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  Speakers Overviews Print

Nadeem Abu- Rustum, USA
RADICAL ABDOMINAL TRACHELECTOMY

Objectives: To describe the surgical and pathologic findings of fertility-sparing radical trachelectomy using a standardized surgical technique.

Methods: A prospectively maintained database of all patients with FIGO stage IA1-IB1 cervical cancer admitted to the operating room for planned fertility-sparing radical abdominal trachelectomy was analyzed.

Results: Between 11/2001 and 5/2010, 98 consecutive patients with FIGO stage IA1-IB1 cervical cancer underwent surgery with the intent to perform a fertility-sparing radical trachelectomy. Median age was 32 years (range 6-45).

The most common histology was adenocarcinoma 54 (55%) and squamous carcinoma 42(43%). Lymph-vascular invasion was seen in 38 (39%) cases. FIGO stages included: IA1(with LVI) 10(10%), IA2 9(9%), and IB1 79 (81%). Only 15 (15%) needed immediate completion radical hysterectomy due to intraoperative findings. Median number of nodes evaluated was 22 (range 3-54); and 16 (16%) patients had positive pelvic nodes on final pathology. Final trachelectomy pathology revealed no residual disease in 44 (45%) cases, dysplasia in 5(5%) and adenocarcinoma in-situ 3(3%). Overall, 27(27%) patients needed hysterectomy or adjuvant pelvic radiation postoperatively. There was one (1%) documented recurrence that was fatal at the time of this report.

Conclusions: Cervical adenocarcinoma and lymph-vascular invasion are common features of patients selected for radical trachelectomy. The majority of patients can undergo the operation successfully with many having no residual invasive disease; however, nearly 27% of all selected cases will require hysterectomy or postoperative chemoradiation for oncologic reasons. Investigating alternative fertility-sparing adjuvant therapy in node positive patients is needed.

Amir Arav, Israel
CRYOTECHNOLOGY - WHAT IS NEXT?

This year we celebrate 75 years since the first vitrification of sperm done by Luyet in 1938.
Since then many techniques in cryobiology have been developed and the preservation of many cells, tissues and even organs has been made possible. What unifies the tissue and cell cryobanking is that they store the samples in liquid nitrogen. Storage of cryopreserved samples, under liquid nitrogen, is very demanding in terms of maintenance, storage space, storage equipment and costs. An alternative that would minimize costs, storage and maintenance has been gaining a foothold in the field of cell preservation in recent years - the dry storage. Drying of cells can be achieved by either convective-drying or freeze-drying. Freeze-drying is achieved by sublimation of the ice after freezing the sample to subzero temperatures. The process is damaging to the cellular membrane and some degree of chromosomal damage may also take place due to endogenous nucleases.
To date, embryonic development after intracytoplasmic sperm injection (ICSI) with freeze-dried sperm heads has been reported in humans, hamsters, cattle, pigs, rhesus macaque and cats, and live offspring were reported in mice, rabbits, rats and fish . We have recently demonstrated the use of sheep freeze-dried somatic cells for somatic cell nuclear transfer. We utilized the directional freezing technology to freeze-dry somatic cells which were kept at room temperature for 3 years. These cells were rehydrated and then used to direct embryonic development following nuclear transfer into in vitro matured enucleated oocytes. Finally, human hematopoietic stem cells that were lyophilized and rehydrated with water were viable and have maintained their clonogenic capacity showing that they were able to develop into all blood lineages. This was the first report to show cells that have undergone complete lyophilization and following rehydration have maintained not only their viability but also their functionality. Recently we have shown that bovine mature oocytes can be lyophilized and maintain the viability after rehydration.
I believe that the future of fertility preservation will be a simple "on shelf preservation" of gametes, embryos, gonad's tissue and stem cells.

Uzi Beller, Israel
HOW TO SELECT THE PATIENTS FOR FERTILITY PRESERVATION

Identifying and selecting the female patient, with gynecological or breast cancer, to whom we can offer Fertility Preservation (FP) depends upon a variety of factors each with significant as well as unique importance.
We should address the following issues prior to recommending any FP modality to our patient:
   1.Oncologic prognosis and quality of life
   2.Fertility and obstetrical history and potential.
   3.Availability and reliability of the FP technologies
   4.Surgical modalities and expertise
   5.Medical and laboratory modalities
   6.Explaining the meaning of success
   7.The psycho-social perspective
   8.The role of couple counseling
   9.Obtaining informed consent

Fertility preservation is by definition a personalized medical therapy; the "one size fits all” approach does not exist in these situations. Therefore, for every woman, a multidisciplinary group of experts should convene and analyze all the related issues prior to recommending the best option for the particular patient and spouse.
One should not provide information or data with exaggerated reassurance of obstetrical success rates. We should definitely not compromise on oncologic proven treatments and outcome on account of possible FP.
We emphasize the need for a carefully obtained informed consent.

