Abstract Irregular cleavage divisions are expected to produce chromosomally deviant embryos. We investigated whether embryos from irregular cleavages could develop into euploid blastocysts, and, if ...so, whether any evidence existed of a self-correction mechanism of the embryo. We also investigated the role of different dynamic aspects of morula compaction in this process. A total of 791 embryos from 141 patients undergoing pre-implantation genetic screening were retrospectively analysed using a time-lapse imaging system, and multiple cell divisions were evaluated. A total of 276 embryos developed into blastocysts suitable for biopsy and chromosome screening through array-comparative genomic hybridization. As well as testing trophectoderm biopsy specimens for aneuploidy, excluded cells of 18 blastocysts, which developed from partially compacted morulas, were also analysed. Unique data on the developmental fate of embryos with cleavage abnormalities are presented, and a potential mechanism of ‘aneuploidy rescue’ is postulated through which mosaic embryos may form partially compacted morulas to exclude aneuploid cells. In addition, this process seems to be less efficient in older women. The data obtained also provide further evidence that excluded cells should not be used to infer the cytogenetic status of the embryo.
Subjects increasing sperm DNA fragmentation (sDF) during Density Gradient Centrifugation (DGC), a common sperm selection procedure in Assisted Reproduction Techniques (ARTs), experience a 50% lower ...probability of pregnancy. Hence, identification of these subjects is of clinical importance. Here, we investigated whether such subjects are identified with higher accuracy detecting DNA fragmentation in viable (viable sDF) instead of total spermatozoa (total sDF) and whether swim up, an alternative procedure to DGC, does not increase sDF. With DGC, we identified 10/20 subjects increasing total sDF, and 2 more subjects using viable sDF. With swim up, we identified 8/40 subjects increasing total sDF, and 8 more subjects using viable sDF. In addition, viable sDF reveals more accurately the increase of the damage when it occurs. Finally, a multivariate analysis demonstrated that the proportional increase of sDF was higher after DGC respect to swim up. In conclusion, viable sDF is a more accurate parameter to reveal the increase of the damage by selection both with swim up and DGC. Swim up increases sDF in some samples, although at a lesser extent than DGC, suggesting that it should be used to select spermatozoa for ARTs when possible.
BACKGROUND: The relationship between early embryo post-implantation development in couples undergoing assisted reproductive techniques (ARTs) and sperm chromatin alterations has not been ...satisfactorily explained. The aim of this study was to assess the relationship between sperm DNA fragmentation in IVF/ICSI patients, sperm parameters (concentration, motility and morphology) and ART outcome, especially with regard to clinical pregnancy and pregnancy loss (spontaneous miscarriage or biochemical pregnancy). METHODS: DNA fragmentation was evaluated by TUNEL assay, performed on sperm suspensions after density gradient separation, in 132 men undergoing an ART cycle (82 IVF and 50 ICSI) and correlated with sperm parameters and ART outcome. RESULTS: A highly significant negative correlation was found between DNA fragmentation and sperm parameters. There was a close relationship between DNA fragmentation and post-implantation development in ICSI patients: the clinical pregnancy and pregnancy loss rates significantly differed between patients with high and low sperm DNA fragmentation (P = 0.007 and P = 0.009, respectively). CONCLUSIONS: Sperm DNA fragmentation seems to affect embryo post-implantation development in ICSI procedures: high sperm DNA fragmentation can compromise ‘embryo viability’, resulting in pregnancy loss.
