ANXA5 and VEGFA Gene Variants in Women with Early Pregnancy Losses from North Macedonia

Early pregnancy loss (EPL), defined as a loss of the conceptus before the 12th week of gestation, is the most common pregnancy complication and is found in approximately 15% of all clinically recognized pregnancies [1]. About 5% of the couples trying for childbirth, experience recurrent pregnancy loss (RPL), a condition defined as two or more consecutive pregnancy losses, according to the European Society of Human Reproduction and Embryology (ESHRE) and American Society for Reproductive Medicine (ASRM) [2, 3], as well as three or more first trimester miscarriages, according to the Royal College of Obstetricians and Gynecologists, UK (RCOG) [4]. Up to now, various studies have reported that maternal as well as fetal factors may lead to RPL. Fetal chromosomal abnormalities are acknowledged as a significant contributor to pregnancy loss in approximately 50% of cases. This pattern is mirrored in our previous study concerning chromosomal abnormalities in early pregnancy losses (EPLs). In that study, chromosomal abnormalities were detected in 56.25% of uncontaminated products of conceptions (POCs), aligning with this observed trend [5]. Additionally, maternal endocrine dysregulations, autoimmune disorders, anatomical abnormalities, maternal thrombophilia, as well as genetic factors can contribute to RPL [6]. Concerning genetic factors, in a recent study that was designed and executed in our laboratory, we detected a high incidence of Joubert Syndrome among a group of EPLs (2.03%), indicating that fetal monogenic diseases can be a common cause of EPLs [7]. Still, in a large portion of RPL cases, the exact cause is not clearly identified, and these cases are referred as idiopathic RPL [8].

Angiogenesis is a physiological process through which new blood vessels are formed. Decreased angiogenesis has been associated with several adverse pregnancy outcomes, such as infertility, preeclampsia, miscarriage, intrauterine fetal distress/growth restriction, and in severe cases, fetal demise. The Annexin A5 protein, encoded by the ANXA5 gene, is found in abundance to hinder coagulation within the placenta and during the early angiogenesis. When calcium is present, Annexin A5 can attach to phosphatidylserine situated on the upper surface of syncytiotrophoblasts in the placenta to prevent clotting of maternal blood in the intervillous space. Additionally, the ANXA5 gene plays a crucial role in promoting epithelial repair, upholding placental integrity [9]. Studies have indicated that the activity of ANXA5 in placental tissue samples collected from patients with RPL was significantly diminished, accompanied by a suppressed ANXA5 expression. These findings imply that a decrease in Annexin A5 protein expression elevates the risk of RPL, especially in early pregnancy [10]. Bogdanova et al. first reported that the combination of certain single nucleotide changes: rs28717001 (c.-210A>C), rs28651243 (c.-184T>C), rs112782763 (c.-229G>A) and rs113588187 (c.-135G>A) in the promoter region of ANXA5 was associated with an increased risk of recurrent miscarriages. All four changes were defined as M2 haplotype, and the first two were designated as M1 haplotype [11]. As mentioned in a recent meta-analysis study [12], and a review article [13], dealing with the effect of the ANXA5 haplotypes on RPL, several case-control studies have shown a favorable correlation between the M2/ANXA5 haplotype and RPL across diverse ethnic populations, including cohorts from Germany [11, 14, 15], Japan [16], the United Kingdom [17] Italy [18] China [19] Malaysia [20] and Greece [21]. However, contrasting results were observed in studies involving the Chinese and Danish/Estonian ethnic groups. These groups did not reveal any association between the M2/ANXA5 haplotype and RPL [22, 23].

The human placenta is rich in angiogenic factors such as VEGF, standing out as the most potent stimulator of angiogenesis. VEGF plays a crucial role in endometrial readiness, implantation, and the development of placental and fetal blood vessels in early pregnancy, and in the vascular adaptation during pregnancy in the mother [24]. Several single nucleotide changes (SNPs) have been identified in the VEGFA gene, affecting VEGF-A activity and expression. The most significant finding in most studies is that presence of the c.-1154G/A (rs1570360) variant is associated with recurrent early pregnancy losses in contrast to normal control groups. The earliest study demonstrating a significant difference goes back to 2005 in Greece [25]. The study involved 52 women with 3 or more recurrent pregnancy losses and 82 controls with live births and no history of pregnancy loss. The analysis of allele frequency for the polymorphic variant c.-1154G/A (rs1570360) yielded a p-value of 0.016, indicating an increased allelic frequency of the mutant allele A in patients with early pregnancy losses. The c.*237C>T variant (rs3025039) increases VEGFA expression as well and acts in a similar direction [26, 27]. Similarly, the effect of the variants in the ANXA5 gene have been shown in some studies to contribute negatively to the rs1570360 and rs3025039 variants in susceptibility to recurrent miscarriages in different geographic groups [27, 28, 29]. Yet others have not confirmed this association [30, 31], therefore the need for further research to see if these relations exist. Hence, these allelic variants are unquestionably noteworthy in investigating the predisposition to RPL development.

In establishing an effective and cost-efficient method for variant detection based on multiplex single-base extension, we aimed to examine the association of the c.-210A>C, c.-184T>C, c.-229G>A, c.-135G>A variants in the ANXA5 gene and c.-1154G>A, c.*237C>T variants in the VEGFA gene in a cohort of women with early pregnancy losses, compared to a control group of women without pregnancy loss and at least one live birth. Moreover, the determination of the status of these variants would provide a direction for future therapies in women with unexplained pregnancy losses, since the large amount of data suggest that the M2/ANXA5 haplotype may contribute to the occurrence of this condition.

