Application of droplet digital PCR for non-invasive prenatal diagnosis of single gene disease in two families / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To assess the value of droplet digital PCR (ddPCR) for non-invasive prenatal diagnosis of single gene disease in two families.</p><p><b>METHODS</b>Paternal mutation in cell-free DNA derived from the maternal blood and amniotic fluid DNA was detected by ddPCR. Suspected mutation in the amniotic fluid DNA was verified with Sanger sequencing.</p><p><b>RESULTS</b>The result of ddPCR and Sanger sequencing indicated that the fetuses have carried pathogenic mutations from the paternal side in both families.</p><p><b>CONCLUSION</b>Droplet digital PCR can accurately detect paternal mutation carried by the fetus, and it is sensitive and reliable for analyzing trace samples. This method may be applied for the diagnosis of single gene diseases caused by paternal mutation using peripheral blood sample derived from the mother.</p>

Do triple test results predict risk for neonatal hyperbilirubinemia?

Objective: Neonatal jaundice is the most common condition that requires hospital admission and outpatient follow-up after discharge in neonates

The values of more than 17 mg/dL in term infants are accepted as neonatal significant hyperbilirubinemia. We aimed to define if there is any relationship between second trimester serum markers and neonatal severe hyperbilirubinemia to protect the neonates from its neurological damage

Methods: Total 1372 pregnant women were enrolled who had done triple test between April 2014 and 2015 and then given birth at our hospital. Our primary outcome was neonatal significant hyperbilirubinemia

Results: The mean age of our study population was 27.9+/-5.6. A total of 59 patients had babies with neonatal hyperbilirubinemia after exclusion of Rh incompatibility. We detected that the presence of in vitro pregnancy, maternal health problems or poor obstetric history had no effect on the risk for neonatal hyperbilirubinemia. Neonatal hyperbilirubinemia was related with low E[3] levels. The ratios of AFP/E[3] and hCG/E[3]were the most helpful to predict the neonatal hyperbilirubinemia

Conclusions: According to our results, low E3 levels in the triple test result can be helpful to predict the development of the neonatal hyperbilirubinemia. However, this is a bit expensive and many developing countries may not afford it

Management of syphilis in pregnant womenand their newborns: is it still a problem? / Manejo de sífilis em gestantes e seus recém-nascidos: ainda um problema?

A sífilis congênita (SC) é um agravo prevenível, mas o Brasil ainda apresenta alta prevalência da doença, com consequente morbimortalidade perinatal. Objetivo: Avaliar a abordagem de sífilis em gestantes e seus recém-nascidos encaminhados para centro de referência. Métodos: Estudo transversal, de março de 2012 a abril de 2013. A coleta de dados foi realizada em prontuários de pacientes referenciados com SC, considerando critérios estabelecidos pelo Ministério da Saúde (MS). Os dados foram analisados pelo Statistical Package for the Social Sciences (SPSS) e o estudo foi aprovado pelo Comitê de Ética. Resultados: Um total de 31 recém-nascidos foi encaminhado devido à triagem materna com Venereal Disease Research Laboratory(VDRL) materno positivo durante a gestação, com 4 mulheres adequadamente tratadas. Treze recém-nascidos apresentaram alteração no hemograma e1 apresentou alteração óssea, 28 deles com tratamento adequado. Discussão: Quando se considera adequação de tratamento de acordo com as diretrizes nacionais, poucos casos de sífilis na gestação são considerados adequadamente tratados. Isso impacta na assistência ao recém-nascido, que, muitas vezes,é submetido a propedêutica invasiva e tratamento extenso, embora na maioria das vezes seja assintomático. Conclusão: O seguimento das recomendações para o tratamento da sífilis na gestante tem sido, frequentemente, considerado inadequado, o que dificulta a eliminação da SC.

Congenital syphilis (CS) is a preventable disease, but its prevalence is still high in Brazil, with consequent perinatal morbidity and mortality.Objective: To evaluate the approach of syphilis in pregnant women and their newborns referred to the referral center of Orestes Diniz, in Belo Horizonte.Methods: A cross-sectional study was carried out from March 2012 to April 2013. Data collection was performed on the medical records of patients referred with CS, considering the criteria established by the Ministry of Health. Data were analyzed using SPSS and the study was approved by the Ethics Committee. Results: A total of 31 newborns were referred due to a positive result in maternal testing with Venereal Disease Research Laboratory during pregnancy. However, only four women have been adequately treated in accordance with the Ministry of Health. Thirteen newborns presented alterations inblood cells count, one had bone rarefactions, and 28 presented proper information of treatment. Discussion: When considering the adequacy of treatmenta ccording to the national guidelines, few cases of syphilis during pregnancy can be considered adequately treated. This affects the assistance to the newborn,who is often subjected to invasive investigation and extensive treatment, although most are asymptomatic. Conclusion: The follow-up of recommendations for the treatment of syphilis in pregnant women has often been considered inadequate, making CS difficult to eliminate

