Mitochondrial DNA Copy Number in Cleavage Stage Human Embryos—Impact on Infertility Outcome

A retrospective case control study was undertaken at the molecular biology department of a private center for reproductive medicine in order to determine whether any correlation exists between mitochondrial DNA (mtDNA) content of cleavage-stage preimplantation embryos and their developmental potential. A total of 69 couples underwent IVF treatment (averaged women age: 36.5, SD 4.9) and produced a total of 314 embryos. A single blastomere was biopsied from each embryo at the cleavage stage (day-3 post-fertilization) subjected to low-pass next generation sequencing (NGS), for the purpose of detecting aneuploidy. For each sample, the number of mtDNA reads obtained after analysis using NGS was divided by the number of reads attributable to the nuclear genome. The mtDNA copy number amount was found to be higher in aneuploid embryos than in those that were euploid (mean mtDNA ratio ± SD: 6.3 ± 7.5 versus 7.1 ± 5.8, p < 0.004; U Mann–Whitney test), whereas no statistically significant differences in mtDNA content were seen in relation to embryo morphology (6.6 ± 4.8 vs. 8.5 ± 13.6, p 0.09), sex (6.6 ± 4.1 vs. 6.2 ± 6.8, p 0.16), maternal age (6.9 ± 7.8 vs. 6.7 ± 4.5, p 0.14) or its ability to implant (7.4 ± 6.6 vs. 5.1 ± 4.6, p 0.18). The mtDNA content cannot serve as a useful biomarker at this point in development. However, further studies investigating both quantitative and qualitative aspects of mtDNA are still required to fully evaluate the relationship between mitochondrial DNA and human reproduction.

Clinical outcomes for Day 3 double cleavage-stage embryo transfers versus Day 5 or 6 single blastocyst transfer in frozen–thawed cycles: a retrospective comparative analysis

Objective This study aimed to compare the clinical outcomes for transfer of Day 3 (D3) double cleavage-stage embryos and Day 5/6 (D5/6) single blastocysts in the frozen embryo transfer (FET) cycle to formulate a more appropriate embryo transplantation strategy. Methods We retrospectively analyzed 609 FET cycles from 518 women from April 2017 to March 2021. All FETs were assigned to the D3-DET group (transfer of a Day 3 double cleavage-stage embryo), D5-SBT group (transfer of a Day 5 single blastocyst), or D6-SBT group (transfer of a Day 6 single blastocyst). Clinical outcomes were comparatively analyzed. Results There were no significant differences in the biochemical pregnancy rate, clinical pregnancy rate, or ongoing pregnancy rate between the D3-DET and D5-SBT groups, but these rates in the two groups were all significantly higher compared with those in the D6-SBT group. The implantation rate in the D5-SBT group was significantly higher than that in the D3-DET group. The twin pregnancy rate in the D5-SBT and D6-SBT groups was significantly lower than that in the D3-DET group. Conclusion This study suggests that D5-SBT is the preferred option for transplantation. D6-SBT reduces the pregnancy rate, making it a more cautious choice for transfer of such embryos.

Hedgehog signaling controls mouth opening in the amphioxus

Introduction The left-sided position of the mouth in amphioxus larvae has fascinated researchers for a long time. Despite the fundamental importance of mouth development in the amphioxus, the molecular regulation of its development is almost unknown. In our previous study, we showed that Hh mutation in the amphioxus leads to no mouth opening, indicating a requirement of Hh signaling for amphioxus mouth formation. Nevertheless, since the Hh mutant also exhibits defects in early left-right (LR) patterning, it remains currently unknown whether the loss of mouth opening is affected directly by Hh deficiency or a secondary effect of its influence on LR establishment. Results We demonstrated that knockout of the Smo gene, another key component of the Hh signaling pathway, in the amphioxus resulted in the absence of mouth opening, but caused no effects on LR asymmetry development. Upregulation of Hh signaling led to a dramatic increase in mouth size. The inability of Smo mutation to affect LR development is due to Smo’s high maternal expression in amphioxus eggs and cleavage-stage embryos. In Smo mutants, Pou4 and Pax2/5/8 expression at the primordial oral site is not altered before mouth opening. Conclusions Based on these results and our previous study, we conclude that Hh signal is necessary for amphioxus mouth formation and that the Hh-mediated regulation of mouth development is specific to the mouth. Our data suggest that Hh signaling regulates mouth formation in the amphioxus in a similar way as that in vertebrates, indicating the conserved role of Hh signaling in mouth formation.

