The Need to Address Sex as a Biological Variable in Neonatal Clinical Studies.

Male sex has been identified as a risk factor associated with mortality in preterm neonates.1Brothwood M. Wolke D. Gamsu H. Benson J. Cooper D. Prognosis of the very low birthweight baby in relation to gender.Arch Dis Child. 1986; 61: 559-564Crossref PubMed Scopus (122) Google Scholar, 2Stevenson D.K. Verter J. Fanaroff A.A. Oh W. Ehrenkranz R.A. Shankaran S. et al.Sex differences in outcomes of very low birthweight infants: the newborn male disadvantage.Arch Dis Child Fetal Neonatal Ed. 2000; 83: F182-F185Crossref PubMed Google Scholar, 3Ito M. Tamura M. Namba F. NRN of JapanRole of sex in morbidity and mortality of very premature neonates.Pediatr Int. 2017; 59: 898-905Crossref PubMed Scopus (41) Google Scholar, 4O'Driscoll D.N. McGovern M. Greene C.M. Molloy E.J. Gender disparities in preterm neonatal outcomes.Acta Paediatr. 2018; 107: 1494-1499Crossref Scopus (71) Google Scholar Even though male preterm neonates have shown faster declines in mortality, respiratory distress syndrome (RDS), and bronchopulmonary dysplasia (BPD), they still have a significantly higher risk of mortality before hospital discharge, RDS, necrotizing enterocolitis, late-onset sepsis, severe intraventricular hemorrhage (IVH), severe retinopathy of prematurity (ROP), and BPD.5Boghossian N.S. Geraci M. Edwards E.M. Horbar J.D. Sex differences in mortality and morbidity of infants born at less than 30 Weeks' gestation.Pediatrics. 2018; 142: e20182352https://doi.org/10.1542/PEDS.2018-2352Crossref PubMed Scopus (0) Google Scholar Similar results of increased mortality in premature male neonates have been reported from neonatal cohorts from Korea, Canada, Japan, Austria, and Switzerland.3Ito M. Tamura M. Namba F. NRN of JapanRole of sex in morbidity and mortality of very premature neonates.Pediatr Int. 2017; 59: 898-905Crossref PubMed Scopus (41) Google Scholar,6Hwang J.H. Jung E. Lee B.S. Kim E.A.R. Kim K.S. Survival and morbidities in infants with birth weight less than 500 g: a Nationwide cohort study.J Korean Med Sci. 2021; 36: 1-9Crossref Google Scholar, 7Garfinkle J. Yoon E.W. Alvaro R. Nwaesei C. Claveau M. Lee S.K. et al.Trends in sex-specific differences in outcomes in extreme preterms: progress or natural barriers?.Arch Dis Child Fetal Neonatal Ed. 2020; 105: F158-F163Crossref PubMed Scopus (31) Google Scholar, 8Steurer M.A. Adams M. Bacchetti P. Schulzke S.M. Roth-Kleiner M. Berger T.M. Swiss medical centres vary significantly when it comes to outcomes of neonates with a very low gestational age.Acta Paediatr. 2015; 104: 872-879Crossref PubMed Scopus (21) Google Scholar, 9Kiechl-Kohlendorfer U. Simma B. Urlesberger B. Maurer-Fellbaum U. Wald M. Wald M. et al.Low mortality and short-term morbidity in very preterm infants in Austria 2011-2016.Acta Paediatr. 2019; 108: 1419-1426Crossref PubMed Scopus (17) Google Scholar In one meta-analysis, 26 of the 32 studies showed increased mortality in premature males, while 6 reported no sex difference.10Vu H.D. Dickinson C. Kandasamy Y. Sex difference in mortality for premature and low birth weight neonates: a systematic review.Am J Perinatol. 2018; 35: 707-715Crossref PubMed Scopus (46) Google Scholar The increasing evidence of the role of sex as a biological variable in disease outcome, pathophysiology, and response to therapy has highlighted the gap in neonatal clinical and translational trials. This commentary highlights studies that have demonstrated sex-specific differences in outcomes. We acknowledge that other studies not discussed here, fail to demonstrate sex differences in the outcomes discussed. However, most neonatal studies are not adequately powered to detect sex-specific differences in key outcomes, which likely lead to underreporting of critical interactions between biological sex and outcomes. We dissect how these observations may result from sex differences at baseline or with adaptation or response to common neonatal therapies such as antenatal and postnatal steroids, and indomethacin. We argue that these data raise the critical need to assess therapeutic responses stratified by biological sex. Biological sex should be recognized as an essential factor that can drive susceptibility, pathophysiology, response to therapy, and outcomes in clinical and translational studies in neonatology. Respiratory morbidity, including the development of BPD, is common in preterm neonates with a long-term impact on quality of life. A propensity score matching study reported that the incidence of RDS, BPD, and moderate to severe BPD in extremely low birth weight males was significantly increased after adjusting for multiple confounding factors.11Su Z. Lin L. Fan X. Jia C. Shi B. Huang X. et al.Increased risk for respiratory complications in male extremely preterm infants: a propensity score matching study.Front Endocrinol. 