Perinatal changes in plasma of type 2 diabetes mellitus pregnancies who delivered newborns with cardiomyopathy, depression of the central nervous system, or hepatomegaly

This is a cross-sectional prospective controlled study. Here, we reported proteomic investigation for several cases of severe types of diabetic fetopathy (cardiomyopathy (CRDM, n = 37), central nervous system depression (CNSD, n = 35) and hepatomegaly (HPMG, n = 35)) diagnosed during 30–35 gestational weeks and conrmed upon delivery by from patients with type 2 diabetes mellitus (T2DM). Control groups were comprised from women in whom T2DM had been ruled out (n = 40) and group of pregnancies with T2DM who delivered healthy newborns (n = 40).


Results
We found a composition of serum-based non-trivial markers capable that are strongly associated with the certain type of fetopathy or anatomical malfunctions in the affected newborns. Signi cant impact on mRNA splicing and DNA reparation has been determined by emerging alterations in CDCL5. Patients of CNSD groups were characterized by utmost depletion (ca. 7% of baseline) of DFP3 neurotrophic factor needed for the proper specialization of cardiomyocytes and oligodendrocytes. Corrupted regulation of non-canonical Wnt-signaling guided by PEDF (in CNSD and HPMG groups) and DAAM2 (in CRDM and HPMG groups) was also proposed. In addition, de ciency in retinoic acid and thyroxine transport was revealed by dramatic increase of TTHY in CNDS group.

Conclusions
We examined peripheral blood plasma and determined a small proportion of proteins indicating the pre- Diabetic fetopathy (DF) is a sever complication de ned in systematic changes of newborns and caused by maternal pre-existing (type 1 or type 2 (T2DM)) or gestational diabetes mellitus (GDM). Maternal hyperglycemia due to onset and duration of glucose intolerance during pregnancy may stipulate diabetic embryopathy resulted in congenital morphogenesis, endocrine impairments and spontaneous abortions [1]. DF can be pre-determined by ultrasound examination and Doppler scanning during pregnancy. However, due to insu cient sensitivity diagnosis of DF occurs in the late gestational age (beyond the 30th gestational week). A phenotypic analysis may yield the highest diagnostic value to reveal the grade of fetal hepatomegaly, cardiomyopathy, and pancreatic enlargement but the eventual results are reported upon delivery [2,3].
The affected newborns may suffer metabolic syndrome, risk of obesity and respiration distress [1,4]. Growing population of infants with metabolic syndrome as a consequence of maternal diabetic condition raises the great concern about cardiovascular disease developed already in adolescents [5]. The most prevalent sign of DF is macrosomia which is a response to maternal diabetes and can be considered as a highly valuable predictor for the later glucose intolerance and growing risk of improper fetal development.
Up to 45% of infants born to diabetic mothers have a macrosomia which is almost 3 times higher than the rate comparing to normoglycemic patients [6].
Adipose tissue which produces adiponectin, TNF-α, IL-6, IL-10 and leptin, makes the main contribution in insulin sensitivity of pregnancies [7,8]. While impaired maternal glycemic control and increased insulin resistance (IR), the level of circulating glucose is raised. The issue is that excessive maternal glucose may cross the placenta whereas insulin none. Starting from the second trimester, the fetal pancreas is already competent to secret insulin. Therefore, the combined condition of maternal hyperglycemia and excessive secretion of insulin in fetus engages the risk of increased fat production leading to macrosomia and malfunction of morphogenesis [6].
Heart defects are the most common type of diabetic fetopathy with prevalence up to 12% [9]. There is evidence arguing that exactly maternal increasing IR and high glucose level are mainly responsible for improper differentiation of cardiomyocytes and heart malformation [10]. Hypertrophic cardiomyopathy symptoms exhibited in asymmetric septal enlargement with a disproportionally hypertrophic septum were routinely found in 12.1% cases of infants affected by the maternal diabetes [10,11].
Neural tube formation is also profoundly affected by maternal diabetic condition through direct in uencing on regulation with a great variability of embryo genes expression, in particular, those involved in Wnt-and TGF-β signaling [12]. Recent studies also demonstrated that the etiology of neural tube defects in infants from diabetic mothers encompasses elevated levels of SOD (superoxide dismutase), inhibition of the pentose phosphate pathway (PPP) and extended oxidative stress [13,14].
Hepatomegaly also holds the prevalent place of the potential infants' injury caused by maternal diabetes and in strict association with macrosomia [15]. It was proposed the great prognostic value of HbA1c for monitoring the risk of hepatomegaly however the indicator cannot be used solely [16]. Ultrasound examination is still the most con dent and reliable instrumental method for indication fetal hepatomegaly [17].
In this report, we provided a cross-sectional study among patients with T2DM who delivered newborns with different types of diabetic fetopathy. We focused on a proteomic assay and evaluated results for control group of non-diabetic pregnant women and patients with T2DM. Based on the obtained results, we performed a quantitative analysis of the small share of proteins and accommodated the most signi cantly altered markers in the reconstructed signaling pathways that determine morphogenesis, differentiation of cardiomyocytes, exhibit neurotrophic properties and activities toward mRNA splicing and DNA repair. It can be a correctly focused view that the majority of discovered mechanisms are generalized for all types of diabetic fetopathy in integrity, but there are small portion of speci c markers that capable to segregate considered pathologies.

