The Effect of Adding Milk Oligosaccharides and Optimizing Multiple Nutrients to Infant Formula on the Growth and Development of Infants Aged 0-1 Years: a Multicenter, Double-blind, Randomized, Controlled Trial
Phase: NA
Conditions: Healthy
Sponsor: Heilongjiang Feihe Dairy Co. Ltd.

Project Number: CTS-MP-2023-01
Version Number: 2.0; Version date: November 17, 2023
Clinical trial protocol
Effect of adding breast milk oligosaccharides and multinutrient
optimized infant formula on the growth and development of
infants aged 0 to 1 years: a multicenter, double-blind,
randomized, controlled trial
Version number: 2.0; Date: November 17,2023
Group leader:
Nanjing Medical University
address:
No.101, Longmian Avenue, Jiangning District,
Nanjing city
Participating units:
The First Affiliated Hospital of Tianjin
University of Traditional Chinese Medicine
address:
No.88, Changling Road, Xiqing District,
Tianjin city
Participating units:
Changzhou Second People's Hospital
address:
No.29, Changzhou city, Jiangsu Province
Participating units:
Linyi City Maternal and Child Health Care
Hospital
address:
No.1, Qinghe South Road, Luozhuang
District, Linyi City
Participating units:
Jiangnan University Affiliated Hospital

Project Number: CTS-MP-2023-01
Version Number: 2.0; Version date: November 17, 2023
address:
No.200, Huihe Road, Binhu District, Wuxi
City, Jiangsu Province
Participating units:
The Seventh Medical Center of the PLA
General Hospital
address:
Nanmen Cang, No.5, East 46, Dongcheng
District, Beijing

Project Number: CTS-MP-2023-01
Version number: 2.0; version dated：17 November 2023
catalogue
1 Summary of the clinical trial protocol............................................................... 1
2 research background.......................................................................................... 6
2.1 Breast milk oligosaccharides................................................................... 6
2.2 lactoferrin...............................................................................................10
2.3 Docosahexaenoic acid and eicosapentetraenoic acid.............................12
3 Study purpose and significance....................................................................... 15
4 research contents..............................................................................................15
5 subject investigated..........................................................................................16
5.1 Inclusion criteria.................................................................................... 16
5.1.1 Breastfeeding group....................................................................16
5.1.2 Artificial feeding group...............................................................17
5.2 Exclusion criteria................................................................................... 17
5.3 Exit standard.......................................................................................... 18
6 research design.................................................................................................19
6.1 Test flow chart........................................................................................19
6.2 recruit.....................................................................................................19
6.3 informed consent....................................................................................20
6.4 dressing by screening.............................................................................20
6.5 randomization........................................................................................ 20
6.5.1 Random group table....................................................................20
6.5.2 Sample size and calculation basis...............................................21
6.6 Interview................................................................................................ 21
6.6.1 Visit 1..........................................................................................21
6.6.2 Visit 2..........................................................................................22
6.6.3 Visit 3..........................................................................................22

Project Number: CTS-MP-2023-01
Version number: 2.0; version dated：17 November 2023
6.6.4 Visit 4..........................................................................................23
6.7 Subjects stopped / withdrew from the trial............................................ 23
7 Research products............................................................................................24
7.1 burden.................................................................................................... 24
7.2 Nutritional composition table................................................................ 26
7.3 Sample management..............................................................................32
8 Outcome indicators..........................................................................................32
8.1 Main end point.......................................................................................32
8.2 Secondary end point...............................................................................32
8.3 Safety evaluation index..........................................................................33
9 Data statistics method...................................................................................... 33
9.1 Data analysis software................................................................................... 33
9.2 General data analysis..................................................................................... 33
9.3 Analyze the crowd......................................................................................... 33
9.4 Subject completion status.............................................................................. 33
9.5 Demographic data..................................................................................34
9.6 adverse event..........................................................................................34
9.7 efficiency analysis..........................................................................................35
9.7.1 Main endpoint indicators............................................................................ 35
9.7.2 statistical analysis technique.......................................................................35
10 Management plan for common / serious adverse events............................... 36
10.1 Common adverse events / serious adverse events............................... 36
10.2 Management of the adverse events......................................................36
10.3 Management of serious adverse events............................................... 37
10.4 Subject safeguards............................................................................... 37
11 Ethical requirements...................................................................................... 37
11.1 Protection of subject privacy................................................................37

Project Number: CTS-MP-2023-01
Version number: 2.0; version dated：17 November 2023
11.2 informed consent..................................................................................37
11.3 Approval of the ethics committee........................................................ 37
11.4 Declaration of Helsinki........................................................................38
12 Quality control and quality assurance............................................................38
13 Study termination...........................................................................................38

Project Number: CTS-MP-2023-01
Version number: 2.0; version dated：17 November 2023
1
1 Summary of the clinical trial protocol
Research
name
Effect of adding breast milk oligosaccharides and multinutrient
optimized infant formula on the growth and development of infants
aged 0 to 1 years: a multicenter, double-blind, randomized, controlled
trial
research
design
Multicenter, randomized, controlled, parallel, double-blind trial
sample
capacity
240
Interventio
n cycle
360 Days
purpose of
research
Compare add milk oligosaccharides and a variety of nutrients
optimization of infant formula and conventional infant formula on the
immune function of infants under 1 year of age, and compared with
breastfeeding infants, explore different infant formula on infant
intestinal health, immune function, growth and development, cognitive
development and tolerance.
Main
end
point
Intestinal indicators (16s r RNA, SCFAs)
Secondary
end point
· Inflammation indicators: serum hsCRP;
· Nutritional index: serum albumin;
· Immune indicators: fecal sIgA and blood lymphocyte subsets;
· Growth
and
development
indicators:
height, weight,
head
circumference and chest circumference;
· Bone development indicators: osteocalcin, alkaline phosphatase;
· Tolerance indicators: survey of infant gastrointestinal comfort,
infant sleep crying, infant diarrhea and upper respiratory tract
infection, infant stool Bristol stool classification;
· Cognitive development: Bailey Infant Development Scale (version

Project Number: CTS-MP-2023-01
Version number: 2.0; version dated：17 November 2023
2
IV), serum amino acids.
Safety
indicators
· Adverse events and serious adverse events.
Inclusion
criteria
Inclusion criteria for the breast-feeding group:
· The 0 ~ 28 days after birth;
· Single-child birth;
· The gestational age was from 37 to 42 weeks;
· Birth weight is 2500 ~ 4000 g;
· Parents or guardians agree to exclude the infant from participating
in other interventional clinical studies during this study;
· Parents or guardians agreed to feed the baby according to the trial
protocol;
Inclusion criteria for the artificial feeding group:
· The 0 ~ 28 days after birth;
· Single-child birth;
· The gestational age was from 37 to 42 weeks;
· Birth weight is 2500 ~ 4000 g;
· Parents or guardians agree to exclude the infant from participating
in other interventional clinical studies during this study;
· Parents or guardians agreed to feed the baby according to the trial
protocol;
· The mothers cannot be exclusively breastfeeding due to subjective
and objective reasons.
Exclusion
criteria
· Artificial assisted reproduction;
· The mother suffers from diseases that may endanger the
intrauterine growth;
· The mother had gestational diabetes mellitus or severe metabolic
disease or chronic disease at the time of pregnancy;
· Having congenital malformations and genetic, chronic, and

Project Number: CTS-MP-2023-01
Version number: 2.0; version dated：17 November 2023
3
congenital diseases that may interfere with the investigation;
· IgE mediated milk protein allergy or factors that increase the risk
of milk protein allergy;
· Having an acute infection or gastroenteritis;
· Functional gastrointestinal diseases, such as gastroparesis;
· Is participating in other clinical trials;
· The investigator was unable to determine the parents' willingness
or ability to comply with the protocol requirements.
Exit
standard
· Ineligible (ignored during screening, occurred during the study
or found by review);
· Serious protocol deviation occurs;
· Serious non to protocol or test requirements;
· Need to terminate the designated formula due to adverse events
or cause failure to continue following the trial procedures;
· Need to discontinue the designated formula or cause the inability
to follow the trial procedures due to disease progression;
· Withdraw informed consent or remove informed consent;
· Misvisit.
Interventio
n method
Test group: Artificial feeding of the experimental formula
0-4 months of age: artificial feeding is not less than 2 times a day;
4~12 months of age: artificial feeding is not less than 5 times a
day;
Control group: artificial feeding of the control formula
0-4 months of age: artificial feeding is not less than 2 times a day;
4~12 months of age: artificial feeding is not less than 5 times a
day;
Breastfeeding group: breastfeeding
0-4 months of age: exclusive breastfeeding (must be fully

Project Number: CTS-MP-2023-01
Version number: 2.0; version dated：17 November 2023
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breastfeeding 1 day before the visit)
4 to 12 months old: breastfeeding at least once a day
Research
implementa
tion
In the artificial feeding group, 160 infants from 0 to 28 days were
randomly divided into trial group and control group, 80 infants in each
group; 80 breastfed infants were included in the same period. The test
group, control group and breastfeeding group were fed the infants.
Based on the time of the last visit, the visit was conducted at 28,90 ± 3
days, 180 ± 5 days and 360 ± 7 days after birth.
metric
dressing
by
screenin
g
Visit 1
(28
Days)
Visit 2
(90
Days)
Visit 3
(180
Days)
Visit 4
(360
Days)
Demographic data
√
√
Bailey
Infant
Development
Scale
(Version IV)
√
Growth
and
development
indicators
√
√
√
√
excrement and urine
· 16S rRNA
· SCFAs
· sIgA
√
√
√
blood
· hsCRP
·
Lymphocyte
subsets
· amino acid
· bone gla protein
·
alkaline
phosphatase
√
√

