First trimester fetal sex determination in maternal serum using real-time PCR

OBJECTIVE: Fetal sex prediction can be achieved using PCR targeted at the SRY gene by analyzing cell-free fetal DNA in maternal serum. Unfortunately, the results reported to date, show lack of sensitivity, especially in the first trimester of pregnancy. Therefore, determination of fetal sex by maternal serum analysis can not replace caryotype analysis following chorionic villus sampling. PATIENTS AND METHODS: A new highly sensitive real-time PCR was developed to detect a SRY gene sequence in maternal serum. Analysis was performed on 121 pregnant women during their first trimester of pregnancy (mean gestational age: 11.8 weeks). Among them, 61 had at least one previous male-bearing pregnancy. Results were compared to fetal sex. RESULTS: SRY PCR analysis of maternal serum was in complete concordance with fetal sex. Among the 121 pregnant women, 61 were bearing a male fetus and 60 a female fetus No false negative results were observed. Furthermore, no false positive results results occurred although 27 women carried female fetus during the current pregnancy, had at least one previous male-bearing pregnancy. DISCUSSION AND CONCLUSION: This study demonstrates that a reliable, non-invasive sex determination can be achieved by PCR analysis of maternal serum during the first trimester of pregnancy. This non-invasive approach for fetal sex prediction should have great implications in the management of pregnant women carriers of an X-linked genetic disorder. Prenatal diagnosis is thus performed for male fetuses only, avoiding invasive procedures and the risk of fetal loss for female fetuses.     

Earliest gestational age for fetal sexing in cell-free maternal plasma

OBJECTIVES: To evaluate at what gestational age fetal DNA can reliably be detected at the earliest in maternal plasma. METHODS: We performed consecutive blood sampling in the first trimester of pregnancy in 17 women who were pregnant after in vitro fertilization (IVF) or intrauterine insemination (IUI). DNA was isolated and the Y-chromosome specific SRY was amplified by real-time polymerase chain reaction (PCR).We likewise studied 31 women prior to invasive prenatal diagnosis procedures for test validation purposes. All test results were compared to cytogenetic sex or sex at birth. RESULTS: The earliest SRY detection was at a gestational age of 5 weeks and 2 days. In none of 4 pregnancies ending in a miscarriage was SRY detected. We detected SRY in maternal plasma in 1 of 2 patients (50%) carrying a male fetus at a gestational age of 5 weeks, in 4 of 5 (80%) at a gestational age of 7 weeks, in 4 of 4 (100%) at a gestational age of 9 weeks. In all 7 women pregnant with a male fetus, the correct fetal sex was detected by 10 weeks. In none of the 6 patients who delivered a girl was SRY detected. In the validation group, SRY was detected in 13 of the 13 male, and none of the 18 female fetuses. CONCLUSIONS: We conclude that real-time PCR of the SRY gene promises to be a reliable technique for early fetal sexing in maternal plasma.     

荧光定量PCR检测孕妇血浆中胎儿DNA的研究

目的探讨荧光定量PCR（fluorescence quantitative PCR，FQ-PCR）方法检测孕妇血浆中胎儿DNA的可行性，探讨胎儿DNA在孕妇血浆中的含量及其在孕期的变化规律。方法以胎儿SRY基因序列作为胎儿DNA在孕妇血浆中的标志，应用荧光MGB（Minor Groove Binder）探针实时定量PCR方法连续测定30例孕妇在不同孕期和产后共237份血浆标本中胎儿DNA的含量。结果使用该方法最低可检测到20000个女性DNA中的一个男性DNA。孕妇血浆中最早检出SRY序列的时间为孕6^＋6周。孕8周起，所有怀男胎孕妇的标本中均可检出SRY序列，其含量随着妊娠的进展而增高，在晚期妊娠达高峰，产后24～48h血浆中SRY序列检测均为阴性。胎儿DNA在孕妇血浆总DNA含量中的相对浓度分别为孕早期4．88％、孕中期6．10％、孕晚期4．77％。全部实验无假阳性和假阴性出现。结论FQ-PCR方法是一种灵敏度和准确性高，特异性强的定量检测孕妇血浆中胎儿游离DNA的方法，孕妇血浆中存在高浓度的胎儿DNA，可用于无创伤性产前基因诊断。     

