IVF in General

In vitro fertilisation

As known, women ovulate once a month; at this time, an egg is released from the ovary and enters the fallopian tube, where it stays for 24-36 hours. Following intercourse, the ovulated egg in the fallopian tube will meet spermatozoa, one of which will “unite” with the egg and fertilize it. The now fertilized egg will leave the fallopian tube within 2-3 days to travel into the uterus, where it is implanted in the uterine lining and grows into an embryo.

If pregnancy is not achieved, the uterine lining (endometrium) is shed as monthly menses and a new ovulation cycle begins.

Ultrasound screening of ovarian follicular development and determination of the day of ovulation may help couples trying to conceive. The screening is performed by a transvaginal ultrasound and no longer requires a full bladder.

If ovulation is not regular, drugs can be given at the beginning of the cycle to stimulate it, which may also induce the development of multiple ovarian follicles. The follicle is a small fluid-filled pouch in which the egg grows and matures.

The production of multiple eggs increases the chance of conception per cycle, and it also increases the odds of a multiple pregnancy (twins, triplets, etc.).

In such cases, the type of treatment, dosage and combination of drugs are tailored to cover the needs of each couple. The doctors will discuss your problem in detail and the recommended treatment, and will answer all your questions.

Read IVF Treatments in detail.


It is called the inability of a couple to fertilize after at least one year of unprotected sexual intercourse.

Infertility may be caused by:

  • Ovulation disorders (15-20%)
  • Abnormalities of the cervix (10%)
  • Disorders of the fallopian tubes and ovaries (30%)
  • Disorders of the uterus and endometrium (5%)
  • Disorders of the male sperm (30-50%)
  • 10-20% despite the thorough testing, no cause is found (unexplained infertility)

For the investigation of infertility many tests are recommended: blood tests, uterine and ovarian ultrasound, hysterosalpingography, diagnostic laparoscopy for woman and semen analysis for the man. Depending on the findings, medication or methods of assisted reproduction are recommended (stimulate ovulation, intrauterine insemination IUI, IVF IVF etc.) with very good results in most cases. Infertility is a unique nosologic entity because it concerns a pair rather than a person.




What does infertility mean?

In earlier years, infertility was defined as the failure to conceive after two years of unprotected sexual intercourse. After two years it is expected that about 95% of couples will achieve conception, while in one year the figure is already 90%. We know that the probability of conception per month is higher in the first three months (25%) and reduced to about 10% after. Therefore in six months the majority of fertile couples are expected to achieve conception. The couples that have not achieved in six months, approximately 50% will not achieve either the next 18 months. For these reasons, the infertile couples are considered to be those that have not achieved conception after 12 months of try, until the age of 35 years. In women older than 35 years, the period is six months.


Is infertility common?

10% of women will receive medical services under infertility. It is known that the most important factor is the age of the mother. Fertility remains high throughout the decade of the 20 to 30 and then decreases gradually and rapidly after 37-38 years.




What are the causes? It is a woman issue?

The causes of infertility remain stable over the last 25 years. A large study of global health organization indicated that female infertility was about 35%, male infertility about 10% and about 35% in combination. The survey showed that a very large proportion, almost half of cases, involved the male factor. Basic rule to investigate the infertile couple is that both man and woman should be evaluated simultaneously.


Is it easy to find what's wrong?

Some causes of infertility are easily recognizable, such as azoospermia, amenorrhea or bilateral tubal occlusion. Other causes, such as endometriosis or cervical and uterine factor are not easy to identify. It seems that in cases of unexplained infertility many factors are not sufficient enough to explain the infertility.


Is there a way to enhance the fertility of the couple itself?

In any case, changes in lifestyle and the two partners such as the pursuit of normal weight, quitting smoking, reducing caffeine intake and alcohol are proposed.


When to start checking infertility?

Studies have shown the majority of fertile couples with regular contacts will achieve conception up to six months; due to this many suggest that the initial investigation should start after six months. This approach is considered to be absolutely correct in women over 35 years. In women over 40 years since the reproductive capacity is rapidly reduced the investigation should begin earlier than six months. It is important not to forget that there may be more than one factors that decrease fertility and therefore investigation should be systematic even if you found a factor. The minimum investigation comprising:

For the man: Sperm examination

For women: documentation ovulation, HSG, FSH and E2 measuring the 3rd day of the cycle for exclusion of decline in ovarian function and ultrasound of inner genitals.

