Jun 29, 2026

The Effect of Probiotic Supplementation on Neonatal Bilirubin Levels and Breastmilk Total Antioxidant Capacity in Gestational Diabetes: A Single-Center Randomized Controlled Trial Protocol

DOI
https://dx.doi.org/10.17504/protocols.io.5qpvojy7zg4o/v1
  • Fatma Bengü Kuyulu Bozdoğan1,
  • Seray Kabaran2,
  • Ferhat Çetin3
  • 1Osmaniye Korkut Ata University, Department of Nutrition and Dietetics;
  • 2Eastern Mediterranean University, Department of Nutrition and Dietetics;
  • 3Private Park Hospital, Obstetrics and Gynecology Clinic
BENGÜ KUYULU
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DOI: https://dx.doi.org/10.17504/protocols.io.5qpvojy7zg4o/v1
Protocol Citation: Fatma Bengü Kuyulu Bozdoğan, Seray Kabaran, Ferhat Çetin 2026. The Effect of Probiotic Supplementation on Neonatal Bilirubin Levels and Breastmilk Total Antioxidant Capacity in Gestational Diabetes: A Single-Center Randomized Controlled Trial Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvojy7zg4o/v1
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 29, 2026
Last Modified: June 29, 2026
Protocol  Integer ID: 319988
Keywords: Gestational Diabetes Mellitus (GDM), Probiotics, Mediterranean Diet, Developmental Origins of Health and Disease (DOHaD), Neonatal Bilirubin, Breast Milk TAS
Abstract
Abstract: Effects of Probiotic Supplementation on Neonatal Health and Breast Milk Antioxidant Capacity in Mothers with Gestational Diabetes
This protocol presents a single-center, randomized controlled clinical trial model aimed at evaluating the therapeutic effects of multi-strain probiotic supplementation on neonatal and maternal health parameters in pregnant women diagnosed with gestational diabetes mellitus (GDM). The study is designed to comprehensively investigate the role of probiotics in GDM management on neonatal bilirubin levels, infant anthropometry, and, for the first time in literature, the Total Antioxidant Status (TAS) of breast milk.
Key Components and Methodology of the Protocol
  • Study Design and Participants: The study includes 68 pregnant women aged 20–40 years diagnosed with GDM at the 24th week of gestation (34 in the intervention group, 34 in the control group). The trial utilizes an open-label design without a placebo for the control group. Participants are monitored from the 24th week until the 36th week of gestation.
  • Dietary and Probiotic Intervention: Both groups receive a personalized medical nutrition therapy based on the principles of the Mediterranean diet. In addition to their diet, the intervention group receives a multi-strain probiotic supplement formulation (L. acidophilus, L. rhamnosus, B. bifidum, B. longum, E. faecium along with FOS, lactulose, and vitamins) to be consumed orally once daily for 12 weeks.
  • Dietary Inflammatory Index (DII): To isolate the inflammatory burden of the maternal diet and evaluate nutritional quality, maternal DII scores are calculated from 72-hour retrospective dietary intake records to divide participants into quartiles.
  • Clinical and Laboratory Measurements: Detailed anthropometric measurements of the newborns (birth weight, length, head circumference, abdominal circumference, MUAC, and skinfold thicknesses) are performed at birth. Neonatal serum bilirubin levels are measured on the 3rd postnatal day. During the lactation period, breast milk samples collected via expression are analyzed for Total Antioxidant Status (TAS) using a colorimetric method.

Strengths and Innovative Contributions of the Protocol

  • Evaluation of Breast Milk TAS: This protocol represents the first clinical trial methodology to rigorously evaluate the impact of probiotic intervention on the total antioxidant capacity of breast milk in women with gestational diabetes.
  • Confounder Control: Major confounding variables that could directly affect metabolic and inflammatory outcomes—such as the inflammatory potential of the maternal diet (DII score), sleep quality (PSQI), and physical activity levels (PPAQ)—are strictly controlled within the protocol.
  • Therapeutic Focus: While existing literature predominantly focuses on the preventive or protective role of probiotics in GDM, this protocol directly tests a "therapeutic and symptom-alleviating" adjunct treatment model after diagnosis
Materials
**Supplement Formulation: Multi-strain synbiotic formulation (NBL Probiotic Gold®).

**Composition (Per Sachet/Capsule): Lactobacillus acidophilus (4.3× 10^8^ CFU), Lactobacillus rhamnosus (4.3× 10^8^ CFU), Bifidobacterium bifidum (4.3× 10^8^ CFU), Bifidobacterium longum (4.3× 10^8^ CFU), and Enterococcus faecium (8.2× 10^8^ CFU). It also contains fructooligosaccharides (FOS, 625 mg), lactulose (400 mg), and vitamins A, B1, B2, B6, E, and C.

