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Immune modulation treatments—where is the evidence?

  • Malene Meisner Hviid
    Affiliations
    Department of Obstetrics and Gynecology, Zealand University Hospital, Roskilde, Denmark
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  • Nick Macklon
    Correspondence
    Reprint requests: Nick Macklon, M.D., Ph.D., Department of Obstetrics and Gynaecology, University of Southampton, Princess Anne Hospital, Coxford Road, Southampton SO16 5YA, United Kingdom.
    Affiliations
    Department of Obstetrics and Gynecology, Zealand University Hospital, Roskilde, Denmark

    Department of Obstetrics and Gynaecology, University of Southampton, Princess Anne Hospital, Southampton, United Kingdom
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      While advances in assisted reproductive techniques have been substantial, failure of the apparently viable embryo to implant remains a source of distress and frustration to patients and specialists alike. The unique maternal immunological response to the embryo and the notion that defects in early placentation underlie the great complications of pregnancy have focused attention on the therapeutic potential of peri-implantation immunomodulation. On the face of it, the rationale for this approach is very attractive. However, as will be argued in this review, the clinical evidence base supporting the use of immunosuppressive treatments is weak and difficult to apply in practice and fails the needs of both doctors and their patients. This evidence gap is filled by justifications that are based largely on meeting patient expectations and commercial imperatives. However, this does not mean that immunomodulation treatments should be written off as ineffective. The literature in this field, while suffering the same challenges of heterogeneity, small studies, and publication bias as other areas of medicine, does hint at the way forward. Recurrent implantation failure and pregnancy loss are not diagnoses but clinical presentations that require appropriate phenotyping and etiological investigation. We are increasingly gaining the tools to make an “endometrial diagnosis,” and these will allow us to design clinical studies of interventions that treat the underlying cause rather than the symptoms of implantation failure. The current evidence base does not support the clinical use of immunomodulation therapies in patients undergoing IVF. However, more discerning phenotyping may identify groups who could benefit.

      Key Words

      Discuss: You can discuss this article with its authors and with other ASRM members at https://www.fertstertdialog.com/users/16110-fertility-and-sterility/posts/16263-24178
      Despite advances in assisted reproductive techniques (ART), the majority of IVF cycles still do not result in an ongoing pregnancy or live birth. The failure of apparently morphologically sound embryos to implant now represents the major limiting step to improving IVF outcomes. Depending on the definition used, up to 10% of couples undergoing IVF will experience recurrent implantation failure (RIF), and among those who do achieve implantation, many will face the disappointment of early pregnancy loss. Both represent a devastating occurrence for patients in whom serial transfers of high-quality embryos fail to result in a pregnancy. The pressures to “do something” to “improve implantation,” which include assertive patient demand and the competitive commercial context in which IVF is increasingly practiced, continue to rise, and empirical treatments have come in to fill the gap between scientific rationale and clinical need (
      • Spencer E.A.
      • Mahtani K.R.
      • Goldacre B.
      • Heneghan C.
      Claims for fertility interventions: a systematic assessment of statements on UK fertility centre websites.
      ,
      • Nardo L.G.
      • El-Toukhy T.
      • Stewart J.
      • Balen A.H.
      • Potdar N.
      British Fertility Society Policy and Practice Committee: adjuvants in IVF: evidence for good clinical practice.
      ).
      Particular interest has focused on modulating the maternal immune response to the implanting embryo. The premise for this would appear compelling, as much research has focused on understanding how the mother tolerates a genetically alien embryo and the mechanisms by which invasion of the maternal tissues by the embryo is permitted but limited. One of the paradigms to emerge has been the concept of a balance between pro- and anti-inflammatory states, characterized by a palette of cytokines and immune cells such as T-helper cells and natural killer (NK) cells, the relative populations of which are proposed to determine the fate of the implanting embryo. While it is becoming clear that such concepts risk oversimplification, the story is readily communicated and understood by the lay press and by patients, and an industry has grown at the interface of IVF and belief in the importance of the immunological determinants of implantation and their therapeutic modulation.
      The drivers to implement new innovations into clinical practice are strong and numerous in IVF, and much of the progress made since the very early days of assisted conception has derived from the willingness of patients to try something new, as well as a measure of serendipity. However, the failure of our field to thus far effectively address the remaining challenges in implantation failure requires us to reassess the data supporting current treatment approaches and, more importantly, the established paradigms of human implantation.
      There is a sizable literature relating to the use of immune modulation treatments in IVF. But the studies exploring the efficacy of different immunomodulation therapies, while numerous, are heterogeneous in design, method, intervention, and study population, making it difficult to interpret them and to design evidence-based rational therapy strategies. If we are to make progress in this challenging field, we need a greater understanding of the mechanisms and modulators of human implantation. In the meantime, immunomodulation therapies remain attractive to both patients and their doctors. This review aims to provide a brief overview of the current evidence supporting their use.

