Quantification of spiral artery remodelling using an Adobe Photoshop-based technique.

Uterine spiral arteries undergo remodelling in normal pregnancy, with replacement of the musculoelastic arterial media by fibrinoid containing extravillous trophoblast cells. Deficient spiral artery remodelling is associated with several adverse pregnancy outcomes. Although there are distinct components of spiral artery remodelling, assessment is subjective and often based on an overall impression of morphology. We aimed to develop a quantitative approach for assessment of uterine spiral artery remodelling. Placental bed biopsies were immunostained using smooth muscle markers, digital images of spiral arteries were captured and Adobe Photoshop was used to analyse positive immunostaining. The method was then used to investigate variation in the same vessel at different levels within a paraffin block, and the effect of parity, pre-eclampsia or miscarriage on vascular smooth muscle cell content. Results were also compared with a more subjective morphology-based assessment system. There was good intra- and interobserver agreement and the method correlated well with the more subjective assessment system. There was an overall reduction in vascular smooth muscle, as detected by caldesmon 1 (h-caldesmon) immunopositivity, with increasing gestational age from 8 weeks to term. A previous pregnancy did not affect the amount of spiral artery smooth muscle. Comparison of pre-eclampsia and late miscarriage samples with controls of the appropriate gestational age demonstrated increased medial smooth muscle in pathological samples. This technique provides a simple, rapid, reproducible and inexpensive approach to quantitative assessment of spiral artery remodelling in normal and pathological human pregnancy, a process which although fundamental for successful pregnancy, is still incompletely understood.

Role of interleukin 8 in uterine natural killer cell regulation of extravillous trophoblast cell invasion.

BACKGROUND: Extravillous trophoblast cell (EVT) invasion of maternal tissues is critical for successful pregnancy. Decidual factors, including uterine natural killer (uNK) and T cell derived cytokines play a role in regulating this process. Interleukin (IL) 8 has been implicated as a regulator of EVT invasion. HYPOTHESIS: uNK cell stimulation of EVT invasion is associated with IL-8 levels. METHODS: CD8+, total decidual and CD56(+) uNK cells (8-10 and 12-14 weeks gestational age) were cultured. IL-8 mRNA and protein levels were determined. IL-8 receptors (IL-8RA and IL-8RB) were localised in first trimester placental bed biopsies. The effect of IL-8 +/- IL-8 neutralising antibodies and CD8+ T cell or uNK cell supernatants +/- IL-8 neutralising antibodies on EVT invasion was assessed. EVT secreted levels of MMP-2, MMP-9, uPA, PAI-1 and PAI-2 were assessed by substrate zymography or Western Blot. RESULTS: High levels of IL-8 protein and mRNA were detected in all samples. IL-8RA and IL-8RB were expressed by EVT. Exogenous IL-8 stimulated EVT invasion in a paracrine manner. uNK cell supernatants, but not CD8+ cell supernatants, stimulated EVT invasion. IL-8 neutralising antibody partially abrogated this uNK cell stimulated invasion. IL-8 increased levels of secreted MMP-2, but did not alter any of the other proteases or protease inhibitors tested. CONCLUSION: uNK cell stimulation of EVT invasion is partially mediated by IL-8. Unstimulated CD8+ T cells do not alter EVT invasion despite secreting similar levels of IL-8 as uNK cells.

Secretion of angiogenic growth factors by villous cytotrophoblast and extravillous trophoblast in early human pregnancy.

Angiogenesis is a key feature of placental and uterine development in early pregnancy. We hypothesized that trophoblast cells produce angiogenic growth factors, and that expression differs between villous cytotrophoblast (CTB) and extravillous trophoblast (EVT) cells. Fourteen angiogenic growth factors were measured by multiplex growth factor analysis or ELISA in tissue culture supernatants from EVT and CTB from pregnancies at 8-10 and 12-14 weeks' gestation. Gestational age and cell type differences were observed. EVT and CTB are major producers of angiogenic growth factors that likely contribute to placental vascular development and spiral artery remodeling.

Decidual leucocyte populations in early to late gestation normal human pregnancy.

