Influence of sperm DNA damage on human preimplantation embryo metabolism

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Original article

Influence of sperm DNA damage on human preimplantation embryo metabolism Shubhashree Uppangalaa , Shivananda Pudakalakattib , Fiona D’souzaa , Sujith Raj Saliana , Guruprasad Kalthura , Pratap Kumard, Hanudatta Atreyab,c,** , Satish Kumar Adigaa,* a

Division of Clinical Embryology, Kasturba Medical College, Manipal University, Manipal, India NMR Research Centre, Indian Institute of Science, Bangalore, India Solid State and Structural Chemistry Unit, Indian Institute of Science, Bangalore, India d Department of Obstetrics & Gynecology, Kasturba Medical College, Manipal University, Manipal, India b c

A R T I C L E I N F O

Article history: Received 17 February 2016 Received in revised form 22 July 2016 Accepted 23 July 2016 Available online xxx Keywords: DNA damage Spermatozoa NMR Metabolism Preimplantation embryo

A B S T R A C T

Understanding the embryo metabolic response to sperm induced specific abnormalities could help in developing the metabolic markers to prevent the transfer of embryos carrying sperm mediated defects. In this study, NMR based metabolic profiling of the embryo spent media was employed in 34 patients undergoing ICSI cycles. Processed ejaculates were tested for DNA damage using comet assay. Relative intensities of the metabolites from 74 embryo spent media samples from 34 patients and 23 medium controls were profiled using 1H NMR and compared between ‘male-factor’ and control groups. Relative intensities in the subgroups which are independent of patients with male factor or tubal factors, but related to the extent of sperm DNA damage were also compared. Sperm characteristics including DNA damage levels (Olive tail moment, OTM) were significantly different between ‘male factor’ and control groups (P < 0.001–0.0001). Of the metabolites analyzed, glutamine intensity was significantly lower in ‘male factor’ group (P < 0.01) whereas, pyruvate intensity was significantly lower in embryos derived from the processed sperm fraction having <1.0 OTM (P = 0.003). In contrast glutamine and alanine intensities were significantly higher in the embryos derived from sperm population having OTM <1.0. (P = 0.03 & 0.005 respectively). Pyruvate to alanine ratio was significantly lower in <1.0 OTM group (P < 0.0001). This study indicates that increased level of sperm DNA damage in the processed ejaculate affects embryo metabolism which could be related to embryonic genetic integrity. ã 2016 Published by Elsevier Sp. z o.o. on behalf of Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn.

0. Introduction A ‘male factor’ is implicated in approximately 50% of couples treated with Assisted Reproductive Technology [1]. Of the factors known, the relationship between poor semen quality, poor embryonic development and repeated miscarriage has been extensively reported [2,3]. Hence, attention is being given to the diagnosis of structural and genetic defects in the spermatozoa to reduce the risk of sperm mediated abnormalities in the conceptus [4].

* Corresponding author at: Division of Clinical Embryology, Kasturba Medical College, Manipal University, Manipal, 576 104, India. ** Corresponding author at: NMR Research Centre, Indian Institute of Science, Bangalore, 560012, India. E-mail addresses: [email protected] (H. Atreya), [email protected] (S.K. Adiga).

The sperm chromatin is an extremely compact and stable structure and its integrity is essential for the accurate transmission of genetic material to the offspring [5]. However, the association between sperm DNA damage and fertility which is defined in various ways and under various circumstances has been contradictory. Though sperm DNA damage impairs natural conception [6], IVF [7,8], ICSI [8,9] and IUI [10] success, the general conclusion from these studies is that the correlations between DNA damage and reproductive outcome are weak and of variable significance. The most significant associations were observed with natural conceptions, IUI and IVF; whereas, ICSI did not show any correlation [11] even though the process of natural selection is completely bypassed in ICSI. Nevertheless, the potential adverse effects of sperm DNA damage on spontaneous abortions are an important concern [12] which awaits the discovery of potential markers to elucidate the association between sperm pathology and pregnancy loss.

http://dx.doi.org/10.1016/j.repbio.2016.07.004 1642-431X/ ã 2016 Published by Elsevier Sp. z o.o. on behalf of Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn.

Please cite this article in press as: S. Uppangala, et al., Influence of sperm DNA damage on human preimplantation embryo metabolism, Reprod Biol (2016), http://dx.doi.org/10.1016/j.repbio.2016.07.004

