Smooth Endoplasmic Reticulum Clusters in Oocytes From Patients Who Received Intracytoplasmic Sperm Injections Negatively Affect Blastocyst Quality and Speed of Blastocyst Development

Findings regarding the relationship between smooth endoplasmic reticulum clusters (SERCs) in oocytes and blastocyst development have been conflicting. In this study, the effects of SERCs on blastocyst quality and the speed of blastocyst development were evaluated. Patients who received intracytoplasmic sperm injections (ICSI) at our reproductive center from 2016 to 2020 were retrospectively analyzed. SERC (+) oocytes (n = 217) and SERC (–) oocytes (n = 822), as well as SERC (+) cycles (n = 146) and SERC (–) cycles (n = 1,951) were compared. There was no significant difference in embryological, clinical, and neonatal outcomes between the SERC (+) and SERC (–) cycles. The fertilization rate (73.9%), good quality blastocyst rate (26.7%) and the speed of blastocyst development (44.4%) were significantly lower (P < 0.05) in SERC (+) oocytes than in unaffected counterparts (86.2%, 44.1% and 63.4%, respectively). Furthermore, the proportion of blastocysts with trophectoderm (TE) grade C was significantly higher in the SERC (+) oocyte group than in the SERC (–) oocyte group (73.3 vs. 55.9%, P < 0.05). After adjusting for age, years of infertility, endometriosis, stimulation protocols (GnRHa), and male infertility, multiple logistic regression analysis revealed that the presence of SERCs in the oocytes significantly affected the speed of blastocyst development (odds ratio, 2.812; 95% CI, 1.257–6.292; P = 0.012). These findings suggest that the presence of SERCs in oocytes may negatively affect blastocyst quality and the speed of blastocyst development.

PSV-5 Relationship between sire conception rate, sperm motility, sperm SERPINA5 relative concentration, and in vitro produced embryos in dairy bulls

Sire conception rate (SCR) is a field measure of fertility among bulls, but it can be influenced by several factors (Sperm transport, sperm-egg binding, early embryo development, etc). The objective of this study was to evaluate the relationship between SCR, sperm motility, SERPINA5 concentrations, and in vitro embryo development. Measurements were performed in 19 bulls with SCR values ranging from -7.7 to 4.45. For each bull, an aliquot of frozen-thawed semen was used for analyses of total (TMOT) and progressive (PROG) motility. Remaining semen was fixed with 2% formaldehyde, and concentration of SERPINA5 was determined by immunolocalization (antibody SERPINA5/Dylight405; PA5-79976-Invitrogen / ab201798-Abcam). Mean fluorescence intensity was determined in ~200 sperm heads/bull. Approximately 149 oocytes/bull were fertilized in vitro for embryo development analysis (cleavage and blastocyst rates). Statistical procedures were performed in SAS (9.4) using the procedures CORR for correlations (SCR, TMOT, PROG, SERPINA5, cleavage and blastocyst) and GLIMMIX for comparison of “field-fertility” (SCR divided in HIGH or LOW) and “field-embryo-fertility” (LOW-SCR sires were divided based on blastocyst rate (HIGH or LOW) resulting in two classifications; LOW-HIGH≥31% and LOW-LOW≤26%, respectively). There were positive correlations (P < 0.05) between cleavage-blastocyst (r=0.50), SERPINA5-cleavage (r=0.48), and TMOT-PROG (r=0.76). Sire SCR was not associated with SERPINA5, TMOT, PROG, cleavage and blastocyst rate (P > 0.52). Among LOW-SCR sires, LOW-LOW sires (-4.83±0.60) tended to have a better SCR score than LOW-HIGH (-6.18±0.42) sires (P = 0.08), but there were no differences (P > 0.43) between LOW-HIGH, LOW-LOW, and HIGH sires for SERPINA5, TMOT, PROG, and cleavage. In conclusion, some LOW SCR sires have good embryo development indicating a different mechanism for their low SCR; however, these differences in SCR could not be explained by TMOT, PROG, SERPINA5, cleavage and blastocyst. There were, however, positive correlations between cleavage-blastocyst rate, and SERPINA5-cleavage rate.

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

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.

EVALUATION OF TWO CULTURE MEDIA IN IN VITRO PRODUCTION OF ALPACA (Vicugna pacos) EMBRYOS

The present study aims to evaluate the effect of two culture media on the production of in vitro embryos in alpacas (Vicugna pacos). The ovaries were transported at 10.52° C in 0.9% saline solution supplemented with gentamicin. The ovaries were transported at 10.52° C in 0.9% physiological saline solution supplemented with gentamicin. 492 ovaries were used throughout the experiment. 2142 oocytes of quality I, II and III were recovered. The oocytes were matured in vitro for 32 h and were subsequently fertilized (incubated for 18 h) with sperm obtained from the tail of the epididymis and selected with a 45/90 percoll gradient. Then, the presumed zygotes were denuded from the cumulus cells, to later be cultured in two culture media: synthetic oviductal fluid medium (SOFaa) and simple optimized potassium medium (KSOMaa) and incubated at 38.5 ° C, 5 % CO2, 5%, O2, and 90% relative humidity for 7 days. Morula and blastocyst rate evaluation was performed at the end of embryo culture. The morula rate at 7 days was 41.49 ± 10.52 and 41.51 ± 6.50% for KSOMaa and SOFaa, respectively (P <0.05). The blastocyst rate for the two culture media KSOMaa and SOFaa, was 14.08 ± 5.17 and 11.73 ± 5.69 %, respectively, and there were no statistical differences (P˃0.05). The embryonic quality in KSOMaa and SOFaa media did not show statistical differences. In conclusion, the KSOMaa and SOFaa culture medium can be used in the production of in vitro embryos of alpacas