Most research on human decidual leucocytes to date has focused on the predominant CD56+ uterine natural killer (uNK) cell population in early pregnancy. Few reports have documented decidual leucocyte populations after 13 weeks gestation and in late pregnancy. Placental bed (decidua basalis) and non-placental bed (decidua parietalis) biopsies from normal pregnancies were taken from women undergoing termination of pregnancy in the 1st and 2nd trimesters and following Caesarean section in the 3rd trimester. Immunohistochemistry was used to quantify the numbers of decidual cells expressing CD56, CD3, CD8, CD94, NKG2A and CD14 and double labelled CD161+CD3+ NKT-like cells. Although a significant reduction in CD56+ uNK cells was found in 3rd trimester samples compared with 1st and 2nd trimester decidua, a substantial residual CD56+ leucocyte population was identified in 3rd trimester decidua. Expression of the KIR CD94/NKG2A mirrored that of CD56 at all gestational ages, providing an explanation for the absence of cytotoxic responses at the fetal-maternal interface. There was no difference in leucocyte populations between decidua basalis and decidua parietalis. Double immunohistochemical labelling revealed small numbers of decidual CD3+CD56+ and CD8+CD56+ cells, which decreased in number at term, and CD161+CD3+ cells, which increased in number at term. No differences in leucocyte populations were detected between decidua parietalis and decidua basalis. In contrast to previous reports, a substantial residual CD56+ cell population was demonstrated in 3rd trimester decidua. Decidual cytotoxic T-lymphocytes did not alter in number during gestation, while in contrast CD14+ macrophages decreased at term, representing the smallest decidual population assessed.

The urokinase plasminogen activator (uPA) system in uterine natural killer cells in the placental bed during early pregnancy.

The urokinase plasminogen activator (uPA) system plays pivotal roles in cell invasion, adhesion and migration. Roles for uterine natural killer (uNK) cells in regulating extravillous trophoblast (EVT) invasion and spiral artery remodeling have been proposed. Placental bed biopsies from early pregnancy were obtained from three gestational age groups (8-10, 12-14 and 15-20 weeks). Total caseinase activity in the placental bed was studied using casein in situ zymography. Localisation of uPA, uPA receptor (uPAR), plasminogen activator inhibitor (PAI)-1 and -2 in the placental bed was investigated by immunohistochemistry. CD56(+) uNK cells were separated from collagenase-digested decidual cells using an immunomagnetic technique, and uPA activity was measured in isolated cell culture supernatants by casein/plasminogen gel zymography (8-10 and 12-14 weeks' gestation, n=10 each group). uPAR in cell lysates and PAI-1 and -2 secretion in supernatants were measured by Western blotting. Caseinase activity was stronger in decidua than myometrium as shown by in situ zymography. uPA localised strongly to uNK cells, especially at 8-10 weeks. Moderate uPAR localisation on uNK cells also observed. There was very weak immunostaining of uNK cells for PAI-1 and PAI-2. In casein gel zymography, uPA activity was similar in uNK cell culture supernatant compared with total unseparated decidual cells. uPAR in uNK cell lysates was significantly stronger than in total decidual cell lysates. PAI-1 and PAI-2 were not detected in uNK cell culture supernatants by Western blot analysis. These results suggest that uNK cells may regulate EVT invasion and spiral artery remodeling via the uPA system.

Effect of low oxygen concentrations on trophoblast-like cell line invasion.

The applicability of trophoblast-like cell lines to the study of trophoblast function has been widely debated. The present study investigated the effect of oxygen on the invasiveness, apoptosis, proliferation and secreted proteases of four different trophoblast cell lines; HTR-8/SVneo, SGHPL-4, JEG3 and JAR. All experiments were performed at 20% and 3% oxygen for 24, 48 and 72h. Immunostaining for integrins alpha1, alpha6 and beta3, cytokeratin 7 and HLA-G was used to determine the phenotype of the different cell lines. Invasion was assessed using the Matrigel invasion assay. Immunostaining for M30 and Ki67 determined levels of apoptosis and proliferation, respectively. Gelatin and casein/plasminogen zymography were performed on conditioned media to determine levels of secreted matrix metalloproteinase (MMP) 2 and MMP9 and urokinase plasminogen activator (uPA), respectively. None of the cell lines immunostained for all markers normally expressed by extravillous trophoblast cells. Invasiveness of HTR-8/SVneo and JEG3 cells cultured in 3% oxygen was increased after 24h but was inhibited by 72h in culture. Invasion of SGHPL-4 cells was inhibited after culture in 3% oxygen for 24h. Invasion by JAR cells was not affected by changes in oxygen concentration. The different cell lines also displayed different responses to culture period in 3% oxygen with respect to apoptosis, proliferation and secreted proteases. Care should be taken before results obtained using cell lines as a model for EVT are extrapolated to extravillous trophoblast cell behaviour in vivo.

