Mean Fertilization Rate Research Articles

High rate of detected variants in male PLCZ1 and ACTL7A genes causing failed fertilization after ICSI.

What is the frequency of PLCZ1, ACTL7A, and ACTL9 variants in male patients showing fertilization failure after ICSI, and how effective is assisted oocyte activation (AOA) for them? Male patients with fertilization failure after ICSI manifest variants in PLCZ1 (29.09%), ACTL7A (14.81%), and ACTL9 (3.70%), which can be efficiently overcome by AOA treatment with ionomycin. Genetic variants in PLCZ1, and more recently, in ACTL7A, and ACTL9 male genes, have been associated with total fertilization failure or low fertilization after ICSI. A larger patient cohort is required to understand the frequency at which these variants occur, and to assess their effect on the calcium ion (Ca2+) release during oocyte activation. AOA, using ionomycin, can restore fertilization and pregnancy rates in patients with PLCZ1 variants, but it remains unknown how efficient this is for patients with ACTL7A and ACTL9 variants. This prospective study involved two patient cohorts. In the first setting, group 1 (N = 28, 2006-2020) underwent only PLCZ1 genetic screening, while group 2 (N = 27, 2020-2023) underwent PLCZ1, ACTL7A, and ACTL9 genetic screening. Patients were only recruited when they had a mean fertilization rate of ≤33.33% in at least one ICSI cycle with at least four MII oocytes. Patients underwent a mouse oocyte activation test (MOAT) and at least one ICSI-AOA cycle using calcium chloride (CaCl2) injection and double ionomycin exposure at our centre. All patients donated a saliva sample for genetic screening and a sperm sample for further diagnostic tests, including Ca2+ imaging. Genetic screening was performed via targeted next-generation sequencing. Identified variants were classified by applying the revised ACMG guidelines into a Bayesian framework and were confirmed by bidirectional Sanger sequencing. If variants of uncertain significance or likely pathogenic or pathogenic variants were found, patients underwent additional determination of the sperm Ca2+-releasing pattern in mouse (MOCA) and in IVM human (HOCA) oocytes. Additionally, ACTL7A immunofluorescence and acrosome ultrastructure analyses by transmission electron microscopy (TEM) were performed for patients with ACTL7A and/or ACTL9 variants. Overall, the frequency rate of PLCZ1 variants was 29.09%. Moreover, 14.81% of patients carried ACTL7A variants and 3.70% carried ACTL9 variants. Seven different PLCZ1 variants were identified (p.Ile74Thr, p.Gln94*, p.Arg141His, p.His233Leu, p.Lys322*, p.Ile379Thr, and p.Ser500Leu), five of which are novel. Interestingly, PLCZ1 variants p.Ser500Leu and p.His233Leu occurred in 14.55% and 9.09% of cases. Five different variants were found in ACTL7A (p.Tyr183His, p.Gly214Ser, p.Val340Met, p.Ser364Glnfs*9, p.Arg373Cys), four of them being identified for the first time. A novel variant in ACTL9 (p.Arg271Pro) was also described. Notably, both heterozygous and homozygous variants were identified.The MOCA and HOCA tests revealed abnormal or absent Ca2+ release during fertilization in all except one patient, including patients with PLCZ1 heterozygous variants. TEM analysis revealed abnormal acrosome ultrastructure in three patients with ACTL7A variants, but only patients with homozygous ACTL7A variants showed reduced fluorescence intensity in comparison to the control.AOA treatment significantly increased the fertilization rate in the 19 patients with detected variants (from 11.24% after conventional ICSI to 61.80% after ICSI-AOA), as well as positive hCG rate (from 10.64% to 60.00%) and live birth rate (from 6.38% to 37.14%), resulting in 13 healthy newborns. In particular, four live births and two ongoing pregnancies were produced using sperm from patients with ACTL7A variants. Genetic screening included exonic and outflanking intronic regions, which implies that deep intronic variants were missed. In addition, other male genes or possible female-related factors affecting the fertilization process remain to be investigated. Genetic screening of PLCZ1, ACTL7A, and ACTL9 offers a fast, cost-efficient, and easily implementable diagnostic test for total fertilization failure or low fertilization after ICSI, eliminating the need for complex diagnostic tests like MOAT or Ca2+ analysis. Nonetheless, HOCA remains the most sensitive functional test to reveal causality of uncertain significance variants. Interestingly, heterozygous PLCZ1 variants are sufficient to cause inadequate Ca2+ release during ICSI. Most importantly, AOA treatment using CaCl2 injection followed by double ionomycin exposure is highly effective for this patient group, including those with ACTL7A variants, who also display a Ca2+-release deficiency. This study was supported by the Flemish Fund for Scientific Research (FWO) (TBM-project grant T002223N awarded to B.H.) and by the Special Research Fund (BOF) (starting grant BOF.STG.2021.0042.01 awarded to B.H.). A.C.B., R.R.G., C.C., E.V.D.V., A.R., D.S., L.L., P.C., S.S., A.B., and F.V.M. have nothing to disclose. B.H. reports a research grant from FWO and BOF, and reports being a board member of the Belgian Ethical Committee on embryo research. N/A.

Assessment of some egg quality parameters and chorion ultrastructure characterization in Atlantic salmon (Salmo salar)

Farmed salmonid production requires oocytes and embryos of high quality to supply the increasing market demand; achieving this depends primarily on the management of females and gametes. The chorion of the fertilized egg is a crucial structure for embryo protection; however, it is still poorly studied in some fish species. The object of the present study was to evaluate some parameters of oocyte quality and embryonic development in S. salar, and to characterize the chorion at the ultrastructural level in oocytes and embryos at different stages up to hatching by scanning electron microscopy (SEM). The samples (oocytes and embryos) were obtained from five mature females (4 years old, 9.75 ± 0.86 kg) of S. salar. The results show 32% (20%; 64%) of oocytes with yolk clots, a hydration percentage of 17% (11.1%; 23.3%) and a significant increase in embryo hardness at 12 accumulated thermal units (ATU). A mean fertility rate of 77% (60%; 93%) was recorded. The survival rates were 72% (20%; 100%) and 73% (32%; 92%) for 130 and 318 ATU, respectively. The values of symmetrical blastomeres were high, 65% (45%; 80%); however, the rate of spine deformities was also high at 42.4% (22%; 71%). Several significant correlations were observed between the parameters assessed: there were high-positive correlations between the hydration percentage and blastomere symmetry (r = 0.89), between oocyte hardness and spine deformities (r = 0.88), and between embryo hardness at 130 and 318 ATU (r = 0.97); there were also strong correlations between symmetry and survival rate at 130 ATU (r = 0.95) and between symmetry and survival rate at 318 ATU (r = 0.96). In the ultrastructural analysis, the SEM images revealed that the outer layer of the chorion of both oocytes and fertilized eggs, through development to hatching, exhibits a granular pattern. In contrast, the inner layer is composed of numerous interconnected fibres that form a complex network, absent in the hatching area. Both outer and inner layers show numerous pore canals. The pores in the outer layer are covered by plugs, and are larger than those in the inner layer. The information generated in this study is useful to complement understanding of the biology of S. salar eggs; however, given the ultrastructural variability of the chorion, further studies are needed to expand on these findings.

