IVF Failure Research Articles

High pregnancy rate after comprehensive chromosomal screening of blastocysts

OBJECTIVE: Traditionally, preimplantation genetic screening has involved the biopsy of a single blastomere at the cleavage stage followed by limited chromosomal assessment using fluorescent in situ hybridization (FISH). The objective of this study was to clinically apply a novel strategy for aneuploidy detection, based upon comprehensive chromosome screening of trophectoderm (TE) cells biopsied at the blastocyst stage. DESIGN: Clinical evaluation of a new approach for embryo viability assessment. MATERIALS AND METHODS: All 23 pairs of chromosomes were screened for aneuploidy in 106 blastocysts derived from 17 patients. The patients were of advanced maternal age (mean 37 years), with a history of multiple failed IVF attempts and/or multiple abortions. 3-10 TE cells were biopsied from each blastocyst and subjected to whole genome amplification and comparative genomic hybridization (CGH). Blastocysts were cryopreserved using vitrification while CGH analysis took place. Chromosomally normal blastocysts were warmed and transferred in a subsequent FET. RESULTS: The CGH approach underwent extensive validation prior to clinical application, confirming accuracy and cytogenetic concordance between the TE and inner cell mass. Clinically, CGH succeeded in producing a full chromosome screen for 91% of blastocysts tested. The aneuploidy rate was 43.8%. Embryo survival after biopsy and warming were excellent (100%). To date, 10 patients have undergone blastocyst FET, resulting in an implantation rate of 55% per embryo and an ongoing clinical pregnancy rate (fetal heart) of 70% per cycle. Further transfers are in progress. CONCLUSIONS: We report the first clinical application of a novel approach for preimplantation genetic screening, involving assessment of all chromosomes at the blastocyst stage. The sampling of several cells resulted in a robust diagnosis and greatly reduces the risk of misdiagnosis due to chromosomal mosaicism. Implantation and clinical pregnancy rates were high (55% and 70%), especially given the poor reproductive history of the patients, and compare very favorably with non-PGS cycles matched for maternal age (30% and 60% respectively). Despite the high implantation potential of blastocysts it is clear that many still harbor lethal aneuploidies. It is likely that accurate, comprehensive chromosomal screening will significantly improve embryo selection and may be key to maintaining high pregnancy rates in cycles involving elective single blastocyst transfer.

The role of aneuploidy in repeated implantation failure: implications for patient counseling and preimplantation genetic screening

OBJECTIVE: It has been suggested that the use of preimplantation genetic screening (PGS) to assist in the identification and preferential transfer of chromosomally normal embryos, may lead to improved IVF outcomes for certain groups of patients. Current PGS strategies, employ FISH analysis of 9-12 chromosomes, and have been shown to increase implantation and pregnancy rates for women of advanced age and women with recurrent spontaneous abortion. However, patients with repeated implantation failure (RIF) have been harder to target. We employed comparative genomic hybridization (CGH) clinically, to examine the entire chromosome complement of oocytes and embryos derived from RIF couples. DESIGN: Investigation of all chromosomes in zygotes and blastocysts using CGH. MATERIALS AND METHODS: First and second polar bodies (PBs) were biopsied from 194 zygotes from 27 women (average age: 40 years) and analyzed by CGH. Additionally, 79 blastocysts from 13 women (average age: 37 years) were also biopsied and examined. All patients had a history of failed IVF attempts (mean 3.9 failed cycles). Zygotes and blastocysts were cryopreserved while CGH analysis took place. Normal embryos were transferred in subsequent cycles. RESULTS: The oocyte and blastocyst aneuploidy rates were 68% and 43% respectively. 27% of aneuploid zygotes were classified as highly abnormal (3 or more chromosome errors), versus 16% of blastocysts. PGS using FISH would have failed to detect 42% of oocyte and 38% of blastocyst aneuploidies. Thus far, 12 women have had an embryo transfer (ET) after PB CGH, leading to one ongoing pregnancy. 6 women have had an ET after blastocyst analysis, resulting in 4 ongoing pregnancies. Further ET's are underway. CONCLUSIONS: Comprehensive screening of oocytes and blastocysts revealed that errors may affect any chromosome in material from RIF patients, including unusual types of abnormality not seen during later gestational stages. Screening of oocytes from RIF patients seems to have little if any impact on pregnancy rates, although such analyses may reveal prognostic information (some patients had multiple abnormalities affecting all oocytes and are unlikely to be able to conceive using their own gametes). Unlike oocyte screening, blastocyst analysis was associated with high pregnancy rates. Hence, the application of comprehensive chromosome screening is likely to assist a subset of RIF patients (those capable of producing blastocysts) in achieving pregnancies.

