Ashok Agarwal
email: agarwaa@ccf.org

Rakesh K Shar

Sajal Gupta

Florence Boitrelle

Renata Finelli

Ne Parekh

Damayanthi Durairajanayag

Ramad Saleh

Moham Arafa

Chak L Cho

Amarnat Ala'a Farkouh

Rambhat

Ralf Henkel

Paraskevi Vogia

Nichol Tadros

Parviz Kavoussi

Edmun Ko

Krist Leisegang

Husse Kandil

Ayad Pala

Gianmaria Sal

Taymour Mosta

Osvald Rajmi

Sale Ali Banihani

Samantha Sch

Tan V Le

Ponco Biro

Gökhan Çek

Juan Alvare

Juan Manuel Corral Moli

Christopher C K Ho

Aldo E Calogero

Mesut Kareim Khalafa

Berkan Duran

Shinnosuke Kurod

Giovanni M Col

Christina Anagnostopoul

Edoardo Pescatori

Eric Chu

Ettore Carop

Fotios Dimitria

Germar-Michael Pingg

Gian Mari Busett

Giancarlo Baler

Haitham Elbard

Hisanori Taniguchi

Hyun Jun Park

Israel Maldonad Rosa

Jean De La Rosette

Jonathan Ramsay

Kasond Bowa

Ma Simopoulou

Marcelo Gabr Rodriguez

Marjan Sabbaghian

Marlon Martinez

Moham Ali

Sadi Gilani

Moham S Al-Marhoon

Raghavender Kosg

Rossella Cannarella

Sav Mici

Shinichiro Fukuha

Sijo Parekat

Sunil Jindal

Abo-Almagd Taha

Abdel-Meguid

Yoshiharu Morimoto

Rupin Shah

Sperm Vitality and Necrozoospermia: Diagnosis, Management, and Results of a Global Survey of Clinical Practice

Keywords: Asthenozoospermia, Eosine Yellowish-(YS), Infertility, Nigrosin, Spermatozoa, Vitality

Sperm vitality testing is a basic semen examination that has been described in the World Health Organization (WHO) Laboratory Manual for the Examination and Processing of Human Semen from its primary edition, 40 years ago. Several methods can be used to test sperm vitality, such as the eosin-nigrosin (E-N) stain or the hypoosmotic swelling (HOS) test. In the 6th (2021) edition of the WHO Laboratory Manual, sperm vitality assessment is mainly recommended if the total motility is less than 40%. Hence, a motile spermatozoon is considered alive, however, in certain conditions an immotile spermatozoon can also be alive. Therefore, the differentiation between asthenozoospermia (pathological decrease in sperm motility) and necrozoospermia (pathological decrease in sperm vitality) is important in directing further investigation and management of infertile patients. The causes leading to necrozoospermia are diverse and can either be local or general, testicular or extra-testicular. The andrological management of necrozoospermia depends on its etiology. However, there is no standardized treatment available presently and practice varies among clinicians. In this study, we report the results of a global survey to understand current practices regarding the physician order of sperm vitality tests as well as the management practices for necrozoospermia. Laboratory and clinical scenarios are presented to guide the reader in the management of necrozoospermia with the overall objective of establishing a benchmark ranging from the diagnosis of necrozoospermia by sperm vitality testing to its clinical management.

INTRODUCTION

Sperm vitality, assessed by eosin-nigrosin (E-N) stain, refers to the integrity and permeability of the cell membrane, while sperm viability, assessed by hypoosmotic swelling (HOS) test, refers to the percentage of live spermatozoa in the semen. In laboratory practice, however, the terms "vitality" and "viability" are commonly used interchangeably [START_REF] Agarwal | Eosin-nigrosin staining procedure[END_REF] as they both refer to sperm membrane function and thereby whether or not the male germ cell is dead or alive. The assessment of human sperm vitality is an important parameter of basic semen analysis [START_REF] Björndahl | Why the WHO recommendations for eosin-nigrosin staining techniques for human sperm vitality assessment must change[END_REF][START_REF] Björndahl | A practical guide to basic laboratory andrology[END_REF][START_REF] Mortimer | Practical laboratory andrology[END_REF]. This test is commonly conducted in andrology laboratories and assisted reproduc-Samsun Vezirköprü State Hospital, Samsun, Turkey, 30 Centro ANDROGEN, La Coruña, Spain, 31 Servicio de Urología, Hospital Clínico de Barcelona. Barcelona, Spain, 32 Department of Surgery, School of Medicine, Faculty of Health and Medical Sciences, Taylor's University, Subang Jaya, Malaysia, 33 Department of Clinical and Experimental Medicine, University of Catania, Catania, Italy, 34 Department of Urology, Samsun Training and Research Hospital, Samsun, Turkey, 35 Andrology and IVF Unit, Procrea Institute, Lugano, Switzerland, 36 Department of Surgery, McGill University, Montreal, QC, Canada, 37 IVF Clinic "Akeso-Embryo ART", Athens, Greece, 38 Andrology and Reproductive Medicine Unit, Gynepro Medical, Bologna, Italy, 39 Department of Urology, Princess Alexandra Hospital, University of Queensland, Brisbane, QLD, Australia, 40 AndroUrology Centre, Brisbane, QLD, Australia, 41 Reproductive and IVF Unit, Andrology Outpatients Clinic, ASL Bari, Conversano (Ba), Italy, 42 1st Urology Department, School of Medicine, Aristotle University, Thessaloniki, Greece, 43 Department of Urology, Innsbruck Medical University, Innsbruck, Austria, 44 Department of Urology and Renal Transplantation, University of Foggia, Ospedali Riuniti of Foggia, Foggia, Italy, 45 Department of Urology and Andrology, Kansai Medical University, Hirakata, Osaka, Japan, tive technology (ART) clinics and the methodology is described in the 5th (2010) edition of the World Health Organization (WHO) Laboratory Manual for the Examination and Processing of Human Semen [START_REF]WHO laboratory manual for the examination and processing of human semen[END_REF]. In this 5th edition, it is stated that sperm vitality testing is possible on all semen samples but is mainly recommended if the progressive motility is lower than 40%. The choice of a progressive motility threshold is surprising and confusing. Indeed, a spermatozoon, even a non-progressive motile one, can be alive. Moreover, the threshold of 40% corresponds to the reference threshold of total motility [START_REF] Björndahl | A practical guide to basic laboratory andrology[END_REF][START_REF]WHO laboratory manual for the examination and processing of human semen[END_REF][START_REF] Barratt | ESHRE special interest group for andrology basic semen analysis course: a continued focus on accuracy, quality, efficiency and clinical relevance[END_REF]. Both these arguments have been addressed in the latest (6th) edition of the WHO manual, available since July 2021. According to the 6th edition, it is not necessary to assess vitality when at least 40% of spermatozoa are motile [START_REF]WHO laboratory manual for the examination and processing of human semen[END_REF]. Thus, in this article, we will describe the sperm vitality test as necessary if total motility is below 40%.

Regarding the definition of necrozoospermia, the WHO 5th edition states that the lower reference threshold of sperm vitality is set at 58%. Normal sperm vitality is applied to samples with ≥58% (5th centile; 95% confidence interval [CI], 55%-63%) alive spermatozoa [START_REF]WHO laboratory manual for the examination and processing of human semen[END_REF]. Thus, necrozoospermia was defined as the presence of less than 58% of alive spermatozoa in semen. The 6th edition of the WHO manual has removed the standards and reference values, arguing that the boundaries between sperm from infertile and fertile men were not as clear [START_REF]WHO laboratory manual for the examination and processing of human semen[END_REF].

Vitality should be examined in conjunction with motility in the same sample. The purpose of assessing both sperm vitality and motility is to clarify whether immotile spermatozoa seen in ejaculates with low sperm motility are dead or alive (Table 1). Vitality testing can also provide a counter-check on the motility evaluation, since the percentage of dead cells should not exceed (within sampling error) the percentage of immotile spermatozoa [START_REF]WHO laboratory manual for the examination and processing of human semen[END_REF]. The importance of a proper diagnosis must be emphasized here as the management of asthenozoospermia is different from that of necrozoospermia in many instances, which is why a clear discrimination should be pursued.

The most commonly used method to assess sperm vitality is the combined E-N stain [START_REF]WHO laboratory manual for the examination and processing of human semen[END_REF]. The prevalence of its use at the College of American Pathologists (CAP) certified clinics and laboratories across the United States is 62.8% (359/572) (CAP Proficiency report, May 2021; Supplement File 1). The E-N test is automati-

Testing for necrozoospermia

Indication: if total sperm motility is less than 40% Importance: in order to distinguish whether the immotile spermatozoa are dead (necrozoospermia) or are alive but with abnormal motility (asthenozoospermia), as this will have clinical implications in terms of approach and management Methods:

• Eosin-nigrosin staining: dead sperm heads are stained dark pink or red -all tested sperm are damaged and can no longer be used for ART • HOS test: live sperm tails become swollen -sperm is not damaged and can be used for ART

Causes and risk factors of necrozoospermia

• Genital tract infections -the most common cause cally conducted by the laboratory technologist within the requirements of a basic semen analysis in cases of asthenozoospermia (<40% total motility). For this test, eosin is used as a vitality dye in the evaluation of the integrity of the sperm membrane. An intact membrane will not be penetrated by eosin, unlike compromised membranes of dead sperm. One of the drawbacks of the E-N test is that stained spermatozoa cannot be used in subsequent procedures, namely in ART. Therefore, in such cases sperm viability should be tested in a manner which is not detrimental to the spermatozoa to be used in ART. The HOS test, for example, is compatible with fertilization procedures [START_REF] Agarwal | Eosin-nigrosin staining procedure[END_REF][START_REF] Jeyendran | Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics[END_REF][START_REF] Esteves | Sperm viability assays--a matter of life and death![END_REF][START_REF] Smikle | Hypo-osmotic swelling can accurately assess the viability of nonmotile sperm[END_REF][START_REF] Simopoulou | Improving ICSI: a review from the spermatozoon perspective[END_REF].

In the current review article, we describe the technical characteristics of sperm vitality tests and discuss the etiology and management of necrozoospermia. We also present laboratory and clinical scenarios on the relevance, utilization, and significance of sperm vitality testing. Additionally, we provide the results of a short global survey on the current practice patterns of sperm vitality testing among infertility specialists.