P-060 When is it the sperm’s fault? The incidence of sperm-related poor fertilization after ICSI in 13,859 matched oocyte donation cycles

Abstract Study question What is the true incidence and impact of male factor-related poor fertilization in ICSI cycles when analyzed using matched pairs of high quality donor oocytes? Summary answer In ICSI cycles with donor oocytes, male factor-related poor fertilization occurs in 3.3% of cases and contributes to 86.7% of all poor (<30%) fertilization outcomes. What is known already Opting for oocyte donation is a complex decision for couples, involving significant financial and long-term implications. This decision is typically prompted by presumed female infertility as male fertility diagnostics remain limited. Although ICSI is a highly effective treatment, cases of poor or complete fertilization failure can still occur, even when sperm parameters appear normal. This suggests the presence of unidentified male-related factors. However, the precise incidence of such factors remains poorly understood. This study aims to address this gap, offering a unique perspective, by employing a matched oocyte donation model to investigate the influence of sperm-related factors on fertilization outcomes. Study design, size, duration This retrospective cohort study analyzed 14,928 oocyte donation ICSI cycles, derived from 7,519 ovarian stimulations (OS) of 2,963 unique oocyte donors. The data were from a single center from January 2015 to December 2022. Sibling oocytes from the same OS were utilized for at least two different recipients (n = 13,859), facilitating comparisons under varying paternal conditions. Participants/materials, setting, methods Cases from the same oocyte lot with poor (<30%) and good (>65%) fertilization rates were matched to isolate sperm-related factors influencing fertilization success. Paired t-tests were used to compare outcomes. Ordinary least squares regression was used to isolate variables associated with poor fertilization, such as oocyte status (fresh versus vitrified), sperm origin (partner versus donor) and the presence of severe male factor (defined as a concentration of < 1 million/mL and/or <1% progressive motility). Main results and the role of chance The mean fertilization rate was 73.0% across all cycles analyzed. The incidence of poor fertilization (≤30%) was found to be 3.8% across the entire cohort (532 out of 13,859 cycles). Of these, 86.7% (461/532) cases using the same oocyte lot could be matched to cases with high fertilization rates (>65%) in other recipient cycles (n = 1,441), indicating that poor fertilization was male-related. We observed comparable numbers of inseminated oocytes between matched cases with high and low fertilization (7.23 ± 1.32 and 7.42 ± 1.42, respectively, p = 0.011), while the mean number of fertilized oocytes (1.44 ± 0.77 versus 6.12 ± 1.36, p = <0.001) and mean fertilization rates (20% versus 83%, p < 0.001) differed significantly. Notably, oocyte vitrification and the presence of a severe male factor negatively affected fertilization outcomes (coeff. -0.0898, p < 0.001 and coeff. -0.1500, p < 0.001, respectively). Nevertheless, the majority of poor fertilization cases (92.7%) occurred in the absence of these factors, underscoring the limitations of conventional semen diagnosis tests as predictors of poor fertilization. Across the entire cohort male factor alone accounted for 3.3% (461/13,859) of cycles with <30% fertilization rates. Furthermore, sperm-related factors were the primary cause of poor fertilization outcomes in oocyte donation cycles, accounting for 86.7% of cases. Limitations, reasons for caution Certain variables were not analyzed due to the retrospective nature and extended timeframe of the study. The matched oocyte model may overlook certain non-male factor-related instances of poor fertilization, such as procedural or technical issues. Wider implications of the findings This extensive analysis emphasizes the clinical relevance of male factor in fertilization outcomes, highlighting the need for improved semen diagnosis. It also indicates the importance of considering male-related factors in treatment decisions and shifting the focus from female-centric to more balanced fertility evaluations. Trial registration number Not applicable

P-027 Sperm motility and calcium ionophore treatment influence fertilization and pregnancy outcomes in TESE-ICSI

Abstract Study question Are there differences in fertilization and pregnancy rates post-TESE-ICSI between motile and immotile sperm, and do these vary in treatments with and without calcium ionophore? Summary answer Post-TESE-ICSI data reveals differences in fertilization and pregnancy rates when comparing motile vs. immotile sperm and treatments with and without calcium ionophore after TESE-ICSI. What is known already Testicular sperm extraction (TESE) followed by intracytoplasmic sperm injection (ICSI) poses a challenge for every IVF laboratory. This is particularly true in patients exhibiting azoospermia, where only a few and mostly immotile sperm are present in testicular tissue. In these cases, a calcium ionophore treatment for activation of the oocyte may help to overcome poor prognosis fertilization outcome, and thereby enhancing the likelihood to achieve a transfer leading to a successful pregnancy. Although both techniques, TESE-ICSI and activating substances are commonly applied, detailed analyses for individual counselling of couples are missing. Study design, size, duration This study analyzed 787 TESE-ICSI cycles (2015-2023) regarding patient age, number of attempts, oocyte maturity, fertilization and pregnancy rate after fresh embryo transfer. Cycles were split into four groups: (A) at least one motile sperm was injected (n = 493); (B) only immotile sperm were used (n = 294); (C) calcium ionophore (n = 474); (D) no calcium ionophore (n = 313). 17 cycles were excluded from the study because no sperm were found on the day of TESE-ICSI and oocytes were vitrified. Participants/materials, setting, methods In our IVF clinic, calcium ionophore treatments are only offered if a pre-screening of TESE samples reveals the absence of motile spermatozoa or if a previously conducted TESE-ICSI attempt resulted in a low fertilization rate (<30%) or a total fertilization failure. Statistical analyses was performed to compare the motile and immotile sperm group and the groups with or without calcium ionophore treatment. Main results and the role of chance No differences were found in all groups (A-D) regarding age of female or male patient and number of collected oocytes per group. However, our results demonstrate a difference in the mean number of ICSI attempts when comparing motile sperm and immotile sperm (2.0±1.3 vs. 2.3±1.6, p < 0.05), and when comparing attempts with calcium ionophore treatment and those without (2.4±1.6 vs. 1.8±1.8, p < 0.0005). The mean fertilization rate (2PN stage) was higher in the motile sperm group (A) compared to the immotile group (B) (57.8% vs. 28.4%, p < 0.0005) and also higher in attempts of group (D) in comparison to attempts of group C (56.8% vs. 40.2%, p < 0.0005). While fertilization failure occurred less often in group (A) (3.2%) it occurred to a higher rate in group (B) (19.4%), followed by group (C) (12.0%). Biochemical (BPR) and clinical pregnancy rates (CPR) showed a significant difference between the motile and immotile sperm group (BCR: 33.2% vs. 12.3%, p < 0.0005; CPR: 25.6% vs. 9.3%, p < 0.0005), but no differences between the two calcium ionophore groups. Limitations, reasons for caution This analysis was done retrospectively. A potential subgroup analysis of groups (A) and (B) into calcium ionophore use and of groups (C) and (D) into motile or immotile sperm will reveal the effect of calcium ionophore on success rates regardless of sperm motility. Wider implications of the findings Our data provides insight into success rates of TESE-ICSI cycles, especially if only immotile sperm were present. This will serve as a helpful guide for physicians when counseling patients about their success rates and to optimize the outcome in cases with poor prognosis by using a calcium ionophore treatment. Trial registration number not applicable