Interleukin-17 expression in the human placenta.

Interleukin (IL)-17 is a proinflammatory cytokine with pleiotropic activities including inducing neovascularization and production of proangiogenic molecules. As pregnancy outcome depends on the balance of Th1-like/Th2-like cytokines and an increased blood supply to the fetoplacental unit, the expression of IL-17 mRNA and protein in human placental tissues was investigated. IL-17 mRNA was expressed by purified cytokeratin-positive term placental trophoblast cells, HLA-G+ extravillous trophoblast cells and placental macrophages (Hofbauer cells). IL-17 localized in both cyto- and syncytiotrophoblasts of normal term pregnancy, spontaneous miscarriage and in molar pregnancy. In spontaneous miscarriage and molar pregnancy extravillous trophoblast cells were consistently immunoreactive for IL-17. IL-17 expression in human placenta may play a key role in angiogenesis and/or immunoregulation in the establishment of pregnancy.

No evidence for apoptosis of decidual leucocytes in normal and molar pregnancy: implications for immune privilege.

Complete hydatidiform moles are totally paternally derived and represent complete allografts that might be expected to provoke maternal immune rejection. Our previous and other studies have shown expression of Fas by increased numbers of activated decidual CD4(+) T cells in both complete and partial molar pregnancy as well as increased FasL(+) expression by molar trophoblasts compared with trophoblasts in normal pregnancies. As the Fas/FasL system represents a major apoptotic pathway that can play a role in immune privilege, the aim of this study was to investigate whether apoptosis of decidual immune cells, particularly T cells, could be responsible for maternal immune tolerance in molar pregnancy. Using terminal deoxynucleotidyl transferase (TdT)-mediated nick end-labelling (TUNEL), a significant increase in TUNEL(+) cells was demonstrated in decidua associated with partial (P = 0.0052) and complete (P = 0.0096) hydatidiform mole compared with normal early pregnancy. Co-labelling immunoperoxidase studies showed that the TUNEL(+) cells in both normal and molar pregnancies were not activated CD45RO(+) immune cells, CD3(+) T cells, CD56(+) uterine natural killer (NK) cells or CD14(+) CD68(+) macrophages. Double immunohistochemical labelling with antiactive caspase-3 and leucocyte markers confirmed the lack of leucocyte apoptosis. Double immunostaining with anticytokeratin to detect trophoblast and M30 CytoDeath, which detects a neoepitope of cytokeratin 18 revealed after caspase-mediated cleavage, revealed apoptotic extravillous trophoblast cells within decidual tissue. We conclude that there is no evidence that apoptosis of decidual leucocytes plays a role in maintaining maternal tolerance in either normal or molar pregnancy.

Interleukin-8 is involved in cervical dilatation but not in prelabour cervical ripening.

Our aim was to determine the amount and source of interleukin (IL)-8 and to study IL-8 receptor expression in the human cervix during pregnancy and after labour. Cervical biopsies were obtained from six non-pregnant women, eight women undergoing pregnancy termination, 17 women undergoing elective caesarean section and 11 women after vaginal delivery. IL-8 levels were compared in women with and without a ripe cervix, as determined by cervical Bishop score and cervicovaginal fetal fibronectin levels. Levels of IL-8 and IL-1beta, a regulator of IL-8 expression, were determined by enzyme-linked immunosorbent assay (ELISA). IL-8, IL-1beta and IL-8 receptor proteins were localized by immunohistochemistry. Compared with late pregnancy, IL-8 levels increased after labour and vaginal delivery (P < 0.01) but there was no correlation with cervical ripening. IL-8 was localized to stromal cells, macrophages and granulocytes. There were no significant differences in IL-1beta levels between groups. IL-8 receptors were expressed primarily on granulocytes and macrophages after vaginal delivery. We conclude that IL-8 is involved in cervical dilatation but not in cervical ripening.

Placental Fas and Fas ligand expression in normal early, term and molar pregnancy.

