Female offspring of mice perinatally exposed to benzophenone-3 showed early subfertility linked to a poor oocyte stockpile.

Previously, we found that the ultraviolet filter benzophenone-3 (BP3) causes fetal growth restriction in mice when is applied when implantation occurs (first week of gestation). However, whether BP3 can affect gestation and fertility after implantation period is unknown. We aimed to study the effects on reproductive physiology of the offspring caused by perinatal exposure to BP3. C57BL/6 pregnant mice were dermally exposed to 50 mg BP3/kg bw.day or olive oil (vehicle) from gestation day 9 (gd9) to postnatal day 21 (pnd1). We observed no differences in mother's weights, duration of gestation, number of pups per mother, onset of puberty or sex ratio. The weights of the pups exposed to benzophenone-3 were transiently lower than those of the control. Estrous cycle was not affected by perinatal exposure to BP3. Besides, we performed a fertility assessment by continuous breeding protocol: at 10 weeks of age, one F1 female and one F1 male mouse from each group was randomly chosen from each litter and housed together for a period of 6 months. We noticed a reduction in the number of deliveries per mother among dams exposed to BP3 during the perinatal period. To see if this decreased fertility could be associated to an early onset of oocytes depletion, we estimated the ovarian reserve of germ cells. We found reduced number of oocytes and primordial follicles in BP3. In conclusion, perinatal exposure to BP3 leads to a decline in the reproductive capacity of female mice in a continuous breeding protocol linked to oocyte depletion.

Dermal exposure to the UV filter benzophenone-3 during early pregnancy affects fetal growth and sex ratio of the progeny in mice.

The aim of this study was to analyze whether dermal exposure to benzophenone 3 (BP-3) during pregnancy affects critical parameters of pregnancy, and whether this exposure may affect the outcome of a second pregnancy in mice. Pregnant mice were exposed to 50-mg BP-3/kg body weight/day or olive oil (vehicle) from gestation day (gd) 0 to gd6 by dermal exposure. High-frequency ultrasound imaging was used to follow up fetal and placental growth in vivo. Blood flow parameters in uterine and umbilical arteries were analyzed by Doppler measurements. Mice were killed at gd5, gd10, and gd14 on the first pregnancy, and at gd10 and 14 on the second pregnancy. The weight of the first and second progenies was recorded, and sex ratio was analyzed. BP-3 levels were analyzed in serum and amniotic fluid. BP-3 reduced the fetal weight at gd14 and feto-placenta index of first pregnancy, with 16.13% of fetuses under the 5th percentile; arteria uterina parameters showed altered pattern at gd10. BP-3 was detected in serum 4 h after the exposure at gd6, and in amniotic fluid at gd14. Offspring weight of first progeny was lower in BP-3 group. Placenta weights of BP-3 group were decreased in second pregnancy. First and second progenies of mothers exposed to BP-3 showed a higher percentage of females (female sex ratio). Dermal exposure to low dose of BP-3 during early pregnancy resulted in an intrauterine growth restriction (IUGR) phenotype, disturbed sex ratio and alterations in the growth curve of the offspring in mouse model.

Absence of Heme Oxygenase-1 Affects Trophoblastic Spheroid Implantation and Provokes Dysregulation of Stress and Angiogenesis Gene Expression in the Uterus.

The enzyme heme oxygenase-1 (HO-1) is pivotal in reproductive processes, particularly in placental and vascular development. This study investigated the role of HO-1 and its byproduct, carbon monoxide (CO), in trophoblastic spheroid implantation. In order to deepen our understanding of the role of HO-1 during implantation, we conducted in vivo experiments on virgin and pregnant mice, aiming to unravel the cellular and molecular mechanisms. Using siRNA, HO-1 was knocked down in JEG-3 and BeWo cells and trophoblastic spheroids were generated with or without CO treatment. Adhesion assays were performed after transferring the spheroids to RL-95 endometrial epithelial cell layers. Additionally, angiogenesis, stress, and toxicity RT2-Profiler™ PCR SuperArray and PCR analyses were performed in uterine murine samples. HO-1 knockdown by siRNA impeded implantation in the 3D culture model, but this effect could be reversed by CO. Uteruses from virgin Hmox1-/- females exhibited altered expression of angiogenesis and stress markers. Furthermore, there was a distinct expression pattern of cytokines and chemokines in uteruses from gestation day 14 in Hmox1-/- females compared to Hmox1+/+ females. This study strongly supports the essential role of HO-1 during implantation. Moreover, CO appears to have the potential to compensate for the lack of HO-1 during the spheroid attachment process. The absence of HO-1 results in dysregulation of angiogenesis and stress-related genes in the uterus, possibly contributing to implantation failure.