SPERM VITALITY ASSESSMENT

Sperm vitality assessment should be performed with an abstinence period of 2 to 7 days [START_REF]WHO laboratory manual for the examination and processing of human semen[END_REF]. The patient collects the specimen into a sterile container. The semen sample should be stored at 37°C and the sperm vitality test must be performed after liquefaction within 30 minutes to one hour after collection. This prevents the deleterious effects of dehydration and temperature on sperm vitality [START_REF]WHO laboratory manual for the examination and processing of human semen[END_REF].

Eosin-nigrosin staining

The most commonly used method to assess sperm vitality is the combined E-N stain. In this method, eosin is a supravital stain and nigrosin is a purple dye that serves as a counterstain [START_REF]WHO laboratory manual for the examination and processing of human semen[END_REF]. Here we describe the stepby-step protocol for performing the vitality test (Fig. 1) along with the reagents, consumables, and equipment required (Fig. 2).

The E-N stain is prepared by dissolving 0.67 g of eosin Y and 0.9 g of sodium chloride (NaCl) in 100 mL of distilled water and this mixture is then placed on a hot plate to undergo gentle heating. Then 10 g of nigrosin is added to 100 mL of eosin Y solution. The suspension is boiled, then allowed to cool to room temperature. It is next filtered through a filter paper to remove coarse and gelatinous precipitates. The E-N stain is stored in a sealed dark-glass bottle.

To perform the vitality test, two frosted slides are labeled with patient's name, medical record number, and date. Fifty microliters of well-mixed semen are placed into a Boerner slide well (or a test-tube) and equal Fig. 1. Schematics of the steps involved in conducting the vitality test. (A) Fifty microliters of well-mixed semen is placed into a Boerner slide well. Equal volume of E-N stain is added. The sample is mixed well with a wooden stirrer, and left to sit for 30 seconds. (B) Immediately after that, ten microliters of the mixture is placed on the labeled frosted slide and a thin smear is made. Perform this step in duplicate. Once the smear is air-dried, a coverslip is placed on the mounting media. (C) A drop of immersion oil is placed on each of the mounted slides. One hundred spermatozoa are observed under bright-field microscopy with 1,000x magnification. This step is performed twice on each slide. The vital (unstained) and non-vital (stained) sperm are counted. Results are recorded in replicate and the average percentage of vital sperm is reported. volume of E-N stain is added, and the sample is mixed well with a wooden stirrer, then it is let to settle for 30 seconds (Fig. 1A).

A B C

The nigrosin stain provides a dark background that makes it easier to distinguish faintly stained spermatozoa. Immediately two thin smears are made by pipetting 10 µL of the mixture and placing on each of the labeled slide. After air-drying the smear, a coverslip is placed on the non-aqueous mounting media (Fig. 1B). A drop of immersion oil (refractive index=1.52) is placed on the mounted slide, and spermatozoa are observed with 100× objective and a 10× eye-piece at a total magnification of 1,000× under a bright-field microscopy (Fig. 1C). White or faint pink sperm heads indicate vital spermatozoa (Fig. 3A,3B), while red or dark pink heads indicate non-vital spermatozoa (Fig. 3C). If the stain is limited only to a part of the neck region, and the rest of the head area is unstained (Fig. 3B), this is considered a "leaky neck membrane", and it is not a sign of cell death and total membrane disintegration. These cells should be assessed as alive. Routine method of counting at least 200 spermatozoa gives sufficiently accurate results for the intended clinical purpose of establishing whether the immotile spermatozoa seen in the ejaculate with low sperm motility are alive or not [START_REF] Mortimer | A technical note on the assessment of human sperm vitality using eosin-nigrosin staining[END_REF]. The percentage of vital sperm is reported.

Hypoosmotic swelling test

This test evaluates the functional integrity of the sperm plasma membrane and also serves as a useful indicator of fertility potential [START_REF] Jeyendran | Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics[END_REF]. Functional integrity can be demonstrated by allowing sperm to react in a hypoosmotic medium. The membrane is semi-permeable in live cells, therefore cells with intact membranes (live cells) will swell in hypotonic solutions. The HOS test has high reproducibility and accuracy and is closely correlated to the in vitro fertilizing ability of spermato- zoa [START_REF] Sallam | The use of the modified hypo-osmotic swelling test for the selection of immotile testicular spermatozoa in patients treated with ICSI: a randomized controlled study[END_REF][START_REF] Charehjooy | Selection of sperm based on hypo-osmotic swelling may improve ICSI outcome: a preliminary prospective clinical trial[END_REF].

For HOS testing, a hypoosmotic solution is prepared by adding 0.735 g sodium citrate dehydrate and 1.351 g D-fructose in 100 mL of distilled water. A well-mixed 0.1 mL aliquot of semen is mixed with 1 mL HOS solution (Fig. 4). The sample is mixed gently by continuous pipette suction and release and incubated at 37 o C for 30 minutes. Following incubation, 1 drop of the semen mixture is placed on a glass slide with a coverslip. This test is performed as a duplicate. The percentage of spermatozoa demonstrating tail swelling (Fig. 4A) are counted under 40× phase contrast lens. A total assessment of 200 spermatozoa for tail swelling/coiling is performed using a 2-channel differential cell counter. The average percentage of live sperm showing tail swelling is calculated. The normal threshold is ≥58% (95% CI, 55%-63%) of spermatozoa with tail swelling (Fig. 4B) according to the guidelines in the WHO 5th edition manual [START_REF]WHO laboratory manual for the examination and processing of human semen[END_REF].

Quality control and quality assurance 1) Quality control of the eosin-nigrosin stain and hypoosmotic swelling reagents

For E-N staining, a monthly patient quality control (QC) is required to check the quality of the stain. A QC step is performed each time new reagents are prepared. This must be performed before a new stain is used to test further patient specimens [START_REF] Agarwal | Andrological evaluation of male infertility[END_REF]. The total motility of the specimen should be assessed prior to the QC protocol. The specimen should be stained with both the old lot of stain as well as the new lot. Both sets of slides should be scored for sperm vitality. The percentage of viable sperm should be greater than or equal to the total motility of the specimen used. The comparison of the 2 lots of reagents should be within 10% of each other. If results are not within the acceptable range, the staining must be repeated using another specimen [START_REF] Agarwal | Andrological evaluation of male infertility[END_REF]. Similarly, when a new HOS reagent is prepared, QC must be performed before they can be used to test further semen samples. A positive control is prepared using a normal semen sample and run monthly or when a fresh reagent is prepared with a result of ≥58% to verify that the HOS reagents meet the criteria and can be used for patient testing. All results need to be verified and recorded when the control is run.

2) Internal quality control

Both vitality and viability slides are scored by two observers (RKS and SG) and the results are compared. A Bland-Altman plot can be used to check the correlation of inter-technician sperm vitality results.

3) External quality control

Two control vitality slides are prepared by accreditation laboratories and are assessed every six months for external QC. The designated technologist scores at least 200 spermatozoa and records results on the data sheet as percent viable. Results should be within ±2 standard deviation of mean for all laboratories as reported by the accreditation agency. Corrective action must be undertaken if the results are not within the specified range. The technologist will have to be retrained in the test and then instructed to re-score the proficiency slides [START_REF] Agarwal | Andrological evaluation of male infertility[END_REF].

LABORATORY SCENARIOS

Some laboratory issues may be encountered during the preparation and scoring of the smears for vitality. Recommendations for troubleshooting are illustrated in the following scenarios.

Case A 1) Scenario

A 28-year-old patient provides a semen sample for laboratory evaluation. Upon examination, the sperm parameters are (1) volume, 3.3 mL; (2) concentration, 37×10 6 /mL; (3) total motility, 15%. How will you proceed?

2) Solution

Total motility is below 40%. Prepare the slides by following the steps for E-N staining. The percentage of vital and non-vital spermatozoa must be counted and compared with motility. The vitality results must be equal to or greater than the sperm total motility.

Case B 1) Scenario

A 30-year-old patient provides a semen sample for laboratory evaluation. Upon examination, the parameters are (1) volume, 0.5 mL; (2) concentration, 5×10 6 / mL; (3) total motility, 10%. How will you proceed?

2) Solution

All three parameters are below the WHO reference values [START_REF]WHO laboratory manual for the examination and processing of human semen[END_REF]. Since the concentration is below 10×10 6 /mL, first centrifuge the sample at 3,000 g for 7 minutes. Gently remove approximately 300 µL of the supernatant. After mixing the pellet thoroughly, perform the E-N test as previously described. Note on the frosted label "spun" indicating that it is a spun smear. The percentage of vital and non-vital sperm must be counted, and vitality compared with motility. The vitality results must be equal to or greater than the sperm total motility. Vitality test can indeed provide a check on the motility evaluation, since the percentage of dead cells should not exceed (within sampling error) the percentage of immotile spermatozoa [START_REF]WHO laboratory manual for the examination and processing of human semen[END_REF]. If the evaluation of sperm vitality and motility are discordant (presence of less live sperm than motile sperm), the evaluation of these two parameters should be redone.

Case C 1) Scenario

A 35-year-old patient provides a semen sample for laboratory evaluation. Upon examination, the param-eters are (1) volume, 2.5 mL; (2) concentration, 20×10 6 / mL; (3) progressive motility, 30%. The sample is examined for vitality and a majority of the spermatozoa show sperm heads that are neither completely white nor completely stained pink. How will you proceed?

2) Solution

This is an example of "leaky" sperm. The neck region is very fragile and is the first to show signs of damage. The membrane breakdown may be very minimal at the initial stages and so there is a minute portion of stain that can enter the base of the head and neck region. Therefore, only a limited amount of pink stain is observed, and the rest of the sperm head is white (Fig. 3B). Spermatozoa with an appearance as described above following staining should be scored as vital. Gradually the damage may spread to the rest of the sperm head. This allows the stain to penetrate the sperm head and the extent of the pink stain may be more than at the base but still some portion of the head may be unstained and appear white. Spermatozoa that are stained pink should be reported as non-vital (Fig. 3C).