Contribution of genotyping for fetal sex determination in maternal serum for preimplantation genetic diagnosis of X-linked diseases

OBJECTIVE: Couples with a risk of transmitting X-linked diseases included in a preimplantation genetic diagnosis (PGD) center need early and rapid fetal sex determination during pregnancy in two situations. The first situation corresponds to control of embryo sexing after PGD, the second one being that of couples in PGD program having a spontaneous pregnancy. Determination of fetal sex can be achieved by karyotyping using invasive procedures such as chorionic villus sampling (CVS), amniocentesis or cordocentesis and by non-invasive procedures such as ultrasound (US) examination. CVS is the earliest invasive procedure for fetal sex determination and molecular analysis of X-linked genetic disorders during the first trimester but it is associated with a risk of fetal loss. US allows reliable fetal sex determination only during the second trimester. Recently, reliable non-invasive fetal sex determination was realized by using SRY gene amplification in maternal serum. PATIENTS AND METHODS: We report the prospective use of fetal sex determination in maternal serum in our PGD center. Management of pregnancies was performed using this non-invasive procedure in four cases of embryo sexing control and nine cases of spontaneous pregnancies in couples included in PGD program for X-linked diseases. RESULTS: Fetal sex results using SRY gene amplification on maternal serum were in complete concordance with fetal sex observed by cytogenetic analysis or US examination, as well as at birth. DISCUSSION AND CONCLUSION: This new strategy allowed rapid sex determination during the first trimester and permitted to avoid performing invasive procedures in nine pregnancies.     

Fetal sex determination from maternal plasma in pregnancies at risk for congenital adrenal hyperplasia

OBJECTIVE: To determine first-trimester fetal sex by isolating free fetal DNA from maternal plasma. METHODS: The index case was a pregnant woman who previously delivered a girl with congenital adrenal hyperplasia. The SRY gene as a marker for the fetal Y chromosome was detected in maternal serum and plasma by quantitative polymerase chain reaction analysis. Simultaneously, we performed the same test in 25 and 19 women in the first and second trimester, respectively, and compared plasma results with fetal gender as assessed by prenatal karyotyping or as seen at ultrasound or birth. RESULTS: In 44 of 45 patients at gestational ages ranging from 8 3/7 to 17 3/7 weeks, we correctly predicted fetal sex using quantitative polymerase chain reaction analysis of the SRY gene in maternal plasma. In one case, the test result was inconclusive. Overall, fetal sex was correctly predicted in 97.8% of cases (95% confidence interval 88.2%, 99.9%). CONCLUSION: Amplification of free fetal DNA in maternal plasma is a valid technique for predicting fetal sex in early pregnancy. In case of pregnancies at risk for congenital adrenal hyperplasia, the technique allows restriction of dexamethasone treatment to female fetuses resulting in a substantial decrease of unnecessary treatment and invasive diagnostic tests.     

Detection of Y chromosome-specific DNA in the plasma and urine of pregnant women using nested polymerase chain reaction

The present study was undertaken to evaluate a nested polymerase chain reaction (PCR) for detection of Y chromosome-specific fetal DNA in maternal plasma and urine of pregnant women during different gestational stages. DNA isolated from plasma and urine samples of 80 pregnant women (between 7 and 40 weeks' gestation) underwent amplification for Y chromosome-specific 198 bp DNA by nested PCR. The postpartum analysis of fetal gender showed that 55 women carried male and 25 female fetuses. Among the 55 women bearing male fetuses, Y chromosome-specific signals were detected in 53 (96%) plasma and 21 (38%) urine samples. Moreover, out of 25 women bearing female fetuses, 3 (12%) and 1 (4%) women had Y chromosome-specific signal in plasma and urine, respectively. Analysis of results with respect to gestational age revealed that there was no significant difference in the detection of Y chromosome-specific DNA between different trimesters in maternal plasma of women bearing male fetuses. These results showed that fetus-specific DNA was detected with high sensitivity (96%) and specificity (88%) in the maternal plasma by nested PCR, and therefore the method could be useful as a non-invasive procedure for fetal sex determination and prenatal diagnosis.     

