Microenvironment tailors nTreg structure and function.

Natural regulatory T cells (nTregs) ensure the control of self-tolerance and are currently used in clinical trials to alleviate autoimmune diseases and graft-versus-host disease after hematopoietic stem cell transfer. Based on CD39/CD26 markers, blood nTreg analysis revealed the presence of five different cell subsets, each representing a distinct stage of maturation. Ex vivo added microenvironmental factors, including IL-2, TGFß, and PGE2, direct the conversion from naive precursor to immature memory and finally from immature to mature memory cells, the latest being a no-return stage. Phenotypic and genetic characteristics of the subsets illustrate the structural parental maturation between subsets, which further correlates with the expression of regulatory factors. Regarding nTreg functional plasticity, both maturation stage and microenvironmental cytokines condition nTreg activities, which include blockade of autoreactive immune cells by cell-cell contact, Th17 and IL-10 Tr1-like activities, or activation of TCR-stimulating dendritic cell tolerization. Importantly, blood nTreg CD39/CD26 profile remained constant over a 2-y period in healthy persons but varied from person to person. Preliminary data on patients with autoimmune diseases or acute myelogenous leukemia illustrate the potential use of the nTreg CD39/CD26 profile as a blood biomarker to monitor chronic inflammatory diseases. Finally, we confirmed that naive conventional CD4 T cells, TCR-stimulated under a tolerogenic conditioned medium, could be ex vivo reprogrammed to FOXP3 lineage Tregs, and further found that these cells were exclusively committed to suppressive function under all microenvironmental contexts.

Endometrial vascularity by three-dimensional power Doppler ultrasound and cytokines: a complementary approach to assess uterine receptivity.

INTRODUCTION: Uterine receptivity was assessed simultaneously by measurement of vasoactive cytokines possibly involved in development of spiral arteries and by assessment of endometrial and uterine arterial blood flow. The objective was to explore the relationship between cytokine-related dysregulation and endometrial vascularisation in women with repeated implantation failures after in vitro fertilisation embryo transfer (IVF-ET). MATERIALS AND METHODS: We studied 40 women with recurrent IVF/ICSI-ET failures, despite replacement of more than 10 embryos of 'good quality', and 8 fertile controls. Three-dimensional ultrasound with power angiography was performed to record the sub-endometrial vascular flow index (VFI) and uterine artery pulsatility index prior to endometrial biopsy at day 21-23 of a monitored natural cycle. Endometrial IL-18, IL-18BP and IL-15 mRNA expression was assessed by real-time PCR, and the number of CD56(+) cells determined by immunochemistry. RESULTS: IL-18 and IL-15 mRNA expression was significantly different between the two groups. The range of variation in vascular imaging data was increased in the implantation failure (IF) group. The mRNA ratio for IL-15, but not the other cytokines, correlated with sub-endometrial vascular flow (r=0.65; p<0.001). This ratio correlated also with the mean number of CD56(+) cells per high-power field (r=0.41; p=0.005). Both IL-18 and IL-18BP mRNA expression was significantly negatively correlated with mean uterine artery pulsatility index (r=-0.37 and -0.43; p=0.02 and 0.01, respectively). CONCLUSION: Comprehensive ultrasonographic indicators appear to be related to various mechanisms, including insufficient or excessive uterine NK cell recruitment and inadequate endothelial vascular remodelling. New molecular tools may be useful in providing greater precision of uterine receptivity than ultrasonic indicators alone.

Immune cells in uteroplacental tissues throughout pregnancy: a brief review.

In a brief introduction, this review states why the presence of immune cells at the interface poses problems for an immunologist (Medawar paradigm). Different types of placentation are then discussed, and the various interactions with leukocytes, the extreme being with the equids where a certain degree of 'attack' is often seen. The limits of animal models when dealing with the human situation are emphasized. It is then stated why the various phases of pregnancy are different, and an analysis made of the cellular movements at the implantation, peri-implantation, immediate post-implantation and resorption windows in rodents. Details of the cellular components involved are given, as are hints for the human situation. The Th1/Th2 paradigm is described, with clinical examples, and its limits. Thus, the newly appraised dual role of natural killer (NK) cells is discussed, with examples in rodents and in humans (pre-eclampsia, implantation failure, abortion systems). Clinical data on the IL-12 /IL-18/NK tripod and implantation failure in humans are detailed.