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Recent years have seen the development of several noninvasive methods for choosing the most optimal embryo for transfer [13–17]. Indeed, several metabolites including glucose [18] and pyruvate [17] in the embryo spent media have been assayed using various approaches and related to embryo viability and implantation potential [19–24]. Increased uptake of energy substrates such as pyruvate from the culture medium is associated with higher implantation potential in human embryos [17,18]. Studies have also suggested that reduced pyruvate depletion in the medium is linked to the loss of viability [25,26]. Hence there is a need to explore the other metabolites in the medium which have the potential to determine the embryo quality non-invasively. Human embryos can alter their metabolic requirements in relation to their genetic status [27]. Since the natural selection process is completely bypassed in ICSI, there is a raised possibility of selecting genetically abnormal sperm for ICSI which may eventually induce defects in the embryos. Morphology based embryo selection fails to address the genetic integrity; hence we hypothesize that the metabolic uptake by the embryos may be predictive of sperm induced abnormalities in the absence of oocyte pathology. Despite the obvious effect of abnormal sperm characteristics on the reproductive outcome, it is currently unknown whether embryo metabolism is affected by sperm mediated abnormalities. Our group has already demonstrated that pyruvate uptake by human embryos is linked to the implantation potential of the embryos [17]. The embryos having high implantation potential are characterized by relatively higher pyruvate uptake from the medium and lower pyruvate to alanine ratio in the medium. Therefore, the current work was designed to determine whether ICSI derived human embryo metabolism is affected by sperm characteristics especially DNA damage carried by the sperm population at the time of fertilization. 1. Material and methods 1.1. Patient selection This study included thirty four patients (with a total of 97 samples) undergoing assisted reproductive techniques at the University infertility clinic. The patient characteristics included in this study are summarized in Table 1. Kasturba Hospital Institutional Ethics Committee approval was obtained before the initiation of the study. Written informed consent was taken from all the subjects to participate in this study. Only patients undergoing Intra Cytoplasmic Sperm Injection (ICSI) were included in the study to avoid possible interference of spermatozoa and cumulus cells on metabolic signatures. The partners of tubal factor patients had normal sperm characteristics according to WHO

Table 1 Patient demographics and semen characteristics.

Male partner Female partner Sperm concentration (Millions/mL) Sperm count(Millions/ejaculate) Total Motility (%) Normal morphology (%) Sperm DNA fragmentation (OTM) Fertilization rate Top quality embryos (%) Clinical Pregnancy (%)

Age

Control (Mean  SD) (N = 13)

Male factor (Mean  SD) (N = 21)

P value

37.7  5.4 32.2  2.6 53.67  17.03 121.41  56.65 64.5  11.73 35  11.39 0.75  0.3 75.5  20.5 90.9 38.5

36.8  4.8 31  3.2 5.7  8.03 7.2  8.39 35.4  23.5 15.53  9.4 1.35  0.6 70.0  20.8 79.6 28.6

0.4 0.075 <0.0001 <0.0001 <0.001 <0.001 <0.001 0.47 0.33 0.71

(2010), and there was no history of varicocele and hormonal disorders (FSH, LH and Testosterone) in these patients. 1.2. Study group Patients were assigned primarily to two experimental groups based on the clinical and laboratory diagnosis. Of the thirty four patients, thirteen had normal semen profile and tubal pathology in their partners hence considered as ‘control group’. Twenty one patients had abnormal sperm characteristics and partners did not have specific pathology contributing to their infertility problems hence considered as ‘male factor’ group. However, patients were later sub-grouped based on the extent of sperm DNA damage but independent of male factor or control groups. The olive tail moment (OTM), a measure of DNA damage in spermatozoa was quantified from the processed sperm fraction of both male factor and control groups. Sixteen patients had <1.0 OTM out of which four patients were from male factor group whereas eighteen patients had OTM of >1.0 in which two patients were from control group. These groups were used to determine the association between the extent of DNA damage and metabolite intensities. 1.3. Semen sample and sperm preparation Ejaculates were obtained on the day of oocyte pick up and subjects were asked to maintain ejaculatory abstinence of 3–5 days. Upon completion of liquefaction, the sample was mixed well and evaluated for physical and microscopic characteristics according to WHO criteria [28]. Sperm preparation was done by swim up or density gradient technique. 1.4. Single cell gel electrophoresis (neutral comet) assay Single cell gel electrophoresis (neutral comet) assay was performed according to the method described by Singh and Stephens [29] with minor modifications. Briefly, the spermatozoa were suspended in phosphate buffer saline (PBS) and the sperm density was kept constant (50,000 spermatozoa per mL) by appropriate dilution in order to maintain uniformity in distribution of the spermatozoa during electrophoresis. The sperm suspension was mixed with equal volume of 1% low melting agarose (Cat No. A 9414, Sigma Chemical Co, USA) and layered on a slide pre-coated with 1% normal agarose (Cat No. 9539, Sigma Chemical Co, USA). A third coat of agarose was layered over the second layer followed by overnight incubation in lysis solution (2.5 M NaCl, 100 mM disodium EDTA, 10 mM Trizma base, pH 10, 1% Triton X-100, 10% DMSO, 20 Mm DTT) under alkaline conditions (pH 10) at 4 C. Sperm DNA unwinding was carried out for 20 min followed by electrophoretic separation in a buffer (300 mM Sodium acetate and 100 Mm Tris base, pH = 9), 20 V (VcM = 0.74 V/cm, 300 mA) for 60 min. The slides were neutralized in 0.4 M Tris HCl buffer for 10 min and dehydrated in chilled absolute alcohol for 10 min. For visualization, the slides were stained with ethidium bromide (2 mg/mL) and observed under a fluorescent microscope (Imager-A1, Zeiss, Germany). Images were captured under the 40 objective. Each slide was coded to avoid observer’s bias and a minimum of 50 images were captured from each sample randomly, avoiding the anode end and the edges of the slides. Damaged sperm attain the shape of a comet with the tail region consisting of fragmented DNA and the head region intact DNA. DNA damage was quantified using Kinetic Imaging software (Komet 5.5, UK) (Supplementary Fig. 1). The software based quantification of sperm DNA damage provides olive tail moment (OTM) for the individual sperm which is defined as the product of the tail length and the fraction of total DNA present in the tail of the comet [30].

