Establishing normal ranges for fetal and neonatal small and large intestinal lengths: results from a prospective postmortem study

Introduction

At present, normal growth for fetal and neonatal intestinal lengths is reported in the literature1–4; however, this research is frequently decades old and may not reflect current fetal growth patterns in a Canadian population.5 Moreover, there seems to be less evidence investigating the impact of factors, such as intrauterine growth restriction (IUGR),6 7 multiple gestation and chromosomal defects, on infant gastrointestinal length as opposed to bowel length.8 Small bowel length is estimated to be 250±40 cm at birth. While its growth has initially been thought of as linear, the small bowel grows rapidly in the last 15 weeks reaching its maximal growth during the first year of life.3 4 Given such rapid changes in fetal bowel growth, the application of reference intervals (RI) based on gestational age in a clinical setting has a significant value.

Creating reliable RIs may have relevance in assisting surgeons to appropriately prognosticate and to form postoperative plans for infants undergoing bowel resection for conditions such as Hirschsprung’s disease and necrotizing enterocolitis.3 9 Length of small bowel, for example, can predict the need for bowel transplantation in infants with small bowel syndrome.10 Determination of normal fetal measurements is additionally important for pathologists conducting fetal postmortem examination and assessing fetal growth and development. Creating reliable RIs may assist pathologists in guiding further screening for congenital anomalies associated with small bowel syndrome11 and in properly defining pathological bowel length, which is often defined in terms of rough estimates without accounting for the gestational age of the fetus.11–13

Statistically, RIs are derived from ‘normal’ defined populations and communicate two limits (upper and lower limits) to distinguish normal versus abnormal cases in a population.14 They are used to examine organ and body weights including linear measurements, which are particularly important during the postmortem examination of fetuses and infants.15 16 The Clinical and Laboratory Standards Institute (CLSI) has established guidelines to ensure accuracy of RIs. The purposes of these guidelines are to identify reference subjects, to provide guidance on statistical methods, to discuss options for detecting and removing outliers, and to describe partitioning according to age, sex or other relevant categories.17 18 Specific challenges in establishing pediatric RIs arise owing to the changing physiology of infants, the confounding relationship between gestational age and body size on intestinal length, and limitations in sample size.19–21 A recent paper, which focused on evaluating optimality of the various statistical methods for estimating RIs, showed that the choice of statistical methods in RI estimation is crucial in ensuring accuracy and precision and that researchers should consider the distribution, sample size and heterogeneity of data in making the optimal choice.21 In the present study, we followed the recommendations provided as well as the steps provided in the CLSI guidelines.

The primary objective of the present study was to establish fetal and neonatal RIs for small and large intestinal lengths using data on the Eastern Ontario and Western Québec population. Sensitivity analysis was applied to correct for a variety of fetal factors and pregnancy complications.

Methods

Results

Study population

Data consisted of a total of 131 human fetuses and neonates, of whom 55% (n=72) were males and 45% (n=59) females. Gestational age ranged from 13 and 41 weeks, with a median of 24 weeks and IQR of 20–32 weeks (table 1). About 20% (n=26) of the fetuses had IUGR and 3.1% (n=4) were large for gestational age (LGA). Congenital anomalies were present on 26.7% (n=35) of the fetuses and neonates, and 13.0% (n=17) had gastrointestinal anomalies, such as Meckel’s diverticulum, bowel malrotation and diaphragmatic hernias. There were 10.7% (n=14) fetuses and neonates from multiple pregnancies (table 1).

Table 1

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Population characteristics and clinical measurements (n=131)

The average length of the small intestine was 147.7 cm (SD=82.7) in comparison to 30.6 cm (SD=17.7) for colon length. It is worth noting that both measurements are highly variable as shown by the value of their corresponding SDs. This is attributed to the wide range of gestational ages and to linear growth of intestinal length with respect to age (figures 1 and 2).

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Figure 1

Plot of small intestinal length by gestational age for male and female (left) and for the combined population (right).

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Figure 2

Plot of large intestinal length by gestational age (A) for male and female (B) combined data.

Discussion

We have established age-specific RIs for small and large intestinal lengths of fetuses and neonates using data in the Eastern Ontario and Western Québec regions. In comparison to our study, prior literature also demonstrates a positive, linear relationship between age and intestinal length RIs.1 2 This holds true for both the length of individual sections of the intestinal tract as well as the total intestinal length. In keeping with other studies, no differences in intestine length were found between sexes, and hence we combined data for males and females to maximize the sample size.4 22 23

