Oocyte Activation Research Articles

Artificial oocyte activation with Ca2+ ionophore improves reproductive outcomes in patients with fertilization failure and poor embryo development in previous ICSI cycles.

Does artificial oocyte activation (AOA) by a calcium ionophore (ionomycin) improve the previous fertilization failure or poor embryo development of intracytoplasmic sperm injection (ICSI) account for male factor infertility or other infertility causes? This retrospective study involved 114 patients receiving ICSI-AOA in Shanghai First Maternity and Infant Hospital with previous ICSI fertilization failure or poor embryo development. The previous ICSI cycles of the same patients without AOA served as the control group. The fertilization rates, cleavage rates, transferable embryo rates and blastocyst formation rates of the two groups were compared. Additionally, the clinical pregnancy, implantation rate and live birth rates were also compared to assess the efficiency and safety of AOA. Furthermore, two subgroup analyses were performed in this study based on the cause of infertility and the reason for AOA. The fertilization rate, embryonic development potential and clinical outcome were compared among groups. Among 114 ICSI-AOA cycles, the fertilization rate, top-quality embryo rate, implantation rate, clinical pregnancy per patient and live birth rate per patient were improved significantly compared with previous ICSI cycles (p<0.05 to P< 0.001), and the miscarriage rate in the AOA group was significantly lower than that of the control group (p<0.001). In the AOA subgroups based on the cause of infertility, the fertilization rates of each subgroup were significantly improved compared with previous control cycles except for the mixed factor infertility subgroup (p<0.05 to p<0.001). In the AOA subgroups based on the reason for AOA, the fertilization rates of each subgroup were significantly increased compared with those in their previous ICSI cycle without AOA (p<0.001); however, there was no significant difference in the top-quality embryo rate. No significant improvement was found in the implantation rates and the clinical pregnancy rate in each subgroup except for the poor embryo development subgroup. In the 114 AOA cycles, 35 healthy infants (21 singletons and 7 twins) were delivered without major congenital birth defects or malformations. This study showed that AOA with the calcium ionophore ionomycin can improve the reproductive outcomes of patients with previous fertilization failure and poor embryo development after ICSI.

NLRP14 Safeguards Calcium Homeostasis via Regulating the K27 Ubiquitination of Nclx in Oocyte-to-Embryo Transition.

Sperm-induced Ca2+ rise is critical for driving oocyte activation and subsequent embryonic development, but little is known about how lasting Ca2+ oscillations are regulated. Here it is shown that NLRP14, a maternal effect factor, is essential for keeping Ca2+ oscillations and early embryonic development. Few embryos lacking maternal NLRP14 can develop beyond the 2-cell stage. The impaired developmental potential of Nlrp14-deficient oocytes is mainly caused by disrupted cytoplasmic function and calcium homeostasis due to altered mitochondrial distribution, morphology, and activity since the calcium oscillations and development of Nlrp14-deficient oocytes can be rescued by substitution of whole cytoplasm by spindle transfer. Proteomics analysis reveal that cytoplasmic UHRF1 (ubiquitin-like, containing PHD and RING finger domains 1) is significantly decreased in Nlrp14-deficient oocytes, and Uhrf1-deficient oocytes also show disrupted calcium homeostasis and developmental arrest. Strikingly, it is found that the mitochondrial Na+ /Ca2+ exchanger (NCLX) encoded by Slc8b1 is significantly decreased in the Nlrp14mNull oocyte. Mechanistically, NLRP14 interacts with the NCLX intrinsically disordered regions (IDRs)domain and maintain its stability by regulating the K27-linked ubiquitination. Thus, the study reveals NLRP14 as a crucial player in calcium homeostasis that is important for early embryonic development.

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

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

Possible role of translation in unfertilized sea urchin eggs.

Unfertilized eggs of animals contain maternal messenger RNAs (mRNAs) and proteins, which are required for the maintenance of metabolism and regulation of development during the initial stages of embryogenesis. Unfertilized eggs are transcriptionally and translationally quiescent. After fertilization, activated translation of maternal mRNAs is one of the major forces that direct the early stages of embryogenesis before activation of the zygotic genome. However, a low rate and level of protein synthesis have been detected in unfertilized sea urchin eggs indicating that translation is not completely inhibited. Analysis of translatomes of unfertilized eggs and early embryos detected three sets of maternal mRNAs translated either before or after fertilization, or both before and after fertilization. Proteins encoded by maternal mRNAs, which are translated in unfertilized eggs, perform many different functions required for homeostasis, fertilization, egg activation, and early development. This suggests that translation in unfertilized sea urchin eggs may be required to renew the pool of proteins involved in these processes. Thus, translation may be necessary to maintain the fertility and developmental potential of sea urchin eggs during the long-term storage of eggs in ovaries until spawning begins.

Serotonin Transporter Activity in Mouse Oocytes Is a Positive Indicator of Follicular Growth and Oocyte Maturity.

