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.

The expression of endothelial HLA-E in the context of the systemic inflammatory response observed in preeclampsia has not been established. An experimental study was designed to determine the effect of the sera of pregnant women on the expression of HLA-E in EA.hy296 endothelial cells. First, measurements of protein fractions were performed in sera from early-onset, severely preeclamptic women without HELLP syndrome, in which there was no significant difference in total proteins between the groups, but a reduced level of plasma albumin and an increase in α1-globulin were observed in both groups of pregnant women compared with non-pregnant women. Measurements of colloid osmotic pressure (COP) using a recalculated albumin/globulin ratio formula determined only a significant decrease in COP in all pregnant groups compared with non-pregnant women. The expression of membrane HLA-E was increased in EA.hy296 endothelial cells stimulated with sera of early-onset, severely preeclamptic women, while recombinant interferon-γ (IFN-γ) significantly reduced the expression of membrane HLA-E. Pro-inflammatory cytokines were measured by Luminex in the serum samples, and increased levels of tumor necrosis factor (TNF) and decreased levels of IFN-γ were observed in early-onset, severe preeclampsia compared with normal pregnancy. Moreover, soluble HLA-E was detected in these serum samples by Western blot and ELISA, but no significant difference was found. This raises the possibility that a systemic inflammatory response promotes a compensatory mechanism of COP balance in severe preeclampsia by release of inflammation-induced factors, including endothelial HLA-E. Evidence is now provided regarding HLA-E expression by EA.hy296 cells.

Preeclampsia involves an exacerbated maternal inflammatory response that suggests a possible role of innate immunity. NK cells can promote this kind of response through cytokine production and the expression of activating or inhibitory receptors. The aims of the present study were to explore cytokine production by peripheral blood mononuclear cells, as well as cytotoxic ability and receptor expression for HLA-E and HLA-G molecules in peripheral natural killer (NK) cells of women with early-onset severe preeclampsia without HELLP (hemolysis, elevated liver enzyme levels and a low platelet count) syndrome. The expression of the ILT2, KIRDL4, NKG2A, and NKG2C receptors and of cytotoxic activity was measured in non-stimulated NK cells, whereas the intracellular expression of IL-4, IL-10, IL-13, IL-12, IFNγ, TNF and VEGF, was assessed in non-stimulated peripheral blood mononuclear cells subsets using flow cytometry. Circulating soluble HLA-G was also determined by ELISA. The intracellular cytokines tested were significantly higher in NK cell subsets from severely preeclamptic women compared with the control group. On the other hand, the percentage of NK cells expressing NKG2A or NKG2C and the cytotoxic activity of NK cells were significantly higher in severely preeclamptic women. Furthermore, there was a significant correlation between urine protein concentration and soluble human leukocyte antigen G (soluble HLA-G) in serum. We conclude that patients with early-onset severe preeclampsia without HELLP syndrome have increased NK cell function related to cytokine production, cytotoxicity and expression of lectin-like receptors such as NKG2.

To optimize the potentiality of pregnancy of patients with a history of repeated and unexplained implantation failures after IVF-ET (RIF) according to their immune endometrial profiles. A prospective ongoing cohort study of 300 RIF patients. An immune uterine check-up is performed followed by recommendations in function of the endometrial state of activation. The outcome is the pregnancy rate occurring at the subsequent embryo transfer. An mild-luteal endometrial biopsy is performed during a non-conceptual cycle (Cormier pipelle). After datation, uterine Natural Killer cells (uNK) mobilization (immunostaining CD56+), endometrial mRNA expression of IL-15 (uNK maturation state), IL-18 (Th-1/Th-2 bivalence) and TWEAK/Fn-14 (TNF related local immune-regulation) are quantified by real time PCR. A low uNK cell mobilization or activation define a poor endometrial state of activation and a putative mechanism of RIF by insufficient adhesion. A high uNK cells activation define an over immune state of activation and a putative mechanism of RIF by embryo rejection. Thresholds of interpretation were previously defined in a fertile population. In case of poor activation, recommendations were to promote adhesion (endometrial injury the cycle prior the attempt, moderate ovarian stimulation, luteal supplementation with HCG, sexual intercourse). In case of over activation, recommendations were no local injury, Prednisolone 20 mg and vit-E from day -1 of the IVF cycle, luteal high dose of progesterone with estrogens and no sexual intercourse. Table 1Immune Endometrial Check-upLow ActivationOver-activationNormal ActivationNumber of RIF patients518341Pregnancy rate55% (28/51)47% (39/83)19% (8/41)Ongoing pregnancy rate51% (26/51)42% (35/83)15% (6/41)**Preliminary results on 175 RIF. Open table in a new tab Preliminary results on 175 RIF. Optimisation of the endometrial immune state may be useful in RIF patients. Deregulation is observed in 77% and resulted in a pregnancy rate of 45% when considered.

