Effect of time post warming to embryo transfer on human blastocyst metabolism and pregnancy outcome.

PURPOSE: This study is aiming to test whether variation in post warming culture time impacts blastocyst metabolism or pregnancy outcome. METHODS: In this single center retrospective cohort study, outcomes of 11,520 single frozen embryo transfer (FET) cycles were analyzed from January 2015 to December 2020. Patient treatments included both natural and programmed cycles. Time categories were determined using the time between blastocyst warming and embryo transfer: 0 (0- <1h), 1 (1-<2h), 2 (2-<3h), 3(3-<4h), 4 (4-<5), 5 (5-<6), 6 (6-<7) and 7 (7-8h). Non-invasive metabolic imaging of discarded human blastocysts for up to 10h was also performed using Fluorescence lifetime imaging microscopy (FLIM) to examine for metabolic perturbations during culture. RESULTS: The mean age of patients across all time categories were comparable (35.6 ± 3.9). Live birth rates (38-52%) and miscarriage rate (5-11%) were not statistically different across post-warming culture time. When assessing pregnancy outcomes based on the use of PGT-A, miscarriage and live birth rates were not statistically different across culture hours in both PGT-A and non-PGT cycles. Further metabolic analysis of blastocysts for the duration of 10h of culture post warming, revealed minimal metabolic changes of embryos in culture. CONCLUSION: Overall, our results show that differences in the time of post warming culture have no significant impact on miscarriage or live birth rate for frozen embryo transfers. This information can be beneficial for clinical practices with either minimal staffing or a high number of patient cases.

Developmental competence and neonatal outcomes of nonpronuclear zygotes following single vitrified-warmed blastocyst transfers using propensity score matching analysis.

PURPOSE: To investigate developmental competence and neonatal outcomes of nonpronuclear (0PN) zygotes following single vitrified-warmed blastocyst transfers (VBT). METHODS: The clinical, laboratorial and neonatal data of 996 patients with ≤ 38 years who underwent blastocyst culture and single VBT were retrospectively analyzed. The pregnancy and neonatal outcomes of VBT were compared between 0PN and 2PN blastocysts using propensity score matching (PSM). Moreover, Day 3 (D3) embryo development and blastocyst formation were compared between 0PN and 2PN zygotes. RESULTS: There were no significant differences in clinical pregnancy rate (CPR), live birth rate (LBR) and neonatal outcomes of VBT between the 0PN and 2PN blastocysts irrespectively of whether PSM was used. However, early abortion rate (EAR) was higher in blastocysts from 0PN D3 embryos > 10 cells (p < 0.05) before PSM. Moreover, the early developmental competence of 0PN zygotes was different from that of 2PN zygotes presenting higher percentages of D3 embryos ≤ 6 cells (p < 0.01) and > 10 cells (p < 0.01), lower available blastocyst formation rate (ABFR) (p < 0.01) and good-quality blastocyst formation rate (GBFR) (p < 0.01) in D3 embryos with 4-6 cells. ABFR and GBFR increased with cell number when compared among embryos with 4-6 cells, 7-10 cells and > 10 cells, irrespectively of 0PN or 2PN embryos. CONCLUSION: The early developmental competence of 0PN zygotes was different from that of 2PN zygotes, but did not influence pregnancy and neonatal outcomes following VBT. ABFR and GBFR increased with cell number, irrespectively of 0PN or 2PN embryos.

Concordance of PGT for aneuploidies between blastocyst biopsies and spent blastocyst culture medium.

