Modern reproductive immunology emerged in the 1950s and there are still several major challenges to address [1]. Among these challenges, we have focused on understanding the causes of recurrent miscarriage and recurrent implantation failure. Recurrent miscarriage affects approximately 1-3% of couples, and the World Health Organization (WHO) defines it as at least three consecutive miscarriages with a gestational age of less than 20 weeks [2,3]. The term “recurrent implantation failure” describes the situation in which embryo transfers in assisted reproductive technology do not lead to clinical pregnancy. Although several definitions exist, recurrent implantation failure is generally defined as the failure to achieve a clinical pregnancy after two to three embryo transfers with good-quality embryos in women of advanced maternal age [4].

In recurrent miscarriage, several causes are thought to play an important role in its pathogenesis. Among these, chromosomal abnormalities, infections such as toxoplasmosis and chlamydia, endocrine diseases, coagulation disorders, uterine anomalies, and autoimmune diseases are considered the main contributing factors [5]. The risk factors for recurrent implantation failure are similar and include auto- and alloimmune factors, uterine anomalies and other uterine pathologies, infectious agents, psychological stress, and maternal age [6,7]. It is important to note, however, that the causes remain unclear in 40-50% of cases despite comprehensive clinical evaluation [2,8].

Several immune interventions are used to treat unexplained reproductive failure. The most important include lymphocyte immunotherapy (LIT), intravenous immunoglobulin administration, corticosteroids, lipid emulsion therapy, granulocyte colony-stimulating factor (G-CSF), and tumor necrosis factor inhibitors [8]. However, there is still no consensus in current official guidelines regarding the clinical use of any of these therapies.

LIT was first proposed as a treatment for recurrent miscarriage in 1981 by Taylor and Faulk [9]. A decade later, Hasegawa et al. [10] also proposed LIT as a treatment for recurrent implantation failure. LIT is based on the isolation of lymphocytes from the male partner. Subsequently, the lymphocytes are injected intradermally into the female partner to induce immune tolerance to the paternal antigens of the embryo. This approach may be important when the female immune system does not tolerate the paternal antigens of the semi-allogeneic embryo. The mechanisms underlying LIT include the induction of immune tolerance by decreasing Th17 cell numbers, NK cell cytotoxicity, and pro-inflammatory cytokine levels, while simultaneously increasing regulatory T cells and anti-inflammatory cytokines [11,12]. Consistent with these proposed immunological mechanisms, several meta-analyses have shown a therapeutic effect of LIT for couples experiencing recurrent miscarriage [8,13,14].

However, the majority of published studies have evaluated LIT as a treatment for recurrent miscarriage. In contrast, LIT has been investigated less frequently as a treatment for recurrent implantation failure, which motivated us to evaluate its efficacy in this patient population. It is important to note that LIT is associated with mild localized adverse reactions, making it a safe and well-tolerated therapeutic intervention [15].

In this study, we present the results of a prospective study investigating the clinical effectiveness of LIT in 268 couples with a history of recurrent implantation failure. The couples were treated with LIT at our Transfusion Medicine Center. The live birth rate and potential influencing factors, such as female age, human leukocyte antigen (HLA) matching between partners, lymphocyte crossmatch reactions, complement-dependent cytotoxicity (CDC) assays after LIT, and additional therapies received by the couples, were analyzed. Most importantly, we observed a live birth rate of 59% 2 to 3 years after LIT in women with recurrent implantation failure.

In the literature, the vast majority of studies on LIT have focused on women with recurrent pregnancy loss [7]. A Cochrane review reported a beneficial effect of LIT in the majority of the studies analyzed. However, only a few studies have evaluated LIT in women with unexplained infertility.

In the study by Günther et al. [16], the live birth rate 3 years after LIT increased to approximately 25% among women with recurrent implantation failure and to approximately 45% among women in the combined recurrent implantation failure and recurrent miscarriage group. In contrast, we observed a live birth rate of almost 60% 3 years after LIT in our cohort of women with recurrent implantation failure. In another study, Wade et al. [17] and colleagues reported a live birth rate of 58.2-68.5% after three IVF cycles. An additional two IVF cycles (cycles 4 and 5) increased the live birth rate to 75.8-80.1%. Thus, they reported a relative increase in the live birth rate of 36.8-42.1% after the third IVF cycle following two additional IVF cycles. In contrast, our data demonstrate a live birth rate of 58.6% after LIT following three unsuccessful IVF cycles.

