Keywords
pregnancy - saliva - sialic acid - α-amylase, alpha-amylases - pH
Introduction
In pregnancy, physiologic changes cause the development of specific demands for both
the pregnant woman and her fetus. Some changes, such as gingivitis, develop during
pregnancy in the oral cavity, which could be related to hormonal alterations and unfavorable
oral hygiene.[1]
[2]
Although some studies revealed a higher prevalence of dental caries and an increase
in decay-missing-filled teeth (DMFT) in the pregnancy, other studies did not report
any remarkable increase in the rate of caries.[3]
[4]
There is a theory mentioned that it is possible that some dental and oral problems
might be related to the biochemical changes in the salivary composition, pH, and buffering
capacity.[5]
[6]
[7] Salivary compositions including proteins and some ions such as calcium and phosphorous
are important factors in the antimicrobial ability of oral cavity.[6]
Some studies showed that total salivary proteins in the pregnant women are more and
calcium and phosphorous levels are less than nonpregnant ones; also, from the first
to third trimesters of pregnancy, protein levels increase, and calcium and phosphorous
fall.[7] Although, another study showed that concurrent with a reduction in pH and neutral
capacity and calcium levels, an increase in phosphorous concentrations was observed
during pregnancy.[6] Moreover, in the third trimester, the flow rate is more in pregnant women; however,
pH and buffering capacity are less than nonpregnant subjects.[8] It seems that concurrent to elevated serum progesterone and estrogen in the pregnant
saliva, pH and buffering capacity decrease.[8] These mentioned changes during pregnancy lead to more enamel demineralization and
a decrease in the remineralization capacity of saliva.
Salivary α-amylase, which is the most important salivary protein, is secreted from
the acinar cell of salivary glands thorough activation of adeno β-receptor 1.[9] The production of α-amylase in the salivary gland is independent of the salivary
flow rate.[9]
[10] Because major salivary glands are innervated by parasympathetic and sympathetic
nerves, measuring salivary proteins such as α-amylase could be a marker for evaluating
autonomic activity.[9]
Some studies show that physical and psychological stress may cause an increase in
salivary α-amylase,[11] since pregnancy is a physiologically stressful event, it could be effective on salivary
composition.[9]
In respect to few studies that have been performed on salivary composition such as
sialic acid, pH, and α-amylase, as well as the differences of some biomarkers in various
races and populations, this study was aimed to assess these salivary markers in the
pregnant subjects in Zahedan city.
Methods and Materials
Thirty-five pregnant (case group) and 35 nonpregnant women (control group), who were
more than 20 years old, enrolled in this case-control study after signing informed
consent forms.
A urine pregnancy test and ultrasonography were used for confirming pregnancy.
Nonpregnant subjects, who were in the follicular phase of the menstrual cycle and
did not use oral contraceptive, enrolled as the control group. They were selected
among the patients who were referred to Zahedan Dental School for routine dental treatments.
Inclusion criteria for both groups were: not having any systemic diseases (especially
diabetes and hypertension), or mental or psychiatric problems, or periodontal or other
chronic diseases or oral lesions; not smoking, no history of abortion; and not using
any systemic drugs.
The check lists were filled out for all subjects, which included demographic data
consisting of age, sex, and the week of pregnancy (for case group).
Saliva Collection
The subjects were asked not to eat, drink, smoke, or brush their teeth for 90 minutes
before collecting saliva. First, all subjects washed their mouths with distilled water
and after resting for 5 minutes, unstimulated saliva was collected by spitting method
at 9 to 11 a.m.[12] After that, all women spat into 50 mL laboratory tubes every 60 seconds for 2 to
10 minutes. The salivary samples were stored with ice and sent to the laboratory,
then the saliva samples were centrifuged at 2,500 g for 10 minutes and were stored at –20°C until analysis time.[13]
In the pregnant group, saliva samples were collected for sialic acid and α-amylase
in the first trimester.
For the determination of sialic acid, a commercial sialic acid kit (Eastbiopharm Co,
China) was used. The salivary samples were centrifuged at 800 g for 10 minutes. After washing the sediment with saline, it was centrifuged at 11,000
g for 5 minutes. After separation of saline supernatant fraction, thiobarbituric acid
was added to the solution to complete this reaction, and sialic acid was assessed
by the method of Shoza and Mphos.[14]
For the assessment of salivary amylase, commercial amylase kit (Bionik Diagnostic
Systems, Iran) was used. The salivary α-amylase activity was evaluated using spectrophotometric
assay. The amount of p-nitrophenol formation as a product of hydrolysis reaction with
α-glucosidase was measured through the absorbance at the wavelength of 405 nm.[15]
For pregnant women, salivary samples were collected in three phases: the first trimester
(9th week), the second trimester (20th week), and the third trimester (36th week).
