Abnormal cleavage up to Day 3 does not compromise live birth and neonatal outcomes of embryos that have achieved full blastulation: a retrospective cohort study.

STUDY QUESTION: Do embryos displaying abnormal cleavage (ABNCL) up to Day 3 have compromised live birth rates and neonatal outcomes if full blastulation has been achieved prior to transfer? SUMMARY ANSWER: ABNCL is associated with reduced full blastulation rates but does not impact live birth rates and neonatal outcomes once full blastulation has been achieved. WHAT IS KNOWN ALREADY?: It is widely accepted that ABNCL is associated with reduced implantation rates of embryos when transferred at the cleavage stage. However, evidence is scarce in the literature reporting birth outcomes from blastocysts arising from ABNCL embryos, likely because they are ranked low priority for transfer. STUDY DESIGN, SIZE, DURATION: This retrospective cohort study included 1562 consecutive autologous in vitro fertilization cycles (maternal age 35.1 ± 4.7 years) performed at Fertility North, Australia between January 2017 and June 2022. Fresh transfers were performed on Day 3 or 5, with remaining embryos cultured up to Day 6 before vitrification. A total of 6019 embryos were subject to blastocyst culture, and a subset of 664 resulting frozen blastocysts was included for live birth and neonatal outcome analyses following single transfers. PARTICIPANTS/MATERIALS, SETTING, METHODS: ABNCL events were annotated from the first mitotic division up to Day 3, including direct cleavage (DC), reverse cleavage (RC) and <6 intercellular contact points at the 4-cell stage (<6ICCP). For DC and RC in combination, the ratios of affected blastomeres over the total number of all blastomeres up to Day 3 were also recorded. All pregnancies were followed up until birth with gestational age, birthweight, and sex of the baby being recorded. MAIN RESULTS AND THE ROLE OF CHANCE: Full blastulation rates for embryos showing DC (19.5%), RC (41.7%), <6ICCP (58.8%), and mixed (≥2) ABNCL types (26.4%) were lower than the rates for those without ABNCL (67.2%, P < 0.01 respectively). Subgroup analysis showed declining full blastulation rates with increasing ratios of combined DC/RC affected blastomeres over all blastomeres up to the 8-cell stage (66.2% when 0 affected, 47.0% when 0.25 affected, 27.4% when 0.5 affected, 14.5% when 0.75 affected, and 7.7% when all affected, P < 0.01). However, once full blastulation had been achieved, no difference was detected between DC, RC, <6ICCP, and no ABNCL blastocysts following single frozen transfers in subsequent live birth rates (25.9%, 33.0%, 36.0% versus 30.8%, P > 0.05, respectively), gestational age (38.7 ± 1.6, 38.5 ± 1.2, 38.3 ± 3.5 versus 38.5 ± 1.8 weeks, P > 0.05, respectively) and birthweight (3343.0 ± 649.1, 3378.2 ± 538.4, 3352.6 ± 841.3 versus 3313.9 ± 509.6 g, P > 0.05, respectively). Multiple regression (logistic or linear as appropriate) confirmed no differences in all of the above measures after accounting for potential confounders. LIMITATIONS, REASONS FOR CAUTION: Our study is limited by its retrospective nature, making it impossible to control every known or unknown confounder. Embryos in our dataset, being surplus after selection for fresh transfer, may not represent the general embryo population. WIDER IMPLICATIONS OF THE FINDINGS: Our findings highlight the incremental impact of ABNCL, depending on the ratio of affected blastomeres up to Day 3, on subsequent full blastulation. The reassuring live birth and neonatal outcomes of ABNCL blastocysts imply a potential self-correction mechanism among those embryos reaching the blastocyst stage, which provides valuable guidance for clinical practice and patient counseling. STUDY FUNDING/COMPETTING INTEREST(S): This research is supported by an Australian Government Research Training Program (RTP) Scholarship. All authors report no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

Indirect markers of oocyte quality in patients with ovarian endometriosis undergoing IVF/ICSI: a systematic review and meta-analysis.

