Previous Fertilization Failure Research Articles

Assisted oocyte activation significantly improves zygote formation, cleavage, and implantation rates in patients with a history of fertilization failures.

Fertilization check performed at the 18th hour following classic in vitro fertilization procedure (IVF) or intracytoplasmic sperm injection (ICSI) is a critical stage in assisted reproduction. The success of the treatment is significantly reliant on the quantity of zygotes exhibiting two pronuclei. Consequently, low fertilization rates or complete fertilization failure are highly undesirable outcomes for both patients and reproductive specialists. Applying additional calcium ionophore for oocyte activation subsequent to ICSI may offer benefits and potentially enhance treatment outcomes, particularly for patients who have experienced low or absent fertilization rates (FR) in previous treatment cycles. The aim of the study is to evaluate the efficacy of Ca2+ ionophore application for oocyte activation. A retrospective analysis of 924 oocytes obtained from 120 patients who underwent ICSI cycles with a history of low or no fertilization as a result of previous unsuccessful treatment rounds. The next ART cycle followed with additional oocyte Ca2+ ionophore activation applied in 57 of the cases in order to optimize the treatment process (Group 1), and 63 patients were included and their outcomes followed as a control group (Group 2).We conducted a comparative analysis of results in both groups. The study's primary outcomes encompassed fertilization, cleavage embryo quality, blastocyst rate, and established clinical pregnancies. At day 1fertilization check we had 274/386 zygotes (71%FR) in group 1 and 132/410 in group 2 (32.2%FR), (P<0.0001). Twenty-two (34.9%) cycles in group 2 resulted in total fertilization failure (TFF). At the cleavage stage top-quality embryos from group 1 were significantly higher (P = 0.0021) in comparison to group 2. Forty-eight embryo transfers (ET) were performed in group 1 resulting in 41.67% clinical pregnancies versus 33 ET and only 4 pregnancies (12.12%) for group 2 (P = 0.0044). The results confirm the appropriateness of assisted oocyte activation as an additional method in cases of previous fertilization failure cycles.

Assisted Oocyte Activation with Calcimycin and Ionomycin After Modified ICSI Technique: A Potential Treatment for Previous Fertilization Failure Cases

Assisted Oocyte Activation with Calcimycin and Ionomycin After Modified ICSI Technique: A Potential Treatment for Previous Fertilization Failure Cases

Aggressive sperm immobilization improves reproductive outcomes in patients with suboptimal semen parameters and previous ICSI fertilization failure

Intracytoplasmic sperm injection (ICSI) is the most effective procedure to resolve male infertility, enhancing overall fertilization and pregnancy outcomes. However, it is important to note that fertilization failure (FF) can still occur in a few cases after ICSI. This study aims to introduce a specialized technique of aggressive sperm immobilization for ICSI and evaluate its impact on reproductive outcomes in cases involving prior fertilization failure. All infertile couples with male partners having suboptimal semen samples and previous ICSI fertilization failure were evaluated using retrospective data from National Taiwan tertiary university hospital (NTUH) between January 2016 and February 2022. Fertilization failure in our study was defined as less than 30% fertilization rate (FR, the number of normally fertilized oocytes divided by the total number of injected mature oocytes). Data involving both standard (routine procedure) and aggressive sperm immobilization (SI) techniques during different ICSI cycles were included in this study. Standard and aggressive SI methods were performed by compressing the distal half tail of the spermatozoa ≦ 5 and 15 times prior to ICSI respectively. Generalized estimating equations analysis were applied to compare the clinical outcomes between two procedures. Overall, data from 23 infertile couples who had undergone 65 ICSI cycles (31 standard SI with low fertilization rate and 34 aggressive SI) were included in the study. The average FR in the ICSI cycles with standard SI and aggressive SI were 23.6 ± 23.1% and 49.5 ± 31.8 respectively (P = 0.0002). The majority of embryos were transferred at the day 3 stage, with an average number transferred of 2.6 ± 0.9 in the aggressive SI group and 1.9 ± 0.9 in the standard group. The number of embryos transferred per transfer cycle was higher in the aggressive SI (P = 0.015), whereas the number of good-quality embryos was similar between the two procedures (P = 0.44). There were one and seven live births from the standard SI cycles and aggressive SI cycles respectively. In conclusion, aggressive SI was associated with a significantly higher FR, resulting in more available embryos for transfer without compromising embryo quality. Therefore, this specialized technique improved pregnancy outcome among infertile couples with a previous ICSI–FF. It can be a safe, economic, and effective method to improve the assisted reproductive technologies outcomes for infertile patients affected by previous ICSI–FF.

