Mosaic Embryos Research Articles

Trophectoderm cells of human mosaic embryos display increased apoptotic levels and impaired differentiation capacity: a molecular clue regarding their reproductive fate?

Are there cell lineage-related differences in the apoptotic rates and differentiation capacity of human blastocysts diagnosed as euploid, mosaic, and aneuploid after preimplantation genetic testing for aneuploidy (PGT-A) based on concurrent copy number and genotyping analysis? Trophectoderm (TE) cells of mosaic and aneuploid blastocysts exhibit significantly higher levels of apoptosis and significantly reduced differentiation capacity compared to those of euploid blastocysts. Embryos diagnosed as mosaic after PGT-A can develop into healthy infants, yet understanding the reasons behind their reproductive potential requires further research. One hypothesis suggests that mosaicism can be normalized through selective apoptosis and reduced proliferation of aneuploid cells, but direct evidence of these mechanisms in human embryos is lacking. Additionally, data interpretation from studies involving mosaic embryos has been hampered by retrospective analysis methods and the high incidence of false-positive mosaic diagnoses stemming from the use of poorly specific PGT-A platforms. Prospective cohort study performing colocalization of cell-lineage and apoptotic markers by immunofluorescence (IF). We included a total of 64 human blastocysts donated to research on Day 5 or 6 post-fertilization (dpf) by 43 couples who underwent in vitro fertilization treatment with PGT-A at IVI-RMA Valencia between September 2019 and October 2022. A total of 27 mosaic blastocysts were analyzed. The study consisted of two phases: Phase I (caspase-3, n = 53 blastocysts): n = 13 euploid, n = 22 mosaic, n = 18 aneuploid. Phase II (terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL), n = 11 blastocysts): n = 2 euploid, n = 5 mosaic, n = 4 aneuploid. Following donation for research, vitrified blastocysts were warmed, cultured until re-expansion, fixed, processed for IF, and imaged using confocal microscopy. For each blastocyst, the following cell counts were conducted: total cells (DAPI+), TE cells (GATA3+), inner cell mass (ICM) cells (GATA3-/NANOG+), and apoptotic cells (caspase-3+ or TUNEL+). The incidence of apoptosis was calculated for each blastocyst by dividing the number of caspase-3+ cells (Phase I) or TUNEL+ cells (Phase II) by the number of TE or ICM cells. Statistical analysis was performed according to data type and distribution (P < 0.05 was considered statistically significant). Phase I: Mosaic blastocysts displayed a similar number of total cells (49.6 ± 15 cells at 5 dpf; 58.8 ± 16.9 cells at 6 dpf), TE cells (38.8 ± 13.7 cells at 5 dpf; 49.2 ± 16.2 cells at 6 dpf), and ICM cells (10.9 ± 4.2 cells at 5 dpf; 9.7 ± 7.1 cells at 6 dpf) compared to euploid and aneuploid blastocysts (P > 0.05). The proportion of TE cells retaining NANOG expression increased gradually from euploid blastocysts (9.7% = 63/651 cells at 5 dpf; 0% = 0/157 cells at 6 dpf) to mosaic blastocysts (13.1% = 104/794 cells at 5 dpf; 3.4% = 12/353 cells at 6 dpf) and aneuploid blastocysts (27.9% = 149/534 cells at 5 dpf; 4.6% = 19/417 cells at 6 dpf) (P < 0.05). At the TE level, caspase-3+ cells were frequently observed (39% = 901/2310 cells). The proportion of caspase-3+ TE cells was significantly higher in mosaic blastocysts (44.1% ± 19.6 at 5 dpf; 43% ± 16.8 at 6 dpf) and aneuploid blastocysts (45.9% ± 16.1 at 5 dpf; 49% ± 15.1 at 6 dpf) compared to euploid blastocysts (26.6% ± 16.6 at 5 dpf; 17.5% ± 14.8 at 6 dpf) (P < 0.05). In contrast, at the ICM level, caspase-3+ cells were rarely observed (1.9% = 11/596 cells), and only detected in mosaic blastocysts (2.6% = 6/232 cells) and aneuploid blastocysts (2.5% = 5/197 cells) (P > 0.05). Phase II: Consistently, TUNEL+ cells were only observed in TE cells (32.4% = 124/383 cells). An increasing trend was identified toward a higher proportion of TUNEL+ cells in the TE of mosaic blastocysts (37.2% ± 21.9) and aneuploid blastocysts (39% ± 41.7), compared to euploid blastocysts (23% ± 32.5), although these differences did not reach statistical significance (P > 0.05). The observed effects on apoptosis and differentiation may not be exclusive to aneuploid cells. Additionally, variations in aneuploidies and unexplored factors related to blastocyst development and karyotype concordance may introduce potential biases and uncertainties in the results. Our findings demonstrate a cell lineage-specific effect of aneuploidy on the apoptotic levels and differentiation capacity of human blastocysts. This contributes to unravelling the biological characteristics of mosaic blastocysts and supports the concept of clonal depletion of aneuploid cells in explaining their reproductive potential. This work was funded by grants from Centro para el Desarrollo Tecnológico Industrial (CDTI) (20190022) and Generalitat Valenciana (APOTIP/2019/009). None of the authors has any conflict of interest to declare. N/A.

