孕婦外周血胎兒有核紅細(xì)胞計(jì)數(shù)變化及其在不良妊娠結(jié)局預(yù)測(cè)中的應(yīng)用

·臨床研究·

孕婦外周血胎兒有核紅細(xì)胞計(jì)數(shù)變化及其在不良妊娠結(jié)局預(yù)測(cè)中的應(yīng)用

劉麗平1，水麗君2，龔瑞龍1，曹江霞1，黃秋香1，張?jiān)?

(1武漢市婦女兒童醫(yī)療保健中心，武漢430016；2安徽醫(yī)科大學(xué)第二附屬醫(yī)院；3武漢大學(xué)中南醫(yī)院)

摘要：目的觀察孕婦外周血胎兒有核紅細(xì)胞計(jì)數(shù)的變化，探討其在妊娠不良結(jié)局預(yù)測(cè)中的應(yīng)用價(jià)值。方法收集87例孕12～18周孕婦的外周血，密度梯度離心法分離單個(gè)核細(xì)胞，采用PE-GPA /FITC-CD71單克隆抗體雙色標(biāo)記胎兒有核紅細(xì)胞后進(jìn)行流式細(xì)胞分析，追蹤妊娠結(jié)局并進(jìn)行分析。結(jié)果妊娠結(jié)局正常者外周血中胎兒有核紅細(xì)胞百分比為0.89%±0.81%，妊娠結(jié)局不良者為3.25%±2.98%，兩者相比，P<0.05。以胎兒有核紅細(xì)胞百分比>3.79%作為截?cái)嘀?，預(yù)測(cè)妊娠不良結(jié)局的敏感度為76.9%、特異度為64.3%。結(jié)論與妊娠結(jié)局正常的孕婦相比，妊娠結(jié)局不良孕婦孕12～18周時(shí)外周血胎兒有核紅細(xì)胞增多；對(duì)孕12～18周孕婦外周血胎兒有核紅細(xì)胞進(jìn)行計(jì)數(shù)有助于不良妊娠結(jié)局的預(yù)測(cè)，其敏感度及特異度均較高。

關(guān)鍵詞：有核紅細(xì)胞；胎兒有核紅細(xì)胞；妊娠；孕婦；妊娠結(jié)局

doi：10.3969/j.issn.1002-266X.2015.43.012

中圖分類號(hào)：R713 文獻(xiàn)標(biāo)志碼：B

基金項(xiàng)目：湖北省武漢市衛(wèi)生和計(jì)劃生育委員會(huì)2014年立項(xiàng)課題。

收稿日期：(2015-07-13)

Fetus nucleated red blood cell count changes in peripheral blood of pregnant

women and its application in prediction of adverse pregnancy outcomes

LIULi-ping1, SHUI Li-jun, GONG Rui-long, CAO Jiang-xia, HUANG Qiu-xiang, ZHANG Yuan-zhen

(1WuhanMedicalandHealthCenterforWomen-Children,Wuhan430016,China)

Abstract:ObjectiveTo observe the fetus nucleated red blood cell (NRBC) count changes in the peripheral blood of pregnant women and to investigate its application in prediction of adverse pregnancy outcomes.MethodsBlood samples were obtained from 87 pregnant women with the gestational age from 12 to 18 weeks, peripheral blood mononuclear cells were isolated by density gradient centrifugation and then we analyzed the cells marked with PE-GPA/FITC-CD71 monoclonal antibodies on the flow cytometer. Tracking the pregnancy outcomes, and their correlation between fetus nucleated red blood cell measurement and adverse pregnancy outcomes was analyzed.ResultsThe percentage of fetus NRBC in women with normal pregnancy outcomes was 0.89%±0.81%, 3.25%±2.98% in women with adverse pregnancy outcomes, and there was a significant difference between these two groups(P<0.05). We took fetus NRBC percentage >3.79% as the cutoff value to predict adverse outcomes of pregnancy, the sensitivity was 76.9%, and the specificity was 64.3%.Conclusions Compared with women with normal pregnancy outcomes, women with adverse pregnancy outcomes have a higher amount of fetus NRBC in the peripheral blood during the gestational age of 12-18 weeks. Measurement of fetus NRBC in maternal peripheral blood has a predictive role in adverse pregnancy outcomes, with a higher sensitivity and specificity.

