Ms. Doel Bose Pande discusses various semen preparation methods and principles. Common techniques include simple wash, swim-up, and density gradient centrifugation. The choice of method depends on factors like semen quality, intended use, and practical considerations. Density gradient is best for separating motile sperm from debris but is more time-consuming than direct swim-up. The goal is to recover high-quality sperm with minimal processing time and damage. Practical issues like sample volume and number of patients may also influence the choice of preparation technique.
ICSI as it is presently performed is far from an ideal solution because the selection of sperm is based on the judgement of an embryologist, who is looking for the most normal appearing sperm available.
Sperm DNA Fragmentation (Oxidative stress, DNA damage and apoptosis, Test, Techniques, Relation to other semen parameters, Relationship to leucocytes, Relation to ICSI outcomes, Clinical applications, significance and limitations)
Sperm Function Tests are the keystones of evaluating functional condition of sperms. The fertility potential of a sperm will be decided not only with the number & motility but with the functional competence which is of utmost importance.
ICSI as it is presently performed is far from an ideal solution because the selection of sperm is based on the judgement of an embryologist, who is looking for the most normal appearing sperm available.
Sperm DNA Fragmentation (Oxidative stress, DNA damage and apoptosis, Test, Techniques, Relation to other semen parameters, Relationship to leucocytes, Relation to ICSI outcomes, Clinical applications, significance and limitations)
Sperm Function Tests are the keystones of evaluating functional condition of sperms. The fertility potential of a sperm will be decided not only with the number & motility but with the functional competence which is of utmost importance.
in this lecture i tried to summarize the most important normal morphological features of oocyte \ Follicle( including process of oogenesis and female mammalian meiosis) then i tried to summarize abnormal oocyte morphology
OVERVIEW
Aim
Definition
Prerequisites
Individualisation of patient.
Ohss free IUI. Clinic
{Strict cancellation of cycle if OHSS is suspected}
Newer trends
Sucess Rates in IUI with COH
PROGNOTIC FACTORS to increase Pregnancy Rates..& discussion
EMBRYO QUALITY ASSESSMENT, WHICH TO SELECT? Rahul Sen
Traditional embryo evaluation systems are simple, non-invasive, cost-effective & mainstay in majority of IVF laboratories. Embryo selection based on combinations of morphology scores at different stages of embryonic development with time may be more effective
PGD is a state-of-the-art procedure used in conjunction with In Vitro Fertilization (IVF) in which the embryo is tested for certain conditions prior to being placed in the womb of the woman.
in this lecture i tried to summarize the most important normal morphological features of oocyte \ Follicle( including process of oogenesis and female mammalian meiosis) then i tried to summarize abnormal oocyte morphology
OVERVIEW
Aim
Definition
Prerequisites
Individualisation of patient.
Ohss free IUI. Clinic
{Strict cancellation of cycle if OHSS is suspected}
Newer trends
Sucess Rates in IUI with COH
PROGNOTIC FACTORS to increase Pregnancy Rates..& discussion
EMBRYO QUALITY ASSESSMENT, WHICH TO SELECT? Rahul Sen
Traditional embryo evaluation systems are simple, non-invasive, cost-effective & mainstay in majority of IVF laboratories. Embryo selection based on combinations of morphology scores at different stages of embryonic development with time may be more effective
PGD is a state-of-the-art procedure used in conjunction with In Vitro Fertilization (IVF) in which the embryo is tested for certain conditions prior to being placed in the womb of the woman.
Intrauterine insemination (IUI) is a fertility treatment that involves placing sperm inside a woman’s uterus to facilitate fertilisation. The goal of IUI is to increase the number of sperm that reach the fallopian tubes and subsequently increase the chance of fertilisation.
This is an important topic of mammalian (Male) reproductive toxicology.By doing this test sperm abnormalities should be cured. This topic is available in net but not like, what a master student try to find out.If there is anything wrong then correct me please.
In this ppt i have included methods of semen analysis and the importance and some agents which create semen abnormalities.
sperm assessment- traditional and novel approaches.pptxDeepekaTS
The latest WHO recommendations,2010 are based on semen parameters from approximately 2000 fertile men, from eight countries and three continents, whose partners achieved pregnancy within 12 months of unprotected sexual intercourse.
