Preconceptional and Periconceptional Folic Acid Supplementation in the Visegrad Group Countries for the Prevention of Neural Tube Defects

Abstract

Neural tube defects (NTDs) are malformations of the central nervous system that represent the second most common cause of congenital morbidity and mortality, following cardiovascular abnormalities. Maternal nutrition, particularly folic acid, a B vitamin, is crucial in the etiology of NTDs. FA plays a key role in DNA methylation, synthesis, and repair, acting as a cofactor in one-carbon transfer reactions essential for neural tube development. Randomized trials have shown that FA supplementation during preconceptional and periconceptional periods reduces the incidence of NTDs by nearly 80%. Consequently, it is recommended that all women of reproductive age take 400 µg of FA daily. Many countries have introduced FA fortification of staple foods to prevent NTDs, addressing the high rate of unplanned pregnancies. These policies have increased FA intake and decreased NTD incidence. Although the precise mechanisms by which FA protects against NTDs remain unclear, compelling evidence supports its efficacy in preventing most NTDs, leading to national recommendations for FA supplementation in women. This review focuses on preconceptional and periconceptional FA supplementation in the female population of the Visegrad Group countries (Slovakia, Czech Republic, Poland, and Hungary). Our findings emphasize the need for a comprehensive approach to NTDs, including FA supplementation programs, tailored counseling, and effective national-level policies.

Full text

Academic Editors: Lijun Wang,
Chunxia Jing, Jiaomei Yang, Dan Liu
and Federica I. Wolf
Received: 7 October 2024
Revised: 20 December 2024
Accepted: 29 December 2024
Published: 31 December 2024
Citation: Rísová, V.; Saade, R.; Jakuš,
V.; Gajdošová, L.; Varga, I.;
Záhumenský, J. Preconceptional and
Periconceptional Folic Acid
Supplementation in the Visegrad
Group Countries for the Prevention of
Neural Tube Defects. Nutrients 2025,
17, 126. https://doi.org/10.3390/
nu17010126
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license
(https://creativecommons.org/
licenses/by/4.0/).
Review
Preconceptional and Periconceptional Folic Acid
Supplementation in the Visegrad Group Countries for the
Prevention of Neural Tube Defects
Vanda Rísová
1
, Rami Saade
1, 2,
* , Vladimír Jakuš
3, †
, Lívia Gajdošová
3
, Ivan Varga
1
and Jozef Záhumenský
2
1
Institute of Histology and Embryology, Faculty of Medicine, Comenius University, 813 72 Bratislava, Slovakia;
vanda.risova@fmed.uniba.sk (V.R.); ivan.varga@fmed.uniba.sk (I.V.)
22nd Department of Gynecology and Obstetrics, University Hospital Bratislava and Comenius University,
821 01 Bratislava, Slovakia; jozef.zahumensky@fmed.uniba.sk
3Institute of Medical Chemistry, Biochemistry and Clinical Biochemistry, Faculty of Medicine,
Comenius University, 813 72 Bratislava, Slovakia; vladimir.jakus@fmed.uniba.sk (V.J.);
livia.gajdosova@fmed.uniba.sk (L.G.)
*Correspondence: rami.saade@fmed.uniba.sk; Tel.: +421-948-013-580
†Passed away on 2 August 2024.
Abstract: Neural tube defects (NTDs) are malformations of the central nervous system
that represent the second most common cause of congenital morbidity and mortality,
following cardiovascular abnormalities. Maternal nutrition, particularly folic acid, a B
vitamin, is crucial in the etiology of NTDs. FA plays a key role in DNA methylation,
synthesis, and repair, acting as a cofactor in one-carbon transfer reactions essential for
neural tube development. Randomized trials have shown that FA supplementation during
preconceptional and periconceptional periods reduces the incidence of NTDs by nearly
80%. Consequently, it is recommended that all women of reproductive age take 400
µ
g
of FA daily. Many countries have introduced FA fortification of staple foods to prevent
NTDs, addressing the high rate of unplanned pregnancies. These policies have increased
FA intake and decreased NTD incidence. Although the precise mechanisms by which
FA protects against NTDs remain unclear, compelling evidence supports its efficacy in
preventing most NTDs, leading to national recommendations for FA supplementation in
women. This review focuses on preconceptional and periconceptional FA supplementation
in the female population of the Visegrad Group countries (Slovakia, Czech Republic,
Poland, and Hungary). Our findings emphasize the need for a comprehensive approach
to NTDs, including FA supplementation programs, tailored counseling, and effective
national-level policies.
Keywords: folic acid; neural tube defects; supplementation; polymorphisms; fortification;
prevention; recommendations
1. Introduction
Neural tube defects (NTDs) constitute a group of congenital malformations resulting
from the failure of the neurulation process, whereby the neural tube closes during the
embryonic period, specifically between 21 and 28 days after fertilization. These disorders
encompass a wide range of abnormalities affecting the structures of the developing central
nervous system (brain and spinal cord), frequently involving the surrounding bone, muscle,
and skin tissue [
1
]. These defects, which include conditions such as spina bifida and
anencephaly, are among the most common congenital malformations worldwide [
2
]. The
Nutrients 2025,17, 126 https://doi.org/10.3390/nu17010126

