Introduction

The genesis of the umbilical cord helices remains an elusive phenomenon in fetal development. The human umbilical cord (hUC) supplies nutrients and oxygen to the fetus and is indispensable for the adequate development of the fetus. The hUC develops 4–6 wk post-conception. It is mainly composed of the connective tissue, Wharton’s jelly, which supports and protects two umbilical arteries, and one umbilical vein (1). The umbilical vein transports nutrients and oxygen from the placenta toward the fetus, whereas in the umbilical arteries, the deoxygenated blood with waste materials flows from the fetus to the placenta (2). The presence of helices in the hUC was noted as early as 1,521 (2). A helix is a twisted, three-dimensional spiral shape. Over the years, synonyms such as coils, spirals, and turns were introduced and used as analogues. Helices in the hUC arise at an early stage of development. In week 7 of gestation, ∼95% of the fetuses have developed the helices, whereas ∼5% of the hUCs remain un- or hypocoiled (1, 3, 4). Even though the hUC elongates, the number of helices does not change for the remaining gestation (4). Thus far, the origin of the helices themselves and the intensity of coiling remain unclear.

Insight into the origin of the helices is important because hypercoiling is strongly associated with adverse pre- and postnatal clinical outcomes (5, 6, 7). In 1994, Strong et al proposed a standardized metric to quantify hUC helices, the umbilical coiling index (UCI), which is defined as the number of helices per centimeter of the hUC and is irrespective of the helix direction (8, 9, 10, 11). In uncomplicated pregnancies, the average UCI lies around 0.17 helices/cm with the 10th and 90th centiles at 0.07 and 0.3 helices/cm, respectively, and the hUC is categorized as normocoiled (1, 12). When the number of helices exceeds 3 helices per 10 cm, the hUC is defined as hypercoiled.

Numerous studies linked an abnormal hUC coiling intensity to pre- and perinatal morbidity and mortality (5, 6, 7, 13, 14). Hypercoiling is associated with fetal growth restriction and fetal distress resulting in (planned) premature delivery, a decreased Apgar score at 5 min, or even fetal demise (6, 7). Hypercoiling can also lead to umbilical vascular compression and reduced fetal blood flow, which in turn may result in parenchymal abnormalities such as fetal thrombosis in the hUC vessels, chorionic plate, and (stem) villi, creating a higher risk of prenatal and perinatal morbidity and mortality (15). These findings underline the urgency to further explore the origin of the helices and thereby the adverse pregnancy outcomes. However, research into the origin of coiling stagnated in the 90s of the last century after several hypotheses on the development of helices were introduced and subsequently shelved or invalidated (1). State-of-the-art imaging and molecular approaches, which were unavailable during the initial wave of studies, have the potential to alter this situation.

In this study, we investigated the histology of hypercoiled hUCs using digital reconstruction and investigated the basis of the hUC helices in newborn identical twin pairs discordant for their hUC coiling index. Identical twin pairs share their genome, sex, parental factors, and most of the intrauterine environmental factors, minimizing confounding when comparing hyper- and normocoiled hUCs. To define the molecular signature and origin of hUC helices, we generated transcriptomic data using RNA-seq and genome-wide epigenetic data using DNA methylation array profiling >700 1000 locations throughout the genome.

Results

Discussion

Several hypotheses regarding the origin of cord coiling have thus far been proposed. Yet, none of these are validated or widely recognized as a plausible explanation for the origin of hUC coiling (1). However, using state-of-the-art technologies including arterial digital reconstruction and transcriptomic analysis to study cord coiling in our unique monozygotic twin cohort, we can now challenge and refute previous explanations of the origin of cord coiling. We were able to visualize the distinct arrangement of the two muscle layers in the tunica media in the umbilical cord arteries and identify genes that are pivotal in the formation of umbilical cord helices.

The most commonly invoked explanation for hUC coiling is that the number of helices would depend on the amount of fetal movement including active and passive torsions (2). However, this is implausible because the helices develop at 7 wk of gestational age, before the ability of the fetus to actively move (2). Also, this explanation would predict that coiling may change during pregnancy, and this has never been documented. In addition, excessive growth is unlikely to underlie hypercoiling, given extensively long umbilical cords (ELUC) were associated with a variety of placental pathologies such as true knots but not with hypercoiling in a previously conducted study (16). Similarly, a hemodynamic origin can be refuted, as in the first trimester before week 7 when helices are being formed, the blood flow is limited and insufficient to induce helices (51, 52). Around week 11, the blood flow velocity becomes measurable within the umbilical cord, and this occurs after the helices are already present in the umbilical cord (51, 52).

