Germ Cell Dysfunction Research Articles

Single‐cell nucleic acid sequencing reinforces to reveal signal‐ dependent pathways contributing to cellular heterogeneity

Single‐cell nucleic acid sequencing reinforces to reveal signal‐ dependent pathways contributing to cellular heterogeneity

Late Effects Following Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) From Alternative Donors In Patients With Fanconi Anemia (FA)

Hematopoietic Stem Cell Transplantation is the only curative approach for the hematological complications of Fanconi Anemia. However, patients with FA remain at increased risk for HSCT-specific late effects and FA-associated complications including solid tumors, which may affect their long-term outcome.We performed a single-institutional, retrospective review of late effects and long-term outcome in patients with FA who underwent HCST from alternative donors at MSKCC from March 1999 to December 2012. This review was approved by MSKCC IRB/Privacy Board. Twenty-two patients including 14 males and 8 females who underwent T-cell depleted HSCT and survived more than one-year post-transplant were eligible for inclusion in this study. The hematologic diagnoses of these patients were: severe aplastic anemia (SAA, n=11), myelodysplastic syndrome (MDS n=6) and acute myelogenous leukemia (AML n=5). Cytoreduction regimens included Total Body Irradiation (TBI), Cyclophosphamide (CY) and Fludarabine (FLU) (n=18) or Busulfan, CY and FLU (n=4). Donors were unrelated matched (n=9), unrelated mismatched (n=5) or related mismatched (n=8).The median post transplant follow-up for the 22 patients was 6.7 years (range 1.1- 15.1 years). Of the 22 patients who survived more than 1 year post HSCT, 20 are alive and 19 are alive disease-free, with overall survival and disease-free survival rates of 90.9% and 86.3% respectively for this patient group. Two female patients died of complications related to squamous cell carcinoma of the tongue and the vulvo-vaginal area respectively; one patient developed a relapse of MDS and is alive following a second transplant. Full donor chimerism was present in 18 of 20 evaluable patients. No chronic GVHD was observed in any of the 22 patients who received grafts from alternative donors. Of the 18 patients who received a TBI based regimen, 4 patients (22%) developed hypothyroidism post transplant, 7 patients (39%) developed insulin resistance (HOMA-IR >2.6) and one patient developed Insulin dependent diabetes mellitus, a year after transplant. Germ cell dysfunction was noted in 8 of 11 evaluable males and 4 of 5 evaluable females following a TBI based regimen. Among 4 patients receiving a Busulfan-based regimen, only one patient developed hypothyroidism. Prior to transplant, 13 patients (59%) had elevated ferritin levels (>500ng/ml) and seven of these 13 patients had received >20 transfusions. Post transplant, 12 patients had persisting high ferritin levels and six underwent phlebotomy. None of the patients had documented adverse cardiac or pulmonary late outcomes post-transplant.In summary, patients with FA are at risk for treatment-related late effects, and multidisciplinary follow up of patients with FA (including cancer screening) is essential for the early detection and management of late complications. Patients with FA who survive greater than one year post transplant have a very good outcome. However, these patients remain at risk for solid tumors of the head and neck and anogenital area post transplant. It is hoped that newer regimens that minimize radiation exposure and the use of T-cell depletion associated with minimal GvHD will further decrease the risks of solid tumors and other adverse late effects in patients with FA post transplant. Disclosures:No relevant conflicts of interest to declare.

Endocrine complications after busulphan and cyclophosphamide based hematopoietic stem cell transplant: A single tertiary care centre experience

