Albrecht Giuliani, M.D., Wolfgang Schoell, M.D., Johann Auner, M.D., and Wolfgang Urdl, M.D.

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1 FERTILITY AND STERILITY VOL. 70, NO. 5, NOVEMBER 1998 Copyright 1998 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. OVULATION INDUCTION Controlled ovarian hyperstimulation in assisted reproduction: effect on the immune system Albrecht Giuliani, M.D., Wolfgang Schoell, M.D., Johann Auner, M.D., and Wolfgang Urdl, M.D. Department of Obstetrics and Gynecology, University of Graz, Graz, Austria Objective: To determine how controlled ovarian hyperstimulation (COH) in assisted reproduction affects the immune system. Design: A prospective, nonrandomized, case-control study. Setting: Academic research setting. Patient(s): Women with regular menstrual cycles undergoing COH in an assisted reproduction program. Intervention(s): Blood samples were collected in the early and late follicular phase, at the time of ovulation, and in the luteal phase during a natural cycle, and at four times during the next cycle, which included COH and IVF. Main Outcome Measure(s): Lymphocyte subpopulations and the differential blood count. Result(s): In the natural cycles, a significant increase in the total numbers of lymphocytes, B cells, natural killer cells, and CD3 HLADR cells was observed in the late follicular phase, whereas the T helper/t suppressor cell ratio declined. In the hyperstimulated cycles, increases were seen in the total numbers of leukocytes and neutrophils on the day of hcg administration; the number of lymphocytes, monocytes, and neutrophils was increased on the day of oocyte retrieval, and the total number of leukocytes and neutrophils increased during the luteal phase. Conclusion(s): Controlled ovarian hyperstimulation with hmg and simultaneous administration of a GnRH antagonist did not affect the immune system. (Fertil Steril 1998;70: by American Society for Reproductive Medicine.) Key Words: Controlled ovarian hyperstimulation, human menopausal gonadotropin, gonadotropin-releasing hormone antagonist, lymphocyte subsets Received February 25, 1998; revised and accepted June 15, Reprint requests: Albrecht Giuliani, M.D., Department of Obstetrics and Gynecology, University of Graz, Auenbruggerplatz 14, A-8036 Graz, Austria (FAX: ; giuliani@balu.kfunigraz.ac.at) /98/$19.00 PII S (98) Sex hormones have varied effects on the immune system (1 4). Many autoimmune conditions, such as Hashimoto s thyroiditis, systemic lupus erythematosus, juvenile diabetes mellitus, and rheumatoid arthritis, predominantly affect premenopausal women (5, 6). This increased susceptibility to autoimmune disorders disappears after menopause. Hormonal changes are likely to be responsible for this predisposition, but precise correlations between sex hormone levels and immune function have not been defined. The present study was designed to investigate parameters of the immunologic status during the follicular, periovulatory, and luteal phases of the normal menstrual cycle and to compare them with corresponding values during controlled ovarian hyperstimulation (COH) in assisted reproduction. MATERIALS AND METHODS We performed a prospective, nonrandomized, case-control study of the immunologic status of 17 white women who were undergoing COH and IVF-ET because of infertility. The study was approved by the institutional review board. Informed consent was obtained from all patients. The women ranged in age between 26 and 38 years (mean, 32 years) and had regular menstrual cycles lasting between 25 and 34 days (mean, 28.9 days) with an intraindividual variation of 3 days. The causes of infertility were tubal occlusion (n 9), male factor (n 5), and idiopathic (n 3). The FSH level between days 3 and 5 of the cycle was 10 miu/l. None of the women had polycystic ovary syndrome, corpus luteum insufficiency, impaired ovarian function, class III or IV endometriosis (7), an in- 831

2 flammatory condition 3 months before the beginning of the study, or submucosal myoma uteri. Other exclusion criteria were a history of low ovarian response to hmg and FSH; contraindications to the use of gonadotropins; any ovarian and/or abdominal abnormality that would interfere with adequate ultrasound (US) investigation; lactation; delivery or pregnancy loss within 2 months; use of an injectable hormonal method of contraception within 6 months; and use of any hormonal contraceptive within 8 weeks before the start of COH. Blood samples were obtained during a natural menstrual cycle and during a subsequent cycle with COH. Controlled ovarian hyperstimulation was performed with the use of a GnRH antagonist (Cetrorelix; ASTA Medica