Our macroscopic inspection revealed that the coiling phenotype is provoked by the coiling of the arteries as hUC Wharton’s jelly including the vein lost its distinctive coiled characteristics after dissecting out the artery. This observation confirms earlier research describing that the artery is fundamental to hUC coiling (2). This has led to various hypotheses on the origin of cord coiling that involve differences in muscle arrangement in the intima–media of the arteries. One hypothesis states that a small bundle of extra muscle fibers at the side of the arteries, the Roach muscle, may be responsible for cord coiling. However, the presence of the Roach muscle is not the key determinant for hUC coiling as the Roach muscle can also be present in both normo- and hypocoiled cords, and on the contrary, it can be absent in hypercoiled cords (53). Using umbilical artery digital reconstruction of consecutively cut serial slides, we found that hUC coiling most likely originates from the two separate muscle cell layers within the arterial muscle wall, the tunica media, where muscle fibers are aligned in opposite directions.

The composition of the muscle layers of umbilical cord arteries is fundamentally different than arteries found in other parts of the human body (54, 55). All other arteries, including the aorta, are histologically composed of one circular muscle layer (tunica media) with fibers aligned in a uniform circular direction. The umbilical artery is an exception and is the only artery that forms helices and consists of two muscle layers in the tunica media. Interestingly, when comparing the hUC to those of other placental mammals (Placentalia), macroscopic and histological properties support our hypothesis that helices are formed by the structural composition of the artery’s muscle cell layers. The UCs of odd-toed ungulates (Perissodactyla) such as horses and alpacas are macroscopically coiled and consist histologically of two muscle layers aligned in opposite directions, similar to human umbilical cords (56, 57, 58, 59). In contrast, the UCs of even-toed ungulates (Artiodactyla) and aquatic mammals (Sirenia) such as buffaloes and Amazonian manatees, respectively, are macroscopically uncoiled and contain arteries, histologically composed of a single muscle layer (60, 61). These observations strengthen our conclusion that coiling can occur if two muscle cell layers are present in the artery that are aligned in opposite directions to form helices.

To gain a deeper understanding of the molecular basis of hUC helices, we generated a transcriptomic and DNA methylation profile of hUC arteries obtained from MZ twin pairs discordant for cord coiling. The MZ twin pairs share their genome, parental factors, and most of the intrauterine environmental conditions during fetal development. Nevertheless, even with these shared characteristics the twin pairs were distinctly discordant for the number of helices. Therefore, genetic, most environmental, and parental influences are unlikely to be the dominant determinants of cord coiling. We observed distinct differences in gene expression within these discordant twin pairs. Specifically, we identified 28 differentially expressed genes and annotation of the function of these genes implicated a role of vascular development, inflammatory response, extracellular matrix, cell–cell adhesion, polarity, and axis formation including left–right (a)symmetry in the origin of cord coiling.

Zooming in on the differentially expressed genes, 12 of the 28 differentially expressed genes were shown to play a role in vascular development, cell–cell adhesion, polarity, and axis formation. Among the differentially up-regulated genes in hypercoiled arteries were two major transcription factors, KLF2 and KLF4, playing a key role in vascular maturation and blood vessel function. Furthermore, we observed the down-regulation of DLL4, a gene expressed in umbilical arteries but not the umbilical vein (34). Deletion of DLL4 has been associated with abnormal and decreased arterial development in mice (36). We also detected up-regulation of CDH2 (N-cadherin) in hypercoiled arteries. CDH2 is known to promote angiogenesis and stabilize blood vessels by acting as a cell–cell adhesion molecule to enhance the interaction between endothelial and mesenchymal cells (24, 25), potentially playing a role in retaining the hypercoiled state of the UCs. Genes encoding for leucine-rich repeat–containing proteins LUM, ASPN, and DCN were found to be up-regulated. Although DCN (42, 43) and ASPN (44, 45) can bind to type I collagen to support cell adhesion, LUM (40) plays a key role in regulating the stromal collagen matrix. Also, LEFTY2 (26) and CDH2 (22), genes encoding for asymmetrical signaling molecules, were down-regulated in hypercoiled arteries. CDH2 was found to play a role in establishing the left–right symmetry during development independent of LEFTY and NODAL (62).

