Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 24  |  Issue : 5  |  Page : 445-450

MACS-annexin V cell sorting of semen samples with high TUNEL values decreases the concentration of cells with abnormal chromosomal content: a pilot study


1 University of Brest, Inserm, UMR U1078, Faculty of Medicine and Health Sciences, Brest 29238, France Laboratory of Human Cytogenetics, Molecular Genetics and Reproductive Biology, Farhat Hached University Hospital, Sousse 4031, Tunisia Departement of Medical Genetics and Reproductive Biology, Brest University Regional Hospital, Brest 29609, France Department of Reproductive Medicine, Farhat Hached Teaching Hospital, Sousse 4031, Tunisia

Date of Submission07-Jul-2021
Date of Acceptance28-Oct-2021
Date of Web Publication31-Dec-2021

Correspondence Address:
Frédéric Morel
University of Brest, Inserm, UMR U1078, Faculty of Medicine and Health Sciences, Brest 29238; Departement of Medical Genetics and Reproductive Biology, Brest University Regional Hospital, Brest 29609
France
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/aja202197

Rights and Permissions
  Abstract 


We question whether, in men with an abnormal rate of sperm DNA fragmentation, the magnetic-activated cell sorting (MACS) could select spermatozoa with lower rates of DNA fragmentation as well as spermatozoa with unbalanced chromosome content. Cryopreserved spermatozoa from six males were separated into nonapoptotic and apoptotic populations. We determined the percentages of spermatozoa with (i) externalization of phosphatidylserine (EPS) by annexin V-Fluorescein isothiocyanate (FITC) labeling, (ii) DNA fragmentation by TdT-mediated-dUTP nick-end labeling (TUNEL), and (iii) numerical abnormalities for chromosomes X, Y, 13, 18, and 21 by fluorescence in situ hybridization (FISH), on the whole ejaculate and selected spermatozoa in the same patient. Compared to the nonapoptotic fraction, the apoptotic fraction statistically showed a higher number of spermatozoa with EPS, with DNA fragmentation, and with numerical chromosomal abnormalities. Compared to the whole ejaculate, we found a significant decrease in the percentage of spermatozoa with EPS and decrease tendencies of the DNA fragmentation rate and the sum of disomy levels in the nonapoptotic fraction. Conversely, we observed statistically significant higher rates of these three parameters in the apoptotic fraction. MACS may help to select spermatozoa with lower rates of DNA fragmentation and unbalanced chromosome content in men with abnormal rates of sperm DNA fragmentation.

Keywords: chromosomal abnormalities; DNA fragmentation; externalization of phosphatidylserine; magnetic-activated cell separation; spermatozoa


How to cite this article:
El Fekih S, Gueganic N, Tous C, Ali HB, Ajina M, Douet-Guilbert N, Drapier H, Beauvillard D, Morel F, Perrin A. MACS-annexin V cell sorting of semen samples with high TUNEL values decreases the concentration of cells with abnormal chromosomal content: a pilot study. Asian J Androl 2022;24:445-50

How to cite this URL:
El Fekih S, Gueganic N, Tous C, Ali HB, Ajina M, Douet-Guilbert N, Drapier H, Beauvillard D, Morel F, Perrin A. MACS-annexin V cell sorting of semen samples with high TUNEL values decreases the concentration of cells with abnormal chromosomal content: a pilot study. Asian J Androl [serial online] 2022 [cited 2022 Sep 26];24:445-50. Available from: https://www.ajandrology.com/text.asp?2022/24/5/445/334656


  Introduction Top


An increased rate of apoptotic markers in ejaculated spermatozoa of infertile men has been reported by several authors.[1],[2],[3],[4],[5] These data suggest a possible relationship between these markers and male infertility.[6],[7] The apoptotic markers are mainly an externalization of phosphatidylserine (EPS), a decrease in mitochondrial membrane potential, a caspase activation and DNA fragmentation.[2],[8],[9] Numerous studies have shown the negative effects of sperm DNA fragmentation on the results of assisted reproduction outcomes concerning embryonic development, and/or fertilization rates, and/or implantation rates, and/or spontaneous miscarriages, and/or pregnancies, and/or births.[10],[11],[12],[13],[14],[15],[16]

Chromosomal abnormalities in spermatozoa also affect male fertility.[17] They mainly concern men with constitutive abnormal karyotypes. However, certain subgroups of infertile males with normal karyotypes also produce spermatozoa with abnormal chromosomal equipment.[18] From a retrospective study in 319 infertile patients, sex chromosome disomy and disomy of chromosome 21 as well as aneuploidy increased by 2- to 3-fold compared to the fertile control group.[19] These chromosomal abnormalities found in the spermatozoa of infertile men are considered to be one of the causes of pregnancy failure, implantation failure after ICSI, miscarriage, and high frequency of aneuploidy in embryos.[20],[21],[22],[23],[24],[25]

Previous studies showed the presence of DNA fragmentation positively correlated with the presence of chromosomal abnormalities in the same spermatozoa of infertile patients with normal semen parameters, abnormal semen parameters, or infertile patients with a structural chromosome abnormality.[26],[27],[28],[29],[30] Muriel et al.[27] suggested that the occurrence of aneuploidy during sperm maturation could trigger DNA fragmentation via an apoptosis-like process mediated by endogenous nucleases.

