Chromosomal missegregation and aberrant embryo development in repro57 female mice with Rnf212 homozygous mutation.

Repro57 mice, induced with N-ethyl-N-nitrosourea and harboring a mutation in the Rnf212 gene, exhibit infertility in both homozygous mutant males and females. Rnf212 plays a crucial role in recombination and crossover designation. In male repro57 homozygous mutants, spermatocytes often degenerate during late prophase, and mature spermatozoa are absent in the seminiferous epithelium, indicating arrested spermatogenesis as the cause of infertility. Despite reports of infertility in Rnf212 knockout female mice, the specific mechanisms underlying infertility in female repro57 homozygous mutants remain elusive. This study investigates the chromosomal and kinetochore patterns of mature oocytes and their developmental potential following in vitro fertilization in female repro57 homozygous mutant mice. While all wild-type oocytes progress to metaphase II and exhibit euploidy, all repro57 homozygous mutant mouse oocytes display aneuploidy. Additionally, kinetochore distances in repro57 homozygous mutant oocytes exceed those observed in wild-type counterparts. Although no significant differences are noted in fertilization and early embryo development rates between wild-type and repro57 homozygous mutant mice, embryos derived from repro57 homozygous mutants exhibit significantly lower morula and blastocyst rates, accompanied by frequent cytokinesis failure and vacuole formation. These findings suggest that premature segregation of sister chromatids in repro57 homozygous mutant mice adversely impacts later stages of embryo development.

Focusing on the accumulation of chromatin/chromosomes around nucleoli and optimizing the timing of ICSI to facilitate the rescue in vitro maturation of denuded GV stage oocytes.

PURPOSE: This study aims to achieve the methodological improvement of rescue IVM by predicting germinal vesicle breakdown (GVBD) and optimizing the timing of ICSI. METHODS: Time lapse analysis was performed retrospectively to evaluated the relationship between the presence of AC around the nucleoli and GVBD. To find the optimal timing of ICSI, the time from the initiation of the first polar body extrusion to ICSI were measured, and the rates of fertilization at each point were calculated. RESULTS: The GVBD rate of GV stage oocytes with AC around the nucleoli was significantly higher than that of GV stage oocytes without AC. The GV stage oocytes required more time for nuclear maturation after polar body extrusion than MI oocytes, with GV stage oocytes taking 400-600 min from polar body extrusion to the optimal timing of ICSI, while the MI stage oocytes took 200-400 min. The GV stage oocytes resulted in the birth of healthy babies with the appropriate timing of ICSI. CONCLUSION: It was found that GV stage oocytes with AC around nucleoli can initiate GVBD and reach the MII stage with a high rate, and that GV stage oocytes required more time than MI stage oocytes to reach the optimal timing of ICSI. Considering these factors, ART laboratories may employ immature GV stage oocytes in routine ART procedures rather than discarding them.

The effect of betaine for mouse sperm cryopreservation.

Sperm cryopreservation is an effective method of preserving male fertility in humans, as well as domestic and experimental animals. However, various factors such as ice crystal formation, osmotic stress, and oxidative stress, negatively influence the motility and viability of post-thawed spermatozoa. Betaine, which works as an osmoprotectant is known to work as a nontoxic cryoprotectant. However, the protective effects during mouse sperm cryopreservation are still unclear. Thus, the purpose of this study was to investigate whether betaine has protective effects during the process of mouse sperm cryopreservation. In this study, betaine was found to be effective in maintaining sperm motility during the freezing procedure and 1% (85.4 mM) betaine was identified as the optimal concentration to be added to cryopreservation solutions. It was also found that betaine improves the integrity of the plasma membranes of sperm tails, suggesting that betaine has a positive effect on sperm motility.

Nuclear-to-cytoplasmic ratios of 1PN and 2PN zygotes after in vitro fertilization of mouse oocytes.

