Advanced sperm selection techniques for assisted reproduction

  • Protocol
  • Intervention

Authors


Abstract

This is the protocol for a review and there is no abstract. The objectives are as follows:

To evaluate the impact of advanced sperm selection techniques on ART outcomes.

Background

Description of the condition

In vitro fertilisation (IVF) is a form of assisted reproductive technology (ART) used for treating infertility, a condition affecting an estimated 15% of the population. IVF usually involves controlled ovarian hyperstimulation, surgical oocyte retrieval, in vitro fertilisation and embryo transfer. Intra-cytoplasmic sperm injection (ICSI) is a form of ART where instead of relying on spontaneous entry of the sperm into the oocyte, a single sperm is injected into the cytoplasm of each oocyte to achieve fertilisation. ICSI is commonly used as a treatment for male factor infertility where semen parameters are poor, when sperm has been surgically retrieved or following repeated failed fertilisation with standard IVF (Palermo 1992).

Successful embryo development and subsequent pregnancy outcome are likely to be impacted by the quality of the sperm which fertilises an oocyte (Sakkas 2000). Ideally only sperm with a high chance of successful fertilisation and subsequent embryo growth would be used for ART. These sperm would be viable, mature, have high DNA integrity and be structurally sound.

Sperm preparation and selection in IVF is limited to semen washing, density gradient centrifugation and the use of the swim-up techniques (Boomsma 2007). In ICSI, routine sperm selection is based on motility and gross morphology (sperm are examined under a microscope at 200 to 400x magnification) after one or more of the above methods of semen preparation. Advanced sperm selection techniques based on alternative characteristics might enable further selection of the most appropriate sperm for use in ART.

Description of the intervention

Advanced sperm selection techniques have developed as a means of improving ART outcomes in certain clinical scenarios. Techniques can be categorised as follows:

1. Surface charge selection

Electrophorectic sperm selection and sperm zeta potential are surface charge selection protocols utilised in both IVF and ICSI. The zeta potential of the sperm is the electric potential between the sperm membrane and its surroundings. The zeta potential decreases with capacitation and normally differentiated sperm are charged electronegatively. Semen is placed into an electrophoretic device and a current applied. Normally differentiated negatively charged sperm are rapidly separated and collected from an adjacent chamber (Ainsworth 2005).

2. Sperm apoptosis

Selection of non-apoptotic sperm for use in ART is based on the presence of phosphatidylserine on the external surface of the sperm membrane in the early stages of apoptosis. Magnetic activated cell sorting (MACS) and glass wool separation columns utilise the magnetic properties of phosphatidylserine to separate apoptotic sperm from non-apoptotic sperm (Grunewald 2001).

3. Hyaluronic acid binding

Hyaluronic acid binding sites on the sperm plasma membrane indicate sperm maturity. Sperm are placed in a PICSITM dish, which is an IVF petri dish that carries a hyaluronic acid spot. Mature sperm preferentially bind to the hyaluronan impregnated sites (Huszar 2007).

4. Sperm birefringence

The mature sperm nucleus has high intrinsic birefringence due to longitudinally orientated subacrosomal protein filaments. Using polarised light microscopy sperm birefringence can be evaluated and a mature sperm selected (Gianaroli 2008).

5. Sperm morphology

Subtle defects in sperm morphology (acrosome, nucleus, mitochondria, tail, postacrosomal lamina and neck) can be observed using ultra-high magnification (6000x) microscopy (motile sperm organelle morphology examination, 'MSOME') (Bartoov 2002). Intracytoplasmic morphologically selected sperm injection (IMSI) is a modification of ICSI utilising this technique (Bartoov 2003).

How the intervention might work

Through the use of advanced sperm selection techniques a structurally intact and mature sperm with high DNA integrity may be selected for fertilisation. Each modality utilises differing characteristics of sperm structure, physiology or function to allow selection of the most normal sperm. Advanced sperm selection protocols aim to improve ART outcomes and may limit the possible deleterious effects on offspring of using sperm with defective DNA (Aitken 2007).

