New Concepts in Diabetic Embryopathy

Synopsis

Diabetes mellitus is responsible for nearly 10% of fetal anomalies in diabetic pregnancies. Although aggressive perinatal care and glycemic control are available in developed countries, the birth defect rate in diabetic pregnancies remains much higher than that in the general population. Major cellular activities--i.e., proliferation and apoptosis--and intracellular metabolic conditions--i.e., nitrosative, oxidative, and endoplasmic reticulum stress--have been demonstrated to be associated with diabetic embryopathy using animal models. Translating advances made in animal studies into clinical applications in humans will require collaborative efforts across the basic research, preclinical, and clinical communities.

Introduction

Diabetes mellitus is a metabolic disease, primarily due to high concentrations of glucose in the circulation (1). Hyperglycemia interrupts normal cellular metabolism and signaling and eventually causes organ dysfunction (2, 3). Nearly two centuries after diabetes was first recognized (4), its association with congenital birth defects and fetal mortality in pregnancy was recognized and referred to as diabetic embryopathy (4, 5). Prior to the introduction of insulin, diabetes-associated fetal and maternal mortality rates were nearly 70% and 40%, respectively (6, 7). Since the administration of insulin to control glycemia in pregnant women, these mortality rates have decreased dramatically to nearly 12% (8–15). In addition to the control of glycemia with insulin, aggressive perinatal care and neonatal management also contributed to the decline of maternal and fetal mortality (10, 16–19).

Unfortunately, the present birth defect rate in diabetic pregnancies (about 10%) is still higher than that in the general population (3%) and appears to be on the rise (7, 12, 13, 15, 20–25). The reasons for this increase in birth defect rates are complex. One reason is that there has been a rapid increase in diabetic patients in the population which includes women of childbearing age (26). It is estimated that approximately 8,000 babies in the United States are born each year with maternal diabetes-associated congenital malformations. The incidence of these malformations also has risen to near-epidemic level in developing countries.

Tackling this issue involves battles at a number of fronts: diagnosing fetal anomalies must be improved; technologies that can recognize developmental malformations as early as the embryogenesis period are needed; and better prenatal and planned pregnancy consultations should be implemented to help reduce diabetes-associated birth defects. These remain ongoing challenges for perinatal care providers.

An important goal in eliminating birth defects is to develop therapeutic interventions that can protect embryos from hyperglycemic insult. This goal can only be achieved by understanding the cellular and molecular mechanisms underlying diabetic embryopathy. Basic research using animal models has contributed a considerable amount of information about the manifestations of fetal abnormalities, but more work is still needed.

Pre-gestational and gestational diabetes

Diabetes mellitus is a chronic disease manifested by hyperglycemia and its associated metabolic factors. This condition is usually diagnosed by measuring the levels of plasma glucose, expressed as mg/dL or mM, and glycosylated hemoglobin A (HbA_1c), indexed as percentage of total hemoglobin A (27–29). Manifestation of diabetes can be the result of insulin deficiency (type 1) or insulin resistance (type 2).

• Type 1 diabetes, or insulin-dependent diabetes, is caused by autoimmune destruction of insulin-producing β-cells in the pancreas (30, 31).

• Type 2 diabetes, or non-insulin-dependent diabetes, is caused by failure in insulin signaling to regulate cellular glucose uptake (32–34).

Diabetes mellitus, either type 1 or type 2, diagnosed in women before pregnancy is referred to as pre-gestational diabetes (20, 35–37). When hyperglycemia is detected after the onset of pregnancy, usually in the third trimester (24–28 weeks), the pregnant woman is considered having gestational diabetes mellitus (GDM) (38–42). According to guidelines from the International Association of Diabetes in Pregnancy Study Group (IADPSG), women who have a fasting plasma glucose ≥ 126 mg/dL and HbA_1c ≥ 6.5% are diagnosed as having GDM (43).