Pitfalls- huge shift in the lower reference values, one sided criteria.
Reference limits shouldn’t be over-interpreted
Interpret along with clinical history and physical examination.
Tolerance to tissue and cell antigens can be
induced by injection of hemopoietic (stem)
cells in neonatal or severely
immunocompromised (by lethal irradiation
or drug treatment) animals.
Also, grafting of allogeneic bone marrow or
thymus in early life results in tolerance to
the donor type cells and tissues. Such
animals are known as chimeras. These
findings are of significant practical
application in bone marrow grafting
micro teaching on communication m.sc nursing.pdfAnurag Sharma
Microteaching is a unique model of practice teaching. It is a viable instrument for the. desired change in the teaching behavior or the behavior potential which, in specified types of real. classroom situations, tends to facilitate the achievement of specified types of objectives.
Ozempic: Preoperative Management of Patients on GLP-1 Receptor Agonists Saeid Safari
Preoperative Management of Patients on GLP-1 Receptor Agonists like Ozempic and Semiglutide
ASA GUIDELINE
NYSORA Guideline
2 Case Reports of Gastric Ultrasound
NVBDCP.pptx Nation vector borne disease control programSapna Thakur
NVBDCP was launched in 2003-2004 . Vector-Borne Disease: Disease that results from an infection transmitted to humans and other animals by blood-feeding arthropods, such as mosquitoes, ticks, and fleas. Examples of vector-borne diseases include Dengue fever, West Nile Virus, Lyme disease, and malaria.
Prix Galien International 2024 Forum ProgramLevi Shapiro
June 20, 2024, Prix Galien International and Jerusalem Ethics Forum in ROME. Detailed agenda including panels:
- ADVANCES IN CARDIOLOGY: A NEW PARADIGM IS COMING
- WOMEN’S HEALTH: FERTILITY PRESERVATION
- WHAT’S NEW IN THE TREATMENT OF INFECTIOUS,
ONCOLOGICAL AND INFLAMMATORY SKIN DISEASES?
- ARTIFICIAL INTELLIGENCE AND ETHICS
- GENE THERAPY
- BEYOND BORDERS: GLOBAL INITIATIVES FOR DEMOCRATIZING LIFE SCIENCE TECHNOLOGIES AND PROMOTING ACCESS TO HEALTHCARE
- ETHICAL CHALLENGES IN LIFE SCIENCES
- Prix Galien International Awards Ceremony
Report Back from SGO 2024: What’s the Latest in Cervical Cancer?bkling
Are you curious about what’s new in cervical cancer research or unsure what the findings mean? Join Dr. Emily Ko, a gynecologic oncologist at Penn Medicine, to learn about the latest updates from the Society of Gynecologic Oncology (SGO) 2024 Annual Meeting on Women’s Cancer. Dr. Ko will discuss what the research presented at the conference means for you and answer your questions about the new developments.
Ethanol (CH3CH2OH), or beverage alcohol, is a two-carbon alcohol
that is rapidly distributed in the body and brain. Ethanol alters many
neurochemical systems and has rewarding and addictive properties. It
is the oldest recreational drug and likely contributes to more morbidity,
mortality, and public health costs than all illicit drugs combined. The
5th edition of the Diagnostic and Statistical Manual of Mental Disorders
(DSM-5) integrates alcohol abuse and alcohol dependence into a single
disorder called alcohol use disorder (AUD), with mild, moderate,
and severe subclassifications (American Psychiatric Association, 2013).
In the DSM-5, all types of substance abuse and dependence have been
combined into a single substance use disorder (SUD) on a continuum
from mild to severe. A diagnosis of AUD requires that at least two of
the 11 DSM-5 behaviors be present within a 12-month period (mild
AUD: 2–3 criteria; moderate AUD: 4–5 criteria; severe AUD: 6–11 criteria).
The four main behavioral effects of AUD are impaired control over
drinking, negative social consequences, risky use, and altered physiological
effects (tolerance, withdrawal). This chapter presents an overview
of the prevalence and harmful consequences of AUD in the U.S.,
the systemic nature of the disease, neurocircuitry and stages of AUD,
comorbidities, fetal alcohol spectrum disorders, genetic risk factors, and
pharmacotherapies for AUD.
