Day 3 Embryo Fragmentation Is Associated With Singleton Birthweight Following Fresh Single Blastocyst Transfer：A Retrospective Study

Background Previous studies arguably associated poor embryo morphology with low birthweight in singletons following single embryo transfer. However, the association between specific morphological features on the cleavage stage and birthweight is still less known. The purpose of the study was to investigate whether embryo morphological features at the cleavage stage affect birthweight following blastocyst transfer Methods The single-center, retrospective cohort study included 4226 singletons derived from fresh single cleavage stage embryo transfer (ET, n=1185), fresh single blastocyst transfer (BT, n=787), or frozen-thawed single blastocyst transfer (FBT, n=2254) between 2016 and 2019. The morphological parameters including early cleavage, day 3 fragmentation, symmetry, blastomere number, and blastocyst morphology were associated with neonatal birthweight and z-score in multivariate regression models. Models were adjusted for maternal age, BMI, parity, peak estradiol level, endometrial thickness, insemination protocol, female etiologies, order of transfer, mode of delivery, and year of treatment. Results Adjusted for confounders, fragmentation was the only morphology feature associated with birthweight and z-score, while early cleavage, symmetry, blastomere number and blastocyst morphology were not. Fragmentation increased the birthweight in both ET group (115.4g, 95% CI: 26.6 to 204.2) and BT group (168.8g, 95%CI: 48.8 to 288.8), but not in FBT group (7.47g, 95%CI: -46.4 to 61.3). The associations of birthweight and morphological parameters were confirmed in analyses for z-score. Adjusted odds of large for gestational age and high birthweight were also significantly greater in singletons following the transfer of fragmented embryos in BT group (OR 3, 95% CI: 1.2 to 7.51, OR 3.65, 95% CI: 1.33 to 10, respectively). The presence of fragmentation at the cleavage stage also affected the association between blastocyst morphology and birthweight. Inner cell mass grades were negatively associated with birthweight in blastocysts with day 3 fragmentation but not in blastocysts without. Conclusions Birthweight following blastocyst transfer is positively associated with fragmentation at the cleavage stage. The data did not support the argument that transferring a poor-looking embryo may increase the risks of low birthweight. However, concerns for LGA infants still remain.

Physical parameters of bovine activated oocytes and zygotes as predictors of development success

Summary The worldwide production of in vitro-produced embryos in livestock species continues to grow. The current gold standard for selecting quality oocytes and embryos is morphologic assessment, yet this method is subjective and varies based on experience. There is a need for a non-invasive, objective method of selecting viable oocytes and embryos. The aim of this study was to determine if ooplasm area, diameter including zona pellucida (ZP), and ZP thickness of artificially activated oocytes and in vitro fertilized (IVF) zygotes are indicative of development success in vitro and correlated with embryo quality, as assessed by total blastomere number. Diameter affected the probability of development to the blastocyst stage in activated oocytes on day 7 (P < 0.01) and day 8 (P < 0.001), and had a tendency to affect IVF zygotes on day 8 (P = 0.08). Zona pellucida thickness affected the probability of development on day 7 (P < 0.01) and day 8 (P < 0.001) in activated oocytes, and day 8 for IVF zygotes (P < 0.05). An interaction between ZP thickness and diameter was observed on days 7 and 8 (P < 0.05) in IVF zygotes. Area did not significantly affect the probability of development, but was positively correlated with blastomere number on day 8 for IVF zygotes (P = 0.01, conditional R 2 = 0.09). Physical parameters of bovine zygotes have the potential for use as a non-invasive, objective selection method. Upon further development, methods used in this study could be integrated into embryo production systems to improve IVF success.

