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The primary objectives were to describe the rate of repeat surgery for stress urinary incontinence (SUI) after release of an obstructing synthetic mid-urethral sling (MUS) and to evaluate the effect of time from sling placement to sling release on repeat surgery rates. The secondary objective was to assess rates of recurrent and persistent SUI.This is a retrospective review of women who underwent synthetic MUS release (incision or excision of segment of sling) from 2005-2013. SUI rates and repeat surgery were ascertained by patient report and urinary distress inventory (UDI-6) questionnaire responses. Data analysis included descriptive statistics and multivariable logistic regression analysis.107 patients were included. Median time to sling release was 22 months (IQR 5-49 months). 43.2% were transobturator slings. 15/107 patients (14%) underwent repeat surgery for SUI. On multivariable analysis, women were significantly less likely to undergo repeat surgery for SUI when sling release was performed24 months after initial sling surgery (OR 0.12, 95% CI 0.02-0.85, P = 0.03) compared to release within 3 months. 49% and 77% reported recurrent and persistent SUI after sling release, of which 83% were significantly bothered.The repeat surgery rate for SUI was 14%. The rate of recurrent SUI was 49%. Most of these women were significantly bothered. On multivariate analysis, longer interval to sling release was associated with decreased likelihood of repeat surgery for SUI. The reason for a low repeat surgery rate for SUI despite a high rate of bothersome SUI should be explored in future studies. Neurourol. Urodynam. 36:349-353, 2017. © 2015 Wiley Periodicals, Inc.

Functional PRL receptors are expressed in the human endometrium during the secretory phase of the menstrual cycle in which PRL stimulates tyrosine phosphorylation of Janus kinase 2 and STAT (signal transducer and activator of transcription) 1 and 5. In this study, we investigated the effect of PRL on the MAPK/ERK pathway in the human endometrium. Human endometrial tissue was collected during the mid to late secretory phase of the menstrual cycle. Western blot analysis performed on proteins, extracted after up to 30 min culture with PRL, demonstrated rapid tyrosine and threonine phosphorylation of ERK 1 and 2 MAPKs. The phosphorylation of ERK, in response to PRL, was localized by immunohistochemistry to glandular epithelial cells and a subset of stromal cells. Using immunofluorescence histochemistry, PRL-induced phosphorylation of ERK in the stromal compartment was localized to the uterine-specific CD56(+) natural killer (NK) cells. We have demonstrated that the PRL receptor is expressed in uterine CD56(+) NK cells in situ by immunofluorescence and in purified decidual CD56(+) NK cells by RT-PCR and Western blotting analysis. We have further demonstrated phosphorylation of ERK 1 and 2 in cultures of purified uterine CD56(+) NK cells, in response to PRL. Our data demonstrate that PRL stimulates the ERK pathway in multiple cellular compartments of the human endometrium and identify uterine CD56(+) NK cells as novel PRL target cells.

Background Chagas disease is a vector-borne parasitic disease of major public health importance. Current prevention efforts are based on triatomine vector control to reduce transmission to humans. Success of vector control interventions depends on their acceptability and value to affected communities. We aimed to identify opportunities for and barriers to improved vector control strategies in the Yucatan peninsula, Mexico. Methodology/principal findings We employed a sequence of qualitative and quantitative research methods to investigate knowledge, attitudes and practices surrounding Chagas disease, triatomines and vector control in three rural communities. Our combined data show that community members are well aware of triatomines and are knowledgeable about their habits. However, most have a limited understanding of the transmission dynamics and clinical manifestations of Chagas disease. While triatomine control is not a priority for community members, they frequently use domestic insecticide products including insecticide spray, mosquito coils and plug-in repellents. Families spend about $32 US per year on these products. Alternative methods such as yard cleaning and window screens are perceived as desirable and potentially more effective. Screens are nonetheless described as unaffordable, in spite of a cost comparable to the average annual spending on insecticide products. Conclusion/Significance Further education campaigns and possibly financing schemes may lead families to redirect their current vector control spending from insecticide products to window screens. Also, synergism with mosquito control efforts should be further explored to motivate community involvement and ensure sustainability of Chagas disease vector control., Author Summary Chagas disease is an important parasitic disease transmitted by triatomine bugs. Current prevention efforts are based on eliminating triatomines from homes to reduce disease transmission to humans. However, the success of these control interventions depends on their acceptability and value to affected communities. We aimed to identify opportunities for and barriers to triatomine control strategies in the Yucatan peninsula, Mexico. We used a sequence of group discussion, interviews, and a survey to investigate the perception and knowledge of communities on Chagas disease and triatomines in three villages from the Yucatan peninsula, Mexico. Inhabitants are rather familiar with triatomine bugs, but do not associate well these bugs with Chagas disease and its clinical manifestations. Mosquito rather than triatomine control is a common preoccupation, and households frequently use insecticide spray, mosquito coils and plug-in repellents, spending about $32 US per year on these products. Alternative methods such as yard cleaning and window screens are perceived as desirable and potentially more effective. Screens are nonetheless described as unaffordable. The promotion of education campaigns and possibly financing schemes could help families to redirect their current spending from insecticide products to window screens. Also, synergism with mosquito control efforts should be further explored to motivate community involvement and ensure sustainability of Chagas disease vector control.

