Novel Splicing Variants in the ARR3 Gene Cause the Female-Limited Early-Onset High Myopia.

Purpose: Variants in the ARR3 gene have been linked to early-onset high myopia (eoHM) with a unique X-linked female-limited inheritance. However, the clinical validity of this gene-disease association has not been systematically evaluated. Methods: We identified two Chinese families with novel ARR3 splicing variants associated with eoHM. Minigene constructs were generated to assess the effects of the variants on splicing. We integrated previous evidence to curate the clinical validity of ARR3 and eoHM using the ClinGen framework. Results: The variants c.39+1G>A and c.100+4A>G were identified in the two families. Minigene analysis showed both variants resulted in abnormal splicing and introduction of premature termination codons. Based on genetic and experimental evidence, the ARR3-eoHM relationship was classified as "definitive." Conclusions: Our study identified two novel splicing variants of the ARR3 gene linked to eoHM and confirmed their functional validity via minigene assay. This research expanded the mutational spectrum of ARR3 and confirmed the minigene assay technique as an effective tool for understanding variant effects on splicing mechanisms.

Aberrant splicing caused by a novel KMT2A variant in Wiedemann-Steiner syndrome.

INTRODUCTION: Wiedemann-Steiner syndrome (WSS) is a rare autosomal-dominant disorder caused by KMT2A variants. The aim of this study was to characterize a novel KMT2A variant in a child with WSS and demonstrate integrated diagnostic approaches. METHODS: A 3-year-old female with developmental delay, distinctive facial features, and anal fistula underwent whole exome sequencing (WES). RNA analysis was performed to assess splicing effects caused by a novel variant. RESULTS: WES identified novel heterozygous KMT2A c.5664+6T>C variant initially classified as a variant of uncertain significance. RNA analysis provided evidence of aberrant splicing (exon 20 skipping), allowing reclassification to likely pathogenic. The patient exhibited typical WSS features along with a potential novel finding of anal fistula. CONCLUSION: This report describes a novel non-canonical splice site variant in KMT2A associated with WSS. RNA analysis was critical for variant reclassification. Detailed phenotypic evaluation revealed common and expanded WSS manifestations. This case highlights the importance of combining clinical assessment, DNA testing, and RNA functional assays for the diagnosis of rare genetic disorders.

The adverse effects of vitrification on mouse embryo development and metabolic phenotype in offspring.

Embryo vitrification is a standard procedure in assisted reproductive technology. Previous studies have shown that frozen embryo transfer is associated with an elevated risk of adverse maternal and neonatal outcomes. This study aimed to explore the effects of mouse blastocyst vitrification on the phenotype of vitrified-warmed blastocysts, their intrauterine and postnatal development, and the long-term metabolic health of the derived offspring. The vitrified-warmed blastocysts (IVF + VT group) exhibited reduced mitochondrial activity, increased apoptotic levels, and decreased cell numbers when compared to the fresh blastocysts (IVF group). Implantation rates, live pup rates, and crown-rump length at E18.5 were not different between the two groups. However, there was a significant decrease in fetal weight and fetal/placental weight ratio in the IVF + VT group. Furthermore, the offspring of the IVF + VT group at an age of 36 weeks had reduced whole energy consumption, impaired glucose and lipid metabolism when compared with the IVF group. Notably, RNA-seq results unveiled disturbed hepatic gene expression in the offspring from vitrified-warmed blastocysts. This study revealed the short-term negative impacts of vitrification on embryo and fetal development and the long-term influence on glucose and lipid metabolism that persist from the prenatal stage into adulthood in mice.

Association between ambient temperature exposure and pregnancy outcomes in patients undergoing in vitro fertilization in Shanghai, China: a retrospective cohort study.

