Therapy options

This application helps to propose an appropriate fertility therapy method and to find the most suitable clinic worldwide based on the price, duration and legislative options of the treatment in various countries.

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Egg donation

Egg donation is the process by which a woman donates eggs for purposes of assisted reproduction or biomedical research. For assisted reproduction purposes, egg donation typically involves IVF technology, with the eggs being fertilized in the laboratory; more rarely, unfertilized eggs may be frozen and stored for later use. Egg donation is a third party reproduction as part of ART.

Egg donor may have several reasons for donate her eggs:

  • Unrelated donors to the recipients – they do it for altruistic and/or monetary reasons. The European Union limits any financial compensation for donors to at most $1500. In some countries, most notably Spain and Cyprus, this has limited donors to the poorest segments of society. In US, donors are paid regardless of how many egg she produces. In most countries (excluding the US and the UK), the law requires such type of donors to be anonymous.
  • Egg sharing – the woman decides to provide unused egg from her own IVF for another patient.
  • Designated donors – couple bring their friend or the donor specifically to help them.

Procedure

First step is choosing the egg donor by a recipient from the profiles on or clinic databases (or, in countries where donors are required to remain anonymous, they are chosen by the recipient's doctor based on recipient woman’s desired trait). This is due to the fact that all of the mentioned examinations are expensive and the agencies/clinics must first confirm that a match is possible or guaranteed before investing in the process. 

Each egg donor is first referred to a psychologist who will evaluate if she is mentally prepared to undertake and complete the donation process. These evaluations are necessary to ensure that the donor is fully prepared and capable of completing the donation cycle in safe and success manner. The donor is then required to undergo a thorough medical examination, including a pelvic exam, blood tests to check hormone levels and to test for infectious diseases, Rh factor, blood type, and drugs and an ultrasound to examine her ovaries, uterus and other pelvic organs. A family history of approximately the past three generations is also required, meaning that adoptees are usually not accepted because of the lack of past health knowledge. Genetic testing is also usually done on donors to ensure that they do not carry mutations (e.g., cystic fibrosis) that could harm the resulting children; however, not all clinics automatically perform such testing and thus recipients must clarify with their clinics whether such testing will be done. During the process, which usually takes several months, the donor must abstain from alcohol, sexual intercourse, cigarettes, and drugs, both prescription and non-prescription.

Once the screening is complete and a legal contract signed, the donor will begin the donation cycle, which typically takes between three and six weeks. An egg retrieval procedure comprises both the egg donor's cycle and the recipient's cycle. Birth control pills are administered during the first few weeks of the egg donation process to synchronize the donor's cycle with her recipient's, followed by a series of injections which halt the normal functioning of the donor's ovaries. These injections may be self-administered on a daily basis for a period of one to three weeks. Next, FSH is given to the donor to stimulate egg production and increases the number of mature eggs produced by the ovaries. Throughout the cycle the donor is monitored often by a physician using blood tests and ultrasound exams to determine the donor's reaction to the hormones and the progress of follicle growth.

Once the doctor decides the follicles are mature, the doctor will establish the date and time for the egg retrieval procedure. Approximately 36 hours before retrieval, the donor must administer one last injection of hCG to ensure that her eggs are ready to be harvested. The egg retrieval itself is a minimally invasive surgical procedure lasting 20-30 minutes, performed under sedation (but sometimes without any). A small ultrasound-guided needle is inserted through the vagina to aspirate the follicles in both ovaries, which extracts the eggs. After resting in a recovery room for an hour or two, the donor is released. Most donors resume regular activities by the next day.

Laws by state

The legal status and compensation of egg donation has several models across states with examples:

  • Totally illegal procedure (Italy, Germany, Austria, Costa Rica, Sunni Muslim countries, Bahrain, Egypt, Hong Kong, Lebanon, Lithuania, Maldives, Norway, Oman, Pakistan, Philippines, Qatar, Saudi Arabia, Syria, Tajikistan, Turkey, Yemen),
  • Legal, no compensation, anonymous donor (France),
  • Legal, no compensation, non-anonymous donor (Canada),
  • Legal, possible compensation, anonymous donor (Spain, Czech Republic, South Africa),
  • Legal, possible compensation, non-anonymous donor (the UK),
  • Legal, possible compensation, anonymous or non-anonymous (the US).