Revisão sistemática dos métodos sorológicos utilizados em gestantes nos programas de triagem diagnóstica pré-natal da toxoplasmose / Systematic review of serological methods used in prenatal screening of toxoplasmosis in pregnant women

Informações sobre a melhor estratégia para triagem sorológica da toxoplasmose em gestantes são escassas e poucos estudos mencionam o uso de amostras de sangue capilar. Realizou-se uma revisão sistemática para pesquisar os métodos sorológicos empregados em programas de triagem pré-natal da toxoplasmose no mundo e as características principais destes programas, com busca nas bases de dados PUBMED e LILACS. Foram selecionados artigos referentes a programas de triagem sorológica pré-natal da toxoplasmose que descrevessem a amostra (sangue capilar ou soro) e o teste sorológico utilizado. Foram encontrados 1554 trabalhos no PUBMED e 242 na LILACS, sendo 58 em duplicata. Foram analisados 47 artigos finais. Os testes sorológicos de triagem citados com maior frequência foram os imunoenzimáticos para detecção de IgG (19 ou 40,4%) e IgM (18 ou 38,3%) e, entre os testes confirmatórios, o mais utilizado foi o teste de avidez de IgG (14 ou 29,8%). Todos os estudos analisados utilizaram amostras de soro para a triagem pré-natal da toxoplasmose.

The best strategy for toxoplasmosis serological screening in pregnant women is not completely defined and few studies mention the use of capillary blood samples. A systematic review of the literature was conducted to investigate the serological methods used in prenatal screening programs of toxoplasmosis in the world and the main features of these programs, with search in PubMed and LILACS databases. We selected articles that described their serological prenatal screening programs, with mention of the sample (capillary blood or serum) and of the serological tests used. We found 1554 articles in PubMed database and 242 articles in LILACS, with 58 duplicates. 47 final articles were analyzed. The serological screening tests most frequently cited were immunoassays for the detection of IgG (19, 40.4%) and IgM (18, 38.3%) and between confirmatory tests, the most used was IgG avidity test (14, 29.8%). All analyzed studies used serum samples for toxoplasmosis prenatal screening. There is need for studies assessing and testing different samples in longitudinal studies.

Incorporación del estudio de ADN fetal en sangre materna al cribado de cromosomopatías / Incorporation of the study of fetal DNA in maternal blood to the screening of chromosomal diseases

OBJETIVO: Evaluar la efectividad del cribado combinado de primer trimestre para la detección prenatal de aneuploidías tras 6 años de implantación en nuestro servicio y su repercusión en la disminución de pruebas diagnósticas invasivas. Se propone establecer un protocolo para incorporar el estudio de ADN fetal en sangre materna a partir de las revisiones bibliográficas publicadas. MÉTODO: Se evaluó el riesgo de anomalía cromosómica fetal en 3177 gestaciones mediante cribado combinado de primer trimestre entre enero de 2011 y diciembre de 2014. Se revisaron las amniocentesis realizadas desde que se instauró el cribado combinado en 2008 comparándolas con las de los 5 años anteriores. RESULTADOS: La tasa de detección del cribado para trisomía 21 fue del 94,4% y la tasa de falsos positivos de 6,4%. En el año 2005 estábamos realizando 194 amniocentesis, tras 6 años de implantación del cribado, en el año 2013 se realizaron 35 amniocentesis lo que implica una disminución del 70%. CONCLUSIONES: El cribado combinado de primer trimestre ha demostrado una mayor tasa de detección para trisomía 21 que el cribado de segundo trimestre y/o la edad materna, además de que ha llevado a una importante reducción en el número de pruebas invasivas. En los próximos años la incorporación del estudio de ADN fetal mejorará la detección de aneuploidías, con una drástica disminución de las pruebas invasivas por lo que se hace necesario la implantación de nuevos protocolos.

