Joanna Liss

Two new automated, compared with two enzyme-linked immunosorbent, antimüllerian hormone assays.

OBJECTIVE To compare new automated antimüllerian hormone (AMH) assay performance characteristics from the new automated Elecsys AMH (Roche; Elecsys) and Access AMH (Beckman Coulter; Access) assays with the existing AMH Gen II ELISA (enzyme-linked immunosorbent assay; Gen II; Beckman Coulter) and AMH ELISA (Ansh Labs) assays. DESIGN Prospective assay evaluation. SETTING University-affiliated clinical chemistry laboratory. PATIENT(S) Patients referred for serum AMH measurement (n = 83) before start of in vitro fertilization cycle between September 2014 and October 2014. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Serum AMH concentration. RESULT(S) Intra-assay coefficients of variation were low; Ansh ≤ 9.0%; Gen II ≤ 5.8%; Access ≤ 10.7%; and Elecsys ≤ 2.8%. The Passing-Bablok regression equations (pmol/L) were y (Access) = 0.128 + (0.781 × Gen II); and y (Access) = 0.302 + (0.742 x Ansh). For y (Elecys) = 0.087 + (0.729 x Gen II) and y (Elecys) = 0.253 + (0.688 x Ansh Labs). For y (Elecys) = 0.943 - (0.037 × Access). For all the assays, AMH exhibited a moderate positive correlation with AFC (r = 0.62-0.64); number of cumulus oocyte complexes (r = 0.60-0.64); and metaphase II oocytes (r = 0.48-0.50). Accuracy of pregnancy prediction, as determined by area under the receiver operating characteristic curve, was uniformly low for all assays (0.62-0.63). CONCLUSION(S) The novel automated assays exhibit strong concordance in calibration, but derived values are substantially lower than those obtained from pre-existing assays, with assay-specific interpretation required for routine clinical use. These results highlight the need for an international standard of measurement of AMH.

Human Papillomavirus Type 16 Status in Cervical Carcinoma Cell DNA Assayed by Multiplex PCR

ABSTRACT Integration of human papillomavirus (HPV) DNA into host genome occurs early in cancer development and is probably an important event in malignant transformation of cervical cancer. The HPV genome integration usually disrupts E2 gene open reading frames. It results in the lack of E2 gene suppressor of the synthesis of E6 and E7 products which, in turn, leads to the overexpression of E6 and E7 genes. The oncogenic HPV types (HPV16, -18, -45, and -58) can be present as episomes or may integrate into human chromosomes. Sixty-six cervical cancer patients positive for HPV16 were tested for the presence of E6, E2, E1, and L1 genes. Multiplex PCR was carried out in all cases. Using cluster analysis, the calculated ratios of E1/E6, E2/E6, L1/E6, E1/E2, and E2/(E1*E6) gene amplification products were divided into two or three statistically different groups. These were used for statistical analysis of the prevalence of specific gene types in histological types of cancer, different levels of clinical staging, and histologically confirmed nodal metastases. The statistical analysis proved a significant correlation in the ratios of E2/E6 and E1/E2 only. The E2/E6 and E1/E2 were higher in carcinoma in situ than in advanced squamous cancers. The E2/E6 ratios were lower in higher clinical stages. The multiplex PCR estimation of the E2/E6 ratio could be a simple method for selecting patients with a high risk of a poor outcome in a standard stage-dependent treatment procedure.

Use of ovarian reserve parameters for predicting live births in women undergoing in vitro fertilization

OBJECTIVE To examine common clinical determinants, including patient age; levels of anti-Müllerian hormone (AMH), inhibin B, and follicle-stimulating hormone (FSH); antral follicle count (AFC); and number of oocytes retrieved, to predict live births in women undergoing in vitro fertilization. STUDY DESIGN Women undergoing cycles of intracytoplasmic sperm injection (ICSI) for the first time were reviewed retrospectively, and serum levels of AMH, inhibin B, and FSH, as well as AFC (days 1 and 4 of pre-ICSI menstrual period) and patient age were analyzed as determinants of live birth rates. RESULTS Of the patients studied, 35.71% (891/2495) became pregnant, with live births achieved in 32.20% (806/2495) of cycles initiated and in 46.37% (806/1738) of embryo transfers. Clinical pregnancy rate was 35.71% (891/2495) for cycles initiated and 51.26% (891/2318) for embryo transfers. Univariate analysis revealed that the odds of live birth significantly decreased with increasing age, declining AMH or inhibin B concentrations, and fewer oocytes retrieved. At AMH levels greater than 5.7 ng/ml, the odds of live birth were 3.18 times greater than for AMH levels less than 1.9 ng/ml [95% confidence interval (CI), 1.89-5.43]. Using multivariate logistic regression, only AMH (OR = 1.89; 95% CI, 1.00-3.60; p < 0.05) and AFC (OR = 1.86; 95% CI, 1.02-3.40; p < 0.05) showed statistically significant associations with live birth. Area under the curve for ROC (ROC(AUC)) indicated that AMH (AUC = 0.60) surpassed AFC (AUC = 0.59), number of oocytes retrieved (AUC = 0.59), inhibin B (AUC = 0.55), FSH (ROC(AUC) = 0.54) and chronological age (ROC(AUC) = 0.53) in predicting live birth. CONCLUSIONS In this assessment of various indices (i.e., age; levels of AMH, inhibin B, and FSH; AFC; and quantity of oocytes retrieved) for predicting live births for IVF patients, AMH, AFC and the quantity of oocytes retrieved constituted the most reliable determinants.

