Introduction
Bad obstetric history (BOH) refers to the occurrence
of repeated adverse pregnancy outcomes, including two or more
spontaneous abortions, intrauterine foetal deaths, preterm births,
stillbirths, congenital anomalies, or early neonatal deaths. It
remains a critical reproductive health issue due to its profound
emotional, medical and social impact on affected women and their
families. Although BOH has multiple causes, its underlying
molecular mechanisms are not yet fully understood (1). The present study is therefore
relevant, as it focuses on oxidative stress and apoptosis, which
are key factors in placental dysfunction, and may aid in the
identification of early biomarkers and potential targets for the
better prediction and management of BOH.
Oxidative stress occurs when the generation of
reactive oxygen species (ROS) exceeds the capacity of antioxidant
defence systems. During pregnancy, balanced and physiological
levels of ROS are essential for normal placental functions,
including trophoblast proliferation, differentiation, invasion and
angiogenesis (2). Elevated levels
of malondialdehyde (MDA), a widely recognised marker of oxidative
stress in maternal blood, reflect increased lipid peroxidation and
cellular damage, and have been consistently associated with BOH
(3). Excessive oxidative stress
can damage cellular components, such as lipids, proteins and DNA,
ultimately disrupting normal cell function. At the materno-foetal
interface, this oxidative injury can trigger apoptotic pathways,
leading to increased trophoblast cell death, impaired placental
development and a higher risk of recurrent pregnancy loss (4,5).
Apoptosis is a tightly regulated physiological
process that is essential for maintaining tissue balance,
particularly at the materno-foetal interface, where controlled
trophoblast turnover supports normal placental development.
However, when this process becomes dysregulated, it can contribute
to pregnancy complications, such as implantation failure and
recurrent pregnancy loss. Excessive oxidative stress is a major
trigger for apoptosis, as elevated levels of ROS can damage
cellular lipids, proteins and DNA, disrupt mitochondrial integrity,
and activate intrinsic apoptotic pathways. At the placental level,
this oxidative injury promotes trophoblast cell death, the
increased shedding of syncytiotrophoblast fragments and heightened
inflammatory responses, ultimately impairing placental function and
increasing the risk of adverse pregnancy outcomes (6).
Caspase-3, the executioner of the cascade, is
recruited during the terminal steps of both the intrinsic and
extrinsic apoptotic pathways (7).
Previous research has demonstrated that caspase-3 expression is
increased in placental tissues from women with BOH, suggesting a
role in the abnormal turnover of trophoblasts and trophectodermal
dysfunction (8). Caspase-3 is not
only a surrogate marker of apoptosis, but also a key functional
component in understanding the development of BOH. As further
knowledge about its role in pregnancy loss is acquired, it may be
possible to identify women at risk earlier and this knowledge may
aid in the development of potential biomarkers for its
detection.
The p53 gene, known as the ‘guardian of the genome’,
is crucial for regulating cell cycle arrest, DNA repair and
apoptosis under stressful conditions. During pregnancy, high levels
of oxidative stress can lead to trophoblastic apoptosis and affect
placental function, which may result in negative outcomes, such as
BOH. p53 functions as a molecular connector by responding to
oxidative stress and activating pro-apoptotic pathways, such as
caspase-3 and BAX, influencing both implantation and foetal
survival (9,10). Changes in p53 expression have been
noted in women with recurrent pregnancy loss, suggesting that it
may serve as a potential biomarker for identifying the condition
(11).
Oxidative stress and apoptosis are key biological
processes involved in normal pregnancy, and their imbalance has
been implicated in the pathogenesis of BOH. Increased oxidative
stress can damage placental cells and trigger excessive apoptosis,
leading to adverse pregnancy outcomes. The p53 gene plays a central
role in linking oxidative stress to apoptosis by regulating cell
cycle arrest and programmed cell death. Although p53 is essential
for normal pregnancy, its dysregulated expression may contribute to
pathological apoptosis in patients with BOH. Therefore, the present
study aimed to evaluate the oxidative stress markers, apoptotic
markers, and p53 gene expression in women with BOH and explore
their interrelationships.