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ICSI

During ICSI just one sperm is injected directly into the egg cytoplasm using a micromanipulative apparatus that transforms imperfect hand movements into fine and precise movements of micromanipulation tools.

Intracytoplasmic Sperm Injection (ICSI) is an assisted reproductive technique (ART) initially developed by Dr. Gianpiero D. Palermo in 1993 to treat male infertility. It is most commonly used in conjunction with in vitro fertilization (IVF). Following IVF procedure, the physician places the fertilized egg into the female’s uterus for implantation. Sperm are obtained by the same methods as with IVF: either through masturbation, by using a collection condom, or by surgically removing sperm from a testicle through a small incision (MESA, TESE). The females are treated with fertility medications for approximately two weeks prior to oocyte retrieval to stimulate superovulation, where the ovaries produce multiple oocytes rather than the normal one oocyte. The oocytes are retrieved by either laparoscopy, or more commonly, transvaginal oocyte retrieval. In the latter procedure, the physician inserts a thin needle through the cervix, guided by a sonogram and pierces the vaginal wall and then the ovaries to extract several mature ova. Before the embryologist can inject the sperm into the oocyte, the sperm must be prepared by washing and exposing it to various chemicals to slow the sperm movement and prevent it from sticking to the injection plate. Also, the oocytes are treated with hyaluronidase to single out the oocyte ready for fertilization by the presence of the first polar body. Then, one prepared sperm is injected into an oocyte with a thin needle. Often, embryologists try to fertilize several eggs so they can implant more than one into the uterus and increase the chance of at least one successful pregnancy. This also allows them to save extra embryos, using cryopreservation, in case later IVF rounds are needed.

After the embryologist manually fertilizes the oocytes, they are incubated for sixteen to eighteen hours and develop into a pronucleate eggs (successfully fertilized eggs about to divide into an embryo). The egg then grows for one to five days in the laboratory before the physician places it in the female’s uterus for implantation.

The chance of fertilization increases dramatically with ICSI compared to simply mixing the oocytes and sperm in a Petri dish and waiting for fertilization to occur unaided (classical IVF procedure). Studies have shown that successful fertilizations occur 50% to 80% of the time. Since the introduction of ICSI, intrauterine insemination (IUI) has decreased in popularity by 80%.

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IMSI

IMSI is based on a method of high magnification motile sperm organelle morphology examination (MSOME). It requires an inverted light microscope equipped with high power Nomarski optics enhanced with digital imaging. Such examination helps to identify spermatozoa with a normal nucleus and nuclear content. The exact indications of IMSI and usefulness are debatable. Finding normal-looking spermatozoa took a minimum of 60 min, and up to 210 min, depending on the quality of the semen sample. The IMSI procedure improved embryo development and the laboratory and clinical outcomes of sperm microinjection in the same infertile couples with male infertility and poor embryo development over the previous ICSI attempts.

Introduced in 2001, intracytoplasmic morphologically selected sperm injection (IMSI) represents a more sophisticated way of ICSI whereby, prior to injection, the spermatozoon is selected at higher magnification. Doing so, the spermatozoon can be evaluated for fine integrity of its nucleus and the injection of a normal spermatozoon with a vacuole-free head can be assured. 

Additional research is needed to unravel the underlying mechanisms responsible for the presence of vacuoles in sperm heads. Vacuoles in human sperm cells appear in various numbers and sizes, both in abnormal-shaped spermatozoa as well as in normal-shaped spermatozoa. Associations with acrosome status, chromatin condensation, DNA fragmentation and sperm aneuploidy have been documented, however, controversy on their nature exists. Spermatozoon shape and large vacuoles are detected and deselected in conventional ICSI as well. However, the detection of subtle small vacuoles depends on the resolving power of the optical system and may impact oocyte fertilization, embryo development and implantation. 

