Aim- To compare fortification of EBM with PTF to that of HMF with respect to anthropometric parameters and body composition among very preterm and small for gestational age neonates

Primary objective-

To compare EBM fortified with PTF vs HMF in terms of weight gain (g per kg per day) (at 40w PMA or discharge, whichever is earlier)

Secondary objectives-

I.             To assess other anthropometric measures in PTF vs HMF group (at baseline and 40w PMA or discharge)- as mentioned in secondary outcomes

II.           To assess body composition by DEXA (for a sub-group reaching 40w PMA during hospital stay) - as mentioned in secondary outcomes

III.         To assess co- morbidities in PTF vs HMF group- as mentioned in secondary outcomes

Study design- Open label non-inferiority randomized controlled trial

Study site- The study will be carried out in NICU and Dept of Endocrinology in AIIMS Rishikesh, a tertiary care teaching institute located in the state of Uttarakhand, India

Study duration- 18 months; after obtaining institutional ethical clearance and registration in Clinical Trial Registry of India (CTRI). Study will be discontinued once the recruitment is over.

Methodology-

Before starting fortification of a potentially eligible neonate, the parents will be approached for consent. Eligible neonates will be enrolled after obtaining written informed consent from the parents.

Randomization, allocation concealment and blinding-

A computer-based variable stratified block, 1 -is-to- 1, random sequence by the statistician not involved in the study

The allocation sequence will be kept in numbered, sealed, opaque envelopes in the NICU. Envelope will be opened sequentially according to strata (less than 1000g vs more than equal to 1000g) when indicated for an eligible neonate.

Blinding to caregivers is not possible due to the nature of intervention.

Intervention-

Neonates reaching feed of 100ml per kg per day enteral feeds and consuming at least 75 percent of feeds as EBM will be enrolled after obtaining written informed consent from one of the parents.

They will be randomized into 2 groups after obtaining due consent-

Group A (Intervention arm)- EBM with PTF

Group B (Control arm)- EBM with HMF

Baseline assessments

Baseline anthropometric details will be recorded prior to initiation of fortification. Fortification will be commenced in accordance with the study’s standard operating procedures (SOPs).

Intervention: Fortification with PTF

In the PTF group, 1 g of preterm formula powder will be added to every 25 ml of expressed breast milk (EBM). Sachets containing the prescribed quantity of PTF will be prepared by the pharmacy for each randomized neonate.

The nursing staff and mothers will be trained to prepare feed as per SOP.

 Comparator: Fortification with HMF

 In the HMF group, fortification will be carried out as per the manufacturer’s instructions (1 sachet per 25 ml of EBM).

Feeding regimen

A uniform target feed volume of 180 ml per kg per day will be maintained across both study groups.

Nutrition audit and supplementation

A pre-study nutrition audit will be conducted, assuming a daily breast milk intake of 180 ml per kg, to identify the need for additional supplementation after fortification.

Based on the audit findings-

PTF group- will receive iron drops, calcium and phosphate, vitamin D, and multivitamin syrup.

HMF group- will receive only vitamin D supplementation as per unit protocol.

This is to ensure that both groups achieve the recommended dietary allowance of micronutrients as per ESPGHAN 2022 guidelines.

Monitoring and adjustments

Weekly nutritional and anthropometric audits will be performed according to unit policy.

If weight gain is less than 10 g per kg per day, neonates will be evaluated for systemic causes (e.g. anemia, late metabolic acidosis). If no cause is identified, feed volume will be increased to 200 ml per kg per day. If weight gain remains less than 10 g per kg per day for two consecutive weeks, crossover to the alternate fortifier will be planned and recorded duly. Fortification will be advised until the neonate is discharged or 40 w postmenstrual age, whichever occurs earlier.

SFT, BMI will be measured at baseline and endpoint of intervention. BMC, BMD, fat mass, lean body mass will be measured for in a sub-group of neonates those reaching 40 w PMA during hospital stay by DEXA.

Occurrence of co-morbidities including: feed intolerance, NEC, mortality and EUGR will be recorded and reported as the event takes place.

All neonates will be monitored for all trial related or non-related adverse events during the hospital stay and appropriate care will be provided to the patient.

In case of any adverse event, treatment of the patient and any compensation related issues would be taken care as per new drugs and clinical trials rules 2019.

Weaning off fortification will be done as per unit protocol.

Sample size calculation

The non-inferiority limit of PTF in comparison to HMF was reported as 2g per kg per day in a previous RCT (Chinappan et al) Assuming a similar non inferiority limit as well as standard deviation of 4g the sample size of 50 per arm was calculated with 5 pc alpha error and 80 pc power. Considering an attrition rate of 10 pc, a total of 110 SGA neonates (55 per arm) will be enrolled in the study.

Data collection

Data will be collected prospectively on baseline characteristics and clinical outcomes (weight gain per day, length, head circumference, skinfold thickness, BMI). Co-morbidities will also be recorded at regular interval and body-composition parameters will be collected at discharge or 40w PMA whichever is earlier.

Statistical methods

Microsoft Excel 2020 will be used to collate all the data sets for participating neonates. STATA version 15.0 will be used for statistical analysis. All analysis will be done on intention to treat basis. A secondary per protocol analysis will also be performed for the primary outcome. Data assessor will remain blinded regarding the group allocation. Categorical data will be presented as numbers and percentages while continuous variables will be presented as mean and standard deviation (SD) or median (IQR), depending on the distribution. The Student t test will be used for normally distributed variables, and the Wilcoxon rank-sum test for skewed variables. A p-value of less than 0.05 will be considered statistically significant.

Consent and Ethical Clearance

A written informed consent will be taken from the parents before enrolling the neonate for the study. Also, ethical clearance from the Institutional Ethical Committee will be sought followed by registration with the Clinical Trials Registry of India (CTRI) prior to study initiation.

Conflict of Interest The investigators have no conflicts of interest.

Financial implications

The cost of fortification in the control arm will be handled as per standard practice according to unit policy. For the intervention arm, we have applied for funding to Medical Research Unit (MRU) in the institute.