Intrauterine Insemination (IUI)
Selecting the spermatozoa with high quality and motility and injecting them into the uterus in called intrauterine insemination. In this procedure, the main aim is to eliminate the factors which block/decrease the sperm motility, thus helping the spermatozoa with superior fertilizing potential to reach the tubes more effectively.

Oocyte Pick-Up (OPU)
Oocyte pickup procedure is performed under anesthesia 36 hours after hCG injection is given. During procedure, follicles containing oocytes with their final maturation stages are aspirated with a special needle, collected in tubes and transfered into the embryology laboratory. There, follicular fluid is screened for the presence of oocytes with the help of a microscope and collected oocytes are transfered into special culture media. In this way, development for these oocytes and embryos outside the human body (in laboratory environment) starts..
The most important point to be considered at this stage is that not all oocytes collected can have the same quality or can be suitable for insemination. The response of the ovaries to the hormones administered during treatment is rather complex, therefore not all follicles can develop at the same phase and some of the oocytes obtained after OPU may be immature. However, unless a special factor is determined, 80% of the oocytes collected after OPU can be at the suitable maturation stage in general.
After OPU procedure, depending on the treatment procedure that had been previously determined after routine follow-up and control examinations that had been experienced with your doctor as well as properties of the semen sample obtained on the day of procedure, either in vitro fertilization (IVF) or microinjection (ICSI) is determined to be the technique of choice. According to the semen analysis results, cases having spermatozoa with high fertilization capacity is selected for IVF, cases showing low concentration and motility is selected for ICSI procedure.

Obtaining spermatozoa for the procedure
 Spermatozoa that are to be processed for either IVF or microinjection are obtained by masturbation on the day of oocyte collection. Before semen sampling, the male partner should have 2-3 days of abstinence (no intercourse during this time or ejaculate for any other reasons); otherwise, quality and quantity of the sperm can affect the programmed treatment plan.
Collected semen sample is then brought to the laboratory and evaluated for basal parameters such as concentration, motility etc. After analysis, spermatozoa are prepared for insemination by the selected treatment strategy that has previously been determined by your doctor.
For couples in which male partner can not be present on the day of insemination or for male partners having difficulty in giving the semen sample, semen sample can be collected several days prior to the day of oocyte collection, frozen and can be thawed and used for insemination on the day of the procedure.
With the help of the new technologies and techniques, for patients showing no spermatozoa in their ejaculate sample, it can now be possible to extract spermatozoa from testicular tissue by surgical intervention (TESA and TESE procedures) and those spermatozoa can be used in microinjection resulting in acceptable pregnancy rates. For the last 2-3 years, several clinics including our centre in the world have been using microdissection TESE procedure, in which a special microscope is being used during operation, and observing an increase in the sperm retrieval rates and pregnancy rates.  

In Vitro Fertilization (IVF)
It is the preferred method for cases in which a physiological female factor (i.e. tubal blockage) is present but no apparent problems related to sperm concentration, motility and morphology are observed. Oocytes obtained after OPU procedure is incubated together with the spermatozoa that have been washed and processed in order to increase their fertilization ability thereby allowing certain number of motile spermatozoa to pass through the outer shell (zona pellucida) and fertilize the oocyte. In cases having low number of oocytes, or severe male factor cases in which sperm parameters are not suitable for IVF, microinjection technique is the preferred treatment method.

Microinjection (ICSI)
Also called as intracytoplasmic sperm injection, microinjection is a technique in which a sperm cell is injected into an oocyte with the help of a micromanipulation system attached to an advanced microscope and the micro needles connected to the system from outside. Especially in cases with low sperm concentration and motility, since fertilization with IVF is extremely low, aspiration of spermatozoa with normal morphology into a micro needle and directly injecting it into the oocyte results in high fertilization and embryo development. Therefore, couples who previously had no chance of having a baby due to severe sperm problems in the past can have this opportunity with microinjection.
(Click this link to watch the ICSI video).

Embryo culture
After OPU procedure, oocytes obtained from the female partner and spermatozoa retrieved from male partner
are inseminated either by IVF or microinjection method. Sixteen to eighteen hours after insemination, in order to determine the fertilization, a routine control is performed by special microscope and visualization systems. Fertilized oocytes at this stage are called pre-embryo or zygote. During the days in vitro, daily development of each zygote is evaluated within certain time limits by laboratory personnel and best embryos are scored. According to certain criteria such as female age, number of oocytes or embryos obtained, embryo quality, endometrial thickness etc., day of embryo transfer and the number of embryos to be transfered are determined by laboratory personnel and the clinician. Pictures below represent the embryo development on each day after OPU procedure.