AG, Frankfurt, Germany) and simultaneous administration of hmg (Menogon; Ferring, Kiel, Germany). The administration of hmg started on day 2 or 3 of the cycle; 6 days later, 0.25 mg of Cetrorelix was administered SC daily. When the leading follicle reached at least 20 mm or the E 2 level reached 1,200 pg/ml, 10,000 IU of hcg (Pregnyl; Organon, Oss, the Netherlands) was administered IM. In patients who had a contraindication to the administration of hcg, the cycle was cancelled. Blood samples were drawn at four times in both cycles: between days 2 and 5 (t1); before ovulation at a follicle size of mm (t2); on the day of ovulation (t3); and 6 8 days after ovulation (t4). The cycle was monitored with US examinations and hormone profiles. The day of ovulation was identified by the onset of an LH surge to twice the basal level. During COH, blood samples were taken on corresponding days: immediately before the start of COH on day 2 3 of the cycle (ti); on the day of hcg administration (tii); 2 days after hcg administration (which also was the day of ET; tiii); and 6 8 days after ET (tiv). All blood samples were drawn between 8 AM and 10 AM. A full blood count and a differential blood count (Helios 3 Diff; ABX Hematologie, Montpellier, France) were performed. The following peripheral leukocyte subpopulations were examined by dual-color flow cytometry (FACSort; Becton Dickinson, Mountain View, CA) within 4 hours of the collection of each sample with the use of a standardized protocol (8): total T cells (CD3 ), total B cells (CD19 ), T helper cells (CD3 CD4 ), T suppressor cells (CD3 CD8 ), T helper/t suppressor cell ratio (CD4 /CD8 ), total natural killer cells (CD3 CD16 CD56 ), cytotoxic T cells (CD3 CD16 CD56 ), CD69 T cells, CD25 T cells, HLA-DR T cells, CD25 B-cells, CD45RA T cells, CD45RO T cells, CD71 lymphocytes, and CD71 neutrophils. Flow cytometric data were analyzed with Becton Dickinson CellQuest software. Serum levels of the soluble interleukin-2 receptor and the soluble transferrin receptor were measured with highly sensitive commercially available ELISA kits (Innotest hil-2rs; Innogenetics N.V., Zwinjnaarde, Belgium, and Quantikine; R&D Systems Europe, Abingdon, UK). Statistical analyses were performed with the use of SPSS/ PC (SPSS Inc., Chicago, IL). Analysis of variance was used to compare groups. Independent sample t-tests were used to compare the means between the stimulated and unstimulated cycles. To detect possible changes during a normal and during a stimulated cycle, t-tests for repeated measures were performed. For each group, baseline data (t1 resp. ti) were compared with the three following observations: t2 - t4; tii - tiv, resp.). Statistical significance was defined as P.05. RESULTS The results for the natural cycles are outlined in Table 1. A statistically significant increase from t1 to t2 was observed for the total number of lymphocytes (P.046), B cells (P.048), and HLA-DR T cells (P.039). The total number of natural killer cells increased significantly from t1 to t2 (P.045) and from t1 to t3 (P.033). The T helper/t suppressor cell ratio dropped significantly from t1 to t2 (P.002) and from t1 to t3 (P.002). There were no changes in all the other examined leukocyte subpopulations and soluble factors. In the stimulated cycles, alterations were found before ovulation, on the day of oocyte retrieval, and in the luteal phase. There was a significant increase from ti to tii for the total numbers of leukocytes (P.020) and neutrophils (P.001). From ti to tiii, lymphocytes (P.002), monocytes (P.050), and neutrophils (P.013) increased significantly. Leukocytes (P.001) and neutrophils (P.003) increased significantly between ti and tiv. All lymphocyte subpopulations and all soluble factors showed stable values during the stimulated cycles. Table 2 shows the results during the stimulated cycles. There were statistically significant differences between the unstimulated and stimulated cycles in the following parameters. Comparing t2 and tii, there was a significantly increased total number of leukocytes (P.005) and monocytes (P.023) in the stimulated cycle, whereas the median number of activated B cells decreased (P.04). In addition, between t3 and tiii, the total numbers of leukocytes (P.004), neutrophils (P.045), and natural killer cells (P.048) were significantly increased. Comparing t4 with tiv, only the total numbers of leukocytes (P.001) and neutrophils (P.004) increased significantly in the stimulated cycles. DISCUSSION During natural cycles, we found an increase in the total numbers of lymphocytes, B cells, natural killer cells, and late activated T cells and a decline in the T helper/t suppressor 832 Giuliani et al. COH and the immune system Vol. 70, No. 5, November 1998

