Embryo Developmental Competence Research Articles

O-215 Embryo developmental competence of de novo male gametes in a novel three-dimensional culture

Abstract Study question Are de novo gametes generated from mouse embryonic stem cells (mESCs) in a novel three-dimensional (3D) culture system capable of supporting full preimplantation development? Summary answer A novel 3D culture system yielded de novo male gametes capable of fertilizing an oocyte and supporting full preimplantation development. What is known already In regenerative medicine, several 3D culture systems have been shown to be capable of producing functional tissue implants. In reproductive biology, recent studies have reported preliminary success in generating functional de novo gametes through soft-agar culture and testicular organoids, in the mouse model. However, a heterologous transplantation technique is required to obtain functional gametes. Study design, size, duration mESCs were first cultured on a gelatin-coated 6-well plate with fibroblasts in monolayer and later spherified using sodium alginate. Spheres were submerged in specifically designed conditioned media to encourage differentiation of the mESCs into germ-like cells. Over the course of differentiation, cells were assessed for germ cell differentiation biomarkers. Considering that normal spermatogenesis occurs in 30 days, utilization of the de novo gametes was planned for days 15, 22, 29, and 36. Participants/materials, setting, methods mESCs were differentiated by submerging the spheres in EpiLC medium containing Activin A, bFGF, and KSR for 3 days followed by PGCLC medium containing BMP4, LIF, SCF, and EGF for up to 36 days. Differentiation was assessed for markers DAZL (spermatogonium), VASA (spermatocyte), BOULE (post-meiotic stage), and acrosin (spermatid). Differentiated cells were then injected into oocytes and activated by calcium ionophore. Embryo development was monitored in a time-lapse incubator. Main results and the role of chance Expression of DAZL in 20% and VASA in 15% of the cells at day 3 demonstrated progression into spermatogenesis. On day 10, DAZL and VASA were assessed again, revealing increases to 45% and 18%. A small proportion of cells expressed post-meiotic biomarkers, BOULE (1%) and acrosin (2%). On day 15, VASA expression plateaued at 17%, BOULE expression peaked at 10%, and acrosin reached 5%. On day 22, expression of VASA increased slightly to 19%, BOULE decreased to 8%, and acrosin peaked at 7%. On day 29, VASA expression peaked at 20%, acrosin expression remained stable at 7%, and BOULE expression dropped to 2%. On day 36, VASA was still expressed at 13%, and few cells expressed acrosin (1%); there was complete loss of BOULE expression. Our mouse ICSI control achieved 89.2% fertilization and 77.8% blastocyst rates. De novo gametes were injected into oocytes on days 15, 22, 29, and 36, achieving fertilization rates of 35.0%, 61.1%, 81.8%, and 75.0%, respectively. Correspondently, hatching blastocysts were obtained at rates of 5.0%, 16.7%, 36.4%, and 8.3%, respectively. Limitations, reasons for caution Despite the ability to fertilize normally, the blastulation rate remained suboptimal. Most importantly, the ability to generate live offspring still needs to be documented. Wider implications of the findings Our novel 3D differentiation model can generate functional gametes and is aimed at obviating the need for allo-/xeno-geneic transplantation. If reproducibility and the ability to obtain healthy offspring are confirmed, this method may represent a tool for achieving neogametogensis in mammals. Trial registration number N/A

P-227 Exposure to triclosan modifies oocyte development and embryo metabolism

Abstract Study question Does the endocrine disruptor, triclosan, affect the function of cumulus-oocyte-complexes (COCs), and if so, do these effects persist in early embryo developmental competence and metabolism? Summary answer Triclosan caused COCs to i) increase progesterone secretion, ii) switch from releasing to consuming pyruvate, and iii) produce fewer blastocysts that were iv) metabolically compromised. What is known already Triclosan (TCS) is used widely as an antimicrobial agent in a range of consumer healthcare products, including hand sanitiser, toothpastes, soaps, and shampoos. Its prevalent use has led to accumulation in the environment and human exposure. Indeed, TCS has been identified in a range of bodily fluids. It is classed as an endocrine-disrupting chemical and there are reports of its detrimental impact on the vertebrate reproductive system. However, the direct effects of TCS on mammalian Cumulus-Oocyte-Complexes’ (COCs) function and subsequent development of the embryo remains unknown. Study design, size, duration COCs, isolated from abattoir-derived bovine ovaries on 5 independent occasions, were exposed of two environmentally-relevant doses of TCS (10nM and 1nM) during in vitro maturation. The TCS-exposed COCs were subsequently fertilised in vitro without further TCS addition, and the cleavage and blastocyst rate were recorded on Day 2 and Day 7/Day 8, respectively. The resulting blastocysts were collected and further cultured individually for 24 hours, and depletion/release of key-metabolites quantified. Participants/materials, setting, methods In vitro production was performed for the maturation of the bovine oocytes and the embryo culture. The production of steroid hormones (17b-estradiol and progesterone) during IVM was determined by ELISA. The concentrations of glucose, pyruvate, lactate during IVM and blastocyst formation were quantified in the spent media using microfluorometric assays. Main results and the role of chance TCS at 1nM had a significant impact on steroidogenesis during COCs in vitro maturation; progesterone production was significantly higher compared to control group (4.784 ±1.69 vs 2.85 ±0.83 pg/hour/COCs, p = 0.035; n = 5), although oestrogen release was not affected. In addition, there was a dramatic shift in pyruvate metabolism. Over the course of in vitro maturation, COCs exposed to TCS depleted pyruvate from the culture media regardless of the administrated dose whilst net pyruvate concentration increased in the medium of COCs in the control groups. The cleavage rate of bovine embryos produced from TCS-exposed COCs was not significantly different to control group. However, COCs-exposed to 1nM TCS produced significantly fewer blastocysts (12.22% vs 29.11% n = 5; p = 0.02). Those blastocysts that did originate from COCs treated with TCS depleted significantly more pyruvate from the culture media compared to controls, regardless of TCS dose (19.86 ±10.54 vs 10.12 ±12.5 pmol/hour/embryo, p = 0.011 and 21.41 ±10.08 vs 10.12 ±12.5 pmol/hour/embryo, p = 0.002, TCS-10nM and TCS-1nM compared to Control, respectively). Additionally, while glucose depletion did not differ, the lactate release from blastocysts derived from COCs exposed to 10nM TCS was approximately halved compared to controls (15.20 ±14.59 vs 31.83 ±22.73 pmol/hour/embryo p = 0.0054). Limitations, reasons for caution The origin of the ovaries may be a limitation since they came from animals at different stages of estrous cycle. Furthermore, pre-exposure to TCS prior to animal slaughter cannot be discounted. Finally, these observations were made in an animal model in a laboratory setting which may differ from natural exposure. Wider implications of the findings TCS modifies oocyte maturation, developmental competence, and metabolic activity of resulting embryos. The differences in pyruvate metabolism and steroidogenesis point to altered mitochondrial function. These data indicate that TCS directly modulates oocyte and embryo physiology which may relate to fertility in domestic livestock species and may be extrapolated to humans. Trial registration number not applicable