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.

ICSI for non-male factor infertility: time to reappraise IVF?

Intracytoplasmic sperm injection (ICSI) was initially introduced to overcome problems due of severe male factor infertility not being solved with conventional in-vitro fertilization (cIVF). However, recent years have witnessed an increasing use of ICSI by most assisted reproductive technique laboratories for non-male factor indications. Examples of the latter include previous fertilization failure after cIVF, few or poor-quality oocytes, immature oocytes, advanced maternal age, preimplantation genetics test (PGT), cryopreserved oocytes, and unexplained infertility. The replacement of cIVF with ICSI in several non-male factor infertility cases is probably because some reproductive specialists consider that ICSI is associated with better reproductive outcomes. Unfortunately, data on reproductive outcomes in favor of ICSI over cIVF are limited or absent. Therefore, the factors that can help define the use of one technique over the other should be identified. These should include the likelihood of fertilization failure, potential risks of the procedure, and its costs. In this review, we aim to highlight the current guidelines, advantages, and limitations of the use of cIVF/ICSI for infertility treatment. Additionally, we provide a comprehensive review of the use of ICSI in indications other than severe male factor infertility.

Assisted Oocyte Activation following Intracytoplasmic Sperm Injection: A Sensible Option for Infertile Couples with Severe Teratozoospermia.

The intracytoplasmic sperm injection (ICSI) has significantly improved male factor infertility treatment; however, complete fertilization failure still occurs in 1-5% of ICSI treatment cycles mainly due to oocyte activation failure. It is estimated that around 40-70% of oocyte activation failure is associated with sperm factors after ICSI. Assisted oocyte activation (AOA) as an effective approach to avoid total fertilization failure (TFF) has been proposed following ICSI. In the literature, several procedures have been described to overcome failed oocyte activation. These include mechanical, electrical, or chemical stimuli initiating artificial Ca2+ rises in the cytoplasm of oocytes. AOA in couples with previous failed fertilization and those with globozoospermia has resulted in varying degrees of success. The aim of this review is to examine the available literature on AOA in teratozoospermic men undergoing ICSI-AOA and determine whether the ICSI-AOA should be considered as an adjunct fertility procedure for these patients.

Relationship between sperm quality and total fertilization failure in intracytoplasmic sperm injection and in vitro fertilization cycles: A cross-sectional study.

BackgroundTotal fertilization failure (TFF) is associated with essential mechanistic and cellular events.ObjectiveThe present study is a comprehensive examination of detrimental effects with well-known assays for predicting TFF in conventional in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) cycles.Materials and MethodsSemen parameters of 90 men, including 60 cases who had experienced IVF/ICSI failure and a control group of 30 individuals, were evaluated. Sperm chromatin/DNA quality assessments were done by aniline blue, toluidine blue, chromomycin A3, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays. A lipid hydroperoxide (LPO) kit was used to measure the LPO, and JC1 staining was used to evaluate mitochondrial membrane potential (MMP).ResultsThere were statistically significant differences found between the IVF, ICSI and control groups by the toluidine blue (p = 0.01), TUNEL (p = 0.02), and chromomycin A3 (p 0.001) tests, but not by the aniline blue staining. Furthermore, there was a significant difference regarding LPO concentration and high MMP in cases of IVF fertilization failure compared to the control group (p = 0.04, p = 0.02, respectively). The logistic regression model showed that sperm viability was predictive for fertilization failure in the ICSI group. Sperm chromatin and DNA quality assays were not predictors for TFF in either group.ConclusionCellular events such as high DNA fragmentation damage, high levels of reactive oxygen species, and low MMP levels can cause TFF in IVF and ICSI programs. Diagnostic tests, especially in cases with previous fertilization failure, showed significant differences in sperm chromatin and DNA quality between groups but could not predict the risk of TFF.

