Early Embryo Loss Research Articles

BaP/BPDE exposure causes human trophoblast cell dysfunctions and induces miscarriage by up-regulating lnc-HZ06-regulated IL1B

Exposure to environmental BaP or its metabolite BPDE causes trophoblast cell dysfunctions to induce miscarriage (abnormal early embryo loss), which might be generally regulated by lncRNAs. IL1B, a critical inflammatory cytokine, is closely associated with adverse pregnancy outcomes. However, whether IL1B might cause dysfunctions of BaP/BPDE-exposed trophoblast cells to induce miscarriage, as well as its specific epigenetic regulatory mechanisms, is completely unexplored. In this study, we find that BPDE-DNA adducts, trophoblast cell dysfunctions, and miscarriage are closely associated. Moreover, we also identify a novel lnc-HZ06 and IL1B, both of which are highly expressed in BPDE-exposed trophoblast cells, in villous tissues of recurrent miscarriage patients, and in placental tissues of BaP-exposed mice with miscarriage. Both lnc-HZ06 and IL1B suppress trophoblast cell migration/invasion and increase apoptosis. In mechanism, lnc-HZ06 promotes STAT4-mediated IL1B mRNA transcription, enhances IL1B mRNA stability by promoting the formation of METTL3/HuR/IL1B mRNA ternary complex, and finally up-regulates IL1B expression levels. BPDE exposure promotes TBP-mediated lnc-HZ06 transcription, and thus up-regulates IL1B levels. Knockdown of either murine lnc-hz06 (which down-regulates Il1b levels) or murine Il1b could alleviate miscarriage in BaP-exposed mice. Collectively, this study not only discovers novel biological mechanisms and pathogenesis of unexplained miscarriage but also provides novel potential targets for treatment against BaP/BPDE-induced miscarriage.

The novel lnc-HZ12 suppresses autophagy degradation of BBC3 by preventing its interactions with HSPA8 to induce trophoblast cell apoptosis

ABSTRACT Abnormal expression of long non-coding RNAs (lncRNAs) is associated with the dysfunctions of human trophoblast cells and the occurrence of miscarriage (abnormal early embryo loss). BBC3/PUMA (BCL2 binding component 3) plays significant roles in regulation of cell apoptosis. However, whether specific lncRNAs might regulate BBC3 in trophoblast cells and further induce apoptosis and miscarriage remains completely unclear. Through screening, we identified a novel lnc-HZ12, which was significantly highly expressed in villous tissues of recurrent miscarriage (RM) patients relative to their healthy control (HC) group. Lnc-HZ12 suppressed chaperone-mediated autophagy (CMA) degradation of BBC3, promoted trophoblast cell apoptosis, and was associated with miscarriage. In mechanism, lnc-HZ12 downregulated the expression levels of chaperone molecules HSPA8 and LAMP2A in trophoblast cells. Meanwhile, lnc-HZ12 (mainly lnc-HZ12-SO2 region in F2 fragment) and HSPA8 competitively bound with the 169RVLYNL174 patch on BBC3, which prevented BBC3 from interactions with HSPA8 and impaired the formation of BBC3-HSPA8-LAMP2A complex for CMA degradation of BBC3. Thus, lnc-HZ12 upregulated the BBC3-CASP9-CASP3 pathway and induced trophoblast cell apoptosis. In villous tissues, lnc-HZ12 was highly expressed, CMA degradation of BBC3 was suppressed, and the apoptosis levels were higher in RM vs HC villous tissues, all of which were associated with miscarriage. Interestingly, knockdown of murine Bbc3 could efficiently suppress placental apoptosis and alleviate miscarriage in a mouse miscarriage model. Taken together, our results indicated that lnc-HZ12 and BBC3 played important roles in trophoblast cell apoptosis and miscarriage and might act as attractive targets for miscarriage treatment. Abbreviation: 7-AAD: 7-aminoactinomycin D; BaP: benzopyrene; BBC3/PUMA: BCL2 binding component 3; ChIP: chromatin immunoprecipitation; CHX: cycloheximide; CMA: chaperone-mediated autophagy; CQ: chloroquine; DMSO: dimethyl sulfoxide; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HC: healthy control; HSPA8: heat shock protein family A (Hsp70) member 8; IP: immunoprecipitation; LAMP2A: lysosomal associated membrane protein 2; LncRNA: long non-coding RNA; mRNA: messenger RNA; MT: mutant-type; NC: negative control; NSO: nonspecific oligonucleotide; PARP1: poly(ADP-ribose) polymerase 1; RIP: RNA immunoprecipitation; RM: recurrent miscarriage; TBP: TATA-box binding protein; WT: wild-type.

