The Relationship between Sperm Oxidative Stress Alterations and IVF/ICSI Outcomes: A Systematic Review from Nonhuman Mammals

Achieving high embryo quality following IVF and ICSI procedures is a key factor in increasing fertility outcomes in human infertile couples. While the male factor is known to underlie infertility in about 50% of cases, studies performed in human infertile couples have not been able to define the precise effect of sperm affectations upon embryo development. This lack of consistency is, in most cases, due to the heterogeneity of the results caused by the multiple male and female factors that mask the concrete effect of a given sperm parameter. These biases can be reduced with the use of animal gametes, being a good approach for basic researchers to design more homogeneous studies analyzing the specific consequences of a certain affectation. Herein, we conducted a systematic review (March 2020) that assessed the relationship between sperm oxidative stress alterations and IVF/ICSI outcomes in nonhumans mammals. The review was conducted according to PRISMA guidelines and using the MEDLINE-PubMed and EMBASE databases. Thirty articles were included: 11 performed IVF, 17 conducted ICSI, and two carried out both fertilization methods. Most articles were conducted in mouse (43%), cattle (30%) and pig models (10%). After IVF treatments, 80% of studies observed a negative effect of sperm oxidative stress on fertilization rates, and 100% of studies observed a negative effect on blastocyst rates. After ICSI treatments, a positive relationship of sperm oxidative stress with fertilization rates (75% of studies) and with blastocyst rates (83% of studies) was found. In conclusion, the present systematic review shows that sperm oxidative stress is associated with a significant reduction in fertilization rates and in vitro embryo development.


Introduction
Infertility is defined as the failure to achieve a clinical pregnancy after 12 months of regular unprotected intercourse [1]. In order to help human infertile couples, assisted reproductive techniques (ART) were developed decades ago; nowadays, all intrauterine insemination (IUI), in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) are methods with the potential to result in a pregnancy and a healthy newborn. Because of that, they are currently accepted worldwide as having a routine medical use. According to the latest report from the European Society of Human Reproduction and Embryology (ESHRE) [2], about 729,000 ART treatments are performed per year in Europe. Among them, 25.8% correspond to IUI, 21.4% to IVF and 52.8% to ICSI. Thus, procedures involving artificial fertilization (i.e., IVF or ICSI) are the most frequent ART, with more than 541,000 cycles a in pigs, cattle, sheep and horses [31][32][33]. In contrast, IVF and ICSI are not usually performed, but are utilized for research or other specific purposes, such as embryo production, sex selection and genetic selection [34][35][36]. In research, the induction of DNA damage to healthy biological samples and the homogeneity of sperm and oocyte samples make it possible to design more homogeneous studies than those performed in infertile humans [19,37,38]. Therefore, experiments mimicking the sperm oxidative damage happening naturally in human infertile males are important to reach firm conclusions regarding the effect of this damage to ART. The aim of the present study is to systematically review studies performing induction of sperm oxidative damage in mammals other than humans in order to elucidate the influence of the male factor on IVF/ICSI outcomes.

Systematic Review Execution and Registration
Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines [39] were followed in this systematic review. The protocol used was registered in the international prospective register of systematic reviews (PROSPERO 2020: CRD42020176683).

Data Sources and Systematic Search Strategy
The inclusion and exclusion criteria defined in the PICOS (Population, Intervention, Comparator, Outcome, Study) design structure (Supplementary Table S1) led to a comprehensive list of keywords and Medical Subject Headings (MeSH) terms, which were combined with keywords related to oxidative stress and IVF/ICSI outcomes, all them resulting in the design of the search strategy (Appendix A). The systematic literature search, performed with both MEDLINE-PubMed (www.ncbi.nlm.nih.gov/pubmed) and EMBASE (www.embase.com/#search) databases, included articles from the earliest date and until 27 March 2020.

Study Eligibility
The eligibility criteria for the studies included in the present systematic review was defined prior to the search in the PICOS design structure (Supplementary Table S1). Research that fulfilled the following criteria was selected to be included in the review: (i) studies performed on mammals other than humans; (ii) studies aimed at elucidating the effect of sperm oxidative affectation on IVF and ICSI; (iii) studies in which the induced sperm oxidative affectation was measured and compared to controls; and (iv) primary outcomes were defined as IVF/ICSI cycle parameters, such as fertilization and blastocyst rates. Alternatively, studies that performed natural mating or IUI, those that did not measure the effect of oxidative treatments on sperm cells, and studies aimed at analyzing the protective effect of components added to cryopreservation/thawing media were excluded from this review. Review articles, systematic reviews/meta-analysis, case reports, letters and commentary articles were also considered as noneligible.