To clarify the Fas and Fas-ligand status of normal and molar trophoblast, the expression of Fas and FasL by placental trophoblast populations in partial and complete hydatidiform moles was compared with that in normal first trimester and term pregnancies using an avidin-biotin peroxidase technique on frozen and formalin-fixed paraffin-embedded placental tissues with both monoclonal and polyclonal antibodies. The TUNEL technique was used to detect apoptotic cells in the same tissues. The immunoreactivity for Fas and Fas-ligand was comparable with both monoclonal and polyclonal antibodies on frozen as well as paraffin-embedded sections. In normal early and molar pregnancy there was strong FasL expression by villous cytotrophoblast and syncytiotrophoblast. However, there were significant differences in FasL expression by trophoblast subpopulations in both early and term normal pregnancy and between the same trophoblast subpopulation at different gestations, with FasL staining generally being weaker at term. Strong FasL staining by cytotrophoblast cells in the distal parts of cell columns contrasted with unstained cytotrophoblast in the proximal part of columns. Distinct trophoblast subpopulations in partial hydatidiform mole also differentially expressed FasL with reduced FasL expression in proliferating syncytiotrophoblast. In contrast there was no differential FasL expression in complete hydatidiform mole, all trophoblast subpopulations strongly expressing FasL. Unlike the differential expression of FasL there were no differences in Fas expression by trophoblast populations in normal early or term placental tissues. Fas expression was reduced in villous cytotrophoblast at term. Differential expression of Fas by different trophoblast subpopulations was noted in partial and complete hydatidiform mole. In complete mole villous cytotrophoblast and syncytiotrophoblast stained strongly compared with proliferating trophoblast. Using TUNEL labelling apoptosis was rarely detected in placental trophoblast. Differential Fas and FasL expression by trophoblast subpopulations in normal and pathological pregnancy does not appear to be related to apoptosis of trophoblast.

Expression of Fas/Fas ligand by decidual leukocytes in hydatidiform mole.

Complete hydatidiform moles are entirely paternally derived and, therefore, represent a complete intrauterine allograft that might be expected to provoke an altered maternal immune response compared with that of normal pregnancy. Uterine decidua contains a large leukocyte population, of which 10%-20% are T lymphocytes. Fas ligand (FasL) expression by placental trophoblast may induce apoptosis of Fas+ lymphocytes, thereby facilitating immune tolerance and survival of the molar trophoblast. Our previous studies have shown an increase in activated CD4+ decidual T cells in molar pregnancy compared with normal pregnancy. This study was designed to characterize and quantitate Fas/FasL expression by decidual leukocytes in complete and partial hydatidiform mole compared with that in normal early pregnancy using single and double immunohistochemical labeling (i.e., avidin-biotin-peroxidase and avidin-biotin-alkaline phosphatase). A significant increase was found in Fas and FasL expression by decidual CD4+ T cells in complete (Fas+, P = 0.0106; FasL+, P = 0.0081) and partial (Fas+, P = 0.0131; FasL+, P = 0.0051) hydatidiform moles, as was a significant decrease in Fas expression by decidual CD8+ T cells in complete (P = 0.0137) and partial (P = 0.0202) hydatidiform mole compared with normal early pregnancy. The implications of altered Fas/FasL status of decidual T-cell subsets in hydatidiform mole are also discussed.

Abnormal TGFbeta levels in the amniotic fluid of Down syndrome pregnancies.

PROBLEM: The status of cytokines in amniotic fluid (AF) from chromosomally abnormal pregnancy is largely undefined. TGFbeta plays a key role in fetal growth and differentiation and is responsible for the immunoregulatory activity of AF in normal pregnancy, but its status in Down syndrome (DS) pregnancies is unknown. In addition we investigated the IL-2 status of AF from DS pregnancies. METHOD OF STUDY: Midtrimester AF from chromosomally normal (n = 25) and abnormal pregnancies with DS (n = 15) were assayed for bioactive and latent TGFbeta levels using the mink lung epithelial cell growth inhibition bioassay and for IL-2 activity by the CTLL-2 cell proliferation bioassay and by ELISA. RESULTS: Levels of bioactive TGFbeta (mean 4.6+/-0.6 U/mL) were significantly increased in DS AF compared with the normal samples (mean 2.8+/-0.3 U/mL, P<0.003) but latent TGFbeta levels did not differ between DS and normal groups. In addition DS AFs showed reduced IL-2 like bioactivity compared with normal samples (P = 0.0006) but IL-2 immunoactivity was undetectable in DS and normal AFs by ELISA. CONCLUSIONS: DS AFs display increased TGFbeta activity and lacked IL-2 immunoactivity. The reduced ability of DS AFs to stimulate CTLL-2 cell proliferation is unrelated to the IL-2 status of AF. Altered TGFbeta levels may prove useful as an additional biochemical index for the detection of DS pregnancies.

