Chromosomal Abnormalities in Early Pregnancy Losses: A Study of 900 Samples

Our study included 900 POC samples from women who experienced EPLs (gestational age ≤12 weeks). POC samples, previously selected by a gynaecologist/pathologist [15] and accompanied with maternal whole blood sample, were referred for analysis of chromosomal abnormalities to the Research Centre for Genetic Engineering and Biotechnology “Georgi D. Efremov”, at the Macedonian Academy of Sciences and Arts, Skopje. Signed informed consent was obtained from all participants in this study. The study has been approved by the ethical committee of the Macedonian Academy of Sciences and Arts (09-1047/6 from 04.05.2016).

Table 1 displays the clinical characteristics of the women with EPLs, including maternal age, ethnic origin, history of previous EPL, previous live birth, maternal ABO blood group, Rhesus (Rh) factor, and the gestational week (gw) of the EPLs studied. The samples were categorized into three groups according to the maternal age: ≤30, 31–35, and ≥36 years. The majority of patients were of Macedonian (n=528) ethnic origin as well as Albanian (n=208). Gestational ages of the POCs consisted of samples from gw=6 to gw=11 (mean gestational age 8.5 weeks).

DNA extraction from the POC samples, as well as from maternal blood was performed using the standard phenol/chloroform method or the automated magnetic bead-based protocol using the MagCore Super instrument (RBC Bioscience). The study primarily used the quantitative fluorescent (QF)-PCR method with STR markers on chromosomes 13, 18, 21 and sex chromosomes. Three markers were located on chromosome 13, four on chromosomes 18 and 21 each and six on the sex chromosomes. Except aneuploidies on the given chromosomes, this method also allowed for the determination of triploid samples, and exclusion of maternal DNA contamination in the analysed samples. This method is described in detail by Noveski et al. [16]. All results for chromosomal aneuploidies obtained by the QF-PCR analyses were confirmed by the subsequent subtelomere MLPA analyses.

Chromosomal gains and losses were detected with the Multiplex Ligation Probe Amplification (MLPA) method, using the SALSA MLPA P036 Subtelomeres mix 1 and SALSA MLPA P070 Subtelomeres mix 2B (MRC-Holland). Each MLPA kit contains two probes for each chromosome. For metacentric chromosomes the two probes were located subtelomerically, while for the acrocentric chromosomes, one probe was located subcentromeric, while the other was subtelomeric. The detailed MLPA protocol as well as the chromosomal location of each probe contained in the kits is available on the MRC-Holland site. Capillary electrophoresis was performed on the AB3500 Genetic Analyser (Life Technologies), and the obtained results were analysed and interpreted using the Coffalyzer software (MRC-Holland). Mean values, standard deviations, percentages, odds ratios, and p-values were determined where appropriate, using the MedCalc software (MedCalc Software Ltd. https://www.medcalc.org/ Version 22.016; accessed November 17, 2023). A p-value below 0.05 was considered statistically significant.

Chromosomal abnormalities were present in 56.25% of uncontaminated POC samples. Chromosomal trisomies were detected in 66.20%, triploidy in 15.28%, and monosomies were present in 10.88% of the positive cases. The group of monosomies consisted of monosomy X which was predominant (91.49%) and monosomy 21 was present in 8.51%.

All chromosomes, apart from chromosomes 1 and 19, were affected by trisomy; the most common was trisomy 16, present in 26.57% of all trisomy samples, followed by trisomy 22 (15.73%), 21 (10.13%) and 15 (9.09%) (Figure 1).

Figure 1.

Partial chromosomal abnormalities (terminal deletions and duplications), multiple chromosomal aneuploidies, and chromosomal aneuploidies accompanied by partial chromosomal abnormality were found in 3.70%, 3.01%, and 0.93% of all positive samples (Table 2). A detailed description of the later three groups of chromosomal abnormalities is provided in Figure 2. Chromosomes 20, 21, 22 and X were most involved in multiple aneuploidies, chromosomes 1, 13 and 18 in partial aneuploidies, while chromosomes 15, 16 and 22 were found in combination with a partial aneuploidy. In cases of partial chromosomal abnormalities, parental karyotyping could not be performed at the time.

