Higher Blastulation Rate Research Articles

P-246 Use of image recognition to quantify oocyte quality improved the accuracy of prediction of blastulation by 26-48%

Abstract Study question Can an AI oocyte quality model improve blastulation prediction accuracy? Summary answer An AI oocyte quality model presented an overall accuracy of 61-74%. Exhibiting an improvement of 26-48% for prediction of blastulation over random. What is known already Nowadays, the assessment of whether sufficient oocytes have been cryopreserved is purely based on patient age and number of oocytes collected. No morphological abnormalities provide quantitative guidance about the reproductive potential of the oocyte. Additionally, interobserver agreement and prediction of blastulation by experienced embryologists has been reported to be worse than chance (Behr et al., 2018). Therefore, there is a need to implement oocyte quality assessment tools to predict blastulation in oocyte donation, cryopreservation programs and IVF treatments. Study design, size, duration A retrospective study using an AI oocyte quality score (CHLOE-OQ, Fairtility Ltd) was performed in three private clinics evaluating 1749 oocytes that were inseminated with ICSI and cultured between October 2019 and December 2021. Overall and per clinic analysis were conducted (Clinic 1 n = 1131; Clinic 2 n = 576; Clinic 3: n = 42). Participants/materials, setting, methods An AI model provided an oocyte quality (OQ) score to assess prediction of blastulation, measured by binary logistic regression (AUC) per clinic and overall. The correlation between the OQ score and blastulation was measured using t-test. OQ score was classified into groups, Group A: 0.9-1.0, Group B: 0.7-0.9, Group C: 0.4-0.7 and Group D: 0-0.4. Blastulation rate was assessed in each OQ score subgroup using chi-square. Main results and the role of chance OQ score prediction of blastulation presented an overall accuracy of 63% (1107/1749). Accuracy per clinic ranged from 61-74%, Clinic 1: 61% (690/1131), Clinic 2: 67% (386/576) and Clinic 3: 74% (31/42). Exhibiting an improvement of 24-48% for prediction of blastulation over random. Oocyte quality score was predictive of blastulation per clinic and overall Clinic 1: AUC 0.62, n = 1130; Clinic 2: AUC 0.62, n = 575; Clinic 3: AUC 0.78, n = 41; Overall: AUC=0.62, p < 0.001, n = 1749). Overall blastocyst development rate was 53% (929/1749). OQ score was higher in oocytes that blastulated than those that did not blastulate (0.85 ± 0.14 vs 0.74 ± 0.2, p < 0.001). There was a significant direct association between OQ score subgroups and blastulation rate. Group A with the highest score subgroup had the highest blastulation rate [61% (554/903), p < 0.05]. The blastulation rates for Group B [53% (283/539) p < 0.001], Group C [38%, (71/188), p < 0.001] and Group D [18% (21/119), p < 0.001] were decreasingly lower. Limitations, reasons for caution This study was done in 3 centers in the same country. Therefore, further studies should focus on diversifying in terms of geographical location to address generalization of this AI assessment. Further studies should focus on comparing the efficacy of blastulation prediction by humans. Wider implications of the findings Using AI to do an objective oocyte assessment aids clinicians in determining the optimal time to transition to oocyte donor programs. It assists the decision-making for oocyte banking, minimizing unnecessary cycles and guiding timely additional oocyte collections. This approach at the point of cryopreservation, mitigates risks associated with delayed decisions. Trial registration number N/A

P-300 Not all DUCs are the same: Impact of DUC type on the blastulation, utilization and ploidy