Homozygous Loss of Septin12, but not its Haploinsufficiency, Leads to Male Infertility and Fertilization Failure.

The SEPTIN12 gene has been associated with male infertility. Male Septin12 +/− chimera mice were infertile, supporting the prevailing view that SEPTIN12 haploinsufficiency causes male infertility. In this study, we identified a heterozygous mutation on SEPTIN12, c.72C>A (p.Cys24Ter) in the male partner of a patient couple, who had a previous fertilization failure (FF) after intracytoplasmic sperm injection (ICSI) and became pregnant after ICSI together with artificial oocyte activation (AOA). To investigate the role of SEPTIN12 in FF and oocyte activation, we constructed Septin12 knockout mice. Surprisingly, Septin12 −/− male mice, but not Septin12 +/− male mice, are infertile, and have reduced sperm counts and abnormal sperm morphology. Importantly, AOA treatment enhances the 2-cell embryo rate of ICSI embryos injected with Septin12 −/− sperm, indicating that FF caused by male Septin12 deficiency is overcome by AOA. Mechanistically, loss of PLCζ around the acrosome might be the reason for FF of Septin12 −/− sperm. Taken together, our data indicated that homozygous knockout of Septin12, but not Septin12 haploinsufficiency, leads to male infertility and FF.

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

Abstract Study question Will artificial activation of oocytes alter the ploidy status of the resultant blastocysts? A sibling-oocytes pilot study Summary answer AOA-ICSI does not increase the risk of having aneuploidy blastocysts and can improve the fertilization rate in patients with sperm factor deficiency. What is known already Despite introducing ICSI as an aid to improve chances of fertilization, fertilization failure can still occur in 2–3% of ICSI cycles. Fertilization is a complex process triggered by a cascade of events following calcium (Ca2+) oscillations. Evidence suggests that the deficiency, localization or altered structure of the sperm-derived protein PLCζ in oocyte activation may be a reason for meiotic II arrest in the oocyte. Artificial oocyte activation has been proposed to compensate for the lack of calcium oscillation and resumes meiotic progression. There are however insufficient studies to determine its effect on the chromosomal status of the resultant blastocysts. Study design, size, duration This is a prospective, randomized study conducted at our Center from August-October 2020. A total of 20 couples intended for ICSI + Preimplatation Genetic Testing for Aneuploidy (PGT-A) cycles were recruited based on fulfilling one of the following criteria: 1) previous total fertilization failure (TFF), 2) history of low fertilization rate (&amp;lt;30%), 3) more than 2 cycles of failed IVF cycles (no implantation) 4) poor embryo development (no blastocysts formed) and 5) severe male factor. Participants/materials, setting, methods A total of 231 MII oocytes underwent randomization in a 1:1 ratio between AOA-ICSI and control group. All oocytes are subjected to ICSI treatment. Oocytes in the AOA-ICSI group are treated in 25μl droplets 10μM ready to use bicarbonate buffered calcium ionophore (Kitazato, Japan) for 15 minutes post-ICSI. The blastocysts were biopsied and subjected to PGT-A. Primary outcome was the aneuploidy rate and secondary outcomes were fertilization rate and blastocyst rate. Main results and the role of chance There were 11 out of 40 (27.5%) aneuploid blastocysts in the AOA-ICSI group and 7 out of 23 aneuploid blastocysts (30.4%) in the control group [odds ratio (OR) = 0.87; 95% confidence interval (CI) 0.28–2.68, p = 0.8040). There was no statistically significant difference between both groups. However, fertilization rate of the AOA- ICSI group was significantly higher than the fertilization rate in the control group (68.6% vs 49.6% respectively, OR = 2.22; 95% CI, 1.31–3.81, p = 0.0034). There were 40 blastocysts formed in the AOA-ICSI group and 23 blastocysts formed in the control group. It was found that the AOA-ICSI group yielded a higher blastocyst rate (49.4%) compared to the control group (41.1%) (OR = 1.40; 95% CI, 0.71 to 2.78, p = 0.3379) but the difference was not statistically significant. Limitations, reasons for caution The possibility of TE cells biopsied may not be representative of the whole blastocyst makes it possible to have false clinical data. The dosage and time were also not evaluated in this study as exposure time was found to be a critical factor of fertilization rate in a previous study. Wider implications of the findings: This study showed that AOA-ICSI does not increase the risk of having aneuploidy blastocysts and can improve the fertilization rate in patients with sperm factor deficiency. Additional studies involving a larger number of patients with more specific indication can further justify the benefits of AOA as a therapeutic application. Trial registration number NA

