Impact of trophoectoderm biopsy for preimplantation genetic testing on serum ß-hCG levels, time of delivery and birthweight following frozen embryo transfer cycles.

Aim: This study investigated whether trophoectoderm (TE) biopsy adversely impacts serum ß-human chorionic gonadotropin (hCG) level on the 15th day of embryo transfer (ET), delivery week and birthweight, between biopsied and unbiopsied embryo groups, in a cohort of women who delivered a singleton baby, following frozen-thawed ET.Methods: All women having had a live birth after blastocyst ETs following frozen ET cycles with preimplantation genetic testing (PGT) were included. A control group was selected among women who had a live birth following single frozen blastocyst transfer without PGT-A at the same period in our clinicResults: One hundred fifteen and 173 cycles with- and without-PGT, respectively, were included. Serum ß-hCG level on the 15th day after ET was comparable between the groups (p = .336). Average birthweight of the babies born following biopsied embryos were significantly lower (3200 vs. 3380; p = .027). Women who received trophectoderm biopsied embryos had a significantly higher probability of having a baby weighing ≤1500 g and 1500-2500 g (p = .022) or ≤2500 g (p = .008). Proportion of preterm delivery was significantly higher in the biopsy group (p = .023). However, after adjusting for potential covariates, trophectoderm biopsy did not seem to increase the risk of preterm birth (OR 1.525; 95% CI, 0,644-3.611; p = .338)Conclusions: TE biopsy does not seem to impact serum ß-hCG level on the 15th day after ET. Average birthweight is lower when a biopsied embryo was transferred. After adjusting for potential covariates, trophectoderm biopsy does not seem to increase the risk of preterm birth.

Blastomere removal affects homeostatic control leading to obesity in male mice.

Preimplantation embryos are particularly vulnerable to environmental perturbations, including those related to assisted reproductive technologies. Invasive embryo manipulations, such as blastomere biopsy, are applied worldwide in clinical settings for preimplantation genetic testing. Mouse models have previously shown that blastomere biopsy may be associated with altered phenotypes in adult offspring. The aim of the present study was to investigate the specific contribution of blastomere removal to the physiological, behavioral, and molecular regulators of energy homeostasis, as compared to sham manipulation (re-introducing the blastomere into the embryo after its removal) and in vitro culture. Mice derived from 8-cell embryos subjected to blastomere removal displayed: (i) higher body weight and adiposity, (ii) increased food intake and sucrose preference, (iii) decreased time of immobility in the tail suspension test, and (iv) resistance to weight loss after social isolation or following 3 days of physical exercise - compared to mice derived from sham biopsy or from in vitro-cultured embryos. Mice generated after blastomere removal also had increased circulating leptin and leptin gene expression in adipose tissue, as well as increased ghrelin receptor gene expression in the hypothalamus, compared to control mice. The effects of blastomere biopsy on offspring phenotype were sexually dimorphic, with females not being affected. These results indicate that blastomere deprivation, rather than other perturbations of the blastomere biopsy procedure, programs male embryos to develop physiological, behavioral, and molecular dysregulation of energy homeostasis, leading to postnatal obesity.

Child health after preimplantation genetic testing.

RESEARCH QUESTION: Despite the increasing use of preimplantation genetic testing (PGT) for aneuploidy and monogenic diseases, for children conceived using PGT there is limited follow-up beyond 2 years of age. This study examined the health, well-being and development of school-aged children (5-8 years old) conceived following PGT. DESIGN: Retrospective cohort study of children conceived after IVF with PGT (exposed cohort) and children conceived after IVF without PGT (unexposed cohort) at two IVF clinics in Melbourne, born between 2000 and 2008, recruited with a 1:2 ratio. Mothers of the children completed a questionnaire asking child-specific questions regarding health and well-being, mental health, development, educational achievement and family-specific questions regarding family functioning and parent-child attachment. RESULTS: A total of 155 participants were recruited to the PGT cohort and 303 participants to the IVF-only cohort. There were no differences between the two cohorts with regards to maternal characteristics, birth defect frequency and pregnancy characteristics, apart from delivery by Caesarean section, which was more frequent in PGT singletons (55%) compared with IVF-only singletons (36%). While no significant differences between the PGT and IVF-only cohorts were found for the majority of general health and psychological scales, there were differences when compared with population data. Children in the exposed cohort appeared to have more positive outcomes in many of the measures. CONCLUSION: The data from this study suggest that PGT does not cause adverse outcomes in children. However, the nature (self-report) and small sample size of the study must be taken into consideration when interpreting the data.

Multiple cryopreservation-warming cycles, coupled with blastocyst biopsy, negatively affect IVF outcomes.