In vitro blastocyst implantation and trophoblast migration are disrupted by the UV filter benzophenone-3 (BP3).

Benzophenone-3 (BP3) is a common ingredient in personal care products (PCPs) due to its well-established effectiveness in absorbing UV radiation. Sunscreen products are among the most widely used PCPs-containing BP3 applied to the skin, resulting in significant human exposure to BP3 primarily through a dermal application. In the present work, we have tested the action of three environmentally relevant concentrations of BP3 (2, 20 and 200 µg/L) on an in vitro model of implantation of murine blastocysts and on migration ability of the human trophoblast cell line Swan 71. We showed that BP3 caused a significant reduction of blastocyst expansion and a delayed hatching in a non-monotonic way. Besides, embryos displayed a delayed attachment in the three BP3 groups, resulting in a smaller implantation area on the 6th day of culture: BP3(2) (0.32 ± 0.07 mm2); BP3(20) (0.30 ± 0.08 mm2) and BP3(200) (0.25 ± 0.06 mm2) in comparison to the control (0.42 ± 0.07 mm2). We also found a reduced migration capacity of the human first-trimester trophoblast cell line Swan 71 in a scratch assay when exposed to BP3: the lowest dose displayed a higher uncovered area (UA) at 6h when compared to the control, whereas a higher UA of the wound was observed for the three BP3 concentrations at 18 and 24 h of exposure. The changes in UA provoked by BP3 restored to normal values in the presence of flutamide, an androgen receptor (AR) inhibitor. These results indicate that a direct impairment on early embryo implantation and a defective migration of extravillous trophoblast cells through the androgen receptor pathway can be postulated as mechanisms of BP3-action on early gestation with potential impact on fetal growth.

Single and combined exposures to bisphenol A and benzophenone-3 during early mouse pregnancy have differential effects on fetal and placental development.

Endocrine disrupting chemicals (EDCs) possess the capability to interfere with the endocrine system by binding to hormone receptors, for example on immune cells. Specific effects have already been described for individual substances, but the impact of exposure to chemical mixtures during pregnancy on maternal immune regulation, placentation and fetal development is not known. In this study, we aimed to investigate the combined effects of two widespread EDCs, bisphenol A (BPA) and benzophenone-3 (BP-3), at allowed concentrations on crucial pregnancy processes such as implantation, placentation, uterine immune cell populations and fetal growth. From gestation day (gd) 0 to gd10, female mice were exposed to 4 µg/kg/d BPA, 50 mg/kg/d BP-3 or a BPA/BP-3 mixture. High frequency ultrasound and Doppler measurements were used to determine intrauterine fetal development and hemodynamic parameters. Furthermore, uterine spiral artery remodeling and placental mRNA expression were studied via histology and CHIP-RT-PCR, respectively. Effects of EDC exposure on multiple uterine immune cell populations were investigated using flow cytometry. We found that exposure to BP-3 caused intrauterine growth restriction in offspring at gd14, while BPA and BPA/BP-3 mixture caused varying effects. Moreover, placental morphology at gd12 and placental efficiency at gd14 were altered upon BP-3 exposure. Placental gene transcription was altered particularly in female offspring after in utero exposure to BP-3. Flow cytometry analyses revealed an increase in uterine T cells and NK cells in BPA and BPA/BP-3-treated dams at gd14. Doppler measurements revealed no effect on uterine hemodynamic parameters and spiral artery remodeling was not affected following EDC exposure. Our results provide evidence that exposure to BPA and BP-3 during early gestation affects fetal development in a sex-dependent manner, placental function and immune cell frequencies at the feto-maternal interface. These results call for inclusion of studies addressing pregnancy in the risk assessment of environmental chemicals.

