The Journal of Maternal-Fetal & Neonatal Medicine

ISSN: 1476-7058 (Print) 1476-4954 (Online) Journal homepage: http://www.tandfonline.com/loi/ijmf20

The effects of probiotic supplementation on

biomarkers of inflammation, oxidative stress and
pregnancy outcomes in gestational diabetes

Bita Badehnoosh, Maryam Karamali, Mitra Zarrati, Mehri Jamilian,

Fereshteh Bahmani, Maryam Tajabadi-Ebrahimi, Parvaneh Jafari, Elham
Rahmani & Zatollah Asemi

To cite this article: Bita Badehnoosh, Maryam Karamali, Mitra Zarrati, Mehri Jamilian, Fereshteh
Bahmani, Maryam Tajabadi-Ebrahimi, Parvaneh Jafari, Elham Rahmani & Zatollah Asemi (2017):
The effects of probiotic supplementation on biomarkers of inflammation, oxidative stress and
pregnancy outcomes in gestational diabetes, The Journal of Maternal-Fetal & Neonatal Medicine,
DOI: 10.1080/14767058.2017.1310193

To link to this article: http://dx.doi.org/10.1080/14767058.2017.1310193

Accepted author version posted online: 22

Mar 2017.

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 The effects of probiotic supplementation on biomarkers of inflammation,

oxidative stress and pregnancy outcomes in gestational diabetes

Bita Badehnoosh1, Maryam Karamali2, Mitra Zarrati3, Mehri Jamilian4, Fereshteh Bahmani5,

Maryam Tajabadi-Ebrahimi6, Parvaneh Jafari7, Elham Rahmani8, Zatollah Asemi5,*

1
Department of Gynecology and Obstetrics, School of Medicine, Alborz University of Medical

Sciences, Karaj, Iran

2
Department of Gynecology and Obstetrics, School of Medicine, Iran University of Medical

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Sciences, Tehran, Iran
3

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Faculty of Nutrition, School of Public Health, Iran University of Medical Sciences, Tehran, Iran
4
Endocrinology and Metabolism Research Center, Arak University of Medical Sciences, Arak,
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Iran
5
Research Center for Biochemistry and Nutrition in Metabolic Diseases, Kashan University of
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Medical Sciences, Kashan, I.R. Iran

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6
Faculty member of Science department, science faculty, Islamic Azad University, Tehran

Central branch, Tehran, Iran

7
Department of Microbiology, science faculty, Islamic Azad University, Arak branch, Arak, Iran
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8
Department of Gynecology and Obstetrics, School of Medicine, Bushehr University of Medical

Sciences, Bushehr, Iran

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Running Title: Probiotic supplementation and gestational diabetes

* Corresponding Author. Research Center for Biochemistry and Nutrition in Metabolic Diseases,
Kashan University of Medical Sciences, Kashan, I.R. Iran. Tel: +98-31-55463378; Fax: +98-31-
55463377. E-mail addresses: [email protected] (Z.Asemi and M.Jamilian).
Number of words (Text): 3714

Number of words (Abstract): 175- Number of Tables: 5-Number of Figure: 1

ABSTRACT

Objective: This study was designed to evaluate the effects of probiotic supplementation on

biomarkers of inflammation, oxidative stress and pregnancy outcomes among subjects with

gestational diabetes (GDM). Methods: This randomized, double-blind, placebo-controlled clinical

trial was done among 60 subjects with GDM who were not on oral hypoglycemic agents. Patients

were randomly allocated to intake either probiotic capsule containing Lactobacillus acidophilus,

Lactobacillus casei and Bifidobacterium bifidum (2×109 CFU/g each) (n=30) or placebo (n=30)

for 6 weeks. Results: Compared with the placebo, probiotic supplementation resulted in

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significant decreases in fasting plasma glucose (FPG) (-5.3±6.7 vs. +0.03±9.0 mg/dL, P=0.01),

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serum high-sensitivity C-reactive protein (hs-CRP) (-2.2±2.7 vs. +0.5±2.4 µg/mL, P<0.001),

plasma malondialdehyde (MDA) concentrations (-0.1±0.8 vs. +0.5±1.5 µmol/L, P=0.03) and
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MDA/TAC ratio (-0.0003±0.0008 vs. +0.0009±0.002, P=0.004), and a significant increase in

total antioxidant capacity (TAC) levels (+65.4±103.3 vs. -37.2±143.7 mmol/L, P=0.002).
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Probiotic supplementation did not affect pregnancy outcomes. Conclusions: Overall, probiotic
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supplementation among women with GDM for 6 weeks had beneficial effects on FPG, serum hs-

