Scholarly article on topic 'Light and electron microscopic study of the effect of L-carnitine on the sperm morphology among sub fertile men'

Light and electron microscopic study of the effect of L-carnitine on the sperm morphology among sub fertile men Academic research paper on "Clinical medicine"

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{L-carnitine / "Sub-fertile men" / "Oligo / atheno and terato-zoospermia" / Spermatozoa / "Head defects" / "Midpiece defects" / "Cytoplasmic droplet" / "Mitochondrial sheath defects" / "DNA content of sperm heads"}

Abstract of research paper on Clinical medicine, author of scientific article — Sohair A. Abd El-baset, Samia M. Abd El-Wahab, Aziza M.A. Mansour, Eman A.H. Mohamed

Abstract Objective This work was conducted to evaluate the effect of L-carnitine on sperm morphology in sub fertile patients who need enhance for intra cytoplasmic sperm injection (ICSI) as a method of infertility treatment. Setting Assisted Reproduction Unit, at the International Islamic Centre for Population Studies and Researchs (IICPSR), Al-Azhar University and Zoology Department Faculty of Science (Girl's Branch), Al-Azhar University. Materials and methods According to the routine semen analysis, 85 patients were divided into: Group 1: Ten normal fertile men. Group 2: 25 oligozoospermic cases with sperm count less than 20 million/ml. Group 3: 25 athenozoospermic cases with reduced sperm motility <40%. Group 4: 25 teratozoospermic cases with more than >40% abnormal forms. L. carnitine therapy in the form of Carnivita forte 1gm /tab. b.i.d. for three months were given to groups 2, 3 and 4. All the patients underwent semen analysis before and 90 days after therapy. Group 1 was subjected to two semen analysis 3 months apart as a control. Smears were made on slides, fixed in absolute ethyl alcohol and stained with H&E and methyl green pyronin for light microscopic study. A second semen sample was processed for electron microscopic study. Results In the fertile control group, there have not been any statistically significant differences among the patients at the beginning of the study and after 3 months regarding all the studied parameters. In Oligo group, highly significant decrease in the mean percent of head defects, cytoplasmic droplet and mitochondrial sheath defects were observed after treatment. In both atheno and terato groups, highly significant decrease in the mean percent of head defects, midpiece defects, cytoplasmic droplet and mitochondrial sheath defects were observed after treatment. The mean of DNA content of sperm heads demonstrated highly significant increase in oligo, atheno and terato-zoospermia groups as compared to the same groups before treatment. Conclusion These results clearly show that L-carnitine treatment has ameliorative impact on the quality of spermatozoa in the infertile men, resulting in a decrease number in morphologically abnormal spermatozoa. These data indicated that this agent affects the quality of spermatozoa in infertile patients who need intra cytoplasmic sperm injection (ICSI) as a method for infertility treatment.

Academic research paper on topic "Light and electron microscopic study of the effect of L-carnitine on the sperm morphology among sub fertile men"

Middle East Fertility Society Journal (2010) 15, 95-105

Middle East Fertility Society Middle East Fertility Society Journal

www.mefsjournal.com www.sciencedirect.com

ORIGINAL ARTICLE

Light and electron microscopic study of the effect of L-carnitine on the sperm morphology among sub fertile men

Sohair A. Abd El-baset a, Samia M. Abd El-Wahab a *, Aziza M.A. Mansour b, Eman A.H. Mohamed b

a Faculty of Science (Girl's Branch), Zoology Department, Al-Azhar University, Cairo, Egypt

b International Islamic Center for Population Studies and Research-Assisted Reproduction Unit, Al-Azhar University, Cairo, Egypt

Received 11 April 2009; accepted 28 June 2009 Available online 23 June 2010

KEYWORDS

L-carnitine; Sub-fertile men; Oligo, atheno and terato-

zoospermia; Spermatozoa; Head defects; Midpiece defects; Cytoplasmic droplet; Mitochondrial sheath

defects; DNA content of sperm heads

Abstract Objective: This work was conducted to evaluate the effect of L-carnitine on sperm morphology in sub fertile patients who need enhance for intra cytoplasmic sperm injection (ICSI) as a method of infertility treatment.

