The importance of pathological examination of the placenta in HIV-seropositive mothers has been recognized by health professionals for some years, as it allows several diseases to be diagnosed and also provides additional information and statistical data. In general, the placentas of mothers who are only HIV-seropositive have a normal macroscopic and microscopic appearance, as was the case in the patients selected for this study.5 However, as observed in our study, when samples from these normal placentas are analyzed using more specific and sensitive methods such as immunohistochemistry and molecular biology, they do not yield normal results.
The morphometric findings of this study also revealed changes in the diameter and perimeter of the placental villi of HIV-seropositive women that were suggestive of changes in villous maturation and may have been caused by the viral infection itself and/or even use of antiretroviral drugs.
The frequency of positive immunohistochemical results for microorganisms that cause intrauterine infections was the same in placentas from HIV-seropositive women and placentas from HIV-seronegative women, a finding that has been reported in the literature, and this fact could be particularly true in the normal placenta samples of HIV-seropositive pregnant.
Our results for immunohistochemical testing revealed the presence of HIV viral proteins (p24) in two samples even in the absence of histological lesions[6, 8]. The low number of cases in which p24 was detected (2/57) may be related to the low expression of this marker, since it can cross-react with endogenous antigens, leading to false positive or false negative results[3, 9]. However, the absence of positive results in the control group indicates that the antibody against p24 used in the test did not yield false positive results. Furthermore, studies have shown that the virus is not detected by the anti-p24 antibody in patients undergoing antiretroviral treatment. Although Lee and Tschening-Casper detected HIV sequences by PCR in placental cells from HIV-seropositive women who were receiving antiretroviral therapy, the placentas did not have any macroscopic or histological abnormalities, and immunohistochemical analysis failed to detect HIV-1 p24 antigens.
In our study we observed positive reactions in some cytotrophoblast cells and Hofbauer cells in the placentas that were positive for p24. When human placentas infected with HIV in utero or in vitro were analyzed by PCR, the HIV-1 virus was found to be distributed mainly in syncytiotrophoblast and Hofbauer cells . In addition, in several studies HIV-1 sequences were detected in both chorionic villi and trophoblasts in all the placentas analyzed, indicating that the virus sequences are always present in the placentas of these patients, even in the absence of morphological alterations and immunohistochemical expression characteristic of HIV[12, 13].
The elevated expression of ICAM-1 in the placentas of HIV-seropositive women observed in this study may indicate that this molecule can act as an inflammatory marker of this disease. Similarly, the elevated expression of this molecule in the vascular endothelium and the cytoplasm of Hofbauer cells in placental villi may indicate that the HIV virus has passed through the placental barrier, since ICAM is the molecule that mediates the entry of the virus into the macrophage. After the virus has penetrated the placental villi, it can adsorb to the surface of the ICAM molecules and mediate infections in T cells and Hofbauer cells. The presence of the virus in the placental villi is also suggested the by large number of CD8+ T cells in the HIV-seropositive women (p = 0.03).
There was no statistically significant difference in the expression of CD4+ cells between the two groups. CD4+ T cells were inconspicuous inside the chorionic villi. However, large numbers of Hofbauer cells were positive for CD4. CD4+ T cells inside the chorionic villi may mediate control of HIV infection and possible another infections in the placenta.
The binding of HIV viral particles to CD4+ receptors on the surface of Hofbauer cells after the particles have adsorbed to ICAM molecules may be the main transmission path for HIV inside the villous stroma in placentas. Once adsorbed to ICAM molecules, the viral particles can bind to Hofbauer cells by means of these molecules and use their migratory ability to reach the fetal vessels and then infect the conceptus's cells. 15 In light of our results for CD4+ count and ICAM-1 expression in placental villi, it is reasonable to suppose that entry of the virus into the fetal circulatory system may be intimately related to the CD4+ Hofbauer cells found in the villous stroma.
CD8+ T cells were present in greater quantities in the placentas of the HIV-seropositive women, probably because they are associated with anti-viral immune responses against HIV. Although the early villitis of unknown etiology are defined by villi injury, presence of maternal CD8+ T cells and presence of hyperplasic Hofbauer cells, there were no morphological changes compatible with villitis in this study. It might represent a very early stage of this entity which only would show immunohistochemical changes. Otherwise, these immunohistochemical findings might represent early changes of a specific villitis by HIV, but not a villitis of unknown etiology.
The increased immunohistochemical expression of CD8 in the placentas of HIV-seropositive patients may be intimately related to the presence of cytotoxic CD8+ T cells specific to HIV. Acute HIV infection is characterized by an increased number of cytotoxic CD8+ T cells involved in the control of the viremia. These lymphocytes play an important role in acute and chronic HIV infection, and an increase in their number may contribute significantly to protection against intrauterine transmission of HIV. Cells that contain HIV gp120 glycoprotein (an envelope antigen) in their plasma membrane are recognized by activated cytotoxic CD8+ T lymphocytes, which destroy not only the virus but also the whole cell by releasing cytolytic substances . As well as mediating cytolytic activity, CD8+ T cells can suppress HIV by secreting a factor, or set of factors, known as CD8 cell antiviral factor (CAF). CAF can block CCR5 and CXCR4 receptors by transcription regulation and consequently prevent viral replication . These CD8+ T cells are able to produce a cytokine response similar to that in adults. In congenital infection, newborns can develop a mature CD8+ T-cell response to cytomegalovirus similar to that detected in adults . Another factor that may be associated with the presence of CD8+ T cells in the placentas of HIV-seropositive women is that these cells are able to produce antiviral factors mediated by HIV-specific IgG stimulation. These specific antibodies are transferred efficiently from the mother to the conceptus through the placenta[16, 21].
The low expression of VCAM in both groups may be associated with the low stimulation of cytokines that mediate the expression of VCAM on the cell surface. If it is assumed that these placentas did not have active infections, then they did not need VCAM to be activated to recruit activated T cells.
Analysis of the viral load counts showed that 72% of the pregnant women had mean viral loads in excess of two thousand copies. This may indicate that the study population had not received effective treatment as in the majority of cases the viral load was higher than 50 copies (Table 4). Our results also showed that the viral loads did not affect expression of ICAM-1, CD8+ T-cell concentrations or the area or perimeter of the placental villi.
When we analyzed the medical records, we separated patients into those who had received treatment for an appropriate period and those who had not. When these two groups were analyzed, no correlation was found with CD8+ cells counts and the area or perimeter of the placental villi. In other words, antiretroviral therapy did not appear to have been responsible for the increased CD8+ cells count or the reduced perimeter and area of placental villi in the HIV-seropositive women. However, a correlation was identified between antiretroviral therapy and immunohistochemical expression of ICAM-1, which appeared to be higher in patients who had received this therapy. There was also a correlation between antiretroviral therapy and mean viral load, which, as expected, was smaller in this group although it was not less than 50 copies, the limit of detection defined by WHO.