The effect of probiotics on gestational diabetes and its complications in pregnant mother and newborn: A systematic review and meta‐analysis during 2010–2020

Abstract This study was aimed to evaluate the effect of probiotics consumption on gestational diabetes (GD) and its complications in pregnant mother and newborn. The study was registered on PROSPERO (CRD42021243409) and all the enrolled articles were collected from four databases (Medline, Scopus, Embase, and Google Scholar) as randomized controlled trials (RCTs) from 2010 to 2020. A total of 4865 study participants from 28 selected studies were included in this review. The present meta‐analysis showed that the consumption of probiotics supplementation has the potential to decrease GD‐predisposing metabolic parameters such as blood glucose level, lipid profile, inflammation, and oxidative markers which may reduce GD occurrence among pregnant women.


| INTRODUC TI ON
Gestational diabetes (GD) refers to glucose intolerance in pregnant women at 24-28 weeks without a history of diabetes that result in hyperglycemia. Pregnant women who suffer from GD show various symptoms including unusual thirst, frequent urination, frequent infections, and weight gain. 1 Lack of mobility and overweight are the main predisposing factors for GD which occurs in 17% of all pregnancies worldwide with a 10-100% increase in rate during the last 20 years. In recent years, Middle East, North Africa, and Europe have had the highest (12.9%) and lowest (5.8%) prevalence of GD, respectively. 2,3 GD can expose the health of the mother and the fetus at risk by complications including neonatal hypoglycemia, polycythemia, respiratory distress, hypocalcemia, gestational hypertension, pre-eclampsia, increased cesarean section rate, 4,5 and type 2 diabetes mellitus (T2DM), as long-term adverse outcome and the most commonly reported complication. 6,7 Therefore, it is important to prevent and control diabetes during pregnancy.
Defects in carbohydrate and lipid metabolism which some researchers have attributed to microbiome changes, as well as genetic disorders play an important role in the development of GD. During pregnancy, secretion of leptin and inflammatory cytokines such as IL-6 and TNFα is directly related to oxidative damages and the levels of estrogen and progesterone as placental hormones, which in turn leads to increased insulin resistance and eventually the development of GD. 1,5,8 Several approaches are suggested to control glucose levels during GD such as insulin injection, changes in lifestyle (diet and exercise), oral medications (e.g., metformin), and consumption of probiotics and vitamin D, although in many cases these strategies may not work. 6,9 Probiotics (Lactobacillus and Bifidobacterium spp.) are live microorganisms that, if prescribed properly, will have significant effects on human health. 10 Probiotics have proven effective in many clinical applications such as the treatment of enterocolitis, diarrhea and cancers. 10 Consumption of food products that carry probiotics, not only show to prevent food spoilage and growth of pathogens, but also have been effective in increasing the quality, taste, and appearance of foods. Probiotics included in the diet of broilers and laying animals led to an increase in growth of pigs, cows, broilers, and their products (egg yolks and milk production). In addition to increasing the percentage of proteins, probiotics in food industry leads to an improvement in color of meats and pH, reducing oxidative stress and lipid oxidation. 11 Interestingly, dairy products are good source for containing probiotics (such ice cream containing Lactobacillus acidophilus and inulin) and improvement of gastrointestinal tract. 12 Gut health was achieved by using probiotics in foods that results in an increase number of probiotic bacteria, reduces the number of fecal pathogens such as coliforms and staphylococci spp, and improves the quality of feces (in terms of the presence of water and reduced acidity). 13 Some study suggests that probiotics are able to overcome insulin resistance in pregnant women with GD by consuming the blood sugar as energy source, improving lipid metabolism in the gut, increasing glutathione (GSH) levels, and reduction of the inflammatory biomarkers like high sensitive C-reactive protein (hs-CRP) and oxidative stress. 5,7 Through pregnancy, the inflammatory conditions of the body affect the number of bacteria such as Bifidobacterium and Bacteroides and their balance in the body, while probiotics are able to induce gut microbiome to reduce the effects of metabolic defects. In addition, microbial imbalance in women with GD which is known as "Gut microbiome dysbiosis," includes an increase in the number of Bacteroides spp. and a decrease in the number of Bifidobacterium spp. and is the main cause of overweight among pregnant woman, while their hypertension which may be related to low dietary fiber. 9,14,15 Moreover, the use of probiotics during pregnancy is not harmful and is well tolerated in the body. 4 Although choosing the best probiotic and the optimal dose for the treatment of GD requires more studies, Bifidobacterium and Lactobacillus spp.
have been commonly used in studies with more than 10 7 CFU/ ml daily, as the suggested dose needed to achieve desirable results on the reduction of metabolic dysfunction. 8,16 Here, the aim of this study was to investigate the inhibitory effects of probiotics supplementation on GD among pregnant women based on Randomized Controlled Trial (RCT) studies during in the last 10 years (2010-2020).