O-311 Evaluating the Reproductive Potential of Azoospermic Men by Profiling the Genome of Surgically Retrieved Spermatozoa

Abstract Study question Can whole exome sequencing of surgically retrieved spermatozoa from azoospermic men pinpoint mutations related to the etiology of their infertility and ability to support pregnancy? Summary answer Identifying key germline mutations involved in spermatogenesis helps explain reproductive failure, regardless of the etiology of the azoospermia. What is known already Azoospermia accounts for approximately 15% of male factor infertility cases. Although it can be caused by pre-testicular factors, the most recognized forms are testicular and post-testicular. Post-testicular azoospermia is mainly attributed to a mechanical obstruction, whereas testicular azoospermia, the most challenging form, is characterized by scattered functional germinal epithelia that may fail to support the meiotic process during sperm development. To elucidate the etiology of this condition, genetic studies on somatic cells have been carried out. Here, we perform whole exome sequencing (WES) on surgically retrieved spermatozoa to preferentially detect germline mutations that may be passed on to offspring. Study design, size, duration Over 3 years, we recruited patients undergoing epididymal sperm aspiration for acquired obstructive azoospermia (OA; n = 19) or testicular biopsy for nonobstructive azoospermia (NOA; n = 11). Eight men were included as fertile controls. Copy number variants (CNVs) and gene mutation profiles were obtained through WES and compared between the OA and NOA cohorts, followed by sub-analyses within those two categories according to whether they generated a clinical pregnancy (fertile) or not (infertile), while controlling for maternal age. Participants/materials, setting, methods Spermatozoal DNA was extracted and amplified from the surgically retrieved specimens (concentration, 742±520 ng/ul; quality, 1.7±0.1 nm). CNVs and gene mutations were detected using CLC Genomic Server 9.0. Genes were considered duplicated or deleted when the read depth was >1.5 or < 0.5 times the median read depth in the control. Common mutations were compared between the OA and NOA cohorts, as well as according to the couples’ clinical outcomes. Female partners had negative infertility workups. Main results and the role of chance Of 30 men (paternal age, 42.3±7yrs), 19 OA men underwent epididymal sperm retrievals (concentration, 1.1±4x106/ml, 9±12% motility), while 11 NOA men underwent testicular biopsies (concentration, 0.03±0.4x106/ml, 0.5±1% motility). WES did not indicate a significant difference in sperm aneuploidy between the two etiologies (OA, 1.7%; NOA, 1.8%) compared to the control (1.1%). In OA patients, only 3 housekeeping-related genes were deleted, while in the NOA cohort, 5 genes involved in RNA transcription (POLR2L), apoptosis (AP5M1), and basic spermiogenic functions (AP1S2, AP1G2, APOE) were deleted. OA patients and their partners (maternal age, 36.8±4yrs) underwent 19 ICSI cycles resulting in a delivery rate of 47.4% (9/19). Those able to reproduce (n = 9) shared a mutation in ZNF749, specifically affecting sperm production. The infertile men (n = 10) all shared a PRB1 deletion, controlling essential DNA replication. NOA men and their partners (maternal age, 38.2±2yrs) underwent 11 ICSI cycles, yielding a delivery rate of 72.7% (8/11). The fertile men (n = 8) all shared a MPIG6B deletion, involved in stem cell lineage differentiation. All of their infertile counterparts (n = 3) presented gene deletions not only involved in spermato/spermio-genesis (n = 4) but, most importantly, also superimposed with those encoding early embryonic development (MBD5, CCAR1, PMEPA1, POLK, REC8, REPIN1, MAPRE3, ARL4C). Limitations, reasons for caution Although maternal age was controlled for, confounding factors related to the female partner cannot entirely be excluded. While men unable to reproduce shared common gene mutations providing information about their condition, these findings still need to be confirmed in larger observations. Wider implications of the findings By performing WES, we were able to identify specific mutations associated with compromised embryo developmental competence of surgically retrieved spermatozoa. DNA sequencing technologies help identify gametes capable of sustaining a pregnancy even in the most severe form of male infertility, laying the groundwork for precision medicine in this field. Trial registration number N/A

O-310 Browsing the Male Genome to Unravel Its Reproductive Potential

Abstract Study question Can whole genome profiling of sperm DNA be used to identify aspects of ART failure and predict embryo developmental competence? Summary answer Whole genome profiling of sperm DNA identifies germline gene mutations associated with reproductive failure and helps characterize subtle male factor infertility. What is known already A routine semen analysis provides limited information on the characteristics of the male gamete and is unable to predict spermatozoa performance in ART. Therefore, ancillary tests may be used to further assess the male gamete’s reproductive potential. Whole exome sequencing (WES) of the male genome carried out on somatic cells has proven to be a powerful technique capable of identifying the genetic roots of infertility. Here, we aim to preferentially detect germline mutations by exclusively sequencing spermatozoal DNA to identify genes related to the different underlying etiologies of reproductive failure. Study design, size, duration Over a 6-year period, 31 consenting couples with negative female infertility workups and normal semen parameters were included in this study. These couples were divided according to whether they reported a successful pregnancy with ART (fertile; n = 10) or not (infertile; n = 21). Sperm aneuploidy assessment by copy number variant (CNV) analysis with WES were carried out on ejaculated spermatozoa. Gene mutation profiles were enlisted and compared between the two patient cohorts to identify genes involved. Participants/materials, setting, methods DNA was extracted and amplified from at least 500 spermatozoa (DNA concentration, 760±486 ng/ul; quality, 1.7±0.1 nm) for CNV analyses by WES. Mutations corresponding to the CNV were then annotated and assessed using the CLC Genomic Server 9.0. Genes were considered duplicated or deleted when their read depth was >1.5 or < 0.5 times the median read depth in the control, respectively. Main results and the role of chance All couples (n = 31) (maternal age, 37.6±3yrs; paternal age, 39.7±5yrs) had adequate semen parameters (concentration, 59.2±30x106/mL, 44.8±18% motility, normal morphology) and normal peripheral karyotypes. The fertile cohort (n = 10) underwent 12 ICSI cycles, achieving an 82.6% (57/69) fertilization rate with 10/12 (83.3%) cycles resulting in live births. The infertile cohort (n = 21) underwent 25 ICSI cycles, achieving a 68.4% (91/133) fertilization rate and 6/14 (42.9%) clinical pregnancies, all resulting in pregnancy loss. CNV analysis indicated lower sperm aneuploidy in the fertile (4.0% vs. 8.4%) cohort (P < 0.00001). In both cohorts, mutations associated with sperm–egg fusion (ADAM3A) and acrosomal development (SPACA1, SPATA16) were identified, justifying ICSI utilization. The infertile cohort included complete fertilization failure, poor early embryo development, implantation failure, or pregnancy loss. Couples with complete fertilization failure (n = 4) had gene deletions (PLCZ1, PIWIL1, ADAM15) indicating sperm-related oocyte-activating deficiency. Those with poor early embryo development (n = 5) had mutations essential for centrosome integrity (HAUS1) and spindle/microtubular stabilization (KIF4A, XRN1). Couples who failed to achieve pregnancy (n = 7) had mutations commonly implicated in embryonic implantation (IL9R) and microtubule/centrosomal integrity (MAP1S). Those with pregnancy losses (n = 5) displayed mutations related to trophoblast development (NLRP7), cell cycle regulation (MARK4, TRIP13, DAB2IP, KIF1C), and a gene linked to recurrent miscarriage (TP53). Limitations, reasons for caution Using WES, we were able to identify germline mutations that appear to be involved in various aspects of human reproduction. These findings are new and should be validated in a larger study population. Moreover, although we attempted to control for maternal age, we still cannot exclude confounding female factors. Wider implications of the findings Evaluating the sperm genome can help identify elusive genetic factors associated with reproductive competence and help guide treatment options for couples unable to conceive who undergo ART. Therefore, screening spermatozoal DNA may serve as an additional tool in precision medicine to identify and treat subtle male factor infertility. Trial registration number N/A

