Impact of oxygen concentration on adult murine pre-antral follicle development in vitro and the corresponding metabolic profile.

Oxygen concentration during in vitro culture has a significant effect on the physiology of embryos, altering metabolic profile and developmental outcome. Although atmospheric oxygen has been used routinely for the culture of ovarian follicles, oxygen concentration may also be critical for follicle growth but the optimal concentration has not been determined. In this study, mechanically isolated primary and secondary follicles (80-140 µm diameter) from adult mouse ovaries were cultured in serum-free conditions for 8 days in either 5 or 20% oxygen to determine growth (follicular diameter), morphology and viability. For each oxygen concentration, half of the medium was replaced on Days 2, 4 and 6 or on Day 4 only. In the latter group, metabolic analysis of spent follicular culture media was performed by (1)H-NMR. The proportion of viable, growing follicles was significantly (P < 0.0001) higher in 5% than in 20% oxygen (59% versus 8%). Reducing the frequency of medium replacement during culture in 5% oxygen resulted in significantly (P < 0.001) more viable follicles (79 versus 46%). In 20% oxygen, poor follicular viability was observed irrespective of the frequency of medium replacement (8 and 10% respectively). Metabolic profiles showed marked differences in amino acid and carbohydrate utilization with respect to both oxygen concentration and between Days 4 and 8 of development. Metabolites which significantly discriminated between oxygen concentration at both time points were glucose consumption, lactate utilization, alanine, alanyl-glutamine, leucine and proline. In conclusion, the poor in vitro follicular development previously observed in minimal culture conditions may reflect the use of 20% oxygen. Frequent medium replenishment is not necessary and does not overcome the detrimental effect of high oxygen on follicle viability. Further optimization of culture conditions would benefit from metabolic analyses and the use of 5% oxygen should be tested further for impact on functional aspects of follicle culture such as steroid production which is currently unknown.

Clinical outcomes following cryopreservation of blastocysts by vitrification or slow freezing: a population-based cohort study.

STUDY QUESTION: What are the clinical efficacy and perinatal outcomes following transfer of vitrified blastocysts compared with transfer of fresh or of slow frozen blastocysts? SUMMARY ANSWER: Compared with slow frozen blastocysts, vitrified blastocysts resulted in significantly higher clinical pregnancy and live delivery rates with similar perinatal outcomes at population level. WHAT IS KNOWN ALREADY: Although vitrification has been reported to be associated with significantly increased post-thaw survival rates compared with slow freezing, there has been a lack of general consensus over which method of cryopreservation (vitrification versus slow freezing) is most appropriate for blastocysts. STUDY DESIGN, SIZE, DURATION: A population-based cohort of autologous fresh and initiated thaw cycles (a cycle where embryos were thawed with intention to transfer) performed between January 2009 and December 2011 in Australia and New Zealand was evaluated retrospectively. A total of 46 890 fresh blastocyst transfer cycles, 12 852 initiated slow frozen blastocyst thaw cycles and 20 887 initiated vitrified blastocyst warming cycles were included in the data analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS: Pairwise comparisons were made between the vitrified blastocyst group and slow frozen or fresh blastocyst group. A Chi-square test was used for categorical variables and t-test was used for continuous variables. Cox regression was used to examine the pregnancy outcomes (clinical pregnancy rate, miscarriage rate and live delivery rate) and perinatal outcomes (preterm delivery, low birthweight births, small for gestational age (SGA) births, large for gestational age (LGA) births and perinatal mortality) following transfer of fresh, slow frozen and vitrified blastocysts. MAIN RESULTS AND THE ROLE OF CHANCE: The 46 890 fresh blastocyst transfers, 11 644 slow frozen blastocyst transfers and 19 978 vitrified blastocyst transfers resulted in 16 845, 2766 and 6537 clinical pregnancies, which led to 13 049, 2065 and 4955 live deliveries, respectively. Compared with slow frozen blastocyst transfer cycles, vitrified blastocyst transfer cycles resulted in a significantly higher clinical pregnancy rate (adjusted relative risk (ARR): 1.47, 95% confidence intervals (CI): 1.39-1.55) and live delivery rate (ARR: 1.41, 95% CI: 1.34-1.49). Compared with singletons born after transfer of fresh blastocysts, singletons born after transfer of vitrified blastocysts were at 14% less risk of being born preterm (ARR: 0.86, 95% CI: 0.77-0.96), 33% less risk of being low birthweight (ARR: 0.67, 95% CI: 0.58-0.78) and 40% less risk of being SGA (ARR: 0.60, 95% CI: 0.53-0.68). LIMITATIONS, REASONS FOR CAUTION: A limitation of this population-based study is the lack of information available on clinic-specific cryopreservation protocols and processes for slow freezing-thaw and vitrification-warm of blastocysts and the potential impact on outcomes. WIDER IMPLICATIONS OF THE FINDINGS: This study presents population-based evidence on clinical efficacy and perinatal outcomes associated with transfer of fresh, slow frozen and vitrified blastocysts. Vitrified blastocyst transfer resulted in significantly higher clinical pregnancy and live delivery rates with similar perinatal outcomes compared with slow frozen blastocyst transfer. Comparably better perinatal outcomes were reported for singletons born after transfer of vitrified blastocysts than singletons born after transfer of fresh blastocysts. Elective vitrification could be considered as an alternative embryo transfer strategy to achieve better perinatal outcomes following Assisted Reproduction Technology (ART) treatment. STUDY FUNDING/COMPETING INTERESTS: No specific funding was obtained. The authors have no conflicts of interest to declare.

