Can time-lapse culture combined with artificial intelligence improve ongoing pregnancy rates in fresh transfer cycles of single cleavage stage embryos?

Purpose: With the rapid advancement of time-lapse culture and artificial intelligence (AI) technologies for embryo screening, pregnancy rates in assisted reproductive technology (ART) have significantly improved. However, clinical pregnancy rates in fresh cycles remain dependent on the number and type of embryos transferred. The selection of embryos with the highest implantation potential is critical for embryologists and influences transfer strategies in fertility centers. The superiority of AI over traditional morphological scoring for ranking cleavage-stage embryos based on their implantation potential remains controversial. Methods: This retrospective study analyzed 105 fresh embryo transfer cycles at the Centre for Reproductive Medicine from August 2023 to March 2024, following IVF/ICSI treatment at the cleavage stage. All embryos were cultured using time-lapse technology and scored using an automated AI model (iDAScore V2.0). Embryos were categorized into three groups based on the iDAScore V2.0: Group A (8 cells, iDA: 1.0-5.7); Group B (8 cells, iDA: 5.8-8.0); and Group C (>8 cells, iDA: 5.8-8.0). Clinical treatment outcomes, embryonic development, and pregnancy outcomes were analyzed and compared across the groups. Results: Baseline characteristics such as patient age, AMH levels, AFC, and basal sex hormones showed no significant differences among the three groups (p > 0.05). The iDAscores were significantly higher in Group C (7.3 ± 0.5) compared to Group B (6.7 ± 0.5) and the iDAscores were significantly higher in Group B (6.7 ± 0.5) compared to Group A (4.8 ± 1.0) (p < 0.001).The mean number of high-quality embryos was highest in Group C (4.7 ± 3.0), followed by Group B (3.6 ± 1.7) and Group A (2.1 ± 1.2) (p < 0.001). There was no statistical difference (p = 0.392) in the ongoing pregnancy rate for single cleavage-stage transfers between Group B (54.5%, 30/55) and Group A (38.1%, 8/21), although there was a tendency for Group B to be higher. Conclusion: Combining time-lapse culture with AI scoring may enhance ongoing pregnancy rates in single cleavage-stage fresh transfer cycles.

Effect of single blastocyst-stage versus single cleavage-stage embryo transfer on cumulative live births in women with good prognosis undergoing in vitro fertilization: Multicenter Randomized Controlled Trial.

In this multicenter, non-inferiority, randomized trial, we randomly assigned 992 women undergoing in-vitro fertilization (IVF) with a good prognosis (aged 20-40, ≥3 transferrable cleavage-stage embryos) to strategies of blastocyst-stage (n = 497) or cleavage-stage (n = 495) single embryo transfer. Primary outcome was cumulative live-birth rate after up to three transfers. Secondary outcomes were cumulative live-births after all embryo transfers within 1 year of randomization, pregnancy outcomes, obstetric-perinatal complications, and livebirths outcomes. Live-birth rates were 74.8% in blastocyst-stage group versus 66.3% in cleavage-stage group (relative risk 1.13, 95%CI:1.04-1.22; Pnon-inferiority < 0.001, Psuperiority = 0.003) (1-year cumulative live birth rates of 75.7% versus 68.9%). Blastocyst transfer increased the risk of spontaneous preterm birth (4.6% vs 2.0%; P = 0.02) and neonatal hospitalization >3 days. Among good prognosis women, a strategy of single blastocyst transfer increases cumulative live-birth rates over single cleavage-stage transfer. Blastocyst transfer resulted in higher preterm birth rates. This information should be used to counsel patients on their choice between cleavage-stage and blastocyst-stage transfer (NCT03152643, https://clinicaltrials.gov/study/NCT03152643 ).

Cumulative live birth rate of a blastocyst versus cleavage stage embryo transfer policy during in vitro fertilisation in women with a good prognosis: multicentre randomised controlled trial.

