A morphological and chromosomal study of blastocysts developing from morphologically suboptimal human pre-embryos compared with control blastocysts

BACKGROUND: IVF laboratories performing embryo transfer at day 2 or 3 after fertilization are currently discarding pre-embryos considered suboptimal using morphological criteria. The objective of this study was to investigate whether blastocysts, cultured from such pre-embryos (surplus), were chromosomally and morphologically normal. As a control group we used morphologically good quality embryos (GQE), cultured to the blastocyst stage. METHODS: Human pre-embryos considered suboptimal were cultured to the blastocyst stage. As a control group, frozen-thawed pre-embryos of good quality were cultured under identical conditions. The chromosomal status of the blastocysts obtained was studied by multi-colour fluorescence in-situ hybridization for chromosomes 13, 16, 18, 21, 22, X and Y. RESULTS: There is, on average, a significantly higher degree of chromosomal aberrations in blastocysts derived from surplus pre-embryos compared to blastocysts derived from GQE, and the chromosomal aberrations are generally found in a higher number of blastomeres per blastocyst. In addition, blastocysts from surplus pre-embryos had significantly poorer morphology compared to GQE. Improvement in morphology and/or developmental rate in surplus pre-embryos between day 2 and day 3 did not predict a morphologically/chromosomally normal blastocyst. However, this study shows that close to half of the surplus pre-embryos that reach the blastocyst stage can be considered chromosomally normal when assessed for these seven chromosomes. Furthermore, we found that chromosomal aberrations were more concentrated in a particular cell population within blastocysts derived from GQE, compared with surplus blastocysts. CONCLUSIONS: The study suggests that even if the IVF laboratory is on average making the correct decision about the potential of a pre-embryo, surplus pre-embryos that might become chromosomally normal blastocysts are still being discarded.     

Factors influencing maternal estimates of genetic risk

The relationship between the objective and subjective estimates of genetic risk was studied in 202 women accepting and 50 women not accepting amniocentesis. All women were at risk of having a child with congenital anomalies either because of maternal age at pregnancy or family history of Down syndrome (DS) or other congenital anomalies. Only 28.6% of the women rejecting and 44.4% of the women accepting amniocentesis remembered correctly their objective odds. The correlations between the objective risk estimates and the subjective risk estimates were low overall (r = 0.089, p = 0.08); for women rejecting (r = 0.024, p = 0.44) or accepting (r = 0.082, p = 0.12) amniocentesis. The psychosocial and sociodemographic variables relating to either objective or subjective risk estimates were different for both groups of women. The study provides information on variables that should be taken into consideration in formulating a general theory to predict individual perceptions of genetic risk.     

Comparative susceptibility to mouse interferons of Rickettsia tsutsugamushi strains with different virulence in mice and of Rickettsia rickettsii.

Three strains of Rickettsia tsutsugamushi (Karp, Gilliam, and TA716, representing three virulence types in mice) were examined for their sensitivity to the inhibitory effects of recombinant gamma interferon (IFN-gamma) and purified IFN-alpha/beta in two cultured mouse fibroblast cell lines. The susceptibilities of another species, Rickettsia rickettsii, and of encephalomyocarditis virus (EMCV) were also tested for comparative purposes. IFN-gamma inhibited rickettsial replication in only one of the six combinations of R. tsutsugamushi strains and mouse cells (strain Gilliam and the BALB/c mouse-derived cell line). In contrast, R. rickettsii and EMCV replication were markedly inhibited in both cell types, but to a greater extent in the BALB/c line than in the C3H cells. IFN-alpha/beta (300 to 450 U/ml) was uniformly ineffective in three of the combinations of R. tsutsugamushi strains and mouse cells (Gilliam in C3H cells and Karp in both C3H and BALB/c cells); in the remaining sets, IFN-alpha/beta-mediated inhibition of rickettsial replication was variable and in no case was it very pronounced. The tests with R. rickettsii in both cell types also indicated slight, variable sensitivity to IFN-alpha/beta. EMCV, on the other hand, was very susceptible to IFN-alpha/beta, confirming the potency of the preparation used; as with IFN-gamma, virus replication was inhibited to a greater degree in the BALB/c cell line than in the C3H cultures. These results are discussed in terms of their relationship to the virulence properties of the R. tsutsugamushi strains in BALB/c and C3H mice and to the known IFN-sensitivities of the more widely studied Rickettsia prowazekii.     

