Higher Blastocyst Rate Research Articles

Production of bovine embryos by piezo-ICSI using capacitated spermatozoa selected by fluorescence-activated cell sorting (FACS-piezo-ICSI)

Intracytoplasmic sperm injection (ICSI) remains inefficient in cattle. One reason could lie in the injection of oocytes with sperm that have not undergone molecular changes associated with in vivo capacitation and fertilizing ability. This study aimed to enhance the efficiency of bovine intracytoplasmic sperm injection (piezo-ICSI) by employing fluorescent-activated cell sorting (FACS) to select the sperm population before injection based on capacitation markers. First, we evaluated the effects of incubating thawed sperm for 2 hours with different capacitating inductors: heparin, methyl-beta-cyclodextrin (MβCD), and dibutyryl cyclic AMP (dbcAMP), alone or in combinations in a basal capacitating (C) medium (Sp-TALP). Sperm capacitation and quality markers were evaluated by flow cytometry, revealing heparin as the most effective inducer of sperm capacitation changes. It, therefore, this treatment was chosen as the sperm pretreatment for FACS-piezo-ICSI. Two cell populations showing high capacitating levels (Heparin-HCL) and low capacitating levels (Heparin-LCL) of the markers associated with sperm capacitation i(Ca2+) levels and acrosome integrity were selected by FACS and used for sperm injection. Pronuclear formation was significantly higher when ICSI was performed with Heparin-HCL sperm than with Heparin-LCL and the control group (Heparin unsorted) groups (50 %, 10 %, and 20 %, respectively). Furthermore, injecting Heparin-HCL sperm resulted in a higher blastocyst rate (22.5 %) than Heparin-LCL (10 %) and the control group (15.2 %). In conclusion, heparin treatment effectively induced changes associated with sperm capacitation. The combination of Heparin-HCL treatment and FACS enabled precise selection of capacitated sperm before ICSI, enhancing the efficiency of this technology in the bovine species.

Regional abundances of binder of sperm (BSP) proteins are negatively associated with the quality of frozen-thawed bovine spermatozoa.

The localization and abundance of the sperm BSP proteins correlate with in vitro fertility in domestic bulls used in artificial insemination service. Binder of sperm (BSP) proteins, secreted mainly by the accessory sex glands, are the major protein family present in bovine seminal plasma and on the sperm surface after ejaculation. In vivo, BSP proteins facilitate sperm capacitation and sperm reservoir formation; however, their impact on sperm function within the in vitro systems is less clear. Therefore, this biomarker-based study aimed to characterize the localization and abundance of BSP proteins from in vitro processed frozen-thawed bovine spermatozoa. Using image-based flow cytometry and Western blotting, BSP protein localization, abundance, membrane and acrosomal integrity were investigated in the supernatant (nonmotile) and pellet (motile) fractions of gradient-separated bull spermatozoa. Spermatozoa from the supernatant fraction had high enrichment of all BSP proteins investigated (BSP1, BSP3, BSP5; P < 0.05) when compared to the pellet fraction. In the pellet fraction, BSP1 and BSP3 bound predominately to the acrosomal region, whereas BSP5 had a high affinity for the midpiece. However, in the supernatant fraction, BSP proteins predominately coated the entire sperm surface resulting in the loss of regional specificity. High BSP protein abundance in the spermatozoa also correlated with acrosome and membrane damage. Whereas a high abundance of BSP5 correlated with low embryo cleavage rates, high abundance of BSP1 on the sperm head coincided with a high blastocyst rate. Therefore, changes in the quantity and localization of specific BSP proteins could act as potential biomarkers of sperm quality and fertility.

MACS-DGC sperm preparation method resulted in high-quality sperm, top-quality embryo, and higher blastocyst rate in male factor infertile couples with high DNA fragmented sperm

Conventional sperm selection based on motility and morphology fails to provide detailed information on sperm functional and molecular status. Magnetic-activated cell sorting (MACS) protocol aims to optimize this process by selecting apoptotic sperm cells. Phospholipase C zeta-1 (PLCz1) is a physiological stimulus for oocyte activation and early embryonic development. The purpose of this study was to examine seminal parameters, DNA fragmentation index (DFI), and PLCz1 expression levels in MACS-DGC sorted specimens (DFI > 30%) and assess early development in resulting embryos. Semen specimens from 60 patients diagnosed with male factor infertility were collected and processed by either density gradient centrifugation (DGC) or MACS-DGC protocols. Pre and post-preparation analysis was performed. PLCz1 expression was assessed using the RT-PCR method. Retrieved eggs from their partners were divided into two groups in which they were injected with different sorted sperm. The fertilization rate and embryonic development were evaluated. While sperm’s progressive motility and morphology significantly improved, there was a substantial decline in DFI following MACS-DGC. Fertilization rates were almost the same between the groups, and the latter resulted in remarkably more top-quality embryos and more blastocysts. PLCz1 expression was considerably higher in the MACS-DGC group. By eliminating apoptotic cells, the MACS-DGC technique could sort highly PLCz1-expressed sperm, optimize sperm selection in individuals with elevated DFI, development of resulting embryos.

