Purpose Total fertilisation failure (TFF), even with intracytoplasmic sperm injection (ICSI), occurs in approximately 3 % of cycles, can be recurrent and the exact cause is difficult to elucidate. Differentiation between oocyte and sperm-related cause of TFF is possible using mouse oocyteactivation techniques, but is not an option within most clinical settings. Therefore, the management of these couples is clinically driven, and the endpoint, if recurrent, is often the use of donor gametes. However, with the invariable lack of a definitive cause of TFF, any decision between the use of donor sperm or oocytes remains an emotive one. We present two case reports demonstrating the importance of appropriate investigation, activation techniques (mechanical and chemical) and clinical management options to develop a clinical algorithm prior to the use of donor gametes. Methods This study is composed of two case reports of assisted reproduction investigation and treatment within an assisted conception unit for couples with recurrent total fertilisation failure. Results Using appropriate investigation (endocrine, urological and embryological) and treatments (ICSI, IMSI, oocyteactivation techniques), a fertilisation rate of 48 % was achieved in two cycles in couples following a total of nine previous cycles (and 200 previously collected eggs) with TFF.Conclusions Oocyte activation requires the triggering of intracellular calcium oscillations by the release of a spermspecific factor (phospholipase C zeta (PLCζ)) into the oocyte cytoplasm. Although, PLCζ deficiencies have been demonstrated as putative causes of failed activation, impaired oocyte responsiveness may also be a factor. The use of donor gametes is often recommended and is often the required endpoint of treatment. However, these reports outline a clinical algorithm that potentially offers success without donation, and also offers a systematic approach to help decide whether donor oocytes or sperm should be recommended.

STUDY QUESTION What is the impact of day after rescue ICSI (r-ICSI) on success of fresh and frozen embryo transfers? SUMMARY ANSWER The use of r-ICSI can virtually allay fears of total fertilization failure (TFF) after conventional IVF (C-IVF) and achieve high live birth rates after frozen blastocyst transfer. WHAT IS KNOWN ALREADY More infertility clinics have resorted to the use of ICSI in place of C-IVF in IVF treatment owing to fear of TFF or a low fertilization rate. r-ICSI has been attempted either on the day of IVF or the day after. Day after r-ICSI has proved unsuccessful in the past. STUDY DESIGN, SIZE, DURATION A retrospective data analysis was performed of 16 608 qualifying cases between April 2010 and July 2021 conducted at a single private academically affiliated fertility clinic. PARTICIPANTS/MATERIALS, SETTING, METHODS r-ICSI was performed principally on patients with >4 metaphase II oocytes, showing no signs of fertilization 18 h after C-IVF. C-IVF was performed on patients who had >4 million total motile sperm after preparation. r-ICSI was then performed 18–24 h after insemination, using the sperm sample from the previous day. r-ICSI fertilization rates, cryopreservation of cleavage and blastocysts embryos, and pregnancy rates after fresh or frozen transfer were then assessed. MAIN RESULTS AND THE ROLE OF CHANCE r-ICSI was performed on 377 patients (2.3% of eligible retrieval cycles) who had a mean (±SD) female and male age of 35.9 ± 4.5 and 38.1 ± 9.1 years, respectively. A total of 5459 oocytes were initially retrieved. Of the oocytes undergoing r-ICSI, 2389 (49.5%) fertilized normally, and 205 (54.4%) patients underwent a fresh embryo transfer. The live birth rates were 23/186 (12.3%) for fresh cleavage and 5/19 (26.3%) for fresh blastocyst stage transfers. In 145 cycles a blastocyst was frozen, and 137 transfers were performed with a 64/137 (46.7%) live birth rate. Of the 377 cycles receiving r-ICSI only, 25 of the qualifying cases failed to have any fertilization, reducing TFF to 25/16 608 (0.15%). LIMITATIONS, REASONS FOR CAUTION This was a single-center retrospective study on a specific subset of patients, which may limit its generalizability to other clinics. WIDER IMPLICATIONS OF THE FINDINGS r-ICSI allows a second opportunity to fertilize oocytes despite poor initial outcomes. Patients who had a frozen blastocyst transfer achieved high live birth rates, indicating that a resynchronization of the embryo with the endometrium can optimize r-ICSI cases. r-ICSI allays fears of TFF when using C-IVF, providing evidence that the overuse of ICSI in patients without male factor may not be warranted. STUDY FUNDING/COMPETING INTEREST(S) The study was internally funded by Boston IVF. The authors declare that they have no conflict of interest in relation to the data published in the article. TRIAL REGISTRATION NUMBER N/A.