Genomic imprinting is the phenomenon whereby mono-allelic expression of certain genes occurs depending on their parental origin. The observation that imprinting only occurs in placental mammals has led to the suggestion that it may play a role in this form of reproduction. In the present study we have investigated the pattern of expression of the human PEG3 gene in the early to term placenta, as well as the uterus and ovary, using RT-PCR, northern blot and in situ hybridization. A comparison is made with the expression of Peg3 in the mouse by histochemical staining in betageo knock out mice. We have demonstrated high levels of PEG3 in the human placenta and have localized the signal to the layer of villous cytotrophoblast cells. In contrast, the pattern of expression of Peg3 in the mouse placenta is less restricted, the message being present in all trophoblast populations. Thus, expression of PEG3/Peg3 in the human and mouse placenta is not directly comparable. We have also detected PEG3 message in the ovarian stroma. We have sequenced the human PEG3 gene from exon 3 to exon 9. By utilizing a polymorphism detected in exon 9, we have established that only the paternal allele is expressed in human placenta. Human PEG3 is therefore maternally imprinted as in mouse.

During implantation, the uterine decidua is invaded by extravillous trophoblast (EVT) cells whose function is to destroy the walls of the uterine spiral arteries in order to provide an adequate blood flow to the fetus. These EVT cells express an unusual combination of HLA class I molecules-HLA-C, HLA-E and HLA-G. The decidua is infiltrated by a population of natural killer (NK) cells with a distinctive phenotype of CD56(bright)CD16(-)CD3(-). These cells are particularly numerous in the decidua basalis at the implantation site where they come into close contact with invading EVT cells. These NK cells express a variety of receptors (CD94/NKG2, KIR and ILT) which are known to recognize HLA class I molecules. We believe that interaction between these NK cells and EVT cells provides the controlling influence for implantation.

In this paper definitive evidence that the classical class I product, HLA-C, is expressed on the surface of normal trophoblast cells is provided. HLA-C transcripts were sequenced from cDNA isolated from first trimester trophoblast cells obtained by flow cytometric sorting. Both paternal and maternal alleles were transcribed. HLA-C proteins were demonstrated by biochemical analysis and found on the cell surface in association with beta(2)-microglobulin. Upregulation of cell surface HLA-C but not HLA-G expression after interferon (IFN)-gamma treatment was demonstrated by flow cytometric analysis. Immunohistology has confirmed HLA-C is expressed by all extravillous subpopulations in vivo. The question of whether trophoblast HLA-C molecules interact with decidual NK cells expressing killer Ig-like receptors (KIR) has also been addressed. Our results demonstrate that extravillous trophoblast expresses at least two HLA class I molecules, HLA-G and HLA-C on the cell surface.

The transformation of endometrium into decidua is an essential feature of normal implantation and pregnancy. There is a close association with an unusual leukocyte population, uterine natural killer (NK) cells, and onset of decidualization. These uterine NK cells are seen in close contact with stromal cells ultrastructurally and are also seen encircling vessels and glands. The possibility that uterine NK cells in the late secretory phase and in early decidua may be important in initiating and maintaining decidualization is raised. In contrast, the death of uterine NK cells could be an early event in the onset of endometrial breakdown at menstruation. The period between implantation and menstruation (7-14 days after luteinizing hormone surge) is the time when implantation is known to be particularly vulnerable. In this review, the possibility that uterine leukocytes might influence the critical decision that the mid- to late secretory endometrium must make either to decidualize or to undergo menstruation is explored.

The immunological relationship beween the mammalian fetus and its mother during pregnancy has been considered similar to that between a transplanted allograft and its recipient ever since Medawar (1953) first proposed the concept of the ‘fetus as an allograft’ in the early 1950s. Based on this analogy, it has been assumed that implantation of the fetal placenta in the uterus would be controlled similarly by a maternal immune response mediated by T-cells recognizing paternally-derived alloantigens expressed by the placenta. Surprisingly, recent evidence suggests that implantation might involve predominantly a novel allogeneic recognition system based on natural killer cells rather than T-cells (Loke & King, 1995). The cellular and molecular basis of this local immune interaction between the fetal placenta and maternal uterus is now the focus of intense research interest. Since aberrant implantation can cause a variety of clinical problems, including miscarriage, intrauterine growth retardation and pre-eclampsia, an understanding of the immunological mechanism by which this process is controlled could lead to the development of regimens to improve fetal growth and development.

During the luteal phase and the early months of pregnancy, there is a dense mucosal infiltration of CD56+ natural killer (NK) cells. These uterine NK cells have a phenotype (CD56bright, CD16-, mCD3-) which distinguishes them from peripheral blood NK cells (CD56dim, CD16bright, mCD3-). The uterine NK cells are in close association with extravillous trophoblast (EVT) cells which infiltrate into the decidua and maternal spiral arteries. This subpopulation of trophoblast expresses two human leukocyte antigen (HLA) class I molecules, HLA-G and HLA-C. Circulating NK cells express receptors for HLA class I molecules. We have recently found evidence that similar receptors are present on decidual NK cells belonging to both the Killer Inhibitory Receptor (KIR) and CD94 families. The repertoire of NK receptors expressed varies between different women. The findings indicate that decidual NK cells do have receptors for trophoblast HLA class I molecules. Experiments are underway to determine the effects of this interaction on NK cell function.