Study Selection Procedure
As shown in Figure 1, the study selection procedure was conducted in different stages and following the aforementioned eligibility criteria. First, the search was performed in both the PubMed and EMBASE databases using a standardized extraction form that collected the following information: reference, digital object identifier (DOI), publication year, title, abstract, authors, article type, affiliations, and the database in which it was found. Once all records were annotated in the database, duplicate articles and those declared as noneligible were excluded. The second step in the study selection was based on a screening according to the title and abstract, excluding articles that did not meet the eligibility criteria. This step was performed by two researchers that are specialists in male (in)fertility (J.R.-M. and A.S.-H); in case of disagreement, a third author (M.Y.) was involved. Afterwards, full texts of all selected articles were downloaded and screened for a third phase of exclusion conducted in parallel by the same two study selection was based on a screening according to the title and abstract, excluding articles that did not meet the eligibility criteria. This step was performed by two researchers that are specialists in male (in)fertility (J.R.-M. and A.S.-H); in case of disagreement, a third author (M.Y.) was involved. Afterwards, full texts of all selected articles were downloaded and screened for a third phase of exclusion conducted in parallel by the same two authors. Discrepancies regarding eligibility were discussed with the third author (M.Y.) to reach a consensus. The exclusion reasons are depicted in Figure 1.

Data Extraction for Systematic Review and Statistics
After completing the eligibility process, the final list of articles included in the systematic review was analyzed in depth to extract the following data: reference, aim of the study, animal species used, treatment applied to induce oxidative damage in sperm cells, sperm parameters measured, effects caused by the treatment to sperm cells, origin of the oocytes used in IVF or ICSI, fertilization procedure (IVF, ICSI or both), effects observed on IVF/ICSI outcomes and conclusion of the study.
During the data extraction process, the studies included in the systematic review were classified according to whether or not their findings supported the fact that sperm oxidative affectation led to a reduction of fertilization rates, blastocyst rates, and implantation and pregnancy rates. These data were divided regarding the fertilization method used (IVF or ICSI), and the results were compared

Data Extraction for Systematic Review and Statistics
After completing the eligibility process, the final list of articles included in the systematic review was analyzed in depth to extract the following data: reference, aim of the study, animal species used, treatment applied to induce oxidative damage in sperm cells, sperm parameters measured, effects caused by the treatment to sperm cells, origin of the oocytes used in IVF or ICSI, fertilization procedure (IVF, ICSI or both), effects observed on IVF/ICSI outcomes and conclusion of the study.
During the data extraction process, the studies included in the systematic review were classified according to whether or not their findings supported the fact that sperm oxidative affectation led to a reduction of fertilization rates, blastocyst rates, and implantation and pregnancy rates. These data were divided regarding the fertilization method used (IVF or ICSI), and the results were compared using the Chi-squared test (Statistical Package for the Social Sciences Ver. 25.0, SPSS, IBM, New York, NY, USA). Figure 1 shows the flowchart used to conduct the search and study selection process. The initial search gave rise to the identification of 1451 articles in the MEDLINE-PubMed database, and 2191 in the EMBASE database, resulting in 3642 records. After including all the records in a database, 47 articles were removed because they were duplicated and 220 were removed because they were reviews, and therefore, did not meet the inclusion criteria. This first database clean up led to the further screening of 3375 articles by title and abstract, performed by two researchers (J.R.M. and A.S.H.). This resulted in the exclusion of 3295 studies, which were performed on humans, presented irrelevant outcomes or did not meet the scope of the present systematic review; the 80 remaining articles were downloaded and analyzed in parallel to determine their eligibility for inclusion according the same criteria. Fifty studies were excluded for the following reasons: 16 did not use IVF or ICSI fertilization methods, 13 did not analyze oxidative damage caused to sperm cells, 12 had outcomes that were irrelevant to the scope of the review, seven studied the effect of additives or extenders to the cryopreservation process, one compared two treated groups but lacked a control group, and one was performed on human subjects. Therefore, after applying all the eligibility parameters, 30 articles remained for qualitative analysis.