Carbon Monoxide Exposure Does Not Improve The In Vitro Fertilization Rate of Oocytes Obtained from Heterozygous Hmox1 Knockout Mice.

In our experimental study we explored the impact of maternal reduced heme oxygenase-1 (HO-1) gene (Hmox1) expression on the in vitro fertilization (IVF) rate through the use of heterozygous Hmox1 knockout mice models (HET/Hmox1+/ -). Also, we hypothesized a beneficial role of gametes exposure during fertilization to carbon monoxide (CO), one of HO-1 by-products, that might be relevant for the improvement of IVF rates. IVF technique was performed by using oocytes obtained from wild-type (WT) or Hmox1+/ - dams fertilized with WT, Hmox1+/ - or Hmox1-/ - mice-derived sperm. The fertilization step occurred either in a conventional incubator (37°C, 5% CO2) or in an incubator implemented with CO (500 ppm). The superovulation yield of WT and Hmox1+/ - mice and the number of fertilized oocytes was assessed using an optical microscope. The dams' Hmox1 heterozygous knockout neither impact the superovulation yield, nor did influence the fertilization success rate. Moreover, CO exposure during fertilization could not significantly improve the outcome. Our study showed that the maternal Hmox1+/ -condition is not affecting the IVF rate in mice. Furthermore, we discovered that CO exposure cannot be exploited to ameliorate this critical step of the IVF protocol.

Haem oxygenase-1 dictates intrauterine fetal survival in mice via carbon monoxide.

Pregnancy establishment implies the existence of a highly vascularized and transient organ, the placenta, which ensures oxygen supply to the fetus via haemoproteins. Haem metabolism, including its catabolism by haem oxygenase-1 (HO-1), should be of importance in maintaining the homeostasis of haemoproteins and controlling the deleterious effects associated with haem release from maternal or fetal haemoglobins, thus ensuring placental function and fetal development. We demonstrate that HO-1 expression is essential to promote placental function and fetal development, thus determining the success of pregnancy. Hmox1 deletion in mice has pathological consequences for pregnancy, namely suboptimal placentation followed by intrauterine fetal growth restriction (IUGR) and fetal lethality. These pathological effects can be mimicked by administration of exogenous haem in wild-type mice. Fetal and maternal HO-1 is required to prevent post-implantation fetal loss through a mechanism that acts independently of maternal adaptive immunity and hormones. The protective HO-1 effects on placentation and fetal growth can be mimicked by the exogenous administration of carbon monoxide (CO), a product of haem catabolism by HO-1 that restores placentation and fetal growth. In a clinical relevant model of IUGR, CO reduces the levels of free haem in circulation and prevents fetal death. We unravel a novel physiological role for HO-1/CO in sustaining pregnancy which aids in understanding the biology of pregnancy and reveals a promising therapeutic application in the treatment of pregnancy pathologies.

Perinatal exposure to Bisphenol A disturbs the early differentiation of male germ cells.

Understanding the effects of Bisphenol A (BPA) on early germ cell differentiation and their consequences in adult life is an area of growing interest in the field of endocrine disruption. Herein, we investigate whether perinatal exposure to BPA affects the differentiation of male germ cells in early life using a transgenic mouse expressing the GFP reporter protein under the Oct4 promoter. In this model, the expression of GFP reflects the expression of the Oct4 gene. This pluripotency gene is required to maintain the spermatogonial stem cells in an undifferentiated stage. Thus, GFP expression was used as a parameter to evaluate the effect of BPA on early germ cell development. Female pregnant transgenic mice were exposed to BPA by oral gavage, from embryonic day 5.5 to postnatal day 7 (PND7). The effects of BPA on male germ cell differentiation were evaluated at PND7, while sperm quality, testicular morphology, and protein expression of androgen receptor and proliferating cell nuclear antigen were studied at PND130. We found that perinatal/lactational exposure to BPA up-regulates the expression of Oct4-driven GFP in testicular cells at PND7. This finding suggests a higher proportion of undifferentiated spermatogonia in BPA-treated animals compared with non-exposed mice. Moreover, in adulthood, the number of spermatozoa per epididymis was reduced in those animals perinatally exposed to BPA. This work shows that developmental exposure to BPA disturbed the normal differentiation of male germ cells early in life, mainly by altering the expression of Oct4 and exerted long-lasting sequelae at the adult stage, affecting sperm count and testis.

