Analysis of Lower Extremity Long Bone Growth During the Fetal Period. Analiza wzrostu długości kości długich kończyny dolnej w okresie prenatalnym

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1 ORIGINAL PAPERS Adv Clin Exp ed 9, 18, 2, ISSN X Copyright by Wroclaw edical University ALICJA KĘDZIA 1, JOWITA WOŹNIAK 1, KRZYSZTO DUDEK 2 Analysis of Lower Extremity Long Bone Growth During the etal Period Analiza wzrostu długości kości długich kończyny dolnej w okresie prenatalnym 1 Department of Normal Anatomy, Wroclaw edical University, Poland 2 Institute of achines Design and Operation, Technical University of Wrocław, Poland Abstract Objectives. The goal of this survey was a morphometric analysis of the lower extremity long bones (femur, tibia, and fibula) in human fetuses. aterial and ethods. The analysis comprised 3 lower extremity anatomical specimens from 15 embryos at morphological ages of 12 3 weeks and within the crown rump length range of mm. There were 69 (46%) female fetuses. The study employed anthropological and preparative methods, image acquisition with a digital camera, a Scion Image for Windows computer measurement system, and statistical methods. Results. Weekly increases in bone length, size symmetry, and sexual dimorphism were assessed. The results were compared with ultrasound and radiological results available in literature. Neither asymmetry nor sexual dimor phism were detected during the survey. However, differential growth rate was found in the lower leg bones com pared with the femur and these bones showed a nonlinear growth ratio. At the beginning of fetal life (12 th week), the lower leg bones constituted about 63% of the femoral length, but in the 3 th week this increased to 72%. Conclusions. The lower extremity long bone lengths were found to be helpful parameters in assessing fetal size and age (Adv Clin Exp ed 9, 18, 2, ). Key words: fetus, development, lower limb. Streszczenie Cel pracy. Analiza morfometrii kości długich kończyny dolnej (udowej, piszczelowej i strzałkowej) człowieka w okresie prenatalnym. ateriał i metody. Analizie poddano 3 preparatów kończyn dolnych, 15 płodów w wieku morfologicznym między 12. a 3. tygodniem, w przedziale długości v tub: mm. Płodów żeńskich było 69 (46%). W pracy posługiwano się metodami: antropologiczną, preparacyjną, akwizycją obrazu za pomocą aparatu cyfrowego, kom puterowym systemem pomiarów Scion Image for Windows oraz metodami statystycznymi. Wykorzystano testy Shapiro Wilka, Smirnowa Kołmogorowa, anna Whitneya oraz Wilcoxona. Wyniki. Oceniono przyrosty tygodniowe długości kości, symetrię wymiarów po lewej i prawej stronie oraz dymor fizm płciowy. Wyniki pomiarów własnych na materiale sekcyjnym porównano z wynikami pomiarów techniką USG i radiologicznych w dostępnej literaturze. Na podstawie przeprowadzonych badań nie stwierdzono asymetrii i dymor fizmu płciowego. Zaobserwowano natomiast zróżnicowane tempo wzrostu kości podudzia w stosunku do kości udo wej, o czym świadczy nieliniowy wzrost proporcji między tymi kościami. W początkowym okresie życia płodowego (12. tydzień) kości podudzia stanowią 63% długości kości udowej, w 3. tygodniu wartość ta zwiększa się do 72%. Wnioski. Długości kości długich kończyny dolnej uznano za parametry pomocne w ocenie wzrostu i wieku płodu (Adv Clin Exp ed 9, 18, 2, ). Słowa kluczowe: płód, rozwój, kończyna dolna. Ultrasound examination is a basic obstetric diagnostic technique allowing the assessment of fetal age and development. This technique inclu des the standard measurements of bi parietal size, head circumference, femur length, abdominal cir cumference, body mass, and thoracic and vertical

