Do Seminal Isoprostanes Have a Role in Assisted Reproduction Outcome?

F2-isoprostanes (F2-IsoPs), stereoisomers of prostaglandin F2α generated by the free radical-induced oxidation of arachidonic acid, have been associated with different male infertility conditions. This study aimed to evaluate the role of seminal isoprostane levels and sperm characteristics in the reproductive outcome and embryo quality of 49 infertile couples. Semen analysis was performed following WHO guidelines. Sperm chromatin maturity was detected using an aniline blue (AB) assay, and DNA integrity was assessed using the acridine orange (AO) test. Seminal F2-IsoP levels were quantified by gas chromatography/negative ion chemical ionization tandem mass spectrometry (GC/NICI–MS/MS) analysis. Correlations among variables and their impact on in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) outcome were investigated. F2-IsoP levels are positively correlated with double-stranded DNA sperm (p < 0.001) and negatively correlated with mature sperm chromatin (p < 0.001). Patients with positive outcomes had an increased percentage of sperm with double-stranded DNA, as did patients producing high-quality embryo, who showed higher F2-IsoP levels compared to those detected in the low-quality embryo group. An intriguing relationship between a mild increase in F2-IsoP levels, DNA integrity, and embryo quality seems to indicate that the non-enzymatic oxidation of arachidonic acid can be also a marker of metabolic activity in human semen.


Introduction
The life and death of mammalian spermatozoa are mostly ruled by oxidative stress (OS). It is well known that spermatozoa are capable producers of reactive oxygen species (ROS), derived from three potential sources, as revised by Aitken [1]: sperm mitochondria, cytosolic L-amino acid oxidases, and plasma membrane nicotinamide adenine dinucleotide phosphate oxidases.
Small amounts of ROS such as the superoxide anion, hydrogen peroxide, and nitric oxide are important for physiological sperm function, including capacitation, hyperactivation, and acrosomal reaction [2].
However, excessive production of ROS can play a negative effect on sperm concentration, motility, and morphology. In addition, high ROS levels may damage sperm DNA, RNA transcripts, and telomeres and, therefore, might represent a common underlying etiology of male infertility and recurrent pregnancy loss [3]. Polyunsaturated fatty acids

Sperm Preparation
In cases of spermatozoa showing almost total absence of progressive motility, the semen was simply washed in Flushing Medium (Origio, Måløv, Denmark) before being used for ICSI.
In other patients, the standard swim-up technique was used to collect motile and active spermatozoa for IVF and ICSI. The sperm samples were washed with 2 mL of a Flushing Medium (Origio, Denmark) and centrifuged at 200× g for 10 min. The supernatant was discarded, and the pellet was gently over-layered with 0.5-0.8 µL of the medium; the tubes were inclined at 45 • and kept at 37 • C and 5% CO 2 for 45-60 min. Then, 0.2 µL of the uppermost medium containing motile sperm was aspirated with a sterile pipette. Concentration, motility, and morphology were recorded, and the sample was kept at 37 • C until use.

Ovarian Stimulation
The women were treated with an antagonist protocol. According to the patients' age and body weight, the initial dose of gonadotropin was determined to be 150-400 IU/day.
Monitoring started on day 5 of the stimulation, and the dose of gonadotropin was adjusted according to the estradiol (E2) serum concentration and ovarian response, which were examined by ultrasound. When the leading follicles reached 13 mm in diameter, 0.25 mg of Cetrorelix (Merck-Serono, Darmstadt, Germany) was subcutaneously added daily until the day of hCG administration. An intramuscular injection of hCG was made when a minimum of three follicles reached a diameter of ≥18 mm. Oocyte pickup was performed by the ultrasound guide 36 h after the hCG injection.

