Embryonic platelet-activating factor: an indicator of embryo viability.

BACKGROUND: A definitive need exists to identify a biomarker of embryonic viability. Platelet-activating factor (PAF) production by human embryos is related to pregnancy potential. METHODS: Conditioned embryo culture media were obtained following conventional IVF on day 3, with PAF levels and pregnancy outcomes correlated. RESULTS: Overall pregnancy rate was 68% (17/25) with a mean of 84.1 (+/- 8.5) pmol/l/embryo PAF level. PAF levels ranged from a 216.4 pmol/l/embryo (pregnant) to a 3.7 pmol/l/embryo (not pregnant). There was a significant difference (P < 0.05) in PAF content between pregnant (92.1 +/- 9.5 pmol/l/embryo) and non-pregnant groups (52.5 +/- 16.6 pmol/l/embryo). Patients were categorized into three groups based upon PAF levels: low (< or= 5 pmol/l/embryo); medium (51-100 pmol/l/embryo) and high (>100 pmol/l/embryo). The low (60%) group had a significantly (P < 0.05) lower pregnancy rate than either the medium (85%) or high (89%) groups. A receiver-operator characteristic curve predicted a cut-off limit of 45 pmol/l/embryo for PAF content in human embryo conditioned culture media. CONCLUSIONS: The data demonstrate a correlation between PAF levels in human embryo conditioned culture media and pregnancy outcome. Additionally, as embryonic PAF levels increase so does the corresponding pregnancy rate. Therefore, PAF may be used as an indicator of embryo viability and for predicting pregnancy outcome.

Interaction of ceramides with phosphatidylcholine, sphingomyelin and sphingomyelin/cholesterol bilayers.

Ceramides (Cers) may exert their biological activity through changes in membrane structure and organization. To understand this mechanism, the effect of Cer on the biophysical properties of phosphatidylcholine, sphingomyelin (SM) and SM/cholesterol bilayers was determined using fluorescence probe techniques. The Cers were bovine brain Cer and synthetic Cers that contained a single acyl chain species. The phospholipids were 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dipalmitoyl-sn-glyero-3-phosphocholine (DPPC) and bovine brain, egg yolk and bovine erythrocyte SM. The addition of Cer to POPC and DPPC bilayers that were in the liquid-crystalline phase resulted in a linear increase in acyl chain order and decrease in membrane polarity. The addition of Cer to DPPC and SM bilayers also resulted in a linear increase in the gel to liquid-crystalline phase transition temperature (T(M)). The magnitude of the change was dependent upon Cer lipid composition and was much higher in SM bilayers than DPPC bilayers. The addition of 33 mol% cholesterol essentially eliminated the thermal transition of SM and SM/Cer bilayers. However, there is still a linear increase in acyl chain order induced by the addition of Cer. The results are interpreted as the formation of DPPC/Cer and SM/Cer lipid complexes. SM/Cer lipid complexes have higher T(M)s than the corresponding SM because the addition of Cer reduces the repulsion between the bulky headgroup and allows closer packing of the acyl chains. The biophysical properties of a SM/Cer-rich bilayer are dependent upon the amount of cholesterol present. In a cholesterol-poor membrane, a sphingomyelinase could catalyze the isothermal conversion of a liquid-crystalline SM bilayer to a gel phase SM/Cer complex at physiological temperature.

Interfacial properties of phosphatidylcholine bilayers containing vitamin E derivatives.

