Establishment Of Pregnancy Research Articles

Characterization of progesterone-induced dendritic cells in metabolic and immunologic reprogramming

The role of maternal-fetal immune tolerance in the establishment and maintenance of pregnancy has been well established. Dendritic cells (DCs) as a crucial part of the decidual microenvironment, have high plasticity in immunogenicity and tolerogenicity. The regulatory mechanisms of DCs phenotype or function at the maternal-fetal interface, however, have not been fully developed. Studies from the field of immunometabolism have highlighted that the metabolic pathways of DCs are closely associated with their immunity. Our previous study showed that progesterone (P4) up-regulated a series of enzymes involved in DCs mitochondrial oxidative phosphorylation and fatty acid metabolism. In this study, we confirmed that P4 induced significant alternations in DCs metabolic pathways, promoting their glycolysis, mitochondrial function, and the dependency and capacity of fatty acids as mitochondrial fuel. Moreover, P4 also increased the inhibitory molecule ILT4 expression on DCs and down-regulated the CD86, which may coordinate their immune tolerance function in pregnancy. Together, our study helps to understand the role of P4 in DCs metabolic and immunologic reprogramming and may provide novel insights into the hormonal immunometabolism regulation of DCs during normal pregnancy.

Unveiling the Role of MX1 and MX2 Gene Expression in Early Pregnancy of Kari Ewes: Insights into INF-tau Signaling and Potential as a Predictive Biomarker for Pregnancy Detection in Domestic Ruminants

Myxovirus resistance (MX) genes are relevant to a family of interferon (IFN)-stimulated genes. In domestic ruminants such as ewes and cattle, the MX genes have two isoforms called the Myxovirus Resistance 1 (MX1) gene and the Myxovirus Resistance 2 (MX2) gene. INF-tau (INFt) is released from embryonic trophoblast cells in the early days of pregnancy and induces expression of MX genes in the uterine endometrium. In the present study, we summarized the relative expression of the MX1 and MX2 genes during the early days of pregnancy in Kari ewes. Blood samples were taken from inseminated and non-inseminated (control) ewes on different days. Peripheral Blood Monocytes (PBMC) were isolated, and with the addition of MX1 and MX2 gene primers, complementary deoxyribonucleic acid (cDNA) was synthesized by Reverse Transcriptase Polymerase Chain Reaction (RT-PCR). The cDNA was then amplified by rounds of PCR amplification. The amplified product was run on a 2% Agarose gel. The results showed expression of the MX1 and MX2 genes only in pregnant ewes, while no expression was observed in non-pregnant or control ewes. Expression in pregnant ewes was observed on days 14, 16, 17, and 18 after insemination. When comparing the MX1 and MX2 genes, the expression of the MX2 gene was relatively higher than that of the MX1 gene on all experimental days. The result suggested that INF-tau MX genes are expressed during the early days of pregnancy and are involved in the establishment of pregnancy. These results justify the development of this biomarker as a predictive tool for the early detection of pregnancy in ewes and other domestic ruminants.

Effect of dietary energy source on pregnancy rates and reproductive physiology of pastured beef heifers

Proper production of steroid hormones, such as progesterone (P4), is essential for maintenance of pregnancy. Provision of gluconeogenic substrates from the diet may alter postabsorptive energy supplies and associated hormone signaling in a manner supportive of elevating P4 concentrations. Crossbred Angus heifers (n=27) were balanced for pre-trial bodyweight (BW) and body condition score (BCS), then assigned to isoenergetic diets leveraging starch (ES) or fat (EF) as the primary source of energy (45.7% starch vs 11.5% fat). The heifers were placed on the assigned diets 8 days prior to the initiation of estrous synchronization and continued the diets through the second pregnancy diagnosis, 52 days after the initial insemination. On day 28 post-breeding, pregnancy status was diagnosed via ultrasonography. Measurements of follicle size and number were collected via rectal palpation and ultrasonography on days −10, −8, −4, and day 0 relative to breeding. Blood samples were collected on days −10, 1 through 7, 10, 14, 18, 21, 24, and 28 to analyze plasma for P4 concentrations and placental-associated glycoprotein (PAG) concentrations. Data were analyzed using a linear mixed effects model with a fixed effect for treatment and a random effect for pen. Observations collected over time were analyzed using a repeated measures approach with fixed effects for treatment, time, initial condition, and a random effect for pen. Interactions between treatment and pregnancy status were also tested for PAG and P4 concentrations. No treatment differences were observed in final BCS (P=0.12) and total dry matter intake (DMI; P=0.51), though heifers on the ES treatment tended to reach an increased BW (P=0.10). Although heifers fed ES diets had numerically higher conception rates compared to heifers fed EF diets (71.4% vs. 66.7%), there was no statistically significant difference. Furthermore, there were no significant differences observed among diets in terms of pregnancy outcomes, follicle size, or number. A treatment by pregnancy status interaction was detected for both PAG and P4 concentrations. These results suggest that short-term dietary starch supplementation provided around the time of breeding can result in shifts to reproductive hormone abundance that may be conducive for pregnancy establishment.

