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Study Question: What is the cost-effectiveness of elective single embryo transfer (eSET) versus double embryo transfer (DET) strategies from a societal perspective, when applying a time horizon of 1, 5 and 18 years?, Summary Answer: From a short-term perspective (1 year) it is cost-effective to replace DET with single embryo transfer; however when intermediate- (5 years) and long-term (18 years) costs and consequences are incorporated, DET becomes the most cost-effective strategy, given a ceiling ratio of €20 000 per quality-adjusted life years (QALY) gained., What Is Already Known: According to previous cost-effectiveness research into embryo transfer strategies, DET is considered cost-effective if society is willing to pay around €20 000 for an extra live birth. However, interpretation of those studies is complicated, as those studies fail to incorporate long-term costs and outcomes and used live birth as a measure of effectiveness instead of QALYs. With this outcome, both multiple and singletons were valued as one live birth, whereas costs of all children of a multiple were incorporated., Study Design, Size, Duration: A Markov model (cycle length: 1 year; time horizon: 1, 5 and 18 years) was developed comparing a maximum of: (i) three cycles of eSET in all patients; (ii) four cycles of eSET in all patients; (iii) five cycles of eSET in all patients; (iv) three cycles of standard treatment policy (STP), i.e. eSET in women <38 years with a good quality embryo, and DET in all other women; and (v) three cycles of DET in all patients., Participants/materials, Setting, Methods: Expected life years (LYs), child QALYs and costs were estimated for all comparators. Input parameters were derived from a retrospective cohort study, in which hospital resource data were collected (n=580) and a parental questionnaire was sent out (431 respondents). Probabilistic sensitivity analysis (5000 iterations) was performed., Main Results and the Role of Chance: With a time horizon of 18 years, DETx3 is most effective (0.54 live births, 10.2 LYs and 9.8 QALYs) and expensive (€37 871) per couple starting IVF. Three cycles of eSET are least effective (0.43 live births, 7.1 LYs and 6.8 QALYs) and expensive (€25 563). We assumed that society is willing to pay €20 000 per QALY gained. With a time horizon of 1 year, eSETx3 was the most cost-effective embryo transfer strategy with a probability of being cost-effective of 99.9%. With a time horizon of 5 or 18 years, DETx3 was most cost-effective, with probabilities of being cost-effective of 77.3 and 93.2%, respectively., Limitations, Reasons for Caution: This is the first study to use QALYs generated by the children in the economic evaluation of embryo transfer strategies. There remains some disagreement on whether QALYs generated by new life should be used in economic evaluations of fertility treatment. A further limitation is that treatment ends when it results in live birth and that only child QALYs were considered as measure of effectiveness. The results for the time horizon of 18 years might be less solid, as the data beyond the age of 8 years are based on extrapolation., Wider Implications of the Findings: The current Markov model indicates that when child QALYs are used as measure of outcome it is not cost-effective on the long term to replace DET with single embryo transfer strategies. However, for a balanced approach, a family-planning perspective would be preferable, including additional treatment cycles for couples who wish to have another child. Furthermore, the analysis should be extended to include QALYs of family members., Study Funding/competing Interests: This study was supported by a research grant (grant number 80-82310-98-09094) from the Netherlands Organization for Health Research and Development (ZonMw). There are no conflicts of interest in connection with this article., Trial Registration Number: Not applicable., (© The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Study Question: Does embryo culture medium influence pregnancy and perinatal outcome in IVF?, Summary Answer: Embryo culture media used in IVF affect treatment efficacy and the birthweight of newborns., What Is Known Already: A wide variety of culture media for human preimplantation embryos in IVF/ICSI treatments currently exists. It is unknown which medium is best in terms of clinical outcomes. Furthermore, it has been suggested that the culture medium used for the in vitro culture of embryos affects birthweight, but this has never been demonstrated by large randomized trials., Study Design, Size, Duration: We conducted a multicenter, double-blind RCT comparing the use of HTF and G5 embryo culture media in IVF. Between July 2010 and May 2012, 836 couples (419 in the HTF group and 417 in the G5 group) were included. The allocated medium (1:1 allocation) was used in all treatment cycles a couple received within 1 year after randomization, including possible transfers with frozen-thawed embryos. The primary outcome was live birth rate., Participants/materials, Setting, Methods: Couples that were scheduled for an IVF or an ICSI treatment at one of the six participating centers in the Netherlands or their affiliated clinics., Main Results and the Role of Chance: The live birth rate was higher, albeit nonsignificantly, in couples assigned to G5 than in couples assigned to HTF (44.1% (184/417) versus 37.9% (159/419); RR: 1.2; 95% confidence interval (CI): 0.99-1.37; P = 0.08). Number of utilizable embryos per cycle (2.8 ± 2.3 versus 2.3 ± 1.8; P < 0.001), implantation rate after fresh embryo transfer (20.2 versus 15.3%; P < 0.001) and clinical pregnancy rate (47.7 versus 40.1%; RR: 1.2; 95% CI: 1.02-1.39; P = 0.03) were significantly higher for couples assigned to G5 compared with those assigned to HTF. Of the 383 live born children in this trial, birthweight data from 380 children (300 singletons (G5: 163, HTF: 137) and 80 twin children (G5: 38, HTF: 42)) were retrieved. Birthweight was significantly lower in the G5 group compared with the HTF group, with a mean difference of 158 g (95% CI: 42-275 g; P = 0.008). More singletons were born preterm in the G5 group (8.6% (14/163) versus 2.2% (3/137), but singleton birthweight adjusted for gestational age and gender (z-score) was also lower in the G5 than in the HTF group (-0.13 ± 0.08 versus 0.17 ± 0.08; P = 0.008)., Limitations, Reasons for Caution: This study was powered to detect a 10% difference in live births while a smaller difference could still be clinically relevant. The effect of other culture media on perinatal outcome remains to be determined., Wider Implications of the Findings: Embryo culture media used in IVF affect not only treatment efficacy but also perinatal outcome. This suggests that the millions of human embryos that are cultured in vitro each year are sensitive to their environment. These findings should lead to increased awareness, mechanistic studies and legislative adaptations to protect IVF offspring during the first few days of their existence., Study Funding/competing Interests: This project was partly funded by The NutsOhra foundation (Grant 1203-061) and March of Dimes (Grant 6-FY13-153). The authors declare no conflict of interest., Trial Registration Number: NTR1979 (Netherlands Trial Registry)., Trial Registration Date: 1 September 2009., Date of First Patient's Enrolment: 18 July 2010., (© The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

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., (© The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

