Title:

A review of co-culture methods in human assisted reproduction.

KEYWORDS: Co-culture/Growth factors/Animal/Human/
Authors:
Waldemar de Carvalho, MD*
Samuel Lee, PhD FIBMS +
* Research Fellow
Universidade Federal Paulista
Sao Paulo
Brazil

+ Scientific Consultant
Portland Hospital Fertility Unit
London W1N 5HG-UK.

ABSTRACT
This paper reviews the use of co-culture in assisted reproduction. It has been suggested that co-culture procedures may have a significant beneficial impact on IVF as it is believed that "factors" released by the co-culture cells. It has been argued that with conventional IVF the absence of various in vivo influences compromises the viability of IVF embryos. We have therefore set out to look at the current status of co-culture by exploring the different methods and examining the rationale behind their use. We have not attempted a priori to answer the question of which is the best co-culture method nor is it clear in the current climate whether co-culture offers superior results to more modern sequential media formulations which have recently been reported to improved percentages of blastocysts for transfer as compared with traditional media.
Our review of the literature leaves us with the following conclusion; the majority of published papers on co-culture, suggest that they have demonstrated significantly improved blastocyst rates (>50%), though noone has yet demonstrated unequivocally, how co-culture exerts this beneficial effect. The concerns over xeno co-culture and the lack in our understanding of the real underlying factors that underpin the apparent improvements mean that for the time being, co-culture is still only an interesting scientific model with which to study interactions between somatic cell cultures and human embryos. Curently defined media systems are beginning to yield comparable results to those demonstrated by the majority of co-culture studies, and the field is developing rapidly. Under the circumstances, although we feel that co-culture has much to offer as a research tool, for the time being, further research is required to characterize the factors that are involved, with a view to the future likelihood that these will be added to defined media, which now appear likely to supercede the need for co-culture in the very near future.

INTRODUCTION
Assisted Reproduction Technology (ART) brings new expectation for subfertile couples. Since the first experiences with animals (Thibault et al.,1949) and the first test tube baby in 1978, many discoveries have been made in an attempt to improve the chances of pregnancy in such couples. Numerous techniques in ART have been developed to improve results, but in the UK the average pregnancy rates (PR) vary between 15 to 20% and approximately 15% take home baby rate (HFEA, 1999). Some of the failings of ART have been blamed on suboptimal conditions in the culture media for human embryo development in vitro (Bongso et al.,1990). It is pertinent to note that the PR with ART in domestic animals are approximately 60% when the embryos are replaced in the uterus at blastocyst stage (Iritani et al.,1988).
In fertile women fertilisation occurs in the ampullary-isthmic region of the fallopian tube. The subsequent cleavage until the blastocyst stage takes approximately 4 to 5 days before the embryo descends to the uterus for implantation (Johnson & Everitt, 1990). In normal in vitro fertilisation (IVF) procedures when the embryo is replaced at 4-6 cells stage into the uterus, it will therefore be there for 90h or so before implantation. Thus IVF results may be affected detrimentally by this asynchronous transfer. On the other hand one factor which may also contribute to poor results are the inadequate culture conditions which seem, to date, to be largely unable to support good embryo development in vitro to the blastocyst stage (Bolton et al., 1989; Bolton et al.,1991; Dokras et al., 1991, 1993; Hardy et al., 1989).