RESEARCH QUESTION: Do multiple cryopreservation-warming cycles, coupled with blastocyst biopsy, negatively affect IVF outcomes? DESIGN: Patients undergoing IVF with homologous single embryo transfer, and who underwent trophectoderm biopsy for preimplantation genetic testing for aneuploidy (PGT-A) between 2013 and 2017, were divided into three groups based on degree of embryonic micromanipulation: once-biopsied, once-cryopreserved (group BC, n = 2603), once-biopsied, twice-cryopreserved (group CBC, n = 95) and twice-biopsied, twice-cryopreserved (group BCBC, n = 15). The primary outcome was live birth; secondary outcomes included positive serum pregnancy test, clinical pregnancy and miscarriage. RESULTS: Group CBC had a significantly lower chance of live birth (adjusted RR 0.57, 95% CI 0.41 to 0.79) and clinical pregnancy (adjusted RR 0.67, 95% CI 0.53 to 0.85) compared with group BC. Miscarriage rates were similar between groups BC and CBC (adjusted RR 1.3, 95% CI 0.64 to 2.7). CONCLUSIONS: Multiple cryopreservation-warming cycles, coupled with blastocyst biopsy, negatively affect IVF outcomes. Although PGT-A is thought to improve reproductive outcomes on a per transfer basis, caution must be exercised in counselling patients on the possibility of diminishing returns owing to further embryonic micromanipulation after an embryo has been cryopreserved.

Clinical outcomes of Preimplantation genetic testing (PGT) application in couples with chromosomal inversion, a study in the Chinese Han population.

BACKGROUND: Chromosomal inversion was considered to have adverse effects on pregnancy outcomes through abnormal gametogenesis. The purpose of this retrospective study was to investigate whether preimplantation genetic testing (PGT) improves pregnancy outcomes for couples with chromosomal inversion. METHODS: A total of 188 cycles from 165 couples with one chromosomal inversion carrier were divided into two groups: PGT (136 cycles, 125 couples) and non-PGT (52 cycles, 50 couples). Biochemical pregnancy, clinical pregnancy, ongoing pregnancy, miscarriage and live birth rates of their first transfer cycles, as well as cumulative live birth rates of each cycle and euploidy rates, were analyzed. RESULTS: There were no statistically significant differences in the pregnancy outcomes between the two groups. The euploidy rate of pericentric inversion carriers was not higher than that of paracentric inversion carriers in PGT group (60.71% vs 50.54%, P = 0.073). Similarly, the euploid rate of male carriers was not higher than that of female carriers (61.2% vs 56.1%, P = 0.256). CONCLUSIONS: Due to limitation of retrospective study and small sample size, our current data showed that PGT cannot provide prominent benefits for inversion carriers in the Chinese Han population. Further prospective randomized controlled trials are needed to evaluate the effects of PGT.

Preimplantation genetic testing as a component of root cause analysis of errors and reassignment of embryos in IVF.

The risks of embryo/gamete mix-up are a threat to the integrity of the IVF process, with significant implications for affected families. The use of preimplantation genetic testing through single-nucleotide polymorphism array or next-generation sequencing technology can help to identify, characterize and ultimately help, in some cases, to find the root cause, and to mitigate the extent of these errors for a given patient or laboratory.

Second-generation preimplantation genetic testing for aneuploidy in assisted reproduction: a SWOT analysis.

Second-generation preimplantation genetic testing for aneuploidy (PGT-A 2.0) in patients with an unfavourable reproductive and IVF prognosis is becoming common practice, with the aim of improving reproductive outcomes. However, there is still no clear evidence on the possible advantages and drawbacks with regard to this procedure. In this discussion paper, based on a SWOT (strengths, weaknesses, opportunities, threats) analysis, the different aspects of this strategy are evaluated. Current evidence suggests that PGT-A 2.0 should not at present have an indiscriminate application, but it might be indicated in cases in which the risk of aneuploidy is increased.

Optimal timing for blastomere biopsy of 8-cell embryos for preimplantation genetic diagnosis.

