Hum. Reprod. Advance Access originally published online on October 11, 2007
Human Reproduction 2007 22(12):3170-3177; doi:10.1093/humrep/dem314
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
High oxygen atmosphere improves human follicle development in organ cultures of ovarian cortical tissues in vitro
1 The Centre for Reproductive Medicine and Infertility, IVF Namba Clinic, 1-17-28, Minami-horie, Nishi-ku, Osaka 550-0015, Japan 2 IVF Osaka Clinic, Osaka, Japan
3 Correspondence address. E-mail: york{at}ivfnamba.com
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
|---|
BACKGROUND: Obtaining mature human follicles from cultured ovarian tissue may be beneficial for clinical use for women who wish to preserve fertile competence. However, the methodology of culture such as culture condition and gas atmosphere has not been well established in humans. Therefore, we investigated the effect of oxygen concentration in organ culture in order to establish an ovarian tissue culture method.
METHODS: Ovarian tissue was obtained from 26–35-year-old women undergoing removal of a benign tumor (n = 12) or caesarean section (n = 16). The ovarian cortical tissues were cultured on a cell culture insert for 15 days under high (100%) and low (air, 20%) oxygen concentrations and then inspected for follicle development with light and electron microscopy. Estradiol and progesterone concentrations in the medium during culture were measured.
RESULTS: The ultrastructure and the function of hormone secretion in the cultured tissues were well preserved after organ culture. The follicles developing under high oxygen were larger and more matured than those developing under low oxygen (P < 0.05).
CONCLUSIONS: Human ovarian tissues can be cultured for 15 days under high oxygen concentration with the organ culture system used here. This technique could make it possible to utilize ovarian tissue for preservation of reproductive competence in cancer patients.
Key words: ovarian tissue/organ culture/in vitro maturation/follicle/oocyte
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
|---|
In vitro culture of gametes has been studied for a long time in animals and human. In animals, many studies on ovarian cortical tissues, follicles and oocytes have been reported. Experiments of follicle culture have been reported in sheep (Cecconi et al., 1999
), mouse (Cortvrindt et al., 1997), pig (Greenwald et al., 1989), cow (Ralph et al., 1995) and mammals (Ksiazkiewicz, 2006). On the other hand, human follicles were cultured by Abir et al. (1997), Lintern-Moore (1974) and Mitchell et al. (1999). The effectiveness of culture methodologies were identified by follicular counts, immunocytochemical method, fluorescent viability method, endocrine assays and electron transmission microscopy.
Much has been learned about the culture of oocytes from ovarian tissue in animals. Eppig et al. showed successful production of pup in mice in 1996, and thereafter they improved the culture method to achieve more effectiveness (O'Brien et al., 2003). However, attempts to culture human ovarian tissue in vitro have met with little success, mainly because of the difficulties of culturing organs and obtaining human tissues.
Several authors have described the culture of frozen ovarian cortical tissue. Sadeu et al. (2006) cultured frozen–thawed human fetal ovarian tissues and obtained the good survival and progressive follicular growth. Scott et al. (2004) reported the effectiveness of extracellular matrix in culture of human ovarian tissues. Isachenko et al. (2006) successfully cultured human ovarian tissue for 2 weeks with a new culture method with agitation of medium. Biron-Shental et al. (2004) succeeded in prolonging the culture period to 4 weeks using frozen–thawed ovarian tissue obtained from human fetuses. However, organ culture of human adult ovarian tissue is still thought to be difficult.
In order to achieve better result in culture of adult tissue, more studies are needed to determine optimal culture conditions such as the type of medium and gas atmosphere. With respect to the gas atmosphere, issues on tension of carbon dioxide and oxygen are important. In particular, oxygen tension, which has not been well studied so far, is essential. We have proved an efficacy of 100% oxygen environment in organ culture of human decidual tissues (Morimoto, 1989). The objective of the present study was to investigate the effect of 100% versus 20% (air) oxygen on the in vitro culture of human ovarian cortical tissues based on light and electron microscopic observation of follicular structures and the estradiol (E2) and progesterone levels in the culture media.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
|---|
The preparation of human ovarian cortical tissues
Ovarian cortical tissues were obtained from 26- to 35-year-old women during an operation to remove a benign ovarian tumor (12 cases) or during a caesarean section (16 cases). The women took no medicine such as steroid hormones before and during operation, which may influence the result of experiment. Fully informed consent was obtained from all patients. This experiment was approved by Sankaky Ethics Committee.