Some authors have tried to follow human embryo development and to keep them until the blastocyst stage for replacement (Bolton et al., 1989; Bolton et al.,1991; Dokras et al., 1991, 1993; Hardy et al., 1989), since with early transfer, abnormalities of oocyte maturation, fertilisation and early cleavage may not be manifested until later stages, following embryo transfer (ET) when it is no longer possible to visually monitor the progress of pre-implantation development (Bolton et al., 1989; Bolton et al.,1991; Dokras et al., 1991, 1993; Gardner & Lane, 1997; Hardy et al., 1989). If the majority of human IVF embryos succeeded in developing to blastocyst stage following embryo transfer; if we assumed that most blastocysts will hatch and then implant in the endometrium, then most pregnancy failures should be associated with positive l3-hCG results (so-called: biochemical
pregnancies). Since, this is usually not the case, either our assumption is invalid or it seems likely that defective embryonic development prior to implantation may be one of the prime causes of failure in IVF-ET (for review see Gardner & Lane, 1997). In many IVF clinics, probably in order to reduce the risk of fragmentation and degeneration of the human embryo they are replaced earlier (before 8-cells) (Gardner & Lane, 1997). As a matter of fact the large percentage of embryos showing a block at the 8 cell stage, when in culture, led Bavister (1988) to investigate the possible presence of artifacts in the culture medium or failure in genomic activation. It remains unclear though whether or not this might be produced by blocking/embryotoxic factors or indeed, whether failure of genome activation per se is a major factor in human IVF failure or not. Nevertheless, the possibile existence of blocking/embryotoxic factors gains credence when considering the findings of Gandolfi et al. (1989a,b) who showed that the culture of sheep embryos on sheep oviductal cells enhanced the development and the viability of the embryos. Since then a number of authors have begun research into co-culture, using a variety of animals such as cow, goat, monkey and rat (for example: bovine kidney cells, monkey kidney cells (Vero Cell) and buffalo rat liver cells). The co-culture technique has also been used in humans including a large variety of feeder cells: oviductal cells, fallopian tube cells, endometrium cells and luteinized granulosa cells. Co-culture of human embryos with any of the above seems to increase the PR and also provides an opportunity to futher evaluate the embryo (since most studies have looked at blastocyst ET) before replacement (Bongso et al., 1993, 1995; Guerin and Nicollet, 1997).
GROWTH FACTORS AND EMBRYONIC BLOCKS
Until recently, the culture of human embryos up to the expanded blastocyst stage has been suboptimal under existing in vitro conditions in most IVF centers (Bavister, 1988, 1992, 1995; Gardner & Lane, 1997). Even under the strictest quality control measures and the best laboratory conditions only 25 - 30% of "excess" embryos cleave normally to the expanded blastocyst stage (Bongso -unpublished data, Bolton et al., 1989; Dokras et al., 1991; Fehilly et al., 1985; Hardy et al., 1989). The objective of the co-culture system, therefore, is to provide an environment that contains beneficial factors which might more closely simulate, than existing culture media, existing conditions in the human fallopian tube for the embryo. There are two hypotheses to explain how the co-culture cells might improve the fertilisation rate and enhance embryo development. First: specific and non-specific embryotrophic factors might add to and improve culture conditions and second, the co-culture cells might remove undesirable factors from the culture medium (Bongso et al., 1991a,b).
In several species of animals including the human, embryotoxic factors can block the embryo development of different stages in vitro (Bavister, 1988, 1992, 1995; Bongso et al., 1989; Gardner & Lane, 1997). This block appears to happen when activation of the genome (Braude et al, 1988) occurs and the co-culture system appears to overcome it in most mammals (Bongso et al, 1993, 1995; Menezo et al., 1995), which, it is generally postulated, may involve non-specific factors such as glycoproteins (which may or may not be growth factors) being secreted by the feeder cells, thereby facilitating /embryonic development and bypassing any block factors ((Bongso et al, 1993, 1995; Menezo et al., 1995). Some specific glycoproteins have been described in animals (Gandolfi et al., 1989a,b) and the molecular weight of proteins varies between 36 and 2l5 kDa and can be found in different stages of the female cycle, whilst moreover, Butzow (1989) has demonstrated the presence of a 36 kDa glycoprotein (placental protein 5) in the secretory phase of the human menstrual cycle. However, as yet, no unequivocal proof has been put forward that any of these factors specifically play a role in the co-culture systems we have discussed to date. More definitive studies are needed.
The mode of action of the putative growth factors need to be determined by studying the release of glycoprotein(s) by the co-culture cells into conditioned medium. Presumably these factors may simply diffuse into the embryo via the zona pellucida or act by direct contact between zona and ampullary cell allowing cross-transfer of glycoprotein(s) and other important metabolites. Bongso et al. (1993) has suggested an explanation for the action of co-culture on embryos in both positive and negative aspects as compared with traditional media. The negative aspects he attributes to non co-culture situations include: 1. Presence of hypoxanthine which is known to cause the two-cell block in mouse embryo. 2. Need for stabilization of the biochemical in vitro environment (for example, pH, 0₂, C0₂) and decreasing oxygen metabolite levels. 3. Need for redesign of the medium by reducing glucose and increasing lactate levels. 4. The need to use patients serum in simple glucose containing media. The positive conditions which he subscribes to be the benefits of co-culture, such as the presence of embryotrophic factors: 1. Possible secretion of antioxidant taurine by the cells which in turn improves embryonic development. 2. The belief that low-molecular weight fractions in cell-conditioned medium induce a positive effect. 3. Postulated glycoproteins released by the cells. 4. Postulated growth factors (such as transforming growth factor TGF l31, insulin like growth factor IGF) released by the cells.