Title: Sense of Smell
Presenter: Dr. Faiza, Assistant Professor of Physiology
Qualifications:
MBBS (Best Graduate, AIMC Lahore)
FCPS Physiology
ICMT, CHPE, DHPE (STMU)
MPH (GC University, Faisalabad)
MBA (Virtual University of Pakistan)
Learning Objectives:
Describe the primary categories of smells and the concept of odor blindness.
Explain the structure and location of the olfactory membrane and mucosa, including the types and roles of cells involved in olfaction.
Describe the pathway and mechanisms of olfactory signal transmission from the olfactory receptors to the brain.
Illustrate the biochemical cascade triggered by odorant binding to olfactory receptors, including the role of G-proteins and second messengers in generating an action potential.
Identify different types of olfactory disorders such as anosmia, hyposmia, hyperosmia, and dysosmia, including their potential causes.
Key Topics:
Olfactory Genes:
3% of the human genome accounts for olfactory genes.
400 genes for odorant receptors.
Olfactory Membrane:
Located in the superior part of the nasal cavity.
Medially: Folds downward along the superior septum.
Laterally: Folds over the superior turbinate and upper surface of the middle turbinate.
Total surface area: 5-10 square centimeters.
Olfactory Mucosa:
Olfactory Cells: Bipolar nerve cells derived from the CNS (100 million), with 4-25 olfactory cilia per cell.
Sustentacular Cells: Produce mucus and maintain ionic and molecular environment.
Basal Cells: Replace worn-out olfactory cells with an average lifespan of 1-2 months.
Bowman’s Gland: Secretes mucus.
Stimulation of Olfactory Cells:
Odorant dissolves in mucus and attaches to receptors on olfactory cilia.
Involves a cascade effect through G-proteins and second messengers, leading to depolarization and action potential generation in the olfactory nerve.
Quality of a Good Odorant:
Small (3-20 Carbon atoms), volatile, water-soluble, and lipid-soluble.
Facilitated by odorant-binding proteins in mucus.
Membrane Potential and Action Potential:
Resting membrane potential: -55mV.
Action potential frequency in the olfactory nerve increases with odorant strength.
Adaptation Towards the Sense of Smell:
Rapid adaptation within the first second, with further slow adaptation.
Psychological adaptation greater than receptor adaptation, involving feedback inhibition from the central nervous system.
Primary Sensations of Smell:
Camphoraceous, Musky, Floral, Pepperminty, Ethereal, Pungent, Putrid.
Odor Detection Threshold:
Examples: Hydrogen sulfide (0.0005 ppm), Methyl-mercaptan (0.002 ppm).
Some toxic substances are odorless at lethal concentrations.
Characteristics of Smell:
Odor blindness for single substances due to lack of appropriate receptor protein.
Behavioral and emotional influences of smell.
Transmission of Olfactory Signals:
From olfactory cells to glomeruli in the olfactory bulb, involving lateral inhibition.
Primitive, less old, and new olfactory systems with different path
Recomendações da OMS sobre cuidados maternos e neonatais para uma experiência pós-natal positiva.
Em consonância com os ODS – Objetivos do Desenvolvimento Sustentável e a Estratégia Global para a Saúde das Mulheres, Crianças e Adolescentes, e aplicando uma abordagem baseada nos direitos humanos, os esforços de cuidados pós-natais devem expandir-se para além da cobertura e da simples sobrevivência, de modo a incluir cuidados de qualidade.
Estas diretrizes visam melhorar a qualidade dos cuidados pós-natais essenciais e de rotina prestados às mulheres e aos recém-nascidos, com o objetivo final de melhorar a saúde e o bem-estar materno e neonatal.
Uma “experiência pós-natal positiva” é um resultado importante para todas as mulheres que dão à luz e para os seus recém-nascidos, estabelecendo as bases para a melhoria da saúde e do bem-estar a curto e longo prazo. Uma experiência pós-natal positiva é definida como aquela em que as mulheres, pessoas que gestam, os recém-nascidos, os casais, os pais, os cuidadores e as famílias recebem informação consistente, garantia e apoio de profissionais de saúde motivados; e onde um sistema de saúde flexível e com recursos reconheça as necessidades das mulheres e dos bebês e respeite o seu contexto cultural.
