Immunosuppressive therapy with tacrolimus is a potential drug candidate for the prevention of unexplained or preeclamptic stillbirths with Th1-dominant immune states: a case series of five patients

Abstract Some of obstetrical complications such as unexplained pregnancy loss and preeclampsia (PE) are associated with maternal-fetal immune abnormalities, leading to uteroplacental dysfunction, insufficient fetal immune tolerance, or fetal rejection. Immunosuppressants with calcineurin inhibitors could be useful for the prevention of these complications by modulating the cellular immune balance by directly inhibiting activated T-helper (Th) 1 and natural killer (NK)/NKT cells. We present our experience with the immunosuppressant tacrolimus in five pregnant women who had a previous pregnancy history of unexplained or preeclamptic stillbirth. Th1 and Th2 cell populations and NK cell activities in peripheral blood were measured as clinical parameters during pregnancy. Case 1–3 achieved suppressions of predominant Th1 immunity and live births without pregnancy-related complications. In case 4, increased tacrolimus dose after a miscarriage resulted in her first live birth; however, she developed PE and severe fetal growth restriction with elevated Th1/Th2 cell ratios at 26 weeks of gestation. Case 5 had a previous history of early onset PE and the hemolysis, elevated liver enzymes, and low platelet count (HELLP) syndrome, and an emergency cesarean section was needed for maternal safety at 20 weeks of gestation. The course of the next pregnancy was stable under tacrolimus treatment; however, the HELLP syndrome recurred after PE at 33 weeks of gestation. Although an imbalance in the Th1/Th2 cell ratio was not observed during pregnancy, NK cell activity was markedly elevated before delivery. In conclusion, tacrolimus is a potential drug candidate for the prevention of unexplained or preeclamptic stillbirth with Th1-dominant immune states.


Introduction
Immune tolerance to the semi-allogeneic fetus while maintaining protection from foreign pathogens is essential to continued pregnancy.During pregnancy, extravillous trophoblasts (EVTs), decidual stromal cells, and different immune cells, such as T cells, macrophages, dendritic cells, natural killer (NK) cells, and NKT cells, form a complex network of cellular connections at the maternal-fetal interface [1,2].It has been previously proposed that the immunological shift from the inflammatory Th1 cytokine pattern to the Th2 pattern is one of maternal immune alterations [2].Immunity in normal pregnancy also tends toward immune tolerance under several other unique conditions, including EVTs expressing human leukocyte antigen (HLA)-C and non-classical HLA-E and HLA-G, seminal plasma priming, and pregnancy-related hormones such as estrogen, progesterone, and human chorionic gonadotropin [3].Decidual NK cells recognize HLA-C on EVTs through the expression of killer cell immunoglobulin-like receptors (KIRs), and an appropriate match between KIR phenotypes and HLA-C is considered necessary for fetal tolerance and placental development [1].The expression of HLA-E [4] and HLA-G [5] on EVTs also prevents maternal NK cell cytotoxicity.Seminal plasma priming [6] and pregnancyrelated hormones [7] affect the expansion and migration of regulatory T (Treg) cells, which play a critical role in mediating maternal tolerance to the fetus.
Stillbirth, defined as fetal death at 20 weeks' gestation or later, is one of the major adverse pregnancy outcomes.A population-based study investigating the causes of stillbirth reported that about one-quarters had unexplained causes and 9.2% had hypertensive disorders including preeclampsia (PE) [8].Some of obstetrical complications, such as unexplained pregnancy loss and PE, are associated with maternal-fetal immune abnormalities, leading to uteroplacental dysfunction, insufficient fetal immune tolerance, or fetal rejection.The pathophysiology of PE has been studied from various perspectives, including inflammation, endothelial dysfunction, poor angiogenesis, and immune maladaptation.Immunological factors include Th1 immune bias induced by interferon-c (IFNc) from activated peripheral NK cells, defective spiral artery remodeling and abnormal EVTs invasion due to lower levels of IFNc secreted by decidual NK cells, transition of macrophage phenotype from M2 to M1, and imbalance of Th17 cells and Treg cells [9,10].Herein, we present our experiences with the immunosuppressant tacrolimus in five pregnant women who had a previous pregnancy history of unexplained or preeclamptic stillbirth.The patients had infertility associated with Th1-dominant immune states defined as a Th1/Th2 ratio � 10.3.Tacrolimus treatment could correct the imbalance of Th1 and Th2 cells before implantation and during pregnancy, thereby not only treating against implantation failures but also preventing stillbirths.

Case presentation
Table 1 shows the clinical characteristics and pregnancy outcomes of the five patients.All patients conceived through in vitro fertilization and immunosuppressive treatment with tacrolimus at the Sugiyama Clinic (Tokyo, Japan).Tacrolimus (Prograf V R ; Astellas Pharma Inc., Tokyo, Japan) was administered at a daily dose of 1-3 mg 2 days before embryo transfer and continued until delivery.Patients were managed during pregnancy at the National Center for Child Health and Development (Tokyo, Japan).None of the patients had high risk factors for developing PE such as chronic hypertension, antiphospholipid syndrome, renal disease, diabetes mellitus, and maternal body mass index > 30 kg/m 2 .We measured Th1 cell populations in peripheral blood CD4-positive lymphocytes and NK cell activities as clinical parameters during pregnancy.