The establishment of the human placenta in early pregnancy is characterized by the presence of large numbers of natural killer (NK) cells within the maternal decidua in close proximity to the fetally-derived invading extravillous trophoblast which expresses at least two HLA class I molecules, HLA-G and HLA-C. These NK cells have an unusual phenotype, CD56(bright) CD16, distinguishing them from adult peripheral blood NK cells. They may control key events in trophoblast migration and therefore placentation. Human NK cells in peripheral blood express receptors for polymorphic HLA class I molecules. This family of receptors, known as killer cell inhibitory receptors (KIR), are expressed on overlapping subsets of NK cells to give an NK cell repertoire which differs between individuals. Using a panel of monoclonal antibodies to several members of the KIR family and analysis by flow cytometry, we have found that KIR are expressed by decidual NK cells. There is variation in both the percentage of cells expressing a particular receptor and the density of receptor expression between decidual NK cells from different individuals. Comparison of NK cells from decidua and peripheral blood of the same individual showed that NK cells from these two different locations express different repertoires of KIR. Receptors are present in individuals who do not possess the relevant class I ligand, raising the possibility that these NK receptors may be involved in recognition of the allogeneic fetus by the mother at the implantation site.

During the process of implantation in humans, fetal trophoblast cells invade and migrate into the maternal decidua. During this migration, trophoblast cells destroy the wall of the maternal spiral arteries, converting them from muscular vessels into flaccid sinusoidal sacs. This vascular transformation is important to ensure an adequate blood supply to the feto-placental unit. Both cell-cell and cell-matrix interactions are important for trophoblast invasion of the decidual stroma and decidual spiral arteries. Cell-matrix adhesions are mediated by specific receptors, mostly belonging to the family of integrins. Signals transduced to the cells from the matrix via integrins could play a pivotal role in the control of cellular behaviour and gene expression, such as metalloproteinases that facilitate matrix degradation and tissue remodelling. This review focuses on the role of integrins and extracellular matrix in trophoblast cell migration, trophoblast invasion of the decidual spiral arteries and matrix degradation by trophoblast during implantation.

Expression of the oestrogen and progesterone receptors on uterine mucosal leukocytes has been examined by dual immunohistology. Neither the oestrogen receptor nor the progesterone receptor was expressed by lymphocytes, macrophages or the distinctive population of uterine natural killer (NK) cells. Although the accumulation and survival of these NK cells appears to be hormonally dependent, the effects must therefore be indirect.

The maternal leukocytes present in human decidua during the early weeks of pregnancy are composed of CD56+ NK cells (approximately 80%), CD3+ T cells (approximately 10%), and CD14+ macrophages (approximately 10%). These leukocytes are present in large numbers at the implantation site where trophoblast invades. The migrating extravillous trophoblast (EVT) cells express HLA class I antigens HLA-G and HLA-C. To examine whether EVT or other allogeneic cells are capable of inducing a local proliferative response in maternal decidual leukocytes, we have assayed [3H]thymidine incorporation by decidual leukocytes co-cultured with irradiated trophoblast or peripheral blood lymphocytes (PBL). No proliferative response was seen in decidual leukocytes either to trophoblast or to PBL as stimulators. In addition trophoblast did not stimulate a proliferative response in PBL. These findings indicate that extravillous trophoblast is not capable of initiating a classical immune response at the implantation site.

At the time of implantation, the decidua is infiltrated by a unique population of NK cells with large granular lymphocyte morphology, which are thought to influence placental trophoblast invasion and differentiation. The mechanisms used by these cells to migrate within decidua are not known, but in other biological processes such as wound healing and tumor invasion cell-matrix interactions are important. These interactions are mediated by specific receptors, mostly belonging to the family of integrins. Decidual NK cells are observed to bind to type IV collagen and fibronectin, but not to laminin. Adhesion to collagen was inhibited with an anti-alpha 1 integrin subunit mAb, whereas adhesion to fibronectin was blocked with anti-alpha 4, -alpha 5, and -beta 1 integrin subunit mAbs. Binding of decidual NK cells to decidual stromal cells was partially blocked with mAbs to the alpha 4 and alpha 5 integrin subunits. These results provide insight into the possible mechanisms utilized by decidual NK cells for migration and retention within the pregnant uterine mucosa.

During the process of placental implantation, sessile villous trophoblast cells migrate from the villi into the decidua as isolated motile extravillous trophoblast cells. There is differential expression of the epidermal growth factor-receptor (EGF-R) and c-erbB2 proteins on villous and extravillous trophoblast populations. Using monoclonal antibodies to EGF-R and c-erbB2, we have obtained highly purified populations of villous and extravillous trophoblast by fluorescence activated cell sorting. These cells were examined by the reverse transcriptase-polymerase chain reaction (RT-PCR) using nested internal primer pairs for the following cytokines: CSF-1, GM-CSF, TNF-alpha, TGF-beta1, IFN-gamma, IL-2, LIF and also for LIF-receptor. TNF-alpha and TGF-beta 1 were present in all trophoblast populations. GM-CSF and CSF-1 were only found in some samples, with preferential expression of CSF-1 in villous populations. IFN-gamma, IL-2 and LIF mRNA were not found, although all samples contained LIF-receptor mRNA. These cytokines (CSF-1, TGF-beta, TNF-alpha and GM-CSF) are likely to influence trophoblast growth and differentiation in an autocrine manner, since their receptors are also present on trophoblast. These results illustrate a quick and simple method to analyse for the presence of cytokine and other transcripts in trophoblast subpopulations during early pregnancy.