Abstract Study question Do blastocysts with a diverse history of compaction pattern (complete or partial) have different chances to be aneuploid and to implant? Summary answer Embryos undergoing ...partial or complete compaction have comparable chances to be aneuploid. Euploid blastocysts with previous history of blastomere extrusion have reduced chances of implantation. What is known already Compaction at the morula stage is an essential requisite for blastocyst formation and involves blastomere flattening and establishment of tenacious cell-to-cell contacts. Incomplete compaction is associated with several upstream and downstream morphokinetic anomalies. It also affects blastocyst yield and quality and clinical outcome. However, evidence on blastocyst chromosomal constitution and implantation potential after preimplantation genetic testing for aneuploidies (PGT-A) is lacking. Study design, size, duration This retrospective cohort study assessed laboratory and clinical outcomes of 1206 blastocysts derived from 483 infertile patients undergoing ART treatment and PGT-A from January 2015 to July 2022. Three age groups (≤34, 35–38 and ≥39 years) were adopted for sub-analyses. Biopsied blastocysts were vitrified and subsequently used in single vitrified-warmed blastocyst transfers (SVBT). Participants/materials, setting, methods Embryo development was monitored by time-lapse technology (TLT), also annotating abnormal cleavages and multinucleation. Patterns of morula compaction were assessed according to previously published categories: (i) full compaction, with all blastomeres undergoing compaction (FCM); partial compaction (partially compacted morula PCM), with (ii) blastomeres excluded from the outset (exc-PCM) or (iii) extruded after start of compaction (ext-PCM). Embryos were assessed in relation to embryonic and clinical outcomes. PGT-A data were obtained from trophectoderm biopsies. Main results and the role of chance Average maternal age of the overall patient population was 39.0 years. The full compaction pattern (FCM) was detected in 35.7% of all embryos. Partially compacted morulae showing excluded (Exc-PCM) or extruded (Ext-PCM) cells were also observed (54.4% and 9.9%, respectively) and collectively (64.3%) were the larger fraction (P < 0.0001, among all groups). Of 1206 analysed blastocysts, 551 were euploid, of which 366 were used in SVBT. Aneuploidy rate in the overall blastocyst population derived from FCM, Exc-PCM and Ext-PCM was 50.8%, 55.6%, and 60.0%, respectively (P > 0.05). However, in the younger maternal age group (≤34 years), the aneuploidy rate of the Exc-PCM group was higher compared with FCM (39.8% vs. 25.3%, P = 0.03). In the overall population, the Ext-PCM phenotype was associated with a lower implantation rate (FCM, Exc-PCM and Ext-PCM:47.8%, 44.7%, 55.6%, and 27,6%, respectively (P = 0.04), while miscarriage rates were comparable. Finally, transferred euploid blastocysts not classified according to maternal age implanted with comparable rates, irrespective of their morphokinetic history (abnormal division, multinucleation, no morphokinetic abnormalities). 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 confirms and extends our previous findings on the implications of partial compaction. Importantly, it further consolidates the notion that the embryo can develop through atypical morphokinetic patterns while often preserving its genomic integrity and implantation ability. Trial registration number Not applicable
Abstract
Study question
Are the IVF laboratory KPIs of the Vienna consensus equally applicable to ICSI cycles performed with ejaculated or testicular sperm aspiration (TESA) samples?
Summary answer
...In addition to standard ICSI cycles, the Vienna KPIs are largely applicable to TESA cases, except for 2PN fertilization and day 3 development rates.
What is known already
The ESHRE SIG Embryology and Alpha Scientists in Reproductive Medicine produced the first international consensus on a systematic set of IVF laboratory KPIs. These efficiency measures can reveal the possible impact of extrinsic factors, such as operator skills or culture media, on the IVF process. However, to control for intrinsic factors – i.e., specific gamete characteristics – the Vienna Consensus focused on a “reference population” as defined by female age ≤39years, exclusion of PGT cases and use of own fresh oocytes and ejaculated sperm. This leaves the relevance of the Vienna KPIs to other patient populations, including TESA cases, uncertain.
Study design, size, duration
This was a retrospective, single-center cohort analysis of 1678 ART ICSI cycles carried out between 2010 and 2022. Treatments involving TESA and ejaculated sperm were 105 and 1573, respectively.
Participants/materials, setting, methods
Inclusion criteria were indication for IVF/ICSI with own ejaculated or TESA spermatozoa and blastocyst culture of all embryos formed in each cohort. Oocyte donation and PGT cycles were not included. Ovarian stimulation was performed with either recombinant-FSH or hMG, combined with GnRH to prevent spontaneous LH surge. Fertilization of collected oocytes was achieved by ICSI.
Main results and the role of chance
Both maternal and paternal age were comparable between the two groups. In the TESA cases, 2PN rate was lower (N = 593/915, 64.8% versus N = 7276/10350, 70.3%; p < 0.01), while 1PN rate was higher (N = 32/915, 3.5% versus N = 246/10350, 2.4%; p = 0.04). Other fertilization and developmental rates were comparable: 3PN (N = 24/915, 2.6% versus N = 341/10350, 3.3%); microinjection damage (N = 52/915, 5.7% versus 632/10350, 6.1%); failed fertilization (N = 4/105, 3.8% versus N = 61/1573, 3.9%); cleavage (N = 579/593, 97.6% versus N = 7105/7276, 97.6%); day 2 development (N = 318/593, 53.6% versus N = 3887/7276, 53.4%); day 3 development (N = 248/593, 41.8% versus N = 3279/7276, 45.1%); total good blastocyst development rate (N = 147/361, 40.7% versus N = 1728/3944, 43.8%). Compared with the Vienna Consensus, all ICSI outcomes were within normal ranges as defined by competency and benchmark values, while in TESA cycles 2PN fertilization and day 3 development rates were slightly below the competency thresholds.
