Validation of array comparative genome hybridization for diagnosis of translocations in preimplantation human embryos.

Fluorescent in-situ hybridization (FISH) for preimplantation genetic diagnosis (PGD) of structural chromosome abnormalities has limitations, including carrier testing, inconclusive results and limited aneuploidy screening. Array comparative genome hybridization (CGH) was used in PGD cases for translocations. Unbalances could be identified if three fragments were detectable. Smallest detectable fragments were ∼6 Mbp and ∼5 Mbp for blastomeres and trophectoderm, respectively. Cases in which three or more fragments were detectable by array CGH underwent PGD by FISH and concordance was obtained in 53/54 (98.1%). The error rate for array CGH was 1.9% (1/54). Of 402 embryos analysed, 81 were normal or balanced, 92 unbalanced but euploid, 123 unbalanced and aneuploid and 106 balanced but aneuploid. FISH with additional probes to detect other aneuploidies would have missed 28 abnormal embryos in the reciprocal group and 10 in the Robertsonian group. PGD cases (926) were retrospectively reviewed for reciprocal translocations performed by FISH to identify which could have been analysed by array CGH. This study validates array CGH in PGD for translocations and shows that it can identify all embryos with unbalanced reciprocal and Robertsonian translocations. Array CGH is a better approach than FISH since it allows simultaneous screening of all chromosomes for aneuploidy.

Pregnancy complicated by triploidy: a comparison of the three karyotypes.

We evaluated triploid pregnancy to determine whether there are clinically important differences between the three karyotypes: 69,XXX, 69,XXY, and 69,XYY. Prospectively maintained cytogenetic databases at five tertiary care centers were retrospectively reviewed over a 10-year period to identify all triploid pregnancies. Targeted ultrasounds were reviewed to identify fetal and placental findings. Sonographic findings were compared by karyotype. There was a total of 549 triploid gestations; preimplantation genetic diagnosis (PGD) detected 413 triploid embryos, and the cytogenetic databases provided 136 clinical pregnancies with triploidy. In triploid embryos with PGD, the frequency of the 69,XYY karyotype was 8.7% (36/413), compared with 0.74% (1/136) during the first trimester of clinical pregnancies (p = 0.002). In clinical pregnancies, 60% (36/60) of 69,XXY fetuses survived the first trimester of development compared with 69% (52/75) of 69,XXX fetuses (p = NS). No clinically important differences were observed between 69,XXX and 69,XXY karyotypes in terms of type, number, or severity of fetal or placental anomalies. Gestations with a 69,XYY karyotype are found less frequently compared with gestations with a 69,XXX or 69,XXY karyotype. The decline in fetal survival of the 69,XYY triploid karyotype needs further investigation. There are significant abnormalities detected during prenatal sonography in most all clinically recognized cases of triploidy. Sonography cannot reliably distinguish between the 69,XXY and 69,XXX karyotypes.

The impact of LH-containing gonadotropins on diploidy rates in preimplantation embryos: long protocol stimulation.

BACKGROUND: The aim of this study was to evaluate the effect of ovarian stimulation with LH-containing gonadotropins (human menopausal gonadotropin, hMG), on ploidy of human cleavage-stage-embryos. METHODS: A total of 104 women, at ages 27-43 years, undergoing one cycle of controlled ovarian hyperstimulation for IVF in combination with preimplantation genetic diagnosis, were eligible for enrollment in this retrospective, controlled cohort study. Ovarian stimulation included down-regulation with long agonist and stimulation with either recombinant FSH or hMG. Since the ploidy of embryos changes with female age, patients were matched for age and dosage of the respective gonadotropin. RESULTS: Despite similar numbers of chromosomally normal embryos in both groups, women undergoing hMG stimulation demonstrated significantly higher percentages of diploid embryos than did the FSH-stimulated patients (69.8 versus 45.3%; P < 0.01). CONCLUSIONS: Long protocol LH-containing ovarian stimulation improves embryonic ploidy in comparison to pure FSH stimulation. This observation may explain higher IVF pregnancy rates, reported for hMG stimulation in some studies.

Increased efficiency of preimplantation genetic diagnosis for infertility using "no result rescue".

OBJECTIVE: To improve preimplantation genetic diagnosis (PGD) accuracy by using "no result rescue" (NRR) consisting of the reanalysis of dubious results with additional probes binding to a locus different from the one previously analyzed. DESIGN: Prospective study of PGD cycles with and without reanalysis of inconclusive results. SETTING: PGD laboratory. PATIENT(S): Patients undergoing PGD for infertility or Robertsonian translocations. INTERVENTION(S): Nuclei from day 3 biopsied embryos were analyzed with fluorescence in situ hybridization for chromosomes X,Y, 13, 15, 16, 17, 18, 21, and 22. When inconclusive results were obtained, NRR was performed. In addition, 100 PGD cycles using NRR were matched to controls according to maternal age, previous failed cycles, number of zygotes, number of eggs, and date of retrieval. MAIN OUTCOME MEASURE(S): Determination of frequency of inconclusive results and error rate after use of additional probes. Comparison of frequency of inconclusive results with prior PGD results when NRR was not used. Assisted reproductive technology outcome was compared between PGD using NRR and controls not using PGD. RESULT(S): After analysis of 34,831 blastomeres from 34,225 embryos, 2,609 blastomeres (7.5%) showed inconclusive results. After NRR on those 2,609 blastomeres, the number of cells with inconclusive results was reduced to 3.1% (P<.001). After the introduction of NRR, fluorescence in situ hybridization errors, measured as discrepancies between the PGD diagnosis and the analysis of the nonreplaced embryo, decreased from 13.6% to 4.7% (P<.001). PGD with NRR significantly improved implantation rates, from 20% to 31%, and reduced spontaneous abortions from 27% to 6%. CONCLUSION(S): The use of NRR has been proven to be a powerful tool to reduce the error rate and the frequency of inconclusive results in PGD, both parameters of high importance to assess quality of PGD laboratories. Indeed, these parameters are two of the few measurable criteria to measure PGD laboratories. In a parallel controlled study, PGD with NRR significantly improved implantation rates and reduced spontaneous abortions, showing that PGD is more efficient in selecting embryos that will reach term.