The tissues were obtained, wrapped with gauze soaked in phosphate-buffered saline (Gibco, Carlsbad, CA, USA) and transferred to the laboratory within 30 min. The tissues from each patient were sliced to the size of 2 x 2 mm by microsurgical scissors. One block of ovarian material from each patient was cut into many pieces and those pieces were divided into three groups for light microscopy, electron microscopy and hormone assay.
Organ culture technique for human ovarian cortical tissue
The sliced tissues were put on cell culture inserts (Falcon 3189, BD Biosciences, Billerica, MA, USA). The cell culture inserts were 10.3 mm diameter, with 0.4 µm pore size and 1.6 x 106 pore density. The cell culture inserts were fixed in a 12 well plate (Falcon 3043).
Five to six tissues from the same patient were arranged on the cell culture insert. One milliliter of culture medium designed for organ culture (see below) was placed in the bottom compartment so that the upper surface of the medium was just under the membrane of the inserts. One drop of culture media was placed on the tissues to prevent dryness, and surplus medium was removed by pipette (Fig. 1). By this delicate procedure, tissues were covered by a thin layer of medium, and prevented from experiencing a shortage of oxygen. The plates were placed in a Bellco Glass Chamber (Bellco Glass Inc., Vineland, NJ, USA) which is designed to maintain a controlled atmosphere, with a valve that controls the inflow and outflow of the air. The chamber was put in an incubator at 37°C under an atmosphere of 5% CO2 in air (Fig. 2). When the tissues were cultured in a high oxygen (100%) atmosphere, the chamber was filled with pure oxygen and the valve was closed once a day. The valve on the chamber was kept open to provide for an atmosphere of 5% CO2 in air (20% oxygen). Distilled water was placed in two of the wells to maintain a high humidity in the culture environment.
|
|
Culture media
The culture media was
-Minimum Essential Medium supplemented with 10% fetal calf serum, 0.29 M pyruvate and Antibiotic-Antimycotic solution (Gibco). Antibiotic antimycotic solution contains 50 IU/l Penicillin G, 50 µg/ml streptomycin sulfate and 0.125 µg/ml amphotericin B. Moreover, 500 mIU recombinant FSH (Org 32 489, Organon, Amsterdam, Netherlands) and 300 mIU HCG (Serono, MA, USA) were added to the culture medium.
Observation by light and electron microscopy
The tissues were taken and prepared for morphological investigation before culture and 3, 5, 7, 9, 11, 13 and 15 days after culture. For light microscopy, the specimens were fixed by paraformaldehyde and stained with hematoxylin eosin. For electronmicrography, the specimens were prefixed in 3% glutaraldehyde and 0.5% paraformaldehyde in 0.1 M phosphate buffer, ph7.4 for 1 h at 4°C, washed in the phosphate buffer for 15 min, post-fixed in 1% OsO4 in the same buffer for 1 h and dehydrated in ethanol and propylene oxide. Tissues were embedded the in Epon-Araldite, ultrathin sectioned and stained with uranyl acetate and lead citrate and observed using a Nihon Denshi electron microscope operated at 75 kV.
Steroid hormone measurements
E2 and progesterone level in the culture medium were measured by Enzyme linked Fluorescent Assay using Mini-Vidas (Biomerieux, Lyon, France) which is an automated multiparametric immunoassay analyzer.
