Bone mineral density in very low birthweight adults—A sibling study

Abstract Background Children and adults born very low birthweight (VLBW, <1500 g) at preterm gestations have lower bone mineral density (BMD) and/or bone mineral content (BMC) than those born at term, but causality remains unknown. Objectives Our aim was to assess BMD and BMC in adults born at VLBW in a sibling comparison setting to account for shared genetic and environmental confounders. Methods We conducted a cohort study of 77 adults born VLBW and 70 same‐sex term‐born siblings at mean age of 29 years. The primary outcome variables were BMD Z‐scores, and BMC, of the femoral neck, lumbar spine, and whole body, measured using dual‐energy X‐ray absorptiometry. We analysed data by linear mixed models. Results The VLBW adults had a 0.25 (95% CI 0.02, 0.47) Z‐score unit lower femoral neck BMD, and 0.35 (95% CI 0.16, 0.54) grams lower femoral neck BMC than their term‐born siblings, after adjustment for sex, age, and maternal smoking. Additional adjustment for adult body size attenuated the results. Lumbar spine, and whole body BMC were also lower in the VLBW group. Conclusions Individuals born at VLBW had lower BMC values at all three measurement sites, as well as lower femoral neck BMD Z‐scores, compared to term‐born siblings, partly explained by their smaller adult body size, but the differences were smaller than those reported previously with unrelated controls. This suggests that genetic or environmental confounders explain partly, but not exclusively, the association between preterm VLBW birth and adult bone mineralisation.


| BACKG ROU N D
Preterm birth is associated with an increased risk of adverse health outcomes, both in the short and long term. This association is more pronounced among those born preterm with very low birthweight (VLBW, <1500 g), the severity of health outcomes increasing in concordance with the degree of prematurity. 1 The third trimester is critical for foetal skeletal development as most of the placental transport of calcium and phosphorous 2 and up to 80% of foetal bone mineralisation occur during this period. 3 Nearly one third of children born at VLBW suffer from metabolic bone disease of prematurity, a condition defined by radiological and biochemical signs of demineralisation. 2,[4][5][6] Previous studies have not clearly established whether preterm birth is associated with long-term detrimental effects on bone health. 2 Comparisons of adolescents or young adults born preterm at VLBW with controls born at term generally report a lower bone mineral density (BMD) and/or bone mineral content (BMC) in those born preterm at VLBW [7][8][9][10][11] while studies with different designs have not observed any such differences. [12][13][14] At least a part of the reported difference is explained by smaller body size of those born preterm at VLBW. [7][8][9]11 The remaining difference could be a consequence of prenatal and neonatal events associated with preterm birth, or due to genetic and environmental characteristics associated with preterm birth and bone mineralisation. This has not been systematically studied.
To obtain more information regarding long-term skeletal consequences of preterm birth, we compared bone health parameters in young adults born at VLBW with their same-sex, term-born siblings. This study design offers an opportunity to account for shared genetic and/or lifestyle factors regarding the association between preterm birth and later bone health.

| Cases and controls
The original study population consisted of 79 adults born at VLBW and 79 same-sex siblings born at term, with less than a 10-year age difference. The sibling pairs were recruited during 2014-2017 from the Helsinki Study of Very Low Birthweight Adults (HeSVA; n = 22), 15 the Adults born Preterm in Northern Finland (ESTER; n = 6) study, 16 and the Finnish Medical Birth Register (FMBR; n = 51). After initial assessment, eleven individuals were excluded: one sibling pair due to both becoming pregnant after recruitment, one pair due to compliance issues, three controls declined further participation, and four controls were found to be born preterm upon chart review. This left us with 77 VLBW participants and 70 controls. The recruitment process is described in detail previously. 17 As part of the present study, the VLBW young adults and their siblings underwent dual-energy X-ray absorptiometry (DXA), and measurement of height and weight at a mean age of 29 years. Perinatal data were available for the HeSVA and ESTER subjects from earlier cohort studies, and hospital and maternity clinic records provided the corresponding data for the remaining VLBW participants and all siblings. The collected data included maternal age, parity, maternal smoking during pregnancy, birthweight, and gestational age.

