Patients Undergoing Preimplantation Genetic Diagnosis Research Articles

Concise Review: Human Embryonic Stem Cells-What Have We Done? What Are We Doing? Where Are We Going?

Human pluripotent stem cells possess remarkable proliferative and developmental capacity and thus have great potential for advancement of cellular therapy, disease modeling, and drug discovery. Twelve years have passed since the first reported isolation of human embryonic stem cell lines (hESC), followed in October 2010 by the first treatment of a patient with hESC-based cellular therapy at the Shepherd Center in Atlanta. Despite seemingly insurmountable challenges and obstacles in the early days, hESC clinical potential reached application in an extraordinarily short time. Eight currently ongoing clinical trials are yielding encouraging results, and these are likely to lead to new trials for other diseases. However, with the discovery of induced pluripotent stem cells (iPSC), disease-specific hESC lines derived from patients undergoing preimplantation genetic diagnosis for single gene disorders fell short of expectations. Lack of ethical controversy made human iPSC (hiPSC) with specific genotypes/phenotypes more appealing than hESC for drug discovery and toxicology-related studies, and in time, lines from HLA-homologous hiPSC banks are likely to take over from hESC in clinical applications. Currently, hESC are indispensable; the results of hESC-based clinical trials will set a gold standard for future iPSC-based cellular therapy. Stem Cells 2017;35:17-25.

Report on first 54 patients undergoing preimplantation genetic diagnosis for genetic disorders together with 24 chromosome aneuploidy screening using microarrays

Report on first 54 patients undergoing preimplantation genetic diagnosis for genetic disorders together with 24 chromosome aneuploidy screening using microarrays

Reducing multiple pregnancy rates in patients under 35 years old by transferring a single embryo in a preimplantation genetic diagnosis program

Reducing multiple pregnancies without affecting the overall pregnancy rates for patients undergoing preimplantation genetic diagnosis (PGD) treatment remains a challenge for IVF clinics. Our objective was to compare the clinical pregnancy and multiple pregnancy rates between the single embryo transfer (SET) and the double embryo transfer (DET) for patients under 35 years old undergoing preimplantation genetic diagnosis.

Myotonic dystrophy type 1 and PGD: ovarian stimulation response and correlation analysis between ovarian reserve and genotype

This study aimed at evaluating parameters and results of ovarian stimulation for myotonic dystrophy type 1 (DM1) female patients undergoing preimplantation genetic diagnosis (PGD) and to assess an eventual association between genotype and ovarian reserve. A retrospective study involved all 17 DM1 patients treated in the study centre's PGD programme. The control group consisted of 22 patients treated for X-linked disorders in the same period. Comparative analysis of ovarian stimulation parameters and results was performed with bivariate and multivariate analysis. Then, among DM1 patients, a correlation between genotype (number of CTG repeats) and ovarian reserve, assessed by antral follicle count, was investigated. Comparative study showed no difference concerning the number of oocytes, embryos and pregnancy rate between the two groups. Multivariate analysis demonstrated that DM1 patients needed a significantly higher dose of gonadotrophins (+544IU, P<0.001) than X-linked disorders patients and suggests a decreased ovarian sensitivity. However, with higher dose of gonadotrophins, PGD for DM1 offers good reproductive outcomes with a clinical pregnancy rate of 35.7%. Genotype was not correlated to ovarian reserve and appeared not to be helpful for the choice of the dose of gonadotrophins.

Derivation of a human embryonic stem cell line from a couple with a known reciprocal translocation

OBJECTIVE: Affected embryos from patients undergoing preimplantation genetic diagnosis (PGD) have been used to derive human embryonic stem cell (hESC) lines. These can be used to investigate normal and abnormal embryogenesis. In particular, hESC lines from chromosomally abnormal embryos provide a unique method to model these rare conditions. We report the derivation and characterization of a hESC derived from a patient with a known reciprocal translocation. Chromosomal translocations are associated with several human disease states including recurrent pregnancy loss, Down's syndrome, and certain malignancies.

