Search

STUDY QUESTION: What are the factors influencing the success rate for couples undergoing preimplantation genetic testing (PGT) for polycystic kidney disease (PKD)? SUMMARY ANSWER: In our study cohort, the live birth delivery rate is significantly associated with female age while the male infertility accompanying autosomal dominant PKD (ADPKD) does not substantially affect the clinical outcome. WHAT IS KNOWN ALREADY: While women with ADPKD have no specific fertility problems, male ADPKD patients may present with reproductive system abnormalities and infertility. STUDY DESIGN, SIZE, DURATION: This retrospective cohort study involves 91 PGT cycles for PKD for 43 couples (33 couples for PKD1, 2 couples for PKD2 and 8 couples for autosomal recessive PKD (ARPKD)) from January 2005 until December 2016 with follow-up of transfers until end of 2017. PARTICIPANTS/MATERIALS, SETTING, METHODS: Sixteen single-cell clinical tests for PKD based on multiplex PCR of short tandem repeat markers, with or without a specific mutation were developed and applied for diagnosis of 584 Day 3 cleavage stage embryos. In 18 couples, the male partner was affected with ADPKD (=Group A) and 12 of them had a documented infertility status. Group A underwent 52 cycles to oocyte retrieval. For 18 other couples, the female partner was affected with ADPKD (=Group B) and four male partners from this group had a documented history of infertility. This group underwent 31 cycles to OR. MAIN RESULTS AND THE ROLE OF CHANCE: Genetic analysis resulted in 545 embryos (93.3%) with a diagnosis, of which 215 (36.8%) were genetically transferable. Transfer of 74 embryos in 53 fresh cycles and of 34 cryopreserved embryos in 33 frozen-warmed embryo transfer cycles resulted in a live birth delivery rate of 38.4% per transfer with 31 singleton live births, two twin live births and one ongoing pregnancy. The observed cumulative delivery rate was 57.8% per couple after five treatment cycles. Thirty cryopreserved embryos still remain available for transfer. The clinical pregnancy rate per transfer (fresh + frozen; 45.9% in group A versus 60.0% in group B, P < 0.05) and the live birth delivery rate per transfer (fresh + frozen; 27.0% in group A versus 42.9% in group B, P < 0.05) was significantly lower for couples with the male partner affected with ADPKD compared with couples with the female partner affected with ADPKD. However, a multivariate logistic regression analysis showed that only female age was associated with live birth delivery rate (odds ratio = 0.87; 95% CI: 0.77-0.99; P = 0.032). LIMITATIONS, REASONS FOR CAUTION: This study is based on retrospective data from a single centre with Day 3 one-cell and two-cell biopsy. Further analysis of a larger cohort of PKD patients undergoing PGT is required to determine the impact of male infertility associated with ADPKD on the cumulative results. WIDER IMPLICATIONS OF THE FINDINGS: Knowledge about factors affecting the clinical outcome after PGT can be a valuable tool for physicians to counsel PKD patients about their reproductive options. Males affected with ADPKD who suffer from infertility should be advised to seek treatment in time to improve their chances of conceiving a child. STUDY FUNDING/COMPETING INTEREST(S): No funding was obtained. There are no competing interests to declare. TRIAL REGISTRATION NUMBER: Not applicable.

Preimplantation genetic testing-human leukocyte antigen '(PGT-HLA) only' refers to the HLA typing of single or few cells biopsied from in vitro fertilized preimplantation embryos. The aim of the procedure is to establish a pregnancy, in which the fetus is HLA compatible with an affected sibling in need of a hematopoietic stem cell transplantation (HSCT). During PGT-M-HLA, the identification of a HLA-compatible embryo is combined with the detection of mutation(s) underlying immunodeficiencies and hemoglobinopathies. We report a combined retrospective and prospective cohort analysis of PGT-(M-)HLA procedures carried out from 1998 until 2017, with follow-up of transplantations to 2019. During the study period, 234 couples from 22 countries were invited for a multidisciplinary consultation. Two couples were rejected and 70 couples declined (various reasons), leaving 162 couples for which 414 clinical cycles were carried out. Cleavage stage biopsy followed by single-cell multiplex PCR for short tandem repeat-based haplotyping was applied in most cases (98.7%). The diagnostic efficiency was high (94.8%) but only 16.5% of the embryos was genetically suitable for transfer. Fresh and frozen-thawed embryo transfer resulted in 67 clinical pregnancies, 63 deliveries, and 74 live births, of which 60 children were HLA compatible. This yielded a live birth delivery rate of 30.3% per transfer. Information on neonatal characteristics of the matching PGT-(M-)HLA children showed reassuring outcomes. So far, HSCT was carried out successfully for 25 out of 26 cases. In conclusion, our data show that PGT-(M-)HLA is a valuable procedure: the high complexity and limited delivery rate are balanced by the successful HSCT outcome and the positive impact on families.

STUDY QUESTION Does Day 3 embryo biopsy for pre-implantation genetic testing for monogenic (PGT-M) and structural chromosomal aberrations (PGT-SR) affect body composition and blood pressure readings of 6-year-old singletons? SUMMARY ANSWER This study of 87 PGT-M and PGT-SR conceived singletons showed no differences in anthropometric measurements and blood pressure readings in comparison with a matched cohort of peers born after ICSI without embryo biopsy. WHAT IS KNOWN ALREADY While neonatal outcomes after PGT conception have been found comparable to those after ICSI without embryo biopsy, only a few studies have reported outcomes after PGT at older ages. Moreover, embryo biopsy is also applied in couples who opt for PGT-M and PGT-SR and hence are not necessarily infertile. Health parameters and in particular body composition data in this group of children are lacking. STUDY DESIGN, SIZE, DURATION This single-centre matched-pair cohort study evaluated body composition of 6-year-old children born after fresh blastocyst embryo transfer with or without embryo biopsy performed at Day 3 for the purpose of PGT-M and PGT-SR. For each child born after embryo biopsy, a singleton born after transfer of a fresh ICSI embryo at the blastocyst stage and reaching the age of 6 years between May 2011 and June 2017 was matched as closely as possible for gender, age, maternal educational level and birth order. PARTICIPANTS/MATERIALS, SETTING, METHODS Anthropometry (weight, height, BMI, skinfold thickness, waist and mid-upper arm circumference) and blood pressure readings in a longitudinally followed cohort of 87 singletons conceived by PGT-M and PGT-SR and a pairwise matched sample of 87 children conceived by ICSI are described. Results are adjusted for current, neonatal and parental characteristics. MAIN RESULTS AND THE ROLE OF CHANCE From the 124 eligible PGT-M and PGT-SR families, 110 could be reached of whom 23 refused and 87 (87/110 = 79%) participated. All anthropometric measurements, including z-scores of BMI, waist and mid-upper arm circumference, were comparable between the PGT-M and PGT-SR (−0.23; 0.27; 0.17, respectively) and ICSI (−0.29; 0.11; 0.11, respectively) groups (all P > 0.05). Furthermore, indices of peripheral (triceps) and central (subscapular) adiposity derived from skinfold thickness were comparable (PGT-M and PGT-SR: 14.7 mm; 11.6 mm and ICSI: 15.5 mm; 11.5 mm) as well as the percentage total body fat mass derived from these (PGT-M and PGT-SR: 13.7% and ICSI: 13.9%) (all P > 0.05). Z-scores for blood pressure were also comparable between the PGT and ICSI groups (all P > 0.05). Results did not change when adjusted for neonatal (birthweight, birth order), current (age) and parental (smoking during pregnancy, parental BMI) characteristics. Hospitalization rate and surgical intervention rate were not different for PGT-M and PGT-SR children compared to matched peers born after ICSI. LIMITATIONS, REASONS FOR CAUTION Although our study describes the largest cohort of singletons born after embryo biopsy worldwide, we were only able to detect moderate differences in anthropometrics and blood pressure with our sample size. WIDER IMPLICATIONS OF THE FINDINGS Although Day 3 embryo biopsy followed by blastocyst transfer is not associated with adverse outcomes regarding anthropometry and blood pressure, future studies should focus on outcomes in children born after trophectoderm biopsy and/or transfer of warmed embryos after vitrification. STUDY FUNDING/COMPETING INTEREST(s) This study was supported by Methusalem grants and by grants from Wetenschappelijk Fonds Willy Gepts; all issued by the Vrije Universiteit Brussel (VUB). All co-authors, except M.B. declared no conflict of interest. M.B. has received consultancy fees from MSD, Serono Symposia and Merck. The Universitair Ziekenhuis Brussel (UZ Brussel) and the Centre for Medical Genetics have received several educational grants from IBSA, Ferring, Organon, Shering-Plough, Merck for establishing the database for follow-up research and organizing the data collection.