Serotonin (5-hydroxytryptamine, 5-HT) is known to be a regulator of oocyte maturation in a large number of animal species. In maturing mammalian oocytes, the accumulation of exogenous, maternal serotonin occurs due to the activity of the membrane transporter SERT. In this work, we investigated how SERT activity in oocytes correlates with indicators of follicular selection and oocyte maturity. An immunohistochemical study showed that the difference in the 5-HT intake activity in oocytes does not correlate with the marker of apoptosis in follicular cells, but positively correlates with markers of follicular growth, such as granulosa proliferation and follicle size. Functional analysis of oocytes at different stages of maturation showed that the expression and activity of SERT increases with oocyte maturation. An in vivo experiment on administration of the selective serotonin reuptake inhibitor fluoxetine (20 mg/kg) for 7 days showed a significant decrease in the content of serotonin in both growing GV-oocytes and ovulated mature MII-oocytes. The data obtained clearly indicate that the mechanism of specific membrane transport of serotonin normally ensures the accumulation of serotonin in maturing oocytes, and can be considered as a promising positive marker of their mature status.

Ovariole-specific Yellow-g and Yellow-g2 proteins are required for fecundity and egg chorion rigidity in the red flour beetle, Tribolium castaneum

Most insects reproduce by laying eggs that have an eggshell/chorion secreted by follicle cells, which serves as a protective barrier for developing embryos. Thus, eggshell formation is vital for reproduction. Insect yellow family genes encode for secreted extracellular proteins that perform different, context-dependent functions in different tissues at various stages of development involving, for example, cuticle/eggshell coloration and morphology, molting, courtship behavior and embryo hatching. In this study we investigated the function of two of this family's genes, yellow-g (TcY-g) and yellow-g2 (TcY-g2), on the formation and morphology of the eggshell of the red flour beetle, Tribolium castaneum. Real-time PCR analysis revealed that both TcY-g and TcY-g2 were specifically expressed in the ovarioles of adult females. Loss of function produced by injection of double-stranded RNA (dsRNA) for either TcY-g or TcY-g2 gene resulted in failure of oviposition. There was no effect on maternal survival. Ovaries dissected from those dsRNA-treated females exhibited ovarioles containing not only developing oocytes but also mature eggs in their egg chambers. However, the ovulated eggs were collapsed and ruptured, resulting in swollen lateral oviducts and calyxes. TEM analysis showed that lateral oviducts were filled with electron-dense material, presumably from some cellular content leakage out of the collapsed eggs. In addition, morphological abnormalities in lateral oviduct epithelial cells and the tubular muscle sheath were evident. These results support the hypothesis that both TcY-g and TcY-g2 proteins are required for maintaining the rigidity and integrity of the chorion, which is critical for resistance to mechanical stress and/or rehydration during ovulation and egg activation in the oviducts of T. castaneum. Because Yellow-g and Yellow-g2 are highly conserved among insect species, both genes are potential targets for development of gene-based insect pest population control methods.

Toxicity of microplastics and triclosan, alone and in combination, to the fertilisation success of a broadcast spawning bivalve Tegillarca granosa

Since most marine invertebrates adopted external fertilisation, their fertilisation process is particularly vulnerable to aquatic pollutants. Both antimicrobial ingredients and microplastics (MPs) are ubiquitous in aquatic environments; however, their synergistic effects on the fertilisation of marine invertebrates remain unclear. Therefore, in this study, the fertilisation toxicity of MPs and triclosan (TCS), alone and in combination, was investigated in the broadcast spawning bivalve Tegillarca granosa. Results showed that MPs and TCS significantly suppressed the fertilisation success of T. granosa. As the fertilisation success of broadcast spawning invertebrates depends on successful gamete collisions, gamete fusion, and egg activation, sperm swimming velocity, viability, gamete collision probability, ATP status, and ion-transport enzyme activities were also analysed to further ascertain the underlying toxicity mechanisms. In summary, our findings indicate that the presence of MPs may enhance the fertilisation toxicity of TCS by hampering sperm-egg collision probability, reducing gamete fusion efficiency, and restricting Ca2+ oscillation formation.

The Influence of Storage and Cooking on the Vitamin D Content of 25-Hydroxyvitamin D3-Enriched Eggs

Food fortification is an effective approach to improve vitamin D (VD) concentrations in foods. Eggs are a useful food vehicle for enrichment with VD via its hydroxylated metabolite, 25-hydroxyvitamin D (25-D3), in hen feed. This study determined the impact of time of lay, storage conditions (ambient and refrigeration) and common cooking methods (boiling, frying, scrambling, poaching and microwaving) on the vitamin D metabolite concentration of eggs enriched with 25-D3. Processed samples were freeze-dried and analysed for D3 and 25-D3 using an HPLC-MS(/MS) method. The results indicated that storage and cooking practices influence VD metabolites, with 25-D3 showing true retention of 72–111% and concentrations of 0.67–0.96 µg/100 g of whole egg. Vitamin D3 showed true retention of 50–152% and concentrations of 0.11–0.61 µg/100 g of whole egg. Depending on the storage and method of cooking applied, the calculated total VD activity of enriched eggs ranged from 3.45 to 5.43 µg/100 g of whole egg and was 22–132% higher in comparison to standardised VD content for non-enriched British eggs. The study suggests that 25-D3 is a stable metabolite in eggs following storage and cooking, and that 25-D3-enriched eggs may serve as a potent dietary source of VD.

