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Author Topic: Current Research Male Infertility  (Read 24620 times)
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Some of us may feel little research is being undertaken for Male Infertility - this may be the case however the following is some that is.

Research Overview (Cleveland clinic USA)

One of the major goals of our Centre is to better understand the causes of infertility and to design studies aimed at understanding and improving semen quality. We hope this line of research will eventually result in strategies that will enable us to use the healthiest spermatozoa for fertilization to improve the chances of pregnancy. The fertilizing potential of sperm depends on the shape of the spermatozoa, the ability of the spermatozoa to perform the functions necessary for fertilizing an egg, and finally, the transfer of intact genetic material (DNA) to the egg at the time of fertilization. Abnormal spermatozoa are not fully mature, and when in the presence of white blood cells, they can produce harmful substances. These substances are very unstable and reactive molecules that are called free radicals. Other substances called antioxidants, which are present in normal healthy seminal ejaculates, reduce the harmful effects of free radicals by neutralizing them. However, infertile patients who have abnormal semen often have antioxidants that are ineffective neutralizers. This results in a preponderance of free radicals, which in turn results in a situation called oxidative stress. Oxidative stress can damage the DNA of the spermatozoa and prevent them from fertilizing an egg.

We are currently involved in projects evaluating oxidative stress-induced nuclear DNA damage and its effects on sperm quality and pregnancy outcome. Studies designed to understand how free radicals cause DNA damage and the possible methods to counteract them are currently being conducted. We are currently evaluating the role of nutritional supplements in reducing oxidative stress in infertile men and increasing the number of successful pregnancies.