| Guidelines
This systematic review and meta-analysis was performed according to the PRISMA 2020 guidelines (File S1). 17 The study was registered on PROSPERO (CRD42021243409).

| Information sources and search strategy
Data from the four international information databases Medline, Scopus, Embase, and Google Scholar were searched during 2010-2020. The search strategy was based on the combination of the following terms: "gestational diabetes" and "probiotics." The search items in each database are also available in the File S2.

| Inclusion and exclusion criteria
RCTs were included if they were well-described, had high quality, and defined outcomes, investigating the effects of probiotics on pregnant women. Non-English articles, nonhuman trials, nonfull text studies, duplicate reports, and trials with insufficient data were excluded from the study. Figure 1 summarizes the search strategy.

| Data extraction and quality assessment
Data were screened and analyzed independently by two authors, and any discrepancies were discussed to obtain consensus. Reference lists of all the related publications were also investigated to find any ignored articles. The publications cited in more than one database were included only once. A third researcher checked the results to ensure that all the eligible articles were evaluated. The initial phase of article selection consisted of the analysis of titles, abstracts, and finally reading the studies to select them based on the eligibility criteria. The information extracted from each study included the first author's last name, country of investigation, sample size (intervention/control), mean age and mean weigh of participants, study design, participants characteristics, intervention (probiotics), probiotics species, intervention dose, period of intervention, duration of following up, and outcome. The quality of the references was evaluated using the Joanna Briggs Institute (JBI; The Joanna Briggs Institute, 2014). 18 RCTs were used to perform the quality assessment. Each component was rated as "yes," "no," "unclear," or "not applicable." A score ranging from 0 to13 points was attributed to each study. Ultimately, the studies with high quality were included in the present meta-analysis. The File S3 shows JBI quality assessment.

| Data analysis
Publication bias (Small study effect) was evaluated using Egger's linear regression test. 19 To mean differences were estimated to compare the outcomes between intervention and control group, and a random-effects model was used to pool results. The statistical analyses were performed using STATA software, version 16.0 (STATA Corporation, College Station, Texas, USA). Heterogeneity between studies was assessed by a Chi squared test and I 2 statistic.
All the statistical interpretations were reported on a 95% confidence F I G U R E 1 Flow diagram of literature search interval (CI) basis. p-values less than 0.05 were considered as statistically significant.

| Search results
A total of 8629 articles were collected by searching the four electronic databases, among which 3145 were excluded due to duplication. After title, abstract, and full text assessment, 28 publications were retained for meta-analysis ( Figure 1).

| Characteristics of the included studies
The methodological quality of the included studies was high for the RCT studies. (File S3). The age range of the pregnant women undergone by probiotics treatment was 18-40. Other factors regarding the included articles are shown in Table 1. According to Blood was the most prevalent specimen obtained from pregnant women in the articles. As shown in Table 2, among the 28 clinical   trials included, 25 (88%) evaluated the effect of probiotics on GD,   while 3 (11%) examined the synbiotics effects. Generally, among a total of 19 different species used as probiotics in the studies (Figure 3), Lactobacillus acidophilus (59.25%) and Bifidobacterium lactis Bb12 (37.03%) were the two most widely used probiotic species. The mean daily dosage of probiotics used for the treatment of GD among different studies was determined as 4.63 × 10 7 colony forming units (CFU), with a minimum and maximum range of 10 5 -4.5 × 10 14 CFU, respectively. This probiotic dosage totally lasted 4 to 40 weeks and in a few cases 9 month after Mother-related secondary measures (also known as maternal measures) included changes in the lipid profiles, inflammatory markers and oxidative stress, preeclampsia, gestational hypertension, hypertensive disorders of pregnancy, incidence of caesarean delivery, excess weight gain, and change in the prevalence of probiotic bacteria in the gut microbiome. Infant-related secondary measures (or neonatal measures) included bone fracture, stillbirth or neonatal death, gestational age at delivery (weeks), the incidence of macrosomia, the incidence of preterm delivery, the incidence of newborns' hyperbilirubinemia, and the incidence of newborns' hypoglycemia. 20

| Effects of probiotic supplementation on the metabolic status of pregnant women
Among the 28 studies included in the current meta-analysis, only 16 contained meta-analysis able data on the effect of probiotics (or synbiotics) supplementation on metabolic (biochemical) parameters.
The data are gathered in Table 2. In all these studies, blood samples were collected from volunteers following probiotics/synbiotics supplementation, at the beginning and the desired week of the trial after which the analysis of metabolic parameters were carried out according to the specified protocols. Metabolic parameters in these studies can be classified in three distinct groups; glycemic status, lipid profiles and inflammatory markers and oxidative stress. These biochemical parameters are regarded in this meta-analysis as primary  According to Figure 6C, there are significant heterogeneity correlations (I 2 = 100%, p = 0.00) among the 5 corresponding studies used in this meta-analysis.

| Colostrum adiponectin levels
Adiponectin, as a protein hormone, plays important roles in obesityassociated diseases such as type 2 diabetes when present at low levels in serum. 25

| Microbiome, maternal and neonatal health
Among the studies included in this meta-analysis, two examined the effect of probiotics on microbiome population, specifically intestinal microbiome, among pregnant women. 15 Because the composition of the microbial flora changes during obesity and overweight, these changes can affect blood pressure and inflammation. For this purpose, the amount of plasminogen activator 1 inhibitor in obese pregnant women was measured in some studies.