2 122 A. KĘDZIA, J. WOźNIAK, K. DUDEK cerebellar size. The literature provides information on new fetometric parameters which enable fetal growth evaluation. The fetal development rate largely depends on maternal, fetal, and placental factors and its growth is the uterus placenta exchange [22]. The present authors previous observations applied a mathematical model of the upper extremity long bones in fetal age evaluation [18]. The available literature contains anthropo metric surveys of fetal material describing extrem ity growth. Partyka [27] examined 68 fetuses aged weeks in which he measured femoral dia physis. The femur s absolute dimensions showed a strong positive correlation with both crown rump length (CRL) and fetal age. However, nei ther sexual dimorphism nor asymmetry were detected. elts [8] showed that femoral diaphysis length in fetal material increases by.21 mm with 1 mm of CRL growth in fetuses in which this value exceeded 31 mm and diaphysis length surpassed 8.4 mm. Gray and Gardner [11] observed a femoral diaphysis increase by.285 mm with a CRL rise by 1 mm. Kelemen et al. [19] observed lower extremity long bone lengths in 193 fetuses aged 7 22 weeks, including 128 femurs and 117 tibias. The correlation between these parame ters and CRL was linear. They proposed mathe matical formulas which could define fetal age based on the bones longitudinal dimensions. Bareggi et al. [2] measured lower extremity long bone lengths using overexposed alizarine stained specimens. The same team [3] calculated the rela tionship between femoral, tibial, and fibular lengths and CRL ( mm) in 58 fetuses aged 8 14 weeks using a logarithmic model. atsushita et al. [21] analyzed the extremity long bones mea suring 21 anthropometric parameters in 122 Ja panese fetuses aged 18 4 weeks. Increases in lower limb bone length showed positive allometry and that of the upper limb negative allometry. In the examined material, the allometric length coef ficients and CRL were close to 1. (i.e. isometric). In other studies of fetal material, lower limb long bone lengths and their correlation with the body s other dimensions (head circumference, bi parietal dimensions, and abdominal circumfer ence) were assessed from measurements from a scanning electron microscope [6], radiographic measurements [1, 1, 28, 3], and ultrasound examinations [4, 13,, 29]. Similar studies car ried out on material from 142 adult corpses were described by Pan [26] in illy et al. [9] eval uated femur ossification between the 28 th and 35 th week of fetal life in 116 fetuses. The goal of the present study was an analysis of femoral, tibial, and fibular growth in the prena tal period and a comparison of the results with ultrasound examinations as well as a mathematical model designed to assess fetal age on the basis of the lengths of these bones. aterial and ethods Statistical analysis was applied to the results for the right (L R ) and left femur (L L ) femur and the right (PL R ) and left (PL L ) lower leg (shank) bones (tibia and fibula) in 15 fetuses aged weeks of fetal life (mean ± SD: 21.1 ± 4. weeks old). etal CRL ranged from 54 to 25 mm. The analyzed group included 69 female fetuses (46%). etal morphologic age was defined on the basis of the interdependence described by Scammon and Calkins [5]. The study methods included prepara tion and anthropological methods, image acquisi tion with a digital camera, and statistical analysis (Statistica Program package and the Shapiro Wilk, Smirnov Kolmogorov, ann Whitney, and Wilcox tests). easurements of the prepared long bones were performed with the Scion Image computer system for Windows v. Alpha easure ment photographs were taken using the Gimp 2.2 and Bimp 1.43 programs. The results were statisti cally analyzed. The analysis considered sexual dimorphism assessment, symmetry evaluation, and observations of the growth rate and changes in the ratio of the shank and femoral lengths (PL/L). Results Table 1 presents the basic statistics of the quantitative features characteristic for the exam ined group of fetuses. The table also includes these features in subgroups divided according to sex. ig. 1 shows the analyzed parameters of the fetuses. As the distribution of the somatic features dif fered significantly from a normal distribution assessed by the Shapiro Wilk and Smirnov Kol mogorov tests, the significance of differences in their mean values was verified with the ann Whitney nonparametric test. Because the female fetuses were significantly younger, their age was considered in the difference analysis of the female and male long bone sizes. The fetuses were divid ed into four age groups (Table 2) which were homogeneous with respect of rump crown length (CRL) and mass (m), which was verified by the ann Whitney nonparametric test at an α =.5 significance level. The lengths of the femurs (L) and shank bones (PL) in the female () and male () fetuses in the age subgroups were not significantly differ ent (p >.5), which was confirmed by the ann

