CoovriPht Q Munkseaard. 1997 AJUl 1997. 37.279-282 Printed in the United States of America - all rights reserved

Amerlcan lournal of Reproductlve Immunology ISSN 8755-8920

EDITORIAL

Controversies in Diagnosis and Management Recurrent Spontaneous Abortion This issue of the journal contains selected papers presented at the S2nd Annual Meeting of the American Society for Reproductive Medicine, November 2-6, 1996, in Boston, Massachusetts. Over the last several years, a remarkable increase in the number presentations related to immunol- ogy has appeared at these annual meetings. This increase in presentations reflects an appreciation of the importance of immunologic factors in reproductive processes. Clini- cal application of reproductive immunology has largely focused on reproductive wastage manifest as infertility, recurrent pregnancy loss, and endometriosis. While much progress in elucidating immunologic events involved in re- productive wastage has been made, all of the mechanisms are not known. As a result, controversy as to appropriate diagnostic testing and treatment of reproductive wastage has occurred. Several presentations at the 52nd Annual Meeting amplified controversy, making decisions concern- ing clinical management more difficult for practitioners.

lished data may not be reliable, and the primary data need to be collected and analyzed by at least two independent teams. Another explanation for the lack of meaning of APA is differences in performance and/or interpretation of the APA assay. To address the question of the contribution of the specific APA assay the American Society of Reproduc- tive Immunology under the leadership of its President, Dr. David A. Clark, has appointed an ad hoc committee on Antiphospholipid Antibody Syndrome. The committee has organized a wet workshop to test coded sera for the pres- ence of APA in laboratories performing the test who wish to participate. Results of detection and interpretation of APA among participating laboratories will be compared and discussed at a consensus conference opened to all in- terested individuals. It is expected that the consensus con- ference will provide insight into the apparent controversial results of APA and IVF failure.

NATURAL KILLER CELLS ANTIPHOSPHOLIPID ANTIBODIES

At the Reproductive Immunology Special Interest Group Meeting, data were presented from a literature review showing an association between the prevalence of circu- lating antiphospholipid antibodies (APA) and pregnancy rates following in vitro fertilization (IVF) and embryo transfer (ET). The frequency of positive APAs among women undergoing IVF ranged from 15 to 48%. This is a large range in apparently similar populations. But more confusing was the summary of published reports of women undergoing IVF/ET who had been treated with heparin and aspirin compared with those receiving standard treatment. When the results of these selected studies were pooled, no differences between total pregnancy and ongoing preg- nancy rates between APA positive women treated with hep- arin and aspirin and those receiving no treatment, but also no differences in total or ongoing pregnancy rates between APA positive and APA negative women were seen. Pos- sible explanations for the controversial results include dif- ferent study designs and publication bias. A previous meta-analysis study in another area has shown that pub-

Natural killer (NK) cells are mononuclear cells that express CD56. Most circulating NK cells and a minority of uter- ine NK cells express CD16 (Fc receptor). CD56+ CD16- cells making TGF-j32 and presenting in early pregnancy decidua have been associated with successful pregnancy

and a lack of these cells and increased num- ber of CD16+ cells in endometria have been associated with subsequent miscarriage^.^.^ A number of studies have associated elevated circulating NK (i.e., percent of NK/to- tal WBC) cells with adverse pregnancy outcome."" At the Reproductive Special Interest Group Session, one presen- tation suggested that elevated percentages of circulating NK (i.e., 100 x CD56+/total WBC) cells were not associ- ated with pregnancy loss. The data which led to this con- clusion was the mean percent NK cells was the same in women experiencing abortion as in these having a live birth. However, the association of elevated percentage of circulating NK and spontaneous abortion has not been lin- ear but rather a threshold exists such that the difference between normal and abnormal percent of circulating NK cells is discontinuous. Since the relationship between cir-

AMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY VOL. 37,1997

280 / COULAM AND CLARK

TABLE I. Effect of Using Different Meta-Analytic Techniques on Significance of Result of Leukocyte lmrnunotherapy

Type of statistical model Name of specific method Result Significance

Fixed effects Mantel Haenzel (odds ratio) 1.56" P I 0.027 Mantel Haenzel (risk difference) 9.8% P I 0.028 Mantel Haenzel (risk ratio) 1.17 P I 0.028 Peto (odds ratio) 1.55 P S 0.028

