Journal of Fertilization: In Vitro - IVF-Worldwide, Reproductive Medicine, Genetics & Stem Cell Biol
Open Access

The Interest of Circulating DNA in the Management of Infertility, Especially in Men

Author info »

Introduction

The use of circulating or free nucleic acids as potential biomarkers in the management of human infertility, particularly in obstetric gynecology, is no longer to be discussed. They constitute of diagnostic and/or prognostic tools of choice in human pathology because they are easily detectable in biological fluids [1], their level varies according to the physiopathological conditions of the body and finally their involvement in many biological processes steroidogenesis permatogenesis. They are commonly used in the management of female infertility as non-invasive biomarkers in the detection and/or monitoring of pathologies of pregnancy, fetal and/or embryonic anomalies and finally in the evaluation. The functional state of the ovary. In this literature review, we will explore the different components of circulating nucleic acids and try to provide the necessary scientific data to justify their interest and their possible applications in PMA.

Free Nucleic Acids

Free nucleic acids constitute a new source of diagnostic and/or prognostic biomarkers in human pathology. Recent data suggests that they play a vital role in the management of male infertility. Free nucleic acids are free DNA (cfDNA ) (They are molecules present in the form of short (70 to 200 base pairs) or long fragments (up to 21kb) in the body) [2], and free RNA (cfRNA) which consists of nucleotides of three large non-coding RNAs: Piwi interacting RNA (piRNA) (of the order of 24 to 31 nucleotides and having the function of blocking the activity of mobile elements present in DNA), micro-RNA (miRNA) (short strands of non-coding RNA of the order of 19 to 25 nucleotides and whose main role is to block the translation of proteins in the mRNA to which they are attached) and Small interfering RNA (siRNA) (they interfere with RNA in the same way as microRNAs and capable of specifically bind to an mRNA sequence) (Figure 1) [3,4].

Figure 1. Summary diagram of free nucleic acids.

Circulating Nucleic Acids and Female Infertility

Even if at present, very few studies have focused on the determination of free nucleic acids in female fertility, it has been shown that there is an increase in the levels of free DNA and/ or microRNAs circulating in the body. Particularly in the plasma and follicular fluids of patients with implantation failures [5-8]. These variations in serum levels could be related to increased cell lysis that is not recognized etiologically. The level of cell-free DNA varies according to the pathophysiological conditions of the organism [9]. However, there appears to be a close relationship between cell-free nucleic acid levels in follicular fluids, maternal plasma, but also embryonic culture media and embryo quality [10]. Thus, the choice of embryos would not only depend on the simple morphological aspect, but also on its environment, which is more or less rich in acellular DNA. Several studies have shown that the expression of microRNAs varies in certain pathologies responsible for female infertility, such as endometriosis [11,12], micropolycystic ovary syndrome (POCS) [13] and premature ovarian failure (POF) [14]. Indeed, Wang et al. reported increased expression of miR-199a and miR-222 and decreased expression of miR-145 and miR-542-3p in patients with endometriosis [15]. In addition, Murri et al, demonstrated that the expression of miR- 21, miR-27b, miR-103 and miR-155 is higher in SOMPK [16]. According to Yang et al, increased expression of miR-146a, miR- 23a, miR-27a and miR-126 and decreased expression of let-7c and miR-144 are observed in the serum of patients with’ POF [17] (Figure 2). This likely impact on fertility could radically alter the practice of reproductive biology. Indeed, one of the major problems of medically assisted reproduction is the low rate of implantation and pregnancy of some patients. It is only in the last fifty years that the existence of non-coding cell-free DNA fragments has been demonstrated in the different biological fluids of the organism. Thus, the determination of these acellular DNA fragments would allow non-invasive prediction of endometrial and oocyte qualities and indirectly embryonic qualities [18-21] of certain patients with implantation failures.

Figure 2. Free nucleic acids, biomarkers of interest in medically assisted procreation.