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1.5. Fertilization and embryo evaluation Controlled ovarian hyperstimulation was performed using antagonist protocol. Patients were monitored as per the established protocol and were judged to have adequate follicular response when they had >2 follicles measuring 18 mm or greater in diameter as evidence by the ultrasonography. Oocytes were collected by transvaginal ultrasound guided needle aspiration of the follicles under sedation. Retrieved oocyte cumulus complexes were rinsed, assessed for maturity, and placed in universal IVF medium (Cat # 10310060A, Origio-Medicult, Denmark) at 37  C under 5.5% CO2 in an incubator. Metaphase-II oocytes were fertilized by ICSI under stable laboratory conditions. Injected oocytes were cultured individually in 25 mL droplet of ISM1 medium (Cat # 10500010, Origio-Medicult, Denmark) and incubated at 37  C under 5.5% CO2. After 16–18 h, oocytes were examined for the evidence of fertilization. Morphology based embryo assessment was performed on day 3 of development to select suitable embryos for transfer [31]. After selecting the embryos for transfer, the spent media (ISM1) were placed individually into labeled sterile cryovials, snap frozen in liquid nitrogen, and then stored at 80  C until analysis. For each patient, a sample containing only the medium (medium blank) was incubated along with the embryos. 1.6. NMR sample preparation and data collection Nuclear Magnetic Resonance is a spectroscopic technique in which the hydrogen atoms (specifically their nuclei) when placed in a magnetic field absorb energy upon irradiation. The hydrogen atoms of different molecules absorb at different frequencies (i.e., different energies), which is plotted as a ‘spectrum’, displaying the frequencies in units of chemical shifts (Fig. 1). Further, more the

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number of atoms present, more is the amount of energy absorbed, which in turn, is related to the concentration of the molecule. This is reflected in the height or intensity of the peak. Hence, NMR spectroscopy is a quantitative technique, which provides accurate information on the quantity of a given molecule present in a sample or a mixture. Each sample was thawed for 30 min at room temperature. 15 mL of spent medium was diluted to 550 mL with a solution of 2H2O containing pre-calculated amount of TSP (Sodium salt of 2,2,3,3 tetradeutero 3-(trimethyl silyl) propionate) and transferred to a 5 mm NMR tube for acquiring the spectrum. The stock solution for diluting the samples was prepared by dissolving 3.8 mg of TSP in 250 ml in 2H2O. All spectra were acquired at 298 K on a Bruker AVANCE NMR spectrometer operating at a 1 H resonance frequency of 500 MHz equipped with a triple resonance (1H, 13C and 15N) room temperature probe having a Z-axis shielded gradient. To suppress signals from the protein component of the media, a Carr-PurcellMeiboom-Gill (CPMG) sequence of 20 ms delay was incorporated into the one-dimensional (1D) radio-frequency (r.f.) pulse scheme [32]. The pre-saturation method was used for suppression of the water (H2O) line (r.f. strength = 50 Hz). The 1D 1H NMR spectra was acquired using a 1 H 900 pulse width of 6.25 ms, relaxation delay (trel) of 7 s between scans, spectral width of 7500 Hz and an acquisition time (tmax) of 1.09 s (16 K complex points). The sum total of trel + tmax  8 s was used to ensure that it exceeds 3*T1 of the metabolites being studied [33]. A total of 2048 transients were collected resulting in a measurement time of 4 h 38 min for each sample. The time domain data was apodized with a cosine window function and zero-filled to 65 K points prior to Fourier transform. A total of 97 1D 1H spectra were acquired from thirty four subjects which included 23 medium controls. The assignments of peaks in the 1D spectrum was validated using a 2D [13C, 1H] HSQC spectrum

Fig. 1. 1D 1H NMR spectrum of the ISM1 culture medium used in the study. Representative figure illustrating the assignment of peaks for different metabolites identified by 500 MHz NMR spectroscopy. X-axis represents the chemical shift in parts per million.

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acquired on Bruker AVANCE NMR spectrometers operating at a 1H resonance frequency of 700 MHz and 800 MHz equipped with a cryogenically cooled probe [34]. All data were analyzed using the Bruker TOPSPIN 2.1 software. All peak integrals or peak areas were measured with respect to the corresponding integral/area of the TSP signal (which was normalized to 1.0).

equally distributed between ‘male factor’ and control groups (Table 1). The average number of embryos transferred on day 3 was 2.17. Oocyte quality and the number of top quality embryos transferred were not significantly different between the two groups (data not shown). The implantation rate observed according to the OTM group they originated is 38% (<1.0 OTM) and 26% (>1.0 OTM). However, no statistical significance was observed due to low sample size.

