Lower HLA-G levels in Extravillous Trophoblasts of Human Term Placenta in Gestational Diabetes Mellitus than in normal controls

Lower HLA-G levels in Extravillous Trophoblasts of Human Term Placenta in Gestational Diabetes Mellitus than in normal controls | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Lower HLA-G levels in Extravillous Trophoblasts of Human Term Placenta in Gestational Diabetes Mellitus than in normal controls Julia Knabl, Rebecca Hüttenbrenner, Sven Mahner, Franz Kainer, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1823402/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract The non-classical HLA (human leucocyte antigen) class I molecule HLA-G is widely known to play a major role in feto-maternal tolerance. We tested the hypothesis that HLA-G expression is altered in placentas of women with gestational diabetes mellitus (GDM) in a specific pattern depending on fetal sex. HLA-G expression was analysed in a total of eighty placentas (40 GDM/ 40 controls) by immunohistochemistry using the semi-quantitative immunoreactive score (IRS). Double-immunofluorescence staining identified the cells expressing HLA-G in the decidua and to evaluate the expression pattern. We found a significant (p<0.001) reduction of HLA-G expression in extravillous cytotrophoblasts in GDM as compared to controls. We could demonstrate that this downregulation is not due to a loss of cell number, but to a loss of expression intensity. We found a special change in cell pattern in extravillous trophoblastic cells: They show an obvious rarefication on HLAG expression on EVT cell surface. No significant differences according to fetal sex were found. These data show that GDM alters HLA-G expression in EVT cells and provide new insights in altered placental function in diabetic placenta. pregnancy humans male female trophoblasts diabetes gestational receptors immune HLA-G Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction During pregnancy, the semi-allogenic fetus needs to induce maternal immunotolerance in order to prevent its rejection. An important role in the special immune suppression system in pregnancy is attributed to the non-classical MHC class I molecule HLA-G (Hunt, Petroff et al. 2005 ). HLA-G is expressed in placental tissue invading the maternal uterine decidua during implantation. Its highest expression levels are recognized in extravillous cytotrophoblast cells (EVT) with little, if any, expression on the syncytiotrophoblast (SCT) (Le Bouteiller and Lenfant 1996 , O'Callaghan and Bell 1998 ). EVTs are positioned near to maternal immune cells at the materno-fetal interface and well known for their contribution to maternal immunotolerance. EVT do not express the classical MHC class I molecules HLA-A and -B. Therefore, the invasive trophoblast is not recognized as non-self by the maternal immune system (Bainbridge, Ellis et al. 1999 , Bainbridge, Ellis et al. 2000 ) resulting in the inability to induce immune responses. As a result of alternative splicing, several HLA-G isoforms exist (Hunt, Petroff et al. 2005 ). These mRNA variants encode one full-length isoform (HLA-G1), three short membrane-bound isoforms (HLA-G2, -G3, -G4) and two soluble isoforms (HLA-G5, -G6). Current evidence suggests that only the full-length, cell-surface bound isoform HLA-G1 and the soluble HLA-G5 and -G6 are biologically active. Among these isoforms, HLA-G1 is expressed on EVT. Although it may act as a classical HLA class I molecule, it also does not induce maternal immune response (Bainbridge, Ellis et al. 1999 ). The soluble isoforms HLA-G5 and -G6 are not synthesized or secreted by human trophoblasts in detectable amounts (Blaschitz, Juch et al. 2005 ) HLA-G gene almost completely lacks polymorphisms, thus, there is little variation between individuals. However, HLA-G expression levels can be altered by disorders of reproduction: Lower HLA-G expression levels were found in early pregnancy failure, i.e., miscarriage and recurrent abortion (Moreau, Flajollet et al. 2009 , Quach, Grover et al. 2014 , Ferreira, Meissner et al. 2016 ) and at time of delivery a significantly reduced expression of HLA-G was seen in placentas from women with pre-eclampsia in comparison to control (Goldman-Wohl, Ariel et al. 2000 , Yie, Li et al. 2004 ). HLA-G has been implicated in the complex network governing EVT invasion through interaction with uterine natural killer cells. Several inhibitory receptors present on natural killer (NK) cells have been shown to bind to HLA-G, which promotes upregulation of inhibitory receptors on NK cells and CD4 + T cells (LeMaoult, Zafaranloo et al. 2005 ). Whether these expression changes in miscarriage and pre-eclampsia influences trophoblast invasion and placentation, or if it is just a consequence of disturbed blood perfusion and placental hypoxia has remained elusive (Rouas-Freiss, Moreau et al. 2021 ). Intriguingly, HLA-G expression has also been detected in tumor lesions, where it may facilitate immune evasion (Rouas-Freiss, Khalil-Daher et al. 1999 , Wiendl, Mitsdoerffer et al. 2002 , Ferreira, Meissner et al. 2016 ). The restrictive expression pattern of HLA-G expression with a very strong expression in invasive EVTs of the placenta (Kovats, Main et al. 1990 , Hackmon, Pinnaduwage et al. 2017 ) makes it a widely accepted marker for the identification of EVT cells in immunhistochochemical stainings. Its mRNA or protein levels can also serve as normalization parameter in analyses of total first trimester trophoblast cell preparations. These contain a mixed population of villous and extravillous cytotrophoblasts as well as of syncytiotrophoblast fragments (Blaschitz, Weiss et al. 2000 ) with variable proportions of each cell type in different preparations. Although purification steps can be added allowing separation into villous and extravillous populations, this comes at the expense of yield (Majali-Martinez, Barth et al. 2018 ). Therefore, it is not a commonly used step. As an alternative option, one may resort to correcting cell type-specific expression levels of any gene for potential variation in EVT proportion in each cell isolation. Gestational diabetes mellitus (GDM) is defined as glucose intolerance first diagnosed during pregnancy. Between 5 and 15% of all pregnancies are currently affected by GDM(McIntyre, Catalano et al. 2019 ).The risk for a woman to develop GDM is higher when she carries a male fetus (Jaskolka, Retnakaran et al. 2015 ). A further risk contributing to the worldwide increasing GDM prevalence is maternal obesity (Ruchat, Houde et al. 2013 ). Due to a pronounced peripheral insulin resistance women fail to maintain normoglycemia (Metzger, Buchanan et al. 2007 ). GDM is associated with short and long-term complications for the offspring: Short-term consequences include perinatal complications due to high birth weight and fetal hyperinsulinemia (Schwartz 1990 , Hawdon 2011 ). Long term consequences include a higher risk to develop obesity, metabolic syndrome and type 2 diabetes in later life (Barnes-Powell 2007 ). GDM induces a proinflammatory environment in the placenta, as reflected by a prominent increase in markers and mediators of inflammation (Radaelli, Varastehpour et al. 2003 ). While alterations in HLA-G in pre-eclampsia at the end of pregnancy have been reported (Goldman-Wohl, Ariel et al. 2000 , Yie, Li et al. 2004 ), a potential effect of derangement of the maternal glucose-insulin axis as in GDM on HLA-G has not been investigated yet. Therefore, the aim of the present study was to localize HLA-G in the human placenta at term of pregnancy and to quantify potential changes associated with GDM. We included villous tissue and decidua. Sex-specific differences are common in placental function and pregnancy disorders, however, it is unknown whether also HLA-G expression shows sex-dichotomy. 2. Subjects, Materials And Methods 2.1. Study cohort After the study design was approved by the LMU ethics committee (approval number 337-06, approval date: 26-01-2010), 40 GDM patients and 40 healthy expectant mothers (control) were chosen to participate with their written informed consent. To be included in the study, all participants had undergone an oral glucose tolerance test (oGTT) between week 24 and 28 of their pregnancy(Carpenter and Coustan 1982 ). GDM diagnosis was based on the criteria of the German Society for Diabetes Mellitus (two measurements above limits: Fasting glucose > 90 mg/dL (5 mmol/L), 1 h > 180 mg/dL(10 mmol/L) and 2 hs > 155 mg/dL (8,6 mmol/L ). All GDM women were managed with insulin and showed a mean HbA1c of 5.8 ± 0.4%. A total of 75% of the patients were under good glucose control according to their mean blood glucose ( \(\le\) 100 mg/dl (5,6 mmol/L)). Detailed clinical and perinatal data of the study group have been published (Knabl, Hiden et al. 2015 , Knabl, Huttenbrenner et al. 2015 ). Clinical data of the present study cohort is shown in Table 1 and stratified by fetal sex in Table 2 . Fetal sex was balanced in the GDM and control group. 