The development of the fetal immune response is a highly complex process. In the present review, we describe the development of the fetal immune response and the role of the maternal gut bacteria in this process. In contrast to the previous belief that the fetal immune response is inert, it is now thought that the fetal immune response is uniquely tolerant to maternal and allo-antigens, but able to respond to infectious agents, such as bacteria. This is accomplished by the development of T cells toward regulatory T cells rather than toward effector T cells, but also by the presence of functional innate immune cells, such as monocytes and NK cells. Moreover, in fetuses there is different programming of CD8 + T cells and memory T cells toward innate immune cells rather than to adaptive immune cells. The maternal gut bacteria are important in shaping the fetal immune response by producing bacterial products and metabolites that pass the placenta into the fetus and influence development of the fetal immune response. Insight into how and when these products affect the fetal immune response may open new treatment options with pre- or probiotics to affect the maternal gut bacteria and therewith the fetal immune response.

Preeclampsia is a hypertensive disorder of pregnancy and is one of the most prevalent causes of maternal and fetal morbidity and mortality. The disease is thought to originate from impaired placental development or restricted villous perfusion, villous constraints and placental senescence at the end of pregnancy, which in turn cause defective placental functioning and eventually systemic endothelial dysfunction. Because the precise pathophysiology of this pregnancy complication is still not clear and diagnosis is only made when it is clinically visible, considerable research is being performed on biomolecules that could play a role in the development of the disease and could have a predictive, diagnostic or prognostic value. In the present review, we focus on two proteins, associated with endothelial dysfunction, which have changed levels in pregnant women during preeclampsia. These two proteins, endothelial specific molecule-1 (ESM-1) and guanylate binding protein-1 (GBP-1), are known to be involved in processes such as angiogenesis, inflammation and endothelial activation. ESM-1 is increased during preeclampsia and GBP-1 is decreased during preeclampsia, and their potential as biomarkers for preeclampsia could therefore be assessed. In addition to assessing their potential to serve as biomarkers, we will go into potential pathophysiological mechanisms of preeclampsia in which these proteins might be involved. We are proposing that ESM-1 and GBP-1 might play a role in impaired angiogenesis and vascular maladaptation, impaired immune regulation and oxidative stress, which ultimately could lead to endothelial dysfunction and preeclampsia.

PROBLEM:During pregnancy, the maternal immune system undergoes different adaptations to establish tolerance toward the fetus. Several factors are known to affect maternal immune adaptations, including fetal allo-antigens. Although, the effects of fetal antigens have clearly been shown in early pregnancy, the influence of these antigens in mid- and late pregnancy are not fully known. METHOD OF STUDY:In this study, we investigated the impact of fetal allo-antigens during pregnancy on maternal immune adaptations by comparing immunological changes in semi-allogeneic and syngeneic pregnancies in mice. Pregnant mice were analyzed at gestation day 10 (GD10) and gestation day 18 (GD18), and immune responses were assessed in the spleen, uterus draining para-aortic lymph nodes (PALN) and peripheral blood analyzing T cells, monocytes and their subsets. In addition, T cells were analyzed after stimulation using PMA or male antigens. RESULTS:Analyses of memory T cells, Tregs and monocytes (and subsets) demonstrated that fetal semi-allogeneity only had a minor influence on the maternal immune parameters in mid and late gestation. Immune stimulation experiments, using either PMA or syngeneic or allogeneic paternal antigens, revealed minor differences in cytokine production between general immune stimulation and paternal specific stimulation. CONCLUSIONS:Our results show that immune adaptations ad mid and late pregnancy are likely not mainly driven by fetal allo-antigens. This study contributes to a deeper understanding of the maternal immune system during pregnancy (either syngeneic or semi-allogeneic).