STUDY QUESTION: What is the optimal timing for blastomere biopsy during the 8-cell stage, at which embryos will have the best implantation potential? SUMMARY ANSWER: Fast-cleaving embryos that are biopsied during the last quarter (Q4) of the 8-cell stage and are less affected by the biopsy procedure, and their implantation potential is better than that of embryos biopsied earlier during the 8-cell stage (Q1-Q3). WHAT IS KNOWN ALREADY: Blastomer biopsy from cleavage-stage embryos is usually performed on the morning of Day 3 when the embryos are at the 6- to 8-cell stage and is still the preferred biopsy method for preimplantation genetic diagnosis (PGD) for monogentic disorders or chromosomal translocations. Human embryos usually remain at the 8-cell stage for a relatively long 'arrest phase' in which cells grow, duplicate their DNA and synthesize various proteins in preparation for the subsequent division. STUDY DESIGN, SIZE, DURATION: This is a retrospective cohort study. The study group (195 embryos) included all 8-cell stage embryos that underwent blastomere biopsy for PGD for monogenetic disorders and chromosomal translocations in our unit between 2012-2014 and cultured in the EmbryoScope until transfer. The control group (115 embryos) included all embryos that underwent intracytoplasmic sperm injection without a biopsy during the same period. PARTICIPANTS/MATERIALS, SETTING, METHODS: The 8-cell stage was divided into four quarters: the first 5 h post-t8 (Q1), 5-10 h post-t8 (Q2), 10-15 h post-t8 (Q3) and at 15-20 h post-t8 (Q4). Non-biopsied control embryos were divided into four equivalent quarters. Embryos were evaluated for timing of developmental events following biopsy including timing of first cleavge after biopsy, timing of comapction (tM) and start of blastulation (tSB). Timing of these events were compared between PGD and control embryos, as well as with 56 PGD implanted embryos with Known Implantation Data (PGD-KID-positive embryos). MAIN RESULTS AND THE ROLE OF CHANCE: Embryos that were biopsied during Q3 (10-15 h from entry into 8-cell stage) were delayed in all three subsequent developmental events, including first cleavage after biopsy, compaction and start of blastulation. In contrast, these events occurred exactly at the same time as in the control group, in embryos that were biopsied during Q1, Q2 or Q4 of the 8-cell stage. The results show also that embryos that were biopsied during Q1, Q2 or Q3 of the 8-cell stage demonstrated a significant delay from the biopsied implanted embryos already in t8 as well as in tM and tSB. However, embryos that were biopsied during Q4 demonstrated dynamics similar to those of the biopsied implanted embryos in t8 and tM, and a delay was noticed only in the last stage of tSB. LIMITATIONS, REASONS FOR CAUTION: This is a retrospective study that is limited to the timing of biopsy that is routinely performed in the IVF lab. A prospective study in which biopsy will be performed at a desired timing is needed in order to differ between the effect of biopsy itself and the cleavage rate of the embryo. WIDER IMPLICATIONS OF THE FINDINGS: Our findings showed that blastomere biopsy can be less harmful to further development if it is carried out during a critical period of embryonic growth, i.e during Q4 of the 8-cell stage. They also demonstrated the added value of time-lapse microscopy for determining the optimal timing for blastomere biopsy. STUDY FUNDING/COMPETING INTEREST(S): The study was funded by the routine budget of our IVF unit. TRIAL REGISTRATION NUMBER: N/A.

Developmental outcome of 9-year-old children born after PGS: follow-up of a randomized trial.