Abstract Study question We have identified six categories of DUCs as identified automatically by CHLOE-EQ. Do these DUC categories differ in embryo competency? Summary answer DUC-1, Major chaotic DUC and Fragmented DUC have compromised blastulation, utilization and ploidy, compared to DUC2, minor chaotic DUC and not-direct DUC. What is known already CHLOE-EQ (Fairtility, an AI-based support tool) automatically annotates embryo morphokinetics and identifies embryo division anomalies, such as Direct Unequal Cleavage(DUCs). DUCs are defined as less than 5 hours from two cells to three cells. DUCs have been associated with being severely compromised, with lower chance of blastulating, being utilized, euploid, implanting or leading to live birth. In some clinics, DUCs are automatically discarded. Other clinics reported euploids and live births from DUC embryos, raising questions as to whether there are different types of DUCs with different competencies. In this study, we identified 6 types of DUCs and assessed their viability. Study design, size, duration Retrospective cohort study that took place between March to July 2022 at a private fertility clinic in Spain. This study included 1032 time-lapse videos of embryos with Direct unequal cleavage (DUCs) as identified by CHLOE-EQ. CHLOE-EQ defines DUCs as (t3-t2)<5 hours. DUCS annotated by CHLOE-EQ were classified into 6 types by embryologists. Participants/materials, setting, methods DUC1(direct division from the 1-cell to 3 or more, without a visible 2-cell stage); DUC2 (Direct Division from 2-cells where either cell divides directly from one to three cells); Minor Chaotic DUC (asynchronous irregular divisions: cells still countable); Major chaotic DUC (asynchronous irregular divisions: cells/fragments are too chaotic to count); Fragmented DUC1 (resembles a DUC1, but the third ‘cell’ is a fragment); Not direct DUC (quick division from 1 cell to 2cells to 3cells). Main results and the role of chance CHLOE-EQ correctly identified 97.4%(875/898) of 2PN DUCs, based on the definition of DUC (t3-t2<5). Most DUCs annotated by CHLOE-EQ, were classified by embryologists as minor chaotic [25%(231/921)], followed by DUC 1 [20.3%(187/921)], Not direct DUCs [18.1%(167/921)], major chaotic DUCs [14.6%(135/921)], DUC 2 [14%(129/921)], and lastly, fragmented DUC1 [7.8%(72/921)]. The average t3-t2 time did not differ between the six groups (1.6,1.5,1.5,1.6,1.5,1.6,respectively,p>0.05). Among DUC embryos, Minor chaotic DUCs [85%(110/129)] and Not direct DUCs [66.9%(109/163)] and DUC2 [57%(73/128)] had similar blastulation rates (p > 0.05) and utilization rates [DUC2:44.2% (57/129), Minor Chaotics: 37.7%(87/231), Not Direct DUCs: 56.3%(94/167), p > 0.05]. These three groups had a higher blastulation rate than DUC1 [20.1% (37/184)], Major chaotic [21.6% (29/134)], Fragmented DUC1 [19.7% (14/71), p < 0.05)] and a higher utilization rate than DUC1 [11.8% (22/187)], Major chaotic [14% (19/135)], Fragmented DUC1 [11.1%(8/72), p < 0.05)]. Type of DUC was not affected by Age(p > 0.05). Four live births from single embryo transfer of DUC embryos were recorded in this dataset. Limitations, reasons for caution DUCs were assessed by a single embryologist, further studies will assess intra and inter-operator variation in DUC classification across various clinics. It was particularly challenging to differentiate between fragments and cells. This study is ongoing to further understand implication of DUC types on clinical outcome. Wider implications of the findings Given that different DUC types have varied competency levels, the results of this study encourage embryologists not to discard DUC embryos simply because they are DUCs. It is important to assess the type of DUC when determining the fate of the embryo and when managing the expectation of affected patients. Trial registration number NOT APPLICABLE

Physiological intracytoplasmic sperm injection does not improve the quality of embryos: A cross-sectional investigation on sibling oocytes.

This study aimed to compare the efficacy of physiological intracytoplasmic sperm injection (PICSI) and intracytoplasmic sperm injection (ICSI) in terms of the fertilization rate and embryo quality using sibling oocyte cycles. This prospective, cross-sectional study collected data from 76 couples who underwent their first cycle at the Hue Center for Reproductive Endocrinology and Infertility, Vietnam, between May 2019 and November 2021. The inclusion criteria were cycles with at least eight oocytes and a sperm concentration of 5×106/mL. Sperm parameters, sperm DNA fragmentation (SDF), fertilization, and the quality of cleavage-stage embryos on day 2 and blastocysts on day 5 were examined. From 76 ICSI cycles, 1,196 metaphase II (MII) oocytes were retrieved, half of which were randomly allocated to either the PICSI (n=592) or ICSI (n=604) treatment group. The results showed no significant difference between the two groups in terms of fertilization (72.80% vs. 75.33%, p=0.32), day 2 cleavage rate (95.13% vs. 96.04%, p=0.51), blastulation rate (52.68% vs. 57.89%), and high-quality blastocyst rate (26.10% vs. 31.13%, p=0.13). However, in cases where SDF was low, 59 cycles consisting of 913 MII oocytes produced a considerably higher blastulation rate with PICSI than with ICSI (50.49% vs. 35.65%, p=0.00). There were no significant differences between the pregnancy outcomes of the PICSI and ICSI embryo groups following embryo transfer. Using variable sperm quality provided no benefit for PICSI versus ICSI in terms of embryo outcomes. When SDF is low, PICSI appears to be able to produce more blastocysts.

P-276 Embryo development of fresh versus vitrified metaphase II oocytes in low prognosis patients: a retrospective sibling-oocyte monocentric study

