Human oocyte calcium analysis predicts the response to assisted oocyte activation in patients experiencing fertilization failure after ICSI

Human oocyte calcium analysis predicts the response to assisted oocyte activation in patients... Abstract STUDY QUESTION Can human oocyte calcium analysis predict fertilization success after assisted oocyte activation (AOA) in patients experiencing fertilization failure after ICSI? SUMMARY ANSWER ICSI-AOA restores the fertilization rate only in patients displaying abnormal Ca2+ oscillations during human oocyte activation. WHAT IS KNOWN ALREADY Patients capable of activating mouse oocytes and who showed abnormal Ca2+ profiles after mouse oocyte Ca2+ analysis (M-OCA), have variable responses to ICSI-AOA. It remains unsettled whether human oocyte Ca2+ analysis (H-OCA) would yield an improved accuracy to predict fertilization success after ICSI-AOA. STUDY DESIGN, SIZE, DURATION Sperm activation potential was first evaluated by MOAT. Subsequently, Ca2+ oscillatory patterns were determined with sperm from patients showing moderate to normal activation potential based on the capacity of human sperm to generate Ca2+ responses upon microinjection in mouse and human oocytes. Altogether, this study includes a total of 255 mouse and 122 human oocytes. M-OCA was performed with 16 different sperm samples before undergoing ICSI-AOA treatment. H-OCA was performed for 11 patients who finally underwent ICSI-AOA treatment. The diagnostic accuracy to predict fertilization success was calculated based on the response to ICSI-AOA. PARTICIPANTS/MATERIALS, SETTING, METHODS Patients experiencing low or total failed fertilization after conventional ICSI were included in the study. All participants showed moderate to high rates of activation after MOAT. Metaphase II (MII) oocytes from B6D2F1 mice were used for M-OCA. Control fertile sperm samples were used to obtain a reference Ca2+ oscillation profile elicited in human oocytes. Donated human oocytes, non-suitable for IVF treatments, were collected and vitrified at MII stage for further analysis by H-OCA. MAIN RESULTS AND THE ROLE OF CHANCE M-OCA and H-OCA predicted the response to ICSI-AOA in 8 out of 11 (73%) patients. Compared to M-OCA, H-OCA detected the presence of sperm activation deficiencies with greater sensitivity (75 vs 100%, respectively). ICSI-AOA never showed benefit to overcome fertilization failure in patients showing normal capacity to generate Ca2+ oscillations in H-OCA and was likely to be beneficial in cases displaying abnormal H-OCA Ca2+ oscillations patterns. LIMITATIONS, REASONS FOR CAUTION The scarce availability of human oocytes donated for research purposes is a limiting factor to perform H-OCA. Ca2+ imaging requires specific equipment to monitor fluorescence changes over time. WIDER IMPLICATIONS OF THE FINDINGS H-OCA is a sensitive test to diagnose gamete-linked fertilization failure. H-OCA allows treatment counseling for couples experiencing ICSI failures to either undergo ICSI-AOA or to participate in gamete donation programs. The present data provide an important template of the Ca2+ signature observed during human fertilization in cases with normal, low and failed fertilization after conventional ICSI. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the Flemish fund for scientific research (FWO-Vlaanderen, G060615N). The authors have no conflict of interest to declare. fertilization failure, ICSI, calcium oscillations, assisted oocyte activation, mouse oocyte activation test Introduction Fertilization failure after ICSI still occurs in 3–5% of total ICSI cycles (Bhattacharya et al., 2013). Experiencing fertilization failure is a distressing event (Rockliff et al., 2014), which not only prematurely ends the current treatment cycle, but offers little perspective for future treatments. The etiologies underlying fertilization failure after ART have been repeatedly investigated. The ultrastructural analysis of oocytes that failed to fertilize after conventional IVF or ICSI have demonstrated that events, such as aberrant meiotic spindle configuration or defective sperm nuclear decondensation are associated with an impaired progress of the oocyte activation, defining this as main the cause of fertilization failures after ICSI (Rawe et al., 2000; Combelles et al., 2010). Oocyte activation in mammals coincides with a series of spatio-temporal intracytoplasmic calcium (Ca2+) oscillations, evoked by the sperm upon release of a soluble factor phospholipase C zeta (PLCζ) (Swann and Lai, 2016). PLCζ promotes the production of inositol trisphosphate (IP3) which, further downstream, stimulates Ca2+ discharge from the endoplasmic reticulum through its cognate receptor (IP3R1) (Wakai et al., 2013). These Ca2+ changes initiate a complex signaling cascade leading to meiotic alleviation. Given the pivotal roles that both oocyte and sperm-related factors play during oocyte activation, deficiencies associated either with the oocyte’s Ca2+ releasing machinery, such as IP3 receptors (Ajduk et al., 2008) or PLCζ (Kashir et al., 2012; Escoffier et al., 2015) are likely to result in fertilization failures (Yeste et al., 2017). Mouse based assays are commonly used to study sperm activation potential in patients experiencing ICSI failures (Rybouchkin et al., 1995; Heindryckx et al., 2005; Yoon et al., 2008). The mouse oocyte activation test (MOAT) is an established diagnostic test performed in patients participating in our fertility program to help decide whether to use assisted oocyte activation (AOA) in subsequent ICSI cycles (Heindryckx et al., 2005; Vanden Meerschaut et al., 2012). MOAT involves the injection of human sperm into mouse oocytes to determine sperm activation capacity. Hence, MOAT allows classifying patients into three groups in comparison to fertile control samples (Heindryckx et al., 2005), from low to high activation potential: MOAT 1 (sperm-related activation deficiency), MOAT 2 (diminished sperm activation capacity) and MOAT 3 (normal sperm activation capacity, hence suspected oocyte-related oocyte activation deficiencies). The benefit of AOA is particularly evident in cases experiencing ICSI failures with diminished MOAT activation rates (Vanden Meerschaut et al., 2012; Kuentz et al., 2013). AOA strategies induce oocyte activation by an increase in intracytoplasmic Ca2+, facilitated by Ca2+ ionophores, such as ionomycin and calcimycin (Vanden Meerschaut et al., 2014b). However, the real benefit of ICSI-AOA in patients with suspected oocyte-related activation deficiencies (MOAT 3 patients) remains inconclusive (Vanden Meerschaut et al., 2012). Furthermore, the mouse oocyte Ca2+ analysis (M-OCA) demonstrated that aberrant Ca2+ patterns are associated with reduced MOAT activation potential. Interestingly, MOAT 2 patients had abnormal Ca2+ oscillatory patterns, supporting the association of MOAT 2 also with sperm-related activation deficiencies (Vanden Meerschaut et al., 2012). However, MOAT group 3 included patients with normal Ca2+ oscillatory patterns, but also others with aberrant Ca2+responses (Vanden Meerschaut et al., 2013). These observations, together with earlier findings describing that human PLCζ shows greater potency than mouse PLCζ to activate mouse oocytes (Saunders et al., 2002; Nomikos et al., 2014), warrant caution regarding the accuracy of heterologous assays in diagnosing human sperm activation capacity. Moreover, the question remains whether the Ca2+ oscillatory patterns observed in mouse are fully representative of the Ca2+ response elicited in human oocytes. The present study aimed to investigate oocyte Ca2+ analysis (OCA) as a novel strategy to reveal the presence of human sperm activation deficiencies in patients who experienced ICSI failures and demonstrated their capacity to activate mouse oocytes, particularly cases with slightly reduced MOAT (high range of MOAT 2) or normal MOAT (MOAT 3). Furthermore, the diagnostic accuracy of heterologous (M-OCA) and homologous (H-OCA) assays based on the response to ICSI-AOA treatment was evaluated. Materials and Methods Participants and study design A total of 16 male patients (37.1 ± 5.6 years old) that consulted our center between January 2009 and February 2017 regarding failed or low fertilization (<33.3%) after ICSI (Vanden Meerschaut et al., 2012) were randomly selected after signing a written informed consent form. All participants underwent MOAT before medical counseling for ICSI-AOA in a following treatment. AOA was advised following the algorithm described by (Vanden Meerschaut et al., 2013). MOAT classifies the sperm activation potential based on the percentage of oocytes at 2-cell stage as follows: MOAT 1 ≤ 20%, MOAT 2 from 21 to 84%, MOAT 3 ≥ 85% oocytes activated after heterologous ICSI. The present study includes cases with activation rates from 68–96%. All patients were analyzed by M-OCA after medical consultation. Moreover, 11 out 16 patients who underwent AOA treatment in our center were further analyzed by H-OCA. Of note, patients were blindly assigned for further Ca2+analysis prior to obtaining the clinical outcome of ICSI-AOA. Control sperm samples were donated after signing a written informed consent by patients with proven normal fertilization potential who participated in our fertility program. Source and culture of human oocytes Human oocytes discarded from IVF treatments were used. Oocytes were donated by patients <37 years old undergoing controlled ovarian hyperstimulation for IVF/ICSI treatments in our fertility program between October 2014 and August 2016 after signing a written informed consent. Oocytes were collected at one of three stages of maturation: in vivo matured metaphase II (MII) oocytes with smooth endoplasmic reticulum aggregates (SERa), in vitro matured (IVM) oocytes retrieved at prophase I (germinal vesicle, GV) or IVM oocytes retrieved at metaphase I (MI). To allow in vitro maturation, oocytes at GV stage were cultured for 24 h, and oocytes at Metaphase I (MI) were cultured for 3 or 24 h, as described elsewhere (Nikiforaki et al., 2014). Oocytes were vitrified only at MII stage following the manufacturer’s protocol (Irvine Scientific, USA) and using an open support for vitrification (Cryotop®, Kitazato, Japan). Before ICSI, oocytes were warmed following manufacturer’s instructions (Irvine Scientific, USA), and randomly assigned to the study and the control groups. Oocytes were cultured under paraffin oil at standard culture conditions (37°C in 6% CO2 and 5% O2) prior to Ca2+ imaging. Source and culture of mouse oocytes Metaphase II oocytes were collected from 6 to 10-week-old B6D2F1 hybrid female mice following follicular hyperstimulation (Vanden Meerschaut et al., 2013). Oocyte collection was performed 14 h following hCG (Chorulon®, Intervet, Boxmeer, The Netherlands) injection. Further manipulations were performed in HEPES buffered potassium simplex optimized medium (KSOM-HEPES) prepared in-house, supplemented with 4 mg/ml bovine serum albumin (BSA, Calbiochem, Belgium). Denudation of cumulus cells was performed by a brief exposure to 200 IU/ml hyaluronidase. Oocytes were cultured in KSOM containing 4 mg/ml BSA under paraffin oil at standard culture conditions prior to Ca2+ imaging. Oocyte preparation for Ca2+ imaging Before ICSI, mouse and human MII oocytes were loaded with a ratiometric Ca2+-sensitive dye. Mouse oocytes were incubated in KSOM containing fura-2 acetoxymethyl-ester (Invitrogen, Belgium) while human oocytes were incubated in cook cleavage (CC) (Cook Ltd, Ireland) medium containing fura-PE3-acetoxymethyl-ester (Teflabs, Texas, USA) both at 7.5 μM and under standard culture conditions for 30 min. Oocytes were extensively washed in culture media prior to microinjection with human sperm. Sperm preparation for ICSI Frozen control and patient sperm samples were warmed at room temperature (RT) for 15 min and sperm selection was further performed by a swim-up method (Mortimer and Mortimer, 1992) or manually selected under an inverted light microscope after the first washing step in samples showing <5 million sperm/ml. ICSI Mouse ICSI was performed according to Vanden Meerschaut et al. (2013) using piezo electrical pulses as described by Yoshida). Sperm was pre-treated with lyso-lecithine (2 mM) to allow acrosome digestion before performing piezo-ICSI. Human oocytes were microinjected following a standard ICSI protocol, performed at 37°C. Of note, sperm were not pre-treated with lyso-lecithine prior to human ICSI. Ca2+ imaging Total recording period was established at 2 h for M-OCA (Vanden Meerschaut et al., 2013) and 10 h for H-OCA (Nikiforaki et al., 2014). Both mouse and human oocytes were placed in culture medium in a glass-bottomed dish (MatTek Corp., Ashland, MA, USA) covered with paraffin oil (Irvine Scientific, USA) 30 min after ICSI. Oocytes were individually monitored under an inverted epifluorescence microscope (Olympus IX71, Olympus Soft Imaging Solutions GmBH, Belgium) equipped to stabilize standard culture conditions (OKO labs, Olympus, GmBH, Belgium) with a ×10 objective and a filter switch (Lambda DG-4 filter switch, Sutter Instrument Company, Novato, CA, USA) to provide excitation at 340 and 380 nm. Data acquisition and analysis Ca2+ measurements were acquired every 10 s for 2 h for M-OCA and every 30 s for 10 h for H-OCA. Data were analyzed by Clampfit 10.2 software (Axon Laboratories, Molecular devices UK Ltd.). Baseline drifting was adjusted before retrieving values for amplitude (A, average at maximum fluorescence intensity per peak determined overall Ca2+ spikes) expressed in arbitrary units (AU) and peak durations (d, time for a Ca2+ spike to return to baseline) expressed in minutes (min). Frequency (F) reflected the total number of Ca2+ spikes