CRP, plasma TAC, MDA and oxidative stress index, but did not affect pregnancy outcomes.
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Keywords: Probiotic supplementation, gestational diabetes, pregnant women

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Introduction

Gestational diabetes mellitus (GDM) is an increasing progress among pregnant women, which

affect up to 3 million US pregnant women every year [1]. The prevalence of this condition in US

[2] and Iran [3] was reported 5.8 and 4.7%, respectively. GDM might have adverse effects on

both mother and the baby, which are categorized into short term and long term morbidities; pre-

eclampsia and delivery by cesarean section are the short term outcomes of GDM , while

hypoglycemia, excessive adiposity, shoulder dystocia, and macrosomia are life threatening short

term consequences of GDM in newborns [4]. In addition, GDM women are exposed to a higher

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risk for the development of type 2 diabetes mellitus (T2DM) in their later years of life [5].

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Alteration in insulin resistance predisposes subjects with GDM to occurrence of inflammation

which leads to increased levels of inflammatory markers like high sensitive C-reactive protein
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(hs-CRP) [6]. The exact mechanisms which contribute to increased oxidative stress in

hyperglycemia, may include increased non-enzymatic glycosylation and auto-oxidative

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glycosylation along with the decreased antioxidant defense potential [7].

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It was reported that probiotics may effectively manipulate the human gut microbial composition

and function to reduce the adverse metabolic effects that are associated with pathogenic microbial
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communities [8]. Furthermore, the administration of probiotics during pregnancy in order to

improve maternal metabolic and pregnancy outcomes has been the topic of recent research [9-
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10]. Previously, the effects of probiotic supplementation on the biomarkers of oxidative stress

and inflammation have also been reported [11-12]. The beneficial effects of probiotics on

metabolic profiles may be due to improving insulin sensitivity [13], enzymatic deconjugation of

bile acids and conversion of cholesterol into coprostanol in the gut [14]. In addition, the

decreased inflammation and oxidative stress status by probiotic administration might be due to

their effects on increasing glutathione (GSH) levels [15], decreasing expression of inflammatory
cytokines in adipocytes and decreasing adiposity [16]. We have previously shown that the

multispecies probiotic supplementation for 8 weeks in diabetic patients resulted in a decrease in

serum hs-CRP and an increase in plasma GSH levels [11].

Pregnancy seems to be the most critical stage for any interventions willing to reduce the risk of

non-communicable diseases in future generations, beyond the immediate dangers imputable to

the health of the mother, labour and the neonate. Specific probiotic interventions during

pregnancy provide an opportunity to promote the health of both mother and the child [17].

Therefore, the aim of the present study was to investigate the effect of probiotic supplementation

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on biomarkers of inflammation and oxidative stress, and pregnancy outcomes among women

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with GDM, who were not treated with any pharmacological therapies.
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Methods

Trial design
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This study was a single-center, double-blind, placebo-controlled, randomized trial with maternal
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written consent.
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Participants

This randomized, double-blind, placebo-controlled, parallel clinical trial was done among 60
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patients with GDM who were referred to the Akbarabadi Clinic in Tehran, Iran, from April 2016

to September 2016. Eligible subjects were primigravida, aged 18-40 years (at weeks 24-28 of

gestation) who were diagnosed with GDM by a "one-step" 2-h 75-g oral glucose tolerance test

(OGTT). Diagnosis of GDM was done based on the American Diabetes Association guidelines

[18]: those whose plasma glucose met one of the following criteria were considered as having

GDM: fasting plasma glucose (FPG)≥92 mg/dL, 1 hour OGTT≥180 mg/dL and 2 hour
OGTT≥153 mg/dL. Patients with clinical characteristics at enrollment including placenta

abruption, pre-eclampsia, eclampsia, hypo and hyperthyroidism, urinary tract infection, smokers,

those with kidney or liver diseases and required commencing insulin therapy during intervention

were our exclusion criteria. Subjects were excluded from the study if they had taken any

probiotic products including probiotic yogurt and kefir during the trial.

Ethics statements

The study was approved by the Ethics Committee of the Iran University of Medical Sciences

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(IUMS). This trial was conducted in accordance with the Declaration of Helsinki Written and

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informed consent was obtained from all subjects. The present was registered at the Iranian

website for registration of clinical trials (www.irct.ir: www.irct.ir: IRCT201611115623N91).

