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IntroductionIn vitro fertilisation (IVF) has evolved as an intervention of choice to help couples with infertility to conceive. In the last decade, a strategy change in the day of embryo transfer has been developed. Many IVF centres choose nowadays to transfer at later stages of embryo development, for example, transferring embryos at blastocyst stage instead of cleavage stage. However, it still is not known which embryo transfer policy in IVF is more efficient in terms of cumulative live birth rate (cLBR), following a fresh and the subsequent frozen–thawed transfers after one oocyte retrieval. Furthermore, studies reporting on obstetric and neonatal outcomes from both transfer policies are limited.Methods and analysisWe have set up a multicentre randomised superiority trial in the Netherlands, named the Three or Fivetrial. We plan to include 1200 women with an indication for IVF with at least four embryos available on day 2 after the oocyte retrieval. Women are randomly allocated to either (1) control group: embryo transfer on day 3 and cryopreservation of supernumerary good-quality embryos on day 3 or 4, or (2) intervention group: embryo transfer on day 5 and cryopreservation of supernumerary good-quality embryos on day 5 or 6. The primary outcome is the cLBR per oocyte retrieval. Secondary outcomes include LBR following fresh transfer, multiple pregnancy rate and time until pregnancy leading a live birth. We will also assess the obstetric and neonatal outcomes, costs and patients’ treatment burden.Ethics and disseminationThe study protocol has been approved by the Central Committee on Research involving Human Subjects in the Netherlands in June 2018 (CCMO NL 64060.000.18). The results of this trial will be submitted for publication in international peer-reviewed and in open access journals.Trial registration numberNetherlands Trial Register (NL 6857).

Purpose We assessed the uptake of fertility preservation (FP), recovery of ovarian function (OFR) after chemotherapy, live birth after breast cancer, and breast cancer outcomes in women with early-stage breast cancer. Methods Women aged below 41 years and referred to our center for FP counseling between 2008 and 2015 were included. Data on patient and tumor characteristics, ovarian function, cryopreservation (embryo/oocyte) and transfer, live birth, and disease-free survival were collected. Kaplan–Meier analyses were performed for time-to-event analyses including competing risk analyses, and patients with versus without FP were compared using the logrank test. Results Of 118 counseled women with a median age of 31 years (range 19–40), 34 (29%) chose FP. Women who chose FP had less often children, more often a male partner and more often favorable tumor characteristics. The 5-year OFR rate was 92% for the total group of counseled patients. In total, 26 women gave birth. The 5-year live birth rate was 27% for the total group of counseled patients. Only three women applied for transfer of their cryopreserved embryo(s), in two combined with preimplantation genetic diagnosis (PGD) because of BRCA1-mutation carrier ship. The 5-year disease-free survival rate was 91% versus 88%, for patients with versus without FP (P = 0.42). Conclusions Remarkably, most women achieved OFR, probably related to the young age at diagnosis. Most pregnancies occurred spontaneously, two of three women applied for embryo transfer because of the opportunity to apply for PGD.

STUDY QUESTION Can reduced representation genome sequencing offer an alternative to single nucleotide polymorphism (SNP) arrays as a generic and genome-wide approach for comprehensive preimplantation genetic testing for monogenic disorders (PGT-M), aneuploidy (PGT-A) and structural rearrangements (PGT-SR) in human embryo biopsy samples? SUMMARY ANSWER Reduced representation genome sequencing, with OnePGT, offers a generic, next-generation sequencing-based approach for automated haplotyping and copy-number assessment, both combined or independently, in human single blastomere and trophectoderm samples. WHAT IS KNOWN ALREADY Genome-wide haplotyping strategies, such as karyomapping and haplarithmisis, have paved the way for comprehensive PGT, i.e. leveraging PGT-M, PGT-A and PGT-SR in a single workflow. These methods are based upon SNP array technology. STUDY DESIGN, SIZE, DURATION This multi-centre verification study evaluated the concordance of PGT results for a total of 225 embryos, including 189 originally tested for a monogenic disorder and 36 tested for a translocation. Concordance for whole chromosome aneuploidies was also evaluated where whole genome copy-number reference data were available. Data analysts were kept blind to the results from the reference PGT method. PARTICIPANTS/MATERIALS, SETTING, METHODS Leftover blastomere/trophectoderm whole genome amplified (WGA) material was used, or secondary trophectoderm biopsies were WGA. A reduced representation library from WGA DNA together with bulk DNA from phasing references was processed across two study sites with the Agilent OnePGT solution. Libraries were sequenced on an Illumina NextSeq500 system, and data were analysed with Agilent Alissa OnePGT software. The embedded PGT-M pipeline utilises the principles of haplarithmisis to deduce haplotype inheritance whereas both the PGT-A and PGT-SR pipelines are based upon read-count analysis in order to evaluate embryonic ploidy. Concordance analysis was performed for both analysis strategies against the reference PGT method. MAIN RESULTS AND THE ROLE OF CHANCE PGT-M analysis was performed on 189 samples. For nine samples, the data quality was too poor to analyse further, and for 20 samples, no result could be obtained mainly due to biological limitations of the haplotyping approach, such as co-localisation of meiotic crossover events and nullisomy for the chromosome of interest. For the remaining 160 samples, 100% concordance was obtained between OnePGT and the reference PGT-M method. Equally for PGT-SR, 100% concordance for all 36 embryos tested was demonstrated. Moreover, with embryos originally analysed for PGT-M or PGT-SR for which genome-wide copy-number reference data were available, 100% concordance was shown for whole chromosome copy-number calls (PGT-A). LIMITATIONS, REASONS FOR CAUTION Inherent to haplotyping methodologies, processing of additional family members is still required. Biological limitations caused inconclusive results in 10% of cases. WIDER IMPLICATIONS OF THE FINDINGS Employment of OnePGT for PGT-M, PGT-SR, PGT-A or combined as comprehensive PGT offers a scalable platform, which is inherently generic and thereby, eliminates the need for family-specific design and optimisation. It can be considered as both an improvement and complement to the current methodologies for PGT. STUDY FUNDING/COMPETING INTEREST(S) Agilent Technologies, the KU Leuven (C1/018 to J.R.V. and T.V.) and the Horizon 2020 WIDENLIFE (692065 to J.R.V. and T.V). H.M. is supported by the Research Foundation Flanders (FWO, 11A7119N). M.Z.E, J.R.V. and T.V. are co-inventors on patent applications: ZL910050-PCT/EP2011/060211- WO/2011/157846 ‘Methods for haplotyping single cells’ and ZL913096-PCT/EP2014/068315 ‘Haplotyping and copy-number typing using polymorphic variant allelic frequencies’. T.V. and J.R.V. are co-inventors on patent application: ZL912076-PCT/EP2013/070858 ‘High-throughput genotyping by sequencing’. Haplarithmisis (‘Haplotyping and copy-number typing using polymorphic variant allelic frequencies’) has been licensed to Agilent Technologies. The following patents are pending for OnePGT: US2016275239, AU2014345516, CA2928013, CN105874081, EP3066213 and WO2015067796. OnePGT is a registered trademark. D.L., J.T. and R.L.R. report personal fees during the conduct of the study and outside the submitted work from Agilent Technologies. S.H. and K.O.F. report personal fees and other during the conduct of the study and outside the submitted work from Agilent Technologies. J.A. reports personal fees and other during the conduct of the study from Agilent Technologies and personal fees from Agilent Technologies and UZ Leuven outside the submitted work. B.D. reports grants from IWT/VLAIO, personal fees during the conduct of the study from Agilent Technologies and personal fees and other outside the submitted work from Agilent Technologies. In addition, B.D. has a patent 20160275239 - Genetic Analysis Method pending. The remaining authors have no conflicts of interest.

