Ammonium accumulation in commercially available embryo culture media and protein supplements during storage at 2-8°C and during incubation at 37°C.

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.

Reducing low-value care in endometriosis between limited evidence and unresolved issues: a proposal.

Quantification of benefits and harms of medical interventions should be based on high-quality evidence, which is not always the case in the endometriosis field. In many clinical circumstances, healthcare decisions in women with endometriosis are taken based on suboptimal evidence or on evidence of coexistence of benefits and harms that must be balanced. In these conditions, it is important to avoid or reduce the use of low-value care, i.e. interventions with defined harms and uncertain benefits, or whose effectiveness is comparable with less expensive alternatives. In particular, we suggest that: (i) non-surgical diagnosis based on symptoms, physical findings and transvaginal ultrasonography is possible in most women with symptomatic endometriosis. Thus, except in doubtful cases, laparoscopy should be intended for surgical treatment, not for diagnostic purposes: early diagnosis and diagnostic laparoscopy are not synonymous; (ii) future trials on new drugs for endometriosis should address those outcomes that are most important to patients, should be designed as superiority trials and should include a progestin or an estrogen-progestin as a comparator. Moreover, limitation of repetitive surgery for recurrent endometriosis is among the objectives of long-term medical treatment; (iii) indications for surgery should be the result of a balance between demonstrated benefits in terms of fertility enhancement and pain relief, specific risks associated with excision of different types of endometriotic lesions, cost-effectiveness and patient preference after detailed information; (iv) physicians, health professionals and policy makers should discriminate between screening for and diagnosis of endometriosis. Limited peritoneal foci, which are frequently observed also in asymptomatic women, regress or remain stable in about two thirds of cases. Therefore, the theoretical premises for a screening campaign are currently unclear; (v) physicians should develop the ability to effectively communicate quantitative information based on international guidelines and systematic literature reviews. This will assist a woman's understanding of the interaction between the evidence and her priorities, facilitating the transition towards value-based medicine.

Differences in gene expression profiles between human preimplantation embryos cultured in two different IVF culture media.

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.

Age of G-1 PLUS v5 embryo culture medium is inversely associated with birthweight of the newborn.

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 (ß = -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.

Altered gene expression in human placentas after IVF/ICSI.

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 ß 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.

IVF culture medium affects post-natal weight in humans during the first 2 years of life.

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.

A comparison of perinatal outcomes in singletons and multiples born after in vitro fertilization or intracytoplasmic sperm injection stratified for neonatal risk criteria.

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.

Lack of transparency of clinical trials on endometriosis.

OBJECTIVE: To estimate patterns of publication of clinical trials of endometriosis registered in ClinicalTrials.gov and their associated characteristics. METHODS: Information on interventional clinical trials on endometriosis that were registered at ClinicalTrials.gov and updated as having been completed by October 25, 2012, was retrieved and the publication status and time to publication in Medline-indexed journals were ascertained by searching PubMed and by sending e-mail inquiries to the principal investigators listed by the registry. RESULTS: Seventy-one interventional trials of endometriosis, testing various drugs and biologicals, were identified. Among them, 49.3% (35/71) were completed by October 25, 2012, 21.1% were either stopped or inactive in the past 2 years, and the remaining 29.6% were ongoing. Among the 35 completed trials, 25 (71.4%) were sponsored by industry and results were published for only 11 (31.4%; five industry-sponsored, and six nonindustry-sponsored). Trials sponsored by industry were nearly four times less likely to publish their results than nonindustry-sponsored trials, even though these trials typically had larger sample sizes and were completed faster. Compared with the publication rate of 20% found 4 years ago, the current rate has increased only marginally but still lies significantly below the reported 66.3% surveyed recently among 546 completed nonendometriosis trials registered at ClinicalTrials.gov (P<.001). CONCLUSION: Despite mounting pressure on more transparency of clinical trials, the current state of transparency or lack thereof of clinical trials on endometriosis is worrisome and does not benefit the trial sponsor or the public. Thus, we again call for more transparency for endometriosis trials.