P-019 Impact of severely altered sperm parameters on ICSI outcomes: A retrospective analysis of embryo morphokinetics and cumulative clinical outcomes of 10,622 embryos

Abstract Study question How do severely altered parameters in both ejaculated and surgically retrieved testicular sperm affect preimplantation embryo morphokinetics, blastocyst quality and cumulative clinical outcomes? Summary answer Severely altered semen parameters impaired fertilization, embryo morphokinetics, and blastocyst quality, but only the use of testicular sperm reduced cumulative outcomes, including live birth rates. What is known already Semen quality is a significant determinant of reproductive success. Sperm play a crucial role in embryonic genome activation and facilitate embryo progression to the blastocyst stage. Yet, comprehensive analyses of embryo developmental outcomes in cases of male factor infertility remain scarce. Limited by small study cohorts, inconsistent findings and a lack of cumulative outcomes, existing studies have failed to provide a clear understanding of the differential impacts of altered semen parameters on embryo development. Uncertainty about outcomes following the use of poor-quality or testicular sperm complicates clinical management, ultimately leaving patients without comprehensive information to make informed treatment decisions. Study design, size, duration This retrospective study evaluated 10,622 embryos derived from 1,680 patients, across 1,837 ICSI cycles. We included both autologous (n = 429) and donor oocyte (n = 1,407) cycles, performed at a single IVF center between January 2019 and August 2023. To assess differences in critical aspects of reproductive success, we compared various embryo developmental and clinical outcomes across cycles using sperm with severely altered parameters (either ejaculated or obtained through testicular sperm extraction, TESE) to those using normospermic samples. Participants/materials, setting, methods Embryos were distributed into four groups according to sperm quality and origin, as per WHO guidelines: normospermia (n = 8,798), severe oligospermia (<5 million/ml; n = 567), severe oligoastenospermia (<5 million/ml and progressive motility <32%; n = 716) and TESE (n = 541). We assessed differences in embryo morphokinetics using adjusted Cox survival curves. We used multivariate analyses to evaluate associations between sperm groups and clinical outcomes, adjusting for relevant confounders pertaining to patient demographics and treatment characteristics. P-values <0.05 were considered significant. Main results and the role of chance Compared to normospermia, altered semen parameters resulted in lower mean fertilization rates (74.5% normospermia versus 69.7% severe oligospermia, 69.1% severe oligoastenospermia and 60.6% TESE). While oligospermia and oligoastenospermia showed a negative correlation with fertilization rates (R2=-0.05, p = 0.02 and R2=-0.07, p < 0.0001, respectively), this trend was more pronounced for TESE (R2= -0.12, p < 0.0001). Embryo morphokinetics were comparable for normospermia, oligospermia and oligoastenospermia. However, TESE resulted in faster early morphokinetic patterns (tPNf, t2, t3, t4 and t5; p < 0.05), and similar t8, tsB, tB, with comparable cell cycle durations (s2, s3, cc2 and cc3; p > 0.05). Compared to normospermia, severe oligospermia had no effect on inner cell mass (ICM) (OR = 0.93, 95%CI: 0.72-1.21) nor trophectoderm (TE) quality (OR = 0.79, 95%CI: 0.58-1.06), while oligoastenospermia reduced the odds of having a good quality ICM (OR = 0.75, 95%CI: 0.58-0.97) but showed no effect on TE (OR = 0.94, 95%CI: 0.71-1.23). Notably, TESE had the most profound effect on blastocyst quality (ICM, OR = 0.68, 95%CI: 0.49-0.92; TE, OR = 0.68, 95%CI: 0.46-0.98). Following the first embryo transfer, ongoing pregnancy rates were similar across all groups, apart from TESE (R2=-0.217, p = 0.0160). Unlike the other groups (p > 0.05), TESE also led to decreased cumulative implantation, ongoing pregnancy, and live birth rates compared to normospermia (R2=-0.19, p = 0.03; R2=-0.23, p = 0.009; R2=-0.22, p = 0.019, respectively). Limitations, reasons for caution The primary limitation of this study is its retrospective design, which may not account for all confounders. Moreover, lifestyle factors, such as smoking or alcohol consumption were not considered. The reliance on data from a single IVF center may also restrict the generalizability of the results to a broader population. Wider implications of the findings Our findings underscore the imperative need for careful consideration of sperm quality for ICSI, highlighting the distinct challenges presented by TESE sperm for achieving reproductive success. While altered semen parameters compromised fertilization rates and embryo quality, TESE cycles were markedly less efficient, ultimately leading to reduced cumulative live birth rates. Trial registration number not applicable

#391 : Oocyte Cryopreservation — A Five Year Follow Up Study on its Utilization and Outcome

Background & Aim: Oocyte cryopreservation is a boon for women undergoing Assisted reproductive technology (ART). Apart from cancer patients it is recently promoted as a mode of fertility insurance to overcome the age-related decline in fertility, endometriosis, primary ovarian insufficiency, etc. The objective of the study was to evaluate the clinical applications of oocyte freezing and its efficacy in terms of pregnancy outcome. Method: Retrospective observational study of all patients undergoing oocyte cryopreservation from 2016 to 2021 in Lilavati Hospital & Research Centre, Mumbai, India. Result: 221 patients undergoing oocyte cryopreservation were included. Oocyte cryopreservation was done for different indications like azoospermia (50.2%, most common), planned oocyte freezing (41.17%), fertility preservation (1.36%), endometriosis (0.45%), ejaculatory dysfunction (1.8%), husband with active hepatitis B infection (3.62%) and unavailability of husband on the day of egg retrieval (3.62%). A total of 1715 mature oocytes were frozen. 41.17% women were normo-responder, 38.91% were poor responder and 19.90% were hyper-responder. The oocytes of 59 patients (return rate-26.7%) have been thawed and 162 women (73.3%) remain in storage. Out of 508 oocytes frozen for these 59 patients utilizing their oocytes, around 474 oocytes were recovered post-warming (recovery rate 93.3%). A total of 59.3% patients created embryos with a partner, and 40.6% used donor sperm. The mean fertilization rate was 65.8% of surviving oocytes. Of 59 patients, 44 (74.5%) planned a fresh embryo transfer, out of which 20 patients tested positive for pregnancy (pregnancy rate 45.45%). Conclusion(s): We report the outcomes of patients opting for oocyte cryopreservation, which is useful for patient counselling. Limited evidence is available in terms of utilization rate, post warming survival of oocytes, fertilization and pregnancy rate. Further studies over long period of time with larger cohorts are needed.