OBJECTIVES: To evaluate whether embryo transfers at blastocyst stage improve the cumulative live birth rate after oocyte retrieval, including both fresh and frozen-thawed transfers, and whether the risk of obstetric and perinatal complications is increased compared with cleavage stage embryo transfers during in vitro fertilisation (IVF) treatment. DESIGN: Multicentre randomised controlled trial. SETTING: 21 hospitals and clinics in the Netherlands, 18 August 2018 to 17 December 2021. PARTICIPANTS: 1202 women with at least four embryos available on day 2 after oocyte retrieval were randomly assigned to either blastocyst stage embryo transfer (n=603) or cleavage stage embryo transfer (n=599). INTERVENTIONS: In the blastocyst group and cleavage group, embryo transfers were performed on day 5 and day 3, respectively, after oocyte retrieval, followed by cryopreservation of surplus embryos. Analysis was on an intention-to-treat basis, with secondary analyses as per protocol. MAIN OUTCOME MEASURES: The primary outcome was the cumulative live birth rate per oocyte retrieval, including results of all frozen-thawed embryo transfers within a year after randomisation. Secondary outcomes included cumulative rates of pregnancy, pregnancy loss, and live birth after fresh embryo transfer, number of embryo transfers needed, number of frozen embryos, and obstetric and perinatal outcomes. RESULTS: The cumulative live birth rate did not differ between the blastocyst group and cleavage group (58.9% (355 of 603) v 58.4% (350 of 599; risk ratio 1.01, 95% confidence interval (CI) 0.84 to 1.22). The blastocyst group showed a higher live birth rate after fresh embryo transfer (1.26, 1.00 to 1.58), lower cumulative pregnancy loss rate (0.68, 0.51 to 0.89), and lower mean number of embryo transfers needed to result in a live birth (1.55 v 1.82; P<0.001). The incidence of moderate preterm birth (32 to <37 weeks) in singletons was higher in the blastocyst group (1.87, 1.05 to 3.34). CONCLUSION: Blastocyst stage embryo transfers resulted in a similar cumulative live birth rate to cleavage stage embryo transfers in women with at least four embryos available during IVF treatment. TRIAL REGISTRATION: International Clinical Trial Registry Platform NTR7034.

Zygotic spindle orientation defines cleavage pattern and nuclear status of human embryos.

The first embryonic division represents a starting point for the development of a new individual. In many species, tight control over the first embryonic division ensures its accuracy. However, the first division in humans is often erroneous and can impair embryo development. To delineate the spatiotemporal organization of the first mitotic division typical for normal human embryo development, we systematically analyzed a unique timelapse dataset of 300 IVF embryos that developed into healthy newborns. The zygotic division pattern of these best-quality embryos was compared to their siblings that failed to implant or arrested during cleavage stage. We show that division at the right angle to the juxtaposed pronuclei is preferential and supports faithful zygotic division. Alternative configurations of the first mitosis are associated with reduced clustering of nucleoli and multinucleation at the 2-cell stage, which are more common in women of advanced age. Collectively, these data imply that orientation of the first division predisposes human embryos to genetic (in)stability and may contribute to aneuploidy and age-related infertility.

Trade-off between double cleavage-stage embryos transfer and single blastocyst-stage embryo transfer in patients with few good quality embryos in antagonist cycles: a retrospective study using a propensity score matching analysis.

OBJECTIVE: This study aimed to compare the per OPU clinical outcomes for transfer of Day 3 double cleavage-stage embryos (DET) and Day 5 single blastocyst-stage (SBT) in patients with five or fewer good quality embryos on day 3 per occyte pick-up cycle (OPU) in antagonist cycles with consideration of blastocyst formation failure. METHODS: This was a retrospective, observational cohort study of 2,116 cases of OPU treated with antagonist protocol in the affiliated Chenggong Hospital of Xiamen University between January 2013 and December 2020. DET was performed in 1,811cycles and SBT was performed in 305 cycles. The DET group was matched to the SBT group by propensity score (PS) matching according to multiple maternal baseline covariates. After PS matching, there were 303 ET cycles in each group. The primary outcomes were the cumulative live birth rate (CLBR), cumulative multiple pregnancy rate(CMPR)per OPU and the number of ET to achieve live birth per OPU. Secondary outcomes were the percentage of clinical pregnancy(CPR), live birth rate(LBR), multiple pregnancy rate(MPR). RESULTS: Following PS mating, the CLBR was slightly higher (48.8% versus 40.3% ; P = 0.041) and the CMPR was significantly higher in the DET group compared to SBT group(44.2% versus 7.9%, P < 0.001). The CPR, LBR and MPR per fresh transfer were higher in DET group compared to SBT group(50.2% versus 28.7%; 41.3% versus 21.5%;29.6% versus 0%, P < 0.001). The number of ET to achieve live birth per OPU in SBT group was obiviously more than in DET group(1.48 ± 0.578 versus 1.22 ± 0.557 ,P < 0.001). CONCLUSION: With a marginal difference cumulative live birth rate, the lower live birth rate per fresh transfer and higher number of ET per OPU in the SBT group suggested that it might take longer time to achieve a live birth with single blastocyst strategy. A trade-off decision should be made between efficiency and safety.

Abnormal cleavage up to Day 3 does not compromise live birth and neonatal outcomes of embryos that have achieved full blastulation: a retrospective cohort study.

