Simulaciones del control basado en la función tangente hiperbólica

1.11. Transcervical Cell (TCC) H istory

In 1971 Shettles reported that chorionic cells were shed into the cervical mucous plug of pregnant women. He used a cotton swab to obtain mucus smear samples from the mid cervix of 30 women in all three trimesters of pregnancy and applied the quinacrine hydrochloride fluorescein dye test to reveal Y chromosome containing cells. He detected Y bearing nuclei in 18 of the samples and thus predicted the sex of fetus in these cases to be male. At the time of reporting from his ongoing study 1 0 pregnancies had come to term, with the sex of all deliveries in concordance

with his findings; 4 female and 6 male. These results could not be repeated by Bobrow

and Lewis (1971) who examined midcervical smears and cervical mucus from 9 pregnant women, 6 carrying a male fetus and 3 a female. Using the same quinacrine

dye they found the proportion of apparent Y positive cells from female pregnancies averaged 2.52% and from male pregnancies averaged 3.04%. Both these percentages were within the known range of false positives in female control material; where fluorescing clumps of condensed chromatin and other artefacts may be mistaken for the 'Y' fluorescent spot.

In 1972 Warren et al reported to have obtained fetal cells from endocervical mucus. In a blind study, the sex of the fetus was correctly predicted in 50 women in all stages of gestation. These claims were disputed by two studies the following year (Tusk & Sasaki, 1973; Goldstein et al., 1973). Tusk and Sasaki studied 16 pregnant women from 7-12 weeks after the first day of the last menstrual period, prior to elective termination. Smears were obtained from midcervical mucus with a cotton swab and visualisation of Y chromosome containing nuclei attempted using quinacrine mustard. No Y containing cells were identified in any sample, despite there being 12

pregnancies with male fetuses (confirmed by examination of aborted material). However, lavage of the uterine cavity with sterile saline carried out on 3 patients, did reveal Y containing clumps of chorionic cells in the 1 case of a male pregnancy. Goldstein et al., (1973) studied mid-cervical smears of 38 patients at various stages of gestation, but were unable to identify any Y containing cells using quinacrine mustard, despite examining at least 100 nuclei from each sample and there being at least 7 male fetuses.

In 1975, Manuel et al. obtained mid-cervical smears from 20 women in the second trimester of pregnancy prior to elective abortion and again used quinacrine mustard staining in an attempt to detect Y containing cells. No relationship between fluorescent spotting and sex of the fetus was observed, however the presence of multiple spots in many nuclei highlighted the unreliability of the staining technique.

Positive results were obtained by Rhine et al., in 1975, who used quinacrine staining to detect male cells in endocervical smears collected by cotton swabs from 36 pregnant women during all 3 trimesters. Between 100-300 nuclei were scored from each case, and samples displaying more than 3% of cells with an apparent Y

chromosome signal were deemed to be trophoblast shed from a developing male fetus. The sex of the fetus was correctly predicted in 31 of the 36 cases with no false positive results (erroneous male predictions). The percentage of cells containing a Y fluorescent body ranged from 4.0% to 22.7% in samples from male fetus pregnancies (mean 8.9%), and 0.0% to 3.0% in female fetus pregnancies (mean 0.8%). The 5 incorrect results were false negatives (males bom, females predicted) and it was suggested that they were due to the collection of smears from a cervical region below the area of fetal cell accumulation.

In 1977 Rhine et al. published data from further studies where efforts were made to collect samples from the area of the internal os, not the mid cervix of pregnant women in the first trimester, using a device entitled the Antenatal Cell Extractor (ACE). (The internal os, or isthmus, is the restricted region between the corpus and cervix). This enabled lavage of the uterine cavity, using less than 5ml sterile saline. Tissue collected with this method was successfully cultured producing cells with trophoblast morphology in 6 of the 32 cases in the first study, and 18 of the 21 samples

in the second. These cells were karyotyped and polymorphic differences from maternal karyotypes were observed in 17 of 36 successful cultures, although no Y chromosome was detected due to the low quality of the metaphase spreads achieved. In the third stage of the study 13 pregnant women had two sequential lavage samples retrieved. Eight samples were examined histologically and seen to contain tertiary villi. The presence of human chorionic gonadotrophin was assayed from the culture media of a further 3 duplicate samples by radioimmunoassay and the levels found to be

significantly en excess of control cultures from skin fibroblasts. In all cases cell sampling was carried out 8 days prior to termination of pregnancy. During this eight

day period no untoward effects of the sampling procedure were seen to affect either the mother or fetus.

In 1977 Vamer et al., also reported successful results with endocervical samples obtained from abortion patients between the 6th and 12th weeks of pregnancy. They

searched their slides for multinucleated syncitial fragments derived from the syncytiotrophoblast of the developing membranes. These were compared to the syncytium from the abortus tissue for the presence of Y bodies. Of the 106 smears examined, 60 had more than 100 syncytial nuclei present. Y-body sex prediction analyses from these 60 patients were in 95% agreement with the Y-body analysis of the abortus trophoblast.

