First babies conceived with Automated Intracytoplasmic Sperm Injection.

RESEARCH QUESTION: Can an automated sperm injection robot perform Automated Intracytoplasmic Sperm Injection (ICSIA) for use in human IVF? DESIGN: The ICSIA robot automated the sperm injection procedure, including injection pipette advancement, zona pellucida and oolemma penetration with piezo pulses, and pipette removal after sperm release. The robot was first tested in mouse, hamster and rabbit oocytes, and subsequently using discarded human oocytes injected with microbeads. A small clinical pilot trial was conducted with donor oocytes to study the feasibility of the robot in a clinical setting. The ICSIA robot was controlled by engineers with no micromanipulation experience. Results were compared with those obtained with manual ICSI conducted by experienced embryologists. RESULTS: The ICSIA robot demonstrated similar results to the manual procedure in the different animal models tested as well as in the pre-clinical validations conducted in discarded human oocytes. In the clinical validation, 13 out of 14 oocytes injected with ICSIA fertilized correctly versus 16 out of 18 in the manual control; eight developed into good-quality blastocysts versus 12 in the manual control; and four were diagnosed as chromosomally normal versus 10 euploid in the manual control. Three euploid blastocysts from the ICSIA robot group have been transferred into two recipients, which resulted in two singleton pregnancies and two babies born. CONCLUSIONS: The ICSIA robot showed high proficiency in injecting animal and human oocytes when operated by inexperienced personnel. The preliminary results obtained in this first clinical pilot trial are within key performance indicators.

Human Uterine Lavage: First Live Births from In Vivo Conceived Genetically Screened Blastocysts.

INTRODUCTION: We wish to report the first live births from genetically screened human euploid blastocysts obtained by uterine lavage. The embryos transferred to infertile women were previously obtained using a novel fully automated uterine lavage catheter and fluid recovery device developed for this indication. The objective of this portion of the research was to confirm embryo implantation and live births with these unique in vivo conceived blastocysts obtained by uterine lavage. METHODS: In vivo conceived embryos recovered by uterine lavage 5 days after intrauterine insemination were available for embryo donation. In vivo embryos were the result of prior controlled ovarian stimulation cycles in oocyte donors and intrauterine insemination with donor sperm. An observational case series of nine embryo transfer procedures was performed at an outpatient fertility center. One to two embryos were transferred to eight infertile women since one woman had two separate embryo transfer procedures. RESULTS: Nine embryo transfer procedures were performed with 14 blastocysts in eight women resulting in a blastocyst implantation rate of 36% (5/14) and live birth rate of 44% (4/9). Five infants have been born from the four delivered pregnancies with one set of twins. CONCLUSIONS: This is the first report of live births from genetically screened human euploid blastocysts obtained by uterine lavage. The nonsurgical uterine lavage office procedure represents the only current approach to obtain in vivo conceived embryos and can provide a benchmark for comparison to standard in vitro cultured blastocysts. Live births of in vivo conceived blastocysts represent the validation that the nonsurgical uterine lavage procedure allows simplified access to naturally conceived embryos without performing the surgical procedure of an oocyte aspiration. Owing to its simplicity, uterine lavage may be useful in screening embryos for preimplantation genetic testing for aneuploidy in fertile and infertile couples. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov (Identifier NCT03426007).

The overall goal of this research was to develop a procedure that would allow collection of naturally conceived human embryos and compare them to embryos that result from the standard process of in vitro fertilization (IVF). IVF is a procedure where eggs are surgically removed from the ovaries and fertilized with sperm in a laboratory. Embryos from IVF are cultured for 3­7 days before they are placed back into a woman's uterus to establish a pregnancy. Uterine lavage is a different procedure where the sperm fertilizes an egg in the normal process of conception and the uterus is rinsed with fluid to recover the embryo before implantation. The embryos reported in this study were the first to be obtained in over 30 years owing to many improvements in the overall uterine lavage procedure. Until our initial study findings reported in 2020, the vast majority of information on embryo development was based on embryos fertilized and cultured in a laboratory. Our prior report of embryos obtained by uterine lavage compared with IVF embryos from the same women demonstrated a significantly better appearance of the embryos recovered by lavage. This current report documents the first live births from these genetically screened naturally conceived human embryos. The live births provide evidence that uterine lavage allows ready access to normal embryos without performing the surgical procedure IVF. Owing to the simplicity of uterine lavage, the procedure may improve access to genetic testing of embryos before pregnancy.

Embryologist agreement when assessing blastocyst implantation probability: is data-driven prediction the solution to embryo assessment subjectivity?

