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F ollow ing the introduction o f the PCR method for the am plification and

identification o f specific D N A sequences, studies on maternal peripheral blood using

PCR for the am plification o f Y -chrom osom e D N A sequences have demonstrated the

presence o f nucleated male cells in the blood o f wom en carrying male fetuses. These

techniques are unable to identify the type o f fetal cell detected, m erely indicate their

presence. Estimates o f the number o f fetal cells present in maternal blood sam ples

vary. Based on D N A studies by PCR amplification or Southern blot hybridisation, the

ratio o f fetal to maternal nucleated cells was estimated to be more than 1 in 10^ at 8-12

w eeks o f gestation (Kao et al., 1992), less than 1 in 2.5x 10^ at 14-16 w eeks o f

gestation (N akagom e et al., 1991), less than 1 in 10^ at 16 w eeks o f gestation

(Schw inger et al., 1989), 1 in 5x10^ at 16 w eeks o f gestation (Bianchi et al., 1990), 1 in

10^ to 1 in 10^ in the first and second trimesters (Price et al., 1991), less than 1 in

7 x 104 at 24-36 (A dinolfi et al., 1989) and less than 1 in 5 x 10^ throughout the entire

gestational period o f 8-40 w eeks (Ganshirt-Ahlert et al., 1990). U sing an avidin-biotin

based im m unoaffinity system . Hall and W illiam s (1992) estimated the frequency o f

fetal cells to be 1:4.75x10^ to 1.6x10^ o f the nucleated cell fraction o f maternal blood.

Lo et al., (1989) collected blood from 19 pregnant w om en, o f whom 12 were

found to have male fetuses. U sing a PCR specific for the Y chrom osom e, the sex o f the

fetus w as predicted in all cases, with no false positives or negatives. It was claim ed that

this technique could detect a male cell in a fem ale population at a dilution o f 1:10^.

However, it was later shown by Nakagom e et al., (1991) that the particular Y

chrom osom e sequence em ployed has autosomal hom ologies and in their next study Lo

et al., (1990) experienced tw o false positives and four false negatives. In a more recent

equivalent of a single male cell in 3x10^ female cells (Lo et al., 1993a). Using this procedure sex prediction was attempted for the fetuses of 75 women through all three trimesters of pregnancy. This was successfully achieved in 8 6%, 67% and 87% of

women in the first, second and third trimesters respectively. Thomas et al., (1994) used Y-specific PCR following the pregnancies of 5 women who became pregnant by in

vitro fertilisation. In the 2 women found to be carrying male fetuses, Y-specific signals

were detected at 33 and 40 days of gestation, and continued to be detectable to term, ceasing to be seen from 8 weeks post-partum. However, Liou et al., (1993; 1994) used

Y-PCR to detect fetal cells from male pregnancies in blood samples collected from 19 women with male fetuses, and could not detect Y sequences until 6-12 weeks’

gestation. Hamanda et al., (1993) again used Y-PCR but could not consistently detect a Y-specific product in women with male fetuses prior to 15 weeks’ gestation, although the proportion of fetal to maternal cells increased with progressing gestation; 0.27x10"^ fetal to maternal cells in the first trimester; 3.52x10"^ in the second trimester; and 8.56x10"5 in the third trimester.

The question of how much fetal blood would need to be present in the maternal circulation for it to be detected was addressed by Fewings and Adinolfi (Published in Adinolfi 1991). They injected male donor blood into non-pregnant female volunteers then, having allowed the blood to disperse in the whole circulation, took a sample of volunteer blood and using Y-PCR attempted to detect Y DNA. The PCR technique could identify Y-specific DNA in a female DNA background at a dilution of 1 to 7xlO\ It was concluded that 0.2ml of male blood was required to detect the Y chromosome. Bowman and Pollock (1987) and Mollison et al., (1987) estimated that there may be up to 0.2ml of fetal blood in the maternal circulation in most cases, and in 0.2 1% of

pregnancies between 28-30 weeks’ gestation, there may be up to 1ml. However, Hamanda et al, (1993) using an approximate quantitative Y-PCR method, estimated that only 0.2pl fetal blood is present in a 2 0ml maternal sample which extrapolates to

only 50pl fetal blood in the total maternal circulation.