Ultrashort flare GnRH agonist combined with flexible multidose GnRH antagonist for patients with repeated IVF failures and poor embryo quality

To evaluate the appropriate controlled ovarian hyperstimulation (COH) protocol in patients with repeated IVF failures and poor embryo quality we compared the stimulation characteristics of ten cycles which included ultrashort flare GnRH agonist combined with flexible multidose GnRH antagonist with the patients' earlier failed IVF attempts. The use of ultrashort GnRH agonist/GnRH antagonist COH protocol resulted in a significantly higher number and proportion of top-quality embryos, with a consequent improvement in clinical pregnancy rate (50%).

GnRH agonist versus GnRH antagonist in controlled ovarian hyperstimulation: their role in patients with an unfavorable prognosis a priori

In an attempt to examine the influence of the type of GnRH analogue used during controlled ovarian hyperstimulation on IVF outcome in patients with an unfavorable outcome a priori, we studied 728 consecutive cycles in patients with repeated IVF failure. In patients with repeated failure, the GnRH agonist group showed significantly higher clinical pregnancy rate compared with the GnRH antagonist group (20.8% vs 14.5%).

Interval double transfer improves treatment success in patients with repeated IVF/ET failures

To examine whether double transfer of embryos on day 2-3 and on day 5 after ovum pick-up (OPU) improves IVF/ET success rates in patients with multiple consecutive IVF failures Consecutive IVF failures patients (7.6 +/- 0.5) undergoing IVF/ET cycles were treated with interval double transfer on day 2-3 and on day 5 after OPU. Matched patients, that had embryos transferred only once on day 2 or 3 served as controls. Baseline and cycle characteristics were similar in the study group and controls. The total pregnancy rate was 44.6% in the study group and 24.2% in the controls (p = 0.001), and the clinical pregnancy rates were 38.5% and 19.6%, respectively (p = 0.001). Patients with multiple consecutive IVF/ET failures, treated with the interval double transfer approach had significantly improved cycle success rates compared with regular day 2 or 3 embryo transfer protocol.

No room for cancellation, coasting, or ovarian hyperstimulation syndrome in oocyte donation cycles

We retrospectively studied 429 IVF donor cycles in which ovulation was triggered with either hCG (175 cycles) or GnRH agonist (254 cycles). Of the donors in whom ovulation was triggered with hCG, 3.2% developed symptoms of moderate (2.2%) or severe (1%) ovarian hyperstimulation syndrome, while none of the IVF donor cycles that were triggered with the GnRH agonist presented ovarian hyperstimulation syndrome, needed coasting, or were cancelled.

The Role of Decidual Natural Killer Cells in Normal Placentation and in the Pathogenesis of Preeclampsia

Adequate invasion of the human placenta during the first weeks of pregnancy is a critical step in ensuring both fetal and maternal health. A rapidly expanding body of evidence suggests that decidual natural killer (dNK) cells, a distinct population of CD56brightCD16- lymphocytes, are key regulators of this complex process. Experiments using murine models and in vitro evidence using human tissue cultures suggest that dNK cells modulate extravillous trophoblast (EVT) invasion and remodelling of maternal spiral arteries via both contact-dependent and contact-independent mechanisms. In addition, the differential expression of surface receptors by dNK cells may have a role in determining reproductive success through modulation of the maternal immune system at the time of implantation and placentation. The roles of cytokines, chemokines, and growth factors secreted by dNK cells and their influence on EVT migration, invasion, and pseudovasculogenesis are of particular interest. We reviewed the available experimental evidence related to the functional relationships between dNK cells and trophoblasts at the time of placentation to elucidate potential clinical correlations with human pathologies, including preeclampsia, recurrent pregnancy loss, IVF failure, and placenta accreta.

