The use of birefringence for human spermatozoa selection before intracytoplasmic sperm injection to improve artificial reproductive technology outcomes: A systematic review and meta-analysis

Fatemeh Dehghanpour; Ali Nabi; Hossein Fallahzadeh

doi:10.5653/cerm.2024.07745

Clin Exp Reprod Med > Epub ahead of print

Dehghanpour, Nabi, and Fallahzadeh: The use of birefringence for human spermatozoa selection before intracytoplasmic sperm injection to improve artificial reproductive technology outcomes: A systematic review and meta-analysis

Review Article

Clinical and Experimental Reproductive Medicine 2025;cerm.2024.07745.

Published online: August 22, 2025

DOI: https://doi.org/10.5653/cerm.2024.07745

The use of birefringence for human spermatozoa selection before intracytoplasmic sperm injection to improve artificial reproductive technology outcomes: A systematic review and meta-analysis

Fatemeh Dehghanpour¹

, Ali Nabi^1,², Hossein Fallahzadeh³

¹Research and Clinical Center for Infertility, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran

²Andrology Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran

³Research Center of Prevention and Epidemiology of Non-Communicable Disease, Department of Biostatistics and Epidemiology, Shahid Sadoughi University of Medical Sciences, Yazd, Iran

Corresponding author: Fatemeh Dehghanpour Department of Reproductive Biology, School of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
Tel: +98-3538247085 Fax: +98-3538247087 E-mail: dehghanpour64@yahoo.com

Received December 7, 2024 Revised February 15, 2025 Accepted February 28, 2025

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (https://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

In an era where male infertility poses significant challenges for many couples, intracytoplasmic sperm injection (ICSI) is becoming increasingly common. Microscopic evaluation of sperm head morphology can contribute to the development of healthier embryos. A comprehensive literature search was conducted using the PubMed, Scopus, Web of Science, and Cochrane databases. A meta-analysis was performed following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols (PRISMA-P) for randomized controlled trials. Eligible studies were selected based on the PICOS (Population, Intervention, Comparison/Comparator, Outcomes, Study type) model. The pregnancy outcomes considered after artificial reproductive technology (ART) procedures included the fertilization rate (FR), high-quality embryo rate (HQER), implantation rate (IR), and clinical pregnancy rate (CPR). This study was registered on PROSPERO (CRD42023423108). Of 105 records evaluated, three studies met the inclusion criteria. Our analysis indicated that birefringence sperm selection significantly improved HQER and IR after ART, although no significant differences were observed for FR and CPR. The use of birefringence for sperm selection in ICSI cycles may represent a straightforward strategy to improve ART outcomes, particularly for couples with abnormal sperm parameters.

Keywords: Artificial reproductive technology outcomes; Fertilization; Implantation; Sperm head birefringence sperm selection; Sperm injections, intracytoplasmic

Introduction

To produce high-quality embryos, it is important to investigate methods for selecting gametes with optimal quality [1]. This has led to studies of oocyte quality, with evaluations conducted during the intracytoplasmic sperm injection (ICSI) procedure to assess both oocyte morphology and chromosomal status. Similarly, sperm analysis and selection methods have advanced, and a strong relationship exists between sperm parameters and male factor infertility [2].

Selection of spermatozoa based on morphological evaluation of their organelles has progressed, leading to improved pregnancy rates and reduced miscarriage rates. In this approach, sperm evaluation is performed during ICSI using an inverted light microscope equipped with high-power Nomarski optics (Nomarski interference contrast [NIC]) capable of magnifications up to 6,300, ensuring that only sperm with normal morphology and high quality are used for insemination [3].

Through a comparative approach that assesses the organellar organization in both the sperm head (including the core and acrosome) and the tail, the use of polarization microscopy in ICSI has emerged as a modern method for sperm evaluation [4]. This technique is based on observing changes in sperm cell appearance attributable to their unique structural features. The mature sperm nucleus exhibits a strong natural phenomenon known as birefringence, caused by arrays of nucleoprotein filaments arranged in parallel and running lengthwise [5]. Additionally, vertically aligned subacrosomal protein filaments within the mature acrosomal complex contribute to its brightness. Furthermore, the microtubular structure present in the tail tissue of certain organisms can affect light transmission. These structural patterns have been confirmed through transmission electron microscopy studies [4].

