Immunological regulation and the role of autophagy in preeclampsia.

Autophagy is a bulk degradation system that maintains cellular homeostasis by producing energy and/or recycling excess proteins. During early placentation, extravillous trophoblasts invade the decidua and uterine myometrium, facing maternal immune cells, which participate in the immune suppression of paternal and fetal antigens. Regulatory T cells will likely increase in response to a specific antigen before and during early pregnancy. Insufficient expansion of antigen-specific Treg cells, which possess the same T cell receptor, is associated with the pathophysiology of preeclampsia, suggesting sterile systemic inflammation. Autophagy is involved in reducing inflammation through the degradation of inflammasomes and in the differentiation and function of regulatory T cells. Autophagy dysregulation induces protein aggregation in trophoblasts, resulting in placental dysfunction. In this review, we discuss the role of regulatory T cells in normal pregnancies. In addition, we discuss the association between autophagy and regulatory T cells in the development of preeclampsia based on reports on the role of autophagy in autoimmune diseases.

T cell immunity and the etiology and pathogenesis of preeclampsia.

Preeclampsia is more common in nulliparous women, their first pregnancies with a new partner in multiparous women, pregnant women with short duration of cohabitation, and in pregnancies with donor eggs, where the fetus is completely foreign to the mother. The epidemiological study findings strongly suggest that inadequate induction of tolerance to paternal/fetal antigens is involved in the pathogenesis of preeclampsia. This review proposes that preeclampsia may be caused by a reduction in paternal/fetal antigen-specific regulatory T (Treg) cells and decreased PD-1 expression on clonally expanded CD8+ effector memory T (TEM) cells, resulting in a breakdown of mother-to-fetus tolerance. The immune environment of preeclampsia is clearly different from that of recurrent pregnancy loss (RPL). In preeclampsia, cloned Treg cells decreases, and PD-1 expression on cloned CD8+TEM decreased. In RPL, the total number of Treg cells decreased, and the total number of clonally expanded CD8+TEM cells increases. In addition to these changes, increased differentiation of Th17 cells has also been observed in preeclampsia. This change is caused by soluble endoglin, that is increased in preeclampsia, neutralizing TGFß. These immunological changes make the fetus more susceptible to attacks from maternal T cells.

Anti-ß2-glycoprotein I/HLA-DR Antibody and Adverse Obstetric Outcomes.

Anti-ß2-glycoprotein I/HLA-DR (anti-ß2GPI/HLA-DR) antibody has been reported to be associated with antiphospholipid syndrome and recurrent pregnancy loss (RPL). We conducted a prospective multicenter cross-sectional study aimed at evaluating whether the anti-ß2GPI/HLA-DR antibody is associated with adverse obstetric outcomes and RPL. From 2019 to 2021, serum anti-ß2GPI/HLA-DR antibody levels (normal, <73.3 U) were measured in 462 women with RPL, 124 with fetal growth restriction (FGR), 138 with hypertensive disorders of pregnancy (HDP), 71 with preterm delivery before 34 gestational weeks (preterm delivery (PD) ≤ 34 GWs), and 488 control women who experienced normal delivery, by flow cytometry analysis. The adjusted odds ratios (aORs) of anti-ß2GPI/HLA-DR antibody positivity for adverse obstetric outcomes and RPL were evaluated on the basis of comparisons between the control and each patient group, using multivariable logistic regression analysis. The following were the positivity rates for the anti-ß2GPI/HLA-DR antibody in the patient and control groups: RPL, 16.9%; FGR, 15.3%; HDP, 17.4%; PD ≤ 34 GWs, 11.3%; and the control, 5.5%. It was demonstrated that anti-ß2GPI/HLA-DR antibody positivity was a significant risk factor for RPL (aOR, 3.3 [95% confidence interval {CI} 1.9-5.6], p < 0.001), FGR (2.7 [1.3-5.3], p < 0.01), and HDP (2.7 [1.4-5.3], p < 0.01) although not for PD ≤ 34 GWs. For the first time, our study demonstrated that the anti-ß2GPI/HLA-DR antibody is involved in the pathophysiology underlying FGR and HDP, as well as RPL.

The Role of Autophagy in the Female Reproduction System: For Beginners to Experts in This Field.