Mosaic embryo transfer versus additional IVF with PGT-A Cycle: a decision model comparing live birth rate and cost.

To evaluate the relative live birth rate and net cost difference between mosaic embryo transfer and an additional cycle of IVF with PGT-A for patients whose only remaining embryos are non-euploid. A decision analytic model was designed with model parameters varying based on discrete age cutoffs (<35, 35-37, 38-39, 40-42, 43-44, >44). Model inputs included probabilities of successful IVF, clinical pregnancy, and live birth as well as costs of IVF with PGT-A, embryo transfer, live birth, amniocentesis, and dilation and curettage. All costs were modeled from the healthcare system perspective and adjusted for inflation to 2023 $USD. Model outcomes were sub-stratified by degree and type of mosaicism. For patients younger than 43, an additional cycle of IVF with PGT-A resulted in a higher relative live birth rate (<35, +20%; 35-37, +15%; 38-39, +17%; 40-42, +6%; average, +14.5%) compared to mosaic embryo transfer with an average additional cost of $16,633. For patients older than 42, mosaic embryo transfer resulted in a higher live birth rate (43-44, +5%; >44, +3%; average, +4%) while on average costing $9572 less than an additional cycle of IVF with PGT-A. Mosaic embryo transfers are a superior alternative to an additional cycle of IVF with PGT-A for patients older than 42 whose only remaining embryos are non-euploid. Mosaic embryo transfers also should be considered for patients younger than 42 who are unable to pursue additional autologous IVF cycles. Counseling and care should be personalized to individual patients and embryos.

The morphokinetic signature of human blastocysts with mosaicism and the clinical outcomes following transfer of embryos with low-level mosaicism

BackgroundGenetic mosaicism is commonly observed in human blastocysts. Embryos’ morphokinetic feature observed from time-lapse monitoring (TLM) is helpful to predict the embryos’ ploidy status in a non-invasive way. However, morphokinetic research on mosaic embryos is extremely limited. Moreover, transfer of mosaic embryos is a new attempt in reproductive medicine, while studies regarding the clinical and neonatal outcomes following transfer of embryos with different levels and types of mosaicism are needed. This study aimed to investigate the morphokinetic characteristics of mosaic blastocysts, uncover clinical outcomes of mosaic embryos, and evaluate the effect of level and type of mosaicism on transfer outcomes.ResultsA total of 923 blastocysts from 229 preimplantation genetic testing cycles were cultured in TLM incubators in a single fertilization center between July 2016 and July 2021. Multivariate logistic regression models showed mosaic embryos had significantly shorter time to reach morula when compared with euploid (P = 0.002), mosaic with aneuploid (P = 0.005), and aneuploid (P = 0.005) embryos after adjusting the potential confounders. KIDScore is an artificial intelligence scoring program from time lapse incubation system to predict embryo implantation potential. Mosaic with aneuploid embryos had significantly lower KIDScore than euploid (P = 6.47e−4), mosaic (P = 0.005), and aneuploid (P = 0.004) embryos after adjustment. Meanwhile, we compared the clinical outcomes following transfer of low-level (< 50%) mosaic embryos (N = 60) with euploid embryos (N = 1301) matched using propensity scoring collected from September 2020 to January 2023. Mosaic embryos had significantly lower clinical pregnancy rate (41.67% vs. 57.65%, P = 0.015) and live birth rate (38.33% vs. 51.35%, P = 0.048) than the euploid embryos. Subgroup analyses showed the whole, segmental, and complex chromosome mosaic embryos had the similar clinical outcomes.ConclusionsThe shortened time to reach morula in mosaic embryos and the low KIDScore in mosaic with aneuploid embryos revealed innovative clues to embryo selection with the non-invasive TLM and provided new insights into biological mechanism of chromosomal abnormality. The analyses of overall and subgroups of mosaic embryo transfer outcomes helped to optimize embryo transfer scheme for in-vitro fertilization procedures. Multi-center prospective studies with large sample sizes are warranted to validate our results in the future.