Introduction:Endocrine complications are common after hematopoietic stem cell transplant (HSCT). Although HSCT is performed at various centers in India, no study is available for endocrine dysfunctions among them. This study was carried out with the objective to evaluate endocrine dysfunction among patients undergone HSCT in the past.Materials and Methods:We carried out a cross-sectional study in a 50 post-HSCT recipients (39 allogenic, 11 autologous). All relevant data were collected from patient's records. Samples for hormonal estimation were collected and stimulation tests for cortisol and growth hormone were interpreted based on peak values achieved during insulin tolerance test.Results:The mean age of patients was 26.3 ± 16.9 years (range 4-74). Adrenal insufficiency (AI) was present in 60%, hypergonadotropic hypogonadism (HH) in 60%, growth hormone deficiency (GHD) in 54%, hypothyroidism in 4%, hyperprolactinemia in 4%, new onset diabetes after transplant in 4%, and impaired fasting glucose in 6%. Multiple endocrine complications were common. GHD was present in 77% of children (n = 22) although height standard deviation score was not statistically different compared to those who didn’t have GHD. HH was present in 36% of children. In adults (n = 28), 36% had GHD, all females had HH, and 89% of males had HH. Germ cell dysfunction with compensated Leydig cell dysfunction was the most common pattern of HH in males. Fifteen patients had graft versus host disease (GVHD). GVHD had no bearing on development of endocrine deficiencies. AI was related to duration after and type of transplant, but was unrelated to steroid intake.Conclusions:Endocrine manifestations are common after HSCT; they can occur as early or late complications. All HSCT recipients should have endocrine evaluation as per prevailing guidelines.

Verification of Male Infertility Biomarkers in Seminal Plasma by Multiplex Selected Reaction Monitoring Assay

Seminal plasma is a promising biological fluid to use for noninvasive clinical diagnostics of male reproductive system disorders. To verify a list of prospective male infertility biomarkers, we developed a multiplex selected reaction monitoring assay and measured the relative abundance of 31 proteins in 30 seminal plasma samples from normal, nonobstructive azoospermia and post-vasectomy individuals. Median levels of some proteins were decreased by more than 100-fold in nonobstructive azoospermia or post-vasectomy samples, in comparison with normal samples. To follow up the most promising candidates and measure their concentrations in seminal plasma, heavy isotope-labeled internal standards were synthesized and used to reanalyze 20 proteins in the same set of samples. Concentrations of candidate proteins in normal seminal plasma were found in the range 0.1-1000 μg/ml but were significantly decreased in nonobstructive azoospermia and post-vasectomy. These data allowed us to select, for the first time, biomarkers to discriminate between normal, nonobstructive azoospermia, and post-vasectomy (simulated obstructive azoospermia) seminal plasma samples. Some testis-specific proteins (LDHC, TEX101, and SPAG11B) performed with absolute or nearly absolute specificities and sensitivities. Cell-specific classification of protein expression indicated that Sertoli or germ cell dysfunction, but not Leydig cell dysfunction, was observed in nonobstructive azoospermia seminal plasma. The proposed panel of biomarkers, pending further validation, could lead to a clinical assay that can eliminate the need for testicular biopsy to diagnose the category of male infertility, thus providing significant benefits to patients as well as decreased costs associated with the differential diagnosis of azoospermia.

Mono‐(2‐ethylhexyl) Phthalate Increases Spermatocyte Mitochondrial Peroxiredoxin 3 and Cyclooxygenase 2

Mono-(2-ethylhexyl) phthalate (MEHP), the biologically active metabolite of the plasticizer di-(2-ethylhexyl) phthalate, is a member of a class of chemical compounds with known adverse effects on the male reproductive system. Recent studies showed that oxidative stress and mitochondrial dysfunction in germ cells may contribute to phthalate-induced disruption of spermatogenesis. To determine whether the redox-protein mitochondrial thioredoxin-dependent peroxidase, peroxiredoxin 3 (Prx3), may be a component of germ cell homeostasis mechanisms, this study first examined the physiologic relevance of Prx3 in the rodent testis by determining its cell-specific expression. Our findings show that prx3 mRNA is expressed in a developmental, cell-specific manner in rat Leydig cells, Sertoli cells, and germ cells; among mouse germ cells, prx3 expression was highest in spermatocytes, findings consistent with those in rat. In mouse meiotic spermatocytes, Prx3 was strikingly localized at the nuclear perimeter and cytoplasm, findings suggestive of a direct role for Prx3 in determining spermatocyte response to toxicants. To better define the mechanisms involved in male germ cell dysfunction following phthalate exposure, an immortalized mouse spermatocyte-derived germ cell line, GC-2spd(ts), was exposed to MEHP (24 hours; 100 and 200 microM). We determined whether Prx3 and cyclooxygenase-2 (COX-2), pivotal proteins involved in oxidative stress responses in spatially restricted subcellular domains, were affected. Mitochondrial Prx3 and mitochondrial and cytosolic COX-2 significantly increased following 200 microM MEHP treatment; proliferation was inhibited without inducing cell death. Using this germ cell model, the data suggest that changes in cellular oxidation-reduction (redox) homeostasis in the germline can accompany MEHP exposure, disrupting mitochondrial antioxidant defenses, despite absence of phthalate-induced apoptosis.

