Embryo-derived Platelet-activating Factor Research Articles

Events Leading to the Establishment of Pregnancy and Placental Formation: The Need to Fine-Tune the Nomenclature on Pregnancy and Gestation.

Today, there is strong and diversified evidence that in humans at least 50% of early embryos do not proceed beyond the pre-implantation period. This evidence comes from clinical investigations, demography, epidemiology, embryology, immunology, and molecular biology. The purpose of this article is to highlight the steps leading to the establishment of pregnancy and placenta formation. These early events document the existence of a clear distinction between embryonic losses during the first two weeks after conception and those occurring during the subsequent months. This review attempts to highlight the nature of the maternal-embryonic dialogue and the major mechanisms active during the pre-implantation period aimed at "selecting" embryos with the ability to proceed to the formation of the placenta and therefore to the completion of pregnancy. This intense molecular cross-talk between the early embryo and the endometrium starts even before the blastocyst reaches the uterine cavity, substantially initiating and conditioning the process of implantation and the formation of the placenta. Today, several factors involved in this dialogue have been identified, although the best-known and overall, the most important, still remains Chorionic Gonadotrophin, indispensable during the first 8 to 10 weeks after fertilization. In addition, there are other substances acting during the first days following fertilization, the Early Pregnancy Factor, believed to be involved in the suppression of the maternal response, thereby allowing the continued viability of the early embryo. The Pre-Implantation Factor, secreted between 2 and 4 days after fertilization. This linear peptide molecule exhibits a self-protective and antitoxic action, is present in maternal blood as early as 7 days after conception, and is absent in the presence of non-viable embryos. The Embryo-Derived Platelet-activating Factor, produced and released by embryos of all mammalian species studied seems to have a role in the ligand-mediated trophic support of the early embryo. The implantation process is also guided by signals from cells in the decidualized endometrium. Various types of cells are involved, among them epithelial, stromal, and trophoblastic, producing a number of cellular molecules, such as cytokines, chemokines, growth factors, and adhesion molecules. Immune cells are also involved, mainly uterine natural killer cells, macrophages, and T cells. In conclusion, events taking place during the first two weeks after fertilization determine whether pregnancy can proceed and therefore whether placenta's formation can proceed. These events represent the scientific basis for a clear distinction between the first two weeks following fertilization and the rest of gestation. For this reason, we propose that a new nomenclature be adopted specifically separating the two periods. In other words, the period from fertilization and birth should be named "gestation", whereas that from the completion of the process of implantation leading to the formation of the placenta, and birth should be named "pregnancy".

Ligand-activated signal transduction in the 2-cell embryo.

Platelet-activating factor (PAF) is an autocrine trophic/survival factor for the preimplantation embryo. PAF induced an increase in intracellular calcium concentration ([Ca2+]i) in the 2-cell embryo that had an absolute requirement for external calcium. L-type calcium channel blockers (diltiazem, verapamil, and nimodipine) significantly inhibited PAF-induced Ca2+ transients, but inhibitors of P/Q type (omega-agatoxin; omega-conotoxin MVIIC), N-type (omega-conotoxin GVIA), T-type (pimozide), and store-operated channels (SKF 96365 and econazole) did not block the transient. mRNA and protein for the alpha1-C subunit of L-type channels was expressed in the 2-cell embryo. The L-type calcium channel agonist (+/-) BAY K 8644 induced [Ca2+]i transients and, PAF and BAY K 8644 each caused mutual heterologous desensitization of each other's responses. Depolarization of the embryo (75 mM KCl) induced a [Ca2+]i transient that was inhibited by diltiazem and verapamil. Whole-cell patch-clamp measurements detected a voltage-gated channel (blocked by diltiazem, verapamil, and nifedipine) that was desensitized by prior responses of embryos to exogenous or embryo-derived PAF. Replacement of media Ca2+ with Mn2+ allowed Mn2+ influx to be observed directly; activation of a diltiazem-sensitive influx channel was an early response to PAF. The activation of a voltage-gated L-type calcium channel in the 2-cell embryo is required for normal signal transduction to an embryonic trophic factor.

Embryonic platelet-activating factor: temporal expression of the ligand and receptor.

