Extravillous trophoblast cell (EVT) invasion in early pregnancy occurs in a relatively low-oxygen environment. The role of
oxygen in regulation of EVT invasion remains controversial. We hypothesized ...that 1) culture in 3% oxygen inhibits EVT invasion
compared with culture at 8% or 20% oxygen and 2) inhibition of invasion is due to alterations in levels of components of the
urokinase plasminogen activator (PLAU, uPA) system rather than through increased apoptosis and/or decreased proliferation.
Placental samples (8â10, 12â14, and 16â20 wk gestation) were obtained from women undergoing elective surgical termination
of pregnancy or after cesarean section delivery (term) at the Royal Victoria Infirmary, Newcastle upon Tyne, U.K. EVT invasion
from placental explants cultured at 3%, 8%, or 20% oxygen was assessed using Matrigel invasion assays. Invasion was assessed
on Day 6, explants were harvested for analysis of apoptosis and proliferation, and medium was stored for analysis of PLAU
system components by ELISA and casein zymography. Culture at 3% oxygen inhibited EVT invasion. PLAU receptor and plasminogen
activator inhibitor-2 protein levels were increased and PLAU activity decreased in these cultures. There was no difference
in the proliferation in explants cultured at the three different oxygen concentrations. Apoptosis, assessed by M30 immunostaining,
was increased in EVT at both 3% and 8% oxygen. The reduction in the invasive capacity of EVT cultured at 3% oxygen appears
to be mediated both by a general inhibition of the PLAU system and a decrease in the number of cells available to invade.
Abstract
Invasion of primary first-trimester extravillous trophoblast cells is inhibited by culture in 3% oxygen via a mechanism associated
with a decrease in the activity of the urokinase plasminogen activator system.
Multiplex cytokine analysis technologies have become readily available in the last five years. Two main formats exist: multiplex sandwich ELISA and bead based assays. While these have each been ...compared to individual ELISAs, there has been no direct comparison between the two formats. We report here the comparison of two multiplex sandwich ELISA procedures (FAST Quant and SearchLight) and a bead based assay (UpState Luminex). All three kits differed from each other for different analytes and there was no clear pattern of one system giving systematically different results than another for any analyte studied. We suggest that each system has merits and several factors including range of analytes available, prospect of development of new analytes, dynamic range of the assay, sensitivity of the assay, cost of equipment, cost of consumables, ease of use and ease of data analysis need to be considered when choosing a system for use. We also suggest that results obtained from different systems cannot be combined.
To clarify the role of leucocytes in human cervical ripening and dilatation, cervical biopsies were obtained from six non-pregnant women, eight women undergoing early termination of pregnancy, 18 ...pregnant women undergoing elective Caesarean section at term (both with and without a ripe cervix as determined by Bishop score) and 11 women after term vaginal delivery. Leucocytes were localised by immunohistochemistry labelling and quantified in subepithelial and deep stromal areas. CD45+ leucocytes were more numerous in the subepithelial area of the cervix than in the deep stroma in all groups (
P
<
0.01). CD14+ macrophages and CD15+ granulocytes were increased in both the subepithelial and deep stromal areas only in the vaginal delivery group (
P
<
0.01). The number of macrophages in the ripening cervix (Bishop score above 4) was higher than in the unripe cervix (Bishop score 4 or less;
P
<
0.05) with no differences in other leucocyte populations. CD3+ CD8+ T cells in the subepithelial area were reduced in late pregnancy and after vaginal delivery (
P
<
0.01), but showed no relationship to Bishop score. Macrophages and granulocytes may be involved in the process of cervical dilatation, but macrophage infiltration into the ripening cervix before labour suggests their role in the ripening process. Reduced numbers of CD3+ CD8+ T-lymphocytes in late pregnancy and after vaginal delivery suggests that local immunity is down-regulated in the late pregnancy period. Regional differences in leucocyte subpopulations in the cervix indicate that leucocyte infiltration is likely to be regulated by local factors.
Humoral hypercalcemia of malignancy (HHM) is a common paraneoplastic disorder usually associated with increased synthesis of parathyroid hormone-related peptide (PTHrP). Unlike non-cancer forms of ...hypercalcemia, HHM does not routinely involve increased circulating levels of the active form of vitamin D, 1,25-dihydroxyvitamin D
3
(1,25(OH)
2
D
3
). Dysgerminomas are a notable exception to this rule, previous reports having described hypercalcemia with elevated serum 1,25(OH)
2
D
3
. To investigate the etiology of this form of HHM we have characterized expression and activity of the enzyme that catalyzes synthesis of 1,25(OH)
2
D
3
, 25-hydroxyvitamin D-1α-hydroxylase (1α-hydroxylase), in a collection of 12 dysgerminomas. RT-PCR analyses indicated that mRNA for 1α-hydroxylase was increased 222-fold in dysgerminomas compared to non-tumor ovarian tissue. Parallel enzyme assays in tissue homogenates showed that dysgerminomas produced fivefold higher levels of 1,25(OH)
2
D
3
compared to normal ovarian tissue. Immunolocalization studies indicated that 1α-hydroxylase was expressed by both tumor cells and by macrophages within the inflammatory cell infiltrate associated with dysgerminomas. The immunological nature of the increased 1,25(OH)
2
D
3
production observed in dysgerminomas was further emphasized by correlation between expression of 1α-hydroxylase and the endotoxin recognition factors CD14 and toll-like receptor 4 (TLR4). These data suggest that inflammatory mechanisms associated with dysgerminomas are the underlying cause of the increased expression and activity of 1α-hydroxylase associated with these tumors. We further postulate that this autocrine/paracrine action of 1α-hydroxylase may lead to increased circulating levels of 1,25(OH)
2
D
3
and a form of HHM which is distinct from that seen with PTHrP-secreting tumors.
