We here report that maternal diet affects placental growth and gene expression in diabetic pregnancies. In the context of maternal diabetes, the diet recommended specifically for breeding and lactating mice was associated with reduced fetal size 
and decreased placental size (as measured by weight), indicating that there is a specific interaction of diet with the diabetic condition on placenta growth. Conceivably, this could potentially be linked to reduced consumption of the diet. When fed breeder diet, diabetic dams had reduced weight gain compared to normal dams on this diet, but diabetic chow-fed dams also had reduced weight gain. Thus, reduced weight gain is attributable to the metabolic condition, independent of diet. Interestingly, normal dams gained similar amounts of weight with either diet, indicating that differences in caloric content of the diets had no noticeable influence on maternal weight under normal conditions. With the metabolic derangements of maternal diabetes, however, the difference between the two diets had a strong influence on the differential decrease in placental weight: in comparison to chow-fed diabetic dams, the breeder diet-fed diabetic dams had significantly reduced placenta growth.
Litter size is known to be inversely correlated to placenta size 
. However, in our experiments, litter size did not significantly differ between diabetic pregnancies when the dam was fed breeder diet compared to the other conditions 
. Thus, litter size is unlikely to serve as an explanation for reduced placenta growth. It should be kept in mind, that the present study included only placentas that were associated with morphologically normal embryos; analyses on placentas associated with abnormal embryonic development in diabetic pregnancies have yet to conducted.
Interestingly, maternal glucose levels were higher at the start of pregnancy when diabetic dams consumed breeder diet, averaging 349.65±97.79 mg/dL compared to 306.23±81.81 mg/dL in diabetic dams consuming the chow diet. By the time of sacrifice, maternal glucose levels exceeded the upper limit of the meter (600 mg/dL) in 9 out of 53 dams on chow diet, and in 29 out of 45 dams on breeder diet (we therefore cannot estimate average levels for the whole group); measurable blood glucose levels in the remaining dams were 457.50±90.61 mg/dL in chow-fed (n
26), and 506±82.13 mg/dL in breeder diet-fed diabetic dams (n
16), respectively (difference is not statistically significant). Also, if we consider the difference between pre-pregnancy glucose levels and those at sacrifice 
as an indicator of diabetes severity, there was only a weak relationship to placenta growth in diabetic pregnancies, regardless of diet. Dam weight at copulation was 22.46±1.8 g (n
27) for breeder diet-fed diabetic dams, which was approximately 1g less than normal dams (23.5±2.1 g; n
48) on breeder diet. However, compared to diabetic dams on chow diet, the weights, and the weight gains, of breeder diet-fed diabetic dams were indistinguishable throughout pregnancy. Thus, we consider it unlikely that solely the degree of maternal hyperglycemia, or maternal size or weight gain, could have been responsible for the reduced placenta size in breeder diet-fed diabetic pregnancies. Because our data do not implicate maternal factors other than diet
in the reduced growth of placentas in diabetic pregnancies, we therefore conclude that diet is the major factor influencing placenta growth in this model.
The gene expression profiles indicate that breeder diet does not simply exacerbate the detrimental effects of maternal diabetes, but that it has distinct effects. While gene expression is clearly misregulated in diabetic placentas, the different diets influence the magnitude and direction of changes, and exert their effects on specific sub-sets of genes. Except for Tgfßi and Il17ß, where gene expression levels in diabetic placentas could be interpreted to correlate with blood glucose levels (magnitude of change is greater in the breeder diet-fed group than in the chow-fed), all other patterns are indicative of interaction of diet and diabetic state, in additive manner, and often also in opposite directions (see ). Examples to illustrate additive effects are Thbs2, Ptgs2, Hpgd, Slc6a4, Mmp15, Pfpl, and Spi 16; examples for opposite direction of the diet effect in normal compared to diabetic dams are Crct1, Mmp1a, Atoh8, Cyp1a1, Pappa2, Ankrd2, Prl5a1, and Rassf4. In addition, our results reveal several genes that can serve as indicators of diet exposure in the absence of and regardless of maternal diabetes, such as Usp24, Adamts6, Frem1, Tm9sf1, Emp1, Ptgs2, Kcnk2, Mmp13 and Mmp15. These patterns, and particularly those of opposite interactions, imply that the adverse effect of breeder diet on placenta growth acts through mechanisms other than hyperglycemia alone. Thus, we have identified diet-dependent targets, of which some interact with maternal diabetes in regulating gene expression in the placenta at midgestation.
