Evidence for contamination as the origin for bacteria found in human placenta rather than a microbiota.

Until recently the in utero environment of pregnant women was considered sterile. Recent high-sensitivity molecular techniques and high-throughput sequencing lead to some evidence for a low-biomass microbiome associated with the healthy placenta. Other studies failed to reveal evidence for a consistent presence of bacteria using either culture or molecular based techniques. Comparing conflicting "placental microbiome" studies is complicated by the use of varied and inconsistent protocols. Given this situation, we undertook an evaluation of the in utero environment sterility using several controlled methods, in the same study, to evaluate the presence or absence of bacteria and to explain contradictions present in the literature. Healthy pregnant women (n = 38) were recruited in three maternity wards. Placenta were collected after cesarean section with or without Alexis® and vaginal delivery births. For this study we sampled fetal membranes, umbilical cord and chorionic villi. Bacterial presence was analyzed using bacterial culture and qPCR on 34 fetal membranes, umbilical cord and chorionic villi samples. Shotgun metagenomics was performed on seven chorionic villi samples. We showed that the isolation of meaningful quantities of viable bacteria or bacterial DNA was possible only outside the placenta (fetal membranes and umbilical cords) highlighting the importance of sampling methods in studying the in utero environment. Bacterial communities described by metagenomics analysis were similar in chorionic villi samples and in negative controls and were dependent on the database chosen for the analysis. We conclude that the placenta does not harbor a specific, consistent and functional microbiota.

Acute Placental Villitis as Evidence of Fetal Sepsis: An Autopsy Case Report.

Acute placental villitis is very rare and believed to reflect overwhelming fetal sepsis in utero, commonly caused by Escherichia coli or group B streptococci. We present a case of intrauterine fetal death associated with acute placental villitis and acute necrotizing chorioamnionitis by early-onset group B streptococcal infection. A 36-year-old woman presented with decreased fetal movement and fever at 21 weeks of gestation. Ultrasound demonstrated intrauterine fetal death. After delivery, the placenta revealed multifocal neutrophilic infiltration in chorionic villi, most prominently beneath the trophoblast basement membrane, which was also accompanied by acute necrotizing chorioamnionitis. Gram-positive microorganisms were detected in villous vessels as well as in the major organs of the fetus, which was consistent with Streptococcus agalactiae (group B) cultured from maternal blood. Acute placental villitis should be recognized as evidence of fetal sepsis that often has lethal clinical outcome, as compared to intra-amniotic infection associated with acute chorioamnionitis alone.

[DYNAMICS OF CHANGES IN THE NUMERICAL DENSITY OF PLACENTAL MACROPHAGES DURING UROGENITAL INFECTION IN EARLY PREGNANCY].

The changes in the numerical density of macrophages of maternal (basal decidua) and fetal (Kashchenko-Hofbauer cells) origin were studied in the placenta of women with opportunistic (Ureaplasma urealyticum and Mycoplasma hominis) and pathogenic (Chlamydia trachomatis) urogenital microflora. Histological study of placenta was performed and CD68-immunoreactive cells were detected immunohistochemically in the basal decidua and in the chorionic villi obtained during artificial abortions for non-medical reasons in the 6-8th week of pregnancy (n=136). The results showed no changes in the numerical density of macrophages of maternal origin and a significant decrease in the numerical density of macrophages in the stroma of the chorionic villi, which was associated in Chlamydial infection with a delayed, and in Ureaplasma and Mycoplasma infection - with an accelerated development of the villous tree.

PI3-kinase activation is critical for host barrier permissiveness to Listeria monocytogenes.

