人类血清中早期妊娠因子的测定

<正> 早期妊娠因子(Early Pregnancy Factor、EPF)是新发现的一种在体外能抑制花结形成在体内具有免疫抑制活性的物质,在妊娠母血中在受精后的48小时之内就可测出,是妊娠的第一个信号,与胎儿不受母体排斥有关。我们利用标准化的花结抑制试验,对94例妊娠母血进行了测定,证明确实存在有EPF,在妊娠早期活性最强,随孕周的增加而降低,至分娩前8周消失。另外还测定过21例流产前后的孕妇血清,流产前血     

孕血清中早孕因子免疫原性的分析

采用双向免疫扩散(DID)、免疫电泳(IEP)及免疫中和试验对所制备的免抗人EPF免疫血清进行分析、鉴定。DID_α、IEP结果均表明,孕血清提取物与兔免疫血清反应后出现两条沉淀线(弧),其中一条为孕血清提取物所特有的(抗体效价为1:8)。且花抑实验证明,所制备的兔免疫血清能中和孕血清及其提取物EPF活性(与阴性对照比P>0.05)。EPF免疫原性的确立为EPF新的检测方法的建立打下了基础。     

孕妇血清提取物—早孕因子样物质的花结抑制活性测定

以花结抑制试验检测50名妊娠3～8周妇女静脉血混合血清及其经DEAE纤维素离子交换层折和经Sephadex-G-100凝胶过滤后,所得各阶段提取物的花抑滴度,并以HCG胶体金试剂盒测定HCG。对照组为50名处女混合血清,结果证明:混合孕血清经DE-52柱层析后未吸附蛋白部分(DE-I)及Sephadex-G100凝胶过滤后洗脱峰DE-I-GⅡ及DE-I-GⅢ均具花抑活性,DE-I-GⅡ为HCG( )而DE-I-GⅢ为HCG(-)°提示DE-I-GⅡ峰为EPF与HCG混合物,而DE-I-GⅢ峰为不含HCG而具花抑活性的EPF粗提物。     

孕血清中早孕因子的纯化

本文用DEAE纤维素离子交换层析、ConA—Sepharose亲和层析、Sephadex G-100凝胶过滤等技术,分离纯化妊娠3～6周正常早孕妇女静脉血混合血清中早孕因子(Early Pregnancy Factor,EPF).以花结抑制实验检测各阶段层析物中EPF活性,以放射免疫法测定hCG含量.结果表明,经凝胶过滤后,提取物中hCG均为阴性(<3.1ng/ml),高EPF活性部分经SDS-聚丙烯酰胺凝胶电泳显示,分子量分别为24KD.和41KD_α。     

早孕因子分离纯化方法的优化

目的对正常早孕妇女血清中早孕因子(EPF)的分离纯化过程进行优化,以提高EPF的回收率。方法依次采用DEAEFast Flow离子交换色谱和Heparin Fast Flow亲和色谱,从妊娠12周内的正常早孕妇女混合血清中提纯EPF,采用活性玫瑰花结抑制试验(Ea-RIT)检测各阶段产物的EPF活性,SDS-聚丙烯酰胺凝胶(SDS-PAGE)电泳鉴定纯化物并测定其相对分子质量,用Western blot鉴定其特异性。结果经Ea-RIT检测后,DEAE Fast Flow离子交换色谱所现2峰中DE-Ⅰ峰为EPF活性峰,HeparinFast Flow亲和色谱所现2峰中H-Ⅱ峰为EPF活性峰。H-Ⅱ峰收集液经SDS-PAGE电泳,结果显示为1条相对分子质量为38 100的蛋白带,Western blot分析表明抗体与抗原匹配性良好。经检测其蛋白含量为0.615 mg,回收率为0.034%,与传统分离纯化四步法的回收率相比明显提高。结论本优化方法对于提纯孕血清中的EPF是可行的,大大提高了纯化效率。     

