It is a painless procedure that mimics the embryo transfer. It allows us to observe the individualities of the cervix in detail so we can be best prepared for the day of the embryo transfer. Usually, on the same day of suppression check in the long protocol or in the beginning of the stimulation in all other protocols (antagonists, short etc.) the direction, the position and the patency of the cervix is checked with an embryo transfer catheter.

Embryo transfer catheters, type Wallace (EUGONIA archive).

• Monitoring during the treatment
• Treatment and Lifestyle
• Psychological support

Monitoring during the treatment

Checks during ovarian stimulation:

• Follicle development and the increasing thickness of the endometrium (using transvaginal ultrasound)
• Hormone levels, such as E2, LH, progesterone (using blood samples)

These will require 4-6 morning visits to our Unit.

Ultrasound monitoring of endometrium thickness, during multiple follicle development treatment. The picture demonstrates a triple-line pattern, with 13.5 mm thickness and a visible cervix. (EUGONIA archive)

Normally, there is a correlation between the number and the size of the follcles and the oestradiol produced, and therefore with the thickness of the endometrium. The echo pattern of the endometrium is also of great importance and the "triple-line" pattern is checked during the stimulation. The ultrasound monitoring and the hormone measurements are usually performed on specific days of the stimulation.

Multiple follicle development. The follicles have matured (transvaginal ultrasound) (EUGONIA archive).

The Director in collaboration with other doctors and midwives of our Unit assess the results and compare them to previous measurements and your medical history. They will decide on the dose of drugs, and other instructions that will be added to your personal file. Our midwives will inform you extensively and responsibly about the course of your treatment program, drug dosage and the date for the repeat of checks.

Treatment and lifestyle

During the drug treatment you do not have to change your way of life or modify your habits concerning work, exercise, diet or sex. If deemed necessary from the results of the preliminary tests, the male partner may have to take antibiotics at the same time as the onset of your ovarian stimulation.

Psychological support

For some couples, the problem of infertility has psychological effects. Emotional distress is usually greater after previous failed attempts or when IVF is regarded as the last chance of having a baby.

Eugonia gives you the option of talking to our collaborating specialised psychologist. It has been proven that psychological support reduces the emotional load. We therefore encourage you to contact our psychologist, who will positively contribute to your IVF attempt.

Usually not all collected oocytes can be fertilized. In order to be fertilized, the oocytes must be mature both chromosomally and cytoplasmically, and be of good quality. During conventional IVF about one third of the oocytes are not fertilized mainly due to the lack of sperm penetration. However, even after ICSI some oocytes are not fertilized and this may be due to the inability of the oocyte to interact with the spermatozoon or the inability of the spermatozoon to induce oocyte activation.

Oocyte maturity

Maturity of the oocytes is a prerequisite for their fertilization. Only mature oocytes have undergone a full meiotic division and are haploid (23 chromosomes). IN this state they are ready to be fertilized by sperm, which are also haploid. Therefore, the resulting embryo will have the normal number 46 of chromosomes and be diploid. The triggering of final oocyte maturation is induced by LH or the administration of hCG.

Metaphase II

Mature oocyte with 1st polar body and the stage of metaphase II.

This is the stage of a mature oocyte that has the potential of being fertilized. The stage of metaphase II (MII) indicates that the first meiotic division has been completed and cell cycle is suspended at the MII stage. The pairs of homologue chromosomes are divided in the two resulting cells, which are uneven in size. The larger cell is the oocytes, and the smaller cell is the 1st polar body that has half the number of chromosomes and little cytoplasm.

Immature oocytes cannot be fertilized because they have 46 chromosomes (diploid) hence fertilization by a haploid sperm would give rise to a zygote with 69 chromosomes. However, in some cases immature oocytes can be matured in vitro in the laboratory. During IVF, only a small percentage of oocytes is usually immature.

There is a correlation of oocyte maturation and follicle diameter, although this is not always true. It is possible to collect mature oocytes from smaller follicles and vice versa.