Project Number: CTS-MP-2023-01
Version number: 2.0; version dated：17 November 2023
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· albumin
Feeding
status,
Intake questionnaire
(breastfeeding
and
formula feeding)
√
√
√
√
Gastrointestinal
comfort
questionnaire
√
√
√
√
Sleep
crying
questionnaire
√
√
√
√
Questionnaire
for
diarrhea and upper
respiratory
tract
infections
√
√
√
√
Bristol
Stool
Classification
√
√
√
√

Project Number: CTS-MP-2023-01
Version number: 2.0; version dated：17 November 2023
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2 research background
Breast milk is widely recognized as the most perfect food for the newborn period.
Breast milk naturally contains the best proportion of various nutrients suitable for the
baby's digestion and absorption[,]12, Such as protein, fats, carbohydrates, vitamins, and
minerals. In addition, breast milk also contains a variety of bioactive ingredients
suitable for infants, such as lactoferrin, immunoglobulin, hormones, oligosaccharides,
probiotics and so on[,]34, These bioactive ingredients play an important role in the
health of infants and young children.
2.1 Breast milk oligosaccharides
Breast milk oligosaccharides (Human Milk Oligosaccharides, HMOs) are a
natural bioactive substance in breast milk, second only to lactose and fat, is the third
largest solid component of breast milk, and have important physiological functions.
Studies showed that the content of HMOs in colostrum was 20 to 23 g / L and
decreased to 12 – 14 g / L in mature milk[]5, Higher levels of HMOs in breast milk
than in term mothers[]6. Traditional infant formula does not add HMOs, but dietary
fiber such as galactose (GOS) and fructose oligosaccharides (FOS) are used as
alternatives[,]78. The HMOs are produced by D-glucose (D-glucose, Glc), D-galactose
(D-galactose, Gal), N-acetylglucosamine (N-acetylglucosamine, GlcNAc), L-fucinose
(L-fucose, Fuc) and sialic acid (sialic acid, Sia) and other five monosaccharides
constitute complex glycans, Each HMOs contains a single lactose (Gal- β -1, 4-Glc) at
the reducing terminus, And based on this, with β -1, 3 or β -1, 6-bond linkage to
galactose β -1, 3-N-acetylglucosamine (Gal- β -1, 3-GlcNAc, This disaccharide
structure is also known as the lacto-N-biose, Or type 1 chain structure) or N-
acetylylactose (N-acetyllactosamine, Gal-β-1, 4-GIcNAc, Type 2 chain structure) of
the extended sugar chain, Formation of a lacto-N-tetrasaccharide (lacto-N-tetrose,
LNT),
lactol-N-new
tetrasaccharide
(lacto-N-neotetraose,
LNnT),
lactol-N-
1 O’Hare, E.M.; Wood, A.; Fiske, E.Human milk banking.Neonatal Netw.2013, 32, 175–183.
2 Bernatowicz-Łojko, U.The role of breast milk in prevention and treatment.Post Neonatol.2008, 2, 142–143.
3 Dieterich, C.M.; Felice, J.P.; O’Sullivan, E.; Rasmussen, K.M.Breastfeeding and health outcomes for the mother-
infant dyad.Pediatr.Clin.N.Am.2013, 60, 31–48.
4 Ballard, O.; Morrow, A.L.Human milk composition: Nutrients and bioactive factors.Pediatr.Clin.N.Am.2013, 60,
49–74.
5 Coppa, G.V.; Pierani, P.; Zampini, L.; Carloni, I.; Carlucci, A.; Gabrielli, O.Oligosaccharides in human milk
during different phases of lactation.Acta Paediatr.Suppl.1999, 88, 89–94.
6 Gabrielli, O.; Zampini, L.; Galeazzi, T.; Padella, L.; Santoro, L.; Peila, C.; Giuliani, F.; Bertino, E.; Fabris, C.;
Coppa, G.V.Preterm milk oligosaccharides during the first month of lactation.Pediatrics 2011, 128, 1520–1531.
7 Vos, A.P.; Haarman, M.; Buco, A.A specific prebiotic oligosaccharide mixture stimulates delayed-type
hypersensitivity in a murine influenza vaccination model.Int.Immunopharmacol.2006, 6, 1277–1286.
8 Espinosa, R.M.; Tamez, M.; Prieto, P.Efforts to emulate human milk oligosaccharides.Br.J.Nutr.2007, 98, 74–79.

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hexasaccharide (lacto-N-hexaose, LNH) and other core structures. The structure of
FOS and GOS is simpler, and the addition of FOS and GOS in formula is considered a
relatively reasonable and cheap scheme to simulate breast milk. The difference
between these two and HMOs in breast milk still need more research to demonstrate.
The 2 '-fucinose-based lactose (2' FL) and LNnT have been used in infant formula.
Some studies showed that supplementing 2 ′ -FL (1 g / L) and LNnT (0.5 g / L) on the
basis of formula, the intestinal flora of infants at 3 months contained more
bifidobacterium and less Echia, digestive Streptococcus, closer to that of breast-fed
infants; the content of important metabolites such as propionate, butyrate and lactate
in feces of infants where HMOs were supplements[,]910。
HMOs are capable of blocking the adhesion of pathogens. Many viruses or
bacteria must attach to the surface of epithelial cells to proliferate and cause disease,
and HMOs act as soluble ligands like blocking adhesion to pathogens. Animal
experiments showed that 2 ′ FL reduced the invasion of Chlamydia jejunae by 80%
and inhibited the release of mucosal pro-inflammatory signals, which subsequently
reduced the number of episodes of diarrhea. LNnT was shown to reduce the number
of S. pneumoniae cells in the lungs of an animal model[]11. The anti-adhesive
properties of HMOs also apply for dissolution. Entamoeba, and histolytica, capable of
destroying epithelial cells of the large intestine, liver, lung or spleen, is regarded as
the third leading cause of parasite death worldwide[]12. Entamoeba histolytica needs to
be attached to the colon mucosa of the host, and endohistolytica will be excreted with
the feces and will not cause disease.
HMOs are able to help establish and maintain a healthy intestinal flora in infants.
From 0 to 3 years of age, the formation of intestinal flora is a developing process. The
bacteria in the gut mainly include Escherichia coli, Klebsiella, Enterobacter,
Bacteroides and Clostridium spp. Bifidobacterium dominated the gut of breastfed
infants, with Clostridium and Enterococcus, and Klebsiella and Enterobacteria being
the least. The gut flora of artificially fed infants is more similar to the adult digestive
tract, and the composition is more complex than that in breast-fed infants.
Bifidobacterium should dominate in the healthy intestinal flora of infants. In recent
years, people have increased the content of bifidobacterium in the intestine by adding
9 Steenhout, P.; Sperisen, P.; Martin, F.-P.; Sprenger, N.; Wernimont, S.; Pecquet, S.; Berger, B.Term infant formula
supplemented with human milk oligosaccharides (2′fucosyllactose and lacto-N-neotetraose) shifts stool microbiota
and metabolic signatures closer to that of breastfed infants.J.Pediatr.Gastroenterol.Nutr.2016, 63, S55.
10 Donovan, S.M.; Comstock, S.S.Human Milk Oligosaccharides Influence Neonatal Mucosal and Systemic
Immunity.Ann.Nutr.Metab.2016, 69, 42–51.
11 Bode, L.Human milk oligosaccharides: Every baby needs a sugar mama.Glycobiology 2012, 22, 1147–1162.
12 Kumari, M.; Kozyrskyj, A.L.Gut microbial metabolism defines host metabolism: An emerging perspective in
obesity and allergic inflammation.Obes.Rev.2017, 18, 18–31.

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prebiotics to formula. HMOs are not destroyed by the gastric acid of the human body,
nor decomposed by digestive enzymes, and can directly reach the large intestine,
stimulate the growth of beneficial bacteria (bifidobacteria and Lactobacillus) in the
intestinal tract, indirectly inhibit the growth of harmful bacteria, and maintain the
balance of intestinal microecology. Therefore, HMOs are regarded as the primary
prebiotics (prebiotics) of human beings[]13. Bifidobacterium in the large intestine can
produce acetic acid, propionate and butyric acid through fermentation, acetic acid can
reduce the pH value in the intestinal tract and inhibit the growth of pathogenic
bacteria; propionate and butyrate have important physiological functions; butyrate is
an important energy source for colon cells. HMOs are able to indirectly increase the
production of short-chain fatty acids (SCFA s), and most SCFAs are rapidly absorbed
by colonic cells or used as energy sources[]14. Research evidence suggests that SCFAs
are able to participate in the regulation of gene expression[]15, Play an important role
in the activation and differentiation of immune cells and is associated with
inflammatory and allergic diseases[,]1617。
HMOs are able to provide protection against multiple viruses[,,]181920. HMOs
promote the maturation of the immune system, produce a more balanced Th 1 / Th 2
cytokine response; stimulate the immune response and maturation of epithelial cells to
protect the host from viral infection; affect the diversity and concentration of the
microbiome and stimulate the growth of commensal bacteria. Another antiviral
activity
of
HMOs
is
based
on
structural
similarity
to
the
glycochains
of
glycoconjugates present on the epithelial surface, mimicking the surface glycans of
epithelial cells, and in turn capturing viruses that do not adhere to the cells. Virus
lectin receptors blocked by HMOs fail to participate in recognizing the glycosites
13 REID G.Probiotics and prebiotics-progress and challenges[J].International Dairy Journal, 2008, 18(10/11): 969-
975. DOI:10.1016/j.idairyj.2007.11.025.
14 Kulinich, A.; Liu, L.Human milk oligosaccharides: The role in the fine-tuning of innate immune
responses.Carbohydr.Res.2016, 432, 62–70.
15 Koh, A.; De Vadder, F.; Kovatcheva-Datchary, P.; Backhed, F.From Dietary Fiber to Host Physiology: Short-
Chain Fatty Acids as Key Bacterial Metabolites.Cell 2016, 165, 1332–1345.
16 Richards, J.L.; Yap, Y.A.; McLeod, K.H.; Mackay, C.R.; Marino, E.Dietary metabolites and the gut microbiota:
An alternative approach to control inflammatory and autoimmune diseases.Clin.Transl.Immunol.
17 Bridgman, S.L.; Azad, M.B.; Field, C.J.; Haqq, A.M.; Becker, A.B.; Mandhane, P.J.Fecal Short-Chain Fatty Acid
Variations by Breastfeeding Status in Infants at 4 Months: Differences in Relative versus Absolute
Concentrations.Front.Nutr.2017, 4, 11.
18 Yang, B.; Chuang, H.; Chen, R.-F.Protection from viral infections by human milk oligosaccharides: Direct
blockade and indirect modulation of intestinal ecology and immune reactions.Open Glycosci.2012, 5, 19–25.
19 Morrow, A.L.; Ruiz-Palacios, G.M.; Jiang, X.; Newburg, D.S.Human-milk glycans that inhibit pathogen binding
protect breast-feeding infants against infectious diarrhea.J.Nutr.2005, 135, 1304–1307.
20 Li, M.; Monaco, M.H.; Wang, M.; Comstock, S.S.; Kuhlenschmidt, T.B.; Fahey, G.C., Jr.; Miller, M.J.;
Kuhlenschmidt, M.S.; Donovan, S.M.Human milk oligosaccharides shorten rotavirus-induced diarrhea and
modulate piglet mucosal immunity and colonic microbiota.ISME J.2014, 8, 1609–1620.

