uterine cytokine production preceding implantation influences several early ... of oocytes and embryos and preparation of spermatozoa were carried out in ...
Human Reproduction vol.10 no.l pp.171-176, 1995
Detection of cytokines (interleukin-1, interleukin-6, transforming growth factor-/?) and soluble tumour necrosis factor receptors in embryo culture fluids during in-vitro fertilization
3 ^ and A- -SnnHp 3
and A.Sunde
University of Trondheim, 'Institute of Cancer Research, University Medical Center, 2Department of Community Medicine and General Practice, University Medical Center and department of Obstetrics and Gynaecology, The Regional Hospital, 7005 Trondheim, Norway 4
To whom correspondence should be addressed at: University of Trondheim, Institute of Cancer Research, University Medical Center, 7005 Trondheim, Norway
Several lines of evidence suggest that a number of immunoactive cytokines participate in early reproductive events such as implantation and placental development. Furthermore, cytokines may influence embryo growth and differentiation. In the present study, the production of tumour necrosis factor (TNF), interleukin-1 (IL1), interleukin-6 (IL6) and transforming growth factor-/? (TGF/3) during the first 48 h after oocyte retrieval during in-vitro fertilization was investigated. In addition, the question was raised whether soluble receptors may contribute to cytokine activity regulation in early reproduction, and concentrations of TNF and IL6 receptors in culture media were determined. Finally, an investigation of whether any association exists between cytokine concentrations and embryo morphology was performed. Media from 256 embryos were analysed. LL1, IL6 and TGF/3 were produced during the 48 h culture period, whereas no TNF was detected. Levels of IL1 and IL6 were significantly higher in media from the first 24 h culture period than from the second period, whereas TGF/3 concentrations in supernatants from the two observation periods did not differ. IL6 receptors were not detected, whereas TNF receptors (p75) appeared in media from the 24-48 h culture period. Granulosa, cumulus and sperm cells are potential sources of cytokine production, especially during the first 24 h period. The contribution of the embryo to cytokine/cytokine receptor production remains an open question. No significant correlation was observed between cytokine/cytokine receptor concentrations and embryo morphological score. Key words: embryo morphology/ILl/IL6/TGF/3/TNF/TNF receptor
Introduction Cytokines are peptide mediators of immunological, inflammatory, and reparative responses. Recently, accumulating evidence has suggested that a number of cytokines have major influence on reproduction (J.A.Hill, 1992). The cyclic endometrium is an © Oxford University Press
active site of cytokine production and action (Tabibzadeh, 1991). The fertilized oocyte signals to the endometrial tissue (Salomonsen, 1992), and an endometrial inflammatory response conducive to implantation and placental development is generated (Robertson et al., 1992). It is assumed that the increased intrauterine cytokine production preceding implantation influences several early reproductive events, such as blastocyst attachment and trophoblast outgrowth and decidualization (J.A.Hill, 1992). In accordance with this assumption, the establishment and continuation of normal pregnancy in mice are facilitated or hindered by administration of various cytokines (J.A.HD1, 1992; Chaouat et al., 1990), suggesting that cytokines have a function in regulating the early fetomaternal relationship. Cytokines are major participants in the regulation of embryo growth and differentiation (D.J.Hill, 1992; Adamson, 1993). Accordingly, it is tempting to assume that cytokines occurring in close proximity to the embryo might influence embryo morphology. However, no significant relationship has been observed between cytokine concentrations in follicular fluid and the morphological parameters of the corresponding embryo (Barak et al., 1992). The presence of various cytokines in embryo conditioned medium during in-vitro fertilization (TVF) has been reported previously (Zolti et al., 1991; Witkin et al., 1991). The present study was performed to analyse (i) embryo culture medium concentrations of tumour necrosis factor (TNF), interleukin-1 (TL1), interleukin-6 (IL6) and transforming growth factor-/? (TGF/3) and (ii) whether any relationship can be detected between cytokine production and embryo morphology, as evaluated according to the criteria given by Staessen et al. (1989). The biological effects of cytokines are mediated through their interaction with receptors on target cells (Old, 1988; Granowitz et al., 1992). Such specific receptors may also exist as soluble molecules (Seckinger et al., 1988), and release of soluble receptors may serve as a mechanism to regulate cytokine activities (Van Zee et al., 1992). Seminal plasma contains soluble TNF receptors (TNFRs) (Liabakk et al., 1993), and thus the presence of soluble cytokine receptors may add a further dimension to the complexity of regulation of the actions of cytokines in reproductive tissues. To study this issue, the possible occurrence of soluble TNFRs and IL6 receptors (IL6Rs) in conditioned media from early embryos was investigated. Materials and methods Ovarian stimulation The protocol used for ovarian stimulation has been described previously (Kahn et al., 1993). For women with regular cycles 171
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R.Austgulen1-4, K.J.Arntzen1, L.J.Vatten2, J.Kahn3
R.Austgulen et al.
