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Sonographic Measurements of the Thymus in Male and Female Fetuses Juan De Leon-Luis, MD, PhD, Francisco Gámez, MD, Pilar Pintado, MD, Eugenia Antolin, MD, Ricardo Pérez, MD, Luis Ortiz-Quintana, MD, Joaquin Santolaya-Forgas, MD, PhD

Objective. The purpose of this study was to determine whether the size of the thymus is different in male and female fetuses. Methods. In this prospective study, the transverse diameter and perimeter of the thymus were measured in healthy fetuses between 24 and 37 weeks’ gestation. The means of the study variables from male and female fetuses were compared by the Student t test, and the relationships between the transverse diameter and perimeter of the thymus and gestational age and other common fetal biometric parameters were determined by linear regression modeling. Results. No differences were noted between male and female fetuses for the means of the study variables. After the relationship between the transverse diameter and perimeter of the thymus and gestational age was confirmed (R 2 = 0.8 and 0.75, respectively; both P < .01), the 95% confidence interval–predicted changes were calculated, and the scatterplots of the measurements suggested that sex did not affect the size of the thymus. Bland-Altman plots were used to analyze intraobserver variability and showed good agreement for both of these thymic measurements for male and female fetuses. Conclusions. These results suggest that fetal sex does not affect the size of the thymus and, together with previous reports, support the hypothesis that a quantitative reduction in fetal thymus size could serve as an indirect marker of abnormal thymopoiesis and congenital thymic insufficiency. Key words: fetal stress; fetal therapy; fetal thymus; in utero cell-based therapy; prenatal diagnosis; stem cells.

Abbreviations GVHD, graft-versus-host disease; HSCT, hematopoietic stem cell transplantation

Received July 7, 2008, from the Department of Obstetrics and Gynecology, Hospital General Universitario Gregorio Marañón, Madrid, Spain (J.D.L.-L., F.G., P.P., E.A., R.P., L.O.-Q.); and Brigham and Women’s Hospital, Boston, Massachusetts USA (J.S.-F.). Revision requested July 24, 2008. Revised manuscript accepted for publication August 18, 2008. We thank Becky Uhlmann for manuscript preparation. Address correspondence to Joaquin SantolayaForgas, MD, PhD, Center for Fetal Medicine and Prenatal Genetics, Department of Obstetrics and Gynecology, Brigham and Women’s Hospital, Harvard Medical School, 75 Francis St, Boston, MA 02115 USA. E-mail: [email protected]

P

renatal allogenic hematopoietic stem cell transplantation (HSCT) offers the prospect of cure for fetuses with a number of inherited disorders.1 The earlier in gestation that stem cell transplantation is attempted, the more likely that (1) the immune system of the potential host will be incapable of rejecting donor cells, and (2) durable engraftment will occur in empty niches within the hematopoietic compartment.1,2 Prenatal allogeneic HSCT, however, could cause death or severe morbidity by the potential effect of graft-versus-host disease (GVHD). In children and adults, the effect of GVHD on thymopoiesis is evaluated by observing trends of changes in the number of CD41, CD81, and T-cell receptor excision circle cells in peripheral blood samples.3 This type of hematologic surveillance is impractical in prenatal life because of the procedure-related risk associated with serial fetal blood samplings.4 However, 2 pieces of information have opened a new opportunity for nonin-

© 2009 by the American Institute of Ultrasound in Medicine • J Ultrasound Med 2009; 28:43–48 • 0278-4297/09/$3.50

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Sonographic Measurements of the Thymus

vasive evaluation of fetal thymopoiesis after in utero HSCT. The first is that thymic size and thymic T-cell output are related to each other.5–8 Indeed, in neonates, volumetric computed tomographic measurements of the thymus have been used to correlate thymus size with the numbers of circulating CD41 and CD45RA1 naive T cells.9 The second is that at least 3 groups of investigators have reported on the feasibility of measuring the fetal thymus using sonography.10–12 Therefore, we hypothesize that involution of the thymus in response to the “stress” caused by GVHD can be detected sonographically. This stress pathway has already been shown in several other clinical situations.13–16 To test this hypothesis, however, normative data of the sonographic measurements of the thymus must be available for male and female fetuses because the thymus is responsive to the hormonal milieu.17 The primary aim of this study was to produce normative data for the perimeter and transverse diameter of the thymus in male and female fetuses during the second half of pregnancy. Our secondary aim was to investigate the reproducibility of these fetal thymic measurements.

Figure 1. Sonogram of the thymus at the level of the 3-vessel view within the fetal thorax allowing measurements of the thymic diameter (A) and thymic perimeter (B). Measurements of the thymus were standardized by placing a line cursor perpendicular to the line connecting the centers of the sternum to the spine.