STUDY QUESTION: Does Day-3 cleavage-stage PGS affect neurodevelopment of 9-year-old IVF offspring? SUMMARY ANSWER: We did not find evidence of adverse consequences of Day-3 cleavage-stage PGS on neurodevelopment of 9-year-old IVF offspring, although children born after IVF with or without PGS often had a non-optimal neurological condition. WHAT IS KNOWN ALREADY: Knowledge on long-term sequelae for development and health of children born following PGS is lacking. This is striking as evidence accumulates that IVF itself is associated with increased risk for impaired health and development in the offspring. STUDY DESIGN SIZE, DURATION: This prospective, assessor-blinded, multicentre, follow-up study evaluated development and health of 9-year-old IVF children born to women who were randomly assigned to IVF with PGS (PGS group) or without PGS (control group). The follow-up examination at 9 years took place between March 2014 and May 2016. PARTICIPANTS/MATERIALS, SETTING, METHODS: In total, 408 women were included and randomly assigned to IVF with or without Day-3 cleavage-stage PGS. This resulted in 52 ongoing pregnancies in the PGS group and 74 in the control group. In the PGS group, 59 children were born alive; in the control group, 85 children were born alive. At the age of 9 years, 43 children born after PGS and 56 control children participated in the study. Our primary outcome was the neurological optimality score, a sensitive measure of neurological condition assessed with a standardized, age-specific test (Touwen test). Secondary outcomes were adverse neurological condition (neurologically abnormal and the complex form of minor neurological dysfunction), cognitive development (intelligence quotient and specific domains), behaviour (parental and teacher's questionnaires), blood pressure and anthropometrics. MAIN RESULTS AND THE ROLE OF CHANCE: Neurodevelopmental outcome of PGS children did not differ from that of controls; the neurological optimality scores (mean values [(95% CI]: PGS children 51.5 [49.3; 53.7], control children 53.1 [50.5; 55.7]) were not significantly different. The prevalences of adverse neurological outcome (in all but one child implying the presence of the complex form of minor neurological dysfunction) did not differ between the groups (PGS group 17/43 [40%], control group 19/56 [34%]), although the prevalence of complex minor neurological dysfunction in both groups was rather high. Also intelligence quotient scores of the two groups were not significantly different (PGS group 114 [108; 120]); control group 117 [109; 125]), and the behaviour, blood pressure and anthropometrics of both groups did not differ. Mean blood pressures of both groups were above the 60th percentile. LIMITATIONS REASONS FOR CAUTION: The power analysis of the study was not based on the number of children needed for the follow-up study, but on the number of women who were needed to detect an increase in ongoing pregnancy rates after PGS. In addition, our study evaluated embryo biopsy in the form of PGS at cleavage stage (Day-3 embryo biopsy), while currently PGS at blastocyst stage (Day-5 embryo biopsy) is recommended and increasingly being used. WIDER IMPLICATIONS OF THE FINDINGS: Our findings indicate that PGS in cleavage stage embryos is not associated with adverse effects on neurological, cognitive and behavioural development, blood pressure and anthropometrics of offspring at 9 years. This is a reassuring finding as embryo biopsy in the forms of PGS and PGD is increasingly applied. However, both groups of IVF offspring showed high prevalences of the clinically relevant form of minor neurological dysfunction, which is a point of concern for the IVF community. In addition, our study confirms findings of others that IVF offspring may be at risk of an unfavourable cardiovascular outcome. These findings are alarming and highlight the importance of research on the underlying mechanisms of unfavourable neurodevelopmental and cardiovascular outcomes in IVF offspring. STUDY FUNDING/COMPETING INTEREST(S): The randomized controlled trial was financially supported by the Organization for Health Research and Development (ZonMw), The Netherlands (Grant number 945-03-013). The follow-up was financially supported by the University Medical Center Groningen (Grant number: 754510), the Cornelia Foundation, and the graduate schools BCN and Share, Groningen, The Netherlands. The sponsors of the study had no role in study design, data collection, data analysis, data interpretation or writing of the report. There are no conflicts of interest. TRIAL REGISTRATION NUMBER: ISRCTN76355836.

Perinatal follow-up of children born after preimplantation genetic diagnosis between 1995 and 2014.

PURPOSE: We aim to evaluate the safety of PGD. We focus on the congenital malformation rate and additionally report on adverse perinatal outcome. METHODS: We collated data from a large group of singletons and multiples born after PGD between 1995 and 2014. Data on congenital malformation rates in live born children and terminated pregnancies, misdiagnosis rate, birth parameters, perinatal mortality, and hospital admissions were prospectively collected by questionnaires. RESULTS: Four hundred thirty-nine pregnancies in 381 women resulted in 364 live born children. Nine children (2.5%) had major malformations. This percentage is consistent with other PGD cohorts and comparable to the prevalence reported by the European Surveillance of Congenital Anomalies (EUROCAT). We reported one misdiagnosis resulting in a spontaneous abortion of a fetus with an unbalanced chromosome pattern. 20% of the children were born premature (< 37 weeks) and less than 15% had a low birth weight. The incidence of hospital admissions is in line with prematurity and low birth weight rate. One child from a twin, one child from a triplet, and one singleton died at 23, 32, and 37 weeks of gestation respectively. CONCLUSIONS: We found no evidence that PGD treatment increases the risk on congenital malformations or adverse perinatal outcome. TRIAL REGISTRATION NUMBER: NCT 2 149485.

Pre-term birth and low birth weight following preimplantation genetic diagnosis: analysis of 88 010 singleton live births following PGD and IVF cycles.

