akusherstvoObstetrics, Gynecology and ReproductionАкушерство, Гинекология и Репродукция2313-73472500-3194IRBIS LLC10.17749/2313-7347/ob.gyn.rep.2021.200akusherstvo-935Research ArticleOriginal articlesОРИГИНАЛЬНЫЕ ИССЛЕДОВАНИЯDiagnostic significance of assessing peripheral natural killer cells in women with recurrent pregnancy lossДиагностическая значимость натуральных киллеров периферической крови при привычном невынашивании беременностиГоциридзеК. Э.GotsiridzeK. E.

Гоциридзе Кетеван Элгуджевна - врач акушер-гинеколог, руководитель Центра репродуктивного здоровья «Клиники Чачава».Тбилиси 0179, ул. Мераба Костава, д. 38.

Ketevan E. Gotsiridze - MD, Obstetrician-Gynecologist, Head of the Center for Reproductive Health, Chachava Clinic.38 Merab Kostava Str., Tbilisi 0179.

k.gotsiridze@chachava.geКинтраяН. П.KintrayaN. P.

Кинтрая Николоз Паликоевич - доктор медицинских наук, профессор, академик АМН Грузии, президент Ассоциации акушеров-гинекологов и перинатологов Грузии, заведующий кафедрой акушерства и гинекологии ТГМУ; руководитель гинекологического сектора «Клиники Чачава».Тбилиси 0179, ул. Мераба Костава, д. 38; 0171 Тбилиси, ул. Михаила Асатиани, д. 7.

Nikoloz P. Kintraya - MD, Dr Sci Med, Academician of the Georgian Academy of Medical Sciences, President of the Association of Obstetricians-Gynecologists and Perinatologists of Georgia, Head of the Department of Obstetrics and Gynecology, Tbilisi State Medical University; Head of the Gynecological Sector, Chachava Clinic.38 Merab Kostava Str., Tbilisi 0179; 7 Mikhail Asatiani Str., Tbilisi 0171.

Клиника ЧачаваГрузияChachava ClinicGeorgiaКлиника Чачава; Тбилисский государственный медицинский университетГрузияChachava Clinic; Tbilisi State Medical UniversityGeorgia2021060320211516169Copyright © Gotsiridze K.E., Kintraya N.P., 20212021Гоциридзе К.Э., Кинтрая Н.П.Gotsiridze K.E., Kintraya N.P.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://www.gynecology.su/jour/article/view/935

Aim: to assess peripheral blood NK cell (pNK) counts in women with recurrent pregnancy loss.Materials and Methods. There were examined 102 women: 28 non-pregnant (immediately after repeatedly terminated pregnancy, group IA) and 34 pregnant women with history of previous recurrent pregnancy loss (group IB). Control and comparison groups were consisted of healthy women lacking any recorded miscarriage: 20 non-pregnant (group IIA) and 20 pregnant women (group IIB), respectively. Along with assessing complete blood count range, all subjects were analyzed percentage of pNK cell CD16bright(+) CD56dim(-) cytotoxic phenotype by using fluorescence microscopy as well as level of cytokines IL-2, IL-6, IL-10, tumor necrosis factor (TNF-α), vascular endothelial growth factor (VEGF) and interferon-gamma (IFN-γ) using ELISA.Results. It was found that the level of NK cell cytotoxic phenotype - percentage of CD16+CD56- was elevated in both groups: groups: group IA - 36.5 % collared with the control group IIA - 27.5% (р < 0.05); in group IB up to 37.0 %, in comparison group IIB - 27.4% (р < 0.01). It was further corroborated by increased level of serum cytokine IL-6 IL-6 comprising in group IA vs. IIA - 28.5 pg/ml vs. 14.2 pg/ml (р < 0.01) as well as in group IB vs. IIB up to 16.7 pg/ml vs. 12.2 pg/ml (р < 0.01), respectively. However, level of serum IL-2 and IL-10 did not significantly differ in all groups examined. Interestingly, level of VEGF in subjects aged 21-30 years was elevated: in non-pregnant (group IA) vs. control (group IIA) - up to 548.5 pg/ml vs. 310.8 pg/ml (р < 0.01); pregnant women (IB) vs. comparison group (IIB) - insignificantly elevated up to 476.1 pg/ml vs. 381.5 pg/ml, respectively. In contrast, no significant changes were observed in 31-40-year group. Concentration of IFN-γ in group IA vs. control group (IIA) was increased up to 9.2 pg/ml vs.8.64 pg/ml; group IB vs. comparison group - up to 8.36 pg/ml vs. 7.56 pg/ml, respectively.Conclusion. Elevated percentage of cytotoxic CD16+CD56- NK cells in peripheral blood results in imbalance of immune-related parameters that directly correlated with increased serum level of IL-2, IL-6, TNF-α that may alter maternal immune tolerance to the fetus and subsequently resulting in recurrent pregnancy loss. Hence, the aforementioned data may be used as diagnostic and prognostic criteria in recurrent pregnancy losses.

