Antenatal thymus volumes in fetuses that delivered <32 weeks' gestation: An MRI pilot study

Introduction Infection and inflammation have been implicated in the etiology and subsequent morbidity associated with preterm birth. At present, there are no tests to assess for fetal compartment infection. The thymus, a gland integral in the fetal immune system, has been shown to involute in animal models of antenatal infection, but its response in human fetuses has not been studied. This study aims: (a) to generate magnetic resonance imaging (MRI) -derived fetal thymus volumes standardized for fetal weight; (b) to compare standardized thymus volumes from fetuses that delivered before 32 weeks of gestation with fetuses that subsequently deliver at term; (c) to assess thymus size as a predictor of preterm birth; and (d) to correlate the presence of chorioamnionitis and funisitis at delivery with thymic volumes in utero in fetuses that subsequently deliver preterm. Material and methods Women at high-risk of preterm birth at 20-32 weeks of gestation were recruited. A control group was obtained from existing data sets acquired as part of three research studies. A fetal MRI was performed on a 1.5T or 3T MRI scanner: T2 weighted images were obtained of the entire uterine content and specifically the fetal thorax. A slice-to-volume registration method was used for reconstruction of three-dimensional images of the thorax. Thymus segmentations were performed manually. Body volumes were calculated by manual segmentation and thymus:body volume ratios were generated. Comparison of groups was performed using multiple regression analysis. Normal ranges were created for thymus volume and thymus:body volume ratios using the control data. Receiver operating curves (ROC) curves were generated for thymus:body volume ratio and gestation-adjusted thymus volume centiles as predictors of preterm birth. Placental histology was analyzed where available from pregnancies that delivered very preterm and the presence of chorioamnionitis/funisitis was noted. Results Normative ranges were created for thymus volume, and thymus volume was standardized for fetal size from fetuses that subsequently delivered at term, but were imaged at 20-32 weeks of gestation. Image data sets from 16 women that delivered <32 weeks of gestation (ten with ruptured membranes and six with intact membranes) and 80 control women that delivered >37 weeks were included. Mean gestation at MRI of the study group was 28+4 weeks (SD 3.2) and for the control group was 25+5 weeks (SD 2.4). Both absolute fetal thymus volumes and thymus:body volume ratios were smaller in fetuses that delivered preterm (P < .001). Of the 16 fetuses that delivered preterm, 13 had placental histology, 11 had chorioamnionitis, and 9 had funisitis. The strongest predictors of prematurity were the thymus volume Z-score and thymus:body volume ratio Z-score (ROC areas 0.915 and 0.870, respectively). Conclusions We have produced MRI-derived normal ranges for fetal thymus and thymus:body volume ratios between 20 and 32 weeks of gestation. Fetuses that deliver very preterm had reduced thymus volumes when standardized for fetal size. A reduced thymus volume was also a predictor of spontaneous preterm delivery. Thymus volume may be a suitable marker of the fetal inflammatory response, although further work is needed to assess this, increasing the sample size to correlate the extent of chorioamnionitis with thymus size.