STUDY QUESTION: Is PGD associated with the risk of adverse perinatal outcomes such as pre-term birth (PTB) and low birth weight (LBW)? SUMMARY ANSWER: There was no increase in the risk of adverse perinatal outcomes of PTB, and LBW following PGD compared with autologous IVF. WHAT IS KNOWN ALREADY: Pregnancies resulting from ART are associated with a higher risk of pregnancy complications compared with spontaneously conceived pregnancies. The possible reason of adverse obstetric outcomes following ART has been attributed to the underlying infertility itself and embryo specific epigenetic modifications due to the IVF techniques. It is of interest whether interventions such as embryo biopsy as performed in PGD affect perinatal outcomes. STUDY DESIGN, SIZE, DURATION: Anonymous data were obtained from the Human Fertilization and Embryology Authority (HFEA), the statutory regulator of ART in the UK. The HFEA has collected data prospectively on all ART performed in the UK since 1991. Data from 1996 to 2011 involving a total of 88 010 singleton live births were analysed including 87 571 following autologous stimulated IVF ± ICSI and 439 following PGD cycles. PARTICIPANTS/MATERIALS, SETTING, METHODS: Data on all women undergoing either a stimulated fresh IVF ± ICSI treatment cycle or a PGD cycle during the period from 1996 to 2011 were analysed to compare perinatal outcomes of PTB and LBW among singleton live births. Logistic regression analysis was performed adjusting for female age category, year of treatment, previous IVF cycles, infertility diagnosis, number of oocytes retrieved, whether IVF or ICSI was used and day of embryo transfer. MAIN RESULTS AND THE ROLE OF CHANCE: There was no increase in the risk of PTB and LBW following PGD versus autologous stimulated IVF ± ICSI treatment, unadjusted odds of PTB (odds ratio (OR) 0.68, 95% CI: 0.46-0.99) and LBW (OR 0.56, 95% CI: 0.37-0.85). After adjusting for the potential confounders, there was again no increase in the risk of the adverse perinatal outcomes following PGD: PTB (adjusted odds ratio (aOR) 0.66, 95% CI: 0.45-0.98) and LBW (aOR 0.58, 95% CI: 0.38-0.88). LIMITATIONS, REASONS FOR CAUTION: Although the analysis was adjusted for a number of important confounders, the data set had no information on confounders such as smoking, body mass index and the medical history of women during pregnancy to allow adjustment. There was no information on the stage of embryo at biopsy, whether blastomere or trophectoderm biopsy. WIDER IMPLICATIONS FOR THE FINDINGS: The demonstration that PGD is not associated with higher risk of PTB and LBW provides reassurance towards its current expanding application. STUDY FUNDING/COMPETING INTERESTS: No funding was obtained. There are no competing interests to declare.

Embryo biopsy and development: the known and the unknown.

Preimplantation genetic diagnosis (PGD) has been introduced in clinical practice as a tool for selecting 'healthy' embryos before their transfer in utero. PGD protocols include biopsy of cleaving embryos (blastomere biopsy (BB)) or blastocysts (trophectoderm biopsy (TB)), followed by genetic analysis to select 'healthy' embryos for transfer in utero. Currently, TB is replacing the use of BB in the clinical practice. However, based on the European Society of Human Reproduction and Embryology Preimplantation Genetic Diagnosis Consortium reports, BB has been used in >87% of PGD cycles for more than 10 years. An exhaustive evaluation of embryo biopsy (both BB and TB) risks and safety is still missing. The few epidemiological studies available are quite controversial and/or are limited to normalcy at birth or early childhood. On the other hand, studies on animals have shown that BB can be a risk factor for impaired development, during both pre- and postnatal life, while little is known on TB. Thus, there is an urgent need of focused researches on BB, as it has contributed to give birth to children for more than 10 years, and on TB, as its application is significantly growing in clinical practice. In this context, the aim of this review is to provide a complete overview of the current knowledge on the short-, medium- and long-term effects of embryo biopsy in the mouse model.

Challenges facing contemporary preimplantation genetic screening.

PURPOSE OF REVIEW: Aneuploidy is a leading cause of pregnancy failure. Although initial attempts to perform preimplantation genetic screening did not improve outcomes, validated techniques were developed to safely and effectively increase pregnancy rates. Still, many embryos designated as euploid do not implant. Current approaches are being refined to provide additional biologic insight into why this is the case. At present, the diagnosis and clinical relevance of segmental aneuploidy and mosaicism are amongst the more heavily investigated. RECENT FINDINGS: Class I data have proven the safety of trophectoderm biopsy and validation studies have shown single nucleotide polymorphism array and quantitative PCR can accurately detect whole chromosome aneuploidy. Similar studies to validate next generation sequencing are underway. Although randomized control trials have demonstrated the clinical utility of preimplantation genetic screening, recent data on the impact of mosaicism and segmental aneuploidy require clarification. SUMMARY: Several well powered randomized control trials have shown preimplantation genetic screening improves implantation rate. Plausible explanations for euploid failures include undetected mosaicism and segmental aneuploidy. However, the true incidence and dispersion of mosaicism within the embryo is unknown. Likewise, the resolution of detection and clinical significance of segmental aneuploidy is unclear. Further research to validate proposed detection algorithms and class I data to determine if detection impacts outcomes is needed.

The clinical effectiveness of preimplantation genetic diagnosis for aneuploidy in all 24 chromosomes (PGD-A): systematic review.

