Recurrent IVF Failures

The unexplained recurrent failures of IVF treatment are a common, distressing, and challenging clinical problem. This article provides a summary of the evidence supporting various tests/treatments, which tests and treatments can be recommended for use in IVF clinics, which ones can be considered, and those that should not be recommended until their utility and safety are confirmed by high-quality research.

Definition

The term ‘recurrent implantation failure’ (RIF) is often used when two or more good-quality embryo transfers fail to result in pregnancy. The ESHRE (European Society of Human Reproduction and Embryology) Recurrent Implantation Failure Study Group has acknowledged the need for a re-evaluation of how RIF is defined and managed, and has proposed the following definition:

Recurrent IVF failure defines the scenario where the transfer of embryos thought to be viable does not result in a positive pregnancy test often enough to warrant consideration of further investigation and/or intervention in a specific patient. This definition introduces the concept of an individualized approach that does not rely on a ‘one size fits all’ criterion (e.g., a fixed number of embryos transferred), but at least takes the situation into account. The key to this concept is the need to determine how many embryos/embryo transfers are expected to be necessary to provide an ‘acceptable’ cumulative chance of successful implantation in a specific patient.

Predicting Implantation Probability

Various factors related to women, such as age, hormonal levels, endometrial lining, uterine condition, and underlying conditions, as well as embryo-related factors like embryonic division rate, euploid embryo, male factors such as genetic disorders, IVF center and laboratory performance, transfer policies, and legal constraints determine the successful implantation probability after IVF. Ideally, a predictive model incorporating all these factors should be used to provide estimates of the cumulative chance of successful implantation over several embryo transfers, but such a model is currently not available. However, observational studies, published data obtained from the European IVF Monitoring Program (EIM) data collection (European IVF Monitoring Consortium for ESHRE and others, 2022), or the IVF center’s own data can be used to derive a model that can provide guidance.

To initiate further research, a threshold of 60% is proposed for the predicted cumulative implantation chance to define RIF. When a couple fails to achieve successful implantation with a specific number of embryo transfers, and the estimated cumulative implantation chance associated with this number exceeds 60%, they should be counseled regarding further investigation and/or treatment options.

Causes of Recurrent IVF Failure (RIF)

IVF treatment is a promising option for couples dreaming of having children. However, in some cases, recurrent failures may occur.

The causes of recurrent IVF failures and whether these causes are linked to the mother, father, embryo, or a combination of these factors are still largely unknown. In an environment where many unknowns exist, healthcare professionals recommend various tests and treatments for patients diagnosed with RIF; however, there is often a lack of clear evidence from studies that these tests or treatments have helped in achieving pregnancy in subsequent attempts.

There can be many reasons for recurrent IVF failures. Lifestyle factors, genetic reasons, anatomical reasons, chronic intrauterine infections, embryo quality, and immunological factors are the most commonly identified reasons for embryo non-implantation.

Lifestyle Factors

During fertility investigations, lifestyle factors are explored, and when faced with RIF, their review and optimization are recommended since patient behaviors can change.

Obesity, especially when accompanied by metabolic syndrome, is associated with low sperm quality. Similarly, lifestyle habits in men, such as smoking, high caffeine intake, alcohol consumption, and drug use, have been shown to negatively affect not only traditional semen parameters but also other molecular aspects such as sperm DNA integrity or redox status.

Lifestyle interventions in men may help improve certain sperm parameters as well as embryo quality, but the effects of such interventions on couples with recurrent IVF failures have not been evaluated.

There is insufficient data to recommend routine measurement of vitamin D levels or treatment of vitamin D deficiency.

Screening for Genetic Factors

It is known that genetic factors play an important role among the causes of recurrent failures during IVF treatment. Genetic factors may arise from problems that couples experience during embryo formation and implantation. Therefore, genetic testing is crucial for couples experiencing recurrent IVF failures.

Karyotype Analysis in Women and Men

In a clinical practice survey, 67% of clinicians reported considering chromosomal abnormalities as a potential risk factor for RIF, and most clinicians evaluate both female and male karyotypes. Embryo-related chromosomal abnormalities represent the main cause of early pregnancy loss in humans. Most embryo-related chromosomal aneuploidies originate from the mother. It is a known fact that abnormal parental karyotype contributes to susceptibility to chromosomal embryonic errors.

Endometrial Receptivity Array (ERA)

The ERA test is a method used to detect endometrial receptivity issues implicated as the cause of recurrent IVF failures and determine treatment methods. Endometrial receptivity refers to the suitability of the uterine lining for embryo attachment and implantation.

The ERA test is a genetic test conducted to determine whether endometrial receptivity occurs at the correct time. This test involves taking an endometrial sample to determine if endometrial receptivity is optimal for embryo transfer. For example, the expression of specific genes in the endometrium is examined, thereby determining whether endometrial receptivity is timely or needs correction.