Embryo morphokinetics analysis and reproductive outcomes with assisted oocyte activation by a calcium ionophore after prior fertilization failure. A multicentric retrospective study

BackgroundFertilization failure or low fertilization rate after ICSI is around 1–3% in IVF treatments. Several strategies have been studied in order to bypass the lack of activation. The aim of this study is to evaluate embryo morphokinetics and reproductive outcomes after intracytoplasmic sperm injection (ICSI) with assisted oocyte activation (AOA) using a calcium ionophore (CaI) in patients with previous fertilization failure or low fertilization rate (under 30%) and severe male factor. MethodsMulticentric retrospective cohort study including 70 patients with fertilization failure or low fertilization rate and severe male factor (sperm concentration under 1million/mL) who underwent ICSI with CaI (756 oocytes), and 76 patients with severe male factor without previous fertilization failure who had standard ICSI (748 oocytes) between January 2011 and December 2016. ResultsCaI Exposed and non-exposed groups differed significantly for normal fertilization rates, pronuclear disappearance timing (tPNf), time to 4cells stage, multinucleation at the 2- and 4-cell stages, and direct cleavage events. Implantation rate was higher in the non-exposed group (p=0.023). Other morphokinetic variables were similar between groups. Pregnancy (higher in the non exposed group), abortion, and live birth rates, were also not statistically different among groups. ConclusionsAlthough the fertilization and implantation rates were higher in the non-exposed group, ICSI-CaI was associated with an increased fertilization rate compared to patient previous attempts and thus with increased pregnancy chances. Because of the specific patient populations involved (patients with severe male factor with or without previous fertilization failure), the results might not generalize for patients with different etiologies.Trial Registration: 1506-VLC-045-MM.

Evaluation of comparison of clinical outcome treated oocyte with ionomycin and strontium chloride in infertile couple with previous failed in vitro fertilization

Introduction: Several factors are involved in failed fertilization following intracytoplasmic sperm injection (ICSI), which could be related to sperm, oocyte factors and/or both. Failure in oocyte activation is considered as the most important factor in failed fertilization after ICSI. To overcome this shortcoming, artificial oocyte activation (AOA) after ICSI has been suggested. Commonly, ionomycin and strontium chloride are used as the most efficient agents for oocyte activation in the clinic. In this study, for the first time, the clinical results of ionomycin and strontium chloride were compared on sister oocytes of couples who previously have had fertilization failure after ICSI. Methods: In this intervention study, 14 couples with male factor infertility and previous failed fertilization after ICSI were included in this study. Oocytes of their wives were divided into two groups. Half of the oocytes after ICSI were treated with ionomicin and other half were treated with strontium chloride. Then, fertilization rate, embryo quality and pregnancy status were compared through SPSS 18 software, Paired sample t-test and chi-square test. Results: Percentage of fertilization rate was significantly higher in oocytes that were activated using strontium chloride compared to those that were activated with ionomycin (p&lt;0.0001). Although, the pregnancy rate was insignificantly higher in strontium chloride group than ionomycin group (p=0.387), but the quality of embryo was similar between the two groups (p=0.924). Conclusion: In infertile men with previous failed fertilization after ICSI, the use of strontium chloride can also be recommended for oocyte activation.

Reduction of truncated Kit Expression in Men with Abnormal Semen Parameters, Globozoospermia and History of Low or Fertilization Failure.

