Time-lapse monitoring of fertilized human oocytes focused on the incidence of 0PN embryos in conventional in vitro fertilization cycles

We aimed to investigate why the incidence of embryos derived from oocytes with no pronuclei (0PN) decreases using time-lapse monitoring (TLM) versus fixed-point assessment in conventional IVF cycles. We analyzed 514 embryos monitored with TLM 6–9 h after insemination and 144 embryos monitored using microscopic assessment 18–21 h after insemination. The primary endpoint of this study was the incidence of 0PN-derived embryos in short insemination followed by TLM. The secondary endpoint was the duration of insemination. As exploratory endpoints, we analyzed the blastulation rate and cryo-warmed blastocyst transfer outcome of embryos with early PN fading, whereby PN disappeared within < 20 h following the initiation of insemination. The incidence of 0PN-derived embryo reduced more significantly through TLM than through fixed-point observation. The microscopic assessment time was more significantly delayed in the 0PN-derived embryo than that in the 2PN-derived embryo. The embryo with early PN fading formed good-quality blastocysts, and their pregnancy outcomes were similar to those of other embryos. Most 0PN-derived embryos in the fixed-point assessment might have resulted from missed observation of PN appearance in the early-cleaved embryos. TLM or strict laboratory schedule management may reduce 0PN-derived embryos by reducing missed PN observations.

Transfer of Cleavage-Stage Embryos May Benefit Normal Responders With Low Rate of Morphologically Good Embryos Formation On Day 3: A Comparison Study

Background Do morphologically good (MG) embryos from patients with high and low rate of MG embryos on day 3 (RMD3) show similar developmental potential (DP)? Methods This respective study finally included a total of 916 fresh cycles and related 1074 FET cycles from Jan 2017 to May 2020 in our reproductive center. Cycles with high RMD3 were defined as the H group, while cycles with low RMD3 were defined as the L group. The basic characteristics of patients and fresh cycles, blastulation rate, and clinical outcomes were compared between the H and L groups in either ET cycles with MG day 3 cleavage embryos (ETC group) or ET cycles with MG blastocysts (ETB group). Results The overall characteristics of patients and cycles were grossly comparable between the H and L groups either in ETC or ETB groups. In ETB group, useable blastocysts formation rate, implantation rate and live birth rate was significantly reduced in the L group, compared to the H group;In ETC group, useable blastocysts formation rate was significantly reduced in the L group. However, implantation rate and livebirth rate was similar between the L and H groups. Conclusion The in vitro DP of MG day 3 embryos and in vivo DP of MG blastocysts were reduced significantly, while a similar in vivo DP of MG day 3 embryos was observed in patients with low RMD3 as compared to patients with high RMD3. It seems that direct transfer of day 3 MG embryos instead of extended culture may benefit patients with RMD3.

P–615 The effect of late-follicular phase progesterone rise on embryo ploidy, embryo quality and cumulative live birth rates following a freeze-only strategy