The maternal gut microbiome is involved in adapting immune responses to the presence of the semiallogeneic foetus. We have previously shown that antibiotics-induced gut dysbiosis, alterations in the maternal immune response and decreased foetal and placental weight. Here, we tested whether Bifidobacterium animalis subsp. lactis BL-99 (BL-99) could improve antibiotics-induced gut dysbiosis, maternal immune responses and foetal and placental weight. To do so, pregnant mice received antibiotics in their drinking water (day 9-16) and BL-99 via oral gavage (day 9-18). After sacrifice (day 18) immune responses were measured using flowcytometry. BL-99 increased placental weight in antibiotics-treated pregnant mice. BL-99 did not significantly change the maternal microbiome, but improved maternal immune responses by decreasing splenic Th1 cells and Treg cells, and increasing FoxP3/RoRγT double-positive cells in the Peyer's patches to levels observed in control pregnant mice. BL-99 also improved monocyte subsets and activation status. Additionally, BL-99 changed foetal monocyte subsets and activational status and increased foetal splenic Th cells. We thus showed that the effect of antibiotics treatment on immune cells and placental weight was mitigated by supplementation of BL-99. We suggest that pregnancy complications associated with a disturbed microbiome and immune responses, such as preeclampsia or obese pregnancies, could benefit from BL-99 supplementation. This should be tested in future studies.

IntroductionPreconceptional and maternal obesity are well-known risk factors for pregnancy and fetal complications including gestational diabetes, hypertensive disorders, and macrosomia. Maternal obesity is associated with offspring obesity and increased healthcare costs. To disrupt the cycle of obesity, we aim to investigate the impact of the composition of the maternal microbiota (bacteria and viruses) throughout preconception and pregnancy and the associations with the immune responses and one-carbon metabolism (1-CM) as an underlying mechanism in the pathophysiology of increased adverse pregnancy outcomes in maternal obesity.Methods and analysisThe PROMOTE study is a subcohort of the Rotterdam Periconceptional Cohort, a hospital-based observational cohort study. We will include 70 women per BMI group: ≥ 30 kg/m2 or 18.5-25 kg/m2, at different time points in each group: 10 preconceptional, 50 in the first trimester (with longitudinal follow-up during pregnancy, delivery and postpartum) and 10 in the third trimester of pregnancy. Which makes a total of 140 inclusions. Vaginal and rectal bacteriome, virome, and blood samples are collected. In the third trimester inclusions, only faecal samples are collected. Microbiota samples will be analysed using 16S rRNA sequencing. Bacteriome and virome profiles are compared between the BMI subgroups, associations with general immune responses and 1-CM markers will be shown.Trial registrationClinicalTrials.gov (NCT05754645).

Placental macrophages, which include maternal decidual macrophages and fetal Hofbauer cells, display a high degree of phenotypical and functional plasticity. This provides these macrophages with a key role in immunologically driven events in pregnancy like host defense, establishing and maintaining maternal-fetal tolerance. Moreover, placental macrophages have an important role in placental development, including implantation of the conceptus and remodeling of the intrauterine vasculature. To facilitate these processes, it is crucial that placental macrophages adapt accordingly to the needs of each phase of pregnancy. Dysregulated functionalities of placental macrophages are related to placental malfunctioning and have been associated with several adverse pregnancy outcomes. Although fetal growth restriction is specifically associated with placental insufficiency, knowledge on the role of macrophages in fetal growth restriction remains limited. This review provides an overview of the distinct functionalities of decidual macrophages and Hofbauer cells in each trimester of a healthy pregnancy and aims to elucidate the mechanisms by which placental macrophages could be involved in the pathogenesis of fetal growth restriction. Additionally, potential immune targeted therapies for fetal growth restriction are discussed.

Maternal obesity is a well-known risk factor for developing premature obesity, metabolic syndrome, cardiovascular disease and type 2 diabetes in the progeny. The development of white adipose tissue is a dynamic process that starts during prenatal life: fat depots laid down in utero are associated with the proportion of fat in children later on. How early this programming takes place is still unknown. However, recent evidence shows that mesenchymal stem cells (MSC), the embryonic adipocyte precursor cells, show signatures of the early setting of an adipogenic committed phenotype when exposed to maternal obesity. This review aims to present current findings on the cellular adaptations of MSCs from the offspring of women with obesity and how the metabolic environment of MSCs could affect the early commitment towards adipocytes. In conclusion, maternal obesity can induce early programming of fetal adipose tissue by conditioning MSCs. These cells have higher expression of adipogenic markers, altered insulin signalling and mitochondrial performance, compared to MSCs of neonates from lean pregnancies. Fetal MSCs imprinting by maternal obesity could help explain the increased risk of childhood obesity and development of further noncommunicable diseases.