Single blastomere biopsy of slow cleaving embryos can support subsequent development and implantation

OBJECTIVE: Blastomere biopsy causes a reduction in total cytoplasmic volume that may affect subsequent development. Biopsy of slowly cleaving embryos results in a proportionately greater loss of embryonic volume. We have investigated the developmental potential of embryos with as few as 4 blastomeres, according to cell number at the time of single cell biopsy. DESIGN: Retrospective analysis. MATERIALS AND METHODS: Implantation rates of 412 embryos from 310 patients undergoing preimplantation genetic selection (PGS) by aneuploidy assessment and 50 patients undergoing preimplantation genetic diagnosis (PGD) for translocation diagnosis were analyzed. Only single embryo transfers or two-embryo transfers in which both embryos were at the same cell stage on Day 3 were considered. Compacted embryos were excluded. Implantation rate was determined as the number of deliveries per embryo transferred. Biopsy occurred early on the morning of Day 3 by removal of a single blastomere, followed by overnight FISH analysis and embryo transfer on Day 4. RESULTS: Slowly cleaving embryos (4-5 cells on Day 3) implanted and developed to term, albeit at a lower rate than 7-10 cell embryos (p<0.001) (Table 1).Table 1Delivery rate according to cell number on Day3Day 3 Cell NumberEmbryos TransferredDelivered4638 (12.7%)5607(11.7)6492 (4.1)76925 (36.2)814741 (27.9)9-10247 (29.2) Open table in a new tab Embryos that continued to develop slowly to Day 4 (<9 cells, no compaction) also displayed the ability to implant after transfer on Day 4 (Table 2).Table 2Delivery rate according to cell number on Day 4Day 4 Cell NumberEmbryos TransferredDelivered41505295 (17.2%)6485 (10.4)7309 (30)8272 (17.4) Open table in a new tab CONCLUSIONS: Embryos containing 4-5 cells on Day 3 implant and develop to term at a rate at least equal to that of six-cell embryos. Embryos such as these should be included during PGD treatment. The removal of no more than 1 blastomere from 4-5 cell embryos would seem to be essential, in order to avoid removal of more than 20% of the embryonic mass. The size of the blastomere selected for biopsy, an indication of whether it is a product of the second or third cleavage division, may also impact subsequent development. The demonstrated ability of slowly cleaving, non-compacted Day 4 embryos to develop to term suggests that standard measures of embryonic fitness may need to be modified for embryos recovering from biopsy. Embryo transfer on Day 4 may be one factor that improves the implantation potential of slowly cleaving embryos post-biopsy.

Reduced Oocyte Damage Rates After ICSI With Blunted Needle

Reduced Oocyte Damage Rates After ICSI With Blunted Needle

Accuracy of FISH analysis in predicting chromosomal status in patients undergoing preimplantation genetic diagnosis

The purpose of this study was to determine the positive predictive value (PPV) and negative predictive value (NPV) of FISH analysis and to determine which chromosomal abnormalities are most frequently confirmed. Prospective observational. IVF laboratory. Two hundred forty-one embryos were analyzed from 98 patients. FISH reanalysis. Embryos that would have been discarded in patients undergoing preimplantation genetic diagnosis (PGD) were fixed and FISH reanalysis was performed. Results of reanalysis were compared with the day 3 diagnosis while PPV and NPV were calculated. Among the 241 embryos, 198 embryos were abnormal and 43 were normal by day 3 FISH analysis. The PPV was 83% and the NPV was 81%. PPV was also determined for specific categories of aneuploidy, and certain abnormalities such as monosomies, trisomies, tetrasomies, and polyploidies were frequently confirmed on reanalysis (PPV >80%), whereas Turner syndrome diagnosis was not (PPV = 17%). FISH analysis offers a PPV of 83% and NPV of 81% when evaluating a single blastomere in conjunction with PGD. FISH errors and mosaicism are primarily responsible for the errors associated with FISH analysis in PGD.

Are Embryos Normal Obtained from Second-Day Intracytoplasmic Sperm Injection (ICSI), Single Pronucleus (1PN), or Cleaved Embryos from No Pronuclei (0PN) Initially Seen?

