Transcervical recovery of fetal cells from the lower uterine pole: reliability of recovery and histological/immunocytochemical analysis of recovered cell populations

David Miller1,4, Jackie Briggs1, Muhammed S. Rahman1, Martin Griffith-Jones1, Vasu Rane1, Marianne Everett1, Richard J. Lilford2 and Judith N. Bulmer3

1 Centre for Reproduction, Growth and Development, Division of Obstetrics, University of Leeds, Level D, Clarendon Wing, Leeds General Infirmary, Belmont Grove, Leeds, LS2 9NS, 2 Department of Health Medicine, Arthur Thomson House, 142 Hagley Road, Birmingham, B16 9PA, and 3 Department of Pathology, University of Newcastle, Royal Victoria Infirmary, Newcastle-upon-Tyne, NE1 4LP, UK


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The aim of this work was to isolate, enumerate and attempt the identification of fetal cells recovered from the lower uterine pole. Immediately before elective termination of pregnancy at 7–17 weeks gestation, samples were recovered by transcervical flushing of the lower uterine pole (n = 108) or transcervical aspiration of mucus from just above the internal os (n = 187), and their contents examined using histological, immunohistochemical and molecular techniques. Syncytiotrophoblasts were identified morphologically in 28 out of 89 (31%) and 50 out of 180 (28%) flushings and aspirates respectively (mean 29%). Immunocytochemistry with monoclonal antibodies (mAbs) recognizing trophoblast or epithelial cell antigens on a smaller number of samples (n = 69) identified putative placental cells in 13 out of 19 (68%) and 25 out of 50 (50%) flushings and aspirates respectively (mean 55%). These included groups of distinctive cells with a small, round, hyperchromatic nucleus, strongly reactive with mAbs PLAP, NDOG1 and FT1.41.1. Smaller groups of larger, amorphous cells, usually containing multiple large, pale staining nuclei, reactive with mAb 340 and to a lesser degree with mAb NDOG5 were also observed. Taking cellular morphology and immunophenotype into consideration, the smaller uninucleate cells were likely to be villous mesenchymal cells, while the larger cells were possibly degrading villous syncytiotrophoblast. There was no significant difference in the frequency of fetal cells obtained by the two recovery methods. Squamous or columnar epithelial cells, labelled strongly with antibodies to cytokeratins or human milk fat globule protein, were observed in 97% (29 out of 30) of aspirates. The use of cervagem in a small number of patients prior to termination of pregnancy did not appear to influence the subsequent recovery of placental cells. Y-specific DNA was detected by polymerase chain reaction (PCR) in 13 out of 26 (50%) flushings and (99 out of 154) 64% aspirates analysed (mean 62%). In-situ hybridization (ISH) revealed Y-specific targets in 40 out of 69 (60%) of aspirates analysed. A comparison of PCR data obtained from transcervical recovered samples and placental tissues showed a concordance of 80% (76 out of 95), with 10 false positives. Comparing the PCR data from tissues with data derived by ISH from 41 aspirates gave a concordance of 90% with two false positives. Although syncytiotrophoblasts were much more likely to be present in samples containing immunoreactive placental cells, the detection rates of fetal-derived DNA were similar regardless of the morphological and/or immunological presence of placental cells. We conclude that the transcervical recovery of fetal cells, while promising, requires considerable additional effort being expended in further research and development, particular in the sampling procedure.

Key words: fetal cells/immunohistology/in-situ hybridization/polymerase chain reaction/preimplantation diagnosis-transcervical sampling


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 Introduction
 Materials and methods
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First trimester prenatal diagnosis of fetal abnormalities is of singular importance to obstetricians who aim to minimize the interventional trauma caused by late therapeutic termination of pregnancy. Amniocentesis at 14 weeks' gestation or later, followed by cell culture and cytogenetic analysis is accepted as one of the most reliable and safe methods of detecting chromosomal abnormalities, although it carries a 0.5–1.0% risk of causing a miscarriage. Moreover, results are rarely obtained before the 15th week of pregnancy due to the time required for cell culture. Chorionic villus biopsy (CVB), although capable of providing information in the first trimester, in addition to a 2–4% procedure-related risk of miscarriage, is thought to be associated with an increased risk of fetal abnormality such as defective limb development, presumably due to haemorrhage or embolism from the aspirated placental tissues (Jahoda et al., 1993Go; Fortuny et al., 1995Go; Lunshof et al., 1995Go).