The primary advantage of using polarizing and analyzing lenses on an inverted microscope with a micromanipulation system is that it permits the selection of sperm cells with specific light refraction properties for ICSI without compromising their viability or motility. This technique enables the assessment of sperm cell birefringence, providing more detailed information about internal cellular structures than phase-contrast microscopy. Polarized light imparts a characteristic birefringence to the anterior portion of healthy sperm cells and certain segments of the tail due to the unique features of their intracellular organelles [6]. Nucleoprotein and subacrosomal protein filaments are primarily responsible for the birefringence observed in the mature sperm nucleus, influencing its light reflection pattern [7]. Conversely, disorganized structural alterations in dysfunctional sperm cells lead to a loss of these light-bending properties. Based on these findings, the presence of head birefringence is recommended as a technique for selecting intact sperm with minimal risk of DNA damage [8]. Moreover, studies have reported that this phenomenon positively impacts ICSI outcomes [9].

Various methods have been proposed to avoid the injection of nonviable, immotile sperm. These methods include the use of pentoxifylline, assessing sperm swelling in a low-salt solution, employing lasers to select active sperm, utilizing magnets for cell separation, and applying specialized microscopy to evaluate sperm polarization [10].

Research has shown that applying the birefringence phenomenon for human sperm selection yields variable outcomes across different studies. While numerous articles have examined the benefits of traditional sperm selection methods, a comprehensive review exploring the relationship between sperm selection via birefringence and clinical outcomes is needed. Consequently, the purpose of this review is to evaluate how sperm selection based on the birefringence phenomenon influences outcomes in fertility procedures such as ICSI and other artificial reproductive technology (ART) techniques.

Methods

1. Sources

A prospectively prepared protocol following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines was registered with PROSPERO (registration number CRD42023423108). An extensive search of the PubMed, Scopus, Web of Science, and Cochrane Reviews databases was conducted from their inception until June 2023. The search strategy combined several Medical Subject Headings (MeSH) terms and keywords. For ART, the asterisk operator was employed to capture both ‘assisted reproductive technology’ and ‘assisted reproductive technique.’ Similarly, it was used to include both ‘birefringence for human spermatozoa’ and ‘human spermatozoa birefringence.’ Additionally, manual searches were performed using reference lists from the identified studies. Only articles published in English and conducted on human subjects were considered. All abstracts and relevant full texts were evaluated. Two authors (Fatemeh Dehghanpour and Hossein Fallahzadeh) independently reviewed the abstracts and selected articles pertinent to the study’s aim. Any disagreements were resolved through discussion with a third investigator (Ali Nabi). Reference lists of identified articles were also examined for additional relevant studies.

All searches were restricted to human subjects. For each identified article, titles and abstracts were screened, and full texts were retrieved when applicable. The primary variables extracted from these studies included fertilization rate (FR; defined as the number of fertilized oocytes per number of oocytes injected), embryo quality (as reported in the respective articles), implantation rate (IR; defined as the ratio of gestational sacs to embryos transferred), and clinical pregnancy rate (CPR; defined by the appearance of a gestational sac and detection of a heartbeat per cycle).

2. Study selection

Eligible studies were selected based on the PICOS (Population, Intervention, Comparison/Comparator, Outcomes, Study type) model (Table 1). Only studies that compared the efficacy of sperm selection without centrifugation versus with extended dropping on ICSI outcomes—including FR, high-quality embryo rate (HQER), IR, and CPR—were included. Case reports, comments, letters to the editor, systematic or narrative reviews, and studies that did not provide extractable outcomes of interest were excluded from the analysis. Table 2 presents the characteristics of the included articles and patients.

3. Data extraction

The following information was extracted from each study: the first author’s name, year of publication, study design, total number of couples, semen characteristics of the male participants (whether normozoospermic or with altered sperm parameters), and ICSI outcomes (FR, HQER, IR, and CPR) following ART.

4. Statistical analysis

Risk ratios (RR) for FR, embryo quality, embryo transfer rate, IR, and CPR were calculated, and the logarithm of RR along with its corresponding standard error was computed for the meta-analysis. Summary effects were derived using a random-effects model to account for between-study variability (DerSimonian & Laird, 1986) [11]. Statistical heterogeneity among studies was assessed using Cochran's Q and I² tests. Sensitivity analyses were performed to evaluate the influence of individual studies on the overall effect. Publication bias was assessed using Begg's test, and asymmetry was further evaluated with Egger's regression asymmetry test and Begg's adjusted rank correlation test. All analyses were conducted using STATA ver. 11.2 (STATA Corp.), with a p-value of ≤0.05 considered statistically significant.

