Embryo Morphokinetic Parameters Research Articles

Advanced Glycation End-Products of Follicular Fluid are Associated with Embryo Morphokinetic Parameters and ART Outcomes.

Advanced glycation end products (AGEs) can disrupt antioxidant system and steroidogenesis, resulting in detrimental effects on assisted reproductive technology (ART) outcomes. This study aimed to investigate the association of AGEs in follicular fluid (FF) with morphokinetic parameters of embryos and ART outcomes. Fifty women undergoing ART treatment were studied. AGEs, glucose, 25(OH) vitamin D, malondialdehyde (MDA) levels and catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPX) activities were evaluated in FF. The expression of 3βHSD, CYP11A1, and CYP19A1 genes were analyzed in granulosa cells (GCs) by qRT-PCR technique. Morphokinetic parameters were evaluated using time-lapse technology. The FF level of AGEs was reversely associated with CAT, SOD, and GPX activities, and total and mature oocytes number, blastocyst formation rate, and high-grade embryos number, while it showed positive correlations with the FF MDA levels, the expression of steroidogenesis genes, number of immature oocytes, morphokinetic parameters, and number of low-grade embryos. Furthermore, the level of vitamin D in FF had an inverse association with AGEs and positive correlations with ART outcomes and morphokinetic parameters. Comparison between the those with positive and negative biochemical pregnancy showed no significant differences in terms of FF factors and just the expression of 3βHSD, CYP11A1, and CYP19A1 genes were higher in pregnant women (p < 0.05). AGEs could delay blastomere division and lead to an increase in the number of low-quality embryos, while vitamin D have an adverse effect on AGEs and a protective function against AGEs negative effects.

Effect of Male Cigarette Smoking on In Vitro Fertilization (IVF) Outcomes and Embryo Morphokinetic Parameters.

This retrospective cohort study examines the association between male smoking status and embryo development in vitro. The study included non-smoking women aged under 40 years who underwent in vitro fertilization (IVF) at Yanaihara Women's Clinic from May 2019 to May 2022, and they were divided into two groups according to the husband's smoking status. The effect of male smoking status on IVF outcomes was compared retrospectively based on embryonic development using a time-lapse incubator. A total of 184 patients were included; 272 oocytes of 45 female non-smokers were cultured with the sperm of male smokers, and 816 oocytes of 139 female non-smokers were cultured with the sperm of male non-smokers. No significant differences were observed between male smokers and non-smokers groups with regard to fertilization and the top-quality embryo on day 3 and day 5 (p > 0.05). The male smoker group's embryos reached theearly cleavage-stageparameters (time of pronuclei appearance to the five-cell stage) significantly earlier than the male non-smoker group's embryos (p < 0.05). However, no significant differences were observed between the two groups in other parameters of top-quality blastocysts (p > 0.05). It was concluded that male smoking has some differences on the timing of early embryonic events on time-lapse examination.

#247 : Correlation Between the Embryo Morphokinetic Parameters Up To Cleavage Stage and Blastocyst Implantation Capability in Frozen Embryo Transfer Cycles

I. BACKGROUND AND AIMS: Many studies have shown predicted value for embryo development potential or morpho-kinetic parameters. This study aims to determine the correlation between the embryo morpho-kinetic parameters up to the cleavage stage and blastocyst implantation capability in frozen embryo transfer (FET) cycles. II. METHODS: A retrospective cohort study was conducted on 383 good-quality blastocysts of FET cycles from March 2020 to December 2022 at Tam Anh General Hospital. The study evaluated the differences in parameters, including tPNa, tPNf, t2 - t8, cc1, cc2, cc3, s2, s3, and abnormal division (direct, reverse cleavage) between the implant and non-implant groups. Using R’s rpart package, a hierarchical implantation model was created using the decision tree approach. The prognostic value of the model was assessed using ROC. III. RESULTS: 383 blastocysts from 368 FET cycles were examined in our study, of which 222 (57.9%) were implanted and 161 (42.1%) were not. Timing parameters of implanted embryos tended to be longer than that in the non-implanted group, and the division process of blastomeres was more synchronized, however not statistically. Embryos with direct cleavage had a significantly reduced implantation rate compared with non-direct groups (29.0% and 61.0%, p &lt; 0.05) Analysis of morpho-kinetic parameters enabled us to develop a hierarchical model that places the direct cleavage, tPNa, and cc1 as the variables with the best prognosis of implantation. The AUC value of the model was 0.641. IV. CONCLUSION: The study showed that morpho-kinetic parameters at the early division stage have the predictive value of blastocyst implantation and can supplement traditional morphological assessment in embryo selection.

