Non-invasive tool for foetal sex determination in early gestational age.

Free foetal DNA in maternal blood during early pregnancy is an ideal source of foetal genetic material for non-invasive prenatal diagnosis. The aim of this study was to evaluate the use of free foetal DNA analysis at early gestational age as pretest for the detection of specific Y-chromosome sequences in maternal plasma of women who are carriers of X-linked disorders, such as haemophilia. Real-time quantitative PCR analysis of maternal plasma was performed for the detection of the SRY or DYS14 sequence. A group of 208 pregnant women, at different gestational periods from 4 to 12 weeks, were tested to identify the optimal period to obtain an adequate amount of foetal DNA for prenatal diagnosis. Foetal gender was determined in 181 pregnant women sampled throughout pregnancy. Pregnancy outcome and foetal gender were confirmed using karyotyping, ultrasonography or after birth. The sensitivity, which was low between 4th and 7th week (mean 73%), increased significantly after 7+1th weeks of gestation (mean 94%). The latter sensitivity after 7+1th week of gestation is associated to a high specificity (100%), with an overall accuracy of 96% for foetal gender determination. This analysis demonstrates that foetal gender determination in maternal plasma is reliable after the 9th week of gestation and it can be used, in association with ultrasonography, for screening to determine the need for chorionic villus sampling for prenatal diagnosis of X-linked disorders, such as haemophilia.

Vertical transmission of HIV-1: maternal immune status and obstetric factors. The European Collaborative Study.

OBJECTIVE: To estimate the effect of maternal factors and events around the time of delivery on HIV-1 vertical transmission risk. DESIGN: Prospective study. SETTING: Twenty-two obstetric and paediatric clinics in seven European countries. PATIENTS OR OTHER PARTICIPANTS: Mothers identified as HIV-infected before or at delivery and their children. MAIN OUTCOME MEASURE: Paediatric HIV infection. RESULTS: By November 1995, 1846 mothers with 1945 children had been enrolled. The vertical transmission rate was 16.4% (95% confidence interval, 14.5-18.3). Parity, maternal age, race, mode of HIV acquisition, injecting drug use and sex of infant were not statistically significantly associated with risk of transmission. Children delivered vaginally were more likely to be infected than those delivered by Caesarean section. However, in vaginal deliveries the procedures used, duration of ruptured membranes or length of second-stage labour were not related to transmission. Transmission increased almost linearly with decreasing CD4 cell count, but there was no such trend for CD8 cell count. Women with CD4 cell counts below 200 x 10(6)/l were significantly more likely to deliver early (chi 2 for trend, 14.02; P < 0.001). Very premature infants were at increased risk of infection, but after about 35 weeks gestation the transmission rate remained stable, with no increase in late pregnancy. This trend was confirmed after allowing for maternal CD4 cell count. CONCLUSIONS: The rate of vertical transmission increases linearly with decreasing maternal CD4 cell count. Women with fewer than 200 x 10(6) CD4 cells/l have an increased risk of premature delivery, which would affect timing of interventions. The stable transmission rate after 35 weeks gestation suggests little acquisition of infection during late pregnancy.

The incidence of complications after caesarean section in 156 HIV-positive women.

OBJECTIVE: To investigate the risks of post-operative complications in HIV-positive mothers who undergo a caesarean section (CS) because the delivery cannot be safely accomplished by the vaginal route or to protect the infant from viral infection. DESIGN: In a multicentre study, we reviewed the incidence and type of post-operative complications in 156 HIV-positive women who underwent a CS. These results were compared with those observed in an equal number of HIV-uninfected women who matched for the indication requiring a caesarean delivery, the stage of labour, the integrity or rupture of membranes, and the use of antibiotic prophylaxis. SETTING: Seven teaching hospitals providing obstetrical care for mothers infected with HIV. RESULTS: We found that six HIV-infected mothers suffered a major complication (two cases of pneumonia, one pleural effusion, two severe anaemia and one sepsis) compared with only one HIV-negative woman who required blood transfusion after surgery. Minor complications like post-operative fever, endometritis, wound and urinary tract infections were significantly more frequent in HIV-positive women than controls. Multivariate analysis revealed that in HIV-infected women the only factor associated with a significant increase in the rate of complications was a CD4 lymphocyte count < 200 x 10(6)/l. CONCLUSIONS: The results of our study indicate that HIV-positive mothers are at an increased risk of post-operative complications when delivered by CS. The risk of post-operative complications is higher in HIV-infected women who are severely immunodepressed.

