Morphometric study of the primary ossification center of the fibular shaft in the human fetus.

PURPOSES: Precise morphometric data on the development of ossification centers in human fetuses may be useful in the early detection of skeletal dysplasias associated with delayed ossification center development and mineralization. The present study was performed to quantitatively examine the primary ossification center of the fibular shaft with respect to its linear, planar and volumetric parameters. MATERIALS AND METHODS: Using methods of CT, digital-image analysis (Osirix 3.9 MD) and statistics (Student's t-test, Shapiro-Wilk, Fisher's test, Tukey's test, Kruskal-Wallis test, regression analysis), the size of the primary ossification center of the fibular shaft in 47 spontaneously aborted human fetuses (25 ♂ and 22 ♀) aged 17-30 weeks was studied. In each fetus, the assessment of linear dimensions (length, transverse diameters for: proximal end, middle part and distal end), projection surface area and volume of the fibular shaft ossification center was carried out. RESULTS: With no sex and laterality differences, the best fit growth dynamics for the primary ossification center of the fibular shaft was modelled by the following functions: y = - 13.241 + 1.567 × age ± 1.556 (R2 = 0.94) for its length, y = - 0.091 + 0.063 × age ± 0.073 (R2 = 0.92) for its proximal transverse diameter, y = - 1.201 + 0.717 × ln(age) ± 0.054 (R2 = 0.83) for its middle transverse diameter, y = - 2.956 + 1.532 × ln(age) ± 0.090 (R2 = 0.89) for its distal transverse diameter, y = - 69.038 + 4.699 × age ± 4.055 (R2 = 0.95) for its projection surface area, and y = - 126.374 + 9.462 × age ± 8.845 (R2 = 0.94) for its volume. CONCLUSIONS: The ossification center in the fibular shaft follows linear functions with respect to its length, proximal transverse diameter, projection surface area and volume, and natural logarithmic functions with respect to its middle and distal transverse diameters. The obtained morphometric data of the fibular shaft ossification center is considered normative for their respective prenatal weeks and may be of relevance in both the estimation of fetal age and the ultrasound diagnostics of congenital defects.

The syndrome of proximal femur, fibula, and midline metatarsal long bone deficiencies.

Human lower limb congenital long bone deficiencies cluster primarily at three distinct skeletal locations. Proximal femoral and fibular reductions are known phenomena. In contrast, midline metatarsal deficiencies have been misrepresented as lateral. The popular term, "fibular hemimelia," is inaccurate and its use is discouraged. All three locations correspond to discrete sites of evolving angiogenesis during transition from a single embryonic axial limb artery to the familiar and complex adult arterial pattern. Initiation of bone formation of cartilaginous primordia of the long bones at all three sites occurs in proximity to, and depends upon, successful invasion by mature nutrient vessels, formed during the 6th and 7th weeks of embryonic development. The adult arterial pattern is fully established by 8th embryonic week. Arterial transitions occur later in development, around the time of cessation of the molecular processes of patterning/specification of the embryonic limb. Evidence of flawed embryonic arterial transitions, involving missing, reduced and/or retained primitive vessels in association with congenital skeletal reductions have been demonstrated at all three sites. Current molecular models of limb development do not explain the distribution of this triad of congenital skeletal reductions. These dysmorphologies are most accurately described as post-specification errors of limb development. Recognition of this distinctive model of limb maldevelopment demands further investigation to create a more exact taxonomy, one consistent with both clinical and molecular criteria. The established terminologies originated by Frantz and O'Rahilly should be reconsidered or abandoned. Designation of this clinical triad as a syndrome of proximal femur, fibula, and midline metatarsal dystrophisms initiates that endeavor.

Evaluation of fibular muscles and prevalence of accessory fibular muscles on fetal cadavers.

PURPOSE: The purpose of this study was to determine clinical importance and morphology of the fibularis longus, brevis, tertius muscles (presented as fibular muscles in this study), and prevalence of accessory fibular muscles (AFM) on fetal cadavers. MATERIALS AND METHODS: In this study, 200 limbs from 100 embalmed fetuses (54 male and 46 female) were studied. Morphology of fibular muscles and the presence of AFM were observed through dissection on fetal legs bilaterally. The absence of fibularis tertius muscle (FT) and the presence of AFM were identified. Length and length of the tendon of AFM were measured. Insertion of AFM was also identified. RESULTS: Mean values and standard deviations of all parameters according to trimesters were calculated. All parameters were increased with age during the fetal period. We determined the absence of FT; no FT was observed in 40 legs of 200 extremities (20%). The AFM was present in 7 of 200 specimens (3.5%). AFM muscles were classified into two types according to their site of origin. CONCLUSION: The present study has revealed the morphology of the fibular muscles and prevalence of the presence of AFM on fetal cadavers. In addition, it has revealed the morphometric development and prevalence of the absence of FT in a large series, and their clinical importance was discussed.

