Middle East Fertility Society Journal xxx (2017) xxx–xxx Contents lists available at ScienceDirect Middle East Fertility Society Journal journal homepage: www.sciencedirect.com Original Article Co-culture of human embryos with autologous cumulus cell clusters and its beneficial impact of secreted growth factors on preimplantation development as compared to standard embryo culture in assisted reproductive technologies (ART) q Alexandros Vithoulkas a, Michael Levanduski b, Vasilios T. Goudas a,⇑, Karl Illmensee a a b Genesis Fertility Center, Patras 264 42, Greece Embryoserv Inc., NJ 07675, USA a r t i c l e i n f o Article history: Received 23 January 2017 Revised 7 May 2017 Accepted 29 May 2017 Available online xxxx Keywords: IVF IVF outcome Human embryo co-culture Standard embryo culture Standard culture media Autologous cumulus cell clusters Embryo culture supernatant Uterine transfer Follicular aspiration Cumulus-oocyte complex Vitrification Preimplantation embryo Human early embryo Human preimplantation embryo development Grade-1 embryo Grade-1 blastocyst Cellular fragmentation a b s t r a c t Objective: To analyze the concentration of growth factors secreted by autologous cumulus cell clusters during in vitro human embryo co-culture and investigate the impact of culture supplementation with autologous cumulus cell clusters on embryo development and pregnancy rate as compared to standard culture procedure. Design: Randomized prospective comparative study. Setting: Private IVF clinic. Patients: 90 IVF patient couples ranging at the age between 35 and 40 (mean age of 36.5) agreed to participate in this study. In this IVF group, 64 first trials, 22 s trials and 4 third trials were included. 48 cases have been terminated at day 3 of culture and 42 of them have been continued up to day 5. Of the 48 cases assigned for day-3 culture, 208 MII-oocytes were randomly assigned for co-culture and 197 MII-oocytes were assigned for standard culture. The selection was done by alternating the oocyte immediately after stripping to co-culture or control, starting always with co-culture. Interventions: All samples were analyzed with Milliplex factor-specific magnetic-bead panels with phycoerythrin fluorescent factor-specific antibodies using immunofluorescence cytometry technology by Lab Supplies Inc. laboratories in Athens, Greece. The MFI (Mean Fluorescence Intensity) values were calibrated and the readings were translated in pg/ml and calculated against the background values of culture medium alone. The statistical analysis of all data were processed with the Two-tailed T-test form. Main outcome measures: 36 IVF cases terminated at day 3 were randomly chosen. Supernatants from 17 cases were analyzed for seven different growth factors, namely, epidermal growth factor (EGF), fibroblast growth factor 1 and 2 (FGF-1 and FGF-2), vascular endothelial growth factor A, C and D (VEGF-A, VEGF-C and VEGF-D) and Leptin. Supernatants from 19 cases were analyzed for Insulin-like Growth Factor I and II (IGF-I and IGF-II). Results: VEGF-A and VEGF-C were found at statistically significant levels (p < 0.005) in culture supernatant samples where the autologous cumulus clusters were present but were not detectable in supernatants of embryos cultured alone. IGF-I was detected at considerable levels in supernatant samples where the autologous cumulus clusters were present. IGF-I was not detectable in supernatant samples of embryos cultured alone. IGF-II was detected in significant amounts (p < 0.05) in samples of all groups. The highest concentration level of IGF-II was detected in supernatant samples of embryos in standard culture. Conclusion(s): The investigated factors, among other substances, may be causally connected to the beneficial effect observed on embryo development. Our findings suggest that co-culture with autologous cumulus cell clusters improves the outcome of embryo culture in IVF programs. Ó 2017 Middle East Fertility Society. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Peer review under responsibility of Middle East Fertility Society. ⇑ Corresponding author. http://dx.doi.org/10.1016/j.mefs.2017.05.009 1110-5690/Ó 2017 Middle East Fertility Society. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Please cite this article in press as: A. Vithoulkas et al., Co-culture of human embryos with autologous cumulus cell clusters and its beneficial impact of secreted growth factors on preimplantation development as compared to standard embryo culture in assisted reproductive technologies (ART), Middle East Fertil Soc J (2017), http://dx.doi.org/10.1016/j.mefs.2017.05.009 2 A. Vithoulkas et al. / Middle East Fertility Society Journal xxx (2017) xxx–xxx 1. Introduction During the past twenty years many scientific reports have been published on the subject of improving in vitro culture of human embryos in ART [1–4]. With the addition of feeder layers or helper cells from various tissue types (e.g. endometrium, fallopian tube, ovarian follicle, oviduct epithelium, vero cells, fibroblasts, granulosa-cumulus cells) a positive effect on quality and quantity of developing embryos has been observed in comparison to standard culture conditions for in vitro fertilization (IVF) cycles [5– 11]. In particular, cumulus cells that support the growing oocyte and are physiologically linked to oocyte maturation are instantly available after follicular aspiration of cumulus-oocyte complexes for IVF. They can be easily harvested and successfully used to assist in embryo culture [12–16]. During such co-culture these cells not only seem to be responsible for ameliorated embryo morphology but also may provide a beneficial effect on embryo implantation and pregnancy rates in IVF programs [17–19]. It has been demonstrated that various growth factors secreted by feeder cells can promote oocyte maturation and support development of early embryos during in vitro culture, analogously, as they take part in cross-cellular processes under in vivo conditions. Significant progress in growth factor-related effectiveness through co-culture has been reported for reduced fragmentation and improved cleavage in early embryos, for increased numbers of developing embryos and elevated pregnancy rates [20–22]. The composition and cooperation of growth factors may vary among different helper cell types by synthesizing these factors in embryo-unrelated or less physiological concentrations. The presence of undesirable growth factors secreted during co-culture can also lead to uncontrolled in vitro conditions resulting in developmental alterations during early embryogenesis [23]. As alternative approach individual growth factors at defined concentrations have been added deliberately to standard culture medium to evaluate their efficacy on embryo development and IVF outcome. The insulin-like growth factor (IGF-I) when provided to the culture medium has reduced cellular apoptosis and increased the proportion of morphologically normal embryos [24,25]. The granulocyte-macrophage colony-stimulating factor (GM-CSF) supplemented to culture medium has improved blastocyst development and IVF outcome [26–28]. Exposure of early human embryos to the platelet activating factor (PAF) during in vitro culture has led to increased birth rates in ART [29,30]. The heparin-binding epidermal growth factor (HB-EGF) significantly improved development and hatching ability of human blastocysts [31]. The leukemia-inhibiting factor (LIF) has increased implantation and pregnancy rates [32]. The addition of only single factors to culture media remains in opposite to the complex mixture of multiple autocrine/paracrine factors interacting with oocytes and embryos in vivo. Therefore, supplementation of several key growth factors to the culture medium has been propagated and resulted in a substantial increase of high quality blastocysts in comparison to standard culture conditions [33]. Such beneficial effect most likely was due to the additive action of multiple growth factors. The detection of multiple receptors for different growth factors on the surface of early embryos documents the importance of manifold exchange and selective impact of stage-specific morphogenetic products between developing embryo and surrounding cells during preimplantation [34]. Such molecular cross-communication can only insufficiently take place in standard culture media that deprive embryos of vital growth factors. This may imply potential risks associated with standard IVF since early embryos via their receptors cannot respond to and interact with the respective growth factors [23,35]. It therefore seems reasonable to assume that follicular cumulus cells with their particular assortment of secreted growth factors more closely resemble and re-enact the physiological environment in support of sustaining embryo culture in vitro [36]. About five years ago we reported that supplementation of autologous cumulus cell clusters to the culture medium has improved embryo morphology and increased the number of embryos available for uterine transfer in ART [37]. Moreover, we detected the presence of several growth factors during co-culture that most likely have contributed to this qualitative and quantitative amelioration of embryogenesis [38,39]. In the study presented here we have further investigated the impact of autologous cumulus clusters co-culture on embryo development and pregnancy rate as compared to standard culture procedure. We also have analyzed the concentration of growth factors secreted by cumulus cells during in vitro culture. We suggest that these factors, among other substances, might be causally connected to the beneficial effect observed on embryo development. Our findings support the concept of co-culture with autologous cumulus cell clusters and will contribute to continuously optimize embryo culture in IVF programs. 