Daniel H. Kort

Human oocytes reprogram adult somatic nuclei of a type 1 diabetic to diploid pluripotent stem cells

The transfer of somatic cell nuclei into oocytes can give rise to pluripotent stem cells that are consistently equivalent to embryonic stem cells, holding promise for autologous cell replacement therapy. Although methods to induce pluripotent stem cells from somatic cells by transcription factors are widely used in basic research, numerous differences between induced pluripotent stem cells and embryonic stem cells have been reported, potentially affecting their clinical use. Because of the therapeutic potential of diploid embryonic stem-cell lines derived from adult cells of diseased human subjects, we have systematically investigated the parameters affecting efficiency of blastocyst development and stem-cell derivation. Here we show that improvements to the oocyte activation protocol, including the use of both kinase and translation inhibitors, and cell culture in the presence of histone deacetylase inhibitors, promote development to the blastocyst stage. Developmental efficiency varied between oocyte donors, and was inversely related to the number of days of hormonal stimulation required for oocyte maturation, whereas the daily dose of gonadotropin or the total number of metaphase II oocytes retrieved did not affect developmental outcome. Because the use of concentrated Sendai virus for cell fusion induced an increase in intracellular calcium concentration, causing premature oocyte activation, we used diluted Sendai virus in calcium-free medium. Using this modified nuclear transfer protocol, we derived diploid pluripotent stem-cell lines from somatic cells of a newborn and, for the first time, an adult, a female with type 1 diabetes.

Preliminary evidence that cinnamon improves menstrual cyclicity in women with polycystic ovary syndrome: a randomized controlled trial

OBJECTIVE To determine the effect of cinnamon on menstrual cyclicity and metabolic dysfunction in women with polycystic ovary syndrome (PCOS). STUDY DESIGN In a prospective, placebo controlled, double-blinded randomized trial, 45 women with PCOS were randomized (1:1) to receive cinnamon supplements (1.5 g/d) or placebo for 6 months. Menstrual cyclicity (average cycles/month) during the 6 months study period was compared between the 2 groups using the Mann-Whitney U test. Changes in menstrual cyclicity and insulin resistance between baseline and the 6 month study period were compared between the 2 groups using Wilcoxon signed rank tests. RESULTS The 45 women were randomized, 26 women completed 3 months of the study, and 17 women completed the entire 6 months of the study. During the 6 month intervention, menstrual cycles were more frequent in patients taking cinnamon compared with patients taking placebo (median, 0.75; interquartile range, 0.5-0.83 vs median, 0.25; interquartile range, 0-0.54; P = .0085; Mann Whitney U). In patients taking cinnamon, menstrual cyclicity improved from baseline (+ 0.23 cycles/month 95% confidence interval, 0.099-0.36), yet did not improve for women taking placebo. (P = .0076, Wilcoxon signed rank). Samples (n = 5) of serum from the luteal phase in different patients within the cinnamon group were thawed and ovulatory progesterone levels (>3 ng/mL) confirmed. Luteal phase progesterone levels (>3 ng/mL, n = 5) confirmed ovulatory menses. Measures of insulin resistance or serum androgen levels did not change for either group. CONCLUSION These preliminary data suggest that cinnamon supplementation improves menstrual cyclicity and may be an effective treatment option for some women with PCOS.

Human embryos commonly form abnormal nuclei during development: a mechanism of DNA damage, embryonic aneuploidy, and developmental arrest.