Setting: Assisted Reproduction Unit, at the International Islamic Centre for Population Studies and Researchs (IICPSR), Al-Azhar University and Zoology Department Faculty of Science (Girl's Branch), Al-Azhar University.

Materials and methods: According to the routine semen analysis, 85 patients were divided into: Group 1: Ten normal fertile men.

Group 2: 25 oligozoospermic cases with sperm count less than 20 million/ml. Group 3: 25 athenozoospermic cases with reduced sperm motility <40%. Group 4: 25 teratozoospermic cases with more than >40% abnormal forms. L. carnitine therapy in the form of Carnivita forte 1 gm /tab. b.i.d. for three months were given to groups 2, 3 and 4. All the patients underwent semen analysis before and 90 days after therapy. Group 1 was subjected to two semen analysis 3 months apart as a control. Smears were made on slides, fixed in absolute ethyl alcohol and stained with H&E and methyl green pyronin for light microscopic study. A second semen sample was processed for electron microscopic study.

Corresponding author. E-mail address: abdelwahabs@hotmail.com (S.M. Abd El-Wahab).

1110-5690 © 2010 Middle East Fertility Society. Production and Hosting by Elsevier B.V. All rights reserved. Peer-review under responsibility of Middle East Fertility Society. doi:10.1016/j.mefs.2010.04.005

Results: In the fertile control group, there have not been any statistically significant differences among the patients at the beginning of the study and after 3 months regarding all the studied parameters. In Oligo group, highly significant decrease in the mean percent of head defects, cytoplasmic droplet and mitochondrial sheath defects were observed after treatment. In both atheno and terato groups, highly significant decrease in the mean percent of head defects, midpiece defects, cytoplasmic droplet and mitochondrial sheath defects were observed after treatment. The mean of DNA content of sperm heads demonstrated highly significant increase in oligo, atheno and terato-zoospermia groups as compared to the same groups before treatment.

Conclusion: These results clearly show that L-carnitine treatment has ameliorative impact on the quality of spermatozoa in the infertile men, resulting in a decrease number in morphologically abnormal spermatozoa. These data indicated that this agent affects the quality of spermatozoa in infertile patients who need intra cytoplasmic sperm injection (ICSI) as a method for infertility treatment.

© 2010 Middle East Fertility Society. Production and Hosting by Elsevier B.V. All rights reserved.

1. Introduction

Infertility affects approximately 15% of all couples trying to conceive, male infertility is the sole or contributing factor in roughly half of these cases (1). About 25% of all cases were caused by sperm defects (2).

Male infertility is a multifactor disease process with a number of potential contributing causes. The majority of male infertility causes are due to deficient sperm production of unknown origin, environmental and nutritional factors

Mammalian spermatozoa acquire the ability to swim during their transit from the testis to the female oviduct under the control of several external and intracellular factors. These factors play a pivotal role in regulating acquisition of hyper activated motility and during the process of spermatogenesis

(4). Under physiological condition, spermatozoa produce small amounts of reactive oxygen species (ROS), which are needed for capacitation, acrosome reaction and fertilization (5,6).

Normally, equilibrium exists between ROS production and antioxidant scavenging activities in the male reproductive tract. However, excessive amounts of free radicals or ROS produced by leukocytes and mature spermatozoa can cause damage to the normal spermatozoa by inducing lipid per oxidation and DNA damage (6). So, oxidative stress (OS) becomes a potential cause of male infertility (7-10).

A variety of medications have been developed in an attempt to improve the sperm quality and in turn modify the male fertility potential. Many drugs are used as a proposed therapy in male infertility without rationale: such therapy are often prescribed without any positive effect and any imagined improvement in semen parameters are without a real basis and may be caused by natural fluctuations in semen quality (11). The purpose of treatment is to amplify spermatogenesis, boost the highest quality sperm populations, acts on sperm maturation and energetic metabolism and on the testicular epididymal-microenvironments (6).