Random effects DerSirnonian and Laird (odds ratio) 1.56" P 5 0.028 DerSirnonian and Laird (risk difference) 9.0% P I 0.028 DerSirnonian and Laird (risk ratio) 1.15 P I 0.047b

"A value of > I or a positive % risk difference favoring paternal (husband) leukocyte immunization. 'Discrepant compared to all other methods tested.

culating NK cells and spontaneous abortion is discontinu- ous, mean value comparisons are not valid. To conclude that elevated percentages of circulating NK cells is not as- sociated with pregnancy outcome, the 95th percentile of percentages of circulating NK cells needs to be determined for their normal population. Then the proportion of women exceeding this threshold from control and aborting women needs to be compared. When this method was employed, significantly elevated levels of circulating NK cells or NK activity were observed in women with a history of recur- rent spontaneous abortion both preconceptually" and postconceptually.69~'' In fact elevated circulating NK cells have been reported in women aborting karyotypically nor- mal concepti compared to those with chromosomal abnor- malities.'.''

IMMUNOTHERAPY FOR TREATMENT OF RECURRENT SPONTANEOUS ABORTION

Both allogenic leukocyte immunization and intravenous immunoglobulin (IVIg) have been used for the treatment of women with unexplained recurrent spontaneous abor- tion. Randomized clinical trials have shown conflicting results of these treatments'*-'' which is not surprising given

their small sample sizes. The current evidence based medi- cine movement grades the strength of evidence from strong or (Level I) to weak or (Level V). Level I evidence is gen- erated by large double blinded randomized trials or by meta-analysis of small trials generating a data set of suf- ficient power. Meta-analysis of trials of leukocyte immu- nization"" and IVIg

The meta-analysis of trials of leukocyte immunotherapy conducted by the Ethics Committee of the American So- ciety of Reproductive Immunology (ASRI) consisted of two independent teams of statisticians. Both teams found significant (but small) increases in the probability of live birth as a result of paternal leukocyte immunization." A subsequent subset analysis by one of the teams involved in the original ASRI meta-analysis has been used to argue that leukocyte immunotherapy is likely ineffective?' But there are serious scientific flaws in the analyses and an updated meta-analysis including one of the author's own data has confirmed a significant beneficial effect."

During the execution of the latter meta-analyses, a very interesting observation was made. There are sev- eral methods for performing meta-analysis, and not all give the same result. Table I shows the outcome using different methods. It can be seen that one version of the

have been conducted.

TABLE II. Effect of Different Meta-Analytic Techniques on Significance of Result of lVlg in Randomized Controlled Trials

Type of statistical model Name of specific method Result Significance

Fixed effects Mantel Haenzel (odds ratio) 2.24a P I 0.016 Mantel Haenzel (risk difference) 17.6% P I 0.016 Mantel Haenzel (risk ratio) 1.38 P I 0.017 Pet0 (odds ratio) 2.24 P I 0.016

Random effects DerSimonian and Laird (odds ratio) 2.24' P I 0.01 7 DerSimonian and Laird (risk difference) 17.6% P I 0.01 6 DerSirnonian and Laird (risk ratio) 1.31 P I 0.067b

"A value of > I or a positive % risk difference favors IVIg over placebo. hDiscrepant compared to all other methods tested.

0 MUNKSGAARD, COPENHAGEN

EDITORIAL / 281

TABLE Ill. Summary of Results of Meta-Analysis of Various Patient Population Treated With IVlg"

Patient Confidence P Population N RR interval value Reference

RSA 152 1.38 1.04-1.84 4 0.02 19 RSA + IVF 163 1.38 1.06-1.81 4 0.02 20,25 IVF failure 71 2.70 1 .16-6.28 40.02 20

"RSA. retwrrent spontaneous abortion; IVF, in vitro fertilization; RR, relative risk: RR > I favors treatment with IVIg , RR < I

95%

favors cwntrol.

random effects model of DerSimonian and Laird" pro- vided a result that has a much higher P value (lower sig- nificance) than all the other methods. This has been a consistent f inding with different da t a sets. The DerSimonian and Laird risk ratio method is therefore more conservative and may be useful to therapeutic ni- hilists in casting doubt on the wisdom or rejecting the nul l hypothesis. On the other hand, such conservatism may overlook effective therapy, and there is published information indicating treatment may be effective if tar- geted to a particular subset of patient^.^^-^^

