Immunodiagnostic Evaluation in Women with Reproductive Failure

Copyright 0 Munksgaard, 1999

ISSN 8755-8920 American journal of Reproductive Immunology

Immunodiagnostic Evaluation in Women with Reproductive Failure AZADEH s. KAIDER, BRIAN D. KAIDER, PATRICK B. JANOWICZ, AND ROUMEN G. ROUSSEV

Kaider AS, Kliider BD, Junowicz PB, Rousseu RG. Irnmunodiugnostic eiuluation in women w$th reproductice jailure. AJRI 1999; 42:335-346 0 Munksgaard, Copenhagen PROBLEM: Several immunological factors have been associated with diagnostic subpopulations of reproductive failure. It is important to determine a trend of immunological abnormalities among these subpopulations. The purpose of this study is to assist in the selection of treatment for patients suspected of having specific diagnoses of reproductive failure. METHOD O F STUDY: Blood samples from 591 patients were evaluated for the presence of antiphospholipid (APA), antinuclear (ANA), and antithyroid (ATA) antibodies, as well as for lupus anticoagulant (LA), embryotoxic factors (ETF), and elevated levels of natural killer (NK) (CD56 + ) cells, and all tests were performed as a panel. The patients were grouped into the following diagnostic categories: recurrent pregnancy loss ( n = 302), IVF/ET failure (IVFf, n = 122), unexplained infertility (n = 97), ovarian dysfunction (n = 47), and endometriosis ( n = 23). The thresholds for positivity and the prevalence of the tested factors among normal healthy populations have been established by testing 100 or more healthy male and female individuals with each one of the tests used (general population control). All tests as panel were performed on 20 normal fertile female individuals as controls (fertile female controls). RESULTS: Of all patients with reproductive failure, 75.6% had at least one abnormal test. The most frequent abnormal result was found to be the elevation of N K (CD56+) cells (37'%1), followed by ANA (34%), APA (24'%), ATA (19'%1), and ETF (1 1%). Of the recurrent pregnancy loss patients, 74.2% had at least one positive abnormal result from all of the tests performed; overall, 70% of women with IVF failure had at least one abnormal test; of patients diagnosed with unexplained infertility, approximately 8 1% had at least one abnormal result; 74.4% of the patients with ovarian dysfunction and 52% of the patients with endometriosis had at least one abnormal result. From normal fertile controls, 10% showed at least one abnormal test result. CONCLUSION: APA, ANA, ATA, ETF, and elevated NK (CD56+) cells are significantly more prevalent among women experiencing reproductive failure than among the control group and normal healthy individuals.

Key words: Autoantibodies, embryotoxins. N K cells, reproductive failure

AZADEH S. KAIDER BRIAN D. KAIDER PATRICK B. JANOWICZ ROUMEN G. ROUSSEV The Center for Human Reproduction, Chicago, IL 60610

Address reprint requests to: Dr. Roumen G. Roussev, The Center for Human Reproduction, 750 North Orleans Street, Chicago, IL 60610.

Submitted June 1, 1998; revised February 24, 1999; accepted March 17, 1999.

INTRODUCTION

It is well known now that the immune system plays an important role in the success of pregnancy. The associations between autoantibodies, such as antiphospholipid (APA),1-2' antinuclear (ANA),1-s.22 and antithyroid anti-

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bodies (ATA)23-26 with reproductive failure have been studied extensively. Circulating APA received the most attention, specifically the lupus anticoagulant (LA) and anticardiolipin antibodies.16-” The association between APA and adverse pregnancy outcome has been reported since 1975.” Although not all studies have found APA to be predictive of fetal loss, the consensus would support the use of APA as a means of identifying women at risk. In 1989, Gleicher et al. found a relationship between autoantibodies not only with recurrent pregnancy loss, but also with unexplained infertility and endometriosis.’ Since then, there have been several studies linking APA with implantation and IVF failure. 12.13.IS.27-37

The role of other factors such as the level of cir- xulating natural killer (NK) (CD56 + ) ~ e l l s ~ ~ - ~ ~ and embryotoxic factors (ETF)5.4S-53 in reproductive failure has also been found important. The percentage of circulating CD56+ of total peripheral blood lymphocytes has been shown to correlate with pregnancy outcome. Elevated levels of circulating CD56+ cells have been reported in women experiencing recurrent pregnancy loss38-42.44 and unexplained infer ti lit^.^.^^ ETF have been identified in sera of women with recurrent pregnancy ~ O S S , ~ ~ ~ ~ ~ ~ ’

endometr io~is ,~~ unexplained infertility, and implantation failure after IVF/ET.5,50,5’ Thus, all of the immunologic factors associated with the various reproductive problems have been reported in different patient populations. The present study was performed to determine the prevalence of the previously reported immunologic markers where all tests (as a panel) were performed on each patient experiencing reproductive failure with the following diagnoses: recurrent pregnancy loss, unexplained infertility, failure after IVF/ET, endometriosis, and ovarian dysfunction.