2022; 13Crossref Scopus (4) Google Scholar Males had higher rates than females for conventional ventilation, high-frequency ventilation, ventilation after continuous positive airway pressure, inhaled nitric oxide, steroids for BPD, and surfactant.5Boghossian N.S. Geraci M. Edwards E.M. Horbar J.D. Sex differences in mortality and morbidity of infants born at less than 30 Weeks' gestation.Pediatrics. 2018; 142: e20182352https://doi.org/10.1542/PEDS.2018-2352Crossref PubMed Scopus (0) Google Scholar Among extremely premature neonates who developed early hypoxic respiratory failure, female sex was associated with intact survival.12Kaelber D.C. Russell Localio A. Ross M. Leon J.B. Pace W.D. Wasserman R.C. et al.Early hypoxic respiratory failure in extreme prematurity: mortality and neurodevelopmental outcomes.Pediatrics. 2020; 146: e20193318Crossref PubMed Scopus (4) Google Scholar Male sex was associated with higher odds of home oxygen use from the California Perinatal Quality Care Collaborative data among infants diagnosed with BPD13Ejiawoko A. Lee H.C. Lu T. Lagatta J. Home oxygen use for preterm infants with bronchopulmonary dysplasia in California.J Pediatr. 2019; 210: 55-62.e1Abstract Full Text Full Text PDF PubMed Google Scholar and was an independent risk factor for tracheostomy, and among infants who received a tracheostomy, male sex was an independent predictor of mortality.14Han S.M. Watters K.F. Hong C.R. Edwards E.M. Knell J. Morrow K.A. et al.Tracheostomy in very low birth weight infants: a prospective multicenter study.Pediatrics. 2020; 145Crossref PubMed Scopus (12) Google Scholar In a longitudinal study from the United Kingdom, deterioration in lung function trajectories was associated with the male sex.15Bisquera A. Harris C. Lunt A. Zivanovic S. Marlow N. Calvert S. et al.Longitudinal changes in lung function in very prematurely born young people receiving high-frequency oscillation or conventional ventilation from birth.Pediatr Pulmonol. 2022; 57: 1489-1496Crossref PubMed Scopus (4) Google Scholar Hospital readmissions were also skewed towards boys in early childhood.16Gäddlin P.O. Finnström O. Wang C. Leijon I. A fifteen-year follow-up of neurological conditions in VLBW children without overt disability: relation to gender, neonatal risk factors, and end stage MRI findings.Early Hum Dev. 2008; 84: 343-349Crossref PubMed Scopus (22) Google Scholar,17Klitkou S.T. Iversen T. Stensvold H.J. Rønnestad A. Use of hospital-based health care services among children aged 1 through 9 years who were born very preterm - a population-based study.BMC Health Serv Res. 2017; 17: 571Crossref PubMed Scopus (13) Google Scholar Several reports of sex-specific differences in long-term respiratory outcomes of premature neonates highlight the importance of biological sex in the recovery response of the injured preterm lung. Male sex was associated with a significant decrease in forced expiratory volume (at 0.5 s) and forced expiratory flows at 75% of forced vital capacity at 1 year of age.18Voynow J.A. Feng R. Ren C.L. Dylag A.M. Kemp J.S. McDowell K. et al.Pulmonary function tests in extremely low gestational age infants at one year of age.Pediatr Pulmonol. 2022; 57: 435-447Crossref PubMed Scopus (3) Google Scholar In other studies, males saw impairment in long-term lung function.19Sanchez-Solis M. Garcia-Marcos L. Bosch-Gimenez V. Pérez-Fernandez V. Pastor-Vivero M.D. Mondéjar-Lopez P. Lung function among infants born preterm, with or without bronchopulmonary dysplasia.Pediatr Pulmonol. 2012; 47: 674-681Crossref PubMed Scopus (42) Google Scholar Among school-aged children (11-14 years of age), who were born extremely preterm, airway function variables were significantly worse in males.20Harris C. Zivanovic S. Lunt A. Calvert S. Bisquera A. Marlow N. et al.Lung function and respiratory outcomes in teenage boys and girls born very prematurely.Pediatr Pulmonol. 2020; 55: 682-689Crossref PubMed Scopus (11) Google Scholar No differences in respiratory morbidities by sex were reported in another long-term study, with females requiring supplemental oxygen longer compared with males.21Collaco J.M. Aherrera A.D. McGrath-Morrow S.A. The influence of gender on respiratory outcomes in children with bronchopulmonary dysplasia during the first 3 years of life.Pediatr Pulmonol. 2017; 52: 217-224Crossref PubMed Scopus (16) Google Scholar In several large national registries, male sex was a risk factor for severe IVH among preterm neonates.22Mohamed M.A. Aly H. Male gender is associated with intraventricular hemorrhage.Pediatrics. 