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Study design
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At the onset of the study, all participants were categorized according to their baseline BMI (<25
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and ≥25 kg/m2) and age (<30 and ≥30 y). Then, participants in each block were randomly

allocated into two treatment groups to take either probiotic supplements (n=30) or placebo (n=30)
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per day for 6 weeks. Subjects were asked not to consume any probiotic-containing food, probiotic

yogurt or its products during the intervention. Although the duration of intervention was 6 weeks,
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all patients were followed up until the end of pregnancy. Patients were requested not to change

their ordinary physical activity or routine dietary intakes throughout the study and not to take any

supplements other than the one provided to them by the investigators. All patients based on

standard protocol consumed 400 µg/d of folic acid starting at the beginning of pregnancy and 60

mg/d ferrous sulfate as of the second trimester. Patients were requested to check their blood

glucose levels weekly (self-monitoring as daily) during the study. Cut-off for starting insulin
treatment was FPG>105 mg/dL and blood sugar 2-hour postprandial>120 mg/dL. For assessment

of dietary micro- and macro-nutrient intakes, patients were instructed to record their daily dietary

intakes for 3-day, including one weekend day and two weekdays at week 1, 3 and 5. Dietary

intakes were then analyzed using Nutritionist IV software (First Databank, San Bruno, CA)

modified for Iranian foods.

Intervention

Intervention group received a probiotic capsule per day containing Lactobacillus acidophilus,

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Lactobacillus casei and Bifidobacterium bifidum (2×109 CFU/g each) strains. Participants in the

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placebo group received capsules containing starch in a similar fashion. All capsules were

produced by the Tak Gen Zist Pharmaceutical Company in Tehran, Iran, and approved by the
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Food and Drug Administration.
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Treatment adherence
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Treatment adherence was examined after two weeks of supplementation by telephone interview

and also at weeks of 4 and 6 visits by counting the remained capsules. To determine the
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compliance the remaining supplements were counted and subtracted from the amount of

supplements provided to the participants. To increase compliance, all patients received short
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messages on their cell phones every day to remind them about taking the capsules by a clinical

personnel who was not directly involved in the participants care.

Assessment of anthropometric variables

Anthropometric measurements were done by a trained midwife at baseline and after the 6-week

intervention. Weight and height were measured by the Seca 713 scale without shoes and in light
clothing nearest 0.1 kg and 0.1 cm, respectively. BMI was calculated as the ratio of the current

body weight/height2 [kg/m2]. Weight and length of all newborns were measured in labor ward

following the birth by a trained midwife by the use of standard methods (Seca 155 Scale,

Hamburg, Germany). Infants' head circumference was calculated to the nearest 1 mm with a Seca

girth measuring tape. We also determined infants' 1- and 5-min Apgar score as another measure

of pregnancy outcome.

Outcomes

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In the present study, the primary outcomes measurements were and inflammatory markers. The

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secondary outcomes measurements were biomarkers of oxidative stress and pregnancy outcomes.
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Clinical assessment

Polyhydramnios was diagnosed using the sonographic estimation method at post-intervention. On

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the basis of this measurement, polyhydramnios was defined as an amniotic fluid index (AFI) in
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excess of 25 cm [19]. Preterm delivery was defined as delivery occurred at <37 weeks of

pregnancy and newborn's macrosomia was defined as birth weight of >4000 g. Large-for-
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gestational-age (LGA) births were live-born infants that were ≥90th percentile of birth weight

according to nomograms based on gender and gestational age from the latest standard [20].
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Assessment of biochemical variables

Five milliliter of fasting blood samples were obtained from each subjects at baseline and the end

of the study, at the IUMS reference laboratory which were immediately centrifuged (3000×g, 10

min, 4ºC); the plasma was then separated and placed into a tube and stored at -70oC until the

analysis for FPG, nitric oxide (NO), malondialdehyde (MDA), total antioxidant capacity (TAC),
and GSH. To determine FPG, we used enzymatic kits (Pars Azmun, Tehran, Iran). Serum for hs-

CRP levels were assessed using ELISA kit (LDN, Nordhorn, Germany) with intra- and inter-

assay coefficient variations (CVs) of 4.2 and 5.9%, respectively. The plasma NO by Griess

method [21], GSH by the method of Beutler et al. [22] and MDA levels by the thiobarbituric acid

reactive substance spectrophotometric test [23] were quantified. Plasma TAC concentrations

were determined using the ferric reducing antioxidant power method developed by Benzie and

Strain [24]. As MDA is the product of free-radical attacks on polyunsaturated fatty acids (PUFA),

and TAC reflects the physiologic effect to protect against this injury, we obtained an index of

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oxidative stress after dividing the individual values of MDA and TAC. CVs for plasma NO,

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TAC, GSH and MDA were lower than 5%. Newborns’ hyperbilirubinemia was considered when

the total serum bilirubin levels were at 15 mg/dL (257 mol/L) or more among infants who were
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25 to 48 hours old, 18 mg/dL (308 mol/L) in infants who were 49 to 72 hours old, and 20 mg/dL

(342 mol/L) in infants older than 72 hours [25].