Key words: nucleated red blood cells; fetus nucleated red blood cells; pregnancy; pregnant women; pregnancy outcome

通信作者：張?jiān)?/p>

正常妊娠的維持有賴于母胎免疫平衡的建立與穩(wěn)定[1]。一旦該平衡失調(diào)，則會(huì)產(chǎn)生免疫排斥反應(yīng)[2]，引起病理性妊娠，導(dǎo)致不良妊娠結(jié)局，包括流產(chǎn)、早產(chǎn)、妊娠并發(fā)癥、圍產(chǎn)期胎兒死亡等[3]。1969年Walknowska等首次報(bào)道從孕婦外周血中提取胎兒細(xì)胞進(jìn)行產(chǎn)前診斷。隨后的研究[4,5]表明，胎兒有核紅細(xì)胞是最好的產(chǎn)前診斷細(xì)胞來(lái)源。但由于胎兒有核紅細(xì)胞在孕婦外周血中比例極低，故研究多集中在有核紅細(xì)胞的富集與分離上。本研究對(duì)部分孕12～18周孕婦的外周血中胎兒有核紅細(xì)胞進(jìn)行了檢測(cè)，觀察其計(jì)數(shù)變化，追蹤妊娠結(jié)局，分析并探討孕婦外周血胎兒有核紅細(xì)胞計(jì)數(shù)在不良妊娠結(jié)局預(yù)測(cè)中的應(yīng)用價(jià)值。

1 資料與方法

1.1臨床資料選取2014年1～12月在武漢市婦女兒童醫(yī)療保健中心婦產(chǎn)科門診建冊(cè)并住院分娩的孕婦87例，年齡18～39歲，均為單胎妊娠，無(wú)其他疾病及合并癥，無(wú)輸血史，除隨機(jī)抽取的孕婦外，盡量納入具有潛在的不良妊娠可能的孕婦(包括肥胖、營(yíng)養(yǎng)不良、抽煙史、不良孕產(chǎn)史、家族史、唐氏篩查高危、低社會(huì)經(jīng)濟(jì)狀況等孕婦)。在孕婦知情同意的情況下，于孕12～18周抽取其外周血6 mL，注入EDTA抗凝管，輕輕搖勻，置4 ℃冰箱保存，2 h內(nèi)進(jìn)行細(xì)胞分離處理。

1.3妊娠結(jié)局觀察方法87例受檢孕婦接受定期產(chǎn)前檢查，對(duì)有潛在不良妊娠可能的孕婦要觀察有無(wú)妊娠期高血壓疾病、早產(chǎn)、胎兒生長(zhǎng)受限、妊娠期糖尿病、胎盤早剝、胎兒窘迫、HELLP綜合征、妊娠期膽汁淤積綜合征、死胎、胎兒畸形、唐氏綜合征等。

1.4統(tǒng)計(jì)學(xué)方法采用SPSS17.0統(tǒng)計(jì)軟件。計(jì)量資料比較用t檢驗(yàn)，截?cái)嘀涤?jì)算采用ROC曲線。P<0.05為差異有統(tǒng)計(jì)學(xué)意義。

2結(jié)果

受檢的87例孕婦中，妊娠結(jié)局正常者25例，妊娠結(jié)局不良者62例(妊娠期高血壓疾病27例、早產(chǎn)14例、胎兒生長(zhǎng)受限8例、妊娠期糖尿病4例、胎盤早剝2例、胎兒窘迫2例、HELLP綜合征1例、妊娠期膽汁淤積綜合征1例、死胎1例、胎兒畸形1例、唐氏綜合征1例)。87例孕婦外周血中均檢出胎兒有核紅細(xì)胞，其百分比為0.03%～9.37%；其中妊娠結(jié)局正常者胎兒有核紅細(xì)胞百分比為0.89%±0.81%(0.03%～3.89%)，妊娠結(jié)局不良者為3.25%±2.98%(3.19%～9.37%)，兩者相比，P<0.05。ROC曲線顯示，以胎兒有核紅細(xì)胞百分比>3.79%作為預(yù)測(cè)不良妊娠結(jié)局的截?cái)嘀?，該截?cái)嘀档拿舾卸葹?6.9%、特異度為64.3%。

