Developmental neuropsychological assessment of 4- to 5-year-old children born following Preimplantation Genetic Diagnosis (PGD): A pilot study.

The purpose of this pilot study was to evaluate developmental neuropsychological profiles of 4- to 5-year-old children born after Preimplantation Genetic Diagnosis (PGD). Twenty-seven participants received a neurological examination and a battery of neuropsychological assessments including Wechsler Preschool & Primary Scale of Intelligence - Third Edition (WPPSI-III; cognitive development), Preschool Language Scale, Fourth Edition (PLS-4; language development), Wide Range Assessment of Visual Motor Abilities (visual motor abilities), Childhood Autism Rating Scales II (a screening test for autistic spectrum disorders), and the Miles ABC Test (ocular dominance). Parental questionnaires included the Behavior Rating Inventory of Executive Function Preschool Version (BRIEF-P; executive function), Child Behavior Checklist (CBCL) and the Carey Temperament Scales Behavioral Style Questionnaire (socioemotional development and temperament), and the Vineland Adaptive Behavior Scales, Interview Edition, Second Edition (general adaptive behavior). Subjects' tests results were compared to each test's norms. Children born after PGD demonstrated scores within the normal or above-normal ranges for all developmental outcomes (mean ± SD): WPPSI-III-VIQ 107.4 ± 14.4 (p = .013), PLS-4-Total 113.2 ± 12.4, p < .001), CBCL-Total 41.1 ± 8.6 (p < .001), BRIEF-P-Global Executive Composite 44.8 ± 9.5 (p = .009). Twelve (44%) of the PGD children had a significant difference between their VIQ and PIQ scores (compared to 27% in the general population). One subject was found to show possible signs of autistic spectrum disorder, although a family history of autism was noted. In conclusion, in this pilot study, children assessed at age 4-5 years and conceived after PGD displayed developmental neuropsychological outcomes within normal limits as compared to their chronologic peers. A larger study is needed to evaluate and follow the neuropsychological development of children born after PGD.

Neonatal outcome after preimplantation genetic diagnosis.

OBJECTIVE: To examine whether embryo biopsy for preimplantation genetic diagnosis (PGD) influences neonatal outcomes. DESIGN: Prospective follow-up cohort. SETTING: Tertiary university-affiliated medical center. PATIENT(S): 242 children born after PGD, 242 children born after intracytoplasmic sperm injection (ICSI) (158 singletons and 42 twins pairs in each group), and 733 children born after a spontaneous conception (SC) (493 singletons, 120 twins pairs), matched for maternal age, parity, and body mass index. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Gestational age, birth weight, prematurity (<37 and <34 weeks), low birth weight (<2,500 g, very low birth weight, <1,500 g), and intrauterine growth restriction (<10th percentile for gestational age). RESULT(S): For singletons, the mean birth weight was higher after SC compared with ICSI but not compared with PGD. Mean gestational ages were lower after PGD and ICSI compared with SC. The low birth weight and intrauterine growth restriction rates were 4.4%, 12.0%, and 5.7% and 5.1%, 9.5%, and 5.5% for PGD, ICSI, and SC, respectively. Similar results were found when controlled for the number of embryos transferred and cryopreservation. The results for twins exhibited similar but less statistically significant trends. Polar body and blastomere biopsies provided similar outcomes. CONCLUSION(S): Embryo biopsy itself did not cause intrauterine growth restriction or low birth weight compared with SC, despite lower gestational ages with PGD. The worsened outcomes in ICSI compared with PGD pregnancies may be due to the infertility itself.

PGD for germline mosaicism.

The aim of this study was to develop and perform a preimplantation genetic diagnosis (PGD) assay discriminating between wild-type and mutant alleles in two families with germline mosaicism. Family 1 had two children affected with severe myoclonic epilepsy (SCNA1A del exons 1-22). Family 2 had two children with tuberous sclerosis (TSC2 C1327T) and two healthy children. Neither mutation was detected in genomic DNA derived from the parents in either family. Informative microsatellite markers flanking SCNA1A and TSC2 along with the identified mutations were used to construct haplotypes. For tuberous sclerosis, single spermatozoa were analysed using a multiplex assay that included six informative markers and the TSC2 mutation. In family 1, deletion in the maternal allele was detected in the affected child. In family 2, both affected children and one healthy child shared the same paternal allele. To confirm mutant paternal transmission, single spermatozoa were analysed for the mutation along with six markers. Of 44 single spermatozoa, four showed the mutant T allele, allowing linkage between the mutation and the genetic markers. Both families delivered healthy children following IVF/PGD. In conclusion, germline mosaicism complicates allele assignment when constructing haplotypes for PGD. Sperm analysis is a useful tool for verifying allelic linkage.