MtDNA content in cumulus cells does not predict development to blastocyst or implantation.

STUDY QUESTIONIs relative mitochondrial DNA (mtDNA) content in cumulus cells (CCs) related to embryo developmental competence in humans and/or the bovine model?SUMMARY ANSWERmtDNA content in CCs provides a poor predictive value of oocyte developmental potential, both in vitro and following embryo transfer.WHAT IS KNOWN ALREADYCCs are closely connected to the oocyte through transzonal projections, serving essential metabolic functions during folliculogenesis. These oocyte-supporting cells are removed and discarded prior to ICSI, thereby providing interesting biological material on which to perform molecular analyses designed to identify markers that predict oocyte developmental competence. Previous studies have positively associated oocyte mtDNA content with developmental potential in animal models and women. However, it remains debatable whether mtDNA content in CCs could be used as a proxy to infer oocyte developmental potentialSTUDY DESIGN, SIZE, DURATIONmtDNA content was analyzed in CCs obtained from 109 human oocytes unable to develop to blastocyst, able to develop to blastocyst but failing to establish pregnancy or able to develop to blastocyst and to establish pregnancy. mtDNA analysis was also performed on bovine cumulus samples collected from 120 oocytes unable to cleave, oocytes developing into cleaved embryos but arresting development prior to the blastocyst stage or oocytes developing to blastocysts.PARTICIPANTS/MATERIALS, SETTING, METHODSHuman CCs samples were obtained from women undergoing IVF. Only unfrozen oocytes and embryos not submitted to preimplantation genetic testing were included in the analysis. Bovine samples were obtained from slaughtered cattle and individually matured, fertilized and cultured in vitro. Relative mtDNA was assessed by quantitative PCR analysis.MAIN RESULTS AND THE ROLE OF CHANCEmtDNA content in human and bovine CCs did not differ according to the developmental potential of their enclosed oocyte. Moreover, mtDNA content in bovine oocytes did not correlate with that of their corresponding CCs.LARGE SCALE DATAN/A.LIMITATIONS, REASONS FOR CAUTIONThe lack of correlation found between mtDNA content in human CCs and oocytes was also assessed in bovine samples. Although bovine folliculogenesis, mono-ovulatory ovulation and early embryo development exhibit considerable similarities with that of humans, they may not be fully comparable.WIDER IMPLICATIONS OF THE FINDINGSThe use of molecular markers for oocyte developmental potential in CCs could be used to enhance success rates following single embryo transfer. However, our data indicate that mtDNA in CCs is not a good proxy for oocyte quality.STUDY FUNDING/COMPETING INTEREST(S)This research was supported by the Industrial Doctorate Project IND2017/BIO-7748 funded by the Madrid Region Government. The authors declare no competing interests.

MicroRNA-101 regulates oocyte maturation in vitro via targeting HAS2 in porcine cumulus cells

RNA-seq technology can be used for the detection of miRNA transcripts in tissues and cells at specific periods and under specific treatment conditions, which can easily and effectively screen out differential transcripts. The purpose of this study was to compare miRNA expression in porcine cumulus cells before and after oocyte maturation, and to investigate the mechanism whereby cumulus cells may influence oocyte maturation. To that aim, cumulus cells surrounding GV- and MII-stage oocytes were isolated, and their differences in miRNA expression were examined using miRNA-seq. 143 differentially expressed miRNAs were identified, among which miR-101 was selected and further verified by qPCR. Moreover, miR-101 was found to target HAS2 through target gene prediction, luciferase-based co-transfection and cumulus cells transfection. Transfection of COCs with miR-101 mimics or inhibitor revealed that the miR-101 could inhibit oocyte IVM by regulating the expanding of CCSs, but had no effect on the embryo development competence. These findings demonstrated that miR-101 regulated oocyte maturation in vitro via targeting HAS2 in porcine cumulus cells.

PTEN expression in human cumulus cells is associated with embryo development competence.

Embryo quality determines the success of in vitro fertilization and embryo transfer (IVF-ET) treatment. Biomarkers for the evaluation of embryo quality have some limitations. Apoptosis in cumulus cells (CCs) is important for ovarian function. PTEN (phosphatase and tensin homolog) is a well known tumour suppressor gene that functions as a mediator of apoptosis and is crucial for mammalian reproduction. In the present study, we analyzed the expression level of PTEN in human CCs and aimed to investigate its association with embryo developmental competence in IVF treatment cycles. The PTEN mRNA level in CCs was measured using real-time fluorescence quantitative PCR. The association of the differential expression of PTEN with embryo quality was analyzed. Our data showed that PTEN mRNA levels were significantly decreased in CCs surrounding mature oocytes compared with immature oocytes. Similar changes were found in the analysis of fertilization and blastocyst formation. The speculation that the measurement of PTEN mRNA levels in human CCs would provide a useful tool for selecting oocytes with greater chances to implant into the uterus needs to be further verified through single-embryo transfer in the future. The proapoptotic mechanism of PTEN in human reproduction needs to be further studied.

Impact of obesity on medically assisted reproductive treatments.

