Early pregnancy sera were earlier shown to modulate T lymphocyte rosetting with sheep erythrocytes. We set out to investigate whether early pregnancysera also modulate the state of activation of another immunologically relevant cell type, the monocyte. As an index of monocyte activation, we measured phorbol ester-triggered oxidative burst activity by a highly sensitive chemiluminescence method. Contrary to our expectations, incubation of mononuclearcells with sera taken early after embryo transfer from patients with a successfully developing pregnancy had no effect. Unexpectedly, such sera from patients from a control group of patients in whom no pregnancy developed after embryo transfer caused enhancement of mononuclear cell chemiluminescence. Stimulatory activity of these sera appeared between days 4 and 6 and was maximal between days 7 and 9 post embryo transfer. Whether this phenomenon is causally related to, or represents a consequence of the failure of embryo transfer, can currently not be decide

Henderson, David

Maly, Friedrich E.

Sterzik, Karl

Week, Alain L.de

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Human Reproduction vol.5 no.3 pp.342-344, 1990Enhancement of mononuclear cell oxidative burst byearly post-transfer sera from human patients afterunsuccessful embryo transferFriedrich E.Maly1, Karl Sterzik2, David Henderson3and Alain L.de WeekInstitute of Clinical Immunology, Inselspital Bern, CH-3010Switzerland, 2Department of Gynaecology, University of Ulm and'Biochemical Pharmacology, Schering AG Berlin, FRG'To whom correspondence should be addressedEarly pregnancy sera were earlier shown to modulate Tlymphocyte rosetting with sheep erythrocytes. We set out toinvestigate whether early pregnancy sera also modulate thestate of activation of another immunologically relevant celltype, the monocyte. As an index of monocyte activation, wemeasured phorbol ester-triggered oxidative burst activity bya highly sensitive chemiluminescence method. Contrary to ourexpectations, incubation of mononuclear cells with sera takenearly after embryo transfer from patients with a successfullydeveloping pregnancy had no effect. Unexpectedly, such serafrom patients from a control group of patients in whom nopregnancy developed after embryo transfer caused enhance-ment of mononuclear cell chemiluminescence. Stimulatoryactivity of these sera appeared between days 4 and 6 and wasmaximal between days 7 and 9 post embryo transfer. Whetherthis phenomenon is causally related to, or represents a conse-quence of the failure of embryo transfer, can currently notbe decided.Key words: embryo transfer failure/monocytes/oxidativeburst/serum factorsIntroductionThe concept of circulating serum factors indicative of the stateof a pre- or peri-implantation embryo (early pregnancy factor,EPF) was originally developed by Morton et al. from theirfinding of serum factors able to co-inhibit rosette formationbetween T lymphocytes and sheep red blood cells (Morton et al.,1976). Since this original observation, results reported from anumber of laboratories led to the conclusion that the signallingsystems between embryo and mother are complex in nature andutilize a wide variety of mediators, some of which are alsoinvolved in immune reactions. Thus, for example, O'Neill et al.showed that in the mouse, successful fertilization is accompaniedby peripheral thrombocytopenia due to the release of a embryo-derived platelet-activating factor (O'Neill et al., 1985). Morerecently, the ovine trophoblast protein OTP-1 was shown to bea member of the interferon super-family and indeed anti-luteolyticactivity of interferons can be demonstrated in some species(Imakawa et al., 1987). These and other mediators appear to be342largely or wholly involved in the control of luteal function. Thepresence of large numbers of infiltrating leukocytes at the siteof implantation and the necessity for explaining the immuno-logically privileged nature of the embryo suggest, however, thatthe implanting embryo may modulate some component(s) of thematernal immune system. In addition to the central role of T-lymphocytes, accessory cells such as monocytes/macrophagesparticipate in the regulation of the immune response. We thereforeinvestigated