The replacement of both amniocentesis and CVB is now a serious proposition following the identification of fetal cells in the maternal circulation. These include nucleated erythroid cells (Liou et al., 1993Go; Zheng et al., 1993Go), granulocytes (Wessman et al., 1992Go) and trophoblasts (Cacheux et al., 1992Go; Johansen et al., 1995Go). More widespread screening for relatively common fetal abnormalities such as Down's syndrome (Elias et al., 1992Go) may become possible. However, all these circulating fetal cells are present in exceptionally low numbers in maternal blood and their isolation is particularly difficult. To date, enrichment methods have included expensive fluorescence activated cell sorting (Bianchi, 1995Go); labour intensive density gradient centrifugation followed by magnetic cell sorting (Ganshirt et al., 1993Go); and most recently, charge flow separation (Wachtel et al., 1996Go).

Using quinacrine staining of the Y chromosome, a number of early reports suggested that fetal cells, shed from the regressing chorion laeve into the lower uterine pole, could be recovered by simple aspiration of endocervical mucus in the first trimester (Shettles, 1971Go; Warren et al., 1973; Rhine et al., 1975Go). Although conflicting reports which endorsed or refuted these findings played a significant role in the abandonment of further research (Bobrow and Lewis, 1971Go; Goldstein et al., 1973Go; Manuel et al., 1974Go), the inability to culture recovered cells in vitro and the consequent exclusion of classical cytogenetic analysis was of crucial importance. Since the advent of molecular techniques, particularly polymerase chain reaction (PCR) and fluorescence in-situ hybridization (FISH) which offer a timely alternative to classical cytogenetic analysis, a number of workers have attempted a reassessment of trans/endocervical fetal cell sampling using several distinct sampling procedures. In a preliminary report, Griffith-Jones et al. (1992) using Y-specific primers, correctly predicted fetal sex by PCR prior to confirmation by classical cytogenetic analysis in 25 out of 26 samples swabbed from the upper cervix. Later reports (Briggs et al., 1995Go; Miller and Briggs, 1996Go) expanded on these findings and included transcervical aspirates and flushings (using 1–5 ml of normal saline) from an additional 124 elective terminations of pregnancy at 7–17 weeks gestational age. Other reports have used PCR and FISH for sex determination or the detection of various chromosomal aneuploidies and single-gene defects in samples obtained by transcervical procedures (Adinolfi et al., 1993Go, 1995aGo,Adinolfi et al., bGo, 1997Go; Bahado-Singh et al., 1995Go; Tustschek et al., 1995; Massari et al., 1996Go). Yet there has been only one report to date describing the cellular composition of samples recovered by these procedures in any detail (Bulmer et al., 1995Go) and there have been few descriptions of the recovery frequency. Such information is essential if the material giving rise to PCR or FISH signals is to be identified and characterized and for possible anomalies arising from sample heterogeneity to be better understood.

The aim of the present report is to address these issues and we report on the analysis of molecular, morphological and immunophenotypic information, amassed over 3 years of study.


    Materials and methods
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Ethical approval was obtained from Leeds Health Authorities (East and West).

Subjects
A total of 295 patients were recruited into this study. All were informed of and gave consent to transcervical sampling under general anaesthesia, prior to the elective termination of pregnancy.

Reagents
All laboratory reagents were of ultrapure quality (molecular biological grade, Sigma Chemical Co, Poole, UK) unless stated otherwise. Proteinase K and Taq DNA polymerase was purchased from Northumbria Biologicals (Cramlington, UK) or Gibco BRL (Paisley, UK). Standard and biotinylated deoxynucleotides were purchased from Promega UK (Southampton, UK). Ultra-pure bovine serum albumin was from BDH (Poole, UK). The secondary antibodies and developer kits used in the study were from Vector Laboratories (Burlingame, CA, USA). Aminopropyltriethoxysilane (APES) slide coating reagent was from Sigma. All other reagent sources are detailed below.

Sample recovery
Two sampling methods were used, the first of which has been described previously (Griffith-Jones et al., 1992Go). Briefly, a hollow flexible embryo transfer trocar (Rocket Ltd, London, UK) was inserted transcervically under ultrasound guidance into the lower uterine pole. Sterile physiological saline (~5 ml) containing 1.0% w/v bovine serum albumin (BSA) was introduced into the trocar and the fluid allowed to enter the internal os prior to recovery by gentle suction. These samples (n = 108) will be referred to as flushings. For the second method, the trocar was carefully positioned just above the internal os, without the aid of ultrasound, and samples (n = 187) were directly aspirated into phosphate-buffered saline (PBS) containing 1.0% bovine serum albumin (PBS-A). These samples will be referred to as aspirates.