Evaluation of Granulocyte Colony-Stimulating Factor Effects on Treatment-Resistant Thin Endometrium in Women Undergoing In Vitro Fertilization

The aim of the study was to assess the granulocyte colony-stimulating factor (G-CSF) effects on unresponsive thin (<7 mm) endometrium in women undergoing in vitro fertilization (IVF). We included thirty-seven subjects who had thin unresponsive endometrium on the day of triggering ovulation. These patients also failed to achieve an adequate endometrial thickness in at least one of their previous IVF cycles. In all the subjects at the time of infusion of G-CSF, endometrial thickness was 6,74 ± 1,75 mm, and, after infusion, it increased significantly to 8,42 ± 1,73 mm. When we divided the group into two subgroups according to whether the examined women conceived, we showed that the endometrium expanded significantly from 6,86 ± 1,65 to 8,80 ± 1,14 mm in the first group (who conceived) and from 6,71 ± 1,80 to 8,33 ± 1,85 mm in the second, respectively. There were no significant differences between the two subgroups in respect to the endometrial thickness both before and after G-CSF infusion. The clinical pregnancy rate was 18,9%. We concluded that the infusion of G-CSF leads to the improvement of endometrium thickness after 72 hours.

Probability of live birth in women with extremely low anti-Müllerian hormone concentrations

The aim of the present study was to investigate the clinical pregnancy and live birth rates in women with extremely low (≤ 0.4 ng/ml) anti-Müllerian hormone (AMH) concentrations. The study included 101 women (188 cycles) with extremely low AMH concentrations undergoing IVF cycles and compared the number of live births in women with low AMH. Moreover, the study compared the number of live births in women with or without endometriosis stage III/IV. Fourteen clinical pregnancies and 14 live births (including one pair of twins) were recorded; one woman miscarried. Significantly higher clinical pregnancy (P = 0.046) and live birth rates (P = 0.018) were found in women aged < 35 years compared with older women. AMH concentration did not differ significantly between women with or without endometriosis and there were six live births in women with endometriosis. This was not significantly different from the rate in healthy women. It is concluded that live births are possible in women with extremely low AMH concentrations. The presence of endometriosis stage III/IV did not affect live birth rates in women with extremely low AMH concentrations although an important limitation of the study is the small number of women included who were affected by that disease.

Routine use of next-generation sequencing for preimplantation genetic diagnosis of blastomeres obtained from embryos on day 3 in fresh in vitro fertilization cycles

OBJECTIVE To determine the usefulness of semiconductor-based next-generation sequencing (NGS) for cleavage-stage preimplantation genetic diagnosis (PGD) of aneuploidy. DESIGN Prospective case-control study. SETTING A private center for reproductive medicine. PATIENT(S) A total of 45 patients underwent day-3 embryo biopsy with PGD and fresh cycle transfer. Additionally, 53 patients, matched according to age, anti-Müllerian hormone levels, antral follicles count, and infertility duration were selected as controls. INTERVENTION(S) Choice of embryos for transfer was based on the PGD NGS results. MAIN OUTCOME MEASURE(S) Clinical pregnancy rate (PR) per embryo transfer (ET) was the primary outcome. Secondary outcomes were implantation and miscarriage rates. RESULT(S) The PR per transfer was higher in the NGS group (84.4% vs. 41.5%). The implantation rate (61.5% vs. 34.8%) was higher in the NGS group. The miscarriage rate was similar in the 2 groups (2.8% vs. 4.6%). CONCLUSION(S) We demonstrate the technical feasibility of NGS-based PGD involving cleavage-stage biopsy and fresh ETs. Encouraging data were obtained from a prospective trial using this approach, arguing that cleavage-stage NGS may represent a valuable addition to current aneuploidy screening methods. These findings require further validation in a well-designed randomized controlled trial. CLINICAL TRIAL REGISTRATION NUMBER ACTRN12614001035617.

Anti-Müllerian hormone (AMH) is a strong predictor of live birth in women undergoing assisted reproductive technology.