whether monocyte activity is influenced by serumobtained in early pregnancy, with the additional aim of developingan alternative to the rosette inhibition test for early pregnancyfactor (EPF). Depending on their activation state, monocytes/macrophages produce a great variety of biologically activemediators, ranging from reactive oxygen species such as super-oxide and lipid mediators such as prostaglandins to peptidemediators, for example GM-CSF and IL-1. In the present study,we investigated whether sera taken from patients early afterembryo transfer have the potential to modulate the activation stateof monocytes, using sera taken before embryo transfer ascontrols. As an index of monocyte activity we measuredsuperoxide-dependent lucigenin-amplified chemiluminescence(Allen etal, 1985).Materials and methodsThe study population comprised 13 patients in whom pregnancydeveloped (ET+) after in-vitro fertilization and embryo transfer,performed as described previously (Sterzik et al., 1988). Twelveother patients, in whom no pregnancy developed after the sameprocedure performed at the same institution and in the same timeperiod (ET-) , were also studied.Sera from patients were collected before and within 18 daysafter the procedure, sterile filtered and stored at —20°C untilassayed.Mononuclear cells (MNC) were prepared from peripheralblood of healthy male volunteers by Ficoll gradient centrifugation(specific gravity of Ficoll = 1.077) and washed twice in Hank'sbalanced salt solution without divalent cations. Finally, MNCwere suspended at a density of 5 x 106/ml in phenol red-freeminimal essential medium (T038, Biochrom/Seromed, Berlin,FRG) buffered to pH 7.2 with 20 mM HEPES (Calbiochem, LaJolla, USA) containing 100 /ig/ml of bovine serum albumin(Behringwerke, Marburg, FRG) (T038/BSA). Flow-cytometricdetermination of cellular composition of MNC was done on anOrtho 50H cytofluorograph, using the following FITC-labelledmurine monoclonal antibodies (Becton Dickinson MonoclonalCenter, Mountain View, CA): anti-Leu 4 (T cells, CD3), anti-Leu 12 (B cells, CD 19), anti-LeuM3 (monocytes/macrophages)© Oxford University PressMonocyte oxidative burst and early pregnancyand anti-Leu 11 (CD16 on natural killer cells and granulocytes).The cellular composition of MNC from donor I (II) thusdetermined was the following: 75(82)% T cells, 6(7)% B cells,15(10)% monocytes, 5(4)% NK cells, < 1(< 1)% granulocytes.Mononuclear cell incubation with patient sera was performedas follows: 100 n\ aliquots of the MNC suspension (= 0.5 x106 MNC) were dispensed in microtitre plate wells (Blackmicrotitre plates, Dynatech, Embrach-Embraport, Switzerland)and incubated with 100 ;tl of patient serum for 60 min at 37°C.Thereafter, serum was removed by three cycles of centrifugation,aspiration of supernatant with a modified ELISA washer andrenewed addition of T038/BSA medium, yielding a final serumconcentration < 1 %. Control experiments showed that no cellswere lost by this procedure.Measurement of chemiluminescence (CL) from samplescontained in microtitre plates was performed as follows. Cellswere stimulated with 20 ng/ml of phorbol myristyl acetate in thepresence of 100 /*M lucigenin (JV,N-dimethyl-acridinium nitrate;both from Sigma, Miinchen, FRG). The 60 min integral of CLwas then measured using a computer-aided microtitre plateluminometer as described in more detail elsewhere (Maly et al.,1988). It was thus possible to complete the evaluation of all serumsamples in triplicates on MNC of one donor within <3 h.Because sera from different individuals affected the CL of testMNC differently, for each patient, the CL value obtained withher serum taken on the transfer day was used as reference andset at 100. Results obtained with the other sera from the individualpatient were expressed relative to this sample, yielding relativeCL values in per cent.To further compare the time course of relative CL valuesbetween the group of patients with successfully establishedpregnancy (ET+) and those in whom no pregnancy developed(ET —), the values of 3-day intervals before and after the dayof embryo transfer where grouped together and averaged. Thoughthis procedure diminished the temporal resolution of the study,it was necessary because for