ORIGINAL ARTICLE: Changes in Endometrial Natural Killer Cell Expression of CD94, CD158a and CD158b...

Changes in Endometrial Natural Killer Cell Expression of CD94,CD158a and CD158b are Associated with InfertilityEmma McGrath1,2,*, Elizabeth J. Ryan1,2,*, Lydia Lynch1, Lucy Golden-Mason1, Eoghan Mooney3, MaeveEogan4, Colm O’Herlihy4, Cliona O’Farrelly1,2

1Education & Research Centre, St. Vincent’s University Hospital Dublin, Dublin 4, Ireland;2Comparative Immunology Group, School of Biochemistry and Immunology, Trinity College Dublin, Dublin, Ireland;3Department of Pathology and Laboratory Medicine, National Maternity Hospital, Dublin 2, Ireland;4Department of Obstetrics and Gynaecology, University College Dublin, National Maternity Hospital, Dublin 2, Ireland

Introduction

Natural killer (NK) cells are the predominant lym-

phocyte population in the endometrium at the time

of implantation and during early pregnancy.1–3

These cells are believed to play a central role in

maintaining maternal tolerance of the semi-allogenic

foetus4 and elevated NK cell activity has been

implicated in pregnancy failure and infertility.5–7

The hypothesis that local endometrial NK cells acting

in an unregulated way could lead to infertility has

stimulated interest in the characterization of the

local endometrial NK cell population.

NK cells not only have a primary role in innate

immune responses against viruses and transformed

cells but also influence adaptive immunity. They are

Keywords

CD158a, CD158b, CD94, infertility, NK cell,

uterine

Correspondence

Cliona O’Farrelly, School of Biochemistry and

Immunology, Trinity College Dublin, Dublin 2,

Ireland.

E-mail: [email protected]

*These authors contributed equally to this

study.

Submitted March 5, 2008;

accepted January 16, 2009.

Citation

McGrath E, Ryan EJ, Lynch L, Golden-Mason L,

Mooney E, Eogan M, O’Herlihy C, O’Farrelly C.

Changes in endometrial natural killer cell

expression of CD94, CD158a and CD158b are

associated with infertility. Am J Reprod

Immunol 2009; 61: 265–276

doi:10.1111/j.1600-0897.2009.00688.x

Problem

Cycle-dependent fluctuations in natural killer (NK) cell populations in

endometrium and circulation may differ, contributing to unexplained

infertility.

Method of study

NK cell phenotypes were determined by flow cytometry in endometrial

biopsies and matched blood samples.

Results

While circulating and endometrial T cell populations remained constant

throughout the menstrual cycle in fertile and infertile women, circulat-

ing NK cells in infertile women increased during the secretory phase.

However, increased expression of CD94, CD158b (secretory phase),

and CD158a (proliferative phase) by endometrial NK cells from infertile

women was observed. These changes were not reflected in the

circulation.

Conclusion

In infertile women, changes in circulating NK cell percentages are found

exclusively during the secretory phase and not in endometrium; cycle-

related changes in NK receptor expression are observed only in infertile

endometrium. While having exciting implications for understanding NK

cell function in fertility, our data emphasize the difficulty in attaching

diagnostic or prognostic significance to NK cell analyses in individual

patients.

ORIGINAL ARTICLE

American Journal of Reproductive Immunology 61 (2009) 265–276 ª 2009 The Authors

Journal compilation ª 2009 John Wiley & Sons A ⁄ S 265

cytolytic towards cells which lack expression of

major histocompatibility complex (MHC) class I mol-

ecules8 and are major producers of cytokines and

chemokines when activated.9–11 The functional plei-

otropy of NK cells reflects their phenotypic heteroge-

neity. NK cell subpopulations are identified by

differential expression of a range of NK receptors

(NKRs). These include CD56 which is an isoform of

the adhesion molecule NCAM (neural cell adhesion

molecule), the Fcc receptor CD16, c-type lectin

molecules, CD94 and CD161 and receptors with

immunoglobulin-like domains, the KIR (Killer cell

Immunoglobulin-like Receptor family of molecules)

family. Some correlation between phenotype and

function has been demonstrated, for example circu-

lating NK cells are primarily CD56dim CD16+ and are

potent cytotoxic cells, whereas decidual NK cells are

mostly CD56bright CD16) NK cells and are less cyto-

toxic than those of the periphery, but are believed to

play a role in tissue remodelling.12

NKRs control NK cell activity, by balancing signals

between activatory and inhibitory receptors. This bal-

ance is thought to be critical in preventing maternal

rejection of the fetus.13 Decidual NK cells express

NKRs belonging to both the KIR family (CD158a,

CD158b) and c-type lectin family (CD94, CD161)

and these molecules are thought to play an impor-

tant role in early pregnancy,14,15 although this fact

remains controversial.16 KIRs with long cytoplasmic

tails are inhibitory and contain an immuno-receptor

tyrosine-based inhibitory motif (ITIM) sequence.

KIRs with an ITIM sequence, including CD158a

(KIR2DL1), CD158b (KIR2DL2 ⁄ L3) and CD158e

(KIR3DL1), are inhibited from lysing target cells that

express MHC class I molecules that bind to that par-

ticular KIR.17 Depending on the NKG2 molecule with

which CD94 dimerizes, either an inhibitory or activa-

tory receptor is formed. NKG2A contains an ITIM in

its cytoplasmic region, which inhibits NK cell cyto-

toxicity.18 CD161 (NKR-P1A) is a c-type lectin recep-

tor expressed by NK cells that plays an important

role in the regulation of NK cytotoxicity.19 The role

of NKRs in maintaining a fine balance in the regula-

tion of NK cytotoxicity may be a key factor in suc-

cessful pregnancy. At the time of implantation,

CD56+ NK cells comprise 70–80% of the total endo-

metrial leukocyte population. Although the target

remains elusive, a correlation between NK cell cyto-

toxicity and infertility has been reported.20

CD56bright NK cells, the primary NK population in

endometrium, are less cytotoxic than CD56dim NK

cells and produce higher levels of cytokines.12 The

influence of cytokines on pregnancy maintenance

has been controversial with recent suggestions that a

balance favoring Th2 cytokines favors successful

pregnancy.21 Although there are conflicting data in

that IFN-c, a Th1 cytokine inhibited trophoblast

growth and induced apoptosis in vitro,22,23 this cyto-

kine was also shown to be necessary for foetal

implantation in mice.24

NK cell distribution and activation at the time of

fertilization and implantation are likely to play a

critical role in pregnancy outcome. However, unlike

decidual NK cells, NK cell populations from non-

pregnant endometrium have not been studied in

detail and, particularly, in relation to fertility. The

aim of this study was to examine endometrial lym-

phocyte subpopulations during the menstrual cycle

and to compare these cells in fertile women and

women with unexplained infertility.