2.2. Tissue Samples Placentas were obtained within 5 mins after birth. Tissue samples (2 × 2 × 2 cm 3 ) were dissected from a central cotyledon of the placentas. The sampled tissues encompassed decidua, villous tissue and amniotic epithelium. Macroscopically, they were sufficiently supplied with blood, while areas with signs of calcification, bleeding or ischemia were avoided. After 24 h fixation in 4% buffered formalin solution, the tissue samples were embedded in paraffin for long-term storage. 2.3. Immunohistochemistry 2.3.1. Staining and Semi-Quantification Formalin-fixed paraffin-embedded sections (3 µm) were deparaffinized in xylol, rehydrated in a descending ethanol gradient and subjected to epitope retrieval in a pressure cooker using sodium citrate buffer (pH 6.0). After returning sections to room temperature, endogenous peroxidase activity of the tissue was blocked with 3% H 2 O 2 in methanol (20 min). Non-specific binding of the primary antibodies was blocked by using the appropriate blocking solution (see Table 3 ), followed by incubation with the primary antibodies. Salient features of the antibodies used are presented in Table 3 . For staining HLA-G antibody 4H84, which recognizes all HLA-G isoforms through an epitope located on the alpha-1 domain of HLA-G, was used. Immunoreactivity was detected by using the Vectastain Elite ABC-Kit (Vector Laboratories, Burlingame, CA, USA) according to the manufacturer’s protocol. Substrate and chromogen (3,3’-diaminobenzidine DAB; Dako, Glostrup, Denmark) were finally added to the slides, which were then counterstained with Mayer’s acidic hematoxylin and covered with cover slips. Placental tissue from first trimester (8 to 12 weeks)of pregnancy was used for positive and negative controls. Primary anti-HLA-G antibodies were replaced by negative control for super sensitive rabbit IgG antibodies (HK4087R, BioGenex, Mainz, Germany). The signals were semi-quantified using the semi-quantitative immunoreactivity score (IRS). Two independent examiners, blinded for group allocation of the sample, graded optical staining intensity and the proportion of stained cells until consensus was reached. Gradings were defined as 0: none, 1: weak, 2: moderate and 3: strong staining and the percentage of stained cells as 0: no stained cells, 1: ≤ 10% of the cells, 2: 11–50% of the cells, 3: 51–80% of the cells and 4: ≥ 80% of the cells). IRS was calculated by multiplying optical staining intensity with percentage of stained cells. 2.3.2 Identification of HLA-G expressing cells in the decidua with double immunofluorescence Double-immunofluorescence staining to identify HLA-G expressing cell types was conducted on decidua of GDM pregnancies and controls. Placental sections were deparaffinized in xylol for 20 mins. After cleansing with ethanol, sections were incubated in ethanol/methanol for 20 min. After rehydrating the slides in an alcohol gradient, they were placed in a pressure cooker with sodium citrate for 5 min (pH 6.0). For fixation, sections were incubated by room temperature with acetone for 5 min. Subsequently, slides were rinsed with PBS followed blocking with ultra-V blocking solution (Labvision) for 15 min. The slides were incubated with polyclonal rabbit CK-7 IgG to identify trophoblasts (Table 3 ) and further incubated with monoclonal anti-HLA-G antibody overnight. Mouse anti Human HLA-G antibody, clone MEM-G/9, specifically recognizes surface expressed native HLA-G1, when associated with beta 2 microglobulin, but not does recognize the isoforms HLA-G2, G3 and G4. Sections were then incubated with the secondary antibodies. The slides were incubated with the Cy-3 labelled goat-anti-rabbit IgG antibody (Dianova), which was diluted 1:500, and the Cy-2-labelled goat-anti-mouse IgG antibody, diluted 1:100. Next, the slides were embedded in DAPI containing mounting buffer (Vector Laboratories). Afterwards, the slides were analysed with a fluorescent Axioskop photomicroscope (Zeiss, Oberkochen, Germany). Pictures were taken with a digital Axiocam camera system (Zeiss). Analysis was restricted to decidual extravillous tissues due to the almost exclusive abundance of HLA-G expressing cells. 2.4. Statistical Analysis Groups were compared using non-parametrical Mann–Whitney U signed rank tests or Kruskal–Wallis test as appropriate. Multiple linear regression models were used to analyze the associations of clinical characteristics (BMI and birthweight) with IRS. IBM SPSS Statistics (Version 22.0. for Windows, Armonk, NY, USA) was used for data collection, analysis, and visualization. Statistical significance was accepted at p-values < 0.05. 3. Results And Discussion The study cohort was fully described in earlier studies about expression changes of nuclear receptors associated with GDM (Knabl, Hiden et al. 2015 , Knabl, Huttenbrenner et al. 2015 , Knabl, de Maiziere et al. 2020 ). A strength of the present study is the objectively assessed absence of GDM in the control group, since all women participating in the study underwent an oral glucose tolerance test (oGTT). GDM women had higher pre-pregnancy BMI and their newborn had a higher birthweight than non-GDM controls (Table 1 ). Multiple linear regression analysis was used to assess potential confounding or interaction with HLA-G IRS. Neither birth weight nor pre-pregnancy BMI were associated with HLA-G IRS (p < 0.05) (see Fig. 1A, B). We first aimed to test for potential changes in cellular HLA-G location in GDM. HLA-G double fluorescence staining was used for the discrimination between total trophoblast tissue (CK7 positive) and HLA-G. The antibody used was specific for isotypes HLA-G1 and HLA-G5, both regarded the most biologically relevant isotypes (Bainbridge, Ellis et al. 2000 ). HLA-G specific-staining in the decidua of GDM placentas is shown in green, while CK7 expression is shown in red (Fig. 3). In both groups we identified HLA-G on the cell surface, thus, only membrane bound HLA-G. Soluble HLA-G cannot be demonstrated by immunohistochemistry. In normal control decidual tissue EVTs were marked evenly with HLA-G in green (Fig. 3E, F) and in general with CK7 in red (Fig. 3C, D). Triple filter excitation (Fig. 3A, B) shows a stable co-expression of HLA-G (EVT) and CK7. Visual impression suggested a reduction of cell surface HLA-G in GDM EVT as compared to non-GDM controls (compare Fig. 3E and F). To substantiate this observation we used semi-quantitative immunohistochemistry employing an antibody reacting with all isoforms. Staining intensity was semi-quantified in a cell-type specific manner by calculating the immunoreactivity score (IRS). It is a measure integrating number of stained cells with staining intensity and allows to semi-quantify overall protein levels in tissues in a cell-type specific manner. It has been successfully used in earlier studies (Knabl, Hiden et al. 2015 , Knabl, Huttenbrenner et al. 2015 , Knabl, de Maiziere et al. 2020 ). IRS for HLA-G immunolabelling was reduced by 66% in EVT of GDM placenta vs non-GDM controls (p < 0.0001; median IRS: GDM 3 vs. control 9) (Fig. 2). Importantly, we could demonstrate that this downregulation is not due to a loss of cell number, but to a loss of protein expression on EVT in general because CK7 expression is equally distributed on EVT in GDM as well as EVT in controls. Since sex-specific differences are common in placental function and the prevalence of pregnancy disorders as well as fetal outcome differ between sexes, we analyzed the potential effect of fetal sex. (Table 2 for clinical details stratified by fetal sex). Again, GDM women had higher pre-pregnancy BMI and their newborn had a higher birthweight then non-GDM controls, found I both, girl and boy offspring. Multiple linear regression analysis was used to assess potential confounding. Neither birth weight nor BMI, as potential confounders, was associated with HLA-G IRS in both girl and boy offspring placentas (see Fig. 1A, B). We did not see sex-differences in the GDM-associated reduction of HLA-G IRS. After stratification for fetal sex, immunostaining showed no sex-specific difference within controls (mean IRS: male 9.0 vs female 10.5, p > 0.05), nor within GDM (mean IRS: male 2.0 vs female 2.0; p > 0.05) (see Fig. 4). The biological consequences of less HLA-G in GDM vs non-GDM controls are unclear. Placental inflammation is often found in pregnancies complicated by GDM (Kleiblova, Dostalova et al. 2010 ). The hyperglycemia produces an inflammatory environment with a high content of inflammatory cytokines and immune cells. Especially in the placental extravillous layer of GDM and Type 2 diabetes mellitus the number of NK cells is increased (Hara Cde, Franca et al. 2016 ). NK cells are immune cells with