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Sperm Vitality and Necrozoospermia: Diagnosis, Management, and Results of a Global Survey of Clinical Practice
Article #52: “Sperm Vitality and Necrozoospermia: Diagnosis, Management, and Results of a Global Survey of Clinical Practice”
Authors: Ashok Agarwal et al. World J Mens Health 2022 Apr 40(2): 228-242
Contributors: David Penning, MD, Salima Daoud, MD, Tan V. Le, MD, Jesus Fernando Solorzano Vazquez, MD, and Abheesh Varma Hegde, MS, MCh Urol
Commentary:
This paper provides an overview of sperm vitality testing methods and necrozoospermia management in infertile men. It also discusses the results of a global survey about necrozoospermia management practices.
Briefly about Sperm Vitality:
In the latest 6th edition of the WHO Laboratory Manual for examination and processing of human semen (2021), sperm vitality assessment is mainly recommended if the total motility is less than 40%. It enables one to distinguish between immotile dead or alive sperm.
The prevalence of necrozoospermia among infertile men is estimated at 0.2 – 0.4%. In semen samples with poor motility, testing vitality is important as it helps clinicians search for etiology such as genital tract infection, oxidative stress, epididymal pathology, and structural defects of the flagellum. A well-conducted medical examination should be performed, and sperm DNA fragmentation (SDF) testing can have an added value in this context.
And Now about the Analysis:
Two methods based on the evaluation of sperm membrane integrity are available: one by dye exclusion (Eosin-Nigrosin staining or EN staining) or another by hypo-osmotic swelling (HOS test).
Dead cells have damaged plasma membranes allowing entry of the dye: spermatozoa with red or dark pink heads are considered dead, whereas white heads are considered alive. However, tested sperm can no longer be used for ART procedures.
The HOS test presumes that only cells with live membranes can indeed swell in hypotonic solutions. This test is useful when staining of sperm must be avoided, for instance, if ICSI is performed in the lab using fresh sperm. Alternate techniques such as pentoxifylline or theophylline test, mechanical and laserassisted immotile sperm selection, etc. are also available; testicular sperm is the ultimate option in the absence of viable ejaculated sperm.
Take Home Message:
The difference 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.
Eosin-Nigrosin staining is a simple and reliable test if both internal and external quality controls are met. The management of necrozoospermia includes appropriate clinical examination, investigation of the underlying causes, and frequent ejaculation. In the absence of an obvious etiology, selection techniques of viable sperm during ART can overcome the problem in most cases, such as the HOS test to perform ICSI using freshly ejaculated sperm. Particular attention should be paid to the development of international guidelines for clinicians to harmonize the management of necrozoospermia worldwide.
Summary:
Sperm vitality staining is a valuable tool for analyzing semen samples with low motility. It can help pinpoint treatable conditions affecting fertility and guide decisions about sperm selection for ICSI in cases of complete immotility. Additionally, this test might be beneficial for patients with low live sperm counts when combined with DNA fragmentation testing.
(Contributor: Ashok Agarwal)
My Viewpoint on Sperm Vitality and Necrozoospermia
Dr. David Penning responds to questions from Ashok
Q1. What are the common causes and risk factors associated with necrozoospermia?
Dr. Penning: Here are some common causes and risk factors associated with a high percentage of dead sperm in the semen: infections, alcohol, and drug use, unhealthy diet, radiation and chemotherapy, genitourinary infections, hormonal disorders, long periods of abstinence, advanced paternal age, anti-sperm antibodies, early testicular cancer. These factors can occur individually or in combination, however, the exact cause of necrozoospermia can vary from person to person.
Dr. Penning: It allows for accurate diagnosis, as false results can lead to misdiagnosis and inappropriate treatment. It helps avoid unnecessary stress and anxiety by preventing unwarranted concerns about infertility and promotes a cost-effective approach by minimizing additional costs through accurate initial testing.
Dr. Penning: In cases of necrozoospermia, there is often a high level of sperm DNA fragmentation. For severe necrozoospermia associated with high levels of SDF, antioxidant therapy may be indicated to lower SDF levels, potentially improving semen parameters. In cases of total motility loss, some authors have suggested the use of testicular sperm which may be associated with lower levels of SDF.
