1.
INTRODUCTION
Recurrent pregnancy loss (RPL) also termed recurrent miscarriage
(RM) is defined as the loss of two or more pregnancies before 20 weeks of
gestation. This condition affects about 0.5–2% of women of reproductive age and
causes a significant clinical and psychological burden for affected couples
(Ford & Schust, 2009; Mohammed et al., 2018). RPL is a
multifactorial condition involving genetic, anatomical, immunological,
hormonal, infectious, and environmental contributors. However, in nearly half
of the cases, no clear underlying cause can be identified, and such cases are
termed idiopathic RPL. Understanding the molecular and genetic components
contributing to these unexplained cases is important to improve prevention,
diagnosis, and management strategies (Ford & Schust, 2009; Luo et al.,
2015; Turesheva et al., 2023).
In the pathophysiology of idiopathic RPL, genetic factors have become
increasingly important in recent years. Among these, polymorphisms influencing
genes related to metabolism of folate and homocysteine regulation have
attracted considerable attention. One such gene is SLC19A1, also known
as the reduced folate carrier 1 (RFC1) gene and encodes the RFC1
protein, which serves as the main transporter of folate into cells (Luo et
al., 2015). Folate is a B-vitamin necessary for DNA synthesis, repair,
methylation, and cell division. Neural tube abnormalities, intrauterine growth
limitation, preeclampsia, and early pregnancy loss have all been linked to
folate deficiency or impaired folate transport (Scholl & Johnson, 2000).
The RFC1 protein affects homocysteine metabolism because it is
essential for preserving intracellular folate levels. Hyperhomocysteinemia or
elevated homocysteine levels are toxic to the endothelium, encourage
thrombosis, and have been linked to unfavorable pregnancy outcomes, including
RPL (Afaq et al., 2023; Eissa et al., 2019; Govindaiah et al.,
2009).
Polymorphisms in the SLC19A1 gene, particularly the c.80G>A
(rs1051266) variant, have been linked to altered RFC1 activity and reduced
folate absorption. This single nucleotide polymorphism (SNP) leads to an amino
acid substitution (Arg27His), which may affect the protein's shape and function
(Chango & Emery-Fillon, 2000). In women with folate transport deficiencies,
early placental development may be compromised due to increased oxidative
stress and thrombosis at the maternal-fetal interface, potentially resulting in
early pregnancy loss (Sato et al., 2011).
Furthermore, folate deficiency can affect DNA methylation
patterns, which are essential for embryogenesis and epigenetic regulation of
gene expression. In this context, polymorphisms in the SLC19A1 gene may
not only alter folate uptake but also contribute to aberrant methylation and
gene silencing, thereby influencing pregnancy outcomes (Saeed et al.,
2019; Souza et al., 2025).
2.
METHODS
Study Design and Patient Selection
This case-control study was conducted
between August, 2024 and April, 2025 in Duhok City, Kurdistan- Region of Iraq.
The study included a total of 70 women diagnosed with idiopathic RPL and 70
age-matched healthy women with no history of miscarriage and at least one
successful full-term pregnancy. Patients with known causes of miscarriage such
as uterine anomalies, hormonal disorders, antiphospholipid syndrome,
chromosomal abnormalities, infectious diseases, or thrombophilias were excluded
from the study. All participants were recruited from gynecology and obstetrics
outpatient clinics at Duhok Maternity Hospital, and detailed medical and
obstetric histories were obtained using a structured questionnaire.
Sample Collection and Molecular Analysis:
Five milliliters of peripheral venous blood were collected from
each participant and placed in sterile EDTA-containing tubes. Samples were
coded and stored at –20°C
until DNA extraction. Genomic DNA was isolated from whole blood using a
modified salting-out method
(Iranpur-Mubarakeh & Esmailizadeh, 2010; Kashmoola et al.,
2015). The quality and quantity of DNA were assessed using a Nanodrop spectrophotometer (Thermo Fisher
Scientific) at 260/280 nm ratios. To amplify the regions
containing the SLC19A1 (RFC1) rs1131596
(-43T>C), rs1051266 (80G>A), rs12659 (696C>T) polymorphisms,
specific primers were designed (Table 1).
·
χ²
values were calculated using the formula: Σ ((Observed – Expected)²/
Expected).