close contact to extravillous trophoblasts cells. They are found abundantly in the uterine decidua. NK cells generally destroy cells which do not express HLA class-la molecules and, but HLA-G protects the extravillous cells by cytolysis of NK cells (Rajagopalan and Long 1999 ). Therefore, our data could provide a possible link between enhanced inflammation often associated with GDM and an altered immune response by a consequent loss of HLA-G expression(Sharma, Banerjee et al. 2021 ). Secondly, HLA-G specific interactions contribute to placental development through secretion of angiogenic and pro-inflammatory factors by decidual NK cells and macrophages (Li, Houser et al. 2009 ). Therefore, HLA-G may be implicated in vascular remodeling and apoptosis of damaged cells, which is an important process to regulate the extent of trophoblast invasion (Rouas-Freiss, Moreau et al. 2021 ). Whether GDM is associated with altered trophoblast invasion remains to be studied. Summary And Conclusion GDM did not change the location of cell-surface HLA-G in term placentas as reflected by similar staining patterns in GDM and non-GDM controls with HLA-G immunoreactivity restricted to EVT. For controls, this is in line with an earlier study on term placentas from uncomplicated pregnancies (O'Callaghan and Bell 1998 ). However, protein levels of HLA-G reflected by IRS were reduced in GDM EVT as compared to non-GDM controls. An important strength of our study is the objective assessment by oGTT of GDM absence in the control group. While potential confounders such as pre-pregnancy BMI and birth weight have been considered in our analysis, residual confounding cannot be excluded. In conclusion, our study identified a specific downregulation of HLA-G in extravillous trophoblast cells in term placentas of GDM pregnancies, which is not caused by cell loss. These results may provide new knowledge about the changes in cellular immunoregulation at the materno-fetal interface in pregnancies with GDM. Whether these changes are specific for GDM or are a general phenomenon of all forms of diabetes mellitus, i.e. also seen in type-1 and type 2 diabetes mellitus, remains to be studied Abbreviations HLA: human leucocyte antigen EVT: extravillous trophoblast SCT: syncytiotrophoblast GDM: gestational diabetes mellitus BMI: body mass index IRS: immunoreactivity score Declarations Statements and Declarations: J.K., R.H., F.K., G.D. and U.J. have no competing interests to declare. S.M.: Research funding, advisory board, honorary or travel expenses: AbbVie, AstraZeneca, Clovis, Eisai, GlaxoSmithKline, Hubro, Medac, MSD, Novartis, Nykode, Olympus, PharmaMar, Pfizer, Roche, Sensor Kinesis, Teva, Tesaro Acknowledgement: The Authors thank Christina Kuhn and Simone Hoffmann for excellent technical support. References (2015). "Committee Opinion No. 644: The Apgar Score." Obstet Gynecol 126 (4): e52-e55. Bainbridge, D. R., S. A. Ellis and I. L. Sargent (1999). 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Tables Table 1 Clinical details of the patients with gestational diabetes mellitus (GDM) and of the normal control group. GDM (n = 40) Controls (n = 40) P-value Maternal age (years) 32.82 ± 4.56 31.15 ± 6.10 ns Maternal BMI (pre-pregnancy, based on mother passport) [kg/m 2 ] 28.1 ± 6.96 23.4 ± 6.21 p < 0.0001* Median gravidity (IQR) 2 (1.9) 2 (1) ns Median parity (IQR) 1 (1.4) 1 (1) ns Gestational age at delivery (weeks) 39.9 ± 1.3 39.8 ± 1.4 ns With labour (%) 85 85 ns Mean Hba1c% 5.8 ± 0.4 n.d. Birth weight (g) 3611 ± 536 3317 ± 502 P = 0.006* Umbilical artery pH 7.30 ± 0.08 7.29 ± 0.09 ns Median 5 min APGAR (min; max) 10 (8;10) 10 (8;10) ns Sex of neonate (f/m) 20/20 20/20 If not indicated otherwise, data are presented as mean ± SD or median [min, max] as appropriate. The Mann-Whitney-U test was applied to compare clinical outcome data of the two groups. Statistically significant differences are marked with an asterisk (*); BMI = Body Mass Index, APGAR score is a method to quickly summarize the health of newborn children (Appearance, Pulse, Grimace, Activity, Respiration) (2015) (2015), n.s. no significance, IQR = interquartile range) Table 2 Clinical details of the patients with gestational diabetes mellitus (GDM) and of the normal control group stratified by fetal sex. GDM Control p-value Male (n = 20) Female (n = 20) Male (n = 20) Female (n = 20) Maternal age (years) 31.5 ± 4.1 33.2 ± 5.33 30.3 ± 6.11 32.0 ± 6.13 ns Maternal BMI (pre-pregnancy) [kg/m 2 ] 29.4 ± 8.03 27.0 ± 4.73 21.9 ± 3.97 25.0 ± 7.90 p < 0.001* Gestational age at delivery (weeks) 39.7 ± 1.30 39.8 ± 1.40 39.8 ± 1.54 39.8 ± 1.16 ns Median gravidity (IQR) 2 (1.75) 2 (2) 2 (1) 2 (1) ns Median parity (IQR) 2 (1.75) 1 (1) 1 (1) 2 (1) ns Mean HbA1c (%) 5.9 ± 0.4 5.7 ± 0.3 n.a. n.a. ns With labor (%) 75 95 85 85 ns Birth weight (g) 3662 ± 562 3636 ± 661 3340 ± 568 3294 ± 440 p < 0.05* Umbilical artery pH 7.30 ± 0.07 7.30 ± 0.10 7.30 ± 0.10 7.30 ± 0.08 ns Median 5 min APGAR (min; max) 10 (9;10) 10 (8;10) 10 (8;10) 10 (8;10) ns If not indicated otherwise, data are presented as mean ± SD or median [min, max] as appropriate. The Kruskal–Wallis test was applied to compare clinical outcome data of all the different groups. Statistically significant differences between the groups are marked with an asterisk (*, GDM versus control independent of fetal sex); BMI = Body Mass Index, APGAR score is a method to quickly summarize the health of newborn children (Appearance, Pulse, Grimace, Activity, Respiration) n.a. = not available, ns = no significance Table 3 Antibodies and dilutions used for immunohistochemistry and double immune fluorescence. Antibody Dilution Incubation Manufacturer Blocking Solution Blocking Condition Ck-7 (Rabbit polyclonal) 1 100 overnight Santa Cruz Ultra V Block 15 min HLA-G (Mouse IgG) 1:50 overnight AbD Serotec (Bio-Rad) Ultra V Block 15 min HLA-G (Mouse IgG) 1:100 in power block; 1-fold dilution overnight Novus Biologicals, Littleton, Colorado Power block1× solution 3 min RT = room temperature, PBS = phosphate buffered saline solution. Additional Declarations Competing interest reported. J.K., R.H., F.K., G.D. and U.J. have no competing interests to declare. S.M.: Research funding, advisory board, honorary or travel expenses: AbbVie, AstraZeneca, Clovis, Eisai, GlaxoSmithKline, Hubro, Medac, MSD, Novartis, Nykode, Olympus, PharmaMar, Pfizer, Roche, Sensor Kinesis, Teva, Tesaro Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major revision 04 Sep, 2022 Reviews received at journal 11 Aug, 2022 Reviewers agreed at journal 09 Aug, 2022 Reviewers agreed at journal 01 Aug, 2022 Reviewers invited by journal 06 Jul, 2022 Editor assigned by journal 06 Jul, 2022 Submission checks completed at journal 06 Jul, 2022 First submitted to journal 04 Jul, 2022 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1823402","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":118919716,"identity":"e8c29141-b303-4764-9cbc-41c6540b21b4","order_by":0,"name":"Julia Knabl","email":"","orcid":"","institution":"University Hospital, LMU Munich","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Julia","middleName":"","lastName":"Knabl","suffix":""},{"id":118919718,"identity":"c936f222-2ea1-40e4-8cba-428af4f89956","order_by":1,"name":"Rebecca Hüttenbrenner","email":"","orcid":"","institution":"University Hospital, LMU Munich","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Rebecca","middleName":"","lastName":"Hüttenbrenner","suffix":""},{"id":118919719,"identity":"3f8be6e5-393e-45ad-9deb-fbabd5e25c97","order_by":2,"name":"Sven Mahner","email":"","orcid":"","institution":"University Hospital, LMU Munich","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sven","middleName":"","lastName":"Mahner","suffix":""},{"id":118919721,"identity":"4f7f967f-cd56-4720-964f-38c7669e08fc","order_by":3,"name":"Franz Kainer","email":"","orcid":"","institution":"University Hospital, LMU Munich","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Franz","middleName":"","lastName":"Kainer","suffix":""},{"id":118919724,"identity":"0b7412c1-edb4-412e-9a5f-0b0a2f0a9e4f","order_by":4,"name":"Gernot Desoye","email":"","orcid":"","institution":"Medical University Graz","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Gernot","middleName":"","lastName":"Desoye","suffix":""},{"id":118919729,"identity":"81de0a83-908c-4148-87c8-c45438973e0d","order_by":5,"name":"Udo Jeschke","email":"data:image/png;base64,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","orcid":"","institution":"University Hospital, LMU Munich","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Udo","middleName":"","lastName":"Jeschke","suffix":""}],"badges":[],"createdAt":"2022-07-04 10:44:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1823402/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1823402/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":23831709,"identity":"2c09e868-609d-4ed4-b551-c2742e7cff6d","added_by":"auto","created_at":"2022-07-13 19:25:18","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":78324,"visible":true,"origin":"","legend":"\u003cp\u003eNeither birth weight (g) (A) nor BMI (kg/m\u003csup\u003e2\u003c/sup\u003e) (B) correlated with HLA-G immunoreactivity score (IRS).