Synthesis of early pregnancy factor using red deer (Cervus elaphus) as a delayed implantation model

Purpose: This study measured serum early pregnancy factor (EPF) in pregnant red deer (Cervus elaphus) and ascertained whether EPF synthesis is associated with implantation. Methods: Serial serum samples were taken from mated hinds up to 42 days postconception and analyzed for EPF activity using the rosette inhibition test. EPF activity was then correlated with calving records and stages of preimplantation development. Results: EPF was detected in all pregnant animals, with a twin pregnancy giving increased EPF activity. Three animals gave an EPF response following fertilization but failed to continue beyond the preimplantation embryo stage. The increase in EPF synthesis previously associated with implantation in other mammals occurred at the blastocyst stage in red deer. Conclusions: EPF synthesis in red deer (Cervus elaphus) is consistent with the preimplantation period, as occurs in other mammals. However, the second phase of the biphasic increase in early pregnancy factor production is associated with blastocyst formation, not implantation.     

Detection of the early pregnancy factor (EPF) in the serum of patients with questionable pregnancy

The early pregnancy factor (EPF) has been detected in sera of 24 women with a doubtful pregnancy by means of the rosette inhibition test using a horse antihuman lymphocyte serum. In all sera of women with an intact early pregnancy the EPF was found, furthermore in a pregnancy ending as abortion and two cases of an Arias Stella phenomenon. In non-pregnant patients the EPF was not detectable with exception of two patients under medication and one patient suffering from dysmenorrhea membranacea.     

Detection of fetal wastage

Production of the pregnancy-specific protein early pregnancy factor (EPF) was monitored by the rosette inhibition test in a group of 13 nulliparous women. EPF could be detected in serum within 48 hours of fertilization; of 28 cycles in which intercourse took place at the time of ovulation, EPF was detected in 18. However, EPF production continued for more than 14 days in only four cases. Successful pregnancy was maintained in two of these while in the other two, disappearance of EPF preceded miscarriage. In the remaining 14 cases, EPF disappeared from the serum before the onset of menstruation. A high incidence of early embryonic loss is suggested.     

Effects of EPF treatment in human mononuclear cells.

Early Pregnancy Factor (EPF) is one of the earliest pregnancy associated signals, communicating the ensuing pregnancy to the maternal organism. Data published by others on the mouse suggest that EPF bound to spleen cells causes the release of two H2-restricted "suppressor factors" responsible for the rosette inhibiting activity of EPF in the rosette inhibition test. Using human material, we were able to detect the release of a second entity from mononuclear cells that is able to suppress rosette formation in the human rosette inhibition test. In an attempt to show an intracellular EPF effect in the target cell, cytosolic free calcium concentrations were measured in EPF-treated mononuclear cells from peripheral blood. Our findings did not, however, show any changes of intracellular free Ca(2+)-concentrations under the chosen conditions.     

妊娠早期绒毛组织培养液和血清对人淋巴细胞早期E花环形成活性的影响

用羊红细胞花结抑制试验,测正常早孕血清、非妊娠血清、妊娠第7周正常绒毛组织培养液及蜕膜培养液的早孕因子活性。发现绒毛组织培养液和早孕血清提高花结抑制滴定度(P<0.05);蜕膜培养液和正常非妊娠血清对羊红细胞花结形成没有影响。正常绒毛组织可能是着床后妊娠血清早孕因子活性的来源之一。     

Rosette inhibition test: A multicentre investigation of early pregnancy factor in humans

The rosette inhibition test for the detection of early pregnancy factor is described in detail. The extended methodology presented here represents the cumulative experience of three independent laboratories. Special reference is made to the effect on the assay of varying the conditions of rosette formation between lymphocytes and sheep red blood cells. Antilymphocyte sera prepared for use in the rosette inhibition test fell into three catagories: (i) with no rosette inhibiting activity, (ii) with rosette inhibiting activity which is not affected by the presence of EPF, and (iii) rosette inhibiting activity which is significantly increased in the presence of EPF. To date, this third reaction has been found to be a specific indication of the presence in serum of early pregnancy factor.     