IVF culture medium affects human intrauterine growth as early as the second trimester of pregnancy.

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 ß-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 ß-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.

Surgery or embolization for varicoceles in subfertile men

A varicocele is a meshwork of distended blood vessels in the scrotum, usually left-sided, due to dilatation of the spermatic vein. Although the concept that a varicocele causes male subfertility has been around for more than 50 years now, the mechanisms by which a varicocele would affect fertility have not yet been satisfactorily explained. Neither is there sufficient evidence to explain the mechanisms by which varicocelectomy would restore fertility. Furthermore, it has been questioned whether a causal relation exists at all between the distension of the pampiniform plexus (a network of many small veins found in the human male spermatic cord) and impairment of fertility. To evaluate the effect of varicocele treatment on live birth and pregnancy rate in subfertile couples where the male has a varicocele.

We searched the Cochrane Menstrual Disorders and Subfertility Group Trials Register (12 September 2003 to January 2012), the Cochrane Central Register of Controlled Trials (CENTRAL) in The Cochrane Library Issue 1, 2012), Medline (January 1966 to January 2012), Embase (January 1985 to January 2012), PsycINFO (to Week 1 2012) and reference lists of articles. In addition, we handsearched specialist journals in the field from their first issue until 2012. We also checked cross-references, references from review articles and contacted researchers in the field.

Randomized controlled trials (RCTs) were included if they were relevant to the clinical question posed. If they reported pregnancy rates or live birth rates as an outcome measure, and if they reported data in treated (surgical ligation or radiological embolization of the internal spermatic vein) compared ...

Placentas from pregnancies conceived by IVF/ICSI have a reduced DNA methylation level at the H19 and MEST differentially methylated regions.

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 ß, 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 ß 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.

Primary ovarian insufficiency in classic galactosemia: role of FSH dysfunction and timing of the lesion.

FSH inactivity due to secondary hypoglycosylation has been suggested as a potential mechanism for primary ovarian insufficiency in classic galactosemia. To investigate the role of FSH and to gain insight in the timing of the damage, ovarian stimulation tests were performed and data on ovarian imaging collected. Fifteen patients with primary ovarian insufficiency underwent ovarian stimulation with gonadotropins. Only one patient showed a normal increase in estradiol level, all the others had a low or no estradiol response. Anti-Müllerian hormone measurement in all girls and women showed levels below the detection limit of 0.10 µg/l. Ovarian volumes were evaluated by MRI in 14 patients and compared to age matched controls, prepubertal controls and postmenopausal controls. The ovarian volumes of the galactosemic girls were smaller than those of the age matched controls (p = 0.001) and the prepubertal ovaries (p = 0.008), and did not differ significantly from postmenopausal ovarian volumes (p = 0.161). In conclusion we found no evidence that FSH inactivity plays a role in primary ovarian insufficiency in classic galactosemia. Moreover, ovarian imaging results point to an early onset of ovarian failure in this disease.

Surgery or embolization for varicoceles in subfertile men.