Population
In total, n = 187 pregnant women at a gestational age of 30-35 weeks were participated in the study between October 2019-March 2020. Of them, n = 147 patients had a history of T2DM for 7.6 ± 3.2 years and insulin therapy of 6.5 ± 2.5 IU/day. Patients were strati ed according the type of diabetic fetopathy manifested in newborns upon delivery as follow: group with cardiomyopathy (CRDM; n = 37, BMI = 28.78 ± 4.42 kg/m 2 ; age 25.4 ± 4.2 years), depression of CNS (CNSD; n = 35, BMI = 29.50 ± 3.21 kg/m 2 , age 25.9 ± 5.1 years) and hepatomegaly (HPMG; n = 35, BMI = 29.78 ± 3.55 kg/m 2 , age 24.7 ± 4.7 years; Table 1). Patients with T2DM who ruled out the DF for newborns were combined in a separate group (n = 40, BMI = 29.69 ± 4.13 kg/m 2 , age 25.4 ± 3.9 years; Table 1). Control group (n = 40, BMI = 21.89 ± 4.29 kg/m 2 , age 25.6 ± 2.6 years; Table 1) comprised pregnancies who gave birth healthy newborns and had no previous history of T2DM or GDM according to the criteria of IADSPG (revision 2010) and adapted criteria of National Association of Obstetrician and Gynecologist (revision 2012). Table 1 Main anthropometric and clinical records for patients with T2DM and control group of patients. Patient with T2DM were subdivided into three groups according the signs of diabetic fetopathy attributed to the delivered newborns: CRDM -cardiomyopathy, CNSD -syndrome of central nervous system depression and HPMG -hepatomegaly. Patients with T2DM, who gave birth of newborns reported as healthy upon delivery and had no signs of diabetic fetopathy indicated by ultrasound examination during pregnancy course, were considered as the second control group. Hepatomegaly Signs recognized using echocardiographic study. Increased size of liver and the diameter of liver was measured by transverse scanning of the body to determine the distance between the most distant points of the right and left lobes. Craniocaudal size and anteroposterior size were determined during longitudinal scanning at mid-clavicular line.

Samples collection and handling
Samples preparation was performed according the protocol described in [18]. Brie y, following overnight fasting peripheral blood was collected into EDTA-2K + tubes. Plasma obtained after centrifugation at 10 o C and 2500 g for 10 minutes. Proteins concentration was measured using BCA assay kit. A total of 100 µg of each sample used for processing and enzymatic digestion with trypsin. Complete details of protocol are given in Appendix A.

High-resolution LC-MS analysis
Instrumental analysis was conducted as described in [18]. Brie y, the analysis was performed on a highresolution Orbitrap Fusion (Thermo Scienti c, Waltham, MA, USA) mass spectrometer equipped with a nano-ow NSI ions source and integrated with an Ultimate 3000RSLC (Thermo Scienti c, Waltham, MA, USA) liquid chromatography system. Complete work ow and details are available in Appendix A.