Dose- and time-dependent effects of interferon tau on bovine endometrial gene expression

Failure by the developing conceptus to secrete sufficient interferon tau (IFNT), required for maternal recognition of pregnancy (MRP), at the appropriate time is related to early pregnancy loss in cattle. We aimed to test the hypothesis that there is a dose- and time-dependent relationship between IFNT and the endometrial expression of key interferon-stimulated genes (ISGs) involved in the signalling cascade leading to MRP in cattle. Candidate genes were identified first through a bioinformatic approach, where integrated transcriptomic data from two previous studies were analyzed to identify endometrial genes induced by IFNT. Next, expression of selected candidate genes was investigated in vitro in endometrial explants. Endometrial explants collected from cows (n = 8) in the late luteal phase of the estrous cycle were cultured in medium without (control) or with recombinant ovine IFNT (1, 10, 100 ng/mL) for 6 h. Simultaneously, endometrial explants were cultured in medium containing 100 ng/mL IFNT for different time periods (15 min, 30 min, 1 h, 3 h, 6 h). Gene expression was analyzed by RT-qPCR. We identified 54 endometrial genes responding to IFNT and to some degree to the conceptus, from which five ISGs (CMPK2, BPNT1, IFI35, TNFSF10 and TRIM38) were further selected for the dose- and time-dependent experiments. Classical ISGs (ISG15, OAS1, MX1 and MX2) were up-regulated (P < 0.05) in endometrium by 1 ng/mL IFNT. However, other selected ISGs (CMPK2, BPNT1, IFI35, TNFSF10 and TRIM38) were induced only by higher concentrations (10 and 100 ng/mL) of IFNT (P < 0.05). In terms of duration of exposure, IFNT at 100 ng/mL induced a significant (P < 0.05) increase in ISG15 and CMPK2 expression after 1 h incubation, while all other studied ISGs in the endometrium were upregulated when cultured for 3 or 6 h, but did not affect expression when the duration of culture was for 1 h or less. These results suggest that IFNT acts on the uterus in both a dose- and time-dependent manner in cattle and that timely exposure of the endometrium to sufficient IFNT is essential for appropriate signalling to ensure successful pregnancy establishment.

Single-cell transcriptomic characterization of sheep conceptus elongation and implantation

Bidirectional communication between the developing conceptus and endometrium is essential for pregnancy recognition and establishment in ruminants. We dissect the transcriptomic dynamics of sheep conceptus and corresponding endometrium at pre- and peri-implantation stages using single-cell RNA sequencing. Spherical blastocysts contain five cell types, with 68.62% trophectoderm cells. Strikingly, elongated conceptuses differentiate into 17 cell types, indicating dramatic cell fate specifications. Cell-type-specific gene expression delineates the features of distinctive trophectoderm lineages and indicates that the transition from polar trophectoderm to trophoblast increases interferon-tau expression and likely drives elongation initiation. We identify 13 endometrium-derived cell types and elucidate their molecular responses to conceptus development. Integrated analyses uncover multiple paired transcripts mediating the dialogues between extraembryonic membrane and endometrium, including IGF2-IGF1R, FGF19-FGFR1, NPY-NPY1R, PROS1-AXL, and ADGRE5-CD55. These data provide insight into the molecular regulation of conceptus elongation and represent a valuable resource for functional investigations of pre- and peri-implantation ruminant development.

Secretory Proteomic Responses of Endometrial Epithelial Cells to Trophoblast-Derived Extracellular Vesicles.

Synchronized crosstalk between the embryo and endometrium during the periconception period is integral to pregnancy establishment. Increasing evidence suggests that the exchange of extracellular vesicles (EVs) of both embryonic and endometrial origin is a critical component of embryo-maternal communication during peri-implantation. Here, we investigated whether embryonic signals in the form of EVs can modulate the endometrial epithelial cell secretome. Receptive endometrial analog RL95-2 cells were supplemented with trophoblast analog JAr cell-derived EVs, and the secretory protein changes occurring in the RL95-2 cells were analyzed using mass spectrometry. EVs of non-trophoblastic origin (HEK 293 cells) were used as the control EV source to supplement endometrial cells. Trophoblast cell-derived EVs enriched endometrial epithelial cell secretions with proteins that support embryo development, attachment, or implantation, whereas control EVs were unable to induce the same effect. The present study suggests that embryonic signals in the form of EVs may prime receptive endometrial epithelial cells to enrich their secretory proteome with critical proteomic molecules with functional importance for periconception milieu formation.