Objective Phospholipase C zeta 1 (PLCζ) is one of the main sperm factor involved in oocyte activation and other factors may assist this factor to induce successful fertilization. Microinjection of recombinant tr-kit, a truncated form of c-kit receptor, into metaphase II-arrested mouse oocytes initiate egg activation. Considering the potential roles of tr- KIT during spermiogenesis and fertilization, we aimed to assess expression of tr-KIT in sperm of men with normal and abnormal parameters and also in infertile men with previous failed fertilization and globozoospermia. Materials and Methods This experimental study was conducted from September 2015 to July 2016 on 30 normozoospermic and 20 abnormozoospermic samples for experiment one, and also was carried out on 10 globozoospermic men, 10 men with a history low or failed fertilization and 13 fertile men for experiment two. Semen parameters and sperm DNA fragmentation were assessed according to WHO protocol, and TUNEL assay. Sperm tr- KIT was evaluated by flow cytometry, immunostaining and western blot. Results The results show that tr-KIT mainly was detected in post-acrosomal, equatorial and tail regions. Percentage of tr-KIT-positive spermatozoa in abnormozoospermic men was significantly lower than normozoospermic men. Also significant correlations were observed between sperm tr-KIT with sperm count (r=0.8, P<0.001), motility (r=0.31, P=0.03) and abnormal morphology (r=-0.6, P<0.001). Expression of tr-KIT protein was significantly lower in infertile men with low/ failed fertilization and globozoospermia compared to fertile men. The significant correlation was also observed between tr-KIT protein with fertilization rate (r=-0.46, P=0.04). In addition, significant correlations were observed between sperm DNA fragmentation with fertilization rate (r=-0.56, P=0.019) and tr-KIT protein (r=-0.38, P=0.04). Conclusiontr-KIT may play a direct or indirect role in fertilization. Therefore, to increase our insight regarding the role of tr-KIT in fertilization further research is warranted.

Evaluation of PAWP and PLC? Expression in Infertile Men with Previous ICSI Fertilization Failure.

The aim of this study was to evaluate postacrosomal sheet WW domain binding protein (PAWP) and phospholipase C ? (PLC?) protein expression in patients with fertilization failure. Semen samples were collected from 15 fertile men (control group) and 15 patients with previous fertilization failure following ICSI (FF group) and were analyzed according to World Health Organization (WHO) criteria. The mean percentages of PAWP and PLC? positive sperm and the total level of PAWP and PLC? proteins were assessed using immunofluorescence staining. A significantly lower level and lower percentage of PAWP positive sperm in patients with fertilization failure was found compared to the control group (P = 0.01 and P = 0.03, respectively). The mean percentage ofPLC? positive sperm and level of PLC? protein were significantly lower in FF group compared to the control group (P = 0.0003 and P = 0.04, respectively). Significant positive correlations was observed between PAWP and PLC? positive sperms (r = 0.4, P = 0.008) and also total level of expression of PLC? and PAWP proteins (r = 0.4, P = 0.02) in all participants in the study. This is the first study that evaluates two main candidates for sperm-borne oocyte activating factors (SOAFs) simultaneously in patients with fertilization failure. Considering lower expression of PAWP and PLC? proteins in such patients, it seems like both factors might have the potential to be considered as SOAFs and diagnostic markers for the oocyte activation ability.

Is ICSI with calcium ionophore affecting human embryo development?

Artificial Oocyte Activation (AOA) with Calcicum Ionophore (Ica), has shown to be a successful alternative choice for those patients with previous total fertilization failure after ICSI. However, the information concerning its impact on the embryo development is scarce. The aim of this study is to analyze the fertilization, pregnancy and implantation rates, as well as direct cleavage rate, blastomere multinucleation incidence, and embryo morphokinetics parameters in patients who underwent ICSI with AOA using an ICa.

PLCζ sequence, protein levels, and distribution in human sperm do not correlate with semen characteristics and fertilization rates after ICSI.

