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1993 - 2008

Apoptosis in Male Infertility

Apoptosis in Male Infertility
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Apoptosis is an essential part of spermatogenesis that keeps the number of proliferating germ cells in check so that their numbers are within the supportive limit of the Sertoli cells. Recent reports suggest that a dysregulated apoptotic pathway can result in poor semen quality in terms of a low sperm concentration, malformed spermatozoa and higher levels of DNA damage.

We are examining whether a connection exists between OS and dysregulated apoptosis factors. Many have been proposed that OS may be responsible for this dysregulated apoptosis and hence cause the DNA damage. This hypothesis is being tested via a series of experiments that would expose subsets of ejaculated spermatozoa to induce OS and determine whether this induces any apoptotic activity in them. The extent of apoptotic activity visualized and the resulting DNA damage could help illustrate a complete molecular pathogenesis of sperm DNA damage and may provide clues to possible strategies for its prevention and treatment.

  • PDF File (agradoc123.pdf 119 Kb)
    Wang X, Sharma RK, Sikka SC, Thomas AJ Jr, Falcone T, Agarwal A (2003):
    Oxidative stress is associated with increased apoptosis leading to spermatozoa DNA damage in patients with male-factor infertility.
    Fertil Steril 80:531-535.
  • PDF File (agrach015.pdf 50 kb)
    Paasch U, Agarwal A, Gupta AK, Sharma RK, Grunewald S, Thomas AJ Jr, Glander H-J (2003):
    Apoptosis signal transduction and the maturity status of human spermatozoa.
    In: Apoptosis: From Signaling Pathways to Therapeutic Tools, (Edited by: Marc Diederich), RSL/Centre Universitaire de Luxembourg, Luxembourg, Ann N Y Acad Sci 1010:486-488.
  • PDF File (agradoc131.pdf 167 Kb)
    Moustafa MH, Sharma RK, Thornton J, Mascha E, Abdel-Hafez MA, Thomas AJ Jr, Agarwal A (2004):
    Relationship between ROS production, apoptosis and DNA denaturation in spermatozoa from patients examined for infertility.
    Hum Reprod 19:129-138.
  • PDF File (agradoc136.pdf 150 Kb)
    Said TM, Paasch U, Glander H-J, Agarwal A (2004):
    Role of caspases in male infertility. Review article.
    Hum Reprod Update 10:39-51.
  • PDF File (agradoc138.pdf 298 Kb)
    Paasch U, Grunewald S, Agarwal A, Glander H-J (2004):
    The activation pattern of caspases in human spermatozoa.
    Fertil Steril 81:802-9.
  • PDF File (agradoc158.pdf 517 Kb)
    Paasch U, Sharma RK, Gupta AK, Grunewald S, Mascha E, Thomas AJ Jr, Glander H-J, Agarwal A (2004):
    Cryopreservation and thawing is associated with varying extent of activation of apoptotic machinery in subsets of ejaculated human spermatozoa.
    Biol Reprod 71:1828-37.
  • PDF File (agradoc164.pdf 122 Kb)
    Agarwal A, Said TM (2005):
    Oxidative stress, DNA damage and apoptosis in male infertility - a clinical approach. Review article.
    BJU International 95:503-7.
  • PDF File (agradoc173.pdf 179 Kb)
    Grunewald S, Paasch U, Said TM, Sharma RK, Glander H-J, Agarwal A (2005):
    Caspase activation in human spermatozoa in response to physiological and pathological stimuli.
    Fertil Steril 83:1106-12.
  • PDF File (agradoc204.pdf 254 Kb)
    Said TM, Agarwal A, Grunewald S, Rasch M, Baumann T, Kriegel C, Li L, Glander H-J, Thomas AJ Jr, Paasch U (2006):
    Selection of non-apoptotic spermatozoa as a new tool for enhancing assisted reproduction outcomes: an in-vitro model.
    Biol Reprod 74:530-7.
  • PDF File (agradoc235.pdf 356 Kb)
    Aziz N, Said T, Paasch U, Agarwal A (2007):
    The relationship between sperm apoptosis, sperm morphology and the sperm deformity index.
    Human Reproduction 22:1413-19.
  • PDF File (agradoc240.pdf 224 Kb)
    Grunewald S, Said T, Paasch U, Baumann T, Agarwal A, Glander H-J (2007):
    Relationship between sperm apoptosis signaling and oocyte penetration capacity.
    Int J of Androl. Jun 15 (Epub ahead of print)
  • Mahfouz R, Sharma R, Said T, Erenpreis J, Agarwal A (2008):
    Association of sperm apoptosis and DNA ploidy with sperm chromatin quality in human spermatozoa.
    Fertil Steril (In Press).
  • Jha R, Agarwal A, Mahfouz R, Paasch U, Grunewald S, Sabanegh, E, Yadav S, Sharma R (2008):
    Determination of Poly (ADP-ribose) polymerase (PARP) homologues in human ejaculated sperm and its correlation with sperm maturation.
    Fertil Steril (In Press).

Development of Diagnostic Composite Scores


We have developed diagnostic scores by combining two or more semen parameters using statistical modeling and are studying the predictive value of these scores for semen quality.