Uterine receptivity and cytokines: new concepts and new applications.

The implantation process, currently thought to be the most critical step in achieving a successful early pregnancy, remains one of the most important unsolved processes in reproductive medicine. It depends on uterine-dependent and embryo-specific events, which need to be critically coordinated. Early embryo signaling following a maternal hormonal or cytokine-mediated preparation phase seems to be involved in stages immediately before, during and just after the apposition step to permit adequate proliferation of the stroma. Our objective is to develop guidelines and diagnostic tools pertinent to appreciate uterine receptivity. We will focus our attention on the uterine luminal environment at the time of oocyte retrieval and on the monitoring of the endometrium using three-dimensional ultrasound associated with digital technology and cytokine quantification by real-time PCR during the implantation window in an IVF/ICSI population. There is an accumulating body of data which strongly suggests that both implantation and uterine receptivity are controlled, primarily, though not exclusively, by locally acting growth factors and cytokines, some under steroid control. Some specific cytokines (IL-12, IL-15 and IL-18) in the luminal environment and in the endometrium allow a distinct pattern of abnormal uterine receptivity. The identification of these distinct patterns of abnormal uterine receptivity and of the mechanisms leading to the abnormal angiogenesis before implantation strongly suggest that no single therapeutic scheme can correct all cases of implantation failure and should be adapted for each patient especially in the case of unexplained infertility.

The Uterine Immune Profile May Help Women With Repeated Unexplained Embryo Implantation Failure After In Vitro Fertilization.

LABELED PROBLEM: Embryo implantation remains the main limiting factor in assisted reproductive medicine (20% success rate). METHODS OF STUDY: An endometrial immune profiling was performed among 394 women with the previous history of repeated embryo implantation failures (RIF). The endometrial immune profile documented the ratio of IL-15/Fn-14 mRNA as a biomarker of uNK cell activation/maturation (together with the uNK cell count) and the IL-18/TWEAK mRNA ratio as a biomarker of both angiogenesis and the Th1/Th2 balance. According to their profile, we recommended personalized care to counteract the documented dysregulation and assessed its effects by the live birth rate (LBR) for the next embryo transfer. RESULTS: Endometrial immune profiles appeared to be dysregulated in 81.7% of the RIF patients compared to control. Overactivation was diagnosed in 56.6% and low activation in 25%. The LBR among these dysregulated/treated patients at the first subsequent embryo transfer was 39.8%. CONCLUSION: Endometrial immune profiling may improve our understanding of RIF and subsequent LBR if treated.

Cytokines and implantation.

This review introduces the field of cytokines and implantation and then recalls the specialized role of the uterus and the notion of the 'implantation window'. The role of inflammatory and angiogenic cytokines is presented, as well as the involvement of cytokines such as -interferons in corpus luteum maintenance in non-chorionic gonadotrophin-producing species. -Interferons are reviewed, before dealing with the more in depth analysis of cytokine networks in the pre- and peri-implantation uterus. The emerging involvement of cytokines in controlling uterine vascularization angiogenesis is reviewed, with emphasis on NK activating factors.

Role of the endometrial tripod interleukin-18, -15, and -12 in inadequate uterine receptivity in patients with a history of repeated in vitro fertilization-embryo transfer failure.