Study question Is late-follicular phase progesterone elevation (PE) associated with a deleterious effect on embryo euploidy, embryo blastulation and cumulative live birth rates (CLBRs)? Summary answer Late-follicular phase PE has no impact on impact on embryo euploidy rate, embryo blastulation rate nor on the CLBR. What is known already The effect of PE in ART outcomes has been extensively studied, yielding so far conflicting results. While some authors claim it is only detrimental to endometrial receptivity, others have suggested that it may also impair oocyte/embryo quality. Moreover, little is known regarding the potential effect PE may have on embryo ploidy and, consequently, CLBR. Study design, size, duration A multicenter retrospective cross-sectional study was performed between August 2017 and December 2019. A total of 1495 ICSI cycles coupled with preimplantation genetic diagnosis for aneuploidies (PGT-A) and deferred frozen embryo transfer (FET) were analyzed. Participants/materials, setting, methods All patients underwent ovarian stimulation with GnRH antagonist protocol and performed a serum progesterone measurement at one of the participating private fertility clinics on the day of trigger. The sample was stratified according to the progesterone levels: normal (≤1.50 ng/ml) and high (&gt;1.50 ng/ml). The primary outcome was the embryo euploidy rate. Secondary outcomes were the number of euploid blastocysts, the blastulation rate and CLBR. Main results and the role of chance Late-follicular phase PE was associated with higher late-follicular estradiol levels (2847.56±1091.10 pg/ml vs. 2240.94± 996.37 pg/ml, p &lt; 0.001) and more oocytes retrieved (17.67±8.86 vs. 12.70±7.00, p &lt; 0.001). The number of euploid embryos was higher in the PE group (2.32±1.74 vs. 1.86±1.42, p &lt; 0.001), whereas the embryo euploidy rate (48.3% [44.9%–51.7%] vs. 49.1% [47.7%–50.6%] and blastulation rate (47.1% [43.7%–50.5%] vs. 51.0% [49.7%–52.4%]) were comparable between the two groups. Likewise, no significant differences were found regarding the live birth rate (LBR) after the first FET (34.1% vs. 31.1%, p = 0.427) nor the CLBRs (38.9% vs. 37.0%, p = 0.637). Mixed-model analysis was performed in order to account for the clustering of cycles in the same patient. Adjusting for patients’ age, PE and BMI, PE failed to demonstrate any effect on the embryo euploidy rate (OR 1.03 [95% CI 0.89–1.20]). Mixed-model analysis for the number of euploid embryos was also performed. After adjusting for PE, age, BMI and ovarian response, PE did not affect the number of euploid embryos (0.02 [95%CI –0.21;0.25]. Multivariate logistic regression adjusted for PE, age, BMI and ovarian response revealed that PE was not associated with the CLBR (adjOR 0.96 [95% CI 0.66–1.38]). Limitations, reasons for caution Limitations of the study include its retrospective nature. Moreover, including only GnRH antagonist protocol and ICSI does not allow the extrapolation of these results to other populations. Wider implications of the findings: Our findings question results from previous studies claiming a detrimental effect of PE on embryo implantation potential. According to our results, PE has no impact on embryo euploidy rate, blastulation rate nor on CLBRs. Trial registration number Not applicable

P–031 The effect of ejaculatory abstinence period on embryological and clinical outcomes in ICSI cycles: A retrospective analysis of 3,353 cycles

Study question Does ejaculatory abstinence period in male affect embryological and pregnancy outcomes following fresh embryo transfers in ICSI cycles? Summary answer Shorter ejaculatory abstinence period is associated with lower triploid zygotes rate per ICSI cycle but it does not affect clinical outcomes after fresh embryo transfers. What is known already Lower sperm quality may negatively impact on fertilisation rate and embryo morphokinetic parameters after ICSI and the effect of the ejaculatory abstinence period before semen collection on seminal parameters and sperm quality has been widely reported. However, the impact of ejaculatory abstinence on clinical outcomes is still controversial. WHO (World Health Organization) guideline recommended that abstinence period should be 2–7 days. Even so, there are no larger prospective trials determining the optimal timing for ejaculatory abstinence period for infertile couples. Study design, size, duration This is a single center retrospective observational study of 3,353 fresh cycles from January 2017 to December 2020. Semen analysis was done according to the WHO criteria. Exclusion criteria for this study were frozen gametes and cycles with no retrieved oocytes. Primary outcomes were fertilization rate and triploid zygotes rate. Secondary outcomes were blastulation rate, ongoing pregnancy rate and live birth rate per fresh embryo transfer. Participants/materials, setting, methods The correlation between ejaculatory abstinence and continuous outcomes was evaluated by Spearman’s correlation analysis in order to detect potential non-linear associations. Generalized linear model and logistic regression were used, respectively for continuous and binary outcomes, in order to adjust for confounders such as female age, male age, number of retrieved oocytes, percentage of mature oocytes, infertility causes, seminal volume, sperm concentration and total progressive sperm motility. A p value &lt;0.05 was considered significant. Main results and the role of chance The male mean age was 40.3±5.5 and mean duration of abstinence was 2.9±1.7 days. The mean age of female patients was 38.2±4.0. Higher ejaculatory abstinence period was associated with a higher sperm concentration (Spearman p = 3.1x10–6) but not with a higher total sperm progressive motility. Even so, no significant correlation with EA were observed when considering fertilization rate, blastulation rate, ongoing pregnancy and live birth rate per transfer in analyzed cycles. Triploid zygote rate was positively associated with a higher ejaculatory abstinence period. For the ejaculatory abstinence period of 1 day (n = 64), 2 days (n = 1523), 3 days (n = 1032), 4 days (n = 408), 5 days (n = 174), 6 days (n = 47) and ≥7 days (n = 105) the mean triploid rate was 2.4%, 2.4%, 2.5%, 4.1%, 3.6%, 5.4% and 4.3%, respectively (Spearman p = 9x10–3). Triploid zygote rate was independent of semen volume, concentration and total progressive motility. Limitations, reasons for caution This is a large observational study with a retrospective data collection. Despite our methodological approach, the presence of biases related to retrospective design can not be excluded and it may be a reason for caution. Wider implications of the findings: Our results demonstrate that ejaculatory abstinence period do not affect blastulation, ongoing pregnancy and live birth rates. The current findings discourage an abstinence time longer than 3 days due to its association with a higher abnormal fertilization rate. Trial registration number Not applicable

