Adverse drug reactions following lymphocyte immunotherapy for the treatment of infertility: A retrospective study

Abstract

Aim

Unexplained infertility is a major burden for couples who want to have children. Lymphocyte immunotherapy (LIT) could be a therapeutic help for these couples. Although LIT has been carried out for decades, the data on the success of therapy are still controversial and there is hardly information on possible adverse drug reactions.

Methods

In this study, we used a questionnaire to determine the frequency of local and systemic adverse drug reactions in our patients who were treated with LIT between 2017 and 2020 (n = 302). In addition, we asked about pregnancies and/or live births after LIT in a 2‐year follow‐up (n = 140).

Results

Most of the patients reported the occurrence of mild local adverse drug reactions in a period of less than 4 weeks: Over 75% reported moderate erythema, itching or swelling, over 10% erythema, itching or swelling as more pronounced adverse drug reaction. Blistering was specified in 10% of the cases. Serious adverse drug reactions or adverse events were not described. In the follow‐up, 69% of our patients stated a pregnancy after LIT, and 50% a life birth.

Conclusions

Overall, LIT represents a well‐tolerated therapy for couples with unexplained infertility, however, more evidence is needed on the benefits.

Introduction

For couples of reproductive age who desire to have a child, repeated pregnancy loss and unexplained infertility are serious health problems with high psychosocial burden, which severely limit the quality of life. The World Health Organization (WHO) defines recurrent miscarriage as three or more consecutive miscarriages before 20 weeks of gestation. ¹ Approximately 1%–3% of all couples trying to conceive worldwide suffer from recurrent miscarriages whereas 9% of couples are considered infertile. ² , ³ There are various causes of recurrent miscarriage including genetic and infectious reasons, autoimmune diseases, or coagulation disorders. In around 40% of these cases, the cause remains unclear whereby immunological factors are discussed. ⁴ A balanced immune reaction and tolerance toward the semi‐allogeneic fetus are crucial for successful implantation and pregnancy. ⁵ During pregnancy, there is a marked change in the immune system and the lymphocyte composition. This provides the basis for lymphocyte immunotherapy (LIT). ⁶ LIT was first described in 1980 and is supposed to modulate women's immune system increasing the probability of a subsequent, successful pregnancy. ⁷ , ⁸ For this purpose, the partner's whole blood is obtained. Lymphocytes are separated under sterile conditions and injected intracutaneously into woman's forearms. Approximately 4 weeks after the first LIT, anti‐paternal anti‐HLA (human leukocyte antigen) antibodies are determined to control woman's immune response. If no antibodies are detected or T‐ and B‐cell crossmatches are negative, a second or even third LIT can be carried out. ⁹ The success of LIT has been the subject of controversy for years. ¹⁰ A large‐scale multicenter study found an increase in the probability of live birth after LIT of 8%–10%. ¹¹ Carp et al. determined pregnancy in 50% of cases when anti‐HLA antibodies were detected, and in 37% of cases without antibodies. ¹² However, other studies and meta‐analyzes showed no positive effect of LIT on the live birth rate. ¹³ , ¹⁴ , ¹⁵ , ¹⁶ To date, not many studies investigated local and systemic adverse drug reactions (ADRs) of LIT. One observational clinical trial showed very few ADRs ¹⁷ : local erythema and itching for about 2 weeks and blistering. Systemic ADRs were only reported in 6%–8% of the cases.

In our center, LIT is carried out on couples with an unfulfilled desire to have children and unexplained sterility or recurrent pregnancy loss (RPL). The aim of our investigation is to fill the gap regarding the ADR profile of LIT. In addition, we tried to get at least an overview of the pregnancies and live births of our patients after LIT therapy. Since 2017, we ask our patients for informed consent for two surveys: In the first survey, performed about 4 weeks after the last immunotherapy, we asked patients for ADRs and adverse events (AE) experienced after LIT. In a second, still ongoing survey we ask for pregnancies and live births at least 2 years after the last immunotherapy.

