Scholarly article on topic 'Oxidative stress and acrosomal morphology: A cause of infertility in patients with normal semen parameters'

Oxidative stress and acrosomal morphology: A cause of infertility in patients with normal semen parameters Academic research paper on "Clinical medicine"

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Abstract of research paper on Clinical medicine, author of scientific article — Moustafa A. El-Taieb, Mohammed A. Ali, Essam A. Nada

Abstract Introduction Acrosome has a vital role in the process of penetration of zona pellucida (ZP). It is highly susceptible to elevated reactive oxygen species (ROS) and oxidative stress (OS). As generally accepted that OS stress has deteriorating effects on sperm functions, we studied the effect of OS on sperm parameters and acrosomal structure in infertile patients with normal semen parameters. Patients and methods 30 infertile patients with normal semen parameters and 20 normal fertile controls were included in this study. Semen analysis was performed according to WHO parameters. Oxidative stress was evaluated by measurement of Malondialdehyde (MDA). Acrosomal anomalies were detected by transmission electron microscopy (TEM). Results Statistically no significant difference was found between infertile patients and controls regarding semen parameters (p value >0.05). MDA values were statistically highly significant in infertile patients than normal controls (p value <0.001). Acrosomal anomalies were statistically highly significant in infertile patients than normal controls (p value <0.001). Acrosomal anomalies were positively correlated with MDA values. Pearson correlation was 0.645 for correlation between redundant or detached acrosome and MDA values and 0.707 for correlation between acrosomal inclusions and MDA values. Conclusion Percentages of MDA values and acrosomal anomalies were higher in infertile patients than normal subjects. The positive correlation between acrosomal anomalies and MDA values means association between OS and acrosomal anomalies which may indicate negative effects of OS on the acrosomal structure.

Academic research paper on topic "Oxidative stress and acrosomal morphology: A cause of infertility in patients with normal semen parameters"

Middle East Fertility Society Journal (2014) xxx, xxx-xxx

Middle East Fertility Society Middle East Fertility Society Journal

www.mefsjournal.org www.sciencedirect.com

ORIGINAL ARTICLE

Oxidative stress and acrosomal morphology: A cause of infertility in patients with normal semen parameters

Moustafa A. El-Taieb a *, Mohammed A. Ali b, Essam A. Nada

a Department of Dermatology, STDs and Andology, Qina Faculty of Medicine, South Valley University, Egypt b Department of Dermatology, STDs and Andology, Sohag Faculty of Medicine, Sohag University, Egypt c Department of Dermatology, STDs and Andology, Aswan Faculty of Medicine, Aswan University, Egypt

Received 2 March 2014; accepted 1 May 2014

KEYWORDS

Acrosome; MDA;

Oxidative stress;

Sperm;

Infertility

Abstract Introduction: Acrosome has a vital role in the process of penetration of zona pellucida (ZP). It is highly susceptible to elevated reactive oxygen species (ROS) and oxidative stress (OS). As generally accepted that OS stress has deteriorating effects on sperm functions, we studied the effect of OS on sperm parameters and acrosomal structure in infertile patients with normal semen parameters.

Patients and methods: 30 infertile patients with normal semen parameters and 20 normal fertile controls were included in this study. Semen analysis was performed according to WHO parameters. Oxidative stress was evaluated by measurement of Malondialdehyde (MDA). Acrosomal anomalies were detected by transmission electron microscopy (TEM).

Results: Statistically no significant difference was found between infertile patients and controls regarding semen parameters (p value >0.05). MDA values were statistically highly significant in infertile patients than normal controls (p value <0.001). Acrosomal anomalies were statistically highly significant in infertile patients than normal controls (p value <0.001). Acrosomal anomalies were positively correlated with MDA values. Pearson correlation was 0.645 for correlation between redundant or detached acrosome and MDA values and 0.707 for correlation between acrosomal inclusions and MDA values.