We studied the mtDNA bottleneck in zebrafish to elucidate size, timing, and variation in germline and non-germline cells. Mature zebrafish oocytes contain, on average, 19.0 × 10(6) mtDNA molecules with high variation between oocytes. During embryogenesis, the mtDNA copy number decreases to ∼170 mtDNA molecules per primordial germ cell (PGC), a number similar to that in mammals, and to ∼50 per non-PGC. These occur at the same developmental stage, implying considerable variation in mtDNA copy number in (non-)PGCs of the same female, dictated by variation in the mature oocyte. The presence of oocytes with low mtDNA numbers, if similar in humans, could explain how (de novo) mutations can reach high mutation loads within a single generation. High mtDNA copy numbers in mature oocytes are established by mtDNA replication during oocyte development. Bottleneck differences between germline and non-germline cells, due to early differentiation of PGCs, may account for different distribution patterns of familial mutations.

Study question Does age of G-1 PLUS v5 embryo culture medium affect IVF outcome? Summary answer Birthweight of singletons born after IVF showed an inverse association with age of the embryo culture medium, while no association was found between age of culture medium and fertilization rate, embryonic development or ongoing pregnancy. What is known already It has been reported that IVF culture media can deteriorate during storage, which suggests that the capacity of culture media to support optimal embryo development decreases over time. Some animal studies showed an effect of storage time on embryo development, in contrast to other studies, while the effect of aging culture medium on IVF outcome in humans is unknown. Study design, size, duration We used data on outcome of 1832 IVF/ICSI cycles with fresh embryo transfer, performed in the period 2008-2012 to evaluate the association of fertilization rate, embryonic development, ongoing pregnancy and birthweight of singletons with age of the culture medium (Vitrolife AB G-1 PLUS v5). Participants/materials, setting, methods Age of the culture medium was calculated by subtracting the production date from the date of ovum retrieval. Data analysis included linear regression and logistic regression on continuous and categorical outcomes, respectively. Main results and the role of chance Age of the culture medium was not associated with fertilization rate (P = 0.543), early cleavage rate (P = 0.155), percentage of embryos containing four or more cells on Day 2 (P = 0.401), percentage of embryos containing eight or more cells on Day 3 (P = 0.175), percentage of embryos with multinucleated blastomeres (P = 0.527), or ongoing pregnancy (P = 0.729). However, birthweight of the newborn was inversely associated with age of the medium (β = -3.6 g, SE: 1.5 g, P = 0.021), after controlling for possible confounders (day of embryo transfer, number of transferred embryos, child's gender, gestational age at birth, parity, pregnancy complications, maternal smoking, height and weight, and paternal height and weight) and the association was not biased by year of treatment, time since first opening of the bottle or batch variations. This indicates a difference of 234 g in birthweight of newborns for media with an age difference of 65 days. Limitations, reasons for caution The results from this study may be specific for the G-1 PLUS v5 culture medium and extrapolation of the results to other media should be done with caution because of the differences in composition and shelf life. Wider implications of the findings Age of G-1 PLUS v5 medium used to culture human embryos affects birthweight of the respective newborn. This could imply that the preimplantation embryo adapts to its in vitro environment with lasting in vivo consequences. Therefore, it is important that companies are transparent about the exact composition of their embryo culture media, which will allow IVF clinics to further investigate the effects of the media or media components on the health of IVF children. Study funding/competing interests No funding and no competing interests declared. Trial registration number Not applicable.