Numerous studies have reported comparisons of the nuclear-to-cytoplasmic (NC) ratio during mitosis. However, little information is known about how the pronuclear size is regulated and determined at the end of meiosis II in mammalian zygotes. The present study aims to analyze the NC ratio of female and male pronuclei, and also to compare the size of single pronuclei using photographs that were obtained during experiments to create chimeric hermaphrodites from 2-cell oocytes. The volume of both the female and the male pronucleus was found to correlate with the volume of the oocyte cytoplasm. The NC ratio of the male pronucleus was greater than that of the female pronucleus. The NC ratio of the average volume of the female and male pronuclei was greater than the NC ratio of the mononucleate oocytes. The occurrence of 1PN oocytes was significantly higher when the volume of cytoplasm was lower than the cut-off value. These results indicated that the NC ratio is retained during pronuclear formation. A higher NC ratio in male compared with the female pronucleus indicated structural and/or molecular difference between the two pronuclei. 1PN formation may occur when sperm enters close to the MII spindle.

Are tri-pronuclear embryos that show two normal-sized pronuclei and additional smaller pronuclei useful for embryo transfer?

Purpose: This study aimed to analyze whether tripronuclear (3PN) zygotes, with two normal-sized PNs and an additional smaller PN (2.1PN), can be used for embryo transfer. Methods: A retrospective embryo cohort study was conducted on 695 patients who underwent intracytoplasmic sperm injection treatment. Blastocyst formation rates were compared between 2.1PN and 2PN zygotes and PGT-A analysis was performed on 15 blastocysts derived from 2.1PN zygotes. Results: Blastocyst formation rates were comparable between 2.1PN (43.8%) and 2PN zygotes (54.8%; p = 0.212). The rates of blastocysts with good morphology derived from 2.1 PN and 2PN zygotes were 18.8% and 25.5%, respectively. No significant differences were detected (p = 0.383). All of the analyzed blastocysts were diploid; however, 13 of these were found to be aneuploid, with a further two being mosaic. Conclusion: Our results suggest that 2.1PN embryos can reach blastocyst stage. These blastocysts were diploid, however, predominantly aneuploid, and therefore could not be used for embryo transfer.

Clinical outcome of intrauterine infusion of platelet-rich plasma in patients with recurrent implantation failure.

PURPOSE: This study aimed to evaluate the effectiveness of intrauterine infusion of platelet-rich plasma (PRP) before embryo transfer (ET) in recurrent implantation failure (RIF) cases. METHODS: The authors retrospectively analyzed 54 ET cycles involving frozen and thawed high-quality blastocysts after intrauterine PRP infusion between September 2019 and November 2020. All patients had a history of at least two times of implantation failure on ET. A total of 54 patients were categorized into two groups: thin endometrium (39 patients) and unexplained implantation failure (15 patients). In the thin-endometrium group, the endometrial thickness (EMT) was <8.0 mm at cycle days 12-14 in the prior ET cycle. RESULTS: Among the 54 ET cycles after PRP infusion, 31 (57.4%) were positive for human chorionic gonadotropin (hCG) and 27 (50%) achieved clinical pregnancy, which was significantly better than that in prior ET cycles without PRP infusion (27.2% and 9.6%, respectively). The EMT was not increased at ET date on the PRP cycle compared with that in the prior ET cycle in both patient groups. Moreover, EMT was not different between the hCG-positive and hCG-negative groups. CONCLUSION: Although intrauterine PRP infusion had no superior effect on increasing the EMT than conventional therapeutic agents, it resulted in high pregnancy rates in patients experiencing RIF with or without thin endometrium.

A novel system based on artificial intelligence for predicting blastocyst viability and visualizing the explanation.

Purpose: The purpose of the study was to invent and evaluate the novel artificial intelligence (AI) system named Fertility image Testing Through Embryo (FiTTE) for predicting blastocyst viability and visualizing the explanations via gradient-based localization. Methods: The authors retrospectively analyzed 19 342 static blastocyst images with related inspection histories from 9961 infertile patients who underwent in vitro fertilization. Among these data, 17 984 cycles of single-blastocyst transfer were used for training, and data from 1358 cycles were used for testing purposes. Results: The prediction accuracy for clinical pregnancy achieved by a control model using conventional Gardner scoring system was 59.8%, and area under the curve (AUC) was 0.62. FiTTE improved the prediction accuracy by using blastocyst images to 62.7% and AUC of 0.68. Additionally, the accuracy achieved by an ensemble model using image plus clinical data was 65.2% and AUC was 0.71, representing an improvement in prediction accuracy. The visualization algorithm showed brighter colors with blastocysts that resulted in clinical pregnancy. Conclusions: The authors invented the novel AI system, FiTTE, which could provide more precise prediction of the probability of clinical pregnancy using blastocyst images secondary to single embryo transfer than the conventional Gardner scoring assessments. FiTTE could also provide explanation of AI prediction using colored blastocyst images.

