Efficient direct reprogramming of mature amniotic cells into endothelial cells by ETS factors and TGFß suppression.

ETS transcription factors ETV2, FLI1, and ERG1 specify pluripotent stem cells into induced vascular endothelial cells (iVECs). However, iVECs are unstable and drift toward nonvascular cells. We show that human midgestation c-Kit(-) lineage-committed amniotic cells (ACs) can be reprogrammed into vascular endothelial cells (rAC-VECs) without transitioning through a pluripotent state. Transient ETV2 expression in ACs generates immature rAC-VECs, whereas coexpression with FLI1/ERG1 endows rAC-VECs with a vascular repertoire and morphology matching mature endothelial cells (ECs). Brief TGFß-inhibition functionalizes VEGFR2 signaling, augmenting specification of ACs into rAC-VECs. Genome-wide transcriptional analyses showed that rAC-VECs are similar to adult ECs in which vascular-specific genes are expressed and nonvascular genes are silenced. Functionally, rAC-VECs form stable vasculature in Matrigel plugs and regenerating livers. Therefore, short-term ETV2 expression and TGFß inhibition with constitutive ERG1/FLI1 coexpression reprogram mature ACs into durable rAC-VECs with clinical-scale expansion potential. Banking of HLA-typed rAC-VECs establishes a vascular inventory for treatment of diverse disorders.

Xenograft model of heterotopic transplantation of human ovarian cortical tissue and its clinical relevance.

In brief: Xenografts of human ovarian cortical tissue provide a tractable model of heterotopic autotransplantation that is used for fertility preservation in patients undergoing ablative chemo/radiotherapy. This study describes the behavior of hundreds of xenografts to establish a framework for the clinical function of ovarian cortex following autotransplantation over short- and long-term intervals. Abstract: More than 200 live births have been achieved using autotransplantation of cryopreserved ovarian cortical fragments, yet challenges remain to be addressed. Ischemia of grafted tissue undermines viability and longevity, typically requiring transplantation of multiple cortical pieces; and the dynamics of recruitment within a graft and the influence of parameters like size and patient age at the time of cryopreservation are not well-defined. Here, we describe results from a series of experiments in which we xenografted frozen/thawed human ovarian tissue (n = 440) from 28 girls and women (age range 32 weeks gestational age to 46 years, median 24.3 ± 4.6). Xenografts were recovered across a broad range of intervals (1-52 weeks post-transplantation) and examined histologically to quantify follicle density and distribution. The number of antral follicles in xenografted cortical fragments correlated positively with the total follicle number and was significantly reduced with increased patient age. Within xenografts, follicles were distributed in focal clusters, similar to the native ovary, but the presence of a leading antral follicle coincided with increased proliferation of surrounding follicles. These results underscore the importance of transplanting ovarian tissue with a high density of follicles and elucidate a potential paracrine influence of leading antral follicles on neighboring follicles of earlier stages. This temporal framework for interpreting the kinetics of follicle growth/mobilization may be useful in setting expectations and guiding the parameters of clinical autotransplantation.

Ovarian Reserve Disorders, Can We Prevent Them? A Review.

The ovarian reserve is finite and begins declining from its peak at mid-gestation until only residual follicles remain as women approach menopause. Reduced ovarian reserve, or its extreme form, premature ovarian insufficiency, stems from multiple factors, including developmental, genetic, environmental exposures, autoimmune disease, or medical/surgical treatment. In many cases, the cause remains unknown and resulting infertility is not ultimately addressed by assisted reproductive technologies. Deciphering the mechanisms that underlie disorders of ovarian reserve could improve the outcomes for patients struggling with infertility, but these disorders are diverse and can be categorized in multiple ways. In this review, we will explore the topic from a perspective that emphasizes the prevention or mitigation of ovarian damage. The most desirable mode of fertoprotection is primary prevention (intervening before ablative influence occurs), as identifying toxic influences and deciphering the mechanisms by which they exert their effect can reduce or eliminate exposure and damage. Secondary prevention in the form of screening is not recommended broadly. Nevertheless, in some instances where a known genetic background exists in discrete families, screening is advised. As part of prenatal care, screening panels include some genetic diseases that can lead to infertility or subfertility. In these patients, early diagnosis could enable fertility preservation or changes in family-building plans. Finally, Tertiary Prevention (managing disease post-diagnosis) is critical. Reduced ovarian reserve has a major influence on physiology beyond fertility, including delayed/absent puberty or premature menopause. In these instances, proper diagnosis and medical therapy can reduce adverse effects. Here, we elaborate on these modes of prevention as well as proposed mechanisms that underlie ovarian reserve disorders.