Congenital birth defects in infants of diabetic mothers have been found to be associated with pre-gestational diabetes that is uncontrolled in the first trimester of pregnancy (44–48) Although a few cases have suggested a link between GDM and fetal birth defects, this association remains unclear and lacks strong supporting evidence (49, 50). One possible reason for the controversy is that some women with early-onset type 2 diabetes are misdiagnosed as having GDM when first screened in the second trimester (39, 51). Nevertheless, it is well established that GDM can lead to many adverse fetal outcomes, including macrosomia, hypoglycemia, hypocalcemia, and hyperbilirubinemia (51–53).

High glucose as a major teratogenic factor

Human studies have demonstrated a strong link between maternal glycemic level, as indicated by the association of plasma glucose and HbA1c levels (54, 55) with the incidence of congenital malformations in offspring (56–61). Other adverse metabolic factors produced in diabetes mellitus, such as ketone bodies, advanced glycation end products, and branched chain amino acids, may have synergetic effects with glucose on disrupting normal embryonic development (62–66). The putative teratogenic effects of hyperglycemia are supported by studies that demonstrate a reduction in the incidence of birth defects following clinical interventions targeted at achieving euglycemia (67–70). Animal studies also have shown that isolated embryos in culture exposed to high concentrations of glucose develop malformations similar to those seen in human diabetic pregnancies (62, 71–73). These observations indicate that high glucose in either type 1 or type 2 diabetes is a major teratogenic factor that disturbs embryonic development.

The major effort to eliminate adverse outcome in pre-gestational diabetes and GDM complicated pregnancies is to control glycemic levels (20, 74–76). Clinical management of diabetic pregnancy with insulin and oral hypoglycemic medications can achieve the euglycemic standards, recommended by Diabetes Control and Complications Trials and International Federation of Clinical Chemistry and Laboratory Medicine (Table 1) (77–79). However, with respect to pre-gestional diabetes, optimal glycemic control prior to conception appears to be a challenge because:

Table 1.

Recommendations for pre-conception HbA1c targets

	USA	UK
DCCT	<7.0%	<6.1%
IFCC	<53 mmol/mol	<43 mmol/mol

DCCT, Diabetes Control and Complications Trials; IFCC, International Federation of Clinical Chemistry and Laboratory Medicine.

• most women with diabetes do not seek preconception care,

• most have unplanned pregnancies (80).

• even in women who plan pregnancies and receive preconception counseling, euglycemia is difficult to achieve and maintain in non-clinical settings.

Therefore, alternative approaches to glucose control to prevent birth defects need to be developed and implemented.

Malformations and Diagnosis

Maternal diabetes-associated fetal anomalies can be seen in any organ system but are most common and severe in the central nervous system (CNS), cardiovascular system (CVS), craniofacial region, and caudal structure (Table 2) (11, 12, 25, 44, 55, 81, 82).

Table 2.

Developmental anomalies in major organ systems in diabetic embryopathy

Central nervous	Craniofacial	Cardiovascular	Skeletal
Anencephaly	Hemifacial	Conus arteriosus defects	Sacral agenesis
Encephalocele	microsomia	Transposition of great vessels	Sacral hypoplasia
Exencephaly	Macrostomia	Tetralogy of Fallot	Limb defects
Microcephaly	Cleft palate	Ventricular septal defects	Vertebral defects
Hydrocephaly	Cleft lip	Pulmonary vavle defects	Caudal regression
Holoprosencephaly	Microtia	Patent ductus arteriosus
Spina bifida	Micrognathia	Hypoplastic left heart syndrome
	Craniosynostosis	Coarctation of the aorta
	Anotia/Microtia	Right ventricular septal defects
	Eye defects	Atrial septal defects
		Heterotaxia

Central nervous system

The most common structural defects resulting from diabetic embryopathy occur in the brain region and spinal cord of the CNS () (12, 25, 83–86). Some of these can be recognized as early as in the first trimester using ultrasonography.