Exposure to high dose of polystyrene nanoplastics causes trophoblast cell apoptosis and induces miscarriage

BackgroundWith rapid increase in the global use of various plastics, microplastics (MPs) and nanoplastics (NPs) pollution and their adverse health effects have attracted global attention. MPs have been detected out in human body and both MPs and NPs showed female reproductive toxicological effects in animal models. Miscarriage (abnormal early embryo loss), accounting for 15-25% pregnant women worldwide, greatly harms human reproduction. However, the adverse effects of NPs on miscarriage have never been explored.ResultsIn this study, we identified that polystyrene (PS) plastics particles were present in women villous tissues. Their levels were higher in villous tissues of unexplained recurrent miscarriage (RM) patients vs. healthy control (HC) group. Furthermore, mouse assays further confirmed that exposure to polystyrene nanoplastics (PS-NPs, 50 nm in diameter, 50 or 100 mg/kg) indeed induced miscarriage. In mechanism, PS-NPs exposure (50, 100, 150, or 200 µg/mL) increased oxidative stress, decreased mitochondrial membrane potential, and increased apoptosis in human trophoblast cells by activating Bcl-2/Cleaved-caspase-2/Cleaved-caspase-3 signaling through mitochondrial pathway. The alteration in this signaling was consistent in placental tissues of PS-NPs-exposed mouse model and in villous tissues of unexplained RM patients. Supplement with Bcl-2 could efficiently suppress apoptosis in PS-NPs-exposed trophoblast cells and reduce apoptosis and alleviate miscarriage in PS-NPs-exposed pregnant mouse model.ConclusionsExposure to PS-NPs activated Bcl-2/Cleaved-caspase-2/Cleaved-caspase-3, leading to excessive apoptosis in human trophoblast cells and in mice placental tissues, further inducing miscarriage.Graphical

Chromosome Division in Early Embryos-Is Everything under Control? And Is the Cell Size Important?

Chromosome segregation in female germ cells and early embryonic blastomeres is known to be highly prone to errors. The resulting aneuploidy is therefore the most frequent cause of termination of early development and embryo loss in mammals. And in specific cases, when the aneuploidy is actually compatible with embryonic and fetal development, it leads to severe developmental disorders. The main surveillance mechanism, which is essential for the fidelity of chromosome segregation, is the Spindle Assembly Checkpoint (SAC). And although all eukaryotic cells carry genes required for SAC, it is not clear whether this pathway is active in all cell types, including blastomeres of early embryos. In this review, we will summarize and discuss the recent progress in our understanding of the mechanisms controlling chromosome segregation and how they might work in embryos and mammalian embryos in particular. Our conclusion from the current literature is that the early mammalian embryos show limited capabilities to react to chromosome segregation defects, which might, at least partially, explain the widespread problem of aneuploidy during the early development in mammals.

Review: Overview of the transcriptomic landscape in bovine blastocysts and elongated conceptuses driving developmental competence.

