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.

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.

Why we succeed and fail in detecting fetal growth restriction: A population-based study.

INTRODUCTION: The objective of this study was to explore the association between detection of fetal growth restriction and maternal-, healthcare provider- and organizational factors. MATERIAL AND METHODS: A historical, observational, multicentre study. All women who gave birth to a child with a birthweight <2.3rd centile from 1 September 2012 to 31 August 2015 in Zealand, Denmark, were included. The population was identified through the Danish Fetal Medicine Database. Medical charts were reviewed to obtain data regarding maternal characteristics and information on the healthcare professionals. Date of authorization for the midwives and obstetricians involved was extracted from the Danish Health Authorization Registry. Multivariable Cox regression models were used to identify predictors of antenatal detection of fetal growth restriction, and analyses were adjusted for hospital, body mass index, parity, the presence of at least one risk factor and experience of the first midwife, number of midwife visits, number of visits to a doctor, the experience of the consultant midwife or the educational level of the doctor, the number of scans and gaps in continuity of midwife-care. Antenatal detection was defined as an ultrasound estimated fetal weight <2.3rd centile (corresponding to -2 standard deviations) prior to delivery. RESULTS: Among 78 544 pregnancies, 3069 (3.9%) had a fetal growth restriction. Detection occurred in 31% of fetal growth-restricted pregnancies. Clinical experience (defined as years since graduation) of the first consultation midwife was positively associated with detection, with a hazard ratio [HR] of 1.15, 95% confidence interval [CI] 1.03-1.28), for every 10 years of additional experience. The hazard of detection increased with the number of midwife consultations (HR 1.15, 95% CI 1.05-1.26) and with multiparity (HR 1.28, 95% CI 1.03-1.58). After adjusting for all covariates, an unexplained difference between hospitals (P = .01) remained. CONCLUSIONS: The low-risk nullipara may constitute an overlooked group of women at increased risk of antenatal non-detection of fetal growth restriction. Being screened by experienced midwives during early pregnancy and having access to multiple midwife consultations may improve future diagnosis.

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.