Factors affecting post-mortem production of equine ICSI blastocysts

In vitro fertilization of equine oocytes by intracytoplasmic sperm injection (ICSI) has enabled post-mortem production of equine embryos. This procedure makes it possible to secure one last offspring from mares that must be suddenly euthanized. Currently, little is known about how clinical factors influence the outcome. The aim of this study was to evaluate the effect of the following clinical factors on the chance of a successful session (≥1 embryo) and blastocyst rates after post-mortem ICSI: Mare age, number of collected oocytes per mare, location of euthanasia (at our clinic/in the field), duration of ovary transport, method of ovary removal (during anaesthesia, after pentobarbital, after bolt gun), reason for euthanasia (acute life-threatening conditions requiring immediate euthanasia vs. non-acute conditions, where euthanasia could be postponed). The study was performed retrospectively and included ovaries from 65 euthanized mares in the period between 2019-2022. The mares, mainly Warmbloods, were aged between 1 to 26 years old (average 14.4 years old). If euthanasia was performed in the field, the ovaries were transported to our clinic (0.5 to 14 hours) in sterile saline at 22-32⁰C. The oocytes were collected by scraping each follicle. After overnight holding, the oocytes were matured for 30 hours. The mature metaphase II oocytes were inseminated by ICSI with the desired semen and after7-10 days of culture, blastocysts were vitrified. In total, 842 oocytes were collected from 65 mares (range: 1 to 47 oocytes per mare, average = 13), 744 oocytes were placed in maturation medium and the remaining degenerated oocytes were discarded. A total of 65% (481/744) of the oocytes reached the MII stage and were injected, and 11.4% (55/481) of these reached the blastocyst stage within 10 days after ICSI. The proportion of successful sessions was 37%. The factors that were shown to affect the chance of a successful session, were the number of collected oocytes per mare and the reason for euthanasia (acute vs. non-acute; multiple logistic regression, p < 0.05). The other examined factors did not affect the chance of a successful session (multiple logistic regression, p > 0.05). When comparing mares euthanized for non-acute reasons to mares euthanized for acute reasons, there was a higher chance of a successful session (50%, 16/38 vs. 20.7%, 6/29, respectively, p = 0.02, Fisher's exact test) and a higher blastocyst rate (16.2%, 44/272 vs. 5.3%, 11/209, p = 0.0001, Fisher's exact test) in mares euthanized for non-acute reasons. This study reports that post-mortem, in vitro embryo production can be performed on mares euthanized both in the field and at the ICSI clinic, but with a lower chance of a successful session for mares euthanized due to acute life-threatening conditions.

Occurrence of late-apoptotic symptoms in porcine preimplantation embryos upon exposure of oocytes to perfluoroalkyl substances (PFASs) under in vitro meiotic maturation.

The objectives of this study were to evaluate the effect of perfluoroalkyl substances on early embryonic development and apoptosis in blastocysts using a porcine in vitro model. Porcine oocytes (N = 855) collected from abattoir ovaries were subjected to perfluorooctane sulfonic acid (PFOS) (0.1 μg/ml) and perfluorohexane sulfonic acid (PFHxS) (40 μg/ml) during in vitro maturation (IVM) for 45 h. The gametes were then fertilized and cultured in vitro, and developmental parameters were recorded. After 6 days of culture, resulting blastocysts (N = 146) were stained using a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and imaged as stacks using confocal laser scanning microscopy. Proportion of apoptotic cells as well as total numbers of nuclei in each blastocyst were analyzed using objective image analysis. The experiment was run in 9 replicates, always with a control present. Effects on developmental parameters were analyzed using logistic regression, and effects on apoptosis and total numbers of nuclei were analyzed using linear regression. Higher cell count was associated with lower proportion of apoptotic cells, i.e., larger blastocysts contained less apoptotic cells. Upon PFAS exposure during IVM, PFHxS tended to result in higher blastocyst rates on day 5 post fertilization (p = 0.07) and on day 6 post fertilization (p = 0.05) as well as in higher apoptosis rates in blastocysts (p = 0.06). PFHxS resulted in higher total cell counts in blastocysts (p = 0.002). No effects attributable to the concentration of PFOS used here was seen. These findings add to the evidence that some perfluoroalkyl substances may affect female reproduction. More studies are needed to better understand potential implications for continued development as well as for human health.

Overnight holding aids in selection of developmentally competent equine oocytes

The demand for equine in vitro produced embryos has increased over the last decade. The aim of this study was to compare the effects of an extended IVM or a prolonged period before fertilization, including holding time, on equine immature oocyte developmental competence. Oocytes, collected from abattoir-derived ovaries, were divided into 4 groups: H0/24 (n = 165) 0 h holding + standard 24–26 h IVM; H8/36 (n = 160) 8 h holding + 36 h IVM; H20/24 (n = 187) 20 h holding + 24 h IVM; H0/44 (n = 164) 0 h holding + 44 h IVM. Oocytes matured to MII were fertilized by intracytoplasmic sperm injection (ICSI) and cultured for 10 days. The oocyte degeneration rate was higher (P < 0.05) for H20/24 than the other groups (H0/24 38.2 %, H8/36 43.1 %, H20/24 54.5 %, H0/44 32.9 %). Cleavage was higher (P < 0.05) in H20/24 (70 %) compared to H0/24 (45 %) and H8/36 (54 %) but not to H0/44 (63 %). No differences among groups were observed in the number of blastocysts per oocyte. Injected oocytes that reached the blastocysts stage were higher (P < 0.05) for H20/24 (20 %) than H0/24 (7 %) and H0/44 (7 %) but not H8/36 (12 %). For cleaved oocytes, a higher blastocyst rate (P < 0.05) was observed for H20/24 (28 %) than H0/44 (11 %), while H0/24 (15 %) and H8/36 (21 %) were not different from any group (P > 0.05). Timing of blastocyst development was not different among groups. Overnight holding of equine immature oocytes followed by a standard IVM interval may induce a pre-selection of the most competent oocytes thereby improving cleavage and embryo development rates after ICSI.