Invasion of nonphagocytic cells, a critical property of Listeria monocytogenes (Lm) that enables it to cross host barriers, is mediated by the interaction of two bacterial surface proteins, InlA and InlB, with their respective receptors E-cadherin and c-Met. Although InlA-E-cadherin interaction is necessary and sufficient for Lm crossing of the intestinal barrier, both InlA and InlB are required for Lm crossing of the placental barrier. The mechanisms underlying these differences are unknown. Phosphoinositide 3-kinase (PI3-K) is involved in both InlA- and InlB-dependent pathways. Indeed, InlA-dependent entry requires PI3-K activity but does not activate it, whereas InlB-c-Met interaction activates PI3-K. We show that Lm intestinal target cells exhibit a constitutive PI3-K activity, rendering InlB dispensable for InlA-dependent Lm intestinal barrier crossing. In contrast, the placental barrier does not exhibit constitutive PI3-K activity, making InlB necessary for InlA-dependent Lm placental invasion. Here, we provide the molecular explanation for the respective contributions of InlA and InlB to Lm host barrier invasion, and reveal the critical role of InlB in rendering cells permissive to InlA-mediated invasion. This study shows that PI3-K activity is critical to host barrier permissiveness to microbes, and that pathogens exploit both similarities and differences of host barriers to disseminate.

Nanobacteria and psammoma bodies: ultrastructural observations in a case of pathological placental calcification.

Nanobacteria are controversial infectious agents with nanometric size, the capacity to nucleate hydroxyapatite and grow in culture, and present in human diseases associated with calcification and psammoma bodies. The authors report a case of pathological placental calcifications associated with nanobacteria. Electron microscopy and electron energy loss spectroscopy imaging were used to recognize 160-nm-sized calcium-free bodies mainly presenting as extracellular fibrillary tangles and 500-nm-sized calcified bodies; they encrusted the syncito-trophoblast basal membrane and aggregated into miniaturized psammoma bodies. Nanobacteria may be composed of a prionoid protein with self-assembling and self-propagating abilities whose growth is associated with the formation of psammoma bodies.

Immunohistochemical study of the morphological changes in placental villi from fetal membranes infectious disease.

UNLABELLED: Many types of infection cause placental changes but sometimes the etiological cause may be difficult to prove. Infections may ascend from endocervical canal or they may reach placenta hematogenously through the maternal blood. Typically, placenta of "the amnionic sac infection syndrome" is an immature placenta. Complex cellular mechanisms are involved in all types of infection that often are associated with placental insufficiency. OBJECTIVES: The aim of this study was to evaluate cellular changes induced by the hypoxic conditions due to infectious disease in the placental villous structures, especially in the trophoblast layer and vascular bed. MATERIAL AND METHODS: In order to label the trophoblast layer we used anti-cytokeratin cocktail AE1/AE3. Antibodies anti-VEGF and anti-c-ErbB4 were used for the evaluation of tissue response under hypoxic conditions and its involvement in villous remodeling. RESULTS: Chorion villi from placentas with histopathological features of insufficiency due to infectious etiology show an intense immunostaining for VEGF in the trophoblast, vessel walls and some stromal cells, namely Hofbauer cells. Villous trophoblast from the infected placenta expresses c-ErbB4 receptor. CONCLUSIONS: Overexpression of VEGF and c-ErbB4 is needed for the involvement of trophoblast layer in villous remodeling processes in order to maintain placental functionality under the effects of the inflammatory cascade.

Possible role of bacterial and viral infections in miscarriages.

OBJECTIVE: To determine the role of infections in miscarriages. Chorionic villi from aborted material were subjected to cytogenetic evaluation and analyzed for the presence of Chlamydia trachomatis, Ureaplasma urealyticum, Mycoplasma hominis, human cytomegalovirus (HCMV), adeno-associated virus (AAV), and human papillomaviruses (HPV). DESIGN: Retrospective study. SETTING: University hospital and academic research institution. MAIN OUTCOME MEASURE(S): Karyotyping and detection of bacterial and viral DNA by means of polymerase chain reaction (PCR) in placenta specimens. RESULT(S): In 54 (50%) of 108 samples the karyotype was normal, in 38 (35%) samples it was abnormal, and in 16 (15%) samples karyotype was undetermined. No U. urealyticum, M. hominis, HCMV, or AAV-2 DNA was detected, while C. trachomatis DNA was detected in one (1%) and HPV DNA in eight (7%) samples. No significant correlation of HPV-positive findings with karyotype status was established. CONCLUSION(S): Our findings do not support a role of C. trachomatis, U. urealyticum, M. hominis, HCMV, or AAV infections in miscarriages during the first trimester of pregnancy. However, further investigation should be made to determine a possible involvement of HPVs in the development of genetic abnormalities of the fetus and in miscarriages.