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., (© The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Study Question: Do in vitro fertilization (IVF) multiples generate higher hospital costs than IVF singletons, from birth up to age 5?, Summary Answer: Hospital costs from birth up to age 5 were significantly higher among IVF/ICSI multiple children compared with IVF/ICSI singletons; however, when excluding the costs incurred during the birth admission period, hospital costs of multiples and singletons were comparable., What Is Known Already: Concern has risen over the long-term outcome of children born after IVF. The increased incidence of multiple births in IVF as a result of double-embryo transfer predisposes children to a poorer neonatal outcome such as preterm birth and low birthweight. As a consequence, IVF multiples require more medical care. Costs and consequences of poorer neonatal outcomes in multiples may also exist later in life., Study Design, Size, Duration: All 5497 children born from IVF in 2003-2005, whose parents received IVF or ICSI treatment in one of five participating Dutch IVF centers, served as a basis for a retrospective cohort study. Based on gestational age, birthweight, Apgar and congenital malformation, children were assigned to one of three risk strata (low-, moderate- or high-risk)., Participants/materials, Setting, Methods: To enhance the efficiency of the data collection, 816 multiples and 584 singletons were selected for 5-year follow-up based on stratified (risk) sampling. Parental informed consent was received of 322 multiples and 293 singletons. Individual-level hospital resource use data (hospitalization, outpatient visits and medical procedures) were retrieved from hospital information systems and patient charts for 302 multiples and 278 singletons., Main Results and the Role of Chance: The risk of hospitalization (OR 4.9, 95% CI 3.3-7.0), outpatient visits (OR 2.6, 95% CI 1.8-3.6) and medical procedures (OR 1.7, 95% CI 1.2-2.2) was higher for multiples compared with singletons. The average hospital costs amounted to €10 018 and €2093 during the birth admission period (P < 0.001), €1131 and €696 after the birth admission period to the first birthday (not significant (n.s.)) and €1084 and €938 from the second to the fifth life year (n.s.) for multiples and singletons, respectively. Hospital costs from birth up to age 5 were 3.3-fold higher for multiples compared with singletons (P < 0.001). Among multiples and singletons, respectively, 90.8 and 76.2% of the total hospital costs were caused by hospital admission days and 8.9 and 25.2% of the total hospital costs during the first 5 years of life occurred after the first year of life., Limitations, Reasons for Caution: Resource use and costs outside the hospital were not included in the analysis., Wider Implications of the Findings: This study confirms the increased use of healthcare resources by IVF/ICSI multiples compared with IVF/ICSI singletons. Single-embryo transfer may result in substantial savings, particularly in the birth admission period. These savings need to be compared with the extra costs of additional embryo transfers needed to achieve a successful pregnancy. Besides costs, health outcomes of children born after single-embryo transfer should be compared with those born after double-embryo transfer., Study Funding/competing Interests: This study was supported by a research grant (grant number 80-82310-98-09094) from the Netherlands Organization for Health Research and Development (ZonMw). There are no conflicts of interest in connection with this article., Trial Registration Number: Not applicable., (© The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

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., (© The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Study Question: Is gene expression in placental tissue of IVF/ICSI patients altered when compared with a spontaneously conceived group, and are these alterations due to loss of imprinting (LOI) in the case of imprinted genes?, Summary Answer: An altered imprinted gene expression of H19 and Pleckstrin homology-like domain family A member 2 (PHLDA2), which was not due to LOI, was observed in human placentas after IVF/ICSI and several biological pathways were significantly overrepresented and mostly up-regulated., What Is Known Already: Genomic imprinting plays an important role in placental biology and in placental adaptive responses triggered by external stimuli. Changes in placental development and function can have dramatic effects on the fetus and its ability to cope with the intrauterine environment. An increased frequency of placenta-related problems as well as an adverse perinatal outcome is seen in IVF/ICSI derived pregnancies, but the role of placental epigenetic deregulation is not clear yet., Study Design and Participants: In this prospective cohort study, a total of 115 IVF/ICSI and 138 control couples were included during pregnancy. After applying several exclusion criteria (i.e. preterm birth or stillbirth, no placental samples, pregnancy complications or birth defects), respectively, 81 and 105 placentas from IVF/ICSI and control pregnancies remained for analysis. Saliva samples were collected from both parents., Methods: We quantitatively analysed the mRNA expression of several growth-related imprinted genes [H19, insulin-like growth factor 2 (IGF2), PHLDA2, cyclin-dependent kinase inhibitor 1C (CDKN1C), mesoderm-specific transcript homolog (MEST) isoform α and β by quantitative PCR] after standardization against three housekeeping genes [Succinate dehydrogenase A (SDHA), YWHAZ and TATA-binding protein (TBP)]. A quantitative allele-specific expression analysis of the differentially expressed imprinted genes was performed to investigate LOI, independent of the mechanism of imprinting. Furthermore, a microarray analysis was carried out (n = 10 in each group) to investigate the expression of non-imprinted genes as well., Main Results and the Role of Chance: Both H19 and PHLDA2 showed a significant change, respectively, a 1.3-fold (P = 0.033) and 1.5-fold (P = 0.002) increase in mRNA expression in the IVF/ICSI versus control group. However, we found no indication that there is an increased frequency of LOI in IVF/ICSI placental samples. Genome-wide mRNA expression revealed 13 significantly overrepresented biological pathways involved in metabolism, immune response, transmembrane signalling and cell cycle control, which were mostly up-regulated in the IVF/ICSI placental samples., Limitations, Reasons for Caution: Only a subset of samples was found to be fully informative, which unavoidably led to lower sample numbers for our LOI analysis. Our study cannot distinguish whether the reported differences in the IVF/ICSI group are exclusively attributable to the IVF/ICSI technique itself or to the underlying subfertility of the patients., Wider Implications of the Findings: Whether these placental adaptations observed in pregnancies conceived by IVF/ICSI might be connected to an adverse perinatal outcome after IVF remains unknown. However, it is possible that these differences affect fetal development and long-term patterns of gene expression, as well as maternal gestational physiology., Study Funding/competing Interests: Partly funded by an unrestricted research grant by Organon BV (now MSD BV) and GROW School for Oncology and Developmental Biology without any role in study design, data collection and analysis or preparation of the manuscript. No conflict of interests to declare., Trial Registration Number: Dutch Trial Registry (NTR) number 1298., (© The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

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.

