Physiology: Human follicular fluid maturity and endothelial cell mitogenesis

Follicular fluid, of varying maturity, (day 5–16 of cycle)was collected from the largest Graafian follicle of each of22 ovulatory patients during laparoscopic procedures. Threesamples were blood-stained and discarded. The mitogenic potentialof each sample was determined using bovine aortic endothelialcells in the CellTiter 96^TM Non-Radioactive Cell Proliferation/CytotoxicityAssay system. Intra- and inter-plate coefficients of variationwere <9%. The follicular fluid samples induced cell doublingtimes which varied from 12–24 h and final cell numberswhich, in the individual wells, ranged from 782–30 900(starting number 2000/well). Follicular fluid total proteincontent was unrelated to the mitogenic potential, (R² = 0%).Serum oestradiol was negatively correlated with the mitogenicpotential (R² = 26%). No correlation was found with day of themenstrual cycle (R² = 4.3%), maximum follicular diameter (R²= 1.8%), or serum concentration of progesterone (R² = 0.7%),luteinizing hormone (LH) (R² = 1.5%) or follicle stimulatinghormone (R² = 0.1%). Five subjects were in ‘early’and six in ‘mid’-follicular phase, six were in ‘early’and two in ‘late’ LH surge. There was no differencein the mitogenic response between these four groups by one-wayanalysis of variance (F = 0.21; P = 0.89). It is concluded thatthe mitogenic potential of human follicular fluid is not relatedto Graafian follicle maturity or, more particularly, to theLH surge.     

Diagnosing and preventing inherited disease: Cell recycling of a single human cell for preimplantation diagnosis of X-linked disease and dual sex determination

We recently described a new procedure, called ‘cell recycling’,which combines the two powerful techniques of polymerase chainreaction (PCR) and fluorescent in-situ hybridization (FISH)on the same single fixed cell. The dual procedure was developedto single cell sensitivity using single blastomeres of preimplantationmouse embryos. We have now extended the procedure to singlehuman cells and demonstrated its potential use in preimplantationdiagnosis to detect Duchenne muscular dystrophy (DMD) by PCR,in addition to sexing the same single cell by both PCR and FISH.Here we report an efficiency of 65% for cell recycling withefficiencies for PCR ampification of a single copy DMD sequenceat 87% and sexing by FISH at 75%. Should PCR diagnosis of theDMD mutation fail, cell recycling would provide two opportunitiesto identify the sex of the embryo, allowing selection of onlythe female embryos for transfer. human embryo/preimplantation diagnosis/sexing/single cell/X-linked disease     

Ovarian stimulation for in-vitro fertilization combining administration of gonadotrophins and blockade of the pituitary with D-Trp6-LHRH microcapsules: pilot studies with two protocols

In women undergoing in-vitro fertilization and embryo transfer(TVF-ET), a total of 408IVF cycles were stimulated using humanmenopausal gonadotrophin (HMG) or pure follicle stimulatinghormone (FSH) plus HMG in combination with a single injectionof D-Trp⁶-LHRH microcapsules in order to enhance the ovarianresponse to gonadotrophins and to avoid spontaneous LH surges.Sixty-seven pregnancies were achieved. Two protocols were employed.In protocol 1 (‘blocking protocol’, n = 268), thepituitary was first inhibited with a full dose (3.75 mg) ofD-Trp⁶-LHRH in microcapsules and ovarian stimulation was startedafter the hypogonadotrophic hypogonadal state was ascertained(Ej >50 pg/ml). In protocol 2 (‘flareup protocol’,n = 140), the treatment with D–Trp⁶LHRH microcapsules(half-dose = 1.80 mg) and the ovarian stimulation with gonadotrophinswere started at the same time. Higher doses of gonadotrophinswere needed (39.5 11.2 ampoules FSH and/or HMG) in protocol1, in which the pituitary was blocked prior to and during thestimulation, than in protocol 2 (209 ampoules) where the exogenousgonadotrophin stimulation appeared to be augmented by the initialagonistic effect of the injection of D-Trp⁶LHRH microcapsules.In patients with purely tubal infertility, under 38 years oldand no male factor, the results obtained with protocols 1 and2 were similar in terms of pregnancy rate per cycle or per embryotransfer: 22.6 versus 20.5% and 28.3 versus 27.4%, respectively.However, considering the cost benefit, ‘flare-up’protocols appeared to be a better choice and could be recommended.     