Patterns of ovarian response to gonadotropin stimulation in female carriers of balanced translocation.

OBJECTIVE: To determine whether maternal balanced translocation is a risk factor for poor ovarian response to controlled ovarian hyperstimulation (COH). DESIGN: A retrospective analysis. SETTING: Private IVF center. PATIENT(S): All couples presenting to a single center for preimplantation genetic diagnosis (PGD) for autosomal balanced translocation in either partner from 1995 through 2001. INTERVENTION(S): Cycle parameters and embryology outcomes were examined and compared between two groups: 61 cycles in 46 women with balanced translocations compared with 42 cycles in 32 women whose male partner had a balanced translocation. MAIN OUTCOME MEASURE(S): Response to ovarian stimulation. RESULT(S): In couples undergoing IVF/PGD to avoid transmission of an unbalanced karyotype, a significantly higher proportion of women carrying balanced translocations (female carrier) responded very poorly (E2 on the day of hCG <1,000 pg/mL) to ovarian stimulation compared to women whose partner had a balanced translocation (male carrier) (23.0% vs. 7.1%). CONCLUSION(S): In couples undergoing IVF/PGD for balanced translocation, the risk for poor response to ovarian stimulation may be increased when the female partner carries the balanced translocation compared to when the male partner carries the translocation. Given significant embryo attrition due to chromosomal imbalance, aggressive stimulation should be considered if the patient does not have risk factors for ovarian hyperstimulation syndrome (OHSS).

Factors associated with disposition of cryopreserved reproductive tissue.

OBJECTIVE: To study patient characteristics associated with the preferences for the disposition of cryopreserved semen and embryos in the event of death or divorce. DESIGN: Retrospective exploratory study. SETTING: Tertiary care academic medical center. PATIENT(S): One hundred twelve men banking sperm, 54 female patients (partners of men banking sperm) undergoing in vitro fertilization (IVF), and 112 women undergoing IVF who were not partners of men banking sperm. INTERVENTION(S): Male patients banking sperm and couples undergoing IVF completed a standard consent form detailing their desired dispositions (disposal or release to a surviving party) of cryopreserved sperm and embryos in the event of death or divorce. MAIN OUTCOME MEASURE(S): Effect of marital status, age, reason for sperm banking, infertility diagnosis, partner status (single or significant-other partner) on sperm, and embryo disposition choice. RESULT(S): In the event of death, married men were 5.5 times more likely to release banked sperm to a surviving party than were single men. The estimated odds of giving sperm to a survivor were 1.07 times greater with every 1-year increase in age. The choice of sperm disposal was significantly related to the banking reason for the case of banking prior to chemotherapy and/or radiation for malignancy. In the case of male death, the decisions of couples undergoing IVF for embryo disposition agreed with choices of male patients for sperm disposition approximately 33% of the time more often than by chance alone. CONCLUSION(S): Decisions surrounding disposition of cryopreserved sperm are significantly associated with marital status, age, and reason for banking sperm. Respective choices of sperm and embryo disposition in couples undergoing IVF were similar in the situation of male death.

Monozygotic twinning following assisted conception: an analysis of 81 consecutive cases.

BACKGROUND: This study attempts to identify risk factors for monozygotic (MZ) twinning following assisted conception. METHODS: Eighty-one MZ twinning pregnancies that occurred over a 7-year period in our IVF/embryo transfer programme were evaluated. These were compared with 4224 non-MZ pregnancies from the same period. RESULTS: The overall incidence of MZ twinning was 1.88% (81/4305) of all clinical pregnancies. A total of 63% (51/81) of the MZ pregnancies included one or more other implantations as well. MZ twinning was unrelated to maternal age, paternal age, gonadotrophin dosage, peak estradiol and progesterone levels, number of oocytes collected, and number of embryos replaced. Patients with MZ twinning had significantly more attempts at assisted reproduction than those with non-MZ multiple pregnancy. A logistic regression analysis suggested a role, albeit not emphatic, for the zona pellucida in MZ twinning. Twenty-four of the 65 MZ cases with known placentation were monoamniotic. This incidence far exceeds that seen following spontaneous conception (P < 0.0001). CONCLUSIONS: The risk of monoamniotic twinning is increased following IVF/embryo transfer. Zona pellucida disruption appears to increase the incidence of MZ twinning. However, the overall micromanipulation data together with the unexpected placentation data suggest that zona-mediated embryo splitting is not the only mechanism of twinning under artificial conditions.