P-039 Pregnancy outcomes and genetic analysis of globozoospermic patients: A case series and review of the literature

Abstract Study question How should men with globozoospermia be managed in Assisted Reproductive Technologies (ART)? Summary answer IMSI (intracytoplasmic morphologically selected sperm injection), without oocyte activators (OA) allows to obtain a live birth, even when there is a mutation of DPY19L2 gene. What is known already Globozoospermia is a rare syndrome found in less than 0.1% of the infertile population. It is characterized by the presence of spermatozoa with round heads devoid of acrosomes. Two main genes are involved in the transmission of the disease: SPATA 16 and DPY19L2. Only Assisted Reproductive Technologies (ART) such as ICSI (intra-cytoplasmic sperm injection) and IMSI, with or without OA allow these couples to obtain pregnancies. There are no live births described after IMSI without OA in patients with a DPY19L2 mutation. Study design, size, duration Four infertile couples followed in the Department of ART at the Poissy Hospital Center, were studied between 2011 and 2022.All patients had total globozoospermia. Three patients were tested for the DPY19L2 gene mutation. Patients were managed in ART with the IMSI technique without the use of OA. Participants/materials, setting, methods The first patient had a homozygous mutation of the DPY19L2 gene. Two live births were obtained after IMSI. The second patient was not DPY19L2 mutated. 2 IMSI were performed, resulting in a biochemical pregnancy. The third patient had a homozygous mutation of the DPY19L2 gene. An IMSI was performed without obtaining a pregnancy, a second attempt is scheduled soon. The fourth patient refused genetic testing and IMSI management. Sperm donation was performed without achieving pregnancy. Main results and the role of chance In this case series, we present the management of four globozoosperm couples in the Reproductive Biology and Andrology Department at the Poissy Hospital Center. We also present the first two live births after performing an IMSI, without OA, in a globozoospermic patient with a homozygous mutation of DPY19L2 gene. In fact, according to our review of the literature, 53 case reports or articles of patients having benefited from ART with or without OA have been reported in globozoospermic patients. In these couples, 60 children were born. However, no live births had been reported until now in couples managed in IMSI without OA, where men had globozoospermia with DPY19L2 mutation. Limitations, reasons for caution The use of OA to increase the membrane permeability of oocyte to calcium and increase fertilization rates. The highest number of live births in the globozoosperm population occurs after ICSI or IMSI with OA. There is no described live birth without the use of OA in a DPY19L2 mutated patient. Wider implications of the findings The use of OA remains debated and is not authorized in some countries. Although our study found low fertilization rates after IMSI, it’s possible to obtain live births. The selection of spermatozoa with an acrosome outline at high magnification is thus an interesting alternative to the use of oocyte activators. Trial registration number not applicable

P-260 A more physiological approach to support oocyte activation in cases with fertilization failure

Abstract Study question Can we use a physiological compound to trigger oocyte activation in couples undergoing ICSI with history of fertilization failure due to sperm activating factor deficiency? Summary answer In a pilot sibling oocyte study, oocyte activation using recombinant human phospholipase C-zeta (rhPLCζ) yielded a comparable fertilization compared to conventional method using ionomycin. What is known already ICSI overcomes most forms of male gamete defects, however partial or total fertilization failure still occurs regardless of adequate semen parameters and optimal oocyte maturity. This has been attributed to the lack of a cytosolic factor identified as PLCζ. To address this, various approaches of assisted oocyte activation have been proposed, including gamete exposure to chemical or electrical stimuli. The most popular approach remains to be ionomycin. Despite its apparent safety, this stimulus is unnatural and may be aggressive on the oocytes. Therefore, we propose a more physiological approach by utilizing a recombinant form of this specific enzyme. Study design, size, duration In the past 2 months, couples with poor fertilization at their initial ICSI cycles were identified and counseled for assisted oocyte activation (AOA) in the subsequent cycle after confirmation of a PLCζ deficiency. Oocytes were separated into 2 equal cohorts activated either by ionomycin or co-injection with recombinant human rhPLCζ during ICSI. Fertilization rates between ionomycin-activated and rhPLCζ-activated cohorts were compared, as well as with the couples’ history cycles. Participants/materials, setting, methods Six couples with suboptimal fertilization outcomes consented for the study (IRB 0712009553) and were offered AOA with their subsequent ICSI cycles. Conventional AOA was carried out by exposing post-ICSI oocytes to 50 mM ionomycin. The rhPLCζ had a confirmed &amp;gt;95% purity. Our physiological rhPLCζ-activation method was performed by co-injecting spermatozoa with 0.4pL of a rhPLCζ (5mg/mL in glycerol) during ICSI. Main results and the role of chance Couples (n = 6, female age: 36.2 ± 6yrs, male age: 38.0 ± 7yrs) had negative infertility workups with normal semen parameters (3.3 ± 3 mL volume, 42.1 ± 35 x106/mL concentration, 30.8 ± 23% motility, 2.0 ± 1% normal morphology). They underwent 7 unsuccessful ICSI cycles with 20.0% (12/60) fertilization rate and yielded a 91.7% cleavage rate, of those, 3 were transferred at cleavage stage but did not yield clinical pregnancy. A total of 2 blastocysts were obtained but were all aneuploid. In their subsequent treatment cycles, overall AGT achieved a significantly improved fertilization rate of 51.6% (32/62) (P&amp;lt;0.001). We then compared the efficiency between the two AGT methods in equally distributed sibling oocyte cohort. Although mathematically insignificant, we observed a trend that rhPLCζ-activation outperforms ionomycin-activation in total activation (67.9% vs 50.0%) as well as fertilization (57.1% vs 47.0%). The new rhPLCζ-activation method yielded a comparable embryo cleavage rate at 87.5% compared to the conventional method at 81.3%. Although fresh embryo transfer at cleavage stage did not yield pregnancy rate yet, rhPLCζ-activated cohort had one euploid blastocyst while the ionomycin-activated cohort did not yield euploid blastocyst. Limitations, reasons for caution While we observed that a more physiological rhPLCζ-activation yields a comparable fertilization compared to the conventional ionomycin method, the study is preliminary, and a confirmatory clinical outcome is not yet available. Wider implications of the findings This is the first attempt to use recombinant sperm-specific protein to mimic physiological oocyte activation for patient treatment. If this data is confirmed in a larger series, this may represent a way to normalize fertilization in cases of sperm activating factor deficiency even in different sources of spermatozoa. Trial registration number N/A

