N-3 polyunsaturated fatty acids effectively protect against neural tube defects in diabetic mice induced by streptozotocin.

Folate cannot prevent all neural tube defects (NTD), indicating that other pathogeneses still exist except for the folate deficiency. Maternal diabetes mellitus during pregnancy can increase the risk of offspring NTD. Our previous study showed that polyunsaturated fatty acids (PUFA) were lower in the placenta of human NTD cases than in healthy controls, and the supplementation of fish oil (rich in long-chain (LC) n-3 PUFA, mainly C20:5n-3 and C22:6n-3) had a better prevention effect against sodium valproate induced NTD than corn oil (rich in C18:2n-6) and flaxseed oil (rich in C18:3n-3). The aim of the present study was to investigate whether PUFA could prevent diabetes-induced NTD in mice. Streptozotocin (STZ)-induced diabetic pregnant mice were fed with a normal diet (DMC), a diet containing a low dose of fish oil (DMLn-3), a diet containing a high dose of fish oil (DMHn-3) or a diet rich in corn oil (DMn-6). Healthy pregnant mice were fed with a normal diet (HC). Compared with the DMC group, the rate of NTD was significantly lower in the DMHn-3 group (4.44% vs. 12.50%), but not in the DMLn-3 (11.11%) or DMn-6 group (12.03%). The NTD rate in the DMHn-3 group was comparable with that in the HC group (1.33%) (p = 0.246), and lower than that in the DMn-6 group (p = 0.052). The NTD rate in DMLn-3 and DMn-6 groups was significantly higher than that in the HC group. No significant difference was observed in NTD rate between DMLn-3 and DMHn-3 groups, and between DMLn-3 and DMn-6 groups. Compared with the HC group, the DMC group had a significantly lower C22:6n-3 in both serum and embryos. Fish oil supplementation ameliorated neuroepithelial cell apoptosis, and the apoptotic rate was comparable between DMHn-3 and HC groups. Although the apoptotic rate was significantly lower in the DMn-6 group than the DMC group, it was still much higher than that in the HC group. The proteins P53 and Bax in embryos were higher, while the proteins Bcl-2 and Pax3 were lower in the DMC group than in the HC group. The disturbance of Pax3, P53 and Bax induced by diabetes was abolished in DMLn-3, DMHn-3 and DMn-6 groups. Importantly, Bcl-2 in embryos was restored to the normal level only in the DMHn-3 group but not in the DMLn-3 or DMn-6 group. In conclusion, LC n-3 PUFA enriched fish oil has a protective effect against NTD in diabetes induced by STZ through improving neuroepithelial cell apoptosis, and the mechanism may be by increasing the anti-apoptosis protein Bcl-2 independently of Pax3 and P53.

Protopanaxadiol improves endometriosis associated infertility and miscarriage in sex hormones receptors-dependent and independent manners.

Background: Patients with endometriosis (EMs) have high risks of infertility and spontaneous abortion. How to remodel the fertility of patients with EMs has always been the hot spot and difficulty in the field of reproductive medicine. As an aglycone of ginsenosides, protopanaxadiol (PPD) possesses pleiotropic biological functions and has high medicinal values. We aimed to investigate the effect and potential mechanism of PPD in the treatment of EMs-associated infertility and spontaneous abortion. Methods: The EMs mice models were constructed by allotransplantation. The pregnancy rates, embryo implantation numbers and embryo resorption rates of control and EMs were counted. RNA sequencing, qRT-PCR, enzyme linked immunosorbent assay (ELISA) and FCM analysis were performed to screen and confirm the expression of endometrial receptivity/decidualization-related molecules, inflammation cytokines and NK cell function-related molecules in vitro and/or in vivo. The SWISS Target Prediction, STRING and Cytoscape were carried out to predict the potential cellular sensory proteins, the protein-protein interaction (PPI) network between sensory proteins and fertility-related molecules, respectively. Micro-CT detection, liver and kidney function tests were used to evaluate the safety. Results: Here, we observe that PPD significantly up-regulates endometrial receptivity-related molecules (e.g., Lif, Igfbp1, Mmps, collagens) and restricts pelvic inflammatory response (low levels of IL-12 and IFN-γ) of macrophage, and further remodel and improve the fertility of EMs mice. Additionally, PPD increases the expression of decidualization-related genes and Collagens, and promotes the proliferation, residence, immune tolerance and anagogic functions of decidual NK cells (low levels of CD16 and NKp30, high levels of Ki67, VEGF, TGF-ß) in pregnant EMs mice, and further triggers decidualization, decidual NK cell-mediated maternal-fetal immune tolerance and angiogenesis, preventing pregnant EMs mice from miscarriage. Mechanically, these effects should be dependent on ESRs, PGR and other sensory proteins (e.g., AR). Compared with GnRHa (the clinic first-line drug for EMs), PPD does not lead to the decline of serum estrogen and bone loss. Conclusion: These data suggest that PPD prevents EMs-associated infertility and miscarriage in sex hormones receptors-dependent and independent manners possibly, and provides a potential therapeutic strategy with high efficiency and low side effects to remodels the fertility of patients with EMs.

