Intracytoplasmic sperm injection (ICSI) improves fertilization rate (FR) in couples with male factor infertility. ICSI is a significant achievement in assisted reproductive technology (ART) and has become probably the most important therapy for male infertility in recent years. Globally, ICSI use is increasing, mainly because of increased ICSI use in cycles carried out for non-male factors, such as frozen oocytes, in vitro maturation of human immature oocytes, preimplantation genetic testing, and infertility with previous fertilization failure. ICSI use has been observed in 70–80% of fresh cycles, with increasing applications encountered [1]. ICSI is considered the most “revolutionary” in vitro insemination technique since an embryologist artificially selects a single spermatozoon for injection, which can fertilize an oocyte notwithstanding its morphology or motility. Additionally, some natural fertilization processes, such as sperm-cumulus interaction, sperm-zone penetration, and acrosome reaction, are circumvented in the ICSI process [2]. During conventional ICSI, sperm are selected based on motility and morphology, which may not reflect sperm quality. Theoretically, the prominent use of ICSI may increase the possibility of injecting spermatozoa that are defective in centrosome integrity, genetic constitution, phospholipase C zeta content, or DNA methylation [3, 4, 5]. Embryo quality is influenced by the quality of gametes, oocytes, and spermatozoa. Natural barriers to fertilization are circumvented in ICSI; therefore, embryo quality is affected. Therefore, ICSI treatments may be optimized by selecting the ideal spermatozoa before injection. However, the shapes of individual spermatozoa do not indicate chromatin integrity and chromosomal aberrations. Visual shape assessment, which selects the best-looking sperm during ICSI, is unreliable and may result in abnormal chromosomes in subsequent developmental stages [6, 7]. Concerns regarding the potential adverse effects of ICSI on subsequent fertilization, embryo development, and offspring health owing to deviations from natural selection exist. Therefore, a practical test for selecting healthy sperm for ICSI is required.

Polyvinylpyrrolidone (PVP) is used in conventional ICSI to reduce sperm velocity and facilitate the smooth injection of oocytes. However, PVP causes submicroscopic changes in sperm structure and damages sperm membrane integrity and sperm nucleus [8, 9]. Moreover, human oocytes are unable to degrade PVP, subsequently affecting pregnancy rates [10, 11, 12].

Recently, several sperm-selection techniques have been developed. These techniques are categorized into methods based on sperm density, morphology, motility, membrane integrity, surface charge, and nuclear integrity. However, these techniques have not demonstrated enhanced clinical outcomes that would support routine clinical application despite their ability to improve sample quality.

Hyaluronan (hyaluronic acid, HA) is a linear anionic polysaccharide containing N-acetyl-D-glucosamine and D-glucuronic acid bound with $\beta$-1,3 and $\beta$-1,4 glycosidic bonds [13]. This macromolecule is a constituent of the cumulus cell matrix in human oocytes. HA is a physiological selector, and only mature spermatozoa with low chromosomal aneuploidy and low fragmentation can bind to it [14]. The head region of mature spermatozoa contains HA receptors that facilitate passage through the extracellular matrix to the oocyte [15]. Sperms without HA receptors may be deselected, resulting in a lower possibility of reaching oocytes. Immature sperms lacking HA-binding sites are also associated with an increased frequency of chromosomal aberrations [14]. However, sperm without HA receptors can be selected and injected into oocytes during conventional ICSI. Sperm morphology is not associated with the selection of haploid spermatozoa [16]; however, the frequency of chromosomal diploidy in HA-selected spermatozoa is reduced by 4- to 6-fold compared to that in semen sperm [17]. This selection process is expected to facilitate the selection of a single mature spermatozoon with desirable morphological characteristics and without cytoplasmic retention, persistent histones, chromosomal aneuploidy, or DNA fragmentation [17, 18].

Theoretically, the selection of HA-bound sperm for ICSI facilitates fertilization and produces high-quality embryos, leading to better clinical outcomes. Studies on intracytoplasmic injection of hyaluronan-bound spermatozoa (HA-ICSI) are insufficient, and the application of HA-ICSI to treat infertility remains controversial, although it has been used for over a decade. HA-ICSI includes HA-coated dishes (PICSI dishes) and HA-containing media (SpermSlow${}^{\text{TM}}$ and SpermCatch${}^{\text{TM}}$). Several studies have documented the practical benefits of HA-ICSI in terms of fertilization, embryo quality, and pregnancy outcomes [19, 20, 21, 22]. However, some studies have challenged the application of HA-ICSI [23, 24]. Therefore, we performed a meta-analysis and systematic review to evaluate the effectiveness of HA-ICSI in couples with infertility.

Our meta-analysis included RCTs and cohort studies. We identified whether HA-ICSI was beneficial for patients compared to conventional ICSI in terms of LBR, CPR, SAR, BPR, IR, FR, cleavage rate, and good-quality embryo rate. We screened 16 publications (10 RCTs, five cohort studies, and one publication, including an RCT and a cohort trial). Our meta-analysis of the cohort studies indicated a significant advantage for HA-ICSI in terms of LBR, CPR, BPR, IR, FR, and good-quality embryo rate, and no differences in SAR and cleavage rate between the HA-ICSI and control groups. Compared with that in the conventional ICSI group, the SAR in the HA-ICSI group significantly decreased in a meta-analysis of all available studies and RCTs. Similar to the RCT results, a meta-analysis of all studies revealed no difference between the HA-ICSI and control groups in terms of LBR, BPR, and good-quality embryo rates. The advantages of CPR, IR, and FR have been recognized in a meta-analysis of all available studies. However, a meta-analysis of RCTs did not demonstrate these advantages. HA-ICSI significantly decreased the cleavage rate in the meta-analysis of all available studies; however, for RCTs, no difference in the cleavage rate between the HA-ICSI and conventional ICSI groups was observed.