·
Degrees
of freedom = 1; HWE is considered valid if χ² < 3.84 (p > 0.05).
·
All
SNPs are in Hardy-Weinberg equilibrium in both study groups, supporting
genotype data quality.
Haplotype analysis, which
combined the three SNPs to identify possible gene-gene interactions or
cumulative effects, did not yield any statistically significant associations.
This may be attributed to the low linkage disequilibrium observed between the SNPs
and the relatively small sample size, which might have inhibited the
statistical power to identify actual associations. Otherwise, in an event of
insufficient power, false-negative results can be obtained. Though the formal
power analysis was not carried out, this is a limitation that should be taken
into account. Nonetheless, haplotype construction remains a useful exploratory
tool in genetic epidemiology.
In summary, while rs1131596
and rs1051266 did not show significant differences between groups, the rs12659
(696C>T) polymorphism showed a weak but statistically notable association
with RPL. This finding warrants further validation in larger studies and
functional analyses to determine its biological relevance in folate transport
and early pregnancy outcomes.
4. DISCUSSION
This study investigated three
polymorphisms in the SLC19A1 gene (rs1131596, rs1051266, rs12659) and their
relationship with idiopathic recurrent pregnancy loss in women from Duhok,
Iraq. While two SNPs (rs1131596 and rs1051266) did not show any significant
association, the 696C>T variant (rs12659) was marginally associated with
increased RPL risk. Although rs12659 is a synonymous variant, it is located in
a non-coding or regulatory area of the SLC19A1 gene, suggesting that it
still has some effect on the expression of the gene through mechanisms such as
regulation of mRNA destruction, secondary structure, or pre-mRNA splicing. The
non-coding SNPs have also been known to regulate as synonymous variants are
increasingly identified, and these SNPs tend to interfere with RNA splicing
process or transcript stability (Baralle & Giudice, 2017; Sauna &
Kimchi-Sarfaty, 2011). More functional analyses are required to determine the
possibility of alternative effects of rs12659 that influences the expression or
activity of SLC19A1 at the transcriptional level.
Our findings on rs1051266
(80G>A) align with several studies conducted in Iranian, Turkish, and Korean
populations, which similarly reported no significant association between this
SNP and RPL or recurrent miscarriage (Chango & Emery-Fillon, 2000; Rah et
al., 2012; Nomair et al., 2024). In a study from China,
polymorphisms in genes related to folate metabolism, including SLC19A1,
significantly impact the risk of unexplained RPL in Chinese women. In their
study, the 80G>A (rs1051266) variant showed a borderline association with
RPL, particularly when combined with MTHFR polymorphisms, suggesting a
possible synergistic effect between multiple folate pathway genes (Luo et al.,
2015). Being unable to find such significance in the current study may be due
to compensatory mechanisms in folate metabolism or gene-environment
interactions that modulate its effect.
Similarly, a study from Iran
assessed the SLC19A1 80G>A polymorphism among women with RPL and
reported a non-significant difference in genotype distribution between cases
and controls, in agreement with the findings arrived at in this study. However,
when stratified by folate intake, women with the AA genotype and low folate
levels had a significantly higher RPL risk, highlighting the importance of
gene–nutrient interactions (Mohtaram et al., 2016).
However, the association of
rs12659 (696C>T) with RPL is noteworthy and consistent with limited evidence
in the literature. While the functional consequences of this SNP remain
unclear, it may reside in a regulatory region influencing SLC19A1 mRNA
stability or splicing efficiency. Rah et al. (2012) found this SNP to be
potentially associated with idiopathic recurrent spontaneous abortion in Korean
women, particularly in TT homozygotes, suggesting possible functional effects
such as altered mRNA stability or splicing (Rah et al., 2012). Moreover,
studies have indicated that RFC1 gene variants may influence folate uptake and
hence methylation status critical for placental function (Luo et al.,
2015; Souza et al., 2025). Variants in non-coding regions of folate
transport genes can modulate homocysteine levels and folate uptake efficiency,
which are critical during placental development (Chango & Emery-Fillon,
2000).
Furthermore, the interplay
between folate transport and DNA methylation pathways is well-documented.