Fragile X syndrome (FXS), the most common inherited intellectual disability syndrome, is caused by expansion and hypermethylation of the CGG repeat in the 5' UTR of the FMR1 gene. This expanded repeat, also known as the rare fragile site FRAXA, causes X chromosome fragility in cultured cells from patients but only when induced by perturbing pyrimidine synthesis. We performed preimplantation genetic diagnosis (PGD) on 595 blastomeres biopsied from 442 cleavage stage embryos at risk for FXS using short tandem repeat (STR) markers. In six blastomeres, from five embryos an incomplete haplotype was observed with loss of all alleles telomeric to the CGG repeat. In all five embryos, the incomplete haplotype corresponded to the haplotype carrying the CGG repeat expansion. Subsequent analysis of additional blastomeres from three embryos by array comparative genomic hybridization (aCGH) confirmed the presence of a terminal deletion with a breakpoint close to the CGG repeat in two blastomeres from one embryo. A blastomere from another embryo showed the complementary duplication. We conclude that a CGG repeat expansion at FRAXA causes X chromosome fragility in early human IVF embryos at risk for FXS. © 2015 Wiley Periodicals, Inc.

Although the possibilities for the treatment of infertility have been improved tremendously, not every couple will be treated successfully.Crude overall pregnancy rates of 50-65% per patient can be achieved nowadays, irrespective of the type of profertility treatment applied first.IVF only accounts for about 20% of the pregnancies achieved. Dropout is an important reason for not reaching the estimated pregnancy rate. Even after failed IVF, spontaneous pregnancies do occur. Sperm and oocyte donation (OD) offer additional chances to subfertile couples. Severity of the male factor (in sperm donation) and young donor age (in OD) are important determinants of success.Analysis of assisted reproduction technology outcomes would benefit from more universally accepted definitions and deserves better statistical analysis. Long-term cumulative live birth rates of 80% may be expected if dropout can be limited. Milder stimulation, a patient-friendlier approach and better counseling may help to keep more patients in the program.

Disruption of ovulation occurs in different types of clinical infertility. The World Health Organization (WHO) has provided a classification of ovulation disorders. This review focuses on WHO group 2 anovulation. Searches were performed in Medline/PubMed and EMBASE. Each subject summary was presented to the European Society of Human Reproduction and Embryology (ESHRE) Workshop Group, where omissions or disagreements were resolved by discussion. Disorders resulting in ovulatory disturbances are a relatively common cause of infertility. They occur most frequently in the context of WHO group 2 anovulation as reflected, for example, in the polycystic ovary syndrome (PCOS). The aetiology of PCOS remains unclear but evidence exists for a multifactorial origin with a genetic predisposition. Women with PCOS show an increased time to pregnancy but their eventual family size is not necessarily reduced. Also their frequency of miscarriage does not appear increased. Clomiphene citrate is still the first-line treatment in subfertile anovulatory patients with PCOS, with gonadotrophins and laparoscopic ovarian surgery as second-line options. Aromatase inhibitors show promising results. Long-term health risks in patients with WHO group 2 anovulation demand their general health be monitored, even after their reproductive needs have been fulfilled. Metabolic and cardiovascular risk prevention in women with PCOS should start as early as possible. It is not easy to analyse the possible role of PCOS, independent of obesity, metabolic syndrome, insulin resistance and diabetes, on long-term health.

The primary aim of this study was to gain more insight into maturation arrest of spermatogenesis (MA) and its relationship with mutations in genes essential for meiosis. The study also investigated the possibility that mutations in human meiosis genes cause a milder phenotype and that, in such cases, meiosis could potentially be completed with the production of mature germ cells having an abnormal chromosomal constitution causing miscarriage. Among 40 patients with MA, five changes were observed that also predicted alterations at the amino acid level. However, since these changes were also present in men with normozoospermia in equal frequencies, it was assumed that these changes are single nucleotide polymorphisms. Among 46 patients with recurrent miscarriages, two additional changes were detected predicting an alteration at the amino acid level. One change was detected in controls. However, the second heterozygous change, detected in a conserved functional domain of the SYCP3 gene, was absent in >200 controls. These preliminary results stress the need to further investigate the relationship between abnormalities in meiosis genes and the formation of gametes with abnormal chromosomal constitution. More research is also necessary to determine the impact and frequency of such changes before implementing mutation screening in genetic counselling. Our primary aim was to gain more insight into maturation arrest of spermatogenesis (MA). Patients with MA are infertile due to the absence of sperm cells in their ejaculates. A biopsy of the testis of these patients showed that the germ cells stopped developing at a level called meiosis. During meiosis the chromosomes are divided into two daughter cells. We looked for the presence of alterations in genes essential for meiosis. We also investigated the possibility that modifications in human meiosis genes are causing a milder effect and that in such cases meiosis could potentially be completed with the production of mature germ cells having an abnormal chromosomal constitution. These abnormal germ cells might cause recurrent miscarriages after fertilization. Several changes in three investigated genes were detected. Most of these alterations were also observed in a control group consisting of men with normal semen parameters or men who fathered at least one child. Therefore, these modifications are most likely not the cause of the problems in the patients. However, one change present in an evolutionary important functional domain of the SYCP3 gene was detected in the male partner of a couple suffering from recurrent miscarriages and was absent in >200 controls. Despite our small patient groups, these preliminary results confirm our assumption that abnormalities in meiosis genes might be involved in the formation of gametes with abnormal chromosomal constitution. Yet, further research is necessary to determine the impact and frequency of such changes before implementing mutation screening in genetic counselling.

Objective The major objective of this study was to determine whether the embryo biopsy procedure might cause growth restriction or affect health outcome of children. Study design Auxological data and physical findings were compared at birth and age 2 for 102 children (70 singletons and 32 twins) born after PGD/PGS and 102 matched children born after intracytoplasmic sperm injection (ICSI) in a prospective study . Results No statistically significant differences regarding weight, height and head circumference standard deviation scores (SDS) at birth and at age two years were observed. At two years of age the mean BMI SDS tended to be lower in PGD/PGS children ( p = 0.058). PGD/PGS babies had been more often breastfed ( p = 0.013), but mostly during a shorter time. The prevalence of major as well as minor congenital anomalies, hospital admissions and surgical interventions was similar. Conclusion Children born after embryo biopsy applied in PGD/PGS present similar prenatal and postnatal growth and health outcome in the first two years of life compared to ICSI children. Up till now, PGD and PGS appear not to be associated with a higher risk for health problems.

