Lupus (2002) 11, 716±721 www.lupus-journal.com PAPER Pregnancy outcome in 100 women with autoimmune diseases and anti-Ro=SSA antibodies: a prospective controlled study A Brucato1*, A Doria3, M Frassi2, G Castellino1, F Franceschini2, D Faden2, M Pia Pisoni1, L Solerte1, M Muscarà1, A Lojacono2, M Motta2, I Cavazzana2, A Ghirardello3, F Vescovi3, V Tombini1, R Cimaz4, PF Gambari3, PL Meroni5, B Canesi1 and A Tincani2 1 Ospedale Niguarda, Milano, Italy; 2Spedali Civili, Brescia, Italy; 3University of Padova, Padova, Italy; 4Istituti Clinici di Perfezionamento, Milano, Italy; and 5Department of Internal Medicine, University of Milan, Allergy and Clinical Immunology Unit, IRCCS Istituto Auxologico Italiano, Milano, Italy Anti-Ro=SSA antibodies are associated with neonatal lupus but are also considered a possible cause for unexplainedpregnancy loss and adverse pregnancy outcome. In a large multicentres cohort study we have prospectivelyfollowed 100 anti-Ro=SSA positive women (53 systemic lupus erythematosus (SLE)) during their 122 pregnancies and 107 anti-Ro=SSA negative women (58 SLE) (140 pregnancies). Anti-Ro=SSA antibodies were tested by immunoblot and counterimmunoelectrophoresis. Mean gestational age at delivery (38 vs 37.9 weeks), prevalence of pregnancy loss (9.9 vs 18.6%), preterm birth (21.3 vs 13.9%), cesarean sections (49.2 vs 53.4%), premature rupture of membranes (4.9 vs 8.1%), preeclampsia (6.6 vs 8.1%), intrauterinegrowth retardation (0 vs 2.3%) and newborns small for gestationalage (11.5 vs 5.8%) were similar in anti-Ro=SSA positive and negative SLE mothers; Žndings were similar in non-SLE women. Two cases of congenital heart block were observed out of 100 anti-Ro=SSA positive women. In conclusion, anti-Ro=SSA antibodies are responsiblefor congenitalheart block but do not affect other pregnancy outcomes, both in SLE and in non-SLE women. The general outcome of these pregnancies is now very good, if prospectively followed by multidisciplinaryteams with ample experience in this Želd. Lupus (2002) 11, 716–721. Key words: anti-Ro=SSA antibodies; congenital heart block; pregnancy; primary Sjogren’s syndrome; systemic lupus erythematosus Introduction Anti-Ro=SSA antibodies are linked to the development of congenital heart block (CHB) in utero and to other clinical manifestations in newborns such as skin rash, liver abnormalities, thrombocytopenia, etc.1 In contrast, it is not clear if these antibodies are linked to other adverse pregnancy outcomes, both in systemic lupus erythematosus (SLE) and in non-SLE women. Most of the available data come from retrospective studies; some of them considered the anti-Ro=SSA antibodies as a possible causative factor for unexplained pregnancy loss in SLE.2–4 On the other hand, Julkunen retrospectively observed that the relative risk of fetal loss was increased in patients with primary Sjogren’s syndrome (SS), but it was not associated with anti-Ro=SSA or La=SSB.5 The largest retrospective study found that anti-Ro=SSA antibodies do not adversely affect pregnancy outcome in SLE patients, while they appear to be associated with recurrent pregnancy loss in non-SLE patients. 6 Interestingly, a prospective study of pregnancy in SLE patients provided conicting results showing that the absence of anti-Ro=SSA antibodies was signiŽcantly related to the occurrence of fetal loss and IUGR.7 Our study focuses the impact of anti-Ro=SSA antibodies on pregnancy outcome in a large cohort of 100 positive women prospectively followed during their pregnancies; data concerning historical pregnancies of the same patients were also retrospectively examined. Patients and methods Patients *Correspondence: A Brucato, Via del Bollo 4, 20123 Milano, Italy. E-mail: abrucato@ospedale-niguarda.it Received 9 May 2002; accepted 8 August 2002 # Arnold2002 Anti-Ro=SSA-positive women Details concerning patients and methods have already been reported.8 1011.91=0961203302lu252oa Anti-Ro=SSA