Material and Methods

Study group and design

Fertility centers and gynecologists referred infertile couples to our department. Patients suffer from either RPL or unexplained sterility. RPL was defined as three or more consecutive miscarriages before 20 weeks of gestation. Unexplained sterility was defined as a couple which had unprotected intercourse for 12 months without pregnancy and if known obstacles to conception such as anovulation, tubal occlusion, hyperandrogenemia, genetic disorders, and anatomical abnormalities in the woman or oligo‐/azoospermia and genetic disorders in the partner have been ruled out. Furthermore, before LIT was considered, three or more IVF (in vitro fertilization) or ICSI (intracytoplasmic sperm injection) attempts failed. The study includes all couples who were treated with LIT in the Department of Transfusion Medicine of the University Medical Center Göttingen between January 31, 2017 and June 12, 2020 (n = 302). Before couples were treated with LIT, they were informed about the chances of success and ADRs reported in the literature. ¹⁷ , ¹⁸ Examinations at the first visit before treatment included blood group determination, HLA typing, HLA antibody screening, HLA crossmatch with unseparated mononuclear cells (MNC), T‐cells and B‐cells, urine tests for pregnancy and tests for infectious disease markers for parenterally transmitted pathogens (hepatitis B, C, HIV, CMV, and syphilis). Positive results in infection serology, an autoimmune disease, existing pregnancy, antiphospholipid antibodies, anti‐HLA antibodies (≥6+ in complement‐dependent cytotoxicity [CDC] and a positive crossmatch with paternal unseparated MNC or T‐cells) were considered as contraindications. Donor eligibility criteria were the same as for unrelated whole blood donors. Anti‐D prophylaxis (300 μg anti‐D‐immunoglobulin) was carried out in female RhD‐negative patients with RhD‐positive donor before LIT.

LIT was performed on the day of blood sampling. Four weeks after LIT, the patient's immune status was examined. If there were no anti‐HLA antibodies detectable against donor lymphocytes in the patient's plasma and if the T‐ and B‐cell crossmatches were negative or week positive (≤4+), a second LIT was recommended. A third LIT only was performed, if the applicated cell dose was below 1 × 10⁷ leukocytes or if irradiation (30 Gy) of the cells was necessary in such cases where no HLA‐A or HLA‐B mismatch in host versus graft direction was observed, based on low resolution typing. Four weeks after the last LIT at the earliest, the patients received a questionnaire about any local or systemic ADR or AE. Patients could categorize AE between “mild” or “severe” and between “less than four weeks” and “more than four weeks” duration. We used anonymized report forms for this survey in order to give the patient the opportunity to make statements about the therapy and possible ADRs or events freely and informally. Consequently, information on ADR or AE could not be correlated with pregnancies or live births in the follow‐up. According to the ICH GCP guideline any untoward medical occurrence that at any dose resulted in death, was life‐threatening, required inpatient hospitalization or prolongation of existing hospitalization, resulted in persistent or significant disability/incapacity or a congenital anomaly/birth defect would have been classified and reported as serious adverse event (SAE) or serious adverse drug reaction (serious ADR). ¹⁹

In this study, we analyzed local and systemic ADRs notices as response to LIT. Patients (n = 302) were also asked to report AE which did not necessarily have a causal relationship with the treatment. Furthermore, we performed an interim analysis on pregnancies and live births after LIT. For this purpose, we sent a questionnaire (not anonymized) in April 2020 to all patients who agreed to participate in this survey before they were treated with LIT between January 31, 2017 and April 30 2018 (n = 140). We asked for the number of pregnancies, births and live births after LIT, as well as, if applicable, the date of birth and possibly other pregnancy‐promoting therapies that were carried out in the meantime. We received responses from 116 patients. There was a loss to follow up in 24 cases due to no feedback, wrong post address or silent declination. The couples were informed about their rights according to the data protection act.

This retrospective analysis of prospectively collected questionnaires on local and systemic ADR as well as AE was conducted according to the World Medical Association Declaration of Helsinki and approved by the Ethics committee of the University Medical Center Göttingen (approval no. 21/7/20).

Laboratory analysis

At the day of LIT, blood samples of both reproduction partners was drawn for blood group determination, small blood counting and testing of infection markers (HBsAg, anti‐HBc‐abs, anti‐HIV‐abs, anti‐HCV‐abs, anti‐syphilis‐abs, HIV‐, HCV‐, HBV‐, HEV‐NAT).

Four weeks after the first LIT, tests for detection of anti‐HLA antibodies in female patients were carried out. Blood of both reproduction partners was used to carry out T‐ and B‐cell CDC crossmatch and HLA antibody screening with CDC (HLA class I). HLA class II antibody specification was performed with solid phase assay (LabScreen Single Antigen HLA Class II, BmT, Meerbusch, Germany) only if the B‐cell crossmatch was clear positive (≥6+).