Materials and methods

Subjects

Contemporaneous peripheral blood samples and

endometrial tissue biopsies (obtained by sharp curet-

tage) were collected from 28 pre-menopausal

women admitted for operative procedures under

general anaesthesia at the National Maternity Hospi-

tal, Dublin. The study was approved by the Hospi-

tal’s Ethics Committee and written informed consent

was obtained from all participants. Women in the

control group (n = 10, for the determination of T

and NK cell populations, n = 9 for the analysis of

NKRs) were previously parous and were attending

for a variety of procedures, mainly tubal ligation.

Women in the infertility group (n = 18 for determi-

nation of T and NK cell populations, four of whom

were omitted for analysis of NKRs) had normal ovu-

latory hormone profiles, normal pelvic ultrasound

and all semen analyses were reported as being

within normal limits. Women were recruited into

the study at the point of being admitted for tubal

patency assessment via laparoscopy and hydrotuba-

tion. Four patients had minimal endometriosis diag-

nosed and treated at laparoscopy. All but one of the

women had patent tubes confirmed at laparoscopy

and that patient subsequently had patent tubes con-

firmed at hysterosalpingogram, suggesting tubal

spasm at the time of laparoscopy. Any women with

factors that could interfere with normal endometrial

MCGRATH ET AL.


266 Journal compilation ª 2009 John Wiley & Sons A ⁄ S

physiology, such as endometrial hyperplasia, severe

endometriosis and women with recurrent miscar-

riages were excluded. None of the women were

undergoing IVF at the time of the study.

The stage of menstrual cycle was determined for

each subject by menstrual data (all women had reg-

ular menstrual cycles) and this was confirmed by

histological assessment.25

For analysis of peripheral blood lymphocyte popu-

lations, eight of the fertile endometrium samples (4

proliferative, 4 secretory) had a matched blood sam-

ple, while 15 of the infertile samples had a matched

blood sample. Of the infertile blood samples, 13

were used for NKR analysis (6 proliferative, 7 secre-

tory). Of the endometrial samples used for NKR

analysis, five fertile endometrium samples were in

the proliferative phase and four were in the secre-

tory phase, while six infertile endometrium samples

were in the proliferative phase and eight were in the

secretory phase.

Preparation of Single Cell Suspensions

Biopsies were collected in Hanks’ Balanced Salt

Solution (HBSS; GIBCO BRL, Paisley, UK) supple-

mented with antibiotics (100 U ⁄ mL penicillin,

100 mg ⁄ mL streptomycin; GIBCO BRL) and 5% fetal

bovine serum (FBS; GIBCO BRL), transported to the

laboratory, drained and weighed. Endometrial single

cell suspensions were prepared as previously

described.26 In brief, the tissue was washed thor-

oughly with HBSS to remove residual blood and was

then minced finely with opposing scalpels and

rotated for 20 min at 37�C in 5 mL of enzyme solu-

tion consisting of Roswell Park Memorial Institute-

1640 (RPMI-1640), 20 mm Hepes buffer (GIBCO

BRL), 1% FBS, 1% bovine serum albumin (BSA;

GIBCO BRL), 200 U ⁄ mL collagenase IV (Worthing-

ton Biochemicals, NJ, USA) and 35 U ⁄ mL DNase I

(Sigma, Poole, UK). In general, 5 mL of enzyme

solution were required per 1 g (or less) of tissue. The

suspension was then passed through 30 lm gauze to

remove tissue fragments and washed twice with

HBSS at 400 g for 10 min and resuspended in 1 mL

of complete RPMI-1640. Cell yields and viability

were assessed using ethydium bromide ⁄ acridine

orange staining. The cell suspension was adjusted to

1 · 106 cells ⁄ mL in RPMI-1640.

Peripheral blood mononuclear cells (PBMCs) were

prepared by standard density gradient centrifugation

over Lymphoprep (Nycomed, Roskilde, Denmark) at

400 · g for 25 min. The cells were then washed

twice with HBSS supplemented with Hepes buffer

solution (GIBCO BRL) and antibiotics. Cell pellets

were resuspended in 1 mL of RPMI-1640 medium

and cell yields were assessed as described before and

adjusted to 1 · 106 cells ⁄ mL in RPMI-1640 medium.

Cell Surface Staining of Cells for Flow Cytometric

Analysis

100 lL aliquots of both PBMCs and endometrial

mononuclear cells (1 · 105 cells) were labelled with

monoclonal antibodies (mAbs) directed against cell

surface markers classically associated with NK cells

(listed below). Optimized concentrations of mAbs

(0.3 lg ⁄ mL) were added to cells, which were then

incubated in the dark at 4�C for 30 min. Cells were

then washed twice with 1 mL PBS ⁄ 1% BSA ⁄ 0.05%

Sodium Azide and fixed in 1% paraformaldehyde

(Sigma Aldrich).

