In-vitro maturation of human germinal vesicle stage oocytes: role of cumulus cells and epidermal growth factor in the culture medium

In-vitro maturation (IVM) of oocytes is a promising technique to reduce the costs and avert the side-effects of gonadotrophin stimulation for in-vitro fertilization (IVF). The pregnancy rates from oocytes matured in vitro are much lower than those of in-vivo stimulation cycles indicating that optimization of IVM remains a challenge. Therefore, we investigated the effect of supplementation of the medium with gonadotrophins, oestradiol and epidermal growth factor (EGF) and the effect of retaining or removing the cumulus cells on nuclear and cytoplasmic maturation of immature oocytes. Human germinal vesicle (GV) oocytes obtained after gonadotrophin stimulation for intracytoplasmic sperm injection (ICSI) were cultured in a complex defined medium either supplemented with gonadotrophins, oestradiol and physiological concentrations of EGF (2 ng/ml) or gonadotrophins and oestradiol alone. The cumulus cells were either removed or kept intact. In GV stage oocytes cultured without cumulus (group I) significantly more oocytes reached the metaphase II (MII) stage at 30 h in media supplemented with EGF (64.3 versus 33.9%, P < 0.003). For oocytes cultured with intact cumulus (group II), more oocytes reached MII at 30 h than in group I, but there was no difference in medium with or without EGF supplementation (81.8 and 79.8% respectively). Cytoplasmic maturation of MII oocytes was judged from their capability to activate and fertilize after ICSI. In group I, the rates of activation and normal fertilization were similar. However, in group II, significantly more oocytes underwent normal fertilization in the EGF-supplemented than the unsupplemented group (71.7 versus 45.6%, P < 0.05). The cleavage rates of the fertilized oocytes were similar in the sibling oocyte subgroups cultured with or without EGF supplementation, but the overall cleavage rates were higher in cumulus-intact compared to cumulus-denuded oocytes (88.9 versus 47.8%, P < 0.001). Thus, supplementation of the maturation medium with EGF and maintenance of the cumulus during culture improve the nuclear and cytoplasmic maturation of human oocytes in vitro.      

Carbohydrate epitopes on Haemonchus contortus antigens

Extracts of infective larvae and adults of the trichostrongylid Haemonchus contortus were studied for the presence of carbohydrate moieties. Several different lectin-binding sites were demonstrated in both stages using a panel of nine lectins. The carbohydrate specificity of the lectins used strongly suggests that α-D-mannose, α-D-glucose, and D-N-acetylglucosamine are the most important carbohydrate epitopes present on H. contortus proteins. Thus, N-linked oligosaccharides form the major part of the carbohydrate moieties on these glycoproteins. Treatment with sodium periodate was performed to investigate the immunoreactivity towards the carbohydrate moieties. This treatment resulted in a reduction in the immunoreactivity of these antigens as demonstrated by enzyme-linked immunosorbent assay (ELISA) and immunoblotting, suggesting that a substantial part of the host immune response against H. contortus is directed against the carbohydrate epitopes on the parasite antigens.     

Measurement of functional ability and health status in the arthritic patient

Chronic arthritis may have great impact on the patient but also on his or her family, relatives and friends. The assessment of the consequences of chronic arthritis and the effect of therapy not only in terms of physical, but also psychological and social dimensions deserves more attention. Functional ability and health status can be measured using a questionnaire or ‘instrument’, high-lighting important aspects not quantified with more traditional measurements. In this paper, arguments to apply such instruments more frequently are given. Health status instruments can be used not only to assess beneficial but also deleterious (side-)effects of therapeutic interventions. The properties are summarized of the most frequently used instruments assessing functional ability and health status. Many of these instruments have been evaluated sufficiently for validity and reliability; their sensitivity to detect change seems to be satisfactory. Therefore it is advisable to choose an internationally accepted, frequently used instrument, reflecting the area of interest.     

