Centromeric AA motif in KIR as an optimal surrogate marker for precision definition of alloimmune reproductive failure.

Throughout pregnancy, the decidua is predominantly populated by NK lymphocytes expressing Killer immunoglobulin-like receptors (KIR) that recognize human leukocyte antigen-C (HLA-C) ligands from trophoblast cells. This study aims to investigate the association of KIR-HLA-C phenotypes in couples facing infertility, particularly recurrent pregnancy loss (RPL) and recurrent implantation failure (RIF), in comparison to a reference population and fertile controls. This observational, non-interventional retrospective case-control study included patients consecutively referred to our Reproductive Immunology Unit from 2015 to 2019. We analyzed the frequencies of KIR and HLA-C genes. As control groups, we analyzed a reference Spanish population for KIR analysis and 29 fertile controls and their male partners for KIR and HLA-C combinations. We studied 397 consecutively referred women with infertility and their male partners. Among women with unexplained RPL (133 women) and RIF (176 women), the centromeric (cen)AA KIR genotype was significantly more prevalent compared to the reference Spanish population (p = 0.001 and 0.02, respectively). Furthermore, cenAA was associated with a 1.51-fold risk of RPL and a 1.2-fold risk of RIF. Conversely, the presence of BB KIR showed a lower risk of reproductive failure compared to non-BB KIR (OR: 0.12, p < 0.001). Women and their partners with HLA-C1C1/C1C1 were significantly less common in the RPL-Group (p < 0.001) and RIF-Group (p = 0.002) compared to the control group. Moreover, the combination of cenAA/C1C1 in women with C1C1 partners was significantly higher in the control group than in the RPL (p = 0.009) and RIF (p = 0.04) groups, associated with a 5-fold increase in successful pregnancy outcomes. In our cohort, the cenAA KIR haplotype proved to be a more accurate biomarker than the classic AA KIR haplotype for assessing the risk of RPL and RIF, and might be particularly useful to identify women at increased risk among the heterogeneous KIR AB or Bx population. The classification of centromeric KIR haplotypes outperforms classical KIR haplotypes, making it a better indicator of potential maternal-fetal KIR-HLA-C mismatch in patients.

Estado actual del estudio de las células NK en pacientes infértiles / Current study of nk cells NK in infertile patients

Las técnicas de reproducción asistida (TRA) se han convertido en una esperanza importante para muchas parejas que no pueden tener descendencia. A pesar de los avances realizados tanto desde el punto de vista técnico como en las tasas de éxito, todavía persiste un porcentaje significativo de parejas que no logran el embarazo o lo pierden. Existe una creciente evidencia de que ciertas alteraciones inmunológicas pueden estar implicadas en la fisiopatología de la infertilidad La mayoría de la información disponible se basa en estudios realizados en pacientes con Fallo Reproductivo Recurrente (FRR), término amplio que incluye abortos de repetición (AR) y fallos de implantación (FI). En estos pacientes se encuentran diferencias en ciertos parámetros inmunológicos con respecto a los controles fértiles. Las alteraciones inmunológicas más frecuentemente descritas relacionadas con infertilidad son: autoinmunidad, desequilibrio Th1 / Th2, expansión de las células naturales asesinas (NK), los receptores (KIR) de NK y su combinación con ciertos HLA embrionarios. La expansión tanto del número, porcentaje o actividad de las células NK representa un factor de riesgo en FRR, destacando el papel de estas células en la tolerancia materno-fetal. Se han propuesto varias terapias inmunomoduladoras en pacientes con FRR de causa inmunológica con distinto grado de evidencia clínica. Las infusiones de inmunoglobulina intravenosa (IgIV) se han utilizado durante mucho tiempo en la prevención de abortos espontáneos de causa inmunológica, ya sea de forma única o en combinación con otros agentes inmunomoduladores. Una adecuada selección de las pacientes en base a sus alteraciones inmunológicas es crítica para que el tratamiento inmunomodulador resulte efectivo aunque existen estudios sin criterios inmunológicos de selección. En la gestación los factores inmunológicos, hematológicos y hormonales juegan un papel relevante. Es necesario consolidar estos conceptos mediante ensayos clínicos y así ser capaces de estandarizar tanto la evaluación como la interpretación de las pruebas inmunológicas

