Sex‐Based Differences in the Sonographic Characterization of Diaphragm Thickness in Preterm Infants With Bronchopulmonary Dysplasia at Term Corrected Age

To determine the sex‐specific diaphragm thickness in infants with bronchopulmonary dysplasia (BPD) as well as in healthy term and near‐term infants.

I nfants born very premature can have significant respiratory morbidity 1 ; however, male infants are disproportionately affected 2,3 by higher rates of respiratory distress syndrome (RDS), bronchopulmonary dysplasia (BPD), respiratory support, and pneumothorax 4,5 compared with female infants.Although optimal prenatal management has improved the survival of the most vulnerable preterm neonates, 6 it has not closed the gap in neonatal outcomes between female and male infants who are born preterm. 7In fact, male infants who are born preterm continue to have higher rates of RDS and BPD despite the widespread use of antenatal steroids and postnatal surfactant administration. 2,3While sex-based differences in the lung and its development account for higher rates of RDS, 8 it is plausible that sex-specific variation exists in other structures, such as the diaphragm, which may play a role in other respiratory morbidities.
The diaphragm is the main muscle for respiration, and it is well-established that there are sexrelated differences in the structure and function of the diaphragm.From an anatomical perspective, the diaphragm and the length of the zone of apposition are shorter in adult females, 9 while females exhibit greater thickness compared with males. 10Additionally, males tend to demonstrate a more predominant diaphragmatic breathing pattern 9 ; in comparison, the adult female diaphragm may have greater resistance to exercise-induced fatigue. 11iaphragm thickness has been assessed in preterm infants 12,13 and children. 14Despite this knowledge, there has been a notable gap in examining sex-specific differences in the diaphragm among preterm infants who have developed BPD.Expiratory diaphragm thickness measures anatomic diaphragm thickness, 12,15,16 whereas inspiratory diaphragm thickness and diaphragm thickness fraction measures thickness during contraction. 12,15ence, the primary objective of this study was to investigate the impact of sex on diaphragm thickness during expiration (diaphragm relaxation phase) in infants with BPD, as well as in healthy near-term and term infants.Secondary objectives included evaluating sex-specific diaphragm thickness during inspiration (diaphragm contraction phase), diaphragmatic thickness fraction (DTF), and diaphragmatic excursion (DE) in infants with BPD and healthy infants.

Study Design
We conducted a secondary analysis of a prospective case-control study on diaphragm ultrasound of preterm infants with bronchopulmonary dysplasia.Details regarding this prospective study (NCT04941963) were previously published. 13Local research ethics approval for this secondary analysis was obtained (Mount Sinai Hospital REB 23-0016-C).

Study Participants
We included all 111 infants from the prospective case control study.Infants born at <32 weeks' gestation who received oxygen or respiratory support at ≥36 weeks' postmenstrual age (PMA) were diagnosed with BPD.Healthy infants born at ≥36 weeks' gestation and matched by PMA to the infants with BPD group were included as controls.Infants who had congenital or chromosomal abnormalities, neuromuscular disease, or whose parents declined to consent were excluded.