Clinical significance of early pregnancy factor

We studied the clinical significance of Early Pregnancy Factor (EPF), which was detected at a very early stage after fertilization in maternal serum, using a new stable assay system. The results obtained were as follows: EPF was useful for cyesiognosis at the earliest stage, as EPF was detected earlier than beta-hCG. Therefore the detection of EPF makes it possible to supply the first information concerning pregnancy. EPF was also useful in judging the potential for fertilization of gametes, especially spermatozoon, and other various factors. Differentiation between obstruction of fertilization and that of implantation was made possibly by measuring EPF. A high incidence of embryo loss was suggested. And we suggested the importance of the serum progesterone level in the preimplantation period as one of the many factors involved in embryo loss. EPF was also able to contribute to the prognostic diagnosis of abortion in early pregnancy, for it was a very sensitive marker of a viable embryo. These findings showed that EPF supplied new and very useful information as a marker of fertilization and a viable embryo, and it possessed clinical significance in the treatment of sterility.     

Immunosuppressive effect of early pregnancy factor on early expression of cell surface membrane IgG

We have adopted a new assay to investigate the influence of early pregnancy factor (EPF) on the modulation of lymphocyte activity. Lymphocytes were attached to the plastic surfaces of microplates in serum-free medium in the presence of Sepharose-Con A. After 2-3 days incubation with EPF, and ELISA assay was used to detect the expression of surface membrane IgG (smIgG); this was done in the same microplates used for the culture, thus avoiding cell manipulation. Using only a few picograms of EPF a significant inhibition (in the range 26-40%) was obtained. The variation in the inhibition observed was mainly due to the different sources of lymphocytes used. Unrelated proteins and hormones, tested at the same concentration as EPF, did not show any inhibitory activity. Using the F(ab)2 fragment of anti-human IgG instead of the whole molecule the same levels of inhibition were obtained, suggesting that the observed inhibition by EPF was not due to a non-specific interaction between the anti-human IgG and the Fc receptors on the cell. Such inhibitory activity detected in vitro by this method provides additional support for a suppressive role for EPF during pregnancy.     

Human early pregnancy factor: serum concentrations before and after therapeutic abortion in comparison with β-hCG and an early pregnancy associated protein

Summary Serum concentrations of the early pregnancy factor (EPF), -hCG and the early pregnancy associated protein (EPAP) were measured in 12 patients before and after therapeutic abortion for social-medical reasons. Detection of EPF was performed by the rosette-inhibition assay, -hCG quantification by radioimmunoassay and EPAP measurements by rocket immunoelectrophoresis using a monospecific polyclonal rabbit antiserum. The disappearance rate of EPF after termination of pregnancy was closely correlated with the decrease of -hCG concentrations and loosely correlated with the decrease of EPAP concentrations . No correlation has been found between hCG and EPAP values.     

Detection of early pregnancy factor (EPF) in pregnant and nonpregnant subjects with the rosette inhibition test

Summary We tested for early pregnancy factor (EPF) using a rosette inhibition test with polyclonal anti-lymphocyte serum from the horse and two monoclonal antibodies specific for the E-receptor of T-lymphocytes. When lymphocytes were preincubated with early human pregnancy sera, rosette inhibition titres were four or more dilutions higher (P<0.01) than when lymphocytes were preincubated with nonpregnant sera.     

Isolation and partial characterization of early pregnancy factor (EPF) from human pregnancy serum

The isolation and partial characterization of an immunosuppressive early pregnancy factor (EPF) present in the serum of pregnant women, between 3 and 8 weeks of gestation, is described. EPF was purified using ion-exchange chromatography, affinity chromatography and HPLC gel permeation techniques. A homogeneous, active fraction containing a single polypeptide of Mr 21,000 was obtained. This 21 kDa polypeptide appears to represent the major form of rosette-inhibiting active material present in maternal serum during early human gestation.     

