Keywords
spontaneous abortion - aneuploidies - QF-PCR - cytogenetics
Palavras-chave
aborto espontâneo - aneuploidias - QF-PCR - citogenética
Introduction
Miscarriage is defined by the World Health Organization (WHO) as the premature loss
of a fetus before the 20th week of pregnancy, or, if the gestational age is unknown,
the loss of an embryo or fetus weighing less than 400 g.[1] Spontaneous pregnancy loss is the most common complication of pregnancy, and occurs
in ∼12–15% of clinically recognized pregnancies. The chance of a couple experiencing
two consecutive losses is of 2 to 4%, but most women who have miscarriages can give
birth to a healthy child later in life.[2]
The etiology of abortion is multifactorial, and may involve endocrine, anatomic, immunological,
infectious, environmental and genetic factors.[3] Chromosomal abnormalities have been reported in 50% of spontaneously aborted fetuses
of clinically recognized pregnancies, and can be divided in two basic groups: numerical
and structural anomalies. These can involve one or more autosomal, sex or both chromosomes
simultaneously.[4]
[5]
The most frequent autosomal anomaly observed in specimens from spontaneous losses
is trisomy 16 (thought to be lethal and incompatible with full fetal development),
followed by other autosomal aneuploidies and X monosomy.[6]
[7] As fetal chromosomal abnormalities are largely responsible for the inefficiency
of human reproduction and its associated burdens, it is necessary to perform laboratory
investigations of the products of conception (POC) using different diagnostic techniques
to help to understand the possible causes of miscarriage and to provide adequate assistance
for future pregnancies.[8]
[9]
[10]
The analysis of POC has been traditionally performed by cytogenetic karyotyping through
the microscope examination of banded chromosomal preparations, detecting numerical
and structural alterations. Molecular cytogenetic diagnostic tests are based on studying
the fetal karyotype directly at the DNA level, and use DNA extracted from fetal cells,
not requiring tissue culture and allowing the analysis of specimens fixed in ethanol,
formaldehyde or included in paraffin.[11]
[12] Some of these techniques are fluorescence in situ hybridization (FISH), multiplex
ligation-dependent probe amplification (MLPA) and quantitative fluorescent polymerase
chain reaction (QF-PCR).
All reproductive losses should be investigated by cytogenetics, considering that conventional
karyotyping has been the gold standard for the chromosomal investigations of POC.
This method allows the detection of structural (translocations, deletions and inversions)
as well as numerical chromosomal aberrations. However, it is a laborious, time consuming
procedure that can lead to a significant amount of cases with no results, as it depends
on human cells in active process of replication. The rate of culture failure is of
10–40%, as POC tissues are frequently macerated, contaminated or fixated in alcohol
or formaldehyde.[12]
[13] Molecular cytogenetic techniques can be used to study POC abnormalities, as they
do not require cell culture; however, the results could be limited because only numerical
chromosomal alterations can be identified on the analyzed chromosomes.[14]
The genetic studies of POC provide important information for the genetic counseling
of couples who experience pregnancy failure, as they help to elucidate the possible
causes of fetal losses, indicating if any chromosomal abnormality was responsible
for the miscarriage. They can also indirectly suggest if one of the parents could
be the carrier of any structural disorder.[12]
[14]
The aim of this study was to describe the frequencies of chromosomal abnormalities
found in abortion material, and to determine if there is a correlation between the
presence of aberrations and maternal age.
Methods
Study Type and Samples
An observational, retrospective study was conducted to describe the results obtained
from miscarriage material analysis performed by a private medical genetics laboratory
in Belo Horizonte, MG, Brazil. The laboratory performs tests in miscarriage material
received from different regions of Brazil, covering the whole country. The results
were obtained from the laboratory databank, and, besides maternal age, no personal
information from the patients was included. As the laboratory receives material from
different medical facilities with limited information, clinical data such as gestational
age at abortion and clinical history of the parents was not available. A total of
884 results from miscarriage material analysis performed between January 2011 and
December 2015 was included.
Sample Analysis
Miscarriage material analysis techniques were performed by professional staff, according
to the laboratory routine, using conventional cytogenetics, or QF-PCR.
Conventional cytogenetics was performed in samples containing tissue that had fetal
origin, using standard culture, harvesting and staining conditions, by an experienced
cytogenetics technician. A total of 204 cytogenetics results were obtained and included
in the study. The aberrations and karyotypes were classified according to the International
System for Human Cytogenetic Nomenclature 2013 (ISCN 2013).[15]
In case of cell growth failure by the cytogenetics technique, and according to the
laboratory demand, the QF-PCR molecular technique was performed, as previously described.[16] For detection of chromosome aberrations, markers for sexual X and Y, and for autosomal
13, 16, 18, 21 and 22 chromosomes were performed by experienced professionals, according
to the laboratory routine. A total of 680 QF-PCR results were available. These included
the samples with cell growth failure in cytogenetics that were investigated by molecular
QF-PCR, and the miscarriage material samples unsuitable for, or in which cytogenetics
was not demanded.
