Pregnancies in humans by fertilization in vitro and embryo transfer in the controlled ovulatory cycle.

Normal pregnancies have been established in four women with tubal infertility by fertilization in vitro, embryo culture, and embryo transfer after stimulation of follicular growth with clomiphene citrate. In three of these women the time of oocyte maturation was controlled by human chorionic gonadotropin. This procedure for the control of ovulatory response has many advantages when compared with the previously successful method of using the natural ovulatory cycle.

Isolation of pluripotent embryonic stem cells from reprogrammed adult mouse somatic cell nuclei.

Pluripotent human stem cells isolated from early embryos represent a potentially unlimited source of many different cell types for cell-based gene and tissue therapies [1-3]. Nevertheless, if the full potential of cell lines derived from donor embryos is to be realised, the problem of donor-recipient tissue matching needs to be overcome. One approach, which avoids the problem of transplant rejection, would be to establish stem cell lines from the patient's own cells through therapeutic cloning [3,4]. Recent studies have shown that it is possible to transfer the nucleus from an adult somatic cell to an unfertilised oocyte that is devoid of maternal chromosomes, and achieve embryonic development under the control of the transferred nucleus [5-7]. Stem cells isolated from such a cloned embryo would be genetically identical to the patient and pose no risk of immune rejection. Here, we report the isolation of pluripotent murine stem cells from reprogrammed adult somatic cell nuclei. Embryos were generated by direct injection of mechanically isolated cumulus cell nuclei into mature oocytes. Embryonic stem (ES) cells isolated from cumulus-cell-derived blastocysts displayed the characteristic morphology and marker expression of conventional ES cells and underwent extensive differentiation into all three embryonic germ layers (endoderm, mesoderm and ectoderm) in tumours and in chimaeric foetuses and pups. The ES cells were also shown to differentiate readily into neurons and muscle in culture. This study shows that pluripotent stem cells can be derived from nuclei of terminally differentiated adult somatic cells and offers a model system for the development of therapies that rely on autologous, human pluripotent stem cells.

In vitro maturation and intracytoplasmic sperm injection of oocytes collected from hormonally stimulated common wombats, Vombatus ursinus.

Porcine FSH/LH stimulation successfully induced development of multiple large (>or=4mm) antral follicles in 10 of 11 common wombats. A mean of 5.5 metaphase II (MII) oocytes were aspirated from wombats that were stimulated during the follicular phase of the oestrous cycle (n=3) or after pouch young removal (n=3). Three subadults (n=3) and two anoestrus adults did not produce MII oocytes despite pFSH/pLH administration. In vitro maturation of immature oocytes at the time of aspiration doubled the number of MII oocytes that could be collected from pFSH/pLH stimulated wombats. Immature oocytes with cumulus attached, matured more readily to the MII stage than immature oocytes without cumulus. Following intracytoplasmic sperm injection (ICSI), approximately 5% of the oocytes that were MII at the time of collection cleaved. Approximately 5% of those that were matured by in vitro maturation (IVM) formed two polar bodies following ICSI, although they not cleave. Parthenogenesis cannot be excluded. This demonstrates that assisted reproductive technologies may be applicable to the common wombat.

Changes in plasma progesterone concentrations around the time of the luteinizing hormone surge in women superovulated for in vitro fertilization.

The purpose of the present study was to examine plasma progesterone (P4) changes around the time of the onset of the LH surge in women superovulated with clomiphene citrate and human Menopausal Gonadotrophin for in vitro fertilization (IVF). It was considered that changes in P4 levels which should be closely associated with the onset of the LH surge may be an accurate indicator for the time of oocyte recovery for IVF and that premature P4 secretion may prevent the establishment of pregnancy after embryo replacement. The plasma P4 concentrations determined at 0800, 1400, and 2100 h during the period 36 h before to 16 h after the onset of the LH surge in 72 women showed a significant diurnal variation with the nadir at 0800 h. The onset of the LH surge was detected in 51%, 32%, and 17% of patients at 0800, 1400, and 2100 h, respectively. The first significant increase in mean P4 concentrations was coincident with the onset of the LH surge at 1400 and 2100 h but because of the diurnal nadir of P4 at 0800 h, the increase in mean P4 levels was delayed until 1400 h when the LH surge began at 0800 h. Elevation of P4 concentrations from 2 to 8 nmol/liter in individual patients at 1400 and 2100 h before the onset of the LH surge did not prevent the establishment of pregnancy after embryo transfer. P4 concentrations before and after the onset of the LH surge were higher with increasing numbers of mature follicles. We conclude that changes in plasma P4 concentrations may be used to determine the time of ovulation or oocyte recovery for IVF because of their close association with the timing of the onset of the LH surge.