1.The relationship between reactive oxygen species in semen and male infertility, RPC #4252, 1993
2.Effect of motility stimulants on human sperm after cryopreservation, RPC #4925, IRB #4925, 1995
3.Maximizing semen quality in spinal cord injured men, RPC #5269, IRB #5269, 1995
4.Improving post-thaw semen quality in cancer patients, RPC #5489, IRB #1441, 1995
5.Assessment of human sperm function after cryopreservation in processed and unprocessed semen, RPC #5490, IRB #1442, 1996
6.Seminal reactive oxygen species in men with varicocele, and the effect of in vitro antioxidant supplementation, RPC #5549, IRB #1514, 1996
7.Designing an algorithm to predict fertilizing capacity of spermatozoa using biochemical and functional assays, RPC #5765, IRB #1727, 1997
8.The effects of mycoplasmas on the semen analysis and the function of spermatozoa, RPC #5825, IRB #1819, 1997
9.The role of reactive oxygen species and total antioxidant capacity in persistent infertility following vasectomy reversal, RPC #6036, IRB #2155, 1997
10.Impact of oxidative stress and IL-6 on fertility in varicocele patients, RPC #6206, 1998
 11.Predicting fertilization in an IVF program by use of mannose receptor assay, RPC #6238, IRB #2988, 1999
12.Changes in frequency of sperm chromosomal anomalies after chemotherapy for testicular cancer and Hodgkin's disease: a longitudinal study, RPC #6265, IRB #2989,1999
13.Role of reactive oxygen species in the pathophysiology of varicoceles and effects on antioxidant therapy alone or in combination with varicocele repair on sperm function and pregnancy outcome, AFUD, 1999-2001, IRB #3273
14.The role of oxidative stress in assisted reproduction, RPC #6362, IRB #3244, 1999
15.Role of oxidative stress and DNA damage in different subsets of ejaculated spermatozoa, RPC #6387, 1999
16.The role of defective spermiogenesis in male infertility: relationship between sperm immaturity and oxidative stress, RPC #6509, IRB #3853, 2000
17.Relevance of leukocytes on semen parameters, oxidative stress, and DNA damage in semen of infertile patients, RPC #6560, IRB #3841, 2000
18.Mechanism of nuclear DNA damage in mature sperm from infertile men, RPC #6770, IRB #4716, 2001
19.Levocarnitine fumerate and acetyl-l-carnitine combined supplementation in subfertile males with high reactive oxygen species, Sigma-Tau Health Sciences, Inc., Protocol STHS 01-01, IRB #4562, 2001
20.Oxidative stress mediated sperm DNA damage in infertility patients - possible role of apoptosis, RPC #07025, IRB #5474, 2002
 21.Assessment of fertilizing potential of mouse germ cells matured in vitro, RPC #7531, ARC #07518, 2004
22.Characterization of intracellular reactive oxygen species in human spermatozoa, RPC#07549, IRB #6967, 2004
23.Assessing the quality of cryopreserved testicular biopsy tissue, it's relation to etiology of infertility and outcomes of assisted reproduction, IRB 7622, 2004
24.Varying patterns of semen analysis in men evaluated for infertility: A retrospective review, IRB 7644, 2004
25.Correlation of apoptotic markers with sperm fertilization potential, RPC #07812, IRB 7546
26.Demographics of male patients seeking intervention for infertility, IRB 6519, 2004
27.Comparison protocol for human semen samples: SQA-V versus microscopic assessment, IRB 8045, 2005
28.A novel glutathione-S-transferase (GST5mu) knockout mouse model for studying oxidative stress, RPC#1066/ARC#08077
29.Utility of PARP alterations following apoptotic and oxidative DNA damage in human spermatozoa, RPC #1067, IRB 8530
30.Effect of post-Katrina contaminants from Lake Pontchartrain on the male reproductive system, RPC #1011/ARC #08110
 31.Antioxidant combinations to minimize human sperm apoptosis and DNA damage during cryopreservation, RPC #1097, IRB #425
32.Optimizing human sperm preparation techniques for selecting reactive oxygen species (ROS)-based high quality spermatozoa in assisted reproductive procedures, RPC #2007-1057
33.Effects of electromagnetic waves exposure from cell phones on semen quality, IRB #06-423
34.Optimizing sperm preparation techniques for selecting high quality spermatozoa in assisted reproductive procedures, RPC #07-949
35.Defining Sperm DNA Damage in Infertile Men with Varicocele and its Correlation with Oxidative Stress, RPC # 2007-1101 , IRB #08-095
36.Is poor sperm motility associated with defects in RhoGTPase in infertile men? RPC# 2007-1024, IRB 205
37.Effect of radiofrequency electromagnetic waves (RF-EMW) from the cell phone on human semen parameters, reactive oxygen species and DNA damage- an in vitro study, IRB # 08-231
38.Efficacy of L Carnitine before sperm processing for assisted reproduction techniques in reducing sperm DNA damage, RPC # 2008-1007, IRB # 08-225
39.Long term effects of electromagnetic waves on human semen, IRB #06-423
40.Effects of Radiofrequency Electromagnetic Waves (RF-EMW) from Cellular Phones on Human Ejaculated Semen, RPC 2008-1025
 41.Smoking and semen parameters: Is the effect of smoking on male fertility a reversible process, RPC 2008-1033, IRB #304
42.Antioxidants Combinations to Minimize Human Sperm Apoptosis and DNA Damage during Cryopreservation, IRB 06-425
43.Comparison of temperature regulated vs. non-regulated bench-top centrifuge for sperm quality improvement, IRB 09-128
44.Comparative study of sperm vitrification and slow freezing, IRB 09-127
45.Characterizing pathophysiologic effects of radiofrequency electromagnetic waves (RF-EMW) emitted by cell phones on human spermatozoa, IRB 09-069
46.Role of Curcumin in reducing oxidative stress-induced sperm damage, RPC #2009-1040
47.Development and validation of a novel sperm vitrification system using the Ohio-Cryo vitrification device, RPC #2009-1038
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Hi to all who are interested in current research - we will endeavour to give a weekly  overview on these research programmes
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Research – 1

