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+.

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.

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.

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.

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.

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.

Evaluation of cytogenetic analysis for clinical preimplantation diagnosis.

OBJECTIVE: To evaluate the feasibility of using cytogenetic analysis in preimplantation diagnosis. DESIGN: Two different biopsy protocols (chemical drilling and zona cutting) and two fixation methods were tested in a mouse model. Afterwards, the efficiency of obtaining chromosome preparations from untransferable human embryos depending on the method used to obtain the blastomeres (embryos biopsy or removal of the zona pellucida and blastomere disaggregation) was determined. The chances of obtaining chromosome preparations depending on the type of embryo (haploid, diploid, triploid, and apparently unfertilized) were also evaluated. RESULTS: Results from the mouse model showed that chemical drilling yields better results than cutting in terms of metaphases per biopsied embryo and surviving rate after biopsy. In human embryos, biopsy of diploid embryos produced 46.6% chromosome preparations, while 29% were obtained after blastomere disaggregation and 20.4% when biopsying triploid embryos. CONCLUSIONS: These results suggest that the disaggregating procedure and triploid embryos cannot be considered as good models to assess the feasibility of cytogenetic analysis in preimplantation diagnosis. Poor chromosome quality and loss during fixation are the main problems to use cytogenetics in preimplantation diagnosis; a combination of cytogenetics and other techniques is suggested in cases of balanced translocations.

The genotoxic effect of beta-propiolactone on mammalian oocytes.

beta-Propiolactone (beta PL) has been tested on preimplantation mouse embryos for possible genotoxic effects. Tests were performed at different stages of meiosis (late prophase I, diakinesis/metaphase I, anaphase I, telophase I/prophase II and metaphase II) by injecting females at various times after the induction of superovulation. Male and female derived chromosome complements from first-cleavage embryos were analysed before syngamy for cytogenetic abnormalities. A higher proportion of diploid oocytes, produced by the non-extrusion of the first or second polar body, was found after fertilization when the compound was administered immediately before metaphase I or II. No obvious effect was detected at any other time of beta PL exposure. Based on these results, several possible modes of action for beta PL are postulated.

Chromosome constitution of highly motile mouse sperm.

In this study, we address the relationship between motility and genetic content of mouse sperm. The chromosome complements of highly motile mouse sperm, selected using the swim-up technique, were analyzed after in vitro fertilization, at the first cleavage state. They were compared to those of unselected sperm. Identification of male and female chromosome sets was possible because of their differential condensation at the first mitotic division. In vitro fertilization, swim-up separation, chromosome preparation, and staining were carried out using standard techniques. The results indicate that highly motile mouse sperm did not differ in types and frequencies of chromosomal abnormalities from those not selected for motility. Moreover, separation of motile sperm does not deviate the sex ratio from the theoretical 1:1.

Does Ca2+/Mg(2+)-free medium have an effect on the survival of the preimplantation mouse embryo after biopsy?

The way in which the different technical aspects of clinical preimplantation genetic diagnosis affect the survival of the biopsied embryo is not well understood. One of these aspects is the influence of Ca2+/Mg(2+)-free medium on the developmental capacity of the biopsied embryo and the biopsied cell itself. Therefore, we used an experimental design involving 4-cell mouse embryos to evaluate the effect of performing the biopsy procedure under such conditions. Our results indicate that the use of Ca2+/Mg(2+)-free medium has no detrimental effect (at least in mouse embryos) on the viability of the biopsied embryos and their biopsied cells when used during relatively short times of exposure.

Sexual immaturity and maternal age: incidence of aneuploidy and polyploidy in first-cleavage mouse embryos.

The influence of maternal age on the incidence of aneuploidy and polyploidy was studied, using C57Bl/6J X CBA/Ca hybrid mice, including immature females, as gamete donors. The age of the females ranged from 3.5-4 wk (immature or prepubertal), to 10-12 wk (young adults), to 24-28 wk (aged females). Ovulation was induced with gonadotrophins, and the differential condensation of paternal and maternal chromosomes was used to elucidate the origin of chromosome abnormalities in first-division metaphase plates. The results indicated a high incidence of aneuploid oocytes in immature and older female mice, as compared to young adult females. Eggs of immature female mice underwent polyspermic fertilization more often than those of young adults and older females, and the production of diploid oocytes was more frequent in immature females than in the other age groups.

Premature chromosome condensation as a sign of oocyte immaturity.