It is well known that female age is an important determinant of pregnancy outcome. The median age of the women who achieved a live birth after LIT was 34 years, whereas the median age of the women who did not achieve a live birth was 36 years. Accordingly, Piette et al. [18] demonstrated that increased female age was associated with significantly lower IVF success rates. They identified an age cut-off between 36 and 37 years, which corresponded with the median age of 36 years observed among women who did not achieve a live birth in our study. Liu et al. [19] also reported a statistically significant effect of younger maternal age on LIT success.

To better understand the potential factors that affect the success rate of LIT, we analyzed several laboratory parameters. The most important laboratory parameter identified in our study was the crossmatch result. Women with at least one positive T- or B-cell crossmatch had a significantly higher likelihood of achieving a live birth than women without a positive T-cell or B-cell crossmatch. Our crossmatch results are consistent with those of other studies, which have demonstrated an association between a positive crossmatch and successful pregnancy outcomes after LIT [20,21].

The outcome of the CDC HLA antibody screening assay did not appear to significantly influence the live birth rate in our study. In addition, we found no evidence of an association between the number of HLA matches between female and male partners and the likelihood of achieving a live birth. Interestingly, Matsuyama et al. [22] observed a higher number of shared HLA antigens in couples with unexplained implantation failure. Similarly, they observed that couples with a higher number of shared HLA antigens had a lower pregnancy rate after LIT. However, the LIT analysis in their study included only 12 couples. Ho et al. [23] also reported an association between a higher degree of HLA antigen sharing in couples with unexplained infertility and unsuccessful IVF outcomes. In contrast, Martín-Villa et al. [24] found no association between the extent of HLA class I, II, or III antigen sharing in couples with unexplained infertility. To further clarify these findings, artificial intelligence-based biostatistical approaches may be beneficial for detailed analyses of the numerous possible HLA combinations between partners. Minor histocompatibility antigens could also play a role in this context and should be investigated in future studies.

In addition, we asked the couples to complete a questionnaire regarding additional therapies they had received to enhance the likelihood of achieving pregnancy. We found only a weak, but statistically nonsignificant, trend toward ASA use among women who achieved a live birth. For other anticoagulants, we found no evidence that they improved outcomes in women with recurrent implantation failure undergoing LIT. In general, prophylactic anticoagulation is frequently used to prevent complications during pregnancy, but careful subgroup analysis of the female patient’s clinical condition is important for predicting treatment outcomes [25].

The other additional therapies evaluated in the questionnaire included lipid infusion, immunoglobulins, corticosteroids, and G-CSF. However, our analysis showed no significant effect of these therapies on pregnancy outcomes after LIT.

Consistent with our findings, Aflatoonian et al. [26] also found no statistical evidence supporting the efficacy of intravenous immunoglobulins for women with unexplained recurrent implantation failure.

Kemp et al. [27] performed a literature review on corticosteroid treatment to improve the probability of live birth. They highlighted the increasing body of evidence indicating that corticosteroid treatment is ineffective or may even be associated with adverse events in women undergoing fertility treatment.

In contrast to our findings, Wu et al. [28] described G-CSF administration as an effective treatment for women with unexplained implantation failure. Kushniruk et al. [29] observed a nonsignificant increase in pregnancy rates after G-CSF treatment in women with recurrent implantation failure.

To identify an appropriate lymphocyte dose for improving live birth rates after LIT, Liu et al. [30] conducted a meta-analysis of several LIT studies. They compared two dosage groups: one receiving more than 100×10⁶ lymphocytes per dose and the other receiving 100×10⁶ lymphocytes per dose or less. The lower-dose LIT group showed significantly higher live birth rates. Consistent with their findings, we observed a nonsignificant trend toward improved outcomes with total cell doses of approximately 60-80×10⁶ lymphocytes across both LIT cycles. Both lower and higher total cell doses than our median dose were associated with lower live birth rates, although these differences were not statistically significant. In a follow-up study, Liu et al. [31] discovered that low-dose LIT was able to restore Th1/Th2/Treg immune homeostasis in women with unexplained recurrent miscarriage. Overall, it is important to note that treatment decisions should be individualized, because several clinical factors may influence the outcome of LIT [32]. Future directions in assisted reproductive technology, such as artificial intelligence-driven embryo selection, will broaden the spectrum of treatment options, and additional studies are required to evaluate the efficacy of LIT in combination with IVF [33].

Finally, we wish to acknowledge the limitations of our study. We did not include a control group of couples who did not receive LIT. Therefore, we cannot infer the efficacy of LIT itself, as temporal trends in IVF success, regression to the mean, and selection bias could have influenced the results. Of the 475 couples who underwent LIT, only 268 responded to the questionnaire. This represents an important limitation that may affect the precision of the live birth rate calculated from our survey. To definitively establish the efficacy of LIT in couples with recurrent implantation failure, a future double-blind, randomized, controlled clinical trial will be required.