Mendelian gene identification through mouse embryo viability screening

The diagnostic rate of Mendelian disorders in sequencing studies continues to increase, along with the pace of novel disease gene discovery. However, variant interpretation in novel genes not currently associated with disease is particularly challenging and strategies combining gene functional evidence with approaches that evaluate the phenotypic similarities between patients and model organisms have proven successful. A full spectrum of intolerance to loss-of-function variation has been previously described, providing evidence that gene essentiality should not be considered as a simple and fixed binary property. Here we further dissected this spectrum by assessing the embryonic stage at which homozygous loss-of-function results in lethality in mice from the International Mouse Phenotyping Consortium, classifying the set of lethal genes into one of three windows of lethality: early, mid or late gestation lethal. We studied the correlation between these windows of lethality and various gene features including expression across development, paralogy and constraint metrics together with human disease phenotypes, and found that the members of the early gestation lethal category show distinctive characteristics and a strong enrichment for genes linked with recessive forms of inherited metabolic disease. Based on these findings, we explored a gene similarity approach for novel gene discovery focused on this subset of lethal genes. Finally, we investigated unsolved cases from the 100,000 Genomes Project recruited under this disease category to look for signs of enrichment of biallelic predicted pathogenic variants among early gestation lethal genes and highlight two novel candidates with phenotypic overlap between the patients and the mouse knockout.

Effects of Vitrified Cryopreservation Duration on the IVF and Neonatal Outcomes

Objective: To evaluate the impact of cryopreservation storage duration on embryo viability, implantation competence, pregnancy outcome and neonatal outcomes.Design: Retrospective study.Setting: Center for Reproductive Medicine，The Third Affiliated Hospital of Guangzhou Medical University.Patient(s): In vitro fertilization patients who had vitrified cryopreserved embryos and following the first frozen embryo transfer cycles from January 2004 to August 2019. A total of 31143 patients met the inclusion criteria and were grouped according to the storage time (20926 patients in Group 1 with storage time <3 months, 6472 patients in Group 2 with storage time between 3 and 6 months, 2237 patients in Group 3 with storage time between 6 and 12 months and 746 in Group 4 with storage time between 12 and 24 months, 762 patients in Group 5 with storage time >24 months).Intervention(s): None.Main Outcome Measure(s): In the total FET cycles, the embryo survival rate was decreased significantly with the increase of cryopreservation time, and the highest rate was 98. 63 % in the 1-3 months group, and the lowest was 71.13% in the >=731 days group (P <0. 01). The HCG positive rate (57.85%) and clinical pregnancy rate (55. 26%) in the 1-3 months group were the highest (P<0. 01). The >=731 group had the lowest sex ratio of 0.96. There were no significant differences in neonatal birth weight, neonatal height and congenital anomalies among the groups (P>0. 05).Result(s): Length of storage time had a significant effect on post-thaw survival and outcomes for IVF cycles. Conclusion(s): With the prolongation of cryopreservation time, the embryonic survival rate and pregnancy rate were decreased significantly. Short-term cryopreservation (<=3 months) can obtain higher clinical pregnancy rate. Therefore, although long-term hryopreservation of the embryo has no effect on the health of the new baby, but hryopreserved embryos should be recovery as soon as possible if condition allows.

Chronic Dietary Exposure of Roosters to a Glyphosate-Based Herbicide Increases Seminal Plasma Glyphosate and AMPA Concentrations, Alters Sperm Parameters, and Induces Metabolic Disorders in the Progeny

The effects of chronic dietary Roundup (RU) exposure on rooster sperm parameters, fertility, and offspring are unknown. We investigated the effects of chronic RU dietary exposure (46.8 mg kg−1 day−1 glyphosate) for 5 weeks in 32-week-old roosters (n = 5 RU-exposed and n = 5 control (CT)). Although the concentrations of glyphosate and its main metabolite AMPA (aminomethylphosphonic acid) increased in blood plasma and seminal fluid during exposure, no significant differences in testis weight and sperm concentrations were observed between RU and CT roosters. However, sperm motility was significantly reduced, associated with decreased calcium and ATP concentrations in RU spermatozoa. Plasma testosterone and oestradiol concentrations increased in RU roosters. These negative effects ceased 14 days after RU removal from the diet. Epigenetic analysis showed a global DNA hypomethylation in RU roosters. After artificial insemination of hens (n = 40) with sperm from CT or RU roosters, eggs were collected and artificially incubated. Embryo viability did not differ, but chicks from RU roosters (n = 118) had a higher food consumption, body weight and subcutaneous adipose tissue content. Chronic dietary RU exposure in roosters reduces sperm motility and increases plasma testosterone levels, growth performance, and fattening in offspring.