Sex selection by preimplantation genetic diagnosis: should it be carried out for social purposes?: Is preimplantation genetic diagnosis for 'social sexing' desirable in today's and tomorrow's society?

Is preimplantation genetic diagnosis for ‘social sexing’ desirable in today’s and tomorrow’s society? Dear Sir, Although we disagree with most of the ideas expressed in DrSeif’s letter, we will not comment each point or haggleover word definitions but we would like to express a few ideasconcerning preimplantation diagnosis for social sexing (PGDSS). One argument used to defend PGDSS is that it is preferable comparedwith abortion, infanticide or abandonment of     

Risks and benefits of steroid replacement therapy

The recommendations of the ESHRE workshop on ‘Risks andBenefits of Steroid Replacement Therapy’, held in Anacapri,September 18–19, 1987 ^*The workshop was sponsored by Schering (Italy).     

A genetically engineered V79 cell line SD1 expressing rat CYP2B1 exhibits chromosomal instability at the integration site of the transfected DNA

The genetically engineered cell line SD1 was constructed byco-transfection of V79 Chinese hamster cells with two plasmids:one containing a full-length cDNA encoding rat CYP2B1 and thesecond incorporating a selective marker gene. This cell linehas been used in gene mutation tests and in cytokinesis-blockmicronucleus assays to identify procarcinogens which are metabolizedby CYP2B1 to reactive metabolites. An elevated frequency ofspontaneous micronuclei was recorded in SD1 cells compared toparental V79 cultures. Karyotypic analysis revealed a chromosomalinstability which was manifested by amplification of the p-armsof a chromosome designated ‘n’ (derived from chromosome8). This chromosome was variable in length and sometimes exhibiteda telomeric fusion which led to the formation of a dicentricchromosome. Fluorescence in situ hybridization with digoxigenin-labelledplasmid DNA showed the presence of pSV450 plasmid DNA co-amplifiedwith genomic DNA sequences located in the terminal region ofchromosome ‘n’. ¹Present address: Molecular Genetics Laboratory, Departmentof Pathology, Royal Devon and Exeter Healthcare NHS Trust, BarrackRoad, Exeter EX2 5DW, UK. ²To whom correspondence should be addressed      

Extremely high levels of mutant mtDNAs co-localize with cytocohrome c oxidase-negative ragged-red fibers in patients harboring a point mutation at nt 3243

A single mtDNA point mutation at nt 3243 has been associatedwith two different clinical phenotypes: mitochondrial encephalomyopathy,lactic acldosis, and stroke-like episodes (‘MELAS³²⁴³’)and progressive external ophthalmoplegia (‘PEO³²⁴³’).It has been shown that there Is a much higher proportion ofragged-red fibers (RRF) with cytochrome c oxldase (COX) deficiencyIn PEO³²⁴³ than in MELAS³²⁴³. Using PCR/RFLP analysis of isolatedindividual skeletal muscle fibers from patients with both syndromes,we found a direct correlation between the localized concentrationof the nt 3243 mutation and Impairment of COX function at thesingle muscle fiber level: we found relatively low levels ofmutant mtDNAs (56±21%) in ‘normal’ fibers;high levels (90±6%) In COX-positive RRF; and an almostcomplete segregation of mutant mtDNAs (95 ±3%) In COX-negativeRRF. Thus, the differential distribution of fibers with extremelyhigh concentrations of mutant mtDNAs characterizes, and probablydistinguishes, the skeletal muscle of PEO and MELAS patientsharboring the same nt-3243 mutations.     

