|Year : 2016 | Volume
| Issue : 1 | Page : 78-82
Relation between uterine natural killer cells and unexplained recurrent miscarriage
Mohamed M Farghali1, Abdel-Latif G El-kholy1, Khaled H Swidan1, Ibrahim A Abdelazim2, Ahmed R Rashed1, Ezzat El-Sobky3, Mostafa F Goma1
1 Department of Obstetrics and Gynecology, Ain Shams University, Cairo, Egypt
2 Department of Obstetrics and Gynecology, Ain Shams University, Cairo, Egypt; Department of Obstetrics and Gynecology, Ahmadi Hospital, Kuwait Oil Company (KOC), Ahmadi, Kuwait
3 Department of Pathology and Genetic, Ain Shams University, Cairo, Egypt
|Date of Web Publication||5-Jul-2017|
Ibrahim A Abdelazim
Professor of Obstetrics and Gynecology, Ain Shams University, Cairo and Ahmadi Kuwait Oil (KOC) Company Hospital
Source of Support: None, Conflict of Interest: None
Objective: To evaluate the relation between uK (uterine Killer) cells and unexplained repeated miscarriage (RM).
Patients and Methods: Eighty (80) women with unexplained repeated miscarriage and missed miscarriage of current pregnancy were studied. Fetal viability and gestational age of current pregnancy were confirmed by ultrasound, followed by suction evacuation to collect abortus specimens and uterine wall curettage to collect decidua specimens. Abortus specimens were collected for long-term monolayer cell culture and subsequent chromosome analysis using conventional G-banding technique. Decidua specimens were subjected to IHC (Immunohistochemical) staining using monoclonal antibodies specific to CD56+ and CD16+ expressed by uK cells.
Results: CD56+ CD16+ uK cells was found in 85% (68/80) of studied decidua specimens of women with unexplained repeated miscarriage, 88.5% (54/61) had normal abortus karyotyping and 73.7% (14/19) had abnormal abortus karyotyping. 73.75% (59/80) of studied women with past history of early miscarriage had CD56+ CD16+ uK cells in their decidua specimens and 66.25% (53/80) of studied women with past history of late miscarriage had CD56+ CD16+ uK cells in their decidua specimens, the association between early and late miscarriage and CD56+ CD16+ uK cells in deciduas specimen was significant.
Conclusion: CD56+ CD16+ uK cells were predominant in decidua specimens of studied women with repeated miscarriage. Significant association was found between presence of CD56+ CD16+ uK cells in studied decidua specimens and unexplained repeated miscarriage.
Keywords: Obstetrics, Uterine killer cells, Repeated, Miscarriage
|How to cite this article:|
Farghali MM, El-kholy ALG, Swidan KH, Abdelazim IA, Rashed AR, El-Sobky E, Goma MF. Relation between uterine natural killer cells and unexplained recurrent miscarriage. Acta Med Int 2016;3:78-82
|How to cite this URL:|
Farghali MM, El-kholy ALG, Swidan KH, Abdelazim IA, Rashed AR, El-Sobky E, Goma MF. Relation between uterine natural killer cells and unexplained recurrent miscarriage. Acta Med Int [serial online] 2016 [cited 2020 Jun 2];3:78-82. Available from: http://www.actamedicainternational.com/text.asp?2016/3/1/78/209726
| Introduction|| |
Repeated miscarriage (RM) defined as two or more failed pregnancies (confirmed by ultrasound or histopathological examination) and is known to affect approximately 0.5–1 % of couples.'
One miscarriage increases risk of miscarriage in future pregnancy to 24%, this risk increases to 26% with previous 2 miscarriages and reaches 32% with previous 3 miscarriages, thus women with two or more consecutive miscarriages deserve meticulous study to detect definite cause and possible treatment.,,
Various factors are implicated in the pathophysiology of repeated miscarriage. Fetal causes as single gene or genomic imprinting defects account for 3.5–5% of the causes of repeated miscarriage, other fetal defects include fetal infections and developmental abnormalities. Maternal causes of repeated miscarriage include; immunological causes accounting for 30% of the cases, with anti-phospholipid antibody syndrome being the most common autoimmune cause.,
Endocrine dysfunction accounts for 48.71% of the causes, while other maternal factors including anatomical detects and sub-clinical endometrial infection account for a minimal number of cases.,
Approximately 50% of repeated miscarriages are unexplained with no definitive etiology. Several authors suggest the cause to be alloimmune rejection of the fetus.