Mitochondrial DNA Content May Not Be a Reliable Screening Biomarker for Live Birth After Single Euploid Blastocyst Transfer

An increasing number of studies have related the mitochondrial DNA (mtDNA) content to embryo viability and transfer outcomes. However, previous studies have focused more on the relationship between mtDNA and embryo implantation, few studies have studied the effect of the mtDNA content on live birth. In the study, we investigated whether mtDNA content is a reliable screening biomarker for live birth after single blastocyst transfer. A total of 233 couples with 316 blastocyst stage embryos undergoing in vitro fertilization treatment and pre-implantation genetic testing analysis were included in the study. All embryos were chromosomally normal and had undergone single-embryo transfers. There was no significant difference observed in the blastocyst mtDNA content among the live birth, miscarriage and non-implanted groups (p=0.999), and the mtDNA content in blastocysts from the miscarriage and live birth groups was similar [median (interquartile range), 1.00*108(7.59*107- 1.39*108) vs 1.01*108 (7.37*107- 1.32*108)]. Similarly, no significant association was observed between mtDNA content and embryo implantation potential (p=0.965). After adjusting for multiple confounders in a logistic regression analysis with generalized estimating equations, no associations between mtDNA content and live birth were observed in all blastocysts, Day-5 and Day-6 blastocysts (p=0.567, p=0.673, p=0.165, respectively). The live birth rate was not significantly different between blastocysts with an elevated mtDNA content and blastocysts with a normal mtDNA content (26.7% vs 33.6% p=0.780). Additionally, there was no linear correlation between the mtDNA content and maternal age (p=0.570). In conclusion, the mtDNA content does not seem to be a potential biomarker for embryo transfer outcomes (i.e., implantation and live birth) based on the existing testing tools. Embryos with an elevated mtDNA content also have development potential for successful live birth.

Temperature-induced changes in egg white antimicrobial concentrations during pre-incubation do not influence bacterial trans-shell penetration but do affect hatchling phenotype in Mallards

Microbiome formation and assemblage are essential processes influencing proper embryonal and early-life development in neonates. In birds, transmission of microbes from the outer environment into the egg’s interior has been found to shape embryo viability and hatchling phenotype. However, microbial transmission may be affected by egg-white antimicrobial proteins (AMPs), whose concentration and antimicrobial action are temperature-modulated. As both partial incubation and clutch covering with nest-lining feathers during the pre-incubation period can significantly alter temperature conditions acting on eggs, we experimentally investigated the effects of these behavioural mechanisms on concentrations of both the primary and most abundant egg-white AMPs (lysozyme and avidin) using mallard (Anas platyrhychos) eggs. In addition, we assessed whether concentrations of egg-white AMPs altered the probability and intensity of bacterial trans-shell penetration, thereby affecting hatchling morphological traits in vivo. We observed higher concentrations of lysozyme in partially incubated eggs. Clutch covering with nest-lining feathers had no effect on egg-white AMP concentration and we observed no association between concentration of egg-white lysozyme and avidin with either the probability or intensity of bacterial trans-shell penetration. The higher egg-white lysozyme concentration was associated with decreased scaled body mass index of hatchlings. These outcomes demonstrate that incubation prior to clutch completion in precocial birds can alter concentrations of particular egg-white AMPs, though with no effect on bacterial transmission into the egg in vivo. Furthermore, a higher egg white lysozyme concentration compromised hatchling body condition, suggesting a potential growth-regulating role of lysozyme during embryogenesis in precocial birds.

PSI-15 Embryo viability based upon energy

The objective of this study was to analyze embryo energy release and whether or not it is indicative of survivability upon successful implantation. In both bovine and equine reproduction, fertility is a lowly heritable trait that has an exceedingly large financial impact on the ability to grow genetic lines. In order to increase pregnancy rates, producers must have access to accurate decision-making tools. Nineteen (n = 19) fertilized bovine embryos collected via flushing by licensed veterinarian, Dr. Russell Killingsworth, DVM, in Shamrock, TX, were assessed for energy release using amplification of video with a Nikon Camera and Iolight Microscope. Once embryos were flushed, they were filmed for a thirty-five (35) second observation period. Embryo dimensions were then taken using Image J software. Embryo release of energy was humanly perceptible and measurable by changes in their morphological dimensions. Measurements of significance were taken on the embryo intercellular mass area (ICM), total embryo area, and on the vertical, diagonal, and horizontal axis of the sub-zonal distance, or the distance between the zona pellucida and outer edge of the ICM. All measurements were taken in microns. Once embryos were successfully measured, all were transferred into recipients. Significant findings presented that embryos with a greater ICM, total embryo area, and sub-zonal vertical, diagonal, and horizontal distance established pregnancies more frequently than embryos with less momentous differences in distance (P &lt; 0.05). This research has shown the relationship between embryo energy and pregnancy had a significance of determining pregnancy viability. This has therefore resulted in enhanced prediction of embryo transfer techniques based on embryo quality making pregnancy rates for producers more predictable and consistent.

The endogenous mex-3 3´UTR is required for germline repression and contributes to optimal fecundity in C. elegans

RNA regulation is essential to successful reproduction. Messenger RNAs delivered from parent to progeny govern early embryonic development. RNA-binding proteins (RBPs) are the key effectors of this process, regulating the translation and stability of parental transcripts to control cell fate specification events prior to zygotic gene activation. The KH-domain RBP MEX-3 is conserved from nematode to human. It was first discovered in Caenorhabditis elegans, where it is essential for anterior cell fate and embryo viability. Here, we show that loss of the endogenous mex-3 3´UTR disrupts its germline expression pattern. An allelic series of 3´UTR deletion variants identify repressing regions of the UTR and demonstrate that repression is not precisely coupled to reproductive success. We also show that several RBPs regulate mex-3 mRNA through its 3´UTR to define its unique germline spatiotemporal expression pattern. Additionally, we find that both poly(A) tail length control and the translation initiation factor IFE-3 contribute to its expression pattern. Together, our results establish the importance of the mex-3 3´UTR to reproductive health and its expression in the germline. Our results suggest that additional mechanisms control MEX-3 function when 3´UTR regulation is compromised.