Towards a more sustainable balance between optimal live birth rate and supernumerary embryos in ART treatments.

PURPOSE: To assess the relation between number of inseminated oocytes and cumulative live birth rate (CLBR) in order to provide guidance for limiting the number of surplus blastocysts. METHODS: The study was a retrospective, single-center cohort analysis of 1223 ART complete cycles. Cycles were stratified according to female age (≤ 34, 35-38, and 39-42 years) and number of inseminated oocytes (1-5, 6-10, and > 10). Inclusion criteria were indication for IVF/ICSI with own spermatozoa and blastocyst culture up to day 6 of all embryos. RESULTS: In patients younger than 35 years, insemination of more than ten oocytes produced an increase in overall blastocyst number, CLBR (40.3%, 54.3%, and 75.8%, respectively, for each oocyte group) and surplus embryo rate (12.9%, 27.8%, and 49.7% of cases for each group). Instead, in the middle age group, the use of more than ten oocytes was solely associated with an increase in the rate of surplus embryos (1.25%, 21.33%, and 28.68% of cases after stratification for oocyte number). In older patients, neither CLBR (9.1%, 23.9%, and 24.7%, respectively) nor rate of surplus embryos (2.0%, 7.1%, and 13.4% of cases for each group) were higher in cycles with more than ten inseminated oocytes. CONCLUSION: In women up to 38 years, sustainable CLBR are achieved while limiting the number of inseminated oocytes and the resulting blastocysts remaining unused. Based on this notion, novel treatment strategies could pursue high outcome rates, while alleviating the problems derived from surplus stored embryos.

The destinies of human embryos reaching blastocyst stage between Day 4 and Day 7 diverge as early as fertilization.

STUDY QUESTION: What clinical and laboratory differences emerge from parallel direct comparison of embryos reaching the blastocyst stage between Days 4, 5, 6, and 7 (Days 4-7)? SUMMARY ANSWER: Increasing times to blastocyst formation are associated with a worse clinical outcome and perturbations in developmental patterns appear as early as the fertilization stage. WHAT IS KNOWN ALREADY: Previous evidence indicates that later times to blastocyst development are associated with a worse clinical outcome. However, the vast majority of these data concern Day 5 and Day 6 blastocysts, while Day 4 and Day 7 blastocysts remain less thoroughly investigated. In addition, studies comparing in parallel the developmental patterns and trajectories of Day 4-7 blastocysts are lacking. This leaves unanswered the question of when and how differences among such embryos emerge. Acquisition of such knowledge would significantly contribute to understanding the relative impact of intrinsic and extrinsic causes of embryo developmental kinetics and competence. STUDY DESIGN, SIZE, DURATION: This retrospective study involved time-lapse technology (TLT) monitoring of Day 4 (N = 70), Day 5 (N = 6147), Day 6 (N = 3243), and Day 7 (N = 149) blastocysts generated in 9450 ICSI cycles. Oocyte retrievals were carried out after clomiphene citrate-based minimal ovarian stimulation, between January 2020 and April 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS: Couples included in the study presented with different diagnoses, mainly male factor and unexplained infertility. Cases involving cryopreserved gametes or surgically retrieved sperm were excluded. Microinjected oocytes were assessed by a combined TLT-culture system. Day 4-7 blastocyst groups were compared in terms of morphokinetics (pronuclear dynamics, cleavage patterns and timings, and embryo quality) and clinical outcome. Clinically usable blastocysts were cryopreserved and transferred in single vitrified-warmed blastocyst transfers (SVBT). MAIN RESULTS AND THE ROLE OF CHANCE: From 19 846 microinjected oocytes, 17 144 zygotes (86.4%) were obtained. Overall, the blastocyst development rate was 56.0%. Rates of blastocysts formation on Days 4, 5, 6, and 7 were 0.7%, 64.0%, 33.8%, and 1.6%, respectively. The average expanded blastocyst development times were 98.4 ± 0.4, 112.4 ± 0.1, 131.6 ± 0.1, and 151.2 ± 0.5 h in the Day 4-7 groups, respectively. Female age was positively associated with longer times to blastocyst development. Rates of both inner cell mass (ICM) and trophectoderm (TE) morphological grade A blastocysts were negatively associated with the day of blastocyst development (P < 0.0001). The differences in development times and intervals increased progressively until blastocyst expansion (P < 0.0001 for all development times). Strikingly, such differences were already markedly evident as early as the time of pronuclear fading (tPNf) (20.6 ± 0.3, 22.5 ± 0.0, 24.0 ± 0.0, 25.5 ± 0.3; Days 4-7, respectively; P < 0.0001). Rates of cleavage anomalies (tri-/multi-chotomous mitosis or rapid cleavage) occurring at the first or second/third division cycles were also positively associated with longer times to blastocyst development. Implantation, ongoing pregnancy, and live birth rates were progressively reduced with increasing blastocyst development times (P < 0.0001), even after stratification for maternal age. When controlled for female age, male age, number of previous embryo transfer cycles, morphological grade of the ICM and TE, and progesterone supplementation, the probabilities of implantation, clinical, and ongoing pregnancy and live birth were significantly decreased in Day 6 blastocysts in comparison to Day 5 blastocysts. Follow-up data on birth length, weight, and malformations were comparable among the four blastocyst groups. LIMITATIONS, REASONS FOR CAUTION: The study is limited by its retrospective design. Having been obtained from a single centre, the data require independent validation. WIDER IMPLICATIONS OF THE FINDINGS: This study extends previous data on the relation between time of blastocyst formation and clinical outcome. It also indicates that differences in developmental times and patterns of Day 4-7 blastocysts occur as early as the fertilization stage, possibly dictated by intrinsic gamete-derived factors. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the participating institutions. The authors have no conflict of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Humidified atmosphere in a time-lapse embryo culture system does not improve ongoing pregnancy rate: a retrospective propensity score model study derived from 496 first ICSI cycles.