3 Analysis of the Lower Extremities 123 Table 1. Basic statistics of features characteristic of the analyzed group of fetuses and the statistical significance of differ ences in the means in the subgroups according to sex Tabela 1. Podstawowe statystyki cech charakteryzujących analizowaną grupę płodów i wyniki testu istotności różnic wartości przeciętnych w podgrupach różniących się płcią Parameters Sex Total Test results (Cechy) (Płeć) (Łącznie) (Wyniki testu) emale () ale () Number N (Liczebność N) etal age days Z = (Wiek płodu dni) p =.3 mean (średnia) x standard deviation (odchylenie standardowe) SD median (mediana) e minimum (wartość minimalna) x min maximum (wartość maksymalna) x max Body length v pl mm Z = (Długość ciała v pl mm) p =.8 mean (średnia) x standard deviation (odchylenie standardowe) SD median (mediana) e minimum (wartość minimalna) x min maximum (wartość maksymalna) x max Body length CRL mm Z = (Długość ciała v tub mm) p =.12 mean (średnia) x standard deviation (odchylenie standardowe) SD median e (mediana e) minimum (wartość minimalna) x min maximum (wartość maksymalna) x max Body mass m g Z = (asa ciała m g) p =.35 mean x (średnia x) standard deviation (odchylenie standardowe) SD median e (mediana e) minimum (wartość minimalna) x min maximum (wartość maksymalna) x max ig. 1. etus, parameters: CRL crown rump length, L length of femur, PL length of fibula; 1 femur, 2 fibula, 3 tibia Ryc. 1. Płód, parametry: v tub vertex tuberale, L długość kości udowej, PL długość kości podudzia; 1 kość udowa, 2 kość strzałkowa, 3 kość piszczelowa Whitney test and demonstrated in ig. 2. There was also no statistically significant difference observed between long bone lengths on the left (L) and right (R) side. The Wilcoxon test for depen dant trials confirmed these bones symmetry (ig. 3). As neither sexual dimorphism nor asym metry were found, the growth analysis of the long bones was based on all the results of the length measurements (both on the left and right sides of male and female fetuses). Diagrams (ig. 4) of the correlation of lower limb long bone length with fetal age indicate that their increase is nonlinear. A fit of a mathematical model to the empirical data was attained using logarithmic models. In the ana lyzed period of fetal life, the increase in the femur was faster than that of the shank bones, which results in a nonlinear change in the PL/L ratio (ig. 5). Comparison of the femoral growth curve elicited on the basis of indirect selective material

4 124 A. KĘDZIA, J. WOźNIAK, K. DUDEK Table 2. edians of features characteristic for the analyzed fetal group in age groups and statistical significance of differ ences in the average values in the subgroups according to sex Tabela 2. ediany cech charakteryzujących analizowaną grupę płodów w rozbiciu na grupy wiekowe oraz wyniki testu istotności różnic wartości przeciętnych w podgrupach różniących się płcią etal age month N Sex V tub mm ass g (Wiek płodu miesiąc) (Płeć) (CRL mm) (asa g) IV 3 91 p = p = Σ 92 5 V p = p = Σ VI p = p = Σ VII p = p = Σ length of femur L [mm] th month 6 th month ann-whitney U test: test anna-whitneya: p >,5 fetal age months miesi¹c ycia p³odowego 5 th month 7 th month length of femur L [mm] Wilcoxon signed-rank test: test kolejnoœci par Wilcoxona: Z =,867; p =,386 L R length of fibula PL [mm] d³ugoœæ koœci podudzia [mm] th month 6 th month ann-whitney U test: test anna-whitneya: p >,5 ig. 2. Comparison of shank and femoral bones in male and female fetuses in four age subgroups and ann Whitney test results Ryc. 2. Porównanie długości kości udowej i podudzia płodów żeńskich i męskich w czterech grupach wiekowych i wynik testu anna Whitneya 5 th month 7 th month length of fibula PL [mm] d³ugoœæ koœci podudzia [mm] Wilcoxon signed-rank test: test kolejnoœci par Wilcoxona: Z =,567; p =,57 median 25%-75% min-max L ig. 3. Comparison of lower limbs long bones on the left (L) and right (R) side in 15 fetuses and Wilcoxon test results Ryc. 3. Porównanie długości kości długich kończyn dolnych po stronie lewej (L) i prawej (R) 15 płodów oraz wyniki testu Wilcoxona R

