A new perspective on semen quality of aged male: The characteristics of metabolomics and proteomics.

Background: Semen quality is negatively correlated with male age and is mainly quantified by a routine semen analysis, which is descriptive and inconclusive. Sperm proteins or semen metabolites are used as the intermediate or end-products, reflecting changes in semen quality, and hold much promise as a new biomarker to predict fertility in advanced-aged males. Objectives: In this study, we sought to assess whether the semen metabolome and proteome of aged males can affect semen quality and serve as biomarkers for predicting semen quality. Materials and methods: We retrospectively analyzed 12825 males that underwent semen routine analysis to understand the age-dependent changes in sperm quality. To identify the difference between aged and young adults, metabolomics (n=60) analyses of semen and proteomics (n=12) analyses of sperm were conducted. Finally, integrated machine learning of metabolomics was conducted to screen biomarkers to identify aging semen. Results: We discovered that male age was positively correlated with sperm concentration as well as DNA fragmentation index(DFI), and negatively with progressive motile sperm count, total sperm count, sperm volume and progressive sperm motility. The differential metabolites were significantly enriched in various metabolic pathways, and four of these differential metabolites (Pipamperone, 2,2-Bis(hydroxymethyl)-2,2',2''-nitrilotriethanol, Arg-Pro and Triethyl phosphate) were utilized to establish a biomarker panel to identify aging semen. Proteomic analysis showed that differential proteins were significantly enriched in protein digestion and absorption and some energy-related pathways. An integrated analysis of the metabolome and proteome identified differential energy metabolism and oxidative stress-related proteins, which could explain the decreased motility and the increased DFI of aging sperm. Discussion and conclusion: We provide compelling evidence that the changes in semen metabolome and sperm proteome are related to the decline of semen quality in aged males. Moreover, a biomarker panel based on four metabolites was established to identify aging semen.

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.

DNA methylation and gene expression changes in mouse pre- and post-implantation embryos generated by intracytoplasmic sperm injection with artificial oocyte activation.

BACKGROUND: The application of artificial oocyte activation (AOA) after intracytoplasmic sperm injection (ICSI) is successful in mitigating fertilization failure problems in assisted reproductive technology (ART). Nevertheless, there is no relevant study to investigate whether AOA procedures increase developmental risk by disturbing subsequent gene expression at different embryonic development stages. METHODS: We used a mouse model to explore the influence of AOA treatment on pre- and post-implantation events. Firstly, the developmental potential of embryos with or without AOA treatment were assessed by the rates of fertilization and blastocyst formation. Secondly, transcriptome high-throughput sequencing was performed among the three groups (ICSI, ICSI-AOA and dICSI-AOA groups). The hierarchical clustering and Principal Component Analysis (PCA) analysis were used. Subsequently, Igf2r/Airn methylation analysis were detected using methylation-specific PCR sequencing following bisulfite treatment. Finally, birth rate and birth weight were examined following mouse embryo transfer. RESULTS: The rates of fertilization and blastocyst formation were significantly lower in oocyte activation-deficient sperm injection group (dICSI group) when compared with the ICSI group (30.8 % vs. 84.4 %, 10.0 % vs. 41.5 %). There were 133 differentially expressed genes (DEGs) between the ICSI-AOA group and ICSI group, and 266 DEGs between the dICSI-AOA group and ICSI group. In addition, the imprinted gene, Igf2r is up regulated in AOA treatment group compared to control group. The Igf2r/Airn imprinted expression model demonstrates that AOA treatment stimulates maternal allele-specific mehtylation spreads at differentially methylated region 2, followed by the initiation of paternal imprinted Airn long non-coding (lnc) RNA, resulting in the up regulated expression of Igf2r. Furthermore, the birth weight of newborn mice originating from AOA group was significantly lower compared to that of ICSI group. The pups born following AOA treatment did not show any other abnormalities during early development. All offspring mated successfully with fertile controls. CONCLUSIONS: AOA treatment affects imprinted gene Igf2r expression and mehtylation states in mouse pre- and post-implantation embryo, which is regulated by the imprinted Airn. Nevertheless, no significant differences were found in post-natal growth of the pups in the present study. It is hoped that this study could provide valuable insights of AOA technology in assisted reproduction biology.

