Human Reproduction vol.5 no.8 pp.893-900, 1990

OPINION

Co-cultures: their relevance to assisted reproduction

Ariff Bongso1, Soon-Chye Ng and Shan Ratnam Department of Obstetrics and Gynaecology, National University of Singapore, Kent Ridge, Singapore 0511 'To whom correspondence should be addressed

Assisted reproductive techniques have contributed significantly to alleviating subfertility in the childless couple. However, the take-home baby rates have been very low throughout the world. One of the contributory causes has been the reduced viability of replaced embryos brought about by suboptimal in vitro conditions. The culture of human embryos in the presence of passaged human tubal ampullary monolayers (co-cultures) is an attractive approach to improving the viability of embryos for assisted reproduction. Seventy per cent of blastocysts can be produced in human ampullary co-culture as compared to 33% in standard culture media. This paper discusses the various roles of human cocultures in assisted reproduction and provides an opinion as to how the transfer of blastocysts produced via the co-culture system could enhance pregnancy rates. Particular emphasis is placed on human oviductal characteristics, the various co-culture systems, screening of co-cultures for microbes, freezing of ampullary cells, growth factors and embryonic blocks, specificity of co-cultures, sperm hyperactivation in co-culture and pregnancy rates. The first patient on a clinical trial who had four of her oocytes fertilized and grown in human ampullary co-culture and then replaced into her uterus became pregnant. The co-culture system may have tremendous potential in supporting human embryonic growth via embryotrophic factors. Key words: assisted-reproduction/co-cultures

Introduction Following the discovery that spermatozoa required capacitation in the female genital tract before fertilization, the first confirmed fertilization of oocytes in vitro was in the rabbit (Chang, 1959). Later, in 1969, Edwards and others reported the first successful in-vitro fertilization (TVF) of human oocytes which led to the birth of the first test tube baby in 1978 (Edwards et al., 1980). Today, IVF has become an important tool for alleviating subfertility in the human. Several modifications of IVF have been developed such as gamete intra-Fallopian transfer (GIFT), tubal embryo transfer (TET) [including pronuclear stage transfer (PROST) and zygote intra-Fallopian transfer (ZIFT)] and microinsemination sperm transfer (MIST). This new area of repro-

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ductive biology is now frequently being referred to as assisted reproductive technology (ART). In spite of the many advances in die field of ART and the mushrooming of many new IVF centres around the world, IVF pregnancy rates are very poor and the overall take-home baby rate for FVF remains < 10% (Voluntary Licensing Authority, 1987). The clinical pregnancy and take-home baby rates for GIFT and TET are, however, significantly higher (Wong et al., 1988; Yovich et al., 1988) but still not equivalent to results in domestic animals, where embryo transfer after in-vivo fertilization has yielded pregnancy rates of >60% with almost this entire percentage resulting in live-born offspring (Iritani, 1988). A large proportion of the losses in human assisted reproduction are due to low fertilization rates from poor sperm motility (Table I), chromosome anomalies in oocytes (Table II) and unquestionably due to poor culture conditions. The culture of human embryos up to the expanded blastocyst stage has been suboptimal under existing in-vitro conditions in most FVF centres. Even with the strictest quality control measures and the best laboratory conditions, only 25-30% of 'excess' embryos cleave normally to the expanded blastocyst stage (Fehilly et al., 1985). Because of this, embryos are immediately replaced into die uterus at the 4- to 8-cell stage to avoid in-vitro fragmentation and degeneration of embryos in later stages. Further, if embryos were kept in vitro for up to 4 - 5 days to cleave to the blastocyst stage, only a small group of patients would have the opportunity of an embryo replacement. Attempts to improve culture conditions for IVF have focused on the formulation and selection of appropriate culture media and additives, gas atmospheres and stage of development of embryos. Additionally, it is important to note that in the normal reproductive physiology of a fertile woman, fertilization occurs in the ampulla of die oviduct, and cleavage continues in the oviduct until die late morula or early blastocyst stage before the embryo descends into die uterus for expansion, hatching and implantation. Thus the 4- to 6-cell stage embryo spends —70-75 h in the oviductal environment cleaving to die blastocyst stage before it enters into the uterus. The time spent in the uterus during embryonic growth is only - 2 0 - 2 4 h before the onset of implantation. In IVF, 4- to 6-cell embryos are replaced into the uterus and not into die oviducts. The embryo thus spends a longer time (90-99 h) in the uterine environment before implantation. As such, many embryos may be degenerating after the 4- to 6-cell stage because of the inappropriate environment mus resulting in low pregnancy rates. It is pertinent to note diat die high embryo transfer pregnancy rates of >60% observed in domestic animals 893