If T‐cell crossmatch or HLA class I antibody‐screening (CDC) was weak positive (≤4+), a second LIT 6–12 weeks after the first LIT was recommended. The second LIT was carried out following exactly the same protocol as the first LIT (pregnancy test, donor examination, blood collection, manufacture of the lymphocyte concentrate, preparation release, injection, anti‐D prophylaxis when indicated). About 4 weeks after the second LIT a final examination was conducted with the same tests as used after the first LIT.

Lymphocyte immunotherapy

On the day of LIT the female patient has to perform a urine hCG pregnancy test in our institution. If the test was positive, the LIT was not carried out. Before blood donation the male reproduction partner has to be tested for his donation eligibility (health questionnaire), suitability (consultation by health professional) and identity (blood type, identity card), since the blood donation for LIT was considered an allogeneic blood donation. For blood typing, small blood count and infection diagnostics, blood was taken from the male partner. With the same blood donation system 100 mL whole blood was collected into perfusor syringes containing heparin (5.000 I.E. heparin‐sodium) to prepare the lymphocyte concentrate. The buffy coat was separated under sterile conditions by density gradient centrifugation (Ficoll Paque Premium, Cytiva Europe, Freiburg, Germany). The separated lymphocytes were washed with washing solution containing ACD‐A (acid‐citrate‐dextrose, Terumo BCT Europe, Garching, Germany) and HBSS (Hanks' balanced salt solution) and centrifuged (10 min, 900 g with brake). For erythrocyte lysis lymphocytes were resuspended in 1–2 mL aqua ad iniectabilia. After adding HBSS, cells were centrifuged (10 min, 150 g without brake) and erythrocyte lysis was performed a second time. The supernatant was decanted, cells were suspended in approximately 45 mL 0.9% NaCl and centrifuged again (10 min, 900 g with brake). This supernatant was used for sterility controls and the cell pellet was resuspended in 2.5 mL 0.9% NaCl; 1.6 mL of this suspension was drawn up into a syringe whereas the rest of this suspension was used for sterility controls and cell counting (Abbott Cell Dyn Ruby). After release by a qualified person the lymphocyte concentrate containing 5 × 10⁷ (1–7 × 10⁷) MNC per dose was injected intracutaneously into patient's forearm at the day of blood collection. Patient's pulse, temperature and blood pressure was recorded for about 30 min.

Statistics

The data are given either as absolute numbers, relation (in %) or as median (min; max). The statistical evaluation was performed with Microsoft Excel and the graphical representations with Microsoft PowerPoint.

Results

Characteristics of study group

The study group consisted of couples who were treated with LIT in our department between January 31, 2017 and June 12, 2020 (Figure 1). All patients treated with LIT between May 18, 2017 and June 12, 2020 answered a questionnaire around 4 weeks after the last LIT about possible local and systemic ADR or AE that they noticed after the therapy (n = 302). In addition, 140 patients from these cohort, who were treated with LIT therapy between January 31, 2017 and April 30, 2018 received a questionnaire in April 2020 and were asked for pregnancies, births and live births after LIT therapy within 2 years. In this group, we received an answer from 116 patients, which corresponds to a response rate of 83% (Table 1). The median age of the patients was 35.3 (27.3; 45.3) years, those of the donors was 37.1 (27.1; 58.1). On average the partners were older than the patients. The main diagnosis of our patients was unexplained sterility (n = 138). In addition, two patients had a RPL; 87% received two LITs because a negative or only weak positive T‐crossmatch was observed after the first LIT and HLA class I antibody screening with CDC was negative or weak positive. Fourteen patients (10%) received only one LIT, either because of an already detectable immune reaction, because the donor did not fulfill the eligibility criteria or because of patient‐related contraindications. Two patients received a third LIT because of a low MNC concentration in the lymphocyte preparation (<1 × 10⁷ cells per dose). The median time interval between first and second LIT was 56 (48–161) days.

FIGURE 1.

Study flow chart. Assessment of patients after lymphocyte immunotherapy for evaluating ADR (adverse drug reaction), AE (adverse events) and follow‐up

TABLE 1.