Preimplantation embryos synthesize platelet-activating factor (PAF) and this embryo-derived PAF is required for development. PAF's signal transduction is receptor-mediated and PAF-receptor mRNA is present in early embryos. The study objective was to determine the relationship between PAF production and PAF-receptor mRNA expression levels in mouse preimplantation embryos. Embryo-derived PAF levels were determined by radioimmunoassay. Embryonic PAF-receptor mRNA levels were determined by semiquantitated reverse transcription-polymerase chain reaction. Embryonic-PAF increased as time progressed at a relatively constant rate (1.4-1.9 x) except between the eight- and morula-cell stages where levels increased sevenfold. Embryonic PAF-receptor expression was highest at the two-cell stage and decreased steadily until the morula stage before increasing again. Regression analysis of embryo-derived PAF on PAF-receptor expression does not demonstrate a significant relationship. PAF-receptor expression (mRNA) levels decrease, while embryo-derived PAF levels decrease, as the preimplantation embryo develops. Embryonic-PAF and PAF-receptor mRNA expression do not appear related. Therefore, embryonic-PAF does not appear to regulate expression of its own receptor in vitro. The data provide a clue to the complicated cell signaling system involving PAF production and receptor expression that may help our understanding of developmental events.

PAF receptor and PAF acetylhydrolase expression in the endosalpinx of the human Fallopian tube: possible role of embryo-derived PAF in the control of embryo transport to the uterus.

Prostaglandin-E(2) and platelet-activating factor (PAF) are embryonic-derived signals that time embryo passage into the uterus in the mare and hamster respectively. PAF-like activity is detectable in the spent media of preimplantation human embryos and it has been suggested that PAF may be the embryonic signal that controls embryo transport to the uterus in our species. The actions of PAF are regulated at the level of its synthesis and degradation as well as the expression of a specific cell surface receptor (PAFr). The enzyme PAF acetylhydrolase (PAF-AH) degrades PAF. This study was undertaken to examine whether or not PAFr and PAF-AH are expressed in the human Fallopian tube and to identify the cell types in which they are expressed. The presence of PAFr mRNA in tissue extracts was investigated using reverse transcription-polymerase chain reaction. We amplified the predicted amplicon for PAFr mRNA from RNA samples extracted from Fallopian tubes. The expression of PAF-AH was detected by Western blot and the localization of PAFr and PAF-AH proteins was detected by immunohistochemistry. Utilizing antibodies against PAFr and PAF-AH, co-localization of the two proteins in the epithelium and stromal cells were demonstrated. These observations show that the human Fallopian tube expresses PAFr and PAF-AH at a location compatible with the proposed paracrine role of early embryo-derived PAF.

Exogenous platelet-activating factor stimulates cell proliferation in mouse pre-implantation embryos prior to the fourth cell cycle and shows isoform-specific stimulatory effects.

Mouse embryos secrete molecules homologous to platelet-activating factor (PAF), termed embryo-derived PAF (EPAF), which act in an autocrine/paracrine fashion to stimulate embryonic development in vitro. Mouse EPAF is thought to consist predominantly of hexadecyl (C16) and octadecyl (C18) PAF-like components. Mouse pre-implantation embryos cultured with exogenous PAF from the early cleavage stages exhibit increased blastocyst cell numbers and rates of mitosis around the 8-cell stage. We investigated whether exogenous PAF could specifically stimulate embryonic cell proliferation prior to the blastocyst stage in the mouse and also compared the biological activities of the C16 and C18 PAF isoforms as follows. Embryos were cultured for either 24 h or 120 h from the 2-cell stage and their total cell numbers were determined or their development assessed in terms of their incidence of successful zona-hatching respectively. In each case, embryos were cultured in unsupplemented medium or in medium supplemented with either C16 or C18 PAF (0.5 microM). Compared with controls, culture with C16 PAF produced a significant stimulation of mean total per number per embryo and a significant increase in the incidence of successful zona-hatching, whilst culture with C18 PAF had no significant effect. We then cultured 1-cell zygotes for 48 h in unsupplemented medium or medium supplemented with either an equimolar mixture of C16 and C18 PF or with either C16 or C18 PF alone (each at 0.2 microM). Embryos were also scored for cell number at 4 h and 30 h of culture. Although no significant effect on mean cell number per embryo was seen following 4 h or 30 h of culture with a mixed C16/C18 PAF preparation, culture for 48 h with a mixed C16/C18 PAF preparation or with C16 PAF alone produced a significant increase in mean cell number per embryo compared with controls - an effect that is likely to be receptor-mediated, since culture with an equivalent concentration of C18 PAF had no significant effect compared with controls. We have demonstrated that mouse zygotes/embryos can respond in a specific manner to exogenous hexadecyl PAF in terms of increased rates of cell proliferation prior to cavitation, and must be capable of doing so at some time between the first and third, and also between the second and fourth, cell cycles. Such embryos presumably express one or more classes of functional PAF-receptor molecule during this period (i.e. as early as during the 1-, 2- or 4-cell stages). We have also demonstrated that embryonic response to exogenous PAF is significantly isoform-specific, which may reflect differences between the two isoforms either in affinity for binding to putative embryonic PAF-receptor molecules or in their ability to elicit a stimulatory response following binding. This observation calls into question the use of preparations containing a mixture of hexadecyl and octadecyl PAF isoforms, particularly in dose-response studies, in the mouse.

