{"paper_id":"e0884856-6fa4-42c1-805f-f3191145dd41","body_text":"Preparation of a sperm population with high chromatin \nnormality is a basic parameter which is strived for in \nintracytoplasmic sperm injection (ICSI) procedures. \nToday, selection of sperm for ICSI is based on sperm \nmorphology and viability ( 1 ). The percentage of sperm \nwith strict normal morphology is believed to be correlated \nwith percentage of sperm with normal DNA content, \nhowever, this concept has been highly debated ( 2 ,  3 ). \nThere are reports that the percentage of sperm with \nfragmented DNA but normal morphology increases in \nsubfertile/infertile individuals compared to fertile men \n( 4 ,  5 ). In this regrade, previous studies have showed that \nthe percentage of DNA fragmentation in sperm from neat \nsemen of infertile men with normal and abnormal semen\nparameters are around 30 and, 20-40%, respectively. \nSperm with fragmented DNA may fertilize an oocyte,\nbut it has a reduce chance of pre- or post-implantation\ndevelopment ( 6 ).\nA recent meta-analysis has suggested that assessment \nof sperm DNA fragmentation is beneficial in predicting \nmale fertility ( 7 ). A plethora of studies have also concluded \nthat routine sperm preparation procedures such as swim up, \nand density gradient centrifugation (DGC) alongside novel \nsperm preparation procedures based on sperm molecular and \ncellular characteristics can separate a higher percentage of \nnormal sperm with intact DNA compared to routine sperm \npreparation procedures, especially in infertile couples with \nsevere male factor infertility ( 1 ,  8 ,  9 ).\nSperm preparation based on surface electrical charge \nor Zeta potential has been introduced as one of the \nnovel sperm preparation procedures. This procedure, \nwas initially introduced by Chan et al. ( 10 ) who showed \nZeta potential is an effective and feasible procedure for \nselecting of sperm with intact DNA structure. Sperm with \na high level of surface negative electrical charge are more \nmature and several studies have shown that the percentage \nof sperm with fragmented DNA were significantly lower \nin sperm selected in this way. Subsequent studies verified \nthat sperm selected based on Zeta potential have a higher \nchance of having normal intact chromatin ( 11 ,  12 ). Later, \nthis procedure was compared to another novel sperm \npreparation method based on hyaluronic acid binding \nand the results showed that although both novel sperm \npreparation procedures can improve the percentage of \nsperm with DNAfragmentation, the Zeta method was more \nefficient ( 13 ). In addition, it was shown that combined \nDGC/Zeta procedures boost the quality of the sperm \nselected for ICSI and lead to higher clinical pregnancy \nrates per embryo transfer cycle ( 14 ,  15 ). Considering the \nneed for further clinical studies to evaluate the impact of \nsperm quality on assisted reproductive technology (ART) \noutcomes, wider multi-center randomized studies are \nrequired to verify the beneficial effects of DGC/Zeta sperm \nselection. Therefore, the aim of this blind randomised \nclinical trial was to evaluate the effectiveness of the DGC/ \nZeta procedure in improving clinical outcomes in infertile \nmen in an independent center.\n\nAll procedures performed involving human participants\nwere in accordance with the ethical standards of the\ninstitutional research committee and with the 1975 Helsinki\ndeclaration and its later amendments or comparable\nstandards. We performed a randomized, single-blind, \nclinical trial study from April 2015 till August 2017 at \nRouyesh Infertility and Fertility Center belonging to the \nRed Crescent (IRCT20180628040270N1), after approval \nby the Ethical Committee of Tehran Royan Institute [Code \nNo: IR.ACECR.Royan.REC.1396.253].\nAll participants received a complete explanation of the trial\nprior to the start of the study. They were especially ensured\nthat their semen samples would not be exposed to chemical \nagents. Following voluntary completion of the questionnaire,\nthe couples signed informed consent forms. Eligible\nindividuals were assured of confidentiality and anonymity, \nand that their decision to participate in, or withdraw from\nthe study would not impact their current or future relation \nwith the clinic or their future treatment. In this parallel blind\nrandomized clinical trial, 205 candidate couples for ICSI \ncycles were randomly assigned to the DGC or DGC/Zeta\ngroups based on a computer generated random table.