Recurrent IVF Treatment Failure

In general terms, three or more (according to some standards, two or more) unsuccessful IVF treatment cycles are defined as recurrent IVF failure. When the causes are evaluated, embryo quality (morphological and genetic), uterine factors, as well as the mother-to-be’s systemic diseases and immune factors come to the forefront. However, it should not be forgotten that even with the most advanced techniques, we cannot talk about complete success. There are still many factors at the molecular level that have not yet been clarified.

Recurrent IVF failure is an extremely stressful situation for couples, and many do not wish to continue treatment. In the evaluation of these cases, an experienced physician should work to identify the cause or causes of failure. If a cause can be found, this problem should first be eliminated in order to aim for a higher success rate.

Many couples with IVF failure often complain that “we were not told why it failed, the same treatment was always applied, we were not informed.” Therefore, before a new treatment plan, the physician must be sincerely committed to this work and be scientifically well-equipped in this field. The methods offered to couples must be evidence-based, free of harmful side effects, and ethically applicable.

To mention the most up-to-date techniques used in cases of recurrent IVF failure:

Techniques used in cases related to male factor:

When infertility cases are analyzed, the male factor stands out in a significant proportion. The analysis of male infertility and the diagnostic and treatment methods used are rapidly advancing, and many couples can achieve positive outcomes. The basic analysis when evaluating the male factor is the examination of the sperm sample. Sperm count, motility, fertilization capacity, and genetic analysis in selected cases are important. Low sperm count, low motility or complete immotility, morphological abnormalities, and a high rate of DNA fragmentation in the sperm sample are causes of infertility. Today, environmental factors, exposure to many toxic substances in working conditions, smoking, and increasing male age reduce sperm quality.

A thorough evaluation of the male partner before treatment is important. In some cases, specific antioxidant and vitamin therapy may be necessary. In more serious cases, hormone therapy and stimulation of sperm production may be required.

Among the difficult-to-treat groups today are cases with very low sperm count and poor quality, and azoospermia cases with no sperm at all. In severe male factor infertility, ICSI, that is, intracytoplasmic sperm injection within IVF techniques, has opened an important new era and provided solutions in many cases. However, progress has not stopped there. In the microinjection technique, a single sperm is injected into each egg. The quality of the selected sperm directly affects the treatment outcome. The latest developments and techniques related to this selection are as follows:

IMSI (high magnification ICSI)

This is a highly effective method recently introduced for infertile couples with severe male factor. This method is still relatively new and is applied in only a few distinguished centers worldwide. Despite being new, its results have been clearly evaluated and its effectiveness has been proven. It allows the sperm to be used in the microinjection procedure to be examined at thousands of times magnification using the most advanced technological means, thus enabling the selection of the best sperm. A special microscope is used for this procedure. In addition to requiring a significant financial investment, this special microscope also requires a distinct training period and experience for its use. 

In the microinjection procedure, each mature female egg is fertilized with a single sperm. Embryo quality is determined by the quality of the egg and sperm. The selection of the sperm to be used for fertilization is of vital importance. If the sperm with the best fertilization capacity and the highest genetic quality is selected, fertilization rates are high, embryo quality is high, pregnancy rates are high, and pregnancy loss rates are low.

IMSI is used in cases with severe sperm problems. In the standard microinjection system, sperm are selected at 100 to 400 times magnification. In high magnification microinjection, that is IMSI, sperm are magnified up to 1,600 to 7,000 times, examined in great detail, especially certain features of the sperm head, and then selected for microinjection.

Another new technique is to examine the sperm under a polarizing microscope during microinjection and select it according to the brightness in the head region. Recent studies have shown a higher rate of brightness in morphologically normal sperm.

Magnetic Sperm Selection (sperm magnet): When all infertile couples are evaluated, male factor infertility accounts for approximately 50% of the underlying causes. In sperm analyses, low sperm count (oligospermia), low motility (asthenospermia), and morphological abnormalities (teratospermia) are the most important parameters playing a role in male infertility. Today, it has been shown that there are other abnormalities in sperm that cannot be detected with standard microscopes. To avoid using sperm cells with low fertilization capacity in microinjection, a technology called Magnetic Activated Cell Sorter (MACS) is used. Although this technology is promising in male infertility cases where microinjection is applied, there are not yet sufficient studies to reach a definitive consensus.