P-740 Monogenic and digenic variants in male PLCZ1, ACTL7A and ACTL9 genes cause fertilization failure after ICSI

Abstract Study question What is the frequency of PLCZ1, ACTL7A and ACTL9 variants in male patients suffering from fertilization failure (FF) after ICSI? Summary answer Male patients with FF after ICSI exhibit variants in PLCZ1 (27,3%), ACTL7A (13,6%) and ACTL9 (4,5%). Some patients present PLCZ1 variants combined with ACTL7A/ACTL9 variants. What is known already FF after ICSI has been often attributed to phospholipase C zeta (PLCζ) deficiency, and consequently to insufficient calcium release, necessary for fertilization. PLCZ1 genetic variants have been detected in one-third of males with FF. Recently, actin-like 7A (ACTL7A) and 9 (ACTL9) variants were also identified, but larger cohort studies are lacking. ACTL7A/ACTL9 variants alter the acrosome structure, resulting in reduced and abnormally localized PLCζ. Assisted oocyte activation (AOA), by calcium ionophore, restores fertilization in patients with PLCZ1 variants, and could also benefit patients with ACTL7A/ACTL9 defects. This study aimed to expand the spectrum of known PLCZ1, ACTL7A and ACTL9 variants. Study design, size, duration A prospective study was conducted from June 2020 to December 2022 including 22 male patients (P1-P22) with FF after ICSI. Patients were only recruited when they had a mean fertilization rate ≤33,33% in at least 1 ICSI cycle. All patients donated a saliva sample for genetic screening of PLCZ1, ACTL7A and ACTL9 genes and a sperm sample for the mouse oocyte activating test (MOAT). All couples with FF after ICSI were offered an AOA treatment. Participants/materials, setting, methods Genetic screening was performed via next-generation sequencing. Identified variants were classified employing Varsome and confirmed by bidirectional Sanger sequencing. Patients in which uncertain significance (VUS), likely pathogenic or pathogenic variants were found underwent additional diagnostic tests, such as the study of the sperm calcium releasing pattern in mouse (MOCA) and in in vitro matured (IVM) human (HOCA) oocytes, immunostaining of PLCζ and ACTL7A, and the analysis of the sperm morphology by transmission electron microscopy (TEM). Main results and the role of chance Genetic screening revealed heterozygous PLCZ1 variants, a novel variant p.Ile379Thr in P10 and the previously published variant p.His233Leu in three patients (P9, P19 and P20). In addition, two novel homozygous ACTL7A variants were detected, p.Ser364GlnfsTer9 in P1 and p.Gly214Ser in P22. Interestingly, digenic variants were observed in P6 (PLCZ1 p.Ile74Thr and ACTL7A p.Tyr183His) and in P8 (PLCZ1 p.His233Leu and ACTL9 p.Arg271Pro). All patients with identified variants showed a high oocyte activation rate (&amp;gt;70%) after MOAT, except P1 and P22 which activation rate was &amp;lt;25%, indicating a more detrimental sperm-related deficiency. While MOCA only detected deficient calcium release in P1 (and not for P6, P8, P9 and P10), HOCA revealed absence of calcium oscillations in all patients analyzed (P1, P8, P9 and P10), except for P6. Immunostaining experiments in P1 revealed no ACTL7A signal and decreased PLCζ signal in patient sperm. Moreover, TEM analysis in P1 confirmed acrosome detachment from nuclear membrane. Overall, AOA increased fertilization rate (from 7,23% to 53,04%) and pregnancy rate (from 7,14% to 46,15%) in patients with identified variants. Specifically, patients with monogenic PLCZ1 (P9 and P19) and ACTL7A (P1) variants, as well as digenic variants (P6 and P8) achieved a live birth after AOA. Limitations, reasons for caution Some patients have not undergone all diagnostic tests yet, thus functional data on all the found variants identified is still ongoing. In addition, patient recruitment for this study is continuing. Genetic screening was carried on exonic and flanking intronic regions, which might have missed additional variants in other intronic regions. Wider implications of the findings The MOAT/MOCA tests cannot detect some subtle sperm-related activation deficiencies. HOCA represents a more sensitive analysis but requires human oocytes. Therefore, genetic screening of PLCZ1, ACTL7A and ACLTL9 could be a faster and more cost-efficient diagnostic test for FF after ICSI. Furthermore, AOA treatment is very efficient for these patients. Trial registration number NA

P-074 In-vitro vitamin D treatment on human sperm improves sperm motility, capacitation, and oocyte activation