In vitro fertilization and embryo transfer or gamete (or zygote) intra-Fallopian transfer after ovum donation were performed in 16 patients with primary or secondary amenorrhea, associated with chromosome abnormalities. The patients showed the wide range of (mostly X) chromosome abnormalities characteristic for women with primary or premature ovarian failure. Four of these patients became pregnant and three of them have delivered healthy infants with a normal karyotype. This pregnancy rate is far superior to the accepted fertility figure in these patients. When these results were compared with the fertility treatment results of three other groups of women with absent ovarian function (1. ovarian dysgenesis; 2. surgical castration; 3. premature menopause) but with a normal 46,XX karyotype, no difference in treatment efficiency could be detected. These results offer a promising approach for the treatment of infertility in agonadal patients with chromosome aberrations.

BACKGROUND: This is the first follow-up study of the cognitive, psychosocial, family-relational and medical development of ICSI children at ages 8 and 10. Second-wave study results on the cognitive and motor development of 10-year-old ICSI children are discussed and compared with the outcome at 8 years. METHODS: Developmental outcomes of 109 10-year-old singletons born through ICSI after at least 32 weeks of gestation were compared with those of 90 singletons of the same age born after spontaneous conception (SC). The outcomes were also compared with the outcomes of the children at age 8. RESULTS: 10-year-old ICSI children obtained total, verbal and performance intelligence scores comparable to those of SC children. No significant differences were found between ICSI and SC children regarding overall motor, manual and ball skills. ICSI children and girls appear to have better balance skills than SC children and boys, respectively. With regard to long-term follow-up, the IQ results of ICSI and SC children at age 10 appear to have converged (from slightly higher scores in the ICSI children at age 8), probably indicating a decreased effect of maternal educational level or stimulating home environment in the ICSI group over time. CONCLUSIONS: In this follow-up study, ICSI and SC children show a comparable cognitive and motor development until the age of 10. These findings are in line with those obtained at age 8.

Feeder-free human embryonic stem cell (hESC) culture is associated with the presence of mesenchymal-like cells appearing at the periphery of the colonies. The aim of this study was to identify this early differentiation process. Long-term feeder-free hESC cultures using matrigel and conditioned medium from mouse and from human origin revealed that the appearance of mesenchymal-like cells was similar regardless of the conditioned medium used. Standard characterization confirmed the preservation of hESC properties, but the feeder-free cultures could not be maintained longer than 37 passages. The early differentiation process was characterized in the short term after switching hESCs cultured on feeders to feeder-free conditions. Transmission electron microscopy showed an epithelium-like structure inside the hESC colonies, whereas the peripheral cells revealed the acquisition of a rather mesenchymal-like phenotype. Immunochemistry analysis showed that cells at the periphery of the colonies had a negative E-cadherin expression and a positive Vimentin expression, suggesting an epithelial-mesenchymal transition (EMT). Nuclear staining of beta-catenin, positive N-cadherin and negative Connexin 43 expression were also found in the mesenchymal-like cell population. After RT-PCR analysis, Slug and Snail, both EMT-related transcription factors, were detected as up-regulated in the mesenchymal-like cell population. Taken together, our data suggest that culturing hESCs in feeder-free conditions enhances an early differentiation process identified as an EMT.

Patients with recurrent IVF failure are defined as patients who are younger than 37 years and who had at least three consecutive unsuccessful IVF/intracytoplasmic sperm injection (ICSI) cycles with good quality embryos. These patients might be predisposed to chromosome errors in their embryos and therefore might benefit from preimplantation genetic diagnosis for aneuploidy screening (PGD-AS). This technique is, however, expensive and some normal embryos might be lost due to the error rate. The aim of this retrospective study was to define those patients who would benefit most from it. One hundred and twenty-one first PGD-AS cycles for recurrent IVF failure were analysed. The aneuploidy rate, 'no embryo transfer' rate, live birth rate per embryo transfer and implantation rate were respectively 48.3, 22.3, 29.7 and 19.5%. A multivariate logistic regression analysis gave us a predictive model demonstrating that to have a 90% probability of having an embryo transfer after PGD-AS, the patient should have at least 10 mature oocytes, eight normally fertilized oocytes and six embryos for biopsy. This study suggests that most patients with recurrent IVF failure may benefit from PGD-AS. Future studies, however, should more strictly define this heterogeneous group of patients, so that comparison is easier.

In humans, the Deleted in Azoospermia Like (DAZL) gene is believed to function in the development of primordial germ cells and in germ cell differentiation and maturation because the expression of DAZL is only found in the germ and non-germ lineage of the reproductive system and in embryonic stem (ES) cells. The present study examined the presence of DAZL transcripts in the last stages of oocyte maturation, in ES cells, and throughout the preimplantation development; the link between gametes and ES cells. The finding of DAZL transcripts in the last stages of oogenesis and during the first two cell cycles of the preimplantation development was expected, because DAZL is a germ cell marker and the transcripts present at that time are generally encoded by the maternal genome. During the third cell cycle, DAZL showed a variable expression pattern, which may point to the maternal to embryonic transition. After the third cell cycle, transcripts were again consistently detected, suggesting embryonic DAZL transcription. In blastocysts, DAZL transcripts were only detected in those of good quality and this as well in the inner cell mass (ICM) as in the trophectoderm (TE). The presence of DAZL transcripts in the ICM and in ES cells was not surprising since both can lead to the formation of germ cells, but TE cells cannot. The quality-related expression of DAZL in blastocysts, and especially its trophectodermal expression, might imply other functions for DAZL beyond germ cell development.

PGD is an alternative to prenatal diagnosis that circumvents therapeutic abortion. Diagnosis is carried out on single cells obtained from three-day-old embryos, and only those that are free of the disease under consideration are transferred to the mother. Neurofibromatosis type 1 (NF1) is a common neurocutaneous disorder, inherited as an autosomal dominant trait and caused by mutations in the NF1 gene. For some patients, PGD may be the only acceptable manner to ensure the birth of unaffected children. Because of the large number of known NF1 mutations, the development of mutation-specific single-cell protocols is impractical, labour-intensive and expensive. This paper discusses the development of five PGD protocols, three of which are based on multiplex PCR for microsatellite-markers linked to the NF1 gene. After a linkage study, the diagnosis can be established through the markers, thereby obviating the need to detect the mutation itself. This not only ensures the accurate diagnosis of the embryos, but also a prompt acceptance of PGD referrals since one protocol can be useful for several couples. In addition, two mutation-specific PCRs were developed for two couples where a marker-based protocol was not applicable. In total, 16 PGD cycles were carried out for six couples, which resulted in one ongoing pregnancy and the delivery of a healthy unaffected boy.

BACKGROUND: It is generally accepted that the age-related increased aneuploidy rate is correlated with reduced implantation and a higher abortion rate. Therefore, advanced maternal age (AMA) couples are a good target group to assess the possible benefit of preimplantation genetic diagnosis for aneuploidy screening (PGD-AS) on the outcome after assisted reproductive technology (ART). METHODS: A prospective randomized controlled clinical trial (RCT) was carried out comparing the outcome after blastocyst transfer combined with PGD-AS using fluorescence in situ hybridization (FISH) for the chromosomes X, Y, 13, 16, 18, 21 and 22 in AMA couples (aged ≥37 years) with a control group without PGD-AS. From the 400 (200 for PGD-AS and 200 controls) couples that were allocated to the trial, an oocyte pick-up was performed effectively in 289 cycles (148 PGD-AS cycles and 141 control cycles). RESULTS: Positive serum HCG rates per transfer and per cycle were the same for PGD-AS and controls: 35.8% (19.6%) [%/per embryo transfer (per cycle)] and 32.2% (27.7%), respectively (NS). Significantly fewer embryos were transferred in the PGD-AS group than in the control group (P < 0.001). The implantation rate (with fetal heart beat) was 17.1% in the PGD-AS group versus 11.5% in the control group (not significant; P = 0.09). We observed a normal diploid status in 36.8% of the embryos. CONCLUSIONS: This RCT provides no arguments in favour of PGD-AS for improving clinical outcome per initiated cycle in patients with AMA when there are no restrictions in the number of embryos to be transferred.