Salivary pH was assessed by a digital pH meter (Electronics India). The pH meter was
calibrated every day with prepared buffer solutions.
This study was approved by the ethical committee of Zahedan University of Medical
Sciences (code: IR;ZAUMS.REC.1395.162).
The collected data was entered into SPSS software (version 19) and the results were
statistically compared between two groups through t-test and analysis of variance (ANOVA) with a confidence interval of 95%. A significant
level of 0.05 was set in this study.
Results
Thirty-five pregnant (mean age: 25 ± 4.8 years) and 35 nonpregnant (mean age: 23 ±
2.5 years) women were enrolled in this study.
Subjects’ body mass index in the control group was between 20 and 20.9.
The pregnant subjects had an increase in weight of 10 to 12 kg during the pregnancy.
The Shapiro–Wilk test was used for assessment of normality of variables, and the distribution
of the data was considered normal, if p > 0.05.
In the present study, distribution of all data was normal for every three variables.
So, parametric tests such as t-test and ANOVA were selected for statistical analysis.
In the present study, there was not any significant difference between pregnant and
nonpregnant women, according to salivary α-amylase and sialic acid (p = 0.963 and 0.138, respectively) ([Table 1]).
Table 1
Comparison of salivary α-amylase, sialic acid, and pH between the case and control
groups
|
Variables
|
Mean ± SD
|
p-Valuea
|
|
Pregnant
|
Nonpregnant
|
|
Abbreviation: SD, standard deviation.
aIndependent t-test.
|
|
Pregnant
|
Nonpregnant
|
|
Alpha-amylase (U/L)
|
2.46 ± 1.86
|
2.43 ± 2.05
|
0.963
|
|
Sialic acid (mg/dL)
|
2.28 ± 1.23
|
2.74 ± 1.32
|
0.138
|
|
pH
|
6.86 ± 0.41
|
7.84 ± 0.43
|
˂ 0.001
|
As shown in [Table 1], a significant different was observed between the two studies groups, according
to salivary pH (p = 0.001).
Also, salivary pH significantly reduced in the pregnant subjects from the first to
the third trimester (p = 0.001) ([Table 2]).
Table 2
Comparison of salivary pH between three trimesters in the pregnant women
|
Trimester
|
Salivary pH
|
p-Valuea
|
|
Mean ± SD
|
|
|
Abbreviation: SD, standard deviation.
aRepeated measures analysis of variance (ANOVA).
|
|
First
|
7.47 ± 0.42
|
˂ 0.001
|
|
Second
|
6.86 ± 0.41
|
|
|
Third
|
6.56 ± 0.38
|
|
Discussion
This study was designed to compare salivary α-amylase, sialic acid, and pH in pregnant
and nonpregnant women.
There is a near correlation between saliva and plasma parameters. One of the most
important salivary proteins is α-amylase, which is mostly secreted from the parotid
gland. Previous studies showed various results. Bakhshi et al revealed an increase
in unstimulated salivary total protein, and a decrease in calcium and phosphorous
concentration[16]; however, the present study did not show any significant difference between the
subjects according to one of the salivary enzymes, α-amylase.
Moreover, Abrao et al reported more salivary α-amylase during pregnancy compared with
nonpregnant subjects,[17] although Rio et al similar to our study, did not find any remarkable difference
in this biomarker between pregnant and other women.[6]
On the other side, Al–Nuaimy and Al–Doski demonstrated that in the pregnant women,
salivary total protein, calcium concentration, and flow rate reduce, which is associated
with an increase in gingival inflammation index and Decayed, Missing, and Filled Teeth
(DMFT).[18]
The most important function of salivary amylase is hydrolysis of starch to maltose,
which could act as a substance for oral bacteria and leads to acid production; consequently,
this process accelerates demineralization of dental enamel.[19] It is confirmed that some factors could have an affect on secretion of α-amylase.
In the present study, α-amylase did not any significantly change during pregnancy.
