Fetal aneuploidy and chromosome structural abnormalities can lead to a spectrum of diseases, ranging from miscarriage, stillbirth, and other adverse pregnancy outcomes to adults with neurological disorders (e.g., copy number variant, CNV) (Shahine and Lathi, 2015; Yilmaz et al., 2017). Many maternal factors contributed to embryonic chromosomal aberrations, such as age, genetic polymorphism, reproductive history, and immune and endocrine dysfunction (Ozawa et al., 2019). It is reported that polymorphisms in folate-homocysteine pathway genes are related to chromosomal breaks and fetal chromosomal aneuploidy (Enciso et al., 2016). Environmental factors were also implicated in embryonic chromosomal aberrations, such as drug and pesticide exposure may increase the risk of embryonic DNA damage (Scheuerle and Aylsworth, 2016). Most embryos are aneuploidy derived from gamete cells, especially oocytes. However, the exact causes and etiopathogenetic mechanisms of teratogenesis are not well demonstrated (Baldacci et al., 2018; Toufaily et al., 2018).

The mitotic checkpoint system is a well-known major mechanism essential for maintaining the cell’s genomic stability. Consequently, an important monitoring system in cells has been found, named the mitotic spindle assembly checkpoint (SAC), which blocks the anaphase initiator stage until all chromatids correct their pairs (Bolanos-Garcia et al., 1969). Meiosis is considered to be a special form of mitosis, in which some mitotic cell cycle proteins play an important role and are crucial to the process of chromosomal disjunction (Avram et al., 2014). One noteworthy regulatory pathway with elevated roles in meiosis is the SAC. The mechanism of the SAC in meiosis has recently been discovered (Sun and Kim, 2012). The function of SAC protein components is similar in mitosis and meiosis. The weaker SAC signaling in human oocytes could lead to the high rate of aneuploidy compared to that in mice, according to kinetochore–microtubule attachment errors in human oocytes until anaphase I (Holubcova et al., 2015; Gruhn et al., 2019). MAD1L1 and MAD2L1 are important SAC protein components. MAD1L1 may contribute to metaphase delay during meiosis by restraining the activity of the anaphase-promoting complex/cyclosome (APC/C) to ensure proper alignment of the homologous chromosomes and sister chromatids. Initial functional and mutant investigations in different species elaborated that the loss of MAD1LI results in misaligned chromosomes, which proceed directly to anaphase I (Zhang et al., 2004; Stein et al., 2007).

MAD2L1 has been reported to play a more important role in meiosis than mitosis. Functional studies of MAD2L1 revealed that the gene regulates meiotic chromosome segregation and APC/C activity. The absence of MAD2L1 can cause the shortened duration of meiosis I, and meiotic spindle disorder, chromosome mispairing, and chromosome non-disjunction in meiosis I were observed in mouse oocytes (Sun and Kim, 2012). Furthermore, aneuploidy gametes can still be developed, although there are fewer cells with chromosome non-disjunction in meiosis I (Cairo et al., 2020).

Changes in MAD1L1 and MAD2L1 levels were detected in many cancer cell lines and tumor biopsy samples. Changes in MAD1L1 and MAD2L1 may serve as biomarkers for cancer as a potential strategy during anti-cancer treatment (Schuyler et al., 2012). The genotypic polymorphisms of MAD1L1 and MAD2L1 genes significantly increased the risk of certain cancers. The single nucleotide polymorphism C>T (rs1801368) transition in the MAD1L1 gene nucleotide 1673 resulted in the transformation of arginine into histone in codon 558, leading to changes in the chromosomal disjunction domain of the MAD2L1-binding leucine zipper domain. This polymorphic change is associated with the chemotherapeutic response in advanced epithelial ovarian cancer patients (Santibanez et al., 2013). The effect of maternal MAD2L1 Leu84Met polymorphism rs1283639804 G>A on fetal chromosome abnormalities is unclear.

HCY is considered an important intrinsic factor in fetal chromosomal abnormalities (James et al., 1999). We evaluated the previous paper and found that HCY was a significant risk factor for fetal chromosomal abnormalities. Whether HCY affects other biological events related to the formation and development of gametes or embryos is worth exploring due to its important role.

Given the important roles of MAD1L1 rs1801368 and MAD2L1 rs1283639804 in the SAC, we hypothesized that the two polymorphisms may disrupt the protein structure and function, which, in turn, may be associated with susceptibility to fetal chromosomal abnormalities. No studies have been conducted to assess the impact of MAD1L1 and MAD2L1 polymorphisms on fetal chromosomal abnormalities. We conducted this hospital-based case–control study, including two loci, aiming to explore the potential association between maternal genetic polymorphisms of MAD1L1 rs1801368 and MAD2L1 rs1283639804 genes with fetal chromosome abnormalities.

In eukaryotic cells, MAD1 controls chromosome segregation via the SAC mechanism. In mitosis, the cell cycle arrest was impaired when MAD1 expression was downregulated by treatment with microtubule stabilizing and microtubule destabilizing agents (Kienitz et al., 2005). Mutation frequencies of the SAC genes remained low, and only rare polymorphisms in MAD1L1, MAD2L1, and BUB1b were found in certain cancers involved in the modulation of the cell cycle arrest (Su et al., 2016; Bandala-Jacques et al., 2020; Sun et al., 2020).

