Cryopreservation of human ovarian tissue using dimethylsulphoxide and propanediol-sucrose as cryoprotectants

Pieces of ovarian cortical tissue (03–2 mm in diameter)were obtained during gynaecological operations by biopsy oras a result of oophorectomy from 19 women aged 19–44 years.The tissue was frozen in a programmable freezer using one oftwo different cryoprotectants, either 1.5 M dimethylsulphoxide(DMSO), or a combination of 1, 2-propanediol (1.5 M) and sucrose(0.1 M). After cryopreservation lasting from 24 h to 5 weeks,the ovarian pieces were thawed and studied histologically. Specimenstaken before and after cryopreservation with either pro-tectantshowed no signs of tissue necrosis. Follicles at similar developmentalstages were found before and after freezing. The proportionsof follicles showing signs of atresia, 27% in the non-frozentissue and 19% in the frozen-thawed tissue, were not significantlydifferent. Oocytes, too, had the same appearance after freezingand thawing with both cryoprotectants as was seen in the specimenstaken before freezing. These results suggest that cryopreservationof human ovarian tissue is feasible. However, the normalityof the oocytes taken from tissue which has been frozen stillneeds to be established. Cryopreservation of ovarian tissuewould be potentially an excellent method for storage of humanoocytes once methods for their maturation in vitro have beendeveloped.     

Extracellular matrix improves survival of both stored and fresh human primordial and primary ovarian follicles in long-term culture

Ovarian cortical tissue was obtained during gynaecological operations by biopsy or after oophorectomy from 20 women aged 25-42 years. It was placed in organ culture, either fresh or following thawing after cryopreservation, for 1-4 months. The tissue was cut in slices 0.1-0.3 mm in diameter and transferred to 12 mm inserts in 24-well culture plates. These slices were cultured for 4-21 days in either alpha minimum essential medium (alpha-MEM) or Earle's balanced salt solution with added pyruvate. Both media were supplemented with 10% human serum, insulin, gonadotrophins and antibiotics. Half of the inserts were precoated with extracellular matrix (Matrigel). Histological samples revealed that there were viable, non-atretic, primordial, primary and secondary follicles in all the cultures. Mitoses were seen in the granulosa cells of the secondary follicles. Although the proportion of atretic follicles increased during culture, non-atretic follicles were still present after 21 days. After 4-11 days the proportion of viable follicles was significantly higher when cultured in Earle's solution supplemented with pyruvate, than when cultured in MEM (77 versus 38%, P < 0.001). In cultures with extracellular matrix the proportion of viable follicles was significantly higher after 10-15 days than it was without matrix (85 versus 19%, P < 0.001). Culture after thawing frozen ovarian tissue did not affect the density or the proportion of the viable follicles. Two-thirds of follicles in cryopreserved tissue were viable after 10-15 days in culture. The results indicate that it is possible to culture human primary and primordial follicles in vitro, and follicles in cryopreserved tissue are viable.      

Turner's syndrome and fertility: current status and possible putative prospects.

Women with Turner's syndrome should be carefully followed throughout life. Growth hormone therapy should be started at age 2-5 years. Hormone replacement therapy for the development of normal female sexual characteristics should be started at age 12-15 years and continued for the long term to prevent coronary artery disease and osteoporosis. Most women with Turner's syndrome have ovarian dysgenesis; therefore, they are usually infertile, and in very rare cases have spontaneous menses followed by early menopause. Only 2% of the women have natural pregnancies, with high rates of miscarriages, stillbirths and malformed babies. Their pregnancy rate in oocyte donation programmes is 24-47%, but even these pregnancies have a high rate of miscarriage, probably due to uterine factors. A possible future prospect is cryopreservation of ovarian tissue containing immature follicles before the onset of early menopause, but methods of replantation and in-vitro maturation still need to be developed. Should these autologous oocytes indeed be used in the future, affected women would need to undergo genetic counselling before conception, followed by prenatal assessment.     

The Role of Arachidonic Acid Regulatory Enzymes in Colorectal Disease

PURPOSE Nonsteroidal anti-inflammatory drugs have a wide ranging effect on diseases of the colon and rectum. Interestingly, nonsteroidal anti-inflammatory drugs seem to play a beneficial role in colorectal cancer chemoprevention and adenoma regression, but may have a deleterious effect in inflammatory bowel disease. Prostaglandin inhibition is central to both the beneficial and toxic effects of this class of drugs. Arachidonic acid metabolism is essential to prostaglandin synthesis.METHODS A Medline search using “nonsteroidal anti-inflammatory drugs,” “colon cancer,” “inflammatory bowel disease,” “colitis,” “COX inhibitors,” “arachidonic acid,” and “chemoprevention” as key words was performed for English-language articles. Further references were obtained through cross-referencing the bibliography cited in each work.RESULTS Based on numerous studies, nonsteroidal anti-inflammatory drugs have a beneficial role in colon cancer and colonic adenomas. However, they have been reported to have a deleterious effect on the colon in inflammatory bowel disease and have been shown to cause colitis. Nonsteroidal anti-inflammatory drugs work via multiple pathways, some well defined, and others unknown. CONCLUSIONS In the new millennium, nonsteroidal anti-inflammatory drugs may be used for chemoprevention of colorectal and other cancers. In addition, they may be used in combination with surgery and chemotherapy to primarily treat colorectal carcinoma. Undoubtedly, the use of novel cyclooxygenase inhibitors with less of a toxicity profile will allow more widespread use of nonsteroidal anti-inflammatory drugs for a variety of diseases. The future of this class of drugs is promising.     

