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.     

Use of Lymphocyte Platelet Binding Assay for Detecting a Preimplantation Factor: A Quantitative Assay

PROBLEM: To evaluate the ability of the lymphocyte/platelet binding assay to identify a preimplantation factor (PIF). METHOD: Percentages of binding of lymphocytes by platelets in the presence of sera from 30 known pregnant and 30 nonpregnant individuals were compared using a novel lymphocyte/platelet binding assay. The assay is performed using a combination of a heat inactivated sera with donor O+ lymphocytes, activated complement and an antibody against CD₂(T11, Ortho Pharmaceuticals). RESULTS: In nonpregnant females (23.6 ± 6.5%) and males (17.7 ± 4.7%) the percentage of lymphocytes bound by platelets was significantly different from pregnant women (56.1 ± 15.9%) (P < 0.0001). Serial sampling of blood in five women undergoing IVF/ET who had normal pregnancies showed the detection of PIF by 4 days after transfer. The lymphocyte/platelet binding assay was not influenced by hCG, progesterone and estradiol. The interassay and intraassay variabilities were <3%. CONCLUSIONS: The lymphocyte/platelet binding assay is a simple, reproducible, specific and cost efficient assay for measurement of PIF. Application of this assay will provide investigative and diagnostic tools for identifying and monitoring early pregnancy events.     

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.     

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。     

Pregnancy and Multiple Sclerosis (MS): A Beneficial Association. Possible therapeutic application of embryo‐specific Pre‐implantation Factor (PIF*)

A typical autoimmune neuro‐inflammatory disease (NID), multiple sclerosis (MS), is more prevalent in women than in men. Majority of patients with MS are of child‐bearing age; therefore, occurrence in pregnancy is common. Herein, we review proposed disease mechanisms and suggest therapeutic interventions, focusing on the remarkable pregnancy‐induced protection against MS – insofar considered as best, albeit temporary therapy for such harsh NID. Current drugs used for MS therapy in pregnancy are described. Role of non‐pregnancy‐specific agents considered involved in amelioration of disease is also presented. This review highlights pregnancy‐derived neuro‐protective agents, proposing that unique pregnancy‐induced immune‐protective environment is because of the conceptus and its direct action. The essential role of pre‐implantation factor (PIF) in pregnancy is delineated. Finally, PIF immune‐modulatory effects and efficacy in chronic model of neuro‐inflammation to reduce inflammation and paralysis coupled with neural regeneration is presented. Overall, we postulate that this embryo‐derived‐compound holds great promise to improve MS and possibly neuro‐inflammation in general.     

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.     

Improvement of the Rosette Inhibition Assay for the Detection of Early Pregnancy Factor in Humans Using the Monoclonal Antibody,Anti-Human-Lyt-3

ABSTRACT: The detection of early pregnancy factor (EPF) is a controversial subject. The findings of independent groups attempting to confirm the phenomenon of EPF have been quite variable. The present article gives details of an improved rosette inhibition assay using a monoclonal antibody (anti-human-Lyt-3), instead of an anti-lymphocyte serum (ALS), in order to increase reproducibility. Results are calculated for the first time in terms of absolute monoclonal anti-Lyt-3 concentrations. It is suggested that results be expressed as the Rosette Inhibition Antibody Concentration (RIAC) or the more accurate extrapolated 25% Rosette Inhibition Antibody Concentration (25% RIAC), in place of the previously used Rosette Inhibition Titer (RIT), when monoclonal antibodies are used. Following a 1.5 h incubation period the mean 25% RIAC for pregnant serum was 15.9 ± 4.0 ng/ml and for control serum 48.0 ±12.7 ng anti-Lyt-3/ml. The improved method described should enable different groups to establish the test and compare results.     

血小板激活因子在小肠缺血再灌注损伤中的意义及山莨菪碱的保护作用

在大鼠小肠原位灌注模型上,发现小肠缺血—再灌注(I-R)损伤时,肠道产生的PAF显著增加;山莨菪碱灌流明显抑制其PAF的产生,并有与PAF受体拮抗剂Kadsurenone相似的抗I-R损伤效应。但山莨菪碱对外源PAF灌流引起的小肠组织损伤无显著防治效果。提示PAF在小肠I-R损伤中具有发病学意义,山莨菪碱可能是通过抑制肠道PAF的产生,而主要不是通过拮抗PAF,以改善小肠的I-R损伤。     

Production of Immunosuppressor Factor(s) by Preimplantation Human Embryos

ABSTRACT: Embryonic loss prior to implantation appears to be a significantly frequent phenomenon and this is further reinforced by the very low pregnancy rates reported by in vitro fertilization and embryo transfer programs. Implantation failure may possibly be the result of rejection of the antigenic embryo by the hostile maternal immune system. The mechanism by which embryos in successful pregnancies escape these rejection responses may depend upon their ability to produce factor(s) that suppress in vitro mitogen-induced lymphocyte proliferation. Only 43% of cleaved embryos demonstrated this ability. We postulate that successful pregnancies are dependent upon the production by embryos of immunosuppressor factor(s) that has a direct suppressive effect on the maternal immune response.     

