Omega-3 Long Chain Polyunsaturated Fatty Acid (LCPUFA) Supplementation in Very Low Birth Weight Infants for The Prevention Retinopathy of Prematurity

Calculated using RNA extraction from blood, then quantitative polymerase chain reaction (qPCR) analysis. Biomarker significance: STAT3: role in hypoxia pathway leading to ROP (retinopathy of prematurity). Higher STAT3=greater ROP risk PPAR-ɣ: protective anti-angiogenic factor. Higher PPAR-ɣ=lower ROP risk Delta Ct meaning: qPCR gene expression analysis outputs Ct values for each genetic sample tested. A Ct value is the number of qPCR amplification cycles required for fluorescence, a proxy of gene expression, to cross a threshold. Lower Ct means less cycles of gene amplification needed for detectable fluorescence, therefore higher gene expression. Then target gene expression is calculated relative to a housekeeping control gene. Delta Ct=Ct(target gene)-Ct(control). Therefore, a HIGHER delta Ct value corresponds to a LOWER gene expression of the gene of interest relative to control.

Time frame: T3 as defined in study protocol: Prior to discharge from hospital coinciding with time that ROP may be present, ≥35 weeks adjusted age.

Population: Due to low RNA yield and significant participant drop-out by the final time point, we only had one participant from the Omegaven group with a T3 blood sample that yielded enough RNA for gene expression analyses. As a result we were limited to analyzing only PPAR-gamma and STAT-3 and were unable to conduct statistical analyses on this time point.~One blood sample was analyzed per participant.

Calculated using RNA extraction from blood, then quantitative polymerase chain reaction (qPCR) analysis. Biomarker significance: STAT3: role in hypoxia pathway leading to ROP (retinopathy of prematurity). Higher STAT3=greater ROP risk PPAR-ɣ: protective anti-angiogenic factor. Higher PPAR-ɣ=lower ROP risk STC-1: stress response protein. Higher STC-1=lower ROP risk Delta Ct meaning: qPCR gene expression analysis outputs Ct values for each genetic sample tested. A Ct value is the number of qPCR amplification cycles required for fluorescence, a proxy of gene expression, to cross a threshold. Lower Ct means less cycles of gene amplification needed for detectable fluorescence, therefore higher gene expression. Then target gene expression is calculated relative to a housekeeping control gene. Delta Ct=Ct(target gene)-Ct(control). Therefore, a HIGHER delta Ct value corresponds to a LOWER gene expression of the gene of interest relative to control.

Time frame: T0 as defined in study protocol: prior to parental nutrition, within first three days of life

Population: Due to low RNA yield, we were unable to analyze all biomarkers of interest and could only analyze up to 3 biomarkers per sample. We chose to analyze STC-1 in the remaining batch of samples post-COVID-19 pandemic after updated literature review. This low RNA yield is why some groups below (e.g. Omegaven STC-1) have fewer analyzed blood samples than the other groups.~One blood sample was analyzed per participant.

Calculated using RNA extraction from blood, then quantitative polymerase chain reaction (qPCR) analysis. Biomarker significance: STAT3: role in hypoxia pathway leading to ROP (retinopathy of prematurity). Higher STAT3=greater ROP risk PPAR-ɣ: protective anti-angiogenic factor. Higher PPAR-ɣ=lower ROP risk STC-1: stress response protein. Higher STC-1=lower ROP risk Delta Ct meaning: qPCR gene expression analysis outputs Ct values for each genetic sample tested. A Ct value is the number of qPCR amplification cycles required for fluorescence, a proxy of gene expression, to cross a threshold. Lower Ct means less cycles of gene amplification needed for detectable fluorescence, therefore higher gene expression. Then target gene expression is calculated relative to a housekeeping control gene. Delta Ct=Ct(target gene)-Ct(control). Therefore, a HIGHER delta Ct value corresponds to a LOWER gene expression of the gene of interest relative to control.

Time frame: T1 as defined in study protocol: 5 days after parenteral nutrition is started; grace period +/-3 days therefore total 2-8 days after parenteral nutrition started.

