Maternal exposure to an enrichment environment promotes uterine vascular remodeling and prevents embryo loss in mice.

Aim: Implantation-related events are crucial for pregnancy success. In particular, defects in vascular remodeling at the maternal-fetal interface are associated with spontaneous miscarriage and recurrent pregnancy loss. Physical activity and therapies oriented to reduce stress improve pregnancy outcomes. In animal models, environmental stimulation and enrichment are associated with enhanced well-being, cognitive function and stress resilience. Here we studied whether exposure of BALB/c mice to an enriched environment (EE) regulates crucial events during early gestation at the maternal-fetal interface. Method: Pregnant BALB/c mice were exposed to the EE that combines non-invasive stimuli from the sensory pathway with voluntary physical activity. The pregnancy rate was evaluated. Implantation sites were investigated microscopically and macroscopically. Vascular adaptation parameters at the maternal-fetal interface were analyzed. Results: We found that exposure to the EE prevented pregnancy loss between gestational days 7 and 15. Also, it increased the diameter of the uterine artery and decreased the wall:lumen ratio of the mesometrial decidual vessels, suggesting that EE exposure promotes vascular remodeling. Moreover, it increased nitric oxide synthase activity and inducible nitric oxide synthase expression, as well as prostaglandin F2α production and endoglin expression in the implantation sites. Conclusion: Exposure of pregnant females to the EE regulates uterine physiology, promoting vascular remodeling during early gestation. These adaptations might contribute to preventing embryo loss. Our results highlight the importance of the maternal environment for pregnancy success. The design of an “EE-like” protocol for humans could be considered as a new non-pharmacologic strategy to prevent implantation failure and recurrent miscarriage.

Implantation failure and embryo loss contribute to subfertility in female mice mutant for chromatin remodeler Cecr2†

Defects in the maternal reproductive system that result in early pregnancy loss are important causes of human female infertility. A wide variety of biological processes are involved in implantation and establishment of a successful pregnancy. While chromatin remodelers have been shown to play an important role in many biological processes, our understanding of the role of chromatin remodelers in female reproduction remains limited. Here, we demonstrate that female mice mutant for chromatin remodeler Cecr2 are subfertile, with defects detected at the peri-implantation stage or early pregnancy. Using both a less severe hypomorphic mutation (Cecr2GT) and a more severe presumptive null mutation (Cecr2Del) we demonstrate a clear difference in the severity of the phenotype depending on the mutation. While neither strain shows detectable defects in folliculogenesis, both Cecr2GT/GT and Cecr2GT/Del dams show defects in pregnancy. Cecr2GT/GT females have a normal number of implantation sites at E5.5, but significant embryo loss by E10.5 accompanied by the presence of vaginal blood. Cecr2GT/Del females show a more severe phenotype, with significantly fewer detectable implantation sites than wild type at E5.5. Some Cecr2GT/Del females also show premature loss of decidual tissue after artificial decidualization. Together, these results suggest a role for Cecr2 in the establishment of a successful pregnancy.

High-fat diet-induced and genetically inherited obesity differentially alters DNA methylation profile in the germline of adult male rats

Background Paternal obesity has been associated with reduced live birth rates. It could lead to inheritance of metabolic disturbances to the offspring through epigenetic mechanisms. However, obesity is a multifactorial disorder with genetic or environmental causes. Earlier we had demonstrated differential effects of high-fat diet-induced obesity (DIO) and genetically inherited obesity (GIO) on metabolic, hormonal profile, male fertility, and spermatogenesis using two rat models. The present study aimed to understand the effect of DIO and GIO on DNA methylation in male germline, and its subsequent effects on the resorbed (post-implantation embryo loss) and normal embryos. First, we assessed the DNA methylation enzymatic machinery in the testis by Real-Time PCR, followed global DNA methylation levels in spermatozoa and testicular cells by ELISA and flow cytometry, respectively. Further, we performed Methylation Sequencing in spermatozoa for both the groups. Sequencing data in spermatozoa from both the groups were validated using Pyrosequencing. Expression of the differentially methylated genes was assessed in the resorbed and normal embryos sired by the DIO group using Real-Time PCR for functional validation. Results We noted a significant decrease in Dnmt transcript and global DNA methylation levels in the DIO group and an increase in the GIO group. Sequencing analysis showed 16,966 and 9113 differentially methylated regions in the spermatozoa of the DIO and GIO groups, respectively. Upon pathway analysis, we observed genes enriched in pathways involved in embryo growth and development namely Wnt, Hedgehog, TGF-beta, and Notch in spermatozoa for both the groups, the methylation status of which partially correlated with the gene expression pattern in resorbed and normal embryos sired by the DIO group. Conclusion Our study reports the mechanism by which diet-induced and genetically inherited obesity causes differential effects on the DNA methylation in the male germline that could be due to a difference in the white adipose tissue accumulation. These differences could either lead to embryo loss or transmit obesity-related traits to the offspring in adult life.