To evaluate the impact of glucorticoid administration on documented hyper-activated uterine killer cells (uNK) in patient with history of unexplained implantation failures after IVF-ET (RIF). Case controlled study including 44 RIF patients. An initial immune uterine check-up detected an over- endometrial activation. A second cycle with concomittant administration of glucorticoids evaluated the impact on the cytokinic environment, the uNK cells mobilization and activation. An mild-luteal endometrial biopsy was performed in a non-conceptual cycle (Cormier pipelle). After datation, we quantified the uNK mobilization (immunostaining CD56+), the endometrial mRNA expression of IL-15 (uNK maturation state), of IL-18 (Th-1/Th-2 balance) and of TWEAK/Fn-14 (TNF related local immune-regulation) by real time PCR. Criteria of inclusion defining an over immune state of activation of the endometrium (able to induce the embryo rejection) were high expression of IL-18/TWEAK and/or IL-15/Fn-14 and/or high uNK cells mobilization compared to control fertile patients. During the second cycle, Prednisolone was administrated 20 mg daily with 1g of Vit. E starting from Day-1 of the cycle and a new endometrial exploration scheduled in the mild luteal phase. If compared to control fertile, the immune profile was completely normalized in 52% (23/44) and partially improved in 70% (31/44) of the cases under prednisolone. IL-15/Fn-14 and IL-18/TWEAK ratio were significantly reduced (p=0.004 and 0.005 respectively). Normalized mRNA TWEAK expression was significantly increased under glucorticoids (p=0.001) while normalised mRNA expression of IL-15 and IL-18 as well as uNK mobilization were not significantly influenced. Prednisolone was able to completely control the uNK cells related activation and normalize the endometrial profile in about 52% of the case. The main mechanism observed is the over-expression of TWEAK, a TNF related immunoregulator.

