Effect of ovarian stimulation on developmental speed of preimplantation embryo in a mouse model.

Ovarian stimulation protocols are widely used to collect oocytes in assisted reproductive technologies (ARTs). Although the influence of ovarian stimulation on embryo quality has been described, this issue remains controversial. Here, we analyzed the influence of ovarian stimulation on developmental speed and chromosome segregation using live cell imaging. Female mice at the proestrus stage were separated by the appearance of the vagina as the non-stimulation (-) group, and other mice were administered pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) as the stimulation (+) groups. The cumulus-oocyte complexes from both groups were inseminated with sperm suspensions from the same male mice. Fertilization rates and developmental capacities were examined, and the developmental speed and frequency of chromosome segregation errors were measured by live-cell imaging using a Histone H2B-mCherry probe. The number of fertilized oocytes obtained was 1.4-fold more frequent in the stimulation (+) group. The developmental rate and chromosome stability did not differ between the groups. Image analysis showed that the mean speed of development in the stimulation (+) group was slightly higher than that in the non-stimulation (-) group. This increase in speed seemed to arise from the slight shortening of the 2- and 4-cell stages and third division lengths and consequent synchronization of cleavage timing in each embryo, not from the emergence of an extremely rapidly developing subpopulation of embryos. In conclusion, ovarian stimulation does not necessarily affect embryo quality but rather increases the chances of obtaining high-quality oocytes in mice.

Beyond appearance: Can morphologically low-grade euploid blastocysts yield successful pregnancies?

Purpose: The primary objective of this investigation is to evaluate how morphological quality affects the pregnancy outcomes in euploid embryos determined by preimplantation genetic testing for aneuploidies (PGT-A). Concurrently, as a secondary objective, we aim to identify which specific aspects of morphological evaluation exert the most significant impact on these outcomes. Methods: A retrospective analysis of 451 single euploid embryo transfer cycles at our clinic was conducted. Embryos were evaluated based on the degree of blastocyst expansion, inner cell mass (ICM), trophectoderm (TE) morphology, and the day of blastocyst vitrification. Outcomes between morphologically low-grade and high-grade embryos were compared. Additionally, the study analyzed which morphological factors most influenced pregnancy outcomes. Results: Pregnancy outcomes were significantly lower in morphologically low-grade blastocysts compared to high-grade ones. Among the morphological evaluations, the ICM assessment was significantly associated with the live birth rate. Conclusion: Our study indicates that the morphological quality of euploid embryos, particularly the evaluation of the ICM, plays a crucial role in IVF-ET success.

Intraperitoneal administration of mouse kisspeptin-10 to mice during estrus stage induces pseudopregnancy.

A simple method for producing pseudopregnant mice supports pup production. In this study, pregnant ICR were obtained mice without mating with vasectomized mice via administration of mouse Kisspeptin-10 (mKp-10) and transferring blastocysts to the uterus. Blastocyst transfer after mKp-10 administration to mice with gapping and reddish pink vagina resulted in 65.2% (15/23) pregnancies, and 39.1% (34/87) of the transferred blastocysts showed full-term growth. Vaginal smears were observed for accurate estrus cycle determination, and subsequent administration of mKp10 to mice during the estrus stage and blastocyst transfer resulted in 95.2% (20/21) pregnancies and 50.7% (104/205) birth rates. Regarding 2-cell transfer after administration of mKp-10, 100% (8/8) of the mice became pregnant, and 45.0% (36/80) of the embryos were born. Administration of mKp-10 to mice during the estrus stage is a convenient way to generate pseudopregnant mice.

Cryptozoospermia: Should we use ejaculated sperm or surgically retrieved sperm for assisted reproductive technology?