Estas diretrizes consolidadas apresentam algumas recomendações novas e já bem fundamentadas sobre cuidados pós-natais de rotina para mulheres e neonatos que recebem cuidados no pós-parto em unidades de saúde ou na comunidade, independentemente dos recursos disponíveis.
É fornecido um conjunto abrangente de recomendações para cuidados durante o período puerperal, com ênfase nos cuidados essenciais que todas as mulheres e recém-nascidos devem receber, e com a devida atenção à qualidade dos cuidados; isto é, a entrega e a experiência do cuidado recebido. Estas diretrizes atualizam e ampliam as recomendações da OMS de 2014 sobre cuidados pós-natais da mãe e do recém-nascido e complementam as atuais diretrizes da OMS sobre a gestão de complicações pós-natais.
O estabelecimento da amamentação e o manejo das principais intercorrências é contemplada.
Recomendamos muito.
Vamos discutir essas recomendações no nosso curso de pós-graduação em Aleitamento no Instituto Ciclos.
Esta publicação só está disponível em inglês até o momento.
Prof. Marcus Renato de Carvalho
www.agostodourado.com
Pulmonary Thromboembolism - etilogy, types, medical- Surgical and nursing man...VarunMahajani
Disruption of blood supply to lung alveoli due to blockage of one or more pulmonary blood vessels is called as Pulmonary thromboembolism. In this presentation we will discuss its causes, types and its management in depth.
New Drug Discovery and Development .....NEHA GUPTA
The "New Drug Discovery and Development" process involves the identification, design, testing, and manufacturing of novel pharmaceutical compounds with the aim of introducing new and improved treatments for various medical conditions. This comprehensive endeavor encompasses various stages, including target identification, preclinical studies, clinical trials, regulatory approval, and post-market surveillance. It involves multidisciplinary collaboration among scientists, researchers, clinicians, regulatory experts, and pharmaceutical companies to bring innovative therapies to market and address unmet medical needs.
Tom Selleck Health: A Comprehensive Look at the Iconic Actor’s Wellness Journeygreendigital
Tom Selleck, an enduring figure in Hollywood. has captivated audiences for decades with his rugged charm, iconic moustache. and memorable roles in television and film. From his breakout role as Thomas Magnum in Magnum P.I. to his current portrayal of Frank Reagan in Blue Bloods. Selleck's career has spanned over 50 years. But beyond his professional achievements. fans have often been curious about Tom Selleck Health. especially as he has aged in the public eye.
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Introduction
Many have been interested in Tom Selleck health. not only because of his enduring presence on screen but also because of the challenges. and lifestyle choices he has faced and made over the years. This article delves into the various aspects of Tom Selleck health. exploring his fitness regimen, diet, mental health. and the challenges he has encountered as he ages. We'll look at how he maintains his well-being. the health issues he has faced, and his approach to ageing .
Early Life and Career
Childhood and Athletic Beginnings
Tom Selleck was born on January 29, 1945, in Detroit, Michigan, and grew up in Sherman Oaks, California. From an early age, he was involved in sports, particularly basketball. which played a significant role in his physical development. His athletic pursuits continued into college. where he attended the University of Southern California (USC) on a basketball scholarship. This early involvement in sports laid a strong foundation for his physical health and disciplined lifestyle.
Transition to Acting
Selleck's transition from an athlete to an actor came with its physical demands. His first significant role in "Magnum P.I." required him to perform various stunts and maintain a fit appearance. This role, which he played from 1980 to 1988. necessitated a rigorous fitness routine to meet the show's demands. setting the stage for his long-term commitment to health and wellness.
Fitness Regimen
Workout Routine
Tom Selleck health and fitness regimen has evolved. adapting to his changing roles and age. During his "Magnum, P.I." days. Selleck's workouts were intense and focused on building and maintaining muscle mass. His routine included weightlifting, cardiovascular exercises. and specific training for the stunts he performed on the show.
Selleck adjusted his fitness routine as he aged to suit his body's needs. Today, his workouts focus on maintaining flexibility, strength, and cardiovascular health. He incorporates low-impact exercises such as swimming, walking, and light weightlifting. This balanced approach helps him stay fit without putting undue strain on his joints and muscles.
