- Email: [email protected]
Divergent immunomodulatory effects of recombinant and urinary-derived FSH, LH, and hCG on human CD4+ T cells
Journal of Reproductive Immunology 85 (2010) 172–179
Contents lists available at ScienceDirect
Journal of Reproductive Immunology journal homepage: www.elsevier.com/locate/jreprimm
Divergent immunomodulatory effects of recombinant and urinary-derived FSH, LH, and hCG on human CD4+ T cells Fortunata Carbone a,b , Claudio Procaccini a,b , Veronica De Rosa a,b , Carlo Alviggi c , Giuseppe De Placido c , Daniel Kramer d , Salvatore Longobardi e , Giuseppe Matarese a,b,∗ a b c d e
Laboratorio di Immunologia, Istituto di Endocrinologia e Oncologia Sperimentale, Consiglio Nazionale delle Ricerche, Napoli, Italy Laboratorio di Immunologia, Dipartimento di Biologia e Patologia Cellulare e Molecolare, Università di Napoli “Federico II”, Napoli, Italy Dipartimento di Scienze Ostetriche, Ginecologiche, Urologiche e Medicina della Riproduzione, Università di Napoli “Federico II”, Napoli, Italy Merck Serono Institute of Drug Metabolism and Pharmacokinetics, Grafing, Germany1 Merck Serono S.p.A., Rome, Italy1
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Article history: Received 21 October 2009 Received in revised form 11 February 2010 Accepted 16 February 2010 Keywords: Follicle-stimulating hormone Luteinizing hormone Human chorionic gonadotropin T cell Immunity Immune response
a b s t r a c t This study investigated the in vitro immune-modulating activities of recombinant versus highly purified urinary follicle-stimulating hormone (FSH), luteinizing hormone (LH), and human chorionic gonadotropin (hCG) at the cellular level. CD4+ T cells were isolated from peripheral blood mononuclear cells obtained from ten healthy women (aged 19–30 years) with regular menstrual cycles during the follicular phase of their cycle. CD4+ T cells were stimulated with anti-CD3/CD28 monoclonal antibodies as a T cell-specific mitogen. Proliferative and cytokine responses were analyzed at standard time points (72 h). Recombinant FSH (r-FSH) and LH (r-LH) alone showed a modest capacity to influence proliferation and cytokine release by CD4+ T cells. Conversely, their addition to T cells in combination with recombinant hCG (r-hCG) induced a powerful down-modulation of T cell proliferation, decreased interferon-␥ (IFN-␥) secretion and increased interleukin-10 (IL-10) production. These immune-modulating activities were not present when CD4+ T cells were stimulated either in the presence of urinary-purified FSH (u-FSH) or human menopausal gonadotropin (HMG), alone or in combination with recombinant hCG. We are the first to suggest that urinary-purified gonadotropins do not display profound immune-modulating activities as compared with the recombinant preparations, despite their endocrine effects. Therefore, the use of the recombinant preparations in assisted reproductive techniques might be relevant not only for their well-documented endocrine actions but also for their impact on the transient immune tolerance known to favour embryo implantation and progression of pregnancy. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
∗ Corresponding author at: Laboratorio di Immunologia, Istituto di Endocrinologia e Oncologia Sperimentale, Consiglio Nazionale delle Ricerche (IEOS-CNR), via S. Pansini 5, 80131 Napoli, Italy. Tel.: +39 081 746 4580; fax: +39 081 746 3252. E-mail addresses: [email protected], [email protected] (G. Matarese). 1 An affiliate of Merck KGaA, Darmstadt, Germany. 0165-0378/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jri.2010.02.009
It is well known that the neuroendocrine and immune systems interact (Whitacre, 2001; Kelley et al., 2007). Gonadotropins such as follicle-stimulating hormone (FSH), luteinizing hormone (LH), and human chorionic gonadotropin (hCG) are involved in the control of the neuroendocrine response crucial for regulation of the menstrual cycle, ovulation, and maintenance of pregnancy. The immune system has long been recognized as a particu-
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larly versatile system that thanks to the expression of a series of hormones and hormone receptors, can be influenced by hormones and neuroendocrine factors (Whitacre, 2001; Kelley et al., 2007). Receptors for FSH, LH, and hCG are expressed on immune cells and their capacity to influence the immune response has been investigated. More specifically, it has been shown that hCG in particular is able to down-regulate immune responses locally and systemically during pregnancy (Schumacher et al., 2009). Indeed, its presence at high concentrations has been linked to maternal tolerance towards the fetus. Furthermore, a series of reports have shown that hCG is able to inhibit T cell proliferation, secretion of inflammatory cytokines, antigen presentation, and activation by dendritic cells (Foster et al., 2010; Schumacher et al., 2009). Although recombinant gonadotropins have been in clinical use since 1995 (Daya, 2004), highly purified gonadotropin preparations have been of great value over the last 20 years in the treatment of chronic anovulation, assisted reproductive techniques, and infertility (Alviggi et al., 2006). Despite the clinical value of these highly purified gonadotropins, recent reports have raised the question of whether their use can have a negative impact on the reproducibility and standardization of protocols. This is because the preparations differ in biological activity between batches, while the purification process leaves a series of heterogeneous contaminants (Lispi et al., 2006; Bassett et al., 2009). While these gonadotropins have been shown to be active at the level of the ovary, little is known of their capacity to exert immune-modulating effects. This could also have an impact on the process of induction of maternal immune tolerance that is crucial for embryo implantation and tolerance of the fetus during pregnancy (van Mourik et al., 2009). In the present study, we have analyzed the in vitro immunomodulating capacity of recombinant versus highly purified gonadotropins. Specifically, we studied the proliferative response and the cytokine profile of CD4+ T cells isolated from women with regular menstrual cycles. The T cells were stimulated with T cell receptor (TCR)-specific activators (anti-CD3 and anti-CD28 monoclonal antibodies) in the presence or absence of increasing concentrations of either recombinant or highly purified gonadotropins. 2. Materials and methods 2.1. Study design This study was approved by our Institutional Review Board, from Università di Napoli “Federico II”. All subjects were healthy women without immunologic or endocrine disease based on clinical examination and routine blood tests. Ten women (age = 22.4 ± 1.2 years; body mass index < 25 kg/m2 ) with regular menstrual cycles (length 26–32 days) and not on oral contraceptives for at least the previous 6 months were enrolled at the Blood Transfusion Center from Università di Napoli “Federico II”. All the women were in the follicular phase of their cycle. Blood samples were collected at 9.00 am in heparinized vacutainers (BD Biosciences, Milan, Italy) and processed within the following 3 h. Blood samples were also subject to routine
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Fig. 1. The effect of increasing concentrations of recombinant human follicle-stimulating hormone (r-hFSH), recombinant human luteinizing hormone (r-hLH), and recombinant human chorionic gonadotropin (rhCG) on CD4+ T cell proliferation from normal healthy women during CD3/CD28 stimulation. (a and c) No significant changes in proliferation were induced by either r-hFSH or r-hCG. (b) On the contrary, r-hLH alone was able to stimulate significant proliferation of CD4+ T cells (*p < 0.05). Results are expressed as mean (SD) from triplicate cultures from ten healthy women.
