Gynaecologist
Fertility IVF Specialist
Urogynaecologist
Natural Killer Cell in Recurrent Miscarriage Harm and Benefit
The potential benefit and harm of natural killer cell analysis in women with recurrent miscarriage
Recurrent Miscarriage (RM) is defined as three or more consecutivespontaneous abortions1. It affects 1% of child-bearing population2. Aetiologies include parental chromosomal abnormalities,uterine anomalies, antiphospholipid syndrome, endometrial infections, endocrine( luteal phase defect, thyroid dysfunction, uncontrolled diabetes mellitus ), inherited thrombophilias and alloimmune causes3,4,5 but only first three are universally accepted3,5. A cause can be identified in 50% of patients6; but, unknown in the remainder3. Natural killer (NK) cells ‘Immunological cause’ for unexplained RM has been proposed.
PBNK-Cells constitute 10% of lymphocytes. Their prototypic cell surface antigens are CD16 and CD56. CD16 receptor is responsible for NK-mediated antibody-dependent cellular cytotoxicity.CD56 is expressed on essentially all NK cells7.Based on intensity of CD56 expression, majority (90%) of PBNK cells are CD56dim and express high levels of CD16 and 10% are CD56bright and express low levels or no CD168. CD56dim cells are more cytotoxic, whereas the CD56bright subset is the main source of NK-cell derive immunoregulatory cytokines8.
NK-cellActivating Receptors include CD16 and killer-Inhibitory-Receptor (KIR) 2DS9. Interaction with IgG for CD16 and human leukocyte antigen (HLA)-C for KIR2DS transduce activating signals through transmembrane proteins10.
NK-cells Inhibitory Receptors interact with major histocompatibility complex (MHC)-I-bearingtargets and deliver inhibitory signals to NK-cells. Such receptors have been recently identified and consist of KIRs, which recognize mainly HLA-A, HLA-B, and HLA-C expressed on any host cell and CD94/NKG2 receptor, which recognizes the non-classical MHC-molecule HLA-E11.
During normal human pregnancyPBNK-cells particularly CD16+ subset decreasein number12,13 and lytic activity with increase of inhibitory receptors reaching a maximum in third month of gestation, with decline to basal levels by end of pregnancy14.
The changes are result of Progesterone direct action on PBNK-cells and/or through promoting Th2 cytokine and Progesterone-Induced-Binding-Factor production by T-cells15. It also facilitates PBNK-cell homing to endometrium, via inducing homing-receptors on PBNK-cells and addressins-receptors on endometrium. Regulation may occur throughdirect actions of oestrogen on ER-β in PBNK-cells or progesterone via an as yet unidentified receptor. Hormonal regulation abnormalities may underlie the changes occurring in the NK-cell populationin RM.
NK-cells have characteristic ability to lysetarget cells without prior sensitization and without restriction by HLA-antigens. The four functions are antiviral;anti-neoplastic, hematopoiesis regulation and graft-vs.-leukemia effect16. PBNK-cells express few cytokines; however when induced express granulocyte-macrophage-colony-stimulating-factor(GM-CSF), macrophage-CSF(M-CSF),IL-3, IFN-γ, TNF-α, and TNF-ß17,18,19. TNF-α induces apoptosisand IFN-γ induces additional immune response16. NK-cell function is mainly regulated by IL-2 and IFN-γ.IL-2 causes both NK-cell proliferation and enhanced cytotoxicity.IFN-γ augments NK cytolytic activity. Both cytokines act synergistically to augment NK cytotoxicity 16.
Leukocytes account for 10% of stromal cells in proliferative phase, 20% in secretory phase, and 30%in early pregnancy-decidua20.Uterine-NK-cells (large granulated lymphocytes)comprise over 70% of endometrial leukocytes in first-trimester-decidua20,25.
uNK-cells resemble PBNK-cells as they express CD56 as well as the killer activatory and inhibitoryreceptors but lack expression of other typical NK markers suchas CD16 or CD5721. In contrast to PBNK-cells, uNk-cells express CD69, an early activation marker 22,23. uNK are KIR+, CD69+, CD62L–,whereas CD56bright PBNK-cells are KIR–, CD69–,and CD62L+24. uNK-cells during pregnancy decrease expression of activation markers CD69, HLA-DR, leukocyte-function-associatedantigen-1 and CD45RA26 and express one or more inhibitory receptors in early pregnancy27.
Given the high levels of CD56 expression in uNK, it has been suggested that they derive from CD56bright PBNK-cells.
Data on uNK-cells numbers in second trimester are conflicting20. uNK-cells numbers decrease at term28.
uNK-cells originate from bone marrow, may enter endometrium via peripheral circulation ‘direct trafficking’23 or self-renew in-situ as 40% are proliferative20 with up-regulation genes 29 or uNK-cell precursors may be recruited from the spleen30.