antibodies and pregnancy outcomes ABrucatoet al. Briey, this prospective study was performed in four referral hospitals: Spedali Civili, Brescia (47 women); Padua University (26 women), Ospedale Niguarda, Milano (17 women), and Milano University (10 women). All four hospitals are tertiary referral centers, with connective tissue diseases pregnancy clinics. All the mothers were under regular observation before becoming pregnant. The only selection criteria was the presence of anti-Ro=SSA antibodies in the mother’s serum before she became pregnant. One hundred women were studied, during their 122 pregnancies (22 repeated pregnancies), that ended with the delivery of 116 live newborns (four twin pregnancies). Fifty-three SLE women and 47 with other connective tissue diseases were studied (see Table 1). Diagnoses were in accordance with ACR criteria for SLE9 and European criteria for SS.10 Mean age at Žrst delivery was 31.2 years (range 25 – 40). Anti-Ro=SSA negative women In the same observation period, the anti-Ro=SSA negative women with an autoimmune disease prospectively followed during their pregnancies in three out of the four centers were enrolled as controls. Women with primary anti-phospholipid syndrome were excluded, because of the negative impact of this condition on pregnancy outcome. One hundred and seven women were studied (Spedali Civili, Brescia, 41 women; Padua University, 36 women; Ospedale Niguarda, Milano, 30 women), during their 140 pregnancies that ended with the delivery of 125 live newborns (two twin pregnancy). Fifty-eight had SLE; their diagnoses are detailed in Table 1. Mean age at Žrst delivery was 29.8 years (range 20 – 41). Table 1 Maternal diagnoses SLE Primary APS Undifferentiated connective tissue disease Rheumatoid arthritis Primary Sjogren’s syndrome Mixed connective tissue disease Idiopathic thrombocytopenic purpura Erythema nodosum Seronegative arthritis Systemic sclerosis Behcet disease Multiple sclerosis Dermatomyositis Total Ro=SSA positive women, n Ro=SSA negative women, n 53 1 19 58 0 21 0 25 1 9 1 0 0 4 0 0 1 0 0 0 4 4 1 1 3 1 100 107 All women (anti-Ro=SSA positive and negative) were closely followed by a high-risk pregnancy obstetrical team, monthly until the 18th week, then every 2 – 4 weeks. Instrumental monitoring included fetal – maternal Doppler velocimetry. The study protocol included one last assessment 4 weeks after delivery. No woman was lost to follow-up. Data collected included (see the list of abbreviations) the number of pregnancy losses before and after 10 weeks of amenorrhea, prematurity, deŽned as delivery before the completion 37 weeks’gestation, and further specifying cases in which delivery occurred before 34 weeks’ gestation, cesarean sections, premature rupture of membranes (PROM), preterm premature rupture of membranes (PPROM; rupture of membranes prior to 37 weeks’ gestation), intrauterine growth retardation (IUGR), preeclampsia deŽned as new onset of hypertension, proteinuria and edema, and number of newborns small for gestational age (SGA).11 In SLE patients, a are of the disease was deŽned as a reactivation leading to hospital admission or to a substantial change in the previous therapy. Obstetric history was also collected from anti-Ro=SSA positive and negative women, and an analysis of these retrospective data was done. 717 Methods Antibodies to Ro=SSA were determined in a single referral laboratory by counterimmunoelectrophoresis (CIE) and immunoblotting using a home-made human spleen extract as substrate prepared according to Clark et al.12 Antibodies to other cellular antigens were detected by CIE using a rabbit thymus extract (PeelFreeze, Rogers, AK, USA) as described.13 The Žne speciŽcity of anti-Ro=SSA 52 and 60 kDa response was determined by immunoblotting: sodium-dodecylsulfate polyacrilamyde gel electrophoresis was done with a commercial slab gel apparatus (Mini proten Dual Slab gel, BioRad Laboratories, Richmond, CA, USA) and 