Is there an optimal window of time for transferring single frozen-thawed euploid blastocysts? A cohort study of 1170 embryo transfers.

STUDY QUESTION: Is there an optimal window of time when the transfer of single frozen-thawed euploid blastocysts is associated with a maximal live birth rate (LBR)? SUMMARY ANSWER: Performing a single frozen-thawed euploid blastocyst transfer at 160 ± 4 h post-hCG trigger in modified-natural frozen-thawed embryo transfer (FET) cycles was independently associated with a higher LBR as compared to transfers outside this window; however, in natural FET cycles, LBRs were comparable across a wider range of time intervals. WHAT IS KNOWN ALREADY: There is compelling evidence for maintaining embryo-endometrial synchrony to optimize clinical outcomes following FETs, which could potentially be achieved by matching the transfer time of an embryo post-ovulation to its developmental age post-oocyte retrieval. For modified-natural cycles, ovulation is widely accepted to occur ∼40 h following the hCG trigger, whilst ovulation following spontaneous LH surge onset is thought to vary from 24 to 56 h. STUDY DESIGN, SIZE, DURATION: This is a multicentered retrospective cohort study analyzing 1170 single frozen-thawed euploid blastocyst transfers following trophectoderm biopsy and preimplantation genetic testing (PGT) between May 2015 and February 2019. Limiting the analysis to single euploid embryo transfers allowed for a more accurate estimation of the endometrial synchrony factor by controlling for the developmental stage of the embryo (full blastocyst or more advanced) and its genetic composition. LBR per FET was the primary outcome measure. PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients underwent natural or gonadotrophin-induced preparation of the endometrium, with serial serum oestradiol, LH and progesterone measurements. Optimally timed transfers were predefined as those conducted 120 ± 4 h post-ovulation since biopsy and subsequent cryopreservation of full blastocysts which is usually performed at 116-124 h post-oocyte retrieval. This was considered the equivalent of 160 ± 4 h post-hCG trigger in modified-natural cycles (n = 253), as ovulation was assumed to occur ∼40 h after the hCG trigger. For natural cycles (n = 917), this was also considered to be, on average, 160 ± 4 h post the spontaneous LH surge. Thus, study groups were determined as those with optimal timing or not, and additional exploratory and subgroup analyses were performed, varying the time window in terms of onset and width, both overall and per endometrial preparation protocol. Statistical analysis was performed using the generalized estimating equations (GEE) framework to control for the clustered nature of the data while adjusting for potential confounders. MAIN RESULTS AND THE ROLE OF CHANCE: Overall, LBRs were significantly higher when the transfer had been performed at 160 ± 4 h post-hCG trigger or LH surge onset compared to when it had been performed outside this window (44.7% vs 36.0%; P = 0.008). A multivariable regression GEE model including the cycle type (natural versus modified-natural), previtrification embryo quality (top versus good quality), embryo stage (fully hatched versus hatching or earlier blastocyst), vitrification day (D5 versus D6) and survival rate (>90% versus <90%) as covariates, confirmed that, overall, embryo transfers conducted 160 ± 4 h post-hCG trigger or LH surge onset (the assumed equivalent of 120 ± 4 h post-ovulation) were associated with a significantly higher LBR (relative risk (RR) 1.21, 95% CI 1.04-1.41). Subgroup exploratory analyses per endometrial preparation protocol demonstrated that these findings were primarily present in the modified-natural cycle group (RR 1.52, 95% CI 1.15-1.99), whilst the natural cycle group showed comparable LBRs across a wider range of time intervals. Moreover, the overall LBR for the natural group (36.8%; 95% CI 33.7-39.9%) was lower than that of the modified-natural group (41.3%; 95% CI 35.4-47.1%), suggesting that there likely remains a greater potential to further optimize the timing of natural cycle embryo transfers. LIMITATIONS, REASONS FOR CAUTION: As with all retrospective studies, the presence of residual unknown bias cannot be excluded. Additionally, patients included in this study were a selected group who underwent PGT for specific reasons and hence the results obtained might not be directly applicable to the general population or embryos that have not undergone embryo biopsy. Furthermore, the criteria utilized to interpret hormonal data from natural cycles were specifically adopted for the present study and need to be validated in further studies. WIDER IMPLICATIONS OF THE FINDINGS: The results of this study highlight the significance of embryo-endometrial synchrony for the optimization of frozen embryo transfer outcome. However, it also clearly supports that the implantation window is in most cases wide and the achievement of live birth is possible with relatively high success rates even outside the optimal window of 160 ± 4 h post-trigger for modified-natural cycles and across a range of time intervals for natural cycles. Additionally, this study suggests that implantation rates could be further optimized in natural cycles by improving methods of assessing embryo-endometrial synchrony. STUDY FUNDING/COMPETING INTEREST(S): C. V. is supported by a National Health and Medical Research Council Early Career Fellowship (GNT1147154). No other funding was received for this study and there are no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Oocyte meiotic spindle morphology is a predictive marker of blastocyst ploidy-a prospective cohort study.

