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.

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.

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.

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.

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.

Co-transplantation of Human Ovarian Tissue with Engineered Endothelial Cells: A Cell-based Strategy Combining Accelerated Perfusion with Direct Paracrine Delivery.

Infertility is a frequent side effect of chemotherapy and/or radiotherapy and for some patients, cryopreservation of oocytes or embryos is not an option. As an alternative, an increasing number of these patients are choosing to cryopreserve ovarian tissue for autograft following recovery and remission. Despite improvements in outcomes among patients undergoing auto-transplantation of cryopreserved ovarian tissue, efficient revascularization of grafted tissue remains a major obstacle. To mitigate ischemia and thus improve outcomes in patients undergoing auto-transplantation, we developed a vascular cell-based strategy for accelerating perfusion of ovarian tissue. We describe a method for co-transplantation of exogenous endothelial cells (ExECs) with cryopreserved ovarian tissue in a mouse xenograft model. We extend this approach to employ ExECs that have been engineered to constitutively express Anti-Mullerian hormone (AMH), thus enabling sustained paracrine signaling input to ovarian grafts. Co-transplantation with ExECs increased follicular volume and improved antral follicle development, and AMH-expressing ExECs promoted retention of quiescent primordial follicles. This combined strategy may be a useful tool for mitigating ischemia and modulating follicular activation in the context of fertility preservation and/or infertility at large.

CGG repeat length and AGG interruptions as indicators of fragile X-associated diminished ovarian reserve.

PURPOSE: Fragile X premutation (PM) carriers may experience difficulties conceiving a child probably due to fragile X-associated diminished ovarian reserve (FXDOR). We investigated which subgroups of carriers with a PM are at higher risk of FXDOR, and whether the number of AGG interruptions within the repeat sequence further ameliorates the risk. METHODS: We compared markers of ovarian reserve, including anti-Müllerian hormone, antral follicle count, and number of oocytes retrieved between different subgroups of patients with a PM. RESULTS: We found that carriers with midrange repeats size (70-90 CGG) demonstrate significantly lower ovarian reserve. Additionally, the number of AGG interruptions directly correlated with parameters of ovarian reserve. Patients with longer uninterrupted CGG repeats post-AGG interruptions had the lowest ovarian reserve. CONCLUSION: This study connects AGG interruptions and certain CGG repeat length to reduced ovarian reserve in carriers with a PM. A possible explanation for our findings is the proposed gonadotoxicity of the FMR1 transcripts. Reduction of AGG interruptions could increase the likelihood that secondary RNA structures in the FMR1 messenger RNA are formed, which could cause cell dysfunction within the ovaries. These findings may provide women with guidance regarding their fertility potential and accordingly assist with their family planning.

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.

Fragile X-Associated Diminished Ovarian Reserve and Primary Ovarian Insufficiency from Molecular Mechanisms to Clinical Manifestations.

Fragile X syndrome (FXS), is caused by a loss-of-function mutation in the FMR1 gene located on the X-chromosome, which leads to the most common cause of inherited intellectual disability in males and the leading single-gene defect associated with autism. A full mutation (FM) is represented by more than 200 CGG repeats within the FMR1 gene, resulting in FXS. A FM is inherited from women carrying a FM or a premutation (PM; 55-200 CGG repeats) allele. PM is associated with phenotypes distinct from those associated with FM. Some manifestations of the PM are unique; fragile-X-associated tremor/ataxia syndrome (FXTAS), and fragile-X-associated primary ovarian insufficiency (FXPOI), while others tend to be non-specific such as intellectual disability. In addition, women carrying a PM may suffer from subfertility or infertility. There is a need to elucidate whether the impairment of ovarian function found in PM carriers arises during the primordial germ cell (PGC) development stage, or due to a rapidly diminishing oocyte pool throughout life or even both. Due to the possibility of expansion into a FM in the next generation, and other ramifications, carrying a PM can have an enormous impact on one's life; therefore, preconception counseling for couples carrying the PM is of paramount importance. In this review, we will elaborate on the clinical manifestations in female PM carriers and propose the definition of fragile-X-associated diminished ovarian reserve (FXDOR), then we will review recent scientific findings regarding possible mechanisms leading to FXDOR and FXPOI. Lastly, we will discuss counseling, preventative measures and interventions available for women carrying a PM regarding different aspects of their reproductive life, fertility treatment, pregnancy, prenatal testing, contraception and fertility preservation options.

Engineered endothelium provides angiogenic and paracrine stimulus to grafted human ovarian tissue.

Despite major advances in tissue cryopreservation and auto-transplantation, reperfusion ischemia and hypoxia have been reported as major obstacles to successful recovery of the follicular pool within grafted ovarian tissue. We demonstrate a benefit to follicular survival and function in human ovarian tissue that is co-transplanted with exogenous endothelial cells (ExEC). ExECs were capable of forming functionally perfused vessels at the host/graft interface and increased both viability and follicular volume in ExEC-assisted grafts with resumption of antral follicle development in long-term grafts. ExECs that were engineered to constitutively express anti-mullerian hormone (AMH) induced a greater proportion of quiescent primordial follicles than control ExECs, indicating suppression of premature mobilization that has been noted in the context of ovarian tissue transplantation. These findings present a cell-based strategy that combines accelerated perfusion with direct paracrine delivery of a bioactive payload to transplanted ovarian tissue.

Notch hyper-activation drives trans-differentiation of hESC-derived endothelium.