Objective: To compare perinatal singleton and multiple outcomes in a large Dutch in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) population and within risk subgroups. Newborns were assigned to a risk category based on gestational age, birthweight, Apgar score and congenital malformation., Design: Register-based retrospective cohort study., Setting: Netherlands Perinatal Registry data., Sample: A total of 3041 singletons and 1788 multiple children born from IVF/ICSI in 2003-2005., Methods: Student's t-test or Mann-Whitney U-test was used to analyze continuous data, chi-squared analyses were used for categorical data. Multivariate logistic and linear regression analysis was performed to analyze whether the risk stratification criteria were associated with neonatal hospital admission and length of stay., Main Outcome Measures: Start of labor, mode of delivery, gestational age, birthweight, 5-min Apgar score, congenital malformation, neonatal hospital admission, neonatal intensive care unit admission and mortality., Results: IVF/ICSI-conceived multiples had considerably poorer outcomes than singletons in terms of cesarean section rate, preterm birth, birthweight, being small-for-gestational-age, Apgar score, neonatal hospital admission, neonatal intensive care unit admission and neonatal mortality. As opposed to the results found in the total study population and the low-risk and moderate-risk populations, high-risk multiples showed better outcomes than high-risk singletons regarding cesarean section rate, birthweight and Apgar score. All risk stratification variables were associated with being hospitalized after birth. Length of stay was associated with all risk stratification criteria except Apgar score., Conclusions: Perinatal outcomes in IVF/ICSI-conceived multiples are considerably poorer than in singletons. This finding mainly pertains to low-risk children. High-risk multiples had significantly better perinatal outcomes than high-risk singletons., (© 2014 Nordic Federation of Societies of Obstetrics and Gynecology.)

Previous studies examining reproductive parameters in men with galactosemia have inconsistently demonstrated abnormalities. We hypothesized that men with galactosemia may demonstrate evidence of reproductive dysfunction. Pubertal history, physical examination, hormone levels and semen analyses were examined in 26 males with galactosemia and compared to those in 46 controls. The prevalence of cryptorchidism was higher in men with galactosemia than in the general population [11.6% vs. 1.0% (95%CI: 0.75-1.26; p <0.001)]. Testosterone (461±125 vs. 532± 33 ng%; p=0.04), inhibin B (144±66 vs. 183±52 pg/mL; p=0.002) and sperm concentration (46±36 vs. 112±75×10(6) spermatozoa/mL; p=0.01) were lower and SHBG was higher (40.7±21.5 vs 26.7±14.6; p=0.002) in men with galactosemia compared to controls. Semen volume was below normal in seven out of 12 men with galactosemia. Men with galactosemia have a higher than expected prevalence of cryptorchidism and low semen volumes. The subtle decrease in testosterone and inhibin B levels and sperm count may indicate mild defects in Sertoli and Leydig cell function, but does not point towards severe infertility causing reproductive impairment. Follow-up studies are needed to further determine the clinical consequences of these abnormalities.

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 IVF/ICSI have an effect on the epigenetic regulation of the human placenta?, Summary Answer: We found a reduced DNA methylation level at the H19 and MEST differentially methylated regions (DMRs), and an increased RNA expression of H19 in placentas from pregnancies conceived by IVF/ICSI when compared with placentas from spontaneous conception., What Is Known Already: Changes in fetal environment are associated with adverse health outcomes. The placenta is pivotal for intrauterine environment. Animal studies show that epigenetic regulation plays an important role in these environment-induced phenotypic effects. Also, the preimplantation embryo environment affects birthweight as well as the risk of chronic adult diseases. Epigenetic processes are sensitive to the environment, especially during the period around conception., Study Design and Participants: Placental tissue was collected from 35 spontaneously conceived pregnancies and 35 IVF/ICSI (5 IVF, 30 ICSI) derived pregnancies. We quantitatively analysed the DNA methylation patterns of a number of consecutive CpGs in the core regions of DMRs and other regulatory regions of imprinted genes, since these are involved in placental and fetal growth and development., Methods: By using pyrosequencing, the DNA methylation at seven germline-derived primary DMRs was analysed quantitatively. Five of these are maternally methylated (MEST isoform α and β, PEG3, KCNQ1OT1 and SNRPN) and two are paternally methylated [H19 DMR and the intergenic region between DLK1 and MEG3 (IG-DMR)]. The post-fertilization-derived secondary DMRs, IGF2 (DMR0 and 2) and IG-DMR (CG7, also called MEG3 DMR), and the MEG3 promoter region were examined as well. In case of differential methylation between the two groups, the effect on gene expression was assessed by quantitative real-time PCR., Main Results and the Role of Chance: Both the promoter region of MEST isoform α and β and the 6th CTCF binding site within the H19 DMR were significantly hypomethylated in the IVF/ICSI group. The phenomenon was consistently observed over all CpG sites analysed and not restricted to single CpG sites. The other primary and secondary DMRs were not affected. Expression of H19 was increased in the IVF/ICSI group, while that of IGF2 and MEST remained similar., Limitations, Reasons for Caution: In the IVF/ICSI group, mostly ICSI pregnancies were investigated. The ICSI technique or male subfertility could be a confounding factor. Therefore, our results are less generalizable to IVF pregnancies., Wider Implications of the Findings: The clinical effects of the observed placental hypomethylations on the developmental programming of the IVF/ICSI progeny, if any, are as yet unknown. Whether the hypomethylation is an adaptation of the placenta to maintain fetal supply and ameliorate the effects of environmental cues, or whether it is a deregulation leading to deranged developmental programming with or without increased vulnerability for disease, consistent with the developmental origins of health and disease hypothesis, needs further investigation., Study Funding/competing Interest(s): Partly funded by an unrestricted research grant by Organon BV (now MSD BV) without any role in study design, data collection and analysis, or preparation of the manuscript. No conflict of interests to declare., Trial Registration Number: Dutch Trial Registry (NTR) number 1298.

Background: Knowledge of patients' preferences for elective single embryo transfer (eSET) or double embryo transfer (DET) and for singletons or twins is of great importance in counselling for embryo transfer (ET) strategies. In this study, the stability of IVF patients' preferences over time for either a healthy single child or healthy twins was measured and we investigated which factors could explain preference shifts., Methods: Infertile women (n = 177) who participated in an RCT comparing one cycle eSET with one cycle DET were included. A satisfaction questionnaire was developed to measure patient preferences and attitudes at two moments in time, i.e. at 2 weeks before ET and at 2 weeks following ET, after the results of the pregnancy test. Regression analysis examined the effect of several variables on preference shifts., Results: Before ET, most patients expressed a preference for a singleton, whereas most patients were indifferent 2 weeks after ET, resulting in an overall preference shift towards twins (P = 0.002; n = 145). Overall, 62% of patients showed a preference shift. Preference shifts were explained by patients' global satisfaction of the information given by the fertility clinic staff received by the fertility clinic staff, and an interaction between the occurrence of pregnancy and transfer policy (eSET or DET)., Conclusions: In general, patients' preferences for a singleton or twins are not stable during IVF treatment. Possible explanations of a shift in preference are that pregnant patients attuned their preferences to what they expect their pregnancy to result in, whereas non-pregnant patients shifted towards a preference for twins in order to be able to fulfil their ultimate child wish.

