Randomized allocation of oocytes to IVF or ICSI for IVF-naïve cases with unexplained infertility in an IVF-ICSI Split protocol favors ICSI to optimize live birth outcomes

In assisted reproduction treatments (ART), applying the ICSI method for fertilization of oocytes rather than traditional IVF method, is regarded as controversial for two reasons, namely utility and safety. Our study examines an IVF-ICSI Split model for couples with unexplained infertility, where male factor is meticulously excluded and ART is conducted by a strict algorithm, a commitment to blastocyst culture, along with single embryo transfers and a high commitment to cryopreservation. From 242 treatment cycles, 3346 oocytes recovered (13.8 per OPU) were randomly allocated to IVF or ICSI and the fertilization rates standardized to the number of 2PNS arising from each group applying the metaphase II oocyte number identified for the ICSI group, as the denominator for both groups. The fertilization rates were significantly higher overall for ICSI (83.2% vs 65.4%; p<0.0001), being most pronounced for women under 40 years. The resultant embryos had equivalent implantation rates in both fresh ET and frozen (FET) cycles with no significant differences in pregnancy rates, miscarriage rates or live birth outcomes indicating equivalent embryo quality. However, there were significantly higher numbers of ICSI-generated embryos cryopreserved and subsequent FET procedures showed higher live birth rates (21 births vs 6 births; p<0.005) and potential livebirths (214 births vs 104 births; p<0.0001). No congenital fetal abnormalities were detected in any of the 199 babies delivered during the study period to December 2020, neither IVF-generated nor ICSI-generated. Whilst the data strongly favors ICSI, there were 2 women (from 26 with fertilization in one arm only) who demonstrated fertilization only in the IVF arm of the study. We conclude that the IVF-ICSI Split model should be undertaken on all IVF-naïve women with unexplained infertility to determine the appropriate fertilization mode, albeit ICSI will be safely preferred for >90% of cases.

Non-Invasive Chromosome Screening for Embryo Preimplantation Using Cell-Free DNA

Preimplantation genetic testing (PGT) is widely adopted to select embryos with normal ploidy but requires invasive embryo biopsy procedures. Therefore, non-invasive PGT (niPGT) detection of cell-free DNA (cfDNA) in blastocyst culture medium has gradually become a hot area in the field of assisted reproduction. This chapter will systematically summarize how researchers use embryonic cfDNA to conduct niPGT detection worldwide. It will also thoroughly review the factors that affect the accuracy of the test and its underlying issues, as well as prospective applications. We hope to provide a useful reference for the standardized operation of non-invasive PGT that can be widely applied in clinical practice.

Use of a new set of key performance indicators for evaluating the performance of an in vitro fertilization laboratory in which blastocyst culture and the freeze-all strategy are the primary treatment in patients with in vitro fertilization

Objective To evaluate the performance of an in vitro fertilization (IVF) laboratory using a new set of key performance indicators (KPIs) when the main treatment of IVF patients had been changed. Methods Patients who underwent fresh embryo transfer and the freeze-all strategy in August, September, and October 2017 were retrospectively studied to evaluate the performance of an IVF laboratory in September when implantation rate of fresh embryo transfer decreased. KPIs associated with blastocyst culture and the first frozen embryo transfer (FET) cycle in patients with the freeze-all strategy were compared over 3 months. Results Day 5 usable blastocyst and good quality blastocyst rates, and day 3 usable/good quality embryo rates were not different among the three periods. The implantation rate and KPIs associated with morphological changes in warmed blastocysts in the first FET cycle in patients with the freeze-all strategy were also not different among the periods. Conclusions KPIs associated with embryo quality, blastocyst culture, and the pregnancy outcome of the first FET cycle in patients with the freeze-all strategy suggested that performance was unaffected in our IVF laboratory in September. These KPIs might be useful for internal quality control analysis of IVF laboratories.