Circulating Nucleic Acids and Male Infertility

In addition to the female side and implantation, the exploration of male fertility has made many advances. Cell-free DNA is detectable in human semen. Its concentration in semen is much higher than in other body fluids [22]. Its presence and concentration are directly correlated with semen parameters such as velocity, morphology and motility [23]. According to Li et al, the level of cfDNA is higher in the seminal plasma of patients with defective sperm parameters [24]. These observations explain the use of cfDNA in the search for biological markers of sperm quality. Steger's team detected elevated levels of Prm1 and Prm2 and mRNA in semen obtained from patients with failed in vitro fertilization (IVF) compared to those with successful in vitro fertilization [25]. Bansal et al, also found a probable correlation between sperm DNA profile and male infertility [26]. The expression of microRNAs present in semen can be a very interesting approach and a valuable aid in the current practice of semen quality prediction. According to the team of Salas-Huetos et al, human semen contains a stable population of microRNAs related to embryogenesis and spermatogenesis [27]. However, Salas-Huetos et al, showed that miR-34-P, miR-132-3P, mir30C-5P and miR-375 play a role in cell cycle progression and sperm differentiation [27]. In human spermatozoa, piRNAs account for 11% of cfRNAs [28]. PiRNAs are used in the assessment of sperm quality and offer new perspectives for diagnosis, prognosis and treatment in the management of male infertility [29]. In a small cohort, Li et al, showed that seminal acellular DNA levels were significantly higher in azoospermic patients than in patients without sperm abnormalities [30].

Information on Free DNA Detection (CfDNAs) Genetics and Epigenetics

Semen is a mixture of fluids resulting from various secretions from both testes, epididymis, seminal vesicles, Cowper's glands and prostate [30]. The epigenetic information contained in seminal DNA plays an important role in spermatogenesis. It is involved in the reorganization and condensation of the germ cell genome during maturation. Indeed, it reflects the epigenetic aberrations of the testes [31], the problems of male infertility [32].

Non-Obstructive Azoospermia

Due to lack of adequate stimulation by intrinsic gonadotropins or testicular insufficiency, non-obstructive azoospermia is diagnosed in about 10% of infertile men [33,34]. According to Drabovich's team, non-obstructive azoospermia is diagnosed in about 10% of infertile men [35]. The concentration of CfDNA is much higher in semen than in other human body fluids, with a mean value of 1.34 pg/ml in normozoospermia and 2.56 pg/ml in azoospermia [36]. In a recent analysis of the different mRNA and microRNA profiles of patients with non-obstructive azoospermia (NOA) and patients with obstructive azoospermia compared to normozoospermic controls, the team of Li et al, noted differences in profiles compared to normozoospermic control [30]. At the same time, Wang et al, performed studies in patients with non-obstructive azoospermia (NOA) compared to fertile men, and noted a strong decrease in the expression of seven microRNAs (miR-346-5p, miR-122, Mir 149 ± 5p, miR181a, miR-374b, miR-509, and miR-513a-5p) in seminal plasma of NOA patients compared to control [36] (Table 1). However, in a study conducted by Gunes' team on patients with azoospermia, they found additional expression of miR-34c-5p, miR-122, miR-146b-5p, miR-181a, 374b, miR-509-5p, and 513a-5P that is strongly increased in asthenozoospermia [36] (Table 1). Similarly, Wu's team analyzed testicular tissue from patients with ADD and found a significant increase in the expression of miR-141, miR-429 and miR-7-1-3p in plasma. seminal NOA compared to fertile controls [37].

Diseases	Sample	MicroRNA	CfDNA	Reference
Azoospermia	Sperm	Figure 1. Surgical training questionnaire		[50]
		Figure 1. Surgical training questionnaire miR-346-5p, miR-122, Mir 149 + -5p, miR181a, miR-374b, miR-509 and miR-513a-5p		[52] [35]
Asthenozoospermia	sperm	miR-141, miR-429, miR-7-1-3p and HSA-miR-27a		[39] [40]
Teratozoospermia	Sperm	SAH-miR-19b-3p, hsa-miR-28- 5p, SAH-miR-148B, 106B and mir-5P		[37]
Oligozoospermia	Sperm	mir-34C-5p, Mir-122, miR-14BB-5P, miR -181A, miR-374b, miR-509-5p, miR-531a-5P, miR-21, miR-22, miR-19b and miR-7bis		[41] [42]

Table 1: Summary table of Changes in the level of free DNA and miRNAs in some pathologies linked to sperm quality.