The CTG repeat at the 3' untranslated region of the dystrophia myotonica protein kinase (DMPK) gene shows marked intergenerational and somatic instability in patients with myotonic dystrophy (DM1), when the repeat is expanded to more than approximately 55 repeats. Intensive research has yielded some insights into the timing and mechanism of these intergenerational changes: (1) increases in expansion sizes occur during gametogenesis but probably not during meiosis, (2) the marked somatic mosaicism becomes apparent from the 2nd trimester of development onward and increases during adult life, and (3) DNA repair mechanisms are involved. We have performed preimplantation genetic diagnosis for DM1 since 1995, which has given us the unique opportunity to study the expanded CTG repeat in affected embryos and in gametes from affected patients. We were able to demonstrate significant increases in the number of repeats in embryos from female patients with DM1 and in their immature and mature oocytes, whereas, in spermatozoa and embryos from male patients with DM1, smaller increases were detected. These data are in concordance with data on other tissues from adults and fetuses and fill a gap in our knowledge of the behavior of CTG triplet expansions in DM1.

Background Beta-Thalassaemia results from co-inheritance of two mutant beta-globin alleles. Allogeneic cord blood cell transplantation (CBT) from an HLA-identical sibling donor is an excellent treatment option for beta-thalassaemia. In families with an affected child and willing to have another child, IVF followed by preimplantation genetic diagnosis (PGD) can be applied to exclude affected embryos. Furthermore, healthy embryos could be HLA matched with the affected child so that cord blood from the future newborn can be used to transplant the affected sibling. Methods We developed an indirect single-cell HLA typing technique based on the use of a bank of seven microsatellite markers within the HLA locus from which four informative and evenly distributed markers were selected. Results The methodology was validated in three beta-thalassaemia families having six ovarian stimulation cycles in view of IVF and PGD. Six PGD cycles were performed in two families. On 58 embryos tested, the combined PCR was successful in 54 (93%). Two transfers were done and one clinical pregnancy was obtained. Using confirmatory analysis on 50 embryos, the accuracy for HLA typing was 100%. Conclusion This strategy offers a new therapeutic option for patients with beta-thalassaemia and other monogenic diseases that can be cured with CBT.

Imprinting is an epigenetic mechanism leading to mono-allelic expression of imprinted genes. In order to inherit the differential epigenetic imprints from one generation to the next, these imprints have to be erased in the primordial germ cells and re-established in a sex-specific manner during gametogenesis. The exact timing of the imprint resetting is not yet known and the use of immature gametes in assisted reproductive technologies may therefore lead to abnormal imprinting and related diseases. Imprinting is associated with differential allelic methylation in a CpG-context. We studied the methylation patterns of the imprint control (IC) region of the human SNRPN-gene in human spermatozoa, oocytes in different developmental stages [germinal vesicle (GV), metaphase I and metaphase II oocytes] and in preimplantation embryos using the bisulphite sequencing technique. In the spermatozoa, almost all potential methylation sites were unmethylated whereas mainly methylated patterns were found in the oocytes at different developmental stages. In the embryos, an average methylation pattern of 53% was found indicating that the imprints, which have been set during gametogenesis, are stably maintained in the preimplantation embryo. Our results indicate that the maternal imprints for the IC-region of the human SNRPN-gene are already re-established at the GV stage and that they are not re-established in a late oocyte stage or after fertilization as previously reported. Recent advances in assisted reproductive technologies raise questions concerning safety and the epigenetic risks involved. Our study was the first to check the methylation imprints in human pre-implantation embryos and oocytes at different developmental stages.

BACKGROUND: Zona pellucida opening for blastomere removal can be done by mechanical or chemical means, or by laser. So far, only limited data on the use of laser systems for zona drilling in cases of PGD are available. METHODS: Results of embryo biopsy and outcome of PGD in two periods were compared. In the first period, acid Tyrode medium was used for zona drilling. In the second period, zona drilling was performed by a 1.48 µm infrared laser. RESULTS: In the first period, 59 cycles resulted in 53 biopsy procedures with 356 biopsied embryos. In the second period, these numbers were 69 cycles, 69 biopsy procedures and 402 biopsied embryos. Fewer blastomeres were intact (95.2%) after zona drilling with acid Tyrode than after laser zona drilling (98.3%, P = 0.02). Rates of positive HCG (37.5% versus 35.5%), ongoing pregnancy rates (31.3% versus 25.0%) and ongoing implantation rates (18.9% versus 14.9%) did not differ. CONCLUSIONS: The use of a laser for zona drilling in cases of PGD is an easier procedure and results in more intact blastomeres. Since similar pregnancy rates are obtained, it is advantageous to use a laser for zona drilling. Further follow-up is necessary to prove the safety of this procedure.

The use of ICSI has been a major breakthrough in the treatment of male infertility. Even azoospermic patients with focal spermatogenesis in the testis, may benefit from the ICSI technique in order to father a child. As ICSI use has become more common, centres have introduced infertility treatment for Klinefelter patients. To date, 34 healthy children have been born using ICSI without PGD, and the conception of one 47,XXY fetus has been reported. In view of the possible risk of an increased gonosome number in the spermatozoa of Klinefelter patients, a safer approach--offering these couples ICSI combined with PGD--has been used, and has resulted in the birth of three healthy children. Couples in which the male suffered from Klinefelter's syndrome were first treated in 1995; these patients were offered ICSI + PGD using FISH technology, notably to enumerate the X and Y chromosomes. ICSI + PGD was performed in 32 cycles of 20 couples with spermatozoa originating from a fresh ejaculate (n = 1), testicular biopsy (n = 21) or frozen-thawed testicular biopsy (n = 10). Normal fertilization occurred in 56.0 +/- 22.4% of the successfully injected oocytes. On day 3 of development, 119 embryos from 29 cycles were of sufficient quality to undergo biopsy and subsequent PGD; a positive result was obtained in 113 embryos. Embryos were available for transfer in 26 cycles, with a mean of 1.6 +/- 0.6 embryos per transfer. Eight pregnancies were obtained, and five resulted in a delivery. A total of 113 embryos from couples with Klinefelter's syndrome was compared with 578 embryos from control couples with X-linked disease where PGD was used to determine gender. A significant fall occurred in the rate of normal embryos for couples with Klinefelter's syndrome (54.0%) compared with controls (77.2%). Moreover, a significantly increased risk of abnormalities was observed for sex chromosomes and autosomes; for each autosome separately, this reached significance level for chromosomes 18 and 21 only. Hence, a cautious approach is warranted in advising couples with non-mosaic Klinefelter's syndrome. Moreover, the use of ICSI + PGD or prenatal diagnosis should be carefully considered.