per recording period. To further refine the Ca2+ spike frequency analysis, we categorized the frequencies as previously described by (Vanden Meerschaut et al., 2013); four different categories were distinguished based on the F: (0) total absence of Ca2+ spikes, (+) 1–2, (++) 3–9 and (+++) ≥10 (Fig. 1). We further defined the term ‘oscillatory activity’ as the presence of at least one Ca2+ spike during the recording period. Finally, the activation potential per sperm sample was scored using the product A × F (AU) determined from the Ca2+ spiking pattern. Figure 1 View largeDownload slide Frequency patterns of Ca2+ oscillations. Representative traces of Ca2+ oscillations observed during human oocyte activation. Ca2+ response pattern was categorized per oocyte according to a Ca2+ spike scoring system as previously used by Vanden Meerschaut et al. (2013). Four (0, +, ++ and +++) categories are defined based on the total number of Ca2+ spikes observed during the recording period of 10 h (see Materials and Methods). Figure 1 View largeDownload slide Frequency patterns of Ca2+ oscillations. Representative traces of Ca2+ oscillations observed during human oocyte activation. Ca2+ response pattern was categorized per oocyte according to a Ca2+ spike scoring system as previously used by Vanden Meerschaut et al. (2013). Four (0, +, ++ and +++) categories are defined based on the total number of Ca2+ spikes observed during the recording period of 10 h (see Materials and Methods). Statistical analyses Distribution of the frequency patterns, amplitudes and durations (presented as SEM) were analyzed applying a Kruskal–Wallis test for independent samples with a significance of P < 0.05. Oscillatory activity (presented as %) was analyzed by t-test (Statistical Package for the Social Sciences (SPSS® Statistics 24, IBM Corp., NY, USA). EasyROC: webtool for receiver operating characteristic (ROC) curve analysis (version 3.1) was used for analyzing the diagnostic test accuracy based on AOA outcomes with a defined confidence interval of 90% by non-parametric Mann–Whitney test for SE stimulation using a Bonferroni correction for multiple comparisons. Ethical approval This study was approved by the Ghent University Hospital Institutional Review Board (reference: B670201423110). Animal studies were approved by the Ghent University Hospital Ethical Committee for Laboratory Animals (reference: ECD number: 15/56). Results Mouse and human oocyte Ca2+ analysis in patients experiencing ICSI failures The sperm-induced Ca2+ oscillatory profile was evaluated in patients showing moderate (MOAT 2, n = 9, 78.3 ± 6%) to normal (MOAT 3, n = 7, 90.0 ± 4%) sperm activation capacity. The ability of sperm to generate normal Ca2+ response activity was first studied by M-OCA. Sperm from MOAT 2 patients triggered what we define as ‘oscillatory activity’ (≥ 1 Ca2+ spike/recording period), in 66.4% of the oocytes compared to 91.2% in the control group (Fig. 2). The frequency analysis furthermore demonstrated a significantly reduced percentage of oocytes in the high frequency range (at least three spikes, i.e. classes ‘++’ or ‘+++’) compared to the control group (Fig. 2). The oscillatory activity in MOAT 3 patients was 79.3%, i.e. intermediate between MOAT group 2 and the 91.2% in the control group; none of the subcategories (‘+’, ‘++’ or ‘+++’) were significantly different between both groups (Fig. 2). Figure 2 View largeDownload slide Mouse (i) and human (ii) oocyte Ca2+ analysis in patients experiencing ICSI failures and fertile individuals (control). Mouse oocyte activation test (MOAT). Frequency pattern of Ca2+ oscillations: ‘+++’ >10; ‘++’ 3–10; ‘+’ 1–2; ‘0’, absence of Ca2+ oscillations. Number (n) of oocytes. (i) Mouse oocyte Ca2+ analysis (M-OCA) outcome: Patients classified as MOAT group 2 revealed a slightly diminished oscillatory activity (66.4%), with no significant difference compared to the control group (91.2%) (P value = 0.142). Distribution of frequency pattern between MOAT group 2 patients and the control group (Vanden Meerschaut et al., 2013) was similar in categories from 0 to ‘++’. However, the proportion of oocytes showing ≥10 Ca2+ oscillations/2 h (+++) was significantly lower (P = 0.02) compared to the control. Results of MOAT group 3 showed that the oscillatory activity (79.3%) and the distribution of frequency pattern were similar to the control group (P > 0.05). (ii) Human oocyte Ca2+ analysis (H-OCA) outcome: Patients classified as MOAT group 2 revealed a prominent decrease in oscillatory activity (5.4%) and frequency of Ca2+ oscillations, with significant differences in the distribution of frequency pattern compared to MOAT group 3 and control group. Mean oscillatory activity (%) of MOAT 2 and 3 group compared to the control group were compared by an independent samples t-test. Distribution of frequency pattern across MOAT groups 2 and 3, and control group were was compared by an independent-samples Kruskal–Wallis test. *Significance level P < 0.05. Figure 2 View largeDownload slide Mouse (i) and human (ii) oocyte Ca2+ analysis in patients experiencing ICSI failures and fertile individuals (control). Mouse oocyte activation test (MOAT). Frequency pattern of Ca2+ oscillations: ‘+++’ >10; ‘++’ 3–10; ‘+’ 1–2; ‘0’, absence of Ca2+ oscillations. Number (n) of oocytes. (i) Mouse oocyte Ca2+ analysis (M-OCA) outcome: Patients classified as MOAT group 2 revealed a slightly diminished oscillatory activity (66.4%), with no significant difference compared to the control group (91.2%) (P value = 0.142). Distribution of frequency pattern between MOAT group 2 patients and the control group (Vanden Meerschaut et al., 2013) was similar in categories from 0 to ‘++’. However, the proportion of oocytes showing ≥10 Ca2+ oscillations/2 h (+++) was significantly lower (P = 0.02) compared to the control. Results of MOAT group 3 showed that the oscillatory activity (79.3%) and the distribution of frequency pattern were similar to the control group (P > 0.05). (ii) Human oocyte Ca2+ analysis (H-OCA) outcome: Patients classified as MOAT group 2 revealed a prominent decrease in oscillatory activity (5.4%) and frequency of Ca2+ oscillations, with significant differences in the distribution of frequency pattern compared to MOAT group 3 and control group. Mean oscillatory activity (%) of MOAT 2 and 3 group compared to the control group were compared by an independent samples t-test. Distribution of frequency pattern across MOAT groups 2 and 3, and control group were was compared by an independent-samples Kruskal–Wallis test. *Significance level P < 0.05. In a next step, 11 out of 16 patients were selected for an additional H-OCA analysis. First, to characterized the total Ca2+ response pattern elicited by human sperm in human oocytes we used three fertile control sperm samples (Supplementary Fig. S1). Strikingly, human oocytes microinjected with sperm from patients classified as MOAT 2 (n = 6; 77.6 ± 6%), had a prominently lower oscillatory activity amounting to 5.4% (at least one Ca2+ spike), compared to the 91.3% observed in the control group (Fig. 2). Frequency analysis in MOAT 2 demonstrated 0% distribution in the high frequency ‘+++’ group compared to 21.7% in control; 94.5% showed the absence of oscillatory activity (‘0’) compared to 8.7% in control (Fig. 2; Supplementary Table S2). In contrast, MOAT 3 patients (n = 5; 90.0 ± 4%) had an oscillatory activity comparable to the control group (Fig. 2; Supplementary Table S2). The distribution of frequency patterns in patients from MOAT group 3 was also comparable to the controls. Diagnostic accuracy of mouse and human oocyte Ca2+ analysis for the response to ICSI-AOA We evaluated the diagnostic accuracy of oocyte Ca2+ analysis tests for predicting AOA outcome, using the amplitude × frequency (A × F) scores calculated by M-OCA and H-OCA in a total of 11 patients. Plotting A × F values against fertilization rates after ICSI-AOA shows that abnormal A × F scores are associated with favorable AOA outcomes (Fig. 3). Therefore, we assumed that AOA would not have a further benefit in cases with normal A × F values. As a result, M-OCA and H-OCA A × F scores calculated in the study group may define a threshold value associated with abnormal fertilization rates after ICSI-AOA. Consequently, we determined cut-off points by calculating the Youden’s index (Supplementary Fig. S2). A × F values associated with abnormal sperm activation capacity were estimated ≤6.72 AU for M-OCA and ≤0.6 AU for H-OCA. M-OCA identified 5 out of 11 patients with abnormal sperm activation capacity. H-OCA reclassified two patients, yielding 7 out of 11 patients with abnormal sperm activation capacity (Fig. 3). Briefly, Patient 7 had complete absence of Ca2+ oscillations in the H-OCA test. Similarly, Patient 10 had a reduced H-OCA A × F score of 0.6 AU (Table I). The 5 remaining cases with abnormal sperm activation capacity revealed by M-OCA, also showed extremely low Ca2+ oscillatory responses with A × F scores estimated between 0.0 to 0.3 AU in human oocytes (Table I). Table I Clinical outcome. Patients experiencing ICSI failures who demonstrated their capacity to activate mouse oocytes. Participants showed fertilization rates (n zygotes with two pronuclei (pn) 16–20 h after ICSI) <33.3% and high MOAT activation rates from groups 2 and 3. M-OCA was performed in 16 patients. Patient  n cycles  ICSI Fertilization rate % (n 2pn/n MII)  MOAT activation rate (%)  M-OCA A × F AU  H-OCA A × F AU  n cycles  ICSI-AOA Fertilization Rate % (n 2pn/n MII)  Embryo transfer (yes/no)  Pregnancy outcome  P1  3  27.3 (6/22)  68  6.72  0.3  2  84.6 (11/13)  Yes  Singleton  P2  2  6.7 (1/15)  72  3.95  0  2  18.8 (3/16)  Yes  Ongoing pregnancy  P3  3  33.3 (11/18)  73  8.75  *  –  –  –  –  P4  1  0.0 (0/5)  78  6.55  0  1  80.0 (8/10)  Yes  Singleton  P5  1  5.6 (1/18)  81  1.4  0  1  0/11  No  –  P6  2  (2/22)  82  0.7  *  2  –  –  –  P7  2  0.0 (0/13)  83  8.01  0  2  30.8 (4/13)  No  –  P8  4  (2/87)  84  2.04  0  1  80.0 (12/15)  Yes  Singleton  P9  3  (3/16)  84  6.13  *  1  –  –  –  P10  1  0.0 (0/7)  85  12.57  0.6  2  71.9 (23/32)  Yes  Singleton  P11  1  0.0 (0/9)  87  16.71  11  1  0.0 (0/8)  No  –  P12  1  0/10  89  3.27  *  –  –  –  –  P13  2  11.8 (2/17)  90  27.8  7.8  1  0.0 (0/9)  No  –  P14  2  0.0 (0/11)  91  23.1  *  –  –  –  –  P15  1  0.0 (0/10)  92  9.87  2.8  1  0.0 (0/9)  No  –  P16  2  0.0 (0/7)  96  8.95  6.8  1  0.0 (0/4)  No  –  Patient  n cycles  ICSI Fertilization rate % (n 2pn/n MII)  MOAT activation rate (%)  M-OCA A × F AU  H-OCA A × F AU  n cycles  ICSI-AOA Fertilization Rate % (n 2pn/n MII)  Embryo transfer (yes/no)  Pregnancy outcome  P1  3  27.3 (6/22)  68  6.72  0.3  2  84.6 (11/13)  Yes  Singleton  P2  2  6.7 (1/15)  72  3.95  0  2  18.8 (3/16)  Yes  Ongoing pregnancy  P3  3  33.3 (11/18)  73  8.75  *  –  –  –  –  P4  1  0.0 (0/5)  78  6.55  0  1  80.0 (8/10)  Yes  Singleton  P5  1  5.6 (1/18)  81  1.4  0  1  0/11  No  –  P6  2  (2/22)  82  0.7  *  2  –  –  –  P7  2  0.0 (0/13)  83  8.01  0  2  30.8 (4/13)  No  –  P8  4  (2/87)  84  2.04  0  1  80.0 (12/15)  Yes  Singleton  P9  3  (3/16)  84  6.13  *  1  –  –  –  P10  1  0.0 (0/7)  85  12.57  0.6  2  71.9 (23/32)  Yes  Singleton  P11  1  0.0 (0/9)  87  16.71  11  1  0.0 (0/8)  No  –  P12  1  0/10  89  3.27  *  –  –  –  –  P13  2  11.8 (2/17)  90  27.8  7.8  1  0.0 (0/9)  No  –  P14  2  0.0 (0/11)  91  23.1  *  –  –  –  –  P15  1  0.0 (0/10)  92  9.87  2.8  1  0.0 (0/9)  No  –  P16  2  0.0 (0/7)  96  8.95  6.8  1  0.0 (0/4)  No  –  Clinical outcome: ICSI-AOA restored fertilization rates to normal in four out of seven patients with abnormal H-OCA A × F score. The four patients achieved a pregnancy after embryo transfer. Additionally, P2, who showed H-OCA A × F score = 0 AU and fertilization rate after ICSI-AOA of 18.8%, also achieved a pregnancy. 2pn, two pronuclei/diploids; MII, Metaphase II; MOAT, mouse oocyte activation test; M-OCA, mouse oocyte calcium analysis. H-OCA, human oocyte calcium analysis; AOA, assisted oocyte activation. (*) H-OCA was performed in patients who underwent AOA in our center (n = 11). Figure 3 View largeDownload slide Representation of M-OCA and H-OCA A × F scores (Y-axis) vs the fertilization rate after ICSI-AOA (X-axis) demonstrating that abnormal A × F scores are associated with favorable AOA outcomes, corresponding to normal fertilization (>70% of diploid zygotes 16–20 h after ICSI-AOA). M-OCA (i) reveals that all the patients studied could generate Ca2+ oscillations in mouse oocytes. In contrast, H-OCA (ii) clearly demonstrate the incapability of some sperm cells to induce Ca2+ release in human oocytes showing A × F values in the 0 AU to 0.6 AU range. Area highlighted in grey include cases showing abnormal A × F and normal fertilization after ICSI-AOA. (*) H-OCA reclassified P7 and P10 as having abnormal sperm activation capacity. Figure 3 View largeDownload slide Representation of M-OCA and H-OCA A × F scores (Y-axis) vs the fertilization rate after ICSI-AOA (X-axis) demonstrating that abnormal A × F scores are associated with favorable AOA outcomes, corresponding to normal fertilization (>70% of diploid zygotes 16–20 h after ICSI-AOA). M-OCA (i) reveals that all the patients studied could generate Ca2+ oscillations in mouse oocytes. In contrast, H-OCA (ii) clearly demonstrate the incapability of some sperm cells to induce Ca2+ release in human oocytes showing A × F values in the 0 AU to 0.6 AU range. Area highlighted in grey include cases showing abnormal A × F and normal fertilization after ICSI-AOA. (*) H-OCA reclassified P7 and P10 as having abnormal sperm activation capacity. We further analyzed several properties of the A × F score to identify patients that could potentially benefit from AOA treatment using formulations to determine sensitivity, specificity and accuracy of the diagnostic test (Supplementary Table S1). Sensitivity of M-OCA was calculated as 0.75 (CI 90%: 0.19–0.99) (Table II) with three cases showing abnormal Ca2+ oscillation activity out of a total of four cases with successful fertilization after ICSI-AOA (Fig. 3). Interestingly, H-OCA revealed a sensitivity of 1.00 (CI 90%: 0.39–1) (Table II), with four cases showing abnormal Ca2+ oscillation activity with further benefit of ICSI-AOA, while none of the cases with normal Ca2+ release activity attained a normal fertilization rate after ICSI-AOA (Fig. 3). Moreover, M-OCA had a specificity of 0.71 (CI 90%: 0.29–0.96) (Table II), showing the presence of two cases with abnormal Ca2+ release activity without further benefit from ICSI-AOA. H-OCA revealed a specificity of 0.57 (CI 90%: 0.18–0.90) (Table II); with four out of seven cases with abnormal Ca2+ release activity which did not respond favorably to ICSI-AOA treatment (Supplementary data, Table S1). We further calculated the accuracy for M-OCA and H-OCA, which was estimated separately as 0.73 for each of the tests (Table II). Table II Diagnostic accuracy of M-OCA and H-OCA based in the response to ICSI-AOA treatment. Data described is used to calculated the sensitivity, specificity and accuracy of M-OCA and H-OCA, respectively. Cases showing abnormal A × F and normal fertilization after ICSI-AOA (a). Cases showing abnormal A × F and abnormal fertilization after ICSI-AOA (b). Cases showing normal A × F and normal fertilization after ICSI-AOA (c). Cases showing normal and abnormal fertilization after ICSI-AOA (d).   