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MESA

MESA was first described in 1985. This surgical technique requires testis delivery through a 2-3-cm transverse scrotal incision. The epididymal tunica is incised, and an enlarged tubule is selected. Then, the epididymal tubule is dissected and opened with sharp microsurgical scissors. The fluid that flows out of the tubule is aspirated with the aid of a silicone tube or a needle attached to a tuberculin syringe (Pic.1) The aspirate is flushed into a tube containing warm sperm medium and is transferred to the laboratory for examination. MESA can be repeated at a different site on the same epididymis (from the cauda to caput regions) and/or the contralateral epididymis until an adequate number of motile sperm is retrieved. 

An embryologist examines the sample for the presence of motile sperm. If no motile spermatozoa are found at the first site, the maneuver is repeated. Typically, only a few microliters of epididymal fluid are retrieved because sperm are highly concentrated in the epididymal fluid (approximately 1x106 sperm/ml). A MESA approach should provide more than adequate numbers of sperm for immediate use, as well as for cryopreservation. 

If MESA fails to retrieve motile sperm, TESA or TESE can be performed as part of the same procedure. However, MESA often provides enough sperm for cryopreservation. A single MESA procedure usually enables the retrieval of a large number of high-quality sperm that can be used for ICSI or intentionally cryopreserved for subsequent ICSI attempts. As reported by Dr. Shlegel and colleagues, who used MESA and ICSI in a group of men with obstructive azoospermia, clinical pregnancies were detected by a fetal heartbeat in 75% (57/ 76) of attempts, and healthy deliveries occurred in 64% (49/ 76) of attempts.

ANESTHESIA FOR SPERM RETRIEVAL PROCEDURES Sperm retrievals are relatively simple surgeries that can be safely performed with general anesthesia or spinal blocks. However, because sperm retrievals are typically outpatient procedures, the latest trend is to employ local or locoregional anesthesia with or without intravenous sedation. In another study of 26 patients undergoing MESA, only 38% of the patients tolerated the procedure solely under spermatic cord block through the infiltration of 5-8 mL of 1% lidocaine; the remaining 62% required intravenous sedation. The percentage of patients who underwent a bilateral procedure and required intravenous sedation was as high as 75%.

General anesthesia may offer comfort and the efficient management of anxiety. However, when performed with inhalational agents such as N2O and halogenated agents, this approach is associated with a high incidence of postoperative nausea and vomiting. These two complaints are among the most frequent causes of hospitaliza-tion and the inability to discharge patients scheduled for ambulatory procedures. Additionally, these symptoms are among the most feared by patients undergoing minor surgery, surpassing even postoperative pain.

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Micro TESE

The concept of micro-TESE is to identify areas of sperm production within the testes with the aid of optical magnification (15-25x) and based on the size and appearance of the seminiferous tubules. Micro-TESE is recommended for the most severe cases of non-obstructive azoospermia (NOA).

MicroTESE yields the highest sperm retrieval rate and causes the least amount of damage to the testis.

Miniinvasive alternative to TESE using microdissection microscope. In microsurgical testicular sperm extraction (microdissection TESE; micro-TESE), the testicular parenchyma is dissected under magnification to search for enlarged seminiferous tubules, which are more likely to contain germ cells and foci of sperm production compared to non-enlarged or collapsed tubules. Such seminiferous tubules are removed rather than proceeding with the large single or multiple biopsies performed in conventional TESE.

For micro-TESE, the scrotal skin is stretched over the anterior surface of the testis, after which a 2 3 cm transverse incision is made. Alternatively, a single midline scrotal incision can be used. The incision extends through the dartos muscle and the tunica vaginalis. The tunica is opened, and identifiable bleeders are cauterized. The testis is delivered extravaginally, and the tunica albuginea is examined. Then, a single, large, mid-portion incision is made in an avascular area of the tunica albuginea under 6-8× magnification, and the testicular parenchyma is widely exposed in its equatorial plane (Pic. 1). The testicular parenchyma is dissected at 16-25× magnification to enable the search and isolation of seminiferous tubules that exhibit larger diameters (which are more likely to contain germ cells and eventually normal sperm production) in comparison to non-enlarged or collapsed counterparts (Pic. 2). If needed, the superficial and deep testicular regions can be examined, and microsurgical-guided testicular biopsies are performed by carefully removing enlarged tubules using microsurgical forceps. If enlarged tubules are not observed, any tubule that differs from the remaining tubules in size is excised. The excised testicular tissue specimens are placed into the inner well of a Petri dish containing sperm media, and are sent to the laboratory for processing and sperm search (Pic. 3). The tunicas albuginea and vaginalis are then closed in a running fashion using non-absorbable and absorbable sutures. The dartos muscle is closed with interrupted absorbable sutures, respectively. Immediately prior to complete closure, 3 cc of 1% xylocaine solution may be injected into the subcuticular layers. The skin is closed using a continuous subcuticular 4-0 vicryl suture. A fluffy-type scrotal dressing and scrotal supporter are placed.