      The rationale for using immunomodulators

      In order for successful implantation to occur, a high-quality embryo must engage with and breach the luminal surface of the endometrium before embedding in the decidualized endometrium. As the embryo differentiates, angiogenesis and remodeling of the spiral arteries are induced, establishing the maternal-fetal circulation. Immune cell populations have been shown to be key players in the maternal response to the embryo, and many studies have illustrated the importance of a balanced cytokine environment. A still prevailing paradigm describes the balance between T-helper 1 (Th1) and T-helper 2 (Th2) produced cytokines as determinants of implantation. A shift in the ratio towards Th1 cells leads to increased production of proinflammatory cytokines such as interferon gamma (IFN-γ), interleukin (IL) 2, and tumor necrosis factor alpha (TNF-α) that mediate a cytotoxic cell-mediated immune response and increase phagocytosis and inflammation. In contrast, Th2 cells produce a range of interleukins involved in the humeral immune response and inhibit several functions of phagocytosis, which together represent an anti-inflammatory response (
      • Kwak-Kim J.Y.H.
      • Chung-Bang H.S.
      • Ng S.C.
      • Ntrivalas E.I.
      • Mangubat C.P.
      • Beaman K.D.
      • et al.
      Increased T helper 1 cytokine responses by circulating T cells are present in women with recurrent pregnancy losses and in infertile women with multiple implantation failures after IVF.
      ). This paradigm is supported by a number of observational studies reporting increased expression of proinflammatory cytokines in women with a history of recurrent pregnancy loss (
      • Raghupathy R.
      • Makhseed M.
      • Azizieh F.
      • Hassan N.
      • Al-Azemi M.
      • Al-Shamali E.
      Maternal Th1- and Th2-type reactivity to placental antigens in normal human pregnancy and unexplained recurrent spontaneous abortions.
      ).
      Disrupted population of peripheral and/or uterine NK (uNK) cells has also been implicated in implantation failure and early pregnancy. After ovulation, uNK cells become the dominant immune cells present in the decidualized endometrium, accounting for >30% of immune cells (
      • Moffett A.
      • Shreeve N.
      First do no harm: uterine natural killer (NK) cells in assisted reproduction.
      ). In early gestation, the uNK cells expand in number and mass around the trophoblast cells in the decidualized endometrium (
      • Moffett A.
      • Shreeve N.
      First do no harm: uterine natural killer (NK) cells in assisted reproduction.
      ). Here the uNK cells are thought to play an important role in the regulation of placentation by maintaining a balance between normal invasion of the trophoblast and excessive invasion. While peripheral blood NK cells play an important part in the innate immune system by recognizing foreign cells not representing HLA-class 1 molecules and early killing of viral pathogens, they are not thought to be key determinants of endometrial function (
      • Moffett A.
      • Shreeve N.
      First do no harm: uterine natural killer (NK) cells in assisted reproduction.
      ). Furthermore they stimulate antigen-presenting cells and thereby promote activation of the adaptive immune system.
      Another theory rests on the local balance between proinflammatory and anti-inflammatory cytokines in the receptive endometrium. The assumption that a pure anti-inflammatory milieu in the maternal fetal interface exists is an oversimplification (
      • Chaouat G.
      • Zourbas S.
      • Ostojic S.
      • Lappree-Delage G.
      • Dubanchet S.
      • Ledee N.
      • et al.
      New insights into maternal-fetal interactions at implantation.
      ,
      • Mekinian A.
      • Cohen J.
      • Alijotas-Reig J.
      • Carbillon L.
      • Nicaise-Roland P.
      • Kayem G.
      • et al.
      Unexplained recurrent miscarriage and recurrent implantation failure: is there a place for immunomodulation?.
      ,
      • Robertson S.A.
      • Jin M.
      • Yu D.
      • Moldenhauer L.M.
      • Davies M.J.
      • Hull M.L.
      • et al.
      Corticosteroid therapy in assisted reproduction—immune suppression is a faulty premise.
      ). Studying the cytokine composition in endometrial secretions aspirated before ET in 210 women, our group demonstrated a positive association between IL-10 and TNF-α on implantation and clinical pregnancy, respectively. Conversely, a negative association was observed between secretions of monocyte chemoattractant protein-1 levels and IL-1β levels on implantation and clinical pregnancy, respectively. This study was the first to show a positive association between the proinflammatory cytokine TNF-α and clinical pregnancy (
      • Boomsma C.M.
      • Kavelaars A.
      • Eijkemans M.J.C.
      • Lentjes E.G.
      • Fauser B.C.J.M.
      • Heijnen C.J.
      • et al.
      Endometrial secretion analysis identifies a cytokine profile predictive of pregnancy in IVF.
      ).
      The widely reported association between increased peripheral blood NK cells and an increased Th1/Th2 ratio (
      • Kwak-Kim J.Y.H.
      • Chung-Bang H.S.
      • Ng S.C.
      • Ntrivalas E.I.
      • Mangubat C.P.
      • Beaman K.D.
      • et al.
      Increased T helper 1 cytokine responses by circulating T cells are present in women with recurrent pregnancy losses and in infertile women with multiple implantation failures after IVF.
      ,
      • Matsubayashi H.
      • Hosaka T.
      • Sugiyama Y.
      • Suzuki T.
      • Arai T.
      • Kondo A.
      • et al.
      Increased natural killer–cell activity is associated with infertile women.
      ,
      • Emmer P.M.
      • Nelen W.L.
      • Steegers E.A.
      • Hendriks J.C.
      • Veerhoek M.
      • Joosten I.
      Peripheral natural killer cytotoxicity and CD56(pos)CD16(pos) cells increase during early pregnancy in women with a history of recurrent spontaneous abortion.
      ,
      • King K.
      • Smith S.
      • Chapman M.
      • Sacks G.
      Detailed analysis of peripheral blood natural killer (NK) cells in women with recurrent miscarriage.
      ) among women with recurrent miscarriage (RM) and RIF has fueled interest in testing peripheral NK cell counts, but these have been shown to bear little correlation to the NK cell populations present within the endometrium (
      • Moffett A.
      • Shreeve N.
      First do no harm: uterine natural killer (NK) cells in assisted reproduction.
      ,
      • Robertson S.A.
      • Jin M.
      • Yu D.
      • Moldenhauer L.M.
      • Davies M.J.
      • Hull M.L.
      • et al.
      Corticosteroid therapy in assisted reproduction—immune suppression is a faulty premise.
      ). Some authors have reported a significantly higher count of uNK cells in the endometrium from women with RIF (
      • Tuckerman E.
      • Mariee N.
      • Prakash A.
      • Li T.C.
      • Laird S.
      Uterine natural killer cells in peri-implantation endometrium from women with repeated implantation failure after IVF.
      ). Both peripheral blood sampling and endometrial biopsies interrogating NK cell counts and types have gained popularity with patients as intuitively attractive means of assessing the maternal factor in RIF and pregnancy loss. Such testing offers a means of diagnosing specific immune “defects” and a rationale for a variety of immune-modulating therapies. While a number of treatments are available, in general they derive from the premise that dampening the immune response to the embryo will improve outcomes.

