Repeated IVF Failure Research Articles

Clinical Indications for Preimplantation Genetic Diagnosis (PGD)—A Lab Perspective and Comparison of the Type of Abnormalities Based on the Clinical Indication

To review the indications used in clinical practice to request PGD and to compare the rate of abnormalities observed by varying clinical indication. Retrospective analysis. Test requisition forms accompanying the PGD samples were reviewed for 303 cases received in the laboratory for 5 probe or 10 probe analysis. Blastomere samples of 2585 embryos from these 303 cases were analyzed for aneuploidy screen using either a 5-probe or a 10 probe fluorescence in situ hybridization (FISH) panel. The 5-probe panel screens for aneuploidy of chromosomes 13, 18, 21, X, and Y using probes RB1 at 13q14, D18Z1 at 18cen, D21S259/D21S341/D21S342 at 21q22.13-q22.2, DXZ1 at Xcen, and DYZ3 at Ycen (Vysis MultiVysion− PGT). The 10-probe set additionally screens for aneuploidy of chromosomes 8, 9, 15, 16, and 22 using probes D8Z2 at 8p11.1-q11.1, CEP9 at 9p11-q11, D15Z4 at 15p11.1-q11.1, D16Z3 at 16q11.2, and BCR at 22q11.2 (Vysis MultiVysion− Custom 5A). The most common referral indication for PGD was advanced maternal age (AMA-group). 62.2 % of cases received in our laboratory for analysis were for patients over 35 years of age. Recurrent miscarriages (SAB group) accounted for only 2.6 % of cases. Other indications (Other group), including male factor infertility, poor oocyte quality, repeated IVF failure, and family history of chromosome abnormalities was a referral indication in 1.6 % cases. Gender identification (Gender group) was specifically requested in about 5.9 % of cases, some of which included family history of X-linked disorders. 27.4 % of cases, our second biggest group were cases referred for aneuploidy screen (AS group) without another specific indication. Most of these cases were patients less than 35 years as based on the date of birth provided on the test requisition. The rate of abnormalities observed varied by the indication. Comparing the AMA group to AS group, the rate of abnormalities for complex and monosomic abnormalities were very similar at 63.2% and 61.7% respectively. The rate of abnormalities for complex and monosomic abnormalities were also very similar (statistically no significant difference was noted, p ≤ 1) in these two groups; but a statistically significant difference was noted for the trisomic and sex chromosome abnormalities. The rate of trisomic and sex chromosome abnormalities was 14.9 % and 8 % respectively in the AMA group as compared to 10.8 % and 6 % in the AS group (p ≤ 0.05). The rate of abnormality observed in the recurrent SAB group of 61.5 % was also similar to the AMA group (p ≤ 1). The rate of abnormality in the Other group was higher at 72% higher, but this was not found to be statistically significant (p ≤ 0.10). In contrast, the rate of abnormalities seen in the gender identification category was only 54 %. PGD has been increasingly used in infertility practices to improve the overall outcome and success rate. As expected, AMA cases are associated with increased incidence of aneuploidy. Therefore, offering PGD to this group of patients is most appropriate. Patients undergoing IVF for other indications may also benefit from aneuploidy screening.

Chromosome Analysis in Supernumerary Embryos Obtained From Patients Undergoing Aneuploidy Screening for Repeated Implantation Failure or Recurrent Miscarriage

To assess the prevalence and types of chromosome abnormality in spare preimplantation embryos from women with repeated IVF failure (RIF) or with unexplained recurrent miscarriage (RM) undergoing preimplantation genetic diagnosis for aneuploidy screening (PGD-AS).

Preimplantation Diagnosis for Aneuploidies in Patients Undergoing In-Vitro Fertilization With a Poor Prognosis: A Systematic Review and Meta-Analysis

Patients with increased maternal age and/ or repeated IVF failures are given a guarded prognosis due to the relatively poor outcomes that occur with these groups. Different techniques have been used to help increase the chance of pregnancy including Assisted Zona Hatching and Preimplantation Genetic Diagnosis (PGD) for aneuploidies.

