The chromosome constitution of human preimplantation embryos fertilized in vitro

The chromosome constitution of five haploid, 178 diploid and 11 triploid embryos fertilized in vitro was determined after fixation on day 2 or day 3 of development. Karyotype analysis of 178 diploid embryos revealed abnormalities in 40 (22.5%) cases: 34 (19.1%) aneuploids, four (2.2%) mosaic embryos and two (1.1%) structural anomalies were identified. The majority of aneuploid karyotypes (28/34) involved a single chromosome but six embryos had aneuploidy of two or three chromosomes. The E group was most frequently involved in aneuploid karyotypes (10/23 hyperdiploid embryos) and trisomy 16, the most common single anomaly in diploid embryos, was detected in 2.2% (4/178) of cases. Only one case of sex chromosome monosomy was identified. An excess of female karyotypes was detected in abnormal cases (sex ratio 0.48); this ratio was significantly (P < 0.05) different from that observed in normal cases (74:64, XY:XX). The incidence of aneuploidy increased with maternal age but this did not reach statistical significance. Embryo morphology and growth rate, assessed by embryo development rating (EDR), did not distinguish between normal (mean score 7.9; mean EDR 96.1) and aneuploid (mean score 8.1; mean EDR, 92.1) embryos. Numbers of hyperploid (n = 17) and hypoploid (n = 11) embryos (non-mosaic cases involving single chromosomes) were not statistically different. The relative proportions of chromosomes involved in trisomic karyotypes showed a remarkable similarity to the pattern in spontaneous abortions. Pronuclear status was an unreliable predictor of ploidy. Small numbers of karyotyped triploid embryos revealed equal proportions of XXX, XXY and XYY embryos.     

Effect of anordrin on the development of mouse preimplantation embryos in vitro

The aim of this study was to establish the feasibility, evaluate the response rate, and assess the impact on local control and survival in locally advanced (bulky nodal) squamous cell carcinoma of the head and neck (SCCHN) patients treated with neoadjuvant chemotherapy consisting of cisplatin followed by continuous infusion of vindesine and fluorouracil with intermittent i.v. folinic acid. Eligibility criteria included histologically proven SCCHN, previously untreated locally advanced stage III-IV with measurable or evaluable disease, no distant metastases, an Eastern Cooperative Oncology Group (ECOG) performance status of less than 2, patient age of at least 18 years, and adequate bone marrow, hepatic, and renal functions. The protocol consisted of three cycles (day 1, day 21, day 42) of Cisplatin (CDDP) 100 mg/m2/day i.v. on day 1 immediately followed by 4 days (96 h) of continuous infusion of vindesine 0.8 mg/m2/day and 5-fluorouracil (5-FU) 600-700 mg/m2/day with folinic acid 150 mg/m2 i.v. every 6 h x 16 doses before locoregional treatment with radiotherapy preceded by radical surgery when appropriate. Twenty-nine patients were enrolled in this study, and 28 were evaluable for activity; an objective response rate of 55% (four complete responses, 12 partial responses) was achieved. Leukopenia and mucositis were the most frequent and severe toxicities. The addition of vindesine did not improve the activity of the CDDP-FU-folinic acid combination, but this may be partly because of the particularly poor prognosis of the present patient population, with 75% of stage IV bulky nodal disease (N2c-N3).     

Quantitative analysis of luciferase activity of viral and hybrid promoters in bovine preimplantation embryos