STUDY QUESTION: Does ambient temperature exposure affect outcomes including clinical pregnancy and live birth in women undergoing IVF? SUMMARY ANSWER: Both extreme cold and hot ambient temperatures were significantly associated with adverse pregnancy outcomes of IVF cycles. WHAT IS KNOWN ALREADY: Heat exposure has been linked to adverse pregnancy outcomes worldwide. However, the effect of ambient temperature on infertile women undergoing IVF treatment is unclear. STUDY DESIGN, SIZE, DURATION: A retrospective cohort study was conducted from a database of 3452 infertile women who underwent their first fresh or frozen embryo transfer in the Shanghai First Maternity and Infant Hospital from April 2016 to December 2020. PARTICIPANTS/MATERIALS, SETTING, METHODS: Daily mean ambient temperature exposure for each patient was obtained based on their residential address. Temperature-stratified multiple logistic regression analysis was performed to investigate associations between temperature exposure and pregnancy outcomes after controlling for confounders. Vulnerable sub-groups were identified using forest plots. MAIN RESULTS AND THE ROLE OF CHANCE: The clinical pregnancy rate and live birth rate were 45.7% and 37.1%, respectively. Regarding clinical pregnancy, a higher temperature during cold weather was significantly associated with a higher pregnancy rate in the period about 11 weeks before ovarian stimulation (adjusted odds ratio (aOR) = 1.102, 95% CI: 1.012-1.201). Regarding live birth, an increased temperature during cold weather was significantly related to a higher live birth rate in the period after confirmation of clinical pregnancy or biochemical pregnancy, with the aORs of 6.299 (95% CI: 3.949-10.047) or 10.486 (95% CI: 5.609-19.620), respectively. However, a higher temperature during hot weather was negatively associated with the live birth rate in the periods after confirmation of clinical pregnancy or biochemical pregnancy, with the aORs at 0.186 (95% CI: 0.121-0.285) or 0.302 (95% CI: 0.224-0.406), respectively. Moreover, the decline in live birth rates during cold and hot weather was accompanied by increased rates of early miscarriage (P < 0.05). Stratified analyses identified susceptibility characteristics among the participants. LIMITATIONS, REASONS FOR CAUTION: Climate monitoring data were used to represent individual temperature exposure levels according to the patient's residential address in the study. We were not able to obtain information of personal outdoor activity and use of indoor air conditioners in this retrospective study, which may affect actual temperature exposure. WIDER IMPLICATIONS OF THE FINDINGS: This study highlights that the ambient temperature exposure should be taken into account during IVF treatment and afterwards. There is a need to be alert to extremes in cold and hot ambient temperatures, especially during the period of follicle development and pregnancy. With this knowledge, clinicians can scientifically determine the timing of IVF treatment and reinforce patients' awareness of self-protection to minimize adverse pregnancy outcomes associated with extreme temperatures. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by a grant from the Clinical Research Plan of Shanghai Hospital Development Center [SHDC2020CR4080], a grant from the Science and Technology Commission of Shanghai Municipality [19411960500], and two grants from the National Natural Science Foundation of China [81871213, 81671468]. B.W.M. is supported by a NHMRC Investigator grant (GNT1176437). B.W.M. reports consultancy for ObsEva, and research grants from Merck KGaA, Ferring and Guerbet. The other authors have no conflict of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Effect of hyaluronic acid-enriched transfer medium on frozen-thawed embryo transfer outcomes in RIF patients: a single-centre retrospective study.

Introduction: Many patients who undergo assisted reproductive technology (ART) suffer from recurrent implantation failure (RIF). The addition of hyaluronic acid (HA) to the transfer medium is one of several methods to improve pregnancy outcomes. We investigated whether HA could improve the live birth and clinical pregnancy rates of RIF patients. Methods: This study included 248 RIF patients, who were divided into two groups: the control (CTL) group (n=137), which received transfer medium without HA, and the HA group (n=111), which received transfer medium with HA. The two groups were compared according to the ART outcome. Results: The primary outcomes were the clinical pregnancy and live birth rates. Secondary outcomes include a positive urine pregnancy test, the implantation, ongoing pregnancy, multiple pregnancy, clinical miscarriage, and ectopic pregnancy rates, foetal or congenital defects, obstetric complications, infant birth weight and any related adverse events. Regarding the primary outcomes, the clinical pregnancy rate was significantly higher in the HA group than in the control group, and there was no significant difference in the live birth rate (LBR) between the HA and control groups. Regarding the secondary outcomes, the implantation, multiple pregnancy and ectopic pregnancy rates were similar between the two groups. Discussion: Our findings supported the conclusion that HA can improve the clinical pregnancy rate of patients with RIF undergoing FET cycles, but the live birth rate was not significantly improved with the addition of HA to the traditional transfer medium.