Female sex bias in human embryonic stem cell lines.

The factors limiting the rather inefficient derivation of human embryonic stem cells (HESCs) are not fully understood. The aim of this study was to analyze the sex ratio in our 42 preimplantation genetic diagnosis (PGD)-HESC lines, in an attempt to verify its affect on the establishment of HESC lines. The ratio between male and female PGD-derived cell lines was compared. We found a significant increase in female cell lines (76%). This finding was further confirmed by a meta-analysis for combining the results of all PGD-derived HESC lines published to date (148) and all normal karyotyped HESC lines derived from spare in vitro fertilization embryos worldwide (397). Further, gender determination of embryos demonstrated that this difference originates from the actual derivation process rather than from unequal representation of male and female embryos. It can therefore be concluded that the clear-cut tendency for female preponderance is attributed to suboptimal culture conditions rather than from a true gender imbalance in embryos used for derivation of HESC lines. We propose a mechanism in which aberrant X chromosome inactivation and/or overexpression of critical metabolic X-linked genes might explain this sex dimorphism.

Preimplantation genetic diagnosis (PGD)--prevention of the birth of children affected with endocrine diseases.

OBJECTIVE: To develop a reliable and accurate preimplantation genetic diagnosis (PGD) method in six families with endocrine diseases: persistent hyperinsulinemic hypoglycemia of infancy (PHHI), congenital adrenal hyperplasia (CAH) salt-wasting form, Sanjat-Sakati syndrome and multiple endocrine neoplasia 2A (MEN 2A). METHODS: For each disease a battery of at least four informative markers surrounding the tested gene were identified and for each family a protocol of multiplex fluorescent markers was developed and performed on single cells. RESULTS: PGD for PHHI was performed in three families. In family 1 two healthy children were born from different cycles, in family 2 three healthy children were born from two cycles, and in family 3 a healthy boy was born. For CAH in one family a healthy girl was born. One PGD cycle for Sanjat-Sakati resulted in a clinical pregnancy that was terminated due to high nuccal translucency (46X0). For one family with MEN 2A disease, the eighth PGD cycle resulted in birth of healthy twins. In all children genetic confirmation of the healthy status was performed. CONCLUSIONS: PGD is an effective method for preventing birth of affected children with endocrine disorders. Increasing the awareness of clinicians to the availability of these methods is most important.

Preventing mucopolysaccharidosis type II (Hunter syndrome): PGD and establishing a Hunter (46, XX) stem cell line.

OBJECTIVES: Preimplantation genetic diagnosis (PGD) enables the identification of affected embryos prior to implantation. We present for the first time three families in which either the oocytes or embryos obtained from female carriers of mutations in the iduronate-2-sulfatase (IDS) gene underwent PGD for mucopolysaccharidosis type II (Hunter syndrome). Furthermore, we report the first ever derivation of a Hunter's syndrome (46, XX) human stem cell line from embryos (HESC) carrying the IDS and oculocutaneus albinism type 2 mutations. METHODS: Combined polar body (PB) 1 and 2 or a single cell of a six- to eight-cell embryo (blastomere) was used for genetic analysis by multiplex polymerase chain reaction assay using six microsatellite polymorphic markers flanking the gene and mutation. RESULTS: One couple underwent four PB-PGD cycles, with birth of a healthy girl; the second couple with one PB-PGD cycle had healthy twins; the third couple underwent seven cycles of double PGD for Hunter and Albinism syndrome with birth of healthy twins. One novel Hunter 46, XX HESC line was established displaying typical characteristics of HESC cells. CONCLUSIONS: PGD is a reliable method to prevent pregnancy of children affected with Hunter syndrome. In addition, derived HESC can be further utilized for drug testing and better understanding of the pathogenesis of this syndrome.