Background Since the introduction of ICSI in 1991, medical outcome studies on ICSI children have been performed, but few have addressed developmental outcome. Hence, this outcome was assessed by performing a standard developmental test on children born after ICSI as compared with children born after IVF, at the age of 2 years. Methods In a prospective study, the medical and developmental outcome of 439 children born after ICSI (378 singletons, 61 twins) were compared with those of 207 children born after IVF (138 singletons, 69 twins), at the age of 24-28 months. These children were part of a cohort of children followed since birth. Of children reaching the age of 24-28 months between May 1995 and March 2002, 44.3% (2375/5356) were examined by a paediatrician who was unaware of the type of treatment used for each couple. Of all the children born, 12.2% (439/3618) in the ICSI group and 11.9% (207/1738) in the IVF group underwent a formal developmental assessment using the Bayley Scale of Infant Development (mental scale) by a paediatrician blinded to the type of treatment. Results There was no significant difference in maternal educational level, maternal age, gestational age, parity, birthweight, neonatal complication rate or malformation rate at 2 years between ICSI and IVF singletons, or between ICSI and IVF twins. No significant difference was observed in the developmental outcome using the Bayley scale at the age of 24-28 months (raw scores or test age) between ICSI children or IVF children. A multivariate regression analysis for the singleton children indicated that parity, sex (boys had lower scores than girls) and age had a significant influence on the test result, but that the fertility procedure (ICSI versus IVF) did not influence the test result. ICSI children from fathers with low sperm concentration, low sperm motility or poor morphology had a similar developmental outcome to that of children from fathers with normal sperm parameters. There were no significant differences between the initial cohort and the group lost to follow-up, nor between the psychologically tested and the non-tested group for a number of variables such as maternal educational level, birthweight in singletons and neonatal malformation rate. Although only some of the cohort of ICSI children were evaluated, a representative sample of both ICSI and IVF children was compared. Conclusions There is no indication that ICSI children have a lower psychomotor development than IVF children. Paternal risk factors associated with male-factor infertility were found not to play a role in developmental outcome.

The calcium-sensing receptor (Ca-R) is a G-protein-coupled surface receptor that plays a crucial role in calcium homeostasis via parathyroid hormone secretion. Mutations of this receptor can cause a gain in, or loss of, function, leading to hypo- or hypercalcemia, respectively. We report here a family with hypocalcemia in whom a heterozygous missense mutation in exon 4 was demonstrated, predicting a proline to leucine substitution (P221L) in the extracellular part of the Ca-R. Clinical symptoms were limited to fatigue. When serum calcium was further lowered via a citrate infusion, a significant increase in circulating iPTH was observed, although with lower peak values than in normal controls, suggesting a gain in function of the Ca-R. Treatment with calcium supplements and calcitriol led to prohibitive hypercalciuria without normalizing serum calcium. The aims of this case report are: (1) to present a mutation in the Ca-R with a gain in function at a codon where previously loss of function was described, and (2) to suggest that measuring circulating iPTH during a citrate infusion in the presence of familial hypocalcemia is an additional test to diagnose this particular form of hypoparathyroidism.

Recently, several requests were made by couples with an affected child who wanted preimplantation genetic diagnosis (PGD) to select embryos in the hope of conceiving an HLA identical donor sibling. This article considers the ethical arguments for and against the application of PGD for this goal. Only embryos HLA matched with an existing sibling in need of a compatible donor of haematopoietic stem cells would be transferred. The main arguments are the instrumentalization of the child, the best-interests standard, the postnatal test for acceptability and the experience of the donor child. It is argued that conceiving a child to save a child is a morally defensible decision on the condition that the operation that will be performed on the future child is acceptable to perform on an existing child. The instrumentalization of the donor child does not demonstrate disrespect for its autonomy or its intrinsic worth.

Genetic imprinting is a mechanism of gene regulation by which only one of the parental copies of a gene is expressed. This process is mediated by the methylation of DNA. As spermatozoa represent exclusively the paternal contribution to a future individual, they are expected to carry the paternal imprint only. For intracytoplasmic sperm injection (ICSI), spermatozoa mostly have to be selected from samples with pathological semen parameters. Correct establishment of the paternal imprint in these spermatozoa has not yet beets demonstrated. In the present study, imprinting analysis was undertaken using DNA extracted from spermatozoa from men with normal semen analysis (group A: n = 30 patients) and from men with an abnormal sperm count (B: n = 30 patients with 5-20 million spermatozoa/mL and C: n = 30 patients with ≤5 million spermatozoa/mL) from the ICSI program. It was performed using firstly a conventional methylation-specific polymerase-chain-reaction (M-PCR) and secondly a more sensitive modified hemi-nested M-PCR technique. In addition, a single cell PCR was performed on a total of 88 single spermatozoa (collected from nine males) and on 25 leucocytes (control group). With the conventional M-PCR, exclusively paternal imprints were found in all groups. Using the more sensitive hemi-nested M-PCR, additional maternal imprints were found in 63% of the samples in A, 57% in B and 60% in C. In the single cell PCR, exclusively paternal imprints were detected. Because of the very small amount of DNA (3 pg), a complete amplification failure occurred in 43% of spermatozoa. The correct paternal and maternal imprints were found in 56% of the analysed leucocytes (complete amplification failure in the other 44%). In conclusion, ejaculated spermatozoa from males with medium or high-grade semen pathology proved to have the same imprinting status as those from males with normal semen parameters. As the additional maternal imprints were never found at the single cell level, they were classified as contamination by diploid cells such as leucocytes or immature germ cells in the processed and purified semen samples, which can be detected by a more sensitive PCR method in contrast to the conventional standard PCR.

We developed single-cell polymerase chain reaction (PCR) assays for preimplantation genetic diagnosis (PGD) in couples carrying mutations in the beta-globin gene. With PGD the genetic status of an embryo obtained after intracytoplasmic sperm injection (ICSI) is determined by PCR analysis in single blastomeres, allowing only healthy embryos to be transferred to the uterus. We carried out nine PGD cycles using fluorescent PCR for two couples in whom the partners carried sickle-cell trait. Both couples achieved pregnancies, one of which was spontaneously aborted. We have developed two beta-thalassemia PGD protocols: one for the analysis of the 25-26delAA and the IVS2+1G>A mutation, and the other for the simultaneous detection of the IVS1+6T>C and the IVS1+110G>A mutations. For the second protocol, both non-labelled PCR and later fluorescent PCR were used. Both protocols were applied in clinical cycles (two non-labelled PCR cycles and one fluorescent PCR cycle) for two couples. The patient with the fluorescent PCR-PGD cycle became pregnant. Overall, the three fluorescent PCR assays were accurate and reliable with amplification efficiencies of minimum 93% and allele dropout (ADO) rates between 0 and 12%.

This study reports on the validation of a diagnostic screening programme for Yq deletions in a population of infertile men. First, an unselected group of 402 intracytoplasmic sperm injection (ICSI) candidate patients was screened prospectively by means of three polymerase chain reactions (PCR) each with one marker in the region AZFa, AZFb or AZFc. With this screening strategy, eight males (2.2%) were found to carry a deletion in Yq11. Secondly, a subgroup of males were further analysed by multiplex PCR with 27 sequence-tagged sites. In this group of 229 cytogenetically normal males with azoospermia, cryptozoospermia or extreme oligozoospermia, including some patients with varicocele or a history of cryptorchidism, only one additional microdeleted patient was found with the multiplex PCR. Hence we obtained a frequency of 2.2% (9/402) or 4% (9/229) in the unselected and selected patient groups respectively. We conclude that in a diagnostic programme for Yq deletions in ICSI candidates it might be sufficient to use only four markers representing the three AZF regions and a more distal region in AZFc. In this way, it is possible to detect most, if not all, Yq deletions which might be the causal factor in the patient's infertility.

