PGDIS position statement on the transfer of mosaic embryos 2021.

Chromosome testing strategies, such as preimplantation genetic testing for aneuploidy (PGT-A), improve initial IVF outcomes by avoiding unwitting transfer of aneuploid embryos in morphology-based selection practices. Newer technologies have revealed that some embryos may appear to have intermediate whole chromosome (or parts of a chromosome termed segmental) copy number results suggesting trophectoderm mosaicism. An embryo with a trophectoderm mosaic-range result may be the only option for transfer for some patients. Recent data suggest that such mosaic embryos can be transferred without added risk of abnormal birth outcomes but may be associated with increased implantation failure and miscarriage rates, with higher values of mosaicism appearing to be less favourable for producing good outcomes. In this Position Statement, we provide guidance to laboratories, clinics, clinicians and counsellors to assist in discussions on the utility and transfer of mosaic embryos.

Obesity significantly alters the human sperm proteome, with potential implications for fertility.

PURPOSE: In men, obesity may lead to poor semen parameters and reduced fertility. However, the causative links between obesity and male infertility are not totally clear, particularly on a molecular level. As such, we investigated how obesity modifies the human sperm proteome, to elucidate any important implications for fertility. METHODS: Sperm protein lysates from 5 men per treatment, classified as a healthy weight (body mass index (BMI) ≤ 25 kg/m2) or obese (BMI ≥ 30 kg/m2), were FASP digested, submitted to liquid chromatography tandem mass spectrometry, and compared by label-free quantification. Findings were confirmed for several proteins by qualitative immunofluorescence and a quantitative protein immunoassay. RESULTS: A total of 2034 proteins were confidently identified, with 24 proteins being significantly (p < 0.05) less abundant (fold change < 0.05) in the spermatozoa of obese men and 3 being more abundant (fold change > 1.5) compared with healthy weight controls. Proteins with altered abundance were involved in a variety of biological processes, including oxidative stress (GSS, NDUFS2, JAGN1, USP14, ADH5), inflammation (SUGT1, LTA4H), translation (EIF3F, EIF4A2, CSNK1G1), DNA damage repair (UBEA4), and sperm function (NAPA, RNPEP, BANF2). CONCLUSION: These results suggest that oxidative stress and inflammation are closely tied to reproductive dysfunction in obese men. These processes likely impact protein translation and folding during spermatogenesis, leading to poor sperm function and subfertility. The observation of these changes in obese men with no overt andrological diagnosis further suggests that traditional clinical semen assessments fail to detect important biochemical changes in spermatozoa which may compromise fertility.

Infertility patients with chromosome inversions are not susceptible to an inter-chromosomal effect.

PURPOSE: The aim of this study was to evaluate the incidence of an inter-chromosomal effect (ICE) in blastocyst-stage embryos from carriers of balanced chromosome inversions. METHODS: Infertility patients (n = 52) with balanced inversions (n = 66 cycles), and maternal age-matched controls that concurrently cycled (n = 66), consented to an IVF cycle with preimplantation genetic testing for aneuploidy (PGT-A). Blastocyst-stage embryos underwent trophectoderm biopsy for PGT-A with only euploid blastocysts transferred in a subsequent frozen embryo transfer. Subtypes of inversions were included in aggregate: paracentric/pericentric, polymorphic/non-polymorphic, male/female carriers, and varying inversion sizes. RESULTS: The incidence of aneuploidy was not significantly higher for the inversion patients compared to the controls (inversion = 48.8% vs. control = 47.2% ns). Following euploid blastocyst transfer, there were excellent live birth outcomes. CONCLUSIONS: Carriers of balanced chromosome inversions did not exhibit higher aneuploidy rates for chromosomes that were not involved in the inversion compared to maternal age-matched controls, signifying the absence of an inter-chromosomal effect for this data set. These results provide the largest investigation of blastocyst embryos regarding the debated existence of an ICE resulting from the presence of an inversion during meiosis. However, further studies are warranted to investigate an ICE among inversions subtypes that were outside the scope of this study.