5. Risk of bias/quality assessment

The Downs and Black Checklist was utilized to evaluate the risk of bias and applicability of the studies (Downs & Black, 1998). This checklist, comprising six quality assessment tools and 27 items, is well-suited for systematic reviews. It addresses methodological aspects including reporting, external validity, internal validity (bias and confounding), and statistical power. Scores range from 0 to 28, with scores closer to 28 indicating higher methodological quality. To ensure rating consistency, each article was independently assessed by two authors (Table 1).

Results

The search strategy initially identified 105 records. After removing 30 duplicate records, 75 articles remained for screening. Forty-five studies were excluded due to irrelevant ART outcomes, and ultimately, three articles were included in the analysis (Figure 1).

1. Effects of sperm selection with birefringence on ART outcomes: qualitative analysis

Three studies assessed the impact of birefringence-based sperm selection on ICSI outcomes, with all three studies conducted as randomized controlled trials (RCTs) [6,12,13].

Ribeiro et al. [13] demonstrated improvements in high-quality embryo formation, IRs, clinical pregnancy per transfer, and clinical pregnancy per initiated cycle using the birefringence sperm selection technique compared to conventional methods. Their study included 181 couples (107 in the conventional group and 74 in the birefringence group) who underwent ICSI between January 2018 and August 2020 [13].

Ghosh et al. [12] selected oocytes with a birefringent meiotic spindle and a zona pellucida thickness of less than 20 µm for ICSI. In their study, 96 ICSI cycles were analyzed and divided into group A (azoospermic, n=58) and group B (complete asthenozoospermic, n=38). Groups A and B were further subdivided into A1, A2 and B1, B2, respectively, based on the type of ICSI performed. In group A1, a motile spermatozoon with normal morphology was injected into a metaphase-II (M-II) oocyte. In group B1, spermatozoa exhibiting tail coiling after a modified hypo-osmotic swelling test were injected into M-II oocytes. In groups A2 and B2, ICSI was performed by injecting a spermatozoon with a birefringent head. Although ICSI outcome measures were higher in group A2 than in group A1, the differences were not statistically significant. However, a significantly higher percentage of Z1 and Z2 zygotes, grade I and grade II embryos, and an increased pregnancy rate were observed in group B2 compared to group B1. The selection of birefringent spermatozoa demonstrated promising results in asthenozoospermic men and in men undergoing testicular sperm aspiration or extraction prior to ICSI [12].

Previously, Gianaroli et al. [6] reported that analyzing birefringence in the sperm head could serve as both a diagnostic tool and a novel method for sperm selection. In their study, birefringent sperm were selected for ICSI in 112 male patients (study group), and the clinical outcomes were compared with those of 119 couples undergoing conventional ICSI (control group). The proportion of birefringent spermatozoa was assessed before and after treatment in relation to overall sperm quality. Normospermic samples exhibited a significantly higher proportion of birefringent spermatozoa compared to oligoasthenoteratospermic samples with no progressive motility and samples obtained via testicular sperm extraction. Although fertilization and cleavage rates did not differ between the study and control groups, in cases of severe male factor infertility (oligoasthenoteratospermic with no progressive motility and testicular sperm extraction), the rates of clinical pregnancy, ongoing pregnancy, and implantation were significantly higher in the study group compared to the controls [6].

2. Effects of sperm selection with birefringence on ART outcomes: quantitative analysis

1) Fertilization rate

The three studies included in the quantitative analysis were all conducted in patients with altered sperm parameters. The FR ranged from 61.29% to 74% in the birefringence (study group) sperm selection group and from 72% to 75.78% in the control group using the conventional method (Table 3). Statistical analysis revealed no significant change in FR following the use of birefringence-based sperm selection (odds ratio [OR], 1.012; 95% confidence interval [CI], 0.784 to 1.305; p=0.928). There was no significant between-study heterogeneity (Cochran's Q test, p=0.000, I²=88.855%), and no evidence of publication bias was detected, as indicated by Egger’s test (p=0.287) and funnel plot symmetry. The meta-analysis was not sensitive to any single study (Figure 2).