Hormonal (sGRnH) Doses Optimization with the Ages of Brood Fish in Induced Breeding of Mystus tengara (Hamilton, 1822)

The age of brood fish is an inevitable factor in induced breeding technology worldwide. This study aimed to determine the optimal age for M. tengara for induced breeding and identify the most suitable age based on hormonal doses. Three age groups (1, 1.5, and 2-year-old) of brood fishes were used as three different treatments (T1, T2, and T3), with 0.8 ml kg-1, 1 ml.kg-1, and 1.2 ml kg-1 body doses of Ovasis (sGRnH) as the replications (R1, R2, and R3) of each treatment. Results showed that the highest mean fertilization rate (77.5 ± 1.73 %) was achieved in T1R2 for 1-year-old fish, while 1 ml kg-1 dose was ideal for them. For 1.5-year-old M. tengara, the highest mean fertilization rate was observed in T2R1 (79.25 ± 2.99 %) with 0.8 ml kg-1 dose being the most suitable. Two-year-old fish had the highest mean fertilization rate in T3R1 and T3R2 (76.25 ± 5.5 %) in June. Based on the fertilization rate and hatching rate, 1 ml kg-1 dose was identified as ideal for 1-year-old, and 0.8 ml kg-1 was suitable for 1.5-year-old M. tengara. Among all the treatments, T2R1 (1.5 year age, 0.8 ml kg-1) showed the highest hatching rate (78.25 ± 2.36 %) and most successful breeding performance over the study period, and June was identified as the best time for induced breeding. This scientific evidence provides valuable insight into the optimal age and hormonal doses for M. tengara in induced breeding technology

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 (>70%) after MOAT, except P1 and P22 which activation rate was <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-640 No impact of Progestin Primed Ovarian Stimulation (PPOS) on euploid blastocyst rate per cohort of metaphase-II oocytes during PGT-A cycles: a case-control study

Abstract Study question Can progestins be used to prevent spontaneous LH surge during ovarian stimulation (OS) without affecting the euploid blastocyst rate (EBR) per metaphase-II (MII) oocytes? Summary answer Progestin Primed Ovarian Stimulation (PPOS) (conducted with norethisterone acetate) and conventional-OS with GnRH-antagonist show similar EBR per MII-oocytes What is known already PPOS is a novel OS protocol based exogenous progesterone to inhibit LH surge and spontaneous ovulation as an alternative to GnRH analogues. The rationale of its use is that multiple follicular waves arise during the ovarian cycle and no spontaneous ovulation occurs during the luteal phase. PPOS effectiveness and safety have been supported lately in the literature. A recent study reported similar number of euploid blastocysts after PPOS (conducted with medroxyprogesterone acetate) versus conventional-OS with GnRH-antagonist. Study design, size, duration Case-control study involving advanced-maternal-age women undergoing ICSI with PGT-A at two private IVF centers (May-2017 to February-2020). 80 PPOS women were matched with 160 control based on maternal-age and ovarian reserve markers. Patients with poor-ovarian-reserve (AFC<3), premature-ovarian-failure, III-IV stage endometriosis, ovarian cyst, severe-male-factor, PGT-SR or PGT-M were excluded. EBR per MII-oocytes was the primary outcome. All other embryological and clinical outcomes were reported. Participants/materials, setting, methods Both groups underwent recombinant-FSH OS with GnRH-agonist ovulation trigger; the only difference was that norethisterone acetate 10mg/day was administered orally to PPOS patients starting from the second day of the menstrual cycle until trigger. The control group, instead, underwent conventional antagonist protocol. The laboratory procedures were similar in both groups: ICSI, blastocyst culture, trophectoderm biopsy, comprehensive-chromosome-testing to report non-mosaic aneuploidies and vitrified-warmed euploid single-embryo-transfers (SETs). Main results and the role of chance The mean fertilization rate per MII-oocytes and blastulation rate per 2PN-zygotes were similar among PPOS and control (75.5%±21.8% versus 71.7±20.7%, p = 0.1; and 56.5%±27.7% versus 53.2%±27.5%, p = 0.4, respectively). No difference was reported for the EBR per MII-oocytes among PPOS and control (15.4%±19.8% versus 14.8%±18.2%, p = 0.9). No difference was reported among the biopsied blastocysts in terms of morphological quality and day of development. A total of 47 and 86 SETs were conducted in the two groups. There was no difference in the live-birth-rates (LBR) per SET in the two groups (N = 19/47, 40.4%, 95%CI 26.7-55.7 versus N = 35/86, 40.7%, 95%CI 30.4-51.8, p = 0.9), nor in the miscarriage rate per clinical pregnancy (N = 1/20, 5%, 95%CI 0.3-26.9 versus N = 3/36, 8.3%, 95%CI 2.2-25.6, p = 0.9). At last, the cumulative-LBR among concluded cycles (i.e., LB achieved or no transferable/all transferred euploid blastocysts) was also similar (N = 19/68, 27.9%, 95%CI 18.1-40.3 versus N = 31/132, 22.0%, 95%CI 15.4-30.2, p = 0.4). Limitations, reasons for caution The main limitation is the retrospective and non-randomized design. More data are required, especially for follicle recruitment, oocyte yield, gestational and perinatal outcomes. Wider implications of the findings Norethisterone acetate-based PPOS protocol has no impact on oocyte competence when compared to conventional GnRH-antagonist OS. These promising data support further investigation on PPOS, especially in view of its reduced cost, and putative increase in patient compliance and decrease in discomfort. Trial registration number none

O-182 The clinical value of rescued MI-oocytes. Retrospective analysis of 625 MI-oocytes versus 2124 sibling MII-oocytes obtained in 285 fresh donor ICSI cycles