Identification and characterization of circRNAs in peri-implantation endometrium between Yorkshire and Erhualian pigs

BackgroundOne of the most critical periods for the loss of pig embryos is the 12th day of gestation when implantation begins. Recent studies have shown that non-coding RNAs (ncRNAs) play important regulatory roles during pregnancy. Circular RNAs (circRNAs) are a kind of ubiquitously expressed ncRNAs that can directly regulate the binding proteins or regulate the expression of target genes by adsorbing micro RNAs (miRNA).ResultsWe used the Illumina Novaseq6,000 technology to analyze the circRNA expression profile in the endometrium of three Erhualian (EH12) and three Yorkshire (YK12) pigs on day 12 of gestation. Overall, a total of 22,108 circRNAs were identified. Of these, 4051 circRNAs were specific to EH12 and 5889 circRNAs were specific to YK12, indicating a high level of breed specificity. Further analysis showed that there were 641 significant differentially expressed circRNAs (SDEcircRNAs) in EH12 compared with YK12 (FDR < 0.05). Functional enrichment of differential circRNA host genes revealed many pathways and genes associated with reproduction and regulation of embryo development. Network analysis of circRNA-miRNA interactions further supported the idea that circRNAs act as sponges for miRNAs to regulate gene expression. The prediction of differential circRNA binding proteins further explored the potential regulatory pathways of circRNAs. Analysis of SDEcircRNAs suggested a possible reason for the difference in embryo survival between the two breeds at the peri-implantation stage.ConclusionsTogether, these data suggest that circRNAs are abundantly expressed in the endometrium during the peri-implantation period in pigs and are important regulators of related genes. The results of this study will help to further understand the differences in molecular pathways between the two breeds during the critical implantation period of pregnancy, and will help to provide insight into the molecular mechanisms that contribute to the establishment of pregnancy and embryo loss in pigs.

Exposure to di-isononyl phthalate during early pregnancy disrupts decidual angiogenesis and placental development in mice

Di-isononyl phthalate (DiNP), an endocrine-disrupting chemical, is found in numerous consumer products and human exposure to this phthalate is becoming inevitable. The impact of DiNP exposure on the establishment and maintenance of pregnancy remains largely unknown. Thus, we conducted studies in which pregnant mice were exposed to an environmentally relevant dose (20 µg/kg BW/day) of DiNP on days 1–7 of gestation, then analyzed the effects of this exposure on pregnancy outcome. Our studies revealed that exposure to DiNP during this window led to fetal loss towards the end of gestation. Further studies showed that, although embryos were able to attach to the uterus, implantation sites in DiNP-exposed uteri exhibited impaired differentiation of stromal cells to decidual cells and an underdeveloped angiogenic network in the decidual bed. We also found that exposure to this phthalate has a significant effect on trophoblast differentiation and causes disorganization of the placental layers. The labyrinth was significantly reduced, resulting in compromised expression of nutrient transporters in the placentas of mice exposed to DiNP. These placental defects in DiNP-exposed females were the cause of fetal loss during the later stages of gestation.

Embryo-derive TNF promotes decidualization via fibroblast activation.

Decidualization is a process in which endometrial stromal fibroblasts differentiate into specialized secretory decidual cells and essential for the successful establishment of pregnancy. The underlying mechanism during decidualization still remains poorly defined. Because decidualization and fibroblast activation share similar characteristics, this study was to examine whether fibroblast activation is involved in decidualization. In our study, fibroblast activation-related markers are obviously detected in pregnant decidua and under in vitro decidualization. ACTIVIN A secreted under fibroblast activation promotes in vitro decidualization. We showed that arachidonic acid released from uterine luminal epithelium can induce fibroblast activation and decidualization through PGI2 and its nuclear receptor PPARδ. Based on the significant difference of fibroblast activation-related markers between pregnant and pseudopregnant mice, we found that embryo-derived TNF promotes CPLA2α phosphorylation and arachidonic acid release from luminal epithelium. Fibroblast activation is also detected under human in vitro decidualization. Similar arachidonic acid-PGI2-PPARδ-ACTIVIN A pathway is conserved in human endometrium. Collectively, our data indicate that embryo-derived TNF promotes CPLA2α phosphorylation and arachidonic acid release from luminal epithelium to induce fibroblast activation and decidualization.

