Conjoined twins are estimated to occur in 1:50 000 pregnancies. Eighteen cases of pregnancies achieved by ART have been published of which three were achieved after single embryo transfer, allowing discussion of embryo characteristics. We report, to the best of our knowledge, the first case of parapagus conjoined twins after ART. Furthermore, this is the first report of conjoined twins with detailed morphokinetics of the earliest embryogenesis from zygote to expanded and hatched blastocyst stage. The case zygote had three refractile bodies, which were all allocated to one blastomere at first cleavage following an asynchronous pronuclei fading. Within 2 h, this blastomere cleaved to four and fragmented. The remaining blastomere cleaved symmetrically and regularly and a blastocyst (score: 4AB) was vitrified 120 h after IVF. Pregnancy was achieved following a frozen-thawed single blastocyst transfer. The etiopathogenetic mechanism of the origin of conjoined twins is unknown and several hypotheses exist. The morphokinetics in the present case and morphology of other reported cases will be discussed in this context.

Bioburden control in the manufacturing facility is a serious concern regarding biologics due to the possibility of an significant economic impact due to batch failure from a bioburden incident. As a case study on effectively establishing a microbiological environmental monitoring program for cleanrooms, we focused on Time-lapse Shadow Image Analysis as a kind of Rapid Microbiological Method. In this study, the superior rapidity and accuracy were indicated for reference strains and environmental microbial on both 90 mm plate and RODAC plate at 25-30 °C. Especially superior performance in the counting was observed for B.subtilis, P.aeruginosa and A.brasiliensis. The first and the median of colony detection speed for environmental microbial were 12 h and 26 h, respectively. The colony detection rate was 90% at 40 h incubation. Additionally, the characterization of swarming behavior was recognized based on time-lapse image acquisition data at 30 min intervals. This case study indicated that the application for environmental monitoring can contribute to reducing the bioburden excursion risk due to both the rapid detection of colonies and real-time detection for swarming behavior. TSIA would be more acceptable and easier option for biologics due to providing simple interpretations for the results and reducing the time consumption.

We report a case of healthy live birth from a couple diagnosed with oligoasthenoteratozoospermia (OAT). They failed three ICSI cycles in the past 11 years. In the last cycle, the ovarian stimulation was done with antagonist protocol and six oocytes were aspirated. Semen sample was prepared by one layer gradient. 10 class I spermatozoa were selected by motile-sperm organelle-morphology examination (MSOME) according to Cassuto criteria. The selected spermatozoa were then injected into the MII oocytes. 16-18 h after ICSI, three zygotes was formed and, then, cultured in the time lapse monitoring (TLM) for 3 days. Two best embryos were selected according to the morphokinetic status for transfer. A singleton pregnancy was achieved, resulting in the birth of a healthy baby. This successful outcome shows that use of high technologies of MSOME and TLM procedures are useful for selection of the best spermatozoa and embryos in case of severe male infertility.

OBJECTIVE: Can detailed scrutiny of time-lapse imaging (TLI) of inner cell mass (ICM) splitting help to reduce the frequency of multiple pregnancies following elective single embryo transfer (eSET)?
METHODS: Retrospective analysis of time-lapse images of an embryo in vitro, which resulted in a monochorionic triamniotic pregnancy following eSET on Day 5 of development.
RESULTS: A 37-year-old female patient underwent a frozen embryo transfer cycle whereby a single vitrified/warmed embryo was transferred at the hatching blastocyst stage. The subsequent pregnancy scan revealed a monochorionic triamniotic pregnancy. Because the blastocyst was cultured in an incubator incorporating TLI, retrospective scrutiny of the digital recordings demonstrated two distinct ICM structures splitting apart, which formed during the \'8-shaped hatching\'.
CONCLUSIONS: Assisted reproductive techniques and in-vitro culture have been associated with an increased frequency of embryo splitting. This has been postulated to be linked to the in-vitro hatching method observed at the blastocyst stage of development. This case report highlights the need to objectively assess any splitting of the ICM, beyond standard grading of the quality of the ICM and the trophectoderm. Such assessments of ICM splitting should be routine practice in clinical embryology when selecting embryos for transfer.