AIMS: To evaluate the effectiveness of first trimester combined screening in the prenatal detection of aneuploidy after 6 years of implantation in our service and its impact in reducing invasive diagnostic tests. It is proposed to establish a protocol to incorporate the study of fetal DNA in maternal blood from published literature reviews. METHODS: The risk of fetal chromosomal anomalies was assessed in 3177 pregnancies with first trimester combined screening between January 2009 and December 2014. The amniocenteses performed were checked against those of the previous 5 years. RESULTS: The detection rate of screening for trisomy 21 was 94.4% and the false-positive rate was 6.4%. In 2005 there were 194 amniocenteses. In 2013, 5 years after the introduction of screening, 68 amniocenteses were performed, representing a 70% reduction in invasive procedures. CONCLUSIONS: First trimester combined screening has shown a higher detection rate for trisomy 21 that the second trimester screening and/or maternal age, and has substantially reduced the use of invasive prenatal diagnostics procedures. In the coming years, the incorporation of the study of fetal DNA improve the detection of aneuploidys with a drastic reduction of invasive tests so that, the implementation of new protocols is necessary.

Non-invasive prenatal test using cell free DNA

Although conventional prenatal screening tests for Down syndrome have been developed over the past 20 years, the positive predictive value of these tests is around 5%. Through these tests, many pregnant women have taken invasive tests including chorionic villi sampling and amniocentesis for confirming Down syndrome. Invasive test carries the risk of fetal loss at a low but significant rate. There is a large amount of evidence that non-invasive prenatal test (NIPT) using cell free DNA in maternal serum is more sensitive and specific than conventional maternal serum and/or ultrasound screening. Therefore implementing NIPT will increase aneuploidy detection rate and concurrently decrease fetal loss rate accompanying invasive test. More than 1,000,000 NIPT were performed globally since 2011. The uptake rate of NIPT is expected to increase more rapidly in the future. Moreover, as a molecular genetic technique advances, NIPT can be used for not only common aneuploidy screening but single gene disorder, microdeletion, and whole fetal genome sequencing. In this review, I will focus on the NIPT for common aneuploidies such as trisomy 13, 18, and 21.

The assessment of combined first trimester screening in women of advanced maternal age in an Asian cohort

<p><b>INTRODUCTION</b>First trimester screening (FTS) is a validated screening tool that has been shown to achieve detection rates of 84%-90% for trisomies 21, 18 and 13. However, its effectiveness for different maternal ages has not been assessed. The present study aimed to assess the performance of FTS in an Asian population, and to compare its effectiveness in older (≥ 35 years) and younger (< 35 years) women. The potential use of noninvasive prenatal test (NIPT) as a contingent screening test is also examined.</p><p><b>METHODS</b>Data on cases of FTS performed on singleton pregnancies over a six-year period was collated from two Singapore maternal centres, National University Hospital and Singapore General Hospital. Cases that had a 1:250 risk of trisomy were considered to be screen-positive. Pregnancy outcomes were obtained from birth records or karyotype test results.</p><p><b>RESULTS</b>From 10,289 FTS cases, we obtained a sensitivity of 87.8%, a specificity of 97.6%, a false positive rate of 2.4% and a false negative rate of 0.06% for the detection of aneuploidy. The overall detection rate for trisomy 21 was 86.5%-85.7% for older women and 87.5% for younger women. The mean number of invasive tests required per case of trisomy 21 was 9.3 in younger women, 8.6 in older women and 13.5 in women with intermediate risk (1:250-1,000).</p><p><b>CONCLUSION</b>While the performance of FTS was similar in younger and older women, more invasive procedures were required to diagnose trisomy 21 in women with intermediate risk. It may be advantageous to offer contingent NIPT to this group of women to reduce the risk of iatrogenic fetal loss.</p>

Rastreamento de aneuploidias no primeiro trimestre de gestação: evolução da idade materna à avaliação do DNA fetal livre no sangue materno / Screening for aneuploidies in the first trimester of pregnancy: evolution from maternal age to cell-free DNA testing in maternal blood