A.Bongso et al.

Table I. Sperm motility versus fertilization rates in human IVF Visual1 scale

Cellsoft motility parameters (mean ± SEM) Velocity Linearity (p.m/s) (%) b

1 2 3

40.79 ± 4.5 54.74 ± 5.1C 77.65 ± 5.3 d

Mean ALH

59 9 ± 12 58.7 ± 9 55.2 ± 8

(jim)

Beat/cross frequency (Hz)

1.96 ± 0.2c 3.29 ± 0.4f 4.91 ± 0.6*

14.37 ± 1.2 13.29 ± 0.9 12.73 ± 0.8

Mature oocytes fertilized (%) 21 (27/128)h 80 (223/280)' 81 (78/%)'

ALH, amplitude lateral head displacement. *1, sluggish; 2, moderately active; 3, very active. Figures in parentheses are mature oocytes. bcd P < 0.01. c f - -*P < 0.01. h 'P < 0.001. Source: Bongso et al. (1989a,b).

Table n. Chromosome constitution of human metaphase II oocytes Number of analysable oocytes

Oocytes (%) Hypohaploid

Haploid

Hyperhaploid

50 39 14 52 251 188 30 65

28.0* 7.7 21.4 51.9 13.0 10.6 13.3 154

68.0 76.9 643 34.6 76.6 81.4 33.3 52.3

2.0 7.7 14.3 5.8 8.0 85 6.7 10.8

Diploid

-

Polyploid

2.6 3.9 2.0

16.7 12.3

10.0 9.2

Structural anomalies

Reference

4.0 5.1 3.9 0.4 20.0 -

b

Martin el al., 1986 Plachot etal., 1986 ^Wramsby etal., 1987 ''Wramsby and Fredga, 1987 b Bongso et al., 1988b b Pellestor and Sele, 1988 b Papadopoulos et al., 1989 b Ma et al., 1989 b

"According to authors may have been artefactual estimate. •"Oocytes failing to fertilize in vitro. c Fresh oocytes.

is after the replacement of blastocyst-stage embryos into the uterus. The development of an in-vitro system that mimics the oviductal environment would therefore be the most ideal system. Such a system may be useful both in yielding higher quality and viable embryos and in yielding a higher percentage of blastocysts for replacement into the uterus. Recent experimental results suggest that a culture system which mimics the oviductal environment significantly improved embryonic development and may well also benefit in-vitro fertilization and assist in studies on implantation. Human oviductal characteristics Recent definitive studies on the cytology of the oviducts of women have been reported. Two distinct cell types (ciliated and secretory) have been observed in the epithelial lining of human oviducts. The secretory cells varied in cell height in a cyclical fashion. These cells increased their height to a maximum in mid-cycle and then diminished to a minimum height in the premenstrual and menstrual phases (Verhage et al., 1979). No changes were observed by some workers in the percentage of ciliation during the cycle (Patek, 1974; Brosens and Vasquez, 1976; Critoph and Dennis, 1977) while others observed ciliogenesis under transmission electron microscopy (Oberti and Gomez-Rogers, 1972). In a more comprehensive and detailed study, Verhage et al. (1979) showed that the epithelial cells attained maximum height and 894 Downloaded from https://academic.oup.com/humrep/article-abstract/5/8/893/675490 by University of Chicago user on 13 April 2018

