Granulocyte‐colony stimulating factor administration for subfertile women undergoing assisted reproduction
Abstract
This is a protocol for a Cochrane Review (Intervention). The objectives are as follows:
To evaluate the effectiveness and safety of granulocyte‐colony stimulating factor in women undergoing ART.
Background
Description of the condition
Assisted reproductive technology (ART) is widely used to treat subfertility, and it is estimated that more than 5 million babies have been born using this treatment worldwide (Adamson 2013). Despite many technological breakthroughs over the years, live‐birth rates following ART remain modest, at between 20% and 29% (Dyer 2016; Kushnir 2017). Embryo implantation (embryo adherence to uterine lining) is a complex process involving interaction between the genetically competent embryo and endometrium and is influenced by local and systemic immune factors. Endometrial receptivity (window of implantation) and the final process of embryo implantation remains the rate‐limiting step for the success of ART (Gnainsky 2014). During an ART treatment cycle, endometrial thickness is commonly measured using an ultrasound to indirectly assess endometrial receptivity. A systematic review suggests that an endometrial thickness of 7 mm or less during ART is suboptimal and associated with lower clinical pregnancy rates (Kasius 2014). A chronically thin endometrium that does not respond to various treatment modalities is a difficult clinical situation. Similarly, in women with two or more in vitro fertilisation (IVF) failures, where there is a failure of implantation despite the transfer of good‐quality embryos, endometrial receptivity remains the main focus for evaluation and intervention (Coughlan 2014; Macklon 2017; Valdes 2017). Treating chronically thin endometrium and repeated IVF failures despite transfer of good‐quality embryos remain a challenge for clinicians. Numerous strategies and interventions have been suggested to improve endometrial receptivity and thereby embryo implantation.
Description of the intervention
Granulocyte‐colony stimulating factor (G‐CSF) is an amino acid polypeptide that belongs to the colony stimulating factor glycoprotein group (Demetri 1991). Other members of the colony stimulating factor group include granulocyte macrophage‐colony stimulating factor (GM‐CSF) and macrophage‐colony stimulating factor (M‐CSF) (McNiece 1989). Granulocyte‐colony stimulating factor is produced by cells of the immune system such as monocytes, macrophages, endothelial and bone marrow cells and is mainly involved in stimulation of neutrophils proliferation and differentiation (Demetri 1991). Granulocyte‐colony stimulating factor binds to a specific receptor expressed on the surfaces of the target cells (such as neutrophils, vascular endothelium, and trophoblastic cells) and triggers growth signals within it (Demetri 1991). Recombinant human G‐CSF became available in the late 1980s and is mainly used to treat haematological disorders (Bonilla 1989; Tabbara 1997). In reproductive medicine, G‐CSF is being administered locally or systemically mainly in women with thin unresponsive endometrium, repeated IVF failures despite transfer of good‐quality embryos, and unexplained recurrent pregnancy losses (Scarpellini 2009; Gleicher 2011; Würfel 2015). In chronically thin endometrium, the most common route of administration of G‐CSF is intrauterine instillation or perfusion, while the subcutaneous route is preferred in recurrent pregnancy loss (Scarpellini 2009; Gleicher 2011; Gleicher 2013).
How the intervention might work
At the endometrial level, G‐CSF seems to play an important role in the process of embryo implantation and continuation of pregnancy. Granulocyte‐colony stimulating factor is involved in controlling gene expression, thereby influencing local embryo adhesions, cell migration, apoptotic (programmed cell death) activity, angiogenesis (formation of newer blood vessels), and endometrial remodelling, which are important for successful implantation (Rahmati 2014). Promotion of endometrial regeneration, anti‐apoptotic activity, and increased vascularisation (formation of blood vessels) are some of the proposed mechanisms for observed beneficial effect of G‐CSF on thin endometrium (Tanaka 2000; Schneider 2005). It also helps in continuation of pregnancy by temporarily modulating response of T‐helper cells (Th‐1 and Th‐2), which play an important role in immunity, helping mediate maternal immune tolerance against the semi‐allogenic foetus, which shares some maternal genes, but not all. The decidua (uterine lining during pregnancy) plays an important role in embryo implantation, and a balanced immunoregulation of different immune cells is crucial. Granulocyte‐colony stimulating factor predominantly promotes Th‐2 response in the foetal‐maternal interface, thereby blocking any maternal Th‐1 cell attacks against the embryo and helping continuation of pregnancy (Rutella 2005). It also promotes generation of interleukin 10‐producing T regulatory cells, which help in transplantation tolerance, an important immunoregulatory event around the peri‐implantation period (Morris 2004).