STUDY QUESTION: Do embryos displaying abnormal cleavage (ABNCL) up to Day 3 have compromised live birth rates and neonatal outcomes if full blastulation has been achieved prior to transfer? SUMMARY ANSWER: ABNCL is associated with reduced full blastulation rates but does not impact live birth rates and neonatal outcomes once full blastulation has been achieved. WHAT IS KNOWN ALREADY?: It is widely accepted that ABNCL is associated with reduced implantation rates of embryos when transferred at the cleavage stage. However, evidence is scarce in the literature reporting birth outcomes from blastocysts arising from ABNCL embryos, likely because they are ranked low priority for transfer. STUDY DESIGN, SIZE, DURATION: This retrospective cohort study included 1562 consecutive autologous in vitro fertilization cycles (maternal age 35.1 ± 4.7 years) performed at Fertility North, Australia between January 2017 and June 2022. Fresh transfers were performed on Day 3 or 5, with remaining embryos cultured up to Day 6 before vitrification. A total of 6019 embryos were subject to blastocyst culture, and a subset of 664 resulting frozen blastocysts was included for live birth and neonatal outcome analyses following single transfers. PARTICIPANTS/MATERIALS, SETTING, METHODS: ABNCL events were annotated from the first mitotic division up to Day 3, including direct cleavage (DC), reverse cleavage (RC) and <6 intercellular contact points at the 4-cell stage (<6ICCP). For DC and RC in combination, the ratios of affected blastomeres over the total number of all blastomeres up to Day 3 were also recorded. All pregnancies were followed up until birth with gestational age, birthweight, and sex of the baby being recorded. MAIN RESULTS AND THE ROLE OF CHANCE: Full blastulation rates for embryos showing DC (19.5%), RC (41.7%), <6ICCP (58.8%), and mixed (≥2) ABNCL types (26.4%) were lower than the rates for those without ABNCL (67.2%, P < 0.01 respectively). Subgroup analysis showed declining full blastulation rates with increasing ratios of combined DC/RC affected blastomeres over all blastomeres up to the 8-cell stage (66.2% when 0 affected, 47.0% when 0.25 affected, 27.4% when 0.5 affected, 14.5% when 0.75 affected, and 7.7% when all affected, P < 0.01). However, once full blastulation had been achieved, no difference was detected between DC, RC, <6ICCP, and no ABNCL blastocysts following single frozen transfers in subsequent live birth rates (25.9%, 33.0%, 36.0% versus 30.8%, P > 0.05, respectively), gestational age (38.7 ± 1.6, 38.5 ± 1.2, 38.3 ± 3.5 versus 38.5 ± 1.8 weeks, P > 0.05, respectively) and birthweight (3343.0 ± 649.1, 3378.2 ± 538.4, 3352.6 ± 841.3 versus 3313.9 ± 509.6 g, P > 0.05, respectively). Multiple regression (logistic or linear as appropriate) confirmed no differences in all of the above measures after accounting for potential confounders. LIMITATIONS, REASONS FOR CAUTION: Our study is limited by its retrospective nature, making it impossible to control every known or unknown confounder. Embryos in our dataset, being surplus after selection for fresh transfer, may not represent the general embryo population. WIDER IMPLICATIONS OF THE FINDINGS: Our findings highlight the incremental impact of ABNCL, depending on the ratio of affected blastomeres up to Day 3, on subsequent full blastulation. The reassuring live birth and neonatal outcomes of ABNCL blastocysts imply a potential self-correction mechanism among those embryos reaching the blastocyst stage, which provides valuable guidance for clinical practice and patient counseling. STUDY FUNDING/COMPETTING INTEREST(S): This research is supported by an Australian Government Research Training Program (RTP) Scholarship. All authors report no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

Manipulation of Embryonic Cleavage Geometry Using Magnetic Tweezers.

The geometry of reductive divisions that mark the development of early embryos instructs cell fates, sizes, and positions, by mechanisms that remain unclear. In that context, new methods to mechanically manipulate these divisions are starting to emerge in different model systems. These are key to develop future innovative approaches and understand developmental mechanisms controlled by cleavage geometry. In particular, how cell cycle pace is regulated in rapidly reducing blastomeres and how fate diversity can arise from blastomere size and position within embryos are fundamental questions that remain at the heart of ongoing research. In this chapter, we provide a detailed protocol to assemble and use magnetic tweezers in the sea urchin model and generate spatially controlled asymmetric and oriented divisions during early embryonic development.

Seeking arrangements: cell contact as a cleavage-stage biomarker.