1.7. Data normalization Each sample was prepared in D2O which contains the reference compound: TSP. The TSP peak in each sample is normalized to 1 and with respect to this all other peaks integration value is calculated. All peaks intensity is calculated by taking ratio of peak/ TSP value. Based on the experimental design, the average and standard deviation of peak intensity in a group was taken for comparison between the groups. 1.8. Statistical analysis Data has been represented as the mean  standard deviation (SD) for all the continuous variables whereas categorical variables were represented in percentage form. The difference between the study groups was evaluated by unpaired two sample t-test (homoscedastic t-test) and percentage data was evaluated by Chi-square test. When data did not follow normal distribution, Mann Whitney test was considered instead of unpaired t-test for statistical evaluation. A p-value <0.05 was considered statistically significant.

2.2. Metabolite intensities of embryos differing in developmental potential A total of 97 1D 1H NMR spectra were acquired from the embryos of thirty four subjects including 23 medium controls. The characteristic NMR signals corresponding to components of ISM1 media were assigned [34]. Fig. 1 shows the assignments of the metabolites in a representative 1D 1H spectrum. Each metabolite present in the spent medium appeared as peaks in the NMR spectrum. The peaks pertaining to glucose, lactate, pyruvate and the amino acids; alanine, glutamine, histidine, lysine and methionine appeared clear and distinct thereby facilitating their precise identification with reference to TSP. To determine whether there is a relationship between metabolite intensity and embryo quality, the spent media profiles were also analyzed according to the quality of embryos recorded prior to the transfer. No significant difference in the relative metabolite intensities was observed in relation to embryo morphology (Supplementary Table 1), implying that embryo morphology not correlated to their metabolic profile. 2.3. Impact of sperm characteristics on metabolite intensity

2. Results 2.1. Patient demographics and sperm characteristics The patient demographics and sperm characteristics of subjects included in this study are summarized in Table 1. Of the 34 subjects, 13 had normal semen profile and tubal pathology in their partners hence considered as ‘control group’. The sperm characteristics were abnormal in 21 subjects and partners did not have specific pathology contributing to their infertility problems hence considered as ‘male factor’ group. Sperm parameters were evaluated according to WHO criteria [28] and compared between two groups. All the sperm parameters were found significantly different from control (P < 0.001 to 0.0001). The olive tail moment (OTM), a measure of DNA damage in spermatozoa was also significantly higher in ‘male factor’ subjects in comparison to control (P < 0.001). Patient baseline characteristics such as age of the women, mean fertilization rate, embryo cleavage rates were

A total of 8 metabolites including energy substrates and amino acids were assessed between two groups. Glutamine intensity was significantly lower in male factor group compared to control group (P = 0.02; Table 2). No significant changes in levels of amino acids, glucose and/or other metabolites in the media across the two groups were observed. A moderate but non-significant decrease in intensity was observed for methionine in ‘male factor’ group (Table 2). 2.4. Comparative metabolite intensities in relation to sperm DNA damage The metabolite intensities in spent medium were analyzed with respect to the olive tail moment (OTM) which is a measure of sperm DNA damage. Comparison was made between the samples having <1.0 (Thirty six spent media samples from sixteen patients) and >1.0 (thirty eight spent media samples from eighteen patients)

Table 2 Relative metabolite intensities in control (tubal factor) and male factor group. A total of 21 samples from 13 patients in control category and 53 samples from 21 patients in male factor category were profiled. Metabolites

Energy substrates Pyruvate Lactate Glucose Amino acids Glutamine Alanine Histidine Methionine Lysine a

P valuea

Metabolite intensity in spent embryo culture media (Mean  SD) Control (N = 21)

Male factor (N = 53)

Medium control (N = 23)

0.17  0.05 0.15  0.03 0.025  0.007

0.18  0.05 0.16  0.05 0.026  0.008

0.24  0.05 0.15  0.04 0.03  0.008

0.57 0.34 0.64

0.071  0.02 0.06  0.04 0.024  0.006 0.12  0.05 0.02  0.009

0.057  0.01 0.06  0.03 0.023  0.005 0.094  0.03 0.02  0.009

0.084  0.02 0.07  0.03 0.026  0.006 0.084  0.04 0.017  0.003

0.02 0.14 0.45 0.07 0.47

Corresponds to Control Vs Male factor groups.

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OTM (Table 3). It is important to note that both ‘male factor’ and control groups were sub grouped based on OTM level to understand the association between sperm DNA damage and metabolite intensities. The implantation rate observed was 38% and 26% when subjects were segregated based on OTM (<1.0 and >1.0). Spent media of the embryos derived from the population of spermatozoa having >1.0 OTM had a significantly lower glutamine intensity than the embryos derived from the population of spermatozoa carrying <1.0 OTM (P = 0.03), (Fig. 2A). In contrast, pyruvate intensity was significantly higher in >1.0 OTM group suggesting decreased pyruvate uptake by the embryos in this group (P = 0.003), (Fig. 2B). On the other hand, alanine intensity in the spent media of the embryos derived from >1.0 OTM was significantly lower than their counterparts from <1.0 OTM (P = 0.005) (Fig. 2C). This could be due to high alanine uptake from the medium by the embryos derived from sperm carrying higher DNA damage (>1.0 OTM). If one considers the ratio of pyruvate to alanine concentration for each sample, a significant difference was demonstrated between the embryos derived from sperm carrying high and low DNA damage (P < 0.0001) (Fig. 2D). However, as shown in Table 3, no significant difference was observed in metabolite intensities of other energy substrates and amino acids between two groups. 3. Discussion Metabolic profiling of spent culture media has been proposed as an opportunity to identify non-invasive fingerprints of the pathophysiology in preimplantation embryos. Though recent years have seen the development of several non-invasive methods for choosing the most suitable embryos for transfer [13–17] there has been no report on sperm mediated changes in embryo metabolism. The present study, for the first time, indicates that sperm DNA damage affects ICSI derived embryo metabolism which may provide a potential insight into the sperm DNA damage induced pathophysiological changes in human embryos. The relative changes in the metabolite levels in comparison to the spent medium without an embryo provided the information on those substrates that embryos consumed or released to the medium compared with total amount available. In the present study, glutamine, pyruvate and alanine intensities were significantly changed between the embryos derived from the sperm population having OTM <1.0 and >1.0. These observations suggest that sperm DNA damage mediated pathological changes have altered the uptake of metabolites from the culture medium. Though ICSI is being used as a successful infertility treatment in ‘male factor’ infertility, there is a concern on increased risk of birth