Granulocyte‐colony stimulating factor is a potential biomarker for oocyte (female egg) competence (ability of an oocyte to develop and sustain as an early embryo). Granulocyte‐colony stimulating factor receptors have been located on granulosa and luteal cells, which are closely associated with female oocyte development. Low levels of G‐CSF in follicular fluid (fluid surrounding the female oocyte) are linked to lower oocyte competence (Lédée 2008). It is hypothesised that the follicular fluid G‐CSF is linked to mRNA content of oocyte and it may influence the production of adhesion molecules involved in the attachment of the future potential embryo to the endometrial cells (Lédée 2008). Granulocyte‐colony stimulating factor may also influence cumulus granulosa cells to produce growth factors that are essential for the development and implantation of the embryo.
The use of GM‐CSF as a supplement in embryo culture medium has also been explored in repeated IVF failures to mimic in vivo conditions (Tevkin 2014), but our current review will focus only on G‐CSF.
Why it is important to do this review
The first published study on the use of G‐CSF in reproductive medicine was a case series in which the authors reported successful IVF outcomes after using intrauterine instillation of G‐CSF in women with thin endometrium that did not respond to standard treatment (Gleicher 2011). The same authors subsequently published a larger, uncontrolled study involving 21 women with chronic unresponsive endometrium and reported an increase in endometrial thickness after intrauterine instillation of G‐CSF (Gleicher 2013). Some randomised trials were published evaluating the effectiveness of G‐CSF in women undergoing IVF with chronically thin endometrium and recurrent implantation failures (RIF) as well as in women with recurrent pregnancy loss (Scarpellini 2009; Kunicki 2014; Aleyasin 2016). The results of these trials varied, with some showing a benefit of G‐CSF and others showing no improvement in outcomes (Barad 2014; Kunicki 2014; Aleyasin 2016; Kunicki 2017). A recently published systematic review evaluated the effectiveness of G‐CSF in women with thin endometrium and RIF and included a total of four trials (two under each subgroup) (Kamath 2017). The review suggested a possible benefit of G‐CSF in women with thin endometrium and RIF undergoing IVF. Another systematic review that included six trials after searching two databases also suggested a possible benefit of G‐CSF in women with thin endometrium and RIF (Li 2017). Both of these reviews conducted limited searches and suggested the need for further validation of their findings before G‐CSF can be used in routine clinical practice for women undergoing IVF.
The supportive evidence favouring the use of G‐CSF in ART is weak, but its use is now increasing in clinical practice. Newer trials have been published (Aleyasin 2016; Eftekhar 2016; Sarvi 2017). Furthermore, some trials have evaluated role of G‐CSF in fresh cycles, while other trials have included only frozen ART cycles (Barad 2014; Eftekhar 2016; Kunicki 2017; Sarvi 2017). The effect of the intervention (G‐CSF) as assessed by clinical pregnancy rate and obstetric outcomes might differ in fresh and frozen ART cycles due to differences in hormonal milieu at the endometrial level (Evans 2014). A more comprehensive search of the literature and appraisal of the current evidence is needed, hence we planned a systematic review for evaluating the role of G‐CSF in women undergoing assisted reproduction.
Objectives
To evaluate the effectiveness and safety of granulocyte‐colony stimulating factor in women undergoing ART.
Methods
Criteria for considering studies for this review
Types of studies
Published and unpublished randomised controlled trials (RCTs) will be eligible for inclusion. We plan to exclude non‐randomised studies as they are associated with a high risk of bias. We will include cross‐over trials if individually randomised women are the unit of analysis; we will only include data from the first phase (pre‐cross‐over data) in the meta‐analyses, as the cross‐over trial is not a valid study design in the context of subfertility.
Types of participants
Subfertile women undergoing fresh or frozen ART‐IVF cycles.
Types of interventions
We will include RCTs comparing administration of G‐CSF versus no treatment or placebo.