PURPOSE: To investigate whether high relative humidity conditions (HC), when using a time-lapse system (TLS) with sequential culture media, are beneficial to embryo culture, improving ongoing pregnancy rates. METHODS: We included patients undergoing their first ICSI cycle treatment from April 2021 to May 2022. Patients assigned to dry conditions (DC) or HC were 278 and 218, respectively. We used a GERI TLS, three chambers configured in humidity conditions and three in dry conditions. The effect of HC on ongoing pregnancy rate was assessed by the propensity matched sample, to reduce potential differences between women undergoing either HC or DC and reduce biased estimation of treatment effect. RESULT: After adjusting for several confounding variables and applying the propensity score (PS), no significant differences were observed in the rates of normal (2PN) and abnormal (1PN and 3PN) fertilization, blastulation, top-quality blastocysts, frozen blastocysts, ongoing pregnancies, and miscarriages. The 2-cell (t2) and 4-cell (t4) stages and cell divisions between such stages occurred earlier and were more synchronous in the in DC. CONCLUSION: These results suggest that HC conditions do not improve the rate of ongoing pregnancy and several embryological outcomes, under the conditions used in this study based on a time-lapse system and sequential culture with day 3 medium change-over.

Fine-tuning IVF laboratory key performance indicators of the Vienna consensus according to female age.

PURPOSE: To test the validity of the Vienna consensus laboratory key performance indicators (KPIs) to monitor the outcome of treatments involving women of different age ranges. METHODS: The retrospective cohort study included 862 complete IVF/ICSI cycles carried out between January 2014 and May 2021. All embryos of each cycle cohort were subject to extended culture. The overall population was divided into two groups according to female age: the Vienna consensus (≤ 39 years) and older female age (≥ 40 years). We compared outcomes of a selection of the Vienna performance indicators (PIs) and KPIs, with a focus on measures relevant to embryo cleavage and blastocyst formation. A possible association between total good blastocyst development rate (TGBDR) and cumulative clinical pregnancy rate (CPR) was also assessed. RESULTS: No differences were observed in fertilization and embryo cleavage KPIs between the Vienna consensus and the older female age group (standard IVF fertilization, 67.2 vs. 67.3; ICSI fertilization, 72.3 vs. 75.3; day 2 development, 57.6% vs 58.7%; day 3 development, 52.4% vs. 50.7%, respectively). TGBDR was lower in the older female age group (45.5% vs. 33.4% p < 0.001). Multivariate logistic regression analysis indicated female age as a factor independently associated with TGBDR. Clinical outcomes significantly decreased with increasing female age. CONCLUSION: The study suggests that, while most laboratory outcome measures are reliably applicable irrespective of female age, KPIs describing extended embryo culture should be fine-tuned in consideration of older female age.

The Association of Kinetic Variables with Blastocyst Development and Ploidy Status.

