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Table of Contents
ORIGINAL ARTICLE
Year : 2022  |  Volume : 71  |  Issue : 2  |  Page : 93-101

Intrauterine Alcohol Exposure Delays Growth and Disturbs Trabecular Morphology in 3-Week-Old Sprague -- Dawley Rat Femur


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Date of Submission09-Nov-2021
Date of Acceptance17-Mar-2022
Date of Web Publication30-Jun-2022

Correspondence Address:
Diana S Pillay
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jasi.jasi_183_21

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  Abstract 


Introduction: The consequence of gestational alcohol exposure ranges from stillbirth to miscarriage and fetal alcohol syndrome (FAS). FAS is one of the deleterious causes of congenital disabilities, mental and growth retardation. Several studies suggest that low birth weight and impaired bone growth, as well as a decrease in mineralization in utero, may reduce peak bone mass and increase the risk of fractures and osteoporosis later in life. Therefore, the aim of this study was to determine the effects of intrauterine alcohol exposure on the internal architecture of the femur. Material and Methods: Time-mated (n = 13) pregnant Sprague − Dawley dams were assigned to either the ethanol (n = 5), saline control (n = 5) or untreated control (n = 3) group. The former two groups were treated with 0.015 ml/g of 25.2% ethanol and 0.9% saline for the first 19 days of gestation, respectively. The untreated group received no treatment. Once born, the pups were weaned at 21 days. These rats were then terminated. From each dam, two pups were collected resulting in ethanol (n = 10), saline controls (n = 10), and untreated controls (n = 6). The femora of the pups were dissected and scanned using a 3D-μCT scanner (Nikon XTH 225 L) at 15 μm resolution. Trabecular and cortical parameters were analyzed using Volume Graphics Studio® software following reconstruction. Results: We found altered trabecular parameters in the alcohol exposed group. The diaphyseal cortical and medullary cavity proportions were also affected, particularly in the midshaft. Discussion and Conclusion: These results indicate that gestational alcohol exposure may lower bone structural quality by disturbing the internal morphology of the osseous tissue.

Keywords: Cortical thickness, femur, gestational alcohol, internal morphology, trabeculae


How to cite this article:
Pillay DS, Ndou R. Intrauterine Alcohol Exposure Delays Growth and Disturbs Trabecular Morphology in 3-Week-Old Sprague -- Dawley Rat Femur. J Anat Soc India 2022;71:93-101

How to cite this URL:
Pillay DS, Ndou R. Intrauterine Alcohol Exposure Delays Growth and Disturbs Trabecular Morphology in 3-Week-Old Sprague -- Dawley Rat Femur. J Anat Soc India [serial online] 2022 [cited 2022 Aug 11];71:93-101. Available from: https://www.jasi.org.in/text.asp?2022/71/2/93/349465




  Introduction Top


The consumption of alcohol during pregnancy has well-known teratogenic consequences on fetal development.[1] These may include spontaneous abortion, fetal growth inhibition, premature delivery, abruption placentae, and breech presentations.[2] An offspring exposed to alcohol in utero can suffer an assortment of disorders and disabilities[3] collectively described as fetal alcohol spectrum disorder. This is a nondiagnostic, umbrella term used to describe a range of effects on an offspring that result from gestational alcohol exposure.[3] These include alcohol-related birth defects, alcohol-related neurodevelopmental disorder, partial fetal alcohol syndrome, and fetal alcohol syndrome (FAS).[3],[4]

There is limited information on changes in the internal architecture of bone following gestational alcohol exposure. However, research shows a postnatal reduction in cortical and trabecular thickness as well as reduced bone formation rate following intrauterine alcohol exposure.[5],[6],[7] A decrease in the bone formation rate may occur due to the disturbance in proliferation and the activity of osteoblasts,[6],[8] which may alter the internal integrity of the bone. Although studies have shown the adverse impact of alcohol consumption on cortical and trabecular parameters, no research has established the effects of gestational alcohol exposure on the internal architecture of postnatal bone development. Knowledge in this area will be useful and will help us understand weaker bones in FAS children.

