Identification and rescue of a novel TUBB8 mutation that causes the first mitotic division defects and infertility.

PURPOSE: Tubulin beta eight class VIII (TUBB8) is essential for oogenesis, fertilization, and pre-implantation embryo development in human. Although TUBB8 mutations were recently discovered in meiosis-arrested oocytes of infertile females, there is no effective therapy for this gene mutation caused infertility. Our study aims to further reveal the infertility-causing gene mutations in the patient's family and to explore whether the infertility could be rescued by optimizing the conditions of embryo culture and finally achieve the purpose of making the patient pregnant. METHODS: Whole-exome sequence analysis and Sanger sequencing were performed on patients' family members to screen and identify candidate mutant genes. Construction of plasmids, in vitro transcription, microinjection of disease-causing gene cRNA, and immunofluorescence staining were used to recapitulate the infertility phenotype observed in patients and to understand the pathogenic principles. Simultaneously, overexpression of mutant and wild-type cRNA of the candidate gene in mouse oocytes at either germinal vesicle (GV) or metaphase II (MII) stage was performed in the rescue experiment. RESULTS: We first identified a novel heritable TUBB8 mutation (c.1041C>A: p.N347K) in the coding region which specifically affects the first mitosis and causes the developmental arrest of early embryos in a three-generation family. We further demonstrated that TUBB8 mutation could lead to abnormal spindle assemble. And moreover, additional expression of wild-type TUBB8 cRNA in the mouse oocytes in which the mutant TUBB8 were expressed can successfully rescue the developmental defects of resulting embryo and produce full-term offspring. CONCLUSIONS: Our study not only defines a novel mutation of TUBB8 causing the early cleavage arrest of embryos, but also provides an important basis for treating such female infertility in the future.

Dosage effects of ZP2 and ZP3 heterozygous mutations cause human infertility.

The zona pellucida (ZP) is an extracellular matrix universally surrounding mammalian eggs, which is essential for oogenesis, fertilization, and pre-implantation embryo development. Here, we identified two novel heritable mutations of ZP2 and ZP3, both occurring in an infertile female patient with ZP-abnormal eggs. Mouse models with the same mutations were generated by CRISPR/Cas9 gene editing system, and oocytes obtained from female mice with either single heterozygous mutation showed approximately half of the normal ZP thickness compared to wild-type oocytes. Importantly, oocytes with both heterozygous mutations showed a much thinner or even missing ZP that could not avoid polyspermy fertilization, following the patient's pedigree. Further analysis confirmed that precursor proteins produced from either mutated ZP2 or ZP3 could not anchor to oocyte membranes. From these, we conclude that ZP mutations have dosage effects which can cause female infertility in humans. Finally, this patient was treated by intracytoplasmic sperm injection (ICSI) with an improved culture system and successfully delivered a healthy baby.

Vitrification of day 3 cleavage-stage embryos yields better clinical outcome in comparison with vitrification of day 2 cleavage-stage embryos.

The objective of this retrospective study was to determine an optimal time point for vitrification of cleavage-stage human embryos. This study included patients who were undergoing day 2 or day 3 vitrified-warmed cleavage-stage embryo transfer at the In Vitro Fertilization (IVF) Programme of the Shanghai First Maternity and Infant Hospital, China, affiliated to the Tongji University School of Medicine, from April 2010 to March 2012. Intervention was made for the entire cohort of vitrified embryos for poor responder patients so as to avoid severe ovarian hyperstimulation syndrome. Embryo survival rate (SR) after vitrification-warming, implantation rate (IR), and clinical pregnancy rate (CPR) were the main outcome measurements. In total, 380 vitrified-warmed cleavage-stage embryo transfer (VWT) cycles were included. We found that the SR after vitrification and warming for day 2 embryos and day 3 embryos were 92.7% and 92.8%, respectively. For poor ovarian responders, the IR of day 2 and day 3 vitrified-warmed embryos was 6.4% and 13.2%, respectively (P = 0.186). The CPR for day 3 vitrified-warmed embryos was significantly higher than that of day 2 vitrified-warmed embryos (17.6 vs. 4.0% per transfer cycle, P = 0.036). For patients who had their entire cohort of embryos vitrified to prevent severe ovarian hyperstimulation syndrome (OHSS), the IR and CPR were not significantly different for day 2 and day 3 vitrified-warmed embryo transfer. In conclusion, for vitrified-warmed embryo transfer, cryopreservation of the entire cohort of embryos on day 3 resulted in better clinical outcomes compared with cryopreservation on day 2. Therefore, it is highly recommended that cleavage-stage embryos should be vitrified on day 3, but not on day 2, particularly for poor ovarian responder patients.

