Assisted oocyte activation effects on the morphokinetic pattern of derived embryos.

OBJECTIVE: Assisted oocyte activation (AOA) can restore fertilization rates after IVF/ICSI cycles with fertilization failure. AOA is an experimental technique, and its downstream effects remain poorly characterized. Clarifying the relationship between AOA and embryo, morphokinetics could offer complementary insights into the quality and viability of the embryos obtained with this technique. The aim of this study is to compare the preimplantation morphokinetic development of embryos derived from ICSI-AOA (experimental group) vs. ICSI cycles (control group). METHODS: A retrospective cohort study was carried out with 141 embryos from fresh oocyte donation cycles performed between 2013 and 2017; 41 embryos were derived from 7 ICSI-AOA cycles and 100 embryos from 18 ICSI cycles. Morphokinetic development of all embryos was followed using a time-lapse system. RESULTS: We show that embryos from both groups develop similarly for most milestones, with the exception of the time of second polar body extrusion (tPB2) and the time to second cell division (t3). CONCLUSIONS: We conclude that ionomycin mediated AOA does not seem to affect the morphokinetic pattern of preimplantation embryo development, despite the alterations found in tPB2 and t3, which could directly reflect the use of a Ca2+ ionophore as a transient and quick non-physiologic increase of free intracytoplasmic Ca2+.

High reliability of morphokinetic annotations among embryologists.

STUDY QUESTION: Are morphokinetic measurements of time lapse-videos of human embryos comparable among operators? SUMMARY ANSWER: There is little variation among morphokinetic measurements taken by different operators when analyzing the same time lapse-videos of human embryos. WHAT IS KNOWN ALREADY: Morphokinetic analysis of preimplantation embryo development is a complementary method of embryo assessment increasingly used in IVF laboratories. Time-lapse videos of embryo development are normally viewed by trained embryologists and annotated with the times when specific developmental events occur. Such annotations form the basis of embryo selection algorithms, used to rank the embryos for transfer. It is unknown whether the reliability of morphokinetic annotations is related to the morphological characteristics of the analyzed embryo or to the ability of the embryologists performing the annotation. One study so far reported the reliability of morphokinetic annotations among three embryologists using the time-lapse system (TLS), but larger studies with different setups are needed to address this issue further. STUDY DESIGN SIZE DURATION: A prospective study was carried out between October 2015 and June 2016. Six embryologists with various degrees of experience in static, morphology-based evaluation, individually annotated the same 93 videos of preimplantation development, corresponding to 18 IVF/ICSI cycles, recorded with a TLS. PARTICIPANTS/MATERIALS SETTING METHODS: Times of second polar body extrusion, appearance and disappearance of pronuclei, and embryo cleavages (times from 2-cell to 5-cell stage: t2, t3, t4, t5) were annotated. Each embryologist was blinded to the annotations of the others. Intra- and inter-observer agreement was evaluated by computing intra-class correlation coefficients (ICCs). MAIN RESULTS AND THE ROLE OF CHANCE: In the inter-observer analysis, most ICCs obtained were higher than 0.80, indicating a high level of agreement: t2: 0.93; t3: 0.80; t4: 0.89; t5: 0.89; disappearance of two pronuclei: 0.98. However, the ICCs obtained for second polar body extrusion and the appearance of two pronuclei annotations was lower: 0.51 and 0.63, respectively, indicating an average level of agreement. The ICCs obtained from the intra-observer analysis were also higher than 0.80 (t2: 0.96; t3: 0.89; t4: 0.88; t5: 0.86; disappearance of two pronuclei: 0.96). The ICCs obtained from second polar body extrusion and the appearance of two pronuclei annotations were 0.77 and 0.66, respectively. These results indicate that developmental timings, annotated in time-lapse videos, are highly reliable both within and among observers. LIMITATIONS REASONS FOR CAUTION: The events at the developmental stages from 6-cells to blastocyst were not evaluated; since some morphokinetic algorithms use times past the 6-cell stage in their calculations, further studies should be carried out to understand the variations among observers in these cases. WIDER IMPLICATIONS OF THE FINDINGS: Time-lapse measurement should be as objective as possible, especially for the first embryo cleavages, because they are often measured to define algorithms to assess the embryonic implantation potential. Our results show that measurements using this particular TLS are consistent and reliable both within and among operators. STUDY FUNDING/COMPETING INTERESTS: None. TRIAL REGISTRATION NUMBER: Not applicable.