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PESA

The technical procedure for PESA involves the insertion of a needle attached to a syringe through the scrotal skin into the epididymis (Pic 1). Originally, the use of a larger butterfly needle was described. Currently, most experts use a fine needle (26 gauge) attached to a tuberculin syringe containing sperm washing medium. After creating negative pressure by pulling the syringe plunger, the tip of the needle is gently and slowly moved in and out inside the epididymis until fluid is aspirated. If motile sperm are not obtained, PESA may be repeated at a different site (from the cauda to caput epididymis) until an adequate number of motile sperm is retrieved. These aspirations are usually performed in the corpus epididymis and then in the caput epididymis if needed, as aspirates from the cauda are often rich in poor-quality senescent spermatozoa, debris and macrophages. Because PESA is a blind procedure, multiple attempts may be needed before high-quality sperm are found. If PESA fails to enable the retrieval of motile sperm, testicular sperm retrieval can be attempted during the same operation.
Craft and Shrivastav, in 1994, first described the use of the percutaneous approach to retrieve sperm from the epididymis. Percutaneous retrievals are usually undertaken under local anesthesia only or in association with intravenous sedation. Percutaneous sperm retrieval can be either diagnostic or therapeutic. In the former, it is used to confirm the presence of viable spermatozoa prior to ICSI. In the latter, it is carried out at the same day of oocyte retrieval or at the day before.

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PICSI

Up to now, different methodologies to select sperm have been described in the hope of selecting a viable sperm without - or with a low level of - DNA damage. In 2005, it was the first group that reported the use of a hyaluronic acid (HA) assay as a method to select a sperm for use with ICSI (Pic.1). HA is a linear polysaccharide present in the extracellular matrix of cumulus oophorus around the oocyte that seems to play an important role in natural human fertilization. The use of this polysaccharide is based on the theory that hyaluronan is a major constituent of the cumulus oophorous matrix and may play a critical role in the selection of mature, functionally competent spermatozoa during in vivo fertilization. The head of a mature sperm holds a hyaluronan-specific ligand receptor that facilitates mature sperm to unite to hyaluronan. Comparatively, immature sperm do not unite. Competent, mature, biochemically active sperm unite to the hyaluronan where they can be selected by the embryologist and used for ICSI procedure.

The principles of this assay are:
1. expression of the protein HspA2, which indicates sperm maturation;
2. cytoplasmic membrane remodeling, which is responsible for the formation of sperm binding sites for the zona pellucida of oocytes and for HA binding sites. 

It was suggested that immature spermatozoa present low HspA2 levels, fail to undergo cytoplasmic membrane remodeling and consequently are unable to bind to HA.
This modus operandi mimics a focal step in the innate fertilization course of action, the binding of mature sperm to the oocyte complex. As a consequence, the opted spermatozoa are indispensable alike as one that would be successful and doing well in the natural reproductive progression. Nevertheless, this advancement does not imitate the genomic integrity of the spermatozoa and its aptitude to deliver the best paternal contribution to the zygote.
It was shown that binding to hyaluronic acid seems to be related to one or more conventional and one or more functional sperm tests, indicating that spermatozoa from patients with abnormal conventional semen parameters have a higher likelihood for multiple functional abnormalities. In addition, freezing and thawing seems not alter the HA-binding properties of the spermatozoa.
Some previous studies on sperm surface markers have demonstrated that HA-bound spermatozoa are mature and devoid of cytoplasmic retention, persistent histones, apoptotic markers and DNA fragmentation. In addition, a normal frequency of chromosomal aneuplodies, normal and normal nucleus morphology criteria have been correlated positively with HA-bound spermatozoa. In contrast to this hypothesis, some researchers found no correlation between the HA-binding assay (PICSI) and a low degree of DNA damage. The HA-bound spermatozoa did not differ from HA-unbound ones as to DNA fragmentation (19.6% versus 21.4%, respectively). Also it was reported no difference in the sperm morphology between HA-bound and HA-unbound spermatozoa. Such controversy may be due to the different HA binding methods used, the PICSI dish, sperm slow medium and also the sperm morphology criteria: normal nucleus morphology and normal spermatozoa morphology. Other important point to emphasize is the possible influence of the sperm preparation on the outcome of HA binding. It is well defined in literature that semen sample preparations improve motility and morphology. Besides, the kind of semen preparation could impact the final sample quality.

