Fetal hemodynamics and placental histopathology in Down syndrome.

OBJECTIVE: To evaluate the association between Doppler patterns in fetuses with Down syndrome (DS) and their placental histopathologic findings. METHODS: A retrospective cross-sectional study was performed by collecting data from medical records of singleton pregnancies between January 2014 and January 2022, whose fetuses had a confirmed diagnosis of DS either prenatally or postnatally. Placental histopathology, maternal characteristics, and prenatal ultrasound (biometric parameters and umbilical artery [UA] Doppler) were evaluated. RESULTS: Of 69 eligible pregnant women, 61 met the inclusion and exclusion criteria. In the sample, 15 fetuses had an estimated fetal weight < 10th percentile for gestational age (GA) and were considered small for gestational age (SGA). Thirty-eight fetuses had increased resistance on the UA Doppler. Histologic changes were detected in 100% of the placentas, the most common being delayed villous maturation, alterations associated with poor fetal vascular perfusion, and villous dysmorphism. More than 50% of the placentas showed alterations related to placental insufficiency. We did not observe a statistically significant association between UA Doppler examination and placental alterations. All placentas analyzed in the SGA subgroup showed findings compatible with placental insufficiency. CONCLUSION: We found no statistically significant association between placental histopathologic findings and UA Doppler abnormalities in fetuses with DS. The placental alterations identified were delayed villous maturation, alterations associated with poor fetal vascular perfusion, and villous dysmorphism.

Discordant Zika Virus Findings in Twin Pregnancies Complicated by Antenatal Zika Virus Exposure: A Prospective Cohort.

BACKGROUND: There are limited data on the natural history of antenatal Zika virus (ZIKV) exposure in twin pregnancies, especially regarding intertwin concordance of prenatal, placental, and infant outcomes. METHODS: This prospective cohort study included twin pregnancies referred to a single institution from September 2015 to June 2016 with maternal ZIKV. Polymerase chain reaction (PCR) testing of maternal, placental, and neonatal samples was performed. Prenatal ultrasounds were completed for each twin, and histomorphologic analysis was performed for each placenta. Abnormal neonatal outcome was defined as abnormal exam and/or abnormal imaging. Two- to three-year follow-up of infants included physical exams, neuroimaging, and Bayley-III developmental assessment. RESULTS: Among 244 pregnancies, 4 twin gestations without coinfection were identified. Zika virus infection occurred at 16-33 weeks gestation. Zika virus PCR testing revealed discordance between dichorionic twins, between placentas in a dichorionic pair, between portions of a monochorionic placenta, and between a neonate and its associated placenta. Of the 8 infants, 3 (38%) had an abnormal neonatal outcome. Of 6 infants with long-term follow-up, 3 (50%) have demonstrated ZIKV-related abnormalities. CONCLUSIONS: Neonatal PCR testing, placental findings, and infant outcomes can be discordant between co-twins with antenatal ZIKV exposure. These findings demonstrate that each twin should be evaluated independently for vertical transmission.

Early Clinical Infancy Outcomes for Microcephaly and/or Small for Gestational Age Zika-Exposed Infants.

BACKGROUND: Zika-exposed infants with microcephaly (proportional or disproportional) and those who are small for gestational age without microcephaly should be closely followed, particularly their growth trajectories. They are at high risk of adverse outcomes in the first year of life.Antenatal Zika virus (ZIKV) exposure may lead to adverse infant outcomes including microcephaly and being small for gestational age (SGA). ZIKV-exposed infants with a diagnosis of microcephaly (proportional [PM] or disproportional [DM]) or SGA at birth were evaluated with anthropometric measurements and health outcomes. METHODS: Infants had laboratory-confirmed ZIKV exposure in Brazil. PM, DM, or SGA classification was based on head circumference and weight. First-year growth parameters and clinical outcomes were recorded with analyses performed. RESULTS: Among the 156 ZIKV-exposed infants, 14 (9.0%) were SGA, 13 (8.3%) PM, 13 (8.3%) DM, and 116 (74.4%) were neither SGA nor had microcephaly (NSNM). High rates of any neurologic, ophthalmologic, and hearing abnormalities were observed for PM (100%), DM (100%), and SGA (42.9%) vs NSNM infants (18.3%; P <.001); odds ratio [OR], 3.4 (95% confidence interval [CI], 1.1-10.7) for SGA vs NSNM. Neuroimaging abnormalities were seen in 100% of PM and DM and in 42.9% of SGA vs NSNM infants 16%; (P <.001); OR 3.9 (95% CI, 1.2-12.8) for SGA vs NSNM. Growth rates by z score, particularly for microcephaly infants, were poor after birth but showed improvement beyond 4 months of life. CONCLUSIONS: ZIKV-exposed infants with microcephaly (PM and DM) had similarly high rates of adverse outcomes but showed improvement in growth measurements beyond 4 months of life. While SGA infants had fewer adverse outcomes compared with microcephaly infants, notable adverse outcomes were observed in some; their odds of having adverse outcomes were 3 to 4 times greater compared to NSNM infants.Zika-exposed infants with microcephaly, irrespective of being proportional or disproportional, and those who are small for gestational age without microcephaly should be closely followed, particularly their growth trajectories. They are at high risk of adverse outcomes in the first year of life.