Nutrients 2025,17, 126 2 of 18
etiology of NTDs is multifactorial and is influenced by genetic (racial diversity, gender
difference) [
3
], epigenetic (metabolic disorders of mother, proper nutrition—folic acid
(FA), vitamin B12, trace elements) [
4
], non-genetic (geographical factors, socioeconomic
status of parents) [
5
] and teratogenic (pesticides, arsenic, polycyclic aromatic hydrocarbons,
antibiotics, anti-seizure medications, infections) [
6
,
7
] factors. Research suggests possible
interactions between disrupted gene regulatory networks, environmental factors, and
epigenetic regulation [8,9].
Low preconceptional and periconceptional folate intake in women of reproductive
age is one of the key risk factors for NTDs. FA, a synthetic form of folate, plays a crucial
role in DNA synthesis, repair, and methylation, processes essential for normal neural
development (Figure 1). Folate deficiency can cause DNA hypomethylation, block synthesis
of 2
′
-deoxythymidine-5
′
-monophosphate and increase misincorporation of uracil. The
exact prevalence of folate deficiency in women of reproductive age is not well-defined,
but it is generally observed that in many lower-income countries, the prevalence may
exceed 20%. In contrast, in higher-income countries, it is typically reported to be less than
5% [
10
]. The two most common genetic polymorphisms affecting the function of the key
enzyme in folate metabolism, methylenetetrahydrofolate reductase (MTHFR), are C677T
and A1298C. In the general population, approximately 60–70% of individuals carry at least
one of the mentioned MTHFR gene variants. Among them, about 8.5% are homozygous
for either the C677T or A1298C mutations, while 2.25% are compound heterozygous for
both variants. Overall, around 10% of the population is either homozygous or compound
heterozygous for the two mentioned polymorphisms [
11
,
12
]. The metabolic disruptions
caused by mutations in MTHFR gene contribute to genomic instability, which increases
the risk of NTDs. Currently, impaired de novo thymidylate biosynthesis (dTMP) and the
accumulation of uracil in DNA are considered the only known metabolic risk factors for
folate-sensitive NTDs [13].
Nutrients 2025, 17, x FOR PEER REVIEW 2 of 19
The etiology of NTDs is multifactorial and is influenced by genetic (racial diversity, gen-
der difference) [3], epigenetic (metabolic disorders of mother, proper nutrition—folic acid
(FA), vitamin B12, trace elements) [4], non-genetic (geographical factors, socioeconomic
status of parents) [5] and teratogenic (pesticides, arsenic, polycyclic aromatic hydrocar-
bons, antibiotics, anti-seizure medications, infections) [6,7] factors. Research suggests pos-
sible interactions between disrupted gene regulatory networks, environmental factors,
and epigenetic regulation [8,9].
Low preconceptional and periconceptional folate intake in women of reproductive
age is one of the key risk factors for NTDs. FA, a synthetic form of folate, plays a crucial
role in DNA synthesis, repair, and methylation, processes essential for normal neural de-
velopment (Figure 1). Folate deficiency can cause DNA hypomethylation, block synthesis
of 2′-deoxythymidine-5′-monophosphate and increase misincorporation of uracil. The ex-
act prevalence of folate deficiency in women of reproductive age is not well-defined, but
it is generally observed that in many lower-income countries, the prevalence may exceed
20%. In contrast, in higher-income countries, it is typically reported to be less than 5% [10].
The two most common genetic polymorphisms affecting the function of the key enzyme
in folate metabolism, methylenetetrahydrofolate reductase (MTHFR), are C677T and
A1298C. In the general population, approximately 60–70% of individuals carry at least
one of the mentioned MTHFR gene variants. Among them, about 8.5% are homozygous
for either the C677T or A1298C mutations, while 2.25% are compound heterozygous for
both variants. Overall, around 10% of the population is either homozygous or compound
heterozygous for the two mentioned polymorphisms [11,12]. The metabolic disruptions
caused by mutations in MTHFR gene contribute to genomic instability, which increases
the risk of NTDs. Currently, impaired de novo thymidylate biosynthesis (dTMP) and the
accumulation of uracil in DNA are considered the only known metabolic risk factors for
folate-sensitive NTDs [13].
Figure 1. Molecular structure of folic acid [14].
NTDs represent a significant public health challenge, affecting thousands of preg-
nancies worldwide and requiring comprehensive prevention strategies [15]. These serious
birth defects, occurring in early pregnancy, have profound implications for healthcare
systems, families, and society, necessitating coordinated policy responses across national
and regional levels. The prevention of NTDs through folic acid supplementation varies
significantly across countries and regions. While some nations have implemented
Figure 1. Molecular structure of folic acid [14].
NTDs represent a significant public health challenge, affecting thousands of pregnan-
cies worldwide and requiring comprehensive prevention strategies [
15
]. These serious
birth defects, occurring in early pregnancy, have profound implications for healthcare
systems, families, and society, necessitating coordinated policy responses across national
and regional levels. The prevention of NTDs through folic acid supplementation varies sig-

Nutrients 2025,17, 126 3 of 18
nificantly across countries and regions. While some nations have implemented mandatory
food fortification programs, others rely on voluntary approaches or educational initiatives.
In the Visegrad Group countries, approaches range from comprehensive national programs
to fragmented implementation strategies.
2. Historical Overview of FA Supplementation in the Visegrad
Group Countries
The Visegrad Group (also known as the Visegrad Four or simply V4) was officially
established on 15 February 1991, when the leaders of former Czechoslovakia, Poland and
Hungary signed a declaration in Visegrad, Hungary. In this declaration, the Czech Repub-
lic, Slovakia, Hungary, and Poland pledged to collaborate in multiple fields of common
national interests. The Visegrad Group countries, which share similar cultural, political,
religious and intellectual values, aim to promote cooperation between the countries of
Central Europe in various areas, including trade, nature conservation and international
partnerships [
16
]. Although these countries are very similar in many aspects, our work aims
to highlight the differences in policies regarding FA supplementation in the preconceptional
and periconceptional periods among these countries.
In Hungary, early research conducted in the 1980s followed research methodology
that explored the potential benefits of FA administration during pregnancy in preventing
most malformations of the nervous system, especially NTDs [
17
]. In 1984, a clinical
trial assessed the extent to which the use of FA or FA-containing multivitamins in the
periconceptional period might contribute to a reduction in the incidence of early NTDs [
18
].
The results showed that a periconceptional multivitamin supplementation containing a
daily dose of 800
µ
g of FA led to a significant reduction in the incidence of NTDs, as well
as abnormalities of the urinary tract (particularly obstructive defects) and cardiovascular
system malformations (especially ventricular septal defects) [19,20].
In the former Czechoslovakia, a study by Tolarováet al. examined the effects of peri-
conceptional FA supplementation or FA-containing multivitamins on congenital anomalies,
such as cleft lip with or without cleft palate [21].
In Poland, initatives launched in the late 1990s aimed to establish primary prevention
programmes for NTDs, emphasizing the health benefits of FA supplementation for women
of reproductive age [
22
]. Evidence demonstrated that the administration of FA resulted
in a 70% reduction in the incidence of NTDs in newborns [
23
,
24
], as well as a decrease in
other congenital defects, including abnormalities of the heart, limbs, urinary tract, digestive
system, and orofacial clefts (Table 1) [25].
Table 1. Association of folate intake deficiency with congenital disorders.
Congenital Disorder Influence of Folate Intake
Neural tube defects
Folate deficiency in pregnancy increases risk of closed and
open NTDs [18].
Heart defects
Research suggests that folate deficiency during pregnancy
may be associated with an increased risk of congenital
heart defects (non-syndromic septal, conotruncal, right or
left-sided obstructive heart defect) [26].
Cleft lip and palate Some studies suggest that sufficient folate intake may
reduce the risk of cleft lip and palate in newborns [27].
Limb deformities
Folate is important for proper limb development; its
deficiency may be associated with a variety of limb
reduction defects [28].