Zeev Blumenfeld, Israel
GNRH TREATMENT BEFORE CHEMOTHERAPY AS AN OVARIAN PROTECTION

Decreased secretion of the pituitary gonadotropins, by decreasing gonadal function, may possibly protect against the sterilizing effects of chemotherapy. Although previous suggestions have been made claiming that primordial germ cells fare better than germ cells that are part of an active cell cycle, this hypothesis has not been seriously tested clinically, until recently. Five prospective randomized studies performed by now, have found that GnRH-a protected the ovary against cyclophosphamide-induced damage in Rhesus monkeys by significantly decreasing the number of follicles lost during the chemotherapeutic insult. A long-term follow-up of 240 children, 15 years of age or younger, treated for Hodgkin lymphoma [HL] showed azoospermia in 83% of the boys, whereas only 13% of the girls suffered POF. Since ovarian function was preserved in most long-term survivors who were treated prepubertally for lymphoma, but only in about half of similarly treated adult patients, it was clinically logical and therefore tempting to create a temporary prepubertal milieu in women in the reproductive age before and during the chemotherapeutic insult. We have administered a monthly depot IM injection of GnRH-agonistic analogue to more than 220 young patients exposed to gonadotoxic chemotherapy for malignant or non-malignant diseases, after informed consent, starting before chemotherapy for up to six months, in parallel and until the end of chemotherapeutic treatment . Less than 7% developed irreversible hypergonadotropic amenorrhea. The remaining patients (>93%) resumed cyclic ovarian function, and 51 patients spontaneously conceived 76 times. These patients were compared to a control group of over 130 patients of comparable age (15-40), who were similarly treated with chemotherapy but without the GnRH-a adjuvant. Neither the age, nor the diagnoses, ratio between HD or non-Hodgkin lymphoma differed between the two groups. Similar doses of radiotherapy exposure and ratios of patients treated by radiotherapy in addition to chemotherapy were experienced by the two groups. Moreover, the cumulative doses of each chemotherapeutic agent and the mean or median radiotherapy exposure did not differ between the groups. Our and others' results support the effectiveness of GnRH-a administration also to patients receiving cyclophosphamide pulses for SLE and other autoimmune diseases. Recently we have experienced the first worldwide reported case of three spontaneous successful deliveries of a healthy neonates after TWO repeated BMT's, concurrently treated with GnRH-a during the gonadotoxic chemotherapy. How can we possibly explain the beneficial effect of the GnRH-a for minimizing the gonadotoxic effect of chemotherapy, in particular that of alkylating agents?

Several explanations may be put forward:
I. The hypogonadotropic state generated by the GnRH-a simulates the prepubertal hormonal milieu. One can conceivably hypothesize that the alkylating agents may bring about an increased rate of destruction/apoptosis of the non-resting follicles, and subsequently a decrease in the secretion of sex steroids and inhibins produced by these follicles, at different stages of maturation and differentiation. The resultant decrease in sex-steroids (estrogen, progesterone, and androgens) and inhibins' secretion will decrease their plasma concentrations and subsequently the negative feedback on the hypothalamus and pituitary, resulting in an increase in FSH secretion. The increased FSH secretion may bring about an increased recruitment of preantral follicles to enter the differentiational one way of maturation, being furthermore exposed to the gonadotoxic effect of the alkylating agents, ending in an increased, exponential rate of follicular apoptosis and degeneration. This vicious cycle may be interrupted by the GnRH-a administration through its ability to prevent the increase in FSH concentrations.

II. Another possible explanatory mechanism to the beneficial effect of GnRH-a on decreasing the chemotherapy-associated gonadotoxicity is the decrease in the utero-ovarian perfusion due to the hypoestrogenic state, generated by the pituitary- gonadal desensitization . High estrogen concentrations significantly increased ovarian perfusion and the vessel endothelial area, in a rat model of ovarian hyperstimulation, and this effect was significantly and dose-dependently inhibited by administration of GnRH-a. The decreased utero-ovarian perfusion induced by the GnRH-a, may result in a decreased total cumulative exposure of the ovaries to the chemotherapeutic agents as compared to a "control” patient, in a normoestrogenic milieu, thus resulting in decreased gonadotoxicity.

III. It has been shown that not only rodents but also primate and human gonads contain GnRH-receptors. In an ovarian carcinoma cell line, GnRH-I and –II receptors' activation may result in decreased apoptosis. Whether the GnRH-a effect is direct on the oocyte cumulus complex, or on the granulosa cell, or possibly on another ovarian compartment in addition to its possible hypogonadotropic effect, is an open question of significant scientific interest. Most recently, a proof of a direct effect of GnRH-a, independent of the hypogonadotropic milieu, has been provided by Imai et al, who have shown a direct, in-vitro protection from the doxorubicin induced granulosa cell damage, by a GnRH-a.

