Scholarly article on topic 'The efficiency of conventional microscopic selection is comparable to the hyaluronic acid binding method in selecting spermatozoa for male infertility patients'

The efficiency of conventional microscopic selection is comparable to the hyaluronic acid binding method in selecting spermatozoa for male infertility patients Academic research paper on "Health sciences"

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{"acridine orange fluorescence test" / "hyaluronic acid selection" / PVP-ICSI / "sperm DNA integrity" / "sperm selection"}

Abstract of research paper on Health sciences, author of scientific article — Meng-Ting Huang, Robert Kuo-Kuang Lee, Chung-Hao Lu, Ying-Jie Chen, Sheng-Hsiang Li, et al.

Abstract Objective To evaluate if hyaluronic acid (HA)-bound spermatozoa surpassed conventional microscopy-selected spermatozoa in the status of sperm DNA integrity by acridine orange (AO) fluorescence staining. Materials and methods Spermatozoa obtained from couples with indication for the intracytoplasmic sperm injection (ICSI) procedure due to male infertility (n = 34) and control males with normal sperm parameters (n = 12) were analyzed using AO fluorescence staining after density-gradient centrifugation (DGC), polyvinylpyrrolidone (PVP)-microscopic selection, and HA-binding selection to determine sperm DNA integrity. Results Percentages of DNA intact spermatozoa with green fluorescence were significantly higher in both PVP-microscopic selected spermatozoa (82.1 ± 24.0%) and HA-bound spermatozoa (83.9 ± 21.1%) than in spermatozoa prepared by DGC (66.8 ± 24.0%). However, there was no significant difference between the PVP-sperm and HA-sperm groups. When the percentage of green fluorescent spermatozoa prepared by DGC fell initially below 68%, both PVP-microscopic and HA-binding selection failed to select over 90% spermatozoa with intact DNA for ICSI in the male infertility group. Compared to control males with normal sperm parameters (99.3 ± 1.8%), the proportion of green fluorescence sperm after HA-binding selection from couples with male infertility (83.9 ± 21.1%) did not reach the range of > 99% reported by Yagci et al. Conclusion The percentages of DNA intact spermatozoa between the PVP-sperm and HA-sperm groups were not significantly different. In an ICSI procedure, a well-trained embryologist will have the same ability to choose sperm with intact DNA by conventional microscopic selection as with HA-bound spermatozoa selection.

Academic research paper on topic "The efficiency of conventional microscopic selection is comparable to the hyaluronic acid binding method in selecting spermatozoa for male infertility patients"

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Taiwanese Journal of Obstetrics & Gynecology

journal homepage: www.tjog-online.com

Original Article

The efficiency of conventional microscopic selection is comparable to the hyaluronic acid binding method in selecting spermatozoa for male infertility patients

CrossMark

Meng-Ting Huang a'1, Robert Kuo-Kuang Lee a'b'c'1, Chung-Hao Lu b, Ying-Jie Chen a, Sheng-Hsiang Li b'd, Yuh-Ming Hwu a'b'd'e' *

a Department of Obstetrics and Gynecology, Mackay Memorial Hospital, Taipei, Taiwan b Department of Medical Research, Mackay Memorial Hospital, Taipei, Taiwan c Department of Obstetrics and Gynecology, Taipei Medical University, Taipei, Taiwan d Mackay Medicine, Nursing and Management College, Taipei, Taiwan e Mackay Medical College, Taipei, Taiwan

ARTICLE INFO

Article history: Accepted 7 March 2014

Keywords:

acridine orange fluorescence test hyaluronic acid selection PVP-ICSI

sperm DNA integrity sperm selection

ABSTRACT

Objective: To evaluate if hyaluronic acid (HA)-bound spermatozoa surpassed conventional microscopy-selected spermatozoa in the status of sperm DNA integrity by acridine orange (AO) fluorescence staining. Materials and methods: Spermatozoa obtained from couples with indication for the intracytoplasmic sperm injection (ICSI) procedure due to male infertility (n = 34) and control males with normal sperm parameters (n = 12) were analyzed using AO fluorescence staining after density-gradient centrifugation (DGC), polyvinylpyrrolidone (PVP)-microscopic selection, and HA-binding selection to determine sperm DNA integrity.

