Failings of the rosette-inhibition test for early pregnancy factor

Attempts were made to reproduce work applying the rosette-inhibition test for early pregnancy factor (EPF) to early detection of pregnancy in sheep and swine. Twenty-one antisera, raised in rabbits, goats and sheep against sheep or pig lymphocytes isolated from spleen, thymus or peripheral blood, were tested using the original method of Morton et al. (1979) but none provided positive results. Procedures were then modified to incorporate changes claimed to eliminate possible interferences with the test, but all results remained negative, even when using an antiserum found elsewhere to allow detection of EPF. Success in applying the test, reported by some laboratories, is countered by reports of failure in other laboratories, suggesting that a critical elusive factor in the test has yet to be identified.     

A role for interferons in early pregnancy

In order to survive, the developing conceptus must interrupt the normal ovarian cycle of the mother and extend the production of progesterone by the corpus luteum. An unusual Type 1 interferon (IFN), related structurally to the IFN-alpha molecule and produced in massive amounts for only a few days by the first epithelium (trophectoderm) of the preimplantation conceptus, has been implicated as the antiluteolytic agent in sheep and cattle. IFN-alpha therapy during this critical period can also improve pregnancy success in sheep. It remains unclear, however, whether the trophoblast IFN have specialized biological properties or whether they are unique merely in the timing, magnitude and site of their expression.     

Detection of early pregnancy factor (EPF) using the rabbit ovary and oviduct perfused in vitro

Two peaks of rabbit serum EPF activity were seen over the course of pregnancy. Rabbit ovaries with or without attached oviducts were perfused in vitro for 5 h beginning 12, 16, 24, 48, 72 and 120 h after mating. Perfused isolated ovaries did not produce EPF in vitro, but significant EPF activity was detected in the perfusate of the ovary together with oviduct. Pseudopregnant animals and those rabbits that did not ovulate exhibited no perfusate EPF activity. Perfusate EPF activity was highest at the time embryos were at the pronuclear stage and continued through the morula stage. Although the location of embryos at 72 h after mating varied between oviduct and uterus, EPF activity was maintained over the perfusion period. The results suggest that EPF release occurs within 3 h of fertilization and that the presence of the preblastocyst embryo is crucial for EPF release.     

Purification and characterization of early pregnancy factor from human pregnancy sera

Early pregnancy factor (EPF) is a pregnancy-associated protein detected in the maternal serum by using the rosette inhibition assay and by evaluating the suppression of adoptive transfer of contact sensitivity. Because of its inhibitory effect on the functional reactivity of immunocompetent cells, EPF is thought to be involved in immunoregulation of the maternal immune system during early pregnancy. EPF was purified six million-fold from the serum of pregnant women between 5 and 12 weeks of gestation. The specific activity of purified EPF was approximately 8 x 10(8) units/mg. The purification scheme involved sequential DEAE-cellulose chromatography, S-Sepharose chromatography, concanavalin A-Sepharose chromatography, heparin-Sepharose chromatography, Mono S fast protein liquid chromatography, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified protein has an apparent molecular weight of 21,500 as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 28,000 by gel permeation high pressure liquid chromatography. The isoelectric point of purified EPF moiety is 6.5. The biological activity was susceptible to the proteolytic enzyme trypsin, acidic pH conditions, organic solvents, and sodium dodecyl sulfate, but stable to heat treatment at 56 degrees C for 30 min and the reducing agent dithiothreitol. The biological and physicochemical properties of EPF appear to be distinct from other pregnancy-associated and immunoregulatory proteins.     

Investigation of the immunocompetent cells that bind early pregnancy factor and preliminary studies of the early pregnancy factor target molecule

Early pregnancy factor (EPF) is a secreted protein with immunosuppressive and growth factor properties. It has been shown to suppress the delayed-type hypersensitivity response in mice as well as acute and chronic forms of experimental autoimmune encephalomyelitis in rats and mice, respectively. In previous studies, we have demonstrated that EPF binds to a population of lymphocytes and we hypothesized that it mediates its suppressive effects by binding to CD4+ T cells. In the present study, we isolated monocytes and subpopulations of lymphocytes and labelled them with fluoresceinated EPF in order to determine which populations bind EPF. We demonstrated that EPF binds specifically to CD4+, CD8+, CD14+ (monocytes) and CD56+ NK cells but not to CD19+ B cells. The identity of the molecule(s) on the cell surface that is targeted by EPF is unknown, but as EPF is an extracellular homologue of the intracellular protein chaperonin 10 (Cpn10), we examined the possibility that the EPF receptor is a membrane-associated form of chaperonin 60 (Cpn60), the functional associate of Cpn10 within the cell. The EPF target molecule on lymphocytes was visualized by chemical cross-linking of exogenous iodinated Cpn10 to cells and probed with anti-Cpn60. The effect of anti-Cpn60 on activity in the EPF bioassay, the rosette inhibition test, was also examined. In both instances, no specific interaction of this antibody and the putative receptor was observed. It was concluded that the cell surface molecule targeted by EPF is unlikely to be a homologue of Cpn60.     