NK (natural killer) cells are part of immune system lymphocytes., uK (uterine killer) cells are short-lived, lymphocytes found in uterine deciduas. Early in pregnancies uK cells produce angiogenic factors and are now considered important for implantation and development.,
uK cells have been linked to human reproductive disorders, including repeated miscarriage, implantation failure, fetal growth restriction and preeclampsia.,
uK cells secrete cytokines and angiogenic factors, important for placental development and establishment of pregnancy.
It has been found that 37.3% of patients presented with repeated miscarriage had mild to moderate increase in NK cells and 14.7% of women with repeated miscarriage had elevated CD56+ NK cells in peripheral blood.,
Other authors concluded that NK cells cytotoxicity are not related to number of peripheral NK cell and it can be estimated by NK cells markers such as KIRs (killer inhibitory receptors) or CD16+ receptor expression.
Because, more research work are needed to establish relation between uK cells and human reproductive disorders.,
This study designed to evaluate the relation between uK cells and unexplained repeated miscarriage.
| Patients and Methods|| |
Eighty (80) women with unexplained repeated miscarriage and missed miscarriage of current pregnancy were included for evacuation and curettage because of current missed miscarriage (> than 8 weeks diagnosed by ultrasound). Women were studied after proper counseling, consent and approval of the ethical committee. Unexplained repeated miscarriages was defined as ≥2 previous miscarriages <20 weeks' gestation. Thorough history and examination were done for all studied women, followed by trans-vaginal ultrasound to confirm fetal viability and gestational age of current pregnancy by sonographer who was blinded to patients' data. Peripheral venous samples were collected from studied women for oral glucose tolerance test, thyroid stimulating hormone assay, prolactin, serum anticardiolipin, lupus anticoagulant assays, prothrombin gene mutations, anti-thrombin III deficiencies and thrombophilia screen. Women with septic miscarriage, documented endocrinopathies (diabetes, thyroid disorders, hyperprolactinaemia), uterine anomalies, polycystic ovary syndrome, anti-phospholipid antibody syndrome, thrombophilia, abnormal karyotype in one or both parents using leucocyte culture, autoimmune disorders, history of hormonal contraception, history of intrauterine contraceptive device application within last 3 months preceding current pregnancy were excluded from this study. Evacuation and curettage was done for all women included in this study under general anesthesia, using suction evacuation to collect abortus specimens after cervical dilatation, followed by uterine wall curettage to collect decidua specimens. Abortus specimens were collected on a special medium for long term monolayer cell culture and subsequent chromosome analysis using conventional G-banding technique. Decidua specimens were subjected to IHC (Immunohistochemical) staining using monoclonal antibodies specific to uK cells CD56+ and CD16+
Reagents and materials used include;
- Primary antibodies; which is liquid MoAb (monoclonal mouse antibody) against CD56+ and CD16+ expressed on NK cells.
- Universal Kits; supersensitive immunodetection system (Biogenex Laboratories, San Francisco, USA), contains the following; (a) Negative control antibody (b) Biotinylated anti-immunoglobulin for mouse antibody (c) Label: streptavidine peroxidase complex (d) Chromogen: 2,3 DAB (Diamenobenzidine chromogen) solution, ready to use substrate buffer and H2O2 substrate for use with liquid DAB chromogen and substrate buffer (e) Blocking reagent to block endogenous peroxidase activity
- Lyophilized pepsin powder, PBS (Phosphate buffer saline), counter stain (Mayer's hematoxylin), distilled water and mounting media (Canada balsam).
