Marked Reduction in T-Cell Receptor Excision Circles Observed in Neonates with Severe Respiratory Distress Syndrome

Neonatal Respiratory Distress Syndrome (RDS) remains among the leading causes of morbidity and mortality in preterm and low birth weight infants. Treatment with mechanical ventilation and early administration of exogeneous surfactant improve outcomes. RDS severity impacts ventilation duration and surfactant dosing, increasing the risk of associated side effects. Better markers are required to predict disease course and determine which neonates could benefit from early, aggressive treatment. In our study we have shown how T cell marker T cell Receptor Excision Circles (TREC), quantified shortly after birth, can predict RDS severity in preterm and very low birth weight infants. TREC copy numbers are significantly lower in neonates who go on to develop severe RDS when compared to those who will suffer from no or mild RDS. This trend persists when accounting for confounders such as gestational age and birth weight. Our findings suggest that TREC could potentially serve as a reliable biomarker for assessing RDS disease severity, thus dictating the timely and appropriate management of Neonatal RDS. Marked Reduction in T-Cell Receptor Excision Circles Observed in Neonates with Severe Respiratory Distress Syndrome Erez Rechavi 1,3*, Liran Tamir-Hostovsky 2,3, Chava Rosen 2,3, Nadav Sagiv 4, Shlomo Almashanu 4, Tzipora Strauss 2,3 1 Pediatrics A Department, Edmond and Lily Safra Children’s Hospital, Sheba Medical Center, Ramat Gan, Israel. 2 Department of Neonatology, Edmond and Lily Safra Children’s Hospital, Sheba Medical Center, Ramat Gan, Israel. 3 Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel. 4 National Newborn Screening Program, Israeli Ministry of Health, Sheba Medical Center, Ramat Gan, Israel. * Correspondence: [email protected], Phone Number: +972-523904737 Mailing Address: Department of Pediatrics A, Sheba Medical Center, Sheba Road 2, Ramat-Gan, Israel.

Neonatal RDS, Biomarker, TREC TREC as a Biomarker for Neonatal RDS

Neonatal Respiratory Distress Syndrome (RDS) remains among the leading causes of morbidity and mortality in preterm and low birth weight infants. Treatment with mechanical ventilation and early administration of exogeneous surfactant improve outcomes. RDS severity impacts ventilation duration and surfactant dosing, increasing the risk of associated side effects. Better markers are required to predict disease course and determine which neonates could benefit from early, aggressive treatment. In our study we have shown how T cell marker T cell Receptor Excision Circles (TREC), quantified shortly after birth, can predict RDS severity in preterm and very low birth weight infants. TREC copy numbers are significantly lower in neonates who go on to develop severe RDS when compared to those who will suffer from no or mild RDS. This trend persists when accounting for confounders such as gestational age and birth weight. Our findings suggest that TREC could potentially serve as a reliable biomarker for assessing RDS disease severity, thus dictating the timely and appropriate management of Neonatal RDS.

