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Immune therapies for women with history of failed implantation undergoing IVF treatment

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Abstract

This is a protocol for a Cochrane Review (Intervention). The objectives are as follows:

To determine whether immunotherapy results in improved live birth rates in women with a history of recurrent implantation failure who are undergoing assisted reproduction.

Background

Description of the condition

Human embryo implantation is a complex initial step in the establishment of a successful pregnancy (Cakmak, 2010). It consists of a three‐stage process (apposition, adhesion and invasion) that requires a reciprocal interaction between a functional blastocyst and a receptive endometrium, culminating in a small window of opportunity during which implantation can occur (Diedrich, 2007). This  implantation window, as a distinct, compartmentalized process in the course of pregnancy, has been explicitly defined by the practice of in vitro fertilization (IVF) (Rinehart, 2007). Women undergoing IVF have embryos transferred into the uterus during the window of implantation. Since known numbers of embryos are transferred there can be certainty as to whether or not implantation has occurred, and this has given rise to the clinical condition of implantation failure.

Failure of implantation is not uncommon in the IVF setting. The average implantation rate in IVF is estimated at around 25% (De los Santos, 2003). Despite the significant advances in assisted reproductive technologies (ART), pregnancy rates are still relatively low and have not increased significantly in the last decade (Nelson, 2011). This suggests that implantation rates in stimulated cycles remain suboptimal (Diedrich, 2007).

Given the optimal use of reproductive technologies (ovarian stimulation, culture conditions and embryo transfer), repeated implantation failure has been attributed to an embryo factor or uterine receptivity, or both (Margalioth, 2006). With regard to the former, assisted hatching (Das, 2009) and pre‐implantation genetic diagnosis (Basille, 2009) have been investigated, both with inconclusive results. Similarly, several medical interventions to overcome decreased uterine receptivity have been proposed, when this is not accounted for by a hostile environment (for example endometriosis, hydrosalpinx, uterine abnormalities) (Margalioth, 2006) or thrombophilic disturbances. These interventions include glucocorticoids, intravenous immune globulin (IVIG), lymphocyte immune therapy (LIT) administration of peripheral blood mononuclear cells (PBMCs), recombinant human leukemia inhibitory factor (r‐hLIF), granulocyte colony‐stimulating factor (G‐CSF), anti‐TNFα therapy and Intralipid.

The rationale for the use of these immune therapies to address inadequate uterine receptivity and improve implantation rates was grounded both on evidence from basic research (animal models, human in vitro systems) and observational data in women with recurrent implantation failures following assisted reproduction. In brief, evidence suggests that implantation failure may be explained by defects in the cellular adhesion molecules (integrins, selectins, cadherins, immunoglobulins) that mediate cell‐to‐cell adhesion and in the network of cytokines (leukemia inhibitory factor (LIF), interleukin (IL)‐6, IL‐1), prostaglandins and mucins, which work in a well‐orchestrated crosstalk with locally‐acting growth factors to coordinate an immune response of the endometrium to the blastocyst (Achache, 2006). In this immune response, uterine natural‐killer (NK) cells may also be involved (Dey, 2004) since increased numbers have been found in women with a history of repeated implantation failures, both in endometrial biopsies (Ledee‐Bataille, 2005) and the periphery (Miko, 2010). 

Description of the intervention

Immune therapies for women with a history of failed implantation who are undergoing IVF treatment consist of glucocorticoids, intravenous immune globulin (IVIG), lymphocyte immune therapy (LIT), administration of peripheral blood mononuclear cells (PBMC) r‐hLIF, G‐CSF, anti‐TNF‐α therapy and Intralipid.

Glucocorticoids have been used in a variety of different protocols, as summarized in a recent Cochrane review (Boomsma, 2007). Oral prednisone (5, 7.5, 10, 15, 30 and 60 mg per day) and methylprednisone (4 and 16 mg per day) are most frequently used, although oral dexamethasone (0.5 and 1 mg per day) and intravenous hydrocortisone (100 mg) have also been utilised. Timing and duration of administration varies significantly across studies.

Similarly, IVIG has been used for early reproductive failure in a variety of schemes, as summarized elsewhere (Clark, 2006). In brief, different preparations (Venoglobin‐S, Gamimune‐N, Gammagard) have been used, at a dose that is adjusted for body weight (0.2 or 0.4 g per kg). IVIG is generally first administered pre‐conceptionally but the exact scheme varies across studies.