2010; 125: e333-e339Crossref PubMed Scopus (51) Google Scholar,23Kent A.L. Wright I.M.R. Abdel-Latif M.E. Bowen J. Bajuk B. Vincent T. Mortality and adverse neurologic outcomes are greater in preterm male infants.Pediatrics. 2012; 129: 124-131Crossref PubMed Scopus (162) Google Scholar In a very large cohort of preterm neonates with RDS, female sex decreased the risk of all IVH and severe IVH.24Doshi H. Moradiya Y. Roth P. Blau J. Variables associated with the decreased risk of intraventricular haemorrhage in a large sample of neonates with respiratory distress syndrome.Arch Dis Child Fetal Neonatal Ed. 2016; 101: F223-F229Crossref PubMed Google Scholar A longitudinal observational study conducted at 16 Neonatal Research Network centers to characterize the outcomes of extremely preterm neonates younger than 27 weeks gestational age showed that male sex was associated with non-hemorrhagic ventriculomegaly, which in turn is associated with increased odds of neurodevelopmental impairment.25Pappas A. Adams-Chapman I. Shankaran S. McDonald S.A. Stoll B.J. Laptook A.R. et al.Neurodevelopmental and behavioral outcomes in extremely premature neonates with ventriculomegaly in the absence of periventricular-intraventricular hemorrhage.JAMA Pediatr. 2018; 172: 32-42Crossref PubMed Scopus (34) Google Scholar White matter abnormalities on brain MRI in ex-preterm neonates at 35-39 weeks postmenstrual age26Parikh N.A. Sharma P. He L. Li H. Altaye M. Priyanka Illapani V.S. et al.Perinatal risk and protective factors in the development of diffuse white matter abnormality on term-equivalent age magnetic resonance imaging in infants born very preterm.J Pediatr. 2021; 233: 58-65.e3Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar and 8 years of age27Reiss A.L. Kesler S.R. Vohr B. Duncan C.C. Katz K.H. Pajot S. et al.Sex differences in cerebral volumes of 8-year-olds born preterm.J Pediatr. 2004; 145: 242-249Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar were skewed towards the male sex. Long-term neurodevelopmental outcomes also display a strong sex bias in preterm neonates.28Synnes A. Luu T.M. Moddemann D. Church P. Lee D. Vincer M. et al.Determinants of developmental outcomes in a very preterm Canadian cohort.Arch Dis Child Fetal Neonatal Ed. 2017; 102 (F235–F234)Crossref Scopus (164) Google Scholar,29Hintz S.R. Kendrick D.E. Vohr B.R. Poole W.K. Higgins R.D. Gender differences in neurodevelopmental outcomes among extremely preterm, extremely-low-birthweight infants.Acta Paediatr. 2006; 95: 1239-1248Crossref PubMed Scopus (226) Google Scholar Motor function at 5 years was negatively associated with male sex.30Leversen K.T. Sommerfelt K. Rønnestad A. Kaaresen P.I. Farstad T. Skranes J. et al.Prediction of neurodevelopmental and sensory outcome at 5 years in Norwegian children born extremely preterm.Pediatrics. 2011; 127: e630-e638Crossref PubMed Scopus (100) Google Scholar Preterm females had better developmental and neurological scores than preterm boys at 9 years of age.31Gäddlin P.O. Finnström O. Wang C. Leijon I. A fifteen-year follow-up of neurological conditions in VLBW children without overt disability: relation to gender, neonatal risk factors, and end stage MRI findings.Early Hum Dev. 2008; 84: 343-349Crossref PubMed Scopus (22) Google Scholar In premature neonates without severe brain injury, males were at a higher risk for developmental coordination disorder than females.32Zwicker J.G. Yoon S.W. MacKay M. Petrie-Thomas J. Rogers M. Synnes A.R. Perinatal and neonatal predictors of developmental coordination disorder in very low birthweight children.Arch Dis Child. 2013; 98: 118-122Crossref PubMed Scopus (69) Google Scholar In a cohort of 342 very low birth weight neonates, deteriorating and persistently delayed gross motor trajectories were significantly higher in male infants.33Su Y.H. Jeng S.F. Hsieh W.S. Tu Y.K. Wu Y.T. Chen L.C. Gross motor trajectories during the first year of life for preterm infants with very low birth weight.Phys Ther. 2017; 97: 365-373Crossref PubMed Scopus (14) Google Scholar The influence of socio-economic variables that predict neurodevelopmental outcomes may also be sex specific. In a Swedish study, high parental education predicted higher cognitive and language scores in girls.34Månsson J. Fellman V. Stjernqvist K. Extremely preterm birth affects boys more and socio-economic and neonatal variables pose sex-specific risks.Acta Paediatr. 2015; 104: 514-521Crossref PubMed Scopus (48) Google Scholar The overall incidence of ROP is similar in male and female premature neonates; however, the severity of the disease shows a male bias in many studies.35Holmström G. van Wijngaarden P. Coster D.J. 