Detection of zona pellucida proteins during human folliculogenesis.

BACKGROUND: The stage of folliculogenesis at which the human zona pellucida (ZP) is initiated and the cells responsible for the origin of the ZP continue to be controversial. This study characterizes the development of the ZP during human folliculogenesis using ovarian samples donated from patients requesting ovarian storage. METHODS: Follicles (from n = 18 patients, 14-40 years old) within fresh tissue and following development in a xenograft system were stained, using immunohistochemical techniques, for the presence of the three human ZP proteins, ZP1, ZP2 and ZP3. Over 500 primordial follicles and >20 follicles at each developmental stage were examined. RESULTS: All three ZP proteins were detected within the oocyte of the primordial follicle. Presence of ZP1 and ZP3 was observed in the majority of primordial oocytes (93% and 95%, respectively), whereas ZP2 was detected in only 32% of these follicles. The three ZP proteins were detected in the cytoplasm of cuboidal granulosa cells and their distribution correlates with developmental stages throughout folliculogenesis. CONCLUSIONS: ZP proteins were detected in both the oocyte and the granulosa cells as early as the primordial follicle stage in the human. The detection of ZP proteins in the quiescent primordial follicle suggests that these proteins have been present since oogenesis.

Inheritance of cellular resistance to the antitumor agent ICRF 159 in somatic cell hybrids.

Two independently derived sublines of Syrian hamster BHK 21 S cells that demonstrate resistance to the antitumor agent ICRF 159 (NSC-129943) were fused to sensitive cells in both intraspecific and interspecific crosses, and the responses of the resultant hybrids to the drug were determined. In intraspecific hybrids from crosses involving either of the sublines, resistance to ICRF 159 was expressed codominantly ith the hybrid response being halfway between the parental responses. In interspecific hybrids from crosses with sensitive mouse cells, resistance was also expressed but to a lesser extent than in the intraspecific hybrids. However, subsequent passaging of an interspecific hybrid resulted in an increase in the degree of resistance, which appeared to be accompanied by a loss of telocentric mouse chromosomes. The results suggested that resistance in these lines was due to an alteration in the cellular target involved in the response to ICRF 159.

The effects of cryopreservation regimens on the morphology of human ovarian tissue.

Human ovarian tissue consisting of stroma, pre-granulosa cells and oocytes, has been frozen using a variety of cooling rates and dehydration regimens. The differential survival of the various cell types under these conditions highlights the difficulty in defining optimum protocols for the cryopreservation of multicellular tissue.

The developmental potential of cryopreserved human embryos.

Using rigorously matched non-frozen controls we have shown that cryopreservation does not alter the implantation potential of early cleavage stage (day 2) human embryos if no blastomere loss occurs. Thawed intact 4-cell embryos have a significantly higher implantation (fetal heart) rate (16.9%) than similar 2-cell embryos (7.2%). This difference is not due to blastomere number per se since increasing the cell number in frozen embryos by allowing an extended period in culture prior to freezing does not alter their intrinsic developmental potential. Blastomere loss, which occurred in almost half of all thawed embryos, is directly related to a reduction in developmental potential. We estimate that approximately 30% of the expected fresh embryo implantations are lost as a consequence of cryopreservation. Both preimplantation and peri-implantation losses may contribute to this outcome.