An integrated approach to uncover quality markers of stir-baking Semen Cuscuta with salt solution preventing recurrent spontaneous abortion based on chemical and metabolomic profiling.

The previous research of clinical big data mining showed that stir-baking Semen Cuscuta with salt solution (YP) ranked the first in the usage rate of treating abortion caused by kidney deficiency. At the same time, pharmacodynamic studies also showed that YP has better effect on improving recurrent spontaneous abortion (RSA) compared to raw products of Semen Cuscuta (SP). However, there were few studies on the biomarkers of YP improving RSA. In this study, the chemical and metabonomic profiling were used to screen the quality markers of YP on improving RSA. Firstly, a metabolomics study was carried out to select representative biomarkers of RSA. The ultra-high performance liquid chromatography coupled with electrospray ionization-quadrupole-time of flight-mass spectrometry (UPLC-ESI-Q-TOF-MS) technique was used to investigate the components of exogenous and endogenous in serum of rats after administrated with YP and SP. As a result, 14 differential compounds were identified between the serum of rats administrated SP and YP. Compared to SP, there was an upward trend in YP of the compounds including kaempferol-3-glucuronide, iso-kaempferol-3-glucuronide, (1S) -11-hydroxyhexadecanoic acid and 3-phenylpropionic acid. Meanwhile, there was a reducing trend in YP of the compounds including kaempferol 3-arabinofuranoside, apigenin-3-O-glucoside, hyperoside, caffeic acid-ß-D glucoside, dicaffeoylquinic acid, linoleic acid, 3,4-dicaffeoylquinic acid, caffeic acid, palmitic acid and methyl myristate. 12 biomarkers for RSA indication were identified. SP and YP have a certain effect on the endogenous biomarker. The regulation effect of YP was higher than that of SP. The main metabolic pathways included phenylalanine, tyrosine and tryptophan biosynthesis, glycerophospholipid metabolism, fatty acid biosynthesis, sphingolipid metabolism, biosynthesis of unsaturated fatty acids. This study demonstrated a promising way to elucidate the active chemical and endogenous material basis of TCM.

Diets enriched in PUFAs at an early postimplantation stage prevent embryo resorptions and impaired mTOR signaling in the decidua from diabetic rats.

Maternal diabetes increases the risk of embryo resorptions and impairs embryo development. Decidualization is crucial for embryo development and regulated by mTOR signaling. However, little is known about how maternal diabetes affects the decidua at early postimplantation stages and whether dietary treatments enriched in polyunsaturated fatty acids (PUFAs) can prevent decidual alterations. Here, we determined resorption rates, decidual mTOR pathways and markers of decidual function and remodeling in diabetic rats fed or not with diets enriched in PUFAs exclusively during the early postimplantation period. Pregestational streptozotocin-induced diabetic Albino Wistar rats and controls were fed or not with diets enriched in 6% sunflower oil or 6% chia oil (enriched in n-6 or n-3 PUFAs, respectively) on days 7, 8 and 9 of pregnancy and evaluated on day 9 of pregnancy. Maternal diabetes induced an 11-fold increase in embryo resorptions, which was prevented by both PUFAs-enriched diets despite no changes in maternal glycemia. The activity of mTOR pathway was decreased in the decidua from diabetic rats, an alteration prevented by the PUFAs-enriched diets. PUFAs-enriched diets prevented increased expression of Foxo1 (a negative regulator of mTOR) and reduced expression of miR-21 (a negative regulator of Foxo1). These diets also prevented reduced markers of decidual function (leukemia inhibitory factor and IGFBP1 expression and MMPs activity) in diabetic rat decidua. We identified the early post implantation as a crucial stage for pregnancy success, in which dietary PUFAs can protect diabetic pregnancies from embryo resorptions, decidual mTOR signaling impairments, and altered markers of decidual function and remodeling.

