http://repub.eur.nl/res/aut/15246/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/15066/ 2009-01-01T00:00:00Z In mouse zygotes, many post-translational histone modifications are asymmetrically present in male and female pronuclei. We investigated whether this principle could be used to determine the genetic composition of monopronuclear human zygotes in conventional IVF and ICSI. First we determined whether male female asymmetry is conserved from mouse to human by staining polypronuclear zygotes with antibodies against a subset of histone N-tail post-translational modifications. To analyze human monopronuclear zygotes, a modification, H3K9me3, was selected that is present in the maternal chromatin. After IVF a total of 45 monopronuclear zygotes were obtained. In 39 (87%) of zygotes a nonuniform staining pattern was observed, proof of a bi-parental origin and assumed to result into a diploid conception. Two zygotes showed no staining for the modification, indicating that the single pronucleus was of paternal origin. Four zygotes contained only maternally derived chromatin. ICSI-derived monopronuclear zygotes (n=33) could also be divided into three groups based on the staining pattern of their chromatin: (1) of maternal origin (n=15), (2) of paternal origin (n=8) or (3) consisting of two chromatin domains as dominating in IVF (n=10). Our data show that monopronuclear zygotes originating from IVF generally arise through fusion of parental chromatin after sperm penetration. Monopronuclear zygotes derived from ICSI in most cases contain uni-parental chromatin. The fact that chromatin was of paternal origin in 24% of ICSI and in 4% of the IVF zygotes confirms earlier results obtained by FISH on cleavage stages. Our findings are of clinical importance in IVF and ICSI practice. http://repub.eur.nl/res/pub/14541/ 2008-11-01T00:00:00Z BACKGROUND: Ovarian stimulation gives rise to supraphysiological estradiol levels, which may affect oocyte quality. This study aims to investigate whether ovarian stimulation deranges the homocysteine pathway thereby affecting the pre-ovulatory follicle. METHODS: Blood samples were collected on cycle day 2 and the day of hCG administration in 181 women undergoing ovarian stimulation for IVF. In each subject, the diameter of the two leading follicles was measured and the corresponding follicular fluids were collected. In blood and follicular fluid samples, total homocysteine (tHcy), folate, cobalamin and pyridoxal'5-phosphate (PLP) were determined. According to the blood folate levels, women were classified as either folic acid supplemented (n = 113) or non-supplemented (n = 32). RESULTS: Ovarian stimulation resulted in a significant decrease in blood tHcy and cobalamin levels (both P ≤ 0.001). The blood concentrations of tHcy, folate, cobalamin and PLP were significantly correlated with the corresponding follicular fluid concentrations (all P ≤ 0.001). Follicular fluid tHcy concentrations were inversely correlated with follicular diameter (P ≤ 0.05). In folic acid supplemented women, follicular fluid folate was inversely correlated with follicular diameter (P ≤ 0.05). CONCLUSIONS: Ovarian stimulation deranges blood and follicular fluid biomarkers of the homocysteine pathway. High ovarian follicular fluid tHcy and folate levels may have detrimental effects on follicular development. http://repub.eur.nl/res/pub/28974/ 2008-06-01T00:00:00Z Objective: To investigate the influence of folic acid supplementation on the follicular fluid concentrations of folate and total homocysteine and their relationship to the diameter of the follicle. Design: Observational study. Setting: Tertiary referral fertility clinic at the Erasmus MC, University Medical Center, Rotterdam, The Netherlands. Patient(s): Thirty-seven women undergoing IVF or intracytoplasmic sperm injection treatment. Intervention(s): No interventions other than routine stimulation treatment and the recommendation of folic acid supplementation. Main Outcome Measure(s): Concentrations of folate and total homocysteine in monofollicular and pooled follicular fluid and the diameter of the follicle. Result(s): Folic acid supplementation significantly increased folate and decreased total homocysteine concentrations in pooled follicular fluid. In monofollicular fluid, folate concentrations only were significantly increased in supplemented women. The total homocysteine concentration appeared to be significantly correlated with the diameter of the follicle (r = 0.27). Samples from single follicles were less prone to artifacts in the measurements of the folate and total homocysteine concentration. Conclusion(s): Preconception folic acid supplementation significantly alters both folate and total homocysteine concentrations in follicular fluid. The correlation between the diameter of the follicle and total homocysteine concentration in follicular fluid warrants further investigation. http://repub.eur.nl/res/pub/30335/ 2008-05-15T00:00:00Z Background. about 15% to 30% of the DNA in human sperm is packed in nucleosomes and transmission of this fraction to the embryo potentially serves as a mechanism to facilitate paternal epigenetic programs during embryonic development. However, hitherto it has not been established whether these nucleosomes are removed like the protamines or indeed contribute to paternal zygotic chromatin, thereby potentially contributing to the epigenome of the embryo. Results. to clarify the fate of sperm-derived nucleosomes we have used the deposition characteristics of histone H3 variants from which follows that H3 replication variants present in zygotic paternal chromatin prior to S-phase