Successful fertilisation of human oocytes in vitro: concentration of estradiol-17 beta, progesterone and androstenedione in the antral fluid of donor follicles.

Oocytes and matched samples of follicular fluid were obtained from 156 pre-ovulatory follicles in 125 women 26--36 h after either administration of hCG or the onset of an endogenous LR surge. Concentrations of estradiol-17 beta (E2), progesterone (P) and androstenedione (A4) in the fluid of individual donor follicles were measured and related to the success of fertilisation of oocytes in vitro and the incidence of pregnancies after embryo transfer. Oocytes which gave rise to successful pregnancies were obtained from follicles which contained greater concentrations of E2 and a higher ratio of E2:P than did oocytes from which pregnancy did not result. These data provide direct evidence in support of the hypothesis that estrogenic follicles are the sole source of ova which undergo fertilisation and subsequently give rise to pregnancy in women.

Effect on plasma gonadotropins of cyclic steroid replacement in women with premature ovarian failure.

A sequential regimen of steroid replacement of oral estradiol valerate and progesterone (P) by intravaginal suppository was developed for women with premature ovarian failure or ovarian agenesis. The regimen, based on a 28-day cycle, resulted in peripheral plasma concentrations of estradiol and P within the normal range of the menstrual cycle and endometrial differentiation consistent with the normal secretory phase. Pregnancy has now been successfully established in four patients following this regimen of steroid treatment and transfer of donated embryos. Plasma concentrations of LH were within the normal range by the end of the first cycle of treatment with exogenous steroids. However, plasma FSH remained above the normal range, even during the third treatment cycle, consistent with the necessity of a gonadal feedback factor (inhibin?) other than estradiol and P for maintaining FSH in the normal range. Although 7/8 patients had a surge of LH at midcycle, only 3/8 patients had concomitant FSH surges, supporting a role for progesterone in facilitating the midcycle FSH surge.

An analysis of plasma estradiol concentrations during clomiphene citrate-human menopausal gonadotropin stimulation in an in vitro fertilization-embryo transfer program.

We report here a range of plasma estradiol (E2) concentrations suitable for use in an in vitro fertilization (IVF) program. This range was derived from nonparametric analysis of plasma E2 levels using plasma E2 measurements beginning 10 days before the anticipated day of the midcycle LH surge (midpoint), as calculated from each patient's six previous menstrual cycles, during which time the patients all received the same ovarian stimulation regimen. The regimen consisted of 100 mg clomiphene citrate/day for 5 days, beginning 10 days before the anticipated midpoint, plus 150 IU human menopausal gonadotropin, commencing the day after clomiphene. A consecutive series of 102 IVF conception cycles induced in this standardized fashion were analyzed in this study. The 5th-95 percentile envelope of plasma E2 concentrations was derived as a valid clinical indicator of satisfactory folliculogenesis during IVF treatment. Five women had plasma E2 concentrations below the 5th percentile of the E2 range on at least 3 consecutive days of ovarian stimulation, while six women had E2 levels above the 95th percentile of this range on at least 3 consecutive days. This plasma E2 range defined objectively the diagnoses of ovarian hyperstimulation and inadequate stimulation in an IVF program. These criteria should help clinicians in managing ovarian responses during IVF superovulation stimulation treatment.

Evaluation of the long-term function of cryopreserved ovarian grafts in the mouse, implications for human applications.

Ovarian tissue storage has several potentially very valuable clinical applications, including the management of young female patients that are at risk of premature menopause. Ovarian tissue collection, used alone or in combination with oocyte and embryo cryopreservation, may help these patients safeguard their own future fertility. All available evidence from animal studies indicates that grafting of frozen ovarian tissue should be feasible in the human. This study on the mouse shows that frozen thawed ovarian tissue grafts can restore long term fertility to previously ovariectomised recipients. This, and other available evidence, indicates that ovarian tissue collection and storage, used alone or in combination with oocyte or embryo collection, may help safeguard the fertility of patients at risk of premature menopause.