It is now well documented that spermatozoa of infertile men have higher levels of DNA damage than their fertile counterparts. The DNA damage is seen in the form of single and double strand breaks, interstrand cross-links, modification of bases etc. When the male partner has >28-30% DFI, there is a 6-10X decreased probability of full term pregnancy. There is also a significantly decreased probability with this DFI value for an IUI pregnancy, as well as an increased probability of spontaneous miscarriage. The odds are reduced for routine IVF but much less so for ICSI when the DFI >30%.
The reason behind this DNA damage is not well understood. There are two prominent factors implicated in various studies - oxidative stress (OS) and dysregulated apoptosis. OS is the imbalance between the production of reactive oxygen species (ROS) by the spermatozoa and leukocytes, and the antioxidant capacity of the seminal plasma. The prime source of ROS production in infertile patients is the immature spermatozoa having residual cytoplasm. The excess nicotinamide adenine diphosphate hydrogen generated via the glucose 6 phosphate dehydrogenase in this cytoplasm triggers ROS production.
Apoptosis is an essential part of spermatogenesis that keeps a check on the number of proliferating germ cells so that they 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 or not there is a connection between OS and dysregulated apoptosis factors. There have been many suggestions 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 will induce any apoptotic activity in them. The extent of apoptotic activity hence 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.
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Endocrinol Nutr. 2013 Apr 2. pii: S1575-0922(13)00068-5. doi: 10.1016/j.endonu.2013.01.006. [Epub ahead of print]

Body weight increase and quality of semen: a controversial association.

[Article in English, Spanish]

Mormandi EA, Otero P, Bertone AL, Calvo M, Astarita G, Kogovsek N, Levalle O.


Source

Laboratorio de Endocrinología, División de Endocrinología, Hospital Carlos G. Durand, Buenos Aires, Argentina. Electronic address: androsite@gmail.com.


Abstract


BACKGROUND:

Infertility is a public health disorder affecting 10% of the population worldwide. Research on the impact of body mass index (BMI) on male fertility is very limited as compared to the multiple studies evaluating the impact of overweight in women's fertility. Although 25%-30% of the cases of couples consulting for infertility are attributable to male factors, studies evaluating the association between semen parameters and BMI are controversial.

OBJECTIVE:

To assess the impact of BMI on semen parameters in a selected group of men with unexplained infertility.

METHOD:

A retrospective analysis of 168 patients during the 2008-2010 period. They all had at least one semen analysis and related studies to rule out known causes of infertility. Median age of patients was 35 years (22-55), and they were divided into three groups: normal weight (BMI: 20-24.9kg/m2), overweight (BMI 25-29.9kg/m2), and obese (BMI ≥30kg/m2).

RESULTS:

There were no significant differences in semen parameters evaluated between the three groups, and no significant correlation was found between the same parameters and BMI.

CONCLUSIONS:

There was no significant association between BMI and conventional semen parameters, but we cannot exclude an impairment in other semen parameters that are not routinely assessed, which could result in a lower potential fertility in these individuals.

Copyright © 2012 SEEN. Published by Elsevier Espana. All rights reserved.


PMID: 23562378  [PubMed - as supplied by publisher]
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Motility is one of the important factors necessary for natural pregnancy to occur. It is a prerequisite for delivery of the genomic material for fertilization. In asthenozoospermic patients (<30% motility), natural pregnancy rate is very poor. Sperm motility is governed by motor and cytoskeleton proteins. These proteins are regulated by a group of small G proteins called RhoGTPase (Ras homologue) (RhoA-B & Rac). These proteins control actin/tubulin cytoskeleton assembly & vesicle transport (e.g. translocation of phospholipase C from the cytosol to the sperm plasma membrane). Defects in these motor/cytoskeleton proteins and their regulators lead to morphological abnormalities (teratozoospermia) and poor motility (asthenozoospermia). Defects in the tail region in these proteins might be associated with sperm immotility while head region specific RhoGTPase could be linked to failure of sperm to undergo hyperactivation (capacitation) acrosome reaction and sperm oocyte fusion. This may be because of improper sperm head structural organization as a result of abnormal cytoskeleton network proteins or their regulators (RhoGTPase). As these events are regulated by RhoGTPase, aberrant expression of these proteins could be associated with alterations either at the gene expression level or during sperm maturation process. Our aim is to investigate the signaling mechanism of RhoGTPase family proteins and their gene expression in relation to asthenozoopermic and teratozoospermic sperm abnormalities and study how they affect sperm functions (e.g. motility, capitation & acrosome reaction). This study will provide insight into the regulation of sperm motility and issues with sperm oocyte fusion failure.
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Cell phones have become indispensable devices in our daily life. These emit electromagnetic waves (EMW) of different frequencies which have been linked to adverse effects in human beings. In United States cell phones operate at frequency 900 -1900 MHz, whereas in most other parts of world the cellular phones work at 900 -1800 MHz frequencies. Electromagnetic waves have been reported to affect neurological, cardiac and endocrine systems. There are preliminary reports that suggest that EMW can reduce the fertilizing potential of spermatozoa and cause sperm DNA damage. Some of the possible mechanisms by which EMW can alter reproductive function are non-thermal EMW-specific effect, a thermal molecular effect, or a combination of these mechanisms. While some authors have found little or no effect with the use of electronic devices on reproductive function, recent research by our group has shown damaging effects of these devices on semen variables in a study involving 361 men.

