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PubMed1997

Detection of thymidylate synthase gene expression levels in formalin-fixed paraffin embedded tissue by semiquantitative, nonradioactive reverse transcriptase polymerase chain reaction.

We describe a new, simple and reliable semiautomated strategy for quantifying mRNA from archival specimens by using oligo(dT)25 paramagnetic beads and the reverse-transcriptase polymerase chain reaction (PCR) coupled with quantitative digital image analysis (Q-DIA). To evaluate the experimental conditions, we examined thymidylate synthase (TS) gene expression in mRNA isolated from both flash-frozen and formalin-fixed paraffin-embedded human biopsy samples using biopsy material obtained from 2 patients prior to chemotherapy with 5-fluorouracil. Following the electrophoretic separation of the PCR products through a 20% polyacrylamide gel, quantitation of the perimeters of the silver-nitrate-stained PCR products will be done by Q-DIA using a video frame-grabber board attached to a CCD camera using Image-Pro+ software. Validation of this approach will involve a comparison of the observed gene expression levels to TS protein levels obtained by tissue homogenization assays of TS, tetrahydrofolate, 5,10-methylenetetrahydrofolate, 2'-deoxyuridine-5'-monophosphate and 5-fluoro-2'-deoxyuridylate (FdUMP), by established [3H]FdUMP ligand-binding assays. The novelty of this method is that it offers a low-cost means whereby Q-DIA is performed directly from the gel to rapidly and accurately determine the level of TS gene expression, which is standardized against the beta-actin housekeeping gene. In the protocol described herein, gene expression studies can be done quickly and without the use of radioactive substances in both normal clinical samples shock frozen at the time of surgical excision and in formalin-fixed paraffin-embedded archival samples, which are commonly available in all hospital pathology departments. To demonstrate the utility of this method, mRNA was extracted from both nonpathological and tumor biopsies originating from both types of material from the same patients. TS gene expression in the flash-frozen and archival materials was compared to the level of TS intracellular enzyme activity in the same samples and a correlation of 89 and 80% between the shock-frozen and archival material relative to TS intracellular enzyme activity levels was observed. These findings suggest that routine semiautomated quantitative analysis of rare mRNA transcripts, e.g. TS, from archival material can be applied for retrospective studies.

DNA, Complementary↗
PubMed1997

Quality management and influential factors for the detection of single metastatic cancer cells by reverse transcriptase polymerase chain reaction.

The sensitive and specific detection of micrometastasis holds great promise for earlier staging of cancer patients. By amplification of tissue-specific gene expression, the reverse transcriptase polymerase chain reaction (rtPCR) readily detects single tumour cells in different tissues. An increasing number of rtPCR assays with possible relevance for routine laboratory diagnostic procedures is currently being reported in the literature. Interestingly, when used in the clinical setting, assays for the same target mRNA perform very differently, despite comparable sensitivities and specificities in-vitro. Using rtPCRs specific for carcinoembryonic antigen (CEA), prostate-specific antigen (PSA) and cytokeratin 18 (CK 18), we have started to systematically investigate, both experimentally and in clinical specimens, a number of factors that contribute to the varying and seemingly implausible test results. Here we have concentrated on sample collection modalities, assay stability and test reproducibility at the sensitivity limit. Our results demonstrate in detail that, at the maximum sensitivity required for micrometastasis detection, preanalytical and statistical influences increasingly become important for the consistency of the assay results. We conclude that the prerequisite for translating the results from highly sensitive and specific rtPCR assays into clinically relevant data is the thorough definition of assay procedures and the number of tests performed on a sample. Addressing questions of standardization and quality control management is a central aspect yet to be emphasized in assay development and application of routine laboratory rtPCR tests in oncology.

Carcinoembryonic Antigen↗
PubMed2001

Circulating neuroblastoma cells detected by reverse transcriptase polymerase chain reaction for tyrosine hydroxylase mRNA are an independent poor prognostic indicator in stage 4 neuroblastoma in children over 1 year.