Studies investigating the effects of gestational alcohol exposure on bone focus on rat fetuses,[1],[9] therefore, such research is limited among adolescents[10] Understanding how gestational alcohol exposure affects bone postnatal development is crucial as peak bone growth occurs during adolescence.[3] Intrauterine alcohol exposure causes shorter bones[1],[9] and delayed fetal skeletal ossification,[9] with osteoporosis and high fracture risk in postnatal life,[9] suggesting inadequate postnatal development.

The rate of alcohol consumption during gestation is among the highest in South Africa at 13%,[11] compared to 9.8% on average in the rest of the world.[11] However, some conservative areas around the world[12] and in South Africa stigmatize alcohol consumption by woman. Therefore, some women tend to drink in secret to evade the detection by their spouses and to avoid the perceived societal stigma associated with women alcohol drinkers.[12] This behavior of secluded alcohol consumption results in binge drinking, defined as consuming four to five glasses of alcohol per occasion.[13] This pattern of drinking poses a high risk for FAS,[14] particularly as women may not know that they are pregnant in the initial 2 months of gestation. Considering this, we employed a model that sought to mimic binge drinking. Binge drinking causes more severe brain damage and behavioral changes than heavy chronic drinking in rats.[15] This is thought to be due to the high peak blood alcohol concentration produced by consuming large alcohol volumes over a short duration as in binge drinking.[12] The effects of alcohol consumption cause negative consequences to the skeleton of adults; however, it remains uncertain if this deleterious effects on the osseous tissue will continue into adolescence and adult life in cases of gestational alcohol exposure. We wondered whether prenatal alcohol exposure would affect trabecular morphological parameters in postnatal life in weight-bearing bone such as the femur. As such, we investigated this question using the femur in 3-week-old rats that were subjected to alcohol during gestation.


  Material and Methods Top


Breeding and animal husbandry

The study received ethics approval from the Animal Ethics Screening Committee (AESC 2015/27/15C). A total of 13 female virgin SpragueDawley rats weighing between 260 and 350 g were used. All study animals were bred and kept at the Central Animal Services, University of Witwatersrand. These animals were maintained under pathogen-free conditions, temperature-controlled environment (23°C ± 2°C) and a 12-h light/dark cycle. Pregnant dams were individually housed in plastic cages (L 430 mm × W 220 mm × H 200 mm), with free movement within the enclosures. The animals had unrestricted access to tap water and standard rodent diet.

Treatment with ethanol or saline

The dams (n = 13) were divided into either the ethanol (n = 5), saline control (n = 5 and untreated control groups (n = 3). Fewer animals were used in this group (n = 3) to reduce the number of animals required for the study and to lower the study cost. The untreated control group received neither ethanol nor saline. The experimental and saline groups were treated with 0.015 ml/g body mass of either 25.2% ethanol or 0.9% saline control, respectively. The ethanol administered was to mimic a chronic binge drinking model as a relatively large dose was given once a day. The treatment was administered by oral gavage for 19 days from the 1st day of gestation as determined by the presence of a vaginal plug.

Determination of maternal blood alcohol levels

To determine blood alcohol levels in the dams, whole blood was collected from the tail vein into heparinized microcapillary tubes, one hour after alcohol administration. Microcapillary tubes were spun in a microhaematocrit centrifuge (Haematokrit 210, Hetich, Germany) at 3000 rpm for 10 min. Plasma alcohol determination was carried out using the BioVision Ethanol Colorimetric Assay Kit (BioVision incorporation, Milpitas, USA) according to the manufacturer's instructions. All reactions and readings were done in an alcohol-free environment using an iMark Bio-rad Microplate Absorbance Reader (Bio-rad Laboratories Inc, USA). All blood samples were centrifuged, and serum stored at -80°c until analyzed for Blood Alcohol Concentration [BioVision Ethanol Colorimetric/Flurometric Assay Kit (catalog #K620-100)] (BAC).

Allocation of pups

The dams were allowed to litter naturally, and the pups remained with their dams until termination at 21 days of age where they were terminated by a lethal pentobarbital intraperitoneal injection. From each dam, two pups were collected resulting in: ethanol (n = 10), saline controls (n = 10), and untreated controls (n = 6).

Skeletal harvesting

Upon termination, the skeletal samples were then individually immersed and stored in 10% buffered formalin to continue fixation until further processing.