Assisted reproduction techniques (ART) have become an important hope to many couples not able to have progeny. Though increasingly successful and technical advances, a significant proportion of couples fail to conceive or lose their pregnancies. There is a growing evidence of the role of several immunological disorders that may be involved in the pathophysiology of infertility. Most of the information available in this respect is based on studies in patients with recurrent reproductive failure (RRF), a broad term that includes recurrent miscarriages (RM) and repeated implantation failures (RIF) after in vitro fertilization (IVF). Several authors report alterations in certain immune parameters in these patients as compared with healthy fertile controls. More frequently changes described in various immune related infertility factors such as autoimmune pathology, Th1 / Th2 imbalance, expansion of natural killer cells (NK) and killer Ig-like receptors (KIRs) and HLA embryo matching have been described. Expanded counts, percentages and activity of NK cells represents a risk factor, highlighting the role of these cells in the maternal tolerance to the fetus. Various immunomodulatory therapies have been proposal in patients with RRF of immunological cause with different clinical evidence. Intravenous Immunoglobulin (IVIg) preparations have been used for a long time in the prevention of miscarriages of immunological cause, either alone or in combination with other immunomodulatory agents. Selection criteria of the patients by the immune alteration is critical in order to test the effectiveness of IVIg therapy although there are some studies in patients without any immune selection criteria. Immunological as well as hematological or hormonal factors play a relevant role in gestation. There is an urgent need of consolidating these data with clinical trials and of standardizing both the evaluation and interpretation of immunological tests

Experience in IVIg therapy for selected women with recurrent reproductive failure and NK cell expansion.

PROBLEM: Recurrent reproductive failure (RRF) has been associated with expansion of circulating NK cells, key cells for maternal tolerance, decidual vasculogenesis and embryo growth. This study reports our experience in intravenous immunoglobulin (IVIg) therapy of a large cohort of women with RRF with expanded circulating NK and/or NKT-like cells (blood NKT cells are a heterogeneous subset of T cells that share properties of both T cells and NK cells). METHOD OF STUDY: Observational study of RRF women with NK or NKT-like expansion (>12% or 10% cutoff levels of total lymphocytes, respectively), treated with IVIg for the next gestation. RESULTS: By multivariant logistic regression analysis after adjusting for age, NK cells subsets and other therapies, IVIg significantly improved the live birth rate to 96.3% in women with recurrent miscarriage (RM) compared with 30.6% in case not receiving IVIg (P < 0.0001). In women with recurrent implantation failure (RIF), in comparison with women not receiving IVIg, treatment increased the pregnancy rate from 26.2 to 93.8% (P ≤ 0.0001) and the live birth rate from 17.9 to 80.0% in RIF (P ≤ 0.0001). CONCLUSIONS: Immunomodulation with IVIg in our selected group of RRF patients with immunologic alterations enhanced clinical pregnancy and live birth rates. Our results may facilitate the design of future clinical trials of IVIg in this pathology.

New decision-tree model for defining the risk of reproductive failure.

PROBLEM: Natural killer (NK) cells play a key role in embryo implantation and pregnancy success, whereas blood and uterine NK expansions have been involved in the pathophysiology of reproductive failure (RF). Our main goal was to design in a large observational study a tree-model decision for interpretation of risk factors for RF. METHODS OF STUDY: A hierarchical multivariate decision model based on a classification and regression tree was developed. NK and NKT-like cell subsets were analyzed by flow cytometry. RESULTS: By multivariate analysis, blood NK cells expansion was an independent risk factor for RF (both recurrent miscarriages and implantation failures). We propose a new decision-tree model for the risk interpretation of women with RF based on a combination of main risk factors. CONCLUSIONS: Women with age above 35 years and >13% CD56⁺CD16⁺ NK cells showed the highest risk of further pregnancy loss (100%).