Cross-sectional study of fetal long-bone length in an Iranian population at 17-25 weeks of gestation.

OBJECTIVE: To construct improved reference charts for fetal long bones in an Iranian setting and to compare them with previous studies. METHODS: The present prospective cross-sectional study included singleton fetuses assessed by ultrasonography at 17-25 weeks of gestation at the Comprehensive Medical Genetics Center, Shahid Soltani, Shiraz, Iran between May 1, 2012, and February 28, 2014. Exclusion criteria included conditions that could affect fetal growth. Fetal long bones (femur, humerus, tibia, fibula, ulna, and radius) were measured with ultrasonography and biometric charts were produced. Regression models were fitted to estimate bone lengths. The models produced were compared with those from previous studies in other populations. RESULTS: There were 660 singleton fetuses included and 660 femur, 633 humerus, 512 tibia, 498 fibula, 505 ulna, and 498 radius biometric measurements were recorded. The models generated to predict the length of the tibia, fibula, ulna, and radius from the length of the femur and humerus demonstrated a high goodness of fit when the predicted lengths were plotted against the actual lengths. Comparisons of mean lengths with previous studies suggested that long-bone length was affected by maternal ethnicity. CONCLUSION: The equations generated could be used to predict long-bone length in an Iranian population and ethnicity should be considered when using fetal long-bone length as a prenatal diagnostic tool.

Foetal fibular hemimelia with focal femoral deficiency following prenatal misoprostol use: A case report.

Misoprostol is a well known abortifacient. It can cause teratogenicity like Mobius sequence and terminal transverse limb defects. We report a rare case of proximal focal femoral deficiency with fibular hemimelia in a woman who had attempted abortion with self-administered misoprostol and later continued the pregnancy. Though the absolute risk of congenital malformations with its use is low ∼1%, this should be clearly communicated to the women requesting abortion to help them make fully informed reproductive health decisions.

Molecular development of fibular reduction in birds and its evolution from dinosaurs.

Birds have a distally reduced, splinter-like fibula that is shorter than the tibia. In embryonic development, both skeletal elements start out with similar lengths. We examined molecular markers of cartilage differentiation in chicken embryos. We found that the distal end of the fibula expresses Indian hedgehog (IHH), undergoing terminal cartilage differentiation, and almost no Parathyroid-related protein (PTHrP), which is required to develop a proliferative growth plate (epiphysis). Reduction of the distal fibula may be influenced earlier by its close contact with the nearby fibulare, which strongly expresses PTHrP. The epiphysis-like fibulare however then separates from the fibula, which fails to maintain a distal growth plate, and fibular reduction ensues. Experimental downregulation of IHH signaling at a postmorphogenetic stage led to a tibia and fibula of equal length: The fibula is longer than in controls and fused to the fibulare, whereas the tibia is shorter and bent. We propose that the presence of a distal fibular epiphysis may constrain greater growth in the tibia. Accordingly, many Mesozoic birds show a fibula that has lost its distal epiphysis, but remains almost as long as the tibia, suggesting that loss of the fibulare preceded and allowed subsequent evolution of great fibulo-tibial disparity.

Heterochronically early decline of Hox expression prior to cartilage formation in the avian hindlimb zeugopod.

The fibula, a zeugopod bone in the hindlimb, exhibits various morphologies in tetrapod species. The fibula in some species has a similar length with the other zeugopod element, the tibia, while other species have obvious differences in the sizes of the two elements. In the avian hindlimb, for example, the fibula is extremely short, thin, and truncated. Basic morphology of the fibula is established during development, and cartilage primordium of the bone emerges in a certain region defined by a distinct combination of expression of Hox genes (Hox code). In order to elucidate how the different morphologies are produced from a region that is defined as the fixed Hox code, we examined spatial and temporal patterns of Hoxd11/Hoxd12 expression in the developing limb bud, which defines the region from which the fibula emerges, in comparison with the sites of precartilaginous mesenchymal condensations representing regions for cartilage formation among chick, mouse, and gecko embryos. We found that in the chick hindlimb, expression of Hoxd11/Hoxd12 decreased and disappeared from the presumptive zeugopod region before cartilage formation. This heterochronically early decline of expression of Hox genes is strongly correlated with the peculiar trait of the fibula in the avian hindlimb, since in the other species examined, expression of those genes continued after the onset of cartilage formation. This is morphological phenotype-related because the early disappearance was not seen in the chick forelimb. Our results suggest that temporal change of the Hox code governs diversification in morphology of homologous structures among related species.

