Sperm cryopreservation is the process of freezing sperm to preserve fertility. It involves adding cryoprotective agents to minimize freezing damage before slowly cooling sperm to -196°C in liquid nitrogen storage. Factors like semen quality, freezing technique, and thawing process can impact sperm survival. While some studies found cryopreservation may damage DNA, properly performed it selectively affects defective sperm and clinical pregnancy rates are similar to fresh sperm. Optimization involves semen preparation, controlled freezing and thawing rates, and cryoprotectant use. Further research is still needed on impacts to DNA and reproductive outcomes.
Human spermatozoa can tolerate a range of temperature. They are not very sensitive to damage caused by cooling possibly because of high membrane fluidity which is used as a technique to preserve spermatozoa in adverse conditions. cryopreservation technology has been a boon in every aspect of infertility & ART practice.
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.
It was while performing SUZI that a single spermatozoon accidentally penetrated into the oolemma and provided the hint that a direct sperm injection would be more efficient.
1st successful birth by ICSI took place on Jan 14, 1992.
Human spermatozoa can tolerate a range of temperature. They are not very sensitive to damage caused by cooling possibly because of high membrane fluidity which is used as a technique to preserve spermatozoa in adverse conditions. cryopreservation technology has been a boon in every aspect of infertility & ART practice.
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.
It was while performing SUZI that a single spermatozoon accidentally penetrated into the oolemma and provided the hint that a direct sperm injection would be more efficient.
1st successful birth by ICSI took place on Jan 14, 1992.
Antisperm antibody, presentation task in Infertility class. Our study program is Andrology, Medical Faculty, Airlangga University.
Visit us in:
Andrologi FK UNAIR: http://spesialis1.andrologi.fk.unair.ac.id/
FK UNAIR: http://fk.unair.ac.id/
UNAIR: http://unair.ac.id/
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-vitro fertilization (IVF) is a process by which oocytes are fertilized by sperm outside the women’s womb, in vitro. It still represents one of the most exciting modern scientific developments and continues to have a tremendous impact on
people's lives.
Here, we will discuss all about the embryo development inside the dish.
Also we discuss which embryo to choose for transferring into female's uterus.
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
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
Egg freezing, also known as oocyte cryopreservation, is a great advancement in technology allowing women to preserve their fertility and their eggs. These eggs, once removed from the body are frozen using special techniques and can be used to make babies by the process of IVF when the woman desires.
Antisperm antibody, presentation task in Infertility class. Our study program is Andrology, Medical Faculty, Airlangga University.
Visit us in:
Andrologi FK UNAIR: http://spesialis1.andrologi.fk.unair.ac.id/
FK UNAIR: http://fk.unair.ac.id/
UNAIR: http://unair.ac.id/
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-vitro fertilization (IVF) is a process by which oocytes are fertilized by sperm outside the women’s womb, in vitro. It still represents one of the most exciting modern scientific developments and continues to have a tremendous impact on
people's lives.
Here, we will discuss all about the embryo development inside the dish.
Also we discuss which embryo to choose for transferring into female's uterus.
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
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
Egg freezing, also known as oocyte cryopreservation, is a great advancement in technology allowing women to preserve their fertility and their eggs. These eggs, once removed from the body are frozen using special techniques and can be used to make babies by the process of IVF when the woman desires.
Cryopreservation and its application to aquaculture.pptxNarsingh Kashyap
What is Cryopreservation ?
Cryopreservation is a process where biological materials such as cells and tissues are preserved by cooling to very low temperatures, usually at -196°C (the temperature of liquid nitrogen), yet remain viable after later warming to temperatures above 0°C.
Cryopreservation in aquatic species goes back 65 years and began about the same time as similar research was performed in livestock (Blaxter 2011).