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Sperm donation

Sperm donation is the donation by a male (known as a sperm donor) of his sperm (known as donor sperm), principally for the purpose of inseminating a female who is not his sexual partner. Sperm donation is a form of third party reproduction including sperm donation, oocyte donation, embryo donation, surrogacy, or adoption. Number of births per donor sample will depend on the actual ART method used, the age and medical condition of the female bearing the child, and the quality of the embryos produced by fertilization. Donor sperm is more commonly used for artificial insemination (IUI or ICI) than for IVF treatments. This is because IVF treatments are usually required only when there is a problem with the female conceiving, or where there is a “male factor problem” involving the female's partner. Donor sperm is also used for IVF in surrogacy arrangements where an embryo may be created in an IVF procedure using donor sperm and this is then implanted in a surrogate. In a case where IVF treatments are employed using donor sperm, surplus embryos may be donated to other women or couples and used in embryo transfer procedures. 

On the other hand, insemination may also be achieved by a donor having sexual intercourse with a female for the sole purpose of initiating conception. This method is known as natural insemination.

The donation
Donor sperm and fertility treatments using donor sperm may be obtained at a sperm bank or fertility clinic. Here, the recipient may select donor sperm on the basis of the donor's characteristics, e.g. looks, personality, academic ability, race, and many other factors. Sperm banks or clinics may be subject to state or professional regulations, including restrictions on donor anonymity and the number of offspring that may be produced, and there may be other legal protections of the rights and responsibilities of both recipient and donor. Some sperm banks, either by choice or regulation, limit the amount of information available to potential recipients; a desire to obtain more information on donors is one reason why recipients may choose to use a known donor and/or private donation.

A sperm donor will usually donate sperm to a sperm bank under a contract, which typically specifies the period during which the donor will be required to produce sperm, which generally ranges from 6–24 months depending on the number of pregnancies which the sperm bank intends to produce from the donor. Donors may or may not be paid for their samples, according to local laws and agreed arrangements. Even in unpaid arrangements, expenses are often reimbursed. Depending on local law and on private arrangements, men may donate anonymously or agree to provide identifying information to their offspring in the future. Private donations facilitated by an agency often use a "directed" donor, when a male directs that his sperm is to be used by a specific person. Non-anonymous donors are also called known donors, open donors or identity disclosure donors.

Donor selection
A sperm donate must generally meet specific requirements regarding age (most often up to 40) and medical history. Potential donors are typically screened for genetic diseases, chromosomal abnormalities and sexually transmitted infections that may be transmitted through sperm. The donor's sperm must also withstand the freezing and thawing process necessary to store and quarantine the sperm. Samples are stored for at least 6 months after which the donor will be re-tested for sexually transmitted infections. This is to ensure no new infections have been acquired or have developed during the period of donation. If the result is negative, the sperm samples can be released from quarantine and used in treatments.

Screening includes:

  • Taking a medical history of the donor, his children, siblings, parents, and grandparents etc. for three to four generations back. This is often done in conjunction with the patient’s family doctor.
  • HIV risk assessment interview, asking about sexual activity and any past drug use.
  • Blood tests and urine tests for infectious diseases, such as: HIV-1/2, HTLV-1/2, Hepatitis B and C, Syphilis, Gonorrhea, Chlamydia, Cytomegalovirus (CMV), not all clinics test for this.
  • Blood and urine tests for blood typing and general health indicators: ABO/Rh typing, CBC, liver panel and urinalysis
  • Complete physical examination including careful examination of the penis, scrotum and testicles.
  • Genetic testing for carrier traits, for example: Cystic Fibrosis, Sickle-cell disease, Thalassemia, other hemoglobin-related blood disorders.
  • General health
  • Semen analysis for: sperm count, morphology, motility, acrosome activity may also be tested

Preparing the samples
A sperm donor is usually advised not to ejaculate for two to three days before providing the sample, to increase sperm count and to maximize the conception rate. A sperm donor produces and collects sperm by masturbation or during sexual intercourse with the use of a collection condom.