Growing evidence suggested that L-carnitine or acetyl-L-carnitine or their combination may be helpful for improving sperm quality and function, thereby benefiting male infertility (12). Among this the free L-carnitine is taken up from the blood plasma, transported into the epididymal fluid and the spermatozoa, and accumulated as both free and acetylated L. carnitine (13). The highest levels of L-carnitine in the

human body are found in the epididymal fluid, where its concentration is 2000 times higher than that in the serum. In the epididymis it is secreted into the seminal plasma and can stimulate human sperm maturation, motility (14) and normal spermatogenesis process (15). In contrast, previous authors (16) suggested that lowering of L-carnitine in the cauda epididymis was attributable to the adverse effect on epididymal function to transport and/or concentrate L-carnitine. Since L-carnitine has been reported to have antioxidant potential, antioxidant defense enzymes in the cauda epididymis such as superoxide dismutase (SOD), catalase, glutathione peroxidase and gluta-thione reductase, so it protects sperm membranes against toxic reactive oxygen species (ROS). It also functions to reduce the availability of lipids for per oxidation by transporting fatty acids into the mitochondria for B-oxidation to generate aden-osine triphosphate (ATP) energy (17). Carnitine also protects the cell membrane and DNA against damage induced by ROS (18).

Thus, the aim of this work is to evaluate the effect of L-car-nitine therapy on semen parameters in subfertile patients with oligo-atheno-terato-zoospermia who need enhance intra cyto-plasmic sperm injection (ICSI) as a method for infertility treatment. The post gonad maturation could be a target for therapy especially at the epididymis where sperm maturation and motility take place.

2. Materials and methods

2.1. Patients

Seventy five infertile men and 10 normal patients (control) were the subjects of the study. All cases were collected from the International Islamic Centre for Population Studies and Research (IICPSR)-ART Unit, Al-Azhar University, Cairo, Egypt. Informed consent was obtained from all subjects.

The patients were subjected to the following criteria:

(a) Age was ranged between 25 and 40 years old.

(b) No medical diseases (e.g., liver and kidney diseases).

(c) Mild male factor (no sever male factor or azoospermia).

(d) No previous testicular or scrotal operation.

(e) No varicocele, genital tract infection, or hormonal disturbances.

The patients were also subjected to:

(1) History: Each patient had full history taken regarding age, period of marriage, work, smoking, drug intake, exposure to radiation, chemicals or heat, operation and previous history of chronic medical illness.

(2) Complete semen analysis: (19).

Patients were classified according to the data obtained from complete semen analysis as follows:

Group 1: Fertile men already have offspring with normal semen parameters (10 cases).

Group 2: Oligo-zoospermia cases have reduced number of sperms; sperm count less than 20 million/ml (25 cases). Group 3: Atheno-zoospermia cases have reduced motility of sperms; sperm motility <40% (25 cases). Group 4: Terato-zoospermia cases have >40% of sperm with abnormal forms (25 cases).

The groups 2-4 treated with L. carnitine therapy in the form of Carnivita forte tablet 1 gm. twice a day morning and evening for three months. The semen samples were collected from the study patients at the beginning and after three months of the study. During this period, the regular intercourse among couples and pregnancy rates were recorded.

2.2. Preparation of smear (20)

The smears were stained with Haematoxylin and Eosin (21) for morphological study of sperms and methyl green pyronin (22) for detection of deoxyribonucleic acids (DNA) content of sper-matozoal heads.

2.3. Light microscopic study

In all patients, 500 spermatozoa were examined under oil immersion lens (x1000 magnification) of the microscope-digital camera-computer system and the percentage of abnormalities such as head, midpiece and tail defects as well as the percentage of cytoplasmic droplet were evaluated. Through digital camera, the images were captured to the computer, saved and the DNA contents of spermatozoal heads per 5 fields for each patient before and after treatment were measured by image analyzer. The obtained measured data were exported to Microsoft excel program and tabulated.