originate from sperm. We have performed heterologous ICSI by injecting human sperm into mouse oocytes. Probing these zygotes with an antibody highly specific for the H3.1/H3.2 replication variants showed a clear signal in the decondensed human sperm chromatin prior to S-phase. In addition, staining of human multipronuclear zygotes also showed the H3.1/H3.2 replication variants in paternal chromatin prior to DNA replication. Conclusion. these findings reveal that sperm-derived nucleosomal chromatin contributes to paternal zygotic chromatin, potentially serving as a template for replication, when epigenetic information can be copied. Hence, the execution of epigenetic programs originating from transmitted paternal chromatin during subsequent embryonic development is a logical consequence of this observation. http://repub.eur.nl/res/pub/35955/ 2007-04-01T00:00:00Z BACKGROUND: To test whether ovarian stimulation for in-vitro fertilization (IVF) affects oocyte quality and thus chromosome segregation behaviour during meiosis and early embryo development, preimplantation genetic screening of embryos was employed in a prospective, randomized controlled trial, comparing two ovarian stimulation regimens. METHODS: Infertile patients under 38 years of age were randomly assigned to undergo a mild stimulation regimen using gonadotrophin-releasing hormone (GnRH) antagonist co-treatment (67 patients), which does not disrupt secondary follicle recruitment, or a conventional high-dose exogenous gonadotrophin regimen and GnRH agonist co-treatment (44 patients). Following IVF, embryos were biopsied at the eight-cell stage and the copy number of 10 chromosomes was analysed in 1 or 2 blastomeres. RESULTS: The study was terminated prematurely, after an unplanned interim analysis (which included 61% of the planned number of patients) found a lower embryo aneuploidy rate following mild stimulation. Compared with conventional stimulation, significantly fewer oocytes and embryos were obtained following mild stimulation (P < 0.01 and < 0.05, respectively). Consequently, both regimens generated on average a similar number (1.8) of chromosomally normal embryos. Differences in rates of mosaic embryos suggest an effect of ovarian stimulation on mitotic segregation errors. CONCLUSIONS: Future ovarian stimulation strategies should avoid maximizing oocyte yield, but aim at generating a sufficient number of chromosomally normal embryos by reduced interference with ovarian physiology. http://repub.eur.nl/res/pub/35867/ 2007-01-01T00:00:00Z Objective: Screening for an increased number of chromosomes may improve the detection of abnormal embryos and thus contribute to the capability of preimplantation genetic screening (PGS) to detect the embryo(s) for transfer in IVF with the best chance for a healthy child. Good-quality day 4 and 5 embryos were analyzed after cryopreservation for the nine chromosomes mostly recommended for screening (13, 14, 15, 16, 18, 21, 22, X and Y), next to six additional chromosomes which are less well studied in this context (1, 2, 7, 6, 10 and 17). Method: The copy numbers of 15 chromosomes were investigated by fluorescence in situ hybridization (FISH) in three consecutive rounds. The proportion of aneuploid and mosaic embryos was determined and compared in retrospect to results in case only the recommended probe set had been analyzed. Results: A total of 52 embryos from 29 infertile women were analyzed. Screening the embryos for six additional chromosomes increased the proportion of abnormal embryos from 67 to 81% (P = 0.03), owing to an increase in mosaic embryos. Conclusion: All but one of the meiotic aneuploidies found in this study would have been detected by the probe set most frequently used in PGS clinics. However, aneuploid cell lines originating from mitotic errors could be detected for almost all chromosomes, so screening of six additional chromosomes mainly increased the proportion of mosaic embryos. The added value of screening for six additional chromosomes in PGS for clinical practice will remain undetermined as long as the fate of mosaic embryos after transfer is unclear. Copyright http://repub.eur.nl/res/pub/13336/ 2004-03-01T00:00:00Z BACKGROUND: Chromosomal mosaicism in human embryos may give rise to false positive or false negative results in preimplantation genetic diagnosis for aneuploidy screening (PGD-AS). Therefore, we have investigated whether the results obtained from a 2-cell biopsy of frozen-thawed embryos and fluorescence in situ hybridization (FISH) analysis are representative for the chromosome constitution of the remaining embryo on day 5. METHODS: Cryopreserved day 3 embryos were thawed and from surviving embryos two blastomeres were biopsied. FISH analysis was performed for chromosomes 1, 7, 13, 15, 16, 18, 21, 22, X and Y. After biopsy, the embryos were cultured until day 5 and further analysed using the same probe panels. RESULTS: In all, 17 embryos were available with a diagnosis based on two blastomeres on day 3 and confirmatory studies on day 5. In 10 of these 17 cases the initial diagnosis could be confirmed. However, in only six cases cytogenetic results were concordant. Besides the 10 cases with a 'correct' diagnosis, there were six false positive results and one false negative, all involving mosaicism. CONCLUSIONS: Investigating the chromosomal constitution of two blastomere nuclei offers a good opportunity to study the incidence of chromosomal mosaicism in early embryo development. The confirmation rate of the results obtained on day 3 depends on the interpretation and is higher when considered from a clinical than from a cytogenetic point of view.