Therefore, the question that arises was: how can chromosomally normal sperm without fragmented DNA be selected? Conventional methods used in assisted reproduction technique (ART) select spermatozoa according to their mobility and morphology, but those methods are not predictive of the quality of the chromosomal equipment. Another technique, magnetic sorting using activated cell sorting (MACS) for annexin V-negative cells, has been proposed as a method for the selection of nonapoptotic spermatozoa.[31],[32] Indeed, annexin V has a high affinity for membrane phosphatidylserines,[33],[34] which are externalized by apoptotic spermatozoa. Therefore, MACS technique involves incubating sperm with magnetic microbeads conjugated with annexin V. After incubation, spermatozoa were selected by going through a column placed under a magnetic field. Therefore, sperm is separated into two populations: nonapoptotic fraction (spermatozoa without EPS, or spermatozoa negative for annexin V) and apoptotic fraction (spermatozoa with EPS, or spermatozoa positive for annexin V).

In this study, we question whether, in men with an abnormal rate of sperm DNA fragmentation, specific MACS by annexinV could select spermatozoa with lower levels of DNA fragmentation as well as unbalanced chromosome content. Furthermore, the levels of spermatozoa with EPS, DNA fragmentation, and chromosomal imbalance after MACS were compared to the total ejaculate population in the same patient.


  Patients and methods Top


Patients

Six samples of cryopreserved sperm from infertile males with an abnormal rate of sperm DNA fragmentation were included in the study. Straws of cryopreserved ejaculated sperm were selected from the sperm bank dedicated to research at the Department of Medical Genetics and Reproductive Biology, Brest, France. The threshold value of DNA fragmentation in our Department of Medical Genetics and Reproductive Biology performed by the TdT-mediated-dUTP nick-end labeling (TUNEL) technique is 7%. A value more than or equal to 7%, in our department, orientated the patients to treatment. All six men were informed of the investigations and gave their written consent. Our study is approved by the ethics committee of Brest University Regional Hospital (ethic code: 29BRC19.0258).

Study design

The study design is presented in [Figure 1]. Briefly, the sperm was thawed for 30 min at room temperature and washed by adding 1 ml binding buffer (1×BB; Miltenyi Biotec SAS, Paris, France) to eliminate the cryoprotectant. The washed sample was then separated into 4 aliquots, for DNA fragmentation analyses by TUNEL, for aneuploidy analyses by fluorescence in situ hybridization (FISH), for externalization of phosphatidylserine determination by annexin V staining, and for MACS.
Figure 1: Flow diagram of the overall experimental design. FITC: fluorescein isothiocyanate; FISH: fluorescence in situ hybridization; BB: binding buffer; MACS: magnetic-activated cell sorting; TUNEL: TdT-mediated-dUTP nick-end labeling.

Click here to view


MACS

The sample was incubated with 100 μl 1×BB and annexin-V microbeads (Miltenyi Biotec SAS) for 15 min at room temperature. An additional aliquot was kept for the study of DNA fragmentation, and the remaining spermatozoa-microbead mix was placed on the separation column containing magnetic beads (MiniMACS, Miltenyi Biotec SAS). The spermatozoa negative for EPS passed through the column and were considered as annexin V (−) fraction. Elution using 1×BB allowed the collection of annexin V (+) spermatozoa, corresponding to sample with EPS. The externalization of phosphatidylserine, DNA fragmentation and aneuploidy rate were carried out on both annexin V (−) and annexin V (+) fractions of each patient.

Annexin V-FITC staining

The externalization of phosphatidylserine was detected by Annexin V-FITC Kit (Miltenyi Biotec SAS). The procedure has been previously published.[35] For each sample, five hundred spermatozoa were analyzed. The spermatozoa without EPS showed blue fluorescence and those with EPS showed green fluorescence.

TUNEL assay

DNA fragmentation was detected by the ApopTag® Red Kit In Situ (Millipore, Saint-Quentin-en-Yvelines, France) according to the manufacturer's recommendations. The TUNEL assay has been described previously.[35],[36] For each sample, five hundred spermatozoa were analyzed. Spermatozoa were considered to contain either fragmented (red fluorescence) or normal (blue fluorescence) DNA.[36]

FISH

Numerical abnormalities of chromosomes 13, 18, 21, X, and Y were evaluated. Triple FISH (chromosomes X, Y, and 18) was carried out with specific alphoid probes for the X chromosome (probe DXZ1, spectrum green; Abbott, Rungis, France) and Y chromosome (probe DYZ3, spectrum orange; Abbott), and a mix of bacterial artificial chromosome (BAC) clones localized in 18q11.1-q11.2 (RP11-676M19, RP11-746M23, RP11-311F3, RP11-446A4, RP11-510P5, and RP11-1076F2) labeled in spectrum aqua (Abbott).

