Development and Validation of an Assay for Measuring Preimplantation Factor (PIF) of Embryonal Origin

PROBLEM: Tests to determine presence of embryos prior to implantation are needed. METHODS: Sera from women after embryo transfer were tested for preimplantation factor (PIF) using the lymphocyte/platelet binding assay. Autorosettes were counted using blood type O+ donor lymphocytes and platelets incubated with blinded serum in the presence of antiCD2 antibody and rabbit complement. Human chorion gonadotropin (hCG) concentrations were determined 7 days later and compared with results of the lymphocyte/platelet assay. Implantation was confirmed by ultrasonographic evidence of presence of an intrauterine gestational sac. The roles of platelet activating factor (PAF) and chaperonin 10 in the observed phenomena were studied experimentally. RESULTS: Significantly more lymphocyte/platelet rosette formations were observed when sera from women who successfully implanted were compared to sera from women who failed to implant. Neither PAF nor chaparonin added to the tested sera controls influenced the percentage of lymphocyte/platelets rosettes. CONCLUSIONS: PIF is a likely candidate to be the next frontier of diagnosing the presence of viable preimplantation embryos in vivo.     

A Novel Bioassay for Detection of Preimplantation Factor (PIF)

PROBLEM: To identify the presence of vital preimplantation embryos in vivo in humans, a newly observed phenomenon based on autorosette formation between lymphocytes and platelets, when treated with pregnant sera, was used as a marker. METHOD: Serum samples were obtained from 65 patients on the fourth day after embryo transfer (ET). Sera from 10 healthy males and 47 nonpregnant women were used as controls. The preimplantation factor (PIF) was detected by using blood group 0+ donor lymphocytes and platelets incubated with blinded serum in the presence of anti-CD2 antibody and rabbit complement. Human chorion gonadotropin (hCG) concentrations were determined 7 days later and compared with results of the lymphocyte-platelets assay. Implantation was confirmed by ultrasonographic evidence of presence of an intrauterine gestational sac. The role of platelet activating factor (PAF) in the observed phenomena was studied experimentally. RESULTS: Significantly more lymphocyte-platelet rosette formations were observed when sera from women who successfully implanted were compared to sera from women who failed to implant. This assay yielded a specificity of 95%, sensitivity of 88%, positive predictive value of 94%, and negative predictability of 90%. PAF added directly to the cell suspension and tested sera controls did not influence the percentage of lymphocyte/platelets rosettes. CONCLUSION: The application of PIF assay will enable the identification and study of early pregnancy events before the implantation occur. PAF by itself is not responsible for the rosette formation.     

Preimplantation Factor (PIF) Predicts Subsequent Pregnancy Loss

PROBLEM : To evaluate the ability of preimplantation factor (PIF) measured in the lymphocyte/platelet binding assay (LPBA) to predict subsequent spontaneous abortion. METHOD : Serum from 57 women experiencing first trimester pregnancy losses were studied using the LPBA (46 women conceived after in vitro fertilization and embryo transfer for treatment of infertility and 11 with a history of unexplained recurrent spontaneous abortion conceived spontaneously). The assay employs a combination of heat inactivated sera with donor O+ lymphocytes and platelets, complement and an antibody against CD2. Chromosome analysis was performed on 32 of the abortuses. Results of PIF assay were compared between karyotypically normal and abnormal abortuses. RESULTS : PIF assay was negative in all 57 women at the time of abortion. Among 12 karyotypically normal abortuses only 1 woman (8%) had an initial positive PIF, 11 (92%) had negative PIF. Serial PIF assays were performed on 15 women. PIF assay became negative a minimum of two weeks prior to demonstration of intrauterine demise at a time when hCG concentrations remained elevated. A trend to subnormal was seen in women with normal when compared to those with abnormal abortus karyotype, but the numbers were too small to reach statistical significance (P = 0.09). CONCLUSION : Measurement of PIF throughout the first trimester of pregnancy predicts subsequent pregnancy loss.     

Progress in characterization of pre-implantation factor in embryo cultures and in vivo.