\u003c/p\u003e","description":"","filename":"figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1823402/v1/aa7daf2176a6d5f371ffe248.jpg"},{"id":23831710,"identity":"7c7b7c87-9422-4ea8-82f5-c32040bbb5b8","added_by":"auto","created_at":"2022-07-13 19:25:18","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":86652,"visible":true,"origin":"","legend":"\u003cp\u003eStrong HLA-G immunoreactivity was found in control EVT (A). In GDM cases EVT immunolabelling was significantly (p\u0026lt;0.001) weaker (B). IRS for each group is shown as box plots for EVT (IRS EVT) (C). The boxes represent the range between the 25th and 75th percentiles with a horizontal line at the median. Groups are labelled as follows: CM:control male; CF: control female; GM: GDM male; GF: GDM female. Arrowheads indicate EVT cells Scale bar = 200 µm.\u003c/p\u003e","description":"","filename":"fig2mitpfeil.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1823402/v1/1925a7e9843ca28c89457688.jpg"},{"id":23831711,"identity":"fd1fe362-2504-42fb-a3b4-7445fdbae920","added_by":"auto","created_at":"2022-07-13 19:25:18","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":79499,"visible":true,"origin":"","legend":"\u003cp\u003eDouble immunofluorescence phenotyping of extravillous tissue. Triple filter excitation showing both, CK7 and HLA-G is shown in (A and B). CK7, bound by Cy-3-labelled secondary antibody, stained red marking EVT and SCT together (C and D). HLA-G, bound by Cy-2-labelled secondary antibody, stained green, marking the extravillous trophoblast (E and F). HLA- G labelling is diminished in GDM (A) compared to control (B) in EVT. Arrowheads indicate HLA-G expressing cells cells\u003c/p\u003e","description":"","filename":"fig3mitpfeil.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1823402/v1/48f811d5d0d03edf1b561009.jpg"},{"id":23831712,"identity":"8ade2b06-e56b-4189-80bd-f54d3630483d","added_by":"auto","created_at":"2022-07-13 19:25:18","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":166363,"visible":true,"origin":"","legend":"\u003cp\u003eHLA-G immunoreactivity is found with similar intensity in control EVT in male (A) and in control female (B). In GDM, we could not identify gender differences as well (GDM male (C) and GDM female (D). (E) shows the negative control in first trimester placenta (8-12 weeks). IRS for each group is shown as box plots for EVT (IRS EVT) (F). Boxes represent the range between the 25th and 75th percentiles with a horizontal line at the median. Groups are labelled as follows: CM: control male; CF: control female; GM: GDM male; GF: GDM female.. Arrowheads indicate EVT cells Scale bar: 100 µm.\u003c/p\u003e","description":"","filename":"abb4mitpfeil.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1823402/v1/80f081cff9104c747b242046.jpg"},{"id":23831713,"identity":"7dfcfd7d-fbce-4c27-8e9c-6fe5c07171d2","added_by":"auto","created_at":"2022-07-13 19:25:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":710113,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1823402/v1/afa50c00-e4e0-4d80-a2bc-062dd5487cb5.pdf"}],"financialInterests":"Competing interest reported. J.K., R.H., F.K., G.D. and U.J. have no competing interests to declare. S.M.: \nResearch funding, advisory board, honorary or travel expenses: AbbVie, AstraZeneca, Clovis, Eisai, GlaxoSmithKline, Hubro, Medac, MSD, Novartis, Nykode, Olympus, PharmaMar, Pfizer, Roche, Sensor Kinesis, Teva, Tesaro","formattedTitle":"Lower HLA-G levels in Extravillous Trophoblasts of Human Term Placenta in Gestational Diabetes Mellitus than in normal controls","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eDuring pregnancy, the semi-allogenic fetus needs to induce maternal immunotolerance in order to prevent its rejection. An important role in the special immune suppression system in pregnancy is attributed to the non-classical MHC class I molecule HLA-G (Hunt, Petroff et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). HLA-G is expressed in placental tissue invading the maternal uterine decidua during implantation. Its highest expression levels are recognized in extravillous cytotrophoblast cells (EVT) with little, if any, expression on the syncytiotrophoblast (SCT) (Le Bouteiller and Lenfant \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1996\u003c/span\u003e, O'Callaghan and Bell \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). EVTs are positioned near to maternal immune cells at the materno-fetal interface and well known for their contribution to maternal immunotolerance. EVT do not express the classical MHC class I molecules HLA-A and -B. Therefore, the invasive trophoblast is not recognized as non-self by the maternal immune system (Bainbridge, Ellis et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1999\u003c/span\u003e, Bainbridge, Ellis et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) resulting in the inability to induce immune responses.\u003c/p\u003e \u003cp\u003eAs a result of alternative splicing, several HLA-G isoforms exist (Hunt, Petroff et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). These mRNA variants encode one full-length isoform (HLA-G1), three short membrane-bound isoforms (HLA-G2, -G3, -G4) and two soluble isoforms (HLA-G5, -G6). Current evidence suggests that only the full-length, cell-surface bound isoform HLA-G1 and the soluble HLA-G5 and -G6 are biologically active. Among these isoforms, HLA-G1 is expressed on EVT. Although it may act as a classical HLA class I molecule, it also does not induce maternal immune response (Bainbridge, Ellis et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). The soluble isoforms HLA-G5 and -G6 are not synthesized or secreted by human trophoblasts in detectable amounts (Blaschitz, Juch et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2005\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eHLA-G gene almost completely lacks polymorphisms, thus, there is little variation between individuals. However, HLA-G expression levels can be altered by disorders of reproduction: Lower HLA-G expression levels were found in early pregnancy failure, i.e., miscarriage and recurrent abortion (Moreau, Flajollet et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2009\u003c/span\u003e, Quach, Grover et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, Ferreira, Meissner et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) and at time of delivery a significantly reduced expression of HLA-G was seen in placentas from women with pre-eclampsia in comparison to control (Goldman-Wohl, Ariel et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2000\u003c/span\u003e, Yie, Li et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). HLA-G has been implicated in the complex network governing EVT invasion through interaction with uterine natural killer cells. Several inhibitory receptors present on natural killer (NK) cells have been shown to bind to HLA-G, which promotes upregulation of inhibitory receptors on NK cells and CD4\u0026thinsp;+\u0026thinsp;T cells (LeMaoult, Zafaranloo et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Whether these expression changes in miscarriage and pre-eclampsia influences trophoblast invasion and placentation, or if it is just a consequence of disturbed blood perfusion and placental hypoxia has remained elusive (Rouas-Freiss, Moreau et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Intriguingly, HLA-G expression has also been detected in tumor lesions, where it may facilitate immune evasion (Rouas-Freiss, Khalil-Daher et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1999\u003c/span\u003e, Wiendl, Mitsdoerffer et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2002\u003c/span\u003e, Ferreira, Meissner et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe restrictive expression pattern of HLA-G expression with a very strong expression in invasive EVTs of the placenta (Kovats, Main et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1990\u003c/span\u003e, Hackmon, Pinnaduwage et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) makes it a widely accepted marker for the identification of EVT cells in immunhistochochemical stainings. Its mRNA or protein levels can also serve as normalization parameter in analyses of total first trimester trophoblast cell preparations. These contain a mixed population of villous and extravillous cytotrophoblasts as well as of syncytiotrophoblast fragments (Blaschitz, Weiss et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) with variable proportions of each cell type in different preparations. Although purification steps can be added allowing separation into villous and extravillous populations, this comes at the expense of yield (Majali-Martinez, Barth et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Therefore, it is not a commonly used step. As an alternative option, one may resort to correcting cell type-specific expression levels of any gene for potential variation in EVT proportion in each cell isolation.