CO-CULTURE SYSTEMS
Much has been written about the evaluation of embryos and it is generally believed that embryos which divide evenly and a at a rate of one cell division every 20-24 hours are the embryos with the highest viability when replaced in the uterus (Bavister, 1992, 1995; Bolton et al., 1989; Dokras et al., 1991, 1993; Gardner & Lane, 1997; Hardy et al., 1989; Hartshorne et al., 1991;Trounson et al., 1982). However, many examples have been noted when retarded and unevenly cleaved embryos develop to term so there are no absolute criteria available at the present to determine which embryos are viable and which are not (Bavister, 1992, 1995; Bolton et al., 1989; Dokras et al., 1991, 1993; Gardner & Lane, 1997; Hardy et al., 1989; Hartshorne et al., 1991;Trounson et al., 1982). In an attempt to determine whether in vitro culture methods might be improved upon, co-culture systems have been developed in which embryos are grown on a layer of somatic helper cells (Bongso & Fong, 1993; Bongso et al., 1995; Gandolfi et al., 1989a,b; Menezo et al., 1995), since it is believed that this approach may provide a stimulus to embryonic development. This approach may, in the long run, help to definine the requirements of embryos during development. Trophoblastic vesicles have been found to be useful only in animals according to Camous et al. (1984). Other studies have also demonstrated the beneficial effect of various cellular monolayers on the development of mammalian embryos, for instance uterine fibroblasts (Wiemer et al., 1989), human tubular cells (Bongso et al., 1990, 1991a,b), and Vero Cells (Huang et al.,1997;.Menezo et al., 1990) have all been used in an attempt to enhance early in vitro human embryo development.
HUMAN CO-CULTURE SYSTEM
A logical choice for co-culture system are cells from the human fallopian tube, although the ability of such cells, cultured as monolayers, to mimic their in vivo function per se, is somewhat doubtful. Verhage et al. (1979) have shown that complex changes occur in these ciliated and secretory epithelial cells during the menstrual cycle. In the early follicular phase, hypertrophy and reciliation occurs, although in the human fallopian tube it is thought that the cilia only play a secondary role in gamete and embryo transport. Then as the cycle progresses, in the late follicular phase, the epithelial cells attain maximum height and ciliation and finally, at the end of the luteal phase atrophy and deciliation occurs. Jansen (1984) observed that around ovulation the isthmus secretions became abundant when estrogen levels were high at the midcycle, and this is possibly an important regulatory role in sperm transport through the isthmus. Oviductal fluid is therefore a crucial environment in which the movement of ovum and spermatozoa, fertilisation, embryo transport and early development takes place. Glucose, lactate and pyruvate support sperm, oocyte and embryo survival (Leese, 1988; Bongso et al., 1993). According to Gardner (Gardner et al., 1996; Gardner and Lane, 1997) the lactate and glucose concentration in the oviduct changes with the day of the cycle and the human embryo is exposed to different metabolite concentrations as it moves along the tract. Furthermore, cumulus cells readily consume glucose, producing lactate. Therefore, the early human embryo is exposed to low glucose and high lactate levels in vivo. Thus, embryos in culture are likely to be in a sub-optimal environment, which even in the presence of human ampullary epithelial cells is unlikely to mimic the in vivo environment. Nevertheless, Bongso (1989) reports that the support of human embryo cleavage and growth with human ampullary epithelial cells in vitro resulted in a reduction in the cleavage abnormalities, producing good quality embryos with equal size blastomeres and minimal fragmentation when compared with conventional medium. Bongso et al. (1992a) have also evaluated embryonic behavior in vitro, examining the pregnancy and implantation rates of embryos grown to blastocysts in co-culture with human ampullary cells in fifty women undergoing a single IVF treatment cycle. This study demonstrated that the expanded blastocyst rates in co-culture were significantly better than in the control patients (44% compared with 23%, p<0.01). In this study a significantly higher percentage of good quality four cell stage embryos (regular blastomeres, absent to slight fragmentation) were also observed. In a similar study (Bongso et al., 1992b) were able to show high success rates with IVF involving co-culture (44% per cycle, implantation rate (IR) of 31.8% ). The authors also reported an 88% rate for embryos of good quality.