PURPOSE: In this prospective, multicenter study, the independent prognostic power of neuroblastoma cells detected by reverse transcriptase polymerase chain reaction (RT-PCR) for tyrosine hydroxylase (TH) mRNA was evaluated. PATIENTS AND METHODS: The clinical significance of disease detected by RT-PCR in peripheral blood from children at diagnosis was compared with established prognostic markers [ie, age, lactate dehydrogenase (LDH), neuron-specific enolase, ferritin, and MYCN gene amplification] by multivariate analysis. The value of disease detection by RT-PCR during treatment and follow-up was also examined. RESULTS: TH mRNA was detected in peripheral blood from 33 of 49 (67%) children with stage 4 neuroblastoma > 1 year old at diagnosis and was a significant predictive factor for overall survival [hazard ratio (HR) = 2.40, 95% confidence interval (CI) 1.19 to 4.84, P =.014) and event-free survival (HR = 2.09, 95% CI 1.06 to 4.17, P =.034) in a multivariate analysis. Detection of disease in blood from clinically disease-free children was related to increased risk of death (HR 2.54, 95% CI 1.42 to 4.55, P =.0014). CONCLUSION: TH mRNA in peripheral blood of children with neuroblastoma is a poor prognostic indicator, reflecting the propensity for dissemination via the bloodstream. When combined with a serum LDH > 1500 IU/L, this is the most powerful poor prognostic model at diagnosis for children > 1 year old with stage 4 disease. The detection of TH mRNA in peripheral blood from clinically disease-free children is related to increased risk of relapse and death.

Age Factors↗
PubMed2001

Reverse transcriptase polymerase chain reaction (RT-PCR) detection of melanoma-related transcripts in the peripheral blood and bone marrow of patients with malignant melanoma. What have we learned?

The detection of circulating tumor cells (CTC) and bone marrow micrometastases (BMM) by reverse transcriptase polymerase chain reaction (RT-PCR) may help predict recurrence and survival in malignant melanoma (MM). Since the appearance of the original article by Smith et al. in 1991 (Lancet 338:1227), several groups have attempted the detection of CTC and BMM in MM using RT-PCR for melanocytic specific markers, mainly tyrosinase mRNA. Most studies show that tyrosinase is not present in the PB and BM of control individuals without MM. The PCR positivity rates in MM are extremely variable, ranging from 0% to 100%. There was a correlation between RT-PCR and stage in some but not all of the studies. These disparate findings could in part be explained by differences in RNA extraction and blood separation techniques, to unrecognized contamination leading to false positive results, or differences in patient selection. Despite these discrepancies, we and others have shown that RT-PCR for tyrosinase mRNA in PB is able to predict overall survival (OS) and disease-free survival (DFS) in a statistically significant manner. In AJCC stage II-IV patients rendered surgically free of disease, we found that blood tyrosinase positivity was an independent predictor of OS and DFS. We also found that BM tyrosinase positivity is an independent predictor of DFS in the same group of patients. RT-PCR may help identify subgroups of patients at high risk for early relapse for more aggressive adjuvant therapy. Large prospective studies and interlaboratory quality assurance initiatives are necessary to confirm the accuracy and prognostic value of these RT-PCR assays.

Biomarkers, Tumor↗
PubMed1999

Detection of prostatic specific membrane antigen messenger RNA using immunobead reverse transcriptase polymerase chain reaction.

