The developmental potential of mature oocytes derived from rescue in vitro maturation.

OBJECTIVE: To examine the developmental competence of immature oocytes in stimulated cycles, that matured after rescue in vitro maturation (IVM) compared with their sibling in vivo matured oocytes. DESIGN: Retrospective cohort study. SETTING: IVF clinic. PATIENTS: A total of 182 patients underwent 200 controlled ovarian stimulation cycles with intracytoplasmic sperm injection cycles in which immature oocytes were retrieved and at least one mature oocyte was obtained through rescue IVM. INTERVENTION: In vitro culture of immature germinal vesicle (GV) and metaphase I (MI) oocytes, retrieved in stimulated cycles. MAIN OUTCOME MEASURES: Fertilization rate, cleavage rate, blastulation rate, ploidy of embryos evaluated using preimplantation genetic testing for aneuploidy, morphokinetic parameters and pregnancy outcomes. RESULTS: In total, 2,288 oocytes were retrieved from 200 cycles. After denudation, 1,056 of the oocytes (46% ± 16%) were classified as metaphase II (MII). A total of 333/375 (89%) of MI oocytes and 292/540 (54%) of GV oocytes matured overnight and underwent intracytoplasmic sperm injection. The fertilization rates of matured oocytes from MI rescue IVM (R-MI) and from GV rescue IVM (R-GV) were comparable with those of their sibling MII oocytes (71% vs. 66%; 66% vs. 63%, respectively). Early cleavage rates (80% ± 35% vs. 92% ± 20%; 80% ± 42% vs. 95% ± 28%, respectively) and blastulation rates (32 ± 40% vs. 62 ± 33%; 24 ± 37% vs. 60 ± 35%, respectively) were significantly decreased in rescue IVM matured oocytes (R-oocytes)-derived zygotes, but the blastocyst (BL) euploidy rate and "good quality" BL rate were comparable with those of MII sibling-derived embryos. In addition, rescue IVM embryos showed significantly higher levels of multinucleation at the 2- and 4-cell stages, as well as higher rates of zygote direct cleavage from one to 3 to 4 cells. Overall, 21 transfers of rescue IVM embryos resulted in 3 healthy live births. CONCLUSIONS: For patients with a low maturation rate and/or low numbers of mature oocytes at retrieval, rescue IVM may contribute more competent oocytes and additional viable BLs for transfer from the same stimulation cycle, maximizing the chances for pregnancy and live birth.

Quantitative selection of single human sperm with high DNA integrity for intracytoplasmic sperm injection.

OBJECTIVE: To study at the single-cell level whether a sperm's motility and morphology parameters reflect its DNA integrity, and to establish a set of quantitative criteria for selecting single sperm with high DNA integrity. DESIGN: Prospective study. SETTING: In vitro fertilization center and university laboratories. PATIENT(S): Male patients undergoing infertility treatments. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The motility and morphology parameters of each sperm were measured with the use of computer vision algorithms. The sperm was then aspirated and transferred for DNA fragmentation measurement by single-cell gel electrophoresis (comet assay). RESULT(S): We adapted the World Health Organization criteria, which were originally defined for semen analysis, and established a set of quantitative criteria for single-sperm selection in intracytoplasmic sperm injection. Sperm satisfying the criteria had significantly lower DNA fragmentation levels than the sample population. Both normal motility and normal morphology were required for a sperm to have low DNA fragmentation. The quantitative criteria were integrated into a software program for sperm selection. In blind tests in which our software and three embryologists selected sperm from the same patient samples, our software outperformed the embryologists and selected sperm with the highest DNA integrity. CONCLUSION(S): At the single-cell level, a sperm's motility and morphology parameters reflect its DNA integrity. The developed technique and criteria hold the potential to mitigate the risk factor of sperm DNA fragmentation in intracytoplasmic sperm injection.

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.

The impact of hyaluronan-enriched culture medium and intrauterine infusion of human chorionic gonadotropin on clinical outcomes in blastocyst transfer cycles.

Over the last few decades, advances in ovarian hormonal stimulation, embryology laboratory technologies and embryo genetic testing, have significantly enhanced clinical outcomes in human assisted reproduction technologies (ART). However, embryo implantation remains a major bottleneck in achieving better pregnancy and live birth rates. Thus, there is growing interest in establishing new approaches to enhance implantation efficiency after embryo transfer. With advanced molecular techniques, many promising biomarkers associated with embryonic and endometrial changes occurring prior to and during embryo implantation have been identified. However, despite the progress in applying novel procedures into IVF practice, clinical evaluation of those biomarkers has so far reached modest predictive value for enhancing blastocyst developmental potential and endometrial receptivity. Therefore, other simpler strategies have also been introduced to increase the rates of successful clinical pregnancies and live births. One of these approaches is to investigate the impact of using embryo transfer medium containing high concentrations of an adherence compound, such as hyaluronic acid (HA), on IVF outcomes. Additionally, intrauterine infusion of a small volume of human chorionic gonadotropin (hCG) at the time of embryo transfer (ET) has also been proposed as a technique that might be advantageous for increasing the clinical outcomes, considering the fact that hCG plays a critical role in synchronizing endometrial and fetal development. However, the current findings from both interventions remain controversial, demonstrating a mixture of positive and indifferent results of these treatments in ART cycles. Further research will be crucial for a better understanding of the molecular mechanism of cross-talk between the blastocyst and the maternal endometrium during the optimal implantation period when using either hyaluronan-enriched medium or hCG infusion before embryo transfers. Therefore, this review aims to present existing literature related to both treatments, emphasizing their effects on blastocyst implantation.Abbreviations: ART: assisted reproduction technologies; HA: hyaluronic acid; hCG: human chorionic gonadotrophin; IVF: in vitro Fertilization; ET: embryo transfer; pH: hydrogen ions; CO2: Carbone dioxide; O2: Oxygen; PGT: pre-implantation genetic testing; FET: frozen embryo transfer; PCOS: Polycystic ovarian syndrome; DNA: deoxyribonucleic acid; miRNA: micro-ribonucleic acid; EVs: extracellular vesicles; ERA: endometrial receptivity array; CD44 and RHAMM: primary hyaluronan surface receptors; RCT: randomized clinical trials; LBR: life birth rate; CPR: clinical pregnancy rate; IR: implantation rate.

Duplication of the sperm genome by human androgenetic embryo production: towards testing the paternal genome prior to fertilization.

There is currently no technique for evaluating the sperm genome before fertilization. However, sperm genome duplication could offer a way forward, whereby one of the sister blastomeres of a 2-cell haploid androgenetic embryo could be analysed. A method was developed for production of human androgenotes by enucleation of oocytes at telophase II (TII) after intracellular sperm injection (ICSI). The results were compared with those obtained via the more usual procedure of oocyte enucleation at metaphase II (MII) prior to ICSI. TII enucleation led to an improvement in the rate of embryo survival, increased the production rate of 1PN-embryos, and also the production of 2- to 8-cell-stage embryos (85.0, 74.9 and 65.8% in TII enucleation, versus 73.8, 48.9 and 33.3% in MII enucleation). Fluorescence in-situ hybridization (FISH) analysis of 30 2- to 5-cell androgenic embryos for two to seven chromosomes revealed the correct chromosome distribution in 76.7% of haploid human androgenotes.