The first clinical application of preimplantation genetic diagnosis (PGD) was reported almost a decade ago. Since then, the range of genetic defects that can be detected at single cell level has increased dramatically. At the 13th Annual Meeting of ESHRE in Edinburgh in 1997, a PGD Consortium was formed to undertake the first systematic and long-term study of the efficacy and clinical outcome of PGD. We report here the first data collection covering the period of January 1997 to September 1998. Referral data on 323 couples have been collected for a variety of monogenic and chromosomal disorders, providing information about which patients, at risk for which genetic diseases, are interested in PGD. Data were collected on 392 PGD cycles, resulting in 302 embryo transfers and 66 clinical pregnancies. Because of the importance of follow-up of the children born after PGD, participating centres were asked to contribute data on the pregnancies achieved and the children born after PGD since the start of their PGD programme. Data on 82 pregnancies and 110 fetal sacs were collected, and information was available on 79 children. Finally, biopsy, fluorescence in-situ hybridization and polymerase chain reaction protocols were collected, clearly showing that no consensus exists on technical aspects such as which culture medium to use, and emphasizing the role the PGD Consortium could play in setting up guidelines for good laboratory practice, In conclusion, it is clear that the effort of gathering data on PGD cycles is worthwhile and will be continued in the future, preferably using electronic data collection.

In this study we compared the pregnancy outcome of 576 pregnancies after prenatal diagnosis with that of 540 pregnancies without prenatal diagnosis in our microinjection programme. Amniocentesis was suggested for singleton pregnancies (n = 465) and chorionic villus sampling (CVS) was proposed for twin pregnancies (n = 111 pregnancies, 222 fetuses). A total of 365 patients with singleton pregnancies and 175 patients with twin pregnancies who did not undergo prenatal diagnosis were selected as controls. Compared with the controls, the odds ratios in the amniocentesis group for preterm delivery, low birthweight, very low birthweight and fetal loss were 0.97 [95% confidence interval (CI): 0.60-1.57], 1.27 (95% CI: 0.78-2.06), 1.57 (95% CI: 0.53-4.66) and 0.86 (95% CI: 0.32-2.37) respectively. Compared with the controls, the odds ratios in the CVS group for preterm delivery, low birthweight, very low birthweight and fetal loss were 0.89 (95% CI: 0.61-1.30), 1.03 (95% CI: 0.74-1.45), 0.79 (95% CI: 0.41-1.53) and 0.47 (95% CI: 0.17-1.30) respectively. We concluded that, in this series of intracytoplasmic sperm injection (ICSI) pregnancies, prenatal testing did not increase the preterm-delivery, the low-birthweight, or the very low-birthweight rates as compared with those of the controls. In the prenatal diagnosis group, the fetal loss rate was comparable to that of the control group. Larger prospective controlled studies are needed in order to inform patients reliably about the risks and the advantages of prenatal testing in ICSI pregnancies.

Preimplantation genetic diagnosis (PGD) is a novel procedure whose use may be considered to obtain a very early prenatal diagnosis for couples at risk for transmitting genetic diseases. Using the polymerase chain reaction (PCR) or fluorescence in-situ hybridization (FISH) the genotype or the sex of biopsied cleavage-stage embryos obtained after in-vitro fertilization can be determined and selected embryos can then be transferred. In-vitro fertilization with intracytoplasmic sperm injection is the method of choice to obtain embryos to be analysed through PCR to reduce contamination by residual sperm DNA. In our series of 61 PGD cycles for 29 couples at risk over a period of 4 years the ongoing pregnancy rate per cycle was 15%, per transfer 19% and per patient 31%. One of the six morphologically normal children born, who is still alive and doing well, weighed 850 g after birth at 25 weeks following a complicated triplet pregnancy. More experience is needed to correctly evaluate the efficiency and safety of this novel technique as well as to determine its place in the prevention of genetic disease.

The aim of this prospective follow-up study of children born after intracytoplasmic sperm injection (ICSI) was to compile data on karyotypes, congenital malformations, growth parameters and developmental milestones in order to evaluate the safety of this new technique. The study design included karyotyping of the parents and their agreement to genetic counselling and prenatal diagnosis and it was based on a physical examination of the child at the Centre for Medical Genetics at the ages of 2 months, 1 year and at 2 years, where major and minor malformations and psychomotor evolution are recorded. Here we describe the first 57 children born from 40 ICSI pregnancies with epididymal spermatozoa (group 1), the first 50 children born from 34 ICSI pregnancies with testicular spermatozoa (group 2) and the first 58 children born from 48 pregnancies after replacement of cryopreserved ICSI embryos (group 3). Parental karyotypes were obtained from only 72/246 (29%) parents and were all normal. Prenatal karyotypes were determined for a total of 70 samples (40%): 21 in group 1, 15 in group 2 and 34 in group 3. In this last group 2 abnormal 47,XXY karyotypes (5.8%) and no structural aberrations were found. This increase in de-novo sex-chromosomal aberrations has already been described with regard to the first 877 children born after ICSI carried out at our Centre and is probably linked directly to the characteristics of the infertile men treated rather than to the ICSI procedure itself. Major malformations, defined as those causing functional impairment or requiring surgical correction, were observed in four children: two born after ICSI with epididymal spermatozoa, one after ICSI with testicular spermatozoa and one after ICSI and cryopreservation. No particular malformation was disproportionally frequent. In the follow-up examinations at 2 months (107/161 or 66.5%) and at 1 year (37/161 or 22.9%), no additional anomalies were observed. Lost for follow-up rate at 2 months was 33.5%. These observations on a limited number of children do not suggest a higher incidence of diseases linked to imprinting, nor do they suggest a higher incidence of congenital malformations. These observations are still limited in number and should be further completed by others and by collaborative efforts. In the meanwhile patients should be told about the available data before any treatment: there appears to be some risk of transmitted chromosomal aberrations, of de-novo, mainly sex-chromosomal aberrations and of transmitting fertility problems to the offspring. Patients should also be reassured that until now there seems to be no higher incidence of congenital malformations in children born after ICSI with epididymal or testicular spermatozoa or after replacement of ICSI embryos.

The pyruvate dehydrogenase complex (PDHc) is a multienzyme complex consisting of three catalytic and two regulatory enzymes, as well as a less well defined subunit called protein X. PDHc deficiency is a common cause of congenital lactic acidosis. Most patients with PDH deficiency have a mutation in the alpha chain of the PDH E1 enzyme. Very few patients have been described in whom the basic defect of a PDH deficiency is situated in the X protein. We studied a boy with severe lactic acidosis and developmental delay in whom a deficiency of PDH activity led to further investigations. Immunochemical analysis with anti-PDHc antibodies demonstrated an absence of the X component. This report is the fourth family in which an abnormal protein X has been found. In cases with PDH deficiency where no mutation of the PDHE1 alpha gene is found, further investigations by means of immunoblotting with specific antibodies against the different subunits should be performed.

