Hyaluronic Acid inclusion in embryo transfer media for assisted reproductive technologies
Abstract
This is a protocol for a Cochrane Review (Intervention). The objectives are as follows:
To determine whether embryo transfer media containing hyaluronic acid (HA) affects the live birth rate in human assisted reproductive technology compared to transfer media without HA.
Background
The birth of the first IVF (in vitro fertilization) baby was in 1978. Much progress has been made in the intervening years in the field of assisted reproductive technology (ART) as ART practitioners are continually striving for higher live‐birth outcomes for their patients and are keen to trial newly‐reported innovations. It is well recognised that embryo implantation into the lining of the uterus is one of the major determining factors in successful human IVF (Gardner 2003). Therefore, much research has focused on the interaction between the embryo and the endometrium at the time of implantation. The composition of the media that surrounds the embryo at the time of IVF transfer is now considered to play an important role at this crucial stage of development. An innovation that could improve the implantation rate may, therefore, lead to a reduction of multiple births and ultimately maximise the chances of subfertile couples having a normal, healthy baby.
One reportedly beneficial component that has been introduced into transfer media is hyaluronic acid (HA). HA is a naturally existing molecule and is one of the major macromolecules present in the female reproductive tract. It is present in the human endometrium (Salamonsen 2001) and has been shown to increase dramatically on the day of implantation in mice (Carson 1987). Although the mechanism by which HA promotes implantation has yet to be elucidated, it does have several properties that make it a potential candidate as an implantation‐enhancing molecule. HA has been demonstrated to increase cell to cell adhesion and cell to matrix adhesion (Turley 1984); it produces a viscous solution that might enhance the embryo transfer process and prohibit expulsion, or it may facilitate diffusion and integration of the embryos in viscous solution that characterizes intrauterine‐secreted fluid (Simon 2003). Its viscosity alone, however, does not explain its involvement in implantation as not all highly viscous solutions, such as human placental collagen, can improve implantation (Menezo 1989). The action of HA during implantation could also be receptor mediated as the primary receptor for HA is CD44, which is expressed both on the pre‐implantation embryo (Campbell 1995) and in the stroma (supporting framework) of the human endometrium (Behzad 1994). Indeed initial studies in mouse embryo transfer showed that inclusion of HA in transfer medium significantly increased both implantation rates and fetal development compared to no HA in the transfer medium (Gardner 1999). Other macromolecules investigated included bovine serum albumin (BSA), polyvinyl alcohol (PVA) and dextran, but none of these were shown to improve implantation rates compared to no macromolecule.
Albumin has traditionally been used as the main macromolecule in most embryo culture media as it is abundant in the female reproductive tract. However, serum albumin which is derived from blood is not a pure substance and it carries a risk of contamination from viruses. In a human trial, Simon et al showed that HA can successfully replace albumin as the sole macromolecule in an embryo transfer medium and result in high pregnancy rates (Simon 2003). Although the risks associated with a biologically‐derived product have partly been overcome with the development of recombinant human serum albumin, HA is preferable to albumin because it is a polysaccharide and can be synthesized and isolated in a pure form (Gardner 1999).
In humans, transfer of the embryo back into the uterus can be performed after two, three, four, five or six days of in vitro culture. The day of transfer could potentially be important as it is not clear whether the small volume of HA in media transferred on days two to four would still be present and able to have an effect on the day of implantation (day 7) (Simon 2003). However, HA in the media may still play an important role at this early stage due to its physical properties and may prohibit expulsion, as mentioned above. Therefore, the day of embryo transfer in this review may need to be assessed in a subgroup analysis. It is also not known whether inclusion of HA in transfer media has any added benefit in frozen embryos compared with fresh embryos, or vice‐versa. Therefore, fresh and frozen‐thawed embryos may also need to be assessed in a subgroup analysis.
Because the rate of human implantation is innately low, it is often difficult to establish small but significant improvements, particularly with the relatively low volume of women that many clinics see in a year. Systematic meta‐analysis of all randomised controlled trials (RCTs) is, therefore, an important tool in assessing if an innovation offers a true advancement. The available literature will be reviewed in an attempt to identify whether inclusion of HA in embryo transfer media benefits women compared to transfer media without HA.
Objectives
To determine whether embryo transfer media containing hyaluronic acid (HA) affects the live birth rate in human assisted reproductive technology compared to transfer media without HA.
Methods
Criteria for considering studies for this review
Types of studies
All randomised controlled trials comparing embryo transfer media containing HA with transfers in media without HA.
Quasi‐randomized trials will not be included.
Types of participants
Inclusion criteria: couples undergoing embryo transfer after IVF, intracytoplasmic sperm injection (ICSI), and an embryo thaw cycle for therapeutic reasons or for oocyte donation.
Types of interventions
All known culture methods for in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) where embryos are grown for between two to six days in vitro or frozen‐thawed, or both, prior to transfer and comparing embryo transfer media containing HA versus embryo transfer media devoid of HA (both commercial and in‐house prepared media).
