Circadian serum progesterone variations on the day of frozen embryo transfer in a modified natural cycle protocol.

STUDY QUESTION: Is there a circadian variation of serum progesterone (P) on the day of frozen embryo transfer (FET) in a modified natural cycle (mNC)? SUMMARY ANSWER: There is a statistically significant diurnal variation of serum P on the day of a FET in an mNC protocol. WHAT IS KNOWN ALREADY: In recent years, the proportion of FET cycles has increased dramatically. To further optimize pregnancy outcomes after FET, recent studies have focused on serum luteal P levels in both natural and artificially prepared FET cycles. Despite the different cut-off values proposed to define low serum P in the NC, it is generally accepted that lower serum P values (<10 ng/ml) around the day of FET are associated with negative reproductive outcomes. However, a single serum P measurement is not reliable given that P levels are prone to diurnal fluctuations and are impacted by patients' characteristics. STUDY DESIGN, SIZE, DURATION: A prospective cohort study was conducted in a single university-affiliated fertility center, including 22 patients performing a single blastocyst mNC-FET from August 2022 to August 2023. Serum P levels were measured on the day of transfer at 08:00h, 12:00h, 16:00h, and 20:00h. Differences between P levels were compared using the Wilcoxon signed-rank test. The sample size was calculated to detect a difference of 15% between the first and last P measurements with a 5% false-positive rate and a 95% CI. PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients with a normal BMI, between 18 and 40 years old, without uterine diseases were eligible. Patients utilizing donated oocytes were excluded. The mNC-FET protocol involved monitoring the normal ovarian cycle and triggering ovulation with an injection of 250 µg of choriogonadotropin alfa when a pre-ovulatory follicle (16-20 mm diameter) was visualized. The blastocyst was transferred seven days later. The patients were not supplemented with exogenous P at any time before the day of the FET. MAIN RESULTS AND THE ROLE OF CHANCE: The mean age and BMI of the study population were 33.6 ± 3.8 years and 22.7 ± 1.8 kg/m2, respectively. Mean P values at 08:00h, 12:00h, 16:00h, and 20:00h were 14.6 ± 4.5, 14.7 ± 4.1, 12.9 ± 3.5, and 14.6 ± 4.3 ng/ml, respectively. The mean P levels at 16:00h were significantly lower compared to all other time points (P < 0.05: P = 0.007 between P at 8:00h and 16:00h; P = 0.003 between P at 12:00h and 16:00h; P = 0.007 between P at 16:00h and 20:00h). No statistically significant difference was observed between P values at the other time points (P > 0.05: P = 0.88 between P at 8:00h and 12:00h; P = 0.96 between P at 8:00h and 20:00h; P = 0.83 between P at 12:00h and 20:00h). LIMITATIONS, REASONS FOR CAUTION: The study's limitations include the small sample size that may cause a bias when the results are extrapolated to a larger subfertile population undergoing mNC-FET. Ideally, larger prospective trials including a more heterogeneous patient population would be necessary to validate our findings. WIDER IMPLICATIONS OF THE FINDINGS: The current study demonstrates the existence of a diurnal fluctuation of serum P on the day of mNC-FET highlighting the importance of a standardized time point for its measurement. This is especially important for considering clinical actions, such as additional exogenous P supplementation, when encountering P values lower than 10 ng/ml on the day of FET. STUDY FUNDING/COMPETING INTEREST(S): No funding was obtained for the study. The authors have no conflicts of interest to declare regarding the content of the study. TRIAL REGISTRATION NUMBER: NCT05511272.

Progestin primed ovarian stimulation using dydrogesterone from day 7 of the cycle onwards in oocyte donation cycles: a longitudinal study.

RESEARCH QUESTION: Does a progestin-primed ovarian stimulation (PPOS) protocol with dydrogesterone from cycle day 7 yield similar outcomes compared with a gonadotrophin-releasing hormone (GnRH) antagonist protocol in the same oocyte donors? DESIGN: This retrospective longitudinal study included 128 cycles from 64 oocyte donors. All oocyte donors had the same type of gonadotrophin and daily dose in both stimulation cycles. The primary outcome was the number of cumulus-oocyte complexes (COC) retrieved. RESULTS: The number of COC retrieved (mean ± SD 19.7 ± 10.8 versus 19.2 ± 8.3; P = 0.5) and the number of metaphase II oocytes (15.5 ± 8.4 versus 16.2 ± 7.0; P = 0.19) were similar for the PPOS and GnRH antagonist protocols, respectively. The duration of stimulation (10.5 ± 1.5 days versus 10.8 ± 1.5 days; P = 0.14) and consumption of gonadotrophins (2271.9 ± 429.7 IU versus 2321.5 ± 403.4 IU; P = 0.2) were also comparable, without any cases of premature ovulation. Nevertheless, there was a significant difference in the total cost of medication per cycle: €898.3 ± 169.9 for the PPOS protocol versus €1196.4 ± 207.5 (P < 0.001) for the GnRH antagonist protocol. CONCLUSION: The number of oocytes retrieved and number of metaphase II oocytes were comparable in both stimulation protocols, with the advantage of significant cost reduction in favour of the PPOS protocol compared with the GnRH antagonist protocol. No cases of premature ovulation were observed, even when progestin was started later in the stimulation.

A 10-year follow-up of reproductive outcomes in women attempting motherhood after elective oocyte cryopreservation.