Metaphase I

Immature oocyte at the stage of metaphase I

Metaphase I (MI) represents an oocyte of intermediate maturity. It is characterized by the absence of the 1st polar body and is surrounded by a dense layer of cumulus cells.

Germinal vesicle (CV)

Immature oocyte at the stage of prophase I with visible germinal vesicle.

An immature oocyte (prophase I of meiosis) is characterized by the absence of the 1st PB and the presence of a germinal vesicle inside the cytoplasm. It is surrounded by a dense layer of cumulus cells. At this stage the nucleus has 46 chromosomes.

Oocyte quality

An oocyte of good quality is characterized by normal spherical shape, clear and homogeneous cytoplasm, small periviteline space and clear zona pelucida. The human oocytes has a diameter of 110-120 μm, and including the zona pelucida it reaches 140-150 μm.

Large oocyte diameter, presence of vacuoles, granulation or large perivitaline space are characteristics related to low oocyte quality or aneuploidies.

Poor quality oocyte with granular and vacuolated cytoplasm.

Up: MII oocyte with abnormal zona pelucida and large perivitaline space with debris
Down: Immature GV oocyte with large periviteline space.

• Is it painful?
• What preparations are necessary?
• Is it dangerous
• Other information

Is it painful?

The procedure of egg collection is practically painless as it is done under intravenous analgesia (sedation) administered by the anaesthetist. Another reason why the egg collection should be done under sedation is to avoid any involuntary movement by the patient that could momentarily move the egg collection needle and cause damage to the ovary or other structures close to the ovary (uterus, intestine, a large blood vessel etc.)

An egg collection is usually a quick procedure, lasting about 10-30 minutes, depending on the number of the follicles, the degree of difficulty of the aspiration etc. The patient usually needs to stay in the recovery room for a further 30 minutes to 1 hour to rest and allow the staff to ensure that they are well enough to leave the Unit.

Before leaving the Unit, the patient is informed about the number of the eggs retrieved and will receive further instructions for the continuation of the treatment.

What preparations are necessary?

A light dinner the evening before the egg collection, shaving and cleansing of the outer genitals.

On the day of egg collection: you must refrain from eating or drinking, and must not have polished nails or perfume. It is advisable that your schedule is free for the morning of the egg collection.

You must arrive at the Unit at the specified time along with your partner, who will provide the sperm.

Is it dangerous?

There is a chance of trauma or inflammation of the internal organs, but it is estimated internationally to be minimal. In the hands of an experienced doctor this possibility is practically negligible or even zero. The surgery theatre is equipped with all the instruments needed for the anaesthetist to deal with every unexpected event in the best possible way.

Other information

Eggs collected from an egg collection. The eggs have been identified by the embryologist and have been placed in a petri dish along with culture media (EUGONIA archive).

Please note that the number of eggs retrieved may be smaller than the number of follicles visible with the ultrasound because some follicles may not contain any eggs (empty follicle syndrome).

The chance of no eggs collected due to a fault is minimal when the procedure is performed by experienced gynaecologists and embryologists. It is also possible for more eggs to be collected than what initially is expected, especially when the number of follicles in more than 8-10 in every ovary. This is due to the way the follicles are depicted: the image with an ultrasound has two dimensions and it is impossible for a follicle that is "behind" the one measured to be shown (however, when the follicle "at the front" has been aspirated, the one "behind" it that was initially covered could then show in the ultrasound).

The egg collection is the recovery of eggs from the ovaries. It is performed in a special room in our Unit, under sterile conditions, at a specific time, usually 35-36 hours after the last injection (Pregnyl, Profasi, Ovitrelle).

Ultrasound guided transvaginal egg collection. In the picture we can see the tip of the aspiration needle inside the follicle (EUGONIA archive).	VIDEO: Egg collection

The egg collection is performed by the doctor transvaginally, under constant ultrasound guidance, which facilitates the doctor's precise manoeuvres. The follicles are punctured one after another using a special needle that traverses the vaginal wall.