This systematic review and meta-analysis aimed to evaluate the impact of ovarian endometriomas (OMA) on indirect markers of oocyte quality in patients undergoing IVF, compared with women without anatomical or functional ovarian abnormalities. The search spanned original randomized controlled trials, case-control studies and cohort studies published in MEDLINE, the Cochrane Controlled Trials Register and the ClinicalTrials.gov database up to October 2023. Thirty-one studies were included in the meta-analysis, showing no significant differences in fertilization (OR 1.10, 95% CI 0.94-1.30), blastulation (OR 0.86, 95% CI 0.64-1.14) and cancellation (OR 1.06, 95% CI 0.78-1.44) rates. However, patients with OMA exhibited significantly lower numbers of total and mature (metaphase II) oocytes retrieved (mean difference -1.59, 95% CI -2.25 to -0.94; mean difference -1.86, 95% CI -2.46 to -1.26, respectively), and lower numbers of top-quality embryos (mean difference -0.49, 95% CI -0.92 to -0.06). The Ovarian Sensitivity Index was similar between the groups (mean difference -1.55, 95% CI -3.27 to 0.18). The lack of data published to date prevented meta-analysis on euploidy rate. In conclusion, although the presence of OMA could decrease the oocyte yield in patients undergoing IVF/intracytoplasmic sperm injection, it does not appear to have an adverse impact on oocyte quality.

The Blastulation Rate Is Negatively Associated With Euploid Rate.

OBJECTIVES: To study the association between the blastulation rate, the presence of 1 pronucleus (1PN) zygotes, and the ploidy of the cohort of blastocysts. METHODS: A cross-sectional study using the existing databases of 2 university fertility centres in Canada. We included 345 cycles from 235 couples who underwent next-generation sequencing preimplantation genetic testing for the detection of aneuploidy in the study. RESULTS: A total of 1456 blastocysts were biopsied. In multivariate analysis, only female age and the number of 1PN/2PN embryos showed a negative association with euploid ratio. Surprisingly, when the analysis was limited to cycles with no delayed blastulation, the blastulation rate was also negatively associated with the euploid ratio. CONCLUSIONS: This study sheds some light on the stages of early embryo development. Further study on the mechanisms governing embryo development and the different cell cycle checkpoints in embryo development is warranted.

Clinical outcome of different embryo transfer strategies after late rescue ICSI procedure: a 10-year total fertilisation failure cohort study.

BACKGROUND: Late rescue intracytoplasmic sperm injection (r-ICSI) has not been widely accepted as an alternative solution for unexpected total fertilisation failure (TFF) after in vitro fertilisation (IVF), due to the time-dependent in vitro deterioration of oocyte quality and endometrial growth not being synchronised with embryo development. This study aimed to evaluate the safety profile and effectiveness of freeze-all blastocyst transfer in combination with late r-ICSI. METHODS: This was a retrospective cohort study carried out at the Reproductive Centre of Peking University Third Hospital, Beijing, China. All participants received treatment between 2009 and 2019. 2,270 patients in the aggregate encountered unexpected TFF during 149,054 cycles of IVF and adopted a late r-ICSI procedure. Among these patients, 263 women did not have cleavage-stage embryos available for evaluation. The remaining patients were grouped according to different embryo transfer (ET) strategies (926 women in Group 1 underwent fresh ET, 365 women in Group 2 underwent freeze-all ET, 716 women in Group 3 experienced blastulation failure). Patients received different ET strategies after r-ICSI, with the main outcome measures included live birth rate (LBR), cumulative live birth rate (cLBR), and conservative cLBR. RESULTS: TFF occurred in 7.4% of all IVF cycles. Group 1 tended to be older at oocyte retrieval, with more infertile years, higher follicle-stimulating hormone (FSH) levels, higher gonadotropin consumption, and fewer oocytes retrieved. Group 2 exhibited considerably better LBRs following the first ET cycle (37.53% vs. 4.64%) and cLBRs (52.60% vs. 8.21%). After adjustment for covariates using binary logistic regression analyses, Group 2 still showed better obstetric performance in LBRs [OR:11.77, 95% CI (8.42-16.45)], cLBRs (OR:11.29, 95% CI (7.84-16.27)], and conservative cLBRs (OR:2.55, 95% CI (1.83-3.55)]. Additionally, the two groups showed similar miscarriage rates, whilst no new-borns with malformations or congenital diseases were reported. CONCLUSIONS: Freeze-all blastocyst stage ET serves as an optimal strategy to support late r-ICSI. However, for women with limited oocytes available for r-ICSI use, weighing the benefits against the costs of the procedure might be prudent before implementing in vitro blastulation.