Human and mouse embryonic development, metabolism and gene expression are altered by an ammonium gradient in vitro.

Ammonium is generated in culture media by the spontaneous deamination of amino acids at 37 °C and through the metabolism of amino acids by human embryos. The appearance of ammonium is a time-dependent phenomenon and can compromise embryo physiology, development and viability. In this study, the effects of a gradient of ammonium on the development, metabolism and transcriptome of human and mouse embryos were investigated. Pronucleate oocytes were cultured in the presence of an ammonium gradient that mimicked the spontaneous deamination of Eagle's amino acids together with 1 mM glutamine. All embryos were cultured in sequential media G1/G2 at 5% O2, 6% CO2 and 89% N2. Human embryo metabolism was assessed through a non-invasive fluorometric analysis of pyruvate consumption. Transcriptome analysis was performed on the resultant blastocysts from both species using a microarray technology. Embryo development prior to compaction was negatively affected by the presence of low levels of ammonium in both species. Human embryo metabolism was significantly inhibited after just 24 and 48 h of culture. Transcriptome analysis of blastocysts from both species revealed significantly altered gene expression profiles, both decreased and increased. Functional annotation of the altered genes revealed the following over represented biological processes: metabolism, cell growth and/or maintenance, transcription, cell communication, transport, development and transcription regulation. These data emphasize the enhanced sensitivity of the cleavage-stage embryo to its environment and highlight the requirement to renew culture media at frequent intervals in order to alleviate the in vitro induced effects of ammonium build-up in the environment surrounding the embryo.

Embryos whose polar bodies contain isolated reciprocal chromosome aneuploidy are almost always euploid.

STUDY QUESTION: When a chromosome aneuploidy is detected in the first polar body and a reciprocal loss or gain of the same chromosome is detected in the second polar body, is the resulting embryo usually aneuploid for that chromosome? SUMMARY ANSWER: When reciprocal aneuploidy occurs in polar bodies, the resulting embryo is usually normal for that chromosome, indicating that premature separation of sister chromatids (PSSC)-not non-disjunction-likely occurred in meiosis I. WHAT IS KNOWN ALREADY: Single-nucleotide polymorphism-based microarray analysis can be used to accurately determine the chromosomal status of polar bodies and embryos. Sometimes, the only abnormality found is a reciprocal gain or loss of one or two chromosomes in the two polar bodies. Prediction of the status of the resulting embryo in these cases is problematic. STUDY DESIGN, SIZE, DURATION: Blinded microarray analysis of previously diagnosed aneuploid embryos that had reciprocal polar body aneuploidy. MATERIALS, SETTING, METHODS: IVF cycles were performed between 2008 and 2011 in patients aged 40 ± 3 years (range 35-47 years) with an indication for polar body-based aneuploidy screening. Thirty-five aneuploid vitrified Day 3 embryos were warmed, cultured to Day 5 and biopsied for microarray analysis. Predictions were made for the ploidy status of the embryo if PSSC or non-disjunction had occurred. The signal intensity for the aneuploid chromosome in the first polar body was compared between those that resulted in euploid and aneuploid embryos. MAIN RESULTS AND THE ROLE OF CHANCE: Among 34 embryos with evaluable results, 31 were euploid on re-analysis. Of 43 chromosomes that had reciprocal aneuploidy in the polar bodies, 41 were disomic in the embryo, indicating that PSSC was likely to have occurred 95% (95% confidence interval 85-99%) of the time. The log 2 ratio signal intensity from the chromosomes that underwent non-disjunction, resulting in unbalanced embryos, were outliers when compared with those that underwent PSSC. LIMITATIONS, REASONS FOR CAUTION: Although most embryos with reciprocal aneuploid polar bodies were euploid, it is unknown whether they maintain equivalent reproductive potential when transferred. Further study is needed to determine whether these embryos should be re-biopsied and considered for transfer. WIDER IMPLICATIONS OF THE FINDINGS: This study is consistent with increasing evidence that PSSC is the primary cause of meiosis I errors in embryos from women of advanced reproductive age. Clinicians should be cautious in interpreting results from polar body aneuploidy screening, especially when only the first polar body is tested.