A contribution to the discussion concerning the variability of the third peroneal muscle: an anatomical analysis on the basis of foetal material.

The aim of the work was to make a systemic study of the variability of the human musculus peroneus tertius during the foetal period. Examination was made of 193 foetuses of ages ranging from 84 to 256 days after conception. The results obtained indicated that the musculus peroneus tertius was present in 83.16% of the human foetuses studied and that its intrauterine development was progressive and almost proportional. Previous studies have not revealed dimorphic or bilateral differences with respect to any of the features examined. On the basis of the examinations and bibliographical data a uniform typology of the musculus peroneus tertius variants was created and three final types were distinguished: the pithecogenic (44% cases), eugenic (34% cases) and progenic (22% cases).

Correlation between first trimester fetal bone length and maternal serum pregnancy-associated plasma protein-A (PAPP-A).

BACKGROUND: Pregnancy-associated plasma protein-A (PAPP-A) is produced by the embryo and placenta during pregnancy, and its maternal serum concentrations are related to subsequent fetal growth. Evidence from animal models and in vitro experiments suggests that PAPP-A is particularly involved in the regulation of bone development. The aim of this study was to assess the correlation between late first trimester fetal bone length and maternal serum levels of PAPP-A. METHODS: In a cross-sectional observational study, ultrasound measurements of fetal long bones and fluorimetric immunoassays for maternal serum PAPP-A were performed in 514 singleton pregnancies at 10-14 weeks of gestation. RESULTS: There were 501 uncomplicated pregnancies. There were significant correlations between PAPP-A values and length of humerus, femur and tibia [r values 0.12 (P = 0.01), 0.11 (P = 0.01) and 0.10 (P = 0.03), respectively]. The association with the length of ulna and foot did not reach statistical significance (r values 0.08 and -0.03, respectively). CONCLUSIONS: Maternal serum PAPP-A levels at 10-14 weeks of gestation are significantly associated with the length of fetal long bones such as humerus, femur and tibia. This provides further evidence that PAPP-A may be involved in the regulation of bone development.

The corpus fibulae in sheep (Ovis ammon f. aries)--ontogeny, persistence, size and shape from the fetal period to adulthood.

Osteological characteristics are often used to identify animal species. In this regard it is believed that the corpus fibulae of the sheep is represented by a tight cord of connective tissue--the so-called "ligamentum fibulare". The results of the present morphometric study indicate, however, that in 67% of the fetuses, 61% of the lambs or 48% of the adult sheep out of the 160 examined animals, the corpus fibulae is represented by a pin-shaped flattened piece of bone. The length of the corpus fibulae increases with age and varies greatly between individuals but in lambs and adult animals its centre is located close to the border between the proximal and intermediate third of the tibia (distal type) or closely attached to the caput fibulae by connective tissue fibres (proximal type). The cartilaginous fetal anlage is either transformed to bone or catabolised during fetal life. Desmal ossification should play an important role in the growth of the corpus fibulae since no cartilage could be detected in juvenile and adult sheep. Since there are many similarities in the expression of the corpus fibulae between sheep, goat and fallow deer, this osteological characteristic may not be used for a rapid specific and simple animal species identification procedure.

Some distal limb structures develop in mice lacking Sonic hedgehog signaling.

Patterning of the limb is coordinated by the complex interplay of three signaling regions: the apical ectodermal ridge (AER), the zone of polarizing activity (ZPA), and the non-ridge limb ectoderm. Complex feedback loops exist between Shh in the ZPA, Bmps and their antagonists in the adjacent mesenchyme, Wnt7a in the dorsal ectoderm and Fgfs in the AER. In contrast to the previously reported complete absence of digits in Shh(-/-) mice, we show that one morphologically distinct digit, with a well-delineated nail and phalanges, forms in Shh(-/-) hindlimbs, while intermediate structures are severely truncated and fused. The presence of distal autopod elements is consistent with weak expression of Hoxd13 in Shh(-/-) hindlimbs. Shh(-/-) forelimbs in contrast have one distal cartilage element, a less-well differentiated nail and fused intermediate bones. Interestingly, Ihh is expressed at the tip of Shh mutant limbs and could account for formation of distal structures. In contrast to previous studies we also demonstrate that Shh signaling is required for maintenance of normal Fgf8 expression, since expression of Fgf8, unlike some other AER marker genes, is rapidly lost from anterior to posterior after E10.5, with only a small domain of Fgf8 expression remaining posteriorly. Furthermore, loss of expanded Fgf8 expression is paralleled by a collapse of the handplate. Our data show that development of most intermediate elements of the hindlimb skeleton are Shh-dependent, and that Shh signaling is required for anterior-posterior expansion of the AER in both limbs and for the subsequent branching of zeugopod and autopod elements. Finally, we show that Shh is also required for outgrowth of the limb ectoderm and thus for the formation of a distinct limb compartment.