EFFECT OF THE USE OF TWO SPERM SELECTION TECHNIQUES FOR IN VITRO PRODUCTION OF ALPACA EMBRYOS

The objective of this research was to evaluate the effect of the use of two sperm selection techniques for in vitro production of alpaca embryos. The ovaries and testis were collected from the local slaughterhouse and transport to 37 ° C in saline solution (0.9%) supplemented with gentamicin. Quality I, II and II oocytes were incubated in a maturation medium for 32 h at 38.5 ° C and 5% O2 and 5% CO2. For in vitro fertilization, sperm from the epididymis were selected using the Percoll gradient and Swim up technique. 18h after the oocytes were incubated with the sperm, these were denuded from the cumulus cells and cultured in SOFaa culture medium for 7 days. Morula and blastocyst rate and their morphological quality are evaluated at day 7 of culture. From a total of 370 ovaries, 1,137 oocytes were recovered, making an average of 3.6 oocytes / ovary. After the maturation and fertilization process and in vitro culture, the blastocyst rate was 8.43 ± 6.04% and 3.89 ± 1.75%, for oocytes fertilized with sperm selected with Percoll gradient and Swim up, respectively, not finding significant statistical differences (p> 0.05), between the groups. In conclusion, the in vitro fertilization of alpaca oocytes with spermatozoa selected with two selection techniques (percoll and swim up) did not significantly influence the quantity and quality of morulae and blastocysts at day 7 of embryo culture.

More blastocysts are produced from fewer oocytes in ICSI compared to IVF – results from a sibling oocytes study and definition of a new key performance indicator

Background Which fertilization method, between ICSI and IVF in split insemination treatments, has the highest clinical efficiency in producing clinically usable blastocyst? Methods 211 infertile couples underwent split insemination treatments for a non-severe male factor. 1300 metaphase II (MII) oocytes were inseminated by conventional IVF and 1302 MII oocytes were micro-injected with the same partner’s semen. Embryo development until blastocyst stage on day V and clinical outcomes were valuated trough conventional key performance indicators (KPI), and new KPIs such as blastocyst rate per used MII oocytes and the number of MII oocytes to produce one clinically usable blastocyst from ICSI and IVF procedures. Results The results were globally analyzed and according to ovarian stimulation protocol, infertility indication, and female age. The conventional KPI were online with the expected values from consensus references. From global results, 2.3 MII oocyte was needed to produce one clinically usable blastocyst after ICSI compared to 2.9 MII oocytes in IVF. On the same way, more blastocysts for clinical use were produced from fewer MII oocytes in ICSI compared to IVF in all sub-groups. Conclusions In split insemination treatments, the yield of clinically usable blastocysts was always superior in ICSI compared to IVF. The new KPI "number of needed oocytes to produce one clinically usable embryo" tests the clinical efficiency of the IVF laboratory.

Laser-assisted hatching in lower grade cleavage stage embryos improves blastocyst formation: results from a retrospective study

Background Laser-assisted hatching (LAH) has been widely applied to facilitate blastocyst hatching in IVF-ET treatment, however, the effect of LAH on subsequent development and clinical outcomes of the lower grade cleavage stage embryos (LGCE) remains unknown. Our study aimed at evaluating the effect of LAH on blastocyst formation and the clinical pregnancy outcomes of LGCE embryos after transfer. Methods A total of 608 cycles of IVF/ICSI treatment from November 2017 to September 2019 were included in our study as follows: 296 in the LAH group and 312 in the N-LAH group. The total blastocyst rate, usable blastocyst rate, good-grade blastocyst rate and clinical pregnancy rate were statistically compared between the two groups. Results The total blastocyst rate (50.7% vs 40.2%, P < 0.001), usable blastocyst rate (31.0% vs 18.6%, P < 0.001) were significantly higher in the LAH group than those in the N-LAH group. After analysis of generalized estimating equations, LAH was positively correlated with the blastocyst rate (B = 0.201, OR 95% CI = 1.074–1.393, P = 0.002), usable blastocyst rate (B = 0.478, OR 95% CI = 1.331–1.955, P < 0.001). However, the clinical pregnancy rate after blastocyst transfer did not differ between LAH group and N-LAH group (49.4% vs 40.0%, P > 0.05, respectively). Conclusions A higher proportion of total blastocysts and usable blastocysts can be obtained by LAH in LGCE, which may be beneficial to the outcome of the IVF/ICSI-ET cycle.

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

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 (&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