Sperm-borne PLCζ protein induces Ca(2+) oscillations in the oocyte and is believed to play a major role during oocyte activation. However, its implication in fertilization failure following ICSI is still debated. We analyzed PLCζ gene sequence, protein expression level, and localization in both patients with previous failed fertilization by ICSI and sperm donors with proven fertility in order to assess the association of PLCζ with both sperm characteristics and ability to fertilize. Semen from 15 patients and 13 sperm donors with proven fertility was included in the study. Analysis of the PLCζ gene sequence, protein expression through Western blot, and protein localization by immunofluorescence were performed. Two patients with total fertilization failure presented mutations in heterozygosis in the PLCζ gene. Comparison with donor sample sequences displayed comparable SNP allele frequency. Distribution pattern of PLCζ did not vary significantly between donor and patient samples. Levels of PLCζ protein in sperm cells showed an interindividual variability both in patient and donor samples. Several SNPs previously reported in infertile patients were also present in fertile men. Failed fertilization occurs even when levels and distribution of PLCζ protein are within normal range. PLCζ seems to be a necessary but not sufficient factor in determining the molecular pathway involved in oocyte activation.

Artificial oocyte activation to improve reproductive outcomes in women with previous fertilization failure: a systematic review and meta-analysis of RCTs.

In couples with previous fertilization failure, are reproductive outcomes improved using ICSI followed by artificial oocyte activation (ICSI-AOA) compared with conventional ICSI? There is insufficient evidence available from RCTs to judge the efficacy and safety of ICSI-AOA for couples with previous fertilization failure. In cases with previous low fertilization rates or total fertilization failure using ICSI due to sperm-related, oocyte activation deficiency, several methods of AOA have been described, which employ mechanical, electrical or chemical stimuli. Reported fertilization and pregnancy rates appear to be improved after ICSI-AOA compared with conventional ICSI; however, the small studies performed to date make it difficult to assess the clinical efficacy or safety of AOA. The present systematic review and meta-analysis identified RCTs that compared ICSI-AOA and conventional ICSI. The last electronic search was conducted in August 2014 and there was no limitation regarding language, publication date, or publication status. We included studies that randomized either oocytes or women and included them in two different parts of this review: a women-based review and an oocyte-based review. For the women-based review, the primary outcome of effectiveness was live birth per randomized woman and the primary outcome for safety was congenital anomalies per clinical pregnancy. For the oocyte-based review, the primary outcome was embryo formation per oocyte randomized. Record screening and data extraction were performed independently by two authors and risk of bias was assessed by three authors. The effects of ICSI-AOA compared with conventional ICSI were summarized as risk ratio (RR) and the precision of the estimates was evaluated by the 95% confidence interval (CI). A total of 14 articles were assessed for eligibility and 9 included in the meta-analysis: 2 studies comprised the woman-based review (n = 168 women) and 7 studies the oocyte-based review (n = 4234 oocytes). Only four studies evaluated AOA due to fertilization failure after conventional ICSI: these were included in the quantitative analysis. In two studies evaluating couples with a history of fertilization failure in a previous cycle, ICSI-AOA was associated with an increase in the proportion of cleavage stage embryos (RR 5.44, 95% CI 2.98-9.91) and top/high quality cleavage stage embryos (RR 10.02, 95% CI 2.45-40.95). There was no evidence of effect on fertilization rate (RR 2.97, 95% CI 0.84-10.48). In the two studies that evaluated ICSI-AOA as a rescue method for unfertilized oocytes after conventional ICSI, ICSI-AOA was associated with an increase in fertilization (RR 8.26, 95% CI 1.28-53.32, P = 0.03) and cleavage rates (RR 8.65, 95% CI 2.28-32.77) although there was no significant effect on the likelihood of blastocyst formation (RR 1.97, 95% CI 0.11-34.99). The remaining five studies evaluated ICSI-AOA for reasons other than fertilization failure and were excluded. The majority of the studies were not considered to be similar enough for meta-analysis due to different AOA methods and patient inclusion criteria, thus limiting the possibility of pooling studies and achieving a more robust conclusion. Only two studies examined ICSI-AOA in couples with previous fertilization failure, and only one of these included couples with proven male-related, oocyte activation deficiency, which is the primary indication for AOA. The resulting evidence was considered to be of very low quality and should be interpreted with caution. There is insufficient evidence available from the currently available RCTs to judge the efficacy or safety of ICSI-AOA on key reproductive outcomes in couples with previous fertilization failure. Such interventions should be further examined by well-designed RCTs before the introduction of ICSI-AOA as a standard treatment. No funding was obtained. No competing interests to declare. PROSPERO CRD42014007445.