Analysis of global gene expression following mouse blastocyst cryopreservation.

BACKGROUND: The aim of this study was to examine the effect of the cryopreservation procedure (slow freezing or vitrification) and cryoprotectants (1,2-propanediol or dimethylsulphoxide) on mouse blastocyst gene expression. METHODS: Cultured mouse blastocysts were cryopreserved with different protocols. Following thawing/warming, total RNA from re-expanded blastocysts was isolated, amplified and then analyzed using mouse whole-genome microarrays. RESULTS: Compared with non-cryopresevered control blastocysts, gene expression was only significantly altered by slow freezing. Slow freezing affected the expression of 115 genes (P < 0.05). Of these, 100 genes exhibited down-regulation and 15 genes were up-regulated. Gene ontology revealed that the majority of these genes are involved in protein metabolism, transcription, cell organization, signal transduction, intracellular transport, macromolecule biosynthesis and development. Neither of the vitrification treatment groups showed statistically different gene expression from the non-cryopreserved control embryos. Hierarchical cluster analysis, did however, reveal that vitrification using 1,2-propanediol could result in a gene expression profile closest to that of non-cryopreserved blastocysts. CONCLUSIONS: Investigating the effects of cryopreservation on cellular biology, such as gene expression, is fundamental to improving techniques and protocols. This study demonstrates that of the cryopreservation regimens employed, slow freezing induced the most changes in gene expression compared with controls.

Blastocysts from patients with polycystic ovaries exhibit altered transcriptome and secretome.

Polycystic ovaries (PCO) is a common phenotype of women presenting for infertility treatment. This study investigated whether blastocysts derived from women with PCO have an altered molecular signature which could be a causative factor contributing to reproductive failure. Morphologically similar blastocysts derived from women with PCO and donor oocyte cycles were analysed for transcription and protein secretion. Unsupervised hierarchical clustering demonstrated that the transcriptome profiles of blastocysts derived from PCO patients and control blastocysts were markedly different with complete branch separation. Statistical analysis revealed 829 genes with significantly different expression: 784 decreased (94.6%) and 45 increased (5.4%) in blastocysts derived from women with PCO compared with controls (P<0.05). Functional annotation of these genes revealed predominant gene ontology biological processes including protein metabolism (30%), transcription (22%), signal transduction (15%), biosynthesis (15%) and cell cycle (14%). Proteomic profiling identified 12 biomarkers that displayed significant decrease in expression in blastocysts derived from women with PCO compared with controls (P<0.05). These data indicate molecular alterations in human blastocysts derived from PCO patients, potentially demonstrating for the first time a link between patient aetiology/phenotype and subsequent embryo development, which in part may explain the observed reduction in reproductive capacity.

The role of proteomics in defining the human embryonic secretome.

Non-invasive gamete and embryo assessment is considered an important focus in assisted reproductive technologies (ART). Currently, the selection of embryos for transfer is based on morphological indices. Though successful, the field of ART would benefit from a non-invasive quantitative method of viability determination. Omics technologies, including transcriptomics, proteomics and metabolomics, have already begun providing evidence that viable gametes and embryos possess unique molecular profiles with potential biomarkers that can be utilized for developmental and/or viability selection. Unlike the human genome that is relatively fixed and steady throughout the human body, the human proteome, estimated at over a million proteins, is more complex, diverse and dynamic. It is the proteins themselves that contribute to the physiological homeostasis in any cell or tissue. Of particular interest in ART is the secretome, those proteins that are produced within the embryo and secreted into the surrounding environment. Defining the human embryonic secretome has the potential to expand our knowledge of embryonic cellular processes, including the complex dialogue between the developing embryo and its maternal environment, and may also assist in identifying those embryos with the highest implantation potential. Advances in proteomic technologies have allowed the non-invasive profiling of the human embryonic secretome with ongoing research focused on correlation with outcome. From a clinical perspective, embryo selection based on morphological assessment and non-invasive analysis of the human embryonic secretome may improve IVF success and lead to routine single embryo transfers.