Background: The placenta is of utmost importance for intrauterine fetal development and growth. Deregulation of placentation can lead to adverse outcomes for both mother and fetus, e.g. gestational trophoblastic disease (GTD), pre-eclampsia and fetal growth retardation. A significant factor in placental development and function is epigenetic regulation., Methods: This review summarizes the current knowledge in the field of epigenetics in relation to placental development and function. Relevant studies were identified by searching PubMed, Medline and reference sections of all relevant studies and reviews., Results: Epigenetic regulation of the placenta evolves during preimplantation development and further gestation. Epigenetic marks, like DNA methylation, histone modifications and non-coding RNAs, affect gene expression patterns. These expression patterns, including the important parent-of-origin-dependent gene expression resulting from genomic imprinting, play a pivotal role in proper fetal and placental development. Disturbed placental epigenetics has been demonstrated in cases of intrauterine growth retardation and small for gestational age, and also appears to be involved in the pathogenesis of pre-eclampsia and GTD. Several environmental effects have been investigated so far, e.g. ethanol, oxygen tension as well as the effect of several aspects of assisted reproduction technologies on placental epigenetics., Conclusions: Studies in both animals and humans have made it increasingly clear that proper epigenetic regulation of both imprinted and non-imprinted genes is important in placental development. Its disturbance, which can be caused by various environmental factors, can lead to abnormal placental development and function with possible consequences for maternal morbidity, fetal development and disease susceptibility in later life.

Objective: To compare the effectiveness of elective single embryo transfer versus double embryo transfer on the outcomes of live birth, multiple live birth, miscarriage, preterm birth, term singleton birth, and low birth weight after fresh embryo transfer, and on the outcomes of cumulative live birth and multiple live birth after fresh and frozen embryo transfers., Design: One stage meta-analysis of individual patient data., Data Sources: A systematic review of English and non-English articles from Medline, Embase, and the Cochrane Central Register of Controlled Trials (up to 2008). Additional studies were identified by contact with clinical experts and searches of bibliographies of all relevant primary articles. Search terms included embryo transfer, randomised controlled trial, controlled clinical trial, single embryo transfer, and double embryo transfer. Review methods Comparisons of the clinical effectiveness of cleavage stage (day 2 or 3) elective single versus double embryo transfer after fresh or frozen in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI) treatments were included. Trials were included if the intervention differed only in terms of the intended number of embryos to be transferred. Trials that involved only blastocyst (day five) transfers were excluded., Results: Individual patient data were received for every patient recruited to all eight eligible trials (n=1367). A total of 683 and 684 women randomised to the single and double embryo transfer arms, respectively, were included in the analysis. Baseline characteristics in the two groups were comparable. The overall live birth rate in a fresh IVF cycle was lower after single (181/683, 27%) than double embryo transfer (285/683, 42%) (adjusted odds ratio 0.50, 95% confidence interval 0.39 to 0.63), as was the multiple birth rate (3/181 (2%) v 84/285 (29%)) (0.04, 0.01 to 0.12). An additional frozen single embryo transfer, however, resulted in a cumulative live birth rate not significantly lower than the rate after one fresh double embryo transfer (132/350 (38%) v 149/353 (42%) (0.85, 0.62 to 1.15), with a minimal cumulative risk of multiple birth (1/132 (1%) v 47/149 (32%)). The odds of a term singleton birth (that is, over 37 weeks) after elective single embryo transfer was almost five times higher than the odds after double embryo transfer (4.93, 2.98 to 8.18)., Conclusions: Elective single embryo transfer results in a higher chance of delivering a term singleton live birth compared with double embryo transfer. Although this strategy yields a lower pregnancy rate than a double embryo transfer in a fresh IVF cycle, this difference is almost completely overcome by an additional frozen single embryo transfer cycle. The multiple pregnancy rate after elective single embryo transfer is comparable with that observed in spontaneous pregnancies.

Background: Pregnancies induced by in vitro fertilisation (IVF) often result in twin gestations, which are associated with both maternal and perinatal complications. An effective way to reduce the number of IVF twin pregnancies is to decrease the number of embryos transferred from two to one. The interpretation of current studies is limited because they used live birth as outcome measure and because they applied limited time horizons. So far, research on long-term outcomes of IVF twins and singletons is scarce and inconclusive. The objective of this study is to investigate the short (1-year) and long-term (5 and 18-year) costs and health outcomes of IVF singleton and twin children and to consider these in estimating the cost-effectiveness of single embryo transfer compared with double embryo transfer, from a societal and a healthcare perspective., Methods/design: A multi-centre cohort study will be performed, in which IVF singletons and IVF twin children born between 2003 and 2005 of whom parents received IVF treatment in one of the five participating Dutch IVF centres, will be compared. Data collection will focus on children at risk of health problems and children in whom health problems actually occurred. First year of life data will be collected in approximately 1,278 children (619 singletons and 659 twin children). Data up to the fifth year of life will be collected in approximately 488 children (200 singletons and 288 twin children). Outcome measures are health status, health-related quality of life and costs. Data will be obtained from hospital information systems, a parent questionnaire and existing registries. Furthermore, a prognostic model will be developed that reflects the short and long-term costs and health outcomes of IVF singleton and twin children. This model will be linked to a Markov model of the short-term cost-effectiveness of single embryo transfer strategies versus double embryo transfer strategies to enable the calculation of the long-term cost-effectiveness., Discussion: This is, to our knowledge, the first study that investigates the long-term costs and health outcomes of IVF singleton and twin children and the long-term cost-effectiveness of single embryo transfer strategies versus double embryo transfer strategies.

Objective: To determine the incidence of couples dropping out of the in vitro fertilization (IVF) waiting list and to describe the couples' reasons., Design: Prospective cohort study., Setting: Fertility center in an academic hospital., Patient(s): 674 women placed consecutively on the IVF waiting list between June 2000 and July 2003., Intervention(s): None., Main Outcome Measure(s): Number of dropouts and reasons for dropping out., Result(s): Follow-up information was collected in 2005 and 2008. Of the 674 couples on the waiting list, 87% started IVF, and 13% dropped out before starting their first IVF cycle. Follow-up data were obtained for 85 of 86 patients (98.8%): 37% dropped out because of spontaneous pregnancy, 36% for personal reasons (passive censoring), and 27% for medical reasons (active censoring). Most of the pregnancies occurred within 3 months after the patient had been placed on the waiting list (30 of 32, 94%). Of the 54 censored couples, four became pregnant., Conclusion(s): On a 6-month waiting list for IVF, 13% of the couples dropped out before starting treatment. The single most important reason for dropout was (spontaneous) pregnancy. Most of these pregnancies occurred within 3 months, which suggests that psychological factors such as stress relief after being placed on the waiting list might be operative., (Copyright © 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Background: 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., Methods: 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., Results: 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., Conclusions: In vitro culture of human embryos can affect birthweight of live born singletons.