STUDY QUESTION: What is the accuracy and agreement of embryologists when assessing the implantation probability of blastocysts using time-lapse imaging (TLI), and can it be improved with a data-driven algorithm? SUMMARY ANSWER: The overall interobserver agreement of a large panel of embryologists was moderate and prediction accuracy was modest, while the purpose-built artificial intelligence model generally resulted in higher performance metrics. WHAT IS KNOWN ALREADY: Previous studies have demonstrated significant interobserver variability amongst embryologists when assessing embryo quality. However, data concerning embryologists' ability to predict implantation probability using TLI is still lacking. Emerging technologies based on data-driven tools have shown great promise for improving embryo selection and predicting clinical outcomes. STUDY DESIGN, SIZE, DURATION: TLI video files of 136 embryos with known implantation data were retrospectively collected from two clinical sites between 2018 and 2019 for the performance assessment of 36 embryologists and comparison with a deep neural network (DNN). PARTICIPANTS/MATERIALS, SETTING, METHODS: We recruited 39 embryologists from 13 different countries. All participants were blinded to clinical outcomes. A total of 136 TLI videos of embryos that reached the blastocyst stage were used for this experiment. Each embryo's likelihood of successfully implanting was assessed by 36 embryologists, providing implantation probability grades (IPGs) from 1 to 5, where 1 indicates a very low likelihood of implantation and 5 indicates a very high likelihood. Subsequently, three embryologists with over 5 years of experience provided Gardner scores. All 136 blastocysts were categorized into three quality groups based on their Gardner scores. Embryologist predictions were then converted into predictions of implantation (IPG ≥ 3) and no implantation (IPG ≤ 2). Embryologists' performance and agreement were assessed using Fleiss kappa coefficient. A 10-fold cross-validation DNN was developed to provide IPGs for TLI video files. The model's performance was compared to that of the embryologists. MAIN RESULTS AND THE ROLE OF CHANCE: Logistic regression was employed for the following confounding variables: country of residence, academic level, embryo scoring system, log years of experience and experience using TLI. None were found to have a statistically significant impact on embryologist performance at α = 0.05. The average implantation prediction accuracy for the embryologists was 51.9% for all embryos (N = 136). The average accuracy of the embryologists when assessing top quality and poor quality embryos (according to the Gardner score categorizations) was 57.5% and 57.4%, respectively, and 44.6% for fair quality embryos. Overall interobserver agreement was moderate (κ = 0.56, N = 136). The best agreement was achieved in the poor + top quality group (κ = 0.65, N = 77), while the agreement in the fair quality group was lower (κ = 0.25, N = 59). The DNN showed an overall accuracy rate of 62.5%, with accuracies of 62.2%, 61% and 65.6% for the poor, fair and top quality groups, respectively. The AUC for the DNN was higher than that of the embryologists overall (0.70 DNN vs 0.61 embryologists) as well as in all of the Gardner groups (DNN vs embryologists-Poor: 0.69 vs 0.62; Fair: 0.67 vs 0.53; Top: 0.77 vs 0.54). LIMITATIONS, REASONS FOR CAUTION: Blastocyst assessment was performed using video files acquired from time-lapse incubators, where each video contained data from a single focal plane. Clinical data regarding the underlying cause of infertility and endometrial thickness before the transfer was not available, yet may explain implantation failure and lower accuracy of IPGs. Implantation was defined as the presence of a gestational sac, whereas the detection of fetal heartbeat is a more robust marker of embryo viability. The raw data were anonymized to the extent that it was not possible to quantify the number of unique patients and cycles included in the study, potentially masking the effect of bias from a limited patient pool. Furthermore, the lack of demographic data makes it difficult to draw conclusions on how representative the dataset was of the wider population. Finally, embryologists were required to assess the implantation potential, not embryo quality. Although this is not the traditional approach to embryo evaluation, morphology/morphokinetics as a means of assessing embryo quality is believed to be strongly correlated with viability and, for some methods, implantation potential. WIDER IMPLICATIONS OF THE FINDINGS: Embryo selection is a key element in IVF success and continues to be a challenge. Improving the predictive ability could assist in optimizing implantation success rates and other clinical outcomes and could minimize the financial and emotional burden on the patient. This study demonstrates moderate agreement rates between embryologists, likely due to the subjective nature of embryo assessment. In particular, we found that average embryologist accuracy and agreement were significantly lower for fair quality embryos when compared with that for top and poor quality embryos. Using data-driven algorithms as an assistive tool may help IVF professionals increase success rates and promote much needed standardization in the IVF clinic. Our results indicate a need for further research regarding technological advancement in this field. STUDY FUNDING/COMPETING INTEREST(S): Embryonics Ltd is an Israel-based company. Funding for the study was partially provided by the Israeli Innovation Authority, grant #74556. TRIAL REGISTRATION NUMBER: N/A.

