Personalization of IVF-ICSI workflow based on patient characteristics improves IVF laboratory outcomes and embryo ploidy by PGT-A.

BACKGROUND: Intracytoplasmic sperm injection (ICSI) has become a common method of fertilization in assisted reproduction worldwide. However, there are still gaps in knowledge of the ideal IVF-ICSI workflow including the optimal duration of time between induction of final oocyte maturation, oocyte denudation and ICSI. The aim of this study was to examine outcomes following different workflow protocols in IVF-ICSI procedures in blastocysts that have undergone undisturbed incubation and preimplantation genetic testing for aneuploidy (PGT-A) prior to transfer. METHODS: Retrospective secondary analysis of 113 patients (179 IVF cycles, 713 embryos), all of whom have gone through IVF-ICSI and PGT-A using undisturbed culture. Predictive test variables were the length of time from: trigger to OPU, OPU to denudation, and denudation to ICSI. Outcome metrics assessed were: maturation, fertilization, blastulation and euploid rates. Generalized Estimated Equations Linear Model was used to examine the relationship between key elements of a given cycle and continuous outcomes and LOESS curves were used to determine the effect over time. RESULTS: In a paired multi-regression analysis, where each patient served as its own control, delaying OPU in patients with unexplained infertility improved both maturation and blastulation rates (b = 29.7, p < 0.0001 and b = 9.1, p = 0.06, respectively). Longer incubation with cumulus cells (CCs) significantly correlated with improved ploidy rates among patients under 37, as well as among patients with unexplained infertility (r = 0.22 and 0.29, respectively), which was also evident in a multiple regression analysis (b = 6.73, p < 0.05), and in a paired analysis (b = 6.0, p < 0.05). Conversely, among patients with a leading infertility diagnosis of male factor, longer incubation of the denuded oocyte prior to ICSI resulted in a significantly higher euploid rate (b = 15.658, p < 0.0001). CONCLUSIONS: In this study we have demonstrated that different IVF-ICSI workflows affect patients differently, depending on their primary infertility diagnosis. Thus, ideally, the IVF-ICSI workflow should be tailored to the individual patient based on the primary infertility diagnosis. This study contributes to our understanding surrounding the impact of IVF laboratory procedures and highlights the importance of not only tracking "classic" IVF outcomes (maturation, fertilization, blastulation rates), but highlights the importance that these procedures have on the ploidy of the embryo.

Is there a correlation between paternal age and aneuploidy rate? An analysis of 3,118 embryos derived from young egg donors.

OBJECTIVE: To investigate a possible correlation between chromosomal aberrations and paternal age, analyzing embryos derived from young oocyte donors, with available preimplantation genetic testing for aneuploidy results from day 5/6 trophectoderm biopsy obtained by next-generation sequencing for all 24 chromosomes. DESIGN: Retrospective cohort study. SETTING: Canadian fertility centre. PATIENT(S): A total of 3,118 embryos from 407 male patients, allocated into three paternal age groups: group A, ≤39 years (n = 203); group B, 40-49 years (n = 161); group C, ≥50 years (n = 43). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The primary outcomes were aneuploidy, euploidy, mosaicism, and blastocyst formation rates. Secondary endpoints were comparison of specific chromosome aneuploidy, segmental and complex (involving two chromosomes + mosaicism >50%) aneuploidy, and analysis of overall percentage of chromosomal gains and losses within each group. RESULT(S): The study included 437 in vitro fertilization (IVF) antagonist cycles using 302 oocyte donors in which preimplantation genetic testing for aneuploidy was performed. Overall, 70.04% of embryos were euploid, 13.9% were aneuploid, and 16.06% were mosaic. No significant differences among paternal age groups A, B, and C were found in euploidy rates (69.2%, 70.6%, 71.4%, respectively), aneuploidy rates (14.7%, 12.8%, 13.9%, respectively) or mosaicism rates (16.1%, 16.6%, 13.6%; respectively). The fertilization rate was lower in group C compared with group B (76.35% vs. 80.09%). No difference was found in blastocyst formation rate between the study groups (median 52% [interquartile range, 41%, 67%] vs. 53% [42%, 65%] vs. 52% [42%, 64%], respectively). A generalized linear mixed model regression analysis for embryo ploidy rates found older oocyte donor age to be independently associated with embryo aneuploidy (odds ratio = 1.041; 95% CI, 1.009-1.074). The rate of segmental aneuploidies was significantly higher in the older versus younger paternal age group (36.6% vs. 19.4%). CONCLUSION(S): No association was found between paternal age and aneuploidy rates in embryos derived from IVF cycles using young oocyte donors, after adjusting for donor, sperm, and IVF cycle characteristics. Advanced paternal age ≥ 50, compared with younger paternal ages, was associated with a lower fertilization rate and increased rate of segmental aberrations.