STUDY QUESTION: Is preimplantation genetic diagnosis for aneuploidy (PGD-A) with analysis of all chromosomes during assisted reproductive technology (ART) clinically and cost effective? SUMMARY ANSWER: The majority of published studies comparing a strategy of PGD-A with morphologically assessed embryos have reported a higher implantation rate per embryo using PGD-A, but insufficient data has been presented to evaluate the clinical and cost-effectiveness of PGD-A in the clinical setting. WHAT IS KNOWN ALREADY: Aneuploidy is a leading cause of implantation failure, miscarriage and congenital abnormalities in humans, and a significant cause of ART failure. Preclinical evidence of PGD-A indicates that the selection and transfer of euploid embryos during ART should improve clinical outcomes. STUDY DESIGN, SIZE AND DURATION: A systematic review of the literature was performed for full text English language articles using MEDLINE, EMBASE, SCOPUS, Cochrane Library databases, NHS Economic Evaluation Database and EconLit. The Downs and Black scoring checklist was used to assess the quality of studies. Clinical effectiveness was measured in terms of pregnancy, live birth and miscarriage rates. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: Nineteen articles meeting the inclusion criteria, comprising three RCTs in young and good prognosis patients and 16 observation studies were identified. Five of the observational studies included a control group of patients where embryos were selected based on morphological criteria (matched cohort studies). MAIN RESULTS AND ROLE OF CHANCE: Of the five studies that included a control group and reported implantation rates, four studies (including two RCTs) demonstrated improved implantation rates in the PGD-A group. Of the eight studies that included a control group, six studies (including two RCTs) reported significantly higher pregnancy rates in the PGD-A group, and in the remaining two studies, equivalent pregnancies rates were reported despite fewer embryos being transferred in the PGD-A group. The three RCTs demonstrated benefit in young and good prognosis patients in terms of clinical pregnancy rates and the use of single embryo transfer. However, studies relating to patients of advanced maternal age, recurrent miscarriage and implantation failure were restricted to matched cohort studies, limiting the ability to draw meaningful conclusions. LIMITATIONS, REASONS FOR CAUTION: Relevant studies may have been missed and findings from RCTs currently being undertaken could not be included. WIDER IMPLICATIONS OF THE FINDINGS: Given the uncertain role of PGD-A techniques, high-quality experimental studies using intention-to-treat analysis and cumulative live birth rates including the comparative outcomes from remaining cryopreserved embryos are needed to evaluate the overall role of PGD-A in the clinical setting. It is only in this way that the true contribution of PGD-A to ART can be understood.

Impact of blastocyst biopsy and comprehensive chromosome screening technology on preimplantation genetic screening: a systematic review of randomized controlled trials.

Embryonic aneuploidy is highly prevalent in IVF cycles and contributes to decreased implantation rates, IVF cycle failure and early pregnancy loss. Preimplantation genetic screening (PGS) selects the most competent (euploid) embryos for transfer, and has been proposed to improve IVF outcomes. Use of PGS with fluorescence-in-situ hybridization technology after day 3 embryo biopsy (PGS-v1) significantly lowers live birth rates and is not recommended for use. Comprehensive chromosome screening technology, which assesses the whole chromosome complement, can be achieved using different genetic platforms. Whether PGS using comprehensive chromosome screening after blastocyst biopsy (PGS-v2) improves IVF outcomes remains to be determined. A systematic review of randomized controlled trials was conducted on PGS-v2. Three trials met full inclusion criteria, comparing PGS-v2 and routine IVF care. PGS-v2 is associated with higher clinical implantation rates, and higher ongoing pregnancy rates when the same number of embryos is transferred in both PGS and control groups. Additionally, PGS-v2 improves embryo selection in eSET practice, maintaining the same ongoing pregnancy rates between PGS and control groups, while sharply decreasing multiple pregnancy rates. These results stem from good-prognosis patients undergoing IVF. Whether these findings can be extrapolated to poor-prognosis patients with decreased ovarian reserve remains to be determined.

Aneuploidy rates and blastocyst formation after biopsy of morulae and early blastocysts on day 5.