LIT is administered in the form of allogenic immunization with paternal lymphocytes (Carp, 1994; Kuhn, 1993). Preparations, procedures, the definition of successful immunization and sample populations vary across studies. PBMCs have been administered into the uterine cavity, in the form of a combination of cultured and freshly isolated PBMCs, before embryo transfer (Yoshioka, 2006).

LIF (Emfilermin) is administered subcutaneously at a dose of 150 μg twice daily for seven days starting before embryo transfer (Brinsden, 2009).

G‐CSF (Filgastrim, recombinant G‐CSF) has been administered subcutaneously on a daily basis at a dose of 1 μg (100000 IU)/kg/day starting on the sixth day after ovulation (Scarpellini, 2009). Different schemes of G‐CSF therapy (13 million IU of lanogrostim every 3 days) have also been reported in the literature (Wurfel, 2010).

TNF‐α inhibitors, either in the form of adalimumab (Winger, 2009) or etarnecept (Jerzak, 2010), have been injected subcutaneously in combination with other interventions  (Winger, 2009).

Finally, Intralipid (20%, fat emulsion) is administered intravenously at a dose of 2‐3 ml in 250 ml sterile saline at approximately 250cm3/hr (Roussev, 2008).

It is notable that all of the above treatment modalities have often been investigated in co‐administration with heparin or aspirin, or both, or in combination with each other, or with luteal phase support.   

How the intervention might work

Glucocorticoids can exert a range of positive effects which would be expected to promote establishment of early pregnancy, such as suppression of uterine NK cells and stimulation of human chorionic gonadotropin (hCG) secretion, as well as promotion of trophoblast proliferation and invasion (Michael, 2008). IVIG is considered to have immunosuppressive properties, acting against the excess of pro‐inflammatory Th1‐type cytokines relative to Th2 and Th3 cytokines (Clark, 2006), promoting the down‐regulation of systemic NK cells and minimizing their cytotoxic effect on the implanting embryo/trophoblast unit (Daya, 1999). LIT promotes immune tolerance and also favours the re‐polarisation of the Th1 and Th2 response. PBMC, under regulation of hCG, can induce functional changes in the endometrium that facilitate embryo implantation (Nakayama, 2002) and has been shown to promote blastocyst spreading in vitro (Yoshioka, 2006). TNF‐α inhibitors counteract the increased production of Th1 cytokines associated with early reproduction failure (Clark, 2010). LIF regulates the differentiation of the endometrium and may also promote development and implantation of the blastocyst (Chen, 2000). Dysfunction of its expression may contribute to implantation failure (Ledee‐Bataille, 2002). G‐CSF is a cytokine which stimulates neutrophilic granulocyte proliferation and differentiation. Experimental findings showed a positive effect on the trophoblast growth and placenta metabolism (McCracken, 1999).TNF‐α inhibitors counteract the increased production of Th1 cytokines, associated with early reproduction failure (Clark, 2010). Finally, Intralipid has been shown effective in suppressing the number and activity of peripheral natural‐killer (NK) cells, implicated in the pathogenesis of recurrent implantation failure (Matsubayashi, 2001), both in vitro (Roussev, 2007) and in vivo (Roussev, 2008).

Why it is important to do this review

Implantation is considered to be the bottleneck of the reproductive process (Achache, 2006). Inadequate uterine receptivity is responsible for approximately two‐thirds of IVF implantation failures, whereas the embryo itself is thought to be responsible for 30% to 50% of these failures (Fragouli, 2011). Thus, any intervention aiming at overcoming decreased uterine receptivity, such as the above immune therapies, may have a considerable impact on IVF outcomes, if proven effective. Since the relevant trials are insufficiently powered to draw conclusion on efficacy (Boomsma, 2007) and their results remain inconclusive (Boomsma, 2008; Clark, 2006), a rigorous systematic review and meta‐analysis of the relevant literature may help in assessing the absolute and comparative effectiveness of all treatment modalities, reveal different response patterns and thus facilitate targeted, informed decision‐making.

Objectives

To determine whether immunotherapy results in improved live birth rates in women with a history of recurrent implantation failure who are undergoing assisted reproduction.

Methods

Criteria for considering studies for this review

Types of studies

Only trials that are either clearly randomised or claim to be randomised, do not have evidence of inadequate sequence generation, such as date of birth or hospital number, and compare immune therapies for women with a history of failed implantation and who are undergoing IVF treatment will be included. Quasi‐ and pseudo‐randomised trials will not be included. Only first‐phase data of crossover trials will be included in meta‐analyses.

Types of participants

Inclusion criteria

Women with a history of recurrent failed implantations who are undergoing IVF treatment and are receiving a form of immune therapy as described under Types of interventions.

Implantation must have failed at least three times.

