An increase in regulatory T cells in peripheral blood correlates with an adverse prognosis for malignant melanoma patients - A study of T cells and natural killer cells.

Malignant melanoma is a highly immunogenic tumour, and the immune profile significantly influences cancer development and response to immunotherapy. The peripheral immune profile may identify high risk patients. The current study showed reduced levels of CD4+ T cells and increased levels of CD8+ T cells in peripheral blood from malignant melanoma patients compared with controls. Percentages of peripheral CD56dimCD16+ NK cells were reduced and CD56brightCD16-KIR3+ NK cells were increased in malignant melanoma patients. Late stage malignant melanoma was correlated with low levels of CD4+ T cells and high levels of CD56brightCD16-KIR3+ NK cells. Finally, high levels of Tregs in peripheral blood were correlated with poor overall survival and disease-free survival. The results indicate that changes in specific immune cell subsets in peripheral blood samples from patients at the time of diagnosis may be potential biomarkers for prognosis and survival. Further studies will enable clarification of independent roles in tumour pathogenesis.

Investigations of leukocyte and inflammatory markers in pregnancies complicated by preeclampsia.

OBJECTIVES: Preeclampsia is a frequent and potentially fatal pregnancy complication. It can be challenging to make a timely diagnosis. Identifying clinically useful biochemical markers would be a remedying tool to support the diagnosis of preeclampsia. The aim was to investigate differential cell counts and acute phase reactants as diagnostic markers of preeclamptic third-trimester pregnancies and in relation to pregnancy term, gravidity and the severity of hypertension. METHODS: Based on a cohort of 421 pregnant women, we included 174 participants (case n = 84, control n = 90) during the third trimester. Peripheral blood was sampled to measure differential white blood cell counts and acute phase reactants on the day of inclusion. RESULTS: The neutrophil-to-lymphocyte ratio and plasma haptoglobin levels were significantly increased in healthy pregnancies compared with preeclamptic pregnancies. Plasma ferritin levels and albumin levels were respectively increased and decreased in cases of preeclampsia compared with controls. Albumin was specific among multigravida. Plasma transferrin and high-sensitivity C-reactive protein (hs-CRP) levels were significantly decreased and increased, respectively, in cases with preterm preeclampsia compared with term preeclampsia. CONCLUSION: Plasma ferritin and albumin levels reflected higher inflammation in cases with preeclampsia compared with healthy pregnancies; the same did plasma transferrin and hs-CRP levels in preterm versus term preeclampsia. When considering the normal ranges plasma albumin and hs-CRP levels identified preeclamptic from healthy third-trimester pregnancies and preterm from term preeclampsia cases, respectively, with near-acceptable diagnostic performances. Further validation of the diagnostic value will require larger sample-sized studies with paired plasma and serum samples.

Opposing impacts of HLA-G haplotypes PROMO-G010104-UTR-3 and PROMO-G010101b/c-UTR-4 on risk of recurrent implantation failure.

RESEARCH QUESTION: The human leukocyte antigen (HLA) class Ib molecules HLA-F and HLA-G are implicated in pregnancy success, but how do HLA-G and HLA-F genetic polymorphisms impact recurrent implantation failure (RIF)? DESIGN: Prospective cohort study at a fertility clinic including a cohort of 84 women experiencing RIF and 35 IVF controls to assess the influence of HLA-G haplotypes and diplotypes and HLA-F single nucleotide polymorphisms (SNP) on RIF. RESULTS: Over-representation trends for HLA-F SNP genotypes rs1362126, rs2523405 and rs2523393, previously linked with a short time-to-pregnancy, were detected in female control groups compared with RIF patients with no identified pathology linked to infertility. The HLA-G promoter haplotype PROMO-G010101b/c linked with the HLA-G 3'-untranslated region (3'UTR) haplotype UTR-4, which previously has been associated with positive IVF outcome and pregnancy success, was less frequent in the RIF group. For RIF patients carrying the UTR-4 haplotype, the odds ratio (OR) was 0.27 (95% CI 0.12-0.66; P = 0.0044, Pc = 0.026). The HLA-G PROMO-G010104-UTR-3 haplotype was associated with an increased risk of RIF. For RIF patients carrying the UTR-3 haplotype, the OR was 5.86 (95% CI 1.52-26.23; P = 0.0115, Pc = 0.069). CONCLUSIONS: These results show that specific HLA-G haplotypes based on the promoter region and the 3'UTR are either associated with an increased risk of reduced fertility, including the manifestation of RIF, and lower chance of achieving pregnancy, or with a reduced risk of experiencing RIF.

