NEWS & VIEWS

This service brings you the latest news on Assisted Reproduction Techniques as they have been reported in the media .

Sources include on-line media, medical data bases, original press releases,

trade journals, national daily newspapers and broadcasts reports, as well as peer-reviewed publications.

see also the last FERTI.NET HIGHLIGHTS (issue 1 /2002) and HUMAN REPRODUCTION UPDATE (October 2001)

NEWS FROM THE FRENCH CORNER:NEWS FROM THE GERMAN CORNER:

last update :October 9th. 2001

Human Reproduction, Vol. 16, No. 10, 2177-2181, October 2001

The predictive value of pronuclear morphology of zygotes in the assessment of human embryo quality

Andres Salumets1,3, Christel Hydén-Granskog2, Anne-Maria Suikkari1, Aila Tiitinen2 and Timo Tuuri1

1 Infertility Clinic, The Family Federation of Finland, Kalevankatu 16, FIN-00100 and

2 Department of Obstetrics and Gynaecology, Helsinki University Central Hospital, Helsinki, Finland

BACKGROUND: Recent studies have shown that zygote morphology could be used for the assessment of human embryo quality. Pronuclear (PN) morphology is based on certain distinct features seen in zygotes 16&endash;18 h after fertilization. In the present study PN stage morphology was assessed and combined with a single embryo transfer in order to investigate whether currently used zygote classifications are able to predict embryo quality and implantation rates. METHODS AND RESULTS: Zygotes were analysed according to two different classification systems. In the first, a total of 764 zygotes was analysed according to the degree of polarization of nucleolar precursor bodies (NPB). Zygotes with unpolarized PN (i.e. scattered localization of NPB) showed significantly slower (P < 0.005) cleavage rates (38.9%) than zygotes having at least one pronucleus polarized (57.3% and 54%). However, there was no difference in the pregnancy rate in 105 single embryo transfers between the groups. The appearance of a cytoplasmic halo was related to embryo morphology. Embryos derived from halo-positive zygotes had significantly better (P < 0.05) morphology (60.9%) compared to halo-negative derived embryos (52.2%), but in terms of pregnancy rates no difference was found. A total of 1520 zygotes was analysed according to a second classification system, which was based on the number and distribution of NPB. In the comparative analysis, none of the six different classes produced superior quality embryos or higher pregnancy rates in 144 single embryo transfers. CONCLUSIONS: Our results indicate that there are no significant differences in embryo quality or implantation/pregnancy rates between proposed zygote classes.

Key Words: embryo quality • human • IVF • zygote

3 To whom correspondence should be addressed. E-mail: andres.salumets@vaestoliitto.fi

Human Reproduction, Vol. 16, No. 10, 2118-2123, October 2001

Tracking of oocyte dysmorphisms for ICSI patients may prove relevant to the outcome in subsequent patient cycles

James S. Meriano, Jennifer Alexis, Shirin Visram-Zaver, Micheal Cruz and Robert F. Casper,1

Division of Reproductive Sciences, Department of Obstetrics and Gynecology, Toronto Centre for Advanced Reproductive Technology, University of Toronto, Toronto, Ontario, Canada

BACKGROUND: We determined whether oocyte dysmorphisms, especially repetition of specific dysmorphisms from cycle to cycle, had a prognostic impact on intracytoplasmic sperm injection (ICSI) outcome. METHODS: ICSI patients (n = 67) were grouped as follows: group 1 >50% phenotypically dysmorphic oocytes per cohort (cytoplasmic and extra-cytoplasmic dysmorphisms) with no repetition of a specific dysmorphism from cycle one to cycle two (36 cycles and 274 oocytes); group 2 >50% dysmorphic oocytes per cohort and repetition of the same dysmorphism from cycle one to cycle two (32 cycles and 313 oocytes); group 3 (control) <30% dysmorphic oocytes (33 cycles and 378 oocytes). RESULTS: In group 2 (repetitive), 47% of oocytes were observed to have organelle clustering versus 20.5% in group 1 and 17.3% in group 3 (P < 0.001). There was no difference between the groups in fertilization rates, cleavage rates or embryo quality. Embryos derived from normal oocytes were transferred in each group (57, 33 and 72% respectively). The clinical pregnancy and implantation rates in group 2 (3.1 and 1.7% respectively) were lower (P < 0.01, P = 0.005) than both group 1 (28 and 15% respectively) and group 3 (45.5 and 26.5% respectively). CONCLUSIONS: The low implantation rate in group 2, even though 33% of transferred embryos were derived from morphologically normal oocytes, suggests that repetitive organelle clustering may be associated with an underlying adverse factor affecting the entire follicular cohort.

Key Words: cytoplasm • ICSI outcome • implantation rates • oocytes dysmorphisms • organelle clustering

1 To whom correspondence should be addressed at: Division of Reproductive Sciences, Department of Obstetrics and Gynecology, Samuel Lunenfeld Research Institute, 600 University Avenue, Toronto, Ontario M5G 1Z5, Canada. E-mail: rfcasper@aol.com

In vitro maturation of oocytes: towards a daily practice?