Female human pluripotent stem cells rapidly lose X chromosome inactivation marks and progress to a skewed methylation pattern during culture.

STUDY HYPOTHESIS: Does a preferential X chromosome inactivation (XCI) pattern exist in female human pluripotent stem cells (hPSCs) and does the pattern change during long-term culture or upon differentiation? STUDY FINDING: We identified two independent phenomena that lead to aberrant XCI patterns in female hPSC: a rapid loss of histone H3 lysine 27 trimethylation (H3K27me3) and long non-coding X-inactive specific transcript (XIST) expression during culture, often accompanied by erosion of XCI-specific methylation, and a frequent loss of random XCI in the cultures. WHAT IS KNOWN ALREADY: Variable XCI patterns have been reported in female hPSC, not only between different hPSC lines, but also between sub-passages of the same cell line, however the reasons for this variability remain unknown. Moreover, while non-random XCI-linked DNA methylation patterns have been previously reported, their origin and extent have not been investigated. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: We investigated the XCI patterns in 23 human pluripotent stem cell (hPSC) lines, during long-term culture and after differentiation, by gene expression analysis, histone modification assessment and study of DNA methylation. The presence and location of H3K27me3 was studied by immunofluorescence, XIST expression by real-time PCR, and mono- or bi-allelic expression of X-linked genes was studied by sequencing of cDNA. XCI-specific DNA methylation was analysed using methylation-sensitive restriction and PCR, and more in depth by massive parallel bisulphite sequencing. MAIN RESULTS AND THE ROLE OF CHANCE: All hPSC lines showed XCI, but we found a rapid loss of XCI marks during the early stages of in vitro culture. While this loss of XCI marks was accompanied in several cases by an extensive erosion of XCI-specific methylation, it did not result in X chromosome reactivation. Moreover, lines without strong erosion of methylation frequently displayed non-random DNA methylation, which occurred independently from the loss of XCI marks. This bias in X chromosome DNA methylation did not appear as a passenger event driven by clonal culture take-over of chromosome abnormalities and was independent of the parental origin of the X chromosome. Therefore, we suggest that a culture advantage conferred by alleles on the X chromosome or by XCI-related mechanisms may be at the basis of this phenomenon. Finally, differentiated populations inherited the aberrant XCI patterns from the undifferentiated cells they were derived from. LIMITATIONS, REASONS FOR CAUTION: All hPSC lines in this study were cultured in highly similar conditions. Our results may therefore be specific for these conditions and alternative culture conditions might lead to different findings. Our findings are only a first step towards elucidating the molecular events leading to the phenomena we observed. WIDER IMPLICATIONS OF THE FINDINGS: Our results highlight the significant extent of aberrant XCI in female hPSC. The fact that these aberrations are inherited by the differentiated progeny may have a significant impact on downstream research and clinical uses of hPSC. In order to achieve the full potential of hPSC, more insight into the XCI status and its stability in hPSC and its effect on the properties of the differentiated progeny is needed. LARGE SCALE DATA: Not applicable. STUDY FUNDING AND COMPETING INTERESTS: Our research is supported by grants from the Research Foundation - Flanders (FWO-Vlaanderen, grant 1502512N), Generalitat de Catalunya (2014SGR-005214) and the Methusalem grant of the Research Council of the Vrije Universiteit Brussel, on name of K.S. L.V.H. is funded by EMBO (ALTF 701-2013). The authors declare no potential conflict of interest.

How does vitrification affect oocyte viability in oocyte donation cycles? A prospective study to compare outcomes achieved with fresh versus vitrified sibling oocytes.