6746例孕妇血清生化二联标记物筛查唐氏综合征分析

目的探讨孕妇血清标志物甲胎蛋白(AFP)和游离绒毛膜促性腺激素(Free-βhCG)对孕中期妇女进行胎儿唐氏综合征(Down's syndrome,DS)为主的先天缺陷筛查的作用.方法对孕15w～19w妇女进行上述二项血清标志物检测,结合孕妇年龄、孕周、体重等因素,经过软件计算风险率,对高风险孕妇进行羊水细胞染色体检查及三维彩超进行确诊.结果6746例孕妇中,发现DS 2例,18-三体综合征2例,其它胎儿异常24例.结论孕中期血清AFP、Free-βhCG二项血清标志物联合检测,可作为孕中期唐氏综合征筛查优选项目;在孕中期用AFP、Free-βhCG检测还可以筛查神经管缺陷(NTD),18-三体综合征等其它胎儿异常.     

孕中期Down＇s综合征产前筛查的血清和血片方法的一致性研究

目的验证血片方法与血清方法应用于孕中期Down’s综合征产前筛查的一致性。方法同时对1096例孕15—20孕周孕中期孕妇血清以及滤纸干血片中的AFP及β-hCG含量进行了检测,对血清以及滤纸干血片中两分析物的浓度、中倍数进行了相关性分析,比较了两种方法的筛查结果。结果血清与血片中AFP及β—hCG含量呈高度相关,相关系数分别为0．979、0．982。血清与血片中AFP及β—hCGMoM呈高度相关,相关系数为0．968、0．975。当筛查切值为1／270时,血片法和血清法筛查假阳性率分别为6．4％和5．6％。结论血片与血清中AFP及β—hCG含量呈高度相关,血片方法和血清方法应用于孕中期Down’s综合征产前筛查一致性好,血片法可以代替血清法应用于孕中期Down’s综合征产前筛查。     

孕中期21三体综合征、18三体综合征的产前筛查与产前诊断结果分析

目的通过检测妊娠中期孕妇血清甲胎蛋白(AFP)、游离-β-绒毛膜促性腺激素(Free-β-HCG),进行孕中期胎儿21三体综合征、18三体综合征的无创伤性产前筛查。方法采用时间分辨免疫荧光分析法(DELFIA),对孕15~20w孕妇血清AFP、Free-β-HCG的浓度进行检测,结合母龄、孕周、体重等因素,经过软件计算风险率,对高风险孕妇在知情的情况下,自愿选择进行羊膜腔穿刺,羊水细胞染色体核型分析。结果在5750例孕妇中筛查出21三体高风险297例,占筛查总数的5.17%,18三体高风险37例,占筛查总数的0.64%;在258例羊水细胞染色体检查中,检测出21三体2例,18三体3例,其它异常10例。结论利用孕妇血清AFP,Pree-β-HCG进行孕中期胎儿21三体综合征、18三体综合征无创伤性产前筛查,结合产前诊断,对减少出生缺陷的发生,具有一定的意义。     

唐氏综合征孕中期产前筛查与产前诊断结果分析

目的检测孕妇血清甲胎蛋白（AFP）,游离-β-绒毛膜促性腺激素（Free-β-HCG）,进行孕中期胎儿唐氏综合征的无创伤性筛查。方法采用金标定量系统对孕中期（14-22w）妇女进行AFP、Free-β-HCG检测,结合母龄、孕周、体重等因素,经专用软件进行分析校正,计算风险率,对高风险孕妇在知情的情况下,自愿选择羊膜腔穿刺,进行羊水细胞染色体核型分析。结果接受筛查的1568名孕妇中筛查出唐氏综合征高风险104例,占筛查总数的6.63%。其中62例接受羊水细胞染色体检查,占高危孕妇的59.62%。检出染色体异常核型14例,占高危孕妇接受羊水细胞培养染色体检查的22.58%。结论利用孕妇血清AFP、Free-β-HCG进行孕中期胎儿无创伤性产前筛查,结合产前诊断,对减少出生缺陷儿的出生,具有重要意义。     

