4-vinylcyclohexene diepoxide induces apoptosis by excessive reactive oxygen species and DNA damage in human ovarian granulosa cells.

4-Vinylcyclohexene diepoxide (VCD) is a hazardous industrial material which is widely used in the production of fragrances, rubber tires, antioxidants, pesticides, flame retardants and plasticizers. Previous studies have shown that exposure to VCD damages the female reproductive system, but the effects and mechanisms of VCD exposure on human granulosa cells are not reported. In this study, we used a human granulosa cell line (SVOG) to explore the effects of VCD exposure and found that VCD exposure had toxic effects on SVOG cells in vitro. VCD exposure led to excessive accumulation of intracellular ROS, caused DNA damage in cells, altered the expression of some key genes related with apoptosis and oxidative stress, and ultimately inhibited the proliferative capacity of granulosa cells, resulting in increased apoptosis. Overall, our findings provide solid evidence showing that VCD exposure produces severe damage to human granulosa cells, which is helpful for understanding the reproductive toxicity of VCD and etiology of infertility.

Continuous light exposure influences luteinization and luteal function of ovary in ICR mice.

With the rapid change of people's lifestyle, more childbearing couples live with irregular schedules (i.e., staying up late) and suffer from decreased fertility and abortion, which can be caused by luteal phase defect (LPD). We used continuous light-exposed mice as a model to observe whether continuous light exposure may affect luteinization and luteal function. We showed that the level of progesterone in serum reduced (p < .001), the number of corpus luteum (CL) decreased (p < .01), and the expressions of luteinization-related genes (Lhcgr, Star, Ptgfr, and Runx2), clock genes (Clock and Per1), and Mt1 were downregulated (p < .05) in the ovaries of mice exposed to continuous light, suggesting that continuous light exposure induces defects in luteinization and luteal functions. Strikingly, injection of melatonin (3 mg/kg) could improve luteal functions in continuous light-exposed mice. Moreover, we found that, after 2 h of hCG injection, the level of pERK1/2 in the ovary decreased in the continuous light group, but increased in the melatonin administration group, suggesting that melatonin can improve LPD caused by continuous light exposure through activating the ERK1/2 pathway. In summary, our data demonstrate that continuous light exposure affects ovary luteinization and luteal function, which can be rescued by melatonin.

Reduction of mtDNA heteroplasmy in mitochondrial replacement therapy by inducing forced mitophagy.

Mitochondrial replacement therapy (MRT) has been used to prevent maternal transmission of disease-causing mutations in mitochondrial DNA (mtDNA). However, because MRT requires nuclear transfer, it carries the risk of mtDNA carryover and hence of the reversion of mtDNA to pathogenic levels owing to selective replication and genetic drift. Here we show in HeLa cells, mouse embryos and human embryos that mtDNA heteroplasmy can be reduced by pre-labelling the mitochondrial outer membrane of a donor zygote via microinjection with an mRNA coding for a transmembrane peptide fused to an autophagy receptor, to induce the degradation of the labelled mitochondria via forced mitophagy. Forced mitophagy reduced mtDNA carryover in newly reconstructed embryos after MRT, and had negligible effects on the growth curve, reproduction, exercise capacity and other behavioural characteristics of the offspring mice. The induction of forced mitophagy to degrade undesired donor mtDNA may increase the clinical feasibility of MRT and could be extended to other nuclear transfer techniques.

Metabolomic and transcriptomic responses of mouse testis to the dextran sulfate sodium induced colitis.