Statistical analysis, quantitative estimation and functional annotation
Bias-correction to exclude outliers was performed by Mann-Whitney U-test at p < 0.05. Proteins shared between analyzed groups were normalized and ranged according their NSAF (normalized spectra abundancy factor) representation for quantitative analysis. Alterations between groups were represented for proteins in log-fold changes with signi cance cut-off p < 0.01 (Kruskal-Wallis test) and were obtained by de ning a linear model for each protein. A two-sample moderated t-test used, and the reported pvalues were corrected for false discovery rate. To extract proteins speci c for each subgroup, we used PCA (principal component analysis) classi cation analysis, and the differences between scores were compared by Kruskal-Wallis test at p < 0.01.
To unveil biological processes and functions associated with meaningful alterations, proteins were submitted in Gene Ontology supported by the overrepresentation test (annotation release 20200407).
Signi cance was estimated using Fisher's exact test (p < 0.001) against the proteins identi ed in the control group, and Bonferroni correction applied for adjusting the FDR-based (false discovery rate) pvalues [19]. Initial (raw) molecular pathways were extracted from the KEGG [20] and the Reactome (version 72) [21].

General description
The assay was performed on 187 patients of whom 147 were pregnancies with a clinical history of T2DM for 7.6 years (average). Of them 37 patients gave newborns with cardiomyopathy (CRDM), 35 newborns with hepatomegaly (HPMG) and 35 newborns with syndrome of central nervous system depression (CNSD). Patients with T2DM did not differed signi cantly in BMI (Table 1) and fasting glucose level (7.8 mmol/L average, Table 1). Maternal weight gain was marginally higher in patients with T2DM compare to the control group, but the group of T2DM patients delivered healthy newborns was comparable to the baseline of the control group in this indicator (p = 0.694, Table 1). The proportion of patients with Caesar way of delivery was almost evenly distributed among T2DM subgroups and ranged from 10% (HPMG groups) to 13% (CNSD group) but the total rate was higher compared to the control group (45% against 30%, p = 0.007, Table 1). Although, patients with T2DM gave newborns with different types of diabetic fetopathy predetermined by sonography examination, the Apgar-1 and Apgar-5 scores reported upon delivery were somewhat comparable with scores reported in the control group ( Table 1). The integrative score of diabetic fetopathy was distinctive between CRDM, CNSD and HPMG groups but the median value was indistinguishable throughout the study groups with antenatal complications (Table 1).

Identi cation of proteins characterizing the condition of antenatal complications
In total, we identi ed 378, of them 296 were shared throughout the study groups. Using Mann-Whitney Utest with the raw p < 0.05 and fold-change > 1.5 or < 0.8, only 42 proteins were determined as signi cantly altered and 30 proteins may have a meaningful value to discriminate T2DM subgroups (healthy and different types of fetopathy under consideration, Table 1 and Appendix B). The signi cant compounds with p < 0.05 were passed to PCA, with PC1 explaining 18% variance, and a PC2 of 11%. PCA score discrimination analysis showed satis ed segregation of T2DM patients who gave birth healthy newborns from the control group, and good separation and dispersion of subgroups bearing signs of diabetic fetopathy from the control group and from T2DM patients with healthy newborns (Fig. 1A). This indicates a meaningful impact of diabetic condition during gestation on the possible risk and complications of fetal morphogenesis.
At the same time, it was established that only n = 20 proteins among 42 can be accounted as signi cant (with p < 0.01) for distinguishing groups of antenatal pathologies (CRDM, CNSD and HPMG) from groups of T2DM patients who gave birth healthy newborns and the control group. This indicates different contribution of the selected proteins in way of antenatal complications. Signi cantly differed proteins (n = 17) were endorsed by the Kruskal-Wallis test with fold changes > 2 and < 0.5 (linear scale) and p < 0.01. These proteins were selected for clustering into a heatmap exhibiting a different pattern between the studied groups in respect of antenatal pathologies ( Fig. 1B and Table 2). Some proteins among the selected are shared between any two types of fetopathy under consideration and showed signi cant changes (for example, PEDF (pigment epithelium-derived factor) and TSP1 (thrombospondin-1) for CNDS and HPMG groups; or DAAM2 (disheveled-associated activator of morphogenesis 2) and LAMB4 (laminin subunit β4) for HPMG and CRDM groups, Table 2) whereas most of the determining markers demonstrated speci city to the certain type of antenatal complication and did not attain a relevance to cognate groups. Probably, such segregation can be underlined by engaging of different mechanisms deployed during CRDM, HPMG or CNSD (Appendix C).
Functional categorization of the identi ed proteins Most listed proteins ( Table 2  We purposed to assemble the detected proteins into molecular network that can disclose differences between types of diabetic fetopathy (CRDM, CNSD, and HPMG) and can determine triggers that severely underline ndings of fetopathy from patients with T2DM.