RESEARCH QUESTION: What can three-dimensional cell contact networks tell us about the developmental potential of cleavage-stage human embryos? DESIGN: This pilot study was a retrospective analysis of two Embryoscope imaging datasets from two clinics. An artificial intelligence system was used to reconstruct the three-dimensional structure of embryos from 11-plane focal stacks. Networks of cell contacts were extracted from the resulting embryo three-dimensional models and each embryo's mean contacts per cell was computed. Unpaired t-tests and receiver operating characteristic curve analysis were used to statistically analyse mean cell contact outcomes. Cell contact networks from different embryos were compared with identical embryos with similar cell arrangements. RESULTS: At t4, a higher mean number of contacts per cell was associated with greater rates of blastulation and blastocyst quality. No associations were found with biochemical pregnancy, live birth, miscarriage or ploidy. At t8, a higher mean number of contacts was associated with increased blastocyst quality, biochemical pregnancy and live birth. No associations were found with miscarriage or aneuploidy. Mean contacts at t4 weakly correlated with those at t8. Four-cell embryos fell into nine distinct cell arrangements; the five most common accounted for 97% of embryos. Eight-cell embryos, however, displayed a greater degree of variation with 59 distinct cell arrangements. CONCLUSIONS: Evidence is provided for the clinical relevance of cleavage-stage cell arrangement in the human preimplantation embryo beyond the four-cell stage, which may improve selection techniques for day-3 transfers. This pilot study provides a strong case for further investigation into spatial biomarkers and three-dimensional morphokinetics.

Evaluation of TUBB8 gene alterations in infertile women with oocyte maturation and cleavage arrest referred to Royan Institute.

RESEARCH QUESTION: Are TUBB8 gene variations present in Iranian infertile women with oocyte maturation arrest or embryo cleavage arrest? DESIGN: TUBB8 gene variations were investigated by polymerase chain reaction sequencing on blood samples from 16 women with oocyte maturation arrest and 12 women with cleavage arrest, collectively referred to as the experimental cohort, as well as 56 fertile women as the control group. The Exome Sequencing Project and dbSNP databases and the Genome Aggregation Database were used to search the frequency of corresponding variants. PolyPhen and SIFT were used to conduct in-silico analysis of gene variations and Align-GVGD was used to predict the effect of missense variants on proteins. The homology modelling and structure evaluation of variations was also checked. RESULTS: Two likely pathogenic variants [c.713C>T (p.Thr238Met), c.1054G>T (p.Ala352Ser)] were identified in patients with oocyte maturation arrest and one likely pathogenic variant [c.G763A, (p.Val255Met)] was identified in a patient with cleavage arrest. These changes were absent in controls. CONCLUSIONS: Three deleterious variants in TUBB8 related to oocyte maturation arrest or cleavage arrest and infertility were identified. TUBB8 variant screening for patients with oocyte maturation and cleavage arrest is recommended.

Optimal number of high-quality cleavage-stage embryos for extended culture to blastocyst-stage for transfer in women 38 years and older.

PURPOSE: We aimed to evaluate the pregnancy outcomes of cleavage-stage embryo transfers (ETs) for the first time and explore optimal number of high-quality cleavage-stage embryos for extended culture to blastocyst-stage in women of advanced maternal age (AMA). METHODS: We retrospectively identified 1646 AMA women ≥ age 38 years for the first fresh ETs between January 2014 and December 2020 at our hospital. Double ETs were divided into three groups as follows: DET-HH (two high-quality embryos), DET-HL (one high-quality and one low-quality embryo), and DET-LL (two low-quality embryos) groups. We mainly analyzed the pregnancy outcomes of double cleavage-stage ETs with different embryo grades and blastocyst-stage ETs with different number of high-quality cleavage-stage embryos on day 3. RESULTS: Our data indicated that the DET-HH group had significantly higher clinical pregnancy, ongoing pregnancy, and live birth rates than DET-HL and DET-LL groups (p < .05). For extended culture to blastocyst-stage with 2 (D3-2H), 3 (D3-3H), and 4 (D3-≥4H) high-quality cleavage-stage embryos, the D3-≥ 4H group had significantly higher ongoing pregnancy and live birth rates than D3-2H and D3-3H groups (p < .05). We observed that the number of high-quality embryos on day 3 was independently associated with live birth rate for blastocyst transfers (OR: 1.133, 95% CI 1.023-1.256, p = .017). There were no significant differences in the clinical pregnancy, ongoing pregnancy and live birth rates among DET-HH, D3-2H and D3-3H groups (p > .05). CONCLUSIONS: Extended culture to blastocyst-stage for transfer was safe and recommended for AMA women with ≥ 4 high-quality embryos on day 3.

