Document Type : Original Article
Authors
1 Abnormal Uterine Bleeding Research Center, Semnan University of Medical Sciences, Semnan, Iran;Department of Infertility, Amir-AL-Momenin Hospital, Semnan University of Medical Sciences, Semnan, Iran
2 Department of Infertility, Amir-AL-Momenin Hospital, Semnan University of Medical Sciences, Semnan, Iran;Student Research Committee, Semnan University of Medical Sciences, Semnan, Iran
3 Student Research Committee, Semnan University of Medical Sciences, Semnan, Iran;4Cancer Immunotherapy and Regenerative Medicine Department, Breast Cancer Research Center, Motamed Cancer Institute, ACECR, Tehran, Iran
4 Student Research Committee, Semnan University of Medical Sciences, Semnan, Iran;5Department of Immunology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
5 6Cancer Research Center, Semnan University of Medical Sciences, Semnan, Iran;7Immune and Gene therapy Lab, CCK, Karolinska University Hospital Solna, Stockholm, Sweden
Abstract
Keywords
Infertility is defined as the failure of a couple to get pregnant
after 12 months or more of having regular unprotected
intercourse. Unexplained infertility is idiopathic and its
cause remains unclear when the standard investigation of
both male and female partner has made other infertility diagnoses
impossible. Recurrent pregnancy loss (RPL), a heterogeneous
circumstance often idiopathic, is described as three
or more sequential miscarriages occurring before 20 weeks
of gestation (
Among these suggested causes, only chromosomal abnormalities,
antiphospholipid syndrome and uterine anatomic
abnormalities are universally approved (
There is increasing evidence that these cases of unexplained
infertility and RPL might have an immunological
background. Natural killer (NK) cells which are
present in the endometrium at the time of implantation
and during early pregnancy seem to play a role in this
regard. NK cells are a section of the innate immune system,
and constitute 5-10% of peripheral blood lymphocytes
(PBL) and 70-90% of uterine lymphocytes. There
are two clearly different subgroups of human NK cells
identified by cell surface density of CD56 (D56bright
or CD56dim). Although both peripheral NK (pNK) and
uterine NK (uNK) cells show the surface CD56, pNK
cells differ from uNK cells in both phenotype and function
and the fact that 10% of pNK cells are similar to
uNK cells (
One study reported that pNK cell levels show changes
in decidual NK cell levels (
A recent study has not indicated any significant difference
in CD96 marker between RPL and controls (
All the samples were taken from patients who came to the clinic of Amir Al-Momenin Hospital, Semnan, Iran from June 2011 to December 2013 for the evaluation of RPL or infertility in a case control study. The Research Council and Ethical Committee of Semnan University of Medical Sciences provided us with the ethical approval and later the informed written consents were collected from patients for this case-controlled study. Seventy five women were included in three age match group in this study (24 with a history of unexplained RPL, 25 with unexplained infertility and 26 healthy women with no history of pregnancy problem, convenient sampling). In the infertility group, women had an infertility history of more than 1 year, normal serum prolactin (PRL) and thyroid function tests (T4 and TSH), documented patent tubes by hysterosalpingography, and had no other infertility factor, and the male partner had a normal sperm count, motility and morphology according to the World Health Organization (WHO 2010) standards. Women with RPL had a history of at least two sequential spontaneous miscarriages.
Unexplained RPL was defined as a history of =2 sequential miscarriages in which all the following results were normal: parental karyotypes, thyroid function, fetal bovine serum (FBS), anti-cardiolipin antibodies, antiphospholipid antibodies, lupus anticoagulant, follicle-stimulating hormone (FSH), prolactin, progesterone, estrogen, testosterone, free androgen index, prothrombotic risk factors including activated protein-C resistance, factor V Leiden and prothrombin mutations, pelvic ultrasonography and hysterosalpingogram. Twenty six healthy parous women had at least one live birth and had no history of miscarriage, preeclampsia, ectopic pregnancy or preterm delivery.
Sampling: 5 ml of heparinized peripheral blood was taken in mid luteal phase and in women with RPL, at least 2 months after the last abortion. The blood samples were immediately taken to the Immunology Laboratory of Semnan University of Medical Sciences. The whole blood sample was separated into peripheral blood mononuclear cells (PBMC) by ficoll separation and then PBMCs were labeled and kept in freezing condition medium: (RPMI1640+10%FCS+10%DMSO) at the -70°C freezer until all patient samples were collected.
After sampling was completed, the stored cells were thawed and subsequently surface and intracellular staining were performed. Surface markers were determined by flow cytometry, using fluorochrome-conjugated monoclonal antibodies, anti CD3, CD69, CD19, CD56, and perforin using permabilization buffer for permabilizing cell membrane to facilitate antibody entry into cells. Antibodies were bought from BD Biosciences (San Jose, CA, USA) or ebioscience. Appropriate concentrations of antibodies in addition to isotype matched control were added to the cells (5×105 cells/tube) in 100 µL staining buffer and incubated for 25 minutes at 4°C in the dark. Analysis were done by using PARTEC, CyFlow® Space device and FlowMax software. At least 50,000 lymphocyte-gated cells were obtained and analyzed for CD56+CD19+, perforin+ cells. The criteria for positive staining were set at a fluorescent intensity displayed by <0.5% of the cells stained by the appropriate fluorochrome-conjugated isotype control monoclonal antibodies (mAb). The results and graphs were analyzed using Flowjo version 10A software (Flowjo, USA).