3討論

母血中存在四種胎兒細(xì)胞，分別為滋養(yǎng)細(xì)胞、有核紅細(xì)胞、淋巴細(xì)胞和粒細(xì)胞。其中胎兒有核紅細(xì)胞具有完整的核物質(zhì)，從而攜帶了完整的胎兒基因，且在妊娠期間持續(xù)存在于母血循環(huán)中。正常成人外周血不含有核紅細(xì)胞，因而胎兒有核紅細(xì)胞被學(xué)者們[7]認(rèn)為是無(wú)創(chuàng)性產(chǎn)前診斷的最佳細(xì)胞來(lái)源。有研究者[8]發(fā)現(xiàn)，患妊娠期高血壓疾病的孕婦血漿中胎兒DNA的量比正常妊娠孕婦高，且在子癇發(fā)作前孕婦血漿中胎兒DNA水平有異常升高。Holzgreve等[9]研究發(fā)現(xiàn)，妊娠期高血壓疾病孕婦外周血中有核紅細(xì)胞數(shù)增多，并隨著疾病的嚴(yán)重程度的上升而升高。

在妊娠過(guò)程中，胎兒細(xì)胞可通過(guò)胎盤屏障進(jìn)入母體外周血液循環(huán)并分布到母體全身各器官組織[10]。在正常妊娠中，含父系抗原的胚胎能夠逃逸母體細(xì)胞和體液免疫的攻擊；而在病理性妊娠中母胎免疫平衡被打破，導(dǎo)致免疫排斥反應(yīng)的發(fā)生，引起血管內(nèi)皮細(xì)胞病變[11]，導(dǎo)致胎盤損傷，更多的胎兒細(xì)胞會(huì)因此漏入母體循環(huán)。孕婦外周血中胎兒有核紅細(xì)胞數(shù)量與胎盤損傷之間存在一定的相關(guān)性[12]。但是胎盤損傷的程度難以量化，故本研究以母外周血中胎兒有核紅細(xì)胞的數(shù)量作為胎盤損傷的生物學(xué)標(biāo)志，分析其與不良妊娠結(jié)局之間的關(guān)系，結(jié)果表明，孕12～18周孕婦的外周血中均可檢出胎兒有核紅細(xì)胞，妊娠結(jié)局不良者的胎兒有核紅細(xì)胞百分比明顯高于妊娠結(jié)局正常者，其中妊娠期糖尿病、胎兒生長(zhǎng)受限、早產(chǎn)、胎盤早剝等孕婦外周血中胎兒有核紅細(xì)胞百分比升高更明顯；以孕婦外周血中胎兒有核紅細(xì)胞百分比>3.79%作為截?cái)嘀?，其預(yù)測(cè)妊娠不良結(jié)局的敏感度為76.9%、特異度為64.3%。

妊娠期高血壓疾病、胎兒生長(zhǎng)受限、胎盤早剝、HELLP綜合征等在發(fā)病機(jī)制方面有相似之處[13]。但是，這些疾病在臨床上發(fā)病較晚，其中較為常見(jiàn)的妊娠期高血壓疾病發(fā)生于妊娠中、晚期，最終累及胎盤、胎兒和孕婦各主要臟器，可導(dǎo)致胎兒發(fā)育遲緩、胎盤早剝、早產(chǎn)、產(chǎn)后出血等嚴(yán)重后果。但其發(fā)病因素可能在妊娠早期胎盤形成過(guò)程中就已存在，若能早期預(yù)測(cè)這些疾病的發(fā)生，或可進(jìn)行早期干預(yù)，改善妊娠結(jié)局。本研究結(jié)果表明，檢測(cè)母體外周血中胎兒有核紅細(xì)胞數(shù)量變化，或可成為臨床上預(yù)測(cè)不良妊娠結(jié)局的有效手段。

此外，本研究還發(fā)現(xiàn)，胎兒有異常(如先天畸形、染色體異常)的孕婦外周血中也出現(xiàn)胎兒有核紅細(xì)胞增多的現(xiàn)象，其機(jī)制尚不清楚，有待進(jìn)一步研究。

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