ETIOLOGIES OF NECROZOOSPERMIA

Proper patient history is essential to eliminate false results of necrozoospermia. For example, the addition of lubricants during masturbation or inadvertent spillage of antiseptic solution, soap or water into the semen collection sample may be responsible for false results. In such cases, another specimen should be collected following detailed instructions and submitted for vitality assessment. Necrozoospermia is reported to have a prevalence of 0.2% to 0.4% in infertile males [START_REF] Nduwayo | Management of necrospermia][END_REF][START_REF] Correa-Pérez | Clinical management of men producing ejaculates characterized by high levels of dead sperm and altered seminal plasma factors consistent with epididymal necrospermia[END_REF][START_REF] Dumont | Necrozoospermia: from etiologic diagnosis to therapeutic management[END_REF]. Causes of necrozoospermia could either be local or general, testicular or extra-testicular, and some of them can be associated in the same patient (Table 1) [START_REF] Nduwayo | Management of necrospermia][END_REF][START_REF] Fang | Male infertility and adult polycystic kidney disease are associated with necrospermia[END_REF][START_REF] Lerda | Study of reproductive function in persons occupationally exposed to 2,4-dichlorophenoxyacetic acid (2,4-D)[END_REF][START_REF] El-Gothamy | Ultrastructure sperm defects in addicts[END_REF][START_REF] Coutinho | Antispermatogenic action of gossypol in men[END_REF][START_REF] Boitrelle | Bacteriospermia in assisted reproductive techniques: effects of bacteria on spermatozoa and seminal plasma, diagnosis and treatment[END_REF][START_REF] Abalovich | Hypothalamic-pituitary-testicular axis and seminal parameters in hyperthyroid males[END_REF][START_REF] Bari | The manifold actions of endocannabinoids on female and male reproductive events[END_REF][START_REF] Jewett | Necrospermia or immotile cilia syndrome as a cause of male infertility[END_REF][START_REF] Brackett | Predictors of necrospermia in men with spinal cord injury[END_REF][START_REF] Chavez-Badiola | Necrospermia, antisperm antibodies, and vasectomy[END_REF].

Infections may account for nearly half of the causes of necrozoospermia [START_REF] Nduwayo | Management of necrospermia][END_REF]. The alteration of sperm vitality can be explained by several mechanisms: direct damage to spermatozoa by infectious organisms, effects of inflammatory mediators and/or possible long-term alteration of the genital tract environment [START_REF] Boitrelle | Bacteriospermia in assisted reproductive techniques: effects of bacteria on spermatozoa and seminal plasma, diagnosis and treatment[END_REF]. With regard to the epididymal environment, the first to describe epididymal necrozoospermia was Wilton in 1988 [START_REF] Wilton | Human male infertility caused by degeneration and death of sperm in the epididymis[END_REF]. The epididymis could indeed be dysfunctional and www.wjmh.org lead to sperm abnormalities such as asthenozoospermia and necrozoospermia. The mechanisms of action involved could be multiple, including exposure of epididymal spermatozoa to oxidative stress. This is why some necrozoospermic cases, previously considered as due to "epididymal" causes [START_REF] Wilton | Human male infertility caused by degeneration and death of sperm in the epididymis[END_REF], are now better explained by the possible action of seminal oxidative stress [START_REF] Mahfouz | Sperm viability, apoptosis, and intracellular reactive oxygen species levels in human spermatozoa before and after induction of oxidative stress[END_REF].

Some necrozoospermia can also be linked to an impairment of spermatogenesis. Testicular heat stress is a well-known mechanism for altering spermatogenesis [START_REF] Ji | Association of heat shock proteins, heat shock factors and male infertility[END_REF], as heat is indeed the basis of thermal male infertility [START_REF] Bujan | Temperature and male fertility[END_REF][START_REF] Mieusset | Scrotal hyperthermia and male infertility[END_REF]. Hence, local or general hyperthermia (prolonged heat exposure, fever, hyperthyroidism, obesity, varicocele, etc.) can lead to necrozoospermia. For instance, Abalovich et al [START_REF] Abalovich | Hypothalamic-pituitary-testicular axis and seminal parameters in hyperthyroid males[END_REF] described a large cohort of patients with hyperthyroidism presenting with necrozoospermia. These abnormalities were corrected in most of the cases, with the establishment of normal thyroid function. The mechanisms of action could be hormonal with a decrease in testosterone, or thermodysregulation as related to the increase in body heat [START_REF] Abalovich | Hypothalamic-pituitary-testicular axis and seminal parameters in hyperthyroid males[END_REF]. For patients with varicocele, the pathophysiological mechanisms of necrozoospermia involve an increase in local temperature as well as the simultaneous formation of reactive oxygen species (ROS) [START_REF] Cho | Novel insights into the pathophysiology of varicocele and its association with reactive oxygen species and sperm DNA fragmentation[END_REF]. More recently, with the appearance of the Coronavirus Disease-2019 (COVID-19) pandemic, it was described that fever, even without the presence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the semen, could induce an alteration in spermatogenesis and a decrease in sperm vitality [START_REF] Hamdi | COVID-19 and andrology: recommendations of the French-speaking society of andrology (Société d'Andrologie de langue Française SALF)[END_REF]. It is therefore important to consider medical and reproductive history throughout the assessment, along with a thorough physical examination in patients with necrozoospermia in view of the wide spectrum of possible etiologies.

Necrozoospermia can also be observed following longterm exposure to toxic substances such as tobacco, cannabis and pesticides, amongst others [START_REF] Lerda | Study of reproductive function in persons occupationally exposed to 2,4-dichlorophenoxyacetic acid (2,4-D)[END_REF][START_REF] El-Gothamy | Ultrastructure sperm defects in addicts[END_REF][START_REF] Coutinho | Antispermatogenic action of gossypol in men[END_REF][START_REF] Bari | The manifold actions of endocannabinoids on female and male reproductive events[END_REF][START_REF] Harlev | Smoking and male infertility: an evidence-based review[END_REF][START_REF] Zhang | Effect of smoking on semen quality of infertile men in Shandong, China[END_REF][START_REF] Du Plessis | Marijuana, phytocannabinoids, the endocannabinoid system, and male fertility[END_REF][START_REF] Daoud | Routine assessment of occupational exposure and its relation to semen quality in infertile men: a crosssectional study[END_REF]. A direct toxic effect of cannabis can be noted via the presence of cannabinoid receptors in testicular tissue (Sertoli and Leydig cells) and this would lead to the inhibition of mitochondrial respiration and the loss of energy in sperm, followed by death [START_REF] Bari | The manifold actions of endocannabinoids on female and male reproductive events[END_REF][START_REF] Du Plessis | Marijuana, phytocannabinoids, the endocannabinoid system, and male fertility[END_REF]. Necrozoospermia is also commonly reported in patients with spinal cord injury [START_REF] Brackett | Predictors of necrospermia in men with spinal cord injury[END_REF][START_REF] Mallidis | Necrospermia and chronic spinal cord injury[END_REF]. For these patients, other causes of necrozoospermia could include the thermal dysregulation induced by a prolonged sitting stance that promotes temperature increase in the male genital area. These patients are also more at risk of urogenital infections.

Certain cytotoxic antibodies can also cause necrozoospermia. In a case report of a patient who underwent vasectomy reversal, the presence of necrozoospermia was associated with a positive test for antisperm antibodies [START_REF] Chavez-Badiola | Necrospermia, antisperm antibodies, and vasectomy[END_REF]. Antisperm antibodies, produced locally in the seminal tract following the disruption of the epithelial tight-junction barrier, affect the overall motility of the samples involved and could be responsible for necrozoospermia. Furthermore, necrozoospermia has been found to be associated with polycystic kidney disease [START_REF] Fang | Male infertility and adult polycystic kidney disease are associated with necrospermia[END_REF]. In these patients, cystic obstructions/dilatations of the seminal vesicles and ejaculatory duct (due to seminal vesicle atony) have been described as responsible for sperm stasis in the genital tract, followed by sperm death [START_REF] Hendry | Seminal megavesicles with adult polycystic kidney disease[END_REF]. Advanced male age may also be accompanied by necrozoospermia. The mechanisms remain unknown at this time but may involve an alteration in accessory gland secretions [START_REF] Siddighi | Male age and sperm necrosis in assisted reproductive technologies[END_REF][START_REF] Demirkol | At what age threshold does the decline in semen parameters begin?[END_REF][START_REF] Condorelli | Bio-functional sperm parameters: does age matter?[END_REF]. However, after a complete andrological evaluation, some patients with necrozoospermia remain idiopathic.

MANAGEMENT OF NECROZOOSPERMIA

The first line of management in necrozoospermia is avoiding any etiological contributory factors mentioned previously (exposure to heat, modification of lifestyle, treatment of urogenital infections, correction of hyperthyroidism, etc.). In addition, if vitality is affected by infrequent ejaculations and prolonged epididymal storage, the parameters of sperm vitality could be improved by repeated ejaculations (60 minutes, 12 hours or 24 hours after the first ejaculate) [START_REF] Wilton | Human male infertility caused by degeneration and death of sperm in the epididymis[END_REF][START_REF] Strassburger | Repetitive ejaculation before intracytoplasmic sperm injection in patients with absolute immotile spermatozoa[END_REF][START_REF] Hanson | The impact of ejaculatory abstinence on semen analysis parameters: a systematic review[END_REF].

Here we describe two conditions when (1) necrozoospermia is not absolute but asthenozoospermia is, and (2) when necrozoospermia is absolute.

Management of necrozoospermia with absolute asthenozoospermia

When no motile spermatozoa are observed, sperm vitality should be tested to differentiate asthenozoospermia from necrozoospermia. The presence of a large proportion of live but immotile spermatozoa may indeed be indicative of structural defects in the flagellum [START_REF]WHO laboratory manual for the examination and processing of human semen[END_REF][START_REF] Chemes | Sperm pathology: a step beyond descriptive morphology. Origin, characterization and fertility potential of abnormal sperm phenotypes in infertile men[END_REF][START_REF] Afzelius | Lack of dynein arms in immotile human spermatozoa[END_REF]. There is no definition in the latest edition of the WHO manual of this "large proportion of im-motile living spermatozoa", but it is considered that "if more than 25%-30% of all spermatozoa are alive and immotile, a genetic ciliary problem may be the cause" [START_REF]WHO laboratory manual for the examination and processing of human semen[END_REF]. If flagellar dyskinesia is suspected, other clinical or sperm elements may help the diagnosis (Table 1). For example, the observation of short, bent or coiled flagella may be suggestive of such flagellar/ciliary problems [START_REF] Agarwal | Sperm morphology assessment in the era of intracytoplasmic sperm injection: reliable results require focus on standardization, quality control, and training[END_REF].