Abdominal Ultrasound-Guided Embryo Transfer Improves Clinical Pregnancy Rates in Repeated IVF Failure Patients

Abdominal Ultrasound-Guided Embryo Transfer Improves Clinical Pregnancy Rates in Repeated IVF Failure Patients

Improved Outcome in Unexplained Repeated IVF Failures After Immunotherapy With Intravenous Immunoglobulin[IVIg] and AntiTNFalpha Drugs

The most common reason that IVF is unsuccessful is the failure of embryos to implant.Although embryo abnormality may be responsible in many cases, problems relating to abnormalities in a woman’s immune system may play a part in a significant proportion of these failures. Evidence is emerging that immunologic and immunophenotypic evidence of local and peripheral natural killer[NK] cell activation and T-helper 1[TH1] dominance may be associated with unexplained repeated embryo implantation failure.We have identified women with these problems and treated them with what we consider appropriate immunotherapy.We present our findings.

Treatment of recurrent IVF failure and human leukocyte antigen similarity by intravenous immunoglobulin

This study sought to assess the efficacy of intravenous immunoglobulin (IVIg) in improving pregnancy rates and outcome, in a select group of patients with repeated IVF failure and human leukocyte antigen (HLA) similarity. Couples suffering from recurrent IVF failure, defined as at least seven attempts at embryo transfer with no successful implantations, who were found to share at least three HLA loci, and a negative cross-match test, were included in the study. The treatment consisted of two 30 g IVIg doses: one before oocyte retrieval, and a second as soon as a fetal pulse was identified on ultrasound. Ten couples comprised the study group. In total, these couples had undergone 98 IVF cycles with no successful pregnancies prior to initiation of the study. Following a total of 18 IVIg courses, seven women conceived, two women twice. Up to date, five women have delivered at least one live fetus, at 27 weeks or later. One woman is currently in the early third trimester of a twin pregnancy, and one woman had a late abortion at 19 weeks. The results suggest that couples with recurrent IVF failure and HLA similarity, may benefit from IVIg treatment.

Preimplantation genetic diagnosis for aneuploidy screening in repeated implantation failure

Chromosomal abnormalities are thought to be responsible for implantation failure, and among chromosomal abnormalities in normally developing embryos, aneuploidy is the most frequent. Genetic testing of preimplantation embryos for chromosomal aneuploidy allows selection of chromosomally normal embryos, and early detection of chromosomal aberration will increase the chance of conceiving. Preimplantation genetic diagnosis for aneuploidy screening (PGD-AS), performed by polar body or blastomere analysis, is used in infertile patients treated with assisted reproduction technologies, especially in those with a poor prognosis, e.g. repeated IVF failure, advanced maternal age, or recurrent spontaneous abortion. The aim of this paper is to clarify the impact of PGD-AS in repeated implantation failure. In this review, the data collected so far regarding PGD-AS in this patient group will be discussed in depth.

Immunological abnormalities and infertility

Immunological abnormalities and infertility

Antiphospholipid antibodies and infertility: The importance of anti-phosphatidylserine antibody