PROBLEM: Pre-implantation factor (PIF), a small, embryo-derived peptide is detected in the maternal serum prior to implantation and is associated with successful pregnancy outcome. However, its identity is not known. METHOD OF STUDY: PIF was isolated from mouse embryo conditioned media and from pregnant porcine sera, using high-performance liquid chromatography (HPLC) followed by mass spectrometry. RESULTS: Conditioned culture media was separated by gel filtration chromatography followed by reversed phase chromatography. At each step, PIF activity was determined by the lymphocyte/platelet binding autorosette assay (LPBA). Mass spectrometry yielded a single peak with a mass of 1300 Da. The peptide is, however, present in very low concentrations (fM), which has so far precluded complete identification. Pregnant porcine sera that exhibit potent PIF activity were deproteinated by acetone and further fractionated by reversed phase HPLC. Active fractions contain peptides of molecular masses 523 and 551 Da. CONCLUSION: PIF, likely to be peptides, represents a novel substance related to pregnancy initiation and maintenance.     

Preimplantation embryology: Embryonic origin of preimplantation factor (PIF): biological activity and partial characterization

Preimplantation factor (PIF) is detected in the serum of womenshortly after fertilization; its origin, however, has not beenestablished. In this study, the embryonal origin of PIF wasinvestigated and partial characterization of the factor wascarried out. Culture media from viable human 2–8-cellstage embryos and mouse 2-cell-blastocyst stage embryos wereanalysed using the lymphocyte/platelet binding assay (LPBA).The assay was performed by combining culture media with donor0+ type blood-derived lymphocytes/platelets, complement andan antibody against CD2. Increased autorosette formation betweenlymphocytes and platelets (>9%) was an indication for thepresence of PIF. In addition, the effect of platelet-activatingfactor (PAF) and chaperonin 10 on PIF activity was determined.Partial purification of PIF was carried out using gel filtrationand reverse-phase high purification liquid chromatography (HPLC),followed by mass spectrometry. Culture media of single humanviable fertilized oocytes were negative for PIF; however, the10-fold concentrated medium was positive for PIF. In mediumin which five or more mouse embryos were cultured, PIF activitywas observed starting at the morula stage and was higher bythe blastocyst stage. Addition of PAF or chaperonin 10 to thePIF assay did not elicit a specific effect on PIF activity.Chromatographic data suggest that PIF activity is due to lowmolecular weight proteins. PIF appears to be a low molecularweight protein which is derived from viable preimplantationembryos. It is different from PAF or chaperonin 10. Its finalcharacterization will be valuable for better understanding ofmaternal recognition of pregnancy and implantation. characterization/culture/embryo/preimplantation factor     

Evidence for a Leukocyte Adhesion Factor Produced by the Early Embryo

PROBLEM : To determine if the embryo may induce adhesive molecules needed for implantation. METHOD : Determination of whether platelet rosetting around lymphocytes might occur when exposed to sera from pregnant, but not nonpregnant patients and from culture fluid from embryos but not oocytes. RESULTS : 90.2% of women with positive sera beta human chorionic gonadotropin (P-hCG) levels taken at least 12 days postovulation demonstrated platelet rosette factor (PRF) vs only 18.7% when β-hCG was negative. Using mid-luteal phase sera in women receiving hCG injection 1 wk before, 64.7% had positive PRF when serial β-hCG levels were positive as did 100% of samples taken from in vitro fertilization (IVF) patients; however, only 15.3% were positive with negative serial hCG levels. Culture media from fertilized oocytes and embryos tested positive for PRF, but follicular fluid and media from unfertilized oocytes were negative. CONCLUSION : The early embryo secretes a factor(s) that gains access to maternal serum and promotes increased lymphocyte/platelet adhesiveness.     

Soluble Intercellular Adhesion Molecule-1 Serum Profile in Physiologic and Preeclamptic Pregnancy

PROBLEM: The aim of this study was to determine serum levels of soluble intercellular adhesion molecule (sICAM)-1, an adhesion receptor that mediates interactions with the immune system, in physiologic and preeclamptic pregnancies. Moreover, we evaluated whether the release of sICAM-1 during pregnancy correlated to plasma fibronectin concentrations. METHOD OF STUDY: Serum was collected from 18 nonpregnant, control women, from 58 normal pregnant women during the first (n = 13), second (n = 15), and third (n = 30) trimesters, and from 25 preeclamptic patients at 27–39 weeks' gestation. All samples were assayed for sICAM-1 by a specific enzyme-linked immunoassay and for fibronectin by a nephelometric system. Serum sICAM-1 levels in preeclamptic patients were compared to those obtained from gestational-matched normal pregnant women. RESULTS: Levels of sICAM-1 were significantly elevated (P < 0.001) in each of the three trimesters of normal pregnancy (I trimester: 390.4 ± 25.7 ng/ml; II trimester: 386.3 ± 15.4 ng/ml; and III trimester: 367.3 ± 15.8 ng/ml) when compared to those of healthy nonpregnant women (263.3 ± 11.6 ng/ml). No significant difference in sICAM-1 concentrations was observed among the three trimesters. Preeclampsia was associated to a significant decrease (P < 0.01) of sICAM-1 levels (309.8 ± 11.6 ng/ml) relative to those observed in gestational-matched pregnant women (367.3 ± 15.8 ng/ml). Fibronectin and sICAM-1 levels did not correlate. CONCLUSION: The increased levels of sICAM-1 found in physiologic pregnancies and its reduction in preeclampsia may account for some of the immunologic alterations demonstrated to be associated with pregnancy.     