Why it is important to do this review

Advanced sperm selection techniques are hypothesised to improve ART outcome through the selection of sperm with a variety of 'beneficial characteristics'. Despite individual small studies suggesting that these techniques have clinical benefit, there remains no comprehensive review of randomised controlled trials (RCTs) in this area. A systematic review on advanced selection methods was published in 2011, however this incorporated non-randomised data (Said 2011). The current review aims to include only RCTs so that the results can better guide clinical practice and further research efforts.

Objectives

To evaluate the impact of advanced sperm selection techniques on ART outcomes.

Methods

Criteria for considering studies for this review

Types of studies

Published and unpublished randomised controlled trials (RCTs) investigating the impact of advanced sperm selection techniques in ART will be eligible for inclusion. Non-randomised studies will be excluded due to a high risk of bias. Cross-over studies are inappropriate in this context and will be excluded.

Types of participants

Women or couples undergoing ART.

Types of interventions

Trials comparing an advanced sperm selection technique with either another advanced sperm selection technique or an advanced sperm selection technique with standard sperm preparation techniques (e.g. semen washing, density gradient centrifugation, swim-up techniques).

Advanced sperm selection techniques include:

  1. Surface charge selection

  2. Sperm apoptosis

  3. Hyaluronic acid binding

  4. Sperm birefringence

  5. Analysis of sperm morphology

Types of outcome measures

Primary outcomes
Effectiveness
  • Live birth per woman randomised

(Live birth is defined as the delivery of a live fetus beyond 20 completed weeks gestation).

Secondary outcomes
Effectiveness
  • Clinical pregnancy per woman randomised

(Clinical pregnancy is defined as identification of a gestational sac on ultrasound at ≥ 7 weeks gestation).

Adverse events
  • Miscarriage, fetal abnormalities per clinical pregnancy

(Miscarriage is defined as pregnancy loss at < 20 completed weeks gestation or where fetus weighs < 500 g, as confirmed by ultrasound and pregnancy test or histology. This includes partial loss of multiple pregnancies).

Fertilisation rates, implantation rates and outcomes relating to embryo development and quality are of importance to the review and will be described under 'Characteristics of included studies'. These outcomes will not be included in the meta-analysis because there is a lack of standardised grading systems for morphology and the denominators for fertilisation and implantation rates differ.

Search methods for identification of studies

All published and unpublished RCTs using the search terms 'IVF', 'ICSI', 'ART', 'sperm selection', 'sperm preparation', 'sperm parameter', 'Hyaluronic acid', 'PICSI', 'apoptosis', 'DNA', 'membrane maturity', 'magnetic cell sorting', 'morphology', 'intracytoplasmic morphologically selected sperm injection', 'polarization microscopy', 'polscope' and 'sperm birefringence'.

The search will have no language restriction and will be designed and conducted by SK and BM in consultation with the Menstrual Disorders and Subfertility Group (MDSG) Trials Search Co-ordinator.

Electronic searches

We will search the following electronic databases, trial registers and websites:

  • Menstrual Disorders and Subfertility Group (MDSG) Specialised Register

  • Cochrane Central Register of Controlled Trials (CENTRAL)

  • MEDLINE

  • EMBASE

  • PsycINFO

  • CINAHL

Other electronic sources of trials will include the following.

Searching other resources

We will search reference lists of articles retrieved by the search and contact experts in the field to obtain additional data if necessary.

Data collection and analysis

Selection of studies

After an initial screen of titles and abstracts retrieved by the search, we will retrieve the full texts of all potentially eligible studies. Two review authors (SM and BK) will independently examine these full-text articles for compliance with the inclusion criteria and select studies eligible 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 discussion with a third review author (AY). We will document the selection process with a 'PRISMA' flow chart.

Data extraction and management

Two review authors will independently extract data from eligible studies. Any disagreements will be resolved by discussion or by a third review author. Data extracted will include study characteristics and outcome data. We will correspond with study investigators for additional information if necessary. Data to be extracted will include details of methods, participants, setting, context, interventions (sperm selection protocols), outcomes, results and publications.

