The Histidinol Phosphate Phosphatase Involved in Histidine Biosynthetic Pathway Is Encoded by SCO5208 (hisN) in Streptomyces coelicolor A3(2)

Through the screening of a Streptomyces coelicolor genomic library, carried out in a histidinol phosphate phosphatase (HolPase) deficient strain, SCO5208 was identified as the last unknown gene involved in histidine biosynthesis. SCO5208 is a phosphatase, and it can restore the growth in minimal medium in this HolPase deficient strain when cloned in a high or low copy number vector. Moreover, it shares sequence homology with other HolPases recently identified in Actinobacteria. During this work a second phosphatase, SCO2771, sharing no homologies with SCO5208 and all so far described phosphatases was identified. It can complement HolPase activity mutation only at high copy number. Sequence analysis of SCO5208 and SCO2771, amplified from the HolPase mutant strain, revealed that SCO5208 shows a mutation in a conserved amino acid, whereas SCO2771 does not show any mutation. All these results show that S. coelicolor SCO5208, recently renamed hisN, is the HolPase involved in histidine biosynthesi

Sucrose concentration influences the rate of human oocytes with normal spindle and chromosome configurations after slow-cooling cryopreservation

BACKGROUND: Recently described slow-cooling cryopreservation protocols involving elevated sucrose concentration have improved survival frequencies of human oocytes, potentially overcoming a major hurdle that has limited the adoption of oocyte storage. Because implantation rates of embryos from frozen oocytes remain generally low, it is still debated whether, irrespective of survival rates, this form of cryopreservation leads inevitably to the disruption or complete loss of the metaphase II (MII) spindle. METHODS: Human oocytes with an extruded polar body I (PBI) were cryopreserved using a slow-cooling method including 1.5 mol/l propane-1,2-diol (PrOH) and alternative sucrose concentrations (either 0.1 or 0.3 mol/l) in the freezing solution. Fresh control and frozen-thawed survived oocytes were analysed by confocal microscopy to evaluate MII spindle and chromosome organizations. RESULTS: Of the 104 oocytes included in the unfrozen group, 76 (73.1%) displayed normal bipolar spindles with equatorially aligned chromosomes. Spindle and chromatin organizations were significantly affected (50.8%) after cryopreservation involving lower sucrose concentration (61 oocytes), whereas these parameters were unchanged (69.7%) using the 0.3 mol/l sucrose protocol (152 oocytes). CONCLUSIONS: Partial disruption of the MII spindle and associated chromosomes accompanies inadequate cryopreservation during slow cooling. However, protocols adopting higher sucrose concentration in the freezing solution promote the retention of an intact chromosome segregation apparatus comparable in incidence to freshly collected oocytes

Permeability of human oocytes to ethylene glycol and their survival and spindle configurations after slow cooling cryopreservation

BACKGROUND: To develop novel cryopreservation methods, we estimated the permeability coefficients Lp (hydraulic conductivity) and P(EG) (cryoprotectant permeability) of mature human oocytes after exposure to ethylene glycol (EG) and tested the efficiency of a multi-step slow cooling protocol based on this cryoprotectant. METHODS: Oocytes were perfused with 1.5 mol/l EG for 10 min. Oocyte volume at each time point was calculated and normalized to the original volume. Slow cooling was conducted by exposing oocytes to increasing EG concentrations (0.5, 1.0 and 1.5 mol/l n = 155) or 1.5 mol/l of propane-1,2-diol (PrOH) n = 102. Oocytes which survived cryopreservation n = 80 and fresh oocytes n = 73 were prepared for confocal microscopy analysis of the meiotic spindle. RESULTS: During EG exposure, oocytes underwent an abrupt 50% volume reduction. Complete recovery of the initial volume was not observed. From the values of a best fit plot, the coefficients Lp = 0.82 +/- 0.29 microm min(-1) atm(-1) (mean +/- SD) and P(EG) 0.10 +/- 0.01 microm s(-1) were generated. Survival rates after freezing with EG were lower than with PrOH (51.6 versus 71.5%, respectively, P < 0.05). The frequencies of normal spindle configuration were lower in frozen EG and frozen PrOH oocytes compared with fresh oocytes (53.8, 50.9 and 66.7%, respectively, P < 0.05). CONCLUSIONS: The oocyte plasmalemma possesses limited permeability to EG and EG exposure causes considerable osmotic stress. However, post-thaw rates of survival and normal meiotic spindle organization may be preserved by protocols which are designed in order to minimize osmotic stress