blood count and immune-phenotypic analyses to exclude any hematologic and immune-related abnormalities. Blood and lymphocyte counts for all subjects were within the normal range (data not shown). 2.2. T cell proliferation and cytokine production Peripheral blood mononuclear cells (PBMCs) from the above subjects were isolated using Ficoll gradients (Lymphoprep, GE-Healthcare, Milan, Italy), and CD4+ T cells were subsequently purified from PBMCs by negative selection utilizing a CD4+ Isolation Kit (Invitrogen, Carlsbad CA, USA; 95% pure by flow cytometry). T cell cultures were performed in triplicate, in 96-well roundbottom plates (1 × 105 cells per well) in RPMI-1640 Glutamax medium (200 L; Invitrogen) supplemented
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Fig. 2. The effect of increasing concentrations of combined recombinant human follicle-stimulating hormone (r-hFSH), recombinant human luteinizing hormone (r-hLH), and recombinant human chorionic gonadotropin (r-hCG) on CD4+ T cell proliferation from normal healthy women during anti-CD3/antiCD28 stimulation. (a–d) r-hFSH and r-hLH, and their combination with r-hCG, induced a significant inhibition of proliferation of CD4+ T cells from healthy women (*p < 0.05; **p < 0.01). Results are expressed as mean (SD) from triplicate cultures from ten healthy women.
Fig. 3. The effect of increasing concentrations of urinary follicle-stimulating hormone (u-FSH) and human menopausal gonadotropin (HMG), alone or combined with recombinant human chorionic gonadotropin (r-hCG), on CD4+ T cell proliferation from normal healthy women during anti-CD3/anti-CD28 stimulation. (a and c) u-FSH and its combination with r-hCG resulted in a significant increase in proliferation of CD4+ T cells (*p < 0.05); (b) conversely, HMG did not induce significant changes in proliferation of CD4+ T cells, whereas when combined with r-hCG (d), there was a biphasic effect that was inhibitory at higher concentrations (*p < 0.05).
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Fig. 4. The effect of increasing concentrations of combined recombinant human follicle-stimulating hormone (r-hFSH), recombinant human luteinizing hormone (r-hLH), and recombinant human chorionic gonadotropin (r-hCG) on CD4+ T cell secretion of interferon (IFN)-␥ and interleukin (IL)-10. (a) The combination of r-hFSH and r-hLH induced a modest but significant reduction in the secretion of IFN-␥ which was associated with an increase in IL-10 secretion (*p < 0.05). The combination of either r-hFSH or r-hLH with r-hCG resulted in a much more powerful inhibition in IFN-␥ secretion (c, e, and g) and enhancement of IL-10 production (d, f, and h). This effect was much higher and more pronounced in r-hFSH plus r-hCG and also when the three compounds were combined together (*p < 0.05; **p < 0.01).
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Fig. 5. The effect of increasing concentrations of combined urinary follicle-stimulating hormone (u-FSH), human menopausal gonadotropin (HMG), and recombinant human chorionic gonadotropin (r-hCG) on CD4+ T cell secretion of interferon (IFN)-␥ and interleukin (IL)-10. (a and b) u-FSH modestly but significantly stimulated IFN-␥ secretion in the absence of significant changes in basal IL-10 production (*p < 0.05). (c and d) HMG did not induce either significant changes in IFN-␥ secretion or IL-10 (NS, not significant). (e and f) The combination of u-FSH and r-hCG modestly but significantly stimulated IFN-␥ secretion in the absence of significant changes in basal IL-10 production (*p < 0.05). (g and h) HMG in combination with r-hCG induced a biphasic secretion in IFN-␥ that resulted in significant inhibition of its secretion at higher concentrations associated with a significant increase in the production of IL-10 (*p < 0.05).