In the present study, we evaluated the clinical value of the following parameters: basal anti-Müllerian hormone (AMH), follicle-stimulating hormone (FSH), inhibin B and antral follicle count (AFC) in predicting live birth outcomes. The study involved 603 women undergoing in vitro fertilization (IVF) using the long protocol for controlled ovarian hyperstimulation (COH). Serum levels of AMH, FSH and inhibin B as well as AFC were measured on the first three days of the menstrual cycle prior to the beginning of stimulation. AMH was the only independent parameter that correlated with the chance of live birth. We found that live birth rates of 46.2% (patient age <35 years), 44.7% (35-37 years), 32.1% (38-39) and 15.3% (>39) were associated with concentrations of AMH>1.4 ng/ml. For the AMH range 0.6-1.4 ng/ml, the live birth rates were 29.3%, 12.5%, 5.6% and 2.7%, respectively, and for AMH concentrations below 0.6 ng/ml the rates were 7.1%, 8.3%, 0% and 5.8%, respectively. Independently of other parameters affecting the chance of live birth, the success rate was the highest when the AMH level was >2 ng/ml, significantly lower when the AMH concentration was about 1 ng/ml, and 0% when the AMH concentration was ∼0.1 ng/ml. In conclusion, this is the first report to demonstrate that AMH level correlated better than age, FSH or inhibin B concentrations or AFC with live birth outcome. Therefore, the basal serum concentration of AMH may become a new, substantial prognostic factor regarding live birth above and beyond other currently available predictors of IVF outcome.

Decreasing quality of the new generations of anti-Mullerian hormone assays.

Anti-Müllerian hormone (AMH) measurements are widely used to optimize the stimulation protocols. First generation AMH kits correlated well with ovarian reserve and response to stimulation. In the present study we aimed to asses if the new generation kits share the same accurate correlations. Retrospective data were collected from 8323 blood samples. For comparison we used Immunotech I generation kit (ImI 4035 samples), Beckman Coulter II generation kit RUO (BCII RUO 3449, samples) and Beckman Coulter II generation kit with IVD certificate (BCII IVD 839 samples). We compared average AMH concentrations measured with different kits, as well as correlation between kits. We also compared average AMH concentrations in sera collected on different cycle days and samples of different quality of preservation. AMH serum concentrations differed for each kit, ranging 4.4 ± 4.12 (mean ± SD) for the ImI, 2.68 ± 3.15 for the BCII RUO, and 1.64 ± 2.85 for BCII IVD. The mean differences from an adjusted regression model were −48.7%, −40%, and −69.2%, respectively. In conclusion, the changes of the BC AMH kits are unpredictable; however, the improvement of them is still possible. It would be very dangerous to use elaborated stimulation protocol (based on the Ist generation AMH results) with the results from the IInd generation assays.

Healthy Baby Born to a Robertsonian Translocation Carrier following Next-Generation Sequencing-Based Preimplantation Genetic Diagnosis: A Case Report.

Preimplantation genetic diagnosis (PGD) is well established method for treatment of genetic problems associated with infertility. Moreover, PGD with next-generation sequencing (NGS) provide new possibilities for diagnosis and new parameters for evaluation in, for example, aneuploidy screening. The aim of the study was to report the successful pregnancy outcome following PGD with NGS as the method for 24 chromosome aneuploidy screening in the case of Robertsonian translocation. Day 3 embryos screening for chromosomal aneuploidy was performed in two consecutive in vitro fertilization (IVF) cycles, first with fluorescent in situ hybridization (FISH), and then with NGS-based protocol. In each IVF attempt, three embryos were biopsied. Short duration of procedures enabled fresh embryo transfer without the need for vitrification. First IVF cycle with the embryo selected using PGD analysis with the FISH method ended with pregnancy loss in week 8. The second attempt with NGS-based aneuploidy screening led to exclusion of the following two embryos: one embryo with 22 monosomy and one with multiple aneuploidies. The transfer of the only euploid blastocyst resulted in the successful pregnancy outcome. The identification of the euploid embryo based on the NGS application was the first successful clinical application of NGS-based PGD in the case of the Robertsonian translocation carrier couple.

Next generation sequencing for preimplantation genetic testing of blastocysts aneuploidies in women of different ages.

Most of the current preimplantation genetic screening of aneuploidies tests are based on the low quality and low density comparative genomic hybridization arrays. The results are based on fewer than 2,700 probes. Our main outcome was the association of aneuploidy rates and the womens age. Between August-December 2013, 198 blastocysts from women (mean age 36.3+-4.6) undergoing in vitro fertilization underwent routine trophectoderm biopsy. NGS was performed on Ion Torrent PGM (Life Technologies). The results were analyzed in five age groups (<31, 31-35, 36-38, 39-40 and >40). 85 blastocysts were normal according to NGS results. The results in the investigated groups were (% of normal blastocyst in each group): <31 (41.9%), 31-35 (47.6%), 36-38 (47.8%), 39-40 (37.7%) and >40 (38.5%). Our study suggests that NGS PGD is applicable for routine preimplantation genetic testing. It allows also for easy customization of the procedure for each individual patient making personalized diagnostics a reality.