How to use Chlamydia antibody testing in subfertility patients

Screening for tubal factor subfertility by means of Chlamydia antibody testing (CAT) was introduced into the initial work-up of subfertile couples several years ago. The results reported, however, are heterogeneous, and no uniformity exists in cut-off levels of titres, or in definitions of tubal factor subfertility. We performed a prospective cohort study to evaluate the implications of varying the definitions of tubal pathology and of modifying the cut-off levels on the clinical impact of CAT in predicting tubal factor subfertility. In 227 consecutive patients who attended our fertility clinic, the Chlamydia IgG antibody titre was determined and related to tuboperitoneal abnormalities at laparoscopy as a reference standard. According to received operating characteristic (ROC) curve analysis, a titre of 16 is the optimum cut-off level. Increasing the cut-off level improves specificity and positive likelihood ratio (LR+), at the expense of sensitivity and negative LR (LR-). Changing the definition of tubal factor subfertility from unspecified tuboperitoneal abnormalities into extensive adhesions and/or bilateral distal tubal occlusion improves LR+, LR- and kappa significantly. We conclude that CAT is more accurate in predicting severe distal tubal pathology than unspecified tuboperitoneal abnormalities. Although from a statistical point of view a titre of 16 is the optimum cut-off level, from a clinical point of view 32 or 64 may be preferable, depending on the aim of screening and the inception cohort.      

Malignancy may adversely influence the quality and behaviour of oocytes

A case series of eight cycles of in-vitro fertilization (IVF) in five women diagnosed with malignant disorders is presented. These patients chose to defer definitive treatment for a chance for preservation of potential fertility. The response of these patients to ovarian stimulation, and the outcome, was compared with 17 IVF cycles in 12 age- matched patients with isolated tubal infertility. An apparent adverse influence of malignant disease on the quality and behaviour of oocytes was observed. Despite a comparable total number of oocytes per cycle in the two groups, a significantly reduced percentage of mature oocytes was retrieved per cycle from patients with malignant diseases. The oocytes from patients with malignant disorders were of a poorer quality and exhibited a significantly impaired fertilization rate compared to the controls. We propose that neoplastic processes, irrespective of the site or cell of origin, may have a detrimental impact on the biology of oocytes, an effect akin to that seen on spermatozoa in men with certain malignancies.      

Distribution of mutations in the PEX gene in families with X-linked hypophosphataemic rickets (HYP)

Mutations in the PEX gene at Xp22.1 (phosphate-regulating gene with homologies to endopeptidases, on the X-chromosome), are responsible for X-linked hypophosphataemic rickets (HYP). Homology of PEX to the M13 family of Zn2+ metallopeptidases which include neprilysin (NEP) as prototype, has raised important questions regarding PEX function at the molecular level. The aim of this study was to analyse 99 HYP families for PEX gene mutations, and to correlate predicted changes in the protein structure with Zn2+ metallopeptidase gene function. Primers flanking 22 characterised exons were used to amplify DNA by PCR, and SSCP was then used to screen for mutations. Deletions, insertions, nonsense mutations, stop codons and splice mutations occurred in 83% of families screened for in all 22 exons, and 51% of a separate set of families screened in 17 PEX gene exons. Missense mutations in four regions of the gene were informative regarding function, with one mutation in the Zn2+-binding site predicted to alter substrate enzyme interaction and catalysis. Computer analysis of the remaining mutations predicted changes in secondary structure, N-glycosylation, protein phosphorylation and catalytic site molecular structure. The wide range of mutations that align with regions required for protease activity in NEP suggests that PEX also functions as a protease, and may act by processing factor(s) involved in bone mineral metabolism.      

Preliminary observations on polar body extrusion and pronuclear formation in human oocytes using time-lapse video cinematography

