Activation of NK cells in women with impaired fertility

Woman infertility can have many causes, including immunity disorders. In the National register of assisted reproduction of the Czech Republic, the immunopathology represents major cause of infertility in approximately 6% of cases, which represents a considerable number of patients. Immunological examination of infertility has become an important topic (1).

Flow cytometry serves as one of the basic methods, that are being used to identify potential immunity disorder regarding woman infertility. It is focused mainly on NK cells and NKT cells, that play important roles not only in anti-cancer and anti-virus immunity, but also in the process of communication between embryo and endometrial tissue. Higher numbers and higher activity of NK cells in the vicinity of chorion can result in unsuccessful pregnancy. Despite repeated discussions about the importance of peripheral NK cell testing in the diagnosis of infertility, several studies have confirmed that increased numbers of NK and NKT cells are associated with reproductive failure and habitual abortion. Although NK cells in the peripheral blood do not fluctuate during the menstrual cycle, their number and activity decrease during pregnancy. In women with repeated abortions, a higher proportion of NK cells and higher cytotoxicity were found at the time of embryo transfer and implantation (3,4).

Cytometric determination of NK cell activation using the NKFlowEx kit can be used to determine the functional activity of NK cells. The test is based on the detection of the activating antigen CD69 on the surface of NK lymphocytes, resp. NKT cells after a peripheral blood sample is cultured in vitro for 24-48 hours with various stimulants (mitogens, trophoblast, sperm, etc.). After incubation, the expression of the CD69 marker is determined and its expression level is compared with that found in a sample incubated under the same conditions but without the presence of a stimulant (2). 

In our study, we focused on comparing the results of the NK and NKT cell activation test in a group of infertile women treated for abnormal numbers of NK / NKT peripheral blood cells at the GENNET and ISCARE assisted reproduction clinics in Prague in 2018-2020 (2). In these women, we compared the results of the NK / NKT cell activation test before and after immunointerventional treatment and also found out whether the patients became pregnant after therapy. Immunomodulatory therapy contributed to the clinical pregnancy of 109 of 184 women (59%), with 25 women becoming pregnant naturally and the remaining women using one of the assisted reproduction techniques. Women who became pregnant had significantly lower numbers of circulating NK cells (p = 0.03) and were found to have significantly lower trophoblast NK cell activation (p <0.001).
    


Fig.1: Example of NK cell activation test results of two women with impaired fertility. First patient (above) showed no reaction of NK cells to the antigens of sperms or to the cells of placenta (simulated by choriocarcinoma JAR cells), although her NK cells were normally activated by PWM in positive control. Second patient (below) showed reaction of NK cells to JAR cells, suggesting that infertility of this woman may be caused by immunity disorder represented by pathological recognition of chorionic antigens as alien ones. NK cell activation test performed by flow cytometry using NKFlowEx kit (cat. no. ED7078).

Conclusion:

Increased number of NK cells in the peripheral blood and their pathological activation by antigens of the trophoblast can be one of causes leading to woman infertility. Adequately chosen immunomodulation increases the chance for successfull pregnancy.
 

REFERENCES:

1. Malíčková K, Ambrusová Z, Belvončíková S, Bergendiová K, Krátká Z, Lešťan M, Luxová Š, Novák J, Tibenská E, Vydláková J, Sedláčková L. Current possibilities of diagnostics and treatment of immunological causes of female infertility (Současné možnosti diagnostiky a léčby imunologických příčin ženské neplodnosti). Cas Lek Cesk. 2021 Winter;160(1):5-13. English. PMID: 33823598.

2. Malíčková K, Luxová Š, Krátká Z, Sedláčková L. Circulating NK and NKT cells in the diagnosis and treatment of immunological causes of female infertility - retrospective data analysis from the tertiary clinical center (Vyšetření NK a NKT buněk v diagnostice a léčbě imunologických příčin ženské neplodnosti - retrospektivní analýza dat terciárního klinického centra). Cas Lek Cesk. 2021 Winter;160(1):27-32. English. PMID: 33823601.

3. Ebina Y, Nishino Y, Deguchi M, et al. Natural killer cell activity in women with recurrent miscarriage: Etiology and pregnancy outcome. J Reprod Immunol. 2017; 120: 42-47. 4. Karami N, Boroujerdnia MG, Nikbakht R, et al. Enhancement of peripheral blood CD56(dim) cell and NK cell cytotoxicity in women with recurrent spontaneous abortion or in vitro fertilization failure. J Reprod Immunol. 2012; 95(1-2): 87-92.

Zuzana Kratka, Ph.D.

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GENNET, s.r.o.
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EXBIO PRODUCTS RELATED TO REPRODUCTION IMMUNOLOGY:
ApoFlowEx kit
SpermFlowEx kit
NKFlowEx kit
TregFlowEx kit
Premixed antibody combinations - Immunophenotyping
Reproductive immunology section