Цель исследования: изучение содержания натуральных киллеров периферической крови (pNK) и определение их диагностической ценности у женщин с привычным невынашиванием беременности.Материалы и методы. Обследовано 102 женщины: 28 небеременных (непосредственно после прерывания беременности, группа IA) и 34 беременных, имевших в анамнезе привычное невынашивание беременности (группа IB); группы контроля и сравнения представлены здоровыми женщинами, не имевшими в анамнезе невынашивание беременности: 20 небеременных (группа НА) и 20 беременных (группа НВ). Всем женщинам кроме общеклинического обследования в периферической крови определено содержание токсического фенотипа pNK клеток CD16bright(+)CD56dim(-) методом флуоресцентной микроскопии, интерлейкинов IL-2, IL-6, IL-10, фактора некроза опухоли (TNF-α), сосудистого эндотелиального фактора роста (VEGF) и интерферона гамма (IFN-γ) методом иммуноферментного анализа.Результаты. Выявлена высокая активность цитотоксического фенотипа NK клеток (CD16+CD56-) в обеих основных группах: в группе IA - 36,5 % по сравнению с группой контроля IIA- 27,5 % (р < 0,05); в группе IB - 37,0 %, в группе сравнения IIB - 27,4 % (р < 0,01). Этот факт подтвердился динамикой других иммунологических параметров, индуцируемых NK клетками. Так, у небеременных и беременных с привычным невынашиванием беременности в анамнезе установлено достоверное повышение содержания IL-6: в группе IA - до 28,5 пг/мл по сравнению с контрольной группой IIA, где его содержание составило 14,0 пг/мл; в группе IB - до 16,7 пг/мл, в группе сравнения IIB - 12,2 пг/мл (р < 0,01). Содержание интерлейкинов IL-2 и IL-10 значимо не различалось во всех исследованных группах. Содержание TNF-a было повышено в обеих группах: в группе IA - 39,0 пг/мл, в контрольной группе IIA - 27,5 пг/мл (р < 0,05); в группе IB - 35,1 пг/мл, в группе сравнения IIB - 23,4 пг/мл (р < 0,01). Содержание VEGF у обследованных в возрасте 21-30 лет было повышено: до 548,5 пг/мл у небеременных (группа IA), в контрольной группе IIA - до 310,8 пг/мл (р < 0,01); у беременных (группа IB) - до 476,1 пг/мл, в группе сравнения IIB - до 381,5 пг/мл (различия несущественны, англ. non significant, NS). Для пациенток в возрасте 32-40 лет различия в содержании VEGF были несущественны (NS). Концентрация IFN-y была увеличена в группе IA до 9,2 пг/мл, в контрольной группе IIA - до 8,64 пг/мл, в группе IB - до 8,36 пг/мл, в группе сравнения - до 7,56 пг/мл, т. е. различия в содержании IFN-γ во всех группах были несущественны (NS).Заключение. Повышение в крови женщин с привычным невынашиванием беременности в анамнезе содержания натуральных киллеров с цитотоксическим фенотипом CD16+CD56- приводит к дисбалансу иммунологических параметров и находится в прямой корреляции с повышением активности интерлейкинов IL-2, IL-6 и TNF-α, что вызывает нарушение иммунологической толерантности по отношению к плоду и может явиться причиной прерывания беременности на ранних этапах. Это дает возможность использовать полученные параметры в качестве диагностического и прогностического критерия при привычном невынашивании беременности.

