Embryo Resorptions Research Articles

Toxicity and Teratogenic Potential of Piplartine from Piper tuberculatum Jacq. during Embryonic Development in Mice (Mus musculus)

Piplartine, also known as piperlongumine, is a natural and biologically active amide alkaloid found in various Piper species within the Piperaceae family. It possesses numerous beneficial properties that can be leveraged in the development of nanotechnological and pharmaceutical products. However, information on the effects of piplartine on mammalian embryonic development is scarce. This study aims to assess the general toxicity and teratogenic potential of piplartine during the embryonic development of mice. Pregnant mice received daily treatments of 25, 50, or 100 mg/kg of piplartine via gavage from the sixth day of gestation (implantation) to the eighteenth. On the eighteenth day, the mice were euthanized, and whole organs, blood samples (for hematological and biochemical analyses), and bone marrow cells (for DNA fragmentation and cell cycle assays) were collected. The uterus was examined for implantation sites and embryo resorptions. Additionally, fetuses were collected to assess for fetal anomalies. Piplartine did not result in maternal or embryo-fetal toxicity, induce fetal anomalies, cause hematological and biochemical alterations, or lead to DNA fragmentation. The oral administration of piplartine is safe and does not exhibit toxicity or teratogenic effects in mice. This finding opens avenues for the development of piplartine-based biotechnological products for therapeutic interventions in disease treatment.

Development of peptides for targeting cell ablation agents concurrently to the Sertoli and Leydig cell populations of the testes: An approach to non-surgical sterilization.

The surgical sterilization of cats and dogs has been used to prevent their unwanted breeding for decades. However, this is an expensive and invasive procedure, and often impractical in wider contexts, for example the control of feral populations. A sterilization agent that could be administered in a single injection, would not only eliminate the risks imposed by surgery but also be a much more cost-effective solution to this worldwide problem. In this study, we sought to develop a targeting peptide that would selectively bind to Leydig cells of the testes. Subsequently, after covalently attaching a cell ablation agent, Auristatin, to this peptide we aimed to apply this conjugated product (LH2Auristatin) to adult male mice in vivo, both alone and together with a previously developed Sertoli cell targeting peptide (FSH2Menadione). The application of LH2Auristatin alone resulted in an increase in sperm DNA damage, reduced mean testes weights and mean seminiferous tubule size, along with extensive germ cell apoptosis and a reduction in litter sizes. Together with FSH2Menadione there was also an increase in embryo resorptions. These promising results were observed in around a third of all treated animals. Given this variability, we discuss how these reagents might be modified in order to increase target cell ablation and improve their efficacy as sterilization agents.

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.

P7 La adición de ácidos grasos poliinsaturados en el período de postimplantación temprana previene reabsorciones embrionarias y anomalías deciduales en ratas gestantes con diabetes

Introducción: la diabetes pregestacional incrementa el riesgo de reabsorciones embrionarias y afecta el desarrollo embrionario, de impacto en la programación fetal. La función decidual es crucial en el desarrollo postimplantatorio temprano y sus posibles anomalías son poco conocidas en la diabetes materna. Previamente evidenciamos que la vía de señalización de mTOR en la decidua es relevante para el crecimiento y desarrollo embrionario, y que interactúa con la vía de los PPAR, cuyos agonistas son nutrientes de naturaleza lipídica como los ácidos grasos poliinsaturados (PUFAs).Objetivos: determinar el índice de reabsorciones y evaluar la regulación de la vía de mTOR y marcadores de la función, y remodelación de la decidua en ratas con diabetes pregestacional que recibieron o no dietas enriquecidas en PUFAs exclusivamente en el período de postimplantación temprana.Materiales y métodos: ratas sanas o con diabetes (inducidas preconcepcionalmente con estreptozotocina, 55 mg/kg) fueron apareadas con machos sanos. En los días 7,8 y 9 de preñez recibieron una dieta enriquecida o no en 6% aceite de girasol o de chía (enriquecidas en PUFAs n-6 o n-3, respectivamente). En el día 9 de preñez se estudió el índice de reabsorción embrionaria y se explantó la decidua para estudiar la vía de mTOR, la expresión de su regulador FoxO1 y del microRNA-21 (miR-21) que regula a FoxO1, como así también marcadores de función y remodelación decidual (expresión de LIF y IGFBP1 y actividad gelatinasa de metaloproteasas (MMPs).