HIV-1 proviral DNA polymerase chain reaction detection in chorionic villi after exclusion of maternal contamination by variable number of tandem repeats analysis.

OBJECTIVE: The study of the placental HIV infection in cases of seropositive pregnant women after exclusion of maternal contamination of chorionic villi samples by variable number of tandem repeats (VNTR) analysis. METHODS: We studied 30 HIV-positive women: 17 terminated their pregnancy (11 in the first trimester and six in the second) and 13 delivered at term (one was a twin gestation). We selected chorionic villi and ruled out maternal contamination by VNTR analysis. DNA from chorionic villi and cord and maternal blood were tested for HIV by PCR. All infants underwent a paediatric follow-up. RESULTS: All maternal blood samples tested positive for HIV-1 by polymerase chain reaction. No maternal contamination was revealed and HIV was found in six out of 11 first trimester placentas, in all second trimester samples, and in 10 out of 14 at term. Cord blood tested positive in all second trimester cases and in seven out of 14 liveborns. In no case was HIV found in cord blood without infection of the corresponding placenta; conversely, three placentas tested positive but cord blood was negative. Two infants were HIV-positive, 11 were uninfected (one case was lost to follow-up). CONCLUSION: Our study indicates that HIV-1 can infect the placenta from first trimester onwards. HIV was found in two-thirds of our cord blood samples but it is possible that some viral DNA in cord blood may have come from infected placental cells. Additional studies are needed to assess the source of HIV in cord blood and the possible contribution of placental or maternal cells infected with HIV to vertical transmission of the virus.

New in vitro findings on the "free" form of apolipoprotein A-1.

Clinical and experimental gerontologists are extremely interested in lipoproteins as well as in new methods for investigating and probing the apolipoprotein pattern. Using immunofixation electrophoresis, we separated free apolipoprotein A-1 from the apo A-1 associated with high-density lipoproteins. Free apolipoprotein A-1 is a low-molecular-mass form of apo A-1 that seems to contain an extremely low quantity of lipids. The use of IFE as a tool for probing free apo A-1 has revealed new and interesting findings, such as its "artificial" increase during serum conservation at temperatures between 0-4 degrees C. From the clinical point of view, we demonstrated a decrease to the point of disappearance of free apo A-1 in some patients with liver cirrhosis. Moreover, one of the main findings here reported is the failure of anti-human apo A-1 murine monoclonal antibody and monoclonal antibody mixture to precipitate free apo A-1 in agarose systems. This discovery has important implications both for basic knowledge on apolipoproteins and for practical reasons concerning variability in those immunoassays (radial immunodiffusion) utilizing monoclonal antibody mixtures.

Treatment with interferon for genital HPV in HIV-positive and HIV-negative women.

The administration of interferons can be resorted to, either on its own or in combination with physical destruction methods, when the extent of genital HPV is widespread. Extensive genital HPV involvement is often seen in HIV-positive patients as a consequence of their immunodeficiency. The extension of these lesions may invalidate treatment by physical destruction, while an underlying immunodeficiency renders interferon therapy less efficacious. We studied HIV-positive and HIV-negative patients with a similar HPV involvement of their genital tract and compared the effectiveness of systemically administered alpha 2b and beta interferons in clearing HPV. Our results confirm that interferon therapy will cure most patients with extensive genital HPV when they are HIV-negative. HIV-positive patients with CD4 counts over 400 lymphocytes/mm3 may expect a similar cure rate, but this halves when this critical threshold is crossed. In these severely immunodeficient patients repeated courses of interferon therapy alone or in combination with physical destruction methods may be required to cure HPV infection.

The changing HIV epidemic in Italian pregnant women.

OBJECTIVE: To describe changes in the characteristics of HIV-pregnant women in Italy and the impact of strategies for prevention of HIV vertical transmission. STUDY DESIGN: Since 1985, HIV-infected women and their children are followed in 23 European centres in the European Collaborative Study (ECS), according to a standard protocol. Eight Italian Obstetric units participating in the ECS enrolled 815 patients. RESULTS: Overall use of zidovudine to reduce HIV vertical transmission has increased significantly since 1994 and between 1995 and 1997, 57% of Italian women were treated. However, 27% of babies received the infant component of the 076 regimen. Over the years, age at delivery has increased and their CD4 count at delivery decreased, most likely reflecting heterosexually infected women with a longer duration of infection. The increasing rate of elective caesarean section (42%) is not related to maternal, foetal or obstetrical indications, but its use as an intervention to reduce HIV vertical transmission. CONCLUSIONS: The identification of HIV-infected women during pregnancy or before delivery ensures the appropriate management of the woman and her child, and clinicians should be aware of the increasing number of women with heterosexual acquisition of HIV-infection who may be less easily identified.