Medical management of early pregnancy failure: how to treat and what to expect

Medical management is gaining acceptance as a treatment option for women with early pregnancy failure (EPF). We reviewed randomized trials comparing misoprostol for EPF with surgical (dilation and curettage) or expectant management. Overall, approximately 85% of women with EPF can expect complete uterine evacuation after one or two doses of 600 or 800 microg misoprostol without surgery. Medical management is safe. As with surgical therapy, serious complications are rare. Women undergoing medical management of EPF may expect moderate pain and bleeding for several days, which may persist for 2 weeks or longer. Side effects associated with misoprostol are common; however, acceptability of medical management remains high.     

重组早孕因子(EPF)的表达、纯化及鉴定

目的:获取大量具有良好生物活性的早孕因子(EPF)重组蛋白。方法:从HeLa细胞中提取总RNA,采用RT-PCR的方法扩增EPF cDNA,将该cDNA插入载体pGEX-5X-1,获得重组表达质粒pGEX-5X-1/EPF,将重组表达质粒pGEX-5X-1/EPF转化大肠杆菌BL21后通过IPTG进行诱导表达,然后用谷胱甘肽-琼脂糖球珠亲和层析方法,分离纯化得到纯重组GST-EPF融合蛋白(rEPF)。再用Xa酶切GST,得到纯化的EPF重组蛋白。用SDS-PAGE鉴定其纯度,用Western blotting鉴定其特异性,用玫瑰花环抑制试验(RIT)检测其生物学活性。结果:HeLa细胞总RNA中有效扩增出EPF cDNA。EPF cDNA以正确的阅读框架插入表达载体pGEX-5X-1,经IPTG诱导后高效表达EPF重组蛋白。Western blotting表明其与抗EPF抗体有特异性结合,RIT检测结果显示目的蛋白具有良好的EPF生物学特性。结论:成功构建了EPF的原核表达载体,并纯化获得了具有生物学特性的重组蛋白,为进一步开展EPF蛋白的相关研究奠定了基础。     

Detection of early pregnancy factor by the rosette inhibition test: use of cyclosporin A

For the detection of the early pregnancy factor (EPF) as early pregnancy signal and as tumour marker only the rosette inhibition test is generally accepted. In the testing of 10 EPF-positive and 10 EPF-negative sera the immunosuppressive agent cyclosporine A was used for the first time. The differences between both groups were highly significant (alpha less than 0.001). Similar results were obtained by using the more expensive anti-human T lymphocyte globulin.     

Detection of early pregnancy factor in the sera of conceived women before nidation

In order to apply the early pregnancy factor (EPF) to early diagnosis of fertilization, the establishment of optimal conditions for assay of EPF was attempted, and then EPF in the sera of contracepted and conceived women 4 to 6 days after ovulation were measured. For assay of EPF, 0.25 ml of 1:2 step diluted anti-human lymphocyte serum (ALS), 0.05ml of guinea pig serum as complement and 0.1ml of lymphocytes suspension (1 X 10(7)/ml) pretreated with test serum were mixed and then incubated at 37 degrees C for 90 min. To this mixture 0.1ml of sheep red blood cell suspension (2 X 10(9)/ml) was added and the rosette formation was counted after centrifugation. The rosette inhibition titer (RIT) was expressed as reciprocal of ALS dilutions which resulted in less than 75% of rosette formation as compared with the control. RITs of the conceived women who were assayed on the 5 th day after ovulation were in the range from 16 to 32 X 10(3), while that of the control contraceptive women who were assayed on the same day was in the range from 2 to 4 X 10(3). The sterile women who received AIH but failed to conceive all showed less than 4 X 10(3) as RIT. These results suggest that the assay of EPF is valuable in detecting the early stage of fertilization and possibly may help to differentiate the impairment of embryo implantation from non-fertilization of the ovum as a cause of sterility.     

