Infertility examination protocol

Questioning medical history

  • Checking clinical evidence, photocopy, documentation
  • Handeling Infromed Consents
  • Gynaecologic examination, STD (Urine culture, Urine Chlamydia, Ureplasma/Mycoplasma examination, Vaginal secretion bacteriological culture,Cervix Chlamydia, Ureoplasma/Mycoplasma, HPV Female genetic panel (Leiden-mutation, prothrombin G20210A, MTHFR C677T, CBS 844ins68)
  • Basis-hormone examinations (on 3rd day of cycle: FSH, LH, Ostradiol, Prolactin, Testosterone, DHEA-SO4, TSH, T3, T4,, AMH, on 21-24th days of cycle: Progesterone
  • TORCH-examination: Toxoplasma gondii antibody Rubeola virus antibody, Cytomegalovirus antibody, Herpes simplex virus 1,2, Varicella zoster virus, Parvovirus B 19, Hepatitis panel (HBsAg, HbsAt,HBc At, anti HCV), Epstein-Barr virus, Chlamydia trachomatis antibody.
  • Spermiogram, STD, male genetic panel (Y chromosome microdeletions, cystic fibrosis, X and Y chromosome aneuploidies)


The Hystero-Salpingo contrast agent ultra-sonography checks the patency of the tubes and the cavity of the uterus using ultrasound and an echogenic contrast agent.

With this simple procedure it is possible to make an ambulant check in the fallopian tubes without anaesthesia or any pain. Although the procedure sounds simple, it can be more complicated. This is because the tubes are not rigid but are organs that contract and relax (like the intestines) and can, therefore, cramp at the moment of injection. This is why, in some cases, a false positive, or false negative result can be expected. In this case further investigation is suggested (hysteroscopy, laparoscopy).

One day infertility diagnosis

There is a possibility to organise the examinations in such a way to find the most probable causes of infertility in the fastest way.

With one single visit the following examinations can be carried out:

  • Spermiogram, TR, STD, Andrologic examination and recommendation
  • Gynaecologic examinations (Cytology, HVTR, STD test, UH, Breast screening, HYCOSY, hormone examinations)

Stages in IVF

The IVF consists of six stages: ovarian stimulation with hormones, oocyte pick-up, insemination, in-vitro culture until embryo is in different stages of development, embryo transfer and freezing and thawing of embryos in some cases.

  1. The ovarian stimulation allows us to obtain several follicles in one single cycle and is necessary since the pregnancy possibilities increase proportionally to the number of embryos transferred, considering that not all the oocytes obtained evolve into embryos suitable for transferring. The stimulation requires subcutaneous injections and several (3 or 4) visits to Versys Clinics where blood tests (Estradiol) and vaginal ultrasound scanning are preformed.
  2. Oocyte pick-up (OPU): The extraction is carried out through a transvaginal puncture under ultrasound control. The average duration of this intervention is about 15-20 minutes, it is done under sedation and the patient will be ready to go home in a few hours.
  3. Insemination. Once we have obtained the oocytes, a semen specimen is required. In order to carry out the insemination there are two alternatives: the classical insemination, placing the oocytes together with the spermatozoa, previously treated and selected; and the intracytoplasmatic spermatic injection (ICSI).
  4. In Vitro Embryo Culture. The fertilised oocytes are checked the following day. From this moment on the embryos are kept in an adequate type of culture for their particular development. The embryos usually stay in the culture for three days. In some occasions, it is convenient to extend the culture in the laboratory till the stage called blastocyst.

The fertilised eggs then develop into embryos:

     Day 1 after the egg puncture = pre-nuclear stage (pronuclei)
Some eggs fertilise and some don't. The fertilised eggs are recognisable because they have two pro-nuclei.

•     Day 2 after the egg puncture = two to four cell stage

The fertilised egg has already developed into a two-to-four-cell group. Now we decide whether to put the embryo(s) straight back into the female uterus (embryo transfer) on day 3 or 5. You will receive a phone call from Versys Clinics  IVF Laboratory to make the date for embryo transfer!

•     Day 3 after the egg puncture = eight cell stage

The fertilised egg may have already divided itself into an eight-cell group. Either we put it (them) into the female uterus today or we wait until day 5.

•     Day 4 after the egg puncture = morula stage

The morula-stage is reached.

•     Day 5 after the egg puncture = blastocyst (expanding or hatching)

The embryo(s) is (are) in the blastocyst stage and is (are) put back in the uterus today at the latest. 

