STUDY QUESTION:Are transfer day, developmental stage and morphology of the competent blastocyst in pregnancies leading to live birth associated with preterm birth, birthweight, length at birth and sex of the child?SUMMARY ANSWER:A high score in blastocyst developmental stage and in trophectoderm (TE) showed a significant association with the sex of the child, while no other associations with obstetric outcomes were observed.WHAT IS KNOWN ALREADY:The association between blastocyst assessment scores and obstetric outcomes have been reported in small single-center studies and the results are conflicting.STUDY DESIGN, SIZE, DURATION:Multicenter historical cohort study based on exposure data (transfer day (blastocyst developmental stage reached by Day 5 or Day 6)) blastocyst developmental stage (1-6) and morphology (TE and inner cell mass (ICM): A, B, C)) and outcome data (preterm birth, birthweight, length at birth, and sex of the child) from women undergoing single blastocyst transfer resulting in a singleton pregnancy and live birth.PARTICIPANTS/MATERIALS, SETTING, METHODS:Data from 16 private and university-based facilities for clinical services and research were used. A total of 7246 women, who in 2014-2018 underwent fresh-embryo transfer with a single blastocyst or frozen-thawed embryo transfer (FET) with a single blastocyst resulting in a singleton pregnancy were identified. Linking to the Danish Medical Birth Registry resulted in a total of 4842 women with a live birth being included. Cycles with pre-implantation genetic testing and donated gametes were excluded. The analyses were adjusted for female age (n = 4842), female BMI (n = 4302), female smoking (n = 4290), parity (n = 4365), infertility diagnosis (n = 4765), type of treatment (n = 4842) and center (n = 4842); some analyses additionally included gestational age (n = 4368) and sex of the child (n = 4833).MAIN RESULTS AND THE ROLE OF CHANCE:No statistically significant associations between blastocyst assessment scores (transfer day, developmental stage, TE, ICM) and preterm birth (8.3%) or birthweight (mean 3461.7 g) were found. The adjusted association between blastocysts with a TE score of C and a TE score of A and length at birth (mean 51.6 cm) were statistically significant (adjusted mean difference 0.4 cm (95% CI: 0.02; 0.77)). Blastocysts transferred with developmental stage score 5 compared to blastocysts transferred with score 3 had a 34% increased probability of being a boy (odds ratio (OR) 1.34 (95% CI: 1.09; 1.64). Further, TE score B blastocysts compared to TE score A blastocysts had a 31% reduced probability of being a boy (OR 0.69 (95% CI: 0.60; 0.80)).LIMITATIONS, REASONS FOR CAUTION:It is possible that some residual confounding remains.WIDER IMPLICATIONS OF THE FINDINGS:Blastocyst selection during ART does not appear to introduce any negative effects on obstetric outcome. Therefore, clinicians and patients can be reassured that the assessment scores of the selected blastocyst will not in themselves pose a risk of preterm birth or affect birthweight and the length at birth.STUDY FUNDING/COMPETING INTEREST(S):Unrestricted grant from Gedeon Richter Nordics AB, Sweden. None of the authors have any competing interest to declare.TRIAL REGISTRATION NUMBER:N/A.

ABSTRACTObjectiveWhen a triploid pregnancy is diagnosed prenatally, gynaecologists have traditionally relied on the histopathological examination of the tissue from the terminated pregnancy to determine if the pregnancy is molar. However, reproducibility is poor and variability is high when diagnosing hydatidiform moles. Triploid pregnancies can have either the chromosomal constitution of two maternal and one paternal set, or two paternal and one maternal set, but only the conceptuses with two paternal sets have the potential to cause maternal complications. Therefore, it would be beneficial to introduce a method that gives the gynaecologist the parental origin of the genome of the triploid conceptus as early as possible, without delaying the process by first collecting parental samples.MethodsUsing methylation‐specific multiplex ligation‐dependent probe amplification, we measured methylation levels at different imprinted sites.ResultsWe were able to correctly determine the parental origin of the genome in all 105 triploid pregnancies analysed.ConclusionsWe present methylation‐specific multiplex ligation‐dependent probe amplification as a method capable of determining the parental origin of the genome of triploid conceptuses within 24 h; it is inexpensive, simple and easy to use, and parental samples are not needed. © 2013 John Wiley & Sons, Ltd.