Electrofusion Stimulation Is an Independent Factor of Chromosome Abnormality in Mice Oocytes Reconstructed via Spindle Transfer

Oocytes reconstructed by spindle transfer (ST) are prone to chromosome abnormality, which is speculated to be caused by mechanical interference or premature activation, the mechanism is controversial. In this study, C57BL/6N oocytes were used as the model, and electrofusion ST was performed under normal conditions, Ca2+ free, and at room temperature, respectively. The effect of enucleation and electrofusion stimulation on MPF activity, spindle morphology, γ-tubulin localization and chromosome arrangement was compared. We found that electrofusion stimulation could induce premature chromosome separation and abnormal spindle morphology and assembly by decreasing the MPF activity, leading to premature activation, and thus resulting in chromosome abnormality in oocytes reconstructed via ST. Electrofusion stimulation was an independent factor of chromosome abnormality in oocytes reconstructed via ST, and was not related to enucleation, fusion status, temperature, or Ca2+. The electrofusion stimulation number should be minimized, with no more than 2 times being appropriate. As the electrofusion stimulation number increased, several typical abnormalities in chromosome arrangement and spindle assembly occurred. Although blastocyst culture could eliminate embryos with chromosomal abnormalities, it would significantly decrease the number of normal embryos and reduce the availability of embryos. The optimum operating condition for electrofusion ST was the 37°C group without Ca2+.

P–560 Comparative analysis of non-invasive preimplantation genetic testing of aneuploidies (niPGT-A), PGT-A and IVF cycles without aneuploidy testing: preliminary results

Study question Can non-invasive preimplantation genetic testing of aneuploidies (niPGT-A) improve the clinical outcome in IVF patients after proper validation? Summary answer We demonstrate the usefulness of the embryonic cell-free DNA (cfDNA) in the blastocyst culture medium to select more objectively the blastocysts with higher implantation potential. What is known already One of the greatest challenges in IVF is accurately selecting viable embryos that are more likely to achieve healthy livebirths following embryo transfer. Trophectoderm (TE) biopsy and PGT-A provide a direct assessment of chromosome status and improve implantation and clinical pregnancy rates per transfer. A non-invasive alternative is to analyse embryonic cfDNA in the blastocyst culture medium. Previous studies have shown that cfDNA testing in culture medium of blastocysts on day 6 of development allows aneuploidy detection with high concordance rates compared to TE biopsy and inner cell mass (Rubio et al., 2020). Study design, size, duration Observational study of the clinical application of niPGT-A (July 2020-December 2020). The clinical application consisted in a first validation phase, comparing TE biopsies with cfDNA in the media of 28 blastocysts. And, in a second phase, niPGT-A was applied and the outcome of 13 single embryo transfers (SETs) compared to 13 PGT-A SETs and 130 IVF/ICSI SETs performed in a period of six months. In the three groups, women and donors age was ≤38 years. Participants/materials, setting, methods Embryos were cultured in a Geri incubator (Merck) up to day 4, and then individually cultured in 10µl drops of CCSS (Fujifilm) until day 6 in a bench-top K-system. At day 6, blastocysts were vitrified, and media collected in sterile PCR tubes after at least 40 hours in culture. After collection, media were immediately frozen and analyzed by NGS analysis in our reference laboratory (Igenomix, Spain). Deferred transfer was performed according to media results. Main results and the role of chance Before the first clinical cases, a validation of the protocol comparing the results of cfDNA with the TE biopsies of the same day–6 blastocyst was performed, and ploidy concordance rates were 87.5%. Similar results were found for niPGT-A and PGT-A in terms of aneuploidy results and in clinical outcomes. The percentages of informative results were 95% and 97% and the aneuploidy rates were 44% and 46%, for niPGT-A and PGT-A, respectively. Clinical pregnancy rates were in both groups of aneuploidy testing, 69.2%, with 8 ongoing pregnancies (61.5%) and 4 tested by prenatal screaning NACE. For untested embryos clinical pregnancy (57.7%) and ongoing pregnancy rates (48.5%) were lower than in the two groups of tested embryos (niPGT-A and PGT-A). In the niPGT-A cycles embryo transfer was performed according to media results and morphology. We did a secondary analysis of which blastocyst we would transfer, if only morphology is considered. We observed that if we only select the embryos by morphology, in 61.5% of the cases we would choose the same embryo than with niPGT-A, and in 30.4% of the cases we would transfer a blastocyst with an aneuploid medium. Limitations, reasons for caution Our results are encouraging but should be interpreted with caution due to the small sample size. Larger and randomized controlled trials are needed to verify and extend our findings in each group. Wider implications of the findings: We observed consistent results for niPGT-A compared to TE biopsies in our internal validation. These results endorse the clinical application of niPGT-A in the routine of the laboratory and can avoid the embryo manipulation also reducing the subjectivity when embryos are selected only by morphology. Trial registration number Sa–16552/19-EC:428

