| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
,
,,
,
From the * Instituto Valenciano de Infertilidad
(IVI), the IVI Foundation, and the
Department of Pediatrics, Obstetrics, and
Gynecology, Valencia University School of Medicine, Valencia, Spain; and the
Hospital Universitario Dr. Peset, Valencia,
Spain.
| Correspondence to: Dr Marcos Meseguer, Andrology Laboratory, Instituto Valenciano de Infertilidad, Policía Local 3, Valencia 46015, Spain. (e-mail: marcos.meseguer{at}ivi.es). |
| Received for publication July 28, 2003; accepted for publication April 21, 2004. |
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
Key words: Oxidative stress, freezing, thawing, sperm motility, assisted reproduction
The plasma membrane of sperm, which contains high levels of polyunsaturated fatty acids, renders it particularly sensitive to free radical–mediated attacks. One of the most common types of damage induced by free radicals is membrane lipid peroxidation (LPO). Spontaneous LPO has been well characterized in human semen samples (Alvarez et al, 1987; Mazzilli et al, 1995). This LPO considerably impairs sperm function by adversely affecting its viability and motility and, ultimately, its fertilizing potential (Critser et al, 1987; Centola et al, 1992; Mc Laughlin et al, 1992). Increased susceptibility to ROS and cell death has been reported in cryopreserved sperm. The freezing and subsequent thawing of sperm is a physically stressful process carried out during routine procedures in assisted reproduction techniques (ARTs), which results in a highly variable and unpredictable reduction in the number of motile sperm cells. Various oxidative defense enzymes, such as superoxide dismutase, glutathione peroxidase (GPx), and glutathione reductase (GR), are crucial for the prevention of LPO (reviewed by Storey, 1997). GPx and GR have provoked special interest, since LPO increases 20-fold following the inhibition of GPx activity with mercaptosuccinate (Alvarez et al, 1987). Additionally, the GPx/GR system has been shown to play a major role in maintaining membrane integrity in human sperm, since inhibition of GPX with mercaptosuccinate increases membrane damage (Alvarez and Storey, 1989).
The complete glutathione system consists of a complex series of interactions. In short, GPx isoforms catalyze the reduction of inorganic and organic hydroperoxides. During catalysis, glutathione acts as a reducing equivalent, inactivating free radicals through the action of GPx isoforms and thereby converting the reduced form (glutathione [GSH]) to the oxidized form (GSSG). Conversely, GR restores glutathione to its reduced form.
GPx isoforms are a family of enzymes whose properties vary slightly, depending on the tissues. The classic intracellular GPx1 is expressed in sperm and in the genital tract, and a direct relationship has been demonstrated with sperm motility (Dandekar et al, 2002). However, knockout mice lacking GPx1 have been found to be fertile (de Haan et al, 1998).
More significantly, a direct relationship has been reported between male fertility and phospholipid hydroperoxide glutathione peroxidase (PHGPx, or GPx4), a selenoprotein that is highly expressed in testicular tissue (Foresta et al, 2002). Furthermore, genetic variations of GPx4 have been detected in humans. Eleven variant sites have been discovered in 25% of all infertile males, yet only 25% of these affect activity or transcription, which rules out a relationship with peroxidase content and fertility-related parameters. However, GPx4 does deserve further attention as a potential cause of infertility in certain cases (Maiorino et al, 2003). Its importance is reflected by a selenium deficiency–related suppression of spermatogenesis in rats (Behne et al, 1996). GPx4 has a dual function as an active peroxidase that may protect rapidly dividing cells against oxidative injury and as a structural protein that cross-links with itself and other proteins and subsequently builds up the mitochondrial capsule (Foresta et al, 2002).
To date, there is a lack of markers for prognosticating the quality of semen samples following the freeze-thaw cycle. Moreover, there are no sperm parameters or biochemical features that provide a reliable predictive value for the success of postthaw recovery.
The goal of the current study was to correlate oxidative stress status with the postthaw recovery of progressive motile cells, both in terms of GSH levels and GPx and GR expression and activity, as a first step in improving sperm cryostorage protocols. Evidently, these measurements could serve as indicators of the postthaw quality of sperm samples.