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defects in children conceived by this technique [35,36]. A potential risk of ICSI is the use of apparently morphologically normal spermatozoa but with chromosomal and sub-chromosomal abnormalities. The sperm mediated genetic and epigenetic defects in the embryos are invisible to the conventional morphological analyses typically employed during the selection of embryo for transfer. Hence the primary goal of this study was to look for differential metabolic signatures between the embryos derived from normal and abnormal semen parameters. Though several energy substrates and amino acids were compared between ‘male factor’ and control groups, only glutamine uptake was significantly higher in ‘male factor’ group. Glutamine is a key amino acid involved in several biological processes such as purine and pyrimidine synthesis [37], ATP production [38] and other roles which may have beneficial effect on cleavage stage embryos [39]. It has been earlier shown that aneuploid cells have a tendency to utilize more glutamine from the medium than the euploid cells [40] which suggests an association between genetic integrity and glutamine uptake. Concurrently, Sturmey et al. [27] found that human embryos show a direct correlation between the amount of DNA damage and glutamine uptake. It is possible that, in response to genetic insult, preimplantation embryos can alter protein synthesis [41] especially by utilizing glutamine from the culture medium (reviewed by Sturmey et al. [42]). These observations prompted us to look into the differential uptake of glutamine and other metabolites by the embryos in relation to sperm DNA damage level. It has previously been shown that DNA damaged sperm can fertilize the egg and repair process in the oocyte starts soon after fertilization [43,44]. Though embryos have the ability to repair the damage introduced by the sperm, the hierarchy in embryonic DNA damage response pathways [43–45] may lead to genomic instability in the offspring if the sperm DNA damage level is high at the time of fertilization [46]. Glutamine intensity between high and low sperm DNA damage group was found significantly different suggesting the possible association between glutamine and genetic integrity as described earlier [27,40]. One striking observation was in the pyruvate and alanine intensities between two groups. Pyruvate intensity was significantly lower in the embryos derived from the group with OTM <1.0 suggesting higher uptake by these embryos which is in agreement with our earlier observations where successfully implanted embryos had utilized more pyruvate from the culture medium than embryos that failed to undergo implantation [17]. Though, the embryos were morphologically similar between two groups, decreased pyruvate uptake by the embryos derived from spermatozoa with OTM >1.0 possibly suggests altered genetic and functional integrity in the

Table 3 Relative metabolite intensity in spent embryo culture media in relation to sperm DNA damage. A total of 36 spent media samples from <1.0 OTM group (16 patients) and 38 spent medium samples from >1.0 OTM group (18 patients) were analyzed. Metabolites

Energy Substrates Pyruvate Lactate Glucose Amino acids Glutamine Alanine Histidine Methionine Lysine Pyruvate/alanine a

P valuea

Metabolite intensity in spent embryo culture media (Mean  SD) <1.0 OTM (N = 36)

>1.0 OTM (N = 38)

Medium control (N = 23)

0.16  0.04 0.16  0.05 0.024  0.009

0.2  0.05 0.16  0.065 0.03  0.009

0.24  0.05 0.15  0.04 0.03  0.008

0.003 0.36 0.16

0.072  0.03 0.11  0.03 0.024  0.007 0.13  0.074 0.032  0.06 2.2  1.3

0.057  0.02 0.06  0.06 0.024  0.006 0.19  0.44 0.021  0.009 3.9  1.2

0.084  0.02 0.07  0.03 0.026  0.006 0.084  0.04 0.017  0.003 2.4  1.2

0.033 0.005 0.97 0.37 0.85 <0.0001

Corresponds to <1.0 OTM Vs >1.0 OTM.