Abstract Study question Do fresh and vitrified/warmed oocytes have the same developmental potential in low prognosis patients defined by POSEIDON criteria? Summary answer Embryos derived from vitrified/warmed oocytes show lower competence for embryo development when compared to those obtained from fresh oocytes in low prognosis patients. What is known already The strategy of oocyte accumulation has been proposed for the management of low-prognosis patients with the aim to increase the number of oocytes available for fertilization and further the number of embryos. The effect of oocyte vitrification/warming on the embryo developmental potential has mainly been studied in oocyte donation programs. Investigations on sibling-oocyte failed to show difference in the morphological embryo development, but the evaluation stopped after 2-3 days of embryo culture. In addition, morphokinetic evaluation revealed delayed development of embryos derived from vitrified oocytes, from the first cleavage to the time of blastulation. Study design, size, duration Single-center sibling-oocyte retrospective monocentric study including low-prognosis patients, defined by POSEIDON criteria, who had undergone an accumulation of oocyte strategy for managing their infertility, between November 2013 and January 2021. Only patients presenting fresh and vitrified/warmed oocytes at the time of insemination were analyzed for the sibling-oocyte comparison. Main study outcomes were fertilization, cleavage, blastulation rates, day-2 and day-5 embryo quality, and the rate of embryos available for transfer/cryopreservation. Participants/materials, setting, methods Forty-five patients were included, with a mean age of 34.8 ± 3.4 years. Oocyte vitrification/warming was performed using the Cryotop method. Top-quality and good-quality embryos were defined at day-2 respectively as 4 and 3-5 adequate-sized blastomeres, without multinucleation and containing <20% of cytoplasmic fragmentation, and at day-5 according to Gardner and Schoolcraft’s classification, top ≥B4 (AB/BA/AA) and good ≥ B3BB. Statistical analyses used mixed effects logistic regression to take into account the sibling-oocyte design. Main results and the role of chance A total of 656 oocytes were inseminated, 225 fresh and 431 vitrified/warmed oocytes. The oocyte survival rate after warming was 82%, 95%CI [76-87%]. There was no difference between fresh and vitrified/warmed oocytes groups regarding the degeneration rate following ICSI (5.0% vs 4.8% respectively, p = 0.95), the fertilization rate (74.6% vs 71.4% respectively, p = 0.42) or the cleavage rate on day-2 (98.3% vs 95.4% respectively, p = 0.15). However, the embryo development was significantly different from day 2, with higher day 2 top-quality and good-quality embryos rates in the fresh oocytes group when compared to the vitrified/warmed oocytes group (top-quality: 38.3% vs 24.8% respectively, p = 0.01; good-quality 58.0% vs 45.2% respectively, p = 0.02). The same results were observed on blastocysts when extended culture was decided, with a higher blastulation rate in the fresh oocyte group (56.7% vs 31.7% respectively, p < 0.001) and higher top-quality and good-quality day 5 blastocyst rate (top-quality: 23.4% vs 5.6% respectively, p = 0.01; good-quality 23.9% vs 7.9%, p = 0.01).The rate of embryos available for transfer/cryostorage was similar in both groups (40,7 % vs 38,8 %, p = 0,71). The cumulative live-birth rate was 33,3% (4/45 from vitrified oocytes group, 5/45 from fresh oocytes group, 6/45 from mixed oocytes group). Limitations, reasons for caution The retrospective nature of the study, on a limited number of limited number of patients represents an inherent limitation. Embryo assessment was subjective, the evaluator knowing the oocyte’s source group. The impact of the morphological alterations observed in embryos derived from vitrified/warmed oocytes need to be confirmed with clinical outcomes. Wider implications of the findings The majority of studies comparing fresh and vitrified/warmed oocytes were performed in egg-donation programs. This study focuses on low-prognosis patients, who reflect perhaps more the patients who consult in ART centers. Further prospective studies are needed to confirm our findings. Trial registration number Not applicable

IDEAL CULTURE CONDITIONS FOR ENHANCING FULL PREIMPLANTATION DEVELOPMENT OF EMBRYOS GENERATED THROUGH SOMATIC CELL HAPLOIDIZATION

To identify the ideal culture conditions for promoting full preimplantation development of artificial embryos generated through somatic cell hapoloidization (SCH). Metaphase II (MII) oocytes from B6D2F1 mice were enucleated under Oosight™ visualization and exposure to cytochalasin B. Nuclear transfer of an individual FVB mouse cumulus cell was conducted by transfer into the perivitelline space of the resulting ooplast and fused using Sendai virus. Development of a pseudo-meiotic spindle was assessed in the reconstructed oocytes 2 hours after transfer, and the oocytes were then piezo-ICSI inseminated. Zygotes were cultured either in KSOM medium (KSOM), KSOM supplemented with Trichostatin A (TSA), or KSOM supplemented with fasudil, scriptaid, and RS-1 (cocktail). Trichostatin A and scriptaid are both histone-deacetylase inhibitors that may improve cellular reprogramming and embryo development after nuclear transfer. Fasudil, a Rho-associated kinase inhibitor, reduces apoptosis, while RS-1 aids in DNA damage repair. Zygotes were cultured in TSA and cocktail for 24 hrs only, before being changed to KSOM. Intact oocytes were ICSI inseminated as controls and cultured in KSOM (control). All four groups were monitored in a time-lapse imaging system for 96hr. A total of 527 MII oocytes were enucleated, resulting in 522 ooplasts (99.0%). Nuclear transfer of these ooplasts resulted in a survival rate of 99.6%, generating 520 reconstructed oocytes. A positive spindle was assessed in 360 artificial oocytes (69.2%), and piezo-ICSI of these spindle-positive oocytes resulted in a survival rate of 75.0%. ICSI insemination of 60 control oocytes had a comparable survival rate (83.3%). Fertilization rates were similar among all culture conditions, as well as to the control, with rates of 91.2% for the cocktail group, 93.8% for the KSOM group, 94.4% for the TSA group, and 94.0% for the control. The cocktail group exhibited significant delays at all stages (P<0.05) except the 2-cell stage when compared to the control. KSOM-cultured embryos had comparable morphokinetic development to the control up to the blastocyst stage, except for a delay in pronuclei appearance (P<0.002). Morphokinetic parameters of TSA embryos were comparable to the controls at all stages, beginning with the pseudo-second polar body extrusion (haploidization) up until the blastocyst stage. The intact oocyte control had an 82.3% blastulation rate compared to all experimental groups (P<0.00001). The cocktail cohort had a blastulation rate of only 9.6%, and the KSOM group had a blastulation rate of 14.5%; the highest blastulation rate was reached by the TSA group at 23.5%. Although the blastocyst development was still suboptimal, among the culture media tested, the addition of a histone-deacetylase inhibitor (TSA) yielded artificial embryos with morphokinetic parameters similar to the control.