Flow Cytometric Analysis

For the detection of T (CD3+, CD56)) and NK

(CD3), CD56+) cells, anti-CD56 phycoerythrin (PE)

(clone MY31) and anti-CD3 peridin chlorophyll pro-

tein (PerCP) (clone SK7) (BD Biosciences, Erem-

bodegem, Belgium) were used. In addition, the

following mAbs were used to determine the pheno-

type of both pNK and endometrial NK cells: anti-

CD158a fluorescein isothiocyanate (FITC) (clone

HP-3E4), anti-CD158b FITC (clone CH-L), anti-

CD158e FITC (clone DX9), anti-CD161 FITC (clone

DX12), anti-CD94 FITC (clone HP-3D9), anti-CD16

FITC (clone NKP15); anti-NKG2A PE (Beckman

Coulter, Fullerton, CA, USA) was used in combina-

tion with CD56-FITC (clone NCAM16.2) (all, except

anti-NKG2A-PE, from BD Biosciences). Isotype

matched FITC, PE and PerCP conjugated controls

were used to exclude non-specific binding. Cells

were stored in the dark at 4�C and acquisition was

performed within 24 hr of staining. Cells were

analysed using a FACScan flow cytometer and Cell-

Quest� software (BD Biosciences). Lymphocytes

were gated by their density and granularity using

forward scatter and side scatter parameters. A total

of 10,000 events in the lymphocyte gate were

acquired for each sample. All samples included in

this study yielded sufficient cells for analysis. A dot

plot of side scatter against CD56 was used to confirm

that all CD56+ cells were in the lymphogate. The

ENDOMETRIAL NK CELLS AND UNEXPLAINED INFERTILITY



proportions of T cells (CD3+CD56)) and NK cells

(CD3+CD56)) as a percentage of lymphocytes were

determined in each sample. In addition, the percent-

age of NK cells expressing each of the NKRs was

determined.

All results are expressed as median (range). Statis-

tical differences between groups were assessed using

the Mann–Whitney U-test (Statview for Windows,

Version 5.0.1; SAS Institute Inc., NC, USA). P < 0.05

was considered significant.

Results

Subjects

Twenty eight pre-menopausal women with an aver-

age age of 34 years (range 29–43) were recruited for

the study. The average age of the group of women

with a diagnosis of unexplained infertility was not

significantly different from that of the parous

women.

Circulating and Endometrial T and NK cell

Populations of Fertile and Infertile Women

We compared the percentage of T (CD3+CD56)) and

NK (CD3)CD56+) cells in the lymphocyte population

of the non-pregnant endometrium and peripheral

blood of fertile and infertile women. When the

phase of the menstrual cycle was ignored, there was

no significant difference in the percentage of these

populations of lymphocytes between infertile and

fertile women in either the endometrium (Fig. 1a)

or circulation (Fig. 1b). After gating on the lymphoid

population, the percentages of T and NK cells in the

endometrium from fertile women were 9.4% (range

2.0–54.4%) and 6.8% (range 2.5–12.8%) respec-

tively, while in infertile endometrial samples, the

percentage of T and NK cells was 6.2% (range

0.03–53.5%) and 4.7% (range 0.17–14.1%) of lym-

phocytes respectively (Fig. 1a). Likewise, there were

no significant differences in the percentages of T and

NK cell populations in peripheral blood from fertile

women 65.8% (range 58.5–76.0%) and 14.2%

(range 4.9–20.9%) of lymphocytes, respectively

when compared with infertile women; 64.3% (range

15–70.9%) and 13.5% (range 8.2–19.6%; Fig. 1b).

These data, when taken with the observation that

there was no difference in NK cell number from

fertile or infertile specimens (Table I), suggest no sig-

nificant change in the distribution of lymphocytes in

the periphery or endometrium in these infertile

women. However, minor trends may be masked by

the large inter-individual variation in percentages of

T and NK cells in the lymphocyte gate of both blood

and endometrial samples for both the fertile and

infertile groups of women (Fig. 1). Variation in NK

cell number between individual samples is shown in

Table I.

Cycle-related Fluctuations in T and NK cell

Populations

T cells (CD3+CD56)) in peripheral blood remained

constant throughout the menstrual cycle in both fer-

tile and infertile women (Fig. 2b). However, during

the secretory phase of the menstrual cycle, infertile

women had a significantly higher percentage of cir-

culating NK cells (16.9%, range 8.2–19.6% of lym-

phocytes) compared with fertile women (7.4%,

Endometrium

% ly

mph

ocyt

es

Blood

T cells T cells

NK cells NK cells

InfertileFertile

6050

40

3020

10

0

–10

1614121086420

–2

22201816141210864

8070

6050

40

30

20–10

(b)(a)

Fig. 1 T and natural killer (NK) cell numbers in (a) endometrium and

(b) blood of fertile and infertile women. (a) The percentages of T and

NK cells were determined in single cell suspensions isolated from

endometrial biopsies of both fertile (n = 10) and infertile (n = 18)

women by flow cytometry. (b) The percentages of T and NK cells were

determined in peripheral blood mononuclear cells of fertile (n = 8) and

infertile (n = 15) women. Results are expressed as a percentage of the

cells in the lymphocyte gate as determined by forward and side scat-

ter analysis.

MCGRATH ET AL.



range 4.9–16.8% lymphocytes) in the same phase of

the menstrual cycle (P < 0.05) (Fig. 2b).

NK Cell Receptor Expression

While we found no difference in the percentages of

endometrial NK cells (CD3)CD56+) of fertile women

when compared with infertile women (Fig. 2a),

expressions of CD158a (P < 0.05), CD158b (P <

0.01) and CD94 (P < 0.05) were all significantly

lower on endometrial NK cells in fertile women

(3.8%, range 0.2–6.7%, 19.5%, range 10.9–36.2%

and 46.3%, range 14.6–79.1% of NK cells respec-

tively) compared with infertile (6.9%, range 0.6–

23.9%, 32.5%, range 20.3–56.7% and 73.3%, range

38.7–88.9% of NK cells respectively) (Fig. 3a). We

found no difference in the expression of CD158e,

CD16, CD161 or NKG2A on endometrial NK cells

isolated from fertile or infertile women (data not

shown). Expression of CD158a, CD158b, CD158e,

CD161, CD94, CD16 and NKG2A on peripheral

blood NK cells did not differ significantly between

fertile and infertile women irrespective of stage of

cycle (Fig. 3b; data not shown). These results indi-

cate that the NKRs, CD158a, CD158b and CD94 are

differentially expressed by endometrial NK cells of

infertile women when compared to fertile women.

