Implicit bias in diagnosing mosaicism amongst preimplantation genetic testing providers: results from a multicenter study of 36 395 blastocysts.

STUDY QUESTION: Does the diagnosis of mosaicism affect ploidy rates across different providers offering preimplantation genetic testing for aneuploidies (PGT-A)? SUMMARY ANSWER: Our analysis of 36 395 blastocyst biopsies across eight genetic testing laboratories revealed that euploidy rates were significantly higher in providers reporting low rates of mosaicism. WHAT IS KNOWN ALREADY: Diagnoses consistent with chromosomal mosaicism have emerged as a third category of possible embryo ploidy outcomes following PGT-A. However, in the era of mosaicism, embryo selection has become increasingly complex. Biological, technical, analytical, and clinical complexities in interpreting such results have led to substantial variability in mosaicism rates across PGT-A providers and clinics. Critically, it remains unknown whether these differences impact the number of euploid embryos available for transfer. Ultimately, this may significantly affect clinical outcomes, with important implications for PGT-A patients. STUDY DESIGN, SIZE, DURATION: In this international, multicenter cohort study, we reviewed 36 395 consecutive PGT-A results, obtained from 10 035 patients across 11 867 treatment cycles, conducted between October 2015 and October 2021. A total of 17 IVF centers, across eight PGT-A providers, five countries and three continents participated in the study. All blastocysts were tested using trophectoderm biopsy and next-generation sequencing. Both autologous and donation cycles were assessed. Cycles using preimplantation genetic testing for structural rearrangements were excluded from the analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS: The PGT-A providers were randomly categorized (A to H). Providers B, C, D, E, F, G, and H all reported mosaicism, whereas Provider A reported embryos as either euploid or aneuploid. Ploidy rates were analyzed using multilevel mixed linear regression. Analyses were adjusted for maternal age, paternal age, oocyte source, number of embryos biopsied, day of biopsy, and PGT-A provider, as appropriate. We compared associations between genetic testing providers and PGT-A outcomes, including the number of chromosomally normal (euploid) embryos determined to be suitable for transfer. MAIN RESULTS AND THE ROLE OF CHANCE: The mean maternal age (±SD) across all providers was 36.2 (±5.2). Our findings reveal a strong association between PGT-A provider and the diagnosis of euploidy and mosaicism. Amongst the seven providers that reported mosaicism, the rates varied from 3.1% to 25.0%. After adjusting for confounders, we observed a significant difference in the likelihood of diagnosing mosaicism across providers (P < 0.001), ranging from 6.5% (95% CI: 5.2-7.4%) for Provider B to 35.6% (95% CI: 32.6-38.7%) for Provider E. Notably, adjusted euploidy rates were highest for providers that reported the lowest rates of mosaicism (Provider B: euploidy, 55.7% (95% CI: 54.1-57.4%), mosaicism, 6.5% (95% CI: 5.2-7.4%); Provider H: euploidy, 44.5% (95% CI: 43.6-45.4%), mosaicism, 9.9% (95% CI: 9.2-10.6%)); and Provider D: euploidy, 43.8% (95% CI: 39.2-48.4%), mosaicism, 11.0% (95% CI: 7.5-14.5%)). Moreover, the overall chance of having at least one euploid blastocyst available for transfer was significantly higher when mosaicism was not reported, when we compared Provider A to all other providers (OR = 1.30, 95% CI: 1.13-1.50). Differences in diagnosing and interpreting mosaic results across PGT-A laboratories raise further concerns regarding the accuracy and relevance of mosaicism predictions. While we confirmed equivalent clinical outcomes following the transfer of mosaic and euploid blastocysts, we found that a significant proportion of mosaic embryos are not used for IVF treatment. LIMITATIONS, REASONS FOR CAUTION: Due to the retrospective nature of the study, associations can be ascertained, however, causality cannot be established. Certain parameters such as blastocyst grade were not available in the dataset. Furthermore, certain platform-related and clinic-specific factors may not be readily quantifiable or explicitly captured in our dataset. As such, a full elucidation of all potential confounders accounting for variability may not be possible. WIDER IMPLICATIONS OF THE FINDINGS: Our findings highlight the strong need for standardization and quality assurance in the industry. The decision not to transfer mosaic embryos may ultimately reduce the chance of success of a PGT-A cycle by limiting the pool of available embryos. Until we can be certain that mosaic diagnoses accurately reflect biological variability, reporting mosaicism warrants utmost caution. A prudent approach is imperative, as it may determine the difference between success or failure for some patients. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Torres Quevedo Grant, awarded to M.P. (PTQ2019-010494) by the Spanish State Research Agency, Ministry of Science and Innovation, Spain. M.P., L.B., A.R.L., A.L.R.d.C.L., N.P.P., M.P., D.S., F.A., A.P., B.M., L.D., F.V.M., D.S., M.R., E.P.d.l.B., A.R., and R.V. have no competing interests to declare. B.L., R.M., and J.A.O. are full time employees of IB Biotech, the genetics company of the Instituto Bernabeu group, which performs preimplantation genetic testing. M.G. is a full time employee of Novagen, the genetics company of Cegyr, which performs preimplantation genetic testing. TRIAL REGISTRATION NUMBER: N/A.

