Recommendations for assessing ovarian health and fertility potential in survivors of childhood cancer.

Most children diagnosed with cancer survive for many years after treatment. However, the fertility potential of these patients may suffer due to their oncologic therapies. Certain chemotherapies and radiation are more likely to be detrimental to gonadal function, and put patients at risk of acute or premature ovarian failure. Prepubertal cancer patients will need different follow-up and testing from their post-pubertal counterparts. This review will present evidence to help patients, family members and physicians determine who is most at risk of ovarian insufficiency and how to monitor childhood cancer survivors. It will discuss the impact of age at diagnosis and cancer therapies on reproductive outcomes, and guide caregivers and patients on monitoring gonadal function after therapy.

Fluorescence- and magnetic-activated cell sorting strategies to isolate and enrich human spermatogonial stem cells.

OBJECTIVE: To determine the molecular characteristics of human spermatogonia and optimize methods to enrich spermatogonial stem cells (SSCs). DESIGN: Laboratory study using human tissues. SETTING: Research institute. PATIENT(S): Healthy adult human testicular tissue. INTERVENTION(S): Human testicular tissue was fixed or digested with enzymes to produce a cell suspension. Human testis cells were fractionated by fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS). MAIN OUTCOME MEASURE(S): Immunostaining for selected markers, human-to-nude mouse xenotransplantation assay. RESULT(S): Immunohistochemistry costaining revealed the relative expression patterns of SALL4, UTF1, ZBTB16, UCHL1, and ENO2 in human undifferentiated spermatogonia as well as the extent of overlap with the differentiation marker KIT. Whole mount analyses revealed that human undifferentiated spermatogonia (UCHL1+) were typically arranged in clones of one to four cells whereas differentiated spermatogonia (KIT+) were typically arranged in clones of eight or more cells. The ratio of undifferentiated-to-differentiated spermatogonia is greater in humans than in rodents. The SSC colonizing activity was enriched in the THY1dim and ITGA6+ fractions of human testes sorted by FACS. ITGA6 was effective for sorting human SSCs by MACS; THY1 and EPCAM were not. CONCLUSION(S): Human spermatogonial differentiation correlates with increased clone size and onset of KIT expression, similar to rodents. The undifferentiated-to-differentiated developmental dynamics in human spermatogonia is different than rodents. THY1, ITGA6, and EPCAM can be used to enrich human SSC colonizing activity by FACS, but only ITGA6 is amenable to high throughput sorting by MACS.

Eliminating malignant contamination from therapeutic human spermatogonial stem cells.

Spermatogonial stem cell (SSC) transplantation has been shown to restore fertility in several species and may have application for treating some cases of male infertility (e.g., secondary to gonadotoxic therapy for cancer). To ensure safety of this fertility preservation strategy, methods are needed to isolate and enrich SSCs from human testis cell suspensions and also remove malignant contamination. We used flow cytometry to characterize cell surface antigen expression on human testicular cells and leukemic cells (MOLT-4 and TF-1a). We demonstrated via FACS that EpCAM is expressed by human spermatogonia but not MOLT-4 cells. In contrast, HLA-ABC and CD49e marked >95% of MOLT-4 cells but were not expressed on human spermatogonia. A multiparameter sort of MOLT-4-contaminated human testicular cell suspensions was performed to isolate EpCAM+/HLA-ABC-/CD49e- (putative spermatogonia) and EpCAM-/HLA-ABC+/CD49e+ (putative MOLT-4) cell fractions. The EpCAM+/HLA-ABC-/CD49e- fraction was enriched for spermatogonial colonizing activity and did not form tumors following human-to-nude mouse xenotransplantation. The EpCAM-/HLA-ABC+/CD49e+ fraction produced tumors following xenotransplantation. This approach could be generalized with slight modification to also remove contaminating TF-1a leukemia cells. Thus, FACS provides a method to isolate and enrich human spermatogonia and remove malignant contamination by exploiting differences in cell surface antigen expression.

Oocyte cryopreservation in a patient with sickle cell disease prior to hematopoietic stem cell transplantation: first report.

PURPOSE: To report the first occurrence of successful ovarian stimulation, oocyte retrieval and oocyte cryopreservation for fertility preservation in an adolescent with severe sickle cell disease scheduled to undergo a hematopoietic stem cell transplant METHODS: Case report. RESULTS: A 19 year old female with severe sickle cell disease presented for fertility preservation counseling prior to hematopoietic stem cell transplantation. She ultimately underwent ovarian stimulation using a minimal stimulation GnRH antagonist protocol resulting in the successful banking of oocytes prior to transplant. The unique hazards associated with ovarian stimulation in patients with sickle cell disease, such as thrombosis and vaso-occlusive events, are discussed and the methods undertaken to minimize these risks are described. CONCLUSIONS: Controlled ovarian hyperstimulation and oocyte banking for fertility preservation is feasible in young women with sickle cell disease requiring hematopoietic stem cell transplant and deserves further investigation. Given the elevated risk of thrombosis and predisposition to painful vaso-occlusive events, controlled ovarian hyperstimulation in patients with sickle cell disease is not straightforward and requires a multi-disciplinary team approach to adequately address and minimize the risks in this unique patient population.

Is a premature rise in luteinizing hormone in the absence of increased progesterone levels detrimental to pregnancy outcome in GnRH antagonist in vitro fertilization cycles.

