Clinical trial NCT05669690: Storage Conditions and Breast Milk Microbiota Composition
Official title: The Effect of Storage Conditions on Microbiota Composition of Breast Milk
Study type: OBSERVATIONAL
Overall status: UNKNOWN
Conditions: Breast Milk Collection
Microbial Colonization
Keywords: breast milk
storage conditions
microbiota
Lead sponsor: Istanbul University
Sponsor class: OTHER
First posted: 2023-01-03
Enrollment: 20
Sex: FEMALE
Minimum age (years): 18
Maximum age (years): 35
Standard ages: ADULT
Accepts healthy volunteers: yes
Observational model: COHORT
Time perspective: PROSPECTIVE
Sampling method: PROBABILITY_SAMPLE
Study population: Healthy young mothers who had vaginal delivery at least one week ago will be included

Brief summary: Breast milk is the mainstay of newborn nutrition, providing all the nutrients and vitamins necessary for optimal growth of newborn infants. Beside its nutritional properties, breast milk also contains bioactive factors such as soluble immune factors, antimicrobial proteins, functional fatty acids, hormones, oligosaccharides, stem cells and microbiota. Although fresh breast milk is the optimal source of nutrition for newborn infants, breast milk must be expressed and stored in some conditions.

Breast milk was thought to be sterile for many years. This idea has changed with the isolation of live bacteria in the breast milk of healthy mothers in the recent past. Thus, it has been shown that breast milk is not actually a sterile body fluid, but has a microbiota of its own. This study aimed to examine the effect of storage conditions of breast milk at different temperatures on the microbial composition of the breast milk.

The prospective experimental study will be conducted under the supervision of Istanbul University, Istanbul Medical Faculty, Department of Pediatrics, Neonatology Department. Informed consent of mothers will be taken. Thirty mililiter (30 mL) breast milk that was obtained from volunteer mothers will be divided into 3 samples, each 10 mL of breast milk will be stored at different temperatures. Ten mililiter will be studied for microbial composition with 16S rRNA amplicon sequencing and bioinformatics analysis immediately, 10 mL will be stored at +4'C for 3 days and will be studied at the end of 3rd day, other 10 mL will be stored at -20'C for 3 months and will be studied at the end of 3rd months. 16S rRNA amplicon sequencing and bioinformatic analysis studies will be performed at Istanbul Medical Faculty Clinical Nutrition and Microbiota Research Laboratory.

This present study is planned to be carried out for 24 months between January 2023 and December 2024

Detailed description: Fresh breast milk is the mainstay of newborn nutrition and provides all the nutrients and vitamins needed for optimal growth of newborn babies. In addition to the nutritional properties of breast milk, there are some bioactive factors such as soluble immune factors, antimicrobial proteins, functional fatty acids, hormones, oligosaccharides, stem cells and microbiota in breast milk. Breastfeeding also strengthens the bond between mother and baby in postpartum period and provides nutrition to the newborn infant. Breast milk improves microbial colonization, improves immune system maturation and supports the metabolic activities of the newborn infant.

Although fresh breast milk is the optimal food source for newborn babies, in some cases, breast milk must be expressed and stored.It is reported that breast milk can be stored in the refrigerator for 3 days without being frozen at +4 degrees Celsius, and can be stored as frozen for 3 months if it is frozen within the first 24 hours after milking. The North American Breast Milk Banking Association, on the other hand, reports that the storage period can be extended up to 12 months at temperatures below -20 degrees Celsius.

While breast milk was thought to be a sterile fluid, this idea has changed with the isolation of live bacteria in the milk of healthy mothers in the recent past. The presence of many bacteria such as Lactobacillus, Lactococcus, Bifidobacterium, Streptococcus, Enterococcus and Staphylococcus has been demonstrated by traditional culture methods of breast milk. The new generation sequencing technologies obtained higher microbial diversity of breast milk and detected mouth-dwelling bacteria such as Veillonella and Prevotella, skin bacteria such as Propionobacterium and gram-negative bacteria such as Pseudomonas, Ralstonia, Klebsiella. Thus, it has been shown that breast milk is not actually a sterile body fluid, but has a microbiota of its own.

