Recent Advances

 

For some patients with severe testicular failure, sometimes, it is not possible to find any sperm at all as even in spite of taking multiple testicular biopsies. In such patients pregnancies have been achieved even by injecting round spermatids (immature precursor cells from which the sperm are formed) from the testis into the egg. This is now an area of intense research all over the world, but the results have been disappointing so far. Other labs are trying to develop methods of in vitro spermatogenesis, in order to mature the spermatids in vitro.

ICSI: In vitro fertilization using introcitoplasmatic microinjection of a spermatozoid

In Vitro fertilization with intracytoplasmic sperm injection (VIF-ICSI). This procedure is called ICSI from English and stands for Intracytoplasmic Sperm Injection. It consists of the insemination of each egg via the injection of a single sperm inside its cytoplasm.
ICSI is carried out together with IVF. The steps before and after the insemination are exactly the same (ovarian stimulation, egg collection and embryo transfer), the only difference is the insemination procedure. Employing this technique less sperm is needed, as only one per egg is required, whereas with IVF 50.000 to 100.000 sperm are necessary. There have been remarkable advances in the treatment of male sterility since the application of ICSI.

ICSI procedure

First step (ovulation stimulation and monitoring) 
In order to obtain the eggs the ovulation has to be stimulated with fertility drugs. The follicle stimulating hormone (FSH) will be administered to the woman as well as GnRh antagonists. A strict control of the menstrual cycle monitoring the Estradiol (a hormone) level in the blood, and an ultrasound of the follicular development in the ovaries is carried out. When the hormone levels and the number and size of the follicles are sufficient, their maturity is triggered by use of a hormone called LH (luteinizing hormone).

Second step (IVF) 
36 hours after administrating the LH, the gynaecologist collects the eggs. This procedure consists of puncturing the follicles by means of a vaginal ultrasound and liquid aspiration on the inside. This liquid is taken to the laboratory where the biologist separates the eggs from the liquid. The eggs are cultivated in culture medium while the sperm is prepared (as with Artificial Insemination). Later on the biologist carries out the insemination which consists of injecting a single sperm into an egg.

With ICSI a sperm has to be selected and injected into the cytoplasm of an egg. A micromanipulator connected to a microscope is used in order to carry out this procedure. The insemination has to be done with all eggs available. The following day a check is made on how many eggs have been fertilized.

Third step (transfer) 
Two or three days later the fertilized eggs (or zygotes) start dividing and become embryos which are ready to be transferred to the uterus. Two or three embryos are selected for the transfer. They are introduced in the uterus together with a small quantity of culture medium in a fine catheter. Under an abdominal ultrasound control, the gynaecologist places the catheter up to the end of the uterus where the embryos are deposited.

Fourth step (cryopreservation) 
The non transferred embryos are frozen in liquid nitrogen (cryopreservation) and stored correctly identified in the embryo bank of the laboratory. These embryos can be used in future cycles if pregnancy does not occur at the first attempt. Obviously, it simplifies the process and makes it cheaper, although the pregnancy rates decrease.

Indications

Insemination using ICSI began in 1992 and was first used in cases of male sterility due to unknown causes or when IVF was not viable. For example, when there is a very small number of spermatozoa in sperm (severe oligozoospermia) or when sperm motility is very low (severe astenozoospermia).

Special cases

When there are no motile sperm in the ejaculate they can be obtained via epididymis aspiration or via a biopsy of the testicular tissue. This technique is used when the man wants to have a child had a vasectomy (a sufficient semen sample cannot be obtained by means of a deferent duct puncture) or in special cases such as malformation of deferent ducts, retrograde ejaculation, paraplegia, tetraplegia and impotence. The possibilities for the fertilization and development of normal embryos with testicular sperm are identical as with ejaculated sperm, and the process is exactly the same.

