Embryo Development Rates Research Articles

Developmental and Molecular Effects of C-Type Natriuretic Peptide Supplementation in In Vitro Culture of Bovine Embryos.

The use of C-type natriuretic peptide (CNP) in the interaction with the oocyte and in the temporary postponement of spontaneous meiosis resumption has already been well described. However, its action in pre-implantation developmental-stage embryos is yet to be understood. Thus, our study aimed to detect the presence of the canonical CNP receptor (natriuretic peptide receptor, NPR2) in germinal vesicle (GV)-, metaphase II (MII)-, presumptive zygote (PZ)-, morula (MO)-, and blastocyst (BL)-stage embryos and, later, to observe possible modulations on the embryos when co-cultured with CNP. In Experiment I, we detected and quantified NPR2 on the abovementioned embryo stages. Further, in Experiment II, we intended to test different concentrations (100, 200, or 400 nM of CNP) at different times of inclusion in the in vitro culture (IVC; inclusion from the beginning, i.e., day 1, or from day 5). In Experiment III, 400 nM of CNP was used on day 1 (D1) in the IVC, which was not demonstrated to be embryotoxic, and it showed potentially promising results in the blastocyst production rate when compared to the control. Thus, we analyzed the embryonic development rates of bovine embryos (D7) and hatching kinetics (D7, D8, and D9). Subsequently, morula and blastocyst were collected and evaluated for transcript abundance of their competence and quality (apoptosis, oxidative stress, proliferation, and differentiation) and lipid metabolism. Differences with probabilities less than p < 0.05, and/or fold change (FC) > 1.5, were considered significant. We demonstrate the presence of NPR2 until the blastocyst development stage, when there was a significant decrease in membrane receptors. There was no statistical difference in the production rate after co-culture with 400 nM CNP. However, when we evaluated the abundance of morula transcripts, there was an upregulated transcription in ADCY6 (p = 0.057) and downregulated transcripts in BMP15 (p = 0.013), ACAT1 (p = 0.040), and CASP3 (p = 0.082). In addition, there was a total of 12 transcriptions in morula that presented variation FC > 1.5. In blastocysts, the treatment with CNP induced upregulation in BID, CASP3, SOX2, and HSPA5 transcripts and downregulation in BDNF, NLRP5, ELOVL1, ELOVL4, IGFBP4, and FDX1 transcripts (FC > 1.5). Thus, our study identified and quantified the presence of NPR2 in bovine pre-implantation embryos. Furthermore, 400 nM of CNP in IVC, a concentration not previously described in the literature, modulated some transcripts related to embryonic metabolism, and this was not embryotoxic morphologically.

7 Role of the preovulatory follicle in oocyte metabolic and developmental competence for pregnancy

Abstract A majority of bovine pregnancy loss occurs in the first 7 d of gestation. The oocyte is a key driver of embryo viability during this time, and its competence for embryo development and pregnancy establishment is greatly influenced by the surrounding microenvironment during oocyte maturation. Assisted reproductive technologies allow for more efficient propagation of superior genetics and improved reproductive management of the cattle herd; however, many assisted reproductive technologies alter the normal microenvironment of the maturing oocyte and negatively impact oocyte competence for early embryo development. For example, oocytes induced to ovulate from preovulatory follicles of lesser physiological maturity (decreased estradiol production and smaller diameter) have reduced blastocyst development, poorer embryo quality, and a hastened rate of embryo development compared with those ovulated from follicles of greater maturity. Our overarching hypothesis is that the intrafollicular milieu of physiologically mature preovulatory follicles provides the necessary microenvironment to metabolically program maturing oocytes for successful embryo development. Studies by our team have demonstrated that the follicular fluid metabolite milieu during in vivo oocyte maturation differs between preovulatory follicles of greater and lesser maturity and that such differences have direct effects on the maturing oocyte. Numerous follicular fluid metabolites involved in glucose and amino acid metabolism were more abundant in preovulatory follicles of greater versus lesser physiological maturity. Additionally, abundance of transcripts encoding glycolytic enzymes was higher in cumulus cells from follicles of greater maturity, and both ATP and levels of key transcripts in the mitochondrial respiratory chain were higher in oocytes collected from preovulatory follicles of greater versus lesser maturity. We supplemented in vitro maturing oocytes with follicular fluid from preovulatory follicles of greater or lesser maturity to determine causality of differing follicular fluid milieu on cumulus-oocyte metabolism and oocyte competence for early embryo development. Follicle maturity status had robust effects on cumulus-oocyte complex consumption or production of key metabolites in glucose, purine, and amino acid metabolism pathways during oocyte maturation. We also observed hastened development rate in embryos arising from in vitro matured oocytes supplemented with lesser maturity follicular fluid, mirroring past in vivo findings by others. Furthermore, RNA-sequencing of resulting blastocysts of identical stage and quality suggested more efficient metabolism and Wnt/β-catenin signaling in blastocysts derived from oocytes matured in preovulatory follicular fluid from greater maturity follicles. In contrast, transcriptome profiles of blastocysts from oocytes matured in preovulatory follicular fluid from lesser maturity follicles indicated oxidative stress and dysregulated cell division. These recent findings by our lab indicate that impacts of follicle maturity and the resulting follicular fluid microenvironment on the maturing oocyte withstand throughout early embryo development and hold promising roles to improve reproductive efficiency in cattle.

Chromosomal missegregation and aberrant embryo development in repro57 female mice with Rnf212 homozygous mutation.