Prenatal fetal sex diagnosis by detecting amelogenin gene in maternal plasma

OBJECTIVES: To provide a new, reliable noninvasive method for fetal sex determination. METHODS: Fetal sex was detected in 32 early pregnant women by identifying the amelogenin gene in maternal plasma using nested PCR analysis. First, the 122/128 bp of X-Y homologous region containing 6 bp deletions in the intron 3 of amelogenin gene in X chromosome was amplified, and then the nested PCR was carried out, whose 3' end of the upstream primer is just located in the deletion region. The fetus was male or female, depending on whether it had the 89-bp nested PCR product or not. RESULTS: The 89 bp of nested PCR product was detected in 19 plasma samples obtained from pregnant women, deducing they bear the male fetus and the remaining pregnant women bear female. When compared with the birth outcome, two samples were pseudo-positive. The coincidence was 93.8%. This method had high sensitivity that even trace amount of target fetal DNA (10 pg) could be detected. CONCLUSIONS: This conventional nested PCR analysis of amelogenin gene promises to be a reliable method for noninvasive fetal sex determination at early pregnancy using maternal plasma DNA.     

Non-invasive fetal sex determination using real-time PCR.

OBJECTIVE: The aim of this study was to evaluate the specificity and sensitivity of the real-time quantitative PCR method for fetal gender determination in early pregnancy. METHODS: Blood samples were collected from 46 pregnant women prior to amniocentesis. DNA was extracted from maternal plasma using a QIAmp DNA Blood Mini Kit. DNA samples were subjected to real-time quantitative PCR amplification of SRY (as a fetus-specific marker) and beta-globin (as a marker for total plasma DNA) genes. RESULTS: The beta-globin gene sequence was detected in all samples. The SRY gene was detected in 25 of 28 plasma samples from women with male fetuses and in none of the 18 samples from women with female fetuses (sensitivity 89.2% and specificity 100%). The fetal gender was correctly determined in 43 (93.5%) of 46 maternal plasma samples. The concentration of the beta-globin gene ranged from 161 to 25,568 genome-equivalents (GE)/mL (median 1051.1), while the concentration of the SRY gene ranged from 5 to 166 GE/mL (median 27.4). The percentage of free fetal DNA ranged from 0.1% to 46.1% (median 2.0%). CONCLUSION: Amplification of fetal DNA from maternal plasma by real-time quantitative PCR is a promising method for fetal sex determination in early pregnancy. However, further studies are necessary before this procedure can be included into a clinical routine.     

Feasibility study of using fetal DNA in maternal plasma for non-invasive prenatal diagnosis