Definition  M-OCA  H-OCA  Sensitivity (a)/(a + c)  Cases with abnormal Ca2+ response activity experiencing successful fertilization after ICSI-AOA divided by all cases experiencing successful fertilization after ICSI-AOA  3/(3 + 1) = 0.75  4/(4 + 0) = 1  Specificity (d)/(d + b)  Cases with normal Ca2+ response activity which did not respond favorably to ICSI-AOA treatment divided by the total of cases which do not show further benefit from ICSI-AOA treatment  5/(5 + 2) = 0.71  4/(4 + 3) = 0.57  Accuracy (a + d)/(a + b + c + d)  Cases with expected responses to ICSI-AOA (abnormal A × F score associated with normal fertilization after AOA and normal A × F score associated with failed fertilization after ICSI-AOA) divided by the total of cases studied  (3 + 5)/11 = 0.73  (4 + 4)/11 = 0.73    Definition  M-OCA  H-OCA  Sensitivity (a)/(a + c)  Cases with abnormal Ca2+ response activity experiencing successful fertilization after ICSI-AOA divided by all cases experiencing successful fertilization after ICSI-AOA  3/(3 + 1) = 0.75  4/(4 + 0) = 1  Specificity (d)/(d + b)  Cases with normal Ca2+ response activity which did not respond favorably to ICSI-AOA treatment divided by the total of cases which do not show further benefit from ICSI-AOA treatment  5/(5 + 2) = 0.71  4/(4 + 3) = 0.57  Accuracy (a + d)/(a + b + c + d)  Cases with expected responses to ICSI-AOA (abnormal A × F score associated with normal fertilization after AOA and normal A × F score associated with failed fertilization after ICSI-AOA) divided by the total of cases studied  (3 + 5)/11 = 0.73  (4 + 4)/11 = 0.73  Discussion We evaluated whether the use of human oocytes would allow the discrimination between sperm or oocyte-related activation deficiencies in patients experiencing fertilization failures after routine ICSI and whose sperm were capable of activating mouse oocytes. Patients showing slightly diminished capacity of activating mouse oocytes (MOAT 2) revealed abnormal Ca2+ oscillatory patterns during mouse oocyte activation. Interestingly, the six MOAT 2 patients, who were further analyzed by H-OCA, showed a pronounced decrease in Ca2+ spiking activity in human oocytes. In support of previous findings, M-OCA revealed abnormal Ca2+ profiles in patients from MOAT group 2, confirming that sperm-related activation deficiencies, rather than oocyte deficiencies, are responsible for fertilization failures in these cases (Vanden Meerschaut et al., 2013). The present study revealed for the first time that sperm from patients showing reduced MOAT and M-OCA outcomes, are totally incapable to induce Ca2+ oscillations in human oocytes. Fertilization failures experienced by patients with normal capacity for activating mouse oocytes (MOAT 3) were initially thought to be associated with oocyte-related oocyte activation deficiencies (Heindryckx et al., 2005). However, later findings demonstrated the presence of abnormal Ca2+ oscillatory patterns after M-OCA also in some patients categorized as MOAT 3 (Vanden Meerschaut et al., 2013). Our results show that the majority of the patients classified as MOAT 3 had normal Ca2+ oscillatory patterns after M-OCA. However, H-OCA demonstrated the incapability of inducing normal Ca2+ responses in two patients who showed high MOAT activation rates and M-OCA A × F scores. Overall, 7 out of 11 patients showed incapability of generating a normal Ca2+ response in human oocytes. Reduced sperm activation potential, particularly in MOAT 1 patients, was previously correlated with favorable responses to ICSI-AOA (Heindryckx et al., 2008, Vanden Meerschaut et al., 2012). Still, the real benefit of the ICSI-AOA treatment in patients from MOAT groups 2 and 3 could not be clearly established. Our analysis reveals that Ca2+ analysis can be used to predict fertilization success after ICSI-AOA, with H-OCA yielding higher sensitivity than M-OCA to detect the presence of human sperm activation deficiencies. The increase in sensitivity of H-OCA over M-OCA results from the absence of cases with normal sperm activation capacity after H-OCA, and hence favorable responses after ICSI-AOA. However, the success of the treatment will be limited to an estimated 57% of the cases presenting with abnormal H-OCA. Interestingly, the non-responding cases will be identified subsequently at the occasion of ICSI-AOA treatment, allowing them to be classified as having an additional oocyte-related oocyte activation deficiency (Fig. 4). Overall, M-OCA and H-OCA showed similar capacity to allocate cases according to ICSI-AOA response. Here, it is interesting to note that H-OCA detected the incapability of human sperm to induce any or few Ca2+ events in cases which showed the ability of generating Ca2+ responses and subsequent oocyte activation in mouse oocytes. In our opinion, H-OCA reveals a clear cut off in the capacity of sperm to generate Ca2+ responses, which supports the use of human oocytes as the most optimal test for the study of sperm activation potential. H-OCA data showed that, four out of the seven cases with reduced A × F score experienced an improvement in fertilization rate after ICSI-AOA, with four successful pregnancies achieved to term. Intriguingly, ICSI-AOA did not overcome failed fertilization in the three cases out of the seven diagnosed with sperm activation deficiencies by all three functional tests (MOAT, M-OCA and H-OCA). As previously mentioned, in these three cases we suspect the presence of an oocyte-related oocyte activation deficiency in addition to a sperm activation incapability (Fig. 4). Given that our department is a reference center for patients experiencing fertilization failure, we may have identified a higher proportion of cases with a combined sperm–oocyte factor in the present study. In our experience, couples suffering from suspected combined sperm–oocyte factors require particular attention. Patients experiencing fertilization failures are frequently redirected to gamete donation programs. Our findings indicate that identifying sperm activation deficiencies could be important in making an informed decision regarding the use of donor oocytes or sperm in future treatments. Our data support that ICSI-AOA should be applied in patients with diagnosed sperm activation deficiencies, in this case irrespective of the origin of the oocytes (Fig. 4). Figure 4 View largeDownload slide Treatment counseling algorithm based on human oocyte Ca2+ analysis (H-OCA) result. H-OCA allows to distinguish between the presence of oocyte or sperm factors responsible of oocyte activation failure. Cases with normal H-OCA (normal sperm activation capacity) are associated with unfavorable responses to ICSI-assisted oocyte activation (AOA) treatment. Treatment advice follows the participation in an oocyte donation program. Cases with abnormal H-OCA (abnormal sperm activation capacity) are associated with favorable responses to ICSI-AOA. The treatment advice follows the use of AOA irrespective of the origin of the oocytes. Figure 4 View largeDownload slide Treatment counseling algorithm based on human oocyte Ca2+ analysis (H-OCA) result. H-OCA allows to distinguish between the presence of oocyte or sperm factors responsible of oocyte activation failure. Cases with normal H-OCA (normal sperm activation capacity) are associated with unfavorable responses to ICSI-assisted oocyte activation (AOA) treatment. Treatment advice follows the participation in an oocyte donation program. Cases with abnormal H-OCA (abnormal sperm activation capacity) are associated with favorable responses to ICSI-AOA. The treatment advice follows the use of AOA irrespective of the origin of the oocytes. Our study further diagnosed 4 out of 11 cases with normal H-OCA A × F scores who experienced ICSI failure after AOA. Since all four patients showed the capacity of generating Ca2+ oscillations in mouse, and most importantly in human oocytes, experiencing a failure in fertilization after ICSI-AOA points to the presence of a sole oocyte-related activation deficiency. These observations indicate that H-OCA can detect the cases who would not benefit from the Ca2+ replacement induced by Ca2+ ionophores during AOA procedure. In this regard, these cases would be candidates for participating in oocyte donation programs (Fig. 4). We hypothesize that the mechanisms involved in supporting Ca2+ oscillatory activity, such as IP3Rs or plasma membrane Ca2+ ATPases (PMCAs), may not be directly responsible for the oocyte activation failure (Yeste et al., 2016). Further investigations are urgently needed to develop AOA techniques aimed at tackling failed fertilization associated with oocyte-related oocyte activation failure. It is worth mentioning that procedures as IVM and cryopreservation have a slight impact on Ca2+ oscillatory patterns, with, for instance, vitrified/warmed oocytes showing variations in amplitudes and frequencies when compared to their fresh counterparts (Nikiforaki et al., 2014). Similarly, the presence of SERa in MII oocytes have a moderate effect on the Ca2+ oscillatory response (De Gheselle et al., 2014). However, accumulating evidence supports the activation competence of these oocytes, with fertilization rates comparable to their fresh counterparts (Cobo et al., 2012; Rienzi et al., 2012). Moreover, sperm cryopreservation also showed to have an influence on PLCz content (Kashir et al., 2011). Since reduced levels of PLCz have been associated with impaired fertilization (Heytens et al., 2009), it is worth taking into account that our results might not fully reflect the sperm activation capacity in our control and study groups. Further investigations are required to elucidate the real impact of cryopreservation in reducing sperm activation potential. Our results indicate that Ca2+ pattern analysis is a sensitive tool to predict the response to AOA. However, it should be noted that Ca2+ imaging has its intrinsic complexity (Swann, 2013), which requires specific equipment to monitor fluorescence changes over time. Interestingly, free cytoplasmic Ca2+ oscillations have been correlated with cytoplasmic movements observed in oocytes during fertilization (Ajduk et al., 2011). These cytoplasmic contractions, provoked by the polymerization of actin–myosin proteins, can be monitored by regular light microscopy, as shown already in mouse (Ajduk et al., 2011) and human (Swann et al., 2012) studies. These observations define a paradigm for the study of the early events of fertilization which could be feasibly adopted in IVF laboratories due to the emergence of time-lapse imaging technologies (Freour et al., 2015). In this regard, the present data provide an important template of the Ca2+ signature observed during human fertilization in cases with normal, low and failed fertilization after conventional ICSI. The use of Ca2+ ionophores for human ICSI-AOA has resulted in normal fertilization rates (Ebner et al., 2015), subsequent pregnancies (Heindryckx et al., 2005) and healthy livebirths (D’haeseleer et al., 2014; Vanden Meerschaut et al., 2014a, 2014b; Sfontouris et al., 2015; Miller et al., 2016). Nevertheless, safety concerns remain regarding the use of Ca2+ ionophores to induce oocyte activation (Santella and Dale, 2015; van Blerkom et al., 2015), which is generated by a single and high Ca2+ transient rather than the natural series of Ca2+ oscillations. Moreover, specific Ca2+ signatures during oocyte activation would likely have an impact on cellular events observed during oocyte activation (Ducibella et al., 2002), on the subsequent embryonic development (Ozil, 1998; Ozil et al., 2006; Ducibella and Fissore, 2008) and they are possibly associated with variations in gene expression profiles (Ozil et al., 2006, Rogers et al., 2006). However, the efficiency of Ca2+ ionophores to induce fertilization is supported by findings that described that mammalian fertilization could be mediated by the total summation of the individual Ca2+ spikes rather than by specific patterns (Ozil et al., 2005; Tóth et al., 2006). Further investigations are required to elucidate the effect of Ca2+ oscillation pattern on pre- and post-implantation events, and most importantly on the off-spring. We showed the importance of diagnosing sperm-related activation capacity prior to the application of ICSI-AOA in patients experiencing ICSI failures. H-OCA is the most sensitive diagnostic test to study human sperm activation potential, and in combination with fertilization outcomes after ICSI-AOA, H-OCA identifies cases with suspected oocyte-related activation deficiencies. Further research is needed to explore oocyte factors associated with failed fertilization and possible treatments to restore fertilization in this challenging group of patients. Supplementary data Supplementary data are available at Human Reproduction online. Author’s contributions B.H and M.F.B conceived the study. M.F.B. and Y.L. conducted experiments, performed data acquisition and analysis. L.D. validated patient’s selection and treatment follow-up. D.B. contributed to patient’s selection and figure design. F.V.M. provided patient’s historical data. M.F.B. wrote the article. All authors edited the article. P.D.S., B.H. and L.L. were involved in interpreting the results, revised the article and approved the final draft. Funding Flemish fund for scientific research (FWO-Vlaanderen, G060615N). Conflict of interest The authors have no conflict of interest to declare. Acknowledgements We would also like to thank Mrs Sylvie Lierman for her assistance in sperm sample preparation and to I.V.F. lab team during the collection of human oocytes. We would also like to thank Dr Fiona Dunlevy for editorial assistance. References Ajduk A, Ilozue T, Windsor S, Yu Y, Seres KB, Bomphrey RJ, Tom BD, Swann K, Thomas A, Graham C et al.  . Rhythmic actomyosin-driven contractions induced by sperm entry predict mammalian embryo viability. Nat Commun  2011; 2: 417. Google Scholar CrossRef Search ADS PubMed  Ajduk A, Małagocki A, Maleszewski M. Cytoplasmic maturation of mammalian oocytes: development of a mechanism responsible for sperm-induced Ca2+ oscillations. Reprod Biol  2008; 8: 3– 22. Google Scholar CrossRef Search ADS PubMed  Bhattacharya S, Maheshwari A, Mollison J. Factors associated with failed treatment: an analysis of 121,744 women embarking on their first IVF cycles. PLoS One  2013; 8: e82249. Google Scholar CrossRef Search ADS PubMed  Cobo A, de los Santos MJ, Castellò D, Gámiz P, Campos P, Remohí J. Outcomes of vitrified early cleavage-stage and blastocyst-stage embryos in a cryopreservation program: evaluation of 3,150 warming cycles. Fertil Steril  2012; 98: 1138– 1146.e1131. Google Scholar CrossRef Search ADS PubMed  Combelles CM, Morozumi K, Yanagimachi R, Zhu L, Fox JH, Racowsky C. Diagnosing cellular defects in an unexplained case of total fertilization failure. Hum Reprod  2010; 25: 1666– 1671. Google Scholar CrossRef Search ADS PubMed  De Gheselle N, Verpoest, De Croo, Lu, Heindryckx, Van den Abbeel, De Sutter. The effect of smooth endoplasmic reticulum aggregates in human oocytes on calcium signalling and the significance for oocyte collection cycle outcome. Abstracts of the 30th Annual Meeting of ESHRE, Munich, Germany, 29 June–2 July, 2014. D’haeseleer E, Vanden Meerschaut F, Bettens K, Luyten A, Gysels H, Thienpont Y, De Witte G, Heindryckx B, Oostra A, Roeyers H et al.  . Language development of children born following intracytoplasmic sperm injection (ICSI) combined with assisted oocyte activation (AOA). Int J Lang Commun Disord  2014; 49: 702– 709. Google Scholar CrossRef Search ADS PubMed  Ducibella T, Fissore R. The roles of Ca2+, downstream protein kinases, and oscillatory signaling in regulating fertilization and the activation of development. Dev Biol  2008; 315: 257– 279. Google Scholar CrossRef Search ADS PubMed  Ducibella T, Huneau D, Angelichio E, Xu Z, Schultz RM, Kopf GS, Fissore R, Madoux S, Ozil JP. Egg-to-embryo transition is driven by differential responses to Ca(2+) oscillation number. Dev Biol  2002; 250: 280– 291. Google Scholar CrossRef Search ADS PubMed  Ebner T, Montag M, Oocyte Activation Study G, Montag M, Van der Ven K, Van der Ven H, Ebner T, Shebl O, Oppelt P, Hirchenhain J et al.  . Live birth after artificial oocyte activation using a ready-to-use ionophore: a prospective multicentre study. Reprod Biomed Online  2015; 30: 359– 365. Google Scholar CrossRef Search ADS PubMed  Escoffier J, Yassine S, Lee HC, Martinez G, Delaroche J, Coutton C, Karaouzene T, Zouari R, Metzler-Guillemain C, Pernet-Gallay K et al.  . Subcellular localization of phospholipase Czeta in human sperm and its absence in DPY19L2-deficient sperm are consistent with its role in oocyte activation. Mol Hum Reprod  2015; 21: 157– 168. Google Scholar CrossRef Search ADS PubMed  Freour T, Basile N, Barriere P, Meseguer M. Systematic review on clinical outcomes following selection of human preimplantation embryos with time-lapse monitoring. Hum Reprod Update  2015; 21: 153– 154. Google Scholar CrossRef Search ADS PubMed  Heindryckx B, De Gheselle S, Gerris J, Dhont M, De Sutter P. Efficiency of assisted oocyte activation as a solution for failed intracytoplasmic sperm injection. Reprod Biomed Online  2008; 17: 662– 668. Google Scholar CrossRef Search ADS PubMed  Heindryckx B, Van der Elst J, De Sutter P, Dhont M. Treatment option for sperm- or oocyte-related fertilization failure: assisted oocyte activation following diagnostic heterologous ICSI. Hum Reprod  2005; 20: 2237– 2241. Google Scholar CrossRef Search ADS PubMed  Heytens E, Parrington J, Coward K, Young C, Lambrecht S, Yoon SY, Fissore RA, Hamer R, Deane CM, Ruas M et al.  . Reduced amounts and abnormal forms of phospholipase C zeta (PLCzeta) in spermatozoa from infertile men. Hum Reprod  2009; 24: 2417– 2428. Google Scholar CrossRef Search ADS PubMed  Kashir J, Heynen A, Jones C, Durrans C, Craig J, Gadea J, Turner K, Parrington J, Coward K. Effects of cryopreservation and density-gradient washing on phospholipase C zeta concentrations in human spermatozoa. Reprod Biomed Online  2011; 23: 263– 267. Google Scholar CrossRef Search ADS PubMed  Kashir J, Konstantinidis M, Jones C, Lemmon B, Lee HC, Hamer R, Heindryckx B, Deane CM, De Sutter P, Fissore RA et al.  . A maternally inherited autosomal point mutation in human phospholipase C zeta (PLCζ) leads to male infertility. Hum Reprod  2012; 27: 222– 231. Google Scholar CrossRef Search ADS PubMed  Kuentz P, Vanden Meerschaut F, Elinati E, Nasr-Esfahani MH, Gurgan T, Iqbal N, Carré-Pigeon F, Brugnon F, Gitlin SA, Velez de la Calle J et al.  . Assisted oocyte activation overcomes fertilization failure in globozoospermic patients regardless of the DPY19L2 status. Hum Reprod  2013; 28: 1054– 1061. Google Scholar CrossRef Search ADS PubMed  Miller N, Biron-Shental T, Sukenik-Halevy R, Klement AH, Sharony R, Berkovitz A. Oocyte activation by calcium ionophore and congenital birth defects: a retrospective cohort study. Fertil Steril  2016; 106: 590– 596.e592. Google Scholar CrossRef Search ADS PubMed  Mortimer D, Mortimer ST. Methods of sperm preparation for assisted reproduction. Ann Acad Med Singapore  1992; 21: 517– 524. Google Scholar PubMed  Nikiforaki D, Vanden Meerschaut F, Qian C, De Croo I, Lu Y, Deroo T, Van den Abbeel E, Heindryckx B, De Sutter P. Oocyte cryopreservation and in vitro culture affect calcium signalling during human fertilization. Hum Reprod  2014; 29: 29– 40. Google Scholar CrossRef Search ADS PubMed  Nomikos M, Theodoridou M, Elgmati K, Parthimos D, Calver BL, Buntwal L, Nounesis G, Swann K, Lai FA. Human PLCζ exhibits superior fertilization potency over mouse PLCζ in triggering the Ca2+ oscillations required for mammalian oocyte activation. Mol Hum Reprod  2014. Ozil JP. Role of calcium oscillations in mammalian egg activation: experimental approach. Biophys Chem  1998; 72: 141– 152. Google Scholar CrossRef Search ADS PubMed  Ozil JP, Banrezes B, Tóth S, Pan H, Schultz RM. Ca2+ oscillatory pattern in fertilized mouse eggs affects gene expression and development to term. Dev Biol  2006; 300: 534– 544. Google Scholar CrossRef Search ADS PubMed  Ozil JP, Markoulaki S, Toth S, Matson S, Banrezes B, Knott JG, Schultz RM, Huneau D, Ducibella T. Egg activation events are regulated by the duration of a sustained [Ca2+]cyt signal in the mouse. Dev Biol  2005; 282: 39– 54. Google Scholar CrossRef Search ADS PubMed  Rawe VY, Olmedo SB, Nodar FN, Doncel GD, Acosta AA, Vitullo AD. Cytoskeletal organization defects and abortive activation in human oocytes after IVF and ICSI failure. Mol Hum Reprod  2000; 6: 510– 516. Google Scholar CrossRef Search ADS PubMed  Rienzi L, Cobo A, Paffoni A, Scarduelli C, Capalbo A, Vajta G, Remohí J, Ragni G, Ubaldi FM. Consistent and predictable delivery rates after oocyte vitrification: an observational longitudinal cohort multicentric study. Hum Reprod  2012; 27: 1606– 1612. Google Scholar CrossRef Search ADS PubMed  Rockliff HE, Lightman SL, Rhidian E, Buchanan H, Gordon U, Vedhara K. A systematic review of psychosocial factors associated with emotional adjustment in in vitro fertilization patients. Hum Reprod Update  2014; 20: 594– 613. Google Scholar CrossRef Search ADS PubMed  Rogers NT, Halet G, Piao Y, Carroll J, Ko MS, Swann K. The absence of a Ca(2+) signal during mouse egg activation can affect parthenogenetic preimplantation development, gene expression patterns, and blastocyst quality. Reproduction  2006; 132: 45– 57. Google Scholar CrossRef Search ADS PubMed  Rybouchkin A, Dozortsev D, de Sutter P, Qian C, Dhont M. Intracytoplasmic injection of human spermatozoa into mouse oocytes: a useful model to investigate the oocyte-activating capacity and the karyotype of human spermatozoa. Hum Reprod  1995; 10: 1130– 1135. Google Scholar CrossRef Search ADS PubMed  Santella L, Dale B. Assisted yes, but where do we draw the line? Reprod Biomed Online  2015; 31: 476– 478. Google Scholar CrossRef Search ADS PubMed  Saunders CM, Larman MG, Parrington J, Cox LJ, Royse J, Blayney LM, Swann K, Lai FA. PLC zeta: a sperm-specific trigger of Ca(2+) oscillations in eggs and embryo development. Development  2002; 129: 3533– 3544. Google Scholar PubMed  Sfontouris IA, Nastri CO, Lima ML, Tahmasbpourmarzouni E, Raine-Fenning N, Martins WP. Artificial oocyte activation to improve reproductive outcomes in women with previous fertilization failure: a systematic review and meta-analysis of RCTs. Hum Reprod  2015; 30: 1831– 1841. Google Scholar CrossRef Search ADS PubMed  Swann K. Measuring Ca2+ oscillations in mammalian eggs. Methods Mol Biol  2013; 957: 231– 248. Google Scholar CrossRef Search ADS PubMed  Swann K, Lai FA. Egg activation at fertilization by a soluble sperm protein. Physiol Rev  2016; 96: 127– 149. Google Scholar CrossRef Search ADS PubMed  Swann K, Windsor S, Campbell K, Elgmati K, Nomikos M, Zernicka-Goetz M, Amso N, Lai FA, Thomas A, Graham C. Phospholipase C-ζ-induced Ca2+ oscillations cause coincident cytoplasmic movements in human oocytes that failed to fertilize after intracytoplasmic sperm injection. Fertil Steril  2012; 97: 742– 747. Google Scholar CrossRef Search ADS PubMed  Tóth S, Huneau D, Banrezes B, Ozil JP. Egg activation is the result of calcium signal summation in the mouse. Reproduction  2006; 131: 27– 34. Google Scholar CrossRef Search ADS PubMed  van Blerkom J, Cohen J, Johnson M. A plea for caution and more research in the ‘experimental’ use of ionophores in ICSI. Reprod Biomed Online  2015; 30: 323– 324. Google Scholar CrossRef Search ADS PubMed  Vanden Meerschaut F, D’Haeseleer E, Gysels H, Thienpont Y, Dewitte G, Heindryckx B, Oostra A, Roeyers H, Van Lierde K, De Sutter P. Neonatal and neurodevelopmental outcome of children aged 3–10 years born following assisted oocyte activation. Reprod Biomed Online  2014a; 28: 54– 63. Google Scholar CrossRef Search ADS PubMed  Vanden Meerschaut F, Leybaert L, Nikiforaki D, Qian C, Heindryckx B, De Sutter P. Diagnostic and prognostic value of calcium oscillatory pattern analysis for patients with ICSI fertilization failure. Hum Reprod  2013; 28: 87– 98. Google Scholar CrossRef Search ADS PubMed  Vanden Meerschaut F, Nikiforaki D, De Gheselle S, Dullaerts V, Van den Abbeel E, Gerris J, Heindryckx B, De Sutter P. Assisted oocyte activation is not beneficial for all patients with a suspected oocyte-related activation deficiency. Hum Reprod  2012; 27: 1977– 1984. Google Scholar CrossRef Search ADS PubMed  Vanden Meerschaut F, Nikiforaki D, Heindryckx B, De Sutter P. Assisted oocyte activation following ICSI fertilization failure. Reprod Biomed Online  2014b; 28: 560– 571. Google Scholar CrossRef Search ADS PubMed  Wakai T, Zhang N, Vangheluwe P, Fissore RA. Regulation of endoplasmic reticulum Ca2+ oscillations in mammalian eggs. J Cell Sci  2013; 126: 5714– 5724. Google Scholar CrossRef Search ADS PubMed  Yeste M, Jones C, Amdani SN, Coward K. Oocyte activation and fertilisation: crucial contributors from the sperm and oocyte. Results Probl Cell Differ  2017; 59: 213– 239. Google Scholar CrossRef Search ADS PubMed  Yeste M, Jones C, Amdani SN, Patel S, Coward K. Oocyte activation deficiency: a role for an oocyte contribution? Hum Reprod Update  2016; 22: 23– 47. Google Scholar CrossRef Search ADS PubMed  Yoon SY, Jellerette T, Salicioni AM, Lee HC, Yoo MS, Coward K, Parrington J, Grow D, Cibelli JB, Visconti PE et al.  . Human sperm devoid of PLC, zeta 1 fail to induce Ca(2+) release and are unable to initiate the first step of embryo development. J Clin Invest  2008; 118: 3671– 3681. Google Scholar CrossRef Search ADS PubMed  Yoshida N, Perry AC. Piezo-actuated mouse intracytoplasmic sperm injection (ICSI). Nat Protoc  2007; 2: 296– 304. Google Scholar CrossRef Search ADS PubMed  © The Author(s) 2018. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Human Reproduction Oxford University Press