Statistical analysis
The significance of differences between follicles produced under low and high oxygen atmosphere was analyzed with Student's t-test. A two–tailed P-value <0.05 was considered statistically significant. The statistical software used was MedCalc (Med Calc Software, Mariakerke, Belgium).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
|---|
Follicle growth during organ culture
By observation of follicles in adult human ovarian cortical tissues, the density of follicles was low (Table I). The follicles observed in the original specimens were mostly immature ones such as primordial follicles and primary follicles. Thirty percentage (60/200) of all the tissues in low and high oxygen groups underwent central necrosis or degeneration after 15 days organ culture. The following categories of follicles appeared during organ culture (Fig. 3). (i) Clusters of early primordial follicles. Five or more very small follicles uncovered by follicular cells. (ii) Primary follicles covered by a monolayer of flat follicular cells. (iii) Primary follicles covered by cuboidal follicular cells. (iv) Primary follicles having expanded cytoplasm and covered by cuboidal follicular cells. (v) Secondary follicles covered by multilayered follicular cells. (vi) Secondary follicles having expanded cytoplasm. (vii) Pre-antral follicles. (viii) Antral follicles. (ix) Atretic follicles (not shown here). The structure of follicles or oocytes was totally or partially degenerated.
|
|
Various type of follicles appeared during the 15 days of culture under high oxygen atmosphere as shown in Table I. Counted total follicle number was 87 and the numbers and percentages of primordial follicle (forming cluster), primary follicle (flat, cuboidal, cuboidal/expand), secondary follicles (multilayer, multilayer/expand), large follicle and atretic follicle were 30 (34.4%), 37 (42.5%), 13 (14.9%), 3 (3.4%) and 4 (4.6%), respectively. Cuboidal expanded and multilayer-expanded follicles appeared after 5 days of culture. Large follicles started to appear after 9 days culture.
Ultrastructural observations
Before organ culture, mitochondria, Golgi apparatus and lysozomes were seen in the oocytes of primordial follicles, and the nucleolonema was clearly identified in the nucleus (Fig. 4). Nucleolonema were also clearly identified in the nucleus. After organ culture of 15 days, secondary follicles with two or three layers of follicular cells showed more developed ultrastructure. The ultrastructures were well preserved after organ culture (Fig. 5). The outer surfaces of the follicles were covered by thick external lamina, and well-developed villi were observed between the oocyte and the follicular cells. The follicular cells were partially connected by desmosomes.
|
|
Steroid hormone analysis
E2 and progesterone levels were measured by enzyme immune assay in the culture medium (Fig. 6). The E2 level in the culture medium increased up to 9 days and then decreased quite dramatically in the high oxygen group. The E2 level was significantly higher in the high oxygen group at 3, 13 and 15 days of culture. Progesterone production increased until day 9 in low oxygen group and then decreased. In contrast, progesterone continued to increase in the high oxygen group until 15 days of culture when levels were significantly higher than in low oxygen.
|
Effect of oxygen concentration on follicle development
More follicles with diameters over 100 µm were seen under high oxygen than under low oxygen atmosphere (Fig. 7). Forty-eight follicles in the low oxygen group and 81 follicles in the high oxygen group were compared in size. Mean diameter of follicles cultured under high oxygen (81 ± 58.5 µm) was significantly larger than in follicles cultured under low oxygen (44.6 ± 17.5 µm) (P < 0.05).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
|---|
In vitro development from primordial to mature follicle has been achieved in baboon (Wandji et al., 1997
; Fortune et al., 1998), hamster (Roy et al., 2000) and mouse (Cortvrindt et al., 1998). Eppig and O'Brien (1995) cultured the ovaries of newborn mice to achieve the full development of mouse oocytes, but the competence of the implanted embryos to grow was initially extremely low. By avoiding the use of a combination of FSH and insulin, which reduced the ability of oocytes to complete pre-implantation development, they were able to produce two pups (O'Brien et al., 2003). Subsequently, using the ovary of newborn mice, they have achieved a high success rate with 5.7% of two-cell stage embryos developing to term. The quality and quantity of immature follicles and oocytes obtained from newborn pups or fetal tissue are far better than those obtained from adult tissues. The number of primordial follicles is remarkably few in adult human tissue and the competence to achieve oocyte maturation, fertilization and embryo development are likewise very low.
