Cytogenetic abnormalities in couples with a history of primary and secondary recurrent miscarriage: a Brazilian Multicentric Study.

Objective: To evaluate the difference between chromosomal abnormalities between the gender of couples affected by Recurrent miscarriage (RM) and if there is an association between previous obstetric history and chromosomal abnormalities of the parents.Methods: Multicenter, retrospective, observational study from seven different RM clinics between 2006 and 2016. We enrolled 707 couples (1014 participants) with a history of RM. We compared the frequency of chromosomal abnormalities between groups of couples with primary and secondary RM and separated between women and their partners. Furthermore, we compared the prevalence of chromosomal abnormalities between groups based on the number of previous spontaneous abortions.Results: The overall prevalence of all cytogenetic abnormalities was 5.59% (n = 1414, women and their partners). Excluding cases of polymorphism and inversion of chromosome 9, which are considered variants of normality, the prevalence in all individuals was 2.26% (n = 32/1414). The comparative analysis of cases of chromosomal abnormalities among couples with primary and secondary RM based on the number of previous miscarriages (PM) revealed a similar frequency between groups. The statistical analysis of the total cases (primary PM + secondary PM) in these three groups were as follows: (a) couple, 2 pm versus 3 pm vs. ≥4 PM, p = .514; (b) women, 2 pm versus 3 pm vs. ≥4 PM, p = .347; and (3) partner, 2 pm versus 3 pm vs. ≥4 PM, p = .959. Chromosomal abnormalities were significantly more prevalent among women than among their partners (6.9 versus 4.2%; p = .027). Moreover, the distribution of leading chromosomal abnormalities among women was different compared with their partners. Among women, we observed these abnormalities in the following frequency order: mosaicism (38.8%), polymorphism (32.6%), translocation (16.3%), and inversion (12.3%). Among their partners, these abnormalities were polymorphism (73.3%), inversion (13.3%), mosaicism (6.7%), and translocation (6.7%).Conclusion: The number of PM and the history of full-term pregnancy does not correlate with an increase or decrease in the prevalence of cytogenetic abnormalities in couples with RM.

Gestational and perinatal outcomes in recurrent miscarriages couples treated with lymphocyte immunotherapy.

OBJECTIVE: This study aims to elucidate which types of recurrent miscarriage (RM) patients experienced a livebirth after paternal lymphocyte immunotherapy (LIT) and to evaluate the perinatal outcome. STUDY DESIGN: Retrospective analysis of a multicenter, observational study which enrolled 1096 couples with a history of two or more spontaneous miscarriages without any intercalated delivery. We conducted an intention-to-treat analysis of couples with RM treated with or without LIT regarding to gestational and perinatal outcomes. We compared groups by using the Student's t-test or Kruskal-Wallis test, Fisher's exact-test and χ 2 test when appropriate. RESULTS: The success of gestation was significantly higher in the LIT group (60.1% vs. 33.1%; p < 0.001). A sub-analysis of four different immune disorder groups revealed a significantly higher success in the LIT group in all immune categories, except in patients who had autoantibodies positive. We observed no significant differences in perinatal outcomes such as gestational age at birth, preterm and extreme preterm birth, and birth weight in successful pregnancy in both groups. The success rate was significantly higher when LIT was administrated before and during pregnancy and only during pregnancy compared to only before pregnancy (p < 0.01). CONCLUSIONS: Careful laboratory test phenotyping of RM patients may identify subgroups most likely to benefit and exclude those with little likelihood of benefit, and LIT during a pregnancy may significantly improve success rates.

Lymphocyte immunotherapy for recurrent miscarriages: Predictors of therapeutic success.

PROBLEM: To evaluate the predictors of successful pregnancies in women with a history of recurrent miscarriages (RMs) having undergone lymphocyte immunotherapy (LIT). METHOD OF STUDY: Retrospective, multicenter, observational study which involved 702 pregnant women with history of RM treated with LIT. Comparative analysis of women with a history of RM having undergone LIT and experienced treatment success vs those having experienced treatment failure along with the analysis of the association between the number of prior miscarriages and the efficacy of LIT. RESULTS: A total of 421 women were able to carry the pregnancy to term, with treatment success rate of 60%. The multivariate analysis showed that age, the association between autoantibodies and thrombophilia, and the number of previous miscarriages were factors associated with LIT failure. Secondary RMs alone were not found to be a factor predictive of LIT success or failure; however, secondary RMs among women with a history of 5 or more RM were found to be a predictor of LIT success (OR: 10.24; 95% CI: 1.9-55.8; P = .007). CONCLUSION: Age, the number of previous miscarriages, and the association between autoantibodies and thrombophilia are associated with LIT failure. A higher number of previous miscarriages in cases of secondary RM resulted in better LIT outcomes.