Wnt inhibition promotes vascular specification of embryonic cardiac progenitors.

Several studies have demonstrated a multiphasic role for Wnt signaling during embryonic cardiogenesis and developed protocols that enrich for cardiac derivatives during in vitro differentiation of human pluripotent stem cells (hPSCs). However, few studies have investigated the role of Wnt signaling in the specification of cardiac progenitor cells (CPCs) toward downstream fates. Using transgenic mice and hPSCs, we tracked endothelial cells (ECs) that originated from CPCs expressing NKX2.5. Analysis of EC-fated CPCs at discrete phenotypic milestones during hPSC differentiation identified reduced Wnt activity as a hallmark of EC specification, and the enforced activation or inhibition of Wnt reduced or increased, respectively, the degree of vascular commitment within the CPC population during both hPSC differentiation and mouse embryogenesis. Wnt5a, which has been shown to exert an inhibitory influence on Wnt signaling during cardiac development, was dynamically expressed during vascular commitment of hPSC-derived CPCs, and ectopic Wnt5a promoted vascular specification of hPSC-derived and mouse embryonic CPCs.

Endothelial deletion of ADAM10, a key regulator of Notch signaling, causes impaired decidualization and reduced fertility in female mice.

During the initiation of pregnancy, the vasculature of the implantation site expands rapidly, yet little is known about this process or its role in fertility. Here, we report that endothelial-specific deletion of a disintegrin and metalloprotease 10 (ADAM10), an essential regulator of Notch signaling, results in severe subfertility in mice. We found that implantation sites develop until 5.5 days post conception (dpc) but are resorbed by 6.5 dpc in A10ΔEC mice. Analysis of the mutant implantation sites showed impaired decidualization and abnormal vascular patterning compared to controls. Moreover, RNA-seq analysis revealed changes in endothelial cell marker expression consistent with defective ADAM10/Notch signaling in samples from A10ΔEC mice, suggesting that this signaling pathways is essential for the physiological function of endometrial endothelial cells during early pregnancy. Our findings raise the possibility that impaired endothelial cell function could be a cause for repeated pregnancy loss (RPL) and infertility in humans.

Reprogramming human endothelial cells to haematopoietic cells requires vascular induction.

Generating engraftable human haematopoietic cells from autologous tissues is a potential route to new therapies for blood diseases. However, directed differentiation of pluripotent stem cells yields haematopoietic cells that engraft poorly. Here, we have devised a method to phenocopy the vascular-niche microenvironment of haemogenic cells, thereby enabling reprogramming of human endothelial cells into engraftable haematopoietic cells without transition through a pluripotent intermediate. Highly purified non-haemogenic human umbilical vein endothelial cells or adult dermal microvascular endothelial cells were transduced with the transcription factors FOSB, GFI1, RUNX1 and SPI1 (hereafter referred to as FGRS), and then propagated on serum-free instructive vascular niche monolayers to induce outgrowth of haematopoietic colonies containing cells with functional and immunophenotypic features of multipotent progenitor cells (MPPs). These endothelial cells that have been reprogrammed into human MPPs (rEC-hMPPs) acquire colony-forming-cell potential and durably engraft into immune-deficient mice after primary and secondary transplantation, producing long-term rEC-hMPP-derived myeloid (granulocytic/monocytic, erythroid, megakaryocytic) and lymphoid (natural killer and B cell) progenies. Conditional expression of FGRS transgenes, combined with vascular induction, activates endogenous FGRS genes, endowing rEC-hMPPs with a transcriptional and functional profile similar to that of self-renewing MPPs. Our approach underscores the role of inductive cues from the vascular niche in coordinating and sustaining haematopoietic specification and may prove useful for engineering autologous haematopoietic grafts to treat inherited and acquired blood disorders.

Inductive angiocrine signals from sinusoidal endothelium are required for liver regeneration.