OBJECTIVE: The aim of the study was to determine if ACH given after NCH followed by RH could decrease the incidence of distant metastases in patients with locally advanced carcinoma of cervix uteri. MATERIAL: 56 pts (34 Ib, 18 IIb, 4 IIIb) with confirmed diagnosis of squamous cervical cancer were enrolled in this phase II trial. The methodology used was: 1) Figo clinical staging; 2) Ultrasonographic determination of tumor volume in < or > 4 cms; 3) V.B.P. scheme: cis-platinum 50 mg/m2/day 1; vincristine 1 mg/m2/day 1; bleomycin 25 mg/m2/days 1-2-3 (3 courses with 10 days interval); 4) Clinical and sonographic tumor response evaluation following U.I.C.C. response criteria; 5) Radical hysterectomy; 6) Pathological risk factor evaluation; 7) ACH with P.M.C. (cis-platinum 50 mg/m2, methotrexate 30 mg/m2, cyclophosphamide 500 mg/m2) 3 courses every 21 days; 8) Comparison and location of recurrences with a neoadjuvant group (NCH + RH + RT to whole pelvis), and with a control group treated with conventional radiotherapy were done. For statistical analysis the Chi-Square was used and D.F.S. and overall survival (O.S.) were calculated according to the Kaplan Meier and Log Rank Test. RESULTS: After a median follow-up of 75 months (range 42-108), O. S. for stage Ib was 88%, Stage IIb 78%, and 50% for IIIb. The recurrences were 12% (4/34) for Stage Ib (3 local and 1 distant); 28% for IIb (5/18) (4 pelvic and 1 distant); 50% (2/4) for IIIb (2 pelvic recurrences). When residual tumor volume was < 2 cm in the surgical specimen (n=39) there were 4 recurrences (3 pelvic and 1 distant), and 7 (6 pelvic and 1 distant) for tumors > 2 cm. (p<0.01 for pelvic recurrences). For the stage Ib with residual tumor <2 cm (n=14) there were no pelvic recurrences and only 1 distant. Comparing for Stage Ib with previous tumor volume >4 cm of the ACH Group (n=17) with a classical NCH (n=51) and control (n=51) groups, there were observed 2 (11.7%) pelvic and 1 (5,8%) distant relapses for the 1st Group, 3 (5.9%) pelvic and 3 (5.9%) distant relapses for the 2nd, and 11 (21.6%) pelvic and 5 (9.8%) distant relapses for the 3rd Group. From the comparison of locally advanced tumors (Stages IIb + IIIb) of ACH group (n=22), with a Stage IIIb surgically removed of classical NCH group (n=38) and with a control group of conventional RT (n=51), there were observed 6 (27%) pelvic and 1 (4.5%) distant relapses for the 1st Group, 4 (11%) pelvic and 7 (18.4%) distant relapses for the 2nd, and 31 (60.7%) pelvic and 5 (9.8%) distant for the 3rd one. CONCLUSION: ACH after NCH + RH could be used for stage Ib with tumor volume > 4 cm, with complete clinical response or residual tumor < 2 cm. The results of this group of tumors suggest the importance of going on phase III trials comparing NCH+RH alone vs. NCH+RH+ACH. ACH could also be used to try to obtain better control of distant metastases in Stages IIb and IIIb. In these cases radiotherapy to the whole pelvis must not be excluded.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) accelerates differentiation of murine preimplantation embryos in vitro

The effects of antioxidant-rich foods as adjuncts to a prudent diet were compared for 12 weeks in a randomized, single-blind and controlled trial in 204 (group A) and 202 (group B) patients with acute myocardial infarction. There was a significant decrease in cardiac end points in group A compared to group B (37 vs 58, p < 0.01) after 12 weeks. Within intervention group A, those 108 patients with greater adherence to the intervention program showed a greater reduction in cardiac end points (14 vs 58, p < 0.001), and a significant decrease in total mortality (6 vs 28, p < 0.001), including cardiac mortality (6 vs 25, p < 0.01) compared to group B. Underlying these beneficial effects, antioxidant-rich foods caused a significantly smaller rise in lactate dehydrogenase (LDH) cardiac enzyme in group A than in group B (427.8 vs 561.6 IU/dL), indicating that the protective influence of such a diet may be observed within 1 week. The subset of group A patients showing reduction in mortality also had a lesser rise in LDH and greater reduction in blood lipids, blood glucose and blood pressures. Antioxidant-rich foods also caused a significant decrease in blood lipids with a lower decrease in high-density lipoprotein cholesterol in group A than in group B. Assay of serum level of antioxidants and long-term follow-up may confirm our observations.     

