thyroid autoimmunity and its association with cellular and humoral immunity in women with...

Thyroid Autoimmunity and its Association with Cellular andHumoral Immunity in Women with Reproductive FailuresNa Young Kim1, Hye Jin Cho1, Heun Yun Kim1, Kwang Moon Yang1,2, Hyun Kyong Ahn1,2, SimonThornton3, Joon Cheol Park1,4, Kenneth Beaman5, Alice Gilman-Sachs5, Joanne Kwak-Kim1,5

1Reproductive Medicine, Department of Obstetrics and Gynecology, The Chicago Medical School at Rosalind Franklin University of Medicine and

Science. North Chicago, IL, USA;2Department of Obstetrics and Gynecology, Cheil General Hospital & Women’s Healthcare Center, Kwandong University, College of Medicine,

Seoul, Korea;3CARE Fertility, Nottingham, UK;4Department of Obstetrics and Gynecology, Dongsan Medical Center, Keimyung University, School of Medicine, Daegu, Korea;5Department of Microbiology and Immunology, The Chicago Medical School at Rosalind Franklin University of Medicine and Science. North

Chicago, IL, USA

Keywords

Cellular immunity, pregnancy, reproductive

failure, thyroid autoimmunity, thyroid function

Correspondence

Joanne Kwak-Kim, Reproductive Medicine,

Department of Obstetrics and Gynecology,

The Chicago Medical School ⁄ Rosalind Franklin

University of Medicine and Science, 830 West

End Ct. Suite 400, Vernon Hills, IL 60061, USA.

E-mail: [email protected]

Submitted May 30, 2010;

accepted July 6, 2010.

Citation

Kim NY, Cho HJ, Kim HY, Yang KM, Ahn HK,

Thornton S, Park JC, Beaman K, Gilman-Sachs

A, Kwak-Kim J. Thyroid autoimmunity and its

association with cellular and humoral

immunity in women with reproductive failures.

Am J Reprod Immunol 2011; 65: 78–87

doi:10.1111/j.1600-0897.2010.00911.x

Problem

Thyroid autoimmunity (TAI), which is T helper (Th)1-cell-mediated

autoimmunity to thyrocytes, is associated with increased risk of miscar-

riages and highly prevalent in women with infertility. We aim at inves-

tigating the prevalence of TAI in women with recurrent spontaneous

abortions (RSA) or unexplained infertility (UI) and its relationship with

cellular and humoral immune abnormalities.

Method of study

Prevalence of antiphospholipid antibodies, anti-nuclear antibody, other

non-organ-specific antibodies (NOSAs; anti-dsDNA, anti-ssDNA, anti-

histone, anti-Scl70), peripheral blood natural killer (NK) cell levels (%)

and cytotoxicity, and CD3+ ⁄ CD4+ Th1 ⁄ Th2 cell ratios were compared in

women with and without TAI. Thyroid functional tests (TFT) were

analyzed in both groups before and after pregnancy.

Results

Tumor necrosis factor-a ⁄ IL-10 expressing CD3+ ⁄ CD4+ cell ratios

(P < 0.05), CD56+ NK cell levels (P < 0.05), the prevalence of anticardi-

olipin antibodies (P < 0.05) and other NOSAs (P < 0.005) were signifi-

cantly higher in women with TAI when compared to women without

TAI. Changes in thyroid-stimulating hormone levels between before and

after pregnancy in women with TAI were significantly higher when

compared to those of women without TAI (P < 0.05).

Conclusion

TAI is associated with impaired cellular and humoral immune responses

in women with RSA or UI. In women with TAI, serial TFT is recom-

mended when pregnancy is established.

ORIGINAL ARTICLE

American Journal of Reproductive Immunology 65 (2011) 78–87

78 ª 2010 John Wiley & Sons A/S

Introduction

As most autoimmune disturbances are activated in

women during their reproductive years, the clinical

impact of autoimmunity on reproductive processes is

important. Thyroid autoimmunity (TAI)—namely

the presence of autoantibodies against thyroid perox-

idase and ⁄ or thyroglobulin—is the most common

autoimmunity affecting 5–20% of normal pregnant

women.1,2 It has been reported that T helper (Th)1-

cell-mediated autoimmune reaction to thyrocytes

results in thyroid dysfunction.3 Women with TAI

may easily develop thyroid dysfunction during

assisted reproductive technology (ART) cycles and

subsequent pregnancy, which leads to infertility

and pregnancy morbidities.4,5 Increased estrogen and

human chorionic gonadotropin levels induce

increased thyroxine binding globulin (TBG), which

in turn reduces circulating free T4 (FT4) and results

in a compensatory increase in serum thyroid-stimu-

lating hormone (TSH). Therefore, euthyroid women

with TAI before pregnancy may develop overt hypo-

thyroidism during ART cycles or pregnancy because

of these hormonal changes.

A significant association between TAI and risk of

reproductive failures, such as miscarriages4,6 and

infertility7,8, has been shown by various studies.