A comparison of culture and cooling for the short term preservation of in vivo derived dromedary camel embryos of varying morphological quality

Despite recent advancements in the cryopreservation of dromedary camel embryos, widespread application of the technique is still limited by the need for specialised vitrification equipment and supplies. Temporary, liquid-phase embryo storage methods provide a useful tool for short-term preservation of camel embryos.In the current study, we compared the use of in vitro embryo culture with cold liquid storage in order to maintain both high- (Grade 1- Excellent and 2-Good) and low- (Grade 3- Moderate and 4-Poor) morphological grade Day-7 dromedary camel embryos in vitro for up to 3 days. Embryos were either cooled and placed in Hams-F10 medium supplemented with HEPES and 10% FBS and then kept at 4 °C; or placed in Hams-F10 supplemented with sodium bicarbonate and 10% FBS and then cultured in a humidified atmosphere of 6% CO2 at 37 °C before being assessed for viability at 24 h. In high-morphological grade embryos, both cold storage and culture supported 100% viability (maintenance of normal morphology) over this period (Cooled n = 22, Cultured n = 20). In low-morphological grade embryos, culture supported higher viability (16/18, 88.9%) than did cooling (4/18, 22.2%). We then evaluated the effect of up to 3 days of cold storage or culture on embryo morphological grade, diameter, and developmental competence following embryo transfer. High-grade embryos were divided between culture and cold storage; low-grade embryos were evaluated only after culture. Over 3 days of culture, both high- and low-grade embryos tended to either maintain or improve upon their initial morphological score (P < 0.05) and increased in diameter (P < 0.001). Embryos subjected to cooling tended to have reduced morphological scores by 48 h of storage and decreased in diameter by 72 h (P < 0.05). No significant influence of storage method (cooling vs. culture), duration (24–72 h), or embryo grade (high vs low) was observed on pregnancy establishment at Day-60 (22.2%–57.2% pregnancy rates for all treatments). Overall, rates of pregnancy establishment were similar for transferred cultured (n = 45) and cooled (n = 45) embryos (pregnancy rates at Day 18, 48% vs 51.1%; at Day 60, 37.7% vs 37.7%). Rates of embryonic loss also were similar (22.7% vs 26%).In conclusion, whilst similar rates of pregnancy and pregnancy loss were observed following the transfer of both cooled and cultured embryos held in vitro for up to 3 days, amongst the two methods, only embryo culture appears to provide a means of effectively preserving Day- 7 dromedary camel embryos with reduced morphological values in vitro. Considering these embryos appear to show poor tolerance to the cooling procedure and are unlikely candidates for vitrification, embryo culture may provide an effective method for deriving pregnancies from low-morphological grade embryos when immediate transfer is not possible on the day of flushing.

Regulation of IkappaB Protein Expression by Early Gestation in the Thymus of Ewes.

The thymus is an essential component of maternal immune systems that play key roles in recognizing the placenta as immunologically foreign. The inhibitor of the NF-κB (IκB) family has essential effects on the NF-κB pathway; however, it is unclear whether early pregnancy modulates the expression of the IκB family in the thymus. In this study, maternal thymuses were sampled on day 16 of nonpregnancy and different gestation stages in the ovine, and the expression of IκB proteins was analyzed. The data showed that B cell leukemia-3 and IκBβ increased; however, IκBα, IκBε, and IKKγ deceased during gestation. Furthermore, there was an increase in IκBNS and IκBζ expression values on day 13 of pregnancy; however, this decreased on day 25 of gestation. In summary, the expression of the IκB family was modulated in the thymus during early gestation, suggesting that the maternal thymus can be associated with maternal immunologic tolerance and pregnancy establishment in ewes.

Endometrial Origins of Stillbirth (EOS), a case–control study of menstrual fluid to understand and prevent preterm stillbirth and associated adverse pregnancy outcomes: study protocol

IntroductionCurrent research aimed at understanding and preventing stillbirth focuses almost exclusively on the role of the placenta. The underlying origins of poor placental function leading to stillbirth, however, remain poorly...

P-526 Modified Applebaum uterine scoring system to predict uterine receptivity in frozen embryo transfer(FET) cycles