TA B L E 2
The outcomes of different clinical trials assessing the probiotics efficacy on gestational diabetes among pregnant women  and the health status of the mother. Probiotic consumption can cause metabolism regulation in mother, leading to a reduction in GD incidence, which can, ultimately affect the health status of the neonate. 26

| Risk of bias assessment
The mean differences and results of the Egger test are displayed in Figure 8 and Table 3. There was a publication bias in the metaanalysis of the FBS, TG, and CRP groups (p < 0.05).

| Level of evidence
The level of evidence based on GRADE was shown in Table 2.
Certainty of results assessed in this meta-analysis/systematic review "The Effect of Probiotics on Gestational Diabetes and its complications in pregnant mother and newborn" were considered as effective and represent low risk of bias. Almost all trials (twenty-five) showed probiotics have improvement effect during pregnancy and early infancy, except for three trials which announced that probiotics has no effect on GD. 9  This meta-analysis revealed that taking probiotic supplements during pregnancy by women with GD has beneficial effects on the metabolic status, colostrum adiponectin levels, microbiome composition, and the maternal and infant health. However, 4 studies reported no significant effect for the probiotic intervention on the incidence of GD. 27,28,30,31 The difference in the results of these studies may be due to different studies included in the meta-analyses, and also the different method of statistical analysis.

| DISCUSS ION
Probiotic supplements may contain either one strain of bacteria or a mixture of two or more strains/species. Studies have shown that the use of multi-strain and/or multi-species probiotics may in some cases be more effective than single-strain probiotics because multiple strains/species may synergistically augment the effects of each other. 32 The administration of synbiotics rather than probiotics seems to increase the overall beneficial outcome due to the synbiotics ability to improve the viability of the probiotic bacteria by supplying them with energy and nutrients. Among the studies investigated in this meta-analysis, the two species Lactobacillus acidophilus and Bifidobacterium lactis Bb12 were widely used to treat women with GD and to better control their metabolic status through pregnancy. Although the optimal probiotics dosage is not yet clear, it is generally accepted that a probiotic dose of >10 6 CFU/g (CFU/mL) can render highly efficient outcome. 36 In trials included in this study, women with GD received daily probiotic doses from 10 5 to 10 14 CFU during pregnancy or after deliver and an average dose of ≥10 7 CFU/day was rec-  cytokines interfere with insulin signaling related to insulin resistance in women with GD resulting in increased inflammatory markers such as C-reactive protein. 54 Several studies have shown that probiotics can increase the activity of anti-oxidative enzymes or modulate the circulatory oxidative stress in women with GD. 7,8,21,41 This review showed a significant increase in MDA and TAC plasma levels, but did not have a substantial impact on GSH, NO, and CRP levels in women with GD after taking probiotics. Only 1 of the 28 trials in- Adiponectin is an adipocyte-derived polypeptide hormone which, following binding to its receptor in the hypothalamus, exerts its anti-diabetic via regulation of glucose and lipid metabolisms. 63 It reduces insulin sensitivity by increase of glucose utilization and fatty acid oxidation in skeletal Muscles and liver and to improve glucose tolerance by decreasing hepatic gluconeogenesis, independent of AMPK, decreasing glucose production and improving glycemia control. 64,65 Adiponectin exerts both anti-and pro-inflammatory effects by expression of proinflammatory cytokines in adipocytes and macrophages. 66 One study in this meta-analysis showed that probiotics increase adiponectin concentration in the colostrum, 22 81 The normal flora of the intestine can modify almost 10% of the host's transcriptome, particularly genes related to immune system response, cellular proliferation, and metabolism. 82 SCFAs and butyrate are generated as end products of fermenta-

| CON CLUS ION
Despite the presence of heterogeneity and intervening factors among the existing studies, we could discreetly declare that probiotic supplementation, through regulation of the gut microbiota, seems to be able to improve the immune system function, glucose and lipid metabolisms, inflammation, and oxidative stress and subsequently reduce the risk of GD among pregnant women. But above findings remain uncertain, due to the heterogeneity among existing studies. However, more homogeneous studies are needed to confidently generalize the results of this study. Therefore, specific probiotics supplementations may be introduced as one of the adjuvant therapies for GD patients.

CO N FLI C T O F I NTE R E S T
Authors declare that there is no conflict of interest.