In this study, we have developed a SNaPshot genotyping technique, which identifies four distinct variants in the ANXA5 gene and two variants in the VEGFA gene. The method efficiently and concurrently identifies all six selected variants and also stands out for its simplicity, accuracy, ease of execution, and cost-effectiveness. By employing this method, we have assessed the occurrence rates of the two haplotypes (M1 and M2) within the ANXA5 gene and the two variants (c.-1154G/A (rs1570360) and c.*237C/T (rs3025039)) within the VEGFA gene among selected group of women of Macedonian and Albanian ethnic origin experiencing early pregnancy loss. Our analysis involved comparing these rates to controls matched by ethnicity and age.

Given the inconsistencies in the literature regarding the impact of the specified variants, as outlined in the introduction, the primary objective of our study was to investigate whether the six selected variants are linked to an elevated risk of early pregnancy loss, particularly within our two major ethnical groups (Macedonian and Albanian). It is important to emphasize that in our examined population fetal aneuploidy as a reason for EPLs was excluded.

Considering that the majority of studies have focused on individuals with subsequent pregnancy losses, and given the predominant nulliparous status among most patients, it became crucial to examine the distribution of haplotypes within various subgroups of our patients. Bogdanova et al. revealed four consecutive single nucleotide changes in the ANXA5 promoter, which are transmitted as a haplotype called M2 that reduces promoter activity, thereby leading to reduced production of Annexin A5 mRNA as well as M1 haplotype covering the first two nucleotide substitutions. Certain analyzes showed that haplotypes M1 and M2 reduce ANXA5 promoter activity by 40% and 60%, respectively, thereby significantly affecting ANXA5 expression. Women with the M2 haplotype are thought to have more than a 2-fold higher risk of fetal loss between 10 and 15 weeks of gestation than non-carriers [11]. As highlighted in a recent meta-analysis [12] and a review [13] examining the impact of the ANXA5 haplotypes on recurrent pregnancy loss (RPL), multiple case-control studies have demonstrated a positive association between the M2/ANXA5 haplotype and RPL across a range of ethnic backgrounds [11, 14,15,16,17,18,19,20,21].

In line with numerous studies, supporting the concept that M2 and M1 ANXA5 haplotypes are more common in patients experiencing pregnancy loss [10,11,12,13,14,15,16,17,18,19,20,21], our study presented higher frequency of the M2 haplotype among the women with EPLs compared to controls (p-value=0.0006).

Furthermore, when we did several sub categorizations, we observed evident statistical significance of the M2 haplotype among: women with recurrent as well as sporadic early pregnancy loss (p-value=0.057 and p-value<0.00001 respectively), women with EPLs and no live birth (p-value=0.0003), women ≤30 and ≥36 years of age (p-value=0.003 and p-value<0.001 respectively), and also in both subgroups of pregnancy loss between 6-9 GW and 10-11GW (p-value=0.008 and p-value<0.007 accordingly). The M1 haplotype was found to be more prevalent in the subgroup of women with EPLs and a live birth (p-value=0.05) and in the subgroup of women with ages between 31 and 35 years with a p-value of 0.01.

Furthermore, there are studies indicating that low-molecular-weight heparin might have a positive effect on miscarriage rate and recurrent implantation failure in treated M2/ANXA5 haplotype carriers [36, 37], so, a research in this direction could be a further step.

VEGFA has gathered significant attention due to its pivotal role in angiogenesis, particularly notable for its implications in embryo development. Compelling evidence underscores its critical involvement in fetal and placental angiogenesis, suggesting that vascular formation irregularities or dysfunction contribute to RPL. Additionally, first-trimester trophoblast VEGFA expression was found to be weaker in placental samples from RPL cases compared to gestational age-matched normal placenta [38, 39]. The most significant finding in most studies is that presence of the c.-1154G/A (rs1570360) variant is associated with recurrent early pregnancy losses in contrast to normal control groups [25,26,27,28,29], while others have not confirmed the association [30, 31], so the need for further research of these relations exists. According to a meta study by Xu et al., [27] the c.-1154G/A (rs1570360) and c.*237C/T (rs3025039) variant demonstrated statistical significance concerning RPL risk across different geographical populations, and discrepancies such as in our present study may be explained by the small sample size and substantial errors from estimation.

In our examined cohort, we observed a general higher prevalence of the heterozygous and mutant homozygous genotypes for the both VEGFA variants, compared to the controls, however without statistical significance. Nevertheless, we noticed borderline statistically significant difference (p-value=0.05) in heterozygotes for the c.-1154G/A (rs1570360) variant between the women with EPLs and a live birth, compared to the controls. Also, when we analyzed the results from the division based on gestational week of the last pregnancy, we noticed statistically higher frequency of the heterozygous genotypes for both VEGFA variants with a p-value<0.00001 for c.-1154G/A (rs1570360) and p-value=0.05 for c.*237C/T (rs3025039).

Our current study introduces a cost-effective genotyping method for selected ANXA5 and VEGFA variants. Additionally, our study is in line with numerous studies, supporting the concept that M2 and M1 ANXA5 haplotypes are more common in patients experiencing pregnancy loss. Moreover, since there are data on some experimental therapies for the individuals carrying primarily the M2/ANXA5 haplotype, a research in that direction and clinical studies for therapy justification could be a further step.