  1. Embryo transfer (ET): The moment for the embryo transfer into the uterus is decided for each particular case. Depending on the embryo characteristics, your doctor upon the embryologists recommendation suggest the most suitable moment between the second and the fifth day after the obtaining and fertilisation of the oocytes. The uterine transfer takes place transcervically, does not require anaesthesia. We usually transfer 2 or 3 embryos, or in some cases 1 (SET: Single Embryo Transfer). Because of the high rate of embryonic implantation it is advised to limit the number of transferred embryos in order to reduce the incidence of multiple pregnancies. The ET could be performed with a special ( ultrasound guided) catheter, which makes your doctor able to put the embryo into the most precise position.
  2. Freezing and thawing of embryos: after the transfer of the right number of embryos for each case, the rest of feasible embryos are submitted to a freezing process in order to preserve them for a time. This procedure allows the availability of these embryos in case they are needed by the couple. If pregnancy was not achieved, or after finishing it, we proceed to thaw and transfer the embryos that survived freezing.

Causes to fail the IVF program

Failure of the stimulation treatment: the stimulation of the ovarian function doesn't occur. Even an increase in the dosage has no effect. In the puncture procedure the eggs are extracted from an average of 80–90% of matured follicles; however, it occasionally happens that no egg is extracted. Causes for this can be: the oocytes are empty (formation of cysts); the follicles ruptured just before the puncture; .

Sometimes an ovary is situated in a position (especially after abdominal surgery) that means it cannot bepunctured without having a high risk of an injury.

Sometimes the fertilisation rate is low. In the cell culture approximately 70–85% of the eggs are fertilised. The percentage of fertilised eggs depends on the maturation condition of the eggs and the sperm quality.

In some rare cases the embryo transfer through the uterine cervix channel in the uterus can be difficult and painful, especially after operations on the cevix. After the insertion inflammations very rarely occur and the embryo transfer seldom fails completely.

The biggest problem in IVF is the embryo's implantation failure after the transfer due to a non-nidation on the uterine mucosa. The causes of this are to a large extent unknown, and therefore there is nothing you or we can do to prevent this occurrence. We shape the luteal phase (the time after the transfer) using hormones in order to reach optimal nidation conditions.

Psychological problems can occur before, during or after the treatment, which is supported by the team and the specialized psychologists.



The couples submitted to ICSI started from a rather more severe primary problem: the difficulty to fertilise with male sperm. So it is important to remark the fact that, through this technique, even with the worst quality semen specimens, only in the 2–5% of cases there is a failure in fertilising all the injected oocytes.

 The procedure is almost identical to conventional IVF, except at the moment of insemination. The oocytes are decumulated, meaning that they are freed of all the cells around them and the mature ones are taken to the microscope, where, using micromanipulation devices, a single sperm is injected into the oocyte using a micropipette of its same thickness.

We leave these plates in the incubator until the following day, when we check the fertilization of the oocytes. 24 hours after the fertilization, the first embryonic division takes place, first into 2 and then into 4 cells. From this moment on, the procedure is the same as that described for conventional IVF.

When the semen specimens are very pathological, we always make complimentary analysis to detect whether there are chromosomal and/or genetic defects which may justify the pathology or even to recommend that specimen not to be used without the appropiate precautions. In these cases, it is also more probable to obtain worse quality embryos.

The ICSI treatment is applied in the following cases:

  • when the number of sperm is limited (less than 10 million/millilitre)
  • an earlier attempt at fertilisation has been unsuccessful
  • when there are morphological disorders: if the sperm's head doesn't have the correct shape to allow it to penetrate and so fertilise the egg.
  • when the zona pellucidum diameter is higher


Only mature and genetically acceptable sperm produce the hyaluronidase enzyme which is located in the sperm's head.This enzyme ensures that when the sperm meets the egg cell the outer egg integument dissolves and the sperm is able to penetrate, so enabling fertilisation.Only sperm that produce the hyaluronidase enzyme are mature are consequently used in our Institute in the P-ICSI procedure


The egg – as well as the embryo – is surrounded and protected by a shell, the zona pellucida. Shortly before the embryo nests in the uterine mucosa the zona pellucida opens – with the help of special enzymes and with the pressure of the embryo's growth – and the embryo "hatches".

Some studies indicate that the in-vitro culture of the embryo – and the cryo-preservation – can lead to hardening of the zona pellucida and so complicate the "hatching" or prevent its occurrence.

A theory exists that by thinning or opening of the zona pellucida, the hatching of the embryo would be eased.

AHA should be offered:

  • to patients with multiple unsuccessful IVF or ICSI attempts, in spite of them having "top grade" embryos;
  • to patients over 35 years of age;
  • if embryos have a thickened zona pellucida


New findings regarding the physiology of embryos have resulted in the development of better culture mediums. This allows the cultivation of embryos to reach a blastocyst stage.These new mediums minimise the risk of losing the embryo's viability and allow the embryos to be kept in culture until the 5th day (blastocyst stage) which is the preferred method.