BACKGROUND: Despite a plethora of studies conducted so far, a debate is still unresolved as to whether TLM can identify predictive kinetic biomarkers or algorithms universally applicable. Therefore, this study aimed to elucidate if there is a relationship between kinetic variables and ploidy status of human embryos or blastocyst developmental potential. METHODS: For conducting this retrospective cohort study, the normal distribution of data was verified using Kolmogorov-Smirnov test with the Lilliefors' amendment and the Shapiro-Wilk test. Kinetic variables were expressed as median and quartiles (Q1, Q2, Q3, Q4). Mann-Whitney U-test was used to compare the median values of parameters. Univariate and multiple logistic regression models were used to assess relationship between blastocyst developmental potential or ploidy status and kinetics. Several confounding factors were also assessed. RESULTS: Blastocyst developmental potential was positively correlated with the t4-t3 interval (s2) (OR=1.417, 95% CI of 1.288-1.560). s2 median value was significantly different between high- and low-quality blastocysts (0.50 and 1.33 hours post-insemination, hpi, respectively; p=0.003). In addition, timing of pronuclear appearance (tPNa) (OR=1.287; 95% CI of 1.131-1.463) had a significant relationship with ploidy changes. The median value of tPNa was statistically different (p=0.03) between euploid and aneuploid blastocysts (Euploid blastocysts=8.9 hpi; aneuploid blastocysts=10.3 hpi). CONCLUSION: The present findings are in line with the study hypothesis that kinetic analysis may reveal associations between cleavage patterns and embryo development to the blastocyst stage and ploidy status.

The paternal toolbox for embryo development and health.

The sperm is essential for reconstitution of embryonic diploidy and highly specialized developmental functions. Immediately after gamete fusion, the sperm-borne PLC-zeta triggers activation, generating intracellular free Ca2+ oscillations. Mutations in the PLC-zeta encoding gene are associated with the absence of this factor in mature sperm and inability to achieve fertilization. Sperm play also a role in the greater game of the choreography of fertilization. In the human, the sperm centrioles are introduced into the oocyte environment with gamete fusion. They interact with the oocyte cytoskeletal apparatus to form a functional pair of centrosomes and ultimately regulate pronuclear juxtaposition in preparation for the first cleavage. As a consequence, the fidelity of chromosome segregation during the first cell divisions depends on the function of sperm centrioles. Sperm DNA integrity is essential for embryo development and health. Damaged DNA does not impact on the sperm fertilization ability following ICSI. However, detrimental effects emerge at pre- and post-implantation stages. Sperm-specific epigenetic factors also play an active role in the regulation of embryonic development, as shown by correlations between reduced embryo morphological quality and incorrect chromatin packaging during spermiogenesis or abnormal methylation of sperm CpG islands. This functional landscape demonstrates that the contribution of the sperm to development goes far beyond its well-established role in fertilization. Clinical studies confirm this view and indicate sperm function as a crucial aspect of research to increase the efficacy of assisted reproduction treatments.

Plasticity of the human preimplantation embryo: developmental dogmas, variations on themes and self-correction.

BACKGROUND: IVF for the treatment of infertility offers unique opportunities to observe human preimplantation development. Progress in time-lapse technology (TLT) and preimplantation genetic testing (PGT) has greatly expanded our knowledge of developmental patterns leading to a healthy pregnancy or developmental failure. These technologies have also revealed unsuspected plastic properties of the preimplantation embryo, at macromolecular, cellular and multicellular levels. OBJECTIVE AND RATIONALE: This review focuses on the emerging concept of plasticity of the human embryo as revealed by recent evidence derived from TLT and PGT, calling for an updated and more precise redefinition of the boundaries between normal and abnormal development. SEARCH METHODS: PubMed was used to search the MEDLINE database for peer-reviewed English-language original articles and reviews concerning human preimplantation development. Cross-searches were performed by adopting 'fertilisation', 'pronucleus', 'cleavage', 'multinucleation', 'compaction', 'embryo', 'preimplantation genetic testing', 'aneuploidy', mosaicism', 'micromanipulation', 'time-lapse microscopy' and 'IVF/assisted reproduction' as main terms. The most relevant publications, i.e. those concerning major phenomena occurring during normal and abnormal development-with a focus on the human species-were assessed and discussed critically. OUTCOMES: Advances in TLT and PGT have revealed an astonishing plasticity and self-correction ability of the human preimplantation embryo in vitro. At fertilisation, an abnormal number of pronuclei do not always result in the formation of an aneuploid blastocyst. Animal studies and preliminary human observations indicate that combining of parental genomes may occur at the early cleavage stage, if not at fertilisation. Multinucleation occurs with much higher prevalence than previously thought and may be corrected at later cleavage stages. Irregular cleavage (multichotomous, direct, rapid and reverse cleavages) can generate chromosome segregation abnormalities that often lead to developmental arrest, but that sporadically may be confined to cells excluded from the blastocyst, and may sometimes result in viable pregnancy. Mitotic errors can generate mosaic blastocysts, but alternatively normal embryos may form from selective death or clonal depletion of aneuploid cells. WIDER IMPLICATIONS: Deviations from developmental dogmas and the increasing evidence of plasticity of the human embryo challenge current embryological notions and suggest the need to write new rules governing cell cycle, cell determination and chromosome segregation during preimplantation development.

