Production of embryos from in vitro-matured primary human oocytes.

Objective: To determine the factors that influence the number and quality of embryos produced from primary oocytes collected from untreated regularly ovulating and irregular or anovulatory polycystic women.

Design: A direct comparison between two patient groups whose oocytes were matured in vitro and a comparison of the embryo development of in vitro-matured oocytes from untreated patients with in vivo-matured oocytes of superovulated IVF-ET patients obtained during the same period.

Setting: The Monash IVF Clinic, involving patients who expressed the desire to avoid super-ovulation with fertility drugs.

Functional integrity of granulosa cells from polycystic ovaries.

Objective: The polycystic ovarian syndrome is frequently associated with human infertility and is a partially characterized syndrome of unknown aetiology. The aim of this study was to describe the functional integrity of granulosa cells from polycystic ovaries.

Patients: Follicular aspirates were collected from polycystic ovaries of ovulatory (n = 24) and anovulatory (n = 7) patients.

Electrofusion parameters for nuclear transfer predicted using isofusion contours produced with bovine embryonic cells.

Electrofusion is a valuable technique for the nuclear transfer procedure. An enucleated oocyte is electrofused with a blastomere to create a nuclear transfer embryo. The present study constructed isofusion contours after the electrofusion of identical coupled cells that characterized all the bovine embryonic cell types used in nuclear transfer.

The sperm centriole: its inheritance, replication and perpetuation in early human embryos.

The inheritance, replication and perpetuation of the sperm centriole in the early human embryo are reported. Both normal monospermic and abnormal dispermic embryos (n = 127) were examined by transmission electron microscopy. Centrioles were traced from fertilization to the hatching blastocyst stage.

Blastocyst development and birth after in-vitro maturation of human primary oocytes, intracytoplasmic sperm injection and assisted hatching.

Immature oocyte recovery followed by in-vitro oocyte maturation and in-vitro fertilization is a promising new technology for the treatment of human infertility. The technology is attractive to potential oocyte donors and infertile couples because of its reduced treatment intervention. Immature oocytes were recovered by ultrasound-guided transvaginal follicular aspiration.

Enucleation by centrifugation of in vitro-matured bovine oocytes for use in nuclear transfer.

Nuclear transfer has the potential to produce large numbers of identical progeny. Current limitations of the technique are associated with the use of micromanipulation for the demanding enucleation and reconstitution procedure. With the overcoming of this limitation, increased numbers of nuclear transfer embryos could be produced.

Effects of epidermal growth factor, transforming growth factor alpha and androstenedione on follicular growth and aromatization in culture.

The effects of epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha) and androstenedione on follicular growth and oestradiol production were studied in cultured mouse preantral follicles. Cultured follicles in the control group showed growth and steroidogenic capacities up to the antral follicle stage at day 4 of culture. However, many of these follicles failed to resume their growth during the culture.

Intracytoplasmic sperm injection in mice: increased fertilization and development to term after induction of the acrosome reaction.

A technique has been developed for intracytoplasmic sperm injection (ICSI) in the mouse with a relatively low rate of lysis of oocytes (range 5-25% across experiments) and pronuclear formation in around one-third of the intact oocytes (range 30-38% across experiments) for untreated spermatozoa. The treatment of spermatozoa with calcium ionophore, to induce the acrosome reaction (increases acrosome-free spermatozoa from 28 to 58%) before ICSI, increased pronuclear formation to approximately 60% (range 59-62% across experiments) in intact oocytes. The pronuclear oocytes developed to blastocysts in vitro and to term when transferred to recipient foster mothers at rates equivalent to zygotes formed after insemination in vitro.

The effect of testosterone on the maturation and developmental capacity of murine oocytes in vitro.

A dose-dependent inhibition of meiotic maturation and embryonic development was observed in both cumulus-enclosed and cumulus-denuded murine oocytes following incubation in the presence of 10, 20, 40, 60 and 80 microM testosterone for 18 h in vitro. Maturation to metaphase II was enhanced in cumulus-enclosed oocytes following maturation in the presence of human pre-ovulatory mural granulosa cells. However, maturation of cumulus-denuded oocytes was enhanced only when oocytes were cultured on a monolayer of human polycystic ovarian granulosa cells.

Evaluation of propanediol, ethylene glycol, sucrose and antifreeze proteins on the survival of slow-cooled mouse pronuclear and 4-cell embryos.

Mouse pronuclear and 4-cell embryos were cryopreserved by slow cooling to -33 degrees C in 1.5 M 1,2-propanediol or 1.5 M ethylene glycol, with or without 0.

Mouse sperm fertilising capacity following subzonal microinjection is dependent on sperm washing and response to solubilised zonae pellucidae.

Spermatozoa obtained from hybrid male mice were treated with solubilised zonae pellucidae after a period of capacitation in vitro to induce the acrosome reaction. Single spermatozoa were selected and microinjected into the perivitelline space of mature oocytes. A high proportion of the spermatozoa acrosome-reacted after treatment with solubilised zonae (63%).

In vitro maturation and the fertilization and developmental competence of oocytes recovered from untreated polycystic ovarian patients.