Importance of Flexibility and Mobility
In recent years, Selleck has emphasized the importance of flexibility and mobility in his fitness regimen. Understanding the natural decline in muscle mass and joint flexibility with age. he includes stretching and yoga in his routine. These practices help prevent injuries, improve posture, and maintain mobilit
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2. Content
• Purpose of Sperm Separation
• Common Techniques
Simple Wash
Swim-up
Density Gradient
• Factors Determining the choice of method
• Efficiency of Sperm Selection Technique
• Preparing TESA/PESA Samples
• Take Home Message
3. Facts
• The spermatozoa of all placental (eutherian) mammals, including
humans, are in a protective, nonliable state at ejaculation and are
incapable or poorly capable of fertilization even if they are placed in
direct contact with an oocyte.
• They must undergo a subsequent period of final maturation
(Capacitation) during which they acquire the capacity to interact
with the oocyte–cumulus complex and achieve fertilization.
4. Why Sperm Separation ?
• Seminal plasma contain one or more factors (e.g. prostaglandins, zinc, leukocytes)
that prevent spontaneous capacitation of spermatozoa upon ejaculation
• Prolonged exposure to seminal plasma has adverse effects on sperm function :
– Like the ability to penetrate cervical mucus (Kremer, 1968)
– undergo the acrosome reaction in vitro, and the fertilization process in general
(Rogers et al, 1983; Mortimer and Mortimer, 1992; Mortimer et al, 1998).
• Consequently, in order for a spermatozoa to have the capacity to fertilize an
oocyte, they must be separated from the seminal plasma.
5. Why Sperm Separation ?
• The World Health Organization recommendation is to process
sperm sample within one hour after ejaculation in order to
prevent permanent damage from leukocytes and other cells
present in the semen.
• Recommended Time – 20 to 30 mins
6. Purpose of Sperm Preparation
• To yield a final preparation that has high percentage of sperms that are:
Motile
Morphologically Normal
Free from debris, non-germ cell and dead sperms
• Efficiency of any sperm preparation method is expressed in terms of :
Absolute Sperm Numbers
Number of Motile Sperms
Recovery of Morphologically normal Motile Sperms
8. Common Techniques
• There are 4 basic approaches to sperm preparation:
Washing (e.g Simple Wash)
Sperm Migration (Migration of healthy sperm based on self propelled movement. e.g. Swim-Up)
Adherence
Filtration
Density Gradients (Combination of the sperms’ own motility and their adherence to filtration
matrices and the retention at phase borders)
Adherence methods to eliminate debris and dead spermatozoa (e.g. Glass Wool, Glass beads, and
Sephadex columns)
10. Simple Wash
• Provides the highest yield of spermatozoa and is adequate if
semen sample is of optimal quality.
• It is often used for preparing IUI samples from cryopreserved
semen.
11. Simple Wash - Method
1) Mix the semen sample well
2) Dilute the entire semen sample 1 + 2
(1:2) with supplemented medium to
promote removal of seminal plasma.
1) Transfer the diluted suspension into multiple centrifuge tubes, with
preferably not more than 3 ml per tube.
2) Centrifuge at 300–500g for 5–10 minutes.
12. Simple Wash - Method
5) Carefully aspirate and discard the
supernatant.
5) Resuspend the combined sperm pellets in
1 ml of supplemented medium by
gentle pipetting.
5) Centrifuge again at 300–500g for 3–5 minutes.
6) Carefully aspirate and discard the supernatant.
7) Resuspend the sperm pellet, by gentle pipetting, in a volume of
supplemented medium appropriate for final insemination
14. Swim-Up
• It is a migration-based technique.
• Swim-up is one of the most commonly used techniques for
sperm preparation.
• In Conventional swim-up, a pre-washed sperm pellet obtained
by a soft spin is placed in an overlaying culture medium in a
conical tube. So it involves swim-up from the cell pellet rather
than direct semen.
16. Direct Swim Up
Direct swim-up (without diluting semen) is the simplest and fastest method for
separating sperm by migration for Oligospermic samples.
