Endometrial dendritic cell populations during the normal menstrual cycle.

BACKGROUND: Dendritic cells (DCs) are specialized antigen presenting cells that are highly involved in the stimulation and modulation of the immune response within mucosal surfaces, including the female reproductive tract. DCs have been poorly characterized in the non-pregnant endometrium. METHODS: Hysterectomy specimens were obtained from premenopausal women (n = 49) with histologically normal endometrium. Endometrial sections were stained immunohistochemically using antibodies for monoclonal mouse anti-human CD1a and CD83, two markers which are specific for populations of immature and mature DCs, respectively. RESULTS: There was a significantly higher density of endometrial CD1a+ DCs than CD83+ DCs throughout the menstrual cycle (P < 0.001). The density of CD1a+ and CD83+ DCs did not vary between the fundus and isthmus of the uterus. There was a significant increase in the density of CD1a+ DCs, but not CD83+ DCs, in the basal layer of the endometrium through the phases of the menstrual cycle. The density of CD83+ was significantly greater in the basal layer compared with the functional layer during both the proliferative (P = 0.004) and secretory phases (P = 0.001), whereas for CD1a+ DCs, the greater density in the basal layer was only observed in the secretory phase (P < 0.001). CONCLUSIONS: The highly coordinated cyclical changes in DC populations during the normal menstrual cycle reported in this study may be important for local regulatory mechanisms relevant to menstruation and implantation; alterations in this normal profile may contribute to the development of disturbances of function, fertility and even benign gynaecological disease.

A three-dimensional study of endometrial microvessels in women using the contraceptive subdermal levonorgestrel implant system, norplant.

Recent evidence points towards a substantial disturbance of the process of angiogenesis within the endometrium in women who are exposed continuously to low dose contraceptive progestogens. This results in the patchy appearance of abnormally small and abnormally large, thin-walled vessels in the superficial regions of exposed endometrium. Three-dimensional pictures were developed from digitised images of serial tissue sections of endometrium in which microvascular endothelial cells were labelled with antibodies to the endothelial cell surface antigen, CD34 and their basement membranes labelled with anti-Collagen IV antibodies. Microvessels from endometrium exposed to continuous low-dose levonorgestrel from a subdermal implant system (Norplant) displayed considerable variations in size and shape. No spiral arterioles were identified. Some microvessels showed considerable dilatation, distortion and variability in the presence of surrounding basement membrane components. Other endothelial structures included narrow, solid cords of endothelial cells, without basement membranes, which often connected with normal or abnormal vessels containing lumens. Some areas, especially deeper in the tissue, contained microvessels of normal size and shape surrounded by basement membrane. These images have revealed an overall picture of great variability in superficial endometrial vascular structures in some women using a low-dose levonorgestrel implant system which appears substantially different from that seen in normal endometrium.

Whole-mount sections displaying microvascular and glandular structures in human uterus using multiphoton excitation microscopy.

There has been considerable interest over many years in the precise structural relationships between microvessels and secretory glands in human endometrium. However, microcirculatory networks have rarely been studied in three-dimensions (3D) using modern computerised technologies, this has been partly due to the late arrival of suitable endothelial cell markers. This study was designed to develop a technique to visualize and to reveal the relationships between microvessels, their glandular environment and epithelial boundaries in 3D, using endometrium from human hysterectomy biopsies. Specimens were carefully selected from women with conditions unlikely to affect the microvascular networks. Monoclonal antibodies (mouse anti-human CD 34 and goat anti-mouse fluorescein (FITC)) were used to visualize the microvessels, and polyclonal antibodies (rabbit anti-human keratin and goat anti-rabbit tetramethylrhodamine (TRITC)) were used to visualize the glandular structures. The samples were studied with a Leica multiphoton system using a titanium-sapphire laser (excitation 800 nm with pulses in the 200 fs range) to obtain a stack of two-dimensional (2D) images to a minimal focus depth of 120 microm. The initial data sets acquired were volume rendered using the integrated software of the Leica system to produce 3D images. This software allowed for the acquisition of data sets from the microscope and for an observational morphological assessment to be made, but was limited in preparing the data for any quantitative analysis. The additional use of ImarisBasic 3.1 visualization software allowed for an observational morphological assessment but also included numerous tools for data manipulation.