Mechanisms behind flare of renal lupus during murine pregnancy.

The outcome of pregnancy in systemic lupus erythematosus is still controversial. The authors recently reported the disappearance of the manifestation of the skin disease but a diminished survival rate in lupus-prone animals undergoing several pregnancies. It was postulated that lupus-prone animals must have subclinical renal symptoms at an early age and that immune and hormonal changes during pregnancy exacerbate immune reactions in the kidneys, leading to a shortened life span. Here, the authors analysed changes at day 14 of pregnancy in lupus-prone LPR (MRL/lpr) mice and MRL controls regarding cytokines, regulatory T (Treg) cells and deposition of immunocomplexes. Worsened kidney function was observed during pregnancy, even in the absence of lupus signs. This was accompanied by renal inflammation and higher interferon-gamma and interleukin-10 levels. C3 and immunoglobulin G deposition was enhanced in kidney and placenta from lupus-prone pregnant animals. Pregnancy enhanced the levels of Treg cells in control animals but not in lupus-prone animals. As pregnancy-induced Treg cells were shown to be specific for paternal antigens it is not to be expected that these Treg cells can help to destroy autoreactive cells. The authors conclude that early subclinical kidney disease in lupus-prone animals exacerbates during pregnancy. Albeit obtained with an experimental animal model, their data are potentially of importance for lupus patients of reproductive age.

Bisphenol A exposure during early pregnancy impairs uterine spiral artery remodeling and provokes intrauterine growth restriction in mice.

Endocrine disrupting chemicals are long suspected to impair reproductive health. Bisphenol A (BPA) has estrogenic activity and therefore the capacity of interfering with endocrine pathways. No studies dissected its short-term effects on pregnancy and possible underlying mechanisms. Here, we studied how BPA exposure around implantation affects pregnancy, particularly concentrating on placentation and uterine remodeling. We exposed pregnant female mice to 50 µg/kg BPA/day or 0.1% ethanol by oral gavage from day 1 to 7 of gestation. High frequency ultrasound was employed to document the presence and size of implantations, placentas and fetuses throughout pregnancy. Blood velocity in the arteria uterina was analyzed by Doppler measurements. The progeny of mothers exposed to BPA was growth-restricted compared to the controls; this was evident in vivo as early as at day 12 as analyzed by ultrasound and confirmed by diminished fetal and placenta weights observed after sacrificing the animals at day 14 of gestation. The remodeling of uterine spiral arteries (SAs) was considerably impaired. We show that short-term exposure to a so-called "safe" BPA dose around implantation has severe consequences. The intrauterine growth restriction observed in more than half of the fetuses from BPA-treated mothers may owe to the direct negative effect of BPA on the remodeling of uterine SAs that limits the blood supply to the fetus. Our work reveals unsuspected short-term effects of BPA on pregnancy and urges to more studies dissecting the mechanisms behind the negative actions of BPA during early pregnancy.

Immunology of pregnancy: cellular mechanisms allowing fetal survival within the maternal uterus.

Pregnancy success remains a fascinating phenomenon to immunologists as it defies the immunological rules of rejection. Although it was previously thought that the maternal immune system does not see the fetus, it is now well documented that fetal cells reach the maternal body and encounter host immune cells. Natural tolerance mechanisms following this interaction remain to be fully elucidated. This article reviews the current literature on mechanisms of adaptive immunity, with emphasis on regulatory T cells and heme oxygenase 1 (HO-1). We propose a scenario in which regulatory T cells create a tolerant microenvironment at the fetal-maternal interface characterised by the presence of tolerance-associated molecules such as HO-1, which has been shown to be of vital importance for fetal survival.