Objective: To describe hatching of human embryos and investigate differences in hatching between IVF and intracytoplasmic sperm injection (ICSI)-fertilized embryos with the use of time-lapse monitoring.Design: Clinical observational study.Setting: University-based fertility clinic.Patient(s): From February 2011 to July 2012, 161 women consented to embryo culture in a time-lapse incubator until day 6 after oocyte retrieval. The mechanism of hatching was recorded and related to method of fertilization (ICSI or IVF) and clinical pregnancy outcome.Intervention(s): IVF or ICSI.Main Outcome Measure(s): Hatching pattern.Result(s): A total of 430 IVF fertilized embryos from 62 patients and 594 ICSI-fertilized embryos from 99 patients were included. We observed spontanous hatching in 165 IVF embryos and 215 ICSI embryos. Two distinct mechanisms of hatching were observed. Type 1 was characterized by penetration of the zona pellucida (ZP) by small trophectoderm projections, whereas type 2 was preceded by a regular rupture of the ZP followed by extrusion of the blastocyst. Type of hatching was significantly different between IVF and ICSI embryos, with type 2 observed more often in IVF embryos than in ICSI embryos. Furthermore, IVF embryos escaped the ZP more readily than ICSI embryos. Regardless of the type of hatching, implantation rates were similar.Conclusion(s): We describe two distinct mechanisms of in vitro hatching related to fertilization method and suggest that hatching pattern is associated with fertilization method. The hatching pattern has, however, no influence on future implantation. (C)2013 by American Society for Reproductive Medicine.

Time-lapse monitoring allows for a flexible embryo evaluation and potentially provides new dynamic markers of embryo competence. Before introducing time-lapse monitoring in a clinical setting, the safety of the instrument must be properly documented. Accordingly, the aim of this study was to evaluate the safety of a commercially available time-lapse incubator.

OBJECTIVE:To evaluate, using time-lapse monitoring, the temporal influence of culture in 5% O2 or 20% O2 on human embryonic development. DESIGN:Retrospective cohort study. SETTING:University-based fertility clinic. PATIENT(S):In vitro fertilized embryos from women aged <38 years with no endometriosis and ≥8 oocytes retrieved. INTERVENTION(S):Culture in 20% O2 exclusively (group 1), 20% and 5% O2 combined (group 2), or 5% O2 exclusively (group 3). MAIN OUTCOME MEASURE(S):Developmental rates and timing of developmental stages. RESULT(S):The timing of the third cleavage cycle was delayed for embryos cultured in 20% O2 (group 1) compared with embryos cultured in 5% O2 (groups 2 and 3). No difference was observed in timing of the early and full blastocyst stages. More embryos in groups 2 and 3 reached the 8-cell, early blastocyst, and full blastocyst stages than in group 1. We found that embryos in group 3 (5% O2) reached the 8-cell stage faster than embryos in group 2 (5% + 20% O2), but none of the other parameters (i.e., other time points, cumulative development, and embryo score) differed between the two groups. CONCLUSION(S):Culture in 20% O2 reduces developmental rates and delays completion of the third cell cycle. The delayed development after culture in atmospheric oxygen was seen in the precompaction embryo only and therefore appears to be stage specific. CLINICAL TRIAL REGISTRATION NUMBER:NCT01139268.