[Correlation between IVF outcomes and Ureaplasma urealyticum infection in male reproductive tract].

OBJECTIVE: To investigate the influence of Ureaplasma urealyticum (Uu) infection in the male reproductive tract on the outcomes of IVF and the clinical significance of preoperative Uu test by analyzing the correlation between the results of Uu culture before IVF-ET and the outcomes of IVF-ET. METHODS: Among 1,059 couples undergoing IVF-ET, we selected 973 after excluding genetic factors and divided them into a Uu negative and a Uu positive group according to the results of culture of Uu in the semen of the males. We compared the rates of IVF fertilization, oocyte cleavage, clinical pregnancy and abortion between the two groups, and analyzed the influence of Uu infection on IVF outcomes. RESULTS: Among the 973 selected subjects, 836 were Uu negative (group A) and 137 Uu positive (group B), and of the latter, 130 were restored to Uu negative after treatment (group B1) and the other 7 remained unchanged (group B2). No significant differences were found between groups A and B in the rates of IVF fertilization (81.6% vs 79.8%, P = 0.13), abnormal fertilization (11.8% vs 12.4%, P = 0.58) and oocyte cleavage (92.0% vs 92.1%, P = 0.94), nor between groups A and B2 (81.6% vs 89.8%, P = 0.10; 11.8% vs 13.2%, P = 0.75; 92.0% vs 92.5%, P = 0.10). Totally, 747 of the patients underwent embryo transfer, including 643 in group A and 104 in group B. There were no significant differences between groups A and B in the rates of clinical pregnancy (38.6% vs 34.7%, P = 0.44) and abortion (16.5% vs 22.2%, P = 0.39), nor between groups A and B2 (38.6% vs 33.3%, P = 0.79; 16.5% vs 0, P = 0.53). CONCLUSION: Uu infection in the male reproductive tract does not significantly affect the rates of IVF fertilization, oocyte cleavage, clinical pregnancy and abortion. However, more investigations with larger sample sizes of the cases restored from Uu positive to Uu negative are needed to lend further support to our findings.

[Progesterone induction keeps a balanced mitochondrial activity and a low ROS productivity in human sperm].

OBJECTIVE: To observe the relative activity of sperm mitochondria and the proportion of ROS-positive sperm before and after capacitation and progesterone (Pg)-induced hyperactivation, and investigate the functional characteristics of sperm mitochondria. METHODS: We collected 20 samples of normal human spermatozoa that met the criteria of WHO Laboratory Manual for the Examination and Processing of Human Semen (5th ed) and cultured them with the swim-up method in a CO2 incubator at 37 degrees C for 1 hour. We divided the sperm into a pre-capacitation and a capacitated group, and further incubated the capacitated sperm in an upright tube with (Pg-induced group) or without (control group) slow-releasing Pg at 37 degrees C for another hour. Then we determined the relative activity of mitochondria and the percentage of ROS-positive cells in the sperm samples using JC-1 and DCF staining. RESULTS: The relative activities of mitochondria were significantly increased in the capacitated, control and Pg-induced groups (6.23, 14.36 and 12.33) as compared with the pre-capacitation group (1.42) (P < 0.05), while the percentages of balanced mitochondria (mitochondria with equal amount of high and low electric potentials) remarkably reduced (4.27%, 5.03% and 8.57% vs 21.64%, P < 0.05). The percentages of ROS-positive sperm in the pre-capacitation, capacitated, control and Pg-induced groups were 2.89%, 0.70%, 4.25% and 1.90%, respectively, significantly lower in the capacitated than in the pre-capacitation group (P < 0.01), but dramatically increased in the control group after another hour of swim-up incubation and markedly higher than in the Pg-induced group (P < 0.01). CONCLUSION: Progesterone induction can hyperactive human sperm motility, inhibit the relative activity of mitochondria, keep mitochondria potential at a more balanced level, and reduce the production of ROS, which may help to raise the rate of in vitro fertilization and improve the quality of embryos.