Protein-protein interactions (PPI)
Protein-protein interaction (PPI) analysis was conducted on n = 30 proteins capable to discriminate T2DM patients from subjects with uncomplicated pregnancy (Appendix C). The assayed set completely covers n = 17 most signi cant markers that segregate groups of patients with T2DM who delivered newborns with different types of diabetic fetopathy (CNSD, CRDM and HPMG, Table 2).
There are n = 9 common (adjacent) markers between three groups of diabetic fetopathy: APOC4 (apolipoprotein C-IV), LG3BP (galectin-3-binding protein), PEDF, TSP1, PHLD (phosphatidylinositol-glycanspeci c phospholipase D), ALS (insulin-like growth factor-binding protein complex acid labile subunit), DPF3 (zinc nger protein DPF3), DAAM2 and LAMB4. PPI analysis showed that these proteins produce a poorly dense core of functional interactions (the coe cient of protein interactions is PPI = 0.389), except for a pair of PEDF and TSP1 and cluster of DAAM2, DFP3 and LAMB4. Assumingly, it indicates involvement of these proteins in the general processes leading to the development and progression of a particular fetopathy. Given the large number of groups of speci c markers, most likely, adjacent markers are a re ection of secondary signs of fetopathy, and encompass peripheral biological process. The multiple molecular functions possessed by adjacent markers also support this proposition; these proteins are characterized by pleiotropic property in the cell's life cycle. It is worth noting that one of the most important proteins is LAMB4, since this protein is a direct participant in the organization of tissues and the process of organogenesis in the embryonic period and seems critical for determining cell migration and proper orientation in the processes of organogenesis and tissue formation, also xing cells in the intercellular matrix. Similar functions are performed by TSP1, which is paired with PEDF, but its role is largely limited to the adhesive functions that determine the intercellular interaction and the interaction of cells with the extracellular space. proteins, or proteins whose main activity is manifested in the extracellular space region (GO: 0044421, p = 7.90e-09), which indicates their high para-and endocrine potentials.

Discussion
The impact of mRNA splicing and DNA reparation during embryogenesis We focused on the most important markers, which can be either cross-speci c for several types of fetopathy or highly speci c like CDC5L (cell division cycle 5-like protein; Table 2). According to the functional analysis, CDC5L generates a local core of interactions through ACTB (actin, cytoplasmic 1), DEFA1B (neutrophil defensin 1), and C2 (complement factor C2) with the local clustering coe cient 0.782; the last two elements (DEFA1B and C2) are not accounted as CNSD-speci c ( Table 2). According to the data of Human Protein Atlas, CDC5L is characterized by low tissue speci city, but its highest expression is traced in brain cortex, cerebellum, hippocampus and thalamus.
It is di cult to overestimate the role of CDC5L in regulation of cell division, DNA repair and activity guiding mRNA splicing and maturation. Recent data highlighted the association of CDC5L with neuronal differentiation during embryogenesis, the implementation of which occurs through the assembly of active complex (CDC5L/14-3-3β/PRP19α) carrying necessary competencies for differentiation of neurons [22].
Phosphorylation of PRP19α (pre-mRNA-processing factor 19) by Akt-kinase is a critical step in complex assembly, whereupon it acquires a conformational ability to bind with 14-3-3β for translocation into the nucleus (Fig. 2) and to meet CDC5L. Obviously, dysregulation of one of these elements leads to an adverse in uence on neuronal differentiation and corrupts cell division, since the complex regulates G2/M phase.
The relationship between CDC5L and some neurodegenerative and neuroimmune diseases is established in plenty researches, most of which operate with transcriptome analysis [23,24,25]. Adverse regulation of mRNA splicing in cerebral cortex through, inter alia, malfunction of CDC5L lead to a decrease of proliferative activity and disruption of the neuronal differentiation [26]. Comparable results were obtained on HeLa after immunoprecipitation of a complex of CDC5L with PLRG1 (pleiotropic regulator 1) with heteronuclear ribonucleoprotein (hnRNP) necessary for the activity of mRNA splicing complex (Fig. 2).
Mutant cells (CDC5 −/− ) displayed inability to build the complex, disruption of splicing process and underwent extensive apoptosis [27].
Evidence exist that translation repression and degradation of mRNA is accomplished by circulating miRNAs tightly associated in different complexes [28]. Recent GWAS (genome-wide associated study) data demonstrated that about 600 different miRNAs are expressed in placenta and play a key regulatory role in embryogenesis and metabolic adaptation during fetal growth. Such molecules are released in maternal circulation and may indicated cautions of placenta dysfunction and GDM [29]. Our proteomic data suggests that increased concentration of CDC5L (Table 2) may re ect a potential malfunction of mRNA splicing caused by a dysregulation in placental miRNA providing instructions for mRNA processing.
DNA repair is another important aspect of CDC5L activity. Embryonic broblast cells undergo active apoptosis through the p53-mediated mechanism in animals de cient in PLRG1 [30]. The fundamental is a decreased ability of PLRG1to assemble with CDC5L and consequent abolishment of DNA repair mechanism (Fig. 2). As has been highlighted, such embryos demonstrated failed cells division and fragmented nuclei and die rather early due to apoptosis and re-localization of CDC5L from nucleus to cytoplasm (Appendix C). Repeated experiments on cultivating cardiomyocytes and neurons showed similar results, where, apart the re-localization of CDC5L, a decrease of the apoptosis regulator Bcl-2 was noted [30,31]. Murine heterozygous by CDC5L demonstrated a survival rate not exceeding 25%, but the survived embryos characterized by a dramatic increase of degraded neurons and cardiomyocytes (Appendix C) which was accompanied with a reduced Bcl-2 and increased level of the phosphorylated p53 but unaltered Bax [30].
Hence, CDC5L is an important regulator of mRNA splicing and DNA repair in differentiating neurons during embryogenesis. Therefore, drastically increased CDC5L in CNSD group (Table 2) may suggest possible dysregulation of placental miRNA and the ongoing accumulation of DNA errors caused by oxidative stress typical for patients with the manifesting T2DM.