Relationship of the levels of reactive oxygen species in the fertilization medium with the outcome of in vitro fertilization following brief incubation.

Embryo selection in in vitro fertilization-embryo transfer (IVF-ET) mostly relies on morphological assessment using a conventional microscope or the time-lapse monitoring system, which is not comprehensive. Inappropriate levels of reactive oxygen species (ROS) in the fertilization medium may cause damage to gametes, eventually leading to adverse IVF outcomes. The present study aimed to identify the optimal oxidation-reduction level in the fertilization medium for IVF outcomes by measuring the static oxidation-reduction potential (sORP) using a highly accurate and sensitive MiOXSYS system. A total of 136 patients undergoing IVF following brief incubation were divided equally into 4 groups in this prospective cohort study. The sORP value in the fertilization medium was detected using the MiOXSYS system, and its relationship with IVF outcomes was analyzed. The primary outcome was pregnancy outcomes, including live birth rate (LBR), clinical pregnancy rate (CPR), biochemical pregnancy rate (BPR), and implantation rate (IR). The secondary outcome was embryo quality, including fertilization rate (FR), cleavage rate (CR), available embryo rate (AER), and good-quality embryo rate (GQER). Group II (sORP: 228.7-235.3 mV) showed a higher LBR, CPR, BPR, and IR compared with Group III (sORP: 235.4-242.7 mV), presented as follows: LBR (32.0% for Group II vs 3.6% for Group III, P = 0.033), CPR (32.0% for Group II vs 3.6% for Group III, P = 0.033), BPR (36.0% for Group II vs 3.6% for Group III, P = 0.019), and IR (31.3% for Group II vs 2.7% for Group III, P = 0.003). The FR in Groups I and II had lower significant differences compared with that in Groups III and IV (71.7% and 70.3% for Groups I and II vs 83.5% and 80.4% for Groups III and IV, P = 0.000). The GQER in Group I to Group IV was 32.7%, 37.4%, 26.5%, and 33.3%, respectively (P = 0.056). This study indicated that the sORP value in the fertilization medium might be a potential indicator of embryo quality and pregnancy outcome.

Clinical outcomes after fresh versus frozen embryo transfer in women with advanced reproductive age undergoing in vitro fertilization: a propensity score-matched cohort study.

This retrospective cohort study aimed to compare clinical outcomes following fresh or frozen embryo transfer (FET) in women with advanced reproductive age (ARA). Women aged 35-45 years who underwent their first autologous fresh or frozen cleavage stage embryo transfer cycle in the Centre for Assisted Reproduction of Shanghai First Maternity and Infant Hospital between January 2016 and December 2020 were included. The primary outcome was live birth after the first embryo transfer of the in vitro fertilization (IVF) cycle. Multiple covariates were used for propensity score matching (PSM) and generalized estimating equations were performed to examine the independent association between FET and live birth. Of the total 1453 patients, 327 patients had FET and 1126 patients had fresh ET. After the PSM procedure, 274 patients were included in each group. The live birth rate was 24.8% in the FET group and 25.2% in the fresh ET group (OR 0.98, 95% CI: 0.67-1.44, P = 0.92). Other pregnancy, perinatal and neonatal outcomes were all comparable between the two groups. This study showed that FET did not improve live birth and other clinical outcomes as compared with fresh embryo transfer in women with ARA who underwent their first IVF cycle.

The alterations of circulating mucosal-associated invariant T cells in polycystic ovary syndrome.

