Josien G. Derhaag

Effect of in vitro culture of human embryos on birthweight of newborns

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.

Further evidence that culture media affect perinatal outcome: findings after transfer of fresh and cryopreserved embryos

BACKGROUND We have previously shown that the medium used for culturing IVF embryos affects the birthweight of the resulting newborns. This observation with potentially far-reaching clinical consequences during later life, was made in singletons conceived during the first IVF treatment cycle after the transfer of fresh embryos. In the present study, we hypothesize that in vitro culture of embryos during the first few days of preimplantation development affects perinatal outcome, not only in singletons conceived in all rank order cycles but also in twins and in children born after transfer of frozen embryos. Furthermore, we investigated the effect of culture medium on gestational age (GA) at birth. METHODS Oocytes and embryos from consecutive treatment cycles were alternately assigned to culture in either medium from Vitrolife or from Cook. Data on a cohort of 294 live born singletons conceived after fresh transfer during any of a patients IVF treatment cycles, as well as data of 67 singletons conceived after frozen embryo transfer (FET) and of 88 children of 44 twin pregnancies after fresh transfer were analysed by means of multiple linear regression. RESULTS In vitro culture in medium from Cook resulted in singletons after fresh transfer with a lower mean birthweight (adjusted mean difference, 112 g, P= 0.03), and in more singletons with low birthweight (LBW) <2500 g (P= 0.006) and LBW for GA ≥ 37 weeks (P= 0.015), when compared with singletons born after culture in medium from Vitrolife AB. GA at birth was not related to the medium used (adjusted difference, 0.05 weeks, P = 0.83). Among twins in the Cook group, higher inter-twin mean birthweight disparity and birthweight discordance were found. Z-scores after FET were -0.04 (± 0.14) in the Cook group compared with 0.18 (± 0.21) in the Vitrolife group (P> 0.05). CONCLUSIONS Our findings support our hypothesis that culture medium influences perinatal outcome of IVF singletons and twins. A similar trend is seen in case of singletons born after FET. GA was not affected by culture medium. These results indicate that in vitro culture might be an important factor explaining the poorer perinatal outcome after assisted reproduction technology (ART). Further research is needed to confirm this culture medium-induced effect in humans and to provide more insight into whether it is caused by epigenetic disturbance of imprinted genes in fetal or placental tissues. Moreover, embryo culture media and their effects need to be investigated thoroughly to select the best embryo culture medium in order to minimize or prevent short-term risks and maybe even long-term disease susceptibility.

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 childrens 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.

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.

Fertility preservation in female classic galactosemia patients.

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

To do or not to do: IVF and ICSI in chronic hepatitis B virus carriers

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

A dual role for endothelial cells in cytomegalovirus infection? A study of cytomegalovirus infection in a series of rat endothelial cell lines.

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

Mast cell-mediated induction of ICAM-1, VCAM-1 and E-selectin in endothelial cells in vitro: constitutive release of inducing mediators but no effect of degranulation

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

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 (β = -3.6 g, SE: 1.5 g, P = 0.021), after controlling for possible confounders (day of embryo transfer, number of transferred embryos, childs 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.

Heart EC Respond Heterogeneous on Cytokine Stimulation in ICAM-1 and VCAM-1, but not in MHC Expression: A Study with 3 Rat Heart Endothelial Cell (RHEC) Lines

Cytokine-induced expression of ICAM-1, VCAM-1, and MHC class I and II was studied at different time points in microvascular endothelial cells (EC) of heart origin, using three different rat endothelial cell (RHEC) lines that were stimulated with TNFalpha and/or IFNgamma. Each of the three RHEC lines responded to TNFalpha as well as to IFNgamma; stimulation with combined cytokines led to increased or even synergistic effects. TNFalpha was most potent in inducing ICAM-1 and VCAM-1, whereas MHC class II was most effectively induced by IFNgamma. The 3 RHEC lines responded similarly regarding induction of MHC class II and upregulation of constitutively expressed MHC class I on the cells. However, the RHEC lines showed remarkable differences with respect to ICAM-1 and VCAM-1 induction, with each line having a unique expression profile. In RHEC-3, both ICAM-1 and VCAM-1 were well inducible, whereas in RHEC-10, no ICAM-1 and only some VCAM-1 could be induced. RHEC-11 showed minimal induction of ICAM-1, but strong induction of VCAM-1. For P-selectin induction, no such differences were found between the RHEC lines. These heterogeneous effects of cytokine stimulation could neither be explained by differences in mobilization of calcium nor by ultra-structural differences between the lines. Stimulation of the RHEC lines for ICAM-1 and VCAM-1 or MHC class II molecule induction resulted in expressing and non-expressing EC. Experiments with selected and subsequently cultured expressing and non-expressing cell populations for either ICAM-1, VCAM-1 or MHC class II, indicated that this selective induction most likely results from intrinsic regulation mechanisms in the cell cultures, and not from the presence of particular EC subpopulations within the lines. We conclude that microvascular heart endothelial cells, as represented by the 3 RHEC lines, demonstrate a selective heterogeneity in expression of ICAM-1 and VCAM-1, but not of MHC class I and II, upon cytokine stimulation. The consequences of this heterogeneity for leukocyte-endothelial cell interactions in heart inflammation and immune reactivity is discussed.Cytokine-induced expression of ICAM-1, VCAM-1, and MHC class I and II was studied at different time points in microvascular endothelial cells (EC) of heart origin, using three different rat endothelial cell (RHEC) lines that were stimulated with TNFalpha and/or IFNgamma. Each of the three RHEC lines responded to TNFalpha as well as to IFNgamma; stimulation with combined cytokines led to increased or even synergistic effects. TNFalpha was most potent in inducing ICAM-1 and VCAM-1, whereas MHC class II was most effectively induced by IFNgamma. The 3 RHEC lines responded similarly regarding induction of MHC class II and upregulation of constitutively expressed MHC class I on the cells. However, the RHEC lines showed remarkable differences with respect to ICAM-1 and VCAM-1 induction, with each line having a unique expression profile. In RHEC-3, both ICAM-1 and VCAM-1 were well inducible, whereas in RHEC-10, no ICAM-1 and only some VCAM-1 could be induced. RHEC-11 showed minimal induction of ICAM-1, but strong induction of VCAM-1. For P-selectin induction, no such differences were found between the RHEC lines. These heterogeneous effects of cytokine stimulation could neither be explained by differences in mobilization of calcium nor by ultra-structural differences between the lines. Stimulation of the RHEC lines for ICAM-1 and VCAM-1 or MHC class II molecule induction resulted in expressing and non-expressing EC. Experiments with selected and subsequently cultured expressing and non-expressing cell populations for either ICAM-1, VCAM-1 or MHC class II, indicated that this selective induction most likely results from intrinsic regulation mechanisms in the cell cultures, and not from the presence of particular EC subpopulations within the lines. We conclude that microvascular heart endothelial cells, as represented by the 3 RHEC lines, demonstrate a selective heterogeneity in expression of ICAM-1 and VCAM-1, but not of MHC class I and II, upon cytokine stimulation. The consequences of this heterogeneity for leukocyte-endothelial cell interactions in heart inflammation and immune reactivity is discussed.