Dual FISH (chromosomes 13 and 21) was carried out with a mix of BAC specific for 13q33.3 (RP11-1122H21, RP11-158B21, RP11-468K20, RP11-1104E18, RP11-974B24, and RP11-183A20) labeled in spectrum green and BAC for 21q22.11 (RP11-698D15, RP11-760B14, RP11-159L9, RP11-30C7, RP11-369E2, and RP11-1071P10) labeled in spectrum orange. The procedure has been previously published.[35],[37] For each sample, a minimum of 1000 spermatozoa was examined using a Zeiss Axioplan microscope (Zeiss, Le Pecq, France).

Statistical analyses

An independent Chi-square test was used to compare the percentages before and after MACS of spermatozoa with EPS, DNA fragmentation, and numerical abnormalities for chromosomes 13, 21, X, Y, and 18. An independent Chi-square test was also used to compare the frequencies of the different chromosomal abnormalities before and after MACS. The data were studied online using the BiostaTGV software (http://marne.u707.jussieu.fr/biostatgv/). P < 0.05 was considered statistically significant.


  Results Top


Annexin V-FITC staining

[Table 1] shows the percentage of spermatozoa presenting an externalization of phosphatidylserine before and after MACS in apoptotic and nonapoptotic fractions. The percentage (mean ± standard deviation [s.d.]) of spermatozoa with EPS after thawing is 36.1% ± 10.1%. After selection, the percentage (mean ± s.d.) of spermatozoa with EPS decreases significantly in nonapoptotic fraction (19.4% ± 1.7%, P < 0.05), while increasing significantly in apoptotic fraction (65.0% ± 13.0%, P < 0.05). We observed a statistically significant difference between the nonapoptotic and apoptotic fractions (P < 0.05).
Table 1: Percentage of spermatozoa in annexin V-FITC (+), in frozen-thawed semen samples, in nonapoptotic and apoptotic fractions for each patient

Click here to view


TUNEL assay

[Table 2] presents the rate of DNA fragmentation in a frozen-thawed semen sample, after incubation, and in apoptotic and nonapoptotic fractions. Regarding the mean value of sperm DNA fragmentation, the percentage (mean ± s.d.) of DNA fragmentation after thawing is 24.8% ± 5.6%; whereas after incubation with microbeads and 1×BB, it is significantly higher at 30.7% ± 7.8% (P < 0.05). After passage through the column, the fragmentation percentage (mean ± s.d.) in the nonapoptotic fraction is 27.4% ± 8.7%. A decreasing trend is observed in the majority of patients in the nonapoptotic fraction. This rate (mean ± s.d.) increases significantly to 44.1% ± 11.3% in the apoptotic fraction (P < 0.05). We also observed a significant difference between the nonapoptotic and apoptotic fractions (P < 0.05).
Table 2: Percentage of DNA fragmentation in frozen-thawed semen samples, after incubation, in nonapoptotic and apoptotic fractions for each patient

Click here to view


FISH

The results for FISH of chromosomes 13 and 21 are shown in [Table 3]. The percentage (mean ± s.d.) of normal spermatozoa after thawing is 96.9% ± 1.9%. Compared to frozen-thawed semen samples, there was a significant increase (P < 0.05) in the percentage (mean ± s.d.) of normal spermatozoa in nonapoptotic fraction (98.4% ± 0.9%). In contrast, we did not observe any significant difference in the apoptotic fraction (mean ± s.d.: 96.1% ± 1.9%). However, we observed a significant difference (P < 0.05) between the nonapoptotic and apoptotic fractions, with a majority of normal spermatozoa in the nonapoptotic fraction versus unbalanced spermatozoa in the apoptotic fraction.
Table 3: Percentage of normal spermatozoa after fluorescence in situ hybridization of chromosomes 13 and 21 in frozen-thawed semen samples, in nonapoptotic and apoptotic fractions for each patient

Click here to view


The results for FISH of chromosomes X, Y and 18 are shown in [Table 4]. The percentage (mean ± s.d.) of normal spermatozoa after thawing is 97.1% ± 1.0%, whereas it is 97.5% ± 1.1% in nonapoptotic fraction. The rate (mean ± s.d.) is 95.8% ± 1.4% in the apoptotic fraction. No significant difference was found between before and after MACS. However, we observed a significant difference between the nonapoptotic and apoptotic fractions (P < 0.05), with a majority of normal spermatozoa in the nonapoptotic fraction versus unbalanced spermatozoa in the apoptotic fraction.
Table 4: Percentage of normal spermatozoa after fluorescence in situ hybridization of chromosomes X, Y and 18 in frozen-thawed semen samples, in nonapoptotic and apoptotic fractions for each patient

Click here to view


The results concerning the frequencies of the different chromosomal abnormalities are shown in [Table 5]. Data from all patients were grouped according to the type of numerical chromosomal abnormality. The frequencies of chromosomal abnormalities are significantly higher in the apoptotic fraction compared to the frozen-thawed semen sample (P < 0.05), except for disomy 13 and disomy 21. Compared to the frozen-thawed semen sample, there was a significant increase in the sum of the disomy and the sum of diploidy in the apoptotic fraction (P < 0.05). Moreover, we observed a significant difference between the nonapoptotic and apoptotic fractions (P < 0.05), with a majority of normal spermatozoa in the nonapoptotic fraction and unbalanced spermatozoa in the apoptotic fraction concerning the levels of disomy Y, disomy XY, disomy 21, diploidy, the sum of disomy, and the sum of diploidy.
Table 5: Percentage of the different chromosomal abnormalities in frozen-thawed semen samples, in nonapoptotic and apoptotic fractions for each patient

Click here to view



  Discussion Top


In this study, we determined the percentages of spermatozoa with EPS, with DNA fragmentation, and with numerical abnormalities of chromosomes 13, 18, 21, X and Y on total ejaculate and on MACS-sorted spermatozoa, from 6 men presenting an abnormal rate of sperm DNA fragmentation.

