@2024 Afarand., IRAN

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ISSN: 2251-8215    Sarem Journal of Reproductive Medicine 2018;2(1):19-23

Background
Abortion is one of the problems that leads to the lack of maintenance of embryo in a mother. Recursive ‎Spontaneous Abortion (RSA) is defined as three or more than three repeated abortions occurring before ‎the 20th week of pregnancy [1].‎
Previous Studies
‎... [2, 3]. Previous studies have shown that during pregnancy, the mother's immune system usually ‎detects human leukocyte antigen (HLA) as a foreign agent and protects the surface of the embryo by ‎inducing alloantibodies that act as blocking antibodies, cover the surface of the fetus and protect it ‎from maternal immune responses [1, 4]. Some studies have pointed out that most women who have ‎spontaneous abortion due to alloimmune reasons have similar HLA with paternal HLA, that this ‎similarity may prevent proper immune response during pregnancy and the production of antibody-‎mediated antibodies such as anti-parietal cell antibodies (APCA), idiotypic antibodies (Ab2), and ‎mixed lymphocyte reaction blocking antibodies (MLR-Bf) antibodies, and the absence of these ‎antibodies is a major cause of abortion [1, 4-7]. ... [8-16].‎ One of the treatment options for recurrent spontaneous abortion in some European and Asian medical ‎centers, including in Iran, is lymphocyte therapy or immunotherapy using fetal lymphocytes that are ‎used to stimulate the mother's specific immune response to fetal alloantigens. Some research groups ‎reported increasing fertility following this treatment [6-8]. The effectiveness of this therapy has been ‎confirmed by some researchers, while others are doubtful [4-6, 17, 18]. In this therapeutic method, a ‎husband's lymphocyte or a third person donor is injected into a woman's immunization protocol and ‎stimulates her immune system to respond to fetal antigens, and examines the result of this ‎immunization with a leukocyte cross-match test.‎
Aim(s)
The aim of this study was to evaluate the results of leukocyte cross-match derived from lymphocyte ‎therapy in women with recurrent spontaneous abortion.‎
Research type
This is a descriptive cross-sectional study.‎
Research society, place and time
This study was conducted in a population of women with a history of repeated abortions that had two ‎or more spontaneous abortions and referred to Sarem Specialized Hospital in Tehran between 2008 ‎and 2012.‎
Sampling method and number
Of the total number of 1,480 women referred to, 704 patients, after having been diagnosed as having ‎abortion, have no karyotype abnormalities, anatomical abnormalities in the uterus, uterine adhesions, ‎sexual and thyroid hormone disorders, infectious agents, and autoimmunity volunteered for treatment ‎of lymphocytic leukemia according to the specified protocol. Other patients or volunteers who were ‎not treated or did not continue treatment until the end, were excluded from the study.‎
Used devices&materials
Before lymphocytic therapy, a leukocyte cross-match test was performed using serum of patients and ‎leukocytes of their husbands, and lymphocytic treatment was proposed only for patients who were ‎negative for their test results (leukocyte cross-match<0.05). This treatment was carried out for the ‎patients with an explanation of its side effects to the patient and after signing the consent form ‎approved by the ethics committee of Sarem Hospital.‎ Lymphocyte treatment: After screening for spouse or third-person donors, in terms of transferrable ‎infectious factors including CMV, EBV, HBV, HCV, HIV, HTLV1 and Treponema pallidum. After ‎determining the blood group and Rh of patients, and their spouses or their donors, A blood sample was ‎taken from a spouse or a donor in two consecutive and three weeks intervals, 25 ml of heparin blood ‎was taken and by maintaining sterile conditions, their peripheral blood mononuclear cells (PBMCs) ‎were isolated using ficole. After 3 times that the cells were washed with ringer serum and their ‎counting was carried out using neobar slide at each turn, 80-100× 106 cells per ml of Ringer serum ‎were injected intra dermally at several points of the patient's arm. Two weeks after the last ‎immunotherapy, serum of patients and PBMCs were isolated, and an APCA examination of leukocyte ‎cross-match was performed [19-21]. These patients typically performed immunotherapy in two ‎rounds, and those who did not have positive result, continued the treatment 2 to 3 other times or by ‎using PBMCs of the third donor.