Assessment of risk of bias in included studies

Two review authors (SM and BK) will independently assess the included studies for risk of bias using the Cochrane 'Risk of bias' assessment tool (Higgins 2011). This will assess: allocation (random sequence generation and allocation concealment); blinding of participants and personnel, blinding of outcome assessors; incomplete outcome data; selective reporting; and other bias. Disagreements will be resolved by discussion or by a third review author (AY). 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.

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 allow inclusion. We will seek published protocols and compare the outcomes between the protocol and the final published study.

Measures of treatment effect

Data to be extracted will be dichotomous (e.g. live birth rate, miscarriage rate). Using RevMan software (RevMan 2011) we will apply the Mantel-Haenszel pooling method to weight the effect measure (e.g. risk ratio, odds ratio). We will use risk ratios in preference to odds ratios as these are a more suitable description of the intervention. We will present 95% confidence intervals for all outcomes. If appropriate we will use number needed to treat to benefit (NNT), number needed to treat to harm (NNH), or both, to describe any significant findings.

Unit of analysis issues

The primary analysis will be per woman randomised; per pregnancy data may also be included for some outcomes (e.g. miscarriage). We will briefly summarise data that do not allow valid analysis (e.g. 'per cycle' data) in an additional table and these will not be meta-analysed. If studies report only 'per cycle' data, we will contact authors and request 'per woman randomised' data.

We will count multiple live births (e.g. twins or triplets) as one live birth event.

Dealing with missing data

We will analyse the data on an intention-to-treat basis as far as possible and make attempts to obtain missing data from the original trialists. Where the data cannot be obtained, we will assume the outcome measure (e.g. live birth, clinical pregnancy) not to have occurred. For other outcomes, we will only analyse the available data. We will subject any imputation undertaken to sensitivity analysis (see below).

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 assess statistical heterogeneity by the measure of the I2 statistic. We will take an I2 measurement greater than 50% to indicate moderate heterogeneity (Higgins 2003; Higgins 2008). If we detect substantial heterogeneity, we will explore possible explanations in sensitivity analyses (see below) and consider a subgroup analysis. We will take any statistical heterogeneity into account when interpreting the results, especially if there is any variation in the direction of effect.

Assessment of reporting biases

The authors will aim to minimise the potential impact of reporting bias by ensuring a comprehensive search for eligible studies. 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

If the studies are sufficiently similar, we will combine the data using a fixed-effect model in the following comparisons.

  1. IVF versus advanced sperm selection technique, stratified by individual sperm selection technique (refer Description of the intervention section above for details).

  2. ICSI versus advanced sperm selection technique, stratified by individual sperm selection technique.

  3. Advanced sperm selection technique versus another advanced sperm selection technique.

We will display an increase in the risk of a particular outcome, which may be beneficial (e.g. live birth) or detrimental (e.g. adverse effects), graphically in the meta-analyses to the right of the centre-line and a decrease in the odds of an outcome to the left of the centre-line.

Subgroup analysis and investigation of heterogeneity

Where data are available, we will conduct subgroup analyses to determine the separate evidence within the following subgroups.

  1. Sperm morphology: where the Kruger score is equal to or lower than 4%.

  2. Increased DNA fragmentation index (according to the study cut-off).

  3. Surgically retrieved sperm.

  4. Female participants aged over 38 years.

Sensitivity analysis

We will conduct sensitivity analyses for all of the outcome measures 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:

  1. eligibility were restricted to studies without high risk of bias;

  2. a random-effects model had been adopted;

  3. alternative imputation strategies had been implemented.

Overall quality of the body of evidence: 'Summary of findings' table

We will generate a 'Summary of findings' table using GRADEpro software. This table will evaluate the overall quality of the body of evidence for the main review outcomes, using GRADE criteria (study limitations (i.e. risk of bias), consistency of effect, imprecision, indirectness and publication bias). We will justify, document and incorporate into the reporting of results for each outcome our judgements about evidence quality (high, moderate or low).