Computer-generated, three-dimensional reconstruction of histological parallel serial sections displaying microvascular and glandular structures in human endometrium.

This paper describes a technique to develop high-resolution three-dimensional (3D) images of microvasculature structures in curettage, hysterectomy or endometrial resection biopsies using parallel histological serial sections. Employing a labelled streptavidin-biotin-alkaline phosphatase (LSAB(+)) method and visualising by using DAB(+) with the primary antibody, mouse anti human Q-Bend-10, the images were directly digitised from a light microscope into the KS400 Universal Image Processing and Analysis software via a CCD colour camera; binary images of the structures were created and the binary images were exported into VoxBlast 3D rendering software to view still and rotating 3D images on a computer monitor. This in turn enabled hard copies of the full sequence to be printed.

The measurement of endometrial perfusion in norplant users: a pilot study.

The use of progestogens without oestrogen is commonly associated with irregular menstrual bleeding. Oestrogens and progestogens are both thought to influence endometrial perfusion; changes in endometrial perfusion may contribute to vascular fragility and breakdown. In this study, endometrial perfusion was measured using laser-Doppler fluxmetry in women in the secretory phase of the menstrual cycle before and 4-6 weeks after insertion of the low-dose long-acting levonorgestrel contraceptive implant system, Norplant. Endometrial perfusion was also measured in women exposed to Norplant for up to 19 months. There was no significant difference between endometrial perfusion in control cycles (27.2 flux units +/- 5.5, SEM) and at 4-6 weeks after Norplant insertion (16.3 flux units +/- 5.0), a time when irregular bleeding and spotting are common. Endometrial perfusion was no different from controls after longer periods of Norplant exposure (35.7 flux units +/- 7.2). No direct relationships between endometrial perfusion and plasma concentrations of ovarian steroid hormones were demonstrated. Short-term endometrial vasomotion was largely abolished during Norplant exposure.

Endometrial lysosomal enzyme activity in ovulatory dysfunctional uterine bleeding, IUCD users and post-partum women.

The aim of this study was to evaluate the role of lysosomal enzymes in excessively heavy menstruation by comparing women with menorrhagia due to dysfunctional bleeding or intrauterine contraceptive device (IUCD) use with those with normal menstrual periods or with amenorrhoea associated with breastfeeding. This was a prospective cohort investigation of the activity of four endometrial lysosomal enzymes in three contrasting groups: (i) women with ovulatory dysfunctional uterine bleeding and users of intrauterine contraceptive devices; (ii) breastfeeding post-partum women in whom there are long periods of amenorrhoea, particularly in the early months post-partum; and (iii) normal cycling women. It was found that the total activity of lysosomal enzymes, particularly acid phosphatase and N-acetyl-beta-D-glucosaminidase, was markedly elevated (P < 0.001) in IUCD-exposed endometrium, and endometrium from women with dysfunctional uterine bleeding when compared with endometrium from women with a history of entirely normal menstrual periods or that in post-partum breastfeeding women. The activity of alpha-L-fucosidase was moderately elevated in IUCD users (P < 0.05) and ovulatory dysfunctional uterine bleeding (P < 0.05), whereas alphaD-mannosidase activity was elevated in ovulatory dysfunctional uterine bleeding (P < 0.05), but decreased in IUCD users (P < 0.01). No significant differences were observed in the lysosomal enzyme activities of breastfeeding post-partum women and normal cycling women. These results show that total endometrial tissue activity of four lysosomal enzymes was substantially increased throughout the cycle in most circumstances in women with two different causes for increased menstrual bleeding. This suggests a contributory role to the increased bleeding.

Culturing endothelial cells of microvascular origin.