Abstract Study question To evaluate modified Applebaum uterine scoring system in predicting the uterine receptivity and clinical pregnancy rate? Summary answer The modified version of Applebaum uterine scoring system has sensitivity of 0.9411 and specificity of 0.5869 in predicting the uterine receptivity in embryo transfer cycle. What is known already The endometrial receptivity have been accepted to be major limiting factors in the establishment of pregnancy. Different techniques have been developed to evaluate endometrial receptivity, such as endometrial receptivity array or histological dating. The advantage of Ultrasound is it’s non-invasiveness, repeatability, real time monitoring and predictability. Study design, size, duration It is a prospective observational study. Study group comprises of 200 female with primary infertility over a period of 6 month. Participants/materials, setting, methods Exclusion criteria was patients associated with male sub-fertility or having uterine anomalies. The study was conducted at D.Y.Patil fertility centre, unit of Bloom IVF. All the patients were given uterine scoring before FET based on Endometrial thickness, morphology, vascularisation, myometrial echogenicity, uterine artery PI and EDF, myometrial blood flow. Main results and the role of chance The overall pregnancy rate was 54%. A perfect score of 20 was associated with 97.4% pregnancy rate. Scores of 17 to 19 was associated with the pregnancy rate of 85.4% and scores of 14 to 16 was associated with the pregnancy rate of 28.6%. Score less than 13 is not associated with any pregnancy. The Modified Applebaum scoring system showed the sensitivity of 0.9411 and specificity of 0.5869 in predicting the outcome of the cycle. On an individual basis, endometrial morphology, end diastolic blood flow(EDF) and Pulsatility index(PI) of uterine artery and myometrial blood flow were found to be significant for a pregnancy on the basis of Z score and p value of &amp;lt; 0.01 Limitations, reasons for caution As there were no case of secondary infertility, Grade III/IV endometriosis, endocrine disorder and uterine anomalies. The scoring system might not replicate the same results. Wider implications of the findings As the sensitivity and specificity of this uterine scoring system is quite good. It could be a good tool in assessing endometrium before embryo transfer. Trial registration number Not applicable

P-159 Impact of the symbiotic metabolites D-amino acids on preimplantation development

Abstract Study question Are the metabolites derived from the symbiotic microbiota of reproductive tract involved in preimplantation development? Summary answer The metabolites derived from the symbiotic microbiota of reproductive tract, D-amino acid（D-AA）, contributed to preimplantation development. What is known already It has been known that Lactobacillus exist in the vagina as symbiotic bacteria. Though it was thought that Lactobacillus keep the pH environment acidic and prevent infection by pathogenic bacteria, thereby keeping the uterus and fallopian tubes sterile in mammal, we have identified Staphylococcus aureus in oviductal lavage fluid from female mice and confirmed that the antibiotics disrupt the implantation pattern. In human, the vaginal microbiota is reported to be dominated by Lactobacillus, which produces D-amino acids (D-AA). These findings suggest the involvement of bacterial metabolites D-AA in the establishment of human pregnancy. Study design, size, duration 23 women undergoing in vitro fertilization (IVF) treatment at Keio University Hospital from 2019 to 2021 were assessed for eligibility. 3 were excluded due to spontaneous pregnancy or infectious disease. Twenty subjects were allocated to the successful pregnancy group (S-group (8); mean ± standard deviation 35.5 ± 2.8 years) defined as gestational sac detected on ultrasound at 5 weeks' gestation; and to the unsuccessful pregnancy group (U- group (12); 35.5 ± 2.7 years)). Participants/materials, setting, methods Genomic DNA (gDNA) extracted from vaginal and intrauterine samples at each menstrual cycle was used for metagenome analysis at the genus and the species levels by next-generation sequencing, and by quantitative real-time PCR. Amino acids were analyzed using a two-dimensional HPLC. Immunocytochemical staining by a polyclonal antibody to D-AA oxidase (DAO), which catabolizes D-AA, was performed using B6D2F1 mice and mice with a natural point-mutation G181R in DAO gene, which abolishes DAO activity. Main results and the role of chance Both vaginal/uterine bacterial counts were higher in the luteal phase than in the follicular phase. Vaginal bacterial counts in the S-group were lower than those in the U- group. The composition of microbiota of the vaginal samples (N = 23) shifted from the follicular to the luteal phase, with Lactobacillus spp. predominating. However, there was no significant difference in the ratio of Lactobacillus spp. between the S- and U-groups. The intrauterine samples of the S-group contained a high abundance of L. crispatus, L. gasseri, and L. ultunensis, while the U-group contained a high abundance of L. jensenii. Next, we analyzed the Lactobacillus metabolite, D-AA, and detected D-Ser, D-Ala, D-Asn, and D-Pro in the vaginal, uterine, and follicular fluid of the S-group. Follicular fluid that developed into blastocysts contained more D-AA than that of arrested embryos (N = 2). To investigate the significance of D-AA in the preimplantation development, mouse embryos were cultured in amino acid-depleted medium. All of them arrested (0.0%: 0/78), whereas 41% (25/61) of the embryos with D-AA added to the amino acid-depleted medium developed into blastocysts. We confirmed the expression of DAO in oocytes and blastocysts. DAO-null mice had fewer births than wild type. Limitations, reasons for caution The main limitation is the differences of symbiotic microbiota in reproductive tract and embryonic development between mice and humans; it may be difficult to apply the results of this analysis to humans. Wider implications of the findings Our results display a correlation between species-level microbiome profiling and IVF outcomes. In addition, microbiota-derived metabolites D-AA are suggested to be crucial mediators of embryo development. These findings could give rise to the development of new diagnostic techniques and drug. Trial registration number Not applicable

P-543 Higher maternal age alters the insulin/IGF system and intracellular lipid metabolism of the endometrium and in the preimplantation embryo – insights from the rabbit model.