In practice, if the spermatozoon is immotile and ART is being considered for the couple, it is necessary to test sperm vitality. In order to select a viable spermatozoon for intracytoplasmic sperm injection (ICSI), the HOS test is very useful in cases where no motile spermatozoa are present through microscopic examination [START_REF] Simopoulou | Improving ICSI: a review from the spermatozoon perspective[END_REF]. At this instance, a modification of the main approach in HOS testing should be adapted for subsequent use for fertilization through ICSI, where the embryologist should aspirate the selected spermatozoon head first with the injecting needle and immerse the flagellum in a hypoosmotic solution for less than 5 minutes [START_REF]WHO laboratory manual for the examination and processing of human semen[END_REF]. As soon as the tail swelling is visualized, the spermatozoon is considered as viable and is removed from the hypo-osmotic solution and rinsed in culture medium allowing osmotic equilibrium. Immotile testicular spermatozoa when selected with HOS and used for ICSI result in higher fertilization and pregnancy rates than immotile sperm selected without being subjected to HOS test [START_REF] Sallam | The use of the modified hypo-osmotic swelling test for the selection of immotile testicular spermatozoa in patients treated with ICSI: a randomized controlled study[END_REF][START_REF] Charehjooy | Selection of sperm based on hypo-osmotic swelling may improve ICSI outcome: a preliminary prospective clinical trial[END_REF]. HOS test could also be useful in cases of previous ICSI failure due to oocyte fertilization failure [START_REF] Pabuccu | Uncommon but devastating event: total fertilisation failure following intracytoplasmic sperm injection[END_REF]. The only situation where the HOS test is not as useful would be in cases of cryopreserved semen samples [START_REF] Hauser | The predictive fertilization value of the hypoosmotic swelling test (HOST) for fresh and cryopreserved sperm[END_REF][START_REF] Esteves | Suitability of the hypo-osmotic swelling test for assessing the viability of cryopreserved sperm[END_REF]. This might be related to the loss of integrity of the sperm membrane after cryopreservation, which can lead to spontaneous swelling of the flagella [START_REF] Nordhoff | How to select immotile but viable spermatozoa on the day of intracytoplasmic sperm injection? An embryologist's view[END_REF].

Several other approaches for selection of viable spermatozoa for ICSI have been presented [START_REF] Ortega | Absolute asthenozoospermia and ICSI: what are the options?[END_REF]. The mechanical technique of "touching" the flagellum, known as the sperm tail flexibility test (STFT), consists of evaluating the flexibility or rigidity of the flagellum by touching it with the ICSI pipette [START_REF] Soares | Sperm tail flexibility test: a simple test for selecting viable spermatozoa for intracytoplasmic sperm injection from semen samples without motile spermatozoa[END_REF]. A dead spermatozoon would have a rigid flagellum. This technique is subjective and difficult to control [START_REF] Nordhoff | How to select immotile but viable spermatozoa on the day of intracytoplasmic sperm injection? An embryologist's view[END_REF][START_REF] Ortega | Absolute asthenozoospermia and ICSI: what are the options?[END_REF]. The laser technique, known as the laser assisted immotile sperm selection (LAISS), consists of performing a laser shot of 129 microjoule for approximately 1.2 milliseconds very close to the flagellum, using a non-contact diode laser system [START_REF] Aktan | Use of a laser to detect viable but immotile spermatozoa[END_REF]. The living spermatozoon then coils its flagellum. Like the HOS test, STFT and LAISS could give false positives for frozen sperm as the flagellum could spontaneously curl after freezing-thawing [START_REF] Nordhoff | How to select immotile but viable spermatozoa on the day of intracytoplasmic sperm injection? An embryologist's view[END_REF][START_REF] Ortega | Absolute asthenozoospermia and ICSI: what are the options?[END_REF]. These two techniques, STFT and LAISS, have not been described in recent years, probably because their costs in terms of learning time (STFT) or in terms of equipment (LAISS) might limit their routine use in ART laboratories [START_REF] Nordhoff | How to select immotile but viable spermatozoa on the day of intracytoplasmic sperm injection? An embryologist's view[END_REF][START_REF] Ortega | Absolute asthenozoospermia and ICSI: what are the options?[END_REF][START_REF] Aydos | Sperm selection procedures for optimizing the outcome of ICSI in patients with NOA[END_REF].

Therefore, there are several techniques to test the vitality of immobile spermatozoa. Most studies have focused on activating substances such as pentoxifylline (PTX) or theophylline describing their role in activating flagellar movement of immotile spermatozoa (for a review see [START_REF] Nordhoff | How to select immotile but viable spermatozoa on the day of intracytoplasmic sperm injection? An embryologist's view[END_REF][START_REF] Ortega | Absolute asthenozoospermia and ICSI: what are the options?[END_REF][START_REF] Aydos | Sperm selection procedures for optimizing the outcome of ICSI in patients with NOA[END_REF]). PTX is a 3′ 5′ nucleotidase phosphodiesterase inhibitor that improves sperm motility by increasing intracellular cyclic adenosine monophosphate [START_REF] Calogero | Correlation between intracellular cAMP content, kinematic parameters and hyperactivation of human spermatozoa after incubation with pentoxifylline[END_REF]. It has been studied in infertile patients since the late 1980s [START_REF] Yovich | Preliminary results using pentoxifylline in a pronuclear stage tubal transfer (PROST) program for severe male factor infertility[END_REF]. Theophylline, which belongs to the same family, has been studied less frequently since its first clinical trial in 2011 [START_REF] Ebner | Pharmacological stimulation of sperm motility in frozen and thawed testicular sperm using the dimethylxanthine theophylline[END_REF]. Since then, several studies have shown the effectiveness of these substances in inducing sperm motility in immotile but alive spermatozoa (for a review see [START_REF] Ortega | Absolute asthenozoospermia and ICSI: what are the options?[END_REF][START_REF] Aydos | Sperm selection procedures for optimizing the outcome of ICSI in patients with NOA[END_REF]). Their safety has been demonstrated in several studies (for a review see [START_REF] Nordhoff | How to select immotile but viable spermatozoa on the day of intracytoplasmic sperm injection? An embryologist's view[END_REF][START_REF] Ortega | Absolute asthenozoospermia and ICSI: what are the options?[END_REF][START_REF] Aydos | Sperm selection procedures for optimizing the outcome of ICSI in patients with NOA[END_REF]). However, given their potential toxicity, it is necessary to rinse the spermatozoon before injecting it into the oocyte [START_REF] Nordhoff | How to select immotile but viable spermatozoa on the day of intracytoplasmic sperm injection? An embryologist's view[END_REF][START_REF] Ortega | Absolute asthenozoospermia and ICSI: what are the options?[END_REF][START_REF] Aydos | Sperm selection procedures for optimizing the outcome of ICSI in patients with NOA[END_REF].

Management of absolute necrozoospermia

If absolute necrozoospermia in the ejaculate is confirmed, then testicular sperm extraction should be considered (Table 1), since it allows the extraction of live spermatozoa [START_REF] Negri | ICSI outcome is significantly better with testicular spermatozoa in patients with necrozoospermia: a retrospective study[END_REF][START_REF] Tournaye | The use of testicular sperm for intracytoplasmic sperm injection in patients with necrozoospermia[END_REF].

Necrozoospermia has been shown to be correlated with sperm DNA fragmentation (SDF). Samplaski et al [START_REF] Samplaski | The relationship between sperm viability and DNA fragmentation rates[END_REF] reported a strong direct correlation between sperm vitality and normal DNA integrity (r=0.83, p<0.001). Moreover, authors observed that SDF measured by spermatozoa chromatin structure assay (SCSA) was ≤30% (normal) when sperm vitality was ≥75% in 95% of samples. Similarly, SDF was ≥30% (high) when vitality was ≤50%. Two other studies showed the same correlations between necrozoospermia and DNA fragmentation tested by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nickend labeling (TUNEL) assay. SDF was significantly higher in sperm with necrozoospermia [START_REF] Aitken | Analysis of the relationships between oxidative stress, DNA damage and sperm vitality in a patient population: development of diagnostic criteria[END_REF][START_REF] Brahem | DNA fragmentation status in patients with necrozoospermia[END_REF]. Derbel et al [START_REF] Derbel | Relationship between nuclear DNA fragmentation, mitochondrial DNA damage and standard sperm parameters in spermatozoa of infertile patients with leukocytospermia[END_REF] found the same results using the sperm chrowww.wjmh.org matin dispersion test in leukocytospermia. Some of the authors report a continuum between DNA damage and sperm death. The loss of viability would be part of a progressive oxidative process that starts with the excessive generation of ROS, leads to the peroxidation of membrane lipids and DNA damage, and ends in DNA fragmentation and ultimately death [START_REF] Aitken | On the possible origins of DNA damage in human spermatozoa[END_REF]. Thus, the question that must now be asked is the following: even if some immobile spermatozoa are alive, can we be confident of their nuclear integrity? The answer to this question is probably a no. Testing for SDF could then be useful in deciding whether to use live ejaculated sperm in ICSI or to use testicular sperm extraction. Nevertheless, interest in using antioxidants to reduce potential SDF in these patients with almost total necrozoospermia is open for discussion. Antioxidants may indeed have a beneficial effect on reducing SDF [START_REF] Dumont | Necrozoospermia: from etiologic diagnosis to therapeutic management[END_REF][START_REF] Agarwal | Utility of antioxidants in the treatment of male infertility: clinical guidelines based on a systematic review and analysis of evidence[END_REF][START_REF] Agarwal | Sperm DNA fragmentation: a critical assessment of clinical practice guidelines[END_REF].

CLINICAL SCENARIOS

There are several clinical and diagnostic conditions or findings which indicate the need to perform the E-N or HOS test to rule out necrozoospermia or asthenozoospermia.