\u003c/p\u003e \u003cp\u003eGestational diabetes mellitus (GDM) is defined as glucose intolerance first diagnosed during pregnancy. Between 5 and 15% of all pregnancies are currently affected by GDM(McIntyre, Catalano et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).The risk for a woman to develop GDM is higher when she carries a male fetus (Jaskolka, Retnakaran et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). A further risk contributing to the worldwide increasing GDM prevalence is maternal obesity (Ruchat, Houde et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Due to a pronounced peripheral insulin resistance women fail to maintain normoglycemia (Metzger, Buchanan et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). GDM is associated with short and long-term complications for the offspring: Short-term consequences include perinatal complications due to high birth weight and fetal hyperinsulinemia (Schwartz \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1990\u003c/span\u003e, Hawdon \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Long term consequences include a higher risk to develop obesity, metabolic syndrome and type 2 diabetes in later life (Barnes-Powell \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). GDM induces a proinflammatory environment in the placenta, as reflected by a prominent increase in markers and mediators of inflammation (Radaelli, Varastehpour et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhile alterations in HLA-G in pre-eclampsia at the end of pregnancy have been reported (Goldman-Wohl, Ariel et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2000\u003c/span\u003e, Yie, Li et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), a potential effect of derangement of the maternal glucose-insulin axis as in GDM on HLA-G has not been investigated yet. Therefore, the aim of the present study was to localize HLA-G in the human placenta at term of pregnancy and to quantify potential changes associated with GDM. We included villous tissue and decidua. Sex-specific differences are common in placental function and pregnancy disorders, however, it is unknown whether also HLA-G expression shows sex-dichotomy.\u003c/p\u003e"},{"header":"2. Subjects, Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Study cohort\u003c/h2\u003e \u003cp\u003e After the study design was approved by the LMU ethics committee (approval number 337-06, approval date: 26-01-2010), 40 GDM patients and 40 healthy expectant mothers (control) were chosen to participate with their written informed consent. To be included in the study, all participants had undergone an oral glucose tolerance test (oGTT) between week 24 and 28 of their pregnancy(Carpenter and Coustan \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1982\u003c/span\u003e). GDM diagnosis was based on the criteria of the German Society for Diabetes Mellitus (two measurements above limits: Fasting glucose\u0026thinsp;\u0026gt;\u0026thinsp;90 mg/dL (5 mmol/L), 1 h\u0026thinsp;\u0026gt;\u0026thinsp;180 mg/dL(10 mmol/L) and 2 hs\u0026thinsp;\u0026gt;\u0026thinsp;155 mg/dL (8,6 mmol/L ). All GDM women were managed with insulin and showed a mean HbA1c of 5.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4%. A total of 75% of the patients were under good glucose control according to their mean blood glucose (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\le\\)\u003c/span\u003e\u003c/span\u003e100 mg/dl (5,6 mmol/L)). Detailed clinical and perinatal data of the study group have been published (Knabl, Hiden et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, Knabl, Huttenbrenner et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Clinical data of the present study cohort is shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and stratified by fetal sex in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Fetal sex was balanced in the GDM and control group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Tissue Samples\u003c/h2\u003e \u003cp\u003ePlacentas were obtained within 5 mins after birth. Tissue samples (2 \u0026times; 2 \u0026times; 2 cm\u003csup\u003e3\u003c/sup\u003e) were dissected from a central cotyledon of the placentas. The sampled tissues encompassed decidua, villous tissue and amniotic epithelium. Macroscopically, they were sufficiently supplied with blood, while areas with signs of calcification, bleeding or ischemia were avoided. After 24 h fixation in 4% buffered formalin solution, the tissue samples were embedded in paraffin for long-term storage.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Immunohistochemistry\u003c/h2\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1. Staining and Semi-Quantification\u003c/h2\u003e \u003cp\u003eFormalin-fixed paraffin-embedded sections (3 \u0026micro;m) were deparaffinized in xylol, rehydrated in a descending ethanol gradient and subjected to epitope retrieval in a pressure cooker using sodium citrate buffer (pH 6.0). After returning sections to room temperature, endogenous peroxidase activity of the tissue was blocked with 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in methanol (20 min). Non-specific binding of the primary antibodies was blocked by using the appropriate blocking solution (see Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), followed by incubation with the primary antibodies. Salient features of the antibodies used are presented in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. For staining HLA-G antibody 4H84, which recognizes all HLA-G isoforms through an epitope located on the alpha-1 domain of HLA-G, was used. Immunoreactivity was detected by using the Vectastain Elite ABC-Kit (Vector Laboratories, Burlingame, CA, USA) according to the manufacturer\u0026rsquo;s protocol. Substrate and chromogen (3,3\u0026rsquo;-diaminobenzidine DAB; Dako, Glostrup, Denmark) were finally added to the slides, which were then counterstained with Mayer\u0026rsquo;s acidic hematoxylin and covered with cover slips.\u003c/p\u003e \u003cp\u003ePlacental tissue from first trimester (8 to 12 weeks)of pregnancy was used for positive and negative controls. Primary anti-HLA-G antibodies were replaced by negative control for super sensitive rabbit IgG antibodies (HK4087R, BioGenex, Mainz, Germany).\u003c/p\u003e \u003cp\u003eThe signals were semi-quantified using the semi-quantitative immunoreactivity score (IRS). Two independent examiners, blinded for group allocation of the sample, graded optical staining intensity and the proportion of stained cells until consensus was reached. Gradings were defined as 0: none, 1: weak, 2: moderate and 3: strong staining and the percentage of stained cells as 0: no stained cells, 1: \u0026le; 10% of the cells, 2: 11\u0026ndash;50% of the cells, 3: 51\u0026ndash;80% of the cells and 4: \u0026ge; 80% of the cells). IRS was calculated by multiplying optical staining intensity with percentage of stained cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.3.2 Identification of HLA-G expressing cells in the decidua with double immunofluorescence\u003c/h2\u003e \u003cp\u003eDouble-immunofluorescence staining to identify HLA-G expressing cell types was conducted on decidua of GDM pregnancies and controls. Placental sections were deparaffinized in xylol for 20 mins. After cleansing with ethanol, sections were incubated in ethanol/methanol for 20 min. After rehydrating the slides in an alcohol gradient, they were placed in a pressure cooker with sodium citrate for 5 min (pH 6.0). For fixation, sections were incubated by room temperature with acetone for 5 min. Subsequently, slides were rinsed with PBS followed blocking with ultra-V blocking solution (Labvision) for 15 min. The slides were incubated with polyclonal rabbit CK-7 IgG to identify trophoblasts (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) and further incubated with monoclonal anti-HLA-G antibody overnight. Mouse anti Human HLA-G antibody, clone MEM-G/9, specifically recognizes surface expressed native HLA-G1, when associated with beta 2 microglobulin, but not does recognize the isoforms HLA-G2, G3 and G4. Sections were then incubated with the secondary antibodies. The slides were incubated with the Cy-3 labelled goat-anti-rabbit IgG antibody (Dianova), which was diluted 1:500, and the Cy-2-labelled goat-anti-mouse IgG antibody, diluted 1:100. Next, the slides were embedded in DAPI containing mounting buffer (Vector Laboratories). Afterwards, the slides were analysed with a fluorescent Axioskop photomicroscope (Zeiss, Oberkochen, Germany). Pictures were taken with a digital Axiocam camera system (Zeiss). Analysis was restricted to decidual extravillous tissues due to the almost exclusive abundance of HLA-G expressing cells.