Impact of the number of retrieved oocytes on IVF outcomes: oocyte maturation, fertilization, embryo quality and implantation rate.

The process of oocyte retrieval represents a key phase during the cycles of in vitro fertilization (IVF). It involves controlled ovarian stimulation to retrieve the highest number of oocytes possible. According to many previous studies, the higher the number of oocytes the higher the chances of obtaining embryos for multiple transfers. In this study, in total, 1987 patients were retrospectively reviewed to investigate the correlations between the number of retrieved oocytes and the subsequent IVF outcomes. Patients were divided into three groups according to the number of retrieved oocytes (Group 1: ≤5 oocytes; Group 2: 6-15 oocytes; Group 3: ≥15 oocytes). The results showed a significant negative correlation between oocyte number and maturation rate as well as fertilization rate. However, a significant positive correlation was found between oocyte number and the blastulation rate. The implantation rate after fresh embryo transfers was higher in group 2 (6-15 oocytes) compared with group 1 (≤5 oocytes). According to our findings, we conclude that oocyte numbers between 6 and 15 oocytes can result in the highest chances of positive IVF outcomes in terms of embryo quality and fresh embryo transfers with lower risks of ovarian hyperstimulation.

Delayed blastocyst development is influenced by the level of progesterone on the day of trigger.

PURPOSE: To evaluate the association between progesterone (P) level on the day of trigger and time to blastulation in IVF cycles. METHODS: This was a retrospective cohort study with autologous IVF cycles performed at our Institution from January 2019 to December 2021. A total of 1109 IVF cycles were included. The primary outcome was to compare time to blastulation in terms of percentage of expanded (grade 3) blastocysts on day 5 according to progesterone level at trigger. RESULTS: A total of 3517 blastocysts were analyzed. After dividing progesterone level in quartiles (Q1, P < 0.50 ng/ml; Q2 0.50 ng/ml ≤ P ≤ 0.78 ng/ml; Q3, 0.79 ng/ml ≤ P ≤ 1.15 ng/ml; Q4, P > 1.15 ng/ml), we observed a delay in blastocyst development according to the increasing level of progesterone at trigger (analysis by rank, P-value = 0.01). After adjusting for confounding factors at the multivariate analysis, the percentage of day 5 blastocysts was reduced for Q3 (- 13.8%, 95% CI from - 20.5 to - 7.0%, p < 0.001) and Q4 (- 7.7%, 95% CI from - 15.5 to 0.0%, p = 0.05) compared to Q1 (reference). CONCLUSIONS: Progesterone levels on day of trigger correlate to the percentage of expanded (grade 3) blastocysts on day 5 and a delayed blastocyst development day 5 is expected for high progesterone levels.

Does conventional morphological evaluation still play a role in predicting blastocyst formation?

BACKGROUND: Advanced models including time-lapse imaging and artificial intelligence technologies have been used to predict blastocyst formation. However, the conventional morphological evaluation of embryos is still widely used. The purpose of the present study was to evaluate the predictive power of conventional morphological evaluation regarding blastocyst formation. METHODS: Retrospective evaluation of data from 15,613 patients receiving blastocyst culture from January 2013 through December 2020 in our institution were reviewed. Generalized estimating equations (GEE) were used to establish the morphology-based model. To estimate whether including more features regarding patient characteristics and cycle parameters improve the predicting power, we also establish models including 27 more features with either LASSO regression or XGbosst. The predicted number of blastocyst were associated with the observed number of the blastocyst and were used to predict the blastocyst transfer cancellation either in fresh or frozen cycles. RESULTS: Based on early cleavage and routine observed morphological parameters (cell number, fragmentation, and symmetry), the GEE model predicted blastocyst formation with an AUC of 0.779(95%CI: 0.77-0.787) and an accuracy of 74.7%(95%CI: 73.9%-75.5%) in the validation set. LASSO regression model and XGboost model based on the combination of cycle characteristics and embryo morphology yielded similar predicting power with AUCs of 0.78(95%CI: 0.771-0.789) and 0.754(95%CI: 0.745-0.763), respectively. For per-cycle blastocyst yield, the predicted number of blastocysts using morphological parameters alone strongly correlated with observed blastocyst number (r = 0.897, P < 0.0001) and predicted blastocyst transfer cancel with an AUC of 0.926((95%CI: 0.911-0.94). CONCLUSION: The data suggested that routine morphology observation remained a feasible tool to support an informed decision regarding the day of transfer. However, models based on the combination of cycle characteristics and embryo morphology do not increase the predicting power significantly.

