Nataki C. Douglas

Changes in markers of ovarian reserve and endocrine function in young women with breast cancer undergoing adjuvant chemotherapy

Premenopausal women undergoing chemotherapy are at risk for amenorrhea and impaired fertility. The objective of the current study was to assess levels of mullerian inhibitory substance (MIS), estradiol (E2), follicle‐stimulating hormone (FSH), and menstrual status, in women undergoing chemotherapy.

Derivation of Two New Human Embryonic Stem Cell Lines from Nonviable Human Embryos

We report the derivation and characterization of two new human embryonic stem cells (hESC) lines (CU1 and CU2) from embryos with an irreversible loss of integrated organismic function. In addition, we analyzed retrospective data of morphological progression from embryonic day (ED) 5 to ED6 for 2480 embryos not suitable for clinical use to assess grading criteria indicative of loss of viability on ED5. Our analysis indicated that a large proportion of in vitro fertilization (IVF) embryos not suitable for clinical use could be used for hESC derivation. Based on these combined findings, we propose that criteria commonly used in IVF clinics to determine optimal embryos for uterine transfer can be employed to predict the potential for hESC derivation from poor quality embryos without the destruction of vital human embryos.

The T-box Transcription Factors TBX2 and TBX3 in Mammary Gland Development and Breast Cancer

TBX2 and TBX3, closely related members of the T-box family of transcription factor genes, are expressed in mammary tissue in both humans and mice. Ulnar mammary syndrome (UMS), an autosomal dominant disorder caused by mutations in TBX3, underscores the importance of TBX3 in human breast development, while abnormal mammary gland development in Tbx2 or Tbx3 mutant mice provides models for experimental investigation. In addition to their roles in mammary development, aberrant expression of TBX2 and TBX3 is associated with breast cancer. TBX2 is preferentially amplified in BRCA1/2-associated breast cancers and TBX3 overexpression has been associated with advanced stage disease and estrogen-receptor-positive breast tumors. The regulation of Tbx2 and Tbx3 and the downstream targets of these genes in development and disease are not as yet fully elucidated. However, it is clear that the two genes play unique, context-dependent roles both in mammary gland development and in mammary tumorigenesis.

Dynamic expression of Tbx2 subfamily genes in development of the mouse reproductive system.

Background: Tbx2, Tbx3, Tbx4, and Tbx5, members of the Tbx2 subfamily of T‐box transcription factor genes, are important for many aspects of embryonic development and mutations in some human TBX2 subfamily genes cause developmental syndromes. In addition, TBX2 and TBX3 are overexpressed in a variety of cancers, including reproductive system cancers. This study characterizes the expression of Tbx2 subfamily genes during development of the reproductive system. Results: We show that these genes are expressed in both the internal and external reproductive systems. Tbx2 is expressed in gonads and genital ducts, the Wolffian and Müllerian ducts, while Tbx3 is only expressed in genital ducts. Tbx4 is expressed in embryonic and postnatal germ cells. All four genes are expressed in mesenchyme in external genitalia, with Tbx3 and Tbx5 expression in the epithelium as well. Conclusion: This study lays the foundation for investigation of functional requirements for Tbx2 subfamily genes in development of the mammalian reproductive system. Developmental Dynamics 241:365–375, 2012.

VEGFR-1 blockade disrupts peri-implantation decidual angiogenesis and macrophage recruitment

BackgroundAngiogenesis and macrophage recruitment to the uterus are key features of uterine decidualization; the progesterone-mediated uterine changes that allow for embryo implantation and initiation of pregnancy. In the current study, we characterized the expression of vascular endothelial growth factor receptor-1 (VEGFR-1) in macrophages and endothelial cells of the peri-implantation uterus and determined if VEGFR-1 function is required for decidual angiogenesis, macrophage recruitment, and/or the establishment of pregnancy.MethodsExpression of VEGFR-1 in uterine endothelial cells and macrophages was determined with immunohistochemistry. To assess the effect of continuous VEGFR-1 blockade, adult female mice were given VEGFR-1 blocking antibody, MF-1, every 3xa0days for 18xa0days. After 6 doses, females were mated and a final dose of MF-1 was given on embryonic day 3.5. Endothelial cells and macrophages were quantified on embryonic day 7.5. Pregnancy was analyzed on embryonic days 7.5 and 10.5.ResultsF4/80+ macrophages are observed throughout the stroma and are abundant adjacent to the endometrial lumen and glands prior to embryo implantation and scatter throughout the decidua post implantation. VEGFR-1 expression is restricted to the uterine endothelial cells. F4/80+ macrophages were often found adjacent to VEGFR-1+ endothelial cells in the primary decidual zone. Continuous VEGFR-1 blockade correlates with a significant reduction in decidual vascular and macrophage density, but does not affect embryo implantation or maintenance of pregnancy up to embryonic day 10.5.ConclusionsWe found that VEGFR-1 functions in both decidual angiogenesis and macrophage recruitment to the implantation site during pregnancy. VEGFR-1 is expressed by endothelial cells, however blocking VEGFR-1 function in endothelial cells results in reduced macrophage recruitment to the uterus. VEGFR-1 blockade did not compromise the establishment and/or maintenance of pregnancy.