2.4. Electron microscopic study

A fresh semen sample was processed in each of the patients for electron microscopy (EM) within one hour of collection,

according to methods described previously (23). In brief, the spermatozoa were washed with phosphate buffer (0.1 mol/l, pH 7.4), pelleted by centrifugation and fixed in 4% glutaralde-hyde followed by 1% osmium tetroxide. The pellets were embedded in Epon Araldite and spermatozoal ultrastructure was analyzed and photographed in a Zeiss electron microscope after staining with Reynold's lead citrate. In all patients, 50 spermatozoa were studied for each cellular structure (microtu-bules, fibrous sheath and mitochondria), and the percentages of mitochondrial sheath, outer dense fibers and axonemal defects were recorded. At least 5 longitudinal and 5 cross-sections per sample were photographed.

2.5. Statistical analysis

The data were analyzed by using statistical analysis software package (24). Paired t-test and Mc Namar's test were used to compare between the studied parameters before and after treatment in each studied group. None paired t-test and Fischer exact test were also used to compare the mean changes in different studied groups regarding the studied parameters. The significant level was set at P < 0.05. The study was approved by Ethics Committee of International Islamic Centre for Population Studies and Research (IICPSR), Al-Azhar University, Cairo, Egypt.

3. Results

The present results revealed no statistically significant differences among the studied oligo, atheno, terato-zoospermia and control groups regarding mean age, infertility duration

Table 2 Characteristics of the studied Group 1 (Fertile

n = 10).

Parameter Distribution

Age 36.8 ± 2.4

Smoking status

non smoker 5 (50%)

smoker 5 (50%)

Volume (ml) 3.2 ± 0.6

Head defects (%) 23 ± 11.5

Mid piece defects (%) 26 ± 10

Cytoplasmic droplet (%) 26 ± 10.7

Tail defects (%) 31 ± 23

DNA content of sperm head 0.985 ± 0.167

Mitochondrial sheath defects (%) 2.5 ± 2.6

Outer dense fibers defects (%) 1 ± 3.1

Axonemal defects (%) 0.5 ± 1.5

Data presented by mean ± SD or n

Table 1 Characteristics of the studied patients groups.

Group Age Infertility years Smoking status No Yes

Oligo-zoospermia (n = 25) 36.4 ± 2.9 4.1 ± 1.6 12 (48) 13 (52)

Atheno-zoospermia (n = 25) 35.0 ± 3.8 3.5 ± 1.9 10 (40) 15 (60)

Terato-zoospermia (n = 25) 33.8 ± 3.7 3.2 ± 1.4 10 (40) 15 (60)

Control (n = 10) 33.3 ± 4.5 4.3 ± 3.1 9(36) 16 (64)

P value 0.18 0.38 0.83

Data presented as mean ± SD or n (%).

Table 3 Comparison between semen parameters before and after treatment among Group 3 (Oligo, n = 25).

Parameter Before treatment After treatment P value

Head defects (%) 21.5 ± 10.8 15.6 ± 11.3 0.001**

Mid piece defects (%) 29.0 ± 14.1 24.2 ± 6.7 0.07

Cytoplasmic droplet% 25.7 ± 7.9 19.0 ± 9.9 0.0003*

Tail defects (%) 23.7 ± 10.4 23.2 ± 9.1 0.32

DNA content of sperm head 0.645 ± 0.14 0.776 ± 0.18 0.0001*'

Mitochondrial sheath defects (%) 20.7 ± 15.5 9.0 ± 11.8 <0.0001*'

Outer dense fibers defects (%) 49.7 ± 20.1 49.7 ± 20.1 1.00

Axonemal defects (%) 28.0 ± 13.6 28.0 ± 13.6 1.00

Data presented by mean ± SD or n (%). Highly significant difference.

and smoking habit (P = 0.18, 0.38 and 0.83, respectively) (Table 1).

Table 2 showed that the mean age of these subjects was 36.8 ± 2.4 and half of them were smokers. The mean volume of semen was 3.2 ± 0.6. The mean of defects was 23% (head), 26% (midpiece) and 31% (tail).The image analysis revealed that the mean DNA content was 0.985 ± 0.167 and EM examination revealed that, the mean percent of dense fibers and axonemal defects were very low.

Table 3 showed that the mean percent of head defects and cytoplasmic droplets had highly significant decrease (P 6 0.001 and 60.0003, respectively) after treatment. However, non significant changes were observed after treatment in this group regarding mid piece and tail defects in comparison to the same group before treatment. The mean percent of

Table 4 Comparison between semen parameters before and after treatment among Group 4 (Atheno, n = 25).