Characterization and Functional Significance of Calcium Transients in the 2-Cell Mouse Embryo Induced by an Autocrine Growth Factor

Growth of preimplantation embryos is influenced by autocrine trophic factors that need to act by the 2-cell stage, but their mode of action is not yet described. This report shows that late zygote and 2-cell stage mouse embryos responded to embryo-derived platelet-activating factor (PAF) with transient increases in intracellular calcium concentration ([Ca(2+)](i)). [Ca(2+)](i) transients were single global events and were specifically induced by embryo-derived PAF. They were blocked by inhibition of phospholipase C (U 73122) and an inositol trisphosphate (IP(3)) receptor antagonist (xestospongin C), indicating the release of calcium from IP(3)-sensitive intracellular stores. Transients were also inhibited by the absence of calcium from extracellular medium and partially inhibited by treatment with dihydropyridine (nifedipine, 10 micrometer), but not pimozide (an inhibitor of an embryonic T-type calcium channel). (+/-)BAY K8644 (an L-type channel agonist) induced [Ca(2+)](i) transients, yet these were completely inhibited by nifedipine (10 micrometer). The complete inhibition of BAY K8644, but only partial inhibition of PAF by nifedipine shows that L-type channels were only partly responsible for the calcium influx. Depolarization of 2-cell embryos by 50 mm K(+) did not inhibit PAF-induced calcium transients, showing that the influx channels were not voltage-dependent. Depletion of intracellular calcium stores by thapsigargin revealed the presence of store-operated channels. The interdependent requirement for IP(3)-sensitive internal calcium stores and extracellular calcium in the generation of PAF-induced transients may be explained by a requirement for capacitative calcium entry via store-operated channels. A functionally important role for the PAF-induced transients is supported by the observation that inhibition of [Ca(2+)](i) transients by a PAF-antagonist (WEB 2086) or an intracellular calcium chelator (1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetrakis-acetoxymethyl ester; BAPTA-AM) caused marked inhibition of early embryo development. Growth inhibition by BAPTA-AM was relieved by addition of exogenous PAF.

Autocrine mediators are required to act on the embryo by the 2-cell stage to promote normal development and survival of mouse preimplantation embryos in vitro.

Autocrine mediators released by the early embryo are implicated in the support of embryo development. Their mechanisms and timing of action, however, are uncertain. This study shows that their action is necessary during the 2-cell stage of development, at which time they do not act as classical growth factors, having no impact on the rate of cell-cycle progression. Rather, they act as survival factors, protecting embryos from subsequent cell death. Culture of zygotes for the first 48 h after insemination (but not the first 24 h only) at a high concentration of 1 embryo/microl before transfer to a concentration of 1 embryo/100 microl significantly improved development compared with culture at the lower concentration for 120 h. By contrast, if the initial 48 h of culture was at 1 embryo/100 microl, subsequent culture at 1 embryo/microl caused no improvement. Platelet-activating factor (PAF) supplementation (0.186 microM) of media for the 48-h period after insemination (but not 24 h only) improved the development of embryos cultured at low embryo concentrations (1 embryo/10 microl) even when transferred to PAF-free media for the subsequent 72-h culture period. The ability of PAF to mimic the effects of higher embryo density implicates embryo-derived PAF as one of the autocrine mediators stimulating early embryo development. The cell death that occurred at low embryo concentration had a mixed phenotype: some cells had lost membrane integrity whereas others had intact membranes but punctate or fragmented chromatin. It is concluded that the action of autocrine diffusible factors, including PAF, are necessary during the 2-cell stage for subsequent survival and normal development of embryos to the blastocyst stage.

