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.

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.

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.

Successful pregnancy following assisted reproduction and transmyometrial embryo transfer in a patient with anatomical distortion of the cervical canal.

Abstract Presented is the case report of a patient noted to have gross distortion of the internal cervical canal during her attempt at embryo transfer following an in vitro fertilization with intracytoplasmic sperm injection (IVF-ICSI) procedure. Multiple attempts at cervical dilation were unsuccessful and the patient was ultimately treated by transmyometrial embryo transfer also known as the Towako method. She successfully achieved a singleton pregnancy and delivered at 41 weeks by primary cesarean section because of arrest of cervical dilation. Transmyometrial embryo transfer represents a viable option for patients with cervical stenosis refractory to conventional methods of navigation or severe anatomical distortion of the internal cervical canal.

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.

Pregnancy after age 50: defining risks for mother and child.

Assisted reproductive technology using donor-egg in vitro fertilization (D-IVF) has enabled women 50 years and above to successfully achieve pregnancy. We examine the safety profile of these pregnancies through a large, single-center case series and retrospective cohort analysis in which all participants were carefully screened medically prior to conception. Consecutive women aged ≥ 50 years (n = 101) who achieved a viable pregnancy via D-IVF were identified and their perinatal outcomes were recorded. These data were compared with control data from younger (≤ 42 years) recipients of D-IVF (n = 41) who also achieved a viable pregnancy at our center during the same period. Compared with the younger women, older recipients had statistically similar rates of hypertensive disorders of pregnancy (23% versus 14%, odds ratio [OR] 1.9 [0.65 to 5.4]), gestational diabetes (4.0% versus 3.0%, OR 1.4 [0.15 to 113.0]), preterm premature rupture of membranes/preterm labor (8.9% versus 14%, OR 0.59 [0.18 to 1.9]), and abnormal placentation (2.1% versus 0%). Cesarean delivery was high in women ≥ 50 (81% of singletons, 100% of multiples). There was one maternal death, which occurred before delivery in a 49-year-old woman who would have been 50 at term had she survived, presumably secondary to myocardial infarction. Primary neonatal outcomes of gestational age and birth weight were excellent and similar between groups. Women ≥ 50 years who achieve pregnancy via D-IVF are at high risk for maternal complications, particularly hypertensive disorders and cesarean section, but at rates similar to those seen in younger recipients.

Does the addition of clomiphene citrate or letrazole to gonadotropin treatment enhance the oocyte yield in poor responders undergoing IVF?

PURPOSE: To evaluate the effect of adjunctive letrazole or clomiphene in IVF stimulation protocols. METHODS: A retrospective analysis of high dose GnRH antagonist IVF cycles (450-600 IU of gonadotropins) that have met poor responder criteria. Selected consecutive cycles in same patients differed solely in presence or absence of adjunctive clomiphene or letrazole. RESULTS: Supplementation with clomiphene citrate in poor responders showed significant improvements (p < 0.05) in estradiol levels (1506 vs. 1044 pg/ml), number of dominant follicles (5.6 vs. 3.9), oocytes retrieved (5.2 vs. 3.4) and number of transferred embryos (1.7 vs. 1.1). It significantly improved biochemical pregnancy rates (18.1% vs. 5.9%) while reducing cycle cancellations (11.7% vs. 32.6%). Letrozole supplementation showed similar effects. CONCLUSION: Both Clomiphene and Letrazole performed similarly and showed significant effects. However, despite increasing oocyte yield and embryo transfer rates, the overall clinical and live birth rate in this population remained low and showed no measurable increase.