Case A 1) Scenario

A couple is undergoing ICSI procedure. On the day of the procedure, a fresh sperm sample is examined, and the embryologist identifies 6 spermatozoa, but none of them display any motility. How should they proceed?

2) Solution

Since the spermatozoa are going to be used for ICSI, assessing sperm viability is very important and hence, E-N stain cannot be used. The solution is to use HOS test, STFT, LAISS, PTX, or theophylline. If the sperm is assessed as viable, it can be rinsed in sperm wash medium and subsequently be used for oocyte injection. If the spermatozoon is not considered as viable, the procedure is repeated until a viable sperm can be retrieved. If absolute necrozoospermia is found (0% vitality), testicular sperm should be retrieved by surgical sperm extraction.

Case B 1) Scenario

A patient presents for semen analysis and has the following results: (1) semen volume, 3.5 mL; (2) sperm concentration, 35×10 6 /mL; (3) sperm motility, 0%; (4) vitality, 0%. How will you manage this patient?

2) Solution

First, rule out spermicidal lubricant use or improper collection (with spillage of antiseptic solution, soap or water into the semen collection container). Repeat semen analysis. If there is no pathological cause, ICSI using testicular sperm improves the chance of conception, otherwise other alternatives should also be considered (e.g., use of donor sperm).

Case C 1) Scenario

A patient presents for semen analysis. Upon analysis the following parameters are reported: (1) semen volume, 4.5 mL; (2) concentration, 70×10 6 /mL; (3) total sperm motility, 0%. His spouse had an early spontaneous miscarriage a few months ago. No cause for necrozoospermia was found on questioning or clinical examination. How will you manage this patient?

2) Management

Since the patient has a history of confirmed natural fertility and no motility, performing a viability test is recommended. Testing for SDF will help in the decision between using testicular and ejaculated sperm. Antioxidants can be proposed as supplements to reduce SDF if it is present.

ONLINE SURVEY

We have thus far described the principles of the sperm vitality test, the causes of necrozoospermia and discussed appropriate patient management. We now present the results of a worldwide survey of andrologists/urologists that was conducted to evaluate practice patterns in the management of necrozoospermia.

Methodology

The American Center for Reproductive Medicine (ACRM) designed and conducted an online survey to investigate the importance, use and clinical implication of sperm vitality testing in male infertile patients. No ethical approval was required for this study. Researchers and clinicians from ACRM drafted the questions (AA, RKS, RF, SG, NP), which were subsequently revised by a pool of experienced andrologists, urologists, and attending physicians with expertise in male infertility selected from all over the world. English was the primary language of the survey and questions were carefully revised to make them understandable for non-native English speakers.

The survey consisted of 15 questions, in 2 different sections: the first section (6 questions) aimed to collect the demographic data of the participants, while the questions in the second section targeted the clinical practice pattern of sperm vitality testing (Supplement File 2). We used SelectSurvey (https://www.classapps. com/product_ssv5.aspx; ClassApps Inc., Kansas City, MO, USA), a tool approved by the Information Technology Department, Cleveland Clinic, to securely populate the survey. A total of 106 international experts in male infertility received the survey link by email on June 19, 2021, and they were asked to complete the survey by June 28, 2021. SelectSurvey automatically

Survey results

The survey was completed by 65 out of 106 experts (response rate: 61.3%). The participants originated from 31 countries (Fig. 5, Supplement File 3). Most participants' ages were between 35 and 54 years old (n=45, 69.2%) (Fig. 6A), with >10 years of experience in clinical practice (n=50, 76.9%) (Fig. 6B). Their primary practice setting was mostly identified as academic hospital/clinic (n=46, 70.8%) or private practice/clinic (n=22, 33.8%).

The majority of participants recommended sperm vitality testing in their clinical practice (n=45, 69.2%). The test is reported to be conducted on a weekly frequency (n=21, 46.7%), but the majority of practitioners prescribe it less than 10 times a month (n=28, 62.2%). More than half of them prescribe the test if the sperm total motility is <25% (n=26, 57.8%), while only one third of the participants conduct sperm vitality testing automatically when sperm motility is <40% (n=15, 33.3%) (Fig. 7).

The E-N test is reportedly the most commonly used stain to test sperm vitality (n=35, 77.8%) and 73.0% of the participants responded to order a test for SDF in case of abnormal vitality (n=33, 73.3%) (Fig. 8A). Few andrologists look for signs of infection or markers of oxidative stress (n=21, 46.7% respectively) (Fig. 8A).

Concerning management, patients with necrozoospermia (0% sperm vitality) are mainly offered surgical sperm retrieval followed by ICSI (n=41, 91.1%) (Fig. 8B). Few andrologists recommended frequent ejaculation (n=22, 48.9%) (Fig. 8B).

This survey highlighted that the WHO guidelines (test vitality if total motility is <40%) are not necessarily applied in clinical practice. Few andrologists look for signs of infection in the presence of necrozoospermia, even though this is a frequent cause of reduced sperm vitality. Regarding management, repetitive ejaculation is a non-invasive option in comparison with surgical sperm extraction. However, it seems to be given much less consideration by the andrologists.

CONCLUSIONS

Sperm vitality staining is a useful test that should be performed when a semen sample shows reduced motility. It helps identify samples with necrozoospermia, and thus points to a number of potentially correctable clinical conditions that may contribute to impaired fertility. When all spermatozoa are immotile, then testing to distinguish between "live-immotile" "dead-immotile" spermatozoa is critical in determining whether ejaculated or testicular spermatozoa should be used for the ICSI procedure. Furthermore, necrozoospermia positively correlates with increased sperm DNA damage. Hence, SDF testing could be useful in patients with low numbers of alive spermatozoa. Our global survey reveals considerable diversity in approaches to vitality testing, and a relatively limited approach to the management of necrozoospermia, thus highlighting the need for guidelines on this relevant topic.

Fig. 2 .

 2 Fig. 2. Equipment, reagents, and consumables, required for components of the eosin-nigrosin test.

Fig. 3 .

 3 Fig. 3. Schematic representation of alive (A, B) and dead sperm (C) after eosin-nigrosin staining.

Fig. 4 .

 4 Fig. 4. (A) Components of the hypoosmotic swelling test. (B) Semen sample observation under a phase contrast microscope 40× objective and 10× eye piece, and sperm differentiation according to tail swelling. (a) Non-swollen tail of a dead spermatozoon. (b-g) Different patterns of swollen tails of alive spermatozoa.

Fig. 5 .Fig. 6 .

 56 Fig. 5. Geographic map illustrating the distribution of participants.

Fig. 7 .Fig. 8 .

 78 Fig. 7. Pie chart reporting the use of sperm vitality in the andrology laboratory.

Table 1 .

 1 Summary of the clinical and laboratory aspects pertaining to necrozoospermia

  Should always rule out false results due to lubricant use or contamination of semen sample with antiseptic solution, soap, or water

	• Testicular hyperthermia -local or systemic (such as fever)
	• Varicocele
	• Hyperthyroidism
	• Spinal cord injury
	• Polycystic kidney disease
	• Antisperm antibodies
	• Advanced male age
	• Toxic substances -such as: tobacco, cannabis, pesticides
	• Idiopathic
	Note: Management of necrozoospermia

• Avoiding and treating underlying risks and conditions • Frequent ejaculation • Absolute asthenozoospermia: consider the diagnosis of flagellar dyskinesia -perform HOS test or use enhancers of sperm motility and select viable sperm for ICSI • Absolute necrozoospermia: consider SDF testing if ejaculated live sperm are to be used in ICSI, testicular sperm extraction followed by ICSI ART: assisted reproductive technology, HOS: hypoosmotic swelling, ICSI: intracytoplasmic sperm injection, SDF: sperm DNA fragmentation. www.wjmh.org

  -separated values (CSV) file with the results, which were analyzed using the MedCalc Software (version 20.009; MedCalc Software, Ostend, Belgium). Incomplete responses were excluded. Results are reported as number of participants and percentage calculated for each category. When more than one option was available, results were expressed in percentage based on the total number of participants.

www.wjmh.org generated a comma

ACKNOWLEDGEMENTS

Authors are thankful to the artists Bernastine Buchanan and Ken Kula from the Cleveland Clinic's Center for Medical Art & Photography for their help with the illustrations. The study was supported by the American Center for Reproductive Medicine.

Conflict of Interest

The authors have nothing to disclose.

Author Contribution

Conceptualization: AA. Data curation: RF, SK. Methodology: AA, RKS, SG, RF. Project administration: AA, RKS, SG, RF, SK.

Writing -original draft: AA, RKS, SG, FB, RF, Rupin Shah.

Writing -review & editing: all authors.

Supplementary Materials

Supplementary materials can be found via https://doi. org/10.5534/wjmh.210149.


Bernard Cazalis

Jean Desquines

Christophe Poussard

Marc Petit

Yann Monerie

Christian Bernaudat

Pascal Yvon

Xavier Averty

The PROMETRA Program: Fuel Cladding Mechanical Behavior under High Strain Rate

come

I. INTRODUCTION

In support of the CABRI REP-Na and CIP programs [START_REF] Papin | Summary and Interpretation of the CABRI REP-Na Program[END_REF][START_REF] Papin | Definition and Status of the CABRI International Program for High Burnup Fuel Studies[END_REF] performed by the French Institut de Radioprotection et de Sûreté Nucléaire ~IRSN! in the framework of an international cooperation @reactivity-initiated accident ~RIA! experiments in the CABRI reactor in Cadarache#, the assessment of highly irradiated pressurized water reactor ~PWR! cladding mechanical behavior under high strain rate has been carried out within the PROME-TRA program. [START_REF] Balourdet | The PROMETRA Programme: Assessment of Mechanical Properties of Zircaloy 4 Fuel Properties During an RIA[END_REF][START_REF] Yvon | Development of New Techniques to Assess the Mechanical Behaviour of Zircaloy-4 During an RIA[END_REF][START_REF] Cazalis | The PROMETRA Program: A Reliable Material Database for Highly Irradiated Zircaloy-4, Zirlo, M5 Fuel Claddings[END_REF][START_REF] Desquines | Zircaloy-4 Fuel Cladding Mechanical Behavior in the Field of RIA Transients Through the PROMETRA Program[END_REF] The PROMETRA program, undertaken by IRSN in collaboration with Electricité de France ~EDF! and Commissariat à l'Energie Atomique ~CEA!, seeks to calibrate models for Zircaloy-4, ZIRLO, and M5 hardening behavior ~stress-strain curves!, including effects of temperature and waterside corrosion such as oxidation and hydriding. These models derived from a large mechanical testing database gathering all the available results of tensile tests performed on ring and longitudinal samples, machined from defueled cladding between 20 and 11008C submitted to high-strain-rate loading ~0.01 to 5 s Ϫ1 !. Clad failure data are also investigated by means of specific tests, such as burst or plane strain tests ~to be started in 2007!. The models are then implemented in the SCANAIR code [START_REF] Federici | Status of Development of the SCANAIR Code for the Description of Fuel Behavior Under Reactivity Initiated Accidents[END_REF][START_REF] Federici | The SCANAIR Code Version 3.2: Main Features and Status of Qualifications[END_REF] to simulate more accurately the fuel rod behavior during a rod ejection accident ~REA! inaPWR.