During embryogenesis, endothelial cells induce organogenesis before the development of circulation. These findings suggest that endothelial cells not only form passive conduits to deliver nutrients and oxygen, but also establish an instructive vascular niche, which through elaboration of paracrine trophogens stimulates organ regeneration, in a manner similar to endothelial-cell-derived angiocrine factors that support haematopoiesis. However, the precise mechanism by which tissue-specific subsets of endothelial cells promote organogenesis in adults is unknown. Here we demonstrate that liver sinusoidal endothelial cells (LSECs) constitute a unique population of phenotypically and functionally defined VEGFR3(+)CD34(-)VEGFR2(+)VE-cadherin(+)FactorVIII(+)CD45(-) endothelial cells, which through the release of angiocrine trophogens initiate and sustain liver regeneration induced by 70% partial hepatectomy. After partial hepatectomy, residual liver vasculature remains intact without experiencing hypoxia or structural damage, which allows study of physiological liver regeneration. Using this model, we show that inducible genetic ablation of vascular endothelial growth factor (VEGF)-A receptor-2 (VEGFR2) in the LSECs impairs the initial burst of hepatocyte proliferation (days 1-3 after partial hepatectomy) and subsequent reconstitution of the hepatovascular mass (days 4-8 after partial hepatectomy) by inhibiting upregulation of the endothelial-cell-specific transcription factor Id1. Accordingly, Id1-deficient mice also manifest defects throughout liver regeneration, owing to diminished expression of LSEC-derived angiocrine factors, including hepatocyte growth factor (HGF) and Wnt2. Notably, in in vitro co-cultures, VEGFR2-Id1 activation in LSECs stimulates hepatocyte proliferation. Indeed, intrasplenic transplantation of Id1(+/+) or Id1(-/-) LSECs transduced with Wnt2 and HGF (Id1(-/-)Wnt2(+)HGF(+) LSECs) re-establishes an inductive vascular niche in the liver sinusoids of the Id1(-/-) mice, initiating and restoring hepatovascular regeneration. Therefore, in the early phases of physiological liver regeneration, VEGFR2-Id1-mediated inductive angiogenesis in LSECs through release of angiocrine factors Wnt2 and HGF provokes hepatic proliferation. Subsequently, VEGFR2-Id1-dependent proliferative angiogenesis reconstitutes liver mass. Therapeutic co-transplantation of inductive VEGFR2(+)Id1(+)Wnt2(+)HGF(+) LSECs with hepatocytes provides an effective strategy to achieve durable liver regeneration.

Human ESC-derived hemogenic endothelial cells undergo distinct waves of endothelial to hematopoietic transition.

UNLABELLED: Several studies have demonstrated that hematopoietic cells originate from endotheliumin early development; however, the phenotypic progression of progenitor cells during human embryonic hemogenesis is not well described. Here, we define the developmental hierarchy among intermediate populations of hematopoietic progenitor cells (HPCs) derived from human embryonic stem cells (hESCs). We genetically modified hESCs to specifically demarcate acquisition of vascular (VE-cadherin) and hematopoietic (CD41a) cell fate and used this dual-reporting transgenic hESC line to observe endothelial to hematopoietic transition by real-time confocal microscopy. Live imaging and clonal analyses revealed a temporal bias in commitment of HPCs that recapitulates discrete waves of lineage differentiation noted during mammalian hemogenesis. Specifically, HPCs isolated at later time points showed reduced capacity to form erythroid/ megakaryocytic cells and exhibited a tendency toward myeloid fate that was enabled by expression of the Notch ligand Dll4 on hESC-derived vascular feeder cells. These data provide a framework for defining HPC lineage potential, elucidate a molecular contribution from the vascular niche in promoting hematopoietic lineage progression, and distinguish unique subpopulations of hemogenic endothelium during hESC differentiation. KEY POINTS: Live imaging of endothelial to hematopoietic conversion identifies distinct subpopulations of hESC-derived hemogenic endothelium. Expression of the Notch ligand DII4 on vascular ECs drives induction of myeloid fate from hESC-derived hematopoietic progenitors.

Akt suppression of TGFß signaling contributes to the maintenance of vascular identity in embryonic stem cell-derived endothelial cells.