Systematic Review: Qualitative Analysis
The 30 studies included in the systematic review were carefully analyzed in order to extract the key data summarizing: (a) their aims; (b) the treatments performed to induce sperm oxidative damage; and (c) the IVF/ICSI outcomes obtained as a result of the treatment, as described in the methods section. This analysis is summarized in Tables 1 and 2. Among the studies, 11 (36.6%) used IVF as a fertilization method, 17 (56.6%) used ICSI, and two (6.6%) used both techniques. Regarding the animal models used, three studies (10%) were performed on pigs, nine (30%) were conducted on cattle, 13 (43.3%) were performed on mice, one (3.3%) compared mice and hamsters, one study (3.3%) was conducted on sheep, one (3.3%) was carried out on rats, one (3.3%) was conducted on horses and one (3.3%) was performed on macaques. The percentage of studies performing IVF or ICSI differed among different animal models. All studies in pigs (3/3) performed IVF; for cattle, 89% (8/9) of studies conducted IVF and one study performed ICSI; and for mice, 54% (7/13) of studies conducted IVF and 46% (6/13) carried out ICSI.
Regarding the origin of the oocytes used, 37% (11/30) of the studies collected the ovaries and performed in vitro maturation (IVM), 53% (16/30) conducted superovulation, one recovered oocytes from induced ovulation and two did not provide this information. These origins also differed between species; thus, whereas porcine and bovine studies mainly used IVM oocytes (100% and 78% of the studies, respectively), superovulation was the main oocyte source in studies on mice (92% of the articles).

Effect of Different Treatments on Spermatozoa
A very wide variety of treatments were tested in different studies. Eighteen studies applied treatments to sperm cells, nine applied treatments to animals or to their testis, and three performed comparisons between cohorts of animals with high and low fertility. With regard to studies performing treatments, three applied heat stress, three applied radiation (X rays or Gamma rays), seven treated sperm with oxidant molecules, such as H 2 O 2 or xanthine oxidase, one applied genetic modifications creating mouse chimeras for Protamine 2 (Prm2) gene, one caused damage through freeze-thawing, and eleven performed treatments with other chemical agents, such as manganese and calcium, lipopolysaccharide, cyclophosphamide, alkaline pH, fluoride, pyridaben, and DAVA, or fed animals with a selenium-deficient diet. To determine whether sperm oxidative damage induced by ROS affects embryo development.

Rhesus Macaque
Oxidation of sperm cells with xanthine oxidase.
-Lipid peroxidation Treatment with xanthine oxidase led to an increase of lipid peroxidation.

Super-ovulated ICSI
-Exposure to high levels of ROS leads to arrest of embryo development before eight-cell stage. Additionally, all stages were affected in the treated group.
Paternal oxidative stress influences early embryo development. 5 Castro et al. 2016 [43] To assess the impact of sperm oxidative stress on embryo development.    17 Li et al. 2020 [54] To assess the extent to which the measurement of DNA fragmentation index in sperm can predict their fertilizing ability.

Mouse
Freeze-thawing -DNA damage: TUNEL DNA fragmentation index is an accurate parameter to determine sperm quality and fertility potential. 18 Llamas Luceño et al. 2020 [55] To address the impact of natural heat stress on bull fertility.

Cattle
Comparison of different bulls exposed to separate levels of heat stress -ROS production -Lipid peroxidation -DNA fragmentation: TUNEL To evaluate the effects of fish oil feeding on sperm parameters and its incidence in IVF.

Sheep
Fish oil diet for 70 days Intracellular ROS Superoxide anion was lower in the treated than in the control group.
IVM IVF -Cleavage rates were higher in the treated group.
Addition of fish oil to ram diet improves sperm quality and in vitro fertilization ability. 21 Mehraban et al. 2019 [58] To evaluate the antioxidant effects of Gallic Acid on apoptotic-like changes in sperm.

Mouse
Cyclophosphamide and gallic acid Apoptotic-like changes: Annexin V staining Cyclophosphamide induces apoptotic-like changes compared to controls. Gallic Acid mitigates that increase.

Super-ovulated IVF
-Cyclophosphamide reduces fertilization rates and proportions of cleaved embryos.
Gallic Acid suppresses ROS induced by Cyclophosphamide and help rescue fertility.   30 Yi et al. 2017 [66] To determine the impact of difructose dianhydride IV (DFA-IV) on in vitro sperm fertilizing ability. Most of these treatments had a detrimental effect upon sperm quality, and only three studies observed no effect on the evaluated parameters. Hence, DNA damage occurred in 17 out of 19 studies analyzing this parameter; ROS production and antioxidant capacity were affected in all of the studies that took these two indicators into consideration (eight and two studies, respectively), and five out of six studies observed affectations related to plasma membrane oxidation, such as lipid peroxidation.