A Classroom Activity Debating the Ethical Issues of Preimplantation Genetic Diagnosis: A Role-Playing Exercise

Almost three decades ago, the birth of Louise Brown England marked the beginning of the vitro fertilization (IVF) era. IVF is done to overcome infertility problems that some couples experience. Sperm and eggs are collected and fertilized the lab. (In vitro means in glass and is used to refer to laboratory conditions.) Fertilized eggs are allowed to divide to form an embryo which is then implanted the mother. IVF begins by stimulating the ovaries with the hormone gonadotropin. Ultrasound is used to monitor the number and size of the follicles. When the follicles are suitable, hormones are used to mature the oocytes (cells from which the egg develops) which are collected after 34 to 38 hours. Oocytes are either inseminated and left to fertilize or have sperm injected directly (Braude et. al., 2002). The fertilized egg begins to divide, forming an embryo. Embryos divide until two to eight cells have formed and are then implanted the mother. IVF has allowed many couples to overcome infertility problems. During the 1980s scientists began developing procedures for characterizing the genetic makeup of embryos created by IVF a process known as preimplantation genetic diagnosis (PDG). Detection of chromosomal abnormalities that may lead to miscarriage and single gene disorders for certain diseases are among the reasons for performing this procedure. Once the genetic makeup of the embryo has been determined, the parents can then decide if they want it to be implanted. Those that are not implanted can be discarded or frozen. PGD is recommended for the following couples: * those who have family histories of inherited genetic disease * those which the female is over 35 and, therefore, has an increased likelihood of producing eggs with an abnormal chromosome number * those which the woman has experienced numerous miscarriages, suggesting an abnormal chromosome set coming from either the mother or father * those which either the mother or father has chromosomal translocations (see below) * those who have experienced repeated IVF failure * those which the male's infertility necessitates sperm injection (Marick, 2005) The first successful PGD procedure was reported 1989 (Marick, 2005). As of 2003, more than 1,000 babies had been born following PGD (Verlinsky et. al, 2004). For PGD, once the embryos have reached the eight-to-ten cell stage (typically after three days), one or two of the cells are removed for genetic testing. The removal of these cells does not adversely affect the embryo. Alternatively, polar bodies (meiotic products that do not become an egg) may be examined prior to fertilization. There are two basic approaches for genetic testing PGD: polymerase chain reaction (PCR) and fluorescent situ hybridization (FISH). With PCR, a small section of the DNA is repeatedly copied (called amplification). This creates billions of copies of a specific section of DNA that can be used for further testing. For PGD, genes associated with different diseases are amplified. PCR is performed by repeating a series of temperature-dependent steps. First, the double-stranded DNA is heated to a temperature where the two strands separate (95[degrees] C). The DNA is then cooled (50[degrees] C) the presence of primers, short pieces of DNA that bind to specific sequences the genomic DNA (i.e., those associated with a specific trait). The temperature is again raised (75[degrees] C), allowing a special DNA polymerase (isolated from a thermophilic bacteria found growing hot springs) to begin synthesizing new DNA from the primers. After a short period, the temperature is again raised, causing the DNA strands to separate, beginning the cycle over again. With each cycle, the desired region of DNA is doubled. After 30 to 35 cycles, billions of copies of specific DNA sequences (i.e., genes that may code for abnormalities) are created for further analysis. …

A retroprospective study comparing three different assisted hatching techniques

The purpose of this study was to determine the effect of four different assisted hatching techniques on pregnancy rates in women with prior IVF failure in fresh IVF cycles. The results suggested that assisted hatching utilizing laser, chemical, or microsurgical techniques increases both implantation and pregnancy rates.

A Comparison of Microdose Flare Protocol Versus AACEP Protocol for IVF Poor Responders

Background: There is presently no uniform consensus defining the best protocol to use for poor-responding IVF patients. At our center, we generally advocate the use of gonadotropin-releasing hormone (GnRH) agonist/antagonist conversion with estradiol valorate priming (AACEP) protocol for poor responders; we have noted much anecdotal success using AACEP even in those who have experienced repeated IVF failures. This protocol, we believe, synchronizes follicular growth while better developing the endometrium for implantation.

Autoimmune response to Chlamydia trachomatis infection and in vitro fertilization outcome

This observational study was conducted in 235 patients undergoing IVF who had a cervical swab positive for Chlamydia trachomatis and who underwent antibiotic treatment until a negative cervical swab before IVF attempt. After oocyte retrieval, follicular fluids of 109 patients out of 228 still showed the presence of IgA antichlamydia antibodies and a significantly lower pregnancy and implantation rate; therefore we conclude that patients should undergo IVF procedure after serum antichlamydia IgA tests negative.