O rastreamento fetal de aneuploidia apresentou uma evolução fantástica a partir da avaliação individual da idade materna até os dias atuais, na qual evidências sugerem que o teste de avaliação do DNA fetal livre no sangue materno detecta mais de 99% dos casos de trissomia do cromossomo 21 e, aproximadamente, 98% dos casos de trissomia do 18 e 92%, do 13, com taxas de falso-positivo de 0,1; 0,1 e 0,3%, respectivamente. Recentemente, o grupo de trabalho em boas práticas médicas da Federação Internacional de Ginecologia e Obstetrícia recomendou que todas as gestantes, independentemente da idade, deveriam realizar uma avaliação de risco para aneuploidias por meio da translucência nucal, do teste combinado ou do teste de DNA fetal livre no sangue materno. O teste invasivo para diagnóstico de aneuploidia não deveria ser realizado considerando apenas a idade materna como fator de risco. O objetivo desta revisão foi apresentar esta nova ferramenta de rastreio, presente em muitos centros, e descrever as estratégias para implementação de tal tecnologia na prática clínica diária.(AU)

Screening for fetal aneuploidy has a tremendous evolution from maternal age to now where recent evidence suggests that cell-free DNA testing in maternal blood can detect more than 99% of cases of trisomy 21, about 98% of trisomy 18, and 92% of trisomy 13, with respective false-positive rates of 0.1, 0.1, and 0.3%. Recently, the working group on the best practice on maternal fetal medicine of the International Federation of Gynecology and Obstetrics has recommended as a good medical practice that pregnant women, regardless of maternal age, be offered prenatal assessment for aneuploidy through nuchal translucency, combined test, or cell-free DNA testing. The invasive procedure for diagnosis of aneuploidy should be avoided taking into account only the maternal age as a risk factor nowadays. The purpose of this review was to present this new screening tool available in most centers and to describe the strategies for implementation of this technology on the daily clinical practice.(AU)

Clinical application of non-invasive prenatal testing using cell free fetal DNA

Non-invasive prenatal testing using next generation sequencing technology with cell free fetal DNA from the blood of pregnant women has been rapidly adopted as a screening test for the detection of disorders involving chromosomal aneuploidy, especially Down syndrome. However as part of a prenatal recommendation in high-risk group, this laboratory assessment should be accompanied by informed counseling at both pre-test and post-test stages. In low-risk group and multifetal pregnancies, only conventional maternal serum screening tests in the first trimester and/or second trimester in addition to measurement of nuchal translucency should be recommended, until this potential tool has been incorporated into current screening strategic modalities on the basis ofsufficient published data.

Eight years incidence of diabetes mellitus in gestational diabetic patients

To evaluate eight years incidence of Gestational Diabetes Mellitus [GDM]. A retrospective study. Primary Healthcare, Bahrain. Five hundred sixty-two patients suffering from GDM were identified in 2002 and were traced in 2010 for laboratory diagnosis of diabetes. Two hundred one [36%] patients were found to be diabetic. One hundred one [18%] were normal, 33 [5.8%] had impaired glucose tolerance [IGT] and 227 [40.4%] were of undetermined status. There is a statistically significant relation between the development of diabetes and the age at diagnosis of GDM. The long-term rate development of diabetes after GDM is high. Age and level of screening test at diagnosis are strong predictive factors

Comparação da coleta das amostras de sangue na triagem pré-natal, utilizando o papel de filtro e punção venosana técnica de ELISA para detecção de sífilis / Comparison of the collection of blood samples in prenatal screening, using the filter paperand venipuncture technique ELISA for the detection of syphilis

A sífilis é uma doença infecciosa sistêmica, de evolução crônica e causada pelo Treponema pallidum, um espiroqueta de transmissão sexuale vertical, que pode produzir, respectivamente, as formas adquirida e congênita da doença. No Brasil, segundo o Ministério da Saúde (MS), embora a subnotificação de casos de sífilis seja alta, alguns dados disponíveis indicam a elevada magnitude deste problema infeccioso. Objetivo: comparar a coletadas amostras de sangue no papel de filtro (PF) e no plasma (padrão-ouro) na triagem pré-natal, utilizando anticorpos antitreponêmicos totais (IgG + IgM) no procedimento imunoquímico ELISA, registrado pelo Imunoscreen, da firma MBiolog. Métodos: foram estudadas 1.142 grávidas de quatro municípiosdo Estado de Rio de Janeiro: Itaboraí (N = 131), Itaguaí (N = 597), Niterói (N = 377) e São João de Meriti (N = 37) a partir do mês de novembro de 2008 até fevereiro de 2009. As grávidas foram submetidas a punção venosa e digital para a rotina da triagem pré-natal, sendo a última aplicada em PF. Foram calculados os limites de especificidade, sensibilidade e valores preditivos positivo e negativo para o estudo. Resultados: os resultados da sorologia parasífilis nas amostras do município de Itaboraí apresentaram ELISA positivo em 4,58%, os municípios de Itaguaí, Niterói e São João de Meriti mostraram positividade em 3,18%, 2,65% e 0%, respectivamente. Os procedimentos realizados tiveram uma sensibilidade e especificidade de 100% e os critérios preditivos positivos e negativos para todas as grávidas, estudados nas 1.142 amostras, foram de 100%. Conclusão: os resultados da sorologia para sífilis no sangue seco coletado em PF foram semelhantes aos da coleta por punção venosa, validando esta técnica.

