NLRP7 is increased in human idiopathic fetal growth restriction and plays a critical role in trophoblast differentiation.

Fetal growth restriction (FGR) the leading cause of perinatal mortality and morbidity is highly related to abnormal placental development, and placentas from FGR pregnancies are often characterized by increased inflammation. However, the mechanisms of FGR-associated inflammation are far from being understood. NLRP7, a member of a family of receptors involved in the innate immune responses, has been shown to be associated with gestational trophoblastic diseases. Here, we characterized the expression and the functional role of NLRP7 in the placenta and investigated its involvement in the pathogenesis of FGR. We used primary trophoblasts and placental explants that were collected during early pregnancy, and established trophoblast-derived cell lines, human placental villi, and serum samples from early pregnancy (n = 38) and from FGR (n = 40) and age-matched controls (n = 32). Our results show that NLRP7 (i) is predominantly expressed in the trophoblasts during the hypoxic period of placental development and its expression is upregulated by hypoxia and (ii) increases trophoblast proliferation ([3H]-thymidine) and controls the precocious differentiation of trophoblasts towards syncytium (syncytin 1 and 2 and ß-hCG production and xCELLigence analysis) and towards invasive extravillous trophoblast (2D and 3D cultures). We have also demonstrated that NLRP7 inflammasome activation in trophoblast cells increases IL-1ß, but not IL-18 secretion. In relation to the FGR, we demonstrated that major components of NLRP7 inflammasome machinery are increased and that IL-1ß but not IL-18 circulating levels are increased in FGR. Altogether, our results identified NLRP7 as a critical placental factor and provided evidence for its deregulation in FGR. NLRP7 inflammasome is abundantly expressed by trophoblast cells. It is regulated by a key parameter of placental development, hypoxia. It controls trophoblast proliferation, migration, and invasion and exhibits anti-apoptotic role. NLRP7 machinery is deregulated in FGR pregnancies. KEY MESSAGES: NLRP7 inflammasome is abundantly expressed by trophoblast cells. It is regulated by a key parameter of placental development, hypoxia. It controls trophoblast proliferation, migration, and invasion and exhibits anti-apoptotic role. NLRP7 machinery is deregulated in FGR pregnancies.

Decorin expression is decreased in first trimester placental tissue from pregnancies with small for gestation age infants at birth.

Fetal growth restriction (FGR) is a leading cause of perinatal morbidity and mortality. FGR pregnancies are often associated with histological evidence of placental vascular thrombosis. The proteoglycans are important components and regulators of vascular homeostasis. Previous studies from our laboratory highlighted mRNA and protein expression differences in placental proteoglycan decorin (DCN), within a clinically well-characterised cohort of third-trimester idiopathic FGR compared with gestation-matched uncomplicated control pregnancies. We also showed that decorin contributes to abnormal angiogenesis and increased thrombin generation in vitro. These observations suggest that DCN gene expression may contribute to the etiology of FGR. Small for gestational age (SGA) is frequently used as a proxy for FGR and is defined as a birth weight below the 10th percentile of a birth weight curve. We therefore made use of a unique resource of first trimester tissues obtained via chorionic villus sampling during the first trimester to investigate the temporal relationship between altered DCN expression and any subsequent development of SGA. We hypothesized that placental DCN expression is decreased early in gestation in SGA pregnancies. Surplus chorionic villus specimens from 15 women subsequently diagnosed with FGR and 50 from women with uncomplicated pregnancies were collected. DCN mRNA and DCN protein were determined using real-time PCR and immunoblotting, respectively. Both DCN mRNA and protein were significantly decreased in placentae from first-trimester SGA-pregnancies compared with controls (p < 0.05). This is the first study to report a temporal relationship between altered placental DCN expression and subsequent development of SGA.

Placental vitamin D receptor expression is decreased in human idiopathic fetal growth restriction.