融合蛋白TAP-SSL5对激活的血小板与人淋巴细胞结合的影响

目的:研究抗炎、抗凝双效融合蛋白蜱抗凝血肽(TAP)-金黄色葡萄球菌超抗原样蛋白5(SSL5)对激活的血小板与人淋巴细胞结合作用的影响。方法:采用免疫磁珠分选法筛选人外周血总淋巴细胞;CCK-8法检测TAP-SSL5对细胞活力的影响;流式细胞术检测Jurkat细胞(人外周血白血病T细胞株)表面CD162(PSGL-1)的表达及TAP-SSL5对小鼠抗人CD162单抗(KPL-1)与Jurkat细胞结合的抑制作用。以20μmol/L ADP激活人血小板,流式细胞术检测血小板与Jurkat细胞或人淋巴细胞的结合情况,并研究TAP-SSL5的干预作用。结果:30mg/L及以下浓度的TAP-SSL5对Jurkat细胞的活力无明显影响。流式细胞术检测显示,10 mg/L的TAP-SSL5能显著抑制KPL-1与Jurkat细胞的结合;20μmol/L ADP激活的血小板与Jurkat细胞或淋巴细胞的结合率分别为(11.86±4.49)%和(8.32±1.00)%;细胞经10 mg/L TAP-SSL5预先处理后,结合率分别降至(6.73±2.71)%和(5.51±0.70)%,差异有统计学意义。结论:TAP-SSL5可与淋巴细胞表面的PSGL-1结合,从而抑制激活的血小板与人淋巴细胞的结合,这可能是抗炎、抗凝双效融合蛋白TAP-SSL5发挥其抗炎作用的机制之一。     

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.     

Lymphocyte Cytotoxicity Assay for Predicting Preterm Delivery of Small for Date Babies

ABSTRACT: Cytotoxic activity of lymphocytes, obtained from 84 pregnant women was tested, using human embryonic fibroblast cells as target cells. Lymphocytes of 8 women who delivered small for date babies before term and those of 3 women who had spontaneous abortions were significantly more cytotoxic (p < 0.001) than lymphocytes of women who delivered small for date babies at term, appropriately grown babies before term, or appropriately grown babies at term. Data suggest that a higher level of in vitro lymphocyte cytotoxic activity of pregnant women forecasts either the preterm delivery of small for date babies or the occurrence of spontaneous abortions.     

Cell-Mediated Immunity to Cytomegalovirus in Pregnant Women

ABSTRACT: Employing the techniques of in vitro lymphocyte transformation (LTF) and complement fixation, cell-mediated immunity (CMI) and antibody to cytomegalovirus (CMV) were studied in pregnant and nonpregnant women. The LTF activity was determined by the whole blood microassay using four strains of CMV (AD-169 and its early antigen [EA], Davis, Veca, and Towne strains), and phytohemagglutinin (PHA). Lymphocyte transformation response to specific CMV antigens at 11–30 weeks of gestation and to nonspecific mitogen (PHA) in all pregnant and postpartum women were found to be significanty depressed compared with the nonpregnant women. The lower LTF responses to CMV antigen and PHA were found in specimens taken from pregnant women at 21–30 weeks of gestation. There were no significant differences in the mean complement-fixing (CF) antibody titers and the percentage of E-rosette-forming T lymphocytes between subjects in various stages of pregnancy. In addition, concanavalin A (Con A)-generated suppressor T cell activity was evaluated in pregnant and nonpregnant women. The suppressor effect of Con A-activated lymphocytes in pregnant women was somewhat higher than in nonpregnant women. These observations suggest that CMV-specific suppression of cellular immunity may play an important role in reactivation of CMV in pregnancy.     