Absence, dysfunction or low levels of cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein result in a broad range of clinical manifestations with CF with pancreatic insufficiency at the severe end of the phenotypic spectrum and, at the other end, relatively mild clinical conditions, including several forms of male infertility. The condition of congenital bilateral absence of the vas deferens (CBAVD) is in 75–80% of the patients associated with defects in the CFTR gene. In the remaining patients, CBAVD is also associated with urinary tract malformations, and this form of CBAVD is not related to CF. Congenital unilateral absence of the vas deferens also seems to be associated with CF except when associated with renal abnormalities at the ipsilateral side of the absent vas. A possible role of the CFTR protein in the aetiology of infertility due to defects in sperm production and maturation has also been suggested recently. In contrast, Young's syndrome is probably not related to CF. The relation between some conditions of male infertility and CF implies appropriate clinical examination of the patients, CFTR mutation analysis and genetic counselling. Because infertility can now in many cases be treated by artificial reproductive technology couples have an increased risk of having children with CF or infertility if the female partner is also a carrier of a CFTR mutation. Couples should be well informed about these risks and risk prevention including pre-implantation diagnosis. Follow-up studies of children born to these couples are mandatory, whether male infertility is linked to CF or not.

BACKGROUNDHuntington's disease (HD) is an autosomal dominant neurodegenerative late onset disorder. This review of reproductive options aims to increase reproductive confidence and to prevent suffering in relation to family planning around HD and possibly other late onset neurodegenerative disorders.METHODSSelected relevant literature and own views and experiences as clinical geneticists, psychologists and ethicists have been used.RESULTSPossible options, with emphasis on prenatal diagnosis (PD) and preimplantation genetic diagnosis (PGD) to prevent the transmission of HD to the next generation, are described and discussed. They are formally presented in a decision tree, taking into account the presence or absence of a fully penetrant allele (FPA), a reduced penetrant allele (RPA) or an intermediate allele (IA). A table compares invasive and non-invasive PD and PGD. From a psychological perspective, the complex process of counselling and decision-making regarding reproductive options is discussed. Special attention is paid to the decision to avoid the transmission of the mutation and to the confrontation and coping of a mutation-free child growing up with a parent developing disease symptoms. From an ethical point of view, reflections on both PD and PGD are brought forward taking into account the difference between FPA, RPA and IA, direct testing or exclusion testing and taking into account the welfare of the child in the context of medically assisted reproduction.CONCLUSIONRecommendations and suggestions for good clinical practice in the reproductive care for HD families are formulated. ispartof: Human Reproduction Update vol:19 issue:3 pages:304-15 ispartof: location:England status: published

A brightfield microscopical in-situ hybridization (ISH) technique was applied to semen samples of two 47,XYY males, one 46,XY/47,XXY/XXY male with fertility problems, and two normal 46,XY men, who served as controls. The use of a standardized nuclear DNA decondensation method, together with double-target ISH and morphological staining, allowed an accurate study of the sex chromosomal content and morphology of spermatozoa. In the males carrying an extra sex chromosome, we detected X- and Y-bearing spermatozoa in a ratio which did not differ significantly from the 1:1 ratio found in normal males. Aneuploidy for the sex chromosomes was found in approximately 15% of the spermatozoa of both XYY males and in 3% of the XXY male. The most striking finding was the relatively low percentage of spermatozoa in these patients, with an average of 65% in the XYY males and 84% in the XXY male. The other cells represented immature germ cells (IGC), including spermatogonia and spermatocytes arrested at various stages of spermatogenesis. Apparently, in XYY or XXY men, these IGC are shed into the semen to an increased extent as compared to normal, fertile men. The sex chromosome constitution of these IGC was heterogeneous. However, the finding that the majority of spermatozoa in semen of 47,XYY and 47,XXY males carried a single sex chromosome strengthens the hypothesis that a 46,XY germ cell line must be present, apparently with a proliferative advantage over the 47,XYY or 47,XXY cells.

Klinefelter's syndrome is generally characterized by hypergonadotrophic hypogonadism and azoospermia. The clinical features, however, are variable, and occasionally severe oligozoospermia may be present. Usually in these cases a 46,XY/47,XXY mosaic karyotype is involved. However, focal spermatogenesis and severe oligozoospermia have been reported in 47,XXY individuals too. In the present study we investigated whether testicular spermatozoa can be recovered in 47,XXY patients with a view to intracytoplasmic sperm injection (ICSI). In four out of nine apparently non-mosaic 47,XXY patients, spermatozoa were recovered from the wet preparations of testicular tissue and ICSI was performed in three couples. In one patient in whom spermatozoa were successfully recovered and used for ICSI, no spermatozoa were retrieved at a second trial. Although these results show that in some 47,XXY individuals testicular spermatozoa can be successfully recovered and even used for ICSI, at present this approach should be considered experimental. There may indeed be some concern about the chromosomal normality of the embryos generated through this infertility treatment. Patients with Klinefelter's syndrome should therefore be counselled about the complexity of this treatment, which involves multiple testicular biopsies from hypogonadal testes, ICSI and preimplantation diagnosis by fluorescence-in-situ hybridization.

Most of the mutations causing deficiency of the pyruvate dehydrogenase (PDH) complex are in the X-linked E ^ gene. We have developed a rapid screening method for the detection of mutations in this gene using reverse transcription of total RNA, polymerase chain reaction amplification of the whole coding region of the gene and single-strand conformation polymorphism (SSCP) analysis. With this method, we studied eight patients with a PDH complex deficiency, using cultured fibroblasts. In all patients, aberrant SSCP patterns were found and, after sequencing of the corresponding fragments, we were able to identify six new mutations and two mutations already described previously. The mutations are point mutations leading to amino acid substitutions (5) and direct repeat insertions (3). The presence of the mutations was confirmed in genomic fibroblast DNA* The 4 female patients were shown to carry both a normal and a mutated Exa gene. © 1996 Wiley^Liss, Inc, k e y w o r d s : Pyruvate dehydrogenase Eta, RT-PCR, SSCP, Mutation analysis

The safety of intracytoplasmic sperm injection (ICSI) as a novel procedure of assisted fertilization may be assessed by the health of the children born. In a prospective follow-up study of children born after assisted procreation, 130 children born consecutively after ICSI were compared with 130 control children born after in-vitro fertilization (IVF). In both groups, mothers were matched for age and had the same standard treatment protocol. There were 74 singleton, 50 twin and six triplet children in each group. Prenatal karyotyping and ultrasound screening, physical examination at birth and developmental milestones, with a follow-up at 2 months and 1 year, were recorded. Prenatal karyotypes were obtained in 100 of the 130 children in the ICSI group compared with 22 of the 130 children in the matched IVF group. All karyotypes were normal except for one prenatally detected mosaicism, which was not confirmed at birth. Four major malformations were detected in the ICSI group (holoprosecencephaly, femur fibula ulna syndrome and palatoschisis in two children), compared with six in the matched IVF group (coarctation of the aorta, palatoschisis, hypospadias, unilateral cryptorchidism, soft tissue syndactily and 11-beta-hydroxylase deficiency). In the ICSI and IVF groups, mean +/- SD birth weights were 2.94 +/- 0.67 and 2.80 +/- 0.73 kg, lengths were 48.46 +/- 3.56) and 47.47 +/- 5.78 cm, and head circumferences were 33.79 +/- 2.20 and 31.19 +/- 8.88 cm respectively. Among the ICSI singletons, the mean +/- SD birth weight was 3.28 +/- 0.58 kg and among the twins it was 2.60 +/- 0.43 kg; for the IVF singletons and matched twins the mean +/- SD birth weights were 3.19 +/- 0.56 and 2.36 +/- 0.61 kg respectively. In conclusion, there was no difference in the paediatric follow-up of 130 children born after ICSI and 130 children born after conventional IVF in age-matched control patients.

Resume La possibilite de proceder a un depistage prenatal de nombreuses maladies a constitue l'un des progres importants des trente dernieres annees. Avec la mise au point de la Fivete (fecondation in vitro et transfert d'embryons) et des methodes d'analyse de l'ADN par PCR ( polymerase chain reaction ) et par hybridation in situ par fluorescence (FISH), la possibilite d'un depistage encore plus precoce, le diagnostic preimplantatoire, prend forme.