ciliation during the late follicular phase in the fimbria and ampulla. At the end of the luteal phase, atrophy and deciliation occurred in the fimbria. In the early follicular phase, hypertrophy and reciliation occurred. During pregnancy and in the postpartum period there was further atrophy and deciliation. Atrophy and deciliation were associated with elevated progesterone levels while hypertrophy and reciliation related to low progesterone and moderate oestrogen levels. Thus there appears to be a ciliation-deciliation cycle in the human oviduct that is hormonally controlled. A more recent study of the human oviduct confirmed these findings (Donnez et al., 1985). Hormonal regulation of the human oviduct secretions has been reviewed by Jansen (1984). He observed that around ovulation, the isthmus secretions became more viscous than that of the ampulla. The secretions became abundant when oestrogen levels were high at mid-cycle. Jansen suggested that these secretions may play an important regulatory role in sperm transport through the isthmus towards the ampullary — isthmic junction where fertilization normally occurs. The mammalian oviduct has been reported to be fundamentally secretory (Leese, 1988). Recent thinking recognized a bidirectional movement of molecules across the epithelial lining of the oviduct. There are fluxes of molecules from the fluid bathing the serosal surfaces to those bathing the mucosal surfaces and vice versa (Leese, 1988). Borland et al. (1980) found that oviduct fluid has a much higher potassium concentration and a much lower calcium ion concentration than plasma. The alkalinity of

Co-cultures in assisted reproduction

Table III. In-vitro behaviour of human ampullary and endometnal epithelial cells Cell type (epithelial)

Days to form primary monolayer (range)

Type of growth (primary culture)

Number of subcultures

Days between subcultures

Type of growth (subculture)

Cell type (subculture)

Anipullary Endometrial

6-7 3-7

E E + F

4-6 5-7

3-4 2-4

F F

S C + S

E. epithelioid; F, fibroblast-like, C, ciliated: S. secretory Source: Bongso et al. (1988a, 1989c)

oviductal fluid (pH 7.5-8.0) was due to the high concentration of bicarbonate ions. The oxygen tension in the rabbit oviductal lumen ranges from 25 to 60 mmHg (Ross and Graves, 1974). All available studies suggest that spermatozoa, ova and early embryos are exposed to an environment that is able to support aerobic metabolism. Much emphasis has been placed on those non-electrolytes that are important to gamete and embryo metabolism (glucose, pyruvate, lactate and amino acids). The concentration of these in oviductal fluid was below plasma concentrations (Leese and Barton, 1985). The same authors confirmed earlier work that pyruvate levels in the vicinity of the cumulus mass were high, thus suggesting that cumulus cells can contribute to the pyruvate pool in the oviductal lumen. Plasma proteins were shown to account for most of the protein content of oviductal fluid, the most abundant being albumin and immunoglobulin G (Leese, 1988). Oviductal fluid is a crucial environment in which the movement of ovum and spermatozoon in the oviduct, fertilization, embryo transport and early development takes place. The electrolytes are primarily responsible for maintaining the osmolarity and pH of the fluid while the high potassium concentration is said to be involved in the inhibition of sperm motility in the lower isthmus. Bicarbonate ions promote the dispersal of corona cells around the oocyte and they also stimulate sperm respiration. Glucose, lactate and pyruvate support sperm, oocyte and embryo survival (Leese, 1988). Co-culture systems An attractive approach to defining the requirements of embryos for development is to co-culture embryos with other cell types that may provide a stimulus for embryonic development. Two co-culture systems that may offer promise are cellular monolayers and trophoblastic vesicles. The use of trophoblastic vesicles has been restricted to data to animal experimentation and not adopted for the human (Camous et al., 1984; Heyman et al., 1987). Early preimplantation embryos have been co-cultured with epithelial cells of the reproductive tract such as ampullary and endometrial cells. Such cells could be used either fresh, as established primary cell cultures or cell lines. The co-culture concept presumes that the cultured monolayer provides some stimulus via specific factors towards the development of the embryo or regulates the embryo's in-vitro environment. It was recently shown that in the first 3 days after fertilization, sheep embryos cleaved normally when co-cultured with sheep oviductal cells (Gandolfi and Moor, 1987). Cells were obtained by scraping the lumenal surface of dissected oviducts