All recovered samples were centrifuged at 500 g for 5 min and resuspended in PBS-A. Aliquots from 26 flushings and 156 aspirates were immediately stored at –20°C for subsequent analysis by PCR. Cytospin preparations from all samples were routinely examined by microscopy following staining with haematoxylin and eosin. Samples of placental chorionic villi obtained from 41 patients following termination of pregnancy (from whom aspirates were obtained) were frozen and stored at –20°C, for separate PCR analysis. Additional small samples of first-trimester placental villi were snap-frozen in liquid nitrogen-cooled isopentane (BDH) for sectioning (see below).

Sample preparation for polymerase chain reaction
Samples were routinely checked for the presence of DNA by sequence-specific gene amplification of proteinase K-digested material, in the presence of primers for ß-actin as described previously (Briggs et al., 1995Go). Actin and Y-specific DNA were amplified using the following PCR programme: denaturation at 95°C for 5 min followed by 30 cycles of annealing at 56°C for 1 min, extension at 72°C for 1 min and denaturation at 95°C for 1 min. A final 5 min extension at 72°C was also included. Products were resolved on 1.8% (w/v) agarose gels in Tris-borate EDTA buffer (25 mM Tris, 0.1 M sodium borate, 1 mM EDTA, pH 8.0) and visualized following staining with ethidium bromide and ultraviolet illumination. Results were compared with those obtained following the implementation of identical procedures on fetal tissues from terminated pregnancies and the in-situ hybridization (ISH) of Y-specific DNA probes to cytocentrifuged samples (see below).

In-situ hybridization
The procedure has been described in detail elsewhere (Briggs et al., 1995Go; Miller and Briggs et al., 1996) where representative illustrations are shown. Briefly, samples were centrifuged at 500 g for 5 min, and cytospun onto slides precoated with APES. After air drying, samples were fixed in PBS containing 4% (w/v) paraformaldehyde for 30 min, washed in PBS for 5 min and transferred into PBS containing 0.25% (v/v) Triton X-100 and 0.25% (v/v) NP-40 for 10 min. After washing twice in PBS, endogenous alkaline phosphatase activity was blocked by 15 s immersion in 20% (v/v) acetic acid at 4°C followed by a 5 min wash in PBS and 30 min incubation in 20% (v/v) glycerol at room temperature. Slides were then rinsed twice in 2x sodium chloride/sodium citrate (SSC) buffer, drained and incubated with 75 µl of a biotinylated Y-probe as described previously (Lewis and Wells, 1992Go) in hybridization buffer [2x SSC, 5% (w/v) dextran sulphate, 0.2% (w/v) dried milk powder containing formamide 50% (v/v)]. The procedure was as follows: after heating to 95°C for 10 min followed by an overnight hybridization at 37°C, the slides were rinsed twice in 2x SSC at room temperature followed by 20 min in 2x SSC at 60°C, 20 min in 0.2x SSC at 42°C and 5 min 0.1x SSC at room temperature. Slides were then transferred into buffer A [0.1 M Tris–Cl pH 7.5, 0.1 M NaCl, 2 mM MgCl2, 0.05% (v/v) Triton X-100] containing 5% (w/v) BSA for 20 min prior to draining and transfer into a humidified box. Streptavidin (10 µg/ml in buffer A) was added to the slides prior to incubation at room temperature for 10 min. Slides were washed in buffer A (2x5 min) prior to incubation in buffer A containing biotinylated alkaline phosphatase (10 µg/ml) for 10 min as above. Slides were washed twice washed in buffer A for 5 min and then equilibrated for 30 min in buffer B (0.1 M Tris–Cl, 0.1 M NaCl, 50 mM MgCl2, pH 9.5) containing bichloroindolylphosphate (BCIP; 0.003% v/v) and nitrotetrazolium blue (0.004% v/v); Gibco–BRL. Development was in the dark at room temperature for 5–10 min. Nuclei were counterstained with 2% (w/v) methyl green (3 min) and mounted in glycerol jelly (Merck, UK).