Repro57 mice, bearing an Rnf212 gene mutation, exhibit infertility in both homozygous mutant males and females, revealing arrested spermatogenesis in males and investigating unclear mechanisms in females. The study highlights aneuploidy and altered kinetochore patterns in repro57 homozygous mutant oocytes, which impact later stages of embryo development. Repro57 mice, induced with N-ethyl-N-nitrosourea and harboring a mutation in the Rnf212 gene, exhibit infertility in both homozygous mutant males and females. Rnf212 plays a crucial role in recombination and crossover designation. In male repro57 homozygous mutants, spermatocytes often degenerate during late prophase, and mature spermatozoa are absent in the seminiferous epithelium, indicating arrested spermatogenesis as the cause of infertility. Despite reports of infertility in Rnf212-knockout female mice, the specific mechanisms underlying infertility in female repro57 homozygous mutants remain elusive. This study investigates the chromosomal and kinetochore patterns of mature oocytes and their developmental potential following in vitro fertilization in female repro57 homozygous mutant mice. While all wild-type oocytes progress to metaphase II and exhibit euploidy, all repro57 homozygous mutant mouse oocytes display aneuploidy. Additionally, kinetochore distances in repro57 homozygous mutant oocytes exceed those observed in wild-type counterparts. Although no significant differences are noted in fertilization and early embryo development rates between wild-type and repro57 homozygous mutant mice, embryos derived from repro57 homozygous mutants exhibit significantly lower morula and blastocyst rates, accompanied by frequent cytokinesis failure and vacuole formation. These findings suggest that the premature segregation of sister chromatids in repro57 homozygous mutant mice adversely impacts the later stages of embryo development.

Effect of Transport Condition on the Structural Integrity of Ovarian Tissue and the Development of Sheep Embryos In-Vitro

The oocyte quality decreases during ovarian tissue transport to the laboratories of in vitro embryo production. To provide additional information on how the conditions of transporting sheep ovaries impact the ovarian tissue and oocytes’ ability to develop into blastocyst stages, we have studied new transport media Ankara University Zootekni (AUZ1, AUZ2) supplemented with antioxidants (melatonin, Vit E, and Vit A), buffer solution, and energy substrates, and compared them with the traditional transport media: Phosphate-Buffered Saline (PBS), and Charles Rosenkrans 1 (CR1), Normal Saline (NS) at different temperatures (-6 to 30 °C). We also studied and compared how well different transport media preserve the ovarian tissue's structural integrity while transporting sheep ovaries at 4°C. Our findings indicated that various temperatures and transport media play critical roles in embryo development. The embryo development rates showed that when sheep ovaries are transported in AUZ1, they produce oocytes with a higher embryo development rate than other transport media at any temperature. In addition, histology examination revealed that the transport of sheep ovarian tissue in any medium at a temperature of 4 °C did not negatively impact the viability and histomorphology of the primordial, primary, and secondary follicles. In contrast to other transport media, the AUZ1 medium maintained the normal morphology of antral follicles, Graafian follicles, and the cumulus oophorus of sheep ovarian tissue. In conclusion, adding melatonin, buffer solution, and energy substrates to the transportation medium of ovarian tissues has a beneficial and positive role in maintaining ovarian tissue and increasing the rates of embryonic development.

Impact of fluazuron on oocyte maturation: May the antiparasitic affect bovine reproduction?

Fluazuron is a novel veterinary pour-on antitick formulation which can be applied simultaneously with bovine reproduction management strategies. Considering the economic importance of the livestock industry in many countries, it is important to know whether antiparasitics such as fluazuron may cause embryonic loss. The aim of this study was to evaluate the toxicological effect of fluazuron on bovine oocytes during in vitro maturation. The best fluazuron concentrations were determined in a preliminary experiment on Chinese hamster ovary (CHO)-K1 cells and further used to compare fluazuron toxicity in both study models. Results of the annexin V and alkaline single cell gel electrophoresis assays demonstrated that fluazuron caused cytotoxicity and genotoxicity in bovine cumulus cells at all the concentrations tested (50, 75 and 100 μg fluazuron/mL). The evaluation of cortical granules and mitochondria distribution showed that cytoplasmic maturation was not affected by fluazuron treatment. However, a decrease in metaphase II + polar body, degenerate oocytes as well as disorganized chromatin in polar body were observed at all concentrations tested. Whereas the fertilization process was not altered by 50 μg/mL fluazuron, the embryo development rate decreased significantly. No significant differences were observed in any of the oxidative stress parameters assessed. This study contributes to a better understanding of fluazuron in bovines, suggesting that the antiparasitic may affect bovine reproduction and might cause embryo loss.

Small molecules, enormous functions: potential approach for overcoming bottlenecks in embryogenic tissue induction and maintenance in conifers.

Somatic embryogenesis (SE) is not only the most effective method among various strategies for the asexual propagation of forest trees but also a basis for genetic improvement. However, some bottlenecks, such as the recalcitrance of initiation, the maintenance of embryogenic potential during proliferation and the low efficiency of maturation as well as high rate of abnormal embryo development remain unresolved. These bottlenecks refer to complex mechanisms, including transcriptional regulatory networks, epigenetic modifications and physiological conditions. In recent years, several small molecules utilized in animal stem cell research have exhibited positive effects on plant regeneration, including conifer species, which offers a potential novel approach to overcome the challenges associated with SE in conifers. In this review, we summarize the small molecules used in conifers, including redox substances, epigenetic regulatory inhibitors and other metabolism-related molecules, which overcome these difficulties without the use of genetic engineering. Moreover, this approach also has the advantages of dynamic reversibility, simple operation, and simultaneous regulation of multiple targets, which might be one of the best choices for optimizing plant regeneration systems including SE.