Syphilis is a systemic disease of chronic evolution and caused by Treponema pallidum, a spirochete of sexual and vertical transmission,which can produce, respectively, the form of acquired and congenital disease. In Brazil, according to the Ministry of Health (MoH), although the underreporting of cases of congenital syphilis is high, some available data indicate the high magnitude of this problem that especially affects the weakness of pregnant women. Congenital syphilis causes great social impact, which results in deterioration of quality of life on a important stratum of the population,and indirect costs to the economy of the country, which, added to the direct costs resulting from hospitalizations and procedures for the treatment of its complications, increasing the total costs of care of public health. Objective: to compare the collection of blood samples on filter paper (FP) and plasma(gold standard) in prenatal screening, using anti-treponema total (IgG + IgM) in ELISA immunochemical. Methods: we studied 1,142 pregnant of the following cities: Itaboraí (N = 131), Itaguaí (N = 597), Niterói (N = 377) and St. João de Meriti (N = 37). Blood samples were collected from the finger and venipuncture of pregnant women in stations of collection of these counties, calculating elapsed time from sample collection to delivery of the report to thecouncil. We calculated the limits of sensitivity, specificity, positive predictive value and negative for the study. Results: Itaboraí showed positive ELISA in4.58%, in Itaguai, Niterói and St. João de Meriti showed, respectively, 3.18%, 2.65% and 0%. The procedures performed had a sensitivity and specificity of 100%, and the positive and negative predictive criteria for all pregnant women studied in 1,142 samples were 100%. All positive cases were reported to the Municipality within 10 days of sample collection. Conclusion: we conclude that the implementation of collection of dried blood on filter paper in pregnantwomen screening, was similar to that collected by venipuncture, validating this technology.

Triploidy in a fetus following amniocentesis referred for maternal serum screening test at second trimester.

Amniocentesis was carried out at 17 weeks gestation in a 27-year-old woman, following an abnormal maternal serum screening (MSS) test. MSS test was carried out primarily to estimate the risk of trisomy for chromosome 21. The maternal serum markers used were alpha-fetoprotein (AFP), human chorionic gonadotrophin (hCG), and unconjugated estriol (uE3), together with maternal age. The fetus was identified as screen-positive for Edward’s syndrome (trisomy 18), with low uE3, normal AFP and hCG levels. The calculated risk for trisomy 18 was more than 1:50. To identify any possible chromosomal abnormality, cytogenetic investigation was carried out on the amniotic fluid sample. The fetus’s karyotype showed triploidy with 69, XXX chromosome complement in all the metaphase spreads obtained from three different cultures, using GTG banding technique. Upon termination of the fetus, gross abnormalities indicative of triploidy were present in the fetus.

Evaluation of Access(R), Automatic Immunoassay Analyzer, for Screening of the Risk of Down's Syndrome in Mother's Serum / 대한진단검사의학회지

INTRODUCTION: It is reported that analytical precision in maternal serum screening tests for Down's syndrome has a direct effect on the precision of the risk estimation. Recently, an automatic immunoassay analyzer, which can measure the alphafetoprotein (AFP), chorionic gonadotropin (CG) and unconjugated estriol (uE3) was introduced, and we evaluated the analytical characteris-tics and defined the median value in healthy pregnant women. METHOD: We measured the AFP, CG and uE3 with Access(R) (Beckman Coulter, Fullerton, CA, USA) and evaluated the precision, the low detection limit, the linearity and we defined the median value in the women who were in 15-20 weeks of pregnant. RESULT: Between-run precisions of AFP, CG, uE3 were 1.12%, 2.01%, and 2.59%, respectively. The lower detection limit of AFP, CG, uE3 was 0.08 ng/mL, 0.16 mIU/mL, and 0.015 ng/mL, respec-tively. All three items passed the lack of fit test of the linear regression. Median values for the gesta-tional period (15- 20 week) of AFP, CG, uE3 were 37.47- 69.01 ng/mL, 41.44- 29.10 IU/mL, and 0.871- 2.008 ng/mL, respectively. CONCLUSION: The automatic immunoassay analyzer used for screening for Down's syndrome was satisfactory for the analytical features and showed median values similar to that of the Wald's. We conclude that Access(R) could be used for screening pregnant women for Down's syndrome with better precision and convenience.

