Cleavage stage versus blastocyst stage embryo transfer in assisted conception.

BACKGROUND: Recent advances in cell culture media have led to a shift in IVF practice from early cleavage embryo transfer to blastocyst stage transfer. The rationale for blastocyst culture is to improve both uterine and embryonic synchronicity and self selection of viable embryos thus resulting in higher implantation rates. OBJECTIVES: To determine if blastocyst stage embryo transfers (ETs) affect live birth rate and associated outcomes compared with cleavage stage ETs and to investigate what factors may influence this. SEARCH STRATEGY: Cochrane Menstrual Disorders and Subfertility Group Specialised Register of controlled trials, Cochrane Controlled Trials Register (CENTRAL) (The Cochrane Library), MEDLINE, EMBASE and Bio extracts. The last search date was January 2007. SELECTION CRITERIA: Trials were included if they were randomised and compared the effectiveness of early cleavage versus blastocyst stage transfers. DATA COLLECTION AND ANALYSIS: Of the 50 trials that were identified, 18 randomised controlled trials (RCTs) met the inclusion criteria and were reviewed. The primary outcome was rate of live birth. Secondary outcomes were rates per couple of clinical pregnancy, multiple pregnancy, high order pregnancy, miscarriage, failure to transfer embryos and cryopreservation. Quality assessment, data extraction and meta-analysis were performed following Cochrane guidelines. MAIN RESULTS: Evidence of a significant difference in live-birth rate per couple between the two treatment groups was detected in favour of blastocyst culture (9 RCTs; OR 1.35, 95% CI 1.05 to 1.74 (Day 2/3: 29.4% versus Day 5/6: 36.0%)). This was particularly for trials with good prognosis patients, equal number of embryos transferred (including single embryo transfer) and those in which the randomisation took place on Day 3. Rates of embryo freezing per couple was significantly higher in Day 2 to 3 transfers (9 RCTs; OR 0.45, 95% CI 0.36 to 0.56). Failure to transfer any embryos per couple was significantly higher in the Day 5 to 6 group (16 RCTs; OR 2.85, 95% CI 1.97 to 4.11 (Day 2/3: 2.8% versus Day 5/6: 8.9%)) but was not significantly different for good prognosis patients (9 RCTs; OR 1.50, 95% CI 0.79 to 2.84). AUTHORS' CONCLUSIONS: This review provides evidence that there is a significant difference in pregnancy and live birth rates in favour of blastocyst transfer with good prognosis patients with high numbers of eight-cell embryos on Day three being the most favoured in subgroup for whom there is no difference in cycle cancellation. There is emerging evidence to suggest that in selected patients, blastocyst culture maybe applicable for single embryo transfer.

Biosynthesis of galactogen: identification of a beta-(1----6)-D-galactosyltransferase in Helix pomatia albumen glands.

A beta-(1----6)-D-galactosyltransferase has been purified over 2000-fold by affinity chromatography on UDP-p-aminophenyl-Sepharose. The enzyme, from a pellet fraction (8000 x g) of Helix pomatia albumen gland, catalyzes transfer of D-galactose from UDP-galactose to a (1----6) linkage on acceptor H. pomatia galactogen. Three other polymers served as acceptors: beef lung galactan, Lymnaea stagnalis galactogen and arabinogalactan from larch wood. To determine the linkage specificity of the enzyme, it was incubated with UDP-D-galactose and acceptor galactogen that had been tritiated previously by treatment with galactose oxidase and [3H]KBH4. The [3H]galactogen reaction product was recovered, methylated, hydrolyzed and acetylated; tritiated derivatives were identified by mass spectroscopy of effluent fractions separated by gas chromatography. This analysis revealed that (1----6)-linked galactosyl groups had been added to the enzyme-treated acceptor galactogen. Also identified was a hydrolytic enzyme that removed terminal alpha 1,2-linked L-galactosyl residues from H. pomatia galactogen.

Fenofibrate subcellular distribution as a rationale for the intracranial delivery through biodegradable carrier.