Platelet-activating factor in the rabbit uterus during early pregnancy

Platelet-activating factor (PAF) concentrations were low in the non-pregnant, oestrous uterus (mean +/- s.e.m.: 2.2 +/- 1.2 pmol/g, n = 3). However, uterine PAF increased dramatically during pregnancy to a maximum of 37.8 +/- 4.90 pmol/g (n = 7) on Day 5. By Day 7, PAF concentrations in the uteri of pregnant rabbits had returned to levels similar to those found at oestrus. In contrast, uterine PAF in pseudopregnant rabbits peaked at 30.6 +/- 2.8 pmol/g (n = 8) on Day 4, declined to 20.5 +/- 2.4 pmol/g (n = 8) on Day 5 and then remained at that concentration through Day 7. Uterine PAF co-migrated with synthetic PAF (1-O-hexadecyl-2-acetyl-sn-glycero-phosphocholine) in both thin-layer and normal-phase high-performance liquid chromatography. PAF activity in the uterus during pregnancy and pseudopregnancy was found almost exclusively in the endometrium; little or no PAF was found in myometrium, uterine flushings or blastocysts. While no PAF was detected in blastocysts on Days 5 and 6 of pregnancy, the presence of the embryo appears to modulate biosynthesis and/or degradation of PAF by the uterus, since PAF decreased significantly in uterine tissue apposed to the implanting embryo (but not in similar areas between such attachment sites). Increased concentrations of PAF in the preimplantation rabbit uterus followed by a dramatic decrease on the day of blastocyst attachment suggest that this potent inflammatory autacoid may play a vital role in implantation.     

Platelet-activating factor induces the expression of early pregnancy factor activity in female mice

When synthetic platelet-activating factor (PAF, 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine) was injected into mature female mice during dioestrus, pro-oestrus or oestrus, it induced the expression of early pregnancy factor (EPF) activity in the sera of these animals within 1 h of injection. The sera of similarly injected males, metoestrous or immature females did not display any EPF activity. The results suggest that embryo-derived PAF may be the ovum factor responsible for triggering the generation of serum EPF activity during the preimplantation stages of pregnancy.     

Cardiac amyloidosis: the need for early diagnosis

Amyloidosis is a collection of systemic diseases characterised by misfolding of previously soluble precursor proteins that become infiltrative depositions, thereby disrupting normal organ structure and function. In the heart, accumulating amyloid fibrils lead to progressive ventricular wall thickening and stiffness, resulting in diastolic dysfunction gradually progressing to a restrictive cardiomyopathy. The main types of cardiac amyloidosis are amyloid light chain (AL) amyloidosis caused by an underlying plasma cell dyscrasia, amyloid transthyretin (TTR) amyloidosis of wild-type (normal) TTR at older age (ATTRwt) and hereditary or mutant amyloid TTR (ATTRm) in which a genetic mutation leads to an unstable TTR protein. Overall survival is poor once heart failure develops, underlining the need for early referral and diagnosis. Treatment for AL amyloidosis has improved markedly over the last decades, and TTR amyloidosis gene silencers and orally available transthyretin stabilisers are ready to enter the clinical arena after recent positive outcome trials. Novel therapies aiming at fibril degradation with monoclonal antibodies are under investigation. In this review, we focus on ‘red flag’ signs and symptoms, diagnosis and management of cardiac amyloidosis which differs considerably from the general management of heart failure. Only by increasing awareness, prognosis for patients with this devastating disease can be improved.