Inflammatory bowel diseases (IBDs), including Crohn's disease (CD) and ulcerative colitis, are widespread in developed countries and gradually increasing in developing countries. Evidences showed that man with CD has a decrease of serum testosterone, but how IBD take effects on testicular testosterone synthesis is not well elucidated. To investigate the effects of IBD on testis, we analyzed testicular metabolome and transcriptome data of the dextran sulfate sodium (DSS) induced IBD mice. As a result, metabolomic data showed that DSS indeed induced androgen decrease in mouse testis. Correspondingly, androgen synthesis associated genes, especially Lhcgr, were down-regulated in DSS testis. From the metabolomic data, we found vitamin intake associated metabolites vitamin B2 and pyridoxamine were significantly decreased, whereas fatty acid metabolism associated molecules N-lauroylglycine and N-decanoylglycine were increased in DSS testis. In addition, we found 8-hydroxy-deoxyguanosine, a DNA oxidative damage marker, and 8-oxoguanine, a molecule responsible for DNA damage repair, were also changed in DSS testis. Simultaneously, our data also showed that DSS up-regulated the expression of meiosis initiation associated gene Stra8 and oxygen transport associated genes in testis. In summary, these results depicted the complex effects of colitis on testis. These metabolites and transcripts changed in DSS testis could be used as potential targets for IBD treatment or symptom relieve.

Correction: Chromosomal variants accumulate in genomes of the spontaneous aborted fetuses revealed by chromosomal microarray analysis.

[This corrects the article DOI: 10.1371/journal.pone.0259518.].

High-Survival Rate After Microinjection of Mouse Oocytes and Early Embryos With mRNA by Combining a Tip Pipette and Piezoelectric-Assisted Micromanipulator.

Utilizing microinjection to introduce biological molecules such as DNA, mRNA, siRNA, and proteins into the cell is well established to study oocyte maturation and early embryo development in vitro. However, microinjection is an empirical technology. The cellular survival after microinjection is mainly dependent on the operator, and an experienced operator should be trained for a long time, from several months to years. Optimizing the microinjection to be highly efficient and quickly learned should be helpful for new operators and some newly established laboratories. Here, we combined the tip pipette and piezo-assisted micromanipulator to microinject the oocyte and early embryos at different stages of mouse. The results showed that the survival rate after microinjection was more than 85% for cumulus-oocyte complex, germinal vesicle oocyte, two-cell, and four-cell embryos, and close to 100% for MII oocyte and zygotes. The high-rate survival of microinjection can save many experimental samples. Thus, it should be helpful in studying some rare animal models such as aging and conditional gene knockout mice. Furthermore, our protocol is much easier to learn for new operators, who can usually master the method proficiently after several training times. Therefore, we would like to publicly share this experience, which will help some novices master microinjection skillfully and save many laboratory animals.

Single-cell RNA sequencing analysis of mouse follicular somatic cells†.

Within the development of ovarian follicle, in addition to cell proliferation and differentiation, sophisticated cell-cell cross talks are established among follicular somatic cells such as granulosa cells (GCs) and theca cells. To systematically reveal the cell differentiation and signal transductions in follicular somatic cells, we collected the mouse follicular somatic cells from secondary to ovulatory stage, and analyzed the single cell transcriptomes. Having data filtered and screened, we found 6883 high variable genes in 4888 single cells. Then follicular somatic cells were clustered into 26 cell clusters, including 18 GC clusters, 4 theca endocrine cell (TEC) clusters, and 4 other somatic cell clusters, which include immune cells and Acta2 positive theca externa cells. From our data, we found there was metabolic reprogramming happened during GC differentiation. We also found both Cyp19a1 and Cyp11a1 could be expressed in TECs. We analyzed the expression patterns of genes associated with cell-cell interactions such as steroid hormone receptor genes, insulin signaling genes, and cytokine/transformation growth factor beta associated genes in all cell clusters. Lastly, we clustered the highly variable genes into 300 gene clusters, which could be used to search new genes involved in follicle development. These transcriptomes of follicular somatic cells provide us potential clues to reveal how mammals regulating follicle development and could help us find targets to improve oocyte quality for women with low fertility.

Double-strand breaks induce short-scale DNA replication and damage amplification in the fully grown mouse oocytes.