The first part of this paper provides an overview of the PROMETRA mechanical testing and its representativeness with regard to the RIA loading conditions.

The second part presents, compares, and discusses the ductile mechanical properties of highly irradiated Zircaloy-4 ~Zry-4!, ZIRLO, and M5 claddings obtained from the hoop tensile and burst tests performed at CEA-Saclay hot laboratories.

Finally, the third part of the paper reports the investigation of the clad brittle behavior, in particular for highly corroded or spalled Zry-4 cladding, by means of a specific program performed at CEA-Grenoble and pursued at CEA-Saclay.

II. OVERVIEW OF THE PROMETRA PROGRAM

II.A. Experimental Testing

The main types of tests performed at CEA hot laboratories are listed in Table I. The experimental procedures used for these mechanical tests have been described elsewhere. [START_REF] Yvon | Development of New Techniques to Assess the Mechanical Behaviour of Zircaloy-4 During an RIA[END_REF][START_REF] Desquines | Zircaloy-4 Fuel Cladding Mechanical Behavior in the Field of RIA Transients Through the PROMETRA Program[END_REF] *E-mail: bernard.cazalis@irsn.fr

The PROMETRA Program: Fuel Cladding Mechanical Behavior under High Strain Rate

Tensile tests ~axial and hoop! are mainly used to provide the clad mechanical behavior laws ~stress-strain curves!. In the hoop tensile test, full three-dimensional finite element analyses have been carried out with the CAST3M code [START_REF] Cast3m | CEA Saclay Finite Element Code[END_REF] to eliminate possible artifacts due to the specimen geometry and test procedure, contact, and friction effects and to estimate local strain and stress fields. Two types of heating techniques, furnace or induction systems, are used depending on the temperature range. For temperatures higher than 4808C, the induction heating system is used to reach heating rates as high as 2008C{s Ϫ1 . These high heating rates allow the prevention of any significant annealing before performing the ring tensile tests. In that case, the induction coil is located around the mandrel. [START_REF] Averty | Induction Heating on Dynamic Tensile Tests in CEA Saclay[END_REF] The mechanical behavior of in-pile spalled cladding has also been addressed through a test campaign initiated at CEA-Grenoble to investigate highly corroded and spalled Zry-4 cladding and to access the ductile-brittle transition.

Moreover, in order to establish a reliable failure criterion, two other types of tests are investigated: the burst test and the plane strain test @also known as the Penn State ~Pennsylvania State University! test#. The burst test is relevant for simulating a fission gas controlled pressure loading of the cladding ~second step of the RIA mechanical loading! up to failure. However, only a few burst tests have been performed at CEA-Saclay because the major concerns for this type of test are its important material consumption ~due to the minimum required sample length!, the difficulties of reaching high temperature levels, and keeping the strain rate constant for high-strainrate loading. Therefore, a new test similar to the annular sample developed by Penn State 11 has been developed on a wider notched ring, which approaches the planestrain conditions ~«zz ϭ 0! in its central ligament. The failure modes obtained with these notched specimens are different from those obtained on standard ring specimens.

The implementation of this test in hot cells is now under progress at CEA-Saclay. The first tests on irradiated Zry-4, ZIRLO, and M5 samples are scheduled for 2006.

II.B. Test Matrix

The overview of the PROMETRA testing program performed on standard Zry-4 is described in Fig. 1, including the number of each type of test ~valid tests only!.

As can be seen, the hoop tensile tests ~ring test! represent most of the tests performed on irradiated samples. These tests provide a great amount of information but a full three-dimensional finite element analysis was required to understand the test results and ensure that this type of test could lead to a relevant material constitutive law.

More specifically, an overview of the hoop tensile tests performed on different types of irradiated cladding alloy ~standard Zry-4, M5, ZIRLO! is shown in Fig. 2. All the tests performed so far on ZIRLO and M5 exhibited a ductile behavior.

II.C. Prototypicality of Mechanical Testing for RIA Transient Analysis

First, it is important to remember that the effective fuel0clad mechanical loading during an RIA transient is not well known yet. However, tendencies can be brought out to position the domain of the applied loading. Thus, the intense fuel0clad contact pressure is at the origin of a coupling between the axial and hoop deformations, and an estimation of the ratio between hoop and axial plastic strain can be given by Eq. ~1!, assuming isotropic plasticity:

« zz p « uu p ϭ 2 s zz s uu Ϫ 1 2 Ϫ s zz s uu . ~1!
Moreover, the ratio between axial and hoop mean stresses is an increasing function of the fuel0clad friction.

The ratio of average axial strains to average hoop strains can be deduced for each CABRI REP-Na test based on posttest measurements. The average residual hoop strain is deduced from posttest profilometries, and the average residual axial strain, from residual rod elongation. Thus, the different CABRI REP-Na test plastic strain ratios can be positioned ~Fig. 3! according to the stress ratio calculated with Eq. ~1!.

The strain ratio was considered to be a potentially influential parameter on total elongation of the specimen, [START_REF] Yunchang | The Influence of Multiaxial States of Stress on the Hydrogen Embrittlement of Zirconium Alloy Sheet[END_REF] the increase of the strain ratio inducing a reduction of total elongation.

The residual strain ratio of CABRI REP-Na tests obtained with this approach varies between 0 and 1. All the tests performed on an axially short ring specimen lead, at the rupture site, to a negative axial deformation, and total elongations are overestimated when compared to RIA loading conditions. In consequence, these tests have to be interpreted by an iterative finite element analy-sis to estimate the behavior laws in spite of the heterogeneous stress and strain fields during the test.

Similarly to the burst test, the Penn State test performed on a Penn State plane strain specimen leads to a zero plastic strain ratio. The burst test leads to high strain rate during the failure end stages, and the strain rate has for effect to decrease the ductility. Therefore, the burst test provides a lower bound of the total elongation under plane-strain displacement controlled loading.

Finally, the equal biaxial test [START_REF] Glendening | The Influence of Strain State on the Failure of Hydrided Zircaloy-4[END_REF] leads to a plastic strain ratio equal to 1. Moreover, the test results have shown that the strain to failure is similar to that obtained in the Penn State test.

We conclude that the Penn State test type appears to be representative of the low values of the deformation ratio obtained in RIA situations ~0.2 to 0.6! and is expected to provide a significant improvement for fracture strain data when compared to the axially short ring tensile tests. Moreover, the orientation of the failure plane is much more typical than those obtained on ring tensile tests.

In conclusion, it has to be mentioned that the approach developed in this section is only qualitative, and its goal is not to favor one test over another or to interpret REP-Na tests but to use each of the mechanical tests in their own domain of prototypicality to provide mechanical data. As the equation used in the section does not exactly represent RIA loading conditions, it is a simple analytical approach that shows that none of the mechanical tests is fully representative of the loading, but each of them can be used for specific data. Some of these tests, as ring tensile tests, are well adapted to conventionnal mechanical characteristics @yield stress ~YS!, ultimate tensile stress ~UTS!, uniform elongation ~UE!, total elongation ~TE!# . Other tests, such as burst or Penn State tests, seem better fit to failure characteristics, according to the more representative failure plane. All these data are then inserted in the SCANAIR code for RIA test simulation or interpretation.

III. DUCTILE MECHANICAL PROPERTIES OF HIGHLY IRRADIATED ZRY-4, ZIRLO, AND M5 CLADDINGS

This section reports the main results of hoop tensile tests and burst tests performed, respectively, on ring and tube specimens machined in M5 irradiated for five and six annual cycles and in ZIRLO 75 GWd0t heavy metal ~HM!, and the comparison to previous results on Zry-4 irradiated for five cycles.

All the results presented in this section are related to the ductile behavior of the irradiated claddings.

III.A. Hoop Tensile Tests Results

The investigated rods are identified in Table II. Test temperatures lie between 280 and 8008C and strain rates between 0.010s and 50s. In the aforementioned validity range, the ductile material strengths have been fitted according to Eq. ~2! and plotted in Figs. 4 and5:

Ys,UTS ~MPa! ϭ a ϩ bT ~8C! 1 ϩ e c~T ~8C!Ϫd! . ~2!
In the case of stress-relieved Zry-4 over 6008C, fits are consistent with the results of tensile tests performed with Joule heating. The fitted law coefficients of YS and UTS are listed in Tables III and IV, respectively.

First, it can be noted that the results observed with induction heating follow continuously the trend observed with conventional heating ~furnace!.

Moreover, the comparison between M5 alloy irradiated for five and six cycles and ZIRLO 75 GWd0tHM and Zry-4 alloy irradiated for five cycles indicates that for temperatures ,6008C, the strengths of ZIRLO and M5 alloy ~YS and UTS! are lower than those of Zry-4 alloy irradiated for five cycles. Over 6008C, the low values and the scatter of the results do not permit the establishment of a real trend. The strength differences between M5 and Zircaloy are consistent with the expected influence of the heat treatments: M5 is a recrystallized alloy and Zircaloy is a stress-relieved alloy.

A small difference between M5 irradiated five-or six-cycle strengths is noticed, but it is difficult to deconvolute the strain rate ~10sto50s! from the burnup effect.