      Glucocorticoids

      Corticosteroid treatment presents a number of appealing characteristics in the context of IVF. The treatment is easy to take and cheap and occurs in short treatment regimens, considered to be safe. As a result they are widely prescribed, often as part of an immunomodulating package that includes other interventions such as aspirin or low molecular weight heparin (LMWH). But do they work? Our group has published a meta-analysis of randomized controlled trials (RCTs) in which the evidence for the efficacy of supplementary systemic administration of glucocorticoids in the peri-implantation period in women undergoing IVF or intracytoplasmic sperm injection (ICSI) was subject to systematic review (
      • Boomsma C.M.
      • Keay S.D.
      • Macklon N.S.
      Peri-implantation glucocorticoid administration for assisted reproductive technology cycles.
      ). The analysis was restricted to women with a standard IVF or ICSI indication, and studies on men or women with autoantibodies were excluded. Thirteen studies were eligible for inclusion in the meta-analysis, involving a total number of 1,759 trial participants.
      Within the three studies that reported the live-birth rate per couple, no significant difference was observed between the intervention and control group odds ratio (OR), 1.21, and 95% confidence interval (CI), 0.67–2.19 (
      • Ando T.
      • Suganuma N.
      • Furuhashi M.
      • Asada Y.
      • Kondo I.
      • Tomoda Y.
      Successful glucocorticoid treatment for patients with abnormal autoimmunity on in vitro fertilization and embryo transfer.
      ,
      • Bider D.
      • Amoday I.
      • Tur-Kaspa I.
      • Livshits A.
      • Dor J.
      The addition of a glucocorticoid to the protocol of programmed oocyte retrieval for in-vitro fertilization—a randomized study.
      ,
      • Moffitt D.
      • Queenan J.T.J.
      • Veeck L.L.
      • Schoolcraft W.
      • Miller C.E.
      • Muasher S.J.
      Low-dose glucocorticoids after in vitro fertilization and embryo transfer have no significant effect on pregnancy rate.
      ). The pregnancy rate per couple was reported in all the included studies, but no significant differences were observed between the intervention and control group (OR, 1.16; 95% CI, 0.94–1.44). However, an analysis of a subgroup of studies that only included couples undergoing IVF rather than ICSI (
      • Ando T.
      • Suganuma N.
      • Furuhashi M.
      • Asada Y.
      • Kondo I.
      • Tomoda Y.
      Successful glucocorticoid treatment for patients with abnormal autoimmunity on in vitro fertilization and embryo transfer.
      ,
      • Bider D.
      • Amoday I.
      • Tur-Kaspa I.
      • Livshits A.
      • Dor J.
      The addition of a glucocorticoid to the protocol of programmed oocyte retrieval for in-vitro fertilization—a randomized study.
      ,
      • Moffitt D.
      • Queenan J.T.J.
      • Veeck L.L.
      • Schoolcraft W.
      • Miller C.E.
      • Muasher S.J.
      Low-dose glucocorticoids after in vitro fertilization and embryo transfer have no significant effect on pregnancy rate.
      ,
      • Kemeter P.
      • Feichtinger W.
      Prednisolone supplementation to Clomid and/or gonadotrophin stimulation for in-vitro fertilization—a prospective randomized trial.
      ,
      • Kim C.H.
      • Chae H.D.
      • Kang B.M.
      • Chang Y.S.
      • Mok J.E.
      The immunotherapy during in vitro fertilization and embryo transfer cycles in infertile patients with endometriosis.
      ,
      • Mottla G.L.
      • Smotrich D.B.
      • Gindoff P.R.
      • Stillman R.J.
      Increasing clinical pregnancy rates after IVF/ET. Can immunosuppression help?.
      ,
      • Tan S.L.
      • Balen A.
      • el Hussein E.
      • Campbell S.
      • Jacobs H.S.
      The administration of glucocorticoids for the prevention of ovarian hyperstimulation syndrome in in vitro fertilization: a prospective randomized study.
      ) indicated a marginally significantly higher pregnancy rate among those women who received glucocorticoid treatment (OR, 1.50; 95% CI, 1.05–2.13).
      Further subgroup analysis specific for cause of infertility and dosage or timing of glucocorticoid administration did not reveal any significant differences in pregnancy rates. Seven studies included in the meta-analysis reported the miscarriage rate per couple (
      • Ando T.
      • Suganuma N.
      • Furuhashi M.
      • Asada Y.
      • Kondo I.
      • Tomoda Y.
      Successful glucocorticoid treatment for patients with abnormal autoimmunity on in vitro fertilization and embryo transfer.
      ,
      • Bider D.
      • Amoday I.
      • Tur-Kaspa I.
      • Livshits A.
      • Dor J.
      The addition of a glucocorticoid to the protocol of programmed oocyte retrieval for in-vitro fertilization—a randomized study.
      ,
      • Moffitt D.
      • Queenan J.T.J.
      • Veeck L.L.
      • Schoolcraft W.
      • Miller C.E.
      • Muasher S.J.
      Low-dose glucocorticoids after in vitro fertilization and embryo transfer have no significant effect on pregnancy rate.
      ,
      • Kemeter P.
      • Feichtinger W.
      Prednisolone supplementation to Clomid and/or gonadotrophin stimulation for in-vitro fertilization—a prospective randomized trial.
      ,
      • Kim C.H.
      • Chae H.D.
      • Kang B.M.
      • Chang Y.S.
      • Mok J.E.
      The immunotherapy during in vitro fertilization and embryo transfer cycles in infertile patients with endometriosis.
      ,
      • Mottla G.L.
      • Smotrich D.B.
      • Gindoff P.R.
      • Stillman R.J.
      Increasing clinical pregnancy rates after IVF/ET. Can immunosuppression help?.
      ,
      • Ubaldi F.
      • Rienzi L.
      • Ferrero S.
      • Anniballo R.
      • Iacobelli M.
      • Cobellis L.
      • et al.
      Low dose prednisolone administration in routine ICSI patients does not improve pregnancy and implantation rates.
      ). Again, no statistically significant impact on early pregnancy outcomes was evident (OR, 1.48; 95% CI, 0.86–2.54). One study was undertaken to address the effectiveness of glucocorticoids to prevent ovarian hyperstimulation syndrome (OHSS) (
      • Tan S.L.
      • Balen A.
      • el Hussein E.
      • Campbell S.
      • Jacobs H.S.
      The administration of glucocorticoids for the prevention of ovarian hyperstimulation syndrome in in vitro fertilization: a prospective randomized study.
      ). Again, no significant difference in the incidence of this complication was observed (OR, 0.93; 95% CI, 0.22–3.91).
      A more recent study investigated a combined therapy of prednisolone 20 mg/day and LMWH 1 mg/kg/day among 295 women with one or more failed ICSI attempts (
      • Fawzy M.
      • El-Refaeey A.A.
      Does combined prednisolone and low molecular weight heparin have a role in unexplained implantation failure?.
      ). Treatment was started on the day of oocyte retrieval and continued until the eighth week of gestation. The authors reported a significant increase in pregnancy, implantation, clinical pregnancy, and ongoing pregnancy rates among the intervention group. However, an important limitation of this study is the mode of “quasi” randomization by alternate numbers. This is subject to a serious risk of bias since the investigator knows to which group the next subject will be allocated (
      • Vickers A.J.
      How to randomize.
      ).
      In addition to routine use, glucocorticoids have been proposed as a therapeutic option for women undergoing IVF treatment who test positive for autoantibodies. Autoantibodies have been associated with implantation failure and repeated pregnancy loss (
      • Stern C.
      • Chamley L.
      • Hale L.
      • Kloss M.
      • Speirs A.
      • Baker H.W.
      Antibodies to beta2 glycoprotein I are associated with in vitro fertilization implantation failure as well as recurrent miscarriage: results of a prevalence study.
      ), endometriosis, and unexplained infertility (
      • Reimand K.
      • Talja I.
      • Metskula K.
      • Kadastik U.
      • Matt K.
      • Uibo R.
      Autoantibody studies of female patients with reproductive failure.
      ,
      • Taylor P.V.
      • Campbell J.M.
      • Scott J.S.
      Presence of autoantibodies in women with unexplained infertility.
      ). Up to 50% lower pregnancy rates have been reported after IVF treatment in women with autoantibodies compared with matched controls (
      • Dmowski W.P.
      • Rana N.
      • Michalowska J.
      • Friberg J.
      • Papierniak C.
      • el-Roeiy A.
      The effect of endometriosis, its stage and activity, and of autoantibodies on in vitro fertilization and embryo transfer success rates.
      ,
      • Taniguchi F.
      Results of prednisolone given to improve the outcome of in vitro fertilization-embryo transfer in women with antinuclear antibodies.
      ). It is proposed that uteroplacental thrombosis and vasoconstriction, resulting from binding of the antibodies to platelet and endothelial membrane phospholipids, may underlie adverse reproductive outcomes in these women. Corticosteroids are known to suppress autoantibodies and therefore may offer a rational means of supporting successful implantation in these women.
      Retrospective analyses have reported beneficial effects of glucocorticoid treatment in autoantibody seropositive women with implantation failure (
      • Geva E.
      • Amit A.
      • Lerner-Geva L.
      • Yaron Y.
      • Daniel Y.
      • Schwartz T.
      • et al.
      Prednisone and aspirin improve pregnancy rate in patients with reproductive failure and autoimmune antibodies: a prospective study.
      ) and in women with antinuclear antibodies (
      • Taniguchi F.
      Results of prednisolone given to improve the outcome of in vitro fertilization-embryo transfer in women with antinuclear antibodies.
      ). Prospective studies have also indicated a possible benefit in this patient group. An RCT (
      • Ando T.
      • Suganuma N.
      • Furuhashi M.
      • Asada Y.
      • Kondo I.
      • Tomoda Y.
      Successful glucocorticoid treatment for patients with abnormal autoimmunity on in vitro fertilization and embryo transfer.
      ) investigating the effect of glucocorticoids in women with antinuclear, antidouble-stranded DNA, anticardiolipin antibodies, or lupus anticoagulant tested the effect of a low daily dose of 5 mg prednisolone given throughout the IVF cycle. Although no significant decrease in autoantibody titers was observed, pregnancy rates in women with autoantibodies were significantly increased in the intervention arm after the use of glucocorticoids. A further RCT investigated the use of glucocorticoids in women with endometriosis, of whom 38% were also seropositive for non-organ-specific autoantibodies (
      • Kim C.H.
      • Chae H.D.
      • Kang B.M.
      • Chang Y.S.
      • Mok J.E.
      The immunotherapy during in vitro fertilization and embryo transfer cycles in infertile patients with endometriosis.
      ). Again, significantly higher pregnancy rates were reported in women allocated to the treatment arm of the study.
      While this overview of the published literature would appear to support the case for prescribing glucocorticoids in certain patient groups, it should be noted that few studies have been double blinded and placebo controlled. Moreover, a funnel plot of precision versus OR in our meta-analysis suggested a significant impact of publication bias. Overall, there is no clear evidence to support empirical administration of glucocorticoids during the peri-implantation period in subfertile women undergoing IVF/ICSI. This does not imply that they are wholly without value, however. The challenge remains to differentiate those women who may benefit from those who will see no effect or indeed a detrimental impact of treatment.