Exclusion criteria

Women with gynaecological cancer.

Types of interventions

The following immune therapies will be considered in women with a history of failed implantation who are undergoing IVF treatment.

  • Glucocorticoids

  • TNF‐α inhibitors

  • Peripheral blood mononuclear cells (PBMCs)

  • Intravenous immune globulin (IVIG)

  • Lymphocyte immune therapy (LIT)

  • Intralipid

  • Granulocyte colony stimulating factor (G‐CSF)

  • Leukcocyte inhibitory factor (LIF)

These immune therapies will be compared with each other or with placebo. Combination therapies are possible.

Types of outcome measures

All outcomes will be per woman.

Primary outcomes

Live birth rate per woman, defined as delivery of a live fetus after 24 completed weeks of gestation

Secondary outcomes
Adverse outcomes

  • Miscarriage, defined as miscarriage prior to 24 gestational weeks

  • Ectopic pregnancy, confirmed by histology and ultrasound

  • Drug side effects

  • Multiple pregnancies

  • Ovarian hyperstimulation syndrome (OHSS) rate

  • Post ovum pick‐up infection

Other secondary outcomes

  • Ongoing pregnancy rate, defined as pregnancies continuing beyond 12 weeks

  • Pregnancy rate, defined as biochemical pregnancy or the presence of one or more gestation sacs with fetal heart activity visualized on ultrasound at 6 to 8 weeks gestation

  • Implantation rate, defined as the number of fetal sacs divided by the number of embryos transferred.

  • Mean number of mature oocytes (follicles > 17 mm) retrieved after therapy

  • Percentage of fertilization

  • Number of embryo transfers

Search methods for identification of studies

All published and unpublished randomised controlled trials (RCTs) of immune treatment versus another immune treatment or placebo will be obtained using the strategies described below.

Electronic searches

The following electronic databases, trial registers and websites will be searched, from inception to present.

The MEDLINE search will be combined with the Cochrane highly sensitive search strategy for identifying randomised trials, which appears in the Cochrane Handbook for Systematic Reviews of Interventions (version 5.1.0, chapter 6, 6.4.11) (Higgins 2011). The EMBASE search will be combined with trial filters developed by the Scottish Intercollegiate Guidelines Network (SIGN). There will be no language restrictions in these searches.

Other electronic sources of trials that will be searched

The search terms that will be used are the following: 'IVF', 'glucocorticoids', 'immunotherapy'.

In The Cochrane Library we will search the DARE database to find reviews with potentially useful RCTs.

Trial registers: we will search for ongoing and registered trials in the following trial registers:

Citation indexes (Citation indexes).

Conference abstracts in the ISI web of Knowledge (ISI web of Knowledge).

Clinical Study results for clinical trial results of marketed pharmaceuticals (Clinical Study Results).

OpenSigle database (OpenSigle).

Google for grey literature.

Searching other resources

The reference list of trials retrieved by the search will be handsearched and personal contact will be made with experts in the field to obtain any additional data.

Any relevant journals and conference abstracts that are not covered in the MDSG Specialised Register will be handsearched in liaison with the Trials Search Coordinator.

Data collection and analysis

Data collection and analysis will be conducted in accordance with the Cochrane Handbook for Systematic Reviews of Interventions (version 5.1.0) (Higgins 2011)

Selection of studies

The titles and abstracts retrieved by the Trials Search Coordinator will be read by one author. The ones that are irrelevant will be removed and the full text of the remaining trials will be retrieved. Two review authors will independently examine the full text articles for compliance with the inclusion criteria and will select trials eligible for inclusion in the review. Authors will correspond with trial investigators if required to clarify trial eligibility. Disagreements will be resolved by consensus or by discussion with a third author.

Data extraction and management

A data extraction form will be designed and pilot‐tested by the authors. This will be used to extract data from eligible trials. When a trial has multiple publications, the main trial report will be used as the reference. Additional details will be sought from secondary papers. The review authors will correspond with trial investigators in order to resolve any data queries and as required. Two review authors will independently extract data and any disagreement between these authors will be resolved by a third author.

Assessment of risk of bias in included studies

The Cochrane risk of bias assessment tool will be used to assess for risk of bias in the included trials. The following will be assessed: sequence generation; allocation concealment; blinding of participants, providers and outcome assessors; completeness of outcome data; selective outcome reporting; and other potential sources of bias. These six domains will be independently assessed by two authors, with any disagreements resolved by consensus or by discussion with a third author. All judgements will be fully described. Conclusions will be presented in a 'Risk of bias table' and when possible they will be discussed through sensitivity analyses. A sensitivity analysis will be conducted when there is selection bias (bias in allocation sequence and allocation concealment). Selective reporting will not be a reason to do a sensitivity analysis based on bias because many fertility trials do not report on the primary outcome live birth, which will be a form of selective reporting. We will accept such selective reporting as long as the trials do report interim outcomes such as clinical pregnancy.