In 1990, Bianchi et al., used quantitative PCR employing radioactive nucleotides, to amplify a region on the Y chromosome. The number of fetal cells present in each sample was estimated by comparing the intensity of the radio-labelled band with that obtained using known aliquots of male cells. Experiments were carried out on 2 0ml of blood collected from women with male fetuses, and it was estimated

that in this volume there were on average 16 Y-bearing cells. The same tests performed on 20ml of blood collected from women with a female fetus indicated there were 1.45

Y containing cells- thought to be either false positive results or residual cells from previous male pregnancies. From these results it was estimated that each 20ml sample contained 0.04% fetal blood, i.e. lOpl. In a later paper blood samples were collected from women with normal euploid pregnancies (n=199) mostly prior to any invasive procedure (190/9), and from women carrying male aneuploid pregnancies, mostly after an invasive test (27/31) (Bianchi et al., 1997). Although no conclusive proof can be derived from these data due to the almost exclusive collection of one group of samples subsequent to an invasive procedure, the mean of detectable cells appeared to increase when the fetus was aneuploid. The mean number of male cells detected from 16ml of blood collected from women with normal male pregnancies was 19; range 0-91 (compared with 2 from normal female gestations [range 0-24]). The mean number of male cells detected from 16ml of blood collected from women with 47 XY+21 pregnancies was 110 [range 0-650].

Diagnosis of fetal disorders has also been achieved using unsorted, whole blood. Camaschella et al., (1990) obtained maternal blood from 3 pregnant women at risk for fetal beta-thalassaemia/haemoglobin Leporegoston- Haemoglobin

Leporegoston is a haemoglobinopathy caused by a 7-kilobase deletion in the beta- globin cluster. Using PCR to amplify haemoglobin Leporegoston fragments from the maternal blood of normal women whose partners carried the deleted gene, the

condition was correctly identified in two fetuses, and confirmed absent in the third. Lo et al., (1993b) used whole blood in an attempt to diagnose the Rh(D) status of fetuses carried by Rh(D) negative mothers. Without any enrichment for fetal cells, the blood from 21 Rh(D) negative pregnant women was collected and subjected to PCR for Rh(D) sequences. Marginal success was achieved; however there were 4 false positives and 2 false negatives. Greifman-Holtzman et al., (1994) also looked to identify the Rh(D) status of an embryo by examining the peripheral blood of pregnant women. Blood was collected from 9 Rh(D) negative women at 10-22 weeks gestation, prior to amniocentesis or CVS. Rh(D) positive cells were identified in 7 of the 9 samples, all of which were found to come from women with Rh(D) negative fetuses. The remaining two women gave birth to Rh(D) negative infants (Rh(D) negative twins in one case) confirming this diagnosis.

All the aforementioned studies were performed on whole blood from the maternal peripheral circulation. For any realistic future diagnosis, fetal cells would have to be identified or isolated. Isolation or enrichment techniques would have to be directed toward a specific cell type. The human placenta has a villous haemochorial

structure. Unlike other forms of placenta, the haemochorial placenta is characterised by the absence of maternal endothelial, stromal and epithelial layers, resulting in direct contact of the maternal blood with the fetal syncytiotrophoblasts. Exposure of

syncytiotrophoblast to the maternal blood flow may result in trophoblast cells being shed into the maternal circulation. In addition, the reduced barrier between the fetal and maternal circulations may allow passage of fetal leucocytes and erythrocytes into the maternal blood stream (Adinolfi 1992b; 1992c). If any of these nucleated cells could be isolated, non-invasive prenatal diagnosis of fetal genetic disorders would be possible.