The impact of endometrioma on in vitro fertilisation/intra-cytoplasmic injection IVF/ICSI reproductive outcomes: a systematic review and meta-analysis.

BACKGROUND: Assisted reproductive technologies (ART) such as in vitro fertilisation (IVF) and intra-cytoplasmic sperm injection (ICSI) are often used to aid fertility in women with endometrioma; however, the implications of endometrioma on ART are unresolved. OBJECTIVE: To determine the effect of endometrioma on reproductive outcomes in women undergoing IVF or ICSI. METHODS: A systematic review and meta-analysis was conducted to identify articles examining women who had endometrioma and had undergone IVF or ICSI. Electronic searches were performed in PubMed, BIOSIS and MEDLINE up to September 2019. The primary outcome was live birth rate (LBR). Secondary outcomes included clinical pregnancy rate (CPR), implantation rate (IR), number of oocytes retrieved, number of metaphase II (MII) oocytes retrieved, number of embryos and top-quality embryos and the duration of gonadotrophin stimulation and dose. RESULTS: Eight studies were included. Where significant heterogeneity between studies was identified, a random-effects model was used. The number of oocytes (weighted means difference; WMD-2.25; 95% CI 3.43 to - 1.06, p = 0.0002) and the number of MII oocytes retrieved (WMD-4.64; 95% CI 5.65 to - 3.63, p < 0.00001) were significantly lower in women with endometrioma versus controls. All other outcomes, including gonadotrophin dose and duration, the total number of embryos, high-quality embryos, CPR, IR and LBR were similar in women with and without endometrioma. CONCLUSION: Even though women with endometriomas had a reduced number of oocytes and MII oocytes retrieved when compared to women without, no other differences in reproductive outcomes were identified. This implies that IVF/ICSI is a beneficial ART approach for women with endometrioma.

SMAD3 directly regulates cell cycle genes to maintain arrest in granulosa cells of mouse primordial follicles.

Primordial follicles, consisting of granulosa cell (GC)-enveloped oocytes are maintained in a state of developmental arrest until activated to grow. The mechanism that operates to maintain this arrested state in GCs is currently unknown. Here, we show the TGFß-activated transcription factor SMAD3 is expressed in primordial GC nuclei alongside the cell cycle proteins, cyclin D2 (CCND2) and P27. Using neonatal C57/Bl6 mouse ovaries densely populated with primordial follicles, CCND2 protein co-localised and was detected in complex with P27 by immunofluorescence and co-immunoprecipitation, respectively. In the same tissue, SMAD3 co-precipitated with DNA sequences upstream of Ccnd2 and Myc transcription start sites implicating both as direct SMAD3 targets. In older ovaries follicle growth was associated with nuclear exclusion of SMAD3 and reduced P27 and CCND2 in GCs, alongside elevated Myc expression. Brief (2 H) exposure of neonatal ovaries to TGFß1 (10 ng/ml) in vitro led to immediate dissociation of SMAD3 from the Ccnd2 and Myc promoters. This coincided with elevated Myc and phospho-S6, an indicator of mTOR signalling, followed by a small increase in mean primordial GC number after 48 H. These findings highlight a concentration-dependent role for TGFß signalling in the maintenance and activation of primordial follicles, through SMAD-dependent and independent signalling pathways, respectively.

Nuclear exclusion of SMAD2/3 in granulosa cells is associated with primordial follicle activation in the mouse ovary.

Maintenance and activation of the limited supply of primordial follicles in the ovary are important determinants of reproductive lifespan. Currently, the molecular programme that maintains the primordial phenotype and the early events associated with follicle activation are not well defined. Here, we have systematically analysed these events using microscopy and detailed image analysis. Using the immature mouse ovary as a model, we demonstrate that the onset of granulosa cell (GC) proliferation results in increased packing density on the oocyte surface and consequent GC cuboidalization. These events precede oocyte growth and nuclear translocation of FOXO3a, a transcription factor important in follicle activation. Immunolabelling of the TGFß signalling mediators and transcription factors SMAD2/3 revealed a striking expression pattern specific to GCs of small follicles. SMAD2/3 were expressed in the nuclei of primordial GCs but were mostly excluded in early growing follicles. In activated follicles, GC nuclei lacking SMAD2/3 generally expressed Ki67. These findings suggest that the first phenotypic changes during follicle activation are observed in GCs, and that TGFß signalling is fundamental for regulating GC arrest and the onset of proliferation.