Limitations, reasons for caution
The study, especially because retrospective and with small sample size, requires external independent validation
Wider implications of the findings
The study confirms the robustness of the Vienna Consensus recommendations for the monitoring of the IVF laboratory performance, while highlighting the need for fine-tuning individual indicators in specific patient populations.
Trial registration number
not applicable
Abstract
Study question
Is the Vienna consensus appropriate to monitor IVF laboratory outcomes of treatments involving women of different age ranges?
Summary answer
Most IVF laboratory key ...performance indicators(KPIs) are reliably applicable irrespective of female age, while total good blastocyst rate should be adjusted according to female age
What is known already
The IVF laboratory is central to ART treatments. This demands methodical and precise monitoring of the performance. To assess laboratory efficiency, the Vienna consensus identified a set of relevant performance indicators(PIs) and KPIs applicable to a “reference population” defined by female age <40years, exclusion of PGT cases and use of own fresh oocytes and ejaculated sperm. It is known that women older than 39years – whose treatment outcome is affected by reduced oocyte quality–represent an increasingly large proportion of IVF patients. Thus, in this subgroup of patients the relevance of the Vienna indicators to treatments of remains to be demonstrated.
Study design, size, duration
Reported data concern a retrospective, single-center cohort analysis of 862 ART cycles carried out between January 2014 and May 2021. Inclusion criteria were indication for IVF/ICSI, blastocyst culture of all embryos formed in each cohort, use of own ejaculated spermatozoa (fresh or frozen), and complete cycles, i.e. those whose all embryos were transferred, cryopreserved or disposed of. Cancelled and PGT cycles were not included
Participants/materials, setting, methods
The overall population was divided into two groups according to female age: Vienna consensus (≤39 years) and older female age(≥40 years). We measured a selection of the Vienna performance indicators and KPIs, with a focus on measures relevant to embryo cleavage and blastocyst formation. Assessment of fertilization, cleavage and blastocyst rates was carried out. To assess more comprehensively blastocyst quality and quantity, we estimated the total usable blastocyst rate(TBUR). Finally, blastocyst cryosurvival rate was assessed.
Main results and the role of chance
No differences were observed in fertilization and embryo cleavage KPIs between the Vienna consensus and the older female age groups (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). TBUR was lower in the older female age group (45.5% vs. 33.4% p < 0.001). This outcome decreased steadily with increasing female age. Clinical outcome significantly decreased with increasing female age. Implantation rate decreased from 34.3% in the Vienna consensus group to 16.9% in the older female age group (p<.001). Cumulative CPR showed a similar trend decreasing from 52.4% to 23.9% (p<.001). In the two populations, TBUR was further assessed after normalization of the number of retrieved oocytes. Rates were comparable in cycles with only few (1- 5 oocytes) collected oocytes (51.9% and 45.5%, p=ns). In cycles with 6-10, 11-15 and>15 collected oocytes TBUR was lower in older patient groups(p < 0.01). Univariate and multivariate logistic regression analysis showed female age as a factors independently associated with TBUR. Higher female age was associated with a reduced probability to achieve a TBUR greater than 40%.
Limitations, reasons for caution
The study design is retrospective and requires further refinement to control for factors that may impact clinical outcome
Wider implications of the findings
The study confirms the general validity of the Vienna Consensus but also indicate a need for fine-tuning in relation to factors intrinsic to gamete quality that can impact laboratory outcomes.
Trial registration number
Not applicable
Abstract
Study question
Does the duration of the interval between pronuclear fading and first cell division affect preimplantation embryo developmental competence?
Summary answer
Larger ...pronuclear-fading to first-division intervals (t2-tPNf) are associated with greater risk of direct unequal cleavage (DUC) and lower blastulation rate, but not of blastocyst aneuploidy.
What is known already
During the last decade, time lapse technology (TLT) has made possible detailed and dynamic observation of in vitro embryo development, offering a powerful non-invasive tool for improving embryo selection. Recent evidence suggests that the phase between pronuclear fading and the first cleavage is a perilous bridge between the zygote and the cleaving embryo. Following pronuclear breakdown, delayed progression through mitosis caused by incomplete DNA replication may result in chromosome lagging, whole and segmental segregation errors, and breakage. In this analysis, we specifically tested the hypothesis that delays in a short final phase of fertilization have an impact on embryo development.