Analysis of Serum Triple-Marker Screening in In Vitro Fertilization and Naturally Conceived Pregnancy / 대한산부인과학회잡지

OBJECTIVE: To compare and analysis the result of second trimester maternal serum triple marker screening test for Down syndrome and open neural tube defects in singleton pregnancies conceived by conventional in vitro fertilization- embryo transfer (IVF-ET) with that of the naturally conceived pregnancies. METHODS: Maternal serum screening tests during the second trimester in 49 singleton pregnancies conceived by IVF-ET and 813 singleton pregnancies conceived naturally of whom delivery outcome was normal in each other were analyzed from April 1997 to June 2000. RESULTS: 4 (8.2%) out of 49 cases of IVF-ET singleton pregnancies compared with 62 (7.6%) out of 813 cases of naturally conceived pregnancies had a positive RESULTS: for Down syndrome or open neural tube defects. The median level of the triple markers were 1.03 0.47 multiples of the median (MoM) in IVF-ET pregnancies vs 1.05+/-0.39 MoM in natural pregnancies for alpha-fetoprotein (AFP), 1.11+/-0.64 vs 1.19+/-1.13 MoM for unconjugated estriol (uE3) and 1.21+/-0.56 vs 1.11+/-0.59 MoM for human chorionic gonadotropin (hCG). CONCLUSION: The positive rate of triple test and the median values of triple markers for Down syndrome and open neural tube defect between two groups were not different in terms of statistical significance. To provide an objective assessment of an individual patient's risk of fetal abnormality, the impact of IVF-ET on triple marker biochemistry should be studied further in larger samples and adjustments made if appropriate.

Clinical Analysis of Midtrimester Amniocentesis / 대한산부인과학회잡지

OBJECTIVES: Amniocentesis is the most commonly used invasive method for prenatal diagnosis of genetic disorders. But this invasive study may induce preterm labor, endanger both the mother and the fetus, cause infection and abortion. This study was performed to clarify the safety and confidency of amniocentesis. MATERIAL AND METHODS: 1,500 amniocentesis cases were performed from 1987 to 1996 at Severance hospital for prenatal diagnosis of genetic disorders. Of 1,500 cases, 499 cases whose pregnancy outcomes were proven were finally analized. We compared incidence of complications such as spontaneous abortion, vaginal spotting, premature rupture of membranes, preterm labor or chorioamnionitis according to placental location, frequency of needle insertion and amniotic fluid color. RESULTS: 1) Indications for amniocentesis were (1) advanced maternal age - 47.3%, (2) positive maternal serum triple test - 13.8%, (3) fetal chromosomal anomaly suspected in ultrasonography - 10.0%. 2) 6.6% of the amniocentesis samples had color changes due to bleedng or meconium passing. 3) 18.8% of the cases were performed at 16th gestational weeks followed by 20th, 18th and 17th weeks. 4) 2.2% had complications such as spontaneous abortion, vaginal bleeding, premature rupture of membranes, preterm labor and chorioamnionitis in 4 weeks after amniocentesis. 5) No correlation was found between the placental location, frequency of needle insertion(below 3 times) and complication rate. The spontaneous abortion rate in the group with amniotic fluid discolorization was 6.1% whose only 0.4% had spontaneous abortion to the group with clear amniotic fluid. 6) Median value of amniotic fluid alphafetoprotein(alphaFP) of pregnant women in midtrimester were 15,769.4ng/ml at 15 weeks, 13,160.3ng/ml at 16 weeks, 11,539.3ng/ml at 17 weeks, 9,569.8 mg/ml at 18 weeks, 8,423.4mg/ml at 19 weeks, 6,527.1ng/ml at 20 weeks, 5,979.3ng/ml at 21 weeks, 4,363.2ng/ml at 22 weeks, 3,555.2ng/ml at 23weeks, respectively. In midtrimester amniotic fluid alphaFP declined gradually as gestational weeks increase. CONCLUSIONS: Fetal loss rate of midtrimester amniocentesis was 0.8%, proving that it outstanding in the aspects of safty and confidency. Since the fetal loss rate significantly increases in case with amniotic fluid discoloration, it is needless to mention the importance of close follow-up.