Tay-Sachs disease is a lysosomal storage disease, which in its most severe form leads inexorably to death during infancy. We have developed a method for preimplantation diagnosis, using polymerase chain reaction (PCR) technology, by which the two most frequent mutations occurring in this disease can be amplified simultaneously. We have tested this method on single blastomeres and have compared four lysis methods: (i) boiling in water at 94 degrees C for 15 or (ii) 30 min, and (iii) incubation in an alkaline lysis buffer for 30 min at 94 degrees C or (iv) at 65 degrees C for 10 min. The amplification percentages were 21, 67, 71 and 91% respectively.

This study provides an overview of 13 years of experience of preimplantation genetic diagnosis (PGD) for Huntington's disease (HD) at three European PGD centres in Brussels, Maastricht and Strasbourg. Information on all 331 PGD intakes for HD, couples' reproductive history, PGD approach, treatment cycles and outcomes between 1995 and 2008 were collected prospectively. Of 331 couples for intake, 68% requested direct testing and 32% exclusion testing (with a preponderance of French couples). At the time of PGD intake, 39% of women had experienced one or more pregnancies. A history of pregnancy termination after prenatal diagnosis was observed more frequently in the direct testing group (25%) than in the exclusion group (10%; P=0.0027). PGD workup was based on two approaches: (1) direct testing of the CAG-triplet repeat and (2) linkage analysis using intragenic or flanking microsatellite markers of the HTT gene. In total, 257 couples had started workup and 174 couples (70% direct testing, 30% exclusion testing) completed at least one PGD cycle. In total, 389 cycles continued to oocyte retrieval (OR). The delivery rates per OR were 19.8%, and per embryo transfer 24.8%, resulting in 77 deliveries and the birth of 90 children. We conclude that PGD is a valuable and safe reproductive option for HD carriers and couples at risk of transmitting HD.

Study question Are human trophectoderm (TE) cells committed or still able to develop into inner cell mass (ICM) cells? Summary answer Human full blastocyst TE cells still have the capacity to develop into ICM cells expressing the pluripotency marker NANOG, thus they are not yet committed. What is known already Human Day 5 full blastocyst TE cells express the pluripotency markers POU5F1, SOX2 and SALL4 as well as the TE markers HLA-G and KRT18 but not yet CDX2, therefore their developmental direction may not yet be definite. Study design, size, duration The potency of human blastocyst TE cells was investigated by determining their in vitro capacity to develop into a blastocyst with ICM cells expressing NANOG; TE cells were isolated either by aspiration under visual control or after labeling with fluorescent 594-wheat germ agglutinin. Further on, aspirated TE cells were also labeled with fluorescent PKH67 and repositioned in the center of the original embryo. Participants/materials, setting, methods Human preimplantation embryos were used for research after obtaining informed consent from IVF patients. The experiments were approved by the Local Ethical Committee and the 'Belgian Federal Committee on medical and scientific research on embryos in vitro'. Outer cells were isolated and reaggregated by micromanipulation. Reconstituted embryos were analyzed by immunocytochemistry. Main results and the role of chance Isolated and reaggregated TE cells from full human blastocysts are able to develop into blastocysts with ICM cells expressing the pluripotency marker NANOG. Moreover, the majority of the isolated TE cells which were repositioned in the center of the embryo do not sort back to their original position but integrate within the ICM and start to express NANOG. Limitations, reasons for caution Owing to legal and ethical restrictions, manipulated human embryos cannot be transferred into the uterus to determine their totipotent capacity. The definitive demonstration that embryos reconstructed with TE cells are a source of pluripotent cells is to obtain human embryonic stem cell 'like' line(s), which will allow full characterization of the cells. Wider implications of the findings Our finding has important implications in reproductive medicine and stem cell biology because TE cells have a greater developmental potential than assumed previously. Study funding/competing interest(s) Scientific Research Foundation-Flanders (FWO-Vlaanderen) and Research Council (OZR) of the Vrije Universiteit Brussel. None of the authors declared a conflict of interest.

We describe a polymerase chain reaction (PCR) assay for the simultaneous detection of X and Y chromosomes in human blastomeres. In a first round of PCR, both X- and Y-specific fragments are amplified with primers which are common to both chromosomes and are derived from the X-linked steroid sulphatase gene and the Y-linked pseudogene. In a second round of PCR, fragments specific to each chromosome are generated: both an X and a Y fragment in male embryos and, due to the absence of a Y chromosome, only an X fragment in females. The efficiency and accuracy of this assay are high; it generates no false positive amplification signals and allows sexing in approximately 6 h after embryo biopsy. We therefore believe that it is suitable for gender determination by preimplantation diagnosis for couples at risk for X-linked genetic diseases.

background: The aim of this study was to analyse the outcome of closed blastocyst vitrification of embryos biopsied at the cleavage stage. methods: Vitrification of supernumerary blastocysts was performed using the closed CBS-VIT High Security straws. Warming cycles (n ¼ 100) for patients with preimplantation genetic diagnosis (PGD) and/or aneuploidy screening in the fresh cycle were analysed. The outcome parameters were morphological survival and transfer rates after warming, clinical pregnancy rate and implantation rate (with fetal heart beat). Clinical outcome was compared with two control groups of (i) vitrified/warming transfer cycles without embryo biopsy and (ii) fresh Day 5 transfer of biopsied embryos. results: In total, 131 blastocysts were warmed with a morphological survival of 83.2% (109/131) and a transfer rate of 79.4% (104/ 131). Day 5 blastocysts survived significantly better (90.4%) than Day 6 blastocysts (70.8%, P , 0.01). No difference in survival rate was observed between early cavitating (89.2%) and full/expanded blastocysts (93.3%). In nine cycles, no blastocyst was available for transfer. The clinical pregnancy rate was 19.2% (15/78) after single-embryo transfer (SET) and 38.5% (5/13) after double-embryo transfer (DET). In SET, the implantation rate for blastocysts frozen on Day 5 was 13.7% (7/51), which was not different from the implantation rate of Day 6 blastocysts (18.5%, 5/27). The overall implantation rate of vitrified PGD biopsied blastocysts (14.4%) was comparable with that of vitrified blastocysts without biopsy (20.4%), but lower than the implantation rate obtained in the fresh PGD cycles (24.4%). conclusion: Blastocysts on Day 5 and Day 6 of development derived from biopsied embryos can be successfully vitrified using a closed

The efficiency of the polymerase chain reaction (PCR) in detecting the cystic fibrosis (CF) delta F508 mutation (which is the most common mutation of CF) was assessed in single human blastomeres. Twenty-one human immature oocytes (germinal-vesicle-stage oocytes) that had been donated for research were matured in vitro and a single spermatozoon from a carrier of the CF delta F508 mutation was injected into the ooplasm. Fourteen embryos were obtained after intracytoplasmic sperm injection (ICSI). PCR analysis was carried out on 70 single blastomeres isolated from these 14 embryos. The results showed that the efficiency of DNA amplification by PCR in single nucleate blastomeres was 94 per cent (59/63). There were no false-positive results since none of the blank samples or the blastomeres without a nucleus showed an amplified signal. We found that nine embryos were homozygous for the unaffected genotype and that four embryos were heterozygous since they contained both the unaffected and the delta F508 genotype. In a four-cell embryo, we observed the homozygous unaffected genotype in one blastomere and a heterozygous delta F508/unaffected genotype in the other three blastomeres.