Results: Percentages of DNA intact spermatozoa with green fluorescence were significantly higher in both PVP-microscopic selected spermatozoa (82.1 ± 24.0%) and HA-bound spermatozoa (83.9 ± 21.1%) than in spermatozoa prepared by DGC (66.8 ± 24.0%). However, there was no significant difference between the PVP-sperm and HA-sperm groups. When the percentage of green fluorescent spermatozoa prepared by DGC fell initially below 68%, both PVP-microscopic and HA-binding selection failed to select over 90% spermatozoa with intact DNA for ICSI in the male infertility group. Compared to control males with normal sperm parameters (99.3 ± 1.8%), the proportion of green fluorescence sperm after HA-binding selection from couples with male infertility (83.9 ± 21.1%) did not reach the range of > 99% reported by Yagci et al.

Conclusion: The percentages of DNA intact spermatozoa between the PVP-sperm and HA-sperm groups were not significantly different. In an ICSI procedure, a well-trained embryologist will have the same ability to choose sperm with intact DNA by conventional microscopic selection as with HA-bound spermatozoa selection.

Copyright © 2014, Taiwan Association of Obstetrics & Gynecology. Published by Elsevier Taiwan LLC. All

rights reserved.

Introduction

The quality of the oocyte and spermatozoon influences subsequent fertilization and embryo development. As spermatogonia transform into mature spermatozoa, a complex process involving meioses and mitoses occurs. Aberrant chromatin packaging and

* Corresponding author. Department of Obstetrics and Gynecology, Mackay Memorial Hospital, Number 92, Section 2, Chung-Shan North Road, Taipei, Taiwan. E-mail address: hwu4416@yahoo.com.tw (Y.-M. Hwu).

1 These authors contributed equally to this work.

defective apoptosis during spermatogenesis [1,2], high levels of reactive oxygen species (ROS) produced by immature spermatozoa [3], and environmental toxicants [4] all result in sperm DNA damage. As soon as spermatozoa with damaged DNA enter the oocytes, fertilization may lead to defects in embryonic development, implantation failure, or unexplained pregnancy loss [5—8]. In the era of assisted reproductive technologies (ART), intracytoplasmic sperm injection (ICSI) has clearly overshadowed conventional in vitro fertilization (IVF) procedures and become the ultimate option for the treatment of severe male factor infertility. This technology bypasses the barriers of natural selection by introducing an

http://dx.doi.org/10.1016/j.tjog.2014.11.006

1028-4559/Copyright © 2014, Taiwan Association of Obstetrics & Gynecology. Published by Elsevier Taiwan LLC. All rights reserved.

apparently intact motile spermatozoon into the ooplasm and may transport the DNA-damaged sperm to the oocyte. Furthermore, males with fertility problems show a higher rate of chromosomal abnormalities [9] and often need ICSI treatment. However, the shape of spermatozoa does not predict the absence of chromosomal aberrations [10].

In the natural fertilization process, hyaluronic acid (HA) in the extracellular matrix of the cumulus oophorus complex surrounding the oocyte seems to be a "physiological" selector of mature spermatozoa. In vitro it has been demonstrated that only mature spermatozoa that have completed plasma membrane remodeling, cytoplasmic extrusion, and nuclear maturity are able to bind with HA because they have a high density of HA receptors [11,12]. These HA-bound spermatozoa show low frequency of chromosomal an-euploidies and DNA fragmentation, have good nuclear morphology, and result in good embryo quality [9,13]. Hence, sperm selection with the HA binding method prior to ICSI may help optimize the outcome of ART.

Sperm DNA damage can be evaluated in a variety of ways, including the in situ nick translation (ISNT) assay, terminal deoxy-nucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, single cell gel electrophoresis (COMET) assay, sperm chro-matin dispersion (SCD) test, aniline s (AB) test, chromomycin-A3 (CMA3) staining, and acridine orange (AO) fluorescence by microscopy or flow cytometry (sperm chromatin structure assay, SCSA). In most infertility laboratories, methods requiring expensive equipment like flow cytometry and dedicated software are often unavailable. The AO fluorescence that is based on the principle that damaged DNA denatures much faster than undamaged DNA when subjected to stresses such as heat and pH changes distinguishes sperm with intact double-stranded DNA (green fluorescence) or single-stranded DNA (red fluorescence) [14]. The AO fluorescence staining is a simple rapid microscopic procedure introduced as the AO test (AOT) by Tejada et al [15] and has been widely used for evaluating male infertility [16].