Micronucleus assays using cytochalasin-blocked MCL-5 cells, a proprietary human cell line expressing five human cytochromesP-450 and microsomal epoxide hydrolase

The MCL-5 cell line is a human lymphoblastoid TK^+/- cell linethat constitutively expresses a relatively high level of nativeCYP1A1, four other human cytochromes (CYP1A2, CYP2A6, CYP3A4and CYP2E1) and microsomal epoxide hydrolase, carried as cDNAsin plasmids. The aim of this study was to evaluate this cellline for its suitability for detecting chromosomal anomalies,employing micronucleus formation in cells blocked at cytokinesisas the indicator of clastogenicity. Results from two laboratories(‘ICR’ and ‘Swansea’) using differentprotocols are reported. In the ICR protocol, aflatoxin B¹, sterigmatocystin,benzo[     

Procarbazine is a potent mutagen at the heterozygous thymidine kinase (tk+/-) locus of mouse lymphoma assay

Procarbazine (Natulan®) is a potent inducer of gene mutationsat the heterozygous tk^+/– locus in L5178Y mouse lymphomacells in the presence of Aroclor-induced rat liver s9 metabolicactivation ({small tilde} 10^–3 mutant frequency at 10µg/ml) while exerting a far weaker effect in the absenceof s9. This mutagenicity is fairly robust with respect to thequantitative composition of the s9 mix and to variations inmouse lymphoma assay protocols (soft agar cloning versus ‘microwell’assays). The high proportion of small colony tk^–/–mutants induced by procarbazine together with the far weakermuta-genic response at the hemizygous hgprt locus in these samecells is interpreted in terms of a chromosomal or mufti-genemutational mechanism. Although procarbazine is clastogenic invivo, it does not appear to be so under standard protocols usingcultured human lymphocytes (±s9). It is not yet clearwhy this should be so, especially in light of its apparent clasto-genicityin mouse lymphoma cells.     

Endocrinology: Follicular fluid insulin-like growth factor-I and insulin-like growth factor-II concentrations vary as a function of day 3 serum follicle stimulating hormone

We determined follicular fluid concentrations of insulin-likegrowth factor (IGF)-I, IGF-II and inhibin as a function of day3 serum follicle stimulating hormone (FSH) in 16 women undergoingfollicular fluid aspiration in preparation for in-vitro fertilizationand embryo transfer. Follicular fluid concentrations of IGF-Iand IGF-II were significantly less in the ‘low’FSH group as compared to the ‘high’ FSH group. Themean IGF-I concentration was 67.6 ng/ml [confidence intervals(CI) 51.6–92.5] in the ‘low’ FSH group comparedto 87.1 ng/ml (CI 72.8–104.2; P < 0.025) in the ‘high’FSH group. Mean IGF-II concentrations were 354.8 ng/ml (CI 297.8–422.9)in the ‘low’ FSH group compared to 489.8 ng/ml (CI384.6–624.5; P < 0.05) in the ‘high’ FSHgroup. Follicular fluid inhibin concentrations did not differbetween groups. These differences in follicular fluid IGF asa function of day 3 FSH may raise questions regarding the rolegrowth factors play in the physiological processes of the ageingfollicle.     

Isothermal whole genome amplification from single and small numbers of cells: a new era for preimplantation genetic diagnosis of inherited disease