OBJECTIVE: The purpose of this study was to investigate the cause of repeated multipronucleus (MPN) formation in zygotes in a patient after both in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI).
METHODS: This is a case study. A patient had unexplained primary infertility with recurring total MPN zygotes after IVF and ICSI cycles. Time-lapse monitoring of pronucleus formation was carried out. Embryos developed from MPN zygotes were analyzed by fluorescence in situ hybridization (FISH). Single-cell RNA-seq analysis was used to identify gene expression profiles of the patient\'s oocyte and zygote, and these were compared to the data from oocytes and zygotes from donors with normal fertilization (patient, n = 1; donors, n = 4). Oocyte-specific genes with differential expression were selected by the Amazonia!
METHODS:
RESULTS: From time-lapse analysis, we observed the formation of multiple micronuclei near the site of the second polar body extrusion. These micronuclei migrated, expanded, and juxtaposed with the male pronucleus leading to a multipronucleus. None of these MPN zygotes could develop to the blastocyst stage, and FISH analysis revealed a chaotic chromosomal complement in the arrested embryos. RNA-seq analysis showed 113 differentially expressed genes (DEGs) between the patient and the donor oocytes and zygotes. Moreover, 25 of the 113 DEGs were unique or highly expressed in oocytes and early embryos. From 25 DEGs, three genes, DYNC2LI1, NEK2, and CCNH, which are involved in meiosis and the chromosome separation process, were further validated by real-time PCR.
CONCLUSIONS: We identified several candidate genes affecting pronucleus formation as a new cause of infertility.

OBJECTIVE: To report time-lapse monitoring of human oocytes in which the damaged zona pellucida was removed, producing zona-free (ZF) oocytes that were cultured until the blastocyst stage in time-lapse incubators.
METHODS: Retrospective case series.
METHODS: Private infertility clinic.
METHODS: Infertile patients (n = 32) undergoing minimal ovarian stimulation or natural cycle IVF treatment between October 2012 and June 2014.
METHODS: Intracytoplasmic sperm injection (ICSI) fertilization of ZF oocytes, prolonged embryo culture in time-lapse incubators, elective vitrification, and subsequent single vitrified-thawed blastocyst transfer (SVBT).
METHODS: Rate of fertilization, cleavage and blastocyst development, live-birth rate per SVBT cycle.
RESULTS: In spite of advanced maternal age (39 ± 4.2; range, 30-46 years), good fertilization (94%), cleavage (94%), and blastocyst development rates (38%) were reached after fertilization and culturing of ZF oocytes/embryos. All thawed ZF blastocysts survived, and up to this date seven SVBT transfers were performed, yielding three (43%) term live births with healthy newborns.
CONCLUSIONS: Time-lapse imagery gives a unique insight into the dynamics of embryo development in ZF embryos. Moreover, our case series demonstrate that an oocyte with a damaged zona pellucida that has been removed could be successfully fertilized with ICSI, cultured until blastocyst stage in a time-lapse incubator and vitrified electively for subsequent use.

BACKGROUND: A standard assessment of embryo morphology at given time points does not always allow to transfer the embryo with the highest implantation potential. The effect of transfer of an improper embryo results in a lack of pregnancy or a miscarriage and, as a consequence, exposes the patient to unnecessary emotional stress and necessity to perform yet another transfer of frozen embryos. We present a case of a patient with earlier IVF failures. The use of time-lapse technique in this case helped to choose two good embryos. The transfer resulted in ongoing twin pregnancy.
METHODS: A 35-year-old woman with history of IVF-ET treatment failure was deemed eligible for an ICSI procedure because of the male factor. Ovarian stimulation was performed according to the agonist long protocol. Eight MII oocytes were fertilized and seven embryos were obtained. Continuous embryo monitoring was performed with the use of Primo Vision system. Forty-four hours after fertilization only 2 correctly developing embryos were identified. They were transferred on day 3. The development of the remaining 5 embryos was arrested. These embryos did not achieve the blastocyst stage on day 5-6 after fertilization. Forty days after embryo transfer a twin pregnancy, confirmed with fetal heart rate of both fetuses, was revealed on ultrasound examination. Currently the patient is at 27 weeks of ongoing twin gestation.
CONCLUSIONS: The system of continuous embryo monitoring introduces new criteria for the examination of embryo development. These new parameters can be useful in clinical practice. However prospective randomized studies are necessary to provide data confirming the usefulness of time-lapse technique in IVF treatment.

Time-lapse imaging is increasingly applied as an adjunct to reproductive medicine. The gained information of the morphological and morphokinetic variables before the onset of transcription are supposed to be good predictors for the selection of the best embryo for transfer and are often seen in line with clinical outcomes. This retrospective case series investigated the outcome of transferred blastocysts that did not fulfil the proposed embryo scores at early cleavage or at later stages of development. The observations were made by time-lapse imaging. This study reports the birth of 16 healthy children after day-5 blastocyst transfer, of which at least one of the transferred embryos originated from deviant morphology and/or kinetic cleavage patterns. This case series suggests that some blastocysts derived from embryos with poor conventional morphological score and/or suboptimal morphokinetics can be successfully transferred and might result in live births. Such results might raise awareness that discarding embryos based only on early events is not a suitable approach to give patients the chance to conceive. In conclusion, to date only the transfer of viable embryos after culturing them until day 5 guarantees optimal embryo selection and helps to prevent embryo wastage.