- Staining jars, microscopic positive charged slides, cover slips for slides and immune-stainer.
- Light microscope: with 100x and 400x Magnification.
IHC procedure; Decidua specimens fixed in buffered formalin (not more than 24 hours), embedded in paraffin wax, 3-micometer sections mounted with 3-aminopropyltriethoxysilane (Sigma Chemical Co, UK).
Serial sections were then stained for uK cells (CD56+ and CD16+) using antibodies antigen-retrieval methods.,
Primary antibodies incubated one hour (60 minutes) for CD56+ and for 120 minutes for CD16+ at room temperature, the staining intensity of brown reaction developed with 2,3 DAB containing 0.0I% H2O2 was noted, haematoxylin was used for counterstaining of sections.
Sections were dehydrated and mounted with DPX (Distrene, Plasticiser, Xylene) standard resin (Lamb Ltd, London, UK), then examined by ordinary light microscopy. Appropriate positive controls (neuroblastoma for CD56+ and tonsils for CD16+) were used in each run to judge the effectiveness of staining technique and mouse Ig-G [Immunoglobulin-G] were used instead of primary antibodies as negative controls.
| Sample Size and Statistical Analysis|| |
Sample size of 80 women was required in this study and calculated using G* Power software for sample size calculation (*Heinrich Heine Universität; Düsseldorf; Germany). Mean and + SD (standard deviation) were used to represents numerical values, while, number (n) and percentage (%) were used to represents categorical values. Comparison between variables was done using Chi-square (x2) test was. A difference with a p value <0.05 considered significant.
| Results|| |
Mean age of studied women was 29.6 ± 6.39 years and mean BMI (body mass index) was 26.9 ± 4.5 kg/m2. Karyotyping study of abortus specimens showed; normal karyotyping in 76.25% (61/80) of studied specimens and abnormal karyotyping in 23.75% (19/80) of studied specimens [Table 1].
CD56+ CD16+ uK cells was found in 85% (68/80) of studied decidua specimens of women with unexplained repeated miscarriage, 88.5% (54/61) had normal abortus karyotyping and 73.7% (14/19) had abnormal abortus karyotyping [Table 2].
|Table 2: Relation between karyotyping of abortus specimens and Immunohistochemical results of decidua specimens|
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73.75% (59/80) of studied women with past history of early miscarriage had CD56+ CD16+ uK cells in their decidua specimens and 66.25% (53/80) of studied women with past history of late miscarriage had CD56+ CD16+ uK cells in their decidua specimens, the association between early and late miscarriage and CD56+ CD16+ uK cells in deciduas specimen was significant [Table 3].
|Table 3: Relation between CD56+CD16+uterine killer cells and miscarriage (early and late)|
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| Discussion|| |
Early in pregnancies uK cells produce angiogenic factors and are now considered important for implantation and development. uK cells have been linked to human reproductive disorders, including repeated miscarriage, repeated implantation failure, fetal growth restriction and preeclampsia,, this study designed to evaluate the relation between uK cells and unexplained repeated miscarriage.
CD16 is expressed by neutrophils, activated macrophages, and natural killer cells. CD56 is expressed by natural killer cells as one of neural cell adhesion molecule.
pNK (peripheral natural killer) cells are known to be found in blood and endometrium. pNK and uNK are phenotypically and functionally different. Both pNK (peripheral natural killer) and uNK (uterine natural killer) express the surface CD56.
Studies have shown that 90% of pNK cells express a CD56dim CD16+ phenotype, while 80% of the uNK cells express a CD56bright CD16- phenotype, with the CD56 cells known to have a regulatory function, while the CD16 cells have a cytotoxic function.,,
In humans, it has been proved that the elevated circulating cytotoxic NK cells (not the count) increase the risk of miscarriage. Women<35 years old with unexplained repeated miscarriage were studied to minimize risk of chromosomal abnormalities and miscarriages associated with advanced maternal age.
BMI of women included in this study was 26.9 ± 4.5 kg/m2, this might be due to our selection criteria as we excluded some risk factors that might predispose to RM such as obesity,, diabetes mellitus and thyroid disorders.