Free radicals called reactive oxygen species (ROS) have been shown to be detrimental to sperm and are involved in the pathophysiology of male infertility. Antioxidants present in the semen can neutralize ROS. Oxidative stress occurs when there is an imbalance in the formation of ROS and the ability of antioxidants to neutralize ROS.

In a large prospective study, we assessed the effects of EMW emitted by cell phones (900 - 1900 MHz) on various markers of sperm quality such as count, motility, morphology, viability etc. Pilot studies conducted by us have shown that exposure of ejaculated (neat) semen samples to commercially available cellular phones for one hour caused a significant decrease in sperm motility and viability, increased ROS levels and decreased ROS-TAC (reactive oxygen species-total antioxidant capacity) score when compared with neat semen from a non-exposed group.

It's important to note that many men carry their cell phone in a trouser pocket (or clipped to their belts on waist) while using Bluetooth. This technology exposes testes to high power cell phone density compared with the cell phone in the stand by mode. The phone and the male reproductive organs are separated by multiple tissue layers. The deleterious effects of RF-EMW exposure from cell phone use on functional markers of spermatozoa from fertile and infertile men are not clear. Furthermore, the effects on spermatozoa of frequency, distance of the phone from source and the talk time are not known.

We have designed a two dimensional anatomical model of the tissue to extrapolate the effects seen in "in vitro" condition to real-life conditions. We aim to examine the effect of specific cell phone RF, distance and talk time on functional markers of oxidative stress in immature and mature spermatozoa. In addition we are also evaluating DNA damage and apoptosis.

Results from our study may help us understand the mechanism of action of RF-EMW from cell phones on sperm quality in infertile men - a population who may already have sperm cells that are susceptible to oxidative stress and, therefore, be more susceptible to the negative cell phone effects. Such knowledge may help modify/ revise guidelines for reducing the adverse effects of EMW in men who may be at increased risk of sperm damage and subsequent infertility.
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Sperm cryopreservation is a major and essential component of any infertility service provided to patients.

Sperm cryopreservation is currently done by slow freezing. After thawing, semen samples are usually compromised during the process of freezing and thawing as evident by a decrease in sperm viability and motility especially in infertility patients with poor semen parameters. One of the detrimental factor of decreased viability during freezing is the ice crystal formation.

Vitrification is the ultra-rapid method of freezing which avoids ice crystal formation by rapid transition from liquid to solid state. We have developed a new device "The Ohio-Cryo" that allows vitrification of larger sample volume suitable for freezing sperm. We are validating the use of this device in the vitrification of semen samples and comparing the outcome of slow freezing to vitrification by measuring sperm motility and effect on DNA damage. If sperm vitrification can be properly achieved, we would expect improvement in quality og post-thaw sperm parameters. This may be extremely beneficial, especially in preserving patient samples.