Study question When does a difference in human intrauterine growth of singletons conceived after IVF and embryo culture in two different culture media appear? Summary answer Differences in fetal development after culture of embryos in one of two IVF media were apparent as early as the second trimester of pregnancy. What is known already Abnormal fetal growth patterns are a major risk factor for the development of chronic diseases in adult life. Previously, we have shown that the medium used for culturing embryos during the first few days after fertilization significantly affects the birthweight of the resulting human singletons. The exact onset of this growth difference was unknown. Study design, size and duration In this retrospective cohort study, all 294 singleton live births after fresh embryo transfer in the period July 2003 to December 2006 were included. These embryos originated from IVF treatments that were part of a previously described clinical trial. Embryos were allocated to culture in either Vitrolife or Cook commercially available sequential culture media. Participants/materials, setting, methods We analysed ultrasound examinations at 8 (n = 290), 12 (n = 83) and 20 weeks' (n = 206) gestation and used first-trimester serum markers [pregnancy-associated plasma protein-A (PAPP-A) and free β-hCG]. Differences between study groups were tested by the Student's t-test, χ(2) test or Fisher's exact test, and linear multivariable regression analysis to adjust for possible confounders (for example, parity, gestational age at the time of ultrasound and fetal gender). Main results and the role of chance A total of 294 singleton pregnancies (Vitrolife group nVL = 168, Cook group: nC = 126) from 294 couples were included. At 8 weeks' gestation, there was no difference between crown-rump length-based and ovum retrieval-based gestational age (ΔGA) (nVL = 163, nC = 122, adjusted mean difference, -0.04 days, P = 0.84). A total of 83 women underwent first-trimester screening at 12 weeks' gestation (nVL = 45, nC = 38). ΔGA, nuchal translucency (multiples of median, MoM) and PAPP-A (MoM) did not differ between the study groups. Free β-hCG (MoM) ± SEM differed significantly (1.55 ± 0.19 in Vitrolife versus 1.06 ± 0.10 in Cook; P = 0.031, Student's t-test). At 20 weeks' gestation, a more advanced GA, reflecting an increased fetal growth, was seen at ultrasound examination in the Vitrolife group (n = 115) when compared with the Cook group (n = 91). After adjustment for confounding factors, both the difference between GA based on three biparietal diameter dating formulas minus the actual (ovum retrieval based) GA (adjusted mean difference + 1.14 days (P = 0.04), +1.14 days (P = 0.04) and +1.36 days (P = 0.048)), as well as head circumference (HC) and trans-cerebellar diameter (TCD) were significantly higher in the Vitrolife group (HCvl 177.3 mm, HCc 175.9 mm, adjusted mean difference 1.8, P = 0.03; TCDvl 20.5 mm, TCDc 20.2 mm, adjusted mean difference 0.4, P = 0.008). Limitations, reasons for caution A first trimester (12 weeks) fetal screening was not yet offered routinely during the study period, therefore only 28% of women in our study participated in this elective screening programme. Although all sonographers were experienced and specially trained to perform these ultrasound examinations and were unaware of the randomization procedure, we cannot totally rule out possible intra- and inter-observer variability. Despite being indispensable in daily practice, sonographic weight formulas have a limited accuracy. Wider implications of the findings According to the fetal origins hypothesis, many adult diseases originate in utero owing to adaptations made by the fetus to the environment it encounters. This study indicates that the embryonic environment is already important for fetal development. Therefore, our study emphasizes the need to investigate fetal growth patterns after assisted reproduction technologies and long-term health outcomes of IVF children, especially in relation to the culture medium used during the first few days of preimplantation development. Trial registration number Not applicable.

Study question Does ammonium accumulate in commercially available culture media and protein supplements used for in vitro development of human pre-implantation embryos during storage and incubation? Summary answer Ammonium accumulates in ready-to-use in vitro fertilization (IVF) culture media during storage at 2-8°C and in ready-to-use IVF culture media and protein supplements during incubation at 37°C. What is known already Both animal and human studies have shown that the presence of ammonium in culture medium has detrimental effects on embryonic development and pregnancy rate. It is, therefore, important to assess the amount of ammonium accumulation in ready-to-use IVF culture media under conditions that are common in daily practice. Study design, size, duration Ammonium accumulation was investigated in 15 ready-to-use media, 11 protein-free media and 8 protein supplements. Participants/materials, setting, methods Ammonium was measured by the use of an enzymatic method with glutamate dehydrogenase. To simulate the storage and incubation conditions during IVF treatments, ammonium concentrations were measured at different time-points during storage at 2-8°C for 6 weeks and during incubation at 37°C for 4 days. Main results and the role of chance All ready-to-use, i.e. protein supplemented, culture media showed ammonium accumulation during storage for 6 weeks (ranging from 9.2 to 99.8 µM) and during incubation for 4 days (ranging from 8.4 to 138.6 µM), resulting in levels that might affect embryo development. The protein supplements also showed ammonium accumulation, while the culture media without protein supplementation did not. The main sources of ammonium buildup in ready-to-use culture media were unstable glutamine and the protein supplements. No additional ammonium buildup was found during incubation when using an oil overlay or with the presence of an embryo in the culture droplet. Limitations, reasons for caution In addition to the unstable glutamine and the protein supplements, other free amino acids might contribute to the ammonium buildup. We did not investigate the deterioration of other components in the media. Wider implications of the findings Break-down of components into ammonium is more pronounced during incubation at 37°C, however, it is not negligible during storage at 2-8°C. This results in increasing ammonium levels in culture media over time that may affect embryo development. Therefore, it is important that the use of free l-glutamine in human embryo culture media is stopped and that the use of protein supplements is thoroughly evaluated. Study funding/competing interests No funding or no competing interests declared. Trial registration number Not applicable.