PGD for fragile X syndrome: ovarian function is the main determinant of success.

BACKGROUND: PGD for fragile X syndrome (FRAX) is inefficient, probably owing to fewer oocytes, poor embryo quality and difficulties in genetic analysis. We investigated IVF-PGD in FRAX mutation carriers compared with controls, looking at the effects of oocyte and embryo number/quality on live birth outcome. METHODS: We performed IVF-PGD in 27 patients with the FRAX mutation and 33 controls with other genetic diseases. Genetic testing was by multiplex PCR. RESULTS: Seventy-nine and 108 IVF-PGD cycles were started in FRAX mutation carriers and controls, respectively. Twenty-two patients had a premutation (CGG repeat number 60-200) and five had a full mutation (300-2000 CGG repeats). FRAX patients required higher doses of gonadotrophins (6788 ± 2379 versus 4360 ± 2330, P< 0.001) but had lower peak serum estradiol levels (8166 ± 5880 versus 10 211 ± 4673, P = 0.03) and fewer oocytes retrieved (9.8 ± 6 versus 14 ± 8, P = 0.01). The cancellation rate (unsatisfactory ovarian response) was higher in the FRAX group than in the control group (13 versus 1%, P < 0.001). When embryos were transferred, ongoing pregnancy/live birth rates per transfer were similar (29 versus 36%, P = 0.54). CONCLUSIONS: Ovarian dysfunction in FRAX carriers is more prevalent and profound than previously appreciated, with a high cancelation rate and reduced efficiency of PGD. The main determinant for successful PGD for FRAX is ovarian dysfunction. When embryo transfer is possible, the results are comparable to PGD for other monogenic diseases.

Preimplantation genetic diagnosis (PGD) for nonsyndromic deafness by polar body and blastomere biopsy.

PURPOSE: Development of an efficient and reliable PGD protocol for nonsyndromic deafness, by polar body (PB) and blastomere PGD. METHODS: The GJB2/GJB6 mutations along with 12 polymorphic markers were used in PGD analysis of blastomeres or polar bodies in 14 couples for 35 cycles. Marker informativity, diagnosis rates, Allele Drop Out (ADO) rates and PB1 heterozygosity rates were assessed. RESULTS: Six cycles were performed by PB biopsy, 27 by blastomere and two combined cycles, resulting in delivery of three unaffected children and five ongoing pregnancies. Diagnosis rates for PB and blastomeres were similar. Only 17% PB1s were heterozygote. ADO rates of 19% were observed in both groups. CONCLUSIONS: We have developed a single cell multiplex PGD protocol for nonsyndromic deafness with a high efficiency of diagnosis. Most PB1 are homozygous, and similar ADO rates were observed; therefore, blastomere biopsy appears to be the method of choice for this autosomal recessive disease.

Similar outcome for cryopreserved embryo transfer following GnRH-antagonist/GnRH-agonist, GnRH-antagonist/HCG or long protocol ovarian stimulation.

The pregnancy rates after triggering of final oocyte maturation with gonadotrophin-releasing hormone (GnRH) agonist in GnRH-antagonist ovarian stimulation protocols are lower than those following triggering with human chorionic gonadotrophin (HCG). Furthermore, lower pregnancy rates following GnRH-antagonist protocols compared with long GnRH-agonist protocols have been reported. The differences might be due to an impact on oocyte number and quality or on the endometrium. If any stimulation protocol had a negative impact on oocyte quality, then further evidence of this effect would be observed following frozen-thawed embryo transfer originating from that stimulation cycle. The outcome of frozen-thawed embryo transfer was retrospectively analysed using the long protocol with triptorelin depot 3.75 mg (n = 215) or 0.1 mg/day (n = 83), or GnRH-antagonist protocol with either HCG (n = 69) or GnRH-agonist (n = 25) for final oocyte maturation. The outcomes measured were implantation rate, clinical pregnancy rate, ongoing pregnancy rate and embryo survival rate. All outcomes were similar in the four groups. It is concluded that the potential for frozen-thawed embryos to implant and develop following transfer is independent of the GnRH-analogue and the final oocyte maturation protocol used in the collection cycle. Lower IVF embryo transfer success using GnRH-antagonist/GnRH-agonist protocol does not appear to be related to an adverse effect on oocyte quality.