Transcriptome analysis of in vivo and in vitro matured bovine MII oocytes.

In vitro maturation (IVM) of mammalian oocytes does not support the same rates of embryo development or pregnancy when compared to oocytes that have matured in vivo. Therefore, environment has a significant influence on the oocyte's ability to complete maturation and acquire the mRNA and proteins required for successful fertilization and normal embryonic development. The aim of this study was to analyze the MII oocyte transcriptome between in vivo and in vitro conditions. Total RNA was extracted, processed and hybridized to the Affymetrix GeneChip Bovine Genome Array. Following normalization of the microarray data, analysis revealed 10 differentially expressed genes after IVM compared to in vivo matured controls, including Aqp3, Sept7, Abhd4 and Siah2 (P<0.05). K-means cluster analysis coupled with associated gene ontology, identified several biological processes affected by IVM, including metabolism, energy pathways, cell organization and biogenesis, and cell growth and maintenance. Quantitative real-time PCR validated the microarray data and also revealed altered expression levels after IVM of specific putatively imprinted genes, Igf2r, Peg3 and Snrpn (P<0.05). Distinct IVM transcription patterns reflected the oocyte's response to its surrounding environment. Monitoring transcription levels of key oocyte maturation genes may subsequently assist in improving IVM success.

Symposium: innovative techniques in human embryo viability assessment. Can proteomics help to shape the future of human assisted conception?

Proteomics describes the changes in all proteins expressed and translated from a single genome. At present little is known regarding either the genome or proteome of human gametes or the preimplantation embryo. The unravelling of this information is fundamental to understanding the complexity of reproductive physiology, including the dialogue between the developing embryo and its maternal environment. To date, a lack of sensitivity has been the main reason behind the inability to introduce proteomics technology into assisted reproduction techniques. Proteomics alone involves several sophisticated techniques including imaging, mass spectrometry and bioinformatics to identify, quantify and characterize a proteome. The recent increased sensitivity of these techniques has allowed for the development of new protocols that are capable of not only profiling the proteome of individual human oocytes and embryos, but also the proteins produced by the embryo into the surrounding medium (the secretome). Hence, the identification of proteins that are involved in oocyte maturation, embryo development and implantation could lead to further improvements in assisted reproduction techniques as well as the development of new diagnostic tests. Furthermore, proteomics may contribute in the design of a non-invasive viability assay to assist in the selection of embryos for transfer in human assisted reproduction.

Comprehensive molecular cytogenetic analysis of the human blastocyst stage.

BACKGROUND: The high frequency of chromosomal abnormalities observed in human gametes and embryos is unlike that seen in other mammalian species and is of great clinical significance, leading to high rates of pregnancy loss, and live-born children with aneuploid syndromes. Although much is known concerning the aneuploidy rates of oocytes, cleavage stage embryos and fetuses during pregnancy, the chromosomal status of blastocysts has been relatively little investigated. METHODS: A total of 158 good quality blastocysts were examined using micromanipulation, whole genome amplification and comparative genomic hybridization. RESULTS: From the obtained data, it was evident that the aneuploidy rate (38.8%) is significantly lower for blastocysts than for embryos at earlier stages (51%). However, in many cases, chromosome errors, including monosomy, imbalance affecting the larger chromosomes and complex aneuploidy persisted to this final stage of preimplantation development. CONCLUSIONS: This study represents the first attempt to gain a detailed insight into the extent and type of chromosome errors seen at the blastocyst stage, using a comprehensive molecular cytogenetic method. Our data suggest that the blastocyst stage does not represent an absolute selective barrier, and that the majority of aneuploid embryos are lost at the time of implantation or shortly thereafter.

Embryology in the era of proteomics.