The Role of Amniocentesis in the Diagnosis of Congenital Zika Syndrome.

There are limited data on amniocentesis as a diagnostic tool for congenital Zika syndrome. Here we report on a prospective cohort of 16 women with suspected Zika virus infection in a highly endemic area, and discuss the role of amniocentesis in the prenatal diagnosis of fetal Zika infection.

The spectrum of neuropathological changes associated with congenital Zika virus infection.

A major concern associated with ZIKV infection is the increased incidence of microcephaly with frequent calcifications in infants born from infected mothers. To date, postmortem analysis of the central nervous system (CNS) in congenital infection is limited to individual reports or small series. We report a comprehensive neuropathological study in ten newborn babies infected with ZIKV during pregnancy, including the spinal cords and dorsal root ganglia (DRG), and also muscle, pituitaries, eye, systemic organs, and placentas. Using in situ hybridization (ISH) and electron microscopy, we investigated the role of direct viral infection in the pathogenesis of the lesions. Nine women had Zika symptoms between the 4th and 18th and one in the 28th gestational week. Two babies were born at 32, one at 34 and 36 weeks each and six at term. The cephalic perimeter was reduced in four, and normal or enlarged in six patients, although the brain weights were lower than expected. All had arthrogryposis, except the patient infected at 28 weeks gestation. We defined three patterns of CNS lesions, with different patterns of destructive, calcification, hypoplasia, and migration disturbances. Ventriculomegaly was severe in the first pattern due to midbrain damage with aqueduct stenosis/distortion. The second pattern had small brains and mild/moderate (ex-vacuo) ventriculomegaly. The third pattern, a well-formed brain with mild calcification, coincided with late infection. The absence of descending fibres resulted in hypoplastic basis pontis, pyramids, and cortico-spinal tracts. Spinal motor cell loss explained the intrauterine akinesia, arthrogryposis, and neurogenic muscle atrophy. DRG, dorsal nerve roots, and columns were normal. Lympho-histiocytic inflammation was mild. ISH showed meningeal, germinal matrix, and neocortical infection, consistent with neural progenitors death leading to proliferation and migration disorders. A secondary ischemic process may explain the destructive lesions. In conclusion, we characterized the destructive and malformative consequences of ZIKV in the nervous system, as reflected in the topography and severity of lesions, anatomic localization of the virus, and timing of infection during gestation. Our findings indicate a developmental vulnerability of the immature CNS, and shed light on possible mechanisms of brain injury of this newly recognized public health threat.

Preclinical Studies Evaluating Subacute Toxicity and Therapeutic Efficacy of LQB-118 in Experimental Visceral Leishmaniasis.