Eighty abortus specimens were cytogenetically analyzed using tissue culture and conventional G-banding technique, because comparative genomic hybridization (without culture) was not introduced in our institute until recently. Cytogenetic analysis using tissue culture and conventional G-banding method has some limitations; contamination, culture failure and maternal cells growwth.
In this study, Karyotyping studies showed; normal karyotyping in 76.25% (61/80) of studied abortus specimens and abnormal karyotyping in 23.75% (19/80) of studied abortus specimens. A 29-57% rate of chromosomal abnormality was previously reported during analysis of miscarried tissue from women suffering RM,,,, and the higher of normal chromosomal study in miscarried tissue of women with RM confirms that there may be other factors other than chromosomal abnormalities associated with RM.
CD56+ CD16+ uNK cells were found in 85% (68/80) of studied decidua specimens of women with unexplained repeated miscarriage. Quenby et al. reported that women with RM had significantly more uNK than controls and Clifford et al., also showed that there were increased CD56+ uK cells in women with unexplained repeated miscarriage.
Increased expression of CD56+ CD16+ uNK was also reported in deciduas obtained after spontaneous miscarriage in women with history of repeated miscarriage.
Quenby et al. used IHC to investigate leukocyte populations in mid-luteal endometrial biopsies of 22 women suffering from RM compared to 9 women without RM, they found that CD4+, CD14+, CD16+ and CD56+ uNK cells were significantly higher in RM group than controls.
Lachapelle et al. compared endometrial specimens from 20 women with RM with endometrial samples collected during the secretory phase from 15 fertile controls. Lachapelle et al. found that the percentage of uK was similar in the two groups although, greater percentage of CD56+ CD16+ uK were found in women with RM.
CD56+ CD16+ uNK cells was found in 85% (68/80) of studied decidua specimens of women with unexplained repeated miscarriage, 88.5% (54/61) had normal abortus karyotyping and 73.7% (14/19) had abnormal abortus karyotyping, this difference was statistically not-significant. Yamamoto et al. also, reported same findings, when they studied uNK cells in decidua specimens of both chromosomally normal and abnormal missed miscarriages.
Although, in this study we found expression of CD56+ CD16+ uNK cells in decidua specimens of women with RM is high, Yamamoto et al. did not found over expression of CD56+ CD16+ uK cells in deciduas of studied women with missed abortions, because their study was limited to sporadic cases of missed abortions, not RM cases.
73.75% (59/80) of studied women with past history of early miscarriage had CD56+ CD16+ uK cells in their decidua specimens and 66.25% (53/80) of studied women with past history of late miscarriage had CD56+ CD16+ uK cells in their decidua specimens, the association between early and late miscarriage and CD56+ CD16+ uK cells in deciduas specimen was significant. Findings of this study suggests that CD56+ CD16+ uK cells are predominant in decidua of women with RM. Women refused to participate in this study and use of tissue culture and conventional G-banding technique for cytogenetic analysis and karyotyping of abortus specimens were faced as limitations during this study.
| Conclusion|| |
Further Large case controlled studies are needed to compare decidua specimens from RM cases with decidua specimen from normal cases without RM to establish relation between uK cells and human reproductive disorders and to improve future treatment for such cases.
| Acknowledgment|| |
The authors are very grateful to studied women and Professor Nahla M. M. Awad, Assistant consultant of pathology, Ain shams university, for her great participation in this research.
All authors declare that they didn't receive any financial support and authors declare that no conflict of interest related to this manuscript.
| References|| |
Practice Committee of the American Society for Reproductive Medicine. Definitions of infertility and recurrent pregnancy loss. Fertil Steril 2008; 89 (6):1603.
Bricker L, Farquharson RG. Types of pregnancy loss in recurrent miscarriage: implications for research and clinical practice. Hum Reprod 2002; 17(5): 1345–1350.
Kiwi R. Recurrent pregnancy loss: evaluation and discussion of the causes and their management. Cleve Clin J Med 2006; 73(10):913–21.