RESEARCH QUESTION: Non-invasive preimplantation genetic testing for aneuploidies (niPGT-A) avoids the possible detrimental impact of invasive PGT-A on embryo development and clinical outcomes. Does cell-free DNA (cfDNA) from spent blastocyst culture medium (BCM) reflect embryonic chromosome status better than trophectoderm (TE) biopsy? DESIGN: In this study, 35 donated embryos were used for research and the BCM, TE biopsy, inner cell mass (ICM) and residual blastocyst (RB) were individually picked up from these embryos. Whole genome amplification (WGA) was performed and amplified DNA was subject to next-generation sequencing. Chromosome status concordance was compared among the groups of samples. RESULTS: The WGA success rates were 97.0% (TE biopsy), 100% (ICM), 97.0% (RB) and 88.6% (BCM). Using ICM as the gold standard, the chromosomal ploidy concordance rates for BCM, TE biopsy and RB were 58.33% (14/24), 68.75% (22/32) and 78.57% (22/28); the diagnostic concordance rates were 83.33% (20/24), 87.50% (28/32) and 92.86% (26/28); and the sex concordance rates were 92.31% (24/26), 100% (32/32) and 100% (28/28), respectively. Considering RB the gold standard, the chromosome ploidy concordance rates for BCM and TE biopsy were 61.90% (13/21) and 81.48% (22/27); the diagnostic concordance rates were 71.43% (15/21) and 88.89% (24/27); and the sex concordance rates were 91.30% (21/23) and 100% (27/27), respectively. CONCLUSIONS: The results of niPGT-A of cfDNA of spent BCM are comparable to those of invasive PGT-A of TE biopsies. Modifications of embryo culture conditions and testing methods will help reduce maternal DNA contamination and improve the reliability of niPGT-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.

Aneuploidy rates and morphokinetic parameters of embryos cultured in distinct culture media: a sibling oocyte study.

STUDY QUESTION: Do embryos from sibling oocytes assigned to distinct single-step media culture systems demonstrate differences in early embryo development, morphokinectics or aneuploidy rates? SUMMARY ANSWER: Embryo quality, morphokinetic parameters and aneuploidy rates from trophectoderm biopsy were similar between sibling embryos cultured in distinct media systems from the time of gamete isolation. WHAT IS KNOWN ALREADY: Studies on the effect of commercially available embryo culture media systems have demonstrated inconsistent impact on human embryonic development, morphokinetics, aneuploidy rates and clinical outcomes. In addition, these studies have been primarily randomized at the level of the embryo or the patient to culture media. STUDY DESIGN, SIZE, DURATION: Prospective sibling oocyte cohort derived from 200 subjects undergoing IVF at a tertiary academic medical center between February 2018 and November 2019. PARTICIPANTS/MATERIALS, SETTING, METHODS: Sibling oocytes were allocated to Global® or SAGE® media system based upon laterality of ovary from which they were retrieved. All embryos were cultured in a time-lapse incubator. Blastocysts underwent trophectoderm biopsy for preimplantation genetic testing for aneuploidy using next-generation sequencing. MAIN RESULTS AND THE ROLE OF CHANCE: One hundred twenty-seven subjects (n = 127) had paired blastocysts for biopsy in each culture media system. There was no difference in top quality blastocyst formation (47.1 ± 31.0 vs 48.1 ± 27.2%; P = 0.87) nor aneuploidy rate (62.3 ± 34.0 vs 56.1 ± 34.4%; P = 0.07) for sibling embryos cultured in Global versus SAGE media system. Embryo morphokinetic parameters including time to each cell division from two cells (t2) to eight cells (t8), time to morula stage (tM), time to blastocele formation (tSB), time to fully formed blastocyst (tB) and time to expansion of the blastocyst (tEB) were similar between paired blastocysts from each culture media system. LIMITATIONS, REASONS FOR CAUTION: Pregnancy outcomes and offspring health data were not available for analysis. WIDER IMPLICATIONS OF THE FINDINGS: Commercially available culture media may not have a differential impact on embryo development and blastocyst aneuploidy rate when patient and stimulation-related factors are held constant. STUDY FUNDING/COMPETING INTEREST(S): There was no external funding for this study. C.H. is owner of a consultancy company, IVF Professionals, Chief Scientific Officer at Apricity, Executive Director at TMRW and co-owner and shareholder of Aria Fertility. She has received speaker fees, consulting fees and travel support from Cooper Surgical and Vitrolife. J.B. is an employee and shareholder of vitrolife. TRIAL REGISTRATION NUMBER: N/A.

Does the cell number of 0PN embryos on day 3 affect pregnancy and neonatal outcomes following single blastocyst transfer?