Studies suggest that uNK-cells are hormonally regulated15. Progesterone was suggested as uNK-cells numbers increase in mid-luteal phase. uNK-cells do not express progesterone receptors but do express prolactin receptor31, oestrogen receptor-β and glucocorticoid receptors32. uNK-cells may be regulated by direct action of oestrogen on its receptor or progesterone via an as yet undiscovered receptor. Alternatively, uNK-cells could be regulated by an indirect mechanism whereby progesterone acts on endometrial T-cells and stromal cells, affecting Vascular-Endothelial-Growth-Factor and Macrophage-Inhibitory-Protein-1β to enhance recruitment of uNK-cells to the uterus, as well as acting through prolactin, via interleukin-15, to increase proliferation and differentiation of uNK-cells15 and production of cytokines and other molecules that support placental and trophoblast development and promote local immunomodulation15.
Perivascular location of uNK-cells may reflect trafficking from peripheral circulation33,34,35 or a role in decidualization of stroma20 or remodelling of the spiral arteries28 or promoting angiogenesis34,35. uNK-cells express GFs that are critical in angiogenesis, including VEGF29, PlaGF, angiopoeitin2 and NKT533. Activated uNK-cells secrete IFN-γ involved in development of spiral arteries36.
HLA-G expression is unique to invading cytotrophoblast. uNK-cells express receptors for HLA-G37. uNK-cells position in early pregnancy and their ability to express KIRs for HLA-G suggests that uNK-cells are involved in the regulation of trophoblast invasion and maternal-trophoblast signalling during early pregnancy. However, HLA-C is the dominant ligand for uNK-cell KIRs and recent evidence suggests that combinations of maternal KIRs on uNK-cells combined with specific polymorphisms for fetal HLA-C may be unfavourable to trophoblast cell invasion38.
In contrast to CD56+ CD16+ NK-cells, CD56+ CD16- NK-cells are potent secretors of cytokines but have low cytolytic ability and cytokine production is therefore another function of these cells.The precise function of CD56+ cells in the endometrium and decidua remains speculative.
In normal pregnancy cell-mediated Th1-immunity towardtrophoblast cells is suppressed. RM is associated with Th1 immunity, for which NK-cells are partly responsible39. Th1-immunity against trophoblast is inhibited by addition of progesterone to in-vitro PBMC/trophoblast co-cultures40. Preconceptional PBNK-cellactivity is increased in women with unexplained RM compared with controls41. PBNK-cell numbersdo not correlate with levels of NK-cell cytotoxicity42. PBNK-cell numbers and activity can fluctuatewith exercise and time ofday43,44. Increase in PBNK-cellnumbers and/or activity in luteal or early pregnancy of women with RM41,45 is of concern but additional studies are needed to confirm these findings.
CD69, an early activation marker, was significantly higher on PBNK-cells in women with RM or infertility of uncertain etiology compared with controls46. This Phenotype difference was significant across all subsets of NK cells studied (CD56dim, CD56bright,CD16neg). Compared with controls, women with RM did show significant decrease inexpression of KIR CD94/NKG2. Phenotypic changes are apparent in PBNK-cells in women with RM, which may account for their increasedactivity46.
It is assumed PBNK-cells and uNK-cells are similar. It is speculated that women with RM and infertility have abnormalities in uNK-cell function. It has been implied that these are discernible from analysis of PBNK-cells45,46,50.
The role of PBNK-cells in implantation failure has been questioned51,52. Firstly, utNK-cells are different from PBNK-cells as discussed earlier and examination of PBNK-cells will not tell us what is happening in the uterus. Secondly >12% NK cells in women with infertility or miscarriage has been arbitrarily defined as abnormally raised and used as an indication for treatment47even though percentage of CD56+ PBNK-cells in normal healthy individuals varies from 5%- 29%48 and affected by sex, ethnicity, stress, and age. Thirdly, PBNK-cells activity is measured by a range of assays and the results will vary in different laboratories. The most commonly used in-vitro assay is cytotoxicity, which may not have much relevance to PBNK-cells function in-vivo49. Certainly, in viral infection, PBNK-cells function mainly by producing cytokines. Furthermore, uNK-cells have much lower cytolytic activity than PBNK-cells. Thus, no clinically relevant information is gained from studying either the percentage or cytotoxicity of PBNK-cells in women with pregnancy failure.
uNK-cells in RM
Women with RM have an endometrial factor contributing to the pregnancy losses53.
There is likely no difference in the percentageof total uNK-cells in women with RMcompared with women without RM54,55. Using flow-cytometry CD16–CD56bright NK-cells were decreased and CD16+CD56dim NK-cells were increased in luteal-endometrium from women who had RM54 which may have important functional implications in women with RM. Immuno-histochemistry detected increased numbers of CD56+cells in midluteal-endometrium in RM compared with fertile controls56,57,58. Increased uNK-cells in women withRM were predictive of and associated with miscarriage in subsequent pregnancy57,58.
