1 1Department of Biomedical Sciences and Advanced Therapy, Section of Obstetrics and Gynaecology, 2 Department of Biology, Section of Evolutionary Biology and 3 Department of Clinical and Experimental Medicine, Section of Nuclear Medicine, University of Ferrara, Ferrara, Italy
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Abstract |
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Key words: amniotic fluid/aneuploidy/cytokines/fetal chromosomal abnormalities/prenatal diagnosis
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Introduction |
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It is known that ~15% of human pregnancies end in spontaneous abortion before 12 weeks gestation. However, the percentage of pregnancy loss is much higher in the presence of aneuploidy (Boué et al., 1985). It can be thought that the chromosomal abnormality itself represents the cause of abortion, but the pathogenic mechanism is unknown. In our attempts to understand the causes of pregnancy loss, we felt that these are not different in euploidy compared with aneuploidy, they are simply much more frequent in the latter, possibly as a consequence of an imbalance of the mediators of placental perfusion and uterine contraction related to a genetic basis. The levels of the cytokines relevant to the establishment and maintenance of a viable pregnancy in the presence of fetal chromosomal abnormalities has not been reported. However, in the presence of fetal Down's syndrome, an increased amniotic level of TGF-ß has been found and this has been proposed as a possible additional biochemical index for the prenatal detection of the disease (Bromage et al., 2000
).
Different behaviour of these mediators in the fetal compartment could represent the mechanism by which, in the absence of infection, the human fetus itself participates in its own demise. In this context, aneuploid pregnancies could be considered as a natural study model for a better understanding of the general causes of pregnancy loss.
On the basis of the above considerations, the present study assessed the amniotic and maternal serum levels of IL-6, IL-8 and TNF-ß in euploid and aneuploid pregnancies.
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Materials and methods |
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The study population was selected among 1082 amniocentesis performed from 1994 to 1998. All the observed 31 cases with chromosomal abnormalities (2.9%) were included, while 92 control cases were randomly chosen among euploid pregnancies. The pathological sample consisted of 15 aneuploidies, representing 1.4% of the study population, and 16 minor chromosomal abnormalities (1.5%). Average maternal age was 35.1 years in the control group, and 33.4 in the group with chromosomal abnormalities. Indication to amniocentesis in the control group was represented by maternal age over 35 years in 60 cases, positive triple test in 24, history of recurrent abortion in two, previous fetal Down's syndrome in three, assumption of teratogenic agents in two, parental chromosomal translocation in one, previous fetal malformation in two. In the pathological group there were 14 cases over 35 years, six with positive triple test, three with mosaicism at the chorionic biopsy performed for maternal age, three with ultrasound abnormalities, four with history of recurrent abortion and one with previous fetal malformation.
All amniocenteses were performed using ultrasonographic guidance. The amniotic fluid was stored within 6 h at 20°C, in the Laboratory of Nuclear Medicine, after the removal of particulate materials by centrifugation (800 g for 10 min). Cytogenetic analysis on the cells was carried out at the Section of Medical Genetics of the University of Ferrara, according to the International Guidelines Association of Cytogenetics Technologies. At the same time as amniocentesis, a maternal blood sample was collected and centrifuged (800 g for 10 min); the serum was then stored at 20°C and kept frozen without interruption until analysis.
Since it is known that intraamniotic infection can exist early in pregnancy, even with intact membranes, and in most cases without any clinical symptom (Goldstein et al., 1990), our standard procedure for amniocentesis included microbiological studies of all the amniotic fluid samples. Two millilitres of amniotic fluid were transferred into two culture bottles, one aerobic and one anaerobic. Positive cultures were identified by means of a computerized Bac T Alert System® (Organon Teknica, Rome, Italy). After Gram stain microscopic examination, further cultures were prepared with the appropriate medium. Final identification of species was obtained by biochemical (Api System®; Montalieu, Verciev, France) or serological methods.
The population was divided into the following three groups (Table I): euploid (n = 92), minor chromosomal abnormalities (n =16) and aneuploid (n = 15), in agreement with our previous study (Vesce et al., 2001
). There were two cases lacking the amniotic fluid sample (aneuploid group) and five cases lacking the maternal serum sample (three patients with aneuploid chromosomal abnormalities and two controls).