Gonadotrophin administration can benefit ovarian tissue grafted to the body wall: implications for human ovarian grafting.

Ovarian grafting provides a strategy for clinical infertility treatment and is starting to be used in conjunction with ovarian tissue storage for patients at risk of early ovarian failure. As patients are starting to return for their frozen stored tissue we need to ascertain how to maximise follicle survival when this tissue is grafted back to the patient. For research purposes ovarian tissue is commonly grafted to the kidney capsule as the rich capillary bed at this site favours rapid graft revascularization. This is however not an ideal site for natural conceptions or for the harvest of mature oocytes for in vitro fertilization. While oocytes would be relatively easy to recover from grafts on the abdominal wall or subcutaneous tissue graft revascularization at these sites is slower and evidence indicates that fewer follicles survive. As gonadotropins can upregulate angiogenic growth factors in the ovary this study was designed to test whether the administration of exogenous gonadotropins would increase the number of surviving follicles in grafts placed at less vascularised sites. We showed that exogenous gonadotrophins, given to either the donor or the recipient, could increase the number of developing follicles but the magnitude of this effect was influenced by the timing of the injections relative to the time of grafting.

Colonization and maintenance of murine embryonic stem cells on poly(alpha-hydroxy esters).

The aim of this study was to determine the ability of various poly(alpha-hydroxy esters) to support the in vitro propagation of murine embryonic stem (ES) cells in an undifferentiated state. To this end, ES cell colonization, growth and Oct-4 immunoreactivity following a 48 h culture period upon poly((D,L)-lactide), poly((L)-lactide), poly(glycolide) and poly((D,L)-lactide-co-glycolide) (PLGA) were assessed. By the analysis of live and dead cell number indices and Oct-4 immunoreactivity, ES cell colonization rate during a 48 h culture period was found to be significantly greater on PLGA compared to all the other unmodified poly(alpha-hydroxy esters) tested. Surface treatment of all polymers with 0.1m potassium hydroxide revealed a significant increase in ES cell live numbers when compared to all unmodified polymers, thus revealing a correlation between polymer content, hydrophilicity and colonization rate. These data suggest that surface treated poly(alpha-hydroxy esters) may be employed for ES cell scale up procedures and in tissue engineering applications requiring the colonization of scaffolds by ES cells in an undifferentiated state. According to such applications, once the designated scaffold has been colonized, ES cell directed differentiation into the desired and fully differentiated, functional adult tissue may then be effected.

Sintered hydroxyfluorapatites--IV: The effect of fluoride substitutions upon colonisation of hydroxyapatites by mouse embryonic stem cells.

Biodegradable scaffolds serve a central role for tissue engineering scaffolds and guiding tissue regeneration. Some of these scaffolds, including apatites, display a significant effect upon cell adhesion and cell proliferation. The incorporation of scaffold technology with the developing embryonic stem (ES) cell field and the capacity of ES cells for self-renewal and differentiation are believed to hold enormous potential for applications in biomedical research and regenerative medicine. The purpose of this work was to determine the effect of hydroxyapatite (HAP) and fluoride substitutions of HAP upon ES cell growth and colonisation. Sintered hydroxyfluorapatite discs were found to support cellular proliferation and colonisation, and the ES cells displayed a tendency for differentiation on the apatite surface as determined by reductions in colony Oct4 immunoreactivity. Fluoride-containing HAPs were found to provide equivalent support to gelatin in terms of cell numbers, yet superior support for cellular colonisation when compared to HAP. This study indicates that fluoride substitutions of HAP may represent a viable strategy for the development of certain engineered tissue replacements and tissue regeneration systems using ES cells.

Monoclonal antibody against a sperm antigen Mr 95,000 inhibits attachment of human spermatozoa to the zona pellucida.

A murine monoclonal antibody raised against hamster spermatozoa was found to cross-react with human spermatozoa. By immunofluorescence, the antigen was visualized over the equatorial segment of human sperm heads. In the presence of antibody, sperm binding to the zona pellucida of salt-stored human oocytes was significantly inhibited (P less than or equal to 0.005) compared with other antibodies or control preparations. Using SDS-PAGE of whole spermatozoa and membrane preparations followed by Western blot analysis, the antigen was identified as a determinant with a relative molecular weight of 95,000.

Cryopreservation of human embryos.