From the Cover: Abnormal development of Purkinje cells and lymphocytes in Atm mutant mice

The autosomal recessive disease ataxia–telangiectasia (A-T) is characterized by the early onset of progressive ataxia, ocular telangiectasias, and susceptibility to infections and tumors (1, 2). A-T is caused by mutations in the ATM gene (3). The ATM protein encodes a serine/threonine kinase and induces p53, c-Abl, and Brca1 activation after DNA damage (4–8). ATM-deficient cells exhibit cell-cycle checkpoint defects, resulting in hypersensitivity to genotoxic stress (9). However, it is less clear whether the abnormal cellular physiology of various ATM-deficient cell types also results from defective DNA repair. For example, potassium-mediated depolarization is attenuated in A-T fibroblasts (10), and calcium mobilization and phospholipase-Cγ activation are reduced in A-T B cells after antigen receptor stimulation (11). Altered cellular physiology may independently contribute to the clinical manifestations of A-T. Several lines of Atm mutant mice recapitulate numerous aspects of A-T (12–15). These Atm mutant mice exhibit growth retardation, immune system defects including defects in T cell maturation, germ cell dysfunction, and increased sensitivity to ionizing radiation (IR). Although behavioral abnormalities were observed (14), altered cerebellar histology or degeneration was not reported (12–15). The diversity of ATM mutations and the variability in the clinical progression of A-T raises questions concerning how individual ATM mutations and genetic factors contribute to the clinical severity of A-T. Likewise, independently generated Atm-deficient mice may also vary in their phenotypic manifestations, reflecting mutagenesis strategies and/or genetic backgrounds. Murine Atm is expressed in both embryonic and adult tissues and likely functions at multiple developmental stages (16). It remains unclear whether Atm deficiency alters developmental processes and how this may contribute to neurodegeneration and immunodeficiency in A-T patients. Clinical studies documenting the early onset of ataxia (17–19) and early abnormalities in Purkinje cell morphology and localization (20, 21) support the hypothesis that ATM function is essential for normal cerebellar development. To further evaluate the potential Atm functions, we have generated and characterized a line of Atm mutant mice.

Gonadal function in young patients successfully treated for Hodgkin disease.

Gonadal function in pediatric and young adult survivors of Hodgkin disease is not very well defined. This study evaluates the outcome following the Multiple Drug Protocol (MDP) and the results are compared to the published experience. Ovarian and testicular function was assessed in 65 patients (36 males) with Hodgkin disease in first or second complete remission after treatment with either radiation (RT, n = 13), chemotherapy (CT, n = 9), or both (n = 43). Chemotherapy consisted of six cycles of the MDP (doxorubicin, procarbazine, prednisone, vincristine, and cyclophosphamide). Median age at diagnosis was 13.1 years (range, 2.4-22.6) and median age at evaluation was 22.6 years (range, 15.1-33.7), which was 6.7 years (range, 2.0-19.8) after the completion of all treatments. For the purpose of analysis, patients were divided into three groups: group A, patients who received only RT that did not include the pelvis (8 females, 5 males); group B, patients who received CT but no pelvic RT (15 females, 25 males); and group C, patients who received CT plus pelvic RT (6 females, 6 males). All patients progressed spontaneously through puberty and evaluable patients were found to be sexually mature (Tanner stage IV and V). Serum follicle stimulating hormone (FSH) was increased in 0/5, 13/25, and 5/6 and testicular volume was decreased in 1/3, 4/11, and 2/3 group A, B, and C male patients, respectively. Leydig cell dysfunction was uncommon; 91% and 88% of males had normal serum concentrations of luteinizing hormone (LH) and testosterone, respectively. FSH and LH were increased in 0/8, 3/15, and 2/6 group A, B, and C female patients, respectively, at last follow-up, indicating a 17% prevalence of ovarian dysfunction. Serial data in seven females whose initial levels of FSH/LH were elevated revealed normalization in four. Six females delivered eight normal children. The majority of males who received CT +/- RT have evidence of germ cell dysfunction, while Leydig cell function is unaffected in most. In females, although abnormal function early after the end of treatment was observed, ovarian function remained or returned to normal in most young women. Thus, in females the results of hormone testing performed early after treatment may not be predictive of their eventual reproductive potential.