     

Detection of activity similar to that of early pregnancy factor after mating sows with a vasectomized boar

Incubation of normal pig lymphocytes in serum samples collected from 10 sows immediately before, and at daily intervals after mating with a vasectomized boar significantly elevated the rosette inhibition titre (RIT) of a standard antilymphocyte serum in 6 animals on the first but not on the 2nd and 3rd day after copulation. Infusion of seminal plasma without mating into 5 sows induced an obvious, but not statistically significant, transient rise of titres in 3 pigs. Neither sodium chloride infusion (N = 5), nor sham copulation with diverted penis (N = 5) influenced serum RITs. Porcine seminal plasma showed an inherent rosette-inhibiting property. A depression of rosette formation was evident in a concentration-dependent fashion up to a dilution of 1 in 320. Similarly, preincubation of lymphocytes in serial dilutions of seminal plasma in a non-pregnancy serum sample led to an amplification of the rosette inhibiting capacity of the antilymphocyte serum. Non-specific activation of the eggs to release a signal which induces the production of early pregnancy factor (EPF) or the resorption of seminal plasma components into the blood circulation are considered as possible explanations for the EPF-like activity after mating with a vasectomized boar.     

Identification of a putative inhibitor of early pregnancy factor in mice

Previous studies have indicated that early pregnancy factor (EPF) produced in the pre- and peri-implantation stage of pregnancy appears to consist of inactive components which combine to produce the active species. This is in contrast with EPF produced later in gestation which appears to consist of a single active species. The original studies on ammonium sulphate fractionation of mouse serum and in-vitro culture of mouse ovaries and oviducts have been repeated but tested in the bioassay for EPF, the rosette inhibition test, over an extended range of dilutions. This revealed that the two components in early pregnancy can be understood as EPF and an inhibitor(s). Once this inhibitor is removed, the active fractions in both early and late pregnancy sera exhibit similar behaviour in the above assay. It was shown also that the ovary alone is the source of activity but that this is modulated by an inhibitory substance(s) from the oviduct. Reversed-phase HPLC studies on purified 'early' EPF confirm that active and inhibitory components are present and demonstrate that the active component exhibits an identical elution pattern to 'late' EPF. Thus as pregnancy proceeds, it is not EPF that alters but rather the inhibitor(s), which disappears from the circulation soon after implantation. This substance(s) is under hormonal control, being present during oestrus as well as the early stages of pregnancy; it may be an important biological regulator of EPF. Its action in the rosette inhibition test has profound implications for further study using this bioassay.     

Low consumption of seafood in early pregnancy as a risk factor for preterm delivery: prospective cohort study

Objective To determine the relation between intake of seafood in pregnancy and risk of preterm delivery and low birth weight.DesignProspective cohort study.Setting Aarhus, Denmark.Participants8729 pregnant women.Results The occurrence of preterm delivery differed significantly across four groups of seafood intake, falling progressively from 7.1% in the group never consuming fish to 1.9% in the group consuming fish as a hot meal and an open sandwich with fish at least once a week. Adjusted odds for preterm delivery were increased by a factor of 3.6 (95% confidence interval 1.2 to 11.2) in the zero consumption group compared with the highest consumption group. Analyses based on quantified intakes indicated that the working range of the dose-response relation is mainly from zero intake up to a daily intake of 15 g fish or 0.15 g n-3 fatty acids. Estimates of risk for low birth weight were similar to those for preterm delivery.Conclusions Low consumption of fish was a strong risk factor for preterm delivery and low birth weight. In women with zero or low intake of fish, small amounts of n-3 fatty acids—provided as fish or fish oil—may confer protection against preterm delivery and low birth weight.

What is already known on this topic

Long chain n-3 fatty acids in amounts above 2 g a day may delay spontaneous delivery and prevent recurrence of preterm deliveryLarge studies have not been carried out to determine to what extent low consumption of n-3 fatty acids is a risk factor for preterm deliveryThe dose-response relation has not been described

What this study adds

Low consumption of fish seems to be a strong risk factor for preterm delivery and low birth weight in Danish womenThis relation is strongest below an estimated daily intake of 0.15 g long chain n-3 fatty acids or 15 g fish     

Protein restriction in early pregnancy alters fetal and placental growth and allantoic fluid proteins in swine

This study was designed to determine the impact of protein malnutrition during early pregnancy on fetal and placental growth and on the protein synthesis capacity of placental and endometrial tissues. Twelve crossbred sows received 1.8 kg/d of a control (13% protein) or protein-restricted (0.5% protein) diet from the day of breeding to Day 63 of pregnancy, when dissections were performed on each conceptus unit. The de novo protein synthetic rate of placental and endometrial explants was measured using (35)S-methionine. These proteins and the proteins from amniotic and allantoic fluids were separated by polyacrylamide gel electrophoresis. Placental weight was significantly reduced in the sows fed the restricted diet, with a tendency for decreased fetal weight as well. No differences were found due to dietary treatment in de novo protein synthesis or in the electrophoretic patterns of secreted proteins of the placenta or endometrium. The apparent quantity of 3 proteins in the allantoic fluid of the restricted diet fetuses decreased, while 1 protein increased in comparison with that of the control fetuses. These data suggest that protein malnutrition in early pregnancy decreases placental growth, thereby decreasing both fetal growth and the opportunity for compensatory growth upon nutritional rehabilitation.     