The study done by Rio et al demonstrated an increase in salivary α-amylase during
pregnancy.[6] However, there was no significant relationship between the amount and activity of
α-amylase and the age of the pregnant women.[20]
[21] It is expressed that rising α-amylase in pregnancy is due to sympathetic activity
during physical and physiological stress.[9] It seems that pregnancy depression leads to an increase in salivary α-amylase activity
as compared to nondepressed pregnant women.[22]
History of early delivery, race, and women’s temper during pregnancy could be reasons
for contradictory results in the studies. Furthermore, psychological factors could
affect secretion of some enzymes.[9]
[21] Giesbrecht et al believe that the sex of the fetus, race of mother, and pregnancy
month is not related to daily α-amylase levels; however, history of miscarriage and
trait anxiety, momentary depression, and positive temper causes an increase in α-amylase
levels. Also, they expressed that chronic fatigue leads to a reduction in α-amylase.
It confirmed that α-amylase is sensitive to emotional stimulation.[21]
The difference between the present study and other studies could be because of mothers’
race and various laboratory methods for analyzing these biomarkers. In the present
study, the subjects did not have any recognized psychiatric problems. Since temper
problems have an affect on secretion of α-amylase, some different results are reasonable.
Also, in this study, there was no significant difference between pregnant women and
other subjects according to sialic acid. Another study reported an increase in sialic
acid during pregnancy.[23] Eliasson et al established that concurrent with rising estrogen after hormone therapy,
salivary labial flow rate and buffering capacity increase that leads to a decrease
in complaint of patients’ dry mouth.[24] It is logical to claim that some salivary alteration occurs due to hormonal changes
during pregnancy.
Salvolini et al reported an increase in sialic acid and a decrease in calcium and
phosphorus at weeks 21th and 40th with an elevated α-amylase at weeks 10 and 21 of
pregnancy. There is an opinion that concurrent with a decrease in salivary pH in the
third trimester, sialic acid increases to improve bacterial clearance, in fact, sialic
acid plays a protective role during pregnancy.[7]
In the present study, a decrease in salivary pH was detected during pregnancy, and
there is a decreasing trend, from the first to the third trimester. Moreover, another
study found a significant decrease in salivary pH in the third trimester as compared
with the first trimester in pregnant women. Also, it was revealed that salivary pH
during postpartum was lower than the control group.[25] Other studies found a decrease in pH and buffering capacity and an increase in gingival
inflammation and DMFT during pregnancy.[18]
[26]
There may be a correlation between gingival inflammation and some alteration in cytokines
such as the decrease in tumor necrosis factor-α levels in the third trimester.[27] Various reasons were mentioned for reducing pH, as well as decreasing buffering
capacity in several studies.
Plasma bicarbonate and CO2 of salivary glands are considered as the origins of salivary bicarbonate. During
pregnancy, salivary protein composition changes, also progesterone causes a reduction
in plasma bicarbonate, which leads to a decrease in pH and buffering capacity.[26] It is confirmed that the salivary flow rate and composition is affected by hormones,
although its exact mechanism is not clear. Rising estrogen during pregnancy is concurrent
with a reduction in sialic acid, pH, and buffering capacity.[26]
[28]
Naveen et al showed that salivary flow rate increases during pregnancy; however, pH
and buffering capacity decrease in pregnant women as compared with nonpregnant subjects.[26] They mentioned that rising salivary flow rate could be due to an increase in estrogen
and progesterone concentration. Moreover, reduced plasma HCO3 and increased α-amylase might lead to decreased salivary pH and buffering capacity
and prepare a suitable substrate for the acidogenic microorganisms.[26] The mentioned mechanism as well as an increase in number of daily meals and morning
sickness contribute to an increase in susceptibility of pregnant women to dental caries.[5]
[28]
Rio et al analyzed biochemical composition of the saliva during pregnancy and demonstrated
that unstimulated salivary pH was in the acidic range and stimulated salivary pH was
in the neutral range. They revealed that pregnancy causes a decrease in calcium levels
and an increase in phosphate levels.[6] Briefly, pregnancy changes oral biochemical environment and makes the mouth suitable
for some oral damages such as dental caries.[6]
It is recommended to evaluate the correlation between salivary pH and other biomarker
levels and oral and psychological alterations during pregnancy to reach comprehensive
results for the oral status during pregnancy.
Conclusion
The present study showed that salivary α-amylase and sialic acid levels do not change;
however, pH significantly decreases in pregnant women, and salivary pH has a reducing
trend in the last months of pregnancy. Alteration in salivary composition could increase
development of some oral conditions.