2) High-quality embryo rate

The HQER was calculated as the percentage of embryos with grades A and B among the total number of embryos [6,12,13]. This rate was 53.22% to 87% in the birefringence (study group) sperm selection group and 68.75% to 84% in the control group, which used the conventional method (Table 3). The statistical analysis included thawed cycles. HQER significantly improved after sperm selection with birefringence (OR, 1.560; 95% CI, 1.193 to 2.040; p=0.001) (Figure 3). No inter-study heterogeneity was observed in this analysis (χ²=3, I²=0.00%). However, evidence of publication bias was observed, as indicated by Egger’s test (p=0.430) and funnel plot asymmetry (Figure 3).

3) Implantation rate

The IR was reported in two studies [6,13]. In the birefringence (study group) sperm selection group, it ranged from 19% to 54.37%, compared to 11.2% to 42.58% in the control group using the conventional method (Table 3). Meta-analytic pooling of the data indicated a statistically significant improvement in IR after sperm selection with birefringence (OR, 1.870; 95% CI, 1.259 to 2.779; p=0.002) (Figure 4). The Cochran test did not reveal significant heterogeneity (Cochran's Q²=1, I²=0.00%).

4) Clinical pregnancy rate

In these studies, the CPR was defined as the number of ultrasound views confirming one or more gestational sacs divided by the number of embryo transfers. In the birefringence (study group) sperm selection group, the CPR ranged from 11.11% to 67.15%, while in the control group using the conventional method it ranged from 22% to 50.54% (Table 3). Statistical analysis showed no significant change in CPR after sperm selection with birefringence (OR, 0.690; 95% CI, 0.468 to 1.017; p=0.061) (Figure 5). Inter-study heterogeneity was observed in this analysis (χ²=3, I²=89.312%), and evidence of publication bias was indicated by Egger’s test (p=0.621) and funnel plot asymmetry. No individual study was influential enough to alter these results.

Discussion

To our knowledge, this systematic review and meta-analysis is the first to assess the influence of sperm selection with birefringence on ART outcomes. An increasing body of data suggests that difficulties in selecting the most capable spermatozoa for injection into oocytes may contribute to the relatively low efficiency of ICSI. Therefore, one of the priorities in ART treatment is to implement the most efficient sperm selection technique based on the available evidence [14]. Various methods—such as sperm washing, swim-up, and density gradient centrifugation—are used in ART clinics for processing and separation [15]. However, conventional methods for selecting functional and capable spermatozoa have proven insufficient for achieving optimal ICSI success.

In daily laboratory routines, embryologists frequently encounter immotile spermatozoa. Although using randomly selected immotile spermatozoa is technically feasible, choosing only viable immotile spermatozoa is preferable, as it increases success rates in severe cases. Consequently, rapid and straightforward laboratory methods to distinguish between dead and viable, albeit immotile, spermatozoa are necessary.

In recent decades, the growing number of infertile couples undergoing ART has led to numerous studies investigating how sperm head birefringence under polarized light can assess sperm health and morphology without compromising viability or motility. Gianaroli et al. [6] investigated sperm head birefringence as a criterion for sperm selection prior to ICSI. Other methods, such as hyaluronic acid binding [16] and sperm morphology (intracytoplasmic morphologically selected sperm injection), have been used for sperm selection prior to ICSI [17]. In parallel, some researchers have argued that sperm selection using birefringence may further improve sperm quality [18]. Advanced sperm selection techniques are thought to enhance the likelihood of selecting structurally intact, mature sperm with high DNA integrity for fertilization [7,19]. Our quantitative analysis supports this hypothesis by showing that a centrifuge-free sperm selection method significantly increased HQER and IR in couples undergoing ART, while no significant changes were found for FR and CPR. This may suggest that while the sperm selection technique does not impact the paternal contribution during the early stages of fertilization, it may help mitigate late paternal effects—such as poor embryo development to the blastocyst stage, implantation failure, and pregnancy loss—that are associated with sperm chromatin abnormalities [11,20]. In contrast, improved sperm quality has been associated with enhanced FRs and embryo quality [21]. This is particularly valuable for infertile couples with older female partners, where selecting spermatozoa with lower rates of DNA damage can be beneficial given the reduced DNA repair capacity of oocytes from older women [22,23]. Furthermore, a direct relationship between sperm DNA fragmentation and aneuploidy rates has been observed in normozoospermic men [24].