The contents of each follicle are aspirated into special tubes and are passed on to the embryology laboratory. The embryologist locates the eggs and places them in dishes containing culture medium.

All the equipment and consumables used fulfill special requirements, ensuring that they are not potentially toxic to the gametes and the embryos.

If the egg has not be collected, the doctor flushes the follicle with an isotonic solution (one to three times, until the egg becomes unattached). For this reason, at Eugonia we now only use a double lumen needle with a special inter-lumen connection system that allows the flushing medium to enter the follicle.

VIDEO: Location and collection of an oocyte during egg collection.

Oocyte surrounded my cumulus cells (EUGONIA archive).

For the follicular fluid aspiration, the needle-lumen system is attached to an electronic aspiration pump with a digital display of the sub-pressure selected. Please note that the eggs are sensitive to the sub-pressure and the adjustment of the pump within certain accepted limits is important.

As we have mentioned before, the contents of each follicle (follicular fluid) along with the oocyte and its cumulus cells are aspirated in special tubes and are passed to the embryology laboratory.

The embryologist locates the oocytes and isolates them in special little vessels called culture dishes, which contain special culture media. All the equipment and consumables used fulfill special requirements, ensuring that they are not potentially toxic to the gametes and the embryos.

The culture dishes are then placed in an incubator under specific culture conditions (darkness, temperature of 37ºC, 5% CO2 atmospheric pressure, 100% humidity). Generally, in every culture dish 1-4 eggs are placed, according to the IVF technique to be used for fertilization (there are certain laboratory variations of this technique that do not affect the events to follow and that is why they are not explained in detail).

According to the thickness, the size and the density of the cumulus cells, the embryologist can estimate the maturity of the eggs collected.

Except for the maturity of its cumulus cells, the oocyte also needs to be mature with regards to its cytoplasmic and nuclear contents. Despite other cells in the human body, oocytes and spermatozoa contain half the chromosomes (i.e. only one copy of each chromosome in their nucleus). A mature oocyte therefore contains 22+X chromosomes and a spermatozoon contains either 22+X, or 22+Y. When the oocyte and the spermatozoon are combined with fertilization, a cell with a normal nuclear constitution is then created (i.e. a cell that has two copies of each chromosome). A mature oocyte has completed nuclear changes that enable normal fertilization meaning that it has extruded one set of its 23 chromosomes in a small structure called the first polar body which is situated just under the zona pellucida. The presence of the first polar body means that the oocyte is truly mature.

During the use of classic in-vitro fertilization (IVF), the presence of cumulus cells around the zona pellucida is necessary and we cannot check for the presence of the first polar body. In this case, we rely mainly on the morphological observation of the cumulus cells after the egg collection. On the contrary, during the use of intra-cytoplasmic sperm injection (ICSI) the removal of the cumulus cells is necessary in order to check for the presence of the first polar body and the identification of the mature eggs that can be fertilised and also for the ease of the oocyte maneuvering during the ICSI process.

Double lumen oocyte collection, Casmed type (EUGONIA archive) .

Electronic aspiration pump (Labotect type) used for egg collection. The sub-pressure level is selected and adjusted automatically in order to protect the oocytes (EUGONIA archive).

Azoospermia is the absence of spermatozoa in the ejaculate, and it is classified as obstructive or non-obstructive. In cases of non-obstructive azoospermia the sperm is recovered surgically.

In obstructive azoospermia, sperm production is normal but no sperm appear in the ejaculate due to an obstruction of the male reproductive tract. The obstructive aetiology includes obstruction of the vas deferens, congenital absence of the vas, or vasectomy. In obstructive azoospermia sperm can be easily obtained surgically using aspiration (FNA) or testicular biopsy (TESE).

