Incidence and Types of Fetal Chromosomal Abnormalities in First Trimester of Thai Pregnant Women between Miscarriages and Intrauterine Survivals

Abortion is a common pregnancy complication. Fetuses with several types of chromosomal abnormalities are aborted during the first trimester, while others have a better chance of surviving. This research aims to study and compare the incidence and types of fetal chromosomal abnormalities during the first trimester of Thai pregnant women between miscarriages and intrauterine survivals. Cytogenetic and BACs-on-Beads™ assays were assessed from 2010 to 2020 in Ramathibodi Hospital using first trimester samples of 265 chorionic villi as a retrospective study. Chromosomal abnormalities were observed in 135 cases (50.94%) including 38.11% miscarriages and 12.83% intrauterine survivals. In total, 75.56% single autosomal trisomies, 18.52% sex chromosome aneuploidies, 5.19% double aneuploidies, and 0.74% structural abnormalities were detected. In miscarriages, all chromosomes were involved in abnormalities except chromosomes 1, 5, 8, 9, 11, and 17, while survivals had only trisomy 13, 18, 21, and sex chromosome aneuploidy. Trisomy 16 and 18 were the most common abnormalities in miscarriages and intrauterine survivals, respectively. The highest rate of chromosomal aberrations was demonstrated in 8–9+6 and 12–13+6 weeks of gestation in miscarriages and intrauterine survivals, respectively. Correlation between chromosomal abnormalities and maternal age <35 years and ≥35 years was significant (p < 0.05) in intrauterine survival and first trimester groups.


Introduction
Chromosome aberrations resulting from errors that occur during embryonic division and growth are an important factor in miscarriage within the first trimester [Hassold et al., 2007;Thomas et al., 2021]. Correlation between chromosomal abnormalities and the occurrence of miscarriage has been observed since the 1960s [Bowen et al., 1969]. Cytogenetic techniques (conventional karyotyping) and many molecular assays have been increasingly applied to investigate chromosomal abnormalities in miscarriages [Grati et al., 2015;Sahoo et al., 2017;Teles et al., 2017]. Several studies determined that 50-60% of This article is licensed under the Creative Commons Attribution 4.0 International License (CC BY) (http://www.karger.com/Services/ OpenAccessLicense). Usage, derivative works and distribution are permitted provided that proper credit is given to the author and the original publisher. DOI: 10.1159/000527977 first-trimester miscarriages were caused by fetal chromosomal abnormalities [Hassold et al., 1980;Eiben et al., 1990;Soler et al., 2017;Pylyp et al., 2018]. In Thailand, a study of fetal chromosomal abnormalities in pregnant women was performed during the second trimester using amniotic fluid samples with incidence rates of 2.5-3.3%. Abnormalities were trisomies 13, 18, 21, sex chromosome aneuploidies, and structural rearrangements [Kongyon and Puangsricharern, 2003;Ratanasiri et al., 2011;Pathompanitrat et al., 2013;Rawangkan et al., 2015]. The present study examined the incidence and types of fetal chromosomal abnormalities during the first trimester of Thai pregnant women. Data were compared between miscarriages and intrauterine survivals. No literature is available on chromosomal abnormalities during the first trimester of pregnancy, especially for first trimester miscarriage of Thai pregnant women. Many reports are available on other populations but the Thai population differs in environmental exposure and maternal health conditions from other countries. Accurate identification of genetic characteristics can provide important information for medical management, reproductive counseling, and supportive patient care.

Prenatal Specimens
This retrospective study was carried out by assessing cytogenetic analyses and the BACs-on-Beads TM (BoBs TM ) assay performed in the Human Genetic Laboratory, Department of Pathology, Faculty of Medicine Ramathibodi Hospital, Mahidol University between 2010 and 2020.
A total of 265 chorionic villus samples from first trimester pregnancies were analyzed, consisting of 162 samples from pregnant women who delivered dead embryos or fetuses, in which BoBs assay was performed. The remaining 103 chorionic villus samples were from pregnant women with a living fetus in utero; 90 samples were analyzed by the cytogenetic technique, while the other 13 were subjected to the BoBs assay (Fig. 1). The Ethical Clearance Committee, Faculty of Medicine, Ramathibodi Hospital, Mahidol University approved this study on human rights related to research involving human subjects (COA. MURA2021/811).

Cytogenetic Technique
The cytogenetic technique using chorionic villus samples was modified from the AGT cytogenetic laboratory manual [Arsham et al., 2016]. At least 10 mg of the aspirated villus samples from first trimester pregnancies were carefully washed and estimated under an inverted microscope. Villus fragments were isolated from maternal tissues and blood clots and then disaggregated by mechanical and enzymatic methods. Mesenchymal cells of the villus core were released, and the fibroblasts were actively proliferative in a sterile 25-cm 2 cell culture flask. Three independent cell cultures were performed. After 9-14 days, metaphase chromosomes were prepared for analysis. Karyotyping was performed by G-banding using the trypsin-Giemsa staining technique. Detailed chromosome analyses of 10 metaphase cells of the 400-550 bands per haploid set level were karyotyped and an additional 15 metaphase cells were examined. The aberrations and karyotypes were classified according to the International System for Human Cytogenomic Nomenclature 2020 (ISCN, 2020) [McGowan-Jordan et al., 2020].

BACs-On-Beads TM Assay
A KaryoLite BoBs kit was used to evaluate arm-specific aneuploidies in all 24 chromosomes in a single assay, according to the manufacturer's instructions (Wallac Oy PerkinElmer, Turku, Finland). Native villus fragments that excluded maternal tissue contamination were selected under an inverted microscope and directly performed for DNA extraction. A total of 50-250 ng genomic DNA was labeled with enzyme-linked biotin-deoxynucleoside triphosphate. The labeled products were purified and hybridized with normal DNA from reference males and females with BoBs probes and subjected to fluorescent hybridization incubation. Fluorescence signals were measured using a Luminex 200 platform, and the results were analyzed by BoBsoft 1.0 software (Perki-nElmer) [Jaranasaksakul et al., 2015].