Materials and methods
Study participants
The present case-control study was conducted at
Genetika, Centre for Advanced Genetic Studies, Thiruvananthapuram,
Kerala, India, between September, 2023 and July, 2025. A total of
200 participants were enrolled, including 100 women with BOH and
100 age-matched controls. The case group consisted of women aged
20-45 years with a history of two or more spontaneous abortions,
intrauterine foetal deaths, stillbirths or early neonatal deaths.
Participants were recruited from obstetrics and gynaecology
outpatient departments across South Kerala and referred to the the
Genetika laboratory for an advanced genetic evaluation. Only women
with documented obstetric records confirming previous adverse
outcomes were included. The control group comprised women aged
20-45 years with at least two successful pregnancies and no history
of pregnancy loss. Women with consanguineous marriages, prolonged
medication use, or severe systemic illnesses were excluded from the
study. Ethical approval was obtained from the Institutional Ethics
Committee of Genetika, Centre for Advanced Genetic Studies
(Reference no. 17/2023/IECG).
After obtaining written informed consent, relevant
clinical and demographic data were collected using a pre-structured
questionnaire. A total of 7 ml fasting venous blood was collected
from each participant. Of this, 4 ml was transferred to a clot
activator tube for serum separation, and 3 ml was collected in a
sodium heparinised vacutainer for gene expression analysis.
Assessment of oxidative stress and
apoptosis
Oxidative stress was assessed by estimating serum
MDA levels using a competitive inhibition ELISA kit (cat. no.
OPK8428; Origin). Apoptosis was evaluated by measuring serum
caspase-3 levels using a sandwich ELISA kit (cat. no. OPK1527;
Origin). All ELISA procedures were carried out according to the
manufacturer's instructions.
Gene expression analysis
For gene expression analysis, total RNA was isolated
from heparinised blood samples using an RNA extraction kit (cat.
no. ODP419; Origin). Complementary DNA (cDNA) was synthesised using
a cDNA synthesis kit (cat. no. ODR41; Origin). Reverse
transcription-quantitative PCR (RT-qPCR) was then performed using a
2X SYBR-Green Master Mix (cat. no. ODQ383-01; Origin). The thermal
cycling conditions included an initial denaturation at 95˚C for 5
min, followed by 40 cycles of denaturation at 94˚C for 1 min,
annealing at 54˚C for 1 min, and extension at 72˚C for 1 min, with
a final extension at 72˚C for 10 min. The primer sequences used for
p53 amplification were 5'-CCTCAGCATCTTATCCGAGTGG-3' (forward) and
5'-TGGATGGTGGTACAGTCAGAGC-3' (reverse), with an annealing
temperature of 54˚C. Gene expression levels were normalized using
GAPDH as the internal control, with primer sequences
5'-CCATGGAGAAGGCTGGGG-3' (forward) and 5'-CAAAGTTGTCATGGATGACC-3'
(reverse). Relative p53 gene expression was calculated using the
2-ΔΔCq method (12).
The expression levels were categorized based on fold-change values
as normal (1) or upregulated
(>1.0).
Statistical analysis
The sample size was determined using the formula
Z²pq/d². A 95% confidence level was used, with an assumed
prevalence of 5.27% and an allowable error of 10%. Based on this
calculation, the minimum required sample size was determined. To
ensure adequate statistical power and account for possible
variability, the sample size was increased in each group (cases and
controls). The Mann-Whitney U test was used to compare
non-parametric variables between groups. Pearson's correlation
analysis was performed to assess the association between p53
expression and the molecular biomarkers (MDA and caspase-3). A
two-tailed P-value <0.01 was considered to indicate a
statistically significant difference. Data management and
statistical analyses were carried out using Microsoft Excel and
Jamovi software (version 2.5.3).
Results
MDA levels (nmol/ml), a marker of oxidative stress,
were significantly higher in the cases than in the controls
(Fig. 1). The cases exhibited both
a higher median value and greater variability, indicating increased
oxidative stress in this group. By contrast, the control group
exhibited lower and more consistent values, although there were
some outliers.
The box plot depicted in Fig. 2 demonstrates the comparison between
the caspase-3 levels between the cases and controls. The case group
exhibited a higher median and greater variability, with one extreme
outlier on the upper end, suggesting increased apoptotic activity
in the cells. By contrast, the controls exhibited generally lower
values and a more compact distribution, although a few mild
outliers were present.
