Minimally Invasive Cell-Free Human Embryo Aneuploidy Testing (miPGT-A) Utilizing Combined Spent Embryo Culture Medium and Blastocoel Fluid -Towards Development of a Clinical Assay.

Preimplantation genetic testing for aneuploidies (PGT-A) using trophectoderm (TE) biopsy samples is labour intensive, invasive, and subject to sampling bias. In this study, we report on the efficacy and factors affecting accuracy of a technique we pioneered for minimally invasive preimplantation genetic testing for aneuploidy (miPGT-A). Our technique uses cell-free embryonic DNA (cfeDNA) in spent embryo culture medium (SEM) combined with blastocoel fluid (BF) to increase the amount of assayable cfeDNA. We compared miPGT-A results (n = 145 embryos) with standard PGT-A analysis of the corresponding trophectoderm biopsy. We found that accuracy of miPGT was not related to blastocyst morphological grade. The overall concordance rate per sample for euploidy/aneuploidy status between miPGT-A and TE biopsy samples was 88/90 (97.8%), and was not different between good 47/48 (97.9%) and moderate/low quality blastocysts 41/42 (97.9%) (p > 0.05). Importantly, we also discovered that for cfeDNA analysis, the SurePlex whole genome amplification (WGA) kit can be utilized without an additional cell lysis/extraction DNA step; this efficiency likely reduces the risk of maternal contamination. Regarding origin of embryonic cfeDNA, the average amount of miPGT-A WGA-DNA we obtained from blastocysts with different morphological grades, as well as the size miPGT-A WGA-DNA fragments, suggest that it is unlikely that apoptosis and necrosis are only mechanisms of DNA release from the inner cell mass (ICM) and TE into BF and SEM.

Towards Improving Embryo Prioritization: Parallel Next Generation Sequencing of DNA and RNA from a Single Trophectoderm Biopsy.

Improved embryo prioritization is crucial in optimizing the results in assisted reproduction, especially in light of increasing utilization of elective single embryo transfers. Embryo prioritization is currently based on morphological criteria and in some cases incorporates preimplantation genetic testing for aneuploidy (PGT-A). Recent technological advances have enabled parallel genomic and transcriptomic assessment of a single cell. Adding transcriptomic analysis to PGT-A holds promise for better understanding early embryonic development and implantation, and for enhancing available embryo prioritization tools. Our aim was to develop a platform for parallel genomic and transcriptomic sequencing of a single trophectoderm (TE) biopsy, that could later be correlated with clinical outcomes. Twenty-five embryos donated for research were utilized; eight for initial development and optimization of our method, and seventeen to demonstrate clinical safety and reproducibility of this method. Our method achieved 100% concordance for ploidy status with that achieved by the classic PGT-A. All sequencing data exceeded quality control metrics. Transcriptomic sequencing data was sufficient for performing differential expression (DE) analysis. All biopsies expressed specific TE markers, further validating the accuracy of our method. Using PCA, samples clustered in euploid and aneuploid aggregates, highlighting the importance of controlling for ploidy in every transcriptomic assessment.

Evaluation of a novel non-invasive preimplantation genetic screening approach.

OBJECTIVE: To assess whether embryonic DNA isolated from blastocyst culture conditioned medium (BCCM) combined with blastocoel fluid (BF) could be used for blastocyst stage non-invasive preimplantation genetic testing for chromosomal aneuploidy (non-invasive preimplantation genetic screening, NIPGS). PATIENTS: 47 embryos from 35 patients undergoing IVF. INTERVENTIONS: DNA analysis of combined BCCM plus BF in comparison with trophectoderm (TE) biopsy and/or whole blastocyst (WB)using next generation sequencing (NGS). RESULTS: Embryonic DNA was successfully amplified in 47/47 NIPGS samples (28 frozen-thawed and 19 fresh culture samples) ranging from 6.3 to 44.0 ng/µl. For frozen-thawed embryos, the concordance rate for whole chromosome copy number per sample was equivalent between NIPGS vs. TE biopsy, NIPGS vs. WB and TE vs. WB samples taken from the same embryo was 87.5%; 96.4% and 91.7% respectively (P>0.05), and the rate of concordance per single chromosome was 99.3%, 99.7% and 99.7%, respectively (P>0.05). In fresh cases (Day 4 to Day 5/6 culture), the concordance rate for whole chromosome copy number per sample between NIPGS vs. TE samples taken from the same embryo was 100%, and the rate of concordance per single chromosome was 98.2% (P>0.05). CONCLUSIONS: A combination of BCCM and BF contains sufficient embryonic DNA for whole genome amplification and accurate aneuploidy screening. Our findings suggest that aneuploidy screening using BCCM combined with BF could potentially serve as a novel NIPGS approach for use in human IVF.

Impact of multinucleated blastomeres on embryo developmental competence, morphokinetics, and aneuploidy.

OBJECTIVE: To study the effect of human embryo multinucleation on the rate of aneuploidy, in vitro developmental morphokinetics, and pregnancy outcome. DESIGN: Retrospective study. SETTING: University-affiliated fertility center. PATIENT(S): A total of 296 patients undergoing IVF cycles. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Rate of multinucleation at the 2- and 4-cell stage, time-lapse morphokinetic parameters from zygote to blastocyst stage, ploidy of embryos analyzed by means of trophectoderm biopsy and array comparative genomic hybridization (PGS), and pregnancy outcome. RESULT(S): A total of 1,055 out of 2,441 (43.2%) embryos evaluated with the use of the Embryoscope time-lapse system showed blastomere multinucleation at the 2-cell stage (MN2). The frequency of this abnormality was substantially reduced in 4-cell-embryos (15.0%). Among all clinical factors analyzed, only maternal age had a positive correlation with multinucleation rate. The timing of cleavage divisions from the pronuclear fading to 5-cell embryo was significantly longer (1.0-2.5 h) in MN2 embryos than in non-MN2 control samples. Of the total embryos tested with the use of PGS (n = 607), the rates of multinucleation were similar in euploid versus aneuploid blastocysts (40.8% and 46.7%, respectively). All 24 chromosomes contributed to aneuploidy of MN2 embryos. There were 61 transfers of MN2 embryos that resulted in 45.9% clinical pregnancies and a 31.6% implantation rate. CONCLUSION(S): The frequency of multinucleation is high in human embryos cultured in vitro and equally affects euploid and aneuploid human embryos. It appears that most MN embryos have the capacity for self-correction during early cleavage divisions and can develop into euploid blastocysts resulting in healthy babies.