Depending on the case, laparoscopy, hysteroscopy, thyroid testing and measurements of ovarian reserve are performed. It seems that it is the age of eggs and not the rest of the genital system that determines success. Women over 40 had nearly the same success rate as those under 30 when they took eggs from new donor.


When should a woman undergone IVF?

Absolute indications for IVF are closed trumpets and serious damage sperm. In other cases it is possible to carry out three circles stimulating ovulation and sperm insemination.


Insemination may be the first step for infertility treatment. The method is simple and affordable. The success rate in homologous insemination reaches 10-15%, while in cases of heterologous insemination, the rate is higher (~25%).

Intrauterine insemination is indicated when:

  • The woman’s fallopian tubes are intact and patent;
  • The woman is under 35 years of age;
  • The husband’s sperm is normal or demonstrates minor problems in terms of quality.

Brief description:

Intrauterine insemination may be performed either in a natural cycle or after the administration of medication (ovulation induction), with the aim to increase the number of follicles (up to three). Chorionic gonadotropin (a hormone contributing to the rupture of the follicles) may be also injected. In this case, the time at which ovulation takes place can be estimated and synchronized with the insemination, for an even more effective process.

The sperm is prepared and enriched at the laboratory with a special process and is then directly deposited inside the uterus using a special, fine catheter. Therefore, the number of spermatozoa that reach the fallopian tubes, where fertilization takes place, is larger and well timed.

The procedure of insemination is painless and lasts for a few minutes.


In vitro fertilization (IVF) is currently the definite choice of infertility treatment when other, simpler methods of assisted reproduction are not able to provide solutions (i.e. women with closed or destroyed fallopian tubes). ICSI (Intra-Cytoplasmic Sperm Injection) is a type of IVF with micro-fertilization. It is indicated in cases of male infertility and may be combined with TESE or FNA (Testicular Sperm Extraction - Fine Needle Aspiration).

Regardless of the cause of infertility, the procedure of in vitro fertilization includes the following seven steps:

  • Preliminary testing;
  • Hormonal stimulation of the ovaries;
  • Oocyte collection;
  • Oocyte fertilization;
  • Embryo culture;
  • Embryo transfer;
  • Embryo vitrification.


Preliminary Testing

Before attempting in vitro fertilization, the couple must undergo the following tests:

For the woman:

  • Preliminary testing;
  • Gynecological ultrasound;
  • Hysterosalpingogram;
  • Trial of embryo transfer
    (In cases of narrow or deformed cervical canal, dilatation of the cervix is recommended);
  • Hysteroscopy (where indicated);
  • Prenatal testing:
  1. General blood and urine tests
  2. Blood type - Rhesus
  3. Hepatitis Β, C
  4. HIV I and II
  5. Syphilis
  6. Hemoglobin electrophoresis
  7. Mycoplasma
  8. Chlamydia
  9. CMV
  10. Toxoplasma
  11. Herpes
  12. Rubella
  13. Vaginal cultures

For the man, respectively:

  • Sperm diagram and culture;
  • Prenatal testing:
  1. General blood and urine tests
  2. Blood type - Rhesus
  3. HIV
  4. Hepatitis Β, C
  5. Syphilis
  6. Hemoglobin electrophoresis

In cases of non-obstructive azoospermia or severe oligoasthenospermia, where the concentration of spermatozoa is less than 5 million/ml, the man should undergo the following tests as well:

  • Cystic fibrosis test;
  • Testing for chromosome Y microdeletions;
  • Karyotype


Hormonal Stimulation Of Ovaries

The entire amount of oocytes for every woman is definite and, at birth, is approximately 1 million. This number is gradually reduced, reaching approximately 450,000 at the age when menstruation begins. Every month the number of premature oocytes that enter the maturation phase is about 1,000 . Finally, only 1 or 2 will reach the final stages of maturation and make it to ovulate, while the remaining ones will stay unruptured and be destroyed by the body.