In India, NBFGR & CIFA are the primary organization carrying out fish sperm cryopreservation for long term gene banking (J. K. Jena 2012)
We discuss here all essential principles of cryoperservation in assisted reproductive techniques:
1. Oocye cryoperservation
2. Embryo cryopersevration
3. Sperm cryopersevration
Introduction
Reason for cryopreservation
Selection of part of plant for cryopreservation
Technique of cryopreservation
Application
Limitation
Conclusion
Protocol for Cryopreservation of snowtrout semenN K Agarwal
Schizothorax richardsonii is an indigenous snow-trout in the Himalayan region of Uttarakhand. It contributes a lot to the fish food basket in the hilly area of the state. In past few years snowtrout capture fishery has registered continuously decreasing trend due to over-exploitation and habitat destruction. For reestablishing its status and propagation of the species, first successful attempt was made to cryopreserve the semen of S. richardsonii to make all time availability of viable sperm to increase hatchery production for river ranching and culture practices.
To develop the cryopreservation protocol for S. richardsonii, initially semen quality was evaluated as per species trait and for sperm motility behaviour. Good quality semen samples (>75% motile sperm after activation) were pooled and frozen within 2 hours of collection. Four extenders and two cryoprotectants (CPA) were tested to cryopreserve the S. richardsonii semen. The extender-Mounib’s medium was found most successful. The DMSO proved better CPA than glycerol. Equilibration time was standardized as 45 min for DMSO and 60 min for glycerol. The semen is diluted with the different diluents (extender + cryoprotectant) before freezing. Two semen dilution ratios 1:4 and 1:10 were tested for better viability after cryopreservation. French medium straws of 0.5 ml capacity were used to store the semen in liquid nitrogen. Thawing temperature was standardized as 20-250C.
The cryopreservation success was evaluated by post-thaw motility and fertilizing ability of cryopreserved semen. Post-thaw motility percentage and duration was significantly reduced. Fertilizing capacity of 375 days old cryopreserved semen at 1:4 dilution ratio was almost equal to that of fresh semen (>98%). Semen cryopreserved in Mounib’s extender with 5% DMSO as CPA, even after 375 days had hatching rate-35.91 ±0.15% and fry survival rate 21.28 ±0.41% while those from fresh semen it was 52.97±0.26% and 30.43±0.50% respectively. Though the hatching and survival rates were significantly superior with fresh semen (P<0.001) than the cryopreserved semen, but these results are still encouraging. This developed cryopreservation protocol for S. richardsonii semen has ensured year round availability of viable sperm for optimal utilization in hatchery production. It would open up new perspective such as establishing genetic material reserves, sperm / gene bank for selective breeding, cross breeding and ex-situ conservation of fish germplasm.
Similar to Spermcryoperservation by Dr.Chandan (20)
Defecation
Normal defecation begins with movement in the left colon, moving stool toward the anus. When stool reaches the rectum, the distention causes relaxation of the internal sphincter and an awareness of the need to defecate. At the time of defecation, the external sphincter relaxes, and abdominal muscles contract, increasing intrarectal pressure and forcing the stool out
The Valsalva maneuver exerts pressure to expel faeces through a voluntary contraction of the abdominal muscles while maintaining forced expiration against a closed airway. Patients with cardiovascular disease, glaucoma, increased intracranial pressure, or a new surgical wound are at greater risk for cardiac dysrhythmias and elevated blood pressure with the Valsalva maneuver and need to avoid straining to pass the stool.
Normal defecation is painless, resulting in passage of soft, formed stool
CONSTIPATION
Constipation is a symptom, not a disease. Improper diet, reduced fluid intake, lack of exercise, and certain medications can cause constipation. For example, patients receiving opiates for pain after surgery often require a stool softener or laxative to prevent constipation. The signs of constipation include infrequent bowel movements (less than every 3 days), difficulty passing stools, excessive straining, inability to defecate at will, and hard feaces
IMPACTION
Fecal impaction results from unrelieved constipation. It is a collection of hardened feces wedged in the rectum that a person cannot expel. In cases of severe impaction the mass extends up into the sigmoid colon.