Secretion of human leukocyte antigen-G by human embryos is associated with a higher in vitro fertilization pregnancy rate.

OBJECTIVE: To investigate whether secretion of soluble human leukocyte antigen-G (HLA-G) by human embryos is associated with embryo development and IVF pregnancy outcome. DESIGN: Retrospective study. SETTING: In vitro fertilization program affiliated with a university research center. PATIENT(S): Infertile couples attending an IVF program were selected. INTERVENTION(S): Embryo culture conditioned medium (72 hours) from cases in which intracytoplasmic sperm injection was used for fertilization. MAIN OUTCOME MEASURE(S): Soluble HLA-G in embryo culture medium samples from IVF patients was assayed and associations between soluble HLA-G secretion and outcome measures were analyzed. RESULT(S): Two hundred seventy of 386 samples had detectable soluble HLA-G. Soluble HLA-G secretion was independent of embryo grade or patients' age. The cleavage rate of embryos secreting soluble HLA-G was significantly higher than that of those lacking it (blastomere number 6.71 +/- 0.09 vs. 5.86 +/- 0.22). The live birth rate from embryos with soluble HLA-G was significantly higher than that of those without (48.4% vs. 17.1%, chi(2) = 9.09). Combining soluble HLA-G detection and cleavage rate was most predictive of pregnancy. CONCLUSION(S): Our five conclusions are as follows: [1] embryonic secretion of soluble HLA-G protein is variable, [2] secretion of HLA-G is correlated with a higher cleavage rate, [3] secretion of HLA-G is associated with a higher pregnancy rate, [4] HLA-G secretion is a better independent predictor than cleavage rate alone, and [5] the combination of soluble HLA-G detection and high cleavage rate was the best predictor of outcome.

Is the zona pellucida thickness of human embryos influenced by women's age and hormonal levels?

OBJECTIVE: To evaluate whether zona pellucida thickness (ZPT) of human embryos is correlated with maternal age, patient's hormonal status, embryo quality, and IVF outcomes. DESIGN: Prospective study. SETTING: University-affiliated IVF clinic. PATIENT(S): Couples undergoing IVF-ET cycles. INTERVENTION(S): Zona measurements, clinical data collection. MAIN OUTCOME MEASURE(S): Correlation between the ZPT and maternal age, basal FSH and E(2) levels, stimulation protocols, cause of infertility, embryo quality, and implantation/pregnancy rates. RESULT(S): The measurements of ZPT were collected from 5,184 day 3 human embryos originated from 744 IVF patients. The overall mean ZPT was 16.18 ± 2.00 µm. No significant correlation was observed between the ZPT and the patient's age, E(2) values on the day of hCG administration, basal concentration of serum FSH, stimulation protocol, infertility diagnosis, and implantation/pregnancy rates. The ZPT was strongly influenced only by the embryo quality: Embryos with good morphology exhibited considerably thinner ZP compared with those of less favorable morphology (mean 15.87 ± 2.48 µm vs. 16.36 ± 2.57 µm, respectively). The ZPT had no significant impact on the implantation and pregnancy rates. CONCLUSION(S): The thickness of the human ZP of day 3 embryos is not influenced by women's age and hormonal levels. The strong correlation between ZPT and embryo quality suggests that thickness of ZP depends on inherent embryo properties. The overall ZPT is not a good predictive indicator for IVF clinical outcomes.

Laser zona thinning in women aged

The objective of this prospective randomized double-blind clinical trial was to evaluate whether laser zona pellucida thinning of human embryos improves clinical outcomes in women

Time-dependent capability of human oocytes for activation and pronuclear formation during metaphase II arrest.

BACKGROUND: The purpose of this study was to investigate the fertilization rate and developmental potential of human oocytes in relation to the duration of their metaphase II (MII) arrest stage following the extrusion of the first polar body (1PB). METHODS: Immature metaphase I oocytes (MI; study oocytes, n = 468) that underwent meiotic maturation during brief in vitro culture and their matured in vivo, MII siblings (control oocytes, n = 3293) were subjected to ICSI. Fertilization and early cleavage were evaluated in both study and control groups. RESULTS: The overall fertilization rate was significantly lower in the oocytes matured in vitro than in those matured in vivo (42 versus 77%, P < 0.0001). A significant relationship was observed between oocyte activation potential and the length of MII arrest. The majority of study oocytes injected soon after PB extrusion remained unfertilized (64%; 98/154 oocytes). The proportion of normally activated oocytes that contained two pronuclei and two PBs gradually increased with prolonged time of MII arrest (43 and 61% at 2 and 3-6 h after 1PB extrusion). Significantly more embryos originating from the study than control oocytes were arrested soon after the first two cleavage divisions (39 and 17%; P < 0.0001) and exhibited multinucleated blastomeres (23 and 13%; P < 0.0001), which suggests the existence of chromosomal abnormalities. CONCLUSIONS: Human oocytes progressively develop the ability for full activation and normal development during the MII arrest stage.