To do this, we determined the expression of GPx1, GPx4, and GR by quantitative fluorescent polymerase chain reaction (qfPCR) and, by using spectrophotometry in controlled reactions, we measured the activity of these enzymes as well as cellular GSH.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
After 10 minutes of liquefaction at 37°C with 5% CO2, semen samples were examined for concentration and motility in a Mackler chamber (Sefi Laboratories, Tel Aviv, Israel) according to World Health Organization (1999) guidelines. Sperm vitality was analyzed using the dye exclusion method with eosin (Sigma Chemical Co, St Louis, Mo). The number of sperm used in the experiments described was standardized to 50 million to provide enough spermatozoa for all the experiments, and the sperm were processed by a 10-minute centrifugation at 400 x g. The supernatant was then discarded, and the pellet was resuspended in an isotonic buffer. Aliquots of these suspensions were used to determine protein concentration, enzymatic activity, and messenger RNA (mRNA) expression. No ejaculate showed significant (>0.1 million/mL) levels of leukocytes, immature germ cells, or other somatic cell contaminants, as demonstrated by Quick Panoptic (QCA, Barcelona, Spain) staining of semen smears.
Semen samples were frozen in pills on the surface of dry ice using a glycerol-based cryoprotectant (Sperm Freezing Medium, MediCult, Jyllinsge, Denmark) as described by Meseguer et al (2004). Aliquots of the samples were transferred to new tubes that were immersed in room temperature water and thawed for 10 minutes, after which they were treated for 10 minutes at 37°C.
Samples were then analyzed for concentration, motility, and vitality as explained above, and determinations were recorded independently by 2 different observers (M.M. and N.G.). Final results are presented as a mean of the 2 observers' recordings and only when there was less than 5% discordance. Samples showing greater differences were disregarded.
RNA Extraction and Reverse Transcription-PCR
Sperm RNA extraction was performed with pure, unadulterated, undiluted
semen (n = 27) using the Trizol reagent (TelTest, Friendswood, Tex). The total
amount of RNA was quantified by spectrophotometry on a BioRad (Durviz,
Valencia, Spain) spectrophotometer. Then, RNA was reverse transcribed using
the Adventage RT-for-PCR Kit (Clontech, Palo Alto, Calif) following the
manufacturer's instructions. The product was diluted to a final volume of 100
µL with diethylpyrocarbonate-treated water and stored at -20°C until
PCR analysis.
Primer sequences that targeted the mRNAs of GPx1 and GR in sperm cells were obtained from previously published articles (Dubrovskaya and Wetterhahn, 1998; El Mouatassim et al, 1999). Sequences for human GPx4 were obtained from GenBank, and primers were acquired from Genotek (Barcelona, Spain) and designed using GeneFisher software.
Prior to the qfPCR experiments, all primers were tested via nonfluorescent PCR in an Eppendorf (Hamburg, Germany) Personal Thermocycler, and their products were run in a 2% agarose gel and stained with ethidium bromide to verify that the amplification targeted the correct sequence and that the product size was adequate.
All of the results of qfPCR were normalized by the analysis of ß-globin expression, and the primers used were those provided by the qfPCR kit, as described in the following paragraphs.
Each nonfluorescent PCR was programmed for 36 cycles, and the complete series of PCR reactions is documented in other publications by our group (Garrido et al, 2001; Meseguer et al, 2001, 2002).
qfPCR was performed with 5 µL of complementary DNA (cDNA) using the LightCycler—FastStart DNA Master SYBR Green I Kit (Roche Diagnostics GmbH, Mannheim, Germany) following the manufacturer's instructions.
A calibration curve was included for each experiment from 5 serial dilutions (1/5) of a sample of endometrial cDNA to interpolate the results.
Green fluorescence was measured during each cycle, and the results were analyzed with the aid of an amplification curve, taking into account the starting point, using Roche Molecular Biochemical Lightcycler Software. The units used were the ratios between the expressions of each gene vs that of ß-globin.