The analysis of p53 expression patterns (Fig. 3) demonstrated a clear difference
between the cases and controls. A normal expression was
predominantly observed in the controls (78%) compared with the
cases (22%). By contrast, upregulation was more frequent in the
cases (58%) than in the controls (15%), while downregulation was
observed in a smaller proportion in both groups. This indicates
that the dysregulation of the p53 gene, particularly its
upregulation, is closely associated with the case group, suggesting
its key role in disease development.
The mean levels of MDA and caspase-3 were
significantly elevated in the cases compared with the controls,
suggesting increased oxidative stress and apoptotic activity in the
affected group (Table I).
Likewise, p53 gene expression, assessed using the 2-ΔΔCq
method, exhibited a significant increase in the cases. All these
variances were statistically significant (P<0.01; Table I).
 | Table IComparison of oxidative stress,
apoptosis and p53 gene expression in cases vs. controls. |
Table I
Comparison of oxidative stress,
apoptosis and p53 gene expression in cases vs. controls.
| | Mean ± SD | |
|---|
| Variable | Cases | Controls | P-valuea |
|---|
| MDA (nmol/ml) | 4.1±1.6 | 1.9±0.8 | <0.01 |
| Caspase-3
(ng/ml) | 3.3±2.3 | 1.9±1.0 | <0.01 |
| p53 gene
(2-ΔΔCq method) | 1.4±0.7 | 1.0±0.2 | <0.01 |
A significant positive correlation was found between
MDA levels (nmol/ml), an indicator of oxidative stress, and p53
gene expression (2-ΔΔCq method). As illustrated in
Fig. 4, the correlation
coefficient was r=0.4584 (P<0.01), indicating a moderate
positive correlation. This suggests that elevated oxidative stress
is linked to the increased expression of p53. The regression line
also illustrated the trend of rising p53 expression with increasing
MDA concentration.
The scatter plot presented in Fig. 5 demonstrated a strong positive
correlation (r=0.7136, P<0.01) between p53 gene expression and
caspase-3 levels. As p53 is a key regulator of apoptosis and
caspase-3 is an executioner caspase, this correlation indicates
that the upregulation of p53 directly enhances caspase-3-mediated
apoptotic activity.
Discussion
The present study demonstrates that oxidative
stress-induced, p53-mediated apoptosis plays a crucial role in the
pathogenesis of BOH. The significantly elevated serum MDA levels
observed in women with BOH indicate increase lipid peroxidation and
excessive ROS generation, reflecting a sustained oxidative burden.
Such an altered intrauterine redox environment is known to disrupt
placental homeostasis and initiate cellular stress responses that
are essential for maintaining normal pregnancy. Consistent with
previous studies linking oxidative stress to recurrent pregnancy
loss and placental dysfunction, the findings of the present study
suggest that oxidative injury functions as an early and key
pathogenic event in BOH rather than merely a secondary consequence
(13,14).
Excessive oxidative stress can damage cellular
lipids, proteins and DNA, thereby triggering intrinsic apoptotic
pathways. In the present study, significantly higher caspase-3
levels in the BOH cases indicate the active execution of apoptosis,
confirming that trophoblastic cell death is a prominent feature in
these patients (15). Increased
apoptotic activity at the materno-foetal interface can impair
trophoblast invasion, placental vascular remodelling and nutrient
exchange, ultimately compromising placental function and
contributing directly to pregnancy failure (16). Thus, oxidative stress-induced
apoptosis appears to be a central mechanism linking cellular injury
to adverse pregnancy outcomes in BOH.
A key finding of the present study was the marked
upregulation in p53 gene expression in women with BOH. The
expression pattern analysis revealed that upregulation was
significantly more frequent in the cases than the controls,
highlighting the role of p53 dysregulation in the development of
disease. Notably, the positive correlation observed between MDA
levels and p53 expression indicates that increased oxidative stress
is closely associated with activation of the p53 pathway (17). This supports the well-established
role of p53 as a stress-responsive regulator that becomes activated
in response to oxidative DNA damage.