Perturbations of morphogenesis at the compaction stage affect blastocyst implantation and live birth rates.

STUDY QUESTION: Do perturbations of embryo morphogenesis at compaction affect blastocyst development and clinical outcomes in assisted reproduction cycles? SUMMARY ANSWER: Cell exclusion and extrusion, i.e. cell disposal occurring respectively before or during morula compaction, affect blastocyst yield and quality, as well as rates of pregnancy and live birth. WHAT IS KNOWN ALREADY: Despite its pivotal role in morphogenesis for blastocyst organisation and cell fate determination, compaction at the morula stage has received little attention in clinical embryology. Time lapse technology (TLT) allows detailed morphokinetic analysis of this developmental stage. However, even in the vast majority of previous TLT studies, compaction was investigated without a specific focus. Recently, we reported that compaction may be affected by two clearly-distinct patterns of cell disposal, exclusion and extrusion, occurring prior to and during compaction, respectively. However, the crucial question of the specific relevance of partial compaction for embryo development and competence in ART has remained unanswered until now. STUDY DESIGN, SIZE, DURATION: This study involved the assessment of laboratory and clinical outcomes of 2,059 morula stage embryos associated with 1,117 ICSI patients, who were treated with minimal stimulation and single vitrified-warmed blastocyst transfer (SVBT) from April 2017 to March 2018. Patterns of morula compaction were assessed and analyzed in relation to embryonic and clinical outcomes. PARTICIPANTS/MATERIALS, SETTING, METHODS: Following ICSI, time-lapse videos were analysed to annotate morphokinetic parameters relevant to both pre- and post-compaction stages. According to their morphokinetic history, morulae were classified as: (I) fully compacted morulae (FCM); (II) partially compacted morulae (PCM), showing cells (a) excluded from the compaction process from the outset (Exc-PCM), (b) extruded from an already compacted morula (Ext-PCM), or (c) showing non-compacted cells arisen from both patterns (Exc/Ext-PCM). The number of excluded/extruded cells was also annotated. Possible correlations of compaction patterns with 13 morphokinetic parameters, abnormal cleavage, blastocyst yield and morphological grade, clinical and ongoing pregnancy rates, and live birth rate were evaluated. Other factors, such as patient and cycle characteristics, possibly associated with compaction patterns and their outcomes, were investigated. MAIN RESULTS AND THE ROLE OF CHANCE: Full compaction was observed in 39.0% of all embryos. However, partially compacted morulae (PCM) showing excluded (Exc-PCM), extruded (Ext-PCM) cells, or indeed both phenotypes (Exc/Ext-PCM) were frequently detected (24.8%, 16.6%, and 19.6%, respectively) and collectively (61%) exceeded fully compacted morulae. Blastomere exclusion or extrusion affected one or several cells, in different proportions. In comparison to FCM, the developmental pace of the three PCM groups, observed at 13 developmental stages starting from pronuclear fading, was progressively slower (P < 0.0001). Developmental delay at post-compaction stages was more pronounced in the group showing both patterns of partial compaction. Blastomere exclusion and/or extrusion had a large negative impact on blastocyst development. In particular, rates of blastocyst formation and cryopreservation were very low in the Ext-PCM and Exc/Ext-PCM groups (P < 0.0001). Rates of blastocysts with ICM or TE of highest quality (Grade A) were severely affected in all PCM groups (P < 0.0001). In 1,083 SVBTs, blastocysts derived from all PCM groups produced much lower clinical pregnancy, ongoing pregnancy, and live birth rates (P < 0.0001). All three patterns of partial compaction emerged as factors independently associated with live birth rate, even after multivariate logistic regression analysis including maternal/paternal age, female BMI, and number of previous embryo transfers as possible confounding factors. LIMITATIONS, REASONS FOR CAUTION: The retrospective design of the study represents a general limitation. WIDER IMPLICATIONS OF THE FINDINGS: This large-scale study represents a further important demonstration of embryo plasticity and above all indicates new robust morphokinetic parameters for improved algorithms of embryo selection. STUDY FUNDING/COMPETING INTEREST(S): This study was exclusively supported by the participating institutions. The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: NA.

