Therapeutic aims of drugs offering only progression-free survival are misunderstood by patients, and oncologists may be overly optimistic about likely benefits.

PURPOSE: The use of novel and often expensive drugs offering limited survival benefit in advanced disease is controversial. Treatment recommendations are influenced by patient characteristics and trial data showing overall response rates (ORR), progression-free survival (PFS) and overall survival (OS). PFS is frequently the primary outcome in licencing studies. PATIENTS AND METHODS: As part of a longitudinal study Assessing the 'VALue' to patients of PROgression Free Survival (AVALPROFS), oncologists completed checklists at baseline following consultations with patients. Questions probed perceived clinical benefits of the drugs to populations in general. Patients completed study-specific interview schedules at baseline, 6 weeks into treatment, and at withdrawal due to toxicity or progression. Patients also completed tumour- and treatment-specific quality of life questionnaires monthly for their time in the study. Only baseline results are reported here. RESULTS: Thirty-two UK oncologists discussed management options with 90 patients with heterogeneous advanced cancers. Oncologists' estimates of medical benefit in general from treatment varied between 10 and 80 %. They expected 46/90 (51 %) of their patients to derive some clinical benefit from the prescribed treatment but were either unsure or expected none for 44/90 (49 %). Predictions of life expectancy were variable but 62 % (56/90) of patients were expected to survive longer with treatment. A majority of patients 51/90 (57 %) had 'no idea' or were 'unclear' what PFS meant and 45/90 (50 %) thought extension of life was the primary therapeutic aim of treatment. CONCLUSION: Discussions between doctors and patients with metastatic disease about future management plans and likely therapeutic gains are challenging. Factors influencing decisions about putative benefits of novel drugs are often applied inconsistently can be overly optimistic and may even contradict published data.

An efficient method of ovarian stimulation and in vitro embryo production from prepubertal lambs.

The production of embryos from prepubertal lambs is inefficient, partly resulting from the low developmental competence of prepubertal lamb oocytes, and partly because a high proportion of lambs fail to respond to hormone stimulation. The development of a hormone stimulation regimen that all lambs respond to would increase the efficiency of breeding from prepubertal animals. Using a hormone stimulation regimen consisting of oestradiol benzoate (50 microg), a norgestomet implant (1.5 mg), pregnant mare serum gonadotrophin (400 IU) and follicle stimulating hormone (130 mg) all lambs (n = 19) responded to hormone stimulation. Uterine and ovarian weight ranged from 2.8 to 7.2 g (11.8 +/- 0.7 g) and from 1.7 to 54.1 (12.5 +/- 2.9 g), respectively. The number of ovarian follicles and oocytes recovered ranged from 20.0 to 500.0 (118.2 +/- 29.2) and from 13.0 to 455.0 (82.0 +/- 24.2), respectively, and oocytes suitable for in vitro production were obtained from all 19 lambs. Uterine weight was related to both bodyweight and growth rate (P < 0.05), although ovarian weight and the number of ovarian follicles were not related to either bodyweight or growth rate. Oocyte cleavage varied between hormone-stimulated lambs (0.0-93.0%; P < 0.05), and 484/775 (62.2%) of the oocytes cultured cleaved. Oocytes from 17 of the 19 lambs (89.5%) developed to the blastocyst stage in vitro , and the proportion of zygotes forming a blastocyst (by Day 7) ranged from 0.0 to 66.7% for individual lambs. Overall, 33.9% of zygotes (n = 164) developed to the blastocyst stage, producing 8.6 +/- 2.8 blastocysts per lamb.

Successful in vitro fertilization of in vivo matured oocytes aspirated laparoscopically from red deer hinds (Cervus elaphus).

Most current protocols of in vitro fertilization in ruminants are based on in vitro maturation of oocytes derived from abattoir material. For application of IVF technology to captive endangered species, however, noninvasive techniques are required which allow repeated collection of oocytes from live females. The aim of this study was to develop a method for embryo production from mature oocytes collected laparoscopically from red deer hinds. Follicular development was synchronized in red deer hinds by the insertion of intravaginal progesterone-releasing devices for 10 d, and ovarian stimulation was induced with 1000 IU, i.m. PMSG 48 h before progesterone device removal. Oocytes were harvested by laparoscopy under xylazine/ketamine sedation 24 h after progesterone device removal and then co-incubated with frozen-thawed red deer spermatozoa for 24 h. In Experiment 1, oocytes and embryos were fixed and stained at different developmental timepoints. Their external morphological changes (cumulus expansion, extrusion of the second polar body and cytokinesis) paralleled their nuclear developmental changes (formation of the 2nd metaphase spindle of meiosis, pronuclear formation and nuclear division, respectively). In Experiment 2, embryos were maintained in vitro until they ceased to undergo cell division. A total of 39 aspiration procedures was carried out on 14 red deer hinds. Forty-four cumulus-oocyte complexes (COC) were aspirated from 95 large Graafian follicles; of these, 27 were classed as mature/nondegenerated on the basis of cumulus/cytoplasmic morphology. Seventeen oocytes cleaved following in vitro fertilization, yielding six 2-cell embryos, six 4-cell embryos, four 8-cell embryos and one 16-cell embryo. The results indicate that laparoscopic aspiration of mature oocytes from hormone-treated females offers a valuable source of genetic material for assisted deer breeding programs.

