Spontaneous abortion (SA) refers to the natural termination of a pregnancy prior to the 20th week of gestation and is recognised as one of the most prevalent complications encountered during pregnancy [1, 2]. Ensuring a healthy maternal-fetal immune balance is crucial for a successful pregnancy, as any disruption to this equilibrium heightens the risk of abortion [3, 4].

Toll-like receptors (TLRs) are essential components of innate immunity, as they identify pathogen-associated molecular patterns and initiate suitable immune responses [5]. Throughout pregnancy, these receptors play a crucial role in modulating the immune response within the placenta and maternal tissues, safeguarding against infections while preserving foetal immune tolerance. Prior investigations have indicated that certain TLRs, including TLR2, TLR4, and TLR7, play a role in responses to viral infections and may affect pregnancy outcomes [6–9].

Single-nucleotide polymorphisms (SNPs) are a prevalent form of genetic variation and may affect the effectiveness of the immune response. Current findings indicate that variations in TLR genes may influence the immune response to Human Cytomegalovirus (HCMV), a prevalent virus that can cause immune disorders during pregnancy. Nevertheless, information concerning the influence of these polymorphisms on the relationship between HCMV and SA is still scarce [10–12].

The HCMV ranks among the most prevalent viruses affecting women of reproductive age [13]. Local studies indicate a significant level of exposure to the virus among pregnant women in Iraq, accompanied by varying antibody levels. Nonetheless, there is a limited number of studies connecting genetic variation in TLR to the immune response to HCMV and the risk of SA within the Iraqi population [14–16].

This study sought to assess the connection between genetic variations in the TLR2, TLR4, and TLR7 genes and the likelihood of SA. Additionally, it aimed to explore the relationship between these variations and HCMV serostatus (IgG and IgM), thereby addressing an important gap in existing knowledge.

This study investigated the association between genetic polymorphisms in the TLR2, TLR4, and TLR7 genes with the risk of SA, highlighting the impact of Cytomegalovirus serostatus on the immunological response during pregnancy. The results arise amid increasing focus on the significance of innate immunity and genetic variation in pregnancy-related diseases.

Women aged 20–40 were selected for the current study because they are the most active reproductive age group. According to this study, the age group (31–40 years old) is 56.0% younger than the abortion group. According to the current findings, SA happens at a higher rate in the first trimester (86.0%) than in normal pregnancies (6.0%), confirming that this time is the riskiest. This data is comparable with research [19], which found that more than 80% of SAs occur before the 12th week of pregnancy, especially in older women or those with a history of abortion Table 1.

HCMV is the most prevalent congenital infection globally, affecting 0.7% to 1% of live births. While 11% exhibit symptoms and 30% to 40% experience developmental delays, routine screening for infections in the womb is not recommended due to the risk of misinterpreting serological data. IgG antibodies indicate past exposure, but variability in prevalence across populations complicates understanding [20, 21]. Additionally, factors such as other illnesses and immune status can affect outcomes, and serological and molecular diagnostics differ significantly [22].

Pattern recognition receptors, particularly TLR2, play a vital role in immune response activation and the pathogenesis of infectious diseases. However, research on the link between TLR gene polymorphisms and SA remains inconclusive. Studies by Kuliczkowska-Płaksej et al. [23] and Semlali et al. [24] found no significant genetic differences between affected and control groups in women with polycystic ovarian syndrome and breast cancer patients, respectively. In contrast, Ying et al. [25] reported increased levels of TLR2 and TLR4 proteins and mRNA in decidua tissue from women with recurrent abortion s, which correlated with higher Th1 cytokine levels. This study found no association between the TLR2 (rs3804100) polymorphism and SA in women (Table 3).

The TLR4 gene (rs1927914) analysis revealed significant differences in genotype prevalence between the SA and control groups, particularly for the conjugate and predominant genotypes in Table 4. The heterozygous (GA) genotype was associated with a lower OR for abortion, indicating a potential heterozygote advantage that enhances immunological balance during pregnancy by mitigating excessive inflammation while maintaining immune defenses. A significant association was found between the TLR4 rs1927914 polymorphism and the risk of SA, with the G allele more prevalent among healthy pregnant women and protective effects in heterozygous GA and GG genotypes (OR = 0.483 and 0.463, P < 0.05). These findings contrast with Jiao et al. [26], who identified rs1927914 as a genetic marker for recurrent abortion in various populations. Altered TLR4 expression has also been linked to increased abortion frequency, while the GA genotype’s association with reduced incidence suggests its role in sustaining a balanced inflammatory response during pregnancy [27].