Preimplantation genetic diagnosis (PGD) of single gene defects following assisted conception typically involves removal of single cells from preimplantation embryos and analysis using highly sensitive PCR amplification methods taking stringent precautions to prevent contamination from foreign or previously amplified DNA. Recently, whole genome amplification has been achieved from small quantities of genomic DNA by isothermal amplification with bacteriophage 29 DNA polymerase- and exonuclease-resistant random hexamer primers. Here we report that isothermal whole genome amplification from single and small numbers of lymphocytes and blastomeres isolated from cleavage stage embryos yielded microgram quantities of amplified DNA, and allowed analysis of 20 different loci, including the DeltaF508 deletion causing cystic fibrosis and polymorphic repeat sequences used in DNA fingerprinting. As with analysis by PCR-based methods, some preferential amplification or allele drop-out at heterozygous loci was detected with single cells. With 2-5 cells, amplification was more consistent and with 10 or 20 cells results were indistinguishable from genomic DNA. The use of isothermal whole genome amplification as a universal first step marks a new era for PGD since, unlike previous PCR-based methods, sufficient DNA is amplified for diagnosis of any known single gene defect by standard methods and conditions.     

Human oocyte activation after intracytoplasmic sperm injection

The concentration of acetoxymethyl ester of fluo-3 given inthe subsection ‘Measurement of [Ca²⁺]_i’ of the ‘Materialsand methods’ on page 512 of the above paper was incorrect.The corresponding sentence should read as follows: Oocytes were loaded with the [Ca²⁺]_i indicator fluo-3 (Mintaet al., 1989) by incubation for 45 min at 37°C with 2 µMacetoxymethyl ester of fluo-3 (fluo-3/AM, Sigma) dissolved inB2 medium.     

Bacterial ribonuclease: mutagenic effect in microbial test-systems

Pure enzyme samples of ribonuclease from Bacillus intermedius7P (known commercially as ‘binase’) were investigatedfor genotoxicity in four microbial tests: the Ames plate incorporationmethod, Ara^R-assay; the prophage induction test; and the DNA-repairtest. The weak mutagenic effect of binase at high concentrations(0.1 mg/plate, 1 mg/plate) was established by induction of forwardAra^R-mutations and histidine-reverse mutations (both frameshiftmutations and base pair substitution). Metabolic activationwith rat or chicken liver, human placenta or plant (from tulipbulbs) microsomal fractions in vitro was seen to abolish thebinase mutagenicity. Bacillus intermedius 7P ribonuclease appearsto possess DNA damaging activity in uvrA^– and polA^–mutants, but not in the recA-deficient Escherichia coli strain,and exhibits an induction of recA-dependent mutagenesis detectedby the 8-fold increase of the prophage-induction level in lysogenicBacillus subtilis culture and by the 5-fold increase of thislevel in the Streptomyces lavendulae 3 lysogenic strain. Theimportance of the roles of both of enzyme catalytic activityand native structure is emphasized. A proposed mechanism forexogenous ribonuclease action is discussed. Bacillus intermedius7P ribonuclease probably does not act as a direct genotoxicagent interacting with DNA, but could provoke nucleotide imbalancethrough its catalytic action on membrane-associated RNAs, whichresults in alteration of DNA replication and, as a consequence,in recA-dependent mutagenesis. ¹To whom correspondence should be addressed     

Evaluation of presence and functional activity of potentially self-reactive T cells in aged mice

Autoimmunity is known to increase in aging. A possible factorcould be an alteration in the T cell repertoire wIth advancingage. Antibodies to the variable region of the ß chainof the TCR activate T cells and can serve as probes for analysisof the T cell repertoire. We have used V_ß3 and V_ß17aantibodies to determine the presence and functionality of normallydeleted T cells bearing potentially self-reactive TCR in peripherallymphoid tissue and blood from aged (SJL/J x BALB/c) F₁ LAF₁and BALB/c mice. Although an occasional 20- to 24-month-oldmouse exhibited V_ß3⁺ or V_ß17a⁺ T cells intheir lymph nodes or peripheral blood lymphocytes (PBL) slightlyabove the range for normal young mice of these I-E⁺ strains,there was no striking ‘escape’ from the normal thymicdeletion process. However, responsiveness to anti-V_ß3and anti-V_ß17a was slightly higher In aged, and particularlyIn aged thymectomlzed (TX), than in young mice. This was incontrast to proliferative responses to stimulation with antibodyto the normally expressed V_ß8 which were lower inthe lymph nodes from aged than from young mice. The PBL of some30- to 36-month-old mice were also examined. Enhanced numbersof ‘forbidden’ V_ß bearing T cells wereseen more frequently at this age. In spite of the age-relateddecrease in overall CD4/CD8 T cell ratios in all organs, themice with relatively high V_ß17a⁺ T cells exhibitedproportionally more CD4⁺ cells in that V_ß population.We conclude that the ‘forbidden’ T cells that respondto anti-V_ß stimulation in the 20- to 24-month-oldmice are most likely of extra-thymic origin, since they weremore readily detectable in aged TX mice. Potentially self-reactiveCD4 (and CD8) single-positive T cells were detectable in PBLonly in very aged (30–36 months old) euthymic mice.     