The present in-vitro study using an enzyme-linked immunosorbent assay has identified the cell types responsible for colony stimulating factor-1 (CSF-1) production at the first trimester human placental uterine interface. The major sources were observed to be decidual stromal cells and decidual CD56 + natural killer (NK) cells, but decidual CD3 + T cells did not produce CSF-1, reflecting functional differences between these two decidual lymphoid populations. Of a variety of cytokines tested, only interleukin-2 (IL-2) was found to augment CSF-1 secretion by decidual NK cells. Trophoblast cells also secreted CSF-1, but the amounts were small relative to decidual stromal cells and NK cells. Therefore, most of the CSF-1 present at the implantation site appears to be maternally derived. Co-culture of decidual NK cells on a monolayer of irradiated trophoblast did not augment CSF-1 secretion by decidual NK cells, indicating that the production of this cytokine is not stimulated by contact with fetal trophoblast. CSF-1 was found to increase [ 3 H]thymidine uptake by trophoblast cultured on laminin for 72 h, but no such response was seen in trophoblast cultured on fibronectin, indicating that these extracellular matrix proteins have differential effects on the response of trophoblast to this cytokine.

Trophoblast cells from normal first trimester pregnancies have been shown to express the nonclassical Class I molecule, HLA-G, which is nonpolymorphic and has a heavy chain of 40 kDa. These HLA-G-expressing trophoblast cells infiltrate into maternal decidua, which contains abundant uterine-specific CD56bright natural killer (NK) cells. We believe HLA-G may act as a protective molecule against decidual NK lysis and thus allow trophoblast survival in the maternal tissues. To test this hypothesis, we have constructed HLA-G and HLA-A2 transfectants using LCL 721.221 HLA-null cells. We observed that both of these antigens protected target cells from NK effectors isolated from decidua or peripheral blood, although the effect of HLA-G is not as marked as that of HLA-A2. Our results, therefore, show that in this experimental system expression of a nonclassical Class 1 HLA molecule is also correlated with NK resistance in the same way as a classical Class I HLA molecule.

Research suggests that midurethral sling placement may have a beneficial effect on urgency (U) and urge incontinence (UUI). This study examined short term U/UUI outcomes in patients undergoing TVT-O placement.One hundred patients undergoing TVT-O placement were prospectively assessed using 3 day bladder diary, combined with multiple validated incontinence questionnaires focusing on UUI, U and QoL.At 6 week assessment, improvements in daily pad use (2.6 to 0.9) and incontinence episodes (3.6 to 0.7) were seen (p0.05, both comparisons). Eighty-two patients reported cure of stress incontinence. Focused outcome scores for UUI improved from 2.0 (± 1.1) to 0.8 (± 1.0) (p value0.001), with 39% and 29% of patients reporting score resolution and improvement, respectively. Similarly, mean Urinary Perception Score improved from 10.1 (± 4.4) to 5.8 (± 3.7) (p value0.001) with cure/improvement identified in 75%. Eighty-four patients did not require anticholinergics postoperatively. Interestingly, patients reporting residual SUI but improved UUI demonstrated QoL scores only slightly worse than patients with SUI cure and improved or persistent UUI.TVT-O placement is associated with cure/improvement in a significant proportion of patients. In addition, significant improvement in QoL is seen following TVT-O placement.

Trophoblast cells from normal first trimester pregnancies have been shown to express the nonclassical class I molecule, HLA-G, which is nonpolymorphic and has a heavy chain of 40 kD. In this study, in situ hybridization experiments were performed using a biotinylated HLA-G specific oligonucleotide on sections of normal placenta including the implantation site. HLA-G mRNA was identified in all extravillous trophoblast populations including the cytotrophoblast cell columns, interstitial trophoblast, endovascular trophoblast, and placental bed giant cells. In addition, villous cytotrophoblast and villous mesenchymal cells also contained HLA-G transcripts, but villous syncytiotrophoblast was always negative.

This study has examined the expression of adhesion molecules by uterine-specific CD56bright CD16-CD3- large granular lymphocytes using flow cytometry and immunohistology. Data are compared with those obtained after IL-2 stimulation and with peripheral blood CD56+ cells. We have found that decidual CD56bright cells strongly express three fibronectin receptors, alpha 4 beta 1, alpha 5 beta 1, and alpha 4 beta 7. The HML-1 antigen is also expressed. The beta 2 integrins are also found on decidual CD56+ cells although, apart from CD11c, these are present at levels lower than those of corresponding cells in blood. After IL-2 stimulation there is a considerable increase in CD11a/CD18 expression on CD56bright cells. Both ICAM-1 and NCAM are found on CD56bright cells at higher levels compared to those of peripheral blood CD56+ cells. This pattern of expression of beta 1 and beta 2 integrins and members of the immunoglobulin superfamily must have important implications in the behaviour of decidual LGL. These adhesion molecules are also likely to be important in the recruitment and retention of CD56bright LGL in the decidual microenvironment particularly as the specialised decidual extracellular matrix and uterine LGL appear concomitantly at the time of implantation.