17. Direct Swim Up - Method
• Allow specimen to completely liquefy for 15 to 30 mins
• Place 1 ml of semen in a sterile 15-ml round bottom
tube, and gently layer 2 ml of medium
over it.
• Incline the tube at an angle of about 45°, to increase the
surface area of the semen–culture medium interface,
and incubate for 30 to 60 minutes at 37 °C.
• Gently return the tube to the upright position and remove the uppermost appx 1.5
ml of medium. This will contain highly motile sperm cells.
18. Direct Swim Up - Method
• Dilute this with 1.5–2.0 ml of medium.
• Centrifuge at 300–500g for 5 minutes & discard the supernatant.
• Resuspend the sperm pellet in 0.5 ml of medium
Note:
• To increase number of motile sperm in final inseminate, use multiple tubes with small
volume which will further increase the interface area.
• Do not use unsupplemented medium as it might result in sperm sticking to the surface of the
test tube leading to a lower yield and motility.
19. Disadvantages of Swim Up
• The swim-up method is simple and relatively inexpensive, yet it has some
disadvantages:
Centrifugation performed in conventional swim-up has been shown to generate ROS.
Many layers of the sperm pellet may cause potentially motile spermatozoa in the lower
levels of the pellet never to reach the interface with the culture medium layer.
Amount of motile spermatozoa retrieved is relatively low (< 20% of motile sperms
retrieved)
Takes twice as long as some other routinely performed techniques.
21. Density Gradient - Principle
• Density gradient centrifugation separates sperm cells based on their density. Thus,
at the end of centrifugation, each spermatozoa is located at the gradient level that
matches its density.
• Morphologically normal & abnormal sperms have different densities:
– Normal Mature Sperm – at least 1.10 g/mL
– Abnormal Immature Sperm – Between 1.06 – 1.09 g/mL
• As a result the leukocytes, cell debris and morphologically abnormal and poor
motility sperms are trapped inside the interphases.
22. Density Gradient - Principle
• Density gradients can either be continuous or discontinuous.
Continuous Density Gradient - There is a gradual increase in density from the
top of the gradient to its bottom.
Discontinuous Density Gradient - There are clear boundaries between lower
and upper gradients. The most widely used discontinuous density gradient
preparation includes 45% (v/v) density (top layer) and 90% (v/v) density (lower
layer).
• Double Density Gradient is the most standard and most widely used density
gradient preparation.
23. Density Gradient - Principles
• Components of the density gradient sperm separation procedure include a
colloidal suspension of silica particles stabilized with covalently bonded
hydrophilic silane supplied in HEPES.
• There are two Gradients:
– Lower Phase (90%)
– Upper Phase (45%)
• Sperm washing medium is used to wash the resuspended final pellet.
24. Discontinuous Density Gradient
• Discontinuous density-gradients can provide the best selection of good-quality
spermatozoa, giving good separation from other cell types and debris.
• It is easy to perform and yields higher sperm concentrations with less preparation
time (approximately 55 minutes)
• It is easier to standardize than the swim-up technique, and thus results are more
consistent.
• This technique is used to recover and prepare spermatozoa for use in IUI, IVF and
ICSI.
26. Discontinuous Density Gradient
• Place all components of the upper and lower phase, sperm wash
and semen sample in incubator at 37°C for 20 mins
• Transfer 1mL of the lower phase into sterile conical bottom tube.
• Layer 1 mL of the upper phase on top of the
lower phase very slowly without disturbing the
lower phase using a transfer pipette.
• A distinct line separating the two layers called ‘Interphase’ will be observed.
This two-layer gradient is stable for up to 1 hour.
27. Discontinuous Density Gradient
• Gently place 1 ml of liquefied well mixed semen sample over the layered gradient.
You may use more than one tube based on the count, motility and quantity of
semen sample to generate a better yield.
• Centrifuge for 12 to 15 mins at 300g.
• Remove most of the supernatant from
the sperm pellet.
• Resuspend the sperm pellet in 2 to 3 ml of
supplemented medium by gentle pipetting and
centrifuge at 200g for 4–10 minutes.
