Author
Number of patients at the time of diagnosis
Controls
Diagnosis
Sperm chromatin integrity assay
Pretreatment DNA integrity in cancer patients versus controls
Meseguer et al. [19]
75
50 fertile sperm donors, 166 men attending infertility clinic
16 Hodgkin’s disease
6 NHL
6 leukemia
47 TGCT
Chromatin dispersion test
Significantly higher
Ribeiro et al. [20]
48
50 proven fertile controls
48 TGCT
TUNEL
Not significantly higher
Said et al. [17]
89
20 fertile sperm donors
39 TGCT
27 lymphoma
8 leukemia
10 colorectal cancer
3 skin cancer
2 brain cancer
SCSA
Significantly higher
McDowell et al. [18]
89
35 fertile sperm donors
SCSA
Not significantly higher
Said and colleagues found significantly higher levels of sperm DNA levels assessed by SCSA in 89 patients diagnosed with TGCT, lymphoma, leukemia, colorectal cancer, skin cancer, and brain cancer compared to 20 fertile sperm donors [17]. Contradictory conclusions were drawn by McDowell et al. who used the same assay to evaluate sperm DNA integrity in 89 cancer patients, in comparison to 35 fertile controls, and found no significant differences [18].
The chromatin dispersion test detected a significant increase in DFI among patients with various malignancies including TGCT [19], contrary to TUNEL which failed to detect any significant changes in DFI among patients with TGCT compared to normal controls [20].
The general conclusions that can be drawn from these studies is that cancer itself may negatively affect the sperm DNA integrity. When the available evidence is critically reviewed, several general limitations can be observed. All studies are performed in patients referred for fertility preservation, resulting in overpresentation of the most prevalent cancer diagnosis in males of reproductive age : TGCT, lymphomas, and leukemia. The contemporary literature compares sperm DNA fragmentation levels in cancer patients before the onset of gonadotoxic treatment to proven fertile controls or subfertile men attending reproduction clinics, although population-based controls may prove to be more appropriate controls. The assays used to evaluate sperm DNA integrity all require sperm concentrations of at least 2 × 106/mL excluding men with severely impaired spermatogenesis at the time of diagnosis. Finally, studied cohort s of cancer patients are relatively small. Even the two largest prospective studies among TGCT patients from the Centres d’Etudes et de Conservation des Oeufs et du Sperme humain CECOS consortium in France [21] and Gandini’s Laboratory of Seminology sperm bank in Italy [22] included, respectively, 129 and 254 TGCT patients but sperm DNA fragmentation analysis at the time of diagnosis was available in only 53 and 139 patients, respectively.
Follow-Up of Sperm Chromatin Integrity in Treated Cancer Patients
Studies that evaluated the sperm DNA damage at various follow-up intervals after gonadotoxic chemotherapy or radiotherapy exposure show inconsistent results (Table 2.2). Increased DNA damage using the comet assay was reported in a prospective study by O’Flaherty et al. [23] and O’Donovan [24], but other authors [21, 22, 25–30] reported only temporary decreased sperm chromatin integrity that improved after 12–24 months posttreatment intervals, acknowledging the variable time interval between exposure with heterogeneous chemo- and radiotherapy regimens and different sperm chromatin integrity assays as the most important limiting issues. The generalized consensus is that the chromatin structure in sperm improves following chemotherapy exposure, with a transient increase in sperm DNA fragmentation at 6 months posttreatment. It has been suggested that this phenomenon can be explained by clonal expansion of quiescent type A spermatogonia that survive a chemotherapeutic assault. These quiescent spermatogonia that repopulate the testicular parenchyma after gonadotoxic exposure may be less prone to sperm DNA damage. Surviving stem spermatogonia that are able to proliferate, differentiate, and produce spermatozoa lead to the recovery or partial recovery of spermatogenesis. The clonal offspring of these unaffected spermatogonia harbor improved chromatin integrity compared to pretreatment sperm produced at the time of cancer diagnosis.