The role of albumin in the release of platelet-activating factor by mouse preimplantation embryos in vitro.

Platelet-activating factor (PAF) produced by embryos remained associated with mouse four-cell embryos after culture in vitro and was also released into the medium. The release of PAF into medium required albumin as a media supplement and the amount of PAF released increased (P < 0.05) with increasing albumin concentration. There was a trend for the amount of PAF remaining associated with embryos to decrease as the extracellular albumin concentration increased. The association of released PAF with albumin was confirmed by size fractionation with size exclusion membranes and high performance gel filtration, and by affinity chromatography (Cibacron blue and anti-BSA) and native PAGE. PAF released from embryos was not degraded by serum PAF:acetylhydrolase (PAF:AH; E.C. 3.1.1.47) after exposure for 24 h to the serum in vitro, while an equivalent concentration of synthetic PAF added to identical media was readily degraded, suggesting that PAF released from the embryo was protected from PAF:AH action. However, when the medium was subjected to organic extraction by the Bligh-Dyer (methanol/chloroform) method and the resulting extract added to equivalent media, embryo-derived PAF was readily degraded by PAF:AH. Furthermore, PAF in embryo-conditioned medium (30 two-cell embryos for 24 h) could not be detected after direct assay of the culture medium by radioimmunoassay or bioassay (platelet aggregation in vitro), yet after extraction, purification and addition to medium with BSA, the embryo-derived PAF was readily detected in either assay. To determine whether the different behaviour of synthetic PAF and embryo-derived PAF resulted from differences in the nature of their binding to albumin, the location to which PAF bound was assessed by limited proteolytic digestion of albumin. Digestion with pepsin or trypsin showed that embryo-derived PAF was located exclusively between amino acids 240 and 386 (domain II) of albumin. Most synthetic PAF added to equivalent medium not exposed to embryos was not at this location, suggesting that PAF released from embryos bound to a site on albumin not generally accessible to synthetic PAF added to similar media.

Expression of platelet-activating factor receptor in the hamster oviduct: localization to the endosalpinx.

Platelet-activating factor (PAF) is a lipid mediator that has a range of biological effects on various cells and tissues. PAF-like activity has been detected in the spent media of two-cell to morula stage hamster embryos, leading to the suggestion that PAF may be the embryonic signal that hastens embryo transport to the uterus in this species. The present study was undertaken to examine whether the PAF receptor (PAFr) gene is expressed in hamster oviduct, and to identify the cell types in which the gene is expressed. DNA fragments complementary to the coding region of mRNA encoding hamster PAFr were cloned by reverse transcription-polymerase chain reaction (RT-PCR), identified by sequencing and used to prepare hamster specific cRNA probes. The presence of mRNA transcripts encoding the PAFr receptor in the oviduct was investigated by subjecting oviduct mRNA to RT-PCR. Southern blot analysis of the RT-PCR products verified the identity of the presumptive PAFr cDNAs. The cloned cDNA fragment of hamster PAFr was found to be highly conserved with respect to the receptor of other species, having 94.3% sequence similarity to the rat PAFr receptor. Hybridization histochemistry demonstrated that PAFr is expressed in the subepithelial cells and occasionally in the epithelium. In conclusion, expression of PAFr in the hamster oviduct is compatible with the proposed paracrine role of early embryo-derived PAF.

Assay of embryo-derived platelet activating factor (EDPAF) by an equine platelet aggregation assay: Preliminary data concerning its presence in bovine embryo culture media

