Zygotes were collected from superovulated Dutch-belted rabbits 19 h after injection of LH and insemination. Oocytes that appeared to be unfertilized were discarded. The zygotes were distributed equally within each donor female across all culture treatments. Culture dishes contained 500 μl of macromolecule-free RD medium consisting of equal parts of RPMI 1640 and low glucose Dulbecco's modified Eagle's medium. Embryos were cultured at 39°C in several gas combinations of N2 plus the following: (1) 1% O2:10% CO2; (2) 5% O2:10% CO2; and (3) 20% O2:10% CO2. The control (4) was 95% air:5% CO2. The experiment was replicated with embryos from 11 donors providing 295 usable zygotes. After 84 h of culture, the percentages of blastocysts formed in treatments 1 to 4, respectively, were 13, 86, 82 and 59 (P < 0.01). The corresponding mean cell counts, including all cleaved embryos cultured (but not degenerate ones), were 55, 183, 118 and 68 (P < 0.01). These results indicate that 10% CO2 combined with 5% O2 is a more effective gas phase for culturing rabbit zygotes in a synthetic medium than is the commonly used 5% CO2, and that 5% O2 is superior to either 1% or 20% O2.
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- Abstract: IVM x
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P. B. Farrell and R. H. Foote
I Parrilla, J M Vázquez, C Cuello, M A Gil, J Roca, D Di Berardino, and E A Martínez
Sex selection by flow cytometry/cell sorting involves the staining of spermatozoa with Hoechst 33342 in combination with the impact of a u.v. laser beam, two potentially mutagenic agents. A phenotypic and cytogenetic study of lymphocytes of piglets born after insemination with spermatozoa stained with Hoechst 33342 and from piglets obtained from stain-sorted spermatozoa was performed to evaluate the genotoxic effect of Hoechst 33342 staining and u.v. laser irradiation on the offspring. Lymphocytes from piglets born after insemination with unstained spermatozoa, but from the same ejaculate, were used as a control group. Peripheral blood lymphocytes from these piglets were cultured following a standard cell culture protocol. Cells were then collected by centrifugation, subjected to hypotonic solution and fixed and dropped onto slides. Sister chromatid exchanges (SCEs) and chromosome aberrations (CAs: including chromosome and chromatid breaks) per cell were scored in 50-s division metaphase spreads from each donor. Reproductive parameters and litter performance of all inseminations performed were also recorded in all groups. Data were analyzed by ANOVA. No significant increase (P > 0.05) of SCE and CA frequencies were observed in piglets born from stained spermatozoa or from stain-sorted spermatozoa with respect to controls (untreated sperm). The results indicated that no mutagenic effect on spermatozoa, expressed as increases in the incidence of abnormalities in the resulting offspring and also as increases in SCE and CA frequencies on lymphocytes from these individuals, was induced by the staining of boar spermatozoa with Hoechst 33342, nor by combination of staining with laser impact during flow cytometry.
Francesca E Duncan, Mary Zelinski, Alexander H Gunn, Jennifer E Pahnke, Conor L O’Neill, Nucharin Songsasen, Ryan I Woodruff, and Teresa K Woodruff
Primordial follicles dictate a female’s reproductive life span and therefore are central to fertility preservation for both endangered species and individuals with fertility-threatening conditions. Ovarian tissue containing primordial follicles can be cryopreserved and later thawed and transplanted back into individuals to restore both endocrine function and fertility. Importantly, increasing numbers of human live births have been reported following ovarian tissue cryopreservation and transplantation. A current limitation of this technology is patient access to sites that are approved or equipped to process and cryopreserve ovarian tissue – especially in larger countries or low resource settings. Here, we review empirical evidence from both animal models and human studies that suggest that ovarian tissue can be transported at cold temperatures for several hours while still maintaining the integrity and reproductive potential of the primordial follicles within the tissue. In fact, several human live births have been reported in European countries using tissue that was transported at cold temperatures for up to 20 h before cryopreservation and transplantation. Ovarian tissue transport, if implemented widely in clinical practice, could therefore expand both patient and provider access to emerging fertility preservation options.