OBJECTIVE: To document in the endometrium the correlation among the interleukin (IL)-12, -15, and -18 mRNA and the correlation between cytokine levels, vascular status, and endometrial natural killer (NK) cell count in the context of recurrent implantation failure. DESIGN: A pilot study. SETTING: Department of Reproductive Immunology. PATIENT(S): Women who failed to become pregnant after repeated IVF-embryo transfer and fertile control subjects. INTERVENTION(S): Ultrasonic evaluation and endometrial biopsy in luteal phase. MAIN OUTCOME MEASURE(S): Uterine artery Doppler, count of uterine CD56 bright cells/field, and quantification by real-time polymerase chain reaction (PCR) to monitor IL-12 family (IL-12p40, IL-12p35, EBI3, IL-23), the IL-18 system (IL-18, IL-18R, IL18BP), and the IL-15 mRNA ratio. RESULT(S): The uterine artery Doppler and the CD56 bright cell counts were significantly different in fertile and infertile patients. The mean uterine artery pulsatility index correlated significantly negatively with the IL-18/actin ratio suggesting a defect of the cytokine-dependent vascular remodeling pathway. The number of uterine CD56 bright cells was significantly correlated with the IL-15/actin and IL-18/IL-18BP ratios. Thus, IL-18 and IL-15 seems to be involved in the local recruitment and the activation of uterine natural killer (uNK) cells. IL-18 was itself correlated with IL-15 and IL-12, suggesting a local control of uNK cells activation. CONCLUSION(S): The assessment of the tripod IL-12/-15/-18 shows distinct immune-related mechanisms that are involved in the broader context of inadequate uterine receptivity.

Colony Stimulating Factors 1, 2, 3 and early pregnancy steps: from bench to bedside.

Reproductive immunology applies general immunology principles to specialised targets, reproduction and development. The involvement of colony-stimulating factors (CSFs) in reproduction illustrates this. The CSF family includes CSF-1 or macrophage CSF (M-CSF), CSF-2 or granulocyte macrophage CSF (GM-CSF), and CSF-3 or granulocyte CSF (G-CSF). Each member has a specific localisation and timed expression in the reproductive tract with specific functions involving them in ovulation, embryo implantation, placentation and further embryonic development. They are used in reproductive medicine, either as biomarkers of oocyte quality and competence (follicular G-CSF), or to supplement embryo culture media with human recombinant GM-CSF, or they are used as an innovative therapy by using human recombinant G-CSF for infertile patients. Given fundamental considerations on CSFs and their strong implication in reproduction, this review aimed to detail the current knowledge for each member of the family to improve our understanding of their implication in the maternal-foetal cytokinic dialogue and in possibly preventing reproductive disorders.

Reproductive immunology 2003: reassessing the Th1/Th2 paradigm?

We briefly review the history of concepts (some of which are still valid) which have lead to the present situation where pregnancy is viewed as being a Th2 phenomenon. We recall some of the early evidence which has been taken as supporting the general validity of this concept in murine and human pregnancy. We then recall some of the recent data dealing with "newer" cytokines and the role of uterine natural killer (NK) cells at the feto-maternal interface which fit neither with a steady-state concept nor with inflammatory cytokines, being solely "bad guys" as the paradigm would predict, nor with the concept of reduction of NK "activity" being required for successful pregnancy. As an example of the newer complexity, we briefly recall some of our recent micro-array studies in mice, and describe briefly our most recent data in human pointing out the importance of the tripod IL-12/IL-18/NK in successful or failed pregnancy in human, perhaps under IL-15 control. We conclude by a repeated warning against the so-called rationales of lymphocyte alloimmunization for therapy of recurrent spontaneous abortion and improvement of implantation rates.

Is there a place for immunomodulation in assisted reproduction techniques?

We briefly review the history of the concepts of the materno foetal relationship, and the (various) rationales which have been used to justify lymphocyte alloimmunisation (LA) as a treatment for recurrent spontaneous abortion of putative immune origins. The effectiveness of the treatment is at best dubious and limited to a small number of women for which there is no real positive selection rationale, at worst it is not efficient. The rationales themselves are rather "evolutive". The present one is to use the Th1:Th2 paradigm and, thus, to propose to "dampen NK activity" in abortion prone women and this concept has been extended by some to implantation failure. We briefly review why the Th1:Th2 paradigms is no longer fully valid, describe briefly why it is inappropriate for implantation, and conclude that alloimmunisation should no longer be proposed for RSA, hence, more for implantation failure. We, however, do not reject immunotherapy, but we believe that molecular and cellular defects specific approaches should be used, tailored for the specific pathway whose disruption cause the clinical symptom.