and these were co-cultured fresh with the embryos. The cell preparations included ciliated and secretory cells. In another study, ovine oviductal epithelial cells were cultured in vitro and the subcultured cells from established cell lines were used for co-culture with ovine embryos (Rexroad and Powell, 1988a). The subculturing was shown to yield a single cell type as compared to the mixed populations in fresh preparations. This was claimed to have an advantage as there was a well-defined fibroblast-like cell line for co-culture. Since the use of animal oviducts to support cleavage of human embryos is not ethical, human ampullary and endometrial primary cell cultures and cell lines were developed from material obtained after hysterectomy (Bongso et al., 1988a, 1989c). Gland and stromal cells from human endometrium were separated and established as cell lines for periods of over one menstrual cycle. The two cell types were separated using 0.25% (w/v) collagenase and several intermittent centrifugations at 100 g for 5 min. The cells were seeded in plastic tissue culture flasks using Chang's medium supplemented with L-glutamine and a few other components (Bongso et al., 1988c). Monolayers were established in 3 - 7 days and in-vitro cell growth was a mixed epithelioidfibroblast-like cell type (Table in). Such monolayers could be maintained alive for periods of up to 3 - 4 weeks. Scanning electron micrographs showed ciliated cells and cells with varying densities of microvilli and apical protrusions. Endometrial cells in culture showed structural features remarkably similar to those described for cells in situ. The method of growing endometrial cell lines is also a useful tool for studying the cellular mechanisms of human implantation. Future studies could also be developed to characterize the exact in-vitro behaviour of the endometrium at the time of embryo replacement in IVF patients where fertilization of oocytes has failed and there are no embryos for replacement. The receptor sites and immune responses involved in implantation could also be investigated using this system. This model is also useful to study the hormones and factors released by endometrial cells into the culture medium and the study of cells derived from malignant tissues of the uterus. Based on similar tissue culture principles, a simple method for the establishment of human ampullary cell cultures was also developed (Bongso et al., 1989c). Ampullary cells dislodged from the epithelial lining of oviducts obtained after hysterectomy were seeded into tissue culture flasks with Chang's medium supplemented with 200 mM L-glutamine, penicillin (100 IU/ml), streptomycin (100 /ig/ml) and fungizone (0.1 ml/ml). Incubation was at 37°C in 5% CO2 in air using an 'open-system'. Confluent monolayers were established in 6 - 7 days irrespective of the patient's age and phase of the 895