STUDY QUESTION: Is gene expression in human preimplantation embryos affected by the medium used for embryo culture in vitro during an IVF treatment? SUMMARY ANSWER: Six days of in vitro culture of human preimplantation embryos resulted in medium-dependent differences in expression level of genes involved in apoptosis, protein degradation, metabolism and cell-cycle regulation. WHAT IS KNOWN ALREADY: Several human studies have shown an effect of culture medium on embryo development, pregnancy outcome and birthweight. However, the underlying mechanisms in human embryos are still unknown. In animal models of human development, it has been demonstrated that culture of preimplantation embryos in vitro affects gene expression. In humans, it has been found that culture medium affects gene expression of cryopreserved embryos that, after thawing, were cultured in two different media for 2 more days. STUDY DESIGN, SIZE, DURATION: In a multicenter trial, women were randomly assigned to two culture medium groups [G5 and human tubal fluid (HTF)]. Data on embryonic development were collected for all embryos. In one center, embryos originating from two pronuclei (2PN) zygotes that were not selected for transfer or cryopreservation on Day 2 or 3 because of lower morphological quality, were cultured until Day 6 and used in this study, if couples consented. PARTICIPANTS/MATERIALS, SETTING, METHODS: Ten blastocysts each from the G5 and HTF study groups, matched for fertilization method, maternal age and blastocyst quality, were selected and their mRNA was isolated and amplified. Embryos were examined individually for genome-wide gene expression using Agilent microarrays and PathVisio was used to identify the pathways that showed a culture medium-dependent activity. MAIN RESULTS AND THE ROLE OF CHANCE: Expression of 951 genes differed significantly (P < 0.01) between the G5 and HTF groups. Eighteen pathways, involved in apoptosis, metabolism, protein processing and cell-cycle regulation, showed a significant overrepresentation of differentially expressed genes. The DNA replication, G1 to S cell-cycle control and oxidative phosphorylation pathways were up-regulated in the G5 group compared with the HTF group. This is in agreement with the morphological assessment of the 1527 embryos (originating from 2PN zygotes), which showed that embryos consisted of more cells on Day 2 (3.73 +/- 1.30 versus 3.40 +/- 1.35, P < 0.001) and Day 3 (7.00 +/- 2.41 versus 5.84 +/- 2.36, P < 0.001) in the G5 group when compared with the HTF group. Furthermore, the implantation rate was significantly higher in the G5 group compared with the HTF group (26.7% versus 14.7%, P = 0.002) after transfer on the second or the third day after fertilization. LIMITATIONS, REASONS FOR CAUTION: Despite careful matching of the embryos, it cannot be excluded that the differences observed between the study groups are caused by factors that we did not investigate. Extrapolation of these results to embryos used for transfer demands caution as in the present study embryos that were not selected for either embryo transfer or cryopreservation have been used for the culture experiment until Day 6. WIDER IMPLICATIONS OF THE FINDINGS: This study shows that gene expression in human preimplantation embryos is altered by the culture medium used during IVF treatment and provides insight into the biological pathways that are affected. Whether these changes in gene expression have any long-term effects on children born after IVF remains unknown. However, it is possible that early adaptations of the preimplantation embryo to its environment persist during fetal and post-natal development. STUDY FUNDING/COMPETING INTERESTS: No funding and no competing interests declared. TRIAL REGISTRATION NUMBER: Not applicable.

BACKGROUND: Cleavage stage embryos as well as postimplantation embryos have been studied extensively over the years. However, our knowledge with respect to the chromosomal constitution of human embryos at the blastocyst stage is still rudimentary. METHODS: In the present paper, a large series of human blastocysts was examined by means of fluorescent in situ hybridization (FISH). RESULTS: It was found that only one in four blastocysts (25%) displayed a normal chromosomal pattern. We defined a group of blastocysts (26%) displaying a simple mosaic chromosome pattern (different cell lines resulting from one chromosomal error), an about equally large group of blastocysts (31%) displaying a complex mosaic chromosome pattern, and a smaller group of blastocysts (11%) showing a chaotic chromosome distribution pattern. Six per cent of all blastocysts analysed could not be assigned one of the previously mentioned chromosomal patterns. CONCLUSION: Anaphase lagging appeared to be the major mechanism through which human embryos acquire a mosaic chromosome pattern during preimplantation development to the blastocyst stage.

Purpose This study aimed to evaluate the uptake of fertility preservation, rate of pregnancy, pregnancy outcome and breast cancer outcome after diagnosis of early breast cancer in young women who were referred to Maastricht University Medical Center, from the regional hospitals in the Southeast part of the Netherlands. Patients and methods We prospectively registered the demographics of patients, who visited our university hospital for counseling on fertility preservation at diagnosis of stage I-III invasive breast cancer in the years 2008-2015. At baseline, tumor and treatment characteristics were registered. During follow-up information on fullfilled childwish and disease status was collected. To compare the fertility preservation group and the non-fertility preservation group independent samples Student t-tests and Chi-square tests were conducted. Results In total 128 women with a median age of 32 years (19 – 43) were referred for fertility preservation counseling before start of chemotherapy, with an increase in referral in the more recent years. Thirty-nine (30.5%) women chose for fertility preservation: in 26 patients embryos were frozen, in seven oocytes, and in one both embryos and oocytes. In four patients the procedure was not succesfull. Patients who had chosen for fertility preservation more often had a male partner (87.2% vs 70.8%, P = 0.05) and had a smaller tumor size (median 19 versus 23 mm, P = 0.04) at the time of diagnosis compared to those who did not chose for fertility preservation. During a median follow-up of 30.3 months (range 0 – 96.9), 27 (21.1%) patients had tried to conceive: 14 (35.9%) women in the fertility versus 13 (14.6%) in the non-fertility preservation group. All of these had recovery of ovarian function after chemotherapy-induced ovarian failure. Only two women used the cryopreserved embryos, both succesfully and combined with preimplantation genetic diagnosis of the embryos because of germline mutations in BRCA1-gene. Eight patients in the fertility preservation group and seven patients in the non-fertility preservation group became at least once pregnant. In the fertility preservation group, eight healthy babies were born, one baby had Morbus Hirschsprung, one women is pregnant at this moment and one woman had a miscarriage. Of the eleven pregnancies in the non-fertility preservation group, nine healthy babies were born and one woman had two miscarriages. Of the referred 128 women, nine (7.0%) had breast cancer recurrence, three in the fertility preservation group versus six in the non-fertility preservation group. Conclusion One third of referred patients choose for fertility preservation before start of chemotherapy. In all of these patients, the ovarian function recovered. However, two couples used their cryopreserved embryos for preimplantation genetic diagnosis and both became pregnant. Since the follow-up time is relatively short, more data are mandatory to make a statement on the effectiveness of fertility preservation techniques in young breast cancers patients. Citation Format: Vriens IJ, Butalid EM, Schepers-van der Sterren EE, van der Poel MH, Jansen-Engelen SL, van Riel A-MM, van de Wouw YJ, Vriens BE, van Haaren ER, Lemaire BM, Dercksen WW, Luiten EJ, de Boer M, de Die-Smulders CE, Derhaag JG, van Golde RJ, Tjan-Heijnen VC. Preserving fertility in young women undergoing chemotherapy for early breast cancer; the Maastricht experience [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr PD6-03.