Parameter Before treatment After treatment P value

Head defects (%) 11.7 ± 3.7 6.3 ± 12.3 0.04**

Mid piece defects (%) 40.2 ± 8.8 32.0 ± 8.9 <0.0001

Cytoplasmic droplet (%) 17.2 ± 5.9 5.2 ± 4.4 <0.0001

Tail defects (%) 31.2 ± 9.1 29.0 ± 6.9 0.08

DNA content of sperm head 0.378 ± 0.1 0.427 ± 0.1 0.01**

Mitochondrial sheath defects (%) 27.0 ± 12.6 9.5 ± 9.1 <0.0001

Outer dense fibers defects (%) 24.0 ± 9.1 23.7 ± 11.1 0.33

Axonemal defects (%) 47.5 ± 15.5 47.5 ± 15.5 1.00

Data presented by mean ± SD or n (%). ** Highly significant difference.

Table 5 Comparison between semen parameters before and after treatment among Group 5 (Terato, n = 25).

Parameter Before treatment After treatment P value

Head defects (%) 50.5 ± 14.6 30.3 ± 17.8 <0.0001*'

Mid piece defects (%) 18.5 ± 14.6 13.5 ± 12.2 0.003**

Cytoplasmic droplet (%) 17.5 ± 7.8 4.0 ± 5.9 <0.0001*

Tail defects (%) 19.0 ± 8.05 13.5 ± 8.2 0.001**

DNA content of sperm head 0.284 ± 0.034 0.447 ± 0.12 <0.0001*

Mitochondrial sheath defects (%) 25.0 ± 11.9 15.5 ± 5.1 0.004**

Outer dense fibers defects (%) 25.0 ± 12.1 25.0 ± 12.1 1.00

Axonemal defects (%) 50.0 ± 14.8 50.0 ± 14.8 1.00

Data presented by mean ± SD or n (%).

** Highly significant difference.

Figure 1 Photomicrograph of normal sperms from healthy fertile man. (H&E x 1000).

DNA content showed highly significant increase (P 6 0.0001). However, the mean percent of mitochondrial sheath defects showed highly significant decrease (P 6 0.0001) in comparison to the same group before treatment. No changes were found

regarding the mean percent of the dense fibers and axonemal defects.

Table 4 showed the highly significant decrease regarding to mid piece defect, mitochondrial sheath defects and cytoplasmic

droplets after treatment (P 6 0.0001). However, no significant changes were observed in this group of patients regarding tail defects in comparison to the same group before treatment. The mean DNA content showed highly significant increases (P 6 0.01). No changes were observed regarding the mean percent of the dense fibers and axonemal defects.

There was highly significant decrease in the head, midpiece, tail and mitochondrial sheath defects and cytoplasmic droplets while, the mean DNA content showed highly significant increase (P 6 0.0001) after treatment in comparison to the same group before treatment (Table 5). No changes were observed regarding the mean percent of the dense fibers and axonemal defects.

3.1. Pregnancy rate

Female spouses of 25 Oligo (12%), of 25 atheno (16%) and of 25 Terato zoospermic patients (8%) achieved pregnancy after three months of treatment with L-carnitine.

3.2. Light microscopic results

In healthy group, the smears showed the normal spermatozoa (Fig. 1). Spermatozoa from the terato group before treatment exhibited distinctive departures from the morphology of spermatozoa from healthy group: Rounded head, triple head, pinucleated head, bent neck, thin midpiece, thick midpiece, double tail, triple tail and stump tail syndrome (Fig. 2A-I, respectively).

3.3. Electron microscopic results

Electron microscopic examination showed the spermatozoa with morphological abnormalities in the longitudinal sections of the spermatozoa before treatment.