Knockout mouse models are predictive of malformations or embryo-fetal death in drug safety evaluations.

Traditionally, understanding potential developmental toxicity from pharmaceutical exposures has been based on the results of ICH guideline studies in two species. However, support is growing for the use of weight of evidence approaches when communicating the risk of developmental toxicity, where the intended pharmacologic mode of action affects fundamental pathways in developmental biology or phenotypic data from genetically modified animals may increasingly be included in the overall assessment. Since some concern surrounds the use of data from knockout (KO) mice to accurately predict the risk for pharmaceutical modulation of a target, a deeper understanding of the relevance and predictivity of adverse developmental effects in KO mice for pharmacological target modulation is needed. To this end, we compared the results of embryo-fetal development (EFD) studies for 86 drugs approved by the FDA from 2017 to 2019 that also had KO mouse data available in the public domain. These comparisons demonstrate that data from KO mouse models are overall highly predictive of malformations or embryo-fetal lethality (MEFL) from EFD studies, but less so of a negative outcome in EFD studies. This information supports the use of embryo-fetal toxicity data in KO models as part of weight of evidence approaches in the communication of developmental toxicity risk of pharmaceutical compounds.

[The intervention effect of N-carbamoyl glutamic acid on embryo implantation disorder induced by carbon disulfide and its possible molecular mechanism].

Objective: To explore the preventive effect and possible molecular mechanism of dietary supplementation of N-carbamylglutamate (NCG) in the implantation of carbon disulfide (CS(2)) into embryo implantation disorders. Methods: embryo implantation disorder model was established by single intraperitoneal exposure to CS(2) on the 3rd, 4th, and 5th days after pregnancy. Endometrial tissues were collected for 24h after exposure to CS(2) for western-blot and immunohistochemical staining. Results: The number of embryo implantation was increased in NCG+CS(2) group, compared with CS(2) alone group. Day 4 of pregnancy when CS(2)-exposed after 24 h, the expression of pAKT protein in NCG+CS(2) group was significantly increased (P<0.05), the expression level of pAMPK protein in NCG+CS(2) group was significantly decreased, compared with CS(2) alone group, respectively. Immunohistochemical results showed that pAKT, pAMPK, AKT and AMPK proteins were expressed in luminal epithelial cells, glandular epithelial cells and stromal cells of endometrium; Day 4 of pregnancy when CS(2)-exposed after 24 h, deep staining of ATK and pAKT protein in NCG+CS(2) group, the AMPK and pAMPK protein staining became lighter. Conclusion: Dietary supplementation of NCG can interfere with the embryo loss induced by CS(2) by altering the total amount of AKT/AMPK molecules.

Safety evaluation of a vaccine: Effect in maternal reproductive outcome and fetal anomaly frequency in rats using a leishmanial vaccine as a model.

While the immunogenic potential of the vaccination against infectious diseases was extensively shown, data on the safety assessment of recombinant proteins in vaccine formulations administered during pregnancy are still scarce. In the current study, the antigenicity of a vaccine against leishmaniasis (based on Leishmania braziliensis recombinant protein peroxidoxin) during pregnancy and possible maternal reproductive outcomes and fetal anomalies after immunization with a leishmanial vaccine or adjuvant alone (Bordetella pertussis derived MPLA adjuvant) were assessed. Rats were mated and allocated in three groups: Control-rats received saline; Adjuvant-rats received the adjuvant MPLA, and Vaccine-rats received the combination of MPLA and peroxidoxin. The administration was subcutaneously at the dorsal region, three times (days 0, 7, 14 of pregnancy). On day 21 of pregnancy, all rats were bled for biochemical and immunological measurements. The gravid uterus was weighed with its contents, and the fetuses were analyzed. The immunization with peroxidoxin induced a significant production of circulating IgG levels compared to other groups but caused a significant in post-implantation loss (14.7%) when compared to Control (5.0%) and Adjuvant (4.4%) groups. Furthermore, a significantly high rate of fetal visceral anomalies, such as hydronephrosis and convoluted ureter, was also observed in animals that received vaccine when compared to Control or Adjuvant groups. These data indicate the importance of safety evaluation of vaccines during pregnancy and the limited use of peroxidoxin administration during pregnancy. More importantly, the safety monitoring of immunization with MPLA derived from Bordetella pertussis demonstrated no reproductive outcomes associated with adjuvant administration, suggesting its safe use during pregnancy.

