The uterine immune profile: A method for individualizing the management of women who have failed to implant an embryo after IVF/ICSI.

A unique endometrial immune reaction should occur to promote the human embryo implantation. We postulated that an immune disequilibrium may impact the initial dialogue between the mother and her embryo. In 2012, we set a method of uterine immune profiling for patients with unexplained repeated implantation failures (RIF). The method documents the local Th-1/ Th-2 equilibrium and the recruitment and state of maturation/activation of uNK cells. In function of the disequilibrium observed, personalization of assisted reproductive treatments was suggested. As the concept of personalization in function of the uterine immune profile had never been proposed, a large cohort study and a controlled cohort study were first conducted in RIF patients. 80 % of the RIF patients showed a local disequilibrium if compared to fertile controls. The local disequilibrium was identified in 3 categories: over-immune activation in 45 %, low- local immune activation in 25 % and mixed profile in 10 %. Personalization of treatments in function of the immune profile allowed to restore a live birth rate by 40 % at the following embryo transfer. RIF patients with endometriosis show some particularities regarding their immune profiles. We also suggested that immunotherapy (corticoids, intralipids) may have targeted indications based on a better understanding of the immune type of disequilibrium documented. Personalization of treatments for RIF patients seems to be essential to promote the subsequent live birth rate. The endometrial immune profiling is an innovative method aiming to detect a local immune disequilibrium and, if present, to test preventively its correction under treatment.

Higher interleukin-18 and mannose-binding lectin are present in uterine lumen of patients with unexplained infertility.

The uterine luminal environment was explored with regard to interleukin-18 (IL-18) and mannose-binding lectin (MBL) and the possibility that the procedure of flushing the uterine cavity would optimize the physiological initial pseudo-inflammatory uterine reaction. Uterine flushings were performed among 175 IVF/intracytoplasmic sperm injection (ICSI) patients at the time of oocyte retrieval and the cycles were compared with a control group matched for age, number of previous attempts and type of assisted reproductive procedure (IVF or ICSI) in which no flushing were performed (n = 175). Samples collected were divided into two groups according to the presence/absence of endometrial cells in samples. IL-18 and MBL expressions were explored by enzyme-linked immunosorbent assay. Implantation rates were significantly higher in those patients who underwent the uterine flushing compared with controls (P = 0.04). Luminal concentrations of IL-18 and MBL were higher if endometrial cells were present in flushings, suggesting endometrial origin of the secretion. Both concentrations of MBL and IL-18 were higher in patients with unexplained infertility compared with patients involved in IVF/ICSI for male or tubal infertility (P = 0.005 and 0.02, respectively). The exploration of the endoluminal environment before oocyte retrieval may enhance pregnancy rates and show distinct features in patients with unexplained infertility.

[Current debates on immunology of preeclampsia. Report of the sixth international workshop of Reunion Island (Indian Ocean, December 2008)]. / Débats actuels sur l'immunologie de la prééclampsie. Comptes rendus du sixième colloque international de La Réunion (décembre 2008).

Hypertensive disorders of pregnancy (HDP) represent globally 10% of human births and their major complication, preeclampsia, 3 to 5%. The etiology of these HDP remains still uncertain, however major advances have been made these last 25 years. The Sixth International Workshop on Reproductive Immunology, Immunological Tolerance and Immunology of Preeclampsia 2008 celebrated its 10th Anniversary in Reunion-island (French overseas Department in the Indian Ocean). Over this decade, these six workshops have contributed extensively to immunological, epidemiological, anthropological and even vascular debates. The defect of trophoblastic invasion encountered in preeclampsia, intra-uterine growth retardation and to some extend also preterm labour has been understood only at the end of the 1970's. On the other hand, clinical and epidemiological findings at the end of the 20th century permitted to apprehend that "preeclampsia disease of primiparae" may in fact well be the disease of first pregnancies at the level of human couples. Among the important advances, immunology of reproduction is certainly the topic where knowledge has literally exploded in the last decade. This paper relates some major steps in comprehension of this disease and focuses on the interest to follow these immunological works and their new concepts. It seems, at the beginning of the 21st century, that we are possibly closer than ever to understand the etiology of this obstetrical enigma. In this quest, the immunology of reproduction will certainly come out as one of the main players.