5 Analysis of the Lower Extremities 125 length of femur L [mm] length of fibula L [mm] d³ugoœæ koœci podudzia [mm] measurement (own studies) and curves based on radiological examinations [28] and ultrasound tests [13, 15, 16] revealed a significant difference in absolute value, but similar character. The lengths measured directly are larger than those determined by the ultrasound technique by 15 mm on average. They are also larger than radiological results by about mm (ig. 6). Regardless of the measurement technique, the femoral growth rate was found to be faster in the initial weeks of the 2 nd trimester (ca. 5 mm/week) and it seemed slower in the first weeks of the 3 rd trimester (2 mm/ /week). Due to the observed strong correlation of long bone length and fetal age (R =.976 for femoral length and R =.936 for the shank bones), it is pos sible to assess the fetuses morphological age (A) reliably on the basis of the regression formulas: and L = * ln( A) fetal age A weeks PL = * ln( A) fetal age A weeks ig. 4. Correlations of lower limb long bone lengths and age and the mathematical growth models Ryc. 4. Diagramy korelacyjne długości kości długich kończyn dolnych z wiekiem oraz modele matematy czne wzrostu (.145 L) A = e A = 1.4 e (.19 PL), where A (weeks) is the fetus morphological age, L (mm) the femoral length, and PL (mm) the shank bone length. fibula/femur ( PL / L ) podudzie/udo ig. 5. Correlation of the lower limb long bone length ratio and age and the mathematical growth model Ryc. 5. Diagram korelacyjny proporcji długości kości długich kończyn dolnych z wiekiem oraz model matematyczny wzrostu length of femur L [mm] 1 Discussion PL /L = * ln( A) fetal age A weeks Jeanty Hadlock Scheuer Kêdzia fetal age A weeks ig. 6. Comparison of mathematical models describ ing femoral length increase which were developed on the basis of ultrasound examinations [13, 15, 16], radi ological tests [28], and direct surveys of selective material Ryc. 6. Porównanie modeli matematycznych wzrostu długości kości udowej uzyskanych na podstawie badań USG: Hadlock [13], Jeanty [15, 16], radiolog icznych: Scheuer [28] i pomiarów bezpośrednich na materiale sekcyjnym: Kędzia et al. Jeanty et al. [15 17] presented detailed geo metric measurements of the lower limbs long bones using the ultrasound method in 45 fetuses. The proposed formulas allow assessing the lengths of the femur, tibia, and fibula and can also be applied in fetal limb malformations. In their two part survey, O Brien et al. [24, 25] carried out ser ial measurements of fetal limb long bones with the ultrasound method in 41 patients. The increase in

6 126 A. KĘDZIA, J. WOźNIAK, K. DUDEK the bones was linear between the 12 th and 22 nd week. The results strongly correlated with morphological age and can be used in skeletal dysplasia assess ment. Hadlock et al. [13] examined 338 fetuses aged 12 4 weeks of fetal life. The standard devi ation in age expected on the basis of the femoral length was ± 9.5 days for fetuses aged weeks and ± 22 days for fetuses aged 23 4 weeks. Other material of Hadlock et al. [12] com prised 361 fetuses aged weeks. The ultra sound method was applied to measure femoral length as well as head and abdomen circumfer ence. The increasing curve character was similar, but the values of the present material were much higher. De Biasio et al. [7] reported femoral length reference values taken in fetuses aged 1 14 weeks (CRL: mm) using ultrasound. Strong