BACKGROUND: 0PN zygotes have a low cleavage rate, and the clinical outcomes of cleavage-stage embryo transfers are unsatisfactory. Blastocyst culturing is used to screen 0PN embryos, but whether the cell number of 0PN embryos on day 3 affects the clinical outcomes following single blastocyst transfer is unknown and would be helpful in evaluating the clinical value of these embryos. METHODS: This retrospective study compared 46,804 0PN zygotes, 242 0PN frozen-thawed single blastocyst transfers, and 92 corresponding 0PN singletons with 232,441 2PN zygotes, 3563 2PN frozen-thawed single blastocyst transfers, and 1250 2PN singletons from January 2015 to October 2019 at a tertiary-care academic medical centre. The 0PN and 2PN embryos were divided into two groups: the group with < 6 cells on day 3 and that with ≥ 6 cells. Embryo development, subsequent pregnancy and neonatal outcomes were compared between the two groups. RESULTS: The cleavage and available blastocyst rates of the 0PN zygotes were much lower than those of the 2PN zygotes (25.9% vs. 97.4%, P < 0.001; 13.9% vs. 23.4%, P < 0.001). In the < 6 cells group, the available blastocyst rate of the cleaved 0PN embryos was significantly lower than that of the 2PN embryos (2.5% vs. 12.7%, P < 0.001). However, in the ≥ 6 cells group, the available blastocyst rate of the 0PN cleaved embryos significantly improved, although it was slightly lower than that of the 2PN embryos (33.9% vs. 35.7%, P = 0.014). Importantly, compared to those of the 2PN single blastocyst transfers, the clinical pregnancy rate, live birth rate, Z-score and malformation rate of the 0PN single blastocyst transfers were not significantly different in either the < 6 cells group (30.4% vs. 39.8%, P = 0.362; 30.4% vs. 31.3%, P = 0.932; 0.89 ± 0.90 vs. 0.42 ± 1.02, P = 0.161; 0% vs. 2.6%, P = 1.000) or the ≥ 6 cells group (50.7% vs. 46.6%, P = 0.246; 39.7% vs. 38.3%, P = 0.677; 0.50 ± 1.23 vs. 0.47 ± 1.11, P = 0.861; 2.4% vs. 1.8%, P = 1.000). CONCLUSIONS: The cell number on day 3 of 0PN embryos affected the subsequent formation of blastocysts but did not influence the subsequent pregnancy and neonatal outcomes of 0PN single blastocyst transfers, which may be beneficial to clinicians counselling patients on the clinical value of 0PN embryos.

Does Post-Warming Extended Culture Duration Affect the Clinical and Obstetric Outcomes of Patients of Advanced Maternal Age? A Single-Center Study.

BACKGROUND: The single vitrified-warmed blastocyst transfer (SVBT) cycle has been increasingly utilized for assisted reproductive technology. Women of advanced maternal age (AMA) comprise a significant portion of patients who have undergone 'freeze-all' cycles. This study investigated the association between the post-warming extended culture duration and pregnancy outcomes in patients of AMA. METHODS: This retrospective cohort study analyzed the outcomes of 697 SVBT cycles between January 2016 and December 2017. The cycles were divided into 3 groups based on the age of the female partners: group I: < 35 years (n = 407), group II: 35-37 years (n = 176); and group III, 38-40 years (n = 114). Data are shown as the mean ± standard error of the mean. Data were analyzed using one-way ANOVA followed by Duncan's multiple range test. Statistical significance was set at P < 0.001. RESULTS: The blastocyst rate, clinical pregnancy rate, and live birth rate (LBR) was significantly lower in the AMA groups. However, there were no significant differences in LBR in the transfer between the AMA and younger groups according to blastocyst morphology and post-warming extended culture duration. CONCLUSION: Post-warming extended culture of blastocysts is not harmful to patients of AMA. It could be a useful parameter in clinical counseling and decision making for fertility treatments.

Modeling human peri-implantation placental development and function†.