P-150 Comparison of morphokinetic parameters of embryos generated from Capacitation In-vitro Maturation and Controlled ovarian stimulation-ICSI

Abstract Study question Is there any difference in the embryo morphokinetic parameters between embryos generated from Capacitation – In-vitro maturation (CAPA-IVM) and Controlled ovarian stimulation – ICSI (COS-ICSI)? Summary answer Embryos generated from CAPA-IVM cycles reached tPNa, t2, t4, t8, tSC, tM, tSB, and tB significantly later than COS-ICSI embryos. What is known already IVF with ovarian hyperstimulation has limitations in some subgroups of women at high risk of ovarian stimulation, such as those with polycystic ovary syndrome. IVM may be a feasible alternative to IVF in women with a high AFC. A recent randomized controlled trial produced statistically inconclusive findings regarding the noninferiority of the CAPA-IVM technique compared with COS-ICSI. Moreover, there is a lack of studies investigating the impact of CAPA-IVM on the morphokinetic parameters of embryos. Study design, size, duration This is a retrospective cohort study conducted from March 2021 to March 2022. The study included 115 couples (68 couples used CAPA-IVM, 47 couples used COS-ICSI) who had their embryos cultured to the blastocyst stage using a time-lapse system. Participants/materials, setting, methods The study was conducted at My Duc Phu Nhuan hospital, Ho Chi Minh City, Vietnam. Couples aged ≤ 40 years old, had blastocyst culture using time-lapse system were included in the study. Couples used oocyte or sperm donation, PGT treatment, had severe male factors, and fertilization failure in previous cycles were excluded. We analyzed 392 blastocysts from 115 couples for morphokinetic parameters including tPNa, tPNf, the onset of t2 to t8, tSC, tM, tSB, and tB. Main results and the role of chance There was no difference in tPNf, t3, and t5 between embryos from CAPA-IVM and COS-ICSI. However, embryos generated from CAPA-IVM reached tPNa, t2, t4, t8, tSC, tM, tSB, and tB significantly later than COS-ICSI embryos [tPNa (9.3 2.0 vs 8.0 1.9; P &amp;lt; 0.001), t2 (27.4 3.7 vs. 25.5 3.1; P &amp;lt; 0.001), t4 (38.7 4.5 vs. 37.3 4.3; P = 0.004), t8 (58.9 9.2 vs. 56.3 8.4; P = 0.011), tSC (76.2 11.1 vs. 71.4 10.4; P &amp;lt; 0.001), tM (90.4 8.6 vs. 85.5 10.0; P &amp;lt; 0.001); tSB (101 8.1 vs. 97.1 8.3; P &amp;lt; 0.001) and tB (108 7.6 vs 106 7.7; P &amp;lt; 0.05)]. The frequency of direct cleavage in the first and the second cell division cycle was similar between two groups (58.6% vs. 30.0%; 41.4% vs. 70%, respectively, P &amp;gt; 0.05). Limitations, reasons for caution The retrospective cohort study design could limit the ability to eliminate sources of bias. The characteristics of participants may influence the study findings, i.e. women in the CAPA-IVM group were PCOS while women in the COS-ICSI were not. In addition, the study population's single-ethnicity nature limits the results' external generalizability. Wider implications of the findings This study found that embryos from CAPA-IVM reached tPNa, tPNf, t2, t4, t8, tSC, tM, tSB, and tB significantly later than COS-ICSI. The findings of this study provide evidence for further studies to evaluate CAPA-IVM embryos and to improve the CAPA-IVM culture system. Trial registration number Not Applicable

MP30-19 HUMAN SPERM IGF2 mRNA LEVELS INVERSELY CORRELATE WITH EMBRYO GROWTH VELOCITY DURING EARLY STAGE OF EMBRYO DEVELOPMENT

You have accessJournal of UrologyCME1 Apr 2023MP30-19 HUMAN SPERM IGF2 mRNA LEVELS INVERSELY CORRELATE WITH EMBRYO GROWTH VELOCITY DURING EARLY STAGE OF EMBRYO DEVELOPMENT Rossella Cannarella, Scott Lundy, Nina Desai, Sarah Vij, and Aldo E. Calogero Rossella CannarellaRossella Cannarella More articles by this author , Scott LundyScott Lundy More articles by this author , Nina DesaiNina Desai More articles by this author , Sarah VijSarah Vij More articles by this author , and Aldo E. CalogeroAldo E. Calogero More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003258.19AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: IGF2 is a potent growth factor capable of influencing cell division and modulating numerous cellular functions. IGF2 mRNA has been identified in human spermatozoa, but little is known about its function in in early embryo development. The aim of this study is to evaluate whether IGF2 levels in spermatozoa is associated with early growth kinetics of human embryos in couples undergoing ICSI. METHODS: This study was conducted on patients undergoing ICSI treatment with fresh oocytes and fresh ejaculated spermatozoa. Couples with infertility due to suspected female factor (e.g., impaired oocyte quality as in patients with severe endometriosis and premature ovarian failure) were excluded from the study. Following clinical use, a surplus aliquot of the semen sample used for ICSI was used to analyze the levels of IGF2 mRNA. The kinetic parameters of each embryo were correlated with the amount of IGF2 mRNA measured in the same swim-up sperm sample used for ICSI. RESULTS: 16 patients were enrolled and a total of 131 embryos were collected. Patients were divided into two groups based on the βhCG test results. We found no statistically significant difference in IGF2 mRNA levels and embryo morphokinetic parameters between the two groups (Table 1). At the correlation analysis, we found a significant negative correlation between sperm IGF2 mRNA levels and the time of pronuclei (tPN) fading (r= -0.2572, p= 0.0078, n=106 embryos), and between sperm IGF2 mRNA and t2 (r= -0.2368, p= 0.0145, n=106 embryos). In contrast, sperm IGF2 mRNA levels were not significantly correlated with tPN, t3, t4, t5, t6, t7, t8, t9, t morula (tM), t blastocyst (tB), and time of expanded blastocyst (tEB) (Table 1). CONCLUSIONS: These results suggest a role for sperm IGF2 mRNA in the very early embryo development (tPN fading -t2). This evidence may help in identify the etiology of apparently idiopathic couple infertility. Indeed, since prolonged t2 has been associated with a poor chance of embryo development into a viable blastocyst, a low level of sperm IGF2 mRNA level can delay t2 and lead to no blastocyst formation. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e398 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Rossella Cannarella More articles by this author Scott Lundy More articles by this author Nina Desai More articles by this author Sarah Vij More articles by this author Aldo E. Calogero More articles by this author Expand All Advertisement PDF downloadLoading ...