David Pening, MD, PhD trainee: Short Biography
David Pening, MD, PhD trainee
OB-GYN Department ULB - Université Libre de Bruxelles
H.U.B. - Hôpital Universitaire de Bruxelles
CUB Hôpital Erasme, Belgium
Email: david.pening@ulb.be
ORCID ID: 0000-0002-7221-4361
Dr. David Pening
graduated as an Obstetrician Gynaecologist in 2018 from the ULB School of Medicine, Brussels. He did the training in Fertility Clinic at CRG, UZ Brussel for 1 year. His clinical and research interests are focused on Andrology, as he graduated in Andrology from CHU Lille in 2021. He is currently PHU at CUB – Erasme Hospital, Fertility Clinic. Dr Pening is a member of several scientific societies namely the Royal College of the French Gynaecologists & Obstetricians (CRGOLFB), the French Society of Andrology (SALF), the American Society of Andrology (ASA), the Network for Young Researchers in Andrology (NYRA), and the Belgian Society for Reproductive Medicine (BSRM) as a Board Member. He has co-authored 5 papers (h-index: 3, citation count: 24) and serves as a reviewer for several international journals dealing with reproductive medicine.
My Viewpoint on Sperm Vitality and Necrozoospermia
Dr. Salima Daoud responds to questions from Ashok
Q1. What are the primary differences between sperm vitality and sperm viability assessments?
Dr. Daoud: These are two different methods to evaluate the health of sperm in a semen sample. Vitality refers to whether sperm are alive regardless of their motility. It can be evaluated by a dye exclusion method such as the eosin staining test, where the dead cells uptake the eosin dye due to perforation in their membrane, but it does not consider the functional capacities of the cell. Viability on other hand assesses the physiological functions of living sperm. The HOS test assesses the functional integrity of the sperm plasma membrane under osmotic stress and is therefore used as a viability test.
Dr. Daoud: The E-N stain is a simple, rapid, inexpensive, and efficient vitality test and has therefore been implemented as a routine test in laboratories. Its major limitation is linked to its toxicity for the sperm, which prevents its use in ART.
Q3. How should a laboratory handle and prepare a semen sample for accurate sperm vitality testing?
Dr. Daoud: According to the WHO manual, sperm vitality testing should be performed no later than an hour after ejaculation to avoid a decrease in vitality due to dehydration or a temperature change. Procedure steps should be performed according to WHO recommendations, such as evaluating at least 200 spermatozoa and considering the partially stained cells as alive.
Q4. What are the recommended procedures for ensuring quality control in sperm vitality testing?
Dr. Daoud: Good staff training and controlling sources of variation in sperm vitality testing are highly important. Adherence to the WHO recommendations is necessary to achieve accurate and reproducible results. Internal quality controls should be routinely performed to assess errors and inter-operator variability. Quality control charts are useful to monitor results daily. Participation in external quality controls quarterly or biannually is recommended. Control vitality slides should be prepared by reference laboratories and results should be within ±2 standard deviation of the mean. Corrective action must be undertaken if the results of internal or external quality controls are outside control limits.
Salima Daoud, MD: Short Biography
Salima Daoud, MD
Associate Professor, Laboratory of Histology, Embryology & Reproductive Biology Faculty of Medicine, Sfax University, Tunisia
Email: daoud_salima@medecinesfax.org
ORCID ID: 0000-0003-4330-7363
Dr. Salima Daoud graduated from The Faculty of Medicine of Sfax in 2007. After finishing her internship in Tunisian and French University hospitals, she joined the Laboratory of Histology, Embryology, and Reproductive Biology, Faculty of Medicine of Sfax, in Tunisia, where she currently serves as an Associate Professor. Dr. Daoud is actively involved in preand post-graduate medical education. She is vice director of medical education at her institution. She is also a member of the Tunisian College of Histo-embryology and the 'Association Tunisienne des Embryologistes Tunisiens' (ATME). Her research interests focus on male infertility, reproductive toxicology, and AI-based tools for reproductive biology. Dr. Daoud has co-authored 8 papers (h-index: 4, citations: 63) and serves as a reviewer for several international journals dealing with reproductive medicine.
My Viewpoint on Sperm Vitality and Necrozoospermia
Dr. Tan V. Le responds to questions from Ashok
Q1. What management strategies are recommended for patients diagnosed with necrozoospermia?
Dr. Le: Management strategies for necrozoospermia include lifestyle changes such as diet and exercise, antioxidant therapy, frequent ejaculation to remove dead sperm from the ejaculatory ducts and assisted reproductive techniques like intracytoplasmic sperm injection (ICSI) using testicular sperm extraction (TESE) if viable sperm cannot be found in the ejaculate.
