aDepartment of Obstetrics and Gynaecology and bLaboratory of Human Genetics, University of Milan Medical School, Milan, Italy; cFondazione Luigi Villa, Centro Studi di Patologia Molecolare Applicata alla Clinica, Ospedale Maggiore, 20122 Milan, Italy; and dDepartment of Epidemiology, Institute of Child Health, London, UK.
Sponsorship: This work was supported by grant nos. 9402-14 and 40A.0.98 from the Istituto Superiore di Sanità, Progetti AIDS 1996–1997.
Received: 8 December 1999; accepted: 22 December 1999.
Caesarean section performed before labour and with intact membranes (elective caesarean section) reduces the risk of vertical transmission of HIV-1 by over 70%, whereas procedures carried out during labour or after the rupture of membranes have no protective effect [1,2]. This suggests that the passage of infected blood from the mother to the fetus occurring during labour could be the most important route of congenital HIV-1 infection. To understand the impact of labour on materno–fetal cellular traffic, we tested cord blood samples obtained after vaginal or elective caesarean delivery for the presence of maternal DNA using variable number of tandem repeats analysis and detection by chemiluminescence, a technique capable of detecting one genetically different cell out of 15003.
We enrolled HIV-negative mothers with singleton vaginal or elective caesarean deliveries taking place at term. The indication for abdominal delivery was a previous caesarean section in all women, except for one twin and one triplet case. The women consented to their peripheral blood sampling (10 ml) as well as cord sampling (5 ml). The cord was doubly clamped immediately after birth or after extraction of the fetus through the uterine incision, repeatedly rinsed to eliminate contamination with maternal blood and sampled between the two clamps. To achieve surgical and collecting standards, all procedures were performed by one author (A.E.S.) and all sampling was carried out by a second clinician (V.S.). We tested 30 samples after spontaneous deliveries and 27 from caesarean sections (including one twin and one triplet case, all with separated placentas). In 13 out of 30 of the spontaneous births and in 11 out of 27 caesarean section deliveries it was possible to sample both venous and arterial cord blood. DNA was extracted from all samples with the standard phenol-chloroform method. We amplified all samples using primers amplifying four polymorphic loci (apolipoprotein B genes 3 ′, D1S80, IG-JH, TP0) searching for the informative loci (one maternal allele different from the child alleles) to be used for the biotinylated polymerase chain reaction (PCR). PCR, electrophoresis, blotting and chemiluminescent detection of PCR products were as described previously [3–5]. To assess the sensitivity of the tests and to determine the magnitude of the contamination of cord blood samples with maternal cells, artificial mixes of DNA from two individuals with alleles of different sizes for each polymorphism were amplified in parallel with the other samples and then gel loaded at the dilutions 1 : 500 and 1 : 1500.
The presence of both maternal alleles was detected in four out of 30 cord blood samples obtained after spontaneous delivery, indicating a contamination rate of 13.3%. In the 30 infants delivered by caesarean section, five cord blood samples tested positive for the presence of maternal DNA, with a not statistically significantly different detection rate of 16.6%. No cord blood sample from the twin and the triplet cases were contaminated with maternal DNA. We also tried to assess the concentration of maternal DNA in the nine positive samples by visually comparing the intensity of the control bands with that of the positive sample as previously reported . In the four cases of vaginal deliveries the mean level of contamination was 1:735 (range 550–1500) and in the five cases from caesarean section 1 : 730 (range 50–1300). In four cases, one vaginal birth and three caesarean sections, with cord blood testing positive for maternal DNA, we were able to analyse both venous and arterial samples and, in all four cases, both vascular districts tested positive for maternal DNA and with identical levels of detection.
Our data indicate that materno–fetal haemorrhage determined in cord blood samples at birth, occurs in 15% of cases and cannot be avoided by delivering the child before labour ensues. Cord sampling at birth cannot determine whether the passage of maternal blood takes place before or during delivery, but the similar rate of detection and the amount of maternal DNA after vaginal and elective caesarean birth indicates that labour does not significantly influence this passage. These data are evidence against the possibility that the contractions of labour may induce placental lesions leading to materno–fetal blood transfusion. Our results indicate that only a fraction of mother-to-child transmissions of HIV-1 may be caused by the transfer of infected maternal cells in the fetal circulation. Our findings suggest that free virus, rather than cell-integrated HIV-1, can be passed on to the fetus or released by infected placental cells during labour and can be the major determinant for fetal infection [6,7].
Augusto E Sempriniad
Chiara De Andreisbc
Silvia M Sirchiabc
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