Nutrients 2025,17, 126 4 of 18
3. Neurulation and Genetic Predisposition to NTDs
The process of neural tube formation and closure that results in the formation of the
spinal cord and brain is called neurulation (Figure 2). This process takes place in the early
stages of embryogenesis. Between the 23rd and 26th day of gestation, the neural tube
definitively closes, and its lumen becomes the neural canal [
29
]. The process of neural
tube closure depends on several biological processes, such as convergent neural plate
extension, neural crest cells migration, and neuroepithelial apoptosis. Any abnormalities
in these processes can interfere with the proper development and closure of the neural
tube, which may promote the development of NTDs [
30
]. NTDs can be classified as
“open NTDs”, where the nerve tissue is exposed and there is cerebrospinal fluid (CSF)
leakage, or “closed NTDs”, where the nerve tissue is covered by tissue and there is no
CSF leakage. Anencephaly and myelomeningocele (MMC) are the two most common
forms of open NTDs. Anencephaly is a condition that arises as a result of the neural
tube not closing in the cranial region, characterized by the absence of the skull, and is
always fatal. Conversely, MMC results from the failure of the neural tube not closing in the
spinal region. Closed NTDs are clinically categorized based on the presence (lipomyelocele,
lipomyelomeningocele, meningocele, myelocystocele) or absence of a subcutaneous mass
(dermal sinus, caudal regression, and segmental spinal dysgenesis) (Table 2) [31].
The genetic predisposition to NTDs arises from multiple metabolic and signaling
pathways. Recent experimental studies have shown that the non-canonical signaling
pathway (Wnt) and planar cell polarity (PCP) are directly involved in the processes of
neurulation and neural tube closure. In addition, sonic hedgehog (Shh) signaling is critical
for many aspects of early embryonic central nervous system development. Variants in PCP
genes are increasingly implicated in NTD risk [32].
Nutrients 2025, 17, x FOR PEER REVIEW 4 of 19
3. Neurulation and Genetic Predisposition to NTDs
The process of neural tube formation and closure that results in the formation of the
spinal cord and brain is called neurulation (Figure 2). This process takes place in the early
stages of embryogenesis. Between the 23rd and 26th day of gestation, the neural tube de-
finitively closes, and its lumen becomes the neural canal [29]. The process of neural tube
closure depends on several biological processes, such as convergent neural plate exten-
sion, neural crest cells migration, and neuroepithelial apoptosis. Any abnormalities in
these processes can interfere with the proper development and closure of the neural tube,
which may promote the development of NTDs [30]. NTDs can be classified as “open
NTDs,” where the nerve tissue is exposed and there is cerebrospinal fluid (CSF) leakage,
or “closed NTDs,” where the nerve tissue is covered by tissue and there is no CSF leakage.
Anencephaly and myelomeningocele (MMC) are the two most common forms of open
NTDs. Anencephaly is a condition that arises as a result of the neural tube not closing in
the cranial region, characterized by the absence of the skull, and is always fatal. Con-
versely, MMC results from the failure of the neural tube not closing in the spinal region.
Closed NTDs are clinically categorized based on the presence (lipomyelocele, lipomye-
lomeningocele, meningocele, myelocystocele) or absence of a subcutaneous mass (dermal
sinus, caudal regression, and segmental spinal dysgenesis) (Table 2) [31].
The genetic predisposition to NTDs arises from multiple metabolic and signaling
pathways. Recent experimental studies have shown that the non-canonical signaling path-
way (Wnt) and planar cell polarity (PCP) are directly involved in the processes of neu-
rulation and neural tube closure. In addition, sonic hedgehog (Shh) signaling is critical for
many aspects of early embryonic central nervous system development. Variants in PCP
genes are increasingly implicated in NTD risk [32].
Figure 2. The three main stages of neurulation are illustrated sequentially (adapted from [33]). (A)
Neural plate: A flat layer of ectoderm begins to bend as cells change their shape, laying the founda-
tion for subsequent structures. (B) Neural folds: The folds elevate and converge toward the midline,
facilitating the closure of the neural tube. (C) Neural tube: Once closed, this structure develops fur-
ther into the central nervous system [33].
Table 2. Overview of causes and consequences of most common open and closed neural tube de-
fects.
Neural Tube Defect Characteristics Causes Consequences
Spina bifida
Incomplete closure o
f
the spinal canal. Se-
vere cases lead to pa-
ralysis of lower limbs
and bladder/bowel
dysfunction.
Genetic factors (e.g.,
MTHFR, FOLR1
SNPs), folate defi-
ciency, infections
during pregnancy,
maternal diabetes,
obesity.
Movement impair-
ment, loss of sensa-
tion, incontinence,
hydrocephalus, cog-
nitive impairments
[34].
Figure 2. The three main stages of neurulation are illustrated sequentially (adapted from [
33
]).
(A) Neural plate: A flat layer of ectoderm begins to bend as cells change their shape, laying the
foundation for subsequent structures. (B) Neural folds: The folds elevate and converge toward
the midline, facilitating the closure of the neural tube. (C) Neural tube: Once closed, this structure
develops further into the central nervous system [33].
Table 2. Overview of causes and consequences of most common open and closed neural tube defects.
Neural Tube Defect Characteristics Causes Consequences
Spina bifida
Incomplete closure of the
spinal canal. Severe cases
lead to paralysis of lower
limbs and bladder/bowel
dysfunction.
Genetic factors (e.g.,
MTHFR, FOLR1 SNPs),
folate deficiency, infections
during pregnancy,
maternal diabetes, obesity.
Movement impairment,
loss of sensation,
incontinence,
hydrocephalus, cognitive
impairments [34].
Anencephaly Absence of the brain and
most of the skull.
Genetic factors, teratogenic
substances (e.g., alcohol,
drugs), maternal folate
deficiency (increased risk
with certain MTHFR SNPs),
environmental toxins.
Often results in fetal death
or death shortly after
birth [35].

