Introduction
The World Health Organization defines abortion as
the termination of pregnancy before 20 weeks of pregnancy or a
fetal birth weight of <500 g (1). Threatened abortion (TA) occurs in
30-40% of pregnancies (2,3), and is diagnosed as a combination of
vaginal bleeding, a closed cervix and the presence of the fetal
heartbeat (4). The probability of
abortion during the first 12 weeks of pregnancy is >80%
(5). Following the development of
China's Three Child Policy, the number of pregnant women has
increased in China, which may increase the incidence of TA
(6). Although TA is not directly
associated with increased maternal morbidity or mortality, it may
be associated with preterm delivery, premature rupture of
membranes, low birth weight, fetal growth limit, placental
abruption and an increase in cesarean sections (1). Furthermore, TA may have a negative
impact on the mental health of patients (7). Notably, preterm delivery is defined
as delivery prior to 37 weeks of pregnancy. Preterm delivery
accounts for 5-10% of all deliveries worldwide and is associated
with ~75% of neonatal mortality and more than 50% of long-term
newborn morbidities, including neurological impairments and chronic
lung diseases, as well as learning disability and, psychological,
behavioral and social troubles (8,9).
The mechanisms underlying TA are complex and
pathogenesis may be associated with infection (10), thyroid issues, endocrinopathy
(11), genital deformity (12) chromosomal mutations (13), immune system collapse, diabetes,
increased maternal age, previous abortion, passive smoking, obesity
and a history of drug or alcohol consumption (14,15).
At present, research is focused on the potential association
between TA and the placenta. The most common source of vaginal
bleeding in early pregnancy is the placenta (16). Placental defects may cause the
placenta to synthesize and release angiogenic and anti-angiogenic
factors in the feto-maternal circulation, such as vascular
endothelial growth factor (VEGF) and placental growth factor (PlGF)
(16). VEGF is an angiogenic
factor that serves a role in full-term pregnancy, contributing to
implantation and placental growth (17). In addition, PlGF is a member of the
VEGF family and acts as a modulator of decidual angiogenesis and a
mediator of trophoblast function (18). However, the specific roles of VEGF
and PlGF in TA are yet to be fully elucidated. Thus, the present
study aimed to determine the serum levels of VEGF and PlGF in
patients with early TA who experienced preterm delivery.
Materials and methods
Patients
The present case-control study included a total of
130 pregnant female patients with or without TA and with or without
preterm delivery. All patients were admitted to The Maternal and
Childcare Hospital of Nantong University (Jiangsu, China) from
January 2019-January 2022, and were aged 20-35 years and all cases
were singleton pregnancies. Patients were divided into two groups:
i) Group A, which included 55 patients diagnosed with TA with
slight vaginal bleeding and closed cervical internal os within the
first 6-12 weeks of pregnancy; and ii) group B, which included 75
patients with healthy asymptomatic pregnancies. Premature delivery
refers to childbirth at 28 weeks of pregnancy but less than 37
weeks. Both groups of patients were evaluated via vaginal
examination. The present study was approved by The Medical Ethics
Committee of The Maternal and Childcare Hospital of Nantong
University (approval no. Y2018036).
The inclusion criteria for the present study were as
follows: i) Gestational age, 6-12 weeks; ii) singleton pregnancy;
iii) patient age, 20-35 years; iv) termly prenatal visits; v)
parity <3; and vi) previous medical abortion due to unexpected
pregnancy.
Patients were excluded from the present study due to
the following criteria: i) Patient age, <20 or >35 years; ii)
reproductive tract malformation; iii) multiple gestations; iv)
chronic systemic illness or acute infectious disease; v) exposure
to high levels of alcohol or nicotine; and vi) a history of natural
abortion or any pregnancy complications, such as gestation-mediated
hypertension, gestational diabetes mellitus, intrauterine growth
limit, intrauterine mortality, preterm delivery, early rupture of
membranes or any previous medical history, such as asthma,
antiphospholipid antibody syndrome, thyroid dysfunction,
tuberculosis and other chronic health disorders.
ELISA
A 3 ml peripheral blood sample was obtained from
each participant and stored in EDTA (1.5 mg/ml). Blood samples were
separated via centrifugation at 670.8 x g for 10 min at room
temperature, and subsequently stored at -80˚C. Levels of VEGF
(catalog no. ZK-2291; Zhenke Biology) and PlGF (catalog no.
ZK-2017; Zhenke Biology) were quantified using commercially
available ELISA kits.
Statistical analysis
Statistical analysis was carried out using SPSS
software (version 20.0; IBM Corp.). Differences between groups were
analyzed using the chi-squared test, unpaired Student's t-tests and
two-way ANOVA followed by Bonferroni's post hoc analysis and
presented as mean ± standard deviation, whereas counting data were
examined using a chi-squared test and presented as percentages.
P<0.05 was considered to indicate a statistically significant
difference.