Micro-focus X-ray computed tomography

Bilateral femora were scanned with a Nikon XTH 225/320 LC X-ray microtomograph. To keep the samples steady during scanning, the bones were placed in plastic tubes and mounted in low density Styrofoam, while allowing the X-rays to get to the sample with negligible absorption. The plastic container with the sample inside was then positioned on a rotating manipulator in the scanning chamber for the scanning. The scanning parameters used in this study were as follows: the scanning voltage was 70kv; the X-ray current was 400 μa, with a frame averaging of 4 and a resolution of 15 μm. Following reconstruction, VG studio Max®3.2 was used for the data analysis as previously described.[16]

Parts of the femora and trabecular morphometry parameters studied

The VG studio software built in caliper was used to determine the femoral osteometric parameters. The full bone length and bicondylar length and bicondylar breadth were measured from the femur [Table 1]. The trabecular morphometric parameters (trabecular number [TbN], thickness, spaces, and volume) were studied in the proximal and distal epiphysis of the bone [Figure 1].
Figure 1: Three-dimensional reconstruction of a 12-week-old femur showing parts investigated. (a), Full length; (b), bicondylar breadth; (c and d), proximal and distal epiphysis. (e-g), 25th (proximal); 50th (midshaft) and 75th (distal) percentile marks, respectively

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Table 1: Osteometric parameter descriptions

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A cross-sectional slice from each percentile mark was then saved for further analysis on Fiji image J. From these slices, the cross-sectional area, cortical area, and medullary canal area of the shaft were measured at 3 positions: 25th (proximal), 50th (midshaft), and 75th (distal) percentile marks [Figure 1].

Statistical analysis

The data were managed in Microsoft Excel 2016 (Microsoft Corporation) and analyzed using SPSS® version 27 (IBM®, SPSS Statistics for Windows, Armonk, NY, USA.). Analysis of variance with least significant difference post hoc was used for the multiple group comparisons of means. Binary logistic regression was used to predict group membership into either the ethanol or saline control group. Significance level was set at P < 0.05.


  Results Top


Blood alcohol concentration

In this assay, serum for the saline control group had BAC values that were negative (or undetectable) and 81.53 mg/dl for ethanol.

Size estimation

Femoral length

All three groups in the study exhibited mean differences in femoral bone length. The ethanol group had the shortest femora (mean = 1 5.65 mm ± 0.84) compared to the saline (mean = 16.01 mm ± 0.74) and untreated controls (mean = 17.16 ± 0.85) [Figure 2]a. However, femora belonging to the ethanol group were only significantly shorter when compared to the untreated controls but not the saline group (P 0.001 and P 0.140, respectively). The saline controls were also significantly shorter than the untreated group (P 0.001).
Figure 2: Size estimation; (a), bone length; (b), shaft volume; (c), bicondylar length; (d), epiphyseal volume. Represented as means for the untreated control, saline control, and ethanol group. Error bars indicate standard deviation

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Femoral shaft volume

The phenomenon of lowest values in the ethanol group was also observed regarding shaft volume as it was lowest in the ethanol group (mean = 36.62 mm ± 6.30). It was significantly lower than the saline (mean = 44.29 mm ± 6.90 control) and untreated controls (mean = 46.90 ± 6.28) (P 0.001 for ethanol compared to both controls). No significant differences were detected between the control groups (P 0.286) [Figure 2]b.

Femoral bicondylar breadth

The mediolateral (ML) dimension varied between the groups investigated with the ethanol group (mean = 3.88 mm ± 0.68) showing a significantly lower bicondylar breadth in comparison to both the controls (saline; mean = 4.34 mm ± 0.41 and untreated mean = 4.45 ± 0.42) (P 0.006 for both comparisons). No significant differences were detected between the control groups (P 0.59) [Figure 2]c.

Femoral proximal and distal epiphysis volume

The size of the epiphysis showed regional similarities in the proximal and distal epiphysis among the study groupings. The proximal epiphysis had a marginally larger volume in the untreated controls (mean = 20.75 mm3 ± 1.24) compared to the saline (mean = 18.73 mm3 ± 3.39) and in the ethanol group (mean = 18.66 mm3 ± 2.86). However, no significance was observed among the three groups (P 0.113). In the distal epiphysis, a greater volume in the untreated controls (mean = 23.75 mm3 ± 3.06) compared to the saline (mean = 20.57 mm3 ± 3.91) and ethanol group (mean = 19.83 mm3 ± 2.90) was found (P 0.013 and P 0.002, respectively) [Figure 2]d.