Probiotic treatment with specific lactobacilli does not improve an unfavorable vaginal microbiota prior to fertility treatment-A randomized, double-blinded, placebo-controlled trial.

Objective: To investigate whether treatment with proprietary lactobacilli-loaded vaginal capsules improves an unfavorable vaginal microbiome diagnosed using a commercially available test and algorithm. Design: A randomized, double-blinded, placebo-controlled study was conducted in 74 women prior to undergoing fertility treatment at a single university fertility clinic between April 2019 and February 2021. The women were randomly assigned in a 1:1 ratio to receive one vaginal capsule per day for 10 days containing either a culture of more than 108 CFU of Lactobacillus gasseri and more than 108 CFU Lactobacillus rhamnosus (lactobacilli group) or no active ingredient (placebo group). Vaginal swabs for microbiota analysis were taken at enrollment, after treatment and in the cycle following treatment. Participants and methods: Women aged 18-40 years who prior to fertility treatment were diagnosed with an unfavorable vaginal microbiota, characterized by either a low relative load of Lactobacillus or a high proportion of disrupting bacteria using the criteria of the IS-pro™ diagnostic system (ARTPred, Amsterdam, the Netherlands), were enrolled in the study. The primary outcome measure was the proportion of women with improvement of the vaginal microbiota after intervention. Results: The vaginal microbiota improved after intervention in 34.2% of all participants (lactobacilli group 28.9%, placebo group 40.0%), with no significant difference in the improvement rate between the lactobacilli and placebo groups, RR = 0.72 (95% CI 0.38-1.38). Conclusion: This study indicates that administering vaginal probiotics may not be an effective means of modulating the vaginal microbiome for clinical purposes in an infertile population. However, a spontaneous improvement rate of 34.2% over a period of one to three months, confirming the dynamic nature of the vaginal microbiota, indicates that a strategy of postponing further IVF treatment to await microbiota improvement may be relevant in some patients, but further research is needed. Clinical trial registration: ClinicalTrials.gov, identifier NCT03843112.

Endometrial HLA-F expression is influenced by genotypes and correlates differently with immune cell infiltration in IVF and recurrent implantation failure patients.

STUDY QUESTION: Is human leukocyte antigen (HLA)-F protein expressed in mid-secretory endometrium, and are its expression levels influenced by HLA-F gene polymorphisms and correlated with the abundance of uterine natural killer (uNK) cells and anti-inflammatory M2 macrophages? SUMMARY ANSWER: HLA-F protein is expressed in mid-secretory endometrium, and levels are correlated with immune cell infiltration, plasma progesterone concentrations and HLA-F single-nucleotide polymorphisms (SNPs), however, women experiencing recurrent implantation failure (RIF) show differences when compared to women attending their first IVF treatment. WHAT IS KNOWN ALREADY: The immunomodulatory HLA class Ib molecules HLA-G and HLA-F are expressed on the extravillous trophoblast cells and interact with receptors on maternal immune cells. Little is known regarding HLA-F expression in endometrial stroma and HLA-F function; furthermore, HLA-F and HLA-G SNP genotypes and haplotypes have been correlated with differences in time-to-pregnancy. STUDY DESIGN, SIZE, DURATION: Primary endometrial stromal cell (ESC) cultures (n = 5) were established from endometrial biopsies from women attending IVF treatment at a fertility clinic. Basic HLA-F and HLA-G protein expression by the ESCs were investigated. A prospective controlled cohort study was performed including 85 women with a history of RIF and 36 control women beginning their first fertility treatment and with no history of RIF. In some analyses, the RIF group was divided into unknown cause, male infertility, female infertility, and both female and male infertility. Endometrial biopsies and blood samples were obtained the day equivalent to embryo transfer in a hormone-substituted cycle. PARTICIPANTS/MATERIALS, SETTING, METHODS: HLA protein expression by ESCs was characterized using flow cytometry and western blot. In the cohort study, the specific immune markers HLA-F and HLA-G, CD56 and CD16 (NK cells), CD163 (M2 macrophages), FOXP3 (regulatory T cells) and CD138 (plasma cells) were analysed by immunohistochemistry and a digital image analysis system in endometrial biopsies. Endometrial receptivity was assessed by an endometrial receptivity array test (the ERA® test). Endometrial biopsies were examined according to modified Noyes' criteria. SNPs at the HLA-F gene and HLA-G haplotypes were determined. MAIN RESULTS AND THE ROLE OF CHANCE: HLA-F protein is expressed in the endometrium at the time of implantation. Furthermore, the HLA-F protein levels were different according to the womens HLA-F SNP genotypes and diplotypes, which have previously been correlated with differences in time-to-pregnancy. Endometrial HLA-F was positively correlated with anti-inflammatory CD163+ M2 macrophage infiltration and CD56+ uNK cell abundance for the entire cohort. However, this was not the case for CD56+ in the female infertility RIF subgroup. HLA-F levels in the endometrial stroma were negatively correlated with plasma progesterone concentrations in the RIF subgroup with known female infertility. Conversely, HLA-F and progesterone were positively correlated in the RIF subgroup with infertility of the male partner and no infertility diagnosis of the woman indicating interconnections between progesterone, HLA-F and immune cell infiltration. Glandular sHLA-G expression was also positively correlated with uNK cell abundance in the RIF subgroup with no female infertility but negatively correlated in the RIF subgroup with a female infertility diagnosis. LARGE SCALE DATA: Immunohistochemistry analyses of endometrial biopsies and DNA sequencing of HLA genes. Data will be shared upon reasonable request to the corresponding author. LIMITATIONS, REASONS FOR CAUTION: The control group of women attending their first IVF treatment had an anticipated good prognosis but was not proven fertile. A significant age difference between the RIF group and the IVF group reflects the longer treatment period for women with a history of RIF. The standardization of hormonal endometrial preparation, which allowed consistent timing of endometrial and blood sampling, might be a strength because a more uniform hormonal background may more clearly show an influence on the immune marker profile and HLA class Ib levels in the endometrium by other factors, for example genetic polymorphisms. However, the immune marker profile might be different during a normal cycle. WIDER IMPLICATIONS OF THE FINDINGS: The findings further highlight the importance of HLA-F and HLA-G at the implantation site and in early pregnancy for pregnancy success. Diagnostic measures and modulation of the complex interactions between HLA class Ib molecules, maternal immune cells and hormonal factors may have potential to improve fertility treatment. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Region Zealand Health Sciences Research Foundation and the Zealand University Hospital through the ReproHealth Research Consortium ZUH. The authors declared there are no conflicts of interest.