Are Early Embryo Cleavage Kinetics Affected by Energy Substrates in Different Culture Media?

OBJECTIVE: This study aimed to investigate the influence of different culture media on early embryonic cleavage kinetics using time-lapse analysis and to determine the possible relationships between energy substrates in culture media and the cleavage kinetics. METHODS: A total of 10 021 embryos from 1310 couples were cultured in time-lapse incubators. Embryos cultured in Vitrolife media were allocated to group I, and those in COOK media to group II. Embryo cleavage time points up to the 8-cell stage (t2-t8) were observed after pronuclei fading. RESULTS: The baseline demographic features, in vitro fertilization indications, ovarian stimulation protocol, oocyte-cumulus complexes, fertilization rate, together with pregnancy and perinatal outcomes were similar (P>0.05) between groups I and II. According to the time-lapse analysis, all embryos in group I showed significantly faster cleavage speed than those in group II (P<0.05). Furthermore, there was better synchrony in division (s3) and a longer length of the third cell cycle duration (cc3) in group II. Interestingly, implanted embryos in group II showed faster cleavage speed than those in group I, especially at t4 and t7. The glucose contents and multiple major amino acids were similar between the two groups. Lactic and pyruvic acid contents were generally higher in group I than those in group II. CONCLUSION: Because different commercial culture media may influence cleavage kinetics of embryos, it is essential for embryologists to take culture media into consideration in selecting a potential embryo when using a time-lapse system before implantation.

Cleavage-stage human embryo arrest, is it embryo genetic composition or others?

Embryo transfer is a crucial step in IVF cycle, with increasing trend during the last decade of transferring a single embryo, preferably at the blastocyst stage. Despite increasing evidence supporting Day 5 blastocyst-stage transfer, the optimal day of embryo transfer remains controversial. The crucial questions are therefore, whether the mechanisms responsible to embryos arrest are embryo aneuploidy or others, and whether those embryos arrested in-vitro between the cleavage to the blastocyst stage would survive in-vivo if transferred on the cleavage-stage. We therefore aim to explore whether aneuploidy can directly contribute to embryo development to the blastocyst stage. Thirty Day-5 embryos, that their Day-3 blastomere biopsy revealed a single-gene defect, were donated by 10 couples undergoing preimplantation genetic testing treatment at our center. Affected high quality Day-3 embryos were cultured to Day-5, and were classified to those that developed to the blastocyst-stage and those that were arrested. Each embryo underwent whole genome amplification. Eighteen (60%) embryos were arrested, did not develop to the blastocyst stage and 12 (40%) have developed to the blastocyst stage. Nineteen embryos (63.3%) were found to be euploid. Of them, 12 (66.6%) were arrested embryos and 7 (58.3%) were those that developed to the blastocyst-stage. These figures were not statistically different (p = 0.644). Our observation demonstrated that the mechanism responsible to embryos arrest in vitro is not embryo aneuploidy, but rather other, such as culture conditions. If further studies will confirm that Day-5 blastocyst transfer might cause losses of embryos that would have been survived in vivo, cleavage-stage embryo transfer would be the preferred timing. This might reduce the cycle cancellations due to failure of embryo to develop to the blastocyst stage and will provide the best cumulative live birth-rate per started cycle.

Longitudinal surface measurements of human blastocysts show that the dynamics of blastocoel expansion are associated with fertilization method and ongoing pregnancy.

BACKGROUND: Despite all research efforts during this era of novel time-lapse morphokinetic parameters, a morphological grading system is still routinely being used for embryo selection at the blastocyst stage. The blastocyst expansion grade, as evaluated during morphological assessment, is associated with clinical pregnancy. However, this assessment is performed without taking the dynamics of blastocoel expansion into account. Here, we studied the dynamics of blastocoel expansion by comparing longitudinal blastocoel surface measurements using time-lapse embryo culture. Our aim was to first assess if this is impacted by fertilization method and second, to study if an association exists between these measurement and ongoing pregnancy. METHODS: This was a retrospective cohort study including 225 couples undergoing 225 cycles of in vitro fertilization (IVF) treatment with time-lapse embryo culture. The fertilization method was either conventional IVF, intracytoplasmic sperm injection (ICSI) with ejaculated sperm or ICSI with sperm derived from testicular sperm extraction (TESE-ICSI). This resulted in 289 IVF embryos, 218 ICSI embryos and 259 TESE-ICSI embryos that reached at least the full blastocyst stage. Blastocoel surface measurements were performed on time-lapse images every hour, starting from full blastocyst formation (tB). Linear mixed model analysis was performed to study the association between blastocoel expansion, the calculated expansion rate (µm2/hour) and both fertilization method and ongoing pregnancy. RESULTS: The blastocoel of both ICSI embryos and TESE-ICSI embryos was significantly smaller than the blastocoel of IVF embryos (beta -1121.6 µm2; 95% CI: -1606.1 to -637.1, beta -646.8 µm2; 95% CI: -1118.7 to 174.8, respectively). Still, the blastocoel of transferred embryos resulting in an ongoing pregnancy was significantly larger (beta 795.4 µm2; 95% CI: 15.4 to 1575.4) and expanded significantly faster (beta 100.9 µm2/hour; 95% CI: 5.7 to 196.2) than the blastocoel of transferred embryos that did not, regardless of the fertilization method. CONCLUSION: Longitudinal blastocyst surface measurements and expansion rates are promising non-invasive quantitative markers that can aid embryo selection for transfer and cryopreservation. TRIAL REGISTRATION: Our study is a retrospective observational study, therefore trial registration is not applicable.