alpha-Tocopherol and alpha-tocopheryl succinate are biologically active lipids. The activity of these lipids may be related to how they affect membrane physical-chemical properties. Utilizing fluorescence methods, we have investigated the effect of alpha-tocopherol, alpha-tocopheryl succinate, and alpha-tocopheryl acetate on the properties of model membranes consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine. In liquid-crystalline phase phospholipid bilayers, alpha-tocopherol decreased acyl chain mobility and decreased the interfacial polarity, but had no effect on the interfacial surface charge. In contrast, alpha-tocopheryl succinate had little effect on acyl chain motion or interfacial hydration, but increased the interfacial surface charge. alpha-Tocopheryl acetate had very little effect on any of the measurements of these bilayer properties. In a gel phase bilayer, alpha-tocopherol decreased acyl chain order, whereas alpha-tocopheryl succinate and alpha-tocopheryl acetate did not. Each alpha-tocopheryl derivative had a different effect on interfacial polarity, however, only alpha-tocopheryl succinate increased the interfacial surface charge. The acylation of alpha-tocopherol abolishes its antioxidant activity and generates molecules with different membrane physical properties. The non-polar acetate group of alpha-tocopheryl acetate locates this compound in a region of the bilayer where it has little effect on bilayer interfacial properties. The free carboxyl group of alpha-tocopheryl succinate is located in the interfacial region of the bilayer where it increases the membrane surface charge.

Differential regulation of cyclooxygenase-2 (COX-2) mRNA stability by interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) in human in vitro differentiated macrophages.

Cyclooxygenase-2 (COX-2) is a highly inducible gene in macrophages by pro-inflammatory cytokines. A major mechanism for cytokine-induced COX-2 expression is stabilization of COX-2 mRNA. In this study, we examined the induction of COX-2 expression by interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) in human primary in vitro differentiated macrophages. IL-1 beta (5 ng/mL) or TNF-alpha (1 ng/mL) induced up to an approximately 40-fold increase of COX-2 mRNA in macrophages during a 2 to 2.5-hr incubation. Run-off experiments demonstrated that cytokine stimulation had only a mild effect on the COX-2 transcription rate (approximately 10-40% increase). The translation blocker cycloheximide (CHM) (10 mg/mL) superinduced COX-2 mRNA during 2 hr of incubation and further stabilized the COX-2 mRNA (T1/2 > 4 hr). The CHM-superinduced COX-2 mRNA was subject to a rapid degradation after removal of CHM (T1/2 < 1 hr). Both IL-1 beta and TNF-alpha stabilized cytokine-induced COX-2 mRNA (T1/2 > or = 2 hr). Maximal stabilization of COX-2 mRNA after a short-term stimulation required the continued presence of IL-1 beta in the medium. Long-term treatment of TNF-alpha destabilized the induced COX-2 mRNA. Cells simultaneously treated with both IL-1 beta and TNF-alpha had a reduced induction of COX-2, IL-1 beta, and IL-6 mRNA. In transcription-arrested cells, the translation blocker puromycin affected the TNF-alpha-induced stabilization and destabilization of COX-2 mRNA, but not the IL-1 beta-induced stabilization. The studies suggest that positive and negative regulation of mRNA stability may play a major role in cytokine-mediated COX-2 induction in human macrophages. TNF-alpha may play both pro-inflammatory and protective roles during inflammation by regulation of pro-inflammatory gene transcripts.

Antiphospholipid antibodies and in vitro fertilization success: a meta-analysis.

OBJECTIVE: To evaluate whether the presence of antiphospholipid antibodies among women undergoing IVF affects the likelihood of IVF success. DESIGN: A meta-analysis of seven eligible studies on antiphospholipid antibodies and IVF outcome. MAIN OUTCOME MEASURE(S): Odds ratios (ORs) and 95% confidence intervals (CIs) of an association between the presence of antiphospholipid antibodies and both clinical pregnancy and live birth from IVF. RESULT(S): There was no significant association between antiphospholipid abnormalities and either clinical pregnancy (OR 0.99; 95% CI 0.64-1.53) or live birth (OR 1.07; 95% CI 0.66-1.75) in IVF patients. CONCLUSION(S): The measurement of antiphospholipid antibodies is not warranted in patients undergoing IVF.