The objective of the present investigation was to evaluate if HA-bound spermatozoa surpassed conventional microscopy-selected spermatozoa in terms of sperm DNA integrity by AO fluorescence staining in the patient with male infertility.

Materials and methods

Patient selection

Thirty-four couples with indication for ICSI procedure due to male infertility were enrolled. Semen samples for investigation were obtained only after the ICSI procedure was completed. Twelve semen samples with normal parameters that were collected from husbands of infertile couples referred for semen analysis were considered the control group. The experiments were started after the semen analysis was completed. The use of discarded spermatozoa was approved by the Institutional Review Board and all participants provided informed consent.

Sperm preparation

Forty-six semen samples were collected by masturbation after 2—3 days abstinence. After liquefaction for at least 30 minutes, routine semen analysis was conducted. The motility and concentration of spermatozoa were evaluated according to the 2010 World Health Organization (WHO) criteria, while sperm morphology was assessed according to Kruger's strict criteria. The semen samples were processed using density-gradient centrifugation (PureSperm 40/80, Nidacon, Sweden). After the ICSI procedure, the remaining semen was collected for the experiments.

Experimental design

The 46 collected spermatozoa that were processed with Pure-Sperm 40/80 were labeled as Pure-sperm. Twenty spermatozoa were chosen from Pure-sperm by a well-trained embryologist using conventional polyvinylpyrrolidone (PVP, SAGE Media, USA)-ICSI procedures and labeled as PVP-sperm. For HA selection of mature spermatozoa, the PICSI dish (PICSI Sperm Selection Device; Mid-Atlantic Diagnostics Inc.) with three micro-dots of HA attached to the interior bottom were used. Approximately 10—20 mL of pure sperm suspension was applied to one micro-dot and incubated for at least 30 minutes. The same embryologist selected 20 HA-bound spermatozoa (HA-sperm) that exhibited no progressive migration despite a vigorous tail cross-beat frequency in addition to intact morphology under a microscope (Olympus SZX7, Tokyo, Japan).

Sperm fixation for AO fluorescence

The Pure-sperm, PVP-sperm, and HA-sperm of each sperm sample were smeared respectively on three precleaned glass slides and air-dried. Carnoy's solution (3:1 ratio of methanol and glacial acetic acid) was used to fix the sperms for 6 hours, which were then stained with freshly prepared 0.19 mg/mL AO (Acridine Orange, Sigma, Aldrich, USA) stain (10 mL of 1% AO in distilled water added to a mixture of 40 mL of 0.1 M citric acid and 2.5 mL of 0.3 M Na2HPO4, 2H2O; Polysciences, Warrington, PA) for 5 minutes in a dark room. Subsequently, the glass slides were rinsed with distilled water, covered with glass cover slips, and immediately evaluated under an epifluorescence microscope (Olympus BX40) equipped with an Olympus DP-70 digital camera at an excitation wavelength of 450—490 nm.

Each slide with 20 sperms was assessed by the same examiner within 40 seconds. Spermatozoa showing green fluorescence were scored as containing intact DNA, whereas those displaying yellow/ orange to red fluorescence were considered as having denatured or relaxed DNA induced by citric acid (Fig. 1). The percentage of spermatozoa with green fluorescence was then recorded.

Statistical analysis

The nonparametric Friedman test was used to examine the differences in the percentages of green fluorescence spermatozoa in Pure-sperm, PVP-sperm, and HA-sperm groups. Statistical significance was set at p < 0.05. All data analyses were performed with MedCalc Version 10.2.0.0.

Results

Characteristics and outcomes of ART

Thirty-four couples with indication for the ICSI procedure due to male infertility were included, but 10 failed to participate in the ICSI procedures due to economic considerations or hesitation regarding the procedure itself, and received conventional IVF treatments instead. Data on ART and clinical outcomes were described in Table 1. The mean age of the females was 35.0 ± 4.1 years (mean ± standard deviation, SD) at oocyte retrieval and the mean number of eggs retrieved was 11.0 ± 7.4. After the ICSI procedure, 71.1% of oocytes were fertilized and 25.2% of embryos were graded as good-quality embryos. The mean number of embryos transferred was 2.8 ± 1.0 and the implantation rate was 24.2%. Clinical pregnancy was defined by ultrasonographic visualization of the gesta-tional sac and the clinical pregnancy rate per embryo transfer cycle reached 41.7% in this series. However, the spontaneous abortion rate was 20%.