Histology and immunocytochemistry (ICC)
Where present, contaminating erythrocytes were lysed in 0.16% (w/v) NH4Cl (Vickers Labs Ltd; Pudsey, UK) in distilled water at room temperature. Samples were concentrated by centrifugation at 500 g for 5 min and resuspended in 0.2–3.0 ml PBS-A, depending on the original cell density. Samples were then cytocentrifuged onto APES-coated slides in 50–100 µl aliquots at 250 g for 10 min. Following air drying, cytospin preparations were fixed for 5 min in acetone at room temperature and either processed immediately or stored wrapped in foil at –20°C. Sample cytospins were routinely stained with Harris's haematoxylin (BDH). For immunocytochemistry, non-specific binding sites were blocked with normal horse serum [Vector Laboratories; 20% (v/v) in Tris-buffered saline (TBS; 0.15 M NaCl, 0.05 M Tris–Cl pH 7.6)] and samples were incubated for 60 min with monoclonal antibodies (mAbs) directed against various trophoblastic and maternal cell antigens, dissolved in PBS containing 0.1% (w/v) BSA; specificities, sources and dilutions are given in Table IGo. After 60 min incubation in a humidified chamber, samples were washed in three changes of TBS and incubated for 60 min with biotinylated anti-mouse immunoglobulin (Vectastain Elite, Vector Laboratories). Following further washing in TBS, an avidin–biotin peroxidase complex (Vectastain Elite) was applied to the slides for 30 min. The reaction was developed with 0.025% (w/v) diaminobenzidine (Sigma) and 0.025% (v/v) H2O2 (BDH) in TBS, counterstained with haematoxylin, cleared in xylene (BDH) and mounted in DPX synthetic resin (BDH). For comparison with recovered transcervical samples and to monitor mAb specificities, cryostat sections (10 µm) of frozen placental tissue were air-dried overnight at room temperature and fixed for 5 min in acetone.


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Table I. Specificities and reactivities of antibodies used in this study
 

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Recovery efficiencies of flushings and aspirates
As most women are referred for termination of pregnancy at a similar stage in their pregnancy, bias in the gestational ages of recovered samples is unavoidable; most flushings and aspirates were obtained at 10 weeks gestation (Figure 1Go). In order to investigate how this bias affected the overall efficiency of fetal cell recovery, the percentage of samples shown to contain syncytiotrophoblast (the most easily recognized placental cell) for each week of gestation was determined for both flushings and aspirates (Figure 2Go). Despite the smaller numbers of flushed samples and the absence of any flushed samples at >14 weeks gestation, it is clear that the relative proportions of samples containing syncytia were similar. Furthermore, similar frequencies of syncytiotrophoblast recovery were obtained in flushings (28 out of 89; 31%) and aspirates (50 out of 180; 28%) despite the application of a cervagem prostaglandin pessary prior to termination of pregnancy in more flushings (10 out of 28) than aspirates (two out of 50) containing syncytia (Table IIGo). Although these data suggested that syncytiotrophoblast recovery was not unduly affected by either the sampling technique or prior prostaglandin application, the flushing procedure was quickly abandoned in favour of aspiration in order to avoid the introduction of fluid into the uterine cavity.



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Figure 1. Frequency of flushings (open bars) and aspirates (shaded bars) obtained at 7–17 weeks gestation. Absolute numbers giving rise to these frequencies are given above each bar.

 


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Figure 2. Frequency of samples containing syncytiotrophoblasts in flushings (open bars) and aspirates (shaded bars) at 7–17 weeks' gestation. Absolute numbers giving rise to these frequencies are given above each bar.

 

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Table II. Summary of immunocytochemical data. Total values are given throughout
 
Immunocytochemistry
As suggested previously from molecular data (Briggs et al., 1995Go), unexpectedly low values obtained for syncytiotrophoblast recovery in both flushings and aspirates may not accurately reflect the presence of placental cells in these samples. Hence, both flushings and aspirates were probed with a panel of mAbs capable of detecting placental antigens. As the same samples were used for several different experimental procedures, it was not possible to test every sample with all antibodies. Thus placental cells were detected in 13 out of 19 (68%) flushings and 25 out of 50 (50%) aspirates respectively (Table IIGo). Displaying the percentage of immunoreactive samples following exposure to particular mAbs (Figure 3Go), it is clear that the identification of placental cells was partly dependent on the choice of mAb used. In our hands, mAb 340 consistently performed better than any of the other reagents, with mAb NCL–PLAP lying close behind. A smaller group of aspirates (n = 30) was challenged with MAbs 5D3, LP34 and HMFG1 and immunoreactive cells were detected in 29 (98%). Cells immunoreactive with the common leukocyte antigen, CD45, were found in half (five out of 10) of the samples probed with the corresponding mAb. As many samples were bloody on recovery, the significance of this observation most likely relates to procedural trauma.