A Monoclonal Antibody to an Early Pregnancy Factor-Induced Suppressor Factor (EPF-S1) Disrupts Implantation in Mice

PROBLEM : The importance of EPF during pregnancy has been established previously but the importance of the EPF-induced suppressor factor EPF-S₁ in pregnancy has to date been unaddressed. Investigations were therefore conducted in order to study this. METHOD : Monoclonal antibodies to EPF-S₁ were produced, and one antibody, designated R2Tγ, was characterized. Mated mice were passively immunized with R2Tγ and the effect on implantation determined. RESULTS : Characterization of anti-EPF-S₁ R2Tγ revealed that it cross-reacted with EPF-S₁ of different MHC restriction but not with EPF or EPF-S₂. When injected into mated mice on days 1 to 4, R2Tγ had no effect on pregnancy but when injections continued to day 5, pregnancy was affected; the number of embryos implanted on day 7 were significantly less than the number of corpora lutea counted, signifying embryonic loss. CONCLUSION : These studies show that anti-EPF-S₁ R2T_γ disrupts implantation in mice when injected on days 1 to 5 of pregnancy but not when injected on days 1 to 4, demonstrating that EPF-S₁ exerts its effects around the time of implantation.     

Preimplantation genetics: A case for prospective action

Preimplantation genetics describes a newly-emerging field in medical genetics, the consequence of the implementation of clinical preimplantation diagnosis and the likely future development of germ-line gene therapy. Given the existing clinical and laboratory difficulties already demonstrated in preimplantation diagnosis and the sensitive ethical issues surrounding genetic manipulation of human embryos, there is a need for 1) critical and objective evaluation of developments in this field by human and medical geneticists and 2) development of guidelines for research and clinical practice in the years ahead. We propose a course of prospective action for preimplantation genetics implemented through the newly-formed American College of Medical Genetics in order to address the ethics, safety, accuracy, cost, and overall merit of preimplantation genetics. © 1994 Wiley-Liss, Inc.     

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.     

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 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.     

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.     

Ovum Factor: A First Signal of Pregnancy?

ABSTRACT: Previous studies have shown that, in the mouse, a factor is produced by the fertilized ovum within 24 h of mating. It cooperates with prolactin to stimulate ovarian production of component B of early pregnancy factor (EPF). This paper presents an initial characterization of the substance, termed ovum facor (OF). An indirect assay based on the rosette inhibition test for EPF has shown that OF is first released upon penetration of the ovum by the fertilizing spermatozoon. OF continues to be produced at least until blastulation. Processes which parthenogenetically activate the ovum are also capable of stimulating OF release from unfertilized ova. Gel filtration studies reveal that OF exists in multiple MW forms of approximately 160,000; 2,800; and 1,500. A substance with these characteristics has not been described previously; it may represent the first embryonic signal to the mother.     

Platelet activating factor (PAF) increases plasma protein extravasation and induces lowering of interstitial fluid pressure (Pif) in rat skin

Aim: To investigate the ability of the microdialysis technique to measure capillary selectivity of different sized plasma proteins induced by local administration of platelet activating factor (PAF). Methods: We used hollow plasmapheresis fibres with 3 cm membrane (cut off 3000 kDa) placed on the back of anaesthetized rats. Results: Platelet activating factor (50 μg mL^?1) administered locally via the fibre, increased extravasation of radiolabelled ¹²⁵I‐HSA from plasma to the microdialysis fibre by approximately 900% compared both to baseline and the control fibre within 70 min (n = 6, P < 0.05). The extravasation in the control fibre did not change over time. HPLC measurement of plasma proteins in the microdialysis perfusate also demonstrated decreased capillary selectivity for proteins in the diameter range of 73 Å, 56 Å and 39 Å after local administration of PAF (n = 6, P < 0.05). PAF also significantly lowered interstitial fluid (P_if) pressure after subcutaneous administration (50 μg mL^?1). Mean arterial pressure (MAP) after intravenous injection of PAF (0.4 μg kg^?1) fell instantly by about 50 mmHg, and stabilized at 50 mmHg after 15 min (n = 6). MAP was unaltered when PAF was given through the microdialysis fibre (n = 4). Both total tissue water (TTW) and extravasation of albumin, measured as the plasma‐to‐tissue clearance (E‐alb) showed a significant increase after PAF (n = 7, P < 0.05). Conclusions: The present study demonstrates that PAF induces plasma protein extravasation and decrease capillary selectivity of different sized plasma proteins. It also increases transcapillary fluid flux, and lowers P_if, indicating a role for PAF in the interstitium for generation of transcapillary transport of water and large molecules followed by formation of oedema.     