• Anencephaly is characterized as absence of the cerebral hemispheres with the brain stem and portions of the midbrain intact. It is readily discernible in the first trimester (Figure 1) (76, 87, 88). In the second trimester, it is more evident as poorly formed cranial bones and symmetric absence of the calvarium (76, 87, 89).

• Holoprosencephaly, the complete or partial absence of the midline echo within the fetal brain, can be diagnosed as early as 14 weeks of gestation, though diagnosis at the 20-week anomaly scan is more common (90–92).

• Excencephaly, the absence of the superior vault and convolution of the brain, can be diagnosed as early as 10 weeks of gestation (Figure 2) (93).

• Ultrasonic diagnosis of CNS defects such as spina bifida usually occurs in conjunction with detection of the second trimester maternal blood biomarker α-fetoprotein (81, 94–97).

Figure 1.

Two-dimensional ultrasonic scanning of anencephaly in the first trimester. Arrows indicate missing forebrain. Modified with permission from; Cameron M, Moran P. Prenatal screening and diagnosis of neural tube defects. Prenat Diagn 2009;29:402–11.

Figure 2.

Two-dimensional ultrasonic scanning of exencephaly in the first trimester. The contours of the brain are irregular (arrows). Modified with permission from; Blaas HG, Eik-Nes SH. Sonoembryology and early prenatal diagnosis of neural anomalies. Prenat Diagn 2009;29:312–25.

Craniofacial structures

In newborns of diabetic mothers the most common anomalies in the craniofacial regions are hemifacial microsomia and microtia in newborns of diabetic mothers (82, 98). Cleft palate and lip also occur in relatively high frequency (99–101). Hearing impairment in children has been found to be associated with diabetic pregnancy (82, 98, 102), likely due to developmental defects in the inner and middle ear (102).

Prenatal diagnose of cleft lip and cleft palate relies a combination of coronal and axial two-dimensional ultrasound scans (103). Cleft lip can be easily recognized from coronal scan images, however, when cleft lip extends into the palate, the axial scan of the maxilla can provide the image of the defects (104). Cases of lateral cleft lip/palate often present a so-called maxillary pseudomass visualized in two-dimensional sonographs (105).

Cardiovascular system

Fetal cardiac defects associated with maternal diabetes have been extensively characterized. Anomalies commonly are present in:

• myocardium-derived structures

  • atria,

  • ventricles

  • interventricular septum.

• endocardium-derived structures

  • conotruncal spetum

  • ventricular septum

  • atrio-ventricular valves (44, 47, 106–108).

Sonographic imaging remains the standard method to diagnosis fetal cardiac abnormalities. Technological advances in imaging, especially in high frequency imaging and improvements in resolution, have aided perinatal care providers in recognizing cardiac defects as early as 10 weeks of gestation.

Most heart anomalies, including double outlet hearts, atrial septal defects, and ventricular septal defects, can be diagnosed using the four-chamber view, a transverse projection through the fetal thorax above the level of the diaphragm (Figure 3) (109). Measurement of the thickness of the ventricular walls in the sonographs can reveal myocardial hypoplasia. Due to the position of the canning plane, the four-chamber view usually misses defects in the structures in the outflow tracts. A so-called base view, with scans superior to the four-chamber view, can reveal abnormalities in the aorta and pulmonary artery. Complex defects such as Tetralogy of Fallot, which involves ventricular septal defects and great vessel defects, may require a combination of four-chambered and base views to diagnose.

Figure 3.

Four-chamber view of fetal hearts. (A) Normal heart. Arrow indicates interventricular septum. Arrowhead indicates interatrial septum. (B) Heart with VSD (arrow) in inlet portion of interventricular septum (arrowhead). LA, left atrium; LV, left ventricle; RA, right atrium; RV, right ventricle. Modified with permission from; Rajiah P, Mak C, Dubinksy TJ, et al. Ultrasound of fetal cardiac anomalies. AJR Am J Roentgenol 2011;197:W747–60.