We conducted a case–control study to explore the potential association of maternal MAD1L1 rs1801368 and MAD2L1 rs1283639804 polymorphisms with a chromosomal abnormality in the fetus. It is worth noting that MAD2L1 rs1283639804 G>A loci in the southwestern Chinese women’s population revealed only a single genotype in our study, suggesting that this polymorphism is conserved in the studied population and the limitation to the small sample size presented for our study. This is a low minor allele frequency (MAF) based on the NCBI database [A = 0.000004/1 (GnomA Dexomes)], but some investigators have found a polymorphism of MAD2L1 rs1283639804 in combination with the polymorphism of MAD1L1 rs1801368 conferring colorectal and lung cancers (Guo et al., 2010; Zhong et al., 2015). Hence, we preliminarily explored the possible association of the polymorphism with fetal chromosome abnormalities. The final result suggested, perhaps, some other polymorphisms in MAD2L1 but not rs1283639804 related to fetal chromosome abnormalities, which need to be further studied.

We then found a significant difference in the frequency for MAD1L1 rs1801368 C>T between cases and controls. In some studies, the polymorphism in the MAD1L1 rs1801368 T allele has been found to be associated with certain kinds of cancer that showed somatic cell aneuploidy (Santibanez et al., 2013; Zhong et al., 2015; Bandala-Jacques et al., 2020). However, interestingly, our result of MAD1L1 rs1801368 C>T was contrary to those reports. We have followed possible explanations that 1) the process of regulating meiosis may be different from mitosis and is a variation of different sorts of species; 2) MAD1L1 rs1801368 is only a very small factor in comparison to the entire SAC array; 3) particularly, when the interactions of gene–gene, gene–environment, or other impact factors are jointly involved (Alamgir et al., 2022), certain environmental factors may play a crucial role in the event of gamete meiosis and subsequent embryo development; and 4) Differences could exist due to genetic heterogeneity in different populations (Baker et al., 2007; Karban et al., 2016).

We also performed an additional analysis combining the MAD1L1 rs1801368 C>T polymorphism with levels of maternal HCY, which is a major related factor for fetal chromosome abnormalities described in our previous article. The lower HCY subgroup [HCY <6.755 μmol/L (median value)] had a significantly higher frequency of both the CC genotype and the C allele in the cases compared to the control (dominant model: OR = 0.74, 95%CI: 0.57–0.95, and p = 0.02; C vs. T allele: OR = 1.75, 95%CI: 0.79–1.92, and p = 0.005), which did not occur in the higher HCY group (p > 0.05, respectively). Suggesting that MAD1L1 rs1801368 likely incorporates physiological environmental factors that affect the biological course of the event, even though MAD1L1 has not been included in the circulation of folate-acid homocysteine, it is well known that methylation modification has been controlled by circulating folate-acid homocysteine and affects the expression of the genes involved (Ma et al., 2022; Wang et al., 2022). However, the mechanism of how MAD1L1 methylation interacts with the level of HCY in the internal environment needs to be explored. Alternatively, the outcome of most fetuses with chromosomal abnormalities was miscarriage during early pregnancy, and over 50% of the tissue from miscarriages was detected as chromosomal aneuploidy, and only a small portion can survive. These issues need to be elucidated if the role of the SAC is to either initiate or maintain the continued development of gametes and chromosomal abnormality embryos.

HCY was an important biomarker in the pathway of folate acid and HCY, which was regarded as a risk factor of pregnancy outcomes, such as neural tube defects, Down’s syndrome, and some adverse pregnancy outcomes (Straaten et al., 2001; Liu et al., 2017). It is well known that age was an independent risk of fetal chromosomal abnormalities. In our case–control study matched with age, the result revealed that HCY was a risk factor of fetal chromosomal abnormalities for the younger pregnant women but not for the advanced pregnant women; perhaps the age was the main risk factor of fetal chromosome abnormalities.

The study is limited in some ways. 1) Some selection bias is introduced by the subjects selected from the prenatal diagnosis group. 2) Stratification analysis according to fetal numerical and structural abnormalities was not performed for the small sample size. 3) Of concern were only two MAD1L1 and MAD2L1 polymorphisms in the genes controlling SACs, and it is necessary to investigate other polymorphisms or genes such as Mad3/BubR1, Bub1, Bub3, and Mps11. 4) The association was in the opposite direction to the other reports, and its function during the mitotic process may lack a rational explanation. Depending on the meiosis complex, the cause of fetal chromosomal abnormalities requires further investigation. 5) HCY levels were tested during their second trimester, missing the value of pre-conception and early pregnancy HCY and potentially missing the best time frame that reflects the influence of HCY on fetal chromosomal stabilities. 6) The case group in this study also included some de novo chromosomal structural abnormalities, and origination was determined neither by the mother nor by the father.

In spite of these limitations, this study has some strengths. This study is the first to access the association between maternal polymorphisms implicated in SAC and fetus’ chromosomal abnormalities. In addition, the results then highlight the point that perhaps MAD1L1 rs1801368 C>T interacts with the level of internal HCY to confer susceptibility to fetal chromosomal abnormalities. All participants had an explicit fetal diagnosis with no confounders.

In conclusion, our study initially found the potential association of the maternal MAD1L1 rs1801368 C>T polymorphism with fetal chromosomal abnormalities. If lower levels of HCY are concerned, CC and C alleles may be linked to the higher risk of fetal chromosomal abnormalities. Additional studies were needed to clarify the genetic effect of the MAD1L1 rs1801368 C>T polymorphism on human chromosomal instability gametes or the occurrence of embryos and ongoing development.