In cattle, pregnancy loss due to early embryonic mortality is a major concern that significantly impacts reproductive efficiency. Given the economic importance of cattle in livestock productivity, much research has been carried out to comprehend the regulatory mechanisms underlying this early embryo loss. Thus, understanding the molecular principles behind the reciprocal communication between the maternal uterus and the developing conceptus is paramount. Measurement of mRNA expression through a variety of techniques is widely used to unravel the complex and dynamic interaction between these two players. Development of high-throughput technologies, such as microarrays and RNA sequencing, have allowed global quantification of the full range of expressed mRNA, or transcriptome, of a biological sample. Therefore, numerous investigators have applied one or the other method to study the bovine embryo transcriptome at different developmental checkpoints and under different conditions. The goal of this article was to review studies involving the use of high-throughput techniques to study the transcriptome of the bovine embryo from the blastocyst (∼day 7) to the elongating conceptus stage (∼days 13-16) in terms of developmental capacity and the impact of procedures for in vitro embryo production. Furthermore, the differentially expressed genes reported by each study and enriched pathways were compared to determine common terms. The studies described here highlight differences in the transcriptome (i) between blastocysts with divergent ability to sustain a pregnancy, (ii) between age-matched elongated conceptuses with divergent developmental fates, and (iii) between blastocysts and elongated conceptuses produced in vitro or in vivo. Comparison between these works, supported by other studies involving transcriptomic data integration presented at the end of this review, highlights the involvement of pathways related to energy metabolism in embryonic competence, which may be altered because of the procedures involved in the in vitro production of embryos.

Tributyltin chloride exposure to post-ejaculatory sperm reduces motility, mitochondrial function and subsequent embryo development.

Male exposure to environmental toxicants can disrupt spermatogenesis and sperm function. However, consequences of environmentally relevant organotin exposure to post-ejaculatory mammalian spermatozoa on fertility are poorly understood. Determine the consequences of tributyltin chloride (TBT) exposure on post-ejaculatory sperm function and subsequent embryo development. Frozen-thawed bovine sperm were exposed to TBT (0.1-100nM) for 90min (acute) and 6h (short-term) followed by quantification of multiple sperm kinematics via computer aided sperm analysis. JC-1 dye was used to measure mitochondrial membrane potential. Sperm were then exposed to TBT for 90min in non-capacitating conditions, washed several times by centrifugation and applied to gamete co-incubation for in vitro embryo production to the blastocyst stage. 100nM TBT decreased total motility (88 vs 79%), progressive motility (80 vs 70%) curvilinear velocity and beat-cross frequency for 90min with similar phenotypes at 6h (P <0.05). Sperm mitochondrial membrane potential was lower in 10 and 100nM groups after 6h (P ≤0.05). Embryos fertilised from TBT-exposed sperm had reduced cleavage rate (80 vs 62%) and 8-16 cell morula development (55 vs 24%) compared to development from unexposed sperm. Exposure of post-ejaculatory mammalian sperm to TBT alters sperm function through lowered motility and mitochondrial membrane potential. Fertilisation of oocytes with TBT-exposed sperm reduces embryo development through mechanisms of paternal origin. Acute and short-term environmental exposure of post-ejaculatory sperm to organotins and endocrine disrupting chemicals such as TBT contribute to idiopathic subfertility and early embryo loss.

Early embryo losses, progesterone and pregnancy associated glycoproteins levels during summer heat stress in dairy cows.

Objectives of this study were to characterize the effects of heat stress on pregnancy associated glycoproteins (PAG) and progesterone and its involvement in embryo survival. In trial 1, blood samples collected from days 29 to 36 post insemination were examined for the comparison of PAG concentrations between winter (n = 3721) and summer (n = 2388). In trial 2, embryo losses were assessed in winter (n = 144) and in summer (n = 133), in days 31 or 32 of pregnancy. Pregnancy diagnosis was carried out by ultrasonography on days 24 or 25, and it was repeated a week later; in the second occasion PAG concentration was also determined. In trial 3 the PAG and progesterone concentrations were assessed in days 33 to 36 in winter and summer. In trial 1 PAG levels did not differ between winter and summer, the conception rate and the proportion of uncertain pregnancies were higher in winter than summer. The likelihood of pregnancy was 10 to 15% higher in winter. In trial 2, the embryo death rate was higher in summer, but the PAG levels of cows that had embryo loss in summer were higher than those in winter. In both seasons, lower PAG levels were associated with higher risk of pregnancy loss, while embryo death was five times more likely to occur in summer than in winter and lower PAG concentrations were positively associated with higher risk of embryo loss. In trial 3, mean PAG levels were higher and of progesterone were lower during the summer than during the winter. We infer that despite the devastating effects of heat stress on cows' fertility, those early embryos that survive under continuous heat stress can form a well-functioning placenta; hence, the high embryo mortality rate observed during the summer months could be mainly attributed to luteal insufficiency.

