First birth following spindle transfer for mitochondrial replacement therapy: hope and trepidation

In this issue, Zhang et al. (2017) report the birth of a healthy boy after mitochondrial replacement therapy (MRT) by spindle transfer to prevent transmission of mitochondrial disease from mother to child. The case was first publicized in the lay press (Hamzelou, 2016; see also editorial by Johnson, 2016) and then presented during the 2016 Annual Meeting of the American Society for Assisted Reproduction (ASRM) in October 2016 (Zhang et al., 2016a). It followed an earlier report of an unsuccessful attempt at MRT by pronuclear transfer by the same group (Zhang et al., 2016b). This world-first birth represents an achievement and a steppingstone, and it has played a role in encouraging the Human Fertilization and Embryology Authority (HFEA) in the UK to issue a final recommendation that the technique ‘be approved for cautious use in specific circumstances’. (http://www.hfea.gov.uk/10559.html) We, the editors, were unanimous in deciding that this paper should be published in RBMO, based on our conviction that the scientific community must be informed of the details of the work in full in order to evaluate it critically and discuss it openly. We decided this despite the fact that the work has weaknesses and limitations in a number of areas. Moreover, although we were able to encourage the authors to include more details of their work in the submission, some uncertainties concerning methodologies and results still remain. Here we outline our concerns regarding the approach and the treatment process described by Zhang and colleagues

Immunofluorescence localization of extracellular matrix component (s) in two species of sea urchin

An indirect immunofluorescence method was used to localize and trace the expression of an aggregation-promoting factor(s) (S-2) in the early stage embryos (1, 2, 4, 8, and 16-cell) of two species of sea urchin, Strongylocentrotus purpuratus and Lytechinus pictus. The S-2 antigen(s) was isolated from the supernatant of calcium-magnesium-free sea water dissociated S. purpuratus blastulae. Whole embryos from which fertilization membranes had been removed were incubated with purified anti-S-2 antibody (AS-2). Bound AS-2 was labeled with a second antibody (goat anti-rabbit IgG) that had been conjugated with fluorescein isothiocyanate. Immunofluorescence was assessed and photographed using a fluorescence microscope. The fertilized eggs and early stage embryos of both species of urchin displayed peripheral fluorescence. The antibody did not bind to the surfaces of unfertilized eggs nor did it penetrate the hardened fertilization membrane of embryos. Embryos incubated with preimmune rabbit serum showed no staining. (See more in text.)Includes bibliographical references (pages 27-29)California State University, Northridge. Department of Biology

Laser-assisted zona pellucida thinning does not facilitate hatching and may disrupt the in vitro hatching process: a morphokinetic study in the mouse

STUDY QUESTION: Does laser-assisted zona thinning of cleavage stage mouse embryos facilitate hatching in vitro? SUMMARY ANSWER: No, unlike laser zona opening, zona thinning does not facilitate embryo hatching. WHAT IS KNOWN ALREADY: Artificial opening of the zona pellucida facilitates hatching of mouse and human embryos. Laser-assisted zona thinning has also been used for the purpose of assisted hatching of human embryos but it has not been properly investigated in an animal model; thinning methods have produced inconsistent clinical results. STUDY DESIGN, SIZE, DURATION: Time-lapse microscopy was used to study the hatching process in the mouse after zona opening and zona thinning; a control group of embryos was not zona-manipulated but exposed to the same laser energy. PARTICIPANTS/MATERIALS, SETTING, METHODS: Eight-cell CB6F1/J mouse embryos were pooled and allocated to three groups (n = 56 per group): A control group of embryos that were exposed to a dose of laser energy focused outside the zona pellucida (zona intact); one experimental group of embryos in which the zona pellucida was opened by complete ablation using the same total number of pulses as the control group; a second experimental group of embryos in which the zona pellucida was thinned to establish a smooth lased area using the same number of pulses as used in the other two groups. The width of the zona opening was 25 mum and width of the thinned area was 35 mum. Development was monitored by time-lapse microscopy. Overall treatment differences for continuous variables were analyzed by analysis of variance and pairwise comparisons using the Student t-test allowing for unequal variances, while for categorical data, a standard chi-squared test was utilized for all pairwise comparisons. MAIN RESULTS AND THE ROLE OF CHANCE: The frequency of complete hatching was 33.9% in the control group, 94.4% after zona opening, and 39.3% after zona thinning (overall group comparison, P \u3c 0.0001). Overall, 60.7% of the zona-thinned embryos did not complete the hatching process and remained trapped within the zona; when they did hatch, they did not necessarily hatch from the zona-thinned area. Hatching in about one-third of the zona-intact embryos began with breaches at multiple sites by small groups of cells. Likewise, 53.6% of zona-thinned embryos had multiple breaches, always involving an area outside the thinned zone. Zona opening decreased multiple breaching and led to blastocyst escape an average of 14 h earlier than zona-thinned embryos and 5.5 h before control embryos (P = 0.0003). LIMITATIONS, REASONS FOR CAUTION: The experiments presented here were limited to in vitro experiments performed in the mouse. Whether human embryos would behave the same way under similar circumstances is unknown. We postulate that zona thinning is not beneficial in human embryos. WIDER IMPLICATIONS OF THE FINDINGS: The experiments demonstrate that zona thinning is not equivalent to zona opening for assisted hatching. The study provides reason for systematic reviews of assisted hatching trials to take the method of assisted hatching into consideration and not combine the results of zona thinning and zona opening procedures. STUDY FUNDING/COMPETING INTERESTS: Institutional funds were used for the study. No competing interests are declared