Analysis of results from a randomized, blinded, placebo- controlled trial showed IVIg is efficacious in the treatment of recurrent spontaneous abortion.16 In this study, IVIg treatment began prior to conception. Two other random- ized, placebo-controlled trials using IVIg to treat recurrent pregnancy loss have been In both of these trials, IVIg was started after diagnosis of pregnancy. In the German trial,17 a significant specific effect of IVIg on live birth rate could not be demonstrated. However, success rates of protocol-eligible patients for both IVIg or placebo were 14% and 70%, respectively, with a significant cor- relation of better outcomes if the IVIg was started earlier rather than later.17 The significant difference in the con- trol success rate would suggest that women in the German study were largely from the best prognosis group of just three prior losses (which, in fact, is correct). In addition, treatment in this study was not begun until after pregnancy was diagnosed (5-8 weeks of ge~tat ion) , '~ excluding pa- tients with NK cell-related pathology, which occurs ear- lier during the peri-implantation period. The study by Christiansen and colleagues'* treated only those women with a previous live birth who subsequently experienced recurrent abortion (secondary aborters) and those with abortions in the second trimester only. In this population, even though treatment was also started after diagnosis of pregnancy, a higher live birth rate was observed in the women treated with IVIg compared to placebo.18 A meta- analysis of the results of three randomized control trials on IVIg therapy for recurrent pregnancy loss revealed on overall benefit with relative risk of 1.38 (95% confidence interval 1.04-1.84, P = 0.02).20 Table I1 shows the out- comes of the meta-analyses when different methods were

used. As with the meta-analyses of treatment of recurrent spontaneous abortion using leukocyte immunization (Table I), one version of the random effects model of Der Simonian and Laird" provided a result that was discrep- ant with all of the other methods. Subsequently, random- ized controlled trial data from de Placido et al." on patients recurrently aborting after IVF/ET was added to the meta-analysis." The results of meta-analysis from clinical trials using IVIg to treat recurrent spontaneous abortions and implantation failure after IVF/ET are sum- marized in Table 111.

Thus, Level 1 evidence for therapeutic benefit exists for both leukocyte immunization and IVIg among women ex- periencing recurrent spontaneous abortion. The result of another randomized clinical trial using IVIg is expected to be available early 1997 and should help resolve any uncertainty related to only 6/7 methods of analysis giv- ing P I 0.05. Indeed, there is better evidence for im- munologic treatment of unexplained RSA than for any other treatment objective primarily explained or unex- plained recurrent pregnancy loss. The real controversy may lie with what has been standard practice for treat- ment of recurrent miscarriage and not with the newer immuno-therapeutic methods.

Carolyn B. Coulam, M.D. David A. Clark, M.D., Ph.D.

REFERENCES

1. Recurrent Miscarriage Immunotherapy Trialist Group. Worldwide collaborative observational study and meta- analysis on allogeneic leukocyte immunotherapy for recur- rent spontaneous abortion. Am J Repro Immunol 1994;

2. Michel M, Underwood J, Clark DA, Mowbray J, Beard RW. Histologic and immunologic study of uterine biopsy tissue of incipiently aborting women. Am J Obstet Gynecol 1989; 16 1 :4094 14.

3. Lachapelle MH, Miron P, Hemmings R, Roy DC. Endome- trial T, B and NK cells in patients with recurrent spontane- ous abortion. J Immunol 1996; 156:40274034.

4. Clark DA, Vinci G, Flanders KC, Rite H, Starkey P. CD56+ lymphoid cells in human first trimester decidua contains novel TGF related immunosuppressive factors. Hum Reprod

32: 55-72.

1994; 9~2271-2277.

AMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY VOL. 37,1997

282 / COULAM AND CLARK

5. Lea RG, Underwood J, Flanders KC, Hirte H, Branwatt D, Michel M, Daya S, Harley C, Mowbray JF, Clark DA. A subset of patients with recurrent spontaneous abortion is de- ficient in transforming growth factor beta 2-producing sup- pressor cells in decidua near the placental attachment site. Am J Reprod Immunol 1995; 3452-64.

6. Makida R, Minami M, Takamizawa M, Juji T, Fujii T, Mizuno M. Natural killer cell activity and immunotherapy for recurrent spontaneous abortion. Lancet 1991; 2579-580.

7. Yokoyama M, Sano M, Sonoda K, Nozaki M, Nakamura GI, Nakano H. Cytotoxic cells directed against placental cells de- tected in human habitual abortions by an in vitro terminal la- beling assay. Am J Reprod Immunol 1994; 31:197-204.

8. Coulam CB, Goodman C, Roussev RG, Thomason EJ, Beaman KG. Systemic CD56+ cells can predict pregnancy outcome. Am J Reprod Immunol 1995; 33:40-46.

9. Kwak FM-Y, Kwak JYH, Ajinbinder SW, Ruiz AM, Beer AE. Elevated peripheral blood natural killer cells are effec- tively suppressed by immunoglobulin G infusions in women with recurrent spontaneous abortions. Am J Reprod Immu- no1 1996; 35:363-369.

10. Aoki K, Kajiura S , Matsumato Y, Ogasawara M, Okada S, Yagammi Y, Gleicher N. Preconceptual natural killer cell activity as a predictor of miscarriage. Lancet 1995; 345:1340-1342.

1 1. Coulam CB, Beaman KD. Reciprocal alteration in circu- lating TJ6+ CD19+ leukocytes in early pregnancy predicts success or miscarriage. Am J Reprod Immunol 1995;

12. Mowbray JF, Lidlee H, Underwood JL, Gibbings C, Reginald PW, Beard RW. Controlled trial of treatment of re- current spontaneous abortion by immunization with pater- nal cells. Lancet 1985; 1:941-949.

13. Ho H, Gill TJ, Hsuish HJ, Jiang JJ, Lee TY, Hsish CY. Im- munotherapy for recurrent spontaneous abortion in Chinese population. Am J Reprod Immunol 1991; 25:10-15.

14. Cauchi MN, Lemi D, Young DE, Klosa M, Pepperell RJ. Treatment of recurrent spontaneous aborters by immuniza- tion with paternal cells controlled trial. Am J Reprod Im- munol 1991; 25:16-17.

34:219-224.

15. Gatenby PA, Cameron K, Simes RJ et al. Treatment of re- current spontaneous abortion by immunization with pater- nal lymphocytes: Results of controlled trial. Am J Reprod Immunol 1993; 29:88-94.

16. Coulam CB, Krysa L, Stern JJ, Bustillo M. Intravenous im- munoglobulin for treatment of recurrent pregnancy loss. Am J Reprod Immunol 1995; 34:333-337.

17. The German RSA/IVIg Group. Intravenous immunoglobu- lin in the prevention of recurrent miscarriage. Br J Obstet Gynecol 1994; 101:1072-1077.

18. Christianson OB, Mathiesen 0, Husta M, et al. Placebo controlled trial of treatment on unexplained secondary re- current spontaneous abortions and recurrent spontaneous abortions with IV immunoglobulin. Human Reprod 1995; 10:3690-3695.

19. Clark DA. Alloimmunity and pregnancy loss in Gleicher N (ed): Principles and Practice of Medical Therapy in Preg- nancy, 3rd edition. Appleton and Lange, Norwalk CN, 1997, in press.

20. Clark DA, Gunby J, Daya S. The use of allogeneic leuko- cytes or IVIg for the treatment of patients with recurrent spontaneous abortion. Trans Med Rev, 1997, in press.

21. Jeng GT, Scott JR, Burmeister LF. A comparison of meta- analysis results using literature versus individual patients data. JAMA 1995; 274:830-836.

22. Daya S , Gunby J. The Recurrent Miscarriage Immuno- therapy Trialist leukocyte immunization in unexplained pri- mary recurrent spontaneous abortion. Am J Repro Immunol 1994; 32:294-302.

23. Coulam CB, Clark DA. Immunotherapy for recurrent mis- carriage. Am J Reprod Immunol 1994; 32:257-260.

24. Clark DA, Daya S , Coulam CB, Gunby J. The Recurrent Miscarriage Immunotherapy Trialist Group. Implication of abnormal human trophoblast karyotype for the evidence- based approach to the understanding, investigation and treat- ment of recurrent spontaneous abortion. Am J Reprod Immunol 1996; 35:495-498.

25. DePlacido G, Zullo F, Mollo A, et al. Intravenous immuno- globulin (IVIg) in the prevention of implantation failure. Ann NY Acad Sci 1994; 734:232-234.

0 MUNKSGAARD, COPENHAGEN