Sperm banks and clinics usually "wash" the sperm sample to extract sperm from the rest of the material in the semen. A cryoprotectant semen extender is added if the sperm is to be placed in frozen storage in liquid nitrogen, and the sample is then frozen in a number of vials or straws. One sample will be divided into 1-20 vials or straws depending on the quantity of the ejaculate and whether the sample is washed or unwashed. Following the necessary quarantine period, the samples are thawed and used to inseminate women through artificial insemination or other ART treatments. Unwashed samples are used for ICI treatments, and washed samples are used in IUI and IVF procedures.

Anonymity
Anonymous sperm donation occurs where the child and/or receiving couple will never learn the identity of the donor, and non-anonymous when they will. Non-anonymous sperm donors are, to a substantially higher degree, driven by altruistic motives for their donations.

Even with anonymous donation, some information about the donor may be released to the female/couple at the time of treatment. Limited donor information includes height, weight, eye, skin and hair color. In Sweden, this is all the information a receiver gets. In the US, on the other hand, additional information may be given, such as a comprehensive biography and sound/video samples.

Information made available by a sperm bank will usually include the race, height, weight, blood group, health, and eye color of the donor. Sometimes information about his age, family history and educational achievements will also be given.

Different factors motivate individuals to seek sperm from outside their home state. For example, some jurisdictions do not allow unmarried women to receive donor sperm. Jurisdictional regulatory choices as well as cultural factors that discourage sperm donation have also led to international fertility tourism and sperm markets.

Legal aspects
A sperm donor is generally not intended to be the legal or de jure father of a child produced from his sperm. Depending on the jurisdiction and its laws, he may or may not later be eligible to seek parental rights or be held responsible for parental obligations. Generally, a male who provides sperm as a sperm donor gives up all legal and other rights over the biological children produced from his sperm. However, in private arrangements, some degree of co-parenting may be agreed, although the enforceability of those agreements varies by jurisdiction.

Laws prohibits sperm donation in several countries: Algeria, Bahrain, Costa Rica, Egypt, Hong Kong, Jordan, Lebanon, Lithuania, Libya, Maldives, Oman, Pakistan, Philippines, Qatar, Saudi Arabia, Syria, Tajikistan, Tunisia, Turkey, UnitedArab Emirates, and Yemen. 

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Sperm freezing

Sperm freezing is a procedure used to safe and storage sperm cell in a liquid nitrogen for a relatively long period of time (up to 23 years with proven fertilization ability retained). The process is also called as cryopreservation of sperm and it may help to accomplish fertilization while specific medical conditions leading to male infertility are present. Therefore, sperm cryopreservation is an important component of fertility management and much of its successful application seems to affect the reproductive outcome of assisted reproduction technologies (ART). Long-term storage of a sperm cell can be achieved by lowering the temperature of the cell below zero Celsius degrees, so its metabolic activity is reduced to minimum until abiosis (the cessation of all living processes) is obtained. To protect the cell from harm done by the freeze in most cases is necessary to use specific chemical substances called cryoprotectants.

Cryoprotectants

Generally, cryoprotectants can be described as chemical substances soluble in the water. Their purpose is to lower the freezing temperature of solutions and to prevent damage done by ice crystallization during freezing process. The cryoprotectants can be divided into two groups. The permeating cryoprotectants are chemical substances of low molecular weight with high capacity of penetration into a cell. The non-permeating cryoprotectants does not enter into a cell, they rather create a hyperosmotic ambient around a cell to drain the water from the cell. Usually these two types of cryoprotectants are combined. There are four main well-known cryoprotectants: glycerol, ethylene glycol, dimethyl sulphoxide, and 1,2-propanediol. Glycerol is the permeating cryoprotectant most widely used for human sperm acting on several aspects of a sperm cell such as: the membrane structure, permeability and stability of the lipid bilayer, the association of surface proteins and the cellular metabolism. Its employment gives an unfavorable outcome on membrane and acrosome structure, although allowing the freezing of poor quality sperm.