Assisted Hatching
In mammals, oocytes and embryos are surrounded with a shell called zona pellucida (ZP). This shell structure, besides controlling the fertilization of the oocyte with a sperm, it also protects the developing embryo physiologically from external forces until implantation. ZP consists of 3 different glycoprotein molecules and during fertilization spermatozoa is attached to the oocyte through these proteins. After attachment, initiation of fertilization also triggers the structural change of these glycoproteins, preventing other sperms to bind the zona pellucida.
The main purpose of zona pellucida is to maintain the integrity of the embryo after fertilization. Cells (blastomeres) that form an embryo are attached together with weak bonds until the blastocyst stage, the stage that the embryo becomes suitable for implantation and ZP structure keep these cells together and maintain their integrity during cleavage stages. However, in order to achieve a pregnancy, it is necessary for a blastocyst-stage embryo to get out of the zona pellucida properly (hatching). Studies have shown that embryos obtained by in vitro fertilization and are grown in vitro can have problems during hatching. For this reason, during treatment it is thought that one of the reasons of implantation and pregnancy failures in some cases can be the inability or the difficulty of an embryo to hatch.
Assisted hatching (AHA) technique is the artificial opening or thinning of zona pellucida so that the probability of an embryo to experience hatching difficulties can be minimized.  Nowadays three different techniques are applied for AHA: Mechanical technique, enzyme technique and laser technique. In our centre, AHA is done by laser system, like many other reputable centers worldwide.

Embryo Transfer
From fertilization through early embryo development stages, embryos are evaluated daily for their developmental characteristics and the ones with higher pregnancy potential are scored and determined. After selection, selected embryos are transfered into the uterus. Depending on the number of embryos developed during the course of the treatment, embryos having good quality can also be frozen and stored in the laboratory for a later use according to the couple’s consent. These embryos can, in the future, be thawed and be used for a second trial in which the use of external hormones and drugs is minimized, hence much more convenient for the female partner.
When the day of oocyte collection is considered as day 0, depending on the fertilization and embryo development, embryo transfer is performed on day 2, day 3 or day 5. Couples with only limited number of developing embryos or cases in which the best embryos can be selected, embryo transfer is done on day 2 and day 3, while for patients with high number of good quality embryos, in order to obtain more information about embryo development and to better select the embryos with highest implantation potential, embryo transfer procedure is performed on day 5 (blastocyst transfer).
Nowadays, blastocyst stage embryo transfer is used in numerous laboratories in order to decrease the multiple pregnancy rates and to increase the implantation rate. Being also termed as blastocyst culture, the main aim of this approach is to better select the embryos with superior developmental and implantation potential, hence decreasing the number of embryos to be transfered whereby decreasing the multiple pregnancies.
Transferring more than one embryo increases the pregnancy rate. However, increasing the number of embryos to be transfered also proportionally increase the multiple pregnancy rates. Besides financial and emotional burden it can cause upon couples, multiple pregnancies can also lead to risks including increased abortion, preterm delivery and developmental retardation.
Prior to ET procedure, selected embryos is loaded into a specially-designed catheter, and delivered to the uterus transvaginally. In terms of patient comfort, embryo transfer is much easier and faster procedure compared to OPU procedure. No anesthesia is required except very special cases.
 
Embryo Cryopreservation
Based upon the legal framework of the country in which assisted reproductive techniques are practiced, gamete cells (oocyte, spermatozoa), fertilized zygotes and embryos can be cryopreserved during the current treatment and used in a subsequent treatment upon thawing in the near future. However, depending upon the changing physiology the cells with different developmental stages, considerable variations exist on survival rates after thawing.
Nowadays two techniques are used in gamete and embryo cryopreservation: slow freezing and vitrification.
In slow freezing method, in order to minimize the damage that can emerge during freezing, gametes/embryos to be frozen are incubated in a special cryoprotectant solution and placed in a special device which decreases the temperature of the embryos in a controlled and programmed manner. Slow freezing method is the most common freezing method worldwide. On the other hand, as the developmental stage of the embryos to be frozen increases, a proportional decrease in the survival rate can be observed, which can (depending on the number of embryos cryopreserved) sometimes lead to the cancellation of the current thawing treatment.  Fort this reason, cryopreservation of blastocyst-stage embryos by vitrification method is recommended as an alternative and numerous clinics now report an increase in the efficiency of blastocyst-stage vitrification upon slow freezing procedures. Also, studies show that vitrification can be used not only for blastocyst stage but also during cleavage stages.

Embryo biopsy procedure
Due to certain indications which require genetic screening of the embryos, approximately 3 days after OPU procedure, removal or aspiration of one or two cells from an embryo with more than 6 cells using micromanipulation techniques is called embryo biopsy. Depending on the nature of the indication, aspirated cells are sent to the Genetics laboratory and analyzed either in chromosome or gene level.  Based on the results, embryo(s) that is diagnosed as healthy are selected for embryo transfer. When it is performed with experienced personnel and suitable equipments, biopsy procedure does not harm the embryos and does not affect their implantation potential. Moreover, it is widely used as a method for increasing the chance of having a healthy pregnancy in cases with repeated implantation failures, recurrent abortions, and cases with structural chromosomal abnormalities.