Purpose: In cryptozoospermic subjects, it may often may be difficult to secure motile sperm for assisted reproductive technology (ART). We examined the results of ART with frozen thawed ejaculated sperm in cryptozoospermic subjects and evaluated whether sperm retrieval surgery is necessary for such patients in our clinic. Methods: Between 2013 and 2021, we evaluated 197 cryptozoospermic patients. Age, endocrine panel at the time of the initial semen analysis, and anti-müllerian hormone levels at the time of the spouse's first egg retrieval were examined. Cryopreservation of ejaculated motile sperm collected essentially weekly over a 3-month period was carried out. ART data recorded was the number of egg retrieval cycles, normal fertilization rate, and clinical pregnancy rate. Results: ART using frozen sperm as well as sperm ejaculated on the day of egg retrieval was possible in all cases. The normal fertilization rate was 70.4%, the clinical pregnancy rate per embryo transferred was achieved in 31.5% (870 cycles), and the live birth rate per case was 73.8%. Conclusions: Intracytoplasmic sperm injection (ICSI) was possible without sperm retrieval surgery in cryptozoospermia, resulting in 73.8% of live births per patient. Sperm identification, sperm processing, and ICSI technique are especially important in cryptozoospermia. Sperm retrieval surgery can be avoided in cryptozoospermic patients.

Evaluation of the serum zinc concentration in male infertility patients: an analysis of 2010 cases.

Zinc is an important trace element, and its importance for male infertility has been reported. The aim of the study was to assess whether the serum zinc concentrations were related to semen quality in male infertility patients. In 2010 subjects who consulted at our male infertility clinic between November 2018 and May 2021, serum zinc concentrations were assessed along with age, sperm concentration, sperm motility, endocrine panel, and body mass index (BMI). A normal zinc concentration was observed in 1069 (53.2%), subclinical deficiency in 845 (42.0%), and deficiency in 79 subjects (3.9%). On the other hand, high a zinc level was observed in only 17 subjects (0.9%). The serum zinc concentration did not relate with age, sperm concentration, sperm motility, luteinizing hormone (LH), follicle stimulating hormone (FSH), testosterone, and body mass index (BMI). However, normozoospermic subjects showed significantly higher zinc concentrations than among azoospermic (included non-obstructive; NOA and obstructive; OA) and cryptozoospermic patients. Furthermore, the zinc concentration was lower in NOA subjects when comparing to oligozoospermia and/or asthenozoospermia. An association between zinc concentration and semen analysis remained unclear. This study was cross-sectional and retrospective, however, this is a largest investigation of the zinc concentration during reproductive life span in Japan. Further accumulation of cases are required to further examine the potential relationship between zinc concentration and semen quality.

Asynchronous division at 4-8-cell stage of preimplantation embryos affects live birth through ICM/TE differentiation.

To improve the performance of assisted reproductive technology, it is necessary to find an indicator that can identify and select embryos that will be born or be aborted. We searched for indicators of embryo selection by comparing born/abort mouse embryos. We found that asynchronous embryos during the 4-8-cell stage were predisposed to be aborted. In asynchronous mouse embryos, the nuclear translocation of YAP1 in some blastomeres and compaction were delayed, and the number of ICMs was reduced. Hence, it is possible that asynchronous embryos have abnormal differentiation. When the synchrony of human embryos was observed, it was confirmed that embryos that did not reach clinical pregnancy had asynchrony as in mice. This could make synchrony a universal indicator common to all animal species.

Micronucleus formation during early cleavage division is a potential hallmark of preimplantation embryonic loss in cattle.

