The Next Frontier in ART: Harnessing the Uterine Immune Profile for Improved Performance.

Assisted reproduction techniques have improved considerably in recent decades, but despite these advances, success rates remain relatively low. Endometrial immune profiling involves the analysis of cytokine biomarkers in the endometrium during the mid-luteal phase. This profiling aims to provide insights into the immune environment of the uterus. The aim is to identify immune disturbances and thus guide the development of personalized therapeutic approaches. The first part of the review looks back at the emergence of innovative concepts, highlighting the specificity of the human uterine environment at the time of implantation. Based on this new knowledge, biomarkers have been selected for endometrial immune profiling. The second part details the results of clinical studies conducted over the last ten years. These clinical results suggest that this approach can increase the rate of live births in patients suffering from repeated implantation failures or repeated pregnancy loss. Uterine immune profiling represents a clinical innovation that can significantly improve the performance of medically assisted reproduction treatments through personalized strategies tailored to the local immune profile. Innovation in personalized medicine for assisted reproduction is crucial to improving the success rates of fertility treatments, while reducing the risks and costs associated with ineffective or unnecessary interventions.

Testing the endometrium: is there enough evidence to justify clinical use?

PURPOSE OF REVIEW: Embryo implantation remains the limiting factor in assisted reproduction outcomes. To date research has mainly focused on improving embryo quality, numbers and selection as the route to improve treatment results. However, with success rates plateauing, interest in the possibility of modulating the endometrial factor is increasing, and a number of biomarkers are now available that offer the possibility of assessing endometrial function. RECENT FINDINGS: In this review, we review recent evidence for the efficacy of a number of these biomarkers, with emphasis on those that aim to enable improvement in embryo/endometrial developmental synchrony endometrium and that offer an assessment of the degree of immune activation of the endometrium. The emerging field of reproductive tract microbiome analysis is also considered. Finally, nascent biomarkers of materno-foetal dialogue, including noncoding RNAs, microvesicles and endometrial glycans are discussed. SUMMARY: Tests of potential clinical value are emerging, but further validation studies are required. The usage of innovative endometrial biomarkers provides the possibility of targeted therapies rather than the blind empirical approaches to face embryo implantation failure. It also enables the possibility of randomized controlled trials of interventions targeting the individual cause rather empirical treatments of undiagnosed recurrent implantation failure.

Specific and extensive endometrial deregulation is present before conception in IVF/ICSI repeated implantation failures (IF) or recurrent miscarriages.

The objective was to examine if IVF/ICSI repeated implantation failures (IF) or recurrent miscarriages (RM) could be related to preconceptional endometrial deregulations. IF was defined as the absence of pregnancy despite the transfer of at least ten IVF/ICSI good quality embryos, and RM as having at least three unexplained miscarriages. Fertile controls (FC) were women who had given birth at least once. Endometrial biopsy was performed in the mild luteal phase of a non-conceptual cycle (five women were selected in each group). Affymetrix chips (GeneChip Human Genome U133 Plus2.0 Array) were used for hybridization. Data were normalized by the gcRMA method, and raw p values adjusted by the Bonferroni procedure (1%). Differential expression of selected genes was analysed using real-time PCR. Gene networks and biological functions were explored using the Ingenuity Pathways Analysis software. Endometrial gene expression profiles at the time of uterine receptivity differ dramatically in the endometrium among FC, RM, and IF patients. Compared to FC, 2126 and 2477 genes are differentially expressed in IF and RM groups, respectively, and 2363 between IF and RM. In both conditions, differential gene expression referred mainly to DNA transcription and expression. Other main cellular functions deregulated in IF conditions correspond to cell morphology, cellular development, cell cycle, and cellular assembly, while in RM conditions, deregulated cellular functions relate to cell signalling (degradation of cyclic AMP and calcium metabolism) and cellular maintenance. In both conditions, there is an over-representation of deregulations related to the haematological system. In the IF condition, cell-mediated immune response and nervous system development and function are highly deregulated, while in RM patients, main deregulations are in organ and tissue development, humoral immune response, and muscular system development and function. Extensive endometrial deregulations are present before conception in patients who experienced IF or RM with both distinct and common deregulation.

The Endometrial Immune Profiling May Positively Affect the Management of Recurrent Pregnancy Loss.