Color Doppler provides an added advantage to practitioners because it can detect minor defects in the heart by exhibiting changes in blood flow pattern. The association of increased first trimester nuchal translucency with fetal cardiac defects has been observed; however, more studies are still needed to establish it as a diagnostic marker (110, 111).

Caudal regression

Caudal regression syndrome, also known as caudal dysplasia, is characterized as the absence or hypoplasia of caudal trunk and limbs (112, 113). Caudal regression syndrome can be diagnosed by noting a shortened spine and abnormal lower limbs. This anomaly can be detected in the fetus as early as second trimester.

Mechanisms of Diabetic Embryopathy

Developmental mechanisms

Central Nervous System

Diabetic embryopathy causes embryonic and fetal abnormalities as a result of a disturbance in normal organogenesis. In the CNS, most anomalies occur because of a failure of early neural tube formation, referred to as neural tube defects (NTDs) (114–116). The formation of the neural tube, or neurulation, occurs in human embryos from weeks 3–6 of gestation (114). Neurulation begins with formation of the neural ectoderm, which develops into the neural folds. The neural folds grow dorsal-laterally and eventually fuse at the dorsal midline along the body axis to form the neural tube (117). In diabetic embryopathy, neurulation is perturbed in young embryos, leading to NTDs such as exencephaly or spina bifida (62, 73, 118, 119). Studies using diabetic rodents with poorly controlled hyperglycemia have recapitulated the same CNS defects observed in human fetuses (Figure 4) (120–122).

Figure 4.

Exencephaly in mouse E15.5 fetus of diabetic pregnancy. (A) Nondiabetic control. (B) Diabetes. Arrow indicates exencephaly. Scale bar=3 mm.

Cardiovascular System

The development of the heart is a complex process, involving multiple tissue types. The atria, ventricles, and interventricular septum are derived from the myocardium, whereas the membranous septa in the atrioventricular channel and the outflow tracts and associated valves are derived from the endocardium (123–126).

The most common abnormality of the ventricles is hypoplastic left heart syndrome (44). It is associated with under development of the myocardium during early cardiogenesis (121). Defects in endocardium-derived structures appear to be associated with endocardial cushions (Figure 5) (121, 127), bulbous structures that develop during early embryogenesis at the atrioventricular junction and the bulbous cortis (outflow tract) as a result of the production of the extracellular matrix (128, 129). During development of the endocardial cushions, endocardial (endothelial) cells differentiate into mesenchymal cells. This process is known as the endothelial-mesenchymal transformation (EMT) (128, 130). These mesenchymal cells migrate into the extracellular matrix to fill the acellular space and promote the growth of the cardiac structures (130). The endocardial cushions develop toward each other and eventually fuse to form a continuous septum (125). After fusion, the endocardial tissues undergo dramatic remodeling to connect with the interventricular and primary atrial septum and form valves (130, 131).

Figure 5.

AVSD in mouse E15.5 fetus of diabetic pregnancy. (A) Heart of nondiabetic control. (B) Heart of diabetic group with VSD (arrow). ao, aorta; ivs, interventricular septum; lv, left ventricle; ra, right atrium. Scale bar=200 μm.

In embryos of diabetic pregnancies, early development of the endocardial cushions is inhibited (121). An impact of maternal hyperglycemia on later endocardial cushion remodeling also has been observed (132). Further research is needed to determine the significance of these developmental processes in cardiac malformation in diabetic embryopathy.

Craniofacial

In the craniofacial region, abnormalities in facial structures may be associated with dysmorphogenesis of cartilages (133, 134). These cartilages, such as the Meckel’s cartilage in the mandibular arch, are not only involved in early morphogenesis of the facial processes, but also give rise to many types of bony structures in the craniofacial regions such as the auditory ossicles (135, 136).