Identification of anti-cancer chemical compounds using Xenopus embryos.

Cancer tissues have biological characteristics similar to those observed in embryos during development. Many types of cancer cells acquire pro-invasive ability through epithelial-mesenchymal transition (EMT). Similar processes (gastrulation and migration of cranial neural crest cells [CNCC]) are observed in the early stages of embryonic development in Xenopus during which cells that originate from epithelial sheets through EMT migrate to their final destinations. The present study examined Xenopus embryonic tissues to identify anti-cancer compounds that prevent cancer invasion. From the initial test of known anti-cancer drugs, AMD3100 (an inhibitor of CXCR4) and paclitaxel (a cytoskeletal drug targeting microtubules) effectively prevented migration during gastrulation or CNCC development. Blind-screening of 100 synthesized chemical compounds was performed, and nine candidates that inhibited migration of these embryonic tissues without embryonic lethality were selected. Of these, C-157 (an analog of podophyllotoxin) and D-572 (which is an indole alkaroid) prevented cancer cell invasion through disruption of interphase microtubules. In addition, these compounds affected progression of mitotic phase and induced apoptosis of SAS oral cancer cells. SAS tumors were reduced in size after intratumoral injection of C-157, and peritoneal dissemination of melanoma cells and intracranial invasion of glioma cells were inhibited by C-157 and D-572. When the other analogues of these chemicals were compared, those with subtle effect on embryos were not tumor suppressive. These results suggest that a novel chemical-screening approach based on Xenopus embryos is an effective method for isolating anti-cancer drugs and, in particular, targeting cancer cell invasion and proliferation.

Knockdown of hypothalamic RFRP3 prevents chronic stress-induced infertility and embryo resorption.

Whereas it is well established that chronic stress induces female reproductive dysfunction, whether stress negatively impacts fertility and fecundity when applied prior to mating and pregnancy has not been explored. In this study, we show that stress that concludes 4 days prior to mating results in persistent and marked reproductive dysfunction, with fewer successful copulation events, fewer pregnancies in those that successfully mated, and increased embryo resorption. Chronic stress exposure led to elevated expression of the hypothalamic inhibitory peptide, RFamide-related peptide-3 (RFRP3), in regularly cycling females. Remarkably, genetic silencing of RFRP3 during stress using an inducible-targeted shRNA completely alleviates stress-induced infertility in female rats, resulting in mating and pregnancy success rates indistinguishable from non-stress controls. We show that chronic stress has long-term effects on pregnancy success, even post-stressor, that are mediated by RFRP3. This points to RFRP3 as a potential clinically relevant single target for stress-induced infertility.

Molecular mechanism of Hoxd13-mediated congenital malformations in rat embryos.

OBJECTIVE: To investigate the molecular mechanism of Hoxd13-mediated congenital malformations in rat embryos. METHODS: SD female rats were mated with male rats in a 1:1 mating scheme. Thirty pregnant female rats were randomly divided into three groups: the control group receiving a normal diet, the model group receiving a vitamin A-deficient diet, and the treatment group receiving a vitamin A-deficient diet supplemented with pcDNA-Hoxd13. The expression of Hoxd13 mRNA and protein in normal embryonic tissue and congenital malformations was determined by RT-PCR and Western blot analysis. At day 20, rats were dissected, and the fetal weight, body and tail length, and the number of live births, absorbed fetus, and stillbirth in each group were recorded. Wnt and Slim1 expression was detected by RT-PCR and Western blot analysis. ß-catenin and c-myc expression was also quantified by Western blot analysis. RESULTS: The expression of Hoxd13 mRNA and protein in congenital malformations was significantly lower compared with normal embryonic tissue (P<0.01). The administration of exogenous Hoxd13 in the treatment group markedly increased the fetal weight, body and tail length (P<0.05), improved the embryonic survival rate, and reduced the embryonic resorption rate and stillbirth rate (P<0.05). Exogenous Hoxd13 markedly promoted the expression of Wnt2, Wnt5a, Wnt7b and Slim1 protein and mRNA (P<0.01), and the expression of ß-catenin and c-myc protein in congenital malformations (P<0.01). CONCLUSION: Hoxd13 expression was decreased in rat embryos with congenital malformations. The administration of exogenous Hoxd13 alleviated fetal malformation probably through stimulation of Slim1 expression and Wnt/ß-catenin signaling pathway.

