The effect of progestins on vascular endothelial growth factor, oestrogen receptor and progesterone receptor immunoreactivity and endothelial cell density in human endometrium.

One common side-effect of contraceptive use is that it often leads to disrupted endometrial bleeding patterns. This may be due to changes in endothelial density and vessel integrity. To investigate whether the level of endometrial immunoreactive vascular endothelial growth factor (VEGF), oestrogen receptor or progesterone receptor (PR) have any role in this, women were treated with either Mircette, a monophasic oral contraceptive, or Implanon, a long-acting gestagen, and immunohistochemistry performed. In addition a small number of endometria were studied from women treated with levonorgestrel released from an intrauterine coil. During the untreated normal cycle, there was a significant increase in glandular VEGF immunoreactivity and a significant decrease in PR immunoreactivity in the midand late secretory phases compared to the proliferative phase. There was a significant positive correlation between stromal VEGF immunoreactivity and endothelial cell density. This correlation was also apparent during treatment with Implanon, but not with Mircette. Disrupted bleeding patterns were associated with Implanon and to a lesser extent with Mircette. Both contraceptives significantly reduced glandular VEGF immunoreactivity but the intrauterine treatment with levonorgestrel resulted in strong glandular epithelial staining and intense staining of decidualized stromal cells. Implanon significantly increased glandular PR staining, but Mircette significantly reduced stromal PR staining when compared to secretory phase before-treatment biopsies. There were no changes in endothelial cell density or glandular or stromal ER during the normal cycle, or with use of either contraceptive. There was no association of the parameters measured with bleeding patterns or histological category.

Cytotoxicity and antibacterial activity of ethanol extract from leaves of a herbal drug, boneset (Eupatorium perfoliatum).

The cytotoxic and antibacterial activity of an ethanol extract of leaves of a herbal drug, boneset (Eupatorium perfoliatum), was investigated. The extract showed potent cytotoxicity with EC(50) values (12-14 microg/mL) comparable to a standard cytotoxic agent, chlorambucil. The extract showed a weak antibacterial activity against gram-positive test organisms (Staphylococcus aureus and Bacillus megaterium).

The effect of etonogestrel on VEGF, oestrogen and progesterone receptor immunoreactivity and endothelial cell number in human endometrium.

Contraceptive use often leads to disrupted endometrial bleeding patterns in women. In this study, two different contraceptive regimes (Mircette, a monophasic oral contraceptive and Implanon, a long-acting gestagen) were used and their effects on the immunoreactivity of vascular endothelial growth factor (VEGF), oestrogen receptor (ER), progesterone receptor (PR) and endothelial cell number were determined. During the untreated normal cycle, there was a significant increase (P = 0.005) in glandular VEGF immunoreactivity and a significant decrease (P < 0.05) in PR immunoreactivity in the mid- and late secretory phases compared with the proliferative phase. There was a significant positive correlation (gamma = 0.38, P = 0.046) between stromal VEGF immunoreactivity and endothelial cell number. This correlation was also apparent during treatment with Implanon, but not with Mircette. Disrupted bleeding patterns were associated with Implanon and, to a lesser extent, with Mircette. Both contraceptives significantly reduced glandular VEGF immunoreactivity. Implanon significantly increased (P = 0.016) glandular PR staining, but Mircette significantly reduced (P = 0.027) stromal PR staining when compared with secretory before-treatment biopsies. There were no changes in endothelial cell number or glandular or stromal ER during the normal cycle, or with use of either contraceptive. There was no association between the parameters measured with bleeding patterns and histological category.

Protection against tetanus toxin in mice nasally immunized with recombinant Lactococcus lactis expressing tetanus toxin fragment C.

Mice inoculated intranasally (i.n.) with a recombinant strain of live Lactococcus lactis expressing tetanus toxin fragment C (TTFC), produced both serum and secretory antibodies to TTFC. Killed bacteria which had accumulated TTFC intracellularly in vitro also elicited protective serum antibody responses. There was no requirement for either colonization or invasion of the mucosa. In addition secretory antibody responses in the lung and nasal tissues were elicited after i.n. inoculation in the presence of an adjuvant.

Factors affecting the immunogenicity of tetanus toxin fragment C expressed in Lactococcus lactis.

The relative immunogenicity of tetanus toxin fragment C (TTFC) has been determined in three different strains of inbred mice when expressed in Lactococcus lactis as a membrane-anchored protein (strain UCP1054), as an intracellular protein (strain UCP1050), or as a secreted protein which is partly retained within the cell wall (strain UCP1052). Protection against toxin challenge (20 x LD50) could be obtained without the induction of anti-lactococcal antibodies. When compared in terms of the dose of expressed tetanus toxin fragment C required to elicit protection against lethal challenge the membrane-anchored form was significantly (10-20 fold) more immunogenic than the alternative forms of the protein.

An immunohistochemical study of the vascularization of the human Graafian follicle.