with 5% autologous human serum (heat-inactivated at 56 ◦ C for 20 min to inactivate complement) and 100 g/mL penicillin/streptomycin (Invitrogen). T cells were stimulated in parallel with anti-CD3/anti-CD28 Dynabeads (0.1 bead/cell; Invitrogen) in the presence or absence of increasing concentrations of the different recombinant or
urinary-derived gonadotropins for 72 h, and were cultured at 37 ◦ C in 100% humidity and 5% CO2 . To measure cytokine secretion, supernatants (100 L) were removed 48–60 h after stimulation and frozen at −80 ◦ C until assay for IFN-␥ and IL-10 using specific ELISAs employing cytokine-specific capture and detection mAbs
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(Becton Dickinson-PharMingen, Franklin Lakes, NJ, USA), according to the manufacturer’s instructions. Standard curves for each assay were generated using recombinant human cytokines (IFN-␥ and IL-10; Becton DickinsonPharMingen) and the concentration of the cytokines in the cell supernatants was determined by extrapolation from the appropriate standard curve. The lower limits of detection for each assay were <2 pg/mL for IFN-␥ and <0.6 pg/mL for IL-10. The remaining cells were incubated for an additional 16 h, pulsed with 0.5 Ci per well of [3 H] thymidine (GE-Healthcare), harvested on glass-fiber filters using a 96-well cell harvester (Tomtec Inc., Orange, CT, USA), and counted in a 1205 Betaplate liquid scintillation counter (Wallac, Gaithersburg, MD, USA). Results are expressed as mean (SD) counts per minute from triplicate cultures. 2.3. Recombinant and purified gonadotropins Recombinant human FSH (r-hFSH), LH (r-hLH), and hCG (r-hCG) were from Merck Serono S.p.A., Rome, Italy (an affiliate of Merck KGaA, Darmstadt, Germany); purified urinary FSH (u-FSH; Fostimon® ) was from AMSA Srl, Milan, Italy; human menopausal gonadotropin (HMG) (Menogon® ) was from Ferring SpA, Milan Italy, as previously described (Baer and Loumaye, 2003). Of note, the hMG preparations contain highly variable levels of LH, and are often augmented with human chorionic gonadotropin (hCG), which mimics LH activity. 2.4. Statistical analysis Data are presented as mean (SD). In order to evaluate changes over time and between groups, an ANOVA for repeated measures with a Tukey post-test for multiple comparisons was performed using StatView® (SAS Institute). p value significance was set at 0.05. 3. Results 3.1. Divergent effects on human CD4+ T cell proliferation of r-hFSH, r-hLH, and r-hCG compared with their highly purified counterparts We compared the capacity of r-hFSH, r-hLH, and r-hCG, alone or in combination, to influence CD4+ T cell proliferation upon TCR stimulation with anti-CD3/anti-CD28 mAbs. Increasing concentrations of r-hFSH, r-hLH, and r-hCG were tested (Fig. 1a–c) alone or in combination (Fig. 2a–d). Neither r-hFSH nor r-hLH added alone had substantial stimulatory activity. The r-hFSH-induced increase in proliferation did not reach statistical significance, whereas the modest increase induced by r-hLH was significant (p < 0.05) (Fig. 1a and b, respectively). r-hCG alone showed little effect on T cell proliferation, with a small tendency towards inhibition of proliferation at higher doses that did not reach statistical significance (Fig. 1c). When the recombinant gonadotropins were combined (r-hFSH/r-hLH), a significant inhibition of T cell proliferation (20% inhibition) was observed (p < 0.05; Fig. 2a). This inhibitory capacity was greatly enhanced (50–70% inhibition) when the recombinant gonadotropins were added
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together with r-hCG (r-hFSH/r-hCG, r-hLH/r-hCG, or rhFSH/r-hLH/r-hCG; p < 0.01; Fig. 2b–d). We next tested the capacity of urinary-purified FSH (uFSH) and HMG, alone or in combination with r-hCG, to alter CD4+ T cell proliferation. u-FSH alone significantly stimulated T cell proliferation at high concentrations (p < 0.05; Fig. 3a), whereas purified HMG showed no significant effect on T cell proliferation at any concentration (Fig. 3b) and the combination of both hormones did not influence CD4+ proliferation (data not shown). When these purified preparations were combined with r-hCG (either u-FSH + r-hCG or purified HMG + r-hCG), a different effect was observed. More specifically, a low but significant increase in proliferation was observed when u-FSH and r-hCG were combined (Fig. 3c). Conversely, when purified HMG and r-hCG were combined, a biphasic effect with a significant (p < 0.05) tendency to stimulate T cell proliferation at lower doses and an inhibition (p = 0.05) in T cell proliferation at higher concentrations was observed (Fig. 3d). 3.2. Divergent effects of r-hFSH, r-hLH, and r-hCG on human CD4+ T cell cytokine secretion compared with their highly purified counterparts The capacity to influence the quality of the immune response in terms of cytokine secretion was also investigated. Specifically, IFN-␥ and IL-10 secretion were evaluated in parallel during CD4+ T cell assays in the presence or absence of recombinant and/or highly purified gonadotropins. Generally, IFN-␥ secretion mirrored the pattern of the proliferative response in T cells cultured with recombinant gonadotropins (Fig. 4a–h). Indeed, when the proliferation was inhibited significantly by the combination of recombinant gonadotropins and r-hCG (p < 0.05 and p < 0.01, respectively), it was associated with a reduction of IFN-␥ secretion and a significant increase in the amount of IL-10 produced (Fig. 4a–h). This specific effect on IL-10 secretion was completely absent when urinarypurified gonadotropins were used alone or in combination (Fig. 5a–f). Finally, when HMG was used in combination with r-hCG, a significant (p < 0.05) biphasic effect on inhibition of IFN-␥ was observed, together with increased IL-10 secretion (Fig. 5g and h). 4. Discussion To the best of our knowledge, this report compares for the first time the capacity of recombinant and urinary-purified gonadotropins to influence the in vitro proliferative and cytokine secretion of human CD4+ T cells isolated from regularly cycling, healthy women. Our experimental system was aimed at investigating the physiological role of gonadotropins during immune activation of CD4+ T cells derived from normal healthy women. This model is commonly used to assess the influence of molecules on immune responses. In addition, to preserve physiological conditions, autologous human serum was utilized in all our assays. This avoided any exogenous interference derived from FCS, which is commonly utilized in CD4+ T cell cultures. A range of concentrations of either recombinant or urinary-purified gonadotropins were used.