Sperm freezing methods

Basically there are two main conventional methods to perform sperm freezing distinguished by freezing speed/rate, one is called slow freezing and the second one is called rapid freezing or the vitrification. The cryopreservation methods can by also divided by the storage medium of sperm cells. Most common storage medium is a straw (Pic. 1) used in straw method, which is a default technique for sperm freezing. With some specific modification of a straw, straw method can be also used in vitrification. Lately some new techniques/storage mediums of semen cryopreservation, were developed offering a new possibilities of usage. For example, there is the cryoloop method or a sperm cryopreservation method of freezing single sperm cell using zona pellucida. A very specific method of freezing a sperm without cryoprotectants is a freezing-drying method, but it is still an empiric method under research.

Slow Freezing

This method consists of progressive sperm cooling over a period of 2 – 4 hours in up to three steps. It can be performed manually or automatically using a device called semiprogrammable freezer. While the temperature is being decreased a cryoprotectant is gradually added to the semen. First the sample of semen is cooled to 5°C with a low cooling rate (0,5 – 1°C/min), in the next step the cooling rate is increased (1-10°C) until the temperature of -80°C is reached. After that the sample is ready to be plunged into liquid nitrogen at -196°C. Since the procedure is quiet complicated to perform manually, the programmable freezer is often used as it can reproduce exact desired conditions of the freezing process.

Vitrification

First thing, semen is mixed with cryoprotectant of same volume as the sample. After that the mixture is filled into straws and left to incubate at 4°C for 10 minutes. Then straws can be put in direct contact with nitrogen vapours in horizontal position for about 10 minutes at -80°C. Right after they are immersed into the liquid nitrogen at -196°C. This procedure has some disadvantages such as low reproducibility, since the drop of temperature in the middle step is hard to control meaning that freezing temperatures can vary relatively a lot.

Straw method

It is the most commonly used method for cryopreservation. The sperm is held in a small straw that is chemically inert (does not undergo any chemical reactions), biocompatible with sperm sample and resistant to ultra-low temperatures. The volume of classical straws used in cryopreservation is 0,25 ml or 0,5 ml. Specially modified mini-straws of about 1 μl volume are used in new technique of sperm cryopreservation called open pulled straw method. The straw method represents sterile, simple and convenient method of cryopreservation. The disadvantage may be the fact that some sperm cells tend to adhere to the vessel (straw). This fact implicates that straw method shouldn’t be used to storage severely impaired specimens of sperm.

Cryopreservation of sperm using the cryoloop method

Another way to freeze very low numbers of sperm cells is to use a very small loop (0,5-0,7mm), that can hold a thin layer of cryoprotectant. It is possible to mount a single sperm cell on the film in the loop by micromanipulation. The cryoloop method has excellent recovery rates of frozen sperm. With a micropipette, sperm cells are loaded onto the film in the loop. The cryoloop is screwed onto the specific kind of vial (cryovial; Pic. 2), and the vial is exposed to liquid nitrogen vapor for up to 30 minutes before being plunged into liquid nitrogen for storage. Same way as the method mentioned above, it takes a lot of time and intensive labour to perform the procedure. It also requires extensive training and experience so it is no wonder that up today it is rarely practiced.

Cryopreservation of a single human sperm using a zona pellucida

This technique consists of the cryopreservation of individual spermatozoa inside an empty zona pellucida. This method requires an intensive labour and a lot of time to perform but on the other hand it offers an opportunity to retrieve and store sperm in extreme cases of male factor infertility. To gain an empty zona pellucida an oocyte must be processed in specific way. The oocyte is exposed to an enzyme called hyaluronidase to remove protective cellular layers, cumulus and corona radiata cells. This stripped oocyte is held by a holding pipette (Pic. 3) so two small holes can be drilled into it. The ooplasm filling the oocyte is aspirated by suction leaving the zona pellucida empty of all its contents. Once the zona pellucida is obtained, healthy sperm cells are selected for freezing. Any healthy sperm cell found in sample is transferred into specific medium that immobilize the cell. After that one or more (up to 15) sperm cells can be inserted into empty zona pellucida using a special kind of very thin needle called intracytoplasmic sperm injection (ICSI) needle (Pic. 3). In the next step the zona pellucida is inserted into a cryoprotectant and each single piece is put into individual straw. The heat-sealed straws are then exposed to liquid nitrogen vapour for about two hours and storage in liquid nitrogen. 