Studies on the cryopreservation of ninety-seven 4-cell and 8-cell human embryos indicate that morphologic survival can be achieved by means of two different cryoprotectants and two different freezing procedures. To date, pregnancies can be achieved after freezing and thawing of 8-cell human embryos cooled at 0.3 degree C per minute to -80 degrees C in the presence of 1.5 molar dimethyl sulfoxide (DMSO) and thawed at +8 degrees C per minute from -80 degrees C to +4 degrees C. By this procedure, 27 of 47 (57%) embryos frozen survived with 50% or more of their blastomeres intact. The transfer of these 27 embryos to 22 patients resulted in five pregnancies (22%). Morphologic survival of 4-cell and 8-cell human embryos after freezing and thawing is not affected by slight irregularities in blastomere size or the presence of small cytoplasmic fragments. Light and electron microscopic examination of fixed specimens indicates a good correlation between the appearance of frozen-thawed embryos at the dissecting microscope level and the extent of cryoinjury.

Experimental models for ovarian tissue and immature follicles.

Primordial and growing follicles are abundant within the ovaries of healthy young female mammals. While our understanding of follicular dynamics is based mainly on studies of normal ovaries in intact animals, techniques such as ovarian grafting and in vitro culture, in particular when used in combination with cryopreservation, have provided both significant additional insights and a source of mature oocytes. Primordial follicles are small, quiescent, and most commonly located within the collagen-rich outer portion of the ovary. Providing that appropriate collection, handling, freezing, and thawing methods are used, they can tolerate cryopreservation very well irrespective of whether they are frozen within a whole ovary (small species), as ovarian pieces, or as individual, isolated, follicles. Animal studies show that grafts of such fresh or frozen materials can, providing that they contain viable follicles, form mature fertilizable oocytes, produce hormones, and support pregnancies to term. Grafts can be returned to the original donor (autograft), but grafting between histocompatible individuals of the same species and between species (xenografts) is also possible. Cryopreservation and grafting are therefore useful both as practical and as experimental tools. Clinically, ovarian tissue has started to be collected and frozen for patients who are at risk of ovarian failure. Very recent case reports show that such frozen ovarian tissue autografts can support the return of menses and antral follicle formation in patients, although as yet no pregnancies have been established.

Simplified technique for differential staining of inner cell mass and trophectoderm cells of mouse and bovine blastocysts.

Histological staining and counting of blastocyst inner cell mass (ICM) and trophectoderm (TE) cells differentially with chromatin-specific dyes is a more accurate indicator of cultured blastocyst quality and normality than total cell number assessment. The aim of this study was to test the effectiveness of a simplified method of chemically-defined differential blastocyst staining. The TE of cultured mouse and bovine blastocysts of different developmental stages was stained when blastocysts were treated with a permeabilizing solution containing the ionic detergent Triton X-100 and the fluorochrome propidium iodide. Blastocysts were then incubated in a second solution containing 100% ethanol (for fixation) and the secondary fluorochrome bisbenzimide. Fixed and stained whole blastocysts were mounted and assessed for cell number using ultraviolet fluorescent microscopy. Using this method, in-vitro cultured mouse blastocysts (day 4.5) were shown to have an ICM:TE ratio of 1:2.63 with an average total cell count of 75.3 +/- 3. While day 7 and 8 in-vitro produced bovine blastocysts were shown to have an ICM:TE ratio of 1:3.42 and 1:3.36 with an average total cell count of 151.3 +/- 5.48 and 217.8 +/- 8.75 respectively. Blastocyst staining patterns indicate that this modified technique represents a simple and reliable alternative to current bichromatic blastocyst staining techniques for the differential assessment of cell numbers and may be useful for the assessment of blastocysts derived from in-vitro maturation, novel culture systems and advanced reproductive technologies such as cloning.

Staining of the inner acrosomal membrane of human spermatozoa with concanavalin A lectin as an indicator of potential egg penetration ability.