In vitro fertilisation (IVF) usually involves controlled ovarian stimulation (COS). There is now increasing emphasis on methods that make IVF safer and more patient-friendly. Modified natural cycle (MNC)-IVF is an example of this. In MNC-IVF spontaneous ovulation is prevented with a minimal amount of hormones and spontaneous monofollicular growth is supported. As a result, there is no risk of ovarian hyperstimulation syndrome, and the risk of a multiple pregnancy is low. There is a 9.1% chance of a pregnancy after one MNC-cycle and the cumulative pregnancy rate after a maximum of 6 MNC-IVF cycles is 33.4%. The cumulative results of a maximum of 6 MNC-IVF cycles are comparable to those of the first COS-IVF treatment cycle including transfer of cryopreserved embryos produced as a result of the treatment (33.4% versus 37.7%). The risk of a twin pregnancy following MNC-IVF is 0.1%, and 18.3% following COS-IVF. This means that MNC-IVF is a good alternative for COS-IVF.

Background: A selective switch to elective single embryo transfer (eSET) in IVF has been suggested to prevent complications of fertility treatment for both mother and infants. We compared seven IVF strategies concerning their cost-effectiveness using a Markov model., Methods: The model was based on a three IVF-attempts time horizon and a societal perspective using real world strategies and data, comparing seven IVF strategies, concerning costs, live births and incremental cost-effectiveness ratios (ICERs)., Results: In order to increase pregnancy probability, one cycle of eSET + one cycle of standard treatment policy [STP, i.e. eSET in patients <38 years of age with at least one good quality embryo and double embryo transfer (DET) in the remainder of patients] + one cycle of DET have an ICER of 16,593 euro compared with three cycles of eSET. Furthermore, three STP cycles have an ICER of 17,636 euro compared with one cycle of eSET + one cycle of STP + one cycle of DET, and three DET cycles have an ICER of 26,729 euro compared with three cycles STP., Conclusions: Our study shows that in patients qualifying for IVF treatment, combining several transfer policies was not cost-effective. A choice has to be made between three cycles of eSET, STP or DET. It depends, however, on society's willingness to pay which strategy is to be preferred from a cost-effectiveness point of view.

Background: Dropouts in IVF-programmes affect cumulative pregnancy rates (CPRs), but it is unknown what the impact of loss to follow-up is., Methods: Data were obtained from 588 couples starting IVF treatment ('as treated group'). Cycle-based and real-time-based CPRs were calculated using three assumptions for dropouts: dropouts having no probability of pregnancy, dropouts having the same probability of pregnancy as those continuing treatment and dropouts stopping because of medical reasons having no chance of pregnancy and those stopping because of other reasons having the same probability of pregnancy as those continuing treatment. CPRs obtained in the 'as treated group' were compared with CPRs calculated using the data set including the follow-up data of the dropouts ('completed group')., Results: In 1.7% of couples, no follow-up could be obtained. The cycle-based CPR after three IVF-cycles ranged from 63% to 71% in the 'as treated group' and was 65% in the 'completed group'. The real-time-based CPR after 9 months ranged from 54% to 59% in the 'as treated group' and was 55% in the 'completed group'. The PR in dropouts was 14% (95% confidence interval 8.22%)., Conclusions: In IVF programmes, outcome data of dropouts remain unknown, and CPRs should be calculated by assuming dropouts to have a PR between no probability and the same probability as those who continue treatment. Our study shows that the most accurate estimate for the PR in dropouts is 14%.

Besides the established selection criteria based on embryo morphology and blastomere number, new parameters for embryo viability are needed to improve the clinical outcome of IVF and more particular of elective single-embryo transfer. Genome-wide gene expression in cumulus cells was studied, since these cells surround the oocyte inside the follicle and therefore possibly reflect oocyte developmental potential. Early cleavage (EC) was chosen as a parameter for embryo viability. Gene expression in cumulus cells from eight oocytes resulting in an EC embryo (EC-CC; n = 8) and from eight oocytes resulting in a non-EC (NEC) embryo (NEC-CC; n = 8) was analysed using microarrays (n = 16). A total of 611 genes were differentially expressed (P < 0.01), mainly involved in cell cycle, angiogenesis, apoptosis, epidermal growth factor, fibroblast growth factor and platelet-derived growth factor signalling, general vesicle transport and chemokine and cytokine signalling. Of the 25 selected differentially expressed genes analysed by quantitative real-time PCR 15 (60%) genes could be validated in the original samples. Of these 8 (53%) could also be validated in 24 (12-EC-CC and 12 NEC-CC) extra independent samples. The most differentially expressed genes among these were CCND2, CXCR4, GPX3, CTNND1 DHCR7, DVL3, HSPB1 and TRIM28, which probably point to hypoxic conditions or a delayed oocyte maturation in NEC-CC samples. This opens up perspectives for new molecular embryo or oocyte selection parameters which might also be useful in countries where the selection has to be made at the oocyte stage before fertilization instead of at the embryonic stage.

Introduction: In several clinics, elective single-embryo transfer (eSET) is applied in a selected group of patients based on age and the availability of a good-quality embryo. Whether or not eSET can be applied irrespective of the presence of a good-quality embryo in the first cycle, to further reduce the twin pregnancy rate, remains to be elucidated., Methods: In patients <38 years two transfer strategies were compared, which differed in the first cycle only: group A (n = 141) received eSET irrespective of the availability of a good-quality embryo, and group B (n = 174) received eSET when a good-quality embryo was available while otherwise they received double embryo transfer (DET; referred to as eSET/DET transfer policy). In any subsequent cycle, in both groups the eSET/DET transfer policy was applied., Results: After completion of their IVF treatment (including a maximum of three fresh cycles and the transfer of frozen-thawed embryos), comparable cumulative live birth rates (62.4% in group A and 62.6% in group B) and twin pregnancy rates (10.1 versus 13.4%) were found. However, patients in group A required significantly more fresh (2.0 versus 1.8) and frozen (0.8 versus 0.5) cycles., Conclusions: The transfer of one embryo in the first cycle, irrespective of the availability of a good-quality embryo, in all patients <38 years, is not an effective transfer policy for reducing the overall twin pregnancy rate.

Multiple pregnancies lead to complications and induce high costs. The most successful way to decrease multiple pregnancies in IVF is to transfer only one embryo, which might reduce the efficacy of treatment. The objective of this review is to determine which embryo-transfer policy is most cost-effective: elective single-embryo transfer (eSET) or double-embryo transfer (DET). Several databases were searched for (cost* or econ*) and (single embryo* or double embryo* or one embryo* or two embryo* or elect* embryo or multip* embryo*). On the basis of five exclusion criteria, titles and abstracts were screened by two individual reviewers. The remaining papers were read for further selection, and data were extracted from the selected studies. A total of 496 titles were identified through the searches and resulted in the selection of one observational study and three randomized studies. Study characteristics, total costs and probability of live births were extracted. Besides this, cost-effectiveness and incremental cost-effectiveness were derived. It can be concluded that DET is the most expensive strategy. DET is also most effective if performed in one fresh cycle. eSET is only preferred from a cost-effectiveness point of view when performed in good prognosis patients and when frozen/thawed cycles are included. If frozen/thawed cycles are excluded, the choice between eSET and DET depends on how much society is willing to pay for one extra successful pregnancy.