Cellular mechanisms

The development of organ systems involves cell proliferation, death, migration, and differentiation. Excessive programmed cell death (apoptosis) has been observed in the dorsal region of the neural tube and is associated with NTDs in diabetic embryopathy (71, 122, 137–142). In the developing heart, cell proliferation at the early stages and apoptosis at the late stages of cardiogenesis appear to be associated with cardiac malformations in the embryos of diabetic pregnancy (121). In addition to cell proliferation, endocardial cell differentiation and migration also are suppressed by maternal hyperglycemia (121, 127).

Development of the cardiac outflow tract requires cell migration from the neural crest in the dorsal region of the neural tube. These neural crest cells contribute significantly to the septation of the outflow segment and the formation of the great vessels (143, 144). Malformations in the outflow segment may be due to impaired neural crest cell migration, which can be caused by increased apoptosis (145, 146).

Craniofacial structure development also requires neural crest cells that migrate from the anterior neural tube during early embryogenesis (147–149). Studies using animal models have shown that maternal diabetes perturbs neural crest cell migration in embryos (133, 150). Increased apoptosis in the mandibular arch has been observed in embryos of diabetic women, though it is uncertain whether those cells are of neural crest-origin (146). The impact of maternal diabetes on the differentiation of the neural crest-origin craniofacial mesenchymal cells remains to be demonstrated.

Molecular mechanisms in promoting apoptosis in diabetic embryopathy

Endoplasmic reticulum stress

When exposed to high glucose, embryonic cells take up glucose via glucose transporters (151, 152). An influx of glucose disturbs intracellular metabolic homeostasis and organelle function. Dysfunction of the endoplasmic reticulum (ER) leads to aberrant protein folding and subsequent accumulation of unfolded and misfolded proteins in its lumen (153–155), which cause ER stress (156–159). Under stress conditions, the ER activates a number of molecular cascades, collectively known as the unfolded protein response (UPR), to increase expression of chaperone protein to resolve protein folding crisis, inhibit protein translation, suppress mitosis, and even trigger apoptosis (Figure 6) (160–162). ER stress has been observed in the embryos from diabetic pregnancies (127, 163, 164). The potential causative role of ER stress in embryonic malformation has been demonstrated with experiments using a chemical chaperone to resolve protein folding crisis (127).

Figure 6.

ER stress in diabetic embryopathy. ER, endoplasmic reticulum; UPR, unfolded protein response.

Oxidative stress

High glucose also changes the morphology and function of mitochondria (165). Changes in mitochondria interrupt the electron transport chain, leading to generation of reactive oxygen species (ROS) (166, 167). High glucose also reduces the level of intracellular antioxidants, including glutathione (GSH) and thioredoxin (168–171). The imbalance of ROS and antioxidative buffering results in oxidative stress, which perturbs intracellular signaling (Figure 7) (172, 173). Treating diabetic pregnant animals or embryos cultured in high glucose with antioxidants decreases embryonic malformation rates (174–184). Moreover, embryos treated with antioxidants overexpress a transgene of superoxide dismutase resist maternal hyperglycemic insult (185, 186).

Figure 7.

Oxidative stress in diabetic embryopathy. GSH, glutathione; ROS, reactive oxygen species.

Nitrosative stress

Under hyperglycemic conditions, embryonic cells produce high levels of nitric oxide (NO). NO is a second messenger that regulates various intracellular signaling pathways (187, 188) and also can react with ROS to produce more toxic radicals, called reactive nitrogen species (RNS), than NO or ROS alone (189, 190). The high levels of RNS, such as peroxynitrite, generate a condition known as nitrosative stress (Figure 8).

Figure 8.

Nitrosative stress in diabetic embryopathy. iNOS, inducible nitric oxide synthase; RNS, reactive nitrogen species.

Synthesis of NO is catalyzed by NO synthase (NOS) enzymes. There are three main forms of NOS enzymes: neuronal (nNOS and NOS1); endothelial (eNOS and NOS3); and inducible (iNOS and NOS2)(191–193). Both nNOS and eNOS are constitutively expressed and do not vigorously respond to extracellular stimulation (194, 195), but iNOS actively responds to extracellular changes with marked upregulation in expression and activity (196–198).