Blastocyst versus cleavage transfers: who benefits?

PURPOSE: This retrospective cohort study determined the relative efficacy of blastocyst and cleavage-stage transfers in patients with differing numbers of zygotes. METHODS: A total of 1116 women whose embryo transfers were planned independently of patient characteristics were included. Cleavage-stage (D3) and blastocyst-stage (D5) transfer outcomes were analyzed per number of zygotes. The D5 group included transfer cancellations as the intention-to-treat population. The effect of the embryo transfer date on the clinical outcomes (clinical pregnancy and implantation rates) was analyzed using multivariate logistic regression. RESULTS: Among the patients, 584 and 532 underwent D3 and D5 embryo transfers, respectively. The clinical pregnancy rates were significantly higher in D5 patients with ≥ 6 zygotes (25.7% vs 48.3%). The multivariate logistic regression analysis for clinical pregnancy did not show significant differences between the blastocyst and cleavage-stage transfers in patients with ≤ 5 zygotes (0.874 [0.635-1.204]). Compared to the cleavage-stage, blastocyst-stage transfers for patients with ≥ 6 zygotes resulted in a three-fold increase in clinical pregnancy rates (3.122 [1.797-5.425]). CONCLUSION: Blastocyst transfers were not inferior to cleavage-stage embryo transfers among patients with few zygotes and were preferable for patients with several zygotes.

Ability and willingness to pay for family planning services in low resource settings: evidence from an operational research

Objective: This paper establishes levels and patterns of ability and willingness to pay (AWTP) for contraceptives, and associated factors. Study design: A three-stage cluster and stratified sampling was applied in selection of enumeration areas, households and individuals in a baseline survey for a 5-year Family planning programme. Multivariable linear and modified Poisson regressions are used to establish factors associated with AWTP. Results: Ability to pay was higher among men (84%) than women (52%). A high proportion of women (96%) and men (82%) were able to pay at least Ug Shs 1000 ($0.27) for FP services while 93% of women and 83% of men who had never used FP services will in future be able to pay for FP services costed at least Shs 2000 ($0.55). The factors independently associated with AWTP were lower age group (<25 years), residence in urban areas, attainment of higher education level, and higher wealth quintiles. Conclusion: AWTP for FP services varied by different measures. Setting the cost of FP services at Shs 1000 ($0.27) will attract almost all women (96%) and most of men (82%). Key determinants of low AWTP include residence in poor regions, being from rural areas and lack of/low education. Implications statement: Private providers should institute price discrimination for FP services by region, gender and socio-economic levels. More economic empowerment for disadvantaged populations is needed if the country is to realise higher contraceptive uptake. More support for total market approach for FP services needed

Impact of oxygen tension according to embryo stage of development: a prospective randomized study.