STUDY QUESTION What is the prevalence and developmental significance of morphologic nuclear abnormalities in human preimplantation embryos? SUMMARY ANSWER Nuclear abnormalities are commonly found in human IVF embryos and are associated with DNA damage, aneuploidy, and decreased developmental potential. WHAT IS KNOWN ALREADY Early human embryonic development is complicated by genomic errors that occur after fertilization. The appearance of extra-nuclear DNA, which has been observed in IVF, may be a result of such errors. However, the mechanism by which abnormal nuclei form and the impact on DNA integrity and embryonic development is not understood. STUDY DESIGN, SIZE, DURATION Cryopreserved human cleavage-stage embryos (n = 150) and cryopreserved blastocysts (n = 105) from clinical IVF cycles performed between 1997 and 2008 were donated for research. Fresh embryos (n = 60) of poor quality that were slated for discard were also used. Immunohistochemical, microscopic and cytogenetic analyses at different developmental stages and morphologic grades were performed. PARTICIPANTS/MATERIALS, SETTING, METHODS Embryos were fixed and stained for DNA, centromeres, mitotic activity and DNA damage and imaged using confocal microscopy. Rates of abnormal nuclear formation were compared between morphologically normal cleavage-stage embryos, morphologically normal blastocysts, and poor quality embryos. To control for clinical and IVF history of oocytes donors, and quality of frozen embryos within our sample, cleavage-stage embryos (n = 52) were thawed and fixed at different stages of development and then analyzed microscopically. Cleavage-stage embryos (n = 9) were thawed and all blastomeres (n = 62) were disaggregated, imaged and analyzed for karyotype. Correlations were made between microscopic and cytogenetic findings of individual blastomeres and whole embryos. MAIN RESULTS AND THE ROLE OF CHANCE The frequency of microscopic nuclear abnormalities was lower in blastocysts (5%; 177/3737 cells) than in cleavage-stage embryos (16%, 103/640 blastomeres, P < 0.05) and highest in arrested embryos (65%; 44/68 blastomeres, P < 0.05). DNA damage was significantly higher in cells with microscopic nuclear abnormalities (γH2AX (phosphorylated (Ser139) histone H2A.X): 87.1%, 74/85; replication protein A: 72.9%, 62/85) relative to cells with normal nuclear morphology (γH2AX: 9.3%, 60/642; RPA: 5.6%, 36/642) (P < 0.05). Blastomeres containing nuclear abnormalities were strongly associated with aneuploidy (Fisher exact test, two-tailed, P < 0.01). LIMITATIONS, REASONS FOR CAUTION The embryos used were de-identified, and the clinical and IVF history was unknown. WIDER IMPLICATIONS OF THE FINDINGS This study explores a mechanism of abnormal embryonic development post-fertilization. While most of the current data have explored abnormal meiotic chromosome segregation in oocytes as a primary mechanism of reproductive failure, abnormal nuclear formation during early mitotic cell division in IVF embryos also plays a significant role. The detection of abnormal nuclear formation may have clinical application in noninvasive embryo selection during IVF. STUDY FUNDING/COMPETING INTERESTS The study was supported by Columbia University and the New York Stem Cell Foundation. Authors declare no competing interest.

Chemerin as a marker of body fat and insulin resistance in women with polycystic ovary syndrome.

Abstract Adipocytokines may alter normal metabolic function and play an important role in the pathophysiology of polycystic ovary syndrome (PCOS). We prospectively evaluated a cohort of obese and non-obese women with PCOS and non-PCOS controls for both novel (chemerin and omentin-1) and established (leptin and adiponectin) adipokines. Compared with age-matched controls, non-obese women with PCOS had decreased serum omentin-1 (191.1 ng/ml versus 269.7 ng/ml, p = 0.0001), while serum chemerin was not significantly altered in women with PCOS (53.95 ng/ml versus 48.61 ng/ml, p = 0.11). The findings were similar in the entire group of women with PCOS. However, in women with PCOS, chemerin correlated with leptin (r = 0.508, p = 0.004), adiponectin (r = −0.36, p = 0.014), and the leptin/adiponectin (L/A) ratio (r = 0.605, p < 0.0001), while there were no such correlations with omentin-1. In women with PCOS, chemerin correlated with BMI (r = 0.317, p = 0.034), abdominal subcutaneous fat (r = 0.451, p = 0.0019), and insulin resistance (HOMA-IR, r = 0.428, p = 0.0034), while omentin-1 did not correlate with any parameter. These data suggest that chemerin although not significantly elevated in women with PCOS correlates with adiposity and insulin resistance, and it is the single best adipokine measured in this regard. Chemerin, through its inflammatory role as a chemo-attractant in adipose tissue, may be an important determinant of insulin resistance in PCOS.

Does the level of serum antimüllerian hormone predict ovulatory function in women with polycystic ovary syndrome with aging

OBJECTIVE To determine possible prediction of regular menses with aging in anovulatory women with polycystic ovary syndrome (PCOS). DESIGN Cohort. SETTING Academic practice. PATIENT(S) A total of 54 anovulatory women with PCOS and 28 age- and weight-matched control subjects. INTERVENTION(S) Blood and ovarian ultrasound at baseline and after 5 years. MAJOR OUTCOME MEASURE(S): Serum antimüllerian hormone (AMH), gonadotropins, androgens, insulin sensitivity, and ovarian ultrasound. RESULT(S) After 5 years, there was a significant decrease in AMH in women with PCOS and control subjects (10 of 54 anovulatory women became ovulatory after 5 years). There was a significant negative correlation between baseline serum AMH and ovulatory function after 5 years. However, baseline serum AMH ≤ 4 ng/mL was associated with ovulatory function. CONCLUSION(S) Serum AMH may help predict ovulatory function with aging in anovulatory women with PCOS.