Nutrients 2025,17, 126 5 of 18
Table 2. Cont.
Neural Tube Defect Characteristics Causes Consequences
Encephalocele External sac containing brain
tissue on the head or neck.
Genetic factors (e.g., defects in
early embryonic
development), maternal
folate deficiency,
environmental factors.
Developmental delays,
neurological abnormalities
depending on sac size and
location [36].
Myelomeningocele
Protrusion of meninges and
part of the spinal cord from
the back.
Incomplete closure of the
neural tube during
development, genetic factors
(MTHFR polymorphisms),
maternal folate
deficiency, infections.
Severe paralysis, incontinence,
pelvic organ dysfunction,
cognitive and motor
impairments [37].
Closed neural tube
defects (lipomyelocele,
lipomyelomeningocele,
meningocele, myelocystocele)
Defects involving the
accumulation of fatty tissue or
abnormal spinal structures.
Less well-defined causes
compared to open defects but
may involve genetic
predispositions (MTHFR and
other folate-related SNPs),
maternal folate deficiency, and
early developmental issues.
Less severe but may lead to
spinal deformities, mild
neurological symptoms and
developmental delays [31].
Author’s note: data provided in the table are incomplete; specific SNPs are not listed.
4. The Role of FA and One-Carbon Metabolism in the Etiology of NTDs
Recent research suggests that NTDs may be caused by multiple genetic mutations.
Key mutations linked to NTDs include the MTHFR C677T polymorphism (locus 1p36.6),
which impairs folate metabolism, and its prevalence varies between 10–30% in different
populations [
38
]. Mutations in the FOLR1 (folate receptor 1) gene on chromosome 11q13.4
affect folate transport into cells, reducing folate availability to developing neural tissues.
Although these mutations are rare, they have been linked to familial NTD cases [
39
].
Additionally, mutations in SLC25A32 (chromosome 8p11), which encodes a mitochondrial
folate transporter are very rare, but have also been associated with NTDs, particularly
when combined with folate deficiency [
40
]. The accumulation of these mutations, along
with environmental factors like folate deficiency increases the risk of NTDs [41].
One-carbon metabolism (OCM) is a critical biochemical pathway that involves the
transfer of one-carbon units for various cellular processes, including DNA synthesis and
methylation (Figure 3). This pathway plays a vital role in fetal development, as it provides
the necessary components for the formation of nucleotides and the regulation of gene
expression, making it essential for neural tube closure and overall embryonic health.
Mutations in genes encoding enzymes involved in OCM, such as MTHFR, methionine
synthase (MTR), and thymidylate synthase (TS), are associated with an increased risk of
NTDs [
42
]. Folate status can also be affected by polymorphisms in genes involved in folate
metabolism. Folate metabolism promotes one-carbon transfer processes that contribute to
the biosynthesis of purines and thymidine, the production of S-adenosylmethionine, the
ubiquitous methyl donor required for the methylation of DNA, RNA, proteins, and lipids.
These processes are carried out through linked reactions in the mitochondria and cytosol.
During fetal development, the demand for one-carbon units is at its highest [
43
]. Choline is
a critical nutrient also involved in OCM and serves as a methyl donor, playing an essential
role in DNA methylation and cellular function. Adequate choline intake is associated
with a reduced risk of NTDs. Given its interrelatedness with folate metabolism, ensuring
sufficient choline levels during pregnancy is vital for optimal fetal development and may
enhance the protective effects of folate against NTDs. Choline, as a one-carbon donor, plays

Nutrients 2025,17, 126 6 of 18
a pivotal role in folate metabolism, facilitating its optimal utilisation and contributing to
the prevention of the development of these developmental disorders.
Nutrients 2025, 17, x FOR PEER REVIEW 6 of 19
Mutations in genes encoding enzymes involved in OCM, such as MTHFR, methionine
synthase (MTR), and thymidylate synthase (TS), are associated with an increased risk of
NTDs [42]. Folate status can also be affected by polymorphisms in genes involved in folate
metabolism. Folate metabolism promotes one-carbon transfer processes that contribute to
the biosynthesis of purines and thymidine, the production of S-adenosylmethionine, the
ubiquitous methyl donor required for the methylation of DNA, RNA, proteins, and lipids.
These processes are carried out through linked reactions in the mitochondria and cytosol.
During fetal development, the demand for one-carbon units is at its highest [43]. Choline
is a critical nutrient also involved in OCM and serves as a methyl donor, playing an es-
sential role in DNA methylation and cellular function. Adequate choline intake is associ-
ated with a reduced risk of NTDs. Given its interrelatedness with folate metabolism, en-
suring sufficient choline levels during pregnancy is vital for optimal fetal development
and may enhance the protective effects of folate against NTDs. Choline, as a one-carbon
donor, plays a pivotal role in folate metabolism, facilitating its optimal utilisation and
contributing to the prevention of the development of these developmental disorders.
Figure 3. The role of folate and choline in one-carbon metabolism (adapted from [44]). 5,10-MTHFR,
5,10-methylenetetrahydrofolate reductase; 5,10-MTHF, 5,10-methylene-THF; 5-MTHF, 5-methyl-
THF; BET, betaine; BHMT, betaine-homocysteine S-methyltransferase; CBS, cystathionine β-syn-
thase; CTH, cystathionine; DHF, dihydrofolate; DHFR, dihydrofolate reductase; DMG, dimethyl-
glycine; dUMP, deoxyuridine monophosphate; dTMP, deoxythymidine monophosphate; GLY, gly-
cine; GNMT, glycine N-methyltransferase; HCY, homocysteine; MET, methionine; MTR, methio-
nine synthase; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine; SAR, sarcosine; SER,
serine; SHMT1, serine hydroxymethyltransferase 1; THF, tetrahydrofolate; TS, thymidylate syn-
thase.
NTDs can be caused by disruption of DNA synthesis, which is essential for cell pro-
liferation [45]. Recent research has identified novel variants of the folate transporter
(SLC19A1) and folate receptors (FOLR1, FOLR2, FOLR3) that impair folate metabolism
and are associated with an increased risk of NTDs [46]. Cai et al. discovered additional
polymorphisms in genes related to the folate metabolic pathway (MTHFR, MTHFD1,
MTRR, RFC1) that act as risk factors for NTDs in mothers [47]. Reduced MTHFR activity
leads to decreased levels of 5-methyltetrahydrofolate (5-MTHF) and increased levels of
homocysteine (HCY), which may contribute to the production of reactive oxygen species
and the development of oxidative stress [48]. However, research has shown that only 13%
of NTDs can be aributed to the MTHFR C677T mutation, which suggests that the
Figure 3. The role of folate and choline in one-carbon metabolism (adapted from [
44
]). 5,10-MTHFR,
5,10-methylenetetrahydrofolate reductase; 5,10-MTHF, 5,10-methylene-THF; 5-MTHF, 5-methylTHF;
BET, betaine; BHMT, betaine-homocysteine S-methyltransferase; CBS, cystathionine
β
-synthase;
CTH, cystathionine; DHF, dihydrofolate; DHFR, dihydrofolate reductase; DMG, dimethylglycine;
dUMP, deoxyuridine monophosphate; dTMP, deoxythymidine monophosphate; GLY, glycine; GNMT,
glycine N-methyltransferase; HCY, homocysteine; MET, methionine; MTR, methionine synthase;
SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine; SAR, sarcosine; SER, serine; SHMT1,
serine hydroxymethyltransferase 1; THF, tetrahydrofolate; TS, thymidylate synthase.
NTDs can be caused by disruption of DNA synthesis, which is essential for cell
proliferation [
45
]. Recent research has identified novel variants of the folate transporter
(SLC19A1) and folate receptors (FOLR1, FOLR2, FOLR3) that impair folate metabolism
and are associated with an increased risk of NTDs [
46
]. Cai et al. discovered additional
polymorphisms in genes related to the folate metabolic pathway (MTHFR, MTHFD1,
MTRR, RFC1) that act as risk factors for NTDs in mothers [
47
]. Reduced MTHFR activity
leads to decreased levels of 5-methyltetrahydrofolate (5-MTHF) and increased levels of
homocysteine (HCY), which may contribute to the production of reactive oxygen species
and the development of oxidative stress [
48
]. However, research has shown that only 13%
of NTDs can be attributed to the MTHFR C677T mutation, which suggests that the MTHFR
C677T polymorphism alone may not be solely responsible for NTDs. It is possible that other
factors such as gene-gene interactions, maternal-fetal interactions, or genetic-nutritional
interactions may also play a role in the development of NTDs [
49
]. This is supported by
a study by Behunova et al. in Slovak population, which found no significant association
between polymorphisms C677T and A1298C of the MTHFR gene and NTDs [50].
5. Preconceptional and Periconceptional FA Supplementation in Visegrad
Group Countries
In 1965, the hypothesis of a possible association between FA and a reduced incidence
of NTDs was first proposed [
51
]. The first cohort-controlled studies demonstrated an
embryoprotective effect of FA [
52
] and corroborated the findings that primary prevention
of some major structural birth defects (NTDs, urinary and cardiovascular defects) by
multivitamin supplementation with FA could be of significant public health importance [
20
].
Folate is essential for the proper functioning of various cellular processes, including amino
acid metabolism, DNA methylation, protein and lipid synthesis, DNA replication, and