BACKGROUND: A varicocele is a meshwork of distended blood vessels in the scrotum, usually left-sided, due to dilatation of the spermatic vein. Although the concept that a varicocele causes male subfertility has been around for more than 50 years now, the mechanisms by which a varicocele would affect fertility have not yet been satisfactorily explained. Neither is there sufficient evidence to explain the mechanisms by which varicocelectomy would restore fertility. Furthermore, it has been questioned whether a causal relation exists at all between the distension of the pampiniform plexus (a network of many small veins found in the human male spermatic cord) and impairment of fertility. OBJECTIVES: To evaluate the effect of varicocele treatment on live birth and pregnancy rate in subfertile couples where the male has a varicocele. SEARCH METHODS: We searched the Cochrane Menstrual Disorders and Subfertility Group Trials Register (12 September 2003 to January 2012), the Cochrane Central Register of Controlled Trials (CENTRAL) in The Cochrane Library Issue 1, 2012), MEDLINE (January 1966 to January 2012), EMBASE (January 1985 to January 2012), PsycINFO (to Week 1 2012) and reference lists of articles. In addition, we handsearched specialist journals in the field from their first issue until 2012. We also checked cross-references, references from review articles and contacted researchers in the field. SELECTION CRITERIA: Randomised controlled trials (RCTs) were included if they were relevant to the clinical question posed. If they reported pregnancy rates or live birth rates as an outcome measure, and if they reported data in treated (surgical ligation or radiological embolization of the internal spermatic vein) compared to untreated or placebo groups. Two authors independently screened potentially relevant trials. Any differences of opinion were resolved by consensus (none occurred for this review). DATA COLLECTION AND ANALYSIS: Ten studies met the inclusion criteria for the review. For one study we had only data from a published abstract. All ten studies only included men from couples with subfertility problems; one excluded men with sperm counts less than 5 million per mL and one excluded men with sperm counts less than 2 million per mL, with or without progressive motility of less than 10%. Two trials involving clinical varicoceles included some men with normal semen analysis. Three studies specifically addressed only men with subclinical varicoceles. Studies were excluded from meta-analysis if they made comparisons other than those specified above. MAIN RESULTS: The meta-analysis included 894 men. No studies reported live birth. The combined fixed-effect odds ratio (OR) of the 10 studies for the outcome of pregnancy was 1.47 (95% confidence interval (CI) 1.05 to 2.05, very low quality evidence), favouring the intervention. The number needed to treat for an additional beneficial outcome was 17, suggesting benefit of varicocele treatment over expectant management for pregnancy rate in subfertile couples in whom varicocele in the man was the only abnormal finding. Omission of the studies including men with normal semen analysis and subclinical varicocele, some of which had semen analysis improvement as the primary outcome rather than live birth or pregnancy rate, was the subject of a planned subgroup analysis. The outcome of the subgroup analysis (five studies) also favoured treatment, with a combined OR 2.39 (95% CI 1.56 to 3.66). The number needed to treat for an additional beneficial outcome was 7. The evidence was suggestive rather than conclusive, as the main analysis was subject to fairly high statistical heterogeneity (I(2) = 67%) and findings were no longer significant when a random-effects model was used or when analysis was restricted to higher quality studies. AUTHORS' CONCLUSIONS: There is evidence suggesting that treatment of a varicocele in men from couples with otherwise unexplained subfertility may improve a couple's chance of pregnancy. However, findings are inconclusive as the quality of the available evidence is very low and more research is needed with live birth or pregnancy rate as the primary outcome.

Towards endometriosis diagnosis by gadofosveset-trisodium enhanced magnetic resonance imaging.

Endometriosis is defined as the presence of endometrial tissue outside the uterus. It affects 10-15% of women during reproductive age and has a big personal and social impact due to chronic pelvic pain, subfertility, loss of work-hours and medical costs. Such conditions are exacerbated by the fact that the correct diagnosis is made as late as 8-11 years after symptom presentation. This is due to the lack of a reliable non-invasive diagnostic test and the fact that the reference diagnostic standard is laparoscopy (invasive, expensive and not without risks). High-molecular weight gadofosveset-trisodium is used as contrast agent in Magnetic Resonance Imaging (MRI). Since it extravasates from hyperpermeable vessels more easily than from mature blood vessels, this contrast agent detects angiogenesis efficiently. Endometriosis has high angiogenic activity. Therefore, we have tested the possibility to detect endometriosis non-invasively using Dynamic Contrast-Enhanced MRI (DCE-MRI) and gadofosveset-trisodium as a contrast agent in a mouse model. Endometriotic lesions were surgically induced in nine mice by autologous transplantation. Three weeks after lesion induction, mice were scanned by DCE-MRI. Dynamic image analysis showed that the rates of uptake (inwash), persistence and outwash of the contrast agent were different between endometriosis and control tissues (large blood vessels and back muscle). Due to the extensive angiogenesis in induced lesions, the contrast agent persisted longer in endometriotic than control tissues, thus enhancing the MRI signal intensity. DCE-MRI was repeated five weeks after lesion induction, and contrast enhancement was similar to that observed three weeks after endometriosis induction. The endothelial-cell marker CD31 and the pericyte marker α-smooth-muscle-actin (mature vessels) were detected with immunohistochemistry and confirmed that endometriotic lesions had significantly higher prevalence of new vessels (CD31 only positive) than the uterus and control tissues. The diagnostic value of gadofosveset-trisodium to detect endometriosis should be tested in human settings.