The genes associated with cell–cell adhesion and vascular development as mentioned earlier are essential for maintaining the structural characteristics of the muscle wall. Luis Martinez-Lemus and Gasser et al extensively described the normal appearance and collagen structure of the arterial tunica media in the human body (63, 64). Each smooth muscle cell is surrounded by a basement membrane, cytoskeleton, and intracellular connections such as integrins, collagenous fibrils, and cadherins (65). Vascular smooth muscle cells of arteries in the human body are typically oriented along the longitudinal axis. The collagen fibers in the tunica media are arranged in a helical structure (64). Muscle fiber alignment of a smooth muscle cell can vary with a maximum variation of 20° (63). This variance may be emphasized by the presence of a double muscle layer of which both layers are aligned in opposite directions resulting in a helix. The observed up-regulation of type I collagen–binding DCN and ASPN, stromal matrix component LUM, and polarity-associated CDH2 (N-cadherin) and LEFTY2 may facilitate structural adaptation in the artery, promoting its helical configuration.

Interestingly, blocking the function of CDH2 with an antibody led to randomized heart looping in the chicken embryo (22), a process relying on muscle fiber alignment in crossing direction, similar to the process we identified to underlie the formation of hUC helices. CDH2 and the association with abnormal heart development are linked to the helical appearance and spatial distribution of overlapping superficial and deep myocardial fibers of the ventricular heart with concentric contraction of the myocardium (66, 67). The up-regulation of CDH2 in hypercoiled hUCs may give an increased muscle fiber alignment, contributing to and maintaining the increased coiling.

Lastly, our analysis revealed six genes involved in the inflammatory response that were found to be differentially regulated, a process known to be closely intertwined with thrombotic disorders. KLF2 and KLF4 were shown to uphold an antithrombotic effect in the vessel wall (17), whereas ZFP36 reduces an inflammatory response by guiding mRNA for degradation (28, 29). Yet, we also observed the down-regulation of the pro-inflammatory CXCL2. We hypothesize that observed down-regulation of the anti-inflammatory genes may be the result of the increased thrombotic risk in hypercoiled arteries. Fetal hUC vascular obstruction has been shown to be an effector of the adverse pregnancy outcomes such as stillbirth in case of hypercoiling (15).

Epigenetic marks, together with transcription factors, largely control the regulation of gene expression. DNA methylation is the most extensively studied epigenetic mark in population cohorts contributing to the regulation of gene expression. We compared the DNA methylation profile of hypercoiled and normocoiled hUC arteries. Nevertheless, no difference in DNA methylation was observed. The expected differences in the methylation level are rather small as compared to gene expression differences, and the correction for multiple testing is stringent because of the large number of tested loci. Therefore, extending this comparison to a larger cohort may increase sensitivity to detect potential differential gene regulation in hUC arteries. Another important factor is that gene expression regulation relies on a highly complex interplay of various epigenetic mechanisms and transcription factors. Therefore, it may also be the case that differences have manifested at another level of the epigenome such as in the binding of histones or other regulatory proteins, thereby affecting DNA accessibility to the transcriptional machinery.

The observed differences in gene expression between hypercoiled and normocoiled hUC arteries likely originated and manifested during early development. This is supported by the fact that the number of helices in a cord is established by week 7 and retained throughout pregnancy (1). We hypothesize that developmental plasticity allows an intrinsic or stochastically occurring trigger to modify gene expression, enabling a change in helix formation and stabilization of the hUC arterial hypercoiling. Yet, the underlying mechanism and causality of gene expression on developmental origins of hypercoiling remain to be uncovered.

In conclusion, our investigation into the origin of hUC coiling shows that the helical structure of the hUC is primarily derived from the hUC arteries rather than the vein and Wharton’s jelly. We demonstrated that the tunica media of the hUC arteries consist of two muscle cell layers with muscle fibers oriented in opposite directions. This unique arrangement is specific to the umbilical artery and is likely to enable coiling of the arteries. Showing that MZ twin pairs can be highly discordant for hUC coiling, it is unlikely that the dominant factor for coiling is of genetic or parental origin. Comparing the cord arterial transcriptomic profile of these MZ twin pairs, we found 28 differentially expressed genes. This includes the up-regulation of genes associated with muscle fiber alignment, cell–cell interaction, regulation of the stromal collagen matrix, polarity, and axis formation that may enhance increased muscle fiber alignment in hUC arteries resulting in an increased number of helices. This gives a new twist on the existing explanations of the origin of hUC helices and provides a basis to further elucidate the biological origin of hUC coiling.

Materials and Methods