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Study question Can accurate mitochondrial DNA (mtDNA) quantification of trophectoderm (TE) biopsy specimens provide insights into the biology and viability of blastocyst-stage human embryos? Summary answer mtDNA quantity in TE cells is correlated with embryo morphology and shows alterations associated with aneuploidy. However, measurement does not significantly improve embryo viability assessment. What is known already Mitochondria are essential organelles, responsible for producing ATP. Changes in the amount of mtDNA in blastocysts biopsy specimens have been reported in association with embryo implantation potential, leading to proposals that mtDNA quantification might serve as a useful biomarker of embryo viability. However, results from clinical studies to explore this possibility have yielded contradictory data, due in part to deficiencies of the molecular methods used for mtDNA measurement. We sought to clarify what quantification of mtDNA can tell us about embryo biology and viability by developing and applying a method that we believe to be the most accurate ever devised. Study design, size, duration This study involved the analysis of samples collected during the course of routine preimplantation genetic testing for aneuploidy (PGT-A). The IVF treatments and embryo biopsies were undertaken at two different clinics, while chromosomal analyses were carried out at a single reference laboratory. Mitochondrial data was subsequently analysed in a university setting. The embryos analysed were derived from a broad population of patients referred for PGT-A (average age 38.7 years; range 25-47). Participants/materials, setting, methods 651 blastocysts from 133 couples underwent trophectoderm biopsy on day-5 or day-6. The specimens were analysed using a highly validated real-time PCR method, which was used to quantify three distinct sites in the mtDNA and 198 loci in the nuclear genome. The measurement of multiple independent loci provided outstanding sensitivity and accuracy. The nuclear loci were used to normalise the mtDNA data, adjusting for differences in the number of cells in the biopsy specimens. Main results and the role of chance The method developed displayed extraordinary sensitivity and accuracy when quantifying mtDNA. Lower mtDNA quantities were associated with day-6 biopsy (p < 0.0001), extent of blastocyst expansion (p < 0.0001), and superior TE morphology, although the latter was not statistically significant (p = 0.09). mtDNA levels were higher in aneuploid embryos (p < 0.0001), independent on patient age. However, the difference was not sufficient to be considered diagnostic. There was no correlation between mtDNA level and the chances of blastocyst implantation in this dataset. Lower mtDNA levels, previously reported to be associated with higher probabilities of embryo implantation, were most often observed in embryos of excellent morphological grade and likely reflect the increased TE cell numbers of such embryos. Little if any mtDNA replication occurs during preimplantation development and consequently the mtDNA content is divided amongst an ever-growing number of cells, meaning less mtDNA per cell. In this context, mtDNA quantification of blastocyst biopsy specimens provides a highly sensitive measure of TE cellularity, but probably provides little additional benefit for embryo selection beyond conventional morphological grading. However, the fact that higher mtDNA quantities were observed in aneuploid embryos, may indicate that subtle differences in TE cellularity exist in abnormal embryos, which are not fully captured by traditional morphological assessment. Limitations, reasons for caution Previous studies suggested that some blastocysts have greatly elevated mtDNA levels and that such embryos are not viable. In this study, only 5% of embryos were considered outliers in terms of mtDNA quantity. Unfortunately, none of these embryos were transferred, so the potential of these embryos could not be assessed. Wider implications of the findings The quantification of mtDNA in trophectoderm biopsies has sometimes been used for the prioritisation of embryos for transfer. While our results confirm existence of biologically interesting associations between mtDNA and aneuploidy, and a relationship with certain aspects of embryo morphology, measurement of mtDNA seems unlikely to significantly improve embryo selection. Trial registration number not applicable

