necrotizing enterocolitis ( nec ) is one of the most destructive diseases associated with conditions of neonatal prematurity . despite advances in the science of newborn care , large numbers of cases are occurring in very low birth weight ( vlbw ) neonates , with an incidence of 5 - 10% . in fact , the more premature the infants , the greater will be the incidence of nec . intestinal perforation or colon strictures can also occur in approximately one - third of the affected neonates . however , nec appears to be a multifactorial disease .  intestinal ischemia , pathologic bacterial colonization , and high protein substrate in the intestinal lumen extensive research has been conducted to determine the risk factors , ways of prevention and treatment of nec . different researchers have attempted to find  a way , which has a serious preventative impact on the incidence of nec . some examples are mentioned in the following : due to the presence of many protective factors in breast - milk , its effect on the incidence of nec has been investigated by several researchers . mcguire and anthony in a meta - analysis of four small randomized controlled trials ( rcts ) found that nec were 4 times more common in infants who were fed formula milk than in those receiving breast - milk . in a rct showed that prolonged time of low volume milk ( i.e. slow feed advancement ) in vlbw infants reduced the incidence of nec . however , kennedy and tyson in a cochrane review of three rcts demonstrated that rapid versus slow advancement of feeds in preterm neonates had no significant effect on nec . live enteral micro - organisms supplementations which have a potential health benefit on the host . lactobacillus and bifidobacterium being the most commonly used . caplan and jilling concluded that enteral augmentation with probiotics in preterm neonates can change the course of intestinal inflammation and necrosis and reduce the incidence of nec . in several other clinical trials , researchers found that incidence of neonatal nec was reduced by probiotic preparations . on the other hand , the prebiotics are  nondigestible food components that affect the host beneficially by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon and thereby improving host health . oligosaccharides that are contained in breast - milk sit on the position of microbial receptors and prevent pathogens from binding with epithelial cell walls of infant 's gastrointestine ( gi ) . they have also been found to protect the growth of lactobacilli and bifidobacteria in the gi of breast - fed infants , and therefore , supplementation with enteral oligosaccharides stimulated a bifidogenic intestinal micro - flora with a decrease of pathogens . but based on our web search , the effect of prebiotics on incidence of nec was investigated by only a few researchers . in their studies on the use of prebiotics in preterm infants showed that none of the neonates in their studies was affected by nec . they observed that prebiotic supplementation increased bifidobacteria and lactobacilli colony counts in the stool of preterm neonates . the majority of other studies have examined the different effects of prebiotics except the effect on incidence of nec . given the importance of the nec and the expected properties of prebiotics , in this study we decided to investigate our hypothesis , that is , the evaluation the effect of milk supplementation with prebiotics on the reduction of incidence of neonatal nec in vlbw premature infants . this single - center rct was conducted between december 2012 and november 2013 at the isfahan university of medical sciences in our tertiary neonatal intensive care units ( nicus ) ( alzahra and shahid beheshti hospital nicus ) . preterm neonates were eligible for participation if they had a bw of 1500 g , gestational age ( ga ) 34 weeks and were not fed with formula . when the volume of breast - milk reached 30 ml / kg / day , vlbw infants were enrolled in a case - controlled study within our trial . newborns with : ( a ) asphyxia , ( b ) major congenital anomalies , ( c ) congenital cyanotic heart disease , ( d ) gi system anomalies , ( e ) proven sepsis or infection immediately before starting the study , ( f ) refusing to participate and ( g ) transmission to other department were not included . the effects of short chain galacto - oligosaccharides / long chain fructo - oligosaccharides ( scgos / lcfos ) ( 9:1 ) mixture on the incidence of nec was investigated in two groups of prebiotic ( group p ) and control ( group c ) . we used unequal randomization as 2:1 in which 2 controls were considered against a case in this trial . the neonates included were randomly allocated to two groups who received either a diet of breast - milk with a supplement of prebiotics ( scgos / lcfos mixture ) ( prebiotic group [ p ] ; included 25 subjects ) or breast - milk with no supplements ( control group [ c ] ; included 50 neonates ) . an independent employee divided the infants into two groups based on their file number . in order to select