Comparison of Automated and Manual DNA Isolation Methods of Liquid-Based Cytology Samples.

Liquid-based cytology (LBC) has been used as a diagnostic tool for cervical cancer for years and is now being adopted for other gynecological cancers. LBC represents an important challenge to ensure that the process yields representative biospecimens for quality control (QC) of diagnostic procedures. In this study, we compare QC parameters (integrity, yield and purity, and polymerase chain reaction [PCR] amplification) of DNA isolated from LBC (N = 296) using two different nucleic acid isolation methods, manual (n = 233) or automated (n = 63). We also evaluated two different types of cytological brushes for sampling from the cervix. Our results suggest that manual isolation (yield 22.81 ± 1.92 µg) resulted in increased DNA recovery when compared with automated isolation (yield 9.96 ± 1.11 µg) from LBC samples, with a p-value of <0.0003. We estimated that 98% (53/54) of the samples preserved the integrity of DNA and were suitable for standard molecular biology analyses. The ß-globin gene was amplified in 100% (296/296) of the DNA samples by endpoint PCR. We found no significant difference between the performance of the cytological brushes (p value of <0.6711) in a general overview. However, when looking at the results from using each brush individually, the manual isolation method was statistically superior to the automated method. Our work illustrates the impact of good QC of preanalytic conditions, which will be important for the application of LBC for developing early detection methods for gynecological cancers.

Age as A Predictor of Embryo Quality Regardless of The Quantitative Ovarian Response.

BACKGROUND: One determining factor of a successful in vitro fertilization (IVF) cycle is embryo quality. The aim of the present study was to evaluate associations of embryo quality and reserve markers like age, FSH and AMH. MATERIALS AND METHODS: In this prospective study, 120 infertile women, aged 21-44 years, undergoing routine exploration during an unstimulated cycle preceding assisted reproductive technology (ART) at our center were studied prospectively, from February 2011 to December 2014. Descriptive parameters and patient characteristics were reported as mean (SD) or median (range) depending on the distribution. Student's t test was performed for continuous variables, Wilcoxon and Pearson's Test were used for not distributed variables and Fisher's Test was performed for categorical variables. P<0.05 was considered statistically significant. RESULTS: Overall, at the time of investigation, patients had a mean age of 33.03 ± 4.15 years old. On cycle day three, serum anti-Mullerian hormone (AMH) level was 3.50 ± 1.54 ng/mL, serum follicle-stimulating hormone (FSH) level was 6.29 ± 1.53 mUI/ mL, at baseline, women had 16.57 ± 7.0 antral follicles. The mean of collected oocytes was 11.80 ± 5.25, embryo I+II was 2.46 ± 2.11. A greater number of embryos I+II was observed in young patients. By evaluating 120 patients, a significant relationship was observed between age and FSH (r=0.24, P=0.01), age with AMH (r=-0.22, P=0.02), age with collected oocytes (r=-0.23, P=0.03) and age with embryo I+II (r=-0.22, P=0.03). A significant relationship was also observed between antral follicle count (AFC) and AMH (r=0.29, P=0.01), AFC and the number of transferred embryo (r=-0.18, P=0.03), AFC and total dose of the drugs (r=-0.23, P=0.03). Significant relationship of FSH with total dose of drugs (r=0.19, P=0.02) was also observed. In addition, we determined significant relationships between AMH and the number of collected oocytes (r=0.38, P=0.01), AMH and the number of metaphase II oocytes (r= 0.35, P=0.01), AMH and the number of embryo (r=0.19, P=0.04) as well as AMH and total dose of the drugs (r=-0.25, P=0.01). CONCLUSION: Commonly used clinical markers of ovarian reserve are reflection of the ovarian reserve, while the outcome measurements of ART and age are the best predictors of embryo quality.

Optimal reproductive competence of oocytes retrieved through follicular flushing in minimal stimulation IVF.