Using outcome data from one thousand mosaic embryo transfers to formulate an embryo ranking system for clinical use.

OBJECTIVE: To study how the attributes of mosaicism identified during preimplantation genetic testing for aneuploidy relate to clinical outcomes, in order to formulate a ranking system of mosaic embryos for intrauterine transfer. DESIGN: Compiled analysis. SETTING: Multi-center. PATIENT(S): A total of 5,561 euploid blastocysts and 1,000 mosaic blastocysts used in clinical transfers in patients undergoing fertility treatment. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Implantation (gestational sac), ongoing pregnancy, birth, and spontaneous abortion (miscarriage before 20 weeks of gestation). RESULT(S): The euploid group had significantly more favorable rates of implantation and ongoing pregnancy/birth (OP/B) compared with the combined mosaic group or the mosaic group affecting only whole chromosomes (implantation: 57.2% vs. 46.5% vs. 41.8%; OP/B: 52.3% vs. 37.0% vs. 31.3%), as well as lower likelihood of spontaneous abortion (8.6% vs. 20.4% vs. 25%). Whole-chromosome mosaic embryos with level (percent aneuploid cells) <50% had significantly more favorable outcomes than the ≥50% group (implantation: 44.5% vs. 30.4%; OP/B: 36.1% vs. 19.3%). Mosaic type (nature of the aneuploidy implicated in mosaicism) affected outcomes, with a significant correlation between number of affected chromosomes and unfavorable outcomes. This ranged from mosaicism involving segmental abnormalities to complex aneuploidies affecting three or more chromosomes (implantation: 51.6% vs. 30.4%; OP/B: 43.1% vs. 20.8%). Combining mosaic level, type, and embryo morphology revealed the order of subcategories regarding likelihood of positive outcome. CONCLUSION(S): This compiled analysis revealed traits of mosaicism identified with preimplantation genetic testing for aneuploidy that affected outcomes in a statistically significant manner, enabling the formulation of an evidence-based prioritization scheme for mosaic embryos in the clinic.

Aneuploidy and recombination in the human preimplantation embryo. Copy number variation analysis and genome-wide polymorphism genotyping.

RESEARCH QUESTION: What are the incidence and patterns of meiotic trisomies and recombination separately and in relation to each other at the blastocyst stage via single nucleotide polymorphism genotyping combined with array comparative genomic hybridization. DESIGN: Single nucleotide polymorphism microarrays were carried out on a total of 1442 blastocyst stage embryos derived from 268 fertile couples undergoing preimplantation genetic diagnosis for the purposes of avoiding transmittance of known single gene disorders to their offspring; 24-chromosome aneuploidy screening via array comparative genomic hybridization was carried out in parallel. RESULTS: One hundred per cent of meiotic trisomies identified in these embryos were of maternal origin and their incidence increased significantly with advancing maternal age (P < 0.0001). A total of 55.8% of meiotic trisomies were meiosis I-type and 44.2% were meiosis II-type. Certain chromosomes were affected more by meiosis I-type errors, whereas others experienced more meiosis II-type errors. A detailed recombination analysis was carried out for 11,476 chromosomes and 17,763 recombination events were recorded. The average number of recombination sites was 24.0 ± 0.3 for male meiosis and 41.2 ± 0.6 for female meiosis (autosomes only). Sex-specific differences were observed in the locations of recombination sites. Comparative analysis conducted between 190 euploid embryos and 69 embryos presenting maternal meiotic trisomies showed similar recombination rates (P = 0.425) and non-recombinant chromatid rates (P = 0.435) between the two categories; differences, however, were observed when analysing embryos affected with specific maternal meiotic trisomies. CONCLUSIONS: This study yielded unique data concerning recombination and the origin of aneuploidies observed during the first few days of life and provides a novel insight into these important biological processes.

Follicle size indicates oocyte maturity and blastocyst formation but not blastocyst euploidy following controlled ovarian hyperstimulation of oocyte donors.