Aberrant methylation due to inadequate intracellular folate levels can affect
the expression of key developmental genes and lead to embryonic loss. As demonstrated
by (Stanisławska-Sachadyn et
al.,2009),
parental hyperhomocysteinemia and folate-pathway polymorphisms, including SLC19A1,
significantly contributed to RPL through impaired methylation and vascular
dysfunction.
Interestingly, no significant
haplotypes emerged in the present study, which may be attributed to the low
linkage disequilibrium observed and the relatively modest sample size. This
aligns with findings that emphasized how single polymorphisms may not fully
explain genetic susceptibility and that multi-locus and gene–environment
interaction models are more informative. Since the sample included 70 cases and
70 controls, the study was potentially underpowered to identify haplotype-level
associations. A formal power calculation was not performed; therefore, future
studies should adopt a multi-gene haplotype approach, along with quantification
of serum folate and homocysteine levels, to provide a clearer mechanistic link
(Luo et al., 2015).
The lack of strong
associations for some SNPs could reflect environmental modifiers such as folate
supplementation, which is widespread in prenatal care in Iraq (Lateef &
Al-Hashimi, 2016). Moreover, the high prevalence of consanguineous marriages in
the region may lead to genomic homozygosity, which can obscure the effect of
dominant alleles and highlight recessive genetic traits (Hawi et al., 2024). Taken together, the data support
a modest but likely contribution of the 696C>T variation to RPL risk, particularly
in genetically different populations.
To the best
of the researchers’ knowledge, this is the first study in the Duhok Governorate—and
among the first in Iraq-to investigate the relationship between SLC19A1 gene
polymorphisms and idiopathic recurrent pregnancy loss (RPL). By focusing on
three specific single nucleotide polymorphisms (rs1131596, rs1051266, and
rs12659) and conducting haplotype analysis in a genetically and environmentally
unique population. This study has provided novel perspectives into the
potential role of folate transporter gene variants in unexplained pregnancy
loss. Importantly,
the majority of research sample was composed of Kurdish women, a relatively
understudied ethnic group that may have different genetic patterns. The identification of a marginally significant
correlation between the 696C>T polymorphism and RPL risk provides a valuable
genetic clue that warrants further research and could support
population-specific diagnostic or preventive strategies.
CONCLUSION
This study indicated that the
SLC19A1 696C>T (rs12659) polymorphism may be linked to a higher risk of
idiopathic recurrent pregnancy loss in Duhok women, while rs1131596 and
rs1051266 showed no significant associations. Considering the Kurdish majority
of the cohort studied, possible results can be discussed as population-specific
genetic trends, and it is important to pay attention to them with the planned
continuation of the research in other ethnicities. These findings highlight the
importance of evaluating folate transporter gene variants in the context of
population-specific genetic backgrounds. Future studies with larger sample
sizes, functional assays, and evaluation of environmental modifiers such as
folate intake and consanguinity are required to validate these preliminary
results and explore potential preventative strategies.
Acknowledgments:
The author gratefully acknowledges the women who participated in
this study for their valuable contribution.
Author
Contributions:
All authors have reviewed the final version to be published and
agreed to be accountable for all aspects of the work.
Concept and design: Adil Abozaid Eissa
Data collection and practical work: Hozheen Abdulrahman Darweesh
Data analysis and interpretation: Adil
Abozaid Eissa, Hozheen
Abdulrahman Darweesh
Drafting of the manuscript: Hozheen Abdulrahman Darweesh, with critical revision by Adil Abozaid Eissa.
Ethical Considerations and Consent:
Ethical approval was obtained
for the study from the Scientific and Ethics Committee of the Duhok Directorate
of health (Approval No: 31072024-6-30, dated July 31, 2024) in conformity with
the Declaration of Helsinki which guarantees compliance with medical research
standards, including patient privacy and moral management of clinical data. Written informed consent was obtained from all
participants prior to sample collection, after explaining the purpose,
procedures, benefits, and risks of the study in their native language.
Declaration:
Ethical approval was obtained from the Scientific and Ethics
Committee of the Duhok Directorate of Health (Approval No: 31072024-6-30).
Written informed consent was obtained from all participants prior to data
and sample collection.
Conflict of interest: The authors declare no conflict of interest.
Funding:
This study was solely self-funded by the first author, Hozheen Abdulrahman Darweesh.
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