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.

Improving access to ART in low-income settings through knowledge transfer: a case study from Zimbabwe.

It may be assumed that infertility is not a problem in resource-poor areas where fertility rates are high. However, evidence overwhelmingly shows that childlessness is highly stigmatized in these settings and that women who are unable to bear children suffer significant social and psychological consequences. The World Health Organization has recommended that infertility be considered a global health problem and stated the need for ART to be adapted to low-resource settings. This paper describes a model for improving access to ART in low-resource settings. Experienced ART health professionals from Australia and Italy representing medical science, embryology, nursing and counselling used knowledge transfer to support a clinician, a laboratory scientist and a nurse to establish an ART service in Harare, Zimbabwe. Support and mentorship provided between October 2016 and December 2017 included: hosting the clinician and the embryologist for the new service in established ART clinics for short periods and providing them with dedicated mentorship and training during their stay; funding an experienced embryologist to travel to Zimbabwe (three times) to oversee the setting up of the lab and provide hands-on embryology training; funding a scientist and a nurse to travel to Zimbabwe to troubleshoot and establish protocols for record keeping and psychosocial care; and contributing approximately AUD $15,000 to the purchase of some equipment. By 31 March 2018, the team at IVF Zimbabwe had performed 166 ART procedures, which at time of writing had resulted in 16 births and 4 ongoing pregnancies. This case study demonstrates that with mentorship and modest financial support from ART experts from high-income settings, health professionals in low-income settings can deliver affordable ART with successful outcomes.

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.

Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro.

We describe the derivation of pluripotent embryonic stem (ES) cells from human blastocysts. Two diploid ES cell lines have been cultivated in vitro for extended periods while maintaining expression of markers characteristic of pluripotent primate cells. Human ES cells express the transcription factor Oct-4, essential for development of pluripotential cells in the mouse. When grafted into SCID mice, both lines give rise to teratomas containing derivatives of all three embryonic germ layers. Both cell lines differentiate in vitro into extraembryonic and somatic cell lineages. Neural progenitor cells may be isolated from differentiating ES cell cultures and induced to form mature neurons. Embryonic stem cells provide a model to study early human embryology, an investigational tool for discovery of novel growth factors and medicines, and a potential source of cells for use in transplantation therapy.

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.

Human embryonic stem cell derivation and directed differentiation.

Human embryonic stem cells (hESCs) are produced from normal, chromosomally aneuploid and mutant human embryos, which are available from in vitro fertilisation (IVF) for infertility or preimplantation diagnosis. These hESC lines are an important resource for functional genomics, drug screening and eventually cell and gene therapy. The methods for deriving hESCs are well established and repeatable, and are relatively successful, with a ratio of 1:10 to 1:2 hESC lines established to embryos used. hESCs can be formed from morula and blastocyst-stage embryos and from isolated inner cell mass cell (ICM) clusters. The hESCs can be formed and maintained on mouse or human somatic cells in serum-free conditions, and for several passages in cell-free cultures. The hESCs can be transfected with DNA constructs. Their gene expression profiles are being described and immunological characteristics determined. They may be grown indefinitely in culture while maintaining their original karyotype but this must be confirmed from time to time. hESCs spontaneously differentiate in the absence of the appropriate cell feeder layer, when overgrown in culture and when isolated from the ESC colony. All three major embryonic lineages are produced in differentiating attachment cultures and in unattached embryoid bodies. Cell progenitors of interest can be identified by markers, expression of reporter genes and characteristic morphology, and the culture thereafter enriched for further culture to more mature cell types. The most advanced directed differentiation pathways have been developed for neural cells and cardiac muscle cells, but many other cell types including haematopoietic progenitors, endothelial cells, lung alveoli, keratinocytes, pigmented retinal epithelium, neural crest cells and motor neurones, hepatic progenitors and cells that have some markers of gut tissue and pancreatic cells have been produced. The prospects for regenerative medicine are significant and there is much optimism for their contribution to human medicine.

Nuclear transfer of adult and genetically modified fetal cells of the rat.