A quantitative immunocytochemical study of large granular lymphocytes (LGLs) in the normal cervix and in human papillomavirus (HPV) associated disease was performed using a panel of monoclonal antibodies which included those for LGL surface markers CD56, CD16, and CD57. Only CD56-positive cells were found within the ectocervical epithelium and these cells increased in number in cervical intraepithelial neoplasia (CIN) in comparison with normal cervix. Examination of serial sections and double labelling suggests that these cells are CD3+, CD8+, CD56+, CD16+. The observed increase in number of this subset was not associated specifically with HPV infection but was related to CIN. Lymphocytes expressing all three LGL markers were found in the stroma and CD16(+)-positive cells clustered around endocervical glands with occasional cells extending into the endocervical epithelium. These results indicate that a small subset of LGLs which express T-cell markers is increased in number in CIN. Cells expressing classical NK markers are restricted to the stroma and are not found within the ectocervical epithelium.

Freshly isolated decidual large granular lymphocytes (LGL) show natural killer (NK) activity against K562 cells but not against normal human trophoblast. We now show that these decidual LGL proliferate in vitro in response to recombinant interleukin-2 (rIL-2) and that these rIL-2-stimulated cells acquire a broad cytolytic potential that is characteristic of lymphokine-activated killer (LAK) cells. Both fetal fibroblasts and JEG-3 choriocarcinoma cells are resistant to lysis by freshly isolated decidual effectors but are readily killed by IL-2-stimulated decidual LGL. The ability to kill these target cells is acquired after only 18 hr exposure to rIL-2. rIL-2-activated decidual LGL also kill cultured normal trophoblast cells but much lower levels of cytolysis were seen even after the effectors had been stimulated with rIL-2 for 4-6 days. The preferential killing of malignant over normal human trophoblast cells raises questions about the potential role of IL-2-activated decidual LGL in the control of unduly invasive or malignant trophoblast populations in vivo.

It will be apparent from this review that the exact role of IFN on human placental development remains largely undefined. However, there is now sufficient data available on the biological functions of this cytokine in other cellular systems to provide a conceptual framework upon which the influence of IFN on the human placenta may be based. We have focused on those areas where, in our opinion, IFN is likely to have an important effect on placental-uterine interactions and have discussed the possible mechanisms involved. We hope that the ideas we have presented will stimulate interest in this aspect of human placental biology.

The main population of lymphocytes found in the human decidua during early pregnancy are NK-like cells with a distinctive phenotype, CD56bright CD16- CD3-. These cells are in close association with invading trophoblast that may be their in vivo target. We have examined three aspects of decidual NK function in vitro: cytotoxicity, proliferation, and cytokine production. The functional assays indicate uterine lymphocytes differ fundamentally from both PBL and even from classical circulating NK cells. Their role in the establishment of normal pregnancy remains unknown.

The remodelling of the maternal uterine spiral arteries during pregnancy, known as physiological change, is critical for the normal growth and development of the fetus. Controversy has surrounded the part played by fetal trophoblast in the transformation of these spiral arteries. To address this debate, a histological and immunochemical comparison of blood vessels from the implantation sites of human pregnancies of early gestation with uterine tissue where trophoblast was absent was performed. Results showed that true physiological change, with the features of medial necrosis and deposition of fibrinoid material, only occurred in the presence of trophoblast. In addition, it was found that subpopulations of trophoblast contribute differently in the process. Interstitial trophoblast-mediated destruction of the arterial media precedes replacement of the endothelial cells by endovascular trophoblast.

At the late secretory phase of the menstrual cycle and in early pregnancy, the uterine mucosa is infiltrated by large numbers of natural killer (NK) cells with a distinctive phenotype (CD56bright CD16- CD3-) and large granular lymphocyte (LGL) morphology. Circulating CD56bright NK cells generally proliferate in the presence of interleukin-2 (IL-2), but it is clear that cofactors besides IL-2 are required for optimal response. In the bone marrow, this co-stimulating signal is provided by stromal cells. In the present study we observe that uterine CD56+ cells from early pregnancy decidua similarly proliferate vigorously when cultured with decidual stromal cells and a suboptimal dose of IL-2. This response is dependent on cell-cell contact, as no proliferation of decidual NK cells was observed when they were separated from stromal cells by a permeable cyclopore membrane. In addition, we have studied the expression of Bcl-2 by decidual CD56+ cells. Our results show that the microenvironment of the uterus is likely to have a significant influence on the proliferation and survival of uterine CD56+ cells.

Although human decidual lymphocytes have been widely studied, their function and possible interaction with trophoblast are still unclear. Here we show that whereas human early (EDL) and term (TDL) decidual lymphocytes were unable to kill human trophoblast by necrosis (assessed by the 51Cr-release assay) or apoptosis (DNA fragment assay), TDL but not EDL decreased trophoblast adhesion to a plastic substrate as determined by a [3H]thymidine assay. This effect, however, was not selective for trophoblast, as TDL also decreased the adhesion to plastic of human decidual stromal cells and HeLa cells. Our results suggest that TDL may play a role in placental detachment during parturition by decreasing trophoblast or decidual stromal cell adhesion.