Trabeculae morphometry

Femoral bone to total volume ratio (bone volume/total volume)

Regarding the proximal region, bone volume total volume (BV/TV) was the highest in the untreated controls (mean = 69.75% ± 4.91) compared to the saline controls (mean = 67.18% ± 11.33) and the ethanol group (mean = 62.02% ± 10.65) [Figure 3]a. This difference was significant for the ethanol and untreated controls (P 0.041), with controls showing no significant differences (P 0.195). In distal region, the saline (mean = 69.78% ± 11.95) and untreated controls (mean = 57.43% ± 3.27) had more BV/TV than the ethanol group (mean = 52.53% ± 12.30) (P 0.004 and P < 0.001, respectively) [Figure 3]a.
Figure 3: Trabeculae morphology. Represented as means for the animal groups (a), bone to total volume; (b), trabeculae thickness; (c), trabeculae number and (d), trabeculae spacing represented at the proximal and distal extremities. Error bars represent standard deviation

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Femoral trabecular thickness

Regional differences were observed in the trabecular thickness among the three groups. In the proximal region, all three groups had similarities in trabecular thickness (ethanol group; mean = 0.11 mm ± 0.05, saline controls; mean = 0.13 mm ± 0.07 and untreated controls; mean = 0.11 mm ± 0.03) (P 0.313) [Figure 3]b. Conversely, the distal region revealed that the ethanol group (mean = 0.08 mm ± 0.04) had thinner trabeculae than the saline controls (mean = 0.15 mm ± 0.07) although similar to the untreated controls (mean = 0.08 mm ± 0.02) (P < 0.001 for ethanol and saline controls) [Figure 3]b.

Femoral trabecular number

Trabecular distribution by the study group showed a varied pattern when stratified by epiphyseal region. The number of TbN in the proximal region was the greatest in untreated controls (mean = 6.26 mm−1 ± 1.00) followed by the saline (mean = 5.76 mm−1 ± 1.24) and the ethanol group (mean = 5.10 mm−1 ± 1.22) [Figure 3]c. However, these differences were only significant between the ethanol and untreated controls (P < 0.009).

In the distal region, the untreated controls (mean = 7.41 mm−1 ± 1.17) had more trabeculae than the saline (mean = 5.50 mm−1 ± 1.21) and ethanol groups (mean = 6.38 mm−1 ± 1.68) (P 0.001 and P 0.05, respectively) [Figure 3]c.

Femoral trabecular spacing

Unlike the other trabecular morphometric parameters, trabeculae spacing (TbSp) showed similarities among the groups in both the proximal and distal epiphyseal regions. In the proximal region, the saline and untreated groups had a mean of 0.056 mm (±0.01), whereas the ethanol group (mean = 0.06 mm ± 0.012) had marginally wider TbSps (P 0.160 among the three groups) [Figure 3]d and [Figure 4]a,[Figure 4]b,[Figure 4]c. In the distal region, all three groups had identical mean TbSp (untreated and saline control mean = 0.06 mm ± 0.007), although the ethanol group had a different standard deviation despite equal means (ethanol; mean = 0.06 ± 0.009) (P 0.97 among the three groups) [Figure 3]d and [Figure 4]d,[Figure 4]e[Figure 4]f.{Figure 3}
Figure 4: Trabecular morphology. Representative slices of (a), proximal end in an untreated control; (b), proximal end in a saline control; (c), proximal end of the ethanol group; (d), distal end of an untreated control; (e), distal end of saline control; (f), distal end of the ethanol group

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Femoral cross-sectional area, cortical area (thickness), and medullary canal area

Proximal (25th percentile mark)