Spontaneous embryo resorption in the mouse is triggered by embryonic apoptosis followed by rapid removal via maternal sterile purulent inflammation

BackgroundIn normal mammalian development a high percentage of implantations is lost by spontaneous resorption. This is a major problem in assisted reproduction and blastocyst transfer. Which embryo will be resorbed is unpredictable. Resorption is very fast, so that with conventional methods only final haemorrhagic stages are encountered.Here we describe the histology and immunohistochemistry of 23 spontaneous embryo resorptions between days 7 and 13 of murine development, which were identified by high-resolution ultrasound (US) in a previous study.ResultsIn the early resorptions detected at day 7, the embryo proper was replaced by maternal haemorrhage and a suppurate focus of maternal neutrophils. In the decidua maternal macrophages transformed to foam cells and formed a second focus of tissue dissolution.In the late resorptions detected at day 9, the embryo underwent apoptosis without involvement of maternal cells. The apoptotic embryonic cells expressed caspase 3 and embryonic blood cells developed a macrophage like phenotype. Subsequently, the wall of the embryonic vesicle ruptured and the apoptotic embryo was aborted into the uterine lumen. Abortion was initiated by degeneration of the embryonic lacunar trophoblast and dissolution of the maternal decidua capsularis via sterile inflammation and accompanied by maternal haemorrhage, invasion of the apoptotic embryo by maternal neutrophils, and contraction rings of the uterine muscle layers.ConclusionsWe conclude that spontaneous resorption starts with endogenous apoptosis of the embryo without maternal contribution. After break down of the foetal-maternal border, the apoptotic embryo is invaded by maternal neutrophils, aborted into the uterine lumen, and rapidly resorbed. We assume that the innate maternal unspecific inflammation is elicited by disintegrating apoptotic embryonic cells.Graphical abstract

206. Identification of new candidate proteins involved in preeclampsia

Preeclampsia is a major disease of pregnancy, affecting 2–8% of pregnant women. It is characterized by arterial hypertension and proteinuria occurring from the 20th week of amenorrhea. The STOX1 overexpressing mice develop a preeclamptic syndrome. This model is very relevant to identify potential new actors in preeclampsia because the phenotype is both precocious and severe. Using this mouse model of severe PE, we aim to identify plasma proteins quantitatively modified during pregnancy with a global proteomics approach in the blood and to validate these proteins on human placental cell models. We provoked preeclamptic gestations in mice by crossing WT females with transgenic STOX1 males. Blood was taken at E6.5 of gestation and plasma was analyzed using ITRAQ mass spectrometry to identify proteins of differential abundance. In parallel, we developed BeWo cells stably overexpressing STOX1. BeWo is a choriocarcinoma cell line in which fusion is induced by forskolin treatment and reproduces therefore the physiology of villous trophoblast that syncytialize into syncytiotrophoblast. We are validating the link between STOX1 overexpression and sLIFR production by qRT-PCR and ELISA in this cell model. We identified 15 proteins differentially present, two of which have human homologs: sLIFR (soluble Leukemia Inhibitory Factor Receptor) and TTR (transthyretin) respectively increased and decreased in the preeclamptic mice. In the BeWo cells, after fusion or in the presence of STOX1, alternative splicing of LIFR mRNA increases, leading to more secretion of sLIFR in the supernatant. An early excess of sLIFR could trap Leukemia Inhibitory Factor (LIF) at early stages of embryo implantation and development. Given the primordial role of LIF in implantation in mice and humans, this could lead to a defective implantation and/or placentation possibly causing embryo resorptions or a preeclamptic phenotype.

Obesity and High-Fat Diet Induce Distinct Changes in Placental Gene Expression and Pregnancy Outcome.

Obese women are at high risk of pregnancy complications, including preeclampsia, miscarriage, preterm birth, stillbirth, and neonatal death. In the current study, we aimed to determine the effects of obesity on pregnancy outcome and placental gene expression in preclinical mouse models of genetic and nutritional obesity. The leptin receptor (LepR) null-reactivatable (LepRloxTB), LepR-deficient (Leprdb/+), and high-fat diet (HFD)-fed mice were assessed for fertility, pregnancy outcome, placental morphology, and placental transcriptome using standard quantitative polymerase chain reaction (qPCR) and qPCR arrays. The restoration of fertility of LepRloxTB was performed by stereotaxic delivery of adeno-associated virus-Cre into the hypothalamic ventral premammillary nucleus. Fertile LepRloxTB females were morbidly obese, whereas the wild-type mice-fed HFD showed only a mild increase in body weight. Approximately 80% of the LepRloxTB females had embryo resorptions (∼40% of the embryos). In HFD mice, the number of resorptions was not different from controls fed a regular diet. Placentas of resorbed embryos from obese mice displayed necrosis and inflammatory infiltrate in the labyrinth and changes in the expression of genes associated with angiogenesis and inflammation (e.g., Vegfa, Hif1a, Nfkbia, Tlr3, Tlr4). In contrast, placentas from embryos of females on HFD showed changes in a different set of genes, mostly associated with cellular growth and response to stress (e.g., Plg, Ang, Igf1, Igfbp1, Fgf2, Tgfb2, Serpinf1). Sexual dimorphism in gene expression was only apparent in placentas from obese LepRloxTB mice. Our findings indicate that an obese environment and HFD have distinct effects on pregnancy outcome and the placental transcriptome.