牡丹江地区4316例孕妇唐氏综合征产前筛查和产前诊断结果分析

目的检测孕中期孕妇血清中甲胎蛋白（AFP）、游离-β-绒毛膜促性腺激素（Free—β—HCG），结合软件分析，进行孕中期胎儿唐氏综合征的无创伤性筛查。方法采用酶联免疫吸附（ELISA）技术检测4316例15w～20^＋6w孕妇血清中AFP和B—HCG浓度，结合孕妇年龄、体重、孕周等因素，使用复旦张江公司提供的贝喜唐氏综合征产前筛查软件计算风险率。对唐氏综合征高危孕妇进行羊水细胞染色体分析。并对高风险妊娠跟踪随访至胎儿出生后。结果共筛查出高风险473例，其中经产前诊断确诊唐氏综合征7例，其它染色体异常2例。结论使用孕中期母血清AFP／f-βHCG双联法筛查出唐氏综合征高风险妊娠进而行产前诊断是减少患儿出生的有效方法。     

Detection of Early Pregnancy Factor in Human Sera

ABSTRACT: The rosette inhibition titers (RIT) for sera from 94 women at various stages of gestation were detected with a standardized rosette inhibition test. The results showed that an immunosuppressive substance, named early pregnancy factor (EPF), did exist in the pregnancy sera. We confirmed that the EPF activity was very high in the early pregnant stage (the mean RIT = 6.30), gradually decreased with the continuance of gestation, and disappeared at 8 weeks before delivery when the mean RIT for sera (<4) had fallen in the RIT nonpregnant range. In addition, observations for the EPF dynamic changes before and at varying stages after the induced abortion in 21 pregnant women showed that the mean RIT was 5.9 ± 0.25 (SE) before the abortion and dropped below 4 (EPF activity could not be detected) at 3–5 days after the abortion.     

A Study of Early Pregnancy Factor Activity in Preimplantation

PROBLEM and METHOD: Early pregnancy factor (EPF), an Immunosuppressive substance, which appears in pregnant women's sera 48 h after fertilization, is a kind of pregnancy-specific protein. To determine whether the EPF activity could be a super early indicator of pregnancy, we used rosette inhibition assay to detect EPF activity in the sera, collected from 70 women 2–7 days after ovulation intending to conceive monitored by ultrasonography. Simultaneously we selected 40 non-pregnant sera and 12 early-pregnant sera as negative control and positive control, respectively. RESULTS: The results of this study demonstrated that EPF activity is detected in 35 women's sera out of 70 women within 2–7 days after ovulation, and 28 women out of the 35 were pregnant, which was known by follow-up, and 7 were not pregnant, possibly due to either false positive results or embryo loss because of preimplantation failure, thus causing no pregnancy. The other 35 out of 70 had no EPF activity and 34 of them were not pregnant, which was known by follow-up, but one case became pregnant, which was false negative result. Our study showed that diagnosis of the super early pregnancy could be made by detecting EPF activity in maternal serum within the time of preimplantation. The accuracy of pregnancy diagnosis by this method is 88.6%, with a false negative rate of 3.4% and a false positive rate of 17.1%. The β-HCG level was measured from the above 70 women's sera in order to contrast EPF activity. All of the sera collected 2–6 days following ovulation indicated that there were lower β-HCG values in very early pregnancy (≥a5 mIU/ml). On the seventh day after ovulation, EPF activity was detected in 11 out of 15 sera with only 2 of them with a b-HCG level that reached or slightly surpassed that of the early pregnancy diagnosis (5 mIU/ml and 5.4 mIU/ml, respectively). This demonstrated that β-HCG is not the earliest signal of pregnancy; otherwise the EPF activity is one that appears 2–6 days earlier than β-HCG appears. We measured the progesterone level of the 48 sera from the 70 collected above within 2–7 days postovulation and found most of them reached the level of progesterone in the luteal phase (7.5–98.3 nmol/L). This indicated that ovulation had taken place in these women, which was in accordance with observations by ultrasonography. CONCLUSIONS: Our study showed that diagnosis (of 88.6%) of super early pregnancy could be made with an accuracy of 88.6% by detecting EPF activity in maternal serum within 2-days after ovulation. This offers a basis for pregnancy diagnosis for the women who attempt to terminate their pregnancy safely or who conceive unexpectedly, and it contributes to family-planning.     