In the past 10 years, there has been much progress in the culture of human material. Hovatta et al. (1996) cryopreserved human ovarian cortical tissues, thawed and cultured them on an extracellular matrix for a maximum of 21 days (Hovatta et al., 1997). They were successful in organ culture and reported that two-thirds of the follicles survived and were non-atretic. In our study, cell culture inserts (Falcon 3180) were used to increase the viability of the tissue. The medium can pass through the insert and provide proper bathing of the tissues. Most studies have used MatrigelTM as the extracellular matrix, which is a solubilized basement membrane preparation extracted from the mouse Engelbroth–Holm–Swarm tumor. Diluted MatrigelTM has been reported to benefit the preservation of viability of ovarian tissue (Scott et al., 2004). Our study demonstrated that use of a cell culture insert also benefited culture of human ovarian tissue, by preserving the ultrastructure and steroid production function. Cell culture inserts are easily placed in the culture chamber and function as extracellular matrix.
In organ culture, it appears to be important to keep the tissue wet, but too much medium on the tissue surface may prevent oxygen absorption from the atmosphere. Oxygen deficiency may cause central necrosis. Central necrosis is thought to be the main cause of damage to the tissue and cells, and may reduce the competence of oocytes to develop.
The organ culture method has been applied for the culture of many tissues Eppig (1977), such as chick embryos for more than a century. Culture was originally carried out in a Petri dish, but nowadays many culture dishes are available. Brain tissue was cultured successfully on a multipore membrane (Yamamoto et al., 1992). In organ culture, the tissues can survive in a condition similar to that in vivo. This may allow the three-dimensional structure of an organ to be maintained, preserving function of the tissue and cells, but prevention of central necrosis is one of the issues still to be solved in this method. It is essential to keep a humid atmosphere and to supply nutrition and oxygen to the surface of the tissue. In order to maintain such delicate conditions during culture, when the medium is exchanged, the surplus medium should be drawn off from the surface with a pipette after addition of new medium. By this technique, the tissue can be covered with a thin layer of medium which may allow oxygen to pass through. High humidity may protect the tissue from dryness caused by evaporation from the medium.
The effect of oxygen tension on follicle and oocyte development has been discussed by many authors. Adam et al. (2004) reported that lower oxygen tension in gas atmosphere for IVM improved cleavage and blastocyst formation rate using mouse oocytes. The same benefit by lowering oxygen tension was reported by Booth et al. (2005) in porcine oocytes. Increasing oxygen tension may affect nuclear maturation and implantation embryo development Eppig and Wigglesworth (1995).
However, some papers reported no improvement for 2 days of embryo development (Dumoulin et al., 1995) and fertilization rate, blastocyst formation rate and pregnancy rate (Kea et al., 2007) by reducing oxygen tension in humans.
On the contrary, there are papers which support the benefit of high oxygen level in gas atmosphere. In long-term culture of mouse follicles, 20% oxygen was found to result in higher quality and higher viability of oocytes than 5% oxygen, which showed a deleterious effect on the cumulus–oocyte complex (Smitz, 1996). Oyamada and Fukui (2004) also supported this opinion in bovine oocytes.
In the culture of placental villi, reduced oxygen concentration caused the reduction of villous outgrowth (James et al., 2006). Ischemia-reperfusion injury may be another effect of a low oxygen atmosphere. Occurrence of the damage by the injury was reported in human skeletal muscle cultured in vitro (Martou et al., 2006). In the culture of cells or oocytes, a high oxygen concentration is thought to cause damage by reactive oxygen species. Reactive oxygen species levels in follicular fluid may affect embryo formation and quality (Das et al., 2006). Oxidative stress may also damage sperm membranes and nuclear DNA (Agarwal et al., 2005).
A high concentration of oxygen (nearly 100%) was applied as the gas atmosphere in this study. From the ultrastructure of cultured tissue, the plasma membrane and organelles were well preserved and no evidence of any damage by high tension of oxygen was detected. Further study, such as a metabolic or genetic or epigenetic approach, is needed to clarify this point; however, the benefit of providing oxygen is greater than the damage by high oxygen tension in organ culture, where there are many more cells compared with cell culture conditions.
