Variedades de cultivo de líquido amniótico para el diagnóstico in vitro de poliploidías / Amniotic fluid culture varietys for poliploydies in vitro diagnosis

RESUMEN Fundamento: El cultivo celular permite el análisis directo de las células vivas mediante un microscopio. El estudio de las células contenidas en el líquido amniótico, mediante técnicas de cultivo, detecta anomalías en número y morfología de los cromosomas, que pueden relacionarse con enfermedades genéticas. Objetivo: Caracterizar las variedades de cultivo de líquido amniótico para el diagnóstico in vitro de poliploidías. Metodología: Se realizó un estudio descriptivo transversal, en el Centro Provincial de Genética Médica de Camagüey, en el periodo de noviembre de 2016 a abril de 2018.La población de estudio estuvo constituida por 1571 muestras útiles de líquido amniótico obtenidas por amniocentesis, en gestantes en el segundo trimestre, evaluadas en consulta multidisciplinaria con criterios clínicos de estudios cromosómicos según lo establecido en el diagnóstico prenatal citogenético, previo consentimiento informado. Se utilizaron 20 mL de líquido amniótico para la siembra de células fetales, y se aplicaron tres variantes de cultivo abierto (directo, centrifugado y expandido). Se determinó el complemento cromosómico en cada variedad. Resultados: Predominó el complemento cromosómico normal. Las tetraploidías prevalecieron en el cultivo expandido. El índice mitótico fue similar en las tres variedades de cultivo y el cultivo directo tuvo el más bajo índice de poliploidías. Conclusiones: El cariotipo normal fue predominante. Las tetrapolidías fueron las alteraciones más frecuentes y prevalecieron en el cultivo expandido. En el cultivo directo se presentó el más bajo índice de errores inducidos in vitro.

ABSTRACT Background: Cell culture allows direct analysis of live cells under a microscope. The cell study contained in amniotic fluid, by culture techniques, detects abnormalities in chromosome number and morphology, which can be related to genetic diseases. Objective: To describe amniotic fluid culture strains for the in vitro diagnosis of polyploidy. Methodology: A cross-sectional descriptive study was conducted at the Camagüey Provincial Center of Medical Genetics, from November 2016 to April 2018.The study population consisted of 1571 useful amniotic fluid samples obtained by amniocentesis, in pregnant women in the second trimester, evaluated by multidisciplinary discussion with clinical criteria for chromosomal studies as established in the cytogenetic prenatal diagnosis, prior informed consent. 20 mL of amniotic fluid were used for fetal cell seeding, and three open culture strains (direct, centrifuged and expanded) were applied. Chromosomal complement was determined in each variety. Results: Normal chromosome complement was predominant. Tetraploidy prevailed in the expanded culture. The mitotic index was similar in the three culture strains and the direct culture had the lowest polyploidy index. Conclusions: Normal karyotype was predominant. Tetraploidy were the most frequent modifications and prevailed in the expanded culture. Direct culture had the lowest rate of the in vitro induced errors.

Assessment of fetal lung maturity using analysis of amniotic fluid from ewes that gave birth prematurely and at term / Avaliação da maturidade pulmonar fetal utilizando a análise de líquido amniótico de ovelhas que deram à luz prematuramente e a termo