P-099 The effect of sperm DNA quality on blastocyst and euploid rates

Abstract Study question Do sperm DNA quality measurements influence the blastocyst rate and/or euploid rates? Summary answer Sperm DNA fragmentation has a negative influence on the rate of blastulation. It does not impact on euploidy rates assessed by pre-implantation genetic testing (PGT-A). What is known already Some studies show that sperm DNA has no influence on embryo development and that its negative influence begins following implantation. Other studies show that every step of the fertility process from fertilization onwards is impacted by the male gamete as well as the female gamete. To date, data has been confounded by different sperm DNA tests and different clinical practices and parameters measured. Study design, size, duration This was a retrospective study using the sensitive Comet assay to analyse whether it’s three sperm DNA fragmentation scores influence embryo development or euploidy. Participants/materials, setting, methods Data from 110 cycles of intracytoplasmic sperm injection (ICSI) where the blastocyst rate (number of blastocysts/number of mature oocytes injected) was obtained from the patient database of a UK single centre. Inclusion criteria: Only outcomes from the first assisted reproductive cycle after a Comet assay had been performed were included. Included cycles were within one year of the test. Main results and the role of chance Blastocysts were ranked in 4 groups- 0.25-0.49, 0.5-0.74, 0.75-1.00. Three Comet scores were analysed: Average Comet Score (damage across the semen sample; ACS), Low Comet Score (proportion of sperm with good DNA; LCS) and High Comet Score ( proportion of sperm with high DNA damage; HCS). Clinical thresholds were calculated by comparison with fertile donor sperm. High ACS and LCS and low HCS were all associated with higher blastocyst rates (ANOVA, p = 0.013; Kruskal Wallis, p = 0.027, Post Hoc pairwise comparisons between blastocyst groups (p = 0.016-0.024). No relationship was found between euploid rate (number of euploid embryos/total embryo number) and sperm DNA damage p = 0.761). Limitations, reasons for caution Retrospective design requiring more data to clarify these associations coupled with the lack of randomisation and low power analysis. Wider implications of the findings This study reports the negative impact of sperm DNA fragmentation on blastocyst formation rates. Male partner diagnostic assessments such as sperm DNA quality testing are useful adjuncts to semen analysis. Further research to determine if there is a relationship between sperm DNA damage and euploid embryo outcomes is ongoing. Trial registration number IRB-001C06-01-22

O-204 Non-invasive AI image analysis unlocks the secrets of oocyte quality and reproductive potential by assigning ‘Magenta’ scores from 2-dimensional (2-D) microscope images

Abstract Study question Can an Artificial Intelligence (AI) software tool, utilizing 2-D image analysis of mature oocytes, prospectively correlate an oocyte score to utilizable blastocyst development? Summary answer Oocyte Magenta scores show a statistically significant difference in blastocyst development between the highest (7.1-10) and lowest (1.0-4.0) scored oocytes [46.1% vs 26.6%; p &amp;lt; 0.005]. What is known already Unlike sperm (WHO 2010) or embryos (Gardner blastocyst grading), there is no validated visual oocyte scoring system used in clinical practice. Embryologists have been unsuccessful in correlating oocyte morphological features to reproductive potential. A valuable oocyte scoring system should be able to correlate higher scores with improved embryological outcomes. Although not possible by the human eye, a non-invasive oocyte AI assessment tool (Magenta) has accomplished this feat in retrospective studies. This study applied the Magenta network at two IVF clinics in real-time; representing one of the few prospective AI studies in our field, and the only one focusing on oocytes. Study design, size, duration This prospective, multi-center study was conducted from September - November 2021 by TRIO Fertility (Toronto, Canada) and CARE Fertility (Sheffield and Nottingham, UK), utilizing the oocyte AI image analysis tool, Magenta. Magenta was created with a convolutional neural network trained on 16,373 oocyte images and corresponding outcomes. Inclusion criteria was all IVF-ICSI patients who consented to participate without severe male factor (testicular or epididymal sources). Results are based on 392 images of oocytes (46 patients). Participants/materials, setting, methods Non-invasive, light microscope images were taken of mature oocytes post-denudation, prior to ICSI, utilizing an image capture software. Images were uploaded and analyzed by Magenta, scoring each oocyte on a scale of 1-10, and remained in a blinded folder to the IVF clinics. De-identified patient outcomes were collected to analyze blastocyst development correlation with Magenta scores. Oocytes were handled as per good laboratory practice, without extended periods outside the incubator or disruption to standard protocols. Main results and the role of chance Oocyte images were analyzed by Magenta to score each oocyte on a scale of 1-10. There was a total of 46 patients representing 392 oocytes from both TRIO (26, 280) and CARE (20, 112). The scoring spectrum was divided into 3 tiers (1.0-4.0: 188 oocytes; 4.1-7.0: 128 oocytes; 7.1-10: 76 oocytes). A utilizable blastocyst was defined as a Gardner grade of 2BB or greater on Day 5 or 3BB or greater by Day 6 of embryo development and of adequate quality for transfer, freezing or PGT-A biopsy. The blastocyst development (positivity) rate was 26.6% (1.0-4.0), 32.0% (4.1-7.0) and 46.1% (7.1-10), with mean Magenta scores of 2.4, 5.5 and 8.2, respectively. The lowest and highest tier of Magenta scores were accordingly found to have the lowest and highest blastocyst rates, which was statistically significant (p-value &amp;lt; 0.005) by a Two-Proportions Z-test. Overall, oocytes that developed into a utilizable blastocyst had a higher mean Magenta score (5.0) than oocytes that did not develop into a utilizable blastocyst (4.3); (p-value &amp;lt;0.05) by a Welch’s Two Sample t-test. Limitations, reasons for caution Sample size is currently limited for this ongoing, prospective study. Therefore, additional male factor (non-surgical sperm sources) and possible poor images have not been removed from the current analysis. Furthermore, AI neural network accuracy is restricted by the amount of data it is trained on. Wider implications of the findings Magenta has enabled visual oocyte assessments that will provide IVF-ICSI patients with insights into their oocyte quality; resulting in counselling benefits and the ability to make more informed, personalized decisions regarding future treatment plans. AI will inevitably improve the IVF process and prospective validation studies are critical in its evolution. Trial registration number Not applicable.