Improving the clinical outcomes by extended culture of day 3 embryos with low blastomere number to blastocyst stage following frozen–thawed embryo transfer

Purpose This study aimed to investigate whether the extended culture of day 3 (D3) embryos with low blastomere number to blastocyst following frozen–thawed embryo transfer improved the clinical outcomes. Methods This was a retrospective study of clinical data of women undergoing in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) cycles in the Tangdu Hospital. The patients were divided into groups with 4–5, 6, 7–9 and > 9 cells based on the blastomere number of D3 embryos. The clinical outcomes were compared. Results In fresh transfer cycles, the implantation and clinical pregnancy rates significantly decreased, while the abortion rate significantly increased in the groups with 4–5 and 6 cells compared with those with 7–9 and > 9 cells. In frozen–thawed transfer cycles, the clinical pregnancy and implantation rates for a single blastocyst transfer cycle showed no significant differences in the groups with 4–5 and 6 cells compared with those with 7–9 and > 9 cells. However, the abortion rate was significantly higher in the group with 4–5 cells than in that with 7–9 and > 9 cells. In the double blastocyst transfer cycle, the clinical pregnancy rate showed no significant differences among the groups with 4–5, 6, and 7–9 cells. Conclusion The implantation and clinical pregnancy rates of D3 embryos with 6 cells significantly decreased; these embryos were not considered as high-quality embryos. Extended culture of D3 embryos with ≤ 6 blastomeres to blastocysts, particularly 6-cell embryos, resulted in a similar clinical pregnancy rate as that of blastocysts derived from D3 embryos with ≥ 7 blastomeres.

The effect of Day 3 cell number on pregnancy outcomes in vitrified-thawed single blastocyst transfer cycles

STUDY QUESTION Does cell number on Day 3 have an impact on pregnancy outcomes in vitrified-thawed single blastocyst transfer cycles? SUMMARY ANSWER A low Day 3 cell number (≤5 cells) was independently associated with decreased live birth rate (LBR) during single blastocyst transfer cycles in young women. WHAT IS KNOWN ALREADY Day 3 cell number is an effective predictor of IVF success rates when transferring cleavage stage embryos. However, the association between Day 3 blastomere number and pregnancy outcomes after blastocyst transfer is still unknown. STUDY DESIGN, SIZE, DURATION A retrospective cohort study of 3543 patients who underwent frozen-thawed single blastocyst transfers from January 2013 to June 2018 at a tertiary-care academic medical center. PARTICIPANTS/MATERIALS, SETTING, METHODS Patients were grouped into six groups according to the Day 3 cell number: ≤4 cells, 5 cells, 6 cells, 7 cells, 8 cells and &gt;8 cells. The primary outcome measure was LBR. A logistic regression analysis was performed to explore the independent association between Day 3 blastomere number and LBR after adjustment for some potential confounders. MAIN RESULTS AND THE ROLE OF CHANCE In women &lt;35 years old, the LBR varied significantly according to Day 3 cell number, with the rate of 31.2%, 34.4%, 41.9%, 45.1%, 48.1% and 48.2% for the ≤4-cell, 5-cell, 6-cell, 7-cell, 8-cell and &gt;8-cell groups, respectively (P &lt; 0.001). This significant difference was also observed in the high- and low-quality blastocyst subgroups of young women. However, for women ≥35 years old, the rate of live birth was similar between groups. Furthermore, after accounting for confounding factors, the LBR was significantly decreased in the ≤4-cell (adjusted odds ratio (aOR): 0.62, 95% CI: 0.48–0.80, P &lt; 0.001) and 5-cell (aOR: 0.73, 95% CI: 0.57–0.92, P = 0.009) groups as compared to the 8-cell group. Likewise, the blastocysts arising from ≤4-cell (aOR: 0.73, 95% CI: 0.57–0.93, P = 0.010) or 5-cell (aOR: 0.77, 95% CI: 0.61–0.97, P = 0.024) embryos were associated with lower clinical pregnancy rate than those from 8-cell embryos. No significant differences were observed in biochemical pregnancy rate and miscarriage rate. LIMITATIONS, REASONS FOR CAUTION A limitation of the current study was its retrospective design. Future prospective studies are needed to confirm our findings. WIDER IMPLICATIONS OF THE FINDINGS Our observations suggested that a low Day 3 cell number was related to decreased LBR after blastocyst transfer in young women, which provided vital information for clinicians in selecting blastocyst during IVF treatment. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by the National Natural Science Foundation of China (NSFC) (31770989 to Y.W.; 81671520 to Q.C.) and the Shanghai Ninth People’s Hospital Foundation of China (JYLJ030 to Y.W.). The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER N/A.