STUDY QUESTION: Which reproductive treatment outcomes are observed in women who underwent elective oocyte cryopreservation (EOC) and who returned to the clinic with a desire for a child? SUMMARY ANSWER: Whether to warm oocytes or to first use fresh own oocytes for ART depends on age upon returning, but both strategies result in favorable reproductive outcomes. WHAT IS KNOWN ALREADY: Most affluent countries have observed a trend toward postponement of childbearing, and EOC is increasingly used based on the assumption that oocytes cryopreserved at a younger age may extend a woman's reproductive lifespan and mitigate her age-related fertility decline. Although most follow-up studies after EOC have focused on women who requested oocyte warming, a substantial proportion of women who do not conceive naturally will embark on fertility treatment without using their cryopreserved oocytes. Reports on reproductive outcomes in past EOC users are scarce, and the lack of reproductive treatment algorithms in this group of women hampers counseling toward the most efficient clinical strategy. STUDY DESIGN, SIZE, DURATION: This retrospective observational single-center study encompasses 843 women who had elective oocyte vitrification between 2009 and 2019 at our fertility clinic. Women who underwent fertility preservation for medical or oncological reasons were excluded. This study describes the outcomes of the diverse reproductive treatment strategies performed until May 2022 in women returning to our clinic to attempt motherhood. PARTICIPANTS/MATERIALS, SETTING, METHODS: Using descriptive statistics, patient characteristics and data of ovarian stimulation (OS) of EOC cycles were analyzed, as well as data related to OS and laboratory data of ART in women who pursued fertility treatment with and/or without using their cryopreserved oocytes. The primary outcome was live birth rate (LBR) per patient after oocyte warming and after ART using fresh oocytes. Secondary outcomes were return rate, utilization rate of the cryopreserved oocytes, laboratory outcomes upon return, and LBR per embryo transfer. A multivariable regression model was developed to identify factors associated with the decision to thaw oocytes as the primary strategy and factors associated with ongoing pregnancy upon return to the clinic. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 1353 EOC cycles (mean ± SD, 1.6 ± 0.9 per patient) were performed. At the time of EOC, the mean age was 36.5 ± 2.8 years, mean anti-Müllerian hormone (AMH) was 2.3 ± 2.0 ng/ml, and 174 (20.6%) women had a partner. On average, 13.9 ± 9.2 mature oocytes were cryopreserved. Two hundred thirty-one (27.4%) women returned to the clinic, an average of 39.9 ± 23.4 months after EOC. Upon returning, their mean age was 40.4 ± 3.1 years, mean AMH was 1.5 ± 1.5 ng/ml, and 158/231 (68.3%) patients had a partner. As a primary approach, 110/231 (47.6%) past EOC users embarked on oocyte warming, 50/231 (21.6%) had intrauterine insemination, and 71/231 (30.7%) had ART using fresh own oocytes. Cumulative LBR (CLBR) was 45.9% (106/231) notwithstanding a miscarriage rate (MR) of 30.7% (51/166) in the entire cohort. In total, 141 women performed oocyte warming at some stage in their treatment trajectory. A subset of 90/231 (39.0%) patients exclusively had oocyte warming (41.6 ± 3.0 years, with 10.0 ± 5.2 oocytes warmed per patient). 52/231 (22.5%) patients exclusively had ART using fresh own oocytes (mean age of 39.0 ± 2.8 years, with 9.9 ± 7.4 mature oocytes retrieved per patient). CLBR was 37/90 (41.1%) in the oocyte warming-only group and 25/52 (48.1%) in the OS-only group. MR/transfer was 25.0% and 29.3% in the oocyte warming-only group and the OS-only group, respectively. LIMITATIONS, REASONS FOR CAUTION: Both sample size and the retrospective design are limitations of this study. The decision to embark on a specific reproductive treatment strategy was based on patient preference, after counseling on their treatment options. This precludes direct comparison of the efficiency of reproductive treatment options in past EOC users in this study. WIDER IMPLICATIONS OF THE FINDINGS: Reporting on clinical outcomes of women who underwent EOC and returned to the clinic to embark on divergent reproductive treatment strategies is mandatory to establish guidelines for best clinical practice in this growing patient population. STUDY FUNDING/COMPETING INTEREST(S): None. TRIAL REGISTRATION NUMBER: N/A.

Enzymatic tissue processing after testicular biopsy in non-obstructive azoospermia enhances sperm retrieval.

STUDY QUESTION: What is the added value of enzymatic processing of testicular biopsies on testicular sperm retrieval (SR) rates for patients with non-obstructive azoospermia (NOA)? SUMMARY ANSWER: In addition to mechanical mincing, enzymatic digestion increased SR rates in testicular biopsies of NOA patients. WHAT IS KNOWN ALREADY: Many studies focus on the surgical approach to optimize recovery of testicular sperm in NOA, and in spite of that, controversy still exists about whether the type of surgery makes any difference as long as multiple biopsies are taken. Few studies, however, focus on the role of the IVF laboratory and the benefit of additional lab procedures, e.g. enzymatic digestion, in order to optimize SR rates. STUDY DESIGN SIZE DURATION: This retrospective single-center cohort study included all patients who underwent their first testicular sperm extraction (TESE) by open multiple-biopsy method between January 2004 and July 2022. Only patients with a normal karyotype, absence of Y-q deletions and a diagnosis of NOA based on histology were included. The primary outcome was SR rate after mincing and/or enzymes. The secondary outcome was cumulative live birth (CLB) after ICSI with fresh TESE and subsequent ICSI cycles with frozen TESE. PARTICIPANTS/MATERIALS SETTING METHODS: Multiple biopsies were obtained from the testis, unilaterally or bilaterally, on the day of oocyte retrieval. Upon mechanical mincing, biopsies were investigated for 30 min; when no or insufficient numbers of spermatozoa were observed, enzymatic treatment was performed using Collagenase type IV. Multivariable regression analysis was performed to predict CLB per TESE by adjusting for the following confounding factors: male FSH level, female age, and requirement of enzymatic digestion to find sperm. MAIN RESULTS AND THE ROLE OF CHANCE: We included 118 patients, of whom 72 (61.0%) had successful SR eventually. Spermatozoa were retrieved after mechanical mincing for 28 patients (23.7%; 28/118) or after additional enzymatic digestion for another 44 patients (37.2%; 44/118). Thus, of the 90 patients requiring enzymatic digestion, sperm were retrieved for 44 (48.9%). Male characteristics were not different between patients with SR after mincing or enzymatic digestion, in regard to mean age (34.5 vs 34.5 years), testis volume (10.2 vs 10.6 ml), FSH (17.8 vs 16.9 IU/l), cryptorchidism (21.4 vs 34.1%), varicocele (3.6 vs 4.6%), or histological diagnosis (Sertoli-cell only 53.6 vs 47.7%, maturation arrest 21.4 vs 38.6%, sclerosis/atrophy 25.0 vs 13.6%).Of the 72 patients with sperm available for ICSI, 23/72 (31.9%) achieved a live birth (LB) after the injection with fresh testicular sperm (and fresh or frozen embryo transfers). Of the remaining 49 patients without LB, 34 (69.4%) had supernumerary testicular sperm frozen. Of these 34 patients, 19 (55.9%) continued ICSI with frozen testicular sperm, and 9/19 (47.4%) had achieved an LB after ICSI with frozen testicular sperm. Thus, the total CLB was 32/118 (27.1%) per TESE or 32/72 (44.4%) per TESE with sperm retrieved.Of the female characteristics (couples with sperm available), only female age (30.3 vs 32.7 years; P = 0.042) was significantly lower in the group with a LB, compared to those without.The CLB with testicular sperm obtained after enzymatic digestion was 31.8% (14/44), while the CLB with sperm obtained after mincing alone was 64.3% (18/28). Multivariable logistic regression analysis showed that when enzymatic digestion was required, it was associated with a significant decrease in CLB per TESE (OR: 0.23 (0.08-0.7); P = 0.01). LIMITATIONS REASONS FOR CAUTION: Limitations of the study are related to the retrospective design. However, the selection of only patients with NOA, and specific characteristics (normal karyotype and absence Y-q deletion) and having their first TESE, strengthens our findings. WIDER IMPLICATIONS OF THE FINDINGS: Enzymatic processing increases the SR rate from testicular biopsies of NOA patients compared to mechanical mincing only, demonstrating the importance of an appropriate laboratory protocol. However, NOA patients should be counseled that when sperm have been found after enzymatic digestion, their chances to father a genetically own child may be lower compared to those not requiring enzymatic digestion. STUDY FUNDING/COMPETING INTERESTS: None reported. TRIAL REGISTRATION NUMBER: N/A.