ObjectiveTo document in patient with severe ovarian insufficiency (OI), the follicular concentration of G-CSF (FF [G-CSF] putative biomarker of oocyte competency) in natural and stimulated cycle and the endometrial immune profile.DesignOngoing cohort observational study including 100 OI patients. During a natural cycle, the dominant follicle is aspirated for FF G-CSF quantification, an endometrial biopsy is performed one week later to investigate uterine natural killer (uNK) cells mobilization and activation. The following cycle, a standard IVF is scheduled with individual follicular aspiration of mature follicles to assess FF [G-CSF].Materials and MethodsInclusion criteria for IO was age less than 40 years old with AMH below 0.1 pg/ml. Assessment of FF [G-CSF] in natural and stimulated cycles were performed with the Luminex XMap technology FF[G-CSF] over 18 pg/ml was interpreted as a conservation of the oocyte potentiality. On the endometrial biopsy, after datation, we quantified the uNK mobilization (immunostaining CD56+), the endometrial mRNA expression of IL-15 (reflecting the uNK maturation state), of IL-18 (reflecting the Th-1/Th-2 balance) and of TWEAK/Fn-14 (reflecting the TNF related local immune-regulation) by real time PCR.Results24 IO have been completely evaluated so far. FF [G-CSF] was higher under ovarian stimulation compared to natural cycle in 50%, lower or equivalent in 50%. Under stimulation, 40% (10/24) had a FF[ G-CSF] below 18 pg/ml, none were pregnant. 60% (14/24) had at least one oocyte with a FF [G-CSF] over 18 pg/ml, 35 % (5/14) were pregnant. All the IO patients showed a severe hyper-activated endometrial immune profile (high ratio of IL-18/TWEAK and IL-15/Fn-14) able to induce embryo rejection.ConclusionPreliminary results suggests that FF [G-CSF] may be useful to select OI patients still having a potential for pregnancy with their own gametes. Uterine Receptivity need to be optimized through a concomittant control of the uNK cells hyper-activation. ObjectiveTo document in patient with severe ovarian insufficiency (OI), the follicular concentration of G-CSF (FF [G-CSF] putative biomarker of oocyte competency) in natural and stimulated cycle and the endometrial immune profile. To document in patient with severe ovarian insufficiency (OI), the follicular concentration of G-CSF (FF [G-CSF] putative biomarker of oocyte competency) in natural and stimulated cycle and the endometrial immune profile. DesignOngoing cohort observational study including 100 OI patients. During a natural cycle, the dominant follicle is aspirated for FF G-CSF quantification, an endometrial biopsy is performed one week later to investigate uterine natural killer (uNK) cells mobilization and activation. The following cycle, a standard IVF is scheduled with individual follicular aspiration of mature follicles to assess FF [G-CSF]. Ongoing cohort observational study including 100 OI patients. During a natural cycle, the dominant follicle is aspirated for FF G-CSF quantification, an endometrial biopsy is performed one week later to investigate uterine natural killer (uNK) cells mobilization and activation. The following cycle, a standard IVF is scheduled with individual follicular aspiration of mature follicles to assess FF [G-CSF]. Materials and MethodsInclusion criteria for IO was age less than 40 years old with AMH below 0.1 pg/ml. Assessment of FF [G-CSF] in natural and stimulated cycles were performed with the Luminex XMap technology FF[G-CSF] over 18 pg/ml was interpreted as a conservation of the oocyte potentiality. On the endometrial biopsy, after datation, we quantified the uNK mobilization (immunostaining CD56+), the endometrial mRNA expression of IL-15 (reflecting the uNK maturation state), of IL-18 (reflecting the Th-1/Th-2 balance) and of TWEAK/Fn-14 (reflecting the TNF related local immune-regulation) by real time PCR. Inclusion criteria for IO was age less than 40 years old with AMH below 0.1 pg/ml. Assessment of FF [G-CSF] in natural and stimulated cycles were performed with the Luminex XMap technology FF[G-CSF] over 18 pg/ml was interpreted as a conservation of the oocyte potentiality. On the endometrial biopsy, after datation, we quantified the uNK mobilization (immunostaining CD56+), the endometrial mRNA expression of IL-15 (reflecting the uNK maturation state), of IL-18 (reflecting the Th-1/Th-2 balance) and of TWEAK/Fn-14 (reflecting the TNF related local immune-regulation) by real time PCR. Results24 IO have been completely evaluated so far. FF [G-CSF] was higher under ovarian stimulation compared to natural cycle in 50%, lower or equivalent in 50%. Under stimulation, 40% (10/24) had a FF[ G-CSF] below 18 pg/ml, none were pregnant. 60% (14/24) had at least one oocyte with a FF [G-CSF] over 18 pg/ml, 35 % (5/14) were pregnant. All the IO patients showed a severe hyper-activated endometrial immune profile (high ratio of IL-18/TWEAK and IL-15/Fn-14) able to induce embryo rejection. 24 IO have been completely evaluated so far. FF [G-CSF] was higher under ovarian stimulation compared to natural cycle in 50%, lower or equivalent in 50%. Under stimulation, 40% (10/24) had a FF[ G-CSF] below 18 pg/ml, none were pregnant. 60% (14/24) had at least one oocyte with a FF [G-CSF] over 18 pg/ml, 35 % (5/14) were pregnant. All the IO patients showed a severe hyper-activated endometrial immune profile (high ratio of IL-18/TWEAK and IL-15/Fn-14) able to induce embryo rejection. ConclusionPreliminary results suggests that FF [G-CSF] may be useful to select OI patients still having a potential for pregnancy with their own gametes. Uterine Receptivity need to be optimized through a concomittant control of the uNK cells hyper-activation. Preliminary results suggests that FF [G-CSF] may be useful to select OI patients still having a potential for pregnancy with their own gametes. Uterine Receptivity need to be optimized through a concomittant control of the uNK cells hyper-activation.