* Corresponding author. Tel.: +20 1143929476; fax: +20 965337371. E-mail addresses: musmus22@yahoo.co.uk (M.A. El-Taieb), Mohammedadva@yahoo.com (M.A. Ali), essamnada2011@yahoo. com (E.A. Nada).

Peer review under responsibility of Middle East Fertility Society.

1110-5690 © 2014 Production and hosting by Elsevier B.V. on behalf of Middle East Fertility Society. http://dx.doi.org/10.1016/j.mefs.2014.05.003

Conclusion: Percentages of MDA values and acrosomal anomalies were higher in infertile patients than normal subjects. The positive correlation between acrosomal anomalies and MDA values means association between OS and acrosomal anomalies which may indicate negative effects of OS on the acrosomal structure.

© 2014 Production and hosting by Elsevier B.V. on behalf of Middle East Fertility Society.

1. Introduction

Acrosome is an exocytotic organelle derived from the Golgi apparatus and located at the tip of the sperm head of many mammalian species including humans. Human spermatozoa must have properly formed acrosomes to be able to penetrate the ova and to proceed in fertilization (1-3).

Biosynthesis of acrosome starts in late spermatogenesis by the formation of proacrosomal vesicles in the perinuclear region near the Golgi apparatus of pachytene spermatocytes (4). During the meiotic cell divisions, these vesicles are distributed to the four daughter spermatids. Vesicles coalesce to form one granule attached to the round spermatid nucleus and continues to enlarge by the added material from Golgi apparatus (5,6). During late spermiogenesis, remodeling of acrosome nucleus interaction occurs to give the characteristic shape of sperm head. At this stage Golgi apparatus stops to contribute in acrosomal synthesis. Further maturation occurs during the epididymal transfer to give the characteristic appearance of acrosome on the anterior two thirds of sperm head (7-9).

The acrosome of normal fully mature spermatozoon is formed of outer acrosomal membrane (OAM), acrosomal matrix and inner acrosomal membrane (IAM) (10). OAM fuses with plasma membrane to start the acrosomal reaction at the start of fertilization. IAM is tightly adherent to the nuclear membrane (11). The most important part of acrosome is the acrosomal matrix (12). It is an electron dense material between acrosomal membranes. It consists mainly of proteins, containing many different proteins including proteases, glyco-sidases, and various zona pellucida (ZP) binding proteins. These proteins are vital for acrosomal reaction and ZP penetration (13,14).

The main function of the acrosome is the acrosomal reaction occurring during gamete interaction. Acrosomal reaction includes fusion of the OAM and plasma membrane to expose the acrosomal matrix and underlying IAM. Once exposed, the acrosomal matrix releases its proteases and other proteins including ZP binding proteins to start the penetration of ZP (15). Acrosome may have other functions such as its involvement in sperm morphogenesis (16,17). The acrosome also anchors the spermatid nucleus to the Sertoli cell through Ser-toli-spermatid junctions, including the apical ectoplasmic specializations until the time of spermiation (18,19).

The specialized structure of acrosome, consisting of membranes and proteins, renders it sensitive to high levels of reactive oxygen species (ROS) an oxidative stress (OS) (15). The effect of OS on male fertility has been extensively investigated in recent years (20). Many reports correlated abnormal sperm parameters to OS (21-23). OS negatively affect sperm membranes and proteins. Oxidative modification of proteins by reactive species, especially ROS, is implicated in the etiology or progression of disorders and diseases. Proteins are oxidized by free radicals, whereby the constituent amino acids are

variously modified or degraded (24). Lipid peroxidation of sperm membranes and oxidation of proteins negatively affect the acrosomal structure and function (25,26).

Men classified as having idiopathic male infertility have an unexplained reduction in semen quality with no history associated with fertility problems and have normal findings on physical examination and endocrine laboratory testing. Their routine semen analysis shows decreased number of spermatozoa (oligozoospermia), decreased motility (asthenozoosper-mia), or an increased proportion of abnormal forms (teratozoospermia). These abnormalities usually occur together and are described as the oligoasthenoteratozoosper-mia syndrome (27). In many cases of male infertility, high level of ROS may be produced and negatively affect sperm functions in the absence of detectable causes and abnormal findings in semen analysis (28-30).