The wall of the largest Graafian follicle or corpus luteum was biopsied in 22 patients at laparoscopy. Both granulosa and theca cells were contained in 18 samples. These samples were classified as pre-luteinizing hormone (LH) surge (n = 3), LH surge (n = 3), early luteal (n = 3), mid-luteal (n = 4), late luteal (n = 3) and menstrual (n = 2). A double-staining immunohistochemical protocol was used to demonstrate proliferating endothelial cells: mouse-anti-rat-proliferating cell nuclear antigen, for proliferating cells; mouse-anti-human-CD34 antibody for endothelial cells. The percentage of endothelial cells proliferating (proliferation index) and the area of tissue occupied by endothelial cells (areal fraction) were determined for granulosa and theca layers. Intra- and inter-slide coefficients of variation were < 15%. The granulosa layer was avascular until the LH surge subsided. Maximum vascularization was achieved by the mid-luteal phase. The theca endothelial cell proliferation index was constant from pre-LH surge to mid-luteal phases. The mean theca endothelial cell proliferation index for these phases was significantly greater than for the late luteal and menstrual phases. From first appearance in the granulosa layer, endothelial cells had the same proliferation index as the theca endothelial cells, the proliferation index decreasing significantly in both after the mid-luteal phase (P = 0.018). It is concluded that endothelial cell proliferation is unchanged throughout the follicular, early and mid-luteal phases, decreasing significantly in the late luteal phase. By contrast, endothelial cell invasion of the membrane granulosa, presumably in response to a chemotactic stimulus, occurs after the LH surge has subsided.

Human follicular fluid maturity and endothelial cell chemotaxis.

Follicular fluid of varying maturity was collected from the largest Graafian follicle of 23 ovulatory patients during laparoscopy (five blood-stained samples were discarded) and from five patients undergoing oocyte collection for assisted reproduction. The endothelial cell (EC) chemotactic potentials of the samples were tested with bovine aortic ECs in modified Boyden Chambers and an EC density score was determined for each sample. Intra-assay coefficients of variation (CV) varied from 2.9-17%; inter-assay CV was 45%. Therefore, cell density scores were expressed in terms of positive and negative control values from their respective plates. Serum progesterone was positively correlated with EC chemotactic potential (R2 = 14.9%). There was no correlation with day of cycle, follicular diameter, serum luteinizing hormone (LH) or oestradiol, or follicular fluid total protein levels (all R2 < or = 6%). Five subjects were in early and four in mid-follicular phase, seven were in early and two in late LH surge, and there were also five in-vitro fertilization (IVF) specimens. There was a statistically significant difference between the EC chemotactic potential of the five groups by analysis of variance (F = 5.98; P = 0.006). There was no significant difference between the late LH surge and the IVF samples (two-sample t-test). The mean cell density was higher for these two groups than the other three (P < 0.01 in all cases; two-sample t-test). It is concluded that the EC chemotactic potential of human follicular fluid increases after the LH surge and before ovulation.

Human follicular fluid maturity and endothelial cell mitogenesis.

Follicular fluid, of varying maturity, (day 5-16 of cycle) was collected from the largest Graafian follicle of each of 22 ovulatory patients during laparoscopic procedures. Three samples were blood-stained and discarded. The mitogenic potential of each sample was determined using bovine aortic endothelial cells in the CellTiter 96 Non-Radioactive Cell Proliferation/Cytotoxicity Assay system. Intra- and inter-plate coefficients of variation were < 9%. The follicular fluid samples induced cell doubling times which varied from approximately 12-24 h and final cell numbers which, in the individual wells, ranged from 7828-30,900 (starting number 2000/well). Follicular fluid total protein content was unrelated to the mitogenic potential, (R2 = 0%). Serum oestradiol was negatively correlated with the mitogenic potential (R2 = 26%). No correlation was found with day of the menstrual cycle (R2 = 4.3%), maximum follicular diameter (R2 = 1.8%), or serum concentration of progesterone (R2 = 0.7%), luteinizing hormone (LH) (R2 = 1.5%) or follicle stimulating hormone (R2 = 0.1%). Five subjects were in 'early' and six in 'mid'-follicular phase, six were in 'early' and two in 'late' LH surge. There was no difference in the mitogenic response between these four groups by one-way analysis of variance (F = 0.21; P = 0.89). It is concluded that the mitogenic potential of human follicular fluid is not related to Graafian follicle maturity or, more particularly, to the LH surge.

Reduction in endometrial neutrophils in proximity to implanting rat blastocysts.

An antiserum to rat neutrophils was raised and used to follow the distribution of endometrial neutrophils during the peri-implantation period. Uteri from four pregnant and four pseudopregnant rats killed at 14:00, 17:00, 20:30 and 23:00 h on day 5 of pregnancy and 09:00 h on day 6 were sectioned. Four sections from each of four implantation sites and four intersites from each rat were immunostained. There was wide variability among rats in the number of endometrial neutrophils, but a nested analysis of variance showed significantly fewer neutrophils at implantation sites than at intersites from 20:30 h onwards. This difference was primarily due to the presence of more neutrophils in intersite regions of the endometrium. The results from this study do not support a role for neutrophils in the implantation-associated increase in microvascular permeability or decidualization in rats.

