Table 4.

Overview of all recommendations on laboratory tests and interventions with their level of evidence, benefit versus harm and other considerations that contributed to their formulation.

Intervention		Benefits versus harms (efficacy versus safety)	Level of evidence for efficacy (LBR/CPR)1	Level of evidence for safety1	Considerations	Recommendation
Artificial oocyte activation		Beneficial in patients with previous fertilization failure or low fertilization rate and embryo developmental problems Safety to be considered (mechanism unclear), but not reported	⊕⊕◯◯	⊕⊕◯◯	Current studies show variation in ionophore stimulus with respect to concentration, exposure time, and number of exposures.	Artificial oocyte activation is currently not recommended for routine clinical use. Artificial oocyte activation is recommended for complete activation failure (0% 2PN), very low fertilization (<30% fertilization), or globozoospermia.
Mitochondrial replacement therapy		Few data, no benefit on LBR No data on safety	⊕◯◯◯	No data	In some cases, the acceptors’ mtDNA haplotype takes over the donors’ mtDNA	Mitochondrial replacement therapy to affect oocyte quality is not recommended.
In vitro activation of dormant follicles		No comparative studies Safety, adverse effects, and long-term effects: no data	⊕◯◯◯	No data	For POI patients, options are limited.	In vitro activation of dormant follicles is not recommended.
IVM	Clinical IVM	No comparative studies Abnormal fertilization and development arrest reported	⊕◯◯◯	No data	It has been suggested that IVM encompasses a lower financial and emotional burden as compared to standard IVF/ICSI.	Clinical IVM and rescue-IVM or natural cycle IVF/M are currently not recommended for routine clinical use.
	Rescue IVM	LBR is lower, effect on miscarriage rate is unclear, most studies report increased miscarriages/decreased implantation. Safety of rescue IVM is questionable, since these oocytes commonly have meiotic defects and are of poor quality.	⊕◯◯◯	⊕◯◯◯	/
Sperm DNA testing and treatment		Diagnostic potential inconclusive No adverse events expected	⊕⊕◯◯	No data	Different assays have different discriminatory capacity, different lab conditions and sperm source can influence the outcome of the test	Sperm DNA damage testing is currently not recommended for routine clinical use.
Artificial sperm activation		Higher LBR/CPR No data on safety Malformations reported in animal studies	⊕◯◯◯	⊕◯◯◯	/	Artificial sperm activation is currently not recommended for routine clinical use. Artificial sperm activation is recommended for patients with primary or secondary total asthenozoospermia which are not the result of axonemal structure defects.
Sperm evaluation and selection	Hyaluronic acid binding assay and physiological ICSI	No evidence benefit on LBR, reduced miscarriage rate No harms reported	⊕⊕◯◯	No data	Hyaluronic acid binding assay has limited standardization.	Sperm hyaluronic binding assay is currently not recommended for routine clinical use. Physiological ICSI is currently not recommended for routine clinical use.
	MACS	No evidence of benefit on LBR or miscarriage rate No harms reported	⊕◯◯◯	⊕◯◯◯	/	Magnetic-activated cell sorting is currently not recommended for routine clinical use.
	Microfluidics	Only one RCT reporting benefit on LBR No harms reported	⊕⊕◯◯	No data	/	Microfluidics for sperm selection and preparation can be considered.
	IMSI	No evidence of benefit on LBR or miscarriage rate No complications reported	⊕◯◯◯	⊕◯◯◯	The method of IMSI can be time-consuming and impacts laboratory workflow.	Intracytoplasmic morphologic sperm injection is currently not recommended for routine clinical use.
Growth factor supplemented embryo culture medium		No evidence of benefit on LBR or miscarriage rate Theoretical harms, but not reported	⊕⊕◯◯	⊕⊕◯◯	As growth factors act in both positive and negative synergy to produce an effect, addition of a single growth factor to embryo culture media is questionable and will not necessarily elicit a beneficial effect.	Growth factor-supplemented embryo culture medium is not recommended.
Assisted hatching		No evidence of benefit on LBR or miscarriage rate Complications: increased multiple pregnancy rate	⊕⊕◯◯	⊕◯◯◯	/	Assisted hatching is not recommended.
Genetic testing and treatments	PGT-A	Most RCTs did not report benefit on LBR, but some suggest reduced miscarriage rate Harms include disposal of viable embryos and IUGR	⊕⊕◯◯	⊕◯◯◯	Lack of standardization in biopsy and analysis method	Pre-implantation genetic testing for aneuploidy is currently not recommended for routine clinical use.
	niPGT-A	No data regarding effect on LBR or miscarriage rate Considered to be safer than PGT-A	No data	No data	/	Non-invasive PGT is currently not recommended for routine clinical use.
	Mitochondrial DNA load measurement	No data regarding effect on LBR or miscarriage rate Expected complications are similar to PGT-A	No data	No data	/	Mitochondrial DNA load measurement is currently not recommended for routine clinical use.
Time-lapse imaging		No evidence of benefit on LBR or miscarriage rate No evidence or rationale for harm	⊕⊕◯◯	No data	/	Time-lapse imaging is not recommended as a tool to improve live birth rates.

¹Quality of Evidence Grades: ⊕⊕⊕⊕, body of evidence is of high quality (at least evidence from RCTs); ⊕⊕⊕◯, body of evidence is of moderate quality (evidence from RCTs or a number of observational studies showing a similar large effect); ⊕⊕◯◯, body of evidence is of low quality (mainly observational data); ⊕◯◯◯, body of evidence is of very low quality (few observational data).

CPR: clinical pregnancy rate; LBR: live birth rate; IMSI: intracytoplasmic morphologically selected sperm injection; IUGR: intra-uterine growth restriction; MACS: magnetic-activated cell sorting; mtDNA: mitochondrial DNA; niPGT-A: non-invasive PGT-A; PGT-A: pre-implantation genetic testing for aneuploidy; RCT: randomized controlled trial.