Full-length transcript amplification and sequencing as universal method to test mRNA integrity and biallelic expression in mismatch repair genes.

In pathogenicity assessment, RNA-based analyses are important for the correct classification of variants, and require gene-specific cut-offs for allelic representation and alternative/aberrant splicing. Beside this, the diagnostic yield of RNA-based techniques capable to detect aberrant splicing or allelic loss due to intronic/regulatory variants has to be elaborated. We established a cDNA analysis for full-length transcripts (FLT) of the four DNA mismatch repair (MMR) genes to investigate the splicing pattern and transcript integrity with active/inhibited nonsense-mediated mRNA-decay (NMD). Validation was based on results from normal controls, samples with premature termination codons (PTC), samples with splice-site defects (SSD), and samples with pathogenic putative missense variants. The method was applied to patients with variants of uncertain significance (VUS) or unexplained immunohistochemical MMR deficiency. We categorized the allelic representation into biallelic (50 ± 10%) or allelic loss (≤10%), and >10% and <40% as unclear. We defined isoforms up to 10% and exon-specific exceptions as alternative splicing, set the cut-off for SSD in cDNA + P to 30-50%, and regard >10% and <30% as unclear. FLT cDNA analyses designated 16% of all putative missense variants and 12% of VUS as SSD, detected MMR-defects in 19% of the unsolved patients, and re-classified >30% of VUS. Our method allows a standardized, systematic cDNA analysis of the MMR FLTs to assess the pathogenicity mechanism of VUS on RNA level, which will gain relevance for precision medicine and gene therapy. Diagnostic accuracy will be enhanced by detecting MMR defects in hitherto unsolved patients. The data generated will help to calibrate a high-throughput NGS-based mRNA-analysis and optimize prediction programs.

Predicting Humoral Alloimmunity from Differences in Donor and Recipient HLA Surface Electrostatic Potential.

In transplantation, development of humoral alloimmunity against donor HLA is a major cause of organ transplant failure, but our ability to assess the immunological risk associated with a potential donor-recipient HLA combination is limited. We hypothesized that the capacity of donor HLA to induce a specific alloantibody response depends on their structural and physicochemical dissimilarity compared with recipient HLA. To test this hypothesis, we first developed a novel computational scoring system that enables quantitative assessment of surface electrostatic potential differences between donor and recipient HLA molecules at the tertiary structure level [three-dimensional electrostatic mismatch score (EMS-3D)]. We then examined humoral alloimmune responses in healthy females subjected to a standardized injection of donor lymphocytes from their male partner. This analysis showed a strong association between the EMS-3D of donor HLA and donor-specific alloantibody development; this relationship was strongest for HLA-DQ alloantigens. In the clinical transplantation setting, the immunogenic potential of HLA-DRB1 and -DQ mismatches expressed on donor kidneys, as assessed by their EMS-3D, was an independent predictor of development of donor-specific alloantibody after graft failure. Collectively, these findings demonstrate the translational potential of our approach to improve immunological risk assessment and to decrease the burden of humoral alloimmunity in organ transplantation.

High-Resolution Genetic and Phenotypic Analysis of KIR2DL1 Alleles and Their Association with Pre-Eclampsia.

Killer-cell Ig-like receptor (KIR) genes are inherited as haplotypes. They are expressed by NK cells and linked to outcomes of infectious diseases and pregnancy in humans. Understanding how genotype relates to phenotype is difficult because of the extensive diversity of the KIR family. Indeed, high-resolution KIR genotyping and phenotyping in single NK cells in the context of disease association is lacking. In this article, we describe a new method to separate NK cells expressing allotypes of the KIR2DL1 gene carried by the KIR A haplotype (KIR2DL1A) from those expressing KIR2DL1 alleles carried by the KIR B haplotype (KIR2DL1B). We find that in KIR AB heterozygous individuals, different KIR2DL1 allotypes can be detected in both peripheral blood and uterine NK cells. Using this new method, we demonstrate that both blood and uterine NK cells codominantly express KIR2DL1A and KIR2DL1B allotypes but with a predominance of KIR2DL1A variants, which associate with enhanced NK cell function. In a case-control study of pre-eclampsia, we show that KIR2DL1A, not KIR2DL1B, associates with increased disease risk. This method will facilitate our understanding of how individual KIR2DL1 allelic variants affect NK cell function and contribute to disease risk.