Advantages of Blastocyst Transfer:

  • The fertilized embryo usually takes 5 days to reach the uterus (in case of implanted embryos on the 2nd or 3rd day after the follicle puncture, these arrive at least 2 days earlier in the uterus cavity).
  • While waiting for the blastocyst stage, only those embryos that present an ideal development potential can be used
  • The risk of chromosome abnormalities increases with the woman's age and abnormal embryos tend not to develop in the first few days transfer.
  • The ejection of implanted embryos is avoided due to the diminished movement of the uterus. This is the big advantage of the transfer taking place on the 5th day


For patients with the risk of a hyper stimulation syndrome of the ovaries (OHSS) the IVM method can be particularly therapeutic.After a short hormone stimulation of the ovaries immature eggs are punctured and cultured in a special culture medium until they reach maturation.Then the fertilisation takes place (ICSI) followed by the embryo transfer.
This process is still in the experimental phase and although some children have been born following IVM there are still some methodical difficulties.

Drugs in IVF

Clomifen leads to an increased distribution of the gonadotropin detachment hormone in the pituitary gland. This leads to an increase of FSH – the follicle-stimulating hormone. FSH stimulates the oocyte's growth in the ovary. In some isolated cases, there have been side effects including hot flushes and sweating. One of the drug's biggest disadvantages is that the uterine mucosa doesn't develop as well as one would desire.

Merional, Fostimon (all human menopausegonadotropin – hMG)

Precursor of the genetically produced FSH, hMG is a combination of urinary FSH and urinary LH. hMG was the first gonadotropin preparation used to stimulate the ovaries.

Decapeptyl depot injections, Decapeptyl 0.1, Suprefact (all GnRH-agonists for down regulations)

These drugs are used to prevent ovulation – "down regulation". The ovulation needs to be prevented because the eggs are extracted from the oocytes (OPU) during an IVF treatment.

Orgalutran and Cetrotide (both GnRH-antagonists)

This is a form for "down regulation" When using these drugs, we also use drugs to block the pituitary gland's function immediately.

Recombinant FSH (Puregon,Gonal F)

Biotechnologically extracted FSH, just like the FSH produced by the pituitary glands, causes a stimulation of the ovaries and leads to increased egg maturation. The ovaries' response to this hormone depends on the dosage and varies from case to case.
Progesterone vaginal suppositories, Utrogestan, Duphastone

Progesterone and other methods used to assist the luteal phase are used after the puncture of the ovaries. They ensure that a fertilised egg has optimal conditions to allow a pregnancy to persist. They need to be inserted into the vagina on the evening of the puncture day. From the following day, they should be inserted according to your personal stimulation timetable

Anti-coagulants (for example, aspirin 100 mg, Fraxiparine) are used following the puncture day according to your personal stimulation timetable

Folic acid: at least 0.4mg should be taken daily beginning at least one week before the commencement of stimulation.

Additional drugs such as Pednisolone, Aspirine should only be taken on the advice of the doctor of Versys Clinics.

Risks, complications, problems of treatment

Over-stimulation of the ovaries (OHSS) can arise due to the hormone stimulation. This rare complication occurs after the puncture. The first symptoms are: thick, inflated belly; pain in the abdominal area; nausea; shortage of breath or a tickle in your throat; reduced quantity of urine.
 The fact that the blood thickens is of clinical concern. This increases the risk of blood clots and also thrombosis or embolisms. These can be treated using anticoagulatory measures. Treatment for these conditions should take place in a hospital as there is a risk to the patient. If the patient is pregnant the symptoms can last until the 10th week of pregnancy. After that most symptoms disappear spontaneously without causing any permanent damage. However, due to the size of the ovaries, they can rotate on their own axis (centre). This is very painful and carries the risk of insufficient blood supply reaching the affected ovaries. When this occurs, a laparoscopy is usually carried out.

The puncture of the oocytes is carried out through the vagina, using (if desired) anaesthesia. However, the anaesthesiacan lead to complications (e.g. nausea, vomiting, circulatory problems, headaches and in rare cases apnea).

Injuries to the blood vessels, the urethra or intestinal loops: During the puncture a thin needle pierces the vagina wall to reach the ovaries. There is a risk during this procedure that neighbouring organs can be harmed. A clean cut of the intestine or the bladder is relatively safe. However, injuries to the pelvic wall's blood vessels or the urethra are problematic as they can lead to intense internal haemorrhages or the leaking of urine into the abdominal area. In these cases surgery (in a hospital) would be required. In extremely rare cases there is also the danger of major haemorrhages or inflammatory reactions.

Another risk is the spreading of germs in the abdominal area followed by infection. This complication, though rare, is easily treated with antibiotics. Only occasionally is surgery necessary ("peritonitis" or abdominal abscess).

Extrauterine pregnancies can also occur after an IVF treatment even though the embryos are transferred directly into the uterus, not to the Fallopian tubes.

In pregnancies resulting from IVF or ICSI the risk of miscarriage (abortion) is unfortunately twice as high as in a normal pregnancy.

