Integrating imaging-based classification and transcriptomics for quality assessment of human oocytes according to their reproductive efficiency.

PURPOSE: Utilising non-invasive imaging parameters to assess human oocyte fertilisation, development and implantation; and their influence on transcriptomic profiles. METHODS: A ranking tool was designed using imaging data from 957 metaphase II stage oocytes retrieved from 102 patients undergoing ART. Hoffman modulation contrast microscopy was conducted with an Olympus IX53 microscope. Images were acquired prior to ICSI and processed using ImageJ for optical density and grey-level co-occurrence matrices texture analysis. Single-cell RNA sequencing of twenty-three mature oocytes classified according to their competence was performed. RESULT(S): Overall fertilisation, blastulation and implantation rates were 73.0%, 62.6% and 50.8%, respectively. Three different algorithms were produced using binary logistic regression methods based on "optimal" quartiles, resulting in an accuracy of prediction of 76.6%, 67% and 80.7% for fertilisation, blastulation and implantation. Optical density, gradient, inverse difference moment (homogeneity) and entropy (structural complexity) were the parameters with highest predictive properties. The ranking tool showed high sensitivity (68.9-90.8%) but with limited specificity (26.5-62.5%) for outcome prediction. Furthermore, five differentially expressed genes were identified when comparing "good" versus "poor" competent oocytes. CONCLUSION(S): Imaging properties can be used as a tool to assess differences in the ooplasm and predict laboratory and clinical outcomes. Transcriptomic analysis suggested that oocytes with lower competence may have compromised cell cycle either by non-reparable DNA damage or insufficient ooplasmic maturation. Further development of algorithms based on image parameters is encouraged, with an increased balanced cohort and validated prospectively in multicentric studies.

Molecular tools for the genomic assessment of oocyte's reproductive competence.

The most important factor associated with oocytes' developmental competence has been widely identified as the presence of chromosomal abnormalities. However, growing application of genome-wide sequencing (GS) in population diagnostics has enabled the identification of multifactorial genetic predispositions to sub-lethal pathologies, including those affecting IVF outcomes and reproductive fitness. Indeed, GS analysis in families with history of isolated infertility has recently led to the discovery of new genes and variants involved in specific human infertility endophenotypes that impact the availability and the functionality of female gametes by altering unique mechanisms necessary for oocyte maturation and early embryo development. Ongoing advancements in analytical and bioinformatic pipelines for the study of the genetic determinants of oocyte competence may provide the biological evidence required not only for improving the diagnosis of isolated female infertility but also for the development of novel preventive and therapeutic approaches for reproductive failure. Here, we provide an updated discussion and review of the progresses made in preconception genomic medicine in the identification of genetic factors associated with oocyte availability, function, and competence.

Does morphological assessment predict oocyte developmental competence? A systematic review and proposed score.

PURPOSE: Does existing scientific literature suggest an impact of oocyte dysmorphisms on biological or clinical outcomes of assisted reproduction treatments? METHODS: Studies of interest were selected from an initial cohort of 6651 potentially relevant records retrieved. PubMed was systematically searched for peer-reviewed original papers and reviews identified by keywords and medical subject heading (MeSH) terms. The most relevant publications were critically evaluated to identify criteria for oocyte morphological evaluation and IVF outcomes. For each morphological abnormality, we generated an oocyte literature score (OLS) through the following procedure: (a) papers showing a negative, absence of, or positive correlation between a given abnormality and IVF outcome were scored 1, 0, and - 1, respectively; (b) the sum of these scores was expressed as a fraction of all analyzed papers; (c) the obtained fraction was multiplied by 10 and converted into decimal number. RESULT: We identified eleven different dysmorphisms, of which six were extracytoplasmic (COC, zona pellucida, perivitelline space, polar body 1, shape, giant size) and five intracytoplasmic (vacuoles, refractile bodies, SER clusters, granularity, color). Among the extracytoplasmic dysmorphisms, abnormal morphology of the COC generated an OLS of 8.33, indicating a large prevalence (5/6) of studies associated with a negative outcome. Three intracytoplasmic dysmorphisms (vacuoles, SER clusters, and granularity) produced OLS of 7.14, 7.78, and 6.25, respectively, suggestive of a majority of studies reporting a negative outcome. CONCLUSION: COC morphology, vacuoles, SER clusters, and granularity produced OLS suggestive of a prevalence of studies reporting a negative outcome.