142 Nulliparity alters gene expression in inner cell mass and trophoblast of equine blastocysts in old mares

An increased incidence in early embryo loss has been observed in aged mares. Moreover, the first foal born to a mare is lighter than her subsequent foals, with reported impaired placental function at term. Because trophoblast function may be affected from the embryo stage, the aim of this project was to determine the effect of parity in aged mares on gene expression in Day-8.5 embryos. Middle-aged (13.5±2.2 years) nulliparous (never foaled) (ON) or multiparous (1.8±1.6 foals) (OM) Saddlebred, non-nursing mares were inseminated with the semen of one unique stallion. At 8 days post-ovulation (10 days post-hCG), embryos were recovered by uterine flushing and bisected to obtain samples of pure (trophectoderm, TE) or inner cell mass enriched (ICM) trophoblast. Paired end, non-oriented RNA sequencing was performed with Illumina (NextSEqn 500) on 5 and 6 TE and ICM collected from ON and OM, respectively. Differential expression was analysed with DESEqn 2. Embryo size was included in the model and a P&amp;lt;0.05 cutoff was used after false discovery rate correction. Gene set enrichment analysis (GSEA) was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) databases. Out of the 13 007 and 12 706 genes expressed in ICM and TE, respectively, only 8 in ICM and 6 in TE were differentially expressed, with 2 genes in common. Nevertheless, 19 gene sets were enriched and 6 depleted in the ICM of ON, whereas 2 gene sets were enriched and 8 depleted in the TE of ON compared with OM. Gene sets involved in ribosomal activity and structure, proteasome, integral component of plasma membrane, and immune response were enriched in ICM from ON embryos, and gene sets linked to sphingolipid metabolism, nucleosome, and constituents of the extracellular matrix (ECM) were depleted. In TE from ON mares, enriched gene sets were involved with ribosomes and depleted gene sets were linked to extracellular matrix, focal adhesion, myosin complex, and sequence-specific DNA binding. Overall, 1 enriched (linked to extracellular matrix) and 1 depleted gene set (involved in ribosomal structure) were common to ICM and TE. Thus, embryos from aged nulliparous mares seem to have higher protein turnover and higher immune response compared with those of OM, whereas the depletion of gene sets associated with extracellular matrix and membrane may indicate differences in cellular organisation into lineages. More work is ongoing to study effects on subsequent development.

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

Abstract 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.

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&amp;lt;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&amp;lt;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).

GRIM-19 Deficiency Promotes Macrophage Polarization to M1 Phenotype Partly Through AKT/mTOR Pathway and Glycolysis in Unexplained Recurrent Spontaneous Abortion

Background: The occurrence of unexplained recurrent spontaneous abortion (URSA) is closely related to the disorder of the immune system, but the mechanisms remain unclear. The purpose of this study was to investigate the expression of GRIM-19 in URSA and its possible pathogenesis according to macrophage polarization. Methods: The correlation of macrophage and pregnancy outcome was determined using GRIM-19+/- mice. GRIM-19 expression in decidua of recurrent abortion patients was analysed using clinical specimens, while THP-1-derived macrophages after downregulation of GRIM-19 was used to explore GRIM-19 roles in regulating macrophage polarization and glycolysis. The mechanism of GRIM-19 regulation was studied using molecular biology and biochemical methods. Findings: First, we found that clodronate-liposome mediated macrophage depletion can reduce the embryo resorption rate in GRIM-19+/- mice, which demonstrated that macrophages play a role in the early embryo loss in GRIM-19+/- mice. We further confirmed that GRIM-19 deficiency promotes macrophage polarization from M2 to M1 phenotype in GRIM-19+/- mice uterus. Moreover, we found that the GRIM-19 deficiency enhances M1 polarization in THP-1-derived macrophages. We also showed that protein levels of p-mTOR and p-AKT decreased significantly after downregulation of GRIM-19 in THP-1-derived macrophages. In parallel to this, the upregulation of the amount of M1 macrophages resulted from GRIM-19 loss was significantly reversed in the cells treated with IGF-1 which is an activator of AKT/mTOR pathway. To provide more direct evidence, we showed that GRIM-19 was downregulated in the uterine decidual macrophages of patients with URSA, and GRIM-19 downregulation is accompanied by increased M1 macrophage polarization. Furthermore, we found that expression of glycolytic enzymes was substantially enhanced in the uterine decidual macrophages of URSA patients, and glycolysis in macrophages can be further enhanced with downregulation of GRIM-19. Interpretation: Our study provides evidence that GRIM-19 deficiency may play a role in regulating macrophage polarization partly through AKT/mTOR signaling axis in URSA and glycolysis may participate in this complicated process. Funding Statement: This work was supported by the National Natural Science Foundation of China (grant numbers 81571511, 81701528, 81370711 and 81601315), the Shandong Provincial Key Research and Development Project (grant number 2019GSF107004), the Shandong Provincial Natural Science Foundation (grant number ZR2017PH052). Declaration of Interests: The authors stated: None declared. Ethics Approval Statement: All of the procedures of the animal experiments were approved by the Ethical Committee of Shandong University. All efforts were made to minimize suffering of mice.