There are few studies evaluating the effect of breast milk storage conditions on microbiota. In a study, it was shown that the number of bacterial colonies in breast milk decreased as the freezing period increased. It has been shown that 98-100% of bacteria in frozen breast milk are lost after 12 weeks of storage.

Although studies evaluating fresh breast milk microbiota have been reported in the literature, there are few studies evaluating the effect of milk storage conditions on microbial diversity. In existing studies, microbial growth was ensured by culture method and diversity was defined by traditional microbiological methods. No study was found that evaluated the microbial composition of breast milk with next-generation sequencing based on the 16S rRNA amplicon sequencing strategy.

This study aimed to evaluate how the generally recommended breast milk storage conditions and duration of storage affect the microbiota composition of breast milk. This study will provide new data and new recommendations about storage of breast milk.

Eligibility criteria: Inclusion Criteria:

* Women who had vaginal delivery
* Women who gave delivery at gestational age above 37 weeks
* Women who have a child older than 1 weeks of age
* Mothers with a body mass index between 18.5-25
* Women who gave consent

Exclusion Criteria:

* Women who had cesarean delivery
* Women who gave premature delivery (\<37 weeks of gestation)
* Women who gave birth within prior 7 days
* Women with premature rupture of membranes
* Women with chorioamnionitis
* Women who had antibiotherapy within last 1 week
* Presence of mastitis
* Presence of maternal co-morbid diseases such as; pregestational diabetes, pregestational hypertension, connective tissue diseases, inflammatory bowel syndrome, solid organ transplantation
* Presence of evidenced congenital infections
* Women who didn't give consent

Primary outcomes:
measure: Changes in microbiota composition of breast milk after storage
timeFrame: 24 months
Secondary outcomes:
Other outcomes:
Locations:
city: Istanbul
country: Turkey
facility: Istanbul University Istanbul Medical Faculty
Study officials:
affiliation: Istanbul University
name: Emine Asuman Coban, Professor
role: PRINCIPAL_INVESTIGATOR
affiliation: Istanbul University
name: Elmas Zeynep Ince, Professor
role: PRINCIPAL_INVESTIGATOR
affiliation: Istanbul University
name: Bülent Saka, Professor
role: PRINCIPAL_INVESTIGATOR
affiliation: Istanbul University
name: Leyla Bilgin, Professor
role: PRINCIPAL_INVESTIGATOR
affiliation: Istanbul University
name: Dilek Sever Kaya, Dr Biologist
role: PRINCIPAL_INVESTIGATOR
affiliation: Istanbul University
name: Aslı Berru Arslan, Med Student
role: PRINCIPAL_INVESTIGATOR
affiliation: Istanbul University
name: Beril Yasa, Ass. Prof.
role: STUDY_DIRECTOR
References:
citation: Selma-Royo M, Calvo Lerma J, Cortes-Macias E, Collado MC. Human milk microbiome: From actual knowledge to future perspective. Semin Perinatol. 2021 Oct;45(6):151450. doi: 10.1016/j.semperi.2021.151450. Epub 2021 Jun 16.
citation: Pandya SP, Doshi H, Codipilly CN, Fireizen Y, Potak D, Schanler RJ. Bacterial stability with freezer storage of human milk. J Perinat Med. 2020 Sep 14;49(2):225-228. doi: 10.1515/jpm-2020-0131. Print 2021 Feb 23.
citation: Fernandez L, Ruiz L, Jara J, Orgaz B, Rodriguez JM. Strategies for the Preservation, Restoration and Modulation of the Human Milk Microbiota. Implications for Human Milk Banks and Neonatal Intensive Care Units. Front Microbiol. 2018 Nov 9;9:2676. doi: 10.3389/fmicb.2018.02676. eCollection 2018.