Sperm count affects cumulative birth rate of assisted reproduction cycles in relation to ovarian response.

PURPOSE: To explore the possible influence of sperm quality, as assessed by prewash total sperm count (TSC), on cumulative success rates in assisted reproduction cycles. METHODS: Retrospective study carried out in private IVF centre. Seven hundred sixty-five couples undergoing complete ICSI cycles, i.e. whose all embryos were transferred or disposed of. Couples were characterised by male infertility and female age younger than 36 years. Couples with a combination of female and male infertility factors were excluded. The primary outcome measure was cumulative live birth rate. Secondary outcomes were cumulative pregnancy and miscarriage rates. No specific interventions were made. RESULTS: Higher TSC values have a positive impact on cumulative success rates in cycles characterised by few retrieved oocytes (1 to 5), while does not influence the outcome of cycles with a normal (6 to 10) or high (> 10) number of retrieved oocytes. CONCLUSIONS: The study highlights the importance of sperm quality for the efficacy of assisted reproduction treatments. This influence may remain relatively cryptic in association with normal or high ovarian response, but emerge decisively in cases of reduced ovarian response, suggesting a relationship between ovarian response and oocyte ability to compensate for paternal-derived deficiencies.

Alternative patterns of partial embryo compaction: prevalence, morphokinetic history and possible implications.

RESEARCH QUESTION: The morula stage is a poorly understood developmental stage. In the morula, cell compaction can involve all or only some blastomeres, with largely unknown implications. Here, the prevalence, underlying morphokinetic mechanisms and possible consequences of partial compaction, were investigated. DESIGN: Preimplantation genetic testing for aneuploidies (PGT-A) cycles of women whose embryos were observed by time-lapse technology were studied. PGT-A data, generated by array comparative genomic hybridization analysis and assessed in three age groups (≤34, 35-39 and ≥40 years), were obtained from trophectoderm biopsies after development to blastocyst stage. RESULTS: Compaction occurred according to three modalities: (i) full compaction, with all blastomeres included (FCM); partial compaction (partially compacted morula [PCM]), with blastomeres (ii) excluded from the outset (excluded-PCM) or (iii) extruded after compaction (extruded-PCM). Partial compaction occurred more frequently than full compaction. Excluded-PCM displayed the slowest morphokinetics at most stages and were most often associated with abnormal cleavage. After compaction, embryo degeneration was more frequently associated with cell extrusion. In excluded-PCM, loss of ≥2 cells impacted blastocyst rate. In embryos of both younger and middle age groups, no statistical differences were observed in the rate of aneuploidy in relation to the three compaction groups, unlike what observed in ≥40 years women. Implantation rates after transfer of euploid blastocysts were not statistically different between the three groups. CONCLUSIONS: Alternative modalities of incomplete compaction were detected. Such patterns are characterized by different morphokinetic behaviours overarching the entire preimplantation development, and by different developmental abilities.

Male factor infertility impacts the rate of mosaic blastocysts in cycles of preimplantation genetic testing for aneuploidy.

PURPOSE: In this study, we tested the hypothesis that, in PGT-A cycles, decreased semen quality is associated with increased rates of mosaic blastocysts. METHODS: In a retrospective analysis, three hundred and forty PGT-A cycles are divided into study groups according to semen quality. Cycles were initially divided into two groups, discerning couples with absence of male factor of infertility (non-male factor: NMF; N = 146 cycles) from couples with a male factor of infertility (MF; N = 173 cycles). Couples with severe male factor (SMF) infertility (n = 22) were assessed separately. Embryos were cultured to the blastocyst stage and chromosomally assessed by array comparative genomic hybridization (aCGH). The study did not involve specific interventions. RESULTS: The reproductive outcome of MF and NMF groups did not indicate statistically significant differences. However, while no differences were found between MF and NMF groups in terms of euploid or aneuploid blastocysts rates, a significantly higher rate of mosaic blastocysts was observed in the MF group (3.6% vs. 0.5%, respectively; P = 0.03). A similar pattern of results was observed in the SMF group when compared with those of the other PGT-A cycles taken together (no SMF). In particular, a significantly higher rate of mosaic blastocysts was observed in the SMF group (7.7% and 1.8%, respectively; P = 0.008). CONCLUSIONS: The study outcome strongly suggests that compromised semen quality is associated with increased rates of mosaic blastocysts analysed in PGT-A cycles. Sperm assessment appears therefore as an important factor in the determination of embryo development and for a more precise prognostic assessment of PGT-A cases.