Studies on TAI and recurrent spontaneous abortions

(RSA) are somewhat mixed, although the majority

of the studies have shown an association.9,10 Multi-

ple studies have confirmed that TAI without overt

thyroid dysfunction is associated with a threefold to

fivefold increase in overall miscarriage rate.5,11,12

With regard to infertility, it has been reported that

TAI has strong associations with female factor infer-

tility like endometriosis, ovarian failure or polycystic

ovarian syndrome, although some discrepancies do

exist in other studies.7,8

There are several possible mechanisms that explain

the relationship between TAI and reproductive fail-

ures: (1) TAI induces thyroid dysfunction, which

affects reproductive outcome; (2) anti-thyroid anti-

bodies (ATA) may directly interfere with trophoblast

differentiation and proliferation. In the mice model,

it has been suggested that ATA might exert a direct

pathogenic effect on pregnancy outcome by directly

binding to the placenta, but not directly on the

embryo;13 (3) TAI may induce enhanced immune

response against fetoplacental unit because of fetal

microchimerism;14,15 and (4) TAI itself is thought to

be a possible inducer of T-cell dysfunction and

studies suggested that TAI may cause an alteration of

the endometrium through a general immune dys-

function that affects implantation.1,9 Alternatively,

TAI may represent a marker for a generalized auto-

immune imbalance that is responsible for an

increased risk of reproductive failures rather than the

actual cause of pregnancy losses.16,17 The association

between organ-specific and non-organ-specific au-

toantibodies and reproductive failure has been

reported.11,18–22 This paradigm may explain the con-

flicting data that failed to find a correlation between

TAI itself and pregnancy losses.1,23

Previously, we reported higher ratios of Th1 ⁄ Th2

cytokine-expressing CD3+ ⁄ CD4+ T cells (TNF-a ⁄ IL-

10, INF-c ⁄ IL-10) in peripheral blood in women with

reproductive failures.24 Whether the prevalence of

dominant Th1 immunity is associated with TAI has

not been investigated yet. In this study, we aim at

studying prevalence of TAI, its relation with other

autoimmunity and possible cellular immunological

impacts of TAI in women with RSA or unexplained

infertility (UI). Additionally, clinical manifestations

of TAI on thyroid function during pregnancy were

investigated in women with RSA or UI who received

immunotherapy.

Materials and methods

Study Design

The medical records of women with a history of RSA

or UI who registered at Reproductive Medicine Pro-

gram, the Chicago Medical School at Rosalind Frank-

lin University of Medicine and Science between

January 1, 2004, and December 31, 2008, were seri-

ally reviewed. This study was approved as an exempt

retrospective cohort study by the University Institu-

tional Review Board.

All of the study populations were reviewed for

the presence of anti-thyroglobulin antibody (ATG)

and anti-thyroid peroxidase antibody (TPO). Other

blood measurements were systemically searched

for antiphospholipid antibodies (APA), anti-nuclear

antibody (ANA), other non-organ-specific antibodies

for nuclear components (NOSAs; anti-dsDNA, anti-

ssDNA, anti-histones, anti-Scl70), peripheral blood

CD56+ natural killer (NK) cell levels and cytotoxicity

and the ratios of Th1 cytokines [tumor necrosis

factor (TNF)-a and interferon (IFN)-c] to Th2 cyto-

kine (IL-10) expressing CD3+ ⁄ CD4+ cells ratios.

Searched variables were stored in a secure server,

THYROID AUTOIMMUNITY AND REPRODUCTIVE FAILURES


ª 2010 John Wiley & Sons A/S 79

and statistical analysis was made to compare these

parameters between women with and without TAI.

In addition, subgroup analysis was made for distribu-

tion or prevalence of each variable in women with

RSA or UI. None of the women in study population

were pregnant or on immune-suppressive ⁄ modula-

tory treatments when they had laboratory assays.

A total of 38 women of study groups were found

who achieved pregnancy and completed thyroid

function test (TFT) including TSH, FT4 and free triio-

dothyronine (FT3) before and after pregnancy. To

analyze thyroid hormonal changes between before

and after pregnancy, pregnant women were divided

into four subgroups: Group I, euthyroid women

without TAI (n = 13); Group II, hypothyroid women

without TAI and treated with levothyroxine (L-T4)

(n = 8); Group III, euthyroid women with TAI

(n = 10) and Group IV, hypothyroid women with

TAI and treated with L-T4 (n = 7). Differences in

TSH levels (DTSH) between before (BTSH) and after

pregnancy (PTSH) were calculated in each patient as

follows: DTSH = [PTSH ) BTSH]. DTSH of each

group was compared with the other study groups.

BTSH was tested without any prednisone or intrave-

nous immunoglobulin G infusion (IVIG) treatment.

PTSH was tested with positive pregnancy test. Preg-

nant study groups were treated with prednisone

10 mg ⁄ day, orally on cycle day 6 because of pres-

ence of either ANA and ⁄ or APA. Of the 38 women,

61% (8 of 13) of Group I, 87% (7 of 8) of Group II,

60% (6 of 10) of Group III and 85% (6 of 7) of

Group IV had IVIG treatment 400 mg ⁄ kg intrave-

nously, between cycle day 6–10 of conception cycle

because of elevated peripheral blood CD56+ NK cells

levels (>12%).

A total of eight women with TAI who delivered a

live born infant were found to have TSH and FT4

levels before and during pregnancy. All eight women

were on prednisone 10 mg ⁄ day started on cycle day

6. Four of them were on IVIG treatment on cycle

day 6 and during pregnancy (400 mg ⁄ kg, intrave-

nously every 3 weeks) as well. Prednisone treatment

was tapered off between 12 and 16 weeks of gesta-

tion. IVIG treatment was continued to the third

trimester.