Positive outcome after preimplantation diagnosis of aneuploidy in human embryos.

usromosomal abnormalities are responsible for a great deal of embryo wastage, which is reflected, at least partially, in decreased implantation and increased miscarriage in older women. To address this problem the transfer of only chromosomally normal embryos previously selected by preimplantation genetic diagnosis (PGD) has been proposed. We designed a multi-centre in-vitro fertilization (IVF) study to compare controls and a test group that underwent embryo biopsy and PGD for aneuploidy. Patients were matched retrospectively, but blindly, for average maternal age, number of previous IVF cycles, duration of stimulation, oestradiol concentrations on day +1, and average mature follicles. All these parameters were similar in test and control groups. Only embryos classified as normal for those chromosomes were transferred after PGD. The results showed that the rates of fetal heart beat (FHB)/embryo transferred between the control and test groups were similar. However, spontaneous abortions, measured as FHB aborted/FHB detected, decreased after PGD (P < 0.05), and ongoing pregnancies and delivered babies increased (P < 0.05) in the PGD group of patients. Two conclusions were obtained: (i) PGD of aneuploidy reduced embryo loss after implantation; (ii) implantation rates were not significantly improved, but the proportion of ongoing and delivered babies was increased.

Clinical application of human egg cryopreservation.

Clinical egg cryopreservation has been applied during a 4-year period with some limited success. Mostly mature and a few immature eggs were frozen slowly and thawed rapidly in 1,2-propanediol and sucrose, and subsequently inseminated by intracytoplasmic sperm injection (ICSI). Three studies were performed in which: (i) it was established that 55% of aged unfertilized mature eggs survive freezing; (ii) in 22 cycles of thawed donated eggs cryosurvival was 24% with 15 cycles reaching transfer, and five pregnancies were initiated, one of which went to term at 39 weeks with fraternal twin boys, and one remains ongoing at 37 weeks; and (iii) in five cycles, where in-vitro fertilization patients had some of their own eggs frozen/ thawed, cryosurvival of mature eggs was poor at only 2.2%, although 44% sibling germinal vesicle (GV) stage eggs survived. A normal female infant delivered at 40 weeks arose from transfer of two embryos where GV eggs underwent in-vitro maturation post-thaw and were fertilized by ICSI. Pregnancies reported here and by others indicate a burgeoning awareness of the potential benefits of egg cryopreservation, prompting cautious optimism for the future of this technology.

Effect of cholesteryl hemisuccinate on the interfacial properties of phosphatidylcholine bilayers.

Cholesteryl hemisuccinate (CHEMS) is an amphipathic lipid that can regulate cell growth. A comparison of the effects of CHEMS and cholesterol on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers was investigated using fluorescence techniques. In liquid-crystalline phase POPC bilayers, CHEMS increased the interfacial surface charge, but was less effective than cholesterol in reducing acyl chain mobility and interfacial hydration. In liquid-crystalline phase DPPC bilayers, CHEMS and cholesterol were equally effective in reducing acyl chain mobility. Similar to the POPC matrix, CHEMS increased the interfacial surface charge and cholesterol decreased the surface hydration. The different effect of cholesterol and CHEMS on acyl chain mobility may be due to a preferential interaction of cholesterol with POPC. In gel phase DPPC bilayers, CHEMS and a succinylated pyrenyl cholesterol analog exhibited different effects on membrane physical-chemical properties than cholesterol. Succinylation also increased the rate of transfer of the pyrenyl cholesterol analog between single unilamellar vesicles approximately seven fold. This process demonstrated first-order kinetics which indicated that transbilayer migration was not a rate-limiting step. The succinylation of cholesterol places a carboxyl group at the lipid-water interface and the sterol ring deeper in the bilayer. For a structural model to explain its biological properties, CHEMS should be considered a bulky fatty acid.

Interaction of alpha-tocopherol with model human high-density lipoproteins.

