I= ROLE OF SPERM DNA INTEGRITY IN HUMAN INFERTILITY FROM PAKISTANI POPULATION
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Title of Thesis
ROLE OF SPERM DNA INTEGRITY IN HUMAN INFERTILITY FROM PAKISTANI POPULATION

Author(s)
Laiq Ahmad
Institute/University/Department Details
Department of Biological Sciences/ Quaid-i-Azam University, Islamabad
Session
2006
Subject
Biological Sciences
Number of Pages
212
Keywords (Extracted from title, table of contents and abstract of thesis)
sperm dna integrity, human infertility, infertility problems, semen, in vitro fertilization, intracytoplasmic sperm injection, assisted reproductive technique, sperm morphology, sperm motility, male factor infertility, semen volume, sperm concentration, sperm motility percentage, motility gradations, sperm morphology, sperm deformity index, teratozoospermic index, oligo-astheno-teratozoospermia

Abstract
Though it is well known that 15% of the couples are facing infertility problems, and more than a quarter of infertility problems due to the male factors. Semen analysis is the most important laboratory investigation for men assessing the infertile couples. The role of semen analysis in modem reproductive practice, such as in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) including all assisted reproductive techniques (ART), can not be neglect able. Sperm morphology and sperm motility is considered to be more important prognostic parameters in male infertility evaluation. The sophistication brought into sperm morphological examination by the inclusion of the Kruger strict morphological criteria, teratozoospermic index (TZI) and sperm deformity index (SDI) is enhancing the predictive power and reproducibility of this parameter.

The present study sought to characterize the association between different categories of infertility and semen quality on the basis of modified semen parameters in Pakistani population. The study population consisted of 34 fertile (without any history of fertility problem) and 187 infertile males (with history of primary infertility). The men provided semen samples and other relevant information, which can predict male factor infertility. Semen volume, sperm concentration, sperm motility percentage, motility gradations, normal and abnormal sperm morphology (Kruger criteria), sperm defects categories, sperm deformity index (SDI) and teratozoospermic index (TZI) were measured. In teratozoospermics (TZs) and idiopathics (IDs) there was no significant difference in sperm concentration and motility percentage compared to fertile subjects. Fertile subjects and IDs showed significantly (P<0.001) the lowest mean morphologically abnormal sperm percentage compared to remaining categories of infertile subjects. IDs and TZs had significantly (P<0.001) the highest mean head defects in sperms compared to remaining categories of infertile subjects. OATs showed significantly (P<0.001) the highest mean head-midpiece-tail defects compared to other categories of infertile subjects and fertile subjects. Overall head, midpiece and tail defects in sperm population was found significantly the highest in OATs compared to fertile and other categories of infertile subjects. Mean sperm deformity index (SDI) and teratozoospermic index (TZI) of ATs and OATs was significantly (P<0.01) higher than TZs, IDs and fertile subjects.

In another set of observations, the role of the age was studied on the seminal parameters of fertile subject and the four categories of infertile subjects. The protocol used for semen analysis was the similar as described earlier. In all age groups the seminal parameters results from fertile subjects and all other categories of infertile subjects were consistent with that of the overall results, which has been described earlier. According to multiple regression model among infertile subjects highly significant (P<0.05) negative trend was observed for sperm concentration and sperm motility percentage but sperm midpiece defects were significantly (P<0.05) increased with advance in age. TZs showed significantly lower sperm count (P<0.001) and motility percentage (P<0.05) in older age (40-49 years) compared to younger ones (20-29 years). Mean normal sperm morphology percentage from fertile subjects and all categories of infertile subjects was non significantly declined with the increase in age.

In third set of experiment, the sperm DNA damage was estimated in the raw, swim up, percoll density gradient centrifugation procedure from fertile and all categories of infertile subjects by the comet assay. In addition, other part of experiment explained the sperm DNA damage trend in three different age groups of fertile and infertile subjects. Results indicated that infertile subjects from the three sperm preparation methods showed significantly (P<0.01) higher sperm DNA damage compared to fertile subjects. However Percoll density gradient centrifugation procedure had significantly lower sperm DNA damage compared to raw and swim up procedures from either fertile or infertile subjects. TZs from three sperm preparation methods had significantly (P<0.001) highest sperm DNA damage compared to fertile and other categories of infertile subjects. Sperm DNA damage indicating comet parameters were higher in IDs compared to fertile subjects. Moreover, within the three sperm preparation methods, Percoll density gradient centrifugation procedure showed the highest sperm DNA integrity indicating comet parameters. Sperm DNA damage indicating parameters including mean comet numbers, comet tail length, comet tail DNA percentage, tail moment and olive tail moment from fertile and all categories of infel1ile subjects was increased with the increase in age.