PSX-A-23 Late-Breaking: Supplementation of Nerve Growth Factor (β-NGF) in the maturation medium and efficiency of in vitro embryo production in cattle

Abstract The aim of this study was to evaluate the supplementation of Nerve Growth Factor (β-NGF) in the maturation medium in in vitro embryo production routines. Antral follicles were aspirated from ovaries of cows obtained from slaughterhouses and then oocytes were selected for quality (grades I and II) for in vitro maturation and subjected to 4 successive in vitro embryo production routines (IVEP). Supplementation of 100 ng of β-NGF was performed in the oocyte maturation medium 22 hours before in vitro fertilization. 48 hours after fertilization of the oocytes, an analysis was made of their cleavage rate by counting blastomeres with the aid of a stereoscopic microscope (cleavage rate = number of embryos / number of initial oocytes). Seven days after fertilization, the blastocyst rate was determined through the relation to the number of oocytes that started cleavage and reached this stage of development (blastocyst rate = number of blastocyst / number of oocytes that started cleavage). To verify the existence of a difference between the supplemented and the non-supplemented groups, the paired T test was applied, using the Excel / Action software (Microsoft). In vitro embryo production routines supplemented with β-NGF in the maturation medium had, on average, a higher cleavage rate (P = 0.0072) and a higher blastocyst rate (P = 0.0033) compared to non-supplemented routines with β-NGF. In this study was demonstrated that Nerve Growth Factor supplementation in the maturation medium improves the efficiency of in vitro embryo production in cattle, and this protein has a probable action in the oocyte maturation process.

PREDICTING WHICH BLASTOCYSTS WILL DEVELOP INTO FETUSES: TIME INTERVAL FROM MORULAE TO BLASTOCYSTS IS A NOVEL AND USEFUL INDICATOR FOR SUCCESSFUL PREGNANCY

The increased use of blastocyst transfer has emphasized the importance of selecting high-quality embryos. Analyses of embryos in a time-lapse incubator system showed that zygotes with the shortest intervals between the first and second or the second and third mitotic divisions were higher blastocyst rate. Additionally, embryos that developed relatively faster to the morulae and blastocysts had higher implantation rates. However, the useful indicators that predict which embryos will develop into fetuses without pregnancy loss have not yet been identified. Do time to morulae and interval between morulae and blastocysts predict which blastocysts will develop into fetuses? We analyzed 208 blastocysts derived from ICSI in 177 patients from September 2019 to September 2020. All embryos were monitored by a time-lapse system. The outcome of vitrified-thawed single blastocyst transfers was classified into pregnancy and non-pregnancy groups. The pregnancy group was divided into ongoing pregnancy and pregnancy loss. Data were analyzed using multiple logistics regressions. According to the logistic analysis of women age, the data were classified as less than or more than 39 years old. Time-lapse analyses provided data on time from pronuclear fading to morulae (tM), time to expanded blastocysts (tEB), interval between morulae and blastocysts (tB-tM), the number of equatorial trophectoderm cells (eTE), and the KIDScore D5 v3 (KS5). Data from pregnancy (hCG > 50 mIU/ml at 4 weeks) versus non-pregnancy were compared, as well those from ongoing pregnancy (a gestational sac at 12 weeks) versus pregnancy loss groups. In this study, 51.4% of total blastocyst transfers resulted in pregnancy. Significant differences were found between pregnancy and non-pregnancy groups: tM, 63.0 vs. 65.8 h; tEB, 92.4 vs. 95.5 h; tM-t8, 29.5 vs. 32.2 h; eTE, 6.5 vs. 5.5; and KS5, 6.7 vs. 5.8. The pregnancy rate in women less than 39 years old was significantly higher than in women over 39 years old (67.4% vs. 38.1%). Under 39-years-old, ongoing pregnancy rate was 79.7%, and pregnancy loss rate was 20.3%. A faster tM, a longer tB-tM, and a more eTE was found in the ongoing pregnancy group compared to the pregnancy loss group. The potential of tM, tB-tM and eTE as indicators for pregnancy was predicted with an AUC of 0.79. Over 39-years-old, ongoing pregnancy rate was 72.1%, and pregnancy loss rate was 27.9%. In ongoing pregnancy group, t2, t4 and tM were significantly different to the pregnancy loss group, which suggested delay of early embryonic development increases pregnancy loss risk. Under 39 years old, tM, tB-tM and eTE are effective predictors for selection of high-quality blastocysts that will be successful pregnant. The time interval tB-tM is a new indicator for identifying high-quality blastocysts. Over 39 years old, faster embryo development is a critical indicator for pregnancy.