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.

The human sperm basal body is a complex centrosome important for embryo preimplantation development.

The mechanism of conversion of the human sperm basal body to a centrosome after fertilization, and its role in supporting human early embryogenesis, has not been directly addressed so far. Using proteomics and immunofluorescence studies, we show here that the human zygote inherits a basal body enriched with centrosomal proteins from the sperm, establishing the first functional centrosome of the new organism. Injection of human sperm tails containing the basal body into human oocytes followed by parthenogenetic activation, showed that the centrosome contributes to the robustness of the early cell divisions, increasing the probability of parthenotes reaching the compaction stage. In the absence of the sperm-derived centrosome, pericentriolar material (PCM) components stored in the oocyte can form de novo structures after genome activation, suggesting a tight PCM expression control in zygotes. Our results reveal that the sperm basal body is a complex organelle which converts to a centrosome after fertilization, ensuring the early steps of embryogenesis and successful compaction. However, more experiments are needed to elucidate the exact molecular mechanisms of centrosome inheritance in humans.

Novel Double Factor PGT strategy analyzing blastocyst stage embryos in a single NGS procedure.

In families at risk from monogenic diseases affected offspring, it is fundamental the development of a suitable Double Factor Preimplantation Genetic Testing (DF-PGT) method for both single-gene analysis and chromosome complement screening. Aneuploidy is not only a major issue in advanced-maternal-age patients and balanced translocation carriers, but also the aneuploidy rate is extremely high in patients undergoing in vitro fertilization (IVF), even in young donors. To adequate NGS technology to the DF-PGT strategy four different whole genome amplification systems (Sureplex, MALBAC, and two multiple displacement amplification systems-MDA) were tested using TruSight One panel on cell lines and blastocyst trophectoderm biopsies-TE. Embryo cytogenetic status was analyzed by Nexus software. Sureplex and MALBAC DNA products were considered not suitable for PGT diagnosis due to inconsistent and poor results on Trusight one (TSO) panel. Results obtained with both MDA based methods (GEH-MDA and RG-MDA) were appropriate for direct mutation detection by TSO NGS platform. Nevertheless, RG-MDA amplification products showed better coverage and lower ADO rates than GEH-MDA. The present work also demonstrates that the same TSO sequencing data is suitable not only for the direct mutation detection, but also for the indirect mutation detection by linkage analysis of informative SNPs. The present work also demonstrates that Nexus software is competent for the detection of CNV by using with TSO sequencing data from RG-MDA products, allowing for the whole cytogenetic characterization of the embryos. In conclusion, successfully development of an innovative and promising DF-PGT strategy using TSO-NGS technology in TE biopsies, performed in-house in a single laboratory experience, has been done in the present work. Additional studies should be performed before it could be used as a diagnostic alternative in order to validate this approach for the detection of chromosomal aneuploidies.

Oxidative stress level in fresh ejaculate is not related to semen parameters or to pregnancy rates in cycles with donor oocytes.

PURPOSE: The purpose of the present study is to study the relationship between oxidative stress (OS) in semen, semen characteristics, and reproductive outcomes in oocyte donation intracytoplasmic sperm injection (ICSI) cycles. METHODS: OS was measured in 132 semen samples. RESULTS: OS levels were as follows: very high (1.5 %), high (43.2 %), low (30.3 %), and very low (25.0 %). Overall seminal parameters were as follows: volume (ml) = 4.2 (SD 2.1), concentration (millions/ml) = 61.6 (SD 59.8), motility (a+b%) = 47.4 (SD 18.0), and normal spermatozoa (%) = 8.2 (SD 5.1). Of the 101 cycles that reached embryo transfer, 55.4 % evolved in biochemical, 46.5 % in clinical, and 43.6 % in ongoing pregnancy. OS level does not relate to seminal parameters, fertilization rate, or pregnancy outcomes. CONCLUSIONS: OS testing by nitro blue tetrazolium (NBT) in fresh ejaculate might not be useful for all patients. Reproductive results with young oocytes and ICSI do not seem to be affected by OS-level semen.

Double-factor preimplantation genetic diagnosis: monogenic and cytogenetic diagnoses analyzing a single blastomere.