How to identify patients who would benefit from delayed-matured oocytes insemination: a sibling oocyte and ploidy outcome study.

STUDY QUESTION: Which patients might benefit from insemination of delayed-matured oocytes? SUMMARY ANSWER: Delayed-matured oocytes had a ≥50% contribution to the available cohort of biopsied blastocysts in patients with advanced maternal age, low maturation, and/or low fertilization rates. WHAT IS KNOWN ALREADY: Retrieved immature oocytes that progress to the MII stage in vitro could increase the number of embryos available during ICSI cycles. However, these delayed-matured oocytes are associated with lower fertilization rates and compromised embryo quality. Data on the ploidy of these embryos are controversial, but studies failed to compare euploidy rates of embryos derived from delayed-matured oocytes to patients' own immediate mature sibling oocytes. This strategy efficiently allows to identify the patient population that would benefit from this approach. STUDY DESIGN, SIZE, DURATION: This observational study was performed between January 2019 and June 2021 including a total of 5449 cumulus oocytes complexes from 469 ovarian stimulation cycles, from which 3455 inseminated matured oocytes from ICSI (n = 2911) and IVF (n = 544) were considered as the sibling controls (MII-D0) to the delayed-matured oocytes (MII-D1) (n = 910). Euploidy rates were assessed between delayed-matured (MII-D1) and mature sibling oocytes (MII-D0) in relation to patients' clinical characteristics such as BMI, AMH, age, sperm origin, and the laboratory outcomes, maturation, fertilization, and blastocyst utilization rates. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 390 patients undergoing IVF/ICSI, who had at least one metaphase I (MI) or germinal-vesicle (GV) oocyte on the day of oocyte collection (Day 0), which matured in 20-28 h after denudation were included. MI and GV oocytes that matured overnight were inseminated on the following day (Day 1, MII-D1) by ICSI. Only cycles planned for preimplantation genetic testing for aneuploidy using fresh own oocytes were included. MAIN RESULTS AND THE ROLE OF CHANCE: Fertilization (FR) and blastocyst utilization rates were significantly higher for MII-D0 compared to delayed-matured oocytes (MII-D1) (69.5% versus 55.9%, P < 0.001; and 59.5% versus 18.5%, P < 0.001, respectively). However, no significant difference was observed in the rate of euploid embryos between MII-D0 and MII-D1 (46.3% versus 39.0%, P = 0.163). For evaluation of the benefit of inseminating MI/GV oocytes on D1 per cycle in relation to the total number of biopsied embryos, cycles were split into three groups based on the proportion of MII-D1 embryos that were biopsied in that cycle (0%, 1-50%, and ≥50%). The results demonstrate that patients who had ≥50% contribution of delayed-matured oocytes to the available cohort of biopsied embryos were those of advanced maternal age (mean age 37.7 years), <10 oocytes retrieved presenting <34% maturation rate, and <60% fertilization rate. Every MII oocyte injected next day significantly increased the chances of obtaining a euploid embryo [odds ratio (OR) = 1.83, CI: 1.50-2.24, P < 0.001] among MII-D1. The odds of enhanced euploidy were slightly higher among the MII-D1-GV matured group (OR = 1.78, CI: 1.42-2.22, P < 0.001) than the MII-D1-MI matured group (OR = 1.54, CI: 1.25-1.89, P < 0.001). Inseminating at least eight MII-D1 would have >50% probability of getting a euploid embryo among the MII-D1 group. LIMITATIONS, REASONS FOR CAUTION: ICSI of MII-D1 was performed with the fresh or frozen ejaculates or testicular samples from the previous day. The exact timing of polar body extrusion of delayed-matured MI/GV was not identified. Furthermore, the time point of the final oocyte maturation to MII for the immature oocytes and for the oocytes inseminated by IVF could not be identified. WIDER IMPLICATIONS OF THE FINDINGS: The results of this study might provide guidance to the IVF laboratories for targeting the patient's population who would benefit from MII-D1 ICSI without adhering to unnecessary costs and workload. STUDY FUNDING/COMPETING INTEREST(S): No external funding was received for this study. There are no conflicts of interest to be declared for any of the authors. There are no patents, products in development, or marketed products to declare. TRIAL REGISTRATION NUMBER: N/A.

Sibling oocytes cultured in a time-lapse versus benchtop incubator: how time-lapse incubators improve blastocyst development and euploid rate.

The aim was to study whether a limited exposure of embryos outside the incubator has an effect on embryo development, blastocyst quality and euploid outcomes. This retrospective study was performed at ART Fertility Clinics, Abu Dhabi, United Arab Emirates (UAE) between March 2018 and April 2020 and included 796 mature sibling oocytes that were split randomly between two incubators after intracytoplasmic sperm injection (ICSI): an EmbryoScope™ (ES) incubator and a benchtop incubator, G185 K-SYSTEMS (KS). The fertilization, cleavage, embryo/blastocyst qualities, useable blastocyst and euploid rates were assessed to evaluate the incubator performance. In total, 503 (63.2%) mature oocytes were cultured in the EmbryoScope and 293 (36.8%) in the K-SYSTEMS. No differences were observed in fertilization rate (79.3% vs 78.8%, P = 0.932), cleavage rate (98.5% vs 99.1%, P = 0.676) and embryo quality on Day 3 (P = 0.543) between both incubators, respectively. Embryos cultured in the EmbryoScope, had a significantly higher chance of being biopsied (64.8% vs 49.6%, P < 0.001). Moreover, a significantly higher blastocyst biopsy rate was observed on Day 5 in the EmbryoScope (67.8% vs 57.0%, P = 0.037), with a highly significant increased euploid rate (63.5% vs 37.4%, P = 0.001) and improved blastocyst quality (P = 0.008). We found that exposure of embryos outside the incubator may negatively affect the in vitro blastocyst development and euploid rate on Day 5.

Antimüllerian hormone (AMH) and age as predictors of preimplantation genetic testing for aneuploidies (PGT-A) cycle outcomes and blastocyst quality on day 5 in women undergoing in vitro fertilization (IVF).