Study Procedure
Ultrasound scans were performed between January 2020 and July 2021 and these scans were securely stored for subsequent analysis.We measured the diaphragmatic thickness (DT) and diaphragmatic excursion (DE) over three respiratory cycles using a bedside ultrasound for all enrolled infants (Figures 1 and 2).Scans were performed 1 hour after feeding to avoid any concerns of a full stomach on diaphragmatic assessment.The bedside ultrasound (US) was recorded with a high-resolution L20-5 MHz linear probe for measurement of DT and a C10-3 MHz curvilinear probe for assessing DE (Z.One-Mindray, Inc.).All US assessments were undertaken while the infants were lying supine.All scans were performed by a trained neonatologist in lung and diaphragmatic ultrasound; images or videos were captured and recorded for analysis.Standard neonatal lung ultrasound (LU) views were obtained, and LU scores were determined as previously described. 17The expiratory and inspiratory DT were calculated by determining the maximum perpendicular distance between the pleural and peritoneal layers, measuring only the distance of the hypoechoic area (Figure 1).
Diaphragmatic thickness fraction (DTF) was calculated as the change in diaphragmatic thickness during expiration (DT exp ) and inspiration (DT ins ) using the following formula: Outcomes and Clinical Parameters The primary outcome was the sex-specific DT exp at ≥36 weeks' PMA for infants born at <32 weeks' gestation who developed BPD.Secondary outcomes included sex-specific diaphragmatic thickness during inspiration (DT ins ), DTF, and DE for BPD infants and healthy age-matched late-preterm and term infants.To account for differences in body parameters for infants with BPD compared with healthy infants, we calculated the diaphragm thickness expressed as a ratio of body surface area (BSA), using the Mostellar's formula 14 to calculate BSA.Along with sonographic measurements, we compared baseline demographic data including gestational age (weeks) at birth and PMA at the time of the US scan, anthropometric measurements at birth and at the time of the scan (weight and length) as well as type and duration of respiratory support stratified by phenotypic sex.

Statistical Analysis
Continuous variables were summarized using means and standard deviation or medians and interquartile range, while categorical variables were described in terms of counts and percentages.Continuous variables were analyzed using a Student T-test or Wilcoxon rank sum test and plotted.Categorical variables were analyzed by a χ 2 or Fisher exact test.To determine interrater agreement for lung ultrasound scores, the intra-class correlation coefficient (ICC) was calculated for variables with continuous measurements under a mixed factorial design.To determine the ICC, 20% of the images were independently scored by another individual who was blinded to the groups.The Kolmogorov-Smirnov Test was used to test for normality.Significance was determined by

Results
Out of the 180 infants assessed for eligibility, we completed a secondary analysis for 111 participants enrolled in the original prospective study.Figure 3 provides a comprehensive overview of the study enrollment.There were 57 infants of male sex (51.4%) and 54 infants of female sex (48.6%).Table 1 presents the baseline characteristics of the two groups.There was no significant difference in the gestational age or the body surface area at the time of diaphragm ultrasound between male and female infants in either the BPD or healthy control groups.In addition, the duration of invasive or non-invasive ventilation was no different between male and female infants with BPD (Table 1).However, the number of infants not requiring respiratory support was higher at the time of the diaphragm ultrasound in female than male infants with BPD (42% vs 13%, P < .01).
The DT exp was significantly lower in male infants with BPD compared with female infants with BPD [1.2 (0.3) mm vs 1.5 (0.4) mm, P = .02,Figure 4, supplemental].When comparing DT exp per BSA in male and female infants with BPD, the results remained  2].Furthermore, DT ins per BSA was found to be significantly lower in male infants with BPD compared with their female counterparts [10.9 (2.6) mm/m 2 vs 12.9 (3.6) mm/m 2 , P = .02,Table 2].
In contrast, there were no significant differences in DT exp or DT ins between sexes within the healthy control group [1.5 (0.4) mm vs 1.5 (0.3) mm, P = .89,and 2.2 (0.5) mm vs 2.2 (0.5) mm, P = .66,respectively].Moreover, we found no significant difference in DTF or DE between males and females in either the BPD or healthy control group (Table 2).
The diaphragm thickness at excursion was thinner in male infants with BPD than healthy males (P < .01);however, we observed no difference in DT exp between female infants with BPD and the healthy controls (P = .43).
To evaluate the reliability of diaphragm ultrasound, we measured and found a high level of agreement among observers when measuring diaphragm thickness and excursion (ICC: 0.93, P < .01 for diaphragm thickness, and 0.92, P < .01 for diaphragm excursion).