IV. Another possibility is that the GnRH-a may up regulate an intragonadal anti-apoptotic molecule such as sphingosine-1-phosphate (S-1-P). S1P has been shown to prevent chemotherapy induced gonadotoxicity both in-vivo and in-vitro. Whether the GnRH-a adjuvant cotreatment positive effect is direct or possibly associated with an intraovarian increase in S-1-P is a question of tremendous scientific interest and clinical impact. It obviously awaits further investigation.

V. Most recently revolutionary data were presented, whereby mouse ovaries or even bone marrow may possess mitotically active germ cells that continuously replenish the pool of immature follicles. These Germline Stem Cells (GSC) may exist in the mouse ovary and/or bone marrow and regenerate the primordial follicle pool. These observations contradict the basic doctrine of reproductive biology whereby most mammalian females lose the capacity for germ-cell renewal during fetal life, such that a fixed reserve of germ cells (oocytes) enclosed within follicles is endowed at birth. One may speculate that the GnRH-a protective effect may possibly be through protection of the undifferentiated GSC, who ultimately generates de-novo primordial follicles. Indeed, the observation of temporary, high, reversible FSH concentrations in a third of our patients, several months after the chemotherapy and GnRH-a co-treatment, even in those who spontaneously conceived later on, may point towards reversible gonadotoxicity. The possible de-novo formation of follicles by the surviving germline stem-cells brings about a decrease in FSH concentration and return of regular cycles, ovulation, and even gestations. Multicenter, prospective, randomized studies are awaited to substantiate the in-vivo effect of GnRH-a as an unequivocal means for minimizing follicular apoptosis. Most recently, a prospective randomized study has validated the protective role of GnRH-a in preservation of fertility and ovarian function despite chemotherapy in young women despite gonadotoxic chemotherapy.

Nicoletta Colombo, Italy
EPITHLIAL OVARIAN CANCER

The majority of epithelial ovarian cancer (EOC) are diagnosed in postmenopausal patients and at an advanced stage. However, ∼25% are limited to the ovaries at diagnosis, and 14% of invasive ovarian cancers are found in women <40 year-old.
Fertility-sparing surgery (FSS) for these young women with early-stage malignant epithelial ovarian cancer (ES/EOC) has been intensely debated in the last two decades. Preservation of the adnexa and uterus is currently recommended in patients with non-epithelial and epithelial borderline ovarian tumors, but is still considered suboptimal for women with invasive EOC.
Most of the published series about FSS of ES/EOC are retrospective . Some authors use restrictive criteria to admit patients to conservative treatment, while others, consider all stage I ovarian cancer patients eligible for this approach.
We recently reviewed the oncological and obstetrical outcome of 240 patients treated with FSS for ES/EOC in two Italian centers from 1982 to 2010. At a median follow-up of 9 years, 27 patients had relapsed (11%) and 11 (5%) had died of progressive disease. Multivariate analysis found only grade 3 negatively affected the prognosis of patients [hazard ratio (HR) for recurrence: 4.2, 95% confidence interval (CI): 1.5–11.7, P = 0.0067; HR for death: 7.6, 95% CI: 2.0–29.3, P = 0.0032]. However, the main reason for this poorer prognosis was not related to the preservation of uterus and ovaries but to the significantly higher associated of Grade 3 with extra-ovarian relapse (P = 0.006). Of the 105 patients (45%) who tried to become pregnant, 84 (80%) were successful. Based on this experience we believe that FSS can be proposed to all young patients when tumor is limited to the ovaries. The isolated ovarian recurrences can always be managed successfully with further surgery. The poorer prognosis of patients with G3 tumors is related to the higher incidence of distant recurrences , which is not a function of the type of surgery but rather of the biology of the tumor.

Marie-Madeleine Dolmans, Belgium
REIMPLANTATION OF CRYOPRESERVED OVARIAN TISSUE TAKEN FROM PATIENTS WITH VARIOUS MALIGNANT DISEASES: IS IT SAFE?!

Reversing treatment-related premature ovarian failure by means of autotransplantation of frozen-thawed ovarian tissue harvested before chemo-radiotherapy is becoming an increasingly realistic prospect for clinical application, with 22 live births already reported using this technique. Our objective is to offer young patients at risk of post-treatment premature ovarian failure, safe fertility preservation options.
One major concern raised by transplantation of ovarian cortical fragments in cancer patients is the potential risk that the cryopreserved ovarian tissue might harbor malignant cells that could induce a recurrence of the disease after reimplantation.
Hematological malignancies and breast cancer are the most frequent indications for ovarian tissue cryopreservation. Both carry the risk of ovarian metastasis.
We therefore decided to conduct a study to evaluate the presence of leukemic cells and breast cancer cells in cryopreserved human ovarian tissue from patients with chronic myeloid leukemia or acute lymphoblastic leukemia and advanced breast cancer.
In each case, histology, polymerase chain reaction for disease-specific markers, and long-term xenografting were used to test the frozen-thawed ovarian tissue.
Results showed that malignant cells may be present in ovarian tissue from acute leukemia patients and give rise to tumor development in mice after xenografting (n=5/12, acute leukemia). For mice grafted with ovarian tissue from patients with advanced breast cancer, PCR and MGB2 gene sequencing were positive in ovarian tissue in 5 out of 10 patients, but none of the xenografted mice developed tumor masses during the 6-month grafting period.
Although the malignant potential of these cells is not yet known, the current study demonstrates that conventional histology and IHC need to be associated with more sensitive screening methods, like PCR and sequencing, before ovarian tissue transplantation can be contemplated.

