Calyculin-A improves chromosome condensation for cytogenetic analysis of blastomeres from bovine and murine eight-cell stage embryos.

This study evaluated the serine/threonine phosphatase inhibitor calyculin-A for rapid, efficient induction of premature chromosome condensation (PCC) in blastomeres obtained from Day 3 bovine and Day 2 murine eight-cell stage embryos, and its potential for use in cytogenetic analysis. Experiment 1 tested calyculin-A duration (0, 60, 120, and 180min) to induce PCC in bovine blastomeres. More blastomeres that underwent PCC had chromosomes suitable for cytogenetic analysis if treated for 120 or 180min (P<0.005). Experiment 2 compared doses of calyculin-A (0, 10, 50, and 100nM) on bovine blastomeres; calyculin-A (50nM, 120min) induced PCC suitable for cytogenetic analysis in the greatest number of blastomeres when compared to other doses (52.5%; P<0.005). Effects of calyculin-A (50nM) on murine blastomeres at durations of 0, 60, 90, and 120min to induce PCC were tested in Experiment 3, with 90min inducing the highest frequency of condensed chromosomes suitable for cytogenetic analysis (34%; P<0.05). Finally, Experiment 4 evaluated calyculin-A treated bovine embryos under optimal conditions (50nM, 120min) for use in gender and cytogenetic analysis. Whole chromosome paint probes were successfully hybridized to chromosomes along with 4',6-diamidino-2-phenylindole dihydrochloride hydrate (DAPI) counterstaining, allowing detection of embryo gender (54% F:46% M) and ploidy of individual blastomeres within embryos (64% diploid:36% mixoploid embryos). In conclusion, we inferred that calyculin-A was useful for rapid induction of PCC, producing chromosome spreads suitable for cytogenetic analysis of blastomeres in G1 or G2/M phase of the cell cycle.

Blood lymphocyte chimerism associated with IVF and monochorionic dizygous twinning: case report.

We report on dizygotic (DZ) twins, conceived by IVF and ICSI with assisted hatching, who each had a mixture of 46,XX and 46,XY cells in blood lymphocytes. The female twin had mild genitalia abnormalities but further study revealed anatomically normal reproductive anatomy. Chromosome and fluorescence in situ hybridization studies of buccal, skin and ovarian tissue were normal, as were buccal tissue DNA studies. Fetal ultrasound and fetal membrane pathology were consistent with a monochorionic, diamniotic placenta (MCDAP). These twins thus have blood chimerism but are not chimeric in the other tissues studied. The mechanism for the chimerism could be due to either placental vascular anastamoses (after the development of the haematoblast stem cells) or due to an admixture of trophoblast cells during early blastocyst development. Such trophoblast cell admixtures would be restricted to the extraembryonic tissues so that general physical development in the fetus is normal and without somatic cell chimerism. This case in combination with others previously reported suggests that in IVF conceptions, the prevalence of blood chimerism associated with twinning, and the occurrence of DZ twinning associated with MCDAP, may be higher than previously thought.

Results on single cell PCR for Huntington's gene and WAVE product analysis for preimplantation genetic diagnosis.

Triple repeat base pair amplification is the basis for a number of prevalent genetic diseases such as Huntington's, Fragile X, Myotonic Dystrophy and others. We have chosen to investigate the use of PCR to amplify a portion of the Huntington's gene in single cells in order to develop a clinical test system for preimplantation genetic diagnosis (PGD). Amplification of CAG triple repeat sequences poses difficulties due to resistance of GC melting for amplification. Special PCR modifications are necessary to carry out the amplification of GC rich areas found in most triple base pair expansions. We have used a modified polymerase chain reaction (PCR) protocol to amplify the expanded repeat sequence of the Huntington's gene with satisfactory efficiency. Detection of the amplified expanded CAG repeats is shown to be possible using both agarose gel electrophoresis and high definition denaturing high pressure liquid (DHPLC) chromatography. The incidence of allele dropout (ADO) is documented.

Primer system for single cell detection of double mutation for Tay-Sachs disease.