Preimplantation genetic testing for complex chromosomal rearrangements: clinical outcomes and potential risk factors.

Complex chromosome rearrangements (CCR) are rare structural abnormalities involving at least three breakpoints, categorized into three types based on their structure: type A (three-way rearrangements), type B (double two-way translocations), and type C (exceptional CCR). However, thus far, limited data exists on preimplantation genetic testing for chromosomal structural rearrangements (PGT-SR) in CCR carriers. This study aims to evaluate the clinical outcomes and influencing factors of PGT-SR in couples with CCR. Fifteen couples with unique CCR recruited from 793 couples following PGT-SR between January 2017 and May 2023. In addition, a total of 54 CCR cases, 39 previously reported as well as 15 newly added, were included in the analysis of factors associate with normal/balanced embryos. A total of 100 blastocysts were biopsied and analyzed in 15 CCR couples after 17 PGT-SR cycles, with 16.0% being euploid, 78.0% aneuploid and 6.0% mosaic. 11 normal/balanced embryos and one mosaic embryo were transferred, resulting in eight live births. Furthermore, based on the combined data from 54 CCR carriers, the proportion of normal/balanced embryos was 10.8%, with a significant decrease observed among female carriers compared to male heterozygotes (6.5% vs. 15.5%, p = 0.002). Type B exhibited the lowest rate of euploid embryos at only 6.7%, followed by type A at 11.6% and type C at 14.0%, although the differences were not significant (p = 0.182). After completing the multivariate generalized estimating equation (GEE) analysis, type B (p = 0.014) and female carrier (p = 0.002) were identified as independent risk factors for fewer euploid embryos. The occurrence of balanced CCR in patients with reproductive abnormalities may be more frequent than we expected. Despite the proportion of normal/balanced embryos being significantly low, which can be influenced by CCR type and carrier's sex, PGT-SR may improve the reproductive outcomes among CCR cases. These findings can optimize the clinical management and genetic counseling of CCR carriers seeking assisted reproductive technology (ART).

Mosaic embryo transfer after pre-implantation genetic testing for structural rearrangement: a case study

Mosaic embryo transfer after pre-implantation genetic testing for structural rearrangement: a case study

Healthy Live Births after the Transfer of Mosaic Embryos: Self-Correction or PGT-A Overestimation?

The implementation of next generation sequencing (NGS) in preimplantation genetic testing for aneuploidy (PGT-A) has led to a higher prevalence of mosaic diagnosis within the trophectoderm (TE) sample. Regardless, mosaicism could potentially increase the rate of live-born children with chromosomic syndromes, though available data from the transfer of embryos with putative PGT-A mosaicism are scarce but reassuring. Even with lower implantation and higher miscarriage rates, mosaic embryos can develop into healthy live births. Therefore, this urges an explanation for the disappearance of aneuploid cells throughout development, to provide guidance in the management of mosaicism in clinical practice. Technical overestimation of mosaicism, together with some sort of "self-correction" mechanisms during the early post-implantation stages, emerged as potential explanations. Unlike the animal model, in which the elimination of genetically abnormal cells from the future fetal lineage has been demonstrated, in human embryos this capability remains unverified even though the germ layer displays an aneuploidy-induced cell death lineage preference with higher rates of apoptosis in the inner cell mass (ICM) than in the TE cells. Moreover, the reported differential dynamics of cell proliferation and apoptosis between euploid, mosaic, and aneuploid embryos, together with pro-apoptosis gene products (cfDNA and mRNA) and extracellular vesicles identified in the blastocoel fluid, may support the hypothesis of apoptosis as a mechanism to purge the preimplantation embryo of aneuploid cells. Alternative hypotheses, like correction of aneuploidy by extrusion of a trisomy chromosome or by monosomic chromosome duplication, are even, though they represent an extremely rare phenomenon. On the other hand, the technical limitations of PGT-A analysis may lead to inaccuracy in embryo diagnoses, identifying as "mosaic" those embryos that are uniformly euploid or aneuploid. NGS assumption of "intermediate copy number profiles" as evidence of a mixture of euploid and aneuploid cells in a single biopsy has been reported to be poorly predictive in cases of mosaicism diagnosis. Additionally, the concordance found between the TE and the ICM in cases of TE biopsies displaying mosaicism is lower than expected, and it correlates differently depending on the type (whole chromosome versus segmental) and the level of mosaicism reported. Thus, in cases of low-/medium-level mosaicism (<50%), aneuploid cells would rarely involve the ICM and other regions. However, in high-level mosaics (≥50%), abnormal cells in the ICM should display higher prevalence, revealing more uniform aneuploidy in most embryos, representing a technical variation in the uniform aneuploidy range, and therefore might impair the live birth rate.