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We observed that when used alone, recombinant gonadotropins had some immunomodulatory effects that were different when they were used in combination. More specifically, we observed that r-hLH alone was able to stimulate T cell proliferation and r-hFSH alone did not, whereas when they were used in combination, there was a trend towards inhibition of cell proliferation. These data suggest that during the follicular phase, when both FSH and LH are present, there may be an immunomodulatory activity that can positively influence CD4+ T cell tolerance towards embryo implantation. Moreover, when r-hCG was combined with the recombinant gonadotropins, this trend towards inhibition of T cell proliferation was dramatically enhanced by 50–70%. This reinforces the evidence that hCG has an immunomodulatory activity, which is enhanced by FSH and LH alone or in combination (Schumacher et al., 2009). The cellular and molecular mechanisms by which this hormonal association can induce inhibition of T cell proliferation could be ascribed to: (1) an induction of cyclic adenosyl monophosphate (cAMP); and (2) the secretion of regulatory-type cytokine IL-10. cAMP displays diphasic effects on the proliferation of T cells and their entry into the cell cycle (Kuklina and Shirshev, 2000; Appleman et al., 2001). Indeed, a cAMP transient spike is needed for the T cell to enter into the cell cycle upon TCR engagement. However, when cAMP levels are maintained at constantly high levels, T cell proliferation is profoundly inhibited (Kuklina and Shirshev, 2000; Appleman et al., 2001). This model can be used to interpret our results—FSH or LH alone are able to induce a transient cAMP spike that could increase T cell proliferation upon TCR engagement (Leung and Steele, 1992; Barrios-De-Tomasi et al., 2002), whereas when rhFSH, r-hLH, and r-hCG are used in combination, they exert a synergistic effect on cAMP synthesis so that the cAMP levels are maintained at a constantly high level, which results in the inhibition of T cell proliferation. These effects were not observed when HMG was used. cAMP activities have also been linked to its capacity to induce CD4+ T cell anergy and induction of regulatory-type cytokines such as IL-10 and TGF- (Kuklina and Shirshev, 2000; Appleman et al., 2001). Recently, it has been shown that CD4+ Foxp3+ regulatory T cells (Treg cells), a cell population involved in self-tolerance and secretion of IL-10 (Matarese et al., 2008), express high levels of LH receptor and hCG receptor (Schumacher et al., 2009). This receptor is able to induce migration and activation of Treg cells in terms of suppressor capacity towards proliferation of conventional CD4+ T cells. These findings could, at least in part, explain our results in which there is an enhanced secretion of IL-10 and a profound inhibition of proliferation of IFN␥ when r-hFSH/r-hLH and r-hCG are added to CD4+ T cell cultures. It should also be noted that in our experimental system, the total CD4+ T cell population contains both effector T cells and Treg cells and therefore, it is difficult to confirm whether the increased IL-10 secretion is derived from Treg cells or from IL-10-secreting CD4+ T cells induced by the different treatments. Additionally, our study supports the concept of ‘extragonadal’ effects of gonadotropins. Reproduction is a multi-compartmental process based on the synchronous
preparation and integration of different functions, including the follicle–oocyte unit, the endometrium, and the immune system. In light of this, it is not surprising that the same molecules can contemporaneously regulate different compartments. In this context, it could be hypothesized that both gonadotropins and steroid hormones play a key role in preparing the immune system to accommodate the embryo semi-allograft. It could be suggested that the presence of both FSH and LH activities contribute to produce a ‘tolerogenic’ micro-environment, which is potentially able to favour implantation. This hypothesis is consistent with data emerging from clinical trials comparing ovarian stimulation with r-hFSH alone or with r-hFSH plus r-hLH protocols (Alviggi et al., 2006; Kovacs et al., 2010). In fact, most of these studies suggest that fertilization and implantation rates increase when r-hFSH and r-hLH are used in combination. Using our assay, we also analyzed purified urinaryderived products. Surprisingly, when the capability of u-FSH and HMG to influence T cell proliferation was tested, a non-significant effect or a modest increase in proliferation was observed with either u-FSH alone or in combination with r-hCG. However, these effects were opposite to the changes induced by the recombinant counterparts; the only combination that closely mirrored the effects induced by recombinant gonadotropins was the combination of HMG with r-hCG. However, this effect was biphasic and relatively modest when compared with the recombinant counterparts. The discrepancies between extracted and recombinant FSH and LH could be related to the presence of other non-gonadotropin contaminants present in the different preparations. These contaminants are estimated to be present and to comprise about 30% of the preparation. These contaminants could reasonably influence the proliferative response of CD4+ T cells and potentially the induction of immune tolerance. These effects require additional investigation (van de Weijer et al., 2003; Lispi et al., 2006; Bassett et al., 2009). As little is known about the characterization and kinetics of expression of the LH receptor and hCG receptor on lymphocytes compared with endocrine and reproductive cell systems, it cannot be excluded that while these urinary-derived preparations still maintain hormonal effects when administered, they may not exert the immunomodulatory activities that are also necessary for implantation and maintenance of pregnancy. In conclusion, the data emerging from the present study strongly support the hypothesis of regulatory effects of gonadotropins on the immune system. It cannot be excluded that these effects are dose-dependent and can be affected by secretion kinetics in vivo and therefore extensive additional investigations are needed. In addition, the presence of small peptides, other protein contaminants, and growth factors in extracted and purified compounds may affect the immunoregulatory activities of gonadotropins. Therefore, this is also the first report that shows that different gonadotropin preparations have divergent immunomodulatory effects in vitro that may have relevance in the overall process of embryo implantation and pregnancy.
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Conflict of interest F.C., C.P., V.D.R., C.A., G.D.P., G.M. have no conflict of interest to disclose; D.K. and S.L. are employed by Merck Serono Grafing, Germany and Merck Serono S.p.A., Rome, Italy, respectively. Acknowledgments The authors are grateful to Monica Lispi for assistance with editing of the manuscript and Salvatore De Simone for technical support. F.C., is supported by the PhD program in Cellular and Molecular Pathology from University of Napoli Federcico II; G.M. is supported by grants from the ERC-Starting Grant no. 202579 and from Telethon-JDRF Grant no. GJT08004. This work is dedicated to the memory of Eugenia Papa and Prof. Serafino Zappacosta. References Alviggi, C., Mollo, A., Clarizia, R., De Placido, G., 2006. Exploiting LH in ovarian stimulation. Reprod. Biomed. Online 12, 221–233. Appleman, L.J., Tzachanis, D., Grader-Beck, T., van Puijenbroek, A.A., Boussiotis, V.A., 2001. Helper T cell anergy: from biochemistry to cancer pathophysiology and therapeutics. J. Mol. Med. 78, 673–683. Baer, G., Loumaye, E., 2003. Comparison of recombinant human luteinising hormone (r-hLH) and human menopausal gonadotropin (hMG) in assisted reproductive technology. Curr. Med. Res. Opin. 19, 83–88. Barrios-De-Tomasi, J., Timossi, C., Merchant, H., Quintanar, A., Avalos, J.M., Andersen, C.Y., Ulloa-Aguirre, A., 2002. Assessment of the in vitro and in vivo biological activities of the human follicle-stimulating isohormones. Mol. Cell. Endocrinol. 186, 189–198. Bassett, R., Lispi, M., Ceccarelli, D., Grimaldi, L., Mancinelli, M., Martelli, F., Van Dorsselaer, A., 2009. Analytical identification of additional impurities in urinary derived gonadotrophins. Reprod. Biomed. Online 19, 300–313.