Endometrial expression of selected genes in patients achieving pregnancy spontaneously or after ICSI and patients failing at least two ICSI cycles.

The objective of this study was to identify the endometrial gene expression profile in receptive phase, which could represent a useful prognostic tool for selecting IVF patients. Endometrial expression of 47 selected genes biopsied during the window of implantation in natural cycles was compared between patients who achieved a successful pregnancy spontaneously or after subsequent intracytoplasmic sperm injection (ICSI) cycles and patients who did not achieve a pregnancy after at least two failed ICSI cycles. The comparative analysis showed significantly different levels of expression in 19 genes, five implicated in apoptosis (CASP8, FADD, CASP10, APAF1, ANXA4), three in immunity (LIF, SPP1, C4BPA), five in transcriptional activity (MSX1, HOXA10, MSX2, HOXA11, GATA2), two in lipid metabolism (LEPR, APOD) and four in oxidative metabolism (AOX1, ALDH1A3, GPX3, NNMT). The evidence for these genes being differently expressed could represent the starting point of identifying the ideal receptive endometrial gene expression profile, which could be used in the future as a prognostic tool for IVF patients. Gene expression analysis technology has opened new important perspectives on the study of the physiological processes of different tissues and organs. Specifically for the endometrium, it would be really interesting to find out an endometrial gene expression profile of receptive phase, which could be used in future as a useful prognostic tool for selecting IVF patients. To achieve this aim, the objective of the present paper was the comparison of endometrial expression in natural cycles of 47 selected genes between the biopsies of patients who achieved a successful pregnancy, either spontaneously or after subsequent ICSI cycles, and those of patients who did not achieve a pregnancy after at least two failed ICSI cycles. The comparative analysis showed a significant different expression in 19 genes: five implicated in programmed cell death, known as apoptosis (CASP8, FADD, CASP10, APAF1, ANXA4), three in immunity (LIF, SPP1, C4BPA), five in transcriptional activity (MSX1, HOXA10, MSX2, HOXA11, GATA2), two in lipid metabolism (LEPR, APOD) and four in oxidative metabolism (AOX1, ALDH1A3, GPX3, NNMT). The evidence of these genes being differently expressed could represent the starting point of identifying the ideal receptive endometrial gene expression profile which could be used in the future as a prognostic tool for IVF patients.

Intravenous immunoglobulin treatment increased live birth rate in a Spanish cohort of women with recurrent reproductive failure and expanded CD56(+) cells.

PROBLEM: Natural killer (NK, CD3(-)CD56(+)/CD16(+)) and NKT-like cells (CD3(+)CD56(+)/CD16(+)) activity is considered among the key factors for reproductive success. In the absence of immunological screening, beneficial effects of intravenous immunoglobulin (IVIG) in preventing recurrent reproductive failure (RRF) have not been reported. Here, we analyse the IVIG influence on pregnancy success in women with RRF and circulating NK or/and NKT-like cells expansion. METHOD OF STUDY: One hundred fifty-seven women with previous recurrent miscarriage and/or recurrent implantation failure after in vitro fertilization were consecutively studied. Sixty-four patients with CD56(+) cell expansion, no apparent underlying disease and who maintained their desire to conceive were selected. Forty of them received IVIG during pregnancy. RESULTS: Overall, the clinical pregnancy rate for the women under IVIG therapy was 92.5% and the live birth rate was 82.5%. Significantly lower pregnancy and live birth rates (25% and 12.5%, respectively) were observed for the patients with recurrent pregnancy loss and NK/NKT-like cells expansion without IVIG. After three cycles of IVIG, NK cell percentages decreased significantly and these values persisted throughout gestation. CONCLUSION: Intravenous immunoglobulin therapy for women with RRF and NK or NKT-like cell expansion was a safe and beneficial therapeutic strategy that associated with high clinical pregnancy and live birth rates.