Cleavage vs blastocyst stage embryos: how are they interrelating?

To assess the variables that may predict which cleavage-stage embryo may develop into a blastocyst, and vice versa, to determine whether the cleavage-stage embryo morphology should be taken into consideration when transferring the embryo at the blastocyst stage. A single center, retrospective cohort study. The study cohort included 3072 patients undergoing 3607 retrieval cycles and 23,124 embryos at the cleavage stage. We assessed the blastulation rate and evaluated which variables impact the ongoing pregnancy rate. High blastulation rate correlates with higher embryos' grading (I > II > III > IV > V) and higher number of blastomeres (8 > 7 > 6 > 5 > 4). 949 patients had fresh single blastocyst transfers. The ongoing pregnancy rate was 28.9% per transfer. Patients with ongoing pregnancies were significantly younger (34.3 vs. 36 years, p < 0.001), had higher number of oocyte yield (9.8 vs. 9, p = 0.02), and an increased rate of good-quality embryos transferred (70.7% vs. 47.7%, p = 0.001). When evaluating embryos progression, we found that whenever embryo developed to a good-quality blastocyst, its appearance at the cleavage stage did not affect ongoing pregnancy rate. Higher the number of blastomeres and better embryo grading were found to correlate with a higher blastulation rate. Nevertheless, if the embryo has already developed to a top-quality blastocyst, its morphology at the cleavage stage did not impact ongoing pregnancy rate.

Comparison of art outcome in frozen embryo transfer cycle using oral estradiol valerate and estradiol transdermal gel

Aim: Endometrial preparation with exogenous estrogen is a common practice in frozen embryo transfer (FET) cycles. The aim of this study was to compare the clinical outcome of oral estradiol valerate versus transdermal estrogen (17-β estradiol) gel in FET cycles. Materials and Methods: A prospective pilot study was carried out at a tertiary fertility clinic after Ethics Committee approval from January 2018 to December 2018. It included 103 infertile women who underwent FET cycles. Either oral estradiol valerate or transdermal 17-beta estradiol was used for endometrial preparation. Combination was used in case of breakthrough bleeding or if optimal endometrial thickness was not achieved. Baseline demographic parameters and details of the stimulation protocol and embryogenesis in fresh cycle were noted. In the FET cycle, the patient was seen on day 2 of menstrual cycle, where baseline ultrasound (USG), estradiol, and progesterone levels were done. If normal, the patients were given either oral or dermal preparation. The patient was seen again on day 9 for endometrial thickness and if required again after 2 days till endometrial thickness was 9 mm. If optimal endometrial thickness was not achieved or there was breakthrough bleeding, combination of both oral and dermal preparation was used. Once the endometrial thickness was 9 mm or more, progesterone was started, and ET was done on day 5. On the day of progesterone initiation, endometrial thickness, endometrial volume by 3D, and Doppler indices [pulsatility index (PI), resistance index (RI), peak systolic velocity (PSV)] were noted. The primary outcome of the study was clinical pregnancy rate (CPR) and live birth rate (LBR). Results: There was no statistical difference in any of the demographic parameters in groups A and B. In group C, the pregnant patients were younger with higher body mass index and follicle-stimulating hormone and lower anti-Mullerian hormone and antral follicle count when compared with those who did not conceive. Demographics of the fresh cycle did not show any significant difference in dose and duration of stimulation, fertilization, cleavage, and blastulation rate in group A. In group B, the fertilization rate was significantly higher in the pregnant group (0.001), whereas the other parameters were similar. In group C, the pregnant group required more dose and days of stimulation and had lower oocytes retrieved but had a higher blastulation rate. In the hormone replacement therapy (HRT) cycle, there was no difference in the mean duration of HRT in groups A and B but was significantly higher in group C when compared with group A. The CPR with oral estradiol valerate, transdermal gel, and combination therapy was 34.85%, 35%, and 52.94%, respectively. The LBR with oral estradiol valerate, transdermal gel, and combination therapy was 25.76%, 30%, and 47.06%, respectively. Though the CPR and LBR were higher in group C, it did not reach statistical significance and this could be due to small sample size. There was no difference in the abortion rate (oral 7.58%, gel 5%, combination 5.88%) between the three groups. The implantation rate (oral 26%, gel 25.8%, combination 29.03%) in the three groups was also similar. There was also no statistical difference in the endometrial thickness, volume, and blood flow between the three groups. The cut-off values for Doppler indices for a positive pregnancy were as follows: Group A—PSV: &gt;8.7, RI: &lt;0.99, PI: &gt;1.54; Group B—PSV: &gt;5, RI: &lt;0.72, PI: &gt;2.1; Group C—PSV: &gt;5.6, RI: &lt;0.64, PI: &gt;1.29. Conclusion: Both the oral estradiol valerate and transdermal 17-beta estradiol were equally effective for optimal outcome in an FET cycle in HRT. Those not responding to single preparation may benefit from combination therapy. Transdermal 17-beta estradiol gel may be of use in those patients who have breakthrough bleeding with oral preparation which may be due to hepatic bypass effect.