Other possibilities for co-culture are granulosa cells, for instance, Dirnfeld et al. (1997) have shown an improvement in embryo quality in the short term development of early stage embryos and concluded that poor quality embryos may be rescued to cleave regularly in the presence of autologous human granulosa cells. Another system for study is human endometrial cells, obtained after hysterectomy (Bongso et al., 1988). Gland and stromal cells can be separated and established as cell lines for periods of over one menstrual cycle. Monolayers may be established in 3-7 days and in vitro cell growth has been found to consist of a mixed growth of epithelial fibroblast-like cell types (Bongso et al., 1988). Future studies could also be developed to characterize the exact in vitro behavior of the endometrium at the time of embryo replacement. The secretory cells from endometrial cell lines may affect the embryo, perhaps through the release of unknown/unspecified implantation factors or signals which it is speculated may eventually yield improved pregnancy rates (Birkenfeld and Navot, 1991).

ANIMAL CO-CULTURE SYSTEM
Co-culture of human embryos with various cellular monolayers has been described in the section beforehand, however, there is often a difficulty in obtaining samples from human cells, and furthermore there remain issues over possible bacterial or viral contamination. In animals, there remains similar potential for contamination, but they represent a more readily available source of cells for co-culture, although it should be borne in mind that the dangers of xeno co-culture remain an unknown quantity and significant problems may yet arise in the future. Animal feeder cells have been tried successfully by Rexroad and Powell (1988) who used ovine oviductal epithelial cells cultured in vitro and the subculture cells from established cell lines for co-culture with ovine embryos. Soon after Wiemer et al. (1989) found markedly improved human embryo morphology when they were co-cultured for one day on monolayer of fetal bovine uterine fibroblast. Increased blastomere diameter was the most evident morphological feature of embryos that developed in the presence of helper cells.
Menezo (1990) used immortalized cell lines of extra genital origin such as Vero Cell, which originate from kidney epithelium of African Green Monkeys. These cells were selected because kidney and genital tract have a common embryological origin (mesoderm). Moreover, Vero Cell are perhaps the most appropriate form of animal cells to use for co-culture with human embryos, since they are highly controlled for viruses and other contaminants because they are used for vaccine production. In this study the authors showed that > 50% of embryos of poor quality could reach the blastocyst stage when co-cultured .The Vero Cell co-culture system with B2 medium gives better results in terms of rate of blastocyst formation, with best grade of embryos. This system appears to have good development potential (61 % vs.3% of the control group). This observation suggests that in this case, the co-culture system improved human embryonic development, rescuing them from degeneration. Zetova et al. (1993) also studied the use of Vero Cell Monolayers (no complications reported) in standard culture medium, achieving a pregnancy rate (PR) of 33.33%, and an IR of 19.1% .
Guerin and Nicollet (1997) reported on a total of 1603 co-cultures performed by 11
groups over a 2 year period. The mean rate of clinical pregnancies per transfer after
co-culture was 32.9% which was significantly higher than the mean value obtained by
using conventional IVF procedures . They concluded that almost half of the cleavage embryos were able to develop into blastocyst during co-culture with Vero Cell monolayers, and the subsequent IR of these blastocysts were approximately twice as high as that of four-cell embryo. They suggest that previous successive failure with IVF was a main indicator for treatment with co-culture.
Buffalo Rat Liver cells (BRL) have also been used for the co-culture of human embryos obtained from poor prognosis patients, and have been reported to have a positive effect on both the implantation and the clinical pregnancy rates (Hu et al., 1997). Hu et al., (1998) have also reported that co-culture with BRL cells and assisted hatching in a population of 200 first-time IVF patients was able to achieve a 58% clinical PR and a 49% live birth rate ( 26% IR). The rate of abnormalities detected in uterus and at birth was not different from that observed in the general population. Although Guerin and Nicollet (1997) found that there was an apparent high frequency of trisomies, mainly trisomy 21 (0.8%), the majority of these cases corresponded to situations in which the mother was aged > 37 years.