Extended activation of the immune response
Due to a high perception to changes in homeostasis and immune response, elements of complement system are typically accounted as non-speci c markers although some of them were meaningful in both CNSD (complement factors C1QC, C5, C4, C1QB) and CRDM (complement factors C1B, C2) groups ( Table 2). As outlined above, in patients with manifesting T2DM activation of complement system is associated with oxidative stress caused by hyperglycemic state [32]. Overproduction of ROS (reactive oxygen species) is aggravated by accumulated blood glucose undergoing auto-oxidation (Fig. 2), which activates synthesis of prostaglandins and deployment of in ammatory reaction. A signi cant contribution is caused by activation of FASN (fatty acids synthase) and, consequently, inclined consumption of NADPH through the enhanced rate of PPP (pentose-phosphate pathway) and boosted G6PDH (glucose-6-phosphate 1-dehydrogenase) [33].
As level of ROS inclines, the rate of TGFβ-mediated signaling enhances toward overproduction of cytokines (IL1, IL6, IL10, TNFα) and including complement factors [32,34]. The accumulated ROS and RNS (reactive nitrogen species) positively affect NF-kB pathway that triggers NOS (NO-synthase) and entails to NO overproduction positive regulation of prostacyclin (PTGIS). Unfortunately, extremely high concentration of NO returns to reaction with ROS and generation of dangerous peroxynitrites (Fig. 2) [33].
Hence, it highlights an odd situation when, on the one hand, there is excessive NO generation, and on the other hand, limited access to NO due to reacting with ROS. In turn, overproduction of cytokines stimulates activation and overproduction of compliment system elements, which are well recognized in the study ( Table 2).
Repressed transition from progenitor to post-mitotic state.
Transcriptional activation and repression of selected genes by chromatin remodeling is a key mechanism guiding tissue and organogenesis where DPF3 takes the role of an important indicator ( Table 2). Evidences suggest that the maximum expression of DPF3 is observed in hypothalamus, neurons, myoblasts and oocytes. Its expression is regulated through Mef2, which recognizes for DFP3 promoter with high speci city [35]. As has been overviewed on the cultivated HEK293 cells and later on myoblasts, soon after switching DFP3 gene off, development and differentiation of cells are aborted [36].
Neurogenesis and myogenesis (including cardiac and skeletal muscle) are orchestrated by DFP3, but the exact mechanism of action is still uncertain. The tacit assumption is action through the binding to certain acetylated and methylated regions of histones, thought, direct participation in the regulation of chromatin remodeling. Accordingly, global estimation of methylome has been proposed as a potential hallmark for the assessment of gestational diabetic conditions and its dire consequences affecting fetal development [29]. Notable, DPF3 is a necessary element of nBAF complex (neuron-speci c chromatin remodeling complex BRG1/BRM associated factor), and upon completion of the progenitor activity of neurogenic cells, the essential for proliferative capacity of npBAF (neural stem/progenitor cells) complex (ACTL6A/BAF53A and PHF10/BAF45A) is replaced by a neuron-speci c complex comprised homologous ACTL6B/BAF53B and DPF3/BAF45C.
Some studies outlined that signi cantly increased DPF3 expression leads to cardiac hypertrophy during embryogenesis, while the same was also shown in adults with chronic cardiac hypertrophy [37]. In both occasions, the process of hypertrophy is associated with activation of embryo-speci c genes, including DPF3a and DPF3b isoforms, and with reprogramming the expression of β-MHC and skeletal actin genes.
Other report indicated that initiation of Hirschsprung's disease is largely due to depletion of DFP3 gene, which causes a decrease in cell migration to the appropriate localization of nerve ganglia in various parts of the intestine, especially, in stenotic segment. [38].
One can note the close interaction of DPF3 with RUNX (runt-related transcription factors) signaling attendant with TGF-β route and increasing proliferative rate [39,40]. The RUNX regulator LRG1 (leucinerich alpha-2-glycoprotein) is located upstream and its increased expression repeatedly reported in context of various diseases due to dysregulation of the TGF-β pathway. However, in our study increased expression of LRG1 is noted only in the CRDM group (Table 2).
Both CRDM and CNSD groups were distinguished in DFP3, but CNSD characterized by the lowest abundance ( Table 2). Our data suggest a distinctive feature of DPF3 in neuron-speci c association with chromatin remodeling being executed during embryogenesis and dramatically dropped in the CNDS group. In CRDM group upregulation of LRG1 and DFP3, assumingly, re ects the impaired proliferation where DFP3 is the nal point (switching to the post-mitotic state) and LRG1 is the starting point, thereby determining expression of RUNX factors and, consequently, TGF-β pathway.