Influence of a hyaluronan-binding system for sperm selection in intracytoplasmic sperm injection cycles on embryo morphokinetic parameters and in vitro fertilization cycle outcomes.

Although the impact of the paternal contribution to embryo quality and blastocyst formation is a well-known phenomenon, the current literature provides insufficient evidence that hyaluronan-binding sperm selection methods improve assisted reproductive treatment outcomes. Thus, we compared the cycle outcomes of morphologically selected intracytoplasmic sperm injection (ICSI) with hyaluronan binding physiological intracytoplasmic sperm injection (PICSI) cycles. A total of 2415 ICSI and 400 PICSI procedures of 1630 patients who underwent in vitro fertilization cycles using a time-lapse monitoring system between 2014 and 2018 were analyzed retrospectively. Fertilization rate, embryo quality, clinical pregnancy rate, biochemical pregnancy rate and miscarriage rate were evaluated, differences in morphokinetic parameters and cycle outcomes were compared. In total, 85.8 and 14.2% of the whole cohort were fertilized with standard ICSI and PICSI, respectively. The proportion of fertilized oocytes did not significantly differ between groups (74.53 ± 1.33 vs. 72.92 ± 2.64, p > 0.05). Similarly, the proportion of good-quality embryos according to the time-lapse parameters and the clinical pregnancy rate did not significantly differ between groups (71.93 ± 4.21 vs. 71.33 ± 2.64, p > 0.05 and 45.55 ± 2.91 vs. 44.96 ± 1.25, p > 0.05). No statistically significant differences were found between groups in clinical pregnancy rates (45.55 ± 2.91 vs. 44.96 ± 1.25, p > 0.05). Biochemical pregnancy rates (11.24 ± 2.12 vs. 10.85 ± 1.83, p > 0.05) and miscarriage rates (24.89 ± 3.74 vs. 27.91 ± 4.91, p > 0.05) were not significantly different between groups. The effects of the PICSI procedure on fertilization rate, biochemical pregnancy rate, miscarriage rate, embryo quality, and clinical pregnancy outcomes were not superior. The effect of the PICSI procedure on embryo morphokinetics was not apparent when all parameters were considered.

Influence of Diminished Ovarian Reserve on Early Embryo Morphokinetics during In Vitro Fertilization: A Time-Lapse Study.

There is great controversy as to whether women with Diminished Ovarian Reserve (DOR) exhibit only a quantitative decrease in ovarian reserve or also impaired oocyte and embryo quality. In this retrospective study, we aimed to evaluate the impact of DOR on embryo morphokinetic parameters with a time-lapse system. 1314 embryos were obtained from 256 couples undergoing IVF or ICSI cycles, with 242 embryos in the DOR group as classified by the Bologna and POSEIDON criteria and 1072 embryos derived from the Normal Ovarian Reserve (NOR) group. For each morphokinetic parameter (t2, t3, t4, t5, t8, tB, ECC2, cc2a, ECC3, s2, s3), a generalized linear mixed model was created to control for female age, BMI, smoking status, method of insemination and correlation between oocytes from a same cohort. No significant association was found between DOR and any of the morphokinetic parameters studied. In a secondary analysis, we evaluated the influence of maternal aging, comparing morphokinetic characteristics between two age groups (<37 and ≥37 years). In the univariate analysis, we found that embryos from older women displayed a slower embryo development (in particular for t3, t4, t5, tB, and ECC2), although without statistical significance in the multivariate analysis. In conclusion, our study did not reveal any substantial impact of ovarian aging on early morphokinetic parameters and suggested potential biases that may be a source of controversy in the literature.