Table 2.2
Summary of studies that evaluated sperm chromatin integrity at the time of semen cryopreservation before the onset of gonadotoxic treatment , compared to controls and posttreatment
|
Author |
N
Diagnosis |
N
Follow-up |
Controls |
Diagnosis |
Sperm chromatin integrity assay |
Interval between pre- and posttreatment |
Pretreatment DNA integrity compared to controls |
Posttreatment DNA integrity compared to pretreatment values |
|---|---|---|---|---|---|---|---|---|
|
O’Donovan [24] |
33 |
12 |
14 (proven fertile) |
8 HD
3 NHD
9 leukemia
13 TGCT |
Comet, chromatin condensation assay by propidium iodide |
3 and 6 months |
Significantly higher |
Impaired following cancer treatment |
|
Spermon et al. [30] |
22 |
22 |
13 (normozoospermic attending fertility clinic) |
22 TGCT |
TUNEL, chromatin condensation by CMA3 |
18–84 months |
Significantly higher |
No significant change within patients
Impaired compared to controls |
|
Smit et al. [31] |
127 |
45 |
22 (proven fertile) |
15 HD
5 NHD
25 TGCT |
SCSA |
6–40 months |
Not statistically higher
Only in NHL patients pretreatment DFI significantly higher compared to controls |
DFI decreased significantly compared to controls.
DFI at follow-up was significantly higher in TGCT patients who were treated with RT compared to patients treated with BEP alone |
|
O’Flaherty et al. [23] |
32 |
32 |
11 (healthy male volunteers) |
16 TGCT
16 HD |
COMET |
6, 12, 18, and 24 months |
Significantly higher |
Significant increase in sperm DNA damage at 6 months posttreatment, which remained elevated up to 18–24 months, compared to pretreatment and controls |
|
Stahl et al. [25] |
121 |
58 |
137 (fertile controls) |
84 TGCT
18 HD
9 NHL
3 CNS
2 sarcoma
5 other |
SCSA |
Median 3 years |
Significantly higher DFI in TGCT and HL patients, compared to controls |
No significant difference in pre- and posttreatment DFI |
|
Bujan et al. [21] |
53 |
41 |
51 (fertile controls) |
53 TGCT |
SCSA, TUNEL |
3, 6, 12, and 24 months in, respectively, 28, 36, 40, and 41 patients |
Significantly higher SCSA values but not TUNEL values |
No increased sperm DNA fragmentation or TUNEL values were found at 24 months posttreatment. Transient defects in chromatin condensation were shown at 6 months in patients treated with radiotherapy |
|
Bujan et al. [27] |
75 |
71 |
51 (fertile controls) |
57 HL
18 NHL |
SCSA, TUNEL |
3, 6, 12, and 24 months in, respectively, 38, 38, 43, and 4 patients |
Significantly higher |
Mean values of DFI decreased from 6 to 24 months after treatment, but always remained higher compared to controls. Mean TUNEL values decreased after treatment and were similar to control values from 6 to 24 months |
|
Paoli et al. [22] |
139 |
82 |
– |
139 TGCT |
SCSA |
3, 6, 9, 12, and 24 months in, respectively, 59, 54, 60, 75, and 75 patients |
– |
Impaired chromatin integrity at 3 and 6 months posttreatment, returning to baseline at 9 months and further improving at 12 and 24 months |
These results seem reassuring, but should be interpreted with caution. Evaluation of the fertility status in patients formerly treated for cancer is hampered by unpredictable recovery rates of spermatogenesis, selection bias, and loss to follow-up. As mentioned previously, sperm chromatin integrity assessment with SCSA can only be evaluated in patients with recovered spermatogenesis with sperm concentrations of at least 2 × 106/mL. Selection bias in former studies is further introduced by therapy-related comorbidity such as transient azoospermia, retrograde ejaculation, anorchia, and cancer-specific mortality that complicate follow-up of post-gonadotoxic exposure fertility. For example, in two recent large prospective studies, sperm chromatin integrity analysis was available at 24 months of follow-up in only 41 out of 129 TGCT patients [21], 42 out of 75 Hodgkin’s and non-Hodgkin’s lymphoma patients [27], and 82 out of 254 testicular cancer patients [22] despite a well-designed study protocol and great effort of the investigators. Results from these studies show consistent results with a significant reduction of sperm DNA damage at 24 months in comparison with baseline. Paoli et al. found the greatest levels of sperm DNA damage at 3 and 6 months posttreatment [22], whereas Bujan et al. found chromatin density to be increased at 6 months posttreatment in patients with seminomas treated with radiotherapy [21].
This finding is in agreement with a study by Smit et al. that suggested a greater induction of sperm chromatin integrity defects following radiotherapy [26].
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