Visceral leishmaniasis (VL) is the most severe form of leishmaniasis and is the second major cause of death by parasites, after malaria. The arsenal of drugs against leishmaniasis is small, and each has a disadvantage in terms of toxicity, efficacy, price, or treatment regimen. Our group has focused on studying new drug candidates as alternatives to current treatments. The pterocarpanquinone LQB-118 was designed and synthesized based on molecular hybridization, and it exhibited antiprotozoal and anti-leukemic cell line activities. Our previous work demonstrated that LQB-118 was an effective treatment for experimental cutaneous leishmaniasis. In this study, we observed that treatment with 10 mg/kg of body weight/day LQB-118 orally inhibited the development of hepatosplenomegaly with a 99% reduction in parasite load. An in vivo toxicological analysis showed no change in the clinical, biochemical, or hematological parameters. Histologically, all of the analyzed organs were normal, with the exception of the liver, where focal points of necrosis with leukocytic infiltration were observed at treatment doses 5 times higher than the therapeutic dose; however, these changes were not accompanied by an increase in transaminases. Our findings indicate that LQB-118 is effective at treating different clinical forms of leishmaniasis and presents no relevant signs of toxicity at therapeutic doses; thus, this framework is demonstrated suitable for developing promising drug candidates for the oral treatment of leishmaniasis.

Roles of CDKN1A gene polymorphisms (rs1801270 and rs1059234) in the development of cervical neoplasia.

The CDKN1A gene product is a p53 downstream effector, which participates in cell differentiation, development process, repair, apoptosis, senescence, migration, and tumorigenesis. The objective of our study was investigated the importance of two polymorphisms in the CDKN1A gene, rs1801270 (31C>A) and rs1059234 (70C>T), for the development of cervical lesions in a Southeastern Brazilian population (283 cases, stratified by lesion severity, and 189 controls). CDKN1A genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and/or DNA sequencing. CDKN1A 31A allele presents a genetic pattern of protection for the development of high-grade cervical lesions (CC vs CA genotype: OR = 0.60; 95 % CI = 0.38-0.95; p = 0.029; CA+AA vs CC genotype: OR = 0.60; 95 % CI = 0.39-0.93; p = 0.021). Allele distributions of the CDKN1A 70C>T polymorphism were also different between the two study groups, with the CDKN1A 70T allele being less prevalent among cases. Moreover, the double heterozygote genotype combination 31CA-70CT decreases the chance of developing high-grade squamous intraepithelial lesion (HSIL) and cancer (OR = 0.55; 95 % CI = 0.32-0.93; p = 0.034) by 50 %, representing a protective factor against the development of more severe cervical lesions.

Antenatal Diagnosis of a Large Immature Abdominal Wall Teratoma by 2D-3D Ultrasound Using HDlive and Magnetic Resonance Imaging.

We describe the first case of prenatally detected teratoma of the fetal abdomen wall using ultrasound and fetal magnetic resonance imaging (MRI). A heterogeneous mass, partly solid and cystic, originating from the anterior abdominal wall of the fetus close to an omphalocele sac was detected by means of 2D/3D ultrasound and MRI. Amniodrainage was performed and due to sign of impending fetal risk, an emergency Cesarean section was performed. A bulky, crumbly and bleeding tumoral mass was confirmed at delivery. Ligation of the supplying artery to the tumor was complicated by uncontrollable hemorrhage and early neonatal death. Pathology identified the tumor as an immature teratoma of the anterior fetal abdominal wall. 2D/3D ultrasound, especially using HDlive application and MRI demonstrated accurate detection and characterization of this congenital tumor.

Effects of MDM2 promoter polymorphisms on the development of cervical neoplasia in a Southeastern Brazilian population.

We investigated the importance of two adjacent functional polymorphisms in the Murine Double Minute 2 (MDM2) gene, SNP285 G > C and SNP309 T > G, for the development of cervical lesions in a Southeastern Brazilian population (293 cases and 184 controls). MDM2 genotyping was performed by PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) and/or DNA sequencing. MDM2 SNP309 has potential as a biomarker of cervical neoplasia in non-smokers, patients with family history of cancer, or those who had late sexual debut (>16 years). Besides, this polymorphism may help identify women at risk of developing severe cervical lesion at a young age (<30 years).

Association of CDKN2A polymorphisms with the severity of cervical neoplasia in a Brazilian population.

Variants of p16(INK4a) and p14(ARF), encoded by the CDKN2A locus, may respond differently to the presence of human papillomavirus (HPV). We investigated the potential association of two CDKN2A polymorphisms, 500C > G (rs11515) and 540C > T (rs3088440), with cervical neoplasia in patients with cervical lesions and healthy controls (n = 492). Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), single-strand conformation polymorphism (SSCP) and/or DNA sequencing techniques were employed for genotyping. The 500G allele was found higher, whereas the 540T/T genotype was less frequent in patients with more severe lesions. The CDKN2A variants may have the potential to be markers for the management of patients with cervical neoplasia.