Over-expression of heme oxygenase-1 by adenoviral gene transfer improves pregnancy outcome in a murine model of abortion.

Mammalian pregnancy is a complex phenomenon allowing the maternal immune system to support its allogeneic fetus. Physiological pathways protecting the fetus from rejection are thought to be comparable with those leading to allograft acceptance. Heme oxygenase (HO)-1 is known to protect locally against rejection in transplantation models due to its anti-oxidant, anti-inflammatory and cytoprotective functions. Based on previous data on low HO-1 levels in placenta from mice undergoing abortion, we hypothesized that an up-regulation of HO-1 during pregnancy would avoid fetal rejection in the murine abortion combination CBA/J x DBA/2J, using BALB/c-mated CBA/J as normal controls. We injected pregnant mice undergoing abortion with 1 x 10(5) PFU of an adenoviral vector containing HO-1 and GFP (AdHO-1/GFP), and compared the pregnancy outcome with PBS- or 1 x 10(5) AdEGFP-treated abortion-prone mice and with PBS-treated normal pregnant mice. The abortion rate diminished significantly after adenoviral gene transfer of AdHO-1/GFP. The systemic and local IL-4/IFN-gamma ratio was augmented in AdHO-1-treated mice compared to abortion-prone mice. Interestingly, the HO-1 treatment up-regulated the ratio IL-10/TNF-alpha in spleen but not in decidual lymphocytes. HO-1-treated mice further showed diminished apoptosis rate and increased Bag-1 mRNA levels at the materno-fetal interface. Thus, we propose HO-1 as a key regulator of pregnancy success. HO-1 would exert its action by locally up-regulating the Th2/Th1 cytokine ratio and by further protecting tissues from apoptosis.

Heme oxygenase as a therapeutic target in immunological pregnancy complications.

The allogeneic fetus has been considered to be an allograft and the tolerance mechanisms involved in pregnancy maintenance resemble those leading to graft acceptance. Up-regulation of Heme Oxygenase-1 (HO-1) promotes graft acceptance. Additionally, HO-1 has been proposed to have tissue-protective properties. Previous studies reported the presence of HO-1 and HO-2 in mammalian placenta and postulated a protective role for HO during pregnancy. Here, we analyze HO-1 and HO-2 expression at the feto-maternal interface from normal pregnant and abortion mice and correlate these findings with inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) expression as well as with Th1/Th2 cytokine production by immune cells. DBA/2-mated CBA/J females undergoing abortion and BALB/c-mated CBA/J females having normal pregnancies were included in our study. The mice received no treatment. On day 14 of pregnancy, the mice were sacrificed, the abortion rate was calculated and the ex vivo Th1/Th2 production by decidual immune cells was analyzed by flow cytometry. The expression of HO-1 and HO-2, iNOS and eNOS was analyzed by immunohistochemistry (IHC) and Western blot in placenta samples. The Th1/Th2 cytokines ratio was augmented in decidua from abortion mice. We further observed a significant down-regulation of HO-1, HO-2, iNOS and eNOS molecules in placental tissue from mice undergoing abortion when compared to normal pregnant mice. Since we found diminished HOs and nitric oxide synthase (NOS) levels at the feto-maternal interface from abortion mice when compared to normal pregnant mice, which were further associated with increased Th1/Th2 cytokine production, we propose HO as a putative therapeutic target in immunological abortions. Up-regulation of HO-1 or HO-2 would favour the Th2-cytokine production, which could avoid abortion onset.

A single monoclonal antibody as probe to detect the entire set of native and partially unfolded rhEPO glycoforms.