In embryos of diabetic animals, eNOS expression is decreased (199); in contrast, iNOS expression is dramatically increased (200, 201). Experiments using an iNOS knockout model clearly demonstrate that high level of NO is detrimental to the embryo (Figure 8) (120).

Stress response

Phospholipid peroxidation

Cellular stress perturbs intracellular metabolic homeostasis. Phospholipid metabolism is initiated by phospholipases (PLs), including PLA, PLC, and PLD (202, 203). Cytosolic phospholipase A2 (cPLA₂) cleaves arachidonic acid from the cell membrane (204–206). In the cytoplasm, arachidonic acid undergoes two major pathways of metabolism: it can be converted into prostaglandin E₂ (PGE₂) by cyclooxygenase-2 (COX-2) (207, 208), or it can be converted into PGE₂-like isoprostanes, such as 8-iso-prostagladin F2 (8-iso-PGF2) and 8-iso-PGF2α, by non-COX-mediated peroxidation involving free radicals (Figure 9) (209, 210).

Figure 9.

Lipoperoxidation in diabetic embryopathy. COX-2, cyclooxygenase-2; cPLA2, cytosolic phospholipase A2; PGE2, prostaglandin E2; ROS, reactive oxygen species.

In embryos of diabetic animals or embryos exposed to high concentrations of glucose in vitro, the level of PGE₂ decreases dramatically (211–215). On the other hand, the level of 8-iso-PGF₂ is dramatically elevated in embryos under hyperglycemic conditions as well as in diabetic patients (209, 216). The reduction in PGE₂ in the embryo may be due to a decrease in COX-2 activity and expression (211, 217, 218), suggesting that a shift in arachidonic acid metabolism has occurred from producing PGE₂ to generating isoprostanes. These PGE₂-like isoprostanes have been shown to have damaging effects in animal models of diabetic pregnancy and embryos exposed to high glucose in culture (216, 219), whereas PGE₂ protects embryos from the damages due to hyperglycemic conditions (Figure 9) (211, 218, 220).

Protein kinase C family

Under cellular stress conditions, a number of intracellular signaling systems are affected, including the ones regulated by members of the protein kinase C (PKC) family. The PKC family of serine/threonine protein kinases consists of 12 members, which can be divided into the following three groups, based on their activation mechanisms (221, 222): 1) PKCα, β1, β2, and γ require calcium and diacylglycerol (DAG) for activation; 2) PKCδ, ε, η, ν, and θ require only DAG; and 3) PKCμ, ξ, and ι/λ do not require calcium or DAG, but instead require distinct lipid cofactors (i.e., ceramide and phosphatidylinositol-4-phosphate) (221).

In embryos of diabetic animals, each PKC isoform responds differently to hyperglycemia. For example, PKCα, β2, and δ are phosphorylated and activated under hyperglycemic conditions (141). Consequently, inhibiting PKCα, β2, and δ with isoform-specific inhibitors reduces malformation rates in embryos cultured in a high concentration of glucose (141, 223). Additionally, embryos from diabetic pkcδ knockout animal models exhibit significantly lower malformation rate than embryos from diabetic mice having the gene (164). These and other similar studies demonstrate that PKCs play critical roles in diabetic embryopathy.

Mitogen-activated protein kinase family

The mitogen-activated protein kinase (MAPK) family also plays an important role in mediating the effects of maternal hyperglycemia on embryonic development. Members of the MAPK family, including extracellular signal-regulated kinases (ERKs) and c-jun N-terminal kinases/stress-activated protein kinases (JNKs/SAPKs), can usually be activated by oxidative stress. ERKs primarily regulate cell proliferation and survival, whereas JNKs act on pro-apoptotic pathways (224–226). In embryos of diabetic animals, the levels of phosphorylated ERK1 and 2 are significantly reduced (Figure 8) (227, 228). In contrast, JNKs are activated in embryos of diabetic animals (227–230). Embryos treated with a JNK inhibitor while simultaneously incubated in a high concentration of glucose have a lower malformation rate, compared with those without JNK inhibition (231). More convincing evidence of the role of JNKs in diabetic embryopathy comes from the experiments using jnk1 and jnk2 knockout mice. Embryos homozygous for either jnk1 or jnk2 deletion show significantly lower malformation rates compared with a wild-type embryos from diabetic animals (231, 232).