Clinical observations of sequential therapy with Chinese medicine and hysteroscopic mechanical stimulation of the endometrium in infertile patients with repeated implantation failure undergoing frozen-thawed embryo transfer.

OBJECTIVES: To investigate the clinical effect of sequential therapeutic intervention Yupei Qisun [compensating for weakness by invigorating Kidney (Shen) and Spleen (Pi) in advance] in Chinese medicine (CM) and hysteroscopic endometrial mechanical stimulation on the treatment of infertile patients with repeated implantation failure (RIF); and to study the differences in patients' endometrial thickness and type on the day of embryo transfer, serum hormone levels on embryo transfer day and clinical pregnancy outcomes. METHODS: In the clinical study, 168 frozen-thawed embryo transfer (FET) cycles for couples with RIF conforming to the research protocol were randomly divided into three groups: a CM group with 56 cycles (CM combined with FET), a hysteroscopy group with 55 cycles (hysteroscopic endometrial mechanical stimulation), and a control group with 57 cycles (conventional FET). Differences in endometrial thickness on the embryo transfer day, levels of serum estradiol (E2) and progesterone (P) on the embryo transfer day, the E2/P ratio on the embryo transfer day, biochemical and clinical pregnancy rates, implantation rate, abnormal pregnancy rate and other indices were compared among the three groups. RESULTS: Endometrial thickness, E2 and P levels, and the E2/P ratio on embryo transfer day and other factors had no significant differences among groups. The biochemical pregnancy, clinical pregnancy, and implantation rates of the CM and hysteroscopy groups were significantly higher than the control group (P<0.05), and there were no significant differences between these two groups. The abnormal pregnancy rate had no significant difference among the three groups. CONCLUSIONS: Sequential therapy of Yupei Qisun could significantly improve the clinical outcomes of rif-fet cycles, being equivalent to hysteroscopic endometrial mechanical stimulation, and provided a reliable method to treat such infertile couples.

Clinical observations of sequential therapy with Chinese medicine and hysteroscopic mechanical stimulation of the endometrium in infertile patients with repeated implantation failure undergoing frozen-thawed embryo transfer / 中国结合医学杂志

<p><b>OBJECTIVES</b>To investigate the clinical effect of sequential therapeutic intervention Yupei Qisun [compensating for weakness by invigorating Kidney (Shen) and Spleen (Pi) in advance] in Chinese medicine (CM) and hysteroscopic endometrial mechanical stimulation on the treatment of infertile patients with repeated implantation failure (RIF); and to study the differences in patients' endometrial thickness and type on the day of embryo transfer, serum hormone levels on embryo transfer day and clinical pregnancy outcomes.</p><p><b>METHODS</b>In the clinical study, 168 frozen-thawed embryo transfer (FET) cycles for couples with RIF conforming to the research protocol were randomly divided into three groups: a CM group with 56 cycles (CM combined with FET), a hysteroscopy group with 55 cycles (hysteroscopic endometrial mechanical stimulation), and a control group with 57 cycles (conventional FET). Differences in endometrial thickness on the embryo transfer day, levels of serum estradiol (E2) and progesterone (P) on the embryo transfer day, the E2/P ratio on the embryo transfer day, biochemical and clinical pregnancy rates, implantation rate, abnormal pregnancy rate and other indices were compared among the three groups.</p><p><b>RESULTS</b>Endometrial thickness, E2 and P levels, and the E2/P ratio on embryo transfer day and other factors had no significant differences among groups. The biochemical pregnancy, clinical pregnancy, and implantation rates of the CM and hysteroscopy groups were significantly higher than the control group (P<0.05), and there were no significant differences between these two groups. The abnormal pregnancy rate had no significant difference among the three groups.</p><p><b>CONCLUSIONS</b>Sequential therapy of Yupei Qisun could significantly improve the clinical outcomes of rif-fet cycles, being equivalent to hysteroscopic endometrial mechanical stimulation, and provided a reliable method to treat such infertile couples.</p>

Does dietary folic acid supplementation in mouse NTD models affect neural tube development or gamete preference at fertilization?