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menstrual cycle. These cells could be maintained alive through four to six subcultures with —3-4 days between subcultures (Table HI). In-vitro behaviour was mainly epithelioid and the cell types were both ciliated and non-ciliated (secretory) as observed by scanning electron microscopy. With continuous subculturing, the cells transformed into the fibroblast-like cell pattern and were mainly of the secretory type with numerous microvilli. Transmission electron micrographs of these secretory cells revealed secretory vesicles which had dark contents. Lipid globules of low electron density were also commonly encountered in such cells. Structural features of the cells in vitro were remarkably similar to those described in vivo. Because of the absence of a physiological role for ovum transport in vitro and absence of peristaltic oviductal movements, the ciliated cells gave up their functions and acquired secretory roles. Such ampullary cell lines are also useful tools in studying the microscopic events surrounding sperm-ovum interaction, early embryonic cleavage, the mysteries of ectopic pregnancies and also serve as feeder layers for the support of human embryos for FVF. In the human co-culture studies carried out thus far (Bongso et al., 1988a, 1989c,d), Chang's medium (Hana Biologies, USA) appeared to be the best, compared to Ham's F 10 and T6 medium, for the establishment and growth of ampullary cell lines. The additional nutritional ingredients in Chang's medium such as 8% newborn calf serum, nucleosides, hormones, vitamins, amino acids and polypeptides may be playing an important role (Bongso et al., 1988c). Attempts to optimize the co-culture system must take into account both the embryo and the monolayer. The role of other factors such as temperature and gas phases have not as yet been investigated for co-cultures. Screening of co-cultures for microbes It is extremely important that before co-cultures are used for assisted reproduction, that the cells and medium nourishing them, should be screened for the presence of viruses, microbes, fungi and mycoplasma. This is to avoid the risk of disease transmission to the embryo because of cross-contact between patient materials. Co-cultures could be easily screened for viruses, fungi and other microbes by using the conventional direct or indirect fluorescent antibody techniques, immunoelectron microscopy and microbiological plating tests. Pregnancy rates after co-culture A critical question for a co-culture system is whether it provides transferable embryos. Very recently, fetal cattle uterine fibroblast

monolayers were used to evaluate in-vitro development and implantation of human embryos (Wiemer et al., 1989). Fetal uterine endometrial linings were obtained from two healthy bovine fetuses at 270 days gestational age, and subcultures after seven passages were used for co-culture. After 24 h, the fragmentation rate of co-cultured human embryos was significantly lower than that of controls (medium alone) (P < 0.05). The human embryos co-cultured with the cattle monolayers were transferred back to patients and the pregnancy rate was significantly greater than embryos cultured in conventional Earle's medium alone (35 versus 17%; P < 0.05). Implantation rates increased with time in culture for co-cultured embryos (43%). These authors reported that they now routinely use cattle cocultures for embryonic cleavage and growth in their human IVF programme (Wiemer et al., 1989). It was also recendy shown that the viability of human embryos could be significantly improved when co-cultured with human ampullary cells (Bongso etai, 1989d; Sathananthan etai, 1990). Subcultured ampullary cells from established primary cultures were used instead of fresh cells. The subcultured cells provided a richer source of secretory cells for optimal cleavage and development. The results are summarized in Table IV. Of embryos co-cultured with ampullary cells in T6 medium + 15% patient's serum, 78% cleaved to the compacted stages and 69% cavitated as compared to 50 and 33 %, respectively, for embryos grown in T6 medium + 15% patient's serum alone (P < 0.01). However, only 30% of co-cultured embryos reached the expanded blastocyst stage and 26% underwent hatching compared to 28% for both stages in controls. In the early stages (2- to 4and 6- to 8-cells), 91 and 87% of co-cultured embryos showed absence or slight fragmentation as compared to 72 to 61%, respectively, for embryos grown in medium alone (P < 0.01). All the good-quality co-cultured embryos in this study were frozen to be replaced at a later date. Although co-cultured embryos also cleaved slightly faster than controls, it was clear that subcultured human ampullary cells increase embryonic viability and yield a reasonable number of blastocysts for replacement. Thus, within this framework, oviductal co-culture seems to be of clear benefit but is not yet the ideal system since its support of blastulation is still inadequate. Development past the expanded blastocyst stage may perhaps be another critical phase with specific requirements such as endometrial co-culture. The results of this study were obtained from only 12 patients and hence only limited conclusions can be drawn at this time. Similar studies on a larger series of patients is urgently required together with the pregnancy rates

Table IV. Behaviour of human embryos in vitro when cultured in two systems* Medium

Two pronuclcar stage

% 2- to 4-cell embryos Fragments Fragments (absent/slight) (moderate/severe)

% 6- to 8-cell embryos Fragments Fragments (absent/slight) (moderate/severe)

Compacted embryo (%)

Cavitated embryo (%)

Fully expanded blastocyst (%)

Hatching blastocyst (%)

Human ampulla T 6 + 15% HS

(23) (18)

91 b (21) 72* (13)

87b (20) 61C (10)

78b 5f/

69*

30 28

26 28

9(2) 28(5)

' D a t a from 12 patients. bc C h i square (P < 0.01). Figures in parentheses are number of embryos Source: Bongso et al. (1989d).