Types of outcome measures
Primary outcome measures
Live birth rate per randomised couple: number of live births per randomised couple
Secondary outcome measures
a) Clinical pregnancy rate per randomised couple: number of couples achieving a clinical pregnancy (defined by the demonstration of a gestational sac on ultrasound scan)
b) Multiple pregnancy (per couple randomised)
Additional outcome measures
Data per cycle, per embryo transfer (ET), or per ovum pick up (OPU) are not able to be pooled (Vail 2003). However, due to the frequency that this form of data is reported in the literature it will be entered into an additional table for the following outcomes:
a) live births per OPU and ET;
b) clinical pregnancy rate per OPU and ET;
c) proportion of women in whom at least one embryo implants.
Search methods for identification of studies
We will search:
1) the Menstrual Disorders and Subfertility Group Trials Register;
2) Cochrane Central Register of Controlled Trials (CENTRAL) (The Cochrane Library current Issue);
3) MEDLINE, EMBASE, PsycInfo; and
4) handsearch ESHRE and ASRM supplements, non‐published papers and reference lists of trials;
5) contact experts for additional data.
We will search electronic databases for studies in all languages using the terms given in:
Appendix 1; Appendix 2; Appendix 3 ; Appendix 4.
Data collection and analysis
Two review authors (DB, EW) will perform the selection of trials for inclusion in the review after employing the search strategy described above. Excluded articles will be detailed in the table 'Characteristics of excluded trials'. Included trials will be assessed against the risk of bias criteria and for methodological details, outlined below. This information will be presented within the table 'Characteristics of included studies' and provides a context for assessing the reliability of results.
Trial characteristics
1. Concealment of allocation:
a) a third party (telephone) or trialist (computer, sealed envelope, or register);
b) unclear;
c) inadequate.
2. Method of randomisation:
a) computer generated;
b) random numbers table;
c) not stated.
3. Blinding:
a) clinician or nurses (possible);
b) scientists (not possible);
c) participant (possible).
4. Time of randomisation during cycle:
a) prior to commencement of treatment cycle;
b) after commencement of treatment and before fertilization check;
c) from fertilization check to embryo transfer.
5. Size of study, as number of women:
a) recruited;
b) randomised;
c) excluded;
d) analysed;
e) lost to follow up.
6. Study setting:
a) single‐centre or multicentre;
b) location;
c) timing.
7. Analyses:
a) power calculation;
b) whether or not by intention to treat.
Characteristics of the study participants
1. Baseline characteristics:
a) age;
b) primary or secondary infertility;
c) cause and duration of infertility;
d) previous treatment;
e) undergoing IVF or ICSI, or both;
b) over the age of 37 years and undergoing IVF or ICSI, or both.
3. Treatment characteristics:
a) embryo transfer policy;
b) day of embryo transfer (2, 3, 4, 5 or 6 day post‐OPU);
c) frozen and thaw protocol;
d) mean number of embryos transferred;
e) pregnancy determination.
Interventions used
1. Ovarian stimulation
2. Luteal support
3. Culture and transfer (with HA and without HA) medium brand
4. Exposure time to HA prior to ET
Outcomes
1. Primary:
a) live birth (per couple randomised).
2. Secondary:
a) clinical pregnancy (per couple randomised);
b) multiple pregnancy (per couple randomised).
3.Additional outcomes not appropriate for statistical pooling:
a) live births per OPU and ET;
b) clinical pregnancy rate per OPU and ET;
c) proportion of women in whom at least one embryo implants.
Sensitivity analysis
Any trials other than at 'low risk' of bias will be excluded.
Analysis
Information will be independently extracted on methodological quality and the outcome data by two review authors (DB, EW) using forms designed according to Cochrane guidelines. Another co‐author (NJ) will be available to resolve any discrepancies. Additional information on trial methodology or actual original trial data will be sought from the principal author of trials that appear to meet eligibility criteria but are unclear in aspects of methodology, or where the data is in a form unsuitable for meta‐analysis. Reminder correspondence will be sent when a reply is not received within three weeks.
Statistical analyses will be performed in accordance with the guidelines for statistical analysis developed by the Cochrane Menstrual Disorders and Subfertility Group. Heterogeneity between the results of different studies will be examined by inspecting the scatter of data points, the overlap in their confidence intervals, and more formally by checking the results of the Chi2 tests. A priori, it is planned to look at the possible contribution of differences in trial design to the heterogeneity identified, such as transfer of embryos on different days. Where possible, the outcomes will be pooled statistically.
Where possible, the data will be analysed using an intention‐to‐treat analysis. We will use the number of women randomised as the denominator even if the authors do not.
For dichotomous data (for example clinical pregnancy rate) results for each study will be expressed as odds ratios (OR) with 95% confidence intervals (CI) and combined for meta‐analysis with RevMan software using the Peto‐modified Mantel‐Haenzel method. The data will be entered on the graphs so that: in positive outcomes (for example pregnancy) points to the left of the line of no effect favour HA treatment, and in negative outcomes (for example multiple pregnancy) points to the right of the line of no effect favour HA treatment.
Subgroup analysis
The following subgroup analyses are planned.
Subgroup A: studies where the day of embryo transfer was early stage (up to and including Day 4) compared to studies where the embryo transfer was late (Days 5 and 6). The length of exposure to HA prior to the day of implantation (Day 7) may have an impact on the outcome.
Subgroup B: studies where the embryos have been frozen and thawed versus studies of fresh embryo transfers.
Time line
Completion of the review is expected within one year of publication on The Cochrane Library.
A search for new trials will be conducted annually and the review updated as and when new trials to be incorporated are found.