Regulation of myelinization and differentiation of cardiomyocytes via competing for canonical and noncanonical Wnt-signaling A neurotrophic factor PEDF was found as an element taking an active part in genesis of neurons and cardiomyocytes ( Table 2). It exhibits pronounced properties of angiogenesis inhibitor and belongs to a serine protease but does not undergo characteristic stress/relaxed conformational changes. The highest expression of PEDF is tracked in retina, thalamus, dendrites, and all neurons irrespective of their topology and adaptation; latest data also suggests expression of PEDF in liver cells [41].
Implication in regulation of differentiation intends PEDF to interact with ECM (extracellular matrix) proteins and proteins responsible for the architecture of organelles and cytoskeleton. Among those TSP1 is the most engaging adhesive glycoprotein intended for establishing the cells-to-extracellular matrix connection and modulation CD36-mediated angiogenesis ( Table 2). The crew of PEDF and TSP1 shows strong implications in the regulation of Wnt-signaling (p = 0.01594, Fig. 3).
Assumingly, PEDF acts as an endogenous inhibitor of Wnt/β-catenin pathway through exposure to LRP6 (low-density lipoprotein receptor-related protein 6) co-receptor. The expression level of PEDF increases signi cantly in response to Wnt3a which employs LRP6 as a co-receptor for signal transmission [42] (Fig. 3). Simultaneously, it decreases in response to non-canonical Wnt5a-induced signaling depleted on utilization of LRP6. Matching results were obtained using siRNA when direct interaction of PEDF with LRP6 (K d =3.7 nM) was abolished forwarding of signal transmission via Wnt-pathway [43]. The mechanism, demolishing ligand (Wnt3a) with receptor (LRP6) dimerization, is accomplished through the reduction of LRP6 phosphorylation by PEDF. (Fig. 3). Apparently, inhibition of canonical Wnt-signaling affects down-streaming elements, including suppression of TSP1 necessary for angiogenesis and associated morphogenesis via TGF-β signaling. Suppression of TSP1 is manifested in 25% decrease of 4-hydroxyproline needed for collagen synthesis and structuring of ECM.
Hence, assumingly that the base of competing experience between PEDF and TSP1 is explained by their antagonizing relationship during angiogenesis. Both proteins characterized by the straight functional interaction but by the inversed regulation: while CNSD group was featured by down-regulation of PEDF and up-regulation of TSP1, the situation looked counter in CRDM group; and HPMG group characterized by the increased TSP1 (Table 2). Thus, comprehensive regulation during embryogenesis is tailored to the compensatory mechanism due to activation of non-canonical signaling. This mechanism requires, instead, Wnt5a and tyrosine-protein kinase transmembrane receptor ROR2, thereby translocating βcatenin to the nucleus and keeping its level essential for signal transduction. [44,45].
Considering the enhanced rate of non-canonical Wnt pathway, one cannot ignore an extremely important DAAM2 protein readily recognized (frequency > 0.8) in HPMG and CRDM groups (Table 2). DAAM2, as a switcher, governs the non-canonical Wnt [46] (Fig. 4). It is an utmost regulator for various processes during embryogenesis including determination of myelination, development of spinal cord, potentiation and clustering of signalosome. There are two isoforms exist, and both, DAAM1 (disheveled-associated activator of morphogenesis 1) and DAAM2, are necessary for the development of myocardial cells and maturation of sarcomeres through the organization of actin cytoskeleton [47]. Disruption of DAAM2mediated transduction leads to cerebral amyloid angiopathy, degeneration of epithelial basement membrane and various oncogenic manifestations [48,49].
Non-canonical Wnt-signaling is not a substituent of the abolished canonical one, but rather, a cognate pathway bypassing β-catenin activation [50] and necessary for the controlling of sympathetic neurons morphogenesis, determination of cell specialization, migration and polarization [51,52,53]. It was found that during differentiation of peripheral axons from embryonic broblasts, Wnt5a binds to ROR2 coreceptor providing phosphorylation of DAAM2 which is prerequisite for the signal transmission [50,51] ( Fig. 4).
As evidenced, knockdown of Wnt5a by siRNA dramatically decreases or completely abolishes DAAM2 phosphorylation [54]. Other reports demonstrated that inhibition of canonical Wnt pathway does not depend on ROR2 co-receptor, since its absence or blocking retains the ability of Wnt5a and DAAM2 to switch and potentiate the non-canonical one [55,56].
Impaired expression of DAAM2 leads to non-compaction cardiomyopathy (NCC). At least in murine models, animals with excessive or, on the contrary, suppressed expression of DAAM1 or DAAM2 were characterized by the failure nucleation of actin brils in embryonic broblasts, excessively deep trabeculae myocardial cavities and left ventricular NCC (Appendix C) [57]. Cardiomyocytes looked round and smaller size with a weak interconnection due to inhibited secretion of collagen and decreased rate of actin polymerization (Appendix C). Later, it was found that the molecular function of DAAM2 is caused by the FH1 domain necessary for capping of actin laments, and by the FH2 domain supporting the binding and assembly of actin subunits [58]. Malfunction of DAAM2 is manifested in lack or weakly articulated Z-bands of sarcomeres and anatomically disorganized M-, Hand I-bands [57,59] which becomes of growing attention in regard of the extremely increased DAAM2 in CRDM group ( Table 2).