In non-obstructive azoospermia there is no production of spermatozoa in the testicles. This complete lack of production or minimal production of sperm (oligoasthenoteratospermia, which is practically as severe as azoospermia) suggests testicular failure. The condition can be idiopathic or be attributed to lack of testicular descent, injury, inflammation, contractible diseases (such as mumps), radiation, chemotherapy or chromosomal abnormalities.

In non-obstructive azoospermia, it is quite rare to retrieve sperm following testicular biopsy (TESE). The chances of retrieving sperm may increase when small areas of spermatogenic activity are detected in the testes, although the number of pregnancies achieved is limited. If there is a high chance of no sperm retrieved, the couple needs to be informed beforehand with regards to their options which can be:

• Use of donor sperm
• Oocyte cryopreservation (vitrification)
• Donation of the oocytes collected, or a combination of these options.

Sperm can be recovered directly from the testes either by needle aspiration (FNA) from the testis or epididymis, or via a surgical biopsy of small testicular pieces (TESE).

Surgical sperm recovery is recommended in cases of azoospermia, inability to collect a semen sample on the day of egg collection or failure of electroejaculation. The procedure can be performed on the day of the egg collection under intravenous or local analgesia. The testicular tissue excised is processed by the embryologist in order to retrieve any sperm. Once motile sperm have been isolated, the mature eggs can be fertilized using intracytoplasmic sperm injection (ICSI). Any excess motile sperm can be cryopreserved for future use.

There are various methods or surgical sperm retrieval, either from the epididymis or from the testes.

Sperm retrieval from the epididymis

• Microsurgical epididymal sperm aspiration (MESA):
  It is recommended in cases of obstractive azoospermia with normal spermatogenesis. The epididymis is surgically opened with the use of a microscope and its fluid is aspirated so that sperm can be retrieved.
• Percutaneous epididymal sperm aspiration (PESA):
  It is recommended in case of obstractive azoospermia with normal spermatogenesis. A 19-21G needle is passed into the epididymis and then fluid is gently aspirated.

Sperm retrieval from the testes

• Fine needle aspiration (FNA):
  It is recommended in cases of obstractive azoospermia with normal spermatogenesis. A 21G needle is passed through the testis.
• Testicular sperm aspiration (TESA):
  It is recommended in cases of obstractive azoospermia with normal spermatogenesis.
• Testis biopsy (testicular sperm extraction – TESE):
  It is recommended in case of both obstractive and non-obstractive azoospermia and it includes performing an open biopsy and excising small pieces of testicular tissue.

The sperm aspiration methods from the epididymis (MESA, PESA) or the testes (TESA) are simple, but are usually "blindly" performed and can cause testicular tissue damage such as haematoma, cysts and testicular atrophy. Testicular biopsy (TESE) is suggested by international scientific literature as the more effective method of sperm retrieval and it is associated with fewer complications. For this reason, in our Unit, the surgical sperm retrieval is performed by testicular biopsy (TESE) by specialized surgeons.

The procedure takes place on the day of egg collection with intravenous or local anaesthesia. The small testicular tissue pieces are processed by the embryologist in order to retrieve any sperm. Following the retrieval of sperm, ICSI can be performed. Any excess sperm can be cryopreserved for future use so that there is no need to perform another biopsy.

Testicular biopsy for diagnostic purposes

Testicular biopsy prior to the day of egg collection is not recommended. Our Unit is in line with this recommendation since according to the scientific literature such a biopsy constitutes an extra surgical procedure that causes extra damage to the testicular tissue and sperm retrieval on the day of the biopsy for diagnostic purposes cannot guarantee that sperm will also be retrieved on the day of egg collection.

Testicular biopsy for diagnostic purposes and the histological evaluation that follows is mainly performed for the assessment of spermatogenesis. The histological evaluation can also be performed on the testicular pieces that are excised on the day of the egg collection.