Maternal obesity during pregnancy is associated with adverse pregnancy outcomes. This might be due to undesired obesity-induced changes in the maternal gut microbiota and related changes in the maternal immune adaptations during pregnancy. The current study examines how obesity affects gut microbiota and immunity in pregnant obese and lean mice during mid-pregnancy (gestational day 12 (GD12)). C57BL/6 mice were fed a high-fat diet or low-fat diet from 8 weeks before mating and during pregnancy. At GD12, we analyzed the gut microbiota composition in the feces and immune responses in the intestine (Peyer’s patches, mesenteric lymph nodes) and the peripheral circulation (spleen and peripheral blood). Maternal obesity reduced beneficial bacteria (e.g., Bifidobacterium and Akkermansia) and changed intestinal and peripheral immune responses (e.g., dendritic cells, Th1/Th2/Th17/Treg axis, monocytes). Numerous correlations were found between obesity-associated bacterial genera and intestinal/peripheral immune anomalies. This study shows that maternal obesity impacts the abundance of specific bacterial gut genera as compared to lean mice and deranges maternal intestinal immune responses that subsequently change peripheral maternal immune responses in mid-pregnancy. Our findings underscore the opportunities for early intervention strategies targeting maternal obesity, ideally starting in the periconceptional period, to mitigate these obesity-related pregnancy effects.

BACKGROUND:Maternal obesity is associated with maternal complications, including hypertensive disorders of pregnancy (HDP), and related fetal complications, such as fetal growth restriction. During pregnancy, the placenta is one of the key regulators of embryonic and fetal growth. Previous studies mainly investigated placental growth by measuring postpartum placental weight. However, the effects of obesity on aberrant placental and fetal growth might occur already in the first trimester. OBJECTIVES:Investigate associations between maternal BMI and first-trimester features of placental size and vascular development. STUDY DESIGN:870 women were included from a prospective cohort study. BMI was measured <10 weeks of gestation. Transvaginal 3D Power Doppler ultrasounds were obtained at 7, 9, and 11 weeks of gestation to measure placental volume (PV) and utero-placental vascular volume (uPVV). Associations between BMI and utero-placental (vascular) volume trajectories were assessed using mixed models, adjusted for covariates. RESULTS:Associations were found between maternal BMI and PV (non-linear model; p = 0.022). A BMI ≥34 kg/m2 showed decreased first-trimester PV compared to normal weight (Δ∛PV=-0.070, 95%CI -0.136 to -0.004, p=0.039). Negative associations were found between maternal BMI and uPVV (β=-0.027, 95%CI -0.041 to -0.014, p<0.001). Exclusion of women developing HDP, attenuated the association with PV (non-linear model; p=0.152), whilst the association between uPVV remained (β=-0.031, 95%CI -0.046 to -0.016, p=0.001). CONCLUSION:Increased maternal BMI is associated with decreasing first-trimester uPVV, moreover, in women with a BMI ≥34 kg/m2 the PV is decreased. These findings suggest tissue-specific changes in the placental development of women with obesity.

Benzo(a)pyrene (BaP) can be detected in the human placenta. However, little is known about the effects of BaP exposure on different placental cells under various conditions. In this study, we aimed to investigate the effects of BaP on mitochondrial function, pyrin domain-containing protein 3 (NLRP3) inflammasome, and apoptosis in three human trophoblast cell lines under normoxia, hypoxia, and inflammatory conditions. JEG-3, BeWo, and HTR-8/SVneo cell lines were exposed to BaP under normoxia, hypoxia, or inflammatory conditions for 24 h. After treatment, we evaluated cell viability, apoptosis, aryl hydrocarbon receptor (AhR) protein and cytochrome P450 (CYP) gene expression, mitochondrial function, including mitochondrial DNA copy number (mtDNAcn), mitochondrial membrane potential (ΔΨm), intracellular adenosine triphosphate (iATP), and extracellular ATP (eATP), nitric oxide (NO), NLPR3 inflammasome proteins, and interleukin (IL)-1β. We found that BaP upregulated the expression of AhR or CYP genes to varying degrees in all three cell lines. Exposure to BaP alone increased ΔΨm in all cell lines but decreased NO in BeWo and HTR-8/SVneo, iATP in HTR-8/SVneo, and cell viability in JEG-3, without affecting apoptosis. Under hypoxic conditions, BaP did not increase the expression of AhR and CYP genes in JEG-3 cells but increased CYP gene expression in two others. Pro-inflammatory conditions did not affect the response of the 3 cell lines to BaP with respect to the expression of CYP genes and changes in the mitochondrial function and NLRP3 inflammasome proteins. In addition, in HTR-8/SVneo cells, BaP increased IL-1β secretion in the presence of hypoxia and poly(I:C). In conclusion, our results showed that BaP affected mitochondrial function in trophoblast cell lines by increasing ΔΨm. This increased ΔΨm may have rescued the trophoblast cells from activation of the NLRP3 inflammasome and apoptosis after BaP treatment. We also observed that different human trophoblast cell lines had cell type-dependent responses to BaP exposure under normoxia, hypoxia, or pro-inflammatory conditions.

