Transcervical Sampling Research Articles

A Novel Paradigm for Non-Invasive Prenatal Genetic Screening: Trophoblast Retrieval and Isolation from the Cervix (TRIC).

As the prevalence of pregnancies with advanced maternal age increases, the risk of fetal chromosomal abnormalities is on the rise. Therefore, prenatal genetic screening and diagnosis have become essential elements in contemporary obstetrical care. Trophoblast retrieval and isolation from the cervix (TRIC) is a non-invasive procedure that can be utilized for prenatal genetic diagnosis. The method involves the isolation of fetal cells (extravillous trophoblasts) by transcervical sampling; along with its non-invasiveness, TRIC exhibits many other advantages such as its usefulness in early pregnancy at 5 weeks of gestation, and no interference by various fetal and maternal factors. Moreover, the trophoblast yields from TRIC can provide valuable information about obstetrical complications related to abnormal placentation even before clinical symptoms arise. The standardization of this clinical tool is still under investigation, and the upcoming advancements in TRIC are expected to meet the increasing need for a safe and accurate option for prenatal diagnosis.

O-259 1.\tComparison of endometrial versus vaginal microbiota in 71 infertile patients

Abstract Study question 1.Mostly, endometrial microbiome probes are acquired by trans-cervical sampling using a “pipelle”. However, it is unclear, whether this approach “contaminates” the endometrial material. Summary answer 1.Vaginal and endometrial microbiome samples are significantly different from each other with respect to (alpha-)diversity showing the endometrial community to be more polymicrobial. What is known already 1. The female genital tract is colonized by a continuum of microbiota, ranging from the typically more diverse, but lower-biomass, bacterial communities in the endometrium to the typically less diverse, but higher-biomass, communities in the vagina (Chen et al., 2017). In reproductive-age women, vaginal bacterial communities are mainly dominated by lactobacilli (Ravel, 2011). By contrast, the composition of the endometrial microbiome has remained less well-characterized. With the more recent introduction of next generation sequencing a more quantitative and comprehensive picture of the composition of endometrial bacterial communities has begun to emerge (Baker et al., 2018). Study design, size, duration 1. The study was conducted at “novum, Zentrum für Reproduktionsmedizin”, Essen / Duisburg, Germany. Of 76 enrolled patients undergoing treatment,73 provided both vaginal swab and endometrial biopsy samples from the same sequencing run between February 2020 and March 2021. All analyses were carried out retrospectively. Information about pregnancy or delivery were extracted from medical records or obtained by telephone or email interviews conducted through June 2022. Participants/materials, setting, methods 1.Amplification of V1-V2 16S rDNA regions was carried out at “dus.ana, Düsseldorf Analytik”, Germany, using the QIAseq 16S screening and regional panels. CLC Microbial Genomic Workbench was used to assess bacterial community composition, alpha diversity, and microbial community structure (PCoA). Pairs of complete corresponding vaginal and endometrial microbiota data were available for 71 patients (two patient was excluded due to too low read counts in the vaginal or the endometrial probe, respectively). Main results and the role of chance 1.Patients exhibiting a mean age of 35 years (range 26 – 42) and a mean body mass index (BMI) of 24,5 (range 18,8 to 38,7). 20 (28,2 %) patients were diagnosed with chronic endometritis (more than 1 plasmacyte per HPF) and 15 (21,1 %) with endometriosis. 40 study participants were found to have undergone past miscarriages, and 11 study participants were found to have given birth to 15 children. 2.Abundance analyses showed differences between vaginal and endometrial samples for the Lactobacillus sp., most pronounced for L. jensenii and L. gasserie. The group of non-lactobacilli was only slightly enlarged in the endometrial group, encompassing mainly G. vaginalis. With regard to diversity (Shannon entropy), both microbiota are significantly different (Kruskal-Wallis test). In addition, distribution of the genital microbial communities illustrated by PCoA indicated that the vaginal samples were clustered together showing variance only in one dimension. However, the endometrial samples were far away from each other and grouped very disperse. Limitations, reasons for caution 1.Group of infertile patients of an IVF center with different underlying clinical diagnosis studied in a retrospective setting. Wider implications of the findings 1.Trans-cervical sampling with a “pipelle” provides an endometrial microbiome which is obviously a more polymicrobial community - with a higher diversity, that may harbor more biological heterogeneity, and hence functionality – than the corresponding vaginal microbiome. Further analyses studying the correlation between sequencing and clinical data are ongoing. Trial registration number 2021-1448