Effects of Early Cumulus Cell Removal on Treatment Outcomes in Patients Undergoing In Vitro Fertilization: A Retrospective Cohort Study.

Background: Early cumulus cell removal combined with early rescue intracytoplasmic sperm injection (ICSI) has been widely practiced in many in vitro fertilization (IVF) centers in China in order to avoid total fertilization failure. However, uncertainty remains whether the pregnancy and neonatal outcomes are associated with early cumulus cell removal. Objectives: To investigate if early cumulus cell removal alone after 4 hours co-incubation of gametes (4 h group), has detrimental effect on the pregnancy and neonatal outcomes in patients undergoing IVF, through a comparison with conventional cumulus cell removal after 20 hours of insemination (20 h group). Methods: This retrospective cohort study included 1784 patients who underwent their first fresh cleavage stage embryo transfer at the Centre for Assisted Reproduction of Shanghai First Maternity and Infant Hospital from June 2016 to December 2018 (4 h group, n=570; 20 h group, n=1214). A logistic regression analysis was performed to examine the independent association between early cumulus cell removal and pregnancy outcomes after adjustment for potential confounders. The neonatal outcomes between the two groups were compared. Results: When compared with the 20 h group, the 4 h group had similar pregnancy outcomes, including rates for biochemical pregnancy, clinical pregnancy, ongoing pregnancy, miscarriage, ectopic pregnancy, multiple pregnancy, live birth. There were 1073 infants delivered after embryo transfer (4 h group, n=337; 20 h group, n=736). Outcomes in both groups were similar for both singleton and twin gestations, including preterm birth rate and very preterm birth rate, mean birth weight, mean gestational age, sex ratio at birth and rate of congenital birth defects. In addition, findings pertaining to singleton gestations were also similar in the two groups for Z-scores (gestational age- and sex-adjusted birth weight), rates of small for gestational age, very small for gestational age, large for gestational age and very large for gestational age infants. Conclusions: In this study early cumulus cell removal alone was not associated with adverse pregnancy and neonatal outcomes. From this perspective, early cumulus cell removal to assess for a potential early rescue ICSI is therefore considered to be a safe option in patients undergoing IVF.

Effect of male age on pregnancy and neonatal outcomes in the first frozen-thawed embryo transfer cycles of IVF/ICSI treatment.

BACKGROUND: The effect of male age on pregnancy outcomes after assisted reproductive technology (ART) treatment shown in the previous literature is controversial. In addition, the influence of male age on neonatal outcomes following ART treatment has less been investigated. OBJECTIVES: The aim of this study was to evaluate the effect of male age on reproductive and neonatal outcomes in couples following ART treatment. MATERIALS AND METHODS: A retrospective cohort study was performed in two centers for assisted reproduction from June 2010 to February 2019. A total of 5512 frozen-thawed embryo transfer (FET) cycles were included according to the criteria. The primary outcome measures were pregnancy and neonatal outcomes. Patients were categorized into five groups according to male age (younger than 30, 31-35, 36-40, 41-45, and older than 45), and the group younger than 30 years old was treated as the reference group. RESULTS: The logistic regression analysis showed that clinical pregnancy and live birth were all no statistic difference among the male age-groups compared with the reference group (p values, 0.743, 0.979, 0.948, 0.28; p values, 0.823, 0.342, 0.817, 0.381, respectively). Furthermore, no significant differences were found in the preterm birth rate, child sex, neonatal malformation, birth weight, and gestational age (p > 0.05). The advanced male age was not associated with a higher risk of adverse neonatal outcomes. DISCUSSION AND CONCLUSION: This study showed that there were no effects of male age on pregnancy or neonatal outcomes in infertile couples following their first FET cycles when females were younger than 36 years old.

Embryo incubation by time-lapse systems versus conventional incubators in Chinese women with diminished ovarian reserve undergoing IVF/ICSI: a study protocol for a randomised controlled trial.