Abstract Study question What are the developmental competence and clinical value in ICSI cycles of retrieved metaphase-I oocytes matured in vitro (rescued-MI) to the metaphase-II (MII) stage? Summary answer Rescued MI-oocytes showed lower developmental but similar post-implantation competence than their sibling MII-oocytes, thereby contributing to a + 9.5% relative-increase in the cumulative-live-birth rate per completed-cycle. What is known already MI-oocytes are typically excluded from ICSI. Nevertheless, some authors reported their use in cancer or poor responder patients. On average, 15-20% of the oocytes retrieved in IVF are immature, but 45% of them may mature within a few hours of culture. These rescued-oocytes showed lower developmental, chromosomal and reproductive competence. Regardless, they might be valuable for some patients. To date, most of the studies were conducted including patients using own oocytes, thus implicitly involving the bias of infertility and poor prognosis. Here we aimed at outlining the clinical value of rescued-MI oocytes in the context of egg donation cycles. Study design, size, duration Observational study conducted at a private IVF center including 284 fresh oocyte donation cycles (Jan-2020 to Dec-2021). Two hours after oocyte-retrieval, all MI-oocytes identified after denudation were cultured for 1-2 additional hour(s). If reaching the MII-stage, they were inseminated via ICSI with ejaculated sperm, as for sibling MII-oocytes. Single culture in continuous media was conducted. MI- and MII-derived embryonic cohorts were monitored and compared. One-hundred-fifty-three cycles in the same period had no MI-oocyte available. Participants/materials, setting, methods Two-hundred-eighty-four cycles were conducted from 272 recipients (42.1±4.0 years) with fresh oocytes retrieved from 215 donors (25.5±5.0 years). All sperm samples were ejaculated (42.3% fresh; 15% oligoasthenoteratozoospermic). The number of MI- and MII-oocytes after denudation was 2.2±1.3 (range:1-7) and 7.5±1.6 per cycle, respectively. 1.7±1.2 (0-6) MI-oocytes matured in vitro (rescue-rate: 78.1±33.4%) and were inseminated. Embryological data were compared between sibling MI- and MII-derived cohorts. Clinical data and relative contributions of MI-oocytes were also reported. Main results and the role of chance The mean fertilization rate per cohort of inseminated MI- and MII-oocytes were 54.3±43.0% and 74.4±18.1%, respectively (paired t-test<0.01). The mean blastulation rate per cohort of MI- and MII-derived zygotes were 53.1±44.4% and 59.1±25.9%, respectively (paired t-test=0.1). Overall, 0.5±0.7 (0-4) MI-derived blastocysts were obtained adding up to the 3.2±1.7 MII-derived ones, thereby resulting in a + 20.8±40.1% (0 to + 300%) relative increase per cycle. Overall, 268 (94%), 247 (89.7%), and 188 (66.2%) cycles had ≥1, ≥2 and ≥3 MII-derived blastocyst(s), respectively. These rates showed a + 1.1% (N = +3), +4.9% (N = +12) and +7.4% (N = +24) relative increase due to MI-derived blastocysts use. To date, 19 out of 63 MI-derived blastocysts transferred resulted in a live-birth (30.2%, 95%CI 19.6-43.2) versus 124 out of 399 MII-derived ones (31.1%, 95%CI 26.6-35.9; p = 0.99). We reported 4 miscarriages out of 27 clinical pregnancies (14.8%, 95%CI 4.8-34.6) versus 38 out 162 (23.5%, 95%CI 17.3-30.9, p = 0.45), respectively. To date, 70% (N = 199/284) of the cycles was concluded, with a cumulative-live-birth-rate of 61.3% (N = 122/199). This rate showed a + 9.5% (N = +18) relative increase due to MI-derived blastocysts use. Among 85 non-completed cycles, 19 (22.4%) have MI-derived blastocysts still available. Limitations, reasons for caution Retrospective study. A cost-effectiveness analysis is warranted, especially because MI-oocytes clinical use involves additional workload and costs. 31% (N = 44/140) of patients with ≥1 live-birth have supernumerary cryopreserved MI-derived blastocysts. Many cycles are not concluded. Gestational/perinatal outcomes were not reported. More data with own eggs and/or including chromosomal testing are needed. Wider implications of the findings Rescuing MI-oocytes might increase the number of blastocysts available per cycle, and possibly the cumulative-live-birth-rate. Nevertheless, this practice involves a higher workload and the production of more supernumerary blastocysts. Mostly poor prognosis couples might benefit from their use; therefore a couple-specific decision-making process is desirable. Trial registration number n/a

Luteal Phase Stimulation in the Same Cycle Is an Effective Strategy to Rescue POSEIDON Poor Responders with No Embryos after the First Follicular Stimulation.

Poor responders may benefit from recruiting a 'second wave' of antral follicles within the same cycle. This concept forms the basis of double stimulation which has been named as 'DuoStim'. This protocol involves ovarian stimulation in both follicular and luteal phases with egg retrieval in each phase, respectively, to increase the number of oocytes and embryos in one menstrual cycle. This can be considered a potentially valuable option for women with poor ovarian reserve/response to maximise the number of oocytes retrieved in a single ovarian cycle in the shortest possible time. The aim of this study was to evaluate the efficacy of the DuoStim protocol in women classified as POSEIDON poor responders undergoing in vitro fertilization by comparing the embryological outcomes between the follicular and luteal phase stimulations in the same menstrual cycle. This was a retrospective cohort study of 131 patients who enrolled to undergo DuoStim cycles from January 2021 to Sept. 2022, at a IVF center in a tertiary care hospital. The follicular phase stimulation used a standard antagonist protocol for the first oocyte retrieval. Thereafter, the luteal phase stimulation was started 3 days after the first retrieval, with the same dose of gonadotropin along with a daily 10 mg medroxyprogesterone acetate tablet, followed by a second oocyte retrieval. Blastocysts produced in both the phases were subsequently vitrified. The paired t-test was used for comparing means and 95% confidence intervals (CIs) for different parameters. McNemar's test was used to compare paired proportions. The analysis was conducted using R statistical environment 4.2. The mean number of oocytes retrieved and the mean number of utilizable blastocysts frozen per stimulation cycle were found to be significantly higher in the luteal phase as compared to the follicular phase (5.71 ± 3.95 vs. 4.87 ± 2.79, P = 0.02, and 1.43 ± 1.22 vs. 0.95 ± 1, P = 0.001, respectively). However, the mean fertilization rate and the mean blastocyst utilization rate were found to be similar between both the phases. The length of stimulation was found to be approximately 3 days longer in the luteal phase (12.63 ± 2.43 vs. 9.75 ± 1.85, P = 0.001). Overall, the odds of obtaining a usable blastocyst in the luteal phase was found to be significantly higher than in the paired follicular phase (73.9% vs. 57.7%, P = 0.012, odds ratio: 2.286 [95% CI: 1.186-4.636]). Also importantly, the luteal phase stimulation was able to rescue 68% (32/47) of patients where no blastocysts were formed in the follicular phase. Our data demonstrate that in women with poor reserve, the addition of luteal stimulation could increase the chances of achieving a pregnancy by significantly increasing the number of eggs and transferable embryos per menstrual cycle compared to follicular stimulation alone. Furthermore, luteal phase stimulation in the same cycle proved to be an effective strategy to rescue POSEIDON poor responders with no embryos after the first stimulation.

Notes on the lifecycle and distribution of Sphodromantis gastrica (Stål, 1858) (Mantodea: Mantidae) in South Africa

Sphodromantis gastrica (Stål) (Mantodea: Mantidae), also known as the African mantis, is suggested to be common and widespread in southern Africa. Limited information is available regarding the distribution of this species in South Africa and no information is available on its biology. The aim of this study was to determine the distribution of S. gastrica in South Africa based on historic insect collection records as well as to study its basic biology and developmental parameters under captive breeding conditions. A total of 153 South African museum records of Sphodromantis spp. were recorded during this study. These records indicated that S. gastrica occurred in all but two provinces of South Africa. The incubation time of the S. gastrica oothecae were approximately 10 weeks and each ootheca contained an average of 84 egg chambers. The mean fertility rate was 54.6% while the survival rate until adulthood was 41.8%. The numbers of nymphal instars until adulthood ranged between four and nine. The mean lifespan of S. gastrica individuals were approximately 332 days and females lived longer than males.