The aim of this study was to evaluate the lung maturity of premature and full-term lambs by analyzing amniotic fluid using the following methods: Clements test, Nile blue cytology test, hematoxylin-Shorr stain, lamellar body count, and radiographic tests. The use of these methods is intended to identify high-risk newborns and provide immediate clinical intervention after birth. Altogether, 56 animals (24 ewes and 32 lambs) were included in the study and divided into 3 groups. Group I consisted of 8 ewes that were at approximately 145 days of gestation; this group delivered 10 lambs naturally. Group II consisted of 8 ewes that were at 138 days' gestation; this group delivered 11 lambs by cesarean section. Group III consisted of 8 ewes at 138 days' gestation; this group was administered intramuscular dexamethasone (16mg/animal) 36 hours prior to a cesarean section. Group III delivered11 lambs. Cytological tests were performed using a microscope with a maximum magnification of 1000x, while the Clements test was visually observed by one of the researchers. Amnioticfluid lamellar body counts were measured using transmission electron microscopy. Among the staining methods, hematoxylin-Shorr was reliable, and Group III had a greater number of orangeophilic cells when compared to Group II, probably due to corticoid administration. The Clements test showed pulmonary maturity in approximately 20% of Group I lambs and Group II showed 9.1% of bubbles; however, Group III had the highest pulmonary maturity percentage (36.4%). The lamellar bodies were measured, and all groups had sizes between 0.019 and 0.590μm. Radiographic evaluation revealed that the majority of lambs presented some level of pulmonary radiodensity, indicating an acinar pattern at birth. These results are in line with the expectations of each group. We found that the normal group showed greater pulmonary maturity, whereas Group II presented pulmonary immaturity, which is expected because this group comprised lambs born prematurely and Group III showed pulmonary maturity almost comparable to the normal delivery group (Group I). This is due to the fact that although these animals are premature, the use of dexamethasone helped in pulmonary maturation. Therefore, these pulmonary maturity tests are considered effective when more than one technique is used and can be used routinely in the care of a pregnant ewe in labor, where a simple collection of amniotic fluid can predict a high-risk pregnancy and alert the veterinarian if the newborn needs intensive supportive treatment.(AU)

O objetivo deste estudo foi avaliar a maturidade pulmonar de cordeiros prematuros e a termo por meio da análise do líquido amniótico utilizando os seguintes métodos: teste de Clements, teste de citologia do azul do Nilo, coloração de hematoxilina-Shorr, contagem de corpos lamelares e testes radiográficos. Um desses métodos tem por objetivo identificar recém-nascidos de alto risco e fornecer intervenção clínica imediata após o nascimento. Ao todo, 56 animais (24 ovelhas e 32 cordeiros) foram incluídos no estudo e divididos em 3 grupos. O grupo I foi composto por 8 ovelhas com aproximadamente 145 dias de gestação; este grupo deu à luz 10 cordeiros naturalmente. O Grupo II foi composto por 8 ovelhas com 138 dias de gestação; este grupo deu à luz 11 cordeiros por cesariana. Grupo III consistiu de 8 ovelhas com 138 dias de gestação; este grupo recebeu dexametasona intramuscular (16 mg / animal) 36 horas antes de uma cesariana. O Grupo III entregou 11 cordeiros. Os testes citológicos foram realizados em microscópio com aumento máximo de 1000x, enquanto o teste de Clements foi observado visualmente por um dos pesquisadores. A contagem de corpos lamelares de líquido amniótico foi medida usando microscopia eletrônica de transmissão. Dentre os métodos de coloração, o hematoxilina-Shorr foi confiável, sendo que o Grupo III apresentou maior número de células orangeofílicas quando comparado ao grupo II, provavelmente devido à administração de corticóide. O teste de Clements mostrou maturidade pulmonar em aproximadamente 20% dos cordeiros do Grupo I e o Grupo II apresentou 9,1% de bolhas; entretanto, o Grupo III apresentou o maior percentual de maturidade pulmonar (36,4%). Os corpos lamelares foram medidos e todos os grupos apresentaram tamanhos entre 0,019 e 0,590μm. A avaliação radiográfica revelou que a maioria dos cordeiros apresentava algum grau de radiodensidade pulmonar, indicando padrão acinar ao nascimento. Esses resultados estão alinhados com as expectativas de cada grupo. Verificamos que o grupo normal apresentou maior maturidade pulmonar, enquanto o Grupo II apresentou imaturidade pulmonar, o que é esperado por se tratar de cordeiros nascidos prematuramente e o Grupo III apresentou maturidade pulmonar quase comparável ao grupo de parto normal (Grupo I). Isso se deve ao fato de que, embora esses animais sejam prematuros, o uso da dexametasona auxiliou na maturação pulmonar. Portanto, esses testes de maturidade pulmonar são considerados eficazes quando mais de uma técnica são utilizadas e podem ser usadas rotineiramente no cuidado de uma ovelha gestante em trabalho de parto, onde uma simples coleta de líquido amniótico pode prever uma gravidez de alto risco e alertar o veterinário se o recém-nascido precisa de tratamento de suporte intensivo.(AU)