Expression of RNA from developmentally important genes in preimplantation bovine embryos produced in TCM supplemented with BSA

Rats given unilateral medial agranular (AGm) cortex ablations show neglect for contralateral multimodal stimuli, symptoms that are reversed by 48 hr of light deprivation. To address processes that contribute to this restorative effect, both the rats' locomotion and basal ganglia c-fos expression were studied. AGm-lesioned rats showed less activity in continuous darkness than in normal (12 hr light/12 hr dark) cycles, and the reduced locomotion correlated with the extent of their subsequent behavioral recovery. The AGm ablation reduced the numbers of amphetamine-stimulated Fos-immunoreactive nuclei in the ipsilateral dorsolateral striatum, where the AGm innervation is normally densest. Light deprivation also reduced Fos in this striatal region and attenuated the lesion-induced hemispheric Fos asymmetry. A restored balance of activity between the 2 hemispheres, especially the basal ganglia, appears central to the action of light deprivation.     

Development of in vitro derived bovine embryos following pronuclear transplantation and in vitro culture

This study was designed to evaluate the survival and development of in vitro derived bovine embryos following pronuclear transplantation and in vitro embryo culture. Bovine zygotes were produced by in vitro maturation and in vitro fertilization. Pronuclei were removed by micromanipulation and either transferred back to the same cell (Group 1) or into a previously enucleated zygote (Group 2) by electrofusion. Micromanipulated and non-micromanipulated (Group 3, control) zygotes were co-cultured with oviductal cells in a sealed modular chamber filled with 5% CO2, 5% O2 and 90% N2 at 39 degrees C for 7-8 days. Fusion rates were similar for Groups 1 and 2 (90.7 and 85.1%, respectively, P > 0.05). The percentage of embryos that cleaved was not different for Groups 1 (82.0%), 2 (90.0%) and 3 (76.9%, P > 0.05). Also, the percentage of embryos developing to the compact morula or blastocyst stage was similar (25.6, 22.5 and 22.3%, respectively, for Groups 1, 2 and 3, P > 0.05). The results of this experiment are the first to demonstrate that pronuclear transfer can be carried out successfully using bovine embryos derived from in vitro oocyte maturation and in vitro fertilization. In addition, pronuclei can be transferred from one bovine embryo to another and the reconstructed embryos develop to the compact morula and blastocyst stage in vitro. This technique, used in combination with oocyte retrieval by ultrasound-guided follicular aspiration and embryo transfer, offers the potential to study cytoplasmic inheritance in cattle directly, and to evaluate the effect of cytoplasmic inheritance on traits of economic importance.     

Normal development and metabolic activity of preimplantation embryos in vitro from patients with polycystic ovaries

Polycystic ovary syndrome (PCOS) is closely associated with high miscarriage rates and, following in-vitro fertilization (IVF), with decreased fertilization rates, suggesting that oocytes and embryos are of poor quality. In this prospective study, we examined the development, metabolic activity and blastocyst cell number of embryos following IVF from 51 patients with either anovulatory PCOS, ovulatory PCOS or tubal disease. The number of oocytes retrieved and the fertilization rates were similar for patients with PCOS and tubal disease. Following embryo transfer, 46% of the patients with PCOS and 36% of patients with tubal disease became pregnant. A similar proportion of surplus embryos from patients with PCOS and tubal disease developed to the blastocyst stage (38% and 43% respectively). Patients with anovulatory PCOS had embryos with less fragmentation which cleaved faster, cavitated earlier and had more cells at the blastocyst stage than embryos from patients with tubal disease. While the profile of glucose uptake and lactate production was similar for all groups throughout preimplantation development, patients with tubal disease who underwent ovulation induction using the 'titrated' regimen optimized for PCOS patients resulted in embryos with reduced pyruvate uptake, in addition to low blastocyst cell numbers. This study demonstrates that with an optimized ovulation induction regimen, embryos from PCOS patients are of good quality and developmental potential.     