It is very difficult to gain a better understanding of the events in human pregnancy that occur during and just after implantation because such pregnancies are not yet clinically detectable. Animal models of human placentation are inadequate. In vitro models that utilize immortalized cell lines and cells derived from trophoblast cancers have multiple limitations. Primary cell and tissue cultures often have limited lifespans and cannot be obtained from the peri-implantation period. We present here two contemporary models of human peri-implantation placental development: extended blastocyst culture and stem-cell derived trophoblast culture. We discuss current research efforts that employ these models and how such models might be used in the future to study the "black box" stage of human pregnancy.

Comparing prediction of ongoing pregnancy and live birth outcomes in patients with advanced and younger maternal age patients using KIDScore™ day 5: a large-cohort retrospective study with single vitrified-warmed blastocyst transfer.

BACKGROUND: The KIDScore™ Day 5 (KS-D5) model, version 3, is a general morphokinetic prediction model (Vitrolife, Sweden) for fetal heartbeat prediction after embryo transfer that was developed based on a large data set that included implantation results from a range of clinics with different patient populations, culture conditions and clinical practices. However, there was no study to comparing their pregnancy and live birth prediction ability among different maternal age. The aim of this study is to analyze the performance of KS-D5 in predicting pregnancy and live birth in various maternal age groups after single vitrified-warmed blastocyst transfer (SVBT). METHODS: A total of 2486 single vitrified-warmed blastocyst transfer (SVBT) cycles were analyzed retrospectively. Confirmed fetal heartbeat positive (FHB+) and live birth (LB+) rates were stratified by Society for Assisted Reproductive Technology (SART) maternal age criteria (< 35, 35-37, 38-40, 41-42 and ≥ 43 years of age). Within each age group, the performance of the prediction model was calculated using the AUC, and the results were compared across the age groups. RESULTS: In all age groups, the FHB+ rates decreased as the KIDScore decreased (P <  0.05). Conversely, the AUCs increased as the maternal age increased. The AUC of the < 35 age group (0.589) was significantly lower than the AUCs of the 41-42 age group (0.673) and the ≥43 age group (0.737), respectively (P <  0.05). In all age groups, the LB+ rates decreased as the KIDScore decreased (P <  0.05). Conversely, the AUCs increased as the maternal age increased. The AUC of the ≥43 age group (0.768) was significantly higher than the AUCs of other age groups (P <  0.05; < 35 age group = 0.596, 35-37 age group = 0.640, 38-40 age group = 0.646, 41-42 age group = 0.679). CONCLUSIONS: In the present study, we determined that the KIDScore model worked well for prediction of pregnancy and live birth outcomes in advanced age patients.

High grade trophectoderm is associated with monozygotic twinning in frozen-thawed single blastocyst transfer.

BACKGROUND: The aim of this study was to explore specific factors that predispose to monozygotic twinning (MZT) at the blastocyst stage. METHODS: This was a retrospective observational study of a cohort of 2863 pregnancies after single blastocyst transfer (SBT) between January 2011 and June 2019 in our hospital. MZT pregnancy was identified as the number of fetuses exceeded the number of gestational sacs (GSs) by transvaginal ultrasound at 6-7 gestational weeks. The incidences of MZT regarding the maternal age at oocyte retrieval, paternal age, ovarian stimulation protocol, fertilization method, endometrium preparation protocol, vitrified day, and the Gardner grading of the blastocyst were calculated. The serum estrogen (E2), progesterone (P) levels, endometrium thickness and serum hCG levels on day 11 after embryo transfer (ET) were compared between the MZT and singleton pregnancies. Statistical analyses were used appropriately. RESULTS: Fifty-one MZT pregnancies (1.78%) were identified. The only significant differences observed between MZT and singleton pregnancies were the proportion of TE grade (P = 0.022) and the hCG levels on day 11 after ET (P = 0.003). Multivariate logistic regression revealed that trophectoderm (TE) grade was an independent factor affecting MZT, the adjusted odds ratios (aORs) of grade A and B TE were 5.46 [95% confidential interval (CI) 1.48-20.16, P = 0.011) and 3.96 (95% CI 1.17-13.40, P = 0.027) compared to grade C respectively. There were no significant associations between the parental age, fertilization method, ovarian stimulation protocol, endometrium preparation protocol, vitrified day, expansion stage, inner cell mass (ICM) grade and MZT. CONCLUSIONS: TE grade is associated with MZT at the blastocyst stage, potentially mediated via increased secretion of hCG from more well developed TE. Increased hCG secretion in turn may prolong the implantation window to support the embryo splitting.