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present on the host cell surface, thereby preventing their adhesion and colonization.
Against rotavirus and norovirus infections, HMOs have been found to be useful as an
alternative to current therapeutic approaches. HA may be used in humans as a HMOs-
based drug to treat influenza. Monosialylated HMOs (3 ′ -SL and 6 ′ -SL) have been
shown to reduce influenza infection in cell culture assays.
HMOs are able to promote immune function development in infants. In the first
6 months of life, breastfeeding was shown to promote the maturation of the immune
system, feeding enough to reduce the incidence of many diseases such as asthma,
allergies, inflammatory bowel disease, type 1 diabetes, celiac disease, and leukemia.
HMOs are very important components in breast milk and can affect the infant
immune system through multiple mechanisms including blocking pathogen adhesion
and promoting bifidobacterium colonization. The composition of the infant gut flora
and epithelial cell responses mediated by HMOs may indirectly affect the infant's
immune system. In vitro studies have shown that HMOs can also directly modulate
the immune response. The HMOs may act locally on the lymphohistiocytes associated
with the mucosa[,]2122. Many immune receptors recognize the oligosaccharide
structures of their glycoprotein ligands. HMOs are structurally similar to ligand, thus
it is speculated that they may bind directly to immune cells[,]2324, This binding can
cause signaling, leading to changes in the nature and function of immune cells.
HMOs can reduce the risk and mortality of necrotizing enterocolitis (NEC). NEC
causes severe and fatal destruction of the gut in infants, with more than 25% of NEC
babies dying, and surviving infants often have long-term neurological complications.
The risks of NEC were 6 to 10 times lower than formula-fed infants[,]2526. Animal
experiments in rat models showed that HMOs prevent the release of NEC. If these
results are translated into NEC in humans, lactose disialate-N-tetrasaccharide
(DSLNT) can be used to prevent or treat NEC in formula-fed infants, and its
concentration in breast milk can be used as a biomarker to identify the risk of breast-
21 Macpherson, A.J.; Geuking, M.B.; McCoy, K.D.Immune responses that adapt the intestinal mucosa to
commensal intestinal bacteria.Immunology 2005, 115, 153–162.
22 Goehring, K.C.; Kennedy, A.D.; Prieto, P.A.; Buck, R.H.Direct evidence for the presence of human milk
oligosaccharides in the circulation of breastfed infants.
23 Eiwegger, T.; Stahl, B.; Haidl, P.; Schmitt, J.; Boehm, G.; Dehlink, E.; Urbanek, R.; Szepfalusi, Z.Prebiotic
oligosaccharides: In vitro evidence for gastrointestinal epithelial transfer and immunomodulatory
properties.Pediatr.Allergy Immunol.2010, 21, 1179–1188.
24 Eiwegger, T.; Stahl, B.; Schmitt, J.; Boehm, G.; Gerstmayr, M.; Pichler, J.; Dehlink, E.; Loibichler, C.; Urbanek,
R.; Szepfalusi, Z.Human milk-derived oligosaccharides and plant-derived oligosaccharides stimulate cytokine
production of cord blood T-cells in vitro.Pediatr.Res.2004, 56, 536–540.
25 Neu, J.; Walker, W.A.Necrotizing enterocolitis.N.Engl.J.Med.2011, 364, 255–264.
26 Hintz, S.R.; Kendrick, D.E.; Stoll, B.J.Neurodevelopmental and growth outcomes of extremely low birth weight
infants after necrotizing enterocolitis.Pediatrics 2005, 115, 696–703.

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fed infants with the disease. It may also facilitate the development of innovative
therapies for NEC[,]2728。
The HMOs are able to promote the early development of the infant brain. Parts
of the HMOs in breast milk are terminal to adhere to high concentrations of sialic
acids. Sialic acid is involved in the composition of gangliosides and glycoproteins in
human brain tissue, and is closely related to nerve synapses and nerve conduction, so
breastfeeding helps to enhance nerve synaptogenesis and promote the development of
infant nervous system[]29。
2.2 lactoferrin
Lactoferrin is an iron-bound glycoprotein with a variety of physiological
functions, with a molecular mass of about 70 – 80 kilodaltons[]30. Lactoferrin is
widely distributed in the human body, in milk, saliva, tears, nasal secretions,
gastrointestinal fluid, urine, semen and other external secretions, among which
lactoferrin is the most abundant in breast milk[,,]313233. Lactoferrin content in the
mature milk of term and preterm infants ranged from 1.25 to 4.59 mg/mL and 1.6 to
6.59 mg / mL, respectively[,,,]34353637，
Lactoferrin has many physiological and biological functions, especially in terms
of immune function has been widely recognized. Lactoferrin for many bacteria have
27 Jantscher-Krenn, E.; Zherebtsov, M.; Nissan, C.; Goth, K.; Guner, Y.S.; Naidu, N.; Choudhury, B.; Grishin, A.V.;
Ford, H.R.; Bode, L.Human milk oligosaccharides are differentially metabolised in neonatal
rats.Br.J.Nutr.2011, 110, 640–650.
28 Autran, C.A.; Kellman, B.P.; Kim, J.H.Human milk oligosaccharide composition predicts risk of necrotising
enterocolitis in preterm infants.Gut 2018, 67, 1064–1070.
29 Wang B，Brand-Miller J.The role and potential of sialic acid in human nutrition［J］.Eur J Clin Nutr，2003，
57 (11) ：1351-1369.
30 Liu Shuan, Li Yukun, Wan Dan, et al. Progress in the biological function of lactoferrin [J]. Journal of Animal
Nutrition, 2020,32 (4): 1508-1515.
31 Gao Yuan, Yang Lijie, Ma Manling. Progress in the immunomodulatory effects of lactoferrin [J]. Pharmacy in
China, 2014,25 (37): 3523-3525.
32 Kang Xinyue, Liu Shicai, Zheng Heng. Multifunctional bioactivity and clinical progression of lactoferrin [J].
Biological Resources, 2018,40 (6): 512-517.
33 Zhang Caixia, Su Yixiang, Yang Yufeng. Progress in lactoferrin and infant health [J]. Chinese Journal of
Children's Health Care, 2016,57 (4): 377-380.
34 Trend S, Strunk T, Hibbert J, et al.Antimicrobial protein and peptide concentrations and activity in human breast
milk consumed by preterm infants at risk of late-onset neonatal sepsis[J].PLos One.2015.10(2):e117038.
35 Ronayne DFP, Baroni A, Sambucetti ME, et al.Lactoferrin levels in term and preterm milk[J].J Am Coll
Nutr.2000, 19(3):370-373.
36 Mastromarino P, Capobianco D, Campagna G, et al.Correlation between lactoferrin and beneficial microbiota in
breast milk and infant's feces[J].Biometals, 2014, 27(5):1077-1086.
37 Hirai Y, Kawakata N, Satoh K, et al.Concentrations of lactoferrin and iron in human milk at different stages of
lactation[J].J Nutr Sci Vitaminol (Tokyo).1990.36(6): 531-544.