The activity of DAAM2 is involved in regeneration of myelin sheath of oligodendrocytes which is inhibited after white matter injury [60]. The process is regulated via activation of DAAM2-PIP2 pathway to achieve complex between PIP5K (phosphatidylinositol 4-phosphate 5-kinase) and DAAM2 (Fig. 4), and downstreamed to excite PLR5/6 receptors [60,61]. Here, the role of DAAM2 is restricted to regulation of oligodendrocytes differentiation and axonal myelination, thereby increased DAAM2 expression during embryogenesis and in adults leads to depression of myelination. In this respect, CNSD was the only group with unaltered abundance of DAAM2 in our research (Table 2). Trials with DAAM2 −/− animal embryos reported about considerably increased degree of axonal myelination and weakly controlled enhanced differentiation of oligodendrocytes [62]. Nevertheless, hypoxia condition promotes activation of myelination, which suggests a negative effect on DAAM2-PIP5K axis in regulation of Wnt signaling [62].
Assumingly, regulation of Wnt signaling is achieved through the competitive binding with LPR6 receptors, whereas binding to ROR2 receptors is necessary for interplay with DAAM2 that encourages launching of morphogenesis, myelination and immune response through potentiating of NF-kB signaling (Fig. 4).
Overexpression of DAAM2 during pregnancy places developing fetus at a risk of cardiomyopathy and irregular tissue development.
Coupled retinoic acid de ciency and thyroxine transport bring forth the deployment of oxidative stress Transthyretin (TTHY) is a secreted protein with a nity for thyroxine an order of magnitude greater than for triiodothyronine which makes him the main carrier of thyroid hormone. Besides, TTHY has an extraordinary function for transporting of retinoic acid (RA). It binds with two molecules of RBP (retinol binding protein) in stable complex thereby reducing the glomerular ultra ltration of RA and stabilizing the complex of RA with RBP [63]. Normally, up to 40% of circulating TTHY is bounded with RBP, while the rest act as a carrier of thyroxine (Fig. 5).
The most prevalent disease associated with TTHY is transthyretin amyloidosis, which occurs due to inability to congregate in regular stable homotetrameric structure and, hence, to transport thyroxine and RA to sites of their further transformation [64]. Clinical ndings are accompanied by formation of manifested amyloid brils due to the accumulation of unstructured TTHY oligomers [65] close to ventricular septum and synaptic region of large neurons [66] causing various types of neuropathy and cardiomyopathy [67].
Serum concentration of TTHY in newborns is varied between 50-200 µg/mL, and rapidly increases over twice in the adults. In patients with severe cardiomyopathy, the concentration of circulating TTHY and fragments is about 250 µg/mL due to chronic transthyretin amyloidosis [68,69]. In patients with neurodegenerative diseases, the concentration and the expression level are increased in order of magnitude [70]. The acceptable range of TTHY is wide enough (69-650 µg/mL) and may vary in response to dietary preferences and immune response, therefore, it is rather di cult to appreciate its diagnostic signi cance [70,71]. Nevertheless, admittedly that the normal concentration of TTHY in blood is 200-400 µg/mL and its half-life is 2 days [68].
The abundance of TTHY directly correlates with estradiol which increases signi cantly during pregnancy. Blocking of estradiol or cortisol receptors is exhibited in signi cant decreasing of circulating TTHY [72].
According to our data, TTHY is up-regulated speci cally in CNSD group assumingly caused by both expectedly increased estradiol and de cient in RA (Table 2).
There are data on the latent proteolytic activity of TTHY, when the protein cleaves β-amyloid into small fragments incapable to aggregate into amyloid plaques [73]. Therefore, relationship between TTHY, lipids metabolism and transport of RA becomes imperative. Up to 2% of TTHY are in complex with APOA1 (apolipoprotein A-I), which exhibits antioxidant activity (Fig. 5). An increased concentration of circulating TTHY due to the raised uncoupling with RBP primes exhibition of proteolytic activity largely focused on APOA1. Recently, combination of the substantially decreased APOA1 with BMI in patients with T2DM and GDM was considered as a risk factor for prediction of diabetic condition [74,75]. Fragments of APOA1 aggregate into apolipoprotein amyloid brils and ultimately causes in ammation, activation of compliment system and augmented oxidative stress [76].
Hence, a de ciency of RA can provoke undesirable consequences as a result of increased concentration of circulating TTHY and consequential declination of APOA1. However, overproduction of NO in T2DM patients also accelerates oligomerization of TTHY through its S-nitrosylation [77] that foster to the extension of oxidative stress (Fig. 5).