The investigation reveals a significant association between the TLR7 gene polymorphism (rs179008, A>T) and a lower risk of abortion without external assistance (Table 5). The T allele is more common in healthy pregnant women, indicating a possible protective effect. The heterozygous (AT vs. AA-TT) genotype shows a marked reduction in abortion risk under both codominant and dominant models, suggesting a heterozygote advantage due to balanced TLR7-mediated immune regulation during pregnancy. The TT genotype did not show a significant correlation, implying the protective advantage might be limited to heterozygotes or influenced by other factors. The rs179008 SNP results in an amino acid substitution in the TLR7 protein, potentially affecting immune tolerance required for pregnancy [28].

The study in Table 6 found a higher prevalence of HCMV IgG positivity among women who experienced abortions compared to a control group, indicating prior exposure to the virus. However, HCMV IgG levels should not be viewed as a sign of active infection but rather as reflecting immunological factors that may influence the correlation between TLR receptor genetic diversity and the immune response during pregnancy. The analysis of HCMV IgM in Table 7 was complicated by a low number of positive cases in the control group. The findings revealed that women with TT and TC genotypes of the TLR2 gene exhibit a significantly increased abortion risk, in line with earlier studies on Iraqi women. TLR4 polymorphisms (rs1927914) were also linked to heightened abortion risk in HCMV IgG-positive women, suggesting that TLR4 variants may affect immune responses to infections. Additionally, women with the AA genotype of TLR7 and positive HCMV IgG status have about a sixfold greater risk of abortion, indicating the importance of TLR7 polymorphisms in virus-related pregnancy loss. Previous research supports an association between HCMV IgG levels and adverse pregnancy outcomes, highlighting the need for further investigation of TLR polymorphisms and HCMV antibodies during pregnancy [29–31].

This study found no strong link between TLR2 genotypes and abortion outcomes but noted that HCMV infection can disrupt gene expression and placental function. The absence of TLR2 in syncytiotrophoblasts may reduce immunological activation in early pregnancy. HCMV IgM positivity is correlated with a higher abortion likelihood in those with the TLR4 AA genotype. Understanding TLR-mediated viral resistance is essential, as TLR polymorphisms impact immune responses to HCMV. TLR4 activation enhances IgM production, which influences defenses against HCMV. Previous research has identified associations between TLR4 polymorphisms and infection susceptibility, highlighting TLRs’ role in immune control. While HCMV IgM positivity is more common in women with abortion histories, no significant link was found between TLR7 genotypes and abortion risk [13, 32, 33].

The combined study of TLR gene variants and HCMV serostatus suggests that the interaction between genetic and environmental/viral factors may be more significant than the effects of each factor alone. This interaction may elucidate the variety noted in the outcomes of previous investigations involving heterogeneous populations, including disparities in genetic backgrounds, viral exposure patterns, and environmental influences.

This study has limitations, including a small sample size in some sub-analyses of the HCMV IgM-positive group and a difference in gestational age between the case and control groups. Also, the PCR-RFLP approach is reliable but may not be as accurate as newer genetic methods. This work is notable as one of the first investigations in Iraq evaluating the correlation between genetic diversity in TLR genes and HCMV infection in connection to SA, hence providing significant exploratory scientific value.

In conclusion, this study's findings suggest that the rs1927914 genetic polymorphism in the TLR4 gene, particularly the heterozygous (GA) genotype, may be associated with a reduced risk of SA. The rs179008 polymorphism in the TLR7 gene may affect the immunological response during pregnancy. The findings suggest that HCMV serostatus may affect the relationship between certain TLR gene polymorphisms and pregnancy, although a direct causative relationship has yet to be confirmed. However, further studies with larger sample sizes and more stringent techniques are needed to confirm these findings and elucidate the underlying mechanisms.