Increasing evidence has demonstrated that obesity impairs female fertility and negatively affects human reproductive outcome following medically assisted reproduction (MAR) treatment. In the United States, 36.5% of women of reproductive age are obese. Obesity results not only in metabolic disorders including type II diabetes and cardiovascular disease, but might also be responsible for chronic inflammation and oxidative stress. Several studies have demonstrated that inflammation and reactive oxygen species (ROS) in the ovary modify steroidogenesis and might induce anovulation, as well as affecting oocyte meiotic maturation, leading to impaired oocyte quality and embryo developmental competence. Although the adverse effect of female obesity on human reproduction has been an object of debate in the past, there is growing evidence showing a link between female obesity and increased risk of infertility. However, further studies need to clarify some gaps in knowledge. We reviewed the recent evidence on the association between female obesity and infertility. In particular, we highlight the association between fat distribution and reproductive outcome, and how the inflammation and oxidative stress mechanisms might reduce ovarian function and oocyte quality. Finally, we evaluate the connection between female obesity and endometrial receptivity.

Effect of Ellagic Acid during In Vitro Culture on Bovine Embryo Development Competence

Effect of Ellagic Acid during In Vitro Culture on Bovine Embryo Development Competence

Effects of triton X-100 pretreatment of lyophilized and frozen-thawed ram sperm on preimplantation embryo developmental competence

In this study, it was aimed to determine the effect of destruction of lyophilized and frozen-thawed ram sperm plasma and acrosomal membrane on development of embryos produced by intracytoplasmic sperm injection (ICSI). Semen samples were divided into two groups for lyophilization (L) and freezing (F). For the removal of the plasma membrane, L and F groups were incubated with Triton X-100 (LTX-100 and FTX-100, respectively). Integrities of the plasma membrane, acrosome and chromatin structure were evaluated. Oocytes were injected with these sperm groups. Although no plasma membrane and acrosome integrities of the L (0.0%) group were detected, the plasma membrane integrity of the F group (69.4%) was significantly higher than the FTX-100 group (23.6%) (p < 0.05). The acrosome integrity of the FTX-100 group (3.80%) was significantly lower than the F group (55.6%) (p < 0.05). The chromatin integrities of L and F groups were higher than the Triton X-100 treated groups (p < 0.05). ICSIs with L, LTX-100, F and FTX-100 sperm were produced similar cleavage and blastocyst rates. In conclusion, data presented here confirm that ram spermatozoa can effectively be lyophilized and injected into oocytes for initiation of embryonic development and Triton X-100 pretreatment is not necessary while using lyophilized and frozen semen.

Role of Cumulus Cells and Follicular Fluid on Oocyte Maturation and Developmental Competence of Embryos: Intact and Reconstructed Oocytes

Role of Cumulus Cells and Follicular Fluid on Oocyte Maturation and Developmental Competence of Embryos: Intact and Reconstructed Oocytes

The function of Nucleoporin 37 on mouse oocyte maturation and preimplantation embryo development.

Nucleoporin 37 (NUP37) has been reported to activate the YAP-TEAD signaling, which is crucial for early embryo development. However, whether NUP37 is involved in oocyte meiosis and embryo development remains largely unknown. The study aimed to clarify the function of Nup37 in oocyte maturation and early embryo development, and to explore the mechanism. The expression level and subcellular localization of NUP37 were explored. After knocking down of Nup37 by microinjecting interfering RNA (siRNA), the oocyte maturation rate, aberrant PB1 extrusion rate, and blastocyst formation rate were evaluated. In addition, the effect of the downregulation of Nup37 on YAP-TEAD signaling was confirmed by immunofluorescence staining and real-time quantitative PCR. NUP37 was highly expressed in oocytes and early embryos; it mainly localized to the nuclear periphery at mice GV stage oocytes and early embryos. Nup37 depletion led to aberrant PB1 extrusion at the MII stage oocyte and a decreased blastocyst formation rate. The reduction of NUP37 caused YAP1 mislocalization and decreased the expression of Tead1, Tead2, and Tead4 during mice embryo development, thus affecting the YAP-TEAD activity and embryo developmental competence. In summary, NUP37 played an important role in mice oocyte maturation and preimplantation embryo development.

Tricostatin A-treated round spermatid enhances preimplantation embryo developmental competency following round spermatid injection in mice.

It has been documented that the inefficacy of round spermatid injection (ROSI) might be caused by abnormal epigenetic modifications. Therefore, this study aimed to evaluate the effect of trichostatin A (TSA) as an epigenetic modifier of preimplantation embryo development in activated ROSI oocytes. Matured oocytes were collected from superovulated female mice. Testes were placed in human tubal fluid medium and masses were then cut into small pieces to disperse spermatogenic cells. Round spermatids were treated with TSA and subsequently injected into oocytes. The expression level of the development-related genes including Oct4, Sox2, Nanog, Dnmt and Hdac transcripts were evaluated using qRT-PCR. Immunohistochemistry was performed to confirm the presence of Oct-4 protein at the blastocyst stage. There was no statistically significant difference in fertilization rate following ROSI/+TSA compared with the non-treated ROSI and intracytoplasmic sperm injection (ICSI) groups. Importantly, TSA treatment increased blastocyst formation from 38% in non-treated ROSI to 68%. The relative expression level of developmentally related genes increased and Dnmt transcripts decreased in ROSI/+TSA-derived embryos, similar to the expression levels observed in the ICSI-derived embryos. In conclusion, our results indicate that spermatid treatment with TSA prior to ROSI would increase the success rate of development to the blastocyst stage and proportion of pluripotent cells.

SERUM ANTI-MÜLLERIAN HORMONE CONCENTRATIONS IMPACTS NOT ONLY THE QUANTITY, BUT ALSO THE QUALITY OF THE OVARIAN RESERVE: THE IMPORTANCE OF TIME-LAPSE IMAGING SYSTEM

