EXPRESSION OF INTERLEUKIN-15 AND GRANULYSIN AT THE MATERNAL-EMBRYONAL INTERFACE OF MISSED ABORTION AND ANEMBRYONIC PREGNANCY

Abstract

Cilj istraživanja ovog doktorskog rada je ispitati izražaj citotoksične molekule granulizina na razini bjelančevine i glasničke ribonukleinske kiseline (gRNK) na spoju majčinih i embrijskih tkiva u anembrijskoj trudnoći i zadržanom pobačaju te rezultate usporediti s decidualnim tkivom elektivno prekinute normalne trudnoće prvog tromjesečja. Materijali i metode: Uzorke bazalne decidue normalne i anembrijske trudnoće, te zadržanog pobačaja dobivali smo s Klinike za ginekologiju i porodništvo, KBC Rijeka. Od imunoloških metoda koristili smo imunohistologijsko obilježavanje antigena u decidualnom tkivu uklopljenom u parafin (citokeratin, granulizin, IL-15, Apaf-1, NF-B, CD56) uz kompjutersko kvantificiranje broja obilježenih stanica/mm2 tkiva i semikvantitativnu analizu jačine obilježavanja (H score). Višestrukom imunofluorescencijom obilježavali smo citokeratin i CD56, IL-15, granulizin, Apaf-1 ili NF-B u decidualnom tkivu uz očitavanje na fluorescentnom mikroskopu i obilježavali smo CD3/CD56 i NKp46, NKp44, CD94, NKG2A ili NKG2D u suspenziji DMS dobivenih enzimatskom razgradnjom protočnom citometrijom. Metodom lančane reakcije polimeraze u stvarnom vremenu (RT-qPCR) određivali smo izražaj gena za granulizin i perforin. Rezultati: Udio CD3ˉCD56⁺ stanica NK u suspenziji DMS, apsolutni broj CD56⁺ stanica i IL-15⁺ stanica/mm2 decidualnog tkiva bio je manji u zadržanom pobačaju Učestalost NKG2A, NKG2D i NKp46 biljega na CD3ˉCD56⁺ stanicama NK u suspenziji DMS iz zadržanog pobačaja je bila statistički značajno manja u odnosu na normalnu trudnoću. U anembrijskoj trudnoći udio stanica NK se nije mijenjao. Učestalost NKG2C, CD94 i NKP44 nije se značajnije mijenjala. Glasnička RNK za granulizin je četverostruko manja u DMS zadržanog pobačaja, a četverostruko veća u DMS anembrijske trudnoće, dok je gRNK za perforin povećana u objema. U normalnoj trudnoći dominiralo je citoplazmatsko obilježavanje granulizina, dok se u patološkim trudnoćama granulizin nalazio u jezgrama stanica. Apsolutni broj NF-B⁺ stanica/mm2 decidualnog tkiva je najveći u anembrijskoj trudnoći a najniža učestalost NF-B⁺ stanica je utvrđena u resicama trofoblasta zadržanog pobačaja. Zaključak: Slabije učinkovito ubijanje trofoblasta granulizinom može biti uključeno u sporije odbacivanje trofoblasta u anembrijskoj trudnoći i zadržanom pobačaju, zbog smanjene dopreme granulizina u jezgre trofoblasta u odnosu na normalnu trudnoću.The research objective of this doctoral thesis was to examine the expression of the cytotoxic molecule of granulysin at the level of protein and messenger ribonucleic acid (mRNA) at the interface of maternal and embryonic tissues in anembryonic pregnancy and missed abortion, and to compare the results with decidual tissue from an electively terminated normal first trimester pregnancy. Materials and methods: Basal decidua samples from a normal and an anembryonic pregnancy as well as a missed abortion were obtained from the Obstetrics and Gynecology Clinic of the Clinical Hospital Center Rijeka Of immunoassays, we used immunohistochemical staining of antigens in decidual tissue embedded in paraffin (cytokeratin, granulysin, IL-15, Apaf-1, NF-B, CD56) with computer quantification of the number of stained cells/mm2 of tissue and semiquantitative analysis of the intensity of staining (H score). We performed multiple immunofluorescence labelling of cytokeratin and CD56, IL-15, granulysin, Apaf-1 or in decidual tissue with fluorescent microscope reading, as well as the staining of CD3/CD56 and NKp46, NKp44, CD94, NKG2A or NKG2D in the suspension of DMCs obtained by enzymatic degradation using flow cytometry. Using the real-time polymerase chain reaction method (RT-qPCR), we determined the gene expression for granulysin and perforin. Results: The content of CD3-CD56⁺ NK cells in the DMC suspension, the absolute number of CD56⁺ cells and IL-15⁺ cells/mm2 of decidual tissue was lower in the missed abortion. The frequency of NKG2A, NKG2D and NKp46 markers on CD3ˉCD56⁺ NK cells in the DMC suspension from the missed abortion was statistically significantly lower than in the normal pregnancy. In anembryonic pregnancy proportion of NK cells did not change. The frequency of NKG2C, CD94 and NKP44 did not differ significantly. The messenger RNA for granulysin was four times lower in the DMC from the missed abortion, and four times higher in the DMC from the anembryonic pregnancy, while the mRNA for perforin was increased in both. In the normal pregnancy, cytoplasmic staining of granulysin was predominant, while, in the pathological pregnancies, granulysin was present in cell nuclei. The absolute number of NF-B⁺ cells/mm2 of decidual tissue was the highest in the anembryonic pregnancy. The lowest frequency of NF-B⁺ cells was determined in the trophoblast villi of the missed abortion. Conclusion: A less efficient killing of trophoblasts with granulysin may be involved in a slower rejection of trophoblasts in anembryonic pregnancy and missed abortion, because of a decreased supply of granulysin to trophoblast nuclei compared to normal pregnancy