#275 : A Comparative Study of Hatching Methods for Biopsy Time and Ploidy Status Assessment

Background and Aims: Preimplantation genetic testing plays a crucial role in assisted reproductive technologies by aiding in the selection of embryos with optimal developmental potential. This study aims to compare the time required for biopsy and evaluate the ploidy status between Day3 and Day4 hatching-based methods and cases without assisted hatching (AH). Method: We retrospectively analyzed data from 257 embryos from 56 patients between November 2022 and March 2023. Embryos were categorized into three groups: Day3 hatching-based method (n=43), and Day4 hatching-based method (n=134), and cases without AH (n=80). We used Kruskal-Wallis tests and chi-square tests to compare the groups with R software (version 4.1.0) at a significance level of 0.05. Results: There was a significant difference in the median biopsy time per embryo between the hatching-based methods (H = 6.01, p = 0.049). The Day4 hatching-based method had a significantly shorter biopsy time per embryo than cases without AH (W = 95, p = 0.037). There were no significant differences in the percentages of euploid, aneuploid, or mosaic embryos between the hatching-based methods. The Chi-square test showed no significant differences between the observed and expected percentages of euploid ([Formula: see text] = 0.22, p = 0.893), aneuploid ([Formula: see text] = 2.35, p = 0.308), or mosaic ([Formula: see text]= 0.58, p = 0.748) embryos across all hatching-based methods. The Chi-square test of independence showed no significant differences in the percentages of euploid ([Formula: see text] = 0.56, p = 0.755), aneuploid ([Formula: see text] = 1.41, p = 0.495), or mosaic ([Formula: see text] = 0.22, p = 0.896) embryos between the hatching-based methods. Conclusion: We suggest that the Day4 hatching-based method may provide a significant advantage in terms of shorter biopsy time per embryo compared to cases without AH, but there were no significant differences in the percentages of euploid, aneuploid, or mosaic embryos between the hatching-based methods.

#347 : Factors Related to Mosaicism of Human Embryo: Conventional Statistics and Machine Learning Analysis

Background and Aims: Mosaicism arises from errors in the cell division process and can lead to cells containing a combination of normal and mutated genes. Mosaic embryos can produce healthy babies. However, it may be associated with a lower implantation and a higher miscarriage rate. Despite extensive research, the scientific understanding of the underlying causes of embryonic mosaicism remains limited, and conclusions remain subject to ongoing debate. Machine learning (ML) can capture complex relationships and automatically learn the most informative features, while conventional statistics (CS) are simple to understand. We aimed to apply both ML and CS to determine the critical factors that may be associated with mosaicism. Method: A retrospective analysis was conducted at the Infertility Department of Hung Vuong Hospital between 2018 and 2022 using medical data from 51 IVF couples. The study utilized both machine learning and conventional statistical methods to identify possible factors associated with mosaicism in human embryos. Results: Out of the 246 embryos studied, 84 (32%) were found to be mosaic. The performance of the classifiers in the machine learning analysis was relatively poor when using all features, with F1-scores ranging from 0.08 to 0.26. While feature selection helped to improve performance, the F1-scores remained low. On the other hand, the conventional statistical analysis revealed that patients with cryptozoospermia showed a positive association with mosaicism (OR 2.68, 95% CI 1.08 to 6.66, p=0.033). Conclusion: According to the machine learning algorithms, the current set of clinical features has no relation to the incidence of mosaicism. However, the conventional statistical analysis suggests that patients with cryptozoospermia may contribute to a higher risk of mosaicism. Further research is needed to fully understand the mechanisms underlying mosaicism in human embryos.