Teratozoospermia, Asthenozoospermia and Oligozoospermia

Male fertility can be affected by abnormalities such as: reduced mobility (asthenozoospermia), abnormal morphology (teratozoospermia), undetectable sperm (azoospermia) or a decrease in sperm count (oligozoospermia).

Teratozoospermia: Teratozoospermia is characterized by the presence of more than 85% morphologically abnormal spermatozoa. In the exploration of useful markers to better compensate for male infertility, the team of Milardi D et al, compared the expression profiles of spermatozoa from men with teratozoospermia and found a decreased expression of SAH-miR-19b-3p, hsa-miR-28- 5p, SAH-miR-148B and 106B-mir- 5P compared to controls (Table 1) [38]. This shows the value of using these miRNAs as biomarkers for the management of male infertility.

Asthenozoospermia: In asthenozoospermic patients, the team of Boissonnas et al, noted an increase in the expression of SAHmiR548c- 5p, SAH-miR548c-5p, and SAH-miR-27a, SAH-mi- 548b -5p -548d-5P compared to normozoospermic controls [39]. Abu-Halima and colleagues noted dysregulation of miR-34b-3p in asthenozoospermic patients compared to normozoospermic patients [40]. Similarly, increased expression of HSA-miR-27a was observed in asthenozoospermic patients (Table 1) [41].

Oligozoospermia: The team of Wang et al, compared the miRNA expression profiles of normal and oligozoospermic patients and observed a significantly lower level of expression of miR-34C-5p, miR-122, miR-14BB-5P, miR-181A, miR-374b, miR-509-5p and miR-531a-5P at the control level compared to oligozoospermic samples [42]. The team of Wu et al, found high expression of miR- 19b and miR-7bis in patients with oligospermia [37]. Assou et al, found a significant increase in the expression level of miR-21 and miR-22. However, finding a cut-off value for the diagnosis and prognosis of male infertility remains a problem (Table 1) [43].

Idiopathic Infertility and Other Functional Sperm Defects (DNA Fragmentation, ROS, Methylation, Proteomics, MI-RNA)

Idiopathic male infertility is defined as the absence of a causal factor in sperm analysis when the sperm has abnormalities such as azoospermia, oligozoospermia, asthenozoospermia or teratozoospermia [44]. It can be affected by other factors such as: DNA fragmentation, oxidative stress, methylation or protein.

DNA fragmentation

The DNA fragmentation index is an important factor in the etiology of male infertility [45] or even a good indicator of the potential of conventional semen parameters [46]. Indeed, spermatozoa with normal parameters may have DNA damage [47]. A study on the etiology of age-related male infertility showed that increased levels of the DNA fragmentation index can lead to reduced sperm motility [48]. Similarly, the team of Twyma-Saint Victor et al, in a published study of three patients with male infertility, found that the proportion of sperm with DNA fragmentation ranged from 4.4 to 28% compared to a DNA fragmentation percentage of 1.20 ± 0.95% in controls [49].

Apart from the DNA fragmentation index, various conditions such as chromatin decondensation index, stress and methylation can affect males [50].

Oxidative stress

Even though a low concentration of reactive oxygen species (ROS) is necessary for the critical steps of fertilization, namely capacitation, acrosome reaction and fusion between oocyte and spermatozoon [51]. It appears that cellular stress in all its forms can lead to an increase in the rate of the DNA fragmentation index and affect male fertility. Moreover, the team of Twigg et al, have shown that seminal reactive oxygen species (ROS) levels can lead to sperm damage resulting in male infertility. However, the lack of consensus on the pathophysiological limits of reactive oxygen species (ROS) remains the crucial problem [52].