MATERIALS AND METHODS

From May 1996 to March 1998, the following panel of tests was performed on 591 patients: APA, ANA, ATA, LA, embyrotoxicity assay (ETA), and reproductive immunophenotype. The tested patients were divided by diagnosis into the following subclasses of reproductive failure: recurrent pregnancy loss (iz = 302), IVF failure (IVFf, 11 = 122), unexplained infertility (n = 97), ovarian dysfunction (M = 47), and endometriosis (12 = 23). Recurrent pregnancy loss was diagnosed for patients experiencing three or more consecutive spontaneous abortions. IVF failure was defined as failure of implantation after transfer of at least eight “good” (regular blastomeres and

no/or minor fragments) embryos. A diagnosis of unexplained infertility was given to patients with at least 1 year of unsuccessful attempts at pregnancy, with normal day 3 serum follicle stimulating hormone (FSH) concentrations, normal hystosonosalp- ingogram, normal semen analysis, and/or normal response to medications, if applicable. Endometriosis was diagnosed by laparoscopy and stage according to the American Fertility Society Revised Classifica- t i ~ n . ~ ~ Ovarian dysfunction was diagnosed for patients experiencing irregular menstrual cycles, amenorrhea, oligomenorrhea, polycystic ovarian syndrome, and/or day 3 FSH concentrations of 2 12 mIU/mL. All tests as a panel were performed on 20 normal healthy fertile female individuals as controls (FF controls). Each one of the tests has been performed on at least 100 normal healthy individuals to establish the thresholds for positivity and prevalence of abnormality among general population (GP controls).

Ant iphospliolipid Antibodies APA (immunoglobulin [Ig] A, IgG, IgM) against cardiolipin (CL), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylcholine (PC), and phosphatidylserine (PS) (Sigma, St. Louis, MO, USA) were detected using enzyme-linked immunosor- bent assay (ELISA) method, as previously described,16 with minor modifications. Each phospholipid was dis- solved in methano1:chloroform (3:1, 30 pL of 50 pgl mL final concentration were applied to microtiter plates; DYNATEK Immulon-1, Chantilly, VA, USA). All plates were dried in an 0,-free chamber with N, flow overnight at 4”C, then washed three times with phosphate-buffered saline (PBS). Plates were blocked to eliminate non-specific binding using lo’% adult bovine serum (ABS) for 1 hr at room temperature. After washing three times with PBS, 50 pL of patient sera, negative, and positive controls (diluted 1:lOO in 10% ABS) were applied to plates in triplicate and incubated for 1 hr at room temperature. After washing three times with PBS, 50 pL of alkaline phosphatase conjugated goat anti-human IgA, IgG, or IgM (Sigma, St. Louis, MO, USA) antibodies (diluted 1:lOOO in 1% ABS) were added to each well and incubated for 1 hr at room temperature. The plates were washed again and developed with 50 pL p-nitro- phenyl phosphate in diethanolamine buffer (Sigma, St. Louis, MO, USA) incubated for 1 hr in the dark at 37°C. The reaction was stopped by 70 pL of 3 M NaOH, and the optical density was measured using Bio-Tek EL 800 ELISA reader (405 nm filter). Positive thresholds for each phospholipid in each isotype were established based on results from 105 normal healthy

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IMMUNODIAGNOSTIC EVALUATION IN WOMEN WITH REPRODUCTIVE FAILURE / 337

individuals. The prevalence of APA among G P controls was 6.7%. IgA, IgG, and IgM calibrators (Louisville APL Diagnostic, Inc., Louisville, KY, USA) with known amounts of IgA, IgG, and IgM antibodies against cardiolipin were used to standardize the positive and negative controls.

Antinuclear Antibodies ANA were detected by Theratest ANA ELISA kit (DiaSorin Corporation, Stillwater, MN, USA). Pa- tient sera were evaluated for antibodies against single- stranded DNA (ssDNA), double-stranded DNA (dsDNA), Smith antigen (Sm), ribonuclear protein (RNP), Sjogren's antigen A (SSA), Sjogren's antigen B (SSB), histone, and scleroderma-70 (Scl-70). The optical density was obtained using Bio-Tek EL 800 ELISA reader with the absorbence measured at 450 nm and reference at 630 nm. Negative and positive controls, as well as calibrators for standardizing the assay, were provided in each ANA ELISA kit and run in each assay. In addition, 112 samples from normal healthy women were run as controls, and had an antibody prevalence of 7.7%.

An tithyro id (Thyroglobulin and Tlzyro id Microsomal) Antibodies Serodia" gel-agglutination assays (Fujirebio, Inc., Japan), distributed by Bayer Corp. (Elkhart, IN), were used to test the presence of thyroglobulin and thyroid microsomal antibodies. These semiquantita- tive assays were performed using microtitration tech- nique. Results were obtained by evaluating the settling patterns of colored gelatin particles presensitized with thyroglobulin or thyroid microsomal antigens. Goat anti-serum containing thyroglobulin or thyroid micro- soma1 antibodies (Serodia") was used as the reactive control for these tests. The positive threshold was established by manufacturer as greater than a titer of 1:300, based on published data of 2- 17% prevalence of ATA in the normal healthy non-pregnant population. This range was validated in our laboratory based on 109 healthy non-pregnant women, with a total ATA prevalence of 12.8'%1.

Lupus An ticougulun t Patient samples were evaluated for presence of LA using DVVTest '' and DVVConfirm" (American Diag- nostica, Inc., Greenwich, CT, USA), in which coagulation was measured in seconds by MLA Electra"800 automatic coagulation timer. The ratio of DVVTest '' to DVVConfirm" clotting times for each patient sample was calculated and evaluated. The presence of circulating lupus-like anticoagulant was defined at a ratio result > 1.2. Established normal and abnormal

controls (American Diagnostica, dVVtroll") were used for each test. Prevalence of LA in 100 normal healthy women was determined to be 0%

Embryotoxicity Assay A mouse embryo culture assay, validated by Roussev et al.,5' was used to ascertain the presence of circulating embryotoxin factors for all test subjects. Patient sera were collected, heat inactivated (56"C, 30 min), and frozen ( - 30°C) in aliquots until use. Heat inactivated (56°C) fetal bovine serum (FBS) was used in each assay as control serum known to support mouse embryo growth. Mice (B6C3F1, female and male), 2-4 months of age, housed in 14 hr lighti10 hr dark cycle, were utilized. Female mice were superovulated by 10 IU (i.p.) pregnant mare serum gonadotropin (PMSG) (Sigma, St. Louis, MO, USA), followed in 48 hr by 10 IU (i.p.) human chorionic gonadotropin (hCG). Immediately after hCG injection, each female was mated with a single male. The following morning, the presence of a vaginal plug was used as an indica- tion of successful copulation. Pregnant mice were placed in a separate cage prior to sacrifice 48 hr after hCG. Fallopian tubes were obtained by sharp dissec- tion. Two-cell embryos were collected from the fallopian tubes and transferred into embryo culture dishes containing human tuba1 fluid (HTF) medium supplemented with 10% (v/v) of tested or control sera and incubated in 5% CO, with balanced nitrogen at 37°C. Each serum sample was incubated with a mini- mum of 15 embryos from three different mice. After 72 hr, the stage of development and frequency of atretic embryos were evaluated. Prevalence of ETF in the normal healthy population was calculated as 2% (n = 99).

Reproductive Inununophenotype Lymphocyte immunophenotyping was performed on whole blood by two-color immunofluorescent labeling with Fluorescein Isothiocyanate and Phos- phatidylethanolamine-labeled antibodies against CD3, CD19, CD4, CD8, and CD56 (Becton-Dickinson, San Jose, CA, USA), as well as CD16 (Dako Corp., Carpinteria, CA, USA). Purity and recovery of cells were controlled by CD45 and CD14 antibodies (Dako). Red blood cells were lysed using Becton-Dick- inson FACS lysing solution. The remaining white blood cells were washed in PBS, then acquired and analyzed on Becton-Dickinson FACSCalibur flow cytometer using CellQuest software. Fluorescent-labeled beads (CaliBRITE Beads; Becton-Dickinson, San Jose, CA, USA) were used to standardize the flow cytometer with each assay using FACSComp software. The normal percentage range for each subpopulation was calculated based on Becton-Dickinson range with

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2000 individuals and our own control group of 52 normal healthy donors, with an elevated CD56+ NK cells prevalence of 5.8%.

STATISTICAL ANALYSIS

The significant differences in frequency values between the study and control groups were calculated by two tail .chi-square test, Fisher exact test, and/or Student’s t-test on SPSS software. Bonferonni’s correction was used for multiple comparisons. Significant differences were considered when P<O.O5. The 95% confidence interval was calculated for the histograms using bino- mial distribution.

RESULTS

Overall Of 591 patients with reproductive failure, 75.6% had at least one abnormal test. The most frequent abnormal test result was found to be elevated level of NK (CD56+) cells (37%), followed by ANA (34%), APA (24%), ATA (19%), and ETA (1 1%). From all the tests performed, elevated circulating NK (CD56 + ) cells

x II RPL

IW FAILURE

5 8 S UNEXPL.INF. OVARDYSF.

E Z Z a ENDOMETR. 0 GP CONTROL - FF CONTROL

TESTS

Fig. 1. nostic populations of reproductive failure (‘!A

Prevalence of all six iinmunological tests in different diag- 95‘%) CI).

most commonly prevailed in patients experiencing recurrent pregnancy loss (39.7%), followed by patients with ovarian dysfunction (38.3%), those with unexplained infertility (36.1’%), patients with failed IVF cycles (35.20/0), and those with endometriosis (26.1%) (Fig. 1). ANA was manifest in patients with recurrent pregnancy loss (35. YO) and unexplained infertility (35. 1”/.), followed by patients with IVF failure (33.6%), ovarian dysfunction (31.9%), and endometriosis (21.7%). APA was most common in patients with IVF failure (27.9%), followed by ovarian dysfunction (25.5%), unexplained infertility (23.7%), recurrent pregnancy loss (22.5‘1/0), and patients diagnosed with endometriosis (21.70/). ATA was most commonly present in patients diagnosed with unexplained infertility (38%), followed by those diagnosed with ovarian dysfunction (25.5%), recurrent pregnancy loss (22.8”/0), patients with failed IVF cycles (1 5.6%), and patients with endometriosis (8.7%). ETF were present in 14.9% of patients with ovarian dysfunction and 14.8% of the IVF failure group, followed by 10.3% of patients with unexplained infertility, and 8.9% of recurrent aborters. None of the patients with endometriosis exhibited the presence of embryotoxins in their circulation. LA was detected in less than 3% of all diagnostic groups. Moreover, we did not find a correlation between LA and presence of APA, or vice versa.

Recurrent Pregnancy Loss Of the recurrent pregnancy loss patients, 74.2% had at least one positive abnormal result from all of the tests performed; 31.5% had at least two positive abnormal results; 8.6% had three abnormal results (Table I). At least one positive APA was found in 22.5% of the recurrent aborters, which was significant when compared with controls (GP controls 6.7%), P < 0.0002; FF controls loo/;,, P < 0.05); 11.6% had at least two APA (Table 11). The prevalence of APA isotypes among this group was as follows: 63.9% IgG, followed by 22.9% IgM, and 13.2%) IgA (Fig. 2). The most

TABLE I.

Diagnosis Recurrent Unexplained Ovarian # of pregnancy loss IVF failure infertility dysfunction Endometriosis FF controls positives n = 2241302 n = 85,122 n = 79197

Percent of Abnormal Results from All Six Tests Applied to Patients with Reproductive Failure

n = 35147 n = 12/23 n = 3 2 0

At least 1 74.2* 69.7* 81.4* 74.4* 52.2 * 15 At least 2 31.5 33.6 34.0 34.0 21.7 10 At least 3 8.6 9.8 6.2 8.5 4.3 0

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IMMUNODIAGNOSTIC EVALUATION IN WOMEN WITH REPRODUCTIVE FAILURE / 339

TABLE II. Percent of Abnormal APA and ANA by Diagnosis

Diagnosis Recurrent Unexplained Ovarian ( # of positives) pregnancy loss IVF failure infertility dysfunction Endometriosis GP control FF control

n = 68'302 n = 3411 22 n = 23/97 n= 12/47 n = 5/23 n= 7,105 n = 2/20 APA

At least 1 At least 2 At least 3

22.5* 11.6* 6.0

27.9* 14.8* 9.0

23.7* 13.4* 2.1

25.5* 17.0* 8.5

21.7* 21.7* 17.4

6.7 2.9 0

10 0 0

n = 138:302 n = 52;122 n = 45/97 n = 191'47 n = 8/23 n=2:112 n = 2/20 ANA

At least 1 At least 2 At least 3

35.1 * 9.1 1.7

33.e 8.2 0.8

35.1* 8.3 3.1

31.9* 8.5 0

21.7* 8.7 4.3

1.8 0 0

10 0 0

A P A , tmtiphospholipid ontibotiies: A N A , antiiiucleav untihodies; FF, jiwile ,fmiule; GP. generrrl population * P < 0 . 0 5 .

commonly positive phospholipid among the recurrent pregnancy loss group was phosphatidylcholine (29.9'!'0), followed by phosphatidylglycerol (1 8.8'!'0), and phosphatidic acid (14.6%). ANA analysis revealed 35.1% of the recurrent pregnancy loss group with at least one positive ANA (GP controls 1.8%,, P < 0.001; FF controls lo%, P < 0.05) (Table 11), 46.3% of which were against ssDNA, followed by 13.4% Sm antigen (Table 111). Thirty-nine percent of the recurrent pregnancy loss patients had elevated NK (CD56+) cells (GP controls 5.8%, P < 0.03)(Fig. 1); the percentage distribution of positive N K (CD56+) cells is illus- trated in Table IV. More than 17% of the recurrent pregnancy loss group tested positive for ATAs (not significant when compared with controls); the percentage distribution of ATA is demonstrated in Table V. Embryotoxins were present in 8.9% of the recurrent pregnancy loss patients (GP controls 2%J, P < 0.03). Less than 1% of the recurrent pregnancy losers re-

vealed presence of circulating LA (not significant when compared with controls).

IVF Failure Overall, 69.7% of women experiencing IVF failure had at least one abnormal test from all of the tests performed, 33.6% had at least two, and 9.8% had at least three positive abnormal results (Table I). Among the IVF failure group, 27.9%) tested positive for at least one APA antibody (controls 6.7%1, P = O.OOOOl), and 14.8'1/0 had at least two positive APA (Table 11). The predominant APA isotype among this group was IgG (60'%), followed by IgM (26.2%), and IgA (13.8%) (Fig. 3). The most commonly positive phospholipid among this group was found to be phosphatidylcholine (24.6%), followed by phosphatidylglycerol (18.5%), and phosphatidic acid (15.4%). Approxi- mately 34% of the IVF failure group displayed at least one positive ANA (GP controls l.8'1/0, P < 0.0001; FF controls lo%, P<O.O5); 8% displayed at least two positive ANA (Table 11). Of the specific ANA, ssDNA antigen was most prevalent (38.5%) among this group, followed by Sm antigen (1 5.4%) (Table 111). Thirty- four percent of the IVF failure group revealed elevation of circulating N K (CD56+) cells (GP controls 5.8'%1, P < 0.001); the percent distribution of CD56+ cells is presented in Table IV. Approximately 15.6'%) of patients with failed IVF cycles had circulating ATA (not significant when compared with controls); the percentage distribution of these antibodies is presented in Table V. ETF were found in 14.8% of the IVF failure population (controls 2%, P = 0.007). Less than 2%) of patients with failed IVF cycles exhibited

% ISOTYPES % SPECIFICITY

501 '"1 50 25

IgA IgG IgM CL PE PI PA PG PC PS 0

Fig. 2. in patients with recurrent pregnancy loss.

Isotype and specificity distribution (% _+ 95% CI) of APA

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TABLE 111. Distribution (%) of Positive ANA by Diagnosis

Recurrent pregnancy Diagnosis loss IVF failure Unexplained infertility Ovarian dysfunction Endometriosis Antigen n = 138 n=52 n = 4 5 n= 19 n = 8

ssDNA dsDNA Sm Sm/RNP SSA SSB Histone Scl-70

63 (46.3) 11 (8.1) 18 (13.2) 10 (7.4) 8 (5.9)

11 (8.1) 8 (5.9) 7 (5.9)

20 (38.5) 25 (55.6) 4 (7.7) 7 (15.6) 8 (15.4) 3 (6.7) 4 (7.7) 2 (4.4) 5 (9.6) 1 (2.2) 3 (5.8) 1 (2.2) 4 (7.7) 2 (4.4) 4 (7.7) 4 (8.9)

10 (52.6) 1 (5.3) 1 (5.3) 0 (0)

0 (0) 1 (5.3) 4 (21.0)

2 (10.5)

4 (40.0) 1 (10.0) 1 (10.0) 0 (0) 1 (10.0) 0 (0) 2 (20.0) 1 (10.0)

presence of circulating LA (not significant when compared with controls).

Unexplained Infertility Approximately 8 1% of patients diagnosed with unexplained infertility had at least one positive abnormal result from all of the tests performed; 34% had at least two, and 6% had at least three different abnormal results (Table I). Of this group, 23.7% had at least one positive APA (GP controls 6.7%, P < 0.0004; FF controls lo%, P < 0.05); 13.4% had at least two positive APA (Table 11). The most common APA isotype among the unexplained infertility group was IgG (38.5%1), followed by IgA (33.3%), and IgM (28.2%) (Fig. 4). The most specifically reactive phospholipid among this group was phosphatidylglycerol (25.6'%~), followed by phosphatidic acid (23.1%), and phosphatidylcholine (20.5'%). ANA analysis revealed 35.1% positive rate of at least one ANA (GP controls 1.8%, P < 0.0001; FF controls lo"/;,, P < 0.05) (Table II), among which antibodies against ssDNA antigen were most predominant (55.6'%1), followed by 15.6'% against dsDNA (Table 111). Thirty-five percent of the patients with unexplained infertility revealed elevations in cir-

culating NK (CD56+) cells (GP controls 5.8'%1, P < 0.002) (percent CD56 + distribution in Table IV), followed by 30.9% with ATA (GP controls 12.8'%~, P < 0.0004) (percent ATA distribution in Table V). Nearly 10.3% of the infertile patients had circulating embryotoxins (GP controls 2% P<O.O2). None of these patients displayed the presence of circulating LA (not significant when compared with controls).

Ovarian Dysfunction Approximately 74% of the patients diagnosed with ovarian dysfunction had at least one abnormal result, 34% had at least two, and 8.5% had at least three abnormal results from all the tests performed (Table I). Of these patients, 25.5% had at least one positive APA (GP controls 6.7'%, P < 0.002); 17% had at least two positive APA (Table 11). The most common APA isotype among the ovarian dysfunction group was IgM (46.7%), followed by IgG (43.3'%1), and IgA (10%) (Fig. 5 ) . The most specifically reactive phospholipids among this group were phosphatidylcholine and phosphatidylglycerol (23.3'%,, respectively) (Fig. 5) . Anti- nuclear antibody analysis revealed 31 .!% positive rate of at least one ANA (GP controls 1.8'%1, P < 0.000

TABLE IV. Distribution (%) of Elevated Natural Killer (CD56) Cells by Diagnosis

Recurrent Unexplained Ovarian Diagnosis pregnancy loss IVF failure infertility dysfunction Endometriosis GP control Phenotype n= 120 n = 4 3 n = 3 5 n= 18 n = 6 n = 2

CD 56+/CD16+ 104 (86.7) 38 (88.4) 32 (91.4) 17 (94.4) 6 (100.0) 2 (100.0) CD56+ CD16- 16 (13.3) 5 (11.6) 3 (8.6) 1 (5.6) 0 (0) 0 (0)

IMMUNODIAGNOSTIC EVALUATION IN WOMEN WITH REPRODUCTIVE FAILURE i 341

TABLE V. Distribution (%) of Positive ATA by Diagnosis

Diagnosis Recurrent Unexplained Ovarian

Antigen n = 6 9 n= 19 n = 3 7 n= 12 n = 2 n= 16 pregnancy loss IVF failure infertility dysfunction Endometriosis GP control

Anti- 22 (31.9) 3 (13.6) 10 (27.0) 4 (33.3) 0 (0) 2 (12.5)

Anti- 47 (68.1) 19 (86.4) 27 (73.0) 8 (66.7) 2 (100.0) 14 (87.5) Thyroglobulin

Microsomal

A TA, rmtitliyoid tintibodies

% ISOTYPE % SPECIFICITY had at least two, and 4.3% had at least three abnor- ma1 results from all of the tests performed (Table I). Among the endometriosis group, 21.7% tested positive for at least one and two APA antibodies (GP controls 6.7%, P<O.O4; not significant to FF controls), 17.2% had at least three positive APA (Table 11). The predominant APA isotype among this group

IgA IgG IgM cL PE PG pc ps was IgM (63.6%), followed by IgG (36.4%); no positive IgA antibodies were found among the endometriosis patients (Fig. 6). The most commonly positive phospholipids among this group were found to be phosphatidylglycerol, phosphatidylserine, and

75 30

50 20

25 10

0

F/g .? in patients with IVF failure

Isotype and specificity distribution ('% i 95'5) CI) of APA

(Table II), among which antibodies against ssDNA antigen were most predominant (52.6%), followed by 21% against Scl-70 antigen (Table 111). Over 38% of the patients with ovarian dysfunction revealed elevations in circulating NK (CD56+) cells (GP controls 5.8"/0, P < 0.002) (CD56 + percentage distribution shown in Table IV), followed by 19%) with ATA (not significant when compared with controls) (ATA percentage distribution in Table V). Approximately 15% of the ovarian dysfunction group had circulating embryotoxins (GP controls 2'%, P < 0.005). Only 2.1% of these patients had circulating LA (not significant when compared with controls).

Endoinetviosis Fifty-two percent of the patients diagnosed with endometriosis had at least one abnormal result. 21.7%

% I S O W E % SPEClFlCrPl

501 T

phosphatidyl-inositol (22.796, respectively), followed by phosphatidic acid (18.2%) (Fig. 6). Approximately 22Y0 percent of the endometriosis group displayed at least one positive ANA (GP controls 1.8Y0, P < 0.001); 8.7% displayed at least two positive ANA (Table 11). Of the specific ANAs, ssDNA antigen was most prevalent (40%) among this group, followed by histone antigens (20%)) (Table 111). Twenty-six percent of the endometriosis group revealed elevation of circulating NK (CD56 +) cells (GP controls 5.8'3'0, P < 0.05) (CD56 + percentage distribution presented in Table IV); 8.7%) of the endometriosis patients had ATA in their circulation (not significant when compared with controls) (percent ATA distribution shown in Table V). None of the patients diagnosed with endometriosis exhibited presence of circulating embryotoxins or LA (not significant when compared with controls).

% SPECIFICITY % ISOTWES

301 T T

50 20

25 10

0 0

20 25

10

CL PE PI PA PG PC PS 0 0

Fig. 4. in patients with unexplained infertility.

Isotype and specificity distribution (Y & 95% CI) of APA Fi<g. 5. i n patients with ovarian dysfunction.

Isotype and specificity distribution ('% i 95% CI) of APA


342 1 WIDER ETAL.

yo SPECIFICKY % ISOTYPES

757

20 50

25 10

IgA IgG IgM CL PE PI PA PG PC PS 0

Fig. 6. in patients with endometriosis.

Isotype and specificity distribution (‘A, 95‘% CI) of APA

DISCUSSION

Since women experiencing reproductive failure are a heterogeneous population, specific markers are neces- sary to identify if the problems are of an immunological nature and who will respond to various treatment options. The results of this investigation support previous reports of an association between reproductive failure and abnormal immunological test results including APA,‘-”. ANA,1-5.”. ATA,23-26. and CD56 +

~ e l l s . ’ ~ - ~ ~ The association of abnormal immunological test results with the occurrence of reproductive failure is at present still largely circ~mstantial .~’-~’ The mechanisms by which these markers interfere with reproduction are currently being studied. The advantage of the present study is that all tests were performed on all patients rather than the prevalence of each abnormal immunological test being determined in different patient populations. Among women experiencing reproductive failure, elevated circulating CD56 + cells was the abnormal test most commonly observed followed by ANA, APA, and ATA. The presence of circulating embryotoxins and LA occurred infrequently.

CD56+ cells were most frequently elevated among women experiencing recurrent pregnancy loss. The prevalence of increased circulating CD56 + cells of 39%) in women with recurrent pregnancy loss in the current study compares with frequencies of 18-52”/0 reported in previous s t ~ d i e s . ~ . ~ ~ . ~ ~ Similarly, the rate of increased CD56 + cells among infertile women, including those undergoing treatment with IVF/ET, of 35% compares with previous reports of 3 9 ‘ % ~ ~ ~ ) , ~ ~

The prevalence of elevated autoantibodies including ANA, APA, and ATA are similar to the frequencies

among women with a history of reproductive problems including recurrent pregnancy loss and infertility. ATA were most prevalent in women with unexplained infertility (Fig. 1).

Autoantibodies have recently been shown to have a direct effect on target^.^^^'^^^^ Further studies have

previously cited in the literature’ ~ 6.9 ~ 16.22.25.84.85.87.88

suggested a spectrum of autoantibody specific-

effectss5 APA have been shown to play direct roles in the pathophysiology of thrombosis,’s interference with i m p l a n t a t i ~ n , ~ ~ . ~ ~ and embryot~xicity.~~.” The mechanism of autoantibody mediated thrombosis has been divided into two general types: 1) autoantibody interference with hemostatic reactions occurring on mem- brane surfaces, and 2) autoantibody engagement of antigens on cell surfaces leading to transduction of a signal altered cell a ~ t i v i t y . ~ ~ . ~ ~ An example of interference with hemostatic reactions on cell membranes is the recent observations that APA inhibit the binding of annexin V to cultured endothelial cells and tro- ph~blasts .~’ When annexin V binds to surfaces it acts as an anticoagulant by preventing the formation of prothrombinase complex, thus inhibiting procoagu- lant activity.58 This action on placental endothelial cells would result in placental insufficiency and subsequent pregnancy wastage. Rote et al.” have shown that APA can remove annexin V from the trophoblastic surface and increase binding to prothrombin. In uitvo studies have shown that direct binding of APA to trophoblast cells inhibit differentiation of cytotro- phoblasts to syncytiotrophoblasts, invasion of trophoblasts, and inhibition of hCG production by trophoblasts.” The clinical manifestations of these actions would include unexplained infertility or failed or defective implantation. In ciuo studies have shown an effect of APA on both the uterus and preem- b r y ~ . ~ ’ . ~ ’ However, the major effect contributing to decreasing implantation rates appeared to be on the preembryo.” APA and ANA, but not ATA, have also been shown to have a direct effect on the preembryo in vit r .~ .~”” It is not clear whether or not ANA works by the same mechanisms as APA.

The frequent association of the presence of elevated APA and ANA in the same individual can be explained by the recently proposed hypothesis that nonorgan specific autoimmune diseases may be sec- ondary to some basic cellular abnormality, such as increased apoptosis.61 Recent reports indicated that most of the prominent ANA (DNA, histones, Sm, SS-A, SS-B, and RNP) are clustered in two populations of surface structures on apoptotic cells. It has been postulated that these surface blebs of apoptotic cells as well as the phospholipids exposed on the outside of these blebs are important immunogenic particles,6’ leading to enhanced or aberrant expression of APA and ANA.6’ The prevalence of ANA (and APA) among women with endometriosis would suggest the endometriosis is a disease involving an increase in apoptosis of cells. Potential regulators of apoptosis include F ~ s , ~ ~ perforin,” and tumor necrosis factor (TNF)y.’* Future studies involving Fas,

ities,55.S7.69.92 i.e., ’ different antibodies have different

0 MUNKSGAARD. COPENHAGEN

I M M UNODIAGNOSTIC EVALUATION IN WOMEN WITH REPRODUCTIVE FAILURE / 343

perforin, and TNFw. in women with endometriosis will help in the understanding of the role of these com- pounds in the pathogenesis of endometriosis and infertility associated with endometriosis.

In organ-specific disease, such as thyroid disease, the pathogenic autoantibodies can be identified (e.g., antithyroglobulin and antithyroid peroxidase) as well as autoreactive T cells.64 A significant increase in the endometrial T-cell population has been observed in women with ATA compared to controls with no ATA.” Moreover, human leukocyte antigens (HLA)- DR expression increased and cytokine production showed lower interleukin (1L)-4 and IL-10 and higher interferon (1FN)-y compared to controls.” These observations indicate that there are activated T cells in the uteri of women with ATA and the activated T cells secrete cytokines that do not promote successful pregnancy. Thus, the presence of ATA may represent and epiphenomenon representing a marker of an un- derlying T-cell dysfunction that directly affects i m p l a n t a t i ~ n . ~ ~ , ~ ’

In the endometrium, the major sources of cytokines are not only T cells, but also N K (CD56+) cells.6’ CD56+ 16- cells present within the decidua at the site of implantation have been associated with successful pregnancy o u t ~ o m e , ~ ~ , ~ ~ and a deficiency of these cells have been observed in placental bed biopsy spec- imens from women with incipient r n i ~ c a r r i a g e s . ~ ~ - ~ ~ These decidual CD56+ cells produce a variety of cytokines that may serve as growth factors during pregnancy7’ and have been proposed to act as suppressor cells by secreting a transforming growth factor (TGF) beta-2 set of molecules that inhibit activation of NK to LAK cells potentially able to kill trophoblastic cells and cause pregnancy loss.” In addition to a role in local suppression at the level of the decidua, CD56+ cells in circulation have been shown to predict pregnancy o u t c ~ m e . ’ ~ - ~ ~ Elevated percent- ages of circulating CD56+ cells predict loss of a karyotypically normal pregnan~y.~’ Cells make cytokines. Some cytokines enhance pregnancy growth and development and some are embryo to xi^.^" Circulating ETF have been associated with recurrent pregnancy loss,5,45-50.S8 unexplained infer ti lit^,^.^^ IVF f a i l ~ r e , ~ ” ~ ~ and infertility associated with endometri~sis.~’ The nature of the embryotoxins have been reported to be varied. Some studies have suggested that the embryotoxicity is caused by an IgG fraction in while others have shown embryotoxic effects by T-cell cyto- toxins such as I F N - Y . ~ ~

Elevated CD56+ cells were most prevalent among women with recurrent pregnancy loss, IVF failure, and unexplained infertility, followed by APA. CD56 +

and APA have been shown to have a direct effect on the embryo b e f ~ r e , ~ ” ~ ~ . ~ ~ d ~ r i n g , ~ ’ . ~ ’ and after’-” im-

plantation. Endothelial cells have also been demonstrated to be a target of cytokines from N K cells and m o n ~ c y t e s , ~ ~ as well as APA.’* Endothelial cell dam- age (and subsequent thrombosis) of placental or fetal stem vessels would affect the pregnancy at a later stage. Thus, the clinical presentation is dependent upon the stage of pregnancy that CD56+ cells and APA exert their effect.

While the mechanisms by which the abnormal immunologic test results cause reproductive failure are being studied, these results can serve as markers, which may assist in selection of treatment for a partic- ular reproductive failure.93 Heparin and aspirin have been used to successfully treat women with elevated APA and recurrent pregnancy 1 0 ~ s . ’ ~ Intravenous immunoglobulin has been reported to suppress elevated levels of circulating CD56+ cellsso and has been used successfully to treat women with elevated CD56 +

experiencing IVF failures’ and recurrent pregnancy loss.*’ It is hoped that further elucidation of the mechanisms involved in abnormal immunologic tests and reproductive failure will enhance the selection of treatment options for reproductive failure.

Acknowledgments We thank Dr. Carolyn B. Coulam for valuable discus- sions and suggestions.

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Immunodiagnostic Evaluation in Women with Reproductive Failure