The studies included in this meta-analysis were considered to be of moderate quality based on the evidence assessment. Nonetheless, several limitations should be acknowledged. First, only one RCT has been conducted on this topic. Designing an RCT in this field is challenging because defining an appropriate control group is difficult. One advantage of splitting the oocytes—when retrieved in sufficient numbers—to assess ART outcomes using spermatozoa selected by the birefringence method is that comparisons can be made between fertilized oocytes using conventional and birefringence selection methods from the same woman. This approach helps eliminate significant biases related to the female factor and reduces biases from the male component. Moreover, substantial heterogeneity was found among the included studies, which may be explained by the retrospective design of many studies, a lack of standardization in ART procedures, and varying definitions of ART outcomes. Additionally, data from one study were used twice in the quantitative analysis for FR, IR, and CPR because the authors differentiated between fresh and frozen-thawed cycles. Finally, the included studies had relatively small sample sizes. Other limitations include the inclusion of studies with varying qualities, characteristics, and populations, as well as broad inclusion and exclusion criteria regarding participant ages and sperm characteristics. Despite these limitations, this study provides a comprehensive qualitative evaluation via meta-analysis to enhance our understanding of the efficacy of birefringence sperm selection methods in improving ICSI outcomes. Numerous confounding factors that may influence our meta-analysis results were noted.

This is the first systematic review and meta-analysis to evaluate the effects of sperm selection with birefringence on ART outcomes. Notably, our analysis demonstrated a significantly positive impact of birefringence sperm selection methods on FR, HQER, IR, and CPR. With the widespread use of ART, improving sperm quality is essential for achieving better success rates. Therefore, optimizing sperm sorting in combination with appropriate diagnostic and therapeutic workups for infertile couples prior to ART can help improve overall outcomes.

For ICSI cycles, these findings suggest that sperm selection using birefringence methods—especially in cases of teratozoospermia—prior to oocyte fertilization can enhance ART outcomes.

Conflict of interest

No potential conflict of interest relevant to this article was reported.

Author contributions

Conceptualization: FD, HF. Methodology: FD, AN, HF. Formal analysis: FD, HF. Data curation: FD, HF. Project administration: FD, AN. Visualization: FD, AN. Software: FD. Validation: FD, AN. Investigation: FD, AN. Writing-original draft: FD, HF. Writing-review & editing: FD, AN. Approval of final manuscript: FD, AN, HF.

Figure 1.

The main characteristics of the three studies included in the systematic review and meta-analysis. ART, artificial reproductive technology.

Figure 2.

Forest plot showing the effects of selection with birefringence on the fertilization rate [6,12,13]. CI, confidence interval.

Figure 3.

Forest plot showing the effects of selection with birefringence on the high-quality embryo rate [6,12,13]. CI, confidence interval.

Figure 4.

Forest plot showing the effects of effects of selection with birefringence on the implantation rate [6,13]. CI, confidence interval.

Figure 5.

Forest plot showing the effects of sperm selection with birefringence on the clinical pregnancy rate [6,12,13]. CI, confidence interval.

Table 1.

PICOS (Population, Intervention, Comparison/Comparator, Outcomes, Study type) inclusion criteria

	Inclusion	Exclusion
Population	Couples who underwent assisted reproduction techniques	-
Intervention	Sperm selection using the birefringence technique	Sperm selection using the conventional method
Comparison	Conventional method	-
Outcome	ICSI outcome: FR, IR, CPR, and HQER	-
Study type	Observational, cohort, cross-sectional, case-control, randomized controlled trials	Case reports; comments; letters to the editor; systematic or narrative reviews; in vitro studies; studies on animals

ICSI, intracytoplasmic sperm injection; FR, fertilization rate; IR, implantation rate; CPR, clinical pregnancy rate; HQER, high-quality embryo rate.

Table 2.