HLA Class Ib-receptor interactions during embryo implantation and early pregnancy.

BACKGROUND: Although the immune system intuitively must have an important role in embryo implantation and in the achievement of a pregnancy, the molecular details have for long been controversial. The role of the human leukocyte antigen (HLA) system has been debated. The unique HLA expression profile of the HLA Class Ia molecule HLA-C and the HLA Class Ib molecules HLA-E, HLA-F and HLA-G at the feto-maternal interface is now recognized. However, HLA Class Ib molecules may also have a role in embryo implantation and pregnancy success. OBJECTIVE AND RATIONALE: The aim of this review was to evaluate the literature and recent discoveries on the role of the non-polymorphic HLA Class Ib molecules with a focus on HLA-F and HLA-G molecules at the time of implantation, including the interaction with uterine immune cells through the specific receptors immunoglobulin-like transcript 2 (ILT2), ILT4 and a number of killer cell immunoglobulin-like receptors (KIRs), and the importance of HLA-F and HLA-G genetic variation that influences fertility and time-to-pregnancy. SEARCH METHODS: Drawing on recent advances in basic and clinical studies, we performed a narrative review of the scientific literature to provide a timely update on the role of HLA Class Ib in embryo implantation, fertility and infertility. Pertinent studies were searched in PubMed/Medline using relevant key words. OUTCOMES: Both HLA-F and HLA-G interact with inhibitory or activating ILT2 or ILT4 receptors and KIRs on uterine immune cells, especially uterine natural killer (NK) cells that are highly abundant in the mid-secretory endometrium and in early pregnancy. The binding of HLA-G to ILT2 stimulates the secretion of growth-promoting factors from decidual NK cells. However, functional aspects of a HLA-F-receptor interaction remain to be clarified. Recent studies indicate that HLA-F and HLA-G are expressed in mid-secretory endometrium and HLA-G is expressed in the blastocyst. HLA-F fluctuates during the menstrual cycle with high levels during the implantation window. The level of HLA-F protein expression correlates with the number of CD56-positive NK cells in the mid-secretory endometrium. HLA-F and HLA-G gene polymorphisms, including a single nucleotide polymorphism (SNP) in a progesterone-responsive element, are associated with time-to-pregnancy. Depending on the SNP genotype, the effect of progesterone varies resulting in differences in HLA-F expression and thereby the interaction with receptors on the uterine NK cells. Studies suggest that the expression of HLA-G and HLA-F, both by the embryonic-derived trophoblast cells and by cells in the endometrium and decidua, and the interaction between HLA-G and HLA-F with specific receptors on uterine immune cells, stimulate and facilitate embryo implantation and placentation by secretion of growth factors, cytokines and angiogenic factors. WIDER IMPLICATIONS: A detailed understanding of the molecular mechanisms controlling the expression of HLA-F and HLA-G periconceptionally and in early pregnancy may improve the success of ART and holds promise for further insight into pathophysiological aspects of certain pregnancy complications.