In vitro and in vivo developmental capacity of oocytes from prepubertal and adult sheep.

Development to the blastocyst stage and survival following embryo transfer were assessed for oocytes obtained from prepubertal and adult sheep matured and fertilized in vitro. The rates of maturation, fertilization and cleavage in vitro did not differ significantly between oocytes from prepubertal and adult sheep. The proportion of cleaved zygotes reaching the blastocyst stage was significantly lower for oocytes derived from prepubertal than for those from adult sheep (15.4% and 34.1% respectively). There were no differences in the pregnancy rate and number of lambs born following transfer of blastocyst stage embryos derived from prepubertal and adult sheep to adult recipients. These data show that embryos derived from prepubertal lamb oocytes have reduced developmental potential in vitro but, of those which do reach the blastocyst stage, they have equal capacity to develop to term as embryos derived from adult sheep.

Birth of a male lamb derived from an in vitro matured oocyte fertilised by intracytoplasmic injection of a single presumptive male sperm.

The developmental competence of in vitro matured ovine oocytes, cytoplasmically injected with single male or female chromosome-bearing sperm, was investigated. Eighty-five unsorted, 92 'female-sorted' and 74 'male-sorted' ram sperm were injected into in vitro matured sheep oocytes and, two to four hours later, placed into the oviducts of 28 oestrous sheep. The sperm were separated according to sex by analysis of their DNA content with a flow cytometer. One pregnancy was diagnosed by ultrasound after 55 days and a 3 kg male lamb was born after 150 days gestation. This lamb was derived from an oocyte injected with 'male-sorted' sperm.

Production of lambs after the transfer of fresh and cryopreserved in vitro produced embryos from prepubertal lamb oocytes and unsorted and sex-sorted frozen-thawed spermatozoa.

Cumulus-oocyte complexes from hormone-stimulated 3-4-week-old (n=43) and 6-7-week-old (n=12) prepubertal lambs were matured in vitro and incubated with unsorted, or X- or Y-spermatozoa separated with a high-speed cell sorter (SX MoFlo)frozen-thawed. Presumptive zygotes were then cultured to the blastocyst stage, and transferred to recipients fresh or after cryopreservation (frozen). Oocyte cleavage was higher (p <0.05) with unsorted (515/926, 55.6%) than X- or Y-spermatozoa (261/672, 38.8% and 229/651, 35.2%, respectively) and blastocyst formation (% zygotes) by Day 9 of in vitro culture was lower (p <0.05) for X- (102/261, 39.1%) than unsorted spermatozoa (249/515, 48.3%), but did not differ between Y-spermatozoa (103/229, 45.0%) and unsorted spermatozoa, or between X- and Y-spermatozoa (p >0.05). For fresh embryos, survival to term was 50.0% (3/6) for unsorted, 0.0% (0/6) for X- and 16.7% (1/6) for Y-spermatozoa-derived embryos (p >0.05), and for frozen embryos was 4.0% (2/50) for unsorted, 9.1% (2/22) for X- and 2.9% (1/34) Y-spermatozoa-derived embryos (p >0.05). Of the two lambs born from X-spermatozoa-derived embryos, one was female (50%), and from the two Y-spermatozoa-derived lambs, both were male (100%), demonstrating that lambs can be produced after the transfer of fresh and cryopreserved IVP embryos derived from prepubertal lamb oocytes and frozen-thawed sex-sorted sperm.

Hoechst staining and exposure to UV laser during flow cytometric sorting does not affect the frequency of detected endogenous DNA nicks in abnormal and normal human spermatozoa.

Controlling the sex of offspring by the separation of X and Y chromosome-bearing spermatozoa using flow cytometry has been reported as a clinical technique aiding prevention of X-linked diseases. Although this technique has resulted in several hundred normal births in animals and at least one human birth, there is still concern over its genetic safety due to the involvement of two potentially mutagenic agents: UV light and the fluorochrome dye, Hoechst 33342 (H33342). Human spermatozoa, particularly those considered abnormal, may be more likely to suffer DNA damage following exposure to mutagenic agents, compared with other mammalian species. The stability of normal fresh and decondensed human spermatozoa were examined after exposure to a range of levels of UV and H33342 staining, using an assay that detects endogenous nicks in the DNA of spermatozoa. The stability of abnormal and normal, fresh and frozen-thawed human spermatozoa was examined following UV laser, H33342 staining and flow cytometry treatments utilizing the same assay. There was an increase in the presence of endogenous nicks when spermatozoa were decondensed compared with fresh spermatozoa. There was no increase in the incidence of nicks in any group of spermatozoa after UV and fluorochrome exposure compared with controls without exposure.