Enzymatic Activity Measurements
Sperm samples destined for total protein experiments (n = 29) were lysed,
and protein fractions were extracted using 350 µL of lysis buffer (20%
perchloric acid in 1 mM EDTA). The suspensions were centrifuged once more, and
the supernatants were stored at -20°C until protein and enzymatic
determinations were carried out.
The readings of enzymatic activity were normalized with the total amount of protein. These determinations were performed according to the Bradford method (BioRad).
One unit of GR activity was defined as the amount of enzyme catalyzing the reduction of 1 µmol of GSSG per minute at pH 7.6, 25°C. This activity was determined indirectly, by measuring the consumption of NADPH and observing the decrease in absorbency at 340 nm as a function of time (25°C), with a Bioxytech GR-340 Spectrophotometric Assay (OxisResearch, Portland, Ore).
Selenium-dependent GPx (GPx1) and PHGPx activities (units) are defined as the amount of enzyme catalyzing the oxidation of 1 µmol of GSH per minute at pH 7, 37°C.
GPx1 and GPx4 activities were again measured indirectly by spectrophotometry, based on the consumption of NADPH as the result of added GR, by the decrease of absorbency at 340 nm as a function of time, with GPx first converting GSH to GSSG through the reduction of cumene hydroxide, which acts as an electron donor for GPx1 (Romero et al, 1999) and PHGPx. Phosphatidylcholine hydroperoxide was used as a specific substrate for PHGPx, mainly GPx4 (Imai et al, 2001).
To summarize, 10 µL of thawed sample was added to 590 µL of solution 1 (1 mM EDTA, 1 mM sodium azide, and 0.1 M potassium phosphate buffer, pH 7; all from Sigma). Later, 100 µL of GR with an activity of 2.4 U/mL (using the previous buffer as a diluent) plus 100 µL of 10 mM GSH were added, and the solution was incubated for 5 minutes at 37°C. Afterward, a 100-µL solution of 1.5 mM NADPH was dissolved in 0.1% NaHCO3, and only the oxidation of NADPH that was not dependent on hydroperoxides was monitored for 3 minutes. Finally, 100 µL of either 1.5 mM cumene hydroperoxide (Sigma) or 40 µM phosphatidylcholine hydroperoxide (kindly provided by Dr Matilde Maiorino, University of Padova, Italy) was added to the reaction, and the decrease in absorbency at 340 nm (37°C) was recorded.
GSH Concentration
To study glutathione concentrations, a similar experiment was carried out.
The acidic media in which samples were suspended as basified by the addition
of known volumes of 40% K2 HPO4, and the samples were
centrifuged at 12 000 x g for 5 minutes to precipitate sodium
perchlorate.
Aliquots of 930 µL of solution 1 (1 mM EDTA, 1 mM sodium azide, and 0.1 M potassium phosphate buffer, pH 7; all from Sigma) were first added to the cuvette, followed by 10 µL of solution 2 (0.02 g of 1-chloro-2,4-dinitrobenzene, from Sigma, in 10 mL of ethanol) and 50 µL of the study sample. The cuvette was then introduced into the spectrophotometer.
The reaction was initiated with the addition of 20 µL of solution 3 (500 IU of GSH-S-transferase per milliliter from Sigma in 0.1 M phosphate buffer, pH 7.0), and we monitored the increase in absorbency at 340 nm (37°C) until it reached a plateau, when all available GSH had reacted.
Calculations were performed on the basis of the volume of the sample, light path length, corresponding dilution factors, absorbency decrease, and molar extinction coefficient.
Statistical Analysis
GR, GPx1, and GPx4 mRNA expression and activity and glutathione
concentrations were correlated with postthaw seminal parameters using linear
regression analysis followed by analysis of variance. Significance was defined
as P < .05.
Thawing was considered successful when the percentage of postthaw total progressive motility was 40% higher than fresh total motility. Our goal was to use the determinations from these experiments as a diagnostic test for predicting the state of semen samples after thawing.