Cleveland Clinic
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Intact genetic material, or DNA, is important for healthy fertilization. A morphologically normal looking motile sperm can have DNA damage, which can result in impaired fertilization, miscarriage or subsequent complications associated with pregnancy. Sperm can be frozen and batched for DNA testing. DNA damage can be examined by staining the DNA by utilizing a reaction catalyzed by exogenous terminal deoxynucleotidyltransferase (tdt) and is termed as ‘end labeling’ or “TUNEL” (terminal deoxynucleotidyltransferase dUTP nick end labeling) assay. It utilizes a robust technique called flow cytometery. This technique is sensitive and requires 2-5 X106 sperm compared to other microscopic methods which are very subjective.

Infertile patients, diagnosed with varicocele, prostatitis or with a history of smoking have been shown to have higher percentage of sperm DNA damage. Furthermore, evaluating sperm DNA damage in men with unexplained infertility or idiopathic infertility or those who have severe oxidative stress-related abnormal semen quality may also be good candidates for evaluating sperm DNA damage. Oxidative stress and sperm apoptosis is a major contributor of sperm DNA damage.

High DNA damage in infertile men can lead to poor ART outcomes and increased miscarriage rates. Based on the extent of DNA damage found, certain assisted reproductive techniques may be recommended

Our goal is to identify key proteins that can serve as biomarkers in identifying the underlying pathology of male infertility and provide alternate approaches for treating these patients.
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Sperm processing (e.g., centrifugation) used in preparation for assisted reproduction can result in excessive generation of reactive oxygen species (ROS) with potential sperm damage. This may be particularly significant in samples from infertile men (compared with those from fertile men) where morphologically abnormal spermatozoa inherently are capable of generating high levels of reactive oxygen species and sperm processing may cause further damage Furthermore, freezing of semen samples is also known to cause sperm damage as a result of production of ROS and oxidative stress. The mechanism behind this damage may be related to cold shock, osmotic stress and intracellular ice crystal formation during cryopreservation. These phenomena induce cell injury and affect organelle functions. Membrane fluidity and mitochondrial functions are affected during sperm cryopreservation. Moreover, freeze thawing of spermatozoa is associated with an increase in reactive oxygen species (ROS). Reactive species which are produced within the mitochondria are generated excessively during the process of cryopreservation/ thawing and could attack the cellular components including DNA.

Lycopene is a red carotene and carotenoid pigment as well as a phytochemical found in tomatoes and other red fruits and vegetables. It has been shown to have antioxidant and pro-oxidant properties. The molecule exhibits a variety of beneficial effects on cardiovascular and liver diseases, cancer, diabetes, osteoporosis, including its detoxifying and antioxidant properties. Even though there are some studies on the outcomes of Lycopene supplementation on reproductive performance in animals and humans, very little information is available on the beneficial in vitro effects of Lycopene in reducing oxidative stress during the preservation of human sperm.

Ongoing studies are examining the optimal concentrations of Lycopene that may be beneficial in reducing or eliminating the oxidative stress induced by hydrogen peroxide as well examine it’s beneficial effects in reducing oxidative stress induced effects on sperm quality. The efficacy of Lycopene use before or after freezing of semen samples is currently being evaluated.
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Efficacy of Freezing Protocols in Preserving Sperm Function and DNA integrity in Normozoospermic and Oligoasthenozoospermic Specimens

Despite the success of sperm cryopreservation, this routinely used procedure in assisted reproduction technology (ART) causes cell damages and impairs sperm functions. Sperm freezing and thawing not only alters sperm motility and vitality but also causes an increase in sperm DNA damage. The mechanism behind this damage may be related to cold shock, osmotic stress and intracellular ice crystal formation during cryopreservation.

Conventional slow freezing causes extensive chemical and physical damage to sperm cell membranes. Studies have reported an average reduction in sperm motility of 50% after slow freezing.

The liquid nitrogen vapor technique is considered as a rapid cooling technique for the freezing of human semen with subsequent storage at -196oC.