The ability to generate and maintain stable in vitro cultures of mouse endothelial cells (ECs) has great potential for genetic dissection of the numerous pathologies involving vascular dysfunction as well as therapeutic applications. However, previous efforts at achieving sustained cultures of primary stable murine vascular cells have fallen short, and the cellular requirements for EC maintenance in vitro remain undefined. In this study, we have generated vascular ECs from mouse embryonic stem (ES) cells and show that active Akt is essential to their survival and propagation as homogeneous monolayers in vitro. These cells harbor the phenotypical, biochemical, and functional characteristics of ECs and expand throughout long-term cultures, while maintaining their angiogenic capacity. Moreover, Akt-transduced embryonic ECs form functional perfused vessels in vivo that anastomose with host blood vessels. We provide evidence for a novel function of Akt in stabilizing EC identity, whereby the activated form of the protein protects mouse ES cell-derived ECs from TGFß-mediated transdifferentiation by downregulating SMAD3. These findings identify a role for Akt in regulating the developmental potential of ES cell-derived ECs and demonstrate that active Akt maintains endothelial identity in embryonic ECs by interfering with active TGFß-mediated processes that would ordinarily usher these cells to alternate fates.

Development of a vascular niche platform for expansion of repopulating human cord blood stem and progenitor cells.

Transplantation of ex vivo expanded human umbilical cord blood cells (hCB) only partially enhances the hematopoietic recovery after myelosuppressive therapy. Incubation of hCB with optimal combinations of cytokines and niche cells, such as endothelial cells (ECs), could augment the efficiency of hCB expansion. We have devised an approach to cultivate primary human ECs (hECs) in serum-free culture conditions. We demonstrate that coculture of CD34(+) hCB in direct cellular contact with hECs and minimal concentrations of thrombopoietin/Kit-ligand/Flt3-ligand resulted in a 400-fold expansion of total hematopoietic cells, 150-fold expansion of CD45(+)CD34(+) progenitor cells, and 23-fold expansion of CD45(+) Lin(-)CD34(hi+)CD45RA(-)CD49f(+) stem and progenitor cells over a 12-day period. Compared with cytokines alone, coculture of hCB with hECs permitted greater expansion of cells capable of multilineage engraftment and serial transplantation, hallmarks of long-term repopulating hematopoietic stem cells. Therefore, hECs establish a cellular platform for expansion of hematopoietic stem and progenitor cells and treatment of hematologic disorders.

Generation of functional multipotent adult stem cells from GPR125+ germline progenitors.

Adult mammalian testis is a source of pluripotent stem cells. However, the lack of specific surface markers has hampered identification and tracking of the unrecognized subset of germ cells that gives rise to multipotent cells. Although embryonic-like cells can be derived from adult testis cultures after only several weeks in vitro, it is not known whether adult self-renewing spermatogonia in long-term culture can generate such stem cells as well. Here, we show that highly proliferative adult spermatogonial progenitor cells (SPCs) can be efficiently obtained by cultivation on mitotically inactivated testicular feeders containing CD34+ stromal cells. SPCs exhibit testicular repopulating activity in vivo and maintain the ability in long-term culture to give rise to multipotent adult spermatogonial-derived stem cells (MASCs). Furthermore, both SPCs and MASCs express GPR125, an orphan adhesion-type G-protein-coupled receptor. In knock-in mice bearing a GPR125-beta-galactosidase (LacZ) fusion protein under control of the native Gpr125 promoter (GPR125-LacZ), expression in the testis was detected exclusively in spermatogonia and not in differentiated germ cells. Primary GPR125-LacZ SPC lines retained GPR125 expression, underwent clonal expansion, maintained the phenotype of germline stem cells, and reconstituted spermatogenesis in busulphan-treated mice. Long-term cultures of GPR125+ SPCs (GSPCs) also converted into GPR125+ MASC colonies. GPR125+ MASCs generated derivatives of the three germ layers and contributed to chimaeric embryos, with concomitant downregulation of GPR125 during differentiation into GPR125- cells. MASCs also differentiated into contractile cardiac tissue in vitro and formed functional blood vessels in vivo. Molecular bookmarking by GPR125 in the adult mouse and, ultimately, in the human testis could enrich for a population of SPCs for derivation of GPR125+ MASCs, which may be employed for genetic manipulation, tissue regeneration and revascularization of ischaemic organs.

Direct differentiation of human pluripotent stem cells into vascular network along with supporting mural cells.