PURPOSE: Nearly 100% of infantile Tay-Sachs disease is produced by two mutations occurring in the alpha chain of the lysosomal enzyme beta-N-acetylhexosaminidase (HEXA) in the Ashkenazi Jewish population. Although others have described primer systems used to amplify both sites simultaneously, few discuss the allele dropout problems inherent in this test. Our goal was to construct a more robust test enabling stronger signal generation for single cell preimplantation genetic diagnosis and to investigate the occurrence of allele dropout. METHODS: New nested primers were designed to optimize detection of both major Tay-Sachs mutations. Four hundred fifty-seven single cells, including normal cells and those carrying mutations of either the 4bp insertion exon 11 or splice-site intron 12 defects, were used to screen a new primer system. RESULTS: Based on PCR amplified product analysis, total efficiency of amplification was 85.3%, (390/457). The allele dropout rate for the 4bp insertion mutation in exon 11 and splice-site mutation in intron 12 was 4.8% and 5.8%, respectively. CONCLUSIONS: Multiple mutation detection and analysis within the Tay-Sachs disease gene (HEXA) is possible using single cells for clinical preimplantation genetic diagnosis. Alternative PCR primers and conditions offer various methods for developing systems compatible to specific program requirements.

Simultaneous single-cell detection of two mutations for cystic fibrosis.

PURPOSE: A single-cell diagnosis procedure using polymerase chain reaction (PCR) technology was developed to simultaneously detect two cystic fibrosis (CF) mutations (DF-508, W1282X). METHODS: The reported test procedures made use of specific cell lines (lymphoblasts, fibroblasts) of known CF mutation status to determine the efficiency of signal generation and prevalence of allele dropout (ADO) during amplification. RESULTS: Using cells carrying the DF-508 mutation, the PCR signal efficiency for the affected homozygous, normal homozygous, and carrier heterozygote cell populations were 91%, 81%, and 92%, respectively. The total combined PCR efficiency was 87.7% and the ADO rate was 5.7%. For W1282X carrier heterozygote cells, the PCR signal efficiency was 82.0% and the ADO rate was 8.7%. CONCLUSIONS: Methods have been developed to detect two common mutations simultaneously for CF in single-cell assays. The high signal efficiencies and low ADO rates obtained in these tests allow those embryos from couples wishing to avert the transmission of this serious genetic disease to their offspring to be screened by preimplantation genetic diagnosis.

Twelve-well culture plate for the efficient collection and culture of human oocytes and embryos.

OBJECTIVE: Our goal was to assess a 12-well oocyte collection and embryo culture plate for use in the IVF laboratory. DESIGN: This was a prospective nonrandomized study. SETTING: The setting was a university in vitro fertilization program. PATIENTS: Eighty-four consecutive infertility couples presenting for IVF were studied. MAIN OUTCOME MEASURES: The main outcome measure was pregnancy (delivered). RESULTS: A 34% delivered pregnancy rate per retrieval was attained using the 12-well collection and culture plate without the use of expensive culture media and special serum supplementation. CONCLUSIONS: The use of a 12-well plate for oocyte collection and embryo culture provides a simple, economical, efficient, and effective means of producing human embryos during in vitro fertilization. This system is capable of supporting high rates of ongoing and delivered pregnancies.

Infarct size reduction with coronary venous retroinfusion of diltiazem in the acute occlusion/reperfusion porcine heart model.

Calcium channel blockers are commonly used for the treatment of ischemic heart disease, but their effects on myocardial infarct size (IS) after reperfusion are not well known. Enflurane-anesthetized open-chest pigs subjected to 60-min left anterior descending coronary artery (LAD) occlusion followed by 3-h reperfusion were referred to one of the four experimental groups. Beginning 10 min before the onset of reperfusion, pigs in group A received diltiazem (7.5 micrograms/kg/min) retrogradely infused into the coronary vein for 30 min. In group B, the same amount of diltiazem was infused into the right atrium. A corresponding volume of saline was infused into the coronary vein in group C or into the right atrium in group D. IS expressed as a percentage of the myocardium at risk was significantly smaller (p < 0.01) in group A (33 +/- 14%; mean +/- SD) than in groups B (58 +/- 11%), C (58 +/- 11%), and D(63 +/- 10%). After reperfusion, functional recovery of the ischemic myocardium, determined by ultrasound crystals, was significantly more improved (p < 0.05) in group A as compared with other groups. The ischemic and nonischemic regional myocardial blood flow (RMBF), determined by radioactive microspheres, did not differ between four groups. Coronary venous retroinfusion of diltiazem had a myocardial protective effect in the porcine experimental model of acute coronary occlusion/reperfusion, whereas intravenous drug administration was not effective. The protective effect could not be attributed to washout of toxic metabolites from the ischemic area or to improved microcirculation. It was probably related to a pronounced accumulation of the calcium-channel blocker diltiazem in the ischemic myocardium achieved by the coronary venous route of delivery.

