Differences in distribution of both the genotypes and alleles within
TLR9 2848 G > A SNP between the infected pregnant women analyzed in the current study, and the reported European populations might be due to a selection bias. Similarly, significantly higher prevalence rates of anti-HCMV IgG and IgM antibodies determined among the infected pregnant women studied in this paper, as compared to the previously reported cohort of Polish pregnant women between 2010 and 2011, as well as to other European populations, are possibly also caused by a classification of the patients to the presented study [
4‐
7]. So far, it has been the first study to reveal a really significant role of
TLR9 2848 G > A SNP in the occurrence of HCMV infection in pregnant women. Another research reported before that the GA heterozygotic status in the analyzed
TLR9 SNP, carried by fetuses and neonates, congenitally infected with HCMV, had significantly increased the risk of the infection [
18]. In turn, the G allele in
TLR9 SNP was more frequent among the infected offsprings than among the uninfected ones, although that difference was statistically non-significant [
18]. The distinct contribution of the analyzed
TLR9 SNP in the occurrence of HCMV infection may have been age-related, as well as may have resulted from the classification bias. The qualification criteria of the pregnant women to the study included their serological status, the presence of HCMV DNA in their body fluids and were also based on confirmed congenital infection with the virus in their fetuses. In turn, the fetuses were diagnosed as congenitally infected with HCMV only on the basis of ultrasound markers and on the presence of HCMV DNA in their body fluids, since the diagnostics towards the infection is not routinely performed in pregnant women. Considering the role of
TLR9 SNP in HCMV infection, the TT homozygotic status in
TLR9 -1237 T > C SNP was reported to have been correlated with a decreased risk of the infection in HCMV-seropositive kidney transplant recipients [
50]. In recipients of allogeneic hematopoietic stem cell transplants, the HCMV infection occurred significantly more frequently among carriers of minor C allele in
TLR9 -1237 T > C, as compared to the patients, carrying the T allele [
51]. On the other hand, the
TLR9 2848 G > A polymorphism was not involved in the incidence of HCMV infection among the examined patients [
51]. However,
TLR9 -1486 T > C and 2848 C > T SNPs were associated with an increased risk of HCMV disease among infants [
33]. Therefore, it seems possible that different SNPs, residing within
TLR9 gene, may have been correlated with various disease types. Regarding the HCMV infection, previous papers showed some contribution of TLR9 to its occurrence [
14,
22,
23]. In case of plasmacytoid dendritic cells (pDCs), the infection with HCMV was significantly associated with increased levels of TLR9 [
52]. The inhibited cytokine expression, observed after treatment of pDCs with CpG, agonists for TLR7 and TLR9, suggested an involvement of the reported TLRs in the development of HCMV infection [
52]. What is more, a significant role in the occurrence of HCMV infection was previously confirmed for TLR2 and TLR4 molecules as well [
30,
53,
54]. TLR2 was found to have been involved in the functional sensing of HCMV through a direct interaction with the viral glycoproteins gB and gH [
20]. In case of TLR4, the molecule was involved in an inhibition of HCMV infection [
21]. The expression levels of both TLR2 and TLR4 were correlated with the levels of HCMV IE1-72 protein [
55]. Considering genetic alterations in the current study, no differences were found in the distribution of distinct genotypes within the range of both
TLR2 and
TLR4 SNPs. The frequencies of the genotypes in the analyzed
TLR2 and
TLR4 polymorphic sites, found among HCMV-infected pregnant women were similar to the frequencies observed among European populations (see
https://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?rs=5743708;
https://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?rs=4986790;
https://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?rs=4986791). Taking into account the allelic variability within
TLR2 2258 G > A and
TLR4 1196 C > T SNPs, the frequencies observed among all pregnant women studied in our research, were similar to the frequencies reported for European populations. In case of
TLR4 896 C > T SNP, similarity to the European populations was found for HCMV-infected pregnant women (see
https://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?rs=4986790). So far, no other study has shown before any possible involvement of
TLR2 2258 G > A,
TLR4 896 A > G and
TLR4 1196 C > T SNPs in the occurrence of HCMV infection during pregnancy. However, in case of congenital HCMV infection, GA heterozygotic status and A allele within
TLR2 2258 G > A, as well as CC genotype in the other
TLR2 + 1350 T > C SNP, was reported to be correlated with the infection [
27,
28]. Regarding presented data, the age-dependent type of the involvement of
TLR2 2258 G > A polymorphism to HCMV infection seems to be quite possible. Considering
TLR4 SNPs, the CC genotype in
TLR4 1196 C > T SNP, GC haplotype in both analyzed
TLR4 SNPs, as well as GCA multiple variants within the range of
TLR4 and
TLR9 2848 G > A SNPs, were found to have been correlated with congenital HCMV infection in fetuses and neonates [
18]. In pregnant women, the frequencies of distinct genotypes in
TLR4 SNPs were similar to those, determined among congenitally infected fetuses and neonates, although no CT genotype at
TLR4 1196 C > T polymorphic site was observed among the infected offsprings [
18]. Among pregnant women, the low genotypic variability within
TLR2 and
TLR4 SNPs seems to be the important cause of the lack of any associations with HCMV infection. Similarly to our study, almost the same frequencies of distinct genotypes in both
TLR2 2258 G > A and
TLR4 896 G > A SNPs were determined in patients with transplants, with and without clinical signs of HCMV infection [
50]. Regarding the outcomes presented in this report,
TLR9 2848 G > A SNP seems to be the major polymorphism, contributing to HCMV infection in pregnant women during pregnancy. However, the studied genetic alteration is not involved, either in amino acid changes in TLR9 molecule or in regulatory site modifications within
TLR9 gene [
56]. Therefore, other molecular changes occurring simultaneously with the analyzed polymorphism, may contribute to the course of immune response after the infection with HCMV.