There is considerable divergence of opinion on the best methods for the isolation and in vitro culture of microvascular endothelium. Reports have either only described the isolation without mentioning culture conditions or have not fully defined the cell population being cultured. Even at the close of the 20th Century, the isolation and in vitro culture of endothelial cells of microvasculature origin still proves to be technically difficult and many questions remain. These questions need to be addressed by improvements to current methods of isolation and culture of Endothelial cells. A number of more 'high-tech' approaches to this isolation are being explored currently. Use of a more definitive panel of antibodies for immunocytochemical characterization, should enable a more confident characterization of the endothelial cell preparations cultured in vitro. Cell adhesion molecules such as ELAM and VCAM can be used to assist in determining cell population purity.

Changes in vascular basement membrane in the endometrium of Norplant users.

Progestogen-only contraception is almost invariably associated with changes in menstrual bleeding patterns. Changes in the endometrial vasculature, and in particular an increase in vascular fragility, may contribute to this bleeding. In this study, endometrial vascular density and endothelial cell basement membrane components were examined using immunohistochemistry before and after insertion of Norplant. Endometrial vascular density was increased from a mean (+/- SEM) of 189.6 +/- 7.0 vessels/mm2 during the control cycle to 253.9 +/- 80.7 vessels/mm2 at 2-13 weeks of Norplant exposure, and to 212.7 +/- 12.9 vessels/mm2 at 14-42 weeks. During the control cycle, a mean of 161.4 +/- 4.5 vessels/mm2 stained for collagen IV (85% of all vessels), while at 2-13 weeks, 144.5 +/- 13.0 vessels/mm2 stained for collagen IV (57% of all vessels) (t ratio = 2.08, P = 0.0057). By 14-42 weeks, 71% of vessels (151.0 +/- 9.8) vessels/mm2 were surrounded by collagen IV. This was not significantly different from control values (t ratio = 2.03). Endometrial vascular laminin was also reduced following Norplant insertion, from a mean of 176.0 +/- 4.2 vessels/mm2 in the control cycle (93% of vessels), to 156.3 +/- 6.7 vessels/mm2 at 2-13 weeks of exposure (57% of vessels) (t ratio = 2.08, P = 0.01). By 14-42 weeks of exposure to Norplant, 162.5 +/- 9 vessels/mm2 (76%) stained for laminin. This was not significantly different from control values (t ratio = 2.04). Endometrial vascular heparan sulphate proteoglycan (HSPG) was reduced from 58.6 +/- 3.0 vessels/mm2 during the control cycle (31% of vessels) to 43.6 +/- 5.6 vessels/mm2 (only 17% of vessels) at 2-13 weeks (t ratio = 2.08, P = 0.025). At 14-42 weeks, only 19% of vessels stained for HSPG (41.3 +/- 5.8 vessels/mm2; t ratio = 2.04, P = 0.009).

Endometrial lysosomal enzyme activity in normal cycling endometrium.

The objective of this study was to evaluate the possible role of four lysosomal enzymes in endometrial function and remodelling during the normal menstrual cycle by fluorimetric measurement (acid phosphatase, N-acetyl-beta-D-glucosaminidase, alpha-L-fucosidase and alpha-D-mannosidase). A prospective study was conducted of 45 endometrial biopsies obtained from women with normal menstrual cycles. Activity of all four enzymes was identified in human endometrium. Activity of acid phosphatase and N-acetyl-beta-D-glucosaminidase was relatively high, whilst that of alpha-L-fucosidase and alpha-D-mannosidase was low. There was no significant change in the activity of any of the four enzymes from the proliferative to the secretory phase of the cycle. This study suggests that the activity of these enzymes remains constant throughout a major portion of the normal cycle.

Mechanisms underlying spinal motor neuron excitability during the cutaneous silent period in humans.

The transient suppression of muscle contraction during the cutaneous silent period (CSP) could be produced either through postsynaptic inhibition of motoneurons or through presynaptic inhibition of the excitatory inputs to motoneurons that sustain voluntary contraction. We sought to delineate the mechanisms underlying the CSP in hand muscles by measuring changes in H-reflexes and motor-evoked potentials (MEPs) produced by transcranial magnetic stimulation (TMS) during the CSP in 10 healthy volunteers. H-reflexes and MEPs both measure the excitability of the motoneuron pool and activate similar subpopulations of motoneurons through different pathways. Inhibition of H-reflexes and MEPs of similar size was maximal at the midpoint of the CSP and gradually returned to baseline. The similar time course of recovery suggests that the H-reflex and MEP are affected by inhibition at a common site, most likely postsynaptic inhibition of the motoneurons.