This study was designed to establish a sensitive bioassay for bovine platelet-activating factor (PAF), to determine if the bovine embryo secretes PAF in vitro and if PAF release is correlated with the embryo's potential to establish a pregnancy. Using an equine platelet aggregation assay, lipid extracted culture media from 33 Day-7 embryos (individually cultured for 18 hours in 1 ml of Ham's F10 containing 0.4% BSA at 37°C in an air: CO 2 mixture of 95:5 prior to their transfer to recipient heifers) and from control media (n=15, Ham's F10 + 0.4% BSA incubated simultaneously without embryos) were investigated. In addition, culture media from Day-6 (n=6) and Day-1 (2-cell, n=12) bovine embryos that were cultured for 4 hours but not transferred were examined. The aggregation assay proved to be sensitive to 5 pg of PAF. The assay proved to be specific, since the PAF receptor antagonist SRI 63–441 inhibited platelet aggregation induced by culture media in dosages comparable to aggregation induced by synthetic PAF 18. From the 15 Day-7 embryos that established a pregnancy 2 contained measurable amounts of PAF in their culture media. No PAF was detected in the culture media from 13 embryos that succeeded, in the 18 embryos that failed to establish a pregnancy, or in the control media. One of 6 Day-6 embryos and 3 of 12 Day-1 (2-cell) embryos secreted detectable amounts of PAF into the culture media. Although the results indicate that some bovine embryos release PAF or a PAF-like substance in vitro, PAF measurements in the culture medium seem not to be a suitable method for the evaluation of bovine embryos prior to transfer.

Prefertilization treatment of mice with platelet activating factor affects pregnancy

Embryo-derived platelet activating factor (EPAF) is thought to be either biologically similar to platelet activating factor (PAF) or responsible for PAF liberation in vivo. We have previously shown that premating PAF treatment in the mouse renders the platelets nonresponsive to EPAF, leading to a reduced implantation rate in these animals. In this study, we have shown that females, injected with PAF before mating, show altered embryo development invivo on day 4 postfertilization. This is manifested as an interruption of compaction, a reduced cell number per embryo, and reduced embryo number per mouse. Results suggest that EPAF represents an early pregnancy signal that supports embryo development. The most likely mechanism is via platelet activation, since only those mice that showed thrombocytopenia after fertilization were found to have normal embryos on day 4 postmating.

Loss of PAF-like activity from human embryo conditioned media (ECM) following HPLC separation

Recently a platelet activating factor (PAF)-like activity has been found in embryo conditioned media (ECM) and consequentially been termed embryo-derived PAF (EPAF). Yet it remains unclear whether the embryo-released molecule is in fact PAF or a PAF precursor or inductor in vivo. In this study we shall show that ECM did not induce platelet aggregation in vitro; however, it was possible to detec PAF-activity using the sensitive splenectomized mouse bioassay (SMB). Following lipid extraction, PAF activity was diminished, and after additional HPLC separation completely lost. We propose that the active fraction of ECM is lipid in nature but that this molecule is not PAF. We would rather suggest that this molecule induces the production of PAF by other cell types in vivo.

Metabolic and developmental responses of preimplantation embryos to platelet activating factor (PAF).

Platelet activating factor (PAF) is an ether phospholipid produced by preimplantation embryos of a number of species. Production of PAF by embryos has been measured by detecting thrombocytopenia in a splenectomized mouse bioassay, platelet aggregation bioassays in vitro and a specific radioimmunoassay. Production is highly variable and is adversely affected by culture in vitro. It has, however, been correlated with morphology, development rates in vitro and the pregnancy potential of embryos following transfer. Investigations using PAF-antagonists have established an essential role for PAF in early pregnancy. Together with studies that have shown PAF to have direct effects on embryonic metabolism during culture in vitro, these observations suggest that PAF acts as an embryonic autocoid. Hence, a major site of action for embryo-derived PAF in vivo is the embryo itself. Supplementation of embryo culture media with PAF had no effect on the rate of development in vitro of 2-cell mouse embryos through to the blastocyst stage. However, PAF increased cell numbers of blastocysts cultured from the 2-cell stage and the mitotic index of embryos at both the 8-cell and blastocyst stages. Supplementation of culture media with PAF has also been shown to increase the implantation potential of both mouse and human embryos cultured in vitro. In the mouse, the effect of PAF in enhancing implantation rates was most evident when the developmental potential of untreated embryos was suboptimal. These observations suggest that the production of embryo-derived PAF is one limiting factor in maintaining the viability of embryos cultured in vitro.