BACKGROUND: The discovery of the presence of fetal genetic material in maternal blood has opened up a new approach to prenatal diagnosis. One approach that has been extensively investigated over the past few decades is the isolation of fetal cells from maternal blood (Herzenberg et al. Proc Natl Acad Sci USA. 1979;76:1453-5; Bianchi et al. Proc Natl Acad Sci USA. 1990;87:3279-83; Cha et al. Prenat Diagn. 2005;25:586-91). As the fetal cells are scarce and the enrichment is of low efficiency, the technique could not be implemented in clinics. In 1997, Lo et al. (Lancet 1997;350(9076):485-7) discovered that cell-free fetal DNA is present in the plasma and serum of pregnant women. This discovery suggests that maternal plasma/serum DNA may be a useful source of material for non-invasive prenatal diagnosis. The objective of our study was to investigate the feasibility of using fetal DNA in maternal plasma for prenatal diagnosis. METHODS: Plasma DNA in 277 blood samples of 40 pregnant women at the gestational period from 5 to 40 weeks and 24 h after delivery were extracted by column separation. FQ-PCR was used to amplify the SRY sequence in 237 plasma samples of 30 pregnant women. Fluorescent PCR was used to amplify 9 short tandem repeat loci simultaneously in 40 plasma samples of 10 pregnant women, and genomic DNA samples from their husbands were amplified by the same method. RESULTS: The fetal SRY sequence could be detected from the 7th week of gestation, with a concentration that increased with progressing gestational age, attaining its highest peak before delivery. Twenty-four hours after delivery, fetal SRY sequence could not be detected in the maternal plasma. The concordance rate of the SRY sequence amplification results of plasma-free DNA, with real fetal gender was 100%. Analysis of maternal plasma samples collected during pregnancy revealed the presence of paternally inherited fetal-specific alleles. Among the 30 collected plasma samples, fetal-specific alleles were detected in 23 plasma DNA samples. The rate of positive results was 76% (23/30), and the frequency of positive results was 6/10 in early pregnancy, 8/10 in middle pregnancy, and 9/10 in late pregnancy. Short tandem repeats could not be detected from the maternal plasma 24 h after delivery. CONCLUSION: Fluorescent PCR can be used for amplification of fetal SRY sequence and STRs in maternal plasma to obtain fetal genetic information, which may have implications for non-invasive prenatal diagnosis of certain hereditary diseases.     

Replicate real-time PCR testing of DNA in maternal plasma increases the sensitivity of non-invasive fetal sex determination

BACKGROUND: We determined fetal sex in pregnancies referred for invasive prenatal diagnosis procedures by analysis of DNA in maternal plasma. METHODS: Twelve pregnancies at risk of X-linked haemophilia and 32 pregnancies at risk of chromosomal aneuploidies at a gestational age ranging from 10 to 18 weeks recruited before chorionic villus sampling or amniocentesis were involved in the study. Male fetal DNA in maternal plasma was detected by using real-time polymerase chain reaction with the SRY gene as a marker. RESULTS: The specificity of the system reached 100% (no Y signal was detected in 17 women pregnant with a female fetus) and the sensitivity reached 100% (SRY amplification in 27 examined samples). CONCLUSIONS: Amplification of free fetal DNA in maternal plasma is a valid and rapid technique for predicting fetal sex in first- and second-trimester pregnancies and could allow the restriction of invasive sampling procedures to male fetuses at risk of X-linked disorders.     

Fetal sex-related differences in maternal serum alpha-fetoprotein during the second trimester of pregnancy

In an analysis of data gathered from routine maternal serum alpha-fetoprotein screening, we determined that the sex of the fetus is significantly correlated with differences in maternal serum alpha-fetoprotein concentration during the second trimester of pregnancy. Between the fifteenth and nineteenth gestational weeks, the mean maternal serum alpha-fetoprotein level is significantly higher for the male fetus than for the female fetus. There was no difference, however, in the rate of increase in maternal serum alpha-fetoprotein concentration with time between male and female fetuses during the weeks of gestation that were studied. No apparent cause for these observations has been demonstrated.     

Non-invasive fetal sex determination using real-time PCR

Objective.?The aim of this study was to evaluate the specificity and sensitivity of the real-time quantitative PCR method for fetal gender determination in early pregnancy.

Methods.?Blood samples were collected from 46 pregnant women prior to amniocentesis. DNA was extracted from maternal plasma using a QIAmp DNA Blood Mini Kit. DNA samples were subjected to real-time quantitative PCR amplification of SRY (as a fetus-specific marker) and β-globin (as a marker for total plasma DNA) genes.