Fenofibrate, a well-known normolipidemic drug, has been shown to exert strong anticancer effects against tumors of neuroectodermal origin including glioblastoma. Although some pharmacokinetic studies were performed in the past, data are still needed about the detailed subcellular and tissue distribution of fenofibrate (FF) and its active metabolite, fenofibric acid (FA), especially in respect to the treatment of intracranial tumors. We used high performance liquid chromatography (HPLC) to elucidate the intracellular, tissue and body fluid distribution of FF and FA after oral administration of the drug to mice bearing intracranial glioblastoma. Following the treatment, FF was quickly cleaved to FA by blood esterases and FA was detected in the blood, urine, liver, kidney, spleen and lungs. We have also detected small amounts of FA in the brains of two out of six mice, but not in the brain tumor tissue. The lack of FF and FA in the intracranial tumors prompted us to develop a new method for intracranial delivery of FF. We have prepared and tested in vitro biodegradable poly-lactic-co-glycolic acid (PLGA) polymer wafers containing FF, which could ultimately be inserted into the brain cavity following resection of the brain tumor. HPLC-based analysis demonstrated a slow and constant diffusion of FF from the wafer, and the released FF abolished clonogenic growth of glioblastoma cells. On the intracellular level, FF and FA were both present in the cytosolic fraction. Surprisingly, we also detected FF, but not FA in the cell membrane fraction. Electron paramagnetic resonance spectroscopy applied to spin-labeled phospholipid model-membranes revealed broadening of lipid phase transitions and decrease of membrane polarity induced by fenofibrate. Our results indicate that the membrane-bound FF could contribute to its exceptional anticancer potential in comparison to other lipid-lowering drugs, and advocate for intracranial delivery of FF in the combined pharmacotherapy against glioblastoma.

Thrombospondin: biosynthesis, distribution, and changes associated with wound repair in corneal endothelium.

Thrombospondin is a cell adhesion molecule which interacts via specific domains with a wide array of extracellular matrix components, including fibrinogen, fibrin, fibronectin, collagen, and heparan sulfate proteoglycan. Although this protein has been localized in several human tissues, its presence in corneal tissues had not been previously established. In the present study, we have demonstrated that cultured bovine corneal endothelial cells synthesize thrombospondin and incorporate it into their extracellular matrix. We have also shown immunofluorescently the presence and distribution of thrombospondin in these cultured cells and in the noninjured and injured corneal endothelium in situ. Ultrastructural immunoperoxidase cytochemistry revealed that thrombospondin could be displaced from the cell surface by heparin, but not by keratan sulfate. Confluent cultures of corneal endothelium synthesize and secrete the three cell adhesion proteins laminin, thrombospondin, and fibronectin in the ratios 1:8.2:51.8. Only the laminin B chains were detected in immunoprecipitates. Immunofluorescent studies of these cultured cells, using a polyclonal antiserum raised against purified thrombospondin, revealed a low level of fluorescence associated with the cell layer but a punctate fluorescent pattern at the level of the extracellular matrix. Noninjured corneal endothelium in situ also demonstrated a low level of fluorescence throughout the cell layer. However, this dramatically changed after a circular freeze injury to the tissue. By 24 h after wounding, cells surrounding the injury zone displayed a prominent fluorescence that was still observed at 48 h post-injury. In addition to its increased intracellular fluorescence, thrombospondin was also localized as migration tracks, oriented in the direction of cellular migration into the wound site. Thus, in corneal endothelium, thrombospondin appears to play a major role in injury-induced cell migration in situ along a natural basement membrane.

Matrix stimulates the proliferation of human corneal endothelial cells in culture.