Human amniotic fluid lacks interleukin-2 and interleukin-15 but can interact with the beta-chain of the interleukin-2 receptor.

The present study investigated whether an explanation for the conflicting reports on the interleukin-2 (IL-2) status of amniotic fluid is due to the presence of IL-15 which shares biological activities with IL-2 and utilizes the IL-2 receptor beta-chain. Amniotic fluids from 45 normally progressing pregnancies between 14 and 16 weeks after the last menstrual period were assayed for IL-2 and IL-15 by bioassay and enzyme-linked immunosorbent assay (ELISA). The ability of amniotic fluids to induce cytotoxic T lymphoblastoid line-2 (CTLL-2) cell proliferation was demonstrated to be dependent upon bioassay culture conditions. In serum-free medium each amniotic fluid stimulated CTLL-2 proliferation with a mean level of IL-2-like bioactivity of 14.7 +/- 2.3 ng/ml but amniotic fluids failed to induce CTLL-2 proliferation in serum-supplemented medium. Treatment with neutralizing anti-IL-2 or anti-IL-15 antibodies failed to inhibit amniotic fluid-induced CTLL cell proliferation in serum-free medium, indicating a lack of IL-2 and IL-15 bioactivity. In contrast, treatment with anti-IL-2 receptor beta-chain antibody significantly reduced amniotic fluid-induced proliferation. The lack of IL-2 and IL-15 activity in amniotic fluids was confirmed using ELISA. Although high levels of IL-15 immunoactivity were detected in all samples, specificity controls showed a lack of specific IL-15 immunoactivity in amniotic fluid. Pretreatment of amniotic fluids with 100-500 ng/ml mouse immunoglobulin G abrogated IL-15 immunoactivity, indicating that amniotic fluid contains molecules binding to Fc regions of immunoglobulins and responsible for false ELISA positivity. These studies unequivocally show that amniotic fluid lacks IL-2 and IL-15 but can stimulate CTLL-2 cell proliferation via the IL-2 receptor beta-chain. The absence of IL-2 and IL-15 in normal mid-trimester amniotic fluid suggests that the cytokine profile of human pregnancy appears to be associated with a bias against type 1 cytokines within the feto-placental unit.

Immunoregulatory activity of decidua in spontaneous early pregnancy loss.

The present study aimed to address whether the immunoregulatory properties of the molecules secreted within decidua were altered in women suffering spontaneous miscarriage, compared with apparently normal fertile women. Unfractionated decidual cells from 22 women undergoing therapeutic pregnancy terminations and 25 women experiencing a sporadic spontaneous early pregnancy loss were isolated, cultured for 24 h and 72 h, and supernatants were collected. The effect of decidual supernatants on phytohaemagglutinin (PHA)-induced peripheral blood lymphocyte proliferation was investigated. Immunosuppressive activity was detected in 24 h cell culture supernatants from 91% of therapeutic abortion cases compared with only 64% of spontaneous abortion samples; 72 h supernatants from all of therapeutic abortion samples and 90% of spontaneous abortion cases suppressed lymphoproliferation. The remaining spontaneous abortion samples (36% of 24 h supernatants; 10% of 72 h supernatants) enhanced or had no effect on lymphocyte proliferation. Enhancement of lymphocyte proliferation was not observed in therapeutic abortion samples, and the association between stimulation of cell proliferation and spontaneous abortion was significant for 24 h decidual cell supernatants at 50% concentration (P = 0.02). These findings suggest that in a subgroup of women experiencing spontaneous early pregnancy loss, soluble factors within decidua display altered immune responses that may be implicated in the complex process of fetal rejection.

Up-regulation of natural killer cell cytotoxicity by interleukin-2: the effect of sex and parity.

PROBLEM: Peripheral blood lymphocytes (PBLs) from some, but not all, female donors showed increased cytotoxicity in response to interleukin (IL)-2. METHOD OF STUDY: The effect of IL-2 on natural killer (NK) cell cytotoxicity was compared in nulliparous females, parous females, and males. Peripheral blood lymphocytes were preincubated for 20 or 72 hr with 5 or 100 U/ml IL-2 and cytotoxicity against K562 targets was then examined. RESULTS: In the parous females, only the 72-hr preincubation with 100 U/ml IL-2 significantly increased NK cell cytotoxicity, whereas nulliparous females also showed significantly increased cytotoxicity after a 20-hr preincubation with 100 U/ml IL-2. Neither female subject group had increased activity after preincubation for 20 or 72 hr with 5 U/ml IL-2. However, male peripheral blood lymphocytes also showed a significant increase in NK cell cytotoxicity when preincubated for 72 hr with 5 U/ml IL-2. CONCLUSIONS: The effect of IL-2 on NK cell cytotoxic activity may be related to sex and the state of parity.