Age-specific blastocyst conversion rates in embryo cryopreservation cycles.

RESEARCH QUESTION: What is the blastocyst conversion rate in embryo cryopreservation cycles, per year of female age? DESIGN: Retrospective cohort study including patients undergoing their first ovarian stimulation cycle at our center with planned freeze-all strategy January 1st, 2014-June 30th, 2020. Primary outcome was blastocyst conversion rate. Secondary outcomes included mature oocyte and fertilization rates. Patients were stratified by year of age to assess oocyte yield and embryo development outcomes. RESULTS: 3,362 patients were included. The median blastocyst conversion rate in patients ≤30 was 66.7% (interquartile range 50.0-86.6) and remained statistically comparable through age 40 with a significant decline among ages ≥41 (41-years: marginal effect (ME) -5.2% (-9.7 to -0.7); 42-years: ME -9.6% (-14.3 to -4.8); 43-years: ME -7.7% (-12.8 to -2.6); ≥44-years: ME -20.8% (-26.5 to -15.1)). For the entire cohort, the median mature oocyte rate was 81.8% and the median fertilization rate was 81.8%. The mature oocyte and fertilization rates remained statistically comparable for each year of age except age ≥44 which had a statistically significantly increased mature oocyte rate (ME 4.4% (1.3 to 7.5)) and statistically significantly decreased fertilization rate (ME -5.8% (-9.8 to -1.9)) CONCLUSIONS: In embryo cryopreservation cycles, the blastocyst conversion rate remained statistically comparable through age 40 followed by a statistically significant decline for patients ≥41; however, the mature oocyte and fertilization rates were not impacted by increasing age until age ≥44. Even in women ≥44, over 40% of fertilized oocytes developed to blastocyst. Overall, this information is useful when counseling patients during the embryo culture stage regarding predicted blastocyst yield.

Prediction of embryo implantation rate using a sole parameter of timing of starting blastulation.

A time-lapse monitoring system provides a complete picture of the dynamic embryonic development process and simultaneously supplies extensive morphokinetic data. The objective of this study was to investigate whether the use of the morphokinetic parameter of time of starting blastulation (tSB) can improve the implantation rate of day-5 transferred blastocyst selected based on morphological parameters. In this retrospective study we analyzed the morphokinetics of 196 day-5 transferred blastocysts, selected solely based on morphological parameters. The interval time from intracytoplasmic sperm injection (ICSI) to time of starting blastocyst formation (tSB) was calculated for each embryo. The overall implantation rate of transferred blastocyst, selected based only on morphological parameters, was 49.2%. Implantation rate, determined retrospectively, was significantly higher (58.8% versus 42.6%, P = 0.02) for embryos with a short interval time to tSB (78-95.9 h) compared with embryos with a longer timeframe (96-114 h). Time of expanded blastocyst (tEB) post-ICSI was also significantly associated with implantation; however, this parameter was not available for all the embryos at time of transfer. When we tested only high ranked KIDScore day-3 sub-group embryos, the implantation rate was significantly higher in short interval time embryos compared with longer interval time embryos (62.2% vs. 45.5%, respectively, P = 0.02).These observations emphasize the importance of the timing of starting blastulation over blastocyst morphological parameters and may provide a preferable criterion for good morphology day-5 blastocyst selection.

Embryo blastulation and quality between days 5 and 6 of extended embryo culture.