Immunofixation electrophoresis (IFE) of serum apolipoproteins: a new tool for probing lipoprotein disorders and the atherosclerosis risk.

An immunofixation electrophoresis (IFE) approach to the detection of the apolipoprotein pattern in human sera is described. IFE for apolipoprotein A-1 and B provides informations complementing those obtained by conventional laboratory immunoassay methods. IFE is specific and sensitive enough to detect the differential distribution of apo A-1 into alpha (HDL)-apo A-I and "free" apo A-I. Therefore, this method allows us to study routinely the "free" apo A-I in the clinical laboratory. This is an important new parameter the clinical and pathophysiological significance of which has not yet been established, but it can now be Investigated systematically. IFE also allows the detection of apo B distribution in beta (LDL) and pre-beta (VLDL) lipoproteins. The possibility to obtain densitometric scanning profiles from IFE, may provide quantitative data useful for a deeper laboratory investigation of lipoprotein disorders and towards a potentially better assessment of the risk of atherosclerosis and coronary heart disease (CHD).

Sources of toxoplasma infection in pregnant women: European multicentre case-control study. European Research Network on Congenital Toxoplasmosis.

OBJECTIVE: To determine the odds ratio and population attributable fraction associated with food and environmental risk factors for acute toxoplasmosis in pregnancy. DESIGN: Case-control study. SETTING: Six large European cities. PARTICIPANTS: Pregnant women with acute infection (cases) detected by seroconversion or positive for anti-Toxoplasma gondii IgM were compared with pregnant women seronegative for toxoplasma (controls). MAIN OUTCOME MEASURES: Odds ratios for acute infection adjusted for confounding variables; the population attributable fraction for risk factors. RESULTS: Risk factors most strongly predictive of acute infection in pregnant women were eating undercooked lamb, beef, or game, contact with soil, and travel outside Europe and the United States and Canada. Contact with cats was not a risk factor. Between 30% and 63% of infections in different centres were attributed to consumption of undercooked or cured meat products and 6% to 17% to soil contact. CONCLUSIONS: Inadequately cooked or cured meat is the main risk factor for infection with toxoplasma in all centres. Preventive strategies should aim to reduce prevalence of infection in meat, improve labelling of meat according to farming and processing methods, and improve the quality and consistency of health information given to pregnant women.

Detection of human immunodeficiency virus-1 RNA and DNA by extractive and in situ PCR in unprocessed semen and seminal fractions isolated by semen-washing procedure.

BACKGROUND: To determine the presence of human immunodeficiency virus-1 (HIV-1) viral RNA/DNA in whole semen, in properly isolated seminal fractions and in spermatozoa after swim-up, by extractive nested PCR and to compare the detection of HIV DNA by in situ PCR (IS-PCR) with the results of nested PCR. METHODS: We tested HIV-1 RNA and DNA by nested PCR in semen and in seminal fractions from 55 patients. Non-spermatic cells and spermatozoa pellet fractions from 10 HIV-1-positive and five HIV-1-negative men were tested for proviral DNA by IS-PCR. RESULTS: All samples of spermatozoa recovered after sperm washing were free of HIV RNA. HIV RNA tested positive in seven (13%) seminal plasma samples and only in two (4.2%) whole semen of these same samples. Of the seven seminal plasma samples testing positive for HIV RNA, four men had elevated blood viral load and three an undetectable viraemia. HIV DNA by IS-PCR turned positive in three of five samples in semen of HIV-noninfected men. CONCLUSION: HIV RNA/DNA detection in the semen of HIV-infected men proves the efficacy of sperm washing with swim-up of spermatozoa. It is recommended that nested PCR be conducted on purified seminal compartments. IS-PCR is inadequate for detecting HIV in semen.

HIV and reproductive care--a review of current practice.

In developed countries, antiretroviral treatment has increased life quality and expectancy of HIV-infected individuals and led to a drop in mother-to-child transmission (MCT) risk to below 1%. Fertility has been shown to be reduced in both men and women with HIV. As a result of these factors, the demand for reproductive care in this population is rising. In discordant couples where the man is positive, sperm washing significantly reduces viral transmission risk to the uninfected female partner over unprotected intercourse. Positive women do not necessarily need specialised fertility treatment but should be monitored closely during pregnancy to minimise MCT risk.