Preimplantation genetic testing with HLA matching: from counseling to birth and beyond.

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.

Factors influencing the clinical outcome of preimplantation genetic testing for polycystic kidney disease.

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.

Expanding the clinical spectrum of biallelic ZNF335 variants.

ZNF335 plays an essential role in neurogenesis and biallelic variants in ZNF335 have been identified as the cause of severe primary autosomal recessive microcephaly in 2 unrelated families. We describe, herein, 2 additional affected individuals with biallelic ZNF335 variants, 1 individual with a homozygous c.1399 T > C, p.(Cys467Arg) variant, and a second individual with compound heterozygous c.2171_2173delTCT, p.(Phe724del) and c.3998A > G, p.(Glu1333Gly) variants with the latter variant predicted to affect splicing. Whereas the first case presented with early death and a severe phenotype characterized by anterior agyria with prominent extra-axial spaces, absent basal ganglia, and hypoplasia of the brainstem and cerebellum, the second case had a milder clinical presentation with hypomyelination and otherwise preserved brain structures on MRI. Our findings expand the clinical spectrum of ZNF335-associated microcephaly.

Recent developments in genetics and medically assisted reproduction: from research to clinical applications.

Two leading European professional societies, the European Society of Human Genetics and the European Society for Human Reproduction and Embryology, have worked together since 2004 to evaluate the impact of fast research advances at the interface of assisted reproduction and genetics, including their application into clinical practice. In September 2016, the expert panel met for the third time. The topics discussed highlighted important issues covering the impacts of expanded carrier screening, direct-to-consumer genetic testing, voiding of the presumed anonymity of gamete donors by advanced genetic testing, advances in the research of genetic causes underlying male and female infertility, utilisation of massively parallel sequencing in preimplantation genetic testing and non-invasive prenatal screening, mitochondrial replacement in human oocytes, and additionally, issues related to cross-generational epigenetic inheritance following IVF and germline genome editing. The resulting paper represents a consensus of both professional societies involved.

Body composition and blood pressure in 6-year-old singletons born after pre-implantation genetic testing for monogenic and structural chromosomal aberrations: a matched cohort study.

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 INTERESTS: 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.

Recent developments in genetics and medically-assisted reproduction: from research to clinical applications†‡.

Two leading European professional societies, the European Society of Human Genetics and the European Society for Human Reproduction and Embryology, have worked together since 2004 to evaluate the impact of fast research advances at the interface of assisted reproduction and genetics, including their application into clinical practice. In September 2016, the expert panel met for the third time. The topics discussed highlighted important issues covering the impacts of expanded carrier screening, direct-to-consumer genetic testing, voiding of the presumed anonymity of gamete donors by advanced genetic testing, advances in the research of genetic causes underlying male and female infertility, utilisation of massively-parallel sequencing in preimplantation genetic testing and non-invasive prenatal screening, mitochondrial replacement in human oocytes, and additionally, issues related to cross-generational epigenetic inheritance following IVF and germline genome editing. The resulting paper represents a consensus of both professional societies involved.

Depression, pregnancy-related anxiety and parental-antenatal attachment in couples using preimplantation genetic diagnosis.