Programmed cell death (Apoptosis)

Apoptosis is precisely regulated by a number of factors, including PKCs, JNKs, and members of the Bcl-2 and caspase families (233). Although the mechanisms by which PKCs and JNKs promote apoptosis in diabetic embryopathy remain to be delineated, much is known about the roles that Bcl-2 and caspase family members play in programmed cell death.

In the Bcl-2 family, some members are pro-apoptotic, such as Bax, Bak, and Bid, whereas other members are anti-apoptotic, such as Bcl-2 and Bcl-xL (234). In the caspase family, some members play a role in executing apoptosis. These members, known as effector or executioner caspases, include casapse-3, -6, and -7 (235–237). Effector caspases can be activated by another group of caspases, known as initiator caspases, including caspase-8, -9, and -10 (235–237).

Bax and Bak form a pore in the outer membrane of mitochondria (238, 239), allowing cytochrome C to be release to cytoplasm to induce apoptosis (240–242). During apoptosis, Bax and Bim expression levels increase in the cell (238, 239). Bax is activated by truncated Bid (tBid), which is produced when Bid is cleaved by serine/threonine proteases such as caspase-8.

In diabetic embryopathy, caspase-8 cleaves Bid into tBid, which stimulates the release of cytochrome C (140). Cytochrome C binds to apoptosis protease-activating factor-1 (Apaf-1), and the resulting complex activates Caspase-9 by forming an apoptosome. Activated Caspase-9 then activates effector caspases, including Caspase-3, 6, and 7, which turn on caspase-activated DNase and other factors, leading to DNA fragmentation and cell death (Figure 10) (240, 241).

Figure 10.

Caspase-8-regulated apoptotic pathway in diabetic embryopathy. Apaf-1, apoptotic protease-activating factors-1; Casp, caspase; Cyto, cytochrome;

In animal studies, suppression of apoptosis using caspase inhibitors can reduce embryonic malformations in the embryos cultured in high glucose (140). The results imply potential interventional strategies to block apoptosis in embryos in diabetic pregnancies.

Interventions and Clinical Challenges

Research conducted in animal models of diabetic pregnancy has revealed some of the major molecular changes and subsequent intracellular metabolic conditions that occur in embryos in response to hyperglycemia. This information has guided efforts to explore interventional approaches to reduce diabetes-induced embryonic malformations (122, 174, 183, 184, 227, 243–247).

Targeting lipid metabolism

One anomaly that occurs in diabetic embryopathy is aberrant phospholipid metabolism, especially lipoperoxidation. Dietary supplementation with arachidonic acid or myo-inositol in diabetic pregnant animals has been shown to reduce embryonic malformations due to lipoperoxidation (183, 184, 244). Treatment of diabetic pregnancy animals with polyunsaturated fatty acids also have been shown to exert similar helpful effects on embryonic development (227, 248).

Antioxidative strategies

Targeting oxidative stress to treat diabetic embryopathy, using lipoic acid, ergothioneine, vitamin C, and vitamin E, has been tested in diabetic pregnant animals and shown to decrease embryonic malformations (174, 181, 183, 184, 243, 249). N-acetylcysteine (NAC), which has been used as an antioxidant in clinical practices to treat acetaminophen poisoning (250, 251), has been observed to reduce malformations in animal embryos in culture exposed to high glucose (252–254); however, its effect in human diabetic pregnancy remains to be demonstrated.