O-303 Recurrent pregnancy loss is associated with unfavorable composition of endometrial and vaginal microbiota

Abstract Study question Does endometrial or vaginal microbiota composition associate with recurrent pregnancy loss (RPL)? Summary answer Reduced relative abundance of Lactobacillus crispatus in endometrium and increased relative abundance of Gardnerella vaginalis in endometrium and vagina were associated with RPL. What is known already Dysbiotic vaginal microbiota associates with sporadic miscarriage and the depletion of lactobacilli in the endometrium has been associated with poor IVF outcomes, but their association with RPL has not been studied. Study design, size, duration A nested case-control study of 47 women investigated for RPL and 39 healthy control women without a history of pregnancy loss were recruited between March 2018 and December 2020 at a university hospital. Endometrial biopsies and vaginal samples were collected from the women 6-8 days after positive ovulation test result. Participants/materials, setting, methods The endometrial and vaginal microbiota compositions, analysed using 16S rRNA gene amplicon sequencing, were compared between RPL women and controls, and between individual vaginal and endometrial samples. The mycobiota composition was analysed using internal transcribed spacer 1 amplicon sequencing for a descriptive summary. The models were adjusted for BMI, age, and parity. False discovery rate -corrected P-values (q-values) were used to define nominal statistical significance at q< 0.05. Main results and the role of chance Lactobacillus crispatus was less abundant in the RPL women’s endometrial samples compared to controls (mean relative abundance 17.2% vs. 45.6%, q= 0.04). Gardnerella vaginalis was more abundant in RPL women than in controls in both endometrial (12.4% vs. 5.8%, q< 0.001) and vaginal samples (8.7% vs. 5.7%, q< 0.01). The individual vaginal and endometrial microbial compositions correlated strongly (R = 0.85, P< 0.001). Fungi, mostly Candida albicans, were detected in 22% of the endometrial and 36% of the vaginal samples. Limitations, reasons for caution Limitations include the transcervical sampling technique with a potential risk of cervicovaginal contamination of endometrial samples, the loss of a proportion of the samples from statistical analyses due to their low read count, and the presentation of the 16S rRNA sequencing results in relative rather than absolute abundances Wider implications of the findings Our findings suggest that an unfavorable endometrial microbiota may be a novel risk factor for RPL. Further studies should elucidate the mechanisms, how microbiota may predispose to RPL, possibilities to modify microbiota by treatments, and the effects of such treatments on the success of future pregnancies in couples having RPL. Trial registration number not applicable

Methods of detection and isolation of trophoblast cells from trans-cervical specimens – a historical overview

Abstract Trophoblast cells can be detected and isolated from the cervical epithelial cells obtained via various techniques of trans-cervical samples collection such as a mucus aspiration, endocervical lavage, or standard cervical brushing in the early first trimester, even from the 5 weeks’ gestation. Isolated fetal cells can be used in the early prediction of fetal sex, prenatal diagnostics of the most common aneuploidies, and any other genetic abnormalities. Nevertheless, the collection of trophoblastic cells has limited efficacy compared to currently used methods of detection of free fetal DNA in maternal circulation or other protocols of invasive prenatal diagnostics available at later stages of pregnancy. In the past years, trans-cervical cell samples were collected mainly in women before planned pregnancy termination. The early trophoblastic cells isolation from women in ongoing pregnancies opens new perspectives for further studies focused on the elucidation of pathophysiology of numerous pregnancy-related complications.

