Free centrifuge sorting for sperm separation improves intracytoplasmic sperm injection outcomes: A systematic review and meta-analysis
Article information
Abstract
In assisted reproductive technology, spermatozoa must be separated from seminal fluid to achieve optimal fertilization capacity. Conventional separation techniques frequently result in elevated reactive oxygen species production and iatrogenic injury due to repeated cell centrifugation. The aim of this systematic review and meta-analysis was to evaluate the effects of free centrifuge sorting (FCS) techniques on intracytoplasmic sperm injection (ICSI) outcomes. A comprehensive literature search was conducted using PubMed, Scopus, Web of Science, and Cochrane databases. The meta-analysis adhered to the Preferred Reporting Items for Systematic reviews and Meta-Analyses Protocols (PRISMA-P) guidelines. All eligible studies were selected using the population, intervention, comparison/comparator, outcomes, and study design (PICOS) methodology. The primary outcomes assessed were fertilization rate (FR), the high-quality embryo rate, implantation rate (IR), and clinical pregnancy rate (CPR). The study is registered in PROSPERO under registration number CRD42023415532. After screening 306 records for eligibility, three studies were ultimately included in the analysis. Our results demonstrate that following ICSI, a very brief period of abstinence significantly increased IR and CPR. However, no significant differences were observed for FR. The FCS technique yielded spermatozoa of superior biological quality following removal of seminal samples, and this purified sperm population improved reproductive outcomes in ICSI programs.
Introduction
Following the birth of the first baby born after in vitro fertilization (IVF) in 1978, assisted reproductive technologies (ARTs) were introduced to offer hope to infertile couples worldwide [1]. Due to ongoing societal changes, shifts in lifestyle, and increasing exposure to environmental pollutants, the use of infertility treatments is expected to rise in coming years [2]. Male factor infertility remains a major contributor to ART cycle failure; thus, in recent decades, the scientific community has increasingly focused on the challenges associated with male infertility [3] .
In the ejaculate, spermatozoa are prevented from undergoing capacitation by decapacitating factors present in the seminal plasma. Prolonged exposure to seminal plasma negatively affects sperm function, impeding their ability to penetrate cervical mucus, initiate the acrosome reaction, and achieve fertilization [4]. Naturally, human sperm reach the oviduct isthmus by active swimming, assisted by passive contractions of the female reproductive tract. In vitro studies have proposed multiple guidance mechanisms for sperm to reach the fertilization site: thermotaxis (migration along a temperature gradient), rheotaxis (movement against fluid flow), and chemotaxis (migration up a chemoattractant gradient) [5]. Therefore, natural selection processes can differentiate spermatozoa quality.
The recovery of a high proportion of high-quality spermatozoa depends on the initial quality of the sperm sample, the selection technique used, and the time and temperature at which the sperm suspension is maintained. Effective sperm preparation methods yield numerous motile spermatozoa with good morphology and quality, which may facilitate replacing more complex intracytoplasmic sperm injection (ICSI) procedures with less invasive options such as IVF or intrauterine insemination (IUI), making treatment more natural and cost-effective. It is equally important to assess sperm DNA integrity after preparation, as sperm with normal morphology may still harbor DNA damage. The principal advantage of the free centrifuge sort (FCS) technique is the complete avoidance of centrifugation, a process known to increase reactive oxygen species (ROS) and cause subcellular injury. Previous research has demonstrated that even brief centrifugation can compromise sperm membrane integrity, disrupt mitochondrial function, and increase DNA fragmentation. In contrast, FCS may help maintain the physiological and genomic integrity of spermatozoa, thereby enhancing their fertilization and implantation potential.
Although traditional density gradient centrifugation is widely used, it is associated with several disadvantages: (1) it differs from the physiological in vivo process; (2) it may contaminate preparations with immature motile cells and leukocytes; (3) it can increase ROS generation and iatrogenic injury; (4) it increases cell–cell contact within the sperm pellet; (5) it often requires repeated centrifugation; and (6) it entails prolonged sample incubation. Centrifugation may raise ROS levels, while removal of seminal plasma eliminates protective antioxidants. An imbalance—excess ROS and insufficient antioxidants—leads to oxidative stress, which diminishes sperm motility, DNA integrity, and viability.
Recently, sperm separation methods based on horizontal migration have been described, which avoid centrifugation and thus prevent DNA damage induced by oxidative stress [6]. Centrifugation and prolonged dropping can yield inconsistent results across studies. While several investigations have assessed the efficacy of conventional sperm selection techniques, there is a lack of systematic reviews examining the relationship between prolonged dropping sperm selection and clinical outcomes. Therefore, the objective of this systematic review and meta-analysis was to evaluate the impact of extended falling sperm selection on ICSI outcomes.