The present study was performed to detect circulating prostatic carcinoma (PC) cells using a novel three-step immunobead reverse transcriptase (RT) polymerase chain reaction (PCR) assay for prostatic specific membrane antigen (PSMA) messenger RNA (mRNA). The sensitivity and specificity of this technique was assessed and the incidence of immunobead RT-PCR positivity correlated with progressive metastatic disease and serum prostatic specific antigen (PSA) levels. Fifty peripheral blood (PB) samples from 46 patients with PC were incubated with magnetic beads coated with Ber-EP4 antibody directed against the human epithelial antigen a membrane antigen widely expressed by epithelial cells. The epithelial cell-enriched magnetic fraction was then subjected to mRNA isolation using oligo-deoxythymidine (dT) magnetic beads. Nested RT-PCR for PSMA was performed on the mRNA oligo-dT complex and the identity of the RT-PCR products was confirmed by Southern blotting. Twenty-one PB samples from 8 control subjects without PC were also evaluated. Three-step immunobead PSMA RT-PCR was able to detect one PC cell per 1 mL of PB. The positivity rate of the RT-PCR assay was significantly higher (11 of 25; 44%) in patients with metastatic tumor than in patients with non-metastatic disease (1 of 21; 5%) (P = 0.003). In patients with metastatic PC, RT-PCR positivity was much higher in patients with progressive disease (10 of 13; 77%) than in patients with responding or stable disease (1 of 12; 8%) (P = 0.001). There was a statistically significant correlation between immunobead PSMA PCR positivity and high levels of serum PSA (P = 0.005). All control subjects without PC tested negative for PSMA PCR. The three-step immunobead RT-PCR for PSMA can detect circulating PC cells with high specificity and sensitivity. Preliminary data show a strong correlation between immunobead PCR positivity, the presence of progressive metastatic disease, and high levels of serum PSA.

Antigens, Surface↗
PubMed2000

[Reverse transcriptase polymerase chain reaction and immunocytochemistry of bone marrow aspiration specimen and peripheral blood for detection of microdisseminated prostatic carcinoma. A comparative analysis].

Reverse transcriptase-polymerase chain reaction (RT-PCR) assay for prostate-specific antigen and immunocytochemistry for cytokeratin-18 (CK-18) are tests for the detection of microdisseminated carcinoma of the prostate. Bone marrow aspirates and peripheral venous blood from 50 patients with clinically organ-confined prostate cancer were examined. The rate of positive results was independent of the pT stage, serum PSA, and previous antiandrogen treatment. RT-PCR and immunocytochemistry have to be tested under standardized conditions in prospective trials, and the results have to be compared to the serum PSA follow-up.

Aged↗
PubMed2000

Tropomyosin Is the Major Mollusk Allergen: Reverse Transcriptase Polymerase Chain Reaction, Expression and IgE Reactivity.

The complementary DNAs encoding tropomyosins of the abalone Haliotis diversicolor, the scallop Chlamys nobilis, and the mussel Perna viridis were amplified by reverse transcriptase polymerase chain reaction and thereafter cloned in plasmid vectors for expression. Immunoblot analysis showed that recombinant proteins of abalone, scallop, and mussel tropomyosin were reactive to serum IgE antibodies from subjects allergic to shellfish but not to nonallergic controls. Nucleotide and amino acid sequences of abalone, scallop, and mussel tropomyosins are highly similar (mostly > 55%) to those of crustacean allergens identified as tropomyosins. Absorption experiments showed that recombinant tropomyosins from the 3 mollusks were able to remove serum IgE reactivity against the 38-kDa tropomyosin of the organisms. These results demonstrate that tropomyosin is the major allergen among various common edible mollusks. Yet a comparison between the amino acid sequences of putative epitopes in crustaceans and mollusks suggests that the epitopes in the two groups may be distinct. Parsimony analysis of the nucleotide sequences of tropomyosin from different mollusks suggests that the tropomyosin gene sequence is a useful tool for phylogenetic analysis of this group of animals.

Journal Article↗
PubMed2004

Prediction of sustained virological response in liver transplant recipients with recurrent hepatitis C virus following combination pegylated interferon alfa-2b and ribavirin therapy using tissue hepatitis C virus reverse transcriptase polymerase chain reaction testing.