technical reasons (outpatientprocedure) it was not possible to obtain a serum sample everyday from each patient.Results and discussionPost-transfer sera from the majority of ET — patients enhancedthe chemiluminescence response of male mononuclear cells toPMA, while post-transfer sera of ET+ patients had only slightstimulatory or inhibitory effects (Figure I). On average, thisstimulatory activity of ET — sera appeared between days 4 and6, was maximal between days 7 and 9 post-transfer (maximalvalue: 172 ± 32%, mean ± SEM, N = 9) and thereafterreturned to baseline levels. In contrast, the group of ET+ patientsshowed on average no significant effects of post-transfer sera onchemiluminescence of indicator MNC (Figure 2). Differencesbetween the relative CL means of E T - and ET+ groups werestatistically significant at days 4 to 6 and 7 to 9 (P = 0.0063for days 4 - 6 and P = 0.065 for days 7 - 9 , Test 1, Student's/-test). Interestingly, in six of eight individual E T - patients fromwhom sera in the period between days 4 and 6 were available,relative CL values greater than the mean +2 SD of the valuesof the ET+ group in this time interval were found. As can alsoRel. CL (%)300 TET*2 0 0 -1 0 0 -o-1-300 T200-100--o-1-ETH h-10 -6 -2 \ 2 6DAYH r- H10 14 18Fig. 1. Time course of MNC CL-modulating activity of individualsera from ET+ (upper panel) and E T - patients (lower panel).For each patient, MNC chemiluminescence values are expressed asa percentage of the value obtained with her serum taken on orshortly before the day of embryo transfer, denoted as day 0(arrow) and are linked by solid lines. Data are means of triplicatedeterminations with SD <20% of the respective mean (Test 1).be seen from Figure 2, these findings could be reproduced withtarget MNC from another donor.The major cell type among blood mononuclear cells capableof producing CL was the monocyte, though a few contaminatinggranulocytes were present (<1%). This makes likely thepossibility that monocytes are the primary target cells of theMNC-CL enhancing activity described (Babior, 1978). However,it is also possible that the effect results from an interactionbetween serum factors, monocytes and other cells present inMNC, for example T lymphocytes or natural killer cells, whichmay modulate the monocyte oxidative burst. Therefore,participation of NADPH oxidase-negative cells, such as T or NKcells, in the mechanism leading to the observed enhancement ofmononuclear cell oxidative activity is to be further investigated.Several soluble mediators are known to enhance the superoxide-forming capacity of monocytes/macrophages, e.g. thelymphokines gamma-interferon and granulocyte-macrophagecolony stimulating factor. However, they are mainly producedand consumed locally and generally not found in the circulation,and it is therefore unlikely that they could be responsible for theobserved phenomenon. Further research is needed to examinethe nature of the factor(s) responsible for the upward modulation343F.E.Maly et al.ET POS TEST 1 ET NEG TEST 1I ISOd- 2 - 1 0 1 2 3 4 53 - Day Time IntervalET POS TEST 2- 2 - 1 0 1 2 3 *3 - Day Tin* IntervalET NEG TEST 2| too- 2 - 1 0 1 2 3 43 - Day Tan, Interval- 2 - 1 0 13 - DayFig. 2. Time course of mean MNC CL modulating serum activity.MNC CL modulation values obtained with MNC from twodifferent donors (Test 1 and 2) were averaged over 3-day periodsas described in Materials and methods. The day of embryo transferis denoted day 0. Data shown are mean ± SEM. P values indicatethe significance of differences between ET+ and E T - .of MNC CL.We had originally expected that sera from patients developinga pregnancy would exert inhibitory effects, in analogy with thefindings of Morton et al. (1976) concerning EPF. We chose thephorbol ester PMA as trigger of the mononuclear cell oxidativeburst because it is one of the most potent activators of theoxidative burst which also bypasses cell surface receptors andactivates cell functions more directly, typically via proteinkinase C. Therefore, effects on PMA-induced responses, like theenhancement described, are likely to be caused at the post-receptor level, e.g. within the protein kinase C-initiated signallingpathway or at the level of the NADPH oxidase itself. From