A brief review of recent data on some cytokine expressions at the materno-foetal interface which might challenge the classical Th1/Th2 dichotomy.

Focussing attention on cytokines at the materno-foetal interface represents one of the major advances made in the field. This owes much to the visionary views of Tom Wegmann, and to the changes brought about in the field by immunotrophism and Th1/Th2 paradigms. We review these briefly and also point out some emerging problems.However, a certain number of newly discovered cytokines do not fit into the classical Th1/Th2 dichotomy. Yet, by their capacity to activate or downregulate NK cells, by their action on adhesion molecules, and by their regulatory effects on the vascularisation process, they are of possible interest within the materno-foetal relationship. Therefore, as a first step, we have undertaken a systematic study of the expression of IL-11, IL-12, IL-13, IL-15, IL-16, IL-17 and IL-18 in the uterus, the peri-implantation embryo, and later on decidual and placental tissues throughout pregnancy. These cytokines were detected in every case, with, in each case, a precise localisation, which will be detailed, and which indeed suggests important regulatory functions, especially during implantation. In some cases, as will be shown in the peri-implantation uterus, those cells are perfectly expressed by uterine GMG-NK-like cells. Comparative ELISAs and quantitative RT-PCR have been or are being conducted, but already the expression patterns that are observed, and the very precise window of appearance that is observed for some of the GMG NK-like cells, either around or in the implanting embryo, as well as the complexity of the respective distributions, strongly suggest that, as useful as it certainly was for a while, the Th1/Th2 paradigm must now be considered as an over-simplification. Rather, the existing data point to sequential windows and are suggestive of a system where an extreme complexity is allied to very precise timing and tuning. They also suggest that the materno-foetal relationship is not simply maternal tolerance of a foreign tissue, but a series of intricate mutual cytokine interactions governing selective immune regulation and also control of the adhesion and vascularisation processes during this dialogue.

Implantation: can immunological parameters of implantation failure be of interest for pre-eclampsia?

We restate briefly why we consider that the Th1/Th2 paradigm, as useful as it has been, is now no longer adequate and is obsolete. We take as an example the role of IL-18, abortifacient at high doses but cardinal for the control of natural killer (NK) cell effects on spiral artery remodelling in mice, and likely also in humans. We then describe briefly our recent studies on cytokine defects and implantation failure in humans, a key feature being the link between uterine cytokine dysregulation and abnormal uterine vascular scores. We draw lessons for preeclampsia, and describe features of a model for its immune aetiology.

New insights into maternal-fetal interactions at implantation.

The immunotrophic theory was enunciated by Tom Wegmann. Since then, the involvement of cytokines in implantation and materno-fetal tolerance has emerged as a central topic in reproductive immunology. This brief survey covers the historical background leading to the specification of the crucial role of cytokines at the feto-maternal interface, and the present known patterns of their function. Focus is addressed to the most recent concept, e.g. pregnancy as a Th2 phenomenon (the immune response of the mother is biased towards the production of anti inflammatory cytokines, amongst them IL-10 which suppress inflammatory responses). A brief description of the role of inflammatory cytokines in implantation is presented to explain why it does not fit into such a scheme, and other recent data, for example on gamma interferon, also fails to accord with the Th2 phenomenon.

Controlled natural in vitro fertilization may be an alternative for patients with repeated unexplained implantation failure and a high uterine natural killer cell count.

Patients with a history of repeated and unexplained implantation failure after IVF-embryo transfer and a high uterine natural killer cell count during estrogen-progestin treatment may benefit from controlled natural IVF-ET.

