Laboratory Procedures

Embryology Laboratory

Homapage / Embryology Laboratory

Assisted reproductive technologies aim to mimic the conditions of the uterine environment and thus to create the most appropriate setting for embryo culture in a laboratory, from oocyte pick-up to embryo transfer, in order to create the highest chance of pregnancy...

Collecting oocytes: OPU

The ovaries are stimulated with hormonal preparations to promote the growth of follicules containing eggs (oocytes). When follicules reach the desired size, hCG (Human Chorionic Gonodotrophine) is injected in order to ensure the final oocyte maturation. 35-36 hours after this hCG injection, oocyte pick up (OPU) is performed under anesthesia, by a physician, with the guidance of ultrasound. The timing of OPU is crucial since, if ovulation occurs too early, the oocytes will enter the fallopian tubes, where they cannot be retrieved. Eggs are aspirated from the ovarian follicles by the gynecologist, using special needles, and given immediately to the embryologist, who retrieves from the follicular fluid the oocytes, surrounded by their protective and nourishing cell cloud, the cumulus. The procedure lasts 10-20 minutes. Oocyte collection can be tolerated rather well, does not cause serious pain and can be repeated. Patients are required to fast for 8-9 hours before coming to their appointment and are allowed to leave the center 2-3 hours after the procedure.

Ovum collection is generally performed with a needle passed through the vagina under the guidance of ultrasonography.
Uterus, Fallopian tubes, Ovary, Ultrasound probe, Aspirating needle, Ovarian follicles

  Ovarian follicules containing oocytes 

Preparing eggs for injection: denudation

Oocytes are aspirated from follicles with a cloud of cells called cumulus, which have a role in their maturation. Oocytes, surrounded with their cumulus cells, are therefore allowed to continue to mature for 2-3 hours after OPU. At the end of this incubation period, cumulus cells are loosened by an enzyme called hyaluronidase and removed mechanically with pipettes of fixed tip diameters. After denudation, these “naked” oocytes are evaluated in terms of maturation and morphology, incubated for a supplementary 30 minutes, and finally microinjected (ICSI or IMSI).

In the case of IVF (incubation of the two gametes, oocyte and sperm, in the same environment to allow “natural” fertilization, without microinjection), denudation is performed after fertilization, 22-24 hours after OPU, because the sperm needs the cumulus cells to be able to attach to and penetrate the oocyte.  

Immature oocyte (Prophase I stage)
Immature oocyte (Metaphase I stage)


Mature oocyte (Metaphase II stage) 

Fertilizing eggs

IVF (In vitro Fertilization)

IVF was first practiced in 1978 in England. Louise Brown, the first IVF baby, has become a source of hope for many couples. In IVF, the oocyte and the sperm cells obtained from the spouses are placed in the same environment, the aim being for the sperm to penetrate the oocyte unaided. IVF may be chosen as the appropriate technique for certain patients after the evaluation of several factors: female age, number and quality of oocytes, cause of infertility, sperm quality (number, motility, morphology), whether IUI (intrauterine insemination) or ART (Assisted Reproductive Technology) have been tried previously, whether the patient has secondary infertility (having a previous pregnancy but unable to conceive another child) and PGD (preimplantation genetic diagnosis) indications. To minimize the risk of fertilization failure, IVF is generally practiced in combination with ICSI (intracytoplasmic sperm injection).

IVF (In vitro Fertilization)  

ICSI (Intracytoplasmic Sperm Injection) 

In ICSI, sperm is injected into the oocyte under a microscope using a micropipette. This microinjection is a method developed initially for the treatment of serious male infertility. Sperm which is defective in terms of number, motility, and/ or morphology is incapable of fertilizing the oocyte unaided. The direct injection of the sperm into the oocyte enables fertilization. The embryologist selects the best sperm from the ejaculate, whenever possible a sperm with normal morphology and high progressive motility, immobilizes it by positioning the microinjection pipette onto the middle part of the tail and then injects it into the oocyte. Microinjection is especially important for sperm obtained from the epididymis and testes because in these cases it is the only way of achieving fertilization. 

The first live births in Turkey through ICSI, either with micro-TESE, TESA sperm or with ejaculate, were achieved in our center.  