Study of early pregnancy factor (EPF) in equine (Equus caballus)

PROBLEM: Early pregnancy factor (EPF) is an immunosuppressive protein detected in the early pregnancy serum. We have already reported that we developed the rosette inhibition test for mare EPF and detected EPF in thoroughbreds. The aim of this study was to determine whether or not our method could be used clinically. METHODS OF STUDY: The rosette inhibition test for equine EPF was carried out on serum from six nonpregnant and six pregnant Shetland ponies, a female and a male Chinese pony, and four nonpregnant and 13 pregnant thoroughbred mares. In the thoroughbreds sera were collected during the pregnancy period. Furthermore, we measured progesterone and detected pregnant mare serum gonadotrophin (PMSG) in order to confirm pregnancy of the Chinese pony 3 and 6 months after mating. RESULTS: In the nonpregnant Shetland ponies, the rosette inhibition titre (RIT) was 6.0+/-1.0 and EPF was negative. In contrast, in the pregnant ponies, the RIT was 9.2+/-0.4 and EPF was positive. Based on these results, we diagnosed pregnancy of the Chinese pony. The RIT of the female Chinese pony (3 months after mating) was above 10 and EPF was positive. Furthermore, we detected PMSG and progesterone in the serum of this pony. EPF appeared in the maternal blood circulation at 24-72 hr after mating, it was detected until the second trimester, and after that it disappeared from the maternal serum. CONCLUSIONS: The pony's EPF was detected by using the same rosette inhibition test as in the thoroughbred and was present from 24 to 72 hr after mating until the second trimester. The results indicated that our method was useful for pregnancy diagnosis of Equine.     

Detection of early pregnancy factor in fetal sera

Utilizing rosette inhibition test recommended by Morton et al., (Morton H, Tinneberg HR, Rolfe M, Mettier L. Rosette inhibition test: a multicenter investigation of early pregnancy factor in humans, J Reprod Immunol. 1982; 4:251-261) we have examined 44 fetal sera of abortion induced by water bag in the second and third trimester pregnancy, 4 umbilical sera of newborns, and their 48 maternal sera for detecting early pregnancy factor (EPF), which activity was expressed as rosette inhibition titre (RIT). The mean RIT value of EPF in fetal sera was 6.00 +/- 0.31 (SD) during 16-17 weeks of gestation, gradually decreased along with gestational weeks, got to 4.25 +/- 0.25 by term. The mean RITs of EPF activity in maternal sera were 5.86 +/- 0.26 and 5.89 +/- 0.35 during 5-7 weeks and 18-19 weeks, respectively, decreased afterwards, and then fell into nonpregnant range (RIT: 4.00 +/- 0.40) after 31 weeks of gestation. There was a close correlation between RITs of 44 fetal and those of their maternal sera (r = 0.615; P less than 0.001). The results revealed the presence of EPF in fetal serum during gestation. The significance of EPF distribution and its possible role of immunological regulation on survival of semiallogeneic conceptus have thus been discussed.     

Detection of early pregnancy factor-like activity in human amniotic fluid

Early pregnancy factor (EPF), an immunosuppressive substance, appears in mammalian maternal serum at a very early stage after fertilization. To investigate the normal distribution of EPF in the human body, the rosette inhibition test has been used to detect EPF in human amniotic fluid. The mean value of rosette inhibition titer (RIT) was significantly higher (5.93 +/- 0.43 SE) in 52 amniotic fluid samples (16-30 weeks' gestation) than in 23 nonpregnant sera (3.54 +/- 0.11), indicating that something in amniotic fluid inhibits rosette formation. This inhibitory factor is recognized as EPF or EPF-like substance for its activity significantly correlated with that of EPF in serum from the same donor (r = 0.756: P less than 10(-6]. The immunological significance of the EPF-like substance in amniotic fluid may be important to the survival of semiallogeneic conceptus.     