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Statistical Analysis\u003c/h2\u003e \u003cp\u003eGroups were compared using non-parametrical Mann\u0026ndash;Whitney U signed rank tests or Kruskal\u0026ndash;Wallis test as appropriate. Multiple linear regression models were used to analyze the associations of clinical characteristics (BMI and birthweight) with IRS. IBM SPSS Statistics (Version 22.0. for Windows, Armonk, NY, USA) was used for data collection, analysis, and visualization. Statistical significance was accepted at p-values\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results And Discussion","content":"\u003cp\u003eThe study cohort was fully described in earlier studies about expression changes of nuclear receptors associated with GDM (Knabl, Hiden et al. \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e, Knabl, Huttenbrenner et al. \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e, Knabl, de Maiziere et al. \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e). A strength of the present study is the objectively assessed absence of GDM in the control group, since all women participating in the study underwent an oral glucose tolerance test (oGTT). GDM women had higher pre-pregnancy BMI and their newborn had a higher birthweight than non-GDM controls (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Multiple linear regression analysis was used to assess potential confounding or interaction with HLA-G IRS. Neither birth weight nor pre-pregnancy BMI were associated with HLA-G IRS (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (see Fig.\u0026nbsp;1A, B).\u003c/p\u003e\n\u003cp\u003eWe first aimed to test for potential changes in cellular HLA-G location in GDM. HLA-G double fluorescence staining was used for the discrimination between total trophoblast tissue (CK7 positive) and HLA-G. The antibody used was specific for isotypes HLA-G1 and HLA-G5, both regarded the most biologically relevant isotypes (Bainbridge, Ellis et al. \u003cspan class=\"CitationRef\"\u003e2000\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eHLA-G specific-staining in the decidua of GDM placentas is shown in green, while CK7 expression is shown in red (Fig.\u0026nbsp;3). In both groups we identified HLA-G on the cell surface, thus, only membrane bound HLA-G. Soluble HLA-G cannot be demonstrated by immunohistochemistry. In normal control decidual tissue EVTs were marked evenly with HLA-G in green (Fig.\u0026nbsp;3E, F) and in general with CK7 in red (Fig.\u0026nbsp;3C, D). Triple filter excitation (Fig.\u0026nbsp;3A, B) shows a stable co-expression of HLA-G (EVT) and CK7. Visual impression suggested a reduction of cell surface HLA-G in GDM EVT as compared to non-GDM controls (compare Fig.\u0026nbsp;3E and F).\u003c/p\u003e\n\u003cp\u003eTo substantiate this observation we used semi-quantitative immunohistochemistry employing an antibody reacting with all isoforms. Staining intensity was semi-quantified in a cell-type specific manner by calculating the immunoreactivity score (IRS). It is a measure integrating number of stained cells with staining intensity and allows to semi-quantify overall protein levels in tissues in a cell-type specific manner. It has been successfully used in earlier studies (Knabl, Hiden et al. \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e, Knabl, Huttenbrenner et al. \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e, Knabl, de Maiziere et al. \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eIRS for HLA-G immunolabelling was reduced by 66% in EVT of GDM placenta vs non-GDM controls (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; median IRS: GDM 3 vs. control 9) (Fig.\u0026nbsp;2). Importantly, we could demonstrate that this downregulation is not due to a loss of cell number, but to a loss of protein expression on EVT in general because CK7 expression is equally distributed on EVT in GDM as well as EVT in controls.\u003c/p\u003e\n\u003cp\u003eSince sex-specific differences are common in placental function and the prevalence of pregnancy disorders as well as fetal outcome differ between sexes, we analyzed the potential effect of fetal sex. (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e for clinical details stratified by fetal sex). Again, GDM women had higher pre-pregnancy BMI and their newborn had a higher birthweight then non-GDM controls, found I both, girl and boy offspring. Multiple linear regression analysis was used to assess potential confounding. Neither birth weight nor BMI, as potential confounders, was associated with HLA-G IRS in both girl and boy offspring placentas (see Fig.\u0026nbsp;1A, B). We did not see sex-differences in the GDM-associated reduction of HLA-G IRS. After stratification for fetal sex, immunostaining showed no sex-specific difference within controls (mean IRS: male 9.0 vs female 10.5, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05), nor within GDM (mean IRS: male 2.0 vs female 2.0; p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (see Fig.\u0026nbsp;4).\u003c/p\u003e\n\u003cp\u003eThe biological consequences of less HLA-G in GDM vs non-GDM controls are unclear. Placental inflammation is often found in pregnancies complicated by GDM (Kleiblova, Dostalova et al. \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e). The hyperglycemia produces an inflammatory environment with a high content of inflammatory cytokines and immune cells. Especially in the placental extravillous layer of GDM and Type 2 diabetes mellitus the number of NK cells is increased (Hara Cde, Franca et al. \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e). NK cells are immune cells with close contact to extravillous trophoblasts cells. They are found abundantly in the uterine decidua. NK cells generally destroy cells which do not express HLA class-la molecules and, but HLA-G protects the extravillous cells by cytolysis of NK cells (Rajagopalan and Long \u003cspan class=\"CitationRef\"\u003e1999\u003c/span\u003e). Therefore, our data could provide a possible link between enhanced inflammation often associated with GDM and an altered immune response by a consequent loss of HLA-G expression(Sharma, Banerjee et al. \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eSecondly, HLA-G specific interactions contribute to placental development through secretion of angiogenic and pro-inflammatory factors by decidual NK cells and macrophages (Li, Houser et al. \u003cspan class=\"CitationRef\"\u003e2009\u003c/span\u003e). Therefore, HLA-G may be implicated in vascular remodeling and apoptosis of damaged cells, which is an important process to regulate the extent of trophoblast invasion (Rouas-Freiss, Moreau et al. \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). Whether GDM is associated with altered trophoblast invasion remains to be studied.\u003c/p\u003e"},{"header":"Summary And Conclusion","content":"\u003cp\u003eGDM did not change the location of cell-surface HLA-G in term placentas as reflected by similar staining patterns in GDM and non-GDM controls with HLA-G immunoreactivity restricted to EVT. For controls, this is in line with an earlier study on term placentas from uncomplicated pregnancies (O'Callaghan and Bell \u003cspan class=\"CitationRef\"\u003e1998\u003c/span\u003e). However, protein levels of HLA-G reflected by IRS were reduced in GDM EVT as compared to non-GDM controls. An important strength of our study is the objective assessment by oGTT of GDM absence in the control group. While potential confounders such as pre-pregnancy BMI and birth weight have been considered in our analysis, residual confounding cannot be excluded.\u003c/p\u003e\n\u003cp\u003eIn conclusion, our study identified a specific downregulation of HLA-G in extravillous trophoblast cells in term placentas of GDM pregnancies, which is not caused by cell loss. These results may provide new knowledge about the changes in cellular immunoregulation at the materno-fetal interface in pregnancies with GDM. Whether these changes are specific for GDM or are a general phenomenon of all forms of diabetes mellitus, i.e. also seen in type-1 and type 2 diabetes mellitus, remains to be studied\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eHLA: human leucocyte antigen\u003c/p\u003e\n\u003cp\u003eEVT: extravillous trophoblast\u003c/p\u003e\n\u003cp\u003eSCT: syncytiotrophoblast\u003c/p\u003e\n\u003cp\u003eGDM: gestational diabetes mellitus\u003c/p\u003e\n\u003cp\u003eBMI: body mass index\u003c/p\u003e\n\u003cp\u003eIRS: immunoreactivity score\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eStatements and Declarations:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJ.K., R.H., F.K., G.D. and U.J. have no competing interests to declare. S.M.: Research funding, advisory board, honorary or travel expenses: AbbVie, AstraZeneca, Clovis, Eisai, GlaxoSmithKline, Hubro, Medac, MSD, Novartis, Nykode, Olympus, PharmaMar, Pfizer, Roche, Sensor Kinesis, Teva, Tesaro\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Authors thank Christina Kuhn and Simone Hoffmann for excellent technical support.