In assisted reproductive technology (ART)-derived embryos of non-rodent mammals, including humans and cattle, chromosome segregation errors are highly likely to occur during early cleavage division, resulting in aneuploidy, including mosaicism. However, the relationship between chromosomal segregation errors during early cleavage and subsequent embryonic development has not been detailed in these mammals. In the present study, we developed non-invasive live-cell imaging of chromosome segregation dynamics using a histone H2B-mCherry mRNA probe in bovine preimplantation embryos. Chromosome segregation errors in early cleavage affected blastocyst formation. Especially, embryos that underwent abnormal chromosome segregation (ACS) with multiple or large micronucleus formation rarely developed into blastocysts. Embryos with the severe ACS had prolonged cell cycle duration. After transfer of blastocysts with live-cell imaging of chromosome segregation to ten cows, six became pregnant and four of them gave full-term offspring. Interestingly, two of them were derived from blastocysts with ACS. Hence, chromosomal segregation errors with micronucleus formation during early cleavage can be a fatal hallmark of preimplantation embryogenesis in cattle. This technique has shown potential for understanding the relationship between chromosome segregation error and subsequent embryo development, and for selecting viable ART-derived embryos for medical and livestock production.

Search for morphological indicators that predict implantation by principal component analysis using images of blastocyst.

Background: Although the current evaluation of human blastocysts is based on the Gardner criteria, there may be other notable parameters. The purpose of our study was to clarify whether the morphology of blastocysts has notable indicators other than the Gardner criteria. Methods: To find such indicators, we compared blastocysts that showed elevated human chorionic gonadotropin (hCG) levels after transplantation (hCG-positive group; n = 129) and those that did not (hCG-negative group; n = 105) using principal component analysis of pixel brightness of the images. Results: The comparison revealed that the hCG-positive group had grainy morphology and the hCG-negative group had non-grainy morphology. Classification of the blastocysts by this indicator did not make a difference in Gardner score. Interestingly, all embryos with ≥20% fragmentation were non-grainy. The visual classification based on this analysis was significantly more accurate than the prediction of implantation using the Gardner score ≥3BB. As graininess can be used in combination with the Gardner score, this indicator will enhance current reproductive technologies.

Oocyte cytoplasmic diameter of ≥130 µm can be used to determine human giant oocytes.

OBJECTIVE: To determine if a cytoplasmic diameter of ≥130 µm can help identify human giant oocytes (GOs) in clinical practice and confirm the presence of genetic abnormalities in GOs by assessing the spindle length and centromere numbers. DESIGN: Case-control study. SETTING: Private in vitro fertilization clinic. PATIENT(S): The subjects were women aged 20-49 years who underwent oocyte retrieval after ovarian stimulation from January 2014 to December 2020. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The oocyte diameter was measured; immunofluorescent staining was performed to assess the spindle diameter and centromere numbers. RESULT(S): Among the 254,337 oocytes examined, 561 (0.22%) had a diameter of ≥130 µm. The mean diameter ranges in the normal-sized metaphase II (MII) oocytes (MII group) and GO group were 103.0-119.0 and 132.3-175.9 µm. Spindle size could be measured in 6 GOs with 1 spindle (GO1), 10 GOs with 2 spindles (GO2), and 16 MII groups. The equatorial plane and pole-to-pole distance in the GO1 were significantly longer than in the GO2 and MII groups. The median numbers of centromeres were 86 in GOs with 1 spindle and 42 in each spindle for GOs with 2 spindles among 11 GO1s and 5 GO2s. CONCLUSION(S): This study is the first to define GOs as oocytes with a diameter of ≥130 µm and is a large-scale study surveying the incidence of GO. It is also the first study to analyze and elucidate the relationship between spindle numbers within the cytoplasm of GOs and spindle size and centromeres.

DNA replication fork speed underlies cell fate changes and promotes reprogramming.

Totipotency emerges in early embryogenesis, but its molecular underpinnings remain poorly characterized. In the present study, we employed DNA fiber analysis to investigate how pluripotent stem cells are reprogrammed into totipotent-like 2-cell-like cells (2CLCs). We show that totipotent cells of the early mouse embryo have slow DNA replication fork speed and that 2CLCs recapitulate this feature, suggesting that fork speed underlies the transition to a totipotent-like state. 2CLCs emerge concomitant with DNA replication and display changes in replication timing (RT), particularly during the early S-phase. RT changes occur prior to 2CLC emergence, suggesting that RT may predispose to gene expression changes and consequent reprogramming of cell fate. Slowing down replication fork speed experimentally induces 2CLCs. In vivo, slowing fork speed improves the reprogramming efficiency of somatic cell nuclear transfer. Our data suggest that fork speed regulates cellular plasticity and that remodeling of replication features leads to changes in cell fate and reprogramming.