OBJECTIVE: To determine whether a premature rise in the level of luteinizing hormone (LH) without a concomitant increase in the level of progesterone is detrimental to pregnancy outcome in flexible gonadotropin-releasing hormone (GnRH) antagonist in vitro fertilization (IVF) cycles. DESIGN: Retrospective cohort study. SETTING: Large academic IVF practice. PATIENT(S): All women undergoing their first GnRH-antagonist in vitro fertilization cycle at the University of Pittsburgh Medical Center between January 1, 2007, and October 1, 2010. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Primary outcome measures included the overall pregnancy rate, live-birth/ongoing pregnancy rate, and early pregnancy loss compared between cycles with and without a premature rise in the LH level; secondary outcome measures included the number of oocytes retrieved and embryos obtained during the cycle. RESULT(S): Overall, 451cycles were included in the analysis. Patients who experienced a premature rise in LH had improved ovarian reserve parameters and had more oocytes retrieved compared with patients who did not experience an LH rise. However, the premature rise in LH was associated with a decreased ongoing pregnancy rate despite these improvements in cycle parameters. CONCLUSION(S): The incidence of premature LH rise without a concomitant progesterone increase is more common than previously reported and is associated with decreased rates of ongoing pregnancy.

Endometriosis and the adolescent.

The majority of women with endometriosis report symptoms starting in adolescence, yet endometriosis is often a delayed diagnosis in this patient population. Given that endometriosis is felt to be a progressive disease with increasing morbidities over time, such as structural defects and infertility, being more aggressive with pursuing the diagnosis is warranted. Once the diagnosis of endometriosis is made, various medical and surgical treatment modalities are available, and this article will review the most current treatment recommendations.

Clomiphene citrate and intrauterine insemination: analysis of more than 4100 cycles.

OBJECTIVE: To evaluate the outcomes of a large cohort of patients undergoing fertility treatment with clomiphene citrate and intrauterine insemination. DESIGN: A retrospective cohort study. SETTING: Boston IVF, a large university-affiliated reproductive medicine practice. PATIENT(S): A total of 4,199 cycles performed in 1,738 infertility patients between September 2002 and July 2007. INTERVENTION(S): All patients received oral clomiphene citrate, and patients with completed cycles had intrauterine insemination performed. MAIN OUTCOME MEASURE(S): Cumulative and per cycle pregnancy rates achieved among subsets of patients defined by age, completed cycles, and intention to treat (ITT). RESULT(S): For women under age 35 years, 2,351 cycles were initiated in 983 patients. A total of 238 pregnancies ensued, yielding a pregnancy rate (PR) per completed cycle of 11.5% and 10.1% per cycle initiated with ITT. In women aged 35-37 years, 947 cycles in 422 women lead to a PR per completed cycle and ITT of 9.2% and 8.2%, respectively. For patients aged 38-40 years, 614 cycles in 265 women lead to a PR per completed cycle and ITT of 7.3% and 6.5%, respectively. In women aged 41-42 years, 166 cycles in 81 patients lead to a PR per completed cycle and ITT of 4.3% and 3.6%, respectively. For women above age 42 years, 120 cycles in 55 patients lead to a PR per completed cycle and ITT of 1.0% and 0.8%, respectively. On a per-patient treated basis, cumulative PRs were 24.2% under age 35, 18.5% ages 35-37, and 15.1% ages 38-40, whereas only 7.4% ages 41-42 and 1.8% above 42 became pregnant (one pregnancy in 55 patients). CONCLUSION(S): As anticipated, younger patients have a higher PR per cycle than older patients. The PR per cycle for patients who initiate only one or only two treatment cycles is notably higher than the corresponding per cycle rates for cycles 3 through 9. The drop in success per patient among 41- and 42-year-olds is sharp, but the exceptionally low success rate above age 42 suggests that CC with IUI has virtually no place in their treatment.

Nitric-oxide-induced depolarization of neuronal mitochondria: implications for neuronal cell death.

Nitric oxide (NO(*)) has known toxic effects on central nervous system neurons. This study characterized the effect of NO(*) on mitochondrial membrane changes by exploring the relationship among NO(*), excitatory receptor activation, and the induction of peroxynitrite, a highly toxic NO(*) reactant, to neuronal injury. Cultured rat cortical neurons were exposed to the NO(*) generator, diethylenetriamine/nitric oxide adduct, and were examined for signs of cell death, mitochondrial membrane potential changes (Deltapsi(m)), and the induction of a mitochondrial permeability transition (MPT). Neurons were also examined for nitrotyrosine (NT) immunoreactivity, a marker of reactive nitrogen species (RNS) formation. Neurons exposed to NO(*) or to N-methyl-D-aspartate (NMDA) exhibited similar rapid depolarization of mitochondria, which was prevented by an NMDA receptor antagonist. Electrophysiological studies demonstrated NO(*) potentiation of NMDA-induced NMDA receptor currents. NO(*) and NMDA-treated neurons had evidence of mitochondrial-specific NT immunoreactivity that was prevented by a SOD/catalase mimetic (EUK-134). EUK-134 treatment reduced both NO(*) and NMDA-induced NT formation and neuronal cell death. EUK-134 did not prevent NO-induced Deltapsi(m) but partially prevented NMDA-induced Deltapsi(m) loss. Although NO(*) and NMDA both induced MPT and MPT inhibitors prevented NO-induced Deltapsi(m), they did not result in significant neuroprotection, in contrast to treatment designed to decrease peroxynitrite formation. These data suggest that NO-induced NMDA receptor activation is closely linked to intramitochondrial NO-peroxynitrite/RNS formation and thereby acts as a major mediator of neuronal cell death.