Methods
1. Sources
A prospectively designed protocol adhering to the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) guidelines for meta-analyses was registered with PROSPERO (CRD42023415532). Articles were identified via an extensive search of PubMed, Scopus, Web of Science, and Cochrane Reviews from their inception to February 2025. The search strategy utilized combinations of Medical Subject Headings (MeSH) terms and keywords. The asterisk operator was used to capture variations such as ‘ART,’ ‘centrifuge-free,’ and ‘centrifugation free.’ Additionally, manual searches of reference lists supplemented the database search. Inclusion criteria limited studies to those in English and conducted on humans. Two authors independently (Fatemeh Dehghanpour and Leila Motamedzadeh) reviewed abstracts and selected relevant articles; reference lists were further screened for additional eligible studies.
The primary variables included the fertilization rate (FR; number of fertilized oocytes per number of microinjected oocytes), embryo quality (as reported in the articles), implantation rate (IR; ratio of gestational sacs to embryos transferred), and clinical pregnancy rate (CPR; identification of gestational sac and heartbeat per number of cycles).
2. Study selection
All eligible studies were identified using the population, intervention, comparison/comparator, outcomes, and study design (PICOS) model (Table 1). Only studies comparing the efficacy of sperm selection methods without centrifugation (specifically, extended dropping) against conventional centrifuge-based techniques on ICSI outcomes were included. The outcomes considered were FR, embryo cleavage rate, IR, and CPR after ART.
PICOS inclusion criteria
3. Data extraction
Extracted data included the first author’s name, year of publication, study design, total number of couples, semen characteristics of enrolled male partners (normozoospermic or with altered parameters), and ICSI outcomes (FR, IR, and CPR).
4. Statistical analysis
We calculated odds ratios (ORs) and their 95% confidence intervals (CIs) for fertilization, embryo quality, embryo transfer (ET), implantation, and clinical pregnancy rates (CPRs) to determine the log OR and corresponding standard error for meta-analysis. Summary effects were estimated using a random-effects model, which accounts for between-study variability. Statistical heterogeneity among studies was assessed using the Cochrane Q and I² tests. Sensitivity analyses explored the influence of individual studies on overall effects. Publication bias was evaluated using the Begg test, as well as the Egger regression asymmetry test and the Begg adjusted rank correlation test. Analyses were conducted with STATA ver. 11.2 (STATA Corp.). A p≤0.05 was considered statistically significant.
5. Risk of bias/Quality assessment
The Downs and Black Checklist was used to assess the risk of bias and methodological quality [7]. This tool, with six quality domains and 28 items, is appropriate for systematic reviews. It addresses reporting, external validity, internal validity (bias and confounding), and statistical power. Scores range from 0 to 28, with higher scores indicating better methodological quality. Table 2 presents the characteristics of included studies and patient populations.
Characteristics of the studies included in the systematic review
Results
The search strategy identified 306 records. After excluding 295 duplicates, 50 articles remained for screening. Forty-five studies were excluded due to irrelevant ART outcomes. Ultimately, three articles were included in the analysis (Figure 1, Table 3).
Flowchart of study inclusion. ART, assisted reproductive technology.
Characteristics of the treatment cycles
1. Effect of the FCS method without centrifugation and extended dropping on ART outcomes: a qualitative analysis
Three studies evaluated the effects of FCS methods without centrifugation and extended dropping on ICSI outcomes [6]. Among these, two studies assessed ICSI treatments within randomized controlled trials (RCTs) [6,8], while the remaining study used a sibling oocyte design (Figure 2) [9].
Schematic of three different designs of sperm sorting methods using dropping. (A) The free centrifuge sorting technique. Two 80 μL droplets of 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES)-buffered human tubal fluid medium supplemented with 5 mg/mL human serum albumin (HSA) (designated as droplets A and B) were placed at both ends of a 60 mm Falcon dish, maintaining a minimum distance of 2 cm between them. These droplets were connected by a narrow (approximately 1 mm wide) channel of medium, and a third droplet C of culture medium was added to the plate at a precise distance and angle, aligned with droplets A and B. The outlet droplet C was overlaid with 9 mL of mineral oil (Irvine Scientific) and incubated at 37 °C for 20 minutes. Next, 60 μL of droplets A and B were replaced with 50 μL of semen. The dish was then incubated for 10 minutes at 37 °C. During incubation, the larger volume of droplets A and B and the resulting ‘slope’ facilitated fluid movement towards droplet C. This fluid dynamic blocked the movement of particles, contaminants, and non-sperm cells, so that only progressively motile sperm could swim toward the exit reservoir (droplet C). (B) Droplets A and B (each 80 μL Ham’s F10 medium with 5 mg/mL HSA) were placed at opposite ends of a dish, at least 2 cm apart, and connected by a narrow channel created with a pipette tip. Sixty microliters of droplet A were replaced with 50 μL of liquefied semen, and the system was incubated for 45 minutes. Because of the larger volume of droplet B, fluid flowed from B to A, while motile spermatozoa swam toward droplet B, which served as the sperm collection point (A: semen input point; B: sperm collection point). (C) Schematic design of a ICSI plate. For intracytoplasmic sperm injection (ICSI), the preparation involved three additional 50 μL droplets of G-MOPS® (Vitrolife), connected by a small volume of culture medium using a stripper pipette. Depending on sperm concentration and motility, 1 to 5 μL of ejaculate were injected into the proximal droplet 10 minutes before oocyte presentation in the separated droplets. During ICSI, an adequate number of spermatozoa reached the distal edge of droplet A; some were then recovered with a needle, transferred into a polyvinylpyrrolidone (PVP) droplet, and subsequently selected for injection.