The aim of the hormonal stimulation of the ovaries is the maturation of more than one or two oocytes. For this purpose, it is necessary to administer injectable hormones (FSH and LH), subcutaneously or intramuscularly. These hormones are secreted by the pituitary gland during the normal menstrual cycle. An important factor affecting the “response” of an ovary, namely the number of maturing oocytes, is the woman’s age. The phase of hormonal stimulation usually lasts from 9 to 12 days.

In less than >1% of women ovarian hyperstimulation may occur. In this case, more than 20 follicles mature and oestradiol levels are increased.

Preventive measures for OHSS are:

  1. administration of smaller doses of hormones during ovarian stimulation;
  2. "coasting" i.e. interruption of hormonal stimulation for some days prior to oocyte collection, in order to reduce oestradiol levels;
  3. collect, fertilize oocytes but vitrify them (i.e. freeze embryos and transfer them in a subsequent cycle) and
  4. in serious conditions, completely interrupt treatment and “cancel” the stimulation cycle.


Oocyte Collection

No general anesthesia is required for oocyte aspiration. The anesthesiologist intravenously injects a light anesthetic (sedation) while the gynecologist administers local analgesia with xylocaine in the cervical area. If the woman wishes so, a deeper anesthesia can be used. The collection of oocytes from the ovary is transvaginal by the use of a special needle. This needle is ultrasound-guided to the follicle by penetrating the vaginal wall. The special needle aspirates the content of the follicle and the follicular liquid is directly checked by the embryologist for the presence of oocytes.

The average duration of this procedure is 10-15 minutes. After that, the woman stays in bed for about one hour for rest and monitoring. The couple is then informed by the embryologist about the number and quality of collected oocytes. Thereafter the specialist personnel discuss the details of the treatment that should be followed. Following information, the couple can return home.



On the day of oocyte collection, the couple deliver a sperm sample of the husband at the laboratory to proceed with fertilization. Alternatively, cryopreserved sperm sample of the husband can be used, which was previously collected (only in special cases). The fertilization technique that should be used depends on the sperm sample characteristics and the couple’s history.

  • Fertilization Using The Conventional Method (Standard IVF)

This method is indicated when all sperm parameters (concentration, motility, morphology) are within normal range.

In this method, oocytes are cultured together with some hundreds of thousands of spermatozoa during the night.

  • Fertilization Using The ICSI Method

This is indicated when the values of one or more sperm parameters deviates from normal levels (oligospermia, asthenospermia, azoospermia), as well as in cases of previous inexplicable non-fertilization of oocytes with the conventional IVF method. This is an alternative oocyte fertilization method in which a spermatozoon is injected in the cytoplasm of each oocyte, by using a special fine needle. Approximately 17 hours after fertilization, embryologists examine the number of fertilized oocytes.

In cases of azoospermia, testicular biopsy is performed using FNA (fine needle biopsy) or TESE (open biopsy) under local analgesia and light anesthesia (sedation). The biopsy material is examined for immature or mature spermatozoa which can be used to fertilize the oocytes. Our laboratory has been one of the first centers in the world to use immature forms of spermatozoa (spermatids) and achieve the birth of the 4th child in the world from a spermatid. These results have been published in the Human Reproduction medical journal.


Embryo Culture

The culture of fertilized oocytes is performed in special nutritional material and the resulting embryos are kept in special incubators, where they are preserved under fine conditions, until the day of transfer in the woman’s uterus.

The number of zygotes (fertilized oocytes) is checked 16-20 hours after fertilization. Fertilized oocytes are those which contain 2 prenucleolar bodies (one with the mother’s DNA and the other with the father’s DNA). Subsequently, zygotes are cultivated in the lab until the day of embryo transfer which is determined on the basis of the number and morphology of available embryos. In cases that embryos are three or less, embryo transfer is scheduled for the 2nd day of development, at the stage of 3-4 cells. Otherwise, the embryos are transferred to the woman on the 3rd day of development, at the stage of 6-8 cells, while in cases with 6 or more embryos, it is possible to cultivate them until the 5th day of development. This stage of embryonic development is called blastocyst.


