Male fertility and sperm quality are negatively impacted by obesity. Furthermore, recent evidence has shown that male offspring from obese rat mothers also have reduced sperm quality and fertility. Here, we extend work in this area by comparing the effects of both maternal obesity and offspring post-weaning diet-induced obesity, as well as their combination, on sperm quality in mice. We additionally tested whether administration of the NAD+-booster nicotinamide mononucleotide (NMN) can ameliorate the negative effects of obesity and maternal obesity on sperm quality. We previously showed that intraperitoneal (i.p.) injection of NMN can reduce the metabolic deficits induced by maternal obesity or post-weaning dietary obesity in mice. In this study, female mice were fed a high-fat diet (HFD) for 6 weeks until they were 18% heavier than a control diet group. Thereafter, HFD and control female mice were mated with control diet males, and male offspring were weaned into groups receiving control or HFD. At 30 weeks of age, mice received 500 mg/kg body weight NMN or vehicle PBS i.p. for 21 days. As expected, adiposity was increased by both maternal and post-weaning HFD but reduced by NMN supplementation. Post-weaning HFD reduced sperm count and motility, while maternal HFD increased offspring sperm DNA fragmentation and levels of aberrant sperm chromatin. There was no evidence that the combination of post-weaning and maternal HFD exacerbated the impacts in sperm quality suggesting that they impact spermatogenesis through different mechanisms. Surprisingly NMN reduced sperm count, vitality and increased sperm oxidative DNA damage, which was associated with increased NAD+ in testes. A subsequent experiment using oral NMN at 400 mg/kg body weight was not associated with reduced sperm viability, oxidative stress, mitochondrial dysfunction or increased NAD+ in testes, suggesting that the negative impacts on sperm could be dependent on dose or mode of administration.
Neil A Youngson, G Mezbah Uddin, Abhirup Das, Carl Martinez, Haley S Connaughton, Sara Whiting, Josephine Yu, David A Sinclair, R John Aitken and Margaret J Morris
V A van der Weijden, J T Bick, S Bauersachs, G J Arnold, T Fröhlich, B Drews and S E Ulbrich
The uterine microenvironment during pre-implantation presents a pro-survival milieu and is essential for embryo elongation in ruminants. The European roe deer (Careolus capreolus) pre-implantation embryo development is characterised by a 4-month period of reduced development, embryonic diapause, after which the embryo rapidly elongates and implants. We investigated the uterine fluid proteome by label-free liquid chromatography tandem mass spectrometry at four defined stages covering the phase of reduced developmental pace (early diapause, mid-diapause and late diapause) and embryo elongation. We hypothesised that embryo development during diapause is halted by the lack of signals that support progression past the blastocyst stage. Three clusters of differentially abundant proteins were identified by a self-organising tree algorithm: (1) gradual reduction over development; (2) stable abundance during diapause, followed by a sharp rise at elongation; and (3) gradual increase over development. Proteins in the different clusters were subjected to gene ontology analysis. ‘Cellular detoxification’ in cluster 1 was represented by alcohol dehydrogenase, glutathione S-transferase and peroxiredoxin-2. ATP-citrate synthase, nucleolin, lamin A/C, and purine phosphorylase as cell proliferation regulators were found in cluster 2 and ‘cortical cytoskeleton’, ‘regulation of substrate adhesion-dependent cell spreading’ and ‘melanosome’ were present in cluster 3. Cell cycle promoters were higher abundant at elongation than during diapause, and polyamines presence indicates their role in diapause regulation. This study provides a comprehensive overview of proteins in the roe deer uterine fluid during diapause and forms a basis for studies aiming at understanding the impact of the lack of cell cycle promoters during diapause.
Jing Liu, Yang Wang, Peng Chen, Yue Ma, Shuo Wang, Ye Tian, Anna Wang and Danbo Wang
Previous lncRNA microarray screening found that the AC002454.1 gene was highly expressed in endometriosis (EMS), and these expression levels were highly correlated with cyclin-dependent kinase-6 (CDK6). This study investigated the expression level and correlation between AC002454.1 and CDK6 in endometrium tissues and the influence of these changes in expression upon the biological behavior of eutopic endometrial cells. We confirmed AC002454.1 and CDK6 mRNA and protein were highly expressed in ectopic and eutopic endometrial tissue from patients with EMS and were clearly correlated. In vitro, both AC002454.1 and CDK6 positively regulated the proliferation, migration and invasion ability of eutopic endometrial cells and could promote the transformation of cells from G0/G1 phase to S phase. AC002454.1 and CDK6 may have synergistic effects, thereby affecting the biological behavior of endometrial cells, and thus promote the progression of EMS.