Cycle-related Fluctuations in NK Cell Receptor

Expression

No significant fluctuations in expression of CD16,

CD161, CD158e and NKG2A on endometrial NK

cells throughout the menstrual cycle in either fertile

or infertile endometrium were observed (data not

shown). However, NK cells isolated from endome-

trial samples taken during the proliferative phase of

the menstrual cycle from infertile women showed

significantly (P < 0.05) higher expression of CD158a

Table I Absolute Numbers of NK Cells Obtained from

Endometrium or Matched Blood Samples

No. of NK cells ⁄ g tissue

Fertile

proliferative

aInfertile

proliferative

Fertile

secretory

cInfertile

secretory

69269 8570 4209 3196

3280 15972 12952 68643

48383 283082 14674 42188

64429 3092 52256 8136

5674

7939

No. of NK cells ⁄ ml blood

Fertile

proliferative

bInfertile

proliferative

Fertile

secretory

Infertile

secretory

7932 3668 3250 474

8542 7562 8278 3070

14894 8044 4482 4360

9618 3776 8154 6950

13808

11002

16904

Data from 4 ⁄ 6 endometrium samples in the proliferative phasea

and 4 ⁄ 6 matched blood samplesb from infertile women were

available.cData from 6 ⁄ 7 endometrium samples were available from infer-

tile women in the secretory phase.

*******

Endometrium(a) (b)

% ly

mph

ocyt

es

Blood

T cells

NK cells

Fertile, Proliferative Fertile, Secretory

Infertile, Proliferative Infertile, Secretory

Pro Sec

Pro

Pro Sec

Sec Pro Sec

60

50

40

30

20

10

0

25

20

15

10

5

0

80

70

60

50

40

30

20

10

0

16

14

12

10

8

6

4

2

0

Fig. 2 Cycle-related fluctuations in lymphocyte populations of (a)

endometrium and (b) peripheral blood. The percentages of T and natu-

ral killer cells in the (a) endometrial lymphocyte population and (b)

peripheral blood mononuclear cells of fertile women were determined

by flow cytometry. Results are expressed as a percentage of the cells

in the lymphocyte gate as determined by forward and side scatter

analysis. Individual results are displayed as circles and each bar repre-

sents the median of that group. *P < 0.05, by Mann–Whitney U-test.




(5.6%, range 0.8–10.9% of NK cells) compared with

that in fertile endometrium (2.1%, range 0.6–4.2%

of NK cells) (Fig. 4a). In addition, CD158b expres-

sion on NK cells was significantly higher in infertile

endometrium (37.0%, range 24.7–56.7% of NK

cells) in the secretory phase of the cycle compared

with that of fertile endometrium (21.4%, range

10.9–36.2% of NK cells) during the same phase of

the cycle (P < 0.05) (Fig. 4b). Expression of CD94

was significantly higher on NK cells in infertile

endometrium (72.2%, range 38.7–88.9% of NK

cells) in the secretory phase of the cycle compared

with that of fertile women (48.8%, range 29.7–

67.7% of NK cells, P < 0.05) (Fig. 4c).

The expression of CD158a (Fig. 4a) in the prolif-

erative phase and CD158b (Fig. 4b) and CD94

(Fig. 4c) in the secretory phase on CD3)CD56+ NK

cells were higher in infertile endometrium than in

fertile endometrium. These trends were also appar-

ent in the secretory phase regarding CD158a and in

the proliferative phase regarding CD158b and CD94.

However, these differences were not statistically sig-

nificant due to the large degree of variation between

individuals (Fig. 4).

We also assessed the expression of NKG2A on

CD3)CD56+ NK cells and found that there was no

significant difference in NKG2A expression between

fertile and infertile endometrium during the men-

strual cycle (data not shown).

Expression of the NKRs CD158a, CD158b,

CD158e, CD161, CD94, CD16 and NKG2A by

peripheral blood NK cells did not differ between fer-

tile and infertile women when the stage of cycle was

ignored (Fig. 3b; data not shown). Neither were

there any cycle-related changes in NKR expression

by peripheral blood NK cells (Fig. 5a-c and data not

shown).

CD56bright and CD56dim Populations

The relative proportions of CD56bright (�65%) and

CD56dim (�35%) NK cells in the endometrium dif-

fered from peripheral blood where �10% of NK cells

were CD56bright and �90% of NK cells CD56dim

(Figs 4, 5; data not shown). However, these relative

proportions did not change with the cycle nor was

there a significant difference between these cell pop-

ulations when comparing fertile and infertile endo-

metrium. The expression levels of CD158a, CD158b

and CD94 on the CD56bright and CD56dim endome-

trial NK cell populations in fertile and infertile

women in both the proliferative and secretory phase

of the menstrual cycle are summarized in Table II.

In infertile women, CD56dim endometrial NK cells

express significantly lower levels of CD158a

(P < 0.01), CD158b (P < 0.05) and CD94 (P < 0.05)

in the secretory phase compared with the expression

of these receptors on CD56dim endometrial cells in

the proliferative phase of the menstrual cycle. Fur-

thermore, we found that the expression of CD158b

was significantly higher on CD56bright endometrial

NK cells of infertile women in the proliferative phase

of the menstrual cycle compared to the CD56bright

cells of fertile women (P < 0.05, Table II). Whereas,

the expression of CD158a (P < 0.05), CD158b

(P < 0.05) and CD94 (P < 0.05) by CD56dim endo-

metrial NK cells of infertile women is significantly

lower than fertile controls, also in the proliferative

phase of the menstrual cycle. This analysis reveals

that the overall increase in NKRs observed in

InfertileFertile

% N

K C

ells

*

Blood

CD158a CD158b CD94

80

–100

20

40

60

100

Endometrium(a)

(b)

CD158a CD158b CD94

80

–100

20

40

60

100

*

**

Fig. 3 Expression of CD158a, CD158b and CD94 in (a) the endome-

trium and (b) the peripheral blood of fertile and infertile women, irre-

spective of stage of cycle. The percentage of NK cells (CD3)CD56+)

expressing CD158a, CD158b and CD94 was determined by multi-

parameter flow cytometry (a) in single cell suspensions isolated from

endometrial biopsies of fertile and infertile women and (b) peripheral

blood mononuclear cells were obtained at the same time as the

biopsy. Individual results are displayed as circles and each bar repre-

sents the median of that group. *P < 0.05 and **P < 0.01, respectively

by Mann–Whitney U-test.