STUDY QUESTION: Do preimplantation genetic diagnosis (PGD) couples experience higher levels of stress during pregnancy and the perinatal period compared with couples who conceive spontaneously (SC) or with ICSI? SUMMARY ANSWER: PGD couples did not experience more psychological stress during pregnancy and beyond than ICSI or SC couples. WHAT IS ALREADY KNOWN: Previous studies have shown that assisted reproduction technology (ART) couples are more prone to pregnancy-related anxieties than SC couples, but display depressed feelings to an equal or lesser extent. However, only one study has focused on a female PGD sample, which may be a more vulnerable group than other ART groups, due to the potentially complex hereditary background, adverse childhood experiences and losses. In that study, PGD women experienced a reduction in state anxiety, and maternal-antenatal attachment did not differ from normative data. Unfortunately, no data exist on pregnancy-related anxiety, depression and parental-antenatal attachment. Valuable information from both parents (e.g.: couples) is also lacking. STUDY DESIGN, SIZE, DURATION: For this longitudinal prospective study questionnaire, data from 185 women and 157 men (157 couples) were collected between February 2012 until April 2014. Data were analysed using multilevel analysis. The couples conceiving after PGD, ICSI or SC were followed from the first trimester of the pregnancy until the third month post-partum. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 60 PGD, 58 ICSI and 69 SC couples were initially recruited by various departments of Universitair Ziekenhuis Brussel (UZ Brussel). At each trimester (T1: 12-14 weeks, T2: 20-22 weeks, T3: 30-32 weeks) of pregnancy, depression (EPDS), pregnancy-related anxieties (PRAQ) and parental-antenatal attachment (M/PAAS) were recorded. At T4 (3 months post-partum), depression (EPDS) was assessed again. In the first trimester (T1) broad socio-demographic data and at T4 perinatal health data of both mother and child were recorded. Differences between conception groups over time were analysed using multilevel analyses, taking into account covariation between measurements and within couples. Several perinatal covariates as well as social desirability, coping and adult attachment style were controlled for. MAIN RESULTS AND THE ROLE OF CHANCE: All three conception groups had similar scores for depression during pregnancy and beyond. Also, pregnancy-related anxiety scales did not differ among the three groups. All groups also followed a similar trajectory in time regarding their scores for anxiety, depression and parental-antenatal attachment. ART groups did not give more socially desirable answers than SC controls. The subsequent moderators: coping and adult attachment style did not add any relevant information. No interaction effects occurred between gender and conception groups. LIMITATIONS, REASONS FOR CAUTION: The participants were Caucasian, Dutch-speaking couples, with medium to high socio-economic status, from a single centre. Our data should be replicated by multicultural and multicentre studies. Furthermore, the inclusion of an additional control group of couples who did not opt for PGD but for prenatal diagnosis may point to the most beneficial strategy for the couple. WIDER IMPLICATIONS OF THE FINDINGS: PGD parents invest a similar amount of time and emotion in their future children compared with controls. This implies that successful PGD treatment makes an important psychological contribution towards the well-being of couples given their complex hereditary and family backgrounds. STUDY FUNDING/COMPETING INTERESTS: This research project was funded by grants from the internal research council of the Vrije Universiteit Brussel (OZR), the Flemish Fonds Wetenschappelijk Onderzoek (FWO) and the Wetenschappelijk Fonds Willy Gepts (WGFG). UZ Brussel and the Centre for Medical Genetics have received several educational grants for organizing the data collection, from IBSA, Ferring, Organon, Shering-Plough, Merck and Merck Belgium. M.B. has received consultancy and speaker's fees from Organon, Serono Symposia and Merck.

Reproductive options for prospective parents in families with Huntington's disease: clinical, psychological and ethical reflections.

BACKGROUND Huntington'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. METHODS Selected relevant literature and own views and experiences as clinical geneticists, psychologists and ethicists have been used. RESULTS Possible 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. CONCLUSION Recommendations and suggestions for good clinical practice in the reproductive care for HD families are formulated.

Neonatal follow-up of 995 consecutively born children after embryo biopsy for PGD.

BACKGROUND: Outcome data on children born after assisted reproduction treatments are important for both patients and health-care providers. The objective of this study was to determine whether embryo biopsy as performed in PGD has an impact on the health of infants up to 2 months of age. METHODS: A prospective comparative follow-up study of children born after PGD and children born after ICSI by collecting written reports and performing a physical examination at 2 months was performed. Auxological data at birth and physical findings up to 2 months of age were compared for 995 children consecutively live born after embryo biopsy (1994-2009) and for a control group of 1507 children born after ICSI with embryo transfer on Day 5. RESULTS: No differences regarding mean term, prematurity (term <32 w and <37 w), mean birthweight, very low birthweight (<1500 g), perinatal death, major malformations and neonatal hospitalizations in singletons and multiples born following PGD versus ICSI were observed. Compared with ICSI, fewer multiples born following PGD presented a low birthweight (<2500 g) (P = 0.005). CONCLUSIONS: Embryo biopsy for PGD does not introduce extra risk to the overall medical condition of newborn children. Multiples born following embryo biopsy appear to be at lower risk for low birthweight compared with multiples born following ICSI.

Neonatal outcome of 724 children born after ICSI using non-ejaculated sperm.

BACKGROUND: Safety concerns have been expressed regarding the use of immature non-ejaculated spermatozoa for ICSI. Therefore, adverse health outcomes, birth parameters, major anomaly rates and chromosomal aberrations in children born after ICSI using testicular and epididymal sperm were investigated. METHODS: Questionnaire data and results of physical examinations of 530 children born after ICSI with testicular sperm and of 194 children born after ICSI with epididymal sperm were compared with data on 2516 ICSI children born using ejaculated sperm. RESULTS: Birth parameters, stillborn rates, prematurity rates and rates of low birthweight and very low birthweight were comparable between the non-ejaculated and the ejaculated sperm groups. The perinatal death rate was higher for twins but not for singletons in the non-ejaculated sperm group in comparison to the control cohort of children born using ejaculated sperm. A non-significant increase in major anomalies was reported in the non-ejaculated sperm group in comparison to the ejaculated sperm group. No more anomalies were observed in pre- and post-natal karyotypes from viable pregnancies established using non-ejaculated sperm versus ejaculated sperm. CONCLUSION: Overall neonatal health in terms of birth parameters, major anomalies and chromosomal aberrations in our large cohort of children born by the use of non-ejaculated sperm seems reassuring in comparison to the outcome of children born after the use of ejaculated sperm.