correlation between CRL and particular bone lengths was observed: for the femur R =.77, p<.1, and for the tibia R =.69, p<.1. erz et al. [23] examined 515 femurs and 385 tibias and fibulas in fetuses aged 12 4 weeks. They described the bones growth with a hyperbolic tan gent. A similar function was applied in studies of the upper limb bone longitude in the present authors own material [18]. Issel [14] compared bone lengths measured by two methods (ultra sound and sectional) in the same 4 stillborn fetus es aged weeks. The difference in the mea surements amounted to ± 2 mm. emoral growth rate proved to be much faster than that of the other examined long bones. The author claimed that the mathematical correlation between fetal age and bone length is best described by a polynomial as the decrease in the growth indexes varies from 4 mm/week in the second trimester to 1 mm/week in the third trimester. The material of the present study revealed nei ther asymmetry (ig. 3) nor sexual dimorphism (ig. 2) and the male and female fetal bone length measurements were identical (p >.5). In the period from the 12 th to 3 th week of fetal life, femoral length increased on average from 1 to 73 mm. In the initial period of fetal life (12 th 13 th week), the increase amounted to 5.8 mm and in the final period (29 th 3 th week) it was.9 mm. This corresponds to a decrease in relative growth from 49% to 1%. The average rate of femoral length increase was 6.9% in the analyzed period. The shank bones (fibula and tibia) average length increased from 7 to 53 mm in this period. In the initial period, the shank bone length increase amounted to 4.1 mm and in the final one 1.1 mm. The average shank bone length increase was 8.1% in the analyzed period. The differential growth rates of the compared bones can also be demon strated by the nonlinear increase in their ratios (ig. 5). In the initial stage of fetal life (12 th week) the shank bones constituted 63% of the femoral length and this value increased to 72% in the 3 th week. In the present authors own material, femoral lengths (ig. 6), assessed with a direct technique, significantly diverged from the results presented by Jeanty [15, 16] and Hadlock et al. [12, 13], who used ultrasound, and Scheurer et al [28], using radiological results. The difference may be caused by systematic measurement errors common in both methods or by the inaccuracy of indirect mea surements such as ultrasound examination. The authors concluded that the longitude dimensions of the lower limb long bones are important parameters helpful in fetal CRL and age assessment, which are of clinical importance in evaluating fetal development. The suggested regression formulas allow fetal morphological age assessment and can be successfully applied in forensic medicine or archaeology. References [1] Bagnall K, Jones PR: Estimating the age of the human foetus from crown rump measurements. Ann Hum Biol 1975, 2, [2] Bareggi R, Grill V, Zweyer, Sandrucci A, Narducci P, orabosco A: The growth of long bones in human embryological and fetal upper limbs and its relationship to other developmental patterns. Anat Embyol 1994, 189, [3] Bareggi R, Grill V, Zweyer, Sandrucci A, Narducci P, orabosco A: On the assessment of the growth pat terns in human fetal limbs: longitudinal measurements and allometric analysis. Early Hum Dev 1996, 45, [4] Benson CB, Doubilet P: Sonographic prediction of gestational age: accuracy of second and third trimester fetal measurements. AJR Am J Roentgenol 1991, 157, [5] Carlson B: Human embryology and developmental Biology. osby, St Louis 1999, 45. [6] Chiarisini D, Barbet J, Copin H, aillet, Pompidou A: Scanning electron microscopy of femoral ossifica tion in the human fetus. Bull Assoc Anat (Nancy) 1992, 233, [7] De Biasio P, Prefumo, Lantieri PB, Venturini PL: Reference values for fetal limb biometry at 1 14 weeks of gestation. Ultrasound Obstet Gynecol 2, 19, [8] elts WJL: The prenatal development of the human femur. Am J Anat 1954, 94, 1 44.

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