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Sample size

We did not find a similar study about probiotic supplementation in GDM patients for determining
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the sample size based on main outcome (hs-CRP); therefore, the sample size was calculated

based on probiotic supplementation in pregnant women. However, the effects of probiotic

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supplementation on insulin metabolism and lipid profiles in GDM women have previously

evaluated [26], to the best of our knowledge, data on the effects of probiotic supplementation on

inflammatory factors in GDM women are limited. To calculate the sample size, we used the

standard formula suggested for parallel clinical trials by considering type one error (α) of 0.05

and type two error (β) of 0.20 (power=80%). Based on a previous study [27], we used a standard

deviation (SD) of 2.6 mg/L and a difference in mean (d) of 2.5 mg/L, considering hs-CRP as the
key variable. Based on this, we needed 25 persons in each group. Assuming 20% dropouts in

each group, the final sample size was determined to be 30 persons per group.

Randomization

Randomization assignment was done using computer-generated random numbers. Randomization

and allocation were concealed from the researchers and subjects until the final analyses were

completed. The randomized allocation sequence, enrolling patients and allocating them to

interventions were done by a trained midwife at the gynecology clinic.

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Statistical analysis

We applied the Kolmogorov-Smirnov test to assess normal distribution of variables. Changes in

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general characteristics and dietary intakes between the two groups were compared using an

independent sample t-test. Differences at the onset of the study and the end of the intervention
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were determined by the paired t test. To determine the effects of probiotic on biomarkers of
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inflammation and oxidative stress, we used one-way repeated measures analysis of variance. To

control some confounding variables including baseline values, maternal age and baseline BMI,
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we used ANCOVA test using general linear models. Differences in proportions were evaluated

by Fisher’s exact test. P<0.05 was considered as statistically significant. All statistical analyses
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were conducted using the SPSS Software (version 18.0, SPSS Inc., Chicago, Illinois, USA).

Results

As revealed in the study flow diagram (Fig. 1), 60 participants [probiotic (n=30) and placebo

(n=28)] completed the trial. On average, the compliance rate in the current study was high, such

that 100% of capsules were consumed throughout the study in both groups.
The mean age, height, baseline weight and BMI as well as their means after the 6-week

intervention were not significant between probiotic supplements and placebo groups (Table 1).

Based on the 3-day dietary records obtained baseline, end-of-trial and throughout the trial (week

3 and 5 of the treatment), we observed no significant change in dietary macro- and micro-nutrient

intakes between the two groups (Table 2).

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After 6 weeks of intervention, probiotic administration, compared with the placebo, resulted in

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significant decreases in FPG (-5.3±6.7 vs. +0.03±9.0 mg/dL, P=0.01), serum hs-CRP (-2.2±2.7

vs. +0.5±2.4 µg/mL, P<0.001), plasma MDA concentrations (-0.1±0.8 vs. +0.5±1.5 µmol/L,
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P=0.03) and MDA/TAC ratio (-0.0003±0.0008 vs. +0.0009±0.002, P=0.004), and a significant

increase in TAC levels (+65.4±103.3 vs. -37.2±143.7 mmol/L, P=0.002) (Table 3).
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Supplementation with probiotic showed no detectable changes in plasma NO and GSH levels.
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There was a significant difference in baseline levels of TAC (P=0.02) between the two groups.
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Therefore, we adjusted he analysis for baseline values of biochemical variables, maternal age and

BMI at baseline. When we adjusted the analysis for baseline values of biochemical parameters,
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maternal age and baseline BMI, plasma MDA (P=0.05) became non-significant, and other

findings did not alter (Table 4). In addition, when we controlled the analysis for BMI at baseline,

gestational weight gain and baseline values of FPG, plasma MDA (P=0.08) became non-

significant, and other findings did not alter.