O Gaspard, Liège, Belgium

In an IVF cycle, about 15% of retrieved oocytes are at the germinal vesicle (GV) stage. In some patients, however, all retrieved oocytes are at the GV stage, and for them in vitro maturation (IVM) is the only option. The competence of GV oocytes to mature and undergo fertilisation in classical fertilisation medium is not known, nor has the influence of in vitro maturation on fertilisation been determined. Dr Gaspard described the results of a study to determine whether IVM in an IVM medium plus intracytoplasmic sperm injection (ICSI) improves maturation, fertilisation, cleavage and blastulation rates compared with IVM without specific medium plus standard IVF.

A group of 59 GV-stage cumulus-oocyte complexes collected from 16 patients were matured in tissue culture medium supplemented with follicle-stimulating hormone, human chorionic gonadotrophin, penicillin/streptomycin and patient heat-inactivated serum. All oocytes that matured to metaphase II underwent ICSI and were then cultured in the appropriate fertilisation medium until day 6. A second group of 14 GV-stage cumulus-oocyte complexes were kept in fertilisation medium with spermatozoa for 18 hours.

In group 1, nearly 63% of GV oocytes matured within 24 hours and an additional 15% within 48 hours, to give an overall maturation rate of 78%. The overall fertilisation and cleavage rates were 78% and 97%, respectively after ICSI of matured oocytes. The fertilisation rates for oocytes matured within 24 hours and within 48 hours were significantly different (89% versus 33%; p < 0.02). After 2 days of culture, 41% of embryos contained eight cells or more, but at 3 days the blastulation rate was 16%. In contrast, the maturation rate was significantly lower in group 2 embryos (19%; p < 0.001). None of the 14 oocytes was fertilised during incubation in fertilisation medium with spermatozoa.

The results of this study indicate that IVM in specific maturation medium achieves significantly higher maturation and fertilisation rates than culture of GV-stage oocytes in standard fertilisation medium followed by IVF. It also appears that oocytes that resume meiosis within 24 hours are more competent in terms of fertilisation than those that matured within 48 hours. Dr Gaspard's group is now moving on to attempts to improve the low blastulation rate of IVM by upgrading the culture conditions (adding growth factors and other cytokines, altering the oxygen content of the culture atmosphere) and to studying the effects of hormones and cytokines. They believe that this procedure will eventually allow IVM to be incorporated into the current range of assisted reproduction techniques.

Establishing a quality management system (International Standards Organisation ISO 9001/2000) in a reproductive medicine unit's clinical programme

SJ Brown, Adelaide, Australia

Although quality management systems are used routinely in industry and are increasingly used in medicine, they have been slow to make an impact on clinical medicine programmes, said Brown. So far, quality assurance has been largely confined to IVF laboratories, and has not reached clinical practice, perhaps because they are little known or understood. Yet the advantages of quality assurance are many, including risk reduction, improved customer service, and efficiency gains, and Brown felt that it was essential to meet increasing demand from patients for reassurance about quality. The aim of this nursing staff-initiated project was to introduce an effective and efficient management system to all areas of the business of a reproductive medicine unit. A small committee was established to oversee the project and a project manager was seconded to supervise the project and adapt the ISO system from an industry standard. The adapted ISO system needed to cover all areas of the business in three separate fully functional infertility units. Over a period of 12 months the unit underwent a quality accreditation process that resulted in Quality Assurance Service (QAS) granting ISO 9001/2000-quality accreditation to the unit. Brown pointed out that accreditation was not the end but the beginning of an ongoing process. The accreditation has had a major impact in several areas, including cost savings, increased staff collaboration and morale, improved documentation, reduced medico-legal risk, improved customer service, and reassurance that the unit meets high standards. Compliance with the audit has been rapid and sustained and it has enabled staff to quickly spot and rectify any problems. In terms of clinical outcomes, pregnancy rates have increased rapidly and have been sustained, which Brown feels is partly due to ISO. Summing up the benefits, Brown said she feels that a quality management system does ensure correct procedures are followed throughout the business. It is an ongoing process and she is proud that it has led to an increase in the quality of care. It is the first reproductive medicine unit in Australia to become quality accredited under the ISO standard and the first in the world to receive this new ISO 9001/2000 standard. Brown concluded that a quality management system is worthwhile because it works.

Assessment of DNA integrity and morphology of ejaculated spermatozoa from fertile and infertile men before and after cryopreservation.

Donnelly ET, Steele EK, McClure N, Lewis SE. Department of Obstetrics & Gynaecology, The Queen's University of Belfast, Northern Ireland, UK.

Cryopreservation of human spermatozoa is extensively used in artificial insemination and IVF programmes. Despite various advances in cryopreservation methodology, the recovery rate of functional post-thaw spermatozoa remains mediocre, with sperm motility being significantly decreased after freezing. This aim of this study was to investigate the effects of cryopreservation on both DNA integrity and morphology of spermatozoa from fertile and infertile men. Semen samples were obtained from 17 fertile and 40 infertile men. All samples were prepared by discontinuous Percoll density centrifugation (95.0:47.5). Samples were divided into aliquots to allow direct comparison of fresh and frozen spermatozoa from the same ejaculate. Aliquots for cryopreservation were mixed with a commercial cryoprotectant and frozen by static phase vapour cooling before plunging into liquid nitrogen. Thawing was carried out slowly at room temperature. Sperm DNA integrity was determined using a modified alkaline single cell gel electrophoresis (comet) assay and sperm morphology analysed using the Tygerberg criteria. DNA of semen and prepared spermatozoa from fertile men was found to be unaffected by cryopreservation. In marked contrast, spermatozoa from infertile men were significantly damaged by freeze-thawing. Cryopreservation had a detrimental effect on morphology of semen and prepared samples from fertile and infertile men.