Previous live birth does not reduces uNK-cells to <5%58. Women with RM, luteal phase defect ‘LPD’ may be involved53, however; increased numbers of uNK-cells in RM were thought to be independent of LPD59. Detailed genetic study found no association between polymorphisms or numbers of KIRs on uNK-cells and RM60.
uNK-cells in RM were thought to be either hostile to invading trophoblast, or facilitating implantation of abnormal blastocysts leading to clinical presentation of RM61. It is doubtful that high numbers of uNk-cells are harmful to the trophoblast as uNk-cells are needed for normal pregnancy to occur, at least in mice62. uNK-cells are more likely to facilitate implantation being more numerous in deciduas from chromosomally abnormal miscarriages / RM than in chromosomally normal miscarriages /RM63,64.
Endometrial CD56+ cells in peri-implantation endometrium did not appear to have any useful prognostic value on the outcome of a subsequent pregnancy65 and questions the usefulness of measurement of uNK-cell numbers into routine clinical practice.
A recent study suggested that increased endometrial uNK-cell density noted in recurrent reproductive failure contributes to increased preimplantation angiogenesis leading to early maternal circulation and miscarriage by final common pathway of excessive oxidative stress66.
In recurrent IVF failure ‘with recurrent implantation failure’ preimplantation endometrium did show higher numbers of uNK-cells67,68, but high numbers of uNK-cells are more likely part of a complex array of immune and vascular abnormalities68.
In spite of the exact mechanism of unexplained RM is lacking; progesterone,heparin, aspirin, hCG, prednisone, Anti-TNF-alpha, leukocyte immunization, and IVIGare advocated69,70,71.
Progesterone treatment ‘early in pregnancy’ has beneficial effectin women with RM72. Women with Th1 immunity to trophoblastcan be treated effectively with progesteronebeginning 3d after ovulation70. Progesterone,can inhibit Th1 cytokine release and reduce embryotoxicity bytrophoblast-activated PBMC cultures from women with RM40. Ovarian stimulation with gonadotropins and progesteronein women with unexplained infertility results in a decrease in Th1 CD4+ cells, NK-cells, and NK-cellactivity73, with decrease inthe levels of plasma IFN-γ and IL-2 and an increase in TGF-ß173; higher numbers of successful pregnancies, however, remains to be determined through RCTs.
Heparin ‘anticoagulant’ suppresses NK-cell cytotoxicity74,75 and antagonizes IFN-γ action by inhibiting its bindingto the cell surface76,77.
It is interesting that Progesterone and Heparin two of the most commonly used the rapiesin RM affect NK-cell function.
Preconceptional prednisolone produced life birth in a woman with 10 miscarriages when given intrauterine78 and in a woman with elevated uNK-cells with 19 consecutive miscarriages 79. Prednisolone from day1-day21 reduces high numbers of uNK-cells in preimplantation endometrium in RM58. Prednisolone for 3 days reduces peripheral NK cell numbers80. A RCT of prednisolone for 4-weeks prior to ET reduced miscarriage rates (18 versus 23%)81.
A retrospective study of prednisolone and aspirin given for 5 weeks prior to IVF and ET found an apparent improvement in pregnancy rates in women with a possible autoimmune aetiology to their subfertility82. Prednisolone given at egg collection or ET did not improve pregnancy rates83,84. While immunomodulation of preimplantation endometrium-decidua opens new avenues for treatment of RM, RCTs are needed before prednisolone is used to treat RM52.
Immunotherapy for RM is controversial85,86,87,88.
Paternal Leukocyte immunizationand IV Immunoglobulins, have been tried as different approaches of immunomodulationto down-regulate the maternal immune response to the embryo.
Antipaternal lymphocytotoxic antibodies were proposed as a cause of RM89, but were found to be more prevalent in fertile couple90,91 with conclusion that antipaternal lymphocytotoxic antibodies have no effect on subsequent pregnancy outcome91,92.
Paternal Leukocyte immunization is based on the beneficial effects that donor or third party leukocytes have on allograft rejection in transplant patients and evidence that it may decrease the number circulating NK-cells in women with recurrent pregnancy loss93. Studies suggested small treatment benefit ‘8-10% improvement in live birth rate’ weighed against costs and risks including injection site reaction, myalgia, platelets and erythrocyte alloimunization94. Large RCT found no evidence that paternal Leukocyte immunization was effective in the treatment of unexplained RM95 and meta-analysis concluded the same87.