Anbari et al. [8] demonstrated improvements in progressive motility, the proportion of class I sperm morphology, high-quality embryo formation rates, IRs, and CPRs, along with reductions in DNA fragmentation and immotile spermatozoa, when using a practical and accessible microfluidic sperm sorting (MSS) technique compared to the direct swim up (DSU) group. Patients were randomly assigned to two groups for ICSI: one group (n=45) using MSS-prepared sperm, and another group (n=50) using DSU-prepared sperm [8].
Baldini et al. [6] introduced a novel sperm preparation technique based on horizontal sperm migration in injection dishes. In a randomized controlled study involving 1,034 ICSI cycles, they assessed both timing and reproductive outcomes. Couples were divided into two groups: the traditional swim-up method was used in group A, while horizontal sperm migration was conducted in injection dishes. While fertilization, implantation, and CPRs were similar between groups, the rates of cleavage and blastocyst formation were significantly higher in group B [6].
We previously described a sperm sorting system that used sperm rheotaxis in a microfluidic channel where the sperm swim against the fluid flow. Sperm rheotaxis, along with chemotaxis and thermotaxis, is a key factor enabling sperm to navigate the female reproductive tract [10]. The FCS method, when initial sperm counts are high, yields final preparations with superior progressive motility, a higher fraction of class I morphology, and greater mitochondrial membrane potential compared to DSU. Additionally, lipid peroxidation and DNA fragmentation were lower in FCS-prepared sperm. After ICSI, rates of high-quality embryo formation, implantation, and clinical pregnancy were significantly higher in the FCS group.
2. Effects of the FCS method without centrifugation and extended dropping on ART outcomes: a quantitative analysis
1) Fertilization rate
The three included studies, all conducted in patients with altered sperm parameters, reported FRs ranging from 75.4% to 78.9% in the dropping (FCS method) group, and from 72.6% to 80.0% in the conventional method group. The analysis showed no significant differences in FR between groups (odds ratio [OR], 0.985; 95% CI, 0.925 to 1.048; p=0.63) (Figure 3). There was no significant heterogeneity between studies (Cochran’s Q test, p=0.18; I²=42.32%).
2) Implantation rate
The IR was reported in three studies [6,8,9], with values ranging from 24.6% to 48.7% in the dropping (FCS) group, and 20.3% to 32.7% in the conventional control group. Meta-analytic pooling revealed a significant improvement in the IR after sperm processing with the dropping (FCS) method (OR, 1.28; 95% CI, 1.09 to 1.51; p<0.001) (Figure 4). No significant heterogeneity was detected (Cochran’s Q test=0.55, I²=0.00%).
3) Clinical pregnancy rate
The CPR was defined as the number of ultrasound detections of one or more gestational sacs divided by the number of ETs. CPR ranged from 38.9% to 63.0% in the FCS group and from 23.1% to 35.7% in the control group. The analysis showed a statistically significant improvement in the CPR following a very short abstinence period (OR, 1.28; 95% CI, 1.09 to 1.51; p<0.001) (Figure 5). No inter-study heterogeneity was observed (Cochran’s Q test=0.55, I²=0.00%).
Discussion
Due to the increasing number of infertile couples undergoing ART, many studies have focused on the impact of frequent centrifugation during semen processing. One of the primary goals of ART therapy is to implement the most effective sperm-sorting technology. In ART clinics, several technologies, including sperm washing, swim-up, and density gradient centrifugation, are used for sperm separation. Meanwhile, some researchers have advocated for the potential improvement in sperm quality achieved through centrifugation and prolonged dropping procedures [11].
The quality of the prepared sperm suspension influences outcomes in all insemination methods, including IUI, IVF, and ICSI [12]. The recovery of motile spermatozoa after preparation determines the most appropriate insemination procedure. If the sperm suspension contains a high proportion of motile sperm with good morphology and low DNA fragmentation index, more natural techniques such as IUI or IVF are feasible [13]. In contrast, borderline samples or those with male factor infertility are better suited for ICSI [14]. The handling of the sperm sample, the preparation method, and storage temperature affect sperm recovery rates, fertilization ability, DNA integrity, and overall fertilizing capacity [15,16].