Embryo Transfer

Embryo transfer takes place two to five days after oocyte collection and fertilization. With this procedure embryos are transferred in the intrauterine cavity of the woman.

The day of embryo transfer is determined according to the number and morphology of available embryos.

During embryo transfer, embryologists evaluate and select the best embryos to transfer. The number of transferred embryos depends on woman’s age, embryo quality and stage of development. Legislation in Greece permits the transfer of up to three embryos to women up to 40 years of age, and four embryos to women over 40. The remaining embryos, provided they reach the blastocyst stage, can be vitrified, in order to be used by the couple in a subsequent cycle.

When embryologists complete the embryo evaluation and selection, the embryos are aspirated in a special catheter and handed to the gynecologist. Gynecologist enters the catheter in the uterine cavity through the cervix and deposits its content under direct ultrasound-guidance.

This procedure is similar to the intrauterine insemination and is painless.

The transfer of embryos in the uterine cavity is always performed by means of a directly ultrasound-guided procedure to ensure the right position of the catheter in the intrauterine cavity. The use of ultrasounds in embryo transfer improves the results of assisted reproduction, and our centre was one of the first internationally that established this method. This technique was published in the Acta Europea Fertilitatis medical journal in 1996, and the results of our centre were later published in the Human Reproduction medical journal. Currently, the majority of assisted reproduction centers worldwide use this technique during embryo transfer.


Embryo Freezing And Thawing

After embryo transfer, surplus embryos are cultured until the 5th day of development, and those reaching the blastocyst stage are frozen by using the vitrification method. Frozen embryos may be used by the couple in a subsequent cycle. Usually, 30-40% of embryos make it to the blastocyst stage.

During thawing, the percentage of good quality embryos reaches 80%, while the percentage of pregnancies by means of transferring cryopreserved embryos is ~40%. According to Greek legislation, frozen embryos may be preserved for a period of 5 years.






Male infertility covers a wide range from complete absence of germ cells till mild forms of astheno-oligo-teratospermia.

More frequent causes of male infertility are:

  1. idiopathic insufficiency of the germinal epithelium;
  2. varicocele;
  3. infections of auxiliary glands;
  4. chromosomal abnormalities;
  5. infection + varicocele;
  6. cryptorchism;
  7. obstructive azoospermia;
  8. endocrinal causes;
  9. sexual disorders;
  10. post-parotitis orchitis;
  11. immunological causes;
  12. systemic diseases;
  13. testicular tumors.


Male Infertility Treatment

Intrauterine insemination (IUI) is the proposed treatment in cases of mild oligoasthenospermia and/ or asthenospermia.

In more severe forms Intra-Cytoplasmic Sperm Injection (ICSI) is indicated. This is an alternative method of oocyte fertilization in which a spermatozoon is injected in the cytoplasm of each oocyte, by using a special fine needle.

In cases of obstructive azoospermia (normal production of spermatozoa from the testicles, which, however, do not appear in the semen after ejaculate, due to obstruction of the seminal ducts), fine needle aspiration (FNA) of spermatozoa directly from the testicles or even the epidydimides is possible. In cases of non-obstructive azoospermia due to testicular damage (idiopathic or in cryptorchism, injuries, inflammations, infectious diseases, radiation, chemotherapy or chromosomal disorders), testicular sperm extraction (TESE) is recommended.


Sperm donation is the only solution in cases when methods for the treatment of male infertility (IUI, ICSI, FNA, TESE) cannot be applied.

Sperm donors, 18-35 years of age, are examined by an Internal Medicine Physician and are subject to a number of laboratory tests.

  • Blood type - Rhesus
  • Sperm diagram
  • Chromosome test (Karyotype)
  • Hepatitis B and C
  • HIV
  • Syphilis
  • Hemoglobin electrophoresis for thalassemia and Sickle Cell anemia
  • Mycoplasma, Chlamydia, Cytomegalovirus, Toxoplasma, Listeria
  • Cystic fibrosis
  • Herpes types I and II

Sperm samples are frozen and are released after a period of 6-months only after the donor is re-examined for transmitted diseases.