Serum anti-müllerian hormone (AMH) concentration has been implicated as the most valuable marker of the ovarian reserve, as it correlates highly with the baseline antral follicle count. The AMH has been assigned as a marker of ovarian function mainly in the assessment of the quantitative aspect of the ovarian reserve. On the other side, the predictive value of AMH on oocyte quality and embryo developmental competence is controversial. Time-lapse imaging (TLI) systems allow for the assessment of morphological changes with the exact time-point of occurrence, and may be a valuable tool to evaluate the correlation between serum AMH concentration and the competences of both oocyte and developing embryo. The goal for the present study was to evaluate whether the serum AMH concentration impacts morphokinetic events in a TLI system. This retrospective cohort study was performed in a private university-affiliated IVF center from March 2019 to December 2020 and included 902 injected oocytes from 114 ICSI cycles, cultured in a TLI incubator until the fifth day of development. The effects of serum AMH concentrations on morphokinetic events and ICSI outcomes were evaluated by taking into account clustering of data (multiple embryos per cycle), using generalized mixed models. Evaluated kinetic markers were: pronuclei appearance (tPNa), timing to pronuclei fading (tPNf), timing to two (t2), three (t3), four (t4), five (t5), six (t6), seven (t7), and eight cells (t8), and timing to blastulation (tB). Durations of the second (t3-t2) and third (t5-t3) cell cycles (cc2 and cc3, respectively) and timing to complete synchronous divisions s1 (t2-tPNf), s2 (t4-t3), and s3 (t8-t5) were also evaluated. The post hoc achieved power was > 98.0%. Inverse relationships were observed between serum AMH concentrations and faster tPNf (B: -0.047, CI: -0.071 – -0.024, p<0.001), t3 (B: -0.070, CI: -0.102 – -0.039, p<0.001), t4 (B: -0.080, CI: -0.114 – -0.046, p<0.001), t5 (B: -0.075, CI: -0.123 – -0.028, p=0.002), t6 (B: -0.105, CI: -0.147 – -0.064, p<0.001), t7 (B: -0.120, CI: -0.165 – -0.075, p<0.001), t8 (B: -0.170, CI: -0.220 – -0.119, p<0.001), and tB (B: -0.153, CI: -0.224 – -0.082, p<0.001), cc2 (B: -0.041, CI: -0.062 – -0.019, p<0.001), and s3 (B: -0.086, CI: -0.129 - -0.044, p<0.001). There was a positive association between serum AMH concentration and implantation rate (B: -19.11, CI: -29.12 - -9.11, p<0.001). No significant associations were found between serum AMH concentrations and other morphokinetic parameters or clinical outcomes. Increased serum AMH concentrations correlate with faster embryo development in a TLI incubator and implantation rates.

GENOMIC PROFILING OF SURGICALLY RETRIEVED SPERMATOZOA TO EVALUATE THE REPRODUCTIVE POTENTIAL OF AZOOSPERMIC MEN

To identify, by whole exome sequencing (WES), gene mutations affecting the embryo developmental competence of spermatozoa from azoospermic men. Over 2 years, we recruited patients undergoing epididymal sperm aspiration for acquired obstructive azoospermia (OA; n=19) or testicular retrieval for nonobstructive azoospermia (NOA; n=11). Eight additional men were included as fertile controls. Spermatozoal DNA was extracted and amplified from the surgically retrieved specimens (concentration, 742±520 ng/ul; quality, 1.7±0.1 nm). Copy number variants (CNVs) and gene mutations were detected using CLC Genomic Server 9.0 and compared between the OA and NOA cohorts, followed by sub-analyses within those two categories according to whether they generated a clinical pregnancy (fertile) or not (infertile), while controlling for maternal age. Of 30 men (paternal age, 42.3±7yrs), 19 OA men underwent epididymal sperm retrieval (1.1±4x106/ml concentration, 9±12% motility), while 11 NOA men underwent testicular biopsy (0.03±0.4x106/ml concentration, 0.5±1% motility). WES did not reveal a significant difference in sperm aneuploidy between the two etiologies (OA, 1.7%; NOA, 1.8%) compared to the control (1.1%). When assessing the origin of azoospermia, we found that the OA group had only 3 housekeeping genes deleted, while 5 genes in the NOA cohort were deleted; these 5 are involved in RNA transcription (POLR2L), apoptosis (AP5M1), and spermiogenic functions (AP1S2, AP1G2, APOE). We then assessed the reproductive potential of these men. The OA group underwent 19 ICSI cycles with their female partners (maternal age, 36.8±4yrs), resulting in a clinical pregnancy/delivery rate of 47.4% (9/19). Of the couples who delivered (n=9), all the men shared a mutation only in ZNF749, a transcriptional regulation gene. Of those OA men who remained infertile (n=10), all had a deletion on PRB1, which is associated with controlling essential DNA replication. When we assessed the NOA men who underwent 11 ICSI cycles with their female partners (maternal age, 38.2±2yrs), yielding a delivery rate of 72.7% (8/11), the fertile men (n=8) had only a gene deletion involved in stem cell lineage differentiation (MPIG6B) in common. However, their infertile counterparts (n=3) all had commonly deleted genes not only involved in spermato/spermio-genesis (n=6) but, most importantly, also associated with encoding early embryonic development (MBD5, CCAR1, PMEPA1, POLK, REC8, REPIN1, MAPRE3, ARL4C). DNA sequencing of the male gamete can identify germline mutations associated with azoospermia. For individuals capable of reproducing, we found mutations that were mainly limited to spermatogenic function. However, for men unable to sustain a pregnancy, particularly in the NOA cohort, we found gene mutations related to impaired embryo development.

O-119 Evaluating the reproductive potential of azoospermic men by germline mutation profiling

Abstract Study question Can whole exome sequencing (WES) of spermatozoa from azoospermic men identify mutations related to the etiology of their infertility and ability to support a pregnancy? Summary answer Key de novo germline mutations that affect sperm production and/or embryo developmental competence may explain reproductive failure in azoospermic men, regardless of the etiology. What is known already Azoospermia accounts for approximately 15% of male factor infertility cases. Although it can be caused by pre-testicular factors, the most recognized forms are testicular and post-testicular. While post-testicular azoospermia is mainly due to a mechanical obstruction, testicular azoospermia, the most severe form, is characterized by scattered functional germinal epithelia that strive to support the meiotic process during gamete development. To shed light on the etiology of this condition, genetic studies have been performed, albeit exclusively on peripheral blood. We chose to perform a genomic assessment of spermatozoa to preferentially detect germline mutations that may be passed to the progeny. Study design, size, duration In a 2-year period, we recruited infertile men undergoing epididymal aspiration for acquired obstructive azoospermia (OA; n = 19) or testicular retrieval for nonobstructive azoospermia (NOA; n = 10). Four additional men were included as fertile controls. Following WES, copy number variants (CNVs) and gene mutation profiles were compared between the OA and NOA patients, and within those two categories, in relation to whether they generated a clinical pregnancy (fertile) or not (infertile). Participants/materials, setting, methods Spermatozoal DNA was extracted and amplified from the surgically retrieved specimens of consenting men (DNA concentration, 762±492 ng/ul; quality, 1.7±0.1 nm). CNVs, gene mutations, duplications, and deletions were detected using the CLC Genomic Server 9.0. Genes were considered duplicated or deleted when the read depth was &amp;gt;1.5 or &amp;lt; 0.5 times the median read depth in the control. Common mutations in the OA and NOA cohorts were assessed according to the couples’ clinical outcome. Main results and the role of chance Of 29 couples (maternal age, 41.9±7yrs; paternal age, 42.5±7yrs), 19 OA men underwent epididymal sperm retrieval (1.1±4x106/ml concentration, 9±12% motility) while 10 NOA men underwent testicular biopsy (0.03±0.2x106/ml concentration, 0.5±1% motility). WES did not reveal a significant difference in sperm aneuploidy between the two etiologies (OA, 1.8%; NOA, 1.9%). In OA patients, only 3 genes were deleted, mainly housekeeping-related, while in the NOA cohort, 5 genes were deleted, involved in RNA transcription (POLR2L) and apoptosis (AP5M1), in addition to spermiogenic functions (AP1S2, AP1G2, APOE). OA patients and their partners (maternal age, 36.8±4yrs) underwent 19 ICSI cycles that resulted in a pregnancy and delivery rate of 47.4% (9/19). Those able to reproduce (n = 9) shared a mutation in ZNF749, a gene affecting only sperm production. The infertile individuals (n = 10) all had a deletion on PRB1, controlling essential DNA replication. NOA men and their partners (maternal age, 38.2±2yrs) underwent 10 ICSI cycles, yielding a clinical pregnancy rate of 70% (7/10). The fertile men (n = 7) had a concurrent gene deletion involved in stem cell lineage differentiation (MPIG6B). Their infertile counterparts (n = 3) had deleted genes involved in spermato/spermio-genesis (n = 6) and, most importantly, in early embryonic development (MBD5, CCAR1, PMEPA1, POLK, REC9, REPIN1, MAPRE3, and ARL4C). Limitations, reasons for caution This is a novel study with limited observations. The presence of housekeeping-related mutations in fertile OA men as well as the DNA replication mutation in infertile OA patients, considering the acquired condition, remains puzzling. Although maternal age was controlled for, confounding factors related to the female partner cannot be excluded. Wider implications of the findings Screening men for germline mutations provides valuable information on their ability to reproduce, regardless of the etiology of azoospermia. Genome profiling was able to identify reasons for failed reproductive performance in azoospermic men, particularly those individuals with secretory azoospermia (NOA). Genomic profiling may identify gametes with retained embryo developmental competence. Trial registration number n/a