In the final set of experiment, sperm DNA damage was estimated in the cryopreserved raw, swim up and Percoll density gradient centrifugation preparation from fertile (n=34) and infertile subjects (n=166) with their categories. Samples were cryopreserved after sperm preparation and thawed after several weeks for comet analysis. Other part of experiment explained the sperm DNA damage trend in three different age groups of fertile and all categories of infertile subjects. Sperm DNA damage parameters, including mean comet number, comet length, comet tail length, comet tail DNA percentage, tail moment and olive tail moment, from infertile subjects with cryopreserved three sperm preparation methods were significantly (P<0.001) higher compared to fertile subjects. However, Percoll density gradient centrifugation preparation either from fertile or infertile subjects showed lowest sperm DNA damage compared to other sperm preparation methods. TZs, once again, with three sperm preparation methods had (P<0.001) highest sperm DNA damage compared to other categories of infertile subjects and fertile subjects. Sperm DNA damage indicating parameters from fertile and all categories of infertile subjects were increased with the increase in age. Moreover, trend of sperm DNA damage in fertile and infertile subjects with cryopreserved samples was consistent with that of fresh samples as explained earlier with some exceptions. The level of sperm DNA damage (mean comet number) was significantly (P<0.01) increased in cryopreserved infertile subject's samples compared to fresh samples.

In conclusion, the present study suggests that

Oligo-astheno-teratozoospermia is the most sever type of infertility with the least semen parameters

Teratozoospermic and IDs had higher number of sperms with head defects and may reflect the cause of infertility. Whereas OATs had the highest number of sperms with head-midpeice-tail defects.

Semen quality especially sperm morphology parameters is decline with the increase in age.

Higher sperm DNA damage was estimated in infertile subjects as compared to fertile subjects. Percoll density gradient centrifugation sperm preparation procedure was found best to separate the sperm with higher DNA integrity either from fertile subjects or from infertile subjects. TZs had the lowest level of sperm DNA integrity may be due to the highest level of the sperm head defects. Sperm DNA damage was found higher in IDs as compared to fertile subjects which may give the notion for cause of infertility and can not be explained by conventional semen analysis. According to current results, aging may elevate the level of sperm DNA damage.

Cryopreservation process may decrease the sperm DNA integrity in infertile subjects and may consider the additive factor in causing sperm DNA damage.

The results obtained with comet assay using the cryopreserved sperm were reproducible and comparable with those obtained from fresh human sperm.

Cryopreservation may be good tool in ART procedures.

Comet analysis parameters including comet number, comet tail DNA percentage, tail moment and olive tail moment may have the equal prognostic ability to measure the sperm DNA damage.

No single parameter of the semen analysis can predict the human male factor fertility or infertility. Semen analysis alone can not predict the cause of male infertility or miscarriages after the usage of ART like IVF or ICSI treatments. Hence, sperm DNA integrity evaluation may be a better prognostic or diagnostic and cost effective tool for the selection of patients for ART treatment as well as may help in prediction of success of the treatment.

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S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
649.79 KB
2 1 Chapter One 5
880.93 KB
  1.1 Abstract 5
  1.2 Introduction 6
  1.3 Materials And Methods 10
  1.4 Results 16
  1.5 Discussion 32
3 2 Chapter Two 39
739.18 KB
  2.1 Abstract 39
  2.2 Introduction 40
  2.3 Materials And Methods 44
  2.4 Results 45
  2.5 Discussion 61
4 3 Chapter Three 66
1957.58 KB
  3.1 Abstract 66
  3.2 Introduction 67
  3.3 Materials And Methods 74
  3.4 Results 82
  3.5 Discussion 122
5 4 Chapter Four 129
2637.19 KB
  4.1 Abstract 129
  4.2 Introduction 130
  4.3 Materials And Methods 134
  4.4 Results 139
  4.5 Discussion 175
  4.6 General Discussion 183
  4.7 References 190