Autophagy is a fundamental process involved in regulating cellular homeostasis. Autophagy has been classically discovered as a cellular process that degrades cytoplasmic components non-selectively to produce energy. Over the past few decades, this process has been shown to work in energy production, as well as in the reduction of excessive proteins, damaged organelles, and membrane trafficking. It contributes to many human diseases, such as neurodegenerative diseases, carcinogenesis, diabetes mellitus, development, longevity, and reproduction. In this review, we provide important information for interpreting results related to autophagic experiments and present the role of autophagy in this field.

Dynamics of neuropilin1 (Nrp1)-positive thymus-derived and Nrp1-negative peripherally induced paternal antigen specific regulatory T cells in the uterus and spleen during pregnancy in mice.

Paternal antigen-specific regulatory T (PA-Treg) cells suppress the immune response against the fetus. Naturally occurring Treg (nTreg) cells derived from the thymus and peripherally induced Treg (iTreg) cells are functional for sustaining pregnancy. This study aimed to compare the variation in PA-Treg cells between the feto-maternal interface and the spleen and to elucidate the dynamics of nTreg and iTreg cells during the gestational period. PA-Treg cells, defined as Treg cells with paternally derived Mls-1a antigen-specific T cell receptors Vß6, from allogeneic pregnant mice on days 3.5, 5.5, 11.5, and 18.5 post-coitum (pc) were evaluated by flow cytometry. The percentage of Vß6+ Ki67+ PA-Treg cells activated by the paternal antigen increased on day 11.5 pc in the decidua (p < 0.05) compared to non-pregnant mice. On day 18.5 pc, this percentage in the decidua parietalis decreased to the level of the non-pregnant state but was significantly higher (p < 0.05) in the decidua basalis. No changes were observed in the spleens. We used two nTreg cell markers, neuropilin1 (Nrp1) and Helios, to distinguish between nTreg cells and iTreg cells. Nrp1+ PA-Treg cell levels decreased in late pregnancy compared to those observed in early pregnancy (day 3.5 pc: 57.14 ± 6.16% vs. day 18.5 pc: 30.43 ± 3.09%; p < 0.05), whereas Helios+ cell levels did not change. In conclusion, PA immune tolerance is induced by Nrp1+ nTreg cells in early pregnancy and Nrp1-negative Treg cells in late pregnancy.

Chloroquine is a safe autophagy inhibitor for sustaining the expression of antioxidant enzymes in trophoblasts.

Inhibition of autophagy contributes to the pathophysiology of preeclampsia. Although chloroquine (CHQ) is an autophagy inhibitor, it can reduce the occurrence of preeclampsia in women with systemic lupus erythematosus. To clarify this important clinical question, this study aimed to address the safety of CHQ in trophoblast cells from the viewpoint of homeostasis, in which the anti-oxidative stress (OS) response and autophagy are involved. We used Western blotting to evaluate the protein levels in the trophoblast cells. The expression levels of heme oxygenase-1 (HO-1), an anti-OS enzyme, mediate resistance to OS induced by hydrogen peroxide (H2O2) in trophoblast cell lines. Among the autophagy modulators, bafilomycin A1 (BAF), an autophagy inhibitor, but not autophagy activators, suppressed HO-1 expression in BeWo cells; CHQ did not suppress HO-1 expression in BeWo cells. To clarify the role of autophagy in HO-1 induction, we observed no difference in HO-1 induction by H2O2 between autophagy-normal and autophagy-deficient cells. As for the mechanism of HO-1 induction by OS, BAF suppressed HO-1 induction by downregulating the expression of neighbor of BRCA1 gene 1 (NBR1) in the selective p62-NBR1-nuclear factor erythroid 2-related factor 2 (Nrf2) autophagy pathway. CHQ did not inhibit HO-1 expression by sustaining NBR1 expression in human villous tissues compared to BAF treatment. In conclusion, CHQ is a safer medicine than BAF for sustaining NBR1, which resist against OS in trophoblasts by connecting selective autophagy and the anti-OS response.

Probiotics including Clostridium butyricum, Enterococcus faecium, and Bacillus subtilis may prevent recurrent spontaneous preterm delivery.