**Neonatal Blood Bilirubin Level: Total serum bilirubin levels (mg/dL) are measured on the 3rd postnatal day via routine clinical blood sampling.

**Breast Milk TAS Analysis: Human colostrum/milk samples are collected, and Total Antioxidant Status (TAS) levels are analyzed colorimetrically using commercial Rel Assay kits on a fully automated analyzer. Results are reported as mmol Trolox Equivalent per liter.
3. Interventions and Dietary Protocols
Daily energy requirements are estimated using predictive equations derived from the Henry Equations, based on pre-pregnancy weight and height, adjusted for individual physical activity levels (PA coefficients).
Because all participants are in their third trimester, an additional +452 kcal is incorporated into the daily energy target.
For participants classified as pre-obese (3e25 kg/m^2^ or those who have already achieved the optimal gestational weight gain recommended by the IOM, the estimated energy requirement is reduced by 30%.
Macronutrient composition is tailored to match GDM clinical guidelines: approximately 45% carbohydrates (strictly from low-glycemic index foods), 30% fat, and 25% protein, aligned with the principles of the Mediterranean Diet.
3.2. Probiotic Supplementation Protocol (Intervention Group Only)
Supplement Formulation: Multi-strain synbiotic formulation (NBL Probiotic Gold®).
Composition (Per Sachet/Capsule): Lactobacillus acidophilus (4.3× 10^8^ CFU), Lactobacillus rhamnosus (4.3× 10^8^ CFU), Bifidobacterium bifidum (4.3× 10^8^ CFU), Bifidobacterium longum (4.3× 10^8^ CFU), and Enterococcus faecium (8.2× 10^8^ CFU). It also contains fructooligosaccharides (FOS, 625 mg), lactulose (400 mg), and vitamins A, B1, B2, B6, E, and C.
Dosage and Duration: One capsule per day for 12 weeks, from the 24th to the 36th week of gestation.
Administration Instructions: Taken orally with 100 mL of water on an empty stomach in the morning. Supplements must be stored at room temperature in their original packaging.
4. Data Collection and Clinical Measurements
Phase 1: Gestational Follow-up (Weeks 24 to 36)
4.1. Phase 1: Gestational Follow-up (Weeks 24 to 36)
Socio-Demographic and Medical History: Data on maternal age, education, occupation, family history of diabetes, previous delivery modes, and postpartum breastfeeding preferences are recorded.
Dietary Inflammatory Index (DII) Assessment: A 72-hour retrospective dietary intake record is obtained using the Food and Meal Photograph Catalog to standardize portion sizes. Literature-derived inflammatory effect scores (+1 pro-inflammatory, -1 anti-inflammatory, 0 neutral) are assigned to food parameters to compute the maternal DII score and stratify participants into quartiles (Q1–Q4).
4.2. Phase 2: Birth and Neonatal Outcomes
Anthropometric Measurements: At birth, infant body weight (g) is measured using an infant scale, and birth length (cm) is assessed with an infantometer. Head circumference, abdominal circumference, and mid-upper arm circumference (MUAC) are measured using a non-stretch measuring tape. Subscapular and triceps skinfold thicknesses are evaluated with Harpenden skinfold calipers.
Clinical Metrics: Apgar scores, delivery mode, crying status immediately after birth, and the exact time of the first breastfeeding session are retrieved from the hospital information system.
4.3. Phase 3: Postnatal Laboratory Analyses
Neonatal Blood Bilirubin Level: Total serum bilirubin levels (mg/dL) are measured on the 3rd postnatal day via routine clinical blood sampling.
Breast Milk TAS Analysis: Human colostrum/milk samples are collected, and Total Antioxidant Status (TAS) levels are analyzed colorimetrically using commercial Rel Assay kits on a fully automated analyzer. Results are reported as mmol Trolox Equivalent per liter.
5. Data Management and Statistical Analysis Plan
Software: All data calculations and analyses are executed using the SPSS (Statistical Package for the Social Sciences) software package.
Descriptive Data: Quantitative variables are expressed as mean ± standard deviation, minimum, and maximum values. Qualitative/categorical variables are summarized using frequencies and percentages.
Hypothesis Testing:
The Chi-Square test is applied for comparisons involving categorical variables.
For continuous variables, independent groups are compared using the Student's t-test (for two groups) or One-Way ANOVA (for more than two groups) if parametric assumptions are satisfied.
If data violate normality assumptions, the non-parametric Mann-Whitney U test or Kruskal-Wallis test is utilized.
Correlation and Regression: The relationship between breast milk TAS and infant bilirubin is examined using Pearson's Correlation Coefficient. Multiple linear regression models are constructed to evaluate the predictors of neonatal blood bilirubin levels while controlling for the DII score and probiotic intervention.
Significance Level: A two-sided p 3c 0.05 is established as the threshold for statistical significance across all analyses.