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Standard IVF

In vitro fertilization (IVF) is a process by which an egg is fertilised by sperm outside the body: in vitro . The process involves monitoring and stimulating a woman's ovulatory process, removing an ovum or ova (egg or eggs) from the woman's ovaries and letting sperm fertilise them in a liquid in a laboratory. The fertilised egg (zygote) is cultured for 2–6 days in a growth medium and is then implanted in the same or another woman's uterus, with the intention of establishing a successful pregnancy.

IVF techniques can be used in different types of situations. It is a technique of assisted reproductive technology for treatment of infertility. IVF techniques are also employed in gestational surrogacy, in which case the fertilised egg is implanted into a surrogate's uterus, and the resulting child is genetically unrelated to the surrogate. In some situations, donated eggs or sperms may be used. Some countries ban or otherwise regulate the availability of IVF treatment, giving raise to fertility tourism. Restrictions on availability of IVF include to single females, to lesbians and to surrogacy arrangements. Due to the costs of the procedure, IVF is mostly attempted only after less expensive options have failed.

The first successful birth of a "test tube baby", Louise Brown, occurred in 1978. Louise Brown was born as a result of natural cycle IVF where no stimulation was made. Robert G. Edwards, the physiologist who developed the treatment, was awarded the Nobel Prize in Physiology or Medicine in 2010. With egg donation and IVF, women who are past their reproductive years or menopause can still become pregnant. Adriana Iliescu held the record as the oldest woman to give birth using IVF and donated egg, when she gave birth in 2004 at the age of 66, a record passed in 2006.

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TESE

Testicular sperm extraction (TESE) is the process of removing a small portion of tissue from the testicle under local anesthesia and extracting the few viable sperm cells present in that tissue for intracytoplasmic sperm injection (ICSI).

The testicular sperm extraction process is recommended to men who cannot produce sperm by ejaculation due to azoospermia, such as that caused by primary testicular failure, congenital absence of the vas deferens or non-reconstructed vasectomy.

The introduction of the technique of intracytoplasmic sperm injection to achieve fertilization, especially using surgically retrieved testicular or epididymal sperm from men with obstructive or non-obstructive azoospermia, has revolutionized the field of assisted reproduction. Testicular sperm retrieval techniques associated with intracytoplasmic sperm injection have reduced the need for donor sperm and given many azoospermic men the chance to become biological fathers.

The extraction of the testicular parenchyma for sperm search and isolation was first described in 1995. For conventional TESE, a standard open surgical biopsy technique is used to remove the testicular parenchyma without the aid of optical magnification. This procedure is usually carried out without delivering the testis. Briefly, a 2-cm transverse incision is made through the anterior scrotal skin, dartos and tunica vaginalis. A small self-retaining retractor can be used to ensure proper exposure of the tunica albuginea. A 1-cm incision is made in the albuginea, and gentle pressure is applied to the testis to aid the extrusion of the testicular parenchyma. A fragment of approximately 5x5 mm is excised with sharp scissors and placed in sperm culture media. Single or multiple specimens can be extracted from the same incision. Alternatively, individual albuginea incisions can be made in the upper, middle and lower testicular poles in an organized manner for the sampling of different areas. The testicular specimens are sent to the laboratory for processing and immediate microscopic examination. The tunica albuginea is closed with a running, non-absorbable suture.


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How can Hydrocele testis affect fertility

Scientists believe that main impact of hydrocele on fertility is when hydrocele oppresses testes, which affects blood circulation and then affects process of spermatogenesis. The testicular blood supply could be affected by an extremely large hydrocele. The result is testicular ischemia which could leads to testicular atrophy and subsequent impairment of fertility. Also when hydrocele is too large, it leads to enveloping of penile scrotal skin and it does not conducive to the normal sexual intercourse.

Pic. 1: Tunica vaginalis
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