Effects of high dose progestogens on white cells and necrosis in human endometrium.

Endometrium was collected from 20 high-dose progestogen-treated patients and examined for leukocyte populations by immunohistochemistry and phloxine-tartrazine staining. A labelled streptavidin-biotin-alkaline phosphatase technique was used with antibodies against leukocyte common antigen (LCA), T cells (CD3), neutrophils (NP57), macrophages] (CD68, KP1) and B cells (CD20). The numbers of LCA (1070 +/- 117/mm2), CD3 (459 +/- 60/mm2), CD68 (129 +/- 21/mm2) positive cells and endometrial granulated lymphocytes (EGL) (236 +/- 41/mm2) were significantly higher than those in the control group (P < 0.001). Of these, EGL increased most (6.7 times). NP57 positive (NP57+) neutrophils were present in five out of 20 progestogen-treated samples and NP57 negative (NP57-) neutrophils in another six out of 20; while a neutrophil was only identified in one control tissue (P = 0.002). Three progestogen-exposed endometrial samples had either focal or extensive necrosis, and many NP57+ and NP57- neutrophils were present in the necrotic areas. EGL, neutrophils and macrophages are known to release a number of cytolytic and cell toxic molecules which may play a role in the initiation or acceleration of progestational endometrial necrosis.

[A case of watermelon stomach]. / Su un caso di "Watermelon Stomach".

The authors describe a case of antral Watermelon Stomach (WS) in a seventy-eight year old woman with severe iron-deficient chronic anemia, liver cirrhosis and diabetes mellitus. Endoscopy was diagnostic on the 3rd-4th examination because of the disease's rarity and the concomitance of systemic pathologies, such as portal hypertension, in which often a congestive gastropathy with similar aspects is associated. Whether out of clinical evolution, or capillary thrombosis, or vertical fibromuscular hyperplasia of the lamina propria were considered distinctive elements. By means of literature review it wasn't possible to establish the portal hypertension's prevalence out of the WS cases, but it could be a chance factor. In this way some polycentric prospective trials could be useful. The endoscopic practice is important not only for diagnosis but also for therapeutical means, even if in our case surgery was the chosen treatment.

Neuropsychological, psychiatric, and physical manifestations in 149 members from 18 fragile X families.

One hundred forty-nine subjects from 18 families with fragile X [fra(X)] syndrome were evaluated for their neuropsychological, psychiatric, and physical characteristics. The 36 fra(X) males had intelligence quotients ranging from less than 20 to 61, which prevented the delineation of a reliable neuropsychological profile. Behaviour fitted DSM-III-R and ADI diagnostic criteria of autism in only 2 subjects, both with very low intelligence level (IQ less than 20). Of 36 heterozygotes (HZ), 22 had an IQ between 20 and 80 and 14 between 81 and 99. The neuropsychological profile of the latter was compared with IQ-age-environment-matched 14 normal females and 14 normal males. Significantly poorer results in HZ were found on immediate digit memory and on Raven's progressive matrices (a visuo-spatial test of logical capabilities). The latter result, in conjunction with those results on the Bender visual-motor gestalt test and on some WAIS subtests, suggests a frequent deficit in spatial capabilities in such subjects. Such results tended to be confirmed by the profiles of the 22 HZ with IQ 20-80. No psychiatric abnormalities were found in HZ, except in one subject with IQ less than 20 which fitted DSM-III-R and ADI criteria for autism. Typical physical manifestations, especially cranio-facial, were more frequently present in the HZ group with lower IQ. Subnormal IQ was probably the most reliable abnormality for the detection of HZ in 49 females at 50% and 25% risk of heterozygosity.