The diagnostic capacity of a test, or its ability to discriminate between situations, is evaluated using receiver operating characteristic (ROC) curve analysis (Zweig and Campbell, 1993). ROC curves can also be used to compare the diagnostic performance of 2 or more diagnostic tests. In this way, we compared ROC analyses of successful and unsuccessful thawing for all the parameters. By analyzing the area under the ROC curve (AUC), we can be advised about the accuracy of the parameters (their ability to discriminate between the 2 conditions). Such analyses also permit us to determine the optimum discriminating point of each parameter as well as its sensitivity and specificity.
We determined the positive and negative predictive values of GPx1, GPx4, and GR expression and activity and GSH concentration. These values illustrate the probability of samples thawing successfully when the test for GPx1 and GPx4, GR, and GSH is positive and the probability of samples thawing poorly when the same test is negative.
Also, statistical significance was compared between the ROC curves for GPx1, GPx4, GR, and GSH to determine which parameter should be used as the most effective diagnostic tool.
Statistical analysis was performed using the Statistical Package for Social Sciences (SPSS Inc, Chicago, Ill) and MedCalc (Ghent, Belgium) statistical software.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
|
|
Relationship Between GPx1 and GPx4 and PTRR
We identified a statistically significant positive correlation between GPx1
and GPx4 activity and mRNA expression and PTRR: for GPx1 activity, P
= .013; for PHGPx activity, P = .022; for GPx1 gene expression,
P = .019; and for GPx4 gene expression, P = .020.
The correlation coefficient (r) was .493 for the GPx1 assay, .431 for the PHGPx assays (enzyme activity), .471 for the qfPCR GPx1 experiments, and .585 for the qfPCR GPx4 experiments (mRNA) (Figure 1).
|
Relationship Between GSH and Postthaw Motile Sperm Recovery
A contrary pattern was discovered when GSH concentrations were analyzed in
sperm cell lysates. There was a statistically significant negative correlation
between GSH and PTRR, with a significance of P = .011 and an
r value of -.472 (Figure
2).
|
Relationship Between GR and Postthaw Motile Sperm Recovery
When GR activity and gene expression were studied, no correlation of
statistical significance was initially found. Indeed, GR activity was
considerably low when compared to that of GPx. Thus, GR is unlikely to be an
enzyme implicated in cellular metabolism. Significance was P = .104
when we performed mRNA analysis by quantitative PCR, and significance was
P = .062 when we carried out spectrometry enzymatic activity assays.
Nevertheless, as in the GSH study, there seems to be a negative correlation
between GR expression and activity and PTRR (r = -.320 vs r
= -.351) (Figure 3).
|
Predictive Value of Oxidative Stress Status and Postthaw Sperm Quality
To further investigate the possibility of forecasting successful or
unsuccessful sperm thawing, we determined AUCROC curves, using ROC
analysis, for sperm GPx and GR mRNA, GPx and GR activity, and GSH, and these
are presented in Table 3. In
this way, we clearly demonstrate that the most accurate diagnoses provided by
the molecules analyzed are those for GPx4 and GPx1 mRNA expression, followed
by GPx1 and PHGPx enzymatic activity and GSH concentrations. The higher AUCs
for these parameters indicate the greater likelihood of a good postthaw
recovery of semen samples (with a total motile sperm recovery of >40%).
|
The highest AUC values that discriminate between successful and unsuccessful thawing of semen samples were more than 7.2 (sensitivity, 80.0%; specificity, 100%) for GPx4 mRNA and more than 445 U (sensitivity, 45.5%; specificity, 94.1%) for GPx1 activity (Table 3).
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
The glutathione system has a determinant role in the protection of sperm
cells because it inactivates free radicals. The pivotal component is the
tripeptide glutathione
(
-L-glutamyl-L-cysteinylglycine), whose free
thiol group in the cysteine residue is extremely reactive
(Meister and Anderson, 1983).
This molecule exists in a thiol-reduced form (GSH) and in an oxidized form
(GSSG).