PURPOSE: Extracellular matrices were tested for their ability to support the adhesion and proliferation of human corneal endothelial cells. METHODS: Human corneal endothelial cells were plated onto tissue culture dishes coated with purified fibronectin or a matrix elaborated by cultured bovine corneal endothelial cells. The presence of human cells in the cultures was confirmed by karyotyping. Cell size at increasing passage number was analyzed, and cellular response to growth factors was assessed using a 96-well microtiter plate assay. RESULTS: When tissue culture dishes were coated with fibronectin, the cells attached to the dish but grew slowly. Human corneal endothelial cells plated onto the matrices elaborated by bovine corneal endothelial cells attached to the culture dish and grew to fill the flask. At confluence, the cells had a hexagonal morphology similar to that seen in vivo. Karyotype analysis showed that the cells were of human and not bovine origin. The time required for senescence in culture was dependent on the age of the donor cornea. The bovine matrices enhanced the proliferative response of human corneal endothelial cell cultures to endothelial cell growth supplement and keratinocyte growth factor. Epidermal growth factor and hepatocyte growth factor stimulated human cell proliferation in a dose-dependent fashion, regardless of the substratum on which the cells were plated. CONCLUSIONS: The use of substratum elaborated by bovine corneal endothelial cells has proved useful in the preparation of human endothelial cell cultures from juvenile and adult donors. The method has been used to establish cultures from more than 50 donors from age 1 day to 76 years.

Inhibition of retinal angiogenesis by peptides derived from thrombospondin-1.

PURPOSE: Thrombospondin (TSP)1 is a tumor suppressor with activity that is associated with its ability to inhibit neovascularization. Previous studies have mapped this antiangiogenic activity to the type 1 repeats and the amino-terminal portion of the molecule within the procollagen-like domain. The present study was performed to investigate the ability of TSP-1 and peptides derived from the type 1 repeats to inhibit retinal angiogenesis. METHODS: TSP-1 and peptides with tryptophan-rich, heparin-binding sequences and transforming growth factor (TGF)-beta1 activation sequences were evaluated in two models of retinal angiogenesis: a retinal explant assay and a rat model of retinopathy of prematurity (ROP). RESULTS: Platelet-derived TSP-1 inhibited angiogenesis in both experimental models. Peptides from the native TSP-1 sequence, which contained both the tryptophan-rich repeat and the TGF-beta1 activation sequence, were the most potent inhibitors of endothelial cell outgrowth in the retinal explant assay. In contrast, a peptide containing only the tryptophan-rich, heparin-binding sequence was most active in inhibiting neovascular disease in the rat ROP model. CONCLUSIONS: These results indicate that the type 1 repeats of TSP-1 contain two subdomains that may independently influence the process of neovascularization, and that peptides derived from these type 1 repeats may be promising pharmacologic agents for treatment of retinal angiogenesis.

Adverse effects of cardiovascular drug therapy on the fetus and neonate.

Possible adverse effects of cardiovascular medications on the fetus and the neonate have been reviewed. The major classes discussed were diuretics, antihypertensives, antiarrhythmics, cardiac glycosides, and anticoagulants. The recommendations given must not be considered definitive because they are based on flawed or incomplete information. It is to be hoped that further investigation will improve this situation and will also improve our understanding of the ways in which these drugs act.

The effects of chronic gastrointestinal medication on the fetus and neonate.

The adverse fetal and neonatal effects of chronic gastrointestinal medication have been reviewed. The major classes discussed are antiemetics, laxatives and antidiarrheal agents, antacids and cimetidine, drugs for irritable bowel syndrome, and drugs for inflammatory bowel disease. The evaluation given are tentative due to the lack of information on most of these drugs. It is hoped that research into the use of this diverse group of medications during pregnancy will be stimulated, so that the large gaps in our knowledge about them can be filled.

Carbamazepine levels in pregnancy and lactation.

An epileptic patient whose seizures were controlled with carbamazepine and primidone was followed throughout pregnancy and lactation. Blood levels of primidone decreased during pregnancy and rose postpartum requiring dosage adjustments. Pharmacologically insignificant amounts of the drug were detected in breast milk. A review of the literature revealed 94 infants exposed to carbamazepine in utero and no evidence to date that this drug carries a teratogenic risk.

The possible deleterious effects of the intramyometrial injection of hypertonic urea.

Hypertonic urea has been shown to be an effective midtrimester abortifacient. Although safer than hypertonic saline when injected intravascularly, it has not been compared to saline in the case of inadvertent intramyometrial injection. This report documents that intramyometrial injection of hypertonic urea will result in the same type of muscle necrosis as that produced by saline.