Antiphosphplipid antibodies (aPL) have been linked to poor reproductive performance for almost 20 years. Although many different types of antibodies may be present in the so-called antiphospholipid syndrome, the majority of the studies have been focused on anti-cardiolipin and lupus anticoagulant antibodies. However, many other antibodies have been described, among which anti-phosphatidylserine (aPS) is known to have adhesive properties essential for placentation. Therefore, previously unidentified patients with aPS+ may be at increased risk, even in the absence of the classical aPL. The aim of this study was to describe the incidence of aPS in patients with poor reproductive history and compare the results with other abnormal immunological findings. 102 patients with history of either repeated IVF failures (group 1) or recurrent miscarriage (group 2) were submitted to a protocol of immunological evaluation. Recurrent miscarriage was defined as antecedent of ≥ 2 spontaneous previous miscarriages and repeated IVF failures was defined as ≥2 previous failed IVF attempts. The patients were submitted to the following immunological evaluation: group 1: anti-nuclear antibody (ANA), anti-phospholipid antibodies—anti-cardiolipin (aCL) and anti-phosphatidylserine (aPS)—anti-thyroid antibodies (anti-tireoglobulin and anti-microsomal) and natural killer (CD3- CD16+ CD56+) cells count; group 2: the above mentioned exams for group 1 plus cross-match for HLA-sharing assessment, homocysteine level and genetic tests for detection of inherited thrombophilias (data not shown). Fisher’s exact test was used for statistical analysis and a p<0.05 was considered significant. Fifty-three patients (52%) had positive anti-phospholipid tests.Thirty-three patients (32.4%) had aCL and 41 patients (40.2%) presented positive aPS. When the 2 groups were compared, we found an equivalent prevalence of positive aCL: 30.6% (19/62) in group 1 and 35% (14/40) in group 2; p=0.67. The prevalence of positive aPS, however, was significantly higher in group 2: 55% (22/40) vs 30.6% (19/62); p=0.022. Also, 36.4% (12/33) of patients with aCL were negative for aPS. Contrarily, 53.7% (22/41) of patients with positive tests for aPS were negative for aCL. When other immunological abnormalities were studied for aPS positive patients (n=41), we found association with high NK cells in 36.6% (15/41), anti-thyroid antibodies in 9.8% (4/41) and ANA in 29.3% (12/41). Positive aPS and aCL were the only abnormal findings in 8.8% (9/102) and 3.9% (4/102) of all patients, respectively. We found a high prevalence of positive aPS in patients with poor reproductive history (40.2%). Our findings of a higher percentage of aPS positive women in group 2 (55% vs 30.6%) strengthens the importance of phosphatidylserine as a trophoblastic constitutive phospholipid. Autoantibodies against PS might therefore impair placentation and lead to miscarriage. It is noteworthy that neither aCL nor aPS shall be performed alone, because there is a significant number of patients in whom only one or another will be found. Finally, the significant association with other abnormalities in patients aPS+, especially high activated NK cells count (36.6%) and ANA (29.3%), highlights the need of a global immunological evaluation for patients with poor reproductive history, since different abnormalities may indicate distinct and specific treatments.

Increased rates of thrombophilia in women with repeated IVF failures.

We investigated whether hereditary thrombophilia is more prevalent in women with recurrent IVF-embryo transfer failures. This case-control study was conducted in an academic tertiary care hospital and compared 45 women with a history of four or more failed IVF cycles (group A) with 44 apparently healthy women matched for age and ethnic origin (group B). All participants were tested for inherited thrombophilias: mutations of prothrombin, factor V Leiden and methylene tetrahydrofolate reductase (MTHFR), and protein C, protein S and antithrombin III deficiencies. Excluding homozygotic MTHFR, the incidence of thrombophilia in group A, was 26.7% compared with 9.1% in group B (P = 0.003; odds ratio 2.9; 95% confidence interval 1.02-8.4). The incidence of thrombophilia in women with unexplained infertility in group A was 42.9% (9/21), compared with 18.2% in group B (P < 0.002). These data suggest that inherited thrombophilia may play a role in the aetiology of repeated IVF failures, particularly in the subgroup with unexplained fertility.

Preimplantation Genetic Diagnosis and Human Implantation—A Review

By selecting chromosomally normal embryos for replacement, PGD for aneuploidy can (i) increase implantation rates, (ii) reduce spontaneous abortion rates, and (iii) avoid aneuploid conceptions. When eight chromosomes are analysed, a significant increase in implantation is achieved. PGD is also found to significantly reduce the incidence of spontaneous abortion and chromosomally abnormal conceptions. PGD for patients of advanced maternal age with an adequate number of embryos will improve their chances of childbirth via improved implantation and sustained gestation. PGD has also been used to help other specific groups of patients with high rates of chromosome abnormalities such as patients with recurrent spontaneous abortions, non-obstructive azoospermia, repeated IVF failure, and patients previous trisomic offspring. Recently, PGD has been performed using comparative genome hybridization, which counts all chromosomes; but time constraints require embryo cryopreservation, which reduces the potential of improved implantation

Vaginal sildenafil improves ART outcome in a large cohort of patients with multiple prior ART failures attributed to poor endometrial development

Objective: Vaginally administered Sildenafil (SDF), in combination with controlled ovarian hyperstimulation, improved uterine artery blood flow and sonographic endometrial appearance in 4 women with repeated IVF failure attributed to inadequate endometrial development. These findings, while interesting, were inconclusive due to the low number of patients studied. This follow-up study evaluated the effect of vaginally administered SDF on peak endometrial thickness and ART outcome in a large cohort of infertile women with prior ART failures attributed to a thin endometrial lining.