Types of outcome measures
Primary outcomes
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Effectiveness: the live‐birth rate or ongoing pregnancy rate per woman randomised. We defined live birth as delivery of a live foetus after 20 completed weeks of gestational age (duration of pregnancy). We will count the delivery of single, twin, or multiple pregnancies as one live birth. We will use ongoing pregnancy, defined as a clinical pregnancy of 12 or more weeks of gestation, instead of live birth in cases where studies do not report live birth but report ongoing pregnancy.
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Adverse events: the miscarriage rate per woman randomised defined as the spontaneous loss of a clinical pregnancy that occurs before 20 completed weeks of gestation.
Secondary outcomes
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Effectiveness: the clinical pregnancy rate per woman randomised (clinical pregnancy defined as evidence of gestational sac on ultrasound).
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Adverse events: the multiple pregnancy rate per woman randomised.
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Adverse events: the incidence of side effects or adverse reaction due to administration G‐CSF, analysed as a composite measure of any adverse events (including anaphylactic reaction (serious allergic reaction), fever, headache, etc.).
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Adverse events: the ectopic pregnancy rate.
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Adverse events: small for gestational age at birth (defined as birthweight less than the 10th percentile for gestational age and infant sex).
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Adverse events: abnormally adherent placenta (e.g. placenta accreta, increta, or percreta).
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Adverse events: the congenital anomalies (or birth defects) rate.
We will avoid excluding studies based on their reported outcome measures. We will review eligible studies that could have measured the outcomes of interest. We will report in the final review any lack of data for the key outcomes.
Search methods for identification of studies
We will search for all published and unpublished RCTs evaluating the effectiveness of G‐CSF in infertile women undergoing IVF applying no language restriction and in consultation with the Cochrane Gynaecology and Fertilty Group (CGF) Information Specialist.
Electronic searches
We will search the following electronic databases and websites, from their inception to the present.
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Cochrane Gynaecology and Fertility Group Specialised Register of controlled trials; ProCite platform (Appendix 1).
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Cochrane Central Register of Controlled Trials (CENTRAL) (Cochrane Register of Studies Online (CRSO)); web platform (Appendix 2).
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MEDLINE; Ovid platform (Appendix 3).
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Embase; Ovid platform (Appendix 4).
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CINAHL (Cumulative Index to Nursing and Allied Health Literature); EBSCO platform (Appendix 5).
The MEDLINE search will be combined with the Cochrane Highly Sensitive Search Strategy for identifying randomised trials, described in Section 6.4.11 of the Cochrane Handbook for Systematic Reviews of Interventions (Lefebvre 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.
In the Cochrane Library we will search DARE (Database of Abstracts of Reviews of Effects) to identify reviews with potentially relevant RCTs.
We will search for ongoing and registered trials in the following trial registers:
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World Health Organization International Trials Registry Platform (WHO ICTRP) (www.who.int/trialsearch/default.aspx);
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US National Institutes of Health Ongoing Trials Register ClinicalTrials.gov (clinicaltrials.gov);
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Current Controlled Trials (www.controlled‐trials.com).
We will also search:
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Citation indexes (scientific.thomson.com/products/sci/);
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Conference abstracts in the ISI Web of Knowledge (isiwebofknowledge.com/);
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OpenSigle database for grey literature (opensigle.inist.fr/);
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PubMed and Google for any recently published trials not yet indexed in the major databases.
Searching other resources
Two review authors (MSK and SKS) will handsearch reference lists of articles retrieved by the search and contact experts in the field to obtain additional data. We will also handsearch relevant journals and conference abstracts that are not covered in the CGF register, in liaison with the Information Specialist.
Data collection and analysis
We will format a data extraction sheet to retrieve data from the included studies. Two review authors (MSK and SKS) will independently extract the data onto the data extraction sheet. Any discrepancies will be resolved by involving the third review author (RK).
Selection of studies
Two review authors (MSK and SKS) will initially screen the titles and abstracts retrieved by the search for potentially relevant studies. We will retrieve the full texts of all potentially eligible studies, and two review authors (MSK and SKS) will independently examine these full text articles for compliance with the inclusion criteria and select eligible studies for inclusion in the review. We will correspond with study investigators as required to clarify study eligibility. Disagreements as to study eligibility will be resolved by discussion or by involving a third review author (RK). We will document the selection process with a PRISMA flow chart.