Predictive factors influencing pregnancy rate in frozen embryo transfer.

PURPOSE: To evaluate the outcomes of embryo transfer (ET) and to identify the parameters influencing pregnancy outcomes. METHODS: This study included 938 ET cycles involving single frozen and thawed good-quality blastocyst (Gardner grade ≥3BB) between August 2017 and January 2018. The significance of several parameters including endometrial thickness, position of the transferred air bubble, self-evaluation score by physicians, and uterus direction at ET as predictors of clinical pregnancy was evaluated using univariate and multivariate analyses. RESULTS: Among 938 ET cycles, 462 (49.3%) resulted in a clinical pregnancy. Endometrial thickness was positively associated with clinical pregnancy in a linear trend. Between the variable position of the transferred air bubble and clinical pregnancy rate showed a curvilinear relationship. Clinical pregnancy rate was higher in cases with good self-evaluation score, whereas there was no difference between groups with different uterus directions. Univariate analysis of predictive parameters identified endometrial thickness, self-evaluation score by physicians, and position of air bubbles as significant predictors of clinical pregnancy, of which endometrial thickness and position of air bubbles appeared to be independently related to clinical pregnancy. CONCLUSION: Endometrial thickness and the position of transferred air bubbles influenced clinical pregnancy in ET cycles.

Clinical outcomes of MII oocytes with refractile bodies in patients undergoing ICSI and single frozen embryo transfer.

PURPOSE: This study aimed to analyze whether the presence of refractile bodies (RFs) negatively affects fertilization, embryo development, and/or implantation rates following intracytoplasmic sperm injection (ICSI). METHODS: This retrospective embryo cohort study involved a total of 272 patients undergoing ICSI treatment of blastocyst cryopreservation. RESULTS: In the study, no significant differences were found regarding 2PN formation rates between RF(+) (76.5%) and RF(-) oocytes (77.2%). However, the blastocyst formation rate on Day 5 in RF(+) oocytes was 45.8%, which was significantly lower than that of 52.2% in RF(-) oocytes (aOR 0.74, 95% CI 0.59-0.93, P = .011). Implantation rates were also significantly lower in RF(+) oocytes (24.2%) as compared to RF(-) oocytes (42.2%) (aOR 0.46, 95% CI 0.26-0.78, P = .005). Furthermore, the implantation rate of RF(+) oocytes (28.6%), when high-quality blastocysts were transferred, was significantly lower than that of RF(-) oocytes (46.1%) (aOR 0.50, 95% CI 0.25-0.96, P = .043). CONCLUSION: Our results suggest that oocytes with the presence of RFs have a lower potential for blastocyst development. Even when they develop into high-quality blastocysts, the chances of implantation are reduced.

A higher incidence of smooth endoplasmic reticulum clusters with aromatase inhibitors.

PURPOSE: This study aimed to analyze whether a regimen of aromatase inhibitor (AI) could reduce the occurrence of smooth endoplasmic reticulum clusters (sERCs) in oocytes. METHODS: The AI and the clomiphene citrate (CC) regimens were compared, regarding the sERC (+) rates and the serum estradiol and progesterone levels on the date of hCG administration, and the duration of AI, CC, and hMG administration. RESULTS: The occurrence of sERCs in oocytes from patients treated with AI was significantly higher than that in oocytes from those treated with CC. Both the serum estradiol and progesterone levels were found to be significantly higher in sERC (+) than in sERC (-) cycles. With regard to the CC cycles, no significant differences were detected. The duration of AI and hMG administration was longer for sERC (+) than for sERC (-) cycles. CONCLUSION: As AI did not reduce the occurrence of sERCs, the elevation of estradiol may not be the cause of sERC occurrence but a consequence. Considering the higher levels of progesterone and longer duration of hMG in sERC (+) cycles, the negative effects of premature luteinization, which frequently occur with the AI protocol, should be investigated further.