BACKGROUND Blastomere biopsy of human embryos is performed for preimplantation genetic diagnosis (PGD). The impact on further development is largely unexplored, though studies on mice suggest an influence on the hatching process. The objective of this study was to evaluate the effect of blastomere biopsy on early human embryonic development using time-lapse analysis. METHODS Embryos from couples undergoing PGD treatment or IVF/ICSI were included. In the PGD group, 56 human embryos had one blastomere biopsied. As controls, 53 non-biopsied IVF/ICSI embryos were selected. All embryos were cultured until 5 days after fertilization in a time-lapse incubator (EmbryoScope™). Images of embryos were acquired every 20 min. Time-points of key embryonic events were registered, and development in the two groups was compared. RESULTS Duration of the biopsied cell-stage in the PGD group was longer than in the control group (P < 0.001), causing biopsied embryos to reach subsequent embryonic stages until hatching at significantly later time-points (P(compaction) < 0.001; P(morula) < 0.001; P(earlyblast) < 0.001; P(fullblast) = 0.01), but with unchanged intervals. Embryos in the PGD group started hatching at the same time-point as the control group, but had a smaller diameter (P < 0.001), and a thicker zona pellucida (P < 0.001) when hatching. Time-lapse videos revealed that in the control group, expansion of the blastocyst caused continuous thinning of zona pellucida until the blastocyst hatched, whereas in the PGD group the blastocyst hatched through the opening in zona pellucida artificially introduced prior to the biopsy. CONCLUSIONS We find that blastomere biopsy prolongs the biopsied cell-stage, possibly caused by a delayed compaction and alters the mechanism of hatching.

Chromosome painting refers to the complete decoration of specific metaphase chromosomes with complex probe mixtures. The painting signal is obtained by fluorescence in situ hybridization (FISH) of such mixtures established from a number of different sources (1–4). This technique is a valuable supplemental method in the detailed analysis of specific chromosome aberrations, complex chromosome rearrangements, and chromosomes of poor quality, which have been unresolvable with the traditional chromosome banding techniques. In traditional chromosome painting, complex probe mixtures representing specific chromosomes are hybridized to metaphase spreads containing the chromosome aberration in question. If the hybridized probe mixtures are fluorescence labeled, the composition of the aberrant chromosome is revealed by the distribution of the fluorescence signals obtained with the different probes. The combination of different labeled probes and multicolor techniques simplifies the evaluation.

Objective. To compare a new density gradient medium, SpermFilter, for purifying spermatozoa in assisted reproduction with the more established medium, PureSperm.Design. Part 1, a multicenter study on 225 semen samples purified using either PureSperm (115 semen samples) or SpermFilter (110 semen samples). Part 2, a retrospective, single center study on a total of 898 assisted reproductive cycles (245 insemination cycles using husband semen, 58 insemination cycles using donor semen and 595 in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) cycles.Setting. Part 1, three fertility clinics in Denmark (two university-affiliated fertility clinics and one private clinic). Part 2, one university-affiliated fertility clinic in Denmark.Main outcome parameters. Part 1, purity of purified spermatozoa (% motile), motility index and recovery of motile spermatozoa. Part 2, malformation and baby take-home rates (insemination cycles), fertilization, cleavage, implantation, malformation and baby take-home rates (IVF/ICSI cycles).Results. No statistical differences were observed in any of the parameters investigated.Conclusion. SpermFilter is a valid alternative to PureSperm in assisted reproduction technology (ART).

Publisher Summary Primed in situ labeling (PRINS) is a fast and sensitive technique for sequence specific in situ detection of DNA. An unlabeled oligonucleotide probe is hybridized and used as primer for chain elongation in situ catalyzed by a DNA polymerase. Thus, the oligonucleotide primer binds by sequence-specific base pairing to its target sequence, which is subsequently labeled when labeled nucleotides are incorporated by the DNA polymerase, using the oligonucleotide as primer and the cellular DNA as template. Using unlabeled probes (primers) in the PRINS reaction means that high concentrations can be used, because probe bound to cell-structures cannot function as a primer and therefore does not give rise to background signals—only probe hybridized correctly to DNA can function as primer for chain elongation. Thus, using high probe concentrations the hybridization is very fast. A PRINS reaction normally runs for only 5–30 min. Owing to the speed of the reaction the morphology of chromosomes is very well preserved, which makes it possible to obtain chromosome banding of good quality after a PRINS reaction.

