@2024 Afarand., IRAN

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ISSN: 2251-8215    Sarem Journal of Reproductive Medicine 2019;3(1):9-12

Background
The number of couple who enter the IVF process, despite having a high quality of embryo, repeatedly encounter the problem of implantation of the embryo which is called Repeated Implementation Failure (RIF). RIF is one of the most important and most difficult conditions for the couples entering IVF treatment cycle, defined by the occurrence of 2 or 3 time of failure in the IVF cycle[1], or failure in transmission of 10 fetuses or more[2-5].
Previous Studies
… [6]Several causes including decreased endometrial admission, fetal defects, or multifactorial causes are responsible for the incidence of RIF. In general, failure in implantation (especially in the presence of unknown causes) may be due to local or systematic immunological impairments which play an important role in reducing allogeneic immune responses to blastocysts, and as a result, facilitating the embryonic implementation, part of which is alien for immune system and makes pregnancy easier [7]. Immunotherapy such as lymphocyte immunotherapy (LIT) or lymphocyte therapy for managing such problems in couples is one of the major treatments. … [8-12].
Aim(s)
The aim of this study was to evaluate the success rate of treatment of patients with repeated failure of IVF with unknown cause using Lymphocyte Immunization Therapy (LIT).
Research type
This study was a not-experimental LIT study.
Research society, place and time
This study was conducted on patients (infertile couples) with a history of 2 defeats in IVF or more during a two-year period from the beginning year 2010 to 2012 in the Sarem Specialist Hospital of Tehran.
Sampling method and number
Criteria for entering the study were not having known causes such as fetal abnormalities and genetic problems in these couples, and negative outcome of the leukocyte cross-match test for anti-paternal cytotoxic antibodies (APCA). Accordingly, 61 patients were enrolled in the study.
Used devices&materials
The LIT process began by conducting infectious tests from a person’s husband. In terms of safety considerations at the beginning of this process, the tests for BGRh, VDRL, CMV (IgM and IgG), Hbs Ag, Hbs Ab, HCV, HTLV1,2, HIV, HBC Ab, and EBV (IgG and IgM) for the male (hymphoctye) were studied. If a woman has Rh negative blood type, then the lymphoid dermatologist should also have negative Rh, and if her husband has no negative Rh, then a third party can be used. When the results of the infectious tests were prepared, and the lymphocyte donor blood safety was assured, the lymphocyte therapy process began. How to implement and evaluate lymphocyte therapy and its evaluation: Different methods for lymphocyte therapy are used in different countries[13]. The protocol used in this research in Sarem Specialized Hospital is as following: Preparation of lymphocytes for injection: 20cc of blood was injected into the heparin syringe from the donor and about 3 to 4 cc of the ficoll solution was added to each sample for the isolation of lymphocytes. The density of ficoll is 1.077 which is exactly the same as the molecular mass of the lymphocytes, and the rest of the blood factors are heavier than that. Therefore, the ficoll is used to separate the lymphocytes. After centrifugation, the superficial fluid was discarded and this was repeated several times. After the last centrifugation and discharging of the supernatant solution, about one hundred ringers were added to each tube and mixed well. One drop of the prepared mixture was placed on the slide for counting the lymphocytes. The number of lymphocytes should be about 100 * 106 cells. The lymphocytes were then injected intraperitoneally into sterile syringe. For immunization, each women was given a two-step injection, which was about 0.5 cc injecting intraperitoneally at intervals of 3 weeks. Procedure for cross-match test: The immune system was evaluated by performing leukocyte cross-match, 2 weeks after the last injection and was evaluated by the APCA level[13]. For testing, about 20 cc blood was taken from the husband and poured into the 30 ml Erlenmeyer flask containing glass bullets. The Erlenmeyer flask task was rotated for about 10-15 minutes in order to the change the color of blood (lighter red), and a small clot is made inside the Erlenmeyer Flask or the blood is defibrillated and there would be no more clot. 10 cc of the wife’s blood was also taken and the serum was removed. About 3 to 4 cc of ficoll solution was added to each tube and then 6-7 cc of defibrillated blood was added from Erlenmeyer flask to the ficoll-containing tubes to be placed on the ficoll solution and not be mixed with it. After centrifugation, the detached supernatant was removed; the remaining milky solution contained husbands` lymphocytes. On the tubes containing