INTRODUCTION: The time-lapse imaging system (TLS) is a newly developed non-invasive embryo assessment system. Compared with conventional incubators, a TLS provides stable culture conditions and consistent observations of embryo development, thereby potentially improving embryo quality and selection of the best quality embryo. Although TLSs have been routinely used in many in vitro fertilisation (IVF) centres globally, there is insufficient evidence to indicate that TLSs result in higher cumulative live birth rates over conventional incubators. The purpose of this study is to compare the cumulative live birth rates and safety including miscarriage in infertile patients with diminished ovarian reserve (DOR) from both TLSs and conventional incubators. METHODS AND ANALYSIS: This study is a double-blind randomised controlled clinical trial (1:1 treatment ratio of TLSs vs conventional incubator). A total of 730 patients with DOR undergoing the first or second cycle of IVF or intracytoplasmic sperm injection (ICSI) will be enrolled and randomised into two parallel groups. Participants will undergo embryo culture in the TLSs (group A) or the conventional incubators (group B), respectively. Embryos are selected for transfer in both groups by the morphological characteristics. The embryo selection algorithm software is not used in the TLSs. The primary outcome is the cumulative live birth rate of the trial IVF/ICSI cycle within 12 months after randomisation. This study is powered to detect an absolute difference of 10% (35% vs 25%) at the significance level of 0.05% and 80% statistical power based on a two-sided test. ETHICS AND DISSEMINATION: This trial has been approved by the Institutional Ethical Committee of Shanghai First Maternity and Infant Hospital (KS1958). All participants in the trial will provide written informed consent. The study will be conducted according to the principles outlined in the Declaration of Helsinki and its amendments. Results of this study will be disseminated in peer-reviewed scientific journals. TRIAL REGISTRATION NUMBER: Chinese Clinical Trial Registry (ChiCTR1900027746).

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.

Depletion of exosomal circLDLR in follicle fluid derepresses miR-1294 function and inhibits estradiol production via CYP19A1 in polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a common endocrine and metabolic disorder in reproductive women and is characterized by polycystic ovaries, hyperandrogenism and chronic anovulation. Abnormal folliculogenesis is considered as a common characteristic of PCOS. Our aim is to identify the altered circRNA expression profile in exosomes isolated from follicular fluid (FF) of PCOS patients to investigate the molecular function of exosomal circRNA, as a vital mediator in follicular microenvironment, in the aetiology and pathobiology of PCOS. In this study, the circRNA expression profile of FF exosomes were compared between PCOS and control patients by RNA sequencing (N=5 vs 5). Sixteen circRNAs showed significantly different expression. GO and KEGG pathway analyses indicated that their parental genes were enriched in PCOS-related pathways, including ovarian steroidogenesis, aldosterone synthesis and secretion, and Jak-STAT signaling. Among sixteen differentially expressed circRNAs, hsa_circ_0006877 (circLDLR) was processed from its parental LDLR (low density lipoprotein receptor) transcript, which participated in ovarian steroidogenesis. Its depletion in PCOS FF exosomes was further verified in an additional cohort (N=25 vs 25) by qRT-PCR. And a circLDLR-miR-1294-CYP19A1 competing endogenous RNA (ceRNA) network was predicted by cytoscape software, and confirmed by luciferase assay and correlative expression in the cumulus cells of PCOS patients. Mechanistically, the intercellular transfer of functional circLDLR assay and its withdrawal experiments in KGN cells showed that depleting circLDLR in exosomes increased miR-1294 expression and inhibited CYP19A1 expression in recipient cells, as well as reduced their estrogen (E2) secretion. Our findings revealed a ceRNA network of circLDLR and provided new information on abnormal follicle development in PCOS.

Effects of the histone deacetylase inhibitor 'Scriptaid' on the developmental competence of mouse embryos generated through round spermatid injection.