In reproductive medicine, Granulocyte-Colony Stimulating Factor (G-CSF) has been described as promising to reduce early pregnancy loss or increase embryo implantation rate, without clearly established effects on embryo implantation. Our aim was to identify G-CSF effects on subsequent embryo implantation and early pregnancy loss in pro-abortive and fertile murine models. We tested systemic G-CSF supplementation on a murine model obtained by crossing CBA/J females with DBA/2 males, described as a pro-abortive model because of its high foetal resorption rate and compared it to a fertile murine crossing (CBA/J x Balb/c). G-CSF was injected to CBA/J female mice, from the pro-abortive crossing (CBA/J x DBA/2) or from the fertile crossing (CBA/J x Balb/c), at different stage and different doses during pre-implantation period. Embryo implantation rates and embryo resorption rates (equivalent to early pregnancy loss in Humans) were compared to the control groups receiving placebo injections on hysterectomies performed at day 10 post-coitum. High doses of G-CSF significantly increased embryo implantation sites in both models, but increased the embryo resorption rate in the pro-abortive model. Low doses of G-CSF in the pro-abortive model decreased implantation sites and also decreased resorption rate. Unexpectedly, same low doses of G-CSF in the fertile model decreased implantation sites without modifying the resorption rate. G-CSF seems to affect the embryo selection by modifying the endometrial biosensor in these murine models. But the timing and the dose of injection are crucial. Moreover, this supplementation doesn't seem trivial when considering fertile cases and highlights the necessity to define specific indication through a better understanding of the local mechanism induced.

Identification of biomarkers of optimal uterine receptivity to the implanting embryo as well as biomarkers of oocyte competence would undoubtedly improve the efficiency of assisted reproductive technology (ART). Expression of IL-15 and IL-18 has been shown to be different in patients with failed implantation after IVF/ICSI compared with fertile controls and both correlate with local uNK (CD56+) recruitment and angiogenesis. Tumor necrosis factor weak inducer of apoptosis (TWEAK) has been described in mice as a potent early immune regulator able to protect the conceptus. The results of our studies in human suggest that TWEAK modulates the IL-18 related cytotoxicity of uNK cells. Quantification of IL-18, TWEAK and IL-15 mRNA expression by real-time PCR in endometrial tissue collected in mid-luteal phase of non-conception cycles allowed documentation of physiological events that occur at the time of uterine receptivity. Such information may be useful for the physician especially in patients where embryos fail to implant. Cytokine quantification may assist in understanding the mechanisms leading to repeated IVF/ICSI failure: either depletion of cytokines necessary for the apposition-adhesion, or an excess of cytokines leading to local cytotoxicity, may impair the implantation of the embryo. Other new data suggest that a pre-conception dialogue mediated by the oocyte and the follicular fluid and the oocyte may contribute to later implantation success. Follicular concentration of G-CSF appears as a useful biomarker of oocyte competence before fertilization. Moreover both in human and animal models, evidence of a role of the endometrium as a biosensor of the embryo is emerging.

Recurrent Miscarriage is an early pregnancy complication which affects about 1–3 % of child-bearing couples. The mechanisms involved in the occurrence of recurrent miscarriages are not clearly understood.In the last decade Natural Killer cells have been studied in peripheral blood and uterus in order to determine if there are specific characteristics of Natural Killer cells associated with miscarriage. Different authors have described an increased number of uterine and peripheral blood Natural Killer cells in women with recurrent miscarriages compared to control women. However, its relationship with miscarriage has not been confirmed.In patients with recurrent miscarriage a lack of inhibition of decidua Natural Killer cells can be observed, which leads to a more activated state characterized by higher levels of proinflammatory cytokines. In peripheral blood, it has been also reported a dysfunctional cytokine production by Natural Killer cells, with an increase of interferon-γ levels and a decrease of Interleukin-4.Significant progress has been made in the last decade in understanding the biology of Natural Killer cells, including the identification of new receptors that also contribute to the activation and regulation of Natural Killer cells. In this review, we summarize the current progress in the study of Natural Killer cells in recurrent miscarriage.

Small regulatory RNAs can not only guide post-transcriptional repression of target genes, but some of them can also direct heterochromatin formation of specific genomic loci. Here we review the published literature on small RNA-guided epigenetic regulation in insects. The recent development of novel analytical technologies (deep sequencing and RNAi screens) has led to the identification of some of the factors involved in these processes, as well as their molecular mechanism and subcellular localization. Other findings uncovered an additional mode of epigenetic control, where maternally inherited small RNAs can affect phenotypes in a stable, transgenerational manner. The evolutive history of small RNA effector proteins in insects suggests that these two modes of regulation are variably conserved among species.