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13 (3) 39(8)

33 C

Co-cultures in assisted reproduction

using the human ampullary cell co-culture system. Clinical trials on a large group of patients are in progress to evaluate pregnancy rates using the human co-culture system. In the meantime it is interesting to note that increased pregnancy rates have already been reported for the human using animal oviductal co-culture systems as discussed earlier in this section (Wiemer et al., 1989). The data available in domestic animals also confirms that viable embryos do result from co-culture (Rexroad, 1989). An increased pregnancy rate was obtained when bovine embryos co-cultured with bovine cumulus cells were replaced into recipient cows. The results conclude that embryo viability improved in co-culture (Goto et al., 1988). Very recently, a 66.7% pregnancy rate was reported in cattle following transfer of bovine embryos produced by in-vitro maturation, fertilization and co-culture with bovine oviductal epithelial cells. The authors claimed their results were comparable to those obtained with similar embryos produced in vivo (Xu etal., 1990). Freezing of oviductal epithelial cells If co-cultures are to be used routinely in IVF centres in the future, ampullary cell-lines established from cells collected from human oviducts after hysterectomy may have to be maintained in a tissue culture laboratory. Such cell lines have a definite life-span and establishment of new cell cultures may have to go on continuously. Alternatively, cultured ampullary cells may be frozen, later thawed and cultures established for immediate use as cocultures. Frozen—thawed ampullary and endometrial cells may have to be studied to find out whether they maintain their same activity in vitro as in vivo. Preliminary results showed that human ampullary cells could be successfully frozen using the slow programmed freezing method with dimethyl sulphoxide (DMSO) as cryoprotectant. After rapid thawing to 37°C, monolayers were established in —7—10 days which was slightly longer than for unfrozen cells (Bongso etal., unpublished data). Growth factors and embryonic blocks In-vitro embryonic blocks have been well recognized for different embryonic stages in various species of animals. The co-culture system appears to overcome such blocks (Rexroad, 1989). The fact that a large percentage of co-cultured embryos cavitated to the early blastocyst stage is evidence for a bypassing of the critical 8-cell and morula stages in the human. These embryonic blocks have been claimed to be due to artefacts in the culture environment and probably occur at the time the embryonic genome is activated (Bavister, 1988). It has also been observed that the blocks seem to occur at the stage when the embryo would be changing from oviductal to uterine environments (Bavister, 1988). The role of the monolayer in the co-culture system is not clear. The first of two major possibilities is the provision of required metabolites and specific growth stimulators. The second possibility is the detoxification of medium. Specific glycoproteins facilitating embryonic development have been demonstrated in the sheep, mouse and pig oviductal epithelium (Kapur and Johnson, 1986; Brown and Cheng, 1986; Gandolfi et al., 1989). It has been shown that in the immediate post-oestrus period in sheep, the secretion of oviductal fluid is