Biological activity of follistatin isoforms and follistatin-like-3 is dependent on differential cell surface binding and specificity for activin, myostatin, and bone morphogenetic proteins

Follistatin (FST) and FST-like-3 (FSTL3) are activin-binding and neutralization proteins that also bind myostatin. Three FST isoforms have been described that differ in tissue distribution and cell-surface binding activity, suggesting that the FST isoforms and FSTL3 may have some nonoverlapping biological actions. We produced recombinant FST isoforms and FSTL3 and compared their biochemical and biological properties. Activin-binding affinities and kinetics were comparable between the isoforms and FSTL3, whereas cell-surface binding differed markedly (FST288 > FST303 > FST315 > FSTL3). Inhibition of endogenous activin bioactivity, whether the FST isoforms were administered endogenously or exogenously, correlated closely with surface binding activity, whereas neutralization of exogenous activin when FST and FSTL3 were also exogenous was consistent with their equivalent activin-binding affinities. This difference in activin inhibition was also evident in an in vitro bioassay because FST288 suppressed, whereas FST315 enhanced, activin-dependent TT cell proliferation. Moreover, when FSTL3, which does not associate with cell membranes, was expressed as a membrane-anchored protein, its endogenous activin inhibitory activity was dramatically increased. In competitive binding assays, myostatin was more potent than bone morphogenetic proteins (BMPs) 6 and 7, and BMPs 2 and 4 were inactive in binding to FST isoforms, whereas none of the BMPs tested competed with activin for binding to FSTL3. Neutralization of exogenous BMP or myostatin bioactivity correlated with the relative abilities of the isoforms to bind cell-surface proteoglycans. These results indicate that the differential biological actions among the FST isoforms and FSTL3 are primarily dependent on their relative cell-surface binding ability and ligand specificity.     

Osteogenesis imperfecta and Ledderhose disease: Is there a link? A report of two Tunisian siblings

IntroductionOsteogenesis imperfecta (OI) or “brittle bone disease” is a rare genetic disorder tissue due to an abnormal production of type I collagen. It can cause hearing loss, dentinogenesis imperfecta, heart failure, spinal cord problems and permanent deformities. Ledderhose’s disease or Plantar fibromatosis (PF) is also a rare condition that may be caused by an abnormal proliferation of collagen tissue. It is a benign fibroblastic proliferative disorder in which fibrous nodules may develop in the plantar aponeurosis, more specifically on the medial plantar side of the foot arch and on the forefoot region. Rare cases of association of polyfibromatosis with keloids or arthritis have been reported.Case reportWe report two Tunisian brothers aged 17 and 14 years old, from a first-degree consanguineous marriage and with medical history of multiple bone fractures. The elder brother has medical history of juvenile arthritis treated with methotrexate and etanercept. Clinical examination of both patients showed blue sclera, bone deformities with humeral and femoral curvature and unequal leg length. They were diagnosed with OI and received bisphosphonate to prevent further fractures. One year later, they developed lumps under the sole skin of their feet. They were diagnosed with PF and received conservative treatment including non-steroidal anti-inflammatory drugs, physiotherapy and orthotic support.ConclusionTo the best of our knowledge, the association of PF with OI has never been reported. Both diseases suggest an association with abnormalities involving collagen. A question remains currently with no answer: is it a fortuitous association?     

NADPH oxidase DUOX1 promotes long-term persistence of oxidative stress after an exposure to irradiation