Further components of this system are the molecules implicated in glutathione metabolism, which are responsible for their redox state in physiological conditions.
GPx isoforms are a family of selenium-containing enzymes that are present in all mammals and are particularly active in the liver, where there is a relatively high exposure to reactive agents. Their importance to sperm function has been shown in elaborate experiments by Alvarez and Storey (1989), in which enzyme inhibition with mercaptosuccinate (permeate to plasma membrane) increased the LPO process 20-fold and in which glutathione was present in its GSH form, thus indicating that functioning GPx, together with a reductive substrate, is necessary for preventing LPO. Depleting GSH or inhibiting GPx dramatically increases LPO.
GR renews the GSH, which is required by GPx; thus, they are all of equal importance. The reductive substrate employed by this enzyme in human sperm is NADPH (Storey, 1997).
Oxidative stress–induced LPO is undoubtedly related to sperm function in physiological conditions, such as high leukocyte levels and immature sperm, as well as in nonphysiological conditions, such as semen freezing (Gomez et al, 1998; Aitken et al, 1999; Schuffner et al, 2001).
Protection against the harsh process of cryopreservation is necessary to preserve sperm viability and fertilization potential. To this end, GPx isoforms must be relatively abundant and functional in sperm.
The interest in developing a satisfactory system for recovering as many healthy sperm as possible after thawing is, without doubt, a primary objective. Optimal sperm recovery following cryopreservation is especially relevant when samples are subject to elevated oxidative stress levels, as in the case of testicular biopsies and prechemotherapy, prevasectomy, and sperm donor samples (Meseguer et al, 2003).
Through our results, we can conclude that GR mRNA expression and activity are not related to sperm susceptibility to cryodamage, as shown by the lack of correlation between the quality of the sperm sample after thawing and both the gene expression and activity inside sperm cells. GR activity was considerably low in all the samples tested. Therefore, its role in sperm function does not seem to be significant.
Conversely, the activity of GPx and its ability to eliminate free radicals via GSH seems to be essential for post-thaw recovery. A stressful oxidative environment seems to sensitize sperm to the traumatic procedure of freezing and thawing. The impact of PHGPx on sperm survival is less easily explained. In early spermatogenesis, as an active peroxidase, it may protect the rapidly dividing cells against oxidative injury, but it is difficult to attribute this antioxidant function, widely assumed to be mandatory in the protection of sperm against abundant oxidants, to PHGPx in mature spermatozoa. When overwhelming proportions of sperm in the capsule are inactive, the nuclear variant is cross-linked to protamine, and the residual active enzyme is unable to function as an antioxidant system. This is because it lacks the major reducing substrate, GSH, the loss of which has been attributed to processes that occur during final sperm maturation (Foresta et al, 2002). Subsequently, PHPGx adopts the peculiar function of a structural protein. In this way, the lower postfreeze-thaw survival rate could be associated with sperm alterations, such as impaired motility—a fuzzy appearance to the midpiece, which is most likely a result of impaired PHGPx biosynthesis and which affects the sperm's survival potential.
GSH concentrations are inversely correlated with PTRR, which could be related to GPx function; that is to say, lower GPx activity increases GSH reserves, and higher GPx activity reduces GSH levels. Thus, it would seem that, during sperm metabolism, GSH does not inactivate free radicals alone (as occurs during cellular metabolism), but requires the mediation of GPx in order to reduce oxidative components present in the sperm cells.
It also has been demonstrated that GPx and PHGPx activity, gene expression of GPx1 and GPx4, and concentrations of GSH are reliable predictors of postthaw semen quality, as observed in the recovery of progressive motile sperm cells after thawing when compared to the initial seminal analysis.
Both of the GPx isoforms analyzed in the present study showed similar levels of expression and activity and similar predictive values.
Although further experiments are required, given the implications that oxidative stress has on the quality of sperm cells after thawing, oxidative-protective substances (ie, enzyme enhancers) could be added to the medium used to improve postthaw recovery. It seems that anti-oxidants alone do not inactivate free radicals, as observed with GSH. Subsequently, it is unlikely that the addition of these molecules to the medium would improve the sperm quality for the purpose of ART procedures. Indeed, we suspect that a direct effect on antioxidative enzymes would be more effective.