Respiratory Distress Syndrome (RDS) is among the leading causes of morbidity and mortality in preterm infants(1). RDS occurs primarily in preterm and low birth weight infants but can also affect term neonates with risk factors including maternal diabetes or congenital hypothyroidism. RDS is caused by alveolar surfactant deficiency which maintains alveolar surface tension and prevents micro-atelectasis that could lead to a decrease in lung volume and pulmonary edema (2,3) RDS usually occurs shortly after birth and affected newborns usually require oxygen supplementation and in severe cases mechanical ventilation. Administration of exogenic surfactant is the treatment of choice. Within a few days, endogenic production of surfactant is usually accelerated and resolution of RDS commences(4,5). Prenatal steroids administered to mothers at risk of preterm delivery have proven highly effective in preventing RDS and have subsequently decreased morbidity and mortality(6). Most glucocorticoids cross the placenta and promote surfactant production and secretion into fetal alveoli. Prenatal steroid treatment for the prevention of RDS is therefore standard of care, with two doses of IM betamethasone administered to the mother 12 hours apart. Currently there are no good biomarkers for predicting RDS severity and its complications – bronchopulmonary dysplasia (BPD) or mortality(1). For a lab marker to be effective in guiding management of RDS in the neonatal intensive care unit (NICU), it needs to be readily testable at or immediately after birth, standardized for gestational age and birth weight and have some mechanistical link to the etiology of RDS, so that it correlates with its severity. In this study we evaluate T cell receptor excision circles, TRECs, as a possible biomarker for RDS severity. During T cell receptor (TCR) production, a unique TCR is created in each individual T cell via a mechanism termed “somatic recombination” involving semi-random selection of gene segments within the TCR locus. Gene segments not selected are excised from the genome and enclosed into an episomal DNA circle termed T cell Receptor Excision Circle (TREC). Quantitative Polymerase Chain Reaction (qPCR) of TREC enables simple, accurate quantification of naive T cell production and is increasingly being used for clinical assessment of various conditions(7–9). In many countries, TREC measurement in dried blood spots (DBS) is routinely performed for every newborn at 48-72 hours post birth, as part of national newborn screening programs, to detect neonates born with a severe, life-threatening immunodeficiency(10–12) The fetal adaptive immune system, like its respiratory system, develops throughout pregnancy. TREC increases incrementally from gestational week to week, with a “maturational leap” around 28 weeks gestation. We have previously established normal TREC values in DBS for newborns born at different gestational ages and with different birth weights(13). The rationale for using TREC as a marker for RDS severity is based on the similar time frame of development of the immune and respiratory systems in the fetus, both are affected by the presence or absence of steroids, and TREC measurements are readily available through neonatal screening as early as 48-72 hours after birth. Using TREC data from the Israeli newborn screening program (NBS) and clinical data collected in a tertiary hospital NICU, we examine the correlation between neonatal TREC copy numbers and RDS related clinical outcomes in extremely premature and low birth weight neonates.

Clinical Data Collection The study population included extremely premature (gestational age =< 27 weeks gestation) or extremely low birth weight (birth weight =< 1,000 grams) infants, born at Sheba Medical Center between October 2015 and August 2020. Epidemiologic and clinical data regarding the neonate, the pregnancy and the mother was collected. For twin and triplet pregnancies, one infant was randomly selected for the study and its siblings excluded. Neonates born to mothers with proven amnionitis were excluded because of the possible effects of infection on TREC counts. Overall, 101 preterm infants were included in the study. The study was approved by the Sheba Medical Center institutional review board, IRB number SMC7454-20. Patient consent was not required for this study, after de-identification. TREC quantification in DBS TREC copy numbers for preterm infants included in the study were extracted from the computer archives of the Israeli National Center for Newborn Screening. Data entries with missing information or containing apparent typing errors were removed from analysis, as were entries for samples with poor DNA amplification. The Israeli SCID NBS program uses the commercial EnLiteTM Neonatal TREC kit (Wallac Oy, Mustionkatu 6, FI-20750 Turku, Finland). Briefly, DBS punches of 1.5 mm diameter are inserted into a black, 96-well PCR plate. DNA is eluted without extraction. Next, PCR amplification of TREC and beta-actin, an internal control for each specimen, is performed. Four PCR plates are processed in parallel, each plate containing a standard TREC curve in triplicates, as well as positive and negative controls for both targets(14). TREC adjusted to gestational age and birth weight As both TREC and our primary end-point, RDS, are significantly affected by gestational age and birth weight, we adjusted TREC copy numbers to these factors. Using our previously published results delineating TREC values in over 180,000 healthy newborns as a standard curve (15), TREC copy numbers for each infant in the current cohort were divided by mean TREC copy number for their gestational age (week) to receive a value termed ”TREC adjusted for GA”. Similarly, TREC was divided by mean TREC copy number for birth weight range (each group range consisting of 100 grams, i.e. 800-900 grams, 700-800 grams etc.) to receive a value termed ”TREC adjusted for BW”. Clinical Definitions RDS was defined based on clinical and radiological assessment. RDS was further classified as severe if mechanical ventilation over 72 hours was required. Statistical Analysis Statistical analyses were performed using SPSS software (IBM Corp. Released 2016. IBM SPSS Statistics for Windows, Version 24.0. Armonk, NY: IBM Corp). Given the skewed distribution of the data, the Mann-Whitney U test and the Kruskal-Wallis test were used to compare continuous variables between groups. Correlation between continuous variables was assessed using the Spearman’s rank correlation coefficient. All statistical tests were two-tailed. Differences were considered statistically significant when the p value was less than 0.05\RL.