The comparison between M5 alloy irradiated for five and six cycles, ZIRLO irradiated for five cycles, and Zry-4 alloy irradiated for five cycles does not show any important evolution on engineering strain up to 5008C. Uniform elongation value is ;1.5% 6 0.5%. Over 5008C, a strain rate dependency on UE occurs ~Fig. 6!.

In the absence of local clip gauge, an average ductility can be estimated from the TE measured with the jack displacement. This approach may indeed lead to an apparent ductility but has to be complemented with some necking measurements. The average ductility estimated on the basis of the TE remains slightly higher for M5 and ZIRLO alloys than for Zry-4. A slight effect of strain rate on M5 results can be noticed over 5008C ~Fig. 7!.

For both alloys, one of the two samples tested near 8008C presents a very low value of TE close to the UE ~5.39% compared to 5.33% in a six-cycle M5 sample!.

This important apparent reduction of ductility observed in these M5 and ZIRLO specimens has been investigated by means of further posttest examinations performed at CEA-Saclay.

Figure 8 exhibits low-magnification visual examinations of the six-cycle M5 ring samples taken at different test temperatures, which show the specific nature of the ring damage near 8008C, with a very weak necking in the broken leg, giving a brittle-like appearance of the failure ~Fig. 9!. At the same time, the through-thickness visual examination ~Fig. 10! showed a very important necking ~;80 to 100%!. Thus, the reduction of area is obtained in the width of the gauge length for temperature ,6008C and through the thickness of the sample for temperatures .6008C, which suggests that a change in the mode of deformation occurs. Further posttest scanning electron microscopy ~SEM! analyses and metallographic observations have been achieved in order to understand and explain this behavior. A total through-thickness reduction of area was con-firmed with the SEM analyses, as shown in Figs. 11 and 12 for both the M5 and the ZIRLO samples, respectively. For the M5, equiaxial grains ~3t o8mm! with some hydrides located at the grain boundaries as well as inside the grains ~Fig. 13! could be observed. In contrast to the as-received alloy, which has circumferentially oriented hydrides, they were found to be uniformly distributed without a preferential orientation after the test. For ZIRLO, a fine microstructure was observed with elongated hydrides, as shown in Fig. 14.

For both alloys, there is no evidence of an incipient phase transformation ~a r b!. The ex-beta phase should be visible on the micrographs, if any, as a bright thin layer located preferentially at the grain boundaries. However, the present experimental methods cannot resolve low ex-beta phase contents ~,10%! and especially for fast transients.

Also, different deformation mechanisms could be activated at high temperature, or the critical resolved shear stress could evolve, inducing the activation of different slip systems. These questions are under investigation.

III.B. Burst Test Results

Burst tests on chemically defueled M5 and ZIRLO samples were performed at 2808C and at a controlled strain rate of 3 ϫ 10 Ϫ3 s Ϫ1 . The tested samples are presented in Table V.

During the test, the diametral deformation is monitored in situ using displacement transducers. However, as the rupture of the tube does not usually occur at the exact location of the displacement transducers, measurements only provide access to the UE. To evaluate the ductility of the material, the value of the TE has to be determined. Therefore, the hoop TE was measured from image analysis on posttest visual examination samples for ZIRLO and M5.

The results of the tests in terms of conventional mechanical characteristics ~YS at 0.2% plastic strain, ultimate hoop strength, UE, TE! are presented in Table V. A quite good reproducibility of the results is obtained for both alloys.

The ZIRLO alloy shows a mean strength of 690 MPa to be compared to 622 MPa for the M5 alloy. The experimental results show a fairly low hoop UE ~0.5 to 0.6%! for both alloys.

Figures 15 and16 show the samples after burst tests. These pictures reveal that the clad deformation is more pronounced for the M5 alloy than for the ZIRLO alloy: The M5 alloy shows a large ballooning and short axial crack ~the crack length is ;9 mm for a maximum For the ZIRLO samples, the crack is elongated and straight. Zirconia spalling occurs in the vicinity of the cracks. Lower total hoop elongations are measured ~1.6% on average, which is close to the hoop UE considering the accuracy of 2%!. The crack length reaches 18 mm for a maximum pressure of 82.6 MPa at ultimate hoop strength.

This difference of ductility between both alloys is explained by the well-known influence of the material corrosion, higher for the ZIRLO alloy than for the M5 alloy.

IV. INVESTIGATION OF THE BRITTLE BEHAVIOR OF HIGH-BURNUP STRESS-RELIEVED ZRY-4 CLADDING

IV.A. Overview of the PROMETRA Testing on Highly Corroded Spalled Claddings

The case of clad brittle behavior, in particular for highly corroded or spalled cladding, deserves a different treatment. Thus, the investigation of the ductile-brittle transition has been performed by means of a specific program consisting mainly in two special series of hoop tensile tests performed in CEA laboratories:

1. a first series ~see The failure mode, especially brittle failure with cracks orthogonal to the main loading direction, is best determined from the posttest appearance of the specimen as seen globally by visual examination and locally by optical microscopy or by SEM. Moreover, analysis of the information deduced from the fractographies and metallographies of the CEA-Saclay ring specimen allows one to determine parameters, such as the initiated crack depth, characterizing the clad embrittlement.

IV.B. Analysis of Hydride-Related Embrittlement in Oxide-Spalled Cladding

It has been observed on high-burnup fuel cladding that applying very similar loading conditions to apparently similar claddings led to very different test results. To illustrate this statement, a set of load-displacement curves obtained on three CEA-Saclay hoop tensile tests are plotted in Fig. 17

IV.B.1. Metallographic Aspect of Hydride Embrittlement

The brittle behavior of highly corroded, stressrelieved Zry-4 claddings has been related to hydride embrittlement in the PROMETRA program. Two main kinds of hydride damages have been evidenced: a Deduced from a correlation between the average oxide thickness and the hydrogen content. b Out-of-gauge section failure, UTS approximately corrected. 1. hydride blisters illustrated in Fig. 18 2. hydride rims illustrated in Fig. 19.

In the framework of the PROMETRA program, for stress-relieved Zry-4, pure zirconium hydrides with lenticular shape, called hydride blisters or hydride lenses, have been frequently observed, but only on spalled claddings. The usually invoked mechanism ~see Ref. 14, for example! to generate these hydride lenses is that in the spalled area of a highly corroded cladding, there is a cold spot attracting the hydrogen dissolved in the surrounding hot area by a diffusion mechanism. This mechanism is the one used to create artificial hydride blisters in Zircaloy. [START_REF] Dommizzi | Blister Growth in Zirconium Alloys: Experimentation and Modeling[END_REF] Hydride-rich rims sometimes appear at the outer diameter of the cladding at a pellet0pellet interface. The colder region surrounding this interface also attracts hydrogen, enhancing cladding corrosion. [START_REF] Blat | Detrimental Role of Hydrogen on the Corrosion Rate of Zirconium Alloys[END_REF] Thus, there is also usually a thicker corrosion layer at pellet0pellet interfaces. [START_REF] Garde | Effects of Hydride Precipitate Localization and Neutron Fluence on the Ductility of Irradiated Zircaloy-4[END_REF] This mechanism corresponds to axially short hydride rims. Axially extensive hydride rims are also sometimes observed on fractographic examinations after burst tests; axial extensions of 100 mm are not unusual. The high corrosion level and the colder outer diameter might alone explain the generation of such extended hydride rims, which are observed not only on spalled claddings but also on highly corroded, unspalled claddings. The inner diameter region is always hot and usually hydride depleted ~thermally driven hydrogen diffusion toward the cold regions!. A very tiny hydride rim is sometimes observed close to the inner diameter wall; this can be linked to the presence of the inner diameter wall zirconium oxide layer.

The hydride-induced embrittlement is exacerbated by oxide-layer spallation during in-reactor operation, but it is sometimes observed on very oxidized cladding samples without any spallation. This result is consistent with previous studies. [START_REF] Garde | Effects of Hydride Precipitate Localization and Neutron Fluence on the Ductility of Irradiated Zircaloy-4[END_REF]

IV.B.2. Fractographic Aspect of Hydride Embrittlement

Before identifying hydride embrittlement fingerprints on SEM observations, it is necessary to establish the reference fracture aspect of a ductile failure.

Figures 20 and21 present the usual posttest fractographic aspect of a ductile specimen ~PROMETRA 2403, on the M05 rod span 5!. A low-magnification SEM image is shown in Fig. 20. In this figure, the failure plane is orientated at 45 deg to the loading direction; this is one of the macroscopic indications of a ductile failure. Some necking can be observed in Fig. 20c; here, four locations are indicated and high-magnification SEM examinations of each of these locations appear in Fig. 21.

In Fig. 21, the fracture aspect is fully transgranular with ductile dimples at all locations. Figure 21c shows the onset of ductile failure; microvoid coalescence nucleated a penny-shaped crack in the middle of the specimen. This crack failed in a ductile manner in a highly porous ~many microvoids nucleated! material.

The SEM observations performed on CEA-Saclay hoop tensile test 2400 ~Table VII! are shown in Fig. 22. The fracture aspect is typical of a specimen with a hydride rim located at the outer diameter. The outer region with a pseudocleavage fracture aspect is included in a radial-axial plane; the inner region with ductile dimples has a 45-deg orientation. The main fracture steps identified in Refs. 17 and 18 can also be derived from this fractography. An incipient crack is initiated in the hydride rim, the crack being in a radial axial plane. There is a potential for radial crack growth and for a sufficiently high tensile stress or strain so that the remaining ligament cannot carry the load anymore and the specimen fails with a 45-deg rotated fracture angle. The lessons learned from the PROMETRA fractographic examinations can be summarized as follows.

The fingerprint of a brittle failure caused by a hydride blister is

IV.B.3. Size of Hydride Damage

The observed depth of hydride lenses ranged from nearly zero up to the entire cladding thickness. In the case of PROMETRA test 2468, performed on the highly corroded P07 rod with 130-mm oxide layer thickness and oxide spallation, a through-wall hydride lens has been evidenced and is illustrated in Fig. 23. This specimen failed out of the gauge section, and a rough estimate of the failure stress, ;145 MPa, could be determined. It is the lowest failure stress measured within the entire PROMETRA database on high-burnup stress-relieved Zry-4. Very low failure stress values were also obtained on some other ring tests or burst tests that failed at higher but similar stress levels.