Conclusion
In this study, we assumed different mechanisms guiding the morphogenesis out of proportion toward different type of fetopathy. However, obviously, the key issue is emerged from the diabetic condition during pregnancy. Assumingly, that depending on personal clinical history, the outcome of progressing diabetic conditions on the growing fetus may vary, but the grade of in uence can be vague and outcome in cardiomyopathy, neuropathy of liver enlargement, or combination of several types fetopathy. It is hard to propose the nal outcome without clinically relevant and robust markers using only currently employed instrumental methods despite their e ciency.

Consent for publication
This paper does not contain any personal data in any form. All subjects gave their signed written consent to participate in the study. Consent of participation and data personal data security were obtained according the National Legislation of the Russian Federation "On Personal Data" No. 152 of July 27, 2006 (refer to o cial government link to acquire: http://www.kremlin.ru/acts/bank/24154). All subjects participated in the study were anonymized. Authors complied ethical standards and privacy policy and did not disclose any personal data of participated subjects in the paper or in the Supplementary materials in any form.

Availability of data
Supplemental materials are available along with this article: Appendix A -details of sample preparation protocol and LC-MS analysis; Appendix B -details for the most signi cantly altered proteins among study groups and general description of newborns upon delivery; Appendix C -details of statistical analysis, protein-protein interaction analysis and commentaries to CDC5L and DAAM2 proteins.
The datasets generated during and/or analyzed during the current study are available from the corresponding author on request.