Elevated expression of activation molecules by decidual lymphocytes in women suffering spontaneous early pregnancy loss.

The purpose of this study was to investigate, quantify and compare the expression of activation markers by decidual leukocytes in sporadic spontaneous early pregnancy loss and apparently normal first trimester human pregnancy. Decidua was obtained from 18 therapeutic abortions and 20 sporadic spontaneous abortions at 8-12 weeks gestational age. Cryostat sections were labelled by the avidin-biotin complex-peroxidase method using monoclonal antibodies specific for CD45, CD56, CD3, human leukocyte antigen (HLA) DR, CD69, CD25 and very late antigen (VLA)1. Positive cells were quantified and the results were analysed using the Mann-Whitney statistical test. Significantly increased numbers of CD69-positive and CD25-positive cells were detected in spontaneous abortion decidua, when compared with therapeutic abortion decidua. Approximately 50% of women experiencing spontaneous miscarriage also contained significantly elevated numbers of HLA DR-positive cells within decidua. Double immunohistochemical labelling studies demonstrated that the CD25-positive and CD69-positive cells in spontaneous abortion decidua were CD3-positive T cells rather than CD56-positive granulated lymphocytes. Immunological dysfunction within endometrium may account for a proportion of sporadic spontaneous abortions.

Phenotypic analysis and proliferative responses of human endometrial granulated lymphocytes during the menstrual cycle.

The in vivo function of the unusual population of CD56+ CD16- endometrial granulated lymphocytes (eGLs) in human endometrium is unknown; their increased numbers in the secretory phase of the menstrual cycle suggests that they may play a role in the immunobiology of nonpregnant endometrium. In the present study, the phenotype and proliferative responses of eGLs at various phases of the menstrual cycle were compared with those in early pregnancy. Endometrial GLs were highly purified (> 98% CD56+) using immunomagnetic separation, and the expression of cell surface antigens was examined in smears using a double immunohistochemical labeling technique. Proliferative responses to mitogens and interleukin 2 (IL-2) were assessed in hanging drops in 60-well Terasaki plates. There was low to no expression of CD3, CD8, CD16, HML-1, L-selectin, and CD25 (IL-2 receptor alpha) on CD56+ cells isolated from nonpregnant and pregnant endometrium. The expression of CD2, CD49a, and CD122 (IL-2 receptor beta, IL-2Rbeta), however, increased from the proliferative to the late secretory phase of the menstrual cycle. In contrast, CD11a, CD69, and CD49d expression was high and did not vary with menstrual cycle phase; CD49d levels were significantly reduced in early pregnancy. Unlike early-pregnancy eGLs, none of the CD56+ eGL cultures throughout the menstrual cycle displayed phytohaemagglutinin (PHA)-induced lymphoproliferation. In contrast, eGLs from nonpregnant endometrium in the presence of 5 or 100 U/ml IL-2 after 48- and 120-h incubation showed significant proliferative responses, as did eGL cultures from early pregnancy. A significantly reduced number of proliferative phase eGL cultures proliferated in response to IL-2 compared to secretory phase and early-pregnancy eGL cultures. The IL-2-induced proliferative responses of CD56+ eGLs were associated with increased IL-2Rbeta (CD122) expression. These findings demonstrate 1) differential eGL expression of CD2, CD49a, and CD122 during the menstrual cycle; 2) differential IL-2-induced eGL proliferative responses during the menstrual cycle; and 3) differences between eGLs from nonpregnant and pregnant endometrium in CD49d expression and their ability to respond to PHA.

Decidual T lymphocyte activation in hydatidiform mole.

