Vitrification of human blastocysts for couples undergoing assisted reproduction: an updated review.

Over the past 40 years there has been a worldwide critical change in the field of assisted reproduction technology (ART), leading to the increased application of single blastocyst transfer, which is extremely important to avoid the risks of multiple pregnancy and associated complications for both mother and babies. Indeed, advancements in ART over the last few decades have been obtained thanks to several improvements, including ovarian stimulation, embryo culture conditions and, of course, progress in cryopreservation methods, especially with the application of vitrification. The ability to cryopreserve human embryos has improved significantly with vitrification compared to the initially adopted slow-freezing procedures. Since the introduction of vitrification, it has become the gold standard method to effectively cryopreserve human blastocysts. However, some new protocols are now being explored, such as the short warming procedure and even shorter exposure to the equilibration solution before vitrification, which seem to provide optimal results. Therefore, the main aim of the current narrative review, will be to illustrate the benefit of vitrification as an effective method to cryopreserve the human blastocyst and to illustrate new protocols and variations which in future may increase the performance of vitrification protocols.

PGT-A mosaicism based on NGS intermediate copy numbers: is it time to stop reporting them?

Mosaicism represents a genuine real phenomenon, but its high prevalence and undisclosed clinical significance, stress the burden on genetic counseling and the management of PGT-A results. Even though the assumption of mosaicism from NGS intermediate chromosome copy number profiles may represent a reasonable interpretation, other potential technical reasons, including amplification bias, contamination, biopsy technique, or the analysis algorithms, may constitute alternative explanations. Thresholds confining mosaicism ranges are established according to models employing mixtures of normal and abnormal cells with steady conditions of quantity and quality which are unable to reflect the full extent of variability present in a trophectoderm (TE) biopsy specimen. When the concordance of TE with the ICM is considered, mosaic TE biopsies poorly correlate with the chromosomal status of the remaining embryo, displaying mostly ICM aneuploidy in cases of TE high-range mosaics diagnosis and euploidy when mosaicism grade in TE is less than 50% (low-mid range mosaicism), which implies an evident overestimation of mosaicism results. Indeed, a binary classification of NGS profiles that excludes mosaic ranges, including only euploid and aneuploid diagnosis, provides higher specificity and accuracy in identifying abnormal embryos and discarding them. As intermediate copy number profiles do not represent strong evidence of mosaicism but only an inaccurate and misleading assumption, and considering that no increased risk has been reported in the offspring, until diagnosis specificity is improved and its clinical implications are determined, laboratories should consider limiting predictions to euploid and aneuploid and stop reporting mosaicism.

Exploring the effect of cryopreservation in assisted reproductive technology and potential epigenetic risk.

Since the birth of the first baby by in vitro fertilization in 1978, more than 9 million children have been born worldwide using medically assisted reproductive treatments. Fertilization naturally takes place in the maternal oviduct where unique physiological conditions enable the early healthy development of the embryo. During this dynamic period of early development major waves of epigenetic reprogramming, crucial for the normal fate of the embryo, take place. Increasingly, over the past 20 years concerns relating to the increased incidence of epigenetic anomalies in general, and genomic-imprinting disorders in particular, have been raised following assisted reproduction technology (ART) treatments. Epigenetic reprogramming is particularly susceptible to environmental conditions during the periconceptional period and non-physiological conditions such as ovarian stimulation, in vitro fertilization and embryo culture, as well as cryopreservation procedure, might have the potential to independently or collectively contribute to epigenetic dysregulation. Therefore, this narrative review offers a critical reappraisal of the evidence relating to the association between embryo cryopreservation and potential epigenetic regulation and the consequences on gene expression together with long-term consequences for offspring health and wellbeing. Current literature suggests that epigenetic and transcriptomic profiles are sensitive to the stress induced by vitrification, in terms of osmotic shock, temperature and pH changes, and toxicity of cryoprotectants, it is therefore, critical to have a more comprehensive understanding and recognition of potential unanticipated iatrogenic-induced perturbations of epigenetic modifications that may or may not be a consequence of vitrification.

Real-time image and time-lapse technology to select the single blastocyst to transfer in assisted reproductive cycles.

The success of an assisted reproduction cycle should be the achievement of a healthy singleton live birth following the replacement of one embryo. Therefore, one of the most critical points for embryologists has been the selection criteria and how to choose the best embryo to transfer with high implantation potential. In this vein, morphological evaluation has been historically the method applied. However, this practice relies on a limited number of single observations and is associated with high operator variability. Recently, a major innovation in embryo culture has been the introduction of a new type of incubator with integrated time-lapse monitoring, which enables the embryologist to analyze the dynamic events of embryo development, from fertilization to blastocyst formation. This novel practice is quickly growing and has been implemented in many IVF clinics worldwide. Therefore, the main aim of this review is to illustrate the benefits of time-lapse technology in a modern embryology laboratory. In particular, we discuss the blastocyst collapse(s) event and morphometric blastocyst assessment and analyse their association with embryo viability and implantation potential.

Total fertilization failure after ICSI: insights into pathophysiology, diagnosis, and management through artificial oocyte activation.