In healthy fertile group, normal organization of the nucleus and acrosome was noticed. The neck region are showing two

Figure 7 Electron photomicrograph of longitudinal section of human sperm of oligo group after treatment demonstrating, normal head (H), normal acrosome cap (A) and well defined tail (T). But the most proximal segment of the flagellum is enveloped by cytoplasmic droplet (CD). x6000.

opposite planes parallel and perpendicular to the long axis of the proximal centriole. The segmented mitochondria with well defined mitochondrial sheath and helicoidally distribution of mid piece mitochondria was observed (Fig. 3).

In oligo group before treatment, the sample showed small and thin mid piece with defected mitochondria (Fig. 4), vacu-olated and degenerated head and degenerated mitochondria (Fig. 5). In another sample from the same group, abnormal head and defected mitochondria were observed (Fig. 6). After treatment, normal head, normal acrosome cap and well defined tail were noticed (Fig. 7).

In atheno group before treatment, the sample showed abnormal rounded head, degenerated acrosome and retention of a big cytoplasmic droplet at variable loci along tail midpiec-es (Fig. 8). After treatment, normal mid piece and regular microtubule doublets of the axoneme were demonstrated (Fig. 9).

Figure 8 Electron photomicrograph of longitudinal section of human sperm of atheno group before treatment showing, abnormal rounded head (H), degenerated acrosome (A), big cytoplasmic droplet (CD) and abnormal axonemal structure (AS). x3000.

In Terato group before treatment, the sample showed hypoplasia (Fig. 10). After treatment, normal head, normal nuclear pattern and normal arrangement of the mitochondrial structure of the mid piece were demonstrated (Fig. 11).

In the cross-section through the midpiece of healthy spermatozoa, the plasma membrane (PM) and mitochondrial sheath (MS) surrounding the nine outer dense fibers (ODFs). Within the ODFs are the components of the axoneme; the nine outer microtubule doublets of the axoneme (OMDA) with associated dynein arms (DA) and radial spokes (RS) and the central pair of microtubule doublets (CP) (Fig. 12A).

In the oligo-zoospermia group before treatment, some of the abnormalities observed in the midpiece-cross section of spermatozoa, included: an abnormal number and malformed outer dense fibers and malformed mitochondrial sheath (Fig. 12B).

Figure 10 Electron photomicrograph of human sperm of terato group before treatment showing hypoplasia; small head (H), abnormal acrosome (A) and sphericity of the nucleus (N). x4600.

(MDA). x13,000.

(MP). X5000.

In the atheno-zoospermia group, deletion of on outer dense fibers and malformed mitochondria were observed (Fig. 12C).in some cases, the 9 + 2 axonemal structure was completely distorted; central pairs were missing.

In the terato-zoospermia group, absence of some parts of mitochondria was observed, outer dense fibers appeared small, malformed or absent as well as absence of 9 microtubules (Fig. 12D).

In addition to defects in the mid piece, increases in the percent of spermatozoa with abnormalities in the principal piece

of flagella were observed in oligo, atheno and terato-zoo-spermia patients before treatment.

In healthy group, the principal piece-cross section showed the PM surrounding seven ODFs. The ODFs (3,8) have been replaced by the two longitudinal columns of the fibrous sheath (LC). The two columns are connected by transverse ribs (TR). The axonemal components are unchanged (Fig. 13A).

In the oligo-zoospermia group, normal axonemal structure was observed (Fig. 13B).

Figure 12 Electron photomicrographs of cross-sections through the midpiece of control spermatozoon, displaying the 9 plus 2 arrangements of microtubules (M), the 9 outer dense fibers (ODF), and the mitochondrial sheath (MT) (A), as well as spermatozoa from oligo, atheno and terato-zoospermia (B)-(D). The midpiece abnormalities include an abnormal number and malformed outer dense fibers, malformed mitochondrial sheath as well as absence of 9 outer microtubules (B)-(D) and absence of axoneme (C). x20,000.

Figure 13 Electron photomicrographs of cross-sections through the principal piece of a normal spermatozoon displaying the ODFs (3 and 8) have been replaced by the two longitudinal columns of the fibrous sheath (LC) (A) as well as spermatozoa from oligo, atheno and terato-zoospermia (B)-(D). Abnormalities include absence of the axoneme (C) and abnormal number or hemilateral absence of the outer dense fibers (D) and x20,000.