Role of T-cell-associated lymphocyte function-associated antigen-1 in the pathogenesis of experimental colitis

The ß₂ integrin lymphocyte function-associated antigen-1(LFA-1; CD11a/CD18) is important for lymphocyte traffickingand activation as well as recruitment to sites of tissue inflammation.The objective of this study was to assess the role of ‘T-cell-associated’LFA-1 in the pathogenesis of chronic colitis in vivo. Transferof CD4⁺CD25^– T cells isolated from wild-type (wt) miceinto immunodeficient recipients [recombinase-activating gene-1-deficient(RAG-1^–/–)] produced moderate to severe colitis,whereas RAG-1^–/– mice injected with CD11a-deficient(CD11a^–/–; LFA-1^–/–) donor T cells displayedminimal macroscopic and histological evidence of colitis. Surfaceexpression of L-selectin, ₄, ₄ß₇ and chemokine receptor-7were similar for wt and CD11a^–/– donor T cells.Attenuated disease in the CD11a^–/– RAG-1^–/–animals was associated with decreased numbers of CD4⁺ T cellsin the mesenteric lymph nodes (MLNs), spleen and intestinallamina propria (LP). In addition, significant reductions inT_h1 cytokines were observed following ex vivo stimulation ofmononuclear cells obtained from the MLNs and colonic LP. Interestingly,mononuclear cells obtained from the spleens of CD11a^–/– RAG-1^–/– exhibited enhanced pro-inflammatory cytokineproduction compared with splenocytes obtained from wt RAG-1^–/–colitic mice. Taken together, our data suggest that T-cell-associatedCD11a (LFA-1) expression plays a dual role in the initiationof chronic gut inflammation by facilitating naive T-cell priming/activationand expansion within MLNs and by augmenting pro-inflammatorycytokine production following secondary stimulation by antigen-presentingcells in the colonic interstitium.     

Cytotoxic and genotoxic effects of cleistanthin B in normal and tumour cells

Cleistanthin B, one of the toxic constituents of Cleistanthuscollinus, was found to be cytotoxic to normal and tumour cells.In comparison with normal cells, tumour cells were sensitiveto lower doses of toxin. The 50 growth inhibition GI₅₀ valuesfor normal cell lines were from 2 x 10^–5 to 4.7x10^–4M and for tumour cells the values ranged from 1.6x10^–6to 4x10^–5 M. Short exposure 30 min of Chinese hamsterovary CHO cells to cleistanthin B at 1–6 µg/ml resultedin extensive chromatid and isochromatid breaks and gaps. Howeverthere was no significant increase in cell death and DNA strandbreaks in cells treated under the above conditions. CleistanthinB induced micronucleus formation in cultured lymphocytes ina dose-dependent manner. CHO cells treated with high doses ofcleistanthin B showed a decrease in cell viability and a concomitantincrease in DNA strand-breaks. The cell death appears to bedue to apoptosis since nucleosome-like ladders were observedin the treated cells when the DNA was electrophorized in agarosegels. ¹To whom correspondence should be addressed     

Single intragenic microsatellite preimplantation genetic diagnosis for cystic fibrosis provides positive allele identification of all CFTR genotypes for informative couples