Maternal blocking antibody theory of RM presume maternal failure to recognize and respond to fetal antigens ‘mother and father are too anti-genically similar’ by production of blocking antibodies leaving the embryo exposed to a lethal cell-mediated immune rejection96. However HLA sharing does not predict pregnancy outcome and testing has no clinical utility97,98.
IV-Immunoglobulins regarded as immunosuppressive were proposed to treat and are prepared from pooled immunoglobulins. The treatment is costly, require multiple infusions during pregnancy and could transmit viruses and cause anaphylaxis99. IVIG has been tried with apparent success in a small number of women with recurrent implantation failure and a high degree of HLA similarity between the parents100.
Randomised trials of IVIG in treatment of unexplained RM failed to demonstrate improved outcome85,94,87. However, RCTs of this approach are needed to substantiate the efficacy of IVIG.
Conclusion
There is no convincing evidencethat reproductive failure occurs as a result of immune rejection. Contemporary concepts in reproductive immunology now emphasise the cooperative nature of the interaction between thematernal immune system and the feto-placental unit in governing pregnancy outcome101.
Abnormal response auto or alloimmune response has been postulated to underlie some cases of ‘unexplained’ infertility, IVF failure and RM. Despite little evidence, a variety of immune tests and treatments for reproductive failure have been introduced into clinical practice.
Measurement of PBNK-cell numbers or activity as a surrogate marker of events at the maternal–fetal interface is inappropriate. A recent large UK study reported PBNK-cell levels in predicting IVF cycle outcome to be ‘little better than tossing a coin’102.
Several studies linked RM with raised uNK-cell number in a preconception cycle and that these levels may be decreased with prednisolone58. However, an association between uNK cell number and function has not been demonstrated.
Autoantibody screening for lupus anticoagulant and anticardiolipin antibodies, are important treatable cause of recurrent miscarriage103. These two antibodies are increased among those with infertility and implantation failure following IVF-ET. However, a meta-analysis reports no relationship between LA and aCL status and implantation or clinical pregnancy rate among those undergoing IVF104. In IVF antiphosphotidyl ethanolamine and serine may be of more relevance than LA and aCL.
Alloimmune testing is not helpful with arguments against increased sharing of HLA between partners can lead to RM is well established105. Production of blocking antibody occurs after 28 weeks of gestation and successful pregnancies can occur despite failure of production of blocking antibody.
Immune treatments
Meta-analyses confirmed aspirin combined with heparin significantly increases live-birth rate among those with antiphospholipid syndrome106; the routine use of peri-implantation glucocorticoids does not increase the live-birth rate among those undergoing IVF107; white cell immunisation does not increase live-birth rate among those with RM105 and IVIG does not improve live birth rate among those with unexplained RM108.
There are no published data that anti-TNF agents infliximab and etanercept use improve IVF outcome. Anti-TNF-alpha agents can cause lymphomas, granulomatous and demyelinating disease.
Glucocorticoids during pregnancy may be associated with gestational diabetes, pre-eclampsia and rupture of membranes with preterm delivery52,109.
With the exception of aPL testing in RM, there is little evidence to support any particular test or immunomodulatory treatment in investigation and treatment of couples with reproductive failure.
We should protect our patients with reproductive failure, who are vulnerable, from the exploitation by those pedalling new tests and treatments that have little scientific validity52.
References
1. Stirrat G. Recurrent miscarriage. Lancet 1990: 336; 673–675.
2. Clifford K, Rai R, Watson H et al. An informative protocol for the investigation of recurrent miscarriage: preliminary experience of 500 consecutive cases. Hum Reprod 1994: 9; 1328–1332.
3. Lee R, Silver R. Recurrent pregnancy loss: summary and clinical recommendations. Semin Reprod Med 2000:18; 433–440.
4. Kutteh W. Recurrent pregnancy loss: an update. Curr Opin Obstet Gynecol 1999: 11; 435–439.
5. Jauniaux E, Farquharson R, Christiansen O et al. Evidence-based guidelines for the investigation and medical treatment of recurrent miscarriage, Human Reproduction 2006: 21; 2216–2222.
6. Stray-Pedersen B, Stray-Pedersen S. Etiologic factors and subsequent reproductive performance in 195 couples with a prior history of habitual abortion. Am J Obstet Gynecol 1984:148; 140–146.
7. Nagler A, Lanier L, Cwirla S et al. Comparative studies of human FcRIII-positive and negative natural killer cells. J Immunol 1989:143; 3183–3191.
8. Cooper M, Fehniger T, Caligiuri M. The biology of human natural killer-cell subsets. Trends Immunol 2001: 22; 633–640.
9. Lanier L. On guard–activating NK cell receptors. Nat Immunol 2001: 2;23–27.
10. Biassoni R, Cantoni C, Marras D et al. Human natural killer cell receptors: insights into their molecular function and structure. J Cell Mol Med 2003: 7; 376–387.
