Association of sperm oxidative stress and mitochondrial DNA mutation in Iraqi infertile men

Other Title(s)

العلاقة بين الإجهاد ألتأكسدي للنطف و الطفرات في دنا الما يتوكندريا في الرجال العراقيين غير الخصبين

Dissertant

Muhsin, Naim Muhammad

Thesis advisor

al-Azzawi, Hasan Fayyd Samir
Salman, Ihab Dawud

Comitee Members

al-Qazzaz, Abd al-Karim A.
al-Alujy, Sabah Nasir
Farid, Yahya Y. Z.

University

University of Baghdad

Faculty

College of Science

Department

Department of Biology

University Country

Iraq

Degree

Master

Degree Date

2013

English Abstract

-This study was conducted on infertile subjects attending to Al-Karama and Al-Zahra Teaching Hospitals in Kut city during the period October 2012 to June 2013.

Sperm analysis and hormone assays were conducted, as part of their investigations for infertility.

Semen specimens of 27 asthenospermic subjects and 23 oligospermic in addition, 30 healthy fertile men with normal semen parameters according to World Health Organization (WHO) criteria were enrolled as controls.

The three groups were similar as regards mean age (21-45 years of age).subjects had no systemic diseases were non smokers and had no alcohol dependence, and none were taking an oral antioxidant supplement for three months prior to the study.

All semen samples were collected by masturbation following 3 days of abstinence.

After liquefaction, semen volume, sperm concentration, total sperm count, morphology , motility grades were determined using WHO standard procedures .All major determinations were carried out in duplicate.

Determination of sperm DNA damage by comet assay using alkaline method revealed that sperm cell of control showed normal DNA integrity with no tail.

Oligospermic group showed very less tail in cells while, in asthenospermic group showed typical comet with long tail indicating heavy damage to the DNA.

These data indicated that male infertility was associated with oxidative stress leading to oxidative sperm DNA damage.

Therefore, sperm DNA damage could be a suitable early bio-marker to monitor the environmental or occupational infertility toxicity.

By using the long-range PCR techniques and the primer sets L3–H6, the existence of large-scale deletions of mtDNAs in human spermatozoa from 50 infertile males and 30 fertile men was for the first time screened in Iraq peoples and results revealed four major bands (with lengths of ~8360, 3383, 1015, and 761 bp, respectively on the gel after ethidium bromide staining.

The 8360 bp band was the full-length PCR product from the Summary II wild type mtDNAs, the 3383 bp band was generated from the 4977 bp-deleted mtDNAs, the 1015 bp band was obtained from the 7345 bp-deleted mtDNAs, and finally the 761 bp band was amplified from the 7599 bp-deleted mtDNAs.

By using the primer-shift PCR technique, the PCR products with sizes in agreement with the shift in the distance of different primer pairs were obtained by using the primer pairs L6–H5, L6–H6, and L5–H4, the PCR products of 295, 354, and 555 bp respectively was observed from the 7345 bp-deleted mtDNAs.

Similarly, the PCR products of 406 and 702 bp from the 7599 bp-deleted mtDNAs using primer sets of L4–H3, L3–H5 respectively were obtained and nothingness the PCR products by using primer L2–H3 were noticed.

The 4977, 7345, and the 7599 bp deletions of mtDNAs were found more frequently in the spermatozoa with low motility.

These three large-scale deletions were found to occur either alone or in different combinations.

Among them, the 7345 bp deletion was the most frequently seen in spermatozoa with poor motility ,in addition 4977 and 7599 bp-deleted mtDNAs in the spermatozoa from Iraqi infertile males.

Analysis of the nucleotide sequences flanking the breakpoints of the 7599 bp deletion revealed a seven nucleotide direct repeat, which was generally found in the large-scale deletions of mtDNAs.

The seven nucleotide repeat (5-CATCAAC-3) was located in the breakpoints at np 8643/ 8637(ATPase6).

The breakpoint of 7599 bp deletions was located in the hot-regions that were prone to large-scale deletions.

The nucleotide sequences within these regions were found to assume unusual structures, such as bent DNA and anti-bent DNA.

These regions may be thus more vulnerable to attack by free radicals or serve as recognition motifs for certain recombination machinery involved in the largescale deletions.

Analysis of the human mtDNAs revealed five CATCAAC direct repeats, which include np 5617–5611, np 8643–8637, np 13296–13290, np 16079–16073, and np Summary III 16242–16236 on the heavy strand Except for np 5617–5611.

The nucleotide sequences flanking the breakpoints of the 7599 bp deletion revealed a seven nucleotide direct repeat, which is generally found in the large-scale deletions of mtDNAs All polymorphism found in the sequenced mtDNAs regions are summarized.

Briefly, 75 different nucleotide substitutions were identified, of which 16 caused an amino acid change and 22 silent mutation(did not cause an amino acid change) Proven by (www.mitomap.org).

In addition, 37 were considered novel mutations which 5 silent mutation and 32 caused an amino acid change.T8639A, C9195A, deletion( C) np 8673,8640 and insertion( A,C) np 9195 novel polymorphisms were found in the ATPase6 gene, and C14801T, G15216T, A15218T, G15575A, deletion( A) np 14801,14823 and insertion( A) 15398,15427 and 15510 novel polymorphisms were found in the Cytb gene.

Also C3414T, C3417A,T3464A,T3470A,C3476A,A3479,G3483T,G3607T,T3623C,C3782T, ,C3425G , A3787T , deletion( C,CG) np 3420,3421 and insertion( T,A)np 4083 and 4181 novel polymorphisms were found in the ND1 gene.

All these novel mutations cause amino acid changes.DNA sequencing representative chromatograms of novel mutations is performed.

Seminal plasma levels of lipid peroxidation marker, malondialdehyde (MDA) ,enzymatic antioxidant glutathione peroxidase (GPx) superoxide dismutase (SOD), catalase (CAT) and non enzymatic antioxidants glutathione (GSH) , vitamin C, vitamin E , and sperm DNA damage by comet assay using alkaline method were measured sperm DNA damage in men with both Oligospermia and Asthenospermia compared with healthy fertile men .

The seminal plasma levels of MDA was significantly increased(p<0.001) in asthenospermic group more than oligospermic group (p<0.01)compared with healthy fertile normospermic group, the levels of GPx ,SOD and CAT were significantly Summary IV decreased in both oligo and asthenospermic groups (p<0.01) compared with healthy fertile normospermic group while the sperm count and sperm motility were correlated negatively with the levels of MDA in all subject groups(r=-0.67 and r= -0.77 respectively).

On the other hand a significant depletion in seminal plasma levels of GSH , vitamin E levels and vitamin C levels were more observed in asthenospermic than ,oligospermic group compared with healthy fertile normospermic group (P<0.01) .

Testosterone infertile patients had some degrees of disorder in their hormonal status, while the remaining 21(42%) were normal.

Most of the patients (78%) had normal levels of FSH, while only 48% had normal levels of LH.

Testosterone and Prolactin levels were abnormal in 58% and 54% of patients respectively.

The study demonstrated that serum AMH levels of the patient groups were lower than those of the control groups but the difference was not significant ant (P > 0.05).In the present study, a decreased serum AMH level was observed in the oligospermic and asthenospermic groups compared with controls

Main Subjects

Religion

No. of Pages

160

Table of Contents

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Language

English

Data Type

Arab Theses

Record ID

BIM-598046