According to ERA test results, special treatment protocols can be applied to patients with endometrial receptivity issues. These treatment protocols may include hormone medications and other treatment methods to prepare the endometrium for embryo implantation. The ERA test is an important tool for identifying the cause of recurrent IVF failures and developing a personalized approach to treatment.

The European Society of Human Reproduction and Embryology (ESHRE) has stated that there is insufficient data to recommend the routine use of any commercially available endometrial receptivity test (such as the ERA test) in identifying the cause of recurrent IVF failures. However, they noted that specific aspects of endometrial function can still be evaluated through testing.

Anatomical Causes

Anatomical studies indicate that 85% of clinicians take into account anatomical and gynecological examinations when diagnosing the cause of RIF. Asherman’s syndrome (adhesions in the uterus due to previous abortions or myomectomy resulting in amenorrhea), hydrosalpinx (blockage and fluid accumulation in the fallopian tube due to tubal inflammation), endometriosis, adenomyosis, congenital uterine abnormalities, thin endometrial lining, and uterine fibroids are significant anatomical factors that can lead to IVF failures. Hysteroscopy is the most commonly used technique for anatomical evaluations, followed by 3D (three-dimensional) and 2D (two-dimensional) transvaginal ultrasound.

Evaluation of adenomyosis, endometriosis, and uterine fibroids should be done before IVF, while further investigations including MRI or diagnostic laparoscopy should be considered if there is suspicion due to clinical or ultrasound findings after recurrent IVF failures.

Hysteroscopy is often recommended when uterine pathology is detected via transvaginal ultrasound and further diagnostics are needed (such as uterine fibroids or intrauterine adhesions). Hysteroscopic surgeries allow for the removal of polyps and fibroids inside the uterus, correction of congenital uterine anomalies, and lysis of intrauterine adhesions.

The impact of adenomyosis treatment on pregnancy or live birth rates in women with recurrent IVF failure is not yet known. Studies evaluating hysterosalpingography (HSG) in the context of recurrent IVF failure are limited, but if there is suspicion of hydrosalpinx after ultrasound, HSG or other imaging methods of the fallopian tubes may be considered.

In investigating the potential causes of recurrent IVF failures concerning endometrial function and endometrial receptivity, 59% of clinicians consider the implantation window (endometrial biopsy). Tests focusing on specific aspects of endometrial function have emerged. These tests involve examining the gene panel associated with endometrial receptivity from a biopsy taken from the uterus at the time considered to be the implantation window. Identifying these genes gives us insight into whether the endometrium is in a suitable period for embryo implantation (pre-receptive, receptive, post-receptive period).

Chronic endometritis (intrauterine infections)

Chronic endometritis has been identified in patients with recurrent IVF failures with bacterial colonization and in women without clinical signs of infection, which can reduce pregnancy rates. This condition can be diagnosed by hysteroscopy, hematoxylin and eosin staining, and CD138 labeling.

Other diagnostic tests for endometritis include chromohysteroscopy (staining of the endometrium with methylene blue during hysteroscopy), bacterial culture, and molecular techniques such as PCR, RT-PCR, and next-generation sequencing (NGS).

Currently, chronic endometritis (and vaginal infections) seem to be routinely investigated by 85% of clinicians in clinical practice. Antibiotics may be considered in treatment.

Reassessment of endometrial thickness

In clinical practice, 90% of clinicians consider the evaluation of endometrial thickness to be relevant to RIF research. A systematic review and meta-analysis investigating the relationship between endometrial thickness and live birth rates reported significantly lower live birth rates in women with thin endometrium (<7mm) compared to those with a thickness of >7mm.

What to Do About Thin Endometrium?

In cases of thin endometrium, ensuring adequate exposure to estradiol remains the cornerstone of treatment, either by applying estrogen patches to the body or by increasing oral estrogen therapy through vaginal treatment.

Intrauterine injection of platelet-rich plasma (PRP) has been investigated as a treatment to thicken the endometrium, with some studies suggesting its effectiveness in improving endometrial proliferation (i.e., thickening the uterus). However, to date, few studies have evaluated its significance for patients with recurrent IVF failures due to thin endometrium.

Similarly, uterine intrauterine granulocyte colony-stimulating factor (G-CSF) infusion has been proposed for patients with thin endometrium in IVF, with limited published studies showing conflicting results.

The value of these and other interventions following the detection of thin endometrium in patients with recurrent IVF failures will be further elucidated by additional studies.

If the endometrium remains thin despite medication, hysteroscopy may be considered to exclude intrauterine adhesions or Asherman’s syndrome.

Microbiome Profiling?

In recent years, the Human Microbiome Project has emphasized the importance of microorganisms and genomes in human health and disease. Nearly 10% of the bacterial population in the body is found in the female genital tract, with Lactobacillus species being part of the physiological flora.