More E2 was measured in the medium of high oxygen group compared with that of low oxygen group. However, the E2 production diminished suddenly after 9 days of culture. It may be that follicular cells increased in number and produced more estrogen until 9 days, but thereafter the function was damaged for some reason, such as a limitation of the culture methodology. The source of progesterone is not known. However, some of the samples were from patients who underwent caesarean section and as the progesterone level decreased from day 9 of culture in the low oxygen group, it may be that the damage of tissue started earlier, under the condition of reduced oxygen tension.
The size of the tissue pieces is also important. In this study, the tissue was cut into 2 mm pieces. Because follicles are not abundant in human adult tissue, pieces smaller than 2 mm may not have any follicles. On the other hand, larger pieces are vulnerable to central necrosis.
In order to grow primordial follicles to mature follicles, culture methods need to be developed for several stages such as organ culture of ovarian cortical tissue, isolation of follicles from the tissue, follicle culture and oocyte culture for maturation. Organ culture methods have been worked out for human ovarian cortical tissue, but methods for the next stage of follicle separation are not well established. There are reports of successful mechanical separation of human small follicles (Abir et al., 1997; Hovatta et al., 1999). On the other hand, other methods use an enzyme, such as collagenase, in order to separate follicles from tissues (Oktay et al., 1997). However, enzymatic isolation resulted in the premature extrusion of oocytes from follicles (Hovatta et al., 1999). Isolation of follicles is much more difficult in humans than in other mammals because the human tissues contain tougher connective tissues.
It is important to observe the morphology, especially the ultrastructure, of the tissue, follicle or oocytes to evaluate the effect of the culture system. The ultrastructure of human oocyte has been described in many studies (Shafie et al., 2000; Sathananthan, 2003; Sadeu et al., 2006). Eppig (1977) performed electron microscopic observation on the tissue after organ culture of mouse ovary. However, no antrum formation has been observed. Ultrastructural observations provide much information for evaluating how tissues are preserved after culture. In this study, organelles such as mitochondria, smooth endoplasmic reticulum, Golgi apparatus and external lamina were well preserved after culture. The present results show that our organ culture system is an effective method for maintaining the ultrastructure of human ovarian cortical tissue.
The last stage of the entire culture from ovarian tissue to oocyte is the IVM of oocytes. Methods for this stage have been worked out by Cha et al. (1991) and Trounson et al. (1994), and now the procedure has become routine, especially for patients with polycystic ovaries. The procedure can help to prevent the high risk of ovarian hyperstimulation syndrome and multiple pregnancy.
Preservation of ovarian tissue is now discussed as an important issue for the quality of life of cancer patients undergoing radiotherapy or chemotherapy. To preserve a woman's ability to conceive, several methods are now available, such as autografting, xenografting and in vitro culture.
Xenografting is an attractive method for preserving ovarian tissue, because it keeps an abundant blood supply around the tissue, but the ethics of this method are controversial. Autografting of ovarian tissue has occasionally been successful, but the rate of success has still been low.
In vitro culture of ovarian tissue is an effective method for preserving fertility with fewer ethical problems than xenografting; however, the method needs more study in order to be applied as a routine clinical technique.
|
Acknowledgements |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
|---|
I thank Mr Masatake Tohnaka for assisting with the culture experiments, Prof. Isamu Sawaragi and Prof. Shuetu Suzuki for advice for electron micrograph and oocyte physiology.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
|---|
Abir R, Franks S, Mobberley MA, Moore PA, Margara RA, Winston RM. Mechanical isolation and in vitro growth of preantral and small antral human follicles. Fertil Steril (1997) 68:716–719.
Adam AA, Takahashi Y, Katagiri S, Nagano M. Effect of oxygen tension in the gas atmosphere during in vitro maturation, in vitro fertilization and in vitro culture on the efficiency of in vitro production of mouse embryos. Jpn J Vet Res (2004) 52:77–84.[Medline]
Agarwal A, Gupta S, Sharma R. Oxidative stress and its implications in female infertility—a clinicians's perspective. Reprod Biomed Online (2005) 11:641–650.[Web of Science][Medline]
Biron-Shental T, Fish B, Van Den Hurk R, Felz C, Feldberg D, Abir R. Survival of frozen –thawed human ovarian fetal follicles in long-term organ culture. Fertil Steril (2004) 81:109–113.