Purpose Today’s diet consists of a substantial proportion of ultra-processed foods (UPF), especially in women with overweight and obesity in the reproductive period. High UPF intake results in an inadequate and unbalanced diet leading to derangements of several metabolic pathways detrimental to pregnancy and birth outcomes. Therefore, we aim to investigate whether UPF intake in the periconceptional period affects total homocysteine plasma levels (tHcy). Methods 1532 participants were included from the prospective Rotterdam Periconceptional Cohort. UPF intake was calculated using Food Frequency Questionnaires including items classified as 4 in the Nova classification, and tHcy was measured by using liquid chromatography-tandem mass spectrometry system, with an interassay coefficient of variation of < 5.5%. Multivariable linear regression modeling was used and adjusted for covariates and significant interaction terms. Results Women with overweight or obesity showed significantly higher percentage of UPF intake (respectively, 50.3 and 51.3%) and higher tHcy (respectively, 6.6 and 6.3 µmol/L, Kruskal–Wallis test; respectively, p < 0.001 and p = 0.04) compared to women with normal BMI (UPF intake: 46.8%, tHcy: 6.1 µmol/L). A 10% higher intake of UPF was associated with an increase in tHcy (adjusted: β = 1.31, 95% CI = 0.38–2.23). Analysis stratified for BMI classification showed comparable associations in normal weight participants (adjusted: β = 1.07, 95% CI = 0.06–2.07); however, no significant association in participants with overweight (adjusted: β = 0.06, 95% CI = − 0.95–1.07) and obesity (adjusted: β = 1.70, 95% CI = − 0.52–3.92) was shown. Conclusion This study showed that a higher intake of UPF is associated with increased tHcy. Better knowledge and awareness of the nutritional quality of the diet in the periconceptional period may contribute to 1-CM and subsequently improve pregnancy course and outcome. Trial registration number and date NTR4356, November 2010.

Understanding the impact of various culturing strategies on the secretome composition of adipose-derived stromal cells (ASC) enhances their therapeutic potential. This study investigated changes in the secretome of perirenal ASC (prASC) under different conditions: normoxia, cytokine exposure, high glucose, hypoxia, and hypoxia with high glucose. Using mass spectrometry and enrichment clustering analysis, we found that normoxia enriched pathways related to extracellular matrix (ECM) organization, platelet degranulation, and insulin-like growth factor (IGF) transport and uptake. Cytokine exposure influenced metabolism, vascular development, and protein processing pathways. High glucose affected the immune system, metabolic processes, and IGF transport and uptake. Hypoxia impacted immune and metabolic processes and protein processing. Combined hypoxia and high glucose influenced the immune system, IGF transport and uptake, and ECM organization. Our findings highlight the potential of manipulating culturing conditions to produce secretomes with distinct protein and functional profiles, tailoring therapeutic strategies accordingly.