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Figure 3. Frequency of samples containing labelled cells from flushings (open bars) and aspirates (shaded bars) following exposure to a range of monoclonal antibodies. Absolute numbers giving rise to these frequencies are printed above each bar.

 
Placental sections
Patterns of placental antigen expression are shown in Figure 4Go. Of the mAbs recognizing placental antigens, PLAP and 340 provided the strongest labelling of both the syncytiotrophoblast and villous cytotrophoblastic layers (Figure 4A and BGo). In contrast, mAb NDOG1 only weakly labelled these layers (Figure 4CGo) and mAb NDOG5 labelling was patchy within the deeper layers of the tissue (arrowheads in Figure 4DGo). mAb FT41.1 appeared to be specific for the syncytiotrophoblast layer and the occasional deeper layer of villous cytotrophoblast (lower left hand corner of Figure 4EGo) including the trophoblast basement membrane (arrowheads). As expected, there was strong labelling of the syncytiotrophoblast and villous cytotrophoblast with 5D3 (Figure 4FGo). LP34 also labelled these layers but with less intensity (Figure 4GGo). HMFG1 faintly labelled these and the deeper layers of tissue (Figure 4HGo).



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Figure 4. Sections of first-trimester placenta were probed with trophoblast-reactive monoclonal antibodies (mAbs) (A) NCL-PLAP; (B) 340; (C) NDOG1; (D) NDOG5 and (E) FT1.41.1. Reactivities to mAbs-5D3 and LP34, which recognize epithelial cell cytokeratins, are shown in (F) and (G) respectively. A section labelled with HMFG1 is shown in (H). Arrowheads in (D) and (E) indicate pockets of cellular and basement membrane labelling, respectively. Original magnification: A, B, C, D, G, H x160; E, F x250.

 
Transcervically recovered samples
Representative examples of samples containing syncytiotrophoblast are shown in Figure 5Go, labelled with PLAP (Figure 5AGo); 340 (Figure 5BGo); NDOG1 (Figure 5CGo); NDOG5 (Figure 5DGo); and FT1.41.1. (Figure 5EGo). A sample stained with haematoxylin alone is shown in Figure 5FGo for comparison. All these mAbs recognized syncytiotrophoblasts, although the intensity of labelling differed between cases. Figure 5AGo–C and Figure 5DGo–F are from samples obtained by flushing and aspiration respectively.



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Figure 5. The presence of syncytiotrophoblasts in transcervically recovered samples (A–C and F = flushings; D, E = aspirates) were assessed following (F) haematoxylin staining alone or following immunocytochemistry with monoclonal antibodies (A) PLAP; (B) 340; (C) NDOG1; (D) NDOG5, and (E) FT1.41.1. Original magnification: all x250.

 
Representative examples of aspirates challenged with trophoblast-specific mAbs are shown in Figure 6Go for mAbs PLAP (Figure 6A,BGo); 340 (Figure 6C,DGo); NDOG1 (Figure 6E,FGo); NDOG5 (Figure 6GoG,H) and FT1.41.1 (Figure 6I,JGo). These samples were studied to look for trophoblast cells other than syncytiotrophoblast and various immunoreactive cells can be seen. Large groupings of well separated cells with uniformly counterstained round nuclei and strongly labelled with mAbs PLAP and FT1.41.1 (Figure 6B,JGo) were frequently observed. Small clumps of more tightly associating cells, strongly labelled with mAbs PLAP (Figure 6AGo) and NDOG1 (Figure 6FGo) were also frequent. Larger cells with more diffuse, multiple, weakly counterstaining nuclei and weakly labelled with mAbs 340 (Figure 6C,DGo) and NDOG5 (Figure 6HGo) were less common. The solitary mAb NDOG1-labelled cell visible in Figure 6EGo, (arrowhead) resembles those present in Figure 6B and JGo and may be the same cell type. The intensely labelled cells present in Figure 6GGo were also found in most samples containing leukocytes and were CD45-positive (not shown). They are included to illustrate the presence of peroxidase-containing cells, most likely eosinophils.



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Figure 6. The presence of other trophoblast cells in aspirated samples was assessed following immunocytochemistry with monoclonal antibodies (A, B) PLAP; (C, D) 340; (E, F) NDOG1; (G, H) NDOG5 and (I, J) FT1.41.1. Arrowheads in E, G and H indicate cells of interest. Original magnification: A, F, H, I x250; B, C, D, E, G, J x400.