Day 3 Embryo Fragmentation Is Associated With Singleton Birthweight Following Fresh Single Blastocyst Transfer：A Retrospective Study

Background Previous studies arguably associated poor embryo morphology with low birthweight in singletons following single embryo transfer. However, the association between specific morphological features on the cleavage stage and birthweight is still less known. The purpose of the study was to investigate whether embryo morphological features at the cleavage stage affect birthweight following blastocyst transfer Methods The single-center, retrospective cohort study included 4226 singletons derived from fresh single cleavage stage embryo transfer (ET, n=1185), fresh single blastocyst transfer (BT, n=787), or frozen-thawed single blastocyst transfer (FBT, n=2254) between 2016 and 2019. The morphological parameters including early cleavage, day 3 fragmentation, symmetry, blastomere number, and blastocyst morphology were associated with neonatal birthweight and z-score in multivariate regression models. Models were adjusted for maternal age, BMI, parity, peak estradiol level, endometrial thickness, insemination protocol, female etiologies, order of transfer, mode of delivery, and year of treatment. Results Adjusted for confounders, fragmentation was the only morphology feature associated with birthweight and z-score, while early cleavage, symmetry, blastomere number and blastocyst morphology were not. Fragmentation increased the birthweight in both ET group (115.4g, 95% CI: 26.6 to 204.2) and BT group (168.8g, 95%CI: 48.8 to 288.8), but not in FBT group (7.47g, 95%CI: -46.4 to 61.3). The associations of birthweight and morphological parameters were confirmed in analyses for z-score. Adjusted odds of large for gestational age and high birthweight were also significantly greater in singletons following the transfer of fragmented embryos in BT group (OR 3, 95% CI: 1.2 to 7.51, OR 3.65, 95% CI: 1.33 to 10, respectively). The presence of fragmentation at the cleavage stage also affected the association between blastocyst morphology and birthweight. Inner cell mass grades were negatively associated with birthweight in blastocysts with day 3 fragmentation but not in blastocysts without. Conclusions Birthweight following blastocyst transfer is positively associated with fragmentation at the cleavage stage. The data did not support the argument that transferring a poor-looking embryo may increase the risks of low birthweight. However, concerns for LGA infants still remain.

Non-invasive Metabolomic Profiling of Embryo Culture Medium Using Raman Spectroscopy With Deep Learning Model Predicts the Blastocyst Development Potential of Embryos

Purpose: This study aimed to establish a non-invasive predicting model via Raman spectroscopy for evaluating the blastocyst development potential of day 3 high-quality cleavage stage embryos.Methods: Raman spectroscopy was used to detect the metabolic spectrum of spent day 3 (D3) embryo culture medium, and a classification model based on deep learning was established to differentiate between embryos that could develop into blastocysts (blastula) and that could not (non-blastula). The full-spectrum data for 80 blastula and 48 non-blastula samples with known blastocyst development potential from 34 patients were collected for this study.Results: The accuracy of the predicting method was 73.53% and the main different Raman shifts between blastula and non-blastula groups were 863.5, 959.5, 1,008, 1,104, 1,200, 1,360, 1,408, and 1,632 cm–1 from 80 blastula and 48 non-blastula samples by the linear discriminant method.Conclusion: This study demonstrated that the developing potential of D3 cleavage stage embryos to the blastocyst stage could be predicted with spent D3 embryo culture medium using Raman spectroscopy with deep learning classification models, and the overall accuracy reached at 73.53%. In the Raman spectroscopy, ribose vibration specific to RNA were found, indicating that the difference between the blastula and non-blastula samples could be due to materials that have similar structure with RNA. This result could be used as a guide for biomarker development of embryo quality assessment in the future.