A monoclonal antibody to HLA-G has been generated by immunizing HLA-A2.1/human beta 2-microglobulin (beta 2 m) double transgenic mice with murine L cells transfected with both human beta 2 m and HLA-G. This monoclonal antibody, designated as G233, has been found not to cross-react with other HLA class I antigens when tested on numerous cell lines by flow cytometry. With immunohistology, all populations of extravillous trophoblast (cell columns, interstitial trophoblast, endovascular trophoblast, placental bed giant cells) were stained. An extensive range of adult and fetal tissues was also tested but none reacted with monoclonal antibody G233, including those previously reported to express HLA-G mRNA, indicating that the protein has a highly restricted distribution. Failure to detect HLA-G in the fetal thymus raises the question as to how T-cell tolerance to this antigen is induced. Immunoprecipitation of trophoblast surface proteins with monoclonal antibody G233 revealed a heavy chain of 39 kDa and a light chain of 12 kDa, indicating that HLA-G expressed on the surface of trophoblast is complexed with beta 2 m. However, sequential immunoprecipitation with monoclonal antibody W6/32 followed by monoclonal antibody G233 continued to detect a residual band of 39 kDa, suggesting that trophoblast surface HLA-G may also occur as free heavy chains not associated with beta 2 m. Immunoprecipitation followed by two dimensional gel electrophoresis showed that monoclonal antibody G233 recognizes several isoforms of HLA-G from trophoblast similar to the characteristic spot array previously described for HLA-G. This monoclonal antibody G233 will be highly useful in future experiments to elucidate the function of HLA-G.

The expression of natural killer (NK) cell receptors specific for HLA class I molecules has been studied in CD56 bright , CD3 − NK cells isolated from the pregnant uterine mucosa, the decidua. RT-PCR was performed on cDNA from uterine NK cells with primers designed to amplify members of the killer inhibitory receptor (KIR)/killer activatory receptor (KAR) gene family. Sequencing of the PCR products revealed that uterine NK cells express KIR/KAR which have two or three extracellular immunoglobulin superfamily (Ig-SF) domains. NK receptors for both groups of HLA-C alleles were found. KIR, characterised by a long cytoplasmic tail containing the immune receptor tyrosine-based inhibitory motif (ITIM), and KAR, characterised by a short cytoplasmic domain with a transmembrane region containing a charged lysine, were both identified. Different individuals appear to have a distinct but overlapping repertoire of KIR/KAR. No new members of this NK receptor gene family were identified in the uterine CD56 bright NK cells. Similar findings were obtained from non-pregnant endometrial tissues representative of different stages of the menstrual cycle. Immunohistology confirmed that the KIR protein products were expressed by decidual NK cells. These results reveal that NK receptors for trophoblast HLA class I molecules are present in maternal uterine NK cells. Fetal trophoblast cells infiltrating the decidua express HLA-G and HLA-C gene products. This suggests that maternal recognition of the fetus may be mediated by an NK allo-recognition system.

There is currently a great deal of interest in the role that cytokines may play in placental development. A variety of cytokines have been demonstrated at the placental-uterine interface, but the exact cellular sources of production have not yet been identified due to the complex tissue topography of the implantation site. In the present study, we have used the enzyme-linked immunosorbent assay (ELISA) to investigate cytokine production in vitro by cells isolated with a high degree of purity from the placenta and decidua. The cytokines assayed were EGF, interleukin 1 beta (IL-1 beta), IL-2, IL-3, interferon alpha (IFN-alpha), IFN-gamma, tumour necrosis factor alpha (TNF-alpha) and transforming growth factor beta 1 (TGF-beta 1). A necessary starting point in the elucidation of cytokine function is to define those cell populations which express relevant cytokine receptors as this would identify the potential cell targets. For this reason, cytokine receptor expression by placental and uterine cells using immunohistology and flow cytometry has been investigated, those being: IGF-1r, PDGF-r alpha/beta, IL-1rII, IL-6r, IL-7r, IFN-gamma r, TNF-rp80 and endoglin. Taken in conjunction with previously published data on other cytokine receptors, a comprehensive picture can begin to be constructed of the putative cytokine-responding cells present in the human placental-uterine interface.

Flow cytometric data were used to compare the phenotype of term decidual lymphocytes and peripheral blood lymphocytes. Unlike peripheral blood lymphocytes, a significant percentage of CD3+, CD4+, CD8+ and CD16+ term decidual lymphocyte populations expressed the CD69 activation marker. The relative proportions of CD38 in CD3+, CD4+ and CD8+ populations were more than twice as large in term decidual lymphocytes as in peripheral blood lymphocytes. As reported for early decidual lymphocytes, the expression of CD38 and CD69 by term decidual lymphocytes suggests that these cells are also regionally activated. However, term decidual lymphocytes showed no spontaneous cytotoxicity against normal trophoblast or its tumoral counterpart, JEG cells. After stimulation with interleukin-2, these lymphocytes became cytotoxic, as did peripheral blood lymphocytes. The relevance of this latter result to the immune control of the physiological and pathological invasion of the decidua by the trophoblast is discussed.