Mitochondrial DNA copy number in cumulus granulosa cells as a predictor for embryo morphokinetics and chromosome status

While morphokinetic evaluation of embryos has become the most commonly used technique in IVF to select embryos for transfer, studies have demonstrated that mitochondrial DNA (mtDNA) copy number is correlated with embryo viability and transfer outcomes. Correspondingly, this cohort study aims to evaluate the association between the mtDNA copy number in cumulus granulosa cells (CGCs) with embryo morphokinetic parameters and chromosomal status. Real-time PCR was employed to measure the mtDNA copy number of the 129 CGCs in samples obtained from 30 patients undergoing the IVF-IMSI program at Morula IVF Jakarta between July and October 2020. Bivariate and multiple analyses were utilized to determine its relationship with embryo morphokinetics, blastocyst yield, and chromosomal status. According to the analysis, there was a significant correlation between the mtDNA copy number and the blastocyst status after adjusting for the maternal age and sperm morphology (coefficient 0.832, p value = 0.032, RR value 2.299). Moreover, a significant link was observed between mtDNA copy number in CGC and early embryo developmental phase M1 (t2–t8), using the equation of M1 is 5.702–0.271 mtDNA copy number of CGCs + 0.017 maternal age + 0.013 sperm motility –0.115 sperm morphology (p value = 0.032). However, no correlation was found between the mtDNA copy number in CGCs with the other morphokinetic parameters (M2: tC–tEB, M3: t2–tEB, DC, RC, MN with p > 0.05), or the chromosomal status of the embryos (euploid: 139.44 ± 133.12, aneuploid: 142.40 ± 111.30, p = 0.806). In conclusion, our study suggests that mtDNA copy number in CGCs can serve as a useful biomarker for blastocyst status and early embryo developmental phase but not for chromosomal status.

P-181 Morphokinetic analysis may reveal associations between embryo development to the blastocyst stage and ploidy status

Abstract Study question Do euploid and aneuploid embryos have differences of KIDScore D5 value? Summary answer Morphokinetic analysis may reveal associations between embryo development to the blastocyst stage and ploidy status. Euploid embryos had higher KIDScoreD5 values. What is known already Time-lapse technology has allowed us to obtain a lot of information about human embryo development. Furthermore many morphokinetic. parameters have been tested in relation to a variety of outcomes including implantation potential, blastocyst development and ploidy status. Conflicting results regarding potential relationship between morphokinetics and embryo ploidy status, between morphokinetics and embryo implantation potential have been published. Vitrolife has developed the KIDScore D5 algorithm using a large multicenter dataset for predicting implantation with time lapse assessment during culturing in the Embryoscope Plus. Study design, size, duration This retrospective study included 94 cycles with aneuploidy screening (PGT-A) performed from March to November 2021. All embryos were cultured in the Embryoscope Plus and all good quality embryos were biopsied on day 5 or 6. Every biopsied embryo was evaluated by the Guided Annotation KIDScore D5 algorithm. Embryos with the highest KIDScoreD5 value were tested for PGT-A first. We have compared the KIDScore D5 values of 280 genetically tested embryos. Participants/materials, setting, methods Genetic testing was carried out using the NGS method. All embryos were cultured in the same culture media in Embryoscope Plus incubator till day 5 or 6. Every biopsied embryo was evaluated by the Guided Annotation KIDScore D5 algorithm, which took into account: pronuclei number, t2, t3, t4, t5, tB, ICM and TE grade. Main results and the role of chance One hundred and thirty (46%) embryos were euploid, 125 (45%) aneuploid and 24 (9%) mosaic. The average KIDScoreD5 value of euploid embryos was significantly higher than of the aneuploid embryos (6,9±0,1 versus 6,1±0,1, p = 0,000012). There were no significant differences between the average KIDScoreD5 value of euploid - mosaic and aneuploid - mosaic embryos (6,9±0,1 and 6,1±0,3; 6,1±0,1 and 6,1±0,3 accordingly). Also we have assessed possible differences of KIDScoreD5 value of genetic tested embryos depending on patient age. There were 52 cycles (175 embryos) in the younger age group (&amp;lt;38y.o.) and 42 cycles (105 embryos) in the older age group (≥38 y.o.) The euploidy rate was 61% in younger age group and 23% in older age group, the levels of aneuploid and mosaic embryos were 33%, 6% and 64%,13% accordingly. The average KIDScoreD5 value of the euploid embryos in the younger age group was statistically significantly higher than of the aneuploid embryos (7,0±0,1 versus 6,3±0,2, p = 0,0038), and didn’t statistically differ from the average KIDScoreD5 value of mosaic embryos (6,5±0,6). There were no statistically significant differences of the average KIDScoreD5 value of euploid, aneuploid and mosaic embryos in aged patients (6.6±0.4, 6.0±0.1 and 5.7±0.38 acordinly). Limitations, reasons for caution We need more data to assess the possible prediction rate of KIDScoreD5value for ploidy status of embryos in each individual case. First we should evaluate the relationship between the embryo morphokinetic parameters and the individual patient features (hormone levels, stimulation protocol, BMI, etc). Wider implications of the findings The combination of PGT-A and time-lapse morphokinetic assessment (KIDScoreD5 particularly) allows us to range the embryos and identify euploid embryos more effective and thus it allows us to decrease the cost of the ART treatment, especially for younger aged patients (&amp;lt;38 y.o.) with a large number of good quality embryos. Trial registration number not applicable