Immunolocalization of the vasoconstrictor endothelin in human endometrium during the menstrual cycle and in umbilical cord at birth.

OBJECTIVE: Our objective was to determine the localization of immunoreactive endothelin in human cyclic endometrium and in umbilical cord during normal delivery and after cesarean section. STUDY DESIGN: Fixed dated endometrial tissue (n = 41) and umbilical cord (n = 6) were subjected to immunohistochemistry with an antiserum cross reacting with endothelin-1, -2 and -3. RESULTS: Low levels of stromal endometrial staining were seen throughout the cycle. The strongest staining was in luminal epithelium throughout the secretory phase and in glandular epithelium in the late-secretory phase. In umbilical cord the most intense immunoreactivity was present on the amnion cells on the outer cord, with some staining in intermittent cells in the Wharton's jelly and in umbilical vein cells. No differences were detected between cord from normal delivery or cesarean section. CONCLUSION: A paracrine role is suggested for endothelin in regulation of endometrial function and a role in vasoconstriction in the umbilical cord at birth.

In-vivo microscopy of the rat endometrial subepithelial capillary plexus during the oestrous cycle and after ovariectomy.

Blood flow through the endometrium was visualized by using incident-light fluorescence microscopy and a video image recorded for later detailed analysis. The subepithelial microvascular density was calculated for each day of the oestrous cycle and at 7 days after ovariectomy. The results showed that the microvasculature was significantly more dense at dioestrus I, pro-oestrus, and after ovariectomy than at oestrus, with dioestrus II being in between. Mean capillary path lengths running from arteriole to venule were longest at pro-oestrus, followed by oestrus, dioestrus II, dioestrus I, and shortest after ovariectomy. The results suggest that endometrial growth and regression precede microvascular growth and regression. The technique of in-vivo microscopy provides an important new avenue for investigating the role of local factors in the control of the endometrial microcirculation.

Vascular response in a non-uterine site to implantation-stage embryos following interspecies transfers between the rat, mouse, and guinea-pig.

Pre-implantation-stage embryos from rats, mice, and guinea-pigs were transferred to a non-uterine site--the anterior chamber of the eye--of female recipients. All 9 combinations of transfers were performed: 3 allogeneic (intraspecies) transfers as controls, and 6 xenogeneic (interspecies) transfers. Implantation, as judged by extravasation from blood vessels of the iris or ciliary body occurred with success rates of 90.4% per transfer in the control rat group. 76.9% in the control mouse group, and 81.8% in the control guinea-pig group. Significantly reduced implantation rates occurred in the rat to guinea-pig (0%), mouse to rat (46.9%), mouse to guinea-pig (6.7%), and guinea-pig to rat (0%) groups compared to controls. Reductions, although not significant, also occurred in the other 2 groups: rat to mouse (77.8%), and guinea-pig to mouse (44.4%). These results together with some ultrastructural and light-microscopical observations suggest a degree of species specificity involved in the vascular response to the implanting embryo. We propose that the peri-implantation embryo produces a signal(s) which is to some extent species specific and which in the normal allogeneic situation is responsible for the early vascular effects seen at implantation in most eutherian mammals.

Vascular response in a non-uterine site to implantation-stage embryos in the rat and guinea-pig: in vivo and ultrastructural studies.

Preimplantation-stage embryos were transferred to the anterior eye chamber of recipient rats and guinea-pigs. After implantation had occurred the influence of the embryo on the iris vasculature was examined ultrastructurally. In both species, the earliest effect of embryonic implantation was an increased stromal oedema. Under increasing embryonic influence the vascular endothelial cells showed an increased number of projections into the vascular lumen, while in the rat, endothelial projections were also found pushing back into the basement membrane. In the rat, the endothelium became very irregular in thickness prior to complete disintegration and loss during more advanced stages of implantation. Rat embryonic trophoblast was found invading iris vasculature, particularly in areas where the iridial endothelium was partially or completely missing. Other cells in the iris, including the stroma, appeared to be less affected. In the guinea-pig, however, trophoblast cells appeared to be capable of invading the vasculature by displacing endothelial cells that still appeared morphologically normal.

Embryo implantation in the anterior chamber of the eye. Effects on uterine allografts and the microvasculature.

A model suitable for the in vivo study of embryo implantation in the presence or absence of uterine tissue has been developed. After transplantation of a uterine allograft to the anterior chamber of rat eyes, embryos were transferred to control, normally cycling, and pseudopregnant recipients. Implantation rates were 86%, 46% and 49%, respectively, for the three groups. Despite embryo growth rates being delayed by 3-4 days, histologic studies showed relatively normal egg cylinder stage embryos equivalent to those of day 8 of normal development. Uterine allografts varied greatly in appearance, from inert-looking pieces of connective tissue to recognizable uterine structures with an epithelium and a degree of decidual response. Reduced implantation rates in the presence of a uterine allograft may be due either to the uterine production of blastotoxic substances or the presence of a chronic inflammatory reaction resulting from the rejection of the allograft by the recipient. This model provides a unique system for continuous, noninvasive in vivo observations of the implantation process.