DIARRHEA
Diarrhea is an increase in the number of stools and the passage of liquid, unformed feces. It is associated with disorders affecting digestion, absorption, and secretion in the GI tract. Intestinal contents pass through the small and large intestine too quickly to allow for the usual absorption of fluid and nutrients. Irritation within the colon results in increased mucus secretion. As a result, feces become watery, and the patient is unable to control the urge to defecate. Normally an anal bag is safe and effective in long-term treatment of patients with fecal incontinence at home, in hospice, or in the hospital. Fecal incontinence is expensive and a potentially dangerous condition in terms of contamination and risk of skin ulceration
HEMORRHOIDS
Hemorrhoids are dilated, engorged veins in the lining of the rectum. They are either external or internal.
FLATULENCE
As gas accumulates in the lumen of the intestines, the bowel wall stretches and distends (flatulence). It is a common cause of abdominal fullness, pain, and cramping. Normally intestinal gas escapes through the mouth (belching) or the anus (passing of flatus)
FECAL INCONTINENCE
Fecal incontinence is the inability to control passage of feces and gas from the anus. Incontinence harms a patient’s body image
PREPARATION AND GIVING OF LAXATIVESACCORDING TO POTTER AND PERRY,
An enema is the instillation of a solution into the rectum and sig
Navigating the Health Insurance Market_ Understanding Trends and Options.pdfEnterprise Wired
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How many patients does case series should have In comparison to case reports.pdfpubrica101
Pubrica’s team of researchers and writers create scientific and medical research articles, which may be important resources for authors and practitioners. Pubrica medical writers assist you in creating and revising the introduction by alerting the reader to gaps in the chosen study subject. Our professionals understand the order in which the hypothesis topic is followed by the broad subject, the issue, and the backdrop.
https://pubrica.com/academy/case-study-or-series/how-many-patients-does-case-series-should-have-in-comparison-to-case-reports/
CHAPTER 1 SEMESTER V - ROLE OF PEADIATRIC NURSE.pdfSachin Sharma
Pediatric nurses play a vital role in the health and well-being of children. Their responsibilities are wide-ranging, and their objectives can be categorized into several key areas:
1. Direct Patient Care:
Objective: Provide comprehensive and compassionate care to infants, children, and adolescents in various healthcare settings (hospitals, clinics, etc.).
This includes tasks like:
Monitoring vital signs and physical condition.
Administering medications and treatments.
Performing procedures as directed by doctors.
Assisting with daily living activities (bathing, feeding).
Providing emotional support and pain management.
2. Health Promotion and Education:
Objective: Promote healthy behaviors and educate children, families, and communities about preventive healthcare.
This includes tasks like:
Administering vaccinations.
Providing education on nutrition, hygiene, and development.
Offering breastfeeding and childbirth support.
Counseling families on safety and injury prevention.
3. Collaboration and Advocacy:
Objective: Collaborate effectively with doctors, social workers, therapists, and other healthcare professionals to ensure coordinated care for children.
Objective: Advocate for the rights and best interests of their patients, especially when children cannot speak for themselves.
This includes tasks like:
Communicating effectively with healthcare teams.
Identifying and addressing potential risks to child welfare.
Educating families about their child's condition and treatment options.
4. Professional Development and Research:
Objective: Stay up-to-date on the latest advancements in pediatric healthcare through continuing education and research.
Objective: Contribute to improving the quality of care for children by participating in research initiatives.
This includes tasks like:
Attending workshops and conferences on pediatric nursing.
Participating in clinical trials related to child health.
Implementing evidence-based practices into their daily routines.
By fulfilling these objectives, pediatric nurses play a crucial role in ensuring the optimal health and well-being of children throughout all stages of their development.
Navigating Challenges: Mental Health, Legislation, and the Prison System in B...Guillermo Rivera
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Telehealth Psychology Building Trust with Clients.pptxThe Harvest Clinic
Telehealth psychology is a digital approach that offers psychological services and mental health care to clients remotely, using technologies like video conferencing, phone calls, text messaging, and mobile apps for communication.