STUDY QUESTION: Is there is an association between follicle size and the quality of oocytes retrieved from them as judged by ability to achieve the blastocyst stage, blastocyst grades and blastocyst ploidy? SUMMARY ANSWER: Although follicle size is a valuable predictor of oocyte maturity and is a significant predictor of the ability of a fertilized oocyte to become a quality blastocyst, the ploidy of each quality blastocyst is not related to the size of the follicle from which its oocyte was retrieved. WHAT IS KNOWN ALREADY: It is unclear whether the oocytes within larger follicles are the best oocytes of the cohort. Although there have been studies examining follicle size in relation to embryo quality, there has been no study relating the incidence of euploidy in embryos to follicle size. STUDY DESIGN, SIZE, DURATION: The purpose of this study was to examine follicle sizes and the oocytes from those follicles (and the embryos that result from those oocytes) to see if there is an association between follicle size and the quality of oocytes as judged by ability to achieve the blastocyst stage, blastocyst grades and blastocyst ploidy. Follicle sizes for oocytes were assessed both as diameters (mm) and as Z values (expressed as their size relative to the mean and standard deviation of that donor's follicular cohort). Comparisons were made using cumulative histograms, rolling averages and receiver operator characteristic (ROC) curves and its AUC. PARTICIPANTS/MATERIALS, SETTING, METHODS: Twenty-two oocyte donors (ages: 24.5 ± 3.5 years) whose recipients would use ICSI for insemination were enrolled in this study. Follicles were aspirated one-at-a-time to be certain that the aspirated oocyte was from the same follicle measured. The follicle measurement (size) was noted in the embryology records. Oocytes were cultured individually throughout their time in the embryology laboratory so that follicle sizes could be uniquely associated with each oocyte. Oocytes and embryos were analyzed according to the size of the follicle from which the oocyte was retrieved. MAIN RESULTS AND THE ROLE OF CHANCE: Three hundred seventeen oocytes (96.1%) had an associated follicle size. Of the oocytes with follicle sizes, 255 (80.4%) had a polar body (MII), and 60 (18.9%) were immature: 31 (9.8%) with a visible germinal vesicle (GV stage) and 29 (9.1%) with neither a polar body nor a visible germinal vesicle (MI). The incidence of MII oocytes was significantly associated with larger follicle size using either mm (ROC's AUC = 0.87; P < 0.0001) or Z values (ROC's AUC = 0.86; P < 0.0001). Among MII oocytes there was no association with follicle size for the appearance of 228 oocytes with two pronuclei (2 PN). Among 2 PN's, the development of 94 quality blastocysts that underwent trophectoderm biopsy (TE Bx) exhibited a significant association with larger follicles using either mm (ROC's AUC = 0.59; P = 0.01) or Z values (ROC's AUC = 0.57; P = 0.01). The use of follicle diameter as a feature to distinguish between fertilized oocytes that would ultimately become blastocysts versus those that would not become blastocysts resulted in an enrichment for blastocyst formation from 20 to 40%. Of the 94 quality blastocysts, 51 were determined by next generation sequencing (NGS) to be euploid.Although oocyte maturity and the incidence of blastocyst formation were associated with follicle size, the incidence of euploidy among biopsied blastocysts was not. Follicles measured by two different methods (mm or Z values) led to predominantly the same conclusions. LIMITATIONS, REASONS FOR CAUTION: This study investigated the relationship between follicle size and measures of oocyte/embryo quality when donors were treated similarly. Therefore, this study does not investigate the effects of triggering and retrieving oocytes when the follicle cohorts are of different sizes or lead follicles are of different sizes. Although no association was found between follicle size and euploid blastocysts, the fact that blastocyst ploidy is not entirely dependent upon oocyte ploidy (e.g. aneuploidies derived from mitotic errors or from the fertilizing sperm) makes it difficult to infer the relationship between follicle diameter and oocyte ploidy. WIDER IMPLICATIONS OF THE FINDINGS: It is confirmed that follicle diameter is predictive of oocyte maturity. However, once oocyte maturity is known, the diameter of the follicle from which the oocyte was retrieved is not instructive. Embryos generated through fertilization and development of the mature oocytes from any observed follicle diameter were equally likely to become euploid blastocysts. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by ReproART: Georgian American Center for Reproductive Medicine. None of the authors declare any actual conflicts of interest. D.H.M. received compensation from ReproART, Biogenetics Corporation and the Sperm and Embryo Bank of New York and honoraria and travel funding from Ferring Pharmaceuticals and from Granata Bio. S.M. received compensation from Cooper Genomics and an honorarium and travel funding from Ferring Pharmaceuticals. L.C. is the founder of LTD Ovamedi, the organization that represents Cooper Genomics in Georgia, and received travel funding from the European Society for Human Reproduction and Embryology. TRIAL REGISTRATION NUMBER: N/A.

Clinical error rates of next generation sequencing and array comparative genomic hybridization with single thawed euploid embryo transfer.