In the atheno-zoospermia group, deletion of central micro-tubules doublets was observed (Fig. 13C).

In the terato-zoospermia group, abnormal number and hemilateral absence of the outer dense fibers, with or without an intact axoneme, and a missing outer dense fiber were noticed (Fig. 13D).

4. Discussion

The sperm morphology was recently regarded as an important predictive mean for fertilization in coordination with unprotected intercourse among couples. With the advancement of assisted reproductive technology, sperm morphology assessment became much more essential for better in vitro fertilization (IVF) results (25). Changes in morphology impair fertility (26). In the present work, changes in the morphology of spermatozoa were assessed by light and electron microscopy.

The fertile group in the present work formed of 10 fertile men fathered a child within a period less than 2 years; their semen parameters were mostly within the values of strict criteria of sperm morphology. This result was in agreement with the previous result (27). No changes were observed regarding mid piece and tail defects. The DNA content in healthy fertile men ranged from 0.985 to 1.67. EM examinations revealed the mean percent of mitochondrial abnormalities and axonemal defects to be very low.

No studies to date have examined the morphology of spermatozoa from human infertile patients at the electron microscope level. In the present study, at the electron microscopic level, there was an increase in the incidence of morphologically abnormal spermatozoa, as well as a larger variety of abnormalities, in infertile men. Several of the midpiece abnormalities in infertile men have been reported. These abnormalities, however, are extremely rare in healthy group. The flagellum is formed during spermatogenesis in the testis, indicating that the increase in abnormalities in infertile men probably reflects a defect in spermatogenesis, rather than epididymal maturation. This defect may be at the gene expression level and may occur as early as during the pachytene spermatocyte phase of spermatogenesis, as this is when the first genes required for flagellar formation are expressed (28). Nevertheless, a number of the flagellar defects found in infertile men were detected only after the spermatozoa left the caput epididymis (29), indicating that the defects observed may also originate during spermatozoal maturation in the epididymis. In murine models, defects in the flagella of spermazoa are frequently associated with infertility (30,31).

In the infertile groups, the elevation of the percentage of mid piece defects and cytoplasmic droplets are likely due to changes taking place in spermatozoa during the process of spermatogenesis in the testis (e.g., formation of the flagellum), whereas others could occur during sperm maturation in the epididymis (e.g., acquisition of motility) (32). In addition, the previous authors (33) stated that the sperms which retained too much cytoplasm in the midpiece during maturation were associated with excessive generation of ROS and initiation of lipid per oxidation which negatively affect fertilization rate. In the present data, DNA content showed no statistically significant difference. The previous authors (6,34) demonstrated an association between the increased level of oxidative DNA

damage in spermatozoa and male infertility in men with oli-go-zoospermia, atheno-zoospermia and terato-zoospermia. In addition, the optical density of DNA contents was significantly decrease in both atheno-zoospermia and terato-zoospermia (2,35,36). Other results (37,38) showed that, the mitochondrial membrane potential (MMP) was decreased in the spermatozoa of infertile men with elevated levels of ROS production. Excessive ROS may cause ATP to deplete rapidly resulting in decreased phosphorelation of axonemal proteins (6).

In the oligo-zoospermia group, the mean percent of head defects and cytoplasmic droplets has been reduced after treatment with statistically significant decrease. No significant changes were observed after treatment regarding mid piece and tail defects. After treatment, the optical density of DNA contents was with high significant increase. EM examination showed that there was significant decrease in the mitochondrial sheath defects with P value 60.0001. However, no changes were found regarding the mean percent of dense fibers and axonemal defects. Previous work showed that L-carnitine necessary for normal functioning of sperm cells and disturbance in mitochondrial function may contribute to abnormal spermato-genesis (6). Also, there was a good increase in sperm count after L-carnitine therapy in a group of patients with idiopathic atheno-zoospermia and treatment with L-carnitine prevents mitochondrial dysfunction (39,40). In contrast, there was no effect of L-carnitine therapy on sperm count (41). In a similar study to evaluate the effect of L-carnitine or L-acetyl carnitine in nutrition treatment for male infertility, found no significant difference in the sperm concentration (15).