Hang Qi, Guiling Liang, Jin Yu, Xiaofeng Wang, Yan Liang, Xiaoqing He, Tienan Feng and Jian Zhang
MicroRNA (miRNA) expression proﬁles in tubal endometriosis (EM) are still poorly understood. In this study, we analyzed the differential expression of miRNAs and the related gene networks and signaling pathways in tubal EM. Four tubal epithelium samples from tubal EM patients and five normal tubal epithelium samples from uterine leiomyoma patients were collected for miRNA microarray. Bioinformatics analyses, including Ingenuity Pathway Analysis (IPA), Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, were performed. Quantitative real-time polymerase chain reaction (qRT-PCR) validation of five miRNAs was performed in six tubal epithelium samples from tubal EM and six from control. A total of 17 significantly differentially expressed miRNAs and 4343 potential miRNA-target genes involved in tubal EM were identified (fold change >1.5 and FDR-adjusted P value <0.05). IPA indicated connections between miRNAs, target genes and other gynecological diseases like endometrial carcinoma. GO and KEGG analysis revealed that most of the identified genes were involved in the mTOR signaling pathway, SNARE interactions in vesicular transport and endocytosis. We constructed an miRNA-gene-disease network using target gene prediction. Functional analysis showed that the mTOR pathway was connected closely to tubal EM. Our results demonstrate for the first time the differentially expressed miRNAs and the related signal pathways involved in the pathogenesis of tubal EM which contribute to elucidating the pathogenic mechanism of tubal EM-related infertility.
Shou-Bin Tang, Lei-Lei Yang, Ting-Ting Zhang, Qian Wang, Shen Yin, Shi-Ming Luo, Wei Shen, Zhao-Jia Ge and Qing-Yuan Sun
It is demonstrated that repeated superovulation has deleterious effects on mouse ovaries and cumulus cells. However, little is known about the effects of repeated superovulation on early embryos. Epigenetic reprogramming is an important event in early embryonic development and could be easily disrupted by the environment. Thus, we speculated that multiple superovulations may have adverse effects on histone modifications in the early embryos. Female CD1 mice were randomly divided into four groups: (a) spontaneous estrus cycle (R0); (b) with once superovulation (R1); (c) with three times superovulation at a 7-day interval (R3) and (d) with five times superovulation at a 7-day interval (R5). We found that repeated superovulation remarkably decreased the fertilization rate. With the increase of superovulation times, the rate of early embryo development was decreased. The expression of Oct4, Sox2 and Nanog was also affected by superovulation in blastocysts. The immunofluorescence results showed that the acetylation level of histone 4 at lysine 12 (H4K12ac) was significantly reduced by repeated superovulation in mouse early embryos (P < 0.01). Acetylation level of histone 4 at lysine 16 (H4K16ac) was also significantly reduced in pronuclei and blastocyst along with the increase of superovulation times (P < 0.01). H3K9me2 and H3K27me3 were significantly increased in four-cell embryos and blastocysts. We further found that repeated superovulation treatment increased the mRNA level of histone deacetylases Hdac1, Hdac2 and histone methyltransferase G9a, but decreased the expression level of histone demethylase-encoding genes Kdm6a and Kdm6b in early embryos. In a word, multiple superovulations alter histone modifications in early embryos.
Katja Hummitzsch, Nicholas Hatzirodos, Anne M Macpherson, Jeff Schwartz, Raymond J Rodgers and Helen F Irving-Rodgers
The ovary has specialised stromal compartments, including the tunica albuginea, interstitial stroma and theca interna, which develops concurrently with the follicular antrum. To characterise the molecular determinants of these compartments, stroma adjacent to preantral follicles (pre-theca), interstitium and tunica albuginea were laser microdissected (n = 4 per group) and theca interna was dissected from bovine antral follicles (n = 6). RNA microarray analysis showed minimal differences between interstitial stroma and pre-theca, and these were combined for some analyses and referred to as stroma. Genes significantly upregulated in theca interna compared to stroma included INSL3, LHCGR, HSD3B1, CYP17A1, ALDH1A1, OGN, POSTN and ASPN. Quantitative RT-PCR showed significantly greater expression of OGN and LGALS1 in interstitial stroma and theca interna versus tunica and greater expression of ACD in tunica compared to theca interna. PLN was significantly higher in interstitial stroma compared to tunica and theca. Ingenuity pathway, network and upstream regulator analyses were undertaken. Cell survival was also upregulated in theca interna. The tunica albuginea was associated with GPCR and cAMP signalling, suggesting tunica contractility. It was also associated with TGF-β signalling and increased fibrous matrix. Western immunoblotting was positive for OGN, LGALS1, ALDH1A1, ACD and PLN with PLN and OGN highly expressed in tunica and interstitial stroma (each n = 6), but not in theca interna from antral follicles (n = 24). Immunohistochemistry localised LGALS1 and POSTN to extracellular matrix and PLN to smooth muscle cells. These results have identified novel differences between the ovarian stromal compartments.