Ureaplasma urealyticum-derived lipid-associated membrane proteins introduce IL-6, IL-8, and TNF-α cytokines into human amniotic epithelial cells via Toll-like receptor 2 / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

OBJECTIVE@#The purpose of this study was to determine the role of Ureaplasma urealyticum-derived lipid-associated membrane proteins (LAMPs) in the host innate immune system, specifically their effect on Toll-like receptors (TLRs).@*METHODS@#LAMPs were derived from U. urealyticum strains, and human amniotic epithelial cells (HAECs) were isolated from healthy full-term placentas. Cytokine concentrations were determined by enzyme-linked immunosorbent assay (ELISA) and TLR2 mRNA by real-time PCR. Expression of TLR2 was confirmed by Western blotting and immunohistochemistry.@*RESULTS@#LAMPs induced HAECs to produce inflammatory cytokines interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-α. Cytokine production was reduced after blocking TLR2 using TLR2 inhibitor (anti-hTLR2-IgA).@*CONCLUSIONS@#LAMPs isolated from U. urealyticum induced TLR2-dependent up-regulation of inflammatory genes and cytokines in HAECs.

Non-Invasive Prediction of Histologic Chorioamnionitis in Women with Preterm Premature Rupture of Membranes

PURPOSE: To develop a model based on non-invasive clinical and ultrasonographic parameters for predicting the likelihood of subsequent histologic chorioamnionitis in women with preterm premature rupture of membranes (PPROM) and to determine whether the inclusion of invasive test results improves the predictive value of the model. MATERIALS AND METHODS: This retrospective cohort study included 146 consecutive women presenting with PPROM (20-33 weeks). Transvaginal ultrasonographic assessment of cervical length was performed. Maternal serum C-reactive protein (CRP) levels and white blood cell (WBC) counts were measured after amniocentesis. Amniotic fluid (AF) obtained by amniocentesis was cultured, and interleukin-6 (IL-6) levels and WBC counts were determined. The primary outcome measure was histologic chorioamnionitis. RESULTS: Risk scores based on serum CRP concentrations and gestational age (model 1) were calculated for each patient. The model was shown to have adequate goodness of fit and an area under the receiver operating characteristic curve (AUC) of 0.742. When including AF test results (e.g., AF IL-6 levels) in model 1, serum CRP concentrations were found to be insignificant, and thus, were excluded from model 2, comprising AF IL-6 levels and gestational age. No significant difference in AUC was found between models 1 and 2. CONCLUSION: For women with PPROM, the newly developed model incorporating non-invasive parameters (serum CRP and gestational age) was moderately predictive of histologic chorioamnionitis. The inclusion of invasive test results added no predictive information to the model in this setting.

Pre-Clinical Efficacy and Safety Evaluation of Human Amniotic Fluid-Derived Stem Cell Injection in a Mouse Model of Urinary Incontinence

PURPOSE: Stem cell-based therapies represent new promises for the treatment of urinary incontinence. This study was performed to assess optimized cell passage number, cell dose, therapeutic efficacy, feasibility, toxicity, and cell trafficking for the first step of the pre-clinical evaluation of human amniotic fluid stem cell (hAFSC) therapy in a urinary incontinence animal model. MATERIALS AND METHODS: The proper cell passage number was analyzed with hAFSCs at passages 4, 6, and 8 at week 2. The cell dose optimization included 1x10(4), 1x10(5), and 1x10(6) cells at week 2. The in vivo cell toxicity was performed with 0.25x10(6), 0.5x10(6), and 1x10(6) cells at weeks 2 and 4. Cell tracking was performed with 1x10(6) cells at weeks 2 and 4. RESULTS: The selected optimal cell passage number was smaller than 6, and the optimal cell dose was 1x10(6) for the mouse model. In our pre-clinical study, hAFSC-injected animals showed normal values for several parameters. Moreover, the injected cells were found to be non-toxic and non-tumorigenic. Furthermore, the injected hAFSCs were rarely identified by in vivo cell trafficking in the target organs at week 2. CONCLUSION: This study demonstrates for the first time the pre-clinical efficacy and safety of hAFSC injection in the urinary incontinence animal model and provides a basis for future clinical applications.