Basic characteristics, survey results of pregnancies after lymphocyte immunotherapy, additional treatments and immunological side effects in study group

Study group		n	Other treatments used in study group		n
Female age (years)	35.3 (27.3; 45.3)	140	G‐CSF	14 (16%)	87
Age of donor (years)	37.1 (27.7; 58.1)	140	Immunoglobulins	14 (16%)	88
Diagnoses			Lipidinfusion	24 (27%)	89
Unexplained infertility	138	140	Acetylsalicylic acid	28 (33%)	86
Recurrent pregnancy loss	2	140	Other anticoagulants	35 (41%)	86
Number of lymphocyte immunotherapies			Others	40 (54%)	74
One lymphocyte immunotherapy	14	140	Additional reproductive technology measures (EmbryoGlue® methode, polar body diagnostic, ERA test)	18
Two lymphocyte immunotherapies	122	140	Hormone therapy (sex hormones)	16
Three lymphocyte immuntherapies	2	140	Corticosteroides	4
Time between first and second lymphocyte immuntherapy (days)	56 (48; 161)	124	L‐thyroxine	1
			Donation (oocyte or sperm)	3
Survey results of pregnancies after lymphocyte immunotherapy			TCM (e.g. acupuncture)	3
Number of women with at least one pregnancy after lymphocyte immunotherapy	80 (69%)	116	Diet change or nutritional supplements (Diet, Gluthathione, Ubiquinol, Vitamin‐B‐complex)	3
One pregnancy	56	80	Doxycycline	1
Two pregnancies	15	80	Metformin	1
Three pregnancies	1	80	Endometrium‐Scratching	1
Not specified	8	80	Influenca vaccination	1
Live births after lymphocyte immunotherapy	58 (51%)	114
Twins	8	58
Triplets	1	58
Rate between live births and questionnaire feedbacks	50%	116
Time between last lymphocyte immunotherapy and live birth (in months)	13 (8; 31)	58

Immunological reactions			Immunological reactions
Anti‐HLA‐antibodies against donor		140	Lymphocyte crossmatch		140
HLA class I and II negative	43		T‐ and B‐lymphocytes negative	49
HLA class I positive	73		T‐lymphocytes positive	65
HLA class II positive	84		B‐lymphocytes positive	86
HLA class I and II positive	62		T‐ and B‐lymphocytes positive	63
Not assessed	2		Not assessed	2

Note: One hundred and forty patients in our department who received lymphocyte immunotherapy between January 2017 and April 2018 were asked about pregnancies and live births 2 years after treatment, 116 patients answers were received (n = 116). If no answer to a specific question was given, this was not taken into consideration for the calculation of the percentage. After the last lymphocyte immunotherapy injection, T‐ and B‐cell crossmatches were carried out and anti‐HLA class I antibodies (CDC) were investigated in all patients (n = 140). Furthermore, in case of a positive B‐cell crossmatch, HLA class II antibodies against donor lymphocytes were investigated using a solid phase assay (microbead array) as well. Two patients did not sent blood tubes after the last immunization (“not assessed”). Of 116 patients, 87 reported having received another treatment after LIT. Median (min; max) was calculated for age and time interval. “Not specified” means that insufficient information on the number of was available.

Abbreviations: ERA, endometrial receptivity array; G‐CSF, granulocyte‐colony stimulating factor; ISCI, intracytoplasmic sperm injection; IVF, In vitro fertilization; TCM: traditional Chinese medicine.

Local and systemic adverse drug reactions after lymphocyte immunotherapy

Four weeks after the last LIT, approximately 88% of the patients stated that they had at least one ADR after LIT (Table 2). Over 75% of the patients had noticed mild erythema, itching (76.5%) or local swelling (71.9%) locally at the injection site. Over 10% of the patients reported erythema (13.3%), itching (13.8%), or swelling (12.2%) as a severe symptom. Another 41.9% had a mild burning sensation and 40.3% had a mild local hematoma; 27.5% reported a local mild pain in the immunized arm. Only 7.5% stated that they had suffered a severe hematoma. Blistering was reported in only 10% of the cases. Local ADR lasting more than 4 weeks were rarely reported, only in 7.3% of the cases erythema and in 3.4% hematomas persisted longer.

TABLE 2.

Local and systemic side effects after lymphocyte immunotherapy

	Not occured (%)	Mild ADR (<4 week duration)	Severe ADR (<4 week duration)	Any ADR >4 week duration	n
Local reactions
Erythema	4.3	75.0	13.3	7.3	300
Itching	8.4	76.5	13.8	1.3	298
Swelling	14.2	71.9	12.2	1.7	295
Burning sensation	54.0	41.9	3.8	0.3	291
Blister formation	88.9	10.0	1.0	0.0	298
Pain in immunized arm	69.3	27.5	2.4	0.7	287
Swelling of the hand	96.5	3.5	0.0	0.0	287
Hematoma	48.8	40.3	7.5	3.4	293
Systemic symptoms
Elevated temperature or fever	91.2	8.1	0.0	0.7	295
Dizziness, circulatory weakness	87.0	11.3	1.0	0.7	293
Fatigue	68.8	28.4	1.7	1.0	292
Feeling of illness	87	10.8	1.7	0.7	293
Headache	84.6	14.3	1.0	0.0	293
Nausea, vomiting, diarrhea	92.9	6.1	0.7	0.3	295
Lymphadenopathy	95.9	3.8	0.0	0.3	290
Erythema (beyond injection area)	96.2	2.4	1.0	0.3	290

Note: At the earliest 4 weeks after final lymphocyte immunotherapy, patients were asked for possible local and systemic ADR, their duration and severity between 05.2017 and 06.2020 (n = 302).