the neonates , randomly , those with an even digit at the end of their file numbers were placed in group p and neonates with their file numbers ending in an odd digit were assigned to group c. care providers were not blinded to an infant 's protocol . the scgos / lcfos mixture was prepared and sterilized by nutricia mmp company ( nutricia mmp , mashhad , iran ) . in both groups , after considering the inclusion and exclusion criteria , the infants were entered in the study . in the prebiotics group ( p ) , 0.5 g / kg / day of scgos / lcfos mixture was initially administered , and was gradually increased until the milk volume reached 70 ml / kg / day . with the volume at 70 - 110 ml / kg / day , the dose of the scgos / lcfos mixture was increased to 1 g / kg / day and with the milk volume at 110 - 150 ml / kg / day , the mixture was increased to 1.5 g / kg / day . next , the scgos / lcfos mixture was added to their diet for one - two additional days . but in control group , no supplements of prebiotics were added to the breast - milk during hospitalization . in both groups , the infants were fed with an initial dose of 20 cc / kg / day when the attending neonatologist decided to initiate enteral feeding . on day 2 , feeding volumes were increased to 40 cc / kg / d ; on the 3 day of the study , volumes were increased to 60 cc / kg / d , and so forth , until a volume of 150 cc / kg / day was achieved . in both groups , the infants were entered in the study when the milk volume reached 30 cc / kg / day . the primary outcome of the study in both groups was the effect of the scgos / lcfos mixture on the incidence of suspected nec . secondary outcomes were feeding characteristics such as milk volumes , feeding intolerance ( gastric residue , e.g. the presence of milk in the stomach 2 h after completion of a feeding ) , abdominal distension , postnatal age when full enteral feeds was achieved , and death of each neonate , which were recorded daily . furthermore , age at the time of discharge from hospital , weight at day 30 and the associations of patent ductus arteriosus ( pda ) and intraventricular hemorrhage ( ivh ) were also determined . modified bell staging criteria for necrotizing enterocolitis this paper is derived from a residency thesis no . the sample size of infants was based on the sample size design for an outcome other than incidence of nec ( no studies were initially designed to assess the effect of prebiotics on the incidence of nec ) , that is , stool colony counts of bifidobacteria and pathogenic bacteria after 7 days of supplementation of a previous study . normally distributed and nonparametric quantitative data were presented as means (  standard deviation [ sd ] ) and median ( range ) , respectively . the numeric variables were compared with the independent t - test or mann  whitney as appropriate . the qualitative variables were presented as frequency ( percent ) . to examine the effect of the intervention on incidence rate of qualitative primary and secondary outcomes , the kaplan meier with log - rank test was used and hazard ratio ( hr ) was calculated . the data were analyzed using the statistical package for the social sciences ( spss ) version 20.0 ( spss inc . , this single - center rct was conducted between december 2012 and november 2013 at the isfahan university of medical sciences in our tertiary neonatal intensive care units ( nicus ) ( alzahra and shahid beheshti hospital nicus ) . preterm neonates were eligible for participation if they had a bw of 1500 g , gestational age ( ga ) 34 weeks and were not fed with formula . when the volume of breast - milk reached 30 ml / kg / day , vlbw infants were enrolled in a case - controlled study within our trial . newborns with : ( a ) asphyxia , ( b ) major congenital anomalies , ( c ) congenital cyanotic heart disease , ( d ) gi system anomalies , ( e ) proven sepsis or infection immediately before starting the study , ( f ) refusing to participate and ( g ) transmission to other department were not included . the effects of short chain galacto - oligosaccharides / long chain fructo - oligosaccharides ( scgos / lcfos ) ( 9:1 ) mixture on the incidence of nec was investigated in two groups of prebiotic ( group p ) and control ( group c ) . we used unequal randomization as 2:1 in which 2 controls were considered against a case in this trial . the neonates included were randomly allocated to two groups who received either a diet of breast - milk with a supplement of prebiotics ( scgos / lcfos mixture ) ( prebiotic group [ p ] ; included 25 subjects ) or breast - milk with no supplements ( control group [ c ] ; included 50 neonates ) . an independent employee divided the infants into two groups based on their file number . in order to select the neonates , randomly , those with an even digit at the end of their file numbers were placed in group p and neonates with their file numbers ending in an odd digit were assigned to group c. care providers were not blinded to an infant 's protocol . the scgos / lcfos mixture