Debate 3: Genetically Tested Embryos Expected to Have Mild Adult Disease Should Not Be Transferred

MOTION: For Next generation sequencing (NGS) technology has been widely used in the field of genetic diseases, mainly focusing on the diagnosis of genetic diseases, newborn screening, prenatal screening, preimplantation genetic test (PGT) and other fields such as the treatment of cancer. In the field of reproductive medicine, there are still some issues that engendered controversy about the application of PGT. On one hand, disputes over whether using PGT to treat late-onset diseases is ethical remain. On the other hand, some tested embryos are “available” to transfer, while others are “discarded” embryos that are “unavailable” because they are detected carrying certain genetic flaws. But there are still some embryos biopsied expected to have some genetic abnormality that might or not cause malfunction, including embryos with copy number variant of uncertain significance (VUS), embryos tested with adult-onset genetically anomalousness, embryos of balanced translocation as well as mosaic embryos. So how to dispose of this group of embryos in clinical practice is a hot issue up to now. In this debate, we reviewed the relevant case reports and original research combined with the joint consensus or committee opinion to state our proposition: embryos tested with possible abnormal genetic characteristics and thus expected to have mild adult disease should not be transferred. Parents undergoing PGT may wish to avoid the lifelong concern caused by the chance that their children may develop adult-onset conditions. The transfer of embryos expected to have mild adult disease is contrary to their original intention of seeking PGT treatment. Whether intentional or incidental, the discovery and request for transfer of embryos likely to result in the birth of offspring with health-affecting conditions pose ethical dilemmas for physicians and their staff, patients, and society. MOTION: AGAINST Preimplantation genetic testing provides couples with powerful reproductive choices. The technology allows individuals to avoid giving birth to children with significant health issues and developmental disability. Additionally, serious familial adult-onset conditions, such as predisposition to cancer or dementia, can be screened out of future generations. Polygenetic risk scores will be the next leap in genetic testing technology. This data can used to determine the chances of an embryo developing adult-onset conditions such as cardiovascular disease, diabetes, mental illness and even gout. This presentation argues that there is a limit at which society should stop screening embryos for disease and draws upon practical observations and well-accepted ethical frameworks to support the case.

Embryonic, genetic and clinical outcomes of fresh versus vitrified oocyte: A retrospective cohort study

Objective: To compare embryonic development, ploidy status and clinical outcomes between fresh and frozen-thawed oocytes. Methods: This retrospective cohort study evaluated 83 fertilization cycles including both fresh and frozen oocytes from 79 patients at the HP Fertility Center of Hai Phong International Hospital of Obstetrics and Pediatrics in Vietnam. The patient underwent several ovarian stimulation cycles to accumulate a certain number of oocytes that would be vitrified. In the last oocyte retrieval, all patient's oocytes including both frozen and fresh would be fertilized. The outcomes included the rates of oocyte survival, cleavage embryo, blastocyst, ploidy status, pregnancy, biochemical pregnancy and clinical pregnancy. Results: The oocyte survival rate after thawing was 96.5%. No statistically significant difference was found when comparing fresh and frozen oocytes regarding fertilization rate (78.1% vs. 75.5%, P=0.461), usable cleavage embryo rate (86.9% vs. 87.2%, P=0.916) but usable blastocyst rate was found higher statistically in the frozen oocyte group (44.4% vs. 54.0%, P=0.049). The percentages of euploid, aneuploid and mosaic embryos between the fresh group and the vitrified group had no significant differences (33.8% vs. 31.6%, P=0.682; 51.0% vs. 54.2%, P=0.569; 15.2% vs. 12.4%, P=0.787; respectively). The rates of pregnancy, biochemical pregnancy and clinical pregnancy had no statistical difference (68.8% vs. 64.8%, P=0.764; 12.5% vs. 3.6%, P=0.258; 37.5% vs. 46.4%, P=0.565). 17 Mature oocytes are the minimum to have at least one euploid embryo. Conclusions: Oocyte vitrification does not affect embryonic, genetic and clinical results. The number of mature oocytes should be considered for fertilization in some cases.