11. King A, Allan D, Bowen M et al. HLA-E is expressed on trophoblast and interacts with CD94/NKG2 receptors on decidual NK cells. Eur J Immunol 2000: 30; 1623–1631.
12.Beer A, Kwak J, Ruiz J. 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.
13.Gregory C, Lee H, Rees G et al. Natural killer cells in normal pregnancy: analysis using monoclonal antibodies and single-cell cytotoxicity assays. Clin Exp Immunol 1985: 62; 121–127.
14.Ponte M, Cantoni C, Biassoni R et al. Inhibitory receptors sensing HLA-G1 molecules in pregnancy: decidua-associated natural killer cells express LIR-1 and CD94/NKG2A and acquire p49, an HLA-G1-specific receptor. Proc Natl Acad Sci USA 1999: 96; 5674–5679.
15.Dosiou C & Giudice L. Natural killer cells in pregnancy and recurrent pregnancy loss: endocrine and immunologic perspectives. Endocrine Reviews 2005: 26; 44–62.
16. Robertson M, Ritz J. Biology and clinical relevance of human natural killer cells. Blood 1990: 76; 2421–2438.
17.Cuturi M, Anegon I, Sherman F et al. Production of hematopoietic colony-stimulating factors by human natural killer cells. J Exp Med 1989: 169; 569–583.
18.Naume B, Johnsen A, Espevik T et al. Gene expression and secretion of cytokines and cytokine receptors from highly purified CD56+ natural killer cells stimulated with interleukin-2, interleukin-7 and interleukin-12. Eur J Immunol 1993: 23; 1831–1838.
19.Biassoni R, Ferrini S, Prigione I et al. Activated CD3-CD16+ natural killer cells express a subset of the lymphokine genes induced in activated αß + and γδ+ T cells. Scand J Immunol 1991: 33; 247–252.
20. Bulmer J, Lash G. Human uterine natural killer cells: a reappraisal. Molecular Immunology 2005: 42; 511–521.
21. Saito S. Cytokine network at the feto-maternal interface. J Reprod Immunol 2000: 47; 87–103.
22. King A, Balendran N, Wooding P et al. CD3-leukocytes present in the human uterus during early placentation: phenotypic and morphologic characterization of the CD56++ population. Dev Immunol 1991: 1; 169–190.
23. King A, Loke Y. On the nature and function of human uterine granular lymphocytes. Immunol Today 1991:12; 432–435.
24. Moffett-King A. Natural killer cells and pregnancy. Nat Rev Immunol 2002: 2; 656–663.
25. Bulmer J, Morrison L, Longfellow M et al. Granulated lymphocytes in human endometrium: histochemical and immunohistochemical studies. Hum Reprod 1991: 6; 791–798.
26. Kodama T, Hara T, Okamoto E et al. 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.
27. Ponte M, Cantoni C, Biassoni R et al. Inhibitory receptors sensing HLA-G1 molecules in pregnancy: decidua-associated natural killer cells express LIR-1 and CD94/NKG2A and acquire p49, an HLA-G1-specific receptor. Proc Natl Acad Sci USA 1999: 96; 5674–5679.
28. Croy B, He H, Esadeg S et al. Uterine natural killer cells: insights into their cellular and molecular biology from mouse modelling. Reproduction 2003:126; 149–160.
29. Lobo S, Huang S, Germeyer A et al. The immune environment in human endometrium during the window of implantation. Am J of Reprod Immunol 2004: 52; 244–251.
30. Chantakru S, Miller C, Roach L et al. Contributions from self-renewal and traffi cking to the uNK cell population in early pregnancy. J of Immunol 2002:168; 22–28.
31. Gubbay O, Bowen J, Critchley H et al. Prolactin induces ERK phosphorylation in epithelial and CD56+ cells of the human endometrium. Journal of Clinical Endocrinology and Metabolism 2002: 87; 2329–2335.
32. Henderson T, Saunders P, Moffett-King A et al. Steroid receptor expression in uterine natural killer cells. Journal of Clinical Endocrinology and Metabolism 2003: 88; 440–449.
33. Trundley A, Moffett A. Human uterine leucocytes and pregnancy. Tissue Antigen 2004: 63; 1–12.
34. Van den Heuvel M, Horrocks J, Bashar S et al. Menstrual cycle hormones induce changes in functional interactions between lymphocytes and decidual vascular endothelial cells. Journal of Clinical Endocrinology and Metabolism 2005a: 90; 2835–2842.
35. Van den Heuvel M, Xie X, Tayade C et al. A review of trafficking and activation of uterine natural killer cells. American Journal of Reproductive Immunology 2005b: 54; 322–331.
36. Croy B, Esadeg S, Chantakru S et al. Update on pathways regulating the activation of uterine natural killer cells, their interactions with decidual spiral arteries and homing precursors to the uterus. J Reprod Immunol 2003: 59;175–191.