P–062 Annexin-V MACS method could be a good choice for sperm selection with high PLCZ1 expression and high blastocyst rate in high DFI male factor

Abstract Study question What effect does Annexin-V MACS method have on sperm selection by pLCZ1 expression and blastocyst rate in male DNA fragmentation? Summary answer AnnexinV-MACS method could be a good choice for sperm selection with high pLCZ1 expression and high blastocyst rate in high DNA fragmentation male factor. What is known already Sperm selection based on morphology and motility in ART techniques, is not enough for choosing the best sperm especially in male factor patients. In Annexin-V magnetic activated cell sorting (MACS) technique, apoptotic sperm are separated from non-apoptotic one by negative selection. So, this method can improved quality of compaction rate in embryo. PLCζ is oocyte activating factors that it starts oscillations of calcium ca2+ in oocyte and it has a significant effect on fertilization and implantation. Study design, size, duration Semen samples from 30 male factor infertile couples with high DFI (DFI&amp;gt;30%) were selected and divided into two group (control and experimental) in each patient. Participants/materials, setting, methods Control was washed with DGC and experimental one was selected by MACS-DGC. Retrieved eggs in each patient, were divided in 2. Both group were injected by DGC and MACS respectively. Semen parameters and DFI (SCD test) were analyzed before and after processing. After ICSI, rate of fertilization, embryo development and blastocyst formation were evaluated. Real time PCR evaluate expression of PLCζ. Comparison between results of two groups was assessed by SPSS analysis. Main results and the role of chance Results showed that, sperm motility and morphology after MACS method (45%, 1.7%) was significantly higher than DGC method (40%, 1.1%) and before washing (35%, 0.9%). Percent of DFI in MACS group (36%) was significantly decreased compared to DGC (45%) and primitive group (55%). The number of oocytes were injected in DGC group was 93 and in MACS group was 111. Fertilization rate in both groups was almost the same (72.07% in MACS vs 73.11 in DGC). Rate of day 3 embryo with good grade in MACS group (72. 5%) was significantly higher than DGC (51.47%) (P &amp;lt; 0.05). The blastocyst rate in MACS-DGC group (69.69%) was significantly increased compared to DGC group (48%). PLCζ gene expression in MACS-DGC was significantly higher than DGC group (p-value=0.046). Limitations, reasons for caution This experiment was performed invivo. Wider implications of the findings: Sperm selection by MACS-DGC method can improve sperm motility, morphology and reduce sperm DFI. No significant difference was observed in fertilization rate, but percent of high-quality embryo on days 3 and 5 was significantly higher by this method,also it can be suggested as a good choice for patients with highDF. Trial registration number Not applicable

P–214 Effect of artificial activation of oocytes (AOA-ICSI) on the ploidy status of the resultant blastocysts. A sibling-oocytes pilot study