The objective of this study was to assess the reproductive competence of oocytes obtained by follicular flushing in poor responder patients. This prospective comparative study, at the University of Paris XI, Assistance Publique des Hopitaux de Paris, INSERM Unit 782, was performed on 165 infertile IVF embryo transfer candidates. A total of 271 consecutive minimal stimulation IVF cycles were studied. Oocyte retrieval was performed 34 h after human chorionic gonadotrophin administration and oocytes were allocated into two groups according to their retrieval method: oocytes obtained in the first follicular aspiration (FA, n = 127); and oocytes retrieved in the subsequent follicular flushing (FF, n = 102). The principal outcome was to evaluate clinical pregnancy and embryo implantation rates. Thus, patient characteristics, fertilization rate and clinical pregnancy rate per oocyte were comparable in both of groups. In contrast, embryo morphology (41 versus 59%, P < 0.01) and implantation rates (20.4 versus 34.8%, P < 0.04) were better in the FF group. In conclusion, an optimal reproductive competence was observed in oocytes retrieved by follicular flushing in minimal stimulation IVF in poor responder patients.

Impact of early cleaved zygote morphology on embryo development and in vitro fertilization-embryo transfer outcome: a prospective study.

OBJECTIVE: To evaluate the impact of the first division morphology on embryo development and IVF-embryo transfer outcome. DESIGN: Prospective study. SETTING: Teaching hospital, France. PATIENT(S): All zygotes from 201 couples were checked for early cleavage. We defined as "even," early cleaved (EC) zygotes with 2 cells of even size; as "uneven," EC zygotes with 2 cells of uneven size; and as "fragmented," EC zygotes with more than 20% fragmentation rate. Day 2 embryo quality was assessed as "top" embryo or "non-top," with the evaluation of multinucleated blastomeres. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Day 2 embryo quality, pregnancy and implantation rates. RESULT(S): Among EC zygotes, 59.1% were even, 13.0% were uneven, and 27.9% were fragmented. Even EC yielded more "top" embryos and less multinucleated blastomere embryos than uneven EC (77.0% vs. 46.3%) and fragmented EC (77.0% vs. 13.9%). The 125 double embryo transfers that comprised at least one embryo derived from even EC zygote led to higher pregnancy rate (PR) (64.0% vs. 43.4%) and implantation rate (42.0% vs. 27.6%) compared to the 76 double embryo transfers with embryos derived from breakdown or 2PN zygotes. CONCLUSION(S): The morphology of the early cleaved zygote is involved in embryo development. Evaluation of this morphology is an effective and valuable method of assessing the embryo quality.

Enlightenment about the new Architect-i2000 estradiol (Abbott Laboratories) immunoassay during in vitro fertilization.

OBJECTIVE: We assessed a new estradiol (E2) immunoassay on the Architect-i2000 (Abbott Laboratories) for monitoring ovulation stimulation for IVF-ET and re-establishing clinical cut-off points. The method has been modified to improve E2 measurements especially at normal and low concentrations. DESIGN AND METHOD: E2 was determined for 552 samples, from 83 women, presenting normal follicular status and undergoing 100 cycles of IVF treatment. We assessed the value of this assay for down-regulation of E2 concentration limit using gonadoliberin-releasing hormone agonist (GnRHa), and monitoring of the ovarian hyperstimulation, expected range of E2 per mature follicle prior to the administration of exogenous hCG and day 3 concentration limit. We compared results with our routine method (E2-6II Advia-Centaur; Siemens-Diagnostics) for which decision-making values were known. RESULTS: Considering E2 concentrations obtained with the new Architect-i2000 assay for patients treated with GnRHa for 2 weeks, the cutoff-point for ovarian down-regulation should be set down at 110 pmol/L to maintain 100% of sensitivity. Considering day 3 concentration limit determination, results were not significantly different from those obtained with our routine method. The mean E2 values per mature follicle fell into the range generally expected. CONCLUSION: E2 determination with the new E2 Architect-i2000 assay could be used to monitor ovulation, in patients undergoing IVF-ET, in combination with transvaginal ultrasound.

Anti-Müllerian hormone concentrations in the follicular fluid of the preovulatory follicle are predictive of the implantation potential of the ensuing embryo obtained by in vitro fertilization.