In this work we report the possibility that oocyte immaturity is associated with premature chromosome condensation (PCC) after in-vitro fertilization (IVF). Using a murine model, we have related PCC and endoreduplicated-like oocytes to oocyte immaturity as a basis for a prognosis in oocyte immaturity problems. The cytogenetic analysis was performed in 511 embryos obtained from immature oocytes that were directly fertilized in vitro and in 1363 embryos obtained from immature oocytes that were matured in vitro with different concentrations of human chorionic gonadotrophin (HCG) added to the culture medium. As a control we used 507 embryos obtained from freshly ovulated oocytes. PCC at the G1-phase-(G1-PCC) was observed only when immature oocytes were immediately fertilized in vitro (45.4%) and PCC at the S-phase (S-PCC) only when using in-vitro matured oocytes with the highest HCG concentration (3.3%). Neither G1-PCC nor S-PCC were found in the control group. Endoreduplicated-like oocytes appeared in a significant percentage (27.3%) only in the immature group. Immature oocytes yielded a low fertilization rate (16.6%) while in-vitro maturation seemed to confer a higher fertilization capacity compared to the control group (90.1% versus 78.2%). The possible correlation between PCC and oocyte immaturity provides new prospects in the determination of human IVF failures of unknown origin. Thus, when a problem of oocyte immaturity is diagnosed through the presence of PCC, a special programme of in vitro oocyte maturation, such as a longer preincubation time or addition of hormones to the media, would be recommended.

Chromosomes of mouse embryos in vivo and in vitro: effect of manipulation, maternal age and gamete ageing.

The use of human material does not allow the determination of the influence of different parameters on the genetic characteristics of the embryos derived from in-vitro fertilization (IVF) techniques. Using a murine model we assessed the effect of gamete manipulation, maternal age and oocyte ageing on the chromosome complements of the embryos. We have found a positive correlation between all these parameters and the incidence of chromosome abnormalities in first-cleavage mouse embryos obtained by IVF.

The chromosome complement of first-cleavage mouse embryos after in vitro fertilization.

A comparison of the first cleavage-stage chromosome complements of 1022 in vivo fertilized mouse embryos and 1033 in vitro fertilized mouse embryos is reported. The chromosome analysis of first-cleavage embryos allows us to study directly the chromosome complement of the sperm and oocyte that contribute to the embryo, since both chromosome clusters remain separate when an antimitotic agent is used to prevent syngamy. In this paper we show that the sex ratio and the incidence of aneuploidy are similar, irrespective of the fertilization system used. Male and female gametes have the same levels of aneuploidy. Triploidy is more frequent in the in vitro fertilized embryos and the difference can be ascribed to a higher incidence of polyspermy and diploid spermatozoa.

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.

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.

Chromosome studies in oocytes and zygotes from an IVF programme.

Chromosome studies have been carried out in 117 oocytes, 17 one-cell zygotes and four, two- to four-cell zygotes in our IVF programme. Three apparently unfertilized oocytes were, in fact, diploid zygotes. Two of 14 apparently polyspermic zygotes were also diploid. One zygote with four pronuclei was pentaploid. This indicates that pronuclei can either be confused with other cytoplasmic structures, like vacuoles, or be eliminated. Endoreduplication was observed in one tetraploid, apparently polyspermic zygote, and in one two-cell degenerated zygote. The incidence of aneuploidy in unfertilized oocytes, taken as twice the level of hyperhaploidy, was 15.4%. Five oocytes showed fragmented metaphase II chromosomes (4.3%). The incidence of unreduced oocytes, due to a lack of extrusion of the first polar body, was 6.8%. Thus the total number of potentially aneuploid, polyploid or non-viable zygotes due to chromosome aberrations in the oocyte was 26.5%.

Zona pellucida surface of immature and in vitro matured mouse oocytes: analysis by scanning electron microscopy.

PURPOSE: The aim of this work was to determine the morphology of the zona pellucida surface of immature and in vitro matured mouse oocytes by scanning electron microscopy. For this purpose two groups of immature oocytes (germinal vesicle group and metaphase I group) were studied either before or after in vitro maturation. RESULTS: Before in vitro maturation, the germinal vesicle immature group showed mainly an unstructured zona pellucida surface with smooth cumulus cells. The metaphase I immature group showed a more structured zona pellucida with smooth or blebbing cumulus cells. After in vitro maturation, development of the zona pellucida toward a mature surface, related to the initial degree of oocyte maturity, was observed in both groups. CONCLUSIONS: These observations show a correlation between the morphology of the zona pellucida surface and the degree of oocyte maturity; the in vitro maturation process can give rise to a proper development of this endowment when immature oocytes are used.

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.