Break-induced replication (BIR) is essential for the repair of DNA double-strand breaks (DSBs) with single ends. DSBs-induced microhomology-mediated BIR (mmBIR) and template-switching can increase the risk of complex genome rearrangement. In addition, DSBs can also induce the multi-invasion-mediated DSB amplification. The mmBIR-induced genomic rearrangement has been identified in cancer cells and patients with rare diseases. However, when and how mmBIR is initiated have not been fully and deeply studied. Furthermore, it is not well understood about the conditions for initiation of multi-invasion-mediated DSB amplification. In the G2 phase oocyte of mouse, we identified a type of short-scale BIR (ssBIR) using the DNA replication indicator 5-ethynyl-2'-deoxyuridine (EdU). These ssBIRs could only be induced in the fully grown oocytes but not the growing oocytes. If the DSB oocytes were treated with Rad51 or Chek1/2 inhibitors, both EdU signals and DSB marker γH2A.X foci would decrease. In addition, the DNA polymerase inhibitor Aphidicolin could inhibit the ssBIR and another inhibitor ddATP could reduce the number of γH2A.X foci in the DSB oocytes. In conclusion, our results showed that DNA DSBs in the fully grown oocytes can initiate ssBIR and be amplified by Rad51 or DNA replication.

Why is oocyte aneuploidy increased with maternal aging?

One of the main causes of pregnancy failure and fetus abortion is oocyte aneuploidy, which is increased with maternal aging. Numerous possible causes of oocyte aneuploidy in aged women have been proposed, including cross-over formation defect, cohesin loss, spindle deformation, spindle assembly checkpoint malfunction, microtubule-kinetochore attachment failure, kinetochore mis-orientation, mitochondria dysfunction-induced increases in reactive oxygen species, protein over-acetylation, and DNA damage. However, it still needs to be answered if these aneuploidization factors have inherent relations, and how to prevent chromosome aneuploidy in aged oocytes. Epidemiologically, oocyte aneuploidy has been found to be weakly associated with higher homocysteine concentrations, obesity, ionizing radiation and even seasonality. In this review, we summarize the research progress and present an integrated view of oocyte aneuploidization.

The repair of endo/exogenous DNA double-strand breaks and its effects on meiotic chromosome segregation in oocytes.

Germ cell-derived genomic structure variants not only drive the evolution of species but also induce developmental defects in offspring. The genomic structure variants have different types, but most of them are originated from DNA double-strand breaks (DSBs). It is still not well known whether DNA DSBs exist in adult mammalian oocytes and how the growing and fully grown oocytes repair their DNA DSBs induced by endogenous or exogenous factors. In this study, we detected the endogenous DNA DSBs in the growing and fully grown mouse oocytes and found that the DNA DSBs mainly localized at the centromere-adjacent regions, which are also copy number variation hotspots. When the exogenous DNA DSBs were introduced by Etoposide, we found that Rad51-mediated homologous recombination (HR) was used to repair the broken DNA. However, the HR repair caused the chromatin intertwined and impaired the homologous chromosome segregation in oocytes. Although we had not detected the indication about HR repair of endogenous centromere-adjacent DNA DSBs, we found that Rad52 and RNA:DNA hybrids colocalized with these DNA DSBs, indicating that a Rad52-dependent DNA repair might exist in oocytes. In summary, our results not only demonstrated an association between endogenous DNA DSBs with genomic structure variants but also revealed one specific DNA DSB repair manner in oocytes.

Motility, progressive motility score and DNA integrity of spermatozoa from cold-preserved mouse cauda epididymis.

This study attempted to investigate and validate whether epididymis cold storage could be a suitable alternative for short-term preservation of spermatozoa. Mouse cauda epididymides and spermatozoa were preserved at 4-8°C from 1 day to 6 weeks. From days 1 to 10, motility and fertility were daily examined when motility loss occurred. From week 1, spermatozoa were used for intracytoplasmic sperm injection (ICSI) at weekly intervals to test their fertility, and spermatozoa DNA integrity was determined by comet assay. We found that motility and progressive motility scores gradually decreased with storage time. In nearly all spermatozoa, DNA integrity was maintained from days 1 to 10, but the percentage of spermatozoa with damaged DNA significantly increased from week 2 to week 6. Spermatozoa retained fertility until day 6, although fertility gradually decreased after day 3. From week 1 to week 5, fertilization rates by ICSI were more than 82.69% but decreased gradually after week 3. We found that spermatozoa preserved in the epididymis at 4-8°C had progressively lower motility, fertility and proportion of undamaged DNA, but could still fertilize oocytes. However, all the parameters of cold-preserved spermatozoa were completely inferior to that from cold-preserved cauda epididymides. The results imply that cold storage of cauda epididymides could be conducive to short-term preservation of spermatozoa, and the cold-stored spermatozoa can resist DNA denaturation, which is necessary for maintaining reproductive ability.