Is intracytoplasmic sperm (ICSI) better than traditional in vitro fertilization (IVF): confirmation of higher blastocyst rates per oocyte using a split insemination design.

To explore the effects of traditional vs. intracytoplasmic sperm injection (ICSI) insemination method on the outcome of high-quality blastocyst development in a split sibling oocyte cohort. In this retrospective cohort study, we analyzed 62 ICSI/IVF split cycles. Sibling oocytes were randomly assigned to ICSI or IVF insemination. Two hundred thirty-four ICSI-only cycles and 152 IVF-only cycles were also analyzed for comparison. Blastocysts were graded by Gardner's embryo grading and were considered a high-quality blastocyst if 3BB or better (Gardner 1999). In the ICSI/IVF split group, (1) ICSI oocytes had a higher fertilization rate per oocyte allocated (73% vs 62%, p < 0.001), (2) more high-quality day 2 embryos (69% vs 55%, p < 0.005), (3) ICSI oocytes had a lower blastulation rate per 2PN (46% vs 54%, p < 0.05), but a higher blastulation rate when calculated per oocyte allocated (40% vs 32%, p < 0.05). The ICSI-only group had a lower fertilization rate (65% vs 70%, p < 0.001) but more high-quality day 2 embryos in comparison to the IVF-only group (68% vs 64%, p < .05). The total high-quality blastulation rate was higher for the IVF-only group per 2PN (49% vs 43%, p < 0.05) and per oocyte retrieved (34% vs 28%, p < 0.05). This distinctive IVF/ICSI sibling oocyte split design demonstrated a higher-quality blastulation rate in the IVF group compared to the ICSI group when calculated per 2PN, but not per oocyte allocated to each insemination procedure.

Patient and in vitro fertilization (IVF) cycle characteristics associated with variable blastulation rates: a retrospective study from the Duke Fertility Center (2013\u20132017)

BackgroundTo evaluate the association of patient and IVF cycle characteristics with blastulation rate and formation of high-quality blastocystsResultsWe 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.ConclusionsHigh 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.

17. RECOMBINANT VERSUS URINARY GONADOTROPHINS: A PILOT STUDY TO EVALUATE PLOIDY STATUS OF EMBRYOS DERIVED FROM IVF

Introduction The efficacy between both recombinant and urinary gonadotrophins in ovarian stimulation have always been actively compared in different perspectives as they are biochemically different. Urinary gonadotrophin is said to have a higher biological activity which may improve the pregnancy outcome. Besides, urinary gonadotrophin contains LH which is usually added to patients with poor prognosis to obtain better quality oocytes. Yet in other studies, better results were obtained with recombinant gonadotrophins. To date the quality of embryos produced from treatment with either gonadotrophins has not been compared. This pilot study aims to evaluate the ploidy status embryos derived from stimulation with recombinant and/or urinary gonadotrophins. Methods & Materials A total of 345 Preimplantation Genetic Testing for Aneuploidy (PGT-A) cases from Sunfert International Fertility Centre in 2018 were retrospectively analysed. Oocyte donor cases were excluded from this study as some of them were stimulated by external IVF centres. All cases were classified into four groups according to the type of prescribed gonadotrophins given throughout the controlled ovulation stimulation: Monotherapy recombinant (MR) such as Gonal F® and Puregon® (n=42); Monotherapy Urinary (MU) such as Menopur® and Humog® (n=43); Combined Recombinant (CR) which is a combination of Gonal F® and Pergoveris® (n=81); and Combined Recombinant Urinary (CRU) such as the addition of Menopur® or Humog® to either Gonal F® or Puregon® (n=179). Intracytoplasmic Sperm Injection (ICSI) was carried out for MII oocytes upon egg retrieval. Fertilized (2PN) oocytes were further cultured to blastocysts (n=1067) for biopsy. Ploidy screening was performed using Next-Generation Sequencing (Veriseq® protocol, Illumina). Results Analysis of fertilisation rates did not show significant difference within the groups of monotherapy (68.8% and 69.0%, p=0.94) and combined therapy (63.8% and 64.8%, p=0.66). The monotherapy groups however have a trend towards higher blastulation rate (BR) and blastocyst utilisation rate (BUR) rates compared to combined therapy groups, with the MU having the highest BR (76.4%) and BUR (57.4%). In terms of embryonic ploidy status, both groups of monotherapy showed a trend towards higher euploid rate and lower aneuploid rate compared to the combined therapy groups. Within the monotherapy groups however, the MU group seemed to do better than MR as it has a similar euploid rate (51.2% vs 50.3%) even though the mean age is higher for this group (36.5 vs 32.0). All groups, both combined and monotherapy have similar mosaic rate. Interestingly, CR group has the lowest euploid (42.6%) and highest aneuploid rate (47.1%) compared to all other groups, and this deserves further investigations. Conclusion In this small pilot study, we have demonstrated that stimulation with different gonadotrophins used either solely or in combination has a possible impact on the ploidy (but not mosaic) status of the resulting blastocysts. This may however be impacted by different stimulation policies of individual centres. In this study, adding LH into the stimulation protocol seemed to improve on the euploid rate, especially in older women but this seemed to be true in urinary gonadotrophins only. Further studies are necessary to confirm this.