Acridine Orange (AO) Assay to Evaluate Sperm DNA Susceptibility to Damage
Acridine orange (AO, 3, 6-bis (dimethylamino) acridine, hemi (zinc chloride) salt, BDH Chemicals Ltd., Poole, England) treatment assays are used to assess the sperm DNA vulnerability to acid-induced denaturation in situ by quantifying the metachromatic shift of AO fluorescence from green (double-stranded DNA) to red (denatured DNA). The AO test was carried out as described by Moretti et al. [19]. The slides were observed and scored with a Leitz Aristoplan fluorescence Microscope (Leica, Wetzlar, Germany) equipped with a 490 nm excitation light and 530 nm barrier filter. Three hundred sperm nuclei for each sample were analyzed and classified as green or red (sometimes, orange yellow) depending on fluorescence. The sperm heads that exhibited a green stain had double-stranded DNA; the sperm heads showing a spectrum of yellow orange to red fluorescence had singlestranded DNA. The results were expressed as the percentage of spermatozoa that showed normal DNA (green fluorescence).

Aniline Blue (AB) Test
This test verifies the maturity of the sperm chromatin, since AB shows binding affinity with lysine residues, an amino acid in which histones are rich. It is well known that, during the maturation process, spermatozoa replace most histones with protamines, basic proteins rich in arginine accomplishing chromatin compaction. Therefore, the blue staining of sperm after AB treatment indicates the persistence of histones and the immaturity of spermatozoa.
Two hundred microliters of seminal fluid were washed in phosphate buffer saline (PBS), centrifuged at 400× g for 15 min, and resuspended in PBS; the sample, appropriately diluted, was smeared on slides and air-dried. The slides were fixed with 3% glutaraldehyde in PBS, pH 7.2, for 30 min in a humid chamber at room temperature. Then, the slides were treated for 5 min with AB solution (5% aniline powder and 4% acetic acid, with solution made up to volume with distilled water; pH 3.5). After washing in distilled water,

Determination of Total F 2 -IsoPs
Since F 2 -IsoPs are initially formed in situ on phospholipids (esterified F 2 -IsoPs) and later released into biological fluids as unesterified F 2 -IsoPs (free F 2 -IsoPs), the levels of total F 2 -IsoPs represents the sum of free and esterified F 2 -IsoPs.
At the time of total F 2 -IsoP assay, all seminal fluid samples were subjected to basic hydrolysis by incubation at 45 • C for 45 min in the presence of 1N KOH. Subsequently, all samples were acidified to pH 3 by the addition of 1N HCl, and tetradeuterated prostaglandin F2α (PGF2α-d4, 500 pg), used as an internal standard, was added to each sample.
After a period (15 min) of stabilization in the presence of the internal standard, the samples were purified by solid-phase extraction procedures that, by protocol, involve the three sequences of conditioning, washing, and elution. For seminal fluid samples, an octadecylsilane cartridge (C18, 500 mg, 55-105 µm Particle Size, 6cc, Waters, Milford, MA, USA) was used, through which, after an appropriate conditioning step, the samples were passed, followed by cartridge washes and elution.
Eluates from the C18 cartridge were transferred to a previously conditioned aminopropyl cartridge (NH 2 , 500 mg, 55-105 µm particle size, 6cc, Waters, Milford, MA, USA). After loading the eluate, washes and final elution were performed. The final eluates were collected and then subjected to a derivatization procedure after evaporating the solvent mixture under nitrogen blowing. Specifically, working on the dry residue of the extract, the carboxyl groups of F 2 -IsoP and PGF2α-d4 were derivatized to form pentafluorobenzyl esters, while the hydroxyl groups were converted to trimethylsilyl ethers.

Oocyte Preparation and ICSI and IVF Procedure
Oocytes were recovered from follicular fluid immediately after follicle aspiration. They were washed in Flushing Medium (Origio, Denmark) and then in Sage 1-Step TM with HSA (Origio, Denmark) and were finally incubated in 30 µL micro-drops of the same medium under Mineral Oil (Origio, Denmark) at 37 • C and 6% CO 2 . In four to five hours, cumulus cells were removed using hyaluronidase 20 IU (Origio, Denmark) and microinjected as described by Palermo et al. [21] using a Narishige micromanipulator (IM9B, Narishige, Tokyo, Japan). Eighteen hours after ICSI, the presence of two pronuclei and two polar bodies was checked to verify normal fertilization. The embryos were cultured in Sage 1-Step TM with HSA (Origio, Måløv, Denmark) until embryo transfer, 3 days after oocyte retrieval.
In the case of conventional IVF, the oocytes with cumulus cells were recovered in a dish with the selected spermatozoa (250,000 sperm/mL).