BACKGROUND: Neural tube defects (NTDs) are the second most common birth defect in humans. Dietary folic acid (FA) supplementation effectively and safely reduces the incidence of these often debilitating congenital anomalies. FA plays an established role in folate and homocysteine metabolism, but the means by which it suppresses occurrence of NTDs is not understood. In addition, many cases remain resistant to the beneficial effects of folic acid supplementation. To better understand the molecular, biochemical and developmental mechanisms by which FA exerts its effect on NTDs, characterized mouse models are needed that have a defined genetic basis and known response to dietary supplementation. RESULTS: We examined the effect of FA supplementation, at 5-fold the level in the control diet, on the NTD and vertebral phenotypes in Apobtm1Unc and Vangl2Lp mice, hereafter referred to as Apob and Lp respectively. The FA supplemented diet did not reduce the incidence or severity of NTDs in Apob or Lp mutant homozygotes or the loop-tail phenotype in Lp mutant heterozygotes, suggesting that mice with these mutant alleles are resistant to FA supplementation. Folic acid supplementation also did not affect the rate of resorptions or the size of litters, but instead skewed the embryonic genotype distribution in favor of wild-type alleles. CONCLUSION: Similar genotypic biases have been reported for several NTD models, but were interpreted as diet-induced increases in the incidence and severity of NTDs that led to increased embryonic lethality. Absence of differences in resorption rates and litter sizes argue against induced embryonic lethality. We suggest an alternative interpretation, namely that FA supplementation led to strongly skewed allelic inheritance, perhaps from disturbances in polyamine metabolism that biases fertilization in favor of wild-type gametes.

A nonsense mutation in mouse Tardbp affects TDP43 alternative splicing activity and causes limb-clasping and body tone defects.

Mutations in TARDBP, encoding Tar DNA binding protein-43 (TDP43), cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Attempts to model TDP43 dysfunction in mice have used knockouts or transgenic overexpressors, which have revealed the difficulties of manipulating TDP43, whose level is tightly controlled by auto-regulation. In a complementary approach, to create useful mouse models for the dissection of TDP43 function and pathology, we have identified a nonsense mutation in the endogenous mouse Tardbp gene through screening an N-ethyl-N-nitrosourea (ENU) mutant mouse archive. The mutation is predicted to cause a Q101X truncation in TDP43. We have characterised Tardbp(Q101X) mice to investigate this mutation in perturbing TDP43 biology at endogenous expression levels. We found the Tardbp(Q101X) mutation is homozygous embryonic lethal, highlighting the importance of TDP43 in early development. Heterozygotes (Tardbp(+/Q101X) ) have abnormal levels of mutant transcript, but we find no evidence of the truncated protein and mice have similar full-length TDP43 protein levels as wildtype littermates. Nevertheless, Tardbp(+/Q101X) mice have abnormal alternative splicing of downstream gene targets, and limb-clasp and body tone phenotypes. Thus the nonsense mutation in Tardbp causes a mild loss-of-function phenotype and behavioural assessment suggests underlying neurological abnormalities. Due to the role of TDP43 in ALS, we investigated potential interactions with another known causative gene, mutant superoxide dismutase 1 (SOD1). Tardbp(+/Q101X) mice were crossed with the SOD1(G93Adl) transgenic mouse model of ALS. Behavioural and physiological assessment did not reveal modifying effects on the progression of ALS-like symptoms in the double mutant progeny from this cross. In summary, the Tardbp(Q101X) mutant mice are a useful tool for the dissection of TDP43 protein regulation, effects on splicing, embryonic development and neuromuscular phenotypes. These mice are freely available to the community.

Maternal administration of the herbal medicine toki-shakuyaku-san promotes fetal growth and placental gene expression in normal mice.