We investigated clinical error rates with single thawed euploid embryo transfer (STEET) diagnosed by next generation sequencing (NGS) and array comparative genomic hybridization (aCGH). A total of 1997 STEET cycles after IVF with preimplantation genetic testing for aneuploidy (PGT-A) from 2010 to 2017 were identified; 1151 STEET cycles utilized NGS, and 846 STEET cycles utilized aCGH. Any abortions, spontaneous or elective, in which products of conception (POCs) were collected were reviewed. Discrepancies between chorionic villus sampling, amniocentesis, or live birth results and PGT-A diagnosis were also included. Primary outcomes were clinical error rate per: ET, pregnancy with gestational sac, live birth, and spontaneous abortion with POCs available for analysis. Secondary outcomes included implantation rate (IR), spontaneous abortion rate (SABR), and ongoing pregnancy/live birth rate (OPR/LBR). The clinical error rates in the NGS cohort were: 0.7% per embryo, 1% per pregnancy with gestational sac, and 0.1% rate per OP/LB. The error rate per SAB with POCs was 13.3%. The IR was 69.1%, the OPR/LBR was 61.6%, and the spontaneous abortion rate was 10.2%. The clinical error rates in the aCGH cohort were: 1.3% per embryo, 2% per pregnancy with gestational sac, and 0.4% rate per OP/LB. The error rate per SAB with POCs was 23.3%. The IR was 63.8%, the OPR/LBR was 54.6%, and the SAB rate was 12.4%. Our findings demonstrate that, although NGS and aCGH are sensitive platforms for PGT-A, errors still occur. Appropriate patient counseling and routine prenatal screening are recommended for all patients undergoing IVF/PGT-A.

Clinical outcomes after the transfer of blastocysts characterized as mosaic by high resolution Next Generation Sequencing- further insights.

OBJECTIVE: To determine the pregnancy outcome potential of euploid, mosaic and aneuploid embryos. DESIGN: Retrospective study. SETTING: Reference genetics laboratories. PATIENT(S): 2654 PGT-A cycles with euploid characterized embryo transfers, 253 PGT-A cycles with transfer of embryos characterized as mosaic, and 10 PGT-A cycles with fully abnormal embryo transfers. INTERVENTION(S): Blastocysts were assessed by trophectoderm (TE) biopsy followed by PGT-A via array CGH or NGS. MAIN OUTCOME MEASURE(S): Implantation, miscarriage, ongoing implantation rates (OIR), and karyotype if available, were compared between different embryo groups, and between the two PGT-A techniques. RESULTS: The Ongoing Pregnancy Rate (OPR)/transfer was significantly higher for NGS-classified euploid embryos (85%) than for aCGH ones (71%) (p < 0.001), but the OPR/cycle was similar (63% vs 59%). NGS-classified mosaic embryos resulted in 37% OPR/cycle (p < 0.001 compared to euploid). Mosaic aneuploid embryos with <40% abnormal cells in the TE sample had an OIR of 50% compared to 27% for mosaics with 40-80% abnormal cells in the TE, and 9% for complex mosaic embryos. All the karyotyped ongoing pregnancies (n = 29) were euploid. Transfers of embryos classified as aneuploid via aCGH (n = 10) led to one chromosomally abnormal pregnancy. CONCLUSION(S): NGS-classified euploid embryos yielded higher OIRs but similar OPRs/cycle compared to aCGH. NGS-classified mosaic embryos had reduced potential to reach term, compared to euploid embryos. If they did reach term, those with karyotype results available were euploid. Embryos carrying uniform aneuploidies affecting entire chromosomes were mostly unable to implant after transfer, and the one that implanted ended up in a chromosomally abnormal live birth.

First PGT-A using human in vivo blastocysts recovered by uterine lavage: comparison with matched IVF embryo controls†.