The mutagenic potency of 4 agents at the thymidine kinase locus in mouse lymphoma L5178Y cells in vitro: effects of exposure time.

Mutagenic potency at the thymidine kinase (TK) locus in mouse lymphoma L5178Y cells (expressed as induced trifluorothymidine (TFT)-resistant mutants/total dose) was assessed for 4 agents (ethyl methanesulphonate (EMS), benzidine, 1,8-dinitropyrene (1,8-DNP) and ICRF 159) using short (3-4 h) and long (21-24 h) exposure times. The mutagenic potency of EMS was found to be essentially independent of concentration and exposure time when tested over a cytotoxic range consistent with routine testing procedures. Similar results were obtained with benzidine but for both 1,8-DNP and ICRF 159 mutagenic potency was found to be highly dependent on the concentration and exposure time. 1,8-DNP failed to induce any significant increases in mutant frequency when tested at concentrations up to 5 micrograms/ml using short exposure times, whereas the compound was active at concentrations as low as 0.1 microgram/ml when the exposure period was extended to 21 h. Under the latter conditions, however, the molar potency of 1,8-DNP was found to be inversely related to concentration over a range extending from 0.1 to 5 micrograms/ml. ICRF 159 induced increases in the frequency of TFT-resistant mutants using short or long exposure times. When a short exposure time was used, however, the mutagenic potency of the antitumour agent decreased with increasing concentration between 1 and 500 micrograms/ml. Although possible explanations can be offered to account for these observations the results illustrate potential problems which may arise in this system when comparing mutagenic potency values for a range of compounds with a view to assessing relative risk.

Induction of 6-thioguanine resistance, chromosome aberrations and SCE by dinitropyrenes in Chinese hamster ovary cells in vitro.

The ability of Chinese hamster ovary (CHO) cells to convert dinitropyrenes (DNPs) to mutagenic species has been investigated by examining the effects of 1,6-DNP and 1,8-DNP on three distinct end-points in this cell line. At concentrations ranging from 0.05 to 5 micrograms/ml both analogues induced increases in the frequency of 6-thioguanine-resistant mutants in CHO cells when exposure was limited to 3 h. This treatment time was also sufficient to permit the induction of sister-chromatid exchange and chromosome aberrations in CHO cells. The results obtained suggest that CHO cells, unlike mouse lymphoma L5178Y cells, possess an endogenous metabolic activity which is capable of bringing about the conversion of DNPs to their mutagenic form without a requirement for prolonged exposure periods and as such may be a more suitable cell line for the study of this class of compounds.

Cryopreservation of human ovarian tissue.

Approaches to the cryopreservation of human ovarian tissue are typically characterised by the use of slow freezing/rapid thawing methods using dimethyl sulfoxide or 1,2-propanediol (PROH) as cryoprotectants. This paper reviews current experience with these procedures.

Follicle development following cryopreservation of human ovarian tissue.

Despite the recent increase in human ovarian tissue banking, there has been little progress in establishing whether follicles within this tissue are viable and capable of function following cryopreservation. Two methods to assess growth and developmental potential of cryopreserved tissue are evaluated; (1). isolated follicle culture and (2). xenografting of tissue into a host animal. Development of numerous antral follicles following xenografting of cryopreserved tissue indicates that the cryopreservation procedure can preserve the developmental competence of primordial follicles.

Survival and developmental potential of stored human early cleavage stage embryos.

Human early cleavage stage embryos which survive cryopreservation and thawing fully intact demonstrate similar developmental potential to equivalent non frozen embryos when returned to the in vivo environment, whereas blastomere loss is directly related to the loss of potential for subsequent implantation in thawed embryos. This suggests that blastomere lysis during freezing and thawing does not occur preferentially in non viable blastomeres. Prefreeze growth rate rather than prefreeze blastomere number per se correlates with the developmental potential of stored embryos. When blastomere loss occurs as a consequence of cryopreservation, development of thawed early cleavage stage embryos to the blastocyst stage in vitro is impaired and the resultant blastocysts have a reduced total cell content. Blastomere loss is more prevalent in embryos which have been biopsied for preimplantation genetic diagnosis but this increased sensitivity can be circumvented by modification of the standard cryopreservation protocol.

Quantitative and qualitative analysis of the extracellular matrix protein, laminin, in Hirschsprung's disease.