PGD: Genetical diagnosis prior to implant

When there is a history of genetic or chromosomal transmissible family illnesses, it can be of great help to know if an illness is present in the precursor cells (eggs) or in the embryo before the woman becomes pregnant (before the embryo is transferred to the uterus). A technique called Pre-Implantation Genetic Diagnosis is used in cases when it is possible. PGD has to be carried out in conjunction with IVF because to attain the genetic material to be studied, the techniques of the Assisted Reproduction laboratory are needed. The diagnosis can be made in two different ways:

Pre-Implantation Genetic Diagnosis with embryos
The embryos are studied before being transferred into the uterus. The procedure takes place in the laboratory after IVF and when the embryos are at the 4-8 cell stage. This technique allows for the selection and transfer into the uterus of only those embryos which have been diagnosed as normal. Having been informed and after having accepted to be included on an IVF program, the patient undergoes a hormonal induction for follicular stimulation. All the collected mature eggs are inseminated to obtain embryos. After 24 hours a biopsy is performed on all the embryos which meet the necessary requirements, and one of their cells is taken with a micromanipulator. The embryos are identified and kept in an incubator while the study continues. The obtained cells are prepared under special conditions and the genetic material is examined. This examination allows the biologist to find out if the embryo they are taken from is affected or not by the illness. Once the condition of each embryo is known, those which are free of the illness are selected and transferred into the woman’s uterus. The affected embryos have to be destroyed.

Preconception Diagnosis with eggs
In the case of PGD with embryos, the couple has to be aware of the fact that in order to have an illness-free pregnancy, only healthy embryos are selected which necessarily implies that the affected ones will be disposed of. 

Therefore, one step forward in diagnosing these genetic or chromosomal pathologies even before the embryo is formed is the Preconception Diagnosis using female gametes (eggs). In order to do so, a specific egg cell called second polar body is used. This technique cannot be carried out on a spermatozoon and its application is limited to the inherited pathology of maternal origin. 

The couples who may need PGD are those where the woman is a carrier of a chromosomal reorganization, mainly reciprocal and Robersonian translocations, and the male partner has a normal karyotype. In 94.1% of cases with this technique a polar body with a good chromosomal morphology can be obtained.

All the mature eggs are processed for their genetic study. A biopsy is performed on the polar body by means of a micromanipulator making a tiny incision in the zona pellucida surrounding it. The biopsied eggs are identified and kept in an incubator. To ensure the result, it is important to try the reagents called probes in the lymphocyte preparation of the woman (blood cells) before making a Preconception Diagnosis. 

Once the normal eggs are identified they are inseminated via ICSI (intracytoplasmic sperm injection). Therefore, all the resulting embryos are suitable to be transferred and none of them has to be destroyed. The unused ones can be frozen and exploited in future possible attempts. 

Artificial insemination by donor (AID)

This treatment consists of placing the sperm issued from a semen bank inside the uterus or the cervical channel. The woman’s menstrual cycle has to be monitored in order to detect when ovulation takes place. In this case the sperm comes from an anonymous donor and is used when for irreversible reasons the partner’s sperm cannot be used (such as in some cases of cancer or azoospermia) or when the woman does not have a male partner.

Artificial Insemination by a donor is a simple and effective procedure as the semen sample is of optimum quality and quantity. The donor is a healthy male who has been previously checked in order to rule out any illness.

AID procedure

First step (ovulation stimulation and monitoring).
To stimulate the ovulation a follicle stimulating hormone (FSH) is used. The cycle is controlled by ultrasound in order to check the follicular development in the ovaries. When the number and size of the follicles is sufficient (with this technique only one has to be developed) its maturity is triggered by use of another hormone called LH ( a luteinizing hormone).

Second step (semen sample collection).
The sperm is collected after carrying out a complete study of the donor’s health in order to be sure of the good quality of the sperm and to rule out any illness. The potential donors are placed under a series of tests and analyses before being accepted: sperm, blood, urine, general health, sexual transmitted diseases and a psychological examination. All the donors have to be over 18 and sign a consent form agreeing for their sperm to be used in this technique as well as accepting the anonymity therein. The donor sperm is frozen before being used.

Third step (insemination).
The artificial insemination with donor sperm is carried out in exactly the same way as the artificial insemination with the partner’s sperm but with a cryopreserved semen sample from a semen bank. The sperm is placed into a small cannula which is then introduced into the uterus on the precise day when ovulation is expected. It is a simple process, generally painless and very similar to any other routine procedure in a gynecological clinic.

Indications

Before the arrival in 1992 of the intracytoplasmic sperm injection (ICSI), which allows for the injection of a single sperm into each oocyte, this technique was used in cases of severe sperm pathologies. However, these days the regulations have been restricted where there are cases of hereditary illness, testicular failure (testicles cannot produce sperm) or women who have no male partner.

(IVF) In vitro fertilization using eggs and sperm from the couple

IVF with patient’s eggs and donor sperm 
This technique consists of fertilizing the woman’s eggs with the sperm of an anonymous donor in the laboratory. The fertilized eggs will become embryos and will be transferred to the uterus of the patient. The semen sample used for the treatment is of optimum quality and quantity. The donor is a healthy male whose sperm was previously checked to rule out any illness.