PURPOSE: The objective of this study was to investigate whether women with diminished ovarian reserve who planned for PGT-A exhibit a lower number of blastocysts for biopsy, ploidy outcomes, and blastocyst quality on day 5, regardless of age. METHODS: A retrospective analysis was performed between March 2017 and July 2020 at ART Fertility Clinics Abu Dhabi, including couples that were triggered for final oocyte maturation in an ovarian stimulated cycle planned for PGT-A. Patients were stratified into four AMH groups: < 0.65 ng/ml, 0.65-1.29 ng/ml, 1.3-6.25 ng/ml, and > 6.25 ng/ml; four age categories: ≤ 30, 31-35, 36-40, and > 40 years. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 1410 couples with a mean maternal age of 35.2 ± 6.4 years and AMH of 2.7 ± 2.6 ng/ml were included. In a multivariate logistic regression analysis, controlling for age, the chance of having at least one blastocyst biopsied/stimulated cycle (1156/1410), the chance of having at least one euploid blastocyst/stimulated cycle (880/1410), and the chance of having one euploid blastocyst once biopsy was performed (880/1156) were affected in all patients with AMH < 0.65 ng/ml [AdjOR 0.18[0.11-0.31] p = 0.008)], [AdjOR 0.18 [0.11-0.29] p < 0.001], and [AdjOR 0.34 [0.19-0.61] p = 0.015] as well as in patients with AMH 0.65-1.29 ng/ml (AdjOR 0.52 [0.32-0.84] p < 0.001), (AdjOR 0.49 [0.33-0.72] p < 0.001), and (AdjOR 0.57 [0.36-0.90] p < 0.001), respectively. In a multivariate linear regression analysis, AMH values did not affect blastocyst quality (- 0.72 [- 1.03 to - 0.41] p < 0.001). CONCLUSION: Irrespective of age, patients with diminished ovarian reserve (AMH < 1.3 ng/ml) have a lower chance of having at least one blastocyst biopsied and lower chance of having at least one euploid blastocyst per ovarian stimulated cycle. Blastocyst quality was not affected by AMH values.

Segmental duplications and monosomies are linked to in vitro developmental arrest.

PURPOSE: To verify which genetic abnormalities prevent embryos to blastulate in a stage-specific time. METHODS: A single center retrospective study was performed between April 2016 and January 2017. Patients requiring Preimplantation Genetic Testing for Aneuploidies (PGT-A) by Next Generation Sequencing (NGS) were included. All embryos were cultured in a time-lapse imaging system and single blastomere biopsy was performed on day 3 of development. Segmental duplications and deletions as well as whole chromosome monosomies and trisomies were registered. Embryo arrest was defined if the embryo failed to blastulate 118 h post-injection. A logistic regression model was applied using the time to blastulate as the response variable and the different mutations as explanatory variables. A p value < 0.05 was considered significant. RESULTS: Of the 285 biopsied cleavage stage embryos, 103 (36.1%) were euploid, and 182 (63.9%) were aneuploid. There was a significant difference in the developmental arrest between euploid and aneuploid embryos (8.7% versus 42.9%; p = 0.0001). Segmental duplications and whole chromosome monosomies were found to have a significant effect on developmental arrest (p = 0.0163 and p = 0.0075), while trisomies and segmental deletions had no effect on developmental arrest. In case of segmental duplications, an increase of one extra segmental duplication increases the odd of arrest by 159%. For whole chromosome monosomies, the odd will only increase by 29% for every extra chromosomal monosomy. Both chromosomal abnormalities remained significant after adding age as an explanatory variable to the model (p = 0.014 and p = 0.009). CONCLUSION: Day 3 cleavage stage embryos with segmental duplications or monosomies have a significantly decreased chance to reach the blastocyst stage.

Expanding the time interval between ovulation triggering and oocyte injection: does it affect the embryological and clinical outcome?

STUDY QUESTION: Is the time interval between ovulation triggering and oocyte denudation/injection associated with embryological and clinical outcome after ICSI? SUMMARY ANSWER: Expanding the time interval between ovulation triggering and oocyte denudation/injection is not associated with any clinically relevant impact on embryological or clinical outcome. WHAT IS KNOWN ALREADY: The optimal time interval between ovulation triggering and insemination/injection appears to be 38-39 h and most authors agree that an interval of >41 h has a negative influence on embryological and clinical pregnancy outcomes. However, in ART centres with a heavy workload, respecting these exact time intervals is frequently challenging. Therefore, we questioned to what extent a wider time interval between ovulation triggering and oocyte injection would affect embryological and clinical outcome in ICSI cycles. STUDY DESIGN, SIZE, DURATION: A single-centre retrospective cohort analysis was performed including 8811 ICSI cycles from 2010 until 2015. Regarding the time interval between ovulation triggering and oocyte injection, seven categories were considered: <36 h, 36 h, 37 h, 38 h, 39 h, 40 h and ≥41 h. In all cases, denudation was performed immediately prior to injection. The main outcome measures were oocyte maturation, fertilization and embryo utilization rate (embryos adequate for transfer or cryopreservation) per fertilized oocyte. Clinical pregnancy rate (CPR) and live birth rate (LBR) were considered as secondary outcomes. Utilization rate, CPR and LBR were subdivided into two groups according to the day of embryo transfer: Day 3 or Day 5. PARTICIPANTS/MATERIALS, SETTING, METHODS: During the study period, oocyte retrieval was routinely performed 36 h post-triggering except in the <36 h group. The interval of <36 h occurred only if OR was carried out before the planned 36 h trigger interval and was followed by immediate injection. Only cycles with fresh autologous gametes were included. The exclusion criteria were: injection with testicular/epididymal sperm, managed natural cycles, conventional IVF, combined conventional IVF/ICSI, preimplantation genetic testing and IVM cycles. Female age, number of oocytes, pre-preparation sperm concentration, post-preparation sperm concentration and motility, day of transfer, number of embryos transferred and quality of the best embryo transferred were identified as potential confounders. MAIN RESULTS AND THE ROLE OF CHANCE: Among the seven interval groups, adjusted mean maturation rates ranged from 76.4% to 83.2% and differed significantly (P < 0.001). Similarly, there was a significant difference in adjusted mean fertilization rates (range 69.2-79.3%; P < 0.001). The adjusted maturation and fertilization rates were significantly higher when denudation/injection was performed >41 h post-triggering compared to 38 h post-triggering (reference group). Oocyte denudation/injection at <36 h post-triggering had no significant effect on maturation, fertilization or embryo utilization rates compared to injection at 38 h. No effect of the time interval was observed on CPRs and LBRs, after adjusting for potential confounders. When oocyte injection was performed before 36 h the adjusted analysis showed that compared to 38 h after ovulation triggering the chance of having a live birth tends to be lower although the difference was not statistically significant (odds ratio 0.533, 95% CI: 0.252-1.126; P = 0.099). Injection ≥41 h post-triggering did not affect LBR compared to injection at 38 h post-ovulation. LIMITATIONS, REASONS FOR CAUTION: As this is a large retrospective study, the influence of uncontrolled variables cannot be excluded. These results should not be extrapolated to other ART procedures such as IVM, conventional IVF or injection with testicular/epididymal sperm. WIDER IMPLICATIONS OF THE FINDINGS: Our results indicate that the optimal injection time window may be less stringent than previously thought as both embryological and clinical outcome parameters were not significantly affected in our analysis. This is reassuring for busy ART centres that might not always be able to follow strict time intervals. STUDY FUNDING/COMPETING INTEREST(S): No funding. The authors declare no conflict of interest related to the present study. TRIAL REGISTRATION NUMBER: N/A.