STUDY QUESTION: How does vitrification affect oocyte viability? SUMMARY ANSWER: Vitrification does not affect oocyte viability in oocyte donation cycles. WHAT IS KNOWN ALREADY: Oocyte vitrification is performed routinely and successfully in IVF and oocyte donation programs. STUDY DESIGN, SIZE, DURATION: This is a prospective study performed between June 2009 and February 2012 to compare ongoing pregnancy rates and other indices of viability between fresh and vitrified oocytes. A total of 99 donations with more than 16 oocytes (MII) in which oocytes were allocated both to a synchronous recipient (fresh oocytes) and to an asynchronous recipient (vitrified oocytes) were included. PARTICIPANTS/MATERIALS, SETTING, METHODS: The participants were consenting couples (donors and recipients) from the oocyte donation program. On the day of retrieval, the oocytes allocated to the synchronous recipient were inseminated and those allocated for banking were denuded of cumulus and vitrified. Vitrified oocytes were microinjected with spermatozoa 2 h after warming. Embryo transfer was performed on Day 2 of development in both groups, and the remaining embryos were cryopreserved on Day 3. Clinical pregnancy was defined by a positive fetal heartbeat at 6 weeks. MAIN RESULTS AND ROLE OF CHANCE: A total of 989 oocytes were warmed and 85.6% survived. No significant differences were observed between fresh and vitrified oocytes: fertilization rate (80.7 versus 78.2%), ongoing embryo rate (71.0 versus 68.2%) or good-quality embryo rate (54.1 versus 49.8%). The mean number of embryos transferred was similar in both groups (1.82 ± 0.44 versus 1.90 ± 0.34). The implantation rate (33.3 versus 34.0%) and the multiple pregnancy rate (27.7 versus 20.8) were also similar between both groups (P > 0.05). The live birth rate per cycle was 38.4% in the recipients of fresh oocytes and 43.4% in the recipients of vitrified oocytes (P > 0.05). Eighty five frozen embryo transfers were also evaluated. Comparing embryos from fresh and vitrified oocytes there were no significant differences in the embryo survival rate (70.1 versus 65.8%), clinical pregnancy rate (40.8 versus 33.3%) or implantation rate (21.8 versus 26.8%). LIMITATIONS, REASONS FOR CAUTION: The oocytes were donated by healthy, young women (≤35 years) and these results cannot be extrapolated to other populations. WIDER IMPLICATIONS OF THE FINDINGS: Outcomes obtained with vitrified oocytes are as good as with fresh oocytes and the use of vitrification can be extended to new applications, e.g. accumulation of oocytes from successive stimulations for preimplantation genetic diagnosis, for patients at risk of ovarian hyperstimulation syndrome or in patients needing to preserve their fertility. STUDY FUNDING/COMPETING INTEREST(S): This work was done under the auspices of the Càtedra d'Investigació en Obstetrícia i Ginecologia of the Universitat Autònoma de Barcelona.

Preimplantation genetic diagnosis in patients with male meiotic abnormalities.

Indications and candidates for preimplantation genetic diagnosis (PGD) have increased in recent years. This study evaluates whether IVF-intracytoplasmic sperm injection (ICSI) results could be improved by selecting embryos through PGD-AS (aneuploidy screening) in couples in whom the male partner presents meiotic abnormalities. Two hundred and fifty-six embryos were biopsied and 183 were suitable for analysis (73.2%). Ninety-two embryos showed normal chromosomal analysis (50.3% of the analysed embryos and 57.5% of the diagnosed embryos). Pregnancy, abortion and implantation rates were compared with 66 IVF-ICSI cycles performed in 44 patients with meiotic abnormalities without PGD (control group). No statistically significant differences in the pregnancy rate (52 versus 43.9%), implantation rate (32.1 versus 23.5%) and miscarriage rate (15.4 versus 10.3%) were observed between the groups. Although the embryos obtained from men with meiotic abnormalities showed a high frequency of chromosome abnormalities, no improvements in pregnancy and implantation rates were obtained after PGD-AS in the series analysed.

Preimplantation genetic screening and human implantation.

In recent years, preimplantation genetic screening (PGS) has been used and recommended to increase the implantation rate in older women or in couples with previous assisted reproduction (ART) failures, to try to increase pregnancy rates in couples with recurrent abortions, to prevent the transmission of chromosome anomalies to the offspring of carriers of balanced chromosomal rearrangements, or even to try to decrease the incidence of trisomic births in older women. So far, PGS has contributed to increase the implantation rate in older women; however, the rate of clinical pregnancies has not increased, either in older women or in couples with previous ART failures. In couples with recurrent abortions, the pregnancy rate seems to increase, but only when the woman is young (< or =35). In carriers of balanced reorganizations, the prognosis is poor. Attempts to decrease the birth of trisomic children to older women are difficult to evaluate. This absence of relevant results is not related to the technique itself, which is quite safe, but to other still largely unknown factors.