PURPOSE: This study aims to know what proportion of culture day 5 pre-blastocyst-stage embryos develop into blastocysts by culture day 6 and what patient and cycle characteristics are associated with delayed blastocyst formation. METHODS: A retrospective observational cohort analysis was performed including a total of 9886 embryos from 1008 IVF cycles in 835 patients, who underwent treatment between January 1, 2016, and December 31, 2018. Autologous fresh in vitro fertilization (IVF) cycles at a single academic center were included in the analysis. Embryos were group-cultured using single-step culture media. Blastulation was defined as the presence of a new blastocyst. Usable blastulation was defined as the presence of a new good or excellent quality, expanded, hatching, or hatched blastocysts. RESULTS: The mean blastulation rate between days 5 and 6 of extended embryo culture was 30.9%. The mean percentage of embryos developing into usable blastocyst-stage embryos was 19.8%. The factors associated with blastulation on day 6 included the total number of embryos and the number of pre-blastocysts on day 5, as well as the use of ICSI. Age, the number of total embryos, those remained in culture and pre-blastocysts, as well as the blastulation rate on day 5 were associated with usable blastulation. CONCLUSION: It is important to know the usable blastocyst development rate between culture days 5 and 6 in order to adequately counsel patients debating whether to proceed with fresh ET on day 5 or forego ET with the expectation that embryos will be biopsied for PGT and/or cryopreserved on culture day 6. Our findings provide evidence to help guide patients in this difficult decision.

Suboptimal trophectoderm mitochondrial DNA level is associated with delayed blastocyst development.

PURPOSE: To provide a comprehensive analysis of mtDNA quantity in D5 and D6 blastocysts, as well as a further insight to the origin of delayed blastocyst development. METHODS: A retrospective cohort analysis of 829 D5 and 472 D6 blastocysts from 460 patients who underwent in vitro fertilization (IVF) with next-generation sequencing (NGS)-based preimplantation genetic testing for aneuploidy (PGT-A). The quantity of trophectoderm mtDNA was extrapolated from the NGS data, followed by the analysis of mean mtDNA levels between D5 and D6 blastocysts of the same ploidy (aneuploid/euploid) and transfer outcomes (positive/negative clinical pregnancy). RESULTS: D5 blastocysts had significantly higher euploidy rate and clinical pregnancy rate when compared with D6 blastocysts. The proportion of blastocysts derived from patients ≧ 40 years old were similar between the D5 and D6 cohorts. When blastocysts with identical ploidy were analyzed, the D5 cohorts all had significantly higher mean mtDNA levels than their D6 counterparts. Similarly, when embryo transfers with identical outcome were analyzed, the D5 cohorts also had significantly higher mean mtDNA levels than the D6 cohorts. Trophectoderm mtDNA level was independent of maternal age and blastocyst morphology grades. CONCLUSIONS: Our data provided further evidence D5 blastocysts contained significantly greater mtDNA quantity than D6 blastocysts, and mtDNA quantity could be a key factor that affects the development rate of blastocysts. Furthermore, one must avoid using an arbitrary threshold when incorporating mtDNA quantity into the embryo selection criteria, as the observed value may have vastly different clinical implication when blastulation rate is also considered.

Being on the side of old findings: progesterone elevation on the day of oocyte maturation induction does not affect embryological parameters throughout the blastocyst culture period.

PURPOSE: To investigate whether there is any detrimental effect of progesterone elevation (PE) on the day of oocyte maturation induction on embryological development potentials. METHODS: This retrospective single-center cohort study included a total of 1485 individual intracytoplasmic sperm injection (ICSI) cycles between January 2014 and December 2018. Serum progesterone (P) levels were measured on the day of oocyte maturation induction following the GnRH antagonist suppression protocol. Embryological parameters such as maturation, fertilization rate (FR), top-quality embryo (TQE) formation rate per 2PN on day 3, and excellent-quality blastocyst (EQB) formation rate per 2PN on day 5/6 were recorded. The inclusion criteria for women were an age ≤ 37 years, a BMI ≤ 30 kg/m2, and access to a total sperm concentration ≥ 2 million. Groups were stratified according to the serum P levels using the cut-off levels of < 0.8 ng/ml; 0.8-1.49 ng/ml; and ≥ 1.5 ng/ml. RESULTS: Peak E2 level and total number of oocytes retrieved were significantly related to PE (p < 0.001). FR did not display a significance difference between groups (p = 0.108). The TQE and the blastulation rates were not affected by PE (p = 0.82 and p = 0.68, respectively). Chi square analysis revealed a significant relationship between PE and the EQB formation rate (p = 0.01). GEE analysis failed to present any statistical significance regarding the effect of PE on neither the TQE nor the EQB formation rates per 2PN [OR 1.07; 95% (0.98-1.16) p = 0.113 and OR 0.93; 95% (0.80-1.07) p = 0.32, respectively]. CONCLUSIONS: In accordance with previously published papers, our study could not find any detrimental effect of PE on embryological outcomes throughout the blastocyst culture period.