Hum Reprod 2001 Jun;16(6):1191-9

Source: humrep.oupjournals.org

The future of human ovarian cryopreservation and transplantation: fertility and beyond

Kim SS, Battaglia DE, Soules MR.

Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, University of Washington, Seattle, Washington, USA

Objective: To review the current progress in ovarian cryopreservation and transplantation and to discuss the obstacles with the clinical application of this technique.Design: The literature on ovarian cryopreservation and transplantation was reviewed to facilitate understanding and predict future directions. The studies related to this topic were identified through MEDLINE and other bibliographic databases, focusing on the most recent developments.Conclusion(s): The experimental evidence for low-temperature storage of ovarian tissue is encouraging. Although restoration of fertility with cryopreserved ovarian grafts has been successful in various animals, there are uncertainties about the optimum use of stored ovarian tissue in humans. Autotransplantation appears to be promising, but the potential risk of transmitting malignant cells in women with cancer is of great concern. The maturation of primordial follicles with xenotransplantation is encouraging, but the efficacy and the safety of this method need further investigation. Furthermore, the quality of oocytes that have been matured in a host animal is unknown. The development of in vitro culture systems for oocyte maturation is still in its infancy. There are many issues to be resolved in ovarian transplantation before the full clinical use of this emerging technique. Most of all, there is an urgent need to optimize the freeze/thaw procedure and to find the means to protect grafts from ischemia-reperfusion injury. Nevertheless, ovarian transplantation should prove to be clinically useful for women at risk for premature ovarian

failure.

Fertil Steril 2001 Jun;75(6):1049-56

Source: www.ncbi.nlm.nih.gov

Download to Citation Manager Human Reproduction, Vol. 16, No. 5, 918-924, May 2001

The effect of intracytoplasmic sperm injection and semen parameters on blastocyst development in vitro

Jane E. Miller and T. Timothy Smith1,

North Hudson IVF Center, 385 Sylvan Avenue, Englewood Cliffs, NJ 07632, USA

The present study compares the development and quality of blastocysts derived from conventional oocyte insemination with those derived from intracytoplasmic sperm injection (ICSI). Oocytes were collected from patients undergoing ovarian stimulation with human menopausal gonadotrophins for IVF. Patients with normal semen were assigned to conventional oocyte insemination while those with progressive motility <20% and/or normal sperm morphology 4% were assigned to ICSI. Resulting embryos were cultured for up to 6 days. The mean number and percentage of embryos reaching the blastocyst stage and the mean number and percentage of blastocysts of high quality on days 5&endash;6 were assessed for both treatment groups and compared. The influence of paternal factors (sperm concentration, motility, progressive motility, morphology) on blastocyst development and quality were assessed by regression analyses. Significantly more ICSI-derived embryos arrested at the 5- to 8-cell stage (P = 0.024) concomitant with the activation of the paternal genome than those derived from conventional oocyte insemination. Significantly fewer ICSI-derived embryos reached the blastocyst stage on days 5&endash;6 (P < 0.001) and significantly fewer ICSI-derived embryos were of high quality (P = 0.002) compared with conventional oocyte insemination. When treatment groups were combined and evaluated by regression analysis, progressive motility and sperm morphology were significantly correlated with diminished blastocyst development and quality (P < 0.05). From these data, we conclude that paternal factors and/or performing ICSI in cases of severe male factor infertility may have a detrimental effect on blastocyst development and their quality.

Key Words: blastocyst • ICSI • paternal effects • spermatozoa

1 To whom correspondence should be addressed. E-mail: twopn@optonline.net

Download to Citation Manager Human Reproduction, Vol. 16, No. 5, 902-908, May 2001

Extended embryo culture in human assisted reproduction treatments

M.T. Langley1,3, D.M. Marek1, D.K. Gardner2, K.M. Doody1 and K.J. Doody1

1 Center for Assisted Reproduction, Bedford, Texas, 76022 and

2 Colorado Center for Reproductive Medicine, Englewood, Colorado, 80110, USA

In order to evaluate the niche of extended embryo culture in an IVF programme, retrospective analysis of non-selected IVF patients, who underwent ovarian stimulation from April 1998 to June 1999 in a single private practice assisted reproductive technology centre, was performed. Embryos were cultured for 48 h in S1/G1.2 medium followed by 48 to 72 h of culture in S2/G2.2 to day 5 or day 6. Only fertilized oocytes exhibiting two pronuclei from donor and non-donor IVF and intracytoplasmic sperm injection (ICSI) cases were examined to determine the relationship between embryo cell number on day 3 and subsequent rate of blastocyst formation. Results indicated that a proportional relationship existed between the number of blastomeres present in day 3 embryos and the rate of blastocyst formation. Fifty-four per cent of embryos that had six cells on day 3 formed blastocysts, while 76% of those embryos with eight cells formed blastocysts. Blastocyst development did not increase further when embryos had more than eight cells on day 3, indicating that embryos with greater cell numbers on day 3 are not always predictive of a greater likelihood of blastocyst formation. Fertilized oocytes exhibiting two pronuclei from donors produced significantly more blastocysts (67%) than those from IVF patients (52%; P < 0.01), and had a significantly higher implantation rate (54%) compared with IVF patients (30%; P < 0.01). Furthermore, blastocyst cryopreservation resulted in significantly higher implantation rates than cryopreserved cleavage stage embryos (P < 0.001).