Toki-shakuyaku-san (TSS), an herbal formula based on traditional Chinese medicine, is commonly used in obstetrics. To examine the effects of TSS on the normal mouse fetus and placenta, TSS was administered to normal pregnant mice and their placentas and fetuses were studied. First, the effects of maternal TSS treatment on implantation were investigated. Administration of TSS from gestation day 0.5 (G0.5) to G6.5 showed that litter size was not altered at embryonic day 11.5 (E11.5), but the number of resorbed fetuses was slightly decreased. Then, to investigate effects on fetal and placental growths after implantation, TSS was administered from G5.5. At E14.5, the body weight of fetuses from TSS-treated dams was significantly increased. Gene expression of insulin-like growth factor 2 (Igf2), one of the most important modulators of fetal growth, was significantly increased in the placentas and fetuses of TSS-treated dams. In addition, the expression of particular placental developmental genes and nutrient transporter genes was significantly increased in TSS-treated placentas. At E18.5, after longer-term administration of TSS, fetal and placental weights were not altered, but the expression of the placental developmental and nutrient transporter genes remained elevated compared with controls. These results suggest that maternal TSS treatment in normal mice enhances the expression of Igf2, placental developmental genes and nutrient transporter genes, resulting in increased fetal weight. No obvious changes were observed in the expression of these genes after longer-term maternal TSS treatment.

Progesterone is essential for protecting against LPS-induced pregnancy loss. LIF as a potential mediator of the anti-inflammatory effect of progesterone.

Lipopolysaccharide (LPS) administration to mice on day 7 of gestation led to 100% embryonic resorption after 24 h. In this model, nitric oxide is fundamental for the resorption process. Progesterone may be responsible, at least in part, for a Th2 switch in the feto-maternal interface, inducing active immune tolerance against fetal antigens. Th2 cells promote the development of T cells, producing leukemia inhibitory factor (LIF), which seems to be important due to its immunomodulatory action during early pregnancy. Our aim was to evaluate the involvement of progesterone in the mechanism of LPS-induced embryonic resorption, and whether LIF can mediate hormonal action. Using in vivo and in vitro models, we provide evidence that circulating progesterone is an important component of the process by which infection causes embryonic resorption in mice. Also, LIF seems to be a mediator of the progesterone effect under inflammatory conditions. We found that serum progesterone fell to very low levels after 24 h of LPS exposure. Moreover, progesterone supplementation prevented embryonic resorption and LPS-induced increase of uterine nitric oxide levels in vivo. Results show that LPS diminished the expression of the nuclear progesterone receptor in the uterus after 6 and 12 h of treatment. We investigated the expression of LIF in uterine tissue from pregnant mice and found that progesterone up-regulates LIF mRNA expression in vitro. We observed that LIF was able to modulate the levels of nitric oxide induced by LPS in vitro, suggesting that it could be a potential mediator of the inflammatory action of progesterone. Our observations support the view that progesterone plays a critical role in a successful pregnancy as an anti-inflammatory agent, and that it could have possible therapeutic applications in the prevention of early reproductive failure associated with inflammatory disorders.

Marked toxicity of Albizia bernieri extracts on embryo-larval development in the medaka fish (Oryzias latipes).

Previous phytochemical studies have shown that the plants of the Albizia genus (Fabaceae) contain bioactive saponins, lignans, spermine alkaloids, flavonoids, glycosides phenols and pyridoxine derivatives. Their extracts sometimes display medical properties, but can have also toxic effects. The purpose of our study was to determine the in vivo toxicity of Albizia bernieri seeds in the experimental model of the medaka fish embryo, which is recommended for use in toxicity studies. Our results show clearly that incubating the embryos or larvae of the medaka fish in a medium containing A. bernieri extracts caused a dose-dependent reduction in embryo or larvae survival. Embryos exposed to an extract of A. bernieri displayed cerebral lesions, such as cell lysis and the emergence of lysosomes in the glial tissue. We conclude that when comparing with data obtained with different plant extracts tested on medaka development in our laboratory, A. bernieri displays an unusually high toxicity. Focussing on the cerebral target as well as the fish behaviour could bring more specific informations.

Moderately high intake of folic acid has a negative impact on mouse embryonic development.