Risk factors for failed chorionic villus sampling: results of a 4-year retrospective study

Objectives Chorionic villus sampling (CVS) allows for earlier results for aneuploidy or genomic abnormalities compared to amniocentesis. Nevertheless, the inability to provide complete results has been described as being more frequent with CVS. This study was conducted in order to identify risk factors for such failures. Study design A retrospective single-center study was performed from January 2014 to December 2018. Participants were divided into two groups depending on whether complete CVS results were issued (“successful CVS group”) or not (“failed CVS group”). Failure affected preliminary short-term cultures, long-term cultures, or both. Results During the study period, 214 CVS were performed, 73 (34%) of which were classified in the failed CVS group. We observed significant intergroup differences between the successful and failed CVS groups for four variables: BMI (respectively 23.9 [±5.88] and 25.9 [±6.13] kg/m2), term at sampling (12.9 [±1.35] and 12.6 [±1.09] weeks gestation), trophoblastic location (posterior in 49 [40%] and 37 [66%] cases), and sampling approach (transcervical in 54 [43%] and 36 [64%] cases) (p < .05). In a stepwise binary logistic regression analysis, higher BMI, posterior trophoblastic location, and transcervical sampling approach were the only variables negatively influencing CVS success, with respective aOR [95% CI] of 0.947 [0.898; 0.996], 0.322 [0.160; 0.634], and 0.466 [0.238; 0.900]. Conclusions In the presence of CVS failure risk factors, a discussion could be initiated regarding a deferred amniocentesis as a first option.

Characterisation of the human uterine microbiome in non-pregnant women through deep sequencing of the V1-2 region of the 16S rRNA gene.

Background. It is widely assumed that the uterine cavity in non-pregnant women is physiologically sterile, also as a premise to the long-held view that human infants develop in a sterile uterine environment, though likely reflecting under-appraisal of the extent of the human bacterial metacommunity. In an exploratory study, we aimed to investigate the putative presence of a uterine microbiome in a selected series of non-pregnant women through deep sequencing of the V1-2 hypervariable region of the 16S ribosomal RNA (rRNA) gene.Methods. Nineteen women with various reproductive conditions, including subfertility, scheduled for hysteroscopy and not showing uterine anomalies were recruited. Subjects were highly diverse with regard to demographic and medical history and included nulliparous and parous women. Endometrial tissue and mucus harvesting was performed by use of a transcervical device designed to obtain endometrial biopsy, while avoiding cervicovaginal contamination. Bacteria were targeted by use of a barcoded Illumina MiSeq paired-end sequencing method targeting the 16S rRNA gene V1-2 region, yielding an average of 41,194 reads per sample after quality filtering. Taxonomic annotation was pursued by comparison with sequences available through the Ribosomal Database Project and the NCBI database.Results. Out of 183 unique 16S rRNA gene amplicon sequences, 15 phylotypes were present in all samples. In some 90% of the women included, community architecture was fairly similar inasmuch B. xylanisolvens, B. thetaiotaomicron, B. fragilis and an undetermined Pelomonas taxon constituted over one third of the endometrial bacterial community. On the singular phylotype level, six women showed predominance of L. crispatus or L. iners in the presence of the Bacteroides core. Two endometrial communities were highly dissimilar, largely lacking the Bacteroides core, one dominated by L. crispatus and another consisting of a highly diverse community, including Prevotella spp., Atopobium vaginae, and Mobiluncus curtisii.Discussion. Our findings are, albeit not necessarily generalizable, consistent with the presence of a unique microbiota dominated by Bacteroides residing on the endometrium of the human non-pregnant uterus. The transcervical sampling approach may be influenced to an unknown extent by endocervical microbiota, which remain uncharacterised, and therefore warrants further validation. Nonetheless, consistent with our understanding of the human microbiome, the uterine microbiota are likely to have a previously unrecognized role in uterine physiology and human reproduction. Further study is therefore warranted to document community ecology and dynamics of the uterine microbiota, as well as the role of the uterine microbiome in health and disease.

Detection of Fetal Aneuploidies by QF-PCR in Transcervical Cell Samples

Objective. To evaluate the accuracy in the diagnosis of aneuploidies of a quantitative fluorescent polymerase chain reaction (QF-PCR) assay on trophoblastic cells recovered from transcervical cells samples (TCCs) collected by intrauterine lavage (IUL). Study Design. DNA analysis was performed on cells of seemingly trophoblastic origin isolated from IUL samples collected prior to first trimester termination of pregnancy. The analysis was performed by multiplex QF-PCR, using a panel of 29 polymorphic short tandem repeats (STRs) for the chromosomes X, Y, 21, 13, and 18. Results. The QF-PCR analysis on placental samples revealed that among the three cases studied there were two cases of trisomy 21 and one case of monosomy X; the comparison of peak profiles obtained from IUL, placental, and maternal samples confirmed the diagnosis of aneuploidy in all three cases. Conclusion. This study suggests that the detection of chromosomal aneuploidies in micromanipulated TCC samples can be achieved by QF-PCR amplification of selected highly polymorphic and chromosome specific markers. With respect to standard karyotype, QF-PCR analysis has the limitation that only numerical abnormalities of selected chromosomes can be detected but retains the advantages of being quicker, less expensive, and less lab demanding.