Abstract Study question Our aim was to analyse the embryo-maternal-interaction, focusing on the insulin/IGF-system and lipid metabolism on day 6 of pregnancy in reproductive young and old rabbits. Summary answer Advanced maternal age has a decisive effect on maternal and embryonic insulin/IGF-signalling and lipid metabolism, leading to higher embryo loss already during preimplantation period. What is known already The reproductive potential in women declines with age. Molecular and biochemical mechanisms involved in age-related infertility and their impact on embryo-maternal-interaction during early pregnancy are still not completely understood. However, establishment of a successful pregnancy depends on the physiological condition of the mother and a continuous dialogue with the developing embryo. The insulin/IGF-system plays a pivotal role in this embryo-maternal crosstalk, connecting maternal insulin/IGF with embryonic metabolism, cell proliferation and differentiation. Study design, size, duration We used the rabbit as reproductive model to investigate maternal age-related alterations in embryo-maternal-interaction during preimplantation period. Compared to humans, the rabbit shares high similarities in early embryo development, gastrulation and lipid metabolism (DOI: 10.1530/REP-12-0091 and DOI: 10.1093/ajcn/62.2.458S). A total of 50 young (16-20 weeks) and 31 old (&amp;lt;108 weeks) sexually mature, female rabbits were used for analysis at day 6 post coitum. Participants/materials, setting, methods We measured mRNA and protein levels of target genes of the insulin/IGF system and lipid metabolism by quantitative PCR, Western Blot and ELISA. Therefore, maternal blood, endometrium and blastocysts were analysed from reproductive young (16 to 20 weeks) and old (over 108 weeks) rabbits on day 6 of pregnancy. Blastocysts and ovulation points were counted and only blastocysts at the pre-gastrulating stage 1, separated in embryoblast (EB) and trophoblast (TB), were used for further analyses. Main results and the role of chance At day 6 of pregnancy, reproductive old rabbits had a lower amount of embryos. Maternal serum insulin and IGF levels were reduced in reproductive old rabbits, accompanied by a paracrine upregulation of IGF1 and its receptors in the endometrium. Preimplantation blastocysts adapted to hormonal changes by reducing IGF1 and IGF2 levels in both embryonic compartments, EB and TB, while the expression of embryonic IGF receptors was unchanged. Furthermore, an increase of fatty acids metabolism was observed in the ageing endometrium, indicated by the higher level of carnitine palmitoyltransferase I B (CPT1B) and elevated expression of fatty acid binding and transport proteins. These results are in line with lower level of fatty acid synthesis (FASN) in the endometrium from old, gravid rabbits, the key enzyme of fatty acid synthesis. Embryonic fatty acid uptake and b-oxidation were increased in EB and TB, too. The observed changes in embryonic and maternal lipid metabolism are caused by the alterations of the transcription factors cAMP-responsive element binding protein (CREB) and peroxisome proliferator-activated receptor (PPAR) α and γ in endometrium and embryos from reproductive old rabbits. Limitations, reasons for caution Rabbit embryogenesis reflects human development only during blastocysts formation. Therefore, statements are limited to the embryogenesis stages investigated. Wider implications of the findings The results of the current study are in accordance with the common literature, showing that advanced maternal age affects developmental competence of embryos. Our study points out that advanced maternal age alters lipid metabolism and insulin/IGF-signalling, which are two crucial components in embryo-maternal-interaction. Trial registration number not applicable

Blastocyst cell number and allocation affect the developmental potential and transcriptome of bovine Somatic Cell Nuclear Transfer embryos.

Cloning cattle using somatic cell nuclear transfer (SCNT) is inefficient. While the rate of development of SCNT embryos in vitro is similar to that of fertilized embryos, most fail to develop into healthy calves. In this study, we aimed to identify developmentally competent embryos according to blastocyst cell composition and perform transcriptome analysis of single embryos. Transgenic SCNT embryos expressing nuclear-localized HcRed gene at day 7 of development were imaged by confocal microscopy for cell counting and individually transferred to recipient heifers. Pregnancy rates were determined by ultrasonography. Embryos capable of establishing pregnancy by day 35 had an average of 117±6 total cells, whereas embryos with an average of 128±5 cells did not establish pregnancy (p0.05). A lesser average number of 41± 3 cells in the inner cell mass (ICM) also resulted in pregnancies (0.05) than a greater number of 48 ± 2 cells in the ICM. Single embryos were then submitted to RNA sequencing for transcriptome analysis. Using weighted gene co-expression network analysis, we identified clusters of genes in which gene expression correlated with the number of total cells or ICM cells. Gene ontology (GO) analysis of these clusters revealed enriched biological processes in the coenzyme metabolic process, intracellular signaling cascade, and glucose catabolic process, among others. We concluded that SCNT embryos with fewer total- and ICM-cell numbers resulted in greater pregnancy establishment rates and that these differences are reflected in the transcriptome of such embryos.