UNLABELLED: Fetal growth restriction (FGR) affects up to 5 % of pregnancies worldwide, and trophoblast function plays a significant role on the outcome. An epidemiological study has linked vitamin D deficiency to adverse perinatal outcomes, which include decreased birth weight. The placenta as an important source of vitamin D regulates its metabolism through the vitamin D receptor (VDR), but the mechanism by which VDR regulates trophoblast function is poorly understood. Our study aimed at determining placental VDR expression in FGR and gestation-matched control (GMC) pregnancies and identifying the actions of VDR in trophoblast differentiation and apoptosis. Placentae were collected from a well-defined cohort of idiopathic FGR and GMC pregnancies. VDR mRNA and protein expressions were determined by PCR, immunohistochemistry and immunoblotting, while functional consequences of VDR inactivation in vitro were determined on BeWo cells by determining changes in differentiation, attachment and apoptosis. Significant decreases in VDR mRNA expression (p = 0.0005) and protein expression (p = 0.0003) were observed in the FGR samples, while VDR inactivation, which showed markers for differentiation, cell attachment and apoptosis, was significantly increased. Thus, decreased placental VDR may contribute to uncontrolled premature differentiation and apoptosis of trophoblasts that are characteristics of idiopathic FGR pregnancies. KEY MESSAGE: Fetal growth restriction (FGR) affects up to 5 % of all pregnancies worldwide. FGR is the second highest cause of perinatal mortality and morbidity. The placenta plays a pivotal role in vitamin D metabolism during pregnancy. Vitamin D deficiency is associated with adverse pregnancy outcomes. Placental vitamin D receptor expression is decreased in FGR.

Decreased STAT3 in human idiopathic fetal growth restriction contributes to trophoblast dysfunction.

Abnormal trophoblast function is associated with fetal growth restriction (FGR). The JAK-STAT pathway is one of the principal signalling mechanisms by which cytokines and growth factors modulate cell proliferation, differentiation, cell migration and apoptosis. The expression of placental JAK-STAT genes in human idiopathic FGR is unknown. In this study, we propose the hypothesis that JAK-STAT pathway genes are differentially expressed in idiopathic FGR-affected pregnancies and contribute to abnormal feto-placental growth by modulating the expression of the amino acid transporter SNAT2, differentiation marker CGB/human chorionic gonadotrophin beta-subunit (ß-hCG) and apoptosis markers caspases 3 and 8, and TP53. Expression profiling of FGR-affected placentae revealed that mRNA levels of STAT3, STAT2 and STAT5B decreased by 69, 52 and 50%, respectively, compared with gestational-age-matched controls. Further validation by real-time PCR and immunoblotting confirmed significantly lower STAT3 mRNA and STAT3 protein (total and phosphorylated) levels in FGR placentae. STAT3 protein was localised to the syncytiotrophoblast (ST) in both FGR and control placentae. ST differentiation was modelled by in vitro differentiation of primary villous trophoblast cells from first-trimester and term placentae, and by treating choriocarcinoma-derived BeWo cells with forskolin in cell culture. Differentiation in these models was associated with increased STAT3 mRNA and protein levels. In BeWo cells treated with siRNA targeting STAT3, the mRNA and protein levels of CGB/ß-hCG, caspases 3 and 8, and TP53 were significantly increased, while that of SNAT2 was significantly decreased compared with the negative control siRNA. In conclusion, we report that decreased STAT3 expression in placentae may contribute to abnormal trophoblast function in idiopathic FGR-affected pregnancies.

Placental CLIC3 is increased in fetal growth restriction and pre-eclampsia affected human pregnancies.