increased and an oestrus-specific protein which was PAS positive with a mol. wt of ~ 80 kd was present in the fluid (Sutton et al., 1984). More recently, Gandolfi et al. (1989) demonstrated that after incubation of sheep oviductal cells in [35S]methionine-containing medium followed by electrophoretic separation, two classes of secretory polypeptides were identified. The first included those secreted uniformly throughout the oestrous cycle and the second included those showing a cyclical pattern of secretion, which were more predominant (Gandolfi et al., 1989). The first and second classes were composed mainly of polypeptides of Mr 92 and 46 X 103, respectively. Both polypeptide species were referred to as sheep oviduct proteins 92 and 46 (SOP 92, SOP 46) and were detected only during the first 4—5 days after oestrus when the embryos were residing in the oviduct (Gandolfi et al., 1989). The interaction between the oviductal proteins and the developing embryos was also studied by the same workers. The results of iodination studies showed that Mr 92 and 46 x 103, were bound to the zona pellucida of oocytes removed from the oviduct but were absent from oocytes that had not had contact with the oviductal epithelium. The authors suggested that these proteins represent SOP92 and 46 based on their electrophoretic mobility and their ability to bind to the zona of oocytes when added in vitro and by the fact that they both disappear from the zonae of embryos after exit from the oviduct. Using monoclonal antibodies the same authors confirmed that SOP92 binds and crosses the zona and becomes associated with the individual blastomeres of the developing embryo. All the above findings demonstrate that the mammalian oviduct probably plays a direct role in supporting embryonic development through specific polypeptides produced by the epithelial lining cells (Gandolfi etal., 1989). Major proteins synthesized by the human oviduct were also recently identified by Verhage etal. (1988). Two proteins, one acidic and the other basic with mol. wts of 120 000-130 000 were detected. These glycoproteins were observed in the medium of midcycle oviducts, at the time when the oviduct participated in gamete transport and embryo development. Simple diffusion of a glycoprotein into the embryo via the zona pellucida alone may not account for the beneficial effects of the co-culture system. Placement of a permeable filter between the embryos and the monolayer prevented the beneficial effects (Allen and Wright, 1984). Further, 'conditioned' medium did not substitute for co-culture in the development of early sheep embryos (Rexroad and Powell, 1988b). It thus appears that contact between the embryo and monolayer is necessary for expression of the co-culture effect. In the co-culture system it is also possible that the oviductal cells remove a detrimental component from the culture medium (Bongso etal., 1989d). Differences in the physico-chemical environment (pH, gas tension) provided by oviductal secretions have been suggested (Bavister, 1988). Further work is necessary to identify, extract and purify specific embryotrophic or pregnancy signalling factors residing in the human oviduct. Specificity of co-cultures An absolute specificity for a reproductive tract source of co-culture cells has not been demonstrated. Sheep oocytes 897

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co-cultured on ovine fibroblasts cleaved as well in vitro as sheep oocytes co-cultured on oviductal cells for a period of 3 days (Gandolfi and Moor, 1987). Also, Rexroad and Powell (1988b) demonstrated that sheep ova transferred after co-culture on uterine and kidney cells for 2 days cleaved as well as those co-cultured for 2 days on oviductal cells. After 3 days of co-culture, ova that had been co-cultured on oviductal cells had a far greater cleavage rate after transfer. It has also been shown that bovine morulae cleaved equally well when co-cultured on uterine fibroblasts as on testicular fibroblasts (Kuzan and Wright, 1982). Pig embryos cleaved as well when co-cultured on pig endometrial fibroblasts as on ovarian fibroblasts (Allen and Wright, 1984). Recently, Kim et al. (1989) showed that cattle fetal spleen cell and chick embryo fibroblast cell monolayers supported the cleavage and growth of precompaction-stage bovine embryos. The same authors concluded that monolayer systems developed from cells other than adult or fetal reproductive tissues are capable of supporting later-stage bovine embryos for up to 72 h during invitro culture. Very recent studies have shown that co-culture systems may be neither species-specific nor cell-specific. Early 2-cell mouse embryos cleaved regularly and reached blastocyst stages as well on mouse and human ampullary, cumulus and muscle fibroblast co-cultures as on controls of medium alone (Bongso et al., 1990). All these observations suggest that coculture on several cell types may be beneficial for early embryos and there are no stringent requirements for a specific stimulus from the reproductive tract.