It is assumed that 145 MPa is approximately the fracture hoop stress of a hydride blister. This value has the same order of magnitude as the one determined in Ref. 17 on fresh cladding with laboratory-created blisters.

IV.B.4. Analysis of the Influence of Hydride Damage Depth on Sample Integrity

The hydride damage depth, defined as the hydride blister depth or hydride rim depth, has been roughly estimated either from metallography ~based on hydride etching! or from fractography ~pseudocleavage-affected depth! of specimens tested between 280 and 4808C. The obtained hydride damage depth values normalized by the sound cladding thickness are presented in Fig. 24. In this figure, two curves are plotted: the normalized fracture stresses and the normalized plastic strains at failure ~actually residual strains divided by ductile TE value at the corresponding temperature!, as derived from these posttest examinations.

Figure 24 illustrates the deleterious influence of hydride damage on cladding mechanical properties. For a hydride damage deeper than 30 to 40% of the sound cladding thickness, no residual strain is expected. Similar curves were obtained on fresh Zircaloy with hydride rims or hydride blisters reported in Refs. 18 and 19 and more recently in Ref. 17. The fracture strain, determined mainly on ring specimens with near-uniaxial tension loading, most probably overpredicts the integrity of an actual irradiated fuel rod submitted to RIA loading under closeto-plane strain loading conditions. 20

V. CONCLUSIONS

Transient mechanical properties of high-burnup PWR claddings have been investigated through the PROME-TRA program. The representativeness of each test with regard to the RIA loading conditions has been addressed and analyzed. Thus, the Penn State test, scheduled in CEA-Saclay hot laboratories, appears to be representative of the low values of the strain ratio obtained in an RIA situation and is expected to provide a significant improvement of the rupture criterion when compared to the axial short-ring tensile tests.

A material database, including all the available results of burst and tensile tests on high-burnup stressrelieved Zry-4, ZIRLO, and M5 alloy, has been developed and now released. Detailed three-dimensional finite element analyses have been achieved to understand the tests results and validate the geometries. All the ductile parameters deduced from PROMETRA are expected to be valid for burnups higher than ;30 GWd0t U up to 60 GWd0t for Zry-4 and 75 GWd0t U for ZIRLO and M5, and for temperatures up to 8008C obtained with the induction heating device.

The ductility of ZIRLO and M5 cladding is clearly higher than that of Zry-4 cladding up to 6008C. Over 6008C, some tested samples exhibit an important reduction of ductility, not fully understood at this time.

For spalled claddings, most of the generated results converge to demonstrate that the failure conditions are difficult to predict. Potentially very deep hydride blisters and hydride rims have been observed on some of the specimens having a spalled outer zirconia layer using fractography or metallography. For thick oxide layers without spallation, only hydride rims have been evidenced on the outer diameter of the cladding using metallography. There is no direct link between oxide layer thickness or spallation state and hydride damage depth ~hydride blister depth or hydride rim depth!. The fracture conditions strongly depend on hydride content and distribution. There is no evidence of specific embrittlement at the inner diameter within all metallographic examinations. The new investigations of the PROMETRA program focus on new experimental techniques ~clad rupture acoustic detection, image analysis, biaxial testing, etc.! and materials of present and future interest, including ZIRLO, M5, and boiling water reactor claddings.

Fig. 1 .

 1 Fig. 1. Tests and analysis on standard Zry-4 performed within the PROMETRA program.

Fig. 2 .

 2 Fig. 2. PROMETRA hoop tests on irradiated cladding alloys.

Fig. 3 .

 3 Fig. 3. Mechanical tests representativeness relative to CABRI REP-Na tests.

Fig. 5 .

 5 Fig. 5. Ultimate tensile stress as a function of test temperature.

Fig. 7 .

 7 Fig. 7. Total elongation as a function of test temperature.

Fig. 8 .Fig. 9 .

 89 Fig. 8. M5 and ZIRLO TE as a function of test temperature with visual examinations of a six-cycle M5 sample.

Fig. 10 .

 10 Fig. 10. Through-thickness visual detail on the six-cycle M5 sample ~ABDJ3-8168C!.

Fig. 11 .

 11 Fig. 11. SEM analysis of the six-cycle M5 broken leg ~ABDJ4-8098C!.

Fig. 12 .

 12 Fig. 12. SEM analysis of the ZIRLO 75 GWd0t HM broken leg ~ABIO16-7068C!.

Fig. 13 .

 13 Fig. 13. Microstructure observed for the six-cycle M5 broken leg ~ABDJ4-8098C!.

Fig. 14 .

 14 Fig. 14. Microstructure in polarized light observed for the ZIRLO 75 GWd0t HM broken leg ~ABIO16-7068C!.

Fig. 15 .

 15 Fig. 15. Burst test on M5 sample ~ABEF!.

Fig. 16 .

 16 Fig. 16. Burst test on ZIRLO sample ~DFIM!.

  . The three tests have comparable outer oxide layer depth ~130 mm!. Oxide layers of tests 2462 and 2463 were subjected to in-pile spallation, and the gauge section was machined in a spalled area and the oxide layer of test 2471 remained unspalled. The behavior of test 2471 is ductile, whereas the behavior of tests 2462 and 2463 is clearly brittle. The usual signs of cladding embrittlement on axially short hoop tensile ring tests are 1. unusually low fracture strain 2. fracture plane not oriented at 45 deg but in a radialaxial direction.

Fig. 17 .

 17 Fig. 17. Cladding embrittlement on CEA-Saclay hoop tensile test at 4008C and 0.010s.

Fig. 18 .

 18 Fig. 18. Hydride blisters on posttest metallography after hydride etching.

Fig. 19 .

 19 Fig. 19. Hydride rim on posttest metallography of CEA-Saclay hoop tensile test 2470 after hydride etching ~70-mm-deep hydride rim!.

1 .

 1 cleavage in the outer-diameter wall at the blister location ~in an axial radial fracture plane! 2. potentially pseudocleavage surrounding this area ~in the same axial radial fracture plane! 3. ductile dimples and failure plane change, out of the brittle area ~in a 45-deg rotated fracture plane!. The fingerprint of a brittle failure caused by an outerdiameter-located hydride rim is 1. pseudocleavage in the hydride-rich rim ~in an axial radial fracture plane! 2. ductile dimples and failure plane change, out of the brittle area ~in a 45-deg-rotated fracture plane!.

Fig. 20 .

 20 Fig. 20. Low-magnification aspect of ductile failure in PROME-TRA test 2403.

Fig. 21 .

 21 Fig. 21. High-magnification aspect of ductile failure in PROMETRA test 2403.

Fig. 22 .

 22 Fig. 22. Brittle failure initiated by a hydride rim on PROME-TRA test specimen 2400.

TABLE I PROMETRA

 I Main Mechanical Testing

	Type of Tests	Geometry	Material	Range	Objectives0Comments
	CEA-Saclay axial tests		Standard Zry-4	280 to 11008C
			Low-tin Zry-4	0.01 to 5 s Ϫ1

TABLE IV Ultimate

 IV

			Tensile Stress Best-Fit Parameters
				UTS
							Standard
		d«0dt					Deviation
	Material	~sϪ1 !	ab	c	d	~MPa!
	CWSR Zry-4	5	1014	Ϫ0.5430	0.00803	588.6	37
	Five-cycle M5	5	670.0	Ϫ0.0189	0.00584	553.7	13
	Six-cycle M5	1	603.0	Ϫ0.1561	0.00744	608.6	14
	75 GWd0t U ZIRLO	1	898.0	Ϫ0.8180	0.02068	705.0	25

Fig. 6. Uniform elongation as a function of test temperature.

TABLE VI CEA

 VI Grenoble Hoop Tensile Test Results Obtained for an Intensively Spalled Cladding

		Oxide	Hydrogen		Testing
		Thickness	Content	Strain Rate	Temperature	TE	UTS
	Specimen a	~mm!	~ppm!	~sϪ1 !	~8C!	~%!	~MPa!
	Q02-555	74	603 b	0.01	350	0	401
	Q02-557	75	610 b	5	350	2	469
	Q02-558	76	618 b	0.01	350	0	418
	Q02-560	86	573	0.01	280	12	624
	Q02-562	79	1882	5	280	0.05	450
	Q02-563	85	1621	0.01	280	1.5	586
	Q02-565	82	665 b	0.01	350	8	554
	Q02-566	80	645 b	0.01	350	1.5	568

a Extracted from REP-Na 1 parent rod, Q02, span 6, with an average burnup of 63.4 MWd0kg U, irradiated in Gravelines 3ϩ2 EDF reactor ~17 ϫ 17 PWR!, stress-relieved Zry-4. b Deduced from a correlation between the average oxide thickness and the hydrogen content.

TABLE VIII CEA

 VIII -Saclay Hoop Tensile Tests on Highly Corroded Spalled Claddings Supported by Fractography or Metallography

			Oxide Thickness	Hydrogen Content	Strain Rate	Testing Temperature	TE	UTS
	Specimen Parent Rod0Span	~mm!	~ppm!	~sϪ1 !	~8C!	~%!	~MPa!
	2400	M0505	90	727 a	0.01	280	2.3	650
	2401	M0505	90	727 a	0.2	400	3.2	618
	2402	M0505	90	727 a	0.2	400	15.0	578
	2403	M0505	90	727 a	5	280	15.3	704
	2404	M0505	90	727 a	5	400	18.2	600
	2405	M0505	90	727 a	5	400	1.7	618
	2468	P0706	130	1038 a	5	480	0.0 ;145 b
	2470	P0706	130	1038 a	0.01	280	0.6	617
	2472	P0706	130	1038 a	5	280	7.1	702

ACKNOWLEDGMENTS

The contribution of C. Duguay, O. Rabouille, P. Bottin, J. P. Pizzanelli, S. Allegre, and J. J. Espinas, research engineers and technicians at CEA-Saclay, is gratefully acknowledged. Moreover, the authors would like to thank H. S. Rosenbaum, who substantially contributed to better understanding the data generated within the PROMETRA program.