The generation of live offspring from vitrified oocytes

Oocyte cryopreservation is extremely beneficial for assisted reproductive technologies, the treatment of infertility and biotechnology and offers a viable alternative to embryo freezing and ovarian grafting approaches for the generation of embryonic stem cells and live offspring. It also offers the potential to store oocytes to rescue endangered species by somatic cell nuclear transfer and for the generation of embryonic stem cells to study development in these species. We vitrified mouse oocytes using a range of concentrations of trehalose (0 to 0.3 M) and demonstrated that 0.1 and 0.3 M trehalose had similar developmental rates, which were significantly different to the 0.2 M cohort (P < 0.05). As mitochondria are important for fertilisation outcome, we observed that the clustering and distribution of mitochondria of the 0.2 M cohort were more affected by vitifrication than the other groups. Nevertheless, all 3 cohorts were able to develop to blastocyst, following in vitro fertilisation, although developmental rates were better for the 0.1 and 0.3 M cohorts than the 0.2 M cohort (P < 0.05). Whilst blastocysts gave rise to embryonic stem-like cells, it was apparent from immunocytochemistry and RT-PCR that these cells did not demonstrate true pluripotency and exhibited abnormal karyotypes. However, they gave rise to teratomas following injection into SCID mice and differentiated into cells of each of the germinal layers following in vitro differentiation. The transfer of 2-cell embryos from the 0.1 and 0.3 M cohorts resulted in the birth of live offspring that had normal karyotypes (9/10). When 2-cell embryos from vitrified oocytes underwent vitrification, and were thawed and transferred, live offspring were obtained that exhibited normal karyotypes, with the exception of one offspring who was larger and died at 7 months. We conclude that these studies highlight the importance of the endometrial environment for the maintenance of genetic stability and thus the propagation of specific genetic traits

Towards Automation in IVF: Pre-Clinical Validation of a Deep Learning-Based Embryo Grading System during PGT-A Cycles.

Preimplantation genetic testing for aneuploidies (PGT-A) is arguably the most effective embryo selection strategy. Nevertheless, it requires greater workload, costs, and expertise. Therefore, a quest towards user-friendly, non-invasive strategies is ongoing. Although insufficient to replace PGT-A, embryo morphological evaluation is significantly associated with embryonic competence, but scarcely reproducible. Recently, artificial intelligence-powered analyses have been proposed to objectify and automate image evaluations. iDAScore v1.0 is a deep-learning model based on a 3D convolutional neural network trained on time-lapse videos from implanted and non-implanted blastocysts. It is a decision support system for ranking blastocysts without manual input. This retrospective, pre-clinical, external validation included 3604 blastocysts and 808 euploid transfers from 1232 cycles. All blastocysts were retrospectively assessed through the iDAScore v1.0; therefore, it did not influence embryologists' decision-making process. iDAScore v1.0 was significantly associated with embryo morphology and competence, although AUCs for euploidy and live-birth prediction were 0.60 and 0.66, respectively, which is rather comparable to embryologists' performance. Nevertheless, iDAScore v1.0 is objective and reproducible, while embryologists' evaluations are not. In a retrospective simulation, iDAScore v1.0 would have ranked euploid blastocysts as top quality in 63% of cases with one or more euploid and aneuploid blastocysts, and it would have questioned embryologists' ranking in 48% of cases with two or more euploid blastocysts and one or more live birth. Therefore, iDAScore v1.0 may objectify embryologists' evaluations, but randomized controlled trials are required to assess its clinical value

Maternal age affects pronuclear and chromatin dynamics, morula compaction and cell polarity, and blastulation of human embryos.

Study question: Does maternal ageing impact early and late morphokinetic and cellular processes of human blastocyst formation? Summary answer: Maternal ageing significantly affects pronuclear size and intra- and extra-nuclear dynamics during fertilization, dysregulates cell polarity during compaction, and reduces blastocoel expansion. What is known already: In ART, advanced maternal age (AMA) affects oocyte yield, fertilization, and overall developmental competence. However, with the exception of chromosome segregation errors occurring during oocyte meiosis, the molecular and biochemical mechanisms responsible for AMA-related subfertility and reduced embryo developmental competence remain unclear. In particular, studies reporting morphokinetics and cellular alterations during the fertilization and pre-implantation period in women of AMA remain limited. Study design, size, duration: A total of 2058 fertilized oocytes were stratified by maternal age according to the Society for Assisted Reproductive Technology classification (42 years) and retrospectively analysed. AMA effects were assessed in relation to: embryo morphokinetics and morphological alterations; and the presence and distribution of cell polarity markers-Yes-associated protein (YAP) and protein kinase C-ζ (PKC-ζ)-involved in blastocyst morphogenesis. Participants/materials, setting, methods: A total of 1050 cycles from 1050 patients met the inclusion criteria and were analysed. Microinjected oocytes were assessed using a time-lapse culture system. Immature oocytes at oocyte retrieval and mature oocytes not suitable for time-lapse monitoring, owing to an excess of residual corona cells or inadequate orientation for correct observation, were not analysed. Phenomena relevant to meiotic resumption, pronuclear dynamics, cytoplasmic/cortical modifications, cleavage patterns and embryo quality were annotated and compared among groups. Furthermore, 20 human embryos donated for research by consenting couples were used for immunofluorescence. Main results and the role of chance: Static microscopic observation revealed that blastocyst formation and expansion were impaired in the 41-42 and >42-year groups (P 42-year group than those in the 42-year groups (P = 0.0010). Female pronuclear area decreased and asynchronous pronuclear breakdown increased in the >42-year group (P = 0.0027 and P 42-year group (P = 0.0002 and P = 0.0047, respectively). Blastulation and blastocyst expansion were also delayed in the 41-42 and >42-year groups (P 42-year group (P < 0.0001 for both). Limitations, reasons for caution: At the cellular level, the investigation was limited to cell polarity markers. Cell components of other developmental pathways should be studied in relation to AMA. Wider implications of the findings: The study indicates that maternal ageing affects the key functions of embryo morphogenesis, irrespective of the well-established influence on the fidelity of oocyte meiosis. Study funding/competing interest(s): This study was supported by the participating institutions. The authors have no conflicts of interest to declare. Trial registration number: N/A