INTRODUCTION:Preimplantation genetic diagnosis (PGD) is a possible alternative to prenatal diagnosis, whereby families with serious inherited diseases can avoid having children with the disease. The genetic diagnosis is performed on embryos before implantation and therefore implies IVF. Hence, PGD offers the possibility of transferring embryos without disease, thereby avoiding termination of pregnancy owing to an affected fetus.MATERIAL AND METHODS:Activities at the Centre for Preimplantation Genetic Diagnosis at Aarhus University Hospital since its opening in February 1999 are described. The fluorescent in situ hybridisation (FISH) technique was used for sex selection (hemophilia A and Duchenne's muscular dystrophy) and translocations. The polymerase chain reaction (PCR) was used for cystic fibrosis.RESULTS:Of 20 PGD cycles started, 15 were successful in terms of transference of healthy or carrier embryos. A positive pregnancy test was found after six of 15 embryo transfers (40%) with two subsequent clinical pregnancies.CONCLUSIONS:The present pregnancy rates with PGD are comparable to those following IVF; the clinical pregnancy rate may seem low, but the cycle numbers are small. Preimplantation genetic diagnosis seems to be a realistic alternative for selected genetic diseases, in cases where the couple find abortion unacceptable.

The efficiency of IVF in unstimulated cycles was compared with that following ovarian stimulation with clomiphene citrate in a simple protocol with ultrasound monitoring only. A total of 132 couples with no previous IVF attempts, selected by female age <35 years, indication for intracytoplasmic sperm injection or infertility caused by tubal factor or unexplained infertility were randomized to the two protocols. Randomization yielded two comparable groups. The clomiphene group (68 couples) performed significantly better than the unstimulated group (64 couples) in terms of number of cycles with oocyte harvest (90/111 or 81% versus 65/114 or 57%; chi(2) = 9.21, P < 0.002), embryo transfers per started cycle (59/111 or 53% versus 29/114 or 25%; chi(2) = 18.14, P < 0.0001), live intrauterine pregnancy rate per started cycle (20/111 or 18% versus 4/114 or 4%; chi(2) = 12.42, P < 0.0001), live intrauterine pregnancy rate per embryo transfer (20/59 or 34% versus 4/29 or 14%; chi(2) = 3.96, P = 0.047), but not in terms of implantation rate (22/85 or 26% versus 4/29 or 14%; chi(2) = 1.65). Only two twin pregnancies occurred. Modest side-effects were recorded following clomiphene. Accordingly, a simple clomiphene citrate protocol, but not IVF in unstimulated cycles, seems compatible with the concept of 'friendly IVF', yielding a fair pregnancy rate both per cycle started and per embryo transfer in selected patients. The results do not substantiate any important negative anti-oestrogenic effects of clomiphene.

In 1997, the ESHRE PGD Consortium was formed as part of the ESHRE Special Interest Group on Reproductive Genetics, in order to undertake a long-term study of the efficacy and clinical outcome of preimplantation genetic diagnosis (PGD). In December 1999, the first PGD Consortium report was published discussing referrals of 323 couples, 392 PGD cycles and 82 pregnancies and 79 children born. In the second round of data collection, contributing centres were asked to send in data from their PGD activities before January 1997, as well as from 1st October 1998 until 1st May 2000, in order to have as complete as possible an overview of PGD practices in these centres. A further 563 referrals were sent in as well as 926 PGD cycles, and data on 89 pregnancies (including seven pregnancies ongoing from the previous group) and 83 children were collected. This has led to a considerable amount of cumulative data being acquired: over a period of 7 years (the oldest PGD cycle reported dates from 1994), referral data on 886 couples, cycle data on 1318 PGD cycles and data on 163 pregnancies and 162 babies were collected. In all, these data are encouraging: they show first, that the practice of PGD is becoming more and more established, and an increasing number of different applications is emerging; and second, that collecting these data is worthwhile, as they will be a valuable source of information for all those involved, e.g. in counselling patients and interacting with governmental bodies.