The concept of the fetus as an allograft, which has been much cherished by reproductive immunologists, is in need of re-appraisal. We speculate that the relationship between the allogeneic conceptus and mother is not governed by the laws of classical transplantation immunity. Instead, this relationship is more akin to the allorecognition system seen in invertebrates.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a classical haematopoietic cytokine which has been implicated in placental growth and development. In this study, we investigated the production of GM-CSF in human first trimester pregnancy by the predominant uterine lymphocyte population of decidual CD56+ NK cells (large granular lymphocytes) and the factors that influence this production using enzyme-linked immunosorbent assays (ELISAs) and bioassays, supplemented by immunocytochemistry. We have also investigated and compared production of GM-CSF by human first trimester trophoblast and by JEG-3 and JAR choriocarcinoma cells. Our data show that appreciable amounts of GM-CSF are produced in first trimester maternal decidua and that a significant component of this secretion was from decidual large granular lymphocytes (LGL). Production of GM-CSF by LGL was constitutive and considerably greater than that of freshly isolated peripheral blood leukocytes. GM-CSF secretion by decidual LGL could be enhanced by co-culture on a monolayer of decidual stromal cells, and could also be increased in a dose-dependent manner by stimulation with interleukin-1 (IL-1) or IL-2. IL-4, IL-6, tumour necrosis factor alpha (TNF alpha), transforming growth factor beta (TGF beta), interferon alpha (IFN alpha) and IFN gamma individually had no effect on GM-CSF secretion, although IL-4, TGF beta and IFN alpha all inhibited the action of IL-2. IFN gamma had no effect on the IL-2-induced GM-CSF secretion, but did antagonize the action of IL-1. Normal human first trimester trophoblast was also found to produce GM-CSF, although no production whatsoever was seen by JEG-3 or JAR choriocarcinoma cells. These results suggest that GM-CSF from uterine lymphocytes, and from trophoblast itself, may influence placental growth and development in both a paracrine and an autocrine manner.

Trophoblast invasion of the maternal uterus during human placentation is a highly complex process involving proliferation, migration and cell differentiation. Tyrosine kinase receptors are known to be involved in such functions. The epidermal growth factor-receptor (EGF-R) and c-erbB2 proto-oncogene products are two closely related tyrosine kinase receptors which are expressed in high amounts in human placenta. In this study, we have investigated the expression of EGF-R and c-erbB2 proteins by different trophoblast populations using immunohistochemistry. EGF-R was expressed by proliferative villous cytotrophoblasts, but not by non-proliferative, invasive, extravillous cytotrophoblasts. In contrast, c-erbB2 was expressed by invasive extravillous trophoblasts (EVT) but not by villous cytotrophoblasts. Expression of both receptors was seen only by the terminally differentiated placental bed giant cell and villous syncytiotrophoblast populations. This precise spatial expression suggests that EGF-R plays an important role in trophoblast proliferation whereas c-erbB2 may be important for trophoblast invasion and differentiation. The surface expression of these receptors on normal isolated first trimester trophoblasts and on choriocarcinoma cell lines was also investigated by flow cytometry, and compared with that of the c-erbB2-overexpressing mammary tumour cell line BT474. Isolated trophoblasts were shown not to overexpress c-erbB2, and no differences in either EGF-R or c-erbB2 expression were seen between normal and malignant trophoblasts.

Kit ligand, or stem cell factor, is a recently identified growth factor, which binds to and activates the c-kit proto-oncogene, and which has been shown to act synergistically with other haematopoietic growth factors in the bone marrow. We have previously shown that several isoforms of kit ligand, which arise due to alternative splicing, are expressed in human placenta. In order to elucidate the role of c-kit and its ligand during human placental development we have investigated the expression of c-kit and kit ligand in human first trimester and term placenta as well as in pregnant and non-pregnant endometrium, by immunocytochemistry and flow cytometric analysis. In non-pregnant endometrium no expression of kit ligand was seen. By contrast, in first trimester decidua, kit ligand was strongly expressed by the arterial media of maternal blood vessels. Kit ligand was also expressed throughout pregnancy by invasive fetal extravillous trophoblast, and by fetal fibroblasts within the placental villi. c-kit was found to be expressed on Hofbauer cells within the chorionic villi, and by decidual macrophages at all stages in pregnancy. c-kit was also detected on the small CD56dim subset of uterine large granular lymphocytes which form the major leukocyte population in human first trimester decidua. Our results suggest that kit ligand may be involved in the regulation of fetal macrophages, and in particular in signalling between invading extravillous trophoblast which expresses kit ligand, and maternal leukocytes bearing the c-kit receptor.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a classical haematopoietic cytokine which has also been implicated in placental growth and development. In this study we have performed a detailed immunohistological localization of the low affinity GM-CSF receptor (GM-CSF-Rα) in human first trimester implantation site and non-pregnant endometrium. We have also investigated receptor expression and GM-CSF binding in vitro by normal first trimester trophoblast using flow cytometric analysis and compared this with JEG-3 and JAR choriocarcinoma cells. In the first trimester, the GM-CSF-R was found to be present on villous cytotrophoblast and all populations of extravillous trophoblast. Expression by villous syncytiotrophoblast was weak or absent, but this increased markedly by term. GM-CSF-R were also expressed by fetal Hofbauer cells within the mesenchyme of the chorionic villi and by uterine glandular epithelium and decidual macrophages within maternal decidua. GM-CSF-R was not expressed by glands in proliferative phase endometrium but began to appear during the secretory phase, suggesting hormonal regulation of the receptor on uterine glandular epithelium. Flow cytometric comparison of normal isolated first trimester trophoblast and JEG-3 and JAR choriocarcinoma cells revealed two- to threefold higher surface expression of GM-CSF-R by choriocarcinoma cells and higher binding capacity for rhGM-CSF than normal trophoblast. These results suggest that GM-CSF may regulate growth and development of human trophoblast. GM-CSF may also influence placental development and function by acting via decidual and fetal macrophages, and uterine glandular epithelium, which are the other cell populations to express the receptor.