Background: Polycystic ovary syndrome (PCOS) is the most common endocrine disorder affecting reproductive age females and an important cause of infertility. Although the etiology is complex and its pathogenesis remains unclear, the pathological process of PCOS is tightly related with the immune dysfunction and gut microbial dysbiosis. Mucosal-associated invariant T (MAIT) cells are a subset of innate-like T cells which can regulate inflammation through the production of cytokines and play a role in regulating the gut microbiota. We aim to evaluate the correlation between characteristics of PCOS and MAIT cells as well as their impact on cytokine secretion. Methods: Peripheral blood samples were taken from PCOS patients (n=33) and healthy controls (n=30) during 2-5 days of the menstrual period. The frequencies of MAIT cells and T cells were measured by flow cytometry. Cytokines interleukin 17 (IL-17), interleukin 22(IL-22), interferon γ (IFN-γ) and granzyme B were determined by Enzyme-linked immunosorbent assay (ELISA). Results: The frequency of MAIT cells was significantly reduced in the blood of PCOS patients compared with the controls, and negatively correlated with Body Mass Index (BMI), Homeostatic model assessment- insulin resistance (HOMA-IR) index, and Anti Miillerian Hormone (AMH). Thus, the frequencies of MAIT cells decreased in PCOS patients with abnormal weight (BMI≥24kg/m2), higher HOMA-IR (≥1.5), and excessive AMH (≥8ng/ml). The Cytokine IL-17 was significantly higher in PCOS patients and negatively correlated with the frequency of MAIT cells. Even though the IL-22 was lower in PCOS Patients, no correlation with MAIT cells was detected. In subgroup, CD4+MAIT cells correlated with BMI, AMH, and testosterone (T) levels. Conclusion: The frequency change of MAIT cells may play a significant role in the pathogenesis of PCOS. Exploring these interactions with MAIT cells may provide a new target for PCOS treatment and prevention.

The efficacy of anastrozole in subfertile men with and without abnormal testosterone to estradiol ratios.

Background: Aromatase inhibitors (AIs), such as anastrozole, have shown effectiveness in treating oligoasthenozoospermia due to abnormal testosterone to estradiol (T/E2) ratio (T/E2 <10). However, its efficacy in subfertile men without abnormal T/E2 ratio (T/E2 >10) remained unevaluated. This retrospective study aimed to investigate whether patients with T/E2 ratio >10 could also benefit from anastrozole treatment. Methods: One hundred and five subfertile patients treated with 1 mg anastrozole daily were included, in which 62 patients had a T/E2 ratio of <10, and 43 patients had this ratio >10. Semen parameters and sex hormone levels (including FSH, LH, PRL, E2 and total T) were measured before and after a three-month treatment. T/E2 ratio and total progressive motility sperm count were calculated from these results. Results: Patients in both groups (T/E2 ratio <10 and >10) showed significant increase in sex hormone levels (FSH, LH and total T), T/E2 ratio and semen parameters (semen volume, sperm concentration, total sperm count, progressive motility and total progressive motility count). The changes of these parameters between two groups were comparable. A subgroup analysis comparing the effect of anastrozole on overweight and normal patients also showed no significant difference. Improvements in semen parameters were seen in some azoospermic and cryptozoospermic patients. Conclusions: The majority of subfertile men with and without abnormal T/E2 ratios responded to anastrozole treatment with significantly improved semen parameters and sex hormone levels. Anastrozole showed potential effectiveness in male subfertile patients with T/E2 >10, to be confirmed by future prospective, randomized, controlled studies.

The impact of different endometrial preparation protocols on obstetric and neonatal complications in frozen-thawed embryo transfer: a retrospective cohort study of 3,458 singleton deliveries.