In this study we examined the acrosomal structure and morphology in patients with idiopathic infertility and compared them with those in the normal controls. Furthermore, we correlated acrosomal anomalies with levels of OS.

2. Patients and methods

2.1. Patients

Of 255 consecutive patients, examined between August 2011 to October 2013 in the department of Dermatology, Venereology and Andrology, Qina university hospital, south valley university, 30 patients (Group 1) with primary infertility without any apparent cause and normal semen parameters with the following criteria: sperm concentration was >15 million per milliliter, forward progressive motility >32% and normal sperm morphology >4% were included in this study (31). A thorough general and genital examination and complete investigations were performed to exclude any known cause of infertility (as Varicocele, smoking, genetic causes, infectious causes and any other known causes that can be detected by routine investigations). 20 subjects (Group 2) with normal semen parameters and proven fertility admitted to the department for andrological evaluation were included as a control group.

2.2. Semen analysis

Semen samples were collected after 3-5 days of sexual abstinence. Samples were collected by masturbation and examined directly after liquefaction. Semen analysis was performed according to WHO guidelines. Ejaculatory volume, pH, viscosity, sperm concentration, sperm motility and sperm morphology were detected under standardized conditions (31). 200 sperms have been counted per sample to detect the percentage of normal spermatozoa, head abnormalities, midpiece abnormalities and tail abnormalities. Leukoscreen test was

used to detect the concentration of leukocytes in seminal plasma; samples with leukocyte concentration one million or more per milliliter were considered leukocytospermia according to WHO definition (31) and were excluded from the study. Samples with positive culture for any organism were also excluded.

2.3. MDA measurement

MDA levels were measured as per Thiobarbituric Acid (TBA) method described by Hsieh et al. (32). 0.1 ml of seminal plasma was added to 0.9 ml of distilled water in a glass tube; to it 0.5 ml of TBA reagent was added and then heated for 1 h in a boiling water bath. After cooling the tube was centrifuged for 10 min at 4000 rpm and the supernatant absorbance was read on a spectrophotometer at 534 nm (33).

2.4. Electron microscopy examination

Samples were fixed in 4% Paraformaldehyde resolved in 0.1 M Cacodylatebuffer + 1 % Glutaraldehyde. Then Agarose - Pellets were made: 1% Agarose (high gelling Agarose resolved in PBS was warmed to 80 0C. The semen samples were washed in PBS 4 times and then samples were warmed up to 60 0C and 1% Agarose was put on the samples and then mixed, centri-fuged and cooled in crush ice for 3 h. Subsequently, pellets were collected in 0.1 M Cacodylatebuffer.

All samples were embedded for one day. Embedding procedure included exchange of the 0,1 M Cacodylatebuffer (15 min), then three hours in 1% Osmiumtetroxide (2% OsO4 resolved in 3% Potassium hexacyanoferrate), then, twice washing for 15 min. Then samples were dehydrated in ascending grades of ethanol (every step: twice for 15 min: 70%, 80%, and 96% alcohol and 100% alcohol), then samples were put in Intermedium 1.2 Propylenoxid twice for 15 min, Then in mixture 1:1.2 Propylenoxide - Epoxy resin for three hours, then in absolute Epoxy resin overnight on the shaker. The following day all samples were embedded in fresh Epoxy resin in special Embedding forms and put into the oven (60 0Q for 24 h (34,35).

All samples were sectioned semi thin (about 1.5 im) and then ultra thin (about 80 nm) with the Ultracut E (Reichert - Jung, Vienna, Austria). The ultrathin sections were put on copper grids (150 mesh, Balzers), and stained with Uranyl acetate and Lead Citrate.

After all, samples were washed and examined under a Jeol Jem 1010 (Jeol/Japan). In each sample, 200 sections in the head of spermatozoa were examined to detect the percentage of acrosomal anomalies in iOAT patients as well as normal controls.