Human oocyte calcium analysis predicts the response to assisted oocyte activation in patients experiencing fertilization failure after ICSI

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Abstract

Abstract STUDY QUESTION Can human oocyte calcium analysis predict fertilization success after assisted oocyte activation (AOA) in patients experiencing fertilization failure after ICSI? SUMMARY ANSWER ICSI-AOA restores the fertilization rate only in patients displaying abnormal Ca2+ oscillations during human oocyte activation. WHAT IS KNOWN ALREADY Patients capable of activating mouse oocytes and who showed abnormal Ca2+ profiles after mouse oocyte Ca2+ analysis (M-OCA), have variable responses to ICSI-AOA. It remains unsettled whether human oocyte Ca2+ analysis (H-OCA) would yield an improved accuracy to predict fertilization success after ICSI-AOA. STUDY DESIGN, SIZE, DURATION Sperm activation potential was first evaluated by MOAT. Subsequently, Ca2+ oscillatory patterns were determined with sperm from patients showing moderate to normal activation potential based on the capacity of human sperm to generate Ca2+ responses upon microinjection in mouse and human oocytes. Altogether, this study includes a total of 255 mouse and 122 human oocytes. M-OCA was performed with 16 different sperm samples before undergoing ICSI-AOA treatment. H-OCA was performed for 11 patients who finally underwent ICSI-AOA treatment. The diagnostic accuracy to predict fertilization success was calculated based on the response to ICSI-AOA. PARTICIPANTS/MATERIALS, SETTING, METHODS Patients experiencing low or total failed fertilization after conventional ICSI were included in the study. All participants showed moderate to high rates of activation after MOAT. Metaphase II (MII) oocytes from B6D2F1 mice were used for M-OCA. Control fertile sperm samples were used to obtain a reference Ca2+ oscillation profile elicited in human oocytes. Donated human oocytes, non-suitable for IVF treatments, were collected and vitrified at MII stage for further analysis by H-OCA. MAIN RESULTS AND THE ROLE OF CHANCE M-OCA and H-OCA predicted the response to ICSI-AOA in 8 out of 11 (73%) patients. Compared to M-OCA, H-OCA detected the presence of sperm activation deficiencies with greater sensitivity (75 vs 100%, respectively). ICSI-AOA never showed benefit to overcome fertilization failure in patients showing normal capacity to generate Ca2+ oscillations in H-OCA and was likely to be beneficial in cases displaying abnormal H-OCA Ca2+ oscillations patterns. LIMITATIONS, REASONS FOR CAUTION The scarce availability of human oocytes donated for research purposes is a limiting factor to perform H-OCA. Ca2+ imaging requires specific equipment to monitor fluorescence changes over time. WIDER IMPLICATIONS OF THE FINDINGS H-OCA is a sensitive test to diagnose gamete-linked fertilization failure. H-OCA allows treatment counseling for couples experiencing ICSI failures to either undergo ICSI-AOA or to participate in gamete donation programs. The present data provide an important template of the Ca2+ signature observed during human fertilization in cases with normal, low and failed fertilization after conventional ICSI. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the Flemish fund for scientific research (FWO-Vlaanderen, G060615N). The authors have no conflict of interest to declare. fertilization failure, ICSI, calcium oscillations, assisted oocyte activation, mouse oocyte activation test Introduction Fertilization failure after ICSI still occurs in 3–5% of total ICSI cycles (Bhattacharya et al., 2013). Experiencing fertilization failure is a distressing event (Rockliff et al., 2014), which not only prematurely ends the current treatment cycle, but offers little perspective for future treatments. The etiologies underlying fertilization failure after ART have been repeatedly investigated. The ultrastructural analysis of oocytes that failed to fertilize after conventional IVF or ICSI have demonstrated that events, such as aberrant meiotic spindle configuration or defective sperm nuclear decondensation are associated with an impaired progress of the oocyte activation, defining this as main the cause of fertilization failures after ICSI (Rawe et al., 2000; Combelles et al., 2010). Oocyte activation in mammals coincides with a series of spatio-temporal intracytoplasmic calcium (Ca2+) oscillations, evoked by the sperm upon release of a soluble factor phospholipase C zeta (PLCζ) (Swann and Lai, 2016). PLCζ promotes the production of inositol trisphosphate (IP3) which, further downstream, stimulates Ca2+ discharge from the endoplasmic reticulum through its cognate receptor (IP3R1) (Wakai et al., 2013). These Ca2+ changes initiate a complex signaling cascade leading to meiotic alleviation. Given the pivotal roles that both oocyte and sperm-related factors play during oocyte activation, deficiencies associated either with the oocyte’s Ca2+ releasing machinery, such as IP3 receptors (Ajduk et al., 2008) or PLCζ (Kashir et al., 2012; Escoffier et al., 2015) are likely to result in fertilization failures (Yeste et al., 2017). Mouse based assays are commonly used to study sperm activation potential in patients experiencing ICSI failures (Rybouchkin et al., 1995; Heindryckx et al., 2005; Yoon et al., 2008). The mouse oocyte activation test (MOAT) is an established diagnostic test performed in patients participating in our fertility program to help decide whether to use assisted oocyte activation (AOA) in subsequent ICSI cycles (Heindryckx et al., 2005; Vanden Meerschaut et al., 2012). MOAT involves the injection of human sperm into mouse oocytes to determine sperm activation capacity. Hence, MOAT allows classifying patients into three groups in comparison to fertile control samples (Heindryckx et al., 2005), from low to high activation potential: MOAT 1 (sperm-related activation deficiency), MOAT 2 (diminished sperm activation capacity) and MOAT 3 (normal sperm activation capacity, hence suspected oocyte-related oocyte activation deficiencies). The benefit of AOA is particularly evident in cases experiencing ICSI failures with diminished MOAT activation rates (Vanden Meerschaut et al., 2012; Kuentz et al., 2013). AOA strategies induce oocyte activation by an increase in intracytoplasmic Ca2+, facilitated by Ca2+ ionophores, such as ionomycin and calcimycin (Vanden Meerschaut et al., 2014b). However, the real benefit of ICSI-AOA in patients with suspected oocyte-related activation deficiencies (MOAT 3 patients) remains inconclusive (Vanden Meerschaut et al., 2012). Furthermore, the mouse oocyte Ca2+ analysis (M-OCA) demonstrated that aberrant Ca2+ patterns are associated with reduced MOAT activation potential. Interestingly, MOAT 2 patients had abnormal Ca2+ oscillatory patterns, supporting the association of MOAT 2 also with sperm-related activation deficiencies (Vanden Meerschaut et al., 2012). However, MOAT group 3 included patients with normal Ca2+ oscillatory patterns, but also others with aberrant Ca2+responses (Vanden Meerschaut et al., 2013). These observations, together with earlier findings describing that human PLCζ shows greater potency than mouse PLCζ to activate mouse oocytes (Saunders et al., 2002; Nomikos et al., 2014), warrant caution regarding the accuracy of heterologous assays in diagnosing human sperm activation capacity. Moreover, the question remains whether the Ca2+ oscillatory patterns observed in mouse are fully representative of the Ca2+ response elicited in human oocytes. The present study aimed to investigate oocyte Ca2+ analysis (OCA) as a novel strategy to reveal the presence of human sperm activation deficiencies in patients who experienced ICSI failures and demonstrated their capacity to activate mouse oocytes, particularly cases with slightly reduced MOAT (high range of MOAT 2) or normal MOAT (MOAT 3). Furthermore, the diagnostic accuracy of heterologous (M-OCA) and homologous (H-OCA) assays based on the response to ICSI-AOA treatment was evaluated. Materials and Methods Participants and study design A total of 16 male patients (37.1 ± 5.6 years old) that consulted our center between January 2009 and February 2017 regarding failed or low fertilization (<33.3%) after ICSI (Vanden Meerschaut et al., 2012) were randomly selected after signing a written informed consent form. All participants underwent MOAT before medical counseling for ICSI-AOA in a following treatment. AOA was advised following the algorithm described by (Vanden Meerschaut et al., 2013). MOAT classifies the sperm activation potential based on the percentage of oocytes at 2-cell stage as follows: MOAT 1 ≤ 20%, MOAT 2 from 21 to 84%, MOAT 3 ≥ 85% oocytes activated after heterologous ICSI. The present study includes cases with activation rates from 68–96%. All patients were analyzed by M-OCA after medical consultation. Moreover, 11 out 16 patients who underwent AOA treatment in our center were further analyzed by H-OCA. Of note, patients were blindly assigned for further Ca2+analysis prior to obtaining the clinical outcome of ICSI-AOA. Control sperm samples were donated after signing a written informed consent by patients with proven normal fertilization potential who participated in our fertility program. Source and culture of human oocytes Human oocytes discarded from IVF treatments were used. Oocytes were donated by patients <37 years old undergoing controlled ovarian hyperstimulation for IVF/ICSI treatments in our fertility program between October 2014 and August 2016 after signing a written informed consent. Oocytes were collected at one of three stages of maturation: in vivo matured metaphase II (MII) oocytes with smooth endoplasmic reticulum aggregates (SERa), in vitro matured (IVM) oocytes retrieved at prophase I (germinal vesicle, GV) or IVM oocytes retrieved at metaphase I (MI). To allow in vitro maturation, oocytes at GV stage were cultured for 24 h, and oocytes at Metaphase I (MI) were cultured for 3 or 24 h, as described elsewhere (Nikiforaki et al., 2014). Oocytes were vitrified only at MII stage following the manufacturer’s protocol (Irvine Scientific, USA) and using an open support for vitrification (Cryotop®, Kitazato, Japan). Before ICSI, oocytes were warmed following manufacturer’s instructions (Irvine Scientific, USA), and randomly assigned to the study and the control groups. Oocytes were cultured under paraffin oil at standard culture conditions (37°C in 6% CO2 and 5% O2) prior to Ca2+ imaging. Source and culture of mouse oocytes Metaphase II oocytes were collected from 6 to 10-week-old B6D2F1 hybrid female mice following follicular hyperstimulation (Vanden Meerschaut et al., 2013). Oocyte collection was performed 14 h following hCG (Chorulon®, Intervet, Boxmeer, The Netherlands) injection. Further manipulations were performed in HEPES buffered potassium simplex optimized medium (KSOM-HEPES) prepared in-house, supplemented with 4 mg/ml bovine serum albumin (BSA, Calbiochem, Belgium). Denudation of cumulus cells was performed by a brief exposure to 200 IU/ml hyaluronidase. Oocytes were cultured in KSOM containing 4 mg/ml BSA under paraffin oil at standard culture conditions prior to Ca2+ imaging. Oocyte preparation for Ca2+ imaging Before ICSI, mouse and human MII oocytes were loaded with a ratiometric Ca2+-sensitive dye. Mouse oocytes were incubated in KSOM containing fura-2 acetoxymethyl-ester (Invitrogen, Belgium) while human oocytes were incubated in cook cleavage (CC) (Cook Ltd, Ireland) medium containing fura-PE3-acetoxymethyl-ester (Teflabs, Texas, USA) both at 7.5 μM and under standard culture conditions for 30 min. Oocytes were extensively washed in culture media prior to microinjection with human sperm. Sperm preparation for ICSI Frozen control and patient sperm samples were warmed at room temperature (RT) for 15 min and sperm selection was further performed by a swim-up method (Mortimer and Mortimer, 1992) or manually selected under an inverted light microscope after the first washing step in samples showing <5 million sperm/ml. ICSI Mouse ICSI was performed according to Vanden Meerschaut et al. (2013) using piezo electrical pulses as described by Yoshida). Sperm was pre-treated with lyso-lecithine (2 mM) to allow acrosome digestion before performing piezo-ICSI. Human oocytes were microinjected following a standard ICSI protocol, performed at 37°C. Of note, sperm were not pre-treated with lyso-lecithine prior to human ICSI. Ca2+ imaging Total recording period was established at 2 h for M-OCA (Vanden Meerschaut et al., 2013) and 10 h for H-OCA (Nikiforaki et al., 2014). Both mouse and human oocytes were placed in culture medium in a glass-bottomed dish (MatTek Corp., Ashland, MA, USA) covered with paraffin oil (Irvine Scientific, USA) 30 min after ICSI. Oocytes were individually monitored under an inverted epifluorescence microscope (Olympus IX71, Olympus Soft Imaging Solutions GmBH, Belgium) equipped to stabilize standard culture conditions (OKO labs, Olympus, GmBH, Belgium) with a ×10 objective and a filter switch (Lambda DG-4 filter switch, Sutter Instrument Company, Novato, CA, USA) to provide excitation at 340 and 380 nm. Data acquisition and analysis Ca2+ measurements were acquired every 10 s for 2 h for M-OCA and every 30 s for 10 h for H-OCA. Data were analyzed by Clampfit 10.2 software (Axon Laboratories, Molecular devices UK Ltd.). Baseline drifting was adjusted before retrieving values for amplitude (A, average at maximum fluorescence intensity per peak determined overall Ca2+ spikes) expressed in arbitrary units (AU) and peak durations (d, time for a Ca2+ spike to return to baseline) expressed in minutes (min). Frequency (F) reflected the total number of Ca2+ spikes per recording period. To further refine the Ca2+ spike frequency analysis, we