Do we need to measure progesterone in oocyte donation cycles? A retrospective analysis evaluating cumulative live birth rates and embryo quality.

STUDY QUESTION: Does late follicular-phase elevated serum progesterone (LFEP) during ovarian stimulation for oocyte donation have an impact on embryo quality (EQ) and cumulative live birth rate (CLBR)? SUMMARY ANSWER: LFEP does not have an influence on EQ nor CLBR in oocyte donation cycles. WHAT IS KNOWN ALREADY: Ovarian stimulation promotes the production of progesterone (P) which, when elevated during the follicular phase, has been demonstrated to have a deleterious effect in autologous fresh IVF outcomes. While there is robust evidence that this elevation results in impaired endometrial receptivity, the impact on EQ remains a matter of debate. The oocyte donation model is an excellent tool to assess the effects of LFEP on EQ from those on endometrium receptivity separately. Previous studies in oocyte donation cycles investigating the influence of elevated P on pregnancy outcomes in oocyte recipients showed conflicting results. STUDY DESIGN, SIZE, DURATION: This is a retrospective analysis including all GnRH antagonist down-regulated cycles for fresh oocyte donation taking place in a tertiary referral university hospital between 2010 and 2017. A total of 397 fresh donor-recipient cycles were included. Each donor was included only once in the analysis and could be associated to a single recipient. PARTICIPANTS/MATERIALS, SETTING, METHODS: The sample was stratified according to serum P levels of ≤1.5 and >1.5 ng/mL on the day of ovulation triggering. The primary endpoint of the study was the top-quality embryo rate on Day 3, and the secondary outcome measure was CLBR defined as a live-born delivery beyond 24 weeks. MAIN RESULTS AND THE ROLE OF CHANCE: Three hundred ninety-seven fresh oocyte donation cycles were included in the analysis, of which 314 (79%) had a serum P ≤ 1.5 ng/mL and 83 (20.9%) had a serum P > 1.5 ng/mL. The average age of the oocyte donors was 31.4 ± 4.7 and 29.9 ± 4.5 years, respectively, for normal and elevated P (P = 0.017). The mean number of oocytes retrieved was significantly higher in the elevated P group with 16.6 ± 10.6 vs 11.5 ± 6.9 in the P ≤ 1.5 group (P < 0.001).In parallel, the total number of embryos on Day 3, as well as the number of good-quality embryos at this stage, was significantly higher in the elevated P group (6.6 ± 5.6 vs 4.15 ± 3.5 and 8.7 ± 6.3 vs 6.1 ± 4.4; respectively, P < 0.001). However, maturation and fertilization rates did not vary significantly between the two study groups and neither did the top- and good-quality embryo rate and the embryo utilization rate, all evaluated on Day 3 (P = 0.384, P = 0.405 and P = 0.645, respectively). A multivariable regression analysis accounting for P groups, age of the donor, number of retrieved oocytes and top-quality embryo rate as potential confounders showed that LFEP negatively influenced neither the top-quality embryo rate nor the CLBR. LIMITATIONS, REASONS FOR CAUTION: This is an observational study based on a retrospective data analysis. Better extrapolation of the results could be validated by performing a prospective trial. Furthermore, this study was focused on oocyte donation cycles and hence the results cannot be generalized to the entire infertile population. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study providing evidence that LFEP does not influence CLBR and is adding strong evidence to the existing literature that LFEP does not harm EQ in oocyte donation programs. STUDY FUNDING/COMPETING INTERESTS: Not applicable.

The clinicians´ dilemma with mosaicism-an insight from inner cell mass biopsies.

STUDY QUESTION: How reliable are cleavage stage and trophectoderm (TE) biopsies compared to inner cell mass (ICM) biopsies? SUMMARY ANSWER: The reliability of TE biopsy compared to ICM biopsy is almost perfect, but only substantial between cleavage stage biopsy and ICM biopsy. WHAT IS KNOWN ALREADY: One of the prevailing reasons for implantation failure is presumed to be chromosomal aneuploidy in human preimplantation embryos. Preimplantation genetic testing for aneuploidies (PGT-A) has been introduced into assisted reproduction in an effort to increase pregnancy rates. Increasing evidence indicates that genetic results obtained following blastomere or TEbiopsy may not accurately reflect the true genetic status of the embryo due to the presence of embryonic mosaicism, and therefore the reliability of PGT is highly controversial. STUDY DESIGN, SIZE, DURATION: This was an observational descriptive study, performed in a private infertility centre from August 2016 to January 2017. PARTICIPANTS/MATERIALS, SETTING, METHODS: The mean female age was 33.9 years, ranging from 24 to 46 years, and the mean number of biopsied embryos per couple was 2.2 (range 1-7 embryos). Blastomere biopsies had been performed at cleavage stage on Day 3 (D3) due to the turnover time of genetic testing and the inability to cryopreserve embryos in accordance with the local law governing ART. To confirm the genetic results in embryos not chosen for transfer, additional biopsies of the TE at blastocyst stage (BLASTO-TE) as well as of the ICM (BLASTO-ICM) were performed on D5. Only surplus blastocysts, which had not been selected for transfer and were not cryopreserved in accordance with the law governing ART, had been included. MAIN RESULTS AND THE ROLE OF CHANCE: Comparison of all biopsies (D3/BLASTO-ICM/BLASTO-TE) per embryo demonstrated that 50 (59.5%) out of 84 embryos showed concordance in all three results (= full concordance). Thirty-four (40.4%) embryos had at least two discordant results between the three biopsies, regardless of whether the embryo diagnosis (aneuploid/euploid) was discordant or not, or in aneuploid embryos, whether the chromosomal patterns were inconsistent. Nine (= 10.7%) embryos had complete discordance between all three biopsies. False positive results between D3/BLASTO-TE, D3/BLASTO-ICM and BLASTO-TE/BLASTO-ICM were 26.4%/30.2% and 7.5%, respectively, while the Kappa agreement between the different approaches was 0.647, 0.553 and 0.857, respectively. Therefore the reliability of D3/BLASTO-TE, D3/BLASTO-ICM and BLASTO-TE/BLASTO-ICM can be interpreted as substantial, as moderate and as almost perfect. LIMITATIONS, REASONS FOR CAUTION: The limitation of this study is the possible bias in the concordance/discordance rate because embryos that had been selected for transfer did not undergo biopsy on D5. WIDER IMPLICATIONS OF THE FINDINGS: The obvious discordance between the three different approaches for PGT-A underlines the limitations of genetic testing and highlights the importance of ongoing research in order to improve the accuracy of PGT-A results. Until then reproductive specialists will continue to make challenging decisions on whether to transfer or discard an embryo in light of current evidence questioning the reliability of genetic results. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by Igenomix. The funder provided support in the form of salary for R.C. The co-author R.C. is an employee of Igenomix. She participated in the blinded analysis of the samples; however the final data collection and statistical analysis of the results, as well as the decision to publish, was taken by B.L, I.E. and H.F. The authors B.L., I.E., A.L., A.B., A.A., N.D. and H.F. have no competing interests. The funder did not have any additional role in the study design, data collection and analysis, decision to publish or preparation of the manuscript. The commercial affiliation of R.C. did not play any role in the study. TRIAL REGISTRATION NUMBER: This study was approved by the Ethics Committee of IVIRMA Middle East Fertility Clinic, Abu Dhabi, UAE (Research Ethics Committee IVI-MEREFA009a/2017).