Figure 2.

As shown in Table 2 the overall frequency of chromosomally abnormal POCs increased with maternal age (43.40% in the women ≤30 years of age, 53.94% in women 31–35 years and 71.37% in woman ≥36 years). The odds for chromosomally abnormal EPLs were 1.52 higher in the group of women 31–35 years of age (95% CI: 1.07–2.16; p=0.018) and 3.25 higher in women ≥36 years (95% CI: 2.26–4.66; p<0.0001), when compared to the group of women ≤30 years of age (Table 3).

The spectrum of the chromosomal abnormalities differed in the three groups of women according to their age. In the group ≥36 years, the trisomies were predominant (79.68%), while triploidy and monosomies were present in small portions (6.42% each). In the group of women ≤30 years of age, a more even distribution of the chromosomal abnormalities was observed, with trisomies present in 48.70%, triploidy in 26.09% and monosomies in 16.52%. The odds for EPLs with trisomy were 1.88 times higher in the group of women 31–35 years of age (95% CI: 1.26–2.81; p=0.0017) and 4.92 higher in women ≥36 years (95% CI: 3.35–7.21; p<0.0001), when compared to the group of women ≤30 years of age (Table 3).

Multiple chromosomal trisomies were more common among the group ≥36 years (5.88%), while partial chromosomal aneuploidies were more frequent in women ≤35 (Table 2).

Considering the individual trisomies, trisomy 16 had the highest incidence in the group ≤30 years (39.29%), and lowest in the group ≥36 years of age (16.78%). Furthermore, trisomies 13 and 14 were also more common among the group ≤30 years (12.50% and 12.50% each), than in the other two groups. By contrast, trisomy 22 was more common in the group ≥36 years (23.49%) than in the group 31–35 years (12.35%). It was entirely absent in the youngest group of women (≤30 years of age). Similarly, the trisomy 15 was most common in the group of women ≥36 (14.77%) than in other groups (0.00% and 4.94% in groups of women ≤30 and 31–35 years, respectively) (Table 4). The odds of EPLs with trisomy 16 were 0.31 lower in the group of women ≥36 years (95% CI: 0.15–0.62; p=0.0009) while for trisomy 22, the odds were 16.59 higher in the group of women 31–35 years of age (95% CI: 0.95–289.31; p=0.054) and 35.03 higher in women ≥36 years (95% CI: 2.11–581.6; p=0.013) when compared to the group of women ≤30 years of age. The odds for trisomy 15 were higher (OR=19.94, 95% CI: 1.18–334.53; p=0.037), while for trisomies 13 and 14 were lower in women ≥36 years (OR=0.29, 95% CI: 0.09–0.91; p=0.034 and OR=0.24, 95% CI: 0.07–0.80; p=0.02, respectively) when compared to the group of women ≤30 years of age (Table 3).

Although not statistically significant, a higher incidence of chromosomally abnormal POCs were detected in the Macedonian ethnic group (58.52%) in comparison to the Albanian group (50.96%; p=0.06) (Table 2). Triploidy and chromosomal monosomies were significantly more common among the Albanian ethnic group (25.47% and 15.09% respectively; p=0.0001) than in the Macedonian group (12.30% and 9.39% respectively). In contrast, chromosomal trisomies were more prevalent in POCs of Macedonian ethnic origin (69.90%) than in the Albanian group (54.72), (p=0.0044).