The optimal duration of therapy for pegylated interferon combined with ribavirin in recurrent Hepatitis C virus (HCV) following liver transplantation is not known. We wanted to determine if testing for HCV in liver tissue by reverse transcriptase polymerase chain reaction (RT-PCR) was superior in predicting sustained virological response (SVR) in comparison to standard HCV ribonucleic acid (RNA) detection in the serum. All recipients received combination pegylated alpha-2b interferon (1.5 mcg/kg) and ribavirin (200-600 mg/d) therapy for at least 48 weeks of therapy and were found to have nondetectable HCV RNA by PCR serum testing at the end of therapy. Sustained virological response (SVR) was defined as nondetectable serum HCV RNA at 6 months post treatment withdrawal. Ten liver transplant recipients were included in the study; mean time from transplantation was 29.2 months. All had nondetectable serum HCV RNA by RT-PCR. In hepatic tissue 7/10 patients HCV RNA was found to be positive by RT-PCR while 3/10 had nondetectable HCV RNA in their liver by RT-PCR. SVR was attained in all 3/10 that were hepatic tissue HCV PCR negative after 12 months of combination therapy. In conclusion, direct detection of HCV RNA by RT-PCR of liver tissue appears to more effectively predict SVR following pegylated interferon and ribavirin therapy than the conventional use of serum.

Antiviral Agents↗
PubMed2001

Real time reverse transcriptase polymerase chain reaction of urinary cytokeratin 20 detects transitional cell carcinoma cells.

PURPOSE: We evaluate the diagnostic use of cytokeratin 20 messenger (m) RNA quantitation in urine as a marker of urothelial transitional cell carcinoma using the real time reverse transcriptase polymerase chain reaction (RT-PCR). MATERIALS AND METHODS: Spontaneously voided urine was obtained from 47 patients with urothelial transitional cell carcinoma (carcinoma group), 19 other urological diseases (noncarcinoma group) and 27 healthy volunteers (control group). Quantification of cytokeratin 20 was performed with mRNA extracted from urine samples with primers and hybridization probes specific for cytokeratin 20 on a LightCycler instrument (Roche Diagnostics Corp., Indianapolis, Indiana). RESULTS: This method allowed reproducible quantitation of 10 to 106 cytokeratin 20 expressing colon carcinoma cells per 107 peripheral blood leukocytes, comparable to the sensitivity of conventional RT-PCR with a wide linear measuring range. Cytokeratin 20 mRNA values in the carcinoma group (mean 35,850) were significantly higher than noncarcinoma (171) and control groups (4.55, p <0.0001 and <0.0001, respectively). Urinary cytokeratin 20 mRNA values significantly correlated with tumor grade, urinary cytological class, immunostaining pattern and depth of tumor invasion. Sensitivity and specificity of real time RT-PCR with a cutoff value of 15 were 81% and 83%, whereas those of conventional cytology were 28% and 100%, respectively. CONCLUSIONS: These results indicate that real time cytokeratin 20 RT-PCR is a sensitive, quantitative, rapid and specific method to detect free cancer cells in the urine, with good potential for monitoring recurrence of urothelial transitional cell carcinoma.

Adult↗
PubMed1999

Improved sensitivity of reverse transcriptase polymerase chain reaction for hepatitis C virus using random hexamer primers.

The use of specific primers and random primers for the detection of hepatitis C virus RNA by reverse transcriptase-polymerase chain reaction was compared. The cDNA obtained from using both methods was amplified by the same protocol. Of the 30 plasma specimens tested, 6 (20%) were hepatitis C virus RNA-positive using specific primers and 19 (63%) were positive using random primers, demonstrating that the use of random hexamer-primed reverse transcriptase-polymerase chain reaction significantly improves the sensitivity (p < 0.01).

DNA Primers↗
PubMed1996

Detection of circulating neoplastic cells by reverse-transcriptase polymerase chain reaction in malignant melanoma: association with clinical stage and prognosis.