#46 : Outcomes of Transfer Abnormally Chromosomal Embryos

Background and Aims: In some couples, there was no normal chromosomal embryos after preimplantation genetic testing (PGT) for aneuploidy (PGT-A), for structural chromosomal rearrangements (PGT-SR) or for monogenic (PGT-M). The couples decided to use the abnormal embryos for transfer after thorough genetic counselling. This study reports the outcomes of transfer abnormally chromosomal embryos. Method: This retrospective study was conducted from January 2020 to December 2022 at IVFMD, My Duc Hospital, Ho Chi Minh City, Vietnam. The abnormally chromosomal embryos that were considered for transfer included aneuploidy related to [Formula: see text]3 chromosomes (except trisomy 13, 18, 21 and monosomy X); or mosaicism has [Formula: see text]50% of aneuploidy after PGT-A, PGT-SR or PGT-M (except mosaic trisomy 13, 18, 21 and monosomy X). Results: 94 patients transferred 94 single abnormally chromosomal embryos. Out of 94 embryos, 62 were mosaic and 32 were aneuploid. Among the group of mosaic embryos, 25 (40.3%) had segmental, 32 (51.6%) had 1 or 2 chromosomal abnormalities and 5 (8.1%) had 3 or more. Among the group of aneuploid embryos, 4 (12.5%) had segmental, 13 (40.6%) had 1 chomosomal abnormalities, 5 (15.6%) had 2 chomosomal abnormalities and 10 (31.3%) complexes (both mosaic and aneuploid chromosome). Among 20 live births, 2 cases have normal fetal karyotype, and 11 cases have normal CNVs blood results. Conclusion: Mosaic embryos can be considered for transfer in cases having no normal embryos after PGT. More than 50% of babies born from chromosomally abnormal embryo transfer have normal chromosomal results.

#161 : Validation and Concordance of Non-Invasive Preimplantation Genetic Testing for Aneuploidy (niPGT-A) Using Cell Free DNA from Spent Culture Media (SCM) with Gold Standard Trophectoderm Biopsy

Background and Aims: Recent studies suggests that cell free(cf)-DNA is secreted in the spent culture medium (SCM) during embryo development, this can be used as non-invasive approach for PGTA. We aimed to validate Non-invasive Preimplantation Genetic Testing for aneuploidies (niPGT-A) using cf-DNA from SCM Methods: Prospective cohort study, tertiary centre from January-August 2021-2022. A total of 23 aneuploid/high mosaic embryos were donated by couples who had Trophectoderm (TE) biopsy for aneuploidy testing. All aneuploid blastocyst (WB) that were donated for research were thawed and transferred to RNAase and DNAase free PCR tubes. DNA extraction and amplification are carried out using Sureplex DNA amplification system. Libraries prepared using VeriSeqTM PGS Library Prep kit. A total of 24 libraries were pooled, denatured subjected to Next Gen Sequencing (NGS) using MiSeq system. Copy Number Variation (CNV) visualization and analysis for each sample was carried out using BlueFuse Multi Software. Results: NGS results from SCM and TE were respectively used against same positive control to find out accuracy with corresponding WB. NGS profiles generated from aneuploid WB, were manually analysed for CNV. Quality control(qc) was performed using Illumina qc requirements, only samples satisfying were used for analysis. Out of 23 embryos, 14 were aneuploid and 9 were high mosaic based on TE biopsy. Out of 14 aneuploid blastocysts on TE biopsy, 9 (64%) showed corresponding aneuploidy (full chromosomal concordance) on WB NGS profile. The rest 5 showed aneuploidy or mosaicism of a different chromosome on WB NGS profile. Only 5 out of 23 (21.7%) had full chromosome concordance between SCM and WB. The ploidy concordance between WB vs TE and WB vs SCM was 100% and 69.5% (16/23), which was statistically significant (p[Formula: see text]0.05). The decreased ploidy concordance with SCM could be due to suspected maternal contamination. Conclusions: Aneuploidy testing using Cf DNA in SCM is a promising technique but needs more research.

#156 : Patient Decision Making and Trends in Embryo Disposal

Background and Aims: Embryo cryopreservation is common in IVF, allowing for surplus embryos to be utilised in subsequent cycles. As such, for a variety of reasons, patients may need to contend with embryo disposition decisions. This study looked at trends surrounding patient decisions to dispose of cryopreserved embryos. Method: A retrospective study was conducted on the consented embryo disposals of 230 patients between 2019 and 2023. Variables analysed included number of live births, number of embryos for disposal, embryo mosaicism, and reasons behind the decision to dispose of their embryos. The length of time between the date the consent was signed and the last IVF cycle, IVF live birth and initial contact with the clinic was also established. Results: A large proportion of patients (43.9%) did not specify why they chose to discard their embryos, however of those who did, there were three main categories (embryos reaching their expiry (27.9%), the return of mosaic PGT results (24.0%), and patients completing their family (22.5%). Of the 57 patients who chose to discard after being contacted regarding their embryo expiry, 19.3% initially chose to extend their storage before opting to discard. Surprisingly, only 62.2% of patients had at least one live birth prior to submitting their disposal consent, and waited, on average, 1.6 years from the date of their most recent live birth to discard. Furthermore, there was an average of 1.8 years between patients’ last IVF cycle and the decision to discard, with an average of 86.5 days elapsing from the point of initial contact with the clinic until the date the disposal consent was signed. Conclusion: Many patients retain their embryos in storage until their expiry, and this point of contact prompts them to make decisions regarding the disposition of their biomaterial, and the reasons behind discard decisions are not solely associated with achieving a live birth.