Heavy metals disrupt mitochondrial function and activate the NOD-like receptor pyrin-containing 3 (NLRP3) inflammasome. We investigated the effect of lead (Pb)/cadmium (Cd) on mitochondrial function and NLRP3 inflammasome activation in human trophoblast under normoxic, hypoxic and pro-inflammatory conditions. JEG-3, BeWo and HTR-8/SVneo cells were exposed to Pb or Cd for 24 h in the absence or presence of hypoxia or pro-inflammatory lipopolysaccharide (LPS) or poly(I:C). Then, we evaluated cell viability, apoptosis, mitochondrial DNA copy number (mtDNAcn), mitochondrial membrane potential (ΔΨ), NLRP3 inflammasome proteins and interleukin (IL)-1β secretion. Although our data showed that Pb, Cd, hypoxia, poly(I:C) and LPS decreased mtDNAcn in the three cell lines, the effects of these treatments on other biomarkers were different in the different cell lines. We found that hypoxia decreased ΔΨ and promoted apoptosis in JEG-3 cells, increased ΔΨ and prevented apoptosis in BeWo cells, and did not change ΔΨ and apoptosis in HTR-8/SVneo cells. Moreover, Pb under hypoxic conditions reduced ΔΨ and promoted apoptosis of BeWo cells. Exposure of BeWo and HTR-8/SVneo cells to hypoxia, Pb or Cd alone upregulated the expression of NLRP3 and pro-caspase 1 but did not activate the NLRP3 inflammasome since cleaved-caspase 1 and IL-1β were not increased. To conclude, Pb and Cd affected trophoblast mitochondrial function and NLRP3 proteins in trophoblast cell lines, but in a cell line-specific way. KEY POINTS: The objective of this work was an understanding of the effect of lead (Pb) and cadmium (Cd) on mitochondrial function and NLRP3 inflammasome activation in human trophoblast cell lines under normoxic, hypoxic and pro-inflammatory conditions. Apoptosis of JEG-3 cells was increased by hypoxia, while in BeWo cells, apoptosis was decreased by hypoxia, and in HTR-8/SVneo, apoptosis was not affected by hypoxic treatment. Exposure to either Pb or Cd decreased mtDNAcn in three human placental trophoblast cell lines. However, Pb under hypoxia induced a decrease of ΔΨ and promoted apoptosis of BeWo cells, but Cd did not induce a reduction in ΔΨ in the three trophoblast cell lines under any conditions. Exposure to hypoxia, Pb or Cd increased NLRP3 and pro-caspase 1 in BeWo and HTR-8/SVneo cells. Our findings highlight that Pb and Cd affected trophoblast mitochondrial function and NLRP3 proteins in trophoblast cell lines but in a cell line-specific way.

IntroductionMaternal obesity poses risks for both mother and offspring during pregnancy, with underlying mechanisms remaining largely unexplored. Obesity is associated with microbial gut dysbiosis and low-grade inflammation, and also the diet has a major impact on these parameters. This study aimed to investigate how maternal obesity and diet contribute to changes in immune responses, exploring potential associations with gut microbiota dysbiosis and adverse pregnancy outcomes in mice.MethodsBefore mating, C57BL/6 mice were assigned to either a high-fat-diet (HFD) or low-fat-diet (LFD) to obtain obese (n=17) and lean (n=10) mice. To distinguish between the effects of obesity and diet, 7 obese mice were switched from the HFD to the LFD from day 7 until day 18 of pregnancy (“switch group”), which was the endpoint of the study. T helper (Th) cell subsets were studied in the spleen, mesenteric lymph nodes (MLN) and Peyer’s patches (PP), while monocyte subsets and activation status were determined in maternal blood (flow cytometry). Feces were collected before and during pregnancy (day 7,14,18) for microbiota analysis (16S rRNA sequencing). Pregnancy outcome included determination of fetal and placental weight.ResultsObesity increased splenic Th1 and regulatory T cells, MLN Th1 and PP Th17 cells and enhanced IFN-γ and IL-17A production by splenic Th cells upon ex vivo stimulation. Switching diet decreased splenic and PP Th2 cells and classical monocytes, increased intermediate monocytes and activation of intermediate/nonclassical monocytes. Obesity and diet independently induced changes in the gut microbiota. Various bacterial genera were increased or decreased by obesity or the diet switch. These changes correlated with the immunological changes. Fetal weight was lower in the obese than the lean group, while placental weight was lower in the switch than the obese group. DiscussionThis study demonstrates that obesity and diet independently impact peripheral and intestinal immune responses at the end of pregnancy. Simultaneously, both factors affect specific bacterial gut genera and lead to reduced fetal or placental weight. Our data suggest that switching diet during pregnancy to improve maternal health is not advisable and it supports pre/probiotic treatment of maternal obesity-induced gut dysbiosis to improve maternal immune responses and pregnancy outcome.