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The specific monoclonal antibodies of the three different kits were able to detect IL-6, IL-8 and TNF-ß in cell culture supernatants, human serum, plasma, amniotic or other body fluids. For a correct quantitative determination of each factor, all the samples were diluted in the following way: (i) IL-6: dilution 1:6 in assay buffer for amniotic fluids; the sera, instead, were used undiluted; (ii) IL-8: dilution 1:2 in assay buffer for amniotic fluids and sera; and (iii) TNF-ß: no dilution for either amniotic fluids or sera.
Optical density (OD) values were performed using an automated Microplate-Reader Model 550® (Bio-Rad, USA) at 450 nm (620 nm as optional reference wavelength). For calculation of IL-6, IL-8 and TNF-ß levels, linear regressions were established between OD (y-axis) and the corresponding standard concentrations (x-axis). All results were expressed as pg/ml.
In amniotic fluids, TNF-ß levels were distributed normally, whereas IL-6 and IL-8 values were normalized using natural logarithmic transformation. To assay differences in parameter levels between the three different groups of patients, a one-way analysis of variance (ANOVA), with Tukey HSD test for post-hoc comparison, was performed (Vesce et al., 2001). For IL-6 and IL-8, values are expressed both as mean ± 95% confidence interval of the antilog of the logarithmic value and as median value and the interquartile range (lowerupper quartile).
In maternal serum, none of the parameters analysed presented a normal distribution, thus the KruskalWallis ANOVA of Ranks test and the MannWhitney U-test were applied to assay differences in parameter levels and the median values and the interquartile ranges (lowerupper quartile) are presented.
To estimate whether levels of the components in amniotic fluid depended on levels in maternal serum, a Spearman's correlation analysis was carried out.
Finally, to assess the predictive values of the parameters of interest, i.e. the likelihood that a woman who shows alteration in the level of one of the components studied actually presents a fetus with chromosomal abnormalities, the Pearson 2 test was used (Sokal and Rohlf, 1995
).
For all tests, significance levels for the rejection of the null hypothesis were set at P value < 0.05. Data is expressed as mean ± standard deviation (SD) of the variable before the logarithmic transformation in tables. In the graphs, the boxes represent the mean of the variable after natural logarithmic transformation (smallest box) ± SEM (large box), while whiskers around the box indicate 95% confidence interval (CI) of the logarithmic mean. Statistical analyses were carried out with the Statistica software package version 4.5® (StatSoft inc., Tulsa, OK, USA).
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Results |
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In amniotic fluids, the concentrations of TNF-ß did not differ between euploidies, minor chromosomal abnormalities and aneuploidies, whereas for IL-6 and IL-8 levels, significant results were observed (P = 0.034 and < 0.0001) (Table II).
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To assess if the correlation observed between IL-8 in amniotic fluid and IL-6 levels in maternal serum was a general trend for all three groups of pregnancies studied, or if a different behaviour was shown by pathological cases, we verified the strength of the correlation within these groups. What we observed was an absence of correlation between these two parameters within both euploid cases (P = NS) and pregnancies presenting minor chromosomal abnormalities (P = NS). Thus, the direct proportionality between IL-8 level in amniotic fluids and IL-6 in maternal serum observed for the whole sample could be attributed almost entirely to the group of pregnancies presenting aneuploidies (P < 0.006).
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Discussion |
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The results of the present study, which was limited to three cytokines IL-6, IL-8 and TNF-ß, did not show any difference either in maternal serum or in the amniotic levels of TNF-ß between the three groups of patients. A significant increase of amniotic IL-6 level was found in the presence of fetal chromosomal abnormalities. However, the main increase was accounted for by the group of minor chromosomal abnormalities, while for the aneuploidy group, also characterized by a higher level of the cytokine, significance was not reached. Since all the microbiological studies on amniotic fluid gave negative results, the increased level of IL-6 was interpreted as a consequence of a genetic feature rather than infection. In contrast, the amniotic level of IL-8 was significantly lower in aneuploid cases compared with either euploid or minor chromosomal abnormality groups.