The quantitative analysis confirmed our prediction that omitting centrifugation significantly increased the IR and CPR, while no significant changes were observed in the FR. This may suggest that the sperm processing technique does not benefit the paternal contribution in the early steps of fertilization. In contrast, the late paternal effect is characterized by poor embryo development to the blastocyst stage, implantation failure, and pregnancy loss, which are often associated with sperm DNA abnormalities [17]. As demonstrated, higher sperm quality enhances fertilization and embryo quality [18]. This can be particularly advantageous in cases with older female partners. Given the limited DNA repair capacity in oocytes from older women, sperm selection techniques yielding lower DNA damage may be especially beneficial in these instances [19]. Furthermore, in normozoospermic men, a correlation between sperm DNA fragmentation and aneuploidy rates has been identified [20].
Moreover, Ma et al. [21] reported that embryos derived from spermatozoa subjected to minimal centrifugation stress may exhibit improved blastocyst development, reflecting the significance of the paternal contribution to embryo genomic integrity. However, embryo development was not evaluated in the present study [21]. Our findings demonstrate that sperm preparation methods influence both sperm motility and DNA integrity. Embryo development can also be impacted by the DNA quality in the sperm head [22]. It has also been suggested that improved sperm quality is associated with higher embryo quality and increased ongoing pregnancy rates [23]. Additionally, Aitken and Clarkson [24] documented that iatrogenic sperm damage and excessive ROS production can impair fertilizing ability. Superoxide radicals promote peroxidation of sperm plasma membrane phospholipids, and excessive ROS production may also be linked to mitochondrial dysfunction, resulting in lipid peroxidation. This, in turn, may lead to decreased sperm motility, increased DNA fragmentation, and apoptosis [3,25]. Nevertheless, it is essential to recognize that ART outcomes are multifactorial. Factors such as oocyte quality, embryo grading, endometrial receptivity, and hormonal profiles also play critical roles. Although patient demographics were matched between groups in our study, potential confounding variables cannot be entirely excluded. Future RCTs are warranted to confirm the efficacy of FCS as a stand-alone technique and to determine whether its benefits are consistent across different patient populations. Notably, the proposed FCS technique resulted in improved sperm biological characteristics and higher ICSI outcomes compared to the conventional DSU method. The FCS technique eliminates the need for centrifugation and allows for the noninvasive, disposable, easy, and cost-effective separation of high-quality spermatozoa during processing [9].
In terms of evidence quality, the studies included in this meta-analysis were of average methodological rigor. Several limitations should be considered. Only one RCT has been published on this topic, and designing RCTs in this context is challenging, particularly due to difficulties in defining the control group. A further advantage of separating oocytes when sufficient numbers are retrieved is the ability to analyze ART outcomes using spermatozoa prepared by both the traditional and FCS methods within the same patient. This approach minimizes significant female-related biases and allows for a more accurate evaluation of male factors. However, considerable variation was present across the included studies. The retrospective design of several studies, lack of uniform ART protocols, and differences in outcome definitions may contribute to this heterogeneity.
Additional information was obtained from single-study data. Notably, one study differentiated between fresh and frozen-thawed cycles and was therefore included twice in the quantitative analyses for FR, IR, and CPR. Ultimately, sample sizes in the included studies were modest. This review encompassed a variety of study features, patient characteristics, and populations. Furthermore, diverse inclusion and exclusion criteria resulted in a broad range of participant ages and sperm characteristics. Despite these limitations, our aim was to provide a comprehensive qualitative and quantitative assessment through meta-analysis to enhance understanding of FCS efficacy. Overall, our findings suggest that extended dropping sperm selection can improve ICSI outcomes.
Conclusion
This is the first systematic review and meta-analysis to investigate the impact of FCS procedures without centrifugation and prolonged dropping on ART outcomes. Our data show that FCS approaches have a significantly beneficial effect on FR, IR, and CPR. Therefore, refining sperm sorting techniques, alongside comprehensive diagnostic and therapeutic evaluation of infertile couples, may further improve assisted reproduction success rates.
Notes
Conflict of interest
No potential conflict of interest relevant to this article was reported.
Acknowledgments
The authors would like to thank the staff of Yazd Reproductive Science Institute.
Author contributions
Conceptualization: FD. Methodology: FD. Formal analysis: FD, ASA. Data curation: FD. Project administration: FD, LM. Visualization: FD. Software: ASA. Validation: FD, ASA. Investigation: FD. Writing-original draft: FD. Writing-review & editing: FD, MAK, ASA, BJW. Approval of final manuscript: FD, MAK, ASA, BJW, LM.