Tim-1 And Tim-3 Expression In Unexplained Recurrent Spontaneous Abortion

Objective:Active immunotherapy by intradermally injection of paternal lymphocyte on patients with unexplained recurrent spontaneous abortion(URSA);to evaluate the effect of active immunotherapy and probe into the roles of Tim-1,Tim-3 as well as APLA in URSA and the mechanism of active immunotherapy.Methods1.Research objectsPatients:35 women who had histories of recurrent abortions and were clinically diagnosed as URSA,aged 23-34 years old.Normal controls:25 healthy pregnant women who had no history of abortion and were in their mid-trimester pregnancies,aged 23-31 years old.2.Active immunotherapyThe PBMCs were separated by the Ficoll density gradient centrifugation method from 20 ml peripheral blood collected from patient's husband with aseptic procedure and then injected into the patient's forearm intradermally.The same procedure was repeated every 21 days and the whole therapy consisted of 4 times of immunizations and lasted about 2 months.After the third injection,the couples were told to be ready to start the next pregnancy.3.Tim-1 and Tim-3TIM-1,TIM-3 and ACTB cDNA sequence were obtained from NCBI and primers of Tim-1,Tim-3 andβ-actin were designed by using the Premier Primer 5.0 software. The relative expression levels of Tim-1 and Tim-3 mRNA were determined by FQ-RT-PCR method according to the instruction of SYBR Premix EX Taq(Perfect Real Time) kit in normal controls and patients before immunotherapy and on the 15^th days after the 4^th immunotherapy,respectively.4.APLAAPLA was tested by complement-dependent micro mixed lymphocyte cytotoxicity(CDC) in normal controls and patients before immunotherapy and on the 15^th days after immunotherapy.Results1.Pregnancy outcomesAmong the 35 URSA patients,4 did not get pregnancy after finishing the whole immunotherapy(11.43%);28 got pregnancy and did not encounter abortion during the early trimester(80%);and 3 got pregnant but suffered abortion again during the early trimester(8.57%).All of the 25 normal controls got full-term pregnancy and live births(100%).2.Tim-1 and Tim-3 expressionBefore immunotherapy both Tim-1 and Tim-3 mRNA were higherly expressied in 35 patients than that of the 25 normal controls(Tim-3,1.22±0.13 vs 1.28±0.06,P＜0.05;Tim-1,1.25±0.07 vs 1.34±0.09 P＜0.05);in 28 patients who maintained pregnancy and 4 patients who did not get pregnancy,the expression levels of both Tim-1 and Tim-3 were down regulated after immunotherapy while that of the 3 patients who suffered abortion again were not down regulated compared with the levels of themselves before immunotherapy.3.Anti-paternal lymphocyte antibody(APLA)As active immunotherapy could only be applied to URSA patients without APLA, 35 selected URSA patients were all negative before immunotherapy.On the 15^th day after the 4^th immunization,13 of 35 patients produced antibodies among that 12 maintained successful pregnancy and 1 had no pregnancy.The 3 ones who later suffered abortion again were negative.3 of the 25 normal controls produced APLA in their serum. Conclusions1.The active immunotherapy by intradermally injection of paternal lymphocyte is an effective therapy to URSA patients(28/35,80%).2.Tim-1and Tim-3 may be involved in URSA and the mechanism of immunotherapy.3.The Th cell immune response in URSA patients is active.After immunotherapy,it was reduced in most of the patients,which benefited the establishment and maintenance of mother's immune tolerance to embryo.4.The expression levels of Tim-1 and Tim-3 mRNA in PBMCs of URSA patients may have some meaning in the timing of pregnancy and the evaluation of immunotherapy.5.After active immunotherapy,some of the patients(13/35) produced APLA that may protect the embryo from mother's immune attack.But it cannot be concluded that without the production of APLA the outcome of next pregnancy would not be good.