Appendices

Appendix 1. Menstrual Disorders and Subfertility Group keyword search

12.03.13

Keywords CONTAINS "IVF" or "in vitro fertilization" or "in-vitro fertilisation" or "ICSI" or"intracytoplasmic sperm injection" or "in-vitro fertilization" or "assisted reproduction" or "subfertility"or "infertility" or "male factor" or"male fertility" or "male infertility" or "male subfertility" or "subfertility-male" or Title CONTAINS"IVF" or "in vitro fertilization" or "in-vitro fertilisation" or "ICSI" or"intracytoplasmic sperm injection" or "in-vitro fertilization" or "assisted reproduction"or "subfertility"or "infertility" or "male factor" or"male fertility" or "male infertility" or "male subfertility" or "subfertility-male"

AND

 Keywords CONTAINS "sperm morphology"or "sperm motility"or "sperm preparation"or"sperm preparation techniques"or "sperm select"or "sperm selection"or "sperm selection techniques"or"sperm separation"or "sperm sorting"or "birefringent sperm"or "Magnetic Activated Sorting Selection"or"magnetic sperm selection"or "hyaluronan-bound (HB) sperm"or "hyaluronan bound sperm"or "hyaluronic acid sperm selection"or "hyaluronic acid intracytoplasmic sperm injection"or "IMSI"or "intracytoplasmic morphologically selected sperm injection" or "apoptosis"or "semen preparation"or "membrane properties" or Title CONTAINS  "sperm morphology"or "sperm motility"or "sperm preparation"or"sperm preparation techniques"or "sperm select"or "sperm selection"or "sperm selection techniques"or"sperm separation"or "sperm sorting"or "birefringent sperm"or "Magnetic Activated Sorting Selection"or"magnetic sperm selection"or "hyaluronan-bound (HB) sperm"or "hyaluronan bound sperm"

Appendix 2. CENTRAL search strategy

12.03.13

1 exp embryo transfer/ or exp fertilization in vitro/ or exp sperm injections, intracytoplasmic/
2 embryo transfer$.tw.
3 vitro fertili?ation.tw.
4 ivf-et.tw.
5 ivf.tw.
6 icsi.tw.
7 intracytoplasmic sperm injection$.tw.
8 (blastocyst adj2 transfer$).tw.
9 assisted reproduct$.tw.
10 ovulation induc$.tw.
11 (ovari$ adj2 stimulat$).tw.
12 superovulat$.tw.
13 ovarian hyperstimulation.tw.
14 COH.tw.
15 infertil$.tw.
16 subfertil$.tw.
17 (ovari$ adj2 induction).tw.
18 exp Reproductive Techniques, Assisted/
19 ART.tw.
20 or/1-19
21 (sperm$ adj7 selection$).tw.
22 (sperm$ adj7 separat$).tw.
23 surface charge.tw.
24 electrophore$.tw.
25 (zeta adj2 potential).tw.
26 magnetic cell sorting.tw.
27 glass wool.tw.
28 membrane matur$.tw.
29 magnetic activated cell sort$.tw.
30 ultramorpholog$.tw.
31 (hyaluronic acid adj2 binding).tw.
32 (sperm$ adj5 birefringence).tw.
33 (sperm$ adj3 morphology).tw.
34 ultra high magnification.tw.
35 motile sperm$ organelle.tw.
36 MSOME.tw.
37 IMSI.tw.
38 Intracytoplasmic morphologically selected sperm injection$.tw.
39 Raman spectroscopy.tw.
40 confocal light absorption.tw.
41 (scattering adj3 microscopy).tw.
42 polarization microscopy.tw.
43 polarisation microscopy.tw.
44 polscope.tw.
45 (sperm$ adj3 apopto$).tw.
46 zeta method.tw.
47 (nonapoptotic$ adj3 sperm$).tw.
48 sperm$ preparation.tw.
49 (sperm$ adj3 prepar$).tw.
50 (semen adj2 prepar$).tw.
51 (sperm$ adj5 chemotaxis).tw.
52 hyaluronan bound.tw.
53 (hyaluronic acid adj2 bound).tw.
54 or/21-53
55 20 and 54