In contrast to local ADR, which occurred frequently, systemic ADR were rather rare (Table 2): Fatigue was most frequently described (28.4%). Headache (14.3%), dizziness, circulatory weakness (11.3%), and feeling of illness (10.8%) were also common. Other systemic ADR occurred in less than 10% of the patients. Severe systemic ADR (<2%) or systemic ADR lasting more than 4 weeks (n = 12) were almost not reported (<1%). Of note, no serious ADR or SAE was reported.

Successful pregnancies after lymphocyte immunotherapy

The evaluation of the responded questionnaires showed that out of the 116 patients, 80 patients had at least one pregnancy within 2 years after LIT, which corresponds to a success rate of 69% (Table 1). Fifty‐six patients had one pregnancy, 15 had two pregnancies, and one even had three pregnancies during this time. Furthermore, eight patients stated that they were pregnant at the time of the survey (we have no information about the course of those pregnancies). After LIT, 58 patients had at least one live birth. Of these, eight had a twin birth and one patient had a triplet. In relation to the study group, this corresponds to a success rate of 50%. In relation to the number of pregnant women, the live birth rate is 73%. The median time between the last LIT and a live birth was 13 (8–31) months.

Remarkably, many patients received additional therapy (n = 89). We have grouped numerous forms of therapies that were given in our questionnaire (Table 1). Most of the patients received various anticoagulant substances (41%) or took acetylsalicylic acid (33%). Lipid infusion was also used frequently (27%), as was therapy with immunoglobulins (16%) or granulocyte‐colony‐stimulating‐factor (16%). Additional reproductive technology such as the EmbryoGlue® method or polar body diagnostic (n = 18) or the use of sexual hormones (n = 16) were less common. In addition, complementary medical methods such as Traditional Chinese Medicine were also used (n = 3).

Anti‐HLA immunization in patients with 2‐year follow‐up

In the aforementioned patients who received a follow‐up (n = 140), we compared the immune status after the last LIT (Table 1). T‐ and B‐cell crossmatches were carried out between patient and donor, and the anti‐HLA class I antibodies against cells of the donor were also determined, before and after LIT. In cases with a positive B‐cell crossmatch, donor‐specific HLA class II antibodies (DSA) were tested. In the preliminary analysis before LIT only one patient showed positive anti‐HLA class I and II antibodies, but without positive T‐ or B‐cell crossmatch. After LIT, about 70% of all patients developed either anti‐HLA class I or class II DSA after LIT or both. Seventy‐three patients had anti‐HLA class I antibodies and 84 anti‐HLA class II antibodies. Of these, 62 were positive for both, anti‐HLA class I and class II. A similar picture emerged in the lymphocyte crossmatch (CDC): in 65 cases donor T‐cells and in 86 donor B‐cells reacted positive with the patient's serum. In 63 out of 65 patients with positive T‐cell crossmatch, the B‐cell crossmatch was also positive.

Discussion

An unfulfilled desire to have children can lead to psychosocial problems in couples and is highly stressful. Reasons could be recurrent miscarriage due to RPL or recurrent implantation failure. LIT with paternal mononuclear cells is discussed as therapy option putative increasing the rate of live births. ²⁰ Although this form of therapy has been carried out since the 1980s, ⁷ there are controversial results on the success rate and hardly studies on the rate of local or systemic ADR. ¹⁰

In the present study, we would like to fill part of this gap, focusing on local or systemic ADR of LIT. For this purpose, we analyzed by questionnaires about the frequency of local and systemic ADR in our patients who received LIT between 2017 and 2020. Additionally, we performed an interim analysis for measuring the possible benefit of the LIT therapy based on pregnancies and live births after 2 years in the follow‐up of our patients.