Results.?The β-globin gene sequence was detected in all samples. The SRY gene was detected in 25 of 28 plasma samples from women with male fetuses and in none of the 18 samples from women with female fetuses (sensitivity 89.2% and specificity 100%). The fetal gender was correctly determined in 43 (93.5%) of 46 maternal plasma samples. The concentration of the β-globin gene ranged from 161 to 25 568 genome-equivalents (GE)/mL (median 1051.1), while the concentration of the SRY gene ranged from 5 to 166 GE/mL (median 27.4). The percentage of free fetal DNA ranged from 0.1% to 46.1% (median 2.0%).

Conclusion.?Amplification of fetal DNA from maternal plasma by real-time quantitative PCR is a promising method for fetal sex determination in early pregnancy. However, further studies are necessary before this procedure can be included into a clinical routine.     

Quantitative analysis of DNA levels in maternal plasma in normal and Down syndrome pregnancies

BACKGROUND: We investigated fetal and total DNA levels in maternal plasma in patients bearing fetuses affected with Down syndrome in comparison to controls carrying fetuses with normal karyotype. METHODS: DNA levels in maternal plasma were measured using real-time quantitative PCR using SRY and beta-globin genes as markers. Twenty-one pregnant women with a singleton fetus at a gestational age ranging from 15 to 19 weeks recruited before amniocentesis (carried out for reasons including material serum screening and advanced material age), and 16 pregnant women bearing fetuses affected with Down syndrome between 17 to 22 weeks of gestation were involved in the study. RESULTS: The specificity of the system reaches 100% (no Y signal was detected in 14 women pregnant with female fetuses) and the sensitivity 91.7% (SRY amplification in 22 of 24 examined samples). The median fetal DNA levels in women carrying Down syndrome (n=11) and the controls (n=13) were 23.3 (range 0-58.5) genome-equivalents/ml and 24.5 (range 0-47.5) genome-equivalents/ml of maternal plasma, respectively (P = 0.62). The total median DNA levels in pregnancies with Down syndrome and the controls were 10165 (range 615-65000) genome-equivalents/ml and 7330 (range 1300-36750) genome-equivalents/ml, respectively (P = 0.32). The fetal DNA proportion in maternal plasma was 0%-6 % (mean 0.8%) in women carrying Down syndrome and 0%-2.6 % (mean 0.7 %) in the controls, respectively (P=0.86). CONCLUSIONS: Our study revealed no difference in fetal DNA levels and fetal DNA: maternal DNA ratio between the patients carrying Down syndrome fetuses and the controls.     

Non-invasive first trimester fetal gender assignment in pregnancies at risk for X-linked recessive diseases

OBJECTIVES: Prenatal diagnosis in families affected by X-linked recessive disorders should ideally be limited to the subjects at increased risk, i.e. male fetuses, in order to avoid the risk of fetal loss due to the invasive procedure in healthy female fetuses. The aim of the study was to assess the fetal sex within the first trimester of gestation by two non-invasive approaches, using ultrasonography and a molecular analysis of fetal DNA extracted from whole maternal blood with specific markers, in order to avoid invasive sampling in female fetuses. METHODS: A total number of 18 fetuses at risk for an X-linked recessive disease were included in the present investigation. Maternal peripheral blood was analysed between 7 and 12 weeks of gestation by nested PCR for the detection of fetal DNA and the prediction of fetal gender. In addition, when the biparietal diameter (BPD) was between 21 and 23 mm, an ultrasonographic examination was carried out to assess the fetal gender. CVS was then performed in male fetuses only. RESULTS: Fetal gender was correctly assigned by ultrasonography between 21 and 23 mm of BPD in all the cases studied, whereas DNA extracted from whole maternal blood accurately predicted the gender in all the female cases (10), but failed in 4 out of 8 male fetuses, erroneously assigned as females. CONCLUSION: The present study shows that sonography is able to accurately predict the fetal gender within the first trimester of pregnancy, whereas the molecular analysis of DNA extracted from whole maternal blood is biased by false-Y-negative results in 50% of the cases.     