Although intracytoplasmic sperm injection can overcome severe male factor infertility and combat poor reproductive results after thawing, there are specific cases in which an improved freezing method is essential, such as in sperm donor management (Garrido et al, 2002).
Redox status determination could also be employed for predicting the total motile cells expected after thawing. This would be especially useful in the selection and management of sperm donors.
Furthermore, our data clearly demonstrate that no predictive values can be derived from the basic parameters of fresh semen samples.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Alvarez JF, Storey BT. Role of glutathione peroxidase in protecting mammalian spermatozoa from loss of motility caused by spontaneous lipid peroxidation. Gamete Res. 1989; 23: 77 -90.[Medline]
Alvarez JF, Touchstone JC, Blasco L, Storey BT. Spontaneous lipid
peroxidation and production of hydrogen peroxide and superoxide in human
spermatozoa: superoxide dismutase as major enzyme protectant against oxygen
toxicity. J Androl. 1987; 8: 338
-348.
Behne D, Weiler H, Kyriakopoulos A. Effects of selenium deficiency on testicular morphology and function in rats. J Reprod Fertil. 1996;106: 291 -297.
Centola GM, Raubertas RM, Mattox H. Cryopreservation of human
semen: comparison of cryoprotectives, sources of variability and prediction of
post-thaw survival. J Androl. 1992; 13: 283
-288.
Critser JK, Arenson BW, Asker DV, Huse-Benda AR, Ball GD. Cryopreservation of human spermatozoa. II. Post thaw chronology on motility and on zona-free hamster ova penetration. Fertil Steril. 1987;47: 980 -984.[Medline]
Dandekar SS, Graham EM, Plant GT. Lipid peroxidation and antioxidant enzymes in male infertility. J Postgrad Med. 2002;48: 186 -190.[Medline]
de Haan JB, Bladier C, Griffiths P, et al. Mice with a homozygous
null mutation for the most abundant glutathione peroxidase, GPx1, show
increase susceptibility to the oxidative stress-inducing agents paraquat and
hydrogen peroxide. J Biol Chem. 1998; 273: 22528
-22534.
Dubrovskaya VA, Wetterhahn KE. Effects of Cr (VI) on the expression
of the oxidative stress genes in human lung cells.
Carcinogenesis. 1998; 19: 1401
-1407.
El Mouatassim S, Guerin P, Menezo Y. Expression of genes encoding
antioxidant enzymes in human and mouse oocytes during the final stages of
maturation. Mol Hum Reprod. 1999; 5: 720
-725.
Foresta C, Flohe L, Garolla A, Roveri A, Ursini F, Maiorino M. Male
fertility is linked to the selenoprotein phospholipid hydroperoxide
glutathione peroxidase. Biol Reprod. 2002; 67: 967
-971.
Garrido N, Albert C, Krussel JS, O'Connor J, Remohí J, Simón C, Pellicer A. No differences in expression, production and secretion of vascular endothelial growth factor (VEGF) and interleukin-6 (IL-6) by granulosa cells in women with endometriosis compared to those without the disease. Fertil Steril. 2001; 75: 568 -575.
Garrido N, Zuzuarregui JL, Meseguer M, Simon C, Remohi J, Pellicer
A. Sperm and oocyte selection and management: experience of a 10 year
follow-up of more than 2100 candidates. Hum Reprod. 2002; 17: 3142
-3148.
Gomez E, Irvine DS, Aitken RJ. Evaluation of a spectrophotometric assay for the measurement of malonylaldehide and 4-hydroxyalkenals in human spermatozoa: relationships with semen quality and sperm function. Int J Androl. 1998; 15: 71 -77.
Imai H, Suzuki K, Ishizaka K, et al. Failure of expression of
phospholipid hydroperoxide glutathione peroxidase in the spermatozoa of human
infertile males. Biol Reprod. 2001; 64: 674
-683.