STUDY QUESTION: After controlled ovarian stimulation (COS) and IUI, is it clinically feasible to recover in vivo conceived and matured human blastocysts by uterine lavage from fertile women for preimplantation genetic testing for aneuploidy (PGT-A) and compare their PGT-A and Gardner scale morphology scores with paired blastocysts from IVF control cycles? SUMMARY ANSWER: In a consecutive series of 134 COS cycles using gonadotrophin stimulation followed by IUI, uterine lavage recovered 136 embryos in 42% (56/134) of study cycles, with comparable in vivo and in vitro euploidy rates but better morphology in in vivo embryos. WHAT IS KNOWN ALREADY: In vivo developed embryos studied in animal models possess different characteristics compared to in vitro developed embryos of similar species. Such comparative studies between in vivo and in vitro human embryos have not been reported owing to lack of a reliable method to recover human embryos. STUDY DESIGN, SIZE, DURATION: We performed a single-site, prospective controlled trial in women (n = 81) to evaluate the safety, efficacy and feasibility of a novel uterine lavage catheter and fluid recovery device. All lavages were performed in a private facility with a specialized fertility unit, from August 2017 to June 2018. Subjects were followed for 30 days post-lavage to monitor for clinical outcomes and delayed complications. In 20 lavage subjects, a single IVF cycle (control group) with the same ovarian stimulation protocol was performed for a comparison of in vivo to in vitro blastocysts. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: Women were stimulated with gonadotrophins for COS. The ovulation trigger was given when there were at least two dominant follicles ≥18 mm, followed by IUI of sperm. Uterine lavage occurred 4-6 days after the IUI. A subset of 20 women had a lavage cycle procedure followed by an IVF cycle (control IVF group). Recovered embryos were characterized morphologically, underwent trophectoderm (TE) biopsy, vitrified and stored in liquid nitrogen. Biopsies were analyzed using the next-generation sequencing technique. After lavage, GnRH antagonist injections were administered to induce menstruation. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 134 lavage cycles were performed in 81 women. Uterine lavage recovered 136 embryos in 56 (42%) cycles. At the time of cryopreservation, there were 40 (30%) multi-cell embryos and 96 (70%) blastocysts. Blastocysts were of good quality, with 74% (70/95) being Gardener grade 3BB or higher grade. Lavage blastocysts had significantly higher morphology scores than the control IVF embryos as determined by chi-square analysis (P < 0.05). This is the first study to recover in vivo derived human blastocysts following ovarian stimulation for embryo genetic characterization. Recovered blastocysts showed rates of chromosome euploidy similar to the rates found in the control IVF embryos. In 11 cycles (8.2%), detectable levels of hCG were present 13 days after IUI, which regressed spontaneously in two cases and declined after an endometrial curettage in two cases. Persistent hCG levels were resolved after methotrexate in three cases and four cases received both curettage and methotrexate. LIMITATIONS, REASON FOR CAUTION: The first objective was to evaluate the feasibility of uterine lavage following ovarian stimulation to recover blastocysts for analysis, and that goal was achieved. However, the uterine lavage system was not completely optimized in our earlier experience to levels that were achieved late in the clinical study and will be expected in clinical service. The frequency of chromosome abnormalities of in vivo and IVF control embryos was similar, but this was a small-size study. However, compared to larger historical datasets of in vitro embryos, the in vivo genetic results are within the range of high-quality in vitro embryos. WIDER IMPLICATIONS OF THE FINDINGS: Uterine lavage offers a nonsurgical, minimally invasive strategy for recovery of embryos from fertile women who do not want or need IVF and who desire PGT, fertility preservation of embryos or reciprocal IVF for lesbian couples. From a research and potential clinical perspective, this technique provides a novel platform for the use of in vivo conceived human embryos as the ultimate benchmark standard for future and current ART methods. STUDY FUNDING/COMPETING INTEREST(S): Previvo Genetics, Inc., is the sole sponsor for the Punta Mita, Mexico, clinical study. S.M. performs consulting for CooperGenomics. J.E.B. and S.A.C. are co-inventors on issued patents and patents owned by Previvo and ownshares of Previvo. S.N. is a co-author on a non-provisional patent application owned by Previvo and holds stock options in Previvo. S.T.N. and M.J.A. report consulting fees from Previvo. S.T.N., S.M., M.V.S., M.J.A., C.N. and J.E.B. are members of the Previvo Scientific Advisory Board (SAB) and hold stock options in Previvo. J.E.B and S. M are members of the Previvo Board of Directors. A.N. and K.C. are employees of Previvo Genetics. L.V.M, T.M.M, J.L.R and S. S have no conflicts to disclose. TRIAL REGISTRATION NUMBER: Protocol Registration and Results System (PRS) Trial Registration Number and Name: Punta Mita Study TD-2104: Clinical Trials NCT03426007.

Preimplantation genetic testing for aneuploidy versus morphology as selection criteria for single frozen-thawed embryo transfer in good-prognosis patients: a multicenter randomized clinical trial.

OBJECTIVE: To evaluate the benefit of next-generation sequencing (NGS)-based preimplantation genetic testing for aneuploidy (PGT-A) for embryo selection in frozen-thawed embryo transfer. DESIGN: Randomized controlled trial. SETTING: Not applicable. PATIENT(S): Women aged 25-40 years undergoing IVF with at least two blastocysts that could be biopsied. INTERVENTION(S): Randomization for single frozen-thawed embryo transfer with embryo selection based on PGT-A euploid status versus morphology. MAIN OUTCOME MEASURE(S): Ongoing pregnancy rate (OPR) at 20 weeks' gestation per embryo transfer. RESULT(S): A total of 661 women (average age 33.7 ± 3.6 years) were randomized to PGT-A (n = 330) or morphology alone (n = 331). The OPR was equivalent between the two arms, with no significant difference per embryo transfer (50% [137/274] vs. 46% [143/313]) or per intention to treat (ITT) at randomization (41.8% [138/330] vs. 43.5% [144/331]). Post hoc analysis of women aged 35-40 years showed a significant increase in OPR per embryo transfer (51% [62/122] vs. 37% [54/145]) but not per ITT. CONCLUSION(S): PGT-A did not improve overall pregnancy outcomes in all women, as analyzed per embryo transfer or per ITT. There was a significant increase in OPR per embryo transfer with the use of PGT-A in the subgroup of women aged 35-40 years who had two or more embryos that could be biopsied, but this was not significant when analyzed by ITT. CLINICAL TRIAL REGISTRATION NUMBER: NCT02268786.