GONADAL FUNCTION AFTER BONE MARROW TRANSPLANTATION (BMT) FOR ACUTE LEUKEMIA: THE IMPACT OF HYPERFRACTIONATED TOTAL BODY IRRADIATION (TBI) BEFORE PUBERTY

There is only limited information on gonadal function after bone marrow transplant (BMT) during childhood. The existing data is based primarily on the use of single dose or fractionated total body irradiation (TBI). In the present study, we evaluated gonadal function in 33 patients (18 females, 15 males with ALL, n = 18 and AML, n=15), treated with hyperlractionaled TBI before puberty. Median age at diagnosis was 4.8 years (0.4-11.2 yrs), median age at BMT was 7.3 years (3.8-11.5 yrs) and median age at last evaluation of gonadal function was 17.1 years (9.3-22 yrs). Transplant cytoreduction consisted of hyperfractionated TBI (125 cGy TID for total dose of 1375 to 1500 cGy) and 120 mg/kg of Cytoxan; male patients received an additional 400 cGy testicular boost. Fifteen of 33 patients had received cranial irradiation as part of previous treatment of their underlying disease (1000-2400 cGy) but none of the males had received testicular irradiation. Five of 15 boys had elevated baseline LH, 9 of 15 had elevated baseline FSH, and 2 of 15 had elevated LH and low testosterone requiring testosterone treatment to complete pubertal development. Fourteen of 18 females had elevated baseline levels of LH and/or FSH; nevertheless, 8 females experienced spontaneous menarche at a mean age of 12.7 years (9.4-15.8 yrs). All 8 patients including 4 with elevated LH/FSH have regular menses. One female presented with precocious puberty. Eight of 18 females have required hormone replacement therapy. In conclusion, our data suggests hyperfractionated TBI results in preservation of Leydig cell function in males, while the incidence of germ cell dysfunction in males and ovarian failure in females appears to be similar to that reported after single dose or fractionated TBI.

Effects of radiation on testicular function in long-term survivors of childhood acute lymphoblastic leukemia: a report from the Children Cancer Study Group.

Testicular function was evaluated in 60 long-term survivors of childhood acute lymphoblastic leukemia (ALL). All the patients were treated on two consecutive Children Cancer Study Group protocols and received identical chemotherapy and either 18 or 24 Gy radiation therapy (RT) to one of the following fields: craniospinal plus 12 Gy abdominal RT including the gonads (group 1); craniospinal (group 2); or cranial (group 3). The median age at the time of their last evaluation was 14.5 years (range, 10.5 to 25.7), which took place a median of 5.0 years (range, 1 to 10.3) after discontinuing therapy. The incidence of primary germ cell dysfunction as judged by raised levels of follicle-stimulating hormone (FSH) and/or reduced testicular volume was significantly associated with field of RT; 55% of group 1, 17% of group 2, and 0% of group 3 were abnormal (P = .002). Leydig cell function, as assessed by plasma concentrations of luteinizing hormone (LH) and testosterone, and pubertal development, was unaffected in the majority of subjects regardless of RT field. These data indicate that in boys undergoing therapy for ALL, germ cell dysfunction is common following testicular irradiation and can occur following exposure to scattered irradiation from craniospinal RT. In contrast, Leydig cell function appears resistant to direct irradiation with doses as high as 12 Gy.

Male Reproductive Dysfunction: Diagnosis and Management of Hypogonadism, Infertility, and Impotence,

Reproductive Dysfunction is the seventh in an excellent series entitled Basic and Clinical Endocrinology . This volume is a compilation of individual articles on a variety of subjects relating to reproductive and sexual dysfunction in the male that has been edited and selected with great care by the two very competent editors. The book is organized into 26 chapters, under five general subject headings. These headings include the following: Normal Anatomy, Physiology, and Biochemistry; Leydig Cell Germ Cell Dysfunction; Psychogenic Sexual and Male Contraception. A majority of the text is devoted to the first three subject categories, and the final two seem to be added for the sake of completion. Certainly, the first three sections are most appropriate to the series theme of basic and clinical endocrinology and provide an excellent and thorough review of those subjects. The defined purpose for this volume was to consolidate accumulated knowledge