AIM: To quantify and compare decidual leucocyte subpopulations in complete and partial hydatidiform molar pregnancy with those in normal early pregnancy. METHODS: Decidual leucocytes were characterised using an avidin-biotin technique and a panel of monoclonal antibodies in formalin fixed, paraffin embedded decidual tissues from 10 normal first trimester pregnancy terminations and from 13 partial and 13 complete hydatidiform moles. Immunostained cells were fully quantitated and differences between subject groups were analysed using the Mann-Whitney test. T lymphocyte populations were further characterised using double immunohistochemical labelling. RESULTS: The numbers and percentages of CD3+ and CD4+ T cells were significantly increased in complete hydatidiform moles compared with partial moles and normal early pregnancy decidua. No differences were found in the number or percentage of CD8+ T cells. The CD8+ to CD4+ T cell ratio decreased significantly in complete mole compared with partial mole and normal decidua. The numbers and percentages of CD45RO+ cells increased significantly in both partial and complete hydatidiform mole compared with normal first trimester decidua. Double labelling confirmed that 50% of CD3+ T cells in complete and partial molar pregnancy coexpressed CD45RO, compared with 30% in normal pregnancy. Other leucocyte populations (eGLs, macrophages, B cells, and classical natural killer cells) did not differ between complete and partial mole and normal pregnancy decidua. CONCLUSIONS: The presence of an increased population of activated decidual CD45RO+ T cells in complete and partial molar pregnancy suggests altered maternal immune responses against molar trophoblast.

Intraepithelial leukocytes in endometriosis and adenomyosis: comparison of eutopic and ectopic endometrium with normal endometrium.

Intraepithelial leukocytes (IEL) are recognized as an important component of most mucosal surfaces but have received scant attention in the human female reproductive tract. The aim of the present study was to characterize, quantify and compare IEL populations in normal endometrium (n = 30) and in eutopic and ectopic (endometriotic or adenomyotic lesions) endometrium from women with endometriosis (n = 30) or adenomyosis (n = 15) at different menstrual cycle phases in order to assess the role of IEL in these common but poorly understood disorders. IEL populations were examined in formalin-fixed, paraffin-embedded sections using a streptavidin-biotin-peroxidase complex technique and quantified in relation to epithelial cell numbers. IEL in control endometrium and eutopic endometrium in endometriosis and adenomyosis varied during the menstrual cycle, with CD45+, CD43+ and CD56+ cells increasing from the proliferative to the late secretory phase. IEL were elevated in surface compared with glandular epithelium in the proliferative and early secretory phases. Throughout the menstrual cycle there were no significant differences in IEL between eutopic and ectopic endometrium in adenomyosis. Endometriotic foci, however, contained elevated levels of CD45+, CD3+ and CD8+ cells and reduced numbers of CD56 + cells compared with the corresponding eutopic endometrium and these did not vary with menstrual cycle phase. In contrast, ectopic endometrium in adenomyosis showed some cyclical changes with CD56+ cells increasing significantly in the late secretory phase. It is possible these differences may play a role in the pathogenesis of endometriosis and the associated complications.

Apoptosis, bcl-2 expression, and proliferative activity in human endometrial stroma and endometrial granulated lymphocytes.

Human endometrial leukocytes undergo regular cyclical changes during the menstrual cycle, with a striking increase in the phenotypically unusual population of CD56+ CD16- endometrial granulated lymphocytes (eGLs) in the late secretory phase and early pregnancy. The factors that regulate this increase in eGL numbers are unclear; their unusual morphology, however, has led to the suggestion that they undergo apoptosis at the end of the menstrual cycle. Apoptosis, bcl-2 expression, and proliferative activity were examined in the stroma of normal cycling, progesterone-treated, and early-pregnancy endometrium. The expression of bcl-2 and the Ki67 proliferation marker by highly purified (> 98% CD56+) eGLs from endometrium during the menstrual cycle and from first-trimester decidua was also studied. Apoptotic cells were rarely observed in the endometrial stroma of any of the samples examined. Stromal bcl-2 expression, however, increased from the proliferative to the premenstrual phase, and double immunohistochemical labeling demonstrated large numbers of bcl-2+ CD56+ eGLs. In contrast, Ki67 expression was high in the endometrial stroma during the proliferative phase, fell during the secretory phase, and rose again premenstrually, because of expression by eGLs. Isolated CD56+ eGLs also showed high bcl-2 and Ki67 expression at the end of the menstrual cycle. Unlike premenstrual endometrium, progesterone-treated endometrium and first-trimester decidua contained few proliferating cells, expressed high levels of bcl-2, and showed no evidence of apoptosis. Thus, eGLs do not undergo apoptosis in premenstrual endometrium, and their regulatory mechanisms remain to be clarified.