Implantation signals from ampullary and endometrial co-cultures The in-vitro morphology and behaviour of human endometrial glandular and stromal cells has enabled recognition of the secretory, postovulatory and proliferative phases of the menstrual cycle when compared to histologically dated specimens (Bongso et al., 1988a). The state of the endometrium has also been assessed from endometrial biopsies collected prior to embryo replacement in an IVF programme. It was concluded that taking of the biopsy at the time of embryo replacement did not prevent the occurrence of a viable pregnancy and that the presence of a proven secretory endometrium was an important prerequisite in determining the successful outcome of IVF (Abate et al., 1987). Thus, the secretory cells from endometrial cell lines may provide a priming of the embryo with unknown implantation factors or signals which may eventually yield improved pregnancy rates. It has been demonstrated that preimplantation mouse embryos took up or bound at least eight proteins synthesized by endometrial cells when such embryos were co-cultured with endometrial cell monolayers. The mol. wt of the proteins ranged from 11 000 to 120 000 daltons and their specific role in in-vitro embryonic development was not known (O'Fallon et al., 1984). Very recently, placental protein 5 (PP5) which is a glycoprotein with properties of a serine protease inhibitor, was found in the human oviduct. The author claimed that this protein might be playing a role in the implantation of the human embryo (Butzow, 1989).

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Sperm hyperactivation in the presence of oviductal co-cultures Preliminary data showed that a significant number of motile human spermatozoa undergo hyperactivation in the presence of passaged co-cultured human ampullary cells as compared to controls of medium alone over a 6-h period. Using computerized semen analysis (Cellsoft, Cryoresources, USA), curvilinear velocity and mean amplitude of lateral head displacement (ALH) were increased. Binding of motile spermatozoa to co-cultured fibroblasts was also observed (Bongso et al., unpublished data). Sperm motiliry has been shown to be the singlemost important criterion determining fertilization rates (Table I) and the acrosome reaction is also an important prerequisite for fertilization. Thus, hyperactivated spermatozoa recovered after co-culture with ampullary cells may be useful in improving fertilization rates in assisted reproductive technology programmes. The occurrence of these two phenomena in the presence of ampullary cells may explain why pregnancy rates are higher in GIFT and TET than IVF. Future aspects A refinement of the existing in-vitro conditions for human IVF is urgently needed. The use of co-cultures for the union of spermatozoon and oocyte in vitro as well as for cleavage and growth to yield good quality 4- to 6-cell stage embryos may replace conventional culture media in the near future. With more laboratories attempting to use human reproductive cells for coculture, a larger percentage of blastocysts per patient for replacement could be expected. This may eventually lead to the replacement of blastocyst stage embryos rather than 4- to 6-cell stage embryos, —4—5 days after oocyte recovery, thus increasing pregnancy rates, as in domestic animals. The coculture system must be optimized and must yield reliable and consistent results to prevent disappointment for patients who may have to wait anxiously 4 - 5 days before any news of an embryo replacement. It is hoped that the replacement of at least one blastocyst-stage embryo per patient may produce significantly higher pregnancy rates than the current replacement of a maximum of three to four 4- to 6-cell stage embryos. The transfer of frozen -thawed 4- to 6-cell stage embryos from stimulated cycles into subsequent natural cycles has boosted IVF pregnancy rates (Dor and Rudak, personal communication). Further, the cryopreservation of human blastocysts using glycerol has been successfully carried out using the slow programmed method of freezing (Fehilly et al., 1985). Thus, once the coculture system is perfected to yield good quality 4- to 6-cell stage embryos and a reasonable percentage of blastocysts, the replacement of frozen—thawed co-cultured 4- to 6-cell stage embryos or blastocysts in natural cycles may perhaps lead to higher pregnancy rates in human IVF. Acknowledgements The authors thank Miss Harjeet Kaur for her excellent secretarial assistance and the National University of Singapore for its financial assistance.

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