PURPOSE: Circulating melanoma cells can be detected in peripheral blood by means of tyrosinase mRNA amplification by reverse-transcriptase polymerase chain reaction (RT-PCR). We conducted a prospective study to evaluate the clinical significance of the presence of circulating neoplastic cells in the blood of patients with malignant melanoma (MM). METHODS: A sensitive RT-PCR assay was used to detect tyrosinase mRNA in the peripheral blood of patients with stages I to IV melanoma. Healthy subjects or patients with other malignancies were used as negative controls. RESULTS: Ninety-one assessable patients were included in the study. There was a statistically significant association between RT-PCR positivity and clinical stage. Circulating melanoma cells were detected in 36% of patients with localized disease (stages I and II), in 45% of patients with regional nodal involvement (stage III), and in 94% of patients with metastatic disease (stage IV) (P < .001). In stage II-III patients who were RT-PCR-positive for mRNA tyrosinase in blood, the recurrence rate and disease-free survival were significantly worse than patients who were RT-PCR-negative. In multivariate analysis, RT-PCR was an independent prognostic factor for recurrence in patients with nonmetastatic disease (P = .002). CONCLUSION: The detection of circulating melanoma cells in peripheral blood by RT-PCR correlated with the clinical stage of patients with melanoma and was an independent prognostic factor for recurrence. Further studies are warranted to better assess the significance of this test in the evaluation of prognosis, early detection of relapse, and in monitoring the effectiveness of systemic therapy.

Adult↗
PubMed1998

RNA extraction from virus-diseased sweet potato for reverse transcriptase polymerase chain reaction analysis.

Sweet potato virus disease (SPVD), which causes severe crop losses in Africa, is caused by a complex of sweet potato feathery mottle potyvirus (SPFMV) and sweet potato chlorotic stunt crinivirus (SPCSV). Extraction of pure RNA (for diagnosis by reverse transcriptase polymerase chain reaction [RT-PCR] methods) from diseased sweet potato proved to be difficult using five different total-RNA extraction procedures: (a) that of Chomczynski and Sacchi (1) (b) an adaptation of Lodhi et al., (2) and three commercially available kits (c) Clonsep (Clontech, Clontech Labs Inc, Palo Alto, CA; (d) RNeasy (Qiagen, West Sussex, UK); and (e) RNA isolator (Genosys Biotechnologies). Four of these methods (b-e) generated sufficient RNA, but it was unsuitable for RT-PCR amplification of SPFMV. When these RNA samples were treated with Wizard DNA extraction resin (Promega), the inhibitors of RT-PCR were consistently removed from three (b-d) of these four samples. Another deproteinating step was needed to allow RT-PCR amplification of sample e. The Wizard DNA column purification in conjunction with one of these total RNA extraction methods facilitates quick and reliable extraction of pure RNA for diagnostic purposes and might be suitable for similar problematic plant material.

Plant Diseases↗
PubMed2006

A combination of cytomorphology, cytogenetic analysis, fluorescence in situ hybridization and reverse transcriptase polymerase chain reaction for establishing clonality in cases of persisting hypereosinophilia.

To evaluate the frequency of clonal abnormalities in patients with unexplained persisting eosinophilia we analyzed 40 patients (27 males, 13 females) using cytomorphology, cytogenetic analysis, interphase fluorescence in situ hybridization (FISH), and reverse transcriptase polymerase chain reaction (RT-PCR). Cytogenetic analysis revealed clonal abnormalities in five patients (four of whom were males) including t(8;9)(p21;p24), ins(9;4)(q34;q12q31), del(6)(q24), and trisomy 8 (n=2). RT-PCR confirmed a PCM1-JAK2 fusion underlying the t(8;9). FISH analysis suggested a rearrangement involving PDGFRA in the ins(9;4). A FIP1L1-PDGFRA fusion gene was identified in four male patients by interphase FISH and RT-PCR. These methods in combination demonstrated clonality in 8/40 patients (20%) with a male predominance (6/8; 75%).

Chromosome Mapping↗
PubMed1996

The role of the reverse-transcriptase polymerase chain reaction assay for prostate-specific antigen in the selection of patients for radical prostatectomy.