7. GENETIC TESTING FOR COPY NUMBER VARIATIONS IN FIRST POLAR BODIES AND OOCYTES BY SINGLE-CELL NEXT-GENERATION SEQUENCING

Introduction Female meiotic errors are the main cause of whole chromosomal aneuploidy in human embryos and significantly contribute to early embryo loss. Meiotic chromosomal malsegregation occurs during divisions of the maturing oocyte in oogenesis concurrently with extrusion of two polar bodies: polar body first (PB1) and polar body second (PB2). Based on meiosis mechanism, genome of a single oocyte can be deduced by analyzing its sibling polar bodies. PB genetic testing can identify abnormalities of maternal meiotic origin and may be the only form of preimplantation genetic testing for aneuploidies (PGT-A) accepted by patients who object embryo biopsy for ideological reasons. In the majority of previous studies, array comparative genomic hybridization (aCGH) was a method of choice for comprehensive chromosome testing in polar bodies. Here, we test the use of single-cell next-generation sequencing (NGS) method in polar body testing for detecting copy number variations (CNVs) in PB1 genome and validate methods for whole genome amplification (WGA) of PB1. Material and methods In this study, single-cell NGS technology was used to asses ploidy status of oocytes and their sibling PB1. A total of 120 single cells (60 first polar bodies and 60 oocytes) were collected from in vitro fertilization (IVF) couples and subjected for WGA using four different kits based on three distinct amplification systems: (i) polymerase chain reaction (PCR-based WGA), (ii) multiple displacement amplification (MDA) or (iii) multiple annealing and looping-based amplification cycles (MALBAC). WGA products were processed and analyzed with VeriSeq PGS MiSeq kit on MiSeq sequencing platform (Illumina) and random samples were validated with the aCGH method. Amplification uniformity and accuracy for CNV detection of the four tested kits were evaluated. Results Ploidy status was determined for 112 of 120 (93,3%) samples (84/112 based on direct analysis and 28/112 based on indirect analysis of the well-amplified sibling PB1 or oocyte). Estimated aneuploidy rate for the studied group which resembles the total oocyte abnormality resulting from meiosis I errors rate was 33% (37/112 samples). In the group of well-amplified PB1-oocyte pairs (33/60) a 97% concordance between the chromosomal status of PB1 and the corresponding oocyte was observed. Both whole chromosome nondisjunction (6 of 37 aneuploid samples) and abnormal chromatid pre-division and segregation were detected (31 of 37 aneuploid samples). The result is concordant with previous studies - abnormal chromatid segregation was the dominant aneuploidy-causing mechanism. Differences in WGA kits performance including amplification uniformity, aneuploidy calling potential and a number of interpretable NGS results were evaluated and the most optimal kit for PB1 genome WGA was pre-selected. Results from aCGH validation of the randomly selected samples showed full consistency with the NGS results. Conclusions The NGS-based method used for PB1 genome analysis showed a high predictive potential of PB1 in deducing ploidy status of the corresponding oocyte and is a promising method for PGT-A of oocytes. The study also contributes to a better understanding of the chromosome segregation patterns and maternal meiosis errors. Supported by: POIR.02.03.02-14- 0092/17

