Cytoplasmic strings in human blastocysts: hypotheses of their role and implications for embryo selection.

STUDY QUESTION: What are the implications of the presence cytoplasmic strings (Cyt-S) and their quantity and dynamics for the pre-implantation development of human blastocysts? SUMMARY ANSWER: Cyt-S are common in human embryos and are associated with faster blastocyst development, larger expansion, and better morphological quality. WHAT IS KNOWN ALREADY: Cyt-S are dynamic cellular projections connecting inner cell mass and trophectoderm (TE) cells, that can be observed during blastocyst expansion. Their prevalence in human embryos has been estimated to be between 44% and 93%. Data relevant to their clinical implications and role in development are lacking, limited, or controversial. STUDY DESIGN, SIZE, DURATION: Retrospective study conducted at a single IVF center between May 2013 and November 2014 and involving 124 pre-implantation genetic testing for aneuploidy cycles in a time-lapse incubator with ≥1 blastocyst biopsied and vitrified (N = 370 embryos assessed). These cycles resulted in 87 vitrified-warmed single-euploid blastocyst transfers. PARTICIPANTS/MATERIALS, SETTING, METHODS: ICSI, continuous blastocyst culture (Days 5-7), TE biopsy of fully expanded blastocysts without Day 3 zona pellucida drilling, qPCR to assess uniform full-chromosome aneuploidies, and vitrification were all performed. Only vitrified-warmed euploid single-embryo-transfers were conducted. Blastocyst morphological quality was defined according to Gardner's criteria. The AI-based software CHLOE™ (Fairtility) automatically registered timings from time of starting blastulation (tSB) to biopsy (t-biopsy, i.e. blastocyst full-expansion) as hours-post-insemination (hpi), embryo area (including zona pellucida in µm2), and spontaneous blastocyst collapses. One senior embryologist manually annotated Cyt-S presence, quantity, timings, and type (thick cell-to-cell connections and/or threads). All significant associations were confirmed through regression analyses. All couples', cycles', and embryos' main features were also tested for associations with Cyt-S presence, quantity, and dynamics. MAIN RESULTS AND THE ROLE OF CHANCE: About 94.3% of the patients (N = 117/124) had ≥1 embryo with Cyt-S. Out of a total of 370 blastocysts, 55 degenerated between blastulation and full-expansion (N = 55/370, 14.9%). The degeneration rate among embryos with ≥1 Cyt-S was 10.8% (N = 33/304), significantly lower than that of embryos without Cyt-S (33.3%, N = 22/66, P < 0.01). Of the remaining 315 viable blastocysts analyzed, 86% (N = 271/315; P < 0.01) had ≥1 Cyt-S, on average 3.5 ± 2.1 per embryo ranging 1-13. The first Cyt-S per viable embryo appeared at 115.3 ± 12.5 hpi (85.7-157.7), corresponding to 10.5 ± 5.8 h (0.5-31) after tSB. Overall, we analyzed 937 Cyt-S showing a mean duration of 3.8 ± 2.7 h (0.3-20.9). Cyt-S were mostly threads (N = 508/937, 54.2%) or thick cell-to-cell connections becoming threads (N = 382/937, 40.8%) than thick bridges (N = 47/937, 5.0%). The presence and quantity of Cyt-S were significantly associated with developmentally faster (on average 6-12 h faster) and more expanded (on average 2700 µm2-larger blastocyst's area at t-biopsy) embryos. Also, the presence and duration of Cyt-S were associated with better morphology. Lastly, while euploidy rates were comparable between blastocysts with and without Cyt-S, all euploid blastocysts transferred from the latter group failed to implant (N = 10). LIMITATIONS, REASONS FOR CAUTION: Cyt-S presence and dynamics were assessed manually on seven focal planes from video frames recorded every 15 min. The patients included were mostly of advanced maternal age. Only associations could be reported, but no causations/consequences. Lastly, larger datasets are required to better assess Cyt-S associations with clinical outcomes. WIDER IMPLICATIONS OF THE FINDINGS: Cyt-S are common during human blastocyst expansion, suggesting their physiological implication in this process. Their presence, quantity and dynamics mirror embryo viability, and morphological quality, yet their role is still unknown. Future basic science studies are encouraged to finally describe Cyt-S molecular nature and biophysical properties, and Artificial Intelligence tools should aid these studies by incorporating Cyt-S assessment. STUDY FUNDING/COMPETING INTEREST(S): None. 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.

Oocyte vitrification for fertility preservation is an evolving practice requiring a new mindset: societal, technical, clinical, and basic science-driven evolutions.

Infertility is a condition with profound social implications. Indeed, it is not surprising that evolutions in both medicine and society affect the way in vitro fertilization is practiced. The keywords in modern medicine are the four principles, which implicitly involve a constant update of our knowledge and our technologies to fulfill the "prediction" and "personalization" tasks, and a continuous reshaping of our mindset in view of all relevant societal changes to fulfill the "prevention" and "participation" tasks. A worldwide aging population whose life priorities are changing requires that we invest in fertility education, spreading actionable information to allow women and men to make meaningful reproductive choices. Fertility preservation for both medical and nonmedical reasons is still very much overlooked in many countries worldwide, demanding a comprehensive update of our approach, starting from academia and in vitro fertilization laboratories, passing through medical offices, and reaching out to social media. Reproduction medicine should evolve from being a clinical practice to treat a condition to being a holistic approach to guarantee patients' reproductive health and well-being. Oocyte vitrification for fertility preservation is the perfect use case for this transition. This tool is acquiring a new identity to comply with novel indications and social needs, persisting technical challenges, brand-new clinical technologies, and novel revolutions coming from academia. This "views and reviews" piece aims at outlining the advancement of oocyte vitrification from all these tightly connected perspectives.