High Amounts of Total and Extracellular Vesicle-Derived Soluble HLA-G are Associated with HLA-G 14-bp Deletion Variant in Women with Embryo Implantation Failure.

PROBLEM: Human leukocyte antigen-G (HLA-G) expression is related to 14-bp insertion/deletion polymorphism at the 3'UTR of the HLA-G gene. Soluble forms of HLA-G are released as free molecules or via extracellular vesicles (EVs). Due to the crucial role of HLA-G during pregnancy, we analyzed the 14-bp polymorphism and the two secreted forms in implantation failure women (IF) and in fertile women (FW). METHOD OF STUDY: For the genetic analysis, 49 IF and 34 FW were genotyped. For sHLA-G quantification, serum samples from 35 IF and 23 FW were available. ExoQuick(™) kit was used for EVs precipitation. The total soluble HLA-G (sHLA-Gtot ) and vesicular sHLA-GEV were quantified by ELISA. The EVs size and concentration were determined by nanoparticle tracking analysis (NTA). RESULTS: An increased proportion of IF presented high levels of sHLA-Gtot (P = 0.02) and vesicular sHLA-GEV (P = 0.0003) compared to FW. The 14-bp deletion allele is more frequent in IF (P = 0.0002) and associated with high levels of sHLA-Gtot and vesicular sHLA-GEV . CONCLUSION: The high expression of sHLA-Gtot and sHLA-GEV , together with the presence of the 14-bp deletion allele, might be involved in implantation failure.

High levels of circulating extracellular vesicles with altered expression and function during pregnancy.

Extracellular vesicles (EVs) are widely considered important modulators of cell-cell communication and may interact with target cells locally and on a systemic level. Several studies had shown that circulating EVs' levels are increased during pregnancy. However, EVs characteristics, composition and biological functions in pregnancy still need to be clarified. This study aims to determine if circulating EVs during pregnancy are modified regarding levels, markers and cytokine profile as well as their reactivity towards peripheral blood cells. 26 pregnant women (PW) being in the second gestational trimester and 59 non-pregnant women (NPW) were investigated. EVs enrichment was performed by ExoQuick™ or ultracentrifugation; nanoparticle tracking analysis, SDS-PAGE followed by Western Blotting and densitometry, and IFN-γ, IL-10 and TGF-ß1 ELISA for EVs characterization; imaging flow cytometry to analyze EVs' uptake by peripheral blood cells and flow cytometry were performed to analyze EVs function regarding induction of caspase-3 activity. Circulating EVs' levels were increased during pregnancy [26.9×10(6)EVs/ml (range: 6.4-46.3); p=0.003] vs NPW [18.9×10(6)EVs/ml (range: 2.5-61.3)]. Importantly, the immunosuppressive TGF-ß1 and IL-10 cytokine cargo were increased in EVs of PW even after normalization to 1 million EVs [TGF-ß1: 0.25pg/10(6)EVs (range: 0.0-2.0); p<0.0001] and [IL-10: 0.21pg/10(6)EVs (range: 0.0-16.8); p=0.006] vs NPW. Although EVs derived from non-pregnant and pregnant women were taken up by NK cells, the latter exclusively enhanced the caspase-3 activity in CD56(dim) NK cells (8.2±0.9; p=0.02). The qualitative and quantitative pregnancy-related alterations of circulating EVs provide first hints for an immune modulating role of circulating EVs during pregnancy.

Analysis of HLA-G polymorphisms in couples with implantation failure.

PROBLEM: HLA-G expression is related as an immune modulator of fetal-maternal tolerance, and its levels was correlated with pregnancy outcome. In a case-control study, we investigate the association between the genetic variability of the HLA-G gene and serum levels of soluble HLA-G in cases of embryo implantation failure. METHOD OF STUDY: Forty couples with at least two unsuccessful fresh embryo transfers (implantation failure; IF) and 83 fertile couples with at least two successful pregnancies was genotyped by sequencing-based typing. HLA-G alleles were defined by nucleotide sequence variations at exon 2, 3, and 4, and the quantification of soluble HLA-G (sHLA-G) was performed by ELISA. RESULTS: There was a significant difference between the HLA-G allelic distributions between IF couples and the control couples. The HLA-G*01:03:01 allele was increased in the IF couples. There were no significant differences in the serum levels of sHLA-G in the IF and control groups. CONCLUSION: The results suggest that the distribution of HLA-G products may play a significant role in the modulation of maternal-fetal immune response.