A higher incidence of cleavage failure in oocytes containing smooth endoplasmic reticulum clusters.

PURPOSE: In human oocytes, sERCs are one of the dysmorphic phenotypes that have been reported. Significantly reduced pregnancy rates and a comparatively higher number of abnormities in live births appear to be associated with the presence of sERCs in oocytes. However, some reports have shown that healthy babies can be born, without any reduced pregnancy rates, from oocytes observed to contain sERCs. Thus, the clinical and scientific significance of oocytes that harbor sERCs remains controversial. METHODS: The presence of sERCs was evaluated using a time-lapse system while studying the dynamic changes within oocytes and embryos. Logistic regression analysis was carried out to explore the independent variables for meiotic and mitotic cleavage failure.. RESULTS: The incidence of mitotic cleavage failure and the incidence of meiotic cleavage failure during the second polar body extrusion in oocytes with sERCs were found to be significantly higher than that in oocytes without sERCs. Furthermore, ICSI was found to have a greater frequency of meiotic failure than IVF. CONCLUSIONS: In cases of cleavage failure, an embryonic cell could become tetraploid and may induce abnormal chromosomal configurations. Some cells exposed to cleavage failure may become trophectoderm cells and form placental abnormalities. Even if they develop into trophectoderm cells, the ICM can be susceptible to further cleavage failure and may in turn cause further aneuploidy. For these reasons, it is important to monitor pregnancies and births derived from oocytes that contained sERCs.

Retrospective comparative study of the factors affecting birthweights in frozen-thawed embryo transfer, compared to fresh embryo transfer.

Aim: Several studies have indicated that the cause of the increased birthweight of frozen-thawed embryos was associated with assisted reproductive technology (ART) procedures, such as cryopreservation. In the present study, the mean birthweight of singletons was compared between the ovulatory and hormone replacement therapy (HRT) cycles in order to investigate the primary factor that leads to higher birthweights from frozen-thawed embryo transfer (FET). Methods: This retrospective study was carried out from January 2011 to December 2014 on 2738 singletons who were born at 37-41 weeks' gestation, following ART in a single facility. The mean birthweight of the singletons who were born after a fresh embryo transfer (ET) was compared to the mean birthweight of the singletons who were born after a FET. In the FET cycles, the mean birthweight of the singletons was compared between the ovulatory and HRT cycles. Results: The mean birthweight of the singletons who were born after a FET was significantly higher than that of the singletons who were born after a fresh ET. In the FET cycles, the birthweight from the HRT cycles was significantly higher than that from the ovulatory cycles. Among the HRT cycles, there was no difference between the birthweight of the singleton who were born from a blastocyst transfer and those who were born from a cleavage-stage ET. Conclusion: The primary factor to affect the birthweight of singletons might be the pre/postET hormonal environment of the endometrium and not the stage of the transferred embryo nor the frozen-thawed procedure itself.

Potential of zygotes to produce live births can be identified by the size of the male and female pronuclei just before their membranes break down.

Aim: To determine whether there are differences in size between the male and female pronuclei immediately before the pronuclear membrane breakdown (PNMBD) and to evaluate whether pronuclear size differences influence normal birth rates. Methods: Time-lapse photography was used to measure the size of each pronucleus, while the outcome of 71 frozen-thawed single blastocyst transfers in patients receiving hormone therapy was analyzed retrospectively. The pronuclear measurements were made 4 hours before the PNMBD, corresponding to 16-20 hours after insemination or intracytoplasmic sperm injection, and immediately before the PNMBD. The differences in the areas between the pronuclei in the zygotes that were associated with the live births were compared with those that were associated with the failed pregnancies. Results: The average difference in the area between the pronuclei 4 hours before and immediately before the PNMBD in the patients with a live birth was significantly smaller than in the patients with a failed birth. In addition, the average area difference in the patients with a successful birth was significantly smaller when the measurements were made immediately before the PNMBD, compared with the measurements 4 hours before the PNMBD. Such differences were not detected among the patients who did not achieve a birth. Conclusion: The birth of healthy babies resulted from zygotes that contained pronuclei of similar size when the measurements were made immediately before the PNMBD. Evaluating the size of each pronucleus immediately before the PNMBD provides an effective indicator of the embryo's potential at an early stage of development.