P-043 Preliminary clinical outcome using the Q300™ device in a reproductive laboratory environment

Abstract Study question How does the Q300™ Device impact clinical outcomes in a reproductive laboratory environment compared to established standards? Summary answer The device showed favorable clinical outcomes and was user-friendly. More research is needed to identify the patient subgroups that can benefit the most from system What is known already Embryologists struggle with accurately assessing sperm morphology without staining, prompting interest in emerging technologies like AI. These methods evaluate motility parameters, offering non-invasive sperm selection. A previous study comparing Q300 to manual staining methods showed high agreement and repeatability, indicating Q300’s accuracy in measuring live sperm cells. It aids in selecting cells per WHO2021 guidelines without staining, potentially revolutionizing sperm assessment. Study design, size, duration The study is an open-label, non-randomized study. Semen samples intended for ICSI were imaged by the Q300™ device before micro-injection to aid in selecting morphologically compliant sperm cells. The data collected was compared to the Key Performance Indicators (KPI) for ART laboratories according to the Vienna Consensus. Up to 5 sites will be enrolled in the study, with up to 100 couples in total from all sites. Study duration: 18 months Participants/materials, setting, methods The Q300™ device is an optical system using quantitative phase microscopy and holographic imaging to analyze human semen morphology. The device aids embryologists in selecting live and motile sperm cells for ICSI by providing automatic, objective analysis. Couples meeting inclusion criteria, including female age ≤40 and availability of motile sperm, were recruited for ICSI procedures. There were no restrictions on previous IVF/ICSI cycles. Endpoints: fertilization, day-3 embryo development, good blastocyst development rates, and implantation rates. Main results and the role of chance 14 couples were recruited from Barzilay Medical Center’s IVF unit in Ashkelon, Israel. The average fertilization rate is 79.1% (Vienna consensus competency is ≥ 65%), the day 3 embryo development rate is 55% (Vienna consensus competency is ≥ 45%), and the good blastocyst development rate is 42.5% (Vienna consensus competency is ≥ 30%). The implantation rate (day 3+day5) is 36.8% (Vienna consensus competency is ≥ 25% for day 3 embryo transfers and ≥ 35% for blastocyst transfers). The intended users performed the procedures with no reported malfunctions. 270 sperm imaging procedures were performed using the Q300 device to select 109 morphologically suitable cells for ICSI. The utilization of the product resulted in approximately a 40% yield of correct selections by the embryologist. Consequently, out of every 10 cells deemed compliant by the embryologist, only 4 were confirmed by the device, with roughly 3 falling within a margin of 10% around the WHO measurement range. Moreover, only about 1 of these selections fell within the boundaries defined by the WHO standards. This observation offers insights into the characteristics of cells routinely chosen for injection into oocytes. Limitations, reasons for caution The study recruited 14 couples from a single site; it is important to validate and statistically power the outcome measures and compare them with clinical control groups in the future. In the next generation of the product, incorporating an objective selection based on sperm motility can further improve its functionality. Wider implications of the findings The findings offer insights into optimizing sperm selection processes, potentially improving fertilization, embryo development, and implantation rates in couples undergoing ICSI. Moreover, the study highlights the importance of continued research and refinement of technological innovations in ART to better serve patient needs and improve treatment outcomes. Trial registration number NCT06232720

P-108 Automated sperm selection software (SiD) provides similar results as human selection for ICSI

Abstract Study question Automated sperm selection or human eye-based sperm selection, does it make a difference on IVF lab outcomes and clinical outcomes? Summary answer Single sperm selection by the AI SiD was as proficient as highly experienced embryologists and embryo development may have improved for inexperienced embryologists What is known already The implementation of artificial intelligence in reproductive medicine has proven effective for a variety of applications. One application for this technology is single sperm selection during ICSI. The computer assisted program SiD, was designed to grade sperm, in real-time, based on motility parameters transforming qualitative sperm assessments into a quantitative score. SiD’s assistance can be used to select the most optimal sperm for ICSI. The application of sperm selection by AI is expected to bypass user bias, fatigue, limited training, while also optimising time. However, there are limited studies correlating the usage of AI for sperm selection with ICSI outcomes. Study design, size, duration 1 year period 608 sibling MII oocytes were randomly divided in two groups; 1-ICSI group (n = 301): ICSI performed with sperm selected by the embryologist and 2- SID-ICSI group (n = 307): ICSI performed with sperm selected using SiD software. Priority was given to sperm classified as “Best” by SID software for ICSI.. Morphology selection was performed before ICSI in both groups. Embryos were cultured during 6 days in the same conditions. Participants/materials, setting, methods Sperm preparation was performed using a microfluidic chamber (Zymot™). Sperm selection was performed as previously described, and ICSI was performed following routine procedures. Biological and clinical outcomes were analysed in both groups and compared including fertilization rate, cleavage rate, embryo and blastocyst development rate, top quality embryo rate, blastocyst development rate and pregnancy rates. Biological outcomes in both groups were also compared based on embryologist seniority. Embryo morphokinetics parameters were analysed using time-lapse system. Main results and the role of chance There was a non significant trend towards better outcomes in the SID-ICSI group for all biological outcomes including fertilization rate (83,1% vs 82,4%), cleavage rate (97,6% vs 97,2%), day 3 embryo development rate (72,9% vs 70,6%), blastocyst development rate at day 5 (49,0% vs 44,8 %), good quality blastocyst development rate at day 5 (45,1% vs 41,5%), and top quality blastocyst development rate at day 5 (25,9% vs 22,2%). Similarly, a non-significant increase was observed in all these outcomes when sperm selection is performed by a junior embryologist. Embryo development was monitored using a timelapse system. Our data show that 2nd polar body extrusion (tPB2) (4,0h vs 4,8 h [p = 0,005]) and early cleavage (t2) (27,3h vs 28,7h [p = 0,05]) happen significantly earlier when SiD is used for ICSI. No significant difference was observed for all other fertilization events (cytoplasmic wave,tPN1,tPN2,presence of cytoplasmic halo,tPNf,and cytoplasmic halo disappearance), and other cleavage timings (t3-&amp;gt;t10,tM,tSC,tEC,tSB), and embryonic cell cycles (ECC1,ECC2,ECC3,s2,s3). Euploid rate obtained with in the 2 groups was comparable (53,3% in SID-ICSI vs 44,4% in ICSI). Although there was a trend toward higher cumulative early and clinical pregnancy rate after ICSI-SID, the difference did not reach significance (43,1%vs34,0% and 27,5%vs21,2%, respectively). Limitations, reasons for caution The ZymotTM microfluidic chamber was used for sperm preparation. This device optimises sperm selection, giving rise to an already optimised population of spermatozoa. Future investigations evaluating the effect of SiD automated sperm selection software on biological outcomes in samples prepared with a less effective technique would potentially yield conversing results. Wider implications of the findings This pilot study demonstrated that the usage of the automated sperm selection software SiD gives rise to similar biological outcomes and embryo morphokinetics, indicating its effectiveness in sperm selection. We expect this program would be useful in the presence of less experienced laboratory members helping by standardizing sperm selection. Trial registration number NA