Retrieval of trophoblast cells from the cervical canal for prediction of abnormal pregnancy: a pilot study

Fetal cells are shed from the regressing chorionic villi and it is possible to retrieve extravillous cytotrophoblast cells by transcervical sampling. The abundance of trophoblast cells in transcervical samples suggests that this non-invasive approach could distinguish between normal and abnormal pregnancies, such as an ectopic pregnancy (EP) and blighted ovum (BO). We aim to identify and quantify fetal trophoblast cells in the cervical canal during the first trimester to assess their usefulness to predict an abnormal pregnancy. Patients, age 18-45, presenting with a normal intrauterine pregnancy (IUP; n = 37), diagnosis of EP (n = 10) or BO (n = 5) were enrolled for collection of transcervical specimens using a cytobrush and fixative rinse. Non-pregnant, nulliparous women (n = 7) were included as negative controls. Cells were cleared of mucus by acidification, prepared on microscope slides and labeled with a monoclonal antibody recognizing the trophoblast marker, human leukocyte antigen (HLA)-G. HLA-G positive and negative cells were counted to calculate the ratio of trophoblast cells to total cervical cells. Trophoblast cells were observed in 35/37 normal IUP, 6/10 EP and 4/5 BO specimens. The average frequency of HLA-G positive cells in the normal IUP cervical samples was approximately 1 in 2000, which was 4-fold higher than samples from patients with EP or BO (P < 0.001). Receiver operating characteristic analysis showed that EP and BO pregnancies were distinguishable from normal pregnancies with 93% sensitivity, 95% specificity, 97% positive predictive value and 87% negative predictive value. This pilot study presents evidence that trophoblast cells can be reliably obtained and identified among cervical cells in the first trimester by immunohistochemical staining for HLA-G, and suggests for the first time that abnormal pregnancies may be predictable based on the abundance of trophoblast cells in the cervical canal.

Prediction of abnormal pregnancy by trophoblast assessment in first trimester transcervical samples

OBJECTIVE: Ectopic pregnancy complicates about 1-2% of all pregnancies and occurs when the developing blastocyst implants at a site other than the fundus of the uterine cavity, most commonly in the fallopian tube. Delayed clinical diagnosis of this abnormality can result in a dismal maternal outcome. The presence of fetal trophoblast cells in the cervical canal during the first trimester suggested a non-invasive approach for predicting abnormal pregnancy through transcervical sampling.

Prenatal Diagnosis of Common Aneuploidies in Transcervical Samples Using Quantitative Fluorescent-PCR Analysis

The aim of this study was to test the feasibility of diagnosing common fetal chromosomal aneuploidies using quantitative fluorescent (QF)-PCR on transcervical cell (TCC) samples collected in the first trimester of pregnancy by means of intrauterine lavage (IUL). A total of 181 TCC samples were retrieved from pregnant women between 5 and 12 weeks of gestation, immediately before elective termination of pregnancy, at which time corresponding placental tissue and maternal blood specimens were also obtained. Isolation of trophoblastic cells by micromanipulation was attempted in all TCC samples. Micromanipulated specimens were analyzed by multiplex QF-PCR, including short tandem repeats for the chromosomes X, Y, 21, 18, and 13. The micromanipulation was successful in 152 of 181 cases (84.8%) where chorionic villous filaments and/or cell clumps of seeming trophoblastic origin could be isolated. All 152 samples were tested by QF-PCR analysis and peaks of paternal origin could be documented in all cases. Two cases of trisomy 21 and two cases of monosomy X0 were detected by means of QF-PCR assay, in accordance with the results obtained in corresponding placental samples. This study provides evidence that the use of multiplex QF-PCR amplification of selected microsatellites could be applied to micromanipulated TCC samples and in particular to IUL samples, which often contain trophoblastic cells, for the detection of chromosomal aneuploidies. The approach described in this study appears, therefore, a very promising tool toward non-invasive prenatal genetic diagnosis in the early stage of gestation.