The proximal cross-sectional areas were similar in all three groups (ethanol group; mean = 4.52 mm2 ± 0.89, saline controls; mean = 4.68 mm2 ± 0.89 and untreated controls; mean = 4.62 mm2 ± 0.72) (P 0.822) [Figure 5]a. Furthermore, the medullary canal area was similar in all three groups as the ethanol group had a mean of 2.21 mm2 (±0.52) and the saline controls had a mean of 2.21 mm2 (±0.40) and untreated controls had a mean of 2.14 mm2 (±0.68) (P 0.913) [Figure 5]a. With respect to cortical area (thickness) in this region, the ethanol group (mean = 2.31 mm2 ± 1.07) exhibited a marginally lower value compared to the saline (mean = 2.47 mm2 ± 0.96) and the untreated controls (mean = 2.48 mm2 ± 0.54). No significance was detected among the three groups (P 0.812) [Figure 5]a.
Figure 5: Cross-sectional area, medullary canal areas, and cortical thickness. Represented as means for the untreated controls, saline controls, and ethanol group; (a), at the 25th percentile mark; (b), 50th percentile mark; (c), 75th percentile mark. Error bars represent standard deviation

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Midshaft (50th percentile mark)

The midshaft cross-sectional area was similar in all three groups studied with the untreated groups having a mean of 4.69 mm2 (±0.82), saline mean = 4.66 mm2 (±0.92) and the ethanol with 4.58 mm2 (±0.68) (P 0.923) [Figure 5]b. The medullary canal area was the same in the controls (untreated and saline; mean of 2.34 mm2 ± 0.42 and 2.34 mm2 ± 0.43, respectively). The ethanol group (mean = 2.81 ± 0.74) had the largest medullary canal area with significant differences detected when compared to the untreated and saline controls (P 0.031 and P 0.008, respectively) [Figure 5]b. Regarding cortical area (thickness) in this region, the untreated (mean = 2.35 mm2 ± 1.00) and saline controls (mean = 2.32 mm2 ± 1.03) had similar values. The ethanol group (mean = 1.78 mm2 ± 0.69) exhibited lower values compared to untreated and saline controls. However, significance was only detected between the ethanol and saline controls (P 0.044) [Figure 5]b.

Distal (75th percentile mark)

The distal cross-sectional area was marginally smaller in the ethanol group (mean = 5.52 mm2 ± 0.97) compared to the saline and untreated controls (mean = 5.90 mm2 ± 0.97 and 5.96 mm2 ± 0.62, respectively). However, no significant differences were observed among the three groups (P 0.26) [Figure 5]c. The medullary canal area was marginally smaller in the ethanol group (mean = 3.89 mm2 ± 0.71) compared to the saline and untreated controls (mean = 3.93 mm2 ± 0.62 and 3.98 mm2 ± 0.93, respectively). No significant differences were observed among the three groups (P 0.95) [Figure 5]c. With respect to cortical area (thickness) in this region, the ethanol group (mean = 1.62 mm2 ± 0.45) exhibited a marginally lower value compared to the saline controls (mean = 1.96 mm2 ± 0.90) and the untreated controls (mean = 1.98 mm2 ± 0.60), although no significant differences were detected among the three groups (P 0.19) [Figure 5]c.

Femur (3 weeks) trabecular morphometric parameters most affected by gestational ethanol

Proximal femur

The effect of gestational ethanol on length, shaft volume, proximal BV/TV, proximal trabecular thickness (TbTh), proximal TbN, proximal trabecular spacing (TbSp), proximal volume, proximal cortical thickness, midshaft cortical thickness were assessed using binary logistic regression in the ethanol and saline control groups [Table 2]. The parameters used could reliably distinguish between the two groups. A test of the full model against a constant only model was statistically significant, indicating that the predictors as a set, reliably distinguished between ethanol or saline control group membership (Chi-square = 24.443, P 0.004 with df = 9). Nagelkerke's R2 of 0.550 indicated a moderately strong relationship between prediction and grouping. The Wald criterion demonstrated that no specific variable predicted membership [Table 3]. Prediction success overall was 78.3% (75.0% for the ethanol group and 81.8% saline controls).
Table 2: Femur osteometric and proximal epiphysis trabecular and cortical morphometric parameters in the equation

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Table 3: Group membership classification from osteometric and proximal epiphysis trabecular morphometric parameters of the femur