 
The presence of maternal cells was assessed by morphological and immunocytochemical criteria and representative examples are shown in Figure 7Go for mAb 5D3 (Figure 7A,BGo); mAb LP34 (Figure 7C,DGo) and mAb HMFG1 (Figure 7E,FGo). These mAbs were particularly useful for identifying squamous (Figure 7A,CGo) and columnar (Figure 7BGo) epithelial cells which could only be of maternal origin. More ambiguously, the cell clumps present in Figure 7AGo resemble those labelled by trophoblast-reactive mAbs and, as shown in placental sections (Figure 5F,GGo), mAbs 5D3 and LP34 react with all trophoblast cells. However, as HMFG is known to recognize epithelial cells (Burchell et al., 1983Go), the cells shown in Figure 7E and FGo are probably of cervical origin.



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Figure 7. The presence of maternal cells in aspirated samples was assessed following immunocytochemistry with monoclonal antibodies (A, B) 5D3; (C, D) LP34 and (E, F) HMFG1. Original magnification: A, B x400; C, D, E, F x250.

 
Comparison between molecular and cellular data
Cellular data suggested that ~50% of samples (flushings and aspirates combined) did not contain placental cells. However, independent estimates of placental cell presence were available, based on determining fetal sex by PCR and ISH. The accuracy of these data was assessed by comparison with corresponding samples of placental tissue obtained following the termination of pregnancy. Y-specific DNA was detected by PCR in 13 out of 26 (50%) and 99 out of 154 (64%) samples recovered by flushing and aspiration respectively (mean 62%). The difference in these values was not statistically significant and compared favourably with the frequency of aspirates (60%) containing Y chromosome targets as determined by ISH (40 out of 69). More importantly, data from placental tissues were 80% (76 out of 95) and 90% (37 out of 41) concordant with the corresponding data derived from aspirates by PCR or ISH respectively. Data for the 41 samples in which a three-way comparison based on PCR and ISH on aspirates with PCR on CV samples was carried out are shown in Table IIIGo. The 80% concordance was due to eight discordant aspirates consisting of five false (male) positives and three false (male) negatives. Only eight out of 41 (20%) aspirates in this grouping contained syncytia. Taking (placental) antigen-positive (Ag+) and negative (Ag) aspirates into consideration where complementary molecular data were available directly, 11 out of 20 Ag+ (55%) and eight out of 14 Ag (57%) samples gave rise to Y-PCR products. Taking data from aspirates shown to contain syncytiotrophoblast by histological analysis and subsequently processed for immunocytochemistry, 12 out of 25 Ag+ (48%) and one out of 29 Ag (3%) aspirates contained syncytiotrophoblast, a statistically significant difference ({chi}: P < 0.05).


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Table III. Concordance of molecular data using polymerase chain reaction (PCR) and in-situ hybridization (ISH). Numerical values for the two- and three-way concordance are given as footnotes
 

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Our main objectives were to examine transcervically recovered samples for the presence of fetal material, describe the consituent cells and determine whether these cells could be separated into fetal and maternal compartments, a prerequiste for the detection of single gene defects. While we were unable to meet the third objective, our findings have a bearing on the potential use of transcervical sampling for prenatal diagnosis in a clinical setting.

Most of the samples recovered during the study contained intact cells of maternal origin; ectocervical squamous and columnar epithelial cells, often in large numbers, were present in 98% aspirates. In contrast, 55% of all samples examined contained cells which were immunoreactive with one or more of the mAbs recognising placental antigens. These findings should be viewed in the context of the considerable variation in reactivities of the panel of mAbs used in this study. mAb 340, for example, recognized cells in 16 out of 22 (73%) samples compared with the next `best' reagent, NCL–PLAP at 14 out of 24 (58%). These data suggest that estimates for placental cell presence may have been underestimated by the poor sensitivity of reagents used for their detection. Detection rates for syncytiotrophoblasts are probably more accurate but reveal a relatively low level of abundance as illustrated by combining the data obtained by their direct visual observation with immunocytochemical data for all putative placental cells (Figure 8Go).



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Figure 8. Frequency of samples containing any trophoblasts or other placental cells, displayed as a stack column chart. ST = syncytiotrophoblasts.