Reproductive outcome of oocyte activation by calcium ionophore a23187 following intracytoplasmic sperm injection (ICSI) in patients with previous complete fertilization failure

Fertilization failure after ICSI may be due to the inability of the spermatozoa to trigger oocyte activation which, is characterized by a rise in intracellular calcium concentration. Treatment with calcium ionophore may increase the free intracellular calcium, thereby mimicking physiological cell-signaling mechanisms resulting in oocyte activation.The aim of this study was to evaluate the impact on reproductive outcome of oocyte activation (OA) by calcium ionophore (A23187) in patients with complete fertilization failure following ICSI during previous In Vitro Fertilization (IVF) cycles. Prospective cohort study. Between January 2011 and December 2013 30 couples who had prior insuccessful ICSI treatments due to complete fertilization failure accepted to enter this study. Following ICSI 217 injected oocytes were activated with calcium ionophore A23187 for twenty minutes. Embryo transfer was performed on day 3. Fertilization and top quality embryos rate, implantation, clinical pregnancy and birth rates were analyzed. Oocyte retrieval in 30 patients resulted in 288 cumulus-oocyte complexes. After denudation, 217 (84.3%) were mature and, consequently, available for ICSI. Fertilization rate was 63.1% (137/217), cleavage rate was 92% (126/137). A mean of 2.9 Day 3 embryos were transferred to 26 patients. Four couples had no embryos to transfer. Clinical pregnancy rate was 38.5% (10/26), miscarriage rate was 20% (2/10). Nine healthy babies were born (one twin pregnancy). The clinical use of ionophores in assisted reproduction is still limited by insufficient knowledge about their potential toxic effect on oocytes and embryos. However, healthy children already born seem not to be affected by ionophore treatment in our study. Assisted oocyte activation with calcium ionophore may become a reasonable and efficient treatment in cases of very low or complete fertilization failure. More prospective controlled studies should be performed to confirm these results.

Total fertilization failure: is it the end of the story?

To study parameters that could predict in-vitro fertilization (IVF) success in patients who experienced total fertilization failure (TFF) with intracytoplasmic sperm injection (ICSI) in their previous cycles. Cycle characteristics of patients with TFF (Group I, n = 136 cycles), cycles resulting in embryo transfer (ET) following TFF (Group II, n = 36 cycles) and recurrent TFF (Group III, n = 25 cycles) and were studied retrospectively. Demographic features, cycle characteristics of three groups were compared. Follicle count measuring 15-17 mm was significantly higher in group II when compared to group I (p = 0.02). Total number of retrieved oocytes and mature oocytes were significantly higher in group II when compared to groups I and III (p = 0.001). Estradiol level at oocyte pick up (OPU) day was significantly higher in group II when compared to group I (p = 0.02). When the characteristics of ET cycles and preceding TFF cycles of the same patient were compared, total number of retrieved oocytes (5.11 ± 0.72 (95% CI 3.69-6.52) vs. 11.44 ± 1.60 (95% CI 5.29-17.59)) and mature oocytes (3.26 ± 3.66 (95% CI 2.04-4.47) vs. 6.92 ± 5.61 (95% CI 5.09-8.75)) were found to be significantly lower in TFF cycles (p = 0.001). Five biochemical and 5 clinical pregnancies occurred while only 2 healthy babies were born, corresponding to a live birth rate 5.5%. Increasing the number of retrieved and mature oocytes may increase the success of fertilization in patients with a history of previous failed fertilization. However, live birth rate is still low in embryo transfer cycles.