Abstract Study question Will artificial activation of oocytes alter the ploidy status of the resultant blastocysts? A sibling-oocytes pilot study Summary answer AOA-ICSI does not increase the risk of having aneuploidy blastocysts and can improve the fertilization rate in patients with sperm factor deficiency. What is known already Despite introducing ICSI as an aid to improve chances of fertilization, fertilization failure can still occur in 2–3% of ICSI cycles. Fertilization is a complex process triggered by a cascade of events following calcium (Ca2+) oscillations. Evidence suggests that the deficiency, localization or altered structure of the sperm-derived protein PLCζ in oocyte activation may be a reason for meiotic II arrest in the oocyte. Artificial oocyte activation has been proposed to compensate for the lack of calcium oscillation and resumes meiotic progression. There are however insufficient studies to determine its effect on the chromosomal status of the resultant blastocysts. Study design, size, duration This is a prospective, randomized study conducted at our Center from August-October 2020. A total of 20 couples intended for ICSI + Preimplatation Genetic Testing for Aneuploidy (PGT-A) cycles were recruited based on fulfilling one of the following criteria: 1) previous total fertilization failure (TFF), 2) history of low fertilization rate (&amp;lt;30%), 3) more than 2 cycles of failed IVF cycles (no implantation) 4) poor embryo development (no blastocysts formed) and 5) severe male factor. Participants/materials, setting, methods A total of 231 MII oocytes underwent randomization in a 1:1 ratio between AOA-ICSI and control group. All oocytes are subjected to ICSI treatment. Oocytes in the AOA-ICSI group are treated in 25μl droplets 10μM ready to use bicarbonate buffered calcium ionophore (Kitazato, Japan) for 15 minutes post-ICSI. The blastocysts were biopsied and subjected to PGT-A. Primary outcome was the aneuploidy rate and secondary outcomes were fertilization rate and blastocyst rate. Main results and the role of chance There were 11 out of 40 (27.5%) aneuploid blastocysts in the AOA-ICSI group and 7 out of 23 aneuploid blastocysts (30.4%) in the control group [odds ratio (OR) = 0.87; 95% confidence interval (CI) 0.28–2.68, p = 0.8040). There was no statistically significant difference between both groups. However, fertilization rate of the AOA- ICSI group was significantly higher than the fertilization rate in the control group (68.6% vs 49.6% respectively, OR = 2.22; 95% CI, 1.31–3.81, p = 0.0034). There were 40 blastocysts formed in the AOA-ICSI group and 23 blastocysts formed in the control group. It was found that the AOA-ICSI group yielded a higher blastocyst rate (49.4%) compared to the control group (41.1%) (OR = 1.40; 95% CI, 0.71 to 2.78, p = 0.3379) but the difference was not statistically significant. Limitations, reasons for caution The possibility of TE cells biopsied may not be representative of the whole blastocyst makes it possible to have false clinical data. The dosage and time were also not evaluated in this study as exposure time was found to be a critical factor of fertilization rate in a previous study. Wider implications of the findings: This study showed that AOA-ICSI does not increase the risk of having aneuploidy blastocysts and can improve the fertilization rate in patients with sperm factor deficiency. Additional studies involving a larger number of patients with more specific indication can further justify the benefits of AOA as a therapeutic application. Trial registration number NA

Development and frozen-thawed transfer of non-pronuclear zygotes-derived embryos in IVF cycles

ObjectiveTo explore the development and pregnancy potential of non-pronuclear (0PN) zygote-derived embryos in conventional in vitro fertilization (IVF) cycles. Study designEmbryonic development in 1039 oocyte retrieval cycles and clinical outcomes of 659 frozen-thawed blastocyst transfer cycles were retrospectively studied. ResultsDevelopmental potential of embryos with different blastomere numbers on day 3 were inconsistent in 0PN and 2PN groups. For 0PN-derived embryos, blastocyst rate of fast developing embryos (75.4%) was similar to that of intermediately developing embryos (72.9%), but good quality blastocyst rate of the former (49.2%) was significantly higher than that of the later (39.6%). In 2PN group, intermediately developing embryos had the highest blastocyst rate (77.9%) and good quality blastocyst rate (51.5%) (statistically significant). Comparison of frozen-thawed transfer was carried out between 0PN- and 2PN-derived blastocysts. For both single (SBT) and double blastocyst transfer (DBT) groups, no statistical differences existed between 0PN- and 2PN-derived blastocysts in clinical pregnancy rates (45.2% and 49.1% in SBT group, 64.7% and 66.4% in DBT group), implantation rates (45.2% and 49.1% in SBT group, 41.2% and 47.7% in DBT group) and live birth rates (35.5% and 36.8% in SBT group, 52.9% and 51.2% in DBT group). ConclusionThe developmental characteristic of 0PN-derived embryos was different from that of 2PN-derived embryos in IVF cycles. 0PN-derived blastocysts could obtain acceptable clinical pregnancy and live birth, but more studies are needed to confirm the safety..

Prediction of oocyte quality using mRNA transcripts screened by RNA sequencing of human granulosa cells

Research questionCan RNA transcripts of granulosa cells be used to assess oocyte quality? The possibility of predicting the developmental competence of oocytes by RNA sequencing analysis of granulosa cells was evaluated. DesignGranulosa cell samples were collected from 29 women undergoing assisted reproductive technology treatment and divided into two groups: 14 samples from the high blastocyst rate group and 15 from the low blastocyst rate group. Ten samples from each group were selected for RNA sequencing. ResultsA total of 129 differentially expressed genes associated with high developmental competence of oocytes were identified. COL1A2, renin and COL1A1 were selected and further examined by quantitative real-time polymerase chain reaction (qRT-PCR). Expression levels of COL1A2 and renin by qRT-PCR were consistent with the results of RNA sequencing. ConclusionRNA sequencing data could provide novel marker genes for the non-invasive evaluation of oocyte quality and embryo developmental competence.