Booth PJ, Holm P, Callesen H. The effect of oxygen tension on porcine embryonic development is dependent on embryo type. Theriogenology (2005) 63:2040–2052.[CrossRef][Web of Science][Medline]
Cecconi S, Barboni B, Coccia M, Mattioli M. In vitro development of sheep preantral follicles. Biol Reprod (1999) 60:594–601.
Cha KY, Koo JJ, Ko JJ, Choi DH, Han SY, Yoon TK. Pregnancy after in vitro fertilization of human follicular oocytes collected from nonstimulated cycles, their culture in vitro and their transfer in a donor oocyte program. Fertil Steril (1991) 55:109–113.[Web of Science][Medline]
Cortvrindt R, Smitz J. Early preantral mouse follicle in vitro maturation: oocyte growth, meiotic maturation and granulosa-cell proliferation. Theriogenology (1998) 49:845–859.[CrossRef][Web of Science][Medline]
Cortvrindt R, Smitz J, Van Steirteghem AC. Assessment of the need for follicle stimulating hormone in early preantral mouse follicle in vitro culture. Hum Reprod (1997) 12:759–768.
Das S, Chattopadhyay R, Ghosh S, Goswami SK, Chakravarty BN, Chaudhury K. Reactive oxygen species level in follicular fluid –embryo quality marker in IVF ? Hum reprod (2006) 21:2403–2407.
Dumoulin JC, Vanvuchelen RC, Land JA, Pieters MH, Geraedts JP, Evers JL. Effect of oxygen concentration on in vitro fertilization and embryo culture in the human and the mouse. Fertil Steril (1995) 63:115–119.[Web of Science][Medline]
Eppig JJ. Mouse oocyte development in vitro with various culture systems. Dev Biol (1977) 60:371–388.[CrossRef][Web of Science][Medline]
Eppig JJ, O'Brien ML. Development in vitro of mouse oocytes from primordial follicles. Biol Reprod (1995) 54:197–207.[CrossRef]
Eppig JJ, Wigglesworth K. Factors affecting the developemental competence of mouse oocytes growth in vitro: oxygen concentration. Mol Reprod Dev (1995) 42:447–456.[CrossRef][Web of Science][Medline]
Fortune JE, Kito S, Wandji SA, Srsen V. Activation of bovine and baboon primordial follicles in vitro. Theriogenology (1998) 15:441–449.
Greenwald GS, Moor RM. Isolation and preliminary characterization of pig primordial follicles. J Reprod Fertil (1989) 87:561–571.
Hovatta O, Silye R, Krausz T, Abir R, Margara R, Trew G, Lass A, Winston RML. Cryopreservation of human ovarian tissue using dimethylsulphoxide and propanediol-sucrose as cryoprotectants. Hum Reprod (1996) 6:1268–1272.
Hovatta O, Silye R, Abir R, Krausz T, Winston RML. Extracellular matrix improves survival of both stored and fresh human primordial and primary ovarian follicles in long–term culture. Hum Reprod (1997) 12:1032–1036.
Hovatta O, Wright C, Krausz T, Hardy K, Winston RML. Human primordial, primary and secondary ovarian follicles in long-term culture: effect of partial isolation. Hum Reprod (1999) 14:2519–2524.
Isachenko V, Montag M, Isachenko E, Van der Ven K, Dorn C, Roesing B, Braun F, Sadek F, Van der Ven H. Effective method for in-vitro culture of cryopreserved human ovarian tissue. Reprod Biomed Online (2006) 13:228–234.[Web of Science][Medline]
James JL, Stone PR, Chamley LW. The effects of oxygen concentration and gestational age on extravillous trophoblast outgrowth in a human first trimester villous explant model. Hum Reprod (2006) 21:2699–2705.
Kea B, Gebhardt J, Watt J, Westphal LM, Lathi RB, Milki AA, Behr B. Effect of reduced oxygen concentrations on the outcome of in vitro fertilization. Fertil Steril (2007) 87:213–216.[CrossRef][Web of Science][Medline]
Ksiazkiewicz LK. Recent achievements in in vitro culture and preservation of ovarian follicles in mammals. Reprod Biol (2006) 6:3–16.[Medline]
Lintern-Moore S, Peters H, Moore GPM, Faber M. Follicular development in the infant human ovary. J Reprod Fertil (1974) 39:53–64.