Jacques Donnez, Belgium
PREGNANCY FROM FROZEN SLICES OF OVARY

The different cryopreservation options available for fertility preservation in cancer patients are embryo cryopreservation, oocyte cryopreservation and ovarian tissue cryopreservation.
Embryo cryopreservation requires the patient to be of pubertal age, have a partner, and be able to undergo a cycle of ovarian stimulation. Oocyte cryopreservation is also effective, but the quality of eggs in women suffering from cancer is so far unproven, and requires also tissue for ovarian stimulation.
Cryopreservation of ovarian tissue is the only option available for prepubertal girls, and for woman who cannot delay the start of chemotherapy.
Around 50-60 cases of orthotopicreimplantation of cryopreserved ovarian tissue have been reported to date and 22 live births have been achieved, yielding a pregnancy rate of more than 25%. In our department, ten women have undergone orthotopicreimplantation of cryopreserved tissue either once or twice. Restoration of ovarian function, proved by follicular development and estradiol secretion, occurred in all cases. A time interval of 3.5 to 5 months was observed. In the literature, pregnancies were naturally obtained in 50% of cases. Graft activity was found to persist for 2.5 to 4 years. In non-pregnant patients, IVF was performed, but the quality of oocytes and embryos was not optimal.
Prognostic factors (age, previous chemotherapy) are therefore discussed.
In conclusion, fertility preservationis now a real possibility for patients whose gonadal function is threatened by radiotherapy or chemotherapy.

Norbert Gleicher, USA
DIMINISHED OVARIAN RESERVE IN BREAST CANCER PATIENTS WITH BRACA1 MUTATION

For the longest time, androgens have been considered detrimental to follicle maturation. Only in recent years has evidence to the contrary been reported. Such evidence was developed in human experience and based on animal experimentation. In humans, higher testosterone levels have been associated with better IVF pregnancy rates. The, likely, most suggestive evidence comes, so far, however, from data of DHEA supplementation in women with low functional ovarian reserve (FOR). The probably most convincing evidence stems, however, from recent experiments in androgen receptor knock out (ARKO) mice, which demonstrated that normal follicle (and oocyte) development during early follicle-growth stages is dependent on AR in granulosa cells. Indeed, androgens and FSH, appear to support follicle growth and maturation at these stages of follicle development synergistic. Since DHEA is to a large
degree converted to testosterone it, therefore, appears likely that reported clinically beneficial DHEA effects are AR-mediated during these early growth stages of follicles. Recent data from the ARKO mouse model are confirmatory of this hypothesis (Sen et al., personal communication), as are human data demonstrating that pregnancy chances in IVF directly correlate to the efficacy of DHEA conversion to testosterone. Further support comes from the recent recognition that low FOR at all age is characterized by low testosterone levels. It, thus, appears that androgen supplementation represents a completely new treatment paradigm in infertility because, for the first time, infertility treatments not only affects the last two weeks of follicle maturation (the gonadotropin-sensitive phase) but early stages of follicle growth. In this sense, androgen treatments may be only a first therapeutic intervention into earlier stages of follicle maturation, where the impact of such intervention can be expected to be better than at late stages, where quality and quantity of the growing follicle pool already have been mostly determined.
We noted that IVF success correlates to how well DHEA is converted to testosterone. It also depends on testosterone levels reached. Moreover, conversion is better in younger than older women and differs between different genotypes of the FMR1 gene. We described a new family of FMR1 genotypes and sub-genotypes, based on a normal range of 26-34 CGG triple repeats, which, amongst other medical associations, appear closely associated with ovarian aging patterns. Especially the so-called het-norm/low sub-genotype appears to convert DHEA to testosterone very poorly. Interestingly, this sub-genotype has also been demonstrated associated with greatly reduced pregnancy chances in IVF. The FMR1 gene, thus, appears to play an increasingly important role in ovarian aging, which in, yet undetermined ways, also may interact with androgen effects on follicle maturation.