53 Effect of anthocyanin supplementation in bovine pre-implantation embryonic development during invitro maturation of oocytes

Oocyte protection against reactive oxygen species (ROS) during invitro maturation (IVM) may play a decisive role in pre-implantation embryonic development. For instance, anthocyanins have shown greater antioxidant effects than vitamins C and E. The objective of this study was to determine the anthocyanin supplementation level that influences quantity and quality of bovine blastocysts development during IVM. Cumulus–oocyte complexes (COC) were recovered from 185 abattoir ovaries in 6 sessions and classified (Grade 1 and 2) for maturation. Oocytes were in IVM in commercial medium (Vitrogen®) supplemented with anthocyanin (pelargonidin chloride) at different concentrations: 0 (control), 1, 10, 20, and 40μM, in droplets of 70μL with 12 to 15 COC at 38.5°C, 5% CO2 and 90% humidity for 22to 24h. Sperm selection was performed by Percoll gradient method (45/90%) with centrifugation at 600×g for 6min. The final concentration for IVF was 2×106 sperm mL−1. A total of 462 oocytes were used in the experiment (6 replicates). Presumptive zygotes were invitro cultured (IVC) in commercial medium (Vitrogen) in droplets of 70µL with 12–15 zygotes at 38°C, 5% CO2, and 90% humidity until the blastocyst stage (Day 7 of culture). The cleavage (Day 2), morulae (Day 4), and blastocyst (Day 7) rates were measured during IVC. The data were processed with non-parametric tests (Kruskal–Wallis test with independent samples, P&amp;lt;0.05) using IBM SPSS Statistics 2.0 for Windows. The results in the control group of cleavage, morulae, and blastocyst rates were 67.3, 27.0, and 22.1%, respectively. Although, numerically, anthocyanin at 1μM resulted in a higher blastocyst rate (28.8%) and anthocyanin at 10μM resulted in a greater number of blastocysts of advanced stages (65.0%), anthocyanin supplementation during IVM did not influence the quantity and quality of bovine blastocyst development (P&amp;gt;0.05). In conclusion, the supplementation of anthocyanin to the maturation medium did not affect invitro development of bovine embryos. Complementary studies at the cellular and gene expression level may be required.

128 Comparison of multiple maturation times on juvenile invitro embryo transfer (JIVET)-derived oocytes and embryo development in the goat

Juvenile invitro embryo transfer (JIVET) is an assisted reproductive technology (ART) with the potential to produce numerous offspring from a single young female goat at 4 to 8 weeks of age. It has been reported in small ruminants that there can be a marked variable response to the administration of exogenous hormones for superovulation, the subsequent number of oocytes generated, and subsequent embryo developmental potential. The industry standard (as well as the recommendation of commercial media suppliers) invitro maturation time is 21 to 24h for conventionally derived oocytes. This study investigated multiple maturation times for JIVET-derived oocytes: 16, 22, and 28h. Oocytes were collected from four JIVET animals at 6 to 8 weeks of age. The hormonal superovulation regimen used on the juvenile animals consisted of 4×40-mg FSH injections at ∼12h apart and a 400IU of PMSG injection given with the first FSH injection. Surgical recovery of the oocytes via a midline laparotomy was performed the day following the last FSH injection. All of the oocytes were collected via aspirating follicles that were 4mm and larger. Oocytes with compact cumulus cells subsequently underwent IVM, IVF, and invitro culture (IVC) utilising IVF Bioscience media and methods. A single straw of identical cryopreserved/thawed semen from the same buck was utilised for each of the IVF procedures. The results were (37/88) 42%, (37/85) 44%, and (39/91) 43% cleaved and (23/88) 26%, (24/85) 28%, and (28/91) 31% blastocyst rate based on respective maturation times for JIVET-derived ova. Development rate during the cleavage stage and blastocyst stage was analysed using a repeated-measures logistic regression model utilising generalized estimating equations (GEE), with maturation time as fixed effect and a compound symmetry within subject (juvenile goat) covariance structure. The main effect of maturation time on the odds of development during the cleavage stage (P=0.8727) and blastocyst stage (P=0.3857) was not significant. These results indicate that the time in maturation media does not have as profound an effect on development to blastocysts as a factor in the variability reported by other laboratories. The development rate of embryos from one juvenile goat produced very high blastocyst rates of (5/12) 42%, (11/12) 92%, and (11/15) 73%, respectively. Additional logistic regression analysis showed that the odds of development in this juvenile donor was significantly different compared with the other donors (pooled) during the cleavage stage at 16h (P=0.0083) and 28h (P=0.0021) maturation times. Likewise, the odds of development in this donor was significantly different than that of the other donors (pooled) during the blastocyst stage at 22h (P=0.0002) and 28h (P=0.0003) maturation times. This further indicates the wide variation of oocyte quality from JIVET-derived oocytes and indicates potential for higher development rates at 22 and 28h in this specific goat.

7 A record of 485 viable cumulus–oocyte complexes recovered and 165 viable embryos produced in a single ovum pickup session from a Senepol breed donor