P-130 Morphokinetic analysis of early human embryo development and its relationship to endometriosis resection: a retrospective time-lapse study with the use of KIDScore D3 and D5

Abstract Study question Does complete resection of endometriosis improve embryo quality as assessed by morphokinetic parameters using time-lapse microscopy? Summary answer Complete resection of endometriosis in affected women undergoing assisted reproduction is shown to have a positive impact on embryo morphokinetic parameters represented in embryo quality. What is known already Endometriosis is one of the most common gynaecological diseases. It is characterized by the presence of cells comparable to those in the endometrium but being located outside of the uterine cavity. About 4-30% women of child-bearing age are affected by endometriosis. In vitro fertilization (IVF) studies have suggested that women with more advanced endometriosis have poor ovarian reserve, low oocyte and embryo quality and poor implantation rates. Moreover inflammatory peritoneal fluid shows a toxic effect on embryos. This might have an impact on morphokinetic timings but there is only limited data available concerning morphokinetics and endometriosis. Study design, size, duration 246 embryos (endometriosis group: n = 85; non-endometriosis control group: n = 115; complete resection of endometriosis= 46) undergoing infertility treatment at our clinics were included in this retrospective study. Inclusion criteria were female patients aged between 18 and 45 years undergoing IVF and / or ICSI treatment. The in vitro culture was performed in a closed time-lapse incubator (EmbryoScope®, Vitrolife) up to day 3 or in a prolonged culture up to day 5. Participants/materials, setting, methods Patient treatment included conventional insemination or intracytoplasmatic sperm injection. IVF inseminated or ICSI injected oocytes were culturd in the TimeLapse monitoring incubator. Embryos were cultured for 3 to 5 days without media change at 6.8% CO2 and 5% O2 at 37°C. During embryo culture morphokinetic parameters and events were constantly annotated. Analysis was performed using the EmbryoViewer software and evaluated applying KIDScoreTM D3 or KIDScoreTM D5 according to the day of embryo transfer. Main results and the role of chance The analysis revealed a median KIDScoreTM D5 of 2.3 (on a scale from 1 to 9.9) for embryos from patients suffering from endometriosis without complete resection. The control group without endometriosis achieved a score of 7.0 (p = 0.001). The median for embryos from endometriosis patients with complete resection was 7.1 which meant a significant increase compared to embryos from patients without complete resection (p = 0.002). Effect size according to Cohen showed a moderate up to strong biological effect (r = 0.4) for complete resection vs. no resection of endometriosis. For the three groups KIDScoreTM D3, pregnancy rates as well as abortion rates showed the same clinical trends. Additionally, we could describe a series of four patients undergoing IVF-cycles before and after a complete resection of their endometriosis. As expected, in three out of four cases they showed a clear increase in their embryo quality after complete resection of endometriosis. Limitations, reasons for caution The main limitation of the study is the relative small sample size of the groups, especially the number of patients with endometriosis resection. Additionally, the study is limited to its retrospective design. Wider implications of the findings Pathogenesis of endometriosis related infertility is multifactorial and not completely understood. But knowing that endometrioid lesions produce inflammatory cytokines which are toxic for oocytes and embryos, resection of the lesions in order to reduce inflammation seems to be a plausible approach. Trial registration number 20191007 01

Effect of paternal smoking on pre-implantation embryonic development: a prospective cohort study.

Recent studies have failed to demonstrate the negative impact of male tobacco smoking on embryo development, raising the question of its actual implication on natural fecundity and assisted reproductive techniques outcomes. To assess the impact of paternal smoking on embryo development. In this prospective cohort study, 252 men from couples undergoing in vitro fertilisation (IVF) were included. Each patient was interviewed and took a carbon monoxide breath-test, creating three groups: non-smokers (n =113), former smokers (n =81) and active smokers (n =58). The Top-grade embryo ratio (primary endpoint), embryo morphokinetic parameters and clinical outcomes were assessed. In a multivariate analyses based on 1521 embryos, no significant difference was found in the top-grade embryo ratio between the groups. Tobacco smoking had no impact on clinical outcomes. Compared to non-smokers the time to the pronuclei fading (tPNf, P =0.006) and the time to the first embryonic cleavage (t2, P =0.002) were shorter in smokers, and the t2 was also slightly shorter in former smokers (P =0.045). No other differences were found in the morphokinetic parameters. Even if a few differences were observed in the first timing of embryonic events, this study did not highlight a major embryonic and clinical impact of the paternal smoking status. The results obtained here are reassuring towards IVF outcomes. As maternal smoking is highly controlled in the IVF patients in this study, we speculate that the sperm selection process may limit the adverse effects of tobacco consumption on embryo development.