Cytokines and chemokines in follicular fluids and potential of the corresponding embryo: the role of granulocyte colony-stimulating factor.

BACKGROUND: The cytokine/chemokine levels of individual follicular fluids (FFs) were measured to determine whether a biomarker could be linked to the developmental potential of the derived embryo. METHODS: Fluid was collected from 132 individual FFs that were the source of oocytes subsequently fertilized and transferred. In each, a bead-based multiplex sandwich immunoassay (Luminex) was used to measure 28 cytokines and chemokines simultaneously. RESULTS: Significantly higher levels of interleukin (IL-2) and interferon (IFN-gamma) were detected in FF for embryos that underwent early cleavage. IL-12 was significantly higher in FF corresponding to highly fragmented embryos and the chemokine CCL5 was significantly higher in FF related to the best quality (Top) embryos. The level of granulocyte colony-stimulating factor (G-CSF) in individual FF samples was correlated with the implantation potential of the corresponding embryo. The area under the receiver operating characteristics curve, which distinguished the embryos that definitely led to delivery from those that did not, was 0.84 (0.75-0.90) (P = 0.0001) for FF G-CSF. FF G-CSF was significantly lower in patients older than 36 years compared with those <30-year old. When the FF G-CSF was 20 pg/ml or higher, the ratio between Top and non-Top embryos was significantly higher than for the group with FF G-CSF below 20 pg/ml (45 versus 20.45%, P = 0.007). CONCLUSIONS: Individual FF composition is related to the development of the corresponding in vitro generated embryo and its potential of implantation. Individual FF G-CSF may provide a non-invasive biomarker of implantation that needs to be evaluated together with in vitro observation to select the oocyte, and hence the embryo, to transfer.

[Physiology of implantation]. / Physiologie de l'implantation.

This article explains why we have had to come to a central role for innate immunity rather than the threat of maternal rejection of the foetal allograft. We encompass briefly the role of inflammation in implantation, not only for invasion adhesion, but also to prepare future "tolerance". In this context, we envisage the role of TWEAK and complement.

Down modulation of TNF-alpha mRNA placental expression by AZT used for the prevention of HIV-1 mother-to-child transmission.

Mechanisms of HIV-1 in utero mother-to-child transmission (MTCT) protection provided by AZT are not completely understood. The placental cytokine network is involved in the control of HIV-1 in utero transmission but the effect of AZT on this network is unknown. To evaluate the effects of AZT on placental cytokine expression, the chorionic villi from HIV-1 uninfected women term placentae were cultured with 0, 100, and 2,000 ng/ml AZT. Tissue fragments were harvested at days 1, 4, and 7 to determine the level of cytokine mRNA by real-time RT-PCR. The viability and morphology of the placental histocultures were monitored by the expression of beta-human chorionic gonadotropin (beta-hCG) gene, lipopolysaccharide (LPS) activation, and microscopic examination. AZT at 2,000 ng/ml significantly down-regulated TNF-alpha mRNA expression at day 1 and day 4, but had no effect on beta-hCG, stromal cell-derived factor 1 (SDF-1), and IL-10 gene expression. AZT did not induce any deleterious impact on placental tissue structure. Furthermore, activation of chorionic villi by LPS for 24 h up-regulated IL-10 and TNF-alpha mRNA expression. Down-regulation of TNF-alpha mRNA could represent a mechanism through which AZT can decrease the risk of HIV-1 MTCT, in addition to its direct effect on HIV-1 replication.

Evaluation of the placental environment with a new in vitro model of histocultures of early and term placentae: determination of cytokine and chemokine expression profiles.