Analysis of the average of externalization of phosphatidylserine showed a significant decrease between the spermatozoa negative for EPS in the nonapoptotic fraction versus the frozen-thawed semen samples. On the contrary, the externalization of phosphatidylserine significantly increased in the apoptotic fraction compared to the frozen-thawed semen samples. Those trends were also identified in our previously published study in men with a structural chromosomal abnormality[35] and were similar to that found by other teams.[35][38],[39],[40] Moreover, our results confirm that MACS technique can effectively eliminate spermatozoa with EPS caused by apoptotic processes or caused by the premature acrosomal reaction.[35],[38],[39],[40] However, in previous studies, we hypothesized that the number of beads or unspecific binding could be limited and these parameters could explain the presence of spermatozoa with EPS in the nonapoptotic fraction and/or the presence of spermatozoa negative for EPS in the apoptotic fraction.[35],[41]

Concerning the analysis of DNA fragmentation, numerous studies have tried to select sperm without fragmented DNA in different types of populations, especially in infertile men,[42],[43] in men with normal and abnormal semen parameters,[44],[45],[46],[47],[48],[49] in cases of high sperm DNA fragmentation levels,[50],[51],[52] in cases of varicocele,[53] and in cases of idiopathic implantation failure.[38] The majority of these studies reported a significant decrease in fragmented spermatozoa levels, but the majority of authors tested the efficiency of MACS in association with the swim-up and/or density gradient.

Few studies have assessed the efficacy of the MACS technique alone.[43],[46],[48],[54] These studies reported a significant decrease in fragmented spermatozoa levels after selection by MACS in the nonapoptotic fraction. However, the decrease of the percentage of DNA fragmentation varies from one study to another. Tavalaee et al.[46] showed that MACS technique reduces the DNA fragmentation rate by 26.9%. More recently, Berteli et al.[43] have shown that MACS technique makes it possible to reduce by 66.7% DNA fragmentation rate.

In our study, as some authors have demonstrated that freezing process could increase sperm fragmentation, to avoid this bias, we decided to use cryopreserved samples for all patients. We observed a significant increase in the detection of DNA fragmentation rate (mean ± s.d.) after incubation with BB (30.7% ± 7.8%) in comparison to the frozen-thawed semen sample (24.8% ± 5.6%). The BB does not induce DNA fragmentation but increases the accessibility and the integration of TdT and labeled dUTP during TUNEL.[35],[41]

The passage through a MACS-annexin V column allowed a moderate enrichment of nonfragmented spermatozoa in the nonapoptotic fraction in the majority of patients (5/6 patients). Conversely, a significant increase in the rate of fragmented DNA is demonstrated in the apoptotic fraction after MACS-annexin V-sorting. Our results are coherent with those published by Martinez et al.[54] These authors examined the rate of sperm DNA fragmentation before and after MACS in patients with low (<30%) and high (≥30%) levels of sperm DNA fragmentation.[54] The proportion of sperm with DNA damage in nonapoptotic fraction was reduced but heterogeneous across the cohort.[54] Sperm DNA fragmentation in nonapoptotic fraction was higher than that observed before MACS for 20% of the samples.[54]

Indeed, DNA fragmentation in mature spermatozoa may not be directly related to an apoptotic process.[1] Vendrell et al.[38] studied the relationship between DNA fragmentation, EPS and mitochondrial membrane potential in samples sorted by MACS; they did not find a correlation between the presence of early apoptotic markers and DNA fragmentation. This suggests that the presence of apoptotic markers in ejaculated spermatozoa could be associated with abnormalities in chromatin remodeling during later stages of spermatogenesis.[1] Other authors suggest that a phenomenon of early necrotic DNA degradation could explain the presence of fragmented spermatozoa in the nonapoptotic fraction.[54]

About chromosome number abnormalities, since 2002, 12 studies[38],[55],[56],[57],[58],[59],[60],[61],[62],[63],[64],[65] analyzed the chromosomal content before and after spermatic selection in fertile and/or infertile men. Reported results vary according to the team, the selection technique used, the chromosomes analyzed, and the number of spermatozoa studied. To our knowledge, only two teams have attempted to select sperm in 46,XY infertile men by MACS.[38],[64] MACS was used in combination with the density gradient (density gradient followed by MACS) for both teams. Vendrell et al.[38] showed a decrease in the rates of nullisomy and disomy 18 and a decrease in the rate of total aneuploidy. Esbert et al.[64] compared the rate of aneuploidy in nonapoptotic and apoptotic fractions, and they concluded that the aneuploid spermatozoa are preferably retained in the MACS column.