Embryo Score
Before the transfer, the embryos were evaluated morphologically and graded according to a scale [22] from 1 (good-quality embryo) to 4 (poor-quality embryo) based on the assessment of the presence/absence of cytoplasmic fragments, number, and symmetry of blastomeres. In this study, the embryos graded on Veeck's scale as 3 and 4 were considered Life 2021, 11, 675 5 of 12 the group with low-quality embryos and those graded as 1 and 2 belonged to the group with good-quality embryos.

Statistical Analysis
The data were analyzed using SPSS v. 23.0 software package. A significance level of p ≤ 0.05 was adopted. Initially, the Kolmogorov-Smirnov test was used to verify the normal distribution of the variables. To assess the equality of variances or homoscedasticity, Levene's test was performed. To compare the means between independent groups Student's t-test was used.
Spearman's correlation coefficient was employed to evaluate the correlation between the variables. For multiple comparison, the Bonferroni correction was adopted (p < 0.002).
Finally, a binary logistic regression stepwise analysis was conducted on a positive fertilization outcome and embryo quality to identify the variables predictors.
All variables considered were correlated (rho Spearman's coefficient), and the results are shown in Table 1. Interestingly, the F 2 -IsoP levels were positively correlated with doublestranded DNA sperm (p = 0.000, Figure 1a) and negatively correlated with mature sperm chromatin (p = 0.000, Figure 1b). As expected, progressive motility was positively correlated with sperm concentration (p = 0.000) and normal morphology (p = 0.001). The percentage of normal sperm morphology was positively correlated with sperm concentration (p = 0.000) and the percentage of sperm with mature chromatin (p = 0.001). Sperm concentration: sperm/mL × 10 6 ; progressive motility %: slow and rapid motility; normal morphology %: normal sperm morphology assessed by the Papanicolaou modified test, double-stranded DNA%: % of sperm green stained after acridine orange assay; mature chromatin %: % of unstained sperm after aniline blue test; and F2-IsoPs: F2 isoprostanes (ng/mL). Exact p-values have been reported in parentheses; ns represents that no significant p-values (after Bonferroni correction) were obtained.  In both groups (data not shown), the F2-IsoP levels were negatively correlated with the percentage of sperm with mature chromatin and positively correlated with the percentage of sperm with double-stranded DNA, as in the whole patient population. To identify the predictors of a positive fertilization outcome, a binary logistic regression analysis was conducted, and the results are reported in Table 2.  In both groups (data not shown), the F 2 -IsoP levels were negatively correlated with the percentage of sperm with mature chromatin and positively correlated with the percentage of sperm with double-stranded DNA, as in the whole patient population. To identify the predictors of a positive fertilization outcome, a binary logistic regression analysis was conducted, and the results are reported in Table 2.  Table 2. Logistic regression analysis of variables with respect to ART outcome. Legend: OR (odds ratio); CI (confidence interval). Finally, we grouped patients according to embryo quality: the group with low-quality embryos (18 patients, Veeck's scale [22] grades 3 and 4, Figure 3a) and the group with good-quality embryos (30 patients, Veeck's scale [22] grades 1 and 2, Figure 3b). One couple did not obtain any embryo.