Stefano Greggi, Italy
EARLY ENDOMETRIAL CANCER

Approximately one fourth of women with endometrial cancer are diagnosed in fertile age. Nevertheless, the current standard of surgical approach is preclusive of fertility. Primary progestin therapy, sometimes combined with local surgical excision, has been used as conservative treatment in early well differentiated tumors. The worldwide experience and data, however, are still limited due to the small number of cases, potential methodological bias, and the use of different therapeutic regimes. By means of different strategies (hormonal therapy ± hysteroscopic resection) and using different drugs, a mean of 72% complete response was reported, with a 23% relapse rate (only 4 disease-related deaths due to recurrence in the abdomen or distance site). Twenty-eight percent of complete responders subsequently conceived with or without assisted reproduction technology, while the pregnancy rates ranging from 32% to 100% in patients attempting to conceive.

In our experience, we have tested the feasibility and efficacy of combined operative hysteroscopy and hormone therapy as fertility-preserving treatment in a cohort of selected young women with FIGO stage IA (intramucous) well differentiated endometrial cancer (n=14). Treatment consisted of hysteroscopic ablation of the lesion and the myometrial tissue below, followed by oral megestrol acetate for 6 months or levonorgestrel-medicated intrauterine device for 12 months. With a median follow-up of 46 months, one patient recurred after 5 months from operative hysteroscopy and underwent definitive surgery, one patient showed an endometrial hyperplasia without atypia at the 3 and 6 month hysteroscopy control, with negative controls thereafter; three patients have attempted to conceive and one of them conceived and term delivered a healthy baby.

Gedis Grudzinskas, UK
WHY IS FERTILITY PRESERVATION NOT YET AVAILABLE TO ALL SUITABLE SURVIVORS?

Announcements of more than 20 babies born following first birth after human successful orthoptic transplantation of cryopreserved thawed ovarian tissue by Donnez et al. (2004), the news of the birth of healthy babies following orthotopic reimplantation appears to be increasing greatly . Although it may be a little too early to say that this rise is exponential, this cautious position may soon be cast aside. Moreover, Andersen and co-workers (2012) suggest that it might also be possible to postpone the normal time of the menopause or to alleviate menopausal symptoms using auto transplanted ovarian tissue. Since the pioneers in this field have generously given free access to their successful technology why is this technique not yet available to all suitable cancer survivors?

Katsuhiko Hayashi, Japan
GENERATION OF EGGS AND SPERMATOZOA FROM PLURIPOTENT STEM CELLS IN MICE

Generation of gametes from pluripotent stem cells, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), in culture is a key goal in developmental/reproductive biology. More practically, gametes from ESCs/iPSCs can be an indefinite source for reproduction and preservation of animals. In mice, a model of mammals, generation of gametes from ESCs/iPSCs in culture has been attempted for a decade. However, despite a lot of effort, a culture system that reconstitutes the entire process of germ cell development has not been established yet.
Toward establishing such culture system, we recently succeeded in producing a robust number of cells whose potential is equivalent to nascent primordial germ cells (PGCs), origin of eggs and spermatozoa. In the culture system, ESCs/iPSCs first differentiate into epiblast-like cells (EpiLCs) and then induce PGC-like cells (PGCLCs). The manner of the step-wise differentiation from ESCs/iPSCs to PGCLCs reproduces that of PGC specification in vivo. Importantly, PGCLCs produced from ESCs/iPSCs are capable of differentiating into eggs and spermatozoa, when transplanted into ovary and testis, respectively. Furthermore, PGCLC-derived eggs and spermatozoa are fully potent, as they give rise to healthy and fertile offspring.
Successful production of functional PGCLCs from ESCs/iPSCs may lead to an idea of applications to other mammalian species including human. There are, however, technical obstacles to apply immediately our culture system to other species. It would be necessary to understand similarity/difference between mice and other species, especially in nature of pluripotent stem cells and PGC development. Nevertheless, our study opens a possibility that pluripotent stem cells are a practical source for reproduction and preservation of animals.

Peter Hillemanns, Germany
PHOTODYNAMIC THERAPY

Current treatments for cervical neoplasia (CIN) are generally extirpative procedures that have undesirable side effects, including risk of stenosis, infertility and future preterm birth. There are clear benefits to use minimally-destructive procedures which lead to the investigation of photodynamic therapy (PDT). A novel intravaginal combination product including hexaminolevulinate (HAL) and a non-coherent light source (LED) offers an easy to use product that may be offered to patients with cervical intraepithelial neoplasia to avoid tedious follow-ups and conisations.

Ungar Laszlo, Hungary
RADICAL ABDOMINAL TRACHELECTOMY FOR STAGE 1B2

Objective Invasive cervical cancer is one of the most common cancers with 500,000 new cases diagnosed annually. Expert opinion has suggested that fertility sparing radical trachelectomy should be limited to those patients diagnosed with cervical cancer <2cm in diameter. Our objective is to report our abdominal radical trachelectomy (ART) experience in those patients diagnosed with early stage cervical cancer measuring >2cm in diameter.