Assessment of the proportion of transgene-bearing sperm by fluorescence in situ hybridization: a novel approach for the detection of germline mosaicism in transgenic male founders.

Genetic mosaicism is frequent among transgenic animals produced by pronuclear microinjection. A successful method for the screening of founder animals for germline mosaicism prior to mating would greatly reduce the costs associated with the propagation of the transgenic lines, and improve the efficiency of transgenic livestock production. With this aim, we have devised a simple method to detect integrated transgenes in individual spermatozoa using fluorescence in situ hybridization (FISH). The experiments reported here were undertaken to investigate the efficiency of this FISH-based approach to accurately evaluate the proportion of transgene-bearing sperm and to be applied for the detection of potential germline mosaics. Sperm samples from mice homozygous and hemizygous for a beta-lactoglobulin transgene were analyzed in a first set of experiments. A high hybridization efficiency was achieved, and the proportions of transgene-positive sperm cells in both homozygous (94.8-98.2%) and hemizygous (49.8-51.9%) animals were close to the expected frequencies (100 and 50%, respectively). To evaluate the sensitivity of the assay more directly, simulated mosaic samples with 5, 10, 15, 20 and 40% of transgene-bearing spermatozoa were then prepared and analyzed by FISH. Significant differences in the frequency of transgene-positive sperm were observed between all mosaic samples, indicating that even small deviations (5%) from the expected 50% transgene transmission rate in a founder animal could be reliably detected with our assay. Therefore, the method proposed represents a novel approach for the identification of germline mosaic founder males in livestock transgenic projects and a much more economic and faster alternative to breeding.

Preimplantation genetic diagnosis.

Preimplantation genetic diagnosis (PGD) includes a variety of techniques that have been developed to detect the transmission to the offspring of genetic diseases or of chromosome abnormalities by couples at risk before a pregnancy is established, to avoid these couples the risk of recurrent abortions and/or of repeated terminations of pregnancy. Candidate couples are carriers of gene mutations or of structural chromosome rearrangements, or with recurrent spontaneous abortions of unknown origin. Diagnostic procedures include different modalities of gene amplification using the polymerase chain reaction (PCR) or of fluorescent in situ hybridization (FISH). Embryo biopsies are carried out at the 6-8 cell stage. Healthy embryos are transferred on day 4 or at the blastocyst stage. By now, several hundred healthy children have been born using PGD, and only one diagnostic error has been reported.

The decision to cancel a preimplantation genetic diagnosis cycle.

It has been suggested that a minimum number (six) of cumulus-oocyte complexes (COCs) should be retrieved for fertilization to offer enough chances to ensure a pregnancy after a preimplantation genetic diagnosis (PGD) procedure. Therefore a decision to cancel a PGD cycle should be adequately weighted to offer the patients the highest chances to obtain a pregnancy. We describe a case where, after retrieving only three COCs suitable for fertilization, a triplet pregnancy was obtained. This case suggests that, although low numbers of COCs can reduce the effectiveness of the PGD procedure, other factors are involved in its final result. Thus, the opportunity of routinely cancelling such cycles should be reconsidered. In addition, this is, to our knowledge, the first case where sex selection was carried out to prevent the birth of carriers of the abnormal gene, and not of affected offspring.

Is there a place for preimplantation genetic diagnosis screening in recurrent miscarriage patients?