Chromosome counting in the mouse zygote using low-invasive super-resolution live-cell imaging.

In preimplantation embryos, an abnormal chromosome number causes developmental failure and a reduction in the pregnancy rate. Conventional chromosome testing methods requiring biopsy reduce the risk of associated genetic diseases; nevertheless, the reduction in cell number also reduces the pregnancy rate. Therefore, we attempted to count the chromosomes in mouse embryos using super-resolution live-cell imaging as a new method of chromosome counting that does not reduce the cell number or viability. We counted the 40 chromosomes at the first mitosis by injecting embryos with histone H2B-mCherry mRNA under conditions by which pups could be obtained; however, the results were often an underestimation of chromosome number and varied by embryo and time point. Therefore, we developed a method to count the chromosomes via CRISPR/dCas-mediated live-cell fluorescence in situ hybridization targeting the sequence of the centromere region, enabling us to count the chromosomes more accurately in mouse embryos. The methodology presented here may provide useful information for assisted reproductive technologies, such as those used in livestock animals/humans, as a technique for assessing the chromosomal integrity of embryos prior to transfer.

Gonadotropin levels at the start of ovarian stimulation predict normal fertilization after hCG re-trigger in GnRH antagonist cycles.

PURPOSE: To assess the appropriateness of human chorionic gonadotropin (hCG) re-trigger in poor responders to gonadotropin-releasing hormone agonist (GnRHa) trigger in controlled ovarian stimulation (COS) cycles. METHODS: The 2251 cycles in 2251 patients triggered with GnRHa for oocyte stimulation, with or without requiring hCG re-trigger between 2013 and 2018, were retrospectively analyzed to compare gonadotropin levels at the start of COS and the rate of normal fertilization between the re-trigger and non-re-trigger group. Furthermore, patients in the re-trigger group were stratified by the rate of normal fertilization (good: ≥60% or poor: <60%) to compare patient demographics, hormone profiles, and clinical outcome between the subgroups. RESULTS: In the re-trigger group, FSH and LH levels at the start of COS were significantly lower in the good fertilization group than in the poor fertilization group (P < .01). Receiver operating characteristic curves identified cutoff values of the FSH and LH levels of 1.30 and 0.35 mIU/mL, respectively, for predicting ≥60% normal fertilization. CONCLUSION: Gonadotropin levels at the start of COS are predictors of response to GnRHa trigger and hCG re-trigger necessity, and may serve as indicators to help clinicians appropriately choose hCG re-trigger rather than abandoning the cycles or continuing the first oocyte aspiration attempt.

Chromosome segregation error during early cleavage in mouse pre-implantation embryo does not necessarily cause developmental failure after blastocyst stage.

In the pre-implantation embryo, aneuploidy resulting from chromosome segregation error is considered responsible for pregnancy loss. However, only a few studies have examined the relationship between chromosome segregation errors during early cleavage and development. Here, we evaluated this relationship by live-cell imaging using the histone H2B-mCherry probe and subsequent single blastocyst transfer using mouse embryos obtained by in vitro fertilization. We showed that some embryos exhibiting early chromosomal segregation error and formation of micronuclei retained their developmental potential; however, the error affected the blastocyst/arrest ratio. Further, single-cell sequencing after live-cell imaging revealed that all embryos exhibiting micronuclei formation during 1st mitosis showed aneuploidy at the 2-cell stage. These results suggest that early chromosome segregation error causing micronuclei formation affects ploidy and development to blastocyst but does not necessarily cause developmental failure after the blastocyst stage. Our result suggests the importance of the selection of embryos that have reached blastocysts.