P–342 Are blastulation and clinical pregnancy rates in women with endometriomas different than those without?

Study question Does the presence of endometrioma during ovarian stimulation affect blastulation and clinical pregnancy rates (CPR)? Summary answer Blastulation rates were similar in women with endometrioma compared to women without. Likewise, CPR were comparable. What is known already Although relationship of endometriosis and subfertility is well-established, its mechanism is still under investigation. Decreased oocyte quality, resulting from anatomical and/or inflammatory factors is one of the prominent culprits. Most studies regarding endometriosis and oocyte quality are highly heterogeneous and effect of endometriosis on oocyte quality is yet to be determined. Blastulation is thought as a surrogate marker for oocyte quality. Thus, it may be possible that detrimental effect of the presence of endometrioma during ovarian stimulation can be indirectly assessed by blastulation. Study design, size, duration Records of all women who underwent assisted reproductive technology treatment at Koc University Hospital Assisted Reproduction Unit between 2016 and October 2020 were screened for this retrospective study. All women who had endometrioma(s) during ovarian stimulation were included in the study group (EG) (n = 71). They were matched with women diagnosed with tubal factor or unexplained infertility who underwent oocyte pickup within the same period to form the control group (CG) (n = 104). Participants/materials, setting, methods All women underwent antagonist or long protocol. All embryos were cultured until blastocyst stage regardless of the number of oocytes or embryos available. Size/location of endometriomas, number of oocytes retrieved, number of available blastocysts, positive pregnancy test per cycle and clinical pregnancy rate per cycle were recorded. Blastulation rate was calculated as number of available blasts divided by the number of metaphase-II oocytes. Embryos were transferred in a fresh or artificially prepared frozen-thawed cycle. Main results and the role of chance There were 71 women in EG and 104 women in CG, which included 30 women with tubal and 74 with unexplained infertility. Median endometrioma size was 26 mm(22–33). Twenty-three patients in EG had history of endometrioma excision (31.3%). Median age [35.0 years (31.0–39.0) vs 34 (32.0–36.0), p = 0.26] and serum AMH levels [1.8 (1.1 - 4.2) vs 2.3 (1.3 - 3.7) ng/dL, p = 0.91] were similar in EG and CG, respectively. Body mass index in kg/m2 [21.8 (20.2–24.6) vs 24 (21.5–27.9), p &lt; 0.01] and infertility duration in years [2 (1–2.6) vs 3 (2–5), p &lt; 0.01] were significantly lower in EG. Number of retrieved oocytes [8 (5–12) vs 12 (7–15.8), p &lt; 0.01)] and metaphase-II oocytes [6 (4–10) vs 8.5 (6–12), p &lt; 0.01] were lower in EG group compared to CG group. However, blastulation rate per MII oocyte were similar between the EG and CG [(0.25 (0.20–0.41) vs 0.30 (0.14–0.50), respectively, p = 0.58]. Adjusted analysis for age and number of MII oocytes revealed similar finding. Positive pregnancy test per cycle was similar at 53.5% vs 61.5% in EG and CG, respectively (p = 0.3). CPR were similar between the EG and CG (45% vs 58%, respectively, p = 0.10). Limitations, reasons for caution Retrospective design, lack of live birth information are the main limitations of our study. Wider implications of the findings: Presence of endometrioma during ovarian stimulation does not seem to adversely affect blastulation rates. While this is reassuring regarding oocyte quality, further research is required to assess its effect on live birth. Trial registration number Not applicable

P–757 The risk of aspirin and prednisone using in women with antithyroid antibodies undergoing assisted reproductive technology