Time course of pronuclear formation and fertilisation after insemination in vitro and intracytoplasmic sperm injection of in vitro matured sheep oocytes.

The time course of sperm decondensation, oocyte activation, pronuclear formation and the possible causes of abnormalities after intracytoplasmic sperm injection (ICSI) and in vitro fertilisation (IVF) were examined. Frozen-thawed and pooled fresh semen from three different rams were washed and capacitated for ICSI or IVF. In vitro matured oocytes were cultured after sperm injection for 0.5, 0.75, 1, 2, 3, 4, 5, 6, 8, 18, 21 and 23 h, and oocytes were cultured after in vitro insemination for the same times other than 18 and 23 h. All oocytes were cultured in bicarbonate-buffered synthetic oviduct fluid medium (BSOF) supplemented with 2% oestrous sheep serum. A total of 746 metaphase II oocytes were injected with a single spermatozoon and 986 oocytes were inseminated for IVF. The earliest oocyte activation after ICSI was observed at 0.5 h, when 14.8% of oocytes were in anaphase II; this was earlier than after IVF, when only 6.4% of the oocytes exhibited anaphase II 1 h after insemination. Decondensing spermatozoa were first observed 1 h after ICSI and 3 h after insemination for IVF. The earliest female and male pronuclei after ICSI were observed at 2 and 3 h respectively, while the female and male pronuclei after IVF were observed at 4 h after insemination. The overall fertilisation rate was lower after ICSI (28.6%) than IVF (70.4%) but the percentage of abnormal fertilisation was not different between ICSI (8.7%) and IVF (15.2%). It was concluded that the fertilisation events were more advanced for ICSI than IVF, using injection and insemination time as reference points. The formation of male and female pronuclei were asynchronous after ICSI, in contrast to IVF when they appeared simultaneously at 4 h. Abnormalities found in fertilisation after ICSI may therefore be induced by the injection technique.

Birth of lambs of a pre-determined sex after in vitro production of embryos using frozen-thawed sex-sorted and re-frozen-thawed ram spermatozoa.

UNLABELLED: The characteristics and functional capacity of ram spermatozoa frozen-thawed prior to and after flow cytometric sorting was assessed after incubation (37 degrees C; 6 h), in vitro fertilisation (IVF), and transfer of fresh and vitrified in vitro produced embryos. Frozen-thawed spermatozoa from two rams were allocated to four treatment groups: (i) non-sorted (CONTROL); (ii) sorted (FS); (iii) sorted then re-frozen (FSF) and (iv) re-frozen control (FCF). Frozen-thawed samples were separated into X- and Y-chromosome bearing spermatozoa using a high-speed sperm sorter after density gradient centrifugation (X: 88 +/- 1.5% and Y: 87 +/- 1.1% purity). After 6 h incubation (37 degrees C), the percentage of motile spermatozoa was higher (P < 0.001) for FS (84 +/- 2.0%) compared with all other treatments ( CONTROL: 36 +/- 3.3%, FSF: 28 +/- 3.1%, FCF: 20 +/- 2.0%). In a sperm migration test greater numbers of FS spermatozoa penetrated 5 mm into the artificial cervical mucus compared with spermatozoa from all other treatments (152 +/- 39.4 vs 31 +/- 9.2 spermatozoa respectively; P < 0.05). Fertilisation and cleavage rates were higher (P < 0.05) for in vitro matured oocytes inseminated with CONTROL compared with FSF spermatozoa. However, the Day 7 blastocyst development rate was higher for oocytes inseminated with FSF (62.2%) than FS and CONTROL spermatozoa (52.7 and 50.0%; P < 0.05). The number of ewes pregnant (Day 60), lambing and the in vivo embryo survival rate was greater (P < 0.01) after the transfer of fresh embryos rather than vitrified embryos derived from X- and Y-spermatozoa (67.6, 64.7 and 41.2% vs 29.6, 25.9 and 14.8% respectively). Twenty-six of the 30 (86.7%) lambs derived from sex-sorted spermatozoa were of the correct sex. These results demonstrate that frozen-thawed ram spermatozoa can be sex-sorted for immediate or future use after re-cryopreservation and, in conjunction with IVF and embryo transfer, can be used to efficiently produce offspring of pre-determined sex.