AIM: A large cohort study of Japanese women reported that the rate of recurrent spontaneous preterm delivery (sPTD) in the next pregnancy was 22.3%; therefore, it is important to prevent recurrent sPTD. The present study investigated the rate of recurrent sPTD in pregnant women treated with probiotics. METHODS: This was a retrospective study. Fifty-one pregnant women with a history of sPTD and who had been taking probiotics before 14 weeks of gestation were selected. The rate of sPTD in the next pregnancy among 255 pregnant women with a history of sPTD who had not taken probiotics was compared with that in the probiotics group. RESULTS: The rate of recurrent sPTD was 9.8% (5/51), which was lower than previously reported values. Furthermore, the rate of recurrent sPTD was significantly lower in the probiotics group (9.8%) than in the nonprobiotics group (31.0% [79/255]; p = 0.002). CONCLUSIONS: Probiotics may reduce the rate of recurrent sPTD.

Type 1 diabetes woman with repeated miscarriages successfully gave birth after introducing an insulin pump with a predictive low glucose suspend feature.

Sensor-augmented insulin pump therapy with a predictive low glucose suspend (SAP-PLGS) feature is a remarkably progressed modality for the glycemic management of patients with type 1 diabetes. This technology avoids nocturnal hypoglycemia and severe hypoglycemia. A Brazilian woman developed type 1 diabetes at age 11 and was treated with multiple daily insulin injections. At age 20, she was admitted to our internal medicine department for her first pregnancy. Her HbA1c was 7.9% in the 6 weeks of gestation. Although the combination of continuous subcutaneous insulin infusion and a sensor-augmented pump was introduced, she had a miscarriage in the next week. After 6 months, she became pregnant again. Despite an HbA1c of 7.2%, she had another miscarriage. Thereafter, she returned to multiple daily insulin injections and began using intermittently scanned continuous glycemic monitoring. At age 22, she had her third pregnancy. Her HbA1c was 7.3%. SAP-PLGS was then introduced, which reduced her frequent hypoglycemic events and blood glucose fluctuations. She gave birth to a 4137 g boy at 39 weeks without significant complications. Successful delivery can be obtained in women with type 1 diabetes following repeated miscarriages after introducing SAP-PLGS. We hypothesize that the modality might contributed to our patient's miscarriage avoidance by reducing her glycemic fluctuations.

Pre-eclampsia Complicated With Maternal Renal Dysfunction Is Associated With Poor Neurological Development at 3 Years Old in Children Born Before 34 Weeks of Gestation.

Objective: The purpose of this study was to investigate perinatal factors associated with a poor neurodevelopmental outcome in preterm infants. Methods: A retrospective study was conducted by searching our clinical database between January 2006 and December 2016. A total of 165 singleton children who were born between 23 and 33 weeks of gestation were included. We defined poor neurological development outcomes as follows: cerebral palsy; intellectual disability; developmental disorder including autism and attention-deficit/hyperactivity disorder; low score (<85 points) on Bayley Scales of Infant and Toddler Development, Third Edition (Bayley-III); or low score of Kyoto Scale of Psychological Development corrected at 3 years old. We diagnosed maternal renal dysfunction according to the Clinical Practice Guideline for chronic kidney disease 2018 and the Best Practice Guide 2015 for Care and Treatment of Hypertension in Pregnancy. Results: The rate of poor neurological development was 25/165 (15.2%): cerebral palsy (n = 1), intellectual disability (n = 1), developmental disorder (n = 2), low score of Bayley-III (n = 20), and low score of Kyoto Scale of Psychological Development (n = 1). Preeclampsia complicated with maternal renal dysfunction (P = 0.045) and delivery at <30 weeks of gestation (P = 0.007) were independent risk factors for poor neurological development. Conclusions: In addition to previous risk factors such as delivery at <30 weeks of gestation, preeclampsia complicated with renal dysfunction was also associated with poor neurodevelopmental outcomes corrected at 3 years old.

Aggrephagy Deficiency in the Placenta: A New Pathogenesis of Preeclampsia.