STUDY QUESTION: Can the histone deacetylase inhibitor Scriptaid improve the efficiency of the development of round spermatid injection (ROSI)-fertilized embryos in a mouse model? SUMMARY ANSWER: Treatment of ROSI mouse zygotes with Scriptaid increased the expression levels of several development-related genes at the blastocyst stage, resulting in more efficient in vitro development of the blastocyst and an increased birth rate of ROSI-derived embryos. WHAT IS KNOWN ALREADY: The full-term development of embryos derived through ROSI is significantly lower than that following ICSI in humans and other species. STUDY DESIGN, SIZE, DURATION: Oocytes, spermatozoa and round spermatids were collected from BDF1 (C57BL/6 × DBA/2) mice. For in vitro development experiments, mouse ROSI-derived zygotes were treated with Scriptaid at different concentrations (0, 125, 250, 500 and 1000 nM) and for different exposure times (0, 6, 10, 16 or 24 h). Next, blastocysts of the optimal Scriptaid-treated group and the non-treated ROSI group were separately transferred into surrogate ICR mice to compare in vivo development with the ICSI group (control). Each experiment was repeated at least three times. PARTICIPANTS/MATERIALS, SETTING, METHODS: Metaphase II (MII) oocytes, spermatozoa and round spermatids were obtained from sexually mature BDF1 female or male mice. The developmental potential of embryos among the three groups (the ICSI, ROSI and optimal Scriptaid-treated ROSI groups) was assessed based on the rates of obtaining zygotes, two-cell stage embryos, four-cell stage embryos, blastocysts and full-term offspring. In addition, the expression levels of development-related genes (Oct4, Nanog, Klf4 and Sox2) were analysed using real-time PCR, and the methylation states of imprinted genes (H19 and Snrpn) in these three groups were detected using methylation-specific PCR (MS-PCR) sequencing following bisulfite treatment. MAIN RESULTS AND THE ROLE OF CHANCE: The in vitro experiments revealed that treating ROSI-derived zygotes with 250 nM Scriptaid for 10 h significantly improved the blastocyst formation rate (59%) compared with the non-treated group (38%) and further increased the birth rates of ROSI-derived embryos from 21% to 40% in vivo. Moreover, in ROSI-derived embryos, the expression of the Oct4, Nanog and Sox2 genes at the blastocyst stage was decreased, but the optimal Scriptaid treatment restored expression to a level similar to their ICSI counterparts. In addition, Scriptaid treatment moderately repaired the abnormal DNA methylation pattern in the imprinting control regions (ICRs) of H19 and Snrpn. LARGE SCALE DATA: N/A LIMITATIONS, REASONS FOR CAUTION: Because of the ethics regarding the use of human gametes for ROSI studies, the mouse model was used as an approach to explore the effects of Scriptaid on the developmental potential of ROSI-derived embryos. However, to determine whether these findings can be applied to humans, further investigation will be required. WIDER IMPLICATIONS OF THE FINDINGS: Scriptaid treatment provides a new means of improving the efficiency and safety of clinical human ROSI. STUDY FUNDING/COMPETING INTERESTS: The study was financially supported through grants from the National Key Research Program of China (No. 2016YFC1304800); the National Natural Science Foundation of China (Nos: 81170756, 81571486); the Natural Science Foundation of Shanghai (Nos: 15140901700, 15ZR1424900) and the Programme for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning. There are no conflicts of interest to declare.

Generation of hypoxanthine phosphoribosyltransferase gene knockout rabbits by homologous recombination and gene trapping through somatic cell nuclear transfer.

The rabbit is a common animal model that has been employed in studies on various human disorders, and the generation of genetically modified rabbit lines is highly desirable. Female rabbits have been successfully cloned from cumulus cells, and the somatic cell nuclear transfer (SCNT) technology is well established. The present study generated hypoxanthine phosphoribosyltransferase (HPRT) gene knockout rabbits using recombinant adeno-associated virus-mediated homologous recombination and SCNT. Gene trap strategies were employed to enhance the gene targeting rates. The male and female gene knockout fibroblast cell lines were derived by different strategies. When male HPRT knockout cells were used for SCNT, no live rabbits were obtained. However, when female HPRT(+/-) cells were used for SCNT, live, healthy rabbits were generated. The cloned HPRT(+/-) rabbits were fertile at maturity. We demonstrate a new technique to produce gene-targeted rabbits. This approach may also be used in the genetic manipulation of different genes or in other species.