Aggregated chromosomes transfer in human oocytes.

Germinal-vesicle (GV) transfer, spindle-chromosome complex transfer in metaphase-II oocytes and two pronuclei transfer have been evaluated as possible treatments for patients who have mitochondrial diseases. However, GV transfers often lead to heteroplasmy while the other two methods are frequently associated with aneuploidy. The present study used a new method based on the transfer of aggregated chromosomes, which occurs in human oocytes, before the metaphase spindle is established.

Insecticidal activity against rice pest of oxazosulfyl, a novel sulfyl insecticide.

The development and commercialization of new chemical classes of insecticides are important for efficient crop protection, particularly for combatting insecticide resistance and providing sustainable agricultural production. This study reports on oxazosulfyl, a novel "sulfyl" class of insecticide, against a wide range of insect pests of rice. In the laboratory assay, oxazosulfyl showed insecticidal activity against all developmental stages of the brown planthopper Nilaparvata lugens (Stål). Phosphor imaging assays and soil drench bioassays demonstrated good systemic distribution in rice plants. Oxazosulfyl showed insecticidal activity against imidacloprid- and fipronil-resistant field populations of N. lugens, the white-backed planthopper Sogatella furcifera (Horváth), and the small brown planthopper Laodelphax striatellus (Fallén), as well as the respective susceptible strains. No cross-resistance was observed among oxazosulfyl, imidacloprid, and fipronil. Oxazosulfyl with a wide insecticidal spectrum is a potentially useful pest management tool for sustainable rice production.

The formation of aggregated chromatin/chromosomes in mouse oocytes treated with high concentration of IBMX as a model for a chromosome transfer in human.

The presence of cyclic adenosine monophosphate (cAMP) has been considered to be a fundamental factor in ensuring meiotic arrest prior to ovulation. cAMP is regarded as a key molecule in the regulation of oocyte maturation. However, it has been reported that increased levels of intracellular cAMP can result in abnormal cytokinesis, with some MI oocytes leading to symmetrically cleaved 2-cell MII oocytes. Consequently, we aimed to investigate the effects of elevated intracellular cAMP levels on abnormal cytokinesis and oocyte maturation during the meiosis of mouse oocytes. This study found that a high concentration of isobutylmethylxanthine (IBMX) also caused chromatin/chromosomes aggregation (AC) after the first meiosis. The rates of AC increased the greater the concentration of IBMX. In addition, AC formation was found to be reversible, showing that the re-formation of the spindle chromosome complex was possible after the IBMX was removed. In human oocytes, the chromosomes aggregate after the germinal vesicle breakdown and following the first and second polar body extrusions (the AC phase), while mouse oocytes do not have this AC phase. The results of our current study may indicate that the AC phase in human oocytes could be related to elevated levels of intracytoplasmic cAMP.

Chromatin Morphology in Human Germinal Vesicle Oocytes and Their Competence to Mature in Stimulated Cycles.

The search for simple morphological predictors of oocyte quality is an important task for assisted reproduction technologies (ARTs). One such predictor may be the morphology of the oocyte nucleus, called the germinal vesicle (GV), including the level of chromatin aggregation around the atypical nucleolus (ANu)-a peculiar nuclear organelle, formerly referred to as the nucleolus-like body. A prospective cohort study allowed distinguishing three classes of GV oocytes among 135 oocytes retrieved from 64 patients: with a non-surrounded ANu and rare chromatin blocks in the nucleoplasm (Class A), with a complete peri-ANu heterochromatic rim assembling all chromatin (Class C), and intermediate variants (Class B). Comparison of the chromatin state and the ability of oocytes to complete meiosis allowed us to conclude that Class B and C oocytes are more capable of resuming meiosis in vitro and completing the first meiotic division, while Class A oocytes can resume maturation but often stop their development either at metaphase I (MI arrest) or before the onset of GV breakdown (GVBD arrest). In addition, oocytes with a low chromatin condensation demonstrated a high level of aneuploidy during the resumption of meiosis. Considering that the degree of chromatin condensation/compaction can be determined in vivo under a light microscope, this characteristic of the GV can be considered a promising criterion for selecting the best-quality GV oocytes in IVM rescue programs.