Nutrients 2025,17, 126 7 of 18
cell division. During pregnancy, maternal folate requirement increases nearly fourfold to
support the formation and cell division of the placenta and embryo, which are critical for
fetal development [
53
]. FA requirement becomes even greater in lactating women than
during pregnancy [54].
It is now common practice to prescribe FA to women in the preconceptual and pericon-
ceptual periods. Increased FA intake can be achieved in three different ways: by increasing
the intake of folate-rich foods, supplementation in tablet form (FA-containing tablets or
multivitamin preparations), and fortified foods (e.g., FA-fortified flour). The primary
source of folate for humans is the diet. Folate occurs as polyglutamate in a variety of foods,
including green leafy vegetables, legumes, liver, nuts, yeast, cereals, cereal sprouts, whole
grain products, and citrus fruits (Table 3) [
55
]. However, supplements and fortified foods
contain synthetic FA, which is found in the form of monoglutamate [
56
,
57
], an inactive
form, which the human body is able to metabolize and must be converted to the active
molecule 5-MTHF in the liver [58].
Table 3. List of food examples and their folate and choline content [59,60].
Food
Folate Content (
µ
g/100 g)
Choline Content (mg/100 g)
High folate content
(≥100 µg/100 g)
Beef liver (raw) 290 333
Spinach (raw) 194 19.3
Lentils (cooked) 181 32.7
Chickpeas (cooked) 172 42.8
Asparagus (cooked) 149 26.1
Moderate folate content
(30–99 µg/100 g)
Avocado (raw) 89 14.2
Peas (raw) 65 28.4
Broccoli (raw) 63 18.7
Kimchi 52 15.5
Red bell pepper (raw) 47 5.6
Eggs (whole, cooked) 44 294
Mango (raw) 43 7.6
Bread (whole wheat) 42 27.2
Low folate content
(<30 µg/100 g)
Salmon (Atlantic,
farmed, raw) 26 78.5
Orange (raw) 25 8.4
Sauerkraut
(fermented cabbage) 23 10.4
Carrots (raw) 19 8.8
Tofu 15 28.8
Potatoes (baked) 9 14.5
Chicken breast (cooked) 4 35
White rice (cooked) 1 2.1