Human embryo culture under 2-8% O2 is recommended by ESHRE revised guidelines for good practices in IVF labs. Nevertheless, notably due to the higher costs of embryo culture under hypoxia, some laboratories perform embryo culture under atmospheric O2 tension (around 20%). Furthermore, recent meta-analyses concluded with low evidence to a superiority of hypoxia on IVF/ICSI outcomes. Interestingly, a study on mice embryos suggested that oxidative stress (OS) might only have an adverse impact on embryos at cleavage stage. Hence, we aimed to demonstrate for the first time in human embryos that OS has a negative impact only at cleavage stage and that sequential culture conditions (5% O2 from Day 0 to Day 2/3, then «conventional¼ conditions at 20% O2 until blastocyst stage) might be a valuable option for human embryo culture. 773 IVF/ICSI cycles were included in this randomized clinical trial from January 2016 to April 2018. At Day 0 (D0), patients were randomized using a 1:2 allocation ratio between group A (20% O2; n = 265) and group B (5% O2; n = 508). Extended culture (EC) was performed when ≥ 5 Day 2-good-quality-embryos were available (n = 88 in group A (20% O2)). In subgroup B, 195 EC cycles were randomized again at Day 2 (using 1:1 ratio) into groups B' (5% O2 until Day 6 (n = 101)) or C (switch to 20% O2 from Day 2 to Day 6 (n = 94). Fertilization rate, cleavage-stage quality Day 2-top-quality-embryo (D2-TQE), blastocyst quality (Day 5-top-quality-blastocyst (D5-TQB) and implantation rate (IR) were compared between groups A and B (= cleavage-stage analysis), or A(20% O2), B'(5% O2) and C(5%-to-20% O2). Overall, characteristics were similar between groups A and B. Significantly higher rates of early-cleaved embryos, top-quality and good-quality embryos on Day 2 were obtained in group B compared to group A (P < 0.05). This association between oxygen tension and embryo quality at D2 was confirmed using an adjusted model (P < 0.05). Regarding blastocyst quality, culture under 20% O2 from Day 0 to Day 6 (group A) resulted in significantly lower Day 5-TQB number and rates (P < 0.05) compared to both groups B' and C. Furthermore, blastocyst quality was statistically equivalent between groups B' and C (P = 0.45). At Day 6, TQB numbers and rates were also significantly higher in groups B' and C compared to group A (P < 0.05). These results were confirmed analyzing adjusted mean differences for number of Day 5 and Day 6 top quality embryos obtained in group A when compared to those respectively in groups B' and C (P < 0.05). No difference in clinical outcomes following blastocyst transfers was observed. These results would encourage to systematically culture embryos under hypoxia at least during early development stages, since OS might be detrimental exclusively before embryonic genome activation.

Early cleaving embryos result in blastocysts with increased aspartate and glucose consumption, which exhibit different metabolic gene expression that persists in placental and fetal tissues.

OBJECTIVES: Using time-lapse microscopy, previous research has shown that IVF mouse embryos that cleave earlier at the first division ('fast') develop into blastocysts with increased glucose consumption and lower likelihood of post-implantation loss as compared to slower cleaving embryos ('slow'). Further, metabolomics analysis employing LC-MS conducted on groups of 'fast' blastocysts revealed that more aspartate was consumed. With the worldwide adoption of single blastocyst transfer as the standard of care, the need for quantifiable biomarkers of viability, such as metabolism of specific nutrients, would greatly assist in embryo selection for transfer. METHODS: Here we describe the development of a targeted enzymatic assay to quantitate aspartate uptake of single blastocysts. RESULTS: Results demonstrate that the rates of aspartate and glucose consumption were significantly higher in individual 'fast' blastocysts. Blastocysts, together with placental and fetal liver tissue collected following transfer, were analysed for the expression of genes involved in aspartate and carbohydrate metabolism. In 'fast' blastocysts, expressions of B3gnt5, Slc2a1, Slc2a3, Got1 and Pkm2 were found to be significantly higher. In placental tissue derived from 'fast' blastocysts, expression of Slc2a1, Got1 and Pkm2 were significantly higher, while levels of Got1 and Pkm2 were lower in fetal liver tissue compared to tissue from 'slow' blastocysts. CONCLUSIONS: Importantly, this study shows that genes regulating aspartate and glucose metabolism were increased in blastocysts that have higher viability, with differences maintained in resultant placentae and fetuses. Consequently, the analysis of aspartate uptake in combination with glucose represents biomarkers of development and may improve embryo selection efficacy and pregnancy rates.

Single blastocyst stage versus single cleavage stage embryo transfer following fresh transfer: A systematic review and meta-analysis.

OBJECTIVES: To compare the available evidence of the effectiveness of single blastocyst stage transfer against the effectiveness of single cleavage stage embryo transfer. STUDY DESIGN: A systematic research based on Pubmed, Embase and the Cochrane Library was performed until May 2, 2020 to identify all relevant studies. The Cochrane Collaboration's Review Manager (RevMan) 5.0.2 software was used for statistical analysis. RESULTS: Five randomized controlled trials (RCTs) were included in analysis, involving 1784 patients in total, who were divided into 2 groups, which were the single blastocyst stage transfer (SBT) group of 932, and the single cleavage stage transfer (SCT) group of 852. Our meta-analysis concluded that SBT group had a significantly higher clinical pregnancy rate (RR 1.26; 95%CI: 1.14-1.39), ongoing pregnancy rate (RR 1.19; 95%CI: 1.05-1.35) and delivery rate (RR 1.4; 95%CI: 1.13-1.75) than SCT group during the fresh transfer. While miscarriage rate (RR 0.93; 95% CI: 0.66-1.33), multiple pregnancy rate (RR, 1.12; 95% CI, 0.51-2.45) and ectopic pregnancy rate (RR, 0.5; 95% CI: 0.13-1.90) between two groups showed no significant difference. However, the SCT group contained notably more cryopreserved embryos than the SBT group. (RR -0.68, 95% CI: -0.95 to -0.41). CONCLUSIONS: Our results indicate that single blastocyst stage transfer is associated with higher ongoing pregnancy rate and delivery rate comparing to single cleavage stage transfer in the clinical practice. Due to the low quality of the evidence of the primary outcomes, other higher-quality lager RCTs are necessary before a fully informed decision is made.