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good-quality embryos (30 patients, Veeck's scale [22] grades 1 and 2, Figure 3b). One couple did not obtain any embryo. The semen variables were compared, and the results are shown in Figure 4. The semen variables were compared, and the results are shown in Figure 4. Semen samples producing good-quality embryos showed a higher percentage of sperm with double-stranded DNA (p = 0.046; Figure 4) and increased F 2 -IsoP levels (p = 0.024; Figure 4) compared to those obtaining low-quality embryos. In the group with good-quality embryos, a positive correlation (data not shown) was highlighted between the percentage of sperm with mature chromatin and semen parameters (sperm concentration, progressive motility, and normal morphology, all p = 0.000). In both groups, the F 2 -IsoP levels negatively correlated with the percentage of sperm with mature chromatin (group with low-quality embryos p = 0.008; group with good-quality embryos p = 0.000) and positively correlated with the percentage of sperm with double-stranded DNA% (group with low-quality embryos p = 0.032; group with good-quality embryos p = 0.000). scale as 1 and 2) (b) on day 3 after fertilization are shown. In (a), the embryo presents uneven-sized blastomeres, non-homogenous cytoplasm, and 35-50% fragments. In (b), the embryo presents even sized blastomeres.
The semen variables were compared, and the results are shown in Figure 4. Semen samples producing good-quality embryos showed a higher percentage of sperm with double-stranded DNA (p = 0.046; Figure 4) and increased F2-IsoP levels (p = 0.024; Figure 4) compared to those obtaining low-quality embryos. In the group with good-quality embryos, a positive correlation (data not shown) was highlighted between A weak association between semen F 2 -IsoP levels (OR 1.08, 95% CI 1.01 to 1.15, p = 0.019) and embryo quality was obtained (Table 3). Table 3. Logistic regression analysis of variables with respect to embryo quality. Legend: OR (odds ratio); CI (confidence interval).