• Sperm collection
• Sperm collection and preparation
• Ejaculation problems and ways of overcoming them

Sperm collection

At the same time as the egg collection, or straight after, the male partner will produce the sperm, preferably by masturbation. It is very important for the entire sample to be collected. If not all the sample is collected or if the sample does not contain enough spermatozoa, the male partner may be asked to provide a second sample a few hours after the first one. The couple will be informed about the day of egg collection, at which time the male partner must have 2-5 days abstinence from ejaculation.

In certain cases that the male partner is unable to collect the sperm sample by masturbation, special silastic non-toxic condoms that do not contain any spermicides (normal condoms that are not designed for this use usually contain spermicides).

Sperm collection and preparation

Sperm preparation laboratory (EUGONIA archive).

After the sperm production, the sample is processed (preparation-condensation) in order to select the motile and morphologically normal sperm. During this process the sample is centrifuged using a high viscosity colloid sample (e.g. pure sperm).

This process mimics the normal spermatozoa activation that occurs in vivo inside the female genital tract and enables the spermatozoa to fertilize the eggs. Following this preparation, the spermatozoa are kept in the laboratory under sterile culture conditions until placed together with the eggs.

Ejaculation problems and ways of overcoming them

These are identified during the preliminary examination of the couple and ways of overcoming them are explored.

In the case of retrograde ejaculation (backward release of semen into the bladder) the male partner needs to drink a sodium bicarbonate solution (to alkalinize the urine) before he collects a urine sample, and the spermatozoa are recovered following a special preparation process.

In patients with ejaculation deficiency, observed in cases of spine injury (paraplegic-tetraplegic), diabetes and neurological conditions, sperm can be collected by induced ejaculation using a special electro-induction device (electroejaculation).

When there is a chance that the male partner may not be able to provide a semen sample on the day of egg collection, the cryopreservation of a semen sample prior to the egg collection is advised.

Performing ICSI using inverted microscope and special microcontrollers.

ICSI is applied when the sperm is incapable of fertilising the egg on its own (as previously described). Using highly specialised equipment, the embryologist deposits a single spermatozoon inside the egg to induce activation and fertilisation of the egg. Only one motile spermatozoon is required for each egg.

The method has been successfully used since 1992 and overcomes almost all the barriers that cause male infertility.

Thanks to ICSI, men with severely compromised sperm quality (reduced number, low motility, bad morphology or ejaculation problems) can now father a child of their own; something that was impossible in the not so distant past.

Until today, thousands of babies have been born from ICSI, with no indication that the method presents a danger for embryo quality and child health (according to a large scale epidemiological study from Belgium).

Except the way that fertilization occurs, the procedure is the same as conventional IVF as far as the man and woman are concerned.

Indications for ICSI

ICSI was developed to treat mainly male infertility. Indications include:

• Severe oligo-asthenozoospermia
• Azoozpermia
• Globozoospermia (absence of acrosome)
• Sperm agglutination
• P revious fertilization failure with conventional IVF in the presence of normal sperm
• ICSI can also be used for other reasons, such as low number or poor quality of oocytes, think or hard zona pelucida, especially in older women. However, this approach is in doubt by many scientists.

During ICSI, a very small percentage of oocytes may be damaged despite all the precautions. However, the oocytes that have endured the process of injection and have regained their initial shape usually develop normally. Those that do not are isolated and are not transferred.

Recent epidemiological studies in a large number of patients showed that the percentage of congenital abnormalities in neonates conceived by ICSI is not different and reaches about 2.7%. This percentage was found to be similar to children from IVF or even natural conception.

Since male infertility may have a genetic basis, there is a possibility of transmission of infertility to the male offspring. This means that these children may need ICSI themselves when they decide to have children. This case concerns a rare form of azoospermia that is caused by microdeletions on the Y chromosome. For this reason, it is recommended to seek genetic consultation for men with severe OAT or azoospermia (obstructive azoospermia due to congenital absence of the vas deferens and possible relation with a mutation in the gene of cystic fibrosis). At a european and international level the research on the safety of ICSI is ongoing.