The procedure of insemination or IVF follows.


This is indicated for couples where both partners (man and woman) are not able to undergo infertility treatments with their own gametes (oocytes and sperm).


Sperm diagram: This test checks the quality of semen, identifying sperm concentration, motility and morphology.

The characteristics of a normal sperm diagram are:

Volume 2.0 – 5.0 cm3
pH 7,2 - 8,0
Concentration < 20 million/ml
Total number < 40 million/ejaculate
1st hour motility < 50 % directly forward moving spermatozoa
Viability < 50 %
Morphology < 30 % normal spermatozoa
White blood cells > 1 million

A semen sample is characterized by:

  • oligospermia when the number of spermatozoa is lower than normal (>20 million/ml)
  • asthenospermia when the percentage of motile spermatozoa is low
  • oligoasthenospermia when both the number and motility are lower than normal
  • teratospermia when a high percentage of malformed spermatozoa is observed
  • azoospermia when spermatozoa are completely absent.

Sperm enrichment: Enrichment is recommended in cases where the value of one or more semen parameters are not within normal range. This is a special process to improve semen characteristics, in order to be used for IUI insemination or for IVF-ICSI.


At the early stages of development, embryos are covered by an outer layer, the transparent zone, which protects embryonic cells. This zone is partially diluted and the embryo “hatches” when it reaches the uterus, so that it can be implanted in the endometrium and keep developing. However, in certain cases of IVF, it is considered necessary to assist hatching by reducing the transparent zone width. The procedure is performed on the day of embryo transfer using a special laser.

Assisted hatching of embryos is recommended in the following cases:

  1. When the width of the transparent zone is large;
  2. In cases of cryopreserved embryos;
  3. When the woman is older than 37 years of age;
  4. In cases with high FSH values.


In this procedure, the genetic material of embryos is checked prior to embryo transfer. This method constitutes a combination of in vitro fertilization and special genetic analysis techniques.


How it is done

At first, couple undergoes standard IVF/ICSI with oocyte collection and fertilization. On the third day of development, embryos that have developed from six to ten cells are subject to biopsy. During biopsy a cell (blastomere) is removed and genetically analysed. The embryo continues its growth until blastocyst stage. Prenatal diagnosis of the removed blastomere will indicate which embryos will be transferred to the uterus, excluding those demonstrating genetic abnormalities.


The technique

Depending on the disease for which the embryo is analysed, the appropriate techniques are applied for chromosome analysis or DNA molecular analysis. DNA molecular analysis techniques are highly precise and sensitive, such as the Polymerase Chain Reaction, which allows multiplication of a pre-selected DNA section up to 1,000,000 times, or the fluorescent in situ hybridisation (FISH).



Genetic diseases

The aforementioned genetic molecular biology techniques (PCR) are applied to diagnose several hereditarily transmitted genetic diseases, such as b-thalassemia, X-linked inherited diseases, Duchenne muscular dystrophy, Retinitis Pigmentosa, etc. In Greece, the most frequent monogenic disease is hemoglobinopathy (10% of the population are b-thalassemia and other hemoglobinopathy carriers).


Numerical chromosomal abnormalities or aneuploidies

The genetic material (each person’s chromosomes and genes) transmitted from generation to generation is organized in structures, the chromosomes, in the cell nucleus. Each human body has 46 chromosomes grouped in 23 pairs, which are inherited from the parents (23 from the mother and 23 from the father) at the time of conception. Lack or surplus of one or more chromosomes in the cells is called aneuploidy or chromosomal abnormality. This disproportion in fetal chromosomes usually prevents uterine implantation, while it constitutes one of the main causes of first trimester spontaneous miscarriage. Numerical chromosomal abnormalities are detected in 50% of spontaneous miscarriages and are correlated with genetic syndromes, e.g. trisomy 21, also known as Down’s syndrome. Similar genetic syndromes are demonstrated in 8% of gestations. However, in couples with infertility problems the frequency of chromosomal abnormalities in a pregnancy is 20%, higher than expected, and also increasing with woman’s age.