Protocol for translabial 3D-ultrasonography for diagnosing levator defects (TRUDIL): a multicentre cohort study for estimating the diagnostic accuracy of translabial 3D-ultrasonography of the pelvic floor as compared to MR imaging.

BACKGROUND: Pelvic organ prolapse (POP) is a condition affecting more than half of the women above age 40. The estimated lifetime risk of needing surgical management for POP is 11%. In patients undergoing POP surgery of the anterior vaginal wall, the re-operation rate is 30%. The recurrence risk is especially high in women with a levator ani defect. Such defect is present if there is a partially or completely detachment of the levator ani from the inferior ramus of the symphysis. Detecting levator ani defects is relevant for counseling, and probably also for treatment. Levator ani defects can be imaged with MRI and also with Translabial 3D ultrasonography of the pelvic floor. The primary aim of this study is to assess the diagnostic accuracy of translabial 3D ultrasonography for diagnosing levator defects in women with POP with Magnetic Resonance Imaging as the reference standard. Secondary goals of this study include quantification of the inter-observer agreement about levator ani defects and determining the association between levator defects and recurrent POP after anterior repair. In addition, the cost-effectiveness of adding translabial ultrasonography to the diagnostic work-up in patients with POP will be estimated in a decision analytic model. METHODS/DESIGN: A multicentre cohort study will be performed in nine Dutch hospitals. 140 consecutive women with a POPQ stage 2 or more anterior vaginal wall prolapse, who are indicated for anterior colporapphy will be included. Patients undergoing additional prolapse procedures will also be included. Prior to surgery, patients will undergo MR imaging and translabial 3D ultrasound examination of the pelvic floor. Patients will be asked to complete validated disease specific quality of life questionnaires before surgery and at six and twelve months after surgery. Pelvic examination will be performed at the same time points. Assuming a sensitivity and specificity of 90% of 3D ultrasound for diagnosing levator defects in a population of 120 women with POP, with a prior probability of levator ani defects of 40%, we will be able to estimate predictive values with good accuracy (i.e. confidence limits of at most 10% below or above the point estimates of positive and negative predictive values).Anticipating 3% unclassifiable diagnostic images because of technical reasons, and a further safety margin of 10% we plan to recruit 140 patients. TRIAL REGISTRATION: Nederlands trial register NTR2220.

Profiles and motives for PGD: a prospective cohort study of couples referred for PGD in the Netherlands.

BACKGROUND: PGD is nowadays a well-established alternative to prenatal diagnosis. However, information with respect to couples' motives and profiles for choosing PGD is scarce. METHODS: A prospective cohort of 264 couples referred for PGD was interviewed semi-structurally after intake, and follow-up data were collated after 6-8 years. Outcome measures were: the primary choice shortly after intake (PGD intention), and their definitive use, until maximum 8 years later (PGD use). Logistic regression analysis was performed with clinical impact of the genetic disorder, couples' experiences, obstetric history and psychosocial factors as putative predictors. RESULTS: About 53.4% of the couples showed PGD intention. The experience of one or more miscarriages, the loss of an affected child and the absence of (acceptable) alternatives for the female partner positively contributed to PGD intention. For PGD use (45.8% of couples), infertility, a history of pregnancy termination(s) and the absence of alternatives according to the female partner were positive determinants. A living affected child reduced PGD use. Mode of inheritance and clinical impact of the disorder did not contribute. CONCLUSIONS: Fewer than 50% of the referred couples actually started PGD treatment. Personal experiences and reproductive history [the presence of a living affected child, infertility or a history of termination of pregnancy (TOP)] were more important determinants of eventual PGD use than the mode of inheritance or the expected clinical impact of the disorder.

During IVF treatment patient preference shifts from singletons towards twins but only a few patients show an actual reversal of preference.

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.