Virtually full-length subtype F and F/D recombinant HIV-1 from Africa and South America

For reliable classification of HIV-1 strains appropriate reference sequences are needed. The HIV-1 genetic subtype F has a wide geographic spread, causing significant epidemics in South America, Africa, and some regions of Europe. Previously only two full-length sequences of each of the HIV-1 subtype F subclusters F1 and F2 have been described. To extend the knowledge of subtype F variation on a complete genome level, three new virtually full-length F1 sequences were cloned and sequenced, two from Africa and one from South America. Comparison of the new and previously described sequences showed that monophyletic clustering of the subcluster F1 of subtype F is consistent and highly supported in all genome regions. Two additional full-length strains were shown to be mosaics of subtypes F and D. These epidemiologically unrelated F/D sequences showed similar chimeric structure, suggesting that they may represent a previously undescribed circulating recombinant form (CRF). This was supported by partial sequences from three additional unlinked F/D recombinants. Genetic distances in the phylogenetic trees suggest that the recombination event leading to the putative CRF occurred relatively long ago, close to the divergence of the F1 and F2 subclusters. Furthermore, all five F/D recombinants are linked to the Democratic Republic of Congo, suggesting that the original recombination event took place in central Africa.     

Aseptic technology of vitrification of human pronuclear oocytes using open-pulled straws

BACKGROUND: The aim of this study was to compare the viability of human pronuclear oocytes subjected to vitrification using cooling by direct submerging of open-pulled straws in liquid nitrogen versus vitrification by cooling of open-pulled straws located inside a closed 0.5 ml straw (aseptic system). METHODS: Two- and three-pronuclei stage oocytes (n=114) were cryopreserved in super-open-pulled straws by vitrification in 20% ethylene glycol +20% dimethylsulphoxide (DMSO) + osmotic active and neutral non-permeable cryoprotectants with a four-step exposure in 20, 33, 50 and 100% vitrification solution for 2, 1 and 1 min, and 30-50 s, respectively at room temperature, and plunging into liquid nitrogen. Oocytes of group 1 (n=42) were rapidly cooled at a speed of 20,000 degrees C/min by direct plunging of open-pulled straws into liquid nitrogen. Oocytes of group 2 (n=44) were first located in 0.5 ml straws, which were closed at both sides by metal balls, and then plunged into liquid nitrogen. This method resulted in a cooling speed of 200 degrees C/min. For both groups, oocytes were thawed rapidly at a speed of 20 000 degrees C/min using an identical protocol. Oocytes subsequently were expelled into a graded series of sucrose solutions (1.0, 0.75, 0.5, 0.25 and 0.12 mol/l) at 2.5 min intervals. RESULTS: Oocyte development up to expanded blastocyst stage after in vitro culture was 15% in group 1, 14% in group 2 and 29% in an untreated control group. CONCLUSION: The deposition of human pronuclear oocytes in open-pulled straws which are placed inside a hermetically closed container guarantees a complete isolation of oocytes from liquid nitrogen and avoids potential contamination by pathogenic microorganisms. The combination of direct plunging of this container into liquid nitrogen and rapid warming makes this process as efficient as conventional vitrification.     

High prevalence of human papillomavirus infections in urine samples from human immunodeficiency virus-infected men

Infection with human immunodeficiency virus (HIV) and the resulting immunosuppression are associated with an increased risk for human papillomavirus (HPV) persistence and related malignancies. In the present study we investigated the prevalence of HPV in urine samples from 104 HIV-infected men with low CD4+ cell counts (<100 per mm(3)) and 115 urine samples from HIV-negative men. A high prevalence of HPV DNA (39.4%) was found in the HIV patients. Most of the HPV types were high risk (81.4%), with HPV 52 as the most prevalent type (12.5%), followed by HPV 18 (6.7%), HPV 35 (5.8%), and HPV 70 (4.8%). Multiple HPV genotypes were observed in 17 (41%) of the 41 HPV- and HIV-positive men. In contrast, only 11 (9.6%) HPV DNA-positive cases were observed among the 115 HIV-uninfected men, and 3 (27.3%) contained multiple genotypes. Quantitative analyses indicated that the HPV viral load, as measured in urine samples, is significantly higher in HIV-positive men compared to HIV-negative men. In the present study we show that urine samples are useful for detecting HPV DNA, there is a high prevalence of HPV in HIV-positive men, and the HPV viral load is substantially higher in HIV-positive than in HIV-negative men. More studies are needed to evaluate the risk and natural development of HPV-related malignancies in HIV-positive men.     