P-371 It’s worth the freeze: outcomes from thawing electively cryopreserved oocytes are comparable in patients with reduced vs normal ovarian reserve at the time of cryopreservation

Abstract Study question For patients coming back to use oocytes derived from elective cryopreservation, does their initial ovarian reserve predict thaw survival, embryos’ developmental milestones and pregnancy success? Summary answer Thaw survival, fertilization rate, embryo development and pregnancy success rates do not vary significantly according to AMH level at initial cryopreservation. What is known already Increasing numbers of patients are electing to cryopreserve oocytes for future use. AMH (Anti Mullerian Hormone) is an ovarian reserve marker often assessed prior to initiation of treatment that predicts response to fertility treatment and the resulting oocyte yield. However, it is not known whether oocytes derived from a patient with compromised ovarian reserve (perhaps over multiple treatment cycles) will fare as well as that of a patient with normal ovarian reserve and a higher per cycle oocyte yield. Our study seeks to shed light on whether a lower initial AMH level negatively impacts the prognosis of the thawed oocytes. Study design, size, duration We included in our retrospective study all oocyte thaw cycles resulting from electively cryopreserved oocytes since 1996 at a single academically affiliated fertility center. 325 patients completed a total of 510 thaw cycles. 315 cycles were excluded from analysis for no AMH information. 6376 oocyte cryopreservation cycles were performed over that timeframe. The primary outcomes were laboratory statistics including oocyte survival, fertilization and embryo blastulation rates, while the secondary outcome was cumulative live birth rates. Participants/materials, setting, methods AMH categories of &amp;lt; 1, 1-2 and &amp;gt; 2 were selected to stratify reduced, low-normal, and normal ovarian reserves, respectively. 70.7% inseminated oocytes with partner sperm while the remaining 29.3% used donor sperm. Mean age at time of oocyte cryopreservation was 36.5 (range 27.5 to 46.2) while mean age at time of oocyte thaw was 39.2 (range 27.7 to 47.5). The statistical analysis was performed using ANOVA for mean comparisons and chi-squared for pregnancy data. Main results and the role of chance Average oocyte yield per cryopreservation cycle was significantly different across AMH groups (7.7 oocytes in AMH &amp;lt; 1, 10.5 oocytes in AMH 1-2, and 17.2 oocytes in AMH &amp;gt; 2, p &amp;lt; 0.01). The number of treatment cycles completed was highest in patients with AMH &amp;lt; 1 at 1.26 cycles. Oocyte survival (mean 89.0%), fertilization rate (mean 75.3%), and blastocyst conversion rate (mean 62.4%) did not vary significantly across AMH groups. Time to oocyte use was not significantly impacted by AMH at the time of initial testing. Those with reduced AMH &amp;lt; 1 came back earlier to utilize the oocytes (787 days), vs 1141 days for patients with AMH 1-2 and 1021 days for patients with AMH &amp;gt; 2. However, this difference was not statistically significant, p = 0.4. The rate of PGT-A euploid embryos did not vary between groups (38.8% in AMH &amp;lt; 1, 45.3% in AMH 1-2, 53.0% in AMH &amp;gt; 2, p = 0.07). Clinical pregnancy, miscarriage rate, and live birth rate in the first transfer cycle was not impacted by AMH. Total cumulative live birth rate was not correlated with AMH (31.6% in AMH &amp;lt; 1, 44.4% in AMH 1-2, 47.8% in AMH &amp;gt; 2, p = 0.48). Limitations, reasons for caution This data was collected at a single fertility center and thus has limited generalizability. 8.0% of patients came back to utilize their oocytes, which varies widely across the published literature. 60.6% of patients did not have AMH assessed as part of their pre-treatment testing and were excluded from analysis. Wider implications of the findings Although reduced ovarian reserve impacts oocyte yield, many studies find no impact on resulting oocyte quality, embryo development and pregnancy success rates. Those with compromised AMH have comparable oocyte thaw outcomes to those with normal ovarian reserve, though more oocyte cryopreservation cycles may be needed to achieve a successful outcome. Trial registration number N/A

P-009 Impact of sperm preparation methods on embryo development rate and embryo ploidy status: ICSI egg donation as a model