Reproductive Outcomes of HIV Seropositive Women Treated by Assisted Reproduction

BACKGROUNDnOver one million Americans are infected with HIV, and approximately 300,000 are women. Overall health in HIV infected persons has improved, and many seropositive women desire children. This study describes the reproductive outcomes of HIV seropositive women treated by assisted reproduction at our center and compares their clinical results with age-matched HIV seronegative controls.nnnMETHODSnFrom January 1, 1998 to December 31, 2011, 36 HIV seropositive women received treatment with in vitro fertilization (IVF) at a single center. The mean age at start of fertility treatment was 37.7±4.8 years. At presentation, 92% of seropositive women were using antiretrovirals and all had undetectable viral loads at time of cycle initiation. Clinical outcomes of seropositive women were compared in a one-to-one ratio to those of randomly selected age-matched seronegative controls undergoing treatment for male factor infertility during the same time period. Comparisons were stratified by age--women less than 35 and greater than 35 years of age.nnnRESULTSnFifteen treatment cycles resulted in live births (21 infants born without HIV infection). HIV seropositive and seronegative women < 35 years of age had nearly identical IVF clinical outcome parameters, including clinical pregnancy rates and live birth rates. For women 35 years of age or older, baseline serum estradiol levels and live birth rates were significantly lower in HIV seropositive women.nnnCONCLUSIONSnThis study demonstrates that the presence of well-controlled HIV infection does not impair fertility treatment in women undergoing IVF. Virally infected women should be encouraged to seek treatment in appropriate cases.

Vascular Notch proteins and Notch signaling in the peri-implantation mouse uterus

BackgroundAngiogenesis is essential for uterine decidualization, the progesterone-mediated transformation of the uterus allowing embryo implantation and initiation of pregnancy. In the current study, we define the vasculature, expression of Notch proteins and Notch ligands, and Notch activity in both endothelial cells and vascular-associated mural cells of blood vessels in the pre-implantation endometrium and post-implantation decidua of the mouse uterus.MethodsWe used immunofluorescence to determine the expression of Notch in endothelial cells and mural cells by co-staining for the endothelial cell marker, CD31, the pan-mural cell marker, platelet-derived growth factor receptor beta (PDGFR-β), the pericyte markers, neural/glial antigen 2 (NG2) and desmin, or the smooth muscle cell marker, alpha smooth muscle actin (SMA). A fluorescein isothiocyanate-labeled dextran tracer, was used to identify functional peri-implantation vasculature. CBF:H2B-Venus Notch reporter transgenic mice were used to determine Notch activity.ResultsNotch signaling is observed in endothelial cells and pericytes in the peri-implantation uterus. Prior to implantation, Notch1, Notch2 and Notch4 and Notch ligand, Delta-like 4 (Dll4) are expressed in capillary endothelial cells, while Notch3 is expressed in the pericytes. Jagged1 is expressed in both capillary endothelial cells and pericytes. After implantation, Notch1, Notch4 and Dll4 are expressed in endothelial cells of newly formed decidual capillaries. Jagged1 is expressed in endothelial cells of spiral arteries and a subset of decidual pericytes. Notch proteins are not expressed in lymphatic vessels or macrophages in the peri-implantation uterus.ConclusionsWe show Notch activity and distinct expression patterns for Notch proteins and ligands, suggesting unique roles for Notch1, Notch4, Dll4, and Jag1 during decidual angiogenesis and early placentation. These data set the stage for loss-of-function and gain-of-function studies that will determine the cell-type specific requirements for Notch proteins in decidual angiogenesis and placentation.

