A deep dive into the morphokinetics and ploidy of low-quality blastocysts.

Objective: To describe morphokinetic parameters and ploidy among low-quality blastocysts not meeting the criteria for clinical use. Design: Prospective cohort study. Setting: Academic medical center. Patients: Two hundred patients undergoing in vitro fertilization between February 2018 and November 2019. Interventions: All embryos were cultured in a time-lapse incubator. All expanded blastocysts underwent preimplantation genetic testing for aneuploidy using next-generation sequencing. Main Outcome Measures: Static blastocyst morphology grading; morphokinetic parameters, including time to each cell division (2-cell formation to 8-cell formation); time to morula formation; time to the start of blastulation; time to blastocyst formation; and preimplantation genetic testing for aneuploidy results. Results: A total of 1,306 embryos progressed to the expanded blastocyst stage; of these, 935 embryos met the criteria for clinical use and were designated as high quality, whereas 371 embryos were graded as low quality and did not meet the criteria for use. In morphokinetic evaluation, low-quality embryos developed more quickly to 5-cell formation (t5) 48.4 [42.4-48.7) vs 50.2 [46.3-50.1] hours, but progressed more slowly thereafter with tM 91.5 [85.9-92.3] vs 88.3 [82.1-88.3] and tB 114.0 [106.4-113.9] vs 106.9 [101.3-107.4] hours. Among the low-quality embryos, 75.5% were aneuploid, 22.4% were euploid, and 2.2% had undetermined chromosome copy number results. Morphokinetic parameters did not differ between the euploid and aneuploid low-quality embryos. Conclusions: Morphokinetic analysis did not distinguish between euploid and aneuploid low-quality embryos.

Microfluidic preparation of spermatozoa for ICSI produces similar embryo quality to density-gradient centrifugation: a pragmatic, randomized controlled trial.

STUDY QUESTION: Does processing of spermatozoa for IVF with ICSI by a microfluidic sperm separation device improve embryo quality compared with density-gradient centrifugation? SUMMARY ANSWER: Patients randomized to microfluidic sperm preparation had similar cleavage- and blastocyst-stage embryo quality and clinical and ongoing pregnancy rates to those who underwent standard sperm processing for IVF with ICSI. WHAT IS KNOWN ALREADY: Microfluidic sperm preparation can isolate spermatozoa for clinical use with minimal DNA fragmentation but with unclear impact on clinical outcomes. STUDY DESIGN, SIZE, DURATION: A prospective randomized controlled trial of 386 patients planning IVF from June 2017 through September 2021 was carried out. PARTICIPANTS/MATERIALS, SETTING, METHODS: One hundred and ninety-two patients were allocated to sperm processing with a microfluidic sperm separation device for ICSI, while 194 patients were allocated to clinical standard density-gradient centrifugation (control) at an academic medical centre. MAIN RESULTS AND THE ROLE OF CHANCE: In an intention to treat analysis, there were no differences in high-quality cleavage-stage embryo fraction [66.0 (25.8)% control versus 68.0 (30.3) microfluidic sperm preparation, P = 0.541, absolute difference -2.0, 95% CI (-8.5, 4.5)], or high-quality blastocyst fraction [37.4 (25.4) control versus 37.4 (26.2) microfluidic sperm preparation, P = 0.985, absolute difference -0.6 95% CI (-6, 5.9)] between groups. There were no differences in the clinical pregnancy or ongoing pregnancy rates between groups. LIMITATIONS, REASONS FOR CAUTION: The population studied was inclusive and did not attempt to isolate male factor infertility cases or patients with a history of elevated sperm DNA fragmentation. WIDER IMPLICATIONS OF THE FINDINGS: Microfluidic sperm separation performs similarly to density-gradient centrifugation in sperm preparation for IVF in an unselected population. STUDY FUNDING/COMPETING INTEREST(S): No external funding to declare. M.P.R. is a member of the Clinical Advisory Board for ZyMot® Fertility, Inc. TRIAL REGISTRATION NUMBER: NCT03085433. TRIAL REGISTRATION DATE: 21 March 2017. DATE OF FIRST PATIENT'S ENROLLMENT: 16 June 2017.

Aneuploidy rates and morphokinetic parameters of embryos cultured in distinct culture media: a sibling oocyte study.