Paraneoplastic anti-N-methyl-d-aspartate-receptor encephalitis from mature cystic teratoma.

A 20-year-old nullipara with no significant medical history presented to a local emergency department with complaint of severe headache. She was diagnosed with migraine headaches, treated, and discharged. Three days later, she re-presented with persistent and worsening headaches, memory deficit, and altered mental status. In the emergency department, she became agitated and confused and was admitted for overnight observation. She then developed generalized tonic clonic seizures and ultimately required intensive care unit (ICU) admission, intubation, sedation, and daily anti-epileptic therapy. Neurologic workup was performed including routine laboratory tests, lumbar puncture, magnetic resonance imaging of the head, and continuous electroencephalogram. Study results were negative for infectious encephalitis, intracranial mass, and epilepsy. Computed tomography of the abdomen and pelvis revealed a 1-cm left adnexal hypodensity consistent with possible dermoid cyst. The patient was transferred to a tertiary care medical center, where her condition continued to deteriorate. She experienced continued seizure activity, without intentional movements, and no response to external stimuli. She was started on lamotrigine, levetiracetam, and topiramate, all without change in her condition. She continued to require ventilatory support and developed complications, including ventilator-associated pneumonia and right femoral deep vein thrombosis. Given the unique presentation of such severe disease, further investigations into uncommon etiologies, such as paraneoplastic encephalitis, were performed. Extensive serum and cerebrospinal fluid (CSF) studies were assessed, including a recently developed assay for anti–N-methyl-D-aspartate-receptor (anti-NMDA-R) antibodies. Anti–NMDA-R antibodies were found in her serum and CSF. The patient was started on plasmaphoresis and intravenous immunoglobulin therapy. A gynecology consult was then initiated (approximately 5 weeks after initial presentation) to evaluate her suspected dermoid cyst as a possible source for these antibodies and her condition. A transvaginal ultrasound examination revealed a 1.77 1.56-cm solid left ovarian mass suggesting a dermoid cyst (Fig. 1). The patient was taken to the operating room for diagnostic laparoscopy. Bilateral multicystic ovaries were noted and explored laparoscopically (Fig. 2). A small cyst with visible hair and serous fluid was identified and removed from the left ovary. Paraffin section of the specimen confirmed the presence of a benign cystic teratoma with neural tissue present. The specimen was not tested for NMDA-R expressing tissue. Postoperatively, the patient remained in the neurological ICU under ventilator support, with modest improvements in mental status and function. Upon discharge to a rehabilitation center, she remained unresponsive and continued to require assisted ventilation and tube feedings. During the past 5 months, she has improved significantly. She is now responsive to auditory and tactile stimuli, breathing spontaneously, and participating in From the Department of Obstetrics and Gynecology, Columbia University College of Physicians and Surgeons, New York, New York.

Optimization of IVF pregnancy outcomes with donor spermatozoa

PurposeTo identify risk factors for suboptimal IVF outcomes using insemination with donor spermatozoa and to define a lower threshold that may signal a conversion to fertilization by ICSI rather than insemination.MethodRetrospective, age-matched, case-control study of women undergoing non-donor oocyte IVF cycles using either freshly ejaculated (N = 138) or cryopreserved donor spermatozoa (N = 69). Associations between method of fertilization, semen sample parameters, and pregnancy rates were analyzed.ResultsIn vitro fertilization of oocytes with donor spermatozoa by insemination results in equivalent fertilization and pregnancy rates compared to those of freshly ejaculated spermatozoa from men with normal semen analyses when the post-processing motility is greater than or equal to 88%. IVF by insemination with donor spermatozoa when the post-processing motility is less than 88% is associated with a 5-fold reduction in pregnancy rates when compared to those of donor spermatozoa above this motility threshold. When the post-processing donor spermatozoa motility is low, fertilization by ICSI is associated with significantly higher pregnancy rates compared to those of insemination.ConclusionWhile ICSI does not need to be categorically instituted when using donor spermatozoa in IVF, patients should be counseled that conversion from insemination to ICSI may be recommended based on low post-processing motility.

Finding Fibers: Intrauterine Synechia of the Lower Uterus after Cesarean Section

Finding Fibers: Intrauterine Synechia of the Lower Uterus after Cesarean Section Intrauterine adhesions, while most commonly the result of uterine curettage, may occur following any uterine surgery. However, the precise incidence of adhesion formation following uterine surgery is unknown. Described is a case of uterine adhesion formation shortly following cesarean delivery diagnosed and treated successfully with hysteroscopy and resection. The precise location of the adhesion and the time of diagnosis in this case suggest that is was an iatrogenic complication of cesarean delivery.