37. King A, Burrows T, Verma S et al. Human uterine lymphocytes. Hum Reprod Update 1998: 4; 480–485.
38. Moffet A, Hiby SE. How does the maternal immune system contribute to the development of pre-eclampsia? Placenta 2007: 28(Suppl A);S51–6,Epub Feb 8.
39. Yamada H, Polgar K, Hill J. Cell-mediated immunity to trophoblast antigens in women with recurrent spontaneous abortion. Am J Obstet Gynecol 1994: 170; 1339–1344.
40. Choi B, Polgar K, Xiao L et al. Progesterone inhibits in-vitro embryotoxic Th1 cytokine production to trophoblast in women with recurrent pregnancy loss. Hum Reprod 2000: 15(Suppl 1); 46–59.
41. Emmer P, Nelen W, Steegers E et al. 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.
42. Gilman-Sachs A, DuChateau B, Aslakson C et al. 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.
43. Nielsen H, Secher N, Christensen N et al. Lymphocytes and NK cell activity during repeated bouts of maximal exercise. Am J Physiol 1996: 271;R222–R227.
44. Terao K, Suzuki J, Ohkura S. Circadian rhythm in circulating CD16-positive natural killer (NK) cells in macaque monkeys, implication of plasma cortisol levels. Primates 2002: 43 ; 329 –338.
45. Aoki K, Kajiura S, Matsumoto Y et al. Preconceptional natural-killer-cell activity as a predictor of miscarriage. Lancet 1995: 345;1340–1342.
46. Ntrivalas E, Kwak-Kim J, Gilman-Sachs A et al. Status of peripheral blood natural killer cells in women with recurrent spontaneous abortions and infertility of unknown aetiology. Hum Reprod 2001: 16; 855–861.
47. Kwak J, Kwak F, Gilman-Sachs A et al. Immunoglobulin G infusion treatment for women with recurrent spontaneous abortions and elevated CD56+ natural killer cells. Early Preg 2000: 4; 154-164.
48. Bisset L, Lung T, Kaelin M et al. Reference values for peripheral blood lymphocyte phenotypes applicable to the healthy adult population in Switzerland. Eur J Haematol 2004:72; 203-12.
49. Biron C, Nguyen K, Pien G. Innate immune responses to LCMV infections: natural killer cells and cytokines. Curr Top Microbiol Immunol 2002: 263; 7-27.
50. Yamada H, Morikawa M, Kato E et al. Pre-conceptual natural killer cell activity and percentage as predictors of biochemical pregnancy and spontaneous abortion with normal chromosome karyotype. American Journal of Reproductive Immunology 2003: 40; 351–354.
51. Moffett A, Regan L, Braude P. Natural killer cells, miscarriage and infertility. British Medical Journal 2004: 329; 1283–1285.
52. Rai R, Sacks G, Trew G. Natural killer cells and reproductive failure-theory practice and prejudice. Human Reproduction 2005: 20; 1123–1126.
53. Li T, Tuckerman E, Laird S. Endometrial factor in recurrent miscarriage Human Reproduction Update 2002: 8; 43–52.
54. Lachapelle M, Miron P, Hemmings R et al. Endometrial T, B, and NK cells in patients with recurrent spontaneous abortion. Altered profile and pregnancy outcome. J Immunol 1996: 156; 4027–4034
55. Shimada S, Kato E, Morikawa M 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.
56. Clifford K, Flanagan A, Regan L. Endometrial CD56+ natural killer cells in women with recurrent miscarriage: a histomorphometric study. Human Reproduction 1999:14; 2727–2730.
57. Quenby S, Bates M, Doig T et al. Pre-implantation endometrial leukocytes in women with recurrent miscarriage. Human Reproduction 1999: 14; 2386–2391.
58. Quenby S, Kalumbi C, Farquharson R et al. Prednisolone reduces pre-conceptual endometrial natural killer cells in women with recurrent miscarriage. Fertility and Sterility 2005: 84; 980–984.
59. Tuckerman E, Laird S, Stewart R et al. Markers of endometrial function in women with unexplained recurrent pregnancy loss: a comparison between morphologically normal and retarded endometrium. Human Reproduction 2004: 19; 196–205.
60. Witt C, Goodridge J, Gerbase-Delima M et al. Maternal KIR is not associated with recurrent spontaneous abortion. Human Reproduction 2004: 19; 2653–2657.
61. Quenby S, Vince G, Farquharson R et al. Recurrent miscarriage: a defect in nature’s quality control? Human Reproduction 2002: 17; 1959–1963.