The present study examines the handling, activation, and micromanipulation of rat eggs in an attempt to produce live young using nuclear transfer (NT) of adult and genetically modified rat fetal cells. Mature rat eggs cultured in calcium-free medium showed reduced rates (24%) of chromosomal dispersion ("spontaneous activation" characteristic of this species) compared with eggs cultured in calcium-containing medium (47%), but failed to survive micromanipulation procedures. High rates of parthenogenetic cleavage were obtained with chemical activation using ethanol/cycloheximide (65%) compared with other standard chemical activation methods (4-28%). This type of activation was also effective in reestablishing cleavage capability (19-71%), in a time-dependent manner, of spontaneously activated eggs arrested at a second prophase-like state. At most, two of four tested micromanipulation procedures were effective in producing NT embryos capable of morula or blastocyst development (14-16%) in vivo following transfer to mouse oviducts. NT blastocysts produced from cumulus cells and transfected rat fetal fibroblasts appeared morphologically and karyotypically normal (2n = 42). Nocodazole-assisted metaphase enucleation and piezoelectric-assisted donor cell injection produced significant and equivocal effects on survival and cleavage rates of reconstructed embryos but failed to significantly improve in vivo morula/blastocyst development rates (16-28%) compared with unassisted micromanipulation (16%). Live births have not yet been obtained from early cleavage stage embryos (n = 269) transferred to pseudopregnant recipient rat oviducts. Improvements in reconstituted NT embryo culture and transfer are required for these methods to be an effective means of transgenic rat production.

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.

Azoospermia associated with a mutation in the ligand-binding domain of an androgen receptor displaying normal ligand binding, but defective trans-activation.

Although male infertility affects a significant proportion of couples trying to conceive, the cause of defective spermatogenesis is not known in a large number of cases. Ligand binding studies indicate that a number of these subjects may have defects of the androgen receptor (AR). Genetic screening in subjects with defective spermatogenesis and in 110 fertile controls identified an azoospermic (no sperm in any ejaculates) patient with an amino acid substitution (Gln-->Glu) in residue 798 of the AR gene. This germline mutation was pathogenic because it was not observed in fertile controls, was associated with features of minimal androgen insensitivity in our patient, has been related to more severe grades of androgen insensitivity, and caused a subtle, but significant, decrease in receptor trans-activation function in vitro that is consistent with the phenotype. Despite being located in the middle of the ligand-binding domain of the receptor, the Q798E mutation did not cause any ligand binding defect, indicating that this highly conserved residue has a trans-activation function but does not directly form part of the ligand binding pocket of the receptor. The trans-activation defect of the mutant receptor can be rectified in vitro with the androgenic drug, fluoxymesterone, but not with mesterolone or nortestosterone. Further studies are required to determine the therapeutic relevance of this finding.

Fine structure of human oogonia in the foetal ovary.

Foetal ovarian tissue is now being cultured or frozen, to generate oocytes for assisted reproduction, an emerging technology. This study examines the ultrastructure of oogonia at 13-15 weeks of gestation, which could be used as a control for culture and freezing of foetal ovaries. Oogonia are largely located in the ovarian cortex, whilst primordial germ cells (PGC) and somatic follicle cells compose the surface epithelium. Oogonia and PGC have large vesicular nuclei with clear cytoplasm, compared to dense follicle cells, which have polymorphic nuclei. Follicle cells intermingle with oogonia and establish close contacts - beginning of folliculogenesis. Nuclei of oogonia contain one to three highly reticulated nucleoli, reflecting high levels of RNA synthesis at the onset of growth. Rough endoplasmic reticulum (RER) form stacks of cisternae associated with numerous ribosomes. Prominent organelles in the ooplasm are elongated mitochondria with dense matrices and tubular cristate presenting a multilocular appearance. Typical Golgi complexes, dense bodies and clear vacuoles are present and microfilaments are located beneath the plasma membrane. The most remarkable feature of oogonia is that they have typical juxtanuclear centrioles (diplosomes) with dense pericentriolar material, which nucleate microtubules, characteristic of functional centrosomes organizing the cytoskeleton. The mature oocyte has no centrioles, since the maternal centrosome is inactivated or reduced, while the paternal is dominant. Centrioles are most likely involved in mitosis of oogonia.

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.

Cell synchronization for the purposes of nuclear transfer in the bovine.

We have examined the reprogramming ability of donor fibroblast nuclei in various phases of the cell cycle, upon transfer to cytoplasts, using a bovine nuclear transfer (NT) model. Bovine fetal fibroblasts were cultured in reduced serum and conditioned medium to induce quiescence (G0) and treated with nocodazole to induce M phase arrest. Unsynchronized actively dividing cells (control) were mainly in G1. Cells synchronized in G0, M, and G1 phase were transferred to enucleated bovine MII oocytes by direct injection using the Piezo-Drill microinjector. NT oocytes were artificially activated following injection. Cells at the M phase were also transferred to enucleated oocytes after artificial activation. Cells induced into quiescence by serum starvation and unsynchronized donor cells produced the highest rates of development to the morula/blastocyst stage (20% and 18%, respectively). Development to blastocyst was significantly higher in parthenogenetic controls compared to NT embryos. The transfer of M phase nuclei to MII cytoplasts was not associated with high development to the blastocyst stage. Nevertheless, determining the viability of these embryos requires transfer to recipient animals and assessment of in vivo development.

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.