Study Population

A total of 265 women with a history of RSA or UI

comprised the study group. Women with UI

(n = 127) were diagnosed when the results of a

standard infertility evaluation, such as a semen anal-

ysis, assessment of ovulation, a hysterosalpingogram,

cycle day 3 estradiol and FSH levels and, if indicated,

laparoscopy, were normal. All had two or more

IVF ⁄ ET failures with at least two or more good-

quality embryos transferred, and no apparent cause

of IVF ⁄ ET failures has been documented in these

women. Sixty-four were primary infertility and 63

were secondary infertility. Women with male factor

infertility or who used donor oocytes were excluded

from this study.

Women with RSA (n = 138) had a history of two

or more consecutive spontaneous abortions (mean

3.58, range: 2–10) prior to 20 weeks gestation and

had no evidence of anatomical, genetic or infectious

causes for pregnancy losses. Patients who had active

autoimmune disease other than autoimmune thy-

roiditis were excluded from both groups. Forty-nine

women had primary RSA and 89 had secondary

RSA. Obstetrical and infertility histories of women

with and without TAI are listed in Table I.

Laboratory Determinations

TPO and ATG were determined as positive or

negative using a commercial qualitative ELISA kit

Table I Obstetrical and infertility history of women with

recurrent spontaneous abortion (RSA) or unexplained infertility (UI)

TAI positive

mean ± S.D.

(ranges)

TAI negative

mean ± S.D.

(ranges)

RSA

No. 26 112

Gravity 3.73 ± 1.75 (2–8) 4.60 ± 2.22 (2–15)

Parity 0.46 ± 0.58 (0–2) 0.47 ± 0.72 (0–5)

No. of living child 0.38 ± 0.57 (0–2) 0.45 ± 0.69 (0–4)

No. of spontaneous

abortion

2.92 ± 1.16 (2–10) 3.65 ± 1.71 (2–6)

Infertility

No. 28 99

Gravity 0.89 ± 0.91 (0–3) 0.74 ± 0.91 (0–3)

Parity 0.18 ± 0.48 (0–2) 0.24 ± 0.47 (0–2)

No. of living child 0.11 ± 0.41 (0–1) 0.22 ± 0.46 (0–1)

No. of spontaneous

abortion

0.36 ± 0.48 (0–1) 0.29 ± 0.46 (0–1)

Duration of infertility

(year)

3.84 ± 2.83 (1–14) 3.66 ± 2.03 (1–8)

No. of IVF trials 3.00 ± 1.83 (0–9) 2.48 ± 1.79 (0–8)

TAI, thyroid autoimmunity.

KIM ET AL.



(INOVA Diagnostics, Inc., San Diego, CA, USA).

TNF-a ⁄ IL-10 and IFN-c ⁄ IL-10 expressing CD3+ ⁄ CD4+

cells ratios were measured by flow cytometric analy-

sis as previously reported.24 Peripheral blood CD56+

NK cell levels (%) and cytotoxicity (%) were calcu-

lated as stated in our previous report using flow

cytometric analysis.17 Immunophenotype assays

were carried out to measure CD3) ⁄ CD56+ NK cells,

and NK cell cytotoxic activities were determined at

effector-to-target cell (E:T) ratios of 50:1, 25:1 and

12.5:1. The ANA, APA and NOSAs were tested using

in-house qualitative ELISA. The parameters of TFT

including TSH, FT4 and FT3 were measured using

chemiluminescence assay (Ortho clinical diagnostics,

Inc., Raritan, NJ, USA).

Statistical Analysis

All patient data were stored on a secure server, and

statistical analysis was performed using spss 16.0

program (SPSS Inc., Chicago, IL, USA). Prevalence

of autoimmunity in women with and without TAI

were analyzed by means of Pearson’s chi-square or

Fisher’s exact test as indicated, and differences

between mean values were determined by student’s

t-test. Statistical tests were considered significant

whenever P-value was <0.05.

Results

Prevalence of TAI

TAI was present in 20% (54 of 265) of all investi-

gated women and 18.8% (26 of 138) of women with

RSA. In RSA group, 9.4% (13 ⁄ 138) had TPO only,

3.6% (5 ⁄ 138) had ATG only and 5.8% (8 ⁄ 138) had

both TPO and ATG positivity. Twenty-two percent

(22.0%) (28 of 127) of women with UI had TAI. In

UI group, 6.2% (8 ⁄ 127) had TPO only, 7.0%

(9 ⁄ 127) had ATG only and 8.6% (11 ⁄ 127) had both

TPO and ATG positivity. There was no statistical

difference in prevalence of TAI between RSA and

UI groups.

As Table II shows, women with TAI were signifi-

cantly older (36.96 ± 4.46) than women without

TAI (35.17 ± 4.46). Age difference between women

with and without TAI was particularly significant

(38.09 ± 4.64 versus 34.19 ± 3.71, P < 0.005) in

women with primary infertility (n = 64) (Table II).

In women with primary infertility, the prevalence of

TAI was significantly higher in women aged 35 or

older (29.0), when compared to women under

35 years of age (6.2%) (P = 0.017). Mean duration

of infertility was 3.7 ± 2.4 (mean ± S.D.) years in

women without TAI and 4.2 ± 3.3 years in women

with TAI (P = 0.463). Although women with TAI

had longer duration of infertility, the difference was

not statistically significant. The prevalence of APA,

ANA and non-organ-specific autoantibodies in

women 35 years of age or older was not different

from that of women under 35 years of age.

Cellular Immunity and TAI

TNF-a ⁄ IL-10 expressing CD3+ ⁄ CD4+ T cell ratios were

significantly increased in women with TAI

(32.35 ± 11.35) when compared to those of women

without TAI (28.20 ± 9.93) (P < 0.05). Women with

TAI had a higher CD56+ NK cell levels (9.48 ± 6.45%)

when compared to women without TAI (8.03 ±

4.56%) (P < 0.05). However, prevalence of elevated

NK cell levels over 12% was not different between

women with and without TAI. NK cytotoxicities (%)

at E:T ratios of 50:1 (16.78 ± 7.17 versus 15.53 ±

Table II Age distribution of women with recurrent spontaneous abortion (RSA) or unexplained infertility (UI) with or without thyroid

autoimmunity (TAI)

Groups

Women without TAI

average age ± S.D. (years)

Women with TAI

average age ± S.D. (years) P value

Total (n = 265) 35.17 ± 4.46 (n = 211) 36.96 ± 4.46 (n = 54) <0.05

RSA (n = 138) 35.47 ± 4.52 (n = 112) 37.27 ± 4.22 (n = 26) NS

UI (n = 127) 34.83 ± 4.38 (n = 99) 36.68 ± 4.72 (n = 28) <0.05

PIa (n = 64) 34.19 ± 3.71 (n = 52) 38.09 ± 4.64 (n = 11) <0.005

SIb (n = 63) 35.65 ± 4.96 (n = 46) 35.76 ± 4.68 (n = 17) NS

aPrimary infertility; bsecondary infertility.




6.64), 25:1 (11.04 ± 5.40 versus 9.91 ± 5.06) and

12.5:1 (6.63 ± 4.13 versus 5.71 ± 3.99) were not dif-

ferent between women with and without TAI (Fig. 1).

In RSA group, TNF-a ⁄ IL-10 expressing CD3+ ⁄ CD4+

T cell ratios (32.53 ± 11.44) and CD56+ NK cell levels

(10.24 ± 4.25%) were significantly increased in

women with TAI when compared to those of women

without TAI (27.78 ± 10.07, 7.87 ± 4.46%) (P < 0.05

each). However, there was no significant difference

in TNF-a ⁄ IL-10 expressing CD3+ ⁄ CD4+ cell ratios

(32.18 ± 11.4 versus 28.67 ± 9.79) or NK cell levels

(8.77 ± 4.25% versus 8.26 ± 4.46%) in women with

UI.

Autoimmunity and TAI

Prevalence of APA was not different between

women with and without TAI. When autoantibodies

to six different phospholipids were analyzed in each

antibody class, there were no significant differences

in prevalence of IgG, IgA or IgM class APA. When

compared to 17.5% (37 of 211) of women without

TAI (P < 0.05), 27.8% (15 of 54) of women with

TAI were found to be positive for IgG, IgA or IgM

anticardiolipin antibodies . There were no differences

between women with and without TAI in the preva-

lence of ANA. However, other NOSAs were highly

prevalent in women with TAI when compared to

that of women without TAI. Women who had one

or more NOSAs occurred in 35.2% (19 of 54) of

women with TAI when compared to 11.4% (24 of

211) in women without TAI (P < 0.005) (Table III).

Pregnancy and TFT in Women with TAI

TSH levels of pregnant women revealed different pat-

terns depending on whether the patient had TAI or

not. Women without TAI (Group I and II) have

decreased TSH levels and women with TAI (Group III

8090

100

404550 *

304050607080

Percen

tage

TAI (–)

TAI (+)

152025303540

Ratio

TAI (–)

TAI (+)

01020

CD3% CD19% CD19/5% NK% NK 50:1 NK 25:1 NK 12.5:105

105

TNF-α /IL-10 IFN-γ /IL-10

*

(a) (b)

Fig. 1 Cellular immunities in women with reproductive failures (recurrent spontaneous abortion or unexplained infertility) with or without thyroid

autoimmunity (TAI). (a) CD3+ T cells (CD3%), CD19+ B cell (CD19%), CD 19+ ⁄ 5+ B1 cells (CD19 ⁄ 5%), CD56+ NK cells levels (NK%) and NK cytotoxicities

at effector-to-target cell ratios of 50:1 (NK 50:1), 25:1 (NK 25:1) and 12.5:1 (NK 12.5:1) are plotted. Women with TAI has significantly higher CD56+

NK cell levels when compared to those of women without TAI (P < 0.05). (b) TH1 ⁄ TH2 cytokine expressing CD3+ ⁄ CD4+ T cell ratios (TNF-a ⁄ IL-10,

IFN-c ⁄ IL-10), in women with and without TAI. TNF-a ⁄ IL-10 expressing CD3+ ⁄ CD4+ T cell ratios are significantly higher in women with TAI when

compared with women without TAI (P < 0.05). Values are mean ± S.D. (standard deviation). TNF, tumor necrosis factor.

Table III Antiphospholipid antibodies (APA), anti-nuclear

antibody (ANA) and other non-organ-specific autoantibodies

(NOSAs) in women with positive and negative thyroid

autoimmunity (TAI) and a history of recurrent pregnancy losses

(RSA) or unexplained infertility

Women

without TAI

(n = 211)

Women

with TAI

(n = 54) P value

APA (IgG, IgM, IgA)a 43.6% 46.3% NS

Cardiolipin 17.5% 27.8% 0.042

Phosphoethanolamine 14.7% 14.8% NS

Phosphoinositol 15.2% 18.5% NS

Phosphatidic acid 12.8% 13.0% NS

Phosphoglycerol 11.4% 13.0% NS

Phosphoserine 11.4% 14.8% NS

NOSAsb 11.4% 35.2% <0.000

Anti-dsDNA 3.4% 11.5% 0.018

Anti-ssDNA 4.8% 18.0% 0.06

Anti-histone 11.0% 14.3% NS

Anti-Scl70 0.5% 0% NS

ANA 28.0% 29.6% NS

aPrevalence of IgG, IgM or IgA antiphospholipid antibody to any

six phospholipid antigens; bprevalence of autoantibodies to

anti-dsDNA, anti-ssDNA, anti-histone or anti-scl70.

KIM ET AL.



and IV) have increased TSH levels with pregnancy.

DTSHs of Group III and IV (P < 0.05 each) were sig-

nificantly higher than that of Group I (Fig. 2).

When TSH (Fig. 3a,b) levels in women with TAI

were traced during pregnancy, interestingly all four

women with combined prednisone and IVIG treat-

ment (Fig. 3a) had TSH levels <2.3 mIU ⁄ L at 4–7

weeks gestation. Contrarily, women with prednisone

only treatment had TSH levels >2.5 mIU ⁄ L at

4–7 weeks gestation (Fig. 3b). TSH levels were seri-

ally monitored to maintain £2.0 mIU ⁄ L by adjusting

L-T4 doses after the first pregnancy test. In women

with combined prednisone and IVIG treatment,

except one woman at 4–7 weeks gestation, all TSH

levels during pregnancy were <2.0 mIU ⁄ L. In women

with TAI and prednisone treatment group, one of

four still had higher than 2.0 mIU ⁄ L of TSH at

11–16 weeks gestation. FT4 levels are plotted during

pregnancy in women with combined prednisone and

IVIG treatment (Fig. 3c) and prednisone treatment

only (Fig. 3d). All were on L-T4 treatment during

pregnancy and able to maintain normal FT4 levels

during pregnancy regardless of treatment protocol.

Discussion

This study aimed at exploring the implication of TAI,

a common and contentious problem encountered

in human reproduction, particularly in the context

of RSA and UI. With this purpose, we reviewed

immunological markers for cellular and autoimmu-

nity and compared those between women with and

without TAI who had a history of RSA or UI. We

report women with TAI and a history of RSA or UI

are significantly older than women without TAI. In

addition, the prevalence of TAI is significantly higher

in women with primary infertility who are 35 years

of age or older when compared to women who are

under 35 years of age. Our findings are consistent

with the previous studies.1,5,8 The duration of

TAIGroup IVGroup IIIGroup IIGroup I

TSH

(m

IU/L

)

6

5

4

3

2

1

0

–1

–2

–3

–4

BTSH

PTSH

DTSH

**

Fig. 2 Mean thyroid-stimulation hormone (TSH) levels and difference

between pre-pregnancy and the first trimester TSH levels. Group I,

euthyroid women without thyroid autoimmunity (TAI) (n = 13); Group

II, hypothyroid women without TAI and treated with levothyroxine

(L-T4) (n = 8); Group III, euthyroid women with TAI (n = 10) and Group

IV, hypothyroid women with TAI and treated with L-T4 (n = 7). BTSH,

basal TSH before pregnancy; PTSH, TSH after pregnancy; DTSH, differ-

ence between before and after pregnancy TSH. Values were

mean ± standard deviation (S.E.), statistical analysis was made by

student’s t-test, and P < 0.05 was considered to be statistically

significant. *P value <0.05.

0123456

Baseline 4–7 11–16 20–24 30–360123456

Baseline 4–7 11–16 20–24 30–36

0

0.5

1

1.5

2

2.5

Baseline 4–7 11–16 20–24 30–360

0.5

1

1.5

2

2.5

Baseline 4–7 11–16 20–24 30–36

(c)

(a) (b)

(d)

Fig. 3 Thyroid-stimulating hormone (TSH) and

free thyroxine (FT4) during pregnancy in

women with thyroid autoimmunity. (a) TSH

changes in four women with prednisone and

intravenous immunogrobulin (IVIG) treatment;

(b) TSH changes in four women with predni-

son only treatment; (c) FT4 changes in four

women with prednisone and IVIG treatment;

(d) FT4 changes in four women with

prednison only treatment.




infertility in women with thyroid abnormalities and

ovulatory dysfunction was reported to be signifi-

cantly longer than that of the control groups; how-

ever, no significant variation in the duration of

infertility was reported in euthyroid women with

TAI.25 This was consistent with our results. In this

study, majority of women with UI and TAI were

euthyroid (either treated hypothyroidism or without

any treatment), and the duration of infertility was

not different between women with and without TAI.

From these findings, it can be inferred that both

TAI and hypothyroid status may have a role in

infertility.

Previously, an association between recurrent mis-

carriages and autoantibodies to TPO, and the com-

bined panel of TPO, ATG and anti-extractable nuclear

antigens, but not with aPL, has been reported.21 Con-

tradictory to this, an increased frequency of TAI was

reported in antiphospholipid syndrome-recurrent

aborters.11 In infertile women with thyroid autoanti-

bodies, a poor association with non-organ-specific

autoantibodies has been also reported.25 In this study,

the prevalence of NOSAs in women with TAI was

significantly higher than that of women without TAI.

However, the prevalence of APA or ANA was not

different between women with and without TAI. In

this study, the prevalence of autoantibodies was com-

pared in women with and without TAI, allowing the

elimination of possible confounding factors related to

underlying diseases. Therefore, conflicting results of

this study may suggest that previously reported rela-

tion between ATA and other autoantibodies is possi-

bly because of a failure of controlling confounding

factors rather than a true relation or selection bias of

study population.

It has been reported that dominant pro-inflamma-

tory Th1 immune responses are related to RSA or

multiple implantation failures.24,26 In this study, we

found that Th1 ⁄ Th2 cytokine-expressing CD3+ ⁄ CD4+

T cell ratios were significantly higher in women with

TAI when compared to those of women without TAI.

Th1 cytokines such as TNF-a or INF-c can induce

apoptosis of thyrocytes and lead to thyroid failure.

Recently, apoptosis has been reported to play an

important role in autoimmune thyroid diseases.

Increased Th1 cytokines in Hashimoto’s thyroiditis

and Th2 cytokines in Graves’ disease have been

reported.27,28 T regulatory cells are believed to play a

critical role in determining the production of Th1

and Th2 cytokines with unknown mechanism.3 In

our study population, there were no active patients

with Graves’ disease who required medication, and

women who had TAI were either hypothyroid (trea-

ted with L-T4) or euthyroid status. The higher ratios

of TNF-a ⁄ IL-10 expressing CD3+ ⁄ CD4+ T cells in

women with TAI and reproductive failures support

the notion that Th1 immunity and pro-inflammatory

status may induce TAI. In women with reproductive

failures, TAI have further negative implications as

thyroid dysfunction alters endocrine milieu during

an ovulatory cycle and at the time of ovulation.

NK cells are the effectors of the innate immune

response, and elevated NK cell levels and cytotoxic-

ity have been associated with reproductive failures,

such as RSA29 and infertility.30,31 It has been

reported that NK cells play an immunoregulatory

role in the prevention of autoimmune disease.32,33

Studies of NK activity in patients with TAI have

produced widely different results: enhanced,34,35

normal36 or decreased NK activities.37,38 Our study

demonstrated that CD56+ NK cell levels were signifi-

cantly higher in women with TAI than those of

women without TAI, although there were no signifi-

cant differences in NK cytotoxic activity. The dis-

crepancies in these reports may be partly because of

different assay techniques or study populations. As

increased NK cell proportion is known to be an iso-

lated risk factor for reproductive failures, elevated

levels of NK cells in women with TAI may have

further implication in reproductive failure.

TAI seems to affect the capacity of thyroid gland

to enhance hormone production. Chemically,

‘euthyroid’ women, therefore, might experience

hypothyroidism in pregnancy, when additional thy-

roid hormone has to be produced for increased both

TBG and iodine renal clearance. Serum TSH level

has been described as a significant predictor of IVF

failures, and pregnant women with hypothyroidism

have an increased risk of early and late obstetric

complications.39–41 Obstetrical complications are

associated with both overt and also subclinical hypo-

thyroidism, and it has been reported that treatment

with L-T4 greatly reduces the frequency of these

complications.42 It has been proposed to control the

upper limit of normal for serum TSH to a level of

2.0 or 2.5 mIU ⁄ L. However, no consensus has been

reached so far.43 In this study, we were able to con-

trol serum TSH level to be <2.0 mIU ⁄ L prior to the

second trimester in majority of cases.

In this study, hypothyroid women with L-T4

treatment (Groups II and IV) also received

prednisone and ⁄ or IVIG treatment, which suppresses

KIM ET AL.



autoantibody production and inflammatory immune

responses. Previously, Rotondi et al. reported 36.4%

of hypothyroid women who did not have modified

L-T4 levels with pregnancy had TSH levels above

3.5 mU ⁄ mL.44 In this study, 20.0% (3 ⁄ 15) of hypo-

thyroid women (Groups II and IV) with L-T4 treat-

ment had serum TSH levels above 3.5 mU ⁄ mL with

pregnancy. This may suggest prednisone and ⁄ or IVIG

treatment affects thyroid function in women with

TAI. A total of eight women with TAI had consecu-

tive TFT throughout pregnancy. As they were on

prednisone only or prednisone and IVIG treatment

prior to and after pregnancy, the degree of TSH ele-

vation might have been significantly attenuated.

However, it is clear that even with immune-

suppression or modulation treatment, TFT changes

before and after pregnancy were significant in

women with TAI when compared to women without

TAI. Our results confirmed that in pregnant women,

TSH levels were significantly increased after preg-

nancy in women with TAI, and a degree of elevation

was even higher in women who already had overt

hypothyroidism and L-T4 treatment.

In this study, we thoroughly address the immuno-

logical implication of TAI in women with reproduc-

tive failure. We report that TAI is associated with

Th1 immunity and NK cell immunity, and women

with TAI have a tendency to have NOSAs, which is

often associated with subclinical or clinical rheu-

matic diseases. In women with TAI and a history of

reproductive failures, evaluation of other auto-

immune diseases should be considered and serial

monitoring of TFT is needed during pregnancy. It is

getting clear that impact of TAI on reproduction is

largely because of an immune dysregulation rather

than a simple endocrinopathy. Further immuno-

logical evaluation should be considered in women

with TAI and reproductive failures.

References

1 Kutteh WH, Yetman DL, Carr AC, Beck LA, Scott RT

Jr: Increased prevalence of antithyroid antibodies

identified in women with recurrent pregnancy loss

but not in women undergoing assisted reproduction.

Fertil Steril 1999; 71:843–848.

2 Cleary-Goldman J, Malone FD, Lambert-Messerlian

G, Sullivan L, Canick J, Porter TF, Luthy D, Gross S,

Bianchi DW, D’Alton ME: Maternal thyroid

hypofunction and pregnancy outcome. Obstet Gynecol

2008; 112:85–92.

3 Wang SH, Baker JR: The role of apoptosis in thyroid

autoimmunity. Thyroid 2007; 17:975–979.

4 Poppe K, Glinoer D, Tournaye H, Devroey P, van

Steirteghem A, Kaufman L, Velkeniers B: Assisted

reproduction and thyroid autoimmunity: an

unfortunate combination? J Clin Endocrinol Metab

2003; 88:4149–4152.

5 Prummel MF, Wiersinga WM: Thyroid autoimmunity

and miscarriage. Eur J Endocrinol 2004; 150:751–755.

6 Negro R, Formoso G, Coppola L, Presicce G, Mangieri

T, Pezzarossa A, Dazzi D: Euthyroid women with

autoimmune disease undergoing assisted reproduction

technologies: the role of autoimmunity and thyroid

function. J Endocrinol Invest 2007; 30:3–8.

7 Poppe K, Glinoer D, Van Steirteghem A, Tournaye H,

Devroey P, Schiettecatte J, Velkeniers B: Thyroid

dysfunction and autoimmunity in infertile women.

Thyroid 2002; 12:997–1001.

8 Janssen OE, Mehlmauer N, Hahn S, Offner AH,

Gartner R: High prevalence of autoimmune thyroiditis

in patients with polycystic ovary syndrome. Eur J

Endocrinol 2004; 150:363–369.

9 Roberts J, Jenkins C, Wilson R, Pearson C, Franklin

IA, MacLean MA, McKillop JH, Walker JJ: Recurrent

miscarriage is associated with increased numbers of

CD5 ⁄ 20 positive lymphocytes and an increased

incidence of thyroid antibodies. Eur J Endocrinol 1996;

134:84–86.

10 Bellver J, Soares SR, Alvarez C, Munoz E, Ramirez A,

Rubio C, Serra V, Remohi J, Pellicer A: The role of

thrombophilia and thyroid autoimmunity in

unexplained infertility, implantation failure and

recurrent spontaneous abortion. Hum Reprod 2008;

23:278–284.

11 De Carolis C, Greco E, Guarino MD, Perricone C, Dal

Lago A, Giacomelli R, Fontana L, Perricone R: Anti-

thyroid antibodies and antiphospholipid syndrome:

evidence of reduced fecundity and of poor pregnancy

outcome in recurrent spontaneous aborters. Am J

Reprod Immunol 2004; 52:263–266.

12 Prummel MF, Strieder T, Wiersinga WM: The

environment and autoimmune thyroid diseases. Eur J

Endocrinol 2004; 150:605–618.

13 Matalon ST, Blank M, Levy Y, Carp HJ, Arad A,

Burek L, Grunebaum E, Sherer Y, Ornoy A, Refetoff

S, Weiss RE, Rose NR, Shoenfeld Y: The pathogenic

role of anti-thyroglobulin antibody on pregnancy:

evidence from an active immunization model in mice.

Hum Reprod 2003; 18:1094–1099.

14 Ando T, Imaizumi M, Graves PN, Unger P, Davies TF:

Intrathyroidal fetal microchimerism in Graves’

disease. J Clin Endocrinol Metab 2002; 87:3315–3320.




15 Ando T, Davies TF: Self-recognition and the role of

fetal microchimerism. Best Pract Res Clin Endocrinol

Metab 2004; 18:197–211.

16 Gianoukakis AG, Khadavi N, Smith TJ: Cytokines,

Graves’ disease, and thyroid-associated

ophthalmopathy. Thyroid 2008; 18:953–958.

17 Gilman-Sachs A, DuChateau BK, Aslakson CJ,

Wohlgemuth GP, Kwak JY, Beer AE, Beaman KD:

Natural killer (NK) cell subsets and NK cell

cytotoxicity in women with histories of recurrent

spontaneous abortions. Am J Reprod Immunol 1999;

41:99–105.

18 Van Voorhis BJ, Stovall DW: Autoantibodies and

infertility: a review of the literature. J Reprod Immunol

1997; 33:239–256.

19 Mecacci F, Parretti E, Cioni R, Lucchetti R, Magrini A,

La Torre P, Mignosa M, Acanfora L, Mello G: Thyroid

autoimmunity and its association with non-organ-

specific antibodies and subclinical alterations of

thyroid function in women with a history of

pregnancy loss or preeclampsia. J Reprod Immunol

2000; 46:39–50.

20 Sherer Y, Tartakover-Matalon S, Blank M, Matsuura

E, Shoenfeldlt Y: Multiple autoantibodies associated

with autoimmune reproductive failure. J Assist Reprod

Genet 2003; 20:53–57.

21 Marai I, Carp H, Shai S, Shabo R, Fishman G,

Shoenfeld Y: Autoantibody panel screening in

recurrent miscarriages. Am J Reprod Immunol 2004;

51:235–240.

22 Shoenfeld Y, Carp HJ, Molina V, Blank M, Cervera R,

Balasch J, Tincani A, Faden D, Lojacono A, Doria A,

Konova E, Meroni PL: Autoantibodies and prediction

of reproductive failure. Am J Reprod Immunol 2006;

56:337–344.

23 Rushworth FH, Backos M, Rai R, Chilcott IT, Baxter

N, Regan L: Prospective pregnancy outcome in

untreated recurrent miscarriers with thyroid

autoantibodies. Hum Reprod 2000; 15:1637–1639.

24 Kwak-Kim JY, Chung-Bang HS, Ng SC, Ntrivalas EI,

Mangubat CP, Beaman KD, Beer AE, Gilman-Sachs

A: Increased T helper 1 cytokine responses by

circulating T cells are present in women with

recurrent pregnancy losses and in infertile women

with multiple implantation failures after IVF. Hum

Reprod 2003; 18:767–773.

25 Grassi G, Balsamo A, Ansaldi C, Balbo A, Massobrio

M, Benedetto C: Thyroid autoimmunity and

infertility. Gynecol Endocrinol 2001; 15:389–396.

26 Raghupathy R, Makhseed M, Azizieh F, Omu A,

Gupta M, Farhat R: Cytokine production by maternal

lymphocytes during normal human pregnancy and in

unexplained recurrent spontaneous abortion. Hum

Reprod 2000; 15:713–718.

27 Heuer M, Aust G, Ode-Hakim S, Scherbaum WA:

Different cytokine mRNA profiles in Graves’ disease,

Hashimoto’s thyroiditis, and nonautoimmune thyroid

disorders determined by quantitative reverse

transcriptase polymerase chain reaction (RT-PCR).

Thyroid 1996; 6:97–106.

28 Stassi G, De Maria R: Autoimmune thyroid disease:

new models of cell death in autoimmunity. Nat Rev

Immunol 2002; 2:195–204.

29 Kwak JY, Beaman KD, Gilman-Sachs A, Ruiz JE,

Schewitz D, Beer AE: Up-regulated expression of

CD56+, CD56+ ⁄ CD16+, and CD19+ cells in peripheral

blood lymphocytes in pregnant women with

recurrent pregnancy losses. Am J Reprod Immunol

1995; 34:93–99.

30 Beer AE, Kwak JY, Ruiz JE: Immunophenotypic

profiles of peripheral blood lymphocytes in women

with recurrent pregnancy losses and in infertile

women with multiple failed in vitro fertilization

cycles. Am J Reprod Immunol 1996; 35:376–382.

31 Coulam CB, Roussev RG: Correlation of NK cell

activation and inhibition markers with NK cytoxicity

among women experiencing immunologic

implantation failure after in vitro fertilization and

embryo transfer. J Assist Reprod Genet 2003; 20:58–62.

32 Baxter AG, Smyth MJ: The role of NK cells in

autoimmune disease. Autoimmunity 2002; 35:1–14.

33 Lunemann A, Lunemann JD, Munz C: Regulatory NK

cell functions in inflammation and autoimmunity.

Mol Med 2009; 15:352–358.

34 Calder EA, Irvine WJ, Davidson NM, Wu F: T, B and

K cells in autoimmune thyroid disease. Clin Exp

Immunol 1976; 25:17–22.

35 Hidaka Y, Amino N, Iwatani Y, Kaneda T, Nasu M,

Mitsuda N, Tanizawa O, Miyai K: Increase in

peripheral natural killer cell activity in patients with

autoimmune thyroid disease. Autoimmunity 1992;

11:239–246.

36 Pedersen BK, Feldt-Rasmussen U, Bech K, Perrild H,

Klarlund K, Hoier-Madsen M: Characterization of the

natural killer cell activity in Hashimoto’s and Graves’

diseases. Allergy 1989; 44:477–481.

37 Wang PW, Luo SF, Huang BY, Lin JD, Huang MJ:

Depressed natural killer activity in Graves’ disease

and during antithyroid medication. Clin Endocrinol

(Oxf) 1988; 28:205–214.

38 Marazuela M, Vargas JA, Alvarez-Mon M, Albarran F,

Lucas T, Durantez A: Impaired natural killer

cytotoxicity in peripheral blood mononuclear cells in

Graves’ disease. Eur J Endocrinol 1995; 132:175–180.

KIM ET AL.



39 Cramer DW, Sluss PM, Powers RD, McShane P,

Ginsburgs ES, Hornstein MD, Vitonis AF, Barbieri RL:

Serum prolactin and TSH in an in vitro fertilization

population: is there a link between fertilization

and thyroid function? J Assist Reprod Genet 2003;

20:210–215.

40 Mandel SJ: Hypothyroidism and chronic autoimmune

thyroiditis in the pregnant state: maternal aspects.

Best Pract Res Clin Endocrinol Metab 2004; 18:213–224.

41 Casey BM, Dashe JS, Wells CE, McIntire DD, Byrd W,

Leveno KJ, Cunningham FG: Subclinical

hypothyroidism and pregnancy outcomes. Obstet

Gynecol 2005; 105:239–245.

42 Negro R, Formoso G, Mangieri T, Pezzarossa A, Dazzi

D, Hassan H: Levothyroxine treatment in euthyroid

pregnant women with autoimmune thyroid disease:

effects on obstetrical complications. J Clin Endocrinol

Metab 2006; 91:2587–2591.

43 Brabant G, Beck-Peccoz P, Jarzab B, Laurberg P,

Orgiazzi J, Szabolcs I, Weetman AP, Wiersinga WM:

Is there a need to redefine the upper normal limit of

TSH? Eur J Endocrinol 2006; 154:633–637.

44 Rotondi M, Mazziotti G, Sorvillo F, Piscopo M, Cioffi

M, Amato G, Carella C: Effects of increased thyroxine

dosage pre-conception on thyroid function during

early pregnancy. Eur J Endocrinol 2004; 151:695–700.




thyroid autoimmunity and its association with cellular and humoral immunity in women with...

Documents