Comparison of real-time PCR and culture for detection of Porphyromonas gingivalis in subgingival plaque samples

Porphyromonas gingivalis is a major pathogen in destructive periodontal disease in humans. Detection and quantification of this microorganism are relevant for diagnosis and treatment planning. The prevalence and quantity of P. gingivalis in subgingival plaque samples of periodontitis patients were determined by anaerobic culture and real-time PCR amplification of the 16S small-subunit rRNA gene. The PCR was performed with primers and a fluorescently labeled probe specific for the P. gingivalis 16S rRNA gene. By the real-time PCR assay, as few as 1 CFU of P. gingivalis could be detected. Subgingival plaque samples from 259 adult patients with severe periodontitis were analyzed. P. gingivalis was detected in 111 (43%) of the 259 subgingival plaque samples by culture and in 138 (53%) samples by PCR. The sensitivity, specificity, and positive and negative predictive values of the real-time PCR were 100, 94, 94, and 100%, respectively. We conclude that real-time PCR confirms the results of quantitative culture of P. gingivalis and offers significant advantages with respect to the rapidity and sensitivity of detection of P. gingivalis in subgingival plaque samples.     

Reproducibility of flow cytometric assessment of follicular tumours of the thyroid.

The reproducibility of the DNA index of paraffin wax sections from 44 follicular tumours of the thyroid (18 follicular adenomas and 26 follicular carcinomas), which had been assessed by flow cytometry was analysed in two laboratories, using consecutive sections of the same specimens and two different commercially available flow cytometers. Two slightly different cell preparation and staining techniques were used in the two laboratories. Using strictly defined criteria the histograms were classified blind as diploid, peritetraploid, aneuploid, or inadequate and insufficient by two independent investigators. Both the concordance between the two different flow cytometers and the agreement of duplicate assessments within the same flow cytometers were assessed. The mean coefficient of variation of the G0/G1 peak of the diploid tumours in the PARTEC flow cytometer was 5.5 (range 2.3-9.8) and in the FACS flow cytometer 5.2 (range 3.7-8.3); this difference was not significant. There was concordance of classification between the two laboratories in 35 of 36 cases. In 25 cases (18 diploid, seven aneuploid) the intralaboratory variation showed a 100% concordance in histogram classification. It is concluded that flow cytometer DNA index assessment of follicular tumours of the thyroid is reproducible and can be used to evaluate the discriminating and prognostic value of this feature.     

Germinal mosaicism increases the recurrence risk for ''new'' Duchenne muscular dystrophy mutations.

In 288 Dutch and Belgian Duchenne and Becker muscular dystrophy families, the parental origin of 41 new deletion or duplication mutations was determined. Twenty seven of the new mutations occurred in the maternal X chromosome and nine in the grandmaternal and five in the grandpaternal X chromosome. The grandparental data are compatible with equal mutation rates for DMD in male and female X chromosomes. New mutations were defined by their presence in one or more progeny and absence in the lymphocytes of the mother or the grandparents. In one family a fraction of the maternal lymphocytes was found to carry the mutation, suggesting somatic mosaicism. In six cases out of 41, the mutation was transmitted more than once by a parent in whom the mutation was absent in lymphocytes, suggesting gonadal mosaicism as the explanation for the multiple transmission. Using our data for the recurrence of the mutations among the total of at risk haplotypes transmitted, we arrive at a recurrence risk of 14% for the at risk haplotype. The observation of this high risk of germinal mosaicism is crucially important for all physicians counselling females in DMD families. Recently, germinal mosaicism has been observed also in a number of other X linked and autosomal disorders. The implications and appropriate diagnostic precautions are discussed.