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.

Improving the clinical outcomes by extended culture of day 3 embryos with low blastomere number to blastocyst stage following frozen-thawed embryo transfer.

PURPOSE: This study aimed to investigate whether the extended culture of day 3 (D3) embryos with low blastomere number to blastocyst following frozen-thawed embryo transfer improved the clinical outcomes. METHODS: This was a retrospective study of clinical data of women undergoing in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) cycles in the Tangdu Hospital. The patients were divided into groups with 4-5, 6, 7-9 and > 9 cells based on the blastomere number of D3 embryos. The clinical outcomes were compared. RESULTS: In fresh transfer cycles, the implantation and clinical pregnancy rates significantly decreased, while the abortion rate significantly increased in the groups with 4-5 and 6 cells compared with those with 7-9 and > 9 cells. In frozen-thawed transfer cycles, the clinical pregnancy and implantation rates for a single blastocyst transfer cycle showed no significant differences in the groups with 4-5 and 6 cells compared with those with 7-9 and > 9 cells. However, the abortion rate was significantly higher in the group with 4-5 cells than in that with 7-9 and > 9 cells. In the double blastocyst transfer cycle, the clinical pregnancy rate showed no significant differences among the groups with 4-5, 6, and 7-9 cells. CONCLUSION: The implantation and clinical pregnancy rates of D3 embryos with 6 cells significantly decreased; these embryos were not considered as high-quality embryos. Extended culture of D3 embryos with ≤ 6 blastomeres to blastocysts, particularly 6-cell embryos, resulted in a similar clinical pregnancy rate as that of blastocysts derived from D3 embryos with ≥ 7 blastomeres.

Peptidomic analysis of blastocyst culture medium and the effect of peptide derived from blastocyst culture medium on blastocyst formation and viability.

High-quality in vitro human embryo culture medium can improve the blastocyst formation rate and blastocyst quality and be beneficial for the clinical application of single blastocyst transfer. Mammalian embryos can secrete protein products into the surrounding medium. As a group of bioactive molecules and degraded proteins, peptides have been shown to participate in various biological processes. Using liquid chromatography-tandem mass spectrometry, we performed comparative peptidomic analysis of human culture medium in blastocyst formation and nonblastocyst-formation groups. A total of 201 differentially expressed peptides originating from 157 precursor proteins were identified. Among these, a peptide derived from HERC2 (peptide derived from blastocyst culture medium [PDBCM]) passed through the zona pellucida, was distributed on the perivitelline space, was absent in arrest embryos and highly expressed in high-quality blastocysts compared with low-quality blastocysts, and significantly promoted blastocyst formation in a concentration-dependent manner. These results indicate that PDBCM may be a novel biomarker for predicting blastocyst formation and viability. The mechanism remains unclear and needs to be explored in the future.

In-vitro development of embryos derived from vitrified-warmed oocytes is delayed compared with embryos derived from fresh oocytes: a time-lapse sibling oocyte study.