OBJECTIVE: To evaluate oocyte meiotic spindle (OMS) morphology at intracytoplasmic sperm injection (ICSI) as a predictor of blastocyst ploidy and whether OMS morphology could aid standard morphology-based blastocyst selection. DESIGN: Prospective cohort study. SETTING: In vitro fertilization clinic. PATIENT(S): Patients undergoing ICSI cycles with an intention to perform preimplantation genetic testing for aneuploidy (PGT-A) from October 2014 to December 2017. INTERVENTION(S): The OMS was visualized with the use of polarized light microscopy at the time of ICSI and the morphology classified as normal, dysmorphic, translucent, not visible, or in telophase. Blastocyst biopsy for PGT-A was performed on embryos with suitable development. MAIN OUTCOME MEASURE(S): The association of OMS morphology with the resulting blastocyst ploidy was evaluated on an "intention-to-treat" (ITT) and an "as-treated analysis" (ATA) basis. RESULT(S): The morphology of 2,056 OMSs were classified. A strong association of OMS morphology with fertilization, cleavage to at least 6 cells on day 3, and good/top-quality blastocyst formation was present. Normal OMS was positively associated with blastocyst euploidy compared with all other OMS types combined, per either ITT or ATA. Even after controlling for female age, blastocyst quality, and developmental stage, the presence of a normal OMS was strongly associated with the probability of blastocyst euploidy. CONCLUSION(S): OMS morphology is a predictive marker of blastocyst ploidy and can potentially aid standard morphology-based blastocyst selection.

Is more better? A higher oocyte yield is independently associated with more day-3 euploid embryos after ICSI.

STUDY QUESTION: Is the number of oocytes retrieved after ovarian stimulation for ICSI independently associated with the number of day-3 euploid embryos (EE)? SUMMARY ANSWER: A larger oocyte yield is independently associated with more day-3 EE, although the expected benefit decreases significantly with advancing age. WHAT IS KNOWN ALREADY: Although traditionally ovarian stimulation aims at collecting more than one oocyte in order to increase the chance of pregnancy, there is evidence suggesting that excessive ovarian response leads to lower live birth rates. Whether a larger oocyte yield after ovarian stimulation is associated with the genetic composition of the resulting embryos and therefore with their reproductive potential is still largely unknown. STUDY DESIGN, SIZE, DURATION: This is a multi-centered retrospective cohort study analyzing 724 cycles of preimplantation genetic testing for aneuploidy (PGT-A) cycles using day-3 biopsy and array-comparative genomic hybridization between March 2011 and December 2016 in three laboratories. PARTICIPANTS/MATERIALS, SETTING, METHODS: The primary outcome measure was the number of EE on day-3. Statistical analysis was performed using the generalized estimating equations (GEE) framework and multivariate regression models to control for the clustered nature of the data while adjusting for potential confounders. MAIN RESULTS AND THE ROLE OF CHANCE: A multivariate regression GEE model including all significant population and stimulation characteristics as covariates as well as an interaction term between female age and number of oocytes revealed that the number of oocytes retrieved was still positively associated with the number of EE (coeff: +0.40, 95% CI: 0.24-0.56). The interaction term was highly significant (coeff: -0.01, P < 0.001) indicating an effect modifying role of female age on the association of oocytes retrieved with the number of EE. The number of oocytes retrieved was also positively associated with cumulative live birth rates (odds ratio: 1.07, 95% CI: 1.03-1.12). LIMITATIONS, REASONS FOR CAUTION: This study is retrospective and the presence of residual unknown bias cannot be excluded. Furthermore, the population analyzed in this study might not be completely representative of the general population undergoing ICSI. WIDER IMPLICATIONS OF THE FINDINGS: These results provide an explanatory mechanism for the recently published positive association between the number of oocytes retrieved and cumulative live birth rates. STUDY FUNDING/COMPETING INTEREST(S): CAV is supported by a NHMRC Early Career Fellowship (GNT1147154)/ No competing interests to declare.

Is oocyte meiotic spindle morphology associated with embryo ploidy? A prospective cohort study.