Q2. How can frequent ejaculation improve sperm vitality in cases of prolonged epididymal storage?
Dr. Le: Frequent ejaculation helps reduce the accumulation of dead or damaged sperm by continuously clearing the epididymis, which can prevent the negative effects of prolonged sperm storage. This can enhance the overall quality and vitality of sperm by ensuring that fresher sperm is available in the ejaculate.
Dr. Le: Current global practices include routine sperm vitality testing using eosin-nigrosin staining or hypo-osmotic swelling tests, and the management of necrozoospermia through lifestyle modifications, antioxidant supplementation, and ART such as ICSI with TESE. There is a focus on improving diagnostic accuracy and individualizing treatment plans based on specific patient needs.
Q4. What are the key indicators for recommending sperm vitality testing in routine andrology laboratory practice?
Dr. Le: Genital tract infections can lead to necrozoospermia with direct damage to the sperm by microorganisms, the effect of inflammatory mediators, and alteration of the genital tract environment. Also, an infection can cause DNA damage by ROS and the production of neutrophils.
Q5. What are the potential benefits of using antioxidants in the treatment of patients with necrozoospermia and high sperm DNA fragmentation?
Dr. Le:
Antioxidants can reduce oxidative stress, which is a significant factor in sperm cell damage and DNA fragmentation. The use of antioxidants may improve sperm vitality, motility, and overall sperm quality, potentially enhancing the outcomes of ART procedures. They can also help reduce the percentage of sperm with DNA fragmentation, improving the chances of successful fertilization and healthy embryo development.
Tan V. Le, MD: Short Biography
Tan V. Le, MD
Andrologist Department of Andrology Binh Dan Hospital Ho Chi Minh City, Vietnam
E-mail: drlevutan@gmail.com
ORCID ID: 0000-0003-1766- 7453
Dr. Tan V. Le is a Vietnamese Urologist and Andrologist with a strong clinical background and a growing interest in clinical research, particularly in the areas of sexual dysfunction and male infertility. After graduating from Pham Ngoc Thach Medical University in Ho Chi Minh City in 2009, he completed his residency at the Urology Department of the University of Medicine and Pharmacy in Ho Chi Minh City starting in 2010. In 2018, Dr. Le served as a research fellow at the Urology Department of Tulane Medical University in Louisiana, USA, under the supervision of Professor Wayne Hellstrom. He is passionate about collaborating with and learning from andrologists worldwide to enhance his expertise. Dr. Le is affiliated with the Department of Andrology at Binh Dan Hospital and the Department of Urology and Andrology at Pham Ngoc Thach University of Medicine in Ho Chi Minh City, Vietnam. His research credentials include a total of 30 publications, an h-index of 9, and 224 citations.
My Viewpoint on Sperm Vitality and Necrozoospermia
Dr. Vazquez responds to questions from Ashok
Dr. Vazquez: The HOS test is a diagnostic method used to evaluate the functional integrity of the sperm plasma membrane. Sperm with intact cell membranes will swell in response to the hypo-osmotic conditions because water enters the cell. The degree of swelling is observed under the microscope. Sperm with tail swelling are considered to have functional cell membranes. Here lies its importance as an assessment of membrane function, and indication of fertility potential. It is beneficial in cases of complete asthenozoospermia, where all sperm are immotile, to select viable sperm for ICSI.
Q2. How does testicular hyperthermia contribute to necrozoospermia, and what are its sources?
Dr. Vazquez: Hyperthermia contributes to necrozoospermia by dysregulating the production of heat shock proteins and heat shock factors, leading to abnormal germ cell apoptosis. The sources of hyperthermia can be local, such as in varicocele and obesity, or general, such as prolonged heat exposure from kitchens, ovens, or sitting on bicycles, as well as conditions like fever and hyperthyroidism.
Q3. What are the benefits and limitations of using the HOS test in selecting viable sperm for ICSI?
Dr. Vazquez: HOS can be used to select viable sperm for ICSI in cases of necrozoospermia with absolute asthenozoospermia. The sperm with vitality can be identified by adding them for less than 5 minutes in a hypoosmotic solution. They should then be rinsed with culture media and prepared to be injected by ICSI. It is noteworthy that in frozen sperm straws, cryopreserved samples may show spontaneous swelling from the freeze-thaw process.