Osteovisceral afferent interrelations

The effects of uterotonic agents (oxytocin and enzaprost) on platelet aggregation in pregnant and nonpregnant women were studied by low-angle light scattering. In nonpregnant women oxytocin produced different effects on ADP-induced platelet aggregation: potentiation at low (<200 nM) and inhibition at high (>400 nM) ADP concentrations. In pregnant women oxytocin did not modulate ADP-induced platelet aggregation or this effect was negligible. Enzaprost competitively inhibited ADP-induced platelet aggregation in all examined women (inhibition constant 84.8±25.7 nM).     

Lymphocyte Subsets in Habitual Abortion

ABSTRACT: Lymphocyte subpopulations were characterized by means of monoclonal antibodies in 25 women with habitual abortion and 21 muciparous normal women. Compared to nonpregnant women (N = 8), pregnant normal women were associated with significantly lower helper-to-suppressor ratios (1.71 ± 0.41 versus 2.37 ± 0.66). In contrast in pregnant women with habitual abortion (N = 13) the ratio remained high (2.32 ± 0.73). Failure to increase the number of suppressors and a significant rise in helpers caused this increased ratio. We discuss the possible mechanisms and etiological importance of this finding in habitual abortion.     

Platelet absorption on the test tray (PATT): A rapid method for the screening of HLA class II antibodies using the two-colour fluorescence method

Several strategies have been proposed for the screening of alloantisera towards HLA class II antigens. Most often the absorption of the sera with platelets is required in order to remove their anti-HLA-A, B, C activity. We have developed a simple micromethod for platelet absorption of sera: platelet absorption on the test tray (PATT); 0.5 microliter of platelet suspension is incubated with 0.5 microliter of the serum to be absorbed in the same tray which is subsequently used for the microlymphocytotoxicity by the two-colour fluorescence method. This technique is proved to be almost as efficient as classical absorption procedures: out of 44 anti-HLA-A, B sera the T-cell activity is completely removed in 43 by a classical procedure as compared to 41 by PATT; only 8% discrepancies were found among 394 reactions. PATT was then used for anti-B lymphocyte screening in 419 anti-HLA-A, B sera; 4% of the sera remained cytotoxic towards T and B lymphocytes while 35% reacted only with B cells, several of them being DR specific. PATT can also be useful for T/B lymphocyte differential cross-matches before kidney transplantation. The method is now routinely used in our laboratory for anti-HLA class II antibody screening.     

特发性血小板减少性紫癜患儿外周血T细胞PKC活性变化的研究

为了探讨ITP患儿外周血T细胞蛋白激酶C(PKC)的活性变化及其与T细胞活化和血小板减少程度之间的关系,无菌采集35例ITP患儿及30例正常儿童外周血,采用T细胞分离富集柱法分离纯化T细胞,分别用非同位素标记法检测T细胞PKC的活性变化,用流式细胞仪检测T细胞活化标志FasL蛋白的表达,血细胞计数仪计数血小板的减少程度。结果ITP患儿T细胞PKC的总活性与正常儿童相比明显增强[(0.97±0.21)nmol/ml·min和(0.55±0.13)nmol/ml·min,x±s,P<0.05],T细胞活化标志FasL蛋白表达与正常儿童比较显著升高(CD4+TFasL:32.7%±3.4%和14.7%±4.2%;CD8+TFasL:17.3%±9.7%和11.6%±8.5%,x±s,P<0.05),并且T细胞PKC的活性变化与CD4+TFasL、CD8+TFasL的表达均为显著正相关(r1=0.68,r2=0.53,P<0.05),与血小板计数成显著负相关(r=-0.75,P<0.05)。上述研究结果表明ITP患儿PKC活性增强可能引起T细胞的活化,活性T细胞增多可导致患儿血小板大量损伤,提示PKC信号转导在ITP的免疫病理机制中发挥重要作用。     