PURPOSE: Studies have demonstrated high implantation rates after trophectoderm biopsy of day 5 expanded blastocysts. However, biopsy of cleavage stage embryos may adversely affect embryo development and implantation. No studies have assessed the utility of day 5 morulae and early blastocyst biopsy. This study sought to better understand these slower embryos' aneuploidy rates and implantation potential. METHODS: This was a retrospective review of all autologous IVF cycles utilizing PGS at a single academic infertility center. RESULTS: The biopsy of day 5 morulae and early blastocysts provided 22 % additional euploid blastocysts available for fresh day 6 transfer compared to day 5 biopsy of only expanded blastocysts. Aneuploidy did correlate with embryo stage on day 5, even after controlling for maternal age, with 16 % of morulae and 35 % of blastocysts being euploid. The majority (83 %) of euploid morulae progressed to the blastocyst stage by day 6. Experience transferring slower developing embryos is limited, but preliminary pregnancy and implantation rates appear similar to euploid embryos biopsied as expanded blastocysts. CONCLUSIONS: The biopsy of all non-arrested embryos on day 5 provides genetic information for all blastocysts on day 6, increasing the pool of euploid blastocysts available for fresh transfer and avoiding the need to cryopreserve developmentally competent embryos without genetic information.

Single blastomere removal from murine embryos is associated with activation of matrix metalloproteinases and Janus kinase/signal transducers and activators of transcription pathways of placental inflammation.

Single blastomere removal from cleavage-stage embryos, a common procedure used in conjunction with preimplantation genetic diagnosis (PGD), may affect reproductive outcomes. We hypothesized that negative pregnancy outcomes associated with PGD may be due to impairment of placental signaling pathways. The goal of this study was to determine the molecular mechanisms through which placental signaling is deregulated by blastomere removal. Four-cell stage murine embryos produced by in vitro fertilization were subjected to removal of a single blastomere (biopsied) or to the same manipulations without the blastomere removal (controls). Placental tissues from term (18.5 day) pregnancies obtained after embryo transfer were tested for levels of nitrosative species, interleukin 6, signal transducers and activators of transcription (STAT) 1 and 3, suppressors of cytokine signaling (SOCS) 1, 2 and 3 and matrix metalloproteinases (MMP) 1, 2, 3 and 9. Significant increases in nitrosative stress (P < 0.05), phosphorylative activation of STAT1 (P < 0.05) but not STAT3, lower levels of the inhibitors SOCS2 (P < 0.01) and SOCS3 (P < 0.001) and activation of MMP9 (P < 0.001) were observed in placentas derived from biopsied embryos, compared with controls. Such effects could contribute to greater levels of premature membrane rupture, incorrect parturition, preterm birth and intrauterine growth restriction associated with PGD. This work has determined signaling mechanisms that may be responsible for blastomere removal effects on placental function, with the potential to become targets for improving obstetric and neonatal outcomes in assisted reproduction.

Effects of blastomere biopsy on post-natal growth and behavior in mice.

STUDY QUESTION: Does blastomere biopsy (BB) of preimplantation embryos induce long-term effects on their growth and post-natal behavior? SUMMARY ANSWER: BB induces long-term effects on body weight and behavior in male mice. WHAT IS KNOWN ALREADY: BB is an essential technique for performing preimplantation genetic diagnosis (PGD), a screening test that can detect genetic abnormalities of embryos before their transfer in utero. There is limited understanding of the post-natal consequences and safety of BB. STUDY DESIGN, SIZE, DURATION: Offspring who had a BB performed as embryos, as well as control offspring, were examined for body and neurological development and subjected to a screening battery of behavioral tests, designed to model symptoms of psychiatric disorders. At least 12 mice were used for each test over the course of 16 weeks. PARTICIPANTS/MATERIALS, SETTING, METHODS: Embryos were subjected to a single BB at the 8-cell stage and then cultured in vitro until the blastocyst stage (BB group). Two control groups were created, one consisting of embryos cultured in vitro without any manipulation (in vitro control (IVC) group) and one of embryos developed entirely in vivo (in vivo group). Embryos from in vitro groups (BB and IVC) were transferred to pseudo-pregnant female mice at the blastocyst stage. Body growth parameters and developmental landmarks of the resulting offspring were observed during their entire lifespan. Furthermore, validated behavioral tests were used to assess early communicative functions, startle reflex, and anxiety- and depression-like behaviors. MAIN RESULTS AND THE ROLE OF CHANCE: We found that male mice derived from BB exhibited peculiar behavioral alterations and changes in body weight. BB-derived male mice showed increased body weight with respect to both controls as early as the second week of life. Adult males displayed decreased times of immobility in the tail suspension test (P < 0.05) and deficits in habituation to, and pre-pulse inhibition of, the startle reflex (P < 0.05). BB did not affect communicative skills and anxiety-like responses. LIMITATIONS, REASONS FOR CAUTION: Extrapolation of these results to humans requires caution as the culture protocols used in human clinics could be better established than in mice research. Furthermore species-specific neurodevelopmental features could be a source of differences between mice and humans in the effects of BB. WIDER IMPLICATIONS OF THE FINDINGS: Our data demonstrate that BB affects long-term programming of post-natal development and behavior in mice, suggesting that PGD procedures could be a risk factor for late-onset, neurodevelopmental and metabolic disease predisposition. Thus, in light of our observations, long-term follow-up in humans or other primates generated after BB is needed. STUDY FUNDING/COMPETING INTERESTS: This work was supported by the European Research Council (FP7/2007-2013)/Programme IDEAS GA no. 210103 to G.E.P. European Research Council - Programme FP7-KBBE-2012.1.3-04, GA no. 312097 Acronym: FECUND to G.E.P.; MIUR/CNR, Programme FIRB. GA n. B81J12002520001 Acronym: GenHome to P.L. The authors are participating in the COST action FA 1201 'Epiconcept' Epigenetic and Periconception Environment. No competing interests are declared.