The effects of alpha-tocopherol on the properties of model high-density lipoproteins (HDLs), composed of human apolipoprotein A-I and dimyristoylphosphatidylcholine, were investigated by physicochemical methods. The intrinsic fluorescence of alpha-tocopherol and its effects on the polarization of fluorescence of 1,6-diphenyl-1,3,5-hexatriene, which probes the hydrocarbon region of the lipids, and 4-heptadecyl-7-hydroxycoumarin, which is a probe of lipid surfaces, suggest that alpha-tocopherol is located at the lipid-water interface. Relative to cholesterol, alpha-tocopherol in lipid surfaces is virtually inert physicochemically. Incorporation of alpha-tocopherol into HDLs induces only a modest increase in particle size, no change in the transition temperature, and little change in lipid polarity and lipid-lipid interactions. Moreover, alpha-tocopherol has only a negligible effect on the kinetic parameters of the lipophilic enzyme lecithin:cholesterol acyltransferase, which binds to phosphatidylcholine surfaces and forms cholesteryl esters. However, alpha-tocopherol has a dramatic inhibitory effect on the rate of association of apolipoprotein A-I with dimyristoylphosphatidylcholine, a process that occurs through the insertion of the protein into preformed defects in the lipid surface. It is proposed that alpha-tocopherol inhibits the rate of association of apolipoprotein A-I with dimyristoylphosphatidylcholine by inserting into defects within the lipid surface, thereby reducing the size and/or number of sites for insertion of apolipoprotein A-I.

Birth after cryopreservation of immature oocytes with subsequent in vitro maturation.

OBJECTIVE: To establish the clinical feasibility of using cryostored germinal vesicle oocytes for IVF and ET. DESIGN: Case report. SETTING: Private infertility clinic. PATIENT(S): A 28-year-old woman with tubal infertility undergoing IVF therapy. INTERVENTION(S): Oocytes collected after ovarian stimulation were frozen without insemination or were inseminated, fertilized, and frozen as cleavage stage embryos. No fresh oocyte or embryo transfer was undertaken. All oocytes were thawed, and those that survived were used for IVF-ET. MAIN OUTCOME MEASURE(S): Oocyte cryosurvival, in vitro maturation, fertilization, embryo development, and pregnancy outcome. RESULT(S): None of 16 mature oocytes survived thawing; however, three of 13 germinal vesicle oocytes survived. After 30 hours in vitro maturation two oocytes had matured and underwent intracytoplasmic sperm injection with the partner's sperm. Both fertilized normally and were transferred to the patient. The woman delivered an apparently healthy female infant at 40 weeks. CONCLUSION(S): This case report proves the feasibility if not the efficiency of using immature oocytes for cryostorage, coupling both cryopreservation and in vitro maturation.

Fertilization potential and qualitative characteristics of human spermatozoa after short-term cryostorage at 5 degrees C in two different TEST-yolk buffer preparations.

Use of the media TEST-yolk buffer (TYB) in semenology today enables the short-term incubation and cryostorage of spermatozoa and its subsequent use in the various assisted reproductive technologies (ART). Preparation of TYB media involves the addition of egg yolk (20% v/v) to a physiological solution of the zwitterion buffers TES and Tris. The TYB is usually thermoprecipitated to remove the majority of the egg yolk globules and other macromolecules from the medium. However, removal of these egg yolk constituents could possibly eliminate or reduce essential factors that could enhance the sperm viability and fertilization potential after short-term dilution and storage. Improvements in the quality of the TYB could add greater benefits to those techniques employed in the various forms of ART. The objectives of the investigation were 1) to study the sperm qualitative characteristics following short-term cryostorage at 5 degrees C in either thermoprecipitated (T-TYB) or non-thermoprecipitated (NT-TYB), and 2) to compare the fertilizing potential of spermatozoa stored for 24 hours at 5 degrees C in the two TYB preparations. In Experiment 1, semen specimens from 15 patients were collected, assessed and split into two aliquots. Sperm specimens were processed by diluting 1:1 (v/v) with the T-TYB or NT-TYB, followed by centrifugation and reconstitution of the specimen to its initial volume with the corresponding TYB medium. Sperm specimens were cryostored for 1, 2, 24, 48 and 72 hours. Samples were taken at each interval and placed in a 37 degrees C water bath and allowed to warm for 15 minutes after each cryostorage interval. Semen specimens were assessed for percentage and grade of motility. The results of this study indicated that, although the NT-TYB yielded better results than the T-TYB, overall those differences were not statistically significant. In Experiment 2, the fertilization potential of spermatozoa recovered after 24 hours of cryostorage in the two TYB preparations and further prepared via filtration, was assessed by the sperm penetration assay (SPA) using zona-free hamster oocytes. The average penetration rate (PR) and penetration index (PI) were significantly better for the NT-TYB than for the T-TYB. The PR was 54% vs. 25%, and the PI 0.78 and 0.27 for spermatozoa incubated in the NT-TYB vs. T-TYB. The range of penetration was also much lower for the T-TYB (6 to 100%) preparation when compared to the NT-TYB (22 to 100%). The highest penetrator showed 100% for both preparations. However, the lowest penetrator showed 6% for the T-TYB and 22% for the NT-TYB. The data obtained in this study suggest that both TYB preparations can be employed in short-term cryostorage (5 degrees C) of human spermatozoa and can adequately maintain the qualitative characteristics of those spermatozoa. The data also showed that the NT-TYB preparation yielded sperm samples of higher fertilization potential, thus possibly establishing the superior usefulness of the NT-TYB in an ART program.