RESEARCH QUESTION: Is there a difference in blastocyst formation between fresh and vitrified-warmed sibling oocytes and can this difference be attributed to changes in embryo morphokinetics? DESIGN: Between February 2016 and December 2017, 472 metaphase II (MII) oocytes in 67 donor-recipient cycles from 27 different healthy anonymous oocyte donors were allocated for fresh transfer (FSHO) (n = 220) to a synchronous recipient (n = 36) or vitrified (VITO) (n = 252) to be warmed and transferred to another recipient (n = 31). Embryos derived from the FSHO and their sibling VITO were analysed for morphokinetic development using time-lapse imaging, blastocyst formation and clinical outcome. RESULTS: Time-lapse analysis showed an overall delay in cleavage rate from the time of pronuclei disappearance up to the time of blastulation in the VITO compared with their sibling FSHO. Twelve morphokinetic variables were significantly different between the groups. On Day 5 significantly more FSHO embryos developed to blastocyst (expansion 1-6) and reached the full blastocyst stage (expansion 3-6) compared with the VITO embryos [53.2% (84/158) versus 40.0% (64/160); P = 0.0244 and 48.1% (76/158) versus 31.3% (50/160); P = 0.0028, respectively]. The embryo utilization rate was similar in both groups at the time of cryopreservation; 51.3% (FSHO) versus 45.0% (VITO) (P = 0.3124). The pregnancy rate per cycle was 47.2% (17/36) in FSHO patients and 48.4% (15/31) in VITO patients (P = 1). Limitations in this study: non-randomized, small study size and not powered to detect differences in clinical outcomes. CONCLUSIONS: Timing of development is altered and blastocyst formation is delayed in embryos derived from vitrified-warmed donor oocytes compared with their fresh sibling counterparts. Although preliminary results suggest that the clinical impact of this delay may be limited, this needs further investigation in larger randomized studies.

Value of transferring embryos derived from monopronucleated (1PN) zygotes at the time of fertilization assessment.

This paper is a retrospective analysis of the sole transfer of monopronucleated zygotes (1PN) embryos both in in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) to determine the value of transferring embryos formed from 1PN. In fresh cycles, 1PN cleavage-stage embryos (1PN cleavage fresh) were transferred. In frozen-thawed cycles, 1PN blastocyst-stage embryos (1PN blast frozen) were transferred. We used comparison groups: for fresh cycles, 2PN cleavage-stage embryos (2PN cleavage fresh) were transferred; and for frozen-thawed cycles, 2PN blastocyst-stage embryos (2PN blast frozen) were transferred. Comparison groups were matched for cycle and patient characteristics to the 1PN group. Finally, for fresh cycles, live birth rates (LBR) in the 1PN cleavage group were significantly lower than those in 2PN cleavage group, both for IVF [LBR = 7.64% vs. pregnancy rate (PR) = 22.12%, P = 0.003, respectively] and ICSI (LBR = 0% vs. LBR = 20.00%, P < 0.001, respectively). For frozen-thawed IVF cycles, the PR in the 1PN blastocyst group were comparable with those of the 2PN blastocyst group (1PN: LBR = 33.14% vs. 2PN: LBR = 37.24%, P = 0.289, respectively), while in ICSI, the PR in the 1PN blastocyst group were lower than those in the 2PN blastocyst group (LBR = 15.25% vs. LBR = 40.68%, P = 0.002, respectively). So, for IVF, blastocyst culture was capable of selecting normal 1PN embryos for transfer and achieves satisfying outcomes. However, for ICSI, blastocyst culture was not effective enough to eliminate abnormal embryos and 1PN embryo transfer needed to be treated with caution.

Relationship between follicular volume and oocyte competence, blastocyst development and live-birth rate: optimal follicle size for oocyte retrieval.