BACKGROUND: The incidence of neural tube defects has diminished considerably since the implementation of food fortification with folic acid (FA). However, the impact of excess FA intake, particularly during pregnancy, requires investigation. In a recent study, we reported that a diet supplemented with 20-fold higher FA than the recommended intake for rodents had adverse effects on embryonic mouse development at embryonic days (E)10.5 and 14.5. In this report, we examined developmental outcomes in E14.5 embryos after administering a diet supplemented with 10-fold higher FA than recommended to pregnant mice with and without a mild deficiency of methylenetetrahydrofolate reductase (MTHFR). METHODS: Pregnant mice with or without a deficiency in MTHFR were fed a control diet (recommended FA intake of 2 mg/kg diet for rodents) or an FA-supplemented diet (FASD; 10-fold higher than the recommended intake [20 mg/kg diet]). At E14.5, mice were examined for embryonic loss and growth retardation, and hearts were assessed for defects and for ventricular wall thickness. RESULTS: Maternal FA supplementation was associated with embryonic loss, embryonic delays, a higher incidence of ventricular septal defects, and thinner left and right ventricular walls, compared to mothers fed control diet. CONCLUSIONS: Our work suggests that even moderately high levels of FA supplementation may adversely affect fetal mouse development. Additional studies are warranted to evaluate the impact of high folate intake in pregnant women. Birth Defects Research (Part A), 2013. © 2012 Wiley Periodicals, Inc.

Temporal candidate gene expression in the sow placenta and embryo during early gestation and effect of maternal Progenos supplementation on embryonic and placental development.

The present study characterised gene expression associated with embryonic muscle development and placental vascularisation during early gestation in the pig and examined effects of Progenos supplementation in early pregnancy. Tissues were collected from commercial multiparous sows (n = 48) from Days 16 to 49 of gestation. In the placenta, qPCR revealed that vascular endothelial growth factor (VEGFA) expression did not change from Day 17 to 49 of gestation; however, KDR receptor and angiopoietin-1 and -2 expression were differentially regulated, with periods of high expression corresponding to two critical phases of angiogenesis in the pig. In the embryo, the pattern of myogenesis-related gene expression was consistent with available literature. A commercially available nutritional supplement Progenos (20 g day⁻¹ L-arginine) added to the diet of sows from either Day 15 to 29 (P15-29; n = 33), Day 30 to 44 (n = 29) or from Day 15 to 44 (n = 76) of gestation tended to increase (P = 0.058) embryonic growth rate compared with non-supplemented controls (n = 79) and angiogenin expression was higher (P = 0.028) at Day 30 of gestation in placentae from sows on the P15-29 Progenos treatment. These results are consistent with proposed beneficial effects of l-arginine on early embryonic development and placental vascularisation.

Maternal Mthfd1 disruption impairs fetal growth but does not cause neural tube defects in mice.

BACKGROUND: MTHFD1 encodes C1-tetrahydrofolate synthase, which is a folate-dependent enzyme that catalyzes the formation and interconversion of folate-activated one-carbon groups for nucleotide biosynthesis and cellular methylation. A polymorphism in MTHFD1 (1958G→A) impairs enzymatic activity and is associated with increased risk of adverse pregnancy outcomes, but the mechanisms are unknown. OBJECTIVE: The objective of this study was to determine whether disruption of the embryonic or maternal Mthfd1 gene or both interacts with impaired folate and choline status to affect neural tube closure, fetal growth, and fertility in mice and to investigate the underlying metabolic disruptions. DESIGN: Dams with a gene-trapped (gt) allele in Mthfd1 and wild-type dams were fed a control or folate- and choline-deficient AIN93G diet (Dyets Inc). Litters were examined for gross morphologic defects, crown-rump length, and resorptions. Folate status and amounts of folate-related metabolites were determined in pregnant dams. RESULTS: Reduced folate and choline status resulted in severe fetal growth restriction (FGR) and impaired fertility in litters harvested from Mthfd1(gt/+) dams, but embryonic Mthfd1(gt/+) genotype did not affect fetal growth. Gestational supplementation of Mthfd1(gt/+) dams with hypoxanthine increased FGR frequency and caused occasional neural tube defects (NTDs) in Mthfd1(gt/+) embryos. Mthfd1(gt/+) dams exhibited lower red blood cell folate and plasma methionine concentrations than did wild-type dams. CONCLUSIONS: Maternal Mthfd1(gt/+) genotype impairs fetal growth but does not cause NTDs when dams are maintained on a folate- and choline-deficient diet. Mthfd1(gt/+) mice exhibit a spectrum of adverse reproductive outcomes previously attributed to the human MTHFD1 1958G→A polymorphism. Mthfd1 heterozygosity impairs folate status in pregnant mice but does not significantly affect homocysteine metabolism.