3 TABLE 1 Immunologic parameters during a normal menstrual cycle in 17 patients. Mean ( SD) no. of immune cells at indicated point in cycle Immune cells Day 2 5 Before ovulation (follicle size of mm) Day of ovulation 6 8 days after ovulation Leukocytes 5, ,870 1,851 6,400 1,310 6, Lymphocytes 1, , * 2, , Monocytes Neutrophils 3, ,961 1,703 3, ,284 1,293 T cells (CD3 ) 1, , , , B cells (CD19 ) * T helper cells (CD3 CD4 ) , T suppressor cells (CD3 CD8 ) T helper/t suppressor cell ratio * * Natural killer cells (CD3 CD16/56 ) * * Cytotoxic T cells (CD3 CD16 /56 ) Early activated T cells (CD3 CD69 ) Median activated T cells (CD3 CD25 ) Late activated T cells (CD3 HLADR ) * Median activated B cells (CD19 CD25 ) CD3 CD45RA cells CD3 CD45RO cells , , CD71 neutrophils CD71 lymphocytes Soluble interleukin-2 receptor Soluble transferrin receptor * Mean of second, third, or fourth point in cycle versus mean of first point; P.05. cell ratio. These alterations were seen mainly in the late follicular phase and at the time of ovulation. In the late luteal phase, there was no difference from the early follicular phase. These results agree with those of Northern et al. (9), who found no difference in the concentrations of white blood cells between day 6 and day 22 of a normal menstrual cycle. During the stimulated cycles, the total numbers of leukocytes and neutrophils reached a peak before ovulation and remained stable until the luteal phase. Lymphocytes and monocytes showed a significant increase on the day of oocyte retrieval. None of the other lymphocyte subpopulations changed significantly. We found minor fluctuations in white blood cells in a normal and a stimulated cycle. It is surprising that these changes were discordant and affected different cell populations. It remains unclear why there was a discordance in the immunologic fluctuations during the cycle between the normal and the stimulated cycles. In the normal cycles, the changes occurred only near ovulation and were confined to lymphocytes. Even cells of specific immunity (HLA-DR T cells and the T helper/t suppressor cell ratio) showed alterations. This shift in lymphocyte subsets is nearly identical to the enumerative immune changes that occur with acute stress, as described by Mills et al. (10). It is unlikely that this observation is of clinical importance in healthy women. Only cells of the differential blood count showed a persistent increase during stimulated cycles. This could show a minor activation of nonspecific cellular immunity. Lymphocyte subpopulations were not affected, and we did not find a down-regulation of CD3 CD25 lymphocytes in peripheral blood, in contrast with the findings of Ho et al. (11). Neutrophils, monocytes, natural killer cells, and cytotoxic (CD16/CD56 ) lymphocytes are potent cells of nonspecific cellular immunity. T helper, T suppressor, and B cells play a key role after antigen stimulation mediated cellular immunity. Lymphocytes that are positive for CD69, CD25, or HLA-DR indicate recent activation of specific cellular immunity. Levels of the soluble interleukin-2 receptor and the soluble transferrin receptor rise after the stimulation of cellular immunity (12, 13). We characterized the status of the immune system by the following factors: the white blood count, the differential blood count, lymphocyte subpopulations, the soluble interleukin-2 receptor, and the soluble transferrin receptor. The menstrual cycle and its influence on the immune system have been examined in several studies (9, 14 18). Most studies show percentages of lymphocyte subtypes and do not measure possible changes in absolute counts (16, 17). The days on which we chose to measure immunologic parameters during the natural and stimulated cycles correspond with significant shifts in the hormone profiles in both FERTILITY & STERILITY 833

4 TABLE 2 Immunologic parameters during a cycle with COH and IVF-ET in 17 patients. Mean ( SD) no. of immune cells at indicated point in cycle Immune cells Day 2 3 Day of hcg administration 2 days after hcg administration 6 8 days after ET Leukocytes 6,529 1,611 9,094 1,811* 8,863 2,089 11,475 3,596* Lymphocytes 2, , , * 2, Monocytes , * 1,083 1,230 Neutrophils 3, ,291 1,931* 5,525 2,447* 7,833 4,088* T cells (CD3 ) 1, , , , B cells (CD19 ) T helper cells (CD3 CD4 ) T suppressor cells (CD3 CD8 ) T helper/t suppressor cell ratio Natural killer cells (CD3 CD16/56 ) Cytotoxic T cells (CD3 CD16 /56 ) Early activated T cells (CD3 CD69 ) Median activated T cells (CD3 CD25 ) Late activated T cells (CD3 HLADR ) Median activated B cells (CD19 CD25 ) CD3 CD45RA