STUDY QUESTION Is periconceptional multiple-micronutrient supplement (MMS) use including folic acid (FA) compared to FA use only associated with increased embryonic growth, development, and birth weight in a high-risk population?SUMMARY ANSWER Women with MMS intake show no significant differences in first-trimester morphological embryo development, but increased first-trimester embryonic growth trajectories and fewer neonates born small for gestational age (SGA), less than the 3rd percentile (<p3), compared to women using only FA.WHAT IS KNOWN ALREADY Periconceptional maternal FA intake in the general population is associated with increased embryonic and fetal growth, and reduced risks of neural tube defects, other congenital malformations, low birth weight, and neonates born SGA.STUDY DESIGN, SIZE, DURATION A prospective tertiary hospital-based cohort study (the Rotterdam Periconceptional Cohort) was conducted from January 2010 to December 2020.PARTICIPANTS/MATERIALS, SETTING, METHODS We included 1076 women from the Rotterdam Periconceptional Cohort, before 10 weeks of pregnancy with follow-up until delivery. Embryonic growth was assessed by measurement of crown-rump length (CRL) and embryonic volume (EV), and embryonic morphology was described by Carnegie stages using longitudinal three-dimensional ultrasound scans and virtual reality techniques. Birth outcomes were extracted from medical records. General characteristics and supplement use were extracted from research questionnaires.MAIN RESULTS AND THE ROLE OF CHANCE This study showed increased embryonic growth trajectories (adjusted models, CRL: beta = 0.052, 95% CI 0.012-0.090, EV: beta = 0.022, 95% CI 0.002-0.042) in women using MMS compared to those using only FA. Moreover, a 45% reduced risk of a neonate-born SGA (<p3) was shown in women using MMS compared to FA users (adjusted OR = 0.546, 95% CI 0.308, 0.969). Embryonic morphological development (Carnegie stages) and the occurrence of miscarriages did not differ between women using MMS or solely FA.LIMITATIONS, REASONS FOR CAUTION Following the heterogeneity of the composition and dose of MMS preparations, it is unclear which specific micronutrient, combination, or dose explains the increased embryonic growth trajectory and reduction in risk for SGA. This also hampers the possibility of differentiating between the effects of FA alone or as a component of MMS.WIDER IMPLICATIONS OF THE FINDINGS Our findings emphasize the importance of periconceptional maternal MMS use as a potential preventative intervention against reduced embryonic growth and neonates born SGA. Therefore, we recommend the periconceptional use of MMS in women at risk of inadequate micronutrient intake. However, awareness of potentially harmful side effects of high doses and combinations of micronutrients is essential, therefore the optimal composition and dose need to be investigated, and careful surveillance is recommended.STUDY FUNDING/COMPETING INTEREST(S) This research was funded by the Department of Obstetrics and Gynaecology, Erasmus MC, University Medical Center, Rotterdam, The Netherlands, and the ZonMw grant Open Competition 2018 (09120011910046). The authors declare that they have no conflict of interest.TRIAL REGISTRATION NUMBER NTR4356.

Fructans are a group of dietary fibers which are known to have many beneficial effects including immune-modulating effects.

Specific pregnancy outcomes have been linked to fetal sex, with in general higher pregnancy complication rates in boy pregnancies. These pregnancy complications have been linked to dysregulated reactions of the maternal immune system. Previous studies show higher levels of pro-inflammatory cytokines in the decidua of boy pregnancies. It could therefore be that differences in pregnancy outcome are explained by fetal sex dependent maternal immune activation. This study aims to compare decidual immune cell subsets between girl and boy pregnancies with normal pregnancy outcomes. Lymphocytes and macrophages were isolated from fresh decidua basalis and parietalis and stained with CD4, CD8, CD69, CD103, CCR7, FOXP3, CD14, CD45, CD50, CD163 and HLA-DR and analyzed using flowcytometry. In addition, decidual tissue sections were stained for T cells (CD3), macrophages (CD68 and CD206) and NK cells (CD56) using immunohistochemistry. Statistical analysis was performed with unpaired T-test and Mann-Whitney U test. In total, 23 girl and 22 boy pregnancies were included. Using flowcytometry, levels of activated CD8+ T cells, CD8+ memory cells, central and tissue resident memory cells, and M1 macrophages were higher in the decidua basalis of boy pregnancies (p<0.05). In the decidua parietalis, M2-like macrophages were lower in boy pregnancies (p<0.05). The IHC cohort showed a relative higher number in FOXP3+ Treg cells (p< 0.05) in boy pregnancies. Higher numbers of activated T cells, memory cells and M1 macrophages in boy pregnancies could indicate more maternal immune activation in boy pregnancies. These findings could explain the higher complication rates in boy pregnancies.