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Contents lists available at ScienceDirect
Journal of Reproductive Immunology journal homepage: www.elsevier.com/locate/jreprimm
Divergent immunomodulatory effects of recombinant and urinary-derived FSH, LH, and hCG on human CD4+ T cells Fortunata Carbone a,b , Claudio Procaccini a,b , Veronica De Rosa a,b , Carlo Alviggi c , Giuseppe De Placido c , Daniel Kramer d , Salvatore Longobardi e , Giuseppe Matarese a,b,∗ a b c d e
Laboratorio di Immunologia, Istituto di Endocrinologia e Oncologia Sperimentale, Consiglio Nazionale delle Ricerche, Napoli, Italy Laboratorio di Immunologia, Dipartimento di Biologia e Patologia Cellulare e Molecolare, Università di Napoli “Federico II”, Napoli, Italy Dipartimento di Scienze Ostetriche, Ginecologiche, Urologiche e Medicina della Riproduzione, Università di Napoli “Federico II”, Napoli, Italy Merck Serono Institute of Drug Metabolism and Pharmacokinetics, Grafing, Germany1 Merck Serono S.p.A., Rome, Italy1
a r t i c l e
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Article history: Received 21 October 2009 Received in revised form 11 February 2010 Accepted 16 February 2010 Keywords: Follicle-stimulating hormone Luteinizing hormone Human chorionic gonadotropin T cell Immunity Immune response
a b s t r a c t This study investigated the in vitro immune-modulating activities of recombinant versus highly purified urinary follicle-stimulating hormone (FSH), luteinizing hormone (LH), and human chorionic gonadotropin (hCG) at the cellular level. CD4+ T cells were isolated from peripheral blood mononuclear cells obtained from ten healthy women (aged 19–30 years) with regular menstrual cycles during the follicular phase of their cycle. CD4+ T cells were stimulated with anti-CD3/CD28 monoclonal antibodies as a T cell-specific mitogen. Proliferative and cytokine responses were analyzed at standard time points (72 h). Recombinant FSH (r-FSH) and LH (r-LH) alone showed a modest capacity to influence proliferation and cytokine release by CD4+ T cells. Conversely, their addition to T cells in combination with recombinant hCG (r-hCG) induced a powerful down-modulation of T cell proliferation, decreased interferon-␥ (IFN-␥) secretion and increased interleukin-10 (IL-10) production. These immune-modulating activities were not present when CD4+ T cells were stimulated either in the presence of urinary-purified FSH (u-FSH) or human menopausal gonadotropin (HMG), alone or in combination with recombinant hCG. We are the first to suggest that urinary-purified gonadotropins do not display profound immune-modulating activities as compared with the recombinant preparations, despite their endocrine effects. Therefore, the use of the recombinant preparations in assisted reproductive techniques might be relevant not only for their well-documented endocrine actions but also for their impact on the transient immune tolerance known to favour embryo implantation and progression of pregnancy. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
∗ Corresponding author at: Laboratorio di Immunologia, Istituto di Endocrinologia e Oncologia Sperimentale, Consiglio Nazionale delle Ricerche (IEOS-CNR), via S. Pansini 5, 80131 Napoli, Italy. Tel.: +39 081 746 4580; fax: +39 081 746 3252. E-mail addresses: [email protected], [email protected] (G. Matarese). 1 An affiliate of Merck KGaA, Darmstadt, Germany. 0165-0378/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jri.2010.02.009
It is well known that the neuroendocrine and immune systems interact (Whitacre, 2001; Kelley et al., 2007). Gonadotropins such as follicle-stimulating hormone (FSH), luteinizing hormone (LH), and human chorionic gonadotropin (hCG) are involved in the control of the neuroendocrine response crucial for regulation of the menstrual cycle, ovulation, and maintenance of pregnancy. The immune system has long been recognized as a particu-
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larly versatile system that thanks to the expression of a series of hormones and hormone receptors, can be influenced by hormones and neuroendocrine factors (Whitacre, 2001; Kelley et al., 2007). Receptors for FSH, LH, and hCG are expressed on immune cells and their capacity to influence the immune response has been investigated. More specifically, it has been shown that hCG in particular is able to down-regulate immune responses locally and systemically during pregnancy (Schumacher et al., 2009). Indeed, its presence at high concentrations has been linked to maternal tolerance towards the fetus. Furthermore, a series of reports have shown that hCG is able to inhibit T cell proliferation, secretion of inflammatory cytokines, antigen presentation, and activation by dendritic cells (Foster et al., 2010; Schumacher et al., 2009). Although recombinant gonadotropins have been in clinical use since 1995 (Daya, 2004), highly purified gonadotropin preparations have been of great value over the last 20 years in the treatment of chronic anovulation, assisted reproductive techniques, and infertility (Alviggi et al., 2006). Despite the clinical value of these highly purified gonadotropins, recent reports have raised the question of whether their use can have a negative impact on the reproducibility and standardization of protocols. This is because the preparations differ in biological activity between batches, while the purification process leaves a series of heterogeneous contaminants (Lispi et al., 2006; Bassett et al., 2009). While these gonadotropins have been shown to be active at the level of the ovary, little is known of their capacity to exert immune-modulating effects. This could also have an impact on the process of induction of maternal immune tolerance that is crucial for embryo implantation and tolerance of the fetus during pregnancy (van Mourik et al., 2009). In the present study, we have analyzed the in vitro immunomodulating capacity of recombinant versus highly purified gonadotropins. Specifically, we studied the proliferative response and the cytokine profile of CD4+ T cells isolated from women with regular menstrual cycles. The T cells were stimulated with T cell receptor (TCR)-specific activators (anti-CD3 and anti-CD28 monoclonal antibodies) in the presence or absence of increasing concentrations of either recombinant or highly purified gonadotropins. 2. Materials and methods 2.1. Study design This study was approved by our Institutional Review Board, from Università di Napoli “Federico II”. All subjects were healthy women without immunologic or endocrine disease based on clinical examination and routine blood tests. Ten women (age = 22.4 ± 1.2 years; body mass index < 25 kg/m2 ) with regular menstrual cycles (length 26–32 days) and not on oral contraceptives for at least the previous 6 months were enrolled at the Blood Transfusion Center from Università di Napoli “Federico II”. All the women were in the follicular phase of their cycle. Blood samples were collected at 9.00 am in heparinized vacutainers (BD Biosciences, Milan, Italy) and processed within the following 3 h. Blood samples were also subject to routine
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Fig. 1. The effect of increasing concentrations of recombinant human follicle-stimulating hormone (r-hFSH), recombinant human luteinizing hormone (r-hLH), and recombinant human chorionic gonadotropin (rhCG) on CD4+ T cell proliferation from normal healthy women during CD3/CD28 stimulation. (a and c) No significant changes in proliferation were induced by either r-hFSH or r-hCG. (b) On the contrary, r-hLH alone was able to stimulate significant proliferation of CD4+ T cells (*p < 0.05). Results are expressed as mean (SD) from triplicate cultures from ten healthy women.