Discussion
The relevance of isoprostanes has been demonstrated in many pathological conditions [23], and their role in semen has received growing interest in the field of male infertility.
Recent surveys have reported that seminal 8-iso-PGF2α levels were significantly higher in infertile patients than in fertile men [10][11][12]24,25]. In particular, the levels of these compounds were particularly high in patients affected by varicocele, a pathology characterized by sperm immaturity [12,25].
In this research, we found that F 2 -IsoPs are a sensitive marker of sperm immaturity, detected with aniline blue assays, which evaluate the replacement of histones with protamines, confirming results previously obtained by transmission electron microscopy (TEM) analysis [12,13,25]. In brief, some ultrastructural characteristics of sperm immaturity are the presence of uncondensed chromatin, the presence of cytoplasmic residues, and coiled tails. A relationship between immaturity and increased F 2 -IsoPs levels was also reported in a case of globozoospermia [13], a genetic condition of male infertility characterized by sperm with round heads, the absence of acrosome, and immature chromatin. Defects in sperm chromatin, in particular alterations in histone/protamine replacement, has been associated with low-quality embryo affecting early embryo development [26], and increased sperm DNA fragmentation [27]. Overall, these results suggest that F 2 -IsoP levels are an efficient marker of defective histone replacement, suggesting that sperm maturation could be considered a process related to PUFA content and oxidative metabolism.
The seminal F 2 -IsoPs levels of the individuals considered in this research were similar to those detected in groups of patients affected by oligoasthenoteratozoospermia (23.42 ± 8.36) [10] and idiopathic infertility (24.30 ± 50.4) [12]. These levels were slightly higher than those detected in fertile men [8,9,12] and lower than those measured in semen samples from patients affected by varicocele [8,9,12].
The novelty of this study was the investigation of the relationship between semen isoprostanes, fertilization outcome, and embryo quality. The literature on this topic is very scant; to the best of our knowledge, only one research reported that the 8-iso-PGF 2α levels, evaluated in the urine of men from couples attending fertility treatment, did not show significant associations with reproductive outcomes [28].
The intriguing results of the present research regards the positive effect of F 2 -IsoPs on DNA susceptibility to the damage tested with an acridine orange assay.
In addition, comparing the variables in the two groups based on embryo quality, we observed that the group with good-quality embryos showed both increased percentages of sperm with double-stranded DNA and, unexpectedly, slightly higher F 2 -IsoP levels than the group with low-quality embryos (both p < 0.05).
Analyzing the odds ratio of the variables, a slight influence of F 2 -IsoP levels on embryo quality was confirmed; however, no significant influence was found for double-stranded DNA. This discordant result could be due to the characteristics of the samples considered in this study. DNA integrity seems to play a role in the positive outcome of fertilization. Numerous studies investigated the relationship between DNA integrity and ART outcome [29], even if the results are contradictory, considering different factors such as patient selection, differences in DNA integrity detection, female age, and others. Agarwal et al. [29] reviewed many studies on this topic and concluded that DNA integrity is detrimental to normal fertilization, embryo development, and the success of ART, suggesting that sperm DNA testing is important in male infertility evaluation.
The determination of F 2 -IsoPs represents one of the most reliable approaches to evaluating a condition of endogenous LPO in vivo, and F 2 -IsoPs are considered markers in many pathologies including reproductive ones in which OS may be involved, such as varicocele and leukocytospermia, suggesting that arachidonic acid oxidation/metabolism may influence different male reproductive pathological conditions. However, the results obtained are oriented in another direction that needs to be discussed. First, F 2 -IsoPs are not mere markers of OS, but they also elicit a wide variety of responses in different cell types [23,30]. Evidence of their biological activities are reported in the literature [31][32][33]. In addition, the F 2 -IsoP receptor, thromboxane A2 receptor [31], has been localized in rat spermatids, cells that undergo relevant chromatin modifications [34], and has been demonstrated to be involved in the regulation of gene expression in murine Leydig cells [35]. Then, a potential biological activity of F 2 -IsoPs in the male reproductive system cannot be excluded.
For these reasons, we can hypothesize that the sperm that obtained better embryo quality are those active from a metabolic point of view, so they produce a certain amount of ROS, leading to a moderate increase in F 2 -IsoPs detected in the semen. Therefore, the role of F 2 -IsoPs toward spermatozoa can be different compared to other cells and tissues; F 2 -IsoPs could trigger relevant physiological events as capacitation, and only high concentrations of these molecules play a detrimental effect. These observations agree with those of Pasqualotto et al. [36], who analyzed LPO and total antioxidant capacity (TAC) in follicular fluids. They found that LPO and TAC were lower in patients who did not become pregnant than in patients who did, concluding that LPO may represent a good marker of metabolic activity within the follicle and may be necessary to establish pregnancy.
In infertile patients without inflammatory pathologies, a mild increase in F 2 -IsoP levels could enhance the activation and regulation of the Cation channel of Sperm, CatSper [37][38][39][40], and could play a relevant physiological role in the fertilization outcome and embryo quality.
In addition, the sperm membrane of infertile men showed an altered lipid profile [41] with consistent amounts of arachidonic acid [25] that can negatively affect the capacitation process. A mild increase in isoprostanes in semen with respect to the data reported from fertile subjects [9] suggested that the presence of moderate OS, probably slightly higher than the physiological one, is able to increase LPO but unable to damage DNA. Moderate ROS levels can also be hypothesized by observing the values of progressive motility in our cases that, although decreased with respect to reference values [18], are not severely reduced.
High ROS concentrations are dangerous to spermatozoa, but these gametes evolved the capacity to generate highly reactive molecules as they need them to undergo capacitation and to activate motility [1]. Zhang et al. [42] demonstrated a close association between mitochondrial function, acrosin activity, and the DNA fragmentation index in human spermatozoa. This evidence seems to suggest that mitochondrial functional integrity is necessary to maintain sperm fertilizing ability, also influencing DNA integrity, which is a prerequisite for a high fertilization rate and embryo quality after IVF and ICSI [43][44][45].

Conclusions
The intriguing relationship between a mild increase in F 2 -IsoP levels, DNA integrity, and good embryo quality seems to indicate that F 2 -IsoPs can be a marker of metabolic activity in human semen. We are aware that these are preliminary observations that must be confirmed by other studies, but this possibility deserves attention as it may represent a new role of F 2 -IsoPs in human semen. Data Availability Statement: The data are contained within this article and are available from the corresponding author upon request.

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