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.     

Supplementation Effect of Early Pregnancy Factor-Positive Serum into Bovine In Vitro Fertilization Culture Medium

PROBLEM: Early pregnancy factor (EPF) has been detected in pregnant bovine serum, and its activity appeared from 24 to 48 hr after insemination. However, in bovine in vitro fertilization (IVF), an EPF-like substance(s) had been detected in the culture medium of fertilized ovum. Therefore, we think that EPF and EPF-like substance(s) are very important materials for the development of the embryo. In this study, we examined the development of the embryo when fertilized bovine ova were cultured with IVF culture medium supplemented with EPF-positive or -negative serum. METHOD OF STUDY: EPF activity of each serum (fetal calf serum [FCS], calf serum [CS], estrus bovine serum, and pregnant bovine serum) was assessed by the bovine-rosette inhibition test. The sera were supplemented in TCM-199 culture medium, and IVF bovine ova were cultured with the media for 6 or 7 days, respectively. The culture media of each group were evaluated for EPF activity by the bovine-rosette inhibition test 48 hr after IVF. The cleavage rate of each group was calculated at 48 hr, and 6 or 7 days after IVF. The culture medium of cumulus cells was also tested for EPF activity. RESULTS: Only the pregnant bovine sera were EPF positive. All the culture media 48 hr after IVF became EPF positive. Additionally, the culture medium of cumulus cells did not have EPF activity. There was no significant difference in the cleavage rate of the EPF-positive and - negative sera 48 hr after IVF. However, the cleavage rate of EPF-positive sera tended to be higher than the negative sera. In contrast, the blastocyst development rates of EPF-positive sera were significantly higher than those of the negative sera 6 to 7 days after IVF (P < 0.05). CONCLUSIONS: The data suggest that an EPF-like substance(s) may be secreted from the in vitro fertilized bovine ovum but not from the cumulus cell, and that the EPF in the pregnant serum may accelerate the development of the bovine embryo in IVF.     

Preimplantation genetic diagnosis for Charcot-Marie-Tooth disease type 1A

Charcot-Marie-Tooth (CMT) disease is the 'common' name for a range of hereditary peripheral neuropathies. CMT1 is the most common form and is transmitted in an autosomal dominant manner. CMT1A maps to chromosome 17p11.2 and is caused, in the majority of cases, by a 1.5 Mb DNA duplication, that includes the peripheral myelin protein 22 (PMP) gene. This paper reports on preimplantation genetic diagnosis (PGD) for CMT1A in five couples. The CMT1A duplication was detected by fluorescent PCR analysis using polymorphic (CA)n markers localized within the duplication. Single-cell PCR on blastomeres allowed genetic analysis of embryos obtained after ICSI. Only healthy unaffected embryos were transferred to the uterus. PCR experiments with single EBV-transformed lymphoblasts or with research blastomeres allowed the evaluation of amplification efficiencies, as well as contamination and allele drop-out (ADO) rates for each PCR protocol. Three simplex PCR protocols (using one primer pair) and two duplex PCR protocols (using two primer pairs) were developed for CMT1A. Additionally, a protocol using all three primer pairs in triplex was also established. Thirteen clinical ICSI-PGD cycles were performed for five couples (12 simplex PCR cycles and one duplex PCR cycle), resulting in seven embryo transfers. Three singleton pregnancies ensued in two couples and three healthy babies were delivered. This report describes different fluorescent PCR-based tests which allow efficient and accurate single-cell level detection of the CMT1A duplication. On the basis of the presence of the healthy allele of the affected parent-to-be (and/or absence of the affected one), healthy embryos can be selected for transfer. The assays are suitable for PGD for other couples who present with the same CMT1A duplication [depending on their informativity for the (CA)n markers available] as described here.