was prepared and sterilized by nutricia mmp company ( nutricia mmp , mashhad , iran ) . in both groups , after considering the inclusion and exclusion criteria , the infants were entered in the study . in the prebiotics group ( p ) , 0.5 g / kg / day of scgos / lcfos mixture was initially administered , and was gradually increased until the milk volume reached 70 ml / kg / day . with the volume at 70 - 110 ml / kg / day , the dose of the scgos / lcfos mixture was increased to 1 g / kg / day and with the milk volume at 110 - 150 ml / kg / day , the mixture was increased to 1.5 g / kg / day . next , the scgos / lcfos mixture was added to their diet for one - two additional days . but in control group , no supplements of prebiotics were added to the breast - milk during hospitalization . in both groups , the infants were fed with an initial dose of 20 cc / kg / day when the attending neonatologist decided to initiate enteral feeding . on day 2 , feeding volumes were increased to 40 cc / kg / d ; on the 3 day of the study , volumes were increased to 60 cc / kg / d , and so forth , until a volume of 150 cc / kg / day was achieved . in both groups , the infants were entered in the study when the milk volume reached 30 cc / kg / day . the primary outcome of the study in both groups was the effect of the scgos / lcfos mixture on the incidence of suspected nec . secondary outcomes were feeding characteristics such as milk volumes , feeding intolerance ( gastric residue , e.g. the presence of milk in the stomach 2 h after completion of a feeding ) , abdominal distension , postnatal age when full enteral feeds was achieved , and death of each neonate , which were recorded daily . furthermore , age at the time of discharge from hospital , weight at day 30 and the associations of patent ductus arteriosus ( pda ) and intraventricular hemorrhage ( ivh ) were also determined . the study was approved by the regional ethical review board at the university . written informed consent was taken from parents . the sample size of infants was based on the sample size design for an outcome other than incidence of nec ( no studies were initially designed to assess the effect of prebiotics on the incidence of nec ) , that is , stool colony counts of bifidobacteria and pathogenic bacteria after 7 days of supplementation of a previous study . normally distributed and nonparametric quantitative data were presented as means (  standard deviation [ sd ] ) and median ( range ) , respectively . the numeric variables were compared with the independent t - test or mann  whitney as appropriate . the qualitative variables were presented as frequency ( percent ) . to examine the effect of the intervention on incidence rate of qualitative primary and secondary outcomes , the kaplan meier with log - rank test was used and hazard ratio ( hr ) was calculated . the data were analyzed using the statistical package for the social sciences ( spss ) version 20.0 ( spss inc . , throughout the trial , a total of 131 infants with a bw < 1500 gr and ga  34 weeks were studied . twenty - four infants were excluded because of major congenital anomalies , gi system anomalies , asphyxia and sepsis prior to the start of the study [ figure 2 ] . there were a total of 107 infants who met the eligibility criteria , and 19 infants who refused to participate or were transmitted to other wards . of the 88 neonates enrolled , 34 were assigned to the prebiotic  group ( p ) and 54 to the  control  group ( c ) . nine and four neonates were transmitted to other departments in group p and c , respectively [ figure 2 ] . in our study , 75 neonates were randomized and completed the study [ figure 2 ] . average gas in group p and c were 30.48  2.31 weeks and 30.38  2.53 , respectively ( p - 0.76 ) . average bw in group p was 1262.80  213.35 g. and in group c , 1205.60  177.23 ( p - 0.10 ) . the mean age at the start of the feeding was 4.24  2.00 days and 3.90  1.99 days in group p and c , respectively ( p - 0.48 ) . adverse events such as weight loss , constipation or diarrhea were not observed ( p > 0.999 ) . flowchart of the participants basic and clinical characteristics of study infants * the primary outcomes of the two groups were clearly different in terms of the incidence of . only one neonate ( 4.0% ) , in the prebiotic group developed nec , as compared to 11 infants ( 22.0% ) who had not received scgos / lcfos mixture [ hr : 0.49 ( 95% confidence interval [ ci ] : 0.29 - 0.84 ) ; p - 0.002 ; table 2 ] . except one neonate who developed proven nec in group c , others developed suspected nec in both groups . the median time of the incidence of nec in the control group was 15 ( 12 - 21 ) day of life . primary and secondary outcomes in our study groups * secondary outcomes such as hospitalization , sepsis and time of reaching full volume of milk were also investigated . the median ( range ) of hospital stay time was 16 ( 9 - 45 ) days ( 95% ci : 15.34 - 24.09 ) and 25 ( 11 - 80 ) days ( 95% ci : 25.52 - 34.39 ) in group p and c respectively ( p - 0.004 ; table 1 ] . four neonates ( 16% ) in the prebiotic group suffered from sepsis , but 17 infants ( 34% ) who had not received scgos / lcfos mixture developed sepsis , although the difference was not statistically significant [ hr : 0.60 ( 95% ci : 0.27 - 2.72 ) ; p = 0.79 ; table 2 ] . time to establish a total milk intake was significantly shorter in the prebiotic group than the control group . the median ( range ) time of reaching full enteral feeds ( 150 ml / kg / day ) in group p was 11 ( 7 - 21 ) days ( 95% ci : 10.69 - 13.14 ) and in group c , 14 ( 8 - 36 ) days ( 95% ci : 13.17 - 16.50 ) [ p - 0.02 ; table 1 ] . the occurrence of milk intolerance ( lavage ) was investigated in both groups . 