An exploration of immunohistochemistry-based prognostic markers in patients undergoing curative resections for colon cancer.

BACKGROUND: The immune system recognizes and destroys cancer cells. However, cancer cells develop mechanisms to avoid detection by expressing cell surface proteins. Specific tumour cell surface proteins (e.g. HLA-G, PD-L1, CDX2) either alone or in combination with the relative presence of immune cells (CD3 and CD8 positive T-cells) in the tumour tissue may describe the cancer cells' ability to escape eradication by the immune system. The aim was to investigate the prognostic value of immunohistochemical markers in patients with colon cancer. METHODS: We conducted a retrospective study including patients diagnosed with pT3 and pT4 colon cancers. Immunohistochemical staining with HLA-G, PD-L1, CDX2, CD3, and CD8 was performed on tissue samples with representation of the invasive margin. PD-L1 expression in tumour cells and immune cells was reported conjointly. The expression of CD3 and CD8 was reported as a merged score based on the expression of both markers in the invasive margin and the tumour centre. Subsequently, a combined marker score was established based on all of the markers. Each marker added one point to the score when unfavourable immunohistochemical features was present, and the score was categorized as low, intermediate or high depending on the number of unfavourable stains. Hazard ratios for recurrence, disease-free survival and mortality were calculated. RESULTS: We included 188 patients undergoing colon cancer resections in 2011-2012. The median follow-up was 41.7 months, during which 41 (21.8%) patients had recurrence and 74 (39.4%) died. In multivariable regression analysis positive HLA-G expression (HR = 3.37, 95%CI [1.64-6.93]) was associated with higher recurrence rates, while a preserved CDX2 expression (HR = 0.23, 95%CI [0.06-0.85]) was associated with a lower risk of recurrence. An intermediate or high combined marker score was associated with increased recurrence rates (HR = 20.53, 95%CI [2.68-157.32] and HR = 7.56, 95%CI [1.06-54.16], respectively). Neither high expression of PD-L1 nor high CD3-CD8 score was significantly associated with recurrence rates. Patients with a high CD3-CD8 score had a significantly longer DFS and OS. CONCLUSIONS: In tumour cells, expression of HLA-G and loss of CDX2 expression were associated with cancer recurrence. In addition, a combination of certain tumour tissue biomarkers was associated with colorectal cancer recurrence.

Peripheral blood immune markers in breast cancer: Differences in regulatory T cell abundance are related to clinical parameters.

BACKGROUND: Cancer development is among other factors driven by tumor immune escape and tumor-mediated changes in the immune response. Investigating systemic immune changes may provide important knowledge for the improvement of patient prognosis and treatment opportunities. METHODS: The systemic immune profile of patients with ER-positive breast cancer (n = 22) and healthy controls (n = 30) was investigated based on complete blood counts, flow cytometric analysis of T cell subsets including regulatory T cells (Tregs), and immune assays investigating soluble (s)HLA-G and the cytokine profile in plasma. We further examined the correlation between the immune markers and clinical parameters including tumor size, tumor grade and lymph node involvement. RESULTS: Results indicated that breast cancer patients possessed a higher amount of neutrophils and monocytes and fewer lymphocytes and eosinophils compared with healthy controls. Breast cancer patients had significantly more CD25+CD127low Tregs than controls, and both lymphocyte and Treg numbers were negatively correlated with tumor size. Furthermore, Treg numbers were elevated in grade I tumors compared with grade II tumors and with healthy controls. No difference in sHLA-G levels was observed between patients and controls. Higher levels of IL-6 and TNF-α were observed in breast cancer patients. Cytokine and sHLA-G levels were not associated with clinical parameters. CONCLUSION: The results of this exploratory study contribute to the elucidation of the systemic immune response in breast cancer indicating a potential use of peripheral immune cell counts and Tregs to distinguish patients from healthy controls and as potential diagnostic and prognostic biomarkers to be investigated in future studies.

Tumour-infiltrating CD4-, CD8- and FOXP3-positive immune cells as predictive markers of mortality in BRCA1- and BRCA2-associated breast cancer.