We aimed to set up and validate a new in vitro model of placental histocultures, for the evaluation of cytokine and chemokine profiles of the placental environment, over a long culture period. Micro-explant cultures from 6 early and 6 term placentae were set up on collagen sponge gel supports at a liquid/air interface. At various times during culture, we analyzed tissue morphology and cell death by microscopy and quantified beta-hCG production and mRNA levels for beta-hCG and insulin-like 4 (INSL4). Levels of IL-6, LIF, TNF alpha, IL-10, IFN-gamma, IL-16 and RANTES in the medium were measured by ELISA on days 1, 4 and 7 of culture. SDF-1 mRNA expression was determined by real-time PCR at the same time points. Histocultures from early and term placentae remained viable until day 10. High levels of IL-6 and LIF production, low levels of TNF alpha, IL-10 and IFN-gamma production and significant SDF-1 expression were observed. These data indicate that placental histoculture is a suitable and reliable in vitro model for studying the placental environment.

Progesterone receptors in lymphocytes of liver-transplanted and transfused patients.

Previous data have shown that lymphocytes from pregnant women, but not from non-pregnant individuals, displayed progesterone receptors. These receptors are inducible in normal human lymphocytes in vitro by mitogenic or allogeneic stimuli. The present study was designed to test the role of in vivo allogeneic stimulation in inducing progesterone receptors in lymphocytes from transplanted and transfused patients. Receptors were detected by immunohistology using a progesterone receptor-specific MoAb and avidin-biotin system. Peripheral blood lymphocytes from 56 healthy pregnant women, 8 liver-transplanted patients and 15 transfused patients contained significantly more receptor-positive cells (P less than 0.001) than those of non-pregnant individuals. In transplanted and transfused patients no correlation was found between the percentage of positive lymphocytes and age, sex or transplant survival. Our results show that in these three groups the percentage of receptor-bearing lymphocytes was higher than in normal subjects.

Cell-free HIV type 1 infection is restricted in the human trophoblast choriocarcinoma BeWo cell line, even with expression of CD4, CXCR4 and CCR5.

The restriction of cell-free HIV-1 infection has been demonstrated in placental trophoblast choriocarcinoma BeWo cells. We tried to determine the level of the viral replication cycle at which this restriction occurs. BeWo cells produce infectious viruses after transfection with HIV-1 plasmids, independently of viral tropism. CCR5 and CXCR4, but not the CD4 molecule, were detected at the cell surface. We therefore derived CD4-expressing clones from transfected BeWo cells. Cell-free virus infection of these clones resulted in neither virus production nor viral sequence integration, indicating that the restriction occurs before integration of the virus. If we used luciferase reporter viruses pseudotyped with HIV-1 Env R5 and X4 for infection, no luciferase activity was detected, even in the BeWo-CD4+ clone, in contrast to what was observed in VSV-G pseudotyped virus infection. Our results show that infection of trophoblast-derived cells with cell-free virus is at least restricted at the level of entry. Thus, BeWo is an interesting human placental cell line that is resistant to HIV-1, even if CD4, CXCR4, and CCR5 are expressed.

An unusual HIV type 1 env sequence embedded in a mosaic virus from Cameroon: identification of a new env clade. European Network on the study of in utero transmission of HIV-1.

An atypical HIV-1 strain (CAM001) was identified in a pregnant Cameroonian woman in 1995. HMA subtyping of the env region was unsuccessful, and sequence analyses were performed. Unique sequence motifs were found at the V3 tip (GAGRALHA and GAGRAWIHA), and phylogenetic studies showed that the env C2-V5 sequence branched within group M but remained distinct from all known HIV-1 subtypes, while p17 gag branched with the subtype F sequences. Four other HIV group M viruses, undetermined by HMA, of African origin were found to cluster with CAM001 in the C2-V5 sequences. With the BLAST method, we found in databases three strains whose V3 sequences also clustered with CAM001. These unusual env sequences from eight HIV-1 strains derived from Cameroon formed a separate cluster in HIV-1 group M, which we designated k.

HIV type 1 Thai subtype E is predominant in South Vietnam.