Long-term outcomes of freeze-all strategy: A retrospective analysis from a single ART center in Japan.

PURPOSE: To demonstrate the benefits of the freeze-all strategy for in vitro fertilization treatment based on retrospective analyses. METHODS: Post-thaw embryo survival rates of slow-frozen embryos in 294 cycles and vitrified embryos in 12 195 cycles were assessed. Progesterone (P4) and estradiol (E2) levels per mature oocyte by age category were assessed in 9081 cycles and pregnancy rates with fresh embryo transfer and frozen-thawed embryo transfer by P4 level were assessed in 1535 cycles. RESULTS: The survival rates of frozen-thawed embryos were 92.5% with slow freezing and 99.1% with vitrification. P4 levels on the day of human chorionic gonadotropin (hCG) injection showed a trend toward an increase with age. The pregnancy rate per mature oocyte with fresh embryo transfer decreased dependently upon P4 level, while that with frozen-thawed embryo transfer was not affected by P4 level. The pregnancy rates with frozen-thawed embryo transfer were higher than those with fresh embryo transfer in patients aged 42 years or younger. CONCLUSIONS: The freeze-all strategy is a valuable treatment option which allows the separation of an embryo transfer cycle from an oocyte retrieval cycle, especially for patients with high P4 levels at oocyte retrieval and patients of advanced maternal age.

Live-cell imaging of nuclear-chromosomal dynamics in bovine in vitro fertilised embryos.

Nuclear/chromosomal integrity is an important prerequisite for the assessment of embryo quality in artificial reproductive technology. However, lipid-rich dark cytoplasm in bovine embryos prevents its observation by visible light microscopy. We performed live-cell imaging using confocal laser microscopy that allowed long-term imaging of nuclear/chromosomal dynamics in bovine in vitro fertilised (IVF) embryos. We analysed the relationship between nuclear/chromosomal aberrations and in vitro embryonic development and morphological blastocyst quality. Three-dimensional live-cell imaging of 369 embryos injected with mRNA encoding histone H2B-mCherry and enhanced green fluorescent protein (EGFP)-α-tubulin was performed from single-cell to blastocyst stage for eight days; 17.9% reached the blastocyst stage. Abnormalities in the number of pronuclei (PN), chromosomal segregation, cytokinesis, and blastomere number at first cleavage were observed at frequencies of 48.0%, 30.6%, 8.1%, and 22.2%, respectively, and 13.0%, 6.2%, 3.3%, and 13.4%, respectively, for abnormal embryos developed into blastocysts. A multivariate analysis showed that abnormal chromosome segregation (ACS) and multiple PN correlated with delayed timing and abnormal blastomere number at first cleavage, respectively. In morphologically transferrable blastocysts, 30-40% of embryos underwent ACS and had abnormal PN. Live-cell imaging may be useful for analysing the association between nuclear/chromosomal dynamics and embryonic development in bovine embryos.

Peroxiredoxin as a functional endogenous antioxidant enzyme in pronuclei of mouse zygotes.

Antioxidant mechanisms to adequately moderate levels of endogenous reactive oxygen species (ROS) are important for oocytes and embryos to obtain and maintain developmental competence, respectively. Immediately after fertilization, ROS levels in zygotes are elevated but the antioxidant mechanisms during the maternal-to-zygotic transition (MZT) are not well understood. First, we identified peroxiredoxin 1 (PRDX1) and PRDX2 by proteomics analysis as two of the most abundant endogenous antioxidant enzymes eliminating hydrogen peroxide (H2O2). We here report the cellular localization of hyperoxidized PRDX and its involvement in the antioxidant mechanisms of freshly fertilized oocytes. Treatment of zygotes at the pronuclear stage with H2O2 enhanced pronuclear localization of hyperoxidized PRDX in zygotes and concurrently impaired the generation of 5-hydroxymethylcytosine (5hmC) on the male genome, which is an epigenetic reprogramming event that occurs at the pronuclear stage. Thus, our results suggest that endogenous PRDX is involved in antioxidant mechanisms and epigenetic reprogramming during MZT.