正常妊娠中人外周血树突状细胞亚群的研究

目的：探讨髓样DC（MDC） 和浆细胞淋巴样DC（PDC）在正常妊娠中母胎免疫耐受机制中的作用。 方法： 选择正常妊娠妇女30例，分别在每个妊娠妇女早期、中期和晚期妊娠时采集外周血。应用流式细胞仪术，检测MDC和PDC占外周血单个核细胞的百分率及MDC/PDC比率，正常未妊娠女性为对照组。 结果： 与未妊娠妇女外周血中MDC和PDC的百分率（MDC, 0.32%±0.08%; PDC, 0.12%±0.05%）及MDC/PDC比率(2.96±1.39)相比：早期妊娠妇女外周血中MDC和PDC的百分率（MDC, 0.29%±0.07%; PDC, 0.11%±0.04%）及MDC/PDC比率(2.95±0.85)无显著差异（P>0.05）；中期妊娠妇女外周血中MDC和PDC的百分率（MDC, 0.11%±0.06%; PDC, 0.07%±0.03%）及MDC/PDC比率(1.52±0.44)降低（P<0.01）；晚期妊娠妇女外周血中MDC和PDC的百分率（MDC, 0.12%±0.06%; PDC, 0.08%±0.03%）及MDC/PDC比率(1.54±0.43)降低（P<0.01）。与正常早期妊娠妇女外周血中MDC和PDC的百分率及MDC/PDC比率相比，中、晚期妊娠显著降低（P<0.01）。 结论： 正常妊娠妇女中期和晚期妊娠时外周血中MDC和PDC的百分率及MDC/PDC比率均降低。     

Progesterone-Prostaglandin Balance Influences Lymphocyte Function in Relation to Pregnancy

ABSTRACT: The effect of progesterone and prostglandin F₂α on cytotoxic activity of lymphocytes was investigated. Thirty-three pregnant women with a clinical diagnosis of threatened premature labour were treated with the prostaglandin synthesis inhibitor acetylsalicylic acid (2.7 gm/day) for 1 week. Cytotoxic activity of the lymphocytes significantly decreased while their progesterone binding capacity increased by the end of the treatment. Ten healthy pregnant women received a single 15 mg dose of prostaglandin F₂α for pregnancy termination. One day after prostaglandin administration lymphocytes showed significantly higher cytotoxic activity and lower progesterone binding capacity than before. Lymphocytes of 44 healthy nonpregnant volunteers were preincubated with prostaglandin F₂α and/or progesterone. Prostaglandin F₂α (8 nmole) significantly decreased cytotoxic activity of 4 × 10⁶ lymphocytes. In the presence of progesterone prostaglandin F₂α failed to stimulate lymphocytes.     

Changes in anti-lymphocyte and anti-Ia antibodies during pregnancy in systemic lupus erythematosus

Anti-lymphocyte antibodies reactive with monocyte-depleted lymphocytes, T cells, or B cells were studied in 43 nonpregnant and 23 pregnant systemic lupus erythematosus (SLE) patients. Anti-Ia specificity was assayed in an enzyme-linked immunosorbent assay system. No difference in mean lymphocytotoxicity was noted between pregnant and nonpregnant SLE patients; however, anti-Ia lymphocyte antibody associated with disease activity was lower (P less than 0.01) in pregnant than in nonpregnant SLE patients. Lymphocytotoxic or anti-Ia antibody activity did not reliably predict the outcome of individual pregnancies.     

REACTIVITY OF LYMPHOCYTE SUBPOPULATIONS IN HUMAN MIXED LYMPHOCYTE CULTURE

Loss of antigenicity in the mixed lymphocyte culture (MLC) reaction of lymphocytes precultured at 22°C for 7–10 days was accompanied by a decrease in bone-marrow-derived lymphocytes (B cells) from 22 ± 1% to 13 ± 1%, and an increase in thymus-derived lymphocytes (T cells) from 65 ± 2% to 83 ± 1% (P < 0.001). Depletion of B cells from a fresh lymphocyte suspension by either antihuman immunoglobulin-coated column fractionation or by sheep red blood cell (SRBC) rosette formation resulted in a significant reduction of the cell's ability to stimulate in MLC (P < 0.001). Coating of lymphocytes with rabbit antihuman brain serum abrogated their ability to respond but not the ability to stimulate in MLC.     

Detection of platelet antibodies by enzyme-linked immunosorbent assay (ELISA). Comparative studies with the indirect immunofluorescence assay

A newly developed enzyme-linked immunosorbent assay (ELISA) for the detection of platelet antibodies was compared with the platelet immunofluorescence test (PIF). A good correlation was found between both assays. However, ELISA seems to be more sensitive than PIF. Some sera reacted only in ELISA whereas no sera were found that were negative in ELISA and positive in PIF.When comparing the antibody titres, ELISA is at least 8 times more sensitive than PIF.