Chromosomal abnormalities are one of the factors known to interfere with normal embryo development; thus preimplantation genetic diagnosis (PGD) for chromosome anomalies may be a new tool for improving the pregnancy rate in selected groups of patients. Embryos from three groups of patients (control, aged and recurrent miscarriage patients) were screened by PGD using specific DNA probes for chromosomes 13, 16, 18, 21, 22, X and Y. The control and aged groups were included in the PGD study because the women carried sex-linked genetic disease. The frequencies of chromosome anomalies observed in older women (46.3%) and in recurrent miscarriage patients (53%) were significantly higher (P < 0.05) than the frequency in the control group (19.3%). After PGD screening and transfer of normal embryos, pregnancies were obtained in women who had undergone repeated abortions (approximate 25% pregnancy rate per transfer) but not in older women. On the basis of these data, it seems that PGD screening of embryos can help some women undergoing repeated abortions, as these techniques allow successful pregnancies to be obtained or, if no pregnancy can be obtained, the results may help the couple to decide whether to enter an embryo or gamete donation programme.

Effect of 6 beta-methylprednisolone on mouse pregnancy rate.

The aim of this study was to evaluate the effect of methyl-prednisolone on the pregnancy rate in mice. For this reason, zona pellucida-intact and zona pellucida-free embryos at the blastocyst stage were transferred to recipient mice at day 2.5 of pseudopregnancy. Embryo transfer was performed into non-immunodepressed and immunodepressed groups of recipient mice using 0.3 or 0.6 microgram/g of 6 beta-methylprednisolone. A higher implantation and developmental rate of zona-free embryos transferred to the immunodepressed group of recipients was observed after using the higher dose of methylprednisolone.

Distribution of alpha3, alpha5 and alpha(v) integrin subunits in mature and immature human oocytes.

The distribution of three integrin subunits, alpha3, alpha5 and alpha(v), in immature and mature human oocytes has been examined using immunofluorescence and confocal microscopy. The results demonstrate that both alpha5 and alpha(v) are present at the germinal vesicle stage, while alpha3 was only detected in oocytes after germinal vesicle breakdown, in metaphase I and II stage oocytes. The cortical concentration of integrin subunits alpha3 and alpha5 is consistent with their localization in the oolemma. In contrast, the homogeneous distribution of alpha(v) throughout the oocyte suggests the existence of cytoplasmic reservoirs of this protein in the oocyte.

FISH preimplantation diagnosis of chromosome aneuploidy in recurrent pregnancy wastage.

PURPOSE: Our purpose was to detect aneuploidy for chromosomes 13, 16, 18, 21, 22, X, and Y in preimplantation embryos from patients with a history of unexplained recurrent miscarriage. METHODS: Three patients with a history of unexplained recurrent spontaneous abortion were included in this study. Embryos were biopsied at the eight-cell stage, individually fixed on slides, and processed for fluorescent in situ hybridization (FISH). A multiple FISH protocol for seven chromosomes pairs (13, 16, 18, 21, 22, X, and Y) has been developed. RESULTS: A total of 39 embryos was studied with the multiple FISH protocol developed. Successful analysis of the biopsied embryos was achieved within the time limits usually allowed in a preimplantation diagnosis program. Analysis of the blastomeres showed that 17 embryos were chromosomally normal for the probes used, 16 embryos were aneuploid, and in 6 embryos no informative results were obtained. CONCLUSIONS: In the patients studied, a large proportion of embryos (41%) exhibited chromosomal abnormalities for the probes used. Preimplantation diagnosis to screen for chromosome abnormalities could be a feasible approach to improve the possibility of successful pregnancy in these couples.

Recurrent in vitro fertilization failure evaluated by fluorescence in situ hybridization: a case report.

OBJECTIVE: To present a case of IVF failure evaluated by fluorescence in situ hybridization (FISH). DESIGN: Case report. SETTING: Research university laboratory and clinical IVF laboratory. PATIENT(S): An infertile couple with recurrent IVF failure. INTERVENTION(S): Fluorescence in situ hybridization study of the complete cohort of "zygotes" obtained at the third IVF attempt. MAIN OUTCOME MEASURE(S): Fluorescence in situ hybridization studies of chromosomes X, Y, 13, 18, and 21. RESULT(S): All the recovered putative zygotes were abnormal for the expected ploidy, presumably as a result of abnormal oocytes. CONCLUSION(S): Fluorescence in situ hybridization techniques represent a promising approach to analyze zygotes that fail to divide normally in vitro and eggs that fail to become fertilized. In cases of recurrent IVF failure, the results of FISH could be used to counsel couples and thus to help them choose among methods other than IVF for assisted reproduction.