OBJECTIVE: Enhancing implantation rates in preimplantation genetic diagnosis (PGD) cycles is still a challenging aspect to address. As aneuploidy can be one of the factors influencing the low implantation rates obtained, the aim of this work was to combine monogenic analysis with comprehensive aneuploidy screening (double factor) in order to transfer the selected (healthy and euploid) embryos in the same in-vitro fertilization (IVF) cycle. METHOD: In the present double-factor PGD (DF-PGD) approach, a single blastomere was biopsied from each embryo, and the whole genome amplification DNA product obtained was successfully used for both monogenic analysis and metaphase comparative genomic hybridization cytogenetic screening. The developed DF-PGD was applied to 62 embryos from seven families at risk for monogenic-inherited diseases in a total of seven IVF-DF-PGD cycles. RESULTS: While 68.2% of the diagnosed embryos were healthy for the monogenic diseases, only 43.3% of them were chromosomally normal considering aneuploidies and/or segmental chromosome imbalances. Six out of seven families had transferrable embryos according to DF-PGD results. Two healthy babies were born from the 11 selected embryo transfers. CONCLUSION: In families at risk for monogenic diseases, the DF-PGD is a useful tool to select healthy and potentially viable embryos for transfer, according to their chromosome complement.

Non-meiotic chromosome instability in human immature oocytes.

Aneuploidy has been a major issue in human gametes and is closely related to fertility problems, as it is known to be present in cleavage stage embryos and gestational losses. Pre-meiotic chromosome abnormalities in women have been previously described. The aim of this study is to assess the whole-chromosome complement in immature oocytes to find those abnormalities caused by mitotic instability. For this purpose, a total of 157 oocytes at the germinal vesicle or metaphase I stage, and discarded from IVF cycles, were analysed by CGH. Fifty-six women, between 18 and 45 years old (mean 32.5 years), including 32 IVF patients (25-45 years of age) and 24 IVF oocyte donors (18-33 years of age), were included in the study. A total of 25/157 (15.9%) of the oocytes analysed, obtained from three IVF clinics, contained chromosome abnormalities, including both aneuploidy (24/157) and structural aberrations (9/157). Independently of the maternal age, the incidence of abnormal oocytes which originated before meiosis is 15.9%, and these imbalances were found in 33.9% of the females studied. This work sheds light on the relevance of mitotic instability responsible for the generation of the abnormalities present in human oocytes.

Detection of unbalanced chromosome segregations in preimplantation genetic diagnosis of translocations by short comparative genomic hibridization.

OBJECTIVE: To apply a comprehensive chromosomal screening through short comparative genomic hybridization (CGH) in the preimplantation genetic diagnosis (PGD) of translocations. DESIGN: Clinical research study. SETTING: A PGD laboratory and two IVF clinics. PATIENT(S): Three Robertsonian translocation carriers, two reciprocal translocation carriers, and a double-translocation carrier. INTERVENTION(S): After using the short-CGH approach in the reanalysis of two unbalanced embryos, discarded from a PGD for a reciprocal translocation carrier, the same method was applied in the PGD of day-3 embryos of translocation carriers. MAIN OUTCOME MEASURE(S): Ability of short CGH to detect partial chromosomal abnormalities in unbalanced embryos, translocation segregation proportions, and proportion of embryos carrying chromosomal abnormalities not related to the translocations. RESULT(S): The short-CGH technique detected errors resulting from the meiotic segregation of the chromosomes involved in the translocations and other abnormalities affecting the remaining chromosomes. Alternate segregation was detected most frequently among Robertsonian translocation cases, whereas unbalanced chromosome segregations were found predominantly in reciprocal ones. Aneuploidy and structural chromosome errors were found more frequently in Robertsonian than in reciprocal translocation carriers. Application of short-CGH PGD achieved pregnancy in two cases. CONCLUSION(S): Short CGH is a reliable approach for PGD of translocations, as it is capable of detecting partial chromosome errors caused by unbalanced segregations simultaneously to the screening of all chromosomes, and it may improve the results after PGD for translocation carriers.

Errors at mitotic segregation early in oogenesis and at first meiotic division in oocytes from donor females: comparative genomic hybridization analyses in metaphase II oocytes and their first polar body.

The aim of this work is to analyze, using the comparative genomic hybridization technique, the frequencies and the mechanisms involved in the production of aneuploidy events in donor oocytes. The results showed that 32.1% of them were aneuploid, with 51.7% of those originating from first meiotic division errors and 48.3% from the presence of aneuploid oogonium.

Triggering with human chorionic gonadotropin or a gonadotropin-releasing hormone agonist in gonadotropin-releasing hormone antagonist-treated oocyte donor cycles: findings of a large retrospective cohort study.