During embryonic development, endothelial cells (ECs) undergo vasculogenesis to form a primitive plexus and assemble into networks comprised of mural cell-stabilized vessels with molecularly distinct artery and vein signatures. This organized vasculature is established prior to the initiation of blood flow and depends on a sequence of complex signaling events elucidated primarily in animal models, but less studied and understood in humans. Here, we have developed a simple vascular differentiation protocol for human pluripotent stem cells that generates ECs, pericytes, and smooth muscle cells simultaneously. When this protocol is applied in a 3D hydrogel, we demonstrate that it recapitulates the dynamic processes of early human vessel formation, including acquisition of distinct arterial and venous fates, resulting in a vasculogenesis angiogenesis model plexus (VAMP). The VAMP captures the major stages of vasculogenesis, angiogenesis, and vascular network formation and is a simple, rapid, scalable model system for studying early human vascular development in vitro.

Human theca arises from ovarian stroma and is comprised of three discrete subtypes.

Theca cells serve multiple essential functions during the growth and maturation of ovarian follicles, providing structural, metabolic, and steroidogenic support. While the function of theca during folliculogenesis is well established, their cellular origins and the differentiation hierarchy that generates distinct theca sub-types, remain unknown. Here, we performed single cell multi-omics analysis of primary cell populations purified from human antral stage follicles (1-3 mm) to define the differentiation trajectory of theca/stroma cells. We then corroborated the temporal emergence and growth kinetics of defined theca/stroma subpopulations using human ovarian tissue samples and xenografts of cryopreserved/thawed ovarian cortex, respectively. We identified three lineage specific derivatives termed structural, androgenic, and perifollicular theca cells, as well as their putative lineage-negative progenitor. These findings provide a framework for understanding the differentiation process that occurs in each primordial follicle and identifies specific cellular/molecular phenotypes that may be relevant to either diagnosis or treatment of ovarian pathologies.

Dual role of lipids for genome stability and pluripotency facilitates full potency of mouse embryonic stem cells.

While Mek1/2 and Gsk3ß inhibition ("2i") supports the maintenance of murine embryonic stem cells (ESCs) in a homogenous naïve state, prolonged culture in 2i results in aneuploidy and DNA hypomethylation that impairs developmental potential. Additionally, 2i fails to support derivation and culture of fully potent female ESCs. Here we find that mouse ESCs cultured in 2i/LIF supplemented with lipid-rich albumin (AlbuMAX) undergo pluripotency transition yet maintain genomic stability and full potency over long-term culture. Mechanistically, lipids in AlbuMAX impact intracellular metabolism including nucleotide biosynthesis, lipid biogenesis, and TCA cycle intermediates, with enhanced expression of DNMT3s that prevent DNA hypomethylation. Lipids induce a formative-like pluripotent state through direct stimulation of Erk2 phosphorylation, which also alleviates X chromosome loss in female ESCs. Importantly, both male and female "all-ESC" mice can be generated from de novo derived ESCs using AlbuMAX-based media. Our findings underscore the importance of lipids to pluripotency and link nutrient cues to genome integrity in early development.

Extraction, Labeling, and Purification of Lineage-Specific Cells from Human Antral Follicles.

The activation, growth, development, and maturation of oocytes is a complex process that is coordinated not just between multiple cell types of the ovary but also across multiple points of control within the hypothalamic/pituitary/ovarian circuit. Within the ovary, multiple specialized cell types grow in close association with the oocyte within the ovarian follicles. The biology of these cells has been well described at the later stages, when they are easily recovered as byproducts of assisted reproductive treatments. However, the in-depth analysis of small antral follicles isolated directly from the ovary is not commonly carried out due to the scarcity of human ovarian tissue and the limited access to the ovary in patients undergoing assisted reproductive treatments. These methods for processing whole ovaries for the cryopreservation of cortical strips with the concurrent identification/isolation of ovary resident cells enable the high-resolution analysis of the early stages of antral follicle development. We demonstrate protocols for isolating discrete cell types by treating antral follicles enzymatically and separating the granulosa, theca, endothelial, hematopoietic, and stromal cells. The isolation of cells from the antral follicles at various sizes and developmental stages enables the comprehensive analysis of the cellular and molecular mechanisms that drive follicle growth and ovarian physiology and provides a source of viable cells that can be cultured in vitro to recapitulate the follicle microenvironment.

