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.

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.

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.

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.

Ovarian transplantation between monozygotic twins discordant for premature ovarian failure.

Monozygotic 24-year-old twins presented with discordant ovarian function. One had had premature ovarian failure at the age of 14 years, whereas her sister had normal ovaries and three naturally conceived children. After unsuccessful egg-donation therapy, the sterile twin received a transplant of ovarian cortical tissue from her sister by means of a minilaparotomy. Within three months after transplantation, the recipient's cycles resumed and serum gonadotropin levels fell to the normal range. During the second cycle, she conceived, and her pregnancy progressed uneventfully. At 38 weeks' gestation, she delivered a healthy-appearing female infant.

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.

Human LINE-1 retrotransposon induces DNA damage and apoptosis in cancer cells.

BACKGROUND: Long interspersed nuclear elements (LINEs), Alu and endogenous retroviruses (ERVs) make up some 45% of human DNA. LINE-1 also called L1, is the most common family of non-LTR retrotransposons in the human genome and comprises about 17% of the genome. L1 elements require the integration into chromosomal target sites using L1-encoded endonuclease which creates staggering DNA breaks allowing the newly transposed L1 copies to integrate into the genome. L1 expression and retrotransposition in cancer cells might cause transcriptional deregulation, insertional mutations, DNA breaks, and an increased frequency of recombinations, contributing to genome instability. There is however little evidence on the mechanism of L1-induced genetic instability and its impact on cancer cell growth and proliferation. RESULTS: We report that L1 has genome-destabilizing effects indicated by an accumulation of gamma-H2AX foci, an early response to DNA strand breaks, in association with an abnormal cell cycle progression through a G2/M accumulation and an induction of apoptosis in breast cancer cells. In addition, we found that adjuvant L1 activation may lead to supra-additive killing when combined with radiation by enhancing the radiation lethality through induction of apoptosis that we have detected through Bax activation. CONCLUSION: L1 retrotransposition is sensed as a DNA damaging event through the creation DNA breaks involving L1-encoded endonuclease. The apparent synergistic interaction between L1 activation and radiation can further be utilized for targeted induction of cancer cell death. Thus, the role of retrotransoposons in general, and of L1 in particular, in DNA damage and repair assumes larger significance both for the understanding of mutagenicity and, potentially, for the control of cell proliferation and apoptosis.

Comparative maturation of cynomolgus monkey oocytes in vivo and in vitro.

BACKGROUND: In vitro maturation (IVM) of oocytes followed by fertilization in vitro (IVF) and embryo transfer offers an alternative to conventional IVF treatment that minimises drug administration and avoids ovarian hyperstimulation. However, the technique is less efficient than maturation in vivo. In the present study, a non-human primate model was used to address the hypothesis that the number of oocytes is increased and their nuclear and cytoplasmic maturity after IVM are improved when maturation is initiated in vivo by priming with hCG. METHODS: Young, adult cynomolgus monkeys were given recombinant human (rh) gonadotropins to stimulate the development of multiple follicles, and oocytes were aspirated 0, 12, 24, or 36 h after injection of an ovulatory dose of rhCG. The nuclear status of oocytes was determined at the time of recovery and after culture for a total elapsed time of 40-44 hours after hCG. RESULTS: Priming with hCG significantly increased the number of oocytes harvested, especially after delaying aspiration for 24 h or longer. Nuclear maturation after the full period in culture was also enhanced by priming: 71.5, 83.6, and 94.6% of oocytes collected at 0, 12, and 24 h hCG had progressed to MII by the end of the culture period, compared to 87.8% of oocytes that were retrieved at 36 h. A large proportion of oocytes reaching the MII stage had either or both abnormal spindles (>40%) and misaligned chromosomes (>60%), judging by immunofluorescence microscopy, but these abnormalities were independent of culture time. The mitochondria were evenly distributed throughout the cytoplasm at all stages of maturation. Importantly, there was no microscopic evidence that the duration of culture had any injurious effects on the cells. CONCLUSION: In conclusion, the evidence supports this non-human primate as a model for human IVM and the practice of priming with hCG to promote developmental potential.

Prospects for oocyte banking and in vitro maturation.

There is little debate about the desirability of human oocyte ("egg") banking but plenty of discussion about its prospects. Egg banking is needed by young cancer patients before they undergo potentially sterilizing treatment and is a desirable alternative to in vitro fertilization and embryo cryopreservation. However, egg banking is inefficient-oocytes are sensitive to chilling, often fail to survive freeze-thawing, and are susceptible to cytoskeletal damage and aneuploidy. Currently, even the most optimistic success rates offer patients only a slim chance of pregnancy if few oocytes are available. Ultra-rapid freezing with vitrification may offer advantages over conventional equilibrium cooling protocols and needs to be investigated further. Likewise, freezing immature oocytes followed by in vitro maturation offers practical and theoretical advantages, but this method is still inefficient. Nevertheless, all these technologies are improving, and egg banking will eventually become an option for patients seeking fertility preservation.

Quantitative assessment of ischemic tissue damage in ovarian cortical tissue with or without antioxidant (ascorbic acid) treatment.

OBJECTIVE: To estimate ischemic tissue damage in ovarian cortex and to evaluate the effectiveness of ascorbic acid, an antioxidant, to protect ovarian tissue from apoptosis caused by ischemia. DESIGN: In vitro laboratory experiments. SETTING: Academic research institute. INTERVENTION(S): Fresh and frozen/thawed cortical sections of bovine ovaries were incubated in MEM medium with or without ascorbic acid for a duration of 3, 24, and 48 hours at 37 degrees C. MAIN OUTCOME MEASURE(S): Oxygen consumption rates, lactate dehydrogenase concentrations, apoptosis rates determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining, and DNA fragmentation analysis. RESULT(S): The oxygen consumption rates were correlated inversely with the duration of incubation. When the rates of apoptosis in primordial follicles with or without ascorbic acid treatment were compared, there was no statistically significant difference between the two groups. However, the ascorbic acid treatment group showed significantly decreased apoptosis in ovarian cortex (stromal cells) with 24 hours of incubation. CONCLUSION(S): The correlation between ischemic tissue damage and the duration of ischemia was verified. Ovarian cortex could tolerate ischemia at least for 3 hours. Ascorbic acid treatment reduced apoptosis in ovarian cortex up to 24 hours of incubation in vitro. It appeared that stromal cells were more vulnerable to ischemia compared to primordial follicles.

Application of traditional Chinese medicine in the treatment of infertility.

The philosophy and practice of traditional Chinese medicine (TCM) have been evolving for thousands of years in China, Japan and other Asian countries. TCM is now generating popular interest worldwide for reproductive health care and disease prevention, including applications for treating infertility and improving sexual function. This review focuses on the application of TCM for infertility patients, and provides a critical reflection on the efficacy and safety of selected Chinese herbal formulas. It has been claimed that some formulas produce high clinical pregnancy rates with few or no side effects, as well as improving the general well-being of patients. The need for randomized control trials and research into possible mechanisms of action, effective doses, contra-indications and toxicity is self-evident. However, the task is enormous in view of the number of herbal products currently available on the market; yet among these products are undoubtedly some that will prove to be safe and beneficial.

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.