P-247 Fresh oocyte donation, the use of donor sperm, and the number of usable blastocysts are associated with higher clinical pregnancy rates: results from 1655 cycles

Abstract Study question What are the factors associated with clinical pregnancy in the first single embryo transfer of an oocyte donation treatment? Summary answer The number of blastocysts and the use of donor sperm were positively correlated with clinical pregnancy, while the use of vitrified/warmed oocytes was negatively correlated. What is known already The use of donor oocytes for in vitro fertilization treatments is often necessary to overcome infertility. The number of donor oocytes allocated to each recipient is a key variable to the cumulative success of these treatments. However, time to pregnancy is another key metric in assisted reproduction, and it is important to achieve a better understanding of the factors influencing the success of the first embryo transfer of an oocyte donation treatment. Study design, size, duration A retrospective study was conducted to analyze the outcome of the first single blastocyst transfer of 1665 oocyte donation cycles, from 8 private IVF units, from July 2018 to July 2021. Patients who underwent multiple cycles were only included in the study once, during their first treatment. The endpoint of the study was the clinical pregnancy rate, defined by the presence of a gestational sac confirmed by ultrasound one month after the transfer. Participants/materials, setting, methods All cycles during the study period, using donor oocytes, resulting in a first fresh or frozen single blastocyst transfer were analyzed. Cases that used PGT-A or sperm from testicular biopsy were excluded. Multiple logistic regression was used to determine the association of the variables: patient age, sperm origin, vitrified/warmed oocytes, fertilization and blastocyst development rate, total number of usable blastocysts obtained, fresh/frozen transfer, and embryonic day at transfer. Main results and the role of chance A total of 972 (58.4%) embryo transfers resulted in a clinical pregnancy in the study population. The mean age of the recipient and male partner was 42±4.4 years and 42.1±6.0 years, respectively, and did not differ between positive and negative transfer groups. Statistically significant higher pregnancy rates were observed for day 5 transfers vs. day 6 (59% vs 46%) and fresh vs. vitrified/thawed oocytes (64% vs. 55%). The mean number of zygotes (5.1 vs. 4.8, p = 0.032), the mean fertilization rate (76.3% vs. 72.8%, p = 0.007), the mean usable blastocyst development rate (60.0% vs. 57.7%, p = 0.006) and the mean total number of usable blastocysts (2.9 vs. 2.6, p < 0.001) were higher in clinical pregnancy group. The results of multiple logistic regression showed that the use of sperm donor increased the chances of achieving a clinical pregnancy rate in the first embryo transfer (OR 1.36, 1.04-1.79), and the use of vitrified/thawed oocytes reduced them (OR 0.69, 0.56-0.85). There was a positive association with a higher number of usable blastocysts obtained in a cycle, and higher pregnancy rates (OR 1.16, 1.07-1.26), presumably by allowing for additional morphological embryo selection. Limitations, reasons for caution Certain confounding factors were not accounted for: variability of results between the different IVF units, endometrial preparation protocols, post-warming embryo morphology, sperm analysis diagnostic, as well as variability in recipients’ and donors’ baseline characteristics. Wider implications of the findings Factors of an oocyte donation treatment influencing the outcome of the first embryo transfer were identified. A better understanding of these factors, and interactions amongst them, is key to maximize the efficacy of these treatments, and achieve an optimal use of a limited resource such as donated oocytes. Trial registration number not applicable

P-044 Abnormal sperm morphology, so what? The clinical outcome of medical assisted reproduction is not affected by 100% teratospermic semen samples

Abstract Study question What is the success rate (clinical outcome) in medical assisted reproduction (MAR) when 0% normal spermatozoa forms are observed using strict morphology criteria (SMC)? Summary answer Strict criteria morphology has no predictive value for the clinical outcome of MAR. Absence of normal morphology spermatozoa does not preclude high cumulative pregnancy rates. What is known already Many studies have questioned the clinical value of sperm morphology since no effect on treatment outcome is apparent when sperm samples with <4% normal forms are used. However, data evaluating treatment outcome when 0% normal sperm at diagnose (evaluated by strict morphology criteria, WHO 2010) scarce. While teratozoospermia values are known to have a negative correlation with fertilization per cycle, data on cumulative ongoing pregnancy rates (COPR) or live birth rates (LBR) are missing in this selected group. Study design, size, duration This is a retrospective analysis (January 2010-June 2021) of the clinical outcome for couples when the male partner presents with 0% normal morphology (WHO 2010) at diagnostic semen analysis. Cases with 100% globozoospermia, cilia abnormalities or other MMAF phenotypes were excluded. Depending on the total progressively motile sperm count (TPMSC), couples were referred for MAR. The primary outcome presented is COPR/treatment and LBR/couple. Secondary outcomes were total fertilization failure (TFF) and mean fertilization rate (FR). Participants/materials, setting, methods All 193 cases included in this study presented with 100% teratozoospermia, as evaluated by two independent technicians following WHO 2010 manual recommendations. Couples were treated with IUI,IVF or ICSI depending on TPMSC at diagnosis. A total of 17 couples underwent 68 IUI treatments, 20 couples underwent 36 IVF ovum-pickups (OPU) (with respectively fresh embryo transfer (ET) and frozen ET, FET); 156 couples underwent 394 ICSI-OPU (with respectively fresh-ET and FET). Cumulative-OPR per treatment/couple is presented. Main results and the role of chance From the 17 couples undergoing IUI (mean 4 IUI cycles/couple; with or without mild ovarian stimulation) 3 ongoing pregnancies were reported (all singletons; 18% LBR per couple). Twenty IVF couples (mean 1.8 OPUs/couple) resulted in a total of 13 ongoing pregnancies (12 singletons, 1 twin pregnancy), therefore, the LBR per IVF-couple was 65%. In six cycles a TFF was observed even with sufficient motile sperm during insemination (5x with TPMSC >5 x 10^6/ml). If TFF was observed after IVF cycle, then ICSI was performed in the next cycle. The IVF mean %FR using 100% teratozoospermic samples was 54%, compared to 58% in cases with >1% normal forms. Regarding ICSI, 156 couples underwent a total of 377 cycles (mean of 2.4 OPUs/couple). These treatments resulted in a total of 357 fresh-ET and 458 FET. A total of 103 couples achieved 154 ongoing pregnancies (146 singletons, 7 twins), therefore the COPR was 39%/cycle and LBR was 66%/couple. In 13 cycles (3.4%) TFF was observed and in 15 cases the fertilization rate was <20%. The mean FR with ICSI using teratozoospermic samples was 55%. Comparingly, using samples with >1% normal morphology, the laboratory mean for FR is 68% and 3.7% for TFF. Limitations, reasons for caution This is a retrospective study and not matched case-control cohort was used in this analysis. Not all couples have completed 3 complete IVF/ICSI cycles. As not all frozen embryos have been transferred, cumulative pregnancy rates might be higher than the rates reported here. Wider implications of the findings The data presented here confirms the limited value of the actual strict criteria for sperm morphology evaluation. Morphology is important for identification of sporadic sperm samples with atypical malformations such as globozoospermia, MMAF and other aberrant forms of a genetic origin, however SMC evaluation and interpretation should be reconsidered Trial registration number N.A.