Fetal gender affects maternal serum total and placental alkaline phosphatase levels during pregnancy

OBJECTIVE: To analyze whether fetal gender affects total alkaline phosphatase (ALP) and placental ALP levels in normal pregnancy, and to determine the gestational age at which the difference occurs. METHODS: In this longitudinal study, serum total and placental ALP measurements were carried out in 30 normal pregnant women during different ranges of gestational weeks. Infant sex was recorded at the delivery for all women included in the study. Total and placental ALP levels were compared between pregnant women bearing female and male fetuses. RESULTS: At all gestational weeks studied, both total and placental ALP levels were higher in pregnant women carrying female fetuses than in male bearing pregnant women. Particularly, both total (260.9+/-110.2 versus 239.9+/-102.3; p=0.03) and placental (73.1+/-22.4 versus 61+/-18.2; p=0.04) ALP levels were significantly higher in the female group than in the male between 24 and 28 weeks, and the significant difference persisted between 32 and 36 weeks (p=0.02). CONCLUSIONS: Fetal gender seems to affect total and placental ALP levels in healthy pregnant women, particularly during the second and third trimester of pregnancy. Higher ALP levels in pregnant women with female fetuses than in those with male fetuses may suggest that knowledge of the fetal gender may be in particular importance for the studies using ALP as a marker for the prediction of variety of diseases and complications seen during pregnancy.     

Effect of fetal gender on maternal serum human chorionic gonadotropin levels throughout pregnancy

Objectives: Maternal serum human chorionic gonadotropin (MshCG) levels are higher in pregnant women with female fetuses than in pregnant women with male fetuses in the third trimester of pregnancy. Our aim was to examine the effect of fetal sex on MshCG levels throughout the pregnancy. Materials and methods: Twenty-nine uncomplicated pregnancies (14 had male fetus and 15 had female fetus) were included in the study. MshCG levels were measured four times between 8–12, 14–18, 24–28 and 32–36 weeks of pregnancy. Results: MshCG levels were significantly higher in both sexes between 8–12 weeks than the other three measurement periods. At 8–12 and 14–18 weeks measurements, there were no sex related differences in the MshCG levels. At 24–28 (P<0.004) and 32–36 (P<0.001) weeks MshCG levels were significantly higher in pregnancies bearing female fetuses than those bearing male fetuses. Conclusions: Fetal gender has a significant effect on MshCG levels in the third trimester of pregnancy. Accordingly, no correlation seems to exist in the first and second trimesters.     

双色共变性荧光原位杂交产前诊断胎儿唐氏综合征

目的 探讨双色共变性荧光原位杂交用于非侵入性产前诊断胎儿唐氏综合征的可行性。方法 对11例孕妇外周血中的胎儿有核红细胞进行抗血型糖蛋白磁珠直接标记,再经磁激活细胞分选法富集,以Y和21号染色体专一探针对分离的胎儿有核红细胞行双色共变性荧光原位杂交,预测胎儿21号染色体倍性和性别,并用羊水染色体核型分析结果,验证预测准确性。结果 11例胎儿21号染色体倍性均正常,与羊水染色体核型分析结果相符。其中5例为男性胎儿,男性胎儿有核红细胞数量为9－65个,平均为25个,男性胎儿有核红细胞纯度为1．4％－18．8％;6例为女性胎儿,孕妇外周血中未见男性胎儿有核红细胞;性别预测结果与羊水染色体型分析结果一致。结论 双色共变性荧光原位杂交用于分析胎儿21号染色体倍性及性别,诊断胎儿唐氏综合征准确、可靠。     

Human chorionic gonadotropin and testosterone in normal and preeclamptic pregnancies in relation to fetal sex