Mitogen-activated protein kinase-activated protein kinase 2 is a critical regulator of pig oocyte meiotic maturation.

Mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MK2), a direct substrate of p38 MAPK, plays key roles in multiple cellular processes. In the present study, we showed that MK2 affected not only cumulus expansion, but also the oocyte meiotic cell cycle in porcine oocytes. Inhibition of MK2 arrested oocytes at the germinal vesicle (GV) stage or the prometaphase I/metaphase I stage. Unlike in mouse oocytes, where phosphorylated (p-) MK2 was localised at the minus end of spindle microtubules and close to the spindle poles, in porcine oocytes p-MK2 was concentrated at the spindle equator and localised at the plus end of spindle microtubules. Knockdown or inhibition of MK2 resulted in spindle defects: spindles were surrounded by irregular chromosome non-disjunction or by chromosomes detached from the spindles. MK2 regulated spindle organisation and chromosome alignment by connecting microtubules with kinetochores. In addition, unlike in mitotic cells and meiotic mouse oocytes, the MK2-p38 MAPK pathway may not play an important role during meiotic cell cycle in porcine oocytes. In conclusion, MK2 is an important regulator of porcine oocyte meiotic maturation.

[Endometrial microvessel density for assessing endometrial receptivity during the peri-implantation period].

OBJECTIVE: To evaluate the value of endometrial microvessel density (MVD) in assessing the endometrial receptivity during the peri-implantation period. METHODS: A total of 104 patients undergoing in vitro fertilization and embryo transfer (IVF-ET) treatment were analyzed retrospectively. The subjects were divided into clinical pregnancy group (50 cases) and nonpregnant group (54 cases) according to the IVF-ET outcome. Endometrial tissues were collected 7 days after the natural ovulation prior to IVF-ET for measurement of the endometrial MVD using electron microscopy, which was analyzed in relation to the clinical outcome of the treatment. RESULTS: The endometrial MVD was significantly higher in the clinical pregnancy group than in the nonpregnant group [(4.12∓1.84)% vs (3.46∓1.26)%, t=-2.127, P=0.036). ROC curve analysis showed that the MVD had an area under the curve slightly over 0.5 (0.598) for predicting clinical pregnancy, suggesting a poor specificity in predicting the clinical outcome of the treatment. CONCLUSION: In IVF-ET cycles, the endometrial MVD during the peri-implantation period is helpful for assessing the endometrial receptivity, but the specificity remains low.

[Frozen embryo transfer combined with intrauterine administration of autologous peripheral blood mononuclear cells for repeated implantation failure: report of 3 cases].

OBJECTIVE: To explore the efficacy of frozen-thawed embryo transfer combined with intrauterine administration of autologous peripheral blood mononuclear cells (PBMCs) in the treatment of repeated implantation failure (RIF). METHODS: PBMCs obtained from 3 patients with RIF on the day of follicle rupture (natural cycle) or when the endometrial thickness reached 8 mm (hormone replacement cycle) were cultured in the presence of HCG for 48 h. The cultured PBMCs, along with freshly isolated PBMCs, were administered into the uterine cavity of the patients. Vitrified cleavage-stage embryos or blastocysts transfer was performed on day 3 or 5, respectively. RESULTS: Vitrified embryo or blastocyst transfer resulted in pregnancy and healthy live births in all the 3 patients. CONCLUSION: Frozen-thawed embryo transfer combined with intrauterine administration of autologous PBMCs may be an effective and safe approach to the treatment of RIF and may improve the outcomes of assisted reproduction.