Background: Twin pregnancies after IVF are still frequent and are considered high-risk pregnancies leading to high costs. Transferring one embryo can reduce the twin pregnancy rate. We compared cost-effectiveness of one fresh cycle elective single embryo transfer (eSET) versus one fresh cycle double embryo transfer (DET) in an unselected patient population., Methods: Patients starting their first IVF cycle were randomized between eSET and DET. Societal costs per couple were determined empirically, from hormonal stimulation up to 42 weeks after embryo transfer. An incremental cost-effectiveness ratio (ICER) was calculated, representing additional costs per successful pregnancy., Results: Successful pregnancy rates were 20.8% for eSET and 39.6% for DET. Societal costs per couple were significantly lower after eSET (7334 euro) compared with DET (10,924 euro). The ICER of DET compared with eSET was 19,096 euro, meaning that each additional successful pregnancy in the DET group will cost 19,096 euro extra., Conclusions: One cycle eSET was less expensive, but also less effective compared to one cycle DET. It depends on the society's willingness to pay for one extra successful pregnancy, whether one cycle DET is preferred from a cost-effectiveness point of view.

Background: Controlled ovarian stimulation (COS) with intrauterine insemination (IUI) is a common treatment in couples with unexplained non-conception. Induction of multifollicular growth is considered to improve pregnancy outcome, but it contains an increased risk of multiple pregnancies and ovarian hyperstimulation syndrome. In this study the impact of the number of follicles (>14 mm) on the ongoing pregnancy rate (PR) and multiple PR was evaluated in the first four treatment cycles., Methods: A retrospective cohort study was performed in all couples with unexplained non-conception undergoing COS-IUI in the Academic Hospital of Maastricht. The main outcome measure was ongoing PR. Secondary outcomes were ongoing multiple PR, number of follicles of >or=14 mm, and order of treatment cycle., Results: Three hundred couples were included. No significant difference was found in ongoing PR between women with one, two, three or four follicles respectively (P=0.54), but in women with two or more follicles 12/73 pregnancies were multiples. Ongoing PR was highest in the first treatment cycle and declined significantly with increasing cycle order (P=0.006), while multiple PR did not change., Conclusions: In COS-IUI for unexplained non-conception, induction of more than one follicle did not improve the ongoing PR, but increased the risk of multiple pregnancies. Multiple PR remained high in the first four cycles with multifollicular stimulation. Therefore, in order to reduce the number of multiple pregnancies, in all IUI cycles for unexplained non-conception monofollicular growth should be aimed at.

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: 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.

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: To reduce the twin pregnancy rate, elective single embryo transfer (eSET) is increasingly implemented. Improvement of the results obtained with eSET can be achieved by better selection of the most viable embryo. This study investigated the predictive value of early cleavage (EC) as an additional parameter for selecting the embryo with the highest implantation potential by using data from SET's., Methods: Data from 165 SET's were retrospectively evaluated. Cleavage to the 2-cell stage was determined 23-26 h after injection or 25-28 h after insemination. Selection of the embryo to be transferred was based on cell morphology and cell number on the day of transfer, not on the EC status. Additional information on the predictive value of EC on developmental potential was obtained by analysing 253 transfers with two embryos (double embryo transfer, DET) and blastocyst formation of 1160 surplus embryos. Logistic regression was used to determine the predictors of pregnancy or blastocyst development., Results: A significantly higher pregnancy rate was observed after transfer of single EC embryos compared to single non-EC embryos (46 versus 18%). This result was confirmed by the significantly higher pregnancy rate after DET with two EC embryos as compared to DET with two non-EC embryos (45 versus 25%) and the blastocyst formation of EC embryos compared to non-EC embryos (66 versus 40%). Logistic regression showed that EC is an independent predictor for both pregnancy and blastocyst development in addition to cell morphology and cell number., Conclusions: In order to improve the selection of the embryo with the highest implantation potential, selection for transfer should not be based on cell number and morphology on the day of transfer alone, but also on early cleavage status.

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.

Spinocerebellar ataxia 3 (SCA3) is an autosomal dominant neurodegenerative disorder characterized by variable expression and a variable age of onset. SCA3/MJD (Machado-Joseph disease) is caused by an expansion of a (CAG)(n) repeat in the MJD1 gene on chromosome 14q32.1. A single cell PCR protocol has been developed for preimplantation genetic diagnosis (PGD) of SCA3 to select unaffected embryos on the basis of the CAG genotype. Single leukocytes and blastomeres served as a single cell amplification test system to determine the percentage of allelic drop-out (ADO) and PCR efficiency. Out of 105 tested heterozygous single leukocytes, 103 (98.1%) showed a positive amplification signal, while five cells (4.9%) showed ADO. Amplification in single blastomeres was obtained in 13 out of a total of 14, and ADO was observed in two out of the 13 single blastomeres. PGD of SCA3 was performed in a couple with paternal transmission of the SCA3 allele. Seven embryos were available for biopsy, all biopsied blastomeres showed amplification and no ADO occurred. One embryo was diagnosed as affected whereas six embryos were diagnosed as unaffected. Two unaffected embryos were transferred and resulted in a singleton pregnancy and the birth of a healthy girl.

Background: Although well defined for embryos at cleavage stages, the occurrence and frequency of chromosomal aberrations in human blastocysts is relatively unknown. It has been reported that only one in four blastocysts is comprised totally of chromosomally normal cells. One of the selection mechanisms for the embryo proper to become free of these chromosomally abnormal cells would be to sequester them to the extra-embryonic compartment during development. The study aim was to investigate whether such a mechanism of selection exists in human preimplantation embryos., Methods: Inner cell mass (ICM)/trophectoderm (TE) differentiation was performed, followed by fluorescence in-situ hybridization (FISH), to study the chromosomal distribution in both populations of cells., Results: Of the 94 successfully analysed blastocysts, 68.8 +/- 1.5% of all analysable nuclei per blastocyst showed a disomic chromosomal content. Only 22.6% of blastocysts analysed were classified as normal. Of the embryos classified as abnormal at the blastocyst stage, 11.9% showed a simple mosaic pattern and 32.1% a complex mosaic pattern. An equally large group of blastocysts showed either a chaotic pattern (16.7%), or the chromosomal pattern could not be classified. The average degree of normal cells in the ICM (67.9%) was similar to the degree observed in the TE (69.5%)., Conclusions: These findings indicate that chromosomally abnormal cells are not preferentially segregating to the extra-embryonic compartment of the human preimplantation embryo at the blastocyst stage. Hence, other mechanisms should be responsible for an absence of chromosomally abnormal cells in the embryo proper at later stages of development. One possible mechanism might be the elimination of the chromosomally abnormal cells by selective cell death activation.