 
Morphologically distinct immunoreactive cell types were observed, including discrete multinucleate syncytiotrophoblasts, larger cells containing more than one nucleus, groupings of loosely or tightly associating cells and the occasional solitary cell. With the exception of syncytiotrophoblast, definitive identification of placental cells was not achieved due to limitations in the reagents used and inter-sample variability in their reactivity with similar cell types (see below). However, some tentative suggestions could be made, based on cellular morphology, the known reactivity of the mAbs and the pattern of mAb reactivities for the `same' cell type. In general, groups of tightly associating cells were mAb PLAP/NDOG1 positive, while the more loosely associating cell clumps were mAb PLAP/FT1.41.1-positive. Hence, the tightly packed PLAP/NDOG1-positive cells could have been derived from the chorionic villous mesenchyme where both alkaline phosphatase and hyaluronic acid are present. The loosely associating PLAP/FT1.41.1-positive cells are more difficult to identify because placental alkaline phosphatase is ubiquitously expressed in the placenta. Since mAb FT1.41.1 recognized the villous cytotrophoblast layer in placental sections, albeit weakly, these isolated cells may be derived from this layer but could equally derive from the villous mesenchyme. In view of these immunophenotypes, it is unlikely that any of the PLAP-reactive cells are cervical in origin, despite the reported low level presence of a placental-like alkaline phosphatase in cervical tissues (McLaughlin et al., 1984Go). The larger cells containing more than one nucleus were usually mAb 340/NDOG5-positive. While we were unable to confirm that these cells were not villous cytotrophoblast, mAb 340 which is known to recognize this cell type did not recognize equivalent groups of cells in any of the samples challenged.

Based on the reactivity of mAb NDOG5 for extravillous cytotrophoblast, the large 340/NDOG5-positive cells could be of this cell type; however, in view of the occasional positive labelling of isolated syncytiotrophoblasts with this mAb and the syncytial appearance of these cells, it is also conceivable that they are fragmented and degrading syncytiotrophoblast. The latter explanation is supported by our earlier work (Briggs et al., 1995Go) showing the presence of naked nuclei in recovered samples. Although NDOG5 is not noted for its recognition of syncytiotrophoblast in sections (as supported by this study) there is evidence for the expression of the corresponding epitope in syncytiotrophoblasts from term placentas (M.Johansen, personal communication) and it is conceivable that these giant cells could alter their pattern of antigen expression following isolation from the main placental mass. Artefacts may be an inevitable consequence of sampling; gain or loss of syncytiotrophoblast antigen expression may occur by the time the material is recovered. Alternatively, fixation conditions can vary between whole sections and isolated cells, masking or revealing epitopes accordingly. NDOG5, for example, recognized syncytia in the two out of six immunoreactive samples containing these cells (both samples obtained at 10 weeks gestation) but not in any of the first trimester control sections (8–10 weeks gestation).

There have been few reports describing the cellular composition of transcervically recovered samples in any detail. Rhine et al. (1975) provided fluorescent micrographs showing clusters of fetal cells with a ragged appearance which, in relation to the much larger squamous cells also present and their similar morphology to cells observed in the present study, were probably from the villous mesenchyme. While agreeing with our findings in relation to squamous and endocervical cells, most samples recovered by aspiration and flushing by Bulmer et al. (1995) contained syncytial fragments and cytotrophoblasts as demonstrated by morphology, histological staining and immunocytochemistry. Rodeck et al. (1995) detected syncytiotrophoblasts in 18% of samples obtained by endocervical cytobrushing and intrauterine lavage and cytotrophoblasts in all samples. The higher levels of fetal cells reported in both studies may have been due to their use of different mAbs or to differences in respective sampling procedures. Rodeck et al. (1995) included two mAbs (IO3 and H315) which may have been more sensitive in this respect. Ultrasound guidance, which was not used to aid the recovery of aspirates in this study, may also have been a contributory factor, although the similar frequency of syncytiotrophoblast recovery in flushings (obtained under ultrasound guidance) and aspirates suggests otherwise. In contrast, Bahado-Singh et al. (1995) identified trophoblasts in only five out of 10 samples obtained by flushing using a mAb recognising human chorionic gonadotrophin.

Alternative explanations for the discrepant data regarding cytotrophoblast recovery involve the choice of sampling procedure and its related mechanical effect on cellularity. While intra-uterine flushing produced the best data with regard to placental cell recovery and cellularity, it is unlikely that invasive introduction of fluid in pregnancies destined for continuation would be acceptable and this led us to abandon this procedure early in the study. However, rather than the perceived compromise to uterine sterility, it now appears that mechanical trauma induced by changes in intra-uterine pressure is a more compelling reason not to flush, following the recent report of severe limb reduction defects in a male neonate after an uneventful pregnancy in which first trimester flushing was carried out (Chou et al., 1997Go). The aspiration data of Rodeck et al. (1995) were derived from the endocervix and external cervical os using different mAbs and therefore are not directly comparable with our aspiration data. In our earlier report (Briggs et al., 1995Go; Miller and Briggs, 1996Go), the presence of hybridising acellular targets to Y-probes, often in close proximity to syncytiotrophoblasts, suggested that mechanical disruption or other physico-biochemical effects were major causes of cell loss during transcervical aspiration.