What this study adds
Preterm very low birthweight adults have lower bone mineral density and bone mineral content than their term siblings. The difference is smaller than in previous studies with unrelated controls suggesting that unmeasured confounders shared within family may explain a part of the difference.
The DXA examination (Hologic Discovery A) included measurement of lumbar spine (L1-L4), femoral neck, and whole body BMD and BMC. BMD values were transformed to Z-scores based on ageand sex-specific reference data for the equipment. We considered a BMD Z-score below -1.0 as decreased. Because bone size influences lumbar spine BMD, we also estimated volumetric density with bone mineral apparent density (BMAD), calculated as BMAD = BMC L1-L4 / bone area L1-L4 1.5 . 18 By pairing with siblings rather than unrelated controls, potential familial confounding was largely accounted for.
All scans were visually inspected, under supervision of an experienced reader (OM), and no significant scoliosis, vertebral compressions in the lumbar region, or foreign bodies, which could lead to measurement errors, were identified. A product term between sex and the dichotomous VLBW variable was calculated to test for potential interaction. Linear regression models of BMD Z-score (femoral neck, lumbar spine, or whole body) as dependent variable, and with sex and VLBW status as independent variables, were compared to equivalent models with the additional sex*VLBW interaction term. No indication of a sex*VLBW-status interaction was found and accordingly, all analyses were performed with sexes combined. SPSS was used for all statistical analyses (IBM SPSS Statistics for Windows, version 27).

| Missing data
In total, 77 VLBW subjects and 70 sibling controls (including 70 complete sibling pairs) underwent DXA analysis. In two subjects the proximal femur DXA assessment deviated from the study protocol and the values were omitted from the final analysis. For eleven subjects (4 VLBW and 7 siblings), some parts of whole body regions remained outside the scan area due to large body size, and equipment-software-derived estimates were used. In a sensitivity analysis, exclusion of these eleven subjects did not influence the associations between the dichotomous VLBW/control variable and whole body BMD and BMC. Therefore, these individuals were retained in analyses.

| Ethics approval
The study was approved by the ethics committee of the Hospital District of Helsinki and Uusimaa. Informed consent was provided by all participants.

| RE SULTS
The study participants were on average 29 years old. VLBW subjects were shorter and lighter than the sibling controls, whose heights were close to the Finnish median heights (men 180 cm, women 165 cm). The proportion of mothers who smoked during pregnancy was similar between groups, around 15.0%. As for parental education, all subjects had at least one parent who had completed at least a secondary education and 61.4% had at least one parent who had completed a tertiary education. Baseline characteristics of the participants are shown in Table 1.
Bone mineral content measurements were lower for all examined locations (femoral neck, lumbar spine, and whole body) in those born at VLBW, as compared to term-born siblings ( Table 2). Additionally, femoral neck BMD Z-score ( Figure 1) and femoral neck area were lower for those born at VLBW. No apparent differences between the VLBW subjects and siblings were seen for BMAD or BMD Z-scores at lumbar spine or whole body.
After initial adjustment for maternal smoking during pregnancy, sex, and age (Table S1, (Table S1 and Figure 2). The differences were attenuated after further adjustment for height ( Note: Means and standard deviations for the VLBW and term sibling groups are presented together with mean differences with 95% confidence intervals. Note: Means and standard deviations for the VLBW and term sibling groups are presented together with mean differences with 95% confidence intervals. BMD, Bone mineral density g/cm 2 ; BMC, Bone mineral content, g; BMAD, Bone mineral apparent density (Bone mineral content L1-L4 divided by area L1-L4 1.5 ).