Item does not contain fulltext STUDY QUESTION: Is post-natal growth during the first 2 years of life in IVF singletons affected by type of medium used for culturing human embryos during an IVF treatment? SUMMARY ANSWER: The in vitro culture of human embryos in medium from Cook resulted in singletons with a lower weight during the first 2 years of life compared with singletons born after embryo culture in medium from Vitrolife. WHAT IS KNOWN ALREADY: In a previous study, we reported that type of medium used for culturing human IVF embryos during the first few days after fertilization until fresh embryo transfer significantly affects fetal growth and consequently birthweight of the resulting singletons. STUDY DESIGN, SIZE, DURATION: From July 2003 to December 2006, a total of 1432 IVF treatment cycles with fresh embryo transfer were randomly allocated to have all embryos cultured in medium from Vitrolife AB (n = 715) or from Cook (n = 717). Two years after delivery, questionnaires were sent to the parents of all children requesting data about weight, height and head circumference around 1, 2, 3, 4, 6, 7.5, 9, 11, 14, 18 and 24 months of age. These measurements were collected as part of the children's health programme at municipal infant welfare centres in the Netherlands by health professionals unaware of this study. PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients requiring donor oocytes or applying for PGD were excluded from the study. From the 294 live born singletons that fulfilled our inclusion criteria, 29 were lost to follow-up. The remaining 265 singletons (Cook group: 117, Vitrolife group: 148) were included in the analysis. Data analysis included linear regression, to compare cross-sectionally weight standard deviation score (SDS), height SDS and head circumference, and the first order Berkey-Reed model for a longitudinal analysis of the growth data. MAIN RESULTS AND THE ROLE OF CHANCE: Singletons in the Vitrolife group were heavier during the first 2 years of life compared with singletons in the Cook group. Cross-sectional analyses showed that adjusted weight SDS differed between groups at 1 (0.35 +/- 0.14, P = 0.010), 2 (0.39 +/- 0.14, P = 0.006), 3 (0.35 +/- 0.14, P = 0.011), 4 (0.30 +/- 0.13, P = 0.020), 11 (0.28 +/- 0.13, P = 0.036), 14 (0.32 +/- 0.13, P = 0.014) and 24 (0.39 +/- 0.15, P = 0.011) months of age, while adjusted height SDS was only significantly different at 1 (0.21 +/- 0.11, P = 0.048) month of age. Head circumference was similar between the two groups at all ages. Longitudinal analyses showed that both post-natal weight (P = 0.005) and height (P = 0.031) differed between the groups throughout the first 2 years of life, while the growth velocity was not significantly different between the two groups. LIMITATIONS, REASONS FOR CAUTION: Factors that might influence post-natal growth were included in the analysis; however, it was not possible to include all such factors, for example childhood diseases or nutrition, as this information was not available. WIDER IMPLICATIONS OF THE FINDINGS: The effect of culture medium during the first few days after fertilization on prenatal growth and birthweight persists during the first 2 years of life. This suggests that the human embryo is sensitive to its very early environment, and that the culture medium used in IVF may have lasting consequences. Further monitoring of the long-term growth, development and health of IVF children is therefore warranted. STUDY FUNDING/COMPETING INTEREST(S): W.V. was funded with an unrestricted research grant from the Stichting Fertility Foundation. The authors declare no conflict of interest. TRIAL REGISTRATION NUMBER: Not applicable.

Classical complement activation is a major effector mechanism in the development of vascular lesions contributing to allograft rejection. We investigated complement activation by alloantibodies reactive with graft endothelial cell (EC) alloantigens in settings of MHC-mismatched and MHC-matched (non-MHC-mismatched) rat heart transplantation (Tx). Allosera and heart allografts were collected at the day of rejection (day 7-8 and day 28-35 in MHC-mismatched and non-MHC-mismatched Tx respectively) or earlier. Allosera reactivity was studied in vitro using rat-heart-endothelial-cell (RHEC) lines expressing the appropriate donor MHC and non-MHC alloantigen profile. Immunohistochemical analysis of rejected heart allografts showed deposition of alloantibodies in both MHC-mismatched and MHC-matched heart allografts, but expression of C3 was only seen in the vasculature of MHC-mismatched grafts. FACS analysis showed that anti MHC as well as anti non-MHC allosera were reactive with donor EC cell surface antigens. Both sera had similar IgG subclass profiles of anti-endothelial cell antibodies. Complement activation by anti MHC and anti non-MHC alloantibodies on EC was measured by FACS analysis of C3 and C5b-9 (MAC) expression. Distinct expression of C3 was noticed for EC incubated with anti-MHC allosera, but hardly for EC incubated with anti non-MHC allosera. C5b-9 was low but showed no difference between the two allosera. However, complement-mediated cytotoxicity experiments showed that functional (lytic) MAC was induced with anti MHC allosera but hardly with anti non-MHC allosera. Our data show that in settings of MHC-matched heart transplantation alloantibodies against endothelial non-MHC alloantigens are generated, but, in contrast to alloantibodies to MHC alloantigens, these alloantibodies have only poor complement-activating and lytic potentials. Whether anti non-MHC allolantibodies effect other biological processes relevant for heart allograft vasculopathy, including development of graft arteriosclerosis, needs further elucidation.