MCGRATH ET AL.



infertile endometrium is due specifically to the

increase in expression on the predominant

CD56bright population.

Discussion

To test the hypothesis that local endometrial NK

cells acting in an unregulated way could lead to

infertility, we compared NKR expression by the local

endometrial NK cell population with expression by

peripheral blood NK cells in fertile and infertile

women. Our results clearly demonstrate compart-

mentalized differences in NK cell phenotype in

women with unexplained infertility compared with

fertile women. Specifically, increased expression of

NKRs, CD94, CD158b (secretory phase) and CD158a

(proliferative phase) by endometrial NK cells from

infertile women was observed; however, these

changes were not reflected in the circulation.

While it has been reported that circulating NK cell

numbers and activity can be predictive of pregnancy

outcome,27,28 the usefulness of enumerating circulat-

ing NK cell numbers as a diagnostic tool in fertility

assessment remains controversial.29,30 NK cell num-

ber in the circulation is influenced by a number of

factors including age and stress and there is a wide

normal range. In addition, there is no evidence that

numbers of NK cells in the circulation reflect num-

bers of endometrial NK cells. Furthermore, the pre-

cise role of endometrial NK cells and the relative

contribution of cytokine secretion or cytotoxicity in

implantation and maintenance of a successful preg-

nancy remain elusive.30

We found that there was wide inter-individual

variation in the percentage of NK cells in the circula-

tion of both fertile and infertile women (Fig. 1b). A

statistically significant elevation in the proportion of

circulating NK cells was observed, but only during

the secretory phase of the menstrual cycle in infer-

tile women when compared with fertile women in

the same stage of the menstrual cycle (Fig. 2b).

However, the percentage of NK cells observed in all

CD

158A

CD

158B

CD

94

% N

K c

ells

Fertile Infertile

010203040506070

Proliferative

0

20

40

60

80

100

Proliferative

CD158b

CD94

Proliferative–2

0

1012

2468

010203040506070

Secretory

Secretory0

20

40

60

80

100

Secretory

CD158aCD158a; Proliferative phase

CD158b; SecretoryPhase

CD94; Secretory phase

2% of NK cells

6% of NK cells

21% of NK Cells

38% of NK Cells

48% of NK cells

74% of NK cells

0

10

20

30

40

50

–10

FS

C

SR1

CD56

E

FIT

C

Isotype control

Fertile Infertile

(a)

(b)

(c)

Fig. 4 Cycle-related fluctuations in CD158a,

CD158b and CD94 expression on endometrial

NK cells of fertile and infertile women. The

percentage of NK cells (CD3)CD56+) express-

ing (a) CD158a, (b) CD158b and (c) CD94 was

determined by flow-cytometry in single cell

suspensions isolated from endometrial biop-

sies of fertile (n = 9; 5 proliferative, 4 secre-

tory) and infertile (n = 14; 6 proliferative, 8

secretory) women. Results are expressed as a

percentage of total NK cells, with box-plots

representing the median and range. Represen-

tative dot-plots of each of the statistically sig-

nificant results are shown. *P < 0.05 by

Mann–Whitney U-Test.




% N

K c

ells

Fertile Infertile

Proliferative–10

0102030405060

–100

102030405060

Secretory

Proliferative–10

0102030405060

–100

102030405060

Secretory

30405060708090

100

Proliferative

CD158a

CD158b

CD94

30405060708090

100

Secretory

32% of NK cells

34% of NK cells

55% of NK cells

63% of NK cells

CD

158A

CD158a; Proliferative phase

CD

158B

CD158b; Secretory phase

CD

94

CD94; Secretory phase

CD56

SSC

FS

C

R1

FIT

C

PE

Isotype control

3% of NK cells

12% of NK cells

Fertile Infertile

(a)

(b)

(c)

Fig. 5 Expression of CD158a, CD158b and

CD94 on NK cells in peripheral blood of fertile

and infertile women is not cycle-dependant.

The percentage of NK cells (CD3)CD56+)

expressing (a) CD158a, (b) CD158b and (c)

CD94 was determined by multi-parameter flow

cytometry in peripheral blood mononuclear

cells isolated from blood from fertile (n = 8; 4

proliferative, 4 secretory) and infertile (n = 13;

6 proliferative, 7 secretory) women. Results

are expressed as a percentage of total NK

cells, with box-plots representing the median

and range of that group. Representative dot-

plots also illustrate that there was no differ-

ence between fertile and infertile samples.