Influence of ultra-low oxygen (2%) tension on in-vitro human embryo development.

STUDY QUESTION: Is a reduction in the oxygen tension from 5 to 2% during extended culture from Day 3 onwards beneficial for human blastocyst development in vitro? SUMMARY ANSWER: A reduction in oxygen concentration from 5 to 2% O2 after Day 3 did not improve embryo development, quality and utilization rate. WHAT IS KNOWN ALREADY: The human embryo leaves the fallopian tube to reach the uterine cavity around Day 3-4 post-ovulation. As the oxygen concentration ranges from 5 to 7% in the fallopian tube and decreases to 2% in the uterus, reducing the oxygen tension during extended culture from Day 3 onwards seems more physiological. We aim to mimic the in-vivo environment during in-vitro embryo culture. Therefore, we compared the effect of extended culture performed at 5% (control arm) or 2% oxygen (O2; study arm) tension on blastocyst formation and quality. STUDY DESIGN, SIZE, DURATION: Between December 2016 and September 2017, in two prospective studies, sibling embryos were randomized on Day 3 to either 5% O2 (control) or 2% O2 (study) for extended culture. In the control arms of both studies 1 and 2, the dishes with blastocyst medium were pre-equilibrated overnight in 5% O2, 6% CO2 and 89% N2 at 37°C. In the 2% study groups, the overnight pre-equilibration of blastocyst media was performed in either 2% O2 (study 1, 99 cycles) or 5% O2 (study 2, 126 cycles). The latter provides a gradual transition from 5 to 2% O2 environment for the study arm. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: Embryo culture until Day 3 was always performed in 5% O2; if at least four embryos of moderate to excellent quality were obtained on Day 3, the sibling embryos were randomized to either 5% O2 or 2% O2 for extended culture. The endpoints were embryo development and quality on Day 5/6 and the utilization rate (embryos transferred and cryopreserved). Statistical analysis was performed using the chi-square test, a P-value of <0.05 was considered significantly different. MAIN RESULTS AND THE ROLE OF CHANCE: In study 1, 811 embryos were randomized on Day 3: 405 to the 2% O2 and 406 to the 5% O2 condition. No differences were observed in the blastulation rate (68.6 versus 71.9%; P = 0.319) and the proportion of good quality blastocysts on Day 5 (55.8 versus 55.2%; P = 0.888), nor in the utilization rate (53.1 versus 53.2%; P = 1.000). In study 2, 1144 embryos were randomized: 572 in each arm. Similarly, no significant difference was demonstrated in terms of the blastulation rate (63.6 versus 64.7%; P = 0.758), the proportion of good quality blastocysts (46.9 versus 48.8%; P = 0.554) or the utilization rate (49.8 versus 48.1%; P = 0.953). LIMITATIONS, REASON FOR CAUTION: This study evaluated embryo development only until Day 5/6. The effect of oxidative stress on the developing embryo may only become evident at later stages (i.e. during implantation) and should therefore be studied in an RCT. The question also remains as to whether the switch to ultra-low oxygen tension from Day 4 onwards, when the embryo arrives in the uterus in vivo, would be preferential. WIDER IMPLICATIONS OF THE FINDINGS: Based on the present study results, there is no benefit in lowering the oxygen tension from 5 to 2% from Day 3 onwards during extended human embryo culture. STUDY FUNDING/COMPETING INTEREST(s): No funding was received for this study and the authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Impact of late-follicular phase elevated serum progesterone on cumulative live birth rates: is there a deleterious effect on embryo quality?

STUDY QUESTION: Is elevated late-follicular phase progesterone (EP) associated with a deleterious impact on embryo quality (EQ) and cumulative live birth rates (LBRs)? SUMMARY ANSWER: EP was associated with a decrease in embryo utilization and cumulative LBRs. WHAT IS KNOWN ALREADY: Ovarian stimulation promotes the production of progesterone (P) which adversely affects IVF pregnancy outcomes. However, evidence regarding a potential association between EP an EQ is lacking. STUDY DESIGN, SIZE, DURATION: A retrospective analysis of all GnRH antagonist down-regulated ICSI cycles followed by a fresh embryo transfer (ET) between 2010 and 2015 was performed. The sample was stratified according to the following P levels on the day of ovulation triggering: ≤0.50, 0.51-1.49 and ≥1.50 ng/ml. The primary outcomes were embryo utilization rates (number of embryos transferred or cryopreserved) and cumulative LBR, defined as the occurrence of the first live-birth after either the fresh or one of the subsequent frozen ET. PARTICIPANTS/MATERIALS, SETTING, METHODS: Overall, 3400 cycles were included in the analysis, using multivariable regression to account for potential confounding. MAIN RESULTS AND THE ROLE OF CHANCE: Female age and the number of oocytes retrieved increased significantly with increasing serum P values. Utilization rates decreased linearly as P increased for Day 3 embryos (72.3, 63.0 and 45.4%, respectively), while for Day 5 embryos only the EP group was associated with a significant decrease (48.8, 47.8 and 38.8%, respectively). EP was also associated with decreased fresh and cumulative LBRs. LIMITATIONS REASONS FOR CAUTION: The main limitations of this study were its retrospective nature and the fact that it was restricted to GnRH antagonist cycles. WIDER IMPLICATIONS OF THE FINDINGS: These results raise the question whether EP may also be associated with a decrease in cumulative pregnancy outcomes by increasing embryo wastage. Further studies may evaluate the potential benefit of additional measures besides the freeze-all strategy to avoid this issue, such as lowering the stimulation dose or applying a step-down protocol. STUDY FUNDING/COMPETING INTEREST(S): None.

Closed oocyte vitrification and storage in an oocyte donation programme: obstetric and neonatal outcome.