Regulation of embryo survival in cattle

Evidence is presented that bovine somatotrophin (bST) treatment of lactating dairy cows enhances both expression of oviductal insulin-like growth factor II (IGF-II) mRNA and endometrial insulin-like growth factor binding protein 3 (IGFBP-3) mRNA between day 3 and day 7 of the oestrous cycle. mRNA encoding growth hormone (GH) receptor in endometrial tissues increased between day 3 and day 7 of the oestrous cycle. The changes induced by bST treatment may contribute to stimulation of embryo development and increase pregnancy rates in lactating dairy cows. Additive effects of bST and rb interferon tau (rbIFN-tau) to inhibit phorbol ester induction of prostaglandin F2alpha secretion in immortalized bovine endometrial cells indicates that there is interplay between their signal transduction pathways. Non-lactating dairy cows were killed at day 17 after oestrus to evaluate the effects of pregnancy status (cyclic versus pregnant) and bST (bST versus control) treatment on endometrial gene expression. Distinctly different mRNA and protein responses were detected between cyclic and pregnant cows that were related to luteolytic-antiluteolytic drive (that is expression of progesterone receptor, oxytocin receptor, oestradiol receptor alpha and prostaglandin GH synthase 2 (PGHS-2)). The bST-induced changes in PGHS-2 protein (+), oxytocin receptor mRNA (+) and oestrogen receptor alpha protein (+) may potentially affect the mechanisms associated with maintenance of pregnancy. Two experiments were conducted to evaluate whether ovarian follicular suppression induced by biodegradable deslorelin implants would reduce either early or late embryo losses. A 450 microg deslorelin implant used to induce ovulation in a timed insemination programme decreased subsequent follicular development and tended to reduce early embryo losses, whereas a 2.1 mg deslorelin implant failed to reduce late embryonic losses when inserted on day 27 of pregnancy.

Single-cell sequencing of primate preimplantation embryos reveals chromosome elimination via cellular fragmentation and blastomere exclusion.

Aneuploidy that arises during meiosis and/or mitosis is a major contributor to early embryo loss. We previously showed that human preimplantation embryos encapsulate missegregated chromosomes into micronuclei while undergoing cellular fragmentation and that fragments can contain chromosomal material, but the source of this DNA was unknown. Here, we leveraged the use of a nonhuman primate model and single-cell DNA-sequencing (scDNA-seq) to examine the chromosomal content of 471 individual samples comprising 254 blastomeres, 42 polar bodies, and 175 cellular fragments from a large number (N = 50) of disassembled rhesus cleavage-stage embryos. Our analysis revealed that the aneuploidy and micronucleation frequency is conserved between humans and macaques, and that fragments encapsulate whole and/or partial chromosomes lost from blastomeres. Single-cell/fragment genotyping showed that these chromosome-containing cellular fragments (CCFs) can be maternally or paternally derived and display double-stranded DNA breaks. DNA breakage was further indicated by reciprocal subchromosomal losses/gains between blastomeres and large segmental errors primarily detected at the terminal ends of chromosomes. By combining time-lapse imaging with scDNA-seq, we determined that multipolar divisions at the zygote or two-cell stage were associated with CCFs and generated a random mixture of chromosomally normal and abnormal blastomeres with uniparental or biparental origins. Despite frequent chromosome missegregation at the cleavage-stage, we show that CCFs and nondividing aneuploid blastomeres showing extensive DNA damage are prevented from incorporation into blastocysts. These findings suggest that embryos respond to chromosomal errors by encapsulation into micronuclei, elimination via cellular fragmentation, and selection against highly aneuploid blastomeres to overcome chromosome instability during preimplantation development.

In vitro production of horse embryos predisposes to micronucleus formation, whereas time to blastocyst formation affects likelihood of pregnancy.