STUDY QUESTION: Do embryos from sibling oocytes assigned to distinct single-step media culture systems demonstrate differences in early embryo development, morphokinectics or aneuploidy rates? SUMMARY ANSWER: Embryo quality, morphokinetic parameters and aneuploidy rates from trophectoderm biopsy were similar between sibling embryos cultured in distinct media systems from the time of gamete isolation. WHAT IS KNOWN ALREADY: Studies on the effect of commercially available embryo culture media systems have demonstrated inconsistent impact on human embryonic development, morphokinetics, aneuploidy rates and clinical outcomes. In addition, these studies have been primarily randomized at the level of the embryo or the patient to culture media. STUDY DESIGN, SIZE, DURATION: Prospective sibling oocyte cohort derived from 200 subjects undergoing IVF at a tertiary academic medical center between February 2018 and November 2019. PARTICIPANTS/MATERIALS, SETTING, METHODS: Sibling oocytes were allocated to Global® or SAGE® media system based upon laterality of ovary from which they were retrieved. All embryos were cultured in a time-lapse incubator. Blastocysts underwent trophectoderm biopsy for preimplantation genetic testing for aneuploidy using next-generation sequencing. MAIN RESULTS AND THE ROLE OF CHANCE: One hundred twenty-seven subjects (n = 127) had paired blastocysts for biopsy in each culture media system. There was no difference in top quality blastocyst formation (47.1 ± 31.0 vs 48.1 ± 27.2%; P = 0.87) nor aneuploidy rate (62.3 ± 34.0 vs 56.1 ± 34.4%; P = 0.07) for sibling embryos cultured in Global versus SAGE media system. Embryo morphokinetic parameters including time to each cell division from two cells (t2) to eight cells (t8), time to morula stage (tM), time to blastocele formation (tSB), time to fully formed blastocyst (tB) and time to expansion of the blastocyst (tEB) were similar between paired blastocysts from each culture media system. LIMITATIONS, REASONS FOR CAUTION: Pregnancy outcomes and offspring health data were not available for analysis. WIDER IMPLICATIONS OF THE FINDINGS: Commercially available culture media may not have a differential impact on embryo development and blastocyst aneuploidy rate when patient and stimulation-related factors are held constant. STUDY FUNDING/COMPETING INTEREST(S): There was no external funding for this study. C.H. is owner of a consultancy company, IVF Professionals, Chief Scientific Officer at Apricity, Executive Director at TMRW and co-owner and shareholder of Aria Fertility. She has received speaker fees, consulting fees and travel support from Cooper Surgical and Vitrolife. J.B. is an employee and shareholder of vitrolife. TRIAL REGISTRATION NUMBER: N/A.

Embryonic stem cells derived from in vivo or in vitro-generated murine blastocysts display similar transcriptome and differentiation potential.

The use of assisted reproductive technologies (ART) such as in vitro fertilization (IVF) has resulted in the birth of more than 5 million children. While children conceived by these technologies are generally healthy, there is conflicting evidence suggesting an increase in adult-onset complications like glucose intolerance and high blood pressure in IVF children. Animal models indicate similar potential risks. It remains unclear what molecular mechanisms may be operating during in vitro culture to predispose the embryo to these diseases. One of the limitations faced by investigators is the paucity of the material in the preimplantation embryo to test for molecular analysis. To address this problem, we generated mouse embryonic stem cells (mESC) from blastocysts conceived after natural mating (mESCFB) or after IVF, using optimal (KSOM + 5% O2; mESCKAA) and suboptimal (Whitten's Medium, + 20% O2, mESCWM) conditions. All three groups of embryos showed similar behavior during both derivation and differentiation into their respective mESC lines. Unsupervised hierarchical clustering of microarray data showed that blastocyst culture does not affect the transcriptome of derived mESCs. Transcriptomic changes previously observed in the inner cell mass (ICM) of embryos derived in the same conditions were not present in mESCs, regardless of method of conception or culture medium, suggesting that mESC do not fully maintain a memory of the events occurring prior to their derivation. We conclude that the fertilization method or culture media used to generate blastocysts does not affect differentiation potential, morphology and transcriptome of mESCs.

Sexually dimorphic effect of in vitro fertilization (IVF) on adult mouse fat and liver metabolomes.