Correction to: Two-year outcome after recurrent implantation failure: prognostic factors and additional interventions.

The article Two-year outcome after recurrent implantation failure: prognostic factors and additional interventions.

Fertility after recurrent miscarriages: results of an observational cohort study.

PURPOSE: Recurrent pregnancy losses (RPL) are considered a pathological condition associated with heterogeneous laboratory and clinical findings, and are also linked to subfertility. We attempt to rank parameters derived from past history and diagnostic results with regard to the prognosis. METHODS: Observational trial on 719 consecutive couples who were referred to a tertiary immunological care centre (2006-2014) after three or more primary miscarriages. Information on past obstetric history and diagnostic procedures at baseline were correlated with cumulative pregnancy and delivery rates using Kaplan-Meier estimation, logistic regression and multivariate analysis. RESULTS: At baseline, median female age was 34.1 years, waiting time 3 years (1-17), number of preceding miscarriages 3 (3-9), 147 women (20.4%) had conceived at least once in ART or AIH cycles. After a median follow-up of 33.7 (1.7-123.0) months, 5-year pregnancy and delivery rates were 86.1 and 64.5%. Female age (< 35 years), waiting time (< 3 years) until baseline, tubal competence, and male factor fertility significantly correlated with favourable outcome (p < 0.001), while body mass index (> 29 kg/m2), number of preceding miscarriages (> 4), late miscarriages, preclinical losses and smoking revealed non-significant negative trends. Mode of conception until baseline (spontaneously or ART/AIH) and classification into idiopathic and non-idiopathic RPL showed no prognostic relevance. CONCLUSION: Although in general, chances to conceive a child are retained after three or more miscarriages, factors related to subfertility of both partners have an important impact on the outcome. Therefore, prolonged time to pregnancy (> 6-9 months) should result in preventive gynaecological care from the first miscarriage on, so that fertility can be preserved as best as possible.

Two-year outcome after recurrent first trimester miscarriages: prognostic value of the past obstetric history.

PURPOSE: Recurrent miscarriage (RM) is a stressful condition which gives rise to extensive diagnostic evaluation and is seen as a potentially curable maternal disease. Nevertheless, epidemiological data have shown that outcome is related to fertility. In addition to maternal age and number of preceding miscarriages, further markers derived from the past history may support counselling. METHODS: Observational trial comprising 228 couples who were referred between 1996 and 2003 for immunological evaluation at maternal ages 20-39 years after three or more spontaneously conceived primary first trimester miscarriages. They were interviewed in 2005, ongoing pregnancies were followed up until birth in 2006. Past obstetric history was correlated with 2 year cumulative pregnancy and delivery rates (CPR, CDR). RESULTS: CPR and CDR were 206/228 (90.4 %) and 174/228 (76.4 %). Duration of infertility was associated with lower CPR (up to 3/>3 years, p < 0.01), whereas age and number of preceding losses inversely correlated with CDR (<35 years/35-39 years, p < 0.002; 3/>3 miscarriages, p < 0.002). Detection of an embryonic heart beat in 2-3 of the first three miscarriages resulted in favourable outcome (CPR: p < 0.02, CDR: p < 0.002). Prognosis was excellent in younger fertile women after three miscarriages where vital signs had been detected; under less favourable conditions not only risks for further miscarriage, but also for secondary infertility were elevated. CONCLUSION: Secondary infertility is a feature of RM. Embryonic vital signs in preceding pregnancies are prognostic markers and should be regarded as a strong confounding factor in trials on therapeutic interventions. Prevention may be more appropriate than treatment.

HCMV seroprevalence in couples under infertility treatment.

PURPOSE: Congenital HCMV infection affects about 0.5-0.9 % of newborns in Europe. Primary prevention could effectively be implemented in screening programmes for couples treated for infertility. By supplying epidemiological data, the proportion of women susceptible to primary HCMV infection is estimated. METHODS: Retrospective evaluation of HCMV IgG of couples with recurrent implantation failure in German IVF programmes referred to a tertiary immunological centre for lymphocyte immunotherapy in 2002 (727 couples) and 2012 (294 male partners only). RESULTS: HCMV seroprevalence was 45 % in women and 33 % in men of childbearing age in 2002 and has not declined any further in the male group (34 %) in 2012. It was higher in women (39.8 %) than in males (28.0 %) of German descent (p < 0.00001). Most couples were concordantly seropositive (20.5 %) or negative (41.8 %). Discordant partnerships comprised of seropositive women with negative partners (24.6 %) and seronegative women with positive partners (13.1 %). CONCLUSIONS: More than 50 % of the women are HCMV seronegative and may benefit from primary prevention. About one fourth of them live with a seropositive partner. Risks to contract HCMV via the sexual route in a long-term discordant partnership may be very low, and the advice of protective safer sex methods in this group of prospective parents is critical since it leads to a conflict of interests.

