Short-time repeat high-risk HPV testing by self-sampling for screening of cervical cancer.

BACKGROUND: Testing for high-risk human papillomavirus (HPV) in primary screening for cervical cancer is considered more sensitive, but less specific, in comparison with Pap-smear cytology. Women with persistent HPV infections have a higher risk of developing cervical intraepithelial neoplasia 2+ (CIN2+) lesions. This study was performed to evaluate the gain in specificity for detection of histologically confirmed CIN2+ lesions achieved by short-time repeat testing for high-risk HPV in women aged 30-65 years, with the primary sample for HPV analysis taken by self-sampling. METHODS: A total of 8000 women in Uppsala County, aged 30-65 years, who had not attended organised screening for 6 years or longer, were offered self-sampling of vaginal fluid at home and the samples sent for HPV typing. Of these, 8% (669) were not possible to contact or had performed hysterectomy. Women positive for high-risk HPV in the self-sampling test were invited for a follow-up HPV test and a cervical biopsy on average 3 months after the initial HPV test. RESULTS: In all, 39% (2850/7331) of invited women chose to perform self-sampling of vaginal fluid at home. High-risk HPV infection was found in 6.6% (188) of the women. In all, 89% of the women testing HPV positive performed a follow-up examination, on average 2.7 months, after the first test and 59% of these women were HPV positive in the follow-up test. The prevalence of CIN2+ lesions in women with an initial HPV-positive test was 23% (95% CI 18-30%) and in women with two consecutive HPV-positive tests was 41% (95% CI 31-51%). In women with two positive HPV tests, the prevalence of CIN2+ lesions varied from 49% in women at age 30-39 years to 24% in women at age 50-65 years. Short-time repeat HPV testing increased the specificity for detection of CIN2+ lesions from about 94.2% to 97.8%. The most prevalent HPV types were HPV16 (32%), followed by HPV18/45 (19%) and HPV 33/52/58 (19%). CONCLUSION: The short-time persistence of high-risk HPV infection in this age group was about 60%. Repeat testing for high-risk HPV using self-sampling of vaginal fluid can be used to increase the specificity in the screening for cervical cancer in women aged 30-65 years.

Temporal trends of HLA genotype frequencies of type 1 diabetes patients in Sweden from 1986 to 2005 suggest altered risk.

The aim of this study was to compare the frequency of human leukocyte antigen (HLA) genotypes in 1-18-year-old patients with type 1 diabetes newly diagnosed in 1986-1987 (n = 430), 1996-2000 (n = 342) and in 2003-2005 (n = 171). We tested the hypothesis that the HLA DQ genotype distribution changes over time. Swedish type 1 diabetes patients and controls were typed for HLA using polymerase chain reaction amplification and allele specific probes for DQ A1* and B1* alleles. The most common type 1 diabetes HLA DQA1*-B1*genotype 0501-0201/0301-0302 was 36% (153/430) in 1986-1987 and 37% (127/342) in 1996-2000, but decreased to 19% (33/171) in 2003-2005 (P \ 0.0001). The 0501-0201/0501-0201 genotype increased from 1% in 1986-1987 to 7% in 1996-2000 (P = 0.0047) and to 5% in 2003-2005 (P > 0.05). This study in 1-18-year-old Swedish type 1 diabetes patients supports the notion that there is a temporal change in HLA risk.

IA-2 autoantibodies in incident type I diabetes patients are associated with a polyadenylation signal polymorphism in GIMAP5.

In a large case-control study of Swedish incident type I diabetes patients and controls, 0-34 years of age, we tested the hypothesis that the GIMAP5 gene, a key genetic factor for lymphopenia in spontaneous BioBreeding rat diabetes, is associated with type I diabetes; with islet autoantibodies in incident type I diabetes patients or with age at clinical onset in incident type I diabetes patients. Initial scans of allelic association were followed by more detailed logistic regression modeling that adjusted for known type I diabetes risk factors and potential confounding variables. The single nucleotide polymorphism (SNP) rs6598, located in a polyadenylation signal of GIMAP5, was associated with the presence of significant levels of IA-2 autoantibodies in the type I diabetes patients. Patients with the minor allele A of rs6598 had an increased prevalence of IA-2 autoantibody levels compared to patients without the minor allele (OR=2.2; Bonferroni-corrected P=0.003), after adjusting for age at clinical onset (P=8.0 x 10(-13)) and the numbers of HLA-DQ A1*0501-B1*0201 haplotypes (P=2.4 x 10(-5)) and DQ A1*0301-B1*0302 haplotypes (P=0.002). GIMAP5 polymorphism was not associated with type I diabetes or with GAD65 or insulin autoantibodies, ICA, or age at clinical onset in patients. These data suggest that the GIMAP5 gene is associated with islet autoimmunity in type I diabetes and add to recent findings implicating the same SNP in another autoimmune disease.