92 Using physical parameters of bovine zygotes to predict invitro development success

The objective of this study was to determine if physical parameters of bovine zygotes are correlated with invitro development success. We examined the relationship between a zygote's outer diameter (OD), cell area, and zona pellucida (ZP) thickness on blastocyst development and blastomere number. Bovine cumulus-oocyte complexes, from abattoir-derived ovaries, were matured in tissue culture medium 199-based maturation medium for 21-23h. Invitro fertilisation was performed with frozen thawed semen containing a pool of 4 Holstein bulls. After incubation with sperm for 16-18h, presumptive zygotes (n=875) were denuded and placed in 5µL droplets of synthetic oviductal fluid-bovine embryo 1 medium (SOF-BE1) under oil to be individually photographed using an inverted scope with a digital camera. Zygotes were then group-cultured in 50µL of synthetic oviductal fluid-BE1 droplets in a polyester micromesh to identify individuals. Development to the blastocyst stage was recorded on Day 7 and Day 8 of culture, and Day 8 embryos were fixed and stained with Hoechst 33342 (n=87). ImageJ was used to measure the OD, area, and ZP from images. Data were analysed with a logistic regression using the lme4 package in R with Day 7 or Day 8 blastocyst development as the response; OD, area, and ZP as predictors; and study replicate (n=11) and culture droplet as random effects. Residual estimates of area and ZP calculated from OD were used to account for collinearity. Average OD was 151.2µm, ranging 130.4-171.6µm. Average ZP was 11.8µm, ranging 7.2-17.5µm. Area averaged 9856.7µm2, ranging from 6421.9 to 13 814.8 µm2. There was a tendency for an effect of OD on probability of development on Day 8 (P=0.08) but not Day 7. There was an effect of ZP on the probability of development on Day 8 (P=0.04) but not Day 7. Blastocyst rates on Day 8 for zygotes with ZP &lt;11.8µm were 24.3%, whereas zygotes with ZP &gt;11.8µm were 21.7%. There was an interaction (P&lt;0.05) between OD and ZP thickness on Day 7 and Day 8. Estimated probability of development of zygotes with ZP &lt;10.7µm and OD &lt;146.9µm averaged 28.5%, whereas those with ZP &gt;12.7µm and OD &gt;155.8µm averaged 43.3%. The observed blastocyst rates for these two groups were 26.2% and 25%, respectively. Zygotes with OD between 146.9-155.8µm and ZP between 10.7-12.7µm had an estimated development probability of 22.2%. The obtained Day 8 blastocyst rate for this group was 22.5%. Area did not affect probability of development, but was positively correlated with total blastomere number at Day 8 (P=0.01; marginal R2=0.09). The observed interaction between ZP thickness and OD may be indicative of an optimum ooplasm area before maturation or fertilisation that may enhance development. Area assessment after fertilisation is not correlated with development success. In addition, these data in comparison with our previous data generated in parthenogenetic embryos indicate that artificial activation of oocytes is not an ideal model in place of IVF for studying development. We continue to demonstrate that physical parameters of zygotes may have potential as a noninvasive, objective selection tool of embryos.