Data extraction and management
Two review authors (one a methodologist (MSK) and one a topic‐area specialist (SKS) will independently extract data from the included studies using a data extraction form designed and pilot‐tested by the review authors. Any disagreements will be resolved by discussion or by involving a third review author (RK). Data extracted will include study characteristics and outcome data (see data extraction table in Appendix 6 for details). Where studies have multiple publications, we will collate multiple reports of the same study, so that each study, rather than each report, is the unit of interest in the review, and such studies will have a single study ID with multiple references. We will correspond with study investigators for further data on methods or results, or both, as required. We will include studies irrespective of whether outcomes are reported in a 'usable' way. In multi‐arm studies, we will exclude data from arms that do not meet the eligibility criteria.
Assessment of risk of bias in included studies
Two review authors (MSK and SKS) will independently assess the included studies for risk of bias using the Cochrane 'Risk of bias' tool following the criteria outlined in the Cochrane Handbook for Systematic Reviews of Interventions (Higgins 2011). We will assess the following items.
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Selection bias (random sequence generation and allocation concealment)
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Performance bias (blinding of participants and personnel)
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Detection bias (blinding of outcome assessors)
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Attrition bias (incomplete outcome data)
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Reporting bias (selective reporting)
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Other bias (including unplanned interim analysis)
We will consider lack of blinding of personnel (clinician or embryologist, or both) as high risk for performance bias. However, we will consider lack of blinding as low risk for detection bias due to the objective nature of outcomes.
Disagreements will be resolved by discussion or by involving a third review author (RK). We will describe all judgements fully and present the conclusions in the 'Risk of bias' table, which will be incorporated into the interpretation of review findings by means of sensitivity analyses (see below).
Selective reporting is a type of reporting bias that affects the internal validity of an individual study. It refers to the selective reporting of some outcomes (e.g. positive outcomes) and the failure to report others (e.g. adverse events). We will take care to search for within‐trial selective reporting, such as trials failing to report obvious outcomes, or reporting them in insufficient detail to permit inclusion. We will seek published protocols and compare the outcomes in the protocol with those in the final published study. Where identified studies failed to report the primary outcome of live birth, but do report interim outcomes such as pregnancy, we will undertake informal assessment as to whether the interim values (e.g. pregnancy rates) are similar to those reported in studies that also report live birth.
Measures of treatment effect
For dichotomous data (e.g. pregnancy or live‐birth rates), we will use the number of events in the control and intervention groups of each study to calculate Mantel‐Haenszel risk ratios. We will reverse the direction of effect of individual studies, if required, to ensure consistency across trials. We will present 95% confidence intervals for all outcomes. Where data to calculate risk ratios are not available, we will utilise the most detailed numerical data available that may facilitate similar analyses of included studies (e.g. test statistics, P values). We will compare the magnitude and direction of effect reported by studies with how they are presented in the review, taking account of legitimate differences.
Unit of analysis issues
The primary analysis will be per woman randomised; per‐pregnancy data will be included for some outcomes as secondary analysis (for the outcome miscarriage). If studies report only per‐cycle data, we will contact the study authors to request per‐woman data. Data that do not permit valid analysis (e.g. per‐cycle data) will be briefly summarised in an additional table and will not be meta‐analysed. Multiple live births (e.g. twins or triplets) will be counted as one live‐birth event. Only first‐phase data from cross‐over trials will be included.
Dealing with missing data
We will analyse data on an intention‐to‐treat basis to the greatest degree possible and attempt to obtain missing data from the original authors. Where these are unobtainable, we will undertake imputation of individual values for the primary outcomes only. We will assume live births not to have occurred in participants without a reported outcome. For other outcomes, we will analyse only the available data.
Assessment of heterogeneity
We will consider whether the clinical and methodological characteristics of the included studies are sufficiently similar for meta‐analysis to provide a clinically meaningful summary. We will use the I² statistic to measure statistical heterogeneity among the trials in each analysis. If we identify substantial unexplained heterogeneity, we will report it and explore possible causes by prespecified subgroup analysis.
If sufficient studies are available, we will conduct subgroup analysis based on:
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Fresh versus frozen ART‐IVF cycles;
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Route of administration: local versus systemic administration of G‐CSF.
