Two human gonosomes or “sex chromosomes” are carried in diploid cells as one copy each of the X- and Y-chromosome in male cells and two copies of the X-chromosome in female cells. In humans, where imprinting of gonosomal genes exists and dosage-compensation appears to exist for only a small subset of genes, the presence of an extra sex chromosome may lead to clinically recognizable phenotypes, including Turner and Klinefelter syndrome, hypogonadism, etc.[9,46,47]. Since gain or loss of a single gonosomes may not fully impair embryonic or fetal survival, prenatal screening procedures have kept a close watch on the sex chromosome make-up of cell specimens [48–50].

As mentioned earlier, the high frequency of spontaneous abortions with trisomy 16 [15] prompted our probe development. We chose the large block of DNA satellite II as a target for our probe development, since this heterochromatic block represents a very highly reiterated sequence (Figure 2).

The map positions of BAC discussed in this communication, as well as the BAC insert sizes and the Genbank accession numbers of insert end sequences, are listed in Table 1. The Y-specific BAC clone RP11-242E13 targets a highly repeated DNA sequence [52], while the X-specific clone RP11-294C12 targets a cluster of repeated alpha satellite DNA [38]. Thus, this probe combination is expected to bind to multiple sites along the long arm of the human Y chromosome and throughout the centromeric region of the X chromosome [31,38].

The probes for the human X and Y chromosome labeled with digoxigenin and Spectrum Green, respectively, were used in a dual color FISH experiments. After incubation of the slide with rhodamine-conjugated antibodies against digoxigenin (Roche Molecular), the results showed unambiguous labeling of the target region on metaphase chromosomes and the expected number of red or green signals (Figure 3).

Results of the in situ hybridization of BAC-derived probes for chromosome 16 is shown in Figures 4 and 5 using metaphase spreads and tissue sections, respectively. Since the four selected probes showed virtually identical hybridization patterns, only one example is displayed here.

Chromosomal aberrations are very frequently seen in human oocytes and embryos and are often responsible for poor pregnancy outcomes in natural, as well as assisted, conceptions [13].

Chromosome 16 trisomy carries specific significance, as it is considered the most common aneuploidy at conception [53], with an incidence of ~1.5% in all clinically recognized pregnancies [12]. Trisomy 16, arising from new non-disjunctional events, is the most common cause of sporadic first trimester miscarriage (Figure 1) and generally not compatible with life [54]. And, as the rate of trisomies increases with maternal age, the proportion of miscarriages with numeric chromosomal errors, such as trisomy 16, increases the older the mother is [55].

In assisted conception, i.e., in vitro fertilization (IVF) and intracytoplasmatic sperm injection (ICSI), trisomy 16 or mosaic trisomy 16 in pre-implantation embryos are responsible for implantation failure, developmental arrest and miscarriage. Here, pre-implantation genetic diagnosis (PGD) represents a significant scientific advance for couples at risk of having children with heritable and debilitating genetic diseases. For couples who carry a balanced chromosomal translocation [25,56,57], PGD significantly decreases the risk of spontaneous miscarriage and significantly increases live-birth rates [58,59]. The technique described in this communication can be adapted to detect balanced translocations, when required by parents faced with such a problem. PGD algorithms often also include important new technologies, such as arrayCGH, for the detection of aneuploidy, balanced translocations and other chromosome anomalies [59].

Pre-implantation embryos not only present meiotically derived aneuploidy, but also post-zygotic chromosome segregation errors in the cleavage stage [56]. Mitotic chromosome mal-segregation may lead to exaggerated mosaicism, with a minority of embryos displaying a mixture of normal and aneuploid cells and most mosaic embryos presenting two or more abnormal cell lineages. Some embryos show failure of cellular mechanisms that control accurate chromosome segregation, becoming karyotypically unstable or “chaotic mosaics” [56,60]. Aneuploidy screening for embryo selection in assisted conception is developing fast, as it translates into improved clinical outcomes for infertile patients [61–63].

Individual BAC clones were grown overnight in up to 10 mL of Luria broth (LB) medium containing 12.5 μg/mL chloramphenicol (Sigma, St. Louis, MO, USA), and the DNA was isolated using a ZR BAC DNA Miniprep Kit (Zymo Research, Irvine, CA, USA). For the preparation of DNA pools, clones were grown individually and pooled prior to DNA isolation. The isolation of high molecular weight BAC DNAs was confirmed on 1% agarose gels and quantitated by spectrophotometry (Nanodrop 2000, Thermo Scientific, Wilmington, DE, USA). Probe DNAs were labeled with biotin-14-dCTP (part of the BioPrime kit, Invitrogen, La Jolla, CA, USA), digoxigenin-11-dUTP (Roche Diagnostics, Indianapolis, IN, USA) or Spectrum Green-dUTP (Abbott, Downers Grove, IL, USA) by random priming using a commercial kit (BioPrime Kit, Invitrogen) [23]. When incorporating fluorochrome-labeled deoxynucleoside triphosphates, such as Spectrum Green-dUTP, the dTTP to dUTP ratio in the reaction was adjusted to 2:1 [26,72–74].

Between 0.5 μL and 1 μL of each probe, along with of 0.25 μL human COT1™ DNA (1 mg/mL, Invitrogen) and 0.25 μL salmon sperm DNA (20 mg/mL, 3′-5′, Boulder, CO), were added to 3.9 μL of hybridization master mix (50% formamide (FA), 20% dextran sulfate, in 2 × SSC and 50mM phosphate buffer pH 7.0) in a total volume of 6.9 μL. Hybridization and detection of bound probes followed our published procedures [37,44,75,76]. Biotinylated and digoxigenin-labeled probes were detected with avidin-fluorescein isothiocyanate (FITC) (Vector, Burlingame, CA, USA; green fluorescence) and rhodamine-conjugated antibodies against digoxigenin (Roche Diagnostics; red fluorescence).

A pretreatment of the slides with RNase and pepsin followed by post-fixation with formalin buffer was required to reduce the background. Slides were soaked in 2 × SSC for 5 min at 21 °C on a shaking platform (20 × SSC is 3 M sodium chloride and 300mM tri-sodium citrate, pH 7.0). Slides were then placed into a Coplin jar with RNase solution (RNase solution: 50 μg/mL in 2 × SSC) and incubated for 15 min at 37 °C, then washed with 2 × SSC for 3 min on a shaker. Tissue sections were then treated with pepsin-buffer at 37 °C for 20 min (without agitation) (pepsin buffer: freshly prepared 50 μg/mL pepsin in 0.01 M HCl, pre-warmed to 37 °C). Sections were then washed twice for 5 min with 50 mM MgCl₂ (in 1 × PBS) at 21 °C before they were dehydrated in an ethanol series (70%, 85%, 100%; 3 min each) and air dried.

Fluorescence microscopy was performed on a Zeiss Axioskop or Axiovert.A1 microscope (Carl Zeiss GmbH, Oberkochen, Germany) equipped with a quadruple filter set for single wavelength excitation and observation of either DAPI, FITC, Texas Red/rhodamine/Cy3.5 or Cy5/Cy5.5 fluorescence (84000v2 Quad, ChromaTechnology, Brattleboro, VT, USA). Images were collected using an Axiocam HR camera (Carl Zeiss GmbH), and image processing was done using the Axiovision software (Carl Zeiss GmbH) or Photoshop software (Adobe, Inc., Mountain View, CA, USA).