Transcervical cell sampling: a need for clear terminology

has for the first time, carefully compared different sam-pling techniques, and is welcomed. We concur that the genericterm ‘transcervical cell sampling’ is correct, but for any subse-quent publications we encourage researchers to carefully detailthe collection method.There is no doubt that micromanipulation techniques based oncell morphology can successfully isolate fetal cells, but semi-automated methods such as laser capture microdissection

Use of the Quantitative Fluorescent-PCR Assay in the Study of Fetal DNA from Micromanipulated Transcervical Samples

The purpose of this study was to evaluate the validity of the combined use of micromanipulation and quantitative fluorescent (QF)-PCR assay for the identification of fetal elements in transcervical cell (TCC) samples collected in early pregnancy. TCC samples were obtained by intrauterine lavage (IUL) in 113 pregnant women who were between 7 and 12 weeks pregnant before termination of pregnancy. All IUL samples were screened under an inverted microscope, at which time the isolation of fetal cells by micromanipulation was attempted. QF-PCR assay, using 9 small tandem repeat (STR) markers for chromosomes 13, 18, 21, X, and Y, was performed in all specimens to identify fetal cells in TCC samples and the corresponding placental tissue and blood specimens. TCC samples from male fetuses in which either the micromanipulation or QF-PCR analysis were unsuccessful, were studied with fluorescent in situ hybridization (FISH), using probes for X and Y chromosomes. Isolation of supposed fetal material from IUL samples was carried out by means of micromanipulation in 93 cases (82.3%), where discernible chorionic villous filaments or cell clumps of probable trophoblastic origin were present. The QF-PCR analysis was performed in all 93 IUL samples and paternal peaks could be documented in 88 cases (94.6%) thus confirming the presence of fetal cells. Thirteen cases negative to micromanipulation and derived from male fetuses and four male cases not informative with QF-PCR analysis, after micromanipulation, were then tested with FISH assay using probes for sexual chromosomes. In six samples, rare (2-3%) male fetal cells were detected. Considering the combined results obtained from QF-PCR and FISH assays, the overall fetal sexing was correct in 83.2% of cases (94 of 113). This study provides evidence that fetal cells are present in a high proportion of IUL samples. Micromanipulation appears to be an extremely efficient method for the isolation of trophoblastic elements. This study also confirms the potential of IUL as a possible alternative to the traditional prenatal diagnostic procedures for the recovery of fetal cells in precocious stage of gestation, and validates the combination of the isolation of such fetal elements by means of micromanipulation and analysis with the QF-PCR assay for the identification of the most frequent prenatal chromosomal aneuploidies.

Use of the Quantitative Fluorescent-PCR Assay in the Study of Fetal DNA from Micromanipulated Transcervical Samples

Use of the Quantitative Fluorescent-PCR Assay in the Study of Fetal DNA from Micromanipulated Transcervical Samples

HLA-G positive trophoblastic cells in transcervical samples and their isolation and analysis by laser microdissection and QF-PCR.

To assess the frequency of cytotrophoblastic cells in endocervical samples collected by lavage at early stages of gestation using a specific anti-HLA-G McAb (G233). From a set of four selected samples, cells identified by immunostaining were collected by laser microdissection and then tested by quantitative fluorescent polymerase chain reaction (QF-PCR) for the presence of paternally derived DNA markers, in order to establish their fetal origin. Syncytial fragments and cytotrophoblastic cells from 23 transcervical samples were identified by immunostaining with McAb G233 reacting against HLA-G antigen and with antibodies against cytokeratin. Slides from the same samples were also tested by fluorescent in situ hybridization (FISH), while selected samples were analysed by QF-PCR. Slides from four samples retrieved from mothers with male fetuses were immunolabelled and then cytotrophoblastic cells, syncytial fragments and maternal epithelial cells were collected by laser microdissection and tested by QF-PCR. All endocervical samples retrieved from mothers with male fetuses were found to contain some cells with chromosome Y-specific signals when tested by FISH. Using McAb anti- HLA-G, cytotrophoblastic cellular elements were detected in about 50% of the samples. From four samples, cellular elements identified by immunostaining as cytotrophoblast or syncytial fragments were collected by laser microdissection and shown to be of fetal origin when tested by QF-PCR for the presence of fetal DNA markers. These results confirm that, during an early phase of gestation, fetal cells are released in the lower uterine cavity and that they can be isolated and analysed for prenatal diagnosis of single gene defects and aneuploidies.