Closed blastocyst vitrification of biopsied embryos: evaluation of 100 consecutive warming cycles.

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 = 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 system.

Mutation analysis of three genes in patients with maturation arrest of spermatogenesis and couples with recurrent miscarriages.

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.

Methylation of the CpG sites in the myotonic dystrophy locus does not correlate with CTG expansion size or with the congenital form of the disease.

We have studied the methylation status of the sequence 152 nucleotides upstream of the CTG repeat of the DM1 locus in patients' peripheral blood. We used the methylation-sensitive endonucleases SacII, HpaII and HhaI, followed by PCR. This allowed to correlate the methylation status of each CTG allele with its size. Contrary to previous findings, only the SacII site is often but not always differentially methylated among expanded CTG alleles. Importantly, this methylation was not restricted to congenital DM1, nor to large expansions, as it was also present in DM1 patients with a classical phenotype and various expansion sizes. On the other hand, we did not find any methylated alleles on the HhaI and HpaII sites, as was reported by Steinbach et al, which is in line with the results of Shaw and collaborators. The size range of the repeat expansions with methylation was from as small as 300 to as large as 2800 repeats.

Report on a consecutive series of 581 children born after blastomere biopsy for preimplantation genetic diagnosis.

BACKGROUND: Preimplantation genetic diagnosis (PGD) and subsequently preimplantation genetic screening (PGS) have been introduced since 1990. The difference from the already existing in vitro fertilization (IVF) technology, using intracytoplasmic sperm injection (ICSI), was the embryo biopsy at day 3 after fertilization. Although healthy children post-PGD/PGS have been born, the question of whether embryo biopsy could have any harmful effects has to be studied on large series in a prospective manner. METHODS: A prospective cohort study was undertaken from 1992 until 2005, using the same approach as for the follow-up of IVF and ICSI children conceived in the same centre. Questionnaires were sent to physicians and parents at conception and at delivery. Children were examined at 2 months of age by trained clinical geneticists whenever possible. RESULTS: Data collected on 581 post-PGD/PGS children showed that term, birthweight and major malformation rates were not statistically different from that of 2889 ICSI children, with overall rates of major malformation among these post-PGD/PGS and ICSI children being 2.13 and 3.38%, respectively (odds ratio [OR]: 0.62; exact 95% confidence limits [95% CL]: 0.31-1.15). However, the overall perinatal death rate was significantly higher among post-PGD/PGS children compared with ICSI children (4.64 versus 1.87%; OR: 2.56; 95% CL: 1.54-4.18). When stratified for multiple births, perinatal death rates among PGD/PGS singleton and ICSI singleton children were similar (1.03 versus 1.30%; OR: 0.83; 95% CL: 0.28-2.44), but significantly more perinatal deaths were seen in post-PGD/PGS multiple pregnancies compared with ICSI multiple pregnancies (11.73 versus 2.54%; OR: 5.09; 95% CL: 2.80-9.90). The overall misdiagnosis rate was below 1%. CONCLUSIONS: Embryo biopsy does not add risk factors to the health of singleton children born after PGD or PGS. The perinatal death rate in multiple pregnancies is such that both caution and long-term follow-up are required.

Impact of cleavage-stage embryo biopsy in view of PGD on human blastocyst implantation: a prospective cohort of single embryo transfers.

BACKGROUND: Human embryo biopsy is performed for preimplantation genetic diagnosis (PGD). The impact of 1- or 2-cell removal at cleavage-stage on future embryo development and implantation capacity is highly debated. METHODS: In order to explore this issue further, a cohort of Day 5 single embryo transfers was analysed prospectively for embryological and clinical outcome. All transferred embryos resulted from 8-cell embryos on Day 3, from which subsequently either one cell (group I, n = 182) or two cells (group II, n = 259) were removed, or on which no invasion by means of embryo biopsy was performed (group III, control group, n = 702). RESULTS Blastocyst formation was significantly better in group III compared with group II, and similar to group I. Group I and group II did not differ in Day 3 nor in Day 5 embryo development. The overall live birth rate was significantly higher in group I (37.4%, CI 29.0-47.4%) than in group II (22.4%, CI 17.0-28.9%), and comparable to the reference ICSI population (35.0%, CI 30.8-39.7%). CONCLUSIONS: The clinical outcome of 1-cell biopsy was significantly better than that of 2-cell biopsy, even when adjusted for availability of genetically transferable embryos.