PIP: The effects of intramyometrial injection of hypertonic urea were stu died in 4 rhesus monkeys in the second trimester of pregnancy. In all cases where there was no efflux of the hypertonic solution, muscle necrosis, histologically similar to that observed with hypertonic saline, was observed. It was also evident that small amounts of urea or saline were able to produce considerable necrosis of the endometrium. An injection procedure that obviates the possibility of myometrial puncture, along with very low levels of injection pressure, are recommended for abortion procedures of this type.

Experience with an adolescent pregnancy program. A preliminary report.

A program has been designed to give comprehensive health care services to pregnant adolescents. The program components include community liason, patient education, counseling and social services, a nurse "on call" program for labor and delivery, and pediatric nurse-practitioner followup. In addition, a drug use identification component screens the patients by interview and urinalysis. Hypertensive disorders of pregnancy occurred in 10% of the first 202 patients. Forty-three percent had anemia (hematocrit less than 35%), and screening cervical cultures for gonorrhea were positive in 3.5%. Cigarettes, alcohol, and marijuana constituted the most common nonmedical drugs used, and aspirin the most common medical drug. Eighty-one percent of the patients attended 7 or more antenatal visits, one-half did not miss a single clinic appointment, and 95% completed a post-partum visit, indicating that the program was well accepted by the adolescents.

Pharmacolinetics of fluorocarbon 11 and 12 in dogs and humans.

Blood levels and exhalation bag contents of FC-11 and FC-12 from dogs and humans were used to elucidate the pharmacokinetic model describing the time-course of these agents. The derived pharmacokinetic parameters were in good agreement with the physicochemical properties of these substances. The model was used to estimate the percentage of dose absorbed, which averaged 77 per cent for FC-11 and 55 per cent for FC-12, and to predict the level of FC-11 and FC-12 under a variety of conditions simulating both short- and long-term exposure to the maximum allowable concentrations of these agents. With similar doses, an 8-hour continuous exposure was estimated to produce levels of FC-11 and FC-12 that are much lower than the corresponding levels reported to induce cardiac sensitization in dogs.

Antibody-based sensors for heavy metal ions.

Competitive immunoassays for Cd(II), Co(II), Pb(II) and U(VI) were developed using identical reagents in two different assay formats, a competitive microwell format and an immunosensor format with the KinExA 3000. Four different monoclonal antibodies specific for complexes of EDTA-Cd(II), DTPA-Co(II), 2,9-dicarboxyl-1,10-phenanthroline-U(VI), or cyclohexyl-DTPA-Pb(II) were incubated with the appropriate soluble metal-chelate complex. In the microwell assay format, the immobilized version of the metal-chelate complex was present simultaneously in the assay mixture. In the KinExA format, the antibody was allowed to pre-equilibrate with the soluble metal-chelate complex, then the incubation mixture was rapidly passed through a microcolumn containing the immobilized metal-chelate complex. In all four assays, the KinExA format yielded an assay with 10-1000-fold greater sensitivity. The enhanced sensitivity of the KinExA format is most likely due to the differences in the affinity of the monoclonal antibodies for the soluble versus the immobilized metal-chelate complex. The KinExA 3000 instrument and the Cd(II)-specific antibody were used to construct a prototype assay that could correctly assess the concentration of cadmium spiked into a groundwater sample. Mean analytical recovery of added Cd(II) was 114.25+/-11.37%. The precision of the assay was satisfactory; coefficients of variation were 0.81-7.77% and 3.62-14.16% for within run and between run precision, respectively.

Pharmacokinetic studies on quinacrine following intrauterine administration to cynomolgus monkeys.

Recent efforts have been made to develop a chemical oviductal occluding agent. Intrauterine quinacrine has been used in certain areas of the world with moderate success in effected tubal closure. This report presents the pharmacokinetics of a quinacrine solution (30 mg) as administered to cynomolgus monkeys via the intrauterine route, compared with intravascular injection. The data show rapid transfer of the drug from the uterine to the vascular compartment and uptake by almost all tissues examined. Although plasma concentrations disappear within 24 hours, levels can be detected in most tissues for at least 1 week following intrauterine injection. After 28 days, however, tissue levels of the drug are absent or near the limit of detection.