Dongmei He, Hong Zeng, Jingfei Chen, Lan Xiao, Yuhao Zhao and Nenghui Liu
Integrin β3 (ITGB3), which is the target gene of the miRNA let-7 that can be antagonized by long noncoding RNA (lncRNA) H19, is well known to have a critical role in endometrium receptivity. However, the regulation of ITGB3 in cell–cell or cell–extracellular matrix adhesion and invasion for the maintenance of early pregnancy remains unknown. This study aimed to explore the role of the H19/let-7/ITGB3 axis in regulating trophoblastic spheroid adhesion and in vitro invasion ability using the HTR-8/SVneo cell line and to investigate the expression levels of lncRNA H19 and ITGB3 in human products of conception. The in vitro knockdown of H19 resulted in decreased expression of ITGB3 at the mRNA and protein levels and reduced the adhesion and invasion ability. In the embryonic chorion tissue of spontaneous abortion (SA), the expressions of H19 and ITGB3 at both the mRNA and protein levels decreased. The results of quantitative RT-PCR, Western blot analysis, dual-luciferase report gene and functional miRNA let-7 rescue experiments, adhesion assay and in vitro transwell invasion assay confirmed that H19 regulated trophoblastic spheroid adhesion with endometrial stromal cells through the H19/let-7/ITGB3 axis, thereby providing an improved understanding of the molecular mechanism of SA.
Song Li, Qi Fan, Yanqiu Xie, Haiyan Lin, Qi Qiu, Yihua Liang and Qingxue Zhang
In vitro activation of primordial follicles is becoming more essential in assisted reproductive technologies. Vasoactive intestinal peptide (VIP) is one of the members of the neurotrophin family which has demonstrated to have an impact on follicle development in recent years. This study aims to investigate the effect of VIP on the activation of primordial follicles in neonatal rat in an in vitro culture system and to determine the relevant molecular mechanism of their activation. Ovaries of 4-day-old rats were examined for the expression of VIP receptors and were cultured in mediums containing VIP with or without inhibitors of the ERK–mTOR signalling pathway. They were then collected for histological analysis or measurement of the molecular expression of this pathway. The receptors of VIP were found in granular cells and oocytes of primordial and early-growing follicles in neonatal ovary. The ratio of growing follicle increased in the presence VIP at different concentrations, with the highest level of increase being observed in the 10−7 mol/L VIP-treated group. The ratio of PCNA-positive granular cells was also increased, while that of the apoptotic oocytes were decreased, and protein analysis showed increased phosphorylation of ERK1/2, mTOR and RPS6 in the VIP-treated group. However, the effect of VIP on the activation of primordial follicle became insignificant with the addition of MEK inhibitor (U0126) or mTORC1 inhibitor (rapamycin). This study indicated that VIP could activate neonatal rat primordial follicle through the ERK-mTOR signalling pathway, suggesting a strategy for in vitro primordial follicle recruitment.
Renjie Wang, Wei Pan, Lei Jin, Yuehan Li, Yudi Geng, Chun Gao, Gang Chen, Hui Wang, Ding Ma and Shujie Liao
Artificial intelligence (AI) has experienced rapid growth over the past few years, moving from the experimental to the implementation phase in various fields, including medicine. Advances in learning algorithms and theories, the availability of large datasets, and improvements in computing power have contributed to breakthroughs in current AI applications. Machine learning (ML), a subset of AI, allows computers to detect patterns from large complex datasets automatically and uses these patterns to make predictions. AI is proving to be increasingly applicable to healthcare, and multiple machine-learning techniques have been used to improve the performance of assisted reproductive technology (ART). Despite various challenges, the integration of AI and reproductive medicine is bound to give an essential direction to medical development in the future. In this review, we discussed the basic aspects of AI and machine learning, and we addressed the applications, potential limitations and challenges of AI. We also highlighted the prospects and future directions in the context of reproductive medicine.
Claire Stenhouse, Charis O Hogg and Cheryl J Ashworth
Integrins regulate adhesion at the foeto-maternal interface by interacting with secreted phosphoprotein 1 (SPP1) and fibronectin (FN). It is hypothesised that impaired foetal growth of ‘runt’ piglets is linked to altered integrin signalling at the foeto-maternal interface. Placental and endometrial samples associated with the lightest and closest to mean litter weight (CTMLW) (gestational day (GD18, 30, 45, 60 and 90), of both sex (GD30, 45, 60 and 90) (n = 5–8 litters/GD), Large White × Landrace conceptuses or foetuses were obtained. The mRNA expression of the integrin subunits (ITG) ITGA2, ITGAV, ITGB1, ITGB3, ITGB5, ITGB6, ITGB8, SPP1 and FN was quantified by qPCR. Temporal changes in mRNA expression were observed, with different profiles in the two tissues. Endometrial ITGB1 (P ≤ 0.05, GD45) and SPP1 (P ≤ 0.05, all GD combined and GD60) expression was decreased in samples supplying the lightest compared to the CTMLW foetuses. Placentas supplying female foetuses had decreased expression of ITGB6 (GD45, P ≤ 0.05) and FN (GD90, P ≤ 0.05) compared to those supplying male foetuses. Endometrial samples supplying females had increased ITGB3 (P ≤ 0.05, GD60) and FN (P ≤ 0.05, GD30) expression and decreased SPP1 (P ≤ 0.05, GD60) expression compared to male foetuses. Correlations between mean within-gilt mRNA expression and percentage prenatal survival, number of live foetuses or conceptuses and percentage male foetuses were observed. This study has highlighted novel and dynamic associations between foetal size, sex and integrin subunit mRNA expression at the porcine foeto-maternal interface. Further studies should be performed to improve the understanding of the mechanisms behind these novel findings.