10.5% slab gel with an acrylamide= bisacrylamide ratio of 19:1 in order to distinguish the anti-La response (48 kDa) from the anti-52 kDa reactivity. The patients’ laboratory work-up for other autoantibodies was done by each center according to its routine determination. Statistical methods The proportions were compared with the chi-square test with continuity correction, and the means with the two-tailed t-test for independent samples. Lupus Anti-Ro=SSA antibodies and pregnancy outcomes ABrucatoet al. 718 Results One hundred anti-Ro=SSA positive women had 122 consecutive pregnancies, and 107 anti-Ro=SSA negative women had 140 consecutive pregnancies. Table 2 summarizes the pregnancy outcomes in these two groups. The general outcomes were similar (see Table 2), as well as the proportion of cesarean sections (39.3 vs 42.9%), the incidence of extrauterine pregnancies (0.8 vs 0%), PROM (4.1 vs 5.7%), PPROM (7.4 vs 4.3%), preeclampsia (4.9 vs 6.4%), IUGR (1.6 vs 2.8%) and newborns SGA (10.7 vs 7.1%; anti-Ro=SSA positive vs negative women). Non-SLE women were very heterogeneous and difŽcult to compare; in particular we had difŽculties in enrolling anti-Ro=SSA negative Sjogren’s women (see Table 1). Table 3 shows the data relative to non-SLE pregnancies. All the outcomes were similar in these two groups; the proportion of total pregnancy losses was slightly greater in non-SLE anti-Ro=SSA positive pregnancies, but the difference was not statistically signiŽcant (P > 0.30). Other pregnancy outcomes in non-SLE women not reported in Table 3 are (anti-Ro=SSA positive vs negative women): the proportion of cesarean sections (29.5 vs 25.9%); the incidence of PROM (3.3 vs 1.8%); PPROM (4.9 vs 3.7%); preeclampsia (3.3 vs 3.7%); IUGR (3.3 vs 3.7%); and newborns SGA (9.8 vs 9.3%). Table 4 summarizes the data in SLE pregnancies. Also, in this case no statistically or clinically relevant difference Table 2 was found. The maternal mean age at Žrst delivery was similar in the two groups. The proportion of total pregnancy losses, both early and late, was greater in anti-Ro=SSA negative pregnancies, but not statistically signiŽcant (P > 0.20). The incidence of deliveries occurring before 37 or 34 completed weeks, mean gestational age at delivery, the proportion of cesarean sections (49.2 vs 53.4%), the incidence of extrauterine pregnancies (1.6 vs 0%), PROM (4.9 vs 8.1%), PPROM (9.9 vs 4.6%), preeclampsia (6.6 vs 8.1%), IUGR (0 vs 2.3%) and newborn SGA (11.5 vs 5.8%) were similar in anti-Ro=SSA positive and negative women. The prevalence of anti-phospolipid antibodies was similar in all the different subsets (see Tables 3 and 4). The Žne speciŽcity of anti-Ro=SSA antibodies (anti-52 or anti-60 kDa) did not affect the pregnancy outcome, both in SLE and in non-SLE anti-Ro=SSA positive women (data not shown). Two cases of complete CHB were observed out of 100 Žrst pregnancies (2%;8 95% conŽdence interval 0.2 – 7%). We also compared the previous obstetrical history of anti-Ro=SSA and anti-Ro=SSA negative women. This retrospective analysis is summarized in the second part of each table (Tables 2 – 4). All the different subsets of women reported a very high prevalence of previous spontaneous pregnancy loss, ranging from 47.7 to 38.6% in the different subsets. This was observed not only in SLE women, but also in non-SLE women, and the trend was similar in both Pregnancy outcome of all anti-Ro=SSA positive vs all anti-Ro=SSA negative women Ro=SSA positive women n Number of women Number of pregnancies Twin pregnancies Living newborns Total spontaneous pregnancy losses Pregnancy losses < 10 weeks Pregnancy losses > 10 weeks Therapeutical abortions Prematurity < 37 weeks Prematurity < 34 weeks Mean gestational age at delivery; weeks (range) Maternal mean age at delivery; years (range) Previous pregnancies (retrospective analysis) Total pregnancies Pregnancy losses < 10 weeks Pregnancy losses > 10 weeks Total spontenous pregnancy