Fig. 1. AO fluorescence staining of (A,B) Pure-sperm, (C,D) PVP-sperm, and (E,F) HA-sperm. Spermatozoa showing green fluorescence (arrowhead) were scored as containing intact DNA, whereas those displaying yellow/orange (arrows) to red (thick arrow) fluorescence were considered as having denatured DNA. The left column showed sperm obtained from couples with indication for an ICSI procedure due to male infertility; the right column was from control men with normal sperm parameters. AO = acridine orange; HA = hyaluronic acid; ICSI = intracytoplasmic sperm injection; PVP = polyvinylpyrrolidone.

Summary statistics of semen samples

Data regarding semen characteristics before density-gradient centrifugation (DGC) were recorded before the following experiments. The 34 semen samples that showed few spermatozoa, poor motility, or morphology were counted repeatedly. In the oligo-zoospermia group (sperm concentration < 15 x 106 per mL), the mean sperm concentration was 6.8 x 106/mL. The total sperm motility in the asthenozoospermia group (total motility < 40%) showed 29.2%. Only 2.5% sperm had normal appearance according to Kruger's strict criteria in the teratozoospermia group (normal forms < 4%).

AO fluorescence staining

The mean percentage (± SD) of spermatozoa with green fluorescence was significantly higher in both PVP-sperm (82.1 ± 24.0%) and HA-sperm (83.9 ± 21.1%) samples than in Pure-sperm (66.8 ± 24.0%) samples in 34 patients with male infertility

(Table 2). The mean proportion of green fluorescent spermatozoa selected by an embryologist after HA-binding was only 83.9%, which was different from the range > 99% reported by Yagci et al [17]. In their experiments on 50 semen samples from husbands of infertile couples referred for semen analysis, there was an almost exclusive presence of spermatozoa with green fluorescence in the regions of the HA spot [17]. The study group was males tested for fertility, therefore the focus was not on male infertility in their study. As for the semen samples collected from couples with indication for the ICSI procedure due to male infertility in the present study, HA-bound sperm selection did not reached the same results.

Thus, another 12 semen samples with normal sperm parameters according to the WHO and Kruger's strict criteria that had been collected for semen analysis from husbands in infertile couples were studied. The mean percentage (± SD) of spermatozoa with green fluorescence in Pure-sperm, PVP-sperm, and HA-sperm was 77.9 ± 12.6%, 98.8 ± 3.0%, and 99.3 ± 1.8%, respectively (Fig. 1). The proportion of sperm samples with green fluorescence that were

Table 1

Data on ART and clinical outcomes in ICSI cycles.

Number of patients 34

Number of cycles 24

Age of women (y, mean ± SD) 35.0 ± 4.1

Number of eggs retrieved (mean ± SD) 11.0 ± 7.4

Fertilization rate (%) 71.1 (143/201)

Good-quality embryo rate (%) 25.2 (34/135)

Number of embryos transferred (mean ± SD) 2.8 ± 1.0

Implantation rate (%) 24.2 (16/66)

Clinical pregnancy rate per ET (%) 41.7 (10/24)

Abortion rate (%) 20 (2/10)

Ongoing pregnancy rate per ET (%) 33.3 (8/24)

ART = assisted reproductive technology; ET = embryo transfer; ICSI = intracytoplasmic sperm injection; SD = standard deviation.

selected by HA-binding assay was > 99% only in the control group with normal sperm parameters.

An interesting phenomenon was that the percentage of green fluorescent spermatozoa in either the PVP-sperm or HA-sperm groups never exceeded 90% when the Pure-sperm with green fluorescence was initially < 68%, but always > 90% when green fluorescent sperm was > 68% in Pure-sperm (Fig. 2). Hence, three class intervals (> 80%, 68-79%, and < 67% green sperm of Pure-sperm for subgroup A, subgroup B, and subgroup C, respectively) were created. The mean percentage (± SD) of green fluorescent spermatozoa in the Pure-sperm, PVP-sperm, and HA-sperm groups was 87.6 ± 4.4%, 97.2 ± 4.2%, and 98.4 ± 3.2%, respectively, in subgroup A; 74.6 ± 3.2%, 96.1 ± 4.6%, and 95.4 ± 4.8%, respectively, in subgroup B; and 42.3 ± 20.8%, 58.5 ± 23.9%, and 62.5 ± 19.8%, respectively, in subgroup C. In the three subgroups and the total group, percentages of green fluorescent spermatozoa increased significantly in the PVP-sperm and HA-sperm groups compared to the Pure-sperm group (p < 0.05). The percentage of green fluorescent spermatozoa between the PVP-sperm and HA-sperm groups was not significantly different (p > 0.05).