Impact of oxygen tension according to embryo stage of development: a prospective randomized study

Human embryo culture under 2–8% O2 is recommended by ESHRE revised guidelines for good practices in IVF labs. Nevertheless, notably due to the higher costs of embryo culture under hypoxia, some laboratories perform embryo culture under atmospheric O2 tension (around 20%). Furthermore, recent meta-analyses concluded with low evidence to a superiority of hypoxia on IVF/ICSI outcomes. Interestingly, a study on mice embryos suggested that oxidative stress (OS) might only have an adverse impact on embryos at cleavage stage. Hence, we aimed to demonstrate for the first time in human embryos that OS has a negative impact only at cleavage stage and that sequential culture conditions (5% O2 from Day 0 to Day 2/3, then «conventional» conditions at 20% O2 until blastocyst stage) might be a valuable option for human embryo culture. 773 IVF/ICSI cycles were included in this randomized clinical trial from January 2016 to April 2018. At Day 0 (D0), patients were randomized using a 1:2 allocation ratio between group A (20% O2; n = 265) and group B (5% O2; n = 508). Extended culture (EC) was performed when ≥ 5 Day 2-good-quality-embryos were available (n = 88 in group A (20% O2)). In subgroup B, 195 EC cycles were randomized again at Day 2 (using 1:1 ratio) into groups B’ (5% O2 until Day 6 (n = 101)) or C (switch to 20% O2 from Day 2 to Day 6 (n = 94). Fertilization rate, cleavage-stage quality Day 2-top-quality-embryo (D2-TQE), blastocyst quality (Day 5-top-quality-blastocyst (D5-TQB) and implantation rate (IR) were compared between groups A and B (= cleavage-stage analysis), or A(20% O2), B’(5% O2) and C(5%-to-20% O2). Overall, characteristics were similar between groups A and B. Significantly higher rates of early-cleaved embryos, top-quality and good-quality embryos on Day 2 were obtained in group B compared to group A (P < 0.05). This association between oxygen tension and embryo quality at D2 was confirmed using an adjusted model (P < 0.05). Regarding blastocyst quality, culture under 20% O2 from Day 0 to Day 6 (group A) resulted in significantly lower Day 5-TQB number and rates (P < 0.05) compared to both groups B’ and C. Furthermore, blastocyst quality was statistically equivalent between groups B’ and C (P = 0.45). At Day 6, TQB numbers and rates were also significantly higher in groups B’ and C compared to group A (P < 0.05). These results were confirmed analyzing adjusted mean differences for number of Day 5 and Day 6 top quality embryos obtained in group A when compared to those respectively in groups B’ and C (P < 0.05). No difference in clinical outcomes following blastocyst transfers was observed. These results would encourage to systematically culture embryos under hypoxia at least during early development stages, since OS might be detrimental exclusively before embryonic genome activation.

Haplotyping-based preimplantation genetic testing reveals parent-of-origin specific mechanisms of aneuploidy formation

Chromosome instability is inherent to human IVF embryos, but the full spectrum and developmental fate of chromosome anomalies remain uncharacterized. Using haplotyping-based preimplantation genetic testing for monogenic diseases (PGT-M), we mapped the parental and mechanistic origin of common and rare genomic abnormalities in 2300 cleavage stage and 361 trophectoderm biopsies. We show that while single whole chromosome aneuploidy arises due to chromosome-specific meiotic errors in the oocyte, segmental imbalances predominantly affect paternal chromosomes, implicating sperm DNA damage in segmental aneuploidy formation. We also show that postzygotic aneuploidy affects multiple chromosomes across the genome and does not discriminate between parental homologs. In addition, 6% of cleavage stage embryos demonstrated signatures of tripolar cell division with excessive chromosome loss, however hypodiploid blastomeres can be excluded from further embryo development. This observation supports the selective-pressure hypothesis in embryos. Finally, considering that ploidy violations may constitute a significant proportion of non-viable embryos, using haplotyping-based approach to map these events might further improve IVF success rate.