Background
Uterine natural killer (uNK) cells and endometrial blood vessel maturation are increased in the luteal phase of the menstrual cycle in a subset of women with recurrent miscarriage (RM). ...uNK cell numbers are reduced after treatment with prednisolone (20 mg/day for 3 weeks).
Hypotheses
Prednisolone treatment reduces endometrial vascular maturation and angiogenic growth factor expression in women with RM with increased uNK cells.
Methods
Endometrial biopsies (n = 18 paired samples) from women with RM at LH + 7 before and during prednisolone treatment (20 mg/day for 3 weeks) were snap frozen. Total RNA and cDNA was prepared and used in a human angiogenesis RT-PCR superarray (84 genes, n = 6 pairs) with results validated using RT-PCR (n = 15 pairs). Immunohistochemistry (n = 15 pairs) was performed for Factor VIII, α-smooth muscle actin (α-SMA) and myosin heavy chain (MyHC) and the total number of vessels and the percentage of vessels completely surrounded by vascular smooth muscle cells (VSMCs) were determined.
Results
During prednisolone treatment there was no change in the total number of endometrial blood vessels but the percentage of vessels completely surrounded by VSMCs was decreased (α-SMA
P
< 0.0001; MyHC
P
< 0.0001). Endometrial EGF and STAB 1 expression was decreased during prednisolone treatment in samples from woman who went on to have a live birth.
Conclusions
The effect of prednisolone therapy for some women with RM may be due to altered endometrial angiogenic growth factor expression and reduced blood vessel maturation.
Punch biopsy of the human placental bed Robson, Stephen C.; Simpson, Helen; Ball, Elizabeth ...
American journal of obstetrics and gynecology,
11/2002, Letnik:
187, Številka:
5
Journal Article
Recenzirano
Objective: The purpose of this study was to report our experience with placental bed biopsy with the use of forceps. Study Design: Placental bed biopsies were undertaken transcervically under ...ultrasound guidance in 313 women who underwent termination of pregnancy between 7 and 20 weeks of gestation, in 104 women with a missed abortion who underwent evacuation of retained products of conception between 7 and 21 weeks of gestation, and in 13 women after vaginal delivery. Placental bed biopsies were also undertaken in 139 women who underwent caesarean delivery. Frozen sections were immunostained with monoclonal antibodies to cytokeratin, factor VIII-related antigen, and desmin. Results: Of the 417 cases attempted at <22 weeks, the placental bed was successfully sampled 281 women (67%); in 229 women (55%), at least one of the biopsy specimens contained a uterine spiral artery. Success was correlated with gestational age. Figures for the late cases that were sampled during caesarean delivery were 108 of 139 cases (78%) and 66 of 139 cases (47%) and after vaginal delivery were 11 of 13 cases (84%) and 6 of 13 cases (46%), respectively. The sampling procedure did not result in any significant morbidity. Conclusion: With the use of forceps, uterine spiral arteries can be sampled successfully throughout pregnancy in approximately 50 % of cases. (Am J Obstet Gynecol 2002;187:1349-55.)
In addition to its role in the prevention of neural tube defects, folic acid has many other physiological functions, including cell proliferation, DNA replication, and antioxidant protection. The aim ...of this study was to determine the role that folic acid has in regulating placental trophoblast development. Placental explants from placentae at gestational age 7 wk (n = 3) were cultured in folic acid at concentrations of 10−6 M, 10−8 M, and 10−10 M. Extravillous trophoblast (EVT) invasion was assessed following 6-day culture, and explants were used for immunohistochemical evaluation of proliferation (MKI67) and apoptosis (active caspase 3). In addition, an array was performed on cell culture supernatants to examine a range of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs). Folic acid increased the invasion of EVT cells in this explant model by between 83% and 19% (P = 0.005), and this was associated with increased MKI67 positivity and decreased active caspase 3 positivity; this effect was concentration dependent and showed a biphasic response. In addition, culture in folic acid increased vascular density, as determined by anti-CD31 immunostaining (P = 0.05). The increase in EVT invasion correlated with increased placental explant secretion of MMP2 (P = 0.01), MMP3 (P = 0.01), and MMP9 (P = 0.02). This study demonstrates that folic acid is potentially important in a number of crucial early stages of placental development, including EVT invasion, angiogenesis, and secretion of MMPs, and highlights the need for further studies to address the benefit of longer-term folic acid supplementation throughout pregnancy to prevent pregnancy disorders associated with deficient placental development, including preeclampsia.
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