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Fig. 2. Bar plot of relative intensities of metabolites in two different sperm DNA damage group (<1.0 OTM and >1.0 OTM). (A) Glutamine intensity was significantly higher in >1.0 OTM group (P = 0.03). B) Pyruvate intensity was significantly higher in >1.0 OTM group (P = 0.003). C) Alanine intensity was significantly lower in >1.0 OTM group (P = 0.005). D) Pyruvate to alanine ratio was significantly perturbed between two groups (P < 0.0001). The data are represented in mean  SD from 36 samples (<1.0 OTM) and 38 samples (>1.0 OTM) groups.

embryos as the pregnancy rate in this group was only 27.8% in comparison to 37.5% observed in the <1.0 OTM group. Based on these results, we speculate that low viability and/or altered genetic integrity in these embryos have affected the uptake of pyruvate. Alanine is readily converted to ammonium in the culture medium. Alanine level in the culture medium showed a strong correlation with developmental potential of the embryos [20]. Hence the level of alanine in the medium can reflect the amino acid turnover and ammonium production. In contrast to pyruvate, alanine intensity in the present study was almost 1.9 fold lower in the group with OTM >1.0 suggesting that these embryos have utilized alanine in large amounts thus its level in the medium was reduced. Since amino acids are used as energy substrates [47] it is possible that the alanine was taken up from the medium in large quantity to meet the energy requirements towards various DNA damage response pathways [41] triggered by fertilization with DNA fragmented sperm in the present situation. Alanine is also known to protect blood cells against oxidative damage and apoptosis [48] which could explain the increased alanine uptake possibly to counteract sperm mediated DNA damage in the embryos. Specific ratios between selected pairs of metabolite levels have been introduced as biomarkers in clinical and experimental medicine. Taking ratios has the advantage that any variability between samples (such concentration and signal-to-noise) is removed. This is because the two metabolites in the same sample will have identical data acquisition parameters. Hence, the ratio of their peak intensities will reflect their genuine relative values with

respect to each other. The metabolite ratio provides several advantages over single metabolite [49] as ratios between related metabolite pairs in the sample reduce the overall biological variability and thereby increase statistical power. Importantly, experimental errors, such as variations in the level of external standards and variations in the relative concentrations of the samples are taken care by this approach and represent biologically the most relevant entity [50]. Recently, it has been shown that pyruvate to alanine ratio can serve as a single biomarker to predict the reproductive potential of human embryos [17]. The increased consumption of alanine and higher levels of pyruvate in the spent medium of embryos with OTM >1.0 (Fig. 2D) demonstrated a significant perturbation in the ratio (P < 0.0001) though embryo quality as assessed morphologically and cell numbers remained unaffected. Hence, instead of individual metabolite intensity, the pyruvate and alanine ratio may serve as a potential marker to select embryos derived from DNA intact sperm. One obvious limitation that must be considered is that the genetic integrity in the embryos was not assessed due to ethical restriction hence correlation between sperm DNA damage level and metabolite intensities was not made. The other limitations that must be considered are (i) lack of information on the quantity of metabolites and turnover, (ii) failure to directly demonstrate the effect of sperm DNA damage on the outcome of embryo transfers due to small sample size and transfer of multiple embryos in our clinical set up, (iii) estimation of DNA fragmentation is restricted to the entire processed fraction as there is no technique to measure DNA damage in a single sperm non-invasively and (iv) unable to

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demonstrate correlation between individual metabolite intensity of implanted embryos with OTM due to low sample size. In summary, for the first time using NMR based approach we found that glutamine, pyruvate and alanine intensities were differentially altered in addition to change in pyruvate/alanine ratio in the spent media samples and this alteration was related to the extent of sperm DNA damage in the processed ejaculates. It is too early to conclude the biological significance behind this interesting observation. Our results suggest that increased levels of sperm DNA damage in the processed ejaculate during ICSI cycles trigger the uptake of glutamine and alanine to counteract the effects of sperm mediated abnormalities in the embryos. These observations provide a potential insight into the sperm mediated pathophysiological changes in human embryo metabolism. It is hoped that the further exploration of this line of research will allow the development of a more efficient non-invasive method to detect embryos carrying sperm mediated abnormalities. Conflict of interest None. Acknowledgements This work was supported by DST-SERC Grant # SR/SO/HS-0080/ 2007. Department of Science and Technology, India. Technical help from Jayalaxmi H. Pai is gratefully acknowledged. HSA acknowledges the facilities provided by NMR Research Centre at IISc, Bangalore supported by Department of Science and Technology (DST), India. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. repbio.2016.07.004. References [1] Seli E, Sakkas D. Spermatozoal nuclear determinants of reproductive outcome: implications for ART. Hum Reprod Update 2005;11(4):337–49. [2] Janny L, Menezo YJ. Evidence for a strong paternal effect on human preimplantation embryo development and blastocyst formation. Mol Reprod Dev 1994;38(1):36–42. [3] Hamamah S, Fignon A, Lansac J. The effect of male factors in repeated spontaneous abortion: lesson from in-vitro fertilization and intracytoplasmic sperm injection. Hum Reprod Update 1997;3(4):393–400. [4] Lansac J. Delayed parenting. Is delayed childbearing a good thing? Hum Reprod 1995;10(5):1033–5. [5] Donnelly ET, O’Connell M, McClure N, Lewis SE. Differences in nuclear DNA damage and mitochondrial integrity of semen and prepared human spermatozoa. Hum Reprod 2000;15(7):1552–61. [6] Giwercman A, Lindstedt L, Larsson M, Bungum M, Spano M, Levine RJ, et al. Sperm chromatin structure assay as an independent predictor of fertility in vivo: a case-control study. Int J Androl 2010;33(1):e221–7. [7] Simon L, Brunborg G, Stevenson M, Lutton D, McManus J, Lewis SE. Clinical significance of sperm DNA damage in assisted reproduction outcome. Hum Reprod 2010;25(7):1594–608. [8] Simon L, Proutski I, Stevenson M, Jennings D, McManus J, Lutton D, et al. damage has a negative association with live-birth rates after IVF. Reprod BioMed Online 2013;26(1):68–78. [9] Zini A, Jamal W, Cowan L, Al-Hathal N. Is sperm DNA damage associated with IVF embryo quality? A systematic review. J Assist Reprod Genet 2011;28 (5):391–7. [10] Bungum M, Humaidan P, Axmon A, Spano M, Bungum L, Erenpreiss J, et al. integrity assessment in prediction of assisted reproduction technology outcome. Hum Reprod 2007;22(1):174–9. [11] Aitken RJ, Bronson R, Smith TB, De Iuliis GN. The source and significance of DNA damage in human spermatozoa; a commentary on diagnostic strategies and straw man fallacies. Mol Hum Reprod 2013;19(8):475–85. [12] Zini A, Meriano J, Kader K, Jarvi K, Laskin CA, Cadesky K. Potential adverse effect of sperm DNA damage on embryo quality after ICSI. Hum Reprod 2005;20(12):3476–80.