BACKGROUND: Frozen-thawed embryo transfer (FET) is thought to be associated with obstetric and neonatal complications after in vitro fertilization/intracytoplasmic single sperm injection (IVF/ICSI) treatment. The study aimed to determine whether the endometrial preparation protocol is an influencing factor for these complications. METHODS: We conducted a retrospective cohort study of 3,458 women who had singleton deliveries after IVF/ICSI-FET treatment at the Centre for Reproductive Medicine of Shanghai First Maternity and Infant Hospital between July 2016 and April 2021. The women were divided into three groups according to the endometrial preparation protocols: 2,029 women with programmed cycles, 959 with natural cycles, and 470 with minimal ovarian stimulation cycles. The primary outcomes were the incidence rates of obstetric and neonatal complications, namely, hypertensive disorders of pregnancy (HDP), gestational diabetes mellitus (GDM), intrahepatic cholestasis of pregnancy (ICP), placenta previa, preterm rupture of membranes (PROM), preterm delivery, postpartum haemorrhage, large for gestational age (LGA), small for gestational age (SGA), and macrosomia. RESULTS: After adjustments for confounding variables by multivariate logistic regression analysis, the results showed that programmed cycles had an increased risk of HDP (aOR = 1.743; 95% CI, 1.110-2.735; P = 0.016) and LGA (aOR = 1.269; 95% CI, 1.011-1.592; P = 0.040) compared with natural cycles. Moreover, programmed cycles also increased the risk of LGA (aOR = 1.459; 95% CI, 1.083-1.965; P = 0.013) but reduced the risk of SGA (aOR = 0.529; 95% CI, 0.348-0.805; P = 0.003) compared with minimal ovarian stimulation cycles. There were no significant differences between natural cycles and minimal ovarian stimulation cycles. CONCLUSIONS: During IVF/ICSI-FET treatment, the risk of HDP and LGA was increased in women with programmed cycles. Therefore, for patients with thin endometrium, irregular menstruation or no spontaneous ovulation, minimal ovarian stimulation cycles may be a relatively safer option than programmed cycles.

Decreased intracellular IL-33 impairs endometrial receptivity in women with adenomyosis.

Adenomyosis is a common benign uterine lesion that is associated with female infertility, reduced clinical pregnancy rate and high miscarriage risk. While it has been known that the impaired endometrial receptivity is implicated in infertility in patients with adenomyosis, the underlying mechanism remains unclear. In the present study, we showed that intracellular protein level of IL-33 was downregulated in the endometrium of patients with adenomyosis, and IL-33 expression status was shown to be positively correlated with that of HOXA10, an endometrial receptivity marker. The subsequent analysis indicated IL-33 overexpression led to the increase of HOXA10 expression and enhancement of embryo implantation in vitro, which was accompanied with induction of STAT3 phosphorylation. Meanwhile, cryptotanshinone, a potent STAT3 inhibitor, was found to significantly suppress the increase of HOXA10 expression and embryo implantation caused by IL-33 overexpression in vitro, revealing the critical role of STAT3 activity. Consistently, the positive relationship between IL33 and HOXA10 expression in the endometrium was verified in the analysis of adenomyosis mouse model.

Discovery of immune-related diagnostic biomarkers and construction of diagnostic model in varies polycystic ovary syndrome.

AIMS: The various diagnostic criteria for polycystic ovary syndrome (PCOS) raised problem for PCOS research worldwide. PCOS has been demonstrated to be significantly associated with immune response. We aimed to identify several immune-related biomarkers and construct a nomogram model for diagnosis in PCOS. METHODS: The mRNA expression data were downloaded from Gene Expression Omnibus (GEO) database. Significant immune-related genes were identified to be the biomarkers for the diagnosis of PCOS using random forest model (RF), support vector machine model (SVM) and generalized linear model (GLM). The key biomarkers were selected from the optimal model and were utilized to construct a diagnostic nomogram. Receiver operating characteristic (ROC) curves was used to evaluate diagnostic ability of nomogram. Moreover, the relative proportion of 22 immune cell types was calculated by CIBERSORT algorithm. RESULTS: Four immune-related biomarkers (cAMP, S100A9, TLR8 and IL6R) were demonstrated to be highly expressed in PCOS. The nomogram constructed on the ground of the four key biomarkers showed perfect performance in diagnosis of PCOS, whose AUC were greater than 0.7. Higher infiltrating abundance of neutrophils, resting NK cells and activated dendritic cells were observed in PCOS and were tightly associated with the four key biomarkers. CONCLUSIONS: This study identified several immune-related diagnostic biomarkers for PCOS patients. The diagnostic nomogram constructed based the biomarkers provide a theory foundation for clinical application. Multiple immune cells were associated with the expression of these four biomarkers and might played a vital role in the procession of PCOS.