Study design, size, duration
Retrospective study of 1316 zygotes cultured until day 5 using TLT and derived from first ICSI cycles (111 no PGT-A and 94 PGT-A) performed between 2018 and 2022. Testicular sperm or cryopreserved gametes cycles were excluded. DUC was defined as the cleavage of one zygote or blastomere into three daughter blastomeres or cell cycle interval shorter than five hours at the first (DUC1), second (DUC2) or third (DUC3) cell division cycle.
Participants/materials, setting, methods
t2-tPNf intervals were clustered into quartiles (Q1, <2hpi; Q2, 2–2.5hpi; Q3, 2.5–3hpi and Q4, > 3hpi), which were compared based on embryo development and live birth by linear and logistic regression. The Q1 cluster was set as control, while all other quartiles groups were considered as dummy variables. Multivariate analysis was conducted using the generalized estimating equations, to control for variable numbers of zygotes from each patient. The primary outcome was DUC rate.
Main results and the role of chance
Overall, rates of DUC1, DUC2 and DUC 3 rates were 10.3%, 8% and 2.9%, respectively. After adjusting for major confounders (maternal age, paternal age, maternal body mass index, cause of infertility, anti-Müllerian hormone, follicle stimulating hormone) we observed a higher DUC 1 and DUC 2 rate with increasing times of t2-tPNf. We observed abnormal distribution of DUC1 and DUC2 rate between quartiles groups. Specifically, 89% of DUC1 was shown among Q3 (21%) and Q4 (78%) and 61% of DUC2 among Q3 (22%) and Q4 (39%). The percentage of DUC1 was higher for Q3 (multivariate-OR = 3.29, 95%CI 0.99-10.85, adjusted-p = 0.01) and Q4 (multivariate-OR = 22.79, 95%CI 7.80-66.62, adjusted-p<0.01. The percentage of DUC2 was higher only in Q4 (multivariate-OR = 2.58, 95%CI 1.13-5-57, adjusted-p = 0.01). Moreover, we observed a reduced blastulation rate in Q3 (multivariate-OR = 0.68, 95%CI 0.49-0.95, adjusted-p = 0.01) and Q4 (multivariate-OR = 0.27, 95%CI 0.19-0.38, adjusted-p<0.01). No significant differences were observed in DUC3 rate, top quality blastocyst, euploidy rate (PGT-A cycles analysis) and live birth rate.
Limitations, reasons for caution
The retrospective single-centre design and the limited sample size are limitations of the study
Wider implications of the findings
The study is consistent with the emerging view that the final phases of fertilization - although short - are crucial for development. Delays in progression towards mitosis probably express chromosome integrity loss and perturbances of chromosome segregation. In most embryos, such perturbances appear to cause trichotomous mitoses and cleavage arrest.
Trial registration number
Not applicable
Numerous studies have shown the presence of DNA strand breaks in human ejaculated spermatozoa. The nature of this nuclear
anomaly and its relationship to patient etiology is however poorly ...understood. The aim of this study was to investigate the
relationship between nuclear DNA damage, assessed using the TUNEL assay and a number of key apoptotic markers, including Fas,
Bcl-x, and p53, in ejaculated human spermatozoa from men with normal and abnormal semen parameters. We also determined the
nature of the DNA damage by examining the percentage of ejaculated spermatozoa exhibiting DNA damage using the comet assay
and by challenging sperm chromatin to attack by micrococcal nuclease S7 and DNase I. We show that TUNEL positivity and apoptotic
markers do not always exist in unison; however, semen samples that had a low sperm concentration and poor morphology were
more likely to show high levels of TUNEL positivity and Fas and p53 expression. In addition, the DNA damage in ejaculated
human sperm is represented by both single- and double-stranded DNA breaks, and access to the DNA is restricted by the compacted
nature of ejaculated spermatozoa. This DNA protection is poorer in men with abnormal semen parameters. We propose that the
presence of DNA damage is not directly linked to an apoptotic process occurring in spermatozoa and arises due to problems
in the nuclear remodeling process. Subsequently, the presence of apoptotic proteins in ejaculated spermatozoa may be linked
to defects in cytoplasmic remodeling during the later stages of spermatogenesis.
We have carried out experiments to determine if human cervical mucus can act as an in vitro selective barrier against spermatozoa morphologically normal that carry genetic structural abnormalities.
...Sperm chromatin abnormalities have been evaluated by Chromomycin A3 and "endogenous" nick translation.
The data obtained have shown that spermatozoa possessing higher levels of DNA protamination are more proficient in crossing the cervical mucus barrier. Moreover, the levels of positivity to endogenous nick translation treatment was practically zero in such spermatozoa.
We suggest that sperm penetration of cervical mucus could be used to select sperm preparations free of fragmented DNA or chromatin structural abnormalities for assisted reproduction.
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