Factors influencing the outcome of intrauterine insemination (IUI): age, clinical variables and significant thresholds.

The aim was to investigate the influence of various biological factors upon the outcome of intrauterine insemination (IUI). The total IUI history (856 cycles) of 352 couples was studied. Live-birth showed a strong negative correlation with female age but no correlation with male age. Antimüllerian hormone (AMH) and antral follicle count (AFC) correlated negatively with female age, and follicle stimulating hormone (FSH) correlated positively. Significant thresholds were found for all three variables, and also for total motile count (TMC) in the prepared sperm. Calculating pregnancy losses per positive pregnancy showed a strong correlation with increasing female age. This was highly significant for biochemical losses but not for fetal heart miscarriages. Male age had no effect on rate of pregnancy loss. In conclusion, female age, FSH, AMH and TMC are good predictive factors for live-birth and therefore relate to essential in vivo steps in the reproductive process.

Fall in implantation rates following ICSI with sperm with high DNA fragmentation.

BACKGROUND: There is considerable uncertainty as to the significance of a high sperm DNA fragmentation index (DFI) for achieving a successful pregnancy. METHODS: The sperm DFI of 124 patients undergoing 192 IVF cycles and of 96 patients undergoing 155 ICSI cycles was determined using the sperm chromatin structure assay on neat sperm. RESULTS: The rate of continuing pregnancies in ICSI cycles (but not in IVF cycles) showed significant negative correlation (r = -0.184, P = 0.022) with the DFI value. A threshold value of DFI which showed a significant difference (P = 0.005) in rate of continuing pregnancies between higher and lower DFI levels was found for ICSI cycles to be > or = 19%, but no such threshold was found for IVF cycles. However, if the threshold of > or = 30% was used for IVF cycles there was a non-significant lowering of the rates of continuing pregnancy and implantation at the higher DFI levels. DFI level had no effect on fertilization rate or on the percentage of embryos having more than 4 cells at Day 3 after fertilization. A high DFI level had a marked significant effect (P = 0.001) on implantation rate in ICSI cycles but not in IVF cycles. A significant positive correlation (r = 0.268, P = 0.001) between DFI and sperm midpiece defects was also noted in the ICSI patients. CONCLUSIONS: These observations may help to resolve the issues about how, and to what extent, sperm DNA damage impacts upon the success of IVF and ICSI procedures.

Preimplantation genetic diagnosis of chromosome abnormalities: implications from the outcome for couples with chromosomal rearrangements.

OBJECTIVES: Chromosomal rearrangements can lead to infertility or repeated spontaneous or induced abortions. The use of preimplantation genetic diagnosis (PGD) allows the selected transfer of chromosomally balanced embryos. The aim of this study was to carry out detailed analysis of the outcome of 11 PGD cycles for 8 patients carrying various chromosomal rearrangements. METHODS: Patients underwent routine in vitro fertilisation with biopsy of embryos on day 3. Specific fluorescent in situ hybridisation protocols were developed for each couple. Embryo transfer was possible in all 11 cycles. RESULTS: The outcome was four pregnancies, leading to three live births and one biochemical pregnancy. Post-zygotic mosaicism was detected in 75% of untransferred embryos, the majority of which were chaotic. Detailed follow-up and analysis provided evidence for the co-existence of chromosomally balanced and abnormal cells in six embryos. The mechanisms involved included chromosome breakage and loss of material. CONCLUSIONS: Biopsy and analysis of two blastomeres, where possible, reduced the risk of misdiagnosis in cases of balanced/aneuploid mosaics. The three live births achieved for the eight couples treated in this series, despite the poor history in almost all cases, is further proof that a policy of biopsying two cells from embryos consisting of six or more cells and a single cell from four- or five-cell embryos is compatible with a positive outcome.

Ferritin as a cytosol iron transport intermediate in human reticulocytes.

The major iron-bearing cytosol components of human reticulocytes identified after incubation with 59 Fe-125I-transferrin have been studied further. Component C previously found to behave consistently as an intermediate in the iron transport pathway to haem is shown to consist entirely of ferritin. After a short pulse of labelled transferrin incubation, chase experiments showed a fall of ferritin label with time and a corresponding increase in haemoglobin-iron incorporation. There was no loss of ferritin to the culture medium. Restriction of iron uptake by reticulocytes using both p-hydroxymercuribenzoate inhibition of uptake and incubation with progressively lower saturations of iron-transferrin gave linearly related incorporation of 59Fe into ferritin and haemoglobin at all levels of iron uptake, thus negating the concept of ferritin as an 'overspill' form of reticulocyte iron. The results suggest that cytosol ferritin is an obligatory intermediate in reticulocyte iron transport.

The assay of iron-poly (sorbitol-gluconic acid) complex (Ferastral) and its separation from transferrin in serum.

A new iron-poly (sorbitol-gluconic acid) complex (Ferastral) has been studied. A method of assay is described. The iron complex may be separated from serum transferrin using a Sephadex DEAE A50 column. This binds the iron complex and elutes iron-transferrin which can then be assayed. It is shown that the assay of serum transferrin unsaturated binding capacity using excess 59FeCl2 and MgCO3 adsorption, is valid in the presence of Ferastral. Serum unsaturated iron binding capacity may therefore be used to follow the binding of Ferastral iron by transferrin. These methods may be used to follow the distribution of iron in plasma after an intramuscular injection of Ferastral.