374 Early embryonic loss is an important mechanism for maximizing litter size in the pig

Litter size of commercial dam lines of pigs has seen a significant increase over the last 20 years. Although the current large litter size of gilts and sows can be attributed to improved genetic selection and nutrition, much of the increase in total number born has occurred through the continued increase in ovulation rate over the last four decades. Ovulation rate has increased from 14 in the 80’s to today’s average of 20–25 in commercial dam lines. However, high ovulation rate not only causes increased competition between embryos (uterine crowding), but the number of potential piglets born can out strip the number available teats. In addition, fetal crowding can reduce fetal growth and comprise health and survivability of lighter weight piglets at birth. Since there is usually minimal embryo loss observed up to blastocyst formation and hatching, the majority (20 to 30%) of embryonic loss occurs during Days 10 to 30 of gestation, which encompasses a critical period of conceptus development and differentiation, pregnancy recognition signaling, and onset of implantation. This period of early embryo loss is actually essential for establishing adequate uterine space to support fetal survival for a large litter. The period of rapid conceptus elongation on Day 12 of pregnancy provides a key “selection point” for regulating potential litter size in the pig. Establishing the fundamental roles of conceptus- and uterine-secreted factors is an essential step to develop strategies to increase placental and fetal growth to improve overall health and survivability of piglets before and after birth. During the peri-implantation period of pregnancy, porcine conceptuses produce interleukin-1B2, estrogens, prostaglandins and interferons. CRISPR/Cas9 genomic engineering technology has provided a direct method to evaluate the role of key pig conceptus genes. Loss-of-function studies have increased our understanding of the multiplicity of uterine/conceptus factors that are involved with maintenance of pregnancy.

Adiponectin Reduces Embryonic Loss Rate and Ameliorates Trophoblast Apoptosis in Early Pregnancy of Mice with Polycystic Ovary Syndrome by Affecting the AMPK/PI3K/Akt/FoxO3a Signaling Pathway

Reports in recent years have suggested that adiponectin (APN) improves insulin resistance and inhibits apoptosis by activating the AMP-activated protein kinase (AMPK) pathway and the PI3K/Akt signaling pathway after binding to its receptor. This study aims to explore the mechanism by which APN reduces embryo loss rate and trophoblast apoptosis in early pregnancy of mice with polycystic ovary syndrome (PCOS). PCOS mice were subcutaneously injected with APN (10 μg mg kg−1 day−1) on 11 consecutive days from the 3rd day of pregnancy onwards to observe the change of the embryo loss rate of PCOS mice induced by APN. Quantitative real-time PCR and Western blot were used to determine the relative expressions of mRNA and the proteins AMPK, PI3K, and Akt in mouse uterine tissue. At the same time, primary cultured mouse villous trophoblast cells were used to further explore the underlying mechanisms in vitro. APN significantly reduces the pregnancy loss rate of PCOS mice. At the same time, APN increases phosphorylation and mRNA expression levels of AMPK, PI3K, and Akt in PCOS mouse uterine tissue. In addition, trophoblast cells of model mice were treated with APN and inhibitors, and APN was found to reduce trophoblast cell apoptosis by affecting the phosphorylation levels of AMPK, PI3K, Akt, and FoxO3a proteins. APN reduces the embryo loss rate and ameliorates trophoblast apoptosis in PCOS mice by affecting the AMPK/PI3K/AKT/FoxO3a signaling pathway.

Integrated Analysis of miRNA-mRNA Network Reveals Different Regulatory Patterns in the Endometrium of Meishan and Duroc Sows during Mid-Late Gestation

Embryo loss is a major factor affecting profitability in the pig industry. Embryonic mortality occurs during peri-implantation and mid-late gestation in pigs. Previous investigations have shown that the embryo loss rate in Meishan pigs is significantly lower than in commercial breeds. Most studies have focused on embryonic mortality during early gestation, but little is known about losses during mid-late gestation. In this study, we performed a transcriptome analysis of endometrial tissue in mid-late gestation sows (gestation days 49 and 72) sampled from two breeds (Meishan (MS) and Duroc (DU)) that have different embryo loss rates. We identified 411, 1113, 697, and 327 differentially expressed genes, and 14, 36, 57, and 43 differentially expressed miRNAs in four comparisons (DU49 vs. DU72, DU49 vs. MS49, DU72 vs. MS72, and MS49 vs. MS72), respectively. Subsequently; seven differentially expressed mRNAs and miRNAs were validated using qPCR. Functional analysis suggested the differentially expressed genes and miRNAs target genes mainly involved in regulation of hormone levels, blood vessel development, developmental process involved in reproduction, embryonic placenta development, and the immune system. A network analysis of potential miRNA-gene interactions revealed that differentially expressed miRNAs in Meishan pigs are involved in the response to estradiol and oxygen levels, and affect angiogenesis and blood vessel development. The binding site on ssc-miR-503 for epidermal growth factor (EGF) and the binding site on ssc-miR-671-5p for estrogen receptor α (ESR1) were identified using a dual luciferase assay. The results of this study will enable further exploration of miRNA-mRNA interactions important in pig pregnancy and will help to uncover molecular mechanisms affecting embryonic mortality in pigs during mid-late gestation.