2. Material and methods 2.1. Co-culture and standard culture 90 IVF patient couples ranging at the age between 35 and 40 (mean age of 36.5) agreed to participate in this study. In this IVF group, 64 first trials, 22 s trials and 4 third trials were included (Table 1). As prerequisite, patients with prospective eight or more oocytes were considered for this study. After follicular aspiration the cumulus-oocyte complexes were kept shortly in follicular fluid in order to cut off equal pieces of cumulus using fine syringe needles. The oocyte complexes together with the isolated cumulus clusters were washed in Human Tubal Fluid buffered with HEPES (HTF-HEPES) (IrvineScientific, Waalwijk, The Netherlands) supplemented with 10% Serum Substitute Supplement (SSS) (Santa Ana, CA, US) and transferred to Human Tubal Fluid (HTF) (IrvineScientific, Waalwijk, The Netherlands) for one hour. Subsequently, oocytes were stripped from their adhering cumulus cells with Hyaluronidase (Cook, Limerick, Ireland), washed in HTF-HEPES supplemented with 10% SSA and kept in Continuous Culture medium (CSC) (IrvineScientific, Waalwijk, The Netherlands) for 15 min. For each case, the prepared MIIoocytes were divided randomly in two groups. Half of the MII-oocytes were assigned to co-culture (group I) and the other half were assigned to culture in regular medium (group II). As control, isolated autologous cumulus clusters were assigned to be cultured alone (group C). This was done by assigning the first oocyte after stripping to the co-culture group and thereafter alternating to the control standard (no co-culture) group. After ICSI treatment, MII-oocytes of group I and II were transferred separately to microdrops of CSC medium covered with FertiCult Mineral Oil (FertiPro NV, Beemem, Belgium). For each microdrop of group I and group C one equally sized piece of isolated cumulus cluster was added. One microdrop of pure culture medium was kept empty as additional background control. For each case, all microdrop samples were kept on the same culture dish. On day-3 of culture, 50.l of supernatants from embryo cultures of group I and II were collected and stored frozen at -20oC. Supernatants from cultured cumulus clusters (group C), as well as microdrops of pure culture medium were similarly collected and stored frozen as controls. At day 3 or day 5 of embryo culture, grade-1 embryos or blastocysts from group I were selected for embryo transfer (ET). The Please cite this article in press as: A. Vithoulkas et al., Co-culture of human embryos with autologous cumulus cell clusters and its beneficial impact of secreted growth factors on preimplantation development as compared to standard embryo culture in assisted reproductive technologies (ART), Middle East Fertil Soc J (2017), http://dx.doi.org/10.1016/j.mefs.2017.05.009 3 A. Vithoulkas et al. / Middle East Fertility Society Journal xxx (2017) xxx–xxx Table 1 Clinical data for the 90 IVF cases assigned for co-culture and standard culture comparison. Transfer day IVF cases, n 3 48 5 42 Patient mean age First trials Second trials Third trials Total embryos transferred Embryos per transfer 38.4 33 11 4 144 3 36.6 31 11 0 97 2 Metaphase II (MII) oocytes 2PN/Fertilization rate Grade 1 embryos/embryo development rate Grade 1 blastocysts/blastulation rate Pregnancies/pregnancy rate Ongoing pregnancies/ongoing pregnancy rate Co-culture Standard culture Co-culture Standard culture 208 186/89.4% 157/84.4% – 19/39.6% 18/37.5% 197 159/80.7% 106/66.7% – – – 232 223/96% 208/93.3% 145/65% 23/55.8% 20/47.6% 228 198/86.8% 164/82.8% 70/35.4% – – remaining grade-1 embryos or blastocysts of group I and II were vitrified using KITAZATO Vitrification Media (KITAZATO BioPharma Co., Ltd, Japan) and stored in liquid nitrogen. group I and II were vitrified and cryostored. The pregnancy rate for day-5 ETs was 55.8% (Table 1). 