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.

Sperm DNA fragmentation index does not correlate with blastocyst euploidy rate in egg donor cycles.

Sperm DNA fragmentation is a known etiology for male infertility. We evaluated the impact of sperm DNA fragmentation index (DFI) on blastocyst euploidy in IVF cycles with egg donors. This observational retrospective study, which was conducted in a university affiliated fertility clinic, included IVF-ICSI-pre-implantation Genetic Screening (PGS) egg donor cycles in which DFI was tested prior to IVF, between January 1st, 2014 and July 31st, 2016. Twenty-seven cycles with DFI > 15% were included in the study group and compared with 18 cycles of DFI < 15% within control group. Research group participants had significantly lower sperm count and motility (55.4*106/ml and 37.4%, respectively) compared with controls (92.5*106/ml and 55.7%, respectively, p < .05). The groups were similar in terms of donors' demography (age, BMI), ovarian reserve (AMH, AFC) and response to hormonal stimulation (E2 level on triggering day and number of retrieved eggs). Embryo development (from 2PN through day 3 embryos to blastocysts) was similar as well. The number of biopsied blastocysts from study and control groups was 171 and 87, respectively. PGS with array comprehensive genomic hybridization revealed comparable euploidy rates of 69.3% and 67.3%, respectively (p > .05). DFI did not have an impact on the blastocyst euploidy rate in IVF cycles with egg donors.

Sperm DNA fragmentation index does not correlate with blastocyst aneuploidy or morphological grading.

High DNA fragmentation index (DFI) may be associated with poor outcome after IVF. Our aim was to determine whether DFI impacts blastocyst quality or clinical outcome. This retrospective study included 134 couples who underwent 177 IVF-ICSI and pre-implantation genetic screening (PGS) cycles during January 1st, 2014-March 31st, 2016 and had documented previous DFI. Group 1 (DFI>30%) encompassed 25 couples who underwent 36 cycles; Group 2 (DFI 15-30%) included 45 couples and 57 cycles; group 3 (DFI<15%) included 64 couples and 83 cycles. Male partners within group 1 were older (45.1 compared to 40.6 and 38.3 years, respectively, p<0.05), had higher BMI (32.4 compared to 26.6 and 25.8 respectively, p<0.05) and lower sperm count and motility (46*106/ml and 35.5%, respectively) compared to groups 2 (61.8*106/ml and 46.6%, respectively) and 3 (75.8*106/ml and 55.1%, respectively, p<0.05). Female parameters including ovarian reserve and response and embryo development were similar. Total numbers of biopsied blastocysts were 116, 175 and 259 in groups 1, 2 and 3, respectively. PGS for 24 chromosomes revealed comparable euploidy rate of 46-50.4%, with a similar morphological classification. No significant differences were found regarding pregnancy rates or pregnancy loss. It seems that DFI doesn't correlate with blastocyst aneuploidy or morphological grading.

The effects of the apoE4 genotype on the developing mouse retina.

Apolipoprotein E4 (apoE4), the most prevalent genetic risk factor for Alzheimer's disease (AD), is associated with neuronal and vascular impairments. The retina, which is as an extension of the central nervous system (CNS), is a particularly suitable model for studying developmental and functional aspects of the neuronal and vascular systems. This study investigates the apoE4-dependent developmental effects on the retinal vasculature and neuronal systems and on the levels of apoE and the vascular endothelial growth factor (VEGF) in the retina. This was performed utilizing retinas of 4, 7, 12, and of 120-day-old human-apoE4-targeted replacement mice and of corresponding mice that express the AD benign isoform, apoE3. The results obtained revealed retinal vascular pathology in the apoE4 mice, which started on the early post-natal days. This includes transient increase in vascular branching, and vascular buds which are round vascular elements representing sprouting or retracting vessels. These effects peaked and ended during the neonatal period. Examination of the synaptic system utilizing the pre-synaptic marker synaptophysin revealed a significant decrease of retinal synaptic density in the apoE4 mice, which was detectable by post-natal day 12 (P12). These morphological changes are associated with neonatal age-dependent elevation in the apoE levels in both apoE3 and apoE4 retinas which is more profound in the apoE4 mice and a corresponding increase in VEGF levels, which is less profound in the apoE4 mice. Additionally, we observed lower levels of retinal VEGF in the apoE4 mice compared to the apoE3 mice retinas on P12. These results show that apoE4 has a transient vascular effect during retinal development that ends in the neonatal period, which is accompanied by a synaptic effect that begins at the end of the neonatal period. These findings show that the apoE4 genotype can have distinct developmental effects on both the retinal vasculature and on neurons and suggest that the vascular effects of apoE4 may be related to reduced levels of VEGF.