OBJECTIVE: The aim of the present study was to evaluate the effects of fetal gender on serum human chorionic gonadotropin (hCG) and testosterone in normotensive and preeclamptic pregnancies. METHODS: The study consisted of 137 women with singleton pregnancies in the third trimester. Seventy-three pregnancies were uncomplicated; among those were 35 male and 38 female fetuses. Sixty-four pregnancies were complicated by preeclampsia; among those were 33 male and 31 female fetuses. Human chorionic gonadotropin and total testosterone were measured in maternal peripheral blood. RESULTS: In male-bearing pregnancies, maternal hCG and testosterone serum levels were significantly higher in preeclamptic than normotensive mothers (P <.001). In female-bearing pregnancies, testosterone levels were significantly higher in preeclamptic than normotensive mothers (P <.001), whereas the hCG levels were not significantly different. Male-bearing preeclamptic women had significantly higher testosterone levels than female-bearing preeclamptic women (P <.02), whereas the hCG levels were not significantly different. In uncomplicated pregnancies the hCG levels were significantly higher in female-bearing than in male-bearing mothers (P <.005), whereas the testosterone levels were not significantly different. CONCLUSION: In preeclamptic pregnancies with male fetuses, the maternal serum hCG levels were significantly higher than in uncomplicated pregnancies. Total testosterone levels were significantly higher in pregnancies with either gender and significantly higher in male-bearing than in female-bearing pregnancies. This may indicate an androgen influence on the pathophysiologic mechanism of preeclampsia.     

应用实时荧光定量PCR技术检测母体血浆中胎儿游离mRNA的研究

目的:探讨孕妇血浆中胎儿游离mRNA含量变化作为产前筛查指标的可行性。方法:选择39例早中孕期(8～14周)孕妇为研究对象,其中15例行人工流产(A组),11例为稽留流产或胚胎发育异常(B组),13例为正常早孕期单胎孕妇(C组),提取其外周血血浆游离mRNA,应用实时荧光定量PCR技术(QF-PCR)检测胎盘来源的hPL,β-HCG,TFPI2基因,以此确定母体血浆中的胎儿游离mRNA含量,同时检测代表母体与胎儿总游离mRNA的18s rRNA基因作为内参。结果:3组孕妇血浆标本中均检测到hPL,β-HCG,TFPI2基因及18s rRNA基因,且不依赖于胎儿性别,3组样本表达量有差异,B组TFPI2基因显著高于A、C组(P<0.01)。结论:实时荧光定量PCR方法的灵敏度和特异性较高,作为非创性产前基因诊断的一种方法可应用于临床。胎儿游离mRNA可能成为产前筛查的有效指标。     

应用表观遗传学方法检测母体循环中游离胎儿DNA

目的:探讨表观遗传学方法检测孕妇血浆中游离胎儿DNA水平的应用价值。方法:随机选择早、中、晚期妊娠孕妇各20例,以非妊娠妇女20例为对照组。提取血浆中的总游离DNA,用甲基化特异性PCR(MSP)方法检测各组血浆标本中目的基因m-maspin(一种肿瘤抑制基因启动子的甲基化序列)、u-maspin(前者的未甲基化序列)的表达水平,再进行相对定量,对妊娠组和对照组样本均数进行统计学分析。结果:(1)m-maspin在妊娠早、中、晚期孕妇血浆中的浓度分别是非妊娠妇女血浆中浓度的1.0、1.1、1.1倍,差异无统计学意义(P>0.05);(2)u-maspin在正常非妊娠妇女血浆中未能检测到,在60例孕妇血浆中57例可检测到,它在孕妇血浆中的浓度随妊娠进展呈升高趋势,中、晚期妊娠妇女血浆中其浓度相对于早期妊娠分别为1.7倍和3.1倍,差异有统计学意义(P<0.01)。结论:m-maspin不是妊娠特异性标志物,u-maspin为妊娠特异性标志物。u-maspin基因可作为孕妇血浆中游离胎儿DNA水平变化的表观遗传学标志,相对于以胎儿性别基因作为遗传学标志的检测方法,它有助于扩大非创伤性产前诊断的临床应用范围。