3.2. Growth factors associated with co-culture 2.2. Growth factor analysis From the 36 randomly chosen IVF cases terminated at culture day-3, supernatants from 17 cases were analyzed for seven different growth factors, namely, epidermal growth factor (EGF), fibroblast growth factor 1 and 2 (FGF-1 and FGF-2), vascular endothelial growth factor A, C and D (VEGFA, VEGF-C and VEGF-D) and Leptin using immunofluorescence cytometry with a factor-specific magnetic-bead panel. Supernatants from 19 cases were analyzed for Insulin-like Growth Factor I and II (IGF-I and IGF-II) with another growth factor-specific magnetic-bead panel. All samples were analyzed with Milliplex factor-specific magnetic-bead panels with phycoerythrin fluorescent factor-specific antibodies by Lab Supplies Inc. laboratories in Athens, Greece. The MFI (Mean Fluorescence Intensity) values for group I, group II and group C supernatants were calibrated and the readings were translated in pg/ml and calculated against the background values of culture medium alone. The statistical analysis of all data were processed with the Two-tailed T-test form. 3. Results 3.1. Embryo co-culture versus standard culture From 90 investigated cases that were considered for co-culture and standard culture, 48 of them have been terminated at day 3 of culture and 42 of them have been continued up to day 5. From 48 cases randomly assigned for day-3 culture, 208 MII-oocytes were assigned for co-culture (group I) and 197 MII-oocytes were assigned for standard culture (group II). After ICSI treatment the fertilization rates were 89.4% (group I) versus 80.7% (group II). The total number of developing grade-1 embryos was 157 (group I) and 106 (group II). For day-3 ET, 144 embryos were selected from co-culture (group I). The remaining 119 embryos from groups I and II were vitrified and cryostored. The pregnancy rate for day-3 ETs was 39.6% (Table 1). From 42 cases randomly assigned for day-5 culture, 232 MII-oocytes were assigned for co-culture (group I) and 228 MII-oocytes were assigned for standard culture (group II). After ICSI treatment the fertilization rates were 96.1% (group I) versus 86.8% (group II). The total number of developing grade-1 blastocysts was 145 (group I) versus 70 (group II). For day-5 ET, 97 blastocysts were selected from co-culture. The remaining 118 blastocysts from From 17 investigated cases, VEGF-A and VEGF-C were found at statistically significant levels in supernatant samples where the autologous cumulus clusters were present (group I and group C) but were not detectable in samples of group II where embryos were cultured alone (Fig 1). For VEGFA, the p values were <0.005 and <0.01 from samples of group I and group C respectively. For VEGF-C, the p values were <0.05 and <0.01 from samples of group I and group C respectively. FGF-2 was detected in considerable concentration levels in samples of group I and C (Fig 1). However, differences in concentration of FGF-2 between group I and C were not statistically significant. Among the 17 cases investigated, variations in concentration levels were detected for the three growth factors. The mean values of VEGF-A, VEGF-C and FGF-2 concentrations for all groups are presented in Table 2. EGF and FGF-1 were only detectable in very low concentrations in group I and C values ranging from 0.5 to 1.5 pg/ml. VEGF-D and Leptin could not be detected in any of the analyzed samples within the concentration sensitivity range of the immunofluorescence cytometry technology used. From 19 investigated cases, IGF-I was detected at considerable levels in samples of group I and group C where cumulus clusters were present. The difference in concentration levels of IGF-I between group I and C was not statistically significant. IGF-I was not detectable in samples of group II, where embryos were cultured alone (Fig 2). IGF-II, on the contrary, was detected in significant amounts in samples of all groups. Unexpectedly, the highest concentration level of IGFII was detected in samples of group II (Fig 2). For IGF-II, the p values of group I (<0.05) and C (<0.04) were at statistically significant lower levels when compared to group II. Among the 19 cases investigated, variations in concentration levels have been observed for both growth factors. The mean values of IGF-I and IGF-II concentrations for all groups are presented in Table 3. 4. Discussion There is currently evidence that standard culture media used for IVF are optimal for human early embryos. Morphometric observations on preimplantation embryos cultured in standard media have revealed that some embryos showed retarded cleavage divisions, reduced growth rates and increased proportion of lesscompacted inner cell mass (ICM) in blastocysts [23]. Disruption of normal genomic imprinting during embryogenesis due to inferior culture conditions may contribute to developmental Please cite this article in press as: A. Vithoulkas et al., Co-culture of human embryos with autologous cumulus cell clusters and its beneficial impact of secreted growth factors on preimplantation development as compared to standard embryo culture in assisted reproductive technologies (ART), Middle East Fertil Soc J (2017), http://dx.doi.org/10.1016/j.mefs.2017.05.009 4 A. Vithoulkas et al. / Middle East Fertility Society Journal xxx (2017) xxx–xxx Fig. 2. Graph showing the concentration distribution of growth factors IGF-I and IGF-II in all studied