Studies on murine embryo-derived platelet-activating factor (EPAF)

Studies were carried out using the splenectomized mouse bioassay (SMB) to investigate the nature of embryo-derived platelet-activating factor (EPAF) and its relationship to synthetic platelet activating factor (PAF). While both C16-PAF and embryo conditioned media (ECM) induced a significant platelet decline in the SMB at 15 min postinjection, C18-PAF induced a similar effect at 30 min postinjection. The degree of EPAF activity in ECM was not altered with increasing embryo number from 2 to 40/ml of media. In contrast, PAF (C16/C18 mixture) induced a linear increase in activity with increasing concentration, leading to lethal effects at high concentrations. While EPAF activity was not significantly altered when ECM was diluted 1/1,000, PAF activity was abolished at 1/10 dilution. EPAF in ECM was not inactivated by mouse plasma; however, lipid extracted ECM, like PAF, underwent rapid inactivation in the presence of plasma. Aggregometer studies using horse platelets showed that ECM and lipid-extracted ECM were unable to induce platelet aggregation, while thin-layer chromatography (TLC) purified ECM (Rf 0.23) successfully aggregated horse platelets in vitro. Results suggested that EPAF and PAF are not homologous. EPAF might consist of PAF bound to a regulatory carrier molecule and appears to be associated with EPAF-inhibitor substance(s) in ECM.

Comparison of a radioimmunoassay and bioassay for embryo-derived platelet-activating factor

The pre-embryos of a number of species produce platelet-activating factor (PAF). This study compared the sensitivity, reproducibility and cross-reactivity of a radioimmunoassay (RIA) for PAF with a quantitative PAF bioassay and assessed the use of both assays for the measurement of murine and human embryo-derived PAF. PAF was extracted and partially purified by thin-layer chromatography (TLC) from medium in which mouse (30 x 2-cell) or individual human embryos [produced by in-vitro fertilization (IVF)] had been cultured for 24 h. Dose-response curves were generated with synthetic PAF standards over a concentration range of 0.3-30 ng/ml. The RIA was slightly more sensitive than the bioassay since the concentration which gave 50% response in the RIA was 3 ng/ml compared to 6 ng/ml in the bioassay. The RIA showed greater reproducibility than the bioassay, since at these concentrations the coefficients of variation were 1.97 and 6.77% intra-assay and 4.96 and 10.93% inter-assay, respectively. Both assays had a detection limit of 0.3 ng/ml. C-PAF, a non-metabolizable bioactive analogue of PAF, showed 5.16 and 0.85% cross-reactivity in the bioassay and RIA, respectively, while lyso-PAF and phosphatidylcholine had no effect. Following extraction and TLC, the average PAF concentration was 14.52 ng/ml (range 0.10-141.09) and 20.06 ng/ml (range 0.07-106.15) for mouse embryos, and 16.15 ng/ml (range 0.40-78.31) and 23.70 ng/ml (range 2.00-119.10) for human embryos, as measured by bioassay and RIA respectively. Therefore, both the bioassay and RIA were capable of the sensitive quantitative measurement of embryo-derived PAF.

10 A consideration of the factors which influence and control the viability and developmental potential of the preimplantation embryo

The advent of the new reproductive technologies (including in vitro fertilization) has led to a revolution in the treatment of infertility. It has not yet led to a marked improvement in our understanding of the control of development and of viability of the early embryo. It is the poor viability of embryos, with consequent implantation failure, which is the major limiting factor to successful outcomes. While much of the research in this area has concentrated on strategies of ovulation induction, experimental models have shown that the major cause of reduced embryo viability is due to the actual process of fertilization in vitro and subsequent culture of the preembryo in synthetic culture medium. It is likely that this is due to the absence of critical nutrients or trophic factors of maternal origin and work with co-culture of embryos with somatic cells suggests improved viability can be achieved. Such co-culture is not an option for routine clinical use, however. It is essential therefore to understand by detailed study of the physiology of embryonic development their requirements for optimal development. The empirical approach of comparing different formulations of culture media is unlikely to be successful because of the vast range of parameters to be tested and the large number of pregnancies required to demonstrate a significant improvement in outcome. The strategy that is most likely to be successful in the future, therefore, is the use of appropriate experimental models, such as the developing rodent embryo, to understand the essential physiological changes in the embryo during its development, the control processes in place, and the effect of the embryo's environment on the processes. This will allow the rational design of culture media which can then be rigorously tested for improved outcome. An example of successful application of this approach is the discovery of embryo-derived platelet activating factor (PAF). The production of embryo-derived PAF was first described and validated in the rodent. In the same species it was shown to have an essential role in pregnancy and to act as an autocrine mediator of embryo viability. This fundamental observation in rodents was then confirmed in humans, and recent work has shown that supplementation of culture human embryo media with PAF results in a dramatic increase in their developmental and pregnancy potential. This example should be the first of many such improvements based on a more fundamental understanding of the embryo's developmental requirements.