Cumulative reproductive outcome after preimplantation genetic diagnosis: a report on 1498 couples.

BACKGROUND: Couples undergoing preimplantation genetic diagnosis (PGD) have a different background and set of treatment characteristics to couples undergoing regular IVF or ICSI. The aim of this study was to analyse the cumulative reproductive outcome of a large cohort of couples undergoing PGD in relation to a number of explanatory variables potentially affecting the prognosis. METHODS: Prospective cohort study, Kaplan-Meier analysis was performed to estimate real (observed) and expected (calculated) cumulative delivery rates, and Cox proportional hazard regression analysis was used to assess the effect of age, number of cumulus oocyte complexes collected, fertility status, parity, genetic category and method of pituitary suppression. RESULTS: Between 1993 and 2005, 2753 unselected consecutive cycles of ICSI and PGD were carried out in 1498 couples. The cumulative observed delivery rate overall per couple with a maximum of six treatment cycles of ICSI and PGD performed was 29%. The expected cumulative delivery rate (max six cycles) overall was 62%. There were no significant differences in cumulative delivery rates between the different genetic categories (i.e. availability of transferable embryos after PGD of 50 or 75%, chromosomal translocations or aneuploidy screening). The cumulative reproductive outcome in this PGD cohort was also not significantly affected by the fertility status of the couple, their parity or the method of pituitary suppression. However, the age of the patient and the number of oocytes contributed significantly to the reproductive results. CONCLUSION: This prospective observational study demonstrates that age has a significantly negative effect on outcome of PGD, due to poor reproductive performance of female partners 40 years of age and older. The number of oocytes collected has a significant and independent effect. The other factors studied did not affect the cumulative reproductive outcome in this PGD cohort.

The incidence of monozygotic twinning following PGD is not increased.

BACKGROUND: Monozygotic (MZ) twin pregnancies are associated with increased perinatal mortality and morbidity, and risk of congenital anomalies. The causes of MZ twinning in humans are unclear but the incidence may increase after PGD, for example, as a result of holes created in the zona pellucida. We compared the incidence of MZ twin pregnancies in ICSI cycles with PGD, versus ICSI cycles without PGD. METHODS: In this retrospective comparative cohort study, we analysed incidence of twin pregnancies in unselected patients undergoing ICSI and PGD (group A; 1992 cycles) with blastocyst transfer at Day 5, versus a period-matched control population of unselected patients undergoing ICSI and blastocyst transfer at Day 5 without PGD (group B; 2429 cycles) from January 2001 to December 2006. RESULTS: Clinical pregnancy per embryo transfer was established in 618/1992 (31.0%) and 947/2429 (39.0%) in group A versus B, respectively (P < 0.01). Overall MZ twin rate was 29/4421 (0.7%) per embryo transfer and 29/1565 (1.9%) per established clinical pregnancy. The incidence of MZ twinning per established clinical pregnancy did not differ between groups (1.5 versus 2.1%, group A and B, respectively). In group A, seven MZ twins were born versus 19 MZ twins in group B. In group B, one MZ twin pregnancy resulted in two stillbirths. In group A, two MZ twins had severe congenital malformations versus none in group B. CONCLUSIONS: The incidence of MZ twinning was not increased in PGD compared with regular ICSI with blastocyst transfer. This information is useful in counselling patients about potential risks of PGD.

Characterization of CD30 expression in human embryonic stem cell lines cultured in serum-free media and passaged mechanically.

BACKGROUND: The presence of chromosomal abnormalities could have a negative impact for human embryonic stem cell (hESC) applications both in regenerative medicine and in research. A biomarker that allows the identification of chromosomal abnormalities induced in hESC in culture before they take over the culture would represent an important tool for defining optimal culture conditions for hESC. Here we investigate the expression of CD30, reported to be a biomarker of hESCs with abnormal karyotype, in undifferentiated and spontaneously differentiated hESC. METHODS AND RESULTS: hESC were derived and cultured on mouse fibroblasts in KO-SR containing medium (serum free media) and passaged mechanically. Our results based on analysis at mRNA (RT-PCR) and protein (fluorescence-activated cell sorting and immunocytochemistry) level show that CD30 is expressed in undifferentiated hESC, even at very early passages, without any correlation with the presence of chromosomal anomalies. We also show that the expression of CD30 is rapidly lost during early spontaneous differentiation of hESC. CONCLUSION: We conclude that CD30 expression in hESC cultures is probably a consequence of culture conditions, and that KO-SR may play a role. In addition, the expression of so-called 'stemness' markers does not change in undifferentiated hESC during long-term culture or when cells acquire chromosomal abnormalities.