62. Guimond M, Wang B, Croy B. Engraftment of bone marrow from severe combined immunodeficient (SCID) mice reverses the reproductive deficits in natural killer cell-deficient tgε26 mice. Journal of Experimental Medicine 1998: 187; 217–223.
63. Yamamoto T, Takahashi Y, Kase N et al. Role of decidual natural killer NK cells in patients with missed abortion: differences between cases with normal and abnormal chromosomes. Clinical and Experimental Immunology 1999: 116; 449–452.
64. Quack K, Vassiliadou N, Pudney J et al. Leukocyte activation in the decidua of chromosomally normal and abnormal foetuses from women with recurrent abortion. Human Reproduction 2001: 16; 949–955.
65. Tuckerman E, Laird S, Prakash A et al. Prognostic value of the measurement of uterine natural killer cells in the endometrium of women with recurrent miscarriage Human Reproduction 2007: 22; 2208–2213.
66. Quenby S, Nik H, Innes B et al. Uterine natural killer cells and angiogenesis in recurrent reproductive failure. Human Reproduction 2009: 24; 45–54.
67. Laird S, Tuckerman E, Prakash A et al. Endometrial CD56+ cells and implantation failure after IVF. Placenta 2005: 26; A26.
68. Ledee-Bataille N, Bonet-Chea K, Hosny G et al. Role of the endometrial tripod interleukin-18, -15 and -12 in inadequate uterine receptivity in patients with a history of repeated in vitro fertilisation−embryo transfer failure. Fertility and Sterility 2005: 83; 598–605.
69. Lee R, Silver R. Recurrent pregnancy loss: summary and clinical recommendations. Semin Reprod Med 2000: 18;433–440.
70. Hill J. Recurrent pregnancy loss. 1999 In: Creasy RK, Resnik R, eds. Maternal-fetal medicine. 4th ed. Philadelphia: W.B. Saunders Company; 423–443
71. Rai R, Regan L. 2002 The endometrium in recurrent miscarriage. In: Glasser S, Aplin J, Giudice L, Tabibzadeh S, eds. The endometrium. New York: Taylor, Francis; 546–555
72. Daya S. Efficacy of progesterone support for pregnancy in women with recurrent miscarriage. A meta-analysis of controlled trials. Br J Obstet Gynaecol 1989: 96; 275–280.
73. Fornari M, Sarto A, Berardi V et al. Effect of ovaric hyper-stimulation on blood lymphocyte subpopulations, cytokines, leptin and nitrite among patients with unexplained infertility. Am J Reprod Immunol 2002: 48; 394–403.
74. Yamamoto H, Fuyama S, Arai S et al. Inhibition of mouse natural killer cytotoxicity by heparin. Cell Immunol 1985: 96; 409–417.
75. Johann S, Zoller C, Haas S et al. Sulfated polysaccharide anticoagulants suppress natural killer cell activity in vitro. Thromb Haemost 1995: 74; 998–1002.
76. Fritchley S, Kirby J, Ali S. The antagonism of interferon-γ (IFN-γ) by heparin: examination of the blockade of class II MHC antigen and heat shock protein-70 expression. Clin Exp Immunol 2000: 120; 247–252.
77. Douglas M, Rix D, Dark J et al. Examination of the mechanism by which heparin antagonizes activation of a model endothelium by interferon-γ (IFN-γ). Clin Exp Immunol 1997: 107; 578–584.
78. Ogasawara M, Aoki K. Successful uterine steroid therapy in a case with a history of ten miscarriages. American Journal of Reproductive Immunology 2000: 44; 253–255.
79. Quenby S, Farquharson R, Young M et al. Successful pregnancy outcome following 19 consecutive miscarriages. Human Reproduction 2003: 18; 2562–2564.
80. Pountain G, Keogan M, Hazleman B et al Effects of single dose compared to three days’ prednisolone treatment of healthy volunteers: contrasting effects on circulating lymphocyte subsets. Journal of Clinical Pathology 1993: 46; 1089–1092.
81. Ubaldi F, Rienzi L, Ferrero S et al. Low dose prednisolone administration in routine ICSI patients does not improve pregnancy and implantation rates. Human Reproduction 2002: 17; 1544–1547.
82. Hasegawa I, Yamanoto Y, Suzuki M et al. Prednisolone plus low-dose aspirin improves the implantation rate in women with autoimmune conditions who are undergoing in vitro fertilization. Fertility and Sterility 1998: 70; 1044–1048.
83. Lee K, Koo J, Yoon T et al. Immunosuppression by corticosteroid has no effect on the pregnancy rate in routine invitro fertilization/embryo transfer patients. Human Reproduction 1994: 9; 1832–1835.
84. Moffitt D, Queenan J Jr, Veeck L et al. Low-dose glucocorticoids after in vitro fertilization and embryo transfer have no signifi cant effect on pregnancy rate. Fertility and Sterility 1995: 63; 571–577.