and normo-endocrine status, clomiphene citrate with human menopausal gonadotrophin were used for stimulation, and ovulation was induced by administration of human chorionic gonadotrophin. Oocyte recovery
Fertilization and embryo culture Sperm cells were prepared as described by Sher etal. (1984). Seminal plasma was spun down and washed three times with culture medium, and finally, sperm cells were added to cultures at a concentration of 1.5 X lO^ml culture medium. All culturing of oocytes and embryos and preparation of spermatozoa were carried out in serum-free media, as described by Kahn et al. (1993). The oocytes collected were placed in 0.5 ml of culture medium and inseminated 4—6 h after oocyte retrieval. Only one single oocyte was added per culture. The oocytes were examined for fertilization 16—18 h after insemination, cumulus and corona cells were removed, and the oocyte/embryo was placed in 0.5 ml of fresh culture medium. Only one oocyte/embryo was kept in each culture. About 52 h after recovery, the oocyte/embryo was examined and given a score according to the relative volume of anuclear fragments as follows: embryos were given a score of 50%. Totally fragmented embryos were given a score of 4.0. Fertilized, but uncleaved zygotes got a score of 5.0, and unfertilized oocytes a score of 6.0. In addition, the cleavage stage (number of cells) of each embryo was noted. Cytokine data were unknown to the investigator performing the morphological evaluations. Preparation of culture media The first culture media were collected —16 — 18 h after insemination (Dl medium) and the second ~ 2 4 h later (D2 medium). The culture medium was transferred to sterile 0.5 ml Eppendorf tubes and centrifuged at 8000 g for 10 min. Culture media from Dl and D2 without oocyte/spermatozoa were included as controls in all assays performed. The supernatants and control media were collected and kept frozen at — 70°C until assayed for TNF, IL1, IL6, TGF/3, TNFR and IL6R. In total, the presence of cytokines and cytokine receptors in conditioned media from 256 embryos from 30 different women was assessed. Cytokine and cytokine receptor concentrations were compared to the morphological evaluation and cleavage stage of the corresponding embryo. Only one cytokine analysis was performed on each medium sample, due to limited volumes, whereas TNFR and IL6R concentrations were determined in the 172
TNF assay The TNF concentrations in supernatants were determined by the cytotoxic effect of TNF on the mouse fibrosarcoma cell line WEHI 164 clone 13 (Espevik and Nissen-Meyer, 1986). The viability was measured after 20 h by the colorimetric MTT (tetrazolium) assay (Mosmann, 1983). Analyses were performed in triplicates. Human recombinant (r) TNF was included as a standard. The detection limit of the TNF assay was 8 pg/ml supernatant. The presence of < 2 ng/ml rTNFR did not influence the TNF activity assessed in the TNF bioassay (Liabakk et al., 1993). IL1 assay IL1 was determined in a two-stage assay by the IL1-responsive T-cell line EL-4 6.1 clone NOB-1 (Gearing etal, 1987) and the interleukin-2 (IL2) dependent mouse T cell line HT-2 (Mosmann et al, 1986), as described previously (Opsjan et al., 1993). The EL4 cells produce IL2 in response to IL1 stimulation, and IL2 production is measured by the proliferation of the IL2-dependent HT-2 cells. Human recombinant (r) IL1 from Glaxo (Geneva, Switzerland) was included as a standard measuring IL1 activity, and human rIL2 from Genzyme (Cambridge, MA, USA) was used as a standard in the IL2 assay to assess IL2 activity. Analyses were performed in triplicates. The detection limit of IL1 in the assay was 90 pg/ml supernatant. To test whether measured activity might be due to IL2 present in the supernatants, some of the supernatants were also added directly to the IL2-dependent HT-2 cells. The specificity of detected activity was tested by monoclonal antibodies (mAbs) against ILla and IL1/3 from Genzyme. IL6 assay The IL6 activity was measured by a hybridoma cell line B 13.29, which depends on IL6 for growth (Aarden etal, 1987). Recombinant IL6 (kindly provided by Dr L. Aarden, University of Amsterdam, The Netherlands) was used as a reference standard. The IL6 assay had a detection limit of 2.5 pg/ml supernatant. Analyses were performed in triplicates. The specificity of detected activity was tested with a mAb IL6 from Genzyme. The presence of < 250 ng/ml of IL6R did not influence assessment of IL6 concentration in the IL6 bioassay (data not shown). TGF0 assay The TGF/3 activity was detected by a bioassay, using the TGF/3sensitive cell line CCL 64, as described previously (Tucker et al, 1984). A porcine TGF/3 standard from British Biotechnology Ltd. (Abingdon, UK) was included. Analyses were performed in triplicates. The detection limit of the TGF/3 assay was 0.25 ng/ml supernatant.
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Oocyte retrieval was performed 34 — 36 h after gonadotrophin administration, using vaginal ultrasound-guided follicular puncture under local anaesthesia and sedation (Kahn et al., 1993). All the follicles were aspirated, and the oocytes were examined direcdy and given a maturity score (Staessen et al, 1989).
same samples. To reduce the effect of potential individual differences and to study the time-course of cytokine production, cytokine concentration assessments were done on pairs (Dl and D2) of samples. Analysis of TNF was done on 14 pairs of samples, IL1 on 31, IL6 on 40, TGF/3 on 32, IL6R on 36 and TNFR on 36.
Cytokines and cytokine receptors in embryo supernatants
TNFR immunoassay ion (pg
to "c