Q4. How do genital tract infections affect sperm vitality, and what are the mechanisms involved?
Dr. Vazquez: Genital tract infections can lead to necrozoospermia with direct damage to the sperm by microorganisms, the effect of inflammatory mediators, and alteration of the genital tract environment. Also, an infection can cause DNA damage by ROS and the production of neutrophils.
Jesus Fernando Solorzano Vazquez, MD: Short Biography
Jesus Fernando Solorzano Vazquez, MD
Gynecologist and Obstetrician, Reproductive Medicine Specialist, Medical Coordinator in CITMER Mexico City, Mexico
e-mail: dr.solorzanobr@gmail.com fsolorzano@gmail.com
ORCID: 0000-0003-4354-8351
Dr. Jesús Fernando Solorzano Vázquez is a Gynecologist and Reproductive Medicine specialist at CITMER Mexico City, Mexico. Dr. Solorzano obtained his medical degree from University of Guanajuato in 2008. He completed his Gynecology and Obstetrics degree at University of Guadalajara in 2013 and finally got his degree in Reproductive Medicine Biology from National Autonomous University of Mexico 2015. Dr. Solórzano is focused on Male Infertility in his clinical practice and is interested in research in this field. He has published 6 research papers in peer-reviewed journals, has 19 citations, and has an h-index of 2 according to Scopus.
My Viewpoint on Sperm Vitality and Necrozoospermia
Dr. Hegde responds to questions from Ashok
Dr. Hegde: The movement of a sperm is considered proof of vitality. However, in asthenozoospermia, the sperm is alive but not motile. Necrozoospermia is a pathological decrease in sperm vitality. It is important to differentiate these two entities to direct further investigation and treatment in infertile patients. The causes for necrozoospermia are numerous and treatment is directed toward the etiology.
Dr. Hegde: Sperm motility rate below 40% needs assessment for sperm vitality (necrozoospermia) to differentiate between necrozoospermia and asthenozoospermia. This has consequences in clinical approach and management. The next automatic step should be an E-N test that detects dead sperms. The sperms used for this test cannot be used for ART.
Q3. What role do anti-sperm antibodies play in causing necrozoospermia?
Dr. Hegde: The anti-sperm antibodies are usually produced in the genital tract due to alteration of the blood-testis barrier. These are known to affect the motility of the sperm and could cause necrozoospermia.
Dr. Hegde: Upto 20% of patients could have idiopathic necrozoospermia. Repeated ejaculations are known to improve both sperm motility and vitality. (60 minutes, 12 hours, or 24 hours after initial ejaculation). Drug treatments are usually ineffective. Modification of sperm processing methods such as utilizing magnetic activated cell sorting (MACS), incubation of sperms with glycerophospholipids, density gradient centrifugation followed by a swim-up procedure, and adding antioxidants like ethylene diamine tetraacetic acid (EDTA), catalase, vitamin E, ellagic acid, alpha-lipoic acid, and L carnitine to the sperm preparation medium could improve sperm vitality. In severe necrozoospermia, vitality testing needs to be done before initiating ICSI. Surgical sperm retrieval using micro TESE can be done in case of nerozoospermia that cannot be corrected otherwise.
Abheesh Varma Hegde, MS, MCh Urol: Short Biography
Dr. Abheesh Varma Hegde
Assistant Professor Department of Urology and Renal Transplantation Father Muller Medical College Mangalore, India
Email: abhi.vhegde@gmail.com
ORCID 0000-0002-2524-6889
Dr. Abheesh Hegde is a Consultant Urologist and Andrologist and an Assistant Professor at the Dept of Urology, Father Muller Medical College, Mangalore, India, a tertiary care hospital with a Urology residency program. He also serves as a visiting Consultant at rural centers around Mangalore, where no urological services are provided. He received his general surgery training at St John's Medical College, Bangalore, and MCh Urology from Topiwala National Medical College, Mumbai. Dr. Hegde is a member of several international Urological societies, including ISSM, SIU, Endourological Society, AUA, and EAU. He has been academically active throughout his career, winning multiple awards from regional, national, and international urology societies. He has presented over 30 papers at conferences and is a regular training faculty member at these events. Dr. Hegde has authored 16 publications (3 PubMed indexed), 6 book chapters, and has an h-index of 1, and a citation count of 1 according to Scopus.