At present a universal freezing protocol is used irrespective of the semen quality. The goal is to evaluate optimal cryo-conditions for human spermatozoa with normal and poor semen parameters by comparing cryoprotective efficiency of three methods of freezing semen: i.e. conventional method, fast programmable freezing and slow programmable freezing.

We want to determine the most efficient cryoprotective protocol for normospermic samples (normal count >15 x106/mL, motility >40% and morphology >4% normal forms) and oligoasthenozoospermic (poor count; <15 x106/mL and poor motility; <30%) samples to assess the protocol that offers the most superior quality of sperm after freezing. This will allow us to customize the protocol according to the semen characteristics and obtain superior quality (improved motility) spermatozoa for ART after freezing.
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Semen analysis does not provide information on the underlying molecular alterations in the seminal ejaculates of infertile men. Oxidative stress (OS) can affect sperm function and results in modification of proteins in the spermatozoa. Proteomics is the study of the protein profile of spermatozoa or seminal plasma and utilizes MALDI-TOF (matrix assisted laser desorption ionization – time of flight) and LC-MS/MS (liquid chromatography –mass spectrometry/mass spectrometry).

Utilizing proteomic analysis with Two-dimensional differential in-gel electrophoresis (2-DIGE) and liquid chromatography –mass spectrometry/mass spectrometry (LC-MS/MS) protein expression in a variety of sperm population was recently examined in difference in intensity were excised from the preparatory gel and identified by liquid chromatography–mass spectrometry. A total of 1,343 protein spots in gel 1 (ROS-) and 1,265 spots in gel 2 (ROS+) were detected. The majority of protein spots had similar expression, with 31 spots were differentially expressed. Six spots were significantly decreased and 25 increased in the ROS- sample compared with the ROS+ sample. Significantly different expression of protective proteins against oxidative stress was found in ROS- compared with ROS+ sample.

Utilizing Mascot and Sequest programs, further identification of proteins is done. The results from these SEQUEST searches are used to determine the spectral counts. Normalization of the spectral count is done using the total number of spectral counts for all proteins in the sample and the number of amino acids present in the protein. A 2-fold change in protein expression is considered significant since the precision of the proteomic analysis has an average error of 10-20%.

We have identified spermatozoal or seminal plasma proteins that are overexpressed or underexpressed in ROS – samples compared to ROS + samples. In addition we have studied proteins that are differentially expressed in patients with abnormal sperm count and sperm morphology compared with those that have normal sperm count and morphology.

Differentially affected processes pathways and cellular distribution as well as protein–protein interactions can be identified utilizing functional bioinformatics analysis available (Gene Ontology (GO) annotations from GO Term Finder and GO Term Mapper, UNIPROT, STRAP, BioGPS and proprietary software packages such as Ingenuity Pathway Analysis, Metacore™ and STRING database and Cytoscape to identify the differentially affected processes, pathways, cellular distribution, and protein-protein interactions amongst proteins in the two study groups as well as for data integration.

In these preliminary studies, we have established a platform to utilize proteomic tools and examine other etiologies in an effort to unravel the underlying mechanisms of male infertility and identify develop appropriate antioxidant therapy to alleviate oxidative stress related infertility.

Further validation through Western Blot is necessary to identify the biomarker status of the proteins in various pathological conditions attributed to oxidative stress or in patients with other etiologies.

The current focus is to study the alterations of major proteins that are overexpressed or underexpressed in the seminal plasma or spermatozoa of infertile men with various clinical diagnoses and compare with those present in normal healthy men who have established pregnancy.

New planned studies include:

1). Fertile versus infertile subjects, 2) primary versus secondary infertility, 3). patients who are presenting with non-obstructive azoospermia, 4) patients diagnosed with testicular cancer, 5) patients before and after varicocele repair, 6) idiopathic and ptients with 7) unexplained male infertility, Cool advanced paternal age (>40y) and 9). obese patients (>25 BMI).

In addition we are also evaluating a subset of patients who present with the following:

1). hyperviscosity, 2). leukocytospermia as one of the underlying characteristics of seminal ejaculate. Another study is aimed at examining the effect of abstinence time on the sperm proteome.
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