Intra-Amniotic Infection/Inflammation as a Risk Factor for Subsequent Ruptured Membranes after Clinically Indicated Amniocentesis in Preterm Labor

The aim of this study was to determine whether intra-amniotic infection/inflammation (IAI) was associated with subsequent ruptured membranes in women with preterm labor and intact membranes who had a clinically indicated amniocentesis. This retrospective cohort study included 237 consecutive women with preterm labor (20-34.6 weeks) who underwent amniocentesis. The clinical and laboratory parameters evaluated included demographic variables, gestational age, C-reactive protein (CRP) and amniotic fluid (AF) white blood cell, interleukin-6 (IL-6) and culture results. IAI was defined as a positive AF culture and/or an elevated AF IL-6 level (>2.6 ng/mL). The primary outcome was ruptured membranes in the absence of active labor occurring within 48 hours of amniocentesis. Preterm premature rupture of membranes subsequently developed in 10 (4.2%) women within 48 hr of amniocentesis. Multivariate analysis demonstrated that only IAI was independently associated with the ruptured membranes occurring within 48 hr of amniocentesis. In the predictive model based on variables assessed before amniocentesis, only CRP level was retained. IAI is an independent risk factor for subsequent ruptured membranes after clinically indicated amniocentesis in preterm labor. Prior to amniocentesis, measurement of serum CRP level can provide a risk assessment for the subsequent development of ruptured membranes after the procedure.

Valor predictivo del índice de líquido amniótico en las complicaciones neonatales / Predictive value of amniotic fluid index in neonatal complications

Determinar el valor predictivo del índice de líquido amniótico en las complicaciones neonatales. Se seleccionaron 120 embarazadas en las que se evaluó el valor del índice de líquido amniótico, complicaciones neonatales y eficacia diagnóstica. Las pacientes fueron divididas según el punto de corte del índice de líquido amniótico (grupo A: índice de líquido amniótico menor de 60 mm y grupo B índice de líquido amniótico igual o mayor a 60 mm). Servicio de Obstetricia y Ginecología. Hospital Central “Dr. Urquinaona”. Maracaibo. Estado Zulia. Las pacientes del grupo A presentaron una duración mayor del trabajo de parto y recién nacidos con menos peso al nacer que las pacientes del grupo B (P < 0,05). Con respecto a las complicaciones perinatales, la frecuencia de recién nacidos con sufrimiento fetal y con puntuación de Apgar menor o igual de 6 puntos al minuto fue estadísticamente superior en las pacientes del grupo A comparado con aquellas del grupo B (P < 0,05). El valor de corte de 60 mm en la predicción de sufrimiento fetal tiene una sensibilidad del 22,2 por ciento, especificidad del 96,4 por ciento, valor predictivo positivo del 72,3 por ciento y valor predictivo negativo del 74,3 por ciento; en la predicción de puntuación de Apgar menor o igual de 6 puntos al minuto tiene una sensibilidad del 25,0 por ciento, especificidad del 96,4 por ciento, valor predictivo positivo del 69,2 por ciento y valor predictivo negativo del 74,7 por ciento. El índice de líquido amniótico tiene valor en la predicción de sufrimiento fetal y puntuación de Apgar

To determine the predictive value of amniotic fluid index in perinatal complications. One hundred and twenty patients were selected. Amniotic fluid index, perinatal complications and diagnostic accuracy was evaluated. Patients were divided according to cut-off point of amniotic fluid index (group A: amniotic fluid index less than 60 mm and group B amniotic fluid index same or higher than 60 mm). Servicio de Obstetricia y Ginecologia. Hospital Central “Dr. Urquinaona”. Maracaibo. Estado Zulia. Patients in group A presented a longer labor and there newborns had less weight than those of patients in group B (P < 0.05). In relation to perinatal complications, the frequency of fetal distress and Apgar Score less than 6 points at minute was statically superior in patients of group A compared with those in group B (P < 0.05). The cut-off value of 60 mm for prediction of fetal distress has a sensivity of 22.2 percent, specificity of 96.4 percent, positive predictive value of 72.3 percent and negative predictive value of 74.3 percent; in the prediction of Apgar score equal or less than 6 points at minute had a sensivity of 25.0 percent, specificity of 96.4 percent, positive predictive value of 69.2 percent and negative predictive value of 74.7 percent. Amniotic fluid index has a value for prediction of fetal distress and Apgar score

