Recurrent pregnancy loss (RPL) is a common though poorly understood condition. Defined as two or more failed clinical pregnancies, RPL affects up to 5% of couples attempting to conceive [1, 2]. In addition to the physical burdens, RPL is often an emotionally and psychologically difficult diagnosis for these women and their partners. Diagnosing the cause of a pregnancy loss is important both to determine whether further interventions are indicated, as well as to provide a sense of closure to the patient and her partner.
Chromosomal abnormalities account for up to 75% of first trimester pregnancy losses [3–10]. Given this high frequency, a critical initial step in the evaluation of recurrent pregnancy loss is to perform cytogenetic analysis of the products of conception (POCs) in order to determine whether aneuploidy was the cause of the loss. If the pregnancy loss was due to aneuploidy, then the likelihood of recurrence returns to the age-adjusted baseline, which increases with advancing maternal age . However, if aneuploidy was not the cause, a further work-up is indicated.
Traditionally, cytogenetic evaluation following a pregnancy loss is performed via Giemsa staining of metaphase spreads. This requires culturing cells obtained from freshly collected chorionic villi, and so POCs must be promptly placed in appropriate culture media before cell death occurs. These requirements create many situations in which conventional karyotyping may not be feasible. Examples include when the pregnancy loss occurred a significant time before the POCs could be placed in appropriate culture media (e.g. patients with a lengthened interval between the demise and the time of tissue collection or when tissue is passed at home or at a location where culturing could not be started), when there may not be the necessary culture media or situational awareness required to send the material for karyotyping (e.g. in the case of emergent dilation and curettage (D&C)) or when samples are lost in transit, become infected, or fail to grow during culture.
Due to the high likelihood of aneuploidy in cases of sporadic pregnancy loss, current recommendations by the American College of Obstetrics and Gynecology as well as the American Society for Reproductive Medicine are to obtain karyotype results only after the second or third loss . Recent cost-analyses have supported this recommendation [13, 14]. Thus, the POCs from a first or second loss are often not sent for karyotyping, even following a D&C. However, if the patient goes on to have subsequent losses, particularly at earlier gestations precluding successful genomic analysis, the karyotypes from the earlier miscarriage would be informative. Therefore, there is a need to establish a standardized method for retrospectively retrieving cytogenetic information from previous pregnancy losses in a robust manner that could be readily employed in a clinical laboratory.
In the context of freshly collected POCs, array-based comparative genomic hybridization (aCGH) may be equally or even more informative than conventional karyotyping [15–23]. When used in prenatal diagnosis and the evaluation of children with developmental delay, aCGH allows for the detection of copy number variants (CNVs) such as deletions and insertions with a 10-fold increased resolution compared with traditional karyotyping [24–27].
Unlike prior approaches that required PCR amplification of extracted genomic DNA , an automated approach has successfully been utilized in tumor genotyping and cytogenetic analysis in oncology. In these cases, aCGH analysis of DNA extracted from paraffin-embedded tumor samples is frequently used, demonstrating the distinct advantage that this technology does not require live cells . Here we sought to extend this approach to analysis of paraffin embedded POCs.
For the purpose of retrospectively or “rescue” karyotyping, we adapted aCGH analysis of DNA extracted from paraffin embedded tissue, a technique widely used in analysis of tumor samples, to analysis of paraffin embedded POCs. This technique provides the potential to obtain detailed cytogenetic information from previously collected paraffin-embedded conceptual tissue. Based on our findings, we propose this technique as a method of obtaining useful cytogenetic information for patients who require karyotype results but have had either no attempts or failed attempts at conventional karyotyping at the time of prior losses.