Targeted reproductive management for lactating Holstein cows: Reducing the reliance on exogenous reproductive hormones

Adoption of automated monitoring devices (AMD) affords the opportunity to tailor reproductive management according to the cow's needs. We hypothesized that a targeted reproductive management (TRM) would reduce the use of reproductive hormones while increasing the percentage of cows pregnant 305 d in milk (DIM). Holstein cows from 2 herds (n = 1,930) were fitted with an AMD at 251.0 ± 0.4 d of gestation. Early-postpartum estrus characteristics (EPEC; intense estrus = heat index ≥70; 0 = minimum, 100 = maximum) of multiparous cows were evaluated at 40 (herd 1) or 41 (herd 2) DIM and EPEC of primiparous cows were evaluated at 54 (herd 1) or 55 (herd 2) DIM. Control cows received the first artificial insemination at fixed time (TAI; primiparous, herd 1 = 82 and herd 2 = 83 DIM; multiparous, herd 1 = 68 and herd 2 = 69 DIM) following the Double-Ovsynch (DOV) protocol. Cows enrolled in the TRM treatment were managed as follows: (1) cows with at least one intense estrus were inseminated upon AMD detected estrus for 42 d and, if not inseminated, were enrolled in the DOV protocol; and (2) cows without an intense estrus were enrolled in the DOV protocol at the same time as cows in the control treatment. Control cows were re-inseminated based on visual or patch aided detection of estrus, whereas TRM cows were re-inseminated as described for control cows with the aid of the AMD. Cows received a GnRH injection 27 ± 3 d after insemination and, if diagnosed as nonpregnant, completed the 5-d Cosynch protocol and received TAI 35 ± 3 d after insemination. Among cows in the TRM treatment, 55.8 and 42.9% of primiparous and multiparous cows, respectively, received the first insemination in spontaneous estrus. The interaction between treatment and parity affected pregnancy 67 d after the first AI (primiparous: control = 37.6%, TRM = 27.4%; multiparous: control = 41.0%, TRM = 44.7%). The TRM treatment increased re-insemination in estrus (control = 48.3%, TRM = 70.5%). Pregnancy 67 d after re-inseminations tended to be affected by the interaction between treatment and EPEC (no intense estrus: control = 25.3%, TRM = 32.0%; intense estrus: control = 32.9%, TRM = 32.2%). The interaction between treatment and EPEC affected pregnancy by 305 DIM (no intense estrus: control = 80.8%, TRM = 88.2%; intense estrus: control = 87.1%, TRM = 86.1%). Treatment did not affect the number of reproductive hormone treatments among cows that had not had an intense estrus (control = 10.5 ± 0.3, TRM = 9.1 ± 0.2 treatments/cow), but cows in the TRM treatment that had an intense estrus received fewer reproductive hormone treatments than cows in the control treatment (2.0 ± 0.1 vs. 9.6 ± 0.2 treatments/cow). Selecting multiparous cows for first AI in estrus based on EPEC reduced the use of reproductive hormones without impairing the likelihood of pregnancy to first AI. The use of AMD for re-insemination expedited the establishment of pregnancy among cows that did not display an intense estrus early postpartum.

Significance Of Nutrition In Preventing Miscarriage, An Ignored But Modifiable Risk Factor.