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.

[Main endocrine disruptors related to female reproductive health: biological basis of their association]. / Principales disruptores endocrinos vinculados con salud reproductiva femenina: bases biológicas de su asociación.

Endocrine disruptors (EDs) are those chemical substances capable of interfering with hormonal homeostasis, altering the synthesis, function, storage and / or metabolism of hormones. These substances are found in the environment and in a wide variety of products for daily use, so human exposure is permanent. Animal experiments have confirmed the capacity of EDs to induce reproductive disorders, which is why it has been suggested that they could be an important factor in causing human subfertility. Bisphenol A, phthalates and persistent organic compounds are three types of EDs present in the environment and associated with reproductive disorders. Consulting the MEDLINE and PubMed databases, in this review, a bibliography of the last 20 years is gathered where the effects caused by exposure to the mentioned EDs in women during adult life are evaluated. The effects on ovarian reserve markers and the results of in vitro fertilization treatments are summarized. On the other hand, the evidence at the molecular level of the effects caused by EDs on reproductive physiology is evaluated in in vitro and in vivo studies.

Se denominan disruptores endocrinos (DEs) a aquellas sustancias químicas capaces de interferir con la homeostasis hormonal, alterando la síntesis, función, almacenamiento y/o metabolismo de las hormonas. Estas sustancias se encuentran en el ambiente y en una amplia variedad de productos de uso diario, por lo que la exposición humana es permanente. Experimentos con animales han confirmado la capacidad de los DEs para inducir desórdenes reproductivos, por lo que se ha sugerido que podrían ser un factor importante como causa de subfertilidad humana. El bisfenol A, los ftalatos y los compuestos orgánicos persistentes son tres tipos de DEs presentes en el medio ambiente y asociados con alteraciones reproductivas. Consultando las bases de datos MEDLINE y PubMed, en la presente revisión, se reúne bibliografía de los últimos 20 años donde se evalúan los efectos provocados por la exposición a los DEs mencionados en mujeres durante la vida adulta. Se resumen los efectos sobre marcadores de reserva ovárica y los resultados de tratamientos de fertilización in vitro. Por otro lado, se evalúa la evidencia a nivel molecular de los efectos provocados por los DEs sobre la fisiología reproductiva en estudios in vitro e in vivo.

A Simple and Efficient Solution to Eliminate Evaporation in Mammalian Embryo Cultures.

The aim of this brief report is to offer a solution for a problem that compromises the quality of in vitro-produced mammalian embryos. The harmful effects of evaporation-induced osmotic changes in mammalian embryo cultures have been recognized only recently. In this technical report, we describe a modified embryo culture dish (Humdish) that provides consistent >97% humidity and fully eliminates osmotic changes in the commonly used drop-under-oil culture systems from day 0 to 6. As an additional benefit, the Humdish also increases the temperature stability of cultures. If subsequent laboratory and clinical experiments prove its value, our suggested approach may help to improve the in vitro environment and quality of all preimplantation stage mammalian embryos, including the most sensitive ones produced from artificial gametes or by somatic cell nuclear transfer.

Principales disruptores endocrinos vinculados con salud reproductiva femenina: bases biológicas de su asociación / Main endocrine disruptors related to female reproductive health: biological basis of their association

Resumen Se denominan disruptores endocrinos (DEs) a aquellas sustancias químicas capaces de interferir con la homeostasis hormonal, alterando la síntesis, función, almacenamiento y/o metabolismo de las hormonas. Estas sustancias se encuentran en el ambiente y en una amplia variedad de productos de uso diario, por lo que la exposición humana es permanente. Experimentos con animales han confirmado la capacidad de los DEs para inducir desórdenes reproductivos, por lo que se ha sugerido que podrían ser un factor importante como causa de subfertilidad humana. El bisfenol A, los ftalatos y los compuestos orgánicos persistentes son tres tipos de DEs presentes en el medio ambiente y asociados con alteraciones reproductivas. Consultando las bases de datos MEDLINE y PubMed, en la presente revisión, se reúne bibliografía de los últimos 20 años donde se evalúan los efectos provocados por la exposición a los DEs mencionados en mujeres durante la vida adulta. Se resumen los efectos sobre marcadores de reserva ovárica y los resultados de tratamientos de fertilización in vitro. Por otro lado, se evalúa la evidencia a nivel molecular de los efectos provocados por los DEs sobre la fisiología reproductiva en estudios in vitro e in vivo.

Abstract Endocrine disruptors (EDs) are those chemical substances capable of interfering with hormonal homeostasis, altering the synthesis, function, storage and / or metabolism of hormones. These substances are found in the environment and in a wide variety of products for daily use, so human exposure is permanent. Animal experiments have confirmed the capacity of EDs to induce reproductive disorders, which is why it has been suggested that they could be an important factor in causing human subfertility. Bisphenol A, phthalates and persistent organic compounds are three types of EDs present in the environment and associated with reproductive disorders. Consulting the MEDLINE and PubMed databases, in this review, a bibliography of the last 20 years is gathered where the effects caused by exposure to the mentioned EDs in women during adult life are evaluated. The effects on ovarian reserve markers and the results of in vitro fertilization treatments are summarized. On the other hand, the evidence at the molecular level of the effects caused by EDs on reproductive physiology is evaluated in in vitro and in vivo studies.