We did not find a significant difference in cesarean section rate, need of insulin therapy after

intervention, polyhydramnios, maternal hospitalization, gestational age, newborn's birth size,

Apgar scores, incidence of hyperbilirubinemia newborns and newborns' hospitalization, when

comparing the two groups (Table 5).

Discussion

This study demonstrated that the 6-week intervention of probiotic supplements among women

with GDM had beneficial effects on FPG, serum hs-CRP, plasma TAC, MDA and oxidative

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stress index, while did not affect plasma NO, GSH levels and pregnancy outcomes. To our

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knowledge, this is the first trial that examined the effects of probiotic supplementation on

biomarkers of inflammation and oxidative stress, and pregnancy outcomes in GDM women. It
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must be taken into account that there was a significant difference in plasma TAC levels between

the probiotic and the placebo groups at study baseline. The diagnosis of GDM in the current
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study was done based on the criteria of the American Diabetes Association. Furthermore, we did
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not randomize patients based on their TAC levels or other biomarkers of inflammation and

oxidative stress because all participants had GDM. Random assignment to two groups was done
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after stratification for pre-intervention BMI (<25 and ≥25 kg/m2) and age (<30 and ≥30 y) and

random assignment was done by the use of computer-generated random numbers. Therefore, the
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difference in TAC between the two groups was occurred by random. In addition, when we

adjusted the analyses for BMI at baseline, gestational weight gain and baseline values of FPG, no

significant changes in our findings were observed except for plasma MDA levels.

Complications during pregnancy are associated with several adverse outcomes for mother and

newborns in the short and long term [4]. In addition, alterations in the gut and vaginal
microbiome [28] might affect the maternal metabolic profiles, biomarkers of inflammation and

oxidative stress which contribute to the metabolic and immunological health of the offspring

[29]. High levels of reactive oxygen species during embryonic, fetal and placental development is

a feature of pregnancy; consequently, oxidative stress has emerged as a likely promoter of several

pregnancy-related disorders, such as embryopathies, spontaneous abortions, preeclampsia, fetal

growth restriction, preterm labor and low birth weight [30]. Oxidative stress not only causes

much pathopysiological complication but also is linked to insulin resistance which in turn results

in diminished glucose uptake in peripheral tissues and increasing glucose production in the liver

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[31].

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Our study demonstrated that the 6-week intervention with probiotic supplements compared with
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placebo, resulted in significant decreases in serum hs-CRP, plasma MDA concentrations and

MDA/TAC ratio, and a significant increase in TAC levels, but did not significantly affect the
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serum NO and GSH levels. When we compared the oxidative stress index (MDA/TAC) between
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the two groups, a significant change was observed. This confirms that patients with GDM in the

placebo group have higher oxidative stress as evidenced by an elevation of MDA/TAC index due
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to an increment of MDA and a reduction of TAC. We propose that the MDA/TAC index may be

a good indicator of oxidative stress in GDM patients compared with healthy individuals. In
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accordance with our results, Jafarnejad et al. [32] showed that a mixture of probiotic (VSL#3)

supplements influenced the inflammatory markers including hs-CRP in women with GDM after 8

weeks. Some studies have found that high serum hs-CRP levels during pregnancy were inversely

associated with insulin resistance [33-34]. Furthermore, consumption of 200 g/day yogurt,

enriched by Lactobacillus acidophilus, Bifidobacterium langum, and Lactobacillus casei 108

CFU/g among overweight and obese persons after 8 weeks decreased inflammatory factors [35].
Kullisaar et al. [36] also reported that goat milk fermented by Lactobacillus fermentum ME-3

increased TAC and decreased lipid peroxidation markers in healthy persons. Likewise, Harisa et

al. [37] showed that treatment with Lactobacillus acidophilus alone or in combination with

acarbose resulted in a significant decrease in MDA concentrations in diabetic rats. Previous

studies have also shown that special strains of lactic acid bacteria have antioxidant properties [38-

39]. In line with our findings, an animal study of Yadav et al. [13] showed that probiotic dahi not

only decreased the oxidative damage but also increased the antioxidant content and activities of

catalase, glutathione peroxidase and superoxide dismutase in diabetic rats. However, no

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significant change in MDA and TAC levels was seen following the consumption of capsule