P–262 Comparison of embryo aneuploidy rate and reproductive outcomes of ART cycles using fresh and vitrified donor oocytes

Abstract Study question Is there any difference in blastocyst morphology, embryo aneuploidy rate and ART clinical outcomes when using fresh or vitrified donor oocytes? Summary answer Frequency of good quality blastocyst obtained from fresh oocytes is significantly higher compared to vitrified.No difference in embryo aneuploidy rates nor clinical outcomes were found. What is known already Oocytes vitrification is an efficient method that allows non only fertility preservation but also the creation of donor oocytes banks, optimizing clinical resources for patients undergoing Assisted Reproductive Technology. Although the benefits of donor oocytes vitrification are well known, some studies have shown that this cryopreservation process can induce spindle abnormalities and chromosomal changes, leading to aneuploidy. Comparative studies between fresh and vitrified oocytes to evaluate embryo developmental competence, aneuploidy and clinical pregnancy rate (CPR) are needed. Study design, size, duration This retrospective study includes ICSI cycles with fresh donor oocytes(N = 2795) and vitrified donor oocytes (N = 1225) between January 2019 and September 2020. Pre-implantation Genetic Testing for Aneuploidy (PGT-A) was performed on Day 5 and Day 6 blastocysts. Fertilization rate, blastocyst morphology, aneuploidy status and CPR were analysed and compared between the groups. Recipients were equally distributed in terms of maternal age (40.86 years) and previous history, sperm samples were also similar in profile and origin (fresh-frozen). Participants/materials, setting, methods A total of 266 subfertile couples participated in the study, ICSI was carried out in all cycles. Vitrification and warming protocols were performed with a commercial kit. All embryos were cultured to blastocyst stage in a Time-Lapse incubator and assessed by Gardner’s blastocyst grading scale. PGT-A testing was performed on trophectoderm biopsies by Next Generation Sequencing (NGS). Single/double embryo transfers were performed in all cases. Odd-ratios were calculated,and Chi-square was performed for the statistical analysis. Main results and the role of chance A total of 266 patients underwent 289 donor oocyte cycles yielding an overall of 4557 oocytes. ICSI was performed on 2795 fresh and 1225 vitrified mature oocytes. Similar fertilization rates were achieved with fresh and vitrified oocytes (75.9% (2122/2795) and 75.2% (921/1225), respectively (P = 0.6)) yielding a significant difference in blastocyst rate of 71.7% (1522/2122) and 62.5% (576/921) (OR 1,519; 95% CI 1,290–1,789; p &amp;lt; 0.001). In addition, when blastocysts morphology was analysed, a significant difference was shown in the frequency of good quality embryos that decreases from 56.6% (861/1522) with fresh oocytes to 51% (294/576) with vitrified oocytes (OR 1,249; 95% CI 1,031–1,514; P &amp;lt; 0.02). PGT-A testing of blastocysts revealed not significant differences in euploidy rates (73.6% in fresh oocytes vs 76.8% vitrified oocytes, P = 0.2). With regards to clinical outcomes, similar results were found between the groups. A total of 322 embryo transfers were performed (237 from fresh and 85 vitrified) achieving a CPR of 48.9% (116/237) with fresh oocytes and 54% (46/85) with vitrified (P = 0.7) and a pregnancy loss of 6.7%(16/237) in fresh oocytes and 11.7%(10/85) vitrified oocytes (P = 0.1). Limitations, reasons for caution The study was conducted on a small number of cases. Further studies are needed to confirm our findings. Moreover, although the same stimulation protocol was used, donors from different background were included. Wider implications of the findings: This study supports the use of vitrified oocytes in the laboratory routine without compromising clinical outcomes. Although oocyte vitrification may have an influence on embryo morphology, blastocyst rate, no impact of this cryopreservation process is seen on embryo aneuploidy, developmental competence and CPR. Trial registration number Not applicable

P–210 Abnormal cleavage patterns during embryo preimplantation development and their effect on blastulation: an overview from IVF patients with multiple IVF cycles in a time-lapse incubator