ICSI (Intracytoplasmic Sperm Injection)  

IMSI (Intracytoplasmic Morphologically Selected Sperm Injection) 

IMSI, one of the latest developments in Assisted Reproductive Techniques, has been carried out by our laboratory’s experienced personnel since 2008.

Many studies have shown that the presence of vacuoles in the head of the sperm, where genetic material is contained, may indicate DNA damage which would result in fertilization failure, or slow or completely blocked embryonic growth, and would thus reduce the chance of an eventual pregnancy.

Classic ICSI (Intracytoplasmic sperm injection) is performed under 200-400x magnification, in order to select morphologically appropriate and motile sperm. However, this magnification is not sufficient to identify all the morphological abnormalities of a sperm in detail. Although special staining techniques performed in the Andrology Laboratory can help to detect abnormalities in sperm, these procedures make sperm cells unsuitable for future injection.

IMSI, on the other hand, is performed under 8050x magnification, using special optics, and can discard morphologically abnormal sperm, thus creating the opportunity to use the sperm with the best-morphology for injection. 

In couples with male factor infertility, repeated unsuccessful ART trials and unexplained infertility, IMSI can help to identify the underlying cause of infertility and select the sperm most likely to lead to a pregnancy. 

IMSI (Intracytoplasmic Morphologically Selected Sperm Injection) 


Spermatozoa under  400x magnification,
using an ICSI microscope.
Head anomalies are invisible.
Spermatozoa under 8050x magnification,
using an IMSI microscope.
Head anomalies are easily detected
(two cells with normal heads on the left and one with
an abnormal head on the right).


IMSI (8050x magnification)    
Large vacuoles in spermatozoa head
IMSI (8050x magnification)
Large vacuoles in all spermatozoa heads



IMSI (8050x magnification)
Sperm classification
IMSI (8050x magnification)
Sperm cells with abnormal heads and multiple

Checking for fertilization: pronuclear evaluation

Oocytes are checked for fertilization 12-18 hours after IVF or ICSI. Fertilization can be summarized as the union of two nuclei within the activated oocyte cytoplasm (one coming from the sperm and one from the oocyte) containing the genetic material of both parents. Two pronuclei (nucleus of a sperm or an oocyte during the process of fertilization, after the sperm enters the egg, but before they fuse) and first and second polar bodies (one of the minute cells arising from the divisions of the oocyte at or near the time of fertilization) are present in a normally fertilized egg.

At this stage the following are assessed (please see pictures below):

  • The positions and the size of the two pronuclei (PN),
  • The number, size and distribution of the nucleolar precursor bodies (NPB),
  • The presence of polar bodies and the existence of the cytoplasmic halo.

Studies indicate that errors or asynchronization occurring during PN formation may correlate with chromosomal abnormalities and aneuploidy.

 Pronuclear scoring
  A normally fertilized, A score egg with two pronuclei (PN) and well positioned nucleolar precursor bodies (NPB). The two extruded polar bodies (PB) are on the left of the oocyte. 

After fertilization: classifying cleavage-stage embryos

22 to 25 hours after fertilization, the zygote (cell produced by the union of the two gametes, before cleavage) undergoes its first division (cleavage) and becomes a two-cell embryo. A normally growing embryo is then expected to have 3-4 cells on the 2nd day (42-44 hours), 6-8 cells on the 3rd day (66-68 hours) and more than 10 cells on the 4th day.

The number of cells is the first parameter that will determine the grade of the embryo. Thus, if the number of blastomeres (any cell produced during cleavage) is unexpectedly low considering its developmental day, the embryo is evaluated as slow-growing. Additionally, cleavage-stage embryos are checked for the following:

  • Size and shape of blastomeres,
  • Degree of fragmentation between blastomeres (uneven, unclean division of the cells of the embryo, resulting in cytoplasmic debris),
  • Nucleus number in each blastomere,
  • Cytoplasmic appearance,
  • Signs of compaction (please see section below “Going Beyond Day 3”).

Grade 1: Embryos with equal-sized blastomeres, including 0-10% fragmentation and with no granular structure in the cytoplasm are ranked as top quality;

Grade 2: Embryos with slightly unequal-sized blastomeres or including 0-10% fragmentation;

Grade 3: Embryos with either slightly unequal-sized blastomeres including 10-20% fragmentation or with significantly unequal-sized blastomeres including 20-25% fragmentation;

Grade 4: Blastomeres cannot be counted or are distinctly different from each other with a fragmentation more than >25%.