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003e(2015). \u0026quot;Committee Opinion No. 644: The Apgar Score.\u0026quot; Obstet Gynecol \u003cstrong\u003e126\u003c/strong\u003e(4): e52-e55.\u003c/li\u003e\n \u003cli\u003eBainbridge, D. R., S. A. Ellis and I. L. Sargent (1999). \u0026quot;Little evidence of HLA-G mRNA polymorphism in Caucasian or Afro-Caribbean populations.\u0026quot; J Immunol \u003cstrong\u003e163\u003c/strong\u003e(4): 2023-2027.\u003c/li\u003e\n \u003cli\u003eBainbridge, D. R., S. A. Ellis and I. L. 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Carosella (2021). \u0026quot;Role of the HLA-G immune checkpoint molecule in pregnancy.\u0026quot; Hum Immunol \u003cstrong\u003e82\u003c/strong\u003e(5): 353-361.\u003c/li\u003e\n \u003cli\u003eRuchat, S. M., A. A. Houde, G. Voisin, J. St-Pierre, P. Perron, J. P. Baillargeon, D. Gaudet, M. F. Hivert, D. Brisson and L. Bouchard (2013). \u0026quot;Gestational diabetes mellitus epigenetically affects genes predominantly involved in metabolic diseases.\u0026quot; Epigenetics \u003cstrong\u003e8\u003c/strong\u003e(9): 935-943.\u003c/li\u003e\n \u003cli\u003eSchwartz, R. (1990). \u0026quot;Hyperinsulinemia and macrosomia.\u0026quot; N Engl J Med \u003cstrong\u003e323\u003c/strong\u003e(5): 340-342.\u003c/li\u003e\n \u003cli\u003eSharma, S., S. Banerjee, P. M. Krueger and S. M. Blois (2021). \u0026quot;Immunobiology of Gestational Diabetes Mellitus in Post-Medawar Era.\u0026quot; Front Immunol \u003cstrong\u003e12\u003c/strong\u003e: 758267.\u003c/li\u003e\n \u003cli\u003eWiendl, H., M. Mitsdoerffer, V. Hofmeister, J. Wischhusen, A. Bornemann, R. Meyermann, E. H. Weiss, A. Melms and M. Weller (2002). \u0026quot;A functional role of HLA-G expression in human gliomas: an alternative strategy of immune escape.\u0026quot; J Immunol \u003cstrong\u003e168\u003c/strong\u003e(9): 4772-4780.\u003c/li\u003e\n \u003cli\u003eYie, S. M., L. H. Li, Y. M. Li and C. Librach (2004). \u0026quot;HLA-G protein concentrations in maternal serum and placental tissue are decreased in preeclampsia.\u0026quot; Am J Obstet Gynecol \u003cstrong\u003e191\u003c/strong\u003e(2): 525-529.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cdiv class=\"gridtable\"\u003e\n \u003ctable border=\"1\" id=\"Tab1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eClinical details of the patients with gestational diabetes mellitus (GDM) and of the normal control group.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGDM\u003c/p\u003e\n \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;40)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eControls\u003c/p\u003e\n \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;40)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eP-value\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eMaternal age (years)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32.82\u0026thinsp;\u0026plusmn;\u0026thinsp;4.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31.15\u0026thinsp;\u0026plusmn;\u0026thinsp;6.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ens\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eMaternal BMI (pre-pregnancy, based on mother passport) [kg/m\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e]\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e28.1\u0026thinsp;\u0026plusmn;\u0026thinsp;6.96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23.4\u0026thinsp;\u0026plusmn;\u0026thinsp;6.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.0001*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eMedian gravidity (IQR)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2 (1.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2 (1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ens\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eMedian parity (IQR)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1 (1.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1 (1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ens\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eGestational age at delivery (weeks)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e39.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e39.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ens\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eWith labour (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ens\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean Hba1c%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en.d.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eBirth weight (g)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3611\u0026thinsp;\u0026plusmn;\u0026thinsp;536\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3317\u0026thinsp;\u0026plusmn;\u0026thinsp;502\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eP\u0026thinsp;=\u0026thinsp;0.006*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eUmbilical artery pH\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ens\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eMedian 5 min APGAR (min; max)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10 (8;10)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10 (8;10)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ens\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eSex of neonate (f/m)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20/20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20/20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\"\u003eIf not indicated otherwise, data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD or median [min, max] as appropriate.\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\"\u003eThe Mann-Whitney-U test was applied to compare clinical outcome data of the two groups. Statistically significant differences are marked with an asterisk (*); BMI\u0026thinsp;=\u0026thinsp;Body Mass Index, APGAR score is a method to quickly summarize the health of newborn children (Appearance, Pulse, Grimace, Activity, Respiration) (2015) (2015), n.s. no significance, IQR\u0026thinsp;=\u0026thinsp;interquartile range)\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable border=\"1\" id=\"Tab2\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eClinical details of the patients with gestational diabetes mellitus (GDM) and of the normal control group stratified by fetal sex.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eGDM\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ep-value\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eMale\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(n\u0026thinsp;=\u0026thinsp;20)\u003c/strong\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eFemale\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(n\u0026thinsp;=\u0026thinsp;20)\u003c/strong\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eMale\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(n\u0026thinsp;=\u0026thinsp;20)\u003c/strong\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eFemale\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(n\u0026thinsp;=\u0026thinsp;20)\u003c/strong\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eMaternal age (years)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31.5\u0026thinsp;\u0026plusmn;\u0026thinsp;4.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e33.2\u0026thinsp;\u0026plusmn;\u0026thinsp;5.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30.3\u0026thinsp;\u0026plusmn;\u0026thinsp;6.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32.0\u0026thinsp;\u0026plusmn;\u0026thinsp;6.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ens\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eMaternal BMI (pre-pregnancy) [kg/m\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e]\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29.4\u0026thinsp;\u0026plusmn;\u0026thinsp;8.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27.0\u0026thinsp;\u0026plusmn;\u0026thinsp;4.73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21.9\u0026thinsp;\u0026plusmn;\u0026thinsp;3.97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25.0\u0026thinsp;\u0026plusmn;\u0026thinsp;7.90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eGestational age at delivery (weeks)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e39.