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2. Aim of this lecture…………….
• Overview of the current technologies and approaches utilized in sperm
cryopreservation.
• Consider the factors affect to results of cryopreservation
• Sperm Cryodamage.
• How to optimize semen cryopreservation.
• Sperm preparation prior cryopreservation
• Is cryopreservation induce DNA damage?
• whether using fresh rather than cryopreserved sperm cells has the same effect
on reproductive outcome in ICSI
3. What is sperm
cryopreservation?
• A procedure to preserve sperm cells (commonly called sperm banking)
• Cryopreservation is the freezing of cells or tissues to sub zero temperatures, typically -196 º C
(boiling point of liquid nitrogen).
• The first successful cryopreservation of spermatozoa was initiated 1960s.
• For human sperm, the longest reported successful storage is 22 years.
• All biological activity is stopped or paused until it is thawed.
• The freezing of sperm needs cryopreservation agents that minimize damage to the cells during the
freezing and thawing process
4. Why sperm cryopreservation is
performed ?
1. Semen containing a very limited number of spermatozoa or abnormal semen parameters.
2. For cancer patients to preserve their fertility prior to gonadotoxic chemotherapy or radiation.
3. Patients undergoing certain types of pelvic or testicular surgeries
4. Patients who suffer from degenerative illnesses such as diabetes or multiple sclerosis; spinal
cord disease or injury.
5. persons in occupations where a significant risk of gonadotoxicity prevails.
6. men undergoing surgical sterilization such as vasectomy.
7. allow donor semen samples to be quarantined while appropriate screening is performed to
prevent the transmission of infectious pathogens during therapeutic donor insemination (TDI).
8. used in combination with ART techniques.
5. How sperm cryopreservation is
performed?
Cryoprotective agents (CPAs):
• low molecular weight chemicals that serve to protect spermatozoa from freezing damage or ice
crystallization by decreasing the freezing point of materials.
• CPAs can be toxic if used at high concentrations.
1. Permeating CPAs (penetrate the plasma membrane)
such as dimethylacetaldehyde; dimethyl sulfoxide, glycerol, glycol, ethylene and methanol
stabilize cell plasma membrane proteins and reduce concentrations of electrolytes.
2. Nonpermeating CPAs (unable to penetrate the plasma membrane)
such as albumins, dextrans, egg yolk citrate, hydroxyethyl, polyethylene glycols, polyvinyl
pyrollidone and sucrose
minimize intracellular crystallization by increasing viscosity of the sample.
6. How sperm cryopreservation is
performed?
Benefits of cryoprotectants:
1. Maintaining sperm viability.
2. Improvement in the recovery of motile sperm by the use of zwitterion
buffers has been attributed to their ability to bind free hydrogen and hydroxy
ions in the surrounding medium, aiding in the dehydration process.
3. Improve sperm membrane fluidity.
4. Increase sperm longevity and percent survival.
5. Increase ability of sperm to penetrate cervical mucus post-thaw.
7. Techniques for Cryopreservation
1- Slow Freezing:
Slow programmable freezing
- Liquid nitrogen is poured into the tank, and the machine, once programmed, uses the software
data logging to obtain cooling from 20°C to −80°C at rate of 1.5°C/min and then at 6°C/min;
at completion of the freezing the straws are removed and stored into liquid nitrogen at
−196°C. This takes about 40 min.
- Simple to use
- does not require continuous operator intervention
- increase the reproducibility of the freezing operations
8. Techniques for Cryopreservation
2- Rapid Freezing:
• requires direct contact between the straws and the nitrogen vapors for 8–10 min and immersion in
liquid nitrogen at −196°C.
• Inside nitrogen vapors there is a thermal gradient, as a function of the distance and the volume of
the liquid below.