OBJECTIVE: To determine the sensitivity and functional significance of the fluorescein isothiocyanate concanavalin A (FITC-ConA) staining method of assessment of acrosomal status. DESIGN: Treatments were assessed for their ability to induce human sperm acrosomal loss. Penetration of zona-free human eggs by treated spermatozoa was subsequently determined. SETTING: Zona-free human eggs were obtained from the Infertility Medical Centre, Richmond, Victoria, Australia. PATIENTS, PARTICIPANTS: None. INTERVENTIONS: None. MAIN OUTCOME MEASURE: Acrosomal loss of human spermatozoa was determined at 4 hours and 10 hours of treatment incubation. Penetration of zona-free human eggs was assessed 16 hours after reinsemination. RESULTS: Human spermatozoa incubated in a strontium- or lanthanum-based medium, or T6 + 10% maternal human serum (HS) supplemented with 12 mM 8-bromo cyclic guanosine 3,5'-monophosphate and 10 mM imidazole for a 4-hour period before transfer to fresh T6 + 10% HS for a further 6 hours, demonstrated a significant increase (P less than 0.05) in acrosomal loss compared with T6 + 10% HS for a total 10-hour incubation. This increase in acrosomal loss with test treatments correlated with an increase in the development of pronuclei of zona-free human eggs (r = +0.98). CONCLUSIONS: The FITC-ConA staining procedure therefore reflects biological function as assessed by the penetration of zona-free human eggs and consequently provides a further research tool for the investigation of the human sperm acrosome reaction.

Relationship of fine structure of sperm head to fertility of frozen human semen.

Damage to the plasma membrane and acrosome of human spermatozoa was quantitated following dilution, freezing, and thawing. Semen was obtained from 23 donors of known fertility attending an artificial insemination program. Dilution of semen with the cryoprotective medium containing glycerol significantly altered all the fine-structural and semen parameters studied. Membranes of the sperm head were severely altered after freezing and thawing. The percentage of spermatozoa with intact plasma membrane, with intact acrosomes, or with intact or swollen acrosomes was significantly reduced in frozen/thawed semen, compared with fresh semen samples. The percentage of spermatozoa with an intact plasma membrane and acrosome was significantly and positively correlated with the fertility of frozen donor's semen used for insemination (r = 0.6, P less than 0.001). The deleterious effects observed in the sperm head membranes following dilution and freezing and thawing may explain why generally the fertility of frozen/thawed semen is lower than that of fresh semen and may also explain individual variation in fertility of donors after insemination of frozen semen.

The influence of seminal characteristics on the success rate of human in vitro fertilization.

The relationship of conventional semen parameters and the limits of these parameters for fertilization in vitro were analyzed from data over a 3-year period (1980 to 1982). Sperm motility was the single most important parameter determining the fertilization rate. Fertilization failed when the initial and final motilities were less than 20% and 30%, respectively. The percentage of abnormal sperm forms was also significantly related to the fertilization rate; but even when there were greater than 60% abnormal spermatozoa, fertilization could be obtained. Sperm concentration in semen had no significant effect on the fertilization rate when the data were controlled for motility or abnormal sperm forms. The fertilization rate increased with reduced sperm numbers used for insemination in vitro but had no effect on the incidence of multiple pronuclei in oocytes.

Removal of the cumulus oophorus from the human oocyte for in vitro fertilization.

Removal of cumulus may increase the chance of fertilization in patients with sperm antibodies, may facilitate fertilization in vitro with a small number of spermatozoa, and is necessary for microsurgical injection procedures in vitro. The aim of this study was to establish whether removal of the cumulus has any detrimental effects on the fertilization rate and embryo viability. Removal of cumulus cells from the human oocyte with bovine testicular hyaluronidase did not interfere with fertilization, early embryonic development, or pregnancy. This suggests that human spermatozoa can spontaneously undergo capacitation and fertilize oocytes in vitro in a chemically defined medium containing 10% preovulatory human serum. In three patients with low-quality semen, removal of the cumulus and the addition of hypotaurine and epinephrine apparently did not improve the motility nor the fertilizing capacity of the spermatozoa. Delayed fertilization and cleavage arrest was observed in one patient when spermatozoa obtained from the body region of the epididymis was used for insemination.

Human seminal lectin. I. Demonstration and association with male infertility.

A lectin-like hemagglutinin, human seminal lectin (HSL), has been demonstrated in human seminal plasma. It appears to be naturally secreted by all parts of the male reproductive system. High HSL activity was associated with infertility. HSL agglutinated erythrocytes from all of the species tested and agglutination was enhanced by trypsinization of erythrocytes. HSL activity was specifically but weakly inhibited by sugars containing a galactose moiety and was sedimentable by ultracentrifugation. As HSL had properties similar to decapacitation factors, it may play a role in fertilization and could be one of the causes of male infertility.