Abstract Study question Does in-vitro vitamin D treatment on fertile and infertile human sperm improves sperm motility and alter protein markers involved in capacitation and oocyte-activation. Summary answer In-vitro vitamin D treatment enhance functionality of sperm by increase intracellular calcium ion, thus increasing sperm motility and capacitation status. What is known already Many studies have been done to investigate the relationship between vitamin D and male infertility. It is known that low serum vitamin D in human contribute to male infertility. However, the effect of in-vitro vitamin D treatment on sperm remains unclear, particularly on the effect of vitamin D following ejaculation into female reproductive tract. This includes processes involved in fertilisation such as capacitation, hyperpolarization, oocyte activation and sperm-oocyte fusion. Therefore, we examined whether in-vitro vitamin D treatment on sperm can improve the sperm parameters, hence will then improve fertilisation and pregnancy. Study design, size, duration Randomised controlled trial with prospective interventions (in-vitro vitamin D treatment). Based on power analysis, 40 subjects both fertile and infertile men who came for semen analysis were recruited at University Malaya Medical Centre from January 2022 to January 2023. All men were consented to participate in this study. Each washed semen sample were aliquoted to serve as control and receive in-vitro vitamin D treatment. Participants/materials, setting, methods Total of 40 samples were included for final analysis. All semen samples were washed and treated with 1nM vitamin D in-vitro for 45 minutes at 37ºC. Motility, intracellular calcium released, capacitation status, mitochondria membrane potential and other basic semen parameters were measured after in-vitro vitamin D treatment for fertile and infertile samples. Furthermore, immunofluorescence of sperm origin oocyte activation factor, Phospholipase C Zeta (PLCζ), was done to investigate fluorescence intensity per cell using Image J. Main results and the role of chance In both fertile and infertile sample group, total motility is increased after in-vitro vitamin D treatment (MD = 8.727%, P &amp;lt; 0.001). Fluo-4 AM stain shows there are increase of intracellular calcium release after in-vitro vitamin D, with mean difference of Corrected Total Cell Fluorescent (CTCF) for Fluo-4 AM (MD = 25043, P = 0.0228. Chlortetracyclin (CTC) test for capacitation status and immunofluorescence of phosphotyrosine shows there are increase CTCF (MD = 0.02595, P = 0.0142) after in-vitro vitamin D treatment. Staining of JC-1 shows there are more hyperpolarised mitochondria membrane in in-vitro vitamin D treatment group compare to the control group. Ratio of red to green fluorescent is 0.639 in control group and 2.22 in in-vitro vitamin D treatment group. Moreover, immunofluorescence of sperm origin oocyte activation factor, Phospholipase C Zeta (PLCζ) show in-vitro vitamin D improve the expression of these protein. Mean difference of CTCF between control and vitamin D treatment group for PLCζ is 8.013x 10-3, P = 0.0226. Thus, in-vitro vitamin D enhance functionality of sperm by increase intracellular calcium ion, thus increasing sperm capacitation status and sperm membrane hyperpolarization. Limitations, reasons for caution Although power analysis is used to calculate sample size, however bigger sample size always needed to increase the accuracy. Moreover, patients’ sample are examined and treated after 3 hours due to limited collection room in the facility. Wider implications of the findings Vitamin D can be used as a tool in in-vitro fertilisation (IVF) procedure and can be added to sperm preparation medium due to its implication in increasing capacitation and hyperpolarised sperm membrane to increase chance of successful IVF. Trial registration number not applicable

P-218 Does Artificial Oocyte Activation (AOA) improve the outcome beyond fertilization rate in ICSI cycles?

Abstract Study question Does CultActive© improve reproductive outcomes of ICSI cycles in cases of low fertilization rate and normal fertilization rate? Summary answer The application of CultActive© after ICSI improves fertilization rates only in clinical indication of fertilization failure. What is known already Oocyte activation deficiency is attributed in the majority of cases to fertilization failure in ICSI cycles, and these can be corrected by increasing initial levels of calcium (Ca2+) using assisted oocyte activation techniques (AOA), such as the use of Ca2+ ionophores. These agents enhance intracellular calcium release and increase the membrane permeability facilitating the influx of extracellular Ca2+, which initiates the activation cascade. Previous results suggest that Ca2+ ionophore treatment can increase the live birth rate after failed ICSI cycles in couples with poor fertilization rates, embryo developmental arrest, and also increase the number of good quality embryos. Study design, size, duration This was a retrospective observational study. There were 30 patients included and the first group consisted of 30 ICSI cycles, 231 oocytes and 56 embryos (without AOA). The second group consisted of the same patients (n = 30) with 30 ICSI cycles using AOA, 219 oocytes and 47 embryos. Subgroups were defined according to clinical indications (normal fertilization rate group: ≥ 65 % and low fertilization rate group: &amp;lt;65% ). All data was collected from January-December 2022. Participants/materials, setting, methods 450 oocytes were assessed in the study. 231 oocytes belonged to the pre-AOA and 219 belonged to the AOA. The oocytes were exposed for 15 minutes after ICSI in a solution containing the Ca2+ ionophore A23187, CultActive© (Gynemed, Germany). Fertilization, good quality blastocyst formation and pregnancy rate were analyzed.Good quality blastocyst were defined as ≥ 3BB. Statistical significance was analyzed using the Paired Student T test (p &amp;lt; 0.05 was considered statistically significant). Main results and the role of chance The cohort of patients with normal fertilization rate (without clinical indications),was not significantly different. The AOA treatment gave a fertilization rate of 61% compared to 71% of the control cycles (p = 0.1398). There was no increase in the number of good quality blastocysts formation when the study group was compared to the control (p = 0.1660). In the cohort of patients with an indication of low fertilization, a significantly higher fertilization rate was recorded compared to the control (51% and 37%, respectively p = 0.0238). There were no significant difference in the quantity of good quality blastocysts compared with the control (p = 0.3328). Regardless of normal or low fertilization rates there was no significant difference in pregnancy rate when CultActive© was used in treatment (p &amp;gt; 0.05). Limitations, reasons for caution There are limitations in our study. First, the retrospective design and limited study population are major limitations. The data should be interpreted cautiously in the subgroup analyses due to potential bias, specifically the small population in the group without clinical indication. Wider implications of the findings AOA was only beneficial for couples with poor fertilization. Embryo quality and pregnancy rate did not improve with CultActiveive©. External variation was minimized by using the same patients in each cohort. This suggests AOA should not be used without clinical indication or for poor embryo quality/blastulation rate. Trial registration number not applicable