Decidual production of interferon lambda in response to ZIKV persistence: Clinical evidence and in vitro modelling.

Zika virus (ZIKV) infections during pregnancy can result in Congenital Zika Syndrome (CZS), a range of severe neurological outcomes in fetuses that primarily occur during early gestational stages possibly due to placental damage. Although some placentas can maintain ZIKV persistence for weeks or months after the initial infection and diagnosis, the impact of this viral persistence is still unknown. Here, we aimed to investigate the immunological repercussion of ZIKV persistence in term placentas. As such, term placentas from 64 pregnant women diagnosed with Zika in different gestational periods were analyzed by ZIKV RT-qPCR, examination of decidua and placental villous histopathology, and expression of inflammation-related genes and IFNL1-4. Subsequently, we explored primary cultures of term decidual Extravillous Trophoblasts (EVTs) and Term Chorionic Villi (TCV) explants, as in vitro models to access the immunological consequences of placental ZIKV infection. Placenta from CZS cases presented low IFNL1-4 expression, evidencing the critical protective role of theses cytokines in the clinical outcome. Term placentas cleared for ZIKV showed increased levels of IFNL1, 3, and 4, whether viral persistence was related with a proinflammatory profile. Conversely, upon ZIKV persistence placentas with decidual inflammation showed high IFNL1-4 levels. In vitro experiments showed that term EVTs are more permissive, and secreted higher levels of IFN-α2 and IFN-λ1 compared to TCV explants. The results suggest that, upon ZIKV persistence, the maternal-skewed decidua contributes to placental inflammatory and antiviral signature, through chronic deciduitis and IFNL upregulation. Although further studies are needed to elucidate the mechanisms underlying the decidual responses against ZIKV. Hence, this study presents unique insights and valuable in vitro models for evaluating the immunological landscape of placentas upon ZIKV persistence.

Immunohistochemical expression of cyclin D1, p16Ink4a, p21WAF1, and Ki-67 correlates with the severity of cervical neoplasia.

High-risk human papillomaviruses are closely associated with cervical cancer and its precursor lesions through interactions between the E6 and E7 oncoproteins and the cell-cycle regulatory proteins, such as p53 and pRb, respectively. As other molecules involved in the cell-cycle control seem to be important for human papillomavirus (HPV)-mediated cervical carcinogenesis, we have analyzed the expression of p53, p21, p16, cyclin D1, and Ki-67 and the presence of HPV (HPV pool and HPV-16) by immunohistochemical studies using tissue microarray in low squamous intraepithelial lesions (n=50), high squamous intraepithelial lesions (n=98), and cervical carcinoma (n=18). We have found a significant increase in the expression of p16 and p21 (P<0.001) from low- to high-grade lesions and cancer. In contrast, cyclin D1 expression showed a significant decrease in more severe lesions (P<0.001). p16, Ki-67, p21, and p53 positivity increased with the cell-layer level and the lesion severity, with stronger correlations being observed for p16 and Ki-67. High positivity for HPV pool (96.3%) and HPV-16 (77.5%) immunostaining was detected in all cases, with an association between p16 and cyclin D1 expression and HPV-16 infection. Our tissue microarray results corroborate the usefulness of the immunohistochemical assessment of cell-cycle biomarkers in distinguishing different groups of precursor lesions of the cervix and cervical carcinoma.

Clinical and serological findings in pregnant women and newborns: patterns of coronavirus disease 2019 placental histopathology.