Effect of the Different Treatments on IVF/ICSI Outcomes
All the included studies aimed to analyze the effects of sperm alterations on IVF/ICSI outcomes, such as fertilization, blastocyst, implantation and live birth rates. Taking IVF and ICSI treatments together, 78% of the studies analyzing fertilization rates (21/27) found an association between sperm oxidation and this parameter, and 92% of the studies analyzing blastocyst rates (22/24) found that it was related to sperm oxidation. Considering only the works that performed IVF, 80% (12/15) found a relationship between sperm oxidation and fertilization rates, while 100% (12/12) found a negative association with blastocyst rates. Studies performing ICSI displayed a similar trend: 75% (9/12) found a relation to fertilization rates and 83% (9/12) found an association with blastocyst rates. Oxidative stress affected IVF and ICSI treatments in a similar percentage, and no differences between fertilization methods were found, either for fertilization rate (p = 0.756) or for blastocyst rate (p = 0.140).
Among all the studies included in this systematic review, only two [53,60] using mice analyzed implantation rates after ICSI procedures, finding a reduction of this parameter. Li et al. [53] and Li and Lloyd [54] also reported similar findings regarding live birth rates; these studies were the only ones which analyzed this parameter.

Discussion
Thanks to several studies analyzing male factor infertility performed by multiple research groups around the globe, the male partner has gathered more attention in routine clinical practice. Nowadays, it is well known that the male factor contributes equally to the female factor to infertility disorders that affect nearly 50 million couples around the world [67]. The present study systematically reviewed the effects of male oxidative damage on the IVF and ICSI outcomes analyzed in animal models, showing that oxidative affectations in sperm are translated into poorer outcomes after ART. The most affected parameters were fertilization rates and blastocyst rates, since male oxidative damage induces an arrest during embryo cleavage.
Different studies have proven that oxidative stress represents an important issue leading to sperm cell defects such as DNA damage, protein modifications, plasma membrane damage and even epigenetic modifications that can be transmitted to the offspring [68,69]. Oxidative DNA damage in mature sperm cells is irreparable, since the nature of the DNA condensation impedes the transcription of DNA repair enzymes; therefore, the integrity of the genetic material is compromised in the male gamete [15]. Also, cell membranes are particular susceptible to oxidative stress due to the presence of polyunsaturated fatty acids. Free radicals react with fatty acids, producing highly reactive aldehydes which are capable of inhibiting antioxidants, such as glucose-6-phosphate dehydrogenase (G6PDH) and glutathione peroxidase (GPX), and also leading to motility loss [13,70]. All these effects represent a reduction in sperm quality, which has been related to increased difficulty achieving natural pregnancy and to male infertility in a number of studies [16,17,21]. Classical sperm analyses, including concentration, motility and morphology, are among the key initial tests in defining male infertility, while sperm DNA damage represents a more advanced test that may help in defining the physiopathology of the male partner [18,23].
Alternatively, oxidative stress also has effects at different levels. Regarding the sperm epigenome, it has been demonstrated that oxidative stress induces tRNA cleavage [71], generating tsRNA and rRNA fragments that are involved in the sperm-mediated inheritance of intergenerational pathologies [68,72]. Moreover, recent insights also indicated that cigarette smoking can induce sperm oxidative stress, generating DNA methylation changes [73]; this suggests that oxidative stress is an important mechanism through which an unhealthy lifestyle may impact the offspring phenotype [74]. In fact, some authors described that the expression of some sperm small RNAs and tsRNAs rapidly changed in relation to male high-sugar diets [75,76]. The profiles of these RNA were related to sperm quality and embryo viability [77], and may also affect histone modifications and tsRNA biogenesis at the testis level [78]. Therefore, epigenetic components are important affectations to be studied when sperm cells are subjected to an oxidative environment.
Assisted reproductive methods such as IVF and ICSI are commonly applied to infertile couples, representing a good alternative to overcome some infertility issues and achieve pregnancy, despite their limited global effectiveness [2]. IVF and ICSI rely on the generation of good quality embryos to be selected and transferred into the female uterus in order to achieve a clinical pregnancy. While oocyte quality is widely known to be crucial for embryo development [79], the data are inconsistent with regard to the relationship between sperm quality and IVF and ICSI outcomes in humans. In search for a consensus, the most widespread conclusion in the most recent meta-analyses is that while DNA damage may have an influence on pregnancy achievement after IVF treatments, this influence has not been clearly established after ICSI procedures [27,28]. These differential results may indicate that the single sperm selection performed in ICSI may be related to ART outcomes, but resulted in a controversy between different reproductive associations regarding the use of sperm DNA fragmentation as a clinical routine test [80,81]. The meta-analyses performed in humans agree with the high heterogeneity of the results, mainly given by the variety of female factors affecting the cycle, the technicians performing the ART techniques, the methodology used for sperm DNA damage analyses, and the different grades of male factor affectations, amongst others [25,27,28]. Testing the effect of the male factor through animal models may help reduce this heterogeneity, since they make it possible to induce precise and homogeneous oxidative damage to sperm cells, and a cohort of homogeneous oocytes can be also obtained. In this sense, despite the application of different treatments among studies, an effect on sperm cells was found in almost 90% of the studies. In this systematic review, we found that these effects at the sperm level are translated into affectations in fertilization rates and blastocyst rates, which is supported by more than 80% of studies on IVF and more than 75% of the studies on ICSI. In addition, these effects were proven for different animal species (including rodents, cattle, pig, sheep and macaque) and for different oocyte origins (IVM or superovulated), showing that the sperm quality in terms of oxidative damage was highly related to the fertilization and blastocyst rates. Interestingly, the findings reported herein are supported by Toyoshima, 2009 [82], who indicated that a P53 and P21-related DNA damage response (S-phase and G2/M) occurs during early embryo development. These responses are activated at different time points; while a p53 dependent S-phase checkpoint is active from the first mitosis to ensure normal embryo division, the G2/M checkpoint is activated before blastocyst, causing an arrest of affected embryos [82,83]. Thus, as DNA breaks caused by irradiation compromise embryo development, oxidative damage may also have an important role in embryo progression.