Simultaneous detection of chromosomes X, Y, 13, 18, and 21 by fluorescence in situ hybridization in blastomeres obtained from preimplantation embryos.

PURPOSE: Simultaneous fluorescence in situ hybridization (FISH) was used in a preimplantation genetic diagnosis program to determine which embryos were normal for chromosomes X, Y, 13, 18, and 21. METHODS: Single blastomeres were disrupted and attached to glass slides using acetic acid and ethanol. Using a ratio mixture of chromosome enumeration DNA probes in combination with locus-specific identifier DNA probes, FISH was performed for the identification of chromosomes X, Y, 13, 18, and 21. RESULTS: Fourteen couples enrolled in IVF produced 134 embryos for biopsy. Blastomeres subjected to five-color FISH revealed that 22% of embryos were normal for chromosomes X, Y, 13, 18, and 21. In addition, 52% were abnormal and no results could be detected for 25%. Twelve couples underwent embryo transfer, two couples did not receive embryos due to lack of any normal embryos, and three couples became pregnant. CONCLUSIONS: The simultaneous detection of five-color FISH is a feasible method to detect aneuploidy in preimplantation embryos from women of advanced maternal age.

Surface properties of native human plasma lipoproteins and lipoprotein models.

Plasma lipoprotein surface properties are important but poorly understood determinants of lipoprotein catabolism. To elucidate the relation between surface properties and surface reactivity, the physical properties of surface monolayers of native lipoproteins and lipoprotein models were investigated by fluorescent probes of surface lipid fluidity, surface lateral diffusion, and interfacial polarity, and by their reactivity to Naja melanoleuca phospholipase A2 (PLA2). Native lipoproteins were human very low, low-, and subclass 3 high-density lipoproteins (VLDL, LDL, and HDL3); models were 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or its ether analog in single-bilayer vesicles, large and small microemulsions of POPC and triolein, and reassembled HDL (apolipoprotein A-I plus phospholipid). Among lipoproteins, surface lipid fluidity increased in the order HDL3 < LDL < VLDL, varying inversely with their (protein + cholesterol)/phospholipid ratios. Models resembled VLDL in fluidity. Both lateral mobility in the surface monolayer and polarity of the interfacial region were lower in native lipoproteins than in models. Among native lipoproteins and models, increased fluidity in the surface monolayer was associated with increased reactivity to PLA2. Addition of cholesterol (up to 20 mol%) to models had little effect on PLA2 activity, whereas the addition of apolipoprotein C-III stimulated it. Single-bilayer vesicles, phospholipid-triolein microemulsions, and VLDL have surface monolayers that are quantitatively similar, and distinct from those of LDL and HDL3. Surface property and enzymatic reactivity differences between lipoproteins and models were associated with differences in surface monolayer protein and cholesterol contents. Thus differences in the surface properties that regulate lipolytic reactivity are a predictable function of surface composition.

