A demographic projection of the contribution of assisted reproductive technologies to world population growth.

Enormous unmet needs for infertility treatment exist because access to assisted reproductive technologies is demographically skewed. Since the first IVF baby in 1978, the number of people conceived by reproductive technology has grown much faster than expected, reaching several million today and rapidly approaching 0.1% of the total world population. As more patients build families, and their children in turn become parents, the number owing their existence to assisted reproductive technologies, either directly or indirectly, will expand tremendously in future decades, but no attempts have been made hitherto to project the magnitude. We have projected growth to the year 2100, along with the fractional contribution to world population. The chief variable driving growth is access to fertility services. If it stagnates at current levels of about 400,000 babies per year, an estimated 157 million people alive at the end of the century will owe their lives to assisted reproductive technologies (1.4% of global population), but at an arbitrary upper limit of 30,000 extra births annually there will be 394 million additional people alive (3.5%). As the conquest of infertility continues, individuals who owe their lives to assisted reproductive technologies will quietly make a significant contribution to demographic growth as well as social progress.

General principles of cryopreservation.

Cryopreservation quickly became a cornerstone technology in assisted reproduction because the banking of gametes, embryos, and gonadal tissues has increased the effectiveness of assisted reproductive technology cycles for infertility treatment as well as fertility preservation for patients at risk of premature sterilization. Cryopreservation protocols, both slow/equilibrium cooling and vitrification methods, have evolved empirically and still depend heavily on operator skill, but further automation promises to improve reproducibility and uniformity of results.

Oocyte development and loss.

Soon after implantation, cell lineages bifurcate into future somatic and germ cells. Canonical expression of germ cell-specific genes continues during and after their migration to the gonadal ridge, where, after further cell cycles, they enter meiosis, form syncytial clusters, and arrest at diplotene for folliculogenesis. The balance between cell survival and death leaves an ovarian follicular reserve as a legacy.

Programmes and prospects for ovotechnology.

Oocytes hold enormous significance in biology, pathology and ageing, but they are exceedingly rare cells in adults. According to a theory almost universally held until recently, expansion of the germline is halted perinatally when oogonial stem cells differentiate to form to primordial follicles. Thus, there is a finite follicle store which becomes exhausted around the time of menopause because the great majority is lost by atresia instead of undergoing ovulation. While FSH treatment can rescue a few follicles that would otherwise degenerate, in-vitro growth together with in-vitro maturation can potentially yield much larger harvests of oocytes, up to the limited number of primordial follicles available. If, however, germline stem cells persist after birth, enabling new oocytes to be generated, oogenesis is no longer bound by an upper limit. Even if they disappeared, as current theory prescribes, proliferative germline cells might be created de novo either from embryonic stem cells or through induced pluripotent stem cell technology. This paper reviews the prospects for a new road map for discovery research aimed at creating technologies to overcome the shortage of oocytes, which would have a revolutionary impact on IVF treatment, egg donation, premature ovarian insufficiency and regenerative medicine.

Memoir of fertility preservation.

Fertility preservation has been practiced for at least 50 years using semen banking, pelvic surgery, and radiation shields, but in the past 20 years it has emerged as a rapidly growing subspecialty of reproductive medicine. A dramatic rise in survivorship of young cancer patients and the widespread postponement of family building to the later years of the female reproductive lifespan have been major driving forces. Throughout the history of fertility preservation, low temperature banking has played a pivotal role, first for gametes and later for embryos and immature germ cells, while ovarian transplantation recently began to contribute and spermatogonial stem cell transfer holds future promise for men and prepubertal boys. But there are significant risks with some diseases from reimplanting residual disease, which hopefully can be eliminated by new methods for purging the tissue and germ cell culture. Since all technologies are interim, cryopreservation as a mainstay in this field will likely be swept aside eventually by a stream of progress aimed at managing fertility preservation in vivo.

Eggs come in from the cold.

Eggs can be 'forever'. A longstanding goal in assisted reproductive technology (ART) has been realized at last, namely, cryopreservation of oocytes by vitrification technology. This breakthrough heralds benefits for infertility treatment, fertility preservation, and even postponement of reproduction but, as so often with ARTs, new waves of technology draw ethical and societal concerns in their wake.

Imatinib mesylate at therapeutic doses has no impact on folliculogenesis or spermatogenesis in a leukaemic mouse model.

Imatinib should be avoided in women planning to become pregnant or during pregnancy, due to a higher risk of congenital malformations. However, it is not known whether imatinib affects future potential for fertility. Here we analysed ovaries and testes from adult mice receiving imatinib, focusing on testicular and ovarian functions. Seven male and 7 female mice were orally treated with 150 mg/kg body weight/day imatinib for two months. No effects on folliculogenesis or spermatogenesis could be observed postmortem by histological examinations, suggesting that, at least in two mouse models of imatinib treatment this tyrosine kinase inhibitor does not reduce fertility.