NAD deficiency due to environmental factors or gene–environment interactions causes congenital malformations and miscarriage in mice

Causes for miscarriages and congenital malformations can be genetic, environmental, or a combination of both. Genetic variants, hypoxia, malnutrition, or other factors individually may not affect embryo development, however, they may do so collectively. Biallelic loss-of-function variants in HAAO or KYNU, two genes of the nicotinamide adenine dinucleotide (NAD) synthesis pathway, are causative of congenital malformation and miscarriage in humans and mice. The variants affect normal embryonic development by disrupting the synthesis of NAD, a key factor in multiple biological processes, from its dietary precursor tryptophan, resulting in NAD deficiency. This study demonstrates that congenital malformations caused by NAD deficiency can occur independent of genetic disruption of NAD biosynthesis. C57BL/6J wild-type mice had offspring exhibiting similar malformations when their supply of the NAD precursors tryptophan and vitamin B3 in the diet was restricted during pregnancy. When the dietary undersupply was combined with a maternal heterozygous variant in Haao, which alone does not cause NAD deficiency or malformations, the incidence of embryo loss and malformations was significantly higher, suggesting a gene–environment interaction. Maternal and embryonic NAD levels were deficient. Mild hypoxia as an additional factor exacerbated the embryo outcome. Our data show that NAD deficiency as a cause of embryo loss and congenital malformation is not restricted to the rare cases of biallelic mutations in NAD synthesis pathway genes. Instead, monoallelic genetic variants and environmental factors can result in similar outcomes. The results expand our understanding of the causes of congenital malformations and the importance of sufficient NAD precursor consumption during pregnancy.

64 Prediction of pregnancy and early embryo loss through OAS-1 expression, concentrations of pregnancy-associated glycoproteins, and Doppler ultrasonography in beef cattle

We aimed to compare three methods to detect pregnancy based on interferon-tau stimulated gene expression in peripheral blood polymorphonuclear cells and Doppler ultrasonography (Doppler-US) 20 days after timed AI and the concentrations of pregnancy associated glycoproteins (PAGs) 25 days after timed AI. Our second objective was to compare interferon-tau stimulated genes and PAGs as early embryo loss (EEL) predictors. Nelore cows (n=144) and heifers (n=103) were submitted to timed AI (Day (D) 0). On D20, polymorphonuclear cells were isolated from blood samples by Ficoll gradient (GE Healthcare), and the RNA was extracted. Expression of the interferon-tau stimulated gene (OAS-1) was quantified by qPCR and normalized to reference genes (GAPDH and ACTB). On D25, blood was collected from the jugular vein and PAG concentrations were measured by an in-house enzyme-linked immunosorbent assay. Pregnancy diagnoses were performed on D20 by luteolysis detection with Doppler-US and on D30 by detection of an embryo by heartbeat. Animals were classified as pregnant (P; fetus on D30), non-P (NP; no active corpus luteum on D20), and EEL (active corpus luteum on D20 but NP on D30). Expression of OAS-1 and PAG concentrations were analysed by analysis of variance using PROC MIXED of SAS considering the effects of group, category, and their interaction. Receiver operating characteristic curves were created, and the area under the curve (AUC), accuracy, specificity, and sensitivity were calculated for pregnancy predictions on D20 (OAS-1 and Doppler-US) and D25 (PAGs) compared with the standard diagnosis method on D30. Expression of OAS-1 and PAG concentrations were greater (P<0.01) in the P (2.4±0.2 and 4.8±0.2ngmL−1, respectively) compared with the NP (0.50±0.1 and 1.2±0.2ngmL−1, respectively) and EEL (0.8±0.2 and 1.0±0.2ngmL−1, respectively) groups. Receiver operating characteristic analysis indicated that OAS-1, Doppler, and PAGs were significant (P<0.01) predictors of pregnancy in heifers (AUC=0.86, 0.92, and 0.94, respectively) and cows (AUC=0.82, 0.94, and 0.95, respectively). The PAGs and Doppler-US presented higher accuracy on diagnosing pregnancy (92 and 89% for heifers, 94 and 93% for cows, respectively) than OAS-1 (81% for heifers and 75% for cows). Doppler-US was 100% sensitive to detect nonpregnant females, which avoided false negative results, whereas PAG concentrations presented a higher specificity (86% for heifers and cows) when compared with Doppler-US and OAS-1 (76 and 65% for heifers, 85 and 75% for cows, respectively). On D25, PAGs were 90% accurate to detect heifers and cows that would experience EEL, whereas on D20, OAS-1 was 50% accurate. In conclusion, Doppler-US can be used as early as D20 with high accuracy and sensitivity. Likely, the use of PAG concentrations on D25 is highly accurate and is more effective at detecting EEL. Pregnancy diagnosis through OAS-1 expression is not as accurate as the other methods and could not efficiently predict EEL. This research was supported by FAPESP (2015/106069; 2017/134729).