Chloride intracellular channel (CLIC) proteins constitute a subgroup of the glutathione-S-transferase (GSTs) superfamily. In humans, the CLIC family of proteins consists of six members, designated CLIC 1-6, which have a conserved C-terminal 240 residue module and one major transmembrane domain. CLIC proteins regulate fundamental cellular processes including regulation of chloride ion concentration, stabilization of cell membrane potential, trans-epithelial transport, regulation of cell volume and stimulation of apoptotic processes in response to cellular stress. Previously, we described the expression profile of a member of the CLIC family of proteins, CLIC3, in human placentae and fetal membranes. In the current study, we determined CLIC3 expression in placentae from pregnancies complicated with either fetal growth restriction (FGR, n=19), pre-eclampsia (PE, n=16) or both FGR and PE combined (n=12) compared to gestation-matched controls (n=13) using real-time PCR and a CLIC3 specific immunoassay. Significantly increased CLIC3 mRNA and protein were detected in placental extracts from pregnancies with FGR, PE and PE with FGR compared to controls. Our results suggest that increased expression of CLIC3 may play a role in abnormal placental function associated with the human pregnancy disorders FGR and PE.

The PAI-1 4G/5G polymorphism is not associated with an increased risk of adverse pregnancy outcome in asymptomatic nulliparous women.

BACKGROUND: Plasminogen activator inhibitor type 1 (PAI-1) is an important regulator of fibrinolysis. A common deletion polymorphism that results in a sequence of 4G instead of 5G in the promoter region of the gene is associated with a small increase in the risk of venous thromboembolism. Its potential association with adverse pregnancy events remains controversial. OBJECTIVE: We aimed to assess the impact of the 4G PAI-1 polymorphism on pregnancy outcomes in women who had no prior history of adverse pregnancy outcomes or personal or family history of venous thromboembolism. PATIENTS/METHODS: This study represents a secondary investigation of a prior prospective cohort study investigating the association between inherited thrombophilias and adverse pregnancy events in Australian women. Healthy nulliparous women were recruited to this study prior to 22 weeks gestation. Genotyping for the 4G/5G PAI-1 gene was performed using Taqman assays in an ABI prism 7700 Sequencer several years after the pregnancy was completed. Pregnancy outcome data were extracted from the medical record. The primary outcome was a composite comprising development of severe pre-eclampsia, fetal growth restriction, major placental abruption, stillbirth or neonatal death. RESULTS: Pregnancy outcome data were available in 1733 women who were successfully genotyped for this polymorphism. The primary composite outcome was experienced by 139 women (8% of the cohort). Four hundred and fifty-nine women (26.5%) were homozygous for the 4G deletion polymorphism, while 890 (51.4%) were heterozygous. Neither homozygosity nor heterozygosity for the PAI-1 4G polymorphism was associated with the primary composite outcome (homozygous OR = 1.30, 95% CI = 0.81-2.09, P = 0.28, heterozygous OR = 0.84, 95% CI = 0.53-1.31, P = 0.44) or with the individual pregnancy complications. CONCLUSION: The PAI-1 4G polymorphism is not associated with an increase in the risk of serious adverse pregnancy events in asymptomatic nulliparous women.

GAPDH, 18S rRNA and YWHAZ are suitable endogenous reference genes for relative gene expression studies in placental tissues from human idiopathic fetal growth restriction.

Comparative gene expression studies in the placenta may provide insights into molecular mechanisms of important genomic alterations in pregnancy disorders. Endogenous reference genes often referred to as housekeeping genes, are routinely used to normalise gene expression levels. For this reason, it is important that these genes be empirically evaluated for stability between placental samples including samples from complicated pregnancies. To address this issue, six candidate housekeeping genes including several commonly used ones (ACTB, GAPDH, 18S rRNA, TBP, SDHA and YWHAZ) were investigated for their expression stability in placentae obtained from pregnancies complicated by idiopathic FGR (n=25) and gestation-matched control pregnancies (n=25). Real-time PCR was performed using pre-validated gene expression assay kits. The geNorm program was used for gene stability measure (M) for the entire housekeeping genes in all control and FGR-affected placental samples. Results showed that GAPDH and 18S rRNA were most stable, with an average expression stability of M=0.441 and 0.443, respectively, followed by YWHAZ (M=0.472). SDHA, ACTB and TBP were the least stable housekeeping genes (M=0.495, 0.548 and 1.737, respectively). We recommend geometric averaging of two or more housekeeping genes to determine relative gene expression levels between FGR-affected and control placentae.