Evidence for the requirement of autocrine growth factors for development of mouse preimplantation embryos in vitro

The mitotic stimuli in the early mammalian embryo have not been unequivocally identified. One hypothesis is that the embryo releases autocrine growth factors (GFs) that have a role in such growth. To determine whether such putative GFs were limited by dilution, and hence secreted, development was observed at various embryo concentrations in culture. Embryos were collected at the zygote or 2-cell stage. Zygotes were produced by fertilization in situ (ISF) or in vitro (IVF). Two-cell-stage embryos had a high rate of development to the blastocyst stage across an embryo concentration range of 1/microl-0.001/microl. By contrast, zygotes produced by either ISF or IVF were adversely affected by reducing the embryo concentration over this range (p < 0.001), with approximately 80% of ISF zygotes developing to blastocysts at the highest concentration but only 26% at the lowest. For IVF zygotes the corresponding results were 64% and 6%. For all three embryo types, the number of cells in each blastocyst was significantly lower with reduced embryo concentration. The major determinant of zygote development was the concentration of embryos in culture rather than the absolute volume of culture medium or the actual number of embryos present. A concentration of 1 embryo/microl (in the form of 10 embryos/10microl) gave the best development rates and highest cell numbers per blastocyst. Varying the albumin concentration influenced development rates; a 10-fold reduction in BSA concentration (to 0.3 mg/ml) resulted in significantly more IVF zygotes developing to the blastocyst stage. Platelet-activating factor (PAF) is released by embryos, and albumin can act as a competitive inhibitor of PAF's action on cells. ISF embryos released more PAF (p < 0.05) into media than did similarly treated IVF embryos. There was no difference in the amount of PAF remaining associated with the resulting 2-cell embryos. The amount of PAF released by both these groups was markedly less (p < 0.001) than the amount released by 2-cell embryos collected fresh from the reproductive tract and cultured for 24 h. PAF supplementation of media caused a significant increase in the rate of blastocyst development of IVF zygotes at embryo concentrations of 0.1/microl (1 ng/ml) and 0.01/microl (100 ng/ml). Insulin-like growth factor (IGF)-I (30 ng/ml) and IGF-II (1 ng/ml) also stimulated development of IVF zygotes when cultured at an embryo concentration of 1/10 microl. Epidermal growth factor was without effect over the range 0.2-2000 ng/ml. Supplementation of media with both PAF and IGF-II gave no additional benefit over that caused by IGF-II alone, but this treatment was marginally better (p < 0.05) than PAF treatment alone. The results show that factors necessary for normal embryo development are diluted to suboptimal levels during culture at low embryo concentration. The ability of PAF, IGF-I, and IGF-II to partially compensate for the adverse effects of low embryo concentration during culture is consistent with their having roles as autocrine embryotrophic factors. The use of IVF and low embryo concentrations in culture may provide a functional multiple ablation model that will help to define the range of GFs required for normal embryo development.     

Polypeptide profiles of human oocytes and preimplantation embryos

The polypeptides that direct fertilization and early development until activation of the embryonic genome occurs, at the 4-8 cell stage in the human, are exclusively maternal in origin, and are either synthesized during oogenesis or translated later from maternal mRNA. Using sodium dodecyl sulphate-polyacrylamide gel electrophoresis and silver stain, we have visualized and compared the polypeptides present in different populations of human oocytes and cleavage stage embryos obtained after superovulation and insemination in vitro. Two polypeptide patterns were resolved, differing in the region of mol. wt 69 kDa. The distribution of these patterns showed no correlation with the ability of individual oocytes to achieve fertilization and develop normally to the 8-cell stage.     

Three year results of in vitro production of bovine embryos in serum-poor bovine oviduct conditioned medium. An overview

This paper presents a synthesis of 3 year results of in vitro production of bovine embryos in medium previously conditioned by bovine oviduct epithelial cells. In Louvain-la-Neuve, Belgium, a total of 18356 oocytes were matured and inseminated in vitro: 13967 (76%) had cleaved at 3 days post-insemination and 3593 (26%) became blastocysts using this culture system. Our data show that conditioned medium can be stored frozen for up to 3 years without significant loss of activity and is resistant to lyophilization. One single batch of conditioned medium was tested within the same period in four different laboratories and yielded variable results: 27 and 37% blastocysts/cleaved embryos in two of them and only 7 and 0% in the two others whereas in each case more than 30% blastocysts were obtained with the local reference co-culture system. In one laboratory, the batch of oil used to overlay the culture drops had a detrimental effect on the blastocyst rate in conditioned medium but not in co-culture.     