Methylation

DNA methylation modifies the genetic material. It is a causal factor in infertility [53]. Increased levels of cyclic adenosine monophosphate (cAMP) are a negative factor for normal sperm motility and morphology. The teams of Poongotha and al, have shown that methylation contributes to the increase in Mesodermal Specific Transcriptase (MEST) in association with abnormal sperm parameters and male infertility [54].

Proteomics

In seminal fluid, sperm accounts for 10% of the total volume of ejaculation while 90% is a diverse molecular composition. The specific protein concentration provides a rich source of potential biomarkers in the assessment of male fertility [53]. The team of Mitsumoto observed in infertile men a decrease in the protein (DJ- 1-A) involved in the regulation of oxidative stress [55]. Diamandis also found a positive correlation between the seminal concentration of Prostaglandin-D-Synthase (PTGDS) with the mobility and normal morphology of spermatozoa [56]. The proteomic study conducted in the search for biological markers of azoospermia conducted by Bieniek showed that proteins such as, PTGDS, ACRV1, LGALS3BP, ECM1 and TEX101 are seminal biomarkers for the evaluation of male infertility [57].

Seminal Technical Approach CFDNAS from the Animal Model to Humans

The development of biomarkers for the diagnosis of male infertility, the provision of assistance for drug development and its application at the human level cannot be possible without having gone through the animal model, including the mouse [58]. The experts in reproductive biology use several animal models. But in this part, we will only talk about the proteomic technology of sperm. The mouse model led to the identification of 52 proteins at the spermatic level [59]. Bleil et al., from the sperm of the boar have identified the surface proteins of the spermatozoa responsible for connecting the spermatozoon to the oocyte [60]. The list of animals used as models is very long. The dog remains the best experimental model for comparative studies in humans because of the similarity of the prostate [61]. The main challenge according to Naughton is the translation of knowledge acquired from its animal models to the male infertility clinic for an improvement of existing treatment, the development of an accurate diagnosis and the formulation of male contraceptives with minimal side effects [62].

Conclusions

The use of nucleic acids as biomarkers of male fertility remains an innovative approach and is extremely promising because it offers new perspectives from a diagnostic as well as prognostic points of view. This is because of the relationship between the level of CfDNA and the presence or absence of spermatozoa. In addition, it is a noninvasive procedure and therefore reduces the risks the patient is exposed to.

Competing Interests

Authors have declared that no competing interests exist.