lymphocytes, about 3 to 4 cc of Hanks solution was poured and centrifuged. Then, the supernatant was discharged and the remaining residue was washed again with Hanksand centrifuged. After the last centrifugation and discharging of the supernatant, about λ20 was added to each tube and well shaken to mix. One drop of mixture was placed on a slide for counting, and the number of lymphocytes was counted in 16 chamber neubauer slides. The number of lymphocytes should be between 11 and 12 lymphocytes. In the following, λ50 was poured from each tube into a 72-chamber micro plate that was previously filled with mineral oil in form of a drop. For each patient, 2 rows (12 chambers)were assigned that for every 3 chambers a tube of serum was placed. In the first row, the plate was inseminated from the positive control serum and the second row was poured with the negative control serum, and the husband’s lymphocyte was added to all two-row plates of the woman, and then the rabbit complement was added to it. Complement is fixed at the level of cells that have specific antibodies to the receptor-derived surface anti-antagonists, and pierces the surface of these cells. Plates were placed in the refrigerator and the responses were read the following day under the invert microscope. An APCA level higher than 30% was considered as a positive test (Figure 1). Positive cases were selected for IVF reassessment and their pregnancy status and outcome were evaluated by -HCG. In the event of a positive pregnancy test, another injection was given in the first trimester of pregnancy. The implications of intervention were based on full success in pregnancy and the birth of a healthy child. Data were analyzed by SPSS 13 using descriptive and inferential statistics.
Findings by Text
The mean age of infertile women was 34.40±4.60 years and the mean age of infertility was 8.07±4.50 years. The prevalence of infertility was 46.2% in primary infertility and 53.8% in secondary infertility. The mean number of failure in previous IVF processes of these people was 2.95±0.85. Of the patients, 5.0% had PCO. 12 (20.0%) patients had endometriosis treatment and 31.25% of them were pregnant. Pregnancy rate per LIT cycle was 19 (31.1%) and among these 19 pregnant women, the amount of their successful term labor was 16 (26.23%). The most successful treatment (based on positive pregnancy test) was related to age group 30-39 years old compared to the two groups under the age of 30 and over 39 years. There was no significant relationship between the positive results of HCG-β test and the duration of infertility, number of acquired follicles, frozen embryo transfer (FET), or embryo transfer (ET) (p>0.05). Embryos which were transferred to 19 pregnant women, in 7 cases were second day embryos (4 cells), in 6 cases of the third day (8 cells) and 6 cases were FET. The mean number of embryos transferred to patient after LIT treatment was 3.17 (range 1 to 6) for fresh embryos and 1.5 (range 1 to 4) for freeze embryos.
Main comparison to the similar studies
In the study of Kling et al., the pregnancy rate was about 39.9% for every 1.5 embryos and 16.9% for women over 39 years[1]. In a RCT study by Chek et al., the incidence of pregnancy after LIT was 38.3% compared to 28.7% compared in the non-experimental group[13]. Both of these studies support the efficacy of lymphocyte therapy in addition to confirming the results of this study. These studies, like our study, suggest that with an increase in the number of IVF failure, the rate of treatment failure also increases. Therefore, in the third IVF cycle, the success rate is reduced 22% to 50%[2-5]. In another similar study by Chaychian et al., leukocyte immunization was carried out in patients with recurrent abortions resulted in 81.7% of the patients treated with lymphocyte therapy being clinically successful (gestational age of at least 28 weeks), and the subsequent production of APCA immunization was associated with an increased likelihood of live birth[14].
Suggestions
It is recommended that the findings of study be approved by a RCT study in order to provide a more accurate and comparable analysis of the effectiveness of this treatment option in comparison with untreated people as well as those treated with other methods (such as IVIG injection).
Limitations
Non-declared by the authors.
Conclusions
The use of lymphocyte immunotherapy can be a useful treatment option for patients with frequent failure in IVF with unknown cause.
Acknowledgments
The study was sponsored by the Cellular-Molecular Research Center and Sarem Cell Research Center (SCRC). Thanks to all colleagues at this center and colleagues at the Sarem Specialist Hospital.
Conflict of interest
Non-declared by the authors.
Ethical Permissions
Non-declared by the authors.
Funding Sources
Non-declared by the authors.

TABLES and CHARTS

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