This study is part of a strategy aimed at using fluorescent polymerase chain reaction (PCR) on informative genetic microsatellite markers as a diagnostic tool in preimplantation genetic diagnosis (PGD) of severe monogenic disease. Two couples, both of whom had previously had children who were compound heterozygote for severe cystic fibrosis mutations, were offered PGD using fluorescent PCR of the highly polymorphic cystic fibrosis transmembrane conductance regulator (CFTR) intragenic microsatellite marker IVS17bTA. Cleavage-stage embryo biopsy followed by PCR resulted in transfer of one unaffected carrier embryo for each couple. This approach eliminates the need for single cell multiplex PCR strategies to detect CF compound heterozygotes. It also provides a control of chromosome 7 ploidy in the blastomeres and a selection against allele dropout by positive detection of each CFTR copy of all genotypes in preimplantation embryos from genetically informative families.     

Allelic drop-out and preferential amplification in single cells and human blastomeres: implications for preimplantation diagnosis of sex and cystic fibrosis

Previously the diagnosis of sex and cystic fibrosis status hasbeen studied on single cells using the polymerase chain reaction(PCR). It has been suggested that allelic drop-out (PCR failureof one allele) and/or preferential amplification (hypo-amplificationof one allele) may contribute to poor reliability and misdiagnosis,although this remains controversial as some reports suggestthat allelic drop-out does not occur. We investigated an improvedmethod of diagnosing sex and cystic fibrosis in single cellsusing a new technology (fluorescent PCR) to determine the baselevel of PCR artefacts (allelic drop-out and preferential amplification)which, in combination with improved sensitivity, should improvePCR reliability and accuracy. Fluorescent PCR gives high reliability(97%) and accuracy rates (97%) in somatic cells for both sexand cystic fibrosis diagnosis and its lower detection thresholdallows allelic drop-out and preferential amplification to beeasily distinguished. We also achieved high reliability andaccuracy in diagnosing cystic fibrosis in human blastomeres.This study confirms earlier reports of both allelic drop-outand preferential amplification in single cell analysis. We demonstratethat both allelic drop-out and preferential amplification occurin somatic cells and suggest these are separate phenomena. Preferentialamplification appeared common in single cell PCR while allelicdropout apparently occurred at random in each allele. Preferentialamplification was mainly amplification of the larger allele.We suggest that some inaccuracy/misdiagnosis may be due to bothpreferential amplification as well as allelic drop-out. Otherfindings were variability in drop-out between PCR and that amplificationof signals from human blastomeres may be linked to embryo quality.We suggest that allelic drop-out is dependent on the numberof cells within the sample. allelic drop-out/cystic fibrosis/preferential amplification/preimplantation diagnosis/sexing     

Fertilization and early embryology: Antioxidative capacity of preimplantation embryo culture medium declines following the incubation of poor quality embryos

Total antioxidative capacity (TAC), a measure of overall free-radicalscavenging potential, was determined by enhanced chemiluminescencein preimplantation embryo culture medium (PECM; pre-equilibratedHam's F-10 medium supplemented with 7.5% patient's serum). Changeswere evaluated in PECM TAC following a 24 h incubation of 66single human embryos, as was TAC of patient's serum alone. ThePECM TAC averaged 8.1% of the same patient's blood serum TAC.The percentage decline of PECM TAC over an incubation periodof 24 h ranged from 0.9 to 41.7%, with a median of 5.5%. Thedecline in PECM TAC in different embryo quality groups was alsostudied. Embryos were categorized as ‘good’, ‘fair’or ‘poor’ according to a scoring system based onan assessment of both the morphological appearance and developmentalspeed of the embryos. Incubation of poor quality embryos wasassociated with a decline in TAC, which was significantly higherthan that observed in ‘good’ and ‘fair’embryos. The findings suggest that impaired embryo developmentmay be associated with an increased generation of reactive oxygenspecies by the embryo.