categorized the frequencies as previously described by (Vanden Meerschaut et al., 2013); four different categories were distinguished based on the F: (0) total absence of Ca2+ spikes, (+) 1–2, (++) 3–9 and (+++) ≥10 (Fig. 1). We further defined the term ‘oscillatory activity’ as the presence of at least one Ca2+ spike during the recording period. Finally, the activation potential per sperm sample was scored using the product A × F (AU) determined from the Ca2+ spiking pattern. Figure 1 View largeDownload slide Frequency patterns of Ca2+ oscillations. Representative traces of Ca2+ oscillations observed during human oocyte activation. Ca2+ response pattern was categorized per oocyte according to a Ca2+ spike scoring system as previously used by Vanden Meerschaut et al. (2013). Four (0, +, ++ and +++) categories are defined based on the total number of Ca2+ spikes observed during the recording period of 10 h (see Materials and Methods). Figure 1 View largeDownload slide Frequency patterns of Ca2+ oscillations. Representative traces of Ca2+ oscillations observed during human oocyte activation. Ca2+ response pattern was categorized per oocyte according to a Ca2+ spike scoring system as previously used by Vanden Meerschaut et al. (2013). Four (0, +, ++ and +++) categories are defined based on the total number of Ca2+ spikes observed during the recording period of 10 h (see Materials and Methods). Statistical analyses Distribution of the frequency patterns, amplitudes and durations (presented as SEM) were analyzed applying a Kruskal–Wallis test for independent samples with a significance of P < 0.05. Oscillatory activity (presented as %) was analyzed by t-test (Statistical Package for the Social Sciences (SPSS® Statistics 24, IBM Corp., NY, USA). EasyROC: webtool for receiver operating characteristic (ROC) curve analysis (version 3.1) was used for analyzing the diagnostic test accuracy based on AOA outcomes with a defined confidence interval of 90% by non-parametric Mann–Whitney test for SE stimulation using a Bonferroni correction for multiple comparisons. Ethical approval This study was approved by the Ghent University Hospital Institutional Review Board (reference: B670201423110). Animal studies were approved by the Ghent University Hospital Ethical Committee for Laboratory Animals (reference: ECD number: 15/56). Results Mouse and human oocyte Ca2+ analysis in patients experiencing ICSI failures The sperm-induced Ca2+ oscillatory profile was evaluated in patients showing moderate (MOAT 2, n = 9, 78.3 ± 6%) to normal (MOAT 3, n = 7, 90.0 ± 4%) sperm activation capacity. The ability of sperm to generate normal Ca2+ response activity was first studied by M-OCA. Sperm from MOAT 2 patients triggered what we define as ‘oscillatory activity’ (≥ 1 Ca2+ spike/recording period), in 66.4% of the oocytes compared to 91.2% in the control group (Fig. 2). The frequency analysis furthermore demonstrated a significantly reduced percentage of oocytes in the high frequency range (at least three spikes, i.e. classes ‘++’ or ‘+++’) compared to the control group (Fig. 2). The oscillatory activity in MOAT 3 patients was 79.3%, i.e. intermediate between MOAT group 2 and the 91.2% in the control group; none of the subcategories (‘+’, ‘++’ or ‘+++’) were significantly different between both groups (Fig. 2). Figure 2 View largeDownload slide Mouse (i) and human (ii) oocyte Ca2+ analysis in patients experiencing ICSI failures and fertile individuals (control). Mouse oocyte activation test (MOAT). Frequency pattern of Ca2+ oscillations: ‘+++’ >10; ‘++’ 3–10; ‘+’ 1–2; ‘0’, absence of Ca2+ oscillations. Number (n) of oocytes. (i) Mouse oocyte Ca2+ analysis (M-OCA) outcome: Patients classified as MOAT group 2 revealed a slightly diminished oscillatory activity (66.4%), with no significant difference compared to the control group (91.2%) (P value = 0.142). Distribution of frequency pattern between MOAT group 2 patients and the control group (Vanden Meerschaut et al., 2013) was similar in categories from 0 to ‘++’. However, the proportion of oocytes showing ≥10 Ca2+ oscillations/2 h (+++) was significantly lower (P = 0.02) compared to the control. Results of MOAT group 3 showed that the oscillatory activity (79.3%) and the distribution of frequency pattern were similar to the control group (P > 0.05). (ii) Human oocyte Ca2+ analysis (H-OCA) outcome: Patients classified as MOAT group 2 revealed a prominent decrease in oscillatory activity (5.4%) and frequency of Ca2+ oscillations, with significant differences in the distribution of frequency pattern compared to MOAT group 3 and control group. Mean oscillatory activity (%) of MOAT 2 and 3 group compared to the control group were compared by an independent samples t-test. Distribution of frequency pattern across MOAT groups 2 and 3, and control group were was compared by an independent-samples Kruskal–Wallis test. *Significance level P < 0.05. Figure 2 View largeDownload slide Mouse (i) and human (ii) oocyte Ca2+ analysis in patients experiencing ICSI failures and fertile individuals (control). Mouse oocyte activation test (MOAT). Frequency pattern of Ca2+ oscillations: ‘+++’ >10; ‘++’ 3–10; ‘+’ 1–2; ‘0’, absence of Ca2+ oscillations. Number (n) of oocytes. (i) Mouse oocyte Ca2+ analysis (M-OCA) outcome: Patients classified as MOAT group 2 revealed a slightly diminished oscillatory activity (66.4%), with no significant difference compared to the control group (91.2%) (P value = 0.142). Distribution of frequency pattern between MOAT group 2 patients and the control group (Vanden Meerschaut et al., 2013) was similar in categories from 0 to ‘++’. However, the proportion of oocytes showing ≥10 Ca2+ oscillations/2 h (+++) was significantly lower (P = 0.02) compared to the control. Results of MOAT group 3 showed that the oscillatory activity (79.3%) and the distribution of frequency pattern were similar to the control group (P > 0.05). (ii) Human oocyte Ca2+ analysis (H-OCA) outcome: Patients classified as MOAT group 2 revealed a prominent decrease in oscillatory activity (5.4%) and frequency of Ca2+ oscillations, with significant differences in the distribution of frequency pattern compared to MOAT group 3 and control group. Mean oscillatory activity (%) of MOAT 2 and 3 group compared to the control group were compared by an independent samples t-test. Distribution of frequency pattern across MOAT groups 2 and 3, and control group were was compared by an independent-samples Kruskal–Wallis test. *Significance level P < 0.05. In a next step, 11 out of 16 patients were selected for an additional H-OCA analysis. First, to characterized the total Ca2+ response pattern elicited by human sperm in human oocytes we used three fertile control sperm samples (Supplementary Fig. S1). Strikingly, human oocytes microinjected with sperm from patients classified as MOAT 2 (n = 6; 77.6 ± 6%), had a prominently lower oscillatory activity amounting to 5.4% (at least one Ca2+ spike), compared to the 91.3% observed in the control group (Fig. 2). Frequency analysis in MOAT 2 demonstrated 0% distribution in the high frequency ‘+++’ group compared to 21.7% in control; 94.5% showed the absence of oscillatory activity (‘0’) compared to 8.7% in control (Fig. 2; Supplementary Table S2). In contrast, MOAT 3 patients (n = 5; 90.0 ± 4%) had an oscillatory activity comparable to the control group (Fig. 2; Supplementary Table S2). The distribution of frequency patterns in patients from MOAT group 3 was also comparable to the controls. Diagnostic accuracy of mouse and human oocyte Ca2+ analysis for the response to ICSI-AOA We evaluated the diagnostic accuracy of oocyte Ca2+ analysis tests for predicting AOA outcome, using the amplitude × frequency (A × F) scores calculated by M-OCA and H-OCA in a total of 11 patients. Plotting A × F values against fertilization rates after ICSI-AOA shows that abnormal A × F scores are associated with favorable AOA outcomes (Fig. 3). Therefore, we assumed that AOA would not have a further benefit in cases with normal A × F values. As a result, M-OCA and H-OCA A × F scores calculated in the study group may define a threshold value associated with abnormal fertilization rates after ICSI-AOA. Consequently, we determined cut-off points by calculating the Youden’s index (Supplementary Fig. S2). A × F values associated with abnormal sperm activation capacity were estimated ≤6.72 AU for M-OCA and ≤0.6 AU for H-OCA. M-OCA identified 5 out of 11 patients with abnormal sperm activation capacity. H-OCA reclassified two patients, yielding 7 out of 11 patients with abnormal sperm activation capacity (Fig. 3). Briefly, Patient 7 had complete absence of Ca2+ oscillations in the H-OCA test. Similarly, Patient 10 had a reduced H-OCA A × F score of 0.6 AU (Table I). The 5 remaining cases with abnormal sperm activation capacity revealed by M-OCA, also showed extremely low Ca2+ oscillatory responses with A × F scores estimated between 0.0 to 0.3 AU in human oocytes (Table I). Table I Clinical outcome. Patients experiencing ICSI failures who demonstrated their capacity to activate mouse oocytes. Participants showed fertilization rates (n zygotes with two pronuclei (pn) 16–20 h after ICSI) <33.3% and high MOAT activation rates from groups 2 and 3. M-OCA was performed in 16 patients. Patient  n cycles  ICSI Fertilization rate % (n 2pn/n MII)  MOAT activation rate (%)  M-OCA A × F AU  H-OCA A × F AU  n cycles  ICSI-AOA Fertilization Rate % (n 2pn/n MII)  Embryo transfer (yes/no)  Pregnancy outcome  P1  3  27.3 (6/22)  68  6.72  0.3  2  84.6 (11/13)  Yes  Singleton  P2  2  6.7 (1/15)  72  3.95  0  2  18.8 (3/16)  Yes  Ongoing pregnancy  P3  3  33.3 (11/18)  73  8.75  *  –  –  –  –  P4  1  0.0 (0/5)  78  6.55  0  1  80.0 (8/10)  Yes  Singleton  P5  1  5.6 (1/18)  81  1.4  0  1  0/11  No  –  P6  2  (2/22)  82  0.7  *  2  –  –  –  P7  2  0.0 (0/13)  83  8.01  0  2  30.8 (4/13)  No  –  P8  4  (2/87)  84  2.04  0  1  80.0 (12/15)  Yes  Singleton  P9  3  (3/16)  84  6.13  *  1  –  –  –  P10  1  0.0 (0/7)  85  12.57  0.6  2  71.9 (23/32)  Yes  Singleton  P11  1  0.0 (0/9)  87  16.71  11  1  0.0 (0/8)  No  –  P12  1  0/10  89  3.27  *  –  –  –  –  P13  2  11.8 (2/17)  90  27.8  7.8  1  0.0 (0/9)  No  –  P14  2  0.0 (0/11)  91  23.1  *  –  –  –  –  P15  1  0.0 (0/10)  92  9.87  2.8  1  0.0 (0/9)  No  –  P16  2  0.0 (0/7)  96  8.95  6.8  1  0.0 (0/4)  No  –  Patient  n cycles  ICSI Fertilization rate % (n 2pn/n MII)  MOAT activation rate (%)  M-OCA A × F AU  H-OCA A × F AU  n cycles  ICSI-AOA Fertilization Rate % (n 2pn/n MII)  Embryo transfer (yes/no)  Pregnancy outcome  P1  3  27.3 (6/22)  68  6.72  0.3  2  84.6 (11/13)  Yes  Singleton  P2  2  6.7 (1/15)  72  3.95  0  2  18.8 (3/16)  Yes  Ongoing pregnancy  P3  3  33.3 (11/18)  73  8.75  *  –  –  –  –  P4  1  0.0 (0/5)  78  6.55  0  1  80.0 (8/10)  Yes  Singleton  P5  1  5.6 (1/18)  81  1.4  0  1  0/11  No  –  P6  2  (2/22)  82  0.7  *  2  –  –  –  P7  2  0.0 (0/13)  83  8.01  0  2  30.8 (4/13)  No  –  P8  4  (2/87)  84  2.04  0  1  80.0 (12/15)  Yes  Singleton  P9  3  (3/16)  84  6.13  *  1  –  –  –  P10  1  0.0 (0/7)  85  12.57  0.6  2  71.9 (23/32)  Yes  Singleton  P11  1  0.0 (0/9)  87  16.71  11  1  0.0 (0/8)  No  –  P12  1  0/10  89  3.27  *  –  –  –  –  P13  2  11.8 (2/17)  90  27.8  7.8  1  0.0 (0/9)  No  –  P14  2  0.0 (0/11)  91  23.1  *  –  –  –  –  P15  1  0.0 (0/10)  92  9.87  2.8  1  0.0 (0/9)  No  –  P16  2  0.0 (0/7)  96  8.95  6.8  1  0.0 (0/4)  No  –  Clinical outcome: ICSI-AOA restored fertilization rates to normal in four out of seven patients with abnormal H-OCA A × F score. The four patients achieved a pregnancy after embryo transfer. Additionally, P2, who showed H-OCA A × F score = 0 AU and fertilization rate after ICSI-AOA of 18.8%, also achieved a pregnancy. 2pn, two pronuclei/diploids; MII, Metaphase II; MOAT, mouse oocyte activation test; M-OCA, mouse oocyte calcium analysis. H-OCA, human oocyte calcium analysis; AOA, assisted oocyte activation. (*) H-OCA was performed in patients who underwent AOA in our center (n = 11). Figure 3 View largeDownload slide Representation of M-OCA and H-OCA A × F scores (Y-axis) vs the fertilization rate after ICSI-AOA (X-axis) demonstrating that abnormal A × F scores are associated with favorable AOA outcomes, corresponding to normal fertilization (>70% of diploid zygotes 16–20 h after ICSI-AOA). M-OCA (i) reveals that all the patients studied could generate Ca2+ oscillations in mouse oocytes. In contrast, H-OCA (ii) clearly demonstrate the incapability of some sperm cells to induce Ca2+ release in human oocytes showing A × F values in the 0 AU to 0.6 AU range. Area highlighted in grey include cases showing abnormal A × F and normal fertilization after ICSI-AOA. (*) H-OCA reclassified P7 and P10 as having abnormal sperm activation capacity. Figure 3 View largeDownload slide Representation of M-OCA and H-OCA A × F scores (Y-axis) vs the fertilization rate after ICSI-AOA (X-axis) demonstrating that abnormal A × F scores are associated with favorable AOA outcomes, corresponding to normal fertilization (>70% of diploid zygotes 16–20 h after ICSI-AOA). M-OCA (i) reveals that all the patients studied could generate Ca2+ oscillations in mouse oocytes. In contrast, H-OCA (ii) clearly demonstrate the incapability of some sperm cells to induce Ca2+ release in human oocytes showing A × F values in the 0 AU to 0.6 AU range. Area highlighted in grey include cases showing abnormal A × F and normal fertilization after ICSI-AOA. (*) H-OCA reclassified P7 and P10 as having abnormal sperm activation capacity. We further analyzed several properties of the A × F score to identify patients that could potentially benefit from AOA treatment using formulations to determine sensitivity, specificity and accuracy of the diagnostic test (Supplementary Table S1). Sensitivity of M-OCA was calculated as 0.75 (CI 90%: 0.19–0.99) (Table II) with three cases showing abnormal Ca2+ oscillation activity out of a total of four cases with successful fertilization after ICSI-AOA (Fig. 3). Interestingly, H-OCA revealed a sensitivity of 1.00 (CI 90%: 0.39–1) (Table II), with four cases showing abnormal Ca2+ oscillation activity with further benefit of ICSI-AOA, while none of the cases with normal Ca2+ release activity attained a normal fertilization rate after ICSI-AOA (Fig. 3). Moreover, M-OCA had a specificity of 0.71 (CI 90%: 0.29–0.96) (Table II), showing the presence of two cases with abnormal Ca2+ release activity without further benefit from ICSI-AOA. H-OCA revealed a specificity of 0.57 (CI 90%: 0.18–0.90) (Table II); with four out of seven cases with abnormal Ca2+ release activity which did not respond favorably to ICSI-AOA treatment (Supplementary data, Table S1). We further calculated the accuracy for M-OCA and H-OCA, which was estimated separately as 0.73 for each of the tests (Table II). Table II Diagnostic accuracy of M-OCA and H-OCA based in the response to ICSI-AOA treatment. Data described is used to calculated the sensitivity, specificity and accuracy of M-OCA and H-OCA, respectively. Cases showing abnormal A × F and normal fertilization after ICSI-AOA (a). Cases showing abnormal A × F and abnormal fertilization after ICSI-AOA (b). Cases showing normal A × F and normal fertilization after ICSI-AOA (c). Cases showing normal and abnormal fertilization after ICSI-AOA (d).   