Characteristics of the studies included in the systematic review

Study	Inclusion criteria	Exclusion criteria	No. of patients or oocytes injected per patient in the related group		Semen quality		Downs and black score
Study	Inclusion criteria	Exclusion criteria	Birefringence	Non-birefringence	Birefringence	Non-birefringence	Downs and black score
Gianaroli et al. (2008) [6]	NM	Female obesity, diabetes, and polycystic ovary	112 Patients undergoing assisted conception cycles (85 with fresh oocytes and 27 with thawed oocytes)	119 Patients undergoing assisted conception cycles (90 with fresh oocytes and 29 with thawed oocytes)	Normospermic (n=9)	Normospermic (n=9)	20
					Oligoasthenoteratospermic with progressive motility (n=46)	Oligoasthenoteratospermic with progressive motility (n=53)
					Oligoasthenoteratospermic without progressive motility (n=43)	Oligoasthenoteratospermic without progressive motility (n=44)
					Testicular sperm retrieved by testicular sperm extraction (TESE) (n=14)	Testicular sperm retrieved by testicular sperm extraction (TESE) (n=13)
Ghosh et al. (2012) [12] ^a)Group A	Female	NM	In Group A2, of 221 oocytes, 174 M-II oocytes injected with testicular sperm aspiration or extraction of spermatozoa showing birefringent sperm head	In Group A1, of 165 oocytes, 126 M-II oocytes were injected with testicular sperm aspiration or extraction of spermatozoa selected were injected with motile spermatozoa or with spermatozoa having normal morphological appearance	Group A2 (testicular sperm aspiration or extraction selected by birefringent sperm head) (n=30)	Group A1 (testicular sperm aspiration or extraction selected by the conventional method) (n=24)	21
Ghosh et al. (2012) [12] ^a)Group A	<37 years of age, euthyroid, normogonadotropic and normoprolactinemic	NM
Ghosh et al. (2012) [12] ^b)Group B	Female	NM	In Group B2, of 170 oocytes, 128 M-II oocytes injected with spermatozoa showing birefringent sperm head	In Group B1, of 158 oocytes, 124 M-II oocytes injected with	Group B2 (asthenozoospermia selected by birefringent sperm head) (n=20)	Group B1 (asthenozoospermia selected by the conventional method) (n=18)
Ghosh et al. (2012) [12] ^b)Group B	<37 years of age, euthyroid, normogonadotropic and normoprolactinemic	NM		spermatozoa selected with motile spermatozoa or with spermatozoa having normal morphological appearance
Ribeiro et al. (2023) [13]	Couples with women under 37 years old who underwent ICSI using their own oocytes and fresh semen samples obtained from the ejaculate	Patients who had more than three previous unsuccessful cycles, who used cryopreserved gametes, who received preimplantation genetic testing on embryos (embryo biopsy)	Of 181 patients, 74 patients were stratified into the birefringence group	Of 181 patients, 107 patients were stratified into the conventional group	Semen parameters:	Semen parameters:	19
					Volume: 2.71±1.51	Volume: 2.73±1.33
					Concentration/mL: 49.71±18.21	Concentration/mL: 23.62±20.38
					Progressive motility: 14.80±14.84	Progressive motility: 50.09±20.53
					DNA fragmentation (%): 37.17±14.09	DNA fragmentation (%): 16.95±10.78

NM, not mentioned; M-II, metaphase-II.

^a)Group A: Azoospermia (testicular sperm aspiration or extraction, including percutaneous epididymal sperm aspiration);

^b)Group B: Complete asthenozoospermia

Table 3.

Characteristics of the treatment cycles

Study	Fertilization rate^a)		High-quality embryo rate^b)		Implantation rate^c)		Clinical pregnancy rate^d)
Study	Birefringence	Non-birefringence	Birefringence	Non-birefringence	Birefringence	Non-birefringence	Birefringence	Non-birefringence
Gianaroli et al. (2008) [6]	74.13 (235/317)	72.51 (248/342)	87.25 (178/204)	84.28 (177/210)	19.02 (35/184)	11.22 (22/196)	30.69 (31/101)	20.19 (21/104)
Ghosh et al. (2012) [12] ^e)Group A	71.42 (90/126)	74.71 (130/174)	63.49 (80/126)	71.26 (124/174)	NM	NM	33.33 (8/24)	46.66 (14/30)
Ghosh et al. (2012) [12] ^f)Group B	61.29 (76/124)	75.78 (97/128)	53.22 (66/124)	68.75 (88/128)	NM	NM	11.11 (2/18)	45 (9/20)
Ribeiro et al. (2023) [13]	7.74±4.79^g)	7.85±4.83^g)	7.56±4.40^h)	7.38±4.68^h)	54.37 (56/103)	42.58 (66/155)	67.16 (45/67)	50.54 (47/93)

Values are presented as percentage (number/total number) or mean±standard deviation.

NM, not mentioned.

^a)Defined as two pronuclei (2PN) divided by the number of metaphase-II oocytes;

^b)Defined as high-quality embryos (grade A and B embryos) divided by the number of embryos;

^c)Defined as the number of gestational sacs observed divided by the number of transferred embryos;

^d)Clinical pregnancy rate per transfer;

^e)Group A: Azoospermia (testicular sperm aspiration or extraction);

^f)Group B: Complete asthenozoospermia;

^g)Number of fertilized oocytes;

^h)Number of cleaved embryos.

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