Human erythropoietin (hEPO) is a highly heterogeneous glycosylated protein that requires well-characterised immunochemical reagents to evaluate the glycoform profile along its biotechnological production as a recombinant hormone. These reagents should be suitable for several assay conditions (like those used for immunoblotting analysis, liquid or solid-phase quantitative assays, immunoaffinity purification) with no glycoform selectivity. Five anti-recombinant hEPO monoclonal antibodies (mAbs) were characterised with the aim of selecting the appropriate reagent. These antibodies mapped two spatially distinct epitopes and neutralised the in vitro biological activity of the cytokine. All of them were able to bind to both, the partially denatured and the native form of the protein. Isoelectric focusing analysis followed by immunoblotting confirmed that all the mAbs, herein described, were able to bind to each glycoform, recognising amino acid sequences of the hEPO. Nevertheless, only mAb 2B2 preserved the ability to bind to soluble recombinant human erythropoietin (rhEPO) when it was coated to polystyrene plates or immobilised on CNBr-activated Sepharose matrix. Besides, mAb 2B2 was able to bind to the complete set of soluble rhEPO glycoforms, showing the same affinity for the glycosylated and deglycosylated cytokine. Thus, mAb 2B2 was useful as a capture antibody to develop a sandwich enzyme-linked immunosorbent assay (ELISA), performing a simple, specific and fast assay to quantify rhEPO with a detection limit of 7 ng ml(-1). mAb 2B2 was also satisfactorily employed as affinity ligand to purify rhEPO. Our work led us to find a suitable and single reagent to perform a variety of immunochemical approaches, where the binding of each glycoform in the native or partially unfolded form of rhEPO is required.

Hormonal Fluctuations during the Estrous Cycle Modulate Heme Oxygenase-1 Expression in the Uterus.

Deletion of the heme oxygenase-1 (HO-1) (Hmox1) locus in mice results in intrauterine lethality. The expression of the heme catabolizing enzyme encoded by this gene, namely HO-1, is required to successfully support reproductive events. We have previously observed that HO-1 acts at several key events in reproduction ensuring pregnancy. HO-1 defines ovulation, positively influences implantation and placentation, and ensures fetal growth and survival. Here, we embarked on a study aimed to determine whether hormonal changes during the estrous cycle in the mouse define HO-1 expression that may influence receptivity. We analyzed the serum levels of progesterone and estrogen by ELISA and HO-1 mRNA expression in uterus by real time RT-PCR at the metestrus, proestrus, estrus, and diestrus phases of the estrous cycle. Further, we studied the HO-1 protein expression by western blot upon hormone addition to cultured uterine AN3 cells. We observed that HO-1 variations in uterine tissue correlated to changes in hormonal levels at different phases of the estrus cycle. In vitro, HO-1 protein levels in AN3 cells augmented after the addition of physiological concentrations of progesterone and estradiol, which confirmed our in vivo observations. Our data suggest an important role for hormones in HO-1 regulation in uterus during receptivity, a process known to have a significant impact in receptivity and later on blastocyst implantation.

Origin of Foxp3(+) cells during pregnancy.

Pregnancy establishment and maintenance represents a challenge for the maternal immune system because it has to be alert against pathogens while tolerating paternal alloantigens expressed in fetal structures. Regulatory T cells (Tregs) are important for successful implantation and involved in allotolerance towards paternal antigens. The origin and mechanisms leading to Treg generation during pregnancy at different stages remain under discussion. We report an accumulation of Helios(+) Tregs in thymus and in the lymph nodes draining the uterus at early pregnancy. At later pregnancy stages an expanded population of Foxp3(+) Tregs was generated in the periphery as we showed in a Rag-1(-/-) model of cell transfer. Our data suggest that Tregs, predominantly of thymic origin, are needed for pregnancy establishment. At later pregnancy stages an extra thymic Treg population contributes to the Treg pool in the periphery. Our data provides new insights in the origin of Tregs during pregnancy that are essential to understand natural mechanisms of tolerance acquisition.

Blockage of heme oxygenase-1 abrogates the protective effect of regulatory T cells on murine pregnancy and promotes the maturation of dendritic cells.