Early HHV-6 replication is associated with morbidity non-related to CMV infection after kidney transplantation.

UNLABELLED: Human herpesvirus type 6-(HHV-6) has been associated with morbidity after liver transplantation. OBJECTIVE: The aim of this study was to determine the HHV-6 seroprevalence among donor-recipient pairs, analyze the incidence of early active infection, its clinical manifestation, interaction with CMV, and the related morbidity in the first year after kidney transplantation. METHODS: 46 donor-recipient pairs had IgG evaluated by ELISA before transplantation: HHV-6(Pambio - USA) and CMV-(Roche - USA). A frozen whole blood sample collected weekly (from the 1st to the 6th week) was retrospectively tested for HHV-6 viral load (VL) determination by real time quantitative PCR (qPCR, Nanogen - Italy). Patients were preemptively surveyed for CMV by pp65 antigenemia (Ag, APAAP, immunohistochemistry, Biotest - Germany) from the 4th to the 12th week after transplantation. Active infection was defined as qPCR-HHV6+ (viral-load/mL-VL) and Ag+ (+cells/100.000 granulocytes), for HHV-6 and CMV, respectively. DCMV was defined as simultaneous positive antigenemia and suggestive signs/symptoms. Concerning +qPCR-HHV6, associated factors, clinical manifestation, interaction with CMV and morbidity were searched. RESULTS: Pre-transplant HHV-6 seroprevalence was significantly higher among kidney recipients compared to their donors (82.6x54.8%; p = 0.005 [3.9 (1.4-10.4)]). Active infection by this virus occurred in 26.1% (12/46), with no association with previous IgG (p = 0.412). Median VL was 125 copies/mL (53-11.264), and the median Ag was 21 +cells (2-740). There was no association between HHV-6 and CMV activation after transplantation (p = 0.441), neither concerning DCMV (p = 0.596). Median highest Ag+ and days of ganciclovir treatment were similar between qPCR-HHV6 + or - (p = 0.206 and p = 0.124, respectively). qPCR-HHV6+ was associated with higher incidence of bacterial (p = 0.009) and fungal (p = 0.001) infections, and higher number (p = 0.001) of hospital admission and longer duration of hospitalization over the first 6 and 12 months post-transplantation (p = 0.033 and p = 0.001). CONCLUSION: Latent HHV-6 infection is more common among recipients than donors before transplantation. Early active infection by this pathogen after transplantation does not increase DCMV incidence or severity during the first 3 months of follow-up. However, early HHV-6 replication is associated with other infections and hospitalizations in the first year.

Early HHV-6 replication is associated with morbidity non-related to CMV infection after kidney transplantation

Human herpesvirus type 6-(HHV-6) has been associated with morbidity after liver transplantation. OBJECTIVE: The aim of this study was to determine the HHV-6 seroprevalence among donor-recipient pairs, analyze the incidence of early active infection, its clinical manifestation, interaction with CMV, and the related morbidity in the first year after kidney transplantation. METHODS: 46 donor-recipient pairs had IgG evaluated by ELISA before transplantation: HHV-6(Pambio - USA) and CMV-(Roche - USA). A frozen whole blood sample collected weekly (from the 1st to the 6th week) was retrospectively tested for HHV-6 viral load (VL) determination by real time quantitative PCR (qPCR, Nanogen - Italy). Patients were preemptively surveyed for CMV by pp65 antigenemia (Ag, APAAP, immunohistochemistry, Biotest - Germany) from the 4th to the 12th week after transplantation. Active infection was defined as qPCR-HHV6+ (viral-load/mL-VL) and Ag+ (+cells/100.000 granulocytes), for HHV-6 and CMV, respectively. DCMV was defined as simultaneous positive antigenemia and suggestive signs/symptoms. Concerning +qPCR-HHV6, associated factors, clinical manifestation, interaction with CMV and morbidity were searched. RESULTS: Pre-transplant HHV-6 seroprevalence was significantly higher among kidney recipients compared to their donors (82.6x54.8%; p = 0.005 [3.9 (1.4-10.4)]). Active infection by this virus occurred in 26.1% (12/46), with no association with previous IgG (p = 0.412). Median VL was 125 copies/mL (53-11.264), and the median Ag was 21 +cells (2-740). There was no association between HHV-6 and CMV activation after transplantation (p = 0.441), neither concerning DCMV (p = 0.596). Median highest Ag+ and days of ganciclovir treatment were similar between qPCR-HHV6 + or - (p = 0.206 and p = 0.124, respectively). qPCR-HHV6+ was associated with higher incidence of bacterial (p = 0.009) and fungal (p = 0.001) infections, and higher number (p = 0.001) of hospital admission and longer duration of hospitalization over the first 6 and 12 months post-transplantation (p = 0.033 and p = 0.001). CONCLUSION: Latent HHV-6 infection is more common among recipients than donors before transplantation. Early active infection by this pathogen after transplantation does not increase DCMV incidence or severity during the first 3 months of follow-up. However, early HHV-6 replication is associated with other infections and hospitalizations in the first year.