Benefits of PGS

The transfer of embryos with normal chromosome numbers not only increases implantation rates in IVF, but also reduces by 15% the frequency of first trimester miscarriages, according to international literature. In addition, the chances that a couple has a child without a genetic syndrome due to chromosomal abnormalities are increased, thus, reducing the psychological stress of a therapeutic interruption of pregnancy after prenatal diagnosis during the 12th or 16th week of gestation.


When is PGS recommended?

  • In cases of multiple first trimester miscarriages;
  • When the candidate mother is over 36 years of age;
  • In cases of multiple IVF failures;
  • In cases of chromosomal abnormalities of any partner;
  • In cases of a previous pregnancy with known genetic syndrome that can be genetically diagnosed and screened in the laboratory;
  • When there is family history of cerebral damage or abnormal development.


Accuracy of the test

This diagnosis is more than 90% accurate, thus, it is recommended to couples to proceed to a standard prenatal diagnosis test during pregnancy to confirm the PGD/PGS result.


Culture media are continuously improved to adapt in the best way to the embryo’s needs in different stages of development and culture conditions in incubators are continuously optimized Thus, recent advances have allowed us embryo culture until the blastocyst stage.

Embryo culture until day 5 of development and blastocyst transfer to the endometrial cavity offer significant advantages:

  • Selection of potentially more capable and genetically healthier embryos, since the blastocyst stage is the higher developmental stage of the pre-implanting embryo;
  • Better synchronization of embryos and endometrium, since embryos are naturally implanting on the 6th-7th day of development;
  • Reduction of multiple gestation rates, since only 1 or 2 embryos are transferred. Nevertheless, blastocyst transfer cannot be generally applied as only 30%-40% of the embryos reach this stage of development. The number and quality of embryos, as well as the woman’s age, are limiting factors.


Oocyte vitrification was first announced in 1987; however, until recently it was an experimental procedure, since gestation rates were disappointing due to the low survival rates of embryos. Recently, an improved vitrification method has been developed, based on faster vitrification, which ensures a higher oocyte survival rate (up to 100%) and a pregnancy rate of 40%.

Vitrification of unfertilized oocytes will bring revolution in the treatment of assisted reproduction as women will be able to keep their oocytes vitrified for a long period of time.

More specifically, it is recommended for:

  • Women that will be subject to ovariectomy or irreversible suspension of the ovarian function (following chemotherapy or radiotherapy);
  • Women who opt to have children at a later age;
  • Couples not wishing cryopreservation of embryos for ethical or religious reasons.


This method is used in the following cases:

When embryo transfer cannot be performed for some reason (e.g. hyperstimulation of the ovaries);

When there are excess embryos with normal development and morphology after the transfer and can be used in a subsequent cycle.

Cryopreserved embryos are stored and preserved in special containers with liquid nitrogen at -196ο C.

Storage expands over a period of 5 years, while, upon the end it is possible to renew storage for another 5 years. After expiry of the aforementioned deadlines, embryos are either used for research and therapeutic purposes or destroyed according to couples’ consent.


Ovarian tissue cryopreservation is, currently at experimental stage. It may be applied in women who will undergo chemotherapy or surgical removal of ovaries. During this procedure, solid parts of the ovary containing several immature follicles are removed by laparoscopy and are frozen.

The transplantation procedure of the thawed ovarian tissue is termed:

A. Orthotopic when the re- location of the transplanted ovary is at the anatomic position of the ovary;

B. Heterotopic when the re- location of transplanted ovary is at different part of the body, e.g. subcutaneously.

The ovarian tissue restores its function, at least for a short period of time, and responds to hormonal stimulation. The standard procedure of oocyte collection, fertilization and embryo transfer follows.


Sperm cryopreservation is a widespread method, frequently applied with great success as survival rates of spermatozoa after thawing are very high.

It applies to the following cases:

In cases of IVF when the male partner is not possible to be present at the IVF unit on the date of oocyte collection;

Before chemotherapy, radiotherapy or any operation which might affect the patient’s future fertility.

In cases of azoospermia it is also possible to perform a testicular biopsy (FNA-TESE) and e of isolate and cryopreserve the sperm.

Samples are stored for five years, that might be extended after written request of samples’ owner.


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