Co-culture Method

Co-culture can be examined in different groups: endometrial co-culture, which was used in previous years and is produced from cells of the uterine lining, and a very new method produced from the cells surrounding the egg (granulosa cell co-culture). It is an additional culture medium. Embryos are developed in the laboratory in special fluids. These fluids serve as a nutrient medium from the fertilization of the egg and sperm to the development of the embryo and its placement in the uterus, and they mimic the fluids in the uterus and fallopian tubes. The co-culture medium, as an additional nutrient environment, contributes to embryo development through the growth factors it secretes. With this method, higher-quality embryos are obtained and pregnancy rates are increased in selected cases. It can be easily applied during a standard IVF treatment cycle and does not create additional cost for the patient. This is very important. In older co-culture methods, cells taken from the uterine lining were used; the patient had to undergo an intrauterine sampling procedure one month before the IVF treatment cycle and pay a significant additional fee. Apart from the cost, co-culture using uterine cells, which was used as an older method, did not provide the expected results in many scientific studies and has been abandoned by many centers. The new co-culture method produced from cells surrounding the egg provides much better results in every respect.

Detailed analyses and scientific studies on the co-culture method, which has been used for a long time, have shown that this method significantly increases pregnancy rates in many groups of cases, especially in recurrent IVF failure. In this field, Assoc. Prof. Dr. Aygül Demirol, French scientists, and Dr. Moncef Benkhalifa from this group have completed important scientific studies. 

This method is applied in certain centers in the USA, France, Turkey, and India.

To achieve success, the combined use of some techniques is also required. For example, with a special microinjection system using laser technology, the egg is less damaged and higher-quality embryos are obtained. When the laser system and co-culture are applied together in a patient-specific manner, maximum success is achieved. Laser-assisted microinjection is another technique whose effectiveness has been scientifically proven in our publications.

Preimplantation Genetic Diagnosis: PGD (Preimplantation Genetic Diagnosis: PGT) is the process during IVF in which embryos are genetically tested for a specific disorder and only healthy ones are selected and transferred to the mother.

Preimplantation Genetic Screening (PGS): is the evaluation of embryos for chromosomal abnormalities called aneuploidy before they are placed in the uterus. With the PGS method, the aim is to identify and transfer embryos in which no anomaly is observed and to achieve the birth of a healthy baby.

Aneuploidy, which is known as having more or fewer chromosomes than normal, is one of the most common chromosomal abnormalities. Preimplantation Genetic Screening (PGS) is the evaluation of embryos for aneuploidy before they are placed in the uterus. Aneuploidy can occur in both the egg and the sperm, or it can arise in the embryo at the fertilization stage. Depending on the type of chromosomal abnormality present, aneuploidy can cause physical and/or mental developmental problems. With the PGS method, the aim is to identify and transfer embryos in which no anomaly is observed and to achieve the birth of a healthy baby.

Some of the embryos obtained in IVF cannot implant in the uterus, some stop developing at an early stage, and some, even if they pass these stages, may not continue to develop until birth. As a result, 20% of pregnancies, as in natural pregnancies, may end in miscarriage. Among the causes of embryo developmental failure, chromosomal irregularities account for the largest share. The most common chromosomal abnormalities seen in miscarriages are trisomy (three copies of a chromosome) of chromosomes 13, 16, 18, 21, and 22, monosomy (having only one copy of a chromosome), and numerical abnormalities in the sex chromosomes (X and Y).

What is PGD?

Preimplantation Genetic Diagnosis (PGD) is applied to couples at risk for cystic fibrosis, thalassemia, or balanced structural translocation. Balanced structural translocation causes the formation of genetically unbalanced gametes (sperm or egg), and if these achieve fertilization, they can lead to deficiencies or excesses in genetic material, resulting in a chromosomally abnormal embryo. Such a situation in the embryo can lead to embryo death, miscarriage, or the birth of a child with serious medical problems.

How and When Are PGS/PGD Performed?

After fertilization, the embryo is monitored in the culture system until it reaches the 8–10 cell stage, which takes 3 days. One or two cells (blastomeres) are taken from these 3-day-old embryos by biopsy, and chromosomal analysis is performed on specific chromosomes. PGS/PGD is applied to the chromosomes on which chromosomal abnormalities are most frequently seen in miscarriages and live births (13, 16, 18, 21, 22, X, and Y).