Furthermore, the strong positive correlation between
p53 expression and caspase-3 levels provides clear evidence that
p53 activation directly promotes apoptotic execution in BOH. Since
p53 functions as a transcriptional regulator of pro-apoptotic
genes, its overexpression likely shifts the balance from cellular
survival toward programmed cell death (18). The coordinated alterations in
oxidative stress, p53 signalling and caspase-3-mediated apoptosis
indicate a potential molecular axis associated with trophoblastic
injury and placental dysfunction. These markers may serve as a
biomarker panel for early risk stratification and clinical
monitoring in BOH, enabling targeted surveillance and supportive
management. However, these applications remain preliminary and
require validation through prospective and interventional studies.
Notably, p53 expression, in conjunction with related markers, may
aid in early risk detection, although causality cannot be inferred
from this case-control study.
The present study had certain limitations, which
should be mentioned. The case-control design may be prone to
selection bias, as participants were recruited from a single
geographic region in South Kerala, India. The single-centre setting
and lack of multivariate adjustment for potential confounders, such
as lifestyle variables and associated clinical conditions may also
affect the generalisability and interpretation of the findings. The
cross-sectional nature of the study limits the ability to establish
causality, making it unclear whether p53 upregulation is a cause or
a consequence of oxidative stress in BOH. The present study also
relied on biomarker measurements without functional or mechanistic
experiments, which limits direct confirmation of the causal role of
the oxidative stress-p53-apoptosis pathway. Larger, multicentre
longitudinal studies with experimental validation are thus required
to strengthen these findings.
In conclusion, the findings of the present study
support an association between oxidative stress, p53-mediated
apoptosis, and BOH. Elevated levels of oxidative stress are
associated with an increased expression of p53, which is associated
with enhanced trophoblastic apoptosis, potentially contributing to
impaired placental function. The coordinated upregulation of
oxidative stress markers, apoptotic indicators, and p53 expression
suggests a possible molecular link underlying BOH. Collectively,
these results indicate that p53 may function as a potential
mediator linking oxidative stress and apoptosis in BOH. The
assessment of p53 expression, along with oxidative stress and
apoptosis markers, may provide insight for early risk
identification and clinical monitoring in susceptible women.
Acknowledgements
The authors would like to sincerely thank GG
Hospital, Pran Hospital, Susrutha Hospital, PRS Hospital and
Credence Hospital for their valuable support and collaboration in
enabling sample collection for the present study (gynecologists
from these hospitals referred subjects to the Genetika laboratory
for advanced genetic testing, where the samples were collected).
The authors also express their gratitude to the Genetika Laboratory
for providing laboratory facilities and technical support for
carrying out the study.
Funding
Funding: No funding was received.
Availability of data and materials
The data generated in the present study may be
requested from the corresponding author.
Authors' contributions
RPR was involved in data curation, formal analysis,
data investigation, project administration, in the provision of
resources (including the purchase of laboratory reagents and
consumables), visualization (figure preparation), and in the
writing of the original draft of the manuscript. DC was involved in
the conceptualization of the study, in the study methodology, data
investigation, project administration, validation, study
supervision, and in the writing, reviewing and editing of the
manuscript. DRD contributed to the conceptualization and design of
the study, study supervision, software support, and formal
analysis. NM provided resources (supported subject recruitment to
Genetika through collaboration with various hospitals) and was
involved in data validation. SM was involved in data validation,
and in the writing, reviewing and editing of the manuscript. RGM
was involved in the conceptualization of the study, in the study
methodology, data validation, in project administration, and in the
writing, reviewing and editing of the manuscript. DC and DRD
confirm the authenticity of all the raw data. All authors have read
and approved the final manuscript.
Ethics approval and consent to
participate
Ethical approval was obtained from the Institutional
Ethics Committee of Genetika, Centre for Advanced Genetic Studies
(Reference No. 17/2023/IECG) on 03.08.2023. All procedures adhered
to the Declaration of Helsinki of 1975, as revised in 2000. Written
informed consent was obtained from all participants included in the
study.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing
interests.
Use of artificial intelligence tools
During the preparation of this work, AI tools
(QuillBot and Grammarly) were used to improve the readability and
language of the manuscript or to generate images, and subsequently,
the authors revised and edited the content produced by the AI tools
as necessary, taking full responsibility for the ultimate content
of the present manuscript.
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