Introduction: The endometrial immune profiling is an innovative approach based on the analysis of the local immune reaction occurring in the endometrium at the time of the embryo implantation. By documenting the local immune activation during the period of uterine receptivity, we aim to detect and correct potential imbalances before and at the very beginning of placentation. The main objective of the study was to analyze in women with a history of repeated pregnancy loss (RPL) the association of personalized strategies based on immune dysregulations with live birth rates. The secondary objective was to highlight the main prognostic factors for live births. Methods: This is an observational retrospective analysis of 104 patients with RPL, included between January 2012 and December 2019. Inclusion criteria included a spontaneous fertility with at least three miscarriages, an assessment including a three-dimension ultrasound scan, an endometrial biopsy for uterine immune profiling and a follow-up over at least 6 months with personalized care if indicated after the complete assessment. We defined as a success if the patients had a live birth after the suggested plan, as a failure if the patient either did not get pregnant or experienced a new miscarriage after the targeted therapies. Results: Uterine immune profiling was the only exploration to be significantly associated with a higher live birth rate (LBR) if a dysregulation was identified and treated accordingly (55% vs 45%, p=0.01). On the contrary, an absence of local dysregulation (resulting in an apparently balanced immune environment) was associated with a higher risk of a new miscarriage, suggesting that the cause inducing RPL still needed to be identified. Independently of age and AMH level, dysregulated immune profile is significatively associated with 3 times higher LBR than a non-deregulated profile (OR=3.4 CI 95%1.27-9.84) or five times in case of an overactive profile treated by immunotherapy (OR=5 CI 95% 1.65-16.5). The usage of ART was significantly associated with lower LBR regardless of the presence of a subfertility factor (p=0.012). Personalization of medical care using natural cycle or simple hormonal stimulation is associated with a significantly higher LBR than personalization including ART treatments regardless of maternal age and AMH level (OR= 2.9 CI 95% 1.03-8.88). Conclusion: Our study suggests that some endometrial immune profiles with targeted management of RPL are associated with a higher rate of LBR. ART may be negatively associated with LBR.

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.

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.

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.

Human decidual macrophages and NK cells differentially express Toll-like receptors and display distinct cytokine profiles upon TLR stimulation.

Maternofetal pathogen transmission is partially controlled at the level of the maternal uterine mucosa at the fetal implantation site (the decidua basalis), where maternal and fetal cells are in close contact. Toll-like receptors (TLRs) may play an important role in initiating rapid immune responses against pathogens in the decidua basalis, however the tolerant microenvironment should be preserved in order to allow fetal development. Here we investigated the expression and functionality of TLRs expressed by decidual macrophages (dMs) and NK cells (dNKs), the major decidual immune cell populations. We report for the first time that both human dMs and dNK cells express mRNAs encoding TLRs 1-9, albeit with a higher expression level in dMs. TLR2, TLR3, and TLR4 protein expression checked by flow cytometry was positive for both dMs and dNK cells. In vitro treatment of primary dMs and dNK cells with specific TLR2, TLR3, TLR4, TLR7/8, and TLR9 agonists enhanced their secretion of pro- and anti-inflammatory cytokines, as well as cytokines and chemokines involved in immune cell crosstalk. Only dNK cells released IFN-γ, whereas only dMs released IL-1ß, IL-10, and IL-12. TLR9 activation of dMs resulted in a distinct pattern of cytokine expression compared to the other TLRs. The cytokine profiles expressed by dMs and dNK cells upon TLR activation are compatible with maintenance of the fetotolerant immune environment during initiation of immune responses to pathogens at the maternofetal interface.

Seminal plasma peptides may determine maternal immune response that alters success or failure of pregnancy in the abortion-prone CBAxDBA/2 model.

Spontaneous abortion (resorption) in the DBA/2-mated CBA/J mouse involves a deficiency in Treg cell activity against paternal antigens at the time of mating. Preimmunization of female CBA/J by BALB/c splenocytes, but not DBA/2 splenocytes, protects against subsequent abortions after a CBAxDBA/2 mating. Previous immunogenetic studies with BALB/cxDBA/2 recombinants have indicated that H-2(d)-restricted presentation of a single minor non-H-2(d) peptide might be responsible for protection, while the product of a second independent allele might promote abortions. Using brefeldin-treated BALB/c and DBA/2 splenocytes, we found that incubation in BALB/c seminal plasma rendered DBA/2 splenocytes protective and DBA/2 seminal plasma eliminated protection. The active protective moiety was <10 kD consistent with a peptide. DBA/2 seminal plasma contained a <10-kD peptide that boosted the abortion rate. Maternal H-2(k) CBA/J splenocytes were unable to present the protective activity. Amicon fractionation also unmasked a <10-kD activity in DBA/2 seminal plasma that could boost abortion rates when presented by BALB/c splenocytes. SELDI-TOF mass spectrometry proteomic analysis of <10-kD filtrates reproducibly detected 1416, 1468, 1774 D peptides in BALB/c that were reduced or absent in DBA/2, and the presence of 2662, 4559 and 5320 D molecules in DBA/2, the latter two definitely not present in BALB/c. Direct antigen presentation of paternal H-2(d)-restricted paternal peptides (600-1800 D) may prevent the rejection of the CBAxDBA/2 embryos, and larger sized peptides may bind to immunizing splenocytes and augment abortion mechanisms.

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.

Tolerance to the foetal allograft?

In this review, we will detail the concept of tolerance and its history in reproductive immunology. We will then consider whether it applies to the foetal-maternal relationship and discuss the mechanisms involved in non-rejection of the foeto-placental unit.