      Aspirin

      Aspirin or acetylsalicylic acid (ASA) is an ancient pharmaceutical that has found its primary uses as a painkilling anti-inflammatory and more recently as a preventative of cardiovascular disease. ASA is known to inhibit the enzyme cyclooxygenase and thereby decrease the production of prostaglandins. In platelets, the inhibition of cyclooxygenase is known to be irreversible and therefore effectively stops the production of thromboxane acting as a vasoconstrictor. The proposed benefit from aspirin in reproduction is reduction of inflammation in the uterine cavity and improvement of uterine and ovarian perfusion, which might improve endometrial receptivity and ovarian responsiveness (
      • Hanevik H.I.
      • Friberg M.
      • Bergh A.
      • Haraldsen C.
      • Kahn J.A.
      Do acetyl salicylic acid and terbutaline in combination increase the probability of a clinical pregnancy in patients undergoing IVF/ICSI?.
      ,
      • Dirckx K.
      • Cabri P.
      • Merien A.
      • Galajdova L.
      • Gerris J.
      • Dhont M.
      • et al.
      Does low-dose aspirin improve pregnancy rate in IVF/ICSI? A randomized double-blind placebo controlled trial.
      ).
      This possible effect from aspirin in ART has been widely investigated through a large number of RCTs. We identified 21 studies, of which 12 were from 2006 and later (Table 1). Rubinstein et al. showed in a double-blinded placebo-controlled randomized trial a significant improvement in pregnancy and implantation rates in the intervention group receiving 100 mg aspirin/day started before ovarian stimulation and continued through 12 weeks of gestation (
      • Rubinstein M.
      • Marazzi A.
      • Polak de Fried E.
      Low-dose aspirin treatment improves ovarian responsiveness, uterine and ovarian blood flow velocity, implantation, and pregnancy rates in patients undergoing in vitro fertilization: a prospective, randomized, double-blind placebo-controlled assay.
      ). The study included 298 unselected women with infertility caused by tubal factor. Along with the significant increase in pregnancy and implantation rates, the authors found significant increased ovarian response and uterine blood flow in the intervention group. Urman et al. conducted a similar trial on 300 unselected women undergoing ICSI and randomizing them to 80 mg aspirin from stimulation until fetal heart activity was detected (
      • Urman B.
      • Mercan R.
      • Alatas C.
      • Balaban B.
      • Isiklar A.
      • Nuhoglu A.
      Low-dose aspirin does not increase implantation rates in patients undergoing intracytoplasmic sperm injection: a prospective randomized study.
      ). Only the physicians were blinded to the intervention. The authors failed to prove any difference between the two groups (
      • Urman B.
      • Mercan R.
      • Alatas C.
      • Balaban B.
      • Isiklar A.
      • Nuhoglu A.
      Low-dose aspirin does not increase implantation rates in patients undergoing intracytoplasmic sperm injection: a prospective randomized study.
      ). Later Stern et al. randomized 143 women with ≥10 failed ETs by IVF or ICSI attempts and at least one autoantibody (anti-phospholipid antibody or anti-nuclear antibodies). A total of 300 cycles were performed randomized to 100 mg aspirin and 5,000 IU heparin or placebo from the day of ET to the time for pregnancy test. There was no significant difference between the groups in implantation and clinical pregnancy rates (
      • Stern C.
      • Chamley L.
      • Norris H.
      • Hale L.
      • Baker H.W.G.
      A randomized, double-blind, placebo-controlled trial of heparin and aspirin for women with in vitro fertilization implantation failure and antiphospholipid or antinuclear antibodies.
      ). However, a study by Waldenström et al. randomizing an unselected cohort of women to 75 mg aspirin from ET to pregnancy test found a borderline significant increased OR of 1.3 in favor of aspirin on clinical pregnancy and OR of 1.2 on live-birth rate (
      • Waldenström U.
      • Hellberg D.
      • Nilsson S.
      Low-dose aspirin in a short regimen as standard treatment in in vitro fertilization: a randomized, prospective study.
      ). No blinding or placebo was used, and randomization was conducted by alternation by day. Päkkilä et al. randomized 374 women with four or fewer previous IVF or ICSI cycles to receive 100 mg/day aspirin or placebo from ET to birth. The study was double blinded, and randomization was performed with sealed envelopes administered by the pharmacist. No significant difference was found between the groups on any outcome measure (
      • Päkkilä M.
      • Rälsänen J.
      • Heinonen S.
      • Tinkanen H.
      • Tuomivaara L.
      • Mäkikallio K.
      • et al.
      Low-dose aspirin does not improve ovarian responsiveness or pregnancy rate in IVF and ICSI patients: a randomized, placebo-controlled double-blind study.
      ). After that trial, a study by Duvan et al. investigated the effect of aspirin alone or in combination with prednisolone on a randomized group of women undergoing their first cycle of ICSI. The 187 women were divided into four groups to receive aspirin 100 mg/day, prednisolone 10 mg/day, aspirin 100 mg/day and prednisolone 10 mg/day, or no intervention (controls). Again no difference was found between the groups with intervention and the control group with respect to implantation, pregnancy, and clinical pregnancy rates (
      • Duvan C.I.
      • Ozmen B.
      • Satiroglu H.
      • Atabekoglu C.S.
      • Berker B.
      Does addition of low-dose aspirin and/or steroid as a standard treatment in nonselected intracytoplasmic sperm injection cycles improve in vitro fertilization success? A randomized, prospective, placebo-controlled study.
      ).
      Table 1Overview of RCTs testing aspirin as intervention.
      StudyMethodParticipantsInterventionMain outcome
      Hanevik et al. (2012)Single-center RCT by envelope randomization ratio 3:2, no placebo/blinding.279 women, <40 y, normal uterine cavity, no hormonal disorders, partner with no azoospermia, only frozen embryos were used.ASA 75 mg/d from ET to 9 wk of gestation if pregnant and terbutaline 5 mg given 3 h before ET, 6 h after ET, and the following morning.Clinical pregnancy rate per cycle was 30.5% vs. 42% in the intervention vs. control group, P=.18.
      Várnagy et al. (2010)Single-center RCT randomized by paired vs. unpaired social number, no placebo/blinding.2,425 women undergoing GnRH agonist cycles, unselected. Cycles were divided into high risk and low risk of OHSS.ASA 100 mg/d from first day of stimulation to menstruation, a negative pregnancy test, or ultrasound-verified embryo heart activity.Among the high-risk group the intervention group had a lower risk of OHSS (0.25% vs. 8.4%), P<.001. No difference was detected in clinical pregnancy rate between the groups.
      Dirckx et al. (2009)Single-center RCT randomized by computer, double blinded.193 women undergoing first or second cycle of IVF/ICSI, no platelet dysfunction, thrombocytopenia, gastric ulcers, gastritis, aspirin hypersensitivity, or anticoagulation treatment.ASA 100 mg/d first day in the prior cycle throughout stimulation to gestational age 6+3.Clinical pregnancy (heart beat/cycle) 32% vs. 31% in the intervention vs. placebo group, respectively. Live-birth rate 25% vs. 28%, respectively, not significantly different.
      Lambers et al. (2009)Single-center RCT, randomized by computer, double blinded.169 women <39 y with >1 failed IVF/ICSI, excluded when body mass index >30 kg/m2, tubal pathology, hypertension, smoking >5 cigarettes/day, systemic disease, or untreated endocrinopathy.ASA 100 mg/d from first day in the prior cycle throughout stimulation and if pregnant, continued to gestational age 12 wk.Clinical pregnancy rate (serum hCG >50 IU/L) 40.5% vs. 39.3% in the intervention group vs. placebo group, not significant. All other outcomes not significantly different between the two groups.