| INTRODUC TI ON
Preterm birth (PTB) defined as delivery at <37 weeks of gestation is a significant health burden projected to cost health services in England and Wales £2.946 billion (€3.279 billion) per year. 1 The most significant adverse outcomes occur in very preterm deliveries, before 32 weeks of gestation, accounting for 1.4% of all deliveries in the UK. 2 Although the etiology of PTB is complex and often multifactorial, infection/inflammation has been implicated, particularly at earlier gestations: at 28 weeks of gestation approximately 80% of cases of PTB have evidence of significant microbial colonization within the placental parenchyma. 3 Neonatal morbidity, including sepsis, cystic periventricular leukomalacia, intraventricular hemorrhage, and later development of cerebral palsy, are significantly higher among preterm pregnancies that are also complicated by infection as assessed by the presence of chorioamnionitis at delivery. 4,5 At present, there is no test routinely used in clinical practice to assess for fetal compartment infection and iatrogenic delivery, particularly for preterm premature rupture of the membranes (PPROM), is usually only instigated following signs of maternal infection, by which stage fetal infection may already be established.
The thymus plays an integral role in the development of the fetal immune system, being the main site of T-cell production and development, and has been suggested as a marker of the fetal inflammatory response, 6 with involution occurring in the presence of acute stress, including malnutrition, trauma and sepsis. 7 Although thymus size has previously been assessed using ultrasound 8 and a reduction in length and perimeter has been associated with the presence of funisitis in pregnancies complicated by PPROM, 6 adequate visualization can be hampered when ultrasound is used for assessment because of unfavorable fetal position, oligohydramnios, and high maternal body mass index. 6 Ultrasound measurements in previous studies of pregnancies at high risk of preterm delivery have also only assessed two-dimensional markers such as the perimeter 6 or cardiothorax:thymus ratios, 9 which do not take into account variations in the true size and shape of the gland, and may not correlate with actual volume. Calculation of volumes of the gland using magnetic resonance imaging (MRI) obviates some of these difficulties and has successfully been used to evaluate the fetal thymus, both in uncomplicated pregnancies and in those with fetal growth restriction. 10 More recent MRI advances have enabled reconstruction of the fetal thorax to further account for unpredictable fetal motion, so improving the accuracy of volumetric assessments. 11 Results: Normative ranges were created for thymus volume, and thymus volume was standardized for fetal size from fetuses that subsequently delivered at term, but were imaged at 20-32 weeks of gestation. Image data sets from 16 women that delivered <32 weeks of gestation (ten with ruptured membranes and six with intact membranes) and 80 control women that delivered >37 weeks were included. Mean gestation at MRI of the study group was 28 +4 weeks (SD 3.2) and for the control group was 25 +5 weeks (SD 2.4). Both absolute fetal thymus volumes and thymus:body volume ratios were smaller in fetuses that delivered preterm (P < .001). Of the 16 fetuses that delivered preterm, 13 had placental histology, 11 had chorioamnionitis, and 9 had funisitis. The strongest predictors of prematurity were the thymus volume Z-score and thymus:body volume ratio Z-score (ROC areas 0.915 and 0.870, respectively).

Conclusions:
We have produced MRI-derived normal ranges for fetal thymus and thymus:body volume ratios between 20 and 32 weeks of gestation. Fetuses that deliver very preterm had reduced thymus volumes when standardized for fetal size. A reduced thymus volume was also a predictor of spontaneous preterm delivery. Thymus volume may be a suitable marker of the fetal inflammatory response, although further work is needed to assess this, increasing the sample size to correlate the extent of chorioamnionitis with thymus size.

K E Y W O R D S
fetal thymus, infection, magnetic resonance imaging, preterm birth, thymus, volume

Key message
Thymus volumes are smaller in fetuses that deliver very preterm when standardized for fetal size. This may represent a marker of the fetal inflammatory response.
This study aims to investigate if alterations in thymus volume occur in fetuses that are delivered preterm and if there are associations between the size of the gland and the presence of chorioamnionitis at delivery. This will be achieved by: (a) generating MRI-derived normal ranges for thymus volume, and thymus volume standardized for fetal size, in uncomplicated pregnancies between 20 and 32 weeks of gestation; (b) comparing thymus size in fetuses that deliver very preterm, standardizing for fetal body volume; (c) assessing thymus size as a predictor of PTB; and (d) correlating the presence of chorioamnionitis and funisitis at delivery with thymic volumes in utero in fetuses that subsequently deliver preterm. The fetal thymus, a bi-lobed structure located in the thorax anterior to the pericardium and posterior to the sternum, was segmented manually from the reconstructed images of the thorax using ITK-SNAP (version 3.6.0 http://itksn ap.org) 16 and 3D Slicer (version 4.10.2 https://www.slicer.org) 17 ( Figure 1). Intra-and inter-observer variability was confirmed by two operators (LS and AE). Semi-automatic segmentation of the fetal body was again performed using ITK-SNAP in a two-step process. Automatic segmentation of the body was based on image contrast while using user-defined thresholds. Fine editing of each segmentation was performed manually to remove incorrectly labeled areas. Good intra-and inter-observer variability had previously been confirmed for body volume calculation. 11 A control group was identified from three other studies: the intelligent Fetal Imaging and Diagnosis project (www.iFIND proje ct.com), the Placental Imaging Project (www.place ntaim aging proje ct.org) and 'Antenatal assessment of fetal infection utilizing advanced MRI protocols'. All women had given informed written consent to allow their imaging data to be used. Cases were selected from F I G U R E 1 Thymus segmentation of a control fetus, at 30 +0 weeks of gestation, that was subsequently delivered at term acquired on a 1. uncomplicated pregnancies where the MRI was performed between 20 +0 and 32 +0 weeks of gestation and delivery occurred >37 weeks of gestation. All fetuses had undergone the same imaging protocol described above on a 1.5 Tesla or 3 Tesla scanner (Phillips) and had thymus and body volumes reconstructed using the same protocols.