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Distal femur

The effect of gestational ethanol on length, shaft volume, midshaft cortical thickness, distal BV/TV, distal trabecular thickness (TbTh), distal TbN, distal TbSp, distal volume, distal ML, and distal cortical thickness was assessed using binary logistic regression in the ethanol and saline control groups [Table 4]. The parameters used could reliably distinguish between the two groups. A test of the full model against a constant only model was statistically significant, indicating that the predictors as a set, reliably distinguished between ethanol or saline control group membership (Chi-square = 36.004, P < 0.001 with df = 10). Nagelkerke's R2 of 0.724 indicated a very strong relationship between prediction and grouping. The Wald criterion demonstrated that no specific variable predicted membership [Table 5]. Prediction success overall was 91.3% (95.8% for the ethanol group and 86.4% saline controls).
Table 4: Femur osteometric and distal epiphysis trabecular and cortical morphometric parameters in the equation

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Table 5: Group membership classification from osteometric and distal epiphysis trabecular morphometric parameters of the femur

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  Discussion Top


The main aim was to test the hypothesis that prenatal ethanol interrupts trabecular parameters. The internal architecture was assessed from the data obtained using micro focus X-ray computed tomography (CT). Altered trabecular parameters in the alcohol exposed group were found. The diaphyseal cortical and medullary cavity proportions were also affected, particularly in the midshaft.

A concern of consuming alcohol during gestation is children considered small for age.[3],[12] Bone growth and development impact bone size and ultimately lead to stunted growth as observed in offspring of mothers who admit to drinking alcohol during gestation.[17] Therefore, understanding bone size in research related to the effects of alcohol intake during pregnancy is crucial. In the present study, we used micro-CT data to determine the shaft volume, epiphyseal volume, length, and bicondylar length. We could not find comparable studies with respect to the shaft and epiphyseal plate volume as well as bicondylar breadth. However, numerous studies report on full bone length.[1],[9] We were concerned that length alone as a single linear measurement would not be adequate to use as a proxy for bone size. Having used micro-CT data, we were able to quantify the bone size in the form of volume in a manner that best or reliably estimates the true bone sizes.

Limb bone elongation takes place through endochondral ossification, which is entirely dependent on the existence and viability of the epiphyseal growth plate.[18] It has been proposed that alcohol exposure during intrauterine life could result in shorter bone due to disturbances in chondrocyte number and differentiation rate as well as the size of the epiphyseal growth plate.[2],[9] Researchers[19] have used length (height) measurements of the growth plate and its various zones for use as a proxy of epiphyseal growth plate size. Since this measurement would be in the vertical direction, size information with respect to the horizontal aspects of the growth plate would not be captured. Furthermore, the vertical measurements of the growth plate and its various zones differ in each part of the growth plate when assessed from the ML aspect. This means that the height depends on where the researcher measures and is prone to difficulties in reproducibility. To overcome this problem, we used the measurements of surface area to gain a better estimate of the sizes of the growth plate and its zones.

We found a lower bone to tissue volume ratio in the ethanol group together with fewer and thinner trabeculae. We could not find applicable literature on gestational alcohol trabecular morphology in the femur of animals exposed to alcohol during intrauterine life. However, numerous studies elucidate the effects of alcohol use on bone strength and trabecular architecture.[5],[20],[21] These studies report altered bone to volume ratio, trabecular thinning as well as fewer trabeculae with larger spaces.[22],[23] Mean TbSp appeared to be unaffected in our study as there were no group differences.

Employing a binary logistic regression showed that shaft volume, length and proximal cortical thickness were the main parameters that determined group membership into either ethanol or saline control. This means that these three parameters are affected the most in gestational alcohol exposure. When using the proximal trabeculae morphometric parameters more saline control were misclassified as belonging to the ethanol group. This suggests that there was less group variation in these parameters. This, in turn, suggests that that intrauterine ethanol effects in the femur may be minimal for the proximal aspect of the bone.


  Conclusion Top


Gestational alcohol exposure had an adverse impact on bone size at 3 weeks in postnatal life. The smaller femora observed following intrauterine alcohol exposure could be due to the altered trabecular parameters and cortical as well as medullary cavity proportions in the femoral midshaft.

Financial support and sponsorship

The study received funding from the National Research Foundation of South Africa; Grant number: TTK160826186551.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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