BACKGROUND: Total fertilization failure (TFF) is the failure of all metaphase II oocytes to fertilize in ART cycles. The phenomenon represents a known cause of infertility, affecting 1-3% of ICSI cycles. Oocyte activation deficiency (OAD) is the leading cause of fertilization failure, attributed to sperm- or oocyte-related issues, although until recently little attention has been given to oocyte-related deficiencies. Different strategies for overcoming TFF have been proposed in clinical settings, mainly using artificial oocyte activation (AOA) by calcium ionophores. Typically, AOA has been blindly applied with no previous diagnosis testing and, therefore, not considering the origin of the deficiency. The scarcity of data available and the heterogeneous population subjected to AOA make it challenging to draw firm conclusions about the efficacy and safety of AOA treatments. OBJECTIVE AND RATIONALE: TFF leads to an unexpected, premature termination of ART, which inflicts a substantial psychological and financial burden on patients. This review aims to provide a substantial update on: the pathophysiology of fertilization failure, focusing both on sperm- and oocyte-related factors; the relevance of diagnostic testing to determine the cause of OAD; and the effectiveness and safety of AOA treatments to overcome fertilization failure. SEARCH METHODS: Relevant studies were identified in the English-language literature using PubMed search terms, including fertilization failure, AOA, phospholipase C zeta (PLCζ), PLCZ1 mutations, oocyte-related factors, wee1-like protein kinase 2 (WEE2) mutations, PAT1 homolog 2 (PATL2) mutations, tubulin beta-8 chain (TUBB8) mutations, and transducin-like enhancer protein 6 (TLE6) mutations. All relevant publications until November 2022 were critically evaluated and discussed. OUTCOMES: Fertilization failure after ART has been predominantly associated with PLCζ deficiencies in sperm. The reason relates to the well-established inability of defective PLCζ to trigger the characteristic pattern of intracellular Ca2+ oscillations responsible for activating specific molecular pathways in the oocyte that lead to meiosis resumption and completion. However, oocyte deficiencies have recently emerged to play critical roles in fertilization failure. Specifically, mutations have been identified in genes such as WEE2, PATL2, TUBB8, and TLE6. Such mutations translate into altered protein synthesis that results in defective transduction of the physiological Ca2+ signal needed for maturation-promoting factor (MPF) inactivation, which is indispensable for oocyte activation. The effectiveness of AOA treatments is closely related to identifying the causal factor of fertilization failure. Various diagnostic tests have been developed to determine the cause of OAD, including heterologous and homologous tests, particle image velocimetry, immunostaining, and genetic tests. On this basis, it has been shown that conventional AOA strategies, based on inducing the calcium oscillations, are highly effective in overcoming fertilization failure caused by PLCζ-sperm deficiencies. In contrast, oocyte-related deficiencies might be successfully managed using alternative AOA promoters that induce MPF inactivation and meiosis resumption. Such agents include cycloheximide, N,N,N',N'-tetrakis(2-pyridylmethyl)ethane-1,2-diamine (TPEN), roscovitine, and WEE2 complementary RNA. In addition, when OAD is caused by oocyte dysmaturity, applying a modified ovarian stimulation protocol and trigger could improve fertilization. WIDER IMPLICATIONS: AOA treatments represent a promising therapy to overcome fertilization failure caused by sperm- and oocyte-related factors. Diagnosing the cause of fertilization failure will be essential to improve the effectiveness and safe utilization of AOA treatments. Even though most data have not shown adverse effects of AOA on pre- and post-implantation embryo development, the literature is scarce on the matter concerned and recent studies, mainly using mice, suggest that AOA might cause epigenetic alterations in the resulting embryos and offspring. Until more robust data are available, and despite the encouraging results obtained, AOA should be applied clinically judiciously and only after appropriate patient counseling. Currently, AOA should be considered an innovative treatment, not an established one.

Healthy Live Births after the Transfer of Mosaic Embryos: Self-Correction or PGT-A Overestimation?

The implementation of next generation sequencing (NGS) in preimplantation genetic testing for aneuploidy (PGT-A) has led to a higher prevalence of mosaic diagnosis within the trophectoderm (TE) sample. Regardless, mosaicism could potentially increase the rate of live-born children with chromosomic syndromes, though available data from the transfer of embryos with putative PGT-A mosaicism are scarce but reassuring. Even with lower implantation and higher miscarriage rates, mosaic embryos can develop into healthy live births. Therefore, this urges an explanation for the disappearance of aneuploid cells throughout development, to provide guidance in the management of mosaicism in clinical practice. Technical overestimation of mosaicism, together with some sort of "self-correction" mechanisms during the early post-implantation stages, emerged as potential explanations. Unlike the animal model, in which the elimination of genetically abnormal cells from the future fetal lineage has been demonstrated, in human embryos this capability remains unverified even though the germ layer displays an aneuploidy-induced cell death lineage preference with higher rates of apoptosis in the inner cell mass (ICM) than in the TE cells. Moreover, the reported differential dynamics of cell proliferation and apoptosis between euploid, mosaic, and aneuploid embryos, together with pro-apoptosis gene products (cfDNA and mRNA) and extracellular vesicles identified in the blastocoel fluid, may support the hypothesis of apoptosis as a mechanism to purge the preimplantation embryo of aneuploid cells. Alternative hypotheses, like correction of aneuploidy by extrusion of a trisomy chromosome or by monosomic chromosome duplication, are even, though they represent an extremely rare phenomenon. On the other hand, the technical limitations of PGT-A analysis may lead to inaccuracy in embryo diagnoses, identifying as "mosaic" those embryos that are uniformly euploid or aneuploid. NGS assumption of "intermediate copy number profiles" as evidence of a mixture of euploid and aneuploid cells in a single biopsy has been reported to be poorly predictive in cases of mosaicism diagnosis. Additionally, the concordance found between the TE and the ICM in cases of TE biopsies displaying mosaicism is lower than expected, and it correlates differently depending on the type (whole chromosome versus segmental) and the level of mosaicism reported. Thus, in cases of low-/medium-level mosaicism (<50%), aneuploid cells would rarely involve the ICM and other regions. However, in high-level mosaics (≥50%), abnormal cells in the ICM should display higher prevalence, revealing more uniform aneuploidy in most embryos, representing a technical variation in the uniform aneuploidy range, and therefore might impair the live birth rate.