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containing 108 CFU/g of Lactobacillus casei among RA patients for 8 weeks [40]. Different

findings of the present study compared with the other ones might be mediated by different study
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designs, different species and dosage of used probiotics as well as the different periods of

interventions. Produced short chain fatty acids (SCFA) by probiotics can result in decreased
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enzymatic synthesis of hepatic CRP [16]. SCFA may lower serum hs-CRP levels through
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blocking the enzymatic synthesis of hepatic CRP. CRP is synthesized by the liver in response to

releasing factors by fat cells such as interleukin 6 (IL-6) [41]. In a study by Hegazy et al. [16]
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was observed that the consumption of probiotic in patients with ulcerative colitis for 8 weeks

significantly ameliorated the inflammation by decreasing concentrations of IL-6, expression of

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tumor necrosis factor-alpha (TNF-α) and nuclear factor kappa-light-chain-enhancer of activated

B cells (NF-κB). Likely, decreasing concentrations of IL-6 indirectly causes decreasing

production of CRP. In addition, decreased serum CRP concentrations might result from

decreased expression of inflammatory factors [16]. The precise mechanisms involved in the

antioxidative effects of probiotics remain largely unknown; these effects may be partly related to

reactive oxygen species scavenging, metal ion chelation, enzyme inhibition, and the reduction
activity and inhibition of ascorbate autoxidation [38]. Probiotics from lactic acid bacteria family

can be potential candidates for the production of functional foods or natural antioxidant

supplements [42]. In addition, some probiotics result in increased activity of antioxidative

enzymes including glutathione-S-transferase, glutathione reductase, glutathione peroxidase,

superoxide dismutase and catalase or modulation of circulatory oxidative stress [43]. Probiotics

also exert their defensive effects against oxidative stress by re-establishment of the gut flora [44].

On the other hand, metabolic activities of probiotics may have shown the antioxidative effect

through the scavenging of oxidant compounds or the prevention of their generation into the

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intestine [45]. Production of bioactive peptides by probiotics has also been considered an

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effective mode of antioxidative activity in foods containing probiotic bacteria [46].
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This study revealed that supplementation with probiotic among GDM women for 6 weeks did not

affect pregnancy outcomes. However, compared with the placebo, probiotic supplementation
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resulted in a decrease in cesarean section rate, but was not significant. This may be related to the
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blood glucose and oxidative stress control of patients in the probiotic group. We did not collect

blood samples at delivery. Therefore, we could not judge about the effects of probiotic on
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pregnancy outcomes due to the effects on blood glucose and oxidative stress at delivery. On the

other hand, in the current study, the intervention was ended around 34 weeks of gestation and
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most women were delivered around 39 weeks of gestation leaving a 5 week window where

women did not receive any intervention. Supplementation with longer duration of probiotic may

result in significant improvement in pregnancy outcomes. In a study, any statistical significant

effects was not found after the ingestion of Lactobacillus in early pregnancy, including the

number of spontaneous abortions, pre-term births and low birth weight newborns [47]. In our

previous study, we did not show any significant alterations in the incidence of newborns’
hyperbilirubinemia and cesarean section following supplementation with synbiotic containing

Lactobacillus sporogenes and inulin in pregnant women [48]. Furthermore, a recent meta-

analysis showed that consumption of probiotic among pregnant women after week 36 of

gestation did not affect the gestational age at birth, the incidence of caesarean section and birth

weight [49]. A few studies which assessed the effect of probiotics on the amount of bilirubin

levels have reported a reduction in the required length of phototherapy. These results were in

agreement with the findings of Demirel et al.[50] who proved that a daily

Saccharomyces boulardii supplementation at a dosage of 250 mg among infants with a

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gestational age of ≤32 weeks and a birth weight of ≤1500 g, could reduce their serum bilirubin

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concentration and the duration of phototherapy. It must be kept in mind that there were no

differences in the rates of pre-eclampsia in the current study. This may be due to the changes in
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oxidative stress markers induced by the intervention which were not sufficient to alter the rates of

pre-eclampsia. The rates of neonatal hospitalization were identical to the incidence of

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hyperbilirubinemia. This may be due to jaundice that is the main reason for hospitalization in
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these infants.
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The current study had few limitations. The sample size was not large enough to report more

detailed outcomes. Future studies with longer duration of the intervention, and larger sample
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sizes are needed to confirm our findings. In addition, we did not assess the effects of probiotic

supplementation on other pregnancy outcomes including the infant respiratory status and the time

in neonatal intensive care unit. The effects of probiotic on metabolic profiles including lipid

profiles were beyond the scope of this project and we haven’t had funding to do so. It must be

considered that the compliance rate in the current study was high, such that 100% of capsules

were consumed throughout the study in both groups. Due to funding limitations, we did not
assess the compliance through quantifying fecal bacteria loads and SCFA. Therefore, this should

be taken into account in the interpretation of our findings.