PIP: Samples of peripheral blood mononuclear cells from 50 HIV-1-infected individuals in South Vietnam were analyzed to determine with which HIV-1 subtype the subjects were infected. Participants were from Ho Chi Minh city and five surrounding provinces; 16 samples from female prostitutes, 32 from IV drug users, and one each from a man and woman not in any HIV risk group. 32 individuals were therefore most likely infected by IV drug use and the rest through sexual contacts. PCR amplification and heteroduplex mobility assay found all but one case to be infected with HIV-1 subtype E. The only nonsubtype E infection was HIV-1 subtype B in a woman sexually infected by her seropositive partner who was most likely exposed to the virus in Europe. HIV-1 subtype E strongly predominates in South Vietnam. The homogeneous geographic distribution of subtype E suggests the recent introduction of the virus into the country. A Thai origin can be considered given the genetic relationship between the Thai and Vietnamese subtypes E. It may be assumed that subtype E infections of Vietnamese prostitutes are related to the progressive entry and spread of HIV-1 subtype E from Thailand to Cambodia and then to southern Vietnam.^ieng

Involvement of cytokines in eating disorders: a critical review of the human literature.

A number of findings from clinical and animal studies indicate that pro-inflammatory cytokines may play roles in eating disorders. The measurement of pro-inflammatory cytokines (IL-1, IL-6, TNFalpha), which are known to decrease food intake, provides highly variable data from which firm conclusions cannot be drawn. In most of the longitudinal studies where pro-inflammatory cytokines have been shown to be impaired in anorexia or bulimia nervosa, a return to normal values was observed after renutrition. However these findings do not exclude the possibility that pro-inflammatory cytokines might be overproduced in specific brain areas and act locally without concomitantly increased serum or immune production. It was also pointed out that the production of the major type-1 cytokines (especially IL-2) was depressed in anorexia nervosa. It remains unclear whether this is due to undernutrition or to a specific underlying cause common to eating disorders. The impaired cytokine profile observed in eating disorders could be related to several factors including impaired nutrition, psychopathological and neuroendocrine factors. More particular attention should be devoted to the deregulation of the anti/pro-inflammatory balance. Deregulation of the cytokine network may be responsible for medical complications in eating disorder patients who are afflicted with chronic underweight.

Placental immunoregulatory factors.

Local immune suppression appears to be a key feature in the success of the fetal allograft. Various placental factors have been described endowed with immunoregulatory activity. Much of the available evidence concerning the biological function and biochemical characteristics of these molecules is reviewed here, as well as the controversial issues surrounding them. In the final section, some outstanding questions relating to these immunoregulators are considered.

Immunoactive products of human placenta. IV. Immunoregulatory factors obtained from cultures of human placenta inhibit in vivo local and systemic allogeneic and graft versus-host reactions in mice.

We have shown in previous reports that human placental supernatants (HPS) display a variety of immunoregulatory properties in vitro. Here we report that these supernatants are able to suppress the ability of murine splenocytes to evoke an allogeneic reaction in the popliteal lymph node (PLN) assay, as well as their capacity to induce both local (popliteal lymph node swelling) as well as systemic chronic or lethal graft-versus-host reactions. Those results are discussed in the light of the potential use of HPS in the control of transplantation immune reactions as well as in the regulation of xenogeneic pregnancies, as the murine system that was used proved that the material exerted an effect across a species barrier.

Immunoactive products of human placenta. II. Direct inhibition of non-MHC restricted cytolytic activity of human CD3 alpha-beta but not CD3 gamma-delta expressing T cell clones.

The effect of human placental supernatant obtained from explant cultures of caesarean delivery placentae was monitored on both alpha-beta human T cell clones, which display both cytotoxic alloreactivity and non-MHC restricted cytotoxicity against K562 target cells, and gamma-delta ones endowed solely with the latter. It was found that, under appropriate experimental conditions, direct inhibition of the cytolytic activity of alpha-beta T cell clones was exerted by the supernatant. In contrast, gamma-delta T cell clones were unaffected. The relevance of these data to the survival of the fetal allograft is discussed.