BACKGROUND: The prognostic value of tumour-infiltrating lymphocytes (TILs) in breast cancer is well-established. However, the investigation of specific T-cell subsets exclusively in BRCA-associated breast cancer is sparse. METHODS: Tumour tissues from 414 BRCA-mutated breast cancer patients were analysed by immunohistochemistry and digital image analysis for expression of CD4, CD8 and FOXP3 immune markers. Distribution of CD4-, CD8- and FOXP3-positive cells and clinicopathological characteristics were assessed according to groups of low or high expression. The prognostic value was evaluated as continuous variables in univariate and multivariate analyses of overall survival and disease-free survival. RESULTS: Both CD4 and CD8 expression are associated with histological diagnosis, tumour grade and oestrogen and progesterone receptor expression status. CD4 expression is associated with BRCA gene status. A high percentage of tumour-infiltrating CD4-, CD8- or FOXP3-positive cells is significantly associated with lower mortality in BRCA1- and BRCA2-associated breast cancer and CD8-positive cells are associated with disease-free survival. No heterogeneity according to BRCA gene status was found for the prognostic value of the immune markers. CONCLUSIONS: The results support a prognostic role of specific T-cell subsets in BRCA-associated breast cancer and the promising potential of targeting the immune system in the treatment of these patients.

Evidence for a shift in placental HLA-G allelic dominance and the HLA-G isoform profile during a healthy pregnancy and preeclampsia†.

Human leukocyte antigen (HLA)-G, which belongs to a nonclassical class Ib major histocompatibility complex gene family expressed by placental trophoblast cells, plays a central role in establishing tolerance to the semiallogeneic fetus and in placentation. HLA-G exists in different soluble or membrane-bound isoforms. Preeclampsia, a major cause of fetal and maternal morbidity and mortality, has been linked to insufficient placentation and an altered immune response in pregnancy, including altered HLA-G expression. The 14 bp insertion/deletion polymorphism in the 3' untranslated region of the gene and the isoform profile may affect HLA-G expression. The aim of the current pilot study was to characterize the expression patterns of HLAG mRNA, protein, and isoform profile in uncomplicated term pregnancies and in cases of preeclampsia. Maternal sHLA-G mRNA and protein levels were slightly reduced in preeclampsia. No difference was found for placental blood, and no correlation between peripheral and placental sHLA-G levels was found. We observed no association between neither fetal nor maternal HLA-G 14 bp insertion/deletion genotypes and preeclampsia, nor a significant difference in isoform profiles. However, in HLA-G 14 bp insertion/deletion heterozygous placental samples, we observed abundant HLA-G1 14 bp insertion allele expression in the term placentae, which is contrary to previous findings in first trimester trophoblast. Increased HLA-G1 14 bp insertion allele expression in the placenta was associated with reduced levels of placental sHLA-G and an altered isoform profile with increased relative levels of HLA-G1 and -G5 and reduced levels of HLA-G3. The results indicate that an allelic shift in heterozygous individuals could represent a novel regulatory pathway.

Maternal HLA Ib Polymorphisms in Pregnancy Allo-Immunization.

During pregnancy the formation of alloreactive anti-human leukocyte antigen (HLA) antibodies are a major cause of acute rejection in organ transplantation and of adverse effects in blood transfusion. The purpose of the study was to identify maternal HLA class Ib genetic factors associated with anti-HLA allo-immunization in pregnancy and the degree of tolerance estimated by IgG4 expression. In total, 86 primiparous women with singleton pregnancies were included in the study. Maternal blood samples and umbilical cord samples were collected at delivery. Clinical data were obtained. Maternal blood serum was screened for HLA class I and II antibodies, identification of Donor Specific Antibody (DSA), activation of complement measured by C1q and IgG4 concentrations. Mothers were genotyped for HLA class Ib (HLA-E, -F and -G). Anti-HLA class I and II antibodies were identified in 24% of the women. The maternal HLA-E*01:06 allele was significantly associated with a higher fraction of anti-HLA I immunization (20.0% vs. 4.8%, p = 0.048). The maternal HLA-G 3'-untranslated region UTR4-HLA-G*01:01:01:05 haplotype and the HLA-F*01:03:01 allele were significantly associated with a low anti-HLA I C1q activation (16.7% vs. 57.1%, p = 0.028; 16.7% vs. 50.0%, p = 0.046; respectively). Both HLA­G and HLA-F*01:03:01 showed significantly higher levels of IgG4 compared with the other haplotypes. The results support an association of certain HLA class Ib alleles with allo-immunization during pregnancy. Further studies are needed to elucidate the roles of HLA-E*01:06, HLA-F*01:03 and HLA­G UTR4 in reducing the risk for allo-immunization.

Are different markers of endometrial receptivity telling us different things about endometrial function?