Madam, Miscarriage or spontaneous abortion is defined as the pregnancy loss occurring before the 24th week of gestation. This term is used when an ultrasound has confirmed an intrauterine pregnancy1. It is the commonest adverse fate of pregnancy, with vaginal bleeding and abdominal pain as alarming symptoms. It is found that around 12-15% of confirmed pregnancies end up in a miscarriage. Although genetic factors (chromosomal abnormalities) top the list, maternal factors, especially nutritional status, can affect the developing embryo and result in early pregnancy foetal demise2. During pregnancy, maternal requirements for energy, macronutrients (carbohydrates, proteins and fats) and micronutrients increase to cope with the physiological changes occurring in the mother’s body and to support adequate foetal development. Two Case control studies have been conducted and have found a preventive effect of milk consumption and dairy products on spontaneous abortion3. There is also an association between decreased animal fat and carotene intake and increased risk of hydatidiform mole, which can ultimately lead to a miscarriage2. Studies proved that not only macronutrients but deficiency of micronutrients also contribute to poor pregnancy outcomes. Some essential micronutrients include iron, magnesium, zinc, vitamin b12 and folic acid., Due to its antioxidant effects, Vitamin C is also required during this stage and contributes to a healthy pregnancy1. Besides deficiencies, even excess of some micronutrients, like caffeine, especially during the pre-pregnancy state contributed to the risk of early pregnancy loss4. Studies have been done to determine the mechanism behind the nutrition’s effect on the outcome of pregnancy. In the pre pregnancy state, nutritional imbalances result in the alteration of germ cell morphology, which hinders the chances of fertilisation of the altered germ cell. In addition to this, nutritional status during the peri-implantation and placental developmental stage (embryonic stage) play a vital role in the establishment of pregnancy and fetal development so any deficiency during this stage increases the risk of miscarriage. Folic acid deficiency during this stage is harmful. Although it mainly results in the development of congenital anomalies in infants, it is seen that by increasing homocysteine levels it can also lead to miscarriage, although the association is still unknown1. Not only undernutrition but overweight and obesity in mothers also lead to poor pregnancy outcomes. ---Continue

Fetal-maternal interactions during pregnancy: a 'three-in-one' perspective.

A successful human pregnancy requires the maternal immune system to recognize and tolerate the semi-allogeneic fetus, allowing for appropriate trophoblasts invasion and protecting the fetus from invading pathogens. Therefore, maternal immunity is critical for the establishment and maintenance of pregnancy, especially at the maternal-fetal interface. Anatomically, the maternal-fetal interface has both maternally- and fetally- derived cells, including fetal originated trophoblasts and maternal derived immune cells and stromal cells. Besides, a commensal microbiota in the uterus was supposed to aid the unique immunity in pregnancy. The appropriate crosstalk between fetal derived and maternal originated cells and uterine microbiota are critical for normal pregnancy. Dysfunctional maternal-fetal interactions might be associated with the development of pregnancy complications. This review elaborates the latest knowledge on the interactions between trophoblasts and decidual immune cells, highlighting their critical roles in maternal-fetal tolerance and pregnancy development. We also characterize the role of commensal bacteria in promoting pregnancy progression. Furthermore, this review may provide new thought on future basic research and the development of clinical applications for pregnancy complications.

Expression of genes for transport of water and angiogenesis, as well as biochemical biomarkers in Holstein dairy cows during the ovsynch program

Changes in expression of genes associated with angiogenesis and transport of water by cells, as well as biomarkers of oxidative stress were determined at specific times during the ovsynch protocol to synchronize estrus and breed Holstein dairy cows. Blood samples were taken from 82 lactating Holstein cows at the time of the 1st GnRH injection (G1), 7 days later at the time of the PGF2a (PG) injection, and 48 h after the PGF2a treatment when the second injection of GnRH was administered (G2). The serum was analyzed for malondialdehyde (MDA), reduced glutathione (GSH), glutathione peroxidase (GPX), nitric oxide (NO), catalase (CAT), and total antioxidant capacity (TAC). The expression of vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor 2 (VEGFR2), endothelial nitric oxide synthase (eNOS3), aquaporin 3 (AQP3), and AQP4 mRNAs in peripheral blood mononuclear cells (PBMCs) was analyzed. The number of copies of each of the mRNAs was quantified using qPCR. Pregnancy status was determining at 32 ± 3 days after insemination using an ultrasound “Sonoscape-5V″ model. Receiver operating curves (ROC) were used to assess the sensitivity and specificity of the biochemical parameters in serum to predict establishment of p The expression of MDA, GPX, and Catalase changed (P < 0·05) between G1, PG and G2 phases of the ovsynch protocol with higher levels at PG than at G1 and G2. The highest levels of NO were detected at G2. The ROC analyses identified NO, TAC and CAT as the most sensitive and specific biomarker for pregnancy with areas under the curve being 0.875 (P < 0.0001), 0.843 (P < 0.03), 0.833 (P < 0.017), sensitivity being 75.3, 42.86, and 26.27%, and specificity being 90, 90 and 85% respectively. The expression for VEGF, VEGFR2, eNOS3, AQP3, and AQP4 mRNAs was upregulated at PG compared to G1 and G2 phases of the ovsynch protocol. The results suggest that following the first injection of GnRH, there is an increase in expression of VEGF, VEGFR2, eNOS3, AQP3, and AQP4 mRNAs by the time of the PGF2a injection and then expression decreased. Further, ROC analyses identified increases in NO, TAC and CAT as the most sensitive and specific biomarkers with the greatest potential to predict establishment of pregnancy in Holstein cows.