Reproductive outcomes with donor sperm in couples with severe male-factor infertility after intracytoplasmic sperm injection failures.

PURPOSE: To evaluate reproductive outcomes of artificial insemination and IVF with donor sperm (AID or IVF-D) for male-factor couples with a history of unsuccessful ICSI attempt. METHODS: This retrospective cohort includes couples with severe male-factor infertility who failed ICSI treatment, and subsequently underwent semen donation treatment. We report the following outcomes: (1) live birth rates in AID and IVF-D treatment for couples with severe male infertility factors and prior ICSI failures; (2) paternal impact on embryo development of the same oocyte cohort; (3) prognostic factors in obtaining a live birth with donor semen. RESULTS: Of 92 women with failed ICSI cycles (26 with multiple attempts), 45 couples underwent AID treatment. Live birth rate per cycle of AID was 18.9%. Fifty-three patients underwent IVF-D including 6 couples who previously did not conceive with AID. Embryological outcomes including fertilization, viable cleavage embryos, and blastocyst formation rates were significantly lower in ICSI cycles with partner sperm compared with IVF-D (P < 0.01). Logistic regression analysis showed that female age and the severity of spermatogenetic disorder are prognostic factors in obtaining a live birth with donated sperm. CONCLUSION: Couples with severe male infertility factor (azoospermia or extreme oligoasthenospermia) and a history of unsuccessful ICSI cycles benefit from treating with donor sperm. ICSI fertilization, embryo viability, and progression of the embryo to the blastocyst stage are significantly deteriorated by semen parameters. The prognostic factors identified may help couples plan their treatment and prepare for their parenthood journey.

Is intracytoplasmic sperm (ICSI) better than traditional in vitro fertilization (IVF): confirmation of higher blastocyst rates per oocyte using a split insemination design.

PURPOSE: To explore the effects of traditional vs. intracytoplasmic sperm injection (ICSI) insemination method on the outcome of high-quality blastocyst development in a split sibling oocyte cohort. METHODS: In this retrospective cohort study, we analyzed 62 ICSI/IVF split cycles. Sibling oocytes were randomly assigned to ICSI or IVF insemination. Two hundred thirty-four ICSI-only cycles and 152 IVF-only cycles were also analyzed for comparison. Blastocysts were graded by Gardner's embryo grading and were considered a high-quality blastocyst if 3BB or better (Gardner 1999). RESULTS: In the ICSI/IVF split group, (1) ICSI oocytes had a higher fertilization rate per oocyte allocated (73% vs 62%, p < 0.001), (2) more high-quality day 2 embryos (69% vs 55%, p < 0.005), (3) ICSI oocytes had a lower blastulation rate per 2PN (46% vs 54%, p < 0.05), but a higher blastulation rate when calculated per oocyte allocated (40% vs 32%, p < 0.05). The ICSI-only group had a lower fertilization rate (65% vs 70%, p < 0.001) but more high-quality day 2 embryos in comparison to the IVF-only group (68% vs 64%, p < .05). The total high-quality blastulation rate was higher for the IVF-only group per 2PN (49% vs 43%, p < 0.05) and per oocyte retrieved (34% vs 28%, p < 0.05). CONCLUSIONS: This distinctive IVF/ICSI sibling oocyte split design demonstrated a higher-quality blastulation rate in the IVF group compared to the ICSI group when calculated per 2PN, but not per oocyte allocated to each insemination procedure.

The impact of follicle-flushing during oocyte collection on embryo development of in-vitro fertilization.