7

[13] Brison DR, Houghton FD, Falconer D, et al. Identification of viable embryos in IVF by non-invasive measurement of amino acid turnover. Hum Reprod 2004;19(10):2319–24. [14] Seli E, Sakkas D, Scott R, Kwok SC, Rosendahl SM, Burns DH. Noninvasive metabolomic profiling of embryo culture media using Raman and nearinfrared spectroscopy correlates with reproductive potential of embryos in women undergoing in vitro fertilization. Fertil Steril 2007;88(5):1350–7. [15] Katz-Jaffe MG, McReynolds S, Gardner DK, Schoolcraft WB. The role of proteomics in defining the human embryonic secretome. Mol Hum Reprod 2009;15(5):271–7. [16] Hardarson T, Ahlström A, Rogberg L, Botros L, Hillensjö T, Westlander G, et al. Non-invasive metabolomic profiling of Day 2 and 5 embryo culture medium: a prospective randomized trial. Hum Reprod. 2012;27(1):89–96. [17] Pudakalakatti SM, Uppangala S, D'Souza F, Kalthur G, Kumar P, Adiga SK, et al. NMR studies of preimplantation embryo metabolism in human assisted reproductive techniques: a new biomarker for assessment of embryo implantation potential. NMR Biomed 2013;26(1):20–7. [18] Gardner DK, Wale PL, Collins R, Lane M. Glucose consumption of single postcompaction human embryos is predictive of embryo sex and live birth outcome. Hum Reprod 2011;26(8):2672–80. [19] Devreker F, Hardy K, Van den Bergh M, Winston J, Biramane J, Englert Y. Noninvasive assessment of glucose and pyruvate uptake by human embryos after intracytoplasmic sperm injection and during the formation of pronuclei. Fertil Steril 2000;73(5):947–54. [20] Houghton FD, Hawkhead JA, Humpherson PG, Hogg JE, Balen AH, Rutherford AJ, et al. Non-invasive amino acid turnover predicts human embryo developmental capacity. Hum Reprod 2002;17(4):999–1005. [21] Seli E, Botros L, Sakkas D, Burns DH. Noninvasive metabolomics profiling of embryo culture media using proton nuclear magnetic resonance correlates with reproductive potential of embryos in women undergoing in vitro fertilization. Fertil Steril 2008;90(6):2183–9. [22] Scott RT, Seli E, Miller K, Sakkas D, Scott K, Burns DH. Non-Invasive metabolomic profiling of human embryo culture media using Raman spectroscopy predicts embryonic reproductive potential: a prospective blinded pilot study. Fertil Steril 2008;90(1):77–83. [23] Vergouw CG, Kieslinger DC, Kostelijk EH, et al. Day 3 embryo selection by metabolomic profiling of culture medium with near-infrared spectroscopy as an adjunct to morphology: a randomized controlled trial. Hum Reprod 2012;27(8):2304–11. [24] Hemmings KE, Leese HJ, Picton HM. Amino acid turnover by bovine oocytes provides an index of oocyte developmental competence in vitro. Biol Reprod 2012;86(5):1–12. [25] Guerif F, McKeegan P, Leese HJ, Sturmey RG. A simple approach for COnsumption and RElease (CORE) analysis of metabolic activity in single mammalian embryos. PLoS One 2013;8(8):e67834. [26] Conaghan J, Hardy K, Handyside AH, Winston RM, Leese HJ. Selection criteria for human embryo transfer: a comparison of pyruvate uptake and morphology. J Assist Reprod Genet 1993;10(1):21–30. [27] Sturmey RG, Hawkhead JA, Barker EA, Leese HJ. DNA damage and metabolic activity in the preimplantation embryo. Hum Reprod 2009;24(1):81–91. [28] World Health Organization. WHO laboratory manual for the examination of human semen and sperm-cervical mucus interaction. 5th ed. Cambridge, UK: Cambridge University Press; 2010. [29] Singh NP, Stephens RE. X-ray induced DNA double-strand breaks in human sperm. Mutagenesis 1998;13(1):75–9. [30] Salian SR, Kalthur G, Uppangala S, Kumar P, Adiga SK. Frozen-thawed spermatozoa from oligozoospermic ejaculates are susceptible to in situ DNA fragmentation in polyvinylpyrrolidone-based sperm-immobilization medium. Fertil Steril 2012;98(2):321–5. [31] Alpha Scientists in Reproductive Medicine ESHRE Special Interest Group of Embryology. The Istanbul consensus workshop on embryo assessment: proceedings of an expert meeting. Hum Reprod 2011;26(6):1270–83. [32] Lindon JC, Nicholson JK, Holmes E, Metabonomics Everett JR. Metabolic processes studied by NMR spectroscopy of biofluids. Concepts Magn Reson 2000;12:289–320. [33] Seagle C, Christie MA, Winnike JH, et al. High-throughput nuclear magnetic resonance metabolomic footprinting for tissue engineering. Tissue Eng Part C Methods 2008;14(2):107–18. [34] Pudakalakatti SM, Dubey A, Jaipuria G, Shubhashree U, Adiga SK, Moskau D, et al. A fast NMR method for resonance assignments: application to metabolomics. J Biomol NMR 2014;58(3):165–73. [35] Hansen M, Kurinczuk JJ, Bower C, Webb S. The risk of major birth defects after intracytoplasmic sperm injection and in vitro fertilization. N Engl J Med 2002;346(10):725–30. [36] Davies MJ, Moore VM, Willson KJ, Van Essen P, Priest K, Scott H, et al. Reproductive technologies and the risk of birth defects. N Engl J Med 2012;366 (19):1803–13. [37] Leese HJ. Energy metabolism in preimplantation development. Serono Symposia USA. New York: Springer-Verlag; 1993. [38] Wu F, Orlefors H, Bergstrom M, et al. Uptake of 14C- and 11C-labeled glutamate, glutamine and aspartate in vitro and in vivo. Anticancer Res 2000;20(1A):251–6. [39] Chatot CL, Ziomek CA, Bavister BD, Lewis JL, Torres I. An improved culture medium supports development of random-bred 1-cell mouse embryos in vitro. J Reprod Fertil 1989;86(2):679–88.