Martou G, O'Blenes CA, Huang N, McAllister SE, Neligan PC, Ashrafpour H, Pang CY, Lipa JE. Development of an in vitro model for study of the efficacy of ischemic preconditioning in human skeletal muscle against ischemia-repurfusion injury. J Appl Physiol (2006) 101:1335–1342.
Mitchell LM, Kennedy CR, Hartshorne GM. Collection and culture of human primordial and primary follicles arising from ovarian aspiration. Mol Cell Endcrinol (1999) 163:149.
Morimoto Y. Ultrastructural and endocrinological study of the human endometrium and decidua using the organ culture method. Acta Obstet Gynaecol Jpn (1989) 40:201–208.
O'Brien MJ, Pendola J K, Eppig JJ. A revised protocol for in vitro development of mouse oocytes from primordial follicles dramatically improves their developmental competence. Biol Reprod (2003) 68:1682–1686.
Oktay K, Briggs D, Gosden RG. Ontogeny of follicle-stimulating hormone receptor gene expression in isolated human ovarian follicles. J Clin Endocrinol Metab (1997) 82:3748–3751.
Oyamada T, Fukui Y. Oxygen tension and medium supplements for in vitro maturation of bovine oocytes cultured individually in a chemically defined medium. J Reprod Dev (2004) 50:107–117.[CrossRef][Web of Science][Medline]
Ralph JH, Wilmut I, Telfer EE. In vitro growth of bovine preantral follicles and the influence of FSH on follicle and oocyte diameter. J Reprod Fertil (1995) 15:6–7.
Roy SK, Albee L. Requirement for follicle-stimulationg hormone action in the formation of primordial follicles during perinatal ovarian development in the hamster. Endocrinology (2000) 141:4449–4456.
Sadeu JC, Cortvrindt R, Ron-EI R, Kasterstein E, Smitz J. Morphological and ultrasuructural evaluation of cultured frozen-thawed human fetal ovarian tissue. Fertil Steril (2006) 85(Suppl 1):1130–1141.[CrossRef][Web of Science][Medline]
Sathananthan AH. Morphology and pathology of the human oocyte. In: Biology and Pathology of the Oocyte—Trounson AO, Gosden RG, eds. (2003) Cambridge, UK: Cambridge University Press. 185–207.
Scott J E, Carlsson IB, Bavister BD, Hovatta O. Human ovarian tissue cultures: extracellular matrix composition, coating density and tissue dimensions. Reprod Biomed Online (2004) 9:287–293.[Web of Science][Medline]
Shafie ME, Windt ML, Kitshoff M, McGregor P, Sousa M, Wrantz PAB, Kruger TF. Ultrastructure of human oocytes: a transmission electron microscopic view. In: An Atlas of the Ultrastructure of Human Oocytes—Shafie ME, Sousa M, Windt ML, Kruger TF, eds. (2000) New York: Parthenon Publishing Group. 83–173.
Smitz J, Cortvrindt R, Steirteghem ACV. Normal oxygen atmosphere is essential for the solitary long-term culture of early preantral mouse follicles. Mol Reprod Dev (1996) 45:466–475.[CrossRef][Web of Science][Medline]
Trounson A, Wood C, Kaushe A. In vitro maturation and the fertilization and developemental competence of oocytes recovered from untreated polycystic ovarian patients. Fertil Steril (1994) 62:353–362.[Web of Science][Medline]
Wandji SA, Srsen V, Nathanielsz PW, Eppig JJ, Fortune JE. Initiation of growth of baboon primordial follicles in vitro. Hum Rrprod (1997) 12:1993–2001.[CrossRef]
Yamamoto N, Yamada K, Kurotani T, Toyama K. Laminar specificity of extrinsic cortical connections studied in coculture preparations. Neuron (1992) 9:217–228.[CrossRef][Web of Science][Medline]
Submitted on May 5, 2007; resubmitted on August 6, 2007; accepted on August 28, 2007.
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||