IVF procedure 

First step (ovulation stimulation and monitoring)
In order to obtain the eggs the ovulation has to be stimulated with fertility drugs. The follicle stimulating hormone will be administered to the patient as well as GnRh antagonists. A strict control of the menstrual cycle monitoring the Estradiol (a hormone) level in the blood, and an ultrasound check-up of the follicular development in the ovaries is carried out. When the hormone levels and the number and size of the follicles are sufficient, their maturity is triggered by use of a hormone called LH (luteinizing hormone).

Second step (semen sample collection) 
The sperm is collected after carrying out a complete study of the donor’s health in order to be sure of the good quality of the sperm and to rule out any illness. The potential donors are placed under a series of tests and analysis before being accepted: sperm, blood, urine, general health, sexual transmitted diseases and a psychological examination. All the donors have to be over 18 and sign a consent form agreeing for their sperm to be used in this technique as well as accepting the anonymity therein. The donor sperm is frozen before being used.

Third step (fertilization and embryo transfer) 
As with partners’ gametes IVF the eggs are cultivated in a culture medium while the sperm is prepared (as with Artificial Insemination). Later on the biologist carries out the insemination which consists of placing the sperm (between 50.000 and 100.000 motile spermatozoa) in the culture medium with the eggs. The following day a check is made on how many eggs have been fertilized and the embryos are transferred to the recipient’s uterus two or three days after the egg collection. Two or three embryos are selected for the transfer. They are introduced in the uterus together with a small quantity of culture medium in a fine catheter. Under an abdominal ultrasound control, the gynaecologist places the catheter up to the end of the uterus where the embryos are deposited.

Fourth step (cryopreservation) 
The non transferred embryos are frozen in liquid nitrogen (cryopreservation) and stored correctly identified in the embryo bank of the laboratory. These embryos can be used in future cycles if pregnancy does not occur at the first attempt. Obviously, it simplifies the process and makes it cheaper, although the pregnancy rates decrease.

Indications

This technique is applied when the woman decides to become a single mother, when her partner is another woman or when several previous IVF attempts with the partner’s sperm were unsuccessful. It is also recommended in the case of azoospermia (the male partner does not produce sperm) or, less frequent, when the male partner is a carrier of a chromosomal anomaly and out of ethical reasons the couple do not consider undergoing the pre-implantation genetic diagnosis (chromosomal study of the embryo before it is transferred to the woman’s uterus). IVF with donor sperm can be carried out in the case of sperm anomalies such as oligozoospermia (low sperm concentration), astenozoospermia (low motility) or teratozoospermia (few sperm with satisfactory morphology), and also if the previous IVF-ICSI (IVF with intracytoplasmic sperm injection) attempts were not successful.
(IVF) In Vitro fertilization using eggs from a donor and sperm from the male partner

It consists of fertilizing the donor eggs with the sperm of the recipient woman’s partner in the laboratory. The embryos obtained as a result of this insemination are transferred to the woman’s uterus. The patient undergoes medical treatment which prepares her uterus to receive the transferred embryos.

According to law the egg donation is anonymous (the donor and the recipient cannot know each other neither in the present nor in the future) and it must be done under their own free will (commercialization of gametes and embryos is banned by law. However, donors receive an economic reward for their commitment and the time they invest in the donation). The law states that donors should be between 18 and 35 years old.

IVF with donor eggs procedure

First step (donor selection) 
The donors are selected according to very strict medical criteria. First of all they undergo a clinical examination to make sure that their ovaries are healthy and they have no transmittable pathologies (infectious or genetic). Before donating her eggs, the donor has a blood test in order to rule out the risk of any transmittable infectious diseases (hepatitis B and C, HIV, cytomegalovirus, gonococcus, chlamydia and syphilis) and in order to determine her blood group, basal hormone level and karyotype. A cytology test is carried out as well as a neck of the uterus smear and a pelvic ultrasound scan.

These results are studied by a doctor who also verifies by means of a questionnaire the absence of any genetic family illnesses while the psychologist evaluates the donor’s mental health. Finally, the donor signs a consent form according to the law in which she agrees to donate her eggs to a couple who want to have a child and whose identity she will never try to find out. 