Case 1-3
Case 1 and 2 experienced unexplained stillbirths at 38 and 22 weeks of gestation, respectively.Both patients did not receive prophylactic anticoagulant therapy such as low-dose aspirin or heparin in this pregnancy.Due to moderate to severe asthma exacerbation at 34 weeks of gestation, case 1 received prednisolone at a daily dose of 5-30 mg until delivery.In addition, insulin was used to improve steroid-induced glucose intolerance.Case 3 experienced preeclamptic stillbirth occurring at 32 weeks of gestation, and received prophylactic low-dose aspirin along with tacrolimus for two pregnancies.In cases 1-3, predominance of Th1 immune responses with elevated Th1/Th2 cell ratios, observed at the early pregnancy, was suppressed throughout pregnancy, and live births were achieved without pregnancy-related complications.However, no blood test including Th1 and Th2 cells population and NK cell activities was performed during the 36-year-old pregnancy in case 3.

Case 4
Case 4 had a previous pregnancy history of unexplained stillbirth and subsequent recurrent miscarriages.Unfortunately, her first pregnancy under tacrolimus treatment resulted in miscarriage at 15 weeks of gestation.In the next pregnancy, the Th1/Th2 cell ratio of 29.6 in the first trimester was even higher than that at the time of the previous miscarriage, and the daily dose of tacrolimus was elevated from 2 to 3 mg.The patient did not receive prophylactic anticoagulant therapy such as low-dose aspirin or heparin in these two pregnancies.Despite the decreased Th1/Th2 cell ratios of 11.4 in the second trimester, PE with severe fetal growth restriction occurred at 26 weeks of gestation.As her blood pressure was poorly controlled under antihypertensive treatment with a-methyldopa and amlodipine, cesarean section was performed at 28 weeks of gestation, resulting in the birth of a male fetus weighing 736 g.The patient presented with low platelet count, elevated liver enzymes, and non-cardiac pulmonary edema immediately after delivery.The Th1/Th2 cell ratio was markedly elevated to 51.4 before childbirth.

Case 5
Case 5 had a previous history of preeclamptic stillbirth and the hemolysis, elevated liver enzymes, and low platelet count (HELLP) syndrome during the first pregnancy.HELLP syndrome and eclampsia occurred after PE onset at 20 weeks of gestation, and an emergency cesarean section was needed for maternal safety.After the delivery, the patient suffered from severe complications, such as non-cardiac pulmonary edema, disseminated intravascular coagulation, and intraperitoneal hemorrhage.The course of the second pregnancy was stable under tacrolimus treatment without emergence of hypertension or proteinuria until 32 weeks of gestation; however, the HELLP syndrome recurred after PE at 33 weeks of gestation.The patient delivered a male fetus weighing 1,490 g via emergency cesarean section.Although an imbalance in the Th1/Th2 cell ratio was not observed during pregnancy, NK cell activity was markedly elevated to 64% (normal range, 18-40%) at 32 weeks of gestation.Low-dose aspirin was not prophylactically administered due to patient refusal.

Discussion
The calcineurin inhibitor tacrolimus is widely used as an immunosuppressant agent in solid organ transplantation and several autoimmune disorders.
Recently, tacrolimus has received attention as a treatment option for infertility associated with maternal-fetal immune abnormalities [3].Possible therapeutic interventions include endometrial maturation during the implantation window, suppression of fetal rejection, and induction of immune tolerance in a semi-allogeneic fetus.The primary effect of tacrolimus is its involvement in the direct inhibition of activated NK/NKT and Th1 cells.We have already reported the clinical efficacy and cellular immune parameter changes in tacrolimustreated patients with unexplained reproductive failure, such as repeated implantation failure and recurrent pregnancy loss [11][12][13].Tacrolimus might have a preventive effect on the pathological conditions suggestive of fetal rejection and uteroplacental dysfunction, including unexplained or preeclamptic stillbirth with Th1dominant immune states.The predominance of Th1 immunity observed in our cases remains one of the critical factors involved in the development of stillbirth and would be targeted using tacrolimus treatment.
In cases 1-3, tacrolimus treatment inhibited predominant Th1 immunity after the embryo transfer; therefore, the patient successfully achieved a live birth without the emergence of fetal growth retardation or maternal complications during pregnancy.In case 4, increased tacrolimus dose after a miscarriage resulted in her first live birth; however, the pregnancy had to be terminated at 28 weeks of gestation due to uncontrollable hypertension and severe fetal growth restriction.Transfer of fetal antigens into the mother increases with time after placental construction and accelerates after mid-pregnancy [3].In case 4, two peaks of immunological reactions due to recognition of fetal antigens could lead to early recurrent miscarriages and stillbirth/early onset PE, respectively.A daily 2-3-mg dose of tacrolimus probably did not adequately suppress these excessive immune responses.In case 5, no blood pressure, urinary finding, and fetal growth abnormalities were observed until 32 weeks of gestation, and changes in Th1/Th2 cell ratios did not predict the development of PE and HELLP syndrome.The elevation of NK cell activity appeared to be the only predictive indicator of these maternal complications.In contrast to the previous pregnancy, certain placental construction is considered to have been achieved via immunosuppressive treatment with tacrolimus; therefore, pregnancy was continued until 33 weeks of gestation in this patient.
In conclusion, tacrolimus is a potential drug candidate for the prevention of unexplained or preeclamptic stillbirth with Th1-dominant immune states.
However, this experience with tacrolimus should be interpreted carefully because we could not compare placental findings before and after the intervention, which are important clues in investigating the cause of stillbirth.Although Th1/Th2 cell ratios and NK cell activities were monitored as immune parameters in these patients, discussing their pathological conditions based on these parameters alone was difficult.Analysis of other CD4þ T cell subsets including Treg, Th17, and T follicular helper cells would be helpful in elucidating maternal-fetal immune abnormalities.In addition, novel biomarkers reflecting the maternalfetal immune status would be needed to determine the indications for tacrolimus treatment or the dose and duration of therapy in the future.

Table 1 .
Characteristics and pregnancy outcomes.