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

Do embryos from sibling oocytes assigned to distinct single-step media culture systems demonstrate differences in early embryo development, morphokinectics or aneuploidy rates? 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. 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. Prospective sibling oocyte cohort derived from 200 subjects undergoing IVF at a tertiary academic medical center between February 2018 and November 2019. 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. 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. Pregnancy outcomes and offspring health data were not available for analysis. 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. 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. N/A.

Morphokinetic parameter comparison between embryos from couples with high or low sperm DNA fragmentation index

To study whether time-lapse imaging can identify morphokinetic events impacted by a high sperm DNA fragmentation index (DFI). Historical cohort study. Private university-affiliated invitro fertilization center. A total of 978 zygotes cultured until day 5 in a time-lapse imaging incubator between March 2019 and August 2020, derived from 118 patients undergoing intracytoplasmic sperm injection as a result of idiopathic male factor infertility. Kinetic markers from the point of insemination were recorded. Generalized linear mixed models adjusted for potential confounders followed by the Bonferroni post hoc test were used to compare the timing of specific events in patients with a low (<30%) or high (≥30%) sperm DFI. The recorded kinetic markers were the following: timing to pronuclei appearance and fading; timing to 2, 3, 4, 5, 6, 7, and 8 cells; and timing to start blastulation and blastulation. Timing to blastulation. Embryos derived from sperm samples with ≥30% DFI showed significantly slower divisions compared with those with <30% DFI (mean differences of 0.7 hours in timing to pronuclei appearance, 1.2 hours in timing to pronuclei fading, 1.5 hours in timing to 2 cells, 2.5 hours in timing to 3 cells, 1.8 hours in timing to 4 cells, 3.3 hours in timing to 5 cells, 3.1 hours in timing to 6 cells, 3.2 hours in timing to 7 cells, 2.7 hours in timing to 8 cells, 8.4 hours in timing to start blastulation, and 3.8 hours in timing to blastulation). The incidences of reverse or direct cleavages (9.3% vs. 4.4%; odds ratio [OR], 2.24; 95% confidence interval [CI], 1.32-3.77) and multinucleation at 2-cell (18.9% vs. 12.0%; OR, 1.70; 95% CI, 1.12-2.58) and 4-cell (14.2% vs. 6.4%; OR, 2.42; 95% CI, 1.57-3.74) stages were significantly higher in embryos deriving from ≥30% DFI than from <30% DFI. The KIDScore ranked significantly different between embryos derived from samples with <30% and ≥30% DFI. Continuous DFI was positively correlated with all timings of specific events and with the incidences of abnormal cleavage patterns (OR, 1.042; 95% CI, 1.025-1.059) and multinucleation at 2-cell stage (OR, 1.053; 95% CI, 1.030-1.076) and inversely correlated with the KIDScore rank (B, -0.218; 95% CI, -0.044 to -0.007). No significant differences were observed in clinical outcomes between the groups. Embryo morphokinetic parameters are negatively impacted by high sperm DFI, resulting in delayed cell cleavage and blastulation.

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

Abstract 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 &amp;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–203 Applying artificial intelligence for ploidy prediction: The concentration of IL–6 in spent culture medium, blastocyst morphological grade and embryo morphokinetics as variables under consideration