Novel homeobox genes are differentially expressed in placental microvascular endothelial cells compared with macrovascular cells.

Angiogenesis is fundamental to normal placental development and aberrant angiogenesis contributes substantially to placental pathologies. The complex process of angiogenesis is regulated by transcription factors leading to the formation of endothelial cells that line the microvasculature. Homeobox genes are important transcription factors that regulate vascular development in embryonic and adult tissues. We have recently shown that placental homeobox genes HLX, DLX3, DLX4, MSX2 and GAX are expressed in placental endothelial cells. Hence, the novel homeobox genes TLX1, TLX2, TGIF, HEX, PHOX1, MEIS2, HOXB7, and LIM6 were detected that have not been reported in endothelial cells previously. Importantly, these homeobox genes have not been previously reported in placental endothelial cells and, with the exception of HEX, PHOX1 and HOXB7, have not been described in any other endothelial cell type. Reverse transcriptase PCR was performed on cDNA from freshly isolated placental microvascular endothelial cells (PLEC), and the human placental microvascular endothelial cell line HPEC. cDNAs prepared from control term placentae, human microvascular endothelial cells (HMVEC) and human umbilical vein macrovascular endothelial cells (HUVEC) were used as controls. PCR analyses showed that all novel homeobox genes tested were expressed by all endothelial cells types. Furthermore, real-time PCR analyses revealed that homeobox genes TLX1, TLX2 and PHOX1 relative mRNA expression levels were significantly decreased in HUVEC compared with microvascular endothelial cells, while the relative mRNA expression levels of MEIS2 and TGIF were significantly increased in macrovascular cells compared with microvascular endothelial cells. Thus we have identified novel homeobox genes in microvascular endothelial cells and have shown that homeobox genes are differentially expressed between micro- and macrovascular endothelial cells.

Epidemiological characteristics of women with high grade CIN who do and do not have human papillomavirus.

OBJECTIVE: Human papillomavirus infection is an important aetiological agent associated with the development of cervical neoplasia. However, even with the most sensitive methods of detection, human papillomavirus DNA has been detected in only 90% of cases of cervical cancer and between 80%-90% of cases of dysplasia. This study aimed to determine if there are epidemiological differences between women who are positive or negative for human papillomavirus, with high grade cervical intraepithelial neoplasia (CIN). DESIGN: Four hundred and sixty women with CIN II and III lesions were studied. To ensure optimal detection of human papillomavirus DNA, two specimens (i.e. tampon and cervical biopsy) were collected from each woman and tested by three techniques: L1-polymerase chain reaction, E6-PCR and low stringency Southern blotting. A detailed questionnaire was completed and blood sample collected for determination of serum levels of beta-carotene, vitamin A and E from each patient. Human leucocyte antigen (HLA)-DQB 1 alleles were also compared between the groups of women who were positive or negative for human papillomavirus. RESULTS: Overall, human papillomavirus DNA analysis was positive in 411 women (89%). Age, number of sexual partners in the last 12 months, past pregnancy and marital status were associated with human papillomavirus detection in the crude analysis. However, in the adjusted analysis no epidemiological features remained significantly different between the human papillomavirus positive and negative patients. Moreover, examination of vitamin A, E and beta-carotene levels did not show a significant difference between the two groups of patients. However, in the HLA-DQB1 allele profile a significantly higher proportion of women who were negative for human papillomavirus had DQB1 *0201, *0603 and *0604 (P = 0.05, 0.001, 0.03, respectively). CONCLUSION: We did not find a significant difference in epidemiological factors between women with human papillomavirus positive and negative high grade CIN. However differences between the frequency of three HLA DQB1 alleles suggest that women with these allele profiles have a higher chance of clearing human papillomavirus, without affecting their chance of developing dysplasia.