Table 2

Results of AO fluorescence in semen samples (n = 34) in three subgroups.

Number of patients

Green fluorescence sperm (%) Pure-sperm PVP-sperm HA-sperm

All semen samples 34 66.8 t 24.0a 82.1 t 24.0b 83.9 t :21.1c

(5.0— 93.0) (8.0— 100.0) (10.0— 100.0)

Subgroup A: 12 87.6 t 4.4a 97.2 t 4.2b 98.4 t 3.2c

Pure-sperm with (80.0 —93.0) (90.0 —100.0) (90.0— 100.0)

green sperm > 80% t 4.6b

Subgroup B: 9 74.6 t 3.2a 96.1 95.4 t 4.8c

Pure-sperm with (70.0 —79.0) (90.0 —100.0) (90.0— 100.0)

green sperm 68—79% t 23.9b

Subgroup C: 13 42.3 t 20.8a 58.5 62.5 t 19.8c

Pure-sperm with (5.0— 67.0) (8.0— 86.0) (10.0— 82.0)

green sperm < 67%

AO fluorescence, which is based on the principle that damaged DNA denatures much faster than undamaged DNA when subjected to stresses, distinguishes sperm with intact double-stranded DNA (green fluorescence) or single-stranded DNA (red fluorescence).

Data are expressed as mean ± SD (range).

a,b p < 0.0001, 0.0003, < 0.0001, and 0.0014, PVP-sperm versus Pure-sperm in each group.

a,c p < 0.0001, < 0.0001, < 0.0001, and 0.0008, HA-sperm versus Pure-sperm in each group.

b,c p = 0.568, 0.867,1.0, and 0.555, HA-sperm versus PVP-sperm in each group. AO = acridine orange; HA = hyaluronic acid; PVP = polyvinylpyrrolidone; SD = standard deviation.

Fig. 2. Comparison of percentages of green sperm among Pure-sperm, PVP-sperm, and HA-sperm. Each line represented an individual subject. HA = hyaluronic acid; PVP = polyvinylpyrrolidone.

Discussion

In the present study, microscopic sperm selection in PVP or by HA-binding selection both showed a significant improvement in the percentage of spermatozoa with intact DNA compared with Pure-sperm. The lack of statistical difference between these two sperm selection methods is demonstrated. In laboratory practice, an embryologist conducts spermatozoa evaluation and selection to minimize the chromosomal aberrations for subsequent ICSI. PVP, which helps immobilize spermatozoa, is most often used in conventional ICSI procedures even though its safety is still debatable [18,19]. Because of this, HA, a naturally occurring glycosamino-glycan, has been introduced. The oocyte can metabolize HA that may be carried with the selected spermatozoa within the ICSI pipette without any adverse effects on fertilization or embryo development [20].

Sperm DNA damage is mostly because of the adverse effects of ROS during spermatogenesis and sperm transport. AO fluorescence staining measures acid-induced DNA denaturation. Sperm DNA with abnormal chromatin structure is more prone to denaturation by heat or low pH in the nuclei than that with normal chromatin. Thus, AO fluorescence staining detects potential DNA damage in terms of susceptibility to DNA denaturation, while the TUNEL and COMET assay detect the actual, existing DNA fragmentation [21,22]. This potential damage of sperm DNA is similar to the mechanism whereby exposure of mature sperm to ROS in vitro induces significant DNA damage, resembling the condition during the process of sperm transport and fertilization.

SCSA using flow cytometry to measure the susceptibility of sperm nuclear DNA to acid-induced denaturation is considered as the gold-standard test [23]. Tejada et al [15] introduced the simplified fluorescent microscopic method of AOT in 1984, which does not require the expensive instrumentation of flow cytometry and is convenient for clinical practice. However, even though AOT is relatively fast compared to SCSA, which is more time-consuming, its use for assessing sperm DNA integrity is still controversial [23,24]. The methods of single sperm selection in this study mimic the ICSI procedure and have the potential for clinical application.