Serine threonine kinase receptor associated protein regulates early follicle development in the mouse ovary.

The molecular mechanisms involved in regulating the development of small, gonadotrophin-independent follicles are poorly understood; however, many studies have highlighted an essential role for TGFB ligands. Canonical TGFB signalling is dependent upon intracellular SMAD proteins that regulate transcription. STRAP has been identified in other tissues as an inhibitor of the TGFB-SMAD signalling pathway. Therefore, in this study we aimed to determine the expression and role of STRAP in the context of early follicle development. Using qPCR, Strap, Smad3 and Smad7 revealed similar expression profiles in immature ovaries from mice aged 4-16 days containing different populations of early growing follicles. STRAP and SMAD2/3 proteins co-localised in granulosa cells of small follicles using immunofluorescence. Using an established culture model, neonatal mouse ovary fragments with a high density of small non-growing follicles were used to examine the effects of Strap knockdown using siRNA and STRAP protein inhibition by immuno-neutralisation. Both interventions caused a reduction in the proportion of small, non-growing follicles and an increase in the proportion and size of growing follicles in comparison to untreated controls, suggesting inhibition of STRAP facilitates follicle activation. Recombinant STRAP protein had no effect on small, non-growing follicles, but increased the mean oocyte size of growing follicles in the neonatal ovary model and also promoted the growth of isolated preantral follicles in vitro Overall findings indicate STRAP is expressed in the mouse ovary and is capable of regulating development of small follicles in a stage-dependent manner.

Investigations of TGF-ß signaling in preantral follicles of female mice reveal differential roles for bone morphogenetic protein 15.

Bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) are 2 closely related TGF-ß ligands implicated as key regulators of follicle development and fertility. Animals harboring mutations of these factors often exhibit a blockage in follicle development beyond the primary stage and therefore little is known about the role of these ligands during subsequent (preantral) stages. Preantral follicles isolated from immature mice were cultured with combinations of BMP15, GDF9, and activin receptor-like kinase (ALK) inhibitors. Individually, GDF9 and BMP15 promoted follicle growth during the first 24 hours, whereas BMP15 subsequently (48-72 h) caused follicle shrinkage and atresia with increased granulosa cell apoptosis. Inhibition of ALK6 prevented the BMP15-induced reduction in follicle size and under basal conditions promoted a rapid increase in granulosa cell proliferation, suggesting BMP15 signals through ALK6, which in turn acts to restrain follicle growth. In the presence of GDF9, BMP15 no longer promoted atresia and in fact follicle growth was increased significantly more than with either ligand alone. This cooperative effect was accompanied by differential expression of Id1-3, Smad6-7, and Has2 and was blocked by the same ALK5 inhibitor used to block GDF9 signaling. Immunostaining for SMAD2/3 and SMAD1/5/8, representing the 2 main branches of TGF-ß signaling, supported the fact that both canonical pathways have the potential to be active in growing follicles, whereas primordial follicles only express SMAD2/3. Overall results highlight differential effects of the 2 main TGF-ß signaling pathways during preantral follicle growth.

Influence of energy balance on the antimicrobial peptides S100A8 and S100A9 in the endometrium of the post-partum dairy cow.