PGT-A: What Is Its Value in Treating Recurrent Pregnancy Loss?

PGT has been widely applied in the world, but its effectiveness for the treatments of recurrent pregnancy loss is still inconclusive. There is currently no consensus over the precise definition of RPL, according to the ESHRE guideline in 2022, a diagnosis of Recurrent Pregnancy Loss (RPL) could be considered after the loss of two or more pregnancies. The etiology of RPL is complex and highly heterogeneous, the higher proportion of embryo aneuploidy is an important reason, which can be affected significantly by maternal age. RPL has been listed as one of the common indications for PGT-A worldwide. However, the new RPL guideline by EHSRE in 2022, indicated that limited evidence for PGT in couples with RPL shows no clear benefit of treatment with very low quality. We reviewed the published studies, three retrospective studies showed that PGT-A may improve live birth rate for RPL patients, while two studies showed negative results. Most importantly, there were no randomized studies published on the application of PGT-A among RPL couples. Our data also showed similar cumulative live birth rates between PGT-A group and the control, regardless of maternal age, but PGT-A may favour in reducing miscarriage recurrence risk for RPL couples over 37 years old who obtained transferrable embryos. The presence of mosaic embryos and false positive/negative results, which can cause a high rate of cycle cancellation, may be the main reasons that compromise the efficacy of PGT-A. The cost-effectiveness of PGT-A is another important concern. In conclusion, there is still no sufficient evidence to support whether PGT-A can effectively improve the pregnancy outcomes for RPL couples, and proper-designed RCT is required in the future.

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

Does the diagnosis of mosaicism affect ploidy rates across different providers offering preimplantation genetic testing for aneuploidies (PGT-A)? 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. 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. 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. 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. 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. 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. 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. 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. N/A.

Evaluation of pre-implantation genetic testing for aneuploidy outcomes in patients without infertility undergoing in vitro fertilization compared to infertile controls.

To evaluate pre-implantation genetic testing for aneuploidy (PGT-A) outcomes in patients without infertility compared to infertile patients. We performed a retrospective cohort study of all patients without an infertility diagnosis ("fertile" patients) who utilized PGT-A at a large university-affiliated fertility center between 2016 and 2021. Fertile patients were 1-to-3 matched to infertile controls by age and number of oocytes retrieved. The primary outcome was blastocyst aneuploidy rate. Secondary outcomes included ovarian reserve markers, laboratory outcomes, and other PGT-A outcomes [rates of euploidy, mosaicism, and potentially transferrable (euploid + mosaic) embryos]. 283 fertile and 849 infertile patients were included. Median age, anti-Mullerian hormone, and day 2 estradiol levels were equivalent among groups; day 2 follicle-stimulating hormone levels were higher in fertile patients (6.9 vs. 6.5IU/mL, p < 0.01). The aneuploidy rate was similar among fertile and infertile patients (33.7% vs. 31.8%, p = 0.11); the euploidy rate was higher (50.8% vs. 47.0%, p < 0.01), and the mosaicism rate was lower in fertile patients (13.3% vs. 19.2%, p < 0.01). The rate of transferrable embryos was similar among groups (64.0% vs. 66.3%, p = 0.07), as was the percentage of patients yielding ≥ 1 euploid embryo (90.1% vs. 87.3%, p = 0.25). When controlling for significant covariates, multiple linear regression showed that aneuploidy rate was equivalent in both cohorts. Aneuploidy rate was similar in fertile and infertile patients. Fertile patients had slightly higher euploidy and lower mosaicism than infertile patients. Still, compared to fertile patients, infertile patients had equivalent rates of transferrable embryos and were just as likely to yield ≥ 1 euploid embryo.

Clinical management of mosaic results from preimplantation genetic testing for aneuploidy of blastocysts: a committee opinion

This revised document incorporates a growing number of published studies about mosaic embryo transfer and provides current evidence-based considerations for the clinical management of embryos with mosaic results on preimplantation genetic testing for aneuploidy. This document replaces the document titled "Clinical management of mosaic results from preimplantation genetic testing for aneuploidy (PGT-A) of blastocysts: a committee opinion," published in 2020 (Fertil Steril 2020;114:246-54).

Conventional IVF is feasible in preimplantation genetic testing for aneuploidy.

