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
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Abstract |
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Key words: fetal cells/immunohistology/in-situ hybridization/polymerase chain reaction/preimplantation diagnosis-transcervical sampling
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Introduction |
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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., 1993; Zheng et al., 1993
), granulocytes (Wessman et al., 1992
) and trophoblasts (Cacheux et al., 1992
; Johansen et al., 1995
). More widespread screening for relatively common fetal abnormalities such as Down's syndrome (Elias et al., 1992
) 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, 1995
); labour intensive density gradient centrifugation followed by magnetic cell sorting (Ganshirt et al., 1993
); and most recently, charge flow separation (Wachtel et al., 1996
).
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, 1971; Warren et al., 1973; Rhine et al., 1975
). Although conflicting reports which endorsed or refuted these findings played a significant role in the abandonment of further research (Bobrow and Lewis, 1971
; Goldstein et al., 1973
; Manuel et al., 1974
), 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., 1995
; Miller and Briggs, 1996
) expanded on these findings and included transcervical aspirates and flushings (using 15 ml of normal saline) from an additional 124 elective terminations of pregnancy at 717 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., 1993
, 1995a
,Adinolfi et al., b
, 1997
; Bahado-Singh et al., 1995
; Tustschek et al., 1995; Massari et al., 1996
). 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., 1995
) 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.
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Materials and methods |
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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., 1992). 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., 1995). 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., 1995; 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, 1992
) 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 TrisCl 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 TrisCl, 0.1 M NaCl, 50 mM MgCl2, pH 9.5) containing bichloroindolylphosphate (BCIP; 0.003% v/v) and nitrotetrazolium blue (0.004% v/v); GibcoBRL. Development was in the dark at room temperature for 510 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 0.23.0 ml PBS-A, depending on the original cell density. Samples were then cytocentrifuged onto APES-coated slides in 50100 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 TrisCl 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 I. 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 avidinbiotin 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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Results |
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Discussion |
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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, NCLPLAP 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 8).
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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., 1995) 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 (810 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., 1997). 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., 1995
; Miller and Briggs, 1996
), 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., 1995; Ishai et al., 1995
), 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., 1995
; Miller and Briggs, 1996
). 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., 1992). 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., 1997), 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.
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Acknowledgments |
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Notes |
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References |
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Submitted on May 13, 1998; accepted on October 28, 1998.