Table II Expression Levels of CD158a, CD158b and CD94 on CD56bright and CD56dim Endometrial NK Cells of Fertile and Infertile Women in

the Proliferative or Secretory Phase of the Menstrual Cycle. Results Are Expressed as %, Range

CD56bright NK cells

Fertile proliferative aInfertile proliferative Fertile secretory Infertile secretory

CD158a 3.06%, 0.61–9.41 11.13%, 0.44–45.44 9.61%, 3.37–14.49 11.47%, 0.38–35.01

CD158b 25.48%, 12.56–44.06 44.93%, 33.14–79.21 35.5%, 41.9–40.68 46.08%, 4.8–58.73

CD94 83.1%, 72.95–96.02 94.75%, 85.85–97.58 78.15%, 49.05–87.79 87.81%, 75.47–95.54

CD56dim NK cells

Fertile proliferative bInfertile proliferative Fertile secretory cInfertile secretory

CD158a 40.84%, 5.373–58.54 22.32%, 5.83–57.36 13.26%, 7.36–20.23 6.85%, 0–37.61

CD158b 48.77%, 9.14–57.48 24.3%, 11.34–51.25 15.49%, 6.95–21.91 10.91%, 0.41–48.4

CD94 43.7%, 11.38–64.54 33.09%, 17.59–58.49 16.74%, 12.33–32.96 13.2%, 0.19–37.36

aCD56bright expression of CD158b differs significantly between fertile and infertile endometrial NK cells in the proliferative phase of the men-

strual cycle (P < 0.05).bCD56dim expression of CD158a (P < 0.05), CD158b (P < 0.05) and CD94 (P < 0.05) differs significantly between fertile and infertile endome-

trial NK cells in the proliferative phase of the menstrual cycle.cEndometrial CD56dim NK cells express lower levels of CD158a (P < 0.01), CD158b (P < 0.05) and CD94 (P < 0.05) in the secretory phase of

the menstrual cycle compared to the proliferative.

MCGRATH ET AL.



patients was within the normal range (2–29%). In

addition, we found that the difference in percentage

did not translate into a difference in the absolute

number of NK cells in the circulation of fertile and

infertile women (Table I). Previously, we demon-

strated an increase in the number of endometrial NK

cells during the late secretory phase of the menstrual

cycle.31 We did not observe this increase in this

study, as the data were not categorized into early

and late secretory phase, due to the small number of

patient samples. Pooling of data is likely to contrib-

ute to the large inter-individual variations observed,

thus masking minor significant differences in this

cell population during the menstrual cycle. Further-

more, we detected no change in the absolute num-

ber of NK cells or the percentage of NK cells in the

endometrium of fertile women compared to infertile

women in the secretory phase of the menstrual

cycle. These results emphasize the difficulty of

attaching any diagnostic or prognostic significance to

NK cell analyses in individual patients.

Significant changes in NKR expression, however,

were observed on endometrial NK cells from infertile

women. We found that the expression of the NKRs,

CD158a, CD158b and CD94 on endometrial NK cells

of infertile women was consistently higher than that

on endometrial NK cells obtained from fertile

women regardless of stage of cycle (Fig. 3a). This dif-

ference was not observed in circulating NK cells iso-

lated at the same time (Fig. 3b). The majority of

previous studies investigating NKR expression in

women with infertility,32,33 recurrent spontaneous

abortions32 or failed in vitro fertilization5 examined

only circulating NK cells or decidual NK cells.13,14

Ntrivalas et al.32 reported a significant decrease in

the expression of CD158a and CD158b on peripheral

blood NK cells in women with implantation failures

compared to normal controls, a result not replicated

in this study (Fig. 3b). This may be because Ntrivalas

et al. studied women with recurrent spontaneous

abortions, who may have a further expansion of NK

cells than the women with unexplained infertility

who were recruited for this study.

Our data show that no correlation exists between

the expression of NKRs on circulating NK cells and

expression on local endometrial NK cell populations

(Fig. 3). Endometrial NK cells from non-pregnant

women have been less studied. It is possible that

these are separate NK cell populations of different

origins. Evidence is accumulating that there are mul-

tiple sites of NK cell differentiation in the healthy

adult.34 Previously, we demonstrated the presence of

cells with haematopoietic stem cell (HSC) phenotype

in human endometrium, a proportion of which

expressed CD56.35 We therefore suggested that NK

cell populations might differentiate in the uterus

from local haematopoietic progenitor cells.

NK cells represent an important class of lympho-

cyte in the uterine epithelium and are thought to

have a tissue remodelling role during the formation

of the placenta.3 Recently, it has been suggested that

decidual NK cells develop from existing endometrial

NK cells.12 The secretion of chemokines and angio-

genic growth factors by decidual NK cells has been

shown to regulate trophoblast invasion.36 Therefore,

higher expression levels of both CD158a and

CD158b, which exert an inhibitory influence on NK

cells, may affect fertility by suppression of local NK

cell activity (e.g. cytokine and growth factor secre-

tion) thereby affecting placental developmental pro-

cesses including trophoblast invasion and vascular

growth.

The heterodimer CD94 ⁄ NKG2A is a ligand for

HLA-E on foetal trophoblasts. As the interaction of

CD94 ⁄ NKG2A with HLA-E leads to inhibition of NK

cell activity, it is possible that changes in CD94

expression alter cytokine secretion, which may have

critical implications for placental development,

maternal uterine spiral arterial remodelling, antiviral

immune response as well as trophoblast survival.37

Additionally, HLA-G, which has been implicated in

the regulation of trophoblast invasion,38 has been

reported to interact with KIRs and CD94 ⁄ NKG2A;

however, there has been some controversy regarding

this relationship.39 Our data show an increase in

CD94 expression, as well as CD158a and CD158b

expression by NK cells isolated from infertile endo-

metrium, thus possibly altering NK cell ability to reg-

ulate trophoblast invasion.

The percentage of NK cells in the endometrium

apparently increases in the later stages of the secre-

tory phase,40 although this is not obvious from our

data as they were not separated into early and late

secretory phase (Fig. 2a). However, we have shown

that CD158b and CD94 expression on endometrial

NK cells isolated from infertile women during the

secretory phase was significantly higher than in par-

ous women (Fig. 4). In contrast, CD158a expression

on endometrial NK cells was significantly higher in

infertile women compared with parous women in

the proliferative phase of the menstrual cycle.

Although not significantly different due to the degree




of variation between individual samples, these trends

were also present in the secretory phase with respect

to CD158a and in the proliferative phase with respect

to CD158b and CD94, thus suggesting a tendency

towards greater expression of these NKRs in the

endometrium of infertile women (Fig. 4). Cycle-

dependent changes in NKR expression by NK cells

from matched peripheral blood samples were not

observed (Fig. 5), emphasizing the compartmentali-

zation of fertility-associated NK cell changes.