Second step (donor stimulation) 
The donors have to undergo a two-week ovarian stimulation treatment, which consists of administering subcutaneous hormonal injections and whose development is controlled by means of ultrasound checks and blood tests (five respectively). The egg collection is carried out by way of puncturing under sedation. 

Third step (fertilization and embryo transfer) 
As with partners’ gametes IVF, the eggs are cultivated in culture medium while the sperm is prepared (as with Artificial Insemination). Later on the biologist carries out the insemination which consists of placing the sperm (between 50.000 and 100.000 motile spermatozoa) in the culture medium with the eggs. The following day a check is made on how many eggs have been fertilized and the embryos are transferred to the recipient woman’s uterus two or three days after the egg collection. Two or three embryos are selected for the transfer. They are introduced in the uterus together with a small quantity of culture medium in a fine catheter. Under an abdominal ultrasound control, the gynaecologist places the catheter up to the end of the uterus where the embryos are deposited.

Fourth step (cryopreservation) 
The non transferred embryos are frozen in liquid nitrogen (cryopreservation) and stored correctly identified in the embryo bank of the laboratory. These embryos can be used in future cycles if pregnancy does not occur at the first attempt. Obviously, it simplifies the process and makes it cheaper, although the pregnancy rates decrease.

Indications

Women with ovarian failure. These are women whose ovaries are not able to produce eggs any more. It can occur for many reasons:
Premature menopause: premature ovulation stoppage (before 40 years old). This situation, which affects approximately half of the egg recipients, has different causes:

  • Indiopathic or unknown (the most common) – Genetic
  • Immunological or autoimmunologic disorders
  • Latrogenic, meaning consequences of medical procedure such as ovarian surgery, chemotherapy or radiotherapy
  • Natural menopause: In this case the egg donation has to be evaluated according to the age and the general health of the woman.

Women without ovarian failure. These are women whose ovaries are able to produce eggs but they are of insufficient quality, or women who are carriers of genetic illnesses or chromosomal anomalies. The egg donation is recommended in the following cases: 

  • Repeated IVF failures. In some cases the pregnancy does not occur in “conventional” Assisted Reproduction Techniques. 

There can be various reasons:

  • Low response to the ovarian stimulation
  • Repeated failure due to poor egg quality
  • Repeated implantation failure of apparently normal embryos
  • Hidden ovarian failure. These are women who have regular periods but suffer from hormonal alterations which indicate an ovarian failure in its initial stage.
  • Women over 43 with a normal menstrual cycle. Fertility has a direct connection to age and therefore in some cases women older than 43 are recommended to undergo IVF treatment with donor eggs. Moreover, at this age miscarriages and foetus chromosomal alterations rates increase.

(IVF) In Vitro fertilization using eggs from the female partner and sperm from a donor

This technique consists of fertilizing the woman’s eggs with the sperm of an anonymous donor in the laboratory. The fertilized eggs will become embryos and will be transferred to the uterus of the patient. The semen sample used for the treatment is of optimum quality and quantity. The donor is a healthy male whose sperm was previously checked to rule out any illness.

IVF procedure

First step (ovulation stimulation and monitoring)
In order to obtain the eggs the ovulation has to be stimulated with fertility drugs. The follicle stimulating hormone will be administered to the patient as well as GnRh antagonists. A strict control of the menstrual cycle monitoring the Estradiol (a hormone) level in the blood, and an ultrasound check-up of the follicular development in the ovaries is carried out. When the hormone levels and the number and size of the follicles are sufficient, their maturity is triggered by use of a hormone called LH (luteinizing hormone).

Second step (semen sample collection). 
The sperm is collected after carrying out a complete study of the donor’s health in order to be sure of the good quality of the sperm and to rule out any illness. The potential donors are placed under a series of tests and analysis before being accepted: sperm, blood, urine, general health, sexual transmitted diseases and a psychological examination. All the donors have to be over 18 and sign a consent form agreeing for their sperm to be used in this technique as well as accepting the anonymity therein. The donor sperm is frozen before being used.

Third step (fertilization and embryo transfer) 
As with partners’ gametes IVF the eggs are cultivated in a culture medium while the sperm is prepared (as with Artificial Insemination). Later on the biologist carries out the insemination which consists of placing the sperm (between 50.000 and 100.000 motile spermatozoa) in the culture medium with the eggs. The following day a check is made on how many eggs have been fertilized and the embryos are transferred to the recipient’s uterus two or three days after the egg collection. Two or three embryos are selected for the transfer. They are introduced in the uterus together with a small quantity of culture medium in a fine catheter. Under an abdominal ultrasound control, the gynaecologist places the catheter up to the end of the uterus where the embryos are deposited.