\nFemales were between 20 and 40 years old, with no\nreport of endometriosis or polycystic ovaries. Presence \nof at least 2 to 3 follicles more than18 mm in diameters\nwith suitable endometrium for embryo transfer in their \nlast ultrasound before administration of human chorionic\ngonadotropin (hCG). Only couples with male factor\ninfertility and at least one abnormal sperm parameter\n(sperm motility, concentration and morphology) below \nworld health organization (WHO, 2010) criteria were \nincluded in this study ( 16 ).\nCouples whose rate of oocytes with abnormal features \n(without polar body, germinal vesicle, granularity, \nrefractile bodies, fragmented, or degenerated polar bodies) \nexceeded 10%, were excluded from the study. Couples \nthat did not meet the above-mentioned ultrasound criteria \nwere also exclude from the study. As were infertile men \nwith varicocele.\nA two-layer density gradient system (40 and 80%) was \nprepared using PureSperm (Nidacon, Göteborg, Sweden). \nFollowing semen liquefaction, each semen sample was \nplaced on the gradients and centrifuged at 300 g for 20 \nminutes. Then, the pellet was collected and re-suspend \ninto 5mlof sperm processing medium supplemented with \n10% human serum albumin (HSA, Octalbin, Switzerland). \nThe sperm suspension was then centrifuged at 300 g for 7 \nminutes. For insemination; the resultant pellet was diluted \ninto 0.3 mL of sperm processing medium containing10% \nHAS albumin ( 14 ).\nFor DGC/Zeta procedure, sperm pellets were washed \nwith sperm processing media without HSA, and \nsubsequently diluted in 4 ml sperm processing media \nwithout HSA, immediately after DGC. Subsequently, the \ntube was exposed to a positive charge using a rubber latex \ntube ( 14 ). The tube was then removed from the latex tube \nand held by the cap for one minute to provide the time \nneeded for the sperm with adequate negative charge to \nattach to the charged tube. Then the sperm processing \nmedia containing unattached sperm was withdrawn from \nthe tube and the tube was washed with sperm processing \nmedium containing HSA in order to detach the attached \nsperm. Ultimately, the content of each tube was centrifuged \nand the pellet was diluted in sperm processing media with \nHSA and used for ICSI. To reduce inter-sample variation, \na single trained individual carried out all the procedures \nfor sperm processing. The embryologist who inseminated \nthe sperm was blind to the allocation of the sperm to the \ntwo groups; DGC and DGC/Zeta.\nStimulation and ovum pick up procedures were\nperformed base on a single standard protocol for all cases\n( 17 ). Around 16-18 hours post-ICSI, the presence of male\nand female pronuclei was considered as a sign of successful\nfertilization and the rate of fertilization was calculated as \nthe percentage of injected oocytes that became fertilized. \nAn embryo was selected and considered to be a top quality \nembryo if there were six to eight blastomeres on day 3 \nwith fragmentation less than 25% and the absence of multi \nnucleated blastomeres at any stage of early development. The \npercentage of top quality embryos was defined as the number \nof top embryos obtained from the total number of cleaved \nembryos. Procedures of embryo transfer were similar in both\ngroups and were carried out by an embryologist who was \nnot aware of the design of the clinical trial. The embryologist\nwas asked to select the best embryos for transfer and a \nminimum of one and a maximum of three embryos were \ntransferred. Based on the internal policy of the center, \nindividuals under the age of 35 can receive a maximum of 2 \nembryos whileindividualsover35 were allowed to request for \nthe transfer of a maximum of 3 embryos if two top quality \nembryos are not available. Blastocyst quality was assessed \non day 5 ( 18 ). All embryos were transferred fresh. Chemical \nand clinical pregnancy were defined as ß-hCG levels higher\nthan 10 IU and the presence of a gestational sac, 5 weeks \nafter embryo transfer, respectively.