Chronic superphysiologic AMH promotes premature luteinization of antral follicles in human ovarian xenografts.

Anti-Müllerian hormone (AMH) is produced by growing ovarian follicles and provides a diagnostic measure of reproductive reserve in women; however, the impact of AMH on folliculogenesis is poorly understood. We cotransplanted human ovarian cortex with control or AMH-expressing endothelial cells in immunocompromised mice and recovered antral follicles for purification and downstream single-cell RNA sequencing of granulosa and theca/stroma cell fractions. A total of 38 antral follicles were observed (19 control and 19 AMH) at long-term intervals (>10 weeks). In the context of exogenous AMH, follicles exhibited a decreased ratio of primordial to growing follicles and antral follicles of increased diameter. Transcriptomic analysis and immunolabeling revealed a marked increase in factors typically noted at more advanced stages of follicle maturation, with granulosa and theca/stroma cells also displaying molecular hallmarks of luteinization. These results suggest that superphysiologic AMH alone may contribute to ovulatory dysfunction by accelerating maturation and/or luteinization of antral-stage follicles.

Exogenous insulin-like growth factor 1 accelerates growth and maturation of follicles in human cortical xenografts and increases ovarian output in mice.

OBJECTIVE: To measure the influence of exogenous insulin-like growth factor 1 (IGF1) on follicle growth and maturation in human ovarian cortical xenografts. DESIGN: Xenotransplantation model. SETTING: University-based research laboratory. PATIENTS/ANIMALS: Ovarian tissue was donated with consent and institutional review board approval by brain-dead organ donors or patients undergoing ovarian tissue cryopreservation for fertility preservation. Cortical fragments were transplanted into immunocompromised mice. INTERVENTIONS: Cryopreserved ovarian cortical fragments from four women (aged 19, 25, 33, and 46 years) were transplanted into the gluteus muscle of immunocompromised mice in a fibrin matrix containing endothelial cells that were transduced with lentiviral particles encoding secreted IGF1. Xenografts were recovered after 3, 8, and 14 weeks. In addition, C57/Bl6 mice underwent intraovarian injection of saline or recombinant IGF1 (60 µg), followed by superovulation, analysis of ethynyl-deoxyuridine incorporation, and ribonucleic acid sequencing of the whole ovaries. MAIN OUTCOME MEASURES: For xenografts: follicle count and distribution; antral follicle count; and corpora lutea/albicans count. For mice: follicle count and distribution; oocyte yield, ethynyl-deoxyuridine incorporation (granulosa cell proliferation); and ovarian transcriptomic signature. RESULTS: At 3 weeks, xenografts in the IGF1 condition revealed a decreased percentage of primary follicles and increased percentage of secondary follicles that were concentrated in the preantral subtype; at 8 weeks, an increase in secondary follicles was concentrated in the simple subtype; after 14 weeks, primordial follicles were reduced, and while the number of advanced follicles did not power the experiment to demonstrate significance, antral follicles reduced and corpora lutea increased. Supporting experiments in mice revealed an increase in normal oocytes following intraovarian injection of recombinant IGF1 (60 µg) as well as increased proliferative index among follicles of secondary and preantral stages. Ribonucleic acid sequencing analysis of the whole ovaries following injection of recombinant IGF1 (25 µg) revealed an acute (24 hours) upregulation of transcripts related to steroidogenesis and luteinization. CONCLUSIONS: Exogenous IGF1 advances the pace of growth among primordial, primary, and secondary stage follicles but results in near absence of antral stage follicles in long-term (14 weeks) xenografts. In mice, acute administration of IGF1 promotes follicle advance and increased oocyte yield. The results suggest that while superphysiological IGF1 alone advances the pace of growth among early/preantral follicles, a sustained and/or later-stage influence undermines antral follicle growth/survival or promotes premature luteinization. These findings provide a temporal framework for interpreting follicle growth/mobilization and may be useful in understanding the clinical application of human growth hormone in the context of assisted reproduction.

Cycle day 2 insulin-like growth factor-1 serum levels as a prognostic tool to predict controlled ovarian hyperstimulation outcomes in poor responders.