| MATERIAL AND ME THODS
Details of maternal demographics, delivery, and neonatal parameters were collected including: gestation at delivery, sex of infant, birthweight, birthweight centile, neonatal unit admission, number of days of invasive ventilation, continuous positive airway pressure, abnormalities on neurological imaging, and confirmed sepsis. In the preterm cohort, placentas were sent for a structured histological assessment 18 as for routine clinical practice and more specifically to detect the presence of chorioamnionitis. 19

| Statistical analyses
Data were assessed for normality using distributional plots of standardized residuals. Demographic and neonatal outcome data were analyzed using a Student t test where data were continuous and chisquared test where the data were categorical. For the control cases, the Wright and Royston xrimel method was used to estimate normal growth trajectory of thymus volume and thymus:body volume ratios between 20 and 32 weeks of gestation. 20 Receiver operating curves (ROC) were generated for low thymus:body volume ratio and low gestation adjusted thymus volume centiles as predictors of PTB.
We estimated the difference between fetuses that delivered preterm and those that delivered at term in thymus and body volumes and thymus:body volume ratios adjusting for the effects of gestation and the strength of the magnet by multiple regression.

| RE SULTS
Recruitment for this study can be seen in Figure 2. Of the 16 pregnancies analyzed, 13 of the women had received steroids for fetal lung maturity before the time of imaging. Ten of the women had ruptured membranes at the time of MRI and six had intact membranes.
Ten examinations were performed on a 1.5T scanner and six on a 3T scanner. Of the 16 fetuses that delivered preterm, 13 had placental histology available. Of these, 11 had evidence of chorioamnionitis in the placenta and nine had funisitis.
In addition, 80 control cases were identified. All of these pregnancies were delivered after 37 weeks of gestation and were F I G U R E 2 Flow chart illustrating study recruitment scanned on a 1.5T (50 cases) or 3T (30 cases) scanner using similar protocols: 3D reconstruction of the fetal body was performed for each of the data sets.
Clinical characteristics of the group that delivered preterm and the control group are denoted in Table 1. Distribution plots can be seen for thymus volumes and thymus:body volume ratios from 20 to 32 weeks of gestation derived from the control group of fetuses in Figure 3.
The gestation-adjusted thymus volume centiles and the thymus:body volume ratio were tested as predictors of PTB (Figure 4). Fetuses that delivered preterm had significantly lower thymus volumes and body volumes at the time of scan than those that delivered at term, accounting for the effects of gestation (Table 2; Figure 5).

Characteristic
When standardized for fetal size, fetuses that delivered preterm had significantly lower thymus:body volume ratios. No effect was demonstrated regarding magnet strength on these findings. As previously reported, fetal body volumes were also significantly smaller in fetuses that delivered preterm (P < .001). 11 Although numbers are small the finding that thymus volume and thymus:body volumes were smaller persisted in the preterm cohort in both fetuses with ruptured membranes and those with membranes intact.
Although the numbers are small, the relation between thymus volume and the presence of funisitis can be seen in Figure 6.
There was one intrapartum death at 22 +0 weeks of gestation with a reduced thymus:body volume ratio of 0.011. The mean number of days from MRI to delivery in the preterm group was 10.5 (SD 14.2). Two fetuses from the control group that delivered >37 weeks were admitted to the neonatal unit, one for a few hours only with suspected respiratory distress syndrome and the second for 2 days with hypoglycemia. In contrast to these findings, Brandt et al prospectively assessed the fetal thymus in 520 pregnancies using 2D ultrasound, measuring the transverse and anterio-posterior thymus diameters and the thymic:thoracic ratio. Of this population, 12.3% underwent PTB; however, there was no correlation between thymus size and premature delivery. 9 It should be noted that the median gestational age at imaging was 20.5 weeks and very few of the PTBs occurred very early in gestation, hence the infective/inflammatory processes may not have commenced at the time of this initial scan.