Fourth step (cryopreservation)
The non transferred embryos are frozen in liquid nitrogen (cryopreservation) and stored correctly identified in the embryo bank of the laboratory. These embryos can be used in future cycles if pregnancy does not occur at the first attempt. Obviously, it simplifies the process and makes it cheaper, although the pregnancy rates decrease.

Indications

This technique is applied when the woman decides to become a single mother, when her partner is another woman or when several previous IVF attempts with the partner’s sperm were unsuccessful. It is also recommended in the case of azoospermia (the male partner does not produce sperm) or, less frequent, when the male partner is a carrier of a chromosomal anomaly and out of ethical reasons the couple do not consider undergoing the pre-implantation genetic diagnosis (chromosomal study of the embryo before it is transferred to the woman’s uterus).

IVF with donor sperm can be carried out in the case of sperm anomalies such as oligozoospermia (low sperm concentration), astenozoospermia (low motility) or teratozoospermia (few sperm with satisfactory morphology), and also if the previous IVF-ICSI (IVF with intracytoplasmic sperm injection) attempts were not successful.

(IVF) In vitro fertilization using eggs from a donor and sperm from the sperm bank

This Assisted Reproduction technique consists of fertilizing an anonymous donor’s eggs with an anonymous donor’s sperm in the laboratory. The embryos obtained as a result of this procedure will be transferred to the uterus of the recipient woman. 

IVF with donor eggs and sperm procedure

First step (donor selection) 
The donors are selected according to very strict medical criteria. First of all they undergo a clinical examination to make sure that their ovaries are healthy and they have no transmittable pathologies (infectious or genetic). Before donating her eggs, the donor has a blood test in order to rule out the risk of any transmittable infectious diseases (hepatitis B and C, HIV, cytomegalovirus, gonococcus, Chlamydia and syphilis) and in order to determine her blood group, basal hormonal level and karyotype. A cytology is carried out as well as a neck of the uterus smear and a pelvic ultrasound scan.

These results are studied by a doctor who also verifies by means of a questionnaire the absence of any genetic family illnesses while the psychologist evaluates the donor’s mental health. Finally, the donor signs a consent form according to the law in which she agrees to donate her eggs to a couple who want to have a child and whose identity she will never try to find out.

Second step (donor stimulation) 
The donors have to undergo a two-week ovarian stimulation treatment, which consists of administering subcutaneous hormonal injections and whose development is controlled by means of ultrasound checks and blood tests (five respectively). The egg collection is carried out by means of a puncture under sedation. 

Third step (semen sample collection)
The sperm is collected after carrying out a complete study of the donor’s health in order to be sure of the good quality of the sperm and to rule out any illness. The potential donors are placed under a series of tests and analyses before being accepted: sperm, blood, urine, general health, sexual transmitted diseases and a psychological examination. All the donors have to be over 18 and sign a consent form agreeing for their sperm to be used in this technique as well as accepting the anonymity therein. The donor sperm is frozen before being used.

Fourth step (fertilization and embryo transfer)
As with partners’ gametes IVF, the eggs are cultivated in a culture medium while the sperm is prepared (as with Artificial Insemination). Later on the biologist carries out the insemination which consists of placing the sperm (between 50.000 and 100.000 motile spermatozoa) in the culture medium with the eggs. The following day a check is made on how many eggs have been fertilized and the embryos are transferred to the recipient’s uterus two or three days after the egg collection. Two or three embryos are selected for the transfer. They are introduced in the uterus together with a small quantity of culture medium in a fine catheter. Under an abdominal ultrasound control, the gynaecologist places the catheter up to the end of the uterus where the embryos are deposited.

Fifth step (cryopreservation) 
The non transferred embryos are frozen in liquid nitrogen (cryopreservation) and stored correctly identified in the embryo bank of the laboratory. These embryos can be used in future cycles if pregnancy does not occur at the first attempt. Obviously, it simplifies the process and makes it cheaper, although the pregnancy rates decrease.

Indications

This technique is carried out when women present ovarian function anomalies both due to their age and unknown reasons. Donor sperm is needed because the woman has decided to become a single parent, because her partner is another woman or because her male partner suffers from azoospermia (absence of sperm) or other sperm anomalies