Nutrients 2025,17, 126 8 of 18
While adequate folate intake through a diverse diet is ideal, it can be challenging to
achieve optimal levels, particularly during periods of increased demand, such as pregnancy.
Lifestyle factors, dietary preferences, and socioeconomic disparities can influence dietary
folate intake [61–63]. To address these challenges, a multifaceted approach is necessary.
While FA supplementation remains a cornerstone of prenatal care, it’s important to
note that some individuals, particularly those with genetic variations in the MTHFR gene,
may have difficulty converting folic acid into its active form. 5-MTHF is a more bioavailable
form of folate, meaning it can be more readily used by the body. As such, it may offer
additional benefits for certain populations, especially those with MTHFR polymorphisms.
However, the optimal form of folate supplementation for individual women may vary, and
it’s crucial to consult with a healthcare provider to determine the best approach [64,65].
Supplementation as a public health intervention, while beneficial, faces several chal-
lenges. One of the main challenges is that more than half of all pregnancies worldwide
are unplanned, making it difficult to consume the necessary supplements before con-
ception [
66
]. Large-scale FA fortification is considered the most successful and effective
intervention program in reducing the incidence of NTDs and associated infant mortality.
Fortification represents a population-level intervention that has multiple benefits. It can
address nutritional deficiencies at the whole population level without the need for individ-
ual dietary changes, as well as address multiple nutritional deficiencies simultaneously,
thereby increasing its effectiveness. Food fortification is a cost-effective strategy to improve
nutritional status and prevent chronic diseases. It is implemented with respect to maximum
tolerable daily intakes of micronutrients, minimizing the risks of excessive intake [
67
].
By July 2023, 69 countries have introduced mandatory FA fortification and 47 countries
have established voluntary fortification. However, 77 countries have not yet introduced
any FA fortification. Populations with mandatory FA fortification had 3- to 4-fold higher
mean plasma folate levels than populations without any FA fortification [
68
]. De Wals et al.
reported a decrease in the incidence of NTDs in seven Canadian provinces from 1.58 per
1000 births before fortification to 0.86 per 1000 births after fortification [
69
]. Recent estimates
in the United States and Canada suggest that FA food fortification has provided additional
intake of approximately 100 to 150
µ
g/day to women of reproductive age. Measurement of
blood folate levels showed that red blood cell folate concentrations, a more robust indicator
of folate status than plasma folate due to their ability to reflect long-term folate stores over
weeks to months, increased by approximately 50% in each age group after the introduction
of FA fortification [
70
]. In addition, data from the US National Birth Defects Prevention
Study (1998–2003) suggest that the use of FA supplements during the periconceptional
period no longer provides an additional reduction in the risk of NTDs. This suggests that
fortification provides the necessary level of FA to prevent most folate-responsive NTDs [
71
].
Mandatory FA fortification programs may be more effective than supplementation pro-
grams in reducing the incidence of NTDs. This is because fortification provides FA to
all food consumers, whereas periconceptional supplementation requires either planned
pregnancy or continuous supplementation for all women of reproductive age and success-
ful social interventions [
72
]. European Union (EU) countries including Poland [
73
], the
Czech Republic, and Slovakia, are among the group of countries with recommended FA
fortification [
74
]. In August 1998, a bread vitamin fortification programme was launched in
Hungary, which resulted in the following nutritional composition of 200 g of bread: 400
µ
g
FA, 25
µ
g vitamin B12, and 3600
µ
g vitamin B6 [
75
]. This strategy represents one of the
earliest European attempts at population-wide FA fortification.

Nutrients 2025,17, 126 9 of 18
The food commissions of the EU countries have not yet agreed on the adoption
of compulsory fortification, arguing that the impact of FA food fortification, whether
positive or negative, on some selected diseases has not been sufficiently studied. Thus, FA
fortification in EU countries remains only in the form of recommendations, while further
research in this area is ongoing in parallel [76].
6. Comprehensive Strategies for NTDs Prevention
Effective NTDs prevention strategies and optimizing the nutritional status of women
of reproductive age requires a comprehensive approach. This section presents evidence-
based recommendations for FA supplementation and implementation strategies across
the Visegrad Group countries. This includes consideration of individual factors such as
recommended daily intake, optimal timing of FA supplementation, targeted counseling in
the preconceptional and periconceptional periods, and strategic plans at the national level.
6.1. Recommended Daily Intake
Several factors related to digestion influence the absorption of vitamins from food.
These factors include various medical conditions that can affect the body’s ability to
absorb vitamins and other nutrients, either by directly impacting the digestive tract or by
causing metabolic and nutrient distribution disorders. For instance, intestinal issues like
Celiac disease [
77
], Crohn’s disease [
78
,
79
], or short bowel syndrome [
80
,
81
] can lead to
malabsorption of vital nutrients, including folate. While a balanced diet, characterized by
the consumption of a variety of nutrient-dense foods such as fruits, vegetables, lean proteins,
and whole grains, generally provides essential nutrients necessary for good health, it often
does not provide sufficient folate for women of childbearing age to meet the recommended
intake levels required for NTD prevention [
82
]. Despite the consumption of folate-rich foods
such as leafy greens, legumes, and fortified grains, studies have shown that dietary folate
alone is frequently inadequate. This is due to several factors, including the heat sensitivity
of folate, which is significantly reduced by cooking methods like boiling and frying [
83
]. The
increasing consumption of processed foods, often low in folate content, further exacerbates
this issue [
84
]. Therefore, folic acid supplementation is recommended to ensure that women
receive the optimal dose of folate needed to support neural tube development in early
pregnancy. The optimal dosage of FA remains a subject of debate. Health organizations
such as the Centers for Disease Control and Prevention (CDC) and the World Health
Organization (WHO) recommend that women who are planning to become pregnant or
are already pregnant should take 400
µ
g of FA daily [
85
,
86
]. During the periconceptional
period, it is recommended to increase the dose to 600
µ
g/day [
63
], while for women with a
history of NTDs or other malformations, a dose of up to 800
µ
g/day, but not more than
1000
µ
g/day, is recommended due to possible adverse effects caused by excessive FA
intake [
87
]. Dolin et al. emphasize the need to reconsider the recommended daily intake
of FA to women at risk for recurrent NTD from 4000
µ
g to 1000
µ
g due to the reduced
absorption rate and possible adverse health effects, including an increased risk of cleft
palate, spontaneous abortion, impaired psychomotor development, and respiratory issues
in children [
88
]. Furthermore, excessive doses of FA may increase the risk of complications
in early pregnancy, as well as the development of insulin resistance, type 2 diabetes mellitus,
and obesity in children [
89
]. The most prevalent complications include the potential
masking of vitamin B12 deficiency, which poses severe risks for pregnant women with
inadequate intake or impaired absorption of this vitamin. This deficiency can result in
neurological impairment in the mother and potentially in the fetus if not properly identified
and addressed [
90
]. On the other hand, Wald et al. asserted that a daily FA intake of
400
µ
g is insufficient to protect pregnant women from NTDs and proposed a daily FA