PROBLEM: To what extent do endocrine, immunological, gene expression and histological markers of endometrial receptivity correlate? METHOD OF STUDY: Between November 2017 and September 2019, 121 women referred to a University Hospitals Fertility Clinic consented to inclusion in this cohort study. The women underwent timed endometrial biopsy followed by blood samples in a hormone-substituted cycle. Of these, 37 women had just started IVF treatment, and the remaining 84 had experienced recurrent implantation failure following IVF/ICSI. The hormone-substituted cycle consisted of initiation with oral oestradiol followed by addition of vaginal progesterone treatment for five full days. Endometrial biopsies were subject to histological examination, immune cell markers by immunohistochemistry (CD56+ , CD16+ , CD163+ , FoxP3) and gene expression microarray analyses with the endometrial receptivity array (ERA® ) test (Igenomix). Plasma progesterone and oestradiol were measured on the day of biopsy. RESULTS: CD56+ uterine natural killer (uNK) cell counts correlate with transcriptional markers of endometrial receptivity assessed by the ERA test. Endometrial maturation, receptivity and immunological markers were not correlated with mid-luteal blood plasma progesterone level. Mid-luteal serum oestradiol level correlated with markers of endometrial maturation and receptivity. The tests were carried out during a standard hormone substitution cycle, and the findings may not apply in the natural cycle. CONCLUSION: CD56+ uNK cell counts and endometrial receptivity assessed by the ERA test appear to be linked. Mid-luteal progesterone levels were not correlated to the tested markers of endometrial receptivity. In contrast, mid-luteal oestradiol level was inversely related to markers of endometrial receptivity and maturation.

Examining extended human leukocyte antigen-G and HLA-F haplotypes: the HLA-G UTR-4 haplotype is associated with shorter time to pregnancy in an infertility treatment setting when both female and male partners are carriers.

OBJECTIVE: To study the impact of extended human leukocyte antigen (HLA)-G and HLA-F haplotypes on time to pregnancy as measured by the number of treatment cycles in a cohort of couples in infertility treatment. DESIGN: Prospective cohort study of couples undergoing infertility treatment. SETTING: University hospital. PATIENT(S): A cohort of 127 couples and four single women in infertility treatment. INTERVENTION(S): Next-generation sequencing of the HLA-G gene and genotyping of three HLA-F locus single-nucleotide polymorphisms (SNPs). MAIN OUTCOME MEASURE(S): Extended HLA-F.HLA-G haplotypes, HLA-G promoter haplotypes and HLA-G 3'UTR haplotypes and their association with time to pregnancy as measured by number of treatment cycles until achievement of pregnancy with a live birth. Linkage disequilibrium between HLA-G variations and three HLA-F locus SNPs that impact time to pregnancy. RESULT(S): The effect of the HLA-G 3'UTR haplotype, UTR-4, was significantly increased, or modified, if the partner was a carrier compared to being a noncarrier. Extended HLA-F.HLA-G haplotypes, HLA-G promoter haplotypes, and the HLA-G 14 bp indel of the female partners were not associated with time to pregnancy. However, a trend for an association of the HLA-G 14bp insertion allele with a higher frequency of miscarriage than the 14bp deletion allele was observed. Certain HLA-G variations are in linkage disequilibrium with three HLA-F locus SNPs that influence time to pregnancy. CONCLUSION(S): HLA-G UTR-4 is significantly associated with time to pregnancy in couples undergoing infertility treatment. The findings could imply that both male and female HLA class Ib genetics have clinical relevance in reproduction.

Variation in the HLA-F gene locus with functional impact is associated with pregnancy success and time-to-pregnancy after fertility treatment.