Statistics were done using SPSS 15 software (SPSS Inc., USA). Data are presented as mean with standard deviation. Pearson test was used for correlation.

3. Results

This study was conducted on two groups; 30 patients with idi-opathic infertility and 20 normal fertile men. The mean ages for both groups were 35.26 ± 10.07 and 33.5 ± 7.69 respectively. There was no statistically significant difference regarding the mean ages between the two groups (p = 0.58).

Figure 1 Mean MDA values in iOAT patients and normal controls.

3.1. MDA measurements

There was a significant difference in the MDA values between infertile patients and normal fertile controls. The mean MDA values were 2.69 ± 0.87 for infertile patients and 1.68 ± 0.4 for normal fertile controls. p < 0.001 (Fig. 1).

3.2. Semen parameters

Semen parameters, as detected under LM, showed no statistically significant difference between infertile patients and normal fertile controls. Sperm concentrations were 34.52 ± 13.63 in infertile patients and 36.92 ± 15.45 in normal fertile controls which was not statistically significant (p > 0.05). Sperm motility showed no statistically significant difference between infertile patients and normal fertile controls with mean values 36.94 ± 4.44 and 37.39 ± 5.74 respectively (p > 0.05). There was no statistically significant difference in percentage of normal sperm morphology between infertile patients and normal fertile controls with mean values 14.41 ± 6.73 and 15.44 ± 3.04 respectively (p > 0.05).

3.3. Acrosomal anomalies

Normal acrosome, as seen in Fig. 2, covers the anterior two thirds of the head. It is divided into three segments; apical, principal and equatorial segments. Normal acrosome is composed of OAM, IAM and the cavity in between is full of homogenous matrix of medium electron density (acrosomal matrix). Acrosomal anomalies examined in this study included acrosomal redundancy or detachment and acrosomal swelling and inclusions which were represented by vacuoles and granules and separation and enfolding of the cell membrane from the acrosome.

Percentage of redundant acrosome was statistically highly significant in infertile patients than in normal controls with mean values 22.26 ± 2.31 and 12.78 ± 2.96 and p value <0.001. Acrosomal swelling and inclusions were also

Figure 2 Longitudinal section in head of a normal spermatozoa as seen under TEM showing normal dense nucleus (N), normal acrosome (arrow) covering the anterior two thirds of the head. (Mag. 12,000).

statistically highly significant in infertile patients than in normal controls with mean values 9.74 ± 1.93 and 6.06 ± 1.73 with p value <0.001 (Figs. 3-6).

Acrosomal abnormalities were positively correlated with the MDA values. Pearson correlation was 0.645 for correlation between redundant or detached acrosome and MDA values and 0.707 for correlation between acrosomal inclusions and MDA values see Table 1.

4. Discussion

Acrosome is the key factor in ZP penetration. During sperm-ovum interaction, OAM fuses with sperm plasma membrane to start acrosomal reaction and release of proteolytic enzymes of the acrosomal matrix facilitates sperm penetration through ZP with subsequent fertilization. In order to start acrosomal reaction, acrosome must have normal structure and morphology (15-19,36).

Figure 3 Transverse section in the head of an abnormal spermatozoon of infertile patients showing abnormal acrosome (arrow) with areas of detachment and redundancy. Notice the vacuole (V) in the context of nucleus (N) (Mag. 15,000).

Figure 4 Longitudinal section in the head of an abnormal spermatozoon showing redundant acrosomal membrane (arrow) and normal nucleus (N) (Mag. 15,000).

Figure 5 Transverse section in the head of an abnormal spermatozoon showing redundant acrosomal membrane (arrow) with tooth like appearance with otherwise normal nucleus (N) (Mag. 25,000).

Figure 6 Longitudinal section in the head of an abnormal spermatozoon of infertile patients abnormal acrosome (arrow) with areas of redundancy. There is a nuclear vacuole (V) within the context of the nucleus (N). (Mag. 12,000).