Human papillomavirus in corneal and conjunctival carcinoma.

PURPOSE: To investigate the role of human papillomavirus (HPV) types, 6, 11, 16 and 18 in corneal and conjunctival carcinoma, we examined 88 dysplastic corneal and conjunctival specimens and 66 controls that had been formalin-fixed and paraffin-embedded. METHODS: Sections were graded for histological abnormality by light microscopy and the presence of HPV DNA was determined by polymerase chain reaction using LI consensus primers. RESULTS: Human papillomavirus DNA was detected in 34 (39%) dysplasias and in five (7.5%) controls. Of dysplasias that were HPV-positive, 20 (59%) contained either types 16 or 18, 13 (38%) contained only types 6/11, while combinations of HPV types were present in 11 (32%). A histological correlation was found with HPV positivity (all genotypes) and unusually large ('epithelioid') dysplastic cells. CONCLUSION: The present study demonstrates a lower incidence of HPV in corneal and conjunctival carcinoma than previously reported, but shows an unexpectedly high incidence of HPV 6/11 in conjunctival carcinomas.

Juvenile laryngeal papillomatosis in a pediatric population: a clinicopathologic study.

A series of 22 children with juvenile laryngeal papillomatosis treated over a 31-year period is presented. The majority of patients were diagnosed when less than 5 years of age. Two patients died from the disease and four patients still had active disease at the completion of the study period. The duration of disease and number of recurrences were extremely variable. The number of recurrences was inversely related to the age of onset. The histologic findings were very similar in all patients, and no particular histologic feature had prognostic significance. In 20 patients, laryngeal biopsies were positive for human papillomavirus (HPV) 6/11 by either in situ hybridization (17) or polymerase chain reaction (19) or both (16). The number of patients who were HPV negative was small (two); interestingly, neither case had aggressive disease. Our findings suggest that age of onset and HPV status may be of prognostic value in determining the clinical course of the disease.

Patient-administered tampon-collected genital cells in the assessment of Chlamydia trachomatis infection using polymerase chain reaction.

BACKGROUND: Diagnosis of genital Chlamydia trachomatis infection in women traditionally requires a speculum examination to collect endocervical cells, followed by cell culture. This method is time consuming, requires stringent transport conditions, and is technically demanding. GOALS: To compare tampons as a patient-administered collection method followed by detection with polymerase chain reaction (PCR) with the traditional endocervical swab culture followed by cell culture detection. STUDY DESIGN: At the emergency department of a hospital for obstetrics and gynecology, 1,000 consecutive women with symptoms suggestive of infection with C. trachomatis were tested for C. trachomatis infection by PCR on both tampon (PCR-T) and swab (PCR-S) specimen and by culture of the swab specimen. RESULTS: Seventeen PCR-T and 16 PCR-S specimens were positive; 16 endocervical specimens were positive by culture, and 14 of the endocervical samples were positive by the three methods. Sixty-one PCR-S samples were inadequate as shown by the lack of amplification of the beta-globin gene segment, indicating poor collection of specimens by endocervical swab for chlamydial testing. CONCLUSIONS: Tampon specimens collected for PCR detection provided an easy and sensitive method of detection of C. trachomatis and overcame the obstacle of endocervical sampling and subsequent stringent transport requirements of culture.

15-Deoxyspergualin inhibits interleukin 6 production in in vitro stimulated human lymphocytes.