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different degrees of antibacterial effect, with broad spectrum antibacterial, it may
benefit from the following mechanism: (1) the antibacterial properties of lactoferrin
from its high iron binding ability, and the growth process of bacteria also need a lot of
iron, lactoferrin can compete and inhibit the use of iron source, achieve the purpose of
antibacterial[]38. (2) for gram-negative bacteria, lactoferrin by positive amino acid
group and bacterial outer membrane anionic lipopolysaccharide (LPS), can inhibit
LPS and other cations (such as Ca 2 +, Mg2 +, etc.), and cause LPS from the cell wall,
bacterial cell wall permeability increase, bacterial cell apoptosis, achieve the effect of
antibacterial, research also proved that the interaction of lactoferrin and LPS can also
enhance natural antibacterial agents (such as lysozyme) antibacterial[,,]394041. (3) For
Gram-positive bacteria, the positive group of lactoferrin can react with the negative
charge brought by the anionic material lipoteichoic acid on the surface of the bacteria,
thus reducing the negative charge of the cell wall, leading to the reaction of lysozyme
and the underlying peptidoglycan, and playing the inhibitory role of lysozyme[]42。
Recent studies have found that lactoferrin has antiviral ability, which may be
achieved through the following ways: (1) lactoferrin interferes with the viral
replication and syncytial process to inhibit the activity of the virus. In vitro results
show that lactoferrin inhibits viral replication in host cells in human plasma and milk,
thereby inhibiting AIDS (AIDS) viral activity[]43. (2) Lactoferrin can inhibit the entry
of viral particles into the host cells by either directly attaching to the virus or by
blocking its cellular receptors. The results show that lactoferrin can interfere with the
transmission of human immunodeficiency virus (HIV-1), blocking its transmission
from dendritic cells to TCD 4 cells by blocking the binding of the virus to epithelial
cellshand over[]44。
Moreover, lactoferrin can play an immunomodulatory role by acting on immune
38 Chilton C H, Crowther G S, Spiewak K, et al.Potential of lactoferrin to prevent antibiotic-induced Clostridium
difficile infection[J].Journal ofAntimicrobial Chemotherapy, 2016, 71(4): 975-985.
39 Di Wei, Jin Haizhu, Du Ming, et al. Status and prospects of lactoferrin research [J]. Jiangxi Journal of
Agriculture, 2012,24 (3): 167-169,175.
40 Kawakami H, Hiratsuka M, Dosako S.Effects of iron－saturated lactoferrin on iron absorption[J].Agricultural
and Biological Chemistry, 1988,52(4): 903-908.
41 Sun Ruiying, Qu Shuqiang. Progress in the effects of lactoferrin supplementation on newborns and infants and
young children [J]. Chinese Journal of Children's Health Care, 2019,27 (2): 168-170.
42 Hagiwara T, Ozawa K, Fukuwatari Y, et al.Effects of Lactoferrin on iron absorption in immature
mice[J].Nutrition Research, 1997, 17(5): 895-906.
43 Viani R M, Gutteberg T, Lathey J, et al.Lactoferrin inhibits HIV-1 replication in vitro and exhibits synergy when
combined with zidovudine[J].Aids, 1999, 13(10): 1273-1274.
44 Laetitia C, Pierre B, Hakim H, et al.Modulation of HIV binding to epithelial cells and HIV transfer from
immature dendritic cells to CD4 Tlymphocytes by human lactoferrin and its major exposed LF-33 peptide[J].Open
Virol J, 2011, 5(1): 27-34.

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cells and cytokines. The results showed that the treatment of recombinant human
lactoferrin (rhLF) vin-120 in enteritis mice increased the number of regulatory T cells
and activated regulatory T cells (Tregs) to secrete IL-17 to relieve inflammation, and
tested the effect of rh LF on CD4 + cells in vitro, which also confirmed that rh LF can
induce the differentiation of CD4 + T cells into Tregs, leading to an increase in the
number of Tregs[]45. In addition, lactoferrin enhances the cytotoxic function of NK
cells and lymphokine-activated killer (LAK) cells[]46. lactoferrinCan directly bind to
the lipidA of LPS embedded in the bacterial surface, And to promote its release,
Disrupt
the
stability
and
permeability
of
the
bacterial
outer
membrane;
Simultaneously in the cells, Free LPS can also bind to sCD 14 on the cell membrane,
The activation of the Toll-like receptor (TLR) 4 signaling pathway to increase the
generation of pro-inflammatory mediators; After the competitive binding of LPS and /
or sCD 14 by lactoferrin, Thus reducing the binding of LPS-sCD 14, Blocking the
activation of the TLR-4 signaling pathway and signaling, A decrease in the secretion
of pro-inflammatory factors; The LF-LPS complex may activate TLR 4 signaling,
Leading to the expression of the proinflammatory components[]47. Lactoferrin can
promote IL-2 synthesis in macrophages, and IL-2 can guide macrophage migration to
sites of inflammation and activate CD4 + T lymphocytes[]48。
2.3 Docosahexaenoic acid and eicosapentetraenoic acid
Docosahexaenoic acid and eicosapentetracenoic acid is two important long
chain polyunsaturated fatty acid (LCPUFA), referred to as DHA and ARA, can be the
precursor fatty acid α -linolenic acid (ALA) and linoleic acid (LA) in the body, the
two is not only constitute two main fatty acids of brain phospholipids, or the central
nervous system development essential nutrients, DHA accounted for 10% of the brain.
It is known that the critical period of brain development is from the beginning of
pregnancy to the second 2 years of birth. During this critical period of development,
brain cells develop and differentiate rapidly, and are extremely sensitive to internal
and external environment changes and stimulation. Dobbing Sands showed that the
weight of the brain increased from about 20g in the second to third months of life to
45 Artym J, Zimecki M, Kruzel M L.Reconstitution of the cellular immune response by lactoferrin in
cyclophosphamide-treated mice is correlated with renewal of T cell compartment[J].Immunobiology, 2003, 207(3):
197-205.
46 Shau H, Kim A, Golub S H.Modulation of natural killer and lymphokine activated killer cell cytotoxicity by
lactoferrin[J].J Leukoc Biol, 1992, 51(2):343-349.
47 Drago-Serrano ME, de la Garza-Amaya M, Luna JS, et al.Lactoferrin-lipopolysaccharide(LPS) binding as key to
antibacterial and antiendotoxiceffects[J].Int Immunopharmacol, 2012, 12(1): 1.
48 Puddu P, Valenti P, Gessani S.Immunomodulatory effects of lactoferrin on antigen presenting cells[J].Biochimie,
2009, 91(1): 11-18.

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about 1200g at 2 years[]49. Martinez et al reported a nearly 30-fold increase in DHA
and ARA content in infant brains from birth to 2 y[,]5051. This also reflects the
importance of DHA and ARA in the growth and development of infants and young
children.
Although fetuses and newborns can also synthesize DHA and ARA by
themselves, these long-chain polyunsaturated fatty acids are not able to meet the
needs of rapid growth[]52. Therefore, early in life growth and development, breast milk
and or breast milk substitutes are the main food sources for DHA and ARA. Brenna
The team analyzed 106 breast milk studies, which selected 65 studies with a total
sample size of 2474 women and found that the mean content of DHA (% ± standard
deviation) was 0.32 ± 0.22% total fatty acids (range: 0.06~1.4%) and ARA was 0.47 ±
0.13% total fatty acids (range: 0.24-1.0%). DHA is less abundant in breast milk than
ARA and more different than ARA. In addition, the results found that the coastal
population had the highest DHA content and was associated with more dietary intake
of seafood, and the lowest DHA content occurred in inland populations and developed
countries, which was associated with low fish intake[]53. Yuhas et al collected 440
breast milk samples from Australia, Canada, Chile, China, Japan, Mexico, Philippines,
the United States and showed that DHA ranged from 0.17 wt% to 0.99 wt%, the
highest value was Japan, the lowest value was samples from Canada and the United
States, and the average DHA content of samples from China was 0.83%. The content
of ARA was stable in several studied countries, from 0.36wt%~0.49wt%, the mean
was 0.41%. The result in China was 0.49% total fatty acids. The mean ± standard
deviation of the absolute content of Chinese DHA is 149.8±10.5mg/L, and the mean ±
standard deviation of the absolute content of ARA was 207.8 ± 12.05 mg / L[]54.
Koletzko B Led his team to review LCPUFA during pregnancy, lactation and infancy,
and showed that the overall average of ARA in breast milk ranged from 0.35% to
0.70% total fatty acids, and the overall mean of DHA ranged from 0.17% to 1.0%
total fatty acids. Based on the results of the literature study, it is recommended that the
amount of DHA is 0.2 to 0.5%, and the minimum amount of ARA should not be lower
49 Dobbing J, Sands J, Quantitative growth and development of human brain[J].Arch.Dis.Child, 1973, 48:757-767.
50 Martinez M, Tissue levels of polyunsaturated fatty acids during early human development [J].J Pediatr.1992,
120(4):S129-38.
51 Martinez M, Mougan I, Fatty acid composition of human brain phospholipids during normal development
[J].J.Neurochem.1998, 71:2528-2533.
52 Lauritzen L, Hansen HS, et al.The essentiality of long chain n-3 fatty acids in relation to development and
function of the brain and retina.Progress in Lipid Research, 2001, 40:1-94.
53 Brenna J T, Varamini B, Jensen RG, et al.Docosahexaenoic and arachidonic acid concentrations in humanbreast
milk worldwide [J].Am.J.Clin.Nutr.2007, 85(6): 1457-1464.
54 Yuhas R, Pramuk K, Lien EL, Human Milk Fatty Acid Composition from Nine Countries Varies Most in
DHA.Lipids, 2006, 41(9):851-858.