      Revelli et al. (2008)Single-center RCT, randomized by computer in a ratio 1:3, no blinding or placebo.395 women, <40 y undergoing the first cycles of IVF/ICSI. No autoantibodies present or autoimmune disease. The women were all normal or good ovarian responders. Only fresh ET's were used.ASA 100 mg/d and prednisolone 10 mg/d from 1. Day of stimulation, prednisolone was increased to 30 mg 5 d from ET. After that again 10 mg/d throughout 10 wk, if positive pregnancy test.Women were subdivided in groups with high risk of OHSS and low risk of OHSS. Implantation rate was 31.5% vs. 26.2% in the high-risk group and 19.4% vs. 17.3% in the low-risk group, intervention vs. nothing, respectively.
      Duvan et al. (2006)Single-center RCT, lottery randomization, using placebo.187 women undergoing their first cycle of ICSI; no other criteria were made. Participants were divided into four groups according to treatment regime.On ET day (A) 100 mg ASA/day, (B) 10 mg prednisolone/day, (C) 100 mg ASA and 10 mg prednisolone/day, and (D) control group with placebo.Pregnancy and clinical pregnancy rates. No statistically significant results were found between the groups.
      Päkkilä et al. (2005)Multicenter RCT, using envelope randomization, double blinded.374 women with <4 failed IVF/ICSI, <40 y of age, and no contradiction to ASA were included.ASA 100 mg/d from stimulation to menstruation, negative pregnancy test, or birth of child.Number of oocytes, quality of embryo, clinical pregnancy rate, and pregnancy rate were outcome measures. No significant difference between the intervention and control group was documented.
      Waldenström et al. (2004)Single-center RCT, using alternation by day as randomization, no blinding or placebo.1,380 cycles in an unselected cohort of women. Excluded if allergy to aspirin.ASA 75 mg from ET to pregnancy test.Positive serum hCG: OR, 1.3; CI, 1.0–1.6; clinical pregnancy: OR, 1.3; CI, 1.0–1.6; birth rate: OR, 1.2; CI, 1.0–1.6. All borderline significant.
      Stern et al. (2003)Multicenter RCT, using computer randomization and double blinding.143 women, total of 300 cycles, with ≥10 failed IVF/ICSI and ≥1 autoantibody present, normal uterus, no hemophilia or thrombotic disorders, no osteoporosis.ASA 100 mg/d and 5,000 IU heparin from ET until results of serum hCG were available.Positive pregnancy test, implantation rate, and live birth rate. No significant difference was found between intervention and placebo group in all main outcomes.
      Urman et al. (2000)Single-center RCT, randomizing by computer and only blinding of the physician300 unselected women undergoing ICSI because of male factor infertility.ASA 80 mg from stimulation to fetal heart activity was detected. Treatment was stopped if pregnancy test was negative.Implantation rate/ET (15.6% vs. 15.1%) and pregnancy rate (39.6% vs. 43.4%) in the intervention vs. control group, respectively.
      Rubinstein et al. (1999)Single-center RCT, using envelope randomization and double blinding298 women undergoing IVF because of tubal factorASA 100 mg/d from 21st day of the preceding menstrual cycle through to 12 wk if tested pregnant.Ovarian responsiveness was measured with three individual indicators; all were significantly increased in the treatment group, implantation rate (17.8% vs 9.2%), P<.05, and pregnancy rate (45% vs. 28%), P<.05.
      Some years later Revelli et al. investigated the effect of aspirin in combination with prednisolone on implantation rate. Women undergoing their first cycle of IVF or ICSI were randomly allocated to receive 100 mg aspirin and 10 mg prednisolone from the first day of stimulation until pregnancy test. Prednisolone was increased to 30 mg for 5 days from ET. No blinding or placebo was used. Primary outcome was implantation rate, which showed no difference between the groups (
      • Revelli A.
      • Dolfin E.
      • Gennarelli G.
      • Lantieri T.
      • Massobrio M.
      • Holte J.G.
      • et al.
      Low-dose acetylsalicylic acid plus prednisolone as an adjuvant treatment in IVF: a prospective, randomized study.
      ). Lambers et al. conducted a randomized placebo-controlled double-blinded trial investigating the effect of aspirin versus placebo on a group of women with more than one previous failed IVF or ICSI as well as the clinical pregnancy rate. The intervention was administered from stimulation to 12 weeks of gestation. The study failed to prove any effect of aspirin on clinical pregnancy rate (
      • Lambers M.J.
      • Hoozemans D.A.
      • Schats R.
      • Homburg R.
      • Lambalk C.B.
      • Hompes P.G.A.
      Low-dose aspirin in non-tubal IVF patients with previous failed conception: a prospective randomized double-blind placebo-controlled trial.
      ). Another study similar in design to that of Lambers et al. randomized 196 women undergoing their first or second cycle of IVF or ICSI to aspirin 100 mg/day from stimulation to 6 weeks of gestation (
      • Revelli A.
      • Dolfin E.
      • Gennarelli G.
      • Lantieri T.
      • Massobrio M.
      • Holte J.G.
      • et al.
      Low-dose acetylsalicylic acid plus prednisolone as an adjuvant treatment in IVF: a prospective, randomized study.
      ). Again no difference in clinical pregnancy rate was detected (
      • Dirckx K.
      • Cabri P.
      • Merien A.
      • Galajdova L.
      • Gerris J.
      • Dhont M.
      • et al.
      Does low-dose aspirin improve pregnancy rate in IVF/ICSI? A randomized double-blind placebo controlled trial.
      ). Várnagy et al. investigated the possible reduced incidence of ovarian hyperstimulation from treatment with aspirin and the effect on clinical pregnancy rate. Initiation and randomization of 3,154 cycles of which 2,425 cycles received hCG were done, of which aspirin was used in 1,503 cycles and no treatment in 922 cycles. The study found no difference in clinical pregnancy rate between the groups, but a significant reduction of OHSS in the treatment arm was found (
      • Várnagy A.
      • Bódis J.
      • Mánfai Z.
      • Wilhelm F.
      • Busznyák C.
      • Koppán M.
      Low-dose aspirin therapy to prevent ovarian hyperstimulation syndrome.
      ). Finally, a study by Hanevik et al. investigated the effect on aspirin in combination with terbutalin on clinical pregnancy rate per cycle. A total of 279 unselected women receiving only frozen ET were randomized to aspirin 75 mg from ET to 9 weeks of gestation combined with terbutalin × 3 within the first 24 hours after ET. A slightly lower, but insignificant, clinical pregnancy rate was detected in the intervention group (
      • Hanevik H.I.
      • Friberg M.
      • Bergh A.
      • Haraldsen C.
      • Kahn J.A.
      Do acetyl salicylic acid and terbutaline in combination increase the probability of a clinical pregnancy in patients undergoing IVF/ICSI?.
      ).
      Considering the published data together, and consistent with the findings of a recent Cochrane review (
      • Siristatidis C.S.
      • Dodd S.R.
      • Drakeley A.J.
      Aspirin for in vitro fertilisation.
      ), it can be concluded that treatment with low-dose aspirin does not improve pregnancy outcome in terms of implantation, clinical pregnancy, ongoing pregnancy, or live-birth rates in an unselected population of women undergoing IVF or ICSI. However, the available studies differ significantly in design, participants, intervention, and outcome measures, making the conclusions drawn from these studies hard to interpret, and again it may well be that certain women could benefit, but the appropriate endometrial diagnostic strategies still elude us.