Prostate cells present in the peripheral circulation can be detected using an enhanced reverse-transcriptase polymerase chain reaction (RT-PCR) for prostate-specific antigen (PSA) assay. In one study, preoperative enhanced RT-PCR for PSA status was a significant predictor of surgical pathology and postoperative biomechanical recurrence. The use of RT-PCR may enhance the urologist's ability to stage potential candidates for radical prostatectomy, as the assay is a more sensitive and specific predictor of microscopic extracapsular extension than conventional staging modalities. This highly adaptable assay also may have roles in screening for recurrence and in staging other solid tumors.

Adenocarcinoma↗
PubMed1996

Experimental evidence against replication or dissemination of hepatitis C virus in mosquitoes (Diptera:Culicidae) using detection by reverse transcriptase polymerase chain reaction.

In 3 laboratory experiments, mosquitoes were fed hepatitis C virus (HCV)-RNA positive blood by using membrane feeders, separated into head, thorax, and abdomen, and tested by a reverse transcriptase polymerase chain reaction for HCV-RNA. HCV did not replicate or disseminate in mosquitoes that had ingested blood from patients that were HCV-viremic positive. When yellow fever mosquitoes, Aedes aegypti (L.), were held for 1, 3, 7, 14, and 21 d after feeding, HCV-RNA was detected in the abdomens of 5/5 mosquitoes at 1 d after feeding; remaining tissues were negative with the exception of a single positive head at 7 d. In agreement, HCV-RNA was detected in Asian tiger mosquito, Aedes albopictus Skuse, and Anopheles stephensi Liston abdomens at 1 d, but not 3 d after feeding no HCV-RNA was detected in heads or thoraces. In addition, HCV-RNA was detected in heads of Ae. aegypti at 10 and 20 min, but not at 30 min, after feeding. The latter results raise the possibility of HCV contamination of mouthparts and, theoretically, mechanical transmission of this virus.

Aedes↗
PubMed1995

Detection of chimeric transcripts in desmoplastic small round cell tumor and related developmental tumors by reverse transcriptase polymerase chain reaction. A specific diagnostic assay.

Desmoplastic small round cell tumor is a recently described entity associated with fusion of the EWS and WT1 genes and with expression of a chimeric transcript. To investigate the structure and potential diagnostic utility of the detection of EWS-WT1 chimeric RNA in desmoplastic small round cell tumor, 12 examples of this entity and 49 other tumors that enter in its differential diagnosis were studied by reverse transcriptase polymerase chain reaction for the presence of EWS-WT1, EWS-FLI-1, PAX3-FKHR, and PAX7-FKHR chimeric transcripts. EWS-WT1 was detected in 11 of 12 desmoplastic small round cell tumors but not in any other tumor type studied, including 17 Wilms' tumors, 10 Ewing's sarcomas/primitive neuroectodermal tumors, 13 alveolar rhabdomyosarcomas, and 9 embryonal rhabdomyosarcomas. One desmoplastic small round cell tumor was found to have a variant EWS-WT1 chimeric product that included exon 8 of EWS EWS-FLI-1 chimeric RNA was present in all Ewing's sarcoma/primitive neuroectodermal tumor and not identified in any other tumor types, including desmoplastic small round cell tumor. PAX3/PAX7-FKHR chimeras were present in 9 of 13 alveolar rhabdomyosarcomas but not in any other tumors. Detection of chimeric transcripts by reverse transcriptase polymerase chain reaction is a very specific aid in differential diagnosis of developmental tumors and further establishes desmoplastic small round cell tumor as a distinct entity.

Adolescent↗
PubMed2002

Preoperative combined nested reverse transcriptase polymerase chain reaction for prostate-specific antigen and prostate-specific membrane antigen does not correlate with pathologic stage or biochemical failure in patients with localized prostate cancer undergoing radical prostatectomy.