Methods Between 1999 and 2006, all women wishing to preserve fertility and having cervical cancer clinical stage IB1-IB2 were offered ART at the three institutions where the authors are based (Budapest, Hungary; London, UK; and New York City, USA). A total of 45 patients with cervical carcinoma FIGO stage IB1-IB2 measuring >2cm in diameter underwent a fertility sparing ART and pelvic lymphadenectomy (Hungary-36 cases; UK-4 cases; USA-5 cases). They were followed for more than five years.

Results Average operative time was 194 min. Mean estimated blood loss (EBL): 900 ml and lymph nodes removed 41 [range 30-55]. Complications included one ureteral injury, one cervical stenosis and ureteral stenosis both side. 14 out of 45 (31%) patients underwent immediate completion of radical hysterectomy based on the intra-operative findings including: positive lymph node metastasis (n=9), endocervical surgical margin positivity (n=3), injury of the ovarian artery (n=1) or parametrial tumour spread (n=1). In 69% (n=31) of patients, completed ART was considered to have been curative and no adjuvant treatment was advised. 30 out of 31 (96.8%) patients who underwent ART were alive at the time of follow up. The patient who died had a stage IB2 glassy cell histology tumour measuring 5cm diameter. She developed a pelvic side wall recurrence five months following ART.

Conclusions The five year survival rate (96,8%) for this >2cm cervical cancer case series is higher or at least equal to rates reported in the literature for patient treated by radical hysterectomy as the initial treatment. Our survival data seems to support the hypothesis that ART is safe for lesions >2cm. ART has not been shown to be inferior to traditional cervical cancer treatment modalities for this group of cervical cancer patients. We suggest that ART should be offered as a fertility-sparing treatment option for carefully selected stage IB cervical cancers >2cm.


Sibylle Loibl, Germany
THE CONTEXT OF FERTILITY PRESERVATION IN BREAST CANCER PATIENTS

Breast cancer in the (very) young women is rare and mostly treated with cytoxic agentsas part of the systemic therapy because they are at higher risk of relapse. The type of (neo)adjuvantchemotherapy however, does not differ by age. Standard (neo)adjuvant chemotherapy regimen today comprise an anthracycline-taxane based therapy, either as combination or as sequential therapy. Dose-dense therapy seems to be superior, especially in hormone-receptor disease.

Ovarian function is impaired by cytotoxic as well as endocrine therapy for breast cancer. Premature ovarian failure (POF) depends very much on the cytotoxic agents, duration as well as the age of the patient. Cyclophosphamide containing regimen and longer regimen have a stronger influence on POF. It seems that women below the age of 35years recover from intermittent POF. But the age of menopause onset is lower, indicating that the ovarian function is generally impaired. Women who receive tamoxifen in addition to chemotherapy have a higher rate of amenorrhea.

One of the main problems in assessing the effects and comparing the data on impaired ovarian function and fertility preservation are the different definitions used for evaluating the treatment impact on POF as well as the preventive measureson POF, i.e. LHRH analogues. There is no unified definition of POF. Measurement of amenorrhea, menstruation, laboratory values, time points of assessment, are discrepant resulting in data difficult to compare and draw conclusions. Long term data are rare. Last but not least it has to be differentiated between menstruation and fertility, which is very often used synonymously.

Philippe Morice, France
BORDERLINE TUMOR AND NONE EPITHELIAL CANCER

Borderline ovarian tumours are the most frequent ovarian malignant or semimalignant disease in women < 40 years old. The conservative approach is the standard managament in such cases at least in early stage. This treatment increases the risk of recurrence without affecting the survival. Spontaneous fertility rate is about 60%.

Most frequent ovarian nonepithelial malignant tumors are germ cell & sex cord tumors. Conservative series concern mainly the first group and demonstrate that this managements is safe and could be also sed in selected cases of patients with extraovarian tumors and/or bilateral disease.

Nicholas Reed, Scotland
FUNCTION OF OVARIES FOLLOWING TRANSPOSITION AND PELVIC IRRADIATION

The management of the younger patient with cervix cancer who is nulliparous or desiring further family presents great challenges. Challenges arise where there is a potential conflict between fertility preserving treatments and any potential reduction in likelihood of cure. Whether the treatment is by surgical or radiotherapeutic intervention it is likely that it will interfere with subsequent fertility. For women with very small tumours, usually less than 2cm, neoadjuvant chemotherapy and trachelectomy may offer an alternative which preserves fertility. However larger tumours will require either radical hysterectomy with lymph node dissection or concomitant chemo/radiation or even combinations of the two which will clearly affect fertility. Attempts to preserve fertility include neoadjuvant chemotherapy and surgery with transposition of the ovaries which may permit adjuvant brachytherapy if indicated. The delivery of full pelvic radiation even with transposition presents far greater challenges. In this presentation the literature will be reviewed and two cases will be presented to illustrate the kind of management that may be offered and the challenges that present themselves. Patients must understand that this approach remains non-evidence based and in some people's views is highly controversial but given the risks involved patients may consider this to be an acceptable option if it allows the chance of preserving fertility at least in terms of ovarian function and surrogate pregnancies.