\nSPSS for Windows Version 18 (SPSS Inc., Chicago, \nIL, USA) was used for all statistical analyses. Data are \npresented as means ± SEM for continuous variables. \nIndependent-samples t test was used for comparisons \nof couples’ age and sperm parameters between DGC/ \nZeta and DGC groups ( Table 1 ). Independent student's \nt test and Chi-square carried out for statically analyzing \nwas used for comparisons of fertilization, good-quality \nembryo, pregnancy, implantation, and miscarriage ( Table 2 ). The value of P<0.05 was considered statistically \nsignificant. This clinical trial study was a continuation of \nthe Nasr-Esfahani group study ( 15 ). To detect the effect \nof DGC/Zeta on clinical outcome in male factor infertility \npatients following ICSI which is in agreement with the \nstudy of Nasr Esfahani et al. ( 15 ) with a power of 80%, \na sample size of 103 patients per group was necessary, \ngiven an anticipated dropout rate of 10%.\nComparison of couples age, and sperm parameters between density gradient centrifugation (DGC)/Zeta and DGC groups\nData are presented as mean ± SEM and analyzed by independent-samples t test. Asterisk indicate significant difference; *; P<0/05, and NS; Non significant.\nComparison of clinical outcomes between density gradient centrifugation (DGC)/Zeta and DGC groups\nData are presented as mean ± SEM and analyzed by Independent studentʼs t test and Chi-square. Asterisks indicate significant difference; **; P<0/01 , *;\nP<0/05, and NS; Non significant.\nIn this randomized clinical trial, a total of 240 infertile \ncouples were recruited and 220 couples agreed to partake \nin this study. These couples were randomly divided into \ntwo groups based on the randomization table generated by \na computer into the DGC/Zeta or DGC groups. Of the 220 \ninfertile couples, 7 couples were excluded from the study \nbased on exclusion criteria (3 and 4 couples in the DGC/ \nZeta and DGC groups, respectively). Of the 213 remaining \ncouples 8 couples decided to leave the study for personal \nreasons. Finally, of the 205 remaining couples 103 and \n102 belonged to DGC/Zeta and DGC groups, respectively. \nBaseline characteristics of the DGC/Zeta and DGC groups \nincluding male age (P=0.25), female age (P=0.23) semen \nparameters including [sperm concentration (P=0.38), total \nmotility (P=0.9) and normal morphology (P=0.11)] were \nanalyzed and compared between the two groups. These \nwere found to be similar in both groups and no significant \ndifferences were observed ( Table 1 ).\nThe clinical outcomes of a total 205 cycles in two groups \nwere evaluated and compared ( Table 2 ). The mean number \nof retrieved oocytes and transferred embryos in the two \ngroups were similar without any significant differences. \nAn obvious drift towards a superior fertilization rate was \nseen in the DGC/Zeta procedure compared to DGC alone\n(64.75 ± 1.67 vs. 58.88 ± 1.83, P=0.67). Although, there \nwas no statistically significant difference in the mean \nnumber of embryos transferred between the two groups\n(P=0.8), but mean percentage of good embryo quality \nat day 3 (41.89 ± 2.01 vs. 30.64 ± 3.51, P=0.04), good \nquality of blastocysts (33.69 ± 1.22 vs. 23.86 ± 1.51, \nP=0.049), expanded blastocysts (48.2 ± 2.11 vs. 39.24 ± \n2.75, P=0.007), and mean of hatching blastocysts (1.2 ±\n0.82 vs. 0.4 ± 0.68, P=0.005) were significantly higher in \nthe DGC/Zeta group compared to the DGC group. The \npercentage of chemical (P=0.005) and clinical (P=0.007) \npregnancy rates in the DGC/Zeta group were 42.47 and \n35.03, respectively. However these rates were 21.10% \nand 20.43% respectively in the DGC group. The mean \npercentage of implantation rate were insignificantly \n(P=0.17) higher in the DGC/Zeta group (20.80) compared \nto the DGC group (11.96). While the mean percentage of \nmissed spontaneous abortion/missed miscarriages were\nsignificantly (P=0.04) lower in the DGC/Zeta group (8.9) \ncompared to the DGC group (19.01).