Overall, 101 extremely preterm or extremely low birth weight infants were included in the study. 81 (80%) infants developed RDS, and 45 (44.5%) infants developed severe RDS. Table 1 shows the statistical correlation between various maternal and pregnancy parameters and severe RDS development. As previously shown, severe RDS was significantly correlated with younger gestational age and with lower birth weight. In our cohort, 49 (48.5%) of infants were males, but male gender was not associated with greater RDS severity. As expected, most infants were delivered via cesarian section (81/101). Vaginal delivery was correlated with an increased risk of severe RDS compared with cesarian delivery. Pre-eclampsia was positively correlated with no- or mild RDS in our cohort, whereas premature rupture of membranes (PROM) was more prevalent in neonates with severe RDS. TREC in extreme prematurity and very low birth weight As previously shown by us and others, TREC copy numbers in DBS from neonates in our study positively correlated with gestational age and birth weight (Fig. 1A,B). In opposition to previous findings, however, in our cohort TREC was more positively correlated with birth weight than with gestational age. As evident from both plots in Figure 1, TREC values were extremely variable within groups of infants born at the same gestational age or birth weight. As previously described, female neonates in our study had higher TREC copy numbers when compared to male neonates, albeit in a non-significant manner (Fig. 2). Correlation between antenatal steroids and TREC 96 of the 101 infants (95%) in our study received at least one course (two doses) of prenatal steroids. As expected, all five infants who did not receive prenatal steroids developed RDS, and 4/5 developed severe RDS. 53 infants received one course of prenatal steroids, of which 47 (89%) developed RDS but only 28 (53%) developed severe RDS. Finally, 43 infants received upwards of a full course (3 or 4 doses), 29 (67%) of whom developed RDS and only 13 (30%) developed severe RDS. We sought to examine the correlation between prenatal steroid treatment and TREC copy numbers. The mean (SEM) TREC copy number for the group of infants who did not receive prenatal steroids was 51.5 (±12.8), compared with 111.3 (±7) in those who received steroids (median 50 vs 97.5, p value = 0.060). This trend, of insignificantly lower TREC copy numbers in those who did not receive steroids compared to those who did, continues when adjusting TREC to gestational age or birthweight. TREC and RDS severity TREC copy number was inversely correlated with RDS severity in a statistically significant manner in our cohort (Fig. 3). Mean TREC copy number in infants with severe RDS was 83.9 (±9.4), compared with 120.7 (±10.8) in infants with mild RDS (p value = 0.012) and 140.8 (±15.6) in infants with no RDS (p value = 0.002). Statistical significance is lost when adjusting TREC copy numbers to gestational age (p value = 0.76 between severe RDS and no RDS), but the trend remains (Fig. 4A). Statistical significance remains when adjusting TREC copy numbers to birth weight (severe RDS and no RDS p value = 0.021, severe RDS and mild RDS p value = 0.032) (Fig. 4B). Analyses of TREC and RDS severity according to gender shows that while female neonates had higher TREC copy numbers overall, those who developed severe RDS had significantly lower TREC copy numbers than female neonates with no or mild RDS (Fig. 5).