blood count and immune-phenotypic analyses to exclude any hematologic and immune-related abnormalities. Blood and lymphocyte counts for all subjects were within the normal range (data not shown). 2.2. T cell proliferation and cytokine production Peripheral blood mononuclear cells (PBMCs) from the above subjects were isolated using Ficoll gradients (Lymphoprep, GE-Healthcare, Milan, Italy), and CD4+ T cells were subsequently purified from PBMCs by negative selection utilizing a CD4+ Isolation Kit (Invitrogen, Carlsbad CA, USA; 95% pure by flow cytometry). T cell cultures were performed in triplicate, in 96-well roundbottom plates (1 × 105 cells per well) in RPMI-1640 Glutamax medium (200 L; Invitrogen) supplemented
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Fig. 2. The effect of increasing concentrations of combined recombinant human follicle-stimulating hormone (r-hFSH), recombinant human luteinizing hormone (r-hLH), and recombinant human chorionic gonadotropin (r-hCG) on CD4+ T cell proliferation from normal healthy women during anti-CD3/antiCD28 stimulation. (a–d) r-hFSH and r-hLH, and their combination with r-hCG, induced a significant inhibition of proliferation of CD4+ T cells from healthy women (*p < 0.05; **p < 0.01). Results are expressed as mean (SD) from triplicate cultures from ten healthy women.
Fig. 3. The effect of increasing concentrations of urinary follicle-stimulating hormone (u-FSH) and human menopausal gonadotropin (HMG), alone or combined with recombinant human chorionic gonadotropin (r-hCG), on CD4+ T cell proliferation from normal healthy women during anti-CD3/anti-CD28 stimulation. (a and c) u-FSH and its combination with r-hCG resulted in a significant increase in proliferation of CD4+ T cells (*p < 0.05); (b) conversely, HMG did not induce significant changes in proliferation of CD4+ T cells, whereas when combined with r-hCG (d), there was a biphasic effect that was inhibitory at higher concentrations (*p < 0.05).
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Fig. 4. The effect of increasing concentrations of combined recombinant human follicle-stimulating hormone (r-hFSH), recombinant human luteinizing hormone (r-hLH), and recombinant human chorionic gonadotropin (r-hCG) on CD4+ T cell secretion of interferon (IFN)-␥ and interleukin (IL)-10. (a) The combination of r-hFSH and r-hLH induced a modest but significant reduction in the secretion of IFN-␥ which was associated with an increase in IL-10 secretion (*p < 0.05). The combination of either r-hFSH or r-hLH with r-hCG resulted in a much more powerful inhibition in IFN-␥ secretion (c, e, and g) and enhancement of IL-10 production (d, f, and h). This effect was much higher and more pronounced in r-hFSH plus r-hCG and also when the three compounds were combined together (*p < 0.05; **p < 0.01).
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Fig. 5. The effect of increasing concentrations of combined urinary follicle-stimulating hormone (u-FSH), human menopausal gonadotropin (HMG), and recombinant human chorionic gonadotropin (r-hCG) on CD4+ T cell secretion of interferon (IFN)-␥ and interleukin (IL)-10. (a and b) u-FSH modestly but significantly stimulated IFN-␥ secretion in the absence of significant changes in basal IL-10 production (*p < 0.05). (c and d) HMG did not induce either significant changes in IFN-␥ secretion or IL-10 (NS, not significant). (e and f) The combination of u-FSH and r-hCG modestly but significantly stimulated IFN-␥ secretion in the absence of significant changes in basal IL-10 production (*p < 0.05). (g and h) HMG in combination with r-hCG induced a biphasic secretion in IFN-␥ that resulted in significant inhibition of its secretion at higher concentrations associated with a significant increase in the production of IL-10 (*p < 0.05).