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than the DHA content[]55. A study of immune factors and fatty acids between the
University of Reading and the Chinese Center for Disease Control and Prevention in
three different geographical areas in China (Urwin HJ, 2013), Healthy singleton
women from 18 to 22 weeks of pregnancy in Jurong, Jiangsu (river and lake region),
Xushui (inland area) and Rizhao (coastal area), A cross-sectional study collected
colostrum (3 to 5 days), transitional milk (14 days) and mature milk (28 days) for
compositional analysis, See the table below, Higher levels of DHA and ARA in breast
milk are associated with higher levels of immune factors in breast milk, It suggests
that these fatty acids can promote the maturation of the infant intestine and the
immune system[]56. The expert group led by the Chinese Nutrition Society combined
14 domestic reports on DHA and ARA content of breast milk from 1995 to 2013, and
concluded that the median content of DHA of Chinese mothers was 0.42% total fatty
acids, and the median content of ARA was 0.61% total fatty acids.
delspray
fatalism
River and lake
area
foreland
boo-ay
ARA
3～5
0.94%
0.85%
0.88%
14
0.85%
0.80%
0.82%
28
0.76%
0.71%
0.70%
DHA
3～5
0.66%
0.61%
0.41%
14
0.53%
0.53%
0.35%
28
0.50%
0.55%
0.27%
Based on the data of numerous breast milk studies at home and abroad and the
daily fatty acid intake of infants, the recommended intake of DHA and ARA in 0 to 6
months developed by the Food and Agriculture Organization (FAO) in 2010 was
0.1%E~0.18%E (58 mg ~ 104 mg/d) and 0.2%E~0.3%E (115mg~173mg / d),
respectively. The Chinese Nutrition Society recommends that the appropriate intake
(AI, adequate intakes) value of DHA is 100 mg/d and 150mg / d for ARA. The AI of
DHA in infants aged 7 to 36 months is 100 mg/d, based on the recommended AI value
of FAO and EFSA for linoleic acid at this age, 3.0%E~4.5%E, which is considered
sufficient to meet the needs of infants for ARA synthesis, so the AI value of ARA is
not recommended.
55Koletzko B, Lien E, Agostoni C, et al.The roles of long-chain polyunsaturated fatty acids in pregnancy, lactation
and infancy: review of current knowledge and consensus recommendations.J.Petrinat.Med.2008, 36(1):5-14.
56 Urwin HJ, Zhang J, Gao Y, et al.Immune factors and fatty acid composition in human milk from river/lake,
coastal and inland regions of China.Br.J.Nutr.2013, 109(11):1949-1961

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3.4 Bifidobacterium long long subspecies BB536
Bifidobacterium BB536 isolated the intestines of healthy infants in 1969 and has
been approved for infant food in Japan and the United States. It is the first affinity
human Bifidobacterium strain (HRB) recognized by the US FDA. At the same time,
in 2022, it was approved to be included in China's list of edible fungus list.
As of March 2022, more than 220 academic papers were published based on
BB536. A number of clinical studies on infants at home and abroad have proved that
the strain has good food safety. Studies have proved that BB536 is effective in
maintaining intestinal health, improving immunity, and preventing infection.
3 Study purpose and significance
To compare the effects of breast milk oligosaccharide and multinutrient-
optimized infant formula and conventional infant formula on the intestinal health of
infants under 1 year of age. In comparison with breast-fed infants, the effects of
different infant formulas on infant growth and development, cognitive development,
immune function, intestinal comfort, crying, and upper respiratory tract infection were
explored.
4 research contents
This trial is a multicenter, randomized, controlled, parallel, double-blind,
exploratory trial to compare the effects of infant formula, conventional infant formula
on infant formula and breast milk in early life.
240 subjects are expected to be required for this trial. From 0 to 28 days after
birth, infants who had signed informed consent by a guardian were screened.
Guardian according to their own will choose to attend breastfeeding group or artificial
feeding
group
(mother
for
subjective
and
objective
reasons
for
exclusive
breastfeeding, can choose to join artificial feeding group, the researchers shall not
interfere in the decision of the guardian or guide), breast feeding group and artificial
feeding group subjects of 80 cases and 160 cases, respectively.160 subjects in the
artificial feeding group were randomly assigned to the experimental group and the
control group in a ratio of 1:1 ratio. The experimental formula and control formula
were used for artificial feeding or mixed feeding. Artificial feeding was conducted no
less than 2 times a day at 0 to 4 months and no less than 5 times a day at 4 to 12
months. Infants in the breastfeeding group need to be breastfed or mixed fed,
exclusively breastfeeding daily at 0 to 4 months of age (must be fully breastfed 1 day
before the visit), and breastfeeding at least once a day at 4 to 12 months of age.
From the perspective of ethical and subject protection, if the infant and guardian

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are eligible and willing to breastfeeding, we will assign the subject to the breastfed
group, rather than against the wishes of the parent. If the parent / legal guardian is
unable to breastfeed exclusively for objective reasons, or clearly and strongly willing
not to breast, these infants will be randomized to the trial or the control group. It
should be noted that during the decision-making process, the investigator should
encourage the parents / guardian to breastfeeding the infant; rather than interfering or
guiding the decision-making process to induce the test infant to participate in the
artificial feeding group.
Visit 1 will be performed on the 28th day after birth and the intervention period
will last 332 days (slightly changed by clinic visit time) consisting of 3 visits: Visit 2
(90 ± 3 days after birth), Visit 3 (180 ± 5 days after birth), and Visit 4 (360 ± 7 days
after birth). Subjects in each group will be enrolled 1:1 and based on an expected
completion rate of 80%, 64 subjects are expected to complete the trial. Participants
who withdrew from the study before Visit 4, failed in three contacts, met the
withdrawal criteria, seriously violated the trial protocol, or met other withdrawal
criteria were excluded by the investigators.
In addition, indicators and incidence of adverse events (e. g. eczema) will be
recorded and compared during the study. For the purposes of this trial, adverse events
were defined as any adverse and unexpected signs (including other abnormal
laboratory results beyond the study plan), symptoms, disease related to participation
in the clinical trial, and the disease related to the trial product.
5 subject investigated
Infants born 0 to 28 days after birth are screened and who meet all inclusion
criteria and do not meet any exclusion criteria will be assigned to the breastfeeding
group or randomized to the trial / control group.
5.1 Inclusion criteria
5.1.1 Breastfeeding group
Subjects in the breastfeeding group included in this study must meet all of the
following criteria:
· The 0 ~ 28 days after birth;
· Single-child birth;
· The gestational age was from 37 to 42 weeks;
· Birth weight is 2500 ~ 4000 g;

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· Parents or guardians agree to exclude the infant from participating in other
interventional clinical studies during this study;
· Parents or guardians agreed to feed the infants according to the trial protocol.
5.1.2 Artificial feeding group
Subjects in the artificial feeding group included in this study must meet all of the
following criteria:
· The 0 ~ 28 days after birth;
· Single-child birth;
· The gestational age was from 37 to 42 weeks;
· Birth weight is 2500 ~ 4000 g;
· Parents or guardians agree to exclude the infant from participating in other
interventional clinical studies during this study;
· Parents or guardians agreed to feed the baby according to the trial protocol;
· No exclusive breastfeeding mothers for subjective and objective reasons.
5.2 Exclusion criteria
Subjects will not be included in this trial if they meet any of the following
criteria:
· Artificial assisted reproduction;
· The mother suffers from diseases that may endanger the intrauterine growth;
· The mother had gestational diabetes or severe metabolic disease, chronic
disease at the time of pregnancy
·
Having congenital malformations and genetic, chronic, and congenital
diseases that may interfere with the investigation;
· IgE mediated milk protein allergy or factors that increase the risk of milk
protein allergy;
· Having an acute infection or gastroenteritis;
· Functional gastrointestinal diseases, such as gastroparesis;
· Is participating in other clinical trials;

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· The investigator was unable to determine the parents' willingness or ability to
comply with the protocol requirements.
5.3 Exit standard
· Ineligible (ignored during screening, occurred during the study or found by
review);
· Serious protocol deviation occurs;
· Serious non to protocol or test requirements;
· Need to terminate the designated formula due to adverse events or cause
failure to continue following the trial procedures;
· Need to discontinue the designated formula or cause the inability to follow
the trial procedures due to disease progression;
· Withdraw informed consent or remove informed consent;
· Misvisit.

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6 research design
6.1 Test flow chart
6.2 recruit
The investigator will identify suitable infants, contact their parents or guardians
by telephone and otherwise, and make an appointment for informed consent and
subject screening.

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6.3 informed consent
The subject's guardian must personally sign the informed consent form and date
before conducting any trial process. If the version is updated, contact the guardian to
sign the latest version of the informed consent.
A written version of the informed consent was provided to the guardian of the
subject, and the investigator should explain the following: the nature of the trial; the
content of the subject; the requirements of the protocol; known side effects and any
risks involved in participation. The guardian should also be clearly informed that the
baby can be withdrawn from the study at any time for any reason, without affecting
the baby's medical treatment, legal rights, and no obligation to provide a reason for
the withdrawal.
The guardian of the subject was allowed enough time to consider the relevant
information and the opportunity to ask the researcher, their GP or other independent
party to decide whether to enroll the infant in the trial. The subject's guardian will
then sign and date the informed consent form, followed by the investigator giving the
informed consent. The investigator who gave the informed consent must have the
appropriate qualifications and experience and must be authorized by the principal
investigator at each institution. A copy of the signed informed consent form will be
given to the subject's guardian for retention and the original remains at the study
institution.
6.4 dressing by screening
The investigator will ask the guardian, investigate the basic information of the
subject infant, fill in the screening record and the Subject Screening and Enrollment
Registration Form, and the infant will be included in the study after meeting the
inclusion and exclusion criteria. If the guardian chooses to exclusively breastfeed the
infant, the infant will be assigned to the breastfeeding group; if due chooses artificial
or mixed feeding, the infant will be randomized to the trial or control group.
6.5 randomization
6.5.1 Random group table
Randtrial block randomization. The randomization tables were generated
simulated on a computer by an unblinded statistician using the SAS software
according to the random number table. Trial or control milk powder were coded
according to the randomization table. Each center will compete for entry. In clinical
operation, the authorized investigator or the research assistant will distribute the test
or control milk powder according to Powder No. Any deviations should be truthfully

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recorded for evaluation before data analysis.
6.5.2 Sample size and calculation basis
AndStenhoutetal[]57The study enrolled 88 test group, 87 control infants, 34 breastfed infants with 1g /
L 2 ′ / FL and 0.5 g / L LNnT to formula-fed infants, control formula-fed infants and
breast-fed infants. All the fecal samples available at 3 months of age were 58 in the
test group, 65 in the control group, and 34 in the breastfeeding group. They evaluated
the microbiota using 16S rRNA gene sequencing and metagenomics, and found that
the microbiota composition of the test group was different from that of the control
group and closer to that of the breastfeeding group. In this study, the experimental
group, control group and breastfeeding group are planned to include 80 subjects,
calculated by the shedding rate (failure rate) of 20%. The effective sample size of
each group is expected to be 64 cases, which can meet the study needs.
6.6 Interview
6.6.1 Visit 1
Visit 1 will be performed on the 28th day of life and the investigator will obtain
the following information:
· Mode of delivery;
· Pregnancy-related complications;
· Parents' physical condition (height, weight, medical history);
· Parents' living habits (smoking history, drinking history, exercise habits);
· Parents' social and economic background (education level, employment status);
· Growth and development parameters (height, head circumference, chest
circumference, body weight);
· Stool samples (16S rRNA, SCFAs, sIgA);
· Gastrointestinal comfort survey and questionnaire;
· Questionnaire on baby crying condition;
· Questionnaire on infant diarrhea;
· Questionnaire on upper respiratory tract infection in infants;
57Steenhout P , Sperisen P , Martin F , et al.Term Infant Formula Supplemented with Human Milk
Oligosaccharides (2′Fucosyllactose and Lacto‐N‐neotetraose) Shifts Stool Microbiota and Metabolic
Signatures Closer to that of Breastfed Infants[J].The FASEB Journal, 2016, 30.