OBJECTIVE: To explore whether an association exists between oocyte meiotic spindle morphology visualized by polarized light microscopy at the time of intracytoplasmic sperm injection and the ploidy of the resulting embryo. DESIGN: Prospective cohort study. SETTING: Private IVF clinic. PATIENT(S): Patients undergoing preimplantation genetic screening/diagnosis (n = 113 patients). INTERVENTION(S): Oocyte meiotic spindles were assessed by polarized light microscopy and classified at the time of intracytoplasmic sperm injection as normal, dysmorphic, translucent, telophase, or no visible spindle. Single blastomere biopsy was performed on day 3 of culture for analysis by array comparative genomic hybridization. MAIN OUTCOME MEASURE(S): Spindle morphology and embryo ploidy association was evaluated by regression methods accounting for non-independence of data. RESULT(S): The frequency of euploidy in embryos derived from oocytes with normal spindle morphology was significantly higher than all other spindle classifications combined (odds ratio [OR] 1.93, 95% confidence interval [CI] 1.33-2.79). Oocytes with translucent (OR 0.25, 95% CI 0.13-0.46) and no visible spindle morphology (OR 0.35, 95% CI 0.19-0.63) were significantly less likely to result in euploid embryos when compared with oocytes with normal spindle morphology. There was no significant difference between normal and dysmorphic spindle morphology (OR 0.73, 95% CI 0.49-1.08), whereas no telophase spindles resulted in euploid embryos (n = 11). Assessment of spindle morphology was found to be independently associated with embryo euploidy after controlling for embryo quality (OR 1.73, 95% CI 1.16-2.60). CONCLUSION(S): Oocyte spindle morphology is associated with the resulting embryo's ploidy. Oocytes with normal spindle morphology are significantly more likely to produce euploid embryos compared with oocytes with meiotic spindles that are translucent or not visible.

Is measurement of progesterone level prior to FSH stimulation useful in GnRH-antagonist cycles?

AIM: To reduce patient inconvenience during in vitro fertilization (IVF) cycles, some protocols delay intensive monitoring until mid-follicular stimulation. Others assess hormone levels prior to follicle-stimulating hormone (FSH) administration, not commencing stimulation until baseline progesterone (P4) levels (< 5 nmol/l) are achieved. Higher P4 levels (> 4.8 nmol/L) on the day of FSH trigger have been implicated in poorer pregnancy rates. This study evaluates the association of P4 levels at day 1-2 in gonadotrophin-releasing hormone (GnRH)-antagonist cycles with pre-trigger P4 levels and clinical pregnancy rates (CPRs). METHOD: All fresh GnRH-antagonist IVF cycles between June 2011 and June 2012, in which pre-FSH P4 levels were not routinely performed (group 1), were retrieved from the IVF Australia database and compared with controls (group 2). RESULTS: There were 163 cycles in each group. P4 levels on the day of trigger were significantly higher in group 1 (3.75 vs. 2.77, p < 0.05). The incidence of pre-trigger P4 levels >4.8 nmol/l was significantly higher in group 1 (30 vs. 16, p < 0.05). The number of oocytes retrieved was higher in group 1 (11.1 vs. 9, p < 0.05), however fertilization rates were significantly lower in that group (53.6% vs. 61.2%, p < 0.05); CPRs were similar between the two groups (27.8% vs. 31.8%, p = ns). Overall, pregnancy rates were lower in cycles with pre-trigger P4 level of > 4.8 nmol/L compared with those with lower levels (15% vs. 32.5%, p < 0.05). CONCLUSION: We found that measurement of P4 level at early follicular phase was associated with significantly lower pre-trigger levels. However, this did not translate into a difference in CPR between the monitored and unmonitored groups. We have confirmed that elevation in pre-trigger P4 level is associated with halving of the CPR, indicating that the most important P4 measurements are those in the late follicular/pre-trigger phase.

Does meiotic spindle normality predict improved blastocyst development, implantation and live birth rates?

OBJECTIVE: To investigate the relationship between spindle normality and: 1) blastocyst formation; 2) implantation rates; and 3) live birth rates. DESIGN: Prospective observational study. SETTING: A private fertility clinic. PATIENT(S): One hundred patients ≤40 years old undergoing intracytoplasmic sperm injection (ICSI). INTERVENTION(S): Meiotic spindles were imaged before ICSI with the use of Oosight microscopy. With the use of specific criteria, spindle normality was independently assessed by two researchers. Blastocysts were chosen for transfer by standard light microscopic morphologic criteria by researchers who were blinded to the spindle assessment. MAIN OUTCOME MEASURE(S): 1) Blastocyst formation; 2) implantation; and 3) live birth rates. RESULT(S): A total of 808/920 oocytes were metaphase II. Of those, 711 (88%) had a visible spindle: 205 (29%) were normal spindles (NS) and 506 (71%) abnormal spindles (AS). Fertilization rates were significantly higher in NS oocytes. Although NS and AS oocytes both formed morphologically good-quality blastocysts, implantation and live birth rates were higher from NS oocytes. All ongoing pregnancies resulted from NS oocytes. CONCLUSION(S): Spindle assessment with polarized light microscopy provides an early predictor of the pregnancy potential of that oocyte. AS oocytes can form good-quality blastocysts, but these appear to have little chance of live birth. Therefore, spindle assessment should improve the selection of the best embryo for single embryo transfer.