Previous immunohistochemical studies have shown an abnormal distribution of extracellular matrix (ECM) proteins, including laminin, in the smooth muscle layer of muscularis externa in Hirschsprung's disease (HD) bowel. These findings supported the hypothesis that an abnormal ECM microenvironment may be responsible for the failure of migration and/or development of the neural crest cells in the gut in HD. In order to determine the cause of the abnormality in laminin distribution, solid-phase enzyme-linked immunosorbent assays and immunoblots were used to quantitate the ECM protein laminin and characterize its subunits, respectively, in extracts of the dissected smooth muscle layer of the muscularis externa. In the aganglionic bowel, laminin (median concentration, 32.4 ng/mg of tissue) was found to be present in significantly greater quantity than in both the normoganglionic bowel of the same specimen (median, 17.2 ng/mg, P less than or equal to .05) and the normal bowel of age-matched controls (median, 9.7 ng/mg, P less than or equal to .05). Laminin concentration was also found to be significantly higher in normoganglionic HD bowel (median, 17.2 ng/mg) than in age-matched control specimens (median, 10.8 ng/mg, P less than or equal to .05). No difference was observed in the subunit composition of laminin in HD and control extracts analysed by immunoblot after polyacrylamide gel electrophoresis. This study demonstrates a quantitative abnormality of laminin in the bowel in HD, supporting the hypothesis that "abnormal microenvironment" may have a role in the pathogenesis of HD.

Time-dependent effects of endothelin-3 on enteric nervous system development in an organ culture model of Hirschsprung's disease.

BACKGROUND/PURPOSE: Terminal colonic aganglionosis (Hirschsprung disease) results from incomplete rostrocaudal colonisation of the embryonic gut by neural crest cells (NCC). Mutations in the genes encoding endothelin-3 (EDN3) or its receptor (EDNRB) have been shown to result in a similar aganglionosis. This article describes the development of an organ culture model using embryonic murine gut to determine how endothelin-3 regulates development of the enteric nervous system. METHODS: Gut explants from mice of different gestational ages were cultured for up to 3 days in the presence or absence of 5 micromol/L of the specific endothelin-B receptor antagonist BQ788. EDN3 and EDNRB mRNA expression were analysed by reverse-transcription polymerase chain reaction (RT-PCR) and whole-mount in situ hybridisation. NCC were localised using immunoreactivity for PGP 9.5, a specific neuronal marker. RESULTS: EDN3 mRNA continued to be expressed by caecal mesenchymal cells and EDNRB mRNA by the migrating NCC in culture. Embryonic day (E)11.5 explants were already colonised by NCC up to the terminal ileum. Complete colonisation occurred in organ culture over the next 72 hours (equivalent to E 14.5). Explants of E 12.5 and E 13.5 showed complete colonisation after 48 and 24 hours culture, respectively. Terminal aganglionosis resulted from treatment of E 11.5 and E 12.5 gut explants with 5 micromol/L BQ788, whereas there was no inhibitory effect on E 13.5 explants. CONCLUSIONS: An organ culture model has been developed in which NCC colonisation of embryonic gut mirrors that described in vivo. Blockade of the EDN3/EDNRB receptor pathway shows that the interaction of endothelin-3 with its receptor is only necessary for NCC colonisation at early time-points, despite the continued expression of endothelin-3 mRNA in the gut.

Ontogeny of interstitial cells of Cajal in the human intestine.

BACKGROUND/PURPOSE: Interstitial cells of Cajal (ICC) recently have been identified as intestinal pacemaker cells. Abnormalities in ICC are increasingly recognized in a number of neonatal disorders such as infantile hypertrophic pyloric stenosis, Hirschsprung's disease, and transient intestinal pseudo-obstruction. The aim of this study was to determine the fetal and postnatal differentiation and development of ICC in the human gastrointestinal tract to aid interpretation of pathological specimens. METHODS: Specimens of human gastrointestinal tract from (1) fetuses (9 to 17 weeks' gestation; n = 12), (2) premature and full-term neonates with non-gut motility-related disorders, (age 26 to 59 weeks' gestation; n = 13), and (3) children (age 4 months to 13 years; n = 7) were immunohistochemically stained with antibodies to c-kit(a marker for ICC) and protein gene product 9.5 (PGP9.5, a marker for neural tissue). RESULTS: (1) C-kit-positive ICC were present throughout the gut in all specimens including those from the earliest gestational ages. C-kit and PGP9.5 immunoreactivities were present in different cell populations. (2) The distribution of ICC varied with gestational age and with region of the gut. (3) Maturation of ICC networks continues postnatally in a region-specific manner. CONCLUSIONS: ICC are present from an early stage in human gut development. Interpretation of apparent abnormalities in ICC distribution as being of pathological significance should be tempered by the knowledge that ICC networks continue to develop postnatally and that ICC development varies throughout the gut.