While PGS/PGD is being performed on chromosomes, the Fluorescence In Situ Hybridization (FISH) method is used, in which special probes (small DNA fragments) bind to specific regions to be analyzed. Each probe is labeled with a different fluorescent dye. These fluorescent probes are brought into contact with the biopsied cells and are expected to hybridize with specific chromosomes. Using a fluorescence microscope, the geneticist evaluates the colored signals (one, two, or three) seen on a given chromosome. A single signal is evaluated as monosomy, two signals as disomy (normal), and three signals as trisomy. In this way, aneuploidy in specific chromosomes can be identified.

What Are the Advantages of PGS/PGD?

·       To increase pregnancy rates by selecting the best embryo to be transferred, and to decide which embryos will be frozen or discarded

·       To reduce the risk of miscarriage

·       To provide better counseling and guidance to patients

·       To encourage patients who have chromosomally normal embryos but have not been able to conceive

        Indications for PGS

·       Advanced maternal age (over 35–37 years)

·       Recurrent miscarriages

·       Recurrent IVF failure

·       History of a previous pregnancy with chromosomal abnormality

·       Application of intracytoplasmic sperm injection (ICSI) with abnormally shaped sperm

·       Presence of aneuploidy in sperm

·       Presence of aneuploid mosaicism in at least one of the partners

·       At least one partner being a carrier of an X-linked disease

Indications for PGD

·       At least one partner being a carrier of a rearranged structural chromosome (translocation, inversion, deletion)

·       To obtain a healthy pregnancy by diagnosing a known genetic disease

Some disadvantages of the PGD method:

·       Technical problems may occur during the preparation or biopsy stages

·       Even if IVF and PGD are successfully performed, pregnancy may not occur after transfer.

·       The analysis results of a single cell may be undiagnosable or limited due to mosaicism (because blastomeres of the embryo may have different contents). Therefore, the result should be confirmed with prenatal diagnosis.

·       Since only a limited number of chromosomes can be checked, not all chromosomal and genetic abnormalities can be diagnosed with PGD.

·       As a result, only a specific test can be performed on the biopsied cell. All genetic problems cannot be screened with a single cell taken for testing.

What Are the Benefits of PGD?

• In appropriate cases, it increases the success of IVF treatment.
• In selected cases, it increases pregnancy rates.
• It reduces the risk of pregnancy ending in abortion (miscarriage).
• It reduces the need for medical termination of pregnancy.
  • It reduces the rate of multiple pregnancies.
• It reduces the economic and psychological burden caused by recurrent unsuccessful IVF attempts.

Who Should Undergo PGD?

• Women of advanced maternal age 
• Couples who have undergone two or more IVF cycles without achieving pregnancy
• Couples with recurrent early pregnancy losses (miscarriages) due to causes other than translocation carrier status
• Couples who are balanced translocation carriers
•  Partners at risk for certain single-gene diseases that can be diagnosed, such as Familial Mediterranean Fever, Sickle Cell Anemia, Cystic Fibrosis, SMA
•  Selection of an embryo that is HLA-compatible with family members
•  Couples who have had a child with a genetic disease in a previous pregnancy
• Mothers with a history of pregnancy with aneuploidy (chromosomal abnormality)
•  Cases of gonadal mosaicism (where, despite normal genetic test results in the parents, there are two or more offspring with the same abnormality)
•  TESE cases (cases with severe male infertility)
•  Poor responders (cases that respond inadequately to ovarian hyperstimulation protocols)
•  Embryonic sex determination in terms of X-linked diseases when direct genetic diagnosis of the disease in question cannot be performed.

Is There a Margin of Error in Embryo Biopsy and PGT?
To perform PGT (Preimplantation Genetic Diagnosis), a cell sample must be taken from each of the patient’s embryos. During PGT, the likelihood of damage to embryos is almost negligible, and the damage rate is reported as 0.3%.

Depending on the method used, the probability of PGT giving an incorrect result ranges between 2% and 7%.

What Are “Single-Gene Diseases”?
Single-gene diseases are a group of genetic disorders that occur as a result of dysfunction of the units called “genes,” which are encoded on DNA. These genetic diseases increase particularly in consanguineous marriages.

Because consanguineous marriages are common in our country, it is very important to diagnose such diseases in the prenatal period and even at the embryo stage (preimplantation diagnosis). It is possible to perform preimplantation diagnosis for all single-gene diseases that can be diagnosed postnatally (after birth) or prenatally (before birth).