Key Words: embryo culture • embryo development • implantation • pregnancy • viability

3 To whom correspondence should be addressed. E-mail: martinl@embryo.net

Human oocyte cryopreservation: new perspectives regarding oocyte survival

R. Fabbri1,3, E. Porcu1, T. Marsella1, G. Rocchetta2, S. Venturoli1 and C. Flamigni1

1 IVF Center, Human Reproductive Medicine Unit, Institute of Obstetrics and Gynecology, and

2 Department of Biology, University of Bologna, 40138 Bologna, Italy

The success of human oocyte cryopreservation depends on morphological and biophysical factors that could influence oocyte survival after thawing. Various attempts to cryopreserve human oocytes have been performed with contrasting results. Therefore the effect of some factors, such as the presence or absence of the cumulus oophorus, the sucrose concentration in the freezing solution and the exposure time to cryoprotectants, on human oocyte survival after thawing were investigated. The oocytes were cryopreserved in 1,2-propanediol added with sucrose, using a slow-freezing-rapid-thawing programme. After thawing, the oocytes were inseminated by intracytoplasmic sperm injection (ICSI) and the outcomes of insemination and subsequent embryo development were also recorded. The post-thaw cryosurvival rate was not different for the oocytes cryopreserved with their cumuli partially removed mechanically (56%) when compared with those cryopreserved with their cumuli totally removed enzymatically (53%). On the contrary, a significantly higher survival rate was obtained when the oocytes were cryopreserved in the presence of a doubled sucrose concentration (0.2 mol/l) in the freezing solution and the survival rate was even higher when the sucrose concentration was tripled (0.3 mol/l) (60 versus 82% P < 0.001). Furthermore, a longer exposure time (from 10.5 to 15 min) to cryoprotectants, before lowering the temperature, significantly increased the oocyte survival rate (P < 0.005). Intracytoplasmic sperm injection produced a good fertilization rate (57%) of thawed oocytes and a high embryo cleavage rate (91%) and a satisfactory embryo morphology was observed (14 and 34% for grade I and grade II embryos respectively).

Key Words: cryoprotectants • exposure time • human oocyte cryopreservation • oocyte survival • sucrose concentration

Human Reproduction, Vol. 16, No. 3, 411-416, March 2001

Human Reproduction, Vol. 16, No. 4, 686-690, April 2001

A postal survey of embryo transfer practice in the UK

Osama H. Salha, Victoria K. Lamb and Adam H. Balen,1

Department of Reproductive Medicine, Clarendon Wing, Leeds General Infirmary, Leeds, UK

In this postal survey a questionnaire was sent to all unit directors in the UK to determine their attitudes to the factors influencing embryo transfer practice. They were requested to rate each step on a scale of 1-10, where 1 was irrelevant and 10 very important. A total of 80 practitioners from 40 units replied. Over 50% of the corresponding practitioners were consultants, 33% were middle-grade clinicians, and 12% were infertility nurse specialists. The factor that got the highest rating was the need for a standardized protocol for all unit staff regarding embryo transfer technique. The second critical factor voted by the respondents was the presence of blood on the embryo transfer catheter. Not touching the uterine fundus was deemed to be the third most important factor while the type of embryo transfer catheter used was a very close fourth. Prolonged bed rest following embryo transfer was voted the least important factor to influence the outcome. The wide variations in practice and choice of catheters encountered in this survey are indications of the divided opinion and lack of concrete evidence on which to base any firm decisions. The need for large clinical studies to assess clearly whether higher pregnancy rates will result from modifications in embryo transfer practice is highlighted.

Key Words: catheter • embryo transfer technique • implantation rates

1 To whom correspondence should be addressed at: Department of Reproductive Medicine, Clarendon Wing, Leeds General Infirmary, Leeds LS2 9NS, UK. E-mail: abalen@ulth.northy.nhs.uk

Pregnancy rates after embryo transfer depend on the provider at embryo transfer

Rhonda M. Hearns-Stokes M.D., Bradley T. Miller M.D., Lynette Scott Ph.D., David Creuss Ph.D., Prabir K. Chakraborty Ph.D. and James H. Segars M.D.

Assisted Reproductive Technologies Program, Walter Reed Army Medical Center, Walter Reed Army Medical Center and National Institute of Child

Health and Human Development, National Institutes of Health, Washington, D.C., USAPediatric and Reproductive Endocrine Branch, National Institute of Child

Health and Human Development, National Institutes of Health, Walter Reed Army Medical Center and National Institute of Child Health and Human

Development, National Institutes of Health, Washington, D.C., USADepartment of Obstetrics and Gynecology, Uniformed Services University

of the Health Sciences, Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA

Abstract

Objective: To evaluate the effect of individual providers on pregnancy outcome after embryo transfer. Design: Retrospective data analysis.

Setting: University-based tertiary-care assisted reproductive technology program with 10 physician-providers. Patient(s): Six hundred and

seventeen women who underwent 854 fresh embryo transfers between January 1996 and January1999. Intervention(s): Pregnancies after embryo transfer

were recorded for each provider. Main Outcome Measure(s): Establishment of a clinical pregnancy. Result(s): Three hundred ninety-three clinical

pregnancies resulted from 854 embryo transfers, for an overall clinical pregnancy rate of 46.0% per embryo transfer. Three hundred forty-seven

(40.6%) pregnancies were ongoing. The clinical pregnancy rate varied significantly between providers: for example, 17.0% (47 transfers) vs.