Study question Is it safe using aspirin (A) and prednisone (P) before pregnancy among women with antithyroid antibodies (ATAbs) undergoing assisted reproductive technology? Summary answer Combination therapy of aspirin and prednisone didn’t improve likelihood of clinical pregnancy, but increased miscarriage rate. What is known already Compared with women with negative-ATAb, women with positive-ATAb had a lower live birth rate and a higher miscarriage rate. Insufficient evidence existed to determine whether aspirin and prednisone therapy improved the success of pregnancy following assisted reproductive technology (ART) in ATAb-positive euthyroid women. Aspirin and prednisone were used frequently in clinical practice, but the use of these medicines before pregnancy during ART process is still controversial, and the risks of these medicines were not well understood. Study design, size, duration A prospective study involving 268 women with unexplained reason for infertility who tested positive for antithyroperoxidase antibody (TPOAb) and/or thyroglobulin antibody (TgAb) were being treated for infertility at the Second Affiliated Hospital of Zhejiang University School of Medicine, Ningbo Women and Children’s Hospital and People’s Hospital of Jinhua from October 2017 to July 2020. Their TSH level ranged from 0.35–4.0mIU/ml and they all underwent fresh embryo transfer. Participants/materials, setting, methods Overall, a total of 268 ATAb-positive women were divided 2 groups: group A: no treatment; B: A+P. Both medicines were used in the lowest effective dose. Between the two groups, we measured oocytes retrieved, fertilization rate, high-quality embryo rate, blastulation rate, cleavage rate,implantation rate, likelihood of clinical pregnancy and miscarriage rate. Kruskal-Wallis test was used in nonnormally distributed variables, and the χ2 test or Fisher exact test was used to compare categorical variables. Main results and the role of chance A total of 268 infertile women with unexplained reason who tested positive for TPOAb and/or TgAb were recruited in our study. According to assignment, they were divided into two groups. All women in different groups had the similar age, BMI, number of miscarriage and duration of infertility. Levels of FSH, AMH, TSH, FT4, FT3, fibrinogen and d-dimer were similar in all groups. The use of A+P reduced cleavage rate (F = 23.982, P &lt; 0.001) and implantation rate (F = 4.388, P = 0.036). The fertilization rate (P = 0.407), high-quality embryo rate (P = 0.208) and blastulation rate (P = 0.157) were not influenced by the use of medication. In this study, likelihood of clinical pregnancy (P = 0.066) did not change significantly after therapy, and miscarriage rate (P = 0.042) increased after medical treatment. Limitations, reasons for caution Firstly, Aspirin is just one representation of anticoagulation therapy, so additional consideration of low molecular heparin should also be considered. Secondly, further randomized controlled trials of aspirin and prednisone alone are needed. Wider implications of the findings: In this study, use of A+P showed no positive effect, and reduced cleavage rate and implantation rate, while increased miscarriage rate. So, the use of medication for interfile women should be cautious. Trial registration number n/a