Ionizing radiation (IR) causes not only acute tissue damage, but also late effects in several cell generations after the initial exposure. The thyroid gland is one of the most sensitive organs to the carcinogenic effects of IR, and we have recently highlighted that an oxidative stress is responsible for the chromosomal rearrangements found in radio-induced papillary thyroid carcinoma. Using both a human thyroid cell line and primary thyrocytes, we investigated the mechanism by which IR induces the generation of reactive oxygen species (ROS) several days after irradiation. We focused on NADPH oxidases, which are specialized ROS-generating enzymes known as NOX/DUOX. Our results show that IR induces delayed NADPH oxidase DUOX1-dependent H₂O₂ production in a dose-dependent manner, which is sustained for several days. We report that p38 MAPK, activated after IR, increased DUOX1 via IL-13 expression, leading to persistent DNA damage and growth arrest. Pretreatment of cells with catalase, a scavenger of H₂O₂, or DUOX1 down-regulation by siRNA abrogated IR-induced DNA damage. Analysis of human thyroid tissues showed that DUOX1 is elevated not only in human radio-induced thyroid tumors, but also in sporadic thyroid tumors. Taken together, our data reveal a key role of DUOX1-dependent H₂O₂ production in long-term persistent radio-induced DNA damage. Our data also show that DUOX1-dependent H₂O₂ production, which induces DNA double-strand breaks, can cause genomic instability and promote the generation of neoplastic cells through its mutagenic effect.Ionizing radiation (IR) can cause various delayed effects in cells, including genomic instability that leads to the accumulation of gene mutations and chromosomal rearrangements, which are thought to play a pivotal role in radiation-induced carcinogenesis. The persistence of such effects in progeny cells has profound implications for long-term health risks, including emergence of a second malignancy after radiotherapy (1). The thyroid gland is one of the most sensitive organs to the carcinogenetic effects of IR. The risk of thyroid tumors is maximal for exposure at a younger age and increases linearly with radiation dose (2). More than 90% of these cancers are papillary, presenting a RET/PTC chromosomal rearrangement in 70% of cases. Thus, the thyroid can serve as a paradigm for analyzing the long-term delayed effects of IR.The mechanism by which radiation exposure is memorized and leads to delayed DNA breakage remains to be determined. Hypoxia and antioxidant therapy reduce the X-ray–induced delayed effects, suggesting that radio-induced oxidative stress plays a significant role in determining the susceptibility of irradiated cells to genetic instability (3–5). We recently showed that H₂O₂ is able to cause RET/PTC1 rearrangement in thyroid cells, indicating that oxidative stress could be responsible for the RET/PTC rearrangement frequently found in radiation-induced thyroid tumors (6).Cells can produce ROS through activation and/or induction of NADPH oxidases, which constitute a family of enzymes known as NOX/DUOX (7). Unlike other oxidoreductases, NADPH oxidases are “professional” ROS producers, whereas the other enzymes produce ROS only as by-products along with their specific catalytic pathways. ROS produced by NOXs participate in the regulation of many cell functions and have been implicated in various pathological conditions, including the late side effects induced by IR and chemotherapy (8–10). Thyroid cells express three of these NADPH oxidases, including two H₂O₂-generating systems located at the apical plasma membrane of the thyroid cells: DUOX2, which is implicated in thyroid hormone biosynthesis, and DUOX1, whose role in the thyroid is still unknown (11, 12). Furthermore, recently NOX4 was found to be expressed inside these cells (13).Because ROS may contribute to the late effects observed after radiation exposure, we hypothesized that IR induces a delayed oxidative stress in thyroid cells via the activation and/or induction of NADPH oxidase. In the present study, we demonstrate that DUOX1 expression, induced via the IL-13 pathway in response to IR, is the primary source of sustained ROS production that causes persistent DNA damage. We show that p38 MAPK activation is required for the increased radio-induced DUOX1 expression. Finally, our analysis of human thyroid tissues shows that DUOX1 is overexpressed in both radio-induced and sporadic tumors, suggesting that radiation exposure by inducing DUOX1-based oxidative stress might favor a neoplastic process that can occur naturally. Our findings assign the NADPH oxidase DUOX1 a previously unidentified role in radio-induced genetic instability.     

Effects of early growth on blood pressure of infants of British European and South Asian origin at one year of age: the Manchester children's growth and vascular health study

OBJECTIVE: The objective of this study was to investigate early influences of postnatal growth on blood pressure (BP) in healthy, British-born South Asian and European origin infants. We tested the hypotheses that South Asian infants would be smaller in all body dimensions (length and weight) with higher relative truncal skinfold thickness at birth, and that increased (central) adiposity and accelerated growth up to 1 year would be associated with higher BP in both ethnic groups. PATIENTS AND METHODS: Five hundred and sixty infants were followed prospectively from birth to 3 and/or 12 months with measures of anthropometry and resting BP, compared against a UK 1990 growth reference, and analysed using regression methods. RESULTS: Marked differences in birth size persisted, as expected, between European and South Asian babies, but with a sexual dichotomy: South Asian boys were smaller in all anthropometric parameters (P < 0.001), including skinfolds (P < 0.05), than European boys, but South Asian girls, although smaller in length and weight, had similar skinfolds to European girls and thus a slightly larger subscapular skinfold thickness relative to birth weight [1.3 versus 1.2, mean difference 0.07, 95% confidence interval (CI) 0.0009-0.14, P = 0.047]. The dichotomy persisted postnatally; South Asian boys showed a striking early increase in weight and length compared with European boys, associated with significant accrual of subscapular fat (6.1 versus 5.3 mm, mean difference 0.8, 95% CI 0.3-1.3, P = 0.003). In gender and ethnicity adjusted regression models, infants with the largest weight standard deviation score (SDS) increases in the first 3 months had the highest 12-month systolic BP (beta = 2.4, 95% CI 0.5-4.2, P = 0.01), while those with the greatest birth length (beta = 0.7, 95% CI 0.05-1.4, P = 0.04) but the smallest changes in length over 3-12 months (beta = -0.57, 95% CI -0.95 to -0.19, P = 0.004) had the highest diastolic BP. CONCLUSIONS: Ethnic and gender differences in growth and adiposity present in early infancy include truncal fat preservation in South Asian girls from birth, which in boys is related to rapid early weight gain. Weight gain during the first 3 months appears to drive the rise in systolic BP to 1 year, itself a likely driver of later BP.