Acrosome is vulnerable to high levels of ROS and OS due to its specialized structure, two membranes surrounding a matrix composed mainly of proteins. In this study we detected acrosomal morphological anomalies in infertile patients with

Table 1 MDA values, sperm parameters and acrosomal anomalies in iOAT patients and normal fertile controls.

Infertile patients Normal control p value

N =30 N =20

Mean ± SD

MDA value (nmol/ml) 2.69 ± 0.87 1.68 ± 0.4 <0.001

Concentration (million/ml) 34.52 ± 13.63 36.92 ± 15.45 >0.05

Forward progressive motility (%) 36.94 ± 4.44 37.39 ± 5.74 >0.05

Normal morphology (%) 14.41 ± 6.73 15.44 ± 3.04 >0.05

Redundant acrosome (%) 22.26 ± 2.31 12.78 ± 2.96 <0.001

Acrosomal inclusions (%) 9.74 ± 1.93 6.06 ± 1.73 <0.001

p value <0.05 was significant.

normal semen parameters in comparison with those of normal fertile controls in order to detect the effect of OS on acrosomal morphology and ultrastructure and the impact of this on fertility.

In the present study, MDA values were found to be significantly higher in infertile patients than in normal controls. MDA was used in our study as the indicator of OS. This agrees with many previously published data that found higher levels of OS in infertile patients than in normal subjects regardless of the methods of OS detection and the used marker (37-41). In contrast to those studies we detected the higher level of OS in infertile patients but with normal semen parameters.

In this study, levels of OS have been found to be non-significantly correlated with sperm concentration, forward progressive sperm motility and sperm morphology. The majority of previous studies on the relation between OS and male infertility found negative impacts of OS on male infertility and sperm parameters (21,39-43).

On examination under TEM, percentage of abnormal acrosomal morphology was higher in infertile patients than in normal fertile controls. The ultrastructural morphological anomalies detected were redundancy or detachment of acro-some and acrosomal swellings or inclusions. These anomalies have been correlated positively with high levels of MDA. This correlation may indicate a negative impact of OS on the acro-somal morphology and ultrastructure. Up to our knowledge, no previous study has been performed to detect the direct effect of ROS and OS on the ultrastructural morphological anomalies of acrosome in infertile patients.

Our results can be explained on the base of a premature acrosomal reaction. This agrees with a recent work detected negative effects of nicotine on acrosomal reaction and other sperm parameters in vitro (44). Several other studies detected negative effects of ROS and OS on acrosomal reaction. OS may lead to premature acrosomal reaction which causes a reduced sperm capability of penetrating zona-intact oocytes. However, this reduction is not seen in zona-free oocytes (45,46). Although low ROS levels in semen may have a role in acrosomal reaction, excessive ROS exert a negative influence on acrosomal reaction (47).

Our results could support the role of epididymal proteins in intact acrosome and proper acrosomal reaction as the negative effect of OS on proteins (48). In contrast to these results, some investigators found that ROS have no negative effects on acrosomal reaction (49).

Based on these findings, we suggest that high levels of ROS and OS can negatively affect acrosomal ultrastructure in many

ways. The first is that OS leads to premature abnormal acrosomal reaction which leads to the abnormal ultrastructure revealed by TEM. The second is that OS directly alter the ultrastructure of the acrosome which in turn leads to the premature acrosomal reaction. Another mechanism is by affecting the protein structure which is important for acrosomal integrity and functions. These effects can lead to decreased ability of sperm to fertilize ovum even in patients with apparently normal semen analysis.

5. Conclusion and recommendation

From our results, we concluded that increased levels of ROS and OS can decrease the fertility potential in patients with normal semen parameters. They can negatively affect the acrosomal ultrastructure and morphology in those patients. Morphologically abnormal acrosome, caused by increased levels of ROS and OS, can be an important underlying cause of infertility in those patients. We recommend measurement of ROS levels and detection of ultrastructural sperm anomalies in cases of idiopathic male infertility.

Conflict of interest

None. References

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