Experimental data show that relatively low concentrations of 15-deoxyspergualin (DSG) inhibit the induction of cytotoxic T lymphocytes (CTL) and the generation of antibody-producing cells. Considerably higher concentrations of DSG are required to inhibit proliferative responses. In this in vitro study, the effects of DSG on CTL induction, on proliferative responses induced by different stimuli, and on the production of interleukins IL-1, IL-2 and IL-6 and IFN-gamma (gamma-interferon) were assessed and compared with the effects of CsA (cyclosporine A) and/or FK506. We confirmed the suppressive action of DSG on the generation of CTL. Quite unexpectedly, however, we found that, although DSG did not affect the proliferative response to allogeneic lymphocytes or a superantigen, it did inhibit proliferation of peripheral blood leucocytes (PBL) stimulated with Staphyloccus aureus. DSG was active even when added on day 2 of in vitro culture, suggesting that DSG does not inhibit early events. The fraction of CD3+ lymphoblasts and the CD4/CD8 ratio was lower in cells stimulated by S. aureus in the presence of DSG, showing a selective effect on CD3+CD4+ responder T lymphocytes. The proportion of IL-2 receptor (CD25) positive cells was also reduced by DSG treatment. Moreover, we found that DSG inhibited the proliferation induced by PHA (phytohaemagglutinin) but not by Con A (concanavalin A). This effect of DSG was time-dependent, since PHA induced proliferation was not affected until day 4 after stimulation, and indicated that DSG may inhibit proliferation induced via a CD2- but not via a CD3-mediated pathway. DSG did not influence the production of IL-2 or IFN-gamma or the lipopolysaccharide induced production of IL-2 or IL-6. In contrast, the production of IL-6 was inhibited when cells were stimulated by allogeneic lymphocytes, S. aureus, PHA or Con A. This suggested to us that the DSG-suppressed IL-6 production could be the basis for the other observed effects. We tried to mimic the DSG effects with antibodies and indeed found that the IL-6 specific antibodies had similar effects. Furthermore, recombinant IL-6 completely overcame the suppressive effects of DSG on S. aureus and PHA induced proliferation, whereas addition of IL-6 to DSG treated PBL only partly restored the cytotoxic activity of lymphoblasts induced by allogeneic cells. Thus, the inhibitory effect of DSG on de novo synthesis of IL-6 could explain some of its immunosuppressive effects, but additional DSG-sensitive steps are obviously involved in CTL induction and differentiation.

Polymerase chain reaction. A sensitive indicator of the prevalence of human papillomavirus DNA in a population with sexually transmitted disease.

A total of 311 cervical samples from first attenders at a sexually transmitted disease clinic assayed for human papillomavirus (HPV) DNA with ViraType (VT) were analyzed with the polymerase chain reaction (PCR) for HPV using HPV L1 consensus primers and typed using L1 type-specific probes for 6/11, 16, 18 and 33. The prevalence of HPV by PCR was almost double that by VT (23.5% as compared to 12.6%, respectively). The increase was due largely to HPV types other than 6/11, 16, 18 and 33 (61.8%), while HPV types 6/11, 16 and 18 were responsible for 5.9%, 2.9% and 11.8%, respectively. Equal numbers of mixed infections of HPV 6/11/18 and HPV 16/18 each contributed to 8.8%. Mixed infection, as determined by VT, was 11% and increased to 40% with PCR. While the increase in the HPV detection rate by PCR was evident in all clinical categories examined (patients with no warts evident and no past history of warts, with no warts but a past history of warts and with clinical condylomata), a statistically significant increase occurred only in the first group, reflecting the increased sensitivity of PCR in detecting latent infection.

Simultaneous detection and typing strategy for human papillomaviruses based on PCR and restriction endonuclease mapping.

The aim of this study was to devise a simple and reliable method for simultaneous detection and typing of genital human papillomaviruses (HPVs). The method comprises three steps (i) amplification of sample DNA with the L1 consensus primers, (ii) digestion of PCR products with restriction endonuclease RsaI and (iii) hybridization of digested PCR products with a unique oligonucleotide probe that detects different fragments of the six most common genital HPV genotypes, namely, HPV types 6, 11, 16, 18, 31 and 33. Seventeen clinical specimens were analyzed by this method and compared to Southern blot using full genomic HPV DNA probes and to PCR using type-specific probes. All three tests agreed with at least one HPV type; however, the new method identified more additional HPV types in cases of mixed infections.