Invitro embryo production is an increasingly popular means of breeding horses. However, success is limited by a high incidence of early embryo loss. Although there are various possible causes of pregnancy failure, chromosomal abnormalities, including aneuploidy, are important potential contributors. This study evaluated the frequency of micronucleus formation as a proxy for aneuploidy in invitro-produced (IVP) and invivo-derived horse blastocysts. Associations between IVP embryo morphology, frequency of nuclear abnormalities and the likelihood of pregnancy were investigated. IVP blastocysts exhibited a higher frequency of cells with micronuclei than invivo-derived embryos (10% vs 1% respectively; P=0.05). This indication of chromosomal instability may explain the higher incidence of pregnancy failure after transfer of IVP embryos. However, the frequency of micronuclei was not correlated with brightfield microscopic morphological characteristics. Nevertheless, IVP embryos reaching the blastocyst stage after Day 9 of invitro culture were less likely to yield a pregnancy than embryos that developed to blastocysts before Day 9 (27% vs 69%), and embryos that had expanded before transfer were more likely to undergo embryonic death than those that had not expanded (44% vs 10%). These findings indicate that current embryo culture conditions are suboptimal and that the speed of embryo development is correlated with pregnancy survival.

Maternal N-Carbamylglutamate Supply during Early Pregnancy Enhanced Pregnancy Outcomes in Sows through Modulations of Targeted Genes and Metabolism Pathways.

Reducing pregnancy loss is important for improving reproductive efficiency for both human and mammalian animals. Our previous study demonstrates that maternal N-carbamylglutamate (NCG) supply during early pregnancy enhances embryonic survival in gilts. However, whether maternal NCG supply improves the pregnancy outcomes is still not known. Here we found maternal NCG supply during early pregnancy in sows significantly increased the numbers of total piglets born alive per litter ( P < 0.05) and significantly changed the levels of metabolites in amniotic fluid and serum involved in metabolism of energy, lipid, and glutathione and immunological regulation. The expression of endometrial progesterone receptor membrane component 1 (PGRMC1) was significantly increased by NCG supplementation ( P < 0.05) as well as the expression of PGRMC1, endothelial nitric oxide synthesases (eNOS), and lamin A/C in fetuses and placentae ( P < 0.05). Among the NCG-associated amino acids, arginine and glutamine, markedly increased PGRMC1 and eNOS expression in porcine trophectoderm cells ( P < 0.05), whereas glutamate could stimulate the expression of vimentin and lamin A/C in porcine trophectoderm (pTr) cells ( P < 0.05) and proline stimulated lamin A/C expression ( P < 0.05). Collectively, these data reveal the mechanisms of NCG in reducing early embryo loss. These findings have important implications that NCG has great potential to improve pregnancy outcomes in human and mammalian animals.

Early pregnancy loss: A critical review of the evidence for embryo “wastage”

Approximately 10% of clinically recognized pregnancies end in miscarriage. Many authors suggest that this number greatly underestimates the death of early human embryos, commonly asserting that in addition to clinical pregnancy loss, as many as 60% of all conceptions are lost before a recognized pregnancy is established. However, a critical review of the literature indicates that previous analyses of early embryo loss have relied on inadequate data, unwarranted speculation and insufficient clarity about what constitutes a human embryo. Anovulation, fertilization failure and non-development (none of which entail embryo demise) make significant contributions to observed fecundity, yet have often been ignored. Although the actual percentage of early human embryo loss is not known with precision, based on multiple lines of evidence, preimplantation embryo loss is likely to be negligible (if it occurs at all). When all early pregnancy loss is considered, embryo wastage does not exceed 35%, and is likely to be lower than this maximum figure, possibly as low as 7%. In light of this conclusion, the relevance of embryo wastage to an assessment of the moral worth of the human embryo is briefly considered.

Likelihood of pregnancy after embryo transfer is reduced in recipient mares with a short preceding oestrus.