SUMO4 M55V polymorphism affects susceptibility to type I diabetes in HLA DR3- and DR4-positive Swedish patients.

SUMO4 M55V, located in IDDM5, has been a focus for debate because of its association to type I diabetes (TIDM) in Asians but not in Caucasians. The current study aims to test the significance of M55V association to TIDM in a large cohort of Swedish Caucasians, and to test whether M55V is associated in those carrying human leukocyte antigen (HLA) class II molecules. A total of 673 TIDM patients and 535 age- and sex-matched healthy controls were included in the study. PCR-RFLP was performed to identify the genotype and allele variations. Our data suggest that SUMO4 M55V is not associated with susceptibility to TIDM by itself. When we stratified our patients and controls based on heterozygosity for HLA-DR3/DR4 and SUMO4 genotypes, we found that presence of SUMO4 GG increased further the relative risk conferred by HLA-DR3/DR4 to TIDM, whereas SUMO4 AA decreased the risk. From the current study, we conclude that SUMO4 M55V is associated with TIDM in association with high-risk HLA-DR3 and DR4, but not by itself.

Multinuclear-multiflagellar sperm defect in a bull--a new sterilizing sperm defect.

The development and use of modern techniques, such as intracytoplasmic sperm injection (ICSI), gene knockout and sperm fluorescence in situ hybridization with chromosome- specific probes, have significantly increased our knowledge about sperm defects. We describe a new oligoasthenoteratozoospermic defect in a bull. Because of its morphological characteristics the defect was named the multinuclear-multiflagellar sperm defect. All spermatozoa in the ejaculate were abnormal. Many of the spermatozoa had multiple nuclei and multiple sperm tails. All spermatozoa lacked an acrosome, and only seldom did spermatozoa have a mitochondrial helix in the midpiece area. Meiosis and spermiogenesis were severely affected in this otherwise phenotypically normal bull. The sperm defects resembled the phenotype of a targeted gene knockout Hrb(-/-) (HIV-1 Rev-binding/interacting protein) mutant mouse strain, which is expressed as sterility in males, while females remain fertile. Since the father of this bull has been extensively used in at least three countries the defective gene has possibly become widespread in the red and white breeds (Ayrshire, Swedish Red and White, Norwegian Red) in the Nordic countries. However, it is not proved that the father of this bull is a carrier of this defect.

High viral loads of human papillomavirus predict risk of invasive cervical carcinoma.

High loads of human papillomavirus (HPV) 16 and HPV 18/45 increase the risk of developing invasive cervical carcinoma, revealing higher risk in percentiles of highest viral loads for HPV 16 (odds ratio (OR) 58.7, 95% confidence interval (CI) 21.9-151.4) compared to HPV 18/45 (OR 3.3, 95% CI 1.5-7.2). Thus, HPV load is a type-dependent risk marker for invasive carcinoma.

Head area measurements of dead, live, X- and Y-bearing bovine spermatozoa.

The head area of bull spermatozoa was measured after viability and acrosome staining using trypan blue and Giemsa stains, followed by X- and Y-chromosome-specific fluorescence in situ hybridisation (FISH). The former staining made possible the categorisation of cells according to morphology and membrane integrity, whereas the latter allowed distinction of spermatozoa bearing X- and Y-chromosomes. Individual spermatozoa could be followed during the consecutive steps of staining, measurement and FISH. Using a high-resolution digital imaging system and measurement software, the head area of more than 3000 cells of five bulls was determined precisely. In all bulls, morphologically normal, viable cells with intact acrosomes were significantly smaller than dead cells with damaged acrosomes. No significant difference in the head area between X- and Y-chromosome-bearing viable, acrosome-intact spermatozoa was found in individual bulls. However, significant between-bull differences were detected in all cell categories.

Detection of water buffalo sex chromosomes in spermatozoa by fluorescence in situ hybridization.

In order to identify X- and Y-bearing spermatozoa in water buffalo by fluorescence in situ hybridization (FISH), some available probes of closely related species were examined. An X- and Y-specific probe set, made from flow sorted yak chromosomes, labelled in somatic metaphases of water buffalo the whole X and Y, respectively, except their centromere regions. A cattle Y-chromosome repeat sequence (BC1.2) showed strong signal on the telomere region of the buffalo Y-chromosome, demonstrating the evolutionary conservation of this locus in water buffalo. In hybridization experiments with spermatozoa from five buffaloes, the yak X-Y paint set demonstrated clear signals in more than 92% (46.8% X and 45.8% Y) of the cells. Using the cattle Y-chromosome specific BC1.2 probe, clear hybridization signal was detected in more than 48% of the cells. Statistical analysis showed that there was no significant difference between bulls or from the expected 50 : 50 ratio of X- and Y-bearing cells. The probes presented here are reliable to assess separation of X- and Y-bearing spermatozoa.