Villitis of known origin: varicella and toxoplasma.

Chronic villitis is a common condition in human placentae. In some cases an infectious cause can be demonstrated, such as infection with cytomegalovirus and rubella virus. Most often it is of unknown aetiology, the so-called VUE (villitis of unknown aetiology). We describe two cases with identification of specific infectious agents, each demonstrating previously unreported findings, i.e. persistent varicella antigen in the villi in case 1, and presence of toxoplasma cysts in Wharton's jelly in case 2. The identification of the pathogens, varicella virus and toxoplasma, would easily have been overlooked in routine study of the placenta and were possible because of clinical suspicion.

Detection of congenital cytomegalovirus infection by using chorionic villi of the early pregnancy and polymerase chain reaction.

OBJECTIVE: To detect congenital cytomegalovirus (CMV) infection of chorionic villi in early pregnancy. METHODS: Extraction of DNA of chorionic villi and amplification of the gene of major immediate-early (MIE) antigen of CMV using a polymerase chain reaction (PCR). RESULTS: Sixty-eight specimens of chorionic villi and 16 specimens were positive for CMV infection by PCR. The incidence of congenital CMV infection in the first trimester of pregnancy was 23.5%. CONCLUSIONS: The risk of transmission of CMV from mother to fetus in early pregnancy is very high and potential CMV carriers may transmit CMV to their fetus in early pregnancy.

Absence of cytomegalovirus in gestational tissue in recurrent spontaneous abortion.

There have been conflicting reports regarding the association of cytomegalovirus (CMV) and recurrent spontaneous abortions. It is difficult to assess the role of CMV in the endometrium by histology alone, since the characteristic cytomegalic virocytes are often scarce or absent in this site. Our purpose was to use the polymerase chain reaction (PCR) to detect cytomegalovirus in gestational tissue of women with recurrent spontaneous abortions. DNA was extracted from 25 samples of paraffin-embedded, formalin-fixed gestational tissue from 21 women with at least three unexplained spontaneous abortions (mean, 3.4). DNA from an unstained paraffin section of each specimen was amplified using nested, multiplex PCR specific for the late antigen and the major immediate early genes of CMV. The assay used has a demonstrated level of sensitivity on the order of 10(-2) virocytes per square centimeter of 4-microM paraffin section. Intact DNA was successfully isolated from 21 specimens in 18 patients. Histologic features of CMV infection were completely absent from these cases, and none of these specimens contained evidence of cytomegalovirus DNA. These findings suggest that CMV infection of gestational tissue is not a common direct cause of recurrent spontaneous abortions.

Biochemical characterization of a reverse transcriptase activity associated with retroviral-like particles isolated from human placental villous tissue.

The presence of budding type-C retroviral-like particles in normal placental trophoblast, particularly at the basal surface of the placental syncytiotrophoblast, is well documented. Retroviral-like particles were isolated from human placental villous tissues using isopycnic sucrose gradient centrifugation. Reverse transcriptase activity (RTase) associated with isolated retroviral-like particles was characterised using a combination of synthetic template-primers. These studies showed that RTase activity was more specific with poly(rC).oligo(dG)12-18 than poly(dC).oligo(dG)12-18. Furthermore, activity was detected with poly(rCm).oligo(dG)12-18, a template-primer which has previously been shown to be specific for retroviral RTase. Maximum activity appeared at a sucrose density between 1.15-1.17 g/ml, characteristic of enveloped retroviral particles. Electron microscopy examination of the gradient purified particles revealed morphology and size similar to other retroviruses. Endogenous retroviral particles were isolated from 26 out of 32 (81%) first-trimester placental villous tissue extracts. These particles are likely to be product of endogenous proviral sequences present in the germline of humans. Although these studies showed presence of intact retroviral particles in placental tissues, it was not possible to propagate the isolated particles in vitro. All attempts to propagate placental retroviral particles by co-cultivation with human cells (U937 and JAr choriocarcinoma cells) and long term placental villous tissue explant cultures were unsuccessful. Subsequently, there was no evidence of retroviral-like particles or RTase activity in these cell cultures, including after stimulation with 5'-azacytidine or dexamethasone, chemical agents known to stimulate particle production in virus-infected lines.