BACKGROUND: To evaluate the impact of follicle-flushing during oocyte collection on embryo development potential retrospectively. METHODS: A total of 1714 cases, including 133 who experienced retrieval difficulty (repeated follicle-flushing) on the day of oocyte retrieval (difficulty group) and the control 1581 cases (control group), were assessed in this retrospective study. The number of oocytes recovered, two pro-nuclei fertilization (2PN-fertilization), day 3 good-quality embryo and day 5/6 blastocyst utilization rates were compared between the difficulty group and control group correspondingly. Embryo implantation, clinical pregnancy and neonatal outcomes were further analyzed between the two groups in the fresh day- 3 embryo transfer cycles. RESULTS: The number of oocytes recovered in the difficulty group (9.08 ± 4.65) were significantly reduced compared with the control group (12.13 ± 5.27),P < 0.001; The 2PN-fertilization, day 3 good-quality embryo and blastocyst utilization rates were significantly lower in the difficulty group compared with controls (71.7% vs. 75.7%; 52.7% vs. 56.5%; 31.9% vs. 37.0%, all P < 0.05). Embryo implantation in the difficulty group was 53.2%, which was lower than the control value of 58.7%, although not reaching statistical significance. The rate of fresh embryo transfer cycles in the difficulty group was lower than normal ones (51.88% vs. 61.99%, P = 0.026). The pregnancy and live birth rates were similar between the two groups. But the rate of spontaneous miscarriages of the difficulty group was higher than the control group, although not reaching statistical significance. The neonatal outcomes had no statistical difference between the two groups. CONCLUSIONS: Oocyte retrieval difficulty, which include repeated flushing and the corresponded extending time required for oocyte recovery, significantly reduced day 3 good-quality embryo and blastocyst utilization rates of these patients. But the live birth rate had no difference between the difficulty group and the normal ones.

Triggering method in assisted reproduction alters the cumulus cell transcriptome.

RESEARCH QUESTION: How does the choice of triggering final oocyte maturation affect the cumulus cell transcriptome? DESIGN: Sixty patients undergoing gonadotrophin-releasing hormone antagonist (GnRH-ant) IVF cycles were recruited for this nested case-control study. Patients were stratified into three subgroups based on their ovarian reserve (high, normal and low). Triggering final oocyte maturation was accomplished by either single trigger (with human chorionic gonadotrophin [HCG] only or gonadotrophin-releasing hormone agonist [GnRH-ag] only) or dual trigger combining HCG and GnRH-ag. The choice of trigger was at the discretion of the treating physician. Within each group patients receiving a dual trigger were matched by demographic and pre-stimulation parameters with patients receiving a single trigger. The matching was performed to minimize the biological variability within each subgroup. Thirty patients were included in the final analysis. Cumulus cells were stripped away from the retrieved oocytes. Cumulus cells from three sibling oocytes were pooled, the RNA extracted and libraries prepared. Next-generation sequencing was performed on all samples. RESULTS: Dual triggering supports key ovarian pathways of oocyte maturation and extracellular matrix remodelling, while attenuating vasculo-endothelial growth and providing antioxidant protection to the growing follicles. CONCLUSIONS: This is the first study to delineate key transcriptomic changes under dual triggering of final oocyte maturation, across different patient populations. The findings underline the need for larger-scale studies validating transcriptomic effects of methods for triggering final oocyte maturation. Furthermore, there is a need for large-scale clinical randomized controlled studies to relate the findings of this study with clinical outcomes.

Is it worth it to cryopreserve embryos with blastulation delay at day 5?

This is a retrospective study over a 5-year period. In total, 3139 embryos were individually cryopreserved (Cryotop®) and warmed using the Kitazato vitrification/warming kit. They were classified into three categories based on their expansion degree. Transfer, implantation and pregnancy rates were assessed for each embryo category and compared using SPSS (Statistical Package for the Social Sciences) software. In total, 1139 couples enrolled in infertility treatment programme benefitted from embryo vitrification at day 5. After warming, embryos belonging to the three categories showed similar success rates. Although there was a trend towards better outcomes when grade 3 embryos were transferred, the differences did not reach statistical significance: implantation rates (n fetal sac/n embryo transferred) grade 1: 21.9%, grade 2: 22.7% and grade 3: 30.3% (=0.19). Pregnancy rate (n clinical pregnancy/n transfer) (21.9%, 22.7%, 30.3%, respectively; P=0.11). Miscarriage rate was not statistically different in the three categories (14.5%, 20.4%, 20%, respectively, P=0.51). Our overall results show that it is worth vitrifying slow kinetics embryos as they provide a non-negligible chance to give rise to a pregnancy.