STUDY QUESTION: The aim of this study was to investigate a possible influence of three single nucleotide polymorphisms (SNPs) in the HLA-F gene locus on time-to-pregnancy and pregnancy success after fertility treatment. SUMMARY ANSWER: HLA-F SNP genotypes and HLA-F diplotypes are associated with the number of fertility treatment cycles needed to achieve pregnancy and live birth. WHAT IS KNOWN ALREADY: HLA class Ib molecules, including HLA-F, which are known to be expressed by extra-villous trophoblast cells have immunomodulatory properties and play a role at the feto-maternal interface. However, a few recent studies suggest that HLA-F expressed in the mid-luteal endometrium may play a part in the establishment of pregnancy as well. Three genetic polymorphisms in the HLA-F gene locus influence the expression of HLA-F in the mid-luteal endometrium and are associated with time-to-pregnancy in healthy women. STUDY DESIGN, SIZE, DURATION: The current study included 102 female patients and 91 male patients attending for ART treatment and recruited between 2009 and 2014 at fertility clinics in a University Hospital setting, and 78 fertile female controls recruited in 2017 and 2018 at a department of Obstetrics and Gynaecology in a University Hospital. All women in the control group conceived naturally, and no other clinical data for the controls were retrieved. PARTICIPANTS/MATERIALS, SETTING, METHODS: Genotyping of genomic DNA from blood samples was performed with Sanger sequencing for the three SNPs of interest in the HLA-F gene locus: rs1362126 (G/A), rs2523405 (T/G) and rs2523393 (A/G). Furthermore, clinical data were collected for the couples in fertility treatment. MAIN RESULTS AND THE ROLE OF CHANCE: There were no significant differences in the distributions of the three HLA-F SNP genotypes and alleles between the female fertile control group and the female infertility group. We considered if the number of treatment cycles was related to the HLA-F SNP genotypes and HLA-F diplotypes in a discrete time to event analyses. A significant association with longer time-to-pregnancy, measured as number of fertility treatment cycles, was observed for women in the ART group who carried the HLA-F genotypes that are associated with a lower amount of HLA-F mRNA expressed in mid-luteal endometrium. For the rs1362126 AA genotype relative to the GG genotype, the odds ratio (OR) was 0.30 (95% CI = 0.10-0.87, P = 0.02); for the rs2523405 GG genotype relative to the TT genotype, the OR was 0.40 (95% CI = 0.15-1.04, P = 0.06); and for the rs2523393 GG genotype relative to the AA genotype, the OR was 0.27 (95% CI = 0.09-0.78, P = 0.01). In addition to comparing the HLA-F genotypes by a standard likelihood-ratio test, a trend test based on the number of G or A alleles were also performed. The HLA-F genotypes associated with longer time-to-pregnancy in these tests were as follows: number of A alleles at rs1362126 (P = 0.01), the OR was 0.56 per A allele (95% CI = 0.35-0.89); number of G alleles at rs2523405 (P = 0.05), OR was 0.65 per G allele (95% CI = 0.42-1.00); and number of G alleles at rs2523393 (P = 0.01), OR was 0.56 per G allele (95% CI = 0.36-0.86). On average, for the rs1362126 SNP, 2.1 more treatment cycles for a woman who carried the AA genotype were needed to achieve pregnancy within the first eight treatment cycles compared with a woman who carried the GG genotype. Likewise, for the rs2523405 SNP, 1.8 more cycles for the GG genotype compared with the TT genotype were needed, and for the rs2523393 SNP, 2.2 more treatment cycles for a woman who carried the GG genotype compared with a woman who carried the AA genotype were needed. Adjustments for the covariates BMI, female age, IVF (yes/no for each cycle), ICSI (yes/no for each cycle), female factor (yes/no) and male factor (yes/no), were also performed modeling the cycle-specific probabilities and the genotypes remained significant and almost unchanged. LIMITATIONS, REASONS FOR CAUTION: Specific types of ART will be chosen from the start of treatment, which means that the chances of achieving pregnancy could differ between the women solely due to their first line of treatment. However, multivariate analyses are performed to adjust for type of ART treatment. WIDER IMPLICATIONS OF THE FINDINGS: To our knowledge, this is the first study that shows associations between, and implications of, HLA-F gene locus variation and time-to-pregnancy and pregnancy success in a clinical setting for fertility treatment/ART. STUDY FUNDING/COMPETING INTEREST(S): Supported by the Region Zealand Health Sciences Research foundation and by Zealand University Hospital through the ReproHealth Research Consortium ZUH. The authors declare no conflict of interest.

A role for both HLA-F and HLA-G in reproduction and during pregnancy?

The human major histocompatibility complex includes a group of non-classical HLA class I genes, HLA-E, -F and -G. While nearly all focus since the discovery of these class Ib molecules have been on basic biochemistry and molecular biology of HLA-G and HLA-E, as well as their expression patterns, functions in immune modulation and during pregnancy, and also possible implications in a range of diseases, in infertility and pregnancy complications, HLA-F has nearly been ignored. However, recent discoveries show that HLA-F can be expressed as both open conformers binding to a number of KIRs on primarily NK cells, as well as peptide-bound HLA-F binding to ILT2 and ILT4. Furthermore, a number of reports indicate a possible involvement of HLA-F in viral infections, in cancer immunology, and in fertility and reproduction, which may initiate more interest in this rather unknown HLA class I molecule. In this short review, we focus on recent discoveries that indicate a functional role for HLA-F in reproduction and during pregnancy, and the role of HLA-F in relation to HLA-G.