Strengths and Limitations
The present systematic review provides a new overview of the relationship between sperm oxidative damage and IVF/ICSI results. To the best of our knowledge, all systematic reviews and meta-analyses performed until the present moment have been focused on human patients; they observed a high heterogeneity in their results, probably due to individual differences attributable to both male and female factors. Here, we place the focus on reviewing studies in animal models (laboratory or production animals), which makes it possible to design more homogeneous studies due to the use of a single sperm sample with different treatments to inseminate multiple oocytes, usually obtained from the same female or group of females. This reduces the presence of confounding factors, and elucidates the effect of the treatment.
In spite of this, as a main limitation, our review included studies performed on different animals, where different oocyte origins had been used. While most studies performed on cattle, pigs and sheep used IVM oocytes, most studies performed on rodents used superovulated oocytes, which represents a source of bias in comparison to human studies. The searches performed using both MEDLINE-PubMed and EMBASE databases were designed to identify all potentially eligible studies following the criteria previously defined in the PICOS table (Supplementary Table S1). However, some small biases may potentially exist if a study aimed at inducing damage other than oxidative stress performed a treatment on animals or sperm that led to oxidative damage as an unintended consequence.
Additionally, our study is not exempt from publication biases. Studies that did not find an association between the studied parameters would be less likely to get published, and therefore, may be underrepresented in scientific databases. Also, the eligibility of articles published during the period between the search and the publication of the present paper was not assessed.

Conclusions
Notwithstanding the controversies existing in human studies regarding the impact of sperm alterations on IVF and ICSI outcomes, the present systematic review supports the hypothesis that, in animals, oxidative affectations at the sperm level decrease fertilization and blastocyst rates. Then, a cause-effect of sperm cells is observed in embryos. Finally, despite the fact that most studies did not assess implantation and pregnancy rates, the data included in our review suggest that these parameters might also be compromised in a scenario where oxidative sperm damage is present.
Supplementary Materials: The following are available online at http://www.mdpi.com/2079-7737/9/7/178/s1, Table S1: PICOS design structure, including the inclusion and exclusion criteria and the keywords were used for the definition of the search strategy and the eligibility of the study.

Conflicts of Interest:
The authors declare no conflict of interest.

Appendix A
Search strategy for MEDLINE-PubMed: Sperm AND (oxidative OR reactive oxygen species OR free radicals OR oxidative stress OR ROS OR DNA damage OR DNA fragmentation OR 8-Oxo-2 -deoxyguanosine OR oxidative damage OR superoxide OR hydrogen peroxide) AND (ICSI OR Intracytoplasmic Sperm Injection OR IVF OR In vitro fertilization OR Insemination) AND (fertilization OR Blastocyst OR embryo OR Pregnancy).