102 Sperm cryopreservation with a soy lecithin-based medium in black-footed cats (Felis nigripes) and sand cats (Felis margarita)

Felid semen has historically been frozen using an egg yolk-based cryopreservation medium (TEY). However, the use of egg introduces several potential concerns, such as variability in composition, microbial contamination, and regulatory issues. Our recent research has focused on developing an animal protein-free medium containing soy lecithin (SOY). Our studies revealed that SOY was superior to TEY for freezing domestic cat sperm and provided similar results for freezing ocelot, Pallas’ cat, and fishing cat sperm. The objective of this study was to compare SOY to the standard TEY for sperm cryopreservation in 2 wild cat species: the black-footed cat and sand cat. Semen was collected from adult male cats (n=6/species) via electroejaculation, split into 2 aliquots, centrifuged, resuspended in either SOY or TEY, slow-cooled, and frozen in straws over nitrogen vapor. Sperm motility [percent progressively motile (PPM); rate of progressive motility on 0-5 scale (RPM)] was evaluated at 0, 1, 3, 6, and 24h post-thaw and acrosome status (AC) was assessed at 0 and 6h post-thaw. Heterologous IVF was performed using oocytes collected laparoscopically from gonadotropin-treated domestic cats. At 48h post-insemination, Hoechst33342 staining was used to determine oocyte stage, number of blastomeres, and number of accessory sperm (AS) bound to the zona pellucida of embryos and mature oocytes. Percent progressively motile, RPM, and AC were analysed with repeated-measures ANOVA; embryo cleavage, blastomere number, and AS number were analysed with one-way ANOVA. All data are reported as least squares means±average standard error. In the black-footed cat, PPM, RPM, and AC of SOY-treated sperm (32.5±4.0% motile, 2.8±0.2 RPM, 41.8±4.1% intact; 0h) did not differ from TEY-treated sperm (44.2±4.0% motile, 2.8±0.2 RPM, 46.8±4.1% intact; 0h) at any post-thaw time point (P &gt; 0.05). Similarly, in the sand cat, post-thaw PPM, RPM, and AC of SOY-treated sperm (36.7±5.2% motile, 2.6±0.2 progression, 53.3±5.8% intact; 0h) did not differ from TEY-treated sperm (45.8±5.2% motile, 2.8±0.2 RPM, 51.0±5.8% intact; 0h) at any time point (P &gt; 0.05). In black-footed cats, neither embryo cleavage (34.1±10.9% SOY; 58.5±10.9% TEY), blastomere number (7.8±0.8 SOY; 6.3±0.8 TEY), nor AS (3.5±0.8 SOY; 1.7±0.8 TEY) differed between treatments (P &gt; 0.05). Sand cat results were similar, with no difference between SOY and TEY for cleavage (44.7±10.8% SOY; 40.6±10.8% TEY) or blastomere number (7.4±2.0 SOY; 6.7±2.0 TEY) (P &gt; 0.05), but AS was higher in SOY-treated sperm (4.3±0.2 SOY; 3.5±0.2 TEY, P=0.0183). These data collectively demonstrate that our SOY medium was an effective substitute to TEY for sperm cryopreservation in the black-footed cat and sand cat. The replacement of an egg yolk-based cryomedium with a chemically defined, animal protein-free alternative represents a significant advance in quality control and biosecurity for felid semen banking and should augment the use of assisted reproduction for population management of imperiled cats. Funded by the Institute of Museum and Library Services.

T-2 mycotoxin slows down the development of mouse blastocysts, decreases their blastomere number and increases chromatin damage

The mycotoxin T-2 has many harmful effects on mammalian cells and reproductive functions. In the present study, the in vitro effect of T-2 toxin on mouse blastocysts was examined. Embryos were cultured in media supplemented with 0.5, 0.75 and 1 ng/ml T-2. Different exposure times were applied [96 h (treatment I) or 24 h following 72 h in toxin-free media (treatment II)]. Blastomere number, nuclear chromatin status and blastocoel formation were investigated in blastocysts. Our data show that the effect of T-2 toxin may vary depending on the stage of the embryo at the start of exposure. At 96 h of exposure, the blastocysts had blastomeres with normal chromatin quality but their developmental potential was decreased. After 24 h of exposure applied following a 72-h culture, blastomeres had a higher level of chromatin damage, although their developmental potential was the same as in the control embryos. In both cases, decreased mitotic rate was found, which resulted in decreased blastomere number even at low toxin concentration.