O-101 Add-ons in the ART lab

Abstract Study question Which laboratory based add-ons are effective and safe to use in ART treatment? Summary answer In total, more than thirty recommendations were formulated on the use of add-ons, with about a third being laboratory based tests or interventions What is known already Treatment add-ons are defined as not essential for the treatment, but optional additional treatments that are sometimes offered on top of routine fertility treatment. There are a wide range of treatment add-ons available, including diagnostic tests, drugs, equipment, holistic or alternative therapies, laboratory and surgical interventions, all claiming to potentiate the effect of the treatment and/or to lessen side effects, improve pregnancy or live birth rate, reduce the risk of miscarriage or shorten the time to pregnancy. Add-ons and can be at extra cost (or not), and evidence on efficacy and safety is often missing. Presumably they can be both beneficial or non-beneficial, as there may be absence of evidence to either. Study design, size, duration The ESHRE working group for good practice recommendations on add-ons in ART has produced recommendations useful for professionals, patients and policy makers. More than 30 add-ons were identified, and evaluated for efficiency and safety, and wherever possible also for cost. The literature was identified in a systematic search, reviewed and critically appraised. In the absence of any clear scientific evidence, judgement was based on the professional experience and consensus of the multidisciplinary development group. The guidelines are thus based on the best available evidence and expert agreement. Prior to publication, the guidelines were reviewed by independent international reviewers. Participants/materials, setting, methods Interventions were characterized in three sections: 1) Diagnosis and diagnostic tests, 2) Laboratory tests and intervention, and 3) Clinical management. Outcomes were efficacy (pregnancy or live birth rate), risk of miscarriage, time to pregnancy, cost, and safety/risk aspects. Some examples of add-ons in the group of laboratory tests and interventions are oocyte and sperm activation, mitochondria load and transfer, sperm selection, assisted hatching, genetic testing and addition of hyaluronic acid to media. Main results and the role of chance The guideline development group formulated more than 30 recommendations. This lecture will focus on the rationale and the recommendations of the laboratory add-ons currently used in fertility clinics. We will discuss the potential beneficial effects on the IVF treatment, its efficacy, and its safety. While most add-ons are not recommended for the general population of ART patients, where specific patient populations might benefit, indications were formulated. The guideline has been in stakeholder review and is prepared for submission in 2023. Limitations, reason for caution Of the recommendations, none are based on high quality evidence and only a few are based on moderate quality evidence. Hence 90% of the recommendations are supported only by low quality RCTs, observational data, professional experience, or consensus of the development group. Wider implications of the findings These recommendations aim to provide guidance to health care professionals and laboratory experts in the field of reproductive medicine, to help towards safe and efficient ART treatments for patients where they can participate in informed decision making with realistic expectations and be fully informed about their chances to pregnancy and live birth. Study funding and competing interest(s) All costs relating to the development process were covered from ESHRE funds. There was no external funding of the development process or manuscript production.

P-027 Absence of perinuclear theca ACTRT1 protein induces sperm head deformation and primary male infertility in humans

