Publications
110 results found
Soussis I, Harper JC, Handyside AH, et al., 1996, Obstetric outcome of pregnancies resulting from embryos biopsied for pre-implantation diagnosis of inherited disease., Br J Obstet Gynaecol, Vol: 103, Pages: 784-788, ISSN: 0306-5456
OBJECTIVE: Pre-implantation diagnosis of inherited disease is now a viable option for some couples at risk of transmitting inherited disorders to their children. Since the pregnancy begins knowing that the embryo is not at risk, the need for repeated terminations is eliminated. Up to 25% of the embryo is removed during the procedure, and so it is important to study the resulting pregnancies. Here we report on the obstetric outcome of our first 16 pregnancies resulting from embryo biopsy and preimplantation diagnosis of inherited disease. SETTING: Teaching hospital. SAMPLE: The first 16 pregnancies (12 singletons and 4 twins) following pre-implantation diagnosis. RESULTS: Three singleton pregnancies were lost in the first trimester. Of the remaining pregnancies, two had no prenatal diagnosis, six cases of X-linked disease had the sex confirmed by ultrasound and chorionic villus sampling was performed in the remaining five. All the singleton pregnancies had an uneventful antenatal course and the birthweights and Apgar scores of the babies were normal. The twin pregnancies presented obstetric complications but these were not unusual. CONCLUSIONS: Fifteen healthy infants were born, but for the foreseeable future pre-implantation diagnosis pregnancies should be closely followed up.
Soussis I, Trew G, Matalliotakis I, et al., 1996, Symptoms after day-case in vitro fertilization, JOURNAL OF ASSISTED REPRODUCTION AND GENETICS, Vol: 13, Pages: 513-516, ISSN: 1058-0468
Nikas G, Ao A, Winston RM, et al., 1996, Compaction and surface polarity in the human embryo in vitro., Biol Reprod, Vol: 55, Pages: 32-37, ISSN: 0006-3363
The surface morphology of the human ovum fertilized and cultured in vitro to the morula stage was studied by scanning electron microscopy with the specific aim of investigating embryo compaction and polarity. Unfertilized oocytes examined one day after attempted insemination (Day 0) were evenly and densely covered by long microvilli. The length and density of microvilli appeared to decrease in fertilized polypronuclear oocytes; a further decrease was observed in Day 2 and Day 3 embryos with 2-12 cells. No evidence of compaction or surface polarity was observed in any of these stages. On Day 4, compaction was evident in the majority of embryos with 10 or more cells, and the microvilli appeared dense again with a polarized distribution over the free surface of the compacted blastomeres. This study provides ultrastructural evidence that the human conceptus undergoes a relatively marked compaction at the morula stage during Day 4 postinsemination development in vitro.
Hovatta O, Silye R, Krausz T, et al., 1996, Cryopreservation of human ovarian tissue using dimethylsulphoxide and propanediol-sucrose as cryoprotectants, HUMAN REPRODUCTION, Vol: 11, Pages: 1268-1272, ISSN: 0268-1161
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- Citations: 266
Ao A, Handyside A, Winston RM, 1996, Preimplantation genetic diagnosis of cystic fibrosis (delta F508)., Eur J Obstet Gynecol Reprod Biol, Vol: 65, Pages: 7-10, ISSN: 0301-2115
Cystic fibrosis is a common autosomal recessive condition caused by mutations in the cystic fibrosis transmembrane regulator gene. The major mutation is a three base pair deletion (delta F508). If both partners carry this deletion, the chance of having an affected child is 1 in 4. In vitro fertilization (IVF) with preimplantation genetic diagnosis allows the selection of the unaffected embryos only to be returned to the uterus. Preimplantation genetic diagnosis was attempted in 14 couples in which both partners carry the delta F508 deletion. A total of 22 cycles resulted in 170 normally fertilized embryos of which, 145 embryos were successfully biopsied and in 18 cycles, one or two unaffected embryos were transferred. A total of five clinical pregnancies established and at birth all five singletons have been confirmed as homozygous for the normal allele. From our experience, cleavage stage biopsy after in vitro fertilization provides sufficient embryos diagnosed as unaffected for transfer in this autosomal recessive disease. Also, pregnancy rates after the preimplantation diagnosis are similar to those with infertile couples. Prospects for applying preimplantation genetic diagnosis to autosomal dominant conditions, where incidences of having affected embryos would be higher, therefore, appear good.