Finally, although comparison between human and animals co-culture are rare, it has been done by Feng et al. (1996) who studied different culture systems including human granulosa monolayer cells, co-culture with bovine oviductal epithelial cells (BOEC) and co-culture with bovine uterine epithelial cells (BUEC). This study indicated that co-culture with BOEC and BUEC is more efficient than co-culture with human granulosa cells in enhancing the development of embryos to the eight cell stage at 72h after egg retrieval. This result only serves to highlight the fact that in vitro behaviour of cell types are unlikely to mimic exactly their in vivo function.
BLASTOCYST CULTURE USING DEFINED MEDIA
So far, we have seen the potential of co-culture techniques, but there are disadvantages, such as the exposure to animal cells, factors released by such cells and the possibility of cross-infection (Bavister, 1992, 1995; Gardner & Lane,1997). With defined media, there is no need for the relatively more complicated culture methods needed with co-culture (Gardner & Lane,1997). Early attempts by Quinn et al., 1985; Hardy et al., 1989; Bolton et al., 1989,1991, yielded relatively poor results (e.g. low rates of blastocyst formation or poor implantation rates), hence the impetus for co-culture (Bongso & Fong, 1993; Bongso et al., 1995; Gandolfi et al., 1989a,b; Menezo et al., 1990, 1995). However, recent developments in formulations of defined media have resulted in a shift in philosophy amongst those carrying out human IVF (Barnes et al., 1995; Jones et al., 1998; Gardner and Lane (1997). Until recently, embryo transfer at the 4-cell stage had been established global practice, in part, tacit acknowledgement of the deficiencies in culture media (Desai et al, 1997; Desai & Goldfarb, 1998; Gardner & Lane 1997). Better understanding of the physiology of preimplantation mammalian embryos has resulted in improvement in the contents of media (Bavister and McKiernan, 1993; Gardner and Lane, 1993; Scholtes and Zeilmaker, 1996; Gardner & Lane 1997). The advances in media formulations, means that the move towards blastocyst stage human embryo culture and transfer has taken a significant step forward. Now, as with co-culture, up to 50% or more of all embryos derived through human IVF may develop on to the blastocyst stage. In particular, the most recent developments suggest that sequential media, which take into account the changing milieu of the human reproductive tract provide good blastocyst culture results (for review see Gardner and Lane 1997). Taking into consideration the changing needs of a precompaction and a postcompaction embryo with regards for their needs for energy (lactate, pyruvate, glucose etc) and the developing need for non-essential amino acids, the use for two defined media has provided data more than comparable with the best of co-culture (Gardner and Lane 1997). Altogether, these results in addition to criticism of co-culture by Van Blerkom (1993) and Sakkas et al (1994) provide evidence that the use of defined culture media, as opposed to co-culture methods for obtaining blastocyst in human IVF may now have edged ahead.
For those who share concerns about co-culture, advances in formulation technology of defined media, allows for a real glimpse of a future where, defined media may provide a simpler and safer method for blastocyst culture.
DISCUSSION AND CONCLUSION
The application of co-culture systems to in vitro developing human embryos has been performed by numerous investigators. The aim of these researchers has been to see if co-culture is able to provide improved results. These studies, suggest that, in certain hands, the use of co-culture procedures may have a significant beneficial impact on IVF (Bongso & Fong, 1993; Bongso et al., 1995; Gandolfi et al., 1989a,b; Guerin and Nicollet , 1997; Menezo et al., 1995). . Many authors also suggest that co-culture offers a significant alternative for patients with previously failed IVF replacement attempts (Bongso & Fong, 1993; Bongso et al., 1995; Guerin and Nicollet , 1997; Menezo et al., 1995; Wiemer et al., 1989). A number of different somatic cells have been used for co-culture, but there is no clear evidence that any particular cell type is more effective than another. For instance some authors have attempted to provide a rationale for the use of oviductal cell co-culture, by suggesting that oviductal epithelial cells in vitro may provide an environment which mimics the natural environment more closely (Bongso & Fong, 1993; Bongso et al., 1995; Gandolfi et al., 1989a,b). Conversely, they argue that the absence of oviductal-like influences in vitro may compromise the viability of IVF embryos. Unfortunately, there is little or no evidence to suggest that oviductal cells in culture are able to behave as they do in vivo. Furthermore, it would seem that co-culture with any somatic cell and even monkey Vero cells are able to provide similar results (Feng et al., 1996; Hu et al., 1997, 1998; Menezo et al., 1995).