P-045 Sperm chromatin structure resilience is related to fertilization and blastocyst rates in ICSI cycles involving both oocyte and sperm donors

Abstract Study question Does the structural maintenance of sperm chromatin have an implication in assisted reproduction outcomes? Summary answer Sperm deprotamination is related to fertilizing capacity. Also, the time needed for the reduction of protamine disulphide bridges is a parameter underlying early embryo development. What is known already How male factor affects embryo development has been a controversial topic for decades, with opposite results. A meta-analysis recently showed that whereas sperm genotoxic damage has an impact on IVF success, its repercussion on ICSI remains unclear. Revealing the factors that may predict embryo development is much warranted to understand the sperm contribution in the ICSI era. Sperm DNA is preferentially protamine-condensed and necessarily needs to be switched to histone-condensed chromatin upon fertilization. This study aims to determine the degree of protamination and the disulphide bond integrity to provide new insights into the influence of chromatin resilience on embryo development. Study design, size, duration The study setting included a public university and a private sperm bank. The design was a blinded retrospective study, including samples from 51 sperm donors that were used for a total of 55 cycles conducted with donated oocytes in the fertility clinic. Sperm quality parameters, including those focused on chromatin structure, were evaluated after 0 min and 240 min post-thawing and compared to embryo outcomes. Participants/materials, setting, methods Sperm samples from donors selected by the germplasm bank were collected and used to fertilize oocytes in double-donation cycles (oocyte donation) with standard intracytoplasmic sperm injection. Cryopreserved samples were used to assess sperm viability (SYBR14/PI), incidence of thiol reduction (Dibromobimane, DBB) and chromatin deprotamination (Chromomycin A3, CMA3) through flow cytometry, at 0 min and 240 min after thawing. Positive controls after incubation with Triton-X100, DTT and DTT+NaCl were used to establish the flow cytometry thresholds. Main results and the role of chance In fresh semen samples, sperm concentration was 34.12±18.13 sperm/mL and progressive motility was 37.49±13.22% (mean±standard deviation). At 0 min and 240 min post-thawing, sperm viability was 24.80±12.12% and 18.65±10.37%, respectively, and the death rate was -1.54±1.24%/hour. Percentages of sperm with reduced thiols were 17.37±11.26% and 11.53±9.16% after 0 min and 240 min post-thawing, the reduction rate being -1.46±2.31%. Percentages of sperm with chromatin deprotamination were 21.12±9.03% and 35.34±14.10% after 0 min and 240 min post-thawing, and the deprotamination rate was 14.22±11.90%/hour. ICSI was conducted with an average of 11.86±7.89 oocytes per sperm sample, of which 8.78±5.95 were fertilized; the mean fertilization rate was 75.77±17.61%. For each sperm sample, 5.98±4.93 blastocysts at day 5 were obtained, leading to an average blastocyst rate of 65.17±22.22%. Interestingly, the deprotamination status at 0 min post-thaw was correlated to fertilization rates (Rs= -0.388; P = 0.005) and the decreasing rate of sperm with reduced thiols was correlated to blastocyst rate (Rs= -0.351; P = 0.012). Reduced thiols and deprotamination were also correlated with each other (Rs= -0.320; P = 0.022). Limitations, reasons for caution The main limitation of the present study was the sample size. More donors are thus needed in order for the promising results found in this study to be confirmed. Wider implications of the findings The research conducted herein has evidenced that the resilience of the sperm chromatin structure is related to embryo development outcomes from ICSI cycles conducted with oocyte donors. Trial registration number N/A

PSXII-17 Fertility prediction model for Nilli-Ravi buffalo bulls through Fourier harmonic analysis of sperm

Abstract A model to predict Nili-ravi buffalo bull fertility was developed based on Fourier harmonic analysis of sperm. Seventeen bulls with 3032 AI records were categorizes based on fertility rate (FR) as low (36.5±0.2, n = 6: SD< ˗1 from mean FR), medium (39.9±0.2, n = 3; SD +1 to -1 from mean FR) and high fertility (41.4±0.1, n = 8; SD > +1 from mean FR). Cryopreserved semen samples from these bulls were investigated for Fourier harmonic analysis of sperm nuclear shape. Hoechst-33342 and YOYO-1 fluorescent stains were used to identify live and dead sperm. Digital images were analyzed to get sperm nuclear perimeter points at different phase angles to generate Fourier functions. Mean harmonic amplitude (HA) 0 was different (P < 0.05) for 1700 live vs. 1294 dead sperm from the 17 bulls, thus live sperm were used for remaining analyses. The mean, variance, skewness and kurtosis values of 100 live sperm nuclei/bull were compared for HA0-5 between high (n = 6) and low (n = 6) fertility groups, considering equal number of bulls in each category. The mean HA2 (0.739±0.01 vs 0.686±0.00) and 4 (0.105±0.001 vs 0.007±0.001) were higher in high vs low fertility group respectively (P < 0.05). Sperm nuclear perimeter among high fertility group sperm was more elongated. There was also an increased skewness of HA0 as fertility increased (P < 0.05). Discriminant analysis defined a fertility model by using mean HA4, skewness HA0 and variance HA2, that resulted in 91.7% bulls into their correct fertility group upon cross-validation (canonical correlation=0.928; P < 0.05). Higher values of mean HA4, skewness HA0 and variance HA2 increase the chance of bulls being placed in the high fertility group. In conclusion, sperm nuclear shape in Nili-ravi buffalo bull is related to in vivo fertility. A fertility model using reported discriminant measures could be used to objectively identify Nili-ravi buffalo bulls of varying fertility.

Producing flat-head grey mullet Mugil cephalus (Linnaeus, 1758) fries in captivity from sexually mature adults collected in Sardinian lagoons

Mugil cephalus has been identified as a suitable species for feeding populations in developing countries, and it represents a traditionally harvested and consumed species in various European countries. A decline in wild populations and a growing demand have promoted new interest in mullet production in hatcheries, considered imperative for the production of a steady supply of fry and the expansion of M. cephalus aquaculture. In the present study, wild adults collected in Sardinian lagoons were induced to spawn during their natural reproductive period by treating them with a single dose of a slow release GnRHa preparation (200µgkgbw-1). In 2016, a protocol for reducing stress in capturing, transporting and selecting wild adults was set up, and 64% of the females with an oocyte diameter larger than 550µm were successfully induced to spawn. Five spawning induction trials were carried out in the period 2016–2019. The mean fertilization rate and the mean hatching rate obtained were 83±14% and 76±13%, respectively. The individuals obtained were reared indoor in a recirculating aquaculture system (RAS) at a density of 40 individuals L-1 for 60 days, and their survival ranged from 10±2 to 19±1%. During the whole larval rearing phase, from 0 to 34 days post hatching (dph), the mean standard growth rates recorded were 5.0±0.7% in total length and 14.4±3.9% in body weight. The rearing density used resulted in an average survival rate comparable or even better than those obtained by other authors, thus indicating the possibility of producing M. cephalus fries in captivity from sexually mature adults captured in Sardinian lagoons, reducing the costs and optimizing the production.