Abstract Study question Would it be possible to predict embryo ploidy by taking into account conventional morphological and morphokinetic parameters together with IL–6 concentration in spent culture medium? Summary answer Our artificial neural network (ANN) trained with blastocyst morphology, embryo morphokinetics and IL–6 concentration distinguished between euploid/aneuploid embryos in 65% of the testing dataset. What is known already The analysis of spent embryo culture media represents the protein and metabolic state of the embryo and could be a non-invasive method of obtaining information about embryo quality. The impact of the presence/absence of several proteins in embryo culture samples over clinical results has been widely studied. The IL–6 is one of the most mentioned protein for its effect on embryo development, implantation and likelihood of achieving a live birth. In this initial attempt, we examined the predictive value for euploidy of a model that took into account the concentration of IL–6 in the spent culture medium. Study design, size, duration This prospective study included 319 embryos with PGT-A results. Out of the total, 127 were euploid and 192 aneuploid embryos. Concentration of IL–6 in spent embryo culture media (collected on the day of trophectoderm biopsy-fifth/sixth day of development), morphokinetic parameters (division time to 2 cells-t2; to 3 cells-t3, to 4 cells-t4; to 5 cells-t5 and time of blastocyst formation-tB) and blastocyst morphological grade (according to ASEBIR criteria) were considered to predict the embryo ploidy. Participants/materials, setting, methods Embryos were cultured in EmbryoScope. The chromosome analysis was performed using next-generation sequence technology. The concentration of IL–6 was measured in 20µL of spent embryo culture media with ELISA kits. Morphokinetic parameters were automatically annotated and the blastocyst morphology was evaluated by senior embryologists based on blastocele expansion, inner cell mass and trophectoderm quality. All the embryos were divided into 70% for training, 15% for validating and 15% for testing our ANN model with MatLab®. Main results and the role of chance The general description for the euploid embryo population was the following: 2% of the embryos were graded as A, 71% were graded as B and 28% were graded as C; the means and standard deviations were 25.32±2.97 hours (h) for t2, 35.33±5.15h for t3, 37.30±5.43h for t4, 48.24±6.62h for t5 and 103.93±12.8h for tB; and the average of IL–6 concentration was 1.51±0.70 pg/ml. The general description for the aneuploid embryo population was the following: 1% of the embryos were graded as A, 48% were graded as B and 51% were graded as C; the means and standard deviations were 26.13±3.51h for t2, 36.70±4.29h for t3, 38.20±4.24h for t4, 49.86±6.89h for t5 and 107.10±8.29h for tB; and the average of IL–6 concentration was 1.47±0.71 pg/ml. Our ANN model showed a higher general success rate as we increased the variables considered in the final prediction of euploid embryos. The accuracy, sensitivity and specificity for the testing dataset were: 0.60, 0.12 and 0.87 with morphokinetic parameters; 0.63, 0.24 and 0.93 with morphokinetics and IL–6 concentration; and 0.65, 0.16 and 0.96 with morphokinetics, IL–6 concentration and blastocyst morphological grade. Limitations, reasons for caution The low sensitivity and high specificity achieved in our models indicated that they were more capable of detecting aneuploid than euploid embryos. As this was a preliminary study, the small number of embryos included in the test (n = 48) was also a limitation. Wider implications of the findings: The results showed that our model tended to classify the embryos as aneuploid. More euploid embryos would be necessary to train our model and achieve better results in the prediction of chromosomally normal embryos. Further studies with large number of embryos and additional variables could improve the non-invasive ploidy prediction. Trial registration number Not applicable

Sperm DNA fragmentation measured by sperm chromatin dispersion impacts morphokinetic parameters, fertilization rate and blastocyst quality in ICSI treatments.

To determine the effects of sperm DNA fragmentation (SDF) on embryo morphokinetic parameters, cleavage patterns and embryo quality, this retrospective study analyzed 151 intracytoplasmic sperm injection (ICSI) cycles (1152 embryos collected) between November 2016 and June 2019. SDF was assessed using sperm chromatin dispersion. The cycles were divided into two groups based on the SDF rate: SDF < 15% (n = 114) and SDF ≥ 15% (n = 37). The embryo morphokinetic parameters, cleavage patterns, and embryo quality were compared between the two groups. The morphokinetic parameters tPNf, t2, t3, t4, t5, t6, and t8 were achieved significantly earlier in the SDF < 15% group compared with in the SDF ≥ 15% group. The fertilization and 2PN rates seemed to be significantly higher in the SDF < 15% group compared with in the SDF ≥ 15% group, while the abnormal cleavage rates were similar. However, a significantly higher rate of chaotic cleavage (CC) was observed in the SDF ≥ 15% group. The D3 high-quality embryo and available embryo rates were similar between the two groups. The blastocyst formation, high-quality blastocyst, and available blastocyst rates in the SDF < 15% group were significantly higher than those in the SDF ≥ 15% group. With an increase in SDF level, the chemical pregnancy, clinical pregnancy and implantation rates tended to decrease, while the miscarriage rate increased. This study demonstrated that SDF ≥ 15% reduces the fertilization rate of ICSI cycles and affects certain morphokinetic parameters. A higher SDF level can also induce a higher rate of CC, with subsequent decreases in the blastocyst formation rate and blastocyst quality.

Morphokinetic evaluation of embryos generated from vitrified oocytes maintaining the meiotic spindle

Vitrification is a technique for preservation of human oocytes. There is still a lack of basic research about the possible effects of vitrification on subsequent embryos following oocyte vitrification. The purpose of this study was to evaluate the embryo morphokinetic parameters formed after fertilization of vitrified-warmed oocytes, where an intact meiotic spindle (MS) was observed pre- and post-cryopreservation. Matured oocytes after in vitro maturation were collected and MS evaluation was performed. The oocytes with MS were divided into two groups: fresh and post vitrification. After intra-cytoplasmic sperm injection, the oocytes were cultured in time lapse monitoring (TLM) and time of second polar body extrusion (SPBE), pronuclei appearance (tPNA), pronuclei fading (tPNF), formation of two to eight cells (t2 to t8), and irregular cleavage events [direct cleavage (DC), reverse cleavage (RC)] and vacuolation were assessed. The fertilization rate was not significantly different between the groups, although the rate of abnormal fertilization was higher in vitrification group compared with fresh group (23.5% VS 7.7%). Analysis of the TLM showed a significant delay in time points, including SPBE, tPNA, tPNF, t 2-cells cleavage in vitrification group (p = 0.02, p = 0.00, p = 0.002, P = 0.00, P = 0.01, respectively). In addition, t3 and t4 time points tended to be delayed in vitrification group (p = 0.05). Moreover, the higher level of DC, RC and vacuolation were noticed in the vitrification group (P˂0.05). In conclusion, despite MS maintenance after warming, TLM evaluation showed both a delay and abnormal cleavage patterns in generated embryos.