Discussion
In this secondary analysis, we report that male neonates with BPD had significantly thinner DT exp than female infants with BPD.However, no differences in DT exp between sex were observed in our healthy control group.
Our findings were consistent with previous reports that there were no significant differences in diaphragm thickness by sex for healthy newborns. 19n contrast, male infants with BPD had significantly lower DT exp compared with healthy males (P < .01);but this difference was not observed with female infants with BPD.DT exp is a measure of diaphragm thickness during the diaphragm relaxation phase.In adult critical care, studies show that it may also be an indicator of diaphragmatic atrophy and weakness 20 ; moreover, adults on respiratory support had decreased success in liberation from mechanical ventilation when there was a reduction in DT exp . 20Likewise, we showed that preterm infants with BPD had a thinner DT exp than healthy age-matched controls. 13ex differences in preterm infants have not been previously reported.In contrast, the diaphragmatic thicknesses of healthy young adults at functional residual capacity were significantly thicker in females than males. 10In addition, the female adult diaphragm is more resistant to exercise-induced fatigue.Due to the persistent sex-specific differences in the incidence of prematurity associated BPD, we suggest that there may be a comparable association to the preterm diaphragm, in which the female diaphragm is less prone to fatigue and better able to maintain ventilation.In comparison, males have a thinner diaphragm, 10 and the predominant breathing pattern in males is diaphragmatic. 9Taken together, the slimmer diaphragm, and the greater dependence on the diaphragm for breathing could explain the higher need for ventilatory support and ultimately higher incidence of BPD in preterm male infants.This study presents the first report of sex-based differences in diaphragm thickness among infants with BPD.By utilizing sonographic characterization, our findings suggest a novel hypothesis that sex-related variations in diaphragm thickness might contribute to the observed disparity in BPD incidence between sexes.Furthermore, our results in healthy newborn infants are comparable to previous studies, 19,21 suggesting that our data may be used to establish reference values for healthy infants and infants with BPD.
However, we acknowledge the limitations of our study.Firstly, our measurements are based on a single time point, which restricts our ability to compare the evolution of diaphragm thickness in preterm male and female infants.That is, we cannot determine whether preterm males are born with thinner diaphragms vs females, or if male diaphragms exhibit similar initial thickness but fail to grow at a comparable rate to their female counterparts in the context of ongoing respiratory comorbidity.Secondly, we did not include a comparative population of preterm infants without BPD, which limits the characterization of the effect of sex and respiratory support on diaphragm muscle growth and development.Variation between obtaining and interpreting diaphragm  ultrasound images have been reported 20 and a potential limitation to measurement of the diaphragm.Finally, infants included in this study did not have any active sepsis, necrotizing enterocolitis, hemodynamically significant PDA, use of sedative, or use of steroids.However, we cannot exclude the residual effects of these co-morbidities on the diaphragm at the time of the US.

Conclusion
In this study, we provide evidence of sex specific differences in the neonatal diaphragm, in which preterm male infants with BPD have lower DT exp compared with female infants.This finding suggests that the increased incidence of BPD in preterm males may be related to diaphragmatic thickness.Future studies will be needed to delineate the evolution of diaphragm thickness in the preterm infants in correlation with respiratory illness severity and to ascertain the true nature of these disparities.

Figure 1 .
Figure 1.Diaphragmatic thickness during inspiration and expiration: Diaphragmatic thickness is measured at the end of expiration and inspiration by using M-mode to demarcate the vertical distance between the bidirectional arrow.

Figure 2 .
Figure 2. Diaphragmatic excursion: Diaphragmatic excursion is measured using M-mode to determine the vertical distance between the bidirectional arrow.

Figure 4 .
Figure 4. Sex-based diaphragm thickness presented with mean values (black dots) overlaying box and whisker plots.A, Diaphragm thickness at expiration and, B, Diaphragm thickness at expiration per body surface area.* Statistically significant.

Table 2 .
Diaphragmatic Thickness and Excursion in Infants With BPD and Healthy Infants by Sex *Statistically significant.