The Src homology 2 domain-containing adapter protein B (SHB) regulates mouse oocyte maturation.

SHB (Src homology 2 domain-containing adapter protein B) is involved in receptor tyrosine kinase signaling. Mice deficient in the Shb gene have been found to exhibit a transmission ratio distortion with respect to inheritance of the Shb null allele among offspring and this phenomenon was linked to female gamete production. Consequently, we postulated that Shb plays a role for oocyte biology and thus decided to investigate oocyte formation, meiotic maturation, and early embryo development in relation to absence of the Shb gene. Oogenesis was apparently accelerated judging from the stages of oocyte development on fetal day 18.5 and one week postnatally in Shb -/- mice; but in adulthood ovarian follicle maturation was impaired in these mice. Completion of meiosis I (first polar body extrusion) was less synchronized, with a fraction of oocytes showing premature polar body extrusion in the absence of Shb. In vitro fertilization of mature oocytes isolated from Shb +/+, +/- and -/- mice revealed impaired early embryo development in the -/- embryos. Moreover, the absence of Shb enhanced ERK (extracellular-signal regulated kinase) and RSK (ribosomal S6 kinase) signaling in oocytes and these effects were paralleled by an increased ribosomal protein S6 phosphorylation and activation. It is concluded that SHB regulates normal oocyte and follicle development and that perturbation of SHB signaling causes defective meiosis I and early embryo development.

Robert T. Morris, M.D.--appreciation of an enlightened surgeon and pioneer of ovarian transplantation.

OBJECTIVE: To show that ovarian transplantation is a promising strategy for preserving fertility in young women and children who have cryopreserved tissue before undergoing potentially sterilizing treatment for cancer and other conditions. CONCLUSION: Credit for the first transplants is because of a forgotten pioneer, Robert Morris of New York, who was an innovative surgeon as well as a model clinical investigator over a century ago.

Character, distribution and biological implications of ice crystallization in cryopreserved rabbit ovarian tissue revealed by cryo-scanning electron microscopy.

BACKGROUND: Ovarian tissue banking is an emerging strategy for fertility preservation which has led to several viable pregnancies after transplantation. However, the standard method of slow cooling was never rigorously optimized for human tissue nor has the extent and location of ice crystals in tissue been investigated. To address this, we used cryo-scanning electron microscopy (cryo-SEM) to study ice formation in cryopreserved ovarian tissue. METHODS: Rabbit ovarian tissue slices were equilibrated in 1,2-propanediol-sucrose solution and cooled at either 0.3 degrees C/min or 3.0 degrees C/min after nucleating ice at -7 degrees C, or snap-frozen by plunging in liquid nitrogen. Frozen tissues were fractured, etched and coated with gold or prepared by freeze substitution and sectioning for cryo-SEM. RESULTS: The size, location and orientation of extracellular ice crystals were revealed as pits and channels that had grown radially between freeze-concentrated cellular materials. They represented 60% of the total volume in slowly cooled samples that were nucleated at -7 degrees C and the crystals, often >30 microm in length, displaced cells without piercing them. Samples cooled more rapidly were much less dehydrated, accounting for the presence of small ice crystals inside cells and possibly in organelles. CONCLUSIONS: Cryo-SEM revealed the internal structure of ovarian tissue in the frozen state was dominated by elongated ice crystals between islands of freeze-concentrated cellular matrix. Despite the grossly distorted anatomy, the greater degree of dehydration and absence of intracellular ice confirmed the superiority of a very slow rate of cooling for optimal cell viability. These ultrastructural methods will be useful for validating and improving new protocols for tissue cryopreservation.

Fertility preservation: definition, history, and prospect.

Exposure to gonadotoxic agents for treating cancer and other diseases or postponement of maternity jeopardize the prospects of genetic parenthood and are major factors driving the development of fertility preservation technologies. Although its mainstays, cryopreservation and vitrification, have been adopted from infertility medicine for fertility preservation purposes, there is a need for additional specialized procedures, protocols, and biomedical devices. As its multidisciplinary character and aims diverge compared with other branches of reproductive medicine, we are witnessing the emergence of a field in its own right.

Pyruvate and oxygen consumption throughout the growth and development of murine oocytes.