The effect of ICSI-related procedural timings and operators on the outcome

STUDY QUESTION Do the ICSI-related procedural timings and operators affect the outcomes of an ART cycle? SUMMARY ANSWER The ICSI-related timings and operators do not associate with the mean blastulation rate per cohort of inseminated oocytes and the cumulative delivery rate per concluded cycle, except for a mild association between the times from induction of ovulation to oocyte denudation and the former outcome. WHAT IS KNOWN ALREADY In ART, specific timings, protocols and conditions must be complied with to preserve gamete developmental and reproductive competence during the required manipulations. ICSI represents a groundbreaking advancement that has been widely implemented. Nevertheless, the studies that examined the putative impact of ICSI-related procedural timings were mainly conducted in old-fashioned settings or in good prognosis patients. No report addressed issues like operators’ skills and experience and uncertainties exist dealing with the effect of cumulus cells in the pre-incubation period in vitro before ICSI. However, all this information is crucial to efficiently plan the daily routine of an IVF lab, fill the existing gaps of knowledge and define proper key performance indicators. STUDY DESIGN, SIZE, DURATION Observational study conducted at a private IVF clinic (January 2016 to January 2018). We included all consecutive ICSI procedures (n = 1084 infertile couples undergoing 1444 cycles with or without preimplantation genetic testing (PGT); mean ± SD maternal age: 38.1 ± 4.0 years) with fresh autologous oocytes (n = 7999 oocytes, 5.5 ± 3.2 per treatment) inseminated with fresh non-donor ejaculated sperm. All operators and critical procedural timings (induction of ovulation to oocyte denudation, denudation and ICSI) were automatically recorded through an electronic witnessing system. The primary outcome measure was the cumulative delivery rate among both non-PGT and PGT-concluded cycles (i.e. delivery achieved or no supernumerary cryopreserved blastocyst available). The secondary outcome measure was the mean blastulation rate per cohort of inseminated oocytes. All confounders were registered and included in generalized linear models and multivariate logistic regression analyses. PARTICIPANTS/MATERIALS, SETTING, METHODS Fourteen and 12 operators were involved in denudation and ICSI procedures, respectively. Denudation was performed after 4.1 ± 1.2 h (2–7) of pre-incubation in vitro after oocyte retrieval, and ICSI was started immediately after. Beyond procedural timings and operators, all the putative confounders (patients’ and cycles’ characteristics) on the primary and/or secondary outcomes were systematically registered and included in the statistical analyses. MAIN RESULTS AND THE ROLE OF CHANCE The mean time from induction of ovulation to oocyte denudation was 39.3 ± 1.3 h. The mean procedural timings for denudation and ICSI were 8.1 ± 3.8 and 12.6 ± 6.4 min; both these variables were significantly dependent on the number of inseminated oocytes and the operators’ skills and experience. The overall mean blastulation rate per cohort of inseminated oocytes was 34.0 ± 27.9%. This outcome was significantly associated with the time from induction of ovulation to oocyte denudation (mean blastulation rate stable in the time interval 38–42 h, but significantly higher for timings &lt;38 h), maternal age (the mean blastulation rate drops especially beyond the age of 40 years) and categorized sperm concentration (highest mean blastulation rate for sperm concentrations ≥15 mil/ml and lowest for cryptozoospermic patients) through a generalized linear model that showed an adjusted r2 = 0.053 (P &lt; 0.01). No association was found for denudation and ICSI timings and operators. Lastly, when adjusted for maternal age and number of inseminated oocytes, both ICSI-related procedural timings and operators did not associate with the cumulative delivery rate among both non-PGT- or PGT-concluded cycles. LIMITATIONS, REASONS FOR CAUTION This is a single private IVF center study. Its reproducibility should be assessed in different laboratory conditions, with different protocols and in the hands of different operators. Moreover, specific studies are warranted to address the beneficial/detrimental effect of the other putative confounders under investigation (e.g. kind of ovulation trigger, culture media, incubator, etc.). WIDER IMPLICATIONS OF THE FINDINGS Proactive communication between the embryologists and the clinicians might contribute to a reasoned and more efficient organization of the daily workload and increase the mean blastulation rate, especially when poor prognosis couples (advanced maternal age, reduced sperm count and/or ovarian reserve) are treated. STUDY FUNDING/COMPETING INTEREST(S) No funding. The authors declare no conflict of interest related to the present study.

Patient and in vitro fertilization (IVF) cycle characteristics associated with variable blastulation rates: a retrospective study from the Duke Fertility Center (2013–2017)

Background To evaluate the association of patient and IVF cycle characteristics with blastulation rate and formation of high-quality blastocysts Results We analyzed autologous blastocyst cycles from 2013 to 2017. Cycles were subdivided into low (< 33%), intermediate (33–66%), and high (> 66%) blastulation rates. Embryo quality was assigned by embryologists using Gardner Criteria. R statistical package was used, and the blastulation groups were compared using analysis of variance (ANOVA) for continuous variables and chi-squared tests for categorical variables. The Bonferroni correction was used to adjust for multiple comparisons. One hundred seventeen IVF cycles met our inclusion criteria. Of these, 20 (17.1%) had low, 74 (63.2%) had intermediate, and 23 (19.7%) had high blastulation rates. Low blastulation rate was associated with a lower number of blastocysts, including fewer high-quality blastocysts. The mean number of oocytes retrieved was highest (18.1) in the group with the lowest blastulation rate, and lowest (13.4) in those with the highest blastulation rate, although this did not reach statistical significance. There were no significant differences between blastulation rates and age, gravidity, prior live birth, anti-mullerian hormone, estradiol and progesterone levels on the day of ovulation trigger, follicle-stimulating hormone dose, or fertility diagnosis. Conclusions High blastulation rate is associated with a greater number of blastocysts, including a greater number of high-quality blastocysts. Higher oocyte yield, however, is not associated with improved blastulation rates. Blastulation rates, blastocyst number, and quality remain difficult to predict based on cycle characteristics alone, and oocyte yield may not be an accurate predictor of either outcome.