Nutrients 2025,17, 126 10 of 18
intake of 500
µ
g, starting from the preconceptional period to enhance protection against
the aforementioned defects [
91
]. The tolerable safe daily dose of FA for adults, including
pregnant women, is 1000
µ
g per day [
92
]. While higher doses, up to 5000
µ
g per day,
may be recommended for women in high-risk group of NTDs during early pregnancy, it
is crucial to consult with a healthcare professional to determine the optimal dosage and
duration of supplementation. Current consensus suggests that a dose of 5000
µ
g or higher
is generally not justified, and a careful consideration of the benefits and risks is necessary
when determining appropriate FA supplementation. Collectively, these findings underscore
the importance of balancing FA intake to maximize NTD prevention while minimizing
associated risks [93].
Following two groundbreaking studies that demonstrated the benefits of FA-
containing supplements, medical and governmental organizations have published rec-
ommendations to promote the use of FA-containing supplements in the prevention of
NTDs. The FA supplementation guidelines indicate considerable variability in the duration
of preconceptional and periconceptional FA supplementation. Clinical studies confirm that
at least 12 weeks of supplementation with 400
µ
g of FA before conception is required to
achieve optimal erythrocyte folate levels for NTD prevention. The dose of preconceptional
and periconceptional FA administration remains consistent in the European countries
studied [
94
]. One of the more significant differences between the nations of the Visegrad
Four concerns the recommended duration of FA supplementation before conception. Most
European guidelines do not explicitly recommend a specific timeframe for initiating FA
supplementation before conception. Crider et al. have shown that after 2–6 months of
daily supplementation with 400
µ
g FA, erythrocyte folate levels reached 1000 nmol/L.
Erythrocyte folate levels exceeding 1000 nmol/L are associated with a significantly reduced
risk of NTDs. In non-supplemented individuals, erythrocyte folate concentrations below
500 nmol/L were linked to a significantly higher risk of NTDs, with an even higher risk
associated with concentrations below 340 nmol/L [
95
]. Another discrepancy between
countries is in the target group for whom FA supplementation is recommended. It is
recommended that European guidelines move towards recommending FA for all women
of childbearing age, or more specifically for all women at potential risk of pregnancy.
This contrasts with the current recommendation of supplementation only to those women
planning a pregnancy.
Due to the lack of approved recommended practices regarding FA supplementation in
the preconceptional and periconceptional period in Slovakia, the Slovak Gynecological and
Obstetric Society follows the recommendations of the American Society of Gynecologists
and Obstetricians. A comparison of Slovak, Czech, Polish, and Hungarian recommen-
dations on FA supplementation in the preconceptional, periconceptional, and lactation
periods is presented in Table 4. While some countries, such as Poland, recommend higher
doses (e.g., 5000
µ
g/day) for high-risk individuals, it’s important to balance the benefits
and risks of such high doses. Higher doses may increase the risk of adverse effects, includ-
ing those discussed earlier in the text. A personalized approach is crucial to ensure optimal
supplementation while minimizing potential adverse effects.

Nutrients 2025,17, 126 11 of 18
Table 4. Comparison of Slovak, Czech, Polish, and Hungarian recommendations for folic acid
supplementation during preconceptional, periconceptional and lactation period.
Country Recommending
Society
Folic Acid Dosage Common
Recommendations
Low Risk * Intermediate Risk ** High Risk ***
Slovakia
The American
Society
of Gynaecologists
and
Obstetricians [54]
400 µg/day
2–3 months prior to
pregnancy and
throughout the 1st
trimester;
600 µg/day is
recommended
during the 2nd and
3rd trimester and
throughout lactation
1000 µg/day 3 months
prior to pregnancy and
throughout the
1st trimester
4000 µg/day
3 months prior to
pregnancy and the
entire 1st trimester
A diet rich in folate
is recommended
for women of
reproductive age.
Vitamin B12
supplementation is
recommended
along with folate.
The dosage of folic
acid is based on
the risk of NTDs.
Czechia
The Czech Society
of Gynaecologists
and
Obstetricians [51]
400–800 µg/day
one month prior to
pregnancy and
throughout the
1st trimester
4000 µg/day in case of
previous NTD
pregnancy, BMI > 30, or
genetic mutations in
folate metabolism
N/A
Poland
The Polish Society
of Gynaecologists
and
Obstetricians [89]
400 µg/day
3 months prior to
pregnancy, during
pregnancy, and
lactation
800 µg/day 3 months
prior to pregnancy,
during pregnancy, and
lactation in case of
pre-existing type 1 or
2 diabetes mellitus, use
of antiepileptic drugs, or
bariatric surgery
5000 µg/day
3 months prior to
pregnancy and
throughout 1st
trimester,
800 µg/day
throughout 2nd
and 3rd trimester
and lactation
Hungary
The National
Institute for Health
Promotion in
Hungary and The
National Council
of Hungarian Gy-
naecologists [94]
400 µg/day
3 months prior to
and during
pregnancy
N/A N/A
* Low Risk: Women without a history of NTDs or other risk factors. ** Intermediate Risk: Women with conditions
such as obesity (BMI > 30), previous NTD-affected pregnancies, or genetic mutations affecting folate metabolism
(e.g., MTHFR mutations). *** High Risk: Women with pre-existing medical conditions such as type 1 or type 2
diabetes, use of antiepileptic drugs, or a history of bariatric surgery.
6.2. Timing of Supplementation
The periconceptional period is the most vulnerable phase of embryonal develop-
ment. During this period, adequate folate levels are particularly important to support
the formation of the neural tube, often before a woman is aware she is pregnant. For this
reason, women of childbearing age, especially those planning to become pregnant, are
encouraged to ensure sufficient folate intake through a combination of a healthy diet and
dietary supplements. While a diet rich in folate-containing foods should be a staple during
pregnancy, it can be challenging for women to meet the recommended folate intake solely
through food. Consequently, supplementation is often advised to guarantee adequate folate
levels, particularly during the preconception period and the initial months of pregnancy.
This proactive approach helps to address potential dietary deficiencies and ensures that
women receive the necessary nutrients to support healthy embryonic development. It may
take up to 20 weeks for a pregnant woman to reach the optimal erythrocyte folate level
(1050–1340 nmol/L)
needed to reduce the risk of NTDs when supplementing 400
µ
g of
FA daily. For this reason, it is advisable to start supplementation 5 to 6 months before
conception [
96
]. When these optimal levels are reached, the risk of NTDs is approximately
4.5 cases per 10,000 births [
58
]. Dong et al. suggest that the optimal time to start FA sup-
plementation is 1.5 months before pregnancy, with an acceptable range of 1.1–1.9 months.
Women in this study who supplemented with FA during the mentioned period had a 1.52%