Predicting the probability of a live birth after a freeze-all based in vitro fertilization-embryo transfer (IVF-ET) treatment strategy.

Background: The predictors for live birth rate (LBR) following one episode of in vitro fertilization (IVF) cycle for patients using a "freeze-all" strategy are not entirely clear. Methods: A retrospective cohort study utilizing a prediction model was developed to assess the relationship to the LBR. Women undergoing IVF with a freeze-all strategy were screened. Univariate models were first fitted for female age at oocytes retrieval/frozen-thawed embryo transfer (FET), body mass index (BMI), duration and etiology of infertility, previous IVF failures, total dose and duration of gonadotrophin, ovarian sensitivity index (OSI), number of oocytes collected, method of fertilization, number of embryos created, number and stage of embryos frozen, type and number of FET cycles, endometrial thickness (EMT)/pattern, hormone level on transplantation day, storage duration, number of embryos thawed and damaged thawed embryos, number and stage of embryos transferred and number of different quality embryos transferred. Variables with P<0.05 in the univariate model were selected for further analysis of the final multivariate discrete-time logistic regression model. Results: A total of 7,602 women undergoing one ovarian stimulation resulted in 9,964 FETs, of whom 3,066 (40.33%) had a live-birth after their first FET and 3,929 (51.68%) after total FETs. The EMT and woman's age at oocyte retrieval were the most important predictors. In the first FET, the LBR of women with an EMT ≤8 mm [27.40%; 95% confidence interval (CI): (21.60-33.81%)] was significantly lower than that of women with EMT between 9 and 11 mm [36.51%; 95% CI: (34.25-38.81%)] and thicker than 12 mm [44.23%; 95% CI: (42.22-46.25%)] (P<0.05). The optimistic and conservative cumulative LBRs of women younger than 31 years [87.5%; 95% CI: (86.32-88.61%) and 63.04%; 95% CI: (61.36-64.69%)] were significantly decreased in women aged 31-35, 36-40 and >40 (P<0.001). Conclusions: Our study provides an effective prediction model for a woman's chance of having a baby after a "freeze-all" policy. The use of EMT and female age as tools to identify LBR are shown to be justified, and repeated FETs cannot reverse the age-dependent decline in fertility.

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.

Non-invasive embryo selection strategy for clinical IVF to avoid wastage of potentially competent embryos.

RESEARCH QUESTION: Can a non-invasive embryo transfer strategy provide a reference for embryo selection to be established? DESIGN: Chromosome sequencing of 345 paired blastocyst culture medium and whole blastocyst samples was carried out and a non-invasive embryo grading system was developed based on the random forest machine learning algorithm to predict blastocyst ploidy. The system was validated in 266 patients, and a blinded prospective observational study was conducted to investigate clinical outcomes between machine learning-guided and traditional non-invasive preimplantation genetic testing for aneuploidy (niPGT-A) analyses. Embryos were graded as A, B or C according to their euploidy probability levels predicted by non-invasive chromosomal screening (NICS). RESULTS: Higher live birth rate was observed in A- versus C-grade embryos (50.4% versus 27.1%, P = 0.006) and B- versus C-grade embryos (45.3% versus 27.1%, P = 0.022) and lower miscarriage rate in A- versus C-grade embryos (15.9% versus 33.3%, P = 0.026) and B- versus C-grade embryos (14.3% versus 33.3%, P = 0.021). The embryo utilization rate was significantly higher through the machine learning strategy than the conventional dichotomic judgment of euploidy or aneuploidy in the niPGT-A analysis (78.8% versus 57.9%, P < 0.001). Better outcomes were observed in A- and B-grade embryos versus C-grade embryos and higher embryo utilization rates through the machine learning strategy compared with traditional niPGT-A analysis. CONCLUSION: A machine learning guided embryo grading system can be used to optimize embryo selection and avoid wastage of potential embryos.

The Key Role of Peroxisomes in Follicular Growth, Oocyte Maturation, Ovulation, and Steroid Biosynthesis.