Study question If a clinic changes the genetic laboratory it works with, should it expect its new PGT-A results to be equivalent to those it received previously? Summary answer Changing the genetic service provider can result in a significant difference in the proportion of embryos classified euploid, with important clinical implications. What is known already Preimplantation genetic testing for aneuploidy (PGT-A) aims to distinguish potentially viable euploid embryos from embryos harbouring lethal chromosome abnormalities. Typically, IVF clinics perform biopsy at the blastocyst stage and send the resulting trophectoderm specimens to specialist genetic laboratories for analysis. However, many commercially available genetic methods have not been subjected to rigorous validation, leading to uncertainty about their accuracy and predictive value. It would be concerning if the classification of embryos, derived from the same patient population, and from the same clinic, varied depending on the company that provides PGT-A services. Such differences would be unlikely to reflect biological reality. Study design, size, duration Our clinic recently began working with a new genetics company. Our previous PGT-A reference laboratory issued results for 1,136 blastocyst biopsy specimens over ∼15 months. Subsequently, the second company tested 784 biopsy samples in 7 months. Patient populations during the two periods were essentially identical (average maternal age 38.0 years). Participants/materials, setting, methods The first company used whole genome amplification followed by next generation sequencing (NGS) to evaluate the relative amount of DNA from each chromosome. The second company’s method was highly validated, involving targeted amplification of thousands of sites in the genome, again followed by NGS and analysis of relative DNA quantity from individual chromosomes. Additionally, they genotyped numerous polymorphisms, which assist in the detection of haploidy/triploidy, and also reveals problems that can compromise accuracy (e.g. contamination). Main results and the role of chance The first PGT-A company classified 44.4% of blastocysts euploid, 4.1% low-level mosaic, 51.5% aneuploid. In contrast, the second company reported significantly fewer embryos aneuploid (44.0%; P = 0.0019). Even if the mosaics reported by the first company are considered for transfer, the second company is still associated with more potentially transferable embryos (relative increase greater than one-sixth). If PGT-A results from the second company are correct, it implies that potentially viable embryos may have been misclassified by the first company, risking their exclusion and loss of the pregnancies they might have produced. Conversely, if the first company is correct, the second company may be failing to detect some aneuploidies, leading to inadvertent transfer of abnormal embryos, lower pregnancy rates and a higher incidence of miscarriage. 299 transfers following PGT-A using the first company produced 171 pregnancies (74.7%), while 64 single embryo transfers have taken place after PGT-A with the second company, resulting in 53 pregnancies (82.8% per transfer). Losses (biochemical or miscarriage) affected 21.6% and 16.9% of pregnancies after PGT-A conducted by the first and second companies, respectively (ongoing pregnancy rates of 58.5% and 68.8% per transfer, respectively) Thus, there is no evidence that aneuploidies are being missed by the second company. Limitations, reasons for caution Although there were no obvious differences between the patients with embryos tested by the two companies, this study was not prospective nor randomized, so a possibility of undetected differences remains. The study was not sufficiently powered to test the significance of apparent differences in implantation, miscarriage and ongoing pregnancy rates. Wider implications of the findings It is important that IVF clinics appreciate that individual PGT-A methods differ significantly, and that some have undergone much more rigorous validation than others. It is recommended that clinics ask PGT providers to share their validation data, especially clinical studies confirming that embryos classified ‘aneuploid’ or ‘abnormal’ are truly non-viable. Trial registration number Not applicable