Symmetrical division of mouse oocytes during meiotic maturation can lead to the development of twin embryos that amalgamate to form a chimeric hermaphrodite.

BACKGROUND: Gentle compression of mouse oocytes during meiosis-1 prevented the usual extrusion of a small polar body and resulted in the symmetrical division of the ooplasm into two cells of similar size within the zona pellucida. The purpose of our study was to determine whether such cells, equivalent to two small oocytes, were capable of embryonic development and would result in birth following transfer to the uterus. METHODS: IVF of the 2-celled oocytes was performed and the twin intra-zonal embryos were observed. In each case, the two embryos that originated from fertilized cells with two pronuclei were observed to amalgamate and form a single morula and subsequent blastocyst that was transferred to the uterus of a recipient of a different mouse strain. FISH analysis was performed on sectioned paraffin-embedded tissue of the offspring. RESULTS: In symmetrically divided oocytes each cell contained a metaphase II spindle. Both cells were fertilizable and cleaved to form twin embryos within the same zona pellucida. Most twin embryos amalgamated to form a single compacted morula, which progressed to hatched blastocysts that contained a single inner cell mass. In total, 104 of these blastocysts were transferred to 19 mice, two of which became pregnant, resulting in the birth of three offspring. FISH analysis showed that one newborn contained both XX and XY cells. CONCLUSIONS: We found that two small oocytes fertilized within the same zona pellucida to form twin embryos that amalgamate to establish a single chimeric embryo. This may be one mechanism that leads to the formation of a chimeric hermaphrodite when an embryo containing XX cells mixes with its intra-zonal twin containing XY cells.

Developmental trajectory of monopronucleated zygotes after in vitro fertilization when they include both male and female genomes.

OBJECTIVE: To examine the cause of monopronucleated zygote (1PN) formation that includes both maternal and paternal genomes. DESIGN: Retrospective cohort study. SETTING: Private fertility clinic. PATIENT(S): A total of 44 1PN and 726 2-pronuclear zygotes from 702 patients were observed using 2 different time-lapse observation systems. INTERVENTION(S): Previously recorded time lapse data were reviewed to examine the mechanism of 1PN formation. MAIN OUTCOME MEASURE(S): The distance between the position of the second polar body extrusion and the fertilization cone or epicenter/starting position of the cytoplasmic wave was measured, and the consequent data were analyzed. Cytoplasmic waves were confirmed using vector analysis software. RESULT(S): The cut-off value for the difference in the distance between the position of the second polar body extrusion and the fertilization cone or the epicenter/starting position of the cytoplasmic wave was 17 µm (AUC: 0.987, 95% CI: 0.976-0.999) for the Embryo Scope and 18 µm (AUC: 0.972, 95% CI: 0.955-0.988) for the iBIS time-lapse observation systems. CONCLUSION(S): In this study, it was found with a high degree of accuracy that a monopronucleus is formed when the fusion of the sperm takes place within 18 µm from the point of the second polar body extrusion. The theoretical chance of 1PN occurrence after in vitro fertilization is 2.7% when the sperm is considered to be fused anywhere in the plasma membrane of an oocyte.

The Thickness and Density of the Ovarian Tunica Albuginea Increases with Age in Transgender Patients.

It is known that the extracellular matrix structure and composition changes with aging in many organs. Despite this, knowledge on how does the extracellular part of the ovary change with increasing age in women and how those changes might be related to women's loss of fertility is still lacking. For this, we propose that recurrent injury and repair events on the outermost layers of the ovary due to ovulation are partly responsible for those changes women experience with aging. The histological analysis of the ovaries from 18 female-to-male transgender patients revealed that the ovarian tunica albuginea (TA) increases its thickness and density correlatively with increasing age of the patient (r = 0.52 and r = 0.55, P < 0.05 respectively). The increase in thickness is independent of the total androgen dose received and occurs because of the appearance of defined fibrotic areas underneath the TA layer which increase the total distance of dense connective tissue from the ovarian surface. In conclusion, the ovarian TA increases in its thickness and density with aging because of the appearance of fibrotic areas underneath the layer in transgender patients. This fact might contribute to reduce oocyte quality and cause ovulation difficulties in older women.