Uterine inflammation occurs after calving in association with extensive endometrial remodelling and bacterial contamination. If the inflammation persists, it leads to reduced fertility. Chronic endometritis is highly prevalent in high-yielding cows that experience negative energy balance (NEB) in early lactation. This study investigated the effect of NEB on the antimicrobial peptides S100A8 and S100A9 in involuting uteri collected 2 weeks post partum. Holstein-Friesian cows (six per treatment) were randomly allocated to two interventions designed to produce mild or severe NEB (MNEB and SNEB) status. Endometrial samples were examined histologically, and the presence of neutrophils, macrophages, lymphocytes and natural killer cells was confirmed using haematoxylin and eosin and immunostaining. SNEB cows had greater signs of uterine inflammation. Samples of previously gravid uterine horn were used to localise S100A8 and S100A9 by immunohistochemistry. Both S100 proteins were present in bovine endometrium with strong staining in epithelial and stromal cells and in infiltrated leucocytes. Immunostaining was significantly higher in SNEB cows along with increased numbers of segmented neutrophils. These results suggest that the metabolic changes of a post-partum cow suffering from NEB delay uterine involution and promote a chronic state of inflammation. We show that upregulation of S100A8 and S100A9 is clearly a key component of the early endometrial response to uterine infection. Further studies are warranted to link the extent of this response after calving to the likelihood of cows developing endometritis and to their subsequent fertility.

Bmi1 facilitates primitive endoderm formation by stabilizing Gata6 during early mouse development.

The transcription factors Nanog and Gata6 are critical to specify the epiblast versus primitive endoderm (PrE) lineages. However, little is known about the mechanisms that regulate the protein stability and activity of these factors in the developing embryo. Here we uncover an early developmental function for the Polycomb group member Bmi1 in supporting PrE lineage formation through Gata6 protein stabilization. We show that Bmi1 is enriched in the extraembryonic (endoderm [XEN] and trophectodermal stem [TS]) compartment and repressed by Nanog in pluripotent embryonic stem (ES) cells. In vivo, Bmi1 overlaps with the nascent Gata6 and Nanog protein from the eight-cell stage onward before it preferentially cosegregates with Gata6 in PrE progenitors. Mechanistically, we demonstrate that Bmi1 interacts with Gata6 in a Ring finger-dependent manner to confer protection against Gata6 ubiquitination and proteasomal degradation. A direct role for Bmi1 in cell fate allocation is established by loss-of-function experiments in chimeric embryoid bodies. We thus propose a novel regulatory pathway by which Bmi1 action on Gata6 stability could alter the balance between Gata6 and Nanog protein levels to introduce a bias toward a PrE identity in a cell-autonomous manner.

Characterization and significance of adhesion and junction-related proteins in mouse ovarian follicles.

In the ovary, initiation of follicle growth is marked by cuboidalization of flattened granulosa cells (GCs). The regulation and cell biology of this shape change remains poorly understood. We propose that characterization of intercellular junctions and associated proteins is key to identifying as yet unknown regulators of this important transition. As GCs are conventionally described as epithelial cells, this study used mouse ovaries and isolated follicles to investigate epithelial junctional complexes (tight junctions [TJ], adherens junctions [AJ], and desmosomes) and associated molecules, as well as classic epithelial markers, by quantitative PCR and immunofluorescence. These junctions were further characterized using ultrastructural, calcium depletion and biotin tracer studies. Junctions observed by transmission electron microscopy between GCs and between GCs and oocyte were identified as AJs by expression of N-cadherin and nectin 2 and by the lack of TJ and desmosome-associated proteins. Follicles were also permeable to biotin, confirming a lack of functional TJs. Surprisingly, GCs lacked all epithelial markers analyzed, including E-cadherin, cytokeratin 8, and zonula occludens (ZO)-1alpha+. Furthermore, vimentin was expressed by GCs, suggesting a more mesenchymal phenotype. Under calcium-free conditions, small follicles maintained oocyte-GC contact, confirming the importance of calcium-independent nectin at this stage. However, in primary and multilayered follicles, lack of calcium resulted in loss of contact between GCs and oocyte, showing that nectin alone cannot maintain attachment between these two cell types. Lack of classic markers suggests that GCs are not epithelial. Identification of AJs during GC cuboidalization highlights the importance of AJs in regulating initiation of follicle growth.