Background: Various studies have been performed in which potential effects of xenoestrogens on fertility or sperm parameters were investigated by comparing groups of subjects exposed to different levels of these chemicals., Methods: In our study we used an alternative approach, as we selected one group of men with very poor semen quality and another group with normal semen quality and determined the blood organochlorine contents in order to determine whether a difference in these levels could be established. Organochlorine compounds, including polychlorinated biphenyls (PCB) and PCB metabolites, were detected using gas chromatography. The concentrations were compared between both groups, and related to semen parameters., Results: A comparison of both groups did not reveal significant differences in organochlorine levels. Linear relationships were found when PCB and metabolite concentrations were related to the age of the volunteers. Focusing on the subgroup of men with normal semen quality showed that sperm count and sperm progressive motility were inversely related to the concentrations of PCB metabolites within this group., Conclusions: The finding of a significantly decreased sperm count in relation to an elevated PCB metabolite level within the subgroup of men with normal semen quality is important. This is the first time that a correlation between exposure to environmental pollutants with endocrine-disrupting capacity and human sperm quality has been observed.

Cystic fibrosis (CF) is the first monogenic disorder for which single cell preimplantation genetic diagnosis (PGD) has been successfully applied. The spectrum of mutations in CF is extremely heterogeneous, and hence, the development of mutation-specific PGD protocols is impracticable. The current study reports the development and evaluation of a general multiplex marker polymerase chain reaction (PCR) protocol for PGD of CF. Four closely linked highly polymorphic (CA)(n) repeat markers D7S523, D7S486, D7S480 and D7S490, flanking the cystic fibrosis transmembrane regulator (CFTR) gene, were used. In 99% of the single cells tested (100 leukocytes and 50 blastomeres), multiplex PCR results were obtained and the overall allelic drop out (ADO) rate varied from 2 to 5%. After validation for the presence of ADO and additional alleles, 95% of the multiplex PCR results were accepted to construct the marker genotypes. Depending on the genotype of the couple, and taking into account the embryos lost for transfer due to validation criteria (5%), ADO (0-2%) and single recombination (1.1-3%), in general >90% of the embryos could be reliably genotyped by PGD using a single blastomere. The risk of misdiagnosis equals the chance of a double recombination between informative flanking markers and is <0.05%. Therefore, this polymorphic and multi-allelic marker system is a reliable and generally applicable alternative for mutation-directed PGD protocols. Furthermore, it provides a test for the origin of the detected genotype and also gives an indication of the chromosomal ploidy status of the blastomere tested.

Preimplantation genetic diagnosis (PGD) was performed in two couples to avoid chromosomally unbalanced progeny in a family in which a brother and a sister carry an identical maternally inherited balanced translocation t(3;11)(q27.3;q24.3). Embryos were biopsied 3 days after fertilization and blastomeres were analysed by fluorescent in-situ hybridization (FISH). Embryos were classified as unbalanced or normal/balanced. In the first case, the male carrier and his wife underwent one IVF/PGD treatment cycle. In all, 18 embryos were analysed. Of those, 15 revealed an unbalanced karyotype. For one embryo, results were not conclusive, from one embryo results were contradictory and one embryo was classified as normal/balanced and subsequently transferred. A singleton pregnancy was achieved. The PGD analysis was confirmed at 16 weeks gestation by amniocentesis. At term, a healthy girl with a balanced karyotype was born. Pregnancy and delivery were without complications. In the second case, the female carrier and her husband underwent two IVF/PGD treatment cycles. During the first cycle, three embryos were analysed. One embryo revealed an unbalanced karyotype and two embryos were designated a normal/balanced karyotype and transferred but no pregnancy was achieved. During the second PGD cycle three embryos were analysed. Of those, none appeared suitable for transfer. The couple decided not to undergo further treatment. Our results indicate that for individuals carrying a reciprocal translocation PGD is a feasible approach to obtain embryos with a normal chromosome balance and to avoid both spontaneous and induced abortion.

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.

In this prospective randomized study on 1380 consecutive in-vitro fertilization (IVF) treatments, the results were compared of culture of human oocytes and embryos for the first 2 or 3 days of development in microdroplets of medium under oil using a gas phase containing either atmospheric (approximately 20%) or reduced (5%) O2 concentrations. No significant differences were found between the two groups cultured under either 5% or 20% O2 in rates of fertilization (60 versus 61%, respectively), embryo development at day 2 or 3, pregnancy (26.6 versus 25.4%, respectively), and implantation (13.4 versus 14.0%, respectively). Culture of surplus embryos under 5% O2 resulted in a significantly higher mean incidence of blastocyst formation per cycle as compared to the 20% O2 group (25.8 +/- 2.0 versus 20.4 +/- 1.9, respectively). The mean number of cells of embryos classified as blastocysts by microscopic observation of a blastocoel was significantly higher in the 5% O2 group as compared to the 20% O2 group, both in blastocysts fixed on day 5 (39.8 +/- 1.7 versus 31.9 +/- 1.9, respectively), as well as those fixed on day 6 (45.6 +/- 2.6 versus 33.7 +/- 3.4, respectively). This difference was due to the fact that significantly more blastocysts of the 20% O2 group had an abnormal low cell number of < 25 as compared to the 5% O2 group, both in blastocysts fixed on day 5 (39 versus 22%, respectively), as well as those fixed on day 6 (43 versus 22%, respectively). To conclude, although culture under 5% O2 leads to slightly improved preimplantation embryonic viability, this effect is either too marginal to result in higher pregnancy rates, or low O2 concentrations exert an effect during the later stages of preimplantation development only.

Objective: To study the fertility potential of individual semen donors with ejaculates of optimal as compared to suboptimal quality., Methods: 363 semen donations were obtained from 11 donors between January 1993 and September 1997. 270 samples were cryopreserved and 1,399 straws obtained from 120 ejaculates were used in 495 insemination cycles., Results: 52 pregnancies were achieved in 128 recipients (40.6%). No significant differences were found between donors of high fecundability and those of low fecundability regarding sperm parameters. The mean donor fecundability index was 10.5%. The optimal and suboptimal samples yielded similar results. Donors with suboptimal semen quality had an increased number of samples rejected after thawing., Conclusion: Traditional semen analysis parameters do not differentiate high from low fecundability donors. Suboptimal results of the semen analysis should not exclude potential donors from an artificial insemination donor (AID) program.

In this study, the use of Ca2+/Mg2+-free medium for biopsy of human embryos at the 4- to 10-cell stage on the third day of development was evaluated. When compared with control medium containing normal concentrations of Ca2+ and Mg2+ ions, the use of Ca2+/Mg2+-free medium allows an easier removal of blastomeres as illustrated by a lower rate of cell lysis as well as by a shorter time needed to perform the procedure. Subsequent embryo development to the blastocyst stage is not affected by the choice of biopsy medium, not even when embryos are exposed to the medium for 45 min. The use of Ca2+/Mg2+-free medium thus allows for an easier biopsy procedure during pre-implantation genetic diagnosis, while it does not result in a loss of developmental potential of the embryo to the blastocyst stage.