TA B L E 2 DXA assessment between VLBW and term sibling participants
BMD and BMC values found in the subjects born preterm at VLBW are partly but not exclusively due to genetic or environmental factors shared within siblings. Moreover, these differences are to a substantial extent explained by the smaller body size of VLBW adults.

| Strengths of the study
The study design has several strengths. Having siblings as controls adjusts for potential familial residual confounders and increases statistical power. Care was taken to collect perinatal records as accurately and completely as possible, and our study has only few unknown maternal data.

| Limitations of the study
The sibling study design confers a risk of bias in overmatching, as behaviourally similar siblings might be more likely to participate. Our design also inherently excludes VLBW subjects without a suitable sibling, which may be a source of bias and decrease generalisability. We do not explore adverse bone health outcomes, and whether decreased BMC and BMD manifests as increased incidence of fractures remains to be seen.

| Interpretation
It remains unestablished whether the lower BMD and BMC values per se could have adverse effects later in life. Preterm birth has been linked to an increased risk of fracture-related hospitalisations with the association being strongest in early childhood. 19 Preliminary epidemiological data suggest that, compared to individuals born at term, those born extremely preterm (<28 weeks) are less likely to suffer osteoporotic fractures during childhood and early adulthood. 20 The shorter adult height among those born at VLBW could therefore be a protective factor, given the positive association between height and the risk of osteoporotic fractures. 21,22 Conversely, BMD seems to be an independent risk factor for fractures 23 and whether the lower BMD in VLBW subjects might confer an elevated risk for fractures, with increasing age, is unknown. Furthermore, low BMI especially in males has been associated with a greater risk for falls 24 and it remains unknown whether this applies to VLBW subjects.
The observed differences in BMD between VLBW and termborn adults are smaller than those previously reported in the Helsinki Study of Very Low Birthweight Adults (overlap with current study was 22 VLBW participants). In that study, at mean age 23 years, These previous studies suggest that VLBW or very preterm born adults display lower BMD and BMC than their term-born peers, which is in part explained by their smaller body size. The association was weaker in the present study, which used siblings as controls, and also in the Montreal study that used friends and siblings as controls. This suggests that the association is partly due to shared genetic or environmental factors within a family. Indeed, twin studies F I G U R E 2 Mean differences (95% CI error bars) in bone mineral density Z-scores and bone mineral content in femoral neck, lumbar vertebrae 1-4, and whole body (95% CIs, error bars) in adults born at VLBW compared to their term-born siblings (zero line

| Conclusions
This study assesses the association between preterm birth and later bone health in a sibling setting. We found that individuals born at VLBW on average had lower femoral bone mineral density and bone mineral content, as well as lower bone mineral content in the lumbar spine, and whole body, than their term-born siblings. These differences attenuated after adjustment for BMI and height indicating that the differences are primarily explained by the smaller body size of the VLBW subjects. Compared to previous studies on the association between preterm birth and later bone health, the differences in this study were of smaller magnitude. This could indicate that familial factors, such as shared genetic and/or lifestyle factors, have a substantial impact on later bone health, in addition to that of gestational length and/or intrauterine growth. The lower BMD among those born at VLBW might also constitute an independent risk factor for later fracture risk, especially considering the decline in BMD with increasing age, which might significantly impact later morbidity and mortality.

CO N FLI C T O F I NTE R E S T
The authors declare no competing interests.

AUTH O R CO NTR I B UTI O N S
SS processed the data, performed the analyses, and was the primary author of the manuscript throughout the process, together with JK.
JK was the primary agent of the sibling study, and was widely responsible for participant recruitment, logistics of the clinical study, and data collection and analysis. JB was instrumental in collecting and processing the data from patient records, and he reviewed and revised the manuscript. PH and OM contributed to the design and conduct of the sibling study and reviewed and revised the manuscript. EK, as a PI of the study, conceptualized and designed the study at large, provided funding, supervised data collection, and reviewed and revised the manuscript. All authors approved the final manuscript as submitted and met the ICMJE criteria for authorship.