STUDY QUESTION: Does closed oocyte vitrification in an oocyte donation programme have an impact on obstetric and neonatal outcome? SUMMARY ANSWER: Obstetric and neonatal outcomes after closed system vitrification of donor oocytes appear to be reassuring. WHAT IS KNOWN ALREADY: The use of fresh oocytes has not been proved to be superior to the use of vitrified donor oocytes in terms of survival, embryo development and clinical pregnancies. Those studies used open devices to prove the non-superiority. Very limited information is available on the comparison of open and closed devices, and the results for survival, embryo development and pregnancy outcomes are conflicting. Data on obstetric and neonatal outcome from vitrified oocytes are scarce. Only one large report is available after the use of donor oocytes vitrified with an open device. STUDY DESIGN, SIZE, DURATION: Retrospective observational study performed at the Centre for Reproductive Medicine, UZ Brussel, Belgium. All 117 oocyte recipient cycles between March 2010 and August 2014 with the use of a closed vitrification device and leading to a pregnancy beyond 20 weeks were included in this study. PARTICIPANTS/MATERIALS, SETTING, METHODS: All recipient warming cycles with a pregnancy beyond 20 weeks from vitrified donor oocytes: results from the fresh embryo transfers. MAIN RESULTS AND THE ROLE OF CHANCE: For 117 recipient cycles, a total of 793 oocytes were warmed of which 657 (82.8%) survived and 499 (76.0%) were fertilized. Nineteen single and 98 double embryo transfers led to 95 singleton and 22 twin pregnancies. Hypertensive disorders, haemorrhages and gestational diabetes were reported in 22/112 (19.6%), 30/112 (26.8%) and 13/112 (11.6%) of the pregnancies, respectively. No major adverse neonatal outcomes were observed. Congenital malformations were observed in 11 out of 139 children; for one an elective termination was performed at 25 weeks. LIMITATIONS, REASONS FOR CAUTION: Since March 2010, almost all oocytes for donation are vitrified in our centre. Therefore, no recent data are available to control the outcomes of fresh oocyte donations. WIDER IMPLICATIONS OF THE FINDINGS: The reassuring results obtained in the current study show that closed system vitrification devices for donor oocytes may be used as an alternative to open devices which have been linked to possible cross-contamination issues. STUDY FUNDING/COMPETING INTERESTS: None. TRIAL REGISTRATION NUMBER: N/A.

Open versus closed oocyte vitrification in an oocyte donation programme: a prospective randomized sibling oocyte study.

STUDY QUESTION: Is the survival of donor oocytes with the CryotopSC device superior to the survival with the closed CBSvit device? SUMMARY ANSWER: The CryotopSC device and the CBSvit device showed similar survival rates. WHAT IS KNOWN ALREADY: Health authorities are cautious about possible cross contamination during liquid nitrogen storage or handling when working with open vitrification devices. At present, the use of open devices is still allowed since little information is available on the efficiency of closed devices. STUDY DESIGN, SIZE, DURATION: A prospective randomized sibling oocyte study was performed in the Centre for Reproductive Medicine (UZBrussel) between January 2014 and July 2015. The survival after warming and the embryological outcome of donor oocytes vitrified using two devices was compared: the CBSvit device (closed vitrification and closed storage) and the CryotopSC device (open vitrification and closed storage). A difference of 10% was defined to prove the superiority of the CryotopSC device. In total, 250 warmed oocytes were needed in each arm. PARTICIPANTS/MATERIALS, SETTING, METHODS: Oocytes from 48 donors were included in the study: 253 vitrified with the CBSvit device and 257 with the CryotopSC device. Equal numbers of oocytes from both devices and originated from the same donor cycle were allocated to each of 78 recipients, in order to exclude donor and recipient (male factor) effects. MAIN RESULTS AND THE ROLE OF CHANCE: There were no differences found between the CBSvit and the CryotopSC in terms of survival after warming (93.7 versus 89.9%) or fertilization per injected oocyte (74.3 versus 81.4%). The degeneration rate after ICSI was significantly higher for the CBSvit device: 11.4 versus 6.1% (P = 0.041). A significantly higher number of zygotes in the CryotopSC group finished their first mitosis 25-27 h post-injection (34.1 versus 52.1%, P = 0.001). On Day 3, the overall embryo quality distribution did not vary between groups, but a significantly higher cell number was obtained in the CryotopSC device: 6.8 ± 2.8 versus 7.6 ± 2.8 (P = 0.01). The utilization rate per mature oocyte, per surviving oocyte or per fertilized oocyte did not differ. The embryos with the highest quality were selected for transfer on Day 3. The clinical pregnancy rate per transfer cycle was 36.5%. LIMITATIONS, REASONS FOR CAUTION: The results of this study should not be extrapolated to other female groups, since oocytes from young fertile donors were used in this study. WIDER IMPLICATIONS OF THE FINDINGS: In many countries, the use of open devices is still allowed due to the limited reports on the efficiency of closed devices. Knowing the caution of health authorities about the use of open devices, there is an urgent need for efficiency studies with closed devices. The results obtained in the current study shows the efficiency of a safe closed vitrification device, leaving behind any concern about possible cross contamination during handling or storage. STUDY FUNDING/COMPETING INTERESTS: No funding was obtained. The authors have no conflict of interest to declare. TRIAL REGISTRATION NUMBER: NCT01952184. TRIAL REGISTRATION DATE: 24 September 2013. DATE OF FIRST PATIENT'S ENROLMENT: 23 January 2014.

A prospective randomized controlled trial investigating the effect of artificial shrinkage (collapse) on the implantation potential of vitrified blastocysts.