References

1. Jing Swaminathan R, Butt, AN. Circulating nucleic acids in plasma and serum: Recent developments. Ann NY Acad Sci. 2006; 1075(1): 1-9.
2. Lichtenstein AV, Melkonyan HS, Tomei LD, Umansky SR. Circulating nucleic acids and apoptosis. Ann NY Acad Sci. 2001; 945(1): 239-249.
3. Sirotkin AV, Ovcharenko D, Grossmann R, Lauková M, Mlyncek M. Identification of MicroRNAs controlling human ovarian cell steroidogenesis via a genome-scale screen. J Cell Physiol. 2009; 219(2): 415-420.
4. Hayashi K, de Sousa Lopes SMC, Kaneda M, Tang F, Hajkova P, Lao K, et al. MicroRNA biogenesis is required for mouse primordial germ cell development and spermatogenesis. PloS One. 2008; 3(3): e1738.
5. Ewigman BG, Crane JP, Frigoletto FD, LeFevre ML, Bain RP, McNellis D, et al. Effect of prenatal ultrasound screening on perinatal outcome. Taiwan J Obstet Gynecol. 1993; 329(12): 821-827.
6. Akers JC, Gonda D, Kim R, Carter BS, Chen CC. Biogenesis of extracellular vesicles (EV): Exosomes, microvesicles, retrovirus-like vesicles, and apoptotic bodies. J Neurooncol. 2013; 113(1): 1-11.
7. Fernandez-Mercado M, Manterola L, Larrea E, Goicoechea I, Arestin M, Armesto M, et al. The circulating transcriptome as a source of non-invasive cancer biomarkers: Concepts andcontroversies of non-coding and coding RNA in body fluids. J Cell Mol Med. 2015; 19(10): 2307-2323.
8. Lo YD, Zhang J, Leung TN, Lau TK, Chang AM, Hjelm NM. Rapid clearance of fetal DNA from maternal plasma. Am J Hum Genet. 1999; 64(1): 218-224.
9. Traver S, Assou S, Scalici E, Haouzi D, Al- Edani T, Belloc S, et al. Cell-free nucleic acids as non-invasive biomarkers of gynecological cancers, ovarian, endometrial and obstetric disorders and fetal aneuploidy. Hum Reprod Update. 2014; 20(6): 905-923.
10. Kopreski MS, Benko FA, Kwak LW, Gocke CD. Detection of tumor messenger RNA in the serum of patients with malignant melanoma. Clin Cancer Res. 1999; 5(8): 1961- 1965.
11. Dasí F, Lledó S, García-Granero E, Ripoll R, Marugán M, Tormo M, et al. Real-timequantification in plasma of human telomerase reverse transcriptase (hTERT) mRNA: A simple blood test to monitor disease in cancer patients. Lab Invest. 2001; 81(5): 767-769.
12. Brass AL, Huang IC, Benita Y, John SP, Krishnan MN, Feeley EM, et al. The IFITM proteins mediate cellular resistance to influenza A H1N1 virus, West Nile virus, and dengue virus. Cell. 2009; 139(7): 1243- 1254.
13. Bartel DP. MicroRNAs: Genomics, biogenesis, mechanism, and function. Cell. 2004; 116(2): 281-297.
14. Wright EL, Eisenhardt PR, Mainzer AK, Ressler ME, Cutri RM, Jarrett T, et al. The wide-field infrared survey explorer (WISE): Mission description and initial on-orbit performance. Astron J. 2010; 140(6): 1868.
15. Ketting RF, Fischer SE, Bernstein E, Sijen T, Hannon GJ, Plasterk RH. Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans. Genes Dev. 2001; 15(20): 2654-2659.
16. Lecellier CH, Dunoyer P, Arar K, Lehmann-Che J, Eyquem S, Himber C, et al. A cellular microRNA mediates antiviral defense in human cells. Science. 2005; 308(5721): 557-560.
17. Levy S, Sutton G, Ng PC, Feuk L, Halpern AL, Walenz BP, et al. The diploid genome sequence of an individual human. PLoS One. 2007; 5(10): e254.
18. Otsuka M, Jing Q, Georgel P, New L, Chen J, Mois J, et al. Hypersusceptibility to vesicular stomatitis virus infection in Dicer1-deficient mice is due to impaired miR24 and miR93 expression. Immunity. 2007; 27(1): 123-134.
19. Pederson EL, Vogel DL. Male gender role conflict and willingness to seek counseling: Testing a mediation model on college-aged men. J Couns Psychol. 2007; 54(4): 373.
20. Calin GA, Liu CG, Ferracin M, Hylop T, Spizzo R, Sevignani C, et al. Ultra-conserved regions encoding ncRNAs are altered in human leukemias and carcinomas. Cancer Cell. 2007; 12(3): 215- 229.