Diagnostic and prognostic value of calcium oscillatory pattern analysis for patients with ICSI fertilization failure

Does calcium oscillatory pattern analysis following heterologous intra-cytoplasmic sperm injection (ICSI) of human sperm into mouse oocytes lead to diagnostic and prognostic information for patients suffering from ICSI fertilization failure? We found that calcium oscillatory pattern analysis following heterologous ICSI has the strength to reveal, for the individual patient, the most probable underlying reason for low or failed fertilization after conventional ICSI. Fertilization failure occurs in 1-3% of the couples undergoing conventional ICSI, for whom the mouse oocyte activation test (MOAT) or a similar heterologous ICSI model is the only diagnostic test available to evaluate the oocyte-activating capacity of human sperm cells. The MOAT classifies the patients into three groups: a low (group 1), an intermediate (group 2) and a high (group 3) activating group. In MOAT group 1 patients, a sperm-related deficiency is likely to be the cause of previous fertilization failures, while in MOAT group 3 patients a sperm-related deficiency can most probably be refuted. For MOAT group 2 patients, the result is called inconclusive; hence, both sperm and oocyte deficiencies may still contribute to the previous ICSI fertilization failure. The calcium-releasing ability of sperm from 26 MOAT patients with a history of zero or low fertilization following conventional ICSI was compared with the calcium-releasing ability of sperm from 4 control patients, with proven oocyte activation potential. Per case an average of 19 mouse oocytes were injected. Calcium imaging started within 5-10 min after ICSI and continued for 2 h. Human sperm were demembranated with 0.02% lysolecithin for 1 min immediately before heterologous piezo-driven ICSI. For calcium imaging, metaphase II oocytes from B6D2/F1 mice were loaded with fura-2 acetoxymethyl ester. The calcium oscillatory patterns following heterologous ICSI were scored per oocyte and per patient individually based on the presence of calcium spikes and their frequency and amplitude. For patients with low or high MOAT activating capacity (MOAT group 1 or 3, respectively), calcium analysis confirmed the MOAT result. For patients with a former inconclusive intermediate MOAT activating capacity result (MOAT group 2), no or strongly dissimilar calcium oscillatory patterns were seen, with significantly lower amplitude and frequency compared with control sperm. When the product of the amplitude and the frequency of the calcium traces was compared between the groups, MOAT group 1 and 2 cases differed significantly from MOAT group 3 cases and the control sperm (P < 0.01). The results of the calcium analysis in mouse oocytes should not be directly extrapolated to human oocytes, since it is well known that human spermatozoa exhibit a greater activating potency in mouse oocytes compared with mouse spermatozoa. Furthermore, not much is known yet about the influence of aberrant calcium oscillatory patterns, such as found in MOAT group 2 patients, on pre- and post-implantation embryo development in the human. Based on the current calcium oscillatory pattern analysis, we found that the product of calcium spike amplitude with its frequency allowed us to create a new threshold value, which can assist in confirming or refuting, on a single patient base, a sperm-borne activation deficiency. The latter is especially interesting for patients with a former intermediate inconclusive MOAT result (MOAT group 2 patients), for whom calcium oscillatory pattern analysis should be considered. F.V.M. is holder of an aspirant clinical research mandate by the Flemish foundation of Scientific Research (FWO-Vlaanderen). B.H. is supported by a Ghent University grant (KAN-BOF E/01321/01). P.D.S. is holder of a fundamental clinical research mandate by the same Flemish foundation of Scientific Research (FWO-Vlaanderen).

Demographic, medical and treatment characteristics associated with couples' decisions to donate fresh spare embryos for research.

Isolation of human embryonic stem cell lines has opened a promising and pioneering area of basic and applied medical research. The issues in relation to a couples' decision in donating their supernumerary embryos for research need to be investigated further. We carried out a prospective study of 300 couples who underwent IVF/ICSI treatment cycles in our unit. We analysed various factors contributing to their decision to consent to donate spare embryos for stem cell or preimplantation genetic diagnosis research. The majority of couples (54%) consented to donate their surplus embryos for research. Couples of ethnic minority origin were less willing to consent for research compared to Caucasian couples. The number of ovarian follicles at pre-hCG scan and the number of embryos obtained were significantly higher in couples consenting for research. The funding source of treatment did not appear to influence the decision to donate. Couples with previous failed fertilization were less likely to consent to embryo research. Physical characteristics based on ovarian response during a treatment cycle may positively influence a couple's decision to donate embryos. Further studies are needed to identify those couples who are likely to agree to research so that counselling for research can be directed efficiently.