10 ( 40% ) and 20 ( 40% ) infants had lavage in prebiotic group and control group respectively [ p - 0.19 ; table 1 ] . nine neonates ( 36% ) were required cutting off milk ( nonprofit organization ) in prebiotic group as compared to 28 neonates ( 56% ) in control group [ hr : 0.63 ( 95% ci : 0.29 - 1.38 ) ; p - 0.26 ; table 2 ] . average body weights at 30 days of life in the prebiotic group was 1702.80  325.42 g and in control group , 1542.40  270.67 g. it appears that , the weight average at 30 days of life , in prebiotic group was marginally slightly greater , although this was not statistically meaningful [ p - 0.06 ; table 1 ] . the incidence of ivh and pda in prebiotic group was 4 ( 16% ) and 1 ( 11.1% ) respectively , while in the control group it was 11 ( 22% ) and 4 ( 8% ) [ p - 0.08 and p - 0.60 , respectively ; table 1 ] . mortality was similar between the two groups ; only one neonate died in each group [ p - 0.66 ; table 1 ] . in vlbw neonates , interestingly , we observed that enteral supplementation of a prebiotic mixture consisting of neutral oligosaccharides ( scgos / lcfos mixture ) was effective in the reduction of nec in vlbw neonates . the present result is in line with the recent studies which show that increasing the beneficial gut flora by supplementation with probiotic bacteria induces protection against nec in preterm infants . for example in a meta - analysis study , nec in preterm infants was shown to be reduced by the consumption of probiotics . however , few studies have investigated the relationship between enteral prebiotics supplementation in preterm infants and nec . indrio et al . investigated the effect of prebiotics on stool viscosity , gi transport and gastric motility in preterm infants . although these trials were not initially designed to assess the effect of prebiotics on the incidence of nec . they found that nec did not occur in any of the neonates in their studies . in two other studies conducted by westerbeek and modi et al . to verify outcomes other than nec , the difference in the occurrence of nec was not statistically significant in the prebiotic versus control groups . in the intervention group of infants using of formula , in other studies ( e.g. 24 , 32 , 35 - 40 ) may had a negative impact on the study results ; since , it could be rolled as confounding factor . the reason that in the present study supplementation with scgos / lcfos mixture significantly reduced the incidence of nec , may be the existence of formula in other studies , and that its complete removal in our study , had a negative effect , as a confounding factor , on statistically meaningful difference in the incidence of nec between the study groups . therefore , more extensive studies are required to evaluate this factor and its effects on preterm infants . inspecting the various recent articles , indicates that given the positive effects of oligosaccharides on gut flora , there was an idea that prebiotic supplementation can confer protection against neonatal sepsis . sepsis was defined , across the studies , as blood culture being positive , regardless of clinical conditions . found that in the prebiotic - supplemented group , 6 of 55 ( 11% ) infants had > 2 serious infectious episodes compared with 12 of 58 ( 21% ) in the placebo group ( odds ratio : 0.47 ; 95% ci : 0.16 - 1.40 ; p = 0.16 ) . niele et al . in a 1-year follow - up found that the incidence of sepsis / meningitis was similar among infants in prebiotic mixture and placebo groups . srinivasjois et al . , in a systematic review and meta - analysis of three trials involving prebiotic versus control groups , have found that late onset sepsis happened with an relative risk ( rr ) of 1.05 ( 95% ci : 0.45 - 2.44 ) in modi et al . , 0.78 ( 95% ci : 0.53 - 1.16 ) in westerbeek et al . and 0.43 ( 95% ci : 0.09 - 1.99 ) in riskin et al . . observed that the incidence of sepsis was lower in the prebiotic than compared with the placebo group ( 9 of 55 [ 16% ] infants vs. 17 of 58 [ 29% ] infants [ rr : 0.4 , 95% ci : 0.17 - 1.16 , p = 0.10 ] ) . also , modi et al . and riskin et al . , found that neonatal sepsis was lower in the prebiotic in comparison with the placebo group ( [ sf : 10 ; scgos / lcfos : 9 ; p = 0.180 ] ) and ( l : 2 [ 13% ] ; dp : 4 [ 31% ] ; p > 0.05 ) , although the difference was not statistically