The recovery of an exceptionally high number of cumulus–oocyte complexes (COC) in a single ovum pickup (OPU) session is rare, and thus seldom reported in the literature. In the present report, we describe one of these cases. The present data refers to an OPU session performed by author A. O. Resende in a commercial beef farm, located in the rural area of the city of Barretos, Brazil, on October 15, 2019. Breed heifers (N=12) underwent OPU at a random stage of oestrous cycle without any hormonal treatment. Follicle aspirations were performed following the standard operation procedures (SOP) established by ABS Pecplan OPU team, using a portable ultrasound (DP2200, Mindray do Brasil) equipped with a 7.5-MHz microconvex probe, a vacuum pump (WTA), and 18 gauge disposable Jelco catheter (Smith Medical). Invitro embryo production (IVP) was performed at Invitro Technologies laboratory, ABS Pecplan Uberaba, located 140km from the farm. On this day, 1312 oocytes were recovered from the 12 donors. However, a single heifer produced 485 viable COC, from over 500 total COC recovered. The COC from this donor were distributed in four groups, which underwent IVF with Y-sorted semen from four Senepol sires. The invitro embryo production was performed following the SOP established by invitro Technologies, ABS Pecplan. Embryo production was compared among sires by the chi-squared test. Of the 4 sires used during IVF, sire number 4 resulted in a higher blastocyst rate compared with the others (sire 1=25.4%, sire 2=30.6%, sire 3=33.9%, and sire 4=46.3%; P&amp;lt;0.01).The blastocyst rate differed (P&amp;lt;0.01) among sires: 25.4%a (31/122), 30.6%a (37/121), 33.9%a (41/121), and 46.3%b (56/121) for bulls A, B, C, and D, respectively. From the total of 165 viable embryos produced (34.0%), 120 were transferred fresh and the remaining 45 were vitrified using the SOP established by IVB Technologies, ABS. These results highlight the huge potential variation in antral follicle population and number of COC recovered from donors within the same population, breed, and reproductive status. It also shows the potential of IVP in the generation of offspring from high genetic indices.

Quinoline Derivative Enhances Human Sperm Motility and Improves the Functional Competence

In this study, we aimed to explore the beneficial properties of novel quinoline derivatives on human sperm motility and its functional competence. Nine novel quinoline derivatives were screened for their effect on motility in human spermatozoa from normozoospermic ejaculates. Compounds with impressive sperm motility enhancement properties were further assessed for their effect on functional competence of human spermatozoa. To determine the effect on the fertilizing ability of spermatozoa processed with quinoline derivatives and to assess developmental competence of embryos derived, in vitro fertilization (IVF) was performed using mouse model. Among the nine quinoline derivatives, 2 compounds (6MQT and 2,6DQT) exhibited significant enhancement in sperm progressive motility and survival at 24 h. Further, non-significant increase in curvilinear velocity (VCL), straight line velocity (VSL), and amplitude of lateral head displacement (ALH) was observed. Capacitation, intracellular cAMP level and tyrosine phosphorylated sperm proteins were significantly higher in 6MQT (P < 0.05) and 2,6DQT (P < 0.001) compared to control. In vitro fertilization (IVF) experiments using Swiss albino mice revealed that spermatozoa processed with 6MQT had non-significantly higher blastocyst rate and a superior blastocyst quality, while, 2,6DQT resulted in significantly lower blastocyst rate (P < 0.05) compared to control. Quinoline derivative 6MQT has significant motility enhancement property under in vitro conditions.Graphical abstract

Production and Characterization of Mouse Diploid Parthenogenetic Blastocyst Developed in Phosphate-Free Medium

Parthenogenesis is an artificial oocytes activation process without paternal contribution. Blastocyst, derived from parthenogenesis, is one of potential source for pluripotent stem cell propagation. Unfortunately, previous studies reported that parthenogenetic embryo did not achieve exhilarating blastocyst rate. One of the component that predicted inhibit parthenogenetic embryo development is phosphate. Therefore, we try to modify culture medium in order to overcome that problem. The aim of this research was to produce and analyze the characteristics of parthenogenetic blastocyst developed in phosphate-free medium. Mouse oocytes obtained from adult female DDY by superovulation. The activator was strontium chloride 10 mM and diploidization with cytochalasin B 5 μg/ml. Medium for activation and culture medium were modified rat 1 cell embryo medium (MR1ECM) which is phosphate free. The results showed that parthenotes that were cultured in phosphate free medium reached higher blastocyst rate compared to the other groups. The increase of phosphate in culture medium lead to impaired parthenogenetic embryos development. Further experiment was made to analyze the differences between fertilized and parthenogenetic embryo in this medium. The experiment showed that diploid parthenogenetic could achieve high blastocyst rate (30.9±1.3%). The quality of diploid parthenogenetic blastocyst, based on cells number, viability, and ICM ratio, was lower than fertilized blastocyst.

Shading effect on physiological parameters and in vitro embryo production of tropical adapted Nellore heifers in integrated crop-livestock-forest systems

The aim of this study was to evaluate the impact of increased shadow supply in integrated crop-livestock-forest systems on in vitro embryonic development and physiological parameters related to stress response in Nellore heifers (Bos indicus). For the study, animals (n = 16) were randomly divided into two groups and kept in areas with different afforestation systems, the integrated crop-livestock-forest (ICLF) and the integrated crop-livestock (ICL) system. The microclimate of the ICLF system provided better comfort conditions than ICL. No differences of respiratory rate, rectal temperature, cortisol, T3, T4, oocyte quality, and cleavage rate between the systems were verified. A higher blastocyst rate was observed in the ICLF (p < 0.05). The results demonstrate that Nellore heifers managed in ICLF during summer in Midwest of Brazil showed higher production of in vitro embryos, without typical changes in its physiological parameters. The results observed in the present study indicate that zebu females are able to respond satisfactorily to the intense heat conditions; however, we believe that the long period to which these animals are exposed to these conditions interferes in the oocyte competence and embryo development.