this,it is probable that ET-sera will cause an enhancement of the MNCoxidative burst also when PKC and NADPH oxidase are activatedvia receptor-mediated mechanisms such as phagocytosis, thoughthis will have to be tested experimentally.Earlier, changes in the rosette-inhibitory activity of earlypregnancy sera were reported which correlated with the outcomeof mating or embryo transfer (early pregnancy factor; Mortonet al., 1976). The finding that no differences in the MNC-CLmodulatory activity of pre- and post-transfer sera of patients whodeveloped a pregnancy were detected suggests that earlypregnancy factor does not modulate monocyte oxidative burst,at least not in the amounts present in such sera. However, thefinding of serum factors enhancing the monocyte oxidative burstin the group of patients in whom embryo transfer was notfollowed by establishment of pregnancy—originally devised asthe control group—was unexpected and merits furtherinvestigation. The time at which these serum effects were notedwould qualify them as associated with the late preimplantationor the peri-implantation period.Elevated oestradiol levels and hence increased oestradiol/pro-gesterone levels have been found to be associated with failed IVFattempts. However, no difference in oestradiol/progesteroneratios was evident between the ET+ and ET— patient groupsinvestigated in this study (data not shown). In addition, oestradioland progesterone have been demonstrated to inhibit thesuperoxide-generating NADPH oxidase in vitro (Buyon et al.,1984). It is therefore unlikely that either of them could, alone,be responsible for the observed enhancement of superoxidegeneration.In summary, the phenomenon we describe in this communica-tion could be connected with the process itself which causes lossof the embryo. Thus, our data could be interpreted as furtherevidence for the existence of peripherally detectable parametersreflecting the fate of the embryo. Alternatively, the upwardmodulation of MNC CL in the patients that did not becomepregnant, might merely be a consequence of the failure ofpregnancy, such as, for instance, resolution of the corpus luteum.A study with serum samples taken throughout the menstrual cycleof normal women not attempting to conceive and taking nocontraceptive medication could provide pertinent information; butdue to obvious difficulties in obtaining such serum samples, thesedata are not yet available. Though it is therefore presently notpossible to conclude which interpretation is correct, our data showthat failure of an embryo to establish a pregnancy is associatedwith peripherally detectable factors effecting changes in immunecell function.ReferencesAllen,R.C., Mead,M.E. and Kelly,J.L. (1985) In Greenwald.R.A. (ed.),CRC Handbook of Methods for Oxygen Radical Research. CRC, BocaRaton, FL, pp. 343-351.Babior,B.M. (1978) Oxygen-dependent microbial killing by phagocytes.A/. Engl. J. Med., 298, 659-669.Buyon,J.P., Korchak,H.M., Rutherford,L.E., Ganguly,M. andWeissmann,G. (1984) Female hormones reduce neutrophilresponsiveness in vitro. Arthritis Rheum., 27, 623-630.lmakawa,K., Anthony,R.V., Kazemi.M., Marotti,K.R., Polites.H.G.and Roberts,R.M. (1987) Interferon-like sequence of ovine trophoblastprotein secreted by embryonic trophectoderm. Nature, 330. 377-379.Maly,F.-E.. Urwyler,A., Rolli,H.P., Dahinden,C.A. and de Weck,A.L.(1988) A single-photon imaging system for simultaneous quantitationof luminescent emissions from multiple samples. Anal. Biochem., 168,462-469.Morton,H., Hegh.V. and Clunie,G.J.A. (1976) Studies of the rosetteinhibition test in pregnant mice: evidence for immunosuppression?Proc. R. Soc. bond. B, 193, 413-419.O'Neill,CO. (1985) Thrombocytopenia is an initial maternal responseto fertilization in mice. J. Reprod. Fertii. 73, 559—566.Sterzik,K., Dallenbach,C, Schneider,V.. Sasse,V. and Dallenbach-Hellweg.G. (1988) In-vitro fertilization: the degree of endometrialinsufficiency varies with the type of ovarian stimulation. Fertii Steril.,50. 457.Received on October 13. 1989; accepted on January 9, 1990344

Enhancement of mononuclear cell oxidative burst by early post-transfer sera from human patients after unsuccessful embryo transfer

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