Less clear at the moment is how these molecular alterations translate into reduced placental growth. We have previously shown that spongiotrophoblast growth is reduced under conditions of diabetic pregnancy, and the labyrinth also remains smaller 
. We also reported reduced levels of Ascl2
(achaete-scute complex homolog 2), which is normally expressed in and required for growth of spongiotrophoblasts and the labyrinth 
. However, Ascl2
is only regulated by diabetes, not by diet (unpublished observations), and thus cannot account for the greater extent of growth reduction of diabetic placentas in breeder diet-fed dams. More plausible candidates are therefore those genes that exhibit a response to both diabetes and diet. Among those diet-dependent genes that could contribute to altered placental growth are genes known to play a role in inflammation. In this context, the upregulation by diet of genes encoding thrombospondin-domain containing proteins (Adamts6
) and the downregulation by diet of proteinase inhibitors Spink8
are noteworthy. Similarly, diet modulates the expression of genes encoding enzymes that are involved in eicosanoid metabolism, such as Prostaglandin endoperoxide synthase 2
), Hydroxyprostaglandin dehydrogenase
) and Cyp1a1
. The enzymes encoded by these genes are involved in production and metabolism of prostaglandin E2, and our working model is that under conditions hyperglycemia and adverse diet, enzymes catalyzing PGE2
degradation are elevated to levels where they create a functional PGE2
deficiency, through increased catabolism of PGE2
. Prostaglandin E2 has been shown to stimulate trophoblast migration 
and cellular invasive behavior 
. Conversely, in experimental animals with reduced PGE2
levels, cell migration is reduced 
. Our previous histological analysis of diabetic placenta revealed aberrant trophoblast migration and reduced growth of the spongiotrophoblast layer 
, which is consistent with impaired PGE2
signaling. Another role of PGE2
is inactivation of Natural Killer Cell activity in the decidua 
. Intriguingly, we observe high NK cell accumulation in the diabetic placenta 
, again consistent with PGE2
deficiency. A second indication for the involvement of inflammatory pathways in reduced placental growth is the upregulation by breeder diet, at least in the diabetic state, of IL17b
, a member of the pro-inflammatory IL17 cytokine family. The role of inflammatory pathways in aberrant placenta development as a consequence of diabetes or diet warrants further investigation. Also intriguing is the upregulation by breeder diet of Ubiquitin-specific gene 24
) and of Endoplasmic reticulum metallopeptidase 1
), which could be reflective of altered protein processing. Taken together, we detect diet influences on genes with plausible roles in stress responses and inflammation; further studies will be required to demonstrate a functional relation to placental development and growth.
It is noteworthy that our -admittedly short- list of 33 diet-responsive genes does not overlap with the gene repertoire changes reported for placentas from protein restricted FVB dams at E17.5 
, or for a comparison of low fat and high fat content diets in NIH Swiss dams at E12.5 
. Non-congruency could be explained by the different times of sampling, use of different diets, and the fact that our breeder diet (Purina 5015) was used as the control diet in the second paradigm, which also encompasses a strain difference. Yet, the most important feature in our study is the presence of maternal diabetes as a second environmental factor that produces the sensitizing condition under which the adverse effects of breeder diet on placental growth, and the novel interactions of diet and diabetes on gene expression that we have identified here, are revealed.
The molecular mechanisms through which diet affects the regulation of genes with altered expression levels are unknown. To date, regulatory elements that confer placenta-specific expression have not been identified for any of the diet targets our work uncovered. Similarly, it is unknown whether microRNAs or other epigenetic mechanisms may be involved. Changes in cellular composition, namely increased frequency of cells expressing the respective gene, appear to be responsible for the increased expression of the Serotonin transporter
) and Cyp1a1
genes in diabetic placenta 
. Yet, we do not currently know to what extent, and if so, how any particular diet affects the cellular make-up of the placenta.
Both the chow, as well as the breeder diet, are formulated to be replete for minerals and micronutrients, but they differ in macronutrient composition. In particular, protein content is higher in the chow diet, while fat is enriched in the breeder diet. From our results, it appears that placental cells can detect this difference, likely through nutrient sensing mechanisms 
. The mTOR system plays a prominent role in nutrient sensing, and it has recently been shown to be present in placenta 
. Although we did not obtain evidence from our microarrays for altered expression of genes in the mTOR pathway in diabetic placenta 
, it would be expected to play a role in response to different diets. It should also be kept in mind that, although “defined” in their composition, both diets are manufactured from natural ingredients, such as soybean-derived products and fish meal, the quality and molecular composition of which can be variable. In this regard, it is important to note that in normal pregnancies, Cyp1a1
expression is decreased by breeder diet, providing evidence that this diet is not simply contaminated with unidentified toxins. Nonetheless, assays with purified ingredient diets are necessary to determine which of the components in the breeder diet is/are responsible for deficient placental development. The 33 diet-responsive genes we have identified in the present study will be most valuable in monitoring exposure to different nutritional conditions and perturbations.
Taken together, our results demonstrate that maternal diet modulates placental gene expression and growth, with a concomitant effect on fetal growth 
. Because deviations from normal birth weight are linked to adult disease risk, the placental alterations we find in response to diet and diabetes may have important implications for developmental programming of susceptibility to disease later in life