[Value of serum estradiol concentration in predicting the clinical outcome of IVF-ET in patients receiving long protocol of GnRHa].

OBJECTIVE: To investigate the value of serum estradiol increment and serum estradiol/follicles on the day of hCG administration in predicting the clinical outcomes of in vitro fertilization-embryo transfer (IVF-ET). METHODS: A retrospective analysis of the IVF-ET data was conducted involving 121 patients who received a long gonadotrophin-releasing hormone agonist (GnRHa) protocol. According to the increment of serum estradiol on the day of hCG administration (relative to the level on the day before hCG administration), the patients were divided into 3 groups (A1, A2 and A3) with a increment ratio below 30%, between 30% and 50%, and over 50%, respectively. In addition, according to the ratio of serum estradiol level on hCG day to mature follicle (diameter ≥ 14 mm) number, these patients were divided into three groups (B1, B2 and B3) with the ratio below 250 pg/ml, between 250 and 350 gp/ml, and over 350 pg/ml, respectively. The hormonal characteristics and clinical outcomes of the IVF-ET cycles were analyzed comparatively. RESULTS: Both the clinical pregnancy rate (71.05%) and embryo implantation rate (52.63%) were significantly higher in group A3 than in groups A1 and A2 (P<0.05). The best clinical pregnancy rate (67.86%) and embryo implantation rate (49.14%) were significantly higher in group B2 than in groups B1 and B3 (P<0.05). CONCLUSION: The variation of serum estradiol shows an important impact on the clinical outcomes of IVF-ET in patients receiving long GnRH-a protocol. Favorable outcomes can be expected with a hCG day serum estradiol increment ratio above 50% and E(2)/follicle ratio between 250 and 350 pg/ml.

[Association between cell apoptosis and the quality of early mouse embryos].

OBJECTIVE: To investigate the relationship between cell apoptosis and the quality of early mouse embryos, understand the significance of apoptosis-regulatory genes in early embryonic development, and explore a new approach to improving the embryo quality. METHODS: The levels of cell apoptosis and proliferation in early mouse embryos in different developmental status (morphologically normal embryos, arrested embryos and fragmented embryos) were analyzed with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), caspase in situ fluorescence and Bcl-2 immunofluorescence, and immunofluorescent detection of proliferating cell nuclear antigen (PCNA). RESULTS: The cells in arrested embryos and embryonic fragments showed positive results in TUNEL assay with enhanced caspase activity and lowered expressions of Bcl-2 and PCNA. CONCLUSION: Cell apoptosis in early mouse embryos may be closely related to embryonic arrest and fragmentation.

[Analysis of the clinical outcomes of 160 patients undergoing repeated IVF/ICSI-ET treatment].

OBJECTIVE: To study the clinical factors affecting the outcomes of repeated assisted reproductive technology (ART) cycles. METHODS: A retrospective analysis of the clinical data and outcomes was conducted among 160 patients undergoing repeated IVF/ICSI-ET treatment between January 2006 and April 2009. RESULTS: The patients with successful clinical pregnancy after two ART cycles (group A) had a younger age and shorter duration of infertility, and had more antral follicles (AFC), more eggs and good-quality embryos with more transferred embryos available and higher good-quality embryo rate (P<0.05) than those who failed to have pregnancy after the cycles (group B). In the second cycle, the patients in group A had higher doses of short-acting GnRHa, r-HCG and HMG and at the same time more good eggs and embryos than in the first cycle. CONCLUSIONS: Female age is one of the most important factors affecting the pregnancy rate after repeated ART cycles. The clinical pregnancy rate can be enhanced by administering short-acting GnRHa, HMG, oral contraceptives and adjusting the dose of Gn as well as changing the culture medium of embryos.

[Clinical application of prefilled pen and conventional syringe during controlled ovarian stimulation for in vitro fertilization].