We will use the rough guide to interpretation as outlined in the Cochrane Handbook for Systematic Reviews of Interventions (Higgins 2011): we will consider an I² measurement greater than 55% as indicative of substantial heterogeneity.
Assessment of reporting biases
In view of the difficulty of detecting and correcting for publication bias and other reporting biases, we will aim to minimise their potential impact by ensuring a comprehensive search for eligible studies and by being alert for duplication of data. If there are 10 or more studies in an analysis, we will use a funnel plot to explore the possibility of small‐study effects (a tendency for estimates of the intervention effect to be more beneficial in smaller studies).
Data synthesis
One review author (MSK) will enter data into and perform statistical analysis using Review Manager 5 (RevMan 2014). If the studies are sufficiently similar, we will combine the data using a fixed‐effect model for the following comparisons.
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G‐CSF versus no treatment or placebo in women undergoing ART.
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G‐CSF versus no treatment or placebo in women with thin endometrium undergoing ART.
We will stratify the first comparison further into two groups:
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unselected or unstated IVF;
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two or more IVF failures.
An increase in the risk of a particular outcome, which may be beneficial (e.g. live birth) or detrimental (e.g. adverse effects of G‐CSF), will be displayed graphically in the meta‐analyses to the right of the centre‐line, and a decrease in the risk of an outcome to the left of the centre‐line. The aim is to define analyses that are comprehensive and mutually exclusive, so that all results can be slotted into one stratum only, and trials within the same stratum can sensibly be pooled. Stratification is not a requirement, but allows consideration of effects within each stratum as well as, or instead of, an overall estimate for the comparison.
If the review includes more than one comparison that cannot be included in the same analysis, we will report the results for each comparison separately.
Subgroup analysis and investigation of heterogeneity
Where data are available, we will conduct subgroup analyses to determine the separate evidence within the following subgroups.
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Fresh versus frozen ART‐IVF cycles.
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Route of administration: local versus systemic administration of G‐CSF.
The interpretation of the statistical analysis for subgroups is difficult, therefore the interpretation of the subgroup analysis will be deliberate. We will mainly use the subgroup analysis to substantiate certain hypotheses concerning the results.
If no RCTs are retrieved for a given subgroup analysis, the absence of literature will be reported and identified knowledge gaps will be described.
Sensitivity analysis
We plan to carry out sensitivity analyses for the primary outcomes to determine whether the conclusions are robust to arbitrary decisions made regarding the eligibility and analysis. These analyses will include consideration of whether the review conclusions would have differed if:
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eligibility was restricted to studies without high risk of bias (not at high risk of bias in any domain and at low risk for randomisation procedures);
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a random‐effects model had been adopted;
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the summary effect measure had been odds ratio rather than risk ratios;
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alternative imputation strategies had been implemented;
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restricting the analysis by excluding any unpublished studies.
Overall quality of the body of evidence: 'Summary of findings' table
We will use the GRADE approach to summarise and interpret findings and GRADEpro GDT to import data from Review Manager 5 to create 'Summary of findings' tables (GRADEpro GDT 2015). These tables provide outcome‐specific information concerning within‐study risk of bias (methodological quality), directness of evidence, heterogeneity, precision of effect estimates, and risk of publication bias, and the sum of available data on all outcomes rated as important to patient care and decision making. The GRADE approach specifies four levels of quality: high, moderate, low, and very low.
We will use the methods and recommendations described in the Cochrane Handbook for Systematic Reviews of Interventions (Higgins 2011), employing GRADEpro GDT software (GRADEpro GDT 2015). We will justify all decisions to down‐ or upgrade the quality of evidence using footnotes and make comments to aid reader's understanding of the review where necessary. Two review authors will independently make decisions about evidence quality, with any disagreements resolved by discussion.
The main comparisons will be:
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G‐CSF versus no treatment or placebo in women undergoing ART;
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G‐CSF versus no treatment or placebo in women with thin endometrium undergoing ART.
We will present separate 'Summary of findings' tables for the two comparisons.
We will include the following outcomes in the 'Summary of findings' tables.
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Live birth/ongoing pregnancy rate per woman randomised.
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Miscarriage rate per woman randomised.
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Clinical pregnancy per woman randomised.
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Multiple pregnancy per woman randomised.
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Adverse events per woman randomised.