groups. Table 3 Mean concentration (pg/ml) of growth factors IGF-I and IGF-II in supernatants of human embryo culture supplemented with (group I) and without (group II) cumulus cell clusters, and in controls (group C). Differences in concentration of IGF-I between group I and C were not statistically significant. For IGF-II, the p values of group I (<0.05) and C (<0.04) were at statistically significant lower levels when compared to group II (Two-tailed T-test). Growth factor IGF-I IGF-II Group I (pg/ml) Group II (pg/ml) Group C (pg/ml) 6 ± 3.5 0.1 ± 0 9 ± 0.6 28 ± 16.4 68 ± 9.3 27 ± 14.6 n = 19. Fig. 1. Graph showing the concentration distribution of growth factors VEGF-A, VEGF-C and FGF-2 in all studied groups. Table 2 Mean concentration (pg/ml) of growth factors VEGF-A, VEGF-C and FGF-2 in supernatants of human embryo culture supplemented with (group I) and without (group II) cumulus cell clusters, and in controls (group C). For VEGF-A, the p values were <0.005 and <0.01 from samples of group I and group C respectively. For VEGF-C, the p values were <0.05 and <0.01 from samples of group I and group C respectively. Differences in concentration of FGF-2 between group I and C were not statistically significant (Two-tailed T-test). Growth factor VEGF-A VEGF-C FGF-2 Group I (pg/ml) Group II (pg/ml) Group C (pg/ml) 2889 ± 418.6 24.4 ± 10.2 1209.5 ± 214.9 67.92 ± 24 0.1 ± 0 15.25 ± 5.9 46 ± 19.5 0.5 ± 0 256.42 ± 92.7 n = 17. abnormalities. Imprinting errors caused by altered DNA methylation could be initiated in preimplantation embryos. There have been studies indicating minor elevated risks for genetic imprinting diseases [40–42] and for two systemic syndromes caused by epige- netic alterations through abnormal imprinting in ART programs [43,44]. Substantial improvements for embryo culture in IVF have been achieved by co-culture with cumulus cells. Positive effects of such co-culture have been observed on accelerated cleavage divisions, reduced cellular apoptosis and increased number of high quality blastocysts. Embryo co-cultures have been shown to increase embryo implantation and pregnancy rates in IVF patients [18]. It also seems to improve blastocyst transfer in IVF patients with repeated implantation failures [19]. Cumulus cells have shown to remain viable up to ten days in culture as estimated from morphological criteria and therefore continue to be viable beyond embryo implantation [18]. Preferably for each IVF case autologous cumulus cells should be selected from the same patient. Isolated cumulus cells are quite homogeneous and their protein expression profiles are very similar between cumulus cells from different oocytes retrieved from the same patient. On the contrary, dissimilarities in protein profiles of cumulus cells have been detected between patients depending on their particular ovarian stimulation protocols [45]. In fact, there seems to be a good correlation between specific synthesis of cumulus cell proteins and oocyte maturity required for normal embryo development. Cumulus Please cite this article in press as: A. Vithoulkas et al., Co-culture of human embryos with autologous cumulus cell clusters and its beneficial impact of secreted growth factors on preimplantation development as compared to standard embryo culture in assisted reproductive technologies (ART), Middle East Fertil Soc J (2017), http://dx.doi.org/10.1016/j.mefs.2017.05.009 A. Vithoulkas et al. / Middle East Fertility Society Journal xxx (2017) xxx–xxx cell-specific gene products can therefore lead to the discovery of new biomarkers predictive of embryo quality and pregnancy outcome in IVF [46,47]. In our comparative study of embryos cultured with (group I) and without (group II) autologous cumulus clusters we found that under co-culture conditions the cleavage divisions of early embryos were accelerated with more equally sized blastomeres, embryo morphology was improved giving rise to increased numbers of grade-1 embryos and blastocysts available for ET and cryo-storage for further IVF trials. On the contrary, embryos cultured in standard medium showed a decreased growth rate with more unequal cleavage divisions and cellular fragmentation. The total number of grade-1 embryos was reduced substantially with increased numbers of arrested embryos. Only embryos and blastocysts of high quality were kept frozen as supplementary reserve for future ETs. In addition, we obtained a significant increase in the number of grade-1 blastocysts derived from co-culture when compared to standard culture conditions (65% versus 35.4%). Previously, under standard culture conditions in IVF, our overall pregnancy rate had been 24%. Since the introduction of coculture with autologous clusters in IVF, our overall pregnancy rate increased to 36%. Therefore, based