Elevated non-esterified fatty acid concentrations during in vitro murine follicle growth alter follicular physiology and reduce oocyte developmental competence.

Objective: To study how long-term elevated non-esterified fatty acid (NEFA) concentrations, typical in metabolic disorders such as obesity or type 2 diabetes, affect murine follicular development, follicle quality, and subsequent oocyte developmental competence in vitro.

Design: Experimental study.

Setting: In vitro culture setting.

Assuring safety without animal testing: the case for the human testis in vitro.

From 15 to 17 June 2011, a dedicated workshop was held on the subject of in vitro models for mammalian spermatogenesis and their applications in toxicological hazard and risk assessment. The workshop was sponsored by the Dutch ASAT initiative (Assuring Safety without Animal Testing), which aims at promoting innovative approaches toward toxicological hazard and risk assessment on the basis of human and in vitro data, and replacement of animal studies. Participants addressed the state of the art regarding human and animal evidence for compound mediated testicular toxicity, reviewed existing alternative assay models, and brainstormed about future approaches, specifically considering tissue engineering.

The ReProTect Feasibility Study, a novel comprehensive in vitro approach to detect reproductive toxicants.

ReProTect is a project within the 6th European Framework Program which has developed alternative methods aimed to reduce or replace animal experimentation in the field of reproductive toxicology. In its final year, a ring trial, named the "Feasibility Study", was conducted, in which 10 blinded chemicals with toxicologically well-documented profiles were analyzed by employing a test battery of 14 in vitro assays. EC(50) (half maximal effective concentration) or equivalent endpoints were determined and the test compounds were ranked relative to chemicals previously assayed in the tests of the battery.

Timing of nuclear maturation and postovulatory aging in oocytes of in vitro-grown mouse follicles with or without oil overlay.

Meiotic maturation of the oocyte is a timed sequence of events induced by the ovulatory LH surge. In vitro maturation of oocytes is known to alter the meiotic time course. This study documented the timing of meiosis in oocytes grown in vitro for 12 days, from the preantral follicle stage onward, and the influence of an oil overlay.

Continuous exposure to bisphenol A during in vitro follicular development induces meiotic abnormalities.

Bisphenol A (BPA), a widely used environmental contaminant, may exert weak estrogenic, anti-androgenic and anti-thyroidic activities. BPA is suspected to possess aneugenic properties that may affect somatic cells and mammalian oocytes. Oocyte growth and maturation depend upon a complex bi-directional signaling between the oocyte and its companion somatic cells.

Trichlorfon-induced polyploidy and nondisjunction in mouse oocytes from preantral follicle culture.

Trichlorfon (TCF), an organophosphate insecticide and potent inhibitor of choline esterases, was previously shown to induce first meiotic nondisjunction and spindle aberrations in isolated, follicle cell-denuded mouse oocytes maturing in vitro. To explore dose-response and direct and indirect, potentially synergistic effects of TCF on the somatic cells and the oocyte within a follicle, we presently employed preantral follicle culture. 100 microg/ml TCF added at the time of hormonally stimulated resumption of meiosis of follicle cell-enclosed mouse oocytes, 16 h before in vitro ovulation, induced significant rises in first meiotic nondisjunction in oocytes from preantral follicle culture.

Principal findings from a multicenter trial investigating the safety of follicular-fluid meiosis-activating sterol for in vitro maturation of human cumulus-enclosed oocytes.

Objective: To evaluate the safety of applying follicular-fluid meiosis-activating sterol (FF-MAS) in vitro to immature human oocytes.

Design: Phase I bicenter, randomized, parallel-group, controlled, partially blinded trial.

Setting: Third-level referral academic centers, including reproductive biology and genetics laboratories.

In vitro effects of dexamethasone on mouse ovarian function and pre-implantation embryo development.

The effect of dexamethasone (5-80 microg/ml) on ovarian function and embryo development was studied in mice. The follicle bio-assay revealed no effects of DEX up to 40 microg/ml on folliculogenesis and oogenesis, whereas 80 microg/ml hampered follicle differentiation and oocyte maturation. Androgen, estrogen and progestin secretion patterns were strongly impaired at all doses levels.

Morphological and ultrastructural evaluation of cultured frozen-thawed human fetal ovarian tissue.

Objective: To investigate a defined culture condition for the culture of frozen-thawed human ovarian tissue.

Design: Prospective laboratory study.

Setting: Reproductive biology laboratories in university hospitals.

