussed.

Accuracy of single measurements of pregnancy-associated plasma protein-A, human chorionic gonadotropin and progesterone in the diagnosis of early pregnancy failure

BACKGROUND Circulating human chorionic gonadotropin (hCG) and progesterone are commonly used as markers of abnormal pregnancies. Previous studies have shown that pregnancy-associated plasma protein-A (PAPP-A) was also depressed in extrauterine pregnancies (EUP). Previously, PAPP-A was measured with polyclonal antibodies which were later shown to recognise also the pro-form of major basic protein (pro-MBP). OBJECTIVE To evaluate the clinical usefulness of PAPP-A measurements in early pregnancy. STUDY DESIGN Circulating PAPP-A, hCG and progesterone were measured in patients with EUP (n=68), abnormal intrauterine pregnancies (abIUP, n=31) and normal intrauterine pregnancies (nIUP, n=72). Gestational age was 30-70 days from the last menstruation. RESULTS For PAPP-A and hCG, a steep increase was observed from day 30 after last menstrual period onwards, this increase being much less important for abIUP and EUP. The values of PAPP-A and hCG were significantly decreased in abIUP and EUP, from 42 days after LMP onwards. There were no significant differences between abIUP and EUP. Progesterone concentration does not vary with amenorrhoea and was significantly lower in abIUP and EUP. Values in abIUP were significantly (P=0.02) lower compared with EUP for amenorrhoea above 42 days. ROC curves were constructed for amenorrhoea above 42 days. For a specificity of 99%, the sensitivity of PAPP-A, hCG and progesterone were 64.5, 93.3 and 76%, respectively. The threshold values were 14.3mIU/l, 10,400IU/l and 10.1ng/ml for PAPP-A, hCG and progesterone. CONCLUSION We confirm the decrease of PAPP-A concentrations in pregnancy failure, but hCG and progesterone remain the best clinical tools.

Effect of hydrosalpinx fluid on secretion of trophoblastic matrix metalloproteinases

OBJECTIVE To determine if hydrosalpinx fluid affects trophoblastic metalloproteinases (MMPs) secretion. DESIGN Measurement of the effect of hydrosalpinx and peritoneal fluids (as controls) added to the medium on the MMPs secreted by cytotrophoblastic cells. SETTING Academic research center. PATIENT(S) Five samples of hydrosalpinx fluid were obtained at the time of ovocyte retrieval. Three samples of peritoneal fluids were collected at laparoscopic sterilization. MAIN OUTCOME MEASURE(S) The concentration and activity of MMP-2 and MMP-9, the concentration of the tissue inhibitor of metalloproteinases (TIMP-1), and the total gelatinolytic activity of the cytotrophoblastic cells were measured in the culture medium. RESULT(S) Hydrosalpinx significantly stimulated MMP-2, MMP-9, and TIMP-1. The net result was a significant stimulation of the total gelatinolytic activity. Peritoneal fluids increased MMP-2, MMP-9, and TIMP-1 concentrations, but the total gelatinolytic activity was not modified. CONCLUSION(S) In contrast to peritoneal fluids, hydrosalpinx stimulates the total gelatinolytic activity of cytotrophoblastic cells. This might indicate that the effect of hydrosalpinx on implantation rates may not be due to an inhibition of the capacity of an embryo to invade the endometrium. However, the stimulatory effect of hydrosalpinx on the net gelatinolytic activity could partly explain the increased incidence of ectopic pregnancies that have been described in the presence of hydrosalpinx.

CA 125 in seminal plasma: correlation with semen parameters

Ovarian cancer marker CA 125 was measured in human seminal plasma, and the concentrations ranged between 22 and 1149 U/ml, and between 39 and 4711 U/ejaculate. This very high patient-to-patient variability was in contrast to a much lower within-patient variability, which was comparable to that of other semen parameters. No significant differences in CA 125 concentration were found in seminal plasma from normospermic patients, patients with male factors, vasectomized men, and in aliquots of samples which led to a pregnancy, via artificial insemination or in-vitro fertilization. The seminal plasma CA 125 concentration was not correlated with sperm count, motility and morphology. In contrast, seminal plasma CA 125 concentrations correlated with the age of the patient (P < 0.001) and inversely with the volume of the ejaculate (P < 0.001). These correlations were independent of each other. CA 125 did not correlate with the prostatic marker zinc, but did do so with the seminal vesicle marker fructose and the epididymal marker carnitine.

Pregnancy-Associated Plasma Protein-A-Induced Inhibition of Human Leukocyte Elastase: An Artifact

Pregnancy-associated plasma protein A (PAPP-A), was reported to be an inhibitor in many in vitro systems. Since it was shown that the inhibition of coagulation and complement activity attributed to PAPP-A was in fact due to a contamination by heparin occurring during the purification process, we undertook the present study to see whether the reported PAPP-A-induced inhibition of human leukocyte elastase (HLE) could also be attributed to heparin contamination. PAPP-A was purified from maternal pregnancy EDTA plasma by a method which was previously shown to eliminate contaminating heparin: this preparation was inactive in the HLE assay. But PAPP-A isolated by heparin-Sepharose chromatography, or a PAPP-A-free washing of the heparin-Sepharose column were both inhibitors of HLE. Furthermore the inactive PAPP-A preparation, when incubated with the PAPP-A-free washing of the heparin-Sepharose column, yielded a high molecular weight preparation which inhibited HLE. It is concluded that PAPP-A is not an inhibitor of HLE and that the inhibition of HLE previously attributed to PAPP-A was due to contaminating heparin.

Secretion of matrix metalloproteinase-2, tissue inhibitor of metalloproteinase-1 and human chorionic gonadotropin by cytotrophoblastic cells: Is there a regulation by relaxin?

The peptide hormone relaxin is a member of the insulin-related gene family comprising insulin, insulin-like growth factors I and II (IGF I and II), Leydig insulin-like peptide (LEY I-L, RLF, INSL3) and peptides encoded by INSL4 (Early placenta insulin-like), INSL5 and INSL6 genes [1-7]. Whereas the physiological role of relaxin has been well assessed in late pregnancy and parturition [8,9], recent studies in primates and humans have shown that relaxin secreted by the corpus luteum and/or endometrium during the luteal phase may be involved in reproductive processes such as endometrial decidualization [10,11], early pregnancy [12] and even embryo implantation [13].