Human decidual large granular lymphocytes are capable of killing both normal trophoblast and choriocarcinoma cells after stimulation with recombinant interleukin-2 (rIL-2) in vitro. We now show that pre-treatment of normal trophoblast and JEG-3 choriocarcinoma cells with interferon-gamma (IFN-gamma) protects these target cells from killing by decidual effectors, although pre-treatment with interferon-alpha (IFN-alpha) had no such effect. In contrast, JAR choriocarcinoma cells were readily killed before and after exposure to both IFN-gamma and IFN-alpha. This protective effect might be due to upregulation of non-classical trophoblast HLA Class I molecules, as IFN-gamma but not IFN-alpha enhances Class I surface expression in both normal trophoblast and JEG-3 choriocarcinoma cells. JAR cells, however, are constitutively and inductively negative for Class I expression.

The phenotype of human decidual leukocytes, composed predominantly of CD3-CD16(-)-CD56bright cells, was examined after culture with IL-2 by immunofluorescence and flow cytometry. After IL-2 stimulation the phenotype became like that found on classical NK cells, with an increased proportion of cells expressing CD16. The IL-2R alpha was absent before and after IL-2 stimulation. However, the intermediate affinity receptor, IL-2R beta, was expressed by CD56bright decidual cells, but this receptor was downregulated after IL-2 stimulation. IL-2-induced proliferation of CD56+ decidual cells could be blocked using TU27, a monoclonal antibody to the IL-2R beta. These findings indicate activation of decidual leukocytes by IL-2 occurs through the IL-2R beta alone.

This paper describes a new Mab BC-1 which is directed at a cell surface antigen expressed by extravillous cytotrophoblast and not by villous cytotrophoblast, so it is useful for distinguishing between the two populations. The antigen recognised by BC-1 is trypsin-resistant, which allows it to be used for flow cytometric analysis of living trophoblast isolated by enzymic disaggregation. However, BC-1 is not trophoblast-specific but cross-reacts with some other tissues, in particular endothelial cells and peripheral blood monocytes. The nature of this antigen has not been established.

The 'foetal allograft' concept of the maternal-foetal relationship is currently being questioned. This is partly due to the increasing awareness of uterine large granular lymphocytes, a subset of natural killer cells found in the human endometrium at the time of placentation. Analogous cells are present in several species. The possible role of these cells in the regulation of the trophoblast-maternal interface is discussed here by Ashley King and Yung Wai Loke.

This review discusses the three important developments that are likely to have the greatest impact on our understanding of human reproductive immunology: the establishment of the trophoblast major histocompatibility complex class I antigen as HLA-G; the identification of the trophoblast-lymphocyte-crossreactive protein as a complement regulatory protein; and elucidation of the lineage and possible functions of decidual large granular lymphocytes.

In this study, the CD3- LGL/NK cells present in the pregnant human uterus have been characterized. Phenotypic and morphologic analyses of decidual LGL revealed many similarities to the minor CD56bright+, CD16-subset in peripheral blood, but there were some important differences. The relative surface density of CD56+is greatly increased on decidual LGL to 22 x that found on the majority of CD56 peripheral blood NK cells. The CD56bright cells in decidua show LGL morphology, whereas in peripheral blood, they are .mainly agranular. Proliferation of CD56+cells occurs predominantly during the nonpregnant secretory (luteal) phase, indicating these CD56+uterine LGL do not migrate as terminally differentiated cells. The appearance of CD56 cells was examined at the ultrastructural level using immunoelectron microscopy. Cells with phenotypic characteristics of decidual LGL occur in a higher percentage (1.11%) in the peripheral blood of women of reproductive age than in men (0.66%). On the basis of these results, it is proposed that the CD56bright+uterine leukocytes represent a distinctive, hormonally regulated subset possibly adapted to control human placentation.

Leukocyte populations were studied at all stages of the menstrual cycle using a panel of monoclonal antibodies to natural killer cells, T cells, B cells, and macrophages on frozen sections of endometrium. At the time of implantation (mid-secretory phase) the majority of leukocytes appear to be Leu19+, CD16-, Leu7-, CD2+, CD3-, CD5-, CD7-, and HLA DR- with the morphology of large granular lymphocytes. The numbers of these cells showed a marked increase in the mid-secretory phase. These cells exhibited similar phenotypic characteristics to those found in decidua. These findings, therefore, suggest that the recruitment of these large granular lymphocytes to the uterus is under hormonal control and is not a local response to the presence of invading trophoblast.

A panel of monoclonal antibodies directed at fucosylated and sialylated carbohydrates on the Type 1 and Type 2 blood group precursor chains was used in an immunohistological study of trophoblast subpopulations. Formalin-fixed paraffin-embedded sections of normal trophoblast throughout pregnancy, hydatidiform moles and gestational choriocarcinoma were examined. Sialyl-Le x was localized in extravillous trophoblast populations in normal and molar pregnancies and choriocarcinoma tumour cells. After neuraminidase treatment, Le x was identified in similar cell populations. Although Le a was identified in syncytial trophoblast cells at the maternofetal interface, no staining was seen for sialyl-Le a . Villous trophoblast did not stain with any of the monoclonal antibodies used. Our results underline the divergent differentiation of trophoblast into villous and invasive extravillous subpopulations. The analogy with tumour cells is discussed.

The susceptibility of cultured first trimester human trophoblast cells to lysis by NK cells has been studied. Trophoblast cells are resistant to lysis by NK cells from both peripheral blood and decidua. However, decidual cells extracted by enzymatic disaggregation do exhibit cytotoxicity against the NK-sensitive cell line K562. The relevance of these findings to successful implantation of the blastocyst is discussed.