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· The Bristol Stool Classification Questionnaire;
· Past medical history and current medical history.
6.6.2 Visit 2
Visit 2 will be conducted on Birth Day 90 with a window of 3 days and the
investigator will measure and obtain the following information:
· Growth and development parameters (height, head circumference, chest
circumference, body weight);
· Gastrointestinal comfort survey and questionnaire;
· Questionnaire on baby crying condition;
· Questionnaire on infant diarrhea;
· Questionnaire on upper respiratory tract infection in infants;
· The Bristol Stool Classification Questionnaire;
· Adverse events and serious adverse events.
6.6.3 Visit 3
Visit 3 will be conducted on Post Birth Day 180 with a window of 5 days and the
investigator will measure and obtain the following information:
· Growth and development parameters (height, head circumference, chest
circumference, body weight);
·
Blood samples (hsCRP, analysis of lymphocyte subsets, amino acids,
osteocalcin, alkaline phosphatase, albumin);
· Stool samples (16S rRNA, SCFAs, sIgA);
· Gastrointestinal comfort survey and questionnaire;
· Questionnaire on baby crying condition;
· Questionnaire on infant diarrhea;
· Questionnaire on upper respiratory tract infection in infants;
· The Bristol Stool Classification Questionnaire;
· Adverse events and serious adverse events.

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6.6.4 Visit 4
Visit 4 will be performed on Post Birth Day 360 with a window of 7 days and the
investigator will measure and obtain the following information:
· Growth and development parameters (height, head circumference, chest
circumference, body weight);
· Bailey Infant Development Scale (Version IV);
· Blood samples (hsCRP, lymphocyte subsets, amino acids, osteocalcin, alkaline
phosphatase, albumin);
· Stool samples (16S rRNA, SCFAs, sIgA);
· Gastrointestinal comfort survey and questionnaire;
· Questionnaire on baby crying condition;
· Questionnaire on infant diarrhea;
· Questionnaire on upper respiratory tract infection in infants;
· The Bristol Stool Classification Questionnaire;
· Adverse events and serious adverse events.
6.7 Subjects stopped / withdrew from the trial
The guardian of the subject has the right to withdraw the infant from the trial at
any time. In addition, the investigator may discontinue the subject at any time if the
investigator considers it necessary for reasons including:
· Ineligible (ignored during screening, occurred during the study or found by
review);
· Serious protocol deviation occurs;
· Serious non to protocol or test requirements;
· Termination of the designated formula or failure to follow the trial procedures
due to an adverse event;
· Need to discontinue the designated formula or cause the inability to follow
the trial procedures due to disease progression;
· Withdraw informed consent or remove informed consent;
· Misvisit.

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If a subject withdraws from the trial due to an adverse event, the investigator will
schedule a follow-up visit or telephone visit until the adverse event is resolved or is
stable. Data collected before withdrawal will be used for analysis. If a subject
withdraws from the trial, the subject will not be replaced. The reason for the
withdrawal will be documented in the case report form.
7 Research products
7.1 burden
(1) Ingredients list of test milk powder
The test milk powder was produced by Feihe (Jilin) Dairy Co., LTD. Three
formulations were used for infants aged 0-3 months, 3-6 months and 6-12 months
respectively. All products are produced in strict accordance with Good Production
Practice (GMP), and all indicators meet the requirements of relevant standards and
regulations. Compared with the control milk powder, 2 ′ -fucinose based lactose,
lactose-N-new tetrosaccharide, bifidobacterium longum BB536 are the added
ingredients unique to the test milk powder, and the ingredients are as follows:
0 to 3 months old infants: raw milk, lactose, edible plant blend oil (1,3-linoleic
acid 2-palmitate triglyceride, sunflower oil, coconut oil, flaxseed oil), desalting whey
powder, oligo galactose, whey protein powder, whey protein powder, 2'-fucoose based
lactose, lactose-N-new four sugar, phospholipids, vitamin A, vitamin D, vitamin E,
vitamin K, vitamin B1, vitamin B2, vitamin B6, vitamin B12, Niacin, folic acid,
pantothenate, vitamin C, biotin, choline, sodium citrate, potassium chloride, copper
sulfate, magnesium sulfate, iron pyrophosphate, zinc sulfate, manganese sulfate,
calcium carbonate, calcium hydrogen phosphate, potassium iodate, sodium selenite,
innositol, taurine, docosahexaenoic acid (DHA), arachidonic acid (ARA), lutein,
nucleotide,
disodium
disdium
5'-monophosphate,
5
′,
disodium),
Bb-12,
Bifidobacterium longum B B536.
3-6 months old infants: raw milk, lactose, edible plant blend oil (1,3-linoleic acid
2-palmitate triglyceride, sunflower oil, coconut oil, flaxseed oil), oligo galactose,
whey protein powder, desalting whey powder, whey protein powder, 2'-fucoidan
based lactose, lactose-N-new four sugar, phospholipids, vitamin A, vitamin D, vitamin
E, vitamin K, vitamin B1, vitamin B2, vitamin B6, vitamin B12, Niacin, folic acid,
pantothenic acid, vitamin C, biotin, biotin, choline, sodium citrate, potassium chloride,
copper sulfate, magnesium sulfate, iron pyrophosphate, zinc sulfate, manganese

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sulfate, calcium carbonate, calcium hydrogen phosphate, potassium iodiite, sodium
selenite, inositol, hexanotide (DHA), arachidonic acid (ARA), lutein, nucleotide,
disodium, 5 ′ -monomonophosphate, disodium), lactoferrin, Bb-12, Bifidobacterium
longum B B536.
6-12 months old infants: raw milk, lactose, edible plant blend oil (1,3-linoleic
acid 2-palmitate triglyceride, sunflower oil, coconut oil, flaxseed oil), oligo galactose,
whey protein powder, milk powder, whey protein powder, 2'-fucose lactose, lactose-
N-new tetrosaccharide, phospholipids, vitamin A, vitamin D, vitamin E, vitamin K,
vitamin B1, vitamin B2, vitamin B6, vitamin B12, Niacin, folic acid, pantothenic acid,
vitamin C, biotin, biotin, choline, sodium citrate, potassium chloride, copper sulfate,
magnesium sulfate, iron pyrophosphate, zinc sulfate, manganese sulfate, calcium
carbonate, calcium hydrogen phosphate, potassium iodiite, sodium selenite, inositol,
hexanotide (DHA), arachidonic acid (ARA), lutein, nucleotide, disodium, 5 ′ -
monomonophosphate, disodium), lactoferrin, Bb-12, Bifidobacterium longum B B536.
(2) Control with the ingredients list of milk powder
The control milk powder is commercially available infant formula, with a total
of two formulas, which are suitable for infants aged 0-6 months and 6-12 months
respectively. All the indicators of the products meet the requirements of the relevant
standards and regulations. The list of ingredients is as follows:
0 to 6 months old baby: raw milk, desalting whey powder, edible plant blend oil
(1,3-linoleic acid 2-palmitate triglyceride, sunflower oil, coconut oil, linseed oil),
lactose, oligo galactose, walnut oil, whey protein powder, whey protein powder,
vitamin A, vitamin D, vitamin E, vitamin K, vitamin B1, vitamin B2, vitamin B6,
vitamin B12, Niacin, folic acid, pantothenate, vitamin C, biotin, choline, sodium citrate,
potassium chloride, copper sulfate, magnesium sulfate, iron pyrophosphate, zinc
sulfate,
manganese
sulfate,
calcium
carbonate,
calcium
hydrogen
phosphate,
potassium iodate, sodium selenite, innositol, taurine, levosnitine, dococarpohexenoic
acid (DHA), arachidonic acid (ARA), lutein, lutein, nucleotide (5 ′ -cytitilate, 5 ′,
5 ′ monophosphate, 5 ′, 5 ′ -inolate), bifidobacterium Bb-12.
6-12 months of older infants: raw milk, lactose, desalting whey powder, edible
plant blend oil (1,3-linoleic acid 2-palmitate glyceride, sunflower oil, coconut oil,
linseed oil), milk powder, oligomic galactose, whey protein powder, whey protein
powder, walnut oil, vitamin A, vitamin D, vitamin E, vitamin K, vitamin B1, vitamin