T helper (Th)1, Th2, and Th17 interleukin pathways in infertile patients with minimal/mild endometriosis.

In the present study, interleukin (IL)-10, IL-12, IL-17, and IL-23 levels were measured in serum and peritoneal fluid of women with minimal or mild endometriosis and compared with levels in controls without endometriosis. Higher IL-23 levels were encountered in the peritoneal fluid of women with endometriosis, suggesting a possible role of this cytokine in these women's infertility.

BKV-infection in kidney graft dysfunction.

INTRODUCTION: BKV nephropathy (BKN) causes kidney graft loss, whose specific diagnosis is invasive and might be predicted by the early detection of active viral infection. OBJECTIVE: Determine the BKV-infection prevalence in late kidney graft dysfunction by urinary decoy cell (DC) and viral DNA detection in urine (viruria) and blood (viremia; active infection). METHODS: Kidney recipients with >1 month follow-up and creatinine >1.5 mg/dL and/or recent increasing >20% (n = 120) had their urine and blood tested for BKV by semi-nested PCR, DC searching, and graft biopsy. PCR-positive patients were classified as 1+, 2+, 3+. DC, viruria and viremia prevalence, sensitivity, specificity, and likelihood ratio (LR) were determined (Table 2x2). Diagnosis efficacy of DC and viruria were compared to viremia. RESULTS: DC prevalence was 25%, viruria 61.7%, and viremia 42.5%. Positive and negative patients in each test had similar clinical, immunosuppressive, and histopathological characteristics. There was no case of viremia with chronic allograft nephropathy and, under treatment with sirolimus, patients had a lower viruria prevalence (p = 0.043). Intense viruria was the single predictive test for active infection (3+; LR = 2.8).1,6-4,9 CONCLUSION: DC, BKV-viruria and -viremia are commun findings under late kidney graft dysfunction. Viremia could only be predicted by intense viruria. These results should be considered under the context of BKN confirmation.

BKV-infection in kidney graft dysfunction

INTRODUCTION: BKV nephropathy (BKN) causes kidney graft loss, whose specific diagnosis is invasive and might be predicted by the early detection of active viral infection. OBJECTIVE: Determine the BKV-infection prevalence in late kidney graft dysfunction by urinary decoy cell (DC) and viral DNA detection in urine (viruria) and blood (viremia; active infection). METHODS: Kidney recipients with >1 month follow-up and creatinine >1.5 mg/dL and/or recent increasing >20 percent (n = 120) had their urine and blood tested for BKV by semi-nested PCR, DC searching, and graft biopsy. PCR-positive patients were classified as 1+, 2+, 3+. DC, viruria and viremia prevalence, sensitivity, specificity, and likelihood ratio (LR) were determined (Table 2x2). Diagnosis efficacy of DC and viruria were compared to viremia. RESULTS: DC prevalence was 25 percent, viruria 61.7 percent, and viremia 42.5 percent. Positive and negative patients in each test had similar clinical, immunossupressive, and histopathological characteristics. There was no case of viremia with chronic allograft nephropathy and, under treatment with sirolimus, patients had a lower viruria prevalence (p = 0.043). Intense viruria was the single predictive test for active infection (3+; LR = 2.8).1,6-4,9 CONCLUSION: DC, BKV-viruria and -viremia are commun findings under late kidney graft dysfunction. Viremia could only be predicted by intense viruria. These results should be considered under the context of BKN confirmation.

The incidence of cytomegalovirus infection in lung transplant recipients under universal prophylaxis with intravenous ganciclovir.