54.3% (57 transfers) (P<.05). Similarly, the ratio of high-grade embryos required to produce a gestational sac differed between providers. The

number or quality of embryos transferred did not differ significantly.Conclusion(s): Significant differences were observed in pregnancy rates

after embryo transfer done by different providers, suggesting that embryo transfer technique may influence pregnancy outcome in assisted reproductive technology.

Fertil Steril 2000 Jul;74(2):80-86

Source: www.sciencedirect.com

Embryo development and chromosomal anomalies after ICSI: effect of the injection procedure

John C.M. Dumoulin, Edith Coonen, Marijke Bras,J.Marij Bergers-Janssen, Rosie C.M. Ignoul-Vanvuchelen,Lucie C.P. van Wissen, Joep P.M. Geraedts and Johannes L.H. Evers

Research Institute of Growth and Development (GROW), University of Maastricht, Department of Obstetrics and Gynaecology and Department of

Molecular Cell Biology and Genetics, Academic Hospital Maastricht, Maastricht, The Netherlands

Intracytoplasmic sperm injection (ICSI) is a delicate procedure requiring considerable skills of the person performing it.

Theoretically, the injection procedure could damage cytoplasmic structures in the oocyte, resulting in sublethal cellular injury and/or

numerical chromosomal abnormalities that could lead to impaired embryonic development. In the present study, features of the injection

procedure were evaluated in a total of 2924 oocytes from 305 cycles. Development to the blastocyst stage was found to be compromised in a

group of surplus embryos originating from oocytes in which >6 pl of cytoplasm was aspirated into the injection pipette during the ICSI

procedure. Characteristics of the injection procedure as well as blastocyst development of surplus embryos was shown to be significantly

different between the four technicians performing the ICSI. Neither the volume of cytoplasm aspirated during the injection procedure, nor the

position of the polar body (6 o'clock or 12 o'clock) influenced the mean incidence of disomic cells per blastocyst as revealed by fluorescence

in-situ hybridization using probes specific for chromosomes X, Y and 18. In conclusion, certain technical aspects of the injection procedure can

affect subsequent embryonic development to the blastocyst stage, but do not seem to influence the rate of chromosomal abnormalities that occur

in human pre-implantation embryos.

Hum Rep 2001 Feb;16(2):306-312

Source: intl-humrep.oupjournals.org

Italian, US Scientists Unveil Human Cloning Effort

CHICAGO (Reuters Health) Jan 29 - An international group of reproductive

experts plans to launch a serious effort to clone humans to providechildren to infertile couples.

A viable cloned embryo could be available for implantation in a woman'suterus within 18 months, said Dr. Panayiotis Zavos of The Andrology

Institute of America and the Kentucky Center for Reproductive Medicine andIn Vitro Fertilization in Lexington, Kentucky.

Dr. Zavos hosted a conference last week in Lexington, where he and Italianfertility expert Dr. Severino Antinori announced plans for a scientific

coalition to clone humans. "This is going to be the first serious effort,"Dr. Zavos said in a telephone interview. "I do know various individual

groups that are acting on their own, but they lack the support."

"As revolutionary as it may sound, as fictional as it may sound, it will bedone," Dr. Zavos continued. "It's a genie that is out of the bottle and

will be controlled." He said that 10 infertile couples have volunteered to participate.

The consortium plans to operate in an unnamed Mediterranean country. Thescientists will use regular cells or stem cells from each male partner and

insert them into an ovacyte. The wives could also be the ones cloned, depending on the couple's choice, Dr. Zavos said.

He announced that his group will hold a conference in Rome in March, to which a cardinal from the Vatican would be invited.

The Roman Catholic Church is opposed to human cloning.

"We have a great deal of knowledge. We can grade embryos, we can do genetic screening, we can do quality control," Dr. Zavos said. "It's not the

easiest thing. The stability of the genetic information is what's important....You don't want to create a monster."

To create animal clones, scientists typically make hundreds of failed attempts to develop viable embryos. Medical ethicists have posed the

possibility of cruel failures in human cloning, where genetic abnormalities result in grotesque fetuses unable to survive outside the womb.

Dr. Antinori has sparked a furor in Italy by helping postmenopausal women give birth, and he has pioneered a technique to help infertile men by

"cultivating" their nascent sperm cells inside the testicles of mice.

1: Hum Reprod 2001 Jan;16(1):153-163 t

Evidence that glucose is not always an inhibitor of mouse preimplantation development in vitro.

Biggers JD, McGinnis LK

Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA.

A factorial experimental design was used to examine the effects of 16 combinations of four concentrations of glucose (0.20, 0.60, 1.8, 5.4 mmol/l) and four concentrations of potassium dihydrogen phosphate (KH(2)PO(4); 0.05, 0.15, 0.45, 1.35 mmol/l) on the development in vitro of outbred CF1 mouse zygotes. Three responses were measured: (i) the number of zona-enclosed blastocysts; (ii) the number of blastocysts that started to hatch; and (iii) the total cell counts in the blastocysts. General linear modelling was used to estimate the most parsimonious two-dimensional concentration-response surfaces that represent the three responses to the different concentrations of glucose and KH(2)PO(4). There were no significant interactions between the effects of glucose and KH(2)PO(4) in all cases. Thus, the effects of glucose and phosphate are independent. No significant effects of glucose on blastocyst formation and the initiation of hatching were observed. Increasing the concentration of KH(2)PO(4) inhibited slightly (</=20%) the development of zygotes into blastocysts and the initiation of hatching. The slight inhibitory effects of KH(2)PO(4) appeared to be due to the inhibition of the development of a few sensitive embryos. No significant effects of glucose and KH(2)PO(4) were observed on the total cell counts.