Controlled ovarian hyperstimulation (COH) parameters associated with euploidy rates in donor oocytes.

Although oocyte donors are young and are expected to provide a high rate of euploid oocytes, significant differences of euploidy rates for donor embryos exist between different IVF centers (1). Laboratory conditions can lead to differences of euploidy (2,3,4,5,6,7); but, the role of COH has not been investigated. In this study, we investigated whether euploidy rates in the embryos created from donor oocytes are influenced by controlled ovarian hyperstimulation parameters used during assisted reproduction. Euploidy rates in egg donor cycles undergoing PGT-A (N = 423) were examined retrospectively for associations with donor age, gonadotropin doses (dose per day), the fraction of gonadotropin provided by hMG (F(hMG)), days of stimulation, estradiol per mature oocyte on day of trigger, number of mature oocytes retrieved, number of embryos biopsied, incidence of euploidy and physician of record. Differences in euploidy rates between physicians were examined using analysis of variance. The proportion of euploid embryos per donor cycle was examined for associations with COH parameters using pairwise post-hoc comparisons, adjusting for multiple testing. The set of variables from this analysis was then submitted to a principal component analysis. Linear regression analysis was used to assess the relationships between stimulation parameters and the incidence of euploidy (the dependent variable). Euploidy rates and cycle parameters varied significantly among treating physicians. Euploidy rates (expressed as a fraction of biopsied embryos) were associated (p = 0.01) only with the F(hMG) but not with the number of MII retrieved or other variables. On the other hand, the number of euploid embryos (in contrast to the euploidy rate) was associated with the number of MII produced. Donor euploidy rates are significantly associated with the fraction of total gonadotropin comprising human menopausal gonadotropin (or F(hMG)) during controlled ovarian hyperstimulation but are not associated with other cycle parameters. The study provides the first suggestion that patient stimulation parameters can affect the incidence of euploidy in embryos generated through the use of standard assisted reproductive techniques. The study is limited by its retrospective approach and because the aCGH analysis used is less sensitive than more recent NGS technology. Further, it provides a suggestion that the use of hMG is beneficial for obtaining euploid embryos.

The cytogenetic constitution of human blastocysts: insights from comprehensive chromosome screening strategies.

BACKGROUND: Embryos that are able to form blastocysts have succeeded in activating their genome and differentiating into two cell types-an external layer of trophectoderm cells, which will go on to form extra-embryonic tissues such as the placenta, and the inner cell mass, which will give rise to the embryo proper. Culturing embryos to the blastocyst stage has become an increasingly popular IVF practice over the past decade. Additionally, it has been proposed that the identification and transfer of euploid blastocysts could significantly improve IVF outcomes. Toward this end, comprehensive molecular cytogenetic methods have been developed. The application of such methods in both clinical and research contexts has yielded cytogenetic data from large numbers of blastocysts. Questions have been raised concerning the implantation potential of blastocysts diagnosed 'euploid' or 'aneuploid', and there has been particular debate over the detection and viability of embryos categorized as 'mosaic'-composed of a mixture of normal and aneuploid cells. OBJECTIVE AND RATIONALE: This review aims to summarize data from studies using comprehensive molecular cytogenetic methods to examine blastocyst-stage embryos, describing current knowledge related to rates of euploidy, uniform aneuploidy and mosaicism. Issues associated with the developmental capacity of blastocysts of different cytogenetic constitutions will also be addressed. Guidelines on the clinical management of blastocysts with varying chromosome complements will be considered. SEARCH METHODS: Rates of euploidy, uniform aneuploidy (in which all cells have the same abnormal karyotype) and mosaicism were determined via a thorough literature search (PubMed). The keywords used in the search were as follows: preimplantation embryo development, blastocyst stage, embryonic aneuploidy, meiotic chromosome malsegregation, post-zygotic chromosome malsegregation, comprehensive chromosome screening, array comparative genomic hybridization, single-nucleotide polymorphism array, next-generation sequencing, embryo mosaicism and implantation of mosaic embryos. Relevant articles written in English and published up to March 2018 were reviewed. OUTCOMES: Different types of aneuploidy, including some complex forms, are capable of persisting to the blastocyst stage. As expected, euploidy rates decreased with advancing female age, whereas uniform aneuploidy increased. Analysis of multiple cells from individual blastocysts revealed that most of those classified 'abnormal' contained no euploid cells (due to meiotic errors arising in the gametes and therefore present in every cell), some having additional mosaic (post-fertilization, mitotic) errors. Blastocysts with a mix of normal and aneuploid cells were observed less frequently than other classes of embryo. The transfer of embryos with diploid-aneuploid mosaic biopsy specimens is reportedly associated with higher miscarriage and lower implantation rates, compared to embryos in which only euploid cells are detected. WIDER IMPLICATIONS: Detailed investigations into the chromosome constitution of human blastocysts suggest that a significant proportion is euploid in every cell, although the exact fraction is strongly influenced by female age. These findings do not support the notion that mosaic chromosome abnormalities are a natural part of embryo development. Mosaic aneuploidies, arising post-zygotically, were detected by various different comprehensive molecular cytogenetic methods, suggesting that the majority of these represent genuine findings. However, it remains possible that certain comprehensive molecular cytogenetic methods may carry a risk of mosaicism being incorrectly assigned, in a minority of samples, as a result of technical artifact. This may be a consequence of degraded DNA in the trophectoderm biopsy or other technical issues. According to published studies, blastocysts considered to have uniform aneuploidy and, to a lesser extent, those with mosaic abnormalities were associated with poorer clinical outcomes in comparison with euploid embryos.