FACS selection of valuable mutant mouse round spermatids and strain rescue via round spermatid injection.

Round spermatid injection (ROSI) into mammalian oocytes can result in the development of viable embryos and offspring. One current limitation to this technique is the identification of suitable round spermatids. In the current paper, round spermatids were selected from testicular cells with phase contrast microscopy (PCM) and fluorescence-activated cell sorting (FACS), and ROSI was performed in two strains of mice. The rates of fertilization, embryonic development and offspring achieved were the same in all strains. Significantly, round spermatids selected by PCM and FACS were effectively used to rescue the infertile Pten-null mouse. The current results indicate that FACS selection of round spermatids can not only provide high-purity and viable round spermatids for use in ROSI, but also has no harmful effects on the developmental capacity of subsequently fertilized embryos. It was concluded that round spermatids selected by FACS are useful for mouse strain rederivation and rescue of infertile males; ROSI should be considered as a powerful addition to the armamentarium of assisted reproduction techniques applicable in the mouse.

Reprogramming of round spermatids by the germinal vesicle cytoplasm in mice.

The birthrate following round spermatid injection (ROSI) remains low in current and evidence suggests that factors in the germinal vesicle (GV) cytoplasm and certain substances in the GV such as the nucleolus might be responsible for genomic reprogramming and embryonic development. However, little is known whether the reprogramming factors in GV oocyte cytoplasm and/or nucleolus in GV are beneficial to the reprogramming of round spermatids and development of ROSI embryos. Here, round spermatids were treated with GV cytolysates and injected this round spermatid alone or co-injected with GV oocyte nucleolus into mature metaphase II oocytes. Subsequent embryonic development was assessed morphologically and by Oct4 expression in blastocysts. There was no significant difference between experimental groups at the zygote to four-cell development stages. Blastocysts derived from oocytes which were injected with cytolysate treated-round spermatid alone or co-injected with nucleoli injection yielded 63.6% and 70.3% high quality embryos, respectively; comparable to blastocysts derived by intracytoplasmic sperm injection (ICSI), but higher than these oocytes which were co-injected with lysis buffer-treated round spermatids and nucleoli or injected with the lysis buffer-treated round spermatids alone. Furthermore, the proportion of live offspring resulting from oocytes which were co-injected with cytolysate treated-round spermatids and nucleoli or injected with cytolysate treated-round spermatids alone was higher than those were injected with lysis buffer treated-round spermaids, but comparable with the ICSI group. Our results demonstrate that factors from the GV cytoplasm improve round spermatid reprogramming, and while injection of the extra nucleolus does not obviously improve reprogramming its potential contribution, although which cannot be definitively excluded. Thus, some reprogramming factors are evidently present in GV oocyte cytoplasm and could significantly facilitate ROSI technology, while the nucleolus in GV seems also having a potential to improve reprogramming of round spermatids.

The Oct4 promoter-EGFP transgenic rabbit: a new model for monitoring the pluripotency of rabbit stem cells.

The rabbit has long been used as a laboratory animal model for developing reproductive and stem cell-related technologies, as well as for studying human disease. The Oct4 transcription factor plays a crucial role in the maintenance and regulation of pluripotency in embryos and stem cells. We constructed a reporter plasmid containing the gene encoding the enhanced green fluorescent protein (EGFP) under the control of the rabbit Oct4 promoter (prOG) and transfected it into E14 mouse stem cells and rabbit ESCs. In addition, prOG transgenic fibroblasts were derived and prOG transgenic rabbits were produced by somatic cell nuclear transfer (SCNT). The pattern of expression of ectopic EGFP was similar in E14 mouse stem cells whether under the control of the rabbit (prOG) or mouse Oct4 promoter (pmOG). EGFP expression was observed in rabbit ESCs following prOG transfection. Both prOG transgenic SCNT embryos and F1 prOG transgenic embryos derived from adult transgenic rabbits expressed green fluorescence at the morula and blastocyst stages. EGFP was clearly detected in gonads isolated from fetuses at 27 dpc. The prOG transgenic rabbit represents a new model for studying the derivation and maintenance of rabbit pluripotent cells, and for investigating rabbit embryo development.