Egg maturity assessment prior to ICSI prevents premature fertilization of late-maturing oocytes.

PROPOSE: The presence of metaphase II (MII) spindle together with the polar body (PB) indicates completion of oocyte maturation. This study was designed to explore if spindle imaging can be used to optimize timing of intracytoplasmic sperm injection (ICSI). METHODS: The study involved 916 oocytes from 234 conventionally stimulated ICSI cycles with an unexpectedly poor ovarian response. All PB-displaying oocytes were subjected to polarized light microscopy (PLM) prior to ICSI. When MII spindle was absent in the majority of oocytes, ICSI was postponed and performed after additional spindle imaging. Fertilization, embryo development, and clinical outcome were evaluated with respect to the observed spindle pattern. RESULTS: The visible spindle was absent in 32.64% of PB-displaying oocytes. The late-maturing oocytes extruding PB in vitro were less likely to exhibit a spindle signal than in vivo matured MII oocytes (38.86% vs. 89.84%). When fertilization was postponed, 59.39% of initially spindle-negative oocytes developed detectable MII spindle. Spindled eggs had significantly higher developmental potential, and the presence of the spindle has been identified as an independent measure for predicting the formation of the blastocyst. Embryos derived from spindle-positive oocytes also showed a higher chance to implant and develop to term. Notably, 11 children were conceived by finely timed fertilization of late-maturing oocytes which are normally discarded. CONCLUSIONS: The study confirms the prognostic value of spindle imaging and demonstrates that immature oocytes can be clinically utilized and give rise to live births when the timing of ICSI is adjusted to their developmental stage.

Mitochondrial DNA Assessment to Determine Oocyte and Embryo Viability.

Mitochondria are the key regulators of multiple vital cellular processes, including apoptosis, calcium homeostasis, and the generation of ATP via the metabolic pathway known as oxidative phosphorylation. Unlike other cellular organelles, mitochondria contain one or more copies of their own genome (mtDNA). The mtDNA encodes a total of 13 genes with critical functions in cellular metabolism. The energy required to support the normal progress of preimplantation embryo development is provided in the form of ATP generated by the mitochondria. It has been suggested that cellular bioenergetic capacity and suboptimal levels of mitochondria-generated ATP could contribute to a variety of embryo developmental defects, and therefore adversely affect in vitro fertilization success rates. During this review, we discuss the role of mitochondria and their genome during oogenesis and early embryo development. We also assess whether analysis of mitochondria and their genome could be used as biomarkers to determine oocyte quality and embryo viability.

The cost of keeping eggs fresh: quantitative genetic variation in females that mate late relative to sexual maturation.

In many species, females abandon mate choice to ensure that eggs are fertilized before they are lost. But why do females not just maintain oocytes longer if there is a benefit to mate choice? We conducted a quantitative genetic study in the cockroach Nauphoeta cinerea to test whether genetic constraints prevent the evolution of oocyte maintenance or selection against oocyte loss is weak when females mate late relative to sexual maturity. We found standing genetic variation within the population and no evidence for genetic constraints. Levels of genetic variation are of the magnitude found for life-history traits in general, suggesting that this trait has been exposed to selection. We unexpectedly found two categories of females: those that delay reproduction and those that reproduce at a normal time when mating late, which could indicate alternative strategies. However, frequency-dependent selection does not maintain this variation as females that delay always reproduce less well. Given these findings, we suggest that there may be advantages to egg degradation. The evolution of maintenance of fertilizable oocytes over time would then be constrained by the need to maintain the mechanism by which females control the distribution of resources between current and future reproductive events.