85. Daya S, Gunby J. The effectiveness of allogeneic leukocyte immunization in unexplained primary recurrent spontaneous abortion. Recurrent Miscarriage Immunotherapy Trialists Group. American Journal of Reproductive Immunology 1994: 32; 294–302.
86. Carp H, Toder V, Torchinsky A et al. Allogenic leukocyte immunization after fi ve or more miscarriages. Recurrent Miscarriage Immunotherapy Trialists Group. Human Reproduction 1997:12; 250–255.
87. Scott J. Immunotherapy for recurrent miscarriage (Cochrane Review). The Cochrane Library. 2003
88. Pandey M, Thakur S, Agrawal S. Lymphocyte immunotherapy and its probable mechanism in the maintenance of pregnancy in women with recurrent spontaneous abortion. Archives of Gynecology and Obstetrics 2004: 269; 161–172.
89. McIntyre J, Faulk W, Nichols-Johnson V et al. Immunologic testing and immunotherapy in recurrent spontaneous abortion. Obstet Gynecol 1986: 67(2); 169–175.
90. Berke J, Johansson K. The formation of HLA antibodies in pregnancy: The antigenicity of aborted and term foetuses. J Obstet Gynaecol Br Commonw 1974: 81; 222.
91. Coulam C. Immunologic tests in the evaluation of reproductive disorders: a critical review.Am J Obstet Gynecol. 1992:167(6); 1844-1851
92. Cowchock F and Smith J. Predictors for livebirth after unexplained spontaneous abortion: correlations between immunologic test results, obstetric histories, and outcome of next pregnancy without treatment. Am J Obstet Gynecol 1992: 167; 1208–1212.
93. KwakJ, Gilman-Sachs A, Moretti M et al. Naturalkiller cell cytotoxicity and paternal lymphocyteimmunizationin womenwithrecurrentspontaneous abortions. AmJReprod Immunol 1998: 40; 352–358.
94. Porter T, Scott J.Alloimmune causes of recurrent pregnancy loss. Semin Reprod Med. 2000:18(4): 393-400.
95. Ober C; Karrison T; Odem R et al. Mononuclear-cell immunisation in prevention of recurrent miscarriages: a randomised trial. Lancet 1999: 354; 365-369.
96. Beer A, Quebbeman J, Ayers J et al. Major histocompatibility complex antigens, maternal and paternal immune responses, and chronic habitual abortions in humans. Am J Obstet Gynecol 1981: 141(8): 987–999.
97. Ober C, Hyslop T, Elias S et al. Human leukocyte antigen matching and fetal loss: Results of a 10 year prospective study. Human Reproduction 1998: 13; 33-38.
98. Sbraci M, Mastrone M, Scarpellini F et al. Influence of histocompatibility antigrens in recurrent spontaneous abortion couples and on their reproductive performances. Am J Reprod Immunol 1996: 35; 85.
99. Thornton C, Ballow M; Safety of intravenous immunoglobulin; Arch Neurol; 1993: 50(2); 135-136.
100. Elram T, Simon A, Isreal S et al. Treatment of recurrent IVF failure and human leucocyte antigen similarity by intravenous immunoglobulin. Reprod Biomed Online 2005:11; 745–749.
101. Parham P. NK cells and trophoblasts: partners in pregnancy. J Exp Med 2004: 200; 951–955.
102. Thum M, Bhaskaran S, Abdalla H et al. 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.
103. Rai R, Cohen H, Dave M et al. Randomised controlled trial of aspirin and aspirin plus heparin in pregnant women with recurrent miscarriage associated with phospholipid
antibodies (or antiphospholipid antibodies). BMJ 1997: 314; 253–257.
104. Hornstein M, Davis O, Massey J et al. Antiphospholipid antibodies and in vitro fertilization success: a meta-analysis. Fertil Steril 2000: 73; 330–333.
105. Porter T, LaCoursiere Y, Scott J. Immunotherapy for recurrent miscarriage. Cochrane Database Syst Rev 2006: (19);CD000112.
106. Empson M, Lassere M, Craig J et al. Prevention of recurrent miscarriage for women with antiphospholipid antibody or lupus anticoagulant. Cochrane Database Syst Rev 2005:(2);CD002859.
107. Boomsma C, Keay S, Macklon N. Peri-implantation glucocorticoid administration for assisted reproductive technology cycles. Cochrane Database Syst Rev 2007:(1);CD005996.
108. Hutton B, Sharma R, Fergusson D et al. Use of intravenous immunoglobulin for treatment of recurrent miscarriage: a systematic review. BJOG 2007:114;134–142.
109. Laskin C, Bombardier C, Hannah M et al. Prednisone and aspirin in women with autoantibodies and unexplained fetal loss. N Engl J Med 1997: 337;148–153.