Aggrephagy is defined as the selective degradation of aggregated proteins by autophagosomes. Protein aggregation in organs and cells has been highlighted as a cause of multiple diseases, including neurodegenerative diseases, cardiac failure, and renal failure. Aggregates could pose a hazard for cell survival. Cells exhibit three main mechanisms against the accumulation of aggregates: protein refolding by upregulation of chaperones, reduction of protein overload by translational inhibition, and protein degradation by the ubiquitin-proteasome and autophagy-lysosome systems. Deletion of autophagy-related genes reportedly contributes to intracellular protein aggregation in vivo. Some proteins recognized in aggregates in preeclamptic placentas include those involved in neurodegenerative diseases. As aggregates are derived both intracellularly and extracellularly, special endocytosis for extracellular aggregates also employs the autophagy machinery. In this review, we discuss how the deficiency of aggrephagy and/or macroautophagy leads to poor placentation, resulting in preeclampsia or fetal growth restriction.

The role of decidual regulatory T cells in the induction and maintenance of fetal antigen-specific tolerance: Imbalance between regulatory and cytotoxic T cells in pregnancy complications.

Fetal antigen-specific tolerance is important for maintaining allogeneic pregnancies. Maternal conventional T cells recognize fetal antigens; however, regulatory T (Treg) cells suppress immune reactions against the fetus. Fetal antigen-specific Treg cells are induced in the decidua upon contact with antigen-presenting cells and extravillous trophoblasts (EVTs). Functional alteration of cytotoxic T cells (CTLs) in the decidua also contributes to maintaining the pregnancy. Reduced, dysfunctional, and imbalanced Treg cell distribution likely contributes to the pathogenesis of pregnancy complications, such as miscarriage and preeclampsia. Recent studies have revealed differences in Treg cell characteristics during preeclampsia and miscarriage. Treg cell reduction in the decidua is likely associated with miscarriage. Insufficient expansion of fetal antigen-specific Treg cells in the decidua probably plays a role in preeclampsia pathogenesis. In addition, the balance between Treg cell-mediated tolerance and functional alteration of CTLs is important. Further investigations of functional molecules in Treg cells will contribute to the development of immunotherapy for pregnancy complications.

Molecular and immunological developments in placentas.

Cytotrophoblasts differentiate in two directions during early placentation: syncytiotrophoblasts (STBs) and extravillous trophoblasts (EVTs). STBs face maternal immune cells in placentas, and EVTs, which invade the decidua and uterine myometrium, face the cells in the uterus. This situation, in which trophoblasts come into contact with maternal immune cells, is known as the maternal-fetal interface. Despite fetuses and fetus-derived trophoblast cells being of the semi-allogeneic conceptus, fetuses and placentas are not rejected by the maternal immune system because of maternal-fetal tolerance. The acquired tolerance develops during normal placentation, resulting in normal fetal development in humans. In this review, we introduce placental development from the viewpoint of molecular biology. In addition, we discuss how the disruption of placental development could lead to complications in pregnancy, such as hypertensive disorder of pregnancy, fetal growth restriction, or miscarriage.

Placental autophagy failure: A risk factor for preeclampsia.

Hypertensive disorders of pregnancy, including preeclampsia, directly affect maternal and perinatal morbidity and mortality. As the pathophysiology of preeclampsia is multi-factorial and has been studied using different approaches, we have demonstrated that impaired autophagy is an intertwined risk factor for preeclampsia. This concept has been verified in both in vitro and in vivo experiments. Autophagy is primarily involved in maintaining cellular homeostasis, and in immune regulation, longevity, cytokines secretion and a variety of other biological functions. Here, we review the role of autophagy in normal embryogenesis and placentation. Once placental autophagy is impaired by metabolic stress such as hypoxia, endoplasmic reticulum stress or starvation, placental development could be disrupted, resulting in functional maladaptations at the maternal-fetal interface. These malfunctions may result in fetal growth restriction or preeclampsia.

Analysis of TCR Repertoire and PD-1 Expression in Decidual and Peripheral CD8+ T Cells Reveals Distinct Immune Mechanisms in Miscarriage and Preeclampsia.