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B2, vitamin B6, vitamin B12, Niacin, folic acid, pantothenate, vitamin C, biotin,
choline, sodium citrate, potassium chloride, copper sulfate, magnesium sulfate, iron
pyrophosphate, zinc sulfate, manganese sulfate, calcium carbonate, calcium hydrogen
phosphate,
potassium
iodate,
sodium
selenite,
innositol,
taurine,
levosnitine,
dococarpohexenoic acid (DHA), arachidonic acid (ARA), lutein, lutein, nucleotide
(5 ′ -cytitilate, 5 ′, 5 ′ monophosphate, 5 ′, 5 ′ -inolate), bifidobacterium Bb-
12.
7.2 Nutritional composition table
（1）Nutrition composition list of test milk powder
Table 1. Nutrient composition list of test samples at 0-3 months of age
project
unit
Every 100 kJ
Every 100g
energy
kJ
100
2097
protein
g
0.60
12.5
fat
g
1.24
26.0
linoleic acid
g
0.20
4.1
α-linolenic acid
mg
20
410
carbohydrate
g
2.5
52.3
vitamin
vitamin A
µg RE
17
360
vitamin D
µg
0.62
13.0
vitamin E
mg α-TE
0.29
6.00
vitamin K1
µg
2.86
60.0
vitamin B1
µg
31
650
vitamin B2
µg
24
500
vitamin B6
µg
19.1
400
vitamin B12
µg
0.072
1.50
niacin
µg
196
4100
folic acid
µg
4.3
90
pantothenic acid
µg
145
3050
vitamin C
mg
3.3
70.0
biotin
µg
0.76
16.0
bilineurine
mg
10.3
215.0
mineral substance
sodium
mg
8
175
potassium
mg
18
385
copper
µg
16.7
350
magnesium
mg
1.5
32
iron
mg
0.24
5.0

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zinc
mg
0.21
4.50
manganese
µg
1.91
40
calcium
mg
17
350
phosphorus
mg
10
220
iodine
µg
5.1
106.0
chlorine
mg
13
280
Se
µg
0.91
19.0
Available ingredients
inose
mg
1.9
40.0
taurine
mg
1.8
38.0
Docosahexaenoic acid
(DHA)
mg
4.6
97.0
Eicosapentetraenoic acid
(ARA)
mg
7.9
164.9
The oligomeric galactose
g
0.14
3.0
2 ′ -fucoose-based lactose
g
0.037
0.78
Lactose-N-
neotetrasaccharide
g
0.019
0.39
1,3-dioleic acid 2-
palmitate triglyceride
g
0.36
7.5
lutein
µg
3.8
80
ribotide
mg
1.4
30.0
lactoferrin
mg
23.8
500.0
Table 2 Nutrient composition list of 3-6 month old test samples
project
unit
Every 100 kJ
Every 100g
energy
kJ
100
2097
protein
g
0.45
9.5
fat
g
1.24
26.0
linoleic acid
g
0.20
4.1
α-linolenic acid
mg
20
410
carbohydrate
g
2.6
55.3
vitamin
vitamin A
µg RE
17
360
vitamin D
µg
0.62
13.0
vitamin E
mg α-TE
0.29
6.00
vitamin K1
µg
2.86
60.0
vitamin B1
µg
31
650
vitamin B2
µg
24
500
vitamin B6
µg
19.1
400
vitamin B12
µg
0.072
1.50
niacin
µg
196
4100
folic acid
µg
4.3
90
pantothenic acid
µg
145
3050

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vitamin C
mg
3.3
70.0
biotin
µg
0.76
16.0
bilineurine
mg
10.3
215.0
mineral substance
sodium
mg
8
175
potassium
mg
18
385
copper
µg
16.7
350
magnesium
mg
1.5
32
iron
mg
0.24
5.0
zinc
mg
0.21
4.50
manganese
µg
1.91
40
calcium
mg
17
350
phosphorus
mg
10
220
iodine
µg
5.1
106.0
chlorine
mg
13
280
Se
µg
0.91
19.0
Available ingredients
inose
mg
1.9
40.0
taurine
mg
1.8
38.0
L-carnitine
mg
0.4
9.0
Docosahexaenoic acid
(DHA)
mg
4.6
97.0
Eicosapentetraenoic acid
(ARA)
mg
7.9
164.9
The oligomeric galactose
g
0.14
3.0
2 ′ -fucoose-based lactose
g
0.037
0.78
Lactose-N-
neotetrasaccharide
g
0.019
0.39
1,3-dioleic acid 2-
palmitate triglyceride
g
0.36
7.5
lutein
µg
3.8
80
ribotide
mg
1.4
30.0
lactoferrin
mg
23.8
500.0
Casein phosphopeptides
mg
6.2
130.0
Table 3 6-12 months of age test samples
project
unit
Every 100kJ
Every 100g
energy
kJ
100
2012
protein
g
0.57
11.5
fat
g
1.07
21.5
linoleic acid
g
0.14
2.8
α-linolenic acid
mg
14
280
carbohydrate
g
2.9
58.3

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vitamin
vitamin A
µg RE
22
450
vitamin D
µg
0.60
12.0
vitamin E
mg α-TE
0.24
4.90
vitamin K1
µg
2.24
45.0
vitamin B1
µg
30
600
vitamin B2
µg
38
760
vitamin B6
µg
14.9
300
vitamin B12
µg
0.060
1.20
niacin
µg
194
3900
folic acid
µg
3.0
60
pantothenic acid
µg
204
4100
vitamin C
mg
3.3
66.0
biotin
µg
0.60
12.0
bilineurine
mg
9.9
200.0
mineral substance
sodium
mg
7
135
potassium
mg
21
420
copper
µg
11.9
240
magnesium
mg
1.6
33
iron
mg
0.30
6.0
zinc
mg
0.20
4.00
manganese
µg
1.89
38
calcium
mg
22
450
phosphorus
mg
13
270
iodine
µg
4.5
90.0
chlorine
mg
14
280
Se
µg
0.60
12.0
Available ingredients
inose
mg
2.0
40.0
taurine
mg
1.9
38.0
L-carnitine
mg
0.5
10.0
Docosahexaenoic acid
(DHA)
mg
4.7
95.0
Eicosapentetraenoic acid
(ARA)
mg
8.0
161.5
The oligomeric galactose
g
0.15
3.0
2 ′ -fucoose-based lactose
g
0.024
0.48
Lactose-N-
neotetrasaccharide
g
0.010
0.21
1,3-dioleic acid 2-
palmitate triglyceride
g
0.25
5.0

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lutein
µg
10.4
210
ribotide
mg
1.5
30.0
lactoferrin
mg
24.9
500.0
Casein phosphopeptides
mg
8.3
167.0
（2）Nutrient composition table of the control samples
Table Nutrient composition list of control samples from 4 0 to 6 months of age
project
unit
Every 100kJ
Every 100g
energy
kJ
100
2173
protein
g
0.48
10.5
fat
g
1.25
27.2
linoleic acid
g
0.17
3.8
α-linolenic acid
mg
19
423
carbohydrate
g
2.6
57.3
vitamin
vitamin A
µg RE
17
360
vitamin D
µg
0.60
13.0
vitamin E
mg α-TE
0.28
6.00
vitamin K1
µg
2.58
56.0
vitamin B1
µg
29
623
vitamin B2
µg
28
600
vitamin B6
µg
18.4
400
vitamin B12
µg
0.069
1.50
niacin
µg
189
4100
folic acid
µg
3.3
72
pantothenic acid
µg
138
3000
vitamin C
mg
3.3
72.0
biotin
µg
0.74
16.0
bilineurine
mg
9.9
215.0
mineral substance
sodium
mg
8
175
potassium
mg
18
400
copper
µg
16.6
360
magnesium
mg
1.4
31
iron
mg
0.21
4.5
zinc
mg
0.18
4.00
manganese
µg
1.98
43
calcium
mg
17
360
phosphorus
mg
10
210

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iodine
µg
4.9
106.0
chlorine
mg
14
297
Se
µg
0.87
19.0
Available ingredients
inose
mg
1.8
40.0
taurine
mg
1.7
38.0
L-carnitine
mg
0.4
9.0
Docosahexaenoic acid
(DHA)
mg
4.5
97.0
Eicosapentetraenoic acid
(ARA)
mg
7.6
164.9
The oligomeric galactose
g
0.08
1.7
1,3-dioleic acid 2-
palmitate triglyceride
g
0.27
5.8
lutein
µg
3.0
66
ribotide
mg
1.4
30.0
Table 5 Nutrient composition list of control samples from 6-to 12 month old
project
unit
Every 100 kJ
Every 100g
energy
kJ
100
2058
protein
g
0.56
11.5
fat
g
1.07
22.0
linoleic acid
g
0.14
2.8
α-linolenic acid
mg
14
280
carbohydrate
g
3.0
61.0
vitamin
vitamin A
µg RE
22
450
vitamin D
µg
0.58
12.0
vitamin E
mg α-TE
0.22
4.50
vitamin K1
µg
1.75
36.0
vitamin B1
µg
30
610
vitamin B2
µg
34
700
vitamin B6
µg
14.6
300
vitamin B12
µg
0.058
1.20
niacin
µg
190
3900
folic acid
µg
2.9
60
pantothenic acid
µg
155
3200
vitamin C
mg
3.3
67.0
biotin
µg
0.78
16.0
bilineurine
mg
9.7
200.0
mineral substance