• The sample is initially mixed in dropwise manner with equal volume of cold cryoprotectant; the
mixture is loaded into the straws and left to incubate at 4°C for 10 minutes.
• The straws are then placed at a distance of 15–20 cm (1 hr WHO) above the level of liquid
nitrogen (−80°C) for 15 min; after this stage, the straws are immersed in liquid nitrogen.
• place the straws in horizontal position to minimize the heat difference between the two ends.
• Low reproducibility
• temperature drop curve cannot be controlled
• and the freezing temperatures may vary from −70, −80, and −99°C.
9. Techniques for Cryopreservation
3. Ultra-rapid freezing (Vitrification)
• Until only recently, vitrification of spermatozoa was unsuccessful, possibly due to high
concentrations of permeable CPAs (30-50% compared to 5-7% with slow freezing) and low
tolerance of spermatozoa to permeable agents.
• Even brief exposure to a high concentration of CPAs can lead to toxic and osmotic shock and
would be lethal for spermatozoa.
• One possible strategy to lower the concentration of CPAs could be to increase the speed of cooling
and warming temperatures as higher rates of cooling and warming, require lower concentrations of
CPAs; these conditions can help eliminate intracellular ice crystallization, and facilitate the
formation of a glassy state .
• Another option is to add non-permeable CPAs--such as carbohydrates--to permeable CPAs to
minimize osmotic shock by decreasing osmotic pressure and stabilizing the nuclear membrane.
10. Straw holder
Straw or vails
Forceps
Syringe
Vistube
Requirments for Cryopreservation
Tanks
11. Thawing Procedure
• The thawing procedure is an equally important
step: the cell must be allowed to recover its normal
biological activities trying to avoid abrupt thermal
changes as far as possible. Generally speaking, the
cryopreservation protocols use a thawing
temperature of 37°C; even if higher thawing
temperatures allow for more rapid heating, they
are not used because of the risks associated to cell
damage.
12. At the present time, several thawing techniques
are used, among which are
• (i) thawing at room temperature for 10 min and
subsequent thermostat pass at 37°C for another
10 min,
• (ii) thawing in a thermostat and water-bath at 37°C
for 10 min,
• (iii) thawing at room temperature for 15 min.
• Once the semen is thawed, it is separated from the
cryopreservation medium by washing in culture
medium and centrifuging
14. How to avoid Negative Impacts
Maintenance of media.
Standard SOP
Trained staff
Equipped lab
15. Semen preparation pre-freeze and
post-thaw
• The quality of ejaculated semen is also related to the outcome of cryopreservation.
• For example, dead spermatozoa or leukocytes in pre-freezing semen detrimentally affect the
sperm survival rate and the fertility potential after thawing through the ROS generation
process
• Sperm preparation before cryopreservation should be considered in routine sperm
cryopreservation.
• Optimizing the concentration of progressive motile sperm cells before the freezing process is
recommended to ameliorate the fertilizing capacity of the frozen spermatozoa.
• Semen preparation before freezing resulted in better sperm quality and fewer apoptotic sperm.
16.
17. How to optimize semen
cryopreservation ?
Factors affecting the optimization of human sperm preservation:
Technical factorsBiological factors
Cryopreservation mediumGenetic factors
Addition/removal of cryoprotectantSexual abstinence
Cooling rateSeminal fluid quality
packingSeminal quality
Storage
Thawing
storage temperature
18. Cryopreservation and DNA Damage
• There is no agreement in the literature neither on whether cryopreservation induces DNA
damage nor on the amount of damage.
Because:
(1) different freezing procedures
(2) different tests to evaluate the DNA integrity
(3) different semen preparation techniques before cryopreservation (i.e., swimup or density
gradient centrifugation).
19. Cryopreservation and Reproductive
Outcome
Testicular Spermatozoa:
• No statistically significant differences in all parameters examined (fertilization rate,
cleavage rate, embryo quality, implantation rate, clinical pregnancy rate, and ongoing
pregnancy rate) between ICSI cycles with fresh or cryopreserved testicular spermatozoa.