Mechanisms of lymphocyte immunotherapy

The mechanisms behind the effect of active immunization with paternal lymphocytes have not yet been fully clarified, but various theories that contribute to its success have been described. ⁶ During pregnancy, there is a tolerogenic maternal immune response ²¹ and changes in the cellular immune system occur. Lymphocyte populations play an important role in pregnancy maintenance, trophoblast invasion, angiogenesis, and homeostasis of the feto‐maternal immune system. ⁵ This includes both immunosuppressive and immune stimulating factors. Maternal T‐cells recognize fetal antigens and are therefore part of feto‐maternal immune tolerance. ²² During pregnancy, there are profound changes in the T‐cell populations: There is a shift from TH1 to TH2 cells, a decrease of TH17 cells and an increase of Treg cells. ⁵ TH2 cells lead to a suppression of the TH1 cells, which are regarded as embryotoxic. ²³ Increased TH1 cells are also correlated with an increased rate of spontaneous abortions. ²⁴ Therefore, the success of IVF could be influenced by the TH1/TH2 ratio ²⁵ and a disturbed change in the T‐lymphocyte population can lead to a disturbed pregnancy. ²⁶ , ²⁷

Natural killer (NK) cells are also reported to be important during pregnancy, their number can increase to over 70% of the uterine lymphocytes and this is beneficial for an undisturbed implantation. ²⁸ The fetal extravillous trophoblast, on the other hand, suppresses a possible cytotoxic activity of the NK‐cells by expressing special HLA antigens (HLA‐E, HLA‐C, and HLA‐G) and induces tolerance. ²⁹ , ³⁰

The aim of the paternal lymphocyte immunization is to modulate the maternal immune response to the semi‐allogeneic fetus. LIT strengthens the maternal immune system against paternal antigens and the formation of antipaternal antibodies can be regarded as a success of immunization ¹² and is an important factor for the mechanism of action. ³¹ In the presence of antipaternal anti‐HLA antibodies, an increased pregnancy rate after LIT was found. ¹² , ³² LIT improves the TH1/TH2 balance, improves the Treg profile and leads to a suppression of NK‐cell activity. ³³ , ³⁴ Furthermore, anti‐idiotypic antibodies (Ab2), ant‐paternal cytotoxic antibodies (APCA), blocking antibodies ³⁵ and mixed lymphocyte reaction blocking factors (MLR‐Bf), which in turn block paternal HLA antigens on the fetus and improve TH2 dominance, rise the number of Treg cells and reduce NK‐cell activity, which causes improved pregnancy rate. ⁶ , ³⁴ If the compatibility of maternal and paternal HLA antigens is too high, the formation of blocking antibodies is suppressed. ³⁶ Couples who share some HLA antigens seem to be more likely to suffer from abortions. ³⁷ In addition, HLA compatibility was found more frequently in couples with RPL than in fertile couples. ³⁸ It could also be shown that certain HLA types are related to the success of LIT. ³⁹ In our study, more than 70% of the patients showed a positive immune reaction of LIT in the form of antibodies against donor HLA antigens.

Local and systemic adverse drug reactions after lymphocyte immunotherapy

Not many studies were conducted about the local and systemic ADR associated with LIT. To our knowledge, there are only two other studies that have actually systematically examined ADR after LIT. ¹¹ , ¹⁷ Most investigators hardly declare any ADR of LIT for mother and child. ⁴⁰ , ⁴¹ , ⁴² However, general transfusion medical risks must be taken into account, such as the transmission of infectious diseases. ⁴³ The first study on possible side effects was carried out in 1994 as an international multicenter study. ¹¹ Maternal ADR only occurred in 2.1%, in contrast to the control group with 0.5%. The most common complications were viral infections (hepatitis, cytomegalovirus), flu‐like symptoms and fever. Fetal complications were not increased in comparison with the control group. Thanks to good manufacturing practice in transfusion medicine using microbiological, immunological and hematological quality control assays, the risk for serious ADR of intradermal LIT can be regarded as very low. ¹⁷

In our study, 88% of patients reported a local ADR after LIT. Mostly these reactions were slight erythema, swelling or itching lasting less than 4 weeks and reported as mild ADR. Only 10% of the patients reported a blistering. Local ADR lasting longer than 4 weeks were described in less than 10% of our cases. Systemic ADR were rarely reported. The most frequent occurrences were short‐term fatigue, feeling of illness or dizziness, and they were described as mild. Patients reported fever in only 8% of the cases. We were not aware of any serious ADR or SAE, especially no infectious diseases were transmitted.