Simultaneous detection of viability and sex of bovine spermatozoa.

The viability and sex of bovine spermatozoa were simultaneously evaluated. After viability and acrosome staining with trypan blue/Giemsa, only live spermatozoa became decondensed by a modified papain-dithiothreitol method. Owing to this specific effect, live sperm heads were easily distinguished by their enlarged size and dark violet colour from small, light blue dead sperm heads. In the same sperm sample, X- and Y-chromosome-bearing sperm were distinguished by their fluorescent signal, using fluorescence in situ hybridization (FISH) with an XY paint set and 4,6-diamino-2-phenylindole counterstaining. The combined staining provides a method for morphological and viability evaluation before FISH and permits identification of the proportions of X- and Y-chromosome-containing live spermatozoa in a semen sample. However, only 25% of the undecondensed dead sperm express signals allowing detection of the sex of the chromosome. The method may be an effective tool in evaluating sex-oriented semen samples.

A new balanced autosomal reciprocal translocation in cattle revealed by banding techniques and human-painting probes.

Three hundred and twenty-two (264 males and 58 females), randomly sampled Grey Alpine cattle individuals from Northeastern Italy, were investigated cytogenetically by both conventional chromosome staining and R-banding. Two hundred and eighty-one (87%) individuals had a normal karyotype and 41 (13%) carried chromosomal aberrations such as (a) rob(1;29) in two individuals, (b) rob(26;29) in 36 individuals, (c) XX/XY-chimerism in two individuals, and (d) an abnormally long chromosome in one individual. All these aberrations except (d) have been described before. GBG-, RBG-, CBA-banding and sequential GBG/CBA- and RBG/CBA-banding techniques revealed that the abnormally long chromosome was the result of a reciprocal translocation between chromosomes 1 (q21-->qter) and 5 (q11-->q33), as confirmed also by chromosome painting with human chromosome 3 and 12 probes. The dam of the carrier bull carried the same translocation, while the grandam showed a normal karyotype. Since the sire of the dam was not available for study, no conclusion about the origin of the chromosome translocation could be drawn. The carrier bull was eliminated because of poor fertility. The dam had three other calves, which all were chromosomally normal. On average the dam had to be served 2.5 times (breed average was 1.2) to be in calf.

60,XY/60,XX chimerism in the germ cell line of mature bulls born in heterosexual twinning.

According to present knowledge there is a germ cell chimerism (XY/XX) in young bulls born in heterosexual twinning due to exchange of primordial germ cells in embryonic life. These germ cells were believed to have been eliminated in the young bull. Two-color fluorescence in situ hybridization (FISH) identification of the sex chromosomes by biotinylated and digoxygenin labeled probes have been used. The material consisted of three bulls born in heterosexual twinning. The results obtained indicated that even mature bulls (more than two years old) demonstrate spermatogonial chimerism. Several authors state that the bulls with blood cell chimerism, originating from dizygous twinning, are characterized by decreased fertility. Changes of the sex ratio of offspring due to proliferation of the female cells have also been proposed. The present observations should give a renewed interest in checking the possibility of survival and differentiation of germ cells from the female partner in the germ cell lines.

Gene expression and tissue concentrations of IGF-I in human myometrium and fibroids under different hormonal conditions.

The expression of insulin-like growth factor-I (IGF-I) was measured at the mRNA and protein level in myometrium and fibroids from women with and without preoperative treatment with a gonadotrophin-releasing hormone (GnRH) agonist for 3 months, from post-menopausal women, from pregnant women and in myometrium from women without fibroid disease. Women with menstrual periods were classified according to the phase of the cycle. In tissues from non-treated premenopausal women, IGF-I mRNA expression was significantly higher in fibroids than in myometrium, with no differences related to phase of the menstrual cycle. In post-menopausal women and in GnRH agonist-treated women responding to treatment, similar mRNA expression was seen in myometrium and fibroids but the concentrations were lower than in untreated premenopausal women. The IGF-I mRNA value in fibroids from pregnant women was higher than in any other group and myometrium from pregnant women exhibited higher mRNA expression than myometrium from non-treated premenopausal women. The IGF-I protein was more abundant in fibroids than in myometrium of non-treated premenopausal and of pregnant women and in both tissues the concentration was significantly higher in the group of pregnant women. The IGF-I protein concentrations in fibroids and myometrium from GnRH agonist-treated and post-menopausal women were similar to those from premenopausal non-treated women. High sex steroid concentrations in pregnant and non-pregnant women of fertile age seem to be associated with a higher expression of IGF-I in fibroids than in myometrium, suggesting that IGF-I contributes to the selective growth advantage of these tumours.

