Maternal parity affects Day 8 embryo gene expression in old mares

As sport career is a priority in most of equine breeds, mares are frequently bred for the first time at an advanced age. Both age and first gestation were shown to have a deleterious effect on reproduction outcomes, respectively on fertility and offspring weight but the effect mare's parity in older mares on embryo quality has never been considered. The aim of this project was to determine the effect of old mare's nulliparity on gene expression in embryos. Day 8 post ovulation embryos were collected from old (10-16 years old) nulliparous (ON, N=5) or multiparous (OM, N=6) non-nursing Saddlebred mares, inseminated with the semen of one stallion. Pure (TE_part) or inner cell mass enriched (ICMandTE) trophoblast were obtained by embryo bisection and paired end, non-oriented RNA sequencing (Illumina, NextSeq500) was performed on each hemi-embryo. To discriminate gene expression in the ICM from that in the TE, deconvolution (DeMixT R package) was used on the ICMandTE dataset. Differential expression was analyzed (DESeq2) with embryo sex and diameter as cofactors using a false discovery rate <0.05 cutoff. Although the expression of only a few genes was altered by mare's nulliparity (33 in ICM and 23 in TE), those genes were related to nutrient exchanges and responses to environment signaling, both in ICM and TE, suggesting that the developing environment from these mares are not optimal for embryo growth. In conclusion, being nulliparous and old does not seem to be the perfect match for embryonic development in mares.

P–170 The secretomy of embryo sex

Study question Does the analysis of the metabolites of the embryonic culture medium can predict the sex of the embryo? Summary answer The presence and quantity of some metabolites in the culture medium can predict the sex of the human embryos. What is known already Advances in analytical techniques for metabolomics have brought the possibility of better tools for the characterization of molecules. Embryonic metabolism can be used as a good indicator of viability, regardless of the morphology of the blastocysts, since differences were observed in the metabolic activities between the days of embryo development and in the rates of live births. Study design, size, duration 16 patients had their embryos biopsied between the months of January to July 2019 in a human reproduction laboratory. All cases had PGT-A indication and after the biopsy, the embryos were frozen. The culture medium samples were individually prepared for metabolites extraction according to the Bligh and Dyer protocol. Controlled ovarian stimulation and dose adjustments according to the response of each patient. The metabolomics analysis was performed by mass spectrometry. Participants/materials, setting, methods Follicular puncture were performed 35 hours after r-hCG. The eggs were kept in individual culture until the blastocyst stage. The blastocysts biopsy was performed (20). After the culture medium was sent to the 337 metabolites analysis by mass spectrometry. 14 molecules with the highest score on the PLS-Da was submitted to the ROC curves showing the power of metabolic analysis to predict the sex of euploid embryos. Besides, we performed the functional enrichment analysis. Main results and the role of chance After the genetic analysis by PGT-a, we obtain 20 euploid embryos, being 12 female embryos and 08 male embryos. Comparing the quantitative target metabolomic analysis of the 337 metabolites in the embryo culture medium, we observed the Asymmetric dimethylarginine, FAD, Malic Acid, Serotonin, increased in female embryos and Adenosine monophosphate, L-Alanine, L-Arginine, Cysteamine, DL-Dopa, Flavin Mononucleotide, Methionine sulfone, Nicotinic acid, L-Tyrosine, Uracil in male embryos. Through the ROC curve, we can verify AUC = 0.937. This result suggests that the metabolomic analysis of the culture medium is valid to be used as a complement of PGT-A to know embryo sex diagnostic. The functional enrichment analysis shows the Asymmetric dimethylarginine and Malic Sulfone metabolism as the principal function alter by female embryos. Limitations, reasons for caution Small number of samples Wider implications of the findings: Further studies are needed to validate these findings for the diagnostic of sex embryos Trial registration number N/A

Determination of single embryo sex in Macaca mulatta and Mus musculus RNA-Seq transcriptome profiles

To account for sex as a biological variable, it is sometimes necessary to identify the sex of an embryo or embryonic cell that was used to generate libraries for RNA sequencing, without the sex being known a priori. The preferred approach for this would take advantage of the mRNA data, rather than relying on other methods that require separation and analysis of genomic DNA or diversion of limiting RNA for other assays. We describe here a method that has been optimized for this purpose in samples of rhesus monkey and mouse embryos. This method is broadly applicable to any species for which a sufficiently well characterized genome and knowledge of polymorphisms are available, and for embryos that are transcriptionally active and expressing their genome.

214 PRE-IMPLANTATION EMBRYO SEX DETERMINATION BY AMPLIFICATION OF AMELOGENIN GENE IN WATER BUFFALOES (BUBALUS BUBALIS)

Sex-specific sequence variability of the amelogenin (AMEL) gene has been observed in a variety of mammalian species. AMEL is a multi-copy gene expressed in abundance in the development of mammalian tooth enamel matrix. Its homologous genes are located on the X- or Y-chromosome, respectively. In our study, a pair of primers was designed to allow amplification of a single fragment of 312 bp from buffalo cows. Because the gene is slightly differently encoded on the Y-chromosome, the same primers were expected to produce 2 fragments of 312 and 249 bp from bulls. The primers were successfully applied to single, in vitro-produced Day-6 to Day-8 water buffalo blastocysts by direct lysis of the whole blastocysts, followed by PCR. The amplification on presumed female and male embryos consistently displayed the same sex-specific pattern. Of 100 blastocysts, 43 were determined to be females, 51 males, and 6 unknown (samples missed). We concluded that PCR amplification of the AMEL gene can be used for early embryo sex identification of water buffalo. This work was supported by the National Natural Science Foundation of China under Grant No. 31160456 and the Guangxi Natural Science Foundation of China under Grants No. 2013GXNSFAA019075 and 2014GXNSFAA118116.