Evelyn E Telfer
Ovarian cryopreservation rapidly developed from basic science to clinical application and can now be used to preserve the fertility of girls and young women at high risk of sterility. Primordial follicles can be cryopreserved in ovarian cortex for long-term storage and subsequently autografted back at an orthotopic or heterotopic site to restore fertility. However, autografting carries a risk of re-introducing cancer cells in patients with blood-born leukaemias or cancers with a high risk of ovarian metastasis. For these women fertility restoration could only be safely achieved in the laboratory by the complete in vitro growth (IVG) and maturation (IVM) of cryopreserved primordial follicles to fertile metaphase II (MII) oocytes. Culture systems to support the development of human oocytes have provided greater insight into the process of human oocyte development as well as having potential applications within the field of fertility preservation. The technology required to culture human follicles is extremely challenging, but significant advances have been made using animal models and translation to human. This review will detail the progress that has been made in developing human in vitro growth systems and consider the steps required to progress this technology towards clinical application.
C Yding Andersen, L S Mamsen, and S G Kristensen
Ovarian tissue cryopreservation (OTC) is mainly used for fertility preservation in girls and women facing a gonadotoxic treatment. If the woman subsequently becomes menopausal, the ovarian tissue may be transplanted to regain ovarian function, including fertility. The method was developed more than two decades ago and today thousands of women worldwide have undergone OTC. Fewer than 500 patients have had tissue transplanted and close to 100% of those regain ovarian function. Several technical aspects of OTC are now becoming more established, including high quantitative follicle survival, defining the size of the tissue resulting in optimal tissue revascularisation and follicle loss resulting from transport of ovarian tissue prior to freezing. We have used OTC to safeguard fertility in patients with genetic diseases, which for some diagnoses is purely experimental, as no transplantations is yet been performed. Usage of OTC beyond fertility is now also being considered; here, the endocrine function of follicles is the focus. It has been suggested that ovarian tissue stored in the reproductive years may be used to avoid premature ovarian insufficiency (POI) when there is a familial disposition or to postpone menopause in patients with an increased risk of osteoporosis or cardiovascular diseases. The benefit of OTC beyond fertility requires, however, actual clinical studies. The current review includes several recent technical aspects with contributions from Denmark building on some of the early work by Roger Gosden.
Federica Zacchini, Roberta Arena, Adam Abramik, and Grazyna E Ptak
Preimplantation genetic diagnosis (PGD) has been introduced in clinical practice as a tool for selecting ‘healthy’ embryos before their transfer in utero. PGD protocols include biopsy of cleaving embryos (blastomere biopsy (BB)) or blastocysts (trophectoderm biopsy (TB)), followed by genetic analysis to select ‘healthy’ embryos for transfer in utero. Currently, TB is replacing the use of BB in the clinical practice. However, based on the European Society of Human Reproduction and Embryology Preimplantation Genetic Diagnosis Consortium reports, BB has been used in >87% of PGD cycles for more than 10 years. An exhaustive evaluation of embryo biopsy (both BB and TB) risks and safety is still missing. The few epidemiological studies available are quite controversial and/or are limited to normalcy at birth or early childhood. On the other hand, studies on animals have shown that BB can be a risk factor for impaired development, during both pre- and postnatal life, while little is known on TB. Thus, there is an urgent need of focused researches on BB, as it has contributed to give birth to children for more than 10 years, and on TB, as its application is significantly growing in clinical practice. In this context, the aim of this review is to provide a complete overview of the current knowledge on the short-, medium- and long-term effects of embryo biopsy in the mouse model.
J. M. Wallace, J. J. Robinson, and R. P. Aitken
Summary. After lambing in late November, oestrus and ovulation were induced by using a CIDR device and PMSG in early weaned (N = 13) or lactating (N = 14) Border Leicester × Scottish Blackface ewes between 23 and 29 days after parturition. Ewes were intrauterine inseminated under laparoscopic visualization 54–55 h after CIDR-device withdrawal and eggs recovered on Day 3 of the cycle. Ovum recovery and fertilization rates were higher in lactating than in early weaned ewes, with fertilization being achieved as early as 24 days post partum in both groups. Of the 7 early weaned and 11 lactating ewes yielding eggs, fertilization occurred in 4 and 7 ewes respectively. A total of 20 embryos were transferred to the normal uterine environment of 15 recipient ewes in which the interval from parturition was > 150 days. Pregnancies were successfully established in 9 recipient ewes, resulting in the birth of 10 viable lambs.