Currently, relatively little is known regarding the protein production of mammalian embryos. Unlike the genome, the proteome itself is dynamic reflecting both internal and external environmental stimuli. Until now the lack of sensitivity has remained a stumbling block for the global introduction of proteomics into the field of mammalian embryology. However, new developments in mass spectrometry have been revolutionary, utilizing protein profiling and peptide sequencing to elucidate underlying biological processes. The sensitivity of these platforms have allowed for the development of new protocols that are capable of profiling the proteome of individual mammalian oocytes and embryos. This information is fundamental to unravelling the complexity of embryo physiology including the dialogue between the developing embryo and its maternal environment. Such proteomic approaches are also assisting in the optimization of ART techniques, including oocyte cryopreservation and in vitro maturation. Embryo selection for transfer is another area of ART that should benefit in this era of proteomics. Currently, mammalian embryos are selected for transfer based on morphological grading systems. Although of great value, analysis of morphology alone cannot determine the embryo's physiological state or chromosomal complement. Subsequently, there is a need to identify in culture those embryos with the highest implantation potential. Proteomic analysis of the embryonic secretome (proteins produced by the embryo and secreted into the surrounding medium) followed by the identification of specific proteins critical for implantation, may lead to the development of a non-invasive viability assay to assist in the selection of embryos for transfer.

1,2-propanediol and the type of cryopreservation procedure adversely affect mouse oocyte physiology.

BACKGROUND: The aim of this work was to examine the effect of 1,2-propanediol (PrOH) and type of cryopreservation procedure (slow freezing and vitrification) on oocyte physiology. METHODS: Intracellular calcium of mouse metaphase II (MII) oocytes was quantified by fluorescence microscopy. The effect of PrOH on cell physiology was further assessed through analysis of zona pellucida hardening and cellular integrity. Protein profiles of cryopreserved oocytes were generated by time-of-flight mass spectrometry (TOF-MS). RESULTS: PrOH caused a protracted increase in calcium, which was sufficient to induce zona pellucida hardening and cellular degeneration. Using 'nominally calcium free' media during PrOH exposure significantly reduced the detrimental effects. Proteomic analysis identified numerous up- and down-regulated proteins after slow freezing when compared with control and vitrified oocytes. CONCLUSIONS: Using such approaches to assess effects on cellular physiology is fundamental to improving assisted reproduction techniques (ART). This study demonstrates that PrOH causes a significant rise in intracellular calcium. Using calcium-free media significantly reduced the increase in calcium and the associated detrimental physiological effects, suggesting that calcium-free media should be used with PrOH. In addition, analysis of the oocyte proteome following cryopreservation revealed that slow freezing has a significant effect on protein expression. In contrast, vitrification had a minimal impact, indicating that it has a fundamental advantage for the cryopreservation of oocytes.

Mitotic errors in chromosome 21 of human preimplantation embryos are associated with non-viability.

Fluorescent in situ hybridization (FISH) studies of human preimplantation embryos have demonstrated a high proportion of chromosomal mosaicism. To investigate the different timings and nature of chromosomal mosaicism, we developed single cell multiplex fluorescent (FL)-PCR to distinguish meiotic and mitotic cell division errors. Chromosome 21 was investigated as the model chromosome as trisomy 21 (Down's syndrome) represents the most common chromosomal aneuploidy that reaches live birth. Sister blastomeres from a total of 25 chromosome 21 aneuploid embryos were analysed. Of these, 13 (52%) comprised cells with concordant DNA fingerprints indicative of meiotic non-disjunction errors. The remaining 12 (48%) aneuploid embryos comprised discordant sister blastomere allelic profiles and thus were mosaic. Errors at all stages including metaphase (MI) (12%) and first (38%), second (31%) and third (19%) mitotic cleavage divisions were identified from the types and proportion of different allelic profiles. In addition, three embryos showed combined meiotic and mitotic cell division errors including non-disjunction and anaphase lag, suggesting that diploid cells had resulted from an aneuploid zygote. However, the majority of the mosaic aneuploid embryos showed mitotic gains and losses from a diploid zygote occurring prior to the activation of the embryonic genome. Allelic profiling of amniocytes from 15 prenatal diagnosis samples displayed only meiotic errors. There appears to be a large difference between the proportion of mosaic mitotic-derived trisomy 21 embryos and fetuses. These findings indicate that mosaic mitotic error of chromosome 21 is associated with non-viability.