Measurement of early pregnancy factor activity for monitoring the viability of the equine embryo

The viability of embryos before flushing from donor mares (n = 5) and after transfer to recipient mares (n = 7) was monitored in mare serum by detecting early pregnancy factor (EPF) using the rosette inhibition test (RIT). The EPF activity was measured in donor mares before and after natural mating at natural estrus; after ovulation on Days 2, 5 and 8; and after embryo flushing (Day 8) on Days 8, 9, 10 and 13 after ovulation. The collected embryos were transferred immediately after flushing. The EPF activity in recipient mares were measured on the day of transfer and after embryo transfer on Days 1, 2, 3 and 5. Pregnancy was confirmed on Day 12 to 14 after embryo transfer. The mean EPF activity of donor mares was increased to the pregnant level (> an RI titer score of 10) on Day 2 after ovulation. Two days after flushing the embryos, the EPF activity of donor mares had decreased to the nonpregnant level. Among the 7 recipient mares, 3 mares were diagnosed pregnant on Day 12 after embryo transfer with ultrasound. The EPF activity of the pregnant recipient mares was increased above the minimum level observed in pregnant mares on Days 2 to 3 after transfer. However, among the nonpregnant recipient mares after embryo transfer, the EPF activity of 3 mares remained at the pregnant level only 2 to 3 d and then declined to the nonpregnant level. In one recipient mare, EPF activity did not reach the pregnant level throughout the sample collection. The results of this study indicated that equine EPF can be detected in serum of pregnant mares as early as Day 2 after ovulation. From our observation, we conclude that the measurement of EPF activity is useful for monitoring the in vivo viability of equine embryos and early detection of embryonic death.     

Production in vitro of mouse early pregnancy factor and purification to homogeneity

Early pregnancy factor (EPF) has been produced in vitro by culture of oestrous mouse oviducts and ovaries in RPMI, with the addition of prolactin and mouse embryo culture medium. The pooled harvested medium was then subjected to immunoabsorption, electrofocusing and gel filtration. A fraction was isolated with pI 6.83 and molecular weight 21 000 which was responsible for 90% of the recovered biological activity. It appeared to be homogeneous when analysed by high-performance gel-permeation chromatography. SDS treatment showed that the molecule could be split into 3 peptides of molecular weights 10 500, 7200 and 3400, the first having activity equivalent to the EPF component EPF-A from the oviduct, while the last two combined to give activity corresponding with the ovarian component EPF-B.     

Relationship between early pregnancy factor, mouse embryo-conditioned medium and platelet-activating factor.

The effects of synthetic platelet-activating factor (PAF-acether) and mouse embryo-conditioned medium (a source of embryo-derived PAF (EPAF)) on production of early pregnancy factor (EPF) were compared. Embryo-conditioned medium, itself inactive in the EPF bioassay, stimulated ovarian production of EPF in vitro but PAF-acether did not. In vivo, embryo-conditioned medium induced EPF activity in serum of oestrous female, but not in male, mice in contrast to PAF-acether, which induced activity in serum of both male and female mice. This PAF-induced activity was transitory, declining significantly by 2 h and disappearing by 3 h after injection. Activity induced by embryo-conditioned medium was first evident at 2 h after injection, serum concentrations increasing up to 6 h after injection. By discriminating between the behaviour of PAF-acether and EPAF, these studies reinforce the conclusions of other workers that the molecule produced by the embryo is not PAF. Further investigations into the mechanism of action of PAF-acether revealed that it is a potent inducer of activity in the EPF bioassay, with an absolute requirement for platelets in the spleen cell suspension used in the assay. This platelet-derived active species was bound specifically by an anti-EPF monoclonal antibody, indicating that it is EPF-like. This is consistent with parallel studies showing that platelets are not required for induction of activity by either pregnancy serum or purified EPF. These studies were applied to the PAF-induced leukotriene-like species, which had been found by others to be active in the EPF bioassay. Pregnancy serum induced the appearance of this substance from the spleen cell suspension used in the assay; thus the leukotriene-like substance may be regarded as an effector molecule in vitro or mediator of the initiating stimulus of EPF in the bioassay.     