21. Abu-halima M, Backes C, Leidinger P, Keller A, Lubbad AM, Hammadeh M, et al. MicroRNA expression profiles in human testicular tissues of infertile men with different histopathologic patterns. Fertil Steril. 2014; 101(1): 78-86.e2.
22. Aravin AA, Sachidanandam R, Girad A Fejes-Toth K, Hannon GJ. Developmentally regulated piRNA clusters implicate MILI in transposon control. Science. 2007; 316(5825): 744-47.
23. Mustoe AM, Brooks CL, Al-hashimi HM. Hierarchy of RNA functional dynamics. Annu Rev Biochem. 2014; 83(9): 441-466.
24. Spindler KLG, Pallisgaard N, Vogelius I, Jakobsen A. Quantitative cell free DNA, KRAS and BRAF mutations in plasma from patients with metastatic colorectal cancer during treatment with cetuximab and irinotecan. Int J Cancer. 2012; 18(4): 1177-1185.
25. Steger K, Fink L, Failing K, Bohle RM, Kliesch S, Weidner W, et al. Decreased protamine-1 transcript levels in testes from infertile men. Mol Hum Reprod. 2003; 9(6): 331-336.
26. Bansal SK, Gupta N, Sankhwar SN, Rajender S. Differential genes expression between fertile and infertile spermatozoa revealed by transcriptome analysis. PloS One. 2015; 10(5): e0127007.
27. Iversen F, Yang C, Dagnaes-Hansen F, Schaffert DH, Kjems J, Gao S. Optimized siRNA-PEG conjugates for extended blood circulation and reduced urine excretion in mice. Theranostics. 2013; 3(3): 201-209.
28. Liu L, Liu C, Lou F, Zhang G, Wang X, Fan Y, et al. Activation of telomerase by seminal plasma in malignant and normal cervical epithelial cells. J Pathol. 2011; 225(2): 203-211.
29. Tsang J, Zhu J, Van Oudenaarden A. MicroRNA-mediated feedback and feed forward loops are recurrent network motifs in mammals. Mol Cell. 2007; 26(5): 753-767.
30. Salas-Huetos A, Blanco J, Vidal F, Godo A, Grossmann M, Pons MC, et al. Spermatozoa in patients with seminal alterations have a differential microribonucleic acid profile. FertilSteril. 2015; 104(3): 591-601.
31. Gormally E, Caboux E, Vineis P, Hainaut P. Circulating free DNA in plasma or serum as biomarker of carcinogenesis: Practical aspects and biological significance. Mutat Res. 2007; 635(2-3): 105-117.
32. Salvi S, Gurioli G, De Giorgi U, Conteduca V, Tedaldi G, Calistri D, et al. Cell-free DNA as a diagnostic marker for cancer: Current insights. Onco Targets Ther. 2016; 96(5): 6549-6559.
33. Drabovich AP, Sara P, Järvi K, Diamandis EP. Seminal plasma as a diagnostic fluid for disorders of the male reproductive system. Nat Rev Urol. 2014; 11(5): 278-288.
34. Gunes S, Arslan MA, Hekim GNT, Asques R. The role of epigenetics in idiopathic male infertility. J Repro Ass Gene. 2016; 33(5): 553-569.
35. Wu Y, Liang D, Wang Y, Bai N, Tang W, Bao S, et al. Correction of a genetic disease in mouse via use of CRISPRCas9. Cell Stem Cell. 2013; 13(6): 659- 662.
36. Stroun M, Maurice P, Vasiooukhin V, Lyautey J, Lederrey C, Lefort F, et al. The origin and mechanism of circulating DNA. Ann NY Acad Sci. 2000; 906(197): 161-168.
37. Milardi D, Grande G, Vincenzoni F, Castagonla M, Marana R. Proteomics of human seminal plasma: Identification of biomarker candidates for fertility and infertility and the evolution of technology. Mol Reprod Dev. 2013; 80(5): 350-357.
38. Boissonnas CC, Jouannet P, Jammes H. Epigenetic disorders and male subfertility. Fertil Steril. 2013; 99(3): 624-631.
39. Abu-Halima M, Ludwig N, Hart M, Leidinger P, Backes C, Keller A, et al. Altered micro-ribonucleic acid expression profiles of extracellular microvesicles in seminal plasma of patients with oligoasthenozoospermia. Fertil Steril. 2016; 106(5): 1061-1069.e3.
40. Wang C, Yang C, Chen X, Yao B, Yang C, Zhu C, et al. Altered profile of seminal plasma in the molecular diagnosis of male infertility. Clin Chem. 2011; 57(12): 1722-1731.