      Heparin

      A number of biological rationales have been proposed to support the use of heparin cotreatment in IVF. Initially, interest developed primarily because of an antithrombotic effect, but treatment is now justified by other observed actions that may modulate early implantation (
      • Nelson S.M.
      • Greer I.A.
      The potential role of heparin in assisted conception.
      ). Heparin prevents compliment activation in pregnant patients with antiphospholipid antibodies (
      • Berker B.
      • Taşkin S.
      • Kahraman K.
      • Taşkn E.A.
      • Atabekoglu C.
      • Sönmezer M.
      The role of low-molecular-weight heparin in recurrent implantation failure: a prospective, quasi-randomized, controlled study.
      ). The complex consisting of heparin-binding epidermal growth factor has been shown to facilitate an invasive phenotype of the throphoblast and inhibit apoptosis (
      • Berker B.
      • Taşkin S.
      • Kahraman K.
      • Taşkn E.A.
      • Atabekoglu C.
      • Sönmezer M.
      The role of low-molecular-weight heparin in recurrent implantation failure: a prospective, quasi-randomized, controlled study.
      ). Heparin also increases the free level of insulin-like growth factor (IGF) I and IGF II, which increase trophoblast invasion (
      • Nelson S.M.
      • Greer I.A.
      The potential role of heparin in assisted conception.
      ). Heparin has been shown to induce transcription of matrix metalloproteinases, which is known to regulate cell-cell interactions including breakdown of the decidua's basement membrane, facilitating trophoblast invasion. Add to this heparin's relative safety, low cost, and simple administration, and an attractive proposition emerges.
      We identified five randomized controlled studies investigating the possible positive effects from heparin on ART (
      • Fawzy M.
      • El-Refaeey A.A.
      Does combined prednisolone and low molecular weight heparin have a role in unexplained implantation failure?.
      ,
      • Berker B.
      • Taşkin S.
      • Kahraman K.
      • Taşkn E.A.
      • Atabekoglu C.
      • Sönmezer M.
      The role of low-molecular-weight heparin in recurrent implantation failure: a prospective, quasi-randomized, controlled study.
      ,
      • Noci I.
      • Milanini M.N.
      • Ruggiero M.
      • Papini F.
      • Fuzzi B.
      • Artini P.G.
      Effect of dalteparin sodium administration on IVF outcome in non-thrombophilic young women: a pilot study.
      ,
      • Urman B.
      • Ata B.
      • Yakin K.
      • Alatas C.
      • Aksoy S.
      • Mercan R.
      • et al.
      Luteal phase empirical low molecular weight heparin administration in patients with failed ICSI embryo transfer cycles: a randomized open-labeled pilot trial.
      ,
      • Qublan H.
      • Amarin Z.
      • Dabbas M.
      • Farraj A.-E.
      • Beni-Merei Z.
      • Al-Akash H.
      • et al.
      Low-molecular-weight heparin in the treatment of recurrent IVF-ET failure and thrombophilia: a prospective randomized placebo-controlled trial.
      ) (Table 2). Qublan et al. investigated the effect of heparin on a population with three or more failed IVF attempts and at least one thrombophilia disorder. The women were between 19 and 35 years, with body mass index between 19 and 29 m2/kg, no medical or hormonal illnesses, and a normal uterine cavity. A total of 83 women reached randomization. The authors reported a significantly higher implantation rate (19.8% vs. 6.1%), pregnancy rate (31% vs. 9.6%), and live-birth rate (23.4% vs. 2.4%) and a reduced miscarriage rate in the heparin intervention group (
      • Qublan H.
      • Amarin Z.
      • Dabbas M.
      • Farraj A.-E.
      • Beni-Merei Z.
      • Al-Akash H.
      • et al.
      Low-molecular-weight heparin in the treatment of recurrent IVF-ET failure and thrombophilia: a prospective randomized placebo-controlled trial.
      ). Urman et al. conducted an open-label, randomized controlled pilot study, including 150 consecutive women with RIF defined as two or more failed ETs; age ≤38 years; absence of any hormonal, coagulation, or immunological disorder; and normal uterine cavity. Subjects were randomized to receive luteal phase support with or without cotreatment with LMWH. The intervention was continued until 12 weeks of gestation and stopped if the woman miscarried. The study failed to show any significant increase in clinical pregnancy, ongoing pregnancy, live-birth, or implantation rates in the intervention group (
      • Urman B.
      • Ata B.
      • Yakin K.
      • Alatas C.
      • Aksoy S.
      • Mercan R.
      • et al.
      Luteal phase empirical low molecular weight heparin administration in patients with failed ICSI embryo transfer cycles: a randomized open-labeled pilot trial.
      ). Noci et al. reported a similar randomized control study of 172 women under 40 years of age undergoing their first IVF or ICSI cycle (
      • Noci I.
      • Milanini M.N.
      • Ruggiero M.
      • Papini F.
      • Fuzzi B.
      • Artini P.G.
      Effect of dalteparin sodium administration on IVF outcome in non-thrombophilic young women: a pilot study.
      ). The subjects, who had no diagnosed coagulation disorder and a normal uterine cavity, were randomized by computer to take fragmin 2,500 IU SC or no intervention from the day of oocyte retrieval until 9 weeks of pregnancy. Both groups received luteal phase support. The primary outcome was live-birth rate per embryo, and the secondary outcomes included implantation and clinical pregnancy rates. No significant differences were found between the groups.
      Table 2Overview over RCTs using heparin as intervention.
      StudyMethodParticipantsInterventionMain outcome
      Fawzy et al. (2014)Single-center RCT, quasi-randomized by alternation, no blinding, or placebo.295 women contributed with one cycle. All had one or two prior ICSI failures, <39 y, normal uterus, no hormonal disorders, no contradiction to planned treatment.20 mg prednisolone/day and heparin 1 mg/kg/d from oocyte retrieval to 8 wk. Treatment was stopped if the pregnancy test was negative.Total pregnancy (42.8 vs. 30.3), P=.028; clinical pregnancy (40.7% vs. 27.5%), P=.018; ongoing pregnancy (38.6% vs. 24.6%), P=.016; and implantation rates (23.9% vs. 14.7%), P<.001, treatment vs. control, respectively.
      Noci et al. (2011)Single-center pilot RCT, randomized by computer, no blinding or placebo.172 women undergoing their first IVF/ICSI cycle. All <40 y, normal tuba and uterus, no endocrine, coagulation, or chronic disorders.2,500 IU SC fragmin from oocyte retrieval to 9 wk pregnant. Treatment was stopped if tested negative or no live pregnancy was found.Live-birth rate/ET (21% vs. 16%), clinical pregnancy rate/ET (26% vs. 20%), implantation rate (15% vs. 12%) in the treatment vs. controls, respectively. All results not significant.
      Berker et al. (2011)Single-center, randomization dependent on which physician participants saw, no blinding or placebo.219 women with ≥2 prior ICSI failures, normal uterus, no endocrine, coagulation, or autoantibody disorder.4,000 IU Clexane from oocyte retrieval to 12 wk of gestation. Treatment was discontinued if pregnancy test was negative.Clinical pregnancy rate (34.6% vs. 33.9%), live-birth rate (30.7% vs. 29.1%), and implantation rate (22.6% vs. 21.1%) treatment vs. control, respectively. All results not significant.
      Urman et al. (2009)Single-center pilot RCT, randomized by computer, no blinding or placebo.150 women with ≥2 prior IVF/ICSI failures, age ≤38, no hormonal, immunological, or coagulatory diseases, and a normal uterus were included.1mg/kg/d from oocyte retrieval to 12 wk of gestation if tested positive 12 d after ET.Clinical pregnancy (45.3% vs. 38.7%), implantation (24.5% vs. 19.8%), ongoing pregnancy (>20 wk; 37.3% vs. 26.7%), and live birth rates (34.7% vs. 26.7%) in the treatment vs. control group, respectively. All results not significant.
      Qublan et al. (2008)Single-center RCT, randomized by random table, only blinding of subjects, placebo used.83 women with ≥3 prior IVF/ICSI failures and ≥1 coagulation disorder, age between 19 and 35 y, normal ovarian response, no systemic or hormonal diseases, no endometriosis, no family history of thrombosis, and a normal uterus were included.Enoxaparin 40 mg/d from ET to end of pregnancy. Treatment was discontinued if negative pregnancy test 14 d after ET or fetal diminish was diagnosed.Implantation rate (19.8% vs. 6.1%), P<.001; pregnancy rate (fetal heart activity) (31.0% vs. 9.6%), P<.05; and live-birth rate (23.8 vs. 2.4), P<.01 in the treatment vs. control group, respectively.
      A larger single-center study reported outcomes in 219 women who had undergone two or more failed ICSI attempts and had no coagulation or uterine cavity disorders. A total of 110 women were in the treatment arm and received Clexane 4,000 IU per day starting after oocyte retrieval and continued until 12 weeks of gestation or discontinued if no pregnancy was detected. Despite allocation being dependent on which clinician they saw, and hence subject to significant risk of bias, no significant differences in outcomes were observed between the groups.
      Evaluating the results from these RCTs of heparin and that discussed earlier in which cotreatment with glucocorticoids was reported (
      • Fawzy M.
      • El-Refaeey A.A.
      Does combined prednisolone and low molecular weight heparin have a role in unexplained implantation failure?.
      ) again requires the conclusion to be drawn that the evidence supporting the value of heparin as a putative effective immunomodulating treatment in IVF is weak. There may be some support for its use in a younger group of women with three or more failed attempts and at least one thrombophilia disorder, but further high-quality trials are required before this can be advocated.