The morphology of human pronuclear embryos is positively related to blastocyst development and implantation*

Lynette Scott1,4, Ruben Alvero2, Mark Leondires2 and Bradley Miller3

1 The A.R.T. Institute of Washington, Inc. at Walter Reed Army Medical Center,

2 Division of Reproductive Endocrinology, Department of Obstetrics and Gynaecology, Walter Reed Army Medical Center, Washington DC, and

3 Department of Obstetrics and Gynaecology, National Naval Medical Center, Bethesda, MD, USA

Human embryos are selected for transfer using morphology at the cleaving and blastocyst stages. Zygote morphology has been related to implantation and pregnancy. The aim of this study was to relate pronuclear morphology to blastocyst development. Zygotes were scored according to distribution and size of nucleoli within each nucleus. Zygotes displaying equality between the nuclei had 49.5% blastocyst formation and those with unequal sizes, numbers or distribution of nucleoli had 28% blastocyst formation. Cleaving embryos that were selected initially by zygote morphology and secondarily by morphology on day 3 had increased implantation (IR) and pregnancy rates (PR; 31 and 57%), compared with those selected by morphology alone (19 and 33% respectively; P < 0.01). There was a significant difference between zygote-scored and non-scored cycles on day 3 (PR: 57 versus 33%; IR: 31 versus 19%) and on day 5 (PR: 73 versus 58%; IR; 52 versus 39%). Zygote scoring can maintain pregnancy rates for both day 3 and day 5 transfers, increase implantation rates and reduce the numbers of embryos required to achieve a pregnancy.

Key Words: blastocyst • nucleoli • pronuclear morphology • zygote scoring

4 To whom correspondence should be addressed at: The A.R.T. Institute Inc. at Walter Reed Army Medical Center, PO Box 59727, Washington, DC 20012, USA. E-mail: lynette.scott@na.amedd.army.mil

see also Zygote scoring

BIOETHIC REVOLUTION STARTS IN FRANCE.

France will review its 1994 Bioethic law for Spring 2001. What a progress!

Research on frozen "forgotten embryos ", i.e. abandonned by the parents , will be allowed.This should permitt basic research on ES Cells ( embryonic stamm cells also called totipotent cells).

Moreover cloning for therapeutics aims will be also allowed, but reproductive cloning stays fortunately forbidden. Good scientific perspectives for France .

Relationship between granular cytoplasm of oocytes and pregnancy outcome following intracytoplasmic sperm injection

Kahraman S, Yakin K, Donmez E, Samli H, Bahce M, Cengiz G, Sertyel S, Samli M, Imirzalioglu N

Sevgi Hospital, Reproductive Endocrinology and ART Unit, Sevgi Hospital, Genetic Division, G.A.T.A. Genetic Division and Sevgi Hospital Andrology Unit, Turkey

Couples undergoing intracytoplasmic sperm injection (ICSI) for male infertility using oocytes with centrally located granular cytoplasm (CLCG) were evaluated for fertilization, embryo development, implantation and pregnancy rate. CLCG is a rare morphological feature of the oocyte, that is diagnosed as a larger, dark, spongy granular area in the cytoplasm. Severity is based on both the diameter of granular area and the depth of the lesion. Twenty-seven couples with 39 cycles presenting CLCG in >50% of retrieved oocytes were evaluated. A total of 489 oocytes was retrieved, out of which 392 were at MII. CLCG was observed in 258 of the MII oocytes (65.8%); 66.7% of these oocytes had slight and 33.3% had severe CLCG. The overall fertilization rate was 72.2% and no statistical significant difference was found between normal and CLCG oocytes and between the oocytes representing slight and severe CLCG. The development and quality of embryos was the same in normal and CLCG oocytes. In nine cycles, preimplantation genetic diagnosis was executed to evaluate a possible accompanying chromosomal abnormality. Out of 44 blastomeres biopsied, 23 had chromosomal abnormality (52.3%). Eleven pregnancies were achieved in 39 cycles (28.2%), six pregnancies resulted in abortion (54.5%). The implantation rate was found to be 4.2%. Only five ongoing pregnancies were achieved in 39 cycles (12.8%). Couples with CLCG oocytes should be informed about poor on-going pregnancy rates even if fertilization, embryo quality and total pregnancy rates are normal. Furthermore, a high aneuploidy rate may be linked to a high abortion rate.