Appendix 3. EMBASE search strategy

12.03.13

1 (sperm$ adj7 selection$).tw.
2 (sperm$ adj7 separat$).tw.
3 surface charge.tw.
4 electrophore$.tw.
5 (zeta adj2 potential).tw.
6 magnetic cell sorting.tw.
7 glass wool.tw.
8 membrane matur$.tw.
9 magnetic activated cell sort$.tw.
10 ultramorpholog$.tw.
11 (hyaluronic acid adj2 binding).tw.
12 (sperm$ adj5 birefringence).tw.
13 (sperm$ adj3 morphology).tw.
14 ultra high magnification.tw.
15 motile sperm$ organelle.tw.
16 MSOME.tw.
17 IMSI.tw.
18 Intracytoplasmic morphologically selected sperm injection$.tw.
19 Raman spectroscopy.tw.
20 confocal light absorption.tw.
21 (scattering adj3 microscopy).tw.
22 polarization microscopy.tw.
23 polarisation microscopy.tw.
24 polscope.tw.
25 (sperm$ adj3 apopto$).tw.
26 zeta method.tw.
27 (nonapoptotic$ adj3 sperm$).tw.
28 sperm$ preparation.tw.
29 (sperm$ adj3 prepar$).tw.
30 (semen adj2 prepar$).tw.
31 (sperm$ adj5 chemotaxis).tw.
32 hyaluronan bound.tw.
33 (hyaluronic acid adj2 bound).tw.
34 or/1-33
35 exp embryo transfer/ or exp fertilization in vitro/ or exp intracytoplasmic sperm injection/
36 embryo$ transfer$.tw.
37 in vitro fertili?ation.tw.
38 icsi.tw.
39 intracytoplasmic sperm injection$.tw.
40 (blastocyst adj2 transfer$).tw.
41 ivf.tw.
42 assisted reproduct$.tw.
43 ovulation induc$.tw.
44 (ovari$ adj2 stimulat$).tw.
45 superovulat$.tw.
46 ovarian hyperstimulation.tw.
47 COH.tw.
48 infertil$.tw.
49 subfertil$.tw.
50 (ovari$ adj2 induction).tw.
51 exp infertility therapy/
52 or/35-51
53 Clinical Trial/
54 Randomized Controlled Trial/
55 exp randomization/
56 Single Blind Procedure/
57 Double Blind Procedure/
58 Crossover Procedure/
59 Placebo/
60 Randomi?ed controlled trial$.tw.
61 Rct.tw.
62 random allocation.tw.
63 randomly allocated.tw.
64 allocated randomly.tw.
65 (allocated adj2 random).tw.
66 Single blind$.tw.
67 Double blind$.tw.
68 ((treble or triple) adj blind$).tw.
69 placebo$.tw.
70 prospective study/
71 or/53-70
72 case study/
73 case report.tw.
74 abstract report/ or letter/
75 or/72-74
76 71 not 75
77 34 and 52 and 76

