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Dependable methods were developed for preimplantation sexing of human IVF embryos, for use in clinical settings where prospective parents are at high risk for transmission of X-linked diseases. Using single cultured cells and blastomeres from human embryos as model systems, a multiplex protocol was developed for rapid analysis via nested polymerase chain reaction (PCR). Reliability was enhanced by co-amplification of conserved amelogenin gene segments from both X and Y chromosomes, as well as Y-linked DYZ1 repetitive elements. Each cell was manually isolated and individually washed to avoid potential contaminants. Multiplex amplification allowed recognition of spurious amplification failures specific to particular amelogenin single-copy targets. The X-linked internal control and multiple Y-linked markers allowed recognition and exclusion of most aberrant samples, thus averting potential misdiagnosis. The optimized single-cell protocol reduced experimental sexing errors to < 2% (1/60), but also revealed potential pitfalls of single-cell analysis. With human triploid embryos, separate sampling of individual blastomeres provided concordant female or male signals. Slight modification adapted the procedure for diagnosis of biopsy material from blastocyst stage embryos, allowing separate analysis of multiple tubes containing multiple cells for improved reliability.

We report the world's first clinical pregnancy resulting from DNA-based enrichment for X-bearing human spermatozoa, for prevention of X-linked hydrocephalus. Sperm separation was followed by embryo biopsy and nested multiplex polymerase chain reaction (PCR) for gender determination. Enriched populations of X-bearing spermatozoa ranging from 80 to 89% pure as determined by fluorescence in-situ hybridization (FISH) resulted in in-vitro fertilization (IVF) rates indistinguishable from normal IVF procedures (65%). In two separate biopsy procedures, 7/9 and 15/16 of the resulting embryos were determined to be female by multiplex PCR. Embryo transfer resulted in a karyotypically normal female fetus. This technique should be widely applicable to gender selection for the prevention of genetic disorders.

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A total of 47 women were allocated, 23 were assigned to the GnRHa arm and 24 were assigned to estrogen and progesterone treatment arm. Patients' characteristics including age, BMI, gravidity, parity as well as basal FSH levels didn't differ significantly between the study groups. Treatment's characteristics including the FSH dosage, duration of stimulation, peak estradiol levels, number of oocytes retrieved, fertilization rates and number of embryo transferred also didn't differ significantly between the study groups. The implantation rate was 56.5% and 37.5% in the GnRHa arm and in the estrogen and progesterone arm, respectively (P¼ 0.1). The clinical pregnancy rate was higher in the GnRHa treatment group compared to the estrogen and progesterone group although the difference was not statistically significant (60.8% vs. 50%, P¼ 0.45). Of note, no cases of OHSS were observed in both study groups.CONCLUSIONS: Luteal support using GnRHa alone is as effective and safe as using intensive estrogen and progesterone supplementation following GnRHa triggering in high responders. This new approach in fresh embryo transfer in high responders after GnRHa triggering offer a more convenient luteal support without compromising implantation and clinical pregnancy rates.

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Objective: The vaginal and placental microbiomes are partially characterized and impact obstetric outcomes. The uterine microbiome is largely uncharacterized given the limitations of cultivationdependent analysis. Culture-free approaches to bacterial identification focus on sequencing of 16S ribosomal gene and allows for increased dynamic range in assessment. Here, we assess two Next Generation Sequencing (NGS) platforms for 16S sequencing, determine their advantages and disadvantages for 16S metagenomics, and study the endometrial microbial environment at the time of embryo transfer (ET). Design: Method validation and cohort study. Materials and Methods: The method validation of NGS 16S ribosomal gene sequencing was performed on known samples (Escherichia coli, Staphylococcus epidermidis, Cyanobacterium Synechococcus). The Ion 16S metagenomics workflow analyzed seven of nine hypervariable regions (V2,3,4,6,7,8,9) of 16S rRNA gene versus targeted sequences used in Illumina 16S workflow (V3-V4 and V4 only). Customized bioinformatics data analysis was used to improve the taxonomic assignments. Illumina V4 metagenomics workflow was further validated on Microbial Mock Community of 20 bacterial strains. The endometrial microbiome at the time of ET was characterized by analyzing transfer catheter tips with Illumina V4 metagenomics for 70 patients. Results: The built-in analysis from both Ion PGM and Illumina MiSeq metagenomics failed to generate correct taxonomic classification for genus or species for known single-or poly-microbial samples. The customized bioinformatics improved alignment, however for Ion 16S metagenomics workflow, the majority reads were family level. For both amplicons V3-V4 and V4, Illumina metagenomics workflow identified the genus or species level in the single-and poly-microbial samples. V4 had higher Operational Taxonomic Unit (OTU) counts than V3-V4. Subsequently, Illumina V4 metagenomics workflow with customized bioinformatics detected the genus or species level for all the 20 bacterial strains in the Microbial Mock Community. Finally, the endometrial microbiome of the 70 patients was assessed with Illumina V4 metagenomics and customized bioinformatics. Lactobacillus was detected in all the 70 samples along with other bacteria native to the reproductive tract (Corynebacterium, Bifidobacterium, Staphylococcus, Streptococcus). Conclusions: The Illumina V4 metagenomics workflow with customized bioinformatics provided a rapid and sensitive method for the identification of bacterial genus or species in single-or poly-microbial samples. Despite the limited starting material when analyzing clinical ET specimens, the Illumina V4 metagenomics approach provided adequate taxonomic identification. This sets the stage for a larger trial analyzing the relationship between endometrium microbial structure and implantation after ET. Disclosures: Nothing to disclose. Funding: None.