Prenatal ultrasonographic diagnosis of posteromedial bowing of the leg: two case reports.

Congenital posteromedial bowing of the leg was prenatally detected in two pregnancies, at 20 and 31 weeks of gestation. Posteromedial bowing is a rare anomaly of unknown etiology. The prenatal course, monitored by ultrasonography, and the postnatal clinical and radiographic outcomes are discussed and show a complex differential diagnosis. The initial postnatal therapy is conservative. Leg length discrepancy can eventually be treated by lengthening or epiphysiodesis on the contralateral side.

Role of bone bark during growth in width of tubular bones. A study in human fetuses.

The morphologic features of bone bark, a structure surrounding the distal and proximal ends of long bones, were studied in the distal femur, proximal tibia, and proximal fibula of 77 spontaneously aborted human fetuses varying in gestational age from 10 to 20 weeks. Standard histologic techniques used in addition to in situ immunohistochemical staining allowed the examination of the structure of the bone bark and localization of Types 1, 2, and 3 collagens at different gestational ages. The bone bark was shaped like a cylindrical sheath of bone lamellae of varying thickness. The epiphyseal end of the bone bark, known as the groove of Ranvier, was covered outwardly by a fibrous layer and inwardly by the epiphyseal cartilage and contained mesenchymal cells, chondroblastic precursor cells, and densely packed cells differentiating into osteoblasts. Neither the cell density in the groove nor the thickness of the bone bark were identical circumferentially, indicating an unequal growth in width. In addition, the presence of periosteal apposition and endosteal resorption of the bone bark on one side and of endosteal bone deposition accompanied by periosteal resorption of the bone bark on the opposite side support the concept of a spatial drift of bones. These observations furnish histologic proof that groove and bone bark, although assuring an equal growth in length, contribute to an unequal and eccentric growth in width.

Prenatal ultrasound diagnosis of a femur-fibula-ulna complex during the first half of pregnancy.

A case of fetal femur-fibula-ulna (FFU) complex diagnosed by ultrasound is presented. Ultrasonographic features of a fetus displaying bilateral femoral hypoplasia, aplasia of the right forearm and the right hand, ray defects of the left hand are described. The importance of an early diagnosis of this malformation is emphasized with respect to parental counselling concerning prognosis and further prenatal management.

Ossification and growth rates of the limb long bones during the prehatching period in the quail (Coturnix coturnix japonica).

The timing of ossification and the growth of six long bones of the prehatching period in the quail was studied. Ninety-nine quail eggs were incubated and in total nine fetuses were selected daily from the sixth to the sixteenth day of incubation. The fetuses were stained with alizarine and alcian blue double colouration. The fetuses were studied under the stereoscopic microscope and linear measurements were obtained from the humerus, ulna, radius, femur, tibia and fibula. The first appearance of the primary ossification centres in the diaphysis of the studied bones was found to occur between the sixth and the seventh day of incubation. Different growth patterns between the bones of the leg and of the wing were observed. Humerus and tibia showed the greatest growth rate while the radius and fibula showed the lowest.

On the assessment of the growth patterns in human fetal limbs: longitudinal measurements and allometric analysis.

The total length (TL) and length of the ossified part (OL) of some long bones of the upper (humerus, ulna, radius) and lower limb (femur, tibia, fibula) were evaluated in 58 aborted human fetuses (crown-rump length, CRL, between 38 and 116 mm, developmental age from 8 to 14 weeks). The specimens, without any detectable malformation, were cleared and double-stained with alcian blue and alizarin red S to obtain a differential detection of the ossified part within the comprehensive outline between the cartilaginous epiphyses. The correlation between the values of TL and OL and those of CRL emphasized that the systematic OL measurement in limb long bones correlated better than TL with development age, since OL increased faster than TL. TL and OL also correlated with the CRL by bivariate allometry (ln y = ln a + b ln x) and the data obtained showed that they grew with positive allometry. The comparison between the cumulative values of the bones examined in each limb showed that both TL and OL grew relatively faster in the lower limb than the upper; the greatest growth rate was found for OL in the lower limb. These results many provide a tool for a comprehensive assessment of long bone growth patterns and may be useful in determining fetal growth even in incomplete specimens, in which one or some long bones can still be measured.