привычное невынашиваниенатуральные киллеры CD16bright(+)CD56dim(-)интерлейкиныфактор некроза опухолиTNF-αсосудистый эндотелиальный фактор ростаVEGFинтерферон гаммаIFN-γrecurrent pregnancy lossCD16bright(+)CD56dim(-) NK cellsinterleukinstumor necrosis factorTNF-αvascular endothelial growth factorVEGFinterferon-gammaIFN-γReferencesСидельникова В.М., Сухих Г.Т. Невынашивание беременности. М.: МИА, 2010. 534 с.Sidelnikova V.M., Sukhikh G.T. Miscarriage. Guideline. [Nevynashivanie beremennosti]. Moscow: MIA, 2010. 534 p. (In Russ.).Radovic Janoevic D., Popovic J., Kkrstic M. et al. The structure of immunocompetent decidual cells in recurrent missed abortions. Vojnosanit Pregl. 2016;73(4):306-11. https://doi.org/10.2298/VSP141226018R.Radovic Janoevic D., Popovic J., Kkrstic M. et al. The structure of immunocompetent decidual cells in recurrent missed abortions. Vojnosanit Pregl. 2016;73(4):306-11. https://doi.org/10.2298/VSP141226018R.Sharma S. Natural killer cells and regulatory T cells in early pregnancy loss. Int J Der Biol. 2014;58:219-29. https://doi.org/10.1387/ijdb.140109ss.Sharma S. Natural killer cells and regulatory T cells in early pregnancy loss. Int J Der Biol. 2014;58:219-29. https://doi.org/10.1387/ijdb.140109ss.Kuon R.J., Vomstein K., Weber M et al. The “killer cell story” in recurrent miscarriage: association between activated peripheral lymphocytes and uterine natural killer cells. J Reprod Immunol. 2017;119:9-14. https://doi.org/10.1016/j.jri.2016.11.002.Kuon R.J., Vomstein K., Weber M et al. The “killer cell story” in recurrent miscarriage: association between activated peripheral lymphocytes and uterine natural killer cells. J Reprod Immunol. 2017;119:9-14. https://doi.org/10.1016/j.jri.2016.11.002.Fukuli A., Finamizu A., Fikuhara R., Shibahara H. Expression of natural cytotoxicity receptors and cytokine production on endometrial natural killer cells in women with recurrent pregnancy loss or implantation failure and the expression of natural cytotoxicity receptors on peripheral blood natural killer in pregnant women with history of recurrent pregnancy loss. J Obstret Gynaecol Res. 2017;43(11):1678-86. https://doi.org/10.1111/jog13448.Fukuli A., Finamizu A., Fikuhara R., Shibahara H. Expression of natural cytotoxicity receptors and cytokine production on endometrial natural killer cells in women with recurrent pregnancy loss or implantation failure and the expression of natural cytotoxicity receptors on peripheral blood natural killer in pregnant women with history of recurrent pregnancy loss. J Obstret Gynaecol Res. 2017;43(11):1678-86. https://doi.org/10.1111/jog13448.Carayannopolos L., Barks J.L., Yokoyama W.M., Riley J.K. Murine trophoblast cells induce NK cell interferon-gamma production through KLRK1. Biol Reprod. 2010;83(3):404-14. https://doi.org/10.1095/biolreprod.110.084509.Carayannopolos L., Barks J.L., Yokoyama W.M., Riley J.K. Murine trophoblast cells induce NK cell interferon-gamma production through KLRK1. Biol Reprod. 2010;83(3):404-14. https://doi.org/10.1095/biolreprod.110.084509.Ratsep M.T., Felker A.M., Kay V.R. et al. Uterine natural killer cells; supervisors of vasculature construction in early delidua basalis. Reproduction. 2015;149(2):R91-102. https://doi.org/10.1530/REP-14-0271.Ratsep M.T., Felker A.M., Kay V.R. et al. Uterine natural killer cells; supervisors of vasculature construction in early delidua basalis. Reproduction. 2015;149(2):R91-102. https://doi.org/10.1530/REP-14-0271.Абакушина Е.В., Кузьмина Е.Г., Коваленко Е.И. Основные свойства и функции NK-клеток человека. Иммунология. 2012;33(4):220-4.Abakushina E.V., Kuzmina E.G., Kovalenko E.I. The maim characteristics of human natural killer cells. [Osnovnye svojstva i funkcii NK kletok cheloveka]. Immunologiya. 2012;33(4):220-4. (In Russ.).Prado-Draver A., Teppa J., Sonshez P., Cameio M.I. Immunophenotype of peripheral T lymphocytes, NK cells and expression of CD69 activation marker in patients with recurrent spontaneous abortions, during the mid-luteal phase. Am J Reprod Immunol. 