• Only, De Croo and colleagues stated that fertilization, implantation, and live-birth rates per
embryo transfer are significantly lower after ICSI.
Epididymal Spermatozoa:
• Tournaye and colleagues reported that the clinical pregnancy rate in ICSI cycles was
comparable between fresh and frozen-thawed epididymal spermatozoa.
• Sukcharoen and colleagues came to the same conclusion; also Cayan and colleagues [64]
supported the same opinion.
20. Cryopreservation and Reproductive
Outcome
Ejaculated Spermatozoa:
• Kucznynski and colleagues did not report of any statistically significant differences in
fertilization rate between fresh and frozen semen patients.
• ongoing pregnancies are significantly higher in ICSI patients when human sperm samples are
cryopreserved.
this suggests that properly performed cryopreservation selectively affects defective rather
than normal spermatozoa
The freezing-thawing procedure causes more damage in patients with alterations in semen
quality than that in patients with normal semen. However, once the oocyte is fertilized,
implantation and pregnancy rates are similar in patients with or without sperm anomalies.
21. Future issues
• Cryopreservation of human semen is extremely important to the field of male infertility.
However, there is dissension regarding the best cryopreservation protocol for human semen.
• To date there is no agreement in the literature on whether or not cryopreservation affects
sperm chromatin integrity or on the use of a unique and functional protocol for the freezing-
thawing procedure.
• Further investigations are needed to fully understand the real influence of cryopreservation on
sperm DNA integrity and the impact of the use of cryopreserved spermatozoa on the
reproductive outcome, technical measures should be applied to provide maximum protection
to the male gametes: appropriate use of cryoprotectants before and sperm selection
technologies after cryopreservation seems to have the greatest impact on preventing DNA
fragmentation, thus improving sperm cryosurvival rates.
22. References
[1] Marlea Di Santo, Nicoletta Tarozzi, Marco Nadalini, and Andrea Borini. Human Sperm
Cryopreservation: Update on Techniques, Effect on DNA Integrity, and Implications for ART.
Advances in Urology. Volume 2012 (2012), Article ID 854837, 12 pages.
[2] 2014 Andrology and Embryology Review Course Manual of the American Board of
Bioanalysis (ABB).
[3] World Health Organisation: Department of Reproductive Health and Research WHO
laboratory manual for the examination and processing of human semen. 5th edition. 2010.
[4] Gardner DK, Weissman A, Howles CM, Shoham Z. Textbook of Assisted Reproductive
Techniques. 3rd ed. Vol. 1. In: Agarwal A, Erenpreiss J, Sharma R. Sperm chromatin assessment.
United Kingdom: Informa healthcare, 2009:67-84.
1-Removing or reduction the water content of spermatozoa by adding glycerol as acryoprotectant to minimize intracellular ice formation during freezing ,without the addition of cryoprotectant during freezing ,human spermatozoa are damaged by plasma membrane swelling as water expands during freezing ,acrosomal leakage and breakdown.
2-The cryoprotectant penetrate s the cell and displaces the intracellular water,an osmotic equilibrium is reached and the sperm cell returns to almost its original volume.this is accompanied by reduction in temperature reaches to -5 to -15 degree c. the inside of sperm cell remains unfrozen but super cooled intercellular water has higher chemical potential and diffuses out of the cell osmotic ally . Freezing continues extracellularly,resulting hyper tonicity and further redution in water from sperm cell ,leading to almost complete dehydration .
Some authors argue that conventional slow freezing, either manual or automated, causes extensive chemical-physical damage to the sperm probably because of ice crystallization.
Since the intracellular matrix of human spermatozoa contains large amounts of proteins and sugars, they can be successfully frozen in the absence of permeable CPAs using protein- and sugar-rich non-permeable agents
This observation seems to indicate that cryopreservation before ICSI might be helpful to eliminate senescent or deficient spermatozoa, thus, improving reproductive outcome