A new role for natural killer cells, interleukin (IL)-12, and IL-18 in repeated implantation failure after in vitro fertilization.

OBJECTIVE: To investigate the endometrial immunohistochemical staining of interleukin (IL)-12 and IL-18 and to quantify the CD56 bright natural killer (NK) cells in relation to Doppler vascular disorders. DESIGN: Controlled clinical study. SETTING: Research unit of a university hospital. PATIENT(S)": Thirty-five women with repeated implantation failure after ET in IVF and 12 fertile control patients.Ultrasound evaluation and endometrial biopsy on day 20. MAIN OUTCOME MEASURE(S): The balance between IL-12 and IL-18, the number of NK cells, and the vascular status among fertile and implantation failure patients. RESULT(S): The control patients displayed normal vascular parameters, a weak anti-IL-12 staining, a consistent moderate stromal anti-IL-18 staining, and fewer than 15 NK cells/field. This pattern was observed among only 17% (6/35) of the implantation failure group. The remaining patients fit into one of two patterns: [1] 37% (13/35) had more than 40 NK cells/field with a strong anti-IL-12 and/or anti-IL-18 staining, and [2] the remaining 46% (16/35) had a marked local depletion of IL-18 and IL-12. Respectively, 85% and 31% of two groups displayed abnormal vascular parameters. CONCLUSION(S): Distinctions between the different local dysregulations of the cytokine network may provide clues for further exploration and treatment.

Assessment of leukemia inhibitory factor levels by uterine flushing at the time of egg retrieval does not adversely affect pregnancy rates with in vitro fertilization.

OBJECTIVE: To assess intrauterine levels of leukemia inhibitory factor (LIF) by uterine flushing at the time of egg retrieval and to confirm that the procedure has no detrimental effect on pregnancy rates. DESIGN: Prospective study. SETTING: Assisted reproductive unit of a university hospital. PATIENT(S): Uterine flushing was performed in 148 IVF patients. The first 100 patients were compared with a matched control group. INTERVENTION(S): Uterine flushing at the time of egg retrieval. MAIN OUTCOME MEASURE(S): IVP-ET results, pregnancy rates, and intrauterine LIF levels. RESULT(S): Pregnancy rates were not different in the group of patients with (27%) or without uterine flushing (28%). Leukemia inhibitory factor was detected in 60 patients (46%). Pregnancy rates did not differ between patients' detectable LIF and those in whom LIF was undetectable. Mean levels of LIF were 30.1 +/- 49.3 pg/mL and 28.6 +/- 51.2 pg/mL in pregnant and nonpregnant patients respectively. CONCLUSION(S): The flushing procedure at the time of egg retrieval did not adversely affect pregnancy rates. Leukemia inhibitory factor was detected in 46% of patients at the time of egg retrieval, but no correlation were observed with better pregnancy rates in patients with detectable LIF. Mean LIF levels did not differ in pregnant and nonpregnant women. Access to endoluminal secretions of the endometrium during IVF-ET may represent a new research in human implantation.

Granulocyte-Colony Stimulating Factor related pathways tested on an endometrial ex-vivo model.

