Research Article: Increasing Live Birth Rate by Preimplantation Genetic Screening of Pooled Polar Bodies Using Array Comparative Genomic Hybridization

Date Published: May 29, 2015

Publisher: Public Library of Science

Author(s): Michael Feichtinger, Tina Stopp, Christian Göbl, Elisabeth Feichtinger, Enrico Vaccari, Ulrike Mädel, Franco Laccone, Monika Stroh-Weigert, Markus Hengstschläger, Wilfried Feichtinger, Jürgen Neesen, Cheng-Guang Liang.

http://doi.org/10.1371/journal.pone.0128317

Abstract

Meiotic errors during oocyte maturation are considered the major contributors to embryonic aneuploidy and failures in human IVF treatment. Various technologies have been developed to screen polar bodies, blastomeres and trophectoderm cells for chromosomal aberrations. Array-CGH analysis using bacterial artificial chromosome (BAC) arrays is widely applied for preimplantation genetic diagnosis (PGD) using single cells. Recently, an increase in the pregnancy rate has been demonstrated using array-CGH to evaluate trophectoderm cells. However, in some countries, the analysis of embryonic cells is restricted by law. Therefore, we used BAC array-CGH to assess the impact of polar body analysis on the live birth rate. A disadvantage of polar body aneuploidy screening is the necessity of the analysis of both the first and second polar bodies, resulting in increases in costs for the patient and complex data interpretation. Aneuploidy screening results may sometimes be ambiguous if the first and second polar bodies show reciprocal chromosomal aberrations. To overcome this disadvantage, we tested a strategy involving the pooling of DNA from both polar bodies before DNA amplification. We retrospectively studied 351 patients, of whom 111 underwent polar body array-CGH before embryo transfer. In the group receiving pooled polar body array-CGH (aCGH) analysis, 110 embryos were transferred, and 29 babies were born, corresponding to live birth rates of 26.4% per embryo and 35.7% per patient. In contrast, in the control group, the IVF treatment was performed without preimplantation genetic screening (PGS). For this group, 403 embryos were transferred, and 60 babies were born, resulting in live birth rates of 14.9% per embryo and 22.7% per patient. In conclusion, our data show that in the aCGH group, the use of aneuploidy screening resulted in a significantly higher live birth rate compared with the control group, supporting the benefit of PGS for IVF couples in addition to the suitability and effectiveness of our polar body pooling strategy.

Partial Text

The success of an infertility treatment is strongly associated with the age of the female partner, mainly due to the rapid increase in aneuploidies that occurs in the oocytes of women aged 35 years and older. Additionally, aneuploidy rates in the oocytes of infertile female patients seem to be even higher than those in the oocytes of women of the same age without fertility problems [1,2]. Therefore, it is reasonable to assume that the identification of such oocytes or embryos without chromosomal aberrations in women over 35 years of age may improve pregnancy rates and consequently, live birth rates. Unfortunately, no consistent relationship appears to exist between the embryo karyotype and its morphology [3].

In the present study, 351 women between 35 and 45 years of age were included. The patients were treated using standard IVF/ICSI protocols. In the study group (aCGH group), 111 patients with a mean age of 39.5 years underwent BAC array-CGH-based aneuploidy screening (PGS) before embryo transfer using DNA obtained from pooled polar bodies. The indication for polar body screening was either repeated implantation failure or advanced maternal age.

The results of this study strongly suggest that array-CGH analysis using amplified DNA from pooled polar bodies improves the live birth rate compared with that observed in the absence of aneuploidy screening. A number of studies have demonstrated that chromosome aberrations are strongly increased in women older than 35 years and that aneuploidy may be the main reason for infertility in couples with an advanced maternal age. Aneuploid oocytes can be identified with high sensitivity by the comprehensive chromosome screening of amplified DNA from polar bodies. In former studies, polar bodies have been evaluated to predict the majority of aneuploidies in resulting embryos [24,25]. However, in these studies, first and second polar bodies were extracted successively from oocytes and amplified separately. After hybridization, the polar bodies were analyzed using software with specific settings for the first and second polar bodies. Because the BlueFuse Multi software that we used has no setting for pooled polar bodies, we plotted our samples with an adjustment for first polar body analysis. Previous studies have shown that BlueFuse Multi software is able to detect mosaicism for aneuploidy at levels as low as 25–37% with high confidence [21,22]. Our approach to pool the first and second polar bodies corresponds to an aneuploidy mosaicism of 50–60%. Therefore, if the quality of the hybridization signals is sufficient, the BlueFuse Multi software will detect chromatid gains or losses in pooled polar bodies with high efficiency. However, it should be noted that a certain amount of false positives have been reported in previous studies involving sequential and complementary assessments of polar bodies [25]. These discordant results may be due to trisomic rescue, which occurs in the embryo at a very early stage of cleavage and can result in either a normal embryo or an embryo with uniparental disomy or isodisomy [24–27]. Depending on the involved chromosome, disomy is expected to have clinical consequences in the child. In contrast with polar body analysis, isodisomic embryos can be identified by analysis of trophectoderm cells using SNP arrays.

 

Source:

http://doi.org/10.1371/journal.pone.0128317