OBJECTIVE: To investigate the efficacy, convenience and costs of recombinant follitropin alpha administered by a prefilled pen device and conventional syringe in Chinese women undergoing controlled ovarian stimulation for in vitro fertilization (IVF). METHODS: A total of 184 patients undergoing IVF treatment were enrolled in this study. According to a long-term recombinant follicle-stimulating hormone (rFSH) protocol, ovarian stimulation was performed with the prefilled pen and conventional syringe at random in these subjects, and the dose of follitropin, number of oocytes and embryo parameters and IVF-ET outcome were compared between the two groups. RESULTS: The total rFSH dose, cost, and frequency of hospital visits were significantly lower in the pen protocol group, but the residual rFSH amount was higher. Compared with conventional injections, the prefilled pen was associated with significantly lowered rate of local redness, high rate of local bruise, more frequent follitropin dose modulation and lower serum oestradiol levels on HCG day. No significant difference was found in the endometrial thickness, numbers of oocytes retrieved, MII oocytes, transferred embryo, or the clinical pregnancy rates between the two groups. The ratio of MII oocytes, good quality embryo rates and implantation rates was significantly higher in the pen group with lower incidences of moderate and severe ovarian hyperstimulation syndrome. CONCLUSION: The prefilled pen provides an easy, safe, effective and more patient-friendly means for controlled ovarian stimulation procedure in Chinese women, but more attention should be given to protocol optimization and patient education.

[Clinical study of Osaka formula and improved multiparameter ultrasonic measurement for fetal weight estimation].

OBJECTIVE: To analyze the factors affecting the accuracy of Osaka formula multiparameter ultrasound-based fetal mass estimation, thereby establishing new formulas to improve the accuracy of the estimation. METHODS: A retrospective review was conducted among 519 healthy women with singleton pregnancy. Three days before the delivery (between 37 and 42 weeks' gestation), ultrasonic measurement of the fetal weight and other indices of the fetus was routinely performed. Correlation and multiple linear stepwise regression analysis were used to correct the 3 equations, which, along with Osaka University formula, were used to predict another 219 fetuses' birth weight. The coincidence rate of the predicted value and with the actual birth weight, and the absolute error and relative error were compared between the equations. RESULTS: The fetal abdominal area (AA) and abdominal circumference (AC) showed the most conspicuous influence on the estimated fetal birth weight, and fetal humerus length (HL) was more sensitive than femur length (FL) for the estimation. Three new regression equations were established, among which the equation 2 (fetal birth weight=1082.859+4.116xAAxHL) showed the best accuracy in clinical prediction. CONCLUSION: AA,AC and HL are more sensitive indices for estimation of the fetal birth weight, and the equation 2 established in this study still awaits further verification for its clinical value.

[Clinical analysis of pregnancy in second and third trimesters complicated severe acute respiratory syndrome].

OBJECTIVE: To explore and analyze the clinical manifestation, diagnosis, management and maternal-perinatal prognosis of severe acute respiratory syndrome (SARS)-complicated pregnancy in second and third trimesters. METHODS: Clinical data of 5 inpatients with SARS-complicated pregnancy in second and third trimesters from 4(th) February to 17(th) March 2003 were analyzed retrospectively. RESULTS: Five patients were all primigravida (including 2 twins). Two were infected in second trimester while the other 3 in third trimester with 2 hospital-acquired infections and 3 community-acquired infections. All patients had fever (5/5), 3 chills or rigor, 4 cough; 2 with decreased lymphocyte, 2 decreased platelet, 3 elevated alanine aminotransferase (ALT), 4 hypoalbuminemia, 5 abnormal chest radiographs. All 5 patients were cured with 1 requiring intensive care. 5 neonates including 1 twins have been followed up without evidence of SARS infections up to now. In a twin-pregnancy 1 fetus was lost while the pregnant's situation is stable. CONCLUSIONS: Common diagnostic criteria were Suitable for SARS-complicated pregnancy in second and third trimesters, but attention should be paid to the interaction between SARS and special pathological changes during pregnancy. Patients should be isolated and monitored intensively with timely cesarean section in severe cases, which could significantly decrease the maternal-perinatal mortality. The use of corticosteroids and psychological supports need further study.