Regarding the individual trisomies, the most evident difference was observed in trisomy 16, being more common among Albanians (32.76%, p=0.207) than among Macedonians (24.54%). Trisomy 22 was found to be more frequent in the Macedonian group compared to the Albanian (18.52% vs. 6.90%, respectively, p=0.035) (Table 4). However, these differences can be explained by the age distribution of samples with different ethnicity. Namely, most Macedonians (41.47%) belonged to the ≥36 years group and only 24.81% were in the group ≤30, while the majority of Albanians (58.17%) belonged to the group ≤30, and only 16.82% were in the group ≥36. This is in accordance with the tendency of earlier childbearing among Albanian women.

The frequency of chromosomal abnormalities in POCs from women with sporadic EPLs was higher than in those with RPLs (61.19% vs. 55.21%, respectively, p=0.164), this difference, however, was not statistically significant (Table 2). No evident difference was observed between these two groups regarding the presence of different classes of chromosomal abnormalities and chromosomal trisomies (Tables 2 and 4).

In the group of women with a previous live birth, a slightly higher, but not significantly different, incidence of chromosomally abnormal POCs was found compared to the group of women without a live birth (61.97% vs. 56.92%, respectively, p=0.297) (Table 2). However, this is most probably due to the higher mean age of the women with a live birth (34.66 years) in comparison to that of women with no live birth (32.57 years). Triploidy and trisomies did not differ much among these groups, while monosomies were more common in the group with (15.91%, p=0.119) compared to the group without live births (9.63%), but without statistical significance.

Trisomy 16 and 21 were more prevalent in the group without live births (27.97% and 8.39%, respectively) than in the group with live births (15.00%, p=0.049 and 3.33%, p=0.195; respectively), while trisomy 15 was more common in the group with live births (18.33%, p=0,041) vs those without (8.39%) (Table 4). These findings were statistically significant for trisomies 16 and 15, but not for trisomy 21.

The highest incidence of chromosomally abnormal POCs was detected in women with the AB blood group (63.89%), followed by A (62.84%), O (56.28%) and B (50.75%) (Table 2). The O blood group had the highest incidence of monosomies (14.56%) in contrast to the B with the lowest rate of monosomies in our study (8.82%) (p=0.312). The opposite was observed concerning the trisomies, where B had the highest, and O the lowest incidence (76.47% and 61.17%, respectively) (p=0.176).

Higher presence of trisomy 22 was observed among women of the AB blood group (43.75%), in comparison to women of other (combined B, A and O) blood groups (17.46%) (p=0.012). Furthermore, trisomy 21 was more frequently observed among women in the O blood group (9.52%; 6/63), compared to other groups (2.52%) (p=0.034) (Table 4).

Women with positive RhD positive (RhD+) had slightly higher incidence of abnormal POCs (59.51%) than the RhD negative (RhD−) women (52.54) (p=0.310).

Overall, increased incidence of chromosomal abnormalities was present in advanced gestational age. The POCs eliminated in gestational week (gw) 10 showed the highest incidence of chromosomal abnormalities (66.67%), followed by gw 9 (63.08%), while the lowest rate was observed in the POCs eliminated in the gw 6 (52.78%).

Higher incidences of triploidy and monosomies were observed in POCs eliminated between gw 9 and gw 11 (p=0.135 and p=0.0015, respectively), while trisomies were more common among studied POCs from gw 6 to 8 (p=0.0026) (Table 2).

Among the 768 POCs, 379 were male sex (49.34%) and 389 were of female sex (50.65%). A slightly lower number of chromosomally abnormal male POCs was observed (53.56%) in comparison to the female POCs (58.86%). Triploidies were more common among male POCs in contrast to the females (25.56% vs. 13.75%, respectively; p=0.026). Chromosomal monosomies were more prevalent in female than in male POCs (28.12% vs. 1.7%; p<0.0001), but this was expected since monosomy X was predominant (95.55%) in this group, and there were only 2 cases with monosomy 21 (4.44%). Regarding the chromosomal trisomies, no significant difference was observed between males and females (Tables 2 and 4). In the group of RPL, a higher percentage of male POCs were euploid in comparison to the female POCs (50% vs. 39.85%), but the difference was not significant (p=0.1).