Plasma membrane and cytocortex alterations in frozen/thawed mouse embryos deprived of the zona pellucida.

We analyzed the morphological and ultrastructural abnormalities present in two-cell mouse embryos frozen/thawed without the zona pellucida (ZP). Transmission electron microscopy revealed that these embryos had plasma membrane abnormalities that were not observed in the embryos frozen/thawed with an intact ZP. The most frequent anomalies were a decreased number of microvilli with a nonhomogeneous distribution and showing an abnormal morphology and the presence of an increased number of vesicles in the periphery of the cell. The distribution of actin filaments, studied by fluorescence microscopy revealed alterations in both embryos, frozen/ thawed with ZP and embryos frozen/thawed without ZP: an increased staining in some regions of the peripheral actin band, discontinuities or gaps in the band, or the presence of a second actin band connected to the peripheral one.

Characteristics of actin fibers and ultrastructure of the contact regions involved in the separation of blastomeres of two-cell mouse embryos, frozen-thawed without the zona pellucida.

Freezing of embryos deprived of the zona pellucida (ZP) decreases their survival rate immediately after thawing, and gives rise to the separation of their blastomeres in a high percentage of cases. We have studied the ultrastructure and the characteristics of actin fibers in the cell-to-cell contact region in mouse embryos frozen-thawed without the ZP at the two-cell stage. Our results indicate that most of the embryos that retain their blastomeres united after freezing and thawing show either the presence of a midbody, or a contact region with a close apposition of the plasma membranes but without an organized actin cortex in their contact region. Only a small percentage of embryos that retain their blastomeres united after freezing and thawing show a contact region with widely separated plasma membranes and an organized actin cytocortex.

Expression of caprine beta-lactoglobulin in the milk of transgenic mice.

A 14.5 kb-long transgene containing the complete caprine beta-lactoglobulin gene transcription unit as well as 6.1 kb and 3.7 kb of the 5'- and 3'-flanking regions, respectively, was microinjected into pronuclear stage mouse embryos. Four lines of transgenic mice were obtained, three of them expressing the transgene in their mammary glands during lactation. Western blot analysis of caprine beta-lactoglobulin in the milk of hemizygous transgenic animals demonstrated the presence of the exogenous protein at concentrations up to 0.5 mg ml-1 of mouse milk.

Cryopreservation of caprine beta-lactoglobulin transgenic mouse embryos.

As an economical and safer alternative to the maintenance of transgenic animals as live stocks, transgenic embryo cryobanks can be generated and maintained indefinitely. Two-cell embryos obtained from four lines of caprine beta-lactoglobulin transgenic mice homozygous for the transgene were cryopreserved, and their response to cryopreservation-related stress was investigated. Significant differences between transgenic lines were found in the viability of frozen/thawed transgenic embryos and also in two-cell embryo production after superovulatory treatment of transgenic females. The results of this study suggest that cryopreservation protocols should be assessed on each transgenic line before the generation of transgenic embryo banks.

Laser blastocyst biopsy for preimplantation diagnosis in the human.

A new methodology for blastocyst biopsy that uses a 1.48 microm diode laser is described. Trophectoderm cells are biopsied after laster zona drilling and culture, fixed and processed for fluorescent in situ hybridisation (FISH) analysis. Preliminary results on the efficiency of the procedure and blastocyst recovery rate are promising. Blastocyst laser biopsy is a useful tool in preimplantation genetic diagnosis (PGD) as it allows a more reliable diagnosis and widens the diagnostic possibilities on account of the higher number of cells obtained in the biopsy.

Origin of tripronucleate zygotes after intracytoplasmic sperm injection.

Zygotes morphologically classified as tripronuclear (3PN) after intracytoplasmic sperm injection (ICSI), which are thought to be digynic in their origin, were studied by fluorescent in-situ hybridization (FISH). FISH results allowed us to assess the suspected ploidy after morphological evaluation of the zygote and to determine the origin of the third pronuclei. Our results show that, firstly, 36% of those zygotes classified as 3PN following their morphological evaluation were, in fact, diploid, and secondly, the main cause for triploidy after ICSI is the non-extrusion of the second polar body.