Increased translocation of Escherichia coli and development of arthritis in vitamin A-deficient rats.

We studied the immune response and the colonization pattern in vitamin A-deficient rats that were colonized with the Escherichia coli O6 K13 pOmp 21 strain, which is genetically manipulated to produce ovalbumin and to be resistant to ampicillin. In the vitamin A-deficient rats, the number of bacteria per gram of feces was about five times higher than in the paired fed control rats 4 weeks after colonization. In the control rats, the colon and the lower part of the ileum were colonized, while in the vitamin A-deficient rats all parts of the small intestine, as well as the colon, were heavily inhabited by bacteria. Furthermore, in 75% of the vitamin A-deficient rats, the E. coli bacteria were found in the mesenteric lymph nodes, and in 50% of the rats E. coli were found in the kidneys. These animals also developed severe arthritis. The levels of serum immunoglobulin G (IgG), IgM, IgE, and biliary IgA antibodies against the bacterial antigens were significantly higher in the vitamin A-deficient rats than in the control rats. The number of IgA-producing cells in the lamina propria of the small intestine was significantly lower in the vitamin A-deficient rats than in the control rats; however, there was an increase in the number of CD8+ cells and transforming growth factor beta-producing cells in the lamina propria of the vitamin A-deficient rats. Disturbances in T-cell function were demonstrated, since spleen cells from the vitamin A-deficient rats produced more gamma interferon and interleukin-2 in vitro than control spleen cells. In summary, vitamin A deficiency led to a decrease in the ability to control the localization of intestinal bacteria and an increase in translocation, which was followed by development of arthritis regardless of substantial levels of antibacterial antibodies. The bacterial invasion made the animals hyperresponsive to the bacterial antigens, despite the fact that vitamin A deficiency is normally associated with suppressed antibody production, as previously shown by us and others.     

The secretory antibody response in milk and bile against fimbriae and LPS in rats monocolonized or immunized in the Peyer's patches with Escherichia coli.

The homing of lymphoid cells to mucosa-associated lymphoid tissue is, amongst other factors, influenced by the nature of the antigen used to induce an immune response. To study this phenomenon we have monocolonized rats with a type 1 fimbriated Escherichia coli O6K13H1 strain and compared the secretory antibody response to colonization with the primary and secondary response obtained in rats immunized in the Peyer's patches (PP). Samples were tested with respect to the titres of antibodies against two antigens present on the E. coli strain: O6 lipopolysaccharide (LPS) and type 1 fimbrial antigen. In the primary immunized animals, IgA anti-fimbrial antibodies were mainly seen in milk and IgA anti-LPS antibodies mostly found in bile. In the booster immunized, and in the monocolonized, animals there was a shift of the antibody response towards the bile. Thus anti-fimbrial antibodies appeared in milk at approximately the same or at a lower level than in bile and the IgA anti-LPS antibodies were almost completely absent in the milk. The IgG antibody response of the animals immunized in the PP was primarily confined to milk for both anti-LPS and anti-fimbrial antibodies, while the colonized animals responded with higher levels in bile than in milk. IgM antibodies were only seen in the milk, except in primary immunized animals in which biliary IgM antibodies also were found. The data illustrate that: (i) primary stimulated cells predestined to produce IgA anti-LPS antibodies home mainly to the intestine, while cells predestined to anti-fimbrial antibody production have a greater tendency to populate the mammary gland; (ii) after repeated antigen stimulation and maturation of the immune response the cells are directed from the mammary gland to the intestine. We thus conclude that the nature of the antigen and the stage of lymphocyte maturation influences the homing of the cells and the appearance of various antibodies in different secretions.     