Measures of treatment effect

For continuous data such as estradiol concentrations, mean differences (MD) between treatment groups will be calculated if all trials report exactly the same outcomes. For dichotomous data such as live birth, the number of events in the control and intervention groups of each trial will be used to calculate Peto odds ratios (OR). If similar outcomes are reported on different scales, the standardized mean difference (SMD) will be calculated. Every outcome will be presented with 95% confidence intervals and the data will be entered into RevMan 5 software.

Unit of analysis issues

The primary analysis will be per woman randomised. Reported data that do not allow valid analysis, such as 'per cycle' instead of 'per woman', will be summarized in an additional table but will not be included in a meta‐analysis. The number of participants randomised will be used to calculate confidence intervals, not the number of treatment attempts.

Only first‐phase data from crossover trials will be included.

Multiple live births in one pregnancy will be counted as one live birth event.

Dealing with missing data

As far as possible, all data will be analysed on an intention‐to‐treat basis. Attempts will be made to obtain missing data from the original investigators. Live birth will be assumed not to have occurred in participants with unreported outcomes.

If trials report sufficient detail to calculate mean differences but no information is given on associated standard deviations (SD), the outcome will be assumed to have a standard deviation equal to the highest SD from other trials within the same analysis.

For other outcomes only the available data will be analysed. Any imputation that is undertaken will be subjected to sensitivity analysis.

Assessment of heterogeneity

The authors will consider whether the clinical and methodological characteristics of the included trials are sufficiently similar for meta‐analysis to provide a meaningful summary. Statistical heterogeneity will be examined by inspecting the scatter in the data points on the graphs and the overlap in their confidence intervals. In addition, it will be assessed by the use of the I2 statistic. An I2 statistic greater than 50% will be taken to indicate substantial heterogeneity (Higgins 2011). If substantial heterogeneity is detected, possible explanations may be explored in a sensitivity analysis, subgroup analyses and meta‐regression.

Assessment of reporting biases

In view of the difficulty in detecting and correcting for publication bias and other reporting biases, the authors will aim to minimize their potential impact by ensuring a comprehensive search for eligible trials and by being alert for duplication of data. If there are 10 or more trials in an analysis, a funnel plot will be used to explore the possibility of small trial effects. We will be attentive to looking for within trial reporting bias, such as trials failing to report obvious outcomes or reporting them in insufficient detail to allow inclusion.

Data synthesis

The data from primary trials will be combined using a fixed‐effect model for the interventions shown above (Types of interventions). A sensitivity analysis will be done when there is heterogeneity that cannot readily be explained.

Results of dichotomous variables will be expressed as Peto odds ratios (OR) with 95% confidence intervals and combined for meta‐analysis where appropriate. An increase in the odds of a particular outcome, which may be beneficial (live birth) or detrimental (adverse event such as miscarriage), will be displayed graphically in the meta‐analyses to the right of the centre‐line. A decrease in the odds of an outcome will be displayed to the left of the centre‐line.

Results of continuous outcome data will be expressed as a difference in means with 95% confidence intervals and combined for meta‐analysis to calculate a weighted mean difference (WMD). A standardized mean difference (SMD) will be calculated when different scales are used.

For categorical outcomes we will relate the numbers reporting an outcome to each group. Results for each trial will be expressed as an odds ratio with 95% confidence interval and combined for meta‐analysis with RevMan software using the Peto‐modified Mantel‐Haenszel method.

Subgroup analysis and investigation of heterogeneity

Where data are available and substantial heterogeneity is present (I2 > 50%), subgroup analyses or meta‐regression will be conducted to determine the evidence within the following subgroups.

  • Trials including women with an average age over 40 years.

  • Trials including women with different numbers of IVF failure.

  • Trials using different methods of administration (parenteral versus oral).

  • Trials using different dosages and timing of administration.

Sensitivity analysis

Sensitivity analysis will be conducted for the primary outcome live birth to determine whether the conclusions are robust to decisions made regarding eligibility and analysis. These analyses will include consideration of whether conclusions would have differed if:

  • eligibility were restricted to studies without high risk of bias;

  • trials with outlying results were excluded;

  • a random‐effects model had been adopted;

  • alternative imputation strategies had been adopted.