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e39.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e39.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e39.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ens\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eMedian gravidity (IQR)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2 (1.75)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2 (2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2 (1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2 (1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ens\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eMedian parity (IQR)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2 (1.75)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1 (1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1 (1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2 (1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ens\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean HbA1c (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en.a.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en.a.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ens\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eWith labor (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ens\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eBirth weight (g)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3662\u0026thinsp;\u0026plusmn;\u0026thinsp;562\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3636\u0026thinsp;\u0026plusmn;\u0026thinsp;661\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3340\u0026thinsp;\u0026plusmn;\u0026thinsp;568\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3294\u0026thinsp;\u0026plusmn;\u0026thinsp;440\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eUmbilical artery pH\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ens\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eMedian 5 min APGAR (min; max)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10 (9;10)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10 (8;10)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10 (8;10)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10 (8;10)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ens\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"6\"\u003eIf not indicated otherwise, data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD or median [min, max] as appropriate.\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"6\"\u003eThe Kruskal\u0026ndash;Wallis test was applied to compare clinical outcome data of all the different groups. Statistically significant differences between the groups are marked with an asterisk (*, GDM versus control independent of fetal sex); BMI\u0026thinsp;=\u0026thinsp;Body Mass Index, APGAR score is a method to quickly summarize the health of newborn children (Appearance, Pulse, Grimace, Activity, Respiration) n.a. = not available, ns\u0026thinsp;=\u0026thinsp;no significance\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable border=\"1\" id=\"Tab3\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eAntibodies and dilutions used for immunohistochemistry and double immune fluorescence.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAntibody\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDilution\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eIncubation\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eManufacturer\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eBlocking Solution\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eBlocking Condition\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCk-7\u003c/p\u003e\n \u003cp\u003e(Rabbit polyclonal)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1 100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eovernight\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSanta Cruz\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUltra V Block\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15 min\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHLA-G\u003c/p\u003e\n \u003cp\u003e(Mouse IgG)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1:50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eovernight\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAbD Serotec (Bio-Rad)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUltra V Block\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15 min\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHLA-G\u003c/p\u003e\n \u003cp\u003e(Mouse IgG)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1:100 in power block; 1-fold dilution\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eovernight\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNovus Biologicals, Littleton, Colorado\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePower block1\u0026times; solution\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3 min\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"6\"\u003e\n \u003cp\u003eRT\u0026thinsp;=\u0026thinsp;room temperature, PBS\u0026thinsp;=\u0026thinsp;phosphate buffered saline solution.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"histochemistry-and-cell-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"hacb","sideBox":"Learn more about [Histochemistry and Cell Biology](http://link.springer.com/journal/418)","snPcode":"418","submissionUrl":"https://submission.nature.com/new-submission/418/3","title":"Histochemistry and Cell Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"pregnancy, humans, male, female, trophoblasts, diabetes, gestational, receptors, immune, HLA-G","lastPublishedDoi":"10.21203/rs.3.rs-1823402/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1823402/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe non-classical HLA (human leucocyte antigen) class I molecule HLA-G is widely known to play a major role in feto-maternal tolerance. We tested the hypothesis that HLA-G expression is altered in placentas of women with gestational diabetes mellitus (GDM) in a specific pattern depending on fetal sex.\u003c/p\u003e\u003cp\u003eHLA-G expression was analysed in a total of eighty placentas (40 GDM/ 40 controls) by immunohistochemistry using the semi-quantitative immunoreactive score (IRS). Double-immunofluorescence staining identified the cells expressing HLA-G in the decidua and to evaluate the expression pattern. We found a significant (p\u0026lt;0.001) reduction of HLA-G expression in extravillous cytotrophoblasts in GDM as compared to controls. We could demonstrate that this downregulation is not due to a loss of cell number, but to a loss of expression intensity. We found a special change in cell pattern in extravillous trophoblastic cells: They show an obvious rarefication on HLAG expression on EVT cell surface. No significant differences according to fetal sex were found.\u003c/p\u003e\u003cp\u003eThese data show that GDM alters HLA-G expression in EVT cells and provide new insights in altered placental function in diabetic placenta.\u0026nbsp;\u003c/p\u003e","manuscriptTitle":"Lower HLA-G levels in Extravillous Trophoblasts of Human Term Placenta in Gestational Diabetes Mellitus than in normal controls","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-07-13 19:25:16","doi":"10.21203/rs.3.rs-1823402/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2022-09-04T16:24:03+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-08-11T12:14:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"2a3a983e-8fdb-4568-b1a9-2c7e6f7bead0","date":"2022-08-09T09:15:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"5e8f4956-de36-4ecc-bd51-00a34886fca8","date":"2022-08-01T16:00:48+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-07-06T12:13:09+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-07-06T10:35:26+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2022-07-06T07:08:52+00:00","index":"","fulltext":""},{"type":"submitted","content":"Histochemistry and Cell Biology","date":"2022-07-04T10:37:37+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"histochemistry-and-cell-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"hacb","sideBox":"Learn more about [Histochemistry and Cell Biology](http://link.springer.com/journal/418)","snPcode":"418","submissionUrl":"https://submission.nature.com/new-submission/418/3","title":"Histochemistry and Cell Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"5aefb4e2-a1a7-458e-8acf-216b992a5ec3","owner":[],"postedDate":"July 13th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2022-10-29T14:59:22+00:00","versionOfRecord":[],"versionCreatedAt":"2022-07-13 19:25:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1823402","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1823402","identity":"rs-1823402","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}