Glycine rectifies vascular dysfunction induced by dietary protein imbalance during pregnancy

Protein restriction in rat pregnancy programmes the development of elevated systolic blood pressure and vascular dysfunction in the offspring. A recent study has shown that hypertension is reversed by maternal glycine supplementation. Whether this protective effect is exerted directly on the embryo and fetus, or indirectly via effects on the mother, is unknown although we have previously shown abnormalities in the maternal vasculature. We tested the hypothesis that dietary glycine repletion would reverse endothelial dysfunction in protein-restricted pregnant rat dams using wire myography. Impaired acetylcholine- ( P < 0.01) and isoprenaline-induced ( P < 0.05) vasodilatation in isolated mesenteric arteries (MA) from protein-restricted pregnant dams was accompanied by reduced vascular nitric oxide (NO) release ( P < 0.05). Dietary glycine supplementation reversed vascular dysfunction in MA ( P < 0.05) and improved NO release thus potentially protecting the maternal circulation. The impaired NO release in the MA of low protein diet dams was not accompanied by reduced eNOS mRNA expression, suggesting that eNOS activity was altered. Protein restriction did not alter the vascular function of a conduit artery, the thoracic aorta. These results provide evidence that adequate provision of glycine, a conditionally essential amino acid in pregnancy, may play a role in the vascular adaptations to pregnancy, protecting the fetus from abnormal programming of the cardiovascular system.     

Effect of oestradiol on the secretory immune system in the rat: an increase in biliary IgM antibodies against a T-cell independent antigen.

The effect of sex hormones on the secretory immune system was studied in rats ooforectomized and substituted with oestradiol in permeable capsules deposited subcutaneously. Ooforectomized rats and sham-operated rats without oestradiol substitution served as controls. Two weeks after the ooforectomy the rats were immunized in the Peyer's patches with Escherichia coli O6 carrying type 1 fimbriae. Some rats were given a booster dose with the same antigen at the same site 3 weeks later. Bile and serum were taken 7 days after the last immunization. The oestradiol treatment did not influence the total level of IgA or IgG or the level of specific IgA or IgG antibodies in bile or serum. Instead there was a specific increase in biliary IgM antibodies against lipopolysaccharide (LPS) as well as a rise in the total IgM concentration in the bile in the oestradiol-treated rats. Despite this there was no difference in the biliary IgM anti-fimbrial antibody level between the different groups. The oestradiol treatment did not change the levels of total immunoglobulins or antibodies against fimbriae and LPS in serum. An oestradiol-induced increase similar to the one seen in biliary IgM anti-LPS antibodies in primary immunized animals was not seen during the secondary response in booster immunized rats. Thus it seemed as if the effect of oestradiol on the secretory immune system in the bile was mainly due to an influence on primary stimulated B-cell clones in the liver, producing IgM antibodies against a T-cell-independent antigen. The effect may be mediated through a direct action of oestradiol on the B lymphocytes.     

Differences in level and avidity of secretory IgA antibodies in breast milk of Swedish, Indian and Japanese mothers to soybean protein

Colostrum was collected from Swedish, Indian and Japanese mothers. The samples were as a mean, collected 4.00-4.25 days after delivery of term infants. The level of specific IgA antibody to 2S, 7S and crude soybean antigen were measured by the enzyme-linked immunosorbent assay (ELISA). The avidity of the IgA antibodies to 7S soybean antigen was also measured with an ELISA system using different molarities of potassium thiocyanate for elution of the specific IgA antibody from solid phase-bound antigen. The level of specific IgA antibody to 7S and crude soybean antigen in the milk of the Indian mothers was significantly higher than in the milk of the Japanese mothers (p less than or equal to 0.01). In contrast, the avidity expressed as the molarity of KSCN for 50% elution of IgA antibody to 7S soybean antigen in the milk of the Japanese mothers was significantly higher than in the milk of the Indian mothers (p less than 0.01).     

Detection of soy protein in soy lecithin, margarine and, occasionally, soy oil

Samples of soy lecithin, soy oil and margarine were tested for the presence of soy proteins by an inhibition technique using ELISA. All but one of the soy lecithin samples contained soy protein, as did some of the soy oil and margarine samples. The positive margarines contained only about 25% as much soy protein as the soy lecithin preparations. The presence of soy proteins in these soy products might account for hitherto unrecognized exposure to soy proteins in various foods.