Gerald Schatten, USA
SCIENCE FICTION OR FUTURE REALITY? PRODUCING GAMETES FROM STEM CELLS AND SOMATIC CELLS

The 2012 Nobel Prizes to Sir John Gurdon and Shinya Yamanaka for their seminal work in nuclear transfer and induced pluripotency, coupled with the 2010 Nobel Prize to Bob Edwards for pioneering In Vitro Fertilization in humans, attests to the contemporary importance of stem cells and regenerative medicine for ART. This lecture will consider pluripotent stem cells, their differentiation into both sperm and oocytes, as well as their role of PSCs in cancers and epigenetics.

Pluripotent stem cells from humans (hPSCs), including human embryonic stem cells (hESCs) and by induced pluripotency (hiPSCs) captivate medical attention due to their unique properties of unlimited self-renewal and differentiation. ESC lines have only been established robustly and investigated intensively in mice (mESCs) and more recently in humans after derivations from fertilized-blastocysts and after induced pluripotency (iPSCs). Scientists around the World are now asking whether these cells might treat or even cure juvenile diabetes with insulin secreting β-islet cells responsive to circulating glucose; cerebral palsy treatments with neuroprogenitors to repair white matter injuries due to premature births; heart muscle repair with cardiomyocytes; spinal cord regeneration with peripheral motor neurons; multiple sclerosis with neuroprogenitor cells or astrocytes for Schwann cell; Parkinson's disease using dopaminergic neurons; amyotrophic lateral sclerosis with neuronal lineages; reduction or replacement of whole organ transplantation by single cell transplantation of hepatocytes for diseased livers; renal cells in place of kidney transplants, and many others.

Our report entitled: Direct Differentiation of Human Pluripotent Stem Cells into Haploid Spermatogenic Cells ((Easley et al., Cell Reports 2, 440–446, September 27, 2012; http://dx.doi.org/10.1016/j.celrep.2012.07.015) notes that: ‘Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) have been shown to differentiate into primordial germ cells (PGCs) but not into spermatogonia, haploid spermatocytes, or spermatids. Here, we show that hESCs and hiPSCs differentiate directly into advanced male germ cell lineages, including postmeiotic, spermatid-like cells, in vitro without genetic manipulation. Furthermore, our procedure mirrors spermatogenesis in vivo by differentiating PSCs into UTF1-, PLZF-, and CDH1-positive spermatogonia-like cells; HIWI- and HILI-positive spermatocyte-like cells; and haploid cells expressing acrosin, transition protein 1, and protamine 1 (proteins that are uniquely found in spermatids and/or sperm). These spermatids show uniparental genomic imprints similar to those of human sperm on two loci: H19 and IGF2. These results demonstrate that male PSCs have the ability to differentiate directly into advanced germ cell lineages and may represent a novel strategy for studying spermatogenesis in vitro.' Understanding gametogenesis is vital for improving infertility therapies and contraceptives. Patient-specific stem cells undergoing gametogenesis in vitro represent models for mechanistic investigations and potential therapies. Here, we show that human embryonic and induced pluripotent stem cells differentiate into advanced germ cell lineages including spermatogonia, spermatocytes, and haploid spermatids with parent-of-origin genomic imprints similar to fertile human sperm. Developing an in vitro spermatogenesis model may prove critical for understanding the mechanistic causes of male infertility.'

FERTILE OFFSPRING GENERATED FROM OOCYTES DERIVED FROM iPSCs. In their paper, Drs. Hayashi et al. from the Prof Saitou lab have reported in SCIENCE that: "Reconstitution of female germ-cell development in vitro is a key challenge in reproductive biology and medicine. We show here that female (XX) embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) in mice are induced into primordial germ cell–like cells (PGCLCs), which, when aggregated with female gonadal somatic cells as reconstituted ovaries, undergo X-reactivation, imprint erasure, cyst formation, and exhibit meiotic potential. Upon transplantation under mouse ovarian bursa, PGCLCs in the reconstituted ovaries mature into germinal vesicle-stage oocytes, which then contribute to fertile offspring after in vitro maturation and fertilization. Our culture system serves as a robust foundation for the investigation of key properties of female germ cells, including the acquisition of totipotency, and for the reconstitution of whole female germ-cell development in vitro.”Please recognize though that extrapolations to ART clinic are highly speculative. This work teaches us features about oogenesis IN MICE. So unless your patients are rodents, please do not let ‘fertile' imaginations run wild yet…

Cancer Stem Cell Hypothesis: Notwithstanding decades of remarkably powerful chemotherapeutic and radiotherapeutic treatments for various cancers, combined with skillful removal of the tumors, cancer remains a devastating disease with largely unfavorable long-term outcomes. The scientific discoveries on stem cells, and especially the teratoma assay used to demonstrate pluripotency to stem cell scientists, has influenced thinking among oncologists and cancer biologists. The ‘Cancer Stem Cell' Hypothesis proposes that most treatments used today to destroy cancers are actually only targeting the differentiated cells, which comprise the bulk of the tumor. They miss however, the earliest precursor cells, which is the root cause of the disease – the cancer stem cell – since these cells are rare, are highly resistance to drugs, and mutate rapidly to evolve tolerance. This lecture will consider the strengths and weaknesses of the hypothesis as well as how emerging new therapeutic strategies are being developed for the next generation of cancer treatments.