Abstract Study question Given that Actrt1 knockout induces teratospermia, fertilization failure and severe male infertility in mice, will ACTRT1 deficiency affect sperm morphology and male fertility in humans? Summary answer The ∼110 kb microdeletion of X chromosome only including ACTRT1 gene was identified inducing sperm head deformation and fertilization failure in an infertile Chinese man. What is known already The actin-related proteins, ACTRT1, ACTRT2, ACTL7A, and ACTL9, interact to form a multimeric complex in the sperm subacrosomal region and is vital for the attachment of acrosome to nucleus. The disruption of ACTL7A or ACTL9 have been identified to cause acrosomal detachment, total fertilization failure and male infertility in humans and mice. Actrt1-knockout mice are severely subfertile also because of malformed sperm heads with detached acrosomes and a partial deficiency in fertilization. Loss/reduced expression and/or abnormal localization of PLCζ, a well-known sperm-borne activating factor, are identified in ACTL7A/ACTL9 mutant patients as well as Actrt1-, Actl7a-, and Actl9-deficient mice. Study design, size, duration We recruited a cohort of 85 infertile men with teratospermia which is characterized by deformed sperm heads in the center for reproductive medicine from August 2019 to June 2022. Genomic DNA (gDNA) of patients was extracted from peripheral blood, then whole-exome sequencing and in silico analyses were performed to identify gene mutations. Morphological analysis, functional assays, and assisted reproductive therapy were performed in 2022. Participants/materials, setting, methods The ACTRT1 deficiency was identified by whole-exome sequencing and confirmed by whole-genome sequencing, PCR and qPCR. Family members’ gDNA was collected to define the hereditary mode. Papanicolaou-staining, scanning and transmission electronic microscopy were performed to reveal sperm morphologies. Western blot and immunostaining of spermatozoa were conducted to explore the pathological mechanism of the ACTRT1 deficiency. Intracytoplasmic sperm injection (ICSI) combined with artificial oocyte activation (AOA) was applied for the assisted reproductive therapy of ACTRT1-deletion patient. Main results and the role of chance We identified a whole-gene deletion mutation of ACTRT1 on Chromosome X in an infertile male with teratospermia, which was inherited from his mother. Papanicolaou-staining, scanning electronic microscopy, and transmission electronic microscopy showed the sperm head deformation owing to acrosome detachment, which mimicked the previously reported phenotype of Actrt1-knockout mice. The results of western blot and immunostaining suggested that ACTRT1 deficiency induced a down-regulated expression of ACTL7A and PLCζ proteins, but not ACTRT2, in human sperm samples. The detached acrosomes induced fertilization failure in assisted reproductive therapy of both Actrt1-knockout mice and the ACTRT1-deletion patient, which could be effectively rescued by ICSI combined with AOA. Limitations, reasons for caution Additional cases are needed to confirm the genetic contribution of ACTRT1 mutations to male infertility with teratospermia and fertilization failure. In addition, the proband with ACTRT1 deletion was with severe oligozoospermia and mild asthenozoospermia. The effect of ACTRT1 mutations on sperm count and motility still need more investigation. Wider implications of the findings Our results reveal a gene–disease relationship between ACTRT1 deficiency and human male infertility owing to teratospermia and fertilization failure. This report also describe a good fertility outcome of assisted reproductive therapy with ICSI and AOA for the ACTRT1-deficient patient. Trial registration number not applicable

P-058 Defects in phospholipase C zeta cause polyspermy and low fertilization after conventional IVF: not just ICSI failure

Abstract Study question How does sperm phospholipase C zeta (PLCζ) affect fertilization in conventional in vitro fertilization (cIVF)? Summary answer Absence of PLCζ causes polyspermy, and a decline in the proportion of sperm expressing PLCζ is correlated with a low fertilization rate (FR) after cIVF. What is known already PLCζ is a key sperm-borne factor that triggers Ca2+ oscillations and the subsequent oocyte activation following gamete fusion. Mutations in PLCZ1, the gene encoding PLCζ, cause male infertility and intra-cytoplasmic sperm injection (ICSI) fertilization failure; and PLCζ expression and localization patterns are significantly correlated with ICSI FR. However, few studies have been published on the relationship between PLCζ and cIVF, an insemination procedure involving several key events that are bypassed in ICSI, e.g., sperm-zona binding and penetration, as well as polyspermy blocking. It is possible that sperm PLCζ may affect fertilization in a different manner from that in ICSI. Study design, size, duration Sixty couples who underwent cIVF treatments were recruited from the Reproductive and Genetic Hospital of CITIC-Xiangya between February 2019 and January 2022. Participants/materials, setting, methods We performed whole-exome sequencing in two unrelated males who exhibited infertility and polyspermy in cIVF. Immunofluorescence staining and oocyte-activation testing were employed to evaluate the effects of the candidate variants on PLCζ protein. Subsequent PLCζ analysis was performed in additional 58 males who underwent cIVF. The Pearson correlation coefficient was performed to evaluate the relationships between PLCζ and FRs. The receiver operating characteristic curve and Youden’s index analysis were used to indicate the cutoff value. Main results and the role of chance We identified one previously reported and two novel PLCZ1 variants in two males that were associated with male infertility and polyspermy characterized by excessive sperm–zona binding and a delay in pronuclear (PN) formation. In vitro functional analyses revealed that virtually all sperm from patients lacked functional PLCζ and were thus unable to evoke the physiologic pattern of Ca2+ oscillations. ICSI with an artificial oocyte-activation treatment successfully rescued the polyspermic phenotype and resulted in a live birth. Subsequent PLCζ analysis were performed in additional 58 males, who were allocated to three groups according to fertilization outcomes: a multiple pronuclear (MPN) group (no. of PN ≥ 3, MPN rate ≥ 50%, n = 9) and low fertilization (LF, total FR ≤ 30%, n = 18) and normal fertilization groups (NF, 2PN ≥ 50%, 1PN + MPN &amp;lt; 30%, n = 31). We found that the proportion of sperm that expressed PLCζ was positively correlated with both 2PN rate (P = 0.0005) and total FR (P = 0.0008) after cIVF, and that the optimal cutoff value below which males were likely to experience low FR (total FR ≤ 30%) after cIVF was 56.7% (area under the ROC curve, 0.705; P = 0.0176). Limitations, reasons for caution Precise mechanisms that underly the roles of PLCζ in cIVF outcomes are still uncovered. Follow-up studies with larger numbers of patients are required to validate the sensitivity, specificity, and accuracy of PLCζ as a diagnostic biomarker in cIVF outcomes. Wider implications of the findings Our study identified PLCZ1 as a novel causative gene of polyspermy in humans,highlighting the potential value of PLCζ as a fertility marker for cIVF outcomes. Trial registration number nor applicable

Protective effect of onion peel extract on ageing mouse oocytes.