An investigation into the effect of potential confounding patient and treatment parameters on human embryo morphokinetics

To determine the effect of patient and treatment parameters on 19 embryo morphokinetic parameters using pronuclear fading as time zero. Single-site, retrospective cohort analysis. Fertility treatment center. Patients undergoing treatment between September 2014 and January 2016 (n = 639) whose embryos were cultured in the EmbryoScope for 6 days (n = 2,376). None. Multiple regression analysis of body mass index; maternal age; infertility diagnosis; treatment type; suppression protocol on time to each cellular division (tn): t2, t3, t4, t5, t6, t7, t8, t9, time to start of compaction (tM), start of blastulation (tSB), full blastocyst (tB); and interval measurements: s2, s3, cc2, cc3, cc4, t9-tM, tM-tSB, and tSB-tB. Beta coefficients were analyzed to quantify any significant effects. Embryos appeared to be subtly affected by patient and treatment parameters, exhibiting complex relationships between various morphokinetic parameters and specific patient and treatment factors, rather than a systemic effect. These findings outline the need for the consideration of confounding factors when assessing an embryo's ability to achieve implantation. Although morphokinetic parameters have been related to embryo viability, it is likely that this will vary depending on the embryo's origin.

Downregulation of gene expression and the outcome of ICSI in severe oligozoospermic patients: A preliminary study.

Preimplantation embryo development might be influenced by a specific set of transcripts that are delivered to the oocyte by the sperm. The aim of the study was to determine the relationship between the level of selected transcripts in spermatozoa and preimplantation development of the embryos in couples with severe oligozoospermia undergoing intracytoplasmic sperm injection (ICSI) procedure. Therefore, we assessed messenger RNA (mRNA) levels of genes involved in fertilization events, oocyte activation, chromatin remodeling, and DNA repair in severe oligozoospermic compared with normozoospermic men as well as morphokinetic parameters of embryos using the time-lapse imaging system. mRNA profiling (44 genes), in mature sperm, was carried out with custom-designed 384-well TLDA Cards. The morphokinetic parameters of zygotes and embryos were recorded by using a time-lapse imaging system. The transcript levels of 21 genes were significantly decreased in the severe oligozoospermic group. Most were associated with fertilization events, oocyte activation and embryonic genome activation. Among them, mRNA of AKAP4 and PTK7 was greatly reduced, moreover, the transcripts of PLCζ and POU5F1, essential for OA and EGA, were not detected at all in patients with severe oligozoospermia. Moreover, the reduced expression of genes important for spermatogenesis, chromatin remodeling and DNA repair was also observed in this group. Time-lapse analysis revealed that fertilization failure occurred in 14% of retrieved oocytes and 90% of all degenerated embryos did not reach morula stage. This study provides preliminary results indicating a significant decrease in transcripts of genes important for spermatogenesis and early preimplantation development in the mature sperm of men with severe oligozoospermia.

Can embryo morphokinetic parameters predict euploid pregnancy loss?

To use time-lapse imaging to compare embryo morphokinetic parameters between embryos resulting in euploid pregnancy loss and euploid embryos resulting in live birth. Retrospective cohort study. Single academic fertility center. All euploid single embryo transfers between October 2015 and January2018. Collection and analysis of baseline characteristics, cycle parameters, and outcomes. Embryo morphokinetic measurements assessed with time-lapse imaging for time to syngamy (TPNf), time to two cells, time to three cells, time to four cells, time to eight cells, time to morula, and time to blastocyst. The study included 192 euploid single-embryo transfers. Of these, the pregnancy rate was 78% (150 of 193) and the live-birth rate was 63% (121 of 193). There were 43 transfers that did not result in pregnancy, 15 biochemical pregnancy losses, 13 clinical losses, and 121 live births. There was no statistically significant difference in age, body mass index, or number of oocytes retrieved between the groups. Unadjusted and adjusted models revealed no differences in the morphokinetics of embryos resulting in euploid miscarriage compared with those resulting in live birth. Embryos that resulted in a euploid miscarriage did not display evidence of abnormal morphokinetics on time-lapse imaging. Euploid pregnancy loss is likely multifactorial, including both embryo and endometrial factors. Further research is needed to identify factors that can predict and prevent euploid loss.