Results
Patient information and preterm
delivery rates of patients with or without TA
There were not statistically significant differences
in the maternal age, BMI prior to pregnancy, BMI at study
enrollment, gravidity or parity between patients with TA and
patients with healthy pregnancies (Table I). The preterm birth rate in
patients with TA was 21.8% and was 12.0% in patients with healthy
pregnancies. Notably, there were no significant differences between
the two groups of patients (Table
I).
 | Table IPatient information and preterm
delivery rates and VEGF, PIGF levels of patients with or without
TA. |
Table I
Patient information and preterm
delivery rates and VEGF, PIGF levels of patients with or without
TA.
| Patient
information | Patients with TA
(n=55) | Patients without TA
(n=75) | P-value |
|---|
| Age, years | 29.35±3.23 | 28.71±3.31 | >0.05 |
| BMI before pregnancy,
kg/m2 | 21.50±2.10 | 21.39±1.87 | >0.05 |
| Enrolled pregnancy
BMI, kg/m2 | 27.88±3.15 | 28.68±3.00 | >0.05 |
| Gravidity | 1.67±0.75 | 1.59±0.74 | >0.05 |
| Parity | 0.38±0.68 | 0.39±0.64 | >0.05 |
| Preterm labor, n
(%) | | | |
|
Yes | 12 (21.8%) | 9 (12.0%) | >0.05 |
|
No | 43 (78.2%) | 66 (88.0%) | >0.05 |
| VEGF levels,
pg/ml | 62.15±10.87 | 84.39±10.00 | <0.01 |
| PlGF levels,
pg/ml | 39.71±20.70 | 110.88±37.76 | <0.01 |
Comparison of VEGF levels (pg/ml) in
patients with or without TA and preterm delivery
Patients with TA had significantly lower VEGF levels
compared with patients with healthy pregnancies (Table I). There were no significant
differences in the levels of VEGF between patients with and without
preterm delivery in the patients with or without TA (Table II). No interaction effect was
revealed between the two main effects (TA and preterm
delivery).
| Table IIComparison of VEGF levels (pg/ml) in
patients with or without TA and preterm delivery. |
Table II
Comparison of VEGF levels (pg/ml) in
patients with or without TA and preterm delivery.
| | TA main effect | Preterm delivery main
effect |
|---|
| Patient group | Preterm delivery | No preterm
delivery | P-value | F-value | P-value | F-value | P-value |
|---|
| TA | 63.43±4.40 | 61.79±12.10 | >0.05 | 75.23 | <0.01 | 0.14 | >0.05 |
| No TA | 84.60±10.46 | 84.37±10.02 | >0.05 | | | | |
| P-value | <0.01 | <0.01 | | | | | |
Comparison of PlGF levels (pg/ml) in
patients with or without TA and preterm delivery
The levels of PlGF were significantly lower in
patients with TA compared with patients with healthy pregnancies
(Table I). In patients with or
without TA, the levels of serum PlGF in the preterm delivery group
were significantly decreased compared with the group that did not
experience preterm delivery (Table
III). A statistically significant interaction effect between
the two main effects was demonstrated (F=8.45, P<0.01) and
further cross-comparisons were performed. In the case of preterm
delivery, the simple effect of TA was statistically significant
(F=7.38, P<0.05). In the case of no preterm delivery, the simple
effect of TA was statistically significant (F=176.10, P<0.01).
In the case of TA, the simple effect of preterm delivery was not
statistically significant (F=2.14, P>0.05). In the case of no
TA, the simple effect of preterm delivery was statistically
significant (F=27.99, P<0.01).
| Table IIIComparison of PlGF levels (pg/ml) in
patients with or without TA and preterm delivery. |
Table III
Comparison of PlGF levels (pg/ml) in
patients with or without TA and preterm delivery.
| | TA main effect | Preterm delivery main
effect |
|---|
| Patient outcome | TA | No TA | P-value | F-value | P-value | F-value | P-value |
|---|
| Preterm delivery | 28.99±17.89 | 63.39±19.58 | <0.01 | 61.79 | <0.01 | 23.86 | <0.01 |
| No preterm
delivery | 42.70±20.64 | 117.36±34.95 | <0.01 | | | | |
| P-value | <0.01 | <0.01 | | | | | |
Discussion
VEGF serves a role in the physiological or
pathological anti-placental formation and f previous studies have
reported that VEGF is involved in placental angiogenesis (19,20).
In the present study, the mean serum level of VEGF in patients with
TA was 62.15±10.87 pg/ml, compared with 84.39±10.00 pg/ml in
patients with healthy pregnancies. These results demonstrated that
the levels of VEGF were lower in patients with TA. Furthermore, the
mean serum level of VEGF in patients that experienced preterm
delivery was 63.43±4.40 pg/ml, compared with 61.79±12.10 pg/ml in
patients that did not experience preterm delivery. The mean serum
level of VEGF in patients with healthy pregnancies that experienced
preterm delivery was 84.60±10.46 pg/ml, compared with 84.37±10.02
pg/ml in patients that did not experience preterm delivery.