Congenital HCMV and assisted reproduction: Why not use the chance for primary screening?

HCMV is the leading cause of congenital infection, with 0.5-0.9% of infants affected in Europe, and primary maternal infection from the preconceptional phase to the first half of pregnancy bears the highest risk for long-term sequelae-like mental retardation, visual impairment, and progressive sensorineural hearing loss. As compared to couples conceiving spontaneously those under infertility treatment are well accessible to primary HCMV prevention. Since they face higher risk pregnancies this chance should be considered. The concept comprises serological screening for HCMV-IgG, including the partner where appropriate, defining individual risk factors, and counselling on hygiene at the initial assessment of infertility treatment. If seroconversion occurs, the subsequent treatment cycles should be postponed by 6 months. Uncertainties of diagnosis in early pregnancy which may lead to precautious elective termination can be prevented. A newborn at risk of congenital HCMV infection can be identified and scheduled for laboratory and paediatric evaluation within the first 2 weeks of life.

Two-year outcome after recurrent implantation failure: prognostic factors and additional interventions.

OBJECTIVES: After recurrent implantation failure (RIF), empirical figures on further prospects are essential for counselling but difficult to estimate within single IVF centres due to high drop-out rates. Alternatively, couples referred to a tertiary unit for RIF were evaluated. MATERIALS AND METHODS: Multi-centre 2-year observational trial of 1,174 eligible couples treated consecutively with adjuvant lymphocyte immunotherapy (LIT) in a university immunological department from 1999 to 2002 after three or more unsuccessful fresh embryo transfers. Acquisition of data was completed in 2005. RESULTS: With another 1.5 oocyte retrievals, delivery rate per couple depended on age (39.3% at <30 years, 16.9% at >39 years, P < 0.005). Prognosis was favourable when frozen embryo transfers had been conducted before (34.4 vs. 25.8%, P < 0.005). The outcome was slightly better in ICSI couples as compared to conventional IVF (31.0 vs. 24.8%, P < 0.05). Birth rates per fresh embryo transfer from the fourth to eighth retrieval were 17.4-18.3-15.0-12.9-12.9% (decline not significant). Apart from LIT, further additional interventions were given more often to couples who had had frozen embryo transfers before (49 vs. 40%, P < 0.005). CONCLUSIONS: Female age and ovarian response are crucial for further IVF prognosis. Previous frozen embryo transfers indicate better chances. Couples with male factor infertility may benefit from intracytoplasmatic sperm injection (ICSI) because underlying female factors are less prevalent. Cycle rank had comparatively little impact. Additional interventions are preferentially offered to couples who have a favourable prognosis anyway. Their multiple use is common practice in RIF, but its value should be considered limited.

Transfusion-related risks of intradermal allogeneic lymphocyte immunotherapy: single cases in a large cohort and review of the literature.

PROBLEM: Lymphocyte immunotherapy (LIT) is applied in infertility treatment. Moreover, it has been suggested for prevention of rhesus D-hemolytic disease and as a vaccine for reduction of human immunodeficiency virus-1 susceptibility. Although transfusion-related problems have been rarely reported they were a matter of debate. Here we discuss extensive single-center experience with intradermal LIT for implantation failure and recurrent miscarriages. METHOD OF STUDY: Retrospective 2- to 3-year follow-up of in vitro fertilization couples treated during 1996-2002 (feedback 2,848/3,041 = 93%), registering 930 deliveries. Prospective survey for acute reactions for 2000-2003 (feedback 2,687/3,246 = 83%). Review of the literature. RESULTS: Infections of the patient and transplant rejection later in life are minor residual risks. Post-transfusion purpura was suspected once but not verified. Anaphylaxis or malignancy were not promoted. Fetal/newborn alloimmune disease (severe hemolytic disease, thrombocytopenia, neutropenia) were not observed. CONCLUSION: Based on microbiological, immunological, and hematological testing the risks of intradermal LIT are low.