Abstract Study question Do different sperm separation techniques result in distinguishable contributions to embryo development rates and ploidy status? Summary answer CA0 sperm separation device, devoid of centrifugation, yielded a higher euploidy rate than conventional swim-up (50% vs. 74%, p = 0.02), notably observed in abnormozoospermic cases. What is known already Sperm DNA fragmentation (SDF) emerges as a key paternal risk factor associated with embryo aneuploidy, increasing the risk of ART failures. Factors like advanced male age, prolonged ejaculatory abstinence, and the detrimental effects of centrifugation during sperm separation jeopardize sperm DNA integrity. A recent centrifugation-free sperm separation device (CA0), employing live sperm sorting technology, exhibits efficiency in producing highly motile sperm with minimized SDF. This retrospective cohort study solely focusing on ICSI egg donation cycles to assess the influence of sperm separation methods (conventional swim-up vs. CA0) on early embryo development, and embryo ploidy. Study design, size, duration This retrospective study included 83 couples who underwent ICSI egg donation cycles at Chachava Clinic, Tbilisi, Georgia. Thirty-three semen specimens were processed using conventional swim-up (SU) and 427 Mii were inseminated. Forty-four semen samples were separated by CA0 and 519 Mii were inseminated. Clinical outcome measures included fertilization rate (number of 2PN over number of inseminated oocyte), good embryo rate (embryo number with more than 8 cells over inseminated oocyte), and euploidy rate using PGT-A. Participants/materials, setting, methods Male age and basic semen parameters via CASA (CEROS II, Hamilton-Thorne) were recorded. SU involved layering 1 mL neat semen to 1.2 mL HTF medium, inclining the tube at 45° for 60 min at 37 °C, collecting 1 mL uppermost, adding 2 mL HTF for centrifugation at 500×g for 5 min, and resuspending sperm pellet with 0.5 mL HTF. Semen samples were sorted using LensHooke® CA0 device following the manufacturer’s instructions. Main results and the role of chance In comparing demographic characteristics between SU and CA0 arms, CA0 exhibited higher median total motility (SU: 40% vs. CA0: 54%, p = 0.02), with no significant differences in female age and Mii oocyte count. This study assessed the impact of sperm separation methods on early embryo development, finding no significant differences in fertilization rate (SU: 79% vs. CA0: 77%) and good embryo rate (SU: 64% vs. CA0: 62%). However, CA0 demonstrated a significantly higher euploidy rate than SU (SU: 59% vs. CA0: 69%, p = 0.03). Further analysis within subpopulations, including normozoospermic and asthenozoospermic (total motility &amp;lt;40%), revealed equivalent euploidy rates between SU and CA0 for normospermic specimens (SU: 66% vs. CA0: 68%). However, a significant differentiation in euploidy rate emerged for asthenozoospermic cases (SU: 59% vs. CA0: 74%, p = 0.02). Specifically, comparing outcomes in normozoospermic, a significant reduction was observed in asthenozoospermic cases using SU (66% vs. 50%, p = 0.02), whereas CA0 provided improved euploidy rates regardless of semen quality (68% for normozoospermic and 74% for asthenozoospermic). In conclusion, this study suggests that the non-invasive live sperm sorting device enhances embryo euploidy rates, serving as a reliable sperm preparation method, ensuring selection consistency despite sample variations. Limitations, reasons for caution This study’s limitations include its retrospective nature and single-center trial design. To more effectively assess CA0 efficiency, a randomized controlled trial targeting diverse patient eligibility criteria is warranted. Wider implications of the findings Attributed to the improved embryo euploidy with CA0, we anticipate enhanced ART outcomes, such as increased embryo transfer rate and prevention of ART failures related to embryo aneuploidy. Additionally, CA0 offers practical benefits by streamlining and standardizing the sperm separation process with only three steps involved. Trial registration number not applicable

O-077 Depletion of MFF from oocytes impairs mitochondrial dynamics, leading to inhibited oocyte maturation and early embryonic development in mice

Abstract Study question To investigate the effect of mitochondrial fission factor (MFF) on oocyte competence and female fertility using an oocyte-specific Mff knockout mouse model. Summary answer Oocyte-specific Mff deficiency hindered oocyte maturation and early embryonic development via regulation of mitochondrial dynamics, resulting in female subfertility. What is known already Emerging studies have demonstrated that mitochondrial dynamics play a vital role in oocyte maturation and early embryo development. MFF is one of the crucial proteins regulating mitochondrial dynamics. Our previous studies have indicated that Mff is necessary for oocyte competence and fertility using a global Mff knockout mouse model. However, further investigation is needed to determine whether the deletion of Mff in oocytes could affect oocyte competence and early embryonic development by regulating mitochondrial dynamics. Study design, size, duration The oocyte-specific Mff knockout (Mfffl/fl; Gdf9-Cre) mice were generated using the Cre-LoxP conditional knockout system. The 2-month-old female mice from each group (Mfffl/fl; Gdf9-Cre or Mfffl/fl, n = 6) were mated to assess the fecundity over 10 months. The maturation and mitochondrial dynamics of germinal vesicle (GV) oocytes were determined. The spindle and chromosome morphologies of ovulated oocytes were also analyzed. Additionally, the 2-cell embryos were collected and cultured in vitro to assess early embryonic development. Participants/materials, setting, methods Female mice from each group were mated with adult males to assess the fecundity. GV oocytes, ovulated oocytes, and 2-cell embryos were collected and cultured in vitro after superovulation, as indicated. Mitochondria, spindle, and chromosome were stained and then imaged using confocal microscopy. The microscopic morphology of mitochondria was assessed using electron microscopy. Main results and the role of chance The Mfffl/fl; Gdf9-Cre mice consistently exhibited reduced fertility with a significant decrease in litter size (3.00 vs. 5.42, p &amp;lt; 0.001), number of litters per female (4.33 vs. 7.50, p &amp;lt; 0.001), and number of pups per female (13.00 vs. 40.67, p &amp;lt; 0.001) compared to the Mfffl/fl mice. The numbers of GV oocytes (29.37 vs. 39.10, p &amp;lt; 0.05), ovulated oocytes (9.81 vs. 20.91, p &amp;lt; 0.001) and 2-cell embryos (3.50 vs. 10.17, p &amp;lt; 0.05) were significantly lower in the Mfffl/fl; Gdf9-Cre mice. The blastocyst (32.32% vs. 68.44%, p &amp;lt; 0.05) embryo development rate per 2-cell embryos was also significantly lower. In vitro maturation revealed that of Mfffl/fl; Gdf9-Cre GV oocytes had significantly lower rates of GV break-down (68.89% vs. 91.57%, p &amp;lt; 0.05) and polar body extrusion (42.76% vs. 81.87%, p &amp;lt; 0.05). The ratios of aberrant spindle (71.56% vs. 23.74%, p &amp;lt; 0.001) and misaligned chromosomes (71.82% vs. 23.14%, p &amp;lt; 0.01) in ovulated oocytes from Mfffl/fl; Gdf9-Cre mice were significantly increased. In terms of mitochondrial dynamics, the mitochondrial size (0.59 µm2 vs. 0.15µm2, p &amp;lt; 0.001) and the ratio (62.50% vs. 18.67%, p &amp;lt; 0.01) of abnormal mitochondrial distribution (unevenly aggregated) in Mfffl/fl; Gdf9-Cre oocytes significantly elevated. This study found that the Mff played a crucial role in oocyte maturation and early embryonic development. Limitations, reasons for caution Further research is needed to determine the downstream targets or pathways of Mff in oocytes. Wider implications of the findings These results shed light on the important role played by MFF in ensuring proper mitochondrial dynamics in oocytes and underscore the significance of this protein in female fertility. This study provides insights into potential therapeutic targets for infertility and related diseases in humans. Trial registration number not applicable