Status of preimplantation genetic testing and embryo selection.

At the recent 2018 PGDIS congress, a review of randomized controlled trials of preimplantation genetic testing for aneuploidies (PGT-A) showed improved ongoing pregnancy rates per transfer in experienced centres and in women aged 35 years and older. Young women produce 40% abnormal embryos (20-60% range), but not all centres see a selection advantage; this indicates the need for more emphasis in improving biopsy and case management. Some chromosome abnormalities are iatrogenic; PGT-A could, therefore, be used as assisted reproductive technology (ART) quality control. Great improvements in non-invasive PGT by testing spent media have been reported, ranging from 80-95% concordance with trophectoderm biopsy, probably precluding the need for biopsy soon. Mosaicism was widely discussed, with PGDIS agreeing to update their guidelines, but continuing to recommend prioritizing euploid, followed by mosaic embryos. Techniques to allow simultaneous single sample analysis of aneuploidy and inherited mutations are improving, but this does not extend to de-novo mutations. Convincing data were presented on the efficacy of using endometrial receptivity tests to improve ART outcomes adjuvant or independently of PGT-A. Imprinting, CRISPR and cloning were also discussed, with a concluding presentation on the first extensive data (aneuploidy and morphology) on in-vivo conceived embryos.

The cost of a euploid embryo identified from preimplantation genetic testing for aneuploidy (PGT-A): a counseling tool.

PURPOSE: To determine the expected out-of-pocket costs of IVF with preimplantation genetic testing for aneuploidy (PGT-A) to attain a 50%, 75%, or 90% likelihood of a euploid blastocyst based on individual age and AMH, and develop a personalized counseling tool. METHODS: A cost analysis was performed and a counseling tool was developed using retrospective data from IVF cycles intended for PGT or blastocyst freeze-all between January 1, 2014 and August 31, 2017 (n = 330) and aggregate statistics on euploidy rates of > 149,000 embryos from CooperGenomics. Poisson regression was used to determine the number of biopsiable blastocysts obtained per cycle, based on age and AMH. The expected costs of attaining a 50%, 75%, and 90% likelihood of a euploid blastocyst were determined via 10,000 Monte Carlo simulations for each age and AMH combination, incorporating age-based euploidy rates and IVF/PGT-A cost assumptions. RESULTS: The cost to attain a 50% likelihood of a euploid blastocyst ranges from approximately $15,000 U.S. dollars (USD) for younger women with higher AMH values (≥ 2 ng/mL) to > $150,000 for the oldest women (44 years) with the lowest AMH values (< 0.1 ng/mL) in this cohort. The cost to attain a 75% versus 90% likelihood of a euploid blastocyst is similar (~ $16,000) for younger women with higher AMH values, but varies for the oldest women with low AMH values (~ $280,000 and > $450,000, respectively). A typical patient (36-37 years, AMH 2.5 ng/mL) should expect to spend ~ $30,000 for a 90% likelihood of attaining a euploid embryo. CONCLUSIONS: This tool can serve as a counseling adjunct by providing individualized cost information for patients regarding PGT-A.

Forty years of IVF.

This monograph, written by the pioneers of IVF and reproductive medicine, celebrates the history, achievements, and medical advancements made over the last 40 years in this rapidly growing field.