Overall, probiotic supplementation among women with GDM for 6 weeks had beneficial effects

on FPG, serum hs-CRP, plasma TAC, MDA, oxidative stress index, cesarean section, incidence

of newborn's hyperbilirubinemia and newborns' hospitalization.

Acknowledgement

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The present study was founded by a grant from the Vice-chancellor for Research, IUMS, Tehran,

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Iran. EP
Conflicts of interest

No conflicted.
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Authors' contributions: BB, MJ, MZ, MJ, FB, MT-E, PJ and ER contributed in data collection

and manuscript drafting. ZA assisted in conception, design, statistical analysis and drafting of the
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manuscript. All authors confirmed the final version of the paper.

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Clinical trial registration number

http://www.irct.ir: www.irct.ir: IRCT201611115623N91.

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Table 1. General characteristics of the study participantsa

Placebo group Probiotic group Pb

(n=30) (n=30)
Maternal age (y) 27.8±3.7 28.8±5.4 0.40

Height (cm) 162.2±4.4 162.1±5.1 0.91

Weight at study baseline (kg) 74.5±7.6 74.2±9.5 0.89

Weight at end-of-trial (kg) 77.4±7.5 77.4±9.1 0.98

Weight change (kg) 2.9±0.9 3.2±1.8 0.35

Weight at delivery (kg) 80.2±7.3 80.4±8.7 0.84

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Weight gain during pregnancy (kg) 11.6±0.8 11.9±1.4 0.25

BMI at study baseline (kg/m2) 28.4±3.6 28.3±3.9 0.92

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BMI at end-of-trial (kg/m2) 29.5±3.6 29.6±3.7 0.96

BMI change (kg/m2) 1.1±0.3 1.2±0.7 0.33

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Gestational age before intervention (weeks) 25.6±1.2 25.7±1.0 0.81
a
Data are means± standard deviation.
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b
Obtained from independent t test.
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 Table 2. Dietary intakes of study participants throughout the study

Placebo group Probiotic group (n=30) Pa

(n=30)
Energy (kcal/d) 2521.6±173.5 2545.9±153.7 0.56
Carbohydrates (g/d) 347.7±35.3 361.8±40.5 0.15
Protein (g/d) 90.5±11.7 90.3±12.4 0.95
Fat (g/d) 89.0±12.8 85.7±13.2 0.34
SFAs (g/d) 26.4±5.0 26.4±4.9 0.95
PUFAs (g/d) 27.5±6.9 27.8±6.5 0.85
MUFAs (g/d) 24.2±6.6 24.0±5.2 0.89

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Cholesterol (mg/d) 212.6±97.5 201.4±91.0 0.64

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TDF (g/d) 19.9±4.7 20.8±5.5 0.48
Data are means± SDs.
a
Obtained from independent t test.
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SFAs, saturated fatty acid; PUFAs, polyunsaturated fatty acid; MUFAs,
monounsaturated fatty acid; TDF: total dietary fiber.
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Table 3. Biomarkers of inflammation and oxidative stress at the study baseline and after 6-wk
intervention in women with GDM that received either probiotic supplements or placeboa

Placebo group (n=30) Probiotic group (n=30)

Wk0 Wk6 Change Wk0 Wk6 Change Pb

FPG 91.8±7.5 91.8±8.7 0.03±9.0 94.0±5.5 88.7±7.1 -5.3±6.7 0.01

(mg/dL)

hs-CRP 6.5±3.8 7.0±3.9 0.5±2.4 6.7±2.0 4.5±2.4 -2.2±2.7 <0.001

(µg/mL)

NO 46.8±20.1 45.2±26.9 -1.5±22.8 43.5±2.3 43.0±2.1 -0.5±3.1 0.81

(μmol/L)

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TAC 872.6±245.6 835.4±255.7 -37.2±143.7 985.2±93.0 1050.5±119.7 65.4±103.2 0.002

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(mmol/L)