Statistical Analysis
Incidence and types of fetal chromosomal abnormalities were compared between miscarriages and intrauterine survivals. Correlations between frequency of chromosomal aberrations and maternal age groups were analyzed by a χ 2 test using SPSS18.0 software (SPSS Inc., Chicago, IL, USA). Statistical significance was determined at p < 0.05.
Maternal age ranged from 21 to 47 years in miscarriages and 13-45 years in intrauterine survivals. Chromosomal abnormalities by maternal age are shown in Table 3. The frequency of total chromosomal abnormalities in first trimester pregnancies and intrauterine survivals showed a statistically significant correlation with maternal age ≥35 years but it was not significant in miscarriages (p < 0.05) ( Table 4).

Discussion
Maternal age-related risk of a fetus with chromosomal abnormality remains a problem in maternal-fetal medicine. Nowadays, fetal chromosomal assessment is conducted by both noninvasive and invasive methods. Noninvasive methods include maternal factors and history, fetal ultrasound imaging, and maternal serum analyte or cell-free fetal DNA screening. The most popular method of noninvasive prenatal screening for chromosomal aneuploidy involves using circulating cell-free fetal DNA in maternal plasma. Because cell-free fetal DNA is mainly of  placental trophoblastic origin, false positive and false negative results are possible. Moreover, at the early gestational age in which the test is performed, the quantity of circulating fetal DNA is very low, increasing the possibility of false negative results. However, invasive diagnostic methods performed by CVS or amniocentesis can provide definitive results. Chromosomal abnormalities are the most frequent fetal assessment problem during the first trimester of pregnant women. Chorionic villus sampling can be performed much earlier in pregnancy than amniocentesis. When both NIPT/CVS tests showed mosaicism results, amniocentesis was performed to follow up on the definitive survivor karyotypes. The traditional cytogenetic assay offers advantages of detection of additional fetal chromosomal abnormalities and some particular types of chromosomal abnormalities not identifiable through cell-free fetal DNA-based methods. Standard routine prenatal testing of noninvasive methods involves screening for common aneuploidies including trisomy 21, trisomy 18, trisomy 13, and sex chromosome abnormalities. Several laboratory methods have been developed to screen the most common chromosome aneuploidies but the cytogenetic assay is still the gold standard.
Thailand is a middle-income country and faces economic problems in accessing prenatal genetic services of cellfree fetal DNA-based methods. Therefore, cytogenetic analysis is required to reduce the burden of genetic disorders and congenital disabilities that cause significant postnatal functional impairment. The incidence of chromosomal abnormalities during first trimester miscarriages in previously published studies ranged considerably from 40 to 70% [Hassold et al., 1980;Eiben et al., 1990;Ljunger et al., 2005;Soler et al., 2017;Pylyp et al., 2018;Gu et al., 2021]. The types of cytogenetic alteration included single autosomal trisomy, sex chromosome aneuploidy, double aneuploidy, polyploidy, structural abnormality, and placental mosaicism. The correlation between chromosomal abnormalities and maternal age ≥35 years was significant [Hassold et al., 1980;Eiben et al., 1990;Ljunger et al., 2005;Soler et al., 2017;Pylyp et al., 2018;Gu et al., 2021].
However, in this study, fetal chromosomal abnormalities were detected in 62.35% of miscarriages. Single autosomal trisomy, double aneuploidy, sex chromosome aneuploidy, and structural abnormality were observed as chromosomal abnormality types. Interestingly, polyploidy and placental mosaicism were not found in this miscarriage group. The incidence of 62.35% may be underestimated as many miscarriages occur very early before a woman realizes she is pregnant. Limitations of the BoBs assay are its ability to detect only genomic imbalances with a certain resolution but not all chromosomal aberrations. This technique cannot detect balanced structural aberrations, ploidy changes, and mosaicism which can only be detected by conventional karyotyping.
Our results showed chromosomal abnormalities in intrauterine survivals at only 10-13 +6 weeks of gestation, with the highest rate found in 12-13 +6 weeks of gestation, possibly because CVS is usually carried out between the 11th and 14th week of pregnancy and is only offered if there is a high chance of genetic or chromosomal condition. The frequency of chromosomal abnormalities in miscarriages was not significantly correlated with maternal age ≥35 years (p < 0.05). Therefore, other factors apart from age may cause chromosomal nondisjunction in younger women [Ahmad et al., 2010]. A possible explanation for our data involves environmental toxic substance exposure. Economic development and rapid urbanization in Thailand have resulted in environmental degradation and air pollution from traffic in inner cities, chemical hazards from pesticides in countryside areas, heavy metal contaminants from industrial zones and mining, and electronic appliance waste pose a risk to maternal and fetal health [Sinitkul et al., 2018;Waeyeng and Yimthiang, 2021]. The incidence and types of fetal chromosomal abnormalities during first trimester miscarriages in this study concur with previous reports, except for the correlation between chromosomal abnormalities and maternal age ≥35 years.
To the best of our knowledge, no data are available on prenatal chromosomal abnormalities during the first trimester of Thai pregnancies for miscarriages and intrauterine survivals. This may be due to the difficulty of conducting chorionic villus sampling and tissue culture techniques. Obstetricians and technicians must be experienced and possess excellent skills to conduct the medical procedure. Proper handling of first trimester chorionic villus samples in a cytogenetic laboratory requires well-trained cytogeneticists. The genetic characteristics outlined in this study provide vital information for medical management, reproductive counseling, and supportive patient care.