STUDY QUESTION: What is the effect of artificial shrinkage by laser-induced collapse before vitrification on the implantation potential after transfer of vitrified-warmed blastocysts? SUMMARY ANSWER: The artificial shrinkage by laser-induced collapse did not significantly increase the implantation rate per transferred collapsed blastocyst (37.6%) compared with non-collapsed blastocysts (28.9%) [odds ratio (OR): 1.48, 95% confidence interval (CI): 0.78-2.83]. WHAT IS KNOWN ALREADY: Retrospective studies have demonstrated that artificial shrinkage of the blastocyst prior to vitrification can have a positive effect on blastocyst survival after warming. A recent study found a similar survival rate but higher implantation rate for collapsed blastocysts. So far, no randomized controlled trial has been conducted to investigate the implantation potential of collapsed blastocysts. STUDY DESIGN, SIZE, DURATION: Prospective randomized trial. Patients were recruited from December 2011 until April 2014 and warming cycles were included until July 2014. Patients were randomized in the fresh cycle if blastocysts were available for vitrification and were allocated to the study or control arm according to a computer-generated list. In the study group, blastocysts underwent laser-induced collapse before vitrification. In the control group, blastocysts were vitrified without collapsing. PARTICIPANTS/MATERIALS, SETTING, METHODS: In total, 443 patients signed informed consent and 270 patients had blastocysts vitrified. One-hundred and thirty-five patients were allocated to the study group and 135 to the control group. Sixty-nine patients from the study group and 69 from the control group returned for at least one warming cycle in which 85 and 93 blastocysts were warmed in the first cycle, respectively. Primary outcome was implantation rate per embryo transferred in the first warming cycle. Secondary outcomes were survival and transfer rates, blastocyst quality after warming, clinical pregnancy rate and implantation rate per warmed blastocyst. Blastocysts were vitrified-warmed one by one using closed vitrification and one or two blastocysts were transferred per warming cycle. MAIN RESULTS AND THE ROLE OF CHANCE: We calculated that the group sample sizes of 80 embryos in the collapse group and 80 embryos in the control group were needed to achieve 80% power to detect a difference between the group proportions of +20% with P < 0.05. In the study group, 69 first warming cycles resulted in 69 transfers with 1.2 blastocysts (n = 85) transferred. In the control group, an average of 1.3 blastocysts (n = 83) were transferred in 67 out of 69 warming cycles. Implantation rates per embryo transferred in the first warming cycle were not different between both groups (38 versus 29%, OR: 1.48; 95% CI: 0.78-2.83), neither was the implantation rate per warmed embryo (38 versus 26%, OR: 1.74; 95% CI: 0.92-3.29). When all warming cycles were considered (n = 135 in each group), survival rate after collapse was significantly higher compared with the control group (98.0 versus 92.0%, OR: 4.25; 95% CI: 1.19-15.21). Furthermore, a higher percentage of high-quality blastocysts (36.3 versus 23.5%, OR: 1.86; 95% CI: 1.12-3.08) and hatching blastocysts (19.2 versus 5.4%, OR: 4.18; 95% CI: 1.84-9.52) were found compared with the control group. LIMITATIONS, REASONS FOR CAUTION: The study lasted more than 2.5 years since fewer patients than expected returned for a warming cycle because of the high ongoing pregnancy rates in the fresh IVF/ICSI cycle. WIDER IMPLICATIONS OF THE FINDINGS: Although no significant higher implantation rate was found after collapse, the better survival and post-warm embryo quality convinced us to recognize a clinical benefit of artificial shrinkage and to implement it in routine vitrification practice. TRIAL REGISTRATION NUMBER: NCT01980225, www.clinicaltrials.gov. The first patient was included November 2011 and the study was registered October 2013.

Reduced blastocyst formation in reduced culture volume.

PURPOSE: The aim of this prospective sibling oocyte study was to evaluate whether reduced culture volume improves blastocyst formation. METHODS: Twenty-three patients with extended embryo culture until day 5 were selected for the study. After injection, 345 sibling oocytes were individually cultured in either 25 or 7 µl droplets of Origio cleavage medium under oil. On day 3 of development, embryos were transferred to droplets with the corresponding volume of Origio blastocyst culture medium. Fertilization and embryo quality on day 3 and day 5/6 were evaluated. RESULTS: No statistically significant difference (p = 0.326) in fertilization rate was observed (81.3 versus 83.0 %). There was no significant difference in terms of the number of excellent and good-quality embryos obtained on day 3 between both groups (p = 0.655). Embryo culture in 25 µl droplets led to more embryos with a higher cell number when compared to 7 µl culture (p = 0.024). On day 3, 132 and 131 embryos were considered for further culture until day 5/6. Blastulation rates were significantly higher in the 25 µl group (75.0 versus 61.6 %; p = 0.017) and significantly more day 5 embryos with excellent and good quality were found in this group (54.5 versus 40.5 %; p = 0.026). Finally, the utilization rates expressed per mature oocyte (41.4 versus 29.8 %; p = 0.043), per fertilized oocyte (50.7 versus 36.6 %; p = 0.023), and per day 3 embryo undergoing extended culture to day 5/6 (54.5 versus 39.7 %; p = 0.019) were all significantly higher in the 25 µl group. CONCLUSION: Reduced culture volume (7 µl) negatively impacts early development by reducing the cell number on day 3 and both blastocyst formation and quality.

Chromosomal meiotic segregation, embryonic developmental kinetics and DNA (hydroxy)methylation analysis consolidate the safety of human oocyte vitrification.

Oocyte vitrification has been introduced into clinical settings without extensive pre-clinical safety testing. In this study, we analysed major safety aspects of human oocyte vitrification in a high security closed system: (i) chromosomal meiotic segregation, (ii) embryonic developmental kinetics and (iii) DNA (hydroxy)methylation status. Fresh and vitrified sibling oocytes from young donors after intracytoplasmic sperm injection (ICSI) were compared in three different assays. Firstly, the chromosomal constitution of the fertilized zygotes was deduced from array comparative genomic hybridization results obtained from both polar bodies biopsied at Day 1. Secondly, embryo development up to Day 3 was analysed by time-lapse imaging. Ten specific time points, six morphokinetic time intervals and the average cell number on Day 3 were recorded. Thirdly, global DNA methylation and hydroxymethylation patterns were analysed by immunostaining on Day 3 embryos. The nuclear fluorescence intensity was measured by Volocity imaging software. Comprehensive chromosomal screening of the polar bodies demonstrated that at least half of the zygotes obtained after ICSI of fresh and vitrified oocytes were euploid. Time-lapse analysis showed that there was no significant difference in cleavage timings, the predictive morphokinetic time intervals nor the average cell number between embryos developed from fresh and vitrified oocytes. Finally, global DNA (hydroxy)methylation patterns were not significantly different between Day 3 embryos obtained from fresh and from vitrified oocytes. Our data further consolidate the safety of the oocyte vitrification technique. Nevertheless, additional testing in young and older sub-fertile/infertile patients and sound follow-up studies of children born after oocyte cryopreservation remain mandatory.

Survival and post-warming in vitro competence of human oocytes after high security closed system vitrification.

PURPOSE: To compare two vitrification methods and two warming methods for human oocyte vitrification using a high security closed device in terms of survival, fertilization and embryo development. METHODS: For vitrification, oocytes were (1) immediately placed in equilibration solution or (2) they were gradually exposed to the cryoprotectants. For warming, oocytes were placed (1) in a 25 µl preheated (37 °C) thawing solution droplet that was put at room temperature for 1 min once the oocytes were inside or (2) in a 150 µl droplet for 1 minute at 37 °C. RESULTS: Survival and preimplantation development were significantly lower when warming was performed in a small preheated droplet. There was no significant difference in survival and embryo development between the gradual or direct exposure to cryoprotectants. CONCLUSIONS: Using this high security closed vitrification device a 90 % survival rate can be achieved when the oocytes are immediately warmed in a large volume at 37 °C.