Limor Man | Weill Cornell Medicine (original) (raw)

Papers by Limor Man

Communications Biology

Theca cells serve multiple essential functions during the growth and maturation of ovarian follic... more 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 proce...

Fertility and Sterility, Oct 1, 2022

Science Advances, 2022

Anti-Müllerian hormone (AMH) is produced by growing ovarian follicles and provides a diagnostic m... more 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 A...

Reproduction

More than 200 live births have been achieved using auto-transplantation of cryopreserved ovarian ... more More than 200 live births have been achieved using auto-transplantation 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 to 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 total follicle number and was significantly reduced with increased patient ...

F&S Science, 2021

Objective To measure the influence of exogenous insulin-like growth factor 1 (IGF1) on follicle g... more 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.

Cell Reports, 2020

Highlights d Labeling and sorting of granulosa and theca/stroma subsets in human ovarian follicle... more Highlights d Labeling and sorting of granulosa and theca/stroma subsets in human ovarian follicles d PVRL1 distinguishes the oophorus compartment of antralstage follicles d scRNA-seq indicates atypical molecular signature of granulosa cells within xenografts d Analysis of theca/stroma subsets reveals heterogeneity and differentiation hierarchy

Fertility and Sterility, 2021

Journal of the American Society of Nephrology, 2001

Limited data are available concerning the interaction between lymphocytes and human peritoneal me... more 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

Fertility and Sterility, 2020

To study whether patients exhibiting poor ovarian response have abnormal levels of serum insulin-... more 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 (R18 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. (Fertil Steril Ò 2020;113:1205-14. Ó2020 by American Society for Reproductive Medicine.) El resumen está disponible en Español al final del artículo.

Fertility and Sterility, 2018

in the human ovary remains unknown. The purpose of our study was to determine the role of hMSC se... more in the human ovary remains unknown. The purpose of our study was to determine the role of hMSC secretome on immortalized unluteinized gonadotropin dependent human granulosa cell line (GC). DESIGN: Prospective in vitro studies using immortalized unluteinized gonadotropin dependent human granulosa cell line. MATERIALS AND METHODS: GCs were seeded on culture dishes precoated with extracellular matrix at a density of 6x104 cells/ml and cultured for 24 hours before treatment. Media was then collected, and either replaced by control media (C) or conditioned media (Cm). Cm was previously prepared by collecting the media of hMSC when those reached 90% confluence in their culture plate. A subset of those two groups was treated with recombinant follicle stimulating hormone (FSH) at a concentration of 50 ng/ml. Forty eight hours after media change and FSH treatment, cells and supernatant were collected for analysis. Proliferation assay based on Ki67 analysis with flow cytometry was performed. Genes mRNA and protein expression levels were quantified by real-time PCR and Western blot, respectively. Estrogen levels were measured by ELISA. RESULTS: Human GCs cultured in hMSC Cm showed significantly higher proliferation rates when treated with Cm compared to the control group based on the percentage of gated Ki67 + cells (4.02 % AE0.78 vs 30.9 AE 4.62, P<0.001). The addition of FSH to both groups showed a trend of increased proliferation in the Cm group, however this did not reach statistical significance (52.7 AE 2.45 vs 53.1 AE 2.6, P>0.05). Human aromatase mRNA expression was 20 fold increased with Cm compared to the C group (P < 0.05), and 30 fold increase with CmF compared to the CF group (P < 0.05). This was also shown at the level of human StAR mRNA without FSH (14 fold increase, P<0.05), and with FSH (30 fold increase, P < 0.05). Protein studies and other gene expression profiles, in addition to results with molecular pathway inhibitors are being analyzed and will be presented during the meeting in October 2018. CONCLUSIONS: These findings show, for the first time, that hMSC secretome promotes human granulosa cell proliferation and regulates gene expression involved in folliculogenesis, such as aromatase. Further investigation, including co-culture and analysis of Cm is warranted to fully understand the effect of hMSC on human granulosa cells.