Sequential versus Monophasic Media Impact Trial (SuMMIT): a paired randomized controlled trial comparing a sequential media system to a monophasic medium

To determine whether sequential or monophasic media is the more optimal formulation for blastocyst development and sustained implantation rates (SIR) in IVF. Paired randomized controlled trials. Academic. Infertile couples (N = 192) with female partner ≤42years old and normal ovarian reserve. Fertilized zygotes from each patient were randomly divided into two groups: [1] cultured in sequential media and [2] cultured in monophasic medium. Sequential media consisted of Quinn's Advantage Cleavage Medium (SAGE) followed by Blast Assist (Origio). The monophasic medium used was Continuous Single Culture (Irvine Scientific). Paired ETs were accomplished by transferring the best euploid blastocyst from each media group. DNA fingerprinting was used to link outcomes. The primary outcome measure was the proportion of blastocysts suitable for clinical use. Secondary outcome measures included timing of blastulation, aneuploidy rates, and SIR. Sustained implantation rate is defined as the number fetal heart beats at 8-9weeks of gestation, divided by the number of embryos transferred. A total of 192 patients had their 2PN embryos (N = 2,257) randomized to each culture system. Sequential media had higher blastulation rate than monophasic medium (55.2% vs. 46.9%). No differences were found in the day of blastulation or aneuploidy rate. Of the 168 patients who had euploid blastocysts suitable for transfer, 126 completed a paired ET. Among the double ETs, there was no difference in implantation between groups. This is the first randomized controlled trial to examine paired euploid transfers of sibling zygotes cultured in sequential versus monophasic media. This study demonstrates that the usable blastocyst rate is greatest after culture in the sequential media tested in comparison with the monophasic formulation selected for study. However, no difference exists in timing of blastulation, aneuploidy, or SIR. Whether these observations are generalizable to other media systems remains to be determined. NCT01917240.

Supplementing single step culture media with insulin for continuous uninterrupted in vitro culture of human embryos and monitoring the outcome: prospective randomized clinical trial

ulation and are less expensive. Both formulations have attained good outcomes, but an adequately powered assessment including usable blastulation rates (USBR), ploidy risk, and SIR is lacking. DESIGN: Paired RCT. MATERIALS AND METHODS: Patients with normal ovarian reserve were recruited. A paired design allowed each patient to serve as their own control, eliminating many confounding variables seen in prior studies. After confirming fertilization, patients’ zygotes were randomized (1:1 ratio) to either Seq media (Quinn’s Advantage Cleavage Medium, Sage then Blast Assist, Origio) or Mono media (Continuous Single Culture; Irvine Scientific). Each culture system used separate incubators. Assessed endpoints for all embryos included USBR, blastulation timing (arrest vs day 5 vs 6) and ploidy status. Paired euploid blastocyst transfers, one from each group, were performed. DNA fingerprinting of concepti was used as needed to link each embryo to a definitive outcome. SIR was defined as the presence of a fetal heart beat at 8-9 weeks gestation. Statistical analysis performed via McNemar’s Chi Square and Wilcoxon sum rank tests. RESULTS: 186 patients had their 2PN embryos (N1⁄42257) randomized to each culture system. Seq media had a higher blastulation rate then Mono media (p1⁄40.001, 55.5% vs. 46.0%). No differences were found in the day of blastulation (p1⁄40.4063) or in the aneuploidy rate (p1⁄40.5518). Of the 168 patients who had euploid blastocysts suitable for transfer, 126 completed a paired embryo transfer and 42 had a SET. Amongst the SETs, the SIR was equivalent (p1⁄41.0). Of the 126 double embryo transfers, 36 patients had one fetal heart beat at discharge, however there was no statistical difference in the likelihood of implantation between groups (p1⁄40.8642). CONCLUSIONS: This is the first randomized controlled trial to systematically examine paired euploid transfers of sibling zygotes cultured in in Seq vs. Mono media. This study demonstrates that the usable blastocyst rate is greatest after culture in Seq media in comparison to a Mono formulation; however no difference exists in SIR. Supported by: Irvine scientific provided Mono media.

Effects of group culture on the development of discarded human embryos and the construction of human embryonic stem cell lines.