CD8+ T cells, the most abundant T cell subset in the decidua, play a critical role in the maintenance of pregnancy. The majority of decidual CD8+ T cells have an effector memory phenotype, while those in the peripheral blood display a naive phenotype. An increased amount of highly differentiated CD8+ T cells in the decidua indicates local antigen stimulation and expansion, albeit these CD8+ T cells are suppressed. In decidual CD8+ T cells, co-inhibitory molecules such as PD-1, TIM-3, LAG-3, and CTLA-4 are upregulated, reflecting the suppression of cytotoxicity. Previous studies established the importance of the PD-1/PD-L1 interaction for feto-maternal tolerance. CD8+ T cells could directly recognize fetal-specific antigens, such as HLA-C, expressed by trophoblasts. However, although fetal-specific CD8+ T cells have been reported, their TCR repertoires have not been identified. In this study, we analyzed the TCR repertoires of effector memory CD8+ T cells (CD8+ EM cells) and naive CD8+ T cells (CD8+ N cells) in the decidua and peripheral blood of women with normal or complicated pregnancy and examined PD-1 expression at a single-cell level to verify whether antigen-specific CD8+ T cells accumulate in the decidua and to identify immunological differences related to the suppression of antigen-specific CD8+ T cells between normal pregnancy, miscarriage, and preeclampsia. We observed that some TCRß repertoires, which might recognize fetal or placental antigens, were clonally expanded. The population size of clonally expanded CD8+ EM cells was higher in the decidua than in the peripheral blood. CD8+ EM cells began to express PD-1 during the course of normal pregnancy. We found that the total proportion of decidual CD8+ EM cells not expressing PD-1 was increased both in miscarriage and in preeclampsia cases, although a different mechanism was responsible for this increase. The amount of cytotoxic CD8+ EM cells increased in cases of miscarriage, whereas the expression of PD-1 in clonally expanded CD8+ EM cells was downregulated in preeclampsia cases. These results demonstrated that decidual CD8+ EM cells were able to recognize fetal-specific antigens at the feto-maternal interface and could easily induce fetal rejection.

Uterine CD11c+ cells induce the development of paternal antigen-specific Tregs via seminal plasma priming.

Tolerogenic dendritic cells (tDCs) play a central role in the development of paternal antigen-specific regulatory T cells (Tregs) during pregnancy. We examined whether uterine CD11c+ antigen presenting cells (APC) induced paternal antigen-specific tolerance in allogeneic pregnant mice. Female BALB/c mice were mated with male DBA/2 mice, and their surface markers of APCs were studied using flow cytometry. After allogeneic mating, the uterine APCs exhibited significantly decreased expression of major histocompatibility complex (MHC) class II on day 3.5 post-coitus (pc) and day 5.5 pc. To analyze how seminal fluid affects surface markers of APCs, female BALB/c mice were mated with male mice that had undergone seminal vesicle excision (SVX). No reductions of MHC class II expression on APCs were seen in these mice. To analyze APC functions, a mixed lymphoid reaction (MLR) assay to paternal splenocytes was performed. Uterine APCs from allogeneic pregnant mice significantly suppressed the MLR reaction, but APCs from SVX mated mice did not suppress the MLR reaction Uterine APCs induced paternal antigen (Mls1a)-specific Treg development in vitro, but not in mice that mated with allogeneic SVX mice. These findings suggest that seminal fluid priming expands the paternal antigen-specific Treg population by inducing APCs development.

Disruption of Placental Homeostasis Leads to Preeclampsia.

Placental homeostasis is directly linked to fetal well-being and normal fetal growth. Placentas are sensitive to various environmental stressors, including hypoxia, endoplasmic reticulum stress, and oxidative stress. Once placental homeostasis is disrupted, the placenta may rebel against the mother and fetus. Autophagy is an evolutionally conservative mechanism for the maintenance of cellular and organic homeostasis. Evidence suggests that autophagy plays a crucial role throughout pregnancy, including fertilization, placentation, and delivery in human and mouse models. This study reviews the available literature discussing the role of autophagy in preeclampsia.

Pre-conception status, obstetric outcome and use of medications during pregnancy of systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and inflammatory bowel disease (IBD) in Japan: Multi-center retrospective descriptive study.