The enigmatic morula: mechanisms of development, cell fate determination, self-correction and implications for ART.

BACKGROUND: Assisted reproduction technology offers the opportunity to observe the very early stages of human development. However, due to practical constraints, for decades morphological examination of embryo development has been undertaken at a few isolated time points at the stages of fertilisation (Day 1), cleavage (Day 2-3) and blastocyst (Day 5-6). Rather surprisingly, the morula stage (Day 3-4) has been so far neglected, despite its involvement in crucial cellular processes and developmental decisions. OBJECTIVE AND RATIONALE: The objective of this review is to collate novel and unsuspected insights into developmental processes occurring during formation of the morula, highlighting the key importance of this stage for a better understanding of preimplantation development and an improvement of ART. SEARCH METHODS: PubMed was used to search the MEDLINE database for peer-reviewed English-language original articles and reviews concerning the morula stage in mammals. Searches were performed by adopting 'embryo', 'morula', 'compaction', 'cell fate' and 'IVF/assisted reproduction' as main terms, in association with other keywords expressing concepts relevant to the subject (e.g. cell polarity). The most relevant publications, i.e. those concerning major phenomena occurring during formation of the morula in established experimental models and the human species, were assessed and discussed critically. OUTCOMES: Novel live cell imaging technologies and cell biology studies have extended our understanding of morula formation as a key stage for the development of the blastocyst and determination of the inner cell mass (ICM) and the trophectoderm (TE). Cellular processes, such as dynamic formation of filopodia and cytoskeleton-mediated zippering cell-to-cell interactions, intervene to allow cell compaction (a geometrical requisite essential for development) and formation of the blastocoel, respectively. At the same time, differential orientation of cleavage planes, cell polarity and cortical tensile forces interact and cooperate to position blastomeres either internally or externally, thereby influencing their cellular fate. Recent time lapse microscopy (TLM) observations also suggest that in the human the process of compaction may represent an important checkpoint for embryo viability, through which chromosomally abnormal blastomeres are sensed and eliminated by the embryo. WIDER IMPLICATIONS: In clinical embryology, the morula stage has been always perceived as a 'black box' in the continuum of preimplantation development. This has dictated its virtual exclusion from mainstream ART procedures. Recent findings described in this review indicate that the morula, and the associated process of compaction, as a crucial stage not only for the formation of the blastocyst, but also for the health of the conceptus. This understanding may open new avenues for innovative approaches to embryo manipulation, assessment and treatment.

Embryo morphokinetic characteristics and euploidy.

PURPOSE OF REVIEW: The purpose of the current review is to provide an update on time-lapse morphokinetic assessment related to embryo ploidy status. RECENT FINDINGS: The main limitation of the available studies regarding correlation between morphokinetic variables and ploidy is that each embryo is considered as an independent unit whereas recent findings show that embryo kinetics may be affected by patient and ovarian stimulation-related factors, so that clustered data analysis is more appropriate. Moreover, some experimental evidences show how embryos with irregular developmental patterns, often used as deselection criteria, can evolve into usable embryos and give pregnancy. SUMMARY: Time lapse technology has allowed us to obtain a lot of information about human embryo development through the characterization of events that are otherwise not visible using static morphological observations. Many morphokinetic parameters have been tested in relation to a variety of outcomes including implantation potential, blastocyst development and ploidy status. Regarding to this last point, most efforts aim to unravel this relationship with conflicting results in their predictive ability. Furthermore, embryos originating from anomalous behaviour, although with a reduced developmental potential, may result in euploid and transferrable blastocysts.

A quantitative approach to blastocyst quality evaluation: morphometric analysis and related IVF outcomes.