Cancer risk: it has been suspected that the ovarian stimulation increases the risk of cancer. A retrospective study was carried out to investigate the risks. Neither the type of sterility nor the number of IVF cycles (nore their results) had any significant influence on the carcinoma risk.

The risk of malformations

A report from a group from Brussels showed a rate of significant congenital malformations of 4.2% in a group of 2889 children born following ICSI and 4.6% in a group of 2995 children born following IVF. This rate was the same as that expected in children born from spontaneous conception.

A New York working group reported a rate of significant deformities in a group of 2059 children born following ICSI of 1.8%. No difference was found in those children born following IVF or ICSI treatment.

Multiple pregnancies

Multiple pregnancies are often associated with a range of complications.
Twins are born prematurely four times more often than single babies and have to stay in intensive care sixteen times more often. The death rate for twins is three times higher than that for single children and the death rate for triplets is four times higher. The economic impact of multiples pregnancies also has to be considered.

The probability of having a multiple pregnancy is significantly higher after IVF treatment than with naturally conceived pregnancies. Approximately 15–25% of all IVF treatments resulted in a multiple pregnancy compared with a naturally conceived rate of just under 2–4%.

Our goal is to achieve a good pregnancy rate with a low rate of complications. The ideal scenario is for one single pregnancy to result from the transfer of one single embryo.


As part of our policy about providing our patients with true information on success rates we have created a section on our website where we inform each quarter of the year. We consider this information to be essential to help couples decide on one centre or another.

 Biochemical Pregnancy (BP): Are such pregnancies in which the level of hCG (human chorionic gonadotropin, a peptide hormone) is increased on the 14th day after the embryo transfer, indicating the adherence and growing of the embryo, although later neither a gestational sack nor fetal heartsounds can be found by ultrasound which means that the conceived pregnancy is not developing anymore.

 Clinical Pregnancy (CP): Are such pregnancies in which the level of hCG indicates pregnancy, the ultrasound shows the gestational sack and later on the heartsounds which facts clinically prove the pregnancy.

Homologue and donor insemination

Insemination is a method by which the pre-treated sperms are injected directly into the uterine cavity by a thin catheter around the time of ovulation. It is used when the male participant of the couple has very low sperm number or the mobility of sperms is not adequate or when the mucus of the uterine cervix and the sperms are incompatible. In these cases the insemination increases the chance of pregnancy because the sperms can "jump" through the uterine cervix and get into the uterine cavity so they can approach the egg with better chances.

There are two kinds of insemination. In the homologue insemination (AIH) the male participant's own sperms are put into the uterine cavity, while in the heterologue insemination (AID) the sperms of a donor are used.

Before the treatment couples have to avoid sex for 2–3 days. In case of irregular menstrual cycle, an ovulation induction might be needed before insemination to ensure the proper follicular maturation and ovulation. The seed goes through a pre-treatment prior to the injection so the seminal fluid is removed and the sperms are separated. The sperms are injected directly into the uterus by a thin catheter through the uterine cervix. This method causes only a slight feeling of discomfort and lying in bed is needed for only a couple of hours right after the insemination.

Gamete (sperm, egg) and embryo donation

Egg, sperm, embryo donation

Egg Donation

The oocyte donation programme is aimed at those patients whose ovarian function is unable, for different reasons, to produce oocytes of sufficient quality to achieve pregnancy. The oocyte donation programme involves endometrial preparation of the patient, so that when oocytes from a donor with compatible blood group, Rh factor and physical appearance become available, these donated oocytes are inseminated using semen from the receptor couple or, if necessary, from a donor, and the embryos resulting from this fertilization are transferred to the receptor to achieve a pregnancy.

The following are the criteria for taking part in this programme:

– Inactive ovarian function or ovarian function deficient in quantity and quality.
– Maintained endometrial quality.

Main Indications

– Premature ovarian failure (POF).

– Surgical menopause.

– Hereditary illnesses that prevent use of the patient's oocytes.

– Bad oocyte or embryo quality dependent on the female gamete.

Sperm donation:

Treatment using donor sperm from the sperm bank is advised if:

  • the male partner is unable to procreate
  • psychological disease
  • other fertilisation treatments have failed ( TESE/MESE)
  • other reasons exist, such as the male partner carrying a genetic disease that makes using donor sperm the best option.


Gamete, embryo freezing and storing


After finishing an in vitro fertilization treatment, and once the chosen embryos have been transferred, it is possible for there to be extra good quality embryos. These embryos are cryopreserved for use if the fresh transfer pregnancy should fail, or if a second pregnancy is being considered. This process takes place in a complex computerized freezer that reduces the temperature at a constant rate that we programme and it allows us to maintain constant times and temperatures for all freezings, so guaranteeing the best treatment of the frozen embryos to obtain maximum viability after thawing.

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