However, it is original and clinically relevant to study specifically men with elevated DNA fragmentation levels. In our study, we have studied the rate of aneuploidy in this population, and we have identified a significant increase in the rate of disomy Y, disomy XY, disomy 21, diploidy (18, X, and Y), diploidy (13, 21), the sum of disomy, and the sum of diploidy in the apoptotic fraction with respect to the nonapoptotic fraction. In addition, we have also found a tendency toward a decrease of the sum of disomy in the nonapoptotic fraction and a significant increase in the frequencies of different chromosomal numerical anomalies in the apoptotic fraction compared to frozen-thawed semen samples with the exception of disomy 13 and disomy 21. These encouraging results were obtained after the study of only 5 chromosomes (X, Y, 18, 13, and 21) and the analysis of other chromosomes would be interesting. Nevertheless, we have to keep in mind that aneuploidy evaluation is particularly tedious.


  conclusion Top


In conclusion, despite the limited number of cases in this study, we consider that cell sorting using annexin V-conjugated magnetic microbeads is a promising technique to efficiently select spermatozoa with lower levels of DNA fragmentation and unbalanced chromosome content in men with an abnormal rate of sperm DNA fragmentation. These findings could have the potential to affect clinical practice.


  Author contributions Top


SEF performed technical studies, analyzed data, and wrote the manuscript. NG and CT participated in the technical studies. HBA, MA, NDG, HD, and DB participated in the study design and coordination. AP participated in the study design and result analysis, and helped to draft the manuscript. FM conceived and supervised the study, designed and analyzed data, and wrote the manuscript. All authors read and approved the final manuscript.


  Competing Interests Top


All authors declared no competing interests.


  Acknowledgments Top


We thank the French Agency of Biomedicine for its financial support. The authors also thank Dr. Marie-Bérengère TROADEC for her help with the translation of the manuscript.

 
  References Top

1.
Sakkas D, Moffatt O, Manicardi GC, Mariethoz E, Tarozzi N, et al. Nature of DNA damage in ejaculated human spermatozoa and the possible involvement of apoptosis. Biol Reprod 2002; 66: 1061–7.  Back to cited text no. 1
    
2.
Brugnon F, Janny L, Communal Y, Darcha C, Szczepaniak C, et al. Apoptosis and meiotic segregation in ejaculated sperm from Robertsonian translocation carrier patients. Hum Reprod 2010; 25: 1631–42.  Back to cited text no. 2
    
3.
Shukla KK, Mahdi AA, Rajender S. Apoptosis, spermatogenesis and male infertility. Front Biosci (Elite Ed) 2012; 4: 746–54.  Back to cited text no. 3
    
4.
Le-Du A, Lelorc'h M, Frydman N, Benkhalifa M, Romana S, et al. Apoptosis and meiotic segregation in sperm from men with chromosomal translocations. Andrologie 2004; 14: 186–92.  Back to cited text no. 4
    
5.
Zini A, Agarwal A. A Clinician's Guide to Sperm DNA and Chromatin Damage. Cham: Springer International Publishing Basel; 2018. pXIX, 605.  Back to cited text no. 5
    
6.
Brugnon F, Van Assche E, Verheyen G, Sion B, Boucher D, et al. Study of two markers of apoptosis and meiotic segregation in ejaculated sperm of chromosomal translocation carrier patients. Hum Reprod 2006; 21: 685–93.  Back to cited text no. 6
    
7.
Gunes S, Al-Sadaan M, Agarwal A. Spermatogenesis, DNA damage and DNA repair mechanisms in male infertility. Reprod Biomed Online 2015; 31: 309–19.  Back to cited text no. 7
    
8.
Oehninger S, Morshedi M, Weng SL, Taylor S, Duran H, et al. Presence and significance of somatic cell apoptosis markers in human ejaculated spermatozoa. Reprod Biomed Online 2003; 7: 469–76.  Back to cited text no. 8
    
9.
Said TM, Paasch U, Glander HJ, Agarwal A. Role of caspases in male infertility. Hum Reprod Update 2004; 10: 39–51.  Back to cited text no. 9
    
10.
Li Z, Wang L, Cai J, Huang H. Correlation of sperm DNA damage with IVF and ICSI outcomes: a systematic review and meta-analysis. J Assist Reprod Genet 2006; 23: 367–76.  Back to cited text no. 10
    
11.
Avendano C, Franchi A, Duran H, Oehninger S. DNA fragmentation of normal spermatozoa negatively impacts embryo quality and intracytoplasmic sperm injection outcome. Fertil Steril 2010; 94: 549–57.  Back to cited text no. 11
    
12.
Alvarez Sedo C, Bilinski M, Lorenzi D, Uriondo H, Noblia F, et al. Effect of sperm DNA fragmentation on embryo development: clinical and biological aspects. JBRA Assist Reprod 2017; 21: 343–50.  Back to cited text no. 12
    