It has been argued that the improvement(s) offered by co-culture centre on improved quality and/or development of embryos, arising as a consequence perhaps from the following two reasons: (i) empirical use of co-culture to improve embryo development and/or viability and (ii) the belief that the co-culture technology itself has an embryotrophic effect through some secretory or buffering property of the somatic cells (Bavister, 1992; Bongso & Fong, 1993; Bongso et al., 1995; Gandolfi et al., 1989a,b ). A further issue arising from research done on co-culture, is that it might offer the possibility of enhanced embryo viability and consequently the development of routine blastocyst transfer in IVF programmes (Bongso & Fong, 1993; Bongso et al., 1995). When transferring human blastocysts, it is believed that there may be a synchrony between blastocyst transfer and endometrial receptivity (Hartshorne et al., 1991; Olivennes et al., 1994; Lelaidier et al., 1995). Other advantages of such a procedure include the possible selection of embryos with a higher implantation potential (Nakayama et al., 1995) supporting the concept that some (but not necessarily all) embryos with chromosomal and genetic abnormalities may be excluded by their failure to reach the blastocyst stage. Finally, another additional benefit of co-culture, is supposed to be the availability of larger numbers of supernumerary embryos for freezing, since it is believed that fewer blastocysts are needed for embryo transfer because of their supposed enhanced viability. Since cryopreservation of human embryos fertilised in vitro, has become an established clinical procedure (Trounson and Mohr, 1983; Lassalle et al., 1985; Testart et al., 1986; Cohen et al., 1988), the replacement therefore of cryopreserved spare human embryos provides infertility patients with further chances of having a live baby.
As a counter to the idea of co-culture, it is important to consider that a number of improvements in culture techniques that have taken place over the last decade, allowing acceptable rates of development of human embryos to blastocyst stage (Gardner and Lane, 1997). Although the question of which is the best method remains unanswered, it is clear that several techniques are able to provide a good percentage of blastocyst formation after the use of defined medium culture. Moreover, Gardner et al., (1998) have demonstrated the successful use of two sequential serum-free media formulated for the culture of human zygotes to the blastocyst stage. Designated G1 and G2, these media were formulated around the levels of carbohydrates present in the human oviduct and uterine fluids at the time when the embryo is present. They found that viable human blastocysts can be obtained in such sequential culture media in the absence of co-culture and serum and an improvement in PRs were observed when day 5 transfer occurred.
This observation opens again the question of whether co-culture is really necessary to improve results or if other important considerations related to implantation rate like:age, quality of embryos replaced, quality and duration of culture, previous IVF cycle pregnancies, uterine anomalies and general health problems, are more important with respect to treatment outcome (Bavister, 1992, 1995; Van Blerkom, 1993; Sakkas et al., 1994). Bavister (1992, 1995) has argued that a great deal of the enthusiasm for co-culture as a means to improve embryo development is probably misplaced. In light of recent reports (see Bavister and McKiernan, 1993; Gardner and Lane, 1997; Gardner et al.,1998; Jones et al., 1998; Scholtes and Zeilmaker, 1996) his arguments are finding increasing support worldwide. Co-culture, it seems, remains somewhat of an enigma. Clearly in some hands, it produces excellent results; certainly comparable with those obtained with defined media (Bongso & Fong, 1993; Bongso et al., 1995; Gardner & Lane, 1997, Guerin & Nicolett, 1997; Menezo et al., 1995), however its role in the wider world may rest in its use as an experimental clinical tool, whose use will perhaps in time, allow us to characterize the factor(s) that might be beneficial to human embryos developing in culture, with a view to the future likelihood that these will subsequently be added to defined media, which now appear likely to supercede the need for co-culture.
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ACKNOWLEDGEMENTS

WC would like to acknowledge the following: the support by way of a travelling
bursary from the Botucatu Medical School (for WC), the kind help and advice from
Professor JG Franco Jr, Sue Smith and Kathryn Parkinson of the Portland Hospital for
allowing me access to the excellent facilities and lastly, but by no means least, all the
staff at the Portland Hospital Fertility Unit for their kindness and hospitality.

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