PURPOSE: We report a prospective study examining the ability of preoperative nested reverse transcriptase polymerase chain reaction (RT-PCR) for prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSM) to predict pathologic stage and biochemical recurrence in patients with clinically localized prostate cancer treated with radical prostatectomy. PATIENTS AND METHODS: One hundred forty-one patients were entered onto the study. Preoperative evaluation included clinical T stage, serum PSA, biopsy Gleason score, and serum RT-PCR for PSA/PSM. Univariate and multivariate logistic regression models, Kaplan-Meier estimates, and Cox proportional hazards modeling were used to identify predictors of pathologic stage and biochemical failure. RESULTS: Seventy-three patients (51.8%) were RT-PCR positive for PSA, PSM, or both. In the multivariate logistic regression model, only initial PSA was an independent predictor of pathologic stage as defined by organ-confined disease (odds ratio [OR], 1.06; 95% confidence interval [CI], 1.00 to 1.13; P =.026) or organ-/specimen-confined disease (OR, 1.09; 95% CI, 1.02 to 1.16; P =.009). Overall Kaplan-Meier biochemical relapse-free survival (bRFS) was 85% at 59 months. Multivariate analysis of predictors for bRFS with the Cox proportional hazards model indicated that only initial PSA (OR, 1.05; 95% CI, 1.02 to 1.09; P =.004) and biopsy Gleason score (OR, 3.57; 95% CI, 1.37 to 9.58; P =.009) were independent predictors of biochemical failure. RT-PCR status did not predict pathologic stage or biochemical failure. Repeat analysis excluding 27 patients who received preoperative androgen-deprivation therapy did not change the results. CONCLUSION: Combined nested RT-PCR for PSA and PSM is not an independent predictor of pathologic stage or biochemical failure in patients with localized prostate cancer undergoing radical prostatectomy. This assay has no clinical utility in this patient population.

Antigens, Surface↗
PubMed1999

Type-1 and type-2 cytokines in human decidual tissue and trophoblasts from normal and abnormal pregnancies detected by reverse transcriptase polymerase chain reaction (RT-PCR).

PROBLEM: Cytokine expression at the maternal fetal interface has been well documented in rodents, but data in the human are scanty and controversial. METHOD OF STUDY: We examined cytokine expression of human decidua and trophoblasts by semiquantitative visual grading of reverse transcriptase polymerase chain reaction (RT-PCR) products in five groups of patients: ten patients with uncomplicated term pregnancies undergoing elective cesarean section (Group 1); ten women having normal pregnancies at term and vaginal delivery (Group 2); ten patients having intrauterine growth-retarded infants of unknown cause after a spontaneous vaginal delivery at term (Group 3); ten childless women having their first, first-trimester spontaneous abortion (Group 4); and ten childless women with a history of one or more previous first-trimester spontaneous abortions and having a new miscarriage (Group 5). RESULTS: Results favoring the T-helper 1 (Th1)/T-helper 2 (Th2) model during pregnancy were: significantly higher expression of interferon gamma (IFN-gamma) in trophoblast samples from Group 3 versus 2 and in decidual tissue from Group 5 versus 4; stronger positivity of interleukin (IL)-10 in decidual tissue samples from Group 1 versus Groups 2 and 5; and higher expression levels of tumor necrosis factor-beta (TNF)-beta by the trophoblast in Group 5 versus 1. Against the Th1/Th2 paradigm were the following findings: the significantly increased expression of IFN-gamma by decidual or trophoblast samples in Groups 1 versus 2, 2 versus 3, and 1 versus 5; and the significantly higher expression of TNF-alpha in decidual samples from patients in Group 1 (but also Group 4) as compared with Group 5. IL-2 mRNA and IL-4 mRNA could not be detected. CONCLUSIONS: Overall, our findings suggest that there is a balance between type-1 and type-2 cytokines during pregnancy, which is mainly characterized by the expression of IFN-gamma (a type-1 cytokine) and IL-10 (a type-2 cytokine) at the maternal fetal interface.

Cytokines↗