significant . in our study , there was a trend towards lower neonatal sepsis in the prebiotic group ( p - 4 [ 16% ] ; c : 17 [ 34% ] ; 1.66 [ 95% ci : 0.37 - 3.67 ] ; p = 0.79 ) . accordingly , a larger cohort study , with the large sample size , is required to evaluate the actual relationship between prebiotic and neonatal sepsis in preterm infants . two other interesting results observed in the present study were shorter time to establish a total milk intake and shorter hospitalization period . perhaps , complete removal of formula in our study , was the reason that the time of reaching full feeding volume was meaningfully different between the two groups ( in prebiotic group vs. control group was 11.92  2.97 vs. 14.84  5.85 with a hr 1.24 [ 95% ci : 5.40.44 ] ; p - 0.02 ) . however , in other studies , attendance of formula was serious or semi - serious . defined full enteral feeding volume as 150 ml / kg / day , while westerbeek et al . time to full enteral feeding volume ( median [ range ] ) in prebiotic versus control group was 6 ( 5 - 8 ) versus 7 ( 6 - 9 ) days , p = 0.10 in modi et al . while westerbeek et al . observed that the time required was 10 ( 4 - 48 ) versus 11 ( 7 - 50 ) days , p = 0.47 . stated that the average time of full enteral feeding ( mean  sd ) was 41.0  32.0 versus 54.2  31.9 days , in prebiotic versus control group respectively . with reviewing the literatures , we found only two studies that examined the relationship between prebiotics and length of hospital stay , where no difference was found between the two study groups . indrio et al . found a weight gain per day of 34.90  6.90 versus 34.60  9.46 g in prebiotic versus control group . kapiki et al . reported 22.8  6.0 versus 27.4  7.0 g / day in prebiotic versus control group . moreover , riskin et al . shown that weight at discharge had a mean  sd of 2567  355 versus 2846  667 g. none of the above studies showed any statistically meaningful difference between the prebiotic and control groups . also in other studies , such as mihatsch et al . , average body weights at 30 days of life in prebiotic and control group was 1702.80  325.42 g. and 1542.40  270.67 g , respectively ( p - 0.06 ) . the strengths of the study include the rct design in high risk neonates ( i.e. , vlbw infants ) and the complete removal of formula . the major limitation of this study could be the rather small number of the infants included ( 75 premature neonates ) , even though the results clearly indicated a significant difference between the prebiotic and control groups . another limitation of this study was that supply of prebiotics in our country was very difficult . enteral supplementation with prebiotic significantly reduced the incidence of nec in vlbw infants who were fed exclusively breast - milk . this finding suggests that it might have been the complete removal of formula which caused a synergistic effect between nonhuman neutral oligosaccharides ( prebiotic ) and human oligosaccharides , which , in turn , reduced the incidence of nec and decreased the time to full enteral feed as well as the hospitalization time .
background : necrotizing enterocolitis ( nec ) is one of the most destructive diseases associated with conditions of neonatal prematurity . 
 supplementation with enteral prebiotics may reduce the incidence of nec , especially in infants who fed exclusively with breast - milk . 
 therefore , we compared the efficacy and safety of enteral supplementation of a prebiotic mixture ( short chain galacto - oligosaccharides / long chain fructo - oligosaccharides [ scgos / lcfos ] ) versus no intervention on incidence of nec in preterm infants.methods:in a single - center randomized control trial 75 preterm infants ( birth weight [ bw ] 1500 g , gestational age 34 weeks and were not fed with formula ) on 30 ml / kg / day volume of breast - milk were randomly allocated to have enteral supplementation with a prebiotic mixture ( scgos / lcfos ; 9:1 ) or not receive any prebiotic . the incidence of suspected nec , feeding intolerance , time to full enteral feeds , duration of hospitalization were investigated.results:differences in demographic characteristics were not statistically important . 
 scgos / lcfos mixture significantly reduced the incidence of suspected nec , ( 1 [ 4.0% ] vs. 11 [ 22.0% ] ; hazard ratio : 0.49 [ 95% confidence interval : 0.29 - 0.84 ] ; p = 0.002 ) , and time to full enteral feeds ( 11 [ 7 - 21 ] vs. 14 [ 8 - 36 ] days ; p - 0.02 ] . 
 also duration of hospitalization was meaningfully shorter in the prebiotic group ( 16 [ 9 - 45 ] vs. 25 [ 11 - 80 ] ; p - 0.004 ] . 
 prebiotic oligosaccharides were well tolerated by very low bw ( vlbw ) infants.conclusions:enteral supplementation with prebiotic significantly reduced the incidence of nec in vlbw infants who were fed exclusively breast - milk . 
 this finding suggests that it might have been the complete removal of formula which caused a synergistic effect between nonhuman neutral oligosaccharides ( prebiotic ) and human oligosaccharides .