OBJECTIVE: The objective of this study was to evaluate the correlation between clinical and serological findings of pregnant women and newborns with patterns of histopathologic changes of the placenta diagnosed with coronavirus disease 2019. METHODS: A prospective descriptive study was conducted with pregnant women who were positive for SARS-CoV-2 by reverse transcription polymerase chain reaction or serology (IgG and IgM). Clinical analyses were performed using ELISA to detect anti-SARS-CoV-2 IgG and IgA antibodies using the S1 spike protein domain with the Euroimmun kit. Histopathologic analyses of placentas were performed by two expert pathologists. RESULTS: Maternal SARS-CoV-2 infection was associated with increased neonatal hospital length of stay (p=0.03), increased preterm birth (p=0.04), and Apgar score<7 at 1st min (p=0.00) and 5th min (p=0.02). Pregnant women with positive IgG and/or IgA at delivery had a higher incidence of placental histopathologic changes in addition to a greater likelihood of having an IgG-positive fetus (p<0.0001). Placentas with positive reverse transcription polymerase chain reaction for SARS-CoV-2 had a higher incidence of histopathologic changes such as maternal vascular hypoperfusion changes (p=0.00). CONCLUSION: Maternal SARS-CoV-2 infection was associated with adverse perinatal outcomes. Pregnant women with positive IgG at delivery had a higher incidence of placental histopathologic changes. Placentas with positive reverse transcription polymerase chain reaction for SARS-CoV-2 had a higher incidence of histopathologic changes such as maternal vascular hypoperfusion.

TP53 and p21 (CDKN1A) polymorphisms and the risk of systemic lupus erythematosus.

BACKGROUND: The p53 and p21 proteins are important regulators of cell cycle and apoptosis and may contribute to autoimmune diseases, such as systemic lupus erythematosus (SLE). As genetic polymorphisms may cause changes in protein levels and functions, we investigated associations of TP53 and p21 (CDKN1A) polymorphisms (p53 72 G > C-rs1042522; p53 PIN3-rs17878362; p21 31 C > A-rs1801270; p21 70 C > T-rs1059234) with the development of systemic lupus erythematosus (SLE) in a Southeastern Brazilian population. METHODS: Genotyping of 353 female volunteers (cases, n = 145; controls, n = 208) was performed by polymerase chain reaction, restriction fragment length polymorphism and/or DNA sequencing. Associations between TP53 and p21 polymorphisms and SLE susceptibility and clinical manifestations of SLE patients were assessed by logistic regression analysis. RESULTS: Protective effect was observed for the genotype combinations p53 PIN3 A1/A1-p21 31 C/A, in the total study population (OR 0.45), and p53 PIN3 A1/A2-p21 31 C/C, in non-white women (OR 0.28). In Whites, p53 72 C-containing (OR 3.06) and p53 PIN3 A2-containing (OR 6.93) genotypes were associated with SLE risk, and higher OR value was observed for the combined genotype p53 72 G/C-p53 PIN3 A1/A2 (OR 9.00). Further, p53 PIN3 A1/A2 genotype was associated with serositis (OR 2.82), while p53 PIN3 A2/A2 and p53 72 C/C genotypes were associated with neurological disorders (OR 4.69 and OR 3.34, respectively). CONCLUSIONS: Our findings showed that the TP53 and p21 polymorphisms included in this study may have potential to emerge as SLE susceptibility markers for specific groups of patients. Significant interactions of the TP53 polymorphisms with serositis and neurological disorders were also observed in SLE patients.

Cervical Intraepithelial Neoplasia grade 2 biopsy: Do p16INK4a and Ki-67 biomarkers contribute to the decision to treat? A cross-sectional study.

BACKGROUND: Managing cervical intraepithelial neoplasia grade 2 (CIN2) is challenging, considering the CIN2 regression rate, perinatal risks associated with excisional procedures, and insufficient well-established risk factors to predict progression. OBJECTIVES: To determine the ability of p16INK4a and Ki-67 staining in biopsies diagnosed with CIN2 to identify patients with higher-grade lesions (CIN3 or carcinoma). DESIGN AND SETTING: Cross-sectional study conducted at a referral center for treating uterine cervical lesions. METHODS: In 79 women, we analyzed the correlation of p16INK4a and Ki-67 expression in CIN2 biopsies with the presence of a higher-grade lesions, as determined via histopathology in surgical specimens from treated women or via two colposcopies and two cytological tests during follow-up for untreated women with at least a 6-month interval. The expression of these two biomarkers was verified by at least two independent pathologists and quantified using digital algorithms. RESULTS: Thirteen (16.8%) women with CIN2 biopsy exhibited higher-grade lesions on the surgical excision specimen or during follow-up. p16INK4a expression positively and negatively predicted the presence of higher-grade lesions in 17.19% and 86.67% patients, respectively. Ki-67 expression positively and negatively predicted the presence of higher-grade lesions in 40% and 88.24% patients, respectively. CONCLUSIONS: Negative p16INK4a and Ki67 immunohistochemical staining can assure absence of a higher-grade lesion in more than 85% of patients with CIN2 biopsies and can be used to prevent overtreatment of these patients. Positive IHC staining for p16INK4a and Ki-67 did not predict CIN3 in patients with CIN2 biopsies.