Studies of early pregnancy factor by rosette inhibition test using monoclonal antibody

We established a new stable rosette inhibition test using anti-human T-cell and anti-mouse Thy-1 monoclonal antibody, instead of antilymphocyte serum for the testing of immunosuppressive early pregnancy factor (EPF). The results obtained with this experimental system are as follows. Serum of pregnant mouse and woman indicated the immunosuppressive activity as inhibition of rosette formation. Serum of pregnant mouse inhibited rosette formation between human lymphocytes and heterologous erythrocytes as well as mouse spleen cells. It indicated that EPF, at least partially, is species-independent. In mouse serum, EPF became evident within 8 hours after mating, and declined at 4 days before term. In human pregnancy, EPF was detected in the 1st and 2nd trimesters but the rosette inhibition declined to the nonpregnant range in the 3rd trimester. In patients with threatened miscarriage and poor prognosis, the rosette inhibition declined. Surgical abortion led to normalization of the rosette formation within 18 to 24 hours. To investigate the origin of EPF, an in vitro fertilization system for mouse was employed. Immunosuppression was demonstrated in the culture medium of a two cell-stage embryo.     

Early pregnancy factor as a monitor for fertilization in women wearing intrauterine devices

Measurement of early pregnancy factor (EPF) by the rosette inhibition test was performed on serum samples from 14 women using intrauterine devices (IUDs). Serial blood samples taken during the luteal phase of 23 cycles demonstrated in six of the cycles a transient appearance of EPF during the 6- to 8-day period following ovulation. In contrast, no EPF activity was found in sera obtained from women in whom fertilization was prevented, either by sexual abstinence or tubal ligation. These observations support the postulate that the IUD functions by the prevention of implantation of the fertilized ovum, rather than by preventing fertilization.     

Early pregnancy factor: Tissues involved in its production in the mouse

The mouse, 24 h after mating, has been used as an experimental model to determine the site of production of early pregnancy factor (EPF). Results have shown that EPF is formed as two separate components, which appear similar to component A and component B formed after dissociation of EPF by 40% ammonium sulphate. Component A is produced by the oviduct during oestrus and pregnancy. It is present in serum during oestrus in an inactive form, that is, not capable of binding with lymphocytes. Component B can be produced in culture from the ovaries of 24-h pregnant mice. Incubation experiments described in this paper show that the production of component B can also be initiated from non-pregnant mouse ovaries, during oestrus or dioestrus, in the presence of both fertilised ovum and non-pregnancy pituitary but not in the presence of fertilised ovum alone. The pituitary from a 24-h pregnant mouse alone can also stimulate the production of component B from oestrous or dioestrous mice.To summarize, component A is derived from the oviduct and is oestrus-dependent. Component B, the pregnancy-dependent component of EPF, is produced by the ovary; its production can be initiated from non-pregnancy ovaries by the fertilised ovum in cooperation with the pituitary.     

Partial characterisation of early pregnancy factor in the sheep

Early pregnancy factor (EPF), a pregnancy-associated substance, has been detected in sheep serum in multiple molecular weight forms. Within the first 24 h after mating two forms have been found, with apparent M.W.s approximately 250 000 and 20 000. As gestation proceeds, a third M.W. form of approximately 50 000 predominates. These three forms of EPF appear to be closely related to one another. Firstly, the 250 000 M.W. form can be converted to a 50 000 M.W. form by the removal of an apparent carrier molecule, present in pregnant and non-pregnant sheep serum. Secondly, all three forms appear to be produced by different combinations of two basic components, termed A and B. These components can be distinguished from each other by ammonium sulphate fractionation, gel filtration behaviour and ability to interact with lymphocytes.The physical properties, behaviour and kinetics differentiate EPF from other pregnancy-associated substances with putative immunosuppressive properties; evidence is presented in this paper which suggests that EPF is a novel substance.