Christiansen OB, Nybo Andersen AM, Bosch E, Daya S, Delves PJ, Hviid TV, et al. Evidenced-based investigations and treatments of recurrent pregnancy loss. Fertil Steril 2005; 83(4): 821–839.
Egozcue S, Balanco J, Vendrell JM. Human male infertility: chromosomal abnormalities, meiotic disorders, abnormal spermatozoa and recurrent abortion. Hum Reprod 2000; 6; 93–105.
Wilcox AJ, Weinberg CR, O'Connor JF. Incidence of early loss of pregnancy. N Engl J Med1988; 319 (4):189–194.
Rai R, Regan L. Recurent miscarriage. Lancet 2006; 368 (9535):601–611.
Summers PR. Microbiology relevant to recurrent miscarriage. Clin Obstet Gyneco1994l; 37: 722–729.
Matovina M, Husnjak K, Milutin N, Ciglar S, Grce M. Possible role of bacterial and viral infections in miscarriages. Fertil Steril 2004; 81: 662–669.
Abramson J, Stagnaro-Green A. Thyroid antibodies and fetal loss: an evolving story. Thyroid 2001; 11:57–63.
Moffett-King A. Natural Killer cells and pregnancy. Nat Rev Immunol 2002; 2:656–663.
Yovel G, Shakhar K, Ben-Eliyahu S. The effects of sex, menstrual cycle, and oral contraceptives on the number and activity of natural killer cells. Gynecol Oncol 2001; 81:254–262.
Bilinski MJ, Thorne JG, Oh MJ, Leonard S, Murrant C, Tayade C, et al. Uterine NK cells in murine pregnancy. Reprod Biomed Online 2008; 16(2):218–26.
Murphy SP, Fast LD, Hanna NN, Sharma S. Uterine natural killer cells mediate inflammation-induced fetal demise in IL-10 null mice. Journal of immunology 2005; 175: 4084–90.
Lash GE, Bulmer JN. Do uterine natural killer (uNK) cells contribute to female reproductive disorders? J Reprod Immunol 2011; 88(2):156–64.
Quenby S, Farquharson R. Uterine natural killer cells, implantation failure and recurrent miscarriage. Reprod Biomed Online 2006; 13(1):24–8.
Emmer PM, Nelen WL, Steegers EA, Hendriks JC, Veerhoek M, Joosten L. Peripheral natural killer cytotoxicity and CD56(pos) CD16(pos) cells increase during early pregnancy in women with a history of recurrent spontaneous abortion. Hum Reprod 2000; 15:1163–1169.
Thum MY, Haskaran SB, Bansal AS, Shehata H, Ford B, Sumar N, et al. Simple enumerations of peripheral blood natural killer (CD56+
NK) cells, B cells and T cells have no predictive value in IVF treatment outcome. Hum Reprod 2005; 20:1272–1276.
Gilman-Sachs A, Duchateau BK, AslaKson CJ, Wohlgmuth GP, Kwak JY, Beer AE, et al. Natural Killer (NK) cell subsets and NK cell cytotoxicity in women with histories of recurrent spontaneous abortions. Am J Reprod Immuno1999l; 41:99–105.
Quenby S, Kalumbi C, Bates M, Farquharson RG, Vince G. Prednisolone reduces preconceptual endometrial natural killer cells in women with recurrent miscarriage. Fertil Steril; 2005 84: 980–984.
Shimada S, Kato EH, Morikawa M, Iwabuchi K, Nishida R, Kishi R, et al. No difference in natural killer or natural killer T-cell population, but aberrant T-helper cell population in the endometrium of women with repeated miscarriage. Hum Reprod 2004; 19:1018–1024.
Quenby S, Nik H, Innes B, Lash G, Turner M, Drury J, et al. Uterine natural killer cells and angiogenesis in recurrent reproductive failure. Hum Reprod 2009; 24 (1): 45–54.