Please cite this article in press as: S. Uppangala, et al., Influence of sperm DNA damage on human preimplantation embryo metabolism, Reprod Biol (2016), http://dx.doi.org/10.1016/j.repbio.2016.07.004

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[40] Williams BR, Prabhu VR, Hunter KE, et al. Aneuploidy affects proliferation and spontaneous immortalization in mammalian cells. Science 2008;322 (5902):703–9. [41] Baumann CG, Morris DG, Sreenan JM, Leese HJ. The quiet embryo hypothesis: molecular characteristics favoring viability. Mol Reprod Dev 2007;74 (10):1345–53. [42] Sturmey RG, Brison DR, Leese HJ. Assessing embryo viability by measurement of amino acid turnover. Reprod BioMed Online 2008;17(4):486–96. [43] Shimura T, Toyoshima M, Taga M, Shiraishi K, Uematsu N, Inoue M, et al. The novel surveillance mechanism of the Trp53-dependent s-phase checkpoint ensures chromosome damage repair and preimplantation-stage development of mouse embryos fertilized with x-irradiated sperm. Radiat Res 2002;158 (6):735–42. [44] Toyoshima M, Shimura T, Adiga SK, et al. Transcription-independent suppression of DNA synthesis by p53 in sperm-irradiated mouse zygotes. Oncogene 2005;24(20):3229–35. [45] Adiga SK, Toyoshima M, Shiraishi K, Shimura T, Takeda J, Taga M, et al. p21 provides stage specific DNA damage control to preimplantation embryos. Oncogene 2007;26(42):6141–9.

[46] Adiga SK, Upadhya D, Kalthur G, Bola Sadashiva SR, Kumar P. Transgenerational changes in somatic and germ line genetic integrity of first-generation offspring derived from the DNA damaged sperm. Fertil Steril 2010;93(8):2486–90. [47] Collado-Fernandez E, Picton HM, Dumollard R. Metabolism throughout follicle and oocyte development in mammals. Int J Dev Biol 2012;56(10–12):799–808. [48] Li HT, Feng L, Jiang WD, Liu Y, Jiang J, Li SH, et al. Oxidative stress parameters and anti-apoptotic response to hydroxyl radicals in fish erythrocytes: protective effects of glutamine, alanine, citrulline and proline. Aquat Toxicol 2013;126:169–79. [49] Altmaier E, Ramsay SL, Graber A, Mewes HW, Weinberger KM, Suhre K. Bioinformatics analysis of targeted metabolomics—uncovering old and new tales of diabetic mice under medication. Endocrinology 2008;149(7):3478–89. [50] Petersen AK, Krumsiek J, Wägele B, Theis FJ, Wichmann HE, Gieger C, et al. On the hypothesis-free testing of metabolite ratios in genome-wide and metabolome-wide association studies. BMC Bioinf 2012;13:120.

Please cite this article in press as: S. Uppangala, et al., Influence of sperm DNA damage on human preimplantation embryo metabolism, Reprod Biol (2016), http://dx.doi.org/10.1016/j.repbio.2016.07.004