To investigate the feasibility of the application of conventional in vitro fertilization (cIVF) for couples undergoing preimplantation genetic testing for aneuploidies (PGT-A) with non-male factor infertility. To evaluate the efficiency of sperm whole-genome amplification (WGA), spermatozoa were subjected to three WGA protocols: Picoplex, ChromInst, and multiple displacement amplification (MDA). In the clinical studies, 641 couples who underwent PGT-A treatment for frozen embryos between January 2016 and December 2021 were included to retrospectively compare the chromosomal and clinical outcomes of cIVF and intracytoplasmic sperm injection (ICSI). Twenty-six couples were prospectively recruited for cIVF and PGT-A treatment between April 2021 and April 2022; parental contamination was analyzed in biopsied samples; and 12 aneuploid embryos were donated to validate the PGT-A results. Sperm DNA failed to amplify under Picoplex and ChromInst conditions but could be amplified using MDA. In frozen PGT-A cycles, no significant differences in the average rates of euploid, mosaic, and aneuploid embryos per cycle between the cIVF-PGT-A and ICSI-PGT-A groups were observed. The results of the prospective study that recruited couples for cIVF-PGT-A treatment showed no paternal contamination and one case of maternal contamination in 150 biopsied trophectoderm samples. Among the 12 donated embryos with whole-chromosome aneuploidy, 11 (91.7%) presented uniform chromosomal aberrations, which were in agreement with the original biopsy results. Under the Picoplex and ChromInst WGA protocols, the risk of parental contamination in the cIVF-PGT-A cycles was low. Therefore, applying cIVF to couples with non-male factor infertility who are undergoing PGT-A is feasible.

Embryonic mosaicism: What does it mean clinically?

Embryonic mosaicism continues to represent a complex phenomenon that is considered by many authors as a limiting factor in the interpretation of PGT-A cycles results.&#x0D; A mosaic embryo is characterized by the presence of cell populations with at least two different karyotypes. There are controversial aspects related to embryonic mosaicism, such as the great variability in the incidence reported between laboratories and clinics, which in turn is related to the existence of false positives during the diagnosis. These false positives can also affect the results after the transfer of these embryos. Numerous publications have analyzed the results of transferring this type of embryo. The majority agrees that although worse clinical results are achieved that, when transferring a euploid embryo, it is possible to achieve an evolutionary pregnancy with a healthy birth. The objective of this article is to provide tools that allow the professional to advise those patients who perform a PGT-A cycle about this conflictive aspect of it.

Challenges experienced by genetic counselors while they provided counseling about mosaic embryos

To survey genetic counselors (GCs) who have counseled about mosaic embryos regarding the challenges they faced in counseling this patient population and assess their need for more resources to support their practice. Self-administered online survey. Academic university. Seventy-eight GCs primarily from the United States and Canada. Genetic counselors completed a quantitative survey with an embedded qualitative component. Quantitative data were analyzed by descriptive statistics. An inductive thematic analysis was performed on open-text responses. Genetic counselors were asked what clinical activities relating to mosaic embryos they performed. They were then asked to rate how challenging each activity was to perform using a 5-point scale; a rating of 4 or 5 was defined as highly challenging. Open-text questions enabled GCs to describe factors that they felt contributed to these challenges. The challenges reported by GCs included the uncertainty of outcomes in offspring after mosaic embryo transfer, limited guidelines available to assist clinicians with counseling about mosaic embryos, and ranking mosaic embryos by suitability for transfer. The contributing factors suggested by participants included limited outcome data, limited GC involvement in pretest counseling for preimplantation genetic testing for aneuploidy (PGT-A), and perceived inconsistency in counseling practices across clinics. Genetic counselors differed in their genetic testing recommendations for pregnancies conceived after mosaic embryo transfer. Amniocentesis and postnatal assessment were recommended by 85% and 49% of GCs, respectively, and 15% recommended chorionic villus sampling and noninvasive prenatal testing. Almost all (92%) reported a need for more resources, such as standardized guidelines, more outcome data, and continuing education on PGT-A and mosaicism. This study describes challenges experienced by GCs while they counseled about mosaic embryos. Our findings demonstrate a need for more outcome data on mosaic embryo pregnancies and for evidence-based clinical guidelines. The differing recommendations for prenatal genetic testing among GCs in the study warrant further research into contributing factors. We strongly recommend that pretest counseling, including a discussion regarding mosaicism, is provided to all couples considering PGT-A to reduce counseling challenges and to promote patients' informed decision-making.