When the quality of the embryo is reduced, its potential to develop to sustain its vitality and to implant decreases. 

4-cell embryo
(44th hour of development)
8-cell embryo with no compaction
(72nd hour of development)
  8-cell embryo with compaction (72nd hour of development) 

Going beyond day3: assessing late-stage embryos

Compaction starts on the 4th day past fertilization, after approximately 96 hours, when the number of cells in the embryo reaches 16 to 20. Intercellular tight junctions (desmosomes and gap junctions) are formed between cells, creating a compact mass called a morula. Cavitation occurs next where the outer layer of cells (trophectoderm) secrete fluid into the morula. Thus, a central, fluid-filled cavity (blastocoele) is formed (please see the pictures below).  

The quality of 4th day embryos is assessed as follows:

Grade 1: Early blastocyst, cavitated embryo or morula without any anomaly (such as fragmentation or vacuolization);

Grade 2: Morula or compacted embryo with one anomaly (such as fragmentation or vacuolization);

Grade 3: Morula or compacted embryo with 2 or 3 anomalies or embryo with no signs of compaction (10 or more blastomeres); 

Grade 4: Embryo with no compaction (10 or fewer blastomeres).

Ten-cell embryos with compaction on the 4th day of development (grade III)
Morula on the 4th day of development (96th hour) (grade I)

A cavitating embryo on the 4th day of development (96th hour) (grade I) 

Blastocyst culture

Blastocyst formation occurs at day 5 or day 6 after insemination. One of the greatest advantages of blastocyst culture is that it can identify slow growing or growth-arrested embryos which ultimately give a low chance of pregnancy. In our center, to select the best embryo with the best survival capacity, zygotes are cultured until blastocyst stage and the transfer is usually done on day 5, resulting in a pregnancy rate of 65-70%

Another advantage of blastocyst transfer is the reduction in the number of multiple pregnancies. The main reason for this complication is the practice of transferring more than one embryo, in order to increase pregnancy chances after inadequate embryo selection on day 3. In the past 7 years, our laboratory has successfully established blastocyst culture, allowing the selection of the embryo with the best morphology and the best chance of implanting in the uterus and producing a healthy pregnancy. 

Classifying blastocysts

The blastocyst is composed of two differentiated cell groups: the inner cell mass that will later develop into the fetus and the outer cell mass, or trophectoderm, which will produce the placenta and embryonic sac. To transfer the embryo with the best morphology, each blastocyst is assessed regarding:

  • the development (from cavitation to hatching –through a series of expansion-contraction cycles the embryo bulges out and emerges from the rigid envelop, the zona pellucida–),
  • the inner cell mass
  • the trophectoderm
Score Blastocyst development
1 Starting cavitation; volume of blastocoele <50% of the whole embryo
2 Blastocoele volume > 50% of the embryo
3 Blastocoele reaches the volume of the whole embryo
4 Blastocoele exceeds the embryo in size, thinning the zona pellucida
5 Hatching starts
6 Complete hatching, the embryo leaves the zona pellucida 


Score Trophectoderm quality
A Epithelial structure with many closely attached cells
B Epithelial structure with some loose cells
C Epithelial structure with many loose cells 


Score Inner cell mass quality
A Many cells packed together
B Many cells but loosely packed
C Few cells



5th day 5AA, top quality blastocyst
  Orrange: inner cell mass (ICM)
Green: trophectoderm (TE)
5th day 6AA, top quality blastocyst (the
embryo start to leave the
zona pellucida, ZP) 

Continuous embryo monitoring system in assisted reproductive technology: Embryoscope

In a natural cycle, fertilization occurs in the fallopian tubes. During the migration of the zygote from the tube to the uterus, the development of the embryo is supported by nutrients present in the uterine environment with constant pH and temperature until implantation into to the uterine wall occurs.

Assisted reproductive technologies aim to mimic the conditions of the uterine environment and thus to create the most appropriate setting for embryo culture in a laboratory, from oocyte pick-up to embryo transfer, in order to create the highest chance of pregnancy. For this reason, a constant temperature (37°C) is provided by specifically designed incubators, nutrients are supplied by the culture media in which the embryos are floating, and finally, optimal pH is assured by the amount of carbon dioxide gas inside the incubator and by the culture medium.