with 5% autologous human serum (heat-inactivated at 56 ◦ C for 20 min to inactivate complement) and 100 g/mL penicillin/streptomycin (Invitrogen). T cells were stimulated in parallel with anti-CD3/anti-CD28 Dynabeads (0.1 bead/cell; Invitrogen) in the presence or absence of increasing concentrations of the different recombinant or
urinary-derived gonadotropins for 72 h, and were cultured at 37 ◦ C in 100% humidity and 5% CO2 . To measure cytokine secretion, supernatants (100 L) were removed 48–60 h after stimulation and frozen at −80 ◦ C until assay for IFN-␥ and IL-10 using specific ELISAs employing cytokine-specific capture and detection mAbs
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(Becton Dickinson-PharMingen, Franklin Lakes, NJ, USA), according to the manufacturer’s instructions. Standard curves for each assay were generated using recombinant human cytokines (IFN-␥ and IL-10; Becton DickinsonPharMingen) and the concentration of the cytokines in the cell supernatants was determined by extrapolation from the appropriate standard curve. The lower limits of detection for each assay were <2 pg/mL for IFN-␥ and <0.6 pg/mL for IL-10. The remaining cells were incubated for an additional 16 h, pulsed with 0.5 Ci per well of [3 H] thymidine (GE-Healthcare), harvested on glass-fiber filters using a 96-well cell harvester (Tomtec Inc., Orange, CT, USA), and counted in a 1205 Betaplate liquid scintillation counter (Wallac, Gaithersburg, MD, USA). Results are expressed as mean (SD) counts per minute from triplicate cultures. 2.3. Recombinant and purified gonadotropins Recombinant human FSH (r-hFSH), LH (r-hLH), and hCG (r-hCG) were from Merck Serono S.p.A., Rome, Italy (an affiliate of Merck KGaA, Darmstadt, Germany); purified urinary FSH (u-FSH; Fostimon® ) was from AMSA Srl, Milan, Italy; human menopausal gonadotropin (HMG) (Menogon® ) was from Ferring SpA, Milan Italy, as previously described (Baer and Loumaye, 2003). Of note, the hMG preparations contain highly variable levels of LH, and are often augmented with human chorionic gonadotropin (hCG), which mimics LH activity. 2.4. Statistical analysis Data are presented as mean (SD). In order to evaluate changes over time and between groups, an ANOVA for repeated measures with a Tukey post-test for multiple comparisons was performed using StatView® (SAS Institute). p value significance was set at 0.05. 3. Results 3.1. Divergent effects on human CD4+ T cell proliferation of r-hFSH, r-hLH, and r-hCG compared with their highly purified counterparts We compared the capacity of r-hFSH, r-hLH, and r-hCG, alone or in combination, to influence CD4+ T cell proliferation upon TCR stimulation with anti-CD3/anti-CD28 mAbs. Increasing concentrations of r-hFSH, r-hLH, and r-hCG were tested (Fig. 1a–c) alone or in combination (Fig. 2a–d). Neither r-hFSH nor r-hLH added alone had substantial stimulatory activity. The r-hFSH-induced increase in proliferation did not reach statistical significance, whereas the modest increase induced by r-hLH was significant (p < 0.05) (Fig. 1a and b, respectively). r-hCG alone showed little effect on T cell proliferation, with a small tendency towards inhibition of proliferation at higher doses that did not reach statistical significance (Fig. 1c). When the recombinant gonadotropins were combined (r-hFSH/r-hLH), a significant inhibition of T cell proliferation (20% inhibition) was observed (p < 0.05; Fig. 2a). This inhibitory capacity was greatly enhanced (50–70% inhibition) when the recombinant gonadotropins were added
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together with r-hCG (r-hFSH/r-hCG, r-hLH/r-hCG, or rhFSH/r-hLH/r-hCG; p < 0.01; Fig. 2b–d). We next tested the capacity of urinary-purified FSH (uFSH) and HMG, alone or in combination with r-hCG, to alter CD4+ T cell proliferation. u-FSH alone significantly stimulated T cell proliferation at high concentrations (p < 0.05; Fig. 3a), whereas purified HMG showed no significant effect on T cell proliferation at any concentration (Fig. 3b) and the combination of both hormones did not influence CD4+ proliferation (data not shown). When these purified preparations were combined with r-hCG (either u-FSH + r-hCG or purified HMG + r-hCG), a different effect was observed. More specifically, a low but significant increase in proliferation was observed when u-FSH and r-hCG were combined (Fig. 3c). Conversely, when purified HMG and r-hCG were combined, a biphasic effect with a significant (p < 0.05) tendency to stimulate T cell proliferation at lower doses and an inhibition (p = 0.05) in T cell proliferation at higher concentrations was observed (Fig. 3d). 3.2. Divergent effects of r-hFSH, r-hLH, and r-hCG on human CD4+ T cell cytokine secretion compared with their highly purified counterparts The capacity to influence the quality of the immune response in terms of cytokine secretion was also investigated. Specifically, IFN-␥ and IL-10 secretion were evaluated in parallel during CD4+ T cell assays in the presence or absence of recombinant and/or highly purified gonadotropins. Generally, IFN-␥ secretion mirrored the pattern of the proliferative response in T cells cultured with recombinant gonadotropins (Fig. 4a–h). Indeed, when the proliferation was inhibited significantly by the combination of recombinant gonadotropins and r-hCG (p < 0.05 and p < 0.01, respectively), it was associated with a reduction of IFN-␥ secretion and a significant increase in the amount of IL-10 produced (Fig. 4a–h). This specific effect on IL-10 secretion was completely absent when urinarypurified gonadotropins were used alone or in combination (Fig. 5a–f). Finally, when HMG was used in combination with r-hCG, a significant (p < 0.05) biphasic effect on inhibition of IFN-␥ was observed, together with increased IL-10 secretion (Fig. 5g and h). 4. Discussion To the best of our knowledge, this report compares for the first time the capacity of recombinant and urinary-purified gonadotropins to influence the in vitro proliferative and cytokine secretion of human CD4+ T cells isolated from regularly cycling, healthy women. Our experimental system was aimed at investigating the physiological role of gonadotropins during immune activation of CD4+ T cells derived from normal healthy women. This model is commonly used to assess the influence of molecules on immune responses. In addition, to preserve physiological conditions, autologous human serum was utilized in all our assays. This avoided any exogenous interference derived from FCS, which is commonly utilized in CD4+ T cell cultures. A range of concentrations of either recombinant or urinary-purified gonadotropins were used.