Establishment of in vitro maturation system (IVM-IVF) as a routine ART procedure by combination of HCG administration and frozen-thawed embryo transfer

Objective: In vitro fertilization of in vitro matured oocytes (IVM-IVF) is hardly used as a routine ART procedure due to low pregnancy outcome. IVM-IVF is a relatively new method of ART and its methodology should be improved. However, it has numerous benefits such as less stressful physically, mentally and economically, less time consuming and no risks of gonadotropins administrated. The present study was conducted to establish IVM-IVF as a standard ART procedure by selecting either fresh or frozen-thawed embryo transfer depending on endometrium thickness at oocyte retrieval and by administrating HCG on previous number of immature oocytes retrieved. Design: IVM-IVF was applied on the patients with PCOS or having at least unilateral ovary appearing PCO like echo image. Repeated IVF failure cases that desired to have this treatment were another indication. Pregnancy outcomes from Oct 1999 to Dec 2001 were analyzed. Materials/Methods: Immature oocytes were retrieved when patients had at least two small follicles more than 8 mm in diameter and before development of a dominant follicle more than 14 mm. When endometrial thickness was 8 mm or more at oocyte retrieval, embryos were transferred in fresh cycles. In case of endometrial thickness less than 8 mm, two-pronuclear oocytes or cleaved embryos were frozen for future frozen-thawed transfer in hormone-supplemented cycles. Ten thousand unit of HCG was administered 36 hours before retrievals for the patients who did not get more than 2 eggs. Immature oocytes were cultured for 24 hrs and ICSI was performed on matured oocytes. Day three embryos were transferred after biochemical assisted hatching either in fresh or in frozen-thawed cycles. Pregnancy outcomes and other parameters were analyzed. Results: Maturation rate was 43% (870/2018) in 24 hours and 80% (695/870) of matured eggs were fertilized by ICSI. A total of 26 pregnancies (15% per 178 transfers and 17% per 149 patients) were achieved in either fresh (17) or frozen-thawed transfer (9). Miscarriage rate was 31% (8/26) overall. As of Jan 2002, 14 normal babies (1 set of twin) were born from 13 deliveries. The PCOS patient ratio in pregnant cases from fresh transfer (82%) was significantly higher compared to frozen-thawed transfer cycles (33%). Two pregnancies in fresh transfer were achieved after HCG administration. Conclusions: IVM-IVF is enrolled into our routine ART program. The pregnancy rate of IVM-IVF is lower compare to stimulation cycles, but this rate is acceptable when numerous benefits of unstimulated cycles were taken into account. The present study suggests that IVM-IVF can be one of the routine ART procedures and will be potentially an alternative for stimulation IVF cycles. The development of IVM culture media needs to be strongly pursued. Supported by: None.

Dissociation between embyro morphology and euploidy in IVF patients with poor prognosis

It is well recognized that embryo morphology is predictive of successful outcome in IVF cycles. Good quality embryos are preferentially selected for transfer. Recent studies have demonstrated increased aneuploidy in embryos from older patients undergoing IVF treatment. The purpose of this study was to determine if good embryo morphology was predictive of euploidy in older patients or other IVF patients with a poor prognosis. Patients undergoing preimplantation genetic diagnosis (PGD) for advanced maternal age, repeated IVF failure, recurrent pregnancy loss or severe male factor were placed into either a young group (Y; <39 yrs; n = 14) or old group (O; ≥39 yrs; n = 17) at the time of embryo biopsy (Day 3 following oocyte retrieval). Biopsy was performed on all suitable embryos. The 4 embryos with the best morphology were ranked in order based on identical criteria as was used for embryo selection and transfer in non-PGD patients. Single cells were analyzed for chromosomes 13, 15, 16, 17, 18, 21, 22, X and Y. The following afternoon (Day 4) as many as 3 normal embryos were transferred. All patients in the study had at least 1 normal embryo available for transfer.

Changing indications for preimplantation genetic diagnosis (PGD)

Preimplantation genetic diagnosis (PGD) is an attractive addition to prenatal genetic diagnosis. Not only were traditional PGD indications valid, but newer indications should be envisioned. The major new indication is aneuploidy testing by PGD for transfer of euploid embryos. This could increase fertilization success in ART, and extend to couples experiencing repeated IVF failures or repeated spontaneous abortions. Other novel indications can be envisioned for the future.