[Vitrification of oocytes for in vitro fertilization and embryo transfer].

OBJECTIVE: To explore the feasibility, indication and method of oocyte vitrification during the IVF - ET procedure, so as to increase the utilization of oocytes and reduce oocyte waste. METHODS: This study included the patients whose husbands failed to provide sperm samples at the time of oocyte pickup or from whom more than 25 oocytes were obtained. With the patients' consent, some of their oocytes were subjected to cryopreservation by vitrification, and used for IVF - ET after thawed. RESULTS: Totally, 53 oocytes from 7 patients were thawed, and 44 (83.02%) survived, of which 41 M II oocytes were subjected to ICSI and 32 (72.73%) were fertilized. Thirty embryos were formed, with a cleavage rate of 93.75%. Sixteen embryos were transferred in 9 cycles, with achievement of 2 clinical pregnancies and delivery of 3 healthy babies. The implantation rate was 18.75% and the live birth rate 22.22%. Seven of the embryos were still cryopreserved. CONCLUSION: Cryopreservation of oocytes by vitrification effects satisfactory rates of survival and fertilization, and that of surplus oocytes can increase oocyte utilization and adds to the alternatives for IVF - ET.

Aberrant H3K4me3 modification of epiblast genes of extraembryonic tissue causes placental defects and implantation failure in mouse IVF embryos.

Assisted reproductive technology has been widely applied in the treatment of human infertility. However, accumulating evidence indicates that in vitro fertilization (IVF) is associated with a low pregnancy rate, placental defects, and metabolic diseases in offspring. Here, we find that IVF manipulation notably disrupts extraembryonic tissue-specific gene expression, and 334 epiblast (Epi)-specific genes and 24 Epi-specific transcription factors are abnormally expressed in extraembryonic ectoderm (ExE) of IVF embryos at embryonic day 7.5. Combined histone modification analysis reveals that aberrant H3K4me3 modification at the Epi active promoters results in increased expression of these genes in ExE. Importantly, we demonstrate that knockdown of the H3K4me3-recruited regulator Kmt2e, which is highly expressed in IVF embryos, greatly improves the development of IVF embryos and reduces abnormal gene expression in ExE. Our study therefore identifies that abnormal H3K4me3 modification in extraembryonic tissue is a major cause of implantation failure and abnormal placental development of IVF embryos.

Raman Spectrum of Follicular Fluid: A Potential Biomarker for Oocyte Developmental Competence in Polycystic Ovary Syndrome.

Polycystic ovary syndrome (PCOS) is a common endocrine and metabolic disorder in reproductive women where abnormal folliculogenesis is considered as a common characteristic. Our aim is to evaluate the potential of follicular fluid (FF) Raman spectra to predict embryo development and pregnancy outcome, so as to prioritize the best promising embryo for implantation, reducing both physiological and economical burdens of PCOS patients. In addition, the altered metabolic profiles will be identified to explore the aetiology and pathobiology of PCOS. In this study, follicular fluid samples obtained from 150 PCOS and 150 non-PCOS women were measured with Raman spectroscopy. Individual Raman spectrum was analyzed to find biologic components contributing to the occurrence of PCOS. More importantly, the Raman spectra of follicular fluid from the 150 PCOS patients were analyzed via machine-learning algorithms to evaluate their predictive value for oocyte development potential and clinical pregnancy. Mean-centered Raman spectra and principal component analysis (PCA) showed global differences in the footprints of follicular fluid between PCOS and non-PCOS women. Two Raman zones (993-1,165 cm-1 and 1,439-1,678 cm-1) were identified for describing the largest variances between the two groups, with the former higher and the latter lower in PCOS FF. The tentative assignments of corresponding Raman bands included phenylalanine and ß -carotene. Moreover, it was found that FF, in which oocytes would develop into high-quality blastocysts and obtain high clinical pregnancy rate, were detected with lower quantification of the integration at 993-1,165 cm-1 and higher quantification of the integration at 1,439-1,678 cm-1 in PCOS. In addition, based on Raman spectra of PCOS FF, the machine-learning algorithms via the fully connected artificial neural network (ANN) achieved the overall accuracies of 90 and 74% in correctly assigning oocyte developmental potential and clinical pregnancy, respectively. The study suggests that the PCOS displays unique metabolic profiles in follicular fluid which could be detected by Raman spectroscopy. Specific bands in Raman spectra have the biomarker potential to predict the embryo development and pregnancy outcome for PCOS patients. Importantly, these data may provide some valuable biochemical information and metabolic signatures that will help us to understand the abnormal follicular development in PCOS.