Antibodies to early pregnancy factor retard embryonic development in mice in vivo

Previous work in this laboratory has shown that passive immunization of mice against early pregnancy factor (EPF) leads to failure to maintain pregnancy. The findings presented in this paper demonstrate that this treatment affects the development of the embryos very early in gestation. By Day 3, 54 and 25% of embryos in the 2 groups treated with anti-EPF immunoglobulin (Ig)G and IgM, respectively, had not developed to the 4-cell stage, compared with 12 and 1% in the control groups. None of the embryos in the mice treated with anti-EPF had developed beyond the 8-cell stage. A similar delay in development after treatment was observed on Day 4. The effect apparent during the early stages of cleavage is an indirect rather than a direct one, as 2-cell embryos (32-36 h post coitum), cultured in vitro in the presence of anti-EPF antibodies, developed uninterrupted to the morula and blastocyst stage. The delay in development did not appear to be caused by a disruption of the normal pattern of circulating progesterone, as progesterone concentrations on Day 4 were within the normal range for Quackenbush mice.     

Development and evaluation of a direct enzyme immunoassay for oestrone sulphate in urine as a tool for diagnosis of early pregnancy in swine

A competitive inhibition-type enzymeimmunoassay (EIA) has been developed using 3-hemisuccinate-oestrone-peroxidase as conjugate for direct measurement of the hormone in swine urine. The method has a minimum detection level at 0.3 ng ml(-1) and satisfactory specificity, recovery and reproducibility. In a field trial with a group of 387 sows (7 in oestrus, 16 non-pregnant and 364 pregnant sows at several stages post service), it was shown that the assay is potentially an accurate pregnancy test in assessing the viability of the fetoplacental unit from day 23 up to day 30 post service. The assay is well suited for routine testing, particularly as a swine early pregnancy diagnosis test since urine sampling is easier and does not disturb the animal, while in the present assay there is no restriction in the time of sampling and the sample storage conditions.     

Proteome of the early embryo-maternal dialogue in the cattle uterus

We analyzed embryo-maternal interactions in the bovine uterus on day 8 of development. Proteomic profiles were obtained by two-dimensional difference gel electrophoresis from 8 paired samples of uterine fluid (UF) from the same animal with and without embryos in the uterus. Results were contrasted with UF obtained after artificial insemination. We detected 50 differential protein spots (t test, p < 0.05). Subsequent protein characterization by nano-LC-ESI-MS/MS enabled us to identify 38 proteins, obtaining for first time the earliest evidence of involvement of the down-regulated NFkB system in cattle as a pregnancy signature pathway. Embryos enhanced the embryotrophic ability of UF and decreased uterine protein, while blood progesterone was unaltered. Twinfilin, hepatoma-derived growth factor, and synaptotagmin-binding cytoplasmic RNA interacting protein have not previously been identified in the mammalian uterus. TNFα and IL-1B were localized to embryos by immunocytochemistry, and other proteins were validated by Western blot in UF. Glycosylated-TNFα, IL-1B, insulin, lactotransferrin, nonphosphorylated-peroxiredoxin, albumin, purine nucleoside phosphorylase, HSPA5, and NFkB were down-regulated, while phosphorylated-peroxiredoxin, annexin A4, and nonglycosylated-TNFα were up-regulated. The embryonic signaling agents involved could be TNFα and IL-1B, either alone or in a collective dialogue with other proteins. Such molecules might explain the immune privilege during early bovine development.     

Ovulation and early embryogenesis in swine

Thirty gilts were used to examine if the sequence in which oocytes were released at ovulation contributed to differences in embryonic development and uterine secretions by Day 12 (Day 0 = onset of estrus). Oocytes of follicles destined to ovulate last were recovered 42 h after injecting proestrous gilts with hCG, incubated with a fluorescent stain, and returned to the donor's oviduct. These later-maturing oocytes subsequently became the lesser-developed (p less than 0.01) embryos on Day 4. In a second experiment, lesser- vs. more-developed Day 4 embryos from additional gilts were transferred to ligated uterine horns of nonpregnant gilts. Subsequently, the lesser-developed Day 4 embryos became the smaller (p less than 0.01) blastocysts within a litter on Day 12. Uterine flushings associated with lesser-developed embryos on Day 12 contained less estradiol (p less than 0.01), less total protein (p less than 0.10), and less acid phosphatase activity (p less than 0.05), but total content of calcium was not different compared to flushings that contained more-developed embryos. Analysis of uterine flushings with two-dimensional PAGE procedures indicated advanced uteroferrin-associated glycoprotein secretion from the horn that contained more-developed embryos. Results of these experiments suggested that oocytes of later-ovulating follicles were progenitors of smaller embryos, which probably stimulated uterine secretion later than more advanced littermates on Day 12.