INTRODUCTION: Recombinant human Granulocyte-Colony Stimulating Factor (rhG-CSF) supplementation seems to be a promising innovative therapy in reproductive medicine, used in case of recurrent miscarriage, embryo implantation failure or thin endometrium, although its mechanisms of action remain unknown. Our aim was to identify possible endometrial pathways influenced by rhG-CSF. MATERIALS AND METHODS: Hypothetical molecular interactions regulated by G-CSF were designed through a previous large scale endometrial microarray study. The variation of endometrial expression of selected target genes was confirmed in control and infertile patients. G-CSF supplementation influence on these targets was tested on an endometrial ex-vivo culture. Middle luteal phase endometrial biopsies were cultured on collagen sponge with or without rhG-CSF supplementation during 3 consecutive days. Variations of endometrial mRNA expression for the selected targets were studied by RT-PCR. RESULTS: At the highest dose of rhG-CSF stimulation, the mRNA expression of these selected target genes was significantly increased if compared with their expression without addition of rhG-CSF. The selected targets were G-CSF Receptor (G-CSFR), Integrin alpha-V/beta-3 (ITGB3) implicated in cell migration and embryo implantation, Plasminogen Activator Urokinase Receptor (PLAUR) described as interacting with integrins and implicated in cell migration, Thymidine Phosphorylase (TYMP) implicated in local angiogenesis, CD40 and its ligand CD40L involved in cell proliferation control. CONCLUSION: RhG-CSF seems able to influence endometrial expressions crucial for implantation process involving endometrial vascular remodelling, local immune modulation and cellular adhesion pathways. These variations observed in an ex-vivo model should be tested in-vivo. The strict indications or counter indication of rhG-CSF supplementation in reproductive field are not yet established, while the safety of its administration in early pregnancy on early embryogenesis still needs to be demonstrated. Nevertheless, rhG-CSF appears as a promising therapy in some difficult and unsolved cases of reproductive failure. Indications of pre-conceptual rhG-CSF supplementation may derive from a diagnosed lack of endometrial expression of some target genes.

TWEAK appears as a modulator of endometrial IL-18 related cytotoxic activity of uterine natural killers.

BACKGROUND: TWEAK (Tumor necrosis factor like WEAK inducer of apoptosis) is highly expressed by different immune cells and triggers multiple cellular responses, including control of angiogenesis. Our objective was to investigate its role in the human endometrium during the implantation window, using an ex-vivo endometrial microhistoculture model. Indeed, previous results suggested that basic TWEAK expression influences the IL-18 related uNK recruitment and local cytotoxicity. METHODOLOGY/PRINCIPAL FINDINGS: Endometrial biopsies were performed 7 to 9 days after the ovulation surge of women in monitored natural cycles. Biopsies were cut in micro-pieces and cultured on collagen sponge with appropriate medium. Morphology, functionality and cell death were analysed at different time of the culture. We used this ex vivo model to study mRNA expressions of NKp46 (a uNK cytotoxic receptor) and TGF-beta1 (protein which regulates uNK cytokine production) after adjunction of excess of recombinant IL-18 and either recombinant TWEAK or its antibody. NKp46 protein expression was also detailed by immunohistochemistry in selected patients with high basic mRNA level of IL-18 and either low or high mRNA level of TWEAK. The NKp46 immunostaining was stronger in patients with an IL-18 over-expression and a low TWEAK expression, when compared with patients with both IL-18 and TWEAK high expressions. We did not observe any difference for TWEAK expression when recombinant protein IL-18 or its antibody was added, or conversely, for IL-18 expression when TWEAK or its antibody was added in the culture medium. In a pro-inflammatory environment (obtained by an excess of IL-18), inhibition of TWEAK was able to increase significantly NKp46 and TGF-beta1 mRNA expressions. CONCLUSIONS/SIGNIFICANCE: TWEAK doesn't act on IL-18 expression but seems to control IL-18 related cytotoxicity on uNK cells when IL-18 is over-expressed. Thus, TWEAK appears as a crucial physiological modulator to prevent endometrial uNK cytotoxicity in human.

An insight into normal and pathological pregnancies using large-scale microarrays: lessons from microarrays.

In the introduction, we briefly recall old but classic evidence that there is no tolerance to paternal alloantigens in a first pregnancy. Therefore, we performed small- and large-scale microarrays in CBA × DBA/2 and CBA × BALB/c combinations, recently described as a murine model for preeclampsia. Our results are in line with other data suggesting a very early deregulation of local immune vascular events rather than a break of immune tolerance. Other data presented at the Tioman 2010 Preeclampsia Workshop supporting this hypothesis are briefly summarised, as well as indications and caveats from a recent human microarray on implantation failure and recurrent pregnancy loss.