Previous surveys reported a positive association between the length of the follicular phase and subsequent fertility in embryo transfer donor and Thoroughbred mares. However, it is unclear whether a longer oestrus positively influences fertilisation and oviductal development (oocyte quality, oviductal environment), or uterine receptivity and survival of the embryo in the uterus. To determine the effect of length of oestrus (characterised by duration of endometrial oedema) on likelihood of pregnancy and early embryo loss (EEL) in recipient mares after embryo transfer (ET). Retrospective clinical study. A total of 350 embryos recovered from 161 donor mares were transferred into 231 recipient mares during three consecutive breeding seasons. The following variables were analysed via two binary logistic regression models to determine their effect on pregnancy and EEL: 1) year of transfer, 2) season of transfer, 3) age of the recipient mare, 4) age of the donor mare, 5) operator performing the transfer, 6) singleton or twin embryo, 7) embryo size, 8) number of transfers to a given recipient in any one season, the use of 9) d-cloprostenol and 10) hCG in the recipient mare, 11) day of ovulation of the recipient mare at ET, 12) number of corpora lutea (CLs) at ET, and 13) duration of oestrus in the recipient mare. Age of the donor mare (P = 0.01), operator (P = 0.008), number of CLs at ET (P = 0.05) and the number of days of endometrial oedema during the oestrus preceding ET to the recipient mare (P = 0.004) influenced the likelihood of pregnancy. Early embryonic loss was influenced only by the year of transfer (P = 0.014). Retrospective design of the study. The involvement of several veterinary surgeons over the 3-year period could have affected data recording. The likelihood of pregnancy in recipient mares is positively correlated with the duration of endometrial oedema during the oestrus preceding ET. This suggests a role for an adequate duration of oestrogenic priming during oestrus on uterine receptivity and embryo survival.

Early embryo mortality in natural human reproduction: What the data say.

How many human embryos die between fertilisation and birth under natural conditions? It is widely accepted that natural human embryo mortality is high, particularly during the first weeks after fertilisation, with total prenatal losses of 70% and higher frequently claimed. However, the first external sign of pregnancy occurs two weeks after fertilisation with a missed menstrual period, and establishing the fate of embryos before this is challenging. Calculations are additionally hampered by a lack of data on the efficiency of fertilisation under natural conditions. Four distinct sources are used to justify quantitative claims regarding embryo loss: (i) a hypothesis published by Roberts & Lowe in TheLancet is widely cited but has no practical quantitative value; (ii) life table analyses give consistent assessments of clinical pregnancy loss, but cannot illuminate losses at earlier stages of development; (iii) studies that measure human chorionic gonadotrophin (hCG) reveal losses in the second week of development and beyond, but not before; and (iv) the classic studies of Hertig and Rock offer the only direct insight into the fate of human embryos from fertilisation under natural conditions. Re-examination of Hertig's data demonstrates that his estimates for fertilisation rate and early embryo loss are highly imprecise and casts doubt on the validity of his numerical analysis. A recent re-analysis of hCG study data concluded that approximately 40-60% of embryos may be lost between fertilisation and birth, although this will vary substantially between individual women. In conclusion, natural human embryo mortality is lower than often claimed and widely accepted. Estimates for total prenatal mortality of 70% or higher are exaggerated and not supported by the available data.

Early embryo mortality in natural human reproduction: What the data say

How many human embryos die between fertilisation and birth under natural conditions? It is widely accepted that natural human embryo mortality is high, particularly during the first weeks after fertilisation, with total prenatal losses of 70% and higher frequently claimed. However, the first external sign of pregnancy occurs two weeks after fertilisation with a missed menstrual period, and establishing the fate of embryos before this is challenging. Calculations are additionally hampered by a lack of data on the efficiency of fertilisation under natural conditions. Four distinct sources are used to justify quantitative claims regarding embryo loss: (i) a hypothesis published by Roberts & Lowe in The Lancet is widely cited but has no practical quantitative value; (ii) life table analyses give consistent assessments of clinical pregnancy loss, but cannot illuminate losses at earlier stages of development; (iii) studies that measure human chorionic gonadotrophin (hCG) reveal losses in the second week of development and beyond, but not before; and (iv) the classic studies of Hertig and Rock offer the only direct insight into the fate of human embryos from fertilisation under natural conditions. Re-examination of Hertig’s data demonstrates that his estimates for fertilisation rate and early embryo loss are highly imprecise and casts doubt on the validity of his numerical analysis. A recent re-analysis of hCG study data concluded that approximately 40-60% of embryos may be lost between fertilisation and birth, although this will vary substantially between individual women. In conclusion, natural human embryo mortality is lower than often claimed and widely accepted. Estimates for total prenatal mortality of 70% or higher are exaggerated and not supported by the available data.