Effect of prostaglandin synthesis inhibitors on spontaneous and endotoxin-induced abortion in mice.

The putative role of prostaglandin E2 (PGE2) in suppressing rejection of the 'fetal allograft' (resorption) in C3H/HeJ and CBA/J allopregnant mice was tested by administration of the prostaglandin synthesis inhibitors indomethacin (INDO) and acetylsalicylic acid (ASA). When the resorption rate was low, INDO fed at a dose of 15 micrograms/ml in drinking water after implantation had a slight augmenting effect when the endogenous resorption rate was < 30%, but had no effect when the endogenous rate was higher or when bacterial lipopolysaccharide (LPS) was given. ASA fed at 50 micrograms/ml had no augmenting effect and did not increase sensitivity to the abortogen LPS in either CBA/J (LPS sensitive) or C3H/HeJ (LPS resistant) mice. Both INDO and ASA fed to CBA/J mice significantly reduced endogenous PGE2 extractable from the uteri of hormonally pseudopregnant mice after deciduoma induction. Feeding INDO at doses up to 30 micrograms/ml from day 2.5 of pregnancy impaired but failed to completely block implantation in CBA/J mice, and with daily administration, some of the mice became sick: all of the implants in sick mice resorbed. INDO at doses of 150-200 micrograms per day known to inhibit implantation in vivo by sufficiently blocking PGE2 synthesis, was injected on one or more days beginning after the time of implantation. This failed to cause abortion in CBA/J mice and although some mice became ill, provided this happened after day 8.5 of pregnancy when sensitivity to the abortogenic effects of injected LPS decreased substantially in these mice, all implants in the sick mice were 'healthy' (i.e. non-resorbing). We were unable to increase the rate of resorption in syngeneically pregnant CD1 mice above 13% with 15 ml INDO in drinking water. Our data do not support the view that PGE2 represents an important intrauterine suppressor molecular blocking the processes mediating embryo death at the time of abortion. Spontaneous abortion in DBA/2-mated CBA/J mice appears to be determined by the level of bacterial LPS (endotoxin) and treatment with antibiotics or intralipid (which enhances endotoxin clearance), reduces the abortion rate. A sufficient dose of INDO may cause abortion, but the data taken together suggest this may be due to effects on the gut whereby permeability to bacterial LPS is increased.

Vaccination against spontaneous abortion in mice.

We report here that the high rate of spontaneous resorption observed in CBA/J female mice mated with DBA/2 J males can be dramatically reduced by vaccination with Balb/c male spleen cells, but not by CBA/J or DBA/2 J male spleen cells. This effect correlates with the differential ability of Balb/c spleen cells to induce MLR suppressor activity in CBA/J female mice, and should lead to a better understanding of the immunology of the materno-fetal relationship.

Immunoregulatory effects of ovine trophoblastin protein (oTP): all five isoforms suppress PHA-induced lymphocyte proliferation.

The immunosuppressive properties of ovine trophoblastin protein (oTP) isoforms purified to homogeneity by DEAE HPLC have been studied within and across species barriers by in vitro assays. It has been demonstrated that not only the classical oTP 1, but in fact all 5 isoforms, are immunosuppressive in a PHA-induced proliferation assay, whilst being ineffective on IL-2 dependent CTL-L2 cell replication. The significance of these findings is discussed.

Local active suppression and successful vaccination against spontaneous abortion in CBA/J mice.

We report here that vaccination of CBA/J female mice with DBA/2 X BALB/c recombinant line that decreases the spontaneous abortion rate increases local active decidua-associated suppressor cell activity. In contrast, vaccination with a recombinant line that increases the abortion rate decreases suppressor cell activity. No correlation was seen between the effect on the abortion rate and the ability of cells from the fetoplacental unit to inhibit cytolysis by NK cells. Successful vaccination against spontaneous abortion may act primarily by augmenting suppressor cell activity in the decidua at the implantation site.