Identification and regulation of bone morphogenetic protein antagonists associated with preantral follicle development in the ovary.

The TGFß superfamily comprises several bone morphogenetic proteins (BMP) capable of exerting gonadotropin-independent effects on the development of small preantral follicles. In embryonic tissues, BMP concentration gradients, partly formed by antagonistic factors, are essential for establishing phenotypic fate. By examining the expression of candidate genes whose protein products are known to interact with BMP ligands, we set out to determine which antagonists would most likely contribute toward regulation of paracrine signaling during early follicle development. Juvenile mouse ovaries of 4, 8, 12, and 21 d of age enriched with follicles at successive developmental stages were used to assess changes in candidate gene transcripts by quantitative RT-PCR. Although some antagonists were found to be positively associated with the emergence of developing follicles (Nog, Htra1, Fst, Bmper, Vwc2), two (Sostdc1, Chrd) showed a corresponding reduction in expression. At each age, twisted gastrulation homolog 1 (Twsg1), Htra1, Nbl1, and Fst were consistently highly expressed and localization of these genes by in situ hybridization, and immunohistochemistry further highlighted a clear pattern of expression in granulosa cells of developing follicles. Moreover, with the exception of Nbl1, levels of these antagonists did not change in preantral follicles exposed to FSH in vitro, suggesting regulation by local factors. The presence of multiple antagonists in the juvenile ovary and their high level of expression in follicles imply the actions of certain growth factors are subject to local modulation and further highlights another important level of intraovarian regulation of follicle development.

Influence of energy balance on the somatotrophic axis and matrix metalloproteinase expression in the endometrium of the postpartum dairy cow.

Postpartum dairy cows enter a period of negative energy balance (NEB) associated with low circulating IGF1, during which the uterus must undergo extensive repair following calving. This study investigated the effects of NEB on expression of IGF family members and related genes in the involuting uterus. Cows were allocated to two treatments using differential feeding and milking regimes to produce mild NEB or severe NEB (SNEB). Uterine endometrial samples collected 2 weeks post partum were analysed by quantitative PCR. The expression of IGF-binding protein 4 (IGFBP4) mRNA increased in the endometrium of SNEB cows, with trends towards increased IGFBP1 and reduced IGFBP6 expression. There were no significant differences between treatments in mRNA expression of IGF1, IGF2 or of any hormone receptor studied, but significant correlations across all cows in the expression levels of groups of receptors suggested common regulatory mechanisms: type 1 IGF receptor (IGF1R), IGF2R and insulin receptor (INSR); GHR with ESR1; and ESR2 with NR3C1. The expression of IGF1R and INSR also positively correlated with the circulating urea concentration. Matrix metalloproteinases (MMPs) are important in tissue remodelling and can affect IGF signalling via interaction with IGFBPs. The expression levels of MMP1, MMP3, MMP9 and MMP13 mRNAs all showed major upregulation in the endometrium of cows in SNEB and all except MMP9 were highly correlated with expression of IGFBP4. Alpha(2)-HS-glycoprotein (AHSG) and PDK4, two genes implicated in insulin resistance, were also highly expressed in SNEB. These results suggest that cows in SNEB experience alterations to the IGF and insulin signalling pathways in the postpartum endometrium. This may affect the rate of tissue repair with a possible negative impact on subsequent fertility.

IGF1 induces up-regulation of steroidogenic and apoptotic regulatory genes via activation of phosphatidylinositol-dependent kinase/AKT in bovine granulosa cells.