[Genetics and male infertility]. / Genetica van de mannelijke onvruchtbaarheid.

Infertility is a problem affecting many couples with a child wish. In about half of these couples a male factor is (co-) responsible for the fertility concern. For part of these patients a genetic factor will be the underlying cause of the problems. This paper gives an overview of the studies performed in the Department of Embryology and Genetics of the Vrije Universiteit Brussel and the Centre for Medical Genetics of UZ Brussel in order to gain more insight into the genetic causes of male infertility. The studies, focusing on men with fertility problems, can be subdivided into three groups: studies on deletions on the long arm of the Y chromosome, studies on X-linked genes and studies on autosomal genes. It is obvious that Yq microdeletions should be considered as a cause of male infertility. Only for patients with a complete AZFc deletion, a small number of spermatozoa can be retrieved. However, even for these patients assisted reproductive technologies are necessary. Complete AZF deletions are found in 4.6% of the patients visiting the centres for Reproductive Medicine and Medical Genetics of the UZ Brussel and for whom no other cause of the fertility problems have been detected. Taken into consideration this low prevalence of Yq microdeletions, it is obvious that also other factors, including genetic factors, must be causing fertility problems. Potentially, gr/gr deletions (partial deletions of the AZFc region) might influence the fertility status of the patients. It remains, however, unclear which of the genes located in the deleted regions are important for the progression of spermatogenesis, in case of partial or complete AZF deletions. In our studies we have also investigated mutations in genes located on the X chromosome. In analogy to the Y chromosome, the X chromosome is interesting in view of studying male infertility since men only have a single copy of the sex chromosomes. As a consequence, mutations in genes crucial for spermatogenesis will have an immediate impact on the sperm production. The genes NXF2, USP26 and TAF7L were investigated for the presence of mutations. All observed single nucleotide changes were also present in control samples, questioning their relationship with male infertility. We also studied five autosomal genes: SYCP3, MSH4, DNMT3L, STRA8 and ETV5. Only for the genes STRA8 and ETV5, changes were detected that were absent in a control population existing of men with normozoospermia. Functional analysis of the changes in ETV5 and the localization of the change observed in STRA8 showed that also these alterations were probably not the cause of the fertility problems in these men. It can be concluded that mutations are rarely detected in men with fertility problems. This low frequency of mutations has also been confirmed in several published studies. Therefore, further research is necessary to determine the impact of genetic causes on male infertility.

Markers that define stemness in ESC are unable to identify the totipotent cells in human preimplantation embryos.

BACKGROUND: During human preimplantation development, early blastomeres are believed to be totipotent. It is likely, however, that blastomeres are allocated to a specific lineage prior to any morphological differentiation. NANOG, SOX2 and SALL4 are transcription factors that play a key role in controlling stemness in embryonic stem cells (ESC) and are therefore candidate markers for developmental triggers in early embryos. KRT18, a trophoblast-determining gene, may mark early differentiation. Examining the expression pattern of these genes may inform us about when and in which cells totipotency is lost during early human development. METHODS: Thirtheen oocytes, 124 preimplantation embryos and 7 human embryonic stem cell (hESC) lines were examined for the presence of NANOG, SOX2, SALL4 or KRT18 proteins using immunostaining and confocal microscopy. RESULTS: All stemness markers were expressed in the hESC, but none of them was specific for totipotent cells during human preimplantation development, and none of them seemed to mark cells allocated to the inner cell mass (ICM) or trophectoderm. After lineage specification, only the nuclear expression of NANOG and SOX2 became restricted to the ICM, at least to some cells because only a subpopulation expressed NANOG. KRT18 expression was seen for the first time during compaction in some outer cells. KRT18 was not expressed in hESC. CONCLUSION: We conclude that the protein expression patterns of markers that define stemness in ESC do not identify the totipotent cells in human preimplantation embryos. Assessing the presence of KRT18 proteins implied that the outer cells of compacting embryos have probably lost their totipotent competence prior to any visible differentiation.