In the atheno-zoospermia group, significant decrease (P 6 0.0001) in the mean percent of mid piece defect and cyto-plasmic droplet was observed after treatment. The histochem-ical examination revealed statistically significant increase of the mean DNA content. EM examination showed a highly significant decrease (P 6 0.0001) regarding mitochondrial sheath defect after treatment, while no changes were observed regarding the mean percent of dense fibers and axonemal defects. Previous authors (42,43) reported that, the administration of carnitine to patients with idiopathic atheno-zoospermia would provide additive substrate for sperm energy, metabolism and motility.

These results were in agreement with the previous result (44) which provides evidence for the role of carnitine in mito-chondrial respiratory activity by preventing free radical induced damage of the protein. In addition, L-carnitine plays a role in the energy metabolism, which positively affects sperm motility, maturation and the spermatogenic process (15).

Free L-carnitine is necessary in mitochondrial B-oxidation of long-chain fatty acids. Fatty acids must be undergoing activation to inter the mitochondria. They are bound to co-enzyme A (COA), thus forming acyle-COA, and use L-carnitine to cross the internal mitochondrial membrane. After transport of acyle-carnitine into the mitochondria, the acyle group is transferred to the mitochondrial COA, B-oxidation with the product adenosine triphosphate (ATP) leads to the formation of acetyl-COA (15,45).

Post gonad maturation could be an interesting target for therapy especially as it occurs mainly in the epididymal fluid where spermatozoa are far from the complex. In the epididy-mis, the epithelium removes some testicular factors, taken up materials from the blood, and produces specific compounds, which are useful for sperm maturation and motility (46).

In the teratozoospermia group, the results showed that, the observed significant decrease after treatment was for the head defects. Image analysis for DNA content revealed significant increase. L-carnitine may prevent sperm DNA and membrane damage induced by ROS. EM examination showed that the mitochondrial sheath defect was highly statistically significant decrease (P 6 0.0001 and 0.004, respectively). L-carnitine may help in mitochondrial energy production and total antioxidant capacity. No changes were observed regarding the mean percent of dense fibers and axonemal defects. Sperm tail defect, characterized by absence of the fibrous sheath, presence of supplementary axonemes and an abnormally elongated mid-piece. The observations suggest that the spermatozoa showed its defect during spermatocytogenesis and spermiogenesis, including the posttesticular development and maturation of spermatozoa in the epididymis. These defects which occur in the whole sperm population and therefore a genetic origin could be suggested. These results were in agreement with the other results (15,25,47) which showed a statistically significance improvement in atypical sperm cell forms after L-carni-tine therapy (P 6 0.0001) compared with the infertile group before treatment. They also demonstrated that, carnitine was fully effective in reducing ROS production when administrated for three months. In a study on patients with idiopathic and male factor infertility, sperm DNA damage was significantly increased in men with idiopathic and male factor infertility and in men who failed to initiate a pregnancy after assisted reproductive techniques (48,49). Such an increase may be related to high levels of seminal oxidative stress.

In a similar study, the amount of normal mitochondria was significantly increased according to the sequence of the control group and idiopathic atheno-zoospermia treated group and the severity degree of mitochondrial pathological changes (MPCs) increase accompanied with a decrease of sperm viability (15). The same authors concluded that various MPCs exist in idiopathic atheno-spermia. There was a close association between the sperm viability, mitochondrial ultra structure and mitochondrial function.

Supplement containing L-carnitine (an amino acid) can improve sperm function and sperm quality and L-carnitine increases decarboxylation in mitochondria (50,51). In addition, the administration of combined treatment with L-carnitine and acetyl-L-carnitine had no side effects and safe option for treating oligo-atheno-zoospermia by significantly improving sperm kinetic features, as well as increasing pregnant rates (52).

It has been concluded that the results clearly show that L-carnitine treatment has ameliorative impact on the quality of spermatozoa in the infertile men, resulting in a decrease in morphologically abnormal spermatozoa. These data indicate that this agent affects the quality of spermatozoa in infertile patients who need intra cytoplasmic sperm injection (ICSI) as a method for infertility treatment.

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