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However, irrespective of sex hormones, mammalian cells show intrinsic sex-specific differences and respond differently to various stressors.51Penaloza C. Estevez B. Orlanski S. Sikorska M. Walker R. Smith C. et al.Sex of the cell dictates its response: differential gene expression and sensitivity to cell death inducing stress in male and female cells.FASEB J. 2009; 23: 1869-1879Crossref PubMed Scopus (90) Google Scholar Genes on the X and Y chromosomes can be differentially expressed between male and female cells because of X-chromosome inactivation, gene dosage, or genomic imprinting.52Carrel L. Willard H.F. X-inactivation profile reveals extensive variability in X-linked gene expression in females.Nature. 2005; 434: 400-404Crossref PubMed Scopus (1530) Google Scholar Various postnatal biological mechanisms are activated in the preterm neonate that may differ between males and females. For example, compared with the relatively hypoxic in-utero environment, extremely premature neonates are born into a relatively hyperoxic postnatal environment and may need supplemental oxygen to maintain normal oxygen saturations. Thus, these conditions can expose the premature neonate to conditions leading to oxidative stress at the cellular level. In a prospective cohort study, female preterm infants had less oxidative stress, decreased oxidation of DNA and proteins, and better clinical outcomes than male infants, independent of antenatal steroids.53Vento M. Aguar M. Escobar J. Arduini A. Escrig R. Brugada M. et al.Antenatal steroids and antioxidant enzyme activity in preterm infants: influence of gender and timing.Antioxid Redox Signal. 2009; 11: 2945-2955Crossref PubMed Scopus (88) Google Scholar Female premature neonates had higher levels of plasma glutathione peroxidase and glutathione reductase, key enzymes essential for the synthesis and regeneration of glutathione.54Hamon I. Valdes V. Franck P. Buchweiller M.C. Fresson J. Hascoet J.M. [Gender-dependent differences in glutathione (GSH) metabolism in very preterm infants].Arch Pediatr. 2011; 18: 247-252Crossref PubMed Scopus (16) Google Scholar Differences in immune function between male and female neonates may also modulate the pathophysiology of disease processes in the preterm infant.55O’driscoll D.N. Greene C.M. Molloy E.J. Expert Review of Clinical Immunology Immune function? A missing link in the gender disparity in preterm neonatal outcomes Immune function? A missing link in the gender disparity in preterm neonatal outcomes.Clin Immunol. 2017; 13: 1061-1071Google Scholar Microvascular dysregulation due to decreased sympathetic activation in preterm male neonates may increase their risk for hypotension and circulatory failure in the immediate postnatal period with the need for greater respiratory and circulatory support.56Hansen Pupp I. Hellström-Westas L. Elsmén E. Preterm male infants need more initial respiratory and circulatory support than female infants.Acta Paediatr. 2004; 93: 529-533Crossref PubMed Scopus (134) Google Scholar, 57Corbisier De Meautsart C. Dyson R.M. Latter J.L. Berry M.J. Clifton V.L. Wright I.M.R. Influence of sympathetic activity in the control of peripheral microvascular tone in preterm infants.Pediatr Res. 2016; 80: 793-799Crossref PubMed Scopus (8) Google Scholar, 58Stark M.J. Clifton V.L. Wright I.M.R. Sex-specific differences in peripheral microvascular blood flow in preterm infants.Pediatr Res. 2008; 63: 415-419Crossref PubMed Scopus (71) Google Scholar Randomized allocation in trials achieves equal enrollment of male and female neonates in most circumstances. However, some interventions may not have a statistically significant treatment effect in the entire cohort, but when analyzed by sex, they may show benefits in male or female neonates. This may be more likely if the intervention has opposite effects on male and female neonates, and is helpful in one sex and harmful in the other. It is important to consider the limitations of subgroup analyses due to limited statistical power and multiple comparisons leading to false positives. One of the most administered drugs to pregnant persons in preterm labor is antenatal steroids for their overwhelming positive effects on increasing survival in preterm neonates. However, sex-specific differences in response to this therapy have been reported. Following a single course of maternal betamethasone, placental 11β-hydroxysteroid dehydrogenase type 2 decreased in females and increased in males. Decreased 11β-hydroxysteroid dehydrogenase type 2 may lead to increased placental transfer of maternal cortisol in female neonates, improving survivability.59Braun F. Hardt A.K. Ehrlich L. Sloboda D.M. Challis J.R.G. 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