Cell lineage of timed cohorts of Tbx6-expressing cells in wild-type and Tbx6 mutant embryos

ABSTRACT Tbx6 is a T-box transcription factor with multiple roles in embryonic development as evidenced by dramatic effects on mesoderm cell fate determination, left/right axis determination, and somite segmentation in mutant mice. The expression of Tbx6 is restricted to the primitive streak and presomitic mesoderm, but some of the phenotypic features of mutants are not easily explained by this expression pattern. We have used genetically-inducible fate mapping to trace the fate of Tbx6-expressing cells in wild-type and mutant embryos to explain some of the puzzling features of the mutant phenotype. We created an inducible Tbx6-creERT2 transgenic mouse in which cre expression closely recapitulates endogenous Tbx6 expression both temporally and spatially. Using a lacZ-based Cre reporter and timed tamoxifen injections, we followed temporally overlapping cohorts of cells that had expressed Tbx6 and found contributions to virtually all mesodermally-derived embryonic structures as well as the extraembryonic allantois. Contribution to the endothelium of major blood vessels may account for the embryonic death of homozygous mutant embryos. In mutant embryos, Tbx6-creERT2-traced cells contributed to the abnormally segmented anterior somites and formed the characteristic ectopic neural tubes. Retention of cells in the mutant tail bud indicates a deficiency in migratory behavior of the mutant cells and the presence of Tbx6-creERT2-traced cells in the notochord, a node derivative provides a possible explanation for the heterotaxia seen in mutant embryos. Summary: Embryonic cells that transiently express the transcription factor, Tbx6, during the process of gastrulation have been tracked in later development in wild-type and Tbx6 homozygous mutant embryos, where they give rise to the ectopic neural tubes characteristic of the mutant phenotype.

Cyclophosphamide Exposure in Pediatric Systemic Lupus Erythematosus Is Associated with Reduced Serum Anti-Müllerian Hormone Levels

To the Editor: nnThe reproductive risk to girls with pediatric systemic lupus erythematosus (pSLE) of varying disease severity and medication exposure is not well established. Because 15%–20% of patients with SLE are diagnosed before age 19 years1, many young women with pSLE will be affected by disease complications throughout their reproductive years. Although infertility in SLE has been attributed mainly to use of cyclophosphamide (CYC)2,3, further studies are needed to clarify the relationship between disease severity, medication use, and ovarian dysfunction. Because the incidence of premature primary ovarian insufficiency (POI) after CYC exposure in patients with pSLE < 21 years of age is estimated at 0–11%3, biomarkers for morbidities, such as infertility, are important because they may change current treatment strategies.nnAnti-Mullerian hormone (AMH) is produced by granulosa cells of preantral and small antral follicles and has been identified as a sensitive biomarker of ovarian reserve. AMH concentrations are relatively stable throughout the menstrual cycle, are unaffected by hormonal contraceptives, and decline with advancing age, as does ovarian reserve/function4,5. Low to undetectable levels of AMH are found in women ages 25–46 years within 5 years of their final menstrual period, in cancer survivors exposed to chemotherapy and/or radiation therapy-induced follicle depletion, and in women with POI … nnAddress correspondence to Dr. N.C. Douglas, Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology and Infertility, New York Presbyterian Hospital–Columbia University Medical Center, 622 West 168th Street, PH 16-64, New York, NY 10032, USA. E-mail: nd2058{at}columbia.edu

Investigating the Role of Tbx4 in the Female Germline in Mice

ABSTRACT Normal development of germ cells is essential for fertility and mammalian reproduction. Although abnormal development of oocytes or follicles may lead to primary ovarian insufficiency (POI), a disorder that causes infertility in 1% of women less than 40 yr of age, the genes and signaling pathways activated in POI are not as yet fully elucidated. Tbx4, a member of the T-box family of transcription factors, is expressed in embryonic germ cells and postnatal oocytes at all stages of folliculogenesis. To investigate the requirement for Tbx4 in the germline, we analyzed germ cell development in the absence of Tbx4. We show that primordial germ cells (PGCs) are reduced in Tbx4 homozygous null (Tbx4−/−) embryos at Embryonic Day (E) 10.0. Tbx4−/− embryos die by E10.5; to study later time points in vitro, a tamoxifen-inducible estrogen receptor Cre recombinase was used to delete Tbx4 conditional mutant alleles. In addition, Gdf9cre and Zp3cre, two oocyte-specific Cre recombinases, were used to delete Tbx4 from postnatal primordial and primary follicles, respectively. We show that in vitro differentiation of the gonad into morphologically distinct testes and ovaries occurs normally starting at E11.5 when Tbx4 is deleted. In Gdf9cre; Tbx4fl/− and Zp3cre; Tbx4fl/− adult females, primordial, primary, secondary, and antral follicles form, ovulation occurs, corpus luteum formation is normal, and the mice are fertile without any evidence of diminished ovarian reserve. Although postnatal deletion of Tbx4 in oocytes does not obviously impair fertility, it is possible that the reduction in PGCs observed in Tbx4 homozygous null mutant embryos could affect long-term fertility in adults.