Ovarian follicle bioassay reveals adverse effects of diazepam exposure upon follicle development and oocyte quality.

A mouse ovarian follicle bioassay was used to study folliculogenesis and oocyte quality in vitro. Diazepam (DZ) was chosen as test compound to evaluate the system for its ability to detect possible effects of chemicals on reproduction. The bioassay is suitable to analyze the influence of DZ on each of the follicular components at any stage of development.

Batch-to-batch consistency of human-derived gonadotrophin preparations compared with recombinant preparations.

Different gonadotrophin preparations derived from human urine or manufactured by recombinant technology are currently used in clinical practice for the treatment of infertility. It has been widely assumed that gonadotrophin products manufactured by recombinant technology have better batch-to-batch consistency compared with human-derived preparations and that this potentially will be shown to provide a more constant clinical response, but there is little evidence for either statement. This study compared the batch-to-batch consistency between urinary-derived and recombinant manufactured gonadotrophin preparations using standard analytical techniques, as well as a novel in-vitro follicle bioassay to evaluate the consistency of the biological response at the target organ.

Aneuploidy in mouse metaphase II oocytes exposed in vivo and in vitro in preantral follicle culture to nocodazole.

Aneuploidy tests are important in evaluating genetic hazards especially when chemical exposures are suspected to affect the fidelity of chromosome segregation in oocytes and embryos. In the current study, a newly established method, mouse preantral follicle culture, was employed to grow oocytes in vitro within follicles. The sensitivity of in vitro grown follicle enclosed oocytes was compared with oocytes maturing in vivo in the ovary.

A reproducible two-step culture system for isolated primary mouse ovarian follicles as single functional units.

A reproducible two-step culture system for isolated mouse ovarian follicles smaller than 100 microm (type 3a follicles) was designed. First, isolated follicles were grown in single droplets of alpha-minimal essential medium (MEM) without (deoxy)ribonucleosides at a lower concentration of fetal bovine serum (FBS; 1%) for 6 days with mechanical prohibition of thecal cell attachment. Growing follicles reaching at least 100 microm were transferred to alpha-MEM medium enriched with a higher concentration (5%) of FBS to allow attachment and were cultured subsequently for an additional 12 days.

Number of ovarian follicles in human fetuses with the 45,X karyotype.

Objective: To evaluate the numbers of ovarian follicles during fetal life in the gonads of human female fetuses with the 45,X karyotype (Turner syndrome, TS) and to compare them with those from age-matched 46,XX fetuses.

Design: Retrospective study.

Setting: An academic hospital.

Preantral follicle culture as a novel in vitro assay in reproductive toxicology testing in mammalian oocytes.

The most common genetic disorder in humans, trisomy, is caused predominantly by errors in chromosome segregation during oogenesis. Isolated mouse oocytes resuming meiosis and progressing to metaphase II in vitro have recently been used to assess targets, aneugenic potential and sensitivity of oocytes to chemical exposures. In order to extend in vitro maturation tests to earlier stages of oogenesis, an in vitro assay with mouse preantral follicle cultures has been established.

Effects of chilling on structural aspects of early preantral mouse follicles.

Chilling injury is one of the major limiting factors for achieving optimal cryopreservation of gametes. This study aimed to determine potential chilling-induced damage on several structural aspects of early preantral mouse follicles. Mechanically isolated intact early preantral follicles (type 3b-4) were exposed to 0 degrees C for 1, 5, 10, or 30 min.

Human oocytes reversibly arrested in prophase I by phosphodiesterase type 3 inhibitor in vitro.

This study addresses the role of cAMP hydrolytic isoenzyme phosphodiesterase type 3 (PDE 3) modulation on human oocyte maturation in vitro. Presence of phosphodiesterase type 3 A (PDE 3A) mRNA was confirmed in human germinal vesicle-stage (GV) oocytes. Making use of a selective PDE 3 inhibitor, Org 9935 (10 microM), oocytes retrieved from immature follicles were arrested in prophase I with a high efficiency for up to 72 h.

The earliest stages of folliculogenesis in vitro.

In recent years several follicle culture systems have been pioneered in different mammalian species for studying ovarian folliculogenesis and culturing immature oocytes. Applications of these in vitro techniques include fertility preservation for humans, conservation of rare animals and development of oocyte banks for research purposes. Immature female gametes in the ovarian cortex can be cryopreserved for later use if culture techniques are available afterwards to promote growth and maturation.