Prolactin concentrations were significantly higher (P < 0·001) in lactating than in early weaned ewes throughout the study. Nevertheless, normal luteal function (as assessed by daily progesterone concentrations) was exhibited by 12 of 14 lactating and 8 of 13 early weaned ewes. Two post-partum donors in which the corpora lutea completely failed to secrete progesterone yielded fertilized eggs which developed to term when transferred to a normal uterine environment.
The results show that sheep oocytes can be fertilized using laparoscopic intrauterine insemination as early as 24 days after parturition and that the resulting embryos are viable when recovered on Day 3 after oestrus and transferred to a normal uterine environment.
Keywords: post partum; fertilization; embryo viability; pregnancy; sheep
Jianming Li and Robert H. Foote
Embryos were collected from superovulated Dutch rabbits 19 h after injection of LH and insemination. The embryos were at the one-cell stage at that time and those judged to be normal by the absence of granular cytoplasm and regular shape were distributed randomly within donors into culture dishes containing 500 μl of a macromolecule-free medium consisting of RPMI-1640 and low glucose Dulbecco's modified Eagle's medium, 1:1, without a cover of oil. In Expt 1, O2 concentrations of 5, 10 and 15%, with 5% CO2 plus 90, 85 and 80% N2, respectively, were tested. In Expt 2, O2 concentrations of 1, 5 and 20% were combined with 5% CO2 and the remaining gas was N2. After 84 h in culture at 39°C, embryos were examined for stage of development and stained with Hoechst 33342 so that the number of cells could be counted. In Expt 1, the proportion of embryos reaching the hatching blastocyst stage after 84 h in culture in 5, 10 and 15% O2 was 48, 38 and 21% (P < 0.01) and the cell number per embryo averaged 258, 226 and 188, respectively (P < 0.01). In Expt 2, the proportion of hatching embryos after 84 h in culture in 1, 5 and 20% O2 was 67, 72 and 29% (P < 0.01), respectively. Cell numbers in the 1 and 5% O2 concentrations were higher than in the 20% O2 concentration (P < 0.01). These results indicate that reduction of O2 concentration to 5%, well below the frequently used concentration of about 20% O2 in 95% air, is beneficial to rabbit embryo development from the zygote to the blastocyst stage. The O2 concentration may be more critical with simple defined macromolecule-free media than in media containing serum.
Roger G Gosden
Ovarian and uterine transplantation are procedures gaining more attention again because of potential applications in respectively fertility preservation for cancer and other patients and, more tentatively, women with uterine agenesis or hysterectomy. Cryopreservation of tissue slices, and possibly whole organs, is providing opportunities for banking ovaries for indefinite periods before transplanting them back to restore fertility. The natural plasticity of this organ facilitates grafting to different sites where they can be revascularized and rapidly restore the normal physiology of secretion and ovulation. Ischemic damage is a chief limitation because many follicles are lost, at least in avascular grafts, and functional longevity is reduced. Nevertheless, grafts of young ovarian tissue, even after cryopreservation, can be highly fertile in laboratory rodents and, in humans, autografts have functioned for up to 3 years before needing replacement. Transplantation by vascular anastomosis provides potentially longer function but it is technically much more demanding and riskier for the recipient. It is the only practicable method with the uterus, and has enabled successful pregnancies in several species, but not yet in humans. Contrary to claims made many years ago, neither organ is privileged immunologically, and allografts become rapidly rejected except in hosts whose immune system is deficient or suppressed pharmacologically. All in all, transplantation of these organs, especially the ovary, provides a broad platform of opportunities for research and new applications in reproductive medicine and conservation biology.
R A Anderson
Human fertility is dependent on maturation of germ cells through meiosis and their association with supporting cells, which in the female are also the source of sex steroids. These processes are sensitive to both chemotherapy and radiotherapy thus can be damaged by anti-cancer treatments. The uterus is also sensitive to radiotherapy. Our understanding of and the ability to manipulate fertility has increased together with survival rates from many cancers, particularly those affecting children, younger men, and women. The growth of interest in fertility preservation for cancer patients is a natural union of these two fields. Sperm banking has been available for many years, and is a recognized and evidence-based option for men that should be available to all. Options for women and pre-pubertal boys and girls are, however, more experimental, other than for women of committing oocytes to fertilization and cryopreservation as embryos. This Focus Issue of Reproduction aims to address the current status of some of the clinical and laboratory aspects of this burgeoning subspecialty to highlight not only areas of progress but also areas of uncertainty where future developments are required to allow the provision of accurate information, and safe and effective treatments.