Detection of Early Pregnancy Factor-Like Activity in Women With Gestational Trophoblastic Tumors

ABSTRACT: The presence of immunosuppressive early pregnancy factor (EPF) in the maternal serum has so far been associated with gestation. Its presence in the serum of women with gestational trophoblastic tumors was investigated. The results indicate that while EPF activity was detected in the serum of women with choriocarcinoma, no such activity was detected in the serum of women with hydatidiform mole, leading to the novel use of EPF as a marker to distinguish these two clinical situations. Results of the experiments also suggest that EPF moiety present in the maternal serum during pregnancy may be of different molecular entity than that present in the serum of women with choriocarcinoma.     

Partial purification of mare early pregnancy factor

PROBLEM: Early pregnancy factor (EPF) is an immunosuppressive protein detected in the serum in early pregnancy. We have already reported the development of the rosette inhibition test for mare EPF and have detected EPF in thoroughbreds and ponies. Here, we attempted to purify equine EPF from pregnant mare serum. METHODS OF STUDY: Mare EPF was purified by ultrafiltration and ion-exchange chromatography. Purified EPF was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting and a neutralization test. EPF activity was estimated as the rosette inhibition titer (RIT) by the rosette inhibition test. RESULTS: Purified EPF bound to carboxymethyl (CM) sepharose and did not adsorb to diethylaminoethyl (DEAE) sepharose. SDS-PAGE revealed that in the final purified fraction there were many proteins. In the immunoblotting analysis, a protein band of 25.8 kDa was detected as the pregnancy-specific band. Further, antibody gained from the 20 to 30 kDa protein band of the final purified fraction neutralized the RIT activity of pregnant mare serum. CONCLUSIONS: Mare EPF was detected in the final purified fraction and had a molecular weight of 25.8 kDa. EPF in the mare is similar to that obtained from the serum of pregnant cows.     

A Study of Early Pregnancy Factor Activity in the Sera of Patients With Unexplained Spontaneous Abortion

PROBLEM: Seventy-seven sera samples obtained from 51 patients (6–20 wk gestation) with unexplained spontaneous abortions including 20 threatened abortions (TA), 13 inevitable abortions (IA), and 18 intrauterine fetal growth retardation (IFGR), as well as 9 sera from 9 patients (6–20 wk gestation) with hydatidiform mole (HM), were detected for early pregnancy factor (EPF) by rosette inhibition assay. Rosette inhibition titer (RIT) was used to express EPF activity. RESULTS: The RIT value of EPF was 5.95 ± 0.69(mean ± SD) in the TA group; 4.06 ± 0.93 in the IA group; 3.50 ± 0.71 in the IFGR group, and 4.00 ± 0.71 in the HM group. Except for the TA group, in which the mean RIT was in normal pregnancy range, the RIT in the other three groups was significantly lower (P < 0.01) then that of the normal pregnancy group (5.81 ± 0.68). CONCLUSIONS: These results suggest that measurement of EPF is a sensitive method for judging survival of the embryo. It may be used to predict the prognosis of the patient with unexplained spontaneous abortion.     

Early pregnancy factor in cervical mucus of pregnant women

PROBLEM: This study was carried out to determine the presence of early pregnancy factor (EPF) activity in cervical mucus of pregnant women and explore the relationship between EPF activity in sera suggested it and in cervical mucus. METHOD OF STUDY: Cervical mucus and sera were collected from 60 pregnant women in different terms and 25 non-pregnant women, respectively. EPF activity was detected by rosette inhibition assay. RESULTS: EPF activity did exist in cervical mucus of pregnant women and was similar both in cervical mucus and sera. CONCLUSIONS: We report here for the first time the EPF activity in cervical mucus of pregnant women. The parallel change observed in sera suggested it might be another useful index in evaluating embryo viability.