Spontaneous transfer of monoacyl amphiphiles between lipid and protein surfaces.

The kinetics of transfer of natural and fluorescent nonesterified fatty acids (NEFA) and lysolecithins (lysoPC) from phospholipid and protein surfaces were measured. The kinetics of transfer of 12-(1-pyrenyl)dodecanoic acid, from liquid crystalline and gel phase single unilamellar phospholipid vesicles, very low, low, and high density lipoproteins, human serum albumin, and rat liver fatty acid-binding protein, were first-order and characterized by similar rate constants. The halftimes (t1/2) of NEFA transfer from lipids and proteins were dependent on the acyl chain structure according to log t1/2 = -0.62n + 0.59m + 12.0, where n and m, respectively, are the numbers of carbon atoms and double bonds. The structure of the donor surface had a measurable but smaller effect on transfer rates. The kinetics of NEFA and lysoPC transfer are slow relative to the lipolytic processes that liberate them. Therefore, one would predict a transient accumulation of NEFA and lysoPC during lipolysis and an attendant modulation of many metabolic processes within living cells and within the plasma compartment of blood. These data will be useful in the refinement of current models of membrane and lipoprotein function and in the selection of fluorescent NEFA analogs for studying transport in living cells.

Enhancement of outcome from intracytoplasmic sperm injection: does co-culture or assisted hatching improve implantation rates?

In two separate prospectively randomized trials, intracytoplasmic sperm injection (ICSI) cycles were studied in a controlled manner to monitor the effects of either bovine oviductal epithelial cell co-culture (n = 119) or assisted hatching by zona drilling (n = 100). In the first study, immediately following ICSI, all eggs were placed directly either onto partial monolayers of bovine oviductal cells or into regular culture medium. Although the embryo developmental rate was apparently compromised in part by the presence of the co-culture cells, ultimately there were no significant differences in either the viable pregnancy rate (31.6% co-culture versus 29.0% control) or the embryonic implantation rate (11.4% co-culture versus 13.6% control). Assisted hatching also had no significant impact on ICSI cycle outcome in terms of either the viable pregnancy rate (30.0% assisted hatching versus 32.0% control) or the embryonic implantation rate (8.5% assisted hatching versus 13.5% control). However, in female patients aged > or = 35 years, assisted hatching appeared to convey a marginally significant benefit in terms of both the viable pregnancy rate (35.5% assisted hatching versus 11.1% control) and the embryonic implantation rate (10.3% assisted hatching versus 3.1% control). It seems that the overall improvement of ICSI cycle outcome cannot be achieved by the general application of either co-culture or assisted hatching. Nevertheless, it is possible that there remain specific patient groups that might benefit from selected use of either of these modalities.

Structure of human apolipoprotein D: locations of the intermolecular and intramolecular disulfide links.

We have determined the primary structure of human apolipoprotein D (apoD) by aligning peptides derived from digestions by cyanogen bromide, trypsin, and chymotrypsin. Our results confirm the primary structure derived from cDNA [Drayna et al. (1986) J. Biol. Chem. 261, 16535-16539]. ApoD consists of 169 amino acid residues, including 5 cysteines. Tryptic peptide analysis indicated that Cys41 and Cys16 are joined by a disulfide bridge. Using a combination of manual Edman degradations and mass spectrometric analysis on a purified cluster of chymotryptic fragments, we identified an intramolecular disulfide bridge between Cys8 and Cys114 and an intermolecular bridge between Cys116 of apoD and Cys6 of apoA-II. In addition, sites of N-glycosylation were found at Asn45 and Asn78. Because apoD contains two intramolecular disulfide linkages and has a high content of proline to disrupt alpha-helical structures, formation of the amphipathic helical regions that characterize the other soluble apolipoproteins is unlikely. We conclude that apoD binds to lipoprotein surfaces through structures other than alpha-helices, such as disulfide links.