Using time-lapse technology to explore vacuolization in embryos on Day 3 and Day 4.

PURPOSE: To investigate the occurrence and development state of embryo vacuoles between the 8-cell and morula stages, and to explore how vacuoles affected the development of embryos. METHODS: A retrospective study of a cohort of 422 patients undergoing conventional in vitro fertilization or intracytoplasmic sperm injection. With the help of time-lapse imaging, the development processes and outcomes of good quality embryos with or without vacuoles were analyzed. RESULTS: Vacuole positive embryos had significantly lower blastulation rate and good quality blastulation rate than vacuole negative embryos, p < 0.05. Compared to vacuole negative embryos, the number of best and good quality blastocysts was significantly reduced, while the number of fair and discarded ones was significantly increased, p < 0.05. The average starting time of vacuolization was 73.7 ± 9.3 h after insemination. The proportion of blastomeres affected by vacuoles was associated with embryonic developmental potential. CONCLUSIONS: Vacuolization on Day 3 and Day 4 was frequently observed and was detrimental to embryo development. The proportion of blastomeres affected by vacuoles may be an indicator of embryo developmental potential.

Abnormal sperm concentration and motility as well as advanced paternal age compromise early embryonic development but not pregnancy outcomes: a retrospective study of 1266 ICSI cycles.

PURPOSE: To investigate the effect of sperm concentration, motility and advanced paternal age on reproductive outcomes. METHODS: A retrospective analysis of 1266 intracytoplasmic sperm injection (ICSI) cycles between 2013 and 2017. The cohort was divided into four groups according to semen concentration based on the WHO criteria (2010): group A (conc. <1 M/ml), group B (1 ≤ conc. <5 M/ml), group C (5 ≤ conc. < 15 M/ml) and the control group D (conc. ≥15 M/ml). The primary outcome investigated was the blastulation rate. Secondary outcomes were fertilization rate, top quality blastocyst formation rate and ongoing pregnancy rate. RESULTS: After adjustment for maternal age and number of oocytes recovered, a significant difference was observed between group A and group D on the rate of fertilized oocytes [66.7 (40.0-80.0) vs 75.0 (57.1-90.2), adjusted p < 0.001] and the blastocyst formation rate [50.0 (33.3-66.3) vs 55.6 (40.0-75.0), adjusted p < 0.05]. However, the male factor did not affect the top quality blastocyst formation rate nor the ongoing pregnancy rate. Considering the age of the male partner as confounding factor, at the increase of each year of age, a reduction of 0.3% on the fertilization rate was observed but no other outcome was impacted. A negative correlation was also observed between sperm motility and fertilization rate in the group with a motility <5%. CONCLUSION: Male factor infertility and advanced paternal age may compromise fertilization and blastulation rates but not top quality blastocyst formation rate or the establishment of pregnancy in ICSI cycles.

Duration of blastulation may be associated with ongoing pregnancy rate in single euploid blastocyst transfer cycles.

Not all euploid embryos implant, necessitating additional tools to select viable blastocysts in preimplantation genetic screening cycles. In this retrospective cohort study, 129 consecutive patients who underwent 129 single euploid blastocyst transfers in cryopreserved embryo transfer cycles were included. All embryos were individually cultured in a time-lapse incubator from intracytoplasmic sperm injection up to trophoectoderm biopsy. Twenty-three time-lapse morphokinetic variables were tested among patients with (n = 68) or without (n = 61) ongoing pregnancy. All 23 time-lapse morphokinetic variables, apart from duration of blastulation (tB-tSB), were comparable between patients with or without ongoing pregnancy. Duration of blastulation was significantly shorter in patients with ongoing pregnancy (8.1 ± 3.2 versus 9.5 ± 3.4 h; P = 0.014); shorter duration of blastulation remained an independent predictor for ongoing pregnancy, when tested by logistic regression analysis (OR 0.81; 95% CI 0.70 to 0.93). One important limitation of this study, and a reason for caution, is the use of multiple comparisons, which can lead to differences at the 0.05 level simply by chance or random variation. Nonetheless, the study suggests that when more than one euploid blastocyst is available, priority might be given to those with a shorter duration of blastulation.