Embryo-derived platelet activating factor, a marker of embryo quality and viability following ovarian stimulation for in vitro fertilization

Embryo-derived platelet-activating factor (PAF) may be an important mediator of early maternal recognition of pregnancy. PAF was higher in media associated with clinical pregnancies when compared to preclinical pregnancies but not higher in pregnant vs nonpregnant groups. The production of PAF by the preimplantation embryo was not related to follicle size or embryo morphology. However, differences in PAF concentrations in the culture media were related to the age of the embryo culture medium and the developmental stage of the embryo.

Pregnancy Suppression by a Platelet Activating Factor Antagonist, ONO‐6240, in Mice

Mouse embryos produce an embryo-derived platelet activating factor (EDPAF) during their early developmental stages. The present study was aimed at investigating the physiological significance of EDPAF by using a specific PAF antagonist, ONO-6240. EDPAF-induced platelet decrease was antagonized by ONO-6240 in a dose-dependent manner, although this alone did not affect circulating platelet concentrations. ONO-6240, when administered to maternal mice, increased the proportion of mice conceiving small litter sizes, whereas it did not affect in vivo or in vitro development of embryos. EDPAF was suggested to play an important role in establishing pregnancy in mice by promoting implantation.

Measurement of human embryo-derived platelet-activating factor (PAF) using a quantitative bioassay of platelet aggregation

A quantitative bioassay was used to measure the concentration of platelet activating factor (PAF) in medium in which human embryos produced by IVF had been cultured and in various other biological fluids. Following extraction and partial purification, 121 of 228 (53%) media samples in which single human embryos were cultured for 24 h had PAF levels greater than found in corresponding control media. This was assigned as embryo-derived PAF and the corresponding embryos termed 'PAF-positive'. Medium from those PAF-positive embryos transferred to patients who achieved an ongoing pregnancy had a mean PAF concentration of 295 +/- 107 nM (mean +/- SEM, n = 55), which was significantly greater (P less than 0.03) than media of PAF-positive embryos transferred to patients who failed to become pregnant (75 +/- 27 nM, n = 66, t-test). The embryos with the faster cleavage rates tended to secrete more PAF (P less than 0.01). Although a greater proportion of culture media derived from embryos transferred to patients who achieved a pregnancy were PAF-positive (66 out of 121, 54.5%) compared with those transferred to patients who failed to achieve a pregnancy (55 out of 121, 45.4%), this was not significant (P greater than 0.05). It was observed that 13% of women who achieved a pregnancy had embryos transferred which did not produce significant amounts of PAF in vitro. This occurred in 26% of women not achieving pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)

Embryo-derived platelet activating factor: interactions with the arachidonic acid cascade and the establishment and maintenance of pregnancy

Two classes of potent lipid mediators are produced by the mouse and human pre-embryo: platelet activating factor (PAF) and prostaglandins (PGs). This paper reviews the evidence for their production by the pre-embryo and for their role in embryo development and the successful establishment of pregnancy. The biosynthesis of PAF and arachidonic acid may be linked, the synthesis of PAF resulting in the generation of arachidonic acid with its subsequent conversion to prostaglandins. Pharmacological inhibitor studies show that a major site of action of PAF is the embryo itself, acting as an embryonic autocrine growth factor, whereas PGs appear to act primarily on maternal tissues although they do modulate some aspects of early embryo metabolism. It is the maternal tissues that are the primary source of PG production in early pregnancy. Maternal PGs have a variety of functions including involvement in the proinflammatory response of early pregnancy and the control of corpus luteum function. In the ewe, pregnancy is associated with an attenuation of oxytocin-induced production of the luteolysin, prostaglandin F2 alpha (PGF2 alpha). PAF can mimic the effect of pregnancy, preventing the release of PGF2 alpha in response to exogenous oxytocin and, when administered into the uterine lumen, extending the life span of the corpus luteum. Thus, embryo-derived PAF appears to have an essential role in the establishment of pregnancy by acting as an autocrine growth factor for the embryo and by exerting a variety of effects on maternal physiology, including modulating maternal prostaglandin secretion and action.