Summary The alloantigen profile of vascular endothelial cells has been studied in a rat experimental model for MHC-mismatched and MHC-matched (non-MHC mismatched) allogeneic heart transplantation (TX). Hearts were studied for alloantibody deposition in the graft vasculature and the occurrence of vasculopathy. In addition reactivity of allosera with in vitro cultures of graft-type vascular endothelial cells was examined. Alloantibodies were seen deposited in the graft vasculature in MHC-mismatched as well as in MHC-matched TX. In MHC-matched hearts the most dominant vasculopathy consisted of early arteriosclerotic lesions. In vitro studies demonstrated that, in addition to MHC alloantigens, heart vascular endothelial cells express non-MHC alloantigens. These endothelial non-MHC alloantigens are constitutively expressed by heart endothelial cells, and expression is not influenced by cytokine stimulation. We conclude that humoral immune response against these endothelial non-MHC alloantigens may play an important role in development of graft vasculopathy and particularly of arteriosclerotic lesions.

Several assisted reproduction procedures, such as IVF and ICSI, are available for a variety of infertility problems. In fertility clinics, patients are screened for blood-borne viral infections, including hepatitis B virus (HBV). Reasons for screening are prevention of vertical transmission and laboratory safety. We present the case of a 26-year-old female patient with a chronic HBV infection, whose husband tested negative for hepatitis B. She and her husband were referred to our fertility clinic because of subfertility. Analysis of the husband's semen indicated the necessity of an ICSI procedure. The current Dutch guidelines advise against ICSI in chronic HBV carriers, since the risks and effects of chromosomal integration of HBV DNA in the fetus are not well-known. In this article, we review the scientific evidence for the risk of introducing HBV virus into the oocyte and subsequent integration of HBV DNA into the human genome, and debate the question of whether to do, or not to do, IVF and ICSI.

Mast cell (MC)-mediated induction of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) and of E-selectin was studied in cultures of rat heart endothelial cells (EC) and human umbilical vein EC (HUVEC) respectively. MC induced VCAM-1 and E-selectin, but hardly any ICAM-1 in EC. Induction was not dependent on MC degranulation, but seemed to be provoked by constitutively released substances, other than histamine, from MC. Co-incubation of MC and EC, allowing for direct contact between the two cell types, was more potent in induction than MC co-incubated separately from EC using a permeable membrane. MC were less potent in induction than exogenous added cytokines or LPS. Induction of cell adhesion molecules in rat heart EC was MC-specific, since EC incubations with either rat cardiomyocytes or heart fibroblasts had no effect. The data show that rat MC, independent of degranulation, secrete mediators relevant for the induction of a specific set of EC adhesion molecules in vitro. This suggests a (supportive) role for MC in cell-adhesion molecule induction in the endothelium in settings of early or mild inflammation. The results are discussed in the context of inflammatory processes in the heart in vivo.

Almost every female classic galactosemia patient develops primary ovarian insufficiency (POI) as a diet-independent complication of the disease. This is a major concern for patients and their parents, and physicians are often asked about possible options to preserve fertility. Unfortunately, there are no recommendations on fertility preservation in this group. The unique pathophysiology of classic galactosemia with a severely reduced follicle pool at an early age requires an adjusted approach. In this article recommendations for physicians based on current knowledge concerning galactosemia and fertility preservation are made. Fertility preservation is only likely to be successful in very young prepubertal patients. In this group, cryopreservation of ovarian tissue is currently the only available technique. However, this technique is not ready for clinical application, it is considered experimental and reduces the ovarian reserve. Fertility preservation at an early age also raises ethical questions that should be taken into account. In addition, spontaneous conception despite POI is well described in classic galactosemia. The uncertainty surrounding fertility preservation and the significant chance of spontaneous pregnancy warrant counseling towards conservative application of these techniques. We propose that fertility preservation should only be offered with appropriate institutional research ethics approval to classic galactosemia girls at a young prepubertal age.

Several clinical findings point to the involvement of microvascular endothelial cells in cytomegalovirus-related pathology. In this study the interactions of cytomegalovirus (CMV) with microvascular endothelial cells was investigated in an in vitro rat model. A series of rat endothelial cell lines, considered representative for the heterogeneity of heart microvascular endothelium in vivo, were infected with rat CMV (RCMV). The course of infection and production of infectious virus were examined using immunofluorescence staining and plaque titration assays, and was compared with infection of fully permissive rat fibroblasts. These endothelial cell lines diplayed differences in susceptibility to CMV infection. Two endothelial cell lines (RHEC 50 and 191) were practically non-permissive, while four endothelial cell lines (RHEC 3, 10, 11 and 116) were partly permissive for CMV infection. In contrast to CMV infection in fibroblasts, only limited infection of the permissive endothelial cell lines was observed without spreading of CMV infection through the monolayer, although infectious virus was produced. Detachment of infected endothelial cells and recovery of the monolayer with time was observed. The detached endothelial cells were able to transmit CMV infection to fibroblast monolayers, but not to endothelial monolayers. Our in vitro results demonstrate differences in permissiveness for RCMV between the series of rat endothelial cell lines, which is suggestive for endothelial heterogeneity to CMV infection in vivo. Our findings indicate that endothelial cells are relatively resistant to CMV infection and that, upon infection, the endothelial monolayer may dispose of the virus via detachment of the infected cells. This points to a dual role for the endothelium in CMV infection in vivo: a barrier for CMV infection (by the endothelial monolayer) on the one hand and spreading of CMV infection (by detached infected cells) on the other hand.