P-057 Do the reduction of sexual abstinence influence sperm DNA fragmentation? Exploring the relationship between the two parameters

Abstract Study question Is the reduction of sexual abstinence (SA) really beneficial on sperm DNA fragmentation? Summary answer Despite recent studies support short SA’s positive impact on sperm parameters, apparently a reduced SA has not a clear impact on sperm DNA fragmentation. What is known already Sperm DNA fragmentation (sDF) has been linked to male infertility, affecting the success of fertility treatments. It’s strictly associated with failure and/or no longer time to conceive, impaired embryo development or higher miscarriege rates. The exact mechanism for age-dependent patterns of sperm decline is still not fully understood. Several factors, such as the increase in free radicals in conjuction with the rising in SA, have been discussed in literature. Recent studies support short SA’s positive impact on sperm parameters, despite controversial results; hence, we decided to analyze the trend in our facility: Humanitas Fertility Center. Study design, size, duration The present retrospective analysis, performed between 2021 and 2023, aims to investigate whether or not a reduced SA is beneficial on sperm DNA fragmentation in patients undergoing IVF. In our study 828 patients (from 21 to 59 years old) underwent both sperm analysis and sDF evaluation through Sperm Chromatin Structure Assay (SCSA). A DNA fragmentation index (DFI) lower than 30% indicates a sperm population with a good chromatin integrity, according to current knowledge. Participants/materials, setting, methods Patients were split in 4 groups, based on their age (≤38 and &amp;gt;38 years) and days of SA (0-3 and 4-7 days). Group 1 (n = 111/828): ≤38 years, ≤3 days; Group 2 (n = 359/828): ≤38 years, &amp;gt;3 days; Group 3 (n = 83/828): &amp;gt;38 years, ≤3 days; Group 4 (n = 275/828): &amp;gt;38 years, &amp;gt;3 days. Statistics was performed to analyze the variance among DFI values belonging to the four groups analyzed. Main results and the role of chance The results are expressed as mean(±SD). In Group 1, DFI %: 22,5(±14,7); in Group 2, DFI %: 21,5(±14,3); in Group 3, DFI %: 22,3(±14,9); in Group 4, DFI %: 26(±16,3). Surprisingly we observed the same behavior between most of the groups analyzed, except for the one belonging to the older patients. As a matter of fact, we found no statistical difference by comparing the DFI values belonging to samples of the same age stratification (≤38 or &amp;gt; 38 years), indeed Group 1 vs. Group 2: P=0,546; Group 3 vs. Group 4: P=0,1152. Different results were observed by comparing groups with the same SA days (≤3 or &amp;gt; 3), but belonging to a different age stratification, as it follows: Group 1 vs. Group 3: P=0,6384; Group 2 vs. Group 4: P=0,0001. This leads us to think that only the mixture of advanced paternal age (&amp;gt;38 years) and the increase of SA defines a worsening of DFI, according to our patients’ stratification. Limitations, reasons for caution There’s no homogeneity in the number of patients recruited with ≤3 days of SA compared to those with &amp;gt;3 days, as expected. Men undergoing fertility treatments follow strict SA guidelines; moreover, there’s no consensus about to request a second semen sample in clinical practice, in order to eliminate SA. Wider implications of the findings Exploring the inclusion of a broader spectrum of patients with low SA, despite their age, or instructing them to undergo semen analysis with a 0-day SA, could yield interesting insights. This could lead to targeted interventions and treatments, offering further hope for individuals with sDF issues. Trial registration number Not applicable

O-194 Cumulative live birth and neonatal outcomes according to different male factors in IVF/ICSI cycles

Abstract Study question What are the impacts of different male factors on cumulative live birth and neonatal outcomes of IVF/ICSI cycles? Summary answer Severe male infertility factors may be negatively associated with fertilization and egg utilization, embryo development and cumulative live birth rate, but not with neonatal outcomes. What is known already Several studies have explored the effect of semen quality on IVF/ICSI clinical outcomes and have drawn contradictory conclusions because the semen quality of male partners could be fluctuated by many factors and there are many confounding factors from women partner. However, an all-round and definitive conclusion has not been reached yet, and clinicians have not yet determined whether men with abnormal semen parameters should be treated to improve their semen parameters and IVF clinical outcomes. Study design, size, duration The retrospective cohort study involved 6,580 males with abnormal sperm parameters and 17,254 males with normozoospermia who underwent their first IVF/ICSI cycle between January 2018 and September 2022 in the reproductive medicine center at a university hospital. They were divided into five different study groups: normozoospermia (N), moderate male factor (MMF), severe oligoasthenoteratozoospermia (OAT-S), azoospermia-husband (azoospermia-H), and azoospermia-donor (azoospermia-D). We compared fertilization, embryo development, cumulative live birth and neonatal outcome. Participants/materials, setting, methods Reproductive and neonatal outcomes of men with abnormal sperm parameters (MMF, OAT-S, azoospermia-H, and azoospermia-D) were studied through four propensity score matching (PSM) comparisons along with corresponding control groups(N) (match factors: including female age, BMI, Gn time, controlled ovarian stimulation protocol, number of oocytes obtained, endometrial thickness and duration of infertility). Fertilization outcomes were also compared stratified by IVF or ICSI. Main results and the role of chance The median ages of the females in the azoospermia, OAT-S, MMF, and N groups were 29, 30, 32 and 32 years old, respectively, which were comparable among groups after PSM. The fertilization rates were significantly reduced in OAT-S and azoospermia-H compared with N in ICSI cycles (67.05% and 65.05% versus 71.50%, p &amp;lt; 0.001, respectively). The oocyte utilization rates were also significantly declined in OAT-S and azoospermia-H compared with N in IVF/ICSI cycles (28.39% and 29.64% versus 34.83%, p &amp;lt; 0.001, respectively). Azoospermia-H group was divided into obstructive azoospermia (OA) and non-obstructive azoospermia (NOA). The conservative cumulative live birth rate (CLBR) in the OAT-S group with poor spermatogenic function decreased (63.6% vs. 72.9%, P &amp;lt; 0.001), and was also declined in the NOA group with the poorest spermatogenic function (62.4% vs. 72.9%, P &amp;lt; 0.001), compared with that in the OA group with mostly normal spermatogenic function. After binary multifactorial logistic regression, we found that conservative CLBR was lower in the OAT-S (aOR 0.675, 95%CI 0.536-0.850, P = 0.001) and NOA (aOR 0.574, 95%CI 0.384-0.857, P = 0.007) groups than in the OA group. No impact of sperm factor on obstetrical/perinatal outcomes was observed. In IVF/ICSI cycles, reproductive and neonatal outcomes were similar between MMF, azoospermia-D and normal semen groups. Limitations, reasons for caution The main limitation of this study is that it was a single-center, observational and retrospective study. However, the large sample size, use of PSMs and use of a multivariate regression model for a wide array of possible confounding factors rendered the conclusion relatively reliable. Wider implications of the findings This is the first study involving long-term outcomes (obstetrical and neonatal outcomes, cumulative pregnancy and live rates) from IVF/ICSI cycles for different male factors, which can help clinicians identify paternal influence in an all-round way. Trial registration number not applicable