13.
Borini A, Tarozzi N, Bizzaro D, Bonu MA, Fava L, et al. Sperm DNA fragmentation: paternal effect on early post-implantation embryo development in ART. Hum Reprod 2006; 21: 2876–81.  Back to cited text no. 13
    
14.
Benchaib M, Lornage J, Mazoyer C, Lejeune H, Salle B, et al. Sperm deoxyribonucleic acid fragmentation as a prognostic indicator of assisted reproductive technology outcome. Fertil Steril 2007; 87: 93–100.  Back to cited text no. 14
    
15.
Borges E Jr, Zanetti BF, Setti AS, Braga D, Provenza RR, et al. Sperm DNA fragmentation is correlated with poor embryo development, lower implantation rate, and higher miscarriage rate in reproductive cycles of non-male factor infertility. Fertil Steril 2019; 112: 483–90.  Back to cited text no. 15
    
16.
McQueen DB, Zhang J, Robins JC. Sperm DNA fragmentation and recurrent pregnancy loss: a systematic review and meta-analysis. Fertil Steril 2019; 112: 54–60.e3.  Back to cited text no. 16
    
17.
Tempest HG, Griffin DK. The relationship between male infertility and increased levels of sperm disomy. Cytogenet Genome Res 2004; 107: 83–94.  Back to cited text no. 17
    
18.
Templado C, Uroz L, Estop A. New insights on the origin and relevance of aneuploidy in human spermatozoa. Mol Hum Reprod 2013; 19: 634–43.  Back to cited text no. 18
    
19.
Sarrate Z, Vidal F, Blanco J. Role of sperm fluorescent in situ hybridization studies in infertile patients: indications, study approach, and clinical relevance. Fertil Steril 2010; 93: 1892–902.  Back to cited text no. 19
    
20.
Nicopoullos JD, Gilling-Smith C, Almeida PA, Homa S, Nice L, et al. The role of sperm aneuploidy as a predictor of the success of intracytoplasmic sperm injection? Hum Reprod 2008; 23: 240–50.  Back to cited text no. 20
    
21.
Burrello N, Vicari E, Shin P, Agarwal A, De Palma A, et al. Lower sperm aneuploidy frequency is associated with high pregnancy rates in ICSI programmes. Hum Reprod 2003; 18: 1371–6.  Back to cited text no. 21
    
22.
Platteau P, Staessen C, Michiels A, Tournaye H, Van Steirteghem A, et al. Comparison of the aneuploidy frequency in embryos derived from testicular sperm extraction in obstructive and non-obstructive azoospermic men. Hum Reprod 2004; 19: 1570–4.  Back to cited text no. 22
    
23.
Nicolaidis P, Petersen MB. Origin and mechanisms of non-disjunction in human autosomal trisomies. Hum Reprod 1998; 13: 313–9.  Back to cited text no. 23
    
24.
Ioannou D, Fortun J, Tempest HG. Meiotic nondisjunction and sperm aneuploidy in humans. Reproduction 2018; 157: R15–31.  Back to cited text no. 24
    
25.
Esquerre-Lamare C, Walschaerts M, Chansel Debordeaux L, Moreau J, Bretelle F, et al. Sperm aneuploidy and DNA fragmentation in unexplained recurrent pregnancy loss: a multicenter case-control study. Basic Clin Androl 2018; 28: 4.  Back to cited text no. 25
    
26.
Enciso M, Alfarawati S, Wells D. Increased numbers of DNA-damaged spermatozoa in samples presenting an elevated rate of numerical chromosome abnormalities. Hum Reprod 2013; 28: 1707–15.  Back to cited text no. 26
    
27.
Muriel L, Goyanes V, Segrelles E, Gosalvez J, Alvarez JG, et al. Increased aneuploidy rate in sperm with fragmented DNA as determined by the sperm chromatin dispersion (SCD) test and FISH analysis. J Androl 2007; 28: 38–49.  Back to cited text no. 27
    
28.
Ovari L, Sati L, Stronk J, Borsos A, Ward DC, et al. Double probing individual human spermatozoa: aniline blue staining for persistent histones and fluorescence in situ hybridization for aneuploidies. Fertil Steril 2010; 93: 2255–61.  Back to cited text no. 28
    
29.
Perrin A, Nguyen MH, Bujan L, Vialard F, Amice V, et al. DNA fragmentation is higher in spermatozoa with chromosomally unbalanced content in men with a structural chromosomal rearrangement. Andrology 2013; 1: 632–8.  Back to cited text no. 29
    
30.
Perrin A, Basinko A, Douet-Guilbert N, Gueganic N, Le Bris MJ, et al. Aneuploidy and DNA fragmentation in sperm of carriers of a constitutional chromosomal abnormality. Cytogenet Genome Res 2011; 133: 100–6.  Back to cited text no. 30
    
31.
Grunewald S, Paasch U, Glander HJ. Enrichment of non-apoptotic human spermatozoa after cryopreservation by immunomagnetic cell sorting. Cell Tissue Bank 2001; 2: 127–33.  Back to cited text no. 31
    
32.
Glander HJ, Schiller J, Suss R, Paasch U, Grunewald S, et al. Deterioration of spermatozoal plasma membrane is associated with an increase of sperm lyso-phosphatidylcholines. Andrologia 2002; 34: 360–6.  Back to cited text no. 32
    