Nutrients 2025,17, 126 12 of 18
risk of congenital malformations. The recommended duration of FA supplementation is
4 (3.7–4.4) months [97].
Determining the optimal time and duration of FA supplementation
is critical for maximum protection and prevention of side effects that result from excessive
FA supplementation [98].
Prenatal care in gynecological outpatient clinics usually does not begin until after the
7th week of pregnancy, which delays the start of FA supplementation. Some countries, such
as Vietnam, recommend starting FA supplementation from the first antenatal visit, which
usually takes place at 16 weeks of pregnancy [
99
]. Nilsen et al. found that only 48.6% of
Italian women who sought preconception healthcare and planned to become pregnant
were taking FA before pregnancy [
100
]. This suggests that Italian women start taking FA
supplements too late to prevent NTDs. China has one of the highest NTD prevalence rates
in the world, with inadequate dietary folate intake thought to be the main cause. According
to Ren’s study, less than a quarter of women took FA before becoming pregnant, leaving
three-quarters of fetuses at risk during the critical period of neural tube formation [101].
6.3. Preconception Counselling
The lack of uptake of FA in women happens due to a lack of knowledge and awareness
of its protective effect against NTDs. For women, who are planning to become pregnant,
antenatal counselling is a valuable strategy [
102
]. Health care providers play a key role in
providing preconception counselling to women. Counselling should include a comprehen-
sive discussion of the benefits of FA supplementation, the optimal timing and duration
of supplementation, as well as pregnancy planning and addressing specific risk factors a
woman may have. A thorough nutritional assessment to identify the risk of folate defi-
ciency should also be an essential part of the evaluation. This assessment should identify
and evaluate dietary and lifestyle habits (e.g., high alcohol intake [
103
], vegetarianism,
veganism [
104
], smoking [
105
], excessive caffeine consumption [
106
]) that may affect folate
absorption and metabolism. The woman’s diet should be analyzed to determine whether it
contains sufficient natural sources of folate, such as dark green leafy vegetables, legumes,
and citrus fruits. Additionally, factors such as gastrointestinal diseases (e.g., celiac disease,
Crohn’s disease) that may impact folate absorption should also be considered [107].
6.4. National Level Strategic Plan
Many countries have implemented campaigns to encourage women in the precon-
ceptional and periconceptional periods to take regular FA supplementation and thus raise
awareness of the importance of FA intake. Clarification of international recommenda-
tions, fortification of flour, as well as other foods could increase the effectiveness of folate
supplementation at the national level.
National level strategies vary in their approach and policy implementation. The
Polish government introduced the programme called “Primary NTD prophylaxis” in 1998,
recommending a daily FA intake of 400
µ
g for women of childbearing age. The aim of the
programme was to promote awareness of FA and its relationship to NTDs, to influence
attitudes, and to encourage appropriate behaviors in women regarding FA supplementation.
It was implemented in health facilities providing services to pregnant women, integrated
into health education projects, and disseminated through mass media [73].
In 2010, the Czech Republic launched the “Think of Me Before I’m Born” programme,
focusing on the issue of FA and its recommended supplementation. The programme
encouraged women who were trying to conceive or might become pregnant to take dietary
supplements containing FA [108].
While national level strategies for FA supplementation are still under consideration in
Slovakia, recent efforts have focused on raising awareness of the critical role of FA intake

Nutrients 2025,17, 126 13 of 18
in the prevention of NTDs. Educational initiatives targeting women of childbearing age,
as well as partnerships with healthcare providers aim to increase public knowledge of the
benefits of FA intake, particularly during the preconceptional period. Continued efforts,
including potential fortification policies and further public health campaigns will enhance
the effectiveness of FA supplementation at the national level.
7. Conclusions
As FA fortification of foods is still not mandatory in Europe, there is a pressing need
for evidence-based guidelines for FA supplementation before conception and during preg-
nancy. Regional discrepancy in the prevalence of NTDs may be influenced by geographical
differences in dietary folate intake and the distribution of the MTHFR C677T polymorphism
in the population. Preconceptional and periconceptional FA supplementation, as well as
nationwide food fortification programs, have successfully prevented thousands of NTDs.
However, given the genetic complexity of these defects and their persistent interactions
with environmental factors, complete prevention of NTDs remains an unattainable goal.
The optimal approach is early primary prevention, exemplified by the population-wide use
of FA supplements in pregnancy planning.
This review compares and provides practical guidelines and policy recommendations
for implementing effective FA supplementation strategies in the Visegrad Group countries.
Although research on FA supplementation in the preconceptional and periconceptional
periods dates back to 1984, the results obtained from randomized trials have not been
adequately implemented in practice, even after 40 years. It is essential that each country
has clearly defined clinical practice guidelines, based on current evidence-based medicine,
to guide the provision of health care, both in major cities and in more remote urban
and rural areas. Historically and socio-culturally, there are many similarities among the
Visegrad Group countries, but still significant differences in health care delivery can be
found, as in the case of FA supplementation recommendations. As shown in Table 4, there
are considerable differences in folate intake during pregnancy among these countries. In
Slovakia, there is currently no clear standard guideline on FA supplementation in the pre-
and periconceptional periods issued by the Slovak Society of Gynaecology and Obstetrics.
In practice, doctors follow the recommendations of foreign organizations, which are not
uniform and differ in the recommended dosage, as well as the period when FA should be
taken as dietary supplements. Considering the prevalence of MTHFR gene mutations in
the Slovak population, it is necessary to develop a standardized procedure and consider the
introduction of food fortification with FA, either voluntarily or obligatorily, similar to some
other countries. In contrast to Slovakia, the Czech Republic, and Hungary, the Polish Society
of Gynaecologists and Obstetricians has developed comprehensive recommendations for
FA and other vitamin supplementation, serving as a model for other countries in the
Visegrad region.
FA supplementation cannot guarantee 100% prevention of NTDs because up to one-
third of these defects are FA-resistant. Currently, there is a strong emphasis on personalized
medicine, the search for alternative preventive strategies, and the exploration of new
biomarkers using omics technologies supported by artificial intelligence. Precision medicine
strategies that harness the power of human genomics and advanced tools for assessing
genetic risk factors will be essential for future prevention initiatives.
Author Contributions: Conceptualization, V.R.; validation, R.S. and J.Z.; formal analysis, V.R. and
R.S.; investigation, V.R., V.J. and L.G.; writing—original draft preparation, V.R.; writing—review
and editing, R.S. and L.G.; visualization, R.S.; supervision, I.V. and J.Z.; funding acquisition, J.Z. All
authors have read and agreed to the published version of the manuscript.

Nutrients 2025,17, 126 14 of 18
Funding: This research and the APC was funded by the Scientific Grant Agency of the Ministry
of Education, Science, Research and Sport of the Slovak Republic, grant number VEGA 1/0560/22,
project leader: prof. Jozef Záhumenský, MD, PhD.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: No new data were created or analyzed in this study. Data sharing is
not applicable to this article.
Acknowledgments: The authors would like to express their sincere gratitude to the late Vladimír
Jakuš for his invaluable guidance and support.
Conflicts of Interest: The authors declare no conflicts of interest.
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