A. ROS-TAC score

Both ROS and TAC are markers of oxidative stress. Neither of these markers alone has the ability to effectively predict the susceptibility of spermatozoa to oxidative stress. We therefore developed a statistical model using principal components analysis that includes both ROS and TAC values to obtain a composite score, the ROS-TAC score. Healthy male volunteers with normal semen characteristics and ROS and TAC values below the reference values were assigned a score. Patients evaluated with various diagnoses had ROS-TAC scores below the cutoff score assigned to the fertile males. ROS-TAC is a better predictor of oxidative stress than either ROS or TAC alone.

  • PDF File (agradoc71.pdf 169 Kb)
    Sharma RK, Pasqualotto FF, Nelson DR, Thomas AJ Jr, Agarwal A (1999):
    The reactive oxygen species - total antioxidant capacity score is a new measure of oxidative stress to predict male infertility.
    Hum Reprod 14:2801-2807.
  • Pasqualotto FF, Sundaram A, Sharma R, Borges E, Pasqualotto E, Agarwal A (2007):
    Semen quality and oxidative stress scores in fertile and infertile patients with varicocoele.
    Fertil Steril (In Press)
  • Pasqualotto FF, Sharma RK, Pasqualotto EB, Agarwal A (2008):
    Poor semen quality and ROS-TAC scores in patients with idiopathic infertility.
    Urologia Internationalis. (In Press)


B. Semen Quality score

Semen characteristics include both macro and microscopic parameters in evaluating semen quality. In addition, sperm morphology (WHO and strict criteria) and sperm motion characteristics are also evaluated in a clinical lab. However, the significance of these parameters by themselves is not established, especially the sperm motion characteristics. We therefore derived a composite semen quality score to narrow down the ability of a single score to differentiate donors from infertile patients. The semen quality score provides an advantage over individual semen parameters.

  • PDF File (agradoc116.pdf 307 Kb)
    Agarwal A, Sharma RK, Nelson DR (2003):
    New semen quality scores developed by principal component analysis of semen characteristics.
    J Androl 24:343-352.
  • PDF File (agradoc195.pdf 180 Kb)
    Nallella KP, Sharma RK, Allamaneni SSR, Agarwal A (2005):
    Identification of male factor infertility using a novel semen quality score and reactive oxygen species levels.
    Clinics 60:317-24.
  • PDF File (agradoc237.pdf101 Kb)
    Pasqualotto FF, Sundaram A, Sharma RK, Borges E Jr, Pasqualotto EB, Agarwal A (2007):
    Semen quality and oxidative stress scores in fertile and infertile patients with varicocele.
    Fertil Steril. May 5; (Epub ahead of print)


C. Sperm Deformity Index

Sperm morphology assessment is riddled with subjectivity, and several sperm classification systems and study designs have been used. However, the sophistication of sperm morphological examination has increased in recent years with the inclusion of the strict morphological criteria and sperm deformity index (SDI), which have enhanced this parameter's predictive power and reproducibility. SDI is calculated by dividing the total number of deformities observed by the number of sperm that were randomly selected and evaluated, irrespective of their morphological normality. Borderline forms that are considered abnormal include: 1) spermatozoa with slightly elongated head with loss of its oval shape; 2) those with rounded heads and intact acrosome; 3) those with normal heads and a thickened midpiece. We have shown that SDI and acrosomal status are accurate measurements of sperm functional competence and are highly correlated with fertilization in vitro.

  • PDF File (agradoc134.pdf 65 Kb)
    Aziz N, Saleh RA, Sharma RK, Esfandiar, N, Thomas AJ Jr, Agarwal A (2004):
    Novel association between sperm reactive oxygen species production, sperm morphological defects and the sperm deformity index.
    Fertil Steril 81:349-354.
  • PDF File (agradoc181.pdf 85 Kb)
    Said TM, Aziz N, Sharma RK, Lewis-Jone, I, Thomas AJ Jr, Agarwal A (2005):
    Novel association between sperm deformity index and oxidative stress-induced DNA damage in infertile male patients.
    Asian J Androl 7:121-6.
  • PDF File (agradoc235.pdf356 Kb)
    Aziz N, Said T, Paasch U, Agarwal A (2007):
    The relationship between sperm apoptosis, sperm morphology and the sperm deformity index.
    Human Reproduction 22:1413-19.

Intracellular ROS Measurement


Chemiluminescence is a common method to measure ROS in semen, and both extra- and intracellular ROS can be measured using luminal as the probe. Although rapid and easy, the limitation of this assay is the sample volume and the number of sperm necessary to conduct the test. This can be overcome by using new probes specific for hydrogen peroxide and superoxide anion. A total of 10,000 events are measured by flow cytometry. We currently are standardizing the use of specific probes to measure intracellular levels of common free radicals in seminal ejaculates such as hydrogen peroxide and superoxide anion radical. Dichlorodihydro (DCH) and hydroethidium (HE) bromide are being examined as new probes for measurement of intracellular ROS.

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Last Update : December 11, 2008
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American Center for Reproductive Medicine
The Cleveland Clinic
10681 Carnegie Avenue, X11
Cleveland, OH 44195 USA
Tel: (216) 444-9485
Fax: (216) 445-6049
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