Mammalian oocytes not fertilized immediately after ovulation can undergo ageing and a rapid decline in quality. The addition of antioxidants can be an efficient approach to delaying the oocyte ageing process. Onion peel extract (OPE) contains quercetin and other flavonoids with natural antioxidant activities. In this study, we investigated the effect of OPE on mouse oocyte ageing and its mechanism of action. The oocytes were aged in vitro in M16 medium for 16 h after adding OPE at different concentrations (0, 50, 100, 200, and 500 μg/ml). The addition of 100 μg/ml OPE reduced the oocyte fragmentation rate, decreased the reactive oxygen species (ROS) level, increased the glutathione (GSH) level, and improved the mitochondrial membrane potential compared with the control group. The addition of OPE also increased the expression of SOD1, CAT, and GPX3 genes, and the caspase-3 activity in OPE-treated aged oocytes was significantly lower than that in untreated aged oocytes and similar to that in fresh oocytes. These results indicated that OPE delayed mouse oocyte ageing by reducing oxidative stress and apoptosis and enhancing mitochondrial function.

Quality of fish eggs and production of androgenetic and gynogenetic doubled haploids (DHs).

Induced development of haploid embryos (H) with only paternal (androgenesis) or maternal (gynogenesis) chromosomes requires irradiation of eggs before fertilization or activation of eggs with irradiated spermatozoa, respectively. To provide doubled haploids (DHs), androgenetic and gynogenetic haploid zygotes need to be subjected to the thermal or high hydrostatic pressure (HHP) shock to suppress the first mitotic cleavage and todouble paternal or maternal haploid set of chromosomes. Androgenesis and mitotic gynogenesis (mito-gynogenesis) result in the generation of fully homozygous individuals in a single generation. DHs have been utilized in selective breeding programs, in studies concerning the phenotypic consequences of recessive alleles and to evaluate the impact of sex chromosomes on the early ontogeny. Moreover, the use of DHs for the NGS approach radically improves de novo the assembly of the genomes. However, reduced survival of the doubled haploids limits the wide application of androgenotes and gynogenotes. The high mortality of DHs may be only partly explained by the expression of recessive traits. Observed inter-clutch variation in the survival of DHs developing in eggs originating from different females make it necessary to take a closer look at the quality of the eggs used during induced androgenesis and gynogenesis. Moreover, the developmental competence of eggs that are subjected to irradiation before fertilization in order to deactivate maternal chromosomes when undergoing induced androgenesis and exposed to the physical shock after fertilization that leads to the duplication of the zygotes in both mito-gynogenesis and androgenesis may be also altered as irradiation and sublethal values of temperatures and hydrostatic pressure are considered as harmful for the cell organelles and biomolecules. Here, recently provided results concerning the morphological, biochemical, genomic, and transcriptomic characteristics of fish eggs showing high and low competence for androgenesis and mito-gynogenesis are reviewed.

Advances in the study of genetic factors and clinical interventions for fertilization failure.

Fertilization failure refers to the failure in the pronucleus formation, evaluating 16-18 h post in vitro fertilization or intracytoplasmic sperm injection. It can be caused by sperm, oocytes, and sperm-oocyte interaction and lead to great financial and physical stress to the patients. Recent advancements in genetics, molecular biology, and clinical-assisted reproductive technology have greatly enhanced research into the causes and treatment of fertilization failure. Here, we review the causes that have been reported to lead to fertilization failure in fertilization processes, including the sperm acrosome reaction, penetration of the cumulus and zona pellucida, recognition and fusion of the sperm and oocyte membranes, oocyte activation, and pronucleus formation. Additionally, we summarize the progress of corresponding treatment methods of fertilization failure. This review will provide the latest research advances in the genetic aspects of fertilization failure and will benefit both researchers and clinical practitioners in reproduction and genetics.

Yolk granule fusion and microtubule aster formation regulate cortical granule translocation and exocytosis in zebrafish oocytes.

Dynamic reorganization of the cytoplasm is key to many core cellular processes, such as cell division, cell migration, and cell polarization. Cytoskeletal rearrangements are thought to constitute the main drivers of cytoplasmic flows and reorganization. In contrast, remarkably little is known about how dynamic changes in size and shape of cell organelles affect cytoplasmic organization. Here, we show that within the maturing zebrafish oocyte, the surface localization of exocytosis-competent cortical granules (Cgs) upon germinal vesicle breakdown (GVBD) is achieved by the combined activities of yolk granule (Yg) fusion and microtubule aster formation and translocation. We find that Cgs are moved towards the oocyte surface through radially outward cytoplasmic flows induced by Ygs fusing and compacting towards the oocyte center in response to GVBD. We further show that vesicles decorated with the small Rab GTPase Rab11, a master regulator of vesicular trafficking and exocytosis, accumulate together with Cgs at the oocyte surface. This accumulation is achieved by Rab11-positive vesicles being transported by acentrosomal microtubule asters, the formation of which is induced by the release of CyclinB/Cdk1 upon GVBD, and which display a net movement towards the oocyte surface by preferentially binding to the oocyte actin cortex. We finally demonstrate that the decoration of Cgs by Rab11 at the oocyte surface is needed for Cg exocytosis and subsequent chorion elevation, a process central in egg activation. Collectively, these findings unravel a yet unrecognized role of organelle fusion, functioning together with cytoskeletal rearrangements, in orchestrating cytoplasmic organization during oocyte maturation.