CONTEXT: The strong relationship between serum anti-Müllerian hormone (AMH) levels and the number of antral follicles supports the use of AMH measurements as a quantitative marker of the ovarian follicular status. Yet, it still is unclear whether the aptitude of an individual follicle to produce AMH reflects its reproductive competence. OBJECTIVE: This study examined the possible relationship between serum or follicular fluid (FF) AMH concentrations and the fate of the ensuing oocytes and embryos obtained by in vitro fertilization-embryo transfer conducted in monodominant follicle cycles. DESIGN AND SETTING: We conducted a prospective study at the University of Paris XI, Assistance Publique-Hôpitaux de Paris, Institut National de la Santé et de la Recherche Médicale U782. PATIENTS: Patients included 118 infertile in vitro fertilization-embryo transfer candidates. INTERVENTIONS: Concentrations of AMH, progesterone, and estradiol were measured in the serum on cycle d 3 and on the day of oocyte pickup (dOPU), and in FF. Cycles were sorted into three sets of three distinct groups according to whether serum d 3, serum dOPU, and FF AMH concentrations were 30th centile or below (low AMH), between the 31st and the 70th centiles (average AMH), or above the 70th centile (high AMH) of measurements. MAIN OUTCOME MEASURE: Clinical pregnancy and embryo implantation rates were assessed. RESULTS: Clinical pregnancy rates (5.7, 20.0, and 39.5%, respectively; P < 0.002) and embryo implantation rates (11.8, 30.8, and 65.4, respectively; P <0.001) were markedly different among the low, moderate, and high FF AMH groups but not among the serum (d 3 or dOPU) AMH groups. Fertilization rates and embryo morphology remained similar irrespective of AMH concentrations in the serum or in FF. Incidentally, FF AMH concentrations were negatively correlated with FF progesterone (r = -0.27; P <0.003) and FF estradiol (r = -0.21; P <0.02) concentrations. CONCLUSIONS: Concentrations of AMH in the FF, but not in the serum, constitute a useful follicular marker of embryo implantation and are negatively related to FF progesterone and estradiol concentrations.

Anti-Müllerian hormone: clinical insights into a promising biomarker of ovarian follicular status.

In contrast to most hormonal biomarkers of the follicular status, anti-Müllerian hormone (AMH) is exclusively produced by the granulosa cells of a wide range of follicles (primary to the early antral stages), presumably FSH-independently and with little susceptibility to disorders of antral follicle growth during the luteal-follicular transition. This paper summarizes the authors' clinical research on the role of AMH as a marker of ovarian functioning. It shows that the relationship between antral follicle counts and serum AMH concentrations is stronger than that observed with FSH, inhibin B and oestradiol on day 3, and that intercycle reproducibility of AMH measurements is better than the latter parameters. In addition, peripheral AMH concentrations decline during ovarian stimulation, thus confirming that maturing follicles loose progressively their ability to produce AMH. Indeed, follicular fluid (FF) AMH concentrations in small antral follicles are 3-fold as high as AMH in pre-ovulatory follicles. Further, human chorionic gonadotrophin-driven luteinization additionally curtails follicular AMH production. Finally, AMH production measured in FF from individual follicles is increased in women having normal follicular counts and responsiveness to ovarian stimulation. Together, these data reinforce the soundness of AMH measurements as a quantitative and possibly qualitative marker of granulosa cell activity and health.

Coordination of antral follicle growth: basis for innovative concepts of controlled ovarian hyperstimulation.

Among the key objectives of controlled ovarian hyperstimulation (COH) is the achievement of adequate coordination of multiple follicular growth to trigger ovulation when most of follicles have reached concomitant maturation. However, during the early follicular phase, early antral follicles present noticeable size heterogeneities that may be amplified during COH. To challenge the hypothesis that this phenomenon results, at least in part, from the early exposure of antral follicles to gradient follicle-stimulating hormone (FSH) levels during the preceding late luteal phase, we conducted three clinical studies. First, we artificially lowered luteal FSH levels by administering estradiol (E (2)) and measured follicular characteristics on the subsequent day 3. Second, we verified whether luteal E (2) administration could promote the coordination of follicular growth during COH and improve its results. Third, we assessed the effects of premenstrual gonadotropin-releasing hormone (GnRH) antagonist administration on follicular characteristics during the early follicular phase. Our results showed that luteal FSH suppression by either E (2) or GnRH antagonist administration reduces the size and improves the homogeneity of early antral follicles during the early follicular phase, an effect that persists during COH. Coordination of follicular development may optimize ovarian response to short GnRH agonist and antagonist protocols, and constitutes an attractive approach to improving their outcome.

Per-follicle measurements indicate that anti-müllerian hormone secretion is modulated by the extent of follicular development and luteinization and may reflect qualitatively the ovarian follicular status.