33.
Klee CB. Ca2+-dependent phospholipid- (and membrane-) binding proteins. Biochemistry 1988; 27: 6645–53.  Back to cited text no. 33
    
34.
Swairjo MA, Seaton BA. Annexin structure and membrane interactions: a molecular perspective. Annu Rev Biophys Biomol Struct 1994; 23: 193–213.  Back to cited text no. 34
    
35.
El Fekih S, Tous C, Gueganic N, Brugnon F, Ali HB, et al. Decrease of spermatozoa with an unbalanced chromosome content after cell sorting in men carrying a structural chromosomal abnormality. Andrology 2019; 1: 181–90.  Back to cited text no. 35
    
36.
Perrin A, Caer E, Oliver-Bonet M, Navarro J, Benet J, et al. DNA fragmentation and meiotic segregation in sperm of carriers of a chromosomal structural abnormality. Fertil Steril 2009; 92: 583–9.  Back to cited text no. 36
    
37.
Nguyen MH, Morel F, Bujan L, May-Panloup P, De Braekeleer M, et al. A study of aneuploidy and DNA fragmentation in spermatozoa of three men with sex chromosome mosaicism including a 45,X cell line. Hum Fertil (Camb) 2015; 18: 96–9.  Back to cited text no. 37
    
38.
Vendrell X, Ferrer M, Garcia-Mengual E, Munoz P, Trivino JC, et al. Correlation between aneuploidy, apoptotic markers and DNA fragmentation in spermatozoa from normozoospermic patients. Reprod Biomed Online 2014; 28: 492–502.  Back to cited text no. 38
    
39.
Lee TH, Liu CH, Shih YT, Tsao HM, Huang CC, et al. Magnetic-activated cell sorting for sperm preparation reduces spermatozoa with apoptotic markers and improves the acrosome reaction in couples with unexplained infertility. Hum Reprod 2010; 25: 839–46.  Back to cited text no. 39
    
40.
de Vantéry Arrighi C, Lucas H, Chardonnens D, de Agostini A. Removal of spermatozoa with externalized phosphatidylserine from sperm preparation in human assisted medical procreation: effects on viability, motility and mitochondrial membrane potential. Reprod Biol Endocrinol 2009; 7: 1.  Back to cited text no. 40
    
41.
El Fekih S. [Impact of maternal and paternal factors on the results of IVF/ICSI and genetic investigations on the spermatozoa of infertile men]. In: Human Medicine and Pathology. Brest: University of Brest (France); University of Monastir (Tunisia); 2018. pI-301. [Book in French].  Back to cited text no. 41
    
42.
Nadalini M, Tarozzi N, Di Santo M, Borini A. Annexin V magnetic-activated cell sorting versus swim-up for the selection of human sperm in ART: is the new approach better then the traditional one? J Assist Reprod Genet 2014; 31: 1045–51.  Back to cited text no. 42
    
43.
Berteli TS, Da Broi MG, Martins WP, Ferriani RA, Navarro PA. Magnetic-activated cell sorting before density gradient centrifugation improves recovery of high-quality spermatozoa. Andrology 2017; 5: 776–82.  Back to cited text no. 43
    
44.
Said T, Agarwal A, Grunewald S, Rasch M, Baumann T, et al. Selection of nonapoptotic spermatozoa as a new tool for enhancing assisted reproduction outcomes: an in vitro model. Biol Reprod 2006; 74: 530–7.  Back to cited text no. 44
    
45.
Herrero M, Delbes G, Troueng E, Holzer H, Chan P. 28th Annual Meeting of the European Society on Human Reproduction and Embryology (ESHRE). Differential enrichment of sperm with no DNA strand breaks using magnetic activated cell sorting (MACS) in men with various categories of semen parameters. Hum Reprod 2012; 27 Suppl 2: ii121–50.  Back to cited text no. 45
    
46.
Tavalaee M, Deemeh MR, Arbabian M, Nasr-Esfahani MH. Density gradient centrifugation before or after magnetic-activated cell sorting: which technique is more useful for clinical sperm selection? J Assist Reprod Genet 2012; 29: 31–8.  Back to cited text no. 46
    
47.
Cakar Z, Cetinkaya B, Aras D, Koca B, Ozkavukcu S, et al. Does combining magnetic-activated cell sorting with density gradient or swim-up improve sperm selection? J Assist Reprod Genet 2016; 33: 1059–65.  Back to cited text no. 47
    
48.
Chi HJ, Kwak SJ, Kim SG, Kim YY, Park JY, et al. Efficient isolation of sperm with high DNA integrity and stable chromatin packaging by a combination of density-gradient centrifugation and magnetic-activated cell sorting. Clin Exp Reprod Med 2016; 43: 199–206.  Back to cited text no. 48
    
49.
Zhang H, Xuan X, Yang S, Li X, Xu C, et al. Selection of viable human spermatozoa with low levels of DNA fragmentation from an immotile population using density gradient centrifugation and magnetic-activated cell sorting. Andrologia 2018; 50: e12821.  Back to cited text no. 49
    