Definition  M-OCA  H-OCA  Sensitivity (a)/(a + c)  Cases with abnormal Ca2+ response activity experiencing successful fertilization after ICSI-AOA divided by all cases experiencing successful fertilization after ICSI-AOA  3/(3 + 1) = 0.75  4/(4 + 0) = 1  Specificity (d)/(d + b)  Cases with normal Ca2+ response activity which did not respond favorably to ICSI-AOA treatment divided by the total of cases which do not show further benefit from ICSI-AOA treatment  5/(5 + 2) = 0.71  4/(4 + 3) = 0.57  Accuracy (a + d)/(a + b + c + d)  Cases with expected responses to ICSI-AOA (abnormal A × F score associated with normal fertilization after AOA and normal A × F score associated with failed fertilization after ICSI-AOA) divided by the total of cases studied  (3 + 5)/11 = 0.73  (4 + 4)/11 = 0.73    Definition  M-OCA  H-OCA  Sensitivity (a)/(a + c)  Cases with abnormal Ca2+ response activity experiencing successful fertilization after ICSI-AOA divided by all cases experiencing successful fertilization after ICSI-AOA  3/(3 + 1) = 0.75  4/(4 + 0) = 1  Specificity (d)/(d + b)  Cases with normal Ca2+ response activity which did not respond favorably to ICSI-AOA treatment divided by the total of cases which do not show further benefit from ICSI-AOA treatment  5/(5 + 2) = 0.71  4/(4 + 3) = 0.57  Accuracy (a + d)/(a + b + c + d)  Cases with expected responses to ICSI-AOA (abnormal A × F score associated with normal fertilization after AOA and normal A × F score associated with failed fertilization after ICSI-AOA) divided by the total of cases studied  (3 + 5)/11 = 0.73  (4 + 4)/11 = 0.73  Discussion We evaluated whether the use of human oocytes would allow the discrimination between sperm or oocyte-related activation deficiencies in patients experiencing fertilization failures after routine ICSI and whose sperm were capable of activating mouse oocytes. Patients showing slightly diminished capacity of activating mouse oocytes (MOAT 2) revealed abnormal Ca2+ oscillatory patterns during mouse oocyte activation. Interestingly, the six MOAT 2 patients, who were further analyzed by H-OCA, showed a pronounced decrease in Ca2+ spiking activity in human oocytes. In support of previous findings, M-OCA revealed abnormal Ca2+ profiles in patients from MOAT group 2, confirming that sperm-related activation deficiencies, rather than oocyte deficiencies, are responsible for fertilization failures in these cases (Vanden Meerschaut et al., 2013). The present study revealed for the first time that sperm from patients showing reduced MOAT and M-OCA outcomes, are totally incapable to induce Ca2+ oscillations in human oocytes. Fertilization failures experienced by patients with normal capacity for activating mouse oocytes (MOAT 3) were initially thought to be associated with oocyte-related oocyte activation deficiencies (Heindryckx et al., 2005). However, later findings demonstrated the presence of abnormal Ca2+ oscillatory patterns after M-OCA also in some patients categorized as MOAT 3 (Vanden Meerschaut et al., 2013). Our results show that the majority of the patients classified as MOAT 3 had normal Ca2+ oscillatory patterns after M-OCA. However, H-OCA demonstrated the incapability of inducing normal Ca2+ responses in two patients who showed high MOAT activation rates and M-OCA A × F scores. Overall, 7 out of 11 patients showed incapability of generating a normal Ca2+ response in human oocytes. Reduced sperm activation potential, particularly in MOAT 1 patients, was previously correlated with favorable responses to ICSI-AOA (Heindryckx et al., 2008, Vanden Meerschaut et al., 2012). Still, the real benefit of the ICSI-AOA treatment in patients from MOAT groups 2 and 3 could not be clearly established. Our analysis reveals that Ca2+ analysis can be used to predict fertilization success after ICSI-AOA, with H-OCA yielding higher sensitivity than M-OCA to detect the presence of human sperm activation deficiencies. The increase in sensitivity of H-OCA over M-OCA results from the absence of cases with normal sperm activation capacity after H-OCA, and hence favorable responses after ICSI-AOA. However, the success of the treatment will be limited to an estimated 57% of the cases presenting with abnormal H-OCA. Interestingly, the non-responding cases will be identified subsequently at the occasion of ICSI-AOA treatment, allowing them to be classified as having an additional oocyte-related oocyte activation deficiency (Fig. 4). Overall, M-OCA and H-OCA showed similar capacity to allocate cases according to ICSI-AOA response. Here, it is interesting to note that H-OCA detected the incapability of human sperm to induce any or few Ca2+ events in cases which showed the ability of generating Ca2+ responses and subsequent oocyte activation in mouse oocytes. In our opinion, H-OCA reveals a clear cut off in the capacity of sperm to generate Ca2+ responses, which supports the use of human oocytes as the most optimal test for the study of sperm activation potential. H-OCA data showed that, four out of the seven cases with reduced A × F score experienced an improvement in fertilization rate after ICSI-AOA, with four successful pregnancies achieved to term. Intriguingly, ICSI-AOA did not overcome failed fertilization in the three cases out of the seven diagnosed with sperm activation deficiencies by all three functional tests (MOAT, M-OCA and H-OCA). As previously mentioned, in these three cases we suspect the presence of an oocyte-related oocyte activation deficiency in addition to a sperm activation incapability (Fig. 4). Given that our department is a reference center for patients experiencing fertilization failure, we may have identified a higher proportion of cases with a combined sperm–oocyte factor in the present study. In our experience, couples suffering from suspected combined sperm–oocyte factors require particular attention. Patients experiencing fertilization failures are frequently redirected to gamete donation programs. Our findings indicate that identifying sperm activation deficiencies could be important in making an informed decision regarding the use of donor oocytes or sperm in future treatments. Our data support that ICSI-AOA should be applied in patients with diagnosed sperm activation deficiencies, in this case irrespective of the origin of the oocytes (Fig. 4). Figure 4 View largeDownload slide Treatment counseling algorithm based on human oocyte Ca2+ analysis (H-OCA) result. H-OCA allows to distinguish between the presence of oocyte or sperm factors responsible of oocyte activation failure. Cases with normal H-OCA (normal sperm activation capacity) are associated with unfavorable responses to ICSI-assisted oocyte activation (AOA) treatment. Treatment advice follows the participation in an oocyte donation program. Cases with abnormal H-OCA (abnormal sperm activation capacity) are associated with favorable responses to ICSI-AOA. The treatment advice follows the use of AOA irrespective of the origin of the oocytes. Figure 4 View largeDownload slide Treatment counseling algorithm based on human oocyte Ca2+ analysis (H-OCA) result. H-OCA allows to distinguish between the presence of oocyte or sperm factors responsible of oocyte activation failure. Cases with normal H-OCA (normal sperm activation capacity) are associated with unfavorable responses to ICSI-assisted oocyte activation (AOA) treatment. Treatment advice follows the participation in an oocyte donation program. Cases with abnormal H-OCA (abnormal sperm activation capacity) are associated with favorable responses to ICSI-AOA. The treatment advice follows the use of AOA irrespective of the origin of the oocytes. Our study further diagnosed 4 out of 11 cases with normal H-OCA A × F scores who experienced ICSI failure after AOA. Since all four patients showed the capacity of generating Ca2+ oscillations in mouse, and most importantly in human oocytes, experiencing a failure in fertilization after ICSI-AOA points to the presence of a sole oocyte-related activation deficiency. These observations indicate that H-OCA can detect the cases who would not benefit from the Ca2+ replacement induced by Ca2+ ionophores during AOA procedure. In this regard, these cases would be candidates for participating in oocyte donation programs (Fig. 4). We hypothesize that the mechanisms involved in supporting Ca2+ oscillatory activity, such as IP3Rs or plasma membrane Ca2+ ATPases (PMCAs), may not be directly responsible for the oocyte activation failure (Yeste et al., 2016). Further investigations are urgently needed to develop AOA techniques aimed at tackling failed fertilization associated with oocyte-related oocyte activation failure. It is worth mentioning that procedures as IVM and cryopreservation have a slight impact on Ca2+ oscillatory patterns, with, for instance, vitrified/warmed oocytes showing variations in amplitudes and frequencies when compared to their fresh counterparts (Nikiforaki et al., 2014). Similarly, the presence of SERa in MII oocytes have a moderate effect on the Ca2+ oscillatory response (De Gheselle et al., 2014). However, accumulating evidence supports the activation competence of these oocytes, with fertilization rates comparable to their fresh counterparts (Cobo et al., 2012; Rienzi et al., 2012). Moreover, sperm cryopreservation also showed to have an influence on PLCz content (Kashir et al., 2011). Since reduced levels of PLCz have been associated with impaired fertilization (Heytens et al., 2009), it is worth taking into account that our results might not fully reflect the sperm activation capacity in our control and study groups. Further investigations are required to elucidate the real impact of cryopreservation in reducing sperm activation potential. Our results indicate that Ca2+ pattern analysis is a sensitive tool to predict the response to AOA. However, it should be noted that Ca2+ imaging has its intrinsic complexity (Swann, 2013), which requires specific equipment to monitor fluorescence changes over time. Interestingly, free cytoplasmic Ca2+ oscillations have been correlated with cytoplasmic movements observed in oocytes during fertilization (Ajduk et al., 2011). These cytoplasmic contractions, provoked by the polymerization of actin–myosin proteins, can be monitored by regular light microscopy, as shown already in mouse (Ajduk et al., 2011) and human (Swann et al., 2012) studies. These observations define a paradigm for the study of the early events of fertilization which could be feasibly adopted in IVF laboratories due to the emergence of time-lapse imaging technologies (Freour et al., 2015). In this regard, the present data provide an important template of the Ca2+ signature observed during human fertilization in cases with normal, low and failed fertilization after conventional ICSI. The use of Ca2+ ionophores for human ICSI-AOA has resulted in normal fertilization rates (Ebner et al., 2015), subsequent pregnancies (Heindryckx et al., 2005) and healthy livebirths (D’haeseleer et al., 2014; Vanden Meerschaut et al., 2014a, 2014b; Sfontouris et al., 2015; Miller et al., 2016). Nevertheless, safety concerns remain regarding the use of Ca2+ ionophores to induce oocyte activation (Santella and Dale, 2015; van Blerkom et al., 2015), which is generated by a single and high Ca2+ transient rather than the natural series of Ca2+ oscillations. Moreover, specific Ca2+ signatures during oocyte activation would likely have an impact on cellular events observed during oocyte activation (Ducibella et al., 2002), on the subsequent embryonic development (Ozil, 1998; Ozil et al., 2006; Ducibella and Fissore, 2008) and they are possibly associated with variations in gene expression profiles (Ozil et al., 2006, Rogers et al., 2006). However, the efficiency of Ca2+ ionophores to induce fertilization is supported by findings that described that mammalian fertilization could be mediated by the total summation of the individual Ca2+ spikes rather than by specific patterns (Ozil et al., 2005; Tóth et al., 2006). Further investigations are required to elucidate the effect of Ca2+ oscillation pattern on pre- and post-implantation events, and most importantly on the off-spring. We showed the importance of diagnosing sperm-related activation capacity prior to the application of ICSI-AOA in patients experiencing ICSI failures. H-OCA is the most sensitive diagnostic test to study human sperm activation potential, and in combination with fertilization outcomes after ICSI-AOA, H-OCA identifies cases with suspected oocyte-related activation deficiencies. Further research is needed to explore oocyte factors associated with failed fertilization and possible treatments to restore fertilization in this challenging group of patients. Supplementary data Supplementary data are available at Human Reproduction online. Author’s contributions B.H and M.F.B conceived the study. M.F.B. and Y.L. conducted experiments, performed data acquisition and analysis. L.D. validated patient’s selection and treatment follow-up. D.B. contributed to patient’s selection and figure design. F.V.M. provided patient’s historical data. M.F.B. wrote the article. All authors edited the article. P.D.S., B.H. and L.L. were involved in interpreting the results, revised the article and approved the final draft. Funding Flemish fund for scientific research (FWO-Vlaanderen, G060615N). Conflict of interest The authors have no conflict of interest to declare. Acknowledgements We would also like to thank Mrs Sylvie Lierman for her assistance in sperm sample preparation and to I.V.F. lab team during the collection of human oocytes. We would also like to thank Dr Fiona Dunlevy for editorial assistance. References Ajduk A, Ilozue T, Windsor S, Yu Y, Seres KB, Bomphrey RJ, Tom BD, Swann K, Thomas A, Graham C et al.  . Rhythmic actomyosin-driven contractions induced by sperm entry predict mammalian embryo viability. Nat Commun  2011; 2: 417. Google Scholar CrossRef Search ADS PubMed  Ajduk A, Małagocki A, Maleszewski M. Cytoplasmic maturation of mammalian oocytes: development of a mechanism responsible for sperm-induced Ca2+ oscillations. Reprod Biol  2008; 8: 3– 22. Google Scholar CrossRef Search ADS PubMed  Bhattacharya S, Maheshwari A, Mollison J. Factors associated with failed treatment: an analysis of 121,744 women embarking on their first IVF cycles. PLoS One  2013; 8: e82249. Google Scholar CrossRef Search ADS PubMed  Cobo A, de los Santos MJ, Castellò D, Gámiz P, Campos P, Remohí J. Outcomes of vitrified early cleavage-stage and blastocyst-stage embryos in a cryopreservation program: evaluation of 3,150 warming cycles. Fertil Steril  2012; 98: 1138– 1146.e1131. Google Scholar CrossRef Search ADS PubMed  Combelles CM, Morozumi K, Yanagimachi R, Zhu L, Fox JH, Racowsky C. Diagnosing cellular defects in an unexplained case of total fertilization failure. Hum Reprod  2010; 25: 1666– 1671. Google Scholar CrossRef Search ADS PubMed  De Gheselle N, Verpoest, De Croo, Lu, Heindryckx, Van den Abbeel, De Sutter. The effect of smooth endoplasmic reticulum aggregates in human oocytes on calcium signalling and the significance for oocyte collection cycle outcome. Abstracts of the 30th Annual Meeting of ESHRE, Munich, Germany, 29 June–2 July, 2014. D’haeseleer E, Vanden Meerschaut F, Bettens K, Luyten A, Gysels H, Thienpont Y, De Witte G, Heindryckx B, Oostra A, Roeyers H et al.  . Language development of children born following intracytoplasmic sperm injection (ICSI) combined with assisted oocyte activation (AOA). Int J Lang Commun Disord  2014; 49: 702– 709. Google Scholar CrossRef Search ADS PubMed  Ducibella T, Fissore R. The roles of Ca2+, downstream protein kinases, and oscillatory signaling in regulating fertilization and the activation of development. Dev Biol  2008; 315: 257– 279. Google Scholar CrossRef Search ADS PubMed  Ducibella T, Huneau D, Angelichio E, Xu Z, Schultz RM, Kopf GS, Fissore R, Madoux S, Ozil JP. Egg-to-embryo transition is driven by differential responses to Ca(2+) oscillation number. Dev Biol  2002; 250: 280– 291. Google Scholar CrossRef Search ADS PubMed  Ebner T, Montag M, Oocyte Activation Study G, Montag M, Van der Ven K, Van der Ven H, Ebner T, Shebl O, Oppelt P, Hirchenhain J et al.  . Live birth after artificial oocyte activation using a ready-to-use ionophore: a prospective multicentre study. Reprod Biomed Online  2015; 30: 359– 365. Google Scholar CrossRef Search ADS PubMed  Escoffier J, Yassine S, Lee HC, Martinez G, Delaroche J, Coutton C, Karaouzene T, Zouari R, Metzler-Guillemain C, Pernet-Gallay K et al.  . Subcellular localization of phospholipase Czeta in human sperm and its absence in DPY19L2-deficient sperm are consistent with its role in oocyte activation. Mol Hum Reprod  2015; 21: 157– 168. Google Scholar CrossRef Search ADS PubMed  Freour T, Basile N, Barriere P, Meseguer M. Systematic review on clinical outcomes following selection of human preimplantation embryos with time-lapse monitoring. Hum Reprod Update  2015; 21: 153– 154. Google Scholar CrossRef Search ADS PubMed  Heindryckx B, De Gheselle S, Gerris J, Dhont M, De Sutter P. Efficiency of assisted oocyte activation as a solution for failed intracytoplasmic sperm injection. Reprod Biomed Online  2008; 17: 662– 668. Google Scholar CrossRef Search ADS PubMed  Heindryckx B, Van der Elst J, De Sutter P, Dhont M. Treatment option for sperm- or oocyte-related fertilization failure: assisted oocyte activation following diagnostic heterologous ICSI. Hum Reprod  2005; 20: 2237– 2241. Google Scholar CrossRef Search ADS PubMed  Heytens E, Parrington J, Coward K, Young C, Lambrecht S, Yoon SY, Fissore RA, Hamer R, Deane CM, Ruas M et al.  . Reduced amounts and abnormal forms of phospholipase C zeta (PLCzeta) in spermatozoa from infertile men. Hum Reprod  2009; 24: 2417– 2428. Google Scholar CrossRef Search ADS PubMed  Kashir J, Heynen A, Jones C, Durrans C, Craig J, Gadea J, Turner K, Parrington J, Coward K. Effects of cryopreservation and density-gradient washing on phospholipase C zeta concentrations in human spermatozoa. Reprod Biomed Online  2011; 23: 263– 267. Google Scholar CrossRef Search ADS PubMed  Kashir J, Konstantinidis M, Jones C, Lemmon B, Lee HC, Hamer R, Heindryckx B, Deane CM, De Sutter P, Fissore RA et al.  . A maternally inherited autosomal point mutation in human phospholipase C zeta (PLCζ) leads to male infertility. Hum Reprod  2012; 27: 222– 231. Google Scholar CrossRef Search ADS PubMed  Kuentz P, Vanden Meerschaut F, Elinati E, Nasr-Esfahani MH, Gurgan T, Iqbal N, Carré-Pigeon F, Brugnon F, Gitlin SA, Velez de la Calle J et al.  . Assisted oocyte activation overcomes fertilization failure in globozoospermic patients regardless of the DPY19L2 status. Hum Reprod  2013; 28: 1054– 1061. Google Scholar CrossRef Search ADS PubMed  Miller N, Biron-Shental T, Sukenik-Halevy R, Klement AH, Sharony R, Berkovitz A. Oocyte activation by calcium ionophore and congenital birth defects: a retrospective cohort study. Fertil Steril  2016; 106: 590– 596.e592. Google Scholar CrossRef Search ADS PubMed  Mortimer D, Mortimer ST. Methods of sperm preparation for assisted reproduction. Ann Acad Med Singapore  1992; 21: 517– 524. Google Scholar PubMed  Nikiforaki D, Vanden Meerschaut F, Qian C, De Croo I, Lu Y, Deroo T, Van den Abbeel E, Heindryckx B, De Sutter P. Oocyte cryopreservation and in vitro culture affect calcium signalling during human fertilization. Hum Reprod  2014; 29: 29– 40. Google Scholar CrossRef Search ADS PubMed  Nomikos M, Theodoridou M, Elgmati K, Parthimos D, Calver BL, Buntwal L, Nounesis G, Swann K, Lai FA. Human PLCζ exhibits superior fertilization potency over mouse PLCζ in triggering the Ca2+ oscillations required for mammalian oocyte activation. Mol Hum Reprod  2014. Ozil JP. Role of calcium oscillations in mammalian egg activation: experimental approach. Biophys Chem  1998; 72: 141– 152. Google Scholar CrossRef Search ADS PubMed  Ozil JP, Banrezes B, Tóth S, Pan H, Schultz RM. Ca2+ oscillatory pattern in fertilized mouse eggs affects gene expression and development to term. Dev Biol  2006; 300: 534– 544. Google Scholar CrossRef Search ADS PubMed  Ozil JP, Markoulaki S, Toth S, Matson S, Banrezes B, Knott JG, Schultz RM, Huneau D, Ducibella T. Egg activation events are regulated by the duration of a sustained [Ca2+]cyt signal in the mouse. Dev Biol  2005; 282: 39– 54. Google Scholar CrossRef Search ADS PubMed  Rawe VY, Olmedo SB, Nodar FN, Doncel GD, Acosta AA, Vitullo AD. Cytoskeletal organization defects and abortive activation in human oocytes after IVF and ICSI failure. Mol Hum Reprod  2000; 6: 510– 516. Google Scholar CrossRef Search ADS PubMed  Rienzi L, Cobo A, Paffoni A, Scarduelli C, Capalbo A, Vajta G, Remohí J, Ragni G, Ubaldi FM. Consistent and predictable delivery rates after oocyte vitrification: an observational longitudinal cohort multicentric study. Hum Reprod  2012; 27: 1606– 1612. Google Scholar CrossRef Search ADS PubMed  Rockliff HE, Lightman SL, Rhidian E, Buchanan H, Gordon U, Vedhara K. A systematic review of psychosocial factors associated with emotional adjustment in in vitro fertilization patients. Hum Reprod Update  2014; 20: 594– 613. Google Scholar CrossRef Search ADS PubMed  Rogers NT, Halet G, Piao Y, Carroll J, Ko MS, Swann K. The absence of a Ca(2+) signal during mouse egg activation can affect parthenogenetic preimplantation development, gene expression patterns, and blastocyst quality. Reproduction  2006; 132: 45– 57. Google Scholar CrossRef Search ADS PubMed  Rybouchkin A, Dozortsev D, de Sutter P, Qian C, Dhont M. Intracytoplasmic injection of human spermatozoa into mouse oocytes: a useful model to investigate the oocyte-activating capacity and the karyotype of human spermatozoa. Hum Reprod  1995; 10: 1130– 1135. Google Scholar CrossRef Search ADS PubMed  Santella L, Dale B. Assisted yes, but where do we draw the line? Reprod Biomed Online  2015; 31: 476– 478. Google Scholar CrossRef Search ADS PubMed  Saunders CM, Larman MG, Parrington J, Cox LJ, Royse J, Blayney LM, Swann K, Lai FA. PLC zeta: a sperm-specific trigger of Ca(2+) oscillations in eggs and embryo development. Development  2002; 129: 3533– 3544. Google Scholar PubMed  Sfontouris IA, Nastri CO, Lima ML, Tahmasbpourmarzouni E, Raine-Fenning N, Martins WP. Artificial oocyte activation to improve reproductive outcomes in women with previous fertilization failure: a systematic review and meta-analysis of RCTs. Hum Reprod  2015; 30: 1831– 1841. Google Scholar CrossRef Search ADS PubMed  Swann K. Measuring Ca2+ oscillations in mammalian eggs. Methods Mol Biol  2013; 957: 231– 248. Google Scholar CrossRef Search ADS PubMed  Swann K, Lai FA. Egg activation at fertilization by a soluble sperm protein. Physiol Rev  2016; 96: 127– 149. Google Scholar CrossRef Search ADS PubMed  Swann K, Windsor S, Campbell K, Elgmati K, Nomikos M, Zernicka-Goetz M, Amso N, Lai FA, Thomas A, Graham C. Phospholipase C-ζ-induced Ca2+ oscillations cause coincident cytoplasmic movements in human oocytes that failed to fertilize after intracytoplasmic sperm injection. Fertil Steril  2012; 97: 742– 747. Google Scholar CrossRef Search ADS PubMed  Tóth S, Huneau D, Banrezes B, Ozil JP. Egg activation is the result of calcium signal summation in the mouse. Reproduction  2006; 131: 27– 34. Google Scholar CrossRef Search ADS PubMed  van Blerkom J, Cohen J, Johnson M. A plea for caution and more research in the ‘experimental’ use of ionophores in ICSI. Reprod Biomed Online  2015; 30: 323– 324. Google Scholar CrossRef Search ADS PubMed  Vanden Meerschaut F, D’Haeseleer E, Gysels H, Thienpont Y, Dewitte G, Heindryckx B, Oostra A, Roeyers H, Van Lierde K, De Sutter P. Neonatal and neurodevelopmental outcome of children aged 3–10 years born following assisted oocyte activation. Reprod Biomed Online  2014a; 28: 54– 63. Google Scholar CrossRef Search ADS PubMed  Vanden Meerschaut F, Leybaert L, Nikiforaki D, Qian C, Heindryckx B, De Sutter P. Diagnostic and prognostic value of calcium oscillatory pattern analysis for patients with ICSI fertilization failure. Hum Reprod  2013; 28: 87– 98. Google Scholar CrossRef Search ADS PubMed  Vanden Meerschaut F, Nikiforaki D, De Gheselle S, Dullaerts V, Van den Abbeel E, Gerris J, Heindryckx B, De Sutter P. Assisted oocyte activation is not beneficial for all patients with a suspected oocyte-related activation deficiency. Hum Reprod  2012; 27: 1977– 1984. Google Scholar CrossRef Search ADS PubMed  Vanden Meerschaut F, Nikiforaki D, Heindryckx B, De Sutter P. Assisted oocyte activation following ICSI fertilization failure. Reprod Biomed Online  2014b; 28: 560– 571. Google Scholar CrossRef Search ADS PubMed  Wakai T, Zhang N, Vangheluwe P, Fissore RA. Regulation of endoplasmic reticulum Ca2+ oscillations in mammalian eggs. J Cell Sci  2013; 126: 5714– 5724. Google Scholar CrossRef Search ADS PubMed  Yeste M, Jones C, Amdani SN, Coward K. Oocyte activation and fertilisation: crucial contributors from the sperm and oocyte. Results Probl Cell Differ  2017; 59: 213– 239. Google Scholar CrossRef Search ADS PubMed  Yeste M, Jones C, Amdani SN, Patel S, Coward K. Oocyte activation deficiency: a role for an oocyte contribution? Hum Reprod Update  2016; 22: 23– 47. Google Scholar CrossRef Search ADS PubMed  Yoon SY, Jellerette T, Salicioni AM, Lee HC, Yoo MS, Coward K, Parrington J, Grow D, Cibelli JB, Visconti PE et al.  . Human sperm devoid of PLC, zeta 1 fail to induce Ca(2+) release and are unable to initiate the first step of embryo development. J Clin Invest  2008; 118: 3671– 3681. Google Scholar CrossRef Search ADS PubMed  Yoshida N, Perry AC. Piezo-actuated mouse intracytoplasmic sperm injection (ICSI). Nat Protoc  2007; 2: 296– 304. Google Scholar CrossRef Search ADS PubMed  © The Author(s) 2018. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

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Human ReproductionOxford University Press

Published: Mar 1, 2018

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