OBJECTIVE: To analyze oocyte competence in gonadotropin-releasing hormone agonist (GnRHa) stimulation cycles with regard to maturity, fertilization and blastocyst rate, as well as clinical outcome (pregnancy and live-birth rate), in relation to follicular volume, measured by three-dimensional transvaginal sonography (3D-TVS), and follicular fluid composition. METHODS: This was a prospective single-center study conducted between June 2012 and June 2014, including 118 ovum pick-ups with subsequent embryo transfer. Ovarian stimulation was performed using the GnRHa long protocol. Of 1493 follicles aspirated individually, follicular volume was evaluated successfully in 1236 using automated 3D-TVS during oocyte retrieval. Oocyte maturity and blastocyst development were tracked according to follicular volume. Intrafollicular concentrations of estradiol, testosterone, progesterone, luteinizing hormone, follicle-stimulating hormone and granulocyte-colony stimulating factor were quantified by immunoassay. Clinical outcome, in terms of implantation rate, (clinical) pregnancy rate, miscarriage and live-birth rate (LBR), was evaluated. RESULTS: Follicles were categorized, according to their volume, into three arbitrary groups, which included 196 small (8-12 mm/0.3-0.9 mL), 772 medium (13-23 mm/1-6 mL) and 268 large (≥ 24 mm/> 6 mL) follicles. Although oocyte recovery rate was significantly lower in small follicles compared with medium and large ones (63.8% vs 76.6% and 81.3%, respectively; P < 0.001), similar fertilization rates (85.1% vs 75.3% and 81.4%, respectively) and blastocyst rates (40.5% vs 40.6% and 37.2%, respectively) per mature metaphase II oocyte were observed. A trend towards higher LBR after transfer of blastocysts derived from small (< 1 mL) follicles compared with medium (1-6 mL) or large (> 6 mL) follicles (54.5% vs 42.0%, and 41.7%, respectively) was observed. No predictive value of follicular fluid biomarkers was identified. CONCLUSIONS: Our data indicate that the optimal follicular volume for a high yield of good quality blastocysts with good potential to lead to a live birth is 13-23 mm/1-6 mL. However, oocytes derived from small follicles (8-12 mm/0.3-0.9 mL) still have the capacity for normal development and subsequent delivery of healthy children, suggesting that aspiration of these follicles should be encouraged as this would increase the total number of blastocysts retrieved per stimulation. Copyright © 2017 ISUOG. Published by John Wiley & Sons Ltd.

Should all embryos be cultured to blastocyst for advanced maternal age women with low ovarian reserve: a single center retrospective study.

The purpose of this study is to determine whether all-blastocyst-culture can benefit advanced maternal age (AMA) patients with low ovarian reserve. We retrospectively analyze AMA patients with low ovarian reserve undergoing IVF/ICSI in Reproductive Medicine Research Center of the Sixth Affiliated Hospital of Sun Yat-sen University from May 2016 to August 2017. We divide them into group A (all-blastocyst-culture) and group B (none-blastocystculture) based on different culture approach of D3 transferrable embryo. We compared the cumulative biochemical pregnancy rate and cumulative clinical pregnancy rate between them. A total of 261 eligible patients are included, with 151 in group A and 110 in group B. The basic conditions of the two groups show no difference. Cumulative biochemical pregnancy rate/clinical pregnancy rate in group A is higher than that of group B, though not statistically significant (p > .05). After removing patients canceling embryo transfer due to failure in blastocyst culture, the cumulative biochemical pregnancy rate/clinical pregnancy rate in group A is significantly higher than group B (p < .05). Multivariate analysis showed that female age and the number of D3 transferrable embryo are risk factors for the failure of blastocyst culture (OR = 0.672 and 2.270, 95%CI 0.511-0.883 and 1.203-4.284, p = .004 and p = .011, respectively). Hence, we drew to conclusion that all-blastocyst-culture will not adversely affect the pregnancy outcome of AMA patients with low ovarian reserve.

Correlation between the pronucleus size and the potential for human single pronucleus zygotes to develop into blastocysts : 1PN zygotes with large pronuclei can expect an embryo development to the blastocyst stage that is similar to the development of 2PN zygotes.

PURPOSE: In this study, we examined the correlation between pronucleus size and the potential for human single pronucleus (1PN) zygotes to develop into blastocysts after IVF and ICSI. METHODS: This study included 112 patients who underwent a total of 112 cycles of IVF/ICSI. To evaluate embryo development, 1PN zygotes were compared with 2PN zygotes in the same IVF/ICSI cycle (control cycles) using time-lapse live embryo imaging. To assess the potential for blastocyst formation, cutoff values for pronuclear area and diameter were established through receiver operating characteristic curve analysis, after which 1PN zygotes were classified based on those cutoff values. RESULTS: Among 1PN zygotes cultured to day 5/6, the rate of embryo development was significantly lower than from 2PN zygotes. However, the rates of blastocyst formation and good quality blastocysts from 1PN zygotes with large pronuclear areas (≥ 710 µm2) or diameters (≥ 31 µm) were significantly higher than from 1PN zygotes with smaller pronuclear areas (≤ 509, 510-609, and 610-709 µm2) or diameters (≤ 24, 25-27,and 28-30 µm) (P < 0.01). Moreover, the results for 1PN zygotes with large pronuclei were similar to those for 2PN zygotes. CONCLUSIONS: The developmental potential of 1PN zygotes with large pronuclear areas (≥ 710 µm2) or diameters (31 µm) appears to be similar to that of 2PN zygotes, and measurement of pronuclear area or diameter in 1PN zygotes is a simple, potentially useful, clinical method.