50.
Rawe VY, Boudri HU, Alvarez Sedo C, Carro M, Papier S, et al. Healthy baby born after reduction of sperm DNA fragmentation using cell sorting before ICSI. Reprod Biomed Online 2010; 20: 320–3.  Back to cited text no. 50
    
51.
Herrero MB, Delbes G, Chung JT, Son WY, Holzer H, et al. Case report: the use of annexin V coupled with magnetic activated cell sorting in cryopreserved spermatozoa from a male cancer survivor: healthy twin newborns after two previous ICSI failures. J Assist Reprod Genet 2013; 30: 1415–9.  Back to cited text no. 51
    
52.
Curti G, Skowronek F, Vernochi R, Rodriguez-Buzzi AL, Rodriguez-Buzzi JC, et al. Morphological evaluation of sperm from infertile men selected by magnetic activated cell sorting (MACS). Reprod Biol 2014; 14: 289–92.  Back to cited text no. 52
    
53.
Degheidy T, Abdelfattah H, Seif A, Albuz FK, Gazi S, et al. Magnetic activated cell sorting: an effective method for reduction of sperm DNA fragmentation in varicocele men prior to assisted reproductive techniques. Andrologia 2015; 47: 892–6.  Back to cited text no. 53
    
54.
Martinez MG, Sanchez-Martin P, Dorado-Silva M, Fernandez JL, Girones E, et al. Magnetic-activated cell sorting is not completely effective at reducing sperm DNA fragmentation. J Assist Reprod Genet 2018; 35: 2215–21.  Back to cited text no. 54
    
55.
Ong TD, Xun L, Perreaut SD, Robbins WA. Aneuploidy and chromosome breakage in swim-up versus unprocessed semen from twenty healthy men. J Androl 2002; 23: 270–7.  Back to cited text no. 55
    
56.
Jakab A, Sakkas D, Delpiano E, Cayli S, Kovanci E, et al. Intracytoplasmic sperm injection: a novel selection method for sperm with normal frequency of chromosomal aneuploidies. Fertil Steril 2005; 84: 1665–73.  Back to cited text no. 56
    
57.
Garolla A, Fortini D, Menegazzo M, De Toni L, Nicoletti V, et al. High-power microscopy for selecting spermatozoa for ICSI by physiological status. Reprod Biomed Online 2008; 17: 610–6.  Back to cited text no. 57
    
58.
Perdrix A, Travers A, Chelli MH, Escalier D, Do Rego JL, et al. Assessment of acrosome and nuclear abnormalities in human spermatozoa with large vacuoles. Hum Reprod 2011; 26: 47–58.  Back to cited text no. 58
    
59.
Boitrelle F, Ferfouri F, Petit JM, Segretain D, Tourain C, et al. Large human sperm vacuoles observed in motile spermatozoa under high magnification: nuclear thumbprints linked to failure of chromatin condensation. Hum Reprod 2011; 26: 1650–8.  Back to cited text no. 59
    
60.
Brahem S, Letaief K, Ben Ali H, Saad A, Mehdi M. Efficacy of the density gradient centrifugation method in eliminating sperm with aneuploidy. Andrologia 2013; 45: 158–62.  Back to cited text no. 60
    
61.
Levron J, Aviram-Goldring A, Rienstien S, Bider D, Dor J, et al. Aneuploidy rates for chromosomes X/Y and 18 among preselected spermatozoa in men with severe teratospermia. Reprod Biomed Online 2013; 27: 280–5.  Back to cited text no. 61
    
62.
Mongkolchaipak S, Vutyavanich T. No difference in high-magnification morphology and hyaluronic acid binding in the selection of euploid spermatozoa with intact DNA. Asian J Androl 2013; 15: 421–4.  Back to cited text no. 62
    
63.
Seiringer M, Maurer M, Shebl O, Dreier K, Tews G, et al. Efficacy of a sperm-selection chamber in terms of morphology, aneuploidy and DNA packaging. Reprod Biomed Online 2013; 27: 81–8.  Back to cited text no. 63
    
64.
Esbert M, Godo A, Soares SR, Florensa M, Amoros D, et al. Spermatozoa with numerical chromosomal abnormalities are more prone to be retained by Annexin V-MACS columns. Andrology 2017; 5: 807–13.  Back to cited text no. 64
    
65.
Kim SW, Jee BC, Kim SK, Kim SH. Sperm DNA fragmentation and sex chromosome aneuploidy after swim-up versus density gradient centrifugation. Clin Exp Reprod Med 2017; 44: 201–6.  Back to cited text no. 65
    


    Figures

  [Figure 1]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

Top
 
 
  Search
 
Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
Access Statistics
Email Alert *
Add to My List *
* Registration required (free)

 
  In this article
Abstract
Introduction
Patients and methods
Results
Discussion
conclusion
Author contributions
Competing Interests
Acknowledgments
References
Article Figures
Article Tables

 Article Access Statistics
    Viewed2114    
    Printed521    
    Emailed0    
    PDF Downloaded160    
    Comments [Add]    

Recommend this journal


[TAG2]
[TAG3]
[TAG4]