Maiorino M, Bosello V, Ursini F, Foresta C, Garolla A, Scapin M,
Sztajer H, Flohe L. Genetic variations of gpx-4 and males infertility in
humans. Biol Reprod. 2003; 68: 1134
-1141.
Mazzilli F, Rossi T, Sabatini L, Pulcinelli FM, Rapone S, Dondero F, Gazzaniga PP. Human sperm cryopreservation and reactive oxygen species (ROS) production. Acta Eur Fertil. 1995; 26: 145 -148.[Medline]
Mc Laughlin EA, Ford WCL, Hull MGR. Motility characteristics and membrane integrity of cryopreserved human spermatozoa. J Reprod Fertil. 1992;95: 527 -534.
Meister A, Anderson ME. Glutathione. Annu Rev Biochem. 1983;52: 711 -760.[Medline]
Meseguer M, Aplin JD, Caballero-Campo P, O'Connor JE, Martín
JC, Remohí J, Pellicer A, Simón C. Human endometrial MUC1 is
upregulated by progesterone and down-regulated in vitro by the human
blastocyst. Biol Reprod. 2001; 64: 590
-601.
Meseguer M, Garrido N, Martinez-Conejero JA, Simon C, Pellicer A, Remohí J. The role of cholesterol, calcium and mitochondrial activity in the susceptibility for cryodamage after a cycle of freezing and thawing. Fertil Steril. 2004; 81: 588 -594.[Medline]
Meseguer M, Garrido N, Remohí J, Pellicer A, Simon C,
Martínez-Jabaloyas JM, Gil-Salom M. Testicular sperm extraction (TESE)
and ICSI in patients with permanent azoospermia after chemotherapy.
Hum Reprod. 2003; 18: 1281
-1285.
Meseguer M, Garrido N, Simon C, Remohí J, Pellicer A. Comparison of polymerase chain reaction–dependent methods for determining the presence of human immunodeficiency virus and hepatitis C virus in washed sperm. Fertil Steril. 2002; 78: 1199 -1202.[Medline]
Romero FJ, Martinez-Blasco A, Bosch-Morell F, Marin N, Trenor C, Roma J. Glycemic control and not protein kinase C inhibition prevents the early decrease of glutathione peroxidase activity in peripheral nerve of diabetic mice. Peripheral Nerv Syst. 1999; 4: 265 -269.
Saleh RA, Agarwal A. Oxidative stress and male infertility: from
research bench to clinical practice. J Androl. 2002; 23: 737
-752.
Schuffner A, Morshedi M, Oehninger S. Cryopreservation of
fractionated, highly motile human spermatozoa: effect of membrane
phosphatidilserine externalization and lipid peroxidation. Hum
Reprod. 2001;16: 2148
-2153.
Storey BT. Biochemistry of the induction and prevention of
lipoperoxidative damage in human spermatozoa. Mol Hum
Rep. 1997;3: 203
-213.
Zweig MH, Campbell G. Receiver-operating characteristic (ROC)
plots: a fundamental evaluation tool in medicine. Clin
Chem. 1993;39: 561
-577.
This article has been cited by other articles:
|
|
 |
|
  K E Waterhouse, P O Hofmo, A Tverdal, and R R Miller Jr Within and between breed differences in freezing tolerance and plasma membrane fatty acid composition of boar sperm. Reproduction, May 1, 2006; 131(5): 887 - 894. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Muriel, M. Meseguer, J. L. Fernandez, J. Alvarez, J. Remohi, A. Pellicer, and N. Garrido Value of the sperm chromatin dispersion test in predicting pregnancy outcome in intrauterine insemination: a blind prospective study Hum. Reprod., March 1, 2006; 21(3): 738 - 744. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Gadea, D. Gumbao, C. Matas, and R. Romar Supplementation of the Thawing Media With Reduced Glutathione Improves Function and the In Vitro Fertilizing Ability of Boar Spermatozoa After Cryopreservation J Androl, November 1, 2005; 26(6): 749 - 756. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