Epididymal CYP2E1 plays a critical role in acrylamide-induced DNA damage in spermatozoa and paternally mediated embryonic resorptions†.

Acrylamide is a ubiquitous toxicant in human lives, due to its formation in many food products. Acrylamide induces dominant lethal mutations with administration of 25 mg/kg bw/day for 5 days in male mice. Cytochrome P450, family 2, subfamily E, polypeptide 1 (CYP2E1) is responsible for this dominant lethality. CYP2E1 is the only enzyme responsible for the conversion of acrylamide to the highly reactive metabolite glycidamide, which forms adducts with DNA. CYP2E1 is present predominantly in the liver, as well as the brain, kidney, intestines, and spleen. Within the male mouse reproductive tract, CYP2E1 localizes to spermatocytes. However, embryo resorptions have been demonstrated to occur only with exposure of the late stages of spermatogenesis and spermatozoa. It was determined that CYP2E1 is additionally expressed within the mouse epididymal epithelium, and this localization is responsible for acrylamide-induced dominant lethality. Further, an equivalent profile of CYP2E1 expression was identified in the human reproductive tract. While spermatozoa of both species were also established to possess CYP2E1, this did not contribute to acrylamide-induced DNA damage. In vitro studies strengthened these findings further, revealing that acrylamide exposure only induces DNA damage in human and mouse spermatozoa following metabolism by the mouse epididymal epithelial cell line (mECap18) to glycidamide. These findings emphasize, for the first time, the vital role of the epididymis in the reproductive toxicity associated with acute acrylamide exposure.

Folic acid and safflower oil supplementation interacts and protects embryos from maternal diabetes-induced damage

Maternal diabetes increases the risk of embryo malformations. Folic acid and safflower oil supplementations have been shown to reduce embryo malformations in experimental models of diabetes. In this study we here tested whether folic acid and safflower oil supplementations interact to prevent embryo malformations in diabetic rats, and analyzed whether they act through the regulation of matrix metalloproteinases (MMPs), their endogenous inhibitors (TIMPs), and nitric oxide (NO) and reactive oxygen species production. Diabetes was induced by streptozotocin administration prior to mating. From Day 0.5 of pregnancy, rats did or did not receive folic acid (15 mg/kg) and/or a 6% safflower oil-supplemented diet. Embryos and decidua were explanted on Day 10.5 of gestation for further analysis of embryo resorptions and malformations, MMP-2 and MMP-9 activities, TIMP-1 and TIMP-2 levels, NO production and lipid peroxidation. Maternal diabetes induced resorptions and malformations that were prevented by folic acid and safflower oil supplementation. MMP-2 and MMP-9 activities were increased in embryos and decidua from diabetic rats and decreased with safflower oil and folic acid supplementations. In diabetic animals, the embryonic and decidual TIMPs were increased mainly with safflower oil supplementation in decidua and with folic acid in embryos. NO overproduction was decreased in decidua from diabetic rats treated with folic acid alone and in combination with safflower oil. These treatments also prevented increases in embryonic and decidual lipid peroxidation. In conclusion, folic acid and safflower oil supplementations interact and protect the embryos from diabetes-induced damage through several pathways related to a decrease in pro-inflammatory mediators.