Adjusting the risk for trisomy 21 by a simple ultrasound method using fetal long-bone biometry.

OBJECTIVE: To establish the efficacy of second-trimester fetal long-bone biometry (femur, humerus, tibia, and fibula length) in detecting trisomy 21 and to generate tables for adjusting the risk of trisomy 21 according to long-bone biometry. METHODS: Four long-bones--femur, humerus, tibia, and fibula--were measured ultrasonically in singleton fetuses before genetic amniocentesis. Fetuses with normal karyotypes were used to derive regression equations describing predicted lengths on the basis of the biparietal diameter measurement. The efficacy of each abnormally short bone, alone and in combination, was determined in 22 fetuses with trisomy 21 encountered during the study period. After the sensitivity and specificity of long-bone biometry were established, appropriate tables were generated by Bayes' theorem to adjust the risk of trisomy 21 in the second trimester depending on long-bone biometry. RESULTS: Of 515 patients between 14 and 23 weeks' gestation, 493 had normal fetal karyotypes and 22 had trisomy 21. The sensitivity of an abnormal ultrasound, as defined by the presence of one or more short bones, was 63.6% and the specificity was 78.5%. According to Bayes' theorem, genetic amniocentesis may not be recommended for women less than 40 years old in the presence of normal long-bone biometry (ie, all four bones normal). CONCLUSION: Second-trimester fetal long-bone biometry is useful in detecting trisomy 21 and may be used to adjust the a priori risk of both high- and low-risk women for trisomy 21 and, therefore, the need for genetic amniocentesis.

Growth of the tibia and fibula bones in human fetuses.

The growth of the long bones has been a subject of great importance lately, because it provides non invasive diagnosis of prenatal malformations. This study examines 116 tibia and fibula bones of Brazilian fetuses (17 males and 12 females) with gestational age ranging from the 16th to the 34th week postconception. Only fetuses with no visible congenital malformations and with normal biometric parameters were considered. The bones were measured with a 0.1 mm precision caliper and their Total Length (TL), the Ossified Diaphysis Length (ODL), the Diaphysis width (DW), the Proximal Epiphyss length (PEL) and the Distal Epiphyss Length (DEL) were studied. The Total Length (TL) and the Ossified Diaphysis Length (ODL) were correlated with those of the fetal Crown-Rump length (C-R). Our results have shown that there was an increase in all these measures, with no significant statistical difference observed between the left and the right sides. A significant correlation coefficient was found in most parts of the measurements studied. This study aims to provide data to be utilized in monitoring bone growth as well as in the diagnosis of the lower limb deformities during fetal period.

Survival and development of reimplanted fetal epiphyses. An experimental study.

An experiment was undertaken to study the survival and development of fetal epiphyses after excision and reimplantation in rats. The proximal part of fibula in the hindlimb was dissected free from surrounding tissues and then reimplanted again. Of 80 fetuses that were operated on, nine that had been operated on survived to birth. Histological examination on the hindlimbs of these rats at four and six weeks after birth showed that the reimplanted segments survived, and the bony nuclei of the epiphyses were present. Radiographically, at 6 weeks old, secondary centres of ossification at proximal ends of the fibulas could be seen in both operated and normal limbs. These results indicate the clinical possibility of correcting congenital musculoskeletal abnormalities in the future by in utero transplantation of epiphyses.

Multivariate analysis of limb long bone growth during the human prenatal period.

Prenatal development of the human limb bones (humerus, ulna, radius, femur, tibia, fibula) was studied. Cross-sectional data, consisting of 21 anthropometric measures of these bones were collected from 122 Japanese fetuses between the gestational ages of 18 and 40 weeks. Principal component analysis was applied to the data to provide multivariate assessments of morphological patterning among the variables. Three orthogonal components that accounted for 94.2% of the overall sample variation were extracted. The first component accounted for 88.2% of the variation and represented an axis of overall body size that was dependent on gestational age. The second and third components both reflected a trend in shape involving the cartilaginous parts of the humerus and femur. The findings indicated that different growth factors existed between the ossified and cartilaginous parts of fetal long bones. Multivariate allometric coefficients were extracted from the first principal component. The variables that were related to the construction of the articulate showed positive allometry, and the central widths of the diaphysis showed negative allometry compared with total size. In prenatal skeleton, certain shape changes are functionally linked to and required by changes in body size. A comparison among the coefficients of long bone length revealed that lower limb bones grew faster than upper limb bones during the period under study here. Allometric coefficients were equivalent among bones within a limb, whereas homologous bones in the upper and lower limb grew at different rates.