Our data largely coincide with those already published. ⁶ Kling et al. has published studies in larger patient cohorts. ¹⁷ , ²⁰ They have carried out an extensive analysis of possible side effects of LIT in their patients (3246 treatments) and found that typical local symptoms usually lasted about 2 weeks and consisted mainly of erythema and itching. There were no scars or granulomas. The inflammatory response to active immunization occured within 3 weeks. Systemic reactions occurred in 6%–8% of the cases. Fever with 3.1% and rash 0.5% were the most common systemic reactions. Kling et al. describe blistering at the injection site as a characteristic reaction to LIT and were able to rule out an association with HLA mismatches. Fourteen percent of patients developed blistering at the injection site, although it is not clear whether a swelling was also referred to as blistering, so a rate of <6% could probably be present. We were able to confirm these observations.

Only case reports dealed with SAE: In two cases, neonatal alloimmune thrombocytopenia in the newborn after LIT of the mother was described. ⁴⁴ , ⁴⁵ However, they was not able to prove a causal relationship with LIT. Long‐term observation of patients with LIT and their newborns could not determine any negative effects on the immune system of the mother or child. ⁴⁶ As far as we know, no serious ADR has occurred in our patients.

Pregnancies and live births after lymphocyte immunotherapy

In a follow‐up of our patients 2 years after LIT, we used a questionnaire to determine pregnancies and live births after LIT. Of the couples from whom we received an answer, 80% had at least one pregnancy after LIT and 50% had a live birth. In relation to the pregnant women, even 73% were able to carry out the pregnancy successfully. This is within the upper range of the average success rate between 30% and 70% given in other studies. ⁶ It should be noted that our study was nor controlled or randomized, so the success rate cannot be attributed solely to LIT, as many of our patients also received other pregnancy‐promoting therapies or IVF‐therapy after LIT, which we cannot analyze separately. In addition, we only relate our success rates to individuals, not to the number of embryo transfers performed, as is commonly done since we lack this information from our patients. In 2019, around 62 000 women in Germany received IVF treatment. ⁴⁷ Live births per embryo transfer were successful with an average of 29.7%, which means that the success rate for LIT administration needs to be divided by the number of embryo transfer as well. A live birth in median occurred 13 months after the last LIT in our patients. Kling et al. also showed an increase in pregnancies by 25% in the first half of the year after LIT, ⁹ which corresponds to an increase of 10% compared to no IVF‐therapy without LIT. The birth rate was 38% after IVF and 42% spontaneously. In a recently published study by Günther et al. a live birth rate of 43% was found in 148 couples according to LIT. ⁴⁸ Interestingly, the success was strongly dependent on the indication: the live birth rate was significantly higher in women with recurrent miscarriages (53%) in contrast to women with recurrent implantation failure (33%), especially in the first 90 days after LIT, which suggest that LIT appears to have a more pronounced effect on women with recurrent miscarriages.

Despite these observations, the success of LIT in reproductive medicine is controversial: A first meta‐analysis could not find any improvement in the live birth rate. ⁴⁹ An international multicenter study found an increased rate of live births after LIT of up to 8%–10%. ¹¹ However, a Cochrane review could not declare a significant effect on the live birth rate. ¹⁵ Nevertheless, this study is controversial. ⁵⁰ , ⁵¹ , ⁵² The main reason is the consideration of the study of Ober et al. within the review that could not show any effect of LIT. The study published by Ober et al. showed methodological weaknesses, such as the use of overnight stored and frozen lymphocytes for immunization and no exclusion of patients with autoimmune diseases, ¹⁴ which influence the success of LIT. ¹¹ It was found that overnight stored and frozen lymphocytes leads to a reduction in the expression of the CD200 antigen on the cell surfaces, which is reported to be important for immune tolerance and for the efficacy this therapy. ⁵³ , ⁵⁴ Therefore, only freshly removed lymphocytes should be used for LIT, as in our study.

Pandey et al. declared an improved success rate with the administration of LIT, in contrast to untreated patients. ⁵⁰ In addition to age and the number of former abortions in women, autoimmune diseases and thrombophilia are correlated with the failure of LIT. ¹⁸ A recent meta‐analysis showed a significant effect on the live birth rate: 77.8% live births after immunization compared to 41.1% in the control group. ⁵⁵ Cavalcante et al. analyzed six meta‐analyzes: two demonstrated no influence on the life birth rate after LIT and four reported a benefit of LIT and a significant increase of the live birth rate. This most recent meta‐analysis led to the conclusion that there is efficacy for immunotherapy and that the therapy is safe. ¹⁸