After Duchenne muscular dystrophy, spinal muscular atrophy (SMA) is the most common severe neuromuscular disease in childhood. Since 1995, homozygous deletions in exon 7 of the survival motor neuron (SMN) gene have been described in >90-95% of SMA patients. However, the presence of a highly homologous SMN copy gene complicates the detection of exon 7 deletions. This paper describes the adjustment and evaluation of an established SMN exon 7 polymerase chain reaction (PCR) protocol at the single cell level, and the first preimplantation genetic diagnosis (PGD) of SMA with this PCR protocol. To determine PCR efficiency and allelic loss, 200 leukocytes of normal individuals, SMA carriers and patients, and 25 blastomeres were tested. The PCR efficiency of the SMN exon 7 and the adjacent copy gene sequence, tested in the leukocytes, were 90% and 91% respectively. No allelic loss was detected. One out of 25 blastomeres tested revealed a negative PCR signal for the SMN exon 7 sequence. All 25 showed the copy gene sequence. PGD of SMA was offered to a couple with an affected child homozygous for the SMN exon 7 deletion. After intracytoplasmic sperm injection, four and five embryos could be genotyped for the SMN exon 7 in two cycles respectively. After embryo transfer in the second PGD cycle an ongoing gemelli pregnancy was achieved. This study demonstrates that PGD for SMA is feasible when a previous child is homozygous for the SMN exon 7 deletion.

Objective: To evaluate whether the predictive ability of a normal FSH level on cycle day 3 can be enhanced by levels of estradiol-17beta (E2) on cycle day 3., Design: Prospective cohort study., Setting: University hospital-based, tertiary care infertility center., Patient(s): Two hundred thirty-one consecutively seen patients who attended the center for their first IVF attempt., Intervention(s): Blood samples were collected on day 3 of the cycle preceding IVF; IVF was performed in all patients., Main Outcome Measure(s): Patient's age, number of ampules of hMG, cancellation rate, number of oocytes, fertilization rate, and clinical pregnancy rate., Result(s): In patients with elevated FSH levels on cycle day 3, a low oocyte yield was achieved (7 versus 11) and a high number of ampules of hMG was necessary (56 versus 33). Their cancellation rate was high (67% versus 16%). In patients with normal basal FSH levels, high E2 levels predicted a high cancellation rate (56%, versus 13% in patients with low E2 levels) and a low oocyte yield (9, versus 11 in patients with low E2 levels). Patients with both normal FSH levels and low E2 levels on cycle day 3 fared best., Conclusion(s): The basal E2 level on cycle day 3 is a useful prognosticator of response to stimulation in IVF patients with normal basal FSH levels.

A long waiting list for in-vitro fertilization (IVF) offers the possibility to study treatment-independent pregnancy rates in patients with severe reproductive disorders. We performed a retrospective cohort study with a nested case-control design in which the cases achieved a spontaneous pregnancy while on the waiting list for IVF, or for IVF with intracytoplasmic sperm injection (ICSI), and the controls did not become pregnant while on the waiting list. Spontaneous pregnancies occurred in 76 of 1391 patients on the waiting list. Significant differences between pregnant and non-pregnant patients were found for duration of subfertility (couples on the IVF waiting list), and for progressive sperm motility and basal 17beta-oestradiol (couples on the ICSI waiting list). The 12 months cumulative pregnancy rate for patients on the waiting list was 2.4% (95% CI 1.2-3.9%) for tubal subfertility patients, 5.9 % (3.7-8.7%) for longstanding unexplained subfertility patients, and 6.6% (4.5-9.3%) for male subfertility patients. Of the 76 control patients, 21% of tubal subfertility patients, 18% of unexplained subfertility patients, and 17% of male subfertility patients achieved a pregnancy in their first IVF or ICSI treatment cycle. We confirm that the treatment-independent pregnancy rate in patients with severe reproductive disorders is low. More than 75% of the spontaneous pregnancies in the tubal subfertility and unexplained subfertility couples occurred during their first three months on the waiting list, whereas spontaneous pregnancy rate in male subfertility couples showed a more gradual but persisting increase. We conclude that one cycle of IVF or ICSI is superior to 12 months of expectant management in patients with severely impaired fertility due to tubal, unexplained or male factors.

Taurine transport in mouse embryos has been shown to be osmotically regulated. We studied release of taurine from mouse and human oocytes and embryos when exposed to conditions that created osmotic imbalances, either by incubation in anisosmotic media or by inhibition of Na(+)-K(+)-ATPase with ouabain. Furthermore, we studied the effect of cleavage in mouse embryos on release of taurine. When human oocytes that remained unfertilized after in vitro fertilization, human embryos (2- to 8-cell), and mouse 2-cell embryos were loaded with [3H]taurine and subsequently incubated for 4 h in hyposmotic media (200 and 240 mOsm/kg), they showed significantly lower radioactivity as compared to those incubated in media of 280, 320, and 360 mOsm/kg and higher radioactivity of the incubation media. Incubation with 1.5 mM ouabain resulted in decreased radioactivity of mouse embryos and increased radioactivity of incubation medium. When mouse 2-cell embryos were cultured for 24 h after loading with [3H]taurine, radioactivity of embryos that cleaved to the 4-cell stage was significantly lower than that of uncleaved embryos. This finding is in accordance with the theory that cell division induces cell volume-regulatory mechanisms. In contrast, when 1-cell embryos were cultured for 24 h, radioactivity of embryos developing to the 2-cell stage was significantly higher than that of uncleaved embryos. These data support the theory that taurine is released by embryos when they have to adjust their cell volume because of either extracellularly induced or intracellularly occurring osmotic imbalances. When culture is performed without taurine, the resultant taurine depletion of embryos may be disadvantageous, either because the embryo has to rely more on its inorganic osmolytes for volume regulation or because taurine can no longer provide its other protective functions.

Objective: To study the outcome in poor responders to three ampules (225 IU) of hMG per day in subsequent IVF treatment cycles in which six ampules (450 IU)of hMG per day were administered., Design: Retrospective chart review., Setting: Academic tertiary center., Patients: Between January 1988 and May 1995, 126 poor response patients had a first treatment cycle on three ampules and a second cycle on six ampules of hMG per day., Main Outcome Measures: Numbers of follicles, oocytes, and embryos, and pregnancy rates., Results: On six ampules, patients had significantly more follicles and oocytes. The number of embryos did not differ significantly. The pregnancy rate on six ampules were low (3.2% pregnancies per cycle started)., Conclusion: Poor responders do not benefit from high-dose hMG stimulation; their reproduction outcome is poor.