Therefore, there was no significant difference in the levels of
VEGF between patients that experienced preterm delivery and those
that did not.
PlGF is a dimer glycoprotein that is mainly released
by the placenta. PlGF is associated with angiogenesis and serves a
role in the growth and differentiation of trophoblasts. The
circulating level of PlGF increases during pregnancy, and this
serves a role in the progression and maturation of the placental
vascular system (18). In the
first three months of a healthy pregnancy, the levels of PlGF are
low. After this time, levels of PlGF subsequently increase and
plateau at ~30 weeks of pregnancy prior to decreasing again
(21). Results of the present
study demonstrated that the mean serum level of PlGF in patients
with TA was 39.71±20.70 pg/ml, compared with 110.88±37.76 pg/ml in
patients with healthy pregnancies. These results demonstrated that
serum levels of PlGF were significantly reduced in patients with
TA. Furthermore, the mean serum level of PlGF in patients with TA
that experienced preterm delivery was 28.99±17.89 pg/ml, compared
with 42.70±20.64 pg/ml in patients with TA that did not experience
preterm delivery. In addition, the mean serum level of PlGF in
patients with healthy pregnancies that experienced preterm delivery
was 63.39±19.58 pg/ml, compared with 117.36±34.95 pg/ml in patients
with healthy pregnancies that did not experience preterm delivery.
Collectively, these results demonstrated that the levels of PlGF in
patients without TA were lower in patients that experienced preterm
delivery, compared with patients that did not.
The prediction of TA is complex, as predictive
methods are unreliable (22) and
the associated underlying mechanisms remain to be fully elucidated.
A number of previous studies have focused on the association
between human chorionic gonadotropin and estrogen in TA (23,24).
However, the present study aimed to determine the association
between the serum levels of VEGF and PlGF in patients with early TA
that experienced preterm delivery. Notably, the results of previous
studies are inconsistent (25-29).
Eskicioglu et al (25)
investigated the roles of numerous angiogenic factors in pregnancy
losses that occurred during the first trimester, and revealed that
the expression levels of VEGF were lower in patients that
experienced pregnancy loss compared with the women that did not
experience pregnancy loss during the first trimester. Furthermore,
Muttukrishna et al (26)
demonstrated that the levels of PlGF were reduced in patients with
TA that experienced miscarriage, compared with patients with TA
that experienced full-term pregnancy. In addition, patients with TA
that experienced miscarriage had reduced levels of PlGF compared
with asymptomatic gestational patients. In a case-control study by
Dev et al (27), pregnant
patients were divided into three groups according to gestational
age (6-10, 11-15 and 16-20 weeks). The serum levels of VEGF and
PlGF were markedly decreased in patients with TA, compared with
patients with healthy pregnancies. However, there were no
differences in the levels of serum VEGF and PlGF between patients
with different gestational ages. Hussein et al (28) revealed that the concentration of
PlGF was reduced in patients that experienced preterm delivery
compared with women without preterm delivery. Thus, results of the
previous study (28) highlighted
that reduced expression levels of PlGF may act as a potential
biomarker for the prediction of preterm delivery. However, Keskin
et al (29) revealed that
serum levels of VEGF-A were markedly increased in patients with TA,
compared with patients with healthy pregnancies without TA.
However, there were no differences in the levels of PlGF between
the two groups.
In conclusion, the results of the present study
demonstrated that patients with TA had reduced levels of VEGF and
PlGF compared with patients without TA. In addition, patients that
experienced preterm delivery had reduced levels of PlGF compared
with patients that did not experience preterm delivery. However,
the sample size of the present study was small and geographical
factors could have influenced the findings. Predicting and
preventing TA remains a challenge for clinicians; thus, further
investigations into the mechanisms underlying TA are required. In
addition, VEGF and PlGF could potentially be used as biomarkers for
preterm delivery in patients with TA.
Acknowledgements
Not applicable.
Funding
Funding: No funding was received.
Availability of data and materials
The datasets used and/or analyzed during the current
study are available from the corresponding author on reasonable
request.
Authors' contributions
PZ conceptualized the study and methodology,
performed data analysis and investigations and drafted the
manuscript. YJ analyzed and visualized data and wrote and edited
the manuscript. XH interpretated and visualized data, reviewed and
edited the manuscript, supervised the project and conducted project
administration. PZ and YJ confirm the authenticity of all the raw
data. All authors read and approved the final version of the
manuscript.
Ethics approval and consent to
participate
The present study was approved by the Medical Ethics
Committee of the Maternal and Childcare Hospital of Nantong
University (approval no. Y2018036). All patients provided written
informed consent.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing
interests.
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