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.

Regulation of mouse oocyte microtubule and organelle dynamics by PADI6 and the cytoplasmic lattices.

Organelle positioning and movement in oocytes is largely mediated by microtubules (MTs) and their associated motor proteins. While yet to be studied in germ cells, cargo trafficking in somatic cells is also facilitated by specific recognition of acetylated MTs by motor proteins. We have previously shown that oocyte-restricted PADI6 is essential for formation of a novel oocyte-restricted fibrous structure, the cytoplasmic lattices (CPLs). Here, we show that α-tubulin appears to be associated with the PADI6/CPL complex. Next, we demonstrate that organelle positioning and redistribution is defective in PADI6-null oocytes and that alteration of MT polymerization or MT motor activity does not induce organelle redistribution in these oocytes. Finally, we report that levels of acetylated microtubules are dramatically suppressed in the cytoplasm of PADI6-null oocytes, suggesting that the observed organelle redistribution failure is due to defects in stable cytoplasmic MTs. These results demonstrate that the PADI6/CPL superstructure plays a key role in regulating MT-mediated organelle positioning and movement.

Ovulated oocytes in adult mice derive from non-circulating germ cells.

Decades of research in reproductive biology have led to the generally accepted belief that in female mammals, all surviving germ cells enter meiosis at the end of fetal development and as a result, the postnatal ovary harbours a limited supply of oocytes that cannot be replenished or regenerated if lost to injury or disease. However, recent reports have challenged this view, suggesting instead that oocyte production is maintained through continual seeding of the ovary by circulating, bone-marrow-derived germ cells. To test directly the physiological relevance of circulating cells for female fertility, we established transplantation and parabiotic mouse models to assess the capacity of circulating bone marrow cells to generate ovulated oocytes, both in the steady state and after induced damage. Our studies showed no evidence that bone marrow cells, or any other normally circulating cells, contribute to the formation of mature, ovulated oocytes. Instead, cells that travelled to the ovary through the bloodstream exhibited properties characteristic of committed blood leukocytes.

Egg sharing for assisted conception: a window on oocyte quality.

The steep decline in both natural fertility and success after assisted reproduction treatment with increasing maternal age is universally recognized. Large variations in the developmental competence of oocytes collected are seen during assisted cycles,and a link between the biological competence of oocytes retrieved and age has been confirmed. Patients who require donated oocytes can benefit from egg sharing programmes, in which a proportion of oocytes collected from selected patients aged 35 years undergoing conventional assisted reproduction treatment are shared with a matched recipient. The reproductive outcomes of the egg provider and recipient can thus be compared to quantify the significance of oocyte quality. Data gathered from two comparable treatment centres resulted in 285 pairs of egg sharing providers and their recipients that could be analysed statistically. The chief finding was donor pregnancy as a predictor of recipient pregnancy given embryo transfer (odds ratio 2.15, 95% confidence interval 1.33­3.46, P ≈ 0.002), despite an appreciably higher mean age of the recipients. The probability of a recipient pregnancy increased by almost 0.2. Such results strongly indicate the key importance of oocyte quality for a successful clinical outcome in egg sharing practices and assisted reproduction treatment more generally.

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.

Impact of gonadotrophin stimulation for assisted reproductive technology on ovarian ageing and menopause.

This study investigated whether menopausal age is inversely related to the number of assisted reproduction cycles and/or ampoules of FSH used, as an indication of the possibility that gonadotrophin treatment accelerates follicular depletion. A questionnaire addressing reproductive health, history and treatment was mailed to women who had been treated for infertility at Bourn Hall Clinic, Cambridge, between 1981 and 1994. Additional data were extracted from clinical records. Of 739 mailings, 199 responses were obtained and 101 provided full sets of data. No significant associations by multiple regression analysis were found between total number of ampoules of FSH, number of assisted reproduction cycles or pregnancies and menopausal age; the mean of about 50 years is similar to values reported in other menopause studies. No relationship was found between the severity of menopausal symptoms and the number of assisted reproduction cycles or any other covariate. The only significant relationship found was an association between number of IVF cycles and increasing menopausal age (P = 0.001); this may be due to women with later menopause being self-selected for more IVF treatment. This study provides reassuring evidence that ovarian stimulation does not compromise reproductive potential by hastening menopause, an observation consistent with experimental studies showing that primordial follicle recruitment is independent of gonadotrophin stimulation.

Jean Marian Purdy remembered - the hidden life of an IVF pioneer.

Jean Purdy is almost forgotten as one of the British trio that introduced clinical IVF to the world. An unlikely pioneer, she qualified as a nurse but through indefatigable effort and unstinting loyalty to a programme that faced vitriolic opposition she became the clinical embryologist for the first IVF baby. In 1980, she helped to launch fertility services as the 'Technical Director' of Bourn Hall Clinic, near Cambridge. Although Robert Edwards and Patrick Steptoe generously credited her role in research and clinical care, a premature death in 1985 at age 39 robbed her of the reward of witnessing the blossoming of assisted reproductive technologies for patients around the world. This commentary seeks to recognize her contribution and bring her name to the attention of professionals in reproductive medicine and the patients they care for.

The cyclin-like uracil DNA glycosylase (UDG) of murine oocytes and its relationship to human and chimpanzee homologues.

Examination of electrophoretically resolved randomly generated PCR amplicons from mature murine oocytes revealed the presence of a short sequence with partial homology to a cyclin-like human uracil DNA glycosylase (UDG2), a member of an important group of base-excision enzymes that remove misincorporated or cytosine-derived uracil from nascent DNA. A combination of 3' R.A.C.E. and in silico 'cloning' of the 5' region of the original EST uncovered a predicted murine mRNA sequence with limited homology to human UDG2 at both nucleotide and amino acid levels. An alternative contig for human UDG2 showed stronger similarity with the homologous murine and chimp genomic and protein sequences and contained both N and C terminal cyclin box elements. This contig is also an Ensembl annotated alternative splice-site variant of the UDG2 gene (Udg2v2). We suggest that uracil-excision repair in oocytes is likely to be mediated by Udg2v2, or alternatively that Udg2v2 is involved in a process related to oocyte-specific maturation by virtue of its cyclin-like domains.

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.