Appendix 4. MEDLINE search strategy

12.03.13

1 exp embryo transfer/ or exp fertilization in vitro/ or exp sperm injections, intracytoplasmic/
2 embryo transfer$.tw.
3 vitro fertili?ation.tw.
4 ivf-et.tw.
5 ivf.tw.
6 icsi.tw.
7 intracytoplasmic sperm injection$.tw.
8 (blastocyst adj2 transfer$).tw.
9 assisted reproduct$.tw.
10 ovulation induc$.tw.
11 (ovari$ adj2 stimulat$).tw.
12 superovulat$.tw.
13 ovarian hyperstimulation.tw.
14 COH.tw.
15 infertil$.tw.
16 subfertil$.tw.
17 (ovari$ adj2 induction).tw.
18 exp Reproductive Techniques, Assisted/
19 ART.tw.
20 or/1-19
21 (sperm$ adj7 selection$).tw.
22 (sperm$ adj7 separat$).tw.
23 surface charge.tw.
24 electrophore$.tw.
25 (zeta adj2 potential).tw.
26 magnetic cell sorting.tw.
27 glass wool.tw.
28 membrane matur$.tw.
29 magnetic activated cell sort$.tw.
30 ultramorpholog$.tw.
31 (hyaluronic acid adj2 binding).tw.
32 (sperm$ adj5 birefringence).tw.
33 (sperm$ adj3 morphology).tw.
34 ultra high magnification.tw.
35 motile sperm$ organelle.tw.
36 MSOME.tw.
37 IMSI.tw.
38 Intracytoplasmic morphologically selected sperm injection$.tw.
39 Raman spectroscopy.tw.
40 confocal light absorption.tw.
41 (scattering adj3 microscopy).tw.
42 polarization microscopy.tw.
43 polarisation microscopy.tw.
44 polscope.tw.
45 (sperm$ adj3 apopto$).tw.
46 zeta method.tw.
47 (nonapoptotic$ adj3 sperm$).tw.
48 sperm$ preparation.tw.
49 (sperm$ adj3 prepar$).tw.
50 (semen adj2 prepar$).tw.
51 (sperm$ adj5 chemotaxis).tw.
52 hyaluronan bound.tw.
53 (hyaluronic acid adj2 bound).tw.
54 or/21-53
55 randomized controlled trial.pt.
56 controlled clinical trial.pt.
57 randomized.ab.
58 randomised.ab.
59 placebo.tw.
60 clinical trials as topic.sh.
61 randomly.ab.
62 trial.ti.
63 (crossover or cross-over or cross over).tw.
64 or/55-63
65 exp animals/ not humans.sh.
66 64 not 65
67 20 and 54 and 66

Appendix 5. PsycINFO search strategy

12.03.13

1 (sperm$ adj7 selection$).tw.
2 (sperm$ adj7 separat$).tw.
3 surface charge.tw.
4 electrophore$.tw.
5 (zeta adj2 potential).tw.
6 magnetic cell sorting.tw.
7 glass wool.tw.
8 membrane matur$.tw.
9 magnetic activated cell sort$.tw.
10 ultramorpholog$.tw.
11 (hyaluronic acid adj2 binding).tw.
12 (sperm$ adj5 birefringence).tw.
13 (sperm$ adj3 morphology).tw.
14 ultra high magnification.tw.
15 motile sperm$ organelle.tw.
16 MSOME.tw.
17 IMSI.tw.
18 Intracytoplasmic morphologically selected sperm injection$.tw.
19 Raman spectroscopy.tw.
20 confocal light absorption.tw.
21 (scattering adj3 microscopy).tw.
22 polarization microscopy.tw.
23 polarisation microscopy.tw.
24 polscope.tw.
25 (sperm$ adj3 apopto$).tw.
26 zeta method.tw.
27 (nonapoptotic$ adj3 sperm$).tw.
28 sperm$ preparation.tw.
29 (sperm$ adj3 prepar$).tw.
30 (semen adj2 prepar$).tw.
31 (sperm$ adj5 chemotaxis).tw.
32 hyaluronan bound.tw.
33 (hyaluronic acid adj2 bound).tw.
34 or/1-33
35 exp reproductive technology/
36 in vitro fertili?ation.tw.
37 ivf-et.tw.
38 (ivf or et).tw.
39 icsi.tw.
40 intracytoplasmic sperm injection$.tw.
41 (blastocyst adj2 transfer$).tw.
42 assisted reproduct$.tw.
43 ovulation induc$.tw.
44 (ovari$ adj2 stimulat$).tw.
45 ovarian hyperstimulation.tw.
46 COH.tw.
47 superovulat$.tw.
48 infertil$.tw.
49 subfertil$.tw.
50 (ovari$ adj2 induction).tw.
51 or/35-50
52 random.tw.
53 control.tw.
54 double-blind.tw.
55 clinical trials/
56 placebo/
57 exp Treatment/
58 or/52-57
59 34 and 51 and 58

Contributions of authors

SM wrote the first draft of the protocol. BK wrote the revised draft of the protocol. EF contributed to the protocol with methodological and statistical expertise. AY commented on all drafts of the protocol, methods and statistics. DG assisted with revision of the protocol. YH provided technical expertise and will contribute in the analysis phase of the review.

Declarations of interest

There are no conflicts of interest.

Sources of support

Internal sources

  • None, Not specified.

External sources

  • None, Not specified.

Ancillary