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sodium
mg
7
135
potassium
mg
21
430
copper
µg
11.7
240
magnesium
mg
1.6
33
iron
mg
0.29
6.0
zinc
mg
0.19
4.00
manganese
µg
1.85
38
calcium
mg
22
450
phosphorus
mg
13
270
iodine
µg
4.4
90.0
chlorine
mg
12
250
Se
µg
0.58
12.0
Available ingredients
inose
mg
1.2
25.0
taurine
mg
1.8
38.0
L-carnitine
mg
0.4
9.0
Docosahexaenoic acid
(DHA)
mg
4.6
95.0
Eicosapentetraenoic acid
(ARA)
mg
7.8
161.5
The oligomeric galactose
g
0.07
1.4
1,3-dioleic acid 2-
palmitate triglyceride
g
0.18
3.8
lutein
µg
10.2
210
ribotide
mg
1.5
30.0
7.3 Sample management
All trial milk powder for the study will be provided to the subjects free of charge.
The trial and control powder were stored in the central warehouse and distributed by
the investigator by subject number and trial protocol.
8 Outcome indicators
8.1 Main end point
Intestinal indicators (16S rRNA, SCFAs).
8.2 Secondary end point
Inflammation index: serum hsCRP; nutritional index: serum albumin; growth
and development index: height, weight, head circumference, chest circumference;
immune index: fecal sIgA, blood lymphocyte subgroup; bone development index:
osteocalcin, alkaline phosphatase; tolerance index: survey of infant gastrointestinal
comfort, infant sleep crying, infant diarrhea and upper respiratory tract infection,

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infant stool Bristol stool classification; cognitive development index: Bailey Infant
Development Scale (IV edition), serum amino acids.
8.3 Safety evaluation index
Adverse events, serious adverse events.
9 Data statistics method
9.1 Data analysis software
Demographic, safety and efficacy data were analyzed using the SAS (version 9.4)
software.
9.2 General data analysis
Statistical description is mainly used, and statistical inference is used for
reference only. Quantitative data were expressed by n (number of subjects with non-
missing values), mean, median, standard deviation, maximum value, and minimum
value, and qualitative data were expressed by frequency and percentage.
The missing values in the statistical analysis involved in this statistical analysis
plan will not be inserted. Outliers are determined in a unified discussion before the
database lock library.
9.3 Analyze the crowd
The Statistical analysis population data sets were defined as follows:
Screening Set (Screening Set): includes all subjects who signed the informed
consent and participated in trial screening.
Full Analysis Set (Full Analysis Set, FAS): All subjects who participated in the
trial and completed at least Visit 1. The FAS set is the main population for the efficacy
analysis.
Compliance Set (Per-Protocol set, PPS): is a subset of the full analysis set
containing subjects who completed all visits to the trial and had no major protocol
deviations during the trial. The PPS set is a secondary population for the efficacy
analysis.
Safety Analysis Set (Safety Set, SS): The Safety Analysis Set includes all
subjects who participated and received test samples with records of safety indicators.
9.4 Subject completion status
To clarify the implementation of the clinical trial and the status of the clinical

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trial analysis set, a clear summary of the subjects from the completion of the clinical
trial, including the following:
（1）Subject Clinical Trial Completion Form, including
‐
Number of screened subjects;
‐
Number of unlisted subjects;
‐
Number of randomized subjects;
‐
Number and percentage of subjects who have completed the clinical
trial;
‐
Number and percentage of subjects withdrawing early from the
clinical trial;
‐
And
to
distinguish
the
number
and
percentage
of
subjects
withdrawing early from the clinical trial for different reasons.
（2）Number and percentages of the different analysis sets.
（3）Protocol deviation summary.
（4）Subject completion in the clinical trial and the analysis set are provided in
a list form.
9.5 Demographic data
Subject demographic data will be summarized according to the full analysis set.
The summary of demographic data should include the following: (1) age (age year) - -
the difference between the date of signed informed consent and the date of birth /
365.25; (2) gender- -male / female; (3) ethnic group- -Han / other.
9.6 adverse event
All reported adverse event names and terminologies need to be coded in the
latest version of the MedDRA. For all adverse event tables, subjects were counted
only once in each preferred term (Preferred Term, PT) and systemic organoids
(System Organ Class, SOC). Unless specifically defined, adverse events were ranked
in descending order of the preferred term frequency in each system organ class. If
several preferred terms in the same system organclass have the same frequency, then
in the literal order of preferred terms.
All adverse events will be summarized by SOC, PT and CTCAE criteria,
including

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
Number of subjects, percentage of adverse events, and number of adverse
events

Number and percentage of subjects with serious adverse events and number
of adverse events

Number of subjects with early withdrawal due to adverse events and number
of adverse events
All adverse events will be displayed as lists.
9.7 efficiency analysis
9.7.1 Main endpoint indicators
Primary endpoint index: Changes in the intestinal flora of infants compared
between the test group and the control group.
9.7.2 statistical analysis technique
16 The lowest ranked taxon O TUs was obtained from sequencing data for
further analysis.
（1）Analysis of the bacterial species diversity profile

The diluability curve (Rarefaction curve) is used to analyze whether the
existing OTU data can reflect the diversity of the sample flora, and the sequencing
depth is used to evaluate whether the sequencing data amount of the sample is
reasonable. Random sampling of sequences is used to construct the number of
sequences drawn and the number of OTU they can represent. When the curve is flat, it
indicates that the amount of sequencing data is reasonable.

Species composition analysis was conducted, and according to the species
information and abundance information of O TUs, the appropriate taxonomic level
(species <genus <family <order <class <phyla) was selected to draw the colony
columns of different groups of samples.

The Alpha Diversity (Alpha diversity) index was used to quantify the
bacterial community characteristics of the samples, as described by the S obs, C hao,
Ace, Shannon, and S impson index. The S obs Index, Chao, index and Ace index can
estimate the number of species in the sample, and the Shannon index and S impson
index reflect the species richness and species uniformity of the sample.
（2）Analysis of microflora composition differences among samples

Differences in species composition between different groups were analyzed
using W ilcoxon (comparing two groups) and K ruskal-Wallis (three groups and above)
to compare differences in Alpha diversity index between groups, if P <0.05.

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
Beta diversity (Beta diversity) analysis was used to use the species
composition or evolutionary relationship between species and abundance information
to calculate the inter-sample distance, and the similarity or difference of community
composition between different grouped samples was explored. The results are shown
in Heatmap diagram.

Using p rincipal component analysis (p rincipal component analysis, PCA)
to assess whether infant samples from different groups can be distinguished. When the
sample composition is more similar, the closer the distance reflected in the PCA plot
is. Samples from different backgrounds may exhibit a distribution of dispersion and
aggregation.

Using W ilcoxon and K ruskal-Wallis tests by LEfSe software to identify
significantly different species between groups and visually show the distribution of
different species between groups at each taxonomic level.
（3）Association analysis of environmental factors

Correlation analysis was used to explore the associations between infant gut
microbiota and environmental factors (sex, birth weight, etc.) in different groups.
Correlation coefficients were calculated using P earson and S pearman when samples
cannot satisfy a continuous normal distribution and a linear relationship. The results
are presented as the Heatmap plots.
10 Management plan for common / serious adverse events
10.1 Common adverse events / serious adverse events
The test and control milk powder contain lactose and milk protein, and some
infants may have symptoms of lactose intolerance or milk protein allergy. Lactose
intolerant populations were excluded at screening to maximize potential hazards. Trial
and control milk powder generally do not result in the occurrence of serious adverse
events.
10.2 Management of the adverse events
An adverse event is an adverse event after a patient or a clinical study subject
receives an investigational product that does not necessarily be causally related to the
trial. Adverse events are laboratory findings of disease, signs or symptoms, and / or
abnormalities during the course of the study. Adverse events include subjects in
contacting the investigator or their personal physician, being examined or given
medical guidance. This may or may not cause the subject's withdrawal from the study.
Adverse events are generally classified as serious or not serious.

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All adverse events during the study should be reported and recorded, regardless
of whether they are considered serious or not serious and treatment-related. Each case
should include the following: subject and date, description of the event, duration,
frequency, degree, severity, actions taken, results and sequelae, and relationship to the
tested product.
10.3 Management of serious adverse events
Record of serious adverse events (SAEs) requires an independent table,
completed by the investigator for each case.
In addition, serious adverse events were reported to the EC by the investigator
through the project leader. Such events should be informed immediately by telephone
and the SAE form reported within 48 hours.
After an adverse event occurs, if further information examination is required to
evaluate the relationship between the adverse event and the test sample, all tests or
laboratory results must be attached to the CRF or follow-up records.
10.4 Subject safeguards
Subjects participating in the clinical trial will be purchased clinical trial liability
insurance. All the subjects use adverse events or serious adverse events during the
clinical trial, and make economic compensation according to the severity of the
adverse event.
11 Ethical requirements
11.1 Protection of subject privacy
The privacy of all participants will be protected and subjects will be identified by
an identification code.
11.2 informed consent
The investigator receives written informed consent from each subject before each
subject performs enrollment screening. The informed consent form will be signed and
dated by the investigator and the subject. The informed consent form is in duplicate:
the first remains in the investigator folder and the second to the subject. Testing could
not be performed on subjects until written informed consent.
11.3 Approval of the ethics committee
The Investigator is to submit the study protocol to the Institutional Review
Board (IRB) / Independent Ethics Committee (IEC) for review. Clinical studies could
not be performed without written approval from the ethics committee.

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Any additions or changes to the future study protocol will require notification of
the IRB / IEC. The IRB / IEC should also be notified if any serious adverse event
occurs during the clinical study.
11.4 Declaration of Helsinki
This study will be conducted in accordance with the principles and provisions of
the Declaration of Helsinki and the subsidiary articles.
12 Quality control and quality assurance
The implementation process of this trial will refer to the trial protocol, the Good
Laboratory Practice for Drug Clinical Trial issued by the State Food and Drug
Administration and the NHC (2020 edition), and comply with relevant laws and
regulations.
The investigator shall strictly follow the protocol; the hospital will manage and
quality control the trial as required; an independent clinical trial monitor (CRA) will
monitor the quality of the test; the CRA will monitor and report the project weekly.
13 Study termination
If it is clear that the study is be continued early before the completion of the
study, the investigator will notify the Ethics committee of the reason in advance. Data
not released / not used in the study, the test samples will be destroyed or returned.