The pharmacologic inhibition of premature labor.

Oxytocin, elevated estrogen-progesterone ratio, fetal corticosteroids, prostaglandins, catecholamines, and changes in uterine blood flow have all been implicated as triggers of labor. In approximately one-third of cases of threatened premature labor contractions stop spontaneously. Thus placebo-controlled randomized trials of any new drug for inhibition of premature labor are necessary, as the spontaneous cessation of contractions always favors the claimed therapeutic efficacy. Alcohol inhibits the release of endogenous oxytocin and has an additional direct effect on the myometrium. In one study alcohol was more effective than placebo in the postponement of delivery. Isoxsuprine, ritodrine, and terbutaline have also been shown to be better than placebo in the inhibition of premature labor, and the beta adrenergic agents appear to be more effective than alcohol. Prostaglandin inhibitors such as indomethacin are currently under investigation. Success is correlated with early administration of the therapy, which requires treating some patients whose contractions might have stopped spontaneoulsy. As different factors may be involved in triggering premature labor, if one therapeutic approach fails another should be initiated promptly.

South American plants II: taspine isolation and anti-inflammatory activity.

Croton lechleri L. (Euphorbiaceae), a plant from the Upper Amazon Valley of Peru, yielded the alkaloid taspine. The anti-inflammatory activity of taspine hydrochloride was studied using the carrageenan-induced pedal edema method, the cotton pellet-induced granuloma method, and the adjuvant polyarthritis model.

Efficacy of various locally applied chemicals as contragestational agents in rats.

PIP: Test agents were selected because of previous evidence of contragestationaal activity when administered systemically or because of known local effects which would be likely to cause endometrial changes having an adverse effect on pregnancy. A group of virgin female Sprague-Dawley rats were treated on Day 3 of pregnancy (preimplantation) and another group on Day 7 of pregnancy (postimplantation). Injections of .05 ml were made directly into the lumen of each uterine horn. Sodium chloride .9% was used on 1 side and the test agent on the other side. Implantation sites were counted before injections on Day 7. The number of corpora lutea indicated the expected number of conceptions of those injected on Day 3. On Day 15 rats were sacrificed and corpora lutea, viable conceptuses, and absorption sites were counted. Ethanol at 100, 80, 70, and 63% was a highly effective contragestational agent when given on Day 3. Formaldehyde 7-.5% was also highly effective when given on Day 3 but higher concentrations produced maternal toxicity and death. Silver nitrate, iodine, rivanol, cyclizine, urea, and 17beta-bromoacetoxy-19-nortestosterone produced no maternal toxicity but were all effective in reducing the number of viable fetuses. Prostaglandin (PGF2alpha), indomethacin, and ergonovine had no observable effect on preimplantation embryos. Methotrexate reduced survival when injected on Day 3 and more so when given on Day 7 but a systemic toxic effect was also noted. When injected on Day 7 all of the compounds except methotrexate were markedly less effective. Survival of fetuses in the control horns varied from 50% to 100%. Ethanol produced sloughing and necrosis but the endometrium appeared to be normal after 96 hours. Fecundity had not returned after 4-5 estrous cycles. The other compounds produced no histologically evident long-lasting effects. Superficial endometrial damage seemed to be the mechanism of action of compounds that were effective on Day 3. The discrepancies noted between results obtained and the documented efficiency of PGF2alpha and of urea as abortifacients in humans raises the question of the suitability of the rat as a model for predicting abortifacient activity in humans. However, the action of these 2 substances may be different in later gestational phases.^ieng

Intra-cytoplasmic sperm injection versus partial zona dissection, subzonal insemination and conventional techniques for oocyte insemination during in vitro fertilisation.