Ongoing twin pregnancy after rescue intracytoplasmic sperm injection of unfertilized abnormal oocytes

To report a case of an ongoing twin pregnancy resulting from rescue intracytoplasmic sperm injection (ICSI) after failed fertilization of morphologically abnormal oocytes. Case report. Tertiary center for assisted reproductive technology (ART). A 28-year-old woman with primary infertility due to tubal obstruction. Rescue ICSI after failed IVF insemination. Clinical pregnancy. Successful ongoing twin pregnancy. With failed fertilization of oocytes with abnormal morphology after IVF, early rescue ICSI may increase the fertilization rates and result in an ongoing pregnancy.

ANDROLOGY LAB CORNER*: The Clinical Implementation of Sperm Chromosome Aneuploidy Testing: Pitfalls and Promises

Severe male infertility has been shown to be associated with improper meiotic recombination and elevated sperm chromosome aneuploidy. Elevated sperm aneuploidy increases the risk of embryo lethality or fetal anomalies. Although difficulties in interpreting aneuploidy data still exist, advances in fluorescent in situ hybridization (FISH) technology have facilitated the study of sperm from patients with severe spermatogenesis defects, which has demonstrated the prudence of evaluating sperm chromosome aneuploidy in men with severe male factor infertility, such as nonobstructive azoospermia or severe ultrastructure defects, especially in cases of previous repeated in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) failure. Testing is also advisable in men with chromosome translocations and unexplained recurrent pregnancy loss, and it may be beneficial in patients with unexplained, repeated IVF failure. Automated FISH imaging and analysis technology is now available and is beneficial in reducing technician time analyzing sperm aneuploidy. Emerging technologies, such comparative genomic hybridization, may be beneficial in further improving the quality of data derived from aneuploidy analysis and reducing the cost of the assay.

Expression of vascular endothelial growth factor-A and its receptor-1 in a luteal endometrium in patients with repeated in vitro fertilization failure

To investigate temporal and spatial relationships of vascular endothelial growth factor (VEGF)-A and its receptor (VEGFR-1) expression in a luteal endometrium obtained from patients with repeated IVF failure. Immunohistochemical study. University hospital. Thirty-three regular-cycling infertile women (mean age, 34.4 years) who experienced three or more failed IVF cycles (range, 3-14 cycles). Endometrial samplings by silastic suction curette in the luteal phase of a spontaneous unmedicated cycle. Immunohistochemical intensity scoring after staining by monoclonal anti-VEGF-A and polyclonal anti-VEGFR-1. Microvascular density determined after staining by monoclonal anti-CD31 antibody. The data were compared in three phases; early (cycle day [CD]16-19) (n = 12), mid- (CD20-24) (n = 13), and late luteal phase (CD25-27) (n = 8). Expression of VEGF-A tended to be lower in midluteal endometrium at epithelial, stromal, and endothelial cells. Expression of VEGFR-1 at epithelial cells was highest in midluteal endometrium, but VEGFR-1 at stroma and endothelium and microvascular density continuously decreased throughout luteal-phase endometrium. In contrast to previous reports, VEGF-A expression was not highest in the midluteal phase in repeated IVF failure patients who had no apparent endometrial pathology. Further research is needed to determine whether different VEGF-A expression patterns in the luteal phase contribute to repeated IVF failure.

Etiology of sperm immunity in women

Sperm immunity in females can reduce the likelihood of natural conception, and sperm antibodies from female sera have been shown to inhibit IVF in humans and in several animal models. The etiology of sperm immunity in human females is unknown, but several possible mechanisms have been proposed, including cross-reactivity with microbial antigens and interferon gamma-mediated potentiation of the antisperm immune response in women whose male partners have sperm autoantibodies in their semen. This article reviews these ideas and postulates a novel hypothesis based on the potential for the generation of anti-idiotype antibodies in women whose partners have sperm antibodies in their semen.