lossesb Therapeutical abortions 100 122 4 116 10 7 3 0 19 3 38 (30 – 42) 31.2 (25 – 40) 44 16 5 21 3 %a 95.1% 8.2% 5.7% 2.4% 0 15.6% 2.5% 36.3% 11.4% 47.7% 6.8% Ro=SSA negative women n 107 140 2 125 17 10 7 1 16 2 38.4 (33 – 42) 29.8 (20 – 41) 44 9 8 17 5 %a 89.3% 12.1% 7.1% 5.0% 0.7% 11.4% 1.4% 20.4% 18.1% 38.6% 11.4% a Percentages are expressed on the number of pregnancies: prospective, 122 for Ro=SSA positive and 140 for Ro=SSA negative women; 44 retrospective pregnancies for both. b Total spontenous pregnancy losses: in anti-Ro=SSA positive women, prospective (10=122) vs retrospective (21=44), P < 0.001; in anti-Ro=SSA negative women, prospective (17=140) vs retrospective (17=44), P < 0.001. Lupus Anti-Ro=SSA antibodies and pregnancy outcomes ABrucatoet al. Table 3 719 Pregnancy outcome of non-SLE anti-Ro=SSA positive vs non-SLE anti-Ro=SSA negative women Ro=SSA positive women n Number of women Number of pregnancies Twin pregnancies Living newborns Total spontaneous pregnancy losses Pregnancy losses < 10 weeks Pregnancy losses > 10 weeks Therapeutical abortions Prematurity < 37 weeks Prematurity < 34 weeks Mean gestational age at delivery; weeks (range) Maternal mean age at delivery; years (range) Positivity for aPL 47 61 3 60 4 3 1 0 6 1 38 (34 – 42) 30 (24 – 37) 2 Previous pregnancies (retrospective analysis) Total pregnancies Pregnancy losses < 10 weeks Pregnancy losses > 10 weeks Total spontenous pregnancy lossesb Therapeutical abortions 23 9 2 11 2 Ro=SSA negative women %a n %a 98.4% 6.6% 4.9% 1.6% 0 9.8% 1.6% 101.8% 1.8% 0 1.8% 0 7.4% 1.8% 3.3% 49 54 2 55 1 0 1 0 4 1 39.3 (33 – 41) 29.6 (24 – 41) 5 39.1% 8.7% 47.8% 8.7% 22 6 3 9 0 10.2% 27.3% 13.7% 40.9% 0 a Percentages are expressed on the number of pregnancies: prospective, 61 for Ro=SSA positive and 54 for Ro=SSA negative women; retrospective, 23 for Ro=SSA positive and 22 for Ro=SSA negative women. b Total spontenous pregnancy losses: in anti-Ro=SSA positive women, prospective (4=61) vs retrospective (11=23), P < 0.001; in anti-Ro=SSA negative women, prospective (1=54) vs retrospective (9=22), P < 0.001. anti-Ro=SSA positive and anti-Ro=SSA negative women. These retrospective data were in striking contrast with the overall good outcomes of the prospectively followed pregnancies. In almost all the subsets the difference between the proportion of Table 4 spontaneous pregnancy loss in the previous pregnancies as compared with the current prospectively followed pregnancies was signiŽcant (P ranging from < 0.05 to < 0.001 in the different subsets; see Tables 2 – 4). Notably, the incidence of therapeutical Pregnancy outcome of SLE anti-Ro=SSA positive vs SLE anti-Ro=SSA negative women Ro=SSA positive women n Number of women Number of pregnancies Twin pregnancies Living newborns Total spontaneous pregnancy losses Pregnancy losses < 10 weeks Pregnancy losses > 10 weeks Therapeutical abortions Prematurity < 37 weeks Prematurity < 34 weeks Mean gestational age at delivery; weeks (range) Maternal mean age at delivery; years (range) Positivity for aPL SLE plus secondary APS Major ares Previous pregnancies (retrospective analysis) Total pregnancies Pregnancy losses < 10 weeks Pregnancy losses > 10 weeks Total spontenous pregnancy lossesb Therapeutical abortions 53 61 1 56 6 4 2 0 13 2 38 (30 – 41) 30 (23 – 38) 8 4 10 21 7 3 10 1 %a Ro=SSA negative women n 14.3% 7.1% 17.9% 58 86 0 70 16 10 6 1 12 1 37.9 (33 – 42) 30 (20 – 39) 12 9 16 33.3% 14.2% 47.6% 4.8% 22 4 5 9 5 91.9% 9.9% 6.6% 3.3% 0 21.3% 3.3% %a 81.4% 18.6% 11.6% 7.0% 1.1% 13.9% 1.1% 13.9% 10.4% 18.6% 18.1% 22.7% 40.9% 22.7% a Percentages are expressed on the number of pregnancies: prospective, 61 for Ro=SSA positive and 86 for Ro=SSA negative women; retrospective, 21 for Ro=SSA positive and 22 for Ro=SSA negative women. b Total spontenous pregnancy losses: in anti-Ro=SSA positive women, prospective (6=61) vs retrospective (10=21): P < 0.001; in antiRo=SSA negative women, prospective (16=86) vs retrospective (9=22), 0.05 < P < 0.10 ˆ NS Lupus Anti-Ro=SSA antibodies and pregnancy outcomes ABrucatoet al. 720 abortions was also higher in the previous pregnancies, ranging from 0 to 22.7% in the different subsets of women. Discussion We report here the Žrst prospective controlled study on the pregnancy outcome of a large cohort of anti-Ro=SSA positive women. Data in the literature conict regarding the possible contribution of the antiRo=SSA antibodies to reproductive failure of women with autoimmune diseases. Some small or retrospective studies have already addressed this problem, but their retrospective nature is a strong limitation: in fact even data such as the total number of pregnancies might be incorrect in a retrospective analysis. Hull et al. reported on three anti-Ro=SSA positive SLE women with a history of spontaneous abortion;2 Barclay et al. reported on one patient with two spontaneous abortions and a stillbirth, 3 and Watson et al. described a direct correlation of anti-Ro=SSA antibodies with pregnancy loss only in black SLE patients. 4 Julkunen did a retrospective analysis of the fetal outcome in 55 pregnancies in 21 patients with primary SS compared with that in 100 pregnancies in 42 patients with SLE and 94 pregnancies in 42 healthy women. The relative risk for fetal loss in patients with primary SS was 2.7; in SLE it was 2.2. Most pregnancies in women with primary SS occurred before the onset of the disease. Notably, Julkunen observed that the risk of pregnancy loss in primary SS was not associated with the presence of antibodies to Ro=SSA or La=SSB.5 A large retrospective study compared the obstetric histories of 154 anti-Ro=SSA positive women with autoimmune diseases (78 with SLE) with that of both 142 anti-Ro=SSA negative women matched for disease diagnosis and a control group of 180 healthy women.6 The authors found that the overall rate of pregnancy loss and adverse pregnancy outcome did not signiŽcantly differ among the three groups. Anti-Ro=SSA positive SLE women reported a signiŽcantly higher rate (18.0%) of therapeutic abortions compared with anti-Ro=SSA negative women (5.6%, P ˆ 0.0244) and healthy controls (4.6%, P ˆ 0.0013). Anti-Ro=SSA non-SLE positive women reported a signiŽcantly higher rate of recurrent pregnancy loss in comparison to anti-Ro=SSA negative women (23.7 vs 7.04%, P ˆ 0.0063) and healthy controls (6.4, P ˆ 0.0004). The authors concluded that, although anti-Ro=SSA antibodies do not adversely affect pregnancy outcome in SLE patients, they appear to be associated with recurrent pregnancy loss in non-SLE patients.6 In contrast, a prospective study analyzing pregnancy outcome in SLE, clearly Lupus shows that fetal loss and IUGR correlated with the absence of anti-Ro=SSA antibodies.7 Our results show that anti-Ro=SSA antibodies do not affect the pregnancy outcomes except for the risk of the already quoted occurrence of CHB.8 In fact, we did not observe any clinically or statistically signiŽcant difference between anti-Ro=SSA positive and negative women, even if the frequency of pregnancy loss in anti-Ro=SSA positive compared to antiRo=SSA negative women was slightly lower in SLE (9.9 vs 18.6%; relative risk 0.53), in agreement with the data by Le Thi,7 and slightly higher in non-SLE (6.6 vs 1.8%; relative risk 3.7), in agreement with the data by Mavragani.6 Apparently, anti-Ro=SSA antibodies seem to have an inverse effect on pregnancy loss in SLE and non-SLE women. Looking at pregnancy outcome, anti-Ro=SSA seem to have a favorable impact in patients with SLE, which is a severe systemic disease. Conversely, they seem to have an adverse effect in patients with milder autoimmune conditions such as undifferentiated connective tissue disease or Sjogren’s syndrome. However, no signiŽcant differences have been found in our study, therefore this observation should be interpreted with extreme caution. We also found that the Žne speciŽcity of anti-Ro=SSA