A Simple Method for Transportation of Mouse Embryos Using Microtubes and a Warm Box.

Generally, transportation of preimplantation embryos without freezing requires incubators that can maintain an optimal culture environment with a suitable gas phase, temperature, and humidity. Such incubators are expensive to transport. We reported previously that normal offspring were obtained when the gas phase and temperature could be maintained during transportation. However, that system used plastic dishes for embryo culture and is unsuitable for long-distance transport of live embryos. Here, we developed a simple low-cost embryo transportation system. Instead of plastic dishes, several types of microtubes-usually used for molecular analysis-were tested for embryo culture. When they were washed and attached to a gas-permeable film, the rate of embryo development from the 1-cell to blastocyst stage was more than 90%. The quality of these blastocysts and the rate of full-term development after embryo transfer to recipient female mice were similar to those of a dish-cultured control group. Next, we developed a small warm box powered by a battery instead of mains power, which could maintain an optimal temperature for embryo development during transport. When 1-cell embryos derived from BDF1, C57BL/6, C3H/He and ICR mouse strains were transported by a parcel-delivery service over 3 days using microtubes and the box, they developed to blastocysts with rates similar to controls. After the embryos had been transferred into recipient female mice, healthy offspring were obtained without any losses except for the C3H/He strain. Thus, transport of mouse embryos is possible using this very simple method, which might prove useful in the field of reproductive medicine.

Bidirectional developmental potential in reprogrammed cells with acquired pluripotency.

We recently discovered an unexpected phenomenon of somatic cell reprogramming into pluripotent cells by exposure to sublethal stimuli, which we call stimulus-triggered acquisition of pluripotency (STAP). This reprogramming does not require nuclear transfer or genetic manipulation. Here we report that reprogrammed STAP cells, unlike embryonic stem (ES) cells, can contribute to both embryonic and placental tissues, as seen in a blastocyst injection assay. Mouse STAP cells lose the ability to contribute to the placenta as well as trophoblast marker expression on converting into ES-like stem cells by treatment with adrenocorticotropic hormone (ACTH) and leukaemia inhibitory factor (LIF). In contrast, when cultured with Fgf4, STAP cells give rise to proliferative stem cells with enhanced trophoblastic characteristics. Notably, unlike conventional trophoblast stem cells, the Fgf4-induced stem cells from STAP cells contribute to both embryonic and placental tissues in vivo and transform into ES-like cells when cultured with LIF-containing medium. Taken together, the developmental potential of STAP cells, shown by chimaera formation and in vitro cell conversion, indicates that they represent a unique state of pluripotency.

Latrunculin A treatment prevents abnormal chromosome segregation for successful development of cloned embryos.

Somatic cell nuclear transfer to an enucleated oocyte is used for reprogramming somatic cells with the aim of achieving totipotency, but most cloned embryos die in the uterus after transfer. While modifying epigenetic states of cloned embryos can improve their development, the production rate of cloned embryos can also be enhanced by changing other factors. It has already been shown that abnormal chromosome segregation (ACS) is a major cause of the developmental failure of cloned embryos and that Latrunculin A (LatA), an actin polymerization inhibitor, improves F-actin formation and birth rate of cloned embryos. Since F-actin is important for chromosome congression in embryos, here we examined the relation between ACS and F-actin in cloned embryos. Using LatA treatment, the occurrence of ACS decreased significantly whereas cloned embryo-specific epigenetic abnormalities such as dimethylation of histone H3 at lysine 9 (H3K9me2) could not be corrected. In contrast, when H3K9me2 was normalized using the G9a histone methyltransferase inhibitor BIX-01294, the Magea2 gene-essential for normal development but never before expressed in cloned embryos-was expressed. However, this did not increase the cloning success rate. Thus, non-epigenetic factors also play an important role in determining the efficiency of mouse cloning.