IGF1, a potent stimulator of cellular proliferation, differentiation and development, regulates granulosa cell steroidogenesis and apoptosis during follicular development. Depending upon species and stage of follicular growth, IGF1 acts on granulosa cell steroidogenesis either alone or together with FSH. We examined the mechanism of action of IGF1 in bovine granulosa cells in serum-free culture without insulin to determine its potential role in the regulation of steroidogenic and apoptotic regulatory gene expression and to investigate the interaction of FSH with IGF1 on this mechanism. Bovine granulosa cells treated with IGF1 demonstrated a significant increase in 17beta-oestradiol (OE(2)) production, cell number and in mRNA expression of CYP11A1, HSD3B1, CYP19A1, BAX, type 1 IGF receptor (IGF1R) and FSHR, while FSH alone had no significant effects. IGF1 or FSH alone or both together had no effect on BCL2 expression. IGF1 with FSH resulted in a synergistic increase in granulosa cell number and in mRNA expression of CYP19A1 and IGF1R without altering OE(2) production. IGF1 stimulated the phosphoinositide 3'-OH kinase (PI3K) but not the MAPK pathway in granulosa cells, as evidenced by increased phosphorylation of AKT but not extracellular-regulated kinase 1/2. Addition of the PI3K pathway inhibitor LY294002 (but not the MAPK pathway inhibitor PD98059) abrogated the increased expression of genes induced by IGF1. IGF1 therefore up-regulates the steroidogenic and apoptotic regulatory genes via activation of PI3K/AKT in bovine granulosa cells. The synergistic action of IGF1 with FSH is of likely key importance for the development of small antral follicles before selection; subsequently, other factors such as LH may also become necessary for continued cell survival.

Negative energy balance alters global gene expression and immune responses in the uterus of postpartum dairy cows.

Most dairy cows suffer uterine microbial contamination postpartum. Persistent endometritis often develops, associated with reduced fertility. We used a model of differential feeding and milking regimes to produce cows in differing negative energy balance status in early lactation (mild or severe, MNEB or SNEB). Blood hematology was assessed preslaughter at 2 wk postpartum. RNA expression in endometrial samples was compared using bovine Affymetrix arrays. Data were mapped using Ingenuity Pathway Analysis. Circulating concentrations of IGF-I remained lower in the SNEB group, whereas blood nonesterified fatty acid and beta-hydroxybutyrate concentrations were raised. White blood cell count and lymphocyte number were reduced in SNEB cows. Array analysis of endometrial samples identified 274 differentially expressed probes representing 197 recognized genes between the energy balance groups. The main canonical pathways affected related to immunological and inflammatory disease and connective tissue disorders. Inflammatory response genes with major upregulation in SNEB cows included matrix metalloproteinases, chemokines, cytokines, and calgranulins. Expression of several interferon-inducible genes including ISG20, IFIH1, MX1, and MX2 were also significantly increased in the SNEB cows. These results provide evidence that cows in SNEB were still undergoing an active uterine inflammatory response 2 wk postpartum, whereas MNEB cows had more fully recovered from their energy deficit, with their endometrium reaching a more advanced stage of repair. SNEB may therefore prevent cows from mounting an effective immune response to the microbial challenge experienced after calving, prolonging the time required for uterine recovery and compromising subsequent fertility.

Interrelationships between negative energy balance (NEB) and IGF regulation in liver of lactating dairy cows.

In dairy cows, negative energy balance (NEB) during the early post-partum period is associated with major alterations in the growth hormone-insulin-like growth factor (GH-IGF) axis. Since the liver mediates nutrient partitioning during lactation, we aimed to determine how NEB alters the endocrine regulation of the insulin-like growth factor (IGF) system by investigating the expression of IGF family members and related steroid receptors. On the second day of lactation, cows were allocated to one of two treatments designed to produce mild (MNEB) or severe NEB (SNEB). MNEB cows (n=5) were fed ad lib grass silage supplemented with concentrate and milked x1 daily and SNEB cows (n=6) were restricted in dietary intake and milked x3 daily. Energy balance (EB) status was monitored until the second week of lactation when plasma and liver samples revealed a markedly divergent metabolic profile. At this time, plasma protein and hepatic mRNA for IGF-I was reduced in SNEB cows compared with MNEB cows. Both levels of expression correlated highly when data from all animals was pooled (r=0.963; P<0.01). SNEB cows also exhibited reduced hepatic expression for transcripts encoding IGF-1R, IGF-2R, IGF binding proteins (IGFBPs) -3, -4, -5, -6, acid labile subunit, and receptors for oestrogen (ERalpha) and growth hormone (total GHR and 1A variant), while the expression of IGFBP-2 was elevated. Expression of mRNA for IGF-II, IGFBP-1 and receptors for insulin (A/B) and glucocorticoid (alpha) was unaffected by EB. Results demonstrate that SNEB affects hepatic synthesis of IGF-I, and other components known to modulate the bioavailability and stability of circulating IGF-I.