Real-time PCR in the diagnosis of congenital toxoplasmosis.

The diagnosis of congenital toxoplasmosis presents limitations and therefore new options are necessary. The analysis of amniotic fluid by real-time PCR has already proved effective for confirmation of fetal infection. However, its performance in other biological samples is not clear yet. The aim of this study is to better understand the role of real-time PCR in the blood of the mother and newborn as well as in the amniotic fluid and placenta in the diagnosis of congenital toxoplasmosis. This is a descriptive cohort study of pregnant women with toxoplasmosis followed up in Rio de Janeiro, Brazil. Real-time PCR was performed in samples of maternal blood, amniotic fluid, placenta, and blood of newborns. In addition, histopathological examination of placentas was performed, and data collected from babies were collected. 116 pregnant women were followed up and 298 samples were analyzed. One (0.9%) pregnant woman presented positive PCR in the blood, 3 (3.5%) in the amniotic fluid, 1 (2.3%) in the placenta and no newborn had positive PCR in the blood. Histopathological study was suggestive of toxoplasmosis infection in 24 (49%) placentas. Six (5.2%) newborns were diagnosed with congenital toxoplasmosis, and only cases with positive PCR in the amniotic fluid had correlation of the PCR result with the diagnosis of congenital infection. Both maternal and blood samples of newborns and placenta did not prove to be promising in the diagnosis of congenital toxoplasmosis. Further studies are needed to evaluate the real role of molecular diagnosis in other biological materials rather than the amniotic fluid.

Obesity Induces an Impaired Placental Antiviral Immune Response in Pregnant Women Infected with Zika Virus.

Obesity is increasing in incidence worldwide, especially in women, which can affect the outcome of pregnancy. During this period, viral infections represent a risk to the mother, the placental unit, and the fetus. The Zika virus (ZIKV) outbreak in Brazil has been the cause of congenital Zika syndrome (CZS), with devastating consequences such as microcephaly in newborns. Herein, we analyzed the impact of maternal overweight/obesity on the antiviral factors' expression in the placental tissue of Zika-infected mothers. We accessed placentas from women with and without obesity from 34 public health units (São Paulo) and from Zika-infected mothers with and without obesity from the Clinical Cohort Study of ZIKV pregnant women (Rio de Janeiro, Brazil). We first verified that obesity, without infection, did not alter the constitutive transcriptional expression of antiviral factors or IFN type I/III expression. Interestingly, obesity, when associated with ZIKV infection, showed a decreased transcriptional expression of RIG-I and IFIH1 (MDA-5 protein precursor gene). At the protein level, we also verified a decreased RIG-I and IRF-3 expression in the decidual placenta from the Zika-infected obese group, regardless of microcephaly. This finding shows, for the first time, that obesity associated with ZIKV infection leads to an impaired type I IFN downstream signaling pathway in the maternal-fetal interface.

ZIKV replication is differential in explants and cells of human placental which is suppressed by HSV-2 coinfection.

During the Zika fever outbreak in Brazil in 2015-2016, only some babies from infected mothers had teratogenic effects, suggesting that cofactors may influence congenital transmission. We investigated the ZIKV infection profile in explants and isolated cells from full-term human placenta to infection with the Brazilian Zika virus strain (ZIKVBR) and the effect of coinfection with the Brazilian Human alphaherpesvirus 2 strain (HSV-2BR) on ZIKV replication. We found that the ZIKVBR infect the explants of amniotic and chorionic membranes, as well as chorionic villi core, but not the trophoblasts layer. It was also observed that ZIKV replication was higher in amniotic cells than chorionic and trophoblasts cells. Upon coinfection, the replication of ZIKVBR was reduced according to exposed HSV-2BR load in trophoblasts cells and the levels of TNF-α and IL-6 cytokines were also reduced. These findings suggest that the placental cell types and HSV-2BR coinfection may impact on ZIKV replication.