Objective: To describe the pre-conception status, pregnancy outcomes, and medication prevalence in systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), Crohn's disease (CD), and ulcerative colitis (UC).Methods: E-mail-based questionnaire survey for the Japan Maternal Fetal Intensive Care Unit Network hospitals inquiring prevalence and clinical features of SLE, RA, CD and UC complicated pregnancies for 2 years.Results: The number of SLE, RA, CD and UC among 69,810 deliveries was 184, 139, 27 and 178, respectively. Less than half of pregnancies were planned. Assisted reproductive technology (ART) pregnancy rates were higher in SLE, RA and UC than in the general population (11.4, 23.0 and 7.4 vs 5.1%, p < .001 each). Preterm delivery, preeclampsia, and fetal growth restriction (FGR) were more frequent in SLE than in the general population (39.4 vs. 5.6% p < .001, 15.0 vs. 6.0% p < .001, 12.9 vs 4.2% p < .001). Prevalence of preterm delivery in RA and UC (27.5 vs. 5.6% p < .001, 11.3 vs. 5.6% p < .05) and FGR in CD (28.6 vs. 4.2% p < .001) was also higher than that in the general population.Conclusion: SLE, RA, CD, and UC complicated pregnancies were at high risks of obstetric adverse outcome. High ART rates necessitate pre-conception counseling in SLE, RA, and UC pregnancies.

Endoplasmic reticulum stress disrupts lysosomal homeostasis and induces blockade of autophagic flux in human trophoblasts.

Pregnancy is a stress factor culminating into mild endoplasmic reticulum (ER) stress, which is necessary for placental development. However, excessive or chronic ER stress in pre-eclamptic placentas leads to placental dysfunction. The precise mechanisms through which excessive ER stress impacts trophoblasts are not well understood. Here, we showed that ER stress reduces the number of lysosomes, resulting in inhibition of autophagic flux in trophoblast cells. ER stress also disrupted the translocation of lysosomes to the surface of trophoblast cells, and inhibited lysosomal exocytosis, whereby the secretion of lysosomal-associated membrane protein 1 (LAMP1) into culture media was significantly attenuated. In addition, we found that serum LAMP1 and beta-galactosidase levels were significantly decreased in pre-eclampsia patients compared to normal pregnant women, potentially indicating lysosomal dysfunction through ER stress in pre-eclamptic placentas. Thus, we demonstrated that excessive ER stress essentially disrupts homeostasis in trophoblasts in conjunction with autophagy inhibition by lysosomal impairment.

Current Understanding of Autophagy in Pregnancy.

Autophagy is an evolutionarily conserved process in eukaryotes to maintain cellular homeostasis under environmental stress. Intracellular control is exerted to produce energy or maintain intracellular protein quality controls. Autophagy plays an important role in embryogenesis, implantation, and maintenance of pregnancy. This role includes supporting extravillous trophoblasts (EVTs) that invade the decidua (endometrium) until the first third of uterine myometrium and migrate along the lumina of spiral arterioles under hypoxic and low-nutrient conditions in early pregnancy. In addition, autophagy inhibition has been linked to poor placentation-a feature of preeclamptic placentas-in a placenta-specific autophagy knockout mouse model. Studies of autophagy in human placentas have revealed controversial results, especially with regard to preeclampsia and gestational diabetes mellitus (GDM). Without precise estimation of autophagy flux, wrong interpretation would lead to fixed tissues. This paper presents a review of the role of autophagy in pregnancy and elaborates on the interpretation of autophagy in human placental tissues.

New Paradigm in the Role of Regulatory T Cells During Pregnancy.

Semi-allogenic fetuses are not rejected by the maternal immune system because feto-maternal tolerance induced by CD4+CD25+FoxP3+ regulatory T (Treg) cells is established during pregnancy. Paternal antigen-specific Treg cells accumulate during pregnancy, and seminal plasma priming plays an important role in expanding paternal antigen-specific Treg cells in mouse models. Although paternal-antigen specific Treg cells have not been identified in humans, recent studies suggest that antigen-specific Treg cells exist and expand at the feto-maternal interface in humans. Studies have also revealed that reduction of decidual functional Treg cells occurs during miscarriage with normal fetal chromosomal content, whereas insufficient clonal expansion of decidual Treg cells is observed in preeclampsia. In this review, we will discuss the recent advances in the investigation of mechanisms underlying Treg cell-dependent maintenance of feto-maternal tolerance.