Limitations of the study

Some of the limitations of our study should be noted. The evaluation of the side effects that occurred was answered directly by the patient using a questionnaire; a doctor did not check the information. Therefore, the interpretations of the symptoms have only been carried out on the basis of patients' information. It is also not clear whether other illnesses or events could have influenced type and severity of the symptoms. The survey was anonymized in order to facilitate answers without bias. Thus data on ADR or AE could not be compared with a successful or problematic pregnancy or birth in the follow‐up. In addition, we did not ask for AE or ADR in fetuses or newborns. However, as discussed above, no negative effects of LIT on the fetus are reported in the literature. The number of our patients is also rather small compared to larger studies ¹⁷ ; although we had similar results. The rate of pregnancies and live births was only determined by following up a small group of patients (n = 116). Although we had a high response rate, it remains to be feared that some couples have not answered because no pregnancy has occurred. Furthermore, the success rate was not related to the number of pregnancy attempts or embryo transfers, as would be expected in our cohort with primarily implantation failure. Unfortunately, we did not have the information about the number of pregnancy attempts and embryo transfers before and after LIT, mainly because patients were assigned by many fertility centers from all over Germany. In future studies this information needs to be taken into account, especially since it was shown that LIT is more successful in patients with a history of recurrent miscarriages than with recurrent implantation failure. ⁴⁸ This study was neither randomized nor controlled. There was no placebo or control group. From an ethical point of view and taking this very emotionally charged topic into account, a randomized controlled clinical trial is usually problematic to carry out in this context. This makes it difficult to interpret the success rates of the LIT. The success and increase in life birth rates may also have been affected by performing other therapies after LIT. As we have determined, in addition to our LIT therapy, various other therapy options were also carried out by the couples, sometimes at the same time. Some of them also have immunomodulatory mechanisms, such as the administration of glucocorticoids, intralipid infusions, or intravenous immunoglobulin administration. ⁵⁶ , ⁵⁷ , ⁵⁸ Unfortunately, we therefore cannot look at the pregnancies and live births independently for LIT alone in the follow‐up and must also take into account the effect of other therapies.

Overall, from our experience over many years in the treatment of couples with recurrent miscarriage and unexplained sterility, we conclude that active immunization by partner lymphocytes is a safe treatment option. While this treatment has a relatively high rate of overall reported side effects, none of them was categorized as serious ADR or SAE. Ultimately, more data are required on possible side effects and the success rate of LIT. Although placebo‐controlled randomized clinical trials would be desirable, these are difficult to implement due to the special situation of the couples concerned who want to have children and also required funding from public sources.

We will contact the remaining 160 patients 2 years after their last LIT and take the number of embryo transfers into account in future in order to determine the benefit of LIT in our center.

Author contributions

Conceptualization, Abass Eidizadeh, Joachim Riggert, Beatrix Pollok‐Kopp, Tobias J. Legler; Methodology, Abass Eidizadeh, Jakob Valk, Beatrix Pollok‐Kopp, Joachim Riggert, Tobias J. Legler; Software, Abass Eidizadeh, Monika Goldmann, Susanne Papert; Validation, Abass Eidizadeh, Susanne Papert, Beatrix Pollok‐Kopp, Joachim Riggert, Rüdiger Moltrecht, Tobias J. Legler; Formal Analysis, Abass Eidizadeh, Tobias J. Legler, Monika Goldmann; Investigation, Abass Eidizadeh, Jakob Valk, Beatrix Pollok‐Kopp, Tobias J. Legler, Joachim Riggert; Resources, Abass Eidizadeh, Monika Goldmann, Tobias J. Legler, Jakob Valk, Beatrix Pollok‐Kopp, Joachim Riggert; Data Curation, Abass Eidizadeh, Tobias J. Legler, Susanne Papert, Monika Goldmann; Writing—Original Draft Preparation, Abass Eidizadeh, Tobias J. Legler, Susanne Papert; Writing—Review & Editing, Abass Eidizadeh, Susanne Papert, Jakob Valk, Beatrix Pollok‐Kopp, Monika Goldmann, Joachim Riggert, Rüdiger Moltrecht, Tobias J. Legler; Visualization, Abass Eidizadeh, Tobias J. Legler; Supervision, Tobias J. Legler, Abass Eidizadeh; Project Administration, Abass Eidizadeh, Tobias J. Legler, Joachim Riggert, Beatrix Pollok‐Kopp. All authors have read and agreed to the published version of the manuscript. All authors have contributed significantly to this manuscript. And all authors are in agreement with the content of the manuscript.

Conflict of interest

The authors have no conflict of interest to declare.

Data availability statement

The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy and ethical reasons.