Two generation reproduction and teratogenicity studies of feeding cyadox in Wistar rats

To investigate the teratogenic potential and reproductive toxicity of cyadox, a growth promoting agent, Wistar rats (F0) were fed with diets containing cyadox (0, 50, 150 and 2500mg/kg) or olaquindox (150mg/kg), approximately equivalent to cyadox 5, 15, 250 or olaquindox 15mg/kgb.w./day across two generations. Half of the pregnant rats (F0, F1b) were subjected to caesarean section on gestational day 20 for teratogenic examination and the other half produced pups F1a and F2a, respectively. At the 250mg/kgb.w./day cyadox group, body weights of F1b pregnant rats and F2a on day 21 after birth decreased; fetal body lengths and tail lengths decreased; the number of fetal resorptions increased significantly; litter weights, number of viable fetuses decreased; number of embryo resorptions increased significantly; number of liveborn F1a, F1b and F2a decreased. No macroscopic or microscopic change of any significance was found in the reproductive organs. Significant increases in the incidence of cervial ribs or lumbar ribs in F2a pups and significant increases of relative organ weight of testis and epididymis in F1b were observed at the 250mg/kgb.w./day cyadox group. The NOAEL for reproduction/development of cyadox for rats was estimated to be 150mg/kg diet, which was equivalent to approximately 15mg/kgb.w./day.

The Effect of β-Carotene Against Adriamycin Toxicity on the Embryo Formation

Adriamycin is an anthracycline antibiotic widely used for the treatment of many types of cancer. The cytotoxic effect of Adriamycin occurs by a free radical-mediated mechanism. Thus, to prevent or reduce the toxic effect of Adriamycin, it is possible to use it in combination with antioxidants. The aim of this study was to evaluate a potential effect of β-carotene against Adriamycin-induced toxicity on the embryo formation. Pregnant rats were treated with Adriamycin, β-carotene, and their combination during the critical stages of embryogenesis. The first group was control group. Adriamycin was administered on day 9 (group 2a) and day 12 (group 2b) of gestation by a single intraperitoneal injection at a dose of 5 mg/kg. β-Carotene was given at a dosage of 0.6 mg/(kg·day) from day 6 to 10 or from day 9 (group 3a) to 13 (group 3b) of gestation 5 times per os; in the case of their combination, β-carotene was given per os 3 times before Adriamycin injection, one time simultaneously with Adriamycin and one time after its injection (groups 4a and 4b). Animals were euthanized on day 21 of gestation. Embryo resorptions and alive fetuses were counted, weighed, and measured. The embryos of each litter were examined macroscopically after the Buen solution fixation for the embryo defects. In order to render the skeleton visible, the soft tissues were macerated using caustic soda, stained with alizarin red, and cleared with glycerin. Adriamycin induced embryotoxicity; the combination of Adriamycin and β-carotene decreased the number of Adriamycin-induced embryo resorptions about two times. A gavage with Adriamycin alone decreased fetal body weights (P<0.05), while giving it in combination, the fetal body weight was similar to that in the control group. Adriamycin induced the retardation of skeletogenesis and external fetal malformations (microphthalmia, hydrocephaly, anencephaly, and others). After an exposure to β-carotene, external malformations (diaphragmatic hernia) of embryos were found only occasionally. β-Carotene in combination with Adriamycin produced no positive effect on Adriamycin-induced skeletodysgenesis or external malformations. Antioxidant β-carotene in combination with Adriamycin slightly reduced the Adriamycin-induced embryotoxicity, but produced no positive effect on Adriamycin-induced skeleto-dysgenesis or external malformations.

Embryo development alteration in rats treated with lapachol

Lapachol, a naphthoquinone extracted from plants of the genus Tabebuia (family Bignoneaceae), showed multiple therapeutic activities. Pregnant Wistar rats were treated with Lapachol from the 1st to the 4th (pre-implantation period) and from 5th to 7th (implantation period) post insemination day (PID). Mothers were sacrificed on the 5th or on the15th PID. Number of corpora lutea, preimplantation embryo, blastocysts, live and dead fetuses and resorptions were counted. There were no signs of maternal toxicity. The number and the morphology of embryos, during oviduct development (pre-implantation period), did not seem to be affected by this drug, but during the implantation period, lapachol was toxic causing the death of embryos and intrauterine growth retardation.

Cerebellar anomalies in congenital murine toxoplasmosis.