background: We have previously shown that the medium used for culturing IVF embryos affects the birthweight of the resulting newborns. This observation with potentially far-reaching clinical consequences during later life, was made in singletons conceived during the first IVF treatment cycle after the transfer of fresh embryos. In the present study, we hypothesize that in vitro culture of embryos during the first few days of preimplantation development affects perinatal outcome, not only in singletons conceived in all rank order cycles but also in twins and in children born after transfer of frozen embryos. Furthermore, we investigated the effect of culture medium on gestational age (GA) at birth. methods: Oocytes and embryos from consecutive treatment cycles were alternately assigned to culture in either medium from Vitrolife or from Cook. Data on a cohort of 294 live born singletons conceived after fresh transfer during any of a patient’s IVF treatment cycles, as well as data of 67 singletons conceived after frozen embryo transfer (FET) and of 88 children of 44 twin pregnancies after fresh transfer were analysed by means of multiple linear regression. results: In vitro culture in medium from Cook resulted in singletons after fresh transfer with a lower mean birthweight (adjusted mean difference, 112 g, P ¼ 0.03), and in more singletons with low birthweight (LBW) ,2500 g (P ¼ 0.006) and LBW for GA ≥37 weeks (P ¼ 0.015), when compared with singletons born after culture in medium from Vitrolife AB. GA at birth was not related to the medium used (adjusted difference, 0.05 weeks, P ¼ 0.83). Among twins in the Cook group, higher inter-twin mean birthweight disparity and birthweight discordance were found. Z-scores after FET were 20.04 (+0.14) in the Cook group compared with 0.18 (+0.21) in the Vitrolife group (P . 0.05). conclusions: Our findings support our hypothesis that culture medium influences perinatal outcome of IVF singletons and twins. A similar trend is seen in case of singletons born after FET. GA was not affected by culture medium. These results indicate that in vitro culture might be an important factor explaining the poorer perinatal outcome after assisted reproduction technology (ART). Further research is needed to confirm this culture medium-induced effect in humans and to provide more insight into whether it is caused by epigenetic disturbance of imprinted genes in fetal or placental tissues. Moreover, embryo culture media and their effects need to be investigated thoroughly to select the best embryo culture medium in order to minimize or prevent short-term risks and maybe even long-term disease susceptibility.

In animal models, in vitro culture of preimplantation embryos has been shown to be a risk factor for abnormal fetal outcome, including high and low birthweight. In the human, mean birthweight of singletons after in vitro fertilization (IVF) is considerably lower than after natural conception, but it is not known whether culture conditions play a role in this.We compared pregnancy rates and perinatal outcomes from singleton pregnancies resulting from a total of 826 first IVF treatment cycles in which oocytes and embryos were randomly allocated to culture in either of two commercially available sequential media systems.When the 110 live born singletons in the Vitrolife group were compared with the 78 singletons in the Cook group, birthweight +/- SEM (3453 +/- 53 versus 3208 +/- 61 g, P = 0.003), and birthweight adjusted for gestational age and gender (mean z-score +/- SEM: 0.13 +/- 0.09 versus -0.31 +/- 0.10, P = 0.001) were both significantly higher in the Vitrolife group. When analyzed by multiple linear regression together with several other variables that could possibly affect birthweight as covariates, the type of culture medium was significantly (P = 0.01) associated with birthweight.In vitro culture of human embryos can affect birthweight of live born singletons.

BACKGROUND: Elective single embryo transfer (eSET) in a selected group of patients (i.e. young patients with at least one good quality embryo) reduces the number of multiple pregnancies in an IVF programme. However, the reduced overall multiple pregnancy rate (PR) is still unacceptably high. Therefore, a randomized controlled trial (RCT) was conducted comparing eSET and double embryo transfer (DET) in an unselected group of patients (i.e. irrespective of the woman’s age or embryo quality). METHODS: Consenting unselected patients were randomized between eSET (RCT-eSET) (n = 154) or DET (RCT-DET) (n = 154). Randomization was performed just prior to the first embryo transfer, provided that at least two 2PN zygotes were available. Non-participants received our standard transfer policy [SP-eSET in a selected group of patients (n = 100), otherwise SP-DET (n = 122)]. RESULTS: The ongoing PR after RCT-eSET was significantly lower as compared with RCT-DET (21.4 versus 40.3%) and the twin PR was reduced from 21.0% after RCT-DET to 0% after RCT-eSET. The ongoing PRs after SP-eSET and SP-DET did not differ significantly (33.0 versus 30.3%), with an overall twin PR of 12.9%. CONCLUSION: To avoid twin pregnancies resulting from an IVF treatment, eSET should be applied in all patients. The consequence would be a halving of the ongoing PR as compared with applying a DET policy in all patients. The transfer of one embryo in a selected group of good prognosis patients leads to a less drastic reduction in PR but maintains a twin PR of 12.9%.

BACKGROUND: There is concern that IVF and/or ICSI might have an adverse effect on embryonic development via epigenetic alterations. Such alterations might also be involved in the sex-related growth differences in preimplantation embryos found in some animal species. In the present study we analysed cell numbers of human male and female surplus embryos that developed to the blastocyst stage after either IVF or ICSI in order to investigate possible sex-dependent differential growth rates. METHODS: Blastocysts resulting from surplus embryos obtained after either IVF or ICSI during a 5 year study period were analysed using fluorescence in situ hybridization (FISH). RESULTS: The number of cells and sex could be determined in 330 blastocysts collected from 92 IVF cycles and in 322 blastocysts collected from 121 ICSI cycles. Whereas female and male embryos originating from IVF showed comparable mean log cell numbers per embryo ± SEM (3.76 ± 0.05 in 147 female and 3.72 ± 0.04 in 183 male embryos), significant differences were observed in embryos originating from ICSI (3.57 ± 0.05 in 162 female and 3.90 ± 0.03 in 160 male embryos). The sex-related growth difference was significantly greater in ICSI than in IVF embryos. In a subset of 84 embryos, inner cell mass (ICM) and trophectoderm (TE) were analysed separately. A significantly higher mean log cell number of TE cells in ICSI male embryos was found as compared to their female counterparts (3.44 ± 0.12 in 16 female and 3.90 ± 0.11 in 29 male embryos), whereas this difference was not found in IVF embryos. CONCLUSION: A clear sex-related growth difference was found in human blastocysts originating from ICSI, but not in blastocysts originating from IVF. It is as yet unknown which mechanism is responsible for our findings. We hypothesize that the ICSI procedure might interfere with the process of imprinted X-inactivation.