In 1978 Amankwah et al. obtained cotton wool swab midcervical smears from below the mucous plug in 71 pregnant women in all 3 trimesters. Thirty three of these were shown to be carrying male fetuses and 38 female. Atabrine staining was carried out for Y chromosome detection, and between 100-300 nuclei scored in each cases. Fetal sex was correctly predicted in only 55% of cases with extensive false positive and negative results. The following year, Rhine and Milunsky published a further report sampling 53 first trimester patients prior to abortion (Rhine and Milunsky, 1979). They were able to successfully culture 37samples (70%). Fetal chromosomes were

demonstrated to be present in 26 (49%), including a 46,XY male karyotype from an endocervical specimen obtained at 9 weeks of gestation. Attempted culture of samples obtained from second trimester pregnancies were less successful.

In 1980 Goldberg et al., attempted endocervical lavage using 5ml of saline on 30 pregnant women in the first trimester of pregnancy (8-13 weeks) using the ACE apparatus prior to elective termination. Twelve of these terminations were found to be of male fetuses and the lavage samples of these cases were cultured and examined for the presence of XY metaphases. Of the 12 cases, 9 produced successful cultures and were karyotyped. Despite observing cytotrophoblastic elements in the original samples, all cultures contained female karyotypes indicating them to be of maternal origin.

Coleman in 1982 reported the results of an preliminary study collecting intrauterine lavage samples from 1 2 women in the 1st trimester of pregnancy prior to

elective TOP. All samples were seen to contain decidua as well as villus fragments. When cultured however, all metaphases were found to be maternal as adjudged by the absence of XY karyotypes and banding polymorphisms. It was proposed that since intact trophoblast tissue grows slowly in culture because of the inhibiting effect of the trophoectodermal layers, contaminating maternal cells had 'overgrovm' the fetal ones.

With the evolution of relatively safe transcervical chorionic villus sampling, research in this area of prenatal diagnosis halted until 1992 when Griffith-Jones et al..

utilised the technology of the Polymerase Chain Reaction (PCR) to amplify Y

chromosome specific DNA sequences in transcervical samples. Samples were collected from 33 women prior to termination at 9-13 weeks’ gestational age. Samples were obtained with a cotton wool swab from the vagina, cervix, and transcervically 1cm

beyond the internal os in 26 women. In the remaining 7 women, samples were obtained by flushing the lower uterine cavity with 5ml saline from an adapted catheter. Fetal sexing, using PCR to amplify a Y-specific sequence, was performed blind (without prior knowledge of the sex of the fetus) and was correct in 25 out of the 26 initial subjects investigated (9 male) with one false negative. It was concluded that for consistently accurate results, samples needed to be collected from the uterine cavity, beyond the internal os. Collection of cells from the mid-cervix and vagina produced unreliable results by PCR analysis, indicating the region to be unsuitable for shed fetal trophoblast cell accumulation and more prone to spermatozoa contamination. Of the seven samples obtained by intrauterine lavage, all were found to contain syncitial fragments displayed by morphological studies and staining with placenta specific monoclonal antibodies.

Unfortunately these results could not be repeated by Morris & Williamson (1992) who found a Y chromosome-specific sequence using PCR in only 4 of 13 male pregnancies by testing cervical smears, and 7 of the 13 by testing the mucus plug biopsy. They also misdiagnosed some female pregnancies blaming this discrepancy on the presence of sperm from a partner after intercourse. The sampling technique used in this study however did not collect cells from beyond the internal os, and patients were not asked to abstain from sexual intercourse. The authors also highlighted the potential risk of infection of the fetus induced by the procedure.

In 1993 Fluorescent In Situ Hybridisation (FISH) was utilised to highlight the presence of fetal cells in endocervical flushes (Adinolfi et al., 1993). Intrauterine lavage was performed on a mother found to be carrying a fetus with trisomy 18 by conventional CVS at 10 weeks and 2 days gestation. The lavage procedure was carried out 8 days after the CVS. FISH was performed on the transcervical sample with a

chromosome 18 specific centromeric probe. Of the cells examined, 25% were found to contain three chromosomes 18 indicating them to be of fetal origin- the remaining cells containing 2 signals (maternal origin). Trisomy 18 of the fetus was confirmed by performing the same FISH procedure on placental samples following termination of the pregnancy. Unfortunately due to the transcervical sample being obtained subsequent to a CVS biopsy, the possibility that the placental cells were shed into the cervix as a

result of the procedure itself could not be dispelled (Gaudoin,, 1993). In the same study, endocervical lavage was also performed on a further 11 mothers prior to

termination. Primed in situ labelling (PRINS) was performed with a Y specific DNA sequence. Male cells were observed in all 7 pregnancies with male fetuses (2%-33%) with two false positive results in the 4 female fetus cases. Morphological studies were also carried out which showed syncytium-like clumps, some of which reacted to immunohistochemical staining with monoclonal antibodies raised against trophoblastic antigens.