Herniated blastomere following chemically assisted hatching may result in monozygotic twins

Objective: To explore a possible mechanism of the increasing incidence of monozygotic twins following assisted hatching of human embryos. Design: Case report. Setting: Clinical research center in a medical school teaching hospital. Patient(s): A 37-year-old infertile woman with repeated IVF failures. Intervention(s): Assisted hatching of the day 3 embryos using acidic Tyrode’s solution. Main Outcome Measure(s): The morphology of the zona-drilled embryos and the pregnancy outcome. Result(s): After assisted hatching, a herniated blastomere through an oversized opening in the zona pellucida was found in one embryo. The transfer of two zona-drilled embryos resulted in a triplet pregnancy. Conclusion(s): Large openings in the zona pellucida following chemically assisted hatching may cause premature hatching of the blastomeres and may be implicated in the occurrence of monozygotic twins.

Preimplantation diagnosis in older patients--but of course!

Preimplantation diagnosis in older patients--but of course!

Cytogenetic analysis of uncleaved oocytes after intracytoplasmic sperm injection

This work analyzes the causes of cleavage failure after intracytoplasmic sperm injection (ICSI) and the effect of the procedure on the chromosomes of the oocytes. Ninety-seven uncleaved oocytes from 39 patients with severe male infertility or repeated IVF failure were fixed; 79 were analyzable. We checked the decondensation stage of spermatozoa nucleus and the chromosomal abnormalities of the oocytes. Among the fixed oocytes, the spermatozoa nucleus was present in 97% of the cases, and it was undecondensed in 89% of the cases, showing no evolution at all. A low rate (2.6%) of premature chromosome condensation (PCC) of the spermatozoa and a low rate (2.5%) of female diploïdy were observed. Among the oocytes that could be karyotyped, we observed a high rate (45%) of chromosome breakage. ICSI fertilization failure was due mostly to the complete lack of evolution of the spermatozoa nucleus. Oocyte selection before ICSI seemed to lower the PCC rate. The high rate of oocyte chromosomal breakage rate has to be confirmed.

Transfer of six or more embryos improves success rates in patients with repeated in vitro fertilization failures

To examine the results of six or more embryos transferred to patients whose IVF-ET cycles repeatedly met with failure. Prospective clinical evaluation of pregnancy rates and pregnancy outcome. IVF-ET Unit. Seventy-two IVF patients who had failed at least four previous IVF cycles. Forty-one patients (group A) received six or more embryos, and 31 patients (group B) chose to receive five embryos. Per embryo implantation rate, pregnancy rate, multiple pregnancies, and outcome were evaluated. With the transfer of six or more embryos, the pregnancy rate was significantly higher than with the transfer of five embryos (56% versus 29%, respectively). This was associated with a slight, but insignificant, increase in multiple gestations. No difference in pregnancy outcome was noted among the groups. Patients who have had repeated IVF failures may have higher pregnancy rates if six or more embryos are transferred in subsequent cycles.

The role of cumulus cell-secreted proteins in the development of human sperm fertilizing ability: implication in IVF.

Cumulus cells surrounding pre-ovulatory human oocytes were found to secrete a variety of proteins which became firmly associated with the cumulus intercellular material. Antibodies raised against human cumuli oophori completely blocked fertilization in vitro by impairing the sperm-zona pellucida interaction. A group of glycoproteins of high mol. wt were identified as the main cumulus cell secretory products. These proteins showed a marked affinity for human spermatozoa and were potent stimulators of the conversion of human and boar proacrosin into acrosin and of human sperm acrosome reaction. Another fraction of proteins of human cumulus intercellular matrix with an apparent mol. wt of approximately 25,000 daltons was also found to stimulate significantly the acrosome reaction of human spermatozoa, although this fraction had no proacrosin-converting activity. These results indicate that proteins secreted by pre-ovulatory human cumulus cells have an indispensable role in the development of human sperm fertilizing ability. This effect seems to be realized by a concerted action of different types of cumulus-derived proteins just prior to and during the sperm-zona pellucida interaction. Disorders of cumulus cell secretory activity may account for some cases of idiopathic infertility and repeated IVF failures.