The absence of HPV DNA in genital specimens from infants.

OBJECTIVE: To identify the prevalence of HPV DNA using the polymerase chain reaction (PCR) in neonatal foreskin and cervical specimens obtained at necropsy. MATERIALS: Foreskin and cervical specimens were obtained from consecutive neonates who had autopsies performed at The Royal Women's Hospital, Melbourne, from June 1991 to February 1992. Specimens were analysed for HPV DNA using the polymerase chain reaction and the L1 consensus primers and generic probes. RESULTS: Specimens were obtained from 98 neonates, 52 males and 46 female. The mean gestational age of the neonates was 29 weeks (range 20-42). Eighty neonates died in utero, three during labour and 15 following delivery. Ninety four were delivered vaginally whilst four were delivered by caesarean section. Samples were collected a mean of 20 hours (range 2-48) from the time of delivery. In 30 cases there was evidence of autolytic change while in the remaining cases, the histology was well preserved. No evidence of HPV DNA was found in any of the samples using the L1 general primers (95% confidence interval 0-3.6%). Recent cervical cytology was available on 70 of the infant's mothers. Six had cytological evidence of HPV infection while the remainder were normal. CONCLUSIONS: HPV DNA is uncommonly detected (by PCR) in foreskin and cervical specimens obtained from neonates.

Prevalence of HPV in a Melbourne female STD population: comparison of RNA and DNA probes in detecting HPV by dot blot hybridization.

A total of 377 women, consecutively selected as first attenders to a sexually transmitted diseases clinic in Melbourne, Australia, were examined for overt Condylomata acuminata and were screened for genital HPV DNA types 6, 11, 16, 18, 31, 33 and (35) using 2 dot blot hybridization methods. Overall, there was a 90% positivity correlation between the 2 methods with HPV DNA being detected in 12% of ectocervical samples. Overt warts were found in 15% of the women and HPV DNA was detected at the cervix in 35% with cytology predicting HPV with or without dysplasia in 27%. Thirteen percent had a past history of warts but none on examination and HPV DNA was evident in 16% while 18% had cytological features of HPV. Those with no warts evident and no past history of warts had both HPV DNA and cytological features of HPV in 7%.

Detection of human papillomavirus DNA and mRNA using synthetic, type-specific oligonucleotide probes.

Type-specific 30'mer-36'mer oligonucleotide probes complementary to mRNA transcribed from the E6 and E7 open reading frames of HPV 6b/11, 16, 18 and 33 were designed using the published nucleotide sequences. As oligonucleotides are easily and relatively cheaply synthesized in large amounts and are free of vector DNA, they were assessed for potential use in routine clinical detection and typing of HPV. Multiple Southern and dot blots of cloned HPV 6b, 11, 16, 18, 31 and 33 DNA, and of DNA extracted from cell lines carrying integrated HPV 16 and 18 genomes were prepared. In addition, Northern and dot blots of RNA extracted from the HPV-containing cell lines HeLa, CaSki and SiHa, were also prepared. All filters were first probed with the oligonucleotide and then with the corresponding full-genomic HPV DNA probe and their relative sensitivities and specificities compared: both probe types were labelled with 32P. The oligonucleotide probes were all as specific as the full-genomic probes for Southern, DNA and RNA dot blot hybridisations. The HPV 16 and 18 oligonucleotide probes detected HPV transcripts of the appropriate sizes in the cell line RNA. For DNA detection, oligonucleotide probes were up to 10 times less sensitive than the full-genomic probes, but for RNA detection, they were more sensitive. The sensitivity for both HPV DNA and RNA detection could be improved by using two type-specific oligonucleotide probes in combination, without reducing the specificity. The ease of preparation and handling of oligonucleotide probes, together with their lack of contaminating vector DNA, suggests that they may have some advantages over full-genomic probes for the clinical detection and typing of HPV.