The best strategy for control of cytomegalovirus (CMV) infection in lung transplant patients is still not determined. The aim of this study was to document the incidence of CMV infection in a cohort of lung transplant recipients under universal prophylaxis with intravenous ganciclovir. All patients received immunosuppressive regimens consisting of cyclosporine, azathioprine, and prednisone. Regardless of CMV serostatus, intravenous ganciclovir was prescribed for every patient in the first 3 months post-transplantation. CMV infection was defined as the detection of CMV pp65 in leukocytes. Eighty-two lung transplant patients were included over a 5-year period. The incidence of CMV infection in the first year post-transplantation was 68.3%, occurring after a median length of 114 days (range, 26-343 days). This study revealed a high incidence of CMV infection in the first year following lung transplantation despite prolonged universal ganciclovir prophylaxis.

The incidence of cytomegalovirus infection in lung transplant recipients under universal prophylaxis with intravenous ganciclovir

The best strategy for control of cytomegalovirus (CMV) infection in lung transplant patients is still not determined. The aim of this study was to document the incidence of CMV infection in a cohort of lung transplant recipients under universal prophylaxis with intravenous ganciclovir. All patients received immunosuppressive regimens consisting of cyclosporine, azathioprine, and prednisone. Regardless of CMV serostatus, intravenous ganciclovir was prescribed for every patient in the first 3 months post-transplantation. CMV infection was defined as the detection of CMV pp65 in leukocytes. Eighty-two lung transplant patients were included over a 5-year period. The incidence of CMV infection in the first year post-transplantation was 68.3 percent, occurring after a median length of 114 days (range, 26-343 days). This study revealed a high incidence of CMV infection in the first year following lung transplantation despite prolonged universal ganciclovir prophylaxis.

Imunologia da gestação / Pregnancy immunology

Do ponto de vista imunológico, a gestação somente é possível porque uma intrincada rede imunorregulatória é disparada com o objetivo único de desenvolver um estado de tolerância materno-fetal e permitir a implantação e manutenção do concepto até que haja condições de sobrevivência fora da cavidade uterina. Entre os fatores envolvidos nessa complexa rede imunomodulatória para a tolerância e regulação do desenvolvimento fetal e formação da placenta, destacam-se: a influência hormonal sobre o sistema imune materno, o reconhecimento das moléculas do Complexo Principal de Histocompatibilidade paterno (expressas pelo embrião), as citocinas liberadas no meio, o controle da citoxicidade direta das células Natural Killer uterinas e atividade das células T regulatórias. A seguir, uma revisão abrangente e atual da literatura discute de maneira simplificada tais mecanismos.

From the immunological point of view, pregnancy is possible because a complex immunorregulatory network is triggered in order to develop a feto-maternal tolerance from the implantation through the birth. The most important mechanisms involved in the gestational immunotolerance system are debated in this paper and include: maternal hormonal influence on the immune system, the allorecognition of the Human Leukocyte Antigen (HLA) molecules expressed by the embryo, local cytokines profile, Natural Killer cells cytotoxicity and the role of regulatory T cells.

Renal transplants using expanded cadaver donor criteria.

AIMS: to analyze the frequency of transplants using expanded donor criteria (EDC) and the incidence of delayed graft function, acute rejection and the patient and graft survival compared to ideal donors (ID). PATIENTS: retrospective analysis of the 582 cadaver renal transplants performed from Jun 1988 to Mar 2003 in adult recipients. The expanded donor criteria were considered as history of hypertension or evidence of atherosclerosis, diabetes, age less than 5 or more than 55 years old, serum creatinine higher than 2.0 mg/dL, shock and retrieval in cardiac arrest. The statistical analysis used was Student t test, Chi-square test, and Kaplan-Meier method as indicated. RESULTS: 25.4% of our transplants used expanded criteria donor. Comparing, respectively, the EDC and ID we found: the incidence of delayed graft function of 63.9% vs 50.4% (P: 0.007); incidence of acute rejection of 66.1% vs 72.3% (P: 0.203). The patient survival at 1 and 5 years was 87% vs 92% and 81% vs 79%, respectively (P: 0.6809). The graft survival at 1 and 5 year was 74% vs 82% and 57% vs 59% (P: 0.2072), respectively for EDC and ID. CONCLUSIONS: One fourth of our cadaver transplants fulfilled the extended donor criteria. The incidence of delayed graft function was higher in these transplants, but the prevalence of rejection episodes was similar to ideal donors. The patient and graft survival were not statistically different at 1 and 5 year.