As regards selection of spermatozoa with high DNA integrity, Liu and Baker [25] showed that spermatozoa with double-stranded DNA bind to the human zona pellucida (ZP), while sperm with single-stranded or denatured DNA generally do not bind to the ZP.

The sperm selection properties of HA are similar to those of the ZP. Previous studies have shown clearly that HA helps to select sperm with low DNA fragmentation or high DNA chain integrity as determined by either the SCD test, TUNEL assay, or AO fluorescence by microscopy [13,17,26,27].

Since HA-bound spermatozoa have exhibited significantly less DNA fragmentation, the present study attempted to compare the DNA integrity between conventional PVP and HA-mediated ICSI sperm selection by AO fluorescence staining. To mimic the clinical practice of ICSI procedures in the IVF laboratory, all spermatozoa were selected under microscopy using an ICSI pipette and then stained with AO fluorescence to determine the percentage of sperm with intact DNA. The result showed that both conventional PVP selection and HA-bound methods had the ability to choose more sperms with intact DNA from Pure-sperm. When the percentage of green fluorescent spermatozoa in Pure-sperm prepared by DGC was > 68%, the percentage of spermatozoa with intact DNA selected by either conventional microscopy or HA-binding methods was expected to be > 90%. By contrast, when the percentage of green fluorescent spermatozoa in Pure-sperm prepared by DGC was < 68%, the screening efficiency dropped drastically. In other words, both conventional microscopy and HA binding selection provide limited assistance in choosing sperm with intact DNA for ICSI. The assessment of sperm DNA status with AO fluorescence staining in addition to routine semen analysis may be helpful for predicting the DNA integrity of selected sperm prior to ICSI procedures.

As regards the influence of sperm DNA damage on ART outcome, some studies reported a negative effect on fertilization, embryo quality, and pregnancy rates [28—32], while others indicated no detectable effect on pregnancy rate but an increased risk of pregnancy loss [33—35]. In a previous study, neither the DNA fragmentation index (DFI) or the high DNA stainability (HDS) SCSA variables were valuable for predicting embryo quality, fertilization rate, or pregnancy rate. In addition, an obvious relationship between HDS and spontaneous abortion rate in IVF but not in ICSI was observed, indicating that the selection of sperm with normal morphology for ICSI might reduce the potential adverse effects of sperm DNA damage on ART outcome [36]. This also supported the data that microscopic sperm selection in PVP was efficient in decreasing the proportion of spermatozoa with an abnormal chromatin structure for ICSI.

According to Yagci et al [17], the proportion of sperm with green AO fluorescence was 54.9% and 99.1% in semen from husbands in infertile couples who were referred for semen analysis and the regions of the HA spot, where the HA-selected sperm were bound, respectively. Thus, they concluded that the HA-mediated sperm selection reflected the proportion of spermatozoa with high DNA integrity and possessed potential advantages for ICSI treatments. However, semen samples in their study were not collected strictly from male infertility patients. In the present study, the spermatozoa with green AO fluorescence had 77.9% and 66.8% of semen after DGC in the control group with normal sperm parameters and the male infertility group, respectively. After HA-binding sperm selection, the percentage of green fluorescent spermatozoa grew to 99.3% and 83.9%, respectively. There was an important difference between these two groups in that sperm selection with the HA-binding assay did not reach > 99% of spermatozoa with green fluorescence in the study group with male infertility for the ICSI procedures. Sperm DNA integrity assessment before the ICSI treatments may be more valuable to infertile couples with the male factor. When the initial percentage of green fluorescent spermatozoa in Pure-sperm prepared by DGC falls below 68%, the limitation of HA-bound sperm selection and the possibility of higher spontaneous abortion rate should be well considered before proceeding with fertility treatments. Nevertheless, the number of

participants in the study group is relatively small and future research needs additional large-scale investigation to validate the application for clinical practice.

In this investigation, both microscopic sperm selection in PVP and HA-binding selection displayed significant improvements in the percentage of sperm with intact DNA compared to Pure-sperm. Moreover, the lack of statistical difference between these two sperm selection methods is demonstrated. A well-trained embryologist will have the same ability to choose sperm with intact DNA by conventional microscopic selection as with HA-binding sperm selection. However, the HA binding assay may help the personnel of the IVF laboratory who may not have enough experience and skill in micromanipulation techniques to choose DNA intact spermatozoa for ICSI procedures in patients with male infertility.

Conflicts of interest

The authors have no conflicts of interest relevant to this article.

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