Fertility and Sterility, 2019

OBJECTIVE: Obesity has a profound impact on reproductive function, reducing fertility and increas... more OBJECTIVE: Obesity has a profound impact on reproductive function, reducing fertility and increasing the risk of pregnancy complications and birth defects. Obesity in women is associated with hyperlipidemia, hyperinsulinemia, and decreased basal and GnRH-stimulated FSH and LH secretion from gonadotrope cells in the pituitary.We have termed this phenotype 'Reprometabolic Syndrome'. We have previously shown that acute infusions of lipid/insulin into healthy, normal weight, cycling women recapitulates this reprometabolic phenotype of obesity. However, the underlying mechanisms are not understood. We sought to confirm that the decreased FSH and LH were not attributable to differential hemodilution, and investigated if the effects of lipid/insulin infusions were confined to gonadotropes or impacted other anterior pituitary cell types. DESIGN: 8 normal weight, regularly cycling women underwent 6-hour visits with either a saline and heparin (control) infusion, or a hyperinsulinemic-euglycemic clamp with heparin and Liposyn (Abbott laboratories). GnRH stimulation was applied at 240 minutes. MATERIALS AND METHODS: Frequent blood sampling (q10 min) was conducted at each visit, which occurred in random order, between days 2-5 in sequential menstrual cycles. Anterior pituitary hormones TSH and prolactin (PRL), thyroid hormones (free T4, total T3) and cortisol were measured in serum samples. TSH was measured in pooled samples q30 min. PRL, fT4, total T3, and cortisol samples were pooled to measure approximately 0, 30, 160, and 360 min time points. Lastly, creatinine was measured hourly in pooled samples. RESULTS: In contrast to the decrease in gonadotropins, TSH increased in the lipid/insulin-treated samples, with the most dramatic percent change after 160 minutes (28.2% increase), significantly different from TSH levels in the saline infusions (p<0.0005), which slightly decreased (-11.4%). Thyroid hormones (fT4 and total T3), PRL, cortisol, and serum creatinine did not differ between saline or lipid/insulin infusion conditions. CONCLUSIONS: Lack of change in serum creatinine showed there was no hemodilution due to variable infusion volumes. FT4 and total T3 were unchanged, suggesting that the increase in TSH was a thyrotrope cell response to lipid/insulin and not a result of altered thyroid function. Cortisol, an inhibitor of TSH production, was unaffected by infusion condition. Levels of the lactotroph hormone PRL were not impacted by lipid/insulin, confirming that effects on the pituitary are both complex and cell type specific. Our results imply that the impact of obesity on the hypothalamic-pituitary-gonadal axis is not simply suppression, and extends beyond reproductive functions. Further research is needed to elucidate mechanisms underlying the selective modulation of pituitary trophic hormones in response to changes in diet and metabolism.

Genetics in Medicine, 2017

Purpose: Fragile X premutation (PM) carriers may experience difficulties conceiving a child proba... more 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.

Current Opinion in Gynecology and Obstetrics, 2018

Over the last three decades, survival rate from malignancies has steadily increased, turning majo... more Over the last three decades, survival rate from malignancies has steadily increased, turning major focus on quality of life in remission. Unfortunately, a major side effect of medical interventions including chemotherapy, radiotherapy, and/or surgery is premature ovarian failure and infertility for many patients. Ovarian tissue cryopreservation is an experimental technique used to preserve reproductive potential in patients who are pre-pubertal, must urgently undergo gonadotoxic treatment or who have other medical conditions requiring treatment that may undermine ovarian reserve and will lead to sub or infertility. We will review the indications, available techniques of tissue harvesting, and methods for freezing and autotransplanting cortical ovarian tissue. We will discuss outcomes, the potential risk of reintroducing malignancy and lastly, cutting-edge research models being developed in the field.

Development (Cambridge, England), Jan 7, 2017

Several studies have demonstrated a multiphasic role for Wnt signaling during embryonic cardiogen... more Several studies have demonstrated a multiphasic role for Wnt signaling during embryonic cardiogenesis (Naito et al., 2006; Qyang et al., 2007) and developed protocols that enrich for cardiac derivatives during in vitro differentiation of human pluripotent stem cells (hPSC) (Elliott et al., 2011; Iyer et al., 2016; Lian et al., 2012; Paige et al., 2010; Willems et al., 2011; Witty et al., 2014), however, few studies have investigated the role of Wnt signaling in specification of cardiac progenitor cells (CPC) toward downstream fates. Using transgenic mice and hPSCs, we tracked endothelial cells (EC) 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 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 embryog...

Scientific Reports, 2017

Despite major advances in tissue cryopreservation and auto-transplantation, reperfusion ischemia ... more 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.

Fertility and Sterility, 2014

identify failed probes, and the methylation level of each CpG locus (beta value) was calculated i... more identify failed probes, and the methylation level of each CpG locus (beta value) was calculated in GenomeStudio Methylation module. Possible plate/batch effect was assessed visually by Principal Component Analysis, and data was normalized using the Chip Analysis Methylation Pipeline (ChAMP) package (implemented in R). RESULTS: Genomic DNAs isolated from these CVS samples successfully underwent bisulfite conversion. All samples had >99.9% call rate, and two independent technical replicates were clustered tightly. Differentially methylated loci are currently being analyzed in Ingenuity Pathway Analysis (IPA) to identify pathways that may be modified by IVF or non-IVF fertility treatments. CONCLUSION: These results indicate that CVS samples from our Prenatal Biorepository that were preserved in RNAlater can be used for successful isolation of high quality genomic DNA, and that this DNA is suitable for use in epigenetic arrays. Further functional analysis is undergoing, and our findings will determine whether there are epigenetic differences between the groups that may affect placentation and fetal development.

Fertility and Sterility, 2017

Elevated early follicular gonadotropin levels in women with unexplained infertility do not provid... more Elevated early follicular gonadotropin levels in women with unexplained infertility do not provide evidence for disordered gonadotropin-releasing hormone secretion as assessed by luteinizing hormone pulse characteristics. Fertil Steril 2003;80(2):320-7.