Abnormal colonic interstitial cells of Cajal in children with anorectal malformations.

BACKGROUND/PURPOSE: Constipation is a frequent functional problem in children after operation for all types of anorectal malformations. Although this has been assumed to be caused by hypomotility of the rectosigmoid colon, recent studies have demonstrated generalized colonic hypomotility in children with high or intermediate anomalies. The cause of this disorder is unknown. The aim of this study was to determine whether the observed colonic hypomotility seen in patients with anorectal malformations was caused by defects in distribution or density of interstitial cells of Cajal (ICC), recently identified as 'intestinal pacemaker cells'. METHODS: Colostomy specimens from 12 patients with high anorectal anomalies (ARM group; age 0 to 14 months) were compared with colostomy specimens from five control patients with nonmotility-related gastrointestinal pathology (age, 1 to 4 months). Specimens were immunohistochemically labelled with antibodies to PGP9.5, a marker for neural tissue, and antibodies to c-kit, a recently characterized marker for interstitial cells of Cajal (ICC). RESULTS: Ganglion cells were present in all histological specimens. Abnormalities in distribution and density of c-kit-positive ICC were present in 7 of 12 ARM patients. In two ARM patients, ICC were completely absent, and in five patients, ICC density was markedly reduced in circular muscle and at the submucosal border of circular muscle. Only five ARM patients had a distribution of ICC similar to that of control patients. CONCLUSION: Defects in the population of intestinal pacemaker cells may underlie the colonic hypomotility seen in high anorectal malformations and hence may contribute to refractory constipation.

Clinical and biochemical pregnancy in two respective recipients without ovarian function following gamete intrafallopian transfers using oocytes from a single donor.

Infertility resulting from premature ovarian failure in two independent patients was treated using a combination of steroid replacement, oocyte donation and gamete intrafallopian transfer (GIFT). Following ovarian stimulation four oocytes were retrieved from a volunteer donor undergoing simultaneous laparoscopic sterilisation. Two oocytes were subsequently replaced into each recipient's fallopian tube together with capacitated sperm from their respective husbands. In one recipient (Turner's syndrome) an intrauterine sac with fetal heart present was observed by ultrasound six weeks post GIFT whereas in the second recipient (premature menopause) plasma beta-hCG reached a peak value of 954mIU/ml eighteen days after GIFT before decreasing rapidly in the absence of ultrasound evidence of pregnancy. Intramuscular administration of progesterone appeared to be necessary during the post-GIFT period for maintenance of pregnancy. The above treatment was carried out on a predominantly out-patient basis in a small assisted conception unit based in a teaching hospital.

Diagnostic assessment of the developmental potential of human cryopreserved ovarian tissue from multiple patients using xenografting.

BACKGROUND: Although ovarian tissue cryopreservation for women at risk of losing ovarian function is offered by many clinics, there is a lack of evidence relating to the developmental potential of the stored tissue and, therefore, its clinical potential. This study was designed to use xenografting of cryopreserved tissue from multiple patients to assess the reproducibility of preservating developmental potential, the variation in developing follicle profile and the relationship between pre-freeze histology and post-thaw development. METHODS: Using previously published methods, cryopreserved ovarian cortex from nine patients was thawed and grafted under the kidney capsules of immunodeficient mice. Development of follicles was assessed after 26 weeks and compared to histology prior to freezing. RESULTS: Multiple growing follicles including antral stages were observed in multiple grafts of tissue from all patients. Metaphase II oocytes (n=9) were observed in follicles in grafts from five patients. There was no relationship between pre-freeze histology and developing follicle profile in xenografts. CONCLUSIONS: The propanediol freezing method used in this study is capable of reproducibly preserving the developmental potential of human ovarian follicles. The developing follicle profile after cryopreservation cannot be accurately predicted from pre-freeze histology. Xenografting provides a powerful tool for assessing the potential of human cryopreserved ovarian tissue.