Suppression of prolactin-induced signal transducer and activator of transcription 5b signaling and induction of suppressors of cytokine signaling messenger ribonucleic acid in the hypothalamic arcuate nucleus of the rat during late pregnancy and lactation.

During late pregnancy and lactation, the tuberoinfundibular dopamine (TIDA) neurons that regulate prolactin secretion by negative feedback become less able to produce dopamine in response to prolactin, leading to hyperprolactinemia. Because prolactin-induced activation of dopamine synthesis in these neurons requires the Janus kinase/signal transducer and activator of transcription 5b (STAT5b) signaling pathway, we investigated whether prolactin-induced STAT5b signaling is reduced during lactation and whether induction of suppressors of cytokine signaling (SOCS) mRNAs occur at this time and in late pregnancy. During lactation, the ability of exogenous prolactin to induce STAT5 phosphorylation and STAT5b nuclear translocation was markedly reduced when compared with diestrous rats. In nonpregnant female rats, acute treatment with ovine prolactin markedly increased levels of SOCS-1 and -3 and cytokine-inducible SH2-containing protein mRNA in arcuate nucleus micropunches. On gestation d 22, SOCS-1 and SOCS-3 mRNA levels were 10-fold that on G20. SOCS-1 and -3 and cytokine-inducible SH2-containing protein mRNA levels were also elevated on lactation d 7. At these times, dopaminergic activity was decreased and the rats were hyperprolactinemic. The high levels of SOCS mRNA were prevented by bromocriptine pretreatment (gestation d 22) or pup removal (lactation d 7), which suppressed circulating prolactin to basal levels. These results demonstrate that around the end of pregnancy, prolactin loses the ability to activate STAT5b, associated with an increase in SOCS mRNAs. The loss of this stimulating pathway may underlie the reduced tuberoinfundibular dopamine neuron dopamine output and hyperprolactinemia that characterizes late pregnancy and lactation. The high maternal levels of SOCS mRNAs appear to be dependent on prolactin, presumably acting through an alternative signaling pathway to STAT5b.

Caspase-3, TUNEL and ultrastructural studies of small follicles in adult human ovarian biopsies.

BACKGROUND: The aim of this study was to investigate evidence for cell death by apoptosis in small unilaminar ovarian follicles of adult humans. METHODS: Cortical biopsies from 13 healthy donors were either frozen and protein extracted for western blots or fixed for immunohistochemistry (IH) to localize procaspase-3 and active-caspase-3, to detect DNA fragmentation in situ and undertake routine transmission electron microscopy (TEM). RESULTS: Blots identified the presence of the inactive pro-form of caspase-3, and IH localized this in all follicles studied. In contrast, the active form of caspase-3, a major effector of apoptosis, was only detected in large antral follicles that also had microscopic signs of atresia. Active caspase-3 was not detected in primordial (n = 87), primary (n = 8) or secondary follicles. The atretic follicles were also the only ovarian structures with positive evidence of DNA fragmentation after terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) treatment. Confocal microscopy showed dual labelling for both active caspase-3 and TUNEL in individual granulosa cells in large atretic follicles, but no such labelling was evident in any other follicles. No apoptotic bodies were seen by TEM in sections of 39 small follicles from seven patients. CONCLUSION: This study found evidence for TUNEL and active caspase-3 only in human ovarian antral follicles.