The absence of peroxisomes can cause disease in the human reproductive system, including the ovaries. The available peroxisomal gene-knockout female mouse models, which exhibit pathological changes in the ovary and reduced fertility, are listed in this review. Our review article provides the first systematic presentation of peroxisomal regulation and its possible functions in the ovary. Our immunofluorescence results reveal that peroxisomes are present in all cell types in the ovary; however, peroxisomes exhibit different numerical abundances and strong heterogeneity in their protein composition among distinct ovarian cell types. The peroxisomal compartment is strongly altered during follicular development and during oocyte maturation, which suggests that peroxisomes play protective roles in oocytes against oxidative stress and lipotoxicity during ovulation and in the survival of oocytes before conception. In addition, the peroxisomal compartment is involved in steroid synthesis, and peroxisomal dysfunction leads to disorder in the sexual hormone production process. However, an understanding of the cellular and molecular mechanisms underlying these physiological and pathological processes is lacking. To date, no effective treatment for peroxisome-related disease has been developed, and only supportive methods are available. Thus, further investigation is needed to resolve peroxisome deficiency in the ovary and eventually promote female fertility.

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.

Human menstrual blood-derived mesenchymal stem cells regulation of the EGF/Ras p21 pathway as a potential therapeutic target for thin endometrium.

BACKGROUND: Human infertility is caused by many factors, among which thin endometrium is the main reason for poor embryo implantation. Currently, stem cell therapy could be a potential approach in treating human endometrial disorder like thin endometrium. In this study, we aimed to explore the influence of menstrual stem cells from non-thin endometrium (NTE-MenSCs) and thin endometrium (TE-MenSCs) on the phenotype of endometrial epithelial cells (EECs). METHODS: The MenSCs were isolated from women with and without thin endometria, characterized and co-cultured with the EECs. The expression of cytokeratin 7 (CK7) was verified by immunofluorescence while the detection stem cell markers was determined flow cytometry. Osteogenic and adipogenic differentiation were induced in appropriate media. The quantitative real-time PCR and western blotting were respectively used for detecting the mRNA and protein expression levels, respectively. The CCK-8 assay was used for cell viability analysis whereas ELISA was used for the detection of cytokine levels. RESULTS: The results showed that the co-culture of NTE-MenSCs or TE-MenSCs and EECs promoted the proliferation, migration, and angiogenesis of endothelial progenitor cells differently. Furthermore, the TE-MenSCs promoted the expression of inflammation, vascularized adipose, and extracellular matrix related proteins. The epidermal growth factor (EGF)/Ras p21 pathway was found to mediate the influence of MenSCs on EECs. CONCLUSIONS: These findings are vital in that they may promote stem cell therapy of thin endometrium and enable embryo implantation in humans with thin endometrium.

Single-cell analysis defines the lineage plasticity of stem cells in cervix epithelium.

Information about the dynamic change and post-injury regeneration of cervical epithelium is relatively rare, even though it is tightly related to gynecologic malignancy. Here, using a feeder cell-based culturing system, we stably cloned mouse and human P63 and KRT5 expressing cells from the adult cervix as putative cervical stem/progenitor cells (CVSCs). When subjected to differentiation, the cultured cells gave rise to mature cervical epithelium by differentiating into squamous or glandular cells. The ability of endogenous mouse CVSCs to reconstitute cervical epithelium after injury was also evident from the genetic lineage tracing experiments. Single-cell transcriptomic analysis further classified the CVSCs into three subtypes and delineated their bi-lineage differentiation roadmap by pseudo-time analysis. We also tracked the real-time differentiation routes of two representing single CVSC lines in vitro and found that they recapitulated the predicted roadmap in pseudo-time analysis. Signaling pathways including Wnt, TGF-beta, Notch and EGFR were found to regulate the cervical epithelial hierarchy and implicated the different roles of distinct types of cells in tissue homeostasis and tumorigenesis. Collectively, the above data provide a cloning system to achieve stable in vitro culture of a bi-lineage stem/progenitor cell population in the cervix, which has profound implications for our understanding of the cervix stem/progenitor cell function in homeostasis, regeneration, and disease and could be helpful for developing stem cell-based therapies in future.