Preimplantation genetic diagnosis of single-gene diseases is based on DNA analysis of a single cell. Preimplantation genetic diagnoses have been performed for a long list of diseases such as Cystic Fibrosis, Hemophilia A and B, Alpha-1 antitrypsin deficiency, Tay-Sachs and Sickle Cell Anemia, Retinitis Pigmentosa, Thalassemias (Mediterranean anemia), Alport, Gaucher’s disease, Long-chain acyl-CoA dehydrogenase deficiency, Multiple epiphyseal dysplasia, Achondroplasia (dwarfism), Neurofibromatosis, Epidermolysis bullosa, Myotonic dystrophy, X-linked hydrocephalus, cancer predisposition, and Fanconi anemia. 

Embryo Glue

When the embryos selected for transfer are loaded into the transfer catheter, they are taken into the catheter together with special fluids. This special fluid ensures compatibility between the uterine cavity and the embryos. By adding certain supplements to these special nutrient fluids, efforts are made to increase the adhesion of the embryo to the uterus.

Some products have been marketed with the addition of certain supplements to the transfer solution. Embryo glue, as it is called in English, should be used to increase compatibility between the embryo and the uterus and to improve pregnancy rates. 

For the evaluation of uterine factors:

Office Hysteroscopy:

Hysteroscopy is a technique with a gold standard for evaluating the inside of the uterus. With the recently developed office hysteroscopy, this procedure can be performed in a short time without anesthesia in office conditions, and if uterine problems are present, diagnosis and treatment can be carried out at the same time. For example, structural problems such as intrauterine adhesions, polypoid growths, uterine septum cases, and pathological issues such as endometrial hyperplasia and endometritis can be treated, thereby increasing pregnancy rates. Office hysteroscopy is successfully applied to our patients when necessary and is also used for diagnosis and treatment in many patients with recurrent pregnancy loss.

In addition, even if no problem is found during hysteroscopy, some procedures that revitalize and stimulate the uterine lining cells (microtrauma called injury to stimulate stem cells) have a positive effect on increasing pregnancy rates. In a joint project with the University of Sheffield in England, Assoc. Prof. Dr. Aygül Demirol has demonstrated the effectiveness of this treatment. It requires special application.

Detailed evaluation of the couple:

When many recurrent IVF failure cases are evaluated, we encounter deficiencies in detailed systemic analyses. Therefore, a thorough evaluation of the couple in every respect is essential. In particular, hormonal and immune evaluations are very important. When thyroid problems, high prolactin levels, and sometimes subtle, undiagnosed rheumatologic, gastroenterologic, and other systemic problems related to the immune system are resolved, a good response to treatment can be obtained. In addition, systemic coagulation defects should not be overlooked in terms of treatment success and complications. They play an important role, directly or indirectly, in embryo implantation. They also have an important place in pregnancy losses. Within our general concept, we believe that detailed analysis and pre-treatment should be given in selected cases. Meanwhile, we must not forget that psychological evaluation of the couple and preparing them for treatment with confidence and information by a specialized team is also an indispensable factor.

Determining the treatment period that will provide the best response:

The golden key to success in IVF treatments is obtaining a good number of high-quality eggs. A woman does not show the same performance in treatment every month. In other words, in a certain period when you obtain a good number and quality of eggs from the same woman, in another month when the response is not optimal, she may produce fewer and lower-quality eggs. To predict these periods, the patient’s history must be well evaluated and analyzed, a detailed examination must be performed, and her condition must be clarified with some special tests. In addition, not every patient responds in the same way to the same protocol and hormone injections. The dose and treatment details must be reorganized in a different, patient-specific way.

In some special cases, supportive treatments for preparation before IVF are required:

In many disease groups, especially in conditions frequently associated with infertility such as PCOS (Polycystic Ovary Syndrome) and endometriosis, patient-specific preparation and supportive pre-treatments have an extremely positive effect on success. In addition, in cases with low ovarian reserve, that is, low response to treatment, certain special hormonal, herbal, or other supportive therapies tailored to the patient’s underlying condition can have a major impact on the outcome and help achieve success.

In male infertility as well, provided that it is patient-specific, pre-treatment with hormone support when necessary, and antioxidant and vitamin supplements in special doses according to the sperm problem and underlying risk factors, can affect sperm quality, fertilization capacity, and genetic damage in sperm. At this point, we must remember that all these should be prescribed consciously by an expert team. Every day we read and hear about many herbal and other supplements in the media, and unfortunately, these comments can be made by people whose specialty is not obstetrics, infertility, or IVF. Inappropriate treatments, through various interactions, may cause harm instead of benefit, reduce success instead of increasing it by causing other systemic side effects, and even lead to long-term risks.