We have shown here that TREC copy numbers are significantly lower in neonates who develop severe RDS compared to those with no or mild RDS. TREC quantification shortly after birth could therefore potentially be used as a predictive marker for RDS severity. Gestational age, birth weight, gender, mode of delivery and maternal pre-eclampsia were found to affect risk for RDS severity in our cohort. As has been shown before, extreme prematurity and very low birth weight are the most significant risk factors for RDS in general and severe RDS in particular(16). Early gestational age and low birth weight are also major factors in low TREC copy numbers, making it difficult to untangle their impact on the link between low TRECs and RDS severity(13,17,18). However, our data suggests that even after adjusting for gestational age and birth weight, lower TREC copy numbers are indicative of an increased risk for developing severe RDS. In other words, while two neonates born in week 25 of gestation are already at major risk of developing severe RDS, the one with the lower TREC count may be at greater risk than its counterpart. As previously shown, TREC copy numbers are lower in male newborns, compared with female newborns(13). Similarly, severe RDS is more prevalent in male newborns. The correlation between low TREC copy numbers and severe RDS was far more pronounced among female neonates in our study. It is possible that for female infants, who are generally less prone to severe RDS, TREC measurement could prove even more valuable as a predictive tool. Twinhood was more prevalent in the non-severe RDS groups in our cohort, albeit in a non-statistically significant manner. This could be attributed to the slightly more advanced mean gestational age of twins in our cohort compared with singletons (27.4 vs 28.3 GA, weeks), slight majority of female twins in our cohort (56.8% compared with 46% of singletons), or simply small sample size. Neonates born by cesarian delivery had significantly lower rates of severe RDS compared with those born by vaginal delivery. This finding can be attributed to the far lower mean gestational age of neonates born vaginally in our cohort (25.76 mean GA vs 28.43 in the cesarian delivery group, p value < 0.001), as many of those born via cesarian delivery were neonates with intrauterine growth restriction (IUGR), included in the study for their low birth weight and not prematurity. Exogeneous steroid administration, in addition to inducing surfactant production and lowering RDS, is known to have wide ranging effects on the immune system. Glucocorticoids also affect T cell development in myriad, sometimes in contradictory ways(19,20). In this study, TREC copy numbers were much lower (less than half) in neonates who did not receive prenatal steroids when compared to those who did. This finding did not cross the threshold for statistical significance (p=0.6), on account of very few neonates in the group that did not receive prenatal steroids. The main limitation of this study is a sample size that is not large enough to overcome variability of TREC copy numbers among newborns. Additionally, TREC measurement was performed 48-72 hours after birth, after the presentation of RDS, limiting its use as a biomarker for disease severity, rather than disease occurrence. In summary, our findings suggest that TREC could potentially serve as a reliable biomarker for assessing RDS severity, which is crucial for the timely and appropriate management of affected neonates. Currently, TREC quantification as part of national newborn screening surveys is routinely performed several days after birth. For extremely premature and very low birth weight neonates it could be beneficial to perform TREC measurement within the first day of life, to predict disease occurrence and guide interventions to improve outcomes in this vulnerable patient population. Funding No financial assistance was received in support of the study. Author Contributions ER: Conceptualization, Methodology, Formal Analysis and Writing; LTH: Writing – review and editing; CR: Writing – review and editing; NS: Data acquisition and Methodology; SA: Conceptualization, Data acquisition and Methodology; TS: Conceptualization, Formal Analysis, Writing – review and editing. Competing Interests The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Figure Captions Figure 1. (A) TREC according to gestational age. The relationship between gestational age (x-axis, weeks) and TREC copy numbers (y-axis). Each point on the chart represents TREC copy number for an individual newborn. The trend line indicates a mild positive correlation. ( B) TREC according to birth weight. The relationship between birth weight (x-axis, grams) and TREC copy numbers (y-axis). The trend line indicates a significant positive correlation. Figure 2: Box plot comparison of TREC between male and female neonates. The plot illustrates the interquartile range, median, and overall distribution for each group. Statistical analysis indicates no significant difference between the two groups. ns = not significant. Figure 3: Box plot comparison of TREC between neonates with varying RDS severity. The plot illustrates the interquartile range, median, and overall distribution for each group. Statistical analysis indicates significant differences between severe RSD and the two other groups. ns = not significant. * = p value < 0.05. *** = p value < 0.001. Figure 4. Box plot comparison of TREC between neonates with varying RDS severity, after adjusting for (A) gestational age or (B) birth weight. The plot illustrates the interquartile range, median, and overall distribution for each group. Statistical significance is lost after adjusting for gestational age but not for birth weight. ns = not significant. * = p value < 0.05. Figure 5: Box plot comparison of TREC between neonates with varying RDS severity, according to gender. The plot illustrates the interquartile range, median, and overall distribution for each group. (A) For male neonates, no statistical significance between the groups. (B) For female neonates the difference between TREC copy numbers of neonates with severe RDS and the other groups is statistically significant. ns = not significant. *** = p value < 0.001. Supplementary Material File (trec and rds - tables.docx) - Download - 15.83 KB

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Authors Metrics & Citations Metrics Article Usage 149views 93downloads Citations Download citation Erez Rechavi, Liran Tamir Hostovsky, Chava Rosen, et al. Marked Reduction in T-Cell Receptor Excision Circles Observed in Neonates with Severe Respiratory Distress Syndrome. Authorea. 30 May 2025. DOI: https://doi.org/10.22541/au.174862808.85379797/v1 DOI: https://doi.org/10.22541/au.174862808.85379797/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu.