Rate Of Embryonic Development Research Articles

Embryonic heart rate is higher in species that experience greater nest predation risk during incubation.

Avian eggs develop outside of the female body, and therefore embryonic development is subject to multiple internal (physiological) and external (ecological) factors. Embryonic developmental rate has important consequences for survival. Within species, embryos that develop too quickly often experience deformities, disorders, or mortality, while embryos that develop slowly risk inviability and increase the time they are exposed to various sources of mortality in the nest. These contrasting forces may lead to interspecific variation in developmental rates. We investigated potential factors affecting embryonic heart rate (EHR), a proxy of development, across 14 passerine species in the field. More specifically, we investigated if nest predation risk, clutch size, seasonality, and egg volume influenced EHR. From previous research, we expected, and found, that EHR was positively associated with embryonic age and egg temperature. Species with greater nest predation risk had higher EHR, shorter incubation periods, and lower nest temperature variance. EHR increased as the season progressed and with egg volume, while EHR declined with clutch size. Bird species exhibit varying strategies to increase nestling and fledgling survival in response to predation risk, and these results suggest that variation in embryonic development may be related to species-specific differences in nest predation risk.

Acetic acid affects porcine oocyte metabolism and improves oocyte developmental ability

Improvement in vitro maturation culture conditions has been achieved by mimicking in vivo culture environments such as the follicular fluid. Acetic acid is an energy substrate that is abundantly present in the follicular fluid but has not been considered in vitro maturation. This study examined the effects of acetic acid on oocyte quality during nuclear maturation. Cumulus cells and oocyte complexes were collected from the porcine antral follicles of gilt ovaries and matured with 0, 0.1 or 1 mmol/L of acetic acid. After 44 h of in vitro maturation, the energy status, mitochondrial quality and function and embryonic developmental rate following parthenogenetic activation were determined. RNA-sequencing and protein expression analyses were conducted to predict the effects of acetic acid. Supplementation of the in vitro maturation medium with acetic acid (1 mmol/L) improved embryonic development. Oocytes matured with acetic acid had low adenosine triphosphate and lipid contents, mitochondrial membrane potential and reactive oxygen species levels. RNA-sequencing revealed differential expression of genes associated with the adenosine monophosphate-activated protein kinase signalling pathway. Immunostaining revealed that acetic acid increased the levels of phospho-adenosine monophosphate-activated protein kinase, phospho-acetyl-coenzyme A carboxylase, and sirtuin 1 and decreased those of fatty acid synthase and acetyl-coenzyme A synthetase 1. In summary, the use of acetic acid during oocyte maturation improved oocyte developmental ability and metabolism by altering mitochondrial activity and lipid metabolism.

Improvement of survivability and developmental ability in vitrified rat oocytes

Oocyte cryopreservation is useful for human fertility treatment and strain preservation in both experimental and domestic animals. However, the embryonic development of vitrified rat oocytes was lower than that of vitrified embryos. To increase the viability of vitrified oocytes, intracellular ice formation during cooling and warming must be prevented. Rapid warming is important to prevent ice formation. Furthermore, suppressing the spontaneous activation of oocytes is also important because vitrification promotes the spontaneous activation of rat oocytes, and thus compromise developmental competence of the gametes. MG132, a proteasome inhibitor, suppresses the spontaneous activation of rat oocytes. Here, we examined the effects of rapid warming and MG132 treatment on the survival and embryonic development of vitrified rat oocytes. The warming rate was adjusted by changing the vitrification solution volume and warming solution temperature. The survival rate of oocytes vitrified in 10 μL solution and warmed at 50 °C (94%) was significantly higher than that of oocytes vitrified in 100 μL and 10 μL solution and warmed at 37 °C (49% and 81%, respectively). Furthermore, the rate of embryonic development of vitrified oocytes treated with MG132 during vitrification, warming, and intracytoplasmic sperm injection (ICSI) (44%) was significantly higher than that of untreated gametes (10%). Offspring were obtained after transferring embryos derived from MG132-treated vitrified oocytes (14%). Altogether, the survivability of vitrified rat oocytes increased by rapid warming, and MG132 improved embryonic development after ICSI.

Multiple treatments with human embryonic stem cell-derived mesenchymal progenitor cells preserved the fertility and ovarian function of perimenopausal mice undergoing natural aging

ObjectivesCurrently, no approved stem cell-based therapies for preserving ovarian function during aging. To solve this problem, we developed a long-term treatment for human embryonic stem cell-derived mesenchymal progenitor cells (hESC-MPCs). We investigated whether the cells retained their ability to resist ovarian aging, which leads to delayed reproductive senescence.Materials and methodsIn a middle-aged female model undergoing natural aging, we analyzed whether hESC-MPCs benefit the long-term maintenance of reproductive fecundity and ovarian reservoirs and how their transplantation regulates ovarian function.ResultsThe number of primordial follicles and mice with regular estrous cycles were increased in perimenopausal mice who underwent multiple introductions of hESC-MPCs compared to age-matched controls. The estradiol levels in the hESC-MPCs group were restored to those in the young and adult groups. Embryonic development and live birth rates were higher in the hESC-MPC group than in the control group, suggesting that hESC-MPCs delayed ovarian senescence. In addition to their direct effects on the ovary, multiple-treatments with hESC-MPCs reduced ovarian fibrosis by downregulating inflammation and fibrosis-related genes via the suppression of myeloid-derived suppressor cells (MDSCs) produced in the bone marrow.ConclusionsMultiple introductions of hESC-MPCs could be a useful approach to prevent female reproductive senescence and that these cells are promising sources for cell therapy to postpone the ovarian aging and retain fecundity in perimenopausal women.

Studies on the embryonic development and larval infection potential of the stomach bot flies, Gasterophilus pecorum

Endangered Przewalski's horses have faced severe infections from Gasterophilus pecorum (Diptera, Gastrophilidae) in Xinjiang's Kalamaili Nature Reserve (KNR). This study examines G. pecorum's development and infection patterns in embryonic and larval stages, crucial for understanding horse botfly disease in desert grasslands. For the incubation of G. pecorum fertilized eggs, we established the six distinct temperature gradients: 16 °C, 20 °C, 24 °C, 28 °C, 30 °C, and 32 °C. Using the least squares method, we calculated the correlation between the developmental threshold temperature of the eggs and their cumulative effective temperature. Furthermore, we meticulously recorded the survival duration of the larvae across a spectrum of temperature gradients (−20 °C, −10 °C, 4 °C, 10 °C, 20 °C, and 30 °C) and under varying conditions (dark and light). This method allows us to analyze and interpret the impact of these environmental factors on larval survival durations. 1) The formula for predicting the embryonic development period of G. pecorum was N = (182.7 ± 12.03)/[T-(3.191 ± 1.48)], where the developmental threshold temperature was 3.191 ± 1.48 °C, and the effective accumulated temperature was 182.7 ± 12.03 d°C 2) The model describing the relationship between the embryonic development rate and temperature was: y = 0.0001x2+0.0007x+0.0378, demonstrating a positive correlation between the development rate and temperature (R-sq = 0.989, p < 0.001). 3) Larvae in the dark group exhibited a longer survival time, with the longest being 9 months at 4 °C. The adaptation of G. pecorum's embryonic development to cold temperature, combined with the extended survival period of larvae in the egg state, significantly increases the infection potential of G. pecorum in colder climates. This discovery offers essential insights into the predominance of G. pecorum in the KNR region and provides a crucial biological basis for the prevention of myiasis and the conservation of vulnerable species, such as Przewalski's horses.

Ultra-Fast Vitrification: Minimizing the Toxicity of Cryoprotective Agents and Osmotic Stress in Mouse Oocyte Cryopreservation.

Globally, women have been adopting oocyte cryopreservation (OC) for fertility preservation for various reasons, such as inevitable gonadotoxic treatment for specific pathologic states and social preferences. While conventional vitrification (C-VIT) has improved the success rate of OC, challenges of possible toxicities of high-concentration cryoprotective agents and osmotic stress persist. To overcome these challenges, we evaluated the ultra-fast vitrification (UF-VIT) method, which reduces the equilibration solution stage exposure time compared to C-VIT by observing mouse oocyte intracellular organelles and embryonic development. Consequently, compared to fresh mouse oocytes, UF-VIT presented significant differences only in endoplasmic reticulum (ER) intensity and mitochondrial (MT) distribution. Meanwhile, C-VIT showed substantial differences in the survival rate, key ER and MT parameters, and embryonic development rate. UF-VIT exhibited considerably fewer negative effects on key MT parameters and resulted in a notably higher blastocyst formation rate than C-VIT. Meiotic spindle (spindle and chromosomes) morphology showed no significant changes between the groups during vitrification/warming (VW), suggesting that VW did not negatively affect the meiotic spindle of the oocytes. In conclusion, UF-VIT seems more effective in OC owing to efficient cytoplasmic water molecule extraction, osmotic stress reduction, and minimization of cell contraction and expansion amplitude, thus compensating for the drawbacks of C-VIT.

Apigenin delays postovulatory oocyte aging by reducing oxidative stress through SIRT1 upregulation

After ovulation, senescent oocytes inevitably experience reduced quality and defects in embryonic development. Apigenin (API) is a flavonoid with a wide range of pharmacological effects. Therefore, this study examined the protective effects of API on the quality of porcine oocytes during in-vitro ageing and the underlying mechanisms. The results showed that API treatment could reduce the activation rate after aging for 48 h. In addition, API significantly reduced reactive oxygen species, abnormal distribution of mitochondria, early apoptosis in ageing oocytes, increased glutathione, and mitochondrial adenosine triphosphate levels in ageing oocytes. Importantly, API increased the embryonic development rate in aged oocytes. We also examined molecular changes, finding decreased sirtuin 1 expression in in-vitro postovulatory oocytes, but API reversed this effect. Our results suggest that API attenuates the deterioration of oocyte quality during in-vitro ageing, possibly by reducing oxidative stress through the upregulation of sirtuin 1.

An Assessment of the Biological Significance of a Visual Clutch Staging Scheme for Ovigerous Female American Lobster (Homarus americanus).

Qualitative visual clutch staging is a useful tool for rapidly and non-invasively assessing the developmental stage of American lobster, Homarus americanus, embryos. While such a scheme has been used in fisheries monitoring strategies in Canada since the 1980s, the biological relevance of its four visually distinguishable stages is poorly understood. We conducted a laboratory experiment in which 10 ovigerous females were housed and the development of their embryos regularly assessed, both qualitatively and quantitatively, from November until hatching in July/August. We confirmed the biological relevance of the qualitative staging scheme by showing clear quantitative differences in the duration and rate of embryonic development of stages 2-4 (stage 1 was not assessed as the precise spawning date was unknown). Stage 2 represents winter-spring "dormancy". Stage 3 represents a shorter period of rapid development preceding hatch. Stage 4 represents hatching. We also recommend some improvements to the qualitative staging scheme, specifically (1) adding criteria related to the portion of eggs that are occupied by yolk to increase the accuracy of staging, (2) slightly redefining stage 3 to ensure it encompasses the full period of rapid embryonic development pre-hatch, and (3) adding the presence of pre-zoeae as a key indicator of hatching to avoid the misclassification of clutches in the early stages of hatching or those that are completely spent but still have adhesive substance.

Effect of IL-10 and TNF-α on the competence and cryosurvival of in vitro produced Bos indicus embryos

In vitro-produced embryos are constantly exposed to stressful conditions that can lead to the activation of the apoptotic pathway. The nuclear Kappa B factor (NF-κB) is an inflammatory mediator that induces the expression of tumor necrosis factor (TNF-α), a pro-inflammatory cytokine, while interleukin-10 (IL-10), an anti-inflammatory cytokine, inhibits NF-κB activity. This study aimed to investigate the effects of IL-10 and TNF-α on the competence and cryosurvival of in vitro-produced bovine embryos. Embryos were produced in vitro using standard protocols, and Grade I blastocysts were vitrified using the Cryotop method. Non-vitrified and vitrified blastocysts were subjected to the TUNEL assay. In Experiment I, on day 6.5 (156 h post-insemination), the embryos were treated with PBS (control), 50 ng/mL of IL-10, or a combination of 25 ng/mL of TNF-α and 50 ng/mL of IL-10. Embryonic development and apoptotic rates were monitored. In Experiment II, the same groups were set up, with the addition of a group treated with 25 ng/mL of TNF-α alone. Grade I blastocysts were vitrified 5 h after treatment, and cryosurvival was monitored at until 48 h post-warming. The apoptosis rate and total cell number were investigated in the vitrified-hatched blastocysts. IL-10 alone did not affect developmental competence or cryosurvival (P > 0.05). The IL-10-treated embryos, when exposed in combination with TNF-α, presented a detrimental effect (P < 0.05) in the embryonic development of non-vitrified embryos. However, vitrified blastocysts had no negative effect (P > 0.05). The TNF-α treatment reduced (P < 0.05) the re-expansion rate at 6 h post-warming and increased (P < 0.05) the apoptosis rate in vitrified hatched blastocysts, whereas no effect (P > 0.05) of the treatments was detected in the hatching rate and total cell number post-warming. In conclusion, TNF-α has a detrimental effect on embryonic developmental competence and cryosurvival by compromising the development of non-vitrified embryos and apoptotic-related events of vitrified blastocysts, whereas IL-10, when in combination with TNF-α, appears to attenuate the detrimental effects of TNF-α.

Aggregated chromosomes/chromatin transfer: a novel approach for mitochondrial replacement with minimal mitochondrial carryover: the implications of mouse experiments for human aggregated chromosome transfer.

Nuclear transfer techniques, including spindle chromosome complex (SC) transfer and pronuclear transfer, have been employed to mitigate mitochondrial diseases. Nevertheless, the challenge of mitochondrial DNA (mtDNA) carryover remains unresolved. Previously, we introduced a method for aggregated chromosome (AC) transfer in human subjects, offering a potential solution. However, the subsequent rates of embryonic development have remained unexplored owing to legal limitations in Japan, and animal studies have been hindered by a lack of AC formation in other species. Building upon our success in generating ACs within mouse oocytes via utilization of the phosphodiesterase inhibitor 3-isobutyl 1-methylxanthine (IBMX), this study has established a mouse model for AC transfer. Subsequently, a comparative analysis of embryo development rates and mtDNA carryover between AC transfer and SC transfer was conducted. Additionally, the mitochondrial distribution around SC and AC structures was investigated, revealing that in oocytes at the metaphase II stage, the mitochondria exhibited a relatively concentrated arrangement around the spindle apparatus, while the distribution of mitochondria in AC-formed oocytes appeared to be independent of the AC position. The AC transfer approach produced a marked augmentation in rates of fertilization, embryo cleavage, and blastocyst formation, especially as compared to scenarios without AC transfer in IBMX-treated AC-formed oocytes. No significant disparities in fertilization and embryo development rates were observed between AC and SC transfers. However, relative real-time PCR analyses revealed that the mtDNA carryover for AC transfers was one-tenth and therefore significantly lower than that of SC transfers. This study successfully accomplished nuclear transfers with ACs in mouse oocytes, offering an insight into the potential of AC transfers as a solution to heteroplasmy-related challenges. These findings are promising in terms of future investigation with human oocytes, thus advancing AC transfer as an innovative approach in the field of human nuclear transfer methodology.

Identifying optimal puncture position by a real-time image analysis for Piezo-ICSI: a prospective randomized sibling oocyte study

Research questionWould the use of the intracytoplasmic sperm injection (ICSI) position detector (IPD) make it possible to identify the optimal puncture position on oolemma during Piezo-ICSI and reduce oocyte degeneration and unintentional membrane rupture (UMR)? DesignThis sibling oocyte study included 917 inseminated oocytes from 113 infertile patients undergoing Piezo-ICSI. Oocytes were randomly divided into two groups: with or without IPD. The rates of UMR, degeneration, fertilization and embryonic development were compared between the two groups. As a secondary analysis, non-IPD oocytes were retrospectively assessed as appropriate or non-appropriate injection sites and analysed alongside prospective ‘appropriate’ injections. ResultsThe rates of UMR (7.0% versus 12.9%, P = 0.004) and degeneration (2.4% versus 6.1%, P < 0.01 = 0.008) were significantly lower in the IPD group than in the non-IPD group. No significant differences, however, were observed in the rates of fertilization (two pronuclei, 83.8% versus 78.9%), blastocyst formation (48.5% versus 48.8%) or good-quality blastocysts (22.5% versus 20.5%). Additionally, no significant differences were observed in the rates of pregnancy (29.4% versus 35.1%) or live births (26.5% versus 29.7%) in a single embryo transfer setting with or without IPD. Comparing all ‘appropriate’ injections with ‘non-appropriate’ injections also showed a significantly decreased rate of UMR and degeneration (both P ≤ 0.001). ConclusionsThe present study demonstrated that a real-time image analysis during Piezo-ICSI markedly reduced oocyte degeneration by avoiding areas associated with a high risk of UMR. Therefore, IPD may increase the number of embryos available for treatment.

Effect of Campylobacter fetus on in vitro fertilization and embryonic development of preimplantation bovine embryos

Campylobacter fetus is an important veterinary pathogen that causes campylobacteriosis. This disease causes decreased productivity of cattle by inducing reproductive losses. Although several virulence factors have been recognized in C. fetus, including a cytolethal distending toxin (CDT), the exact mechanism responsible for embryonic death remains unknown. The aim of this work was to evaluate the effect of C. fetus subsp. fetus (Cff) and C. fetus subsp. venerealis (Cfv), and their toxin activity on the in vitro fertilization of bovine ova and early embryonic development. Two different experiments were performed. In Experiment 1, a total of 1524 cumulus–oocyte complexes (COCs) were inseminated, distributed into three groups: two of them infected with the microorganism (Cff, Cfv) and a control group. Percentages COCs cleaved were similar among groups (p = 0.1243); however, the embryonic development rate (blastocyst at day 7) in the control group was greater (p < 0.001) than those obtained in Cff and Cfv groups. In experiment 2, a total of 746 COCs were inseminated, divided into three groups: two of them treated with the bacterial-free culture filtrates to test toxin activity (Cff-CDT, Cfv-CDT) and a control group. Both cleavage and embryonic development rates were greater (p < 0.001) in the control group than those obtained in Cff-CDT and Cfv-CDT groups. This study provides evidence that both subspecies of C. fetus do not interfere with fertilization but do affect in vitro embryonic development. It is the first report on the biological effect of the CDT on bovine embryonic development.

P-254 Short-time or overnight co-incubation with sperm in conventional IVF delivers similar pre-implantation development outcomes: a sibling cumulus-oocyte complex study

Abstract Study question Does a short-time co-incubation in conventional IVF improve rates of fertilization, embryo development and euploidy, when compared to overnight co-incubation? Summary answer There is no significant difference in fertilization, embryo development and euploidy rates between sibling oocytes subjected to short co-incubation versus overnight co-incubation. What is known already Conventional IVF usually implies coincubation of cumulus-oocyte complex(es) with sperm for an overnight period. This overnight incubation might create an over exposure of COCs to reactive oxygen species (ROS), which might disturb oocyte homeostasis, and impact on embryonic developmental rate. To this extend, many early studies have compared a short co-incubation time (2-6 hours) with overnight co-incubation, with reported conflicting outcomes. None of the studies combined time-lapse monitoring with short co-incubation, which allows the identification of early morphokinetic events of the inseminated oocytes after conventional IVF, as already known for ICSI oocytes in time-lapse incubators. Study design, size, duration This single center prospective study included 604 COCs in 38 IVF/ICSI cycles with preimplantation genetic testing between December 2020 and November 2022. After oocyte retrieval a min of 6 COCs were assigned for insemination by IVF and equally divided between short(2hrs) and overnight (16-20hrs) co-incubation. Supernumerary oocytes were denuded and inseminated by ICSI. Primary endpoint was fertilization rate; secondary endpoints were maturation rate, ploidy rate after trophectoderm biopsy, embryo development and morphokinetics. Participants/materials, setting, methods Couples with a female age between 18-43 (average:34.4) years, BMI ≤30kg/m2 and normal semen parameters (WHO) in the fresh ejaculate on the day of OR, were eligible for the study. After IVF, all embryos were cultured in a time-lapse imaging system in Global Total LP media. Blastocysts (≥Bl3BCC) were subjected to trophectoderm biopsy on day 5-7 and next generation sequencing (NGS) to determine blastocyst ploidy status. Main results and the role of chance In total, 288 oocytes (47.7%) were denuded for ICSI, whereas 316 (52.3%) oocytes were randomized 1:1 to short and overnight co-incubation groups. Maturation rates (M2/COC) and normal fertilization rates (2PN/COC) in short and overnight co-incubation groups were similar (88.0% vs 83.5%, P = 0.074 and 60.8% vs 62.0%, P = 0.193, respectively). Day-3 embryo cell numbers (P = 0.383), fragmentation rates (P = 0.529), blastulation rates on day 5 (39.9 vs 38.0% P = 0.696), time to blastulation (median: 112.6 vs 111.1 hours after insemination by time lapse, P = 0.544) were similar between short and overnight co-incubation groups. In total, 48 embryos in short and 45 embryos in overnight co-incubation groups were of sufficient quality for biopsy resulting in similar euploidy rates between the groups (39.6 vs 44.4%, P = 0.791). When compared to their siblings in ICSI, short (mean difference: -6.48%, 95% CI: -18.5 to 5.59%, P = 0.290) and overnight co-incubation (mean difference: -0.87%, 95% CI: -12.9 to 11.2%, P = 0.886) attained similar fertilization rates per cycle. No significant differences regarding blastulation and euploidy rates were observed in short or overnight co-incubation compared to ICSI in unadjusted and adjusted analyses. Limitations, reasons for caution This is a prospective study with a limited patient population with a normal ovarian response. Results might not be transferable to patients with high or low ovarian reserve. Wider implications of the findings The present study demonstrated that there is no difference between short and overnight co-incubation in terms of fertilization and euploidy rates. Therefore short co-incubation might allow us to benefit from early denudation, and to investigate early morphokinetics in time lapse monitoring, which are otherwise missed by overnight co-incubation. Trial registration number NCT04627545

P-197 Nuclear transfer with almost nonexistent mitochondrial carryover -a mouse model-

Abstract Study question Can the transfer of aggregated chromosomes (AC) as compared to spindle-chromosome transfer (SCT) reduce mitochondrial DNA (mtDNA) carryover and improve development rates? Summary answer The rate of mtDNA carryover after AC transfer was only 0.1%, which was much less than that of 2.0% after SCT. What is known already Nuclear transfer techniques such as SCT and pronuclear transfer have been applied to prevent mitochondrial diseases and to treat early embryonic arrest, however, the problem of mtDNA carryover has not yet been resolved. We previously reported on an AC transfer (ACT) method in humans which could overcome the above problem, however, consequent embryonic development rates have not been investigated in Japan due to legal restrictions and no animal study has been conducted, as the formation of ACs has not been found in any other species. Study design, size, duration Following our success in the creation of ACs in mouse oocytes using IBMX (3-Isobutyl 1-methylxanthine), a phosphodiesterase inhibitor, a mouse model of ACT was established. This was followed by a comparison of the rates of embryo development and mtDNA carryover in both ACT and SCT in over 100 oocytes. The distribution of mitochondria around SC and AC were also examined by confocal laser microscope (FV3000, Olympus, Japan). Participants/materials, setting, methods GV stage oocytes were collected from 8-12 week B6D2F1 mice. After GVBD, oocytes were incubated in KSOM medium, containing 3 mM IBMX, for 10-14 hours until AC is formed. ACs and SCs were injected into enucleated MII stage donor oocytes and then fertilized at the MII stage. Embryos were cultured for 5 days to compare the rates of embryo development. Enucleated ACs and SCs were subjected to real-time PCR, to analyze the carryover of mtDNA. Main results and the role of chance The rates of fertilization, embryo cleavage and blastocyst formation in AC oocytes without AC transfer (control), in ACT and in SCT were 60.7%, 55.9%, 13.2% (control), 82.5%, 80.8% and 30.8% (ACT), and 81.0%, 74.5% and 31.4% (SCT) respectively. The ACT group showed a significant increase in fertilization (P = 0.007), embryo cleavage (p = 0.008) and blastocyst formation (p = 0.034) rates, as compared to those in the control group. There were no significant differences between ACT and SCT regarding the rates of fertilization, embryo cleavage and blastocyst formation. The relative, real-time PCR showed that mtDNA carryover in the ACT group was 0.1% ± 0.07%, which was significantly lower than that in the SCT group of 2.0% ± 0.6% (p = 0.027). 3D confocal laser microscopy revealed that mitochondria were not localized around either SCs or ACs, as they were around pronuclei. Limitations, reasons for caution Although we succeeded in establishing a mouse model of ACT, validation in human oocytes is required to confirm the benefit of this method. Further experiments are required to confirm if healthy offspring can be obtained after ACT. Wider implications of the findings ACT could reduce the amount of mtDNA carryover and overcome heteroplasmy. As ACT improved the low embryo development rate caused by IBMX and had similar embryo development rates to SCT, the findings of this study may give rise to ACT being employed as a novel method for human nuclear transfer. Trial registration number not applicatable

P-170 A sibling oocyte study using a real-time image analysis system to identify optimal puncture positions in Piezo-ICSI

Abstract Study question Is the newly developed image analysis system capable of visualizing the shape features of the oolemma in real-time, useful for reducing oocyte degeneration during Piezo-ICSI? Summary answer Unintentional membrane rupture (UMR) can be reduced by this real-time image analysis, which lowers the risk of oocyte degeneration following Piezo-ICSI. What is known already UMR in the puncturing process of ICSI often predisposes oocyte degeneration. Identifying the appropriate puncturing positions may decrease the likelihood of UMR and thus degeneration, but this cannot be visualized via a microscope. We have reported that visualization of the shape features of the oolemma using moving image analysis during Piezo-ICSI can locate areas on the oolemma where UMR is likely to occur. It is feasible to assess the incidence of UMR and oocyte degeneration by performing ICSI while selecting appropriate puncturing positions with the aid of the newly developed imaging system. Study design, size, duration Our team have developed an image analysis system called ICSI POSITION DETECTOR (IPD), which can identify areas where rupture is likely to occur and visualize it in real-time using a video monitor. We prospectively evaluated the usefulness of IPD in a sibling oocyte study. From January 2020 to August 2021, a total of 1268 oocytes obtained in 225 oocyte retrieval cycles (average maternal age: 38.3 ± 4.7 years old) was included. Participants/materials, setting, methods The oocytes were randomly assigned to two groups: IPD-using vs. non-IPD-using. In the IPD-using group, Piezo-ICSI was performed at the “appropriate” position with a low chance of UMR indicated by IPD. In the non-IPD-using group, Piezo-ICSI was performed blindly. The rates of UMR, oocyte degeneration, fertilization and embryonic development were compared between the two groups. In addition, in the non-IPD-using group, moving images were recorded during Piezo-ICSI and analyzed retrospectively using IPD. Main results and the role of chance The rates of UMR and degeneration were significantly lower in the IPD-using group compared to the non-IPD-using group (6.0% vs. 11.9%, P &amp;lt; 0.001 and 1.6% vs. 4.6%, P &amp;lt; 0.01, respectively). The rates of fertilization (83.7% vs. 79.7%), blastocyst formation (51.9% vs. 51.0%), and good-quality blastocyst (24.7% vs. 24.3%) were not significantly different. Retrospective analysis of moving images using IPD on the non-IPD-using group showed that in 45.3% of oocytes (286 out of 632) ICSI was performed at a position with a high chance of UMR (inappropriate position). When ICSI was performed at the appropriate positions using IPD, the rates of UMR (6.0% vs. 18.2%, P &amp;lt; 0.001) and degeneration (1.6% vs. 7.3%, P &amp;lt; 0.001) were significantly lower, while the rates of fertilization (83.7% vs. 74.5%, P &amp;lt; 0.01) and blastocyst formation (51.9% vs. 43.0%, P &amp;lt; 0.05) were significantly higher than those when ICSI was performed at an inappropriate position. The rate of good-quality blastocyst (24.7% vs. 19.7%) was also higher, but not statistically significant. These results indicate that IPD can reduce the risk of UMR, thereby lower the degeneration rate. Furthermore, the embryonic development was better when using IPD to identify the appropriate position to perform ICSI, suggesting that UMR seems to be associated with poor ICSI outcome. Limitations, reasons for caution In this study, Piezo-ICSI was performed by only two designated embryologists in a single center. It did not assess clinical outcomes. Further research involving more embryologists is needed. Moreover, it would be feasible to investigate whether the usage of IPD is similarly effective on conventional-ICSI outcome. Wider implications of the findings The application of IPD to perform ICSI at the appropriate position can significantly avoid UMR, and thereby reduce oocyte degeneration and provide better embryonic development. We therefore, consider IPD to be a highly clinically useful tool, which contributes to the production of more embryos that can be used for treatment. Trial registration number not applicable

P-071 L-proline enhances sperm quality and chromatin condensation of normozoospermic men during in vitro incubation

Abstract Study question Does supplementation of human sperm media with L-Proline improve sperm function and chromatin quality during in Vitro Incubation? Summary answer The inclusion of L-proline as an antioxidant in human sperm media improves sperm function and chromatin integrity probably by modulating oxidative stress What is known already Infertility affects millions of people of reproductive age globally. The quality of gametes is one of the most critical aspects that could affect the success rate of infertility therapy. The storage of spermatozoa at the incubator to achieve capacitation could rise sperm DNA fragmentation as a result of prolonged incubation. Although mild and low levels of Reactive Oxygen Species (ROS) could boost fertilization capacity by improving hyperactivation, and capacitation, oxidative stress is one of the major contributors to low sperm quality. L-Proline plays a pivotal role in many aspects of cell metabolism and physiology, including osmotic protection and oxidative stress Study design, size, duration The study was conducted on thirty-five healthy men attending the Motazedi Hospital, Kermanshah of the Division of Andrology, from December 2020 to June 2021. Each semen sample was equally divided into 4 groups: one group as control and three as treatment groups. The control group contained only sperm media (Hams, F10 medium supplemented with 5 mg/ml HSA). In treatment groups, different concentrations of Proline (1, 2, and 4 mmol/L) were also added. Participants/materials, setting, methods 35 normozoospermic men with healthy lifestyles were included. Sperm parameters including motility, viability, and morphology were evaluated. The levels of ROS, lipid peroxidation (LPO), and total antioxidant capacity (TAC) were determined in sperm media. Sperm chromatin integrity was analyzed by Aniline blue (AB), Toluidine blue (TB), and Chromomycin A3 (CMA3) staining tests. DNA fragmentation was measured by the Sperm chromatin dispersion (SCD) test. All assessments were conducted after 1, 4, and 24 hours of incubation. Main results and the role of chance The inclusion of Proline (2 mmol/L) resulted in noticeably improved maintenance of motility and viability after 24 hr of incubation compared to the control group (P &amp;lt; 0.05). Also, 2 mmol/L of Proline was able to significantly maintain normal morphology compared to other groups (P &amp;lt; 0.01). However, the level of ROS production non-significantly diminished in the L-proline groups compared to the control group (P &amp;gt; 0.05). In terms of lipid peroxidation, the presence of 2 mmol/L of Proline kept the MDA production of spermatozoa at lower levels after 4 hr of incubation compared with the control group (P &amp;lt; 0.05). The supplementation of sperm media with 2 mmol/L of Proline led to improved maintenance of TAC level, compared to any other group (P &amp;lt; 0.01). Additionally, the results of AB and TB tests indicated that chromatin quality after supplementation with 2 mmol/L L-proline significantly improved compared to the control group (P &amp;lt; 0.05). In terms of CMA3 assessment, however, there was a minor improvement between the 2 mmol/L Proline group and the control group. Moreover, 2 mmol/L Proline significantly reduced the level of fragmented DNA by comparison to the control group after 24 hr of incubation (P &amp;lt; 0.05). Limitations, reasons for caution Our team was unable to carry out additional studies to verify the impact of L-proline on mitochondrial activity, membrane potential, and apoptosis and to validate the efficiency and safety of L-proline using additional parameters such as the in vitro embryonic development and live birth rate. Wider implications of the findings While further studies are required to uncover the mechanisms underlying the antioxidant properties of Proline, these findings extend our knowledge of human male fertility and pave the way to improving the human sperm culture and in vitro procedure routinely used in infertility clinics for sperm incubation. Trial registration number IR.KUMS.REC.1399.625

Embryonic development, hatchling performance and metabolic profile after egg exposure to environmentally relevant levels of chlorpyrifos in an aquatic turtle

The extensive use of organophosphorus insecticides poses a threat to the survival of non-target organisms. Ecotoxicological outcomes of embryonic exposure to insecticides are rarely evaluated in various oviparous species. In this study, soft-shelled turtle (Pelodiscus sinensis) eggs were incubated in moist substrate containing different levels (0, 2, 20 and 200 μg/kg) of chlorpyrifos to investigate its toxic effects on embryonic development and survival, and hatchling physiological performance. Chlorpyrifos exposure had no significant impacts on embryonic development rate and egg survival in P. sinensis. Similarly, embryonic chlorpyrifos exposure neither obviously affected the size and locomotor performance of hatchlings, nor changed the activities of superoxide dismutase and catalase, and content of malondialdehyde in their erythrocytes. Based on liquid chromatography-mass spectrometry analysis, minor metabolic perturbations related to amino acid, lipid and energy metabolism in hatchlings after embryonic chlorpyrifos exposure were revealed by hepatic metabolite profiling. Overall, our results suggested that embryonic exposure to environmentally relevant levels of chlorpyrifos had only a limited impact on physiological performances of hatchlings, although it would result in a potential risk of hepatotoxicity in P. sinensis.

Biallelic mutations in RNA-binding protein ADAD2 cause spermiogenic failure and non-obstructive azoospermia in humans.

What are some pathogenic mutations for non-obstructive azoospermia (NOA) and their effects on spermatogenesis? Biallelic missense and frameshift mutations in ADAD2 disrupt the differentiation of round spermatids to spermatozoa causing azoospermia in humans and mice. NOA is the most severe cause of male infertility characterized by an absence of sperm in the ejaculate due to impairment of spermatogenesis. In mice, the lack of the RNA-binding protein ADAD2 leads to a complete absence of sperm in epididymides due to failure of spemiogenesis, but the spermatogenic effects of ADAD2 mutations in human NOA-associated infertility require functional verification. Six infertile male patients from three unrelated families were diagnosed with NOA at local hospitals in Pakistan based on infertility history, sex hormone levels, two semen analyses and scrotal ultrasound. Testicular biopsies were performed in two of the six patients. Adad2 mutant mice (Adad2Mut/Mut) carrying mutations similar to those found in NOA patients were generated using the CRISPR/Cas9 genome editing tool. Reproductive phenotypes of Adad2Mut/Mut mice were verified at 2 months of age. Round spermatids from the littermates of wild-type (WT) and Adad2Mut/Mut mice were randomly selected and injected into stimulated WT oocytes. This round spermatid injection (ROSI) procedure was conducted with three biological replicates and >400 ROSI-derived zygotes were evaluated. The fertility of the ROSI-derived progeny was evaluated for three months in four Adad2WT/Mut male mice and six Adad2WT/Mut female mice. A total of 120 Adad2Mut/Mut, Adad2WT/Mut, and WT mice were used in this study. The entire study was conducted over 3 years. Whole-exome sequencing was performed to detect potentially pathogenic mutations in the six NOA-affected patients. The pathogenicity of the identified ADAD2 mutations was assessed and validated in human testicular tissues and in mouse models recapitulating the mutations in the NOA patients using quantitative PCR, western blotting, hematoxylin-eosin staining, Periodic acid-Schiff staining, and immunofluorescence. Round spermatids of WT and Adad2Mut/Mut mice were collected by fluorescence-activated cell sorting and injected into stimulated WT oocytes. The development of ROSI-derived offspring was evaluated in the embryonic and postnatal stages. Three recessive mutations were identified in ADAD2 (MT1: c.G829T, p.G277C; MT2: c.G1192A, p.D398N; MT3: c.917_918del, p.Q306Rfs*43) in patients from three unrelated Pakistani families. MT1 and MT2 dramatically reduced the testicular expression of ADAD2, likely causing spermiogenesis failure in the NOA patients. Immunofluorescence analysis of the Adad2Mut/Mut male mice with the corresponding MT3 mutation showed instability and premature degradation of the ADAD2 protein, resulting in the spermiogenesis deficiency phenotype. Through ROSI, the Adad2Mut/Mut mice could produce pups with comparable embryonic development (46.7% in Adad2Mut/Mut versus 50% in WT) and birth rates (21.45 ± 10.43% in Adad2Mut/Mut versus 27.5 ± 3.536% in WT, P = 0.5044) to WT mice. The Adad2WT/Mut progeny from ROSI (17 pups in total via three ROSI replicates) did not show overt developmental defects and had normal fertility. N/A. This is a preliminary report suggesting that ROSI can be an effective treatment for infertile Adad2Mut/Mut mice. Further assisted reproductive attempts need to be carefully examined in humans during clinical trials. Our work provides functional evidence that mutations in the ADAD2 gene are deleterious and cause consistent spermiogenic defects in both humans and mice. In addition, preliminary results show that ROSI can help Adad2Mut/Mut to produce biological progeny. These findings provide valuable clues for genetic counselling on the ADAD2 mutants-associated infertility in human males. This work was supported by the National Natural Science Foundation of China (32000587, U21A20204, and 32061143006), and the National Key Research and Developmental Program of China (2019YFA0802600 and 2021YFC2700202). This work was also supported by Institute of Health and Medicine, Hefei Comprehensive National Science Center, Hefei, China. The authors declare no competing interests.

17 Using Photobiomodulation to Improve Bovine Oocyte Maturation

Abstract Oocyte cytoplasmic maturation is critical in determining the success of subsequent embryo development. Bovine oocytes matured in vitro yield less embryonic development rates than in vivo matured oocytes and new strategies are needed to improve the culture system. It has been proposed that light in the red-to-near infrared optical region (~600-1000 nm) enhances cellular metabolic activity through activation of the mitochondrial respiratory chain and has the potential to modify the oocyte metabolome. The objective of this work was to affect the cumulus-oocyte-complex metabolic state using photobiomodulation, aiming to enhance cytoplasmic maturation and subsequent embryonic development. A standardized in vitro fertilization (IVF) protocol and commercial media (IVF Bioscience) were used. Briefly, bovine ovaries were obtained at a local slaughterhouse, cumulus oocyte complexes (COCs) were collected by aspiration of small (2-6 mm) follicles. Compacted COCs were cultured in in vitro maturation media (n = 50/well) and treated with no light (control); light (660-665nm) for 10 minutes at hour 16 from start of maturation (L-16), and light for 10 minutes at hour 20 from start of maturation (L-20). At hour 22 from start of maturation, COCs were fertilized by co-culture with bull spermatozoa (1x106 motile sperm/mL) for 18 hours, then subjected to cumulus cell removal. Presumptive zygotes were placed in in vitro culture media (IVC) for 48 hours when cleavage rate was assessed. Embryos with ≥ 8 cells were selected and placed in fresh IVC until evaluation of blastocyst rate at 168 and 192 hours from start of fertilization. The experiment was replicated 6 times. Blastocysts present at 168 hours from start of fertilization were fixed in 5% formalin, stained with Hoechst 33342, imaged with Cytation 1 (Agilent Technologies Inc.), and blastocyst cell number was counted using ImageJ software (ImageJ2 2.9.0) in 4 replicates. For statistical analysis, each well was considered as experimental unit in a model of repeated measures, main effects of time and treatment, blocked by replicate (Mixed procedure, SAS Institute Inc.). One-way ANOVA test was used to analyze the average cell number per well by group, blocked by replicate (JMP Pro 16.1.0). There was a significant (P &amp;lt; 0.05) effect of treatment, replicate, and time for embryo development and there was a tendency (P = 0.6) for treatment effect on blastocyst cell number. Cleavage rates were 81.3 ± 2.5; 88.5 ± 2.5; 87.3 ± 2.5 % (LSM ± SEM), blastocyst rates were 49.1± 2.5; 49.3 ± 2.5; 51.6 ± 2.5 % and cell numbers were 159.9 ± 3.5; 149.3 ± 3.5; 164.0 ±3.5 for groups control, L-16, and L-20 respectively. The positive effect of photobiomodulation treatment on cleavage rate and blastocyst cell number suggests there is a window of sensitivity to 660-665nm light between 16 and 20 hours from start of maturation.

LPS Administration during Fertilization Affects Epigenetic Inheritance during Embryonic Development.

Intrauterine inflammation can cause infertility by disrupting reproductive function. The pathogenesis underlying this process may primarily involve endotoxins from lipopolysaccharides (LPS), which are produced by Gram-negative bacteria. However, the long-term effects of endotoxins in mammalian pregnancy following LPS exposure during fertilization have not been clarified. In this study, we performed experiments to analyze the influence of LPS on early embryonic development and fetal development in mice. Mice uteruses were examined for the expression of genes related to the inflammatory response. The expression of Il-1β and Il-6 increased following the administration of 200 and 1000 µg/kg LPS. Exposure to LPS using in vitro fertilization (IVF) significantly decreased the embryonic developmental rate. A concentration of 100 µg/kg LPS significantly increased the placental weight and fetal crown -rump length (CRL), whereas a concentration of 200 µg/kg LPS significantly decreased the placenta weight and fetal weight in vivo. These findings indicate that maternal LPS during fertilization affects fetal development until the late stage of pregnancy. Thus, maternal endotoxins may affect epigenetic inheritance during embryonic development from the early to late stages of pregnancy.