antibodies did not affect pregnancy outcome. Considering obstetrical history, approximately half of the previous pregnancies had ended with spontaneous or voluntary abortions. The retrospective nature of this analysis does not allow an accurate explanation of these Žndings. Reasons are probably multifactorial: the management of the current pregnancies by highrisk pregnancy obstetrical team; the accurate planning of these prospective pregnancies; a selection bias, by which women with previous negative obstetrical outcomes were more easily referred to centers with experience in the Želd. Moreover, the striking difference emerging suggests how an analysis of data retrospectively reported by the women concerning their previous pregnancies might be of limited value. On the other hand, nowadays the general outcomes of the pregnancies in these autoimmune ‘high-risk’ women can be excellent when carefully followed by dedicated high-risk obstetrical and multidisciplinary teams. In conclusion, we observed in a large prospective controlled multicenter cohort study of anti-Ro=SSA positive women that the presence of these antibodies is associated with complete congenital heart block but does not negatively affect other pregnancy outcomes, both in SLE and in non-SLE women. The general outcome of these pregnancies is now very good, when prospectively followed by multidisciplinary teams with ample experience in this Želd. Anti-Ro=SSA antibodies and pregnancy outcomes ABrucatoet al. Acknowledgement This study was supported in part by MURST-COFIN 2000 (PLM, AD, AT) and by Ricerca Corrente Istituto Auxologico Italiano 2001 (PLM). References 1 Brucato A, Buyon JP, Horsfall AC, Lee LA, Reichlin M. Fourth international workshop on neonatal lupus syndromes and the Ro=SSA-La=SSB System. Clin Exp Rheumatol 1999; 17: 130 – 136. 2 Hull RG, Harris EN, Morgan SH, Hughes GR. Anti-Ro antibodies and abortions in women with SLE. Lancet 1983; 2: 1138. 3 Barclay CS, French MA, Ross LD, Sokol RJ. Successful pregnancy following steroid therapy and plasma exchange in a woman with antiRo (SS-A) antibodies. Case report. Br J Obstet Gynaecol 1987; 94: 369 – 371. 4 Watson RM, Braunstein BL, Watson AJ, Hochberg MC, Provost TT. Fetal wastage in women with anti-Ro(SSA) antibody. J Rheumatol 1986; 13: 90 – 94. 5 Julkunen H, Kaaja R, Kurki P, Palosuo T, Friman C. Fetal outcome in women with primary Sjogren’s syndrome. A retrospective case-control study. Clin Exp Rheumatol 1995; 13: 65 – 71. 6 Mavragani CP, Dafni UG, Tzioufas AG, Moutsopoulos HM. Pregnancy outcome and anti-Ro=SSA in autoimmune diseases: a retrospective cohort study. Br J Rheumatol 1998; 37: 740 – 745. 7 Le Thi HD, Wechsler B, Piette JC, Bletry O, Godeau P. Pregnancy and its outcome in systemic lupus erythematosus. Q J Med 1994; 87: 721 – 729. 8 Brucato A, Frassi M, Franceschini F et al. Risk of congenital complete heart block in newborns of mothers with anti-Ro=SSA antibodies detected by counterimmunoelectrophoresis: a prospective study of 100 women. Arthritis Rheum 2001; 44: 1832 – 1835. 9 Hochberg MC. Updating the American College of Rheumatology revised criteria for the classiŽcation of systemic lupus erythematosus. Arthritis Rheum 1997; 40: 1725. 10 Vitali C, Bombardieri S, Moutsopoulos HM et al. Preliminary criteria for the classiŽcation of Sjogren’s syndrome. Results of a prospective concerted action supported by the European Community. Arthritis Rheum 1993; 36: 340 – 347. 11 Johnson MJ. Obstetric complications and rheumatic disease. Rheum Dis Clin N Am 1997; 23: 169 – 182. 12 Clark G, Reichlin M, Tomasi TB Jr. Characterization of a soluble cytoplasmic antigen reactive with sera from patients with systemic lupus erythmatosus. J Immunol 1969; 102: 117 – 122. 13 Brucato A, Franceschini F, Gasparini M et al. Isolated congenital complete heart block: longterm outcome of mothers, maternal antibody speciŽcity and immunogenetic background. J Rheumatol 1995; 22: 533 – 540. 721 Lupus