During development, endothelial cells (EC) display tissue-specific attributes that are unique to each vascular bed, as well as generic signaling mechanisms that are broadly applied to create a patent circulatory system. We have previously utilized human embryonic stem cells (hESC) to generate tissue-specific EC sub-types (Rafii et al., 2013) and identify pathways that govern growth and trans-differentiation potential of hESC-derived ECs (James et al., 2010). Here, we elucidate a novel Notch-dependent mechanism that induces endothelial to mesenchymal transition (EndMT) in confluent monolayer cultures of hESC-derived ECs. We demonstrate density-dependent induction of EndMT that can be rescued by the Notch signaling inhibitor DAPT and identify a positive feedback signaling mechanism in hESC-ECs whereby trans-activation of Notch by DLL4 ligand induces elevated expression and surface presentation of DLL4. Increased Notch activation in confluent hESC-EC monolayer cultures induces areas of EndMT containing transitional cells that are marked by increased Jagged1 expression and reduced Notch signal integration. Jagged1 loss of function in monolayer hESC-ECs induces accelerated feedback stimulation of Notch signaling, increased expression of cell-autonomous, cis-inhibitory DLL4, and EndMT. These data elucidate a novel interplay of Notch ligands in modulating pathway activation during both expansion and EndMT of hESC-derived ECs.

A Live Birth Subsequent to IVF following Egg Retrieval Only 12 Hours after hCG Priming.

Introduction. To report a live birth following egg retrieval after only 12 hours from hCG priming. Patients. A childless couple with five-years-lasting secondary infertility. Methods. IVF was performed according to the long protocol. Two immature oocytes were retrieved following only 12 hours after hCG priming due to the patient misunderstanding. The eggs were cultured in vitro and ICSI was performed following polar body extruded after 24 hours in culture. After additional 24 hours a 4-cell embryo was developed and ET was performed. Results. A viable pregnancy was achieved and a healthy baby girl was delivered at 38 weeks of gestation. Conclusion. In a rare and unexpected situation when immature oocytes are retrieved following a short hCG priming, the eggs should be cultured in vitro, late ICSI should be performed, and a pregnancy may be expected.

Major involvement of CD40 in the regulation of chemokine secretion from human peritoneal mesothelial cells.

BACKGROUND: CD40 is a member of the tumor necrosis factor (TNF) family of receptors, whose ligand, CD154, is expressed by activated mononuclear cells. CD40 activation is a major immune regulatory pathway and is important for the regulation of chemokine and cytokine secretion. This study investigates the effect of CD40 ligation on the secretion of chemokines from human peritoneal mesothelial cells (HPMC). METHODS: We activated CD40 in HPMC along with combinations of TNF-alpha, interleukin-1 (IL-1), and interferon gamma (IFN-gamma), and evaluated the mRNA levels and protein secretion of regulated upon activation, normal T-cell expressed and secreted (RANTES), monocyte chemoattractant protein-1 (MCP-1), and IL-8. RESULTS: CD40 ligation had a small stimulatory effect on the secretion of all three chemokines, while TNF-alpha, IL-1 and IFN-gamma induced their secretion in a dose-dependent manner. The combination of CD40 ligation with either IL-1 or TNF-alpha increased chemokine secretion additively. IFN-gamma and CD40 ligation acted in synergy to induce the secretion of the mononuclear recruiting chemokines RANTES and MCP-1 (up to approximately 36-fold and approximately threefold, respectively), for which the combination of all three cytokines with CD40 ligation was extremely potent. In contrast, the secretion of the neutrophil chemoattractant IL-8, induced by CD40 ligation or by the combination of IL-1 and TNF-alpha, was reduced in the presence of IFN-gamma. CONCLUSION: In light of our data, it is reasonable to suggest that in the mononuclear phase of peritonitis, IFN-gamma and CD154, expressed by activated mononuclear cells, diminish IL-8 secretion from HPMC and thus inhibit neutrophil recruitment. At the same time, the two act in synergy to induce the secretion of RANTES and MCP-1 from HPMC. Hence, by regulating chemokine secretion, CD40 may be involved in peritonitis and in the development of late phase mononuclear predominance.

CD40 is expressed on human peritoneal mesothelial cells and upregulates the production of interleukin-15 and RANTES.

Limited data are available concerning the interaction between lymphocytes and human peritoneal mesothelial cells (HPMC) during peritonitis. CD40 is a member of the tumor necrosis factor (TNF) family of receptors whose ligand (CD154) is mainly expressed on the membrane of activated CD4-positive lymphocytes. CD154-CD40 cross-linking is a central event in antigen presentation, B-cell activation by T cells, and regulation of cytokine secretion from various types of cells. The goal of this study was to demonstrate in vitro the presence of CD40 on HPMC and to test its functionality in inducing interleukin-15 (IL-15) and RANTES. We assayed the levels of CD40 by reverse transcription-PCR and flow cytometry and IL-15 and RANTES by enzyme-linked immunosorbent assay. Genetically modified L cells that express elevated levels of CD154 (CD40L cells) were used to stimulate CD40. HPMC express CD40 mRNA and protein. After stimulation with interferon-gamma (IFNgamma, 5U/ml) or TNFalpha (1 ng/ml), there was a small increase in CD40 mRNA and protein levels; when both cytokines were applied, the increase in CD40 levels was more than threefold. CD40 ligation induced IL-15 production by HPMC and was additive to IFNgamma stimulation. CD40 ligation was strongly synergistic with IFNgamma in induction of RANTES (20-fold as compared with unstimulated HPMC), whereas neither ligation nor IFNgamma alone could induce RANTES. Pretreatment of HPMC with TNFalpha and IFNgamma increased the response to CD40 ligation in magnitudes that correlated with the elevation of CD40 levels induced by the pretreatment. To conclude, the presence of a functional CD40 on HPMC whose ligation induced IL-15 and RANTES production was detected. It is possible that this receptor acts as a major mediator of T-cell-regulated immune and inflammatory response during peritonitis.