Body: In an effort to determine if embryos obtained from second-day ICSI, 1PN, or 0PN that cleave appropriately are normal and viable for embryo transfer (ET), we retrospectively reviewed 75 patients undergoing preimplantation genetic diagnosis (PGD) from 01/04 through 11/06 who fell into at least one of these groups. All patients reviewed underwent IVF and ICSI, with subsequent PGD for either advanced maternal age (AMA), recurrent pregnancy loss (RPL), two or more failed cycles of IVF (FC), or severe male factor (MF). These indications are known to be associated with increased risk for aneuloidy. Standard stimulation protocols were administered. Ultrasound-guided oocyte retrieval was performed at approximately 35 hours post hCG administration. All mature oocytes underwent ICSI and fertilization was checked the following day. All intermediate and immature oocytes were evaluated 24 hours post oocyte retrieval and, if normal maturation occurred, second-day ICSI was performed. At 72 hours post retrieval, all embryos that were at least 4–8 cells underwent embryo biopsy for PGD. All 0PN and 1PN that cleaved appropriately were also biopsied. Blastomere fixation was performed and slides were sent to Reprogenetics (San Francisco, CA) for flourescence in situ hybridization (FISH). A total of 9–12 chromosomes were evaluated; 8, 13, 14, 15, 16, 17, 18, 20, 21, 22, x, and y. Tabled 1The results are shown below.## Fertilized Normally# Biopsied# Normal1st Day 2PN609609529/609 (86.9%)191/529 (36.1%)2nd Day ICSI6129/61 (47.5%)10/29 (48.3%)01 PN57–24/57 (42.1%)2/24 (8.3%)0 PN33–23/33 (69.7%)1/23 (4.3%) Open table in a new tab In conclusion, the following can be said:1In the high-risk patients for aneuploidy, it was reconfirmed that 2/3 of the embryos were abnormal.2Second-day ICSI in these high-risk patients does not yield chromosomally normal embryos. Perhaps second-day ICSI should not be performed in these patients.3In patients with 1PN and 0PN that cleave appropriately, the rate of aneuploidy is so high that these embryos should be considered abnormal from the outset.

Correlations Between Morphology Grading and Aneuploidy in Patients Undergoing Preimplantation Genetic Diagnosis

In the IVF laboratory, embryos are typically selected for transfer based on morphology criteria from development at the pronuclear, day two and day three stages. Although pregnancy data suggests that these embryos are more likely to be chromosomally normal, it is impossible to determine chromosomal status with morphology. However, trends can be ascertained between morphology and aneuploidy in patients undergoing preimplantation genetic diagnosis (PGD). The goal of this study was to determine if any correlations exist between embryo morphology and aneuploidy status after PGD. Retrospective data analysis Embryo morphology on days one through three of culture was analyzed and compared to aneuploidy status. Embryos from patients undergoing PGD between 2004 through March of 2005 were assessed based on pronuclear morphology; cell number, evenness of cell size and multinucleation on day two and day three cell number. 1310 embryos were biopsied, Of these 1264 had accurate Z scoring, 1040 day 2 cell number, 997 day 2 symmetry; 942, nucleation score and 1310 accurate day 3 score. Percent euploid is calculated from the number scored in each category to estimate the rate of euploidy/ stage-score. Embryos with good morphology on days 2 through 3 were more likely to be euploid after PGD analysis. Tabled 1 Tabled 1*P<0.05 Tabled 1*P<0.05 *P<0.05 *P<0.05 This data suggests that embryos with good morphology with regards to Zscore, day two cell number, cell symmetry and lack of mulitnucleation and day three cell number are more likely to be chromosomally normal and therefore represent a higher implantation and pregnancy potential. The most significant correlations between morphology and aneuploidy appear to be reflected in the day two symmetry and multinucleation categories. Embryos with combined good Z-scores, day 2 morphology and day 3 morphology may be significantly less aneuploid than any others and this can be used in general IVF for transitioning to single embryo transfer.