Successful pregnancies after application of array-comparative genomic hybridization in PGS-aneuploidy screening

Recurrent IVF failure, implantation failure and early embryo demise can be attributed to the high frequency of chromosomal aneuploidy observed in human embryos. Preimplantation genetic screening (PGS) using multiple displacement amplifications (MDA) and array comparative genomic hybridization (aCGH) was successfully performed on eight patients with a minimum of seven recurrent IVF failures with the aim of detecting aneuploidy and ameliorating pregnancy rate. A total of 41 embryos with eight or more cells were biopsied by taking two blastomeres from each embryo. The DNA from these blastomeres were amplified and analysed by aCGH technology. The aCGH results showed a complex panel of chromosomal abnormalities in 60% of the diagnosed embryos. Some abnormalities could not be detected by the seven-probe panel (13, 16, 18, 21, 22, X and Y) used in fluorescence in-situ hybridization. Six out of eight patients had embryos for transfer with five out of those six showing positive pregnancy tests. As far as is known, this report is the first to show a pregnancy after PGS using the aCGH technology. The pregnancy rate obtained here is encouraging and will open the door for enrolment of more patients.

Live births in poor prognosis IVF patients using a novel non-contact human endometrial co-culture system

Patients with repeated implantation failures or poor embryo quality may benefit from embryo culture using the co-culture technique; growth factors secreted by co-culture cells may act as survival factors. Autologous endometrial co-culture has been suggested as a safe alternative to animal cells for co-culture of human embryos. However, the technique is fairly labour intensive and its effectiveness can vary from patient-to-patient. This study presents clinical outcome data on a novel non-contact co-culture system using a human endometrial cell line rather than autologous tissue. Embryos from 316 poor prognosis patients with repeated IVF failures, previous cycles with poor embryo quality or advanced maternal age were cultured in Transwell chambers with a monolayer of endometrial cells. The clinical pregnancy rate in patients less than 39 years of age was 53% and for patients aged between 39 and 42 years it was 33%. To date, 76 patients have delivered 111 healthy infants with no congenital anomalies and 18 pregnancies are ongoing. This is the first report on the potential benefits of a non-contact co-culture system in the IVF laboratory. This study shows that an established human endometrial cell line can be used to obtain the benefits of co-culture without the potential disadvantages associated with using autologous endometrial tissue.

Is preimplantation genetic screening useful in repeated IVF failure group?

Is preimplantation genetic screening useful in repeated IVF failure group?

The current status of preimplantation genetic screening: British Fertility Society Policy and Practice Guidelines

Preimplantation genetic screening (PGS) has been proposed as a method to improve the success rates of assisted conception in certain indications. Most commonly the technique involves analysing part of the karyotype of one or two biopsied blastomeres by fluorescence in situ hybridisation and thus detecting aneuploid embryos which are then not transferred into the uterus. Proposed indications include advanced maternal age, repeated IVF failure and recurrent miscarriage. While there are a considerable number of reports of the use of this technique, there are only two randomised controlled trials reported at present, both for the indication of advanced maternal age. Neither show an increase in live birth rate, and indeed the more recent (using a relatively low age cut-off of 35 years) suggests that live birth rate is reduced by the use of PGS. Methodological aspects of both studies are discussed. It remains possible that PGS may be of benefit under certain circumstances. However at present patients should be informed that there is no robust evidence that PGS for advanced maternal age improves live birth rate per cycle started, and PGS should preferably be offered within the context of robustly designed randomised trials performed in suitably experienced centres.

Impact of preimplantation genetic diagnosis for chromosomal disorders on reproductive outcome

Despite recent controversy, existing experience suggests that aneuploidy testing has had a significant impact on the reproductive outcome of poor prognosis IVF patients, which is based on the experience of over 20,000 cases of PGD for chromosomal disorders. The clinical impact was demonstrated in the improved implantation and pregnancy rates, reduction of spontaneous abortions and improved take-home baby rate in patients of advanced reproductive age, those with repeated IVF failures and recurrent spontaneous abortions. The lack of positive effect of aneuploidy testing in a few smaller series may be due to potential detrimental effect of two blastomere removal, reducing the implantation potential of the biopsied embryos, or exclusion from testing a few key chromosomes and poor outcome of aneuploidy testing, affecting the appropriate pre-selection of embryos for transfer. Despite the need for randomized controlled studies to quantify in more detail the clinical impact of the pre-selection of aneuploidy-free zygotes, the positive impact of PGD is particularly obvious from the comparison of reproductive outcome in the same patients with and without PGD, revealing the actual benefits of PGD.