GSH 422.5±104.2 382.8±126.6 -39.7±130.6 414.4±66.6 409.8±39.0 -4.6±67.0 0.19

(µmol/L)
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MDA 3.5±1.3 4.0±1.7 0.5±1.5 3.5±0.8 3.4±0.8 -0.1±0.8 0.03
(µmol/L)
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MDA/TAC 0.004±0.001 0.005±0.002 0.0009±0.002 0.003±0.0008 0.003±0.0008 -0.0003±0.0008 0.004
ratio
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a
All values are means± SDs.
b
P values represent the time × group interaction (computed by analysis of the repeated measures ANOVA).
FPG, fasting plasma glucose; GDM, gestational diabetes mellitus; GSH, total glutathione; hs-CRP, high-
sensitivity C-reactive protein; MDA, malondialdehyde; NO, nitric oxide; TAC, total antioxidant capacity.
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Table 4. Adjusted changes in biomarkers of oxidative stress and inflammation in women with
GDM that received either probiotic supplements or placeboa

Placebo group Probiotic group Pb

(n=30) (n=30)
TAC (mmol/L)

Model 1* -50.2±23.4 78.3±23.4 <0.001

Model 2** -40.5±23.3 68.7±23.2 0.002

GSH (µmol/L)

Model 1* -37.3±16.6 -6.9±16.6 0.20

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Model 2** -43.3±19.3 -0.9±19.3 0.13

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MDA (µmol/L)

Model 1* 0.5±0.2 -0.1±0.2 0.05

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Model 2** 0.5±0.2 -0.1±0.2 0.08

hs-CRP (µg/mL)
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Model 1* 0.5±0.5 -2.1±0.5 <0.001

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Model 2** 0.5±0.5 -2.1±0.5 <0.001

NO (μmol/L)
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Model 1* -0.8±2.9 -1.3±2.9 0.90

Model 2** -2.1±3.0 0.04±3.0 0.61

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MDA/TAC ratio

Model 1* 0.001±0.00 0.00±0.00 0.007

Model 2** 0.001±0.00 0.00±0.00 0.008

a
All values are means± SE.
b
Obtained from ANCOVA.
*
Model 1: Adjusted based on maternal age, BMI at baseline and baseline values of biochemical
parameters.
*
Model 2: Adjusted based on BMI at baseline, BMI, gestational weight gain and baseline values of
FPG.
GDM, gestational diabetes mellitus; GSH, total glutathione; hs-CRP, high-sensitivity C-reactive
protein; MDA, malondialdehyde; NO, nitric oxide; TAC, total antioxidant capacity.

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Table 5. The association of probiotic supplementation with pregnancy outcomes

Placebo group Probiotic group Pa

(n=30) (n=30)
Cesarean section (%) 14 (46.7) 6 (20.0) 0.054†

Preterm delivery (%) 1 (3.3) 2 (6.7) >0.999†

Need to insulin therapy after intervention 3 (10.0) 2 (6.7) >0.999†

(%)
Pre-eclampsia (%) 2 (6.7) 2 (6.7) >0.999†

Polyhydramnios (%) 1 (3.3) 0 (0) >0.999†

Maternal hospitalization (%) 2 (6.7) 0 (0) 0.492†

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Macrosomia >4000 g (%) 3 (10.0) 0 (0) 0.237†

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Gestational age (weeks) 39.1±1.1 39.1±2.5 0.948

Newborns' weight (g) 3438.0±398.4 3321.7±443.5 0.290

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Newborns' length (cm) 51.2±1.9 50.4±2.8 0.223

Newborns' head circumference (cm) 36.0±1.5 35.8±1.8 0.624

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LGA (%) 9 (30.0) 5 (16.7) 0.360†

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1- min Apgar score 8.93±0.25 8.96±0.18 0.561

5- min Apgar score 9.93±0.18 9.96±0.18 0.561

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Newborns' hyperbilirubinemia (%) 8 (26.7) 2 (6.7) 0.080†

Newborns' hospitalization (%) 8 (26.7) 2 (6.7) 0.080†

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Newborns' hypoglycemia (%) 3 (10.0) 2 (6.7) >0.999†

Values are means± SDs for continuous measures and are number (%) for dichotomous variables.
b
Obtained from independent t test.
†
Obtained from Fisher’s exact test.
LGA, large for gestational age.
 Legend to figure:

Fig. 1. Summary of patient flow diagram.

Screening for GDM among

pregnant women (n=880)

Excluded (n=800)
- Had not GDM (n= 791)
- Needed to start insulin therapy (n= 9)

Assessed for eligibility among

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GDM women (n=80)

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Enrollment

Excluded (n=20)
- Not meeting inclusion criteria (n= 12)
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Randomized (n=60)
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Allocation

Allocated to placebo (n=30) Allocated to intervention (n=30)

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Follow-up

Lost to follow-up (n=0) Lost to follow-up (n=0)

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Analysis

Analyzed (n=30) Analyzed (n=30)