VEGF Mediates ApoE4-Induced Neovascularization and Synaptic Pathology in the Choroid and Retina.

Apolipoprotein E4 (apoE4), the most prevalent genetic risk factor for Alzheimer's disease (AD), is associated with neuronal and vascular impairments. Recent findings suggest that retina of apoE4 mice have synaptic and functional impairments. We presently investigated the effects of apoE4 on retinal and choroidal vasculature and the possible role of VEGF in these effects. There were no histological differences between the retinal and choroidal vasculatures of naïve apoE3 and apoE4 mice. In contrast, laserdriven choroidal injury induced higher levels of choroidal neovascularization (CNV) in apoE4 than in apoE3 mice. These effects were associated with an inflammatory response and with activation of the Muller cells and asrocytic markers gluthatione synthetase and GFAP, all of which were more pronounced in the apoE4 mice. CNV also induced a transient increase in the levels of the synaptic markers synaptophysin and PSD95 which were however similar in the apoE4 and apoE3 naive mice. Retinal and choroidal VEGF and apoE levels were lower in naïve apoE4 than in corresponding apoE3 mice. In contrast, VEGF and apoE levels rose more pronouncedly following laser injury in the apoE4 than in apoE3 mice. Taken together, these findings suggest that the apoE4-induced retinal impairments, under basal conditions, may be related to reduced VEGF levels in the eyes of these mice. The hyper-neovascularization in the apoE4 mice might be driven by increased inflammation and the associated surge in VEGF following injury. Retinal and choroidal VEGF and apoE levels were lower in naïve apoE4 than in corresponding apoE3 mice. In contrast, VEGF and apoE levels rose more pronouncedly following laser injury in the apoE4 than in apoE3 mice. Taken together, these findings suggest that the apoE4-induced retinal impairments, under basal conditions, may be related to reduced VEGF levels in the eyes of these mice. The hyper-neovascularization in the apoE4 mice might be driven by increased inflammation and the associated surge in VEGF following injury.

ApoE4 induces synaptic and ERG impairments in the retina of young targeted replacement apoE4 mice.

The vertebrate retina, which is part of the central nervous system, is a window into the brain. The present study investigated the extent to which the retina can be used as a model for studying the pathological effects of apolipoprotein E4 (apoE4), the most prevalent genetic risk factor for Alzheimer's disease (AD). Immunohistochemical studies of retinas from young (4 months old) apoE4-targeted replacement mice and from corresponding mice which express the AD benign apoE3 allele, revealed that the density of the perikarya of the different classes of retinal neurons was not affected by apoE4. In contrast, the synaptic density of the retinal synaptic layers, which was assessed immunohistochemically and by immunoblot experiments, was significantly lower in the apoE4 than in the apoE3 mice. This was associated with reduced levels of the presynaptic vesicular glutamatergic transporter, VGluT1, but not of either the GABAergic vesicular transporter, VGaT, or the cholinergic vesicular transporter, VAChT, suggesting that the glutamatergic nerve terminals are preferentially affected by apoE4. In contrast, the post synaptic scaffold proteins PSD-95 and Gephyrin, which reside in excitatory and inhibitory synapses, respectively, were both elevated, and their ratio was not affected by apoE4. Electroretinogram (ERG) recordings revealed significant attenuation of mixed rod-cone responses in dark-adapted eyes of apoE4 mice. These findings suggest that the reduced ERG response in the apoE4 mice may be related to the observed decrease in the retinal nerve terminals and that the retina could be used as a novel model for non-invasive monitoring of the effects of apoE4 on the CNS.

Pax6 dosage requirements in iris and ciliary body differentiation.

Pax6 is a highly conserved transcription factor that controls the morphogenesis of various organs. Changes in Pax6 dosage have been shown to affect the formation of multiple tissues. PAX6 haploinsufficiency leads to aniridia, a pan-ocular disease primarily characterized by iris hypoplasia. Herein, we employ a modular system that includes null and overexpressed conditional alleles of Pax6. The use of the Tyrp2-Cre line, active in iris and ciliary body (CB) primordium, enabled us to investigate the effect of varying dosages of Pax6 on the development of these ocular sub-organs. Our findings show that a lack of Pax6 in these regions leads to dysgenesis of the iris and CB, while heterozygosity impedes growth of the iris and maturation of the iris sphincter. Overexpression of the canonical, but not the alternative splice variant of Pax6 results in severe structural aberrations of the CB and hyperplasia of the iris sphincter. A splice variant-specific rescue experiment revealed that both splice variants are able to correct iris hypoplasia, while only the canonical form rescues the sphincter. Overall, these findings demonstrate the dosage-sensitive roles of Pax6 in the formation of both the CB and the iris.