Tissue differences but limited sex steroid responsiveness of c-fos and c-jun in human fibroids and myometrium.

Sex steroids influence the growth of mammalian uterine tissues and the proto-oncogenes c-fos and c-jun have been implicated in the cascade of cellular events induced by the cyclic influence of oestrogen and progesterone. To investigate the role of these proto-oncogenes for fibroid growth their mRNA expression was measured in myometrium and fibroids under different hormonal conditions, using a solution hybridization method. Fibroids and myometrium were collected at surgery from premenopausal, postmenopausal and pregnant women as well as women treated with a gonadotrophin releasing hormone agonist (GnRHa; Goserelin). The phase of the menstrual cycle was determined in all the untreated, premenopausal, non-pregnant women. The mRNA expression of c-fos and c-jun in fibroids was significantly lower than in homologous myometrium. No significant differences in c-fos expression were observed in myometrium, or fibroids, due to menstrual cycle phase, GnRHa treatment, pregnancy or the menopause. The c-jun expression in myometrium from pregnant women without fibroid disease was significantly higher than the corresponding control myometrium from premenopausal, non-pregnant women. These results demonstrate a tissue difference in the expression of c-fos and c-jun between myometrium and fibroids.

Simultaneous detection of X- and Y-bearing bull spermatozoa by double colour fluorescence in situ hybridization.

Double colour fluorescence in situ hybridization with sex chromosome probes was applied on sperm cells of five Swedish Holstein-Friesian bulls. It was demonstrated that cosmids with strong fluorescence signals and scraped chromosomes can successfully be used as markers in this type of study. X and Y segregated as expected according to a 1:1 ratio, and there were no interindividual variations. There was a tendency for there to be more Y- than X-bearing spermatozoa, but this bias was assumed to be due to the markers used. Disomic spermatozoa occurred with a frequency of more than 0.1 % (0.067% XX, 0.029% YY, and 0.029% XY), which is considerably lower than the frequency in humans. Diploid sperm cells occurred with a frequency of 0.05 %.

Sex steroid receptors in human myometrium and fibroids: changes during the menstrual cycle and gonadotropin-releasing hormone treatment.

The content of estrogen and progesterone receptors (ER, PR) is higher in fibroid tissue than in homologous myometrium, and both receptors seem to be regulated by the levels of circulating sex steroids. Myometrial and fibroid tissues were recovered from women undergoing gynecological operations during different phases of the menstrual cycle and during treatment with an analogue of GnRH (GnRHa). Contents of ER and PR in the tissue cytosol were determined by enzyme immunoassay. The ER levels were significantly higher in fibroid tissue than in homologous myometrium in all the endocrine conditions. During the secretory phase, when luteal progesterone production is prominent, the ER levels in the myometrium and fibroids were lower than during the proliferative phase. During GnRHa treatment, the ER levels in both tissues were similar to those in the proliferative phase but significantly higher than in the secretory phase. The PR levels were also significantly higher in fibroids than in myometrium in all the different endocrine conditions. In both tissues, the PR levels were lower in the secretory phase and during GnRHa treatment, compared with the proliferative phase. Our data suggest that, in these categories of women, both ER and PR are overexpressed in fibroid tissue. Apparently, high progesterone levels down-regulate the ER in both fibroids and myometrium, whereas estrogen mediates the up-regulation of the PR during the proliferative phase. Increased knowledge about the mechanisms by which sex steroids regulate their own receptors in uterine tissues might provide a basis for development of new treatment strategies for women with fibroid disease.

Fiber-FISH analysis of the 3'-terminal region of the human L-type Ca2+ channel alpha 1C subunit gene.

Human L-type Ca2+ channel alpha 1C subunit gene (CACNL1A1) maps to the distal region of chromosome 12p13, and is composed of approximately 50 exons spanning over 150 kb of the human genome as estimated by restriction map analysis. However, the structure and the total length of the 3'-end of the gene is not clear because the size of several big introns remains unknown. Here the fiber-FISH technique was used to determine the relative order and size of eight partial genomic DNA clones from the central and 3'-terminal regions of CACNL1A1. The total physical distance of this region, including the size and gap distances between the clones were re-estimated. The results show that the physical order of the tested clones was 5'-g14-5 > g12-2 > g10-8 > g4-5 > g16-7 > g8-3 > g12-5 > g6-20-3'. Their individual sizes vary between 6.7 and 21.9 kb. Clones g6-20 and g12-5, both containing repetitive exon 45/46-like element, were found to be located within 59.1 kb downstream of g8-3 containing earlier identified polyadenylation site, i.e. 229.5 kb away from clone g14-5 (exons 10, 11). The possible implications of this structural complexity is discussed.