on these observations, we have routinely shifted to the co-culture system in our IVF practices for embryo culture in vitro. Our findings are in good agreement to other comparable studies where cumulus cells instead of cumulus clusters have been used for co-culture of human embryos in ART [18,19]. Growth factors secreted by cumulus cells during co-culture have shown to be partially responsible for the beneficial effect observed on human embryo development in IVF [23,35]. In our studies to investigate the presence of growth factors secreted by cumulus clusters during embryo co-culture, we found significant concentrations of VEGF-A and VEGF-C. Their concentration in samples of group I was significantly higher as compared to samples of group C and was not detectable in samples of group II (Fig 1). We assume that embryos in standard culture are not synthesizing these two growth factors whereas embryos under co-culture conditions either seem to be capable of synthesizing VEGF-A and VEGF-C or cumulus cells secreted a higher amount of these two factors. Alternatively embryos together with cumulus cells are synergistically responsible for this concentration increase. Growth factor FGF-2 was not detected in samples of group II but was present in samples of group I and C (Fig 1). We conclude that this growth factor is not synthesized by embryos cultured in standard medium but is secreted in considerable amount by the cumulus clusters alone. Concerning the reduced concentration in samples of group I we can only suspect that either embryos have consumed this growth factor or a substantial amount of FGF-2 has been metabolized under co-culture conditions. For these three growth factors that we have detected under co-culture conditions there is published evidence for their functional role in human preimplantation embryo development and IVF outcome [23,35]. For growth factor IGF-I, we have detected considerable amounts of this factor in samples of group I and C but not in samples of group II (fig 2). We conclude that this factor is not being synthesized by the embryo but secreted by the cumulus cells. The reduced amount of IGF-I in group I versus group C may be due to either metabolic consumption and/or uptake by the embryos during co-culture. It was shown previously that the addition of IGF-I to culture media prevented apoptosis in preimplantation embryos by reducing cellular fragmentation, increasing the number of healthy blastomeres and improving the morphology of the developing blastocysts that resulted in increased implantation and pregnancy rates [24,25,48]. For growth factor IGF- II, we detected significant amounts of this factor in samples of all groups (Fig 2). Whereas the concentra- 5 tion of this factor is almost equally present in group I and C it is significantly increased in samples of group II. We assume that similar levels of IGF-II in co-culture and cumulus-culture reflect the normal amount of this growth factor that is secreted and/or downregulated under co-culture conditions. It has been known for quite some time that IGF-II is being synthesized by the embryo itself [49]. However, when we compare the level of secreted IGF-II in group II with the levels of group I and C we found a statistically significant elevated IGF-II concentration. We postulate that embryos in standard culture (group II) are over synthesizing IGF-II leading to altered concentration of this particular growth factor. On the other hand, under co-culture conditions, secretion of IGF-II seems to reach a more physiological level for embryos (group I) and cumulus cells (group C). Based on these new findings, we suggest that in standard medium early embryos with the appropriate surface receptors are deprived of external molecular cross-reactions with gene products secreted by cumulus cells. Such unbalanced IGF-II concentration may have a negative effect on regular embryogenesis [49,50]. It may also be linked to genetic imprinting diseases and epigenetic abnormalities in ART programs [40–44]. To improve standard culture conditions, attempts have been made by addition of individual growth factors [29–32]. However, supplementation of culture media with individual growth factors can only insufficiently fulfill the manifold metabolic requirements needed for the developing embryos. New approaches to further improve culture conditions have been introduced by supplementing the culture medium with a mixture of several key growth factors that improve embryo morphology and implantation potential [33]. As alternative approach, supplementation of standard culture medium with autologous cumulous clusters creates a physiological environment which more closely support the biological needs for the developing embryo. 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