Vendace (Coregonus albula) Disperse Their Eggs Widely during Spawning

Depending on their reproductive strategy, different fish species either aggregate or disperse eggs and larvae in their reproductive habitat. Because yolk-sac larvae of vendace (Coregonus albula) disperse widely across the littoral and pelagic zones of boreal lakes, it is unclear where the exact spawning and egg incubation locations are. Vendace egg and larvae densities were studied in Lake Southern Konnevesi to clarify its spawning strategy. In autumn 2019, 1–2 weeks prior to spawning, 500 egg samplers were installed in five depth zones in 20 sampling plots. Fertilized eggs were found in 18 plots. The mean density of eggs was 74 eggs m–2 and the mean fertilization rate 85%. During spawning, vendace dispersed their offspring throughout the lake. The sampling-plot-specific egg density in autumn 2019 did not correlate with larval density in the spring next year. The reproduction strategy of vendace reduces the effects of high spatial and temporal fluctuation in their reproduction and nursery habitats.

The effect of rapid and delayed insemination on reproductive outcome in conventional insemination and intracytoplasmic sperm injection in vitro fertilization cycles.

The precise timing of insemination after oocyte retrieval is sometimes challenging. In this study, we have assessed the effect of the variation in insemination timing on reproductive outcome for both conventional insemination (CI) and intracytoplasmic sperm injection (ICSI) cycles. A single-center retrospective cohort data analysis was performed on 6559 patients (9575 oocyte retrievals) from January 2017 to July 2019. The main outcome measured was live birth rates. Secondary outcomes included fertilization rate per all oocytes retrieved, blastocyst utilization, clinical pregnancy, and miscarriage rates. The time interval between oocyte retrieval and insemination was analyzed in eight categories: 0 (0- < 0.5h), 1 (0.5- < 1.5h), 2 (1.5- < 2.5h), 3 (2.5- < 3.5h), 4 (3.5- < 4.5), 5 (4.5- < 5.5), 6 (5.5-6.5), and 7 (6.5- < 8h). The number of retrievals in each group (0-7) was 586, 1594, 1644, 1796, 1836, 1351, 641, and 127 respectively. The mean fertilization rate for CI ranged from 54.1 to 64.9% with a significant difference between time categories 0 and 5 (p < 0.001) and 1 and 5 (p < 0.0.001). The mean fertilization rate for ICSI ranged from 52.8 to 67.3% with no significant difference between time categories. Blastocyst rate for CI and ICSI was not significantly different. Miscarriage and clinical pregnancy rates in CI and ICSI were not significantly different. Live birth rates differed significantly (p < 0.05) in CI with time categories 0 and 7 representing the lowest rates, but not in the ICSI group. If performing CI or ICSI before 1.5h and > 6.5h, any detrimental effects are moderate on fertilization but do not affect blastocyst usage and birth rates. Institutional Review Board Approval from the Beth Israel Deaconess Medical Centre [IRB Protocol #: 2015P000122].

P–169 Does increasing the time interval between Oocyte-Retrieval and Oocyte-Denudation improve the results in ICSI cycles ?

Abstract Study question What should be the optimal time interval which elapses between oocyte retrieval and denudation followed by ICSI , for optimal results in ART cycles ? Summary answer Our study suggests that an optimum interval between oocyte retrieval and oocyte denudation followed by ICSI, leads to better results in ART cycles. What is known already It is widely accepted that the best timing for OPU is 34–39 hours after ovulation trigger. Some studies suggest that preincubation time before ICSI can be beneficial when it comes to fertilization and pregnancy rates while late ICSI (fertilization) may have negative results due to oocyte ageing. Other studies claim that there is no significant difference in ART results when ICSI is performed between 2–6 hours post Oocyte-Retrieval (OR) . Few studies state that 1–3 hours of COC-culture prior to denudation and oocyte injection is better as far as fertilization , embryo quality and improved oocyte cytoplasmic maturity is concerned. Study design, size, duration RCT of 234 ICSI cycles was carried out between 2017–2019. Patients were divided into two groups-: A- Early denudation with ICSI and B- Late denudation with ICSI.Both the groups were comparable in terms of female age, number of oocytes, day of transfer, number of embryos transferred and embryo quality. Fresh or frozen embryos were transferred , which were always derived from the same stimulation cycle. Exclusion criteria were : Severe male factor / TESA / PESA. Participants/materials, setting, methods 234 ICSI cycles with similar ovarian stimulation protocols were analyzed as per time range between triggering, OPU, denudation and ICSI. Patients were divided into two groups: A- Early denudation (1–2 hours after OPU) with ICSI (1–2 hours after denudation) and B- Late denudation (4–6 hours after Oocyte-Retrieval ) with ICSI (1–2 hours after denudation).Primary outcomes were oocyte maturation and fertilization rates and secondary outcomes were clinical pregnancy rate and abortion rates. Main results and the role of chance In group B ( Late denudation and ICSI), the mean fertilization rate was 67% and the Clinical Pregnancy rate was 46%. This was better than the mean fertilization rate of 56% and clinical pregnancy rate of 39% observed in group A ( Early denudation and ICSI). However the difference was not statistically significant. Therefore, ideal maturation rates were observed when denudation ( followed by ICSI ) was delayed and done 4–6 hours after Oocyte-Retrieval. In ICSI cycles in ART , ovarian stimulation is used to induce the simultaneous growth of multiple follicles, followed by final maturation and ovulation triggering with exogenous hCG. or GnRH-Agonist or both. Generally, oocyte retrieval (OR) is performed 34 - 36h later. In addition, 2–4 hours in culture of the cumulus oocyte complexes (COC) prior to oocyte injection is believed beneficial for fertilization and embryo quality, probably due to improved oocyte cytoplasmic maturity. However, in large ART centers with high workloads, following such definite time intervals is frequently very difficult. Limitations, reasons for caution In large busy centers , maintaining meticulous time intervals is difficult . As our study numbers are small, larger multicentric trials are required in order to confirm our findings and to provide more robust data . This data cannot be applied to IVM, TESE / PESE and severe male-factor infertility. Wider implications of the findings: To achieve a successful fertilization, both nuclear and cytoplasmic maturity are required. Our Study indicates that a slight delay in denudation following Oocyte-Retrieval , will yield a higher number of good quality oocytes. A higher success rate can also be expected due to more number of embryos available for transfer. Trial registration number Not applicable