Transfer of iron from Ferastral and other organic complexes to transferrin as measured by reticulocyte uptake.

The transfer of iron from the iron carbohydrate complexes, Ferastral, Imferon, and Jectofer, and from ferric chloride has been studied by the effect of such transfer in reducing reticulocyte uptake of 59Fe from labelled transferrin. There are plasma factors which augment the transfer of iron from complex to transferrin. The pattern of transfer from Ferastral and from Imferon are similar: at concentration of 5000 microgram/100 ml and 1250 microgram/100 ml in plasma these complexes transfer about 0.8% and 1.7%, respectively. Jectofer transfers about four times these amounts under similar conditions. In the case of Ferastral there is evidence of an equilibrium between transferrin-bound and Ferastral-bound iron. The characteristics of Ferastral assessed in this way suggest that it may prove suitable for therapeutic use as a total dose infusion.

Patterns of plasma iron fractions after intramuscular Ferastral.

The distribution of plasma iron fractions after intramuscular Ferastral is described for times up to eight days after injection. Transferrin-bound iron reaches a peak value in 4-8 hours and falls towards normal values while circulating iron-complex concentration remains high. The peak concentration of iron-complex occurs 8-24 hours after injection, and is virtually cleared from the circulation in 6-8 days. An approximate T 1/2 of 30 hours for plasma Ferastral is deduced from the rate of fall from peak values. UIBC values fell as transferrin-bound iron increased, but did not reach zero in any subject studied.

Enzyme-catalysed reactions between some 2-substituted 5-nitrofuran derivatives and glutathione.

1. A glutathione transferase present in rat and human liver supernatant catalyses the reaction of some 2-substituted 5-nitrofuran derivatives with GSH, with formation of a conjugate and release of the nitro group as inorganic nitrite. Some of the substrates undergo the same reaction at a slower rate in the absence of enzyme. Nitrofuran derivatives commonly used as drugs, and five other drugs containing nitro groups, did not react. 2. Substrate activity in the nitrofuran derivatives showed an approximate correlation with the lability of the nitro group to alkali. 3. Optimum pH values ranging from 6.6 to 9.0 were found for the enzymic reaction with various derivatives, the values being influenced by alkali-lability and pK values of the compounds. 4. Tenfold purification of rat liver glutathione S-aryl-transferase resulted in an equal purification of the activities that catalyse the reaction of two of the nitrofuran derivatives with GSH.

The effects of inhibitor mixtures and the specific effects of different anions on the oxidase activity of caeruloplasmin.

1. The interpretation of the effects of mixtures of inhibitors on enzymes is considered. 2. The effects of inhibitor mixtures on caeruloplasmin were determined. 3. Fluoride, chloride and cyanate inhibit at one type of site (alpha), whereas bromide and iodide inhibit at another type (beta) present in the same enzyme intermediate. 4. Effects of inhibitor mixtures containing azide or cyanide are consistent with previous indications (Speyer & Curzon, 1968) that these ligands form inhibited complexes with different enzyme intermediates. 5. Isobols of halides or of cyanate with azide indicate that azide inhibits caeruloplasmin by bridging two alpha sites, these being reduced copper atoms. 6. Iodide and cyanate give hyperbolic plots of 1/v against [I]. 7. It is suggested that in the cyanate-inhibited complex the inhibitor binds to a reduced copper atom (alpha site) but that binding of cyanate at another copper atom is sterically prevented. It is suggested that the less bulky alpha-site inhibitors, fluoride and chloride, cause complete inhibition by binding to both of these copper atoms, which can also be bridged by a single azide group. 8. Each halide shows a pattern of effects on caeruloplasmin that is qualitatively distinct from that of other halides.

The inhibition of caeruloplasmin by cyanide.

1. The reversible inhibition of the oxidase activity of caeruloplasmin by cyanide was investigated. 2. The kinetics are unusual, being competitive but with the inhibited complex formed only during cycling. 3. Inhibitory concentrations of cyanide are comparable with that of caeruloplasmin. 4. One azide group completely inhibits a caeruloplasmin molecule but two cyanide groups are required. 5. The results suggest that azide binds to a half-reduced or fully reduced conformational isomer of the enzyme whereas cyanide binds to completely reoxidized isomers, and that inhibited complexes contain ligand bridges between copper atoms.

Inhibitors of caeruloplasmin.

1. A method is described by which substances inhibiting caeruloplasmin oxidase activity directly may be distinguished from those acting on stimulatory contaminant iron or on the product of enzyme action. 2. Many previously reported inhibitors, including saturated aliphatic carboxylates, hydrazines, 1,10-phenanthroline, borate and various psycho-active drugs, are found either not to act on the enzyme or to inhibit it only weakly. 3. A series of inorganic anions are compared as inhibitors. Anions such as azide and cyanide with strong copper-binding properties are the most effective inhibitors. There is a general inverse relationship between anion size and inhibitory power. Iodide is anomalous, the order of effectiveness of halides being F(-)>I(-)[unk]Cl(-)>Br(-). 4. Multidentate copperchelating ligands have little inhibitory effect. 5. A group of substances containing the structural unit [unk]C=[unk].CO(2)H, including fumarate and benzoate, cause inhibition. 6. Relative inhibitions by a series of mono-substituted benzoates are inversely related to molecular size. 7. Results are discussed in relation to earlier work on the disposition and function of the copper atoms of caeruloplasmin.