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We observed that when used alone, recombinant gonadotropins had some immunomodulatory effects that were different when they were used in combination. More specifically, we observed that r-hLH alone was able to stimulate T cell proliferation and r-hFSH alone did not, whereas when they were used in combination, there was a trend towards inhibition of cell proliferation. These data suggest that during the follicular phase, when both FSH and LH are present, there may be an immunomodulatory activity that can positively influence CD4+ T cell tolerance towards embryo implantation. Moreover, when r-hCG was combined with the recombinant gonadotropins, this trend towards inhibition of T cell proliferation was dramatically enhanced by 50–70%. This reinforces the evidence that hCG has an immunomodulatory activity, which is enhanced by FSH and LH alone or in combination (Schumacher et al., 2009). The cellular and molecular mechanisms by which this hormonal association can induce inhibition of T cell proliferation could be ascribed to: (1) an induction of cyclic adenosyl monophosphate (cAMP); and (2) the secretion of regulatory-type cytokine IL-10. cAMP displays diphasic effects on the proliferation of T cells and their entry into the cell cycle (Kuklina and Shirshev, 2000; Appleman et al., 2001). Indeed, a cAMP transient spike is needed for the T cell to enter into the cell cycle upon TCR engagement. However, when cAMP levels are maintained at constantly high levels, T cell proliferation is profoundly inhibited (Kuklina and Shirshev, 2000; Appleman et al., 2001). This model can be used to interpret our results—FSH or LH alone are able to induce a transient cAMP spike that could increase T cell proliferation upon TCR engagement (Leung and Steele, 1992; Barrios-De-Tomasi et al., 2002), whereas when rhFSH, r-hLH, and r-hCG are used in combination, they exert a synergistic effect on cAMP synthesis so that the cAMP levels are maintained at a constantly high level, which results in the inhibition of T cell proliferation. These effects were not observed when HMG was used. cAMP activities have also been linked to its capacity to induce CD4+ T cell anergy and induction of regulatory-type cytokines such as IL-10 and TGF- (Kuklina and Shirshev, 2000; Appleman et al., 2001). Recently, it has been shown that CD4+ Foxp3+ regulatory T cells (Treg cells), a cell population involved in self-tolerance and secretion of IL-10 (Matarese et al., 2008), express high levels of LH receptor and hCG receptor (Schumacher et al., 2009). This receptor is able to induce migration and activation of Treg cells in terms of suppressor capacity towards proliferation of conventional CD4+ T cells. These findings could, at least in part, explain our results in which there is an enhanced secretion of IL-10 and a profound inhibition of proliferation of IFN␥ when r-hFSH/r-hLH and r-hCG are added to CD4+ T cell cultures. It should also be noted that in our experimental system, the total CD4+ T cell population contains both effector T cells and Treg cells and therefore, it is difficult to confirm whether the increased IL-10 secretion is derived from Treg cells or from IL-10-secreting CD4+ T cells induced by the different treatments. Additionally, our study supports the concept of ‘extragonadal’ effects of gonadotropins. Reproduction is a multi-compartmental process based on the synchronous
preparation and integration of different functions, including the follicle–oocyte unit, the endometrium, and the immune system. In light of this, it is not surprising that the same molecules can contemporaneously regulate different compartments. In this context, it could be hypothesized that both gonadotropins and steroid hormones play a key role in preparing the immune system to accommodate the embryo semi-allograft. It could be suggested that the presence of both FSH and LH activities contribute to produce a ‘tolerogenic’ micro-environment, which is potentially able to favour implantation. This hypothesis is consistent with data emerging from clinical trials comparing ovarian stimulation with r-hFSH alone or with r-hFSH plus r-hLH protocols (Alviggi et al., 2006; Kovacs et al., 2010). In fact, most of these studies suggest that fertilization and implantation rates increase when r-hFSH and r-hLH are used in combination. Using our assay, we also analyzed purified urinaryderived products. Surprisingly, when the capability of u-FSH and HMG to influence T cell proliferation was tested, a non-significant effect or a modest increase in proliferation was observed with either u-FSH alone or in combination with r-hCG. However, these effects were opposite to the changes induced by the recombinant counterparts; the only combination that closely mirrored the effects induced by recombinant gonadotropins was the combination of HMG with r-hCG. However, this effect was biphasic and relatively modest when compared with the recombinant counterparts. The discrepancies between extracted and recombinant FSH and LH could be related to the presence of other non-gonadotropin contaminants present in the different preparations. These contaminants are estimated to be present and to comprise about 30% of the preparation. These contaminants could reasonably influence the proliferative response of CD4+ T cells and potentially the induction of immune tolerance. These effects require additional investigation (van de Weijer et al., 2003; Lispi et al., 2006; Bassett et al., 2009). As little is known about the characterization and kinetics of expression of the LH receptor and hCG receptor on lymphocytes compared with endocrine and reproductive cell systems, it cannot be excluded that while these urinary-derived preparations still maintain hormonal effects when administered, they may not exert the immunomodulatory activities that are also necessary for implantation and maintenance of pregnancy. In conclusion, the data emerging from the present study strongly support the hypothesis of regulatory effects of gonadotropins on the immune system. It cannot be excluded that these effects are dose-dependent and can be affected by secretion kinetics in vivo and therefore extensive additional investigations are needed. In addition, the presence of small peptides, other protein contaminants, and growth factors in extracted and purified compounds may affect the immunoregulatory activities of gonadotropins. Therefore, this is also the first report that shows that different gonadotropin preparations have divergent immunomodulatory effects in vitro that may have relevance in the overall process of embryo implantation and pregnancy.
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Conflict of interest F.C., C.P., V.D.R., C.A., G.D.P., G.M. have no conflict of interest to disclose; D.K. and S.L. are employed by Merck Serono Grafing, Germany and Merck Serono S.p.A., Rome, Italy, respectively. Acknowledgments The authors are grateful to Monica Lispi for assistance with editing of the manuscript and Salvatore De Simone for technical support. F.C., is supported by the PhD program in Cellular and Molecular Pathology from University of Napoli Federcico II; G.M. is supported by grants from the ERC-Starting Grant no. 202579 and from Telethon-JDRF Grant no. GJT08004. This work is dedicated to the memory of Eugenia Papa and Prof. Serafino Zappacosta. References Alviggi, C., Mollo, A., Clarizia, R., De Placido, G., 2006. Exploiting LH in ovarian stimulation. Reprod. Biomed. Online 12, 221–233. Appleman, L.J., Tzachanis, D., Grader-Beck, T., van Puijenbroek, A.A., Boussiotis, V.A., 2001. Helper T cell anergy: from biochemistry to cancer pathophysiology and therapeutics. J. Mol. Med. 78, 673–683. Baer, G., Loumaye, E., 2003. Comparison of recombinant human luteinising hormone (r-hLH) and human menopausal gonadotropin (hMG) in assisted reproductive technology. Curr. Med. Res. Opin. 19, 83–88. Barrios-De-Tomasi, J., Timossi, C., Merchant, H., Quintanar, A., Avalos, J.M., Andersen, C.Y., Ulloa-Aguirre, A., 2002. Assessment of the in vitro and in vivo biological activities of the human follicle-stimulating isohormones. Mol. Cell. Endocrinol. 186, 189–198. Bassett, R., Lispi, M., Ceccarelli, D., Grimaldi, L., Mancinelli, M., Martelli, F., Van Dorsselaer, A., 2009. Analytical identification of additional impurities in urinary derived gonadotrophins. Reprod. Biomed. Online 19, 300–313.
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