Are specific types of aneuploidy more common in high-risk patients undergoing preimplantation genetic diagnosis (PGD)?

Cytogenetic analyses have shown that more than half of all preimplantation embryos contain aneuploidic cells. We have previously published data demonstrating that three groups of patients are at higher risk for aneuploidic embryos. 1.) Patients with recurrent pregnancy loss (RPL); 2.) Patients with advanced maternal age (AMA); 3.) Patients with repeated failed IVF cycles (FC). In an effort to determine if specific types of aneuploidies are more common in these high-risk groups, we retrospectively reviewed karyotypes of 188 embryos biopsied from 40 patients who had been prospectively randomized to undergo PGD from a total of 87 patients enrolled from Aug 1, 2001 − Dec 1, 2003.

High frequency of chromosomal abnormalities in embryos obtained from oocyte donation cycles

High frequency of chromosomal abnormalities in embryos obtained from oocyte donation cycles

Single cell CGH analysis reveals a high degree of mosaicism in human embryos from patients with balanced structural chromosome aberrations

We have performed comparative genomic hybridization (CGH) analysis of single blastomeres from human preimplantation embryos of patients undergoing preimplantation genetic diagnosis (PGD) for inherited structural chromosome aberrations and from embryos of IVF couples without known chromosomal aberrations. The aim was to verify the PGD results for the specific translocation, reveal the overall genetic balance in each cell and visualize the degree of mosaicism regarding all the chromosomes within the embryo. We successfully analysed 94 blastomeres from 28 human embryos generated from 13 couples. The single cell CGH could verify most of the unbalanced translocations detected by PGD. Some of the embryos exhibited a mosaic pattern regarding the chromosomes involved in the translocation, and different segregation could be seen within an embryo. In addition to the translocations, we found a high degree of numerical aberrations including monosomies, trisomies and duplications or deletions of parts of chromosomes. All of the embryos (100%) were mosaic, containing more than one chromosomally uniform cell line, or even chaotic with a different chromosomal content in each blastomere.

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.

Use of fluorescence in situ hybridization to assess the chromosomal status of embryos obtained from cryopreserved oocytes

Objective: To analyze the chromosomal status of human embryos obtained from frozen-thawed oocytes. Design: Fluorescence in situ hybridization analysis of embryos obtained after oocyte cryopreservation. Setting: Department of Obstetrics and Gynecology at the University of Perugia, Italy, and the Instituto Valenciano de Infertilidad, Spain. Patient(s): Oocyte donors ( n = 43). Fertilization, development, and chromosomal status of the embryos were compared with a control group ( n = 18) of patients undergoing preimplantation genetic diagnosis for sex chromosome–linked diseases. Intervention(s): Collection of oocytes after conventional ovarian stimulation and cryopreservation using propanediol as the cryoprotectant and a slow freezing procedure. Microinjection of surviving metaphase II oocytes and evaluation of fertilization and embryo development up to blastocyst stage. Chromosomal analysis after embryo biopsy. Main Outcome Measure(s): Survival, fertilization, and blastocyst rates. Embryo chromosomal analysis employing specific probes for chromosomes 13,18,21, X and Y. Result(s): The overall survival rate was 59.4%. There was no difference between cryopreservation and control groups in fertilization rates (76.5% vs. 90.5%) or blastocyst development (29.6% vs. 35%). The percentage of blastocysts from the original number of cryopreserved oocytes was only 5.6%, comparable to the 5.9% obtained in the control group. The percentage of embryos with abnormal number of chromosomes in the cryopreservation group (28.6%) was comparable to the 26% observed in the controls. Conclusion(s): Fertilization and cleavage rates after oocyte freezing are acceptable. Survival is, however, still poor, leading to overall results that make the technique clinically inefficient. There is no increase in the rate of chromosomal abnormalities, indicating that the technique is, nevertheless, safe enough to be further explored and improved.