Abstract Study question How common abnormal cleavage patterns (ACP) are in IVF and what are their consequences on embryo developmental competence? Summary answer ACP might affect up to 25% of the 2PN-zygotes, independently from patients’/cycles’ characteristics, and mostly cause embryo developmental arrest around the 4-to–8-cell transition. What is known already Since its implementation in IVF, time-lapse-microscopy (TLM) allowed the standardization of embryo culture within undisturbed incubators, but it has not improved embryo selection especially if blastocyst transfer is performed. Nevertheless, TLM holds the potential for boosting our knowledge of embryo preimplantation development. In particular, a continuous observation of embryo morpho-dynamics unveiled peculiar blastomere cleavage patterns previously unidentifiable with a static morphological assessment. These events are possibly associated with massive mitotic errors, affecting both chromosomes and cytoskeletal components, as well as downstream metabolic imbalances. Still, the causes of ACP and their consequences on embryo developmental/reproductive competence require further investigation. Study design, size, duration Observational study including 75 patients (age:38.6±3.7yr, FSH:8.8±3.6IU/l, AMH:1.7±1.3ng/ml; BMI:21.4±2.4) who conducted multiple IVF cycles (N = 160; 8.7±5.0 cumulus-oocyte-complexes and 6.3±3.6 metaphase-II collected; 201±245 days between first and second cycles) in a time-lapse incubator between 2014–2020. All annotations were performed blindly by two operators and confirmed by a third in case of discordance. The outcomes were the blastulation rate after any ACP, their association between each other and with patients’/cycles’ characteristics. Participants/materials, setting, methods We included only ICSI-cycles after ovarian-stimulation with blastocyst culture conducted in the Embryoscope. Overall, 981 metaphase-II were inseminated and 677 2PN-zygotes annotated. The ACP investigated were: (i)cytokinesis-failure, formation of cytoplasmic septa without cell division; (ii)Chaotic-cleavage, disordered and uneven cleavages; (iii)Direct-unequal-cleavage (DUC), cleavage of zygotes or single blastomeres directly into 3; (iv)Rapid-cleavage, t3-t2&amp;lt;5hr; (v)Reverse-cleavage, fusion of 2 blastomeres into 1; (vi)Fragmentation, presence of numerous non-nucleated fragments; (vii)Blastomeres’ exclusion/extrusion, nucleated cells excluded/extruded from the morula. Main results and the role of chance Among the 2PN-zygotes, the prevalence of cytokinesis-failure was 5.9% (N = 40/677), 15.7% for chaotic-cleavage (N = 106/677), 18.6% for DUC (N = 126/677), 4.1% for rapid-cleavage (N = 28/677), 3.5% for reverse-cleavage (N = 24/677) and 24.1% for fragmentation (N = 163/677). Among the morulae, the prevalence of blastomere exclusion/extrusion was 27% (N = 109/410;1.5±1.2 excluded/extruded cells,range:1–7). The risk for reverse-cleavage was higher among 2PN-zygotes facing failed-cytokinesis (N = 8/40,20% versus N = 16/637,2.5%, OR:9.7,95%CI:3.9–24.3,p&amp;lt;0.01). Fragmentation was instead higher among 2PN-zygotes undergoing chaotic cleavage (N = 47/106,44.3% versus N = 116/571,20.3%, OR:3.1,95%CI:2–4.8,p&amp;lt;0.01) or DUC (N = 46/126,36.5% versus N = 117/551,21.2%, OR:2.1,95%CI:1.4–3.2,p&amp;lt;0.01). Lastly, higher prevalence of blastomeres’ exclusion/extrusion were reported among morulae obtained after chaotic-cleavage (N = 17/29,58.6% versus N = 92/381,24.1%, OR:4.4,95%CI:2–9.7,p&amp;lt;0.01), DUC (N = 26/37,70.3% versus N = 83/373,22.3%, OR:8.3,95%CI:3.9–17.4,p&amp;lt;0.01) and in presence of fragmentation (N = 79/195,75.2% versus N = 30/305,9.8%, OR:27.8,95%CI:15.6–49.8,p&amp;lt;0.01); only a higher trend after rapid-/reverse-cleavage. No predictive factor of ACP was identified among patients’ and cycles’ characteristics, except for higher risks of fragmentation (OR:2.6,95%CI:1.1–6.3,p= 0.04) and blastomeres’ exclusion/extrusion (OR:2.7,95%CI:1.1–7.2,p=0.04) among patients with previous experience with these events. The viable-blastocyst rate per 2PN-zygote was 45.1% (N = 305/677). It was lower in case of failed-cytokinesis (N = 12/40,30% versus N = 293/637,46%, OR:0.5,95%CI:0.25–0.99,p=0.05), chaotic cleavage (N = 20/106,18.9% versus N = 285/571,49.9%, OR:0.23,95%CI:0.14–0.39,p&amp;lt;0.01), DUC (N = 27/126,21.4% versus N = 278/551,50.5%, OR:0.27,95%CI:0.17–0.42,p&amp;lt;0.01), rapid-cleavage (N = 6/22,21.4% versus N = 299/649,46.1%, OR:0.32,95%CI:0.13–0.8,p=0.02), and reverse-cleavage (N = 5/19,20.8% versus N = 300/653,45.9%, OR:0.31, 95%CI:0.11–0.84,p=0.02). No difference was instead shown in case of fragmentation and/or blastomeres’ exclusion/extrusion. Limitations, reasons for caution The patients included were poor-prognosis women undergoing ≥2 cycles. We are expanding the sample size to account for all cycles conducted in time-lapse incubators. Larger sample size will provide also statistical-power to investigate the effect of ACP on blastocysts’ chromosomal and implantation competence, and more visualizations of rapid-/reverse-cleavage events. Wider implications of the findings: After ACP,developmental-arrest mostly occurs around the 4-to–8-cell transition (50–70% versus ∼30%), when embryonic-genome-activation takes place. Surviving embryos often fragment and/or exclude/extrude blastomeres at morulation, without further impact on blastulation-rates. Moreover, ACP seem independent from patients’/cycles’ characteristics. These evidence incite future Research on the biological/genetic mechanisms triggering ACP and their consequences. Trial registration number None

P–705 Basal FSH and AMH profiles impact embryo morphokinetics in a maternal age-dependent manner

