Fig 1.
Timing of semen collection showing the stages of spermatogenesis at which the sperm in the ejaculate were during the patients’ treatment with NOVP.
The days noted for exposure of cells as spermatogonia, spermatocytes, spermatids, and mature sperm are based on the timing of cells exposed to an administered agent at these stages to appear as sperm in the ejaculate (see S1 Fig in [42]). This timing is based on the kinetics of spermatogenesis in the testis [59], an updated system for staging cells in human spermatogenesis [60], and the appearance of labeled sperm in the ejaculate [61]. Although the state of knowledge of the kinetics of the human spermatogonial compartments is still in a state of flux [62], we selected the time for sperm exposed as stem cells to appear in the ejaculate (87 days) as the time after chemotherapy when sperm count returns to pretreatment levels after chemotherapies with agents that kill differentiating spermatogonia but not stem cells [40,63]. The individual vertical arrows and brackets are aligned with cell types exposed to NOVP treatment for each sample or groups of patients; samples A1, B1, C1 and D1 were collected before the patients started treatment. Table 1 provides patient-specific information on the start and end of NOVP therapy as well doses and durations of gonadal exposure to ionizing radiation. The black box at the end of the in spermatocytes phase (at ~35 days) represents the two meiotic divisions that occur as the male germ cells differentiate into haploid spermatids and subsequently progress through spermiogenesis.
Fig 2.
DNA probe and multi-color labeling strategy for the human AM8 sperm FISH assay.
This fluorescence labeling strategy was used for the simultaneous detection of sperm carrying duplications or deficiencies in chromosome arm 1p, aneuploidies involving chromosomes 1 and 8, sperm diploidy and complex chromosomal rearrangements (see Methods for scoring details). Approximately 10,000 sperm nuclei were scored per semen sample and nuclei were scored only when a sperm tail could be detected under phase contrast illumination. a (Van Hummelen et al., 1996); b Oncor (Gaithersburg, MD), c Vysis (Downer Grove, IL).
Table 1.
Twenty semen samples from nine Hodgkin Lymphoma patients, their individual therapy plans, timing of semen specimens, and semen parameters.
Fig 3.
Effects of time after NOVP treatment on the fractions of sperm carrying structural aberrations (Panels A, B) or numerical abnormalities (Panel C, D). Six HL patients (C, D, E, F, G, I Table 1) provided repeated semen specimen at various times before, during, and after treatment with NOVP. Zero time represents the end of chemotherapy; the samples plotted at negative times in panels B and D represents that patient’s pretreatment value. The Y-axis represents the frequency of cells with chromosomal abnormalities (per 10,000 cells examined). Straight lines connect values of sperm samples donated by the same patient at various times during and after treatment and the patients are identified by the italicized letters next to the plot. The black symbols in the lower left or right corners represent the average values for historical controls (open triangles) and the pretreatment group (filled circles).
Table 2.
AM8 FISH analyses of sperm carrying structural chromosomal aberrations in Hodgkin Lymphoma patients before, during and after treatment with NOVP chemotherapya.
Fig 4.
Effects of time after NOVP treatment on the fraction of sperm carrying specific classes of chromosomal abnormalities.
Structural chromosomal aberrations (Panel A), sperm with complex chromosomal abnormalities (Panel B), diploid sperm (Panel C), aneuploid sperm (Panel D). Semen specimens were assigned to an exposure group (Fig 1 and Table 1). Statistical analyses are based on one-way ANOVA and post hoc tests with p values: * <0.05, **<0.01, ***<0.005, **** 0.001, no marking = Not significant.
Table 3.
AM8 FISH analyses of sperm carrying complex chromosomal abnormalities or sperm diploidy in Hodgkin lymphoma patients before, during and after treatment with NOVP chemotherapya.
Table 4.
AM8 FISH analyses of sperm aneuploidy in Hodgkin Lymphoma patients before, during and after treatment with NOVP chemotherapya.