\nNumerous methods have been developed to eliminate \nmorphologically normal sperm with damaged DNA from \nbeing inseminated during the ICSI procedure, which is a \nshortcoming of conventional ICSI procedures. Each of \nthese approaches has advantages and disadvantage which \nhave been covered by extensive reviews in this filed ( 1 , \n 8 ,  9 ). The outer surface of the sperm plasma membrane is \nrich in sialic acids. These sialic acids are responsible for \nthe membrane’s negative electrical charge of around -16\nto -20 mV called the \"Zeta potential\" or electrokinetic \npotential ( 19 ,  14 ). Sperm selection based on Zeta potential\nis a new strategy in order to acquire functional sperm in\na manner that optimizes sperm recovery rates specially \nof sperm with normal DNA integrity to improve ICSI \noutcomes ( 20 ,  21 ).\nDespite clear evidence showing benefits of selecting \nsperm based on Zeta potential, low enthusiasm for the \nimplementation of this technique is due to the limited \nnumber of clinical trials. Ainsworth et al. ( 22 - 24 ) and \nFleming et al. ( 25 ) introduced a device based on Zeta \npotential or electrophoresis and their preliminary studies \nshowed that ICSI outcomes can be improved by this \napproach. In this regard, the first pregnancy reported \nusing the electrophoresis method in infertile couples with \nprevious repeated failed fertilization, and high sperm \nDNA fragmentation ( 22 ). In addition, Fleming et al. ( 25 ) \ncompared clinical outcomes of ICSI or IVF between the \nelectrophoretic method and the DGC procedure, and \nconcluded that the mean fertilization rate, and embryo \nquality were similar between the two groups. Then, they \nreported that the electrophoretic method can be harmful \nfor sperm motility. Unlike the electrophoretic method, the \nZeta method is simple, low cost, fast, and no chemicals are \nused during the preparation of the sperm. In this regard, the \nNasr-Esfahani et al. ( 15 ), by inducing positive charge on \nthe surface of a tube showed that ICSI outcome, especially \nclinical pregnancy rate can be improved by this technique \ncompared to DGC alone. Based on their experience, we \nalso decided to assess the efficiency of this technique \nin a sister clinic in different location independently of \nthis group but through their collaboration and with the \ntransfer of experience. Thus, following randomization, a \ntotal of 103 and 102 infertile couples were allocated to the \nDGC/Zeta and DGC groups, respectively and the clinical \noutcomes were evaluated. In this study, higher rates of \ngood embryo quality, blastocysts, expanded blastocysts, \nhatching blastocysts, chemical and clinical pregnancy \nwere seen in the DGC/Zeta group. In addition, the results \nof this study also revealed that the selection of sperm \nthrough the DGC/Zeta procedure did not significantly \naffect the fertilization rate but significantly improves \nembryo quality. This is consistent with the results of \nprevious studies suggesting that sperm DNA damage \ndoes not necessarily preclude sperm from participating \nin the process of fertilization but can significantly \naffect the embryo quality especially during maternal-\nembryonic genomic transition ( 26 ). These results are in \nagreement with a previous randomized trial study by Nasr \nEsfahani et al. ( 15 ) and represented an improvement in \nclinical outcomes after injection of sperm with DGC/ \nZeta processing. Major causes of significant difference in \nclinical consequences between the two procedures, (DGC/ \nZeta vs. DGC alone), may be due to the ability of DGC/ \nZeta for selecting mature sperm with lower DNA damage \ncompared to DGC alone. Indeed, a high rate of sperm \nDNA damage has been associated with reduced clinical \noutcomes following assisted reproduction, increased time \nto conception and high rate of abortion ( 11 ,  26 - 29 ).\n\nThe results of this blind clinical trial along with other \nreports in the literature reveal that selection of sperm based \non Zeta is able to recover a population of mature sperm \nwith intact DNA and eliminate sperm with a high degree \nof DNA fragmentation. Therefore, the improved efficacy \nshould be particularly valuable in ART. Accordingly, \nwe recommend specialists working in field of assisted \nreproduction to evaluate the capacity of Zeta procedure \nespecially for couples with previous implantation failures. \nHowever, use of a device for the selection of sperm based \non Zeta potential may even further improve the efficiency \nof clinical outcomes post ICSI and reduce variations \nbetween different studies.","source_license":"CC-BY-4.0","license_restricted":false}