Pongcharoen S, Searle RF, Bulmer JN. Placental Fas and Fas ligand expression in normal early, term and molar pregnancy, Placenta 2004; 25:32I - 330.
Saito S. Cytokine network at the feto-maternal interface. J Reprod Immunol 2000; 47:87–103.
Bansal AS. Joining the immunological dots in recurrent miscarriage. Am J reprod Immunol 2010; 64:307–315.
Thum M, Bhaskaran S, Abdallah HI, Ford B, Sumar N, Bansal AS. Prednisolone suppresses nK cell cytotoxicity in vitro in women with a history of infertility and elevated nK cell cytotoxicity. Am J reprod Immunol; 2008 59:259–265.
Tang AW, Alfirevic Z, turner MA, Drury JA, Small R, Quenby S. A feasibility trial of screening women with idiopathic recurrent miscarriage for high uterine natural killer cell density and randomizing to prednisolone or placebo when pregnant. Hum Reprod 2013; 28:1743–175
Nybo Anderson AM, Wholfahrt J, Christens P, et al. Maternal age and fetal loss: population based register study. BMJ 2000; 320:1708–12.
Wang JX, Davies MJ, Norman RJ. Infertility Polycystic ovarian syndrome and the risk of spontaneous abortion following assisted reproductive technology treatment. Human Reproduction2001; 16 (12): 2606–2609.
Fedoresak P, Storeng R, Dale PO. Obesity is a risk factor for recurrent pregnancy loss after IVF or ICSI. Acta obstetrica et gynecologica Scandinavica 2000; 79:43–48.
Goddijn, M. and Leschot, NJ. Genetic aspects of miscarriage. Ballie re's Best Pract. Res. Obstet. Gynaecol 2000; 14: 855–865.
Stern JJ, Dorfmann MD, Gutierrez-Najar AJ, Cerrillo M, Coulam CB. Frequency of abnormal karyotypes among abortuses from women with and without a history of recurrent spontaneous abortion. Fertil. Steril 1996; 65: 250–253.
Carp H, Toddler V, Aviram A, Daniely M, Mashiach S, Barkai G. Karyotype of the abortus in recurrent miscarriage. Fertil Steril 2001; 75: 678–682.
Ogasawara M, Aoki K, Okada S, Suzumori K. Embryonic karyotype of abortuses in relation to the number of previous miscarriages. Fertil Steril 2000; 73: 300–304.
Stephenson M, Awartani KA, Robinson WP Cytogenetic analysis of miscarriages from couples with recurrent miscarriage: a case-control study. Hum Reprod 2002.; 17(2): 446–51.
Warburton D, Kline J, Stein Z, Hutzler M, Chin A, Hassold T. Does the karyotype of a spontaneous abortion predict the karyotype of a subsequent abortion? Evidence from 273 women with two karyotyped spontaneous abortions. Am J Hum Genet 1987; 41: 465– 483.
Clifford K, Flanagan AM, Regan L. Endometrial CD56_natural killer cells in women with recurrent miscarriage: a histomorphometric study. Hum Reprod 1999; 14:2727–30.
Emmer PM, Steegers EA, Kerstens HM, Bulten J, Nelen WL, Boer K, et al. Altered phenotype of HLA-G expressing trophoblast and decidual natural killer cells in pathological pregnancies. Hum Reprod 2002; 17:1072–80.
Quenby S, Bates M, Doig T, Brewster J, Lewis-Jones DI, Johnson PM, et al. Pre-implantation endometrial leukocytes in women with recurrent miscarriage. Hum Reprod 1999; 14: 2386–91.
Lachapelle MH, Miron P, Hemmings R, Roy DC. Endometrial TB and NK cells in patients with recurrent spontaneous abortion. Altered profile and pregnancy outcome. J Immunol 1996; 156: 4027–34.
Yamamoto T, Takahashi Y, Kase N, et al. Role of decidual Natural killer cells in patients with missed abortion: differences between cases with normal and abnormal chromosome. Clin Exp Immunol 1999; 116:449–52.
[Table 1], [Table 2], [Table 3]