2008;60(1):66-74. https://doi.org/10.1111/j.1600-0897.2008.00591.x.Prado-Draver A., Teppa J., Sonshez P., Cameio M.I. Immunophenotype of peripheral T lymphocytes, NK cells and expression of CD69 activation marker in patients with recurrent spontaneous abortions, during the mid-luteal phase. Am J Reprod Immunol. 2008;60(1):66-74. https://doi.org/10.1111/j.1600-0897.2008.00591.x.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;120:42-77. https://doi.org/10.1016/j.jri.2017.04.005.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;120:42-77. https://doi.org/10.1016/j.jri.2017.04.005.Seshardi S., Sunkara S.K. Natural killer cells in female infertility and recurrent miscarriage: asystematic review and meta-analysis. Hum Reprod Update. 2014;20(3):429-38. https://doi.org/10.1093/humupd/dmt056.Seshardi S., Sunkara S.K. Natural killer cells in female infertility and recurrent miscarriage: asystematic review and meta-analysis. Hum Reprod Update. 2014;20(3):429-38. https://doi.org/10.1093/humupd/dmt056.Vargas R.G., Bompeixe E.P., Fransa P.P. et al. Activating killer cell immunoglobulin-like receptor genes association with recurrent miscarriage. Am J Reprod Immunol. 2009;62(1):34-43. https://doi.org/10.1111/j.1600-0897.2009.00709.x.Vargas R.G., Bompeixe E.P., Fransa P.P. et al. Activating killer cell immunoglobulin-like receptor genes association with recurrent miscarriage. Am J Reprod Immunol. 2009;62(1):34-43. https://doi.org/10.1111/j.1600-0897.2009.00709.x.Sacks G., Yang Y, Gowen E. et al. Detailed analysis of peripheral blood natural killer cells in women with repeated IVF failure. Am J Reprod Immunol. 2012;67(5):434-43. https://doi.org/10.1111/j.1600-0897.2009.00709.x.Sacks G., Yang Y, Gowen E. et al. Detailed analysis of peripheral blood natural killer cells in women with repeated IVF failure. Am J Reprod Immunol. 2012;67(5):434-43. https://doi.org/10.1111/j.1600-0897.2009.00709.x.Hadinedoushan H., Mirahmadian M., Aflatonian A. Increased natural killer cell cytotoxicity and IL-2 production in recurrent spontaneous abortion. Am J Reprod Immunol. 2007;58(5):409-14. https://doi.org/10.1111/j.1600-0897.2007.00524.x.Hadinedoushan H., Mirahmadian M., Aflatonian A. Increased natural killer cell cytotoxicity and IL-2 production in recurrent spontaneous abortion. Am J Reprod Immunol. 2007;58(5):409-14. https://doi.org/10.1111/j.1600-0897.2007.00524.x.Ivanov P., Lukanov T., Konova E. et al. Activation of peripheral natural killer cells in women with repeated early pregnancy loss. Akush Ginekol (Sofia). 2015;54(8):3-7. [Article in Bulgarian].Ivanov P., Lukanov T., Konova E. et al. Activation of peripheral natural killer cells in women with repeated early pregnancy loss. Akush Ginekol (Sofia). 2015;54(8):3-7. [Article in Bulgarian].Saito S., Nakashima A., Shima T. Future directions of studies for recurrent miscarriage associated with immunol etiologies. J Reprod Immunol. 2011;90(1):91-5. https://doi.org/10.1016/j.jri.2011.03.003.Saito S., Nakashima A., Shima T. Future directions of studies for recurrent miscarriage associated with immunol etiologies. J Reprod Immunol. 2011;90(1):91-5. https://doi.org/10.1016/j.jri.2011.03.003.Yoo J.H., Kwak-Kim J., Han A.R. et al. Peripheral blood NK cell cytotoxicities are negatively correlated with CD8(+) T cells in fertile women, but not in women with a history of recurrent pregnancy loss. Am J Reprod Immunol. 2012;68(1):38-46. https://doi.org/10.1111/j.1600-0897.2012.01133.x.Yoo J.H., Kwak-Kim J., Han A.R. et al. Peripheral blood NK cell cytotoxicities are negatively correlated with CD8(+) T cells in fertile women, but not in women with a history of recurrent pregnancy loss. Am J Reprod Immunol. 2012;68(1):38-46. https://doi.org/10.1111/j.1600-0897.2012.01133.x.Tang A.W., Alfirevic Z., Quenby S. Natural killer cells and pregnancy outcomes in women with recurrent miscarriage and infertility: a systematic review. Hum Reprod. 2011;26(8):1971-80. https://doi.org/10.1093/humrep/der164.Tang A.W., Alfirevic Z., Quenby S. Natural killer cells and pregnancy outcomes in women with recurrent miscarriage and infertility: a systematic review. Hum Reprod. 2011;26(8):1971-80. https://doi.org/10.1093/humrep/der164.Katano K., Suzuki S., Ozaki Y. et al. Peripheral natural killer cell activity as predictor of recurrent pregnancy loss: a large cohort study. Fertil Steril. 