OBJECTIVE: To study whether patients exhibiting poor ovarian response have abnormal levels of serum insulin-like growth factor (IGF)-1 on cycle day 2 when compared with age-matched normal and high responders. DESIGN: Retrospective cohort. SETTING: University-based practice. PATIENT(S): All women between the ages of 21 and 42 years who underwent in vitro fertilization treatment cycle without estrogen pretreatment at our institution between 2013 and 2015. INTERVENTION(S): Patients were separated into three groups: poor responders (≤4 oocytes retrieved/cycle cancellation), normal responders (8-12 oocytes), and high responders (≥18 oocytes). Subanalysis focused on the next cycle for poor responders adjacent to the nonpretreated index cycle, in which estrogen pretreatment was implemented. MAIN OUTCOME MEASURE(S): Serum cycle day 2: IGF-1, insulin-like growth factor-binding protein (IGFBP)-3 levels, and IGF-1:IGFBP3 ratio, number of eggs retrieved, number of two pronuclei embryos, cumulative pregnancy rate, and live birth. RESULT(S): A total of 184 patients met the inclusion criteria. The poor responder group exhibited a more than twofold increase in the cycle day IGF-1 serum levels when compared with normal responders and a threefold increase when compared with the high responders. Cycle day 2 IGF-1 level >72 ng/mL in poor responders had 70% sensitivity and 78% specificity for a negative controlled ovarian hyperstimulation cycle outcome with an area under the curve of 0.83. Luteal estrogen pretreatment in the poor responder group was associated with a significant reduction in IGF-1 levels. Significantly, more retrieved and mature oocytes, as well as two pronuclei embryos, were achieved in the pretreated poor responder group when compared with the yield from their adjacent nonpretreated index cycles. Furthermore, cumulative rates were higher for intrauterine pregnancies, and lower for negative pregnancy outcome. CONCLUSION(S): Patients who respond poorly to controlled ovarian stimulation, despite normal cycle day 2 follicle-stimulating hormone levels, have significantly higher serum cycle day 2 IGF-1 levels when compared with age-matched normal and high responders. Cycle day 2 IGF-1 level >72 ng/mL in poor responders was predictive of a negative cycle outcome. Luteal estrogen pretreatment in the poor responder group was associated with a significant reduction in IGF-1 levels, improved response to stimulation, and higher cumulative rates for intrauterine pregnancies, and lower for negative pregnancy outcome.

Pluripotent stem cells with low differentiation potential contain incompletely reprogrammed DNA replication.

Reprogrammed pluripotent stem cells (PSCs) are valuable for research and potentially for cell replacement therapy. However, only a fraction of reprogrammed PSCs are developmentally competent. Genomic stability and accurate DNA synthesis are fundamental for cell development and critical for safety. We analyzed whether defects in DNA replication contribute to genomic instability and the diverse differentiation potentials of reprogrammed PSCs. Using a unique single-molecule approach, we visualized DNA replication in isogenic PSCs generated by different reprogramming approaches, either somatic cell nuclear transfer (NT-hESCs) or with defined factors (iPSCs). In PSCs with lower differentiation potential, DNA replication was incompletely reprogrammed, and genomic instability increased during replicative stress. Reprogramming of DNA replication did not correlate with DNA methylation. Instead, fewer replication origins and a higher frequency of DNA breaks in PSCs with incompletely reprogrammed DNA replication were found. Given the impact of error-free DNA synthesis on the genomic integrity and differentiation proficiency of PSCs, analyzing DNA replication may be a useful quality control tool.

Comparison of Human Antral Follicles of Xenograft versus Ovarian Origin Reveals Disparate Molecular Signatures.

The activation, growth, and maturation of oocytes to an ovulatory phase, termed folliculogenesis, is governed by the orchestrated activity of multiple specialized cell types within the ovary; yet, the mechanisms governing diversification and behavior of discrete cellular sub-populations within follicles are poorly understood. We use bulk and single-cell RNA sequencing to distinguish the transcriptional signature of prospectively isolated granulosa and theca/stroma cell subsets within human antral follicles derived from xenografts or ovaries. The analysis deconstructs phenotypic diversification within small (<4 mm) antral follicles, identifying secreted factors that are differentially enriched between mural and oophorus granulosa cells, and segregating stromal/support and steroidal activity between theca externa and interna, respectively. Multiple factors are differentially expressed in follicles of xenograft versus ovarian origin. These data capture a high-resolution transcriptional signature of granulosa and theca subpopulations and provide a systems-level portrait of cellular diversification in early antral human follicles.