Effects of ammonium dinitramide on preimplantation embryos in Sprague-Dawley rats

Ammonium dinitramide (ADN) is a class 1.1 oxidizer that may be used in rocket propellants and explosives. Previous studies have shown that ADN is a female reproductive toxicant, causing implantation failure in Sprague-Dawley rats when it is administered during the preimplantation period of gestation. The purpose of this follow-up study was to identify the mechanism(s) associated with implantation failure following exposure to ADN. Mated female rats were treated with 2.0 grams per liter (g l-1) ADN in their drinking water for 24, 48, 72, or 96 h before preimplantation embryos were harvested from the oviducts or uterine horns. On gestation day 1 (GD-1), comparable numbers of morphologically normal two-cell embryos were harvested from the oviducts of the treatment and control groups. On GD-2, the development of the embryos harvested from the treated animals was either slowed or halted when compared to the control embryos. By GD-4, 98% of the embryos harvested from the control group had developed to the morula or blastocyst stage; these were collected from the uterine horns. On GD-4 in the treated group, 41% of the harvested embryos remained at the two- to six-cell stage and 59% were degenerate; 82% of these embryos were collected from the oviducts. These data suggest that the implantation failure seen in animals treated with ADN is due to embryolethality.     

Mitochondrial genotype segregation during preimplantation development in mouse heteroplasmic embryos

The CXCR4 chemokine receptor has been shown to respond to the C-X-C chemokine stromal-derived factor (SDF-1) and has recently been shown to be an important coreceptor for HIV-1 infection. In the present paper we have tested a number of human lymphocyte cell lines, including Jurkat, HUT78, CEM, and Sup-T1 for the presence of CXCR4 receptors. We found that these T cell lines bind SDF-1alpha and SDF-1beta with high affinity. The CXCR4 Ab 12G5 inhibited both SDF-1 binding and HIV-1LAI-mediated fusion of CEM. Scatchard analysis revealed the presence of approximately 150,000 SDF-1alpha-binding sites per cell with a Kd between 5 and 10 nM. Cross-competition experiments using unlabeled SDF-1alpha and SDF-1beta revealed that both chemokines are equally capable of displacing their radiolabeled counterparts. Internalization studies with [125]I-SDF-1alpha revealed that Jurkat cells internalized greater than 90% of the ligand by 2 h at 37 degrees C. SDF-1alpha was also chemotactic for Jurkat cells and caused an increase in the rate of extracellular acidification that was half-maximal at 18 nM SDF-1alpha and could be inhibited by pretreatment with the SDF-1 proteins, pertussis toxin, or the Ab 12G5. Finally, SDF-1alpha also caused an increase in the cytosolic Ca2+ concentration in Sup-T1 cells that was abolished by preincubating the cells with pertussis toxin or PMA and inhibited by the Ab 12G5. This molecular characterization of CXCR4 receptors should prove useful in clarifying receptor interaction with SDF-1 proteins and with HIV-1 glycoprotein, with the ultimate aim of targeting the viral interaction for therapeutic intervention.     

Analysis of glutaminase activity and RNA expression in preimplantation mouse embryos

The efficiency of the American Optical Company (Hardy, Rand and Rittler) (HRR) plates for screening, grading and classifying red-green colour deficiency was examined for 401 male colour deficient subjects previously identified and diagnosed with Nagel anomaloscope. There were 83 protanopes, 30 protanomalous trichromats, 96 deuteranopes and 192 deuteranomalous trichromats. Screening sensitivity was found to be 100% for dichromats and 96.4% for anomalous trichromats based on one screening error (35 subjects, including 7 dichromats, where identified by a single error). Thirty subjects (13.5%) made errors on screening plates only and were identified as having minimal colour deficiency. The HRR grading system did not distinguish dichromats and anomalous trichromats; 54% of dichromats were graded as having moderate rather than severe colour deficiency. Protan/deutan classification was correct for 95% of subjects who failed grading plates. HRR grades for anomalous trichromats were compared with the anomaloscope matching range and with pass or fail of the D15 test. The results show that only two rather than four grading categories can be distinguished by the HRR plates and that both the D15 and the HRR plates are needed in a vocational test battery to establish the severity of colour deficiency.     