Folic acid, or water-soluble vitamin B9, has long been known to reduce NTDs in humans (255, 256). The effect of folic acid in reducing embryonic malformations associated with maternal diabetes has been explored in animal models. Treating cultured rodent embryos with folic acid can reduce neural tube dysmorphogenesis induced by high concentrations of glucose (246). In in vivo experiments, diabetic pregnant rats injected with folic acid also exhibit decreased NTDs in their embryos, compared with diabetic pregnant rats not given the supplement (122, 246, 257).

Combination approaches

The combination of antioxidants and phospholipids that target multiple molecular pathways could have more potent effects in reducing fetal abnormalities compared with monotherapy. Cocktails of vitamin E, arachidonic acid, and myo-inositol have been explored in diabetic animal models and have been shown to decrease embryonic malformations (183, 184). The efficacy of antioxidants to prevent fetal defects in human diabetic pregnancy has not been examined. Antioxidant approaches tested to treat similar diseases, such as preeclampsia, have produced unsatisfactory results (258–262). These results have tampered the enthusiasm in applying similar strategies to treat diabetic embryopathy, but exploration of other strategies is warranted.

Recently, alleviating nitrosative stress via iNOS inhibitors has been tested in diabetic embryopathy (163). Oral treatment of diabetic pregnant animals with an iNOS inhibitor decreased embryonic malformation rates (163). Additional studies are needed to test therapeutic agents or dietary supplements that inhibit iNOS in order to translate these basic research findings into clinical applications for human disease.

Clinical challenges

Diabetic embryopathy involves complex molecular interactions. Extensive understanding of its mechanisms is essential for identifying therapeutic targets and developing effective interventions. Many challenges in developing these approaches lie ahead. First, interventions must be safe for the embryo and mother. The ideal approaches include dietary supplementation of non-toxic agents. Second, because developmental malformations occur early on in the first trimester of pregnancy, the dietary supplements must be easily accessible for women to take before conception or soon thereafter. Third, any therapeutic agents administered via oral treatments or dietary supplements must be able to pass the maternal-fetal barrier to exert potent effects on the developing embryo.

To overcome these challenges, it not only needs scientific research to decipher the molecular mechanisms underlying embryonic malformations and develop prevention strategies, but also requires education of the public to raise awareness about the risk of birth defects in diabetic pregnancy. Preconception counseling and pregnancy planning have been shown to be correlated with reduction in adverse outcomes of pregnancies (263–266). However, more cooperative efforts between perinatal care providers and patients are needed to achieve the goal of eliminating birth defects.

Concluding remarks

Diabetic embryopathy is a global public health issue. Although pregestational screening for maternal diabetes, perinatal care, and postnatal management in developed countries are available to most pregnant women, the rate of birth defects in infants of diabetic mothers remains high. In developing countries, the rates of birth defects and even mortality are high because of unavailable and inadequate care for pregnant women. With the worldwide increase in obesity and type 2 diabetes, diagnosis and management of diabetic pregnancy is a big challenge for the medical community. The increasing public health burden of diabetes in women of childbearing age makes it important to develop interventional approaches to prevent embryonic malformations.

Further understanding of the mechanisms underlying diabetic embryopathy will provide crucial information for developing effective interventions. Clinical approaches must be safe to administer in early pregnancy and accessible to women prior to and soon after conception to be highly effective because diabetes in early pregnancy often goes undetected and many women have unplanned pregnancies. Major cellular activities--i.e., proliferation and apoptosis--and intracellular metabolic conditions--i.e., nitrosative, ER, and oxidative stress--have been demonstrated to be associated with diabetic embryopathy using animal models. Translating advances made in animal studies into clinical applications in humans will require collaborative efforts across the basic research, preclinical, and clinical communities.

Key points.

• Diabetes mellitus in early pregnancy increases the risk of birth defects in infants.

• High glucose induces intracellular stress, increases programmed cell death (apoptosis), and decreases cell proliferation in the embryo.

• Reduction of the risk includes pre-conceptional and early gestational glycemic control.

• Interventions via dietary supplementation remain to be developed.

• Pre-conceptional counseling and planned pregnancy are encouraged for physicians and patients.