OBJECTIVE: To compare pregnancy rates and the incidence of ovarian hyperstimulation syndrome (OHSS) in donor stimulation cycles where final maturation of oocytes was induced with recombinant hCG or GnRH agonist. DESIGN: Retrospective, cohort study. SETTING: Private infertility clinic. PATIENT(S): A total of 1171 egg donors performing 2077 stimulation cycles. INTERVENTION(S): Controlled ovarian hyperstimulation of egg donors with GnRH antagonist protocol triggered with recombinant hCG (rhCG; 250 microg) or GnRH agonist (triptorelin 0.2 mg) based on the physician's decision. MAIN OUTCOME MEASURE(S): Proportion of mature and fertilized oocytes per donor cycle; clinical, ongoing pregnancy and implantation rate in recipients; and incidence of moderate/severe OHSS in oocyte donors. RESULT(S): The proportion of mature oocytes was comparable, whereas the difference in the fertilization rate reached statistical significance (65% vs. 69%). No significant differences were observed in the implantation rate or clinical and ongoing pregnancy rates per ET. The incidence of moderate/severe OHSS was 1.26% (13/1031; 95% confidence interval [CI], 0.74-2.15) and 0% (0/1046; 95% CI, 0.00-0.37) in the rhCG and GnRH agonist groups, respectively. CONCLUSION(S): Recipient outcome was not significantly different when using oocytes from GnRH antagonist-treated donor cycles triggered with hCG or GnRH agonist. However, GnRH agonist triggering was associated with a lower incidence of moderate/severe OHSS in egg donors.

The importance of aneuploidy screening in reciprocal translocation carriers.

The purpose of this study is to investigate the aneuploidy rate and the mosaicism of chromosomes not involved in reciprocal translocations. Aneuploidy screening (AS) (13, 16, 18, 21 and 22) was performed as a re-analysis on fixed blastomeres from 126 embryos already analysed in preimplantation genetic diagnosis (PGD) cycles of eight female and five male reciprocal translocation carriers who had not achieved a pregnancy. A successful diagnosis for AS was achieved in 91.3% of embryos; 30.9% were euploid and 60.3% were aneuploid for the five chromosomes analysed. Of the embryos, 8.7% were euploid for AS and normal-balanced for the translocation and 22.2% were euploid for AS but unbalanced for the translocation; 8% of the embryos were aneuploid for AS but normal-balanced for the translocation and 52.4% were aneuploid for AS and also unbalanced for the translocation. At least 58.7% of the embryos were mosaic regarding mosaicism for the chromosomes involved and not involved in the translocations. Six of the 16 embryos transferred in the PGD cycles were aneuploid for the AS study; four of them were also mosaics. AS should be performed in reciprocal translocation carriers after segregation analysis in PGD.

Karyotyping of human synaptonemal complexes by cenM-FISH.

The purpose of this work was to adapt the recently described centromere-specific multicolour (cenM-) FISH technique to human meiotic cells, and evaluate the usefulness of this multiplex fluorescence method for karyotyping human synaptonemal complex (SC), previously analysed by immunocytogenetic approaches. The results obtained demonstrate that cenM-FISH is a reliable one-single-step method, which allows for the identification of all SC present in pachytene spreads. Moreover, when cenM-FISH is applied after immunocytogenetic analysis, the number and distribution of MLH1 foci per chromosome can be established and recombination analysis for each chromosome can be performed easily.

Analysis of nine chromosome probes in first polar bodies and metaphase II oocytes for the detection of aneuploidies.

We used fluorescent in situ hybridisation (FISH) to detect nine chromosomes (1, 13, 15, 16, 17, 18, 21, 22 and X) in 89 first Polar Bodies (1PBs), from in vitro matured oocytes discarded from IVF cycles. In 54 1PBs, we also analysed the corresponding oocyte in metaphase II (MII) to confirm the results; the other 35 1PBs were analysed alone as when preimplantation genetic diagnosis using 1PB (PGD-1PB) is performed. The frequency of aneuploid oocytes found was 47.5%; if the risk of aneuploidy for 23 chromosomes is estimated, the percentage rises to 57.2%. Missing chromosomes or chromatids found in 1PBs of 1PB/MII doublets were confirmed by MII results in 74.2%, indicating that only 25.8% of them were artefactual. Abnormalities observed in 1PBs were 55.8% whole-chromosome alterations and 44.2% chromatid anomalies. We observed a balanced predivision of chromatids for all chromosomes analysed. Differences between balanced predivision in 1PB and MII were statistically significant (P&<0.0001, chi(2) test); the 1PB was most affected. The mean abnormal segregation frequency for each chromosome was 0.89% (range 0.52-1.70%); so, each of the 23 chromosomes of an oocyte has a risk of 0.89% to be involved in aneuploidy. No significant differences were observed regarding age, type of abnormality (chromosome or chromatid alterations) or frequency of aneuploidy. Nine of the 35 patients (25.7%) whose 1PB and MII were studied presented abnormalities (extra chromosomes) that probably originated in early oogenesis. Analysis of 1PBs to select euploid oocytes could help patients of advanced age undergoing in vitro fertilization (IVF) treatment.