There did not appear to be any difference in per-

centage of CD56bright endometrial NK cells between

fertile endometrium and infertile endometrium. A

similar result was recently reported by Matteo et al,

2007.41 While we show an increase in NKR expres-

sion on CD56+ cells in the endometrium of infertile

women (Fig. 4), interestingly, CD56dim endometrial

NK cells expressed significantly reduced levels of

CD158a, CD158b, CD158e, CD16 and CD94 during

the secretory phase in infertile endometrium

(Table II). This was not observed in fertile endome-

trium. Although the CD56dim cell population of

endometrial NK cells is not generally studied in

detail, being the minority population, it is possible

that changes in this cell population during the secre-

tory phase of the menstrual cycle may impart a neg-

ative influence on maintaining fertility.

To conclude, this study shows that in women with

a diagnosis of unexplained infertility following stan-

dard investigation, fluctuations in peripheral blood

NK cell populations are cycle-dependent and are not

reflected in the local environment of the endome-

trium where NK cell expression of the NKRs; CD158a,

CD158b and CD94 is elevated. We observed wide

inter-individual variation in both NK cell number and

receptor expression. Furthermore, our sample size

was small, which may also contribute to masking

some potentially significant findings. A recent study

has elegantly shown that NK cells with differing

expression of KIR function differently in the context

of viral infection.42 However, further studies are

required both to confirm our findings and to establish

whether differential expression of these molecules is

sufficient to influence endometrial NK cell activity to

the extent that fertility is compromised.

Acknowledgments

We wish to thank the women who participated in

this research which was supported by a grant from

Enterprise Ireland.

References

1 Bulmer JN, Morrison L, Longfellow M, Ritson A, Pace

D: Granulated lymphocytes in human endometrium:

histochemical and immunohistochemical studies. Hum

Reprod 1991; 6:791–798.

2 Kodama T, Hara T, Okamoto E, Kusunoki Y, Ohama

K: Characteristic changes of large granular

lymphocytes that strongly express CD56 in

endometrium during the menstrual cycle and early

pregnancy. Hum Reprod 1998; 13:1036–1043.

3 Moffett-King A, Entrican G, Ellis S, Hutchinson J,

Bainbridge D: Natural killer cells and reproduction.

Trends Immunol 2002; 23:332–333.

4 Santoni A, Zingoni A, Cerboni C, Gismondi A:

Natural killer (NK) cells from killers to regulators:

distinct features between peripheral blood and

decidual NK cells. Am J Reprod Immunol 2007; 58:280–

288.

5 Thum MY, Bhaskaran S, Abdalla HI, Ford B, Sumar

N, Shehata H, Bansal AS: An increase in the absolute

count of CD56dimCD16+CD69+ NK cells in the

peripheral blood is associated with a poorer IVF

treatment and pregnancy outcome. Hum Reprod 2004;

19:2395–2400.

6 Vassiliadou N, Bulmer JN: Immunohistochemical

evidence for increased numbers of ‘classic’ CD57+

natural killer cells in the endometrium of women

suffering spontaneous early pregnancy loss. Hum

Reprod 1996; 11:1569–1574.

7 Kwak JY, Beer AE, Kim SH, Mantouvalos HP:

Immunopathology of the implantation site utilizing

monoclonal antibodies to natural killer cells in

women with recurrent pregnancy losses. Am J Reprod

Immunol 1999; 41:91–98.

8 Yokoyama WM, Kim S: How do natural killer cells

find self to achieve tolerance? Immunity 2006; 24:249–

257.

9 Farag SS, Caligiuri MA: Human natural killer cell

development and biology. Blood Rev 2006; 20:123–

137.

10 Colucci F, Caligiuri MA, Di Santo JP: What does it

take to make a natural killer? Nat Rev 2003; 3:413–

425.

11 Robertson MJ: Role of chemokines in the biology of

natural killer cells. J Leuk Biol 2002; 71:173–183.

12 Manaster I, Mandelboim O: The unique properties of

human NK cells in the uterine mucosa. Placenta 2008;

29:60–66.

13 Kusumi M, Yamashita T, Fujii T, Nagamatsu T,

Kozuma S, Taketani Y: Expression patterns of lectin-

like natural killer receptors, inhibitory CD94 ⁄ NKG2A,

and activating CD94 ⁄ NKG2C on decidual CD56bright

MCGRATH ET AL.



natural killer cells differ from those on peripheral

CD56dim natural killer cells. J Reprod Immunol 2006;

70:33–42.

14 Eidukaite A, Siaurys A, Tamosiunas V: Differential

expression of KIR ⁄ NKAT2 and CD94 molecules on

decidual and peripheral blood CD56bright and

CD56dim natural killer cell subsets. Fertil Steril 2004;

81(Suppl. 1):863–868.

15 Yamada H, Shimada S, Kato EH, Morikawa M,

Iwabuchi K, Kishi R, Onoe K, Minakami H:

Decrease in a specific killer cell immunoglobulin-

like receptor on peripheral natural killer cells in

women with recurrent spontaneous abortion of

unexplained etiology. Am J Reprod Immunol 2004;

51:241–247.

16 Witt CS, Goodridge J, Gerbase-Delima MG, Daher S,

Christiansen FT: Maternal KIR repertoire is not

associated with recurrent spontaneous abortion. Hum

Reprod 2004; 19:2653–2657.

17 Moretta A, Moretta L: HLA class I specific inhibitory

receptors. Curr Opin Immunol 1997; 9:694–701.

18 Le Drean E, Vely F, Olcese L, Cambiaggi A, Guia S,

Krystal G, Gervois N, Moretta A, Jotereau F, Vivier E:

Inhibition of antigen-induced T cell response and

antibody-induced NK cell cytotoxicity by NKG2A:

association of NKG2A with SHP-1 and SHP-2 protein-

tyrosine phosphatases. Eur J Immunol 1998; 28:264–

276.

19 Lanier LL, Chang C, Phillips JH: Human NKR-P1A. A

disulfide-linked homodimer of the C-type lectin

superfamily expressed by a subset of NK and T

lymphocytes. J Immunol 1994; 153:2417–2428.