Strategies for the isolation and detection of fetal cells in transcervical samples.

The aim of the study was to evaluate the detection of fetal cells from transcervical samples, collected in early pregnancy, by means of different molecular techniques. The value of the isolation of trophoblasts using an inverted microscope, also referred to as micromanipulation, is discussed. All the 89 specimens were obtained by intrauterine lavages before termination of pregnancy (TOP), between 7 and 12 weeks of gestation. Micromanipulation was carried out in a subgroup of 57 for the isolation of fetal material. Fetal sexing was achieved by FISH (fluorescent in situ hybridisation) using fluorescently labelled probes for X and Y chromosomes and by polymerase chain reaction (PCR). Male samples were also investigated for aneuploidy of the chromosome 21. Quantitative fluorescent (QF)-PCR using two short tandem repeat (STR) markers for chromosome 21 was carried out in 26 micromanipulated samples. FISH analysis revealed that 45/89 placental samples derived from pregnancies with male fetuses. Correct sexing of the lavage samples from male pregnancies was achieved in 41/45 (91%) using dual-FISH technique, and in 43/45 (95.5%) with PCR. All the samples derived from male pregnancies tested for chromosome 21 were normal. From 57 samples subjected to micromanipulation, 51 (89.5%) showed discernible chorionic villous filaments or cell clumps of possible trophoblastic origin. One case of tetraploidy and two cases of monosomy were recorded. The rate of fetal cells, in the non-micromanipulated samples, was between 4% and 97% (mean 54.3%). In micromanipulated specimens, maternal contaminant cells were absent or extremely rare (1-2%). The efficiency of the QF-PCR analysis in detecting paternal peaks in all lavage samples was only 61.5%. The present study confirms the presence of fetal cells in a very high proportion of both whole and micromanipulated intrauterine lavage samples. The isolation of trophoblastic elements can be achieved in most cases by micromanipulation. FISH and PCR techniques allowed the analysis of the most common fetal aneuploidies, confirming the power of this minimally invasive method.

Fetal cells in transcervical samples at an early stage of gestation.

Several investigations are in progress with the aim of performing prenatal diagnosis of inherited disorders by noninvasive or minimally invasive techniques. The most important approaches are based on the detection of fetal nucleated cells in maternal blood, the analysis of fetal DNA present in maternal plasma, and the identification and isolation of fetal trophoblastic cellular elements shed into the uterine cavity and the endocervical canal. In this review, we discuss the methods that have been employed for the collection of the transcervical samples at an early stage of gestation and the techniques used for the identification of fetal cells. We also report the results of using endocervical cells for the detection of fetal chromosomal disorders by fluorescent in-situ hybridization and for performing prenatal diagnosis of fetal Rh(D) phenotypes. Recent investigations have also shown that--after the isolation of trophoblastic cells from maternal contaminants by micromanipulation--transcervical samples can be employed for the prenatal diagnosis of single gene defects, such as those causing thalassemia and sickle cell anemia. Although the present results are promising, further investigations are required to demonstrate the feasibility of performing accurate diagnosis of fetal diseases by this minimally invasive approach in all transcervical samples retrieved at an early stage of gestation.

Transcervical sampling as a means of detection of fetal cells during the first trimester of pregnancy

Objective: Our aim was to evaluate the detection of fetal cells from transcervical samples by means of fluorescence in situ hybridization analysis. Study Design: Forty-seven women undergoing first-trimester termination of pregnancy were recruited for transcervical sampling and 16 were recruited for endocervical mucus aspiration. Subjects were asked to assess the discomfort of the procedure. Fluorescence in situ hybridization analyses were performed with X and Y chromosome–specific probes. Results were compared with those of conventional chromosomal analysis. Results: Male signals (XY) were observed in 19 of 25 male fetuses (76%), and female signals (XX) were observed in 21 of 22 female fetuses. There was a significant variation between operators in cell retrieval. With mucus aspiration only 37.5% of male fetuses were correctly identified. The subjects did not find the procedure uncomfortable and reported that it was comparable to having a cervical smear taken. Conclusions: Transcervical irrigation correctly identified the sex of 76% of male fetuses, but it still has considerable operator variability. This variability might be reduced by improved catheter design. (Am J Obstet Gynecol 2000;183:752-4.)