      Immunoglobulin G

      Intravenous immunoglobulin G (IVIG) has become established as an effective (if expensive) treatment for autoimmune and inflammatory conditions such as relapsing inflammatory polyneuropathy, Guillain-Barre syndrome, and Kawasaki disease (
      • Clark D.A.
      • Coulam C.B.
      • Stricker R.B.
      Is intravenous immunoglobulins (IVIG) efficacious in early pregnancy failure? A critical review and meta-analysis for patients who fail in vitro fertilization and embryo transfer (IVF).
      ). In ART, the proposed mode of action is by a reduction of peripheral cytotoxic NK cells (
      • Ruiz J.E.
      • Kwak J.Y.
      • Baum L.
      • Gilman-Sachs A.
      • Beaman K.D.
      • Kim Y.B.
      • et al.
      Effect of intravenous immunoglobulin G on natural killer cell cytotoxicity in vitro in women with recurrent spontaneous abortion.
      ), enhancement of regulatory T cells, and down-regulation of antibody-producing B cells (
      • Ramos-Medina R.
      • García-Segovia A.
      • Gil J.
      • Carbone J.
      • Aguarón de la Cruz A.
      • Seyfferth A.
      • et al.
      Experience in IVIg therapy for selected women with recurrent reproductive failure and NK cell expansion.
      ). Proponents of this adjuvant therapy argue that these effects bias the immune system towards a Th2-mediated response by decreasing the Th1 cytotoxic response.
      A Medline search identified only two RCTs, of which only one was conducted as an RCT on humans. In a randomized, placebo-controlled double-blinded trial, women who had undergone two or more failed IVF attempts were randomized to receive 500 mg/kg IVIG at the time of ET and again 4 weeks later if a live fetus was present on ultrasound examination. The study failed to show any positive effect from IVIG treatment on live-birth, ongoing pregnancy, clinical pregnancy, or implantation rates (
      • Stephenson M.D.
      • Fluker M.R.
      Treatment of repeated unexplained in vitro fertilization failure with intravenous immunoglobulin: a randomized, placebo-controlled Canadian trial.
      ).
      Beyond this single RCT, the studies supporting the use of IVIG are a mix of retrospective cohort trials and prospective cohort trials (
      • Ramos-Medina R.
      • García-Segovia A.
      • Gil J.
      • Carbone J.
      • Aguarón de la Cruz A.
      • Seyfferth A.
      • et al.
      Experience in IVIg therapy for selected women with recurrent reproductive failure and NK cell expansion.
      ,
      • Virro M.R.
      • Winger E.E.
      • Reed J.L.
      Intravenous immunoglobulin for repeated IVF failure and unexplained infertility.
      ,
      • Winger E.E.
      • Reed J.L.
      • Ashoush S.
      • El-Toukhy T.
      • Ahuja S.
      • Taranissi M.
      Elevated preconception CD56 +16 + and/or Th1:Th2 levels predict benefit from IVIG therapy in subfertile women undergoing IVF.
      ); all report results that favor IVIG treatment. However, these studies have a number of severe limitations. In an observational retrospective cohort study investigating 428 women with RM or RIF, women with a high count of NK cells or expansion of an NK T-like lymphocyte were treated with IVIG. The women were all treated with IVIG 400 mg/kg on the day of ET, after a positive pregnancy test and again every 3 weeks until 35–36 weeks of gestation. In addition to IVIG, all participants took ASA 100 mg/day, and women with any type of thrombophilia or family history of thrombosis were additionally treated with heparin. The control group consisted of 50 women with proven reproductive health and served as the basis for determination of cutoff levels in peripheral blood samples and NK cell count. The authors reported a significant increase in clinical pregnancy rate and live-birth rate in the intervention IVIG group. In a study of similar design but with an even more comprehensive intervention in which participants also received anti-TNF-alpha, and some were given lymphocyte immunization therapy, while women with inherited or acquired thrombophilia were given Clexane (
      • Virro M.R.
      • Winger E.E.
      • Reed J.L.
      Intravenous immunoglobulin for repeated IVF failure and unexplained infertility.
      ), positive results of this “saturation” strategy were reported. Winger et al. (
      • Winger E.E.
      • Reed J.L.
      • Ashoush S.
      • Ahuja S.
      • El-Toukhy T.
      • Taranissi M.
      Treatment with adalimumab (Humira) and intravenous immunoglobulin improves pregnancy rates in women undergoing IVF.
      ,
      • Winger E.E.
      • Reed J.L.
      • Ashoush S.
      • El-Toukhy T.
      • Ahuja S.
      • Taranissi M.
      Degree of TNF-alpha/IL-10 cytokine elevation correlates with IVF success rates in women undergoing treatment with adalimumab (Humira) and IVIG.
      ) attempted to stratify treatment according to the outcome of putative diagnostic blood tests and identified and concluded from a study in which treatment refusers were considered as controls that a subpopulation of women experiencing IVF failure and increased Th1/Th2 ratio or CD56+ CD3− cells may benefit from IVIG treatment.
      A recent systematic review and meta-analysis of the available evidence has suggested that a positive effect from IVIG on implantation, clinical pregnancy, and live-birth rates may be achievable in selected patients (
      • Li J.
      • Chen Y.
      • Liu C.
      • Hu Y.
      • Li L.
      Intravenous immunoglobulin treatment for repeated IVF/ICSI failure and unexplained infertility: a systematic review and a meta-analysis.
      ). Given the expense and relative inconvenience of treating otherwise healthy women with IVIG, we would suggest that the burden of proof to justify its use is higher when compared with other immunomodulative therapies, and the availability of just one well-designed RCT reporting negative findings means that this is some way from being met.