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Materials and Methods The Institutional Review Board approved the study, and consent was obtained from all patients that participated. The study population was selected from a cohort of women referred for prenatal sonographic evaluation that satisfied the following criteria: date of delivery estimated by first-trimester sonographic evaluation and unremarkable medical, surgical, and obstetric histories. Exclusion criteria were as follows: delivery before 38 or after 42 weeks’ gestation, small or large neonatal weight, and congenital anomalies detected after birth. The thymus was measured by a previously described method,10,13 as shown in Figure 1. Briefly, the thymus occupies the anterior mediastinum in a 3-vessel view of the thorax during a fetal heart evaluation. After identification of the interfaces between the thymus and the lungs, the transverse diameter and perimeter of the thymus were measured. The lateral margins of the thymus were well defined when they were perpendicular to the ultrasound beam. The perimeter and transverse diameter of the thymus were measured by placing a line cursor perpendicular to the line connecting the sternum and the spine. All fetal evaluations were performed between 24 and 37 weeks’ gestation. The fetal thymus was measured by 1 sonologist (F.G.), using a 4- to 8MHz transducer (LOGIQ 9, Voluson Expert; GE Healthcare, Zipf, Austria). All measurements were done in duplicate, allowing for a 5-minute interval between examinations. Possible differences in perinatal outcome variables and prenatal sonographic measurements between male and female fetuses were evaluated by the Student t test. The relationship between the perimeter and transverse diameter of the thymus and gestational age and that of these thymus measurements with other common fetal biometric parameters were determined by linear regression modeling. The predicted changes of the perimeter and transverse diameter of the thymus with gestational age were calculated from the regression equation. A Bland-Altman plot was used to determine the intraobserver bias and agreement for repeated measurements. Data analyses were performed with the SPSS version 15.1 software package (SPSS Inc, Chicago, IL) using default settings. P < .05 was considered statistically significant. J Ultrasound Med 2009; 28:43–48

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Results

Table 1. Perinatal Outcomes and Sonographic Measurements in Male and Female Fetuses

Fifty-nine fetuses, 35 male (53.8%; 95% confidence interval, 49%–58.4%) and 24 female (46.2%; 95% confidence interval, 41.6%–50.9%), fulfilled entry criteria into the study. No statistical differences between groups were noted for gestational age at sonography, gestational age at delivery, newborn weights, or fetal biometric evaluations (Table 1). Table 1 shows that the sonographic measurements of the thymus were similar in male and female fetuses. Tables 2 and 3 depict the equations that predicted the changes of the sonographic measurements of the thymus with gestational age and common fetal biometric variables. Figure 2 shows normal changes of the fetal thymus with gestational age. In addition, analyses of these data points for the perimeter and transverse diameter of the thymus suggest that the size of the thymus is similar for male and female fetuses. Figure 3 shows Bland-Altman plots of intraobserver agreement for the transverse diameter (left) and perimeter (right) of the thymus, suggesting relatively good reproducibility of the measurements without evidence of a systematic error.

Variable/ Measurement

Discussion We used sonography to measure the transverse diameter and perimeter of the thymus and found that the size of the thymus was not affected by fetal sex. We also confirmed that these prenatal sonographic measurements were reproducible. The thymus arises bilaterally from the third and fourth pharyngeal pouches as 2 flask-shaped endodermal diverticula. During the sixth week of development, these thymic diverticula extend laterally and backward into the surrounding mesoderm and neural crest–derived mesenchyme that lies in front of the ventral aorta.5,18 These diverticula eventually fuse in their final position in the anterosuperior mediastinum during the eighth week of development, containing by then elements derived from the 3 germinal layers.10,19,20 By the 10th week of development, hematopoietic lymphoid cells from the fetal liver and bone marrow migrate and are aggregated in the thymus, making a lobulated gland with lymphoid follicles.5,18,21 Further differentiation into J Ultrasound Med 2009; 28:43–48

Gestational age at sonography, wk Gestational age at delivery, wk Birth weight, g Biparietal diameter, mm Abdominal circumference, mm Femur length, mm Thymic diameter, mm Thymic perimeter, mm

Sex

n

Mean

SD

Female Male Female Male Female Male Female Male Female Male Female Male Female Male Female Male

24 35 24 35 24 35 24 35 24 35 24 35 24 35 24 35

31.08 31.5 39.1 38.8 3073 3279 77.97 81.37 248.82 266.51 59.88 61.69 29.63 31.41 85.31 91.16

3.7 3.3 3.8 2.9 652 766 6.2 7.1 56.9 54.3 5.3 7.2 4.8 4.7 14.8 16

P .74 .60 .23 .07 .24 .29 .16 .17

The Student t test was used to compare studied variables depending on fetal sex.

the cortex and medulla is completed by the 14th to 16th weeks.10,20,22 In neonates, a bilobulated thymus that weighs approximately 15 g can be visualized within the thorax immediately under the sternum and proximal sternohyodeus and Table 2. Changes of the Transverse Diameter (Millimeters) of the Fetal Thymus Depending on Gestational Age and 3 Fetal Biometric Measures Variable

Regression Equation

Gestational age (GA) –3.98 × 1.09 GA, wk Biparietal diameter (BPD) –2.5 × 0.39 BPD, mm Abdominal circumference (AC) 3.7 × 0.1 AC, mm Femur length (FL) 1.55 × 0.44 FL, mm

SD

R2

P

0.06 0.04 0.04 0.03

0.8 0.79 0.77 0.76