Statistical Analysis
The results were presented as frequency values according to the different technique
performed (cytogenetics or molecular biology), and distributed according to the chromosomal
alterations found by each method and total sample results. The relation between the
frequency of chromosomal anomalies and maternal age was analyzed by logistic regression
using the statistical software Minitab 17.3.1 (State College, PA, USA). Significance
was set at p < 0.05.
The study was submitted and approved by the Ethics Committee of the institution under
protocol CEP nº 1.346.235, on December 1, 2015.
Results
A total of 884 results from miscarriage material samples was included in the study.
[Table 1] shows the frequency of normal and abnormal results obtained by conventional cytogenetics
and molecular biology QF-PCR technique. From the total sample of 884, 368 (42%) cases
of chromosome abnormalities were detected, while 516 (58%) cases had no detected alterations.
Cytogenetics was able to identify 52% of normal results (106 out of 204 tested), and
QF-PCR, 60% (410 out of 680 tested). Cytogenetics showed 48% (98 out of 204 tested)
of abnormal results, and molecular biology detected 40% (270 out of 680 tested) of
abnormal cases, considering the chromosomes analyzed by this technique.
Table 1
Normal and abnormal results observed by cytogenetics and molecular biology techniques
|
Cytogenetics (n = 204)
|
Molecular biology (n = 680)
|
Total (n = 884)
|
|
Normal
|
106 (52%)
|
410 (60%)
|
516 (58%)
|
|
Abnormal
|
98 (48%)
|
270 (40%)
|
368 (42%)
|
The chromosomal abnormalities identified by each method are demonstrated in [Table 2].
Table 2
Frequency of chromosomal anomalies
|
Anomaly
|
Cytogenetics n (%)
|
Molecular biology n (%)
|
Total n (%)
|
|
X monosomy
|
18 (18.4%)
|
43 (15.9%)
|
61 (16.6%)
|
|
Trisomy 2
|
1 (1%)
|
NA
|
1 (0.3%)
|
|
Trisomy 4
|
2(2%)
|
NA
|
2 (0.5%)
|
|
Trisomy 7
|
1 (1%)
|
NA
|
1 (0.3%)
|
|
Trisomy 8
|
3 (3.1%)
|
NA
|
3 (0.8%)
|
|
Trisomy 10
|
3 (3.1%)
|
NA
|
3 (0.8%)
|
|
Trisomy 13
|
4 (4.1%)
|
25 (9.3%)
|
29 (7.9%)
|
|
Trisomy 14
|
2 (2%)
|
NA
|
2 (0.5%)
|
|
Trisomy 15
|
8 (8.2%)
|
NA
|
8 (2.2%)
|
|
Trisomy 16
|
13 (13.3%)
|
51 (18.9%)
|
64 (17.4%)
|
|
Trisomy 17
|
2 (2%)
|
NA
|
2 (0.5%)
|
|
Trisomy 18
|
5 (5.1%)
|
16 (5.9%)
|
21 (5.7%)
|
|
Trisomy 20
|
1 (1%)
|
NA
|
1 (0.3%)
|
|
Trisomy 21
|
9 (9.2%)
|
23 (8.5%)
|
32 (8.7%)
|
|
Trisomy 22
|
10 (10.2%)
|
53 (19.7%)
|
63 (17.1%)
|
|
47 XXY
|
1 (1%)
|
NA
|
1 (0.3%)
|
|
Triploidy
|
9 (9.2%)
|
56 (20.7%)
|
65 (17.7%)
|
|
Trisomies 16 and 22
|
0
|
2 (0.7%)
|
2 (0.5%)
|
|
Trisomies 16 and 21
|
0
|
1 (0.4%)
|
1 (0.3%)
|
|
Other aneuploidies
|
6 (6.1%)
|
NA
|
6 (1.6%)
|
|
Total
|
98 (100%)
|
270 (100%)
|
368 (100%)
|
Abbreviation: NA, Not analyzed by QF-PCR.
Cytogenetics results showed 18.4% X monosomy, 13.3% trisomy 16, and 10.2% trisomy
22. It also revealed that 8.2% of the abnormalities were trisomy15. Molecular biology
results were 15.9% for X monosomy, 18.9% for trisomy 16, and 19.7% for trisomy 22.
Chromosome 15 was not evaluated by QF-PCR in this study ([Table 2]).