Correction to: The pathogenic intestinal spirochaete Brachyspira pilosicoli forms a diverse recombinant species demonstrating some local clustering of related strains and potential for zoonotic spread

Correction to: Gut Pathogens 5:24 (2013) https://doi.org/10.1186/1757-4749-5-2

Euploidy rates in donor egg cycles significantly differ between fertility centers

STUDY QUESTION: Do external factors affect euploidy in egg donor cycles? SUMMARY ANSWER: The study demonstrates that during human assisted reproduction, embryonic chromosome abnormalities may be partly iatrogenic. WHAT IS KNOWN ALREADY: Chromosome abnormalities have been linked in the past to culture conditions such as temperature and Ph variations, as well as hormonal stimulation. Those reports were performed with older screening techniques (FISH), or ART methods no longer in use, and the subjects studied were not a homogeneous group. STUDY DESIGN, SIZE, DURATION: A total of 1645 donor oocyte cycles and 13 282 blastocyst biopsies from 42 fertility clinics were included in this retrospective cohort study. Samples from donor cycles with PGS attempted between September 2011 and July 2015 were included. PARTICIPANTS/MATERIALS, SETTING, METHODS: PGS cycles from multiple fertility clinics referred to Reprogenetics (Livingston, NJ) that involved only oocyte donation were included in this study. Testing was performed by array comparative genomic hybridization (aCGH). Ploidy data were analyzed using Generalized Linear Mixed Models with logistic regression using a logit link function considering a number of variables that represent fixed and random effects. MAIN RESULTS AND THE ROLE OF CHANCE: Euploidy rate was associated with the referring center and independent of almost all the parameters examined except donor age and testing technology. Average euploidy rate per center ranged from 39.5 to 82.5%. The mean expected rate of euploidy was 68.4%, but there are variations in this rate associated with the center effect. LIMITATIONS, REASONS FOR CAUTION: Data set does not include details of the donor selection process, donor race or ethnic origin, ovarian reserve or ovarian responsiveness. Due to the retrospective nature of the study, associations are apparent, however, causality cannot be established. Discrepancies in regard to completeness and homogeneity of data exist due to data collection from over 40 different clinics. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study to show a strong association between center-specific ART treatment practices and the incidence of chromosome abnormality in human embryos, although the meiotic or mitotic origin of these abnormalities could not be determined using these technologies. Given the widespread applications of ART in both subfertile and fertile populations, our findings should be of interest to the medical community in general as well as the ART community in particular. STUDY FUNDING/COMPETING INTEREST(S): No external funds were used for this study. S. Munne is a founding principle of Reprogenetics/current employee of Cooper Genomics. M Alikani’s spouse is a founding principle of Reprogenetics/current consultant for Cooper Genomics. The remaining authors have no conflicts to declare

Monozygotic multiple gestation following in vitro fertilization: analysis of seven cases from Japan

We present a series of monozygous multiple gestations achieved following in vitro fertilization (IVF): one case of monochorionic triplet pregnancy and six cases of dizygotic triplet pregnancy. From September 2000 to December 2006, all patients achieving clinical pregnancy by ART were reviewed (n = 2433). A 37 year-old woman who delivered a healthy singleton after IVF returned two years later for FET, and a single blastocyst was transferred. This also resulted in pregnancy, but TV-USG revealed a single gestational sac with three distinct amniotic sacs, each containing a distinct fetal pole with cardiac activity. This pregnancy was electively terminated at nine weeks' gestation. An additional six cases of dizygotic triplets established after fresh embryo transfer (no ICSI or assisted hatching) are also described. Of these, one resulted in a miscarriage at eight weeks' gestation and five patients have an ongoing pregnancy. This case series suggests the incidence of dizygotic/monochorionic triplets following IVF is approximately 10 times higher than the expected rate in unassisted conceptions, and underscores the importance of a conservative approach to lower the number of embryos at transfer. The role of embryo transfer technique and in vitro culture media in the twinning process requires further study

Monochorionic-triamniotic triplet pregnancy after intracytoplasmic sperm injection, assisted hatching, and two-embryo transfer: first reported case following IVF

BACKGROUND: We present a case of monochorionic-triamniotic pregnancy that developed after embryo transfer following in vitro fertilization (IVF). METHODS: After controlled ovarian hyperstimulation and transvaginal retrieval of 22 metaphase II oocytes, fertilization was accomplished with intracytoplasmic sperm injection (ICSI). Assisted embryo hatching was performed, and two embryos were transferred in utero. One non-transferred blastocyst was cryopreserved. RESULTS: Fourteen days post-transfer, serum hCG level was 423 mIU/ml and subsequent transvaginal ultrasound revealed a single intrauterine gestational sac with three separate amnion compartments. Three distinct foci of cardiac motion were detected and the diagnosis was revised to monochorionic-triamniotic triplet pregnancy. Antenatal management included cerclage placement at 19 weeks gestation and hospital admission at 28 weeks gestation due to mild preeclampsia. Three viable female infants were delivered via cesarean at 30 5/7 weeks gestation. CONCLUSIONS: The incidence of triplet delivery in humans is approximately 1:6400, and such pregnancies are classified as high-risk for reasons described in this report. We also outline an obstetric management strategy designed to optimize outcomes. The roles of IVF, ICSI, assisted embryo hatching and associated laboratory culture conditions on the subsequent development of monozygotic/monochorionic pregnancy remain controversial. As demonstrated here, even when two-embryo transfer is employed after IVF the statistical probability of monozygotic multiple gestation cannot be reduced to zero. We encourage discussion of this possibility during informed consent for the advanced reproductive technologies