[Placental aspergillosis: myth or reality? Apropos of a case with fetal death in utero]. / L'aspergillose placentaire: mythe ou réalité? A propos d'une observation avec mort foetale in utero.

The authors report an original case-history of massive aspergillosis of the placenta that occurred in a 24 year old primigravida who had had no previous history or clinical changes in pregnancy. It caused fetal death in utero with retention and maceration of the fetus. Macroscopic examination showed that the left lip was cleft and that the placenta was studded with isolated confluent diffuse whitish granulations. Histologic examination made us think that these appearances were those of aspergillar granulomas occurring even in the placental villi and intravillous spaces. Laboratory findings showed that there was Aspergillus Niger in the blood of the mother. A wide search of the literature failed to find any case in humans. On the other hand aspergillosis occurs frequently in animals causing intrauterine growth retardation and prematurity with abortion. There is great economic loss as a result. Why the animal placenta should be susceptible to infection of aspergillosis and how it acquires it is discussed! Finally the association of aspergillosis of the placenta with a cleft lip found in our case, is unique and one wonders if there is any relationship.

Pathology and human immunodeficiency virus expression in placentas of seropositive women.

The pathology of term placentas from seropositive human immunodeficiency virus (HIV)-infected and seronegative women was investigated by routine histologic, immunocytochemical, and in situ hybridization techniques. Placentas were evaluated for evidence of villitis, chorioamnionitis, and funisitis. Membranes, trophoblast, and decidua were also examined by immunohistochemistry using monoclonal HIV p24 antibody. Twenty placentas were evaluated by combined immunochemical and in situ hybridization techniques, using a 35S-labeled RNA probe complementary to the 3' long terminal repeat and envelope region of HIV-1. HIV-seropositive placentas did not show significant villitis; however, the incidence of chorioamnionitis increased (P less than .01). HIV antigens and nucleic acids were identified in the trophoblast of 10% of the placentas that also showed chorionitis. Term HIV-positive placentas may show histologic changes that may or may not be directly related to the virus. Analysis of tissues from earlier gestational placentas may prove more informative in clarifying the mechanism of maternal-fetal HIV transmission.

HIV proteins in normal human placentae.

Cryostat sections of human normal term placentae were studied for evidence of immunopathology by using antibodies to lymphocytes, macrophages, platelets, and coagulation factors. Areas of so-called chronic villitis of unestablished etiology were identified in all placentae. The same tissues were examined for HIV protein antigens gp120, p17, p24, and gp41. No evidence for gp41 was found. Antigens gp120 and p17 were identified in normal chorionic villi in vimentin-positive fibroblast-like cells and in endothelium, respectively. Antigen p24 was localized to HLA-DR positive cells that morphologically resembled macrophages in areas of villitis. The distribution of gp120 and p17 was similar to that observed for tissue factor. These findings prompted speculation that retroviral proto-oncogenes that are known to encode for certain placental receptors could be involved in the presentation of tissue factor, and that gp120 may be a hitherto unrecognized immunobiological mechanism for the blockade of CD4 on maternal lymphocytes if and when such cells gain entrance to chorionic villi.

Diagnosis of foetal rubella virus infection by polymerase chain reaction.

We have used the polymerase chain reaction (PCR) to provide a very sensitive and unequivocal test for diagnosis of foetal rubella virus infection. RNA extracted from biopsy specimens (chorionic villi), placenta or products of conception was reverse-transcribed using a rubella virus-specific oligonucleotide primer and the cDNA was amplified by PCR. The specificity of the amplified fragment was confirmed by Southern blotting. Detection of rubella virus infection in five out of 41 clinical specimens examined by this approach was shown to be entirely consistent with clinical history and other methods of laboratory diagnosis in current use. The sensitivity of the test and the unequivocal nature of the results obtained could be invaluable in providing prenatal counselling following rubella virus infection during pregnancy.