Sensitivity analysis showed that the absence of congenital anomalies, gastrointestinal anomalies, maceration, as well as IUGR and LGA fetuses did not influence intestinal length RIs. Unique to our study were findings that fetuses from multiple pregnancies did not impact small intestinal length. The relationship between singleton pregnancies and the quantifiable impact on fetal intestinal length remains largely unanswered. Evidence suggests that fetuses from twin pregnancies are susceptible to a variety of intrauterine complications, such as IUGR, with the incidence of IUGR approximating 25%–35%.26 27 Whether IUGR impacts fetal intestinal length remains equivocal,3 4 and in our analysis IUGR did not have any impact on intestinal length. Similar to prior literature,4 maceration did not appear to have an impact on the growth of small intestinal lengths, and is in keeping with prior literature demonstrating that maceration does not alter all body measurements.15 The presence of congenital anomalies did not alter the small intestinal length. Though prior literature has demonstrated a reduction in length in Trisomy 21 fetuses,4 we included a range of congenital anomalies, such as Turner syndrome and vermian hypoplasia, in addition to Trisomy 21. Combining the congenital anomalies may have attenuated the veritable impact of Trisomy 21 on fetal intestinal growth. Unfortunately, due to sample size limitations, a subgroup analysis was not possible and represents an area of further research.

Capturing fetal intestinal length as early as 13−41 weeks is useful for both surgeons and pathologists to visualize intestinal length across a wide range of gestational ages. From a clinical standpoint, access to intestinal length RIs across a range of ages may assist in refining prognosis and treatment plans for preterm or term patients where intestinal resection is necessary, such as for Hirschsprung’s disease. Pathologists analyzing biopsies for transition zone between aganglionic and ganglionic cells can communicate residual bowel length to surgeons,28 which could assist surgeons in further planning postoperatively. Residual small bowel length may allow surgeons, for example, to predict the ability of an infant with short bowel syndrome (SBS) to wean from total parenteral nutrition following bowel resection.29 Furthermore, defining accurate small bowel length may provide benefit to pathologists conducting postmortem examinations. As mentioned earlier, there is currently a wide range of definitions being applied to SBS in fetuses and neonates, and establishing RIs may help apply this definition more precisely among this population based on age. Additionally, intervals may assist pathologists in identifying the pathological bowel length and in deciding when to screen for genetic anomalies, such as the CLMP gene in congenital SBS.11 13

Owing to limitations of sample size, determining reliable estimates for intestinal length is difficult. Wide variability in intestinal length observed within partitions in our data may indicate the need for a per-week analysis to capture minute differences over time because intestinal growth can vary widely in fetuses. Marnerides et al established fetal intestinal length intervals in the second trimester and found considerable differences across weekly partitions.4 To overcome these challenges, reference curves may be fitted to data and normal ranges can be obtained by using the functional form of the estimated curves at a given gestational age. We plan to pursue this approach in a subsequent study.

There are several limitations associated with this study. First, bowel measurements were made postmortem after removal of the bowel from the abdomen. Postmortem measurements can overestimate bowel length due to initial muscle contraction shortly after death, when relaxation and loss of tone occur as autolysis begins6; however, we did examine intestinal length in the sensitivity analysis without maceration to account for this. We did not find any impact of maceration on intestinal growth. In contrast, use of silk sutures to measure intestinal length in live infants provides a promising method to accurately measure intestinal length in vivo, although this methodology requires further external validation.19 We also followed a routine protocol as described in similar studies1 by laying out specimens on a moist plastic surface to minimize ‘stretch artifact’, and the same experienced perinatal pathologist measured each specimen twice to reduce error in measurement. Hence, the variations in gut length are not likely to be attributed to measurement inaccuracy. An additional limitation involves our population of study because the results are based on postmortem fetuses. Our results are thus not truly representative of the normal fetus population. However, the RIs could still be applicable to postmortem populations with similar characteristics.4 Further, to develop statistical models that account for the effects of age, sex, congenital anomalies, and other covariates, the Hoq et al’s method could have been used to estimate RIs as a function of age.30 The methods we used by Daly et al21 were valid but did not exploit the linear relationship between age and intestinal length. We also partitioned the age categories in a narrow manner because measurements increased linearly with respect to gestational age. However, this approach may be seen as subjective and contingent on the sample size available in each age category. We will use the discrete age groups commonly reported in the Hoq et al’s30 systematic review to address this limitation in a future study.

To conclude, this study has provided age-specific RIs for normal small and large intestinal lengths of fetuses and neonates. Pathologists can compare our data to other studies that have established reference standards for normal intestinal length. From a surgery perspective, understanding the expected or normal intestinal length might aid surgeons to communicate how much intestine is remaining in infants with parents and to plan postoperative management and prognosis accordingly. Following the standard protocols for conducting linear measurements of gastrointestinal contents,2 31 future studies are encouraged to replicate the study methodology at other health centers for cases in their catchment areas to enable comparisons across provincial institutions. More importantly, further research can be conducted to examine additional factors, such as gestational weight on intestinal length, using the Hoq et al’s30 method and to compare such results to established RIs. This will aid in better understanding of the differences in intestinal length between normal and pathological fetuses.

Lastly, coordination of multicenter, nationwide efforts to collect small and large intestinal length data (eg, similar to the Canadian Laboratory Initiative on Pediatric Reference Intervals)32 and to establish RIs in accordance with CLSI guidelines is needed.