The preimplantation embryo is particularly vulnerable to environmental perturbation, such that nutritional and in vitro stresses restricted exclusively to this stage may alter growth and affect long-term metabolic health. This is particularly relevant to the over 5 million children conceived by in vitro fertilization (IVF). We previously reported that even optimized IVF conditions reprogram mouse postnatal growth, fat deposition, and glucose homeostasis in a sexually dimorphic fashion. To more clearly interrogate the metabolic changes associated with IVF in adulthood, we used nontargeted mass spectrometry to globally profile adult IVF- and in vivo-conceived liver and gonadal adipose tissues. There was a sex- and tissue-specific effect of IVF on adult metabolite signatures indicative of metabolic reprogramming and oxidative stress and reflective of the observed phenotypes. Additionally, we observed a striking effect of IVF on adult sexual dimorphism. Male-female differences in metabolite concentration were exaggerated in hepatic IVF tissue and significantly reduced in IVF adipose tissue, with the majority of changes affecting amino acid and lipid metabolites. We also observed female-specific changes in markers of oxidative stress and adipogenesis, including reduced glutathione, cysteine glutathione disulfide, ophthalmate, urate, and corticosterone. In summary, embryo manipulation and early developmental experiences can affect adult patterns of sexual dimorphism and metabolic physiology.

Use of a mouse in vitro fertilization model to understand the developmental origins of health and disease hypothesis.

The Developmental Origins of Health and Disease hypothesis holds that alterations to homeostasis during critical periods of development can predispose individuals to adult-onset chronic diseases such as diabetes and metabolic syndrome. It remains controversial whether preimplantation embryo manipulation, clinically used to treat patients with infertility, disturbs homeostasis and affects long-term growth and metabolism. To address this controversy, we have assessed the effects of in vitro fertilization (IVF) on postnatal physiology in mice. We demonstrate that IVF and embryo culture, even under conditions considered optimal for mouse embryo culture, alter postnatal growth trajectory, fat accumulation, and glucose metabolism in adult mice. Unbiased metabolic profiling in serum and microarray analysis of pancreatic islets and insulin sensitive tissues (liver, skeletal muscle, and adipose tissue) revealed broad changes in metabolic homeostasis, characterized by systemic oxidative stress and mitochondrial dysfunction. Adopting a candidate approach, we identify thioredoxin-interacting protein (TXNIP), a key molecule involved in integrating cellular nutritional and oxidative states with metabolic response, as a marker for preimplantation stress and demonstrate tissue-specific epigenetic and transcriptional TXNIP misregulation in selected adult tissues. Importantly, dysregulation of TXNIP expression is associated with enrichment for H4 acetylation at the Txnip promoter that persists from the blastocyst stage through adulthood in adipose tissue. Our data support the vulnerability of preimplantation embryos to environmental disturbance and demonstrate that conception by IVF can reprogram metabolic homeostasis through metabolic, transcriptional, and epigenetic mechanisms with lasting effects for adult growth and fitness. This study has wide clinical relevance and underscores the importance of continued follow-up of IVF-conceived offspring.

Effect of substrate stiffness on early mouse embryo development.

It is becoming increasingly clear that cells are remarkably sensitive to the biophysical cues of their microenvironment and that these cues play a significant role in influencing their behaviors. In this study, we investigated whether the early pre-implantation embryo is sensitive to mechanical cues, i.e. the elasticity of the culture environment. To test this, we have developed a new embryo culture system where the mechanical properties of the embryonic environment can be precisely defined. The contemporary standard environment for embryo culture is the polystyrene petri dish (PD), which has a stiffness (1 GPa) that is six orders of magnitude greater than the uterine epithelium (1 kPa). To approximate more closely the mechanical aspects of the in vivo uterine environment we used polydimethyl-siloxane (PDMS) or fabricated 3D type I collagen gels (1 kPa stiffness, Col-1k group). Mouse embryo development on alternate substrates was compared to that seen on the petri dish; percent development, hatching frequency, and cell number were observed. Our results indicated that embryos are sensitive to the mechanical environment on which they are cultured. Embryos cultured on Col-1k showed a significantly greater frequency of development to 2-cell (68 ± 15% vs. 59 ± 18%), blastocyst (64 ± 9.1% vs. 50 ± 18%) and hatching blastocyst stages (54 ± 25% vs. 21 ± 16%) and an increase in the number of trophectodermal cell (TE,65 ± 13 vs. 49 ± 12 cells) compared to control embryos cultured in PD (mean ± S.D.; p<.01). Embryos cultured on Col-1k and PD were transferred to recipient females and observed on embryonic day 12.5. Both groups had the same number of fetuses, however the placentas of the Col-1k fetuses were larger than controls, suggesting a continued effect of the preimplantation environment. In summary, characteristics of the preimplantation microenvironment affect pre- and post-implantation growth.