P-375 Long-term cryostorage of donor semen does not affect egg donor cycles outcomes

Abstract Study question Does prolonged donor semen cryopreservation impact in success rates of egg donor (ED) IVF cycles? Summary answer Prolonged donor semen cryopreservation does not affect good quality embryos or pregnancy rates in ED-IVF cycles. What is known already Births from cryopreserved semen stored for over 20 years have been documented. In 2004, a case of an IVF birth with semen frozen for 21 years was reported, and in 2005 another case with an IUI birth using semen frozen for 28 years was published. Recently, in 2019, a child born through IVF with semen frozen for over 26 years was reported in UK. In 2019, a study found that prolonged storage of these samples did not affect the outcomes of IVF with own eggs or IUI. Study design, size, duration We analysed data from 969 ED-IVF cycles performed at our centre between 2007 and 2022. Initially, cycles were classified into two independent groups of study to eliminate the impact of differences in embryo culture time. Group 1 or short culture (until Day 3) included 511 cycles from 2007 to 2013, and Group 2 or long culture (until Day5/6) included 458 cycles from 2014 to 2022. Participants/materials, setting, methods Donor oocytes were inseminated by ICSI using donor sperm from our bank. We analysed the relationship between the years of semen cryopreservation and the number of good quality embryos in both groups using Pearson correlation coefficient. Afterwards, the groups were divided into three subgroups based on the years of cryopreservation: 0-&amp;lt;3, ≥3-5, ≥5. Pregnancy and term pregnancy rates were calculated, and their relationship with time of semen cryopreservation were analysed using the Chi-square statistic. Main results and the role of chance No relationship was found between the years of cryopreservation and the number of good quality embryos in either group (Group 1 r=-0.05 and Group 2 r = 0.04). Pregnancy rates for the three subgroups 0-&amp;lt;3, ≥3-5, ≥5 were as follows: 219/454(48.24%),17/ 39(43.59%) and 6/18(33.3%) respectively in Group 1 and 141/213 (66.2%), 62/104 (59.62%), and 85/141 (60.28%) in Group 2. No statistically significant relationship was found in either group (Group 1 p-value=0.41 and Group 2 p-value=0.38). Term pregnancy rates for the three subgroups 0-&amp;lt;3, ≥3-5, ≥5 were as follows: 139/219 (63.47%), 10/17 (58.82%) and 4/6 (66.67%) respectively in Group 1 and 89/141 (63.12%), 39/62 (63.12%) and 56/85 (65.88%) in Group 2. No statistically significant relationship was found in either group (Group 1 p-value=0.91 and Group 2 p-value=0.90). The findings of this study indicate that prolonged donor semen cryopreservation does not appear to have a negative impact on success rates in ED-IVF cycles, both in terms of embryo development, and pregnancy and term pregnancy rates. Limitations, reasons for caution In Group 1 there was a big difference in sample size between subgroups, but it was due to the nature of the study. Wider implications of the findings Despite it seems that prolonged cryopreservation of semen does not affect embryonic viability, there is a lack of many molecular studies to ensure that this does not impact in cellular mechanisms or epigenetics. Trial registration number Not Applicable

Effects of Lycopene Treatments on Development, Hatchability, and Heart Rate of Zebrafish Embryos under Heat Stress Exposure

Effects of Lycopene Treatments on Development, Hatchability, and Heart Rate of Zebrafish Embryos under Heat Stress Exposure

In vitro effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on the expression of genes related to sperm motility and energy metabolism and intracytoplasmic sperm injection outcomes in obstructive azoospermic patients.

The presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and its receptor in various testicular cells and spermatozoa suggests a potential role in enhancing spermatogonial and postmeiotic cell development. Moreover, GM-CSF activates the pivotal pathways implicated in sperm motility regulation and glucose metabolism. However, the impact of GM-CSF on testicular biopsies from patients with obstructive azoospermia (OA) remains unexplored. Therefore, this study aimed to investigate the in vitro effects of GM-CSF on the expression of genes related to glucose transporters and signaling pathways, sperm motility, and viability in testicular biopsies. Following testicular sperm extraction from 20 patients diagnosed with OA, each sample was divided into two parts: the experimental samples were incubated with medium containing 2ng/ml GM-CSF at 37°C for 60min, and the control samples were incubated with medium without GM-CSF. Subsequently, the oocytes retrieved from the partner were injected with sperm from the treatment and control groups. The sperm parameters (motility and viability), the expression levels of sperm motility-related genes (PIK3R1, PIK3CA, and AKT1), and the expression levels of sperm energy metabolism-related genes (GLUT1, GLUT3, and GLUT14) were assessed. Furthermore, the fertilization and day 3 embryo development rate and embryo quality were evaluated. Compared with those in the nontreated group, the motility parameters and the mRNA expression levels of PIK3R1, AKT1, and GLUT3 in testicular sperm supplemented with GM-CSF were significantly greater (p < 0.05). However, no significant differences in the mRNA expression of PIK3CA, GLUT1, or GLUT14 were detected. According to the ICSI results, compared with the control group, the GM-CSF treatment group exhibited significantly greater fertilization rates (p = 0.027), Day 3 embryo development rate (p = 0.001), and proportions of good-quality embryos (p = 0.002). GM-CSF increased the expression of genes related to motility and the energy metabolism pathway and effectively promoted the motility of testis-extracted spermatozoa, consequently yielding positive clinical outcomes.

In vitro developmental competence of bovine demi-embryos generated by blastomere separation and blastocyst bisection.

The efficiency of bovine invitro embryo production can be significantly improved by splitting embryos at different stages. However, the blastocyst quality of invitro-produced demi-embryos remains unexplored. The objective of this research was to compare embryo developmental rates and quality of bovine demi-embryos produced by two different strategies: (a) embryo bisection (BSEC) and (b) 2-cell blastomere separation (BSEP). To determine demi-embryos quality, we evaluated total blastocyst cell number and proportion of SOX2+ cells. Additionally, the expression of SOX2, NANOG, OCT4, CDX2, IFNT, BAX and BCL genes and let-7a and miRNA-30c Micro RNAs was analysed. BSEP resulted in improved blastocyst development, higher ICM cells and a significantly higher expression of IFNΤ than demi-embryos produced by BSEC. Let-7a, which is associated with low pregnancy establishment was detected in BSEC, while miRNA-30c expression was observed in all treatments. In conclusion, BSEP of 2-cell embryos is more efficient to improve invitro bovine embryo development and to produce good quality demi-embryos based on ICM cell number and the expression pattern of the genes explored compared to BSEC.

Chemical reversion of age-related oocyte dysfunction fails to enhance embryo development in a bovine model of postovulatory aging

PurposeThere are no clinical treatments to prevent/revert age-related alterations associated with oocyte competence decline in the context of advanced maternal age. Those alterations have been attributed to oxidative stress and mitochondrial dysfunction. Our study aimed to test the hypothesis that in vitro maturation (IVM) medium supplementation with antioxidants (resveratrol or phloretin) may revert age-related oocyte competence decline.MethodsBovine immature oocytes were matured in vitro for 23 h (young) and 30 h (aged). Postovulatory aged oocytes (control group) and embryos obtained after fertilization were examined and compared with oocytes supplemented with either 2 μM of resveratrol or 6 μM phloretin (treatment groups) during IVM.ResultsAged oocytes had a significantly lower mitochondrial mass and proportion of mitochondrial clustered pattern, lower ooplasmic volume, higher ROS, lower sirtuin-1 protein level, and a lower blastocyst rate in comparison to young oocytes, indicating that postovulatory oocytes have a lower quality and developmental competence, thus validating our experimental model. Supplementation of IVM medium with antioxidants prevented the generation of ROS and restored the active mitochondrial mass and pattern characteristic of younger oocytes. Moreover, sirtuin-1 protein levels were also restored but only following incubation with resveratrol. Despite these findings, the blastocyst rate of treatment groups was not significantly different from the control group, indicating that resveratrol and phloretin could not restore the oocyte competence of postovulatory aged oocytes.ConclusionResveratrol and phloretin can both revert the age-related oxidative stress and mitochondrial dysfunction during postovulatory aging but were insufficient to enhance embryo developmental rates under our experimental conditions.

Investigation of Uterine Fluid Extracellular Vesicles' Proteomic Profiles Provides Novel Diagnostic Biomarkers of Bovine Endometritis.

Cow uterine infections pose a challenge in dairy farming, resulting in reproductive disorders. Uterine fluid extracellular vesicles (UF-EVs) play a key role in cell-to-cell communication in the uterus, potentially holding the signs of aetiology for endometritis. We used mass spectrometry-based quantitative shotgun proteomics to compare UF-EV proteomic profiles in healthy cows (H), cows with subclinical (SE) or clinical endometritis (CLE) sampled at 28-35 days postpartum. Functional analysis was performed on embryo cultures with the exposure to different EV types. A total of 248 UF-EV proteins exhibited differential enrichment between the groups. Interestingly, in SE, EV protein signature suggests a slight suppression of inflammatory response compared to CLE-UF-EVs, clustering closer with healthy cows' profile. Furthermore, CLE-UF-EVs proteomic profile highlighted pathways associated with cell apoptosis and active inflammation aimed at pathogen elimination. In SE-UF-EVs, the regulation of normal physiological status was aberrant, showing cell damage and endometrial repair at the same time. Serine peptidase HtrA1 (HTRA1) emerged as a potential biomarker for SE. Supplementation of CLE- and SE-derived UF-EVs reduced the embryo developmental rates and quality. Therefore, further research is warranted to elucidate the precise aetiology of SE in cattle, and HTRA1 should be further explored as a potential diagnostic biomarker.

The potential effect of melatonin on in vitro oocyte maturation and embryo development in animals.

Melatonin is a hormone mainly secreted by the pineal gland during the circadian cycle, with low levels during the daytime and prominent levels during the night. It is involved in numerous physiological functions including the immune system, circadian rhythm, reproduction, fertilization, and embryo development. In addition, melatonin exerts anti-inflammatory and antioxidant effects inside the body by scavenging reactive oxygen and reactive nitrogen species, increasing antioxidant defenses, and blocking the transcription factors of pro-inflammatory cytokines. Its protective activity has been reported to be effective in various reproductive biotechnological processes, including in vitro maturation (IVM), embryo development, and survival rates. In this comprehensive review, our objective is to summarize and debate the potential mechanism and impact of melatonin on oocyte maturation and embryo development through various developmental routes in different mammalian species.