2013;100(6):1629-34. https://doi.org/10.1016/j.fertnstent.2013.07.1996.Katano K., Suzuki S., Ozaki Y. et al. Peripheral natural killer cell activity as predictor of recurrent pregnancy loss: a large cohort study. Fertil Steril. 2013;100(6):1629-34. https://doi.org/10.1016/j.fertnstent.2013.07.1996.Ozcimen E.E., Kiyiei H., Ucknyu A., Yanik F.E. Are CD57+ Natural Killer cells really important in early pregnancy failure? Arch Gynecol Obstet. 2009;279(4):493-7. https://doi.org/10.1007/s00404-008-0736-y.Ozcimen E.E., Kiyiei H., Ucknyu A., Yanik F.E. Are CD57+ Natural Killer cells really important in early pregnancy failure? Arch Gynecol Obstet. 2009;279(4):493-7. https://doi.org/10.1007/s00404-008-0736-y.Yen C.-C., Horng H.-C., Wang P.-H. Recurrent miscarriage: are NK cell subsets a good predictor ? J Chin Med Assoc. 2019;82:443. https://doi.org/10.1097/JCMA.0000000000000097.Yen C.-C., Horng H.-C., Wang P.-H. Recurrent miscarriage: are NK cell subsets a good predictor ? J Chin Med Assoc. 2019;82:443. https://doi.org/10.1097/JCMA.0000000000000097.Hossein S., Zarnani A.-H., Asgarian-Omran H. et al. Comparative analysis of NK cell subsets in menstrual and peripheral blood of patients with unexplained recurrent spontaneous abortion and fertile subjects. J Reprod Immunol. 2014;103:9-17. https://doi.org/10.1016/j.jri.2014.03.002.Hossein S., Zarnani A.-H., Asgarian-Omran H. et al. Comparative analysis of NK cell subsets in menstrual and peripheral blood of patients with unexplained recurrent spontaneous abortion and fertile subjects. J Reprod Immunol. 2014;103:9-17. https://doi.org/10.1016/j.jri.2014.03.002.Giuliani E., Parkin K.L., Lessey B.A. et al. Characterization of uterine NK cell in women with infertility or recurrent pregnancy loss and associated endometriosis. Am J Reprod Immunol. 2014;72(3):262-9. https://doi.org/1111/aji.12259.Giuliani E., Parkin K.L., Lessey B.A. et al. Characterization of uterine NK cell in women with infertility or recurrent pregnancy loss and associated endometriosis. Am J Reprod Immunol. 2014;72(3):262-9. https://doi.org/1111/aji.12259.Zhao X., Jiang Y., Ping Y. et al. Associations between tumor necrosis factor-а and interleukin-6 polymorphisms and unexplained recurrent spontaneous abortion risk: a meta-analysis. Medicine (Baltimore). 2019;98(46):e17919. https://doi.org/10.1097/MD.0000000000017919.Zhao X., Jiang Y., Ping Y. et al. Associations between tumor necrosis factor-а and interleukin-6 polymorphisms and unexplained recurrent spontaneous abortion risk: a meta-analysis. Medicine (Baltimore). 2019;98(46):e17919. https://doi.org/10.1097/MD.0000000000017919.Eskicioglu F., Ozdemir A.T., Ozdemir R.B. et al. The association of HLA-G and immune markers in recurrent miscarriage. J Matern Fetal Neonatal Med. 2016;29(18):3056-60. https://doi.org/10.3109/14767058.2015.1114085.Eskicioglu F., Ozdemir A.T., Ozdemir R.B. et al. The association of HLA-G and immune markers in recurrent miscarriage. J Matern Fetal Neonatal Med. 2016;29(18):3056-60. https://doi.org/10.3109/14767058.2015.1114085.Gao Y., Wang P.L. Increased CD56 (+) cells and enhanced Th1 responses in human unexplained recurrent spontaneous abortion. Genet Mol Res. 2015;14(4):18103-9. https://doi.org/10.4238/2015.December.22.36.Gao Y., Wang P.L. Increased CD56 (+) cells and enhanced Th1 responses in human unexplained recurrent spontaneous abortion. Genet Mol Res. 2015;14(4):18103-9. https://doi.org/10.4238/2015.December.22.36.Fukui A., Kwak-Kim J., Ntrivallas E. et al. Intracellular cytokine expression of peripheral blood natural killer cell subsets in women with recurrent spontaneous abortions and implantation failures. Fertil Steril. 2008;89(1):157-65. https://doi.org/10.1016/j.fertnstert.2007.02.12.Fukui A., Kwak-Kim J., Ntrivallas E. et al. Intracellular cytokine expression of peripheral blood natural killer cell subsets in women with recurrent spontaneous abortions and implantation failures. Fertil Steril. 2008;89(1):157-65. https://doi.org/10.1016/j.fertnstert.2007.02.12.

The authors declare that there are no conflicts of interest present.