Gravid Nya:NYLAR mice, infected with Toxoplasma gondii on gestation day 7, experienced embryo resorptions, abortions, stillbirths, and a reduction in average litter size by one-third. Postnatally, all congenitally infected pups showed growth retardation, cachexia, and hind limb weakness. Some pups developed necrotic petechiae on the ears and tail, and a blood-tinged nasal discharge. Coronal sections of the cerebellum at age 1 month revealed developmental abnormalities including: persistence of remnants of an external granular layer; fragmented and disoriented Bergmann glial foot processes; numerous ectopic granule cells stranded in the molecular layer; focal disorganization and edema of the Purkinje cell layer; and thinning of the internal granular layer. Our working hypothesis is that the cerebellar anomalies originated with parasite invasion of the fetal vascular endothelium leading to vasculitis and microcirculatory dysfunction, perivascular edema, perfusion impairment, and tissue anoxia. In the cerebellar folia, the cellular migration defects are attributed to edema-induced swelling and fragmentation of the Bergmann glial foot processes that guide migrating neurons, whereas the focal loss of Purkinje and granule cells is ascribed to hypoxia-ischemia. Although Toxoplasma cysts were detected in the cerebellum, morphologic evidence of parasite association with neuropathology was not obtained.

Outcome of triplet pregnancies after assisted reproductive techniques: How frequent are the vanishing embryos?

To assess the incidence of spontaneous embryo reduction as well as the obstetric and neonatal outcome of triplet gestations after assisted reproductive techniques (ART). We analyzed the spontaneous outcome of 38 pregnancies in which three gestational sacs were identified with vaginal ultrasound between 21 and 28 days after ART. Weekly follow-up visits were scheduled during the first trimester until referral to a high-risk obstetrician. After delivery, each patient was interviewed individually and, if necessary, the obstetrician was contacted. The triplets delivery rate was 47.4%, whereas 31.6% delivered twins, 18.4% delivered singletons, and only one patient miscarried all three cases (2.6%). Finding three fetal heart beats was associated with a triplet delivery rate of 69.2%, a twin incidence of 19.2%, and a singleton birth rate of 11.6%. Embryo resorptions were observed mainly during the first 7 weeks of gestation and did not occur beyond the 14th week. The mean gestational age at delivery and neonatal birth weight were significantly lower among triplets (32.8 weeks and 1,740 g versus 35.3 weeks and 2,352 g in twins and 39.1 weeks and 3,122 g for singletons). Triplets had a 100% prematurity and cesarean section rate compared with 67% and 75% in twins and 0% and 43% in singletons, respectively. Hospitalization at the Neonatal Intensive Care Unit was required in 83% of newborn triplets, 29% of twins, and 0% of singletons, with a mean stay of 34 and 21 days for triplets and twins, respectively. One stillbirth and no neonatal deaths were reported, with an overall perinatal mortality rate of 11.9 per 1,000. Spontaneously, approximately 50% of triplet pregnancies will experience at least one embryo resorption. The ongoing triplets demand a complex and more expensive perinatal management, a strong argument to consider limiting the number of oocytes-embryos transferred in ART.

Interactions between caffeine and adenosine agonists in producing embryo resorptions and malformations in mice

Caffeine has previously been demonstrated to be teratogenic in mice, causing primarily limb reduction defects and cleft palate. To investigate the possibility that the mechanism of caffeine teratogenesis is related to its central stimulant activity, which is thought to result from its activity as an adenosine antagonist, the effects of the stable adenosine agonists l-phenylisopropyladenosine ( l-PIA) and chloroadenosine on caffeine teratogenicity were examined. It was found that these adenosine analogs have embryolethal effects when administered intraperitoneally on Days 11 and 12 of gestation at extremely low dosages (minimal effect levels of 1 and 10 μmol/kg, respectively). Caffeine at dosages as low as 129 μmol/kg ip protected against the embryolethal effects of 5 μmol/kg l-PIA if administered simultaneously with l-PIA but not if administered 30 to 60 min after l-PIA. The maternotoxicity of either of the adenosine agonists or caffeine alone was ameliorated by coadministration of the other, particularly when the molar excess of caffeine was approximately 20-fold. These results suggest that the embryotoxicity and maternotoxicity of the adenosine agonists and the maternotoxicity of caffeine are mediated by binding to adenosine receptors. When coadministered with teratogenic dosages of caffeine (1160 to 1290 μmol/kg), l-PIA and chloroadenosine at dosage levels of 1 to 100 μmol/kg did not prevent and in some cases potentiated the teratogenic effects of caffeine. This lack of protection by l-PIA and chloroadenosine suggests that adenosine receptors are not the primary site of action for caffeine-induced teratogenicity. The embryolethal effects of l-PIA and chloroadenosine and the teratogenicity of caffeine occurred only at maternotoxic dosages. However, an analysis of the correlation structure indicates that the embryotoxicities of l-PIA and caffeine were not secondary to maternotoxicity as measured by body weight change.