Considering all the samples, trisomy was the most common chromosome aberration, accounting
for 63% (232 out of 368) of the abnormalities. The most frequent was trisomy 16 (17.4%),
followed by trisomy 22 (17.1%). Monosomy accounted for 16.6% (61 out of 368) of the
anomalies, and polyploidies, for 18.8% (69 out of 368) ([Table 2] and [Table 3]).
Table 3
Groups of chromosomal anomalies
|
Anomaly
|
Total n (%)
|
|
Trisomy
|
232 (63%)
|
|
Polyploidy
|
69 (18.8%)
|
|
X monosomy
|
61 (16.6%)
|
|
Others
|
6 (1.6%)
|
|
Total
|
368 (all)
|
The relationship between aneuploidy and maternal age is shown in [Fig. 1]. In the young maternal age group (n = 452), 62.2% (281) had normal results, while abnormalities were found in 171 cases
(37.8%). For the advanced maternal age group (n = 432), 235 (54.4%) normal results were observed, compared with 197 (45.6%) abnormal
findings. This difference was considered statistically significant (p = 0.02) ([Fig. 1]).
Fig. 1 Maternal age and chromosomal abnormality. Numbers between parentheses show the percentage
of the results. The rates of abnormal results were significantly higher for the advanced
maternal age group (n = 432) when compared to the younger maternal age group (n = 452)
(p = 0.02).
Discussion
The analysis of chromosomal abnormalities in POC is useful to determine the possible
causes of miscarriage, and to provide information and counseling for couples regarding
future pregnancies.
This retrospective study showed the presence of aneuploidies in 42% of all the analyzed
samples. When the different techniques were considered, the results were similar (40%
by QF-PCR and 48% by cytogenetics). A recent study by Coelho et al[17] demonstrated the presence of aneuploidies in 54.6% of miscarriage material from
Brazilian patients, analyzed by QF-PCR. Jenderny[15] reported a frequency of 61% abnormal results in POC analyzed by cytogenetics and
QF-PCR in the German population. Other studies found similar (48%) or lower frequencies
(36%) of total chromosomal aberrations.[18]
[19]
The present study revealed that the main chromosomal abnormality detected in abortion
material was trisomy, followed by triploidy and monosomy X. These results corroborate
with other studies that demonstrated similar results, showing that trisomies, especially
those involving chromosomes 16 and 22, are implicated in spontaneous abortion.[17]
[20]
[21]
[22]
[23]
[24]
[25]
An important point noted in the present study was that trisomy 15 was found in 8.2%
of the samples analyzed by conventional cytogenetics. Similar values were observed
by Coelho et al[17] (14.1%), Moraes et al[20] (9%), Subramaniyam et al[21] (13.5%), and Romero et al[25] (7,7%), suggesting that trisomy 15 is recurrent in POC samples. Until the present
moment, chromosome 15 markers have not been included in routine QF-PCR analysis of
abortion material at the laboratory. However, the results showed that a marker for
chromosome 15 should be included in the molecular analysis, improving the quality
of the released technical reports.
Conventional cytogenetics technique remains highly recommended for spontaneous miscarriage
analysis. However, this method has certain disadvantages, such as a long laboratory
cycle, labor intensity and culture failure, especially when the tissues obtained from
patients are not well preserved.[17]
[26]
The QF-PCR technique is regarded as a highly accurate, low cost and rapid diagnostic
method to facilitate the detection of clinically relevant chromosome aberrations.
However, some limitations of the technique are the difficulty to offer a correct diagnosis
for mosaicism, small deletions, translocations and duplications.[15]
[17]
Since our laboratory miscarriage material is received from different medical facilities,
the QF-PCR method plays an important role as a reliable method for the detection of
aneuploidies, when culture fails or when the karyotype is not possible due to inadequate
material preservation.
Many reports have suggested that advanced maternal age is an important factor related
to chromosomal aneuploidies.[22] Our study showed a significant increase in the rate of aneuploidy in the advanced
maternal age group when compared with the young maternal age group (p = 0.02). Bastos et al[6] and Jia et al[22] also suggest a maternal age-related increase in chromosomal anomalies. Hormonal
changes during the aging process, as decreased production of progesterone can lead
to increased rates of miscarriage in women older than 35 years.[27]
In conclusion, chromosomal aberrations are still a major cause of miscarriage, and
the conventional cytogenetics study is highly recommended, as it can detect different
types of chromosomal abnormalities. Molecular biology techniques, such as QF-PCR,
are important complementary methods that can be effective to detect the main chromosomal
anomalies, and may be used in combination with cytogenetics to allow the release of
technical reports with reliable results.