OBJECTIVE: To investigate the possible influence of follicular maturation and luteinization on anti-müllerian hormone (AMH) secretion and the relationship between per-follicle AMH levels, ovarian follicular status, and responsiveness to controlled ovarian hyperstimulation (COH). DESIGN: Prospective study. SETTING: University hospital in France. PATIENT(S): Thirty seven in vitro fertilization/embryo transfer candidates undergoing COH. INTERVENTION(S): On the day of oocyte retrieval, serum samples and follicular fluids from two small (8-12 mm in diameter) and two large (16-20 mm in diameter) follicles were collected for AMH, E2, and progesterone (P4) measurements. MAIN OUTCOME MEASURE(S): Per-follicle AMH levels. RESULT(S): Small follicles secreted AMH levels that were approximately three times as high as large follicles. Follicular fluid AMH and P4 levels were negatively correlated to each other both in small and large follicles. Per-follicle AMH levels in both follicular classes were positively correlated with antral follicle count on cycle day 3 before COH and with growing follicle (> or =12 mm) count and oocytes retrieved, but negatively correlated with FSH requirement. CONCLUSION(S): Both final follicular maturation and luteinization interfere with granulosa cell AMH production. The relationship between intrafollicular AMH content, the surrounding follicular status, and ovarian response to COH indicates that peripheral AMH levels reflect not only follicle count but also per-follicle AMH production.

Hormonal manipulations in the luteal phase to coordinate subsequent antral follicle growth during ovarian stimulation.

During the early follicular phase in the menstrual cycle, antral follicle sizes are often markedly heterogeneous. These follicular size discrepancies may, at least in part, result from the early exposure of FSH-sensitive follicles to gradient FSH concentrations during the preceding luteal phase. In addition, they potentially affect the results of ovarian stimulation. Indeed, pre-existing follicle size discrepancies may encumber coordinated follicular growth during ovarian stimulation, thereby reducing the number of follicles that reach maturation at once. To investigate this issue, three clinical studies were conducted to test the hypothesis that luteal FSH suppression could coordinate follicular growth. First, luteal FSH concentrations were artificially lowered by administering physiological oestradiol doses and measured follicular characteristics on the subsequent day 3. Second, it was verified whether luteal oestradiol administration could promote the coordination of follicular growth during ovarian stimulation and improve its results. Third, the effects of premenstrual gonadotrophin-releasing hormone (GnRH) antagonist administration on follicular characteristics were assessed during the early follicular phase. The results showed that luteal FSH suppression by either oestradiol or GnRH antagonist administration reduces the size and improves the homogeneity of early antral follicles during the early follicular phase, an effect that persists during ovarian stimulation. Coordination of follicular development may optimize ovarian response to short GnRH agonist and antagonist protocols, and constitutes an attractive approach to improving their outcome.

Dynamics of serum anti-Müllerian hormone levels during the luteal phase of controlled ovarian hyperstimulation.

BACKGROUND: To investigate the dynamics of serum anti-Müllerian hormone (AMH) levels during the luteal phase of controlled ovarian hyperstimulation (COH) and its possible association with follicle development. METHODS: We prospectively studied 34 women undergoing COH with GnRH agonist and FSH. On the day of hCG (dhCG), serum AMH, estradiol (E2), progesterone and hCG levels were measured, and ovarian follicles were sorted into three size classes: <12, 12-15 and 16-22 mm. Hormonal measurements were repeated 4 days (hCG + 4) and 7 days (hCG + 7) after hCG. RESULTS: From dhCG to hCG + 4, we observed a decline in serum AMH levels (-64 +/- 3%; P < 0.0001), which paralleled that of E2 levels. From hCG + 4 to hCG + 7, an increase in AMH levels occurred (82 +/- 28%; P < 0.02), whose magnitude was correlated with the number of < 12 mm follicles (r = 0.68; P < 0.0001) but not with other follicle size classes nor with the remaining hormone levels. CONCLUSIONS: After hCG, AMH levels initially decline, presumably as an effect of follicle luteinization, then increase during the mid-luteal phase. Although the mechanisms implicated in the mid-luteal AMH increase are unclear, its positive association with small follicle count, but not with luteal progesterone and E2 levels, supports the hypothesis that AMH levels might reflect luteal follicle development.