The Kolmogorov-Smirnov test was used to examine the normality of the distributions. A one-way analysis of variance and Tukey’s range test for normally distributed data and Kruskal-Wallis analysis for data with non-normal distribution were used to compare study groups. The results were reported to be statistically significant if the P value was<0.05.
Mean age of the study population was 29.2 ± 3.4 (mean ± SD) years in infertile group, 28.9 ± 3.2 (mean ± SD) years in RPL group and 28.8 ± 3.3 (mean ± SD) years in control group. There were no significant differences in age distribution among them (P=0.6).
Mean percentage of CD56+ cells in infertile, RPL and
control groups were respectively: 18.36 ± 7.9, 15.97 ±
5.1, 13.26 ± 5.02. The Mean percentage of peripheral
CD56 + cells in the infertile group was remarkably higher
(P=0.007) than that of the control subjects. There were not
significant differences in the total number of CD56+ cells
between the RPL group and the control group (P=0.2) and
neither between the RPL group and the infertile group
(P=0.36,
The median percentage of CD69+ cells were: 4.5
(1.5-8)% in infertile group, 8 (
The results showed that 15.8% ± 5.9 of total CD56 cells in patients with RPL and 32% ± 14.4 in the infertile group expressed CD69 as compared with 10.6% ± 5.01 in control group.
The percentage of peripheral NK cells (%) and expression of CD69 and perforin levels on these cells in RPL, infertile and controls groups
Cell population | Control | RPL | Infertile | P1 | P2 | P3 |
---|---|---|---|---|---|---|
CD56+ | 13.26 ± 5.02 | 15.97 ± 5.1 | 18.36 ± 7.9 | 0.2 | 0.007 | 0.36 |
CD69+ | 6 (4-11) | 8 (6-10) | 4.5 (1.5-8) | 0.1 | 0.11 | 0.004 |
Perforin+ | 6.4 (4-8) | 16.5 (9-31) | 8 (5-14) | 0.001 | 0.07 | 0.002 |
CD56+CD69+ | 10.6 ± 5.01 | 15.8 ± 5.9 | 32 ± 14.4 | 0.001 | 0.001 | 0.001 |
CD56+Perforin+ | 7 (4-12) | 16 (9-23) | 8 (5-9) | 0.001 | 0.6 | 0.001 |
CD69+Perforin+ | 6 (4-10) | 10 (6-16) | 6.5 (3-9.5) | 0.02 | 0.7 | 0.01 |
Values are presented as mean ± SD and median (interquartile range). NK; Natural killer, RPL; Recurrent pregnancy loss, P1; P value: for the difference between mean value in the RPL group and control group, P2; P value: for the difference between mean value in the infertile group and control group, and P3; P value: for the difference between mean value in the infertile group and RPL group.
CD69 positive population in CD56+ gated cells. A. Control group, B. Recurrent pregnancy loss (RPL) patients, and C. Infertile.
There was a statistically significant difference in the
expression of CD69 in CD56+ cells between the control
group and RPL group (P=0.001), the infertile group
(P=0.001) and between RPL and infertile group (P=0.001,
The triple staining results showed the CD69+Perforin+
population in control group was 6 (
The findings of this study showed that the levels of
CD56+ T cells were remarkably higher in infertility group
than the control group. But there were no important differences
in the total levels of CD56+ cells between the
RPL group and the other two groups. Moreover, there was
a significant increase in the display of CD69 on CD56+
cells in the RPL group and the infertile group compared
with the control group. We also showed that the level of
perforin on CD56+ cells significantly increased in the RPL
group compared with the other two groups. Findings of
this study were similar to those of case-control studies of
Emmer et al. (
King et al. (
There are also contradictory reports regarding the association
of pNK cells with infertility. Some studies have
shown a relationship between pNK cells and infertility
(
They noticed no difference in percentage NK cell and
NK cell subpopulation in infertile women who were unable
to get pregnant and those who became pregnant after
assisted reproductive technology. Tang et al reported
a systemic review and came to this conclusion that there
was no association between the subsequent pregnancy
result and either pNK or uNK cell activity in women
with RPL and infertility (
They recommended that more research should be conducted
before NK cell assessment can be suggested as a
diagnostic method in the area of female infertility or RPL.
There is no clear reason why the results are different when
the information for NK cells is shown as numbers or a
percentage. So, they suggested that NK cell measuring
and immune therapy should not be recommended except
in the area of clinical research (
In a comparative study of activation and inhibition
markers of circulating NK cells, Coulam and Roussev
(
Ghafourian et al. (
The findings of this study showed a significant increase in the percentage of CD56+ pNK cells among the infertility group and also a significantly higher level of CD69 expression on CD56+ NK cells in women with RPL and unexplained infertility in comparison with healthy control group. We also showed that the level of perforin on CD56+ cells significantly increased in the RPL group compared with the other two groups. Although it can be considered as immunological risk markers in these women, the prognostic value of PNK number assessment or activity remains still doubtful. So because of many arguments in this field, further researches are needed to accept or deny the role of NK cell evaluation as a predictive test for screening women with infertility or RPL.