20 Matsubayashi H, Shida M, Kondo A, Suzuki T, Sugi T,

Izumi S, Hosaka T, Makino T: Preconception

peripheral natural killer cell activity as a predictor of

pregnancy outcome in patients with unexplained

infertility. Am J Reprod Immunol 2005; 53:126–131.

21 Chernyshov VP, Tumanova LE, Sudoma IA, Bannikov

VI: Th1 and Th2 in Human IVF Pregnancy with

Allogenic Fetus. Am J Reprod Immunol 2008; 59:352–

358.

22 Ho S, Winkler-Lowen B, Morrish DW, Dakour J, Li H,

Guilbert LJ: The role of Bcl-2 expression in EGF

inhibition of TNF-alpha ⁄ IFN-gamma-induced villous

trophoblast apoptosis. Placenta 1999; 20:423–430.

23 Yui J, Garcia-Lloret M, Wegmann TG, Guilbert LJ:

Cytotoxicity of tumour necrosis factor-alpha and

gamma-interferon against primary human placental

trophoblasts. Placenta 1994; 15:819–835.

24 Ashkar AA, Croy BA: Functions of uterine natural

killer cells are mediated by interferon gamma

production during murine pregnancy. Sem Immunol

2001; 13:235–241.

25 Noyes RW, Herbig AT, Rock J: Dating the endometrial

biopsy. Fertil Steril 1950;1:3–25.

26 Flynn L, Carton J, Byrne B, Kelehan P, O’Herlihy C,

O’Farrelly C: Optimisation of a technique for isolating

lymphocyte subsets from human endometrium.

Immunol Invest 1999; 28:235–246.

27 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.

28 Coulam CB, Beaman KD: Reciprocal alteration in

circulating TJ6+ CD19+ and TJ6+ CD56+ leukocytes

in early pregnancy predicts success or miscarriage. Am

J Reprod Immunol 1995; 34:219–224.

29 Rai R, Sacks G, Trew G: Natural killer cells and

reproductive failure–theory, practice and prejudice.

Hum Reprod 2005; 20:1123–1126.

30 Moffett-King A: Natural killer cells and pregnancy.

Nat Rev 2002; 2:656–663.

31 Flynn L, Byrne B, Carton J, Kelehan P, O’Herlihy C,

O’Farrelly C: Menstrual cycle dependent fluctuations

in NK and T-lymphocyte subsets from non-pregnant

human endometrium. Am J Reprod Immunol 2000;

43:209–217.

32 Ntrivalas EI, Bowser CR, Kwak-Kim J, Beaman KD,

Gilman-Sachs A: Expression of killer

immunoglobulin-like receptors on peripheral blood

NK cell subsets of women with recurrent spontaneous

abortions or implantation failures. Am J Reprod

Immunol 2005; 53:215–221.

33 Michou VI, Kanavaros P, Athanassiou V, Chronis GB,

Stabamas S, Tsilivakos V: Fraction of the peripheral

blood concentration of CD56+ ⁄ CD16- ⁄ CD3- cells in

total natural killer cells as an indication of fertility

and infertility. Fertil Steril 2003; 80(Suppl. 2):691–697.

34 Huntington ND, Vosshenrich CA, Di Santo JP:

Developmental pathways that generate natural-killer-

cell diversity in mice and humans. Nat Rev 2007;

7:703–714.

35 Lynch L, Golden-Mason L, Eogan M, O’Herlihy C,

O’Farrelly C: Cells with haematopoietic stem cell

phenotype in adult human endometrium: relevance

to infertility? Hum Reprod 2007; 22:919–926.

36 Hanna J, Goldman-Wohl D, Hamani Y, Avraham I,

Greenfield C, Natanson-Yaron S, Prus D, Cohen-

Daniel L, Arnon TI, Manaster I, Gazit R, Yutkin V,

Benharroch D, Porgador A, Keshet E, Yagel S,

Mandelboim O: Decidual NK cells regulate key

developmental processes at the human fetal-maternal

interface. Nat Med 2006; 12:1065–1074.

37 Rabot M, Tabiasco J, Polgar B, Aguerre-Girr M,

Berrebi A, Bensussan A, Strbo N, Rukavina D, Le




Bouteiller P: HLA class I ⁄ NK cell receptor interaction

in early human decidua basalis: possible functional

consequences. Chem Immunol Allergy 2005; 89:72–83.

38 Roussev RG, Coulam CB: HLA-G and its role in

implantation (review). J Assist Reprod Gen 2007;

24:288–295.

39 Hunt JS, Petroff MG, Morales P, Sedlmayr P,

Geraghty DE, Ober C: HLA-G in reproduction: studies

on the maternal-fetal interface. Hum Immunol 2000;

61:1113–1117.

40 Mselle TF, Meadows SK, Eriksson M, Smith JM, Shen

L, Wira CR, Sentman CL: Unique characteristics of

NK cells throughout the human female reproductive

tract. Clin Immunol 2007; 124:69–76.

41 Matteo MG, Greco P, Rosenberg P, Mestice A, Baldini

D, Falagario T, Martino V, Santodirocco M, Massenzio

F, Castellana L, Specchia G, Liso A: Normal

percentage of CD56bright natural killer cells in young

patients with a history of repeated unexplained

implantation failure after in vitro fertilization cycles.

Fertil Steril 2007; 88:990–993.

42 Ahlenstiel G, Martin MP, Gao X, Carrington M,

Rehermann B: Distinct KIR ⁄ HLA compound

genotypes affect the kinetics of human antiviral

natural killer cell responses. J Clin Invest 2008;

118:1017–1026.

MCGRATH ET AL.



ORIGINAL ARTICLE: Changes in Endometrial Natural Killer Cell Expression of CD94, CD158a and CD158b...

Documents

Transcript of ORIGINAL ARTICLE: Changes in Endometrial Natural Killer Cell Expression of CD94, CD158a and CD158b...