High levels of maternal cell contamination identified as cervical squamous and columnar epithelial cells, as well as leukocytes presumed to be of maternal origin, were a common feature in both flushed and aspirated samples and was in agreement with Rodeck et al. (1995). Leukocyte contamination is inevitable under conditions which promote bleeding, including trauma and prior application of prostaglandins. While reductions in their presence may have been brought about by the use of alternative sampling tools and techniques, inter-subject differences in upper genital tract physiology may also have been an important and unavoidable factor in maternal cell load.

While it is possible that changes in the handling of samples might minimize cellular damage, poor cellularity due to unavoidable natural reductions in placental cell content, as indicated at least for syncytiotrophoblast recovery, would restrict the use of recovered material to interphase aneuploid detection. Certainly no cell culture of such samples would be possible (Bahado-Singh et al., 1995Go; Ishai et al., 1995Go), effectively excluding any classical cytogenetic analysis. The fate of placental cells in recovered samples is of some concern since the molecular data strongly indicated the presence of fetally-derived DNA in most cases. In this respect, comparison of the molecular and cellular data is revealing and supports a number of conclusions. Firstly, the slightly higher than expected number of designated male aspirates derived by PCR/ISH is probably an artefact of occasional contamination with male DNA, since the number of Y-PCR-positive samples was similar for both antigen positive and negative aspirates (56%) compared with all aspirates tested (64%). Secondly, the detection of fetally-derived DNA was not dependent on the presence of intact placental cells as indicated by the large number of aspirates containing hybridizable targets by ISH, which did not contain immunoreactive placental cells (Briggs et al., 1995Go; Miller and Briggs, 1996Go). This conclusion is supported by the data showing that placental antigen positive samples were more likely to contain syncytiotrophoblasts than antigen negative samples, while the presence or absence of placental cells made little difference to the detection of Y-DNA.

The concordance of our molecular data was 80% comparing data obtained from recovered placental tissues by PCR and aspirates by both PCR and ISH and 90% when considering ISH alone. These data compare with 96% accuracy in our original PCR-based investigation of 26 samples recovered by endocervical swabbing (Griffith-Jones et al., 1992Go). The difference most likely reflects the larger sample size since our original report and the greater likelihood of false-positives when using PCR. Using a similar three-way technique, Adinolfi et al. (1995b) reported 86% concordance between fetal tissues obtained following termination of pregnancy and samples recovered by lavage or cytobrush, using PCR and FISH; 12 out of 22 (54%) samples contained Y-specific DNA as determined by PCR. Three known male samples (one lavage and two cytobrush) were non-informative because of a failure to detect Y-targets by FISH. Interestingly, in the same study, paternally-derived alleles were detected in only four out of 16 (25%) lavages and four out of 29 (14%) aspirates following amplification of chromsome 21-specific short tandem repeats. This is a clear demonstration of the likely requirement for some form of fetal cell enrichment to counteract noise generated by the maternal genome.

In conclusion, the latest data confirm that fetal material, more often in the form of DNA/nuclei than intact cells, is deported into the lower uterine pole during the first trimester of pregnancy and can be recovered by transcervical flushing or aspiration. However, the clinical relevance of this procedure has not yet been proven. All studies have reported considerable variation in both the composition and quality of recovered material, due to sampling errors and operator discontinuities. It is also by no means certain that the procedure is any safer than CVS (Chou et al., 1997Go), despite the small trial carried out by Rodeck et al. (1995), or indeed as reliable, given the heterogeneity of the samples.

A much larger study is now required to establish the application of transcervical sampling as an alternative route to prenatal diagnosis. This would require close collaboration between groups who are currently working in this area, in a number of different settings. At stake is a clearer resolution of the precise niche in which the procedure is likely to be placed, standing as it now does somewhere between impending non-invasive maternal blood sampling on the one hand and existing invasive but highly accurate prenatal diagnostic procedures on the other.


    Acknowledgments
 
This work was supported by the Medical Research Council of the UK. We are grateful to all the patients who participated in this study.


    Notes
 
4 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on May 13, 1998; accepted on October 28, 1998.





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