Precise allele-specific genome editing by spatiotemporal control of CRISPR-Cas9 via pronuclear transplantation.

Gene-targeted animal models that are generated by injecting Cas9 and sgRNAs into zygotes are often accompanied by undesired double-strand break (DSB)-induced byproducts and random biallelic targeting due to uncontrollable Cas9 targeting activity. Here, we establish a parental allele-specific gene-targeting (Past-CRISPR) method, based on the detailed observation that pronuclear transfer-mediated cytoplasmic dilution can effectively terminate Cas9 activity. We apply this method in embryos to efficiently target the given parental alleles of a gene of interest and observed little genomic mosaicism because of the spatiotemporal control of Cas9 activity. This method allows us to rapidly explore the function of individual parent-of-origin effects and to construct animal models with a single genomic change. More importantly, Past-CRISPR could also be used for therapeutic applications or disease model construction.

Genome transfer for the prevention of female infertility caused by maternal gene mutation.

Poor oocyte quality is associated with early embryo developmental arrest and infertility. Maternal gene plays crucial roles in the regulation of oocyte maturation, and its mutation is a common cause of female infertility. However, how to improve oocyte quality and develop effective therapy for maternal gene mutation remains elusive. Here, we use Zar1 as an example to assess the feasibility of genome transfer to cure maternal gene mutation-caused female infertility. We first discover that cytoplasmic deficiency primarily leads to Zar1-null embryo developmental arrest by disturbing maternal transcript degradation and minor zygotic genome activation (ZGA) during the maternal-zygotic transition. We next perform genome transfer at the oocyte (spindle transfer or polar body transfer) and zygote (early pronuclear transfer or late pronuclear transfer) stages to validate the feasibility of preventing Zar1 mutation-caused infertility. We finally demonstrate that genome transfer either at the oocyte or at the early pronuclear stage can support normal preimplantation embryo development and produce live offspring. Moreover, those pups grow to adulthood and show normal fertility. Therefore, our findings provide an effective basis of therapies for the treatment of female infertility caused by maternal gene mutation.

Polarization microscopy imaging for the identification of unfertilized oocytes after short-term insemination.

OBJECTIVE: To develop a unique approach using polarization microscopy (PM) to determine whether the presence of a spindle can be used as an indicator associated with fertilization failure 5 hours after short-term insemination. DESIGN: Observational study. SETTING: Assisted reproduction center. PATIENT(S): Eighty-five patients undergoing short-term insemination. INTERVENTION(S): Oocytes imaged via PM at 4, 5, and 6 hours after standard insemination. MAIN OUTCOME MEASURE(S): Spindle visualization and fertilization rate, with rescue intracytoplasmic sperm injection (ICSI) results determined by rates of normal fertilization, abnormal fertilization, and good-quality embryo formation. RESULT(S): After standard insemination, comparisons of spindle visualization at three time points indicated that the predictive accuracy rates were 84.30% at 5 hours, 86.80% at 6 hours, and 62.20% at 4 hours, with the rates at 5 and 6 hours statistically significantly higher than at 4 hours. A spindle was present in 242 of the 788 metaphase-II oocytes 5 hours after insemination, and there were 204 failed fertilizations on day 1. The positive predictive value was 0.84. After rescue ICSI, the abnormal fertilization rate of the polar body group (assessed using the polar body visualization method) was statistically significantly higher than that of the PM group (assessed using the spindle visualization method) and the regular ICSI group (9.37%, 5.88%, and 4.87%, respectively). CONCLUSION(S): The presence of a spindle 5 hours after insemination in in vitro fertilization is an accurate indicator of unfertilized oocytes. Spindle imaging combined with rescue measures effectively prevents fertilization failure and decreases the polyspermy rate.