To explore the effect of group culture on the developmental potential of discarded embryos in in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) cycles and establish the human embryonic stem cell lines for future research. Fresh discarded embryos were collected from the IVF/ICSI-ET program in the reproductive medical center of the first affiliated hospital of Zhengzhou university in this study. All zygotes were individually cultured from Day 1 to Day 3. On Day 3, discard embryos were then cultured in group of 1-4 embryos per droplet (30 μl/droplets) with a constant culture medium until Day 5 or 6. Mechanical method was used to isolate the inner cell mass (ICM) of blastocyst from the embryo. Then we inoculated the ICM on feeder layer. After identification of those cells, the human embryonic stem cell lines (hESCs) were established. In this study, we collected 1,223 fresh discarded embryos and they were sequential cultured to the blastocysts (18.07 %, 221/1,223), in which good quality blastocysts were 61(4.98 %, 61/1,223). There was no significant difference in the patients. The embryos from 1PN, 2PN, 3PN were sequential cultured to the blastocyst s(39.31 %,92/234;12.87 %,64/497;13.21 %,65/492),in which good quality blastocysts was 13.6 %(32/92),2.61 %(13/64), 3.04 %(15/65).1PN embryo's blastulation rate and quality embryo formation rate was significantly higher than the 2PN and 3PN embryos' (P <0.05). Three embryos group cultivation has the highest blastulation rate and quality embryo formation rate (P <0.05). In total, we successfully established 4 hESCs lines. The group culture of human discard embryos can improve the blastulation rate and blastocyst quality to some extent. Three embryos group cultivate is the better culture number. Human discard embryos are good source for establishment of hESCs.

Developmental capacity and pregnancy rate of tetrahedral- versus non-tetrahedral-shaped 4-cell stage human embryos

The arrangement of the blastomeres within the 4-cell stage embryo reflects the orientation of the cleavage planes during the second division. To examine their relevance, the developmental capacity and the pregnancy rate were compared between tetrahedral-shaped and non-tetrahedral-shaped 4-cell stage human embryos. The study included 3,546 4-cell stage embryos. The arrangement of the blastomeres at the 4-cell stage was annotated as being tetrahedral or non-tetrahedral on day 2 of preimplantation development. Embryo quality was compared on day 3 and day 5. Pregnancy rates were calculated per single embryo transfer on day 3 or day 5. In total, 2,803 4-cell stage embryos (79 %) displayed a tetrahedral arrangement and 743 (21 %) displayed a non-tetrahedral arrangement. Tetrahedral-shaped embryos developed more into high-quality embryos on day 3 (p < 0.001) and day 5 (p = 0.036) and had a higher blastulation rate (p = 0.009). Though, the number of high-quality embryos selected for transfer did not differ between both groups on day 3 (p = 0.167) and day 5 (p ~ 1). Three hundred thirty single embryo transfers were analysed. No significant difference in clinical pregnancy was found between both groups after transfer on day 3 (p = 0.209) and day 5 (p = 0.653). The arrangement of the blastomeres according to their previous cleavage planes was correlated to the developmental potential of the 4-cell stage embryo up to the blastocyst stage. If embryo transfers are performed on day 3 and day 5 of development using embryos of adequate quality, the blastomere arrangement at the 4-cell stage had no predictable value regarding pregnancy success.

Correlation between body mass index (BMI) and serum beta hCG levels post hCG with ART cycle outcomes