Whether unhealthy microbiota is among the determinants of implantation failure is still under investigation, but 47% of clinicians in clinical practice believe it is a relevant factor. Microbiome testing is gaining much interest in the context of fertility treatment, with indications that it holds promise as a potentially treatable factor in embryo implantation.

A recent meta-analysis of six cohort studies involving a total of 1,095 women and several other studies have reported an association between dysbiotic (unhealthy) microbiota and impaired reproductive outcomes. However, this is a dynamic research area, and several questions need to be answered before the appropriate place for microbiome testing in the context of recurrent IVF failures is determined. These include the most suitable tools for microbiome assessment, the stability and spontaneous resolution rate of an inappropriate microbiome, changes during the menstrual cycle and during IVF treatment, and the effectiveness of interventions aimed at improving the microbiome. Finally, it remains uncertain whether an suboptimal microbiome can directly impair implantation or if it serves as an indication of another causative factor. Based on current data and the many unanswered questions regarding the appropriateness of microbiome testing in the context of recurrent IVF failures, the European Society of Human Reproduction and Embryology does not currently recommend profiling of the uterine and vaginal microbiome.

Metabolic and Endocrinological Factors?

82% of clinicians believe that endocrine factors are related to recurrent IVF failure. These endocrine factors mainly include thyroid function (98%), hyperprolactinemia (84%), diabetes (82%), and polycystic ovary syndrome (PCOS) (30-60%).

The latest guidelines from the European Thyroid Association consider serum TSH levels >4 mIU/l (subclinical hypothyroidism) or <0.4 mIU/l (subclinical hyperthyroidism) in the context of IVF as thyroid dysfunction, requiring further investigation and treatment.

Investigating Immunological Factors?

The relationship between immunological factors and IVF failures may vary for each couple. Therefore, conducting immunological tests and evaluating their results play a significant role in treatment planning.

Consulting with a specialist and undergoing appropriate tests are important for determining the cause of recurrent IVF failures and evaluating suitable treatment options.

It’s widely accepted that the mother’s excessive immune response to the embryo implanted in the uterus can disrupt implantation. 69% of clinicians managing recurrent IVF failure find immunological screening appropriate. Among the most commonly used tests are antithyroid antibodies (80%) and antinuclear autoantibodies (ANA) (>60%).

Which Immunological Tests to Use?

Several immunological tests are available and can be used to determine the cause of recurrent IVF failures. These tests include antithyroid antibodies, antinuclear antibody (ANA), and natural killer (NK) cell activity tests.

Antinuclear Antibodies (ANA): ANA are antibodies produced by the immune system against its own cells. High ANA levels in the blood may indicate an immune system-related issue and may hinder embryo implantation. In such cases, the treatment plan may include immunosuppressive therapy or methods of immune modulation. However, the latest reproductive medicine guidelines report insufficient evidence regarding the impact of ANA screening on pregnancy outcomes in infertile women undergoing IVF/ICSI treatment and, consequently, on clinical outcomes in recurrent IVF failure.

Natural Killer Cells (NK Cells): NK cells are an important part of the immune system and are present in the periphery (blood) and the uterus. Normally, NK cells fight infections and destroy cancer cells in the body. However, in some cases, the activity of uterine NK cells may be high, which can prevent embryo implantation. Treatment approaches have been suggested for patients with high uterine NK cell activity or evidence of dysfunction, including intralipid therapy and glucocorticoid administration. However, the European Society of Human Reproduction and Embryology does not recommend NK cell testing until better-validated tests for uterine NK cell function and treatment strategies are available.

Should Thrombophilia Screening Be Done?

Thrombophilia represents a pathological predisposition to form blood clots that can cause regional vascular occlusion, potentially harming embryo implantation. They have been widely implicated as a cause of both recurrent IVF failures and recurrent miscarriages.

In a clinical practice survey, 74% of clinicians considered investigating hematological factors in recurrent IVF failure worthwhile. 96% of these investigated antiphospholipid antibody syndrome (APS), and 75% performed screening tests for hereditary thrombophilia.

What Is Hereditary Thrombophilia?

Hereditary thrombophilia includes conditions where a genetic mutation affects the quantity or function of a protein in the coagulation pathway. Several mutations in different genes have been shown to play a role, including G1619A (Factor V Leiden), R2 H1299R (Factor V Leiden polymorphism), A1298C (methylenetetrahydrofolate reductase (MTHFR) enzyme mutation), C677T (MTHFR polymorphism), V34L (factor XIII polymorphism), G20210A (Prothrombin gene mutation), a/b L33P (MTHFR enzyme ribosomal polymorphism), and 4G/5G (Plasminogen activator inhibitor-1 (PAI-1)).

What is acquired thrombophilia?