Modeling Human Peri-implantation Placental Development and Function

It is very difficult to gain a better understanding of the events in human pregnancy that occur during and just after implantation because such pregnancies are not yet clinically detectable. Animal models of human placentation are inadequate. In vitro models that utilize immortalized cell lines and cells derived from trophoblast cancers have multiple limitations. Primary cell and tissue cultures often have limited lifespans and cannot be obtained from the peri-implantation period. We present here two contemporary models of human peri-implantation placental development: extended blastocyst culture and stem-cell derived trophoblast culture. We discuss current research efforts that employ these models and how such models might be used in the future to study the “black box” stage of human pregnancy.

Lower chromosomal abnormality frequencies in miscarried conceptuses from frozen blastocyst transfers in ART

STUDY QUESTION Are blastocyst culture and cryopreservation in ART associated with chromosomal abnormalities in miscarried products of conception (POC)? SUMMARY ANSWER Frozen blastocyst transfer in women aged 35 years or older and frozen embryo transfer (ET) (including both cleavage-stage embryo and blastocyst) in women aged &lt;35 years are associated with decreased frequencies of embryonic chromosomal abnormalities in miscarried POC. WHAT IS KNOWN ALREADY Blastocyst culture and embryo cryopreservation have been previously associated with favorable ART treatment outcomes and widely applied in clinical practice. However, the association between these embryo manipulation procedures and embryonic chromosomal abnormalities has not been evaluated to the best of our knowledge. STUDY DESIGN, SIZE, DURATION This retrospective study included a total of 720 patients who underwent IVF/ICSI, and the retained POC were obtained. A single-nucleotide polymorphism (SNP)-based chromosomal microarray analysis (CMA) of all miscarried conceptuses was performed. PARTICIPANTS/MATERIALS, SETTING, METHODS This study was based on the Clinical Reproductive Medicine Management System/Electronic Medical Record Cohort Database (CCRM/EMRCD) at our center. In total, 720 miscarried POCs were collected from patients undergoing ART (including fresh cleavage-stage ET, fresh blastocyst transfer, frozen cleavage-stage ET and frozen blastocyst transfer), and the incidences and profiles of cytogenetic abnormalities in the miscarried conceptuses were measured via SNP-based CMA. MAIN RESULTS AND THE ROLE OF CHANCE The chromosomal abnormality rate in POC varied from 33.7% to 66.7% among the different ET strategies. In the patients aged ≥35 years, frozen blastocyst transfer was significantly associated with a lower incidence of chromosomal aberrations in the POCs (adjusted odds ratio (aOR): 0.171 (95% CI: 0.040–0.738); P = 0.018) than fresh blastocyst transfer. In the patients aged &lt;35 years, frozen ET was significantly associated with a lower incidence of chromosomal aberrations than fresh ET in both cleavage-stage ET cycles and blastocyst transfers cycles (aOR: 0.545 (0.338–0.879), P = 0.013; and aOR: 0.357 (0.175–0.730), P = 0.005, respectively). Trisomy was the most frequent abnormal embryonic karyotype in the different ET strategies, and its frequency significantly differed among strategies (P &lt; 0.05). LIMITATIONS, REASONS FOR CAUTION This study was retrospectively designed, and we cannot draw any definite conclusions from our results regarding the adequate safety of embryo cryopreservation in ongoing pregnancy. WIDER IMPLICATIONS OF THE FINDINGS To our knowledge, this is the first study assessing the associations of ET strategies with the probability of miscarriage associated with embryonic chromosomal abnormalities. However, the underlying mechanism of these associations is unknown; this study may promote research concerning ET strategies and promote comprehensive consultations and recommendations for patients. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the Natural Science Foundation of China (Grant No.81571409), Science and Technology Research Project of Henan (Grant No. 172102310009) and Medical Science and Technology Research Project of Henan (Grant No. 201701005). The authors declare no competing interests. TRIAL REGISTRATION NUMBER N/A