cells CD3 CD45RO cells , CD71 neutrophils CD71 lymphocytes Soluble interleukin-2 receptor Soluble transferrin receptor * Mean of second, third, or fourth point in cycle versus mean of first point; P.05. types of cycles. Blood was drawn at the same time each day from all women to avoid differences in circulating white blood cell profiles during the light/dark cycle. Studies on the bioperiodicity of human T and B lymphocytes show fluctuations during the 24-hour sleep-wake cycle, with a low in the morning at approximately 8 AM and a peak at around 00:00 (19). These fluctuations in immunocompetent cells imply that the time of day and the point in the menstrual cycle at which the measurements are made should be considered when evaluating these parameters. For example, if the normal ranges for natural killer cells for a particular laboratory happened to be based on samples obtained from women who were at an early point in the cycle, a patient who was tested at midcycle could be erroneously considered to have abnormal results. In summary, ovulation induction with hmg and simultaneous administration of a GnRH antagonist does not adversely affect the immune system and seems to be safe from an immunologic point of view. As in normal menstrual cycles, it is unlikely that the immunologic fluctuations during COS are of clinical relevance. References 1. Grossman CJ. Interactions between the gonadal steroids and the immune system. Science 1985;227: Ansar Ahmed S, Penhale WJ, Talal N. Sex hormones, immune responses, and autoimmune diseases. Mechanisms of sex hormone. Am J Pathol 1985;121: Lynch EA, Dinarello CA, Cannon JG. Gender differences in IL-1 alpha, IL-1 beta, and IL-1 receptor antagonist secretion from mononuclear cells and urinary excretion. J Immunol 1994;153: Fox HS, Bond BL, Parslow TG. Estrogen regulates the IFN-gamma promoter. J Immunol 1991;146: Giglio T, Imro MA, Filaci G, Scudeletti M, Puppo F, De-Cecco L, et al. Immune cell circulating subsets are affected by gonadal function. Life Sci 1994;54: Chao TC. Female sex hormones and the immune system. Chang Keng I Hsueh 1996;19: American Society for Reproductive Medicine. Revised American Society for Reproductive Medicine classification of endometriosis: Fertil Steril 1997;67: Hamblin A, Taylor M, Bernhagen J, Shakoor Z, Mayall S, Noble G, et al. A method of preparing blood leucocytes for flow cytometry which prevents upregulation of leucocyte integrins. J Immunol Methods 1992; 146: Northern AL, Rutter SM, Peterson CM. Cyclic changes in the concentrations of peripheral blood immune cells during the normal menstrual cycle. Proc Soc Exp Biol Med 1994;207: Mills PJ, Ziegler MG, Dimsdale JE, Parry BL. Enumerative immune changes following acute stress: effect of the menstrual cycle. Brain Behav Immun 1995;9: Ho HN, Chen HF, Chen SU, Chao KH, Yang YS, Huang SC, et al. Gonadotropin releasing hormone (GnRH) agonist induces down-regulation of the CD3 CD25 lymphocyte subpopulation in peripheral blood. Am J Reprod Immunol 1995;33: Roitt MI, Brostoff J, Male DK. Immunology. London: Gower Medical Publishing, Lopez-Karpovitchs X, Larrea F, Cardenas R, Valencia X, Piedras J, Diaz-Sanchez V, et al. Peripheral blood lymphocyte subsets and serum immunoglobulins in Sheehan s syndrome and in normal women during the menstrual cycle. Rev Invest Clin 1993;45: Coulam CB, Silverfield JC, Kazmar RE, Fathman CG. T-lymphocyte subsets during pregnancy and the menstrual cycle. Am J Reprod Immunol 1983;4: Mathur S, Mathur RS, Goust JM, Williamson HO, Fudenberg HH. Cyclic variations in white cell subpopulations in the human menstrual cycle: correlations with progesterone and estradiol. Clin Immunol Immunopathol 1979;13: Giuliani et al. COH and the immune system Vol. 70, No. 5, November 1998

5 16. Chen CK, Huang SC, Chen CL, Yen MR, Hsu HC, Ho HN. Increased expression of CD69 and HLA-DR but not of CD25 or CD71 on endometrial T lymphocytes of nonpregnant women. Hum Immunol 1995;42: Eichler F, Keiling R. Variations in the percentages of lymphocyte subtypes during the menstrual cycle in women. Biomed Pharmacother 1988;42: Lopez-Cabrera M, Santis AG, Fernandez Ruiz E, Blacher R, Esch F, Sanchez-Mateos P, et al. Molecular cloning, expression, and chromosomal localization of the human earliest lymphocyte activation antigen AIM/CD69, a new member of the C-type animal lectin superfamily of signal transmitting receptors. J Exp Med 1993;178: Abo T, Kawate T, Itoh K, Kumagai K. Studies on the bioperiodicity of the immune response. I. Circadian rhythms of human T, B, and K cell traffic in the peripheral blood. J Immunol 1981;126: FERTILITY & STERILITY 835

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