‎ Determination of APCA percentage by leukocyte cross-match test: Husband`s or donor`s PBMCs ‎were removed from their blood using sterile flasks and sterilized under sterile conditions. After ‎washing with Hanks buffer, they were kept on ice and inside the refrigerator until the test was ‎performed. On the other hand, the serum of patient was isolated and 50 μl of dilution 1: 2, 1: 4 and 1: 8 ‎was poured into micro plate of 72 sample wells. Positive and negative control serum were also poured ‎into separate wells. In the wells for each patient, 500 cells of husband`s or donor`s PBMCs (in 50 ‎microliter volume) were added and then the plate was placed at room temperature for half an hour. In ‎the next step, 250 μl of rabbit complement was added to all wells and the plate was placed at 37 ° C for ‎‎1 hour and then 100 μl of eosin was added to all wells. After 5 minutes, the cells were fixed with 12% ‎formalin solution and the plate was examined by introspect microscope. Percentage of dead cells (cells ‎that penetrated the eosin color) was reported as a percentage of leukocyte cross-match [20-23]. In the ‎interpretation of the results, if the percentage of cross-match leukocyte was more than 35%, the result ‎of the test was positive and the lower values were reported as negative.‎
Findings by Text
Cross-match leukocyte test was positive in 319 patients (45.31%) after two rounds of immunotherapy ‎with their spouse PBMCs. The remaining 385 patients (54.96%) who had a negative cross-matched test ‎in previous immunotherapy, continued treatment, and 168 other patients (23.86%) tested positive ‎after their immunotherapy with their husband`s PBMCs. A total of 704 patients who fully treated ‎lymphocytic treatment with their husband`s PBMC, 487 (69.16%) cases were positive in leukocyte ‎cross-match test. The remaining patients who, after several attempts of immunotherapy with their ‎husband's PBMC, were still negative, became volunteers of lymphocytic therapy using PBMC of the ‎third donor, which resulted in 51 (7.24%) cases of positive leukocyte cross-matched test. Of the total ‎number of patients, after this type of test, 538 (76.42%) of the patients had positive APCA after the ‎treatment (Fig. 1).‎
Main comparison to the similar studies
‎… [6, 24, 25]. Studies have shown that in patients with recurrent abortions, these immunologic ‎regulatory mechanisms are impaired, that despite the mention of some studies that the similarity of ‎paternal and maternal HLA antigens is still the main cause of this disorder, it is not well-known and is ‎still being studied by scientists [1, 5, and 6].‎ Experiences and studies from scientists and researchers in the field of infertility and repeated ‎abortions have shown that if women with a history of repeated abortion, are immunized with the ‎husband`s or the third donor white blood cells before the pregnancy, the outcome of the ‎immunological events that occurs after the treatment results in the increases of the chances of ‎maintaining a fetus. New studies explain the change in the regulatory mechanisms of the immune ‎system after the treatment [17, 20, and 26]. One of the methods for assessing the success of this ‎treatment in the development of APCA is to measure these antibodies using leukocyte cross-match test ‎‎(64.5% specificity) that physicians agree with this treatment as a criterion for increasing the chances ‎of success in pregnancy [1, 20, 21]. In this test, if the patient develops APCA, these antibodies react ‎with superficial antigens of the husband's leukocytes and, by adding the complement to the test ‎environment, the death of leukocytes happens, that the percentage of dead cells that absorbed eosin ‎color is considered as the result of test. ‎
Suggestions
The effectiveness and success of this treatment in pregnancy and protection against abortion requires ‎follow up of all patients.‎
Limitations

Conclusions
Lymphocyte therapy is effective in increasing the amount of APCA. The consistency and continuation ‎of lymphocyte therapy can increase the chance of positive leukocyte cross-match in women with ‎spontaneous abortion.‎
Acknowledgments

Conflict of interest

Ethical Permissions
This treatment was done for volunteers by explaining its side effects to the patient and after signing a ‎consent form approved by the morality committee of the Sarem Specialized Hospital.
Funding Sources

TABLES and CHARTS

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