Embryos obtained by assisted reproductive technologies are kept under close observation in order to determine which of them has the highest potential to implant in the uterus and produce a pregnancy. In most cases, embryologists grade the developing embryos under the microscope, outside incubators, at precise intervals (day 1, 2, 3, 4 and 5) in a very short time (maximum 1-2 minutes). In addition, for cases where it may be beneficial to protect the embryo from outside conditions and further minimize changes in temperature and pH, the Embryoscope® has been developed. The Embryoscope allows continuous monitoring of the development of embryos inside the incubator with a special light source and camera, thus eliminating the necessity of taking embryos outside the incubator. Images of the embryo growth are recorded at short time intervals (20 minutes) without disrupting the stable environmental conditions. Using a continuous embryo monitoring system instead of classic ART allows embryologists to accurately evaluate the timeline of embryological development, eg. pronuclei surfacing, cell divisions timings, morula and blastocyst formation. It also allows the simultaneous observation of 12 embryos for each patient. Doctors and embryologists can compare the development of these embryos prior to transfer and pick up the embryo that has the greatest implantation chance. Additionally, patients with unexplained low embryo quality can benefit from this system in future attempts by the possible identification of the underlying defect in the cell cycle. 


Helping embryos to leave the zona: assisted hatching

The term “hatching” refers to the blastocyst budding from and finally leaving the glycoprotein membrane (zona pellucida) enclosing it. Hatching of the blastocyst, occurring on the 5th or 6th day after insemination, is a prerequisite for the implantation of the embryo in the uterus. Assisted hatching (AHA), by either the thinning or the opening of the zona pellucida, is performed in the laboratory before transfer to facilitate the implantation of the embryos. It can be performed mechanically with the help of a glass microneedle, chemically with acid tyrode or with a specially designed laser. Laser is the chosen method in our center because no chemicals are added to the culture medium of the embryo. It is also safer, as the laser is controlled by a computer with microsecond precision.

Mechanical AHA: with the help of a glass microneedle,
a V-shaped or squared opening is made on the zona pellucida.
Chemical AHA: acid Tyrode’s solution is used to
create the opening through the zona pellucida.


AHA with a laser: the zona opening is very quick and easy. This is the method used in our center.


  Assisted Hatching (AHA)

Determining the number of embryos to be transferred

The number of embryos which may be transferred during one treatment is restricted to one for women aged under 35 and two for women aged 35 and over, in accordance with the regulation issued by the Ministry of Health on March 2010. Women under 35 who have had two previous IVF cycles may have two embryos transferred. 

First examination: catheter trial

Embryo transfer, which is the last step of an ART treatment, is the replacing of the cultured embryo(s) into the uterus. To perform the embryo transfer with minimum trauma, a catheter trial is done on the first clinical examination and thereby precautions are taken in cases where the transfer is likely to be difficult. A narrowing or a displacement of the cervix related with previous surgery, congenital anomaly or myoma causing compression may be corrected when diagnosed by hysteroscopy.

Soft embryo transfer catheter 

Replacing the embryos in the womb: embryo transfer

To ensure correct placement of the embryos near the uterine fundus, the transfer procedure is performed under ultrasound guidance, with a full bladder, necessary to straighten the passage to the uterus and enable a safe and easy transfer. A soft catheter is introduced through the cervix and the uterine cavity by the clinician, and the embryos, loaded by the embryologist, are gently .placed into the uterine cavity. As the tip of the catheter is visible under the ultrasound, the most appropriate angle can be achieved, and the region with best implantation potential can be determined without causing any trauma. The catheter is then slowly removed and the embryologist examines under the microscope whether the embryos have been correctly transferred. Anesthesia is often not necessary. 


Replacing the embryos with ultrasound guidance

After embryo transfer: some recommendations

Following the placement of the embryos into the uterus, a half an hour of bed rest is required. The patient is discharged 2-3 hours after the procedure. It is well established that longer rest does not increase the chances of pregnancy. We recommend avoiding strenuous physical activity in the first 24 hours, but the patient can carry on with her normal daily life the next day. However, we recommend avoiding sports, demanding physical tasks and the lifting of heavy objects until the day of the pregnancy test. There is no objection to car and air travel. On 12th and 14th days following oocyte pick-up, a blood pregnancy test is done.