Human blastocyst spontaneous collapse is associated with worse morphological quality and higher degeneration and aneuploidy rates: a comprehensive analysis standardized through artificial intelligence.

Study question: What are the factors associated with human blastocyst spontaneous collapse and the consequences of this event? Summary answer: Approximately 50% of blastocysts collapsed, especially when non-viable, morphologically poor and/or aneuploid. What is known already: Time-lapse microscopy (TLM) is a powerful tool to observe preimplantation development dynamics. Lately, artificial intelligence (AI) has been harnessed to automate and standardize such observations. Here, we adopted AI to comprehensively portray blastocyst spontaneous collapse, namely the phenomenon of reduction in size of the embryo accompanied by efflux of blastocoel fluid and the detachment of the trophectoderm (TE) from the zona pellucida (ZP). Although the underlying causes are unknown, blastocyst spontaneous collapse deserves attention as a possible marker of reduced competence. Study design, size, duration: An observational study was carried out, including 2348 TLM videos recorded during preimplantation genetic testing for aneuploidies (PGT-A, n = 720) cycles performed between January 2013 and December 2020. All embryos in the analysis at least reached the time of starting blastulation (tSB), 1943 of them reached full expansion, and were biopsied and then vitrified. Participants/materials, setting, methods: ICSI, blastocyst culture, TE biopsy without Day 3 ZP drilling, comprehensive chromosome testing and vitrification were performed. The AI software automatically registered tSB and time of expanding blastocyst (tEB), start and end time of each collapse, time between consecutive collapses, embryo proper area, percentage of shrinkage, embryo:ZP ratio at embryo collapse, time of biopsy (t-biopsy) and related area of the fully (re-)expanded blastocyst before biopsy, time between the last collapse and biopsy. Blastocyst morphological quality was defined according to both Gardner's criteria and an AI-generated implantation score. Euploidy rate per biopsied blastocyst and live birth rate (LBR) per euploid single embryo transfer (SET) were the main outcomes. All significant associations were confirmed through regression analyses. All couple, cycle and embryo main features were also investigated for possible associations with blastocyst spontaneous collapse. Main results and the role of chance: At least one collapsing embryo (either viable or subsequently undergoing degeneration) was recorded in 559 cycles (77.6%) and in 498 cycles (69.2%) if considering only viable blastocysts. The prevalence of blastocyst spontaneous collapse after the tSB, but before the achievement of full expansion, was 50% (N = 1168/2348), irrespective of cycle and/or couple characteristics. Blastocyst degeneration was 13% among non-collapsing embryos, while it was 18%, 20%, 26% and 39% among embryos collapsing once, twice, three times or ≥4 times, respectively. The results showed that 47.3% (N = 918/1943) of the viable blastocysts experienced at least one spontaneous collapse (ranging from 1 up to 9). Although starting from similar tSB, the number of spontaneous collapses was associated with a delay in both tEB and time of biopsy. Of note, the worse the quality of a blastocyst, the more and the longer its spontaneous collapses. Blastocyst spontaneous collapse was significantly associated with lower euploidy rates (47% in non-collapsing and 38%, 32%, 31% and 20% in blastocysts collapsing once, twice, three times or ≥4 times, respectively; multivariate odds ratio 0.78, 95%CI 0.62-0.98, adjusted P = 0.03). The difference in the LBR after euploid vitrified-warmed SET was not significant (46% and 39% in non-collapsing and collapsing blastocysts, respectively). Limitations, reasons for caution: An association between chromosomal mosaicism and blastocyst collapse cannot be reliably assessed on a single TE biopsy. Gestational and perinatal outcomes were not evaluated. Other culture strategies and media should be tested for their association with blastocyst spontaneous collapse. Future studies with a larger sample size are needed to investigate putative impacts on clinical outcomes after euploid transfers. Wider implications of the findings: These results demonstrate the synergistic power of TLM and AI to increase the throughput of embryo preimplantation development observation. They also highlight the transition from compaction to full blastocyst as a delicate morphogenetic process. Blastocyst spontaneous collapse is common and associates with inherently lower competence, but additional data are required to deepen our knowledge on its causes and consequences. Study funding/competing interest(s): There is no external funding to report. I.E., A.B.-M., I.H.-V. and B.K. are Fairtility employees. I.E. and B.K. also have stock or stock options of Fairtility. Trial registration number: N/A