      Anti-TNF-α (adalimumab)

      Adalimumab is an established biologic pharmaceutical in the fields of rheumatoid arthritis and inflammatory bowel disease. As described earlier, TNF-α is a cytokine produced by Th1 cells involved in the cell-mediated immune response. Humira interacts with and neutralizes TNF-α, thereby decreasing the inflammatory and cell-mediated immune response from Th1 lymphocytes.
      At present no RCTs have been reported investigating the effect of anti-TNF-α on pregnancy outcome from IVF or ICSI treatment. A Medline search using mesh terms adalimumab and IVF revealed the evidence base to be limited to a few observational studies all executed in a single clinic in London (
      • Winger E.E.
      • Reed J.L.
      • Ashoush S.
      • Ahuja S.
      • El-Toukhy T.
      • Taranissi M.
      Treatment with adalimumab (Humira) and intravenous immunoglobulin improves pregnancy rates in women undergoing IVF.
      ,
      • Winger E.E.
      • Reed J.L.
      • Ashoush S.
      • El-Toukhy T.
      • Ahuja S.
      • Taranissi M.
      Degree of TNF-alpha/IL-10 cytokine elevation correlates with IVF success rates in women undergoing treatment with adalimumab (Humira) and IVIG.
      ,
      • Winger E.E.
      • Reed J.L.
      • Ashoush S.
      • El-Toukhy T.
      • Taranissi M.
      Die-off ratio correlates with increased TNF-alpha: IL-10 ratio and decreased IVF success rates correctable with Humira.
      ). Winger et al. completed a prospective cohort study allocating women with an elevated Th1/Th2 ratio (TNF-α/IL-10 ratio) above 30.6 and/or an elevated IFN-γ/IL-10 ratio above 20.5 into four groups based on the severity of elevated Th1/Th2 ratio, patients’ willingness to undertake immune modulation treatment, and their ability to pay for it. The treatment arm in the four groups was group I, IVIG and TNF-α, 41 patients; group II, IVIG, 23 patients; group III, TNF-α, six patients; group 4, no treatment, five patients. All patients received treatment with LMWH. Only patients <38 years with a negative Heaf test and good embryo development were included. Results showed a significant increase in implantation, clinical pregnancy, and live-birth rates when comparing the treatment groups with the control group (
      • Winger E.E.
      • Reed J.L.
      • Ashoush S.
      • Ahuja S.
      • El-Toukhy T.
      • Taranissi M.
      Treatment with adalimumab (Humira) and intravenous immunoglobulin improves pregnancy rates in women undergoing IVF.
      ).
      A further retrospective study by Winger et al. included women up 40 years of age. Only those with an elevated Th1/Th2 ratio were offered treatment with adalimabab and heparin, while the control group consisted of just eight women without an elevated Th1/Th2 ratio. While the authors reported higher implantation, clinical pregnancy, and live-birth rates in the treatment group, the poor study design makes it impossible to draw any valid conclusions (
      • Winger E.E.
      • Reed J.L.
      • Ashoush S.
      • El-Toukhy T.
      • Ahuja S.
      • Taranissi M.
      Degree of TNF-alpha/IL-10 cytokine elevation correlates with IVF success rates in women undergoing treatment with adalimumab (Humira) and IVIG.
      ).
      Understanding the weaknesses of studies that will often be cited by patients as they make their case for specific treatments is important if the clinician is to advise patients appropriately. At present, the use of anti-TNF-α in ART cannot be recommended without results from well-designed studies.

      Intralipid

      Intralipid is a fat emulsion made from soybean oil, glycerin, and egg phospholipids normally used for parenteral nutrition in patients not able to consume orally. Any therapeutic effect in the context of IVF is proposed to be mediated by a reduction of peripheral blood NK cell activity and suppression of proinflammatory cytokines (
      • Roussev R.G.
      • Ng S.C.
      • Coulam C.B.
      Natural killer cell functional activity suppression by intravenous immunoglobulin, intralipid and soluble human leukocyte antigen-G.
      ).
      Only one randomized placebo-controlled double-blinded trial investigating the effect of intralipid infusion during IVF treatment has been published. A total of 296 women with either unknown fertility or recurrent spontaneous abortion and an increased level of NK cell activity were randomly allocated to receive 250 mL of diluted intralipid infusion or the equivalent dose of saline. The first dose was given after oocyte retrieval, and a second after a positive pregnancy test. Treatment was then repeated every 2 weeks till the end of the first trimester. Women over the age of 40 with an abnormal uterine cavity or endocrine, medical, or thrombophilia disorders were excluded from the study. The primary outcome measured was chemical pregnancy. No difference in chemical pregnancy was found between the two groups. The authors reported a borderline significant difference in ongoing pregnancy and live birth in favor of intralipid (
      • Dakhly D.M.R.
      • Bayoumi Y.A.
      • Sharkawy M.
      • Allah S.H.G.
      • Hassan M.A.
      • Gouda H.M.
      • et al.
      Intralipid supplementation in women with recurrent spontaneous abortion and elevated levels of natural killer cells.
      ), but the study was not sufficiently powered to enable differentiation of this from a chance observation.

      Conclusion

      Reviews of immunomodulation therapies in IVF can make depressing reading. The dismay at the lack of good evidence to support or refute their efficacy is compounded by concerns in some quarters that the motivations for their use may not be wholly Hippocratic. However, it would be wrong to disregard immunotherapies as surrogate activities that address an unmet need for effective treatments to increase the chance of a human embryo to implant. This need is real and keenly felt by both patients and clinicians. While randomized placebo-controlled trials are always advocated by the authors of reviews such as this, it must be emphasized that an empirical approach to testing immunomodulating therapies in IVF can only deliver valid results if they are performed in patients with an underlying disorder that is amenable to correction by the therapy under investigation. The published literature in the field of RIF and RM reveals many studies of excellent design, funded by generous grants and published in high-impact journals. The authority with which they declare their almost uniformly negative findings is strong.
      Yet most fail to address this requirement to properly phenotype ahead of intervening. RIF and RM, which are not clinical diagnoses but clinical presentations. Until we understand and can accurately discern the underlying causes, the value of immunotherapies, and indeed all proposed interventions, is likely to be underestimated.

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