OBJECTIVE: To evaluate if 250 mcg rhCG in women with different BMIs achieved similar serum beta hCG levels for follicular maturation and successful ART outcomes.DESIGN: Retrospective study.MATERIALS AND METHODS: 84 consecutive ART cycles between January - March 2008 were included. Women were treated with GnRH agonist/antagnosists, stimulation began with 150-450 IU rhFSH and 75-150 IU rhLH or hMG added day 6 or 7. Response was assessed by ultrasound after 5 days of treatment; the dose of rhFSH ± rhLH/hMG adjusted as needed. Final follicular maturation was with 250 mcg r-hCG; serum beta hCG levels were measured 12-14 hrs later. Retrieval was done 36-37 hrs post hCG injection. ET was on day 5 or 6. Patients were stratified according to their BMI (kg/m2): 16-20, 21-25, 26-28, and 29-36. Statistical analysis: ANOVA test for continuous data and X2 for categorical data; significance: p<0.05.Table 1Body Mass Index (BMI (kg/m2)16-2021-2526-2829-36P valueN cycles13461411Age (yrs)32.1±4.733.5±4.637.0±3.4∗34.1±4.0∗0.02BMI19.3±1.222.8±1.327.0±0.931.1±2.2<0.001days of stim9.2±3.49.3±1.49.5±1.29.9±1.50.78Total 75 IU FSH24.0±13.832.4±13.034.6±15.341.8±17.2∗∗0.04Total 75 IU LH5.6±1.95.9±3.55.5±3.75.1±2.90.91#foll > 15 mm11.6±7.09.0±4.98.1±4.58.2±3.40.27Peak E2pg/ml2195±14402495±12952186±8321922±7860.48E2 post hCG3219±21662968±14242707±12552583±13850.74serum hCG (IU/L)135±51102±6389±4270±560.05# eggs17.4±11.613.6±6.113.6±6.711.4±4.50.21# mature oocytes15.0±11.111.3±5.712.0±6.77.8±4.30.09MII/total eggs0.810.810.840.660.05# 2PN8.3±8.06.8±4.36.5±2.54.0±2.80.77# blasts transferred1.6±1.01.2±0.81.7±0.71.1±0.80.19# blasts frozen∗∗4.6±1.63.0±2.34.0±1.71.6±0.50.24Clinical pregnancy rate (%)46.1 (6/13)43.8 (20/46)42.8 (6/14)45.4 (5/11)0.61∗ Frozen on day 6. Open table in a new tab CONCLUSIONS: Women with lower BMI (16-20 kg/m2) achieved higher serum levels of beta hCG, had more MII oocytes and blastocysts than those in the highest BMI group. Pregnancy rates were similar for all groups. Patients with a BMI >29 kg/m2 may require a higher dose of rhCG to achieve similar higher serum beta hCG levels which are associated, in women with lower BMIs, with more mature oocytes and higher blastulation rate. OBJECTIVE: To evaluate if 250 mcg rhCG in women with different BMIs achieved similar serum beta hCG levels for follicular maturation and successful ART outcomes. DESIGN: Retrospective study. MATERIALS AND METHODS: 84 consecutive ART cycles between January - March 2008 were included. Women were treated with GnRH agonist/antagnosists, stimulation began with 150-450 IU rhFSH and 75-150 IU rhLH or hMG added day 6 or 7. Response was assessed by ultrasound after 5 days of treatment; the dose of rhFSH ± rhLH/hMG adjusted as needed. Final follicular maturation was with 250 mcg r-hCG; serum beta hCG levels were measured 12-14 hrs later. Retrieval was done 36-37 hrs post hCG injection. ET was on day 5 or 6. Patients were stratified according to their BMI (kg/m2): 16-20, 21-25, 26-28, and 29-36. Statistical analysis: ANOVA test for continuous data and X2 for categorical data; significance: p<0.05. ∗ Frozen on day 6. CONCLUSIONS: Women with lower BMI (16-20 kg/m2) achieved higher serum levels of beta hCG, had more MII oocytes and blastocysts than those in the highest BMI group. Pregnancy rates were similar for all groups. Patients with a BMI >29 kg/m2 may require a higher dose of rhCG to achieve similar higher serum beta hCG levels which are associated, in women with lower BMIs, with more mature oocytes and higher blastulation rate.

Impact of Embryo Quality on Developmental and Aneuploidy Rates After Blastomere Biopsy for PGD

To examine the impact of cleavage stage embryo quality on subsequent embryonic development and aneuploidy rates following blastomere biopsy. Retrospective. Embryology data and PGD records for 189 embryos, which underwent day 3 blastomere biopsy for PGD of aneuploidy at our center were reviewed. Embryos were divided into euploid or aneuploid based on FISH analysis of 9 chromosomes (X, Y, 13, 15, 16, 17, 18, 21 and 22). The Mean patient’s age for all cases in the study (n=22) was 37.0±5.6. Assessment of embryo morphology, blastomere biopsies and nuclei fixation were performed by the same embryologist. All biopsies were performed on day 3 and all FISH analysis were performed at Reprogenetics, N.J. Our laboratory uses a strict embryo morphology assessment system whereby embryos are evaluated systematically once every 24 hr period and assigned a quality grade ranging from A to E. Top quality embryos fall in categories A/B, Mid quality embryos fall in category C, Low quality embryos are assigned scores D/E. All data were compared using the Fisher’s exact test. Out of 189 embryos that were biopsied 88% had one blastomere removed, and 12% required an additional blastomere to be obtained. The distribution of 5 to 8 cell embryos at 60-62 hrs in culture was similar among those that were subsequently reported as aneuploid or euploid (94% vs. 93%, respectively). There was no difference in blastulation rate in embryos with one vs. those with two blastomeres removed. Overall, blastulation rates for Day-3 Top, Mid and Poor quality embryos were 65% (43/66), 56% (62/111), 17% (2/12), respectively. Accurate FISH results were available in 77% of all embryos biopsied. In this cohort, 72% (105/146) were determined aneuploid and 28% (41/146) euploid. Day 3 Top quality euploid embryos had higher blastulation rate, 83% (19/23), as compared to their aneuploid counter parts, 56% (18/32), (P=0.04). There was no significant difference in the blastulation rate of Mid and Low quality Day 3 euploid embryos when compared to their aneuploid cohorts. The table below summarizes aneuploidy / euploidy rates for Top, Mid and Low quality blastocysts. Tabled 1 In our initial experience, biopsy of one or two blastomeres does not appear to affect blastulation rate. However, day 3 embryo quality was associated with blastulation rate following biopsy. Based on FISH results, Top quality day 3 euploid embryos demonstrated greater blastulation potential. Approximately 50% of Top quality blastocysts were aneuploid. Furthermore, the aneuploidy rate was higher in Mid and Low quality blastocysts. Therefore, FISH results are necessary for adequate selection of euploid embryos for transfer.