28. Discontinuous Density Gradient
• Remove most of the supernatant
from the sperm pellet
• Resuspend the final pellet in
supplemented medium by gentle
pipetting so that concentration and
motility can be determined
29. Tips for maximizing Yield
• Avoid overloading the gradient with semen sample as it causes a
phenomenon called ‘rafting’’ i.e aggregation of undesirable
components of semen in the post centrifugation pellet.
• Use the gradient within one hour after layering it as eventually the
two phases will blend over time and sharp interface will cease to
exist.
• A three gradient column (40% -70% - 90%) can be used to remove
the cryopreservatives from thawed semen samples.
31. Not so Common Methods
Test-Yolk Buffer
Migration Sedimentation using Tea-Jondet Tubes
PVP Droplet swim out for ICSI
Glass-wool Filtration, Glass Bead Filteration
Electrophoresis and Flourescence Cell Sorting
Swim-Down
Sephadex Column
Transmembrane Migration
32. Factors Affecting Choice of Method
What Factors determine the choice of
method used for Sperm preparation?
33. Choice of Sperm Preparation Method
• The ideal sperm separation technique should :
be quick, easy and cost-effective
isolate as much motile spermatozoa as possible
not cause sperm damage or non-physiological alterations of the separated
sperm cells
eliminate dead spermatozoa and other cells, including leukocytes and bacteria
eliminate toxic or bioactive substances like decapacitation factors or reactive
oxygen species (ROS)
allow processing of larger volumes of ejaculates
• None of the methods available meets all these requirements
34. Comparison
Technique Used Time Cost Yield Quality
Simple Wash Low Low High Low
Direct Swim Up Highest Low Lowest Highest
Density Gradient High Highest Highest High
*Comparison made for the same volume of liquefied sample prepared.
35. Choice of Sperm Preparation Method
• Depends on nature of semen sample
• Direct Swim Up - Normozoospermic
• Double Density Gradient – Oligozoospermic,
Teratozoospermic, Asthenozoospermic
37. Preparing PESA Samples
• Sperm samples obtained from the epididymis do not contain a
significant amount of non-germ cells such as red blood cells.
• If sufficient numbers of epididymal sperm cells are collected,
density gradient centrifugation can be used to prepare the
spermatozoa for ART.
• If number of spermatozoa aspirated is low, simple wash
technique can be used.
38. Preparing TESA Samples
• Testicular samples contain large numbers of non-germ cells such as red
blood cells. Spermatozoa need to be separated from these non-germ cells.
• To free the testicular spermatozoa from the seminiferous tubules, use
mechanical (Teasing/Mincing) or enzymatic (Collagenase) method.
• Erythrocyte-lysing buffer (ELB) can be used to remove RBC.
• Pentoxyfylline is occasionally used to increase the sperm motility.
39. Preparing TESA Sample
• Use Simple Wash to prepare the specimen obtained by adding 1.5 ml of culture
medium.
• Centrifuge at 300g for 8–10 minutes.
• Remove the supernatant and resuspend the pellet in 0.5 ml of fresh culture
medium.
• Estimate the motility and number of spermatozoa in the pellet.
• (Some specimens with a low number of spermatozoa may need to be resuspended
in a lower volume of medium.)
40. Treating SHV (Semen Hyper Viscosity)
• Viscous semen samples can be treated with following enzymes and
incubated further in order to achieve liquefaction:
Proteolytic Enzyme
Bromelain
Trypsin
N-Acetyl Cysteine (NAC)
• Please follow manufacturers recommendation for processing
viscous semen sample with the enzyme of your choice and process
semen using Density Gradient.
41. Practical Factors
• Practical Factors affecting our choice:
When multiple IUI/IVF/ICSI Sample needs to be
prepared at the same time. How many samples should
you process at the most? Preference to which sample?
Processing Oligospermic sample high in volume ?
Handling sample with high concentration of motile
sperms
42. Take Home Message
• Centrifugal pelleting of unselected populations of human spermatozoa
causes irreversible damage to the spermatozoa that can impair—even
totally destroy—their fertilizing ability
• The method of choice is dependent on the intended ‘use’ of the prepared
sample. e.g. IUI needs a much higher yield as compared to ICSI
• Many practical scenario on day to day basis may affect the choice of
semen preparation method, use your learning wisely to choose the
appropriate method