A multicentre double-blinded randomized controlled trial on the efficacy of laser-assisted hatching in patients with repeated implantation failure undergoing IVF or ICSI.

STUDY QUESTION: Does assisted hatching increase the cumulative live birth rate in subfertile couples with repeated implantation failure? SUMMARY ANSWER: This study showed no evidence of effect for assisted hatching as an add-on in subfertile couples with repeated implantation failure. WHAT IS KNOWN ALREADY: The efficacy of assisted hatching, with regard to the live birth rate has not been convincingly demonstrated in randomized trials nor meta-analyses. It is suggested though that especially poor prognosis women, e.g. women with repeated implantation failure, might benefit most from assisted hatching. STUDY DESIGN, SIZE, DURATION: The study was designed as a double-blinded, multicentre randomized controlled superiority trial. In order to demonstrate a statistically significant absolute increase in live birth rate of 10% after assisted hatching, 294 participants needed to be included per treatment arm, being a total of 588 subfertile couples. Participants were included and randomized from November 2012 until November 2017, 297 were allocated to the assisted hatching arm of the study and 295 to the control arm. Block randomization in blocks of 20 participants was applied and randomization was concealed from participants, treating physicians, and laboratory staff involved in the embryo transfer procedure. Ovarian hyperstimulation, oocyte retrieval, laboratory procedures, embryo selection for transfer and cryopreservation, the transfer itself, and luteal support were performed according to local protocols and were identical in both the intervention and control arm of the study with the exception of the assisted hatching procedure which was only performed in the intervention group. The laboratory staff performing the assisted hatching procedure was not involved in the embryo transfer itself. PARTICIPANTS/MATERIALS, SETTING, METHODS: Participants were eligible for inclusion in the study after having had either at least two consecutive fresh IVF or ICSI embryo transfers, including the transfer of frozen and thawed embryos originating from those fresh cycles, and which did not result in a pregnancy or as having had at least one fresh IVF or ICSI transfer and at least two frozen embryo transfers with embryos originating from that fresh cycle which did not result in a pregnancy. The study was performed at the laboratory sites of three tertiary referral hospitals and two university medical centres in the Netherlands. MAIN RESULTS AND THE ROLE OF CHANCE: The cumulative live birth rate per started cycle, including the transfer of fresh and subsequent frozen/thawed embryos if applicable, resulted in 77 live births in the assisted hatching group (n = 297, 25.9%) and 68 live births in the control group (n = 295, 23.1%). This proved to be statistically not significantly different (relative risk: 1.125, 95% CI: 0.847 to 1.494, P = 0.416). LIMITATIONS, REASONS FOR CAUTION: There was a small cohort of subfertile couples that after not achieving an ongoing pregnancy, still had cryopreserved embryos in storage at the endpoint of the trial, i.e. 1 year after the last randomization. It cannot be excluded that the future transfer of these frozen/thawed embryos increases the cumulative live birth rate in either or both study arms. Next, at the start of this study, there was no international consensus on the definition of repeated implantation failure. Therefore, it cannot be excluded that assisted hatching might be effective in higher order repeated implantation failures. WIDER IMPLICATIONS OF THE FINDINGS: This study demonstrated no evidence of a statistically significant effect for assisted hatching by increasing live birth rates in subfertile couples with repeated implantation failure, i.e. the couples which, based on meta-analyses, are suggested to benefit most from assisted hatching. It is therefore suggested that assisted hatching should only be offered if information on the absence of evidence of effect is provided, at no extra costs and preferably only in the setting of a clinical trial taking cost-effectiveness into account. STUDY FUNDING/COMPETING INTEREST(S): None. TRIAL REGISTRATION NUMBER: Netherlands Trial Register (NTR 3387, NL 3235, https://www.clinicaltrialregister.nl/nl/trial/26138). TRIAL REGISTRATION DATE: 6 April 2012. DATE OF FIRST PATIENT'S ENROLMENT: 28 November 2012.

Preserving fertility in young women undergoing chemotherapy for early breast cancer; the Maastricht experience.

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

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.

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.

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.

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.

Considerations of breast cancer survivors to return for embryo transfer after fertility preservation: A qualitative study.

OBJECTIVE: To gain insight into the considerations of breast cancer survivors to return or not for embryo transfer after the use of fertility preservation. STUDY DESIGN: This is a qualitative study with semi-structured interviews. The interviews were planned until saturation of themes had been achieved. Content analysis was used to analyze the data. Sixteen out of 35 approached women took part in this study. Interviews were conducted with women who had oocytes or embryos cryopreserved prior to breast cancer treatment at the Maastricht University Medical Center between 2008 and 2016. All women who had cryopreservation more than two years ago were invited for the interviews. Women who had recurrence of disease were excluded. In the interviews we hypothesized the situation 'suppose the menses would have been recovered completely' for women who still had chemotherapy-induced menopause or used an GnRH (Gonadotropin-releasing hormone) analogue. RESULTS: Most women had a strong intrinsic motivation to pursue natural conception over the use of earlier cryopreserved oocytes or embryos. Time pressure was the most mentioned consideration to use cryopreserved oocytes or embryos. The wish to use pre-implantation genetic testing (PGT) in the presence of a germline BRCA1/2 mutation was another consideration to opt for embryo transfer. Furthermore, the physician's advice was an important motivation to choose for either natural conception or the use of cryopreserved oocytes or embryos. CONCLUSION: Multiple considerations influence women's decision making on the mode of conception after breast cancer. Although it concerned a single-center study in a highly-selected population, insight into these considerations can help physicians to address these important topics in counseling these women.

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.

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 patient's IVF treatment cycles, as well as data of 67 singletons conceived after frozen embryo transfer (FET) and of 88 children of 44 twin pregnancies after fresh transfer were analysed by means of multiple linear regression. RESULTS: In vitro culture in medium from Cook resulted in singletons after fresh transfer with a lower mean birthweight (adjusted mean difference, 112 g, P= 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.

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.

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 husband's semen indicated the necessity of an ICSI procedure. The current Dutch guidelines advise against ICSI in chronic HBV carriers, since the risks and effects of chromosomal integration of HBV DNA in the fetus are not well-known. In this article, we review the scientific evidence for the risk of introducing HBV virus into the oocyte and subsequent integration of HBV DNA into the human genome, and debate the question of whether to do, or not to do, IVF and ICSI.

Differences in Strength and Timing of the mtDNA Bottleneck between Zebrafish Germline and Non-germline Cells.

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

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.

In unselected patients, elective single embryo transfer prevents all multiples, but results in significantly lower pregnancy rates compared with double embryo transfer: a randomized controlled trial.

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%.

Elective single embryo transfer (eSET) policy in the first three IVF/ICSI treatment cycles.

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.

Growth rate of human preimplantation embryos is sex dependent after ICSI but not after IVF.

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.

Anaphase lagging mainly explains chromosomal mosaicism in human preimplantation embryos.

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.

Chromosomally abnormal cells are not selected for the extra-embryonic compartment of the human preimplantation embryo at the blastocyst stage.

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.