Abstract Study question Do maternal AMH and basal FSH profiles impact embryo morphokinetics and does this relationship change with maternal age? Summary answer Embryo morphokinetics varies with basal FSH and AMH levels and this relationship changes in advanced maternal age (AMA). What is known already Basal FSH and AMH levels have been utilised as markers of ovarian reserve/response and IVF/ICSI outcomes. Basal FSH better reflects post-IVF/ICSI live birth occurrence in pre-AMA patients, while AMH appears more robust in AMA patients. Whilst plasma AMH levels reflect oocyte yield, recent data suggest that plasma basal FSH and intrafollicular AMH levels specifically reflect oocyte quality. Oocyte and embryo developmental competence is associated with faster fertilisation and embryonic morphokinetics. The assessment of the relationship between developmental morphokinetics and maternal basal FSH and AMH plasma profiles shall contribute for a better understanding of their roles as fertility markers and regulators. Study design, size, duration Retrospective cohort study including 1961 first autologous ICSI cycles performed from 2014 to 2020, providing 10774 embryos grouped according to maternal AMH and basal FSH levels in: CF [concordant favourable; AMH&amp;gt;1 (ng/mL), FSH≤10 (IU/L); n = 8055)]; DFA (discordant with favourable AMH; AMH&amp;gt;1, FSH&amp;gt;10; n = 768); DFF (discordant with favourable FSH; AMH≤1, FSH≤10; n = 1362) and CU (concordant unfavourable; AMH≤1, FSH&amp;gt;10; n = 589). Morphokinetic parameters were compared among groups in total, ≤35 (pre-AMA) and &amp;gt;35 (AMA) years old patients, separately. Participants/materials, setting, methods Patients aged 20 to 45 with FSH and AMH levels measured on menstrual cycle day 2 underwent ovarian stimulation, ovum pick-up and ICSI. Embryos were cultured in a time-lapse incubator (Embryoscope). Fertilisation and cleavage morphokinetic parameters [tPNa (time of pronuclei appearance, tPNf (time of pronuclei fading), t2, t3, t4, t5 and t8] were annotated and compared among AMH/FSH groups with the Kruskal-Wallis nonparametric test, followed by a post hoc multiple comparison with Bonferroni correction. Main results and the role of chance In overall embryos, tPNa, tPNf, t2 and t3 varied between AMH/FSH groups. CF were faster than CU embryos for all these parameters [(mean±SEM; hours); tPNa: 6.8±0.02 vs. 7.1±0.08; tPNf: 24.13±0.04 vs. 24.49±0.14; t2: 27.05±0.05 vs. 27.36±0.16; t3: 36.94±0.07 vs. 37.54±0.22 (p &amp;lt; 0.001)]. In addition, CF were faster than DFA (p &amp;lt; 0.001), but not than DFF embryos, for tPNf (CF: 24.13±0.04; DFF: 24.23±0.14; DFA: 24.57±0.14) and t2 (CF: 27.05±0.05; DFF: 27.22±0.12, DFA: 27.42±0.15). In AMA patients, faster morphokinetics was observed when one or both hormonal values were favourable; tPNf, t2 and t3 were reached earlier in CF compared to CU (tPNf: 24.17±0.05 vs. 24.52±0.15; t2: 27.13±0.06 vs. 27.43±0.17; t3: 37.09±0.08 vs. 37.63±0.24; p &amp;lt; 0.05), but not to DFF (tPNf: 24.22±0.12; t2: 27.27±0.14, t3: 36.92±0.19) or DFA embryos (tPNf: 24.39±0.14; t2: 27.34±0.17, t3: 37.26±0.23). Differently, in pre-AMA patients, faster morphokinetics was associated with favourable basal FSH regardless of AMH levels; tPNa and tPNf were reached earlier in CF compared to DFA and CU (p &amp;lt; 0.005), but not to DFF embryos (tPNA: 6.68±0.03, 6.96±0.13, 7.13±0.14, 7.12±0.19; tPNf: 24.05±0.07, 24.27±0.21, 25.14±0.34, 24.37±0.36; for CF, DFF, DFA and CU, respectively). Limitations, reasons for caution Our study is subjected to the intrinsic limitations of a retrospective analysis and the results could have been affected by variables that were not controlled for. Wider implications of the findings: The findings suggest that lower basal FSH levels are associated with faster early morphokinetics likely reflecting superior oocyte quality in pre-AMA patients. The present data may contribute to improve ART prognostic strategies and provide valuable clues for a better understanding of hormonal regulation of oocyte developmental competence. Trial registration number Not applicable

O-083 Non-invasive, label-free optical analysis to detect aneuploidy within the inner cell mass of the preimplantation embryo

Abstract Study question Can we separate between control and reversine-treated cells within the inner cell mass (ICM) of the mouse preimplantation embryo by using label-free and non-invasive hyperspectral microscopy? Summary answer Hyperspectral microscopy is able to discern between control and reversine-treated cells using cellular autofluorescence in the complete absence of fluorescence tags. What is known already Embryo mosaicism (containing cells that are euploid (46 chromosomes) and aneuploid (deviation from the expected number of chromosomes)) affects up to 17.3% of human blastocyst embryos. Current diagnosis of aneuploidy in the IVF clinic involves a biopsy of trophectoderm (TE) cells or spent media followed by sequencing. In some blastocyst embryos these approaches will fail to diagnose of the proportion of aneuploid cells within the fetal lineage (ICM). Study design, size, duration The impact of aneuploidy on cellular metabolism was assessed by using cellular autofluoresence and hyperspectral microscopy (broad spectral profile). Two models were employed: (i) Primary human fibroblast cells with known karyotypes (4-6 independent replicates, euploid n = 467; aneuploid n = 969) and reversine induced aneuploidy in mouse embryos (5-8 independent replicates, 30-44 cells per group). Both models were subjected to hyperspectral imaging to quantify native cell fluorescence. Participants/materials, setting, methods The human model is comprised of euploid (male and female) and aneuploid (triploid and trisomies: 13, 18, 21, XXX, and XXY) primary human fibroblast cells. For the mouse model, we treated embryos with reversine, a reversible spindle assembly checkpoint inhibitor, during the 4- to 8-cell division. Individual blastomeres were dissociated from control and reversine treated 8-cell embryos. Blastomeres were either imaged directly or used to generate chimeric blastocysts with differing ratios of control:reversine-treated cells. Main results and the role of chance Following unsupervised linear unmixing, the relative abundance of metabolic cofactors was quantified: reduced nicotinamide adenine dinucleotide (NAD(P)H) and flavins with the subsequent calculation of the optical redox ratio (ORR: Flavins/[NAD(P)H + Flavins]). Primary human fibroblast cells displayed an increase in the relative abundance of NAD(P)H with a decrease in flavins, leading to a significant reduction in the ORR for aneuploid cells (P &amp;lt; 0.05). The mouse embryos displayed an identical trend as the human model between control and reversine-treated embryos. Mathematical algorithms were applied and able to distinguish between (i) euploid and aneuploid primary human fibroblast cells, (ii) control and reversine-treated mouse blastomeres and (iii) chimeric blastocysts with differing ratios of control and reversine-treated cells. The accuracy of these separations was supported by receiver operating characteristic curves with areas under the curve. We also showed that hyperspectral imaging of the preimplantation embryo does not impact on embryo developmental competence, pregnancy outcome and offspring health in a mouse model. We believe the role of chance is low as both human somatic cells and mouse embryos showed a consistent shift in cellular metabolism in response to human fibroblast cells that are aneuploid and reversine treated mouse embryos. Limitations, reasons for caution Further validation of our approach could include sequencing of the ICM of individual blastocysts to determine the proportion of aneuploid cells in ICM and correlate this with the metabolic profile obtained through hyperspectral imaging. Wider implications of the findings With hyperspectral imaging able to discriminate between (i) euploid and aneuploid human fibroblast cells and (ii) control and reversine-treated mouse embryos, this could be an accurate, non-invasive and label-free optical imaging approach to assess mosaicism within the ICM of mouse embryos, potentially leading to a new diagnostic tool for embryos. Trial registration number Not applicable