Regulatory T cells (Treg) play an important role in fetal protection. They expand during normal pregnancy and protect fetal antigens from maternal effector cells. Their effect is associated with the up-regulation of tolerance-associated molecules at the fetal-maternal interface. Among these, Heme Oxygenase-1 (HO-1, coded by Hmox1) is of special importance as its blockage correlates with increased abortion rates and its up-regulation positively affects pregnancy outcome. Here, we aimed to investigate whether the protective effect of Treg is mediated by HO-1 in a mouse model. HO-1 blockage by Zinc Protoporhyrin (ZnPPIX) abrogated the protective effect of Treg transfer. We found that HO-1 is important in maintaining maternal dendritic cells (DCs) in an immature state, which contributes to the expansion of the peripheral Treg population. This brings to light one essential pathway through which Treg mediates the semi-allogeneic fetus tolerance.

Pregnancy: tolerance and suppression of immune responses.

Presence of foreign tissue in a host's body would immediately lead to a strong immune response directed to destroy the alloantigens present in fetus and placenta. However, during pregnancy, the semiallogeneic fetus is allowed to grow within the maternal uterus due to multiple mechanisms of immune tolerance, which are discussed in this chapter.

The persistence of paternal antigens in the maternal body is involved in regulatory T-cell expansion and fetal-maternal tolerance in murine pregnancy.

PROBLEM: Mammalian pregnancy is a state of immunological tolerance and CD4(+) CD25(+) regulatory T cells (Treg) contribute to its maintenance. Knowing that Treg act in an antigen-specific way during pregnancy, we hypothesized that they are generated after maternal immune cells encounter paternal antigens. METHOD OF STUDY: We mated wild type females with transgenic green fluorescent protein (GFP) males in an allogenic setting and killed them on different days of pregnancy. RESULTS: Presence of paternal and maternal MHC class II(+) cells in vaginal lavage on day 0.5 of pregnancy was confirmed. Thus, antigen presentation may take place early during pregnancy in the periphery either by the direct or indirect pathways. Foxp3(+) cells known to have regulatory activity could be detected on day 2 of pregnancy in lymph nodes and shortly after implantation at the fetal-maternal interface. CONCLUSION: Our data suggest that paternal antigens are processed early during pregnancy, which leads to the generation of Treg. The continuous release of placental antigens into the maternal circulation allows the maintenance of a Treg population which is specific for paternal antigens and mediates tolerance toward the semi-allogeneic fetus until the time point of birth.

Supporting the hypothesis of pregnancy as a tumor: survivin is upregulated in normal pregnant mice and participates in human trophoblast proliferation.

PROBLEM: Survivin, a tumor-promoting antiapoptotic molecule, is expressed in the human placenta. Here, we analyzed its expression during normal and pathological murine pregnancy and investigated its participation in human first trimester trophoblast cell survival and proliferation. METHOD OF STUDY: We first analyzed the expression of survivin on the mRNA and protein level at the fetal-maternal interface of normal pregnant (CBA/J x BALB/c) and abortion-prone (CBA/J x DBA/2J) mice at different pregnancy stages by RT-PCR and immunohistochemistry. We also evaluated apoptosis in murine trophoblasts in both mating combinations by TUNEL technique. Functional studies were carried out by knockdown survivin by means of siRNA methodology in two human first trimester trophoblast cell lines [Swan.71 (Sw.71) and HTR8 (H8)]. RESULTS: We observed a peak in mRNA levels on day 5 and a peak of protein levels on day 8 of pregnancy in both combinations. The level of survivin in animals from the abortion-prone group was decreased compared with normal pregnant mice on day 8, which was accompanied by elevated apoptosis rates. In later pregnancy stages (days 10 and 14), survivin levels decreased to levels comparable to those observed right after fecundation in both groups. Transfection of human first trimester cell lines (H8 and Sw.71) with siRNA targeting the survivin gene led to a 76-82% reduction of its expression leading to reduced trophoblast cell viability and proliferation. CONCLUSION: Our findings suggest an important role of survivin to promote trophoblast cell survival and proliferation during placentation, thus maintaining pregnancy. The pregnancy-associated expression of a cancer molecule such as survivin supports the 'pseudo-malignancy' hypothesis of pregnancy. Our data may contribute to the better understanding of trophoblast cell development during implantation and placentation.