Pregnancy rates of day 4 and day 5 embryos after culture in an integrated time-lapse incubator.

BACKGROUND: The aim of this study was to compare pregnancy rates in patients undergoing IVF/ICSI with embryo transfer after 4 and 5 days of culture in a closed incubation system with integrated time-lapse imaging. METHODS: Out of n = 2207 in vitro fertilization (IVF)/ intracytoplasmic sperm injection (ICSI) cycles performed between January 2011 and April 2016 at a tertiary referral university hospital, a total of n = 599 IVF/ICSI cycles with prolonged embryo culture in an integrated time-lapse system (EmbryoScope© (Vitrolife)) until day 4 or 5 were retrospectively analyzed with regard to embryo morphology and pregnancy rates. RESULTS: A transfer on day 5 compared to a transfer on day 4 did not result in higher implantation and clinical pregnancy rates (IR 29.4% on day 4 versus 33.0% on day 5, p = 0.310; CPR 45.2% on day 4 versus 45.7% on day 5, p = 1.0). The percentage of ideal embryos transferred on day 4 was comparable to the rate of ideal embryos transferred on day 5 (41.6% versus 44.1%, p = 0.508). However, on day 4 a significantly higher number of embryos was transferred (1.92 on day 4 versus 1.84 on day 5, p = 0.023), which did not result in higher rates of multiple pregnancies. CONCLUSIONS: Pregnancy rates in IVF/ICSI cycles with integrated time-lapse incubation and transfer on day 4 and 5 are comparable. This finding provides the clinician, IVF laboratory and patient with more flexibility. TRIAL REGISTRATION: This study was retrospectively registered by the local ethics committee of the University of Heidelberg on December 19, 2016 (registration number S-649/2016).

Optimal embryo transfer strategy in poor response may include freeze-all.

PURPOSE: In this retrospective cohort study, we investigated the best embryo transfer strategy in ICSI cycles with ≤4 oocytes collected at oocyte retrieval. METHODS: Women who underwent antagonist co-treatment COS for ICSI treatment between January 2010 and December 2015 at a private ART clinic (N = 2263). Eight hundred seventy-nine women (group 1) had ≤4 oocytes collected at oocyte retrieval, of whom 645 (group A) had cleavage stage embryo transfer (ET), and 234 (group B) had blastocyst ET. One thousand three hundred eighty-four women (group 2) had 10-15 oocytes collected at oocyte retrieval, of whom 676 (group C) had cleavage stage ET, and 708 women (group D) had blastocyst ET. Blastocyst vitrification was performed using the Cryotop method and FET using artificial cycles. RESULTS: In group 1, the cancellation rate was significantly lower in group A (25.2 vs 38 %). The pregnancy rate (PR), clinical PR, implantation rate (IR), and live birth rate (LBR) per ET and per oocyte retrieval were all lower in group A. The clinical PR, IR, and LBR per ET of vitrified-warmed blastocyst ET were significantly the highest. In group 2, the cycle cancellation rate was significantly lower in group C (3.5 vs 13.4 %). The PR, clinical PR, and IR per ET and per oocyte retrieval were all lower in group C. The LBR per ET was significantly lower, but the LBR per oocyte retrieval was not significantly lower in group C. Again, the PR, clinical PR, and IR per ET of vitrified-warmed blastocyst ET were significantly the highest. CONCLUSIONS: Day 5 ET strategy has been reserved for normal or high responders. The improved pregnancy outcomes from blastocyst culture and cryopreservation may challenge ART to extend this benefit to poor responders.