Growing oocytes in vitro from the most immature stages until they are developmentally competent is a major goal of reproductive technology, requiring fundamental knowledge of metabolic processes. Carbohydrate metabolism and oxygen consumption have been analysed in a series of experiments designed to investigate important energy substrates for mouse oocytes and to reveal any qualitative or quantitative changes between the primordial and ovulatory follicle stages. Primordial follicles were incubated in groups in modified-KSOM medium, whereas growing or ovulated oocytes were studied singly and, in both cases, the depletion or accumulation of metabolites in spent medium were analysed using ultramicrofluorometric assays. The rates of glucose (0.014 +/- 0.006 pmol/hr) and pyruvate (0.028 +/- 0.009 pmol/hr) consumption and l-lactate (0.058 +/- 0.023 pmol/hr) production by primordial follicles suggested that energy production was supported by a combination of metabolic pathways, including glycolysis. Pyruvate and oxygen consumption per oocyte increased two- and ninefold, respectively, between the primary and pre-ovulatory stages (0.82 +/- 0.1 and 1.67 +/- 0.1 pmol pyruvate/hr, respectively and 1.4 +/- 0.3 and 7 +/- 0.6 pmol oxygen/hr) after which oxygen (12.7 +/- 1.1 pmol/hr) utilisation nearly doubled. Oxygen consumption by fully grown oocytes was in excess of oxidation requirements for pyruvate. When pyruvate and oxygen consumption rates were normalised for oocyte cellular volume, which increased over 130-fold during growth, oocyte metabolism was higher in primary follicles than at any subsequent stage, indicating that energy needs are greater during a developmental transition. To conclude, pyruvate and oxygen were consumed throughout oocyte development at increasing rates. When oocyte cellular volume was accounted for, oocytes from primary follicles displayed greatest metabolic rates.

Ovary and uterus transplantation.

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.

Survival of ovarian allografts in an experimental animal model.

Transplantation of ovarian tissue is a promising strategy for fertility preservation in young cancer patients with premature ovarian failure if they have cryopreserved their own tissue before undergoing gonadotoxic treatment. However, extension of ovary donation to children and adults seeking treatment for hypogonadism is controversial unless the tissue does not provoke an immune reaction or specific tolerance can be safely and effectively achieved. The survival of heterotopic ovarian allografts was tested in a mouse model. Isografts were placed under the kidney capsule of ovariectomized animals differing at the H-2 haplotype (H-2d or H-2k). Within three wk, and in contrast to isografts, the allografts were rejected, although their survival was extended when donor and host strains shared the same haplotype (H-2k). Allograft survival was not improved if the tissue was implanted orthotopically. When monoclonal antibodies to CD4 antigens were administered at doses exceeding those effective for long-term tolerance to cardiac allografts, graft survival was prolonged in one of two strain combinations, but they failed to restore fertility. These results indicate that the ovary is not an immunologically privileged organ, as the older literature suggested, and chronic immunosuppression is likely to be required for ovarian allografts in clinical settings.

Carbohydrate metabolism by murine ovarian follicles and oocytes grown in vitro.

Metabolic markers are potentially valuable for assessment of follicle development in vitro. Carbohydrate metabolism of murine preantral follicles grown to maturity over 13 days in vitro has been measured, and metabolism of resulting oocyte-cumulus complexes (OCCs) and denuded oocytes has been compared with in vivo ovulated control counterparts. Spent follicle culture media were analysed for glucose, lactate and pyruvate concentrations. During follicle in vitro growth, glycolysis accounted for a rise from approximately 24 to 60% of all glucose consumed. Ovulation induction caused a significant increase in glucose uptake and lactate production by in vitro-grown follicles to 71.7+/-1.2 and 96.6+/-4.8 nmoles/day respectively. OCCs grown in vitro had significantly higher rates of glucose consumption and lactate and pyruvate production (110.1+/- 3.5, 191.8+/- 8.9 and 31.7+/- 1.7 pmoles/h respectively) than in vivo ovulated controls (67.4+/- 8.1, 113.9+/- 17.1 and 20.2+/- 4.0 pmoles/h respectively), but a reduced capacity for pyruvate consumption (1.13+/- 0.06 vs 1.49+/- 0.06 pmoles/h by in vivo ovulated oocytes). Metabolism of OCCs was affected by the quality of the original follicle. In vitro-grown oocytes had a reduced cytoplasmic volume when compared with controls (168.3+/- 2.0 vs 199.0+/- 3.2 proportionately respectively) but a similar rate of metabolism per unit volume. Meiotic status influenced metabolism of both OCCs and denuded oocytes. In conclusion, glucose consumption and lactate production by cultured follicles increased in tandem with developmental progression and were stimulated prior to ovulation. Additionally, the metabolic profiles of in vitro produced OCCs and the oocytes within them are affected by long-term exposure to the culture environment.