BACKGROUND: In vitro fertilisation (IVF) and embryo transfer as treatment for male factor infertility is associated with lower fertilisation and pregnancy rates than for other indications. Since the late 1980s several assisted fertilisation techniques have emerged and have been rapidly developed to try to enhance results for couples with male factor infertility, or to help couples with severe male factor for whom conventional IVF was not possible. The technique of partial zona dissection (PZD) was developed to increase the probability that a sperm capable of fertilisation comes in contact with the oocyte. Although this method improved conventional IVF results, the improvement was only marginal and relatively large numbers of sperm are still required. This drawback applied less to the subsequent technique of subzonal microinjection of spermatozoa into the perivitelline space (SUZI). However, for all of these techniques fertilisation rates remained low, rates of polyspermic fertilisation were increased, and cases with a very limited number of spermatozoa in the ejaculate could still not be treated. The advent of intra-cytoplasmatic sperm injection (ICSI) of a single sperm (or sperm head or nucleus) into the oocyte appears to be an important breakthrough. OBJECTIVES: To investigate whether ICSI improves fertilisation and/or pregnancy rates in comparison to other fertilisation techniques. SEARCH STRATEGY: The Menstrual Disorders and Subfertility Group search strategy (see Review Group details) was used to identify trials that had compared ICSI with other infertility techniques, such as PZD, SUZI, conventional IVF and additional IVF. SELECTION CRITERIA: Trials were included if they compared the effects of these techniques on fertilisation and pregnancy outcomes. Only randomised studies were included in this review. DATA COLLECTION AND ANALYSIS: Ten studies met the inclusion criteria for this review. Eight studies compared ICSI with conventional IVF. One study compared ICSI with SUZI and one study compared ICSI with additional IVF. Data was extracted independently by two reviewers. Where relevant data was missing or unclear, the authors had been consulted. Male participants were classified according to their semen parameters, i.e. normal semen (concentration >20 million per ml, motility >50%, morphology >14%), borderline semen (concentration 10-20 million per ml, motility 30-50%, morphology 4-14% normal forms) and very poor semen (concentration <10 million per ml, motility <30%, morphology <4% normal forms). MAIN RESULTS: For couples with normal semen there is no evidence of a difference in fertilisation rates per retrieved oocyte or pregnancy rates between ICSI and conventional IVF. On the other hand, for fertilisation rate per inseminated oocyte, ICSI appears to result in better outcomes than IVF for normal semen. For couples with borderline semen ICSI results in higher fertilisation rates (all) than IVF. Couples with very poor semen will have better fertilisation outcomes with ICSI than with SUZI or additional IVF. REVIEWER'S CONCLUSIONS: There is evidence from this systematic review that fertilisation rates are significantly better with ICSI than IVF in couples with borderline semen. When the semen parameters are normal there is insufficient evidence of a difference in effectiveness between ICSI and IVF when retrieved oocytes were the unit of randomisation. However, there was a small but statistically significant increase in fertilisation rate when inseminated oocytes were the unit of randomisation. Total fertilisation rates were significantly reduced in ICSI cycles than IVF but there were no damaged oocytes in IVF cycles regardless of the semen parameters.

Synthesis of N-acetyllactosamine containing a D-[6-3H]galactopyranosyl group.

A simple and convenient method for the introduction of radiolabel onto C-6' of N-acetyllactosamine is described. 1-N-Benzyl-3-O-beta-D-galactopyranosyl-D-arabinosylamine (1) was synthesized from 3-O-beta-D-galactopyranosyl-D-arabinose as described by Lee and Lee. Compound 1 was oxidized with D-galactose oxidase, and the product reduced with KB3H4 to introduce the label at C-6'. After dilution with unlabeled material, the N-benzyl-3-O-beta-D-[6-3H]galactopyranosyl-D-arabinosylamine was converted into 2-(benzylamino)-2-deoxy-4-O-D-[6-3H]galactopyranosyl-D-glucononitrile , which was subjected to simultaneous hydrogenolysis of the benzylamino and nitrile groups. N-Acetylation of the amino group as described by Alais and Veyrières afforded the crystalline title compound in 63% yield.