Our conclusions differ from those of the previous studies. Based on data from four RCTs, Lepine S, et al. [36] discovered that HA-ICSI reduced miscarriage but did not improve LBR or CPR. When our meta-analysis was limited to data sets from RCTs, we obtained similar results. RCTs have superior study designs owing to their internal validity; however, they may lack external validity [37, 38, 39]. Therefore, it is necessary to include cohort studies. Beck-Fruchter R, et al. [40] reviewed six RCTs and two cohort studies. It was concluded that using the HA-ICSI technique yielded no improvement in fertilization and pregnancy rates. A meta-analysis of all available studies showed an improvement in embryo quality and IR, and a meta-analysis of RCTs only revealed an improvement in embryo quality. We conducted a meta-analysis of all cohort studies and recent RCTs, resulting in an extensive dataset for analysis and different results. Our meta-analysis and systematic review, including RCTs and cohort studies, are the most comprehensive analyses of the effects of HA binding on sperm selection in ICSI. In contrast to previous reviews, we thoroughly searched for relevant articles and updated some recent studies.

I${}^{\mathrm{2}}$ was relatively high in most analyses, indicating significant heterogeneity among these analyses. Therefore, a random-effects model was employed to minimize heterogeneity. An absence of heterogeneity in the RCTs or cohort studies in the meta-analysis of LBR, CPR, BPR, IR, and good-quality embryo rates was observed after a subgroup analysis was performed based on the type of study design, suggesting that differences in design and methods affect heterogeneity.

For the cohort studies, none of the outcomes varied significantly with the removal of any study, demonstrating that the results were not highly influenced by any study, suggesting the stability and reliability of the findings. For RCTs, all outcomes, except the SAR, did not vary with the removal of any study, demonstrating that the meta-analysis was thorough and that none of the studies greatly influenced the results. However, in our meta-analysis, the SAR outcome in RCTs was unreliable. Six RCTs reported SARs, excluding the study conducted by Miller D, et al. [23], which led to a different result of no significant difference in the SAR between the two groups, similar to the pooled results of cohort studies. Therefore, we suggest that the pooled SAR result in RCTs was greatly influenced by the study by Miller D, et al. [23], in which 2772 couples undergoing ICSI were randomly assigned to receive either HA-ICSI or conventional ICSI, and a lower miscarriage rate in HA-ICSI was observed. Additionally, the results of most of the included studies were not significant.

Our study had several limitations. First, the significant problem was that the results showed a high degree of statistical heterogeneity in some analyses. We conducted a subgroup analysis and discovered that the design and methodological differences between the RCTs and cohort studies may have contributed to the high heterogeneity observed. However, we were unable to eliminate the effects of other factors, such as the etiology of infertility, ovarian stimulation protocols, and exclusion criteria, which were not available for subgroup analysis owing to insufficient data. Moreover, the use of Embryoscope${}^{\text{TM}}$ for embryo culture in one study [26] is a significant factor that affects heterogeneity. Second, some of the included articles were of low quality, and the number of included articles was insufficient. Third, a potential publication bias was observed. FR and Begg’s funnel plot showed no apparent publication bias for CPR; however, Egger’s linear regression results showed evidence of publication bias for CPR. The language of the included articles was restricted to English; therefore, the presence of another publication bias is possible. Additionally, our search was limited to published articles; as a result, unpublished articles with a possibility of meeting our inclusion criteria might have been excluded. Fourth, our meta-analysis included studies that met different inclusion and exclusion criteria. The enrollment of couples undergoing ICSI from the general in vitro fertilization (IVF) population, unselected for specific indications, might underestimate the potential advantages of HA-ICSI. Hence, the final results should be interpreted carefully and further evaluated using large samples, multiple centers, and high-quality studies.

Notwithstanding these limitations, our analysis included recently published articles, which provided greater detail on ICSI outcomes and focused on subgroup analysis according to the included study designs, leading to an increased significance of our meta-analysis. Additionally, most of the meta-analyzed studies demonstrated improved clinical outcomes with HA-ICSI. No study reported that HA-ICSI adversely affected ICSI outcomes. If a comprehensive multicenter prospective randomized study confirms the positive effects of HA-ICSI, HA-ICSI is a potential first-line infertility treatment for ‘physiological’ sperm selection before ICSI because of its ability to reduce genetic complications and its non-toxic state.

In summary, the cohort studies indicated a statistically significant advantage of HA-ICSI in terms of LBR, CPR, BPR, IR, FR, and good-quality embryo rate. All included studies and RCTs revealed that HA-ICSI significantly decreased SAR compared to conventional ICSI. The advantages of CPR, IR, cleavage rate, and FR have been recognized in a meta-analysis of all available studies. However, a meta-analysis of RCTs did not demonstrate these advantages. HA-ICSI did not have detrimental effects on ICSI outcome parameters. Extensive multicenter prospective randomized studies are required to confirm the beneficial effects of HA-ICSI. Additionally, HA-ICSI should be considered for physiological sperm selection before ICSI.

All data generated or analysed during this study are included in this article. Further enquiries can be directed to the corresponding author.

WF designed and performed the study, collected and analyzed the data, and wrote the paper. WG collected and analyzed the data, reviewed and edited the manuscript. QC collected and analyzed the data and reviewed and edited the manuscript. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

Not applicable.

Thanks to all the peer reviewers for their opinions and suggestions.

This research received no external funding.

The authors declare no conflict of interest.

Supplementary material associated with this article can be found, in the online version, at https://doi.org/10.31083/j.ceog5106147.