Synchronization of cell division in eight-cell bovine embryos produced in vitro: effects of 6-dimethylaminopurine

The effects of NS 1619, a newly developed activator of large-conductance Ca2+-activated K+ channels, were investigated on single smooth muscle fibers dissociated enzymatically from rat vas deferens and on contractions of the epididymal half of vas deferens. K+ currents were recorded using whole-cell patch-clamp methods in near-physiological K+ solutions (5.4 mM extracellular K+/145 mM intracellular K+). When cell membrane voltage was stepped to test potentials (-60 to +60 mV) from a holding potential of -10 mV, NS 1619 increased the outwardly rectifying K+ current in a concentration-dependent manner. The increased portion of the K+ current by NS 1619 was totally abolished by charybdotoxin (100 nM) but not by glibenclamide (3 microM). NS 1619 reduced electrically stimulated contractile responses of rat vas deferens in a concentration-dependent manner, and charybdotoxin but not glibenclamide partially inhibited the effect of NS 1619. NS 1619 (50 microM) inhibited the noradrenaline-induced contraction. Charybdotoxin (100 nM) partially reduced the NS 1619-induced inhibition while glibenclamide (3 microM) had no effect. NS 1619 (10-100 microM) reduced the high K+-induced contractions in a noncompetitive manner. The present results indicate that NS 1619 activates charybdotoxin-sensitive Ca2+-activated K+ channels and probably inhibits Ca2+ influx. These two effects might account largely for the observed mechanical inhibition induced by NS 1619 in the epididymal half of isolated rat vas deferens.     

The expression of beta-glucuronidase during preimplantation development of mouse embryos

The globin mRNAs containing between 30 and 40 polyadenylate residues can be separated from thos mRNAs containing longer poly(A) regions by Millipore filter binding. The molecular weights of the alpha-and beta-globin mRNAs containing this size class of poly(A) have beed determined by lectrophoresis on 3.7% polyacrylamide gels in the presence of 99% formamide. Because the number of adenylic acid residues in these mRNAs is known, the number of non-poly(A) nucleotides can be accurately calculated. The molecular weight of the beta-globin mRNA is 235 000 +/- 28 000 (736 +/- 88 nucleotides) and that of the alpha-globin mRNA is 208 900 +/- 43 870 (653 +/- 78 nucleotides). By subtracting the number of nucleotides in the coding and poly(A) regions, the number of non-coding nucleotides in the beta-globin mRNA were calculated to be 261, 69 more than the 193 present in the alpha-globin mRNA. Comparison of size estimates of newly synthesized globin mRNAs containing longer average lengths of poly(A) shhowed that there is no comparable processin of the 5' termini of the alpha-and beta-globin mRNAs concomitant with the stepwise degradation of the poly(A) regions which occur as the mRNAs mature.     

Role of intracellular calcium in peroxide-induced potentiation of norepinephrine release from bovine isolated irides

PURPOSE: To assess the safety, efficacy, endothelial changes, and risks of pulmonary embolic events after the use of a new thrombolytic brush catheter in mature thrombosed polytetrafluoroethylene (PTFE) dialysis grafts in an animal model. MATERIALS AND METHODS: Loop configuration PTFE grafts were implanted in the femoral vessels of 12 canines 4 weeks before mechanical thrombosis was performed. The thrombus was allowed to consolidate for 24 hours in 10 animals, 72 hours in one animal, and 7 days in one animal. Standard percutaneous criss-cross catheter access was performed, and a soft, low-speed, brush (6 mm in diameter), aided by 250,000 U of periprocedural urokinase, was utilized for thrombolysis. The native vessels, just distal to the anastomosis, and lungs were evaluated macro- and microscopically. RESULTS: Thrombolysis was complete in all grafts with the exception of a small segment between the crossing of the access vascular sheaths. The total thrombolysis time ranged from 8 to 12 minutes; this included 5 minutes of pulse-spray lacing. No difference in thrombolysis time was found with regard to the age or amount of thrombus. Minimal endothelial changes were noted and no evidence of acute pulmonary embolus was found on necropsy or histologic studies. CONCLUSION: This method offers a simple, safe, and efficient means of recanalization of thrombosed PTFE dialysis grafts in this canine model.