Sherman Silber, USA
FRESH AND FROZEN OVARY TRANSPLANT RESULTS

The aim of this lecture is to summarize the state-of-the-art of ovarian tissue cryopreservation and transplantation. This clinical experience will then be applied to understanding 1) the mechanism for initiation of primordial follicle development, 2) controversially why cancer cells metastases do not severely limit the transplantation of frozen ovary tissue back to cancer patients, and 3) what we can learn fro comparing ovarian embryology to testis embryology.
First, all nine fresh ovary transplants were between identical twins all ovulating and menstruating, resulting in eleven healthy babies in seven of the nine recipients. Recipients always reinitiated ovulatory menstrual cycles and normal Day 3 serum FSH levels by 4-1/2 months. Most conceived naturally (three of them twice or three times from the same graft). Duration of function of fresh ovarian grafts, contrary to initial expectation, indicated a very acceptable or minimal oocyte loss from ischemia time. Grafts of just modest portions of ovarian tissue have lasted more than 7 years.
The same surgical techniques were then applied to 4 frozen ovary tissue transplants, up to 14 years after the ovary had been frozen, all resulting in normal ovulation and in 3 more healthy babies. Around the world, the number of healthy babies from ovary grafts has now risen to over 30, with more than 20 from frozen grafts. Although ovary freezing and transplantation has been referred to as "experimental” for preserving fertility in cancer patients, it should be noted that virtually all of the babies born from fertility preservation for cancer patients, have resulted thus far from ovary tissue freezing, rather than from egg freezing.
As to the efficiency of ovarian tissue freezing compared to the use of fresh tissue, slow freeze has resulted in only a 60% loss of oocytes, and vitrification has resulted in no observable loss. For practical purposes therefore both techniques are reliable. However, in vitro studies in humans, and in vivo studies in bovine, show that vitrification of ovarian tissue, may nonetheless be an improvement over slow freeze.
The basic science concept of vitrification is to completely avoid any ice crystal formation by using a very high concentration of cryoprotectant and a very rapid rate (virtually "instant”) of cooling. This is quite different from classic slow freeze cooling which relies on a partial and very gradual removal of water from the cell by encouraging ice crystal formation preferentially on the outside of the cell, drawing the water out.
A comparison has to be made between vitrification for ovarian tissue versus vitrificationofr mature eggs and embryos. For mature eggs and embryos, there is first an equilibration in 7.5% EG and 7.5% DMSO followed by a final solution of 15% EG and 15% DMSO with 0.5 molar sucrose. It is very important to allow enough time for full absorption of this more concentrated cryoprotectant solution usually more than one minute, as these solutions, contrary to myth, are not toxic. However, for ovarian tissue, there must be a longer incubation in 7.5% EG and 7.5% DMSO, and then a later incubation in a denser 20% EG and 20% DMSO with 0.5% sucrose, to make certain there is full absorption of cryoprotectant. In all cases, the eggs or embryos or ovarian tissue must not be frozen in a droplet (even a tiny microdroplet) as this would slow the rate of freeze and thaw. The ovarian tissue must have fully absorbed the cryoprotectant, but be "dry” on the outside. Just as thawed ovarian tissue works as well as fresh, thawed embryos have just as good a success rate as fresh embryos. With vitrification, embryos can be frozen with impunity, whereas with slow freeze (like with ovarian tissue) there is some viability loss.
A comparison of the embryology and anatomy of the ovary and the testis is very instructive for two issues, 1) the initiation of follicle development, and 2) the low risk of neoplastic metastasis to the ovary cortex and, therefore, the safety of ovarian cortical cryopreservation and transplantation in cancer patients. The ovarian cortex is actually identical to the tunica albuginea of the testis, except that in the male the germ cell cords to not invade the testes of boys and men with leukemia, but uncommonly the tunica albuginea of the testis. This phenomenon explains both of these issues.
It is clearly possible to preserve and restore fertility, using ovary and egg or embryo freezing in young women with cancer who are undergoing otherwise sterilizing chemotherapy and radiation. But this approach can also be used for any woman who wishes to prolong her reproductive lifespan. It may thus eventually obviate the growing worldwide epidemic of female age-related decline in fertility, and even could eliminate menopause.

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