Hemeoxygenase expression in human placenta and placental bed implies a role in regulation of trophoblast invasion and placental function.

The purpose of this study was to examine the expression of hemeoxygenases HO-1 and HO-2, which are responsible for the production of carbon monoxide (CO), in the human placenta and placental bed and to determine the role of inhibitors of HO on placental perfusion pressure. We hypothesized that HO is expressed within the placenta and that invading cytotrophoblast cells (CTB) express HO isoforms. The expression of HO-1 and HO-2 was studied on placenta and placental bed biopsies, obtained using a transcervical sampling technique, from normal human pregnancies between 8 and 19 wk gestation and at term. In the placenta, HO-2 immunostaining was prominent in syncytiotrophoblast in the first trimester and reduced toward term (P<0.0005). HO-2 endothelial immunostaining was weak in the first trimester, but increased by term (P<0.0005). Within the placental bed, HO-2 was expressed by CTB in cell columns, the cytotrophoblast shell, and cell islands. Both intravascular CTB and interstitial CTB expressed HO-2. HO-1 immunostaining was low in the placenta but intense on the CTB within the placental bed. A striking feature was the absence of HO-1 from the proximal layers of cell columns, with strong expression on the more distal CTB layers of the cell columns. In placental perfusion studies, a significant dose-dependent increase in perfusion pressure was observed in the presence of zinc protoporphyrin, an inhibitor of HO. These results suggest a role for CO in placental function, trophoblast invasion, and spiral artery transformation. Hemeoxygenase expression in human placenta and placental bed implies a role in regulation of trophoblast invasion and placental function.

Human Trophoblast Invasion and Spiral Artery Transformation: The Role of Nitric Oxide

During early human pregnancy extravillous cytotrophoblasts invade the uterus and also migrate up the spiral arteries, transforming them into large vessels of low resistance. Failure of transformation has been described in pre-eclampsia, fetal growth restriction, and miscarriage. Recent evidence suggests that some maternal vessels undergo structural changes without interaction with cytotrophoblasts. The possibility arises that local vasoactive mediators such as nitric oxide result in spiral artery dilatation before their invasion. In support of this, a recent histological study in the guinea pig suggested that cytotrophoblasts expressed nitric oxide synthase (NOS) as they surrounded vessels. This study tested the hypothesis that invading cytotrophoblasts express NOS and therefore have the potential to induce vasodilatation by releasing nitric oxide. The expression of NOS on extravillous cytotrophoblasts was studied in placental bed biopsies, obtained, using a transcervical sampling technique, from normal human pregnancies between 8 to 19 weeks of gestation and in the third trimester. Whereas eNOS was expressed by syncytiotrophoblast, neither eNOS or iNOS was expressed by extravillous cytotrophoblasts at any time during invasion. The mechanisms controlling spiral artery transformation are pivotal to understanding normal and abnormal placentation. These results suggest that trophoblast-derived nitric oxide is unlikely to contribute to spiral artery dilatation.

Analysis of fetal sex in TCC sample DNA: a contribution to the validation of this approach

We compared two methods of collection of transcervical cell samples, mucus aspiration and cytobrush, with respect to the efficiency in determining fetal sex and we correlated the results with the week of gestation (7-11 weeks) to evaluate if the age of gestation influenced the success of the analysis. DNA extracted from TCC samples recovered by mucus aspiration (n = 27) and cytobrush (n = 36) were analysed by nested PCR to predict fetal sex. The statistical indices of sensitivity, specificity, positive predictive value and negative predictive value were determined, and compared with those of other studies previously performed. No statistically significant difference was found between the two methods of TCC sampling concerning the success of fetal sex prediction which was high for both methods (78 per cent and 89 per cent) and no correlation with the week of gestation was found. Transcervical cell sampling represents an encouraging prospect for first-trimester prenatal diagnosis even when the least invasive techniques are used.