Hum Reprod 2000 Nov;15(11):2390-2393

Source: humrep.oupjournals.org

Prospective randomized study of two cryopreservation policies avoiding embryo selection: the pronucleate stage leads to a higher cumulative delivery rate than the early cleavage stage

Senn A, Vozzi C, Chanson A, De Grandi P, Germond M

Reproductive Medicine Unit, Department of Gynaecology and Obstetrics, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland

Objective: To compare the cumulative live birth rates obtained after cryopreservation of either pronucleate (PN) zygotes or early-cleavage (EC) embryos. Design: Prospective randomized study. Setting: University hospital. Patient(s): Three hundred eighty-two patients, involved in an IVF/ICSI program from January 1993 to December 1995, who had their supernumerary embryos cryopreserved either at the PN (group I) or EC (group II) stage. For 89 patients, cryopreservation of EC embryos was canceled because of poor embryo development (group III). Frozen-thawed embryo transfers performed up to December 1998 were considered. Main Outcome Measure(s): Age, oocytes, zygotes, cryopreserved and transferred embryos, damage after thawing, cumulative embryo scores, implantation, and cumulative live birth rates. Result(s): The clinical pregnancy and live birth rates were similar in all groups after fresh embryo transfers. Significantly higher implantation (10.5% vs. 5.9%) and pregnancy rates (19.5% vs. 10.9%; P</=.02 per transfer after cryopreserved embryo transfers were obtained in group I versus group II, leading to higher cumulative pregnancy (55.5% vs. 38.6%; P</=.002 and live birth rates (46.9% vs. 27.7%; P</=.0001.Conclusion(s): The transfer of a maximum of three unselected embryos and freezing of all supernumerary PN zygotes can be safely done with significantly higher cumulative pregnancy chances than cryopreserving at a later EC stage.

Fertil Steril 2000;74(5):946-952

Source: www.ncbi.nlm.nih.gov

Initial IVF-ET experience with assisted hatching performed 3 days after retrieval followed by day 5 embryo transfer

Graham MC, Hoeger KM, Phipps WR

Department of Obstetrics-Gynecology, University of Rochester, Rochester, New York, USA

Objective: To report our initial IVF-ET experience combining assisted hatching performed 3 days after oocyte retrieval with day 5 embryo transfer (ET).Design: Retrospective review of 110 consecutive IVF cycles not involving donor oocytes, including 16 cycles that involved assisted hatching performed 3 days after oocyte retrieval in combination with day 5 ET. Setting: Academic teaching hospital IVF center. Patient(s): Eighty-six consecutive IVF patients undergoing ET. Intervention(s): Assisted hatching using acid Tyrode's solution performed 3 days after oocyte retrieval in selected cases in combination with day 3 or 5 ETs. Main Outcome Measure(s): Clinical pregnancy rate per ET. Result(s): Of the 16 women undergoing day 5 ET following day 3 assisted hatching, 14 had a clinical pregnancy. These included 11 ongoing/delivered singletons and 2 ongoing/delivered twin pregnancies, neither of which was monochorionic. These clinical and ongoing/delivered pregnancy rates compared very favorably with those of 54% and 46%, respectively, for the 35 patients undergoing day 5 ETs without assisted hatching, even though the latter group appeared to be better IVF candidates based on the prognostic factors commonly used to predict success. Conclusion(s): Our experience suggests that day 3 assisted hatching followed by day 5 ET may be a useful combination in selected patients. Although not seen in our small series, an increased risk of monochorionic pregnancies remains a theoretical concern when such a combination is used, since both assisted hatching and blastocyst transfers have been independently linked to an increased risk in some reports.

Fertil Steril 2000 Oct 1;74(4):668-671

Source: www.ncbi.nlm.nih.gov

Blastocyst score affects implantation and pregnancy outcome: towards a single blastocyst transfer

Gardner DK, Lane M, Stevens J, Schlenker T, Schoolcraft WB

Colorado Center for Reproductive Medicine, Englewood, Colorado, USA

Objective: To determine the relationship between blastocyst score and pregnancy outcome. Design: Retrospective review of blastocyst transfer in an IVF clinic. Patient(s): 107 patients undergoing blastocyst culture and transfer of two embryos. Intervention(s): Culture of all pronucleate embryos in sequential media to the blastocyst stage (day 5), followed by transfer of two blastocysts. Result(s): When a patient received two top-scoring blastocysts (64% of patients), implantation and pregnancy rates were 70% and 87%, respectively. The twinning rate in this group was 61%. When only one top-quality blastocyst was available for transfer (21% of patients), the implantation and pregnancy rates were 50% and 70%. The twinning rate for this group was 50%. In contrast, when only low-scoring blastocysts were available for transfer (15% of patients), implantation and pregnancy rates were 28% and 44%, and the twinning rate was 29%. No monozygotic twins were observed in this group of patients. Conclusion(s): The ability to transfer one high-scoring blastocyst should lead to pregnancy rates greater than 60%, without the complication of twins.

Fertil Steril 2000 Jun 1;73(6):1155-1158

Source: www.ncbi.nlm.nih.gov

First accreditation of an IVF laboratory in the Netherlands

Peter Kastrop & Sjerp Weima

In July 1995 newspapers reported a story which shocked the world of assisted conception: mixed race twins were born as the result of an IVF pregnancy! A mistake with disastrous consequences happened in our IVF laboratory, which raised questions about our professional practice. The hospital instigated an internal inquiry in conjunction with an external ART expert, and the IVF laboratory and procedures were investigated and assessed. The commission concluded that the IVF procedures practised were according to generally accepted standards in IVF laboratories and stated that that protocols were in place and adhered to. Nevertheless, the commission recommended that IVF procedures should be improved and tightened. They took the stand that embryos which are generated in an IVF laboratory can be considered as unique living products, and therefore IVF and ICSI procedures should follow the strict guidelines of Good Manufacturing Practice (GMP), as used by the Pharmaceutical industry. Therefore, in order to optimize the standardization of the laboratory procedures and improve quality assurance and safety, we began to implement GMP regulations into daily practice....at least as far as applicable, since not all GMP rules can be simply introduced into an IVF-laboratory. For instance, regulations about maintaining of reserve samples, random sampling of the endproducts and automatic labelling and packaging of products did not seem to be applicable. On the other hand, several GMP rules did fit perfectly in ART procedures, with IVF/ICSI embryos or prepared semen suspensions regarded as products of an ART laboratory. Amongst the most important GMP regulations implemented were:

1. availability of detailed written standard operating procedures in the laboratory in order to assure the quality of the 'products'

2. validation of critical steps in the procedures

3. adequate labelling of containers to assure unambigious identification

4. performance and recording of every transfer of components by one person to be verified by a second person.

All laboratory operations were thus further detailed and supplemented with procedures concerning the logistics within the laboratory and the use, calibration and maintenance of equipment. All of these operating procedures were drawn up in a standard format and managed according to fixed rules. Process procedures in which critical steps were identified, e.g. all actions in which samples are transferred from one dish or tube to another, all moments where samples of different origin come close to each other as well as all procedures regulating the transfer of samples to or from patients, were carefully detailed. Accompanying laboratory forms were adapted in order to record that a particular procedure had been performed correctly by one person and checked by a second person. Consequently, all physicians, nurses and embryologists involved in the fertility treatment of a certain couple (e.g. from ovum pick up to embryo transfer) are registered on these laboratory forms to guarantee that procedures have been performed accurately and that each gamete and/or embryo was handled properly.

As a result of the publicity about the twin-incident, people working in the field of ART became aware of the responsibility and risks of their work, assuming that similar incidents can occur or might have occurred unnoticed elsewhere. However good protocols and practice in place may be, this can never compensate for negligence or human error...and any error will have far reaching effects.

In the Netherlands, there is an authority responsible for accreditation of clinical laboratories , the Coordinating Committee for the promotion of Quality control of Laboratory research and testing pertaining to the Health Care Sector (CCKL). The Dutch Society of Clinical Embryologists, officially founded in 1991, became a member of CCKL in order to improve laboratory quality control and quality assurance. In cooperation with CCKL, the society developed a 'Model Quality Handbook of Clinical Embryology' (MQHCE) which was based on the CCKL Code of Practice for implementation of a Quality System in Laboratories in the Health Care Sector'. This Code of Practice includes all CCKL guidelines and describes the conditions which quality systems in clinical laboratories must meet if quality is to be assured, according to the EN 45001 and ISO 9000 series of standards.

The Model Quality Handbook for Clinical Embryology, released in December 1996 interpets CCKL guidelines for the field of ART laboratory practice. The Handbook describes the conditions required to fulfill a quality system in general terms: it does not contain protocols, but instead guidelines for setting up protocols in a standardized manner. Every ART laboratory must write its own local quality handbook or manual according to these guidelines. In this way, the Model Handbook forms the basis for setting up an integral quality system in an ART laboratory. Wherever GMP guidelines were not adequate for setting up such a complete quality system, the Model Handbook of our professional association did suffice for accreditation. The more so as CCKL guidelines demand more than '"production and process control' protocols. Clinical laboratories are also expected to meet requirements concerning the supply of goods and services, management and use of means of research, facilities, management of documentation, complaints and deviations and internal and external assessments of the quality system. Furthermore, the organisation of the laboratory should be described at both the management and execution levels, i.e. regulations pertaining to professional expertise and responsibilities of embryologists and technicians have to be laid down in the quality manual.

In order to meet all CCKL requirements and conditions laid down in the Model Quality Handbook, in 1997 we began working towards extending the quality system, based on several applicable GMP regulations. The implementation of those GMP rules gave us a lead over other ART laboratories, which made it possible to establish the complete quality system within two years and to submit an application for accreditation to CCKL.

The Authority recently conferred accreditation upon the IVF laboratoryof the University Medical Center Utrecht, according to the European Norm EN 45001, ISO guide 25, ISO 9001 and ISO 9002. This accreditation involved the complete ART program of our laboratory, i.e. all aspects of the treatment of infertile patients with IUI, IVF or ICSI, cryopreservation of human embryos and sperm, including semen analyses and the organisation and management of the donor bank.

Before the end of this year other ART laboratories in the Netherlands will probably be accredited, and perhaps within a few years all 13 laboratories will have constructed a quality system in acccordance with the standards mentioned above.

The implementation of an integral quality system in our laboratory has improved the assurance of proper identification of all samples, the standardization and transparency of our procedures and the traceability of all our actions in such a way that mistakes can be avoided in future.

It is now our responsibility as an accredited IVF laboratory to maintain the high standards of quality control and assurance of all services and to continue improving and optimizing treatment for all of our patients.

FRANCE: New law for IVF products in preparation.... July 2000

The french agency for the sanitary security for health products recently asked all the IVF centers / laboratories to establish an outstanding list of all products used for laboratory procedures.Aim of this action: to promote a new category of medical products called: PTA i.e.Produits Thérapeutiques Annexes (Annex Therapeutic Products) coming in contact with human gametes or embryos.

these PTA will be estimated for security level and will be given a special status and consequently should be subjected to specific agreement.

further developments of this important items as soon as more informations from France will be available...

CV

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