Population: Due to low RNA yield, we were unable to analyze all biomarkers of interest and could only analyze up to 3 biomarkers per sample. We chose to analyze STC-1 in the remaining batch of samples post-COVID-19 pandemic after updated literature review. This low RNA yield is why some groups below (e.g. Omegaven STC-1) have fewer analyzed blood samples than the other groups.~One blood sample was analyzed per participant.

Calculated using RNA extraction from blood, then quantitative polymerase chain reaction (qPCR) analysis. Biomarker significance: STAT3: role in hypoxia pathway leading to ROP (retinopathy of prematurity). Higher STAT3=greater ROP risk PPAR-ɣ: protective anti-angiogenic factor. Higher PPAR-ɣ=lower ROP risk STC-1: stress response protein. Higher STC-1=lower ROP risk Delta Ct meaning: qPCR gene expression analysis outputs Ct values for each genetic sample tested. A Ct value is the number of qPCR amplification cycles required for fluorescence, a proxy of gene expression, to cross a threshold. Lower Ct means less cycles of gene amplification needed for detectable fluorescence, therefore higher gene expression. Then target gene expression is calculated relative to a housekeeping control gene. Delta Ct=Ct(target gene)-Ct(control). Therefore, a HIGHER delta Ct value corresponds to a LOWER gene expression of the gene of interest relative to control.

Time frame: T2 as defined in study protocol: 5 days after enteral nutrition full feeds have arrived; grace period +/-3 days therefore total 2-8 days after full enteral nutrition arrived.

Population: Due to low RNA yield, we were unable to analyze all biomarkers of interest and could only analyze up to 3 biomarkers per sample. We chose to analyze STC-1 in the remaining batch of samples post-COVID-19 pandemic after updated literature review. This low RNA yield is why some groups below (e.g. Omegaven STC-1) have fewer analyzed blood samples than the other groups.~One blood sample was analyzed per participant.

Number of patients with retinopathy of prematurity severe enough to require laser treatment by the adjusted age of 40 weeks, as assessed by weekly ROP screenings from approximately 31 weeks through 40 weeks adjusted age.

Time frame: approximately 31 to 40 weeks (adjusted age = gestation + post-natal age)

Furthest severity stage of ROP achieved by patients in Arm 1 compared to Arm 2, per eye as assessed by weekly ROP screenings from approximately 31 weeks through 40 weeks adjusted age. Severity staging was determined in an eye exam per accepted clinical guidelines by a trained clinician and retinopathy of prematurity specialist. Briefly, staging is assigned based on the junction of the vascularized and avascular retina when viewed using indirect ophthalmoscopy. The higher the stage, the more severe the ROP. Per the American Association for Pediatric Ophthalmology and Strabismus, * Stage 0: no clear demarcation line between vascularized and non-vascularized retina * Stage 1: demarcation line that separates normal from premature retina * Stage 2: ridge with height and width * Stage 3: growth of fragile new abnormal blood vessels

Time frame: approximately 31 to 40 weeks (adjusted age = gestation + post-natal age)

Population: As noted previously, 21 patients were enrolled in the Omegaven group, however for outcomes only 20 were analyzed because 1 patient was dropped from the study immediately after enrollment (deemed by the physician to be too sick from anemia and never started Omegaven).

We measured concentrations of basic fatty acids in the blood plasma samples: eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and arachidonic acid (AA). Blood samples were processed by the University of California San Diego lipidomics core and fatty acid concentrations in pmol/ml plasma were determined using gas chromatography-mass spectrometry.

Time frame: T2 as defined in study protocol: 5 days after enteral nutrition full feeds have arrived; grace period +/-3 days therefore total 2-8 days after full enteral nutrition arrived.

Population: As stated in limitations or caveats, due to delays caused by the COVID-19 pandemic, funding limitations, and a clinical assessment of ROP outcomes in standard of care versus Omegaven infants, we elected to not conduct full fatty acid analyses for our remaining samples. As a result this data is only a pilot analysis and participant numbers were limited, and we could not conduct reliable statistical analyses for this outcome.