BACKGROUND: Elective single embryo transfer (eSET), applied in the first or second IVF cycle in young patients with good quality embryos, has been demonstrated to lower the twin pregnancy rate, while the overall pregnancy rate is not compromised. It is as yet unclear whether eSET could be the preferred transfer policy in all treatment cycles, or that it should be restricted to the first or first two cycles. METHODS: eSET policy (when two or more embryos were available, at least one of them being of good quality) was offered to patients younger than 38 years in the first three treatment cycles. Retrospectively, treatment cycle outcome was studied. RESULTS: In 326 patients, 586 treatment cycles were performed (326 first, 168 second and 92 third treatment cycles). In 65 cycles (11%), eSET could not be applied because there was either no fertilization, or only one embryo available. In the remaining 521 cycles, eSET was performed in 111 cycles (19%), while in 410 cycles, no good quality embryo was available resulting in the transfer of two embryos (double embryo transfer, DET). No significant differences in ongoing pregnancy rates after transfer of fresh embryos were observed between eSET and DET in the first (both 33%), second (36 and 23%, respectively) and third treatment cycles (20 and 24%, respectively). In significantly more eSET cycles compared to DET cycles, could embryos be frozen. This resulted in a significantly higher cumulative pregnancy rate after eSET compared to DET. CONCLUSIONS: In patients younger than 38 years with at least one top quality embryo, eSET can be the transfer policy of choice in at least the first three treatment cycles, since the pregnancy rates obtained in each treatment cycle are comparable to those after DET.

To gain insight in the pathogenesis of vascular lesions in heart allograft rejection, we investigated effects of allosera reactive with major histocompatibility complex (MHC) or non-MHC alloantigens on graft endothelial cells (EC) in a rat transplantation model.Anti-MHC and anti-non-MHC allosera were obtained from Brown Norway (RT.1(n)) recipients of a Lewis (RT.1(1)) or congenic LEW.1N (RT.1(n)) heart allograft respectively. Reactivity with endothelial alloantigens was studied in vitro using a series of three rat heart endothelial cell (RHEC) lines of Lewis origin. Phenotypic studies of MHC and non-MHC alloantigen expression, and adhesion molecule induction on EC were performed by immunostaining and fluorescence-activated cell sorting analysis. Complement-mediated cytotoxicity of allosera was studied using a 51Cr release assay.Both anti-MHC allosera and anti-non-MHC allosera showed reactivity with all three RHEC lines. EC stimulation with tumor necrosis factor-alpha and interferon-y resulted in increased reactivity of anti-MHC but not of anti-non-MHC allosera. Anti-MHC allosera showed complement-mediated cytotoxicity for EC, which was strongly increased when cytokine-stimulated EC were used. With anti-non-MHC allosera, only minor cytotoxicity was measured, irrespective of the activation of EC. Anti-MHC and anti-non-MHC allosera from the day of rejection (days 7-8 and days 29-35, respectively) had similar subclass profiles of allospecific IgG, except for allospecific IgM, which was only detected in anti-MHC allosera. Complement-mediated cytotoxicity of anti-MHC allosera from the day of rejection was effected mainly by IgM alloantibodies, whereas, in allosera taken 4 days after rejection, a predominance of cytotoxic alloantibodies of the IgG class was observed. No indications were found that either alloantibody reactivity alone or in combination with complement activation led to EC activation processes relevant to intercellular adhesion molecule-1 or vascular cell adhesion molecule-1 induction.Our data show that, in heart allograft rejection, MHC but also non-MHC alloantigens on EC are target structures in the alloantibody response. Alloantibodies reactive with endothelial MHC, but not those reactive with non-MHC alloantigens, may significantly contribute to vasculopathy by complement-mediated cytotoxicity. Although no evidence was found that alloantibodies reactive with graft EC induce adhesion molecule expression, they may trigger other EC mechanisms relevant to graft vasculopathy.

In this retrospective study on 1628 consecutive cycles performed during a period of 4 years, development in vitro is compared of embryos obtained after either conventional in-vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). At 39-42 h after insemination or injection, embryos obtained after ICSI were significantly (P < 0.01) further developed (mean cell number 3.48 +/- 0.03) as compared with those obtained after IVF (3.22 +/- 0.03), whereas after 63-66 h of in-vitro development this difference was no longer present (mean cell number 6.11 +/- 0.15 versus 6.09 +/- 0.13 respectively). Culture of surplus embryos obtained after IVF resulted in a significantly higher (P < 0.001) mean incidence of blastocyst formation per cycle as compared with the ICSI group (31.8 +/- 1.9 versus 23.0 +/- 1.4 respectively). Blastocysts from both groups consisted of comparable numbers of cells. Blastocyst formation was also significantly higher when embryos were cultured in groups (31.2 +/- 1.8) compared to single culture (23.1 +/- 1.5; P < 0.01), in human tubal fluid (HTF) medium (29.2 +/- 1.7) compared with IVF-50(TM) medium (24.2 +/- 1.6; P < 0.01), and when they were cultured under 5% O(2) (30.3 +/- 1.5) compared with 20% O(2) (21.7 +/- 1.7; P < 0.01). In all culture conditions used, the mean incidence of blastocyst formation per cycle showed comparable differences in favour of the IVF group as compared with the ICSI group.

Journal de Gynecologie Obstetrique et Biologie de la Reproduction - Vol. 34 - N° SUP7 - p. 15-16