Human Amniotic Fluid Stem Cell-derived Muscle Progenitor Cell Therapy for Stress Urinary Incontinence

The most promising treatment for stress urinary incontinence can be a cell therapy. We suggest human amniotic fluid stem cells (hAFSCs) as an alternative cell source. We established the optimum in vitro protocol for the differentiation from hAFSCs into muscle progenitors. These progenitors were transplanted into the injured urethral sphincter and their therapeutic effect was analyzed. For the development of an efficient differentiation system in vitro, we examined a commercial medium, co-culture and conditioned medium (CM) systems. After being treated with CM, hAFSCs were effectively developed into a muscle lineage. The progenitors were integrated into the host urethral sphincter and the host cell differentiation was stimulated in vivo. Urodynamic analysis showed significant increase of leak point pressure and closing pressure. Immunohistochemistry revealed the regeneration of circular muscle mass with normal appearance. Molecular analysis observed the expression of a larger number of target markers. In the immunogenicity analysis, the progenitor group had a scant CD8 lymphocyte. In tumorigenicity, the progenitors showed no teratoma formation. These results suggest that hAFSCs can effectively be differentiated into muscle progenitors in CM and that the hAFSC-derived muscle progenitors are an accessible cell source for the regeneration of injured urethral sphincter.

Mesenchymal stem cells from murine amniotic fluid as a model for preclinical investigation

Despite the suitability of a mouse model for preclinical investigations; little is known regarding mesenchymal stem cells derived from murine amniotic fluid. This is the subject of the present study. Amniotic fluid was collected from NMRI mice during the second weeks of pregnancy and plated. The cells that adhered to the culture surfaces were propagated with three successive subcultures and then characterized. To determine the differentiation potential, the cells were cultivated under osteogenic, adipogenic, and chondrogenic conditions, and followed by specific staining and RT-PCR analysis for differentiation. The proliferative potential of the cells were with clonogenic assays, population doubling time and number and by growth curve plotting. Cellular aging was investigated with the senescence-associated beta-galactosidase staining method. The amniotic fluid primary cell culture was composed of round flattened and fibroblastic cells. The latter dominated the culture after several passages. Successful tripotent differentiation of the isolated cells into bone, cartilage and adipose cells were indicative of their mesenchymal stem cells nature. The isolated cells appeared to be relatively proliferative cells as confirmed by the population doubling time value which was equal to about 69 hours. Furthermore, the cells were relatively clonogenic and they tended to initate proliferation immediately after plating [there was no lag phase in their growth curve]. Beta- galactosidase positive cells were first observed at passage 3 and increased in number with subsequent passagers. Collectively it was concluded that murine amniotic fluid contained mesenchymal stem cells with relatively high proliferation property and typical tripotent differentiation potential

Perfil citológico dos líquidos amniótico e alantoidiano nos terços inicial, médio e final da gestação na vaca / Cytology of amniotic and allantoic fluids in the cow at the first, second, and third trimester of pregnancy

The cytology of extraembrionic fluids in cows in the first, second, and third trimester of pregnancy was evaluated. For each trimester, 10 pregnant uteri, collected in the slaughterhouse were used. A volume of 20mL of amniotic and allantoic fluids was collected, and centrifuged at 200g for 10 minutes. The sediment was taken and processed on a slide for cytology examination after staining. The results showed that the total cells number of the amniotic fluid increased during gestation, with the panoptic staining method being efficient to evaluate the amniotic fluid cytology in crossbreed Holstein x Zebu cows.