PURPOSE: To quantify blastocyst morphologic parameters with a feasible and standardized tool, investigating their predictive value on implantation outcome. METHOD: The study retrospectively analyzes 124 blastocysts from 75 patients. Quantitative measurements of blastocyst expansion, inner cell mass and trophoectoderm were taken using digital image analysis software. RESULT(S): Blastocysts areas were found to be ranging from 11626.2 up to 35076.4 µm(2). The area of an early blastocyst is A ≤ 18500 µm(2) with a mean diameter d = 140 ± 9 µm, and the area of an expanded blastocyst is A ≥ 24000 with d = 190 ± 9 µm. While blastocyst mean area was not related to implantation rate, more expanded blastocysts displayed a significantly higher implantation rate. Trophoectoderm cell number is a predictor of positive outcome: since a higher of cells (25.6 ± 11.3 vs 16.3 ± 12.8) `forming a tightly knit epithelium is prognostic of implantation potential. Conversely, inner cell mass size is significantly related to implantation only in expanded blastocysts (3122.7 ± 739.0 vs. 2978.1 ± 366.0 µm(2)). CONCLUSION(S): Evaluation of blastocyst morphology with a digital image system could be a valuable tool to standardize blastocyst grading based on quantitative parameters. Therefore, digital analysis may be helpful in identifying the best blastocyst to transfer.

Cumulative pregnancy rates resulting from the use of fresh and frozen oocytes: 7 years' experience.

Storing supernumerary embryos and transferring them later fully utilizes the reproductive potential of retrieved oocytes, allowing a significant increase in the overall number of pregnancies achieved from a single cycle of ovarian stimulation treatment. As an alternative to embryo cryopreservation, preservation of unfertilized oocytes has been proposed to maximize clinical outcome. This paper presents data concerning the cumulative pregnancy rate after use of fresh and cryopreserved oocytes. In 80 treatment cycles in which patients chose to have only a few fresh oocytes inseminated, 24 pregnancies were obtained (30.0%), with an implantation rate of 22.6%. After cryopreservation with the standard slow-cooling protocol, the survival, fertilization and cleavage rates of 918 frozen oocytes were 43.4, 51.5 and 86.0% respectively. A total of 14 frozen pregnancies were achieved, with pregnancy rate 19.2% per transfer and implantation rate 12.3%. The cumulative pregnancy rate was 47.5% per patient. Therefore, despite a low rate of oocyte post-thaw survival, it appears that oocyte storage appreciably improves the number of pregnancies per treatment cycle in cases in which only a minority of oocytes are destined for the fresh treatment. This outcome provides valuable information for appraising the chances of clinical success when the option of embryo cryopreservation is not available.

Predictive factors for embryo implantation potential.

In spite of recent improvements in IVF, pregnancy rates have not increased significantly and one of the major problems remains the high multiple pregnancy rate. Better criteria are therefore necessary to establish the viability of a transferable embryo. Early prognosis of the developmental fate of the oocyte would help in selecting the best embryos to transfer, but non-invasive selection at the oocyte stage (extracytoplasmic and intracytoplasmic morphology) has proved to be of little prognostic value. Recently, it has been shown that follicular vascularization appears to be predictive of oocyte developmental fate, making it a good first-step approach for selection. Observation of pronuclei patterns at the zygote stage appears to offer an additional prognostic tool, correlating well with IVF outcome. Morphological evaluation of the embryo at days 2-3 remains the most used and valid method of selection, even though it is not sufficient to select embryos with the higher implantation potential. Blastocyst culture is another possible strategy for selecting the best embryos with reduced risk of aneuploidies, though not all major chromosomal aberrations are excluded by prolonged in-vitro culture. In summary, selecting the best embryo for transfer is a decision that should be based on choices made during the different stages of assisted reproductive technologies.

Artificial reproductive technology achievements for optimizing embryo quality.

Evaluation of morphological characteristics, to date, has been the most widely accepted method for the selection of embryos with higher developmental ability and optimization of the outcome of in vitro fertilization (IVF). Improvement in clinical results has also been pursued through attempts to generate embryos of high quality or to identify more reliable selection criteria. This work evaluates the possibility of improving embryo quality and subsequent IVF outcome in situations in which creation of supernumerary embryos is not allowed. Optimization of gamete selection and conditions of insemination, such as hormonal therapy to improve sperm quality of patients undergoing intracytoplasmic sperm injection treatment, use of power Doppler image for follicle selection, short coincubation of gametes during insemination, and use of a polscope for spindle detection, is described.