Cognitive and psychomotor development of 5- to 6-year-old singletons born after PGD: a prospective case-controlled matched study.

STUDY QUESTION: Do preschool preimplantation genetic diagnosis (PGD) children differ in their cognitive and psychomotor development from children born after ICSI and spontaneous conception (SC)? SUMMARY ANSWER: The cognitive development of PGD pre-schoolers was comparable to children born after ICSI and SC but motor development differed between ICSI and SC groups. STUDY DESIGN, SIZE DURATION: The cognitive abilities and motor skills of 5- to 6-year-old singletons born after PGD (n = 47) were assessed in comparison with 49 ICSI and 48 SC children in a prospective, case-controlled, matched follow-up study between April 2011 and May 2013. PARTICIPANTS/MATERIALS, SETTING, METHODS: PGD singletons, ICSI and SC children of preschool age were examined with the Wechsler Preschool and Primary Scale of Intelligence (WPSSI-III-NL) and the Movement ABC (M ABC). The WPSSI-III-NL revealed scores for Full IQ, Verbal IQ and Performance IQ. The M ABC yields a total score and comprising scores for measurements of balance, dexterity and ball skills. Since embryo biopsy is the only technical difference between the PGD and ICSI procedures, ICSI children were included as controls. These children were part of a Dutch-speaking cohort of children conceived after assisted reproduction technology (ART) at the Universitair Ziekenhuis Brussel (UZ Brussel) who received longitudinal follow-up. The SC children acted as a second control group similar to the fertile PGD sample and in contrast to the ICSI group. The SC group was recruited through announcements in a variety of media. The children were matched for age, gender, birth order and educational level of the mother. The assessments carried out for the ART groups were blinded whenever possible. The data were analysed using analysis of covariance (ANCOVA) and partial eta squared (η(2)), which was used as a measurement of effect size. MAIN RESULTS AND THE ROLE OF CHANCE: The overall cognitive development of PGD singletons did not differ from controls [P = 0.647, η(2) = 0.006; 95% confidence interval (CI) (0, 0.043)]. The partial IQ scores for Verbal and Performance intelligence revealed similar results. Analysis of motor development based on the total score as well as subscales did indicate a significant difference between the three conception groups [P = 0.033, η(2) = 0.050, 95% CI (0, 0.124)]. Post hoc analysis indicated that the significant difference was situated between performances of ICSI and SC children. Balance capacities [P = 0.004, η(2) = 0.079, 95% CI (0.025, 0.163)] and its post hoc analysis yielded equivalent results. Motor capacities of PGD singletons, however, did not differ from any of the two other conception groups. LIMITATIONS, REASONS FOR CAUTION: Given that we only assessed Caucasian singletons born after PGD, caution is required when drawing more general inferences from our results. The small sample size may be a limitation. A priori power analysis, however, revealed that at least 52 children per group were needed to detect a medium effect and 80% power using ANCOVA. Originally our sample met this threshold but we had to exclude six cases in order to remove outliers and due to missing data. WIDER IMPLICATIONS OF THE FINDINGS: Long-term follow-up of children born after embryo biopsy, in this case for PGD, is needed to confirm that the development of these children remains comparable to ICSI and SC children. Our findings do support the safety of the PGD technique and will reassure patients with hereditary genetic diseases regarding the health of their future offspring conceived with PGD. STUDY FUNDING/COMPETING INTERESTS: Funding for this study was obtained from the OZR (Research group of the Vrije Universiteit Brussel), the FWO (Fonds Wetenschappelijk Onderzoek) and the Wetenschappelijk Fonds Willy Gepts. The UZ Brussel and the Centre of Medical Genetics received funding from pharmaceutical firms for data collection. UZ Brussel and the Centre for Medical Genetics have received many educational grants for organizing the data collection, from IBSA, Ferring, Organon, Shering-Plough, Merck and Merck Belgium. M.B. has received consultancy and speaker's fees from Organon, Serono Symposia and Merck.