Soluble HLA-G and TGF-ß in couples attending assisted reproduction - A possible role of TGF-ß isoforms in semen?

Soluble isoforms of the non-classical Human Leukocyte Antigen (HLA)-G as well as Transforming Growth Factor (TGF)-ß is expressed in seminal plasma possibly influencing the pregnancy potential. We wanted to examine the association of seminal plasma sHLA-G, TGF-ß1, TGF-ß2 and TGFß3 with pregnancy success in a cohort of 127 couples and 4 single women attending fertility treatment with the use of assisted reproduction technologies (ART). Soluble HLA-G, TGF-ß1, TGF-ß2 and TGF-ß3 in seminal plasma did not fluctuate significantly over time. We did not find any impact of seminal plasma sHLA-G, TGF-ß1, TGF-ß2 and TGF-ß3 on time-to-pregnancy measured as number of treatment cycles. There was a significant association between concentrations of seminal plasma sHLA-G and HLA-G variations in the 3'untranslated region (3'UTR) of the HLA-G gene, supporting and extending previous findings. Furthermore, by comparing seminal plasma concentrations of sHLA-G, TGF-ß1, TGF-ß2 and TGF-ß3 in male subjects with reduced semen quality, male subjects with normal semen quality, and sperm donors, we found that TGF-ß2 was significantly lower, and TGF-ß3 was significantly higher, in seminal plasma from sperm donors. These findings suggest that TGF-ß isoforms may influence semen quality and fertility.

Reflections upon immunological mechanisms involved in fertility, pregnancy and parasite infections.

During a pregnancy, the mother accepts her semi-allogeneic fetus with no signs of immunological rejection. Therefore, some modulation of the maternal immune system must occur. Similarly, changes in the host's immune system occurs during infections with parasites. In a study conducted in an endemic area in Bolivia, it has been reported that women infected with either the helminthic parasite roundworm or hookworm were estimated to give birth to either two more, or three fewer, children than uninfected, endemic women, respectively. Immune regulation by helminthic parasites is a rather well-researched concept, but there are few reports on the effects on human fecundity. The current review focuses on mechanisms of possible importance for especially the increased fertility rates in women infected with roundworm. The host immune response to roundworm has been hypothesized to be more favourable for a successful pregnancy because it bears resemblance to the anti-inflammatory immunological responses observed in pregnancy, steering the immunological response away from a pro-inflammatory state that seem to suppress fecundity. Further research into parasitic worm interactions, fertility, and the molecular mechanisms that they unfold may widen our understanding of the immunomodulatory pathways in both helminthic infections and in fertility and pregnancy.

The Tolerogenic Function of Regulatory T Cells in Pregnancy and Cancer.

Regulatory T cells, a subpopulation of suppressive T cells, are potent mediators of self-tolerance and essential for the suppression of triggered immune responses. The immune modulating capacity of these cells play a major role in both transplantation, autoimmune disease, allergy, cancer and pregnancy. During pregnancy, low numbers of regulatory T cells are associated with pregnancy failure and pregnancy complications such as pre-eclampsia. On the other hand, in cancer, low numbers of immunosuppressive T cells are correlated with better prognosis. Hence, maternal immune tolerance toward the fetus during pregnancy and the escape from host immunosurveillance by cancer seem to be based on similar immunological mechanisms being highly dependent on the balance between immune activation and suppression. As regulatory T cells hold a crucial role in several biological processes, they may also be promising subjects for therapeutic use. Especially in the field of cancer, cell therapy and checkpoint inhibitors have demonstrated that immune-based therapies have a very promising potential in treatment of human malignancies. However, these therapies are often accompanied by adverse autoimmune side effects. Therefore, expanding the knowledge to recognize the complexities of immune regulation pathways shared across different immunological scenarios is extremely important in order to improve and develop new strategies for immune-based therapy. The intent of this review is to highlight the functional characteristics of regulatory T cells in the context of mechanisms of immune regulation in pregnancy and cancer, and how manipulation of these mechanisms potentially may improve therapeutic options.

Expression of Circadian Clock Genes in Human Colorectal Cancer Tissues Using Droplet Digital PCR.

Increasing evidence indicates that disruption of circadian rhythms may be directly linked to cancer. Here we report that the expression levels of the core clock genes Per1 and Per3 measured by droplet digital polymerase chain reaction are significantly decreased in tumour tissue from 16 patients undergoing colorectal cancer surgery compared to paired normal mucosa. No differences were observed in the expression of Per2, Bmal1, and Clock. In conclusion, abnormal expression levels of the clock genes Per1 and Per3 in CRC tissue may be related to tumourigenesis and may provide future diagnostic and prognostic information.