| D ISCUSS I ON
All of the previous studies, however, have only evaluated the thymus using two-dimensional parameters. Its shape is variable and volumetric assessment provides a more complete evaluation F I G U R E 4 Receiver operator curve prediction of prematurity from antenatal magnetic resonance imaging scans: thymus volume, thymus:body volume ratio, thymus volume Z-score, and thymus:body volume ratio Z-score including 95% confidence intervals [Color figure can be viewed at wileyonlinelibrary. com] TA B L E 2 Thymus and body volumes in fetuses that delivered preterm and those that delivered preterm controlling for the effect of gestational age at magnetic resonance imaging scan by multiple regression Although thymic volume has previously been assessed using 3D ultrasound in uncomplicated pregnancies 25,26 and in those with growth restriction, 10,27 to our knowledge our study is the first to assess thymus volume, using either modality, MRI or ultrasound, in pregnancies at high risk of PTB. MRI may be a superior imaging modality in this cohort of fetuses, particularly those with ruptured F I G U R E 5 Graphs illustrating the antenatal thymus (A) and body (B) volumes and the thymus:body volume ratios (C) between 20 and 32 weeks of gestation in fetuses that delivered before 32 weeks of gestation and those that delivered at term membranes as oligohydramnios and unfavorable fetal lie can give inadequate visualization of the gland. 6 Although further work is required to assess the relation in more In contrast, in a sheep model, following lipopolysaccharide-induced chorioamnionitis, no histological changes in the thymus or changes in markers of apoptosis and proliferation in the thymoctes were identified. However, as was the case in our study, thymus:bodyweight ratios were reduced by 40% 5 days after lipopolysaccharide administration. Blood lymphocytes were also found to be 40% lower than the control group after 1 day and elevated at 5 days. 28 However, these findings, and the lack of histological changes in the gland, may be attributable to the fact that the sheep model gave a single dose of lipopolysaccharide whereas the antenatal insult may be more extensive and prolonged in clinical practice.
At present, numbers within this current study are small, the timing between the MRI scan and delivery varies, and placental histology was not available in all cases. The slight difference in the gestation at which the MRI was performed between cases and controls is a limitation of the study; however, it has been adjusted for in the analysis. Furthermore, the presence of confounding factors such as the timing of steroid administration, which has been shown to alter the structure of the gland in an ovine model of chorioamnionitis, needs to be considered in a larger sample. 29 Further work is also required to correlate the imaging findings with markers of infection in both placental histology and umbilical cord blood and both short-term and long-term postnatal outcomes. In this study, the thymus was not the primary focus of all of the investigations and in 24% of cases a thymus volume could not be reconstructed. Acquisition techniques have been optimized F I G U R E 6 Graphs illustrating the antenatal thymus (A) and the thymus:body volume ratios (B) between 20 and 32 weeks of gestation in fetuses that delivered before 32 weeks of gestation, with and without funisitis at delivery and those that delivered at term [Color figure can be viewed at wileyonlinelibrary.com] and we would anticipate this percentage being significantly lower in the future. In addition, MRI studies in the future could include additional sequences to detect microstructural and perfusion alterations within the thymus that may also be associated with a reduction in volume and the presence of funisitis.

| CON CLUS ION
We have reported normal ranges, between 20 and 32 weeks of gestation, for thymus volume and thymus volume standardized for fetal size derived from healthy fetuses that subsequently delivered at term. Thymus volumes are smaller in fetuses that subsequently deliver very preterm, a finding which persisted after standardization for fetal size. Although numbers were too small to compare fetuses with and without chorioamnionitis/funisitis at delivery it may be reflective of thymic involution as a consequence of infective/inflammatory processes in the in utero environment, which are also associated with the etiology of preterm delivery. Thymus volume and thymus:body volume ratio also appeared to be good predictors for preterm delivery. Further evaluation is needed to assess the utility of thymus volume as a marker for the fetal inflammatory response both using MRI and using ultrasound. where there has been instrumentation of the uterine cavity, such as in cases of fetal surgery.

CO N FLI C T O F I NTE R E S T
AHS is the chief investigator on a number of trials funded by NIHR and charity sources related to preterm birth prediction and preven-