As regards maternal serum, the finding of decreased levels of IL-6 concentration in the aneuploid group in the presence of an increased amniotic level of the cytokine represents an apparent discrepancy. However, a strict compartimentalization has been reported for IL-8 and for monocyte chemotactic protein-1 (MCP-1) during the first trimester of pregnancy, with high levels in the extra-embryonic coelomic fluid and very low levels in amniotic fluid and maternal serum (Denison et al., 1998). Furthermore, the absence of correlation between maternal plasma and either fetal plasma or amniotic fluid of IL-6 concentration has been recently reported (Romero et al., 1998
). In Romero's study, the presence of increased fetal plasma concentration of IL-6 was considered as an expression of a fetal inflammatory response to infection. This type of response is supposed to be addressed to trigger uterine contractions, giving rise to labour in order to escape an intrauterine environment that is hostile. It can be confined to the fetus lacking in the amniotic fluid as well as in the maternal plasma, as revealed by lower levels of the cytokine within these compartments. However it is also reported (Gomez et al., 1998
) that in about 13% of the cases the elevation of the fetal plasma concentration of IL-6 is not related to infection, and therefore it is supposed that there is an unknown infection-independent process able to increase the level of the cytokine. Based on our data such a process could be related to genetically controlled factors.
In the search for possible connections between fetal chromosomal abnormalities and alterations in cytokine levels, it must be considered that IL-6 and IL-8 are coded by genes localized on chromosome 7 (Sehgal et al., 1986) and 4 (Modi et al., 1990
) respectively.
Recently, Hughes et al. (Hughes et al., 2000), using whole-genome expression data, discovered an unexpected similarity in transcript profiles for unrelated deletion mutants in Saccharomyces cerevisiae. In fact, in some of the 300 mutants analysed, the observed correlation between gene transcription profiles was mostly due to changes in the expression of genes present on chromosomes other than the aneuploid chromosome, as might be expected when a key transcriptional regulator is affected directly by the aneuploidy. This observation confirms and extends the previous result obtained by Pollack et al. on human transformed cells (Pollack et al., 1999
).
Thus, considering the complexity of gene expression pattern in eucaryotic cells, it seems credible to form the hypothesis that the connection between the fetal chromosomal abnormalities observed in our data, mainly involving chromosomes X, 21, 18 and 13, and variation of IL-6 and IL-8 levels, could lie in a correlation between the expression of genes present in genomic regions localized on chromosomes other than that which determines aneuploidy.
As regards the obstetrical implications of our findings, it should be considered that IL-8 has been reported to stimulate placental prostacyclin (PGI2) production. During pregnancy PGI2 acts as a potent vasodilator and an inhibitor of platelet aggregation, protecting against the vasoconstrictory and platelet aggregatory action of thromboxane A2. Moreover, it has been found that the placentas of women with pre-eclampsia produce seven times less PGI2 than thromboxane A2 (Walsh, 1985), and that decreased PGI2 production correlates with significantly reduced release of IL-8 (Wang et al., 1999
). Therefore, it can be hypothesized that the significantly reduced amniotic level of IL-8, coupled with the increased amniotic level of IL-6 found in our cases, represents a factor that could impair placental perfusion and promote uterine contractions, thus leading to the adverse outcome of pregnancy.
To explain the high incidence of abortion in aneuploid pregnancies compared with euploid, we have previously reported that the former are characterized by a significantly higher amniotic and lower maternal plasma level of endothelin-1 (Vesce et al., 1996), a potent vasoconstrictor peptide, the level of which is also increased in pre-eclampsia (Nova et al., 1991
). Moreover, we have found that the amniotic and maternal serum levels of the components of the plasminogen system, which are involved in the physiological angiogenic processes of pregnancy, are deeply altered in the presence of fetal aneuploidy (Vesce et al., 2001
).
In the present study, it is interesting to highlight the significant positive correlation found between IL-6 levels in serum and amniotic IL-8 values.
All these observations support the suggestion that aneuploidy correlates with a disturbance of the release of some mediators of placental perfusion and uterine contraction that might explain the increased incidence of pregnancy loss.
In the cases of abortion, preterm labour, premature rupture of the membranes and fetal inflammatory response syndrome where infection is not found, the imbalanced levels of inflammatory cytokines could be interpreted as a consequence of a genetic feature whereby the fetus participates in the mechanism of its own distress, death and expulsion. However, in this study the cytokine imbalance apparent in this group of patients might represent a chance finding, and it must be confirmed by extensive investigation before any firm conclusion can be reached. Such an approach could be of value for the correct assessment of some cases with poor outcome of pregnancy as a consequence of a genetic disorder rather than infection.
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Notes |
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Submitted on May 15, 2001; resubmitted on July 27, 2001
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accepted on October 19, 2001.