Effect of heat shock on function of frozen/thawed bull spermatozoa.

Deposition of spermatozoa in the reproductive tract of hyperthermic cows could conceivably result in sperm damage. Accordingly, a series of experiments tested the effects of heat shock on functional characteristics and free radical production of bull spermatozoa. Viability was reduced slightly by short-term (1 to 3 h) culture at 42 and 43 degrees C as compared with culture at 39 degrees C. There was no effect of culture at 42 degrees C on the ability of spermatozoa to undergo swim-up or of 42 degrees C on the percentage of motile spermatozoa. However, exposure to 41 degrees C for 3 h reduced percentage of motile sperm, 41 and 42 degrees C reduced sperm velocity and 43 degrees C decreased the proportion of spermatozoa undergoing swim-up. In other experiments, there was no effect of heat shock (41 or 42 degrees C for 1 to 3 h) on DNA integrity, presence of intact acrosomes, or fertilizing ability of the spermatozoa. Superoxide production by spermatozoa was higher at 42 degrees C than at 39 or 41 degrees C, but there was no detectable hydrogen peroxide production at any temperature. The antioxidant, glutathione, tended to improve the ability of spermatozoa to undergo swim-up at 39 degrees C but not at 43 degrees C. Taken together, these results suggest that heat shock of a magnitude similar to that seen in vivo (41 to 42 degrees C) has little effect on sperm functions that affect fertilizing capability.

Successful implementation of a regional in vitro fertilization program.

In vitro fertilization and other assisted reproductive technologies are becoming widely accepted modalities of treatment for infertile couples. Like other modern technologies, in vitro fertilization requires specially trained physicians and scientists who need specially dedicated equipment and space. In addition, patients having the procedure must undergo timely laboratory tests and sonographic evaluations for monitoring follicular maturation. To make in vitro fertilization available to patients in north central Florida, the Division of Reproductive Endocrinology and Infertility of the Department of Obstetrics and Gynecology at the University of Florida College of Medicine in Gainesville established a system that utilizes local resources in three distant locations. Patients in an assisted reproductive technologies cycle are monitored at satellite locations but coordinated through the University of Florida in vitro fertilization team. Oocyte retrieval, laboratory handling of gametes and embryo transfer are performed at the Shands Teaching Hospital in Gainesville. Micromanipulation for severe male factor infertility and oocyte donation are available when needed. Preimplantation diagnosis for genetic conditions is under development. The ongoing pregnancy and delivery rate is 21% per cycle initiated from July 1, 1992, to July 3, 1993, 62% above the reported national pregnancy rate for 1991. This program may be a useful model for the development of collaborative efforts in delivering highly complex medical services to large populations.

Follicular fluid Lidocaine levels during transvaginal oocyte retrieval.

Lidocaine has been shown to have adverse effects on mouse oocyte fertilization and embryo development. We have demonstrated the presence of pharmacologic levels of lidocaine in human serum and follicular fluid obtained during ultrasound guided transvaginal oocyte retrieval. The significance of this finding is unclear, as four of the eight patients studied became pregnant, including the patient with the highest follicular fluid lidocaine levels. Further evaluation of the effect of lidocaine on human embryos is warranted.

Subjective reports of female orgasmic expulsion of fluid.

Fifty-four percent of a sample of 227 women reported having experienced an orgasmic expulsion of fluid at least one time. The source of the fluid is still not certain.

Method for the preparation of active maturation promoting factor (MPF) from in vitro matured oocytes of Xenopus laevis.

A method for the large scale extraction of Maturation Promoting Factor (MPF) from in vitro matured oocytes of Xenopus laevis is described. MPF has been previously described only as a component(s) of hormone-matured cytoplasm within amphibian oocytes (or eggs) which is able to induce the reinitiation of the meiotic process from late diplotene stage until second metaphase arrest, when microinjected into diplotene arrested (fully grown) recipient oocytes. Standard biochemical methods for the extraction and purification of this factor(s) haven been unsuccessful due to its extreme instability and sensitivity to dilution. The procedure is dependent upon the inclusion of sodium fluoride (NaF) in the extraction medium with its effect presumably due to its ability to inhibit phosphorprotein phosphatases. The successful preservation of MPF activity described in this report permits further attempts to be made to isolate and characterize this, to date, elusive cytoplasmic factor, which plays a key role in the complex cellular processes involved in the hormone-dependent differentiation of an oocyte into an egg.