In this study, we have used time-lapse video cinematography to study fertilization in 50 human oocytes that had undergone intracytoplasmic sperm injection (ICSI). Time-lapse recording commenced shortly after ICSI and proceeded for 17-20 h. Oocytes were cultured in an environmental chamber which was maintained under standard culture conditions. Overall, 38 oocytes (76%) were fertilized normally, and the fertilization rate and embryo quality were not significantly different from 487 sibling oocytes cultured in a conventional incubator. Normal fertilization followed a defined course of events, although the timing of these events varied markedly between oocytes. In 35 of the 38 fertilized oocytes (92%), there were circular waves of granulation within the ooplasm which had a periodicity of 20-53 min. The sperm head decondensed during this granulation phase. The second polar body was then extruded, and this was followed by the central formation of the male pronucleus. The female pronucleus formed in the cytoplasm adjacent to the second polar body at the same time as, or slightly after, the male pronucleus, and was subsequently drawn towards the male pronucleus until the two abutted. Both pronuclei then increased in size, the nucleoli moved around within the pronuclei and some nucleoli coalesced. During pronuclear growth, the organelles contracted from the cortex towards the centre of the oocyte, leaving a clear cortical zone. The oocyte decreased in diameter from 112 to 106 microm (P < 0.0001) during the course of the observation period. The female pronucleus was significantly smaller in diameter than the male pronucleus (24.1 and 22.4 microm respectively, P = 0.008) and contained fewer nucleoli (4.2 and 7.0 respectively, P < 0.0001). After time-lapse recording, oocytes were cultured for 48 h prior to embryo transfer or cryopreservation. Embryo quality was related to fertilization events and periodicity of the cytoplasmic wave, and it was found that good quality embryos arose from oocytes that had more uniform timing from injection to pronuclear abuttal and tended to have a longer cytoplasmic wave. In conclusion, we have shown that time-lapse video cinematography is an excellent tool for studying fertilization and early embryo development, and have demonstrated that human fertilization comprises numerous complex dynamic events.      

Pharmacological analysis of extracellular dopamine and metabolites in the striatum of conscious as/agu rats, mutants with locomotor disorder

The as/agu rat is a spontaneously occurring mutation which exhibits locomotor abnormalities, reduced tyrosine hydroxylase levels in the substantia nigra and lower extracellular levels of dopamine. The animal could represent a model of some human locomotor disorders. High-potassium medium evoked a 460% rise of dopamine levels in control rats but double this in mutants. Amphetamine increased extracellular dopamine by 710% in controls and 1480% in mutants. Clorgyline produced a small increase of dopamine levels in controls but an 1170% increase in mutants. The uptake inhibitor nomifensine increased dopamine levels by 910% in controls but only 270% in mutants. After treatment with benserazide plus L-DOPA, an acute injection of L-DOPA evoked a release of dopamine which was twice as large in the as/agu rats compared with controls. The results show reduced extracellular dopamine in as/agu rats when the locomotor disorder is apparent, but there has been little loss of tyrosine hydroxylase. The responses to drugs are qualitatively different from those obtained using 6-hydroxydopamine.Overall, the effects of compounds affecting aminergic neurons suggest that one possible mechanism for the neuronal abnormality in as/agu rats is a defective regulation of dopamine release from striatal terminals.     

Diagnostic accuracy of cytology and biopsy in primary bronchial carcinoma.

The accuracy of diagnosis in 656 patients with the four common histopathological types of primary lung cancer has been assessed by comparing the cell type diagnosis made on cytological and histological investigation with that determined by examination of the surgically resected or necroscopy specimen. The accuracy of diagnosis achieved by cytological examination of sputum and bronchial aspirate, and by bronchial biopsy histology was over 85%. The least accurate diagnostic procedure was percutaneous needle biopsy (62%). Squamous and small cell tumours were accurately diagnosed by all four investigations but errors were made in the diagnosis of large cell and adenocarcinomas. Nearly half the number of patients (43%) with large cell carcinoma were later reclassified as having squamous carcinoma and of the patients with adenocarcinoma 32% had been predicted to be squamous and 18% large cell carcinoma. We consider such quality control of pretreatment diagnosis mandatory in management of individual patients and before enrollment in clinical trials.     

Evaluation of the single radial haemolysis (SRH) technique for rubella antibody measurement.

Sera from 1258 individuals have been tested by four laboratories for rubella antibody by both the haemagglutination-inhibition and single radial haemolysis techniques. There was good agreement between the results obtained by the two methods. Although sheep red blood cells were used in the single radial haemolysis plates, no problems were encountered with sera from patients with infectious mononucleosis. The single haemolysis technique was found to be simple, convenient, and reliable, and suited to the rapid screening of large numbers of sera to assess susceptibility to rubella in the context of a vaccination campaign. However, since the technique does not detect anti-rubella IgM, it should not be used as the only test to investigate suspected recent infection.