Objective: To determine the maturational and developmental competence of immature oocytes recovered in situ from anovulatory and ovulatory patients with polycystic ovaries (PCO).

Design: A newly designed method for recovery of immature oocytes from 2 to 10 mm follicles by transvaginal ultrasound or laparoscopy was used to compare the recovery and maturation of oocytes from 9 anovulatory polycystic ovarian syndrome (PCOS) patients and 10 ovulatory patients without polycystic ovaries (PCO) (experiment 1). In a second study (experiment 2), we compared the maturation, fertilization, and development of oocytes recovered from another 10 anovulatory PCOS and 13 ovulatory PCO patients.

Stage-specific appearance of the mouse antigen TEC-3 in normal and nuclear transfer bovine embryos: re-expression after nuclear transfer.

Bovine embryos, recovered from the uterus in vivo or derived from in vitro matured and in vitro fertilized oocytes, were investigated for the presence of the developmentally regulated mouse antigen TEC-3 by indirect immunofluorescence. During preimplantation embryo development TEC-3 is expressed on bovine morulae and blastocysts. It is absent from unfertilized and fertilized oocytes, and from all stages before the 32-cell stage.

Microinjection: choice of embryo transfer technique.

Subzonal sperm microinjection (SUZI) is indicated in severe oligoasthenozoospermia, in which the total count of motile sperm is inadequate for in vitro fertilization (IVF), and in cases with repeated failure of fertilization. Sperm for microinjection are selected following centrifugation on a Percoll gradient and stimulation with pentoxifylline and 2-deoxyadenosine. Motile sperm (2-10 per egg) are injected into the perivitelline space and fertilized oocytes are then cultured for two days prior to transfer into the Fallopian tube (tubal embryo stage transfer, TEST) or uterus.

The choice of the most appropriate microfertilization technique for human male factor infertility.

Comparisons were made among techniques used to treat male factor infertility. Patients with semen quality below that recognized by World Health Organization criteria as normal had a better success rate when treated by gamete intrafallopian transfer than by in vitro fertilization (25% v. 7% pregnancy rate per patient).

Embryo development capacity of oocytes fertilized by immature sperm and sperm treated with motility stimulants.

The fertilizing ability of mouse spermatozoa develops during maturation and coincides with the acquisition of motility. The lack of progressive motility of spermatozoa from the testis and precaudal segments of the epididymis interferes with their ability to fertilize oocytes after insemination in vitro. The removal of cumulus cells for insemination in vitro and the use of subzonal injection of a single spermatozoon resulted in a higher number of oocytes fertilized by immature caput and corpus spermatozoa.

Effects of stimulation or inhibition of lipid peroxidation on freezing-thawing of mouse embryos.

This study was conducted to determine whether the tolerance of embryos to the stress of freezing and thawing can be modified by including in the incubation medium stimulators or inhibitors of membrane lipid peroxidation. Mouse zygotes were cultured in medium M16, supplemented or not with FeSO4, apotransferrin, and/or ascorbate. In each culture supplement, 8-cell embryos were randomly allocated to an untreated (nonfrozen) control group, or treatment by freezing using slow or ultra-rapid cooling.

Does the zona pellucida select spermatozoa from the medium with higher fertilizing potential?

The present study was carried out to test whether the zona pellucida selects spermatozoa with higher fertilization potential. Fertilization rates of mouse oocytes after sperm microinjection under the zona pellucida (SMUZ) of zona-bound spermatozoa and of spermatozoa incubated in the absence of oocytes and treated (acid-treated group) or not (control group) with acid Tyrode's solution were compared. SMUZ was performed at 15, 30, 60, and 90 min after the insemination of fresh oocytes required for selecting spermatozoa bound to the zona pellucida.

IVF and related technology. The present and the future.

Objective: To describe the current status of in-vitro fertilisation (IVF) and related technologies, including: the indications for the procedures and the problems associated with the use of stimulated cycles; the use of frozen embryos and donor eggs; and the newer procedures of gamete micromanipulation for male infertility, immature egg collection as a possible alternative to the stimulated cycle, and preimplantation genetic diagnosis.

Data Sources And Study Selection: The clinical experience and research at Monash IVF and the Centre for Early Human Development, Monash University, were reviewed in association with key original or review articles in the world literature.

Data Synthesis: Cumulative pregnancy rates for IVF and the gamete intrafallopian transfer (GIFT) procedure at Monash IVF demonstrate that 29% of IVF patients and 55% of GIFT patients will have a live baby, the average number of treatments pursued being 3.

The effects of the sperm motility activators 2-deoxyadenosine and pentoxifylline used for sperm micro-injection on mouse and human embryo development.

The sperm motility stimulants 2-deoxyadenosine (DOA) and pentoxifylline (PTF), used to improve the success of insemination and sperm micro-injection for low motility sperm samples, were studied for their effects on the developmental capacity of mouse and human oocytes. When human oocytes were micro-injected with spermatozoa in 3 mM DOA 80% of them became blocked at the 1-cell pronuclear stage. However, when spermatozoa in 3 mM PTF were used for micro-injection or when spermatozoa were washed to remove DOA before micro-injection only a few oocytes (9-10%) were blocked.