High ovarian response to ovarian stimulation: effect on morphokinetic milestones and cycle outcomes.

PURPOSE: To assess the effect of high ovarian response on oocyte quality and ovarian stimulation cycle outcomes. METHODS: A retrospective cohort study conducted at three IVF units. The high ovarian response (HOR) and polycystic ovary syndrome (PCOS) with HOR (PCOS HOR) groups included 151 and 13 women who underwent controlled ovarian stimulation (COS) resulting in more than 15 retrieved oocytes, for a total of 1863 and 116 cultured embryos, respectively. The normal ovarian response (NOR) group comprised 741 women with 6-15 retrieved oocytes, resulting in 4907 cultured embryos. Data collected included fresh cycle data and pregnancy rates, in addition to annotation of morphokinetic events from time of pronuclei fading to time of initiation of blastocyst formation of embryos cultured in a time lapse incubator, including occurrence of direct unequal cleavage at first cleavage (DUC-1) (less than 5 h from two to three blastomeres). Comparison was made between morphokinetic parameters between the 3 groups. Cycle outcomes were compared in the high vs. normal ovarian response groups. RESULTS: Oocyte maturation rate was significantly lower in the HOR vs. NOR groups (56.5% vs. 90.0%, p < 0.001), while the fertilization rates were similar (60.2% vs. 58.1%, p = 0.397). The prevalence of DUC-1 embryos was higher in the PCOS HOR and the HOR groups as compared to the NOR group (22.7% vs. 16.2% and 12.0%, respectively, p < 0.001). After exclusion of DUC-1 embryos, remaining embryos from the NOR and HOR groups reached the morphokinetic milestones at similar rates, with comparable implantation and clinical pregnancy rates, while the PCOS HOR showed shorter time to 5 blastomeres compared to the NOR and HOR groups. CONCLUSIONS: High ovarian response might be associated with decreased oocyte quality, manifested as a higher proportion of immature oocytes and higher rate of direct uneven cleavage embryos, while embryos exhibiting normal first cleavage have similar temporal milestones and implantation potential.

Sequential cleavage and blastocyst embryo transfer and IVF outcomes: a systematic review.

BACKGROUND: Sequential embryo transfer has been proposed as a way to improve embryo implantation in women for in vitro fertilization (IVF), but the effect on pregnancy outcomes remains ambiguous. This systematic review was conducted to investigate the efficacy of sequential embryo transfer on IVF outcomes. METHODS: A literature search was performed in the PubMed, Web of Science, Cochrane Library, ScienceDirect and Wanfang databases. Data were pooled using a random- or fixed-effects model according to study heterogeneity. The results are expressed as relative risks (RRs) with 95% confidence intervals (CIs). Heterogeneity was evaluated by the I2 statistic. The study protocol was registered prospectively on INPLASY, ID: INPLASY202180019. RESULTS: Ten eligible studies with 2658 participants compared sequential embryo transfer and cleavage transfer, while four studies with 513 participants compared sequential embryo transfer and blastocyst transfer. The synthesis results showed that the clinical pregnancy rate was higher in the sequential embryo transfer group than in the cleavage embryo transfer group (RR 1.42, 95% CI 1.26-1.60, P< 0.01) for both women who did experience repeated implantation failure (RIF) (RR 1.58, 95% CI 1.17-2.13, P< 0.01) and did not experience RIF (Non-RIF) (RR 1.44, 95% CI 1.20-1.66, P< 0.01). However, sequential embryo transfer showed no significant benefit over blastocyst embryo transfer. CONCLUSION: The current systematic review demonstrates that sequential cleavage and blastocyst embryo transfer improve the clinical pregnancy rate over conventional cleavage embryo transfer. For women with adequate embryos, sequential transfer could be attempted following careful consideration. More high-grade evidence from prospective randomized studies is warranted.