41. Zhang W, Xia W, Lv Z, Xin Y, Ni C, Yang L. Liquid biopsy for cancer: Circulating tumor cells, circulating free DNA or exosomes? Cellular Physiol Biochem. 2017; 41(2): 755-768.
42. Assou S, Al-Edani T, Haouzi D, Philippe N, Lecellier CH, Piquemal D, et al. MicroRNAs: New candidates for the regulation of the human cumulus–oocyte complex. Hum Reprod. 2013; 28(11): 3038- 3049.
43. Singh RP, Shafeeque CM, Sharma SK, Singh R, Mohan J, Sastri KVH, et al. Sperm transcriptome profiling by microarray analysis. Genome. 2015; 59(3): 185-196.
44. Costa F, Barbisan F, Assmann CE, Araujo NKF, de Oliveira AR, Signori GP, et al. Seminal cell-free DNA levels measured by PicoGreen fluorochrome are associated with sperm fertility criteria. Zygote. 2017; 25(2): 111-119.
45. Li HG, Huang SY, Zhou H, Liao AH, Xiong CL. Quick recovery and characterization of cell-free DNA in seminal plasma of normozoospermia and azoospermia: implications for non-invasive genetic utilities. Asian J Androl. 2009; 11(6): 703-709.
46. Twyma-Saint Victor C, Rech AJ, Maity A, Regan R, Pauken KE, Stelekati E, et al. Radiation and dual checkpoint blockade activate non-redundant immune mechanisms in cancer. Nature. 2015; 520(7547): 373-377.
47. Twigg JP, Irvine DS. Aitken RJ. Oxidative damage to DNA in human spermatozoa does not preclude pronucleus formation at intracytoplasmic sperm injection. Hum Reprod. 1998; 13(7): 1864-1871.
48. Mitsumoto A. Nakagawa Y. DJ-1 is an indicator for endogenous reactive oxygen species elicited by endotoxin. Free Radic Res. 2001; 35(6): 885-893.
49. Diamandis EP, Arnett WP, Foussias G. Seminal plasma biochemical markers and their association with semen analysis findings. Urology. 1999; 53(3): 596-603.
50. Poongotha JENS, Gopenath TS, Manoyaki SW. Genetics of human male infertility. Singapore Med J. 2009; 50(4): 336-347.
51. Bleil JD, Wassarman PM. Mammalian sperm-egg interaction: identification of a glycoprotein in mouse egg zonae pellucidae possessing receptor activity for sperm. Cell. 1980; 20(3): 873-882.
52. Agarwal A, Said TM. Role of sperm chromatin abnormalities and DNA damage in male infertility. Hum Reprod Update. 2003; 9(4): 331-345.
53. Naughton CK, Nangia AK. Agarwal A. Varicocele and male infertility: Part II: Pathophysiology of varicoceles in male infertility. Hum Reprod Update. 2001; 7(5): 473-81.
54. Avendano C, Franchi A, Taylor S, Morshedi M, Bocca S, Oehninger S. Fragmentation of DNA in morphologically normal human spermatozoa. Fertil Steril. 2009; 91(4): 1077-1084.
55. Alminana-Brines C. Snooping on a private conversation between the oviduct and gametes/embryos. Mol Reprod Dev. 2015; 12(3): 366-374.
56. Aitken RJ, Koppers AJ. Apoptosis and DNA damage in human spermatozoa. Asian J Androl. 2011; 13(1): 36-42.
57. Handel AE, Ebers GC, Ramagopalan SV. Epigenetics: Molecular mechanisms and implications for disease. Trends Mol Med. 2010; 16(1): 7-16.
58. Arifin D, Aston VJ, Liang X. CoFe2O4 on a porous Al2O3 nanostructure for solar thermochemical CO2 splitting. J Cell Mol Med. 2012; 5(11): 9438-9443.
59. Pilch B, Mann M. Large-scale and high confidence proteomic analysis of human seminal plasma. Genome Biol. 2006; 7(5): R40.
60. Kamath SD, Rahman AMA, Komoda T. Impact of heat processing on the detection of the major shellfish allergen tropomyosin in crustaceans and molluscs using specific monoclonal antibodies. Food Chem. 2013; 141(4): 4031-4039.
61. Ong KT, Perdu J, De Backer J. Effect of celiprolol on prevention of cardiovascular events in vascular Ehlers-Danlos syndrome: A prospective randomized, open, blinded-endpoints trial. Lancet. 2010; 376(9751): 1476-1484.
62. De Jong M, Maina T. Of mice and humans: Are they the same? - Implications in cancer translational research. J Nucl Med. 2010; 51(4): 501-504.