Obstetric outcomes in 102 pregnancies after preimplantation genetic diagnosis

Objective: We sought to determine whether preimplantation genetic diagnosis is associated with particular pregnancy or delivery complications. Study Design: A total of 102 consecutive pregnancies after preimplantation genetic diagnosis by polar body removal performed at Illinois Masonic Medical Center resulting in 114 live births were analyzed. All patients were given a delivery and newborn questionnaire, and attempts were made to contact and question them regarding any pregnancy complications and type of delivery. Permission was obtained to examine medical records and discuss the patient's pregnancy with her obstetrician when questions existed with respect to complications or indication for cesarean delivery. Results: Delivery and newborn questionnaires were completed or telephone contact was achieved for 100 of the 102 pregnancies. There were 85 singleton, 9 twin, and 7 triplet pregnancies. Of the 7 triplet gestations, 3 couples elected multifetal pregnancy reduction to twins and healthy triplets were born to 4 couples between 32 and 36 weeks by cesarean delivery. Of the 80 singleton deliveries, 60 (75%) progressed to term. Of these 60 term singleton deliveries, 34 were vaginal, 23 were cesarean (40%), and 3 delivery types were unknown. The incidence of small-for-gestational-age infants was 3% for neonates in the 60 term singleton deliveries and 7% in the entire cohort of 80 singleton deliveries. Only 3 pregnancy complications (other than premature delivery) were reported more than once. There were 3 instances each of gestational diabetes, intrauterine growth restriction, and pregnancy-induced hypertension. There was 1 case each of HELLP (hemolysis, elevated liver enzymes, and low platelets) syndrome, congestive heart failure, mild oligohydramnios, and abruptio placentae. The indications for cesarean delivery were (in descending order) failure of labor to progress (n = 7), fetal distress (n = 4), placenta previa (n = 4), elective repeat cesarean delivery (n = 4), triplets (n = 3), uterine scarring (n = 3), 1 twin in the breech position (n = 3), failed forceps delivery (n = 2), and a variety of other indications that occurred in only 1 patient each. All preimplantation genetic diagnoses were confirmed by prenatal or postnatal testing. No diagnostic errors were made in this cohort of patients or in any patients undergoing preimplantation genetic diagnosis having polar body removal in our center. Conclusions: Preimplantation genetic diagnosis is associated with a risk of multiple gestations, cesarean delivery, and placenta previa. Cesarean delivery rates and multiple gestation rates are comparable to those of patients undergoing in vitro fertilization in general. The preimplantation genetic diagnosis itself does not seem to cause an increased risk for any particular pregnancy complication, with the possible exception of placenta previa, which was seen in 4% of patients. (Am J Obstet Gynecol 2000;182:1629-32.)

Advantages of day 4 embryo transfer in patients undergoing preimplantation genetic diagnosis of aneuploidy.

Following preimplantation genetic diagnosis of aneuploidy, embryo transfer was executed on day 4, with the aim of providing more time for expanding from six to nine the number of diagnosed chromosomes per single cell (Group 2; 45 cycles). The results obtained were compared to those derived from conventional day 3 transfer (Group 1; 71 cycles). For multicolor fluorescence in situ hybridization analysis, two panels of probes were used: the first, specific for chromosomes XY, 13, 16, 18, and 21, was tested in all patients (Groups 1 and 2); the second was implemented only in Group 2 patients for the detection of chromosomes 14, 15, and 22. A total of 406 embryos underwent fluorescence in situ hybridization analysis in Group 1, and 236 in Group 2. Comparable percentages of both chromosomal abnormalities (61% and 62%) and pregnancy and implantation rates (36% and 24.5% in Group 1, 41% and 23.6% in Group 2) resulted, regardless of the higher mean age in Group 2. The diagnosis of the nine chromosomes which are most frequently associated with aneuploidy in humans could have an immediate impact on the rate of spontaneous abortions. Additional advantages are represented by the more accurate morphological evaluation of euploid embryos; the advanced compaction, which means that embryos are less exposed to damage during the transfer procedure; and the possibility of performing a reanalysis in cases where a fluorescence in situ hybridization diagnosis is not obtained.