Acquired thrombophilia includes acquired deficiencies of protein C, protein S, antithrombin III, APS (antiphospholipid syndrome), and drug-induced thrombophilia. Acquired thrombophilia has been associated with adverse pregnancy outcomes, especially recurrent miscarriages.

Investigating and managing both congenital and acquired predispositions to clotting is a fundamental clinical approach for recurrent IVF failures and recurrent miscarriages. However, the roles underlying both conditions are increasingly being questioned.

In summary, the European Society of Human Reproduction and Embryology currently recommends testing for antiphospholipid antibodies (APA) and evaluating antiphospholipid syndrome (APS) only in women with recurrent IVF failure who have additional risk factors for thrombophilia.

Embryo Quality

Embryo quality is of paramount importance in IVF treatment because it is one of the primary reasons for recurrent IVF failure. Embryo quality is determined based on factors such as the embryo’s genetic structure, cell division, and morphology.

The quality of an embryo directly affects the chances of achieving a successful pregnancy. Low-quality embryos may encounter difficulties during the implantation process and prevent pregnancy formation. Therefore, various methods are used for embryo selection.

Embryo Morphology: One of the most commonly used methods for assessing embryo quality is embryo morphology. Factors such as cell division, cell number, cell symmetry, and cell structure are examined to determine the quality of the embryo.

Genetic Tests: Genetic tests can also be used to assess embryo quality. Tests like Preimplantation Genetic Testing (PGT) examine the genetic structure of the embryo, allowing for the selection of healthy embryos. 

Blastocyst Stage Embryo Transfer

Embryos at the blastocyst stage (Day 5 embryos) may have a lower risk of chromosomal anomalies, better compatibility with the endometrium (uterine lining), and a higher chance of embryo implantation due to less uterine contractions during transfer.

A systematic review of 27 studies in IVF patients, albeit with low-quality evidence, showed that live birth rates after fresh transfer were higher in the group with blastocyst stage embryo transfer compared to the group with cleavage stage embryos.

In summary, transferring embryos with high quality is crucial for successful IVF treatment. Therefore, research on embryo selection and quality contributes to reducing recurrent IVF failures.

PGT (Preimplantation Genetic Testing)

This method aims to minimize the risk of carrying genetic diseases by performing genetic analysis on embryos of couples undergoing IVF treatment.

PGT is a technique used in IVF treatment and is performed before embryo transfer. At this stage, genetic analysis of embryos is conducted to ensure the transfer of healthy embryos.

PGT offers couples the chance to minimize the risk of carrying genetic diseases and achieve a healthy pregnancy. Consequently, it increases the chances of success in IVF treatment and the likelihood of having a healthy baby.

Investigating Male Factors

Investigating factors that may contribute to recurrent IVF failure in the male partner is commonly practiced and deemed significant by approximately 80% of clinicians. Such investigations include inquiries about lifestyle (e.g., smoking, drug use), semen analysis, and sperm DNA fragmentation testing.

Recent guidelines do not recommend routine use of sperm FISH analysis and sperm DNA fragmentation testing in couples experiencing recurrent IVF failure.

Should Endometrial Scratching Be Done?

Endometrial scratching is performed to increase the receptivity of the endometrium to the transferred embryo. The exact biological mechanism is not fully understood, and studies on this topic report different outcomes. However, the latest guidelines published by ESHRE (European Society of Human Reproduction and Embryology) do not recommend endometrial scratching for recurrent IVF failures.

G-CSF (Uterine Wall Healing Vaccine) Application

Also known as filgrastim, G-CSF is administered using an implantation catheter into the uterine cavity on the day of ovulation trigger for patients undergoing fresh embryo transfer. In cases of frozen embryo transfer, if the uterine wall thickness is less than 7mm despite medication, G-CSF can be infused into the uterine cavity when the patient comes for a check-up. Evidence regarding whether uterine G-CSF application improves live birth rates in recurrent IVF failures is contradictory.

The application may have side effects or adverse reactions such as mucosal inflammation, splenic enlargement, hepatic enlargement, transient hypotension, epistaxis, urinary abnormalities, osteoporosis, exacerbation of rheumatoid arthritis, anemia, and pseudo-gout.

Intravenous Intralipid Infusion

Intravenous administration of intralipid helps regulate immunity by reducing clotting factors, decreasing the production of certain proteins crucial for cell signaling, and suppressing NK cell levels and activity. There are limited randomized controlled trials evaluating the efficacy of intravenous intralipid application during IVF in patients with recurrent IVF failures.

Potential benefits of intralipid infusion are countered by reported side effects or adverse events such as hepatic enlargement, jaundice, biliary tract obstruction, splenic enlargement, decreased clotting cell counts, reduced leukocyte counts, and hyperlipidemia syndrome. Therefore, intravenous lipid infusions should be administered with extreme caution.