Soussis I, Harper JC, Kontogianni E, et al., 1996, Pregnancies resulting from embryos biopsied for preimplantation diagnosis of genetic disease: biochemical and ultrasonic studies in the first trimester of pregnancy., J Assist Reprod Genet, Vol: 13, Pages: 254-258, ISSN: 1058-0468
PURPOSE: Our purpose was to investigate early biochemical and ultrasonic measurements of pregnancies resulting from embryos biopsied for preimplantation diagnosis of inherited disease. RESULTS: Singleton pregnancies following biopsy had lower initial hCG levels [10 or 12 days after oocyte recovery (OR)], which rose steeply to match the controls by 16 days after OR. Twin biopsied pregnancies showed hCG levels lower than those of twin control pregnancies, which rose in parallel with the controls but remained lower for a longer period than the singletons. Progesterone levels showed a wide variation. Ultrasound measurements showed that overall the mean sac diameter and crown-rump length at 28 and 42 days after egg collection were similar in biopsied and control pregnancies. CONCLUSIONS: Pregnancies resulting from biopsied embryos behave similarly to control IVF pregnancies. However, the reduction in cell mass following embryo biopsy occasionally results in reduced levels of circulating serum hCG and smaller ultrasound measurements in early pregnancy.
Ray PF, Winston RM, Handyside AH, 1996, Reduced allele dropout in single-cell analysis for preimplantation genetic diagnosis of cystic fibrosis., J Assist Reprod Genet, Vol: 13, Pages: 104-106, ISSN: 1058-0468
BACKGROUND: For couples at risk of transmitting a known single-gene defect, preimplantation genetic diagnosis (PGD) allows the identification and transfer of only unaffected embryos following in vitro fertilisation (IVF), single-cell biopsy at about the eight-cell stage, and genetic analysis by PCR. This technique therefore avoids the risk of terminating an affected pregnancy diagnosed later in gestation. METHODS AND RESULTS: Using nested PCR, the delta F508 mutation causing cystic fibrosis can be detected in single cells and we previously reported successful PGD in a couple in whom both partners carry the delta F508 mutation. To date we have treated 12 couples in a total of 18 cycles. This resulted in five singleton births confirmed to be homozygous normal. Single blastomeres from disaggregated embryos which had not been transferred were analysed to confirm the original diagnosis and assess reliability in clinical practice. Amplification efficiency and accuracy were high, with blastomeres from embryos diagnosed as homozygous normal or affected. In a proportion of blastomeres from presumed carrier embryos, one of the parental alleles failed to amplify, apparently at random (allele dropout, ADO). A possible explanation is the relative inaccessibility of one of the target allele early in the PCR. To test this we have used single lymphocytes from delta F508 carriers and investigated the effects of various denaturation temperatures in the early cycles of amplification. CONCLUSIONS: Increasing the denaturation temperature reduced the rate of ADO without affecting amplification efficiency.
Ao A, Ray P, Harper J, et al., 1996, Clinical experience with preimplantation genetic diagnosis of cystic fibrosis (delta F508)., Prenat Diagn, Vol: 16, Pages: 137-142, ISSN: 0197-3851
Preimplantation genetic diagnosis (PGD) was attempted in 12 couples in whom both parents carry the common delta F508 deletion causing cystic fibrosis (CF). In vitro fertilization (IVF) was followed by cleavage stage biopsy on days 2 and 3 and removal of one or two cells for genetic analysis by nested polymerase chain reaction (PCR) and heteroduplex formation. A total of 18 cycles resulted in 137 normally fertilized embryos, of which 115 developed to cleavage stages and 114 were successfully biopsied. Genetic analysis was successful in 83 embryos (73 per cent). With the remaining embryos, either results from two or more cells were discordant or amplification failed. In 15 cycles, one or two either normal or carrier embryos were transferred and five (33 per cent) clinical pregnancies were established. Five singletons have been born and at birth all five babies have been confirmed as homozygous for the normal allele. Our experience demonstrates that IVF and cleavage stage biopsy consistently provides sufficient embryos, diagnosed as unaffected, for transfer in this autosomal recessive disease and that pregnancy rates are comparable to those following IVF.
HARDY K, ROBINSON FM, PARASCHOS T, et al., 1995, NORMAL DEVELOPMENT AND METABOLIC-ACTIVITY OF PREIMPLANTATION EMBRYOS IN-VITRO FROM PATIENTS WITH POLYCYSTIC OVARIES, HUMAN REPRODUCTION, Vol: 10, Pages: 2125-2135, ISSN: 0268-1161
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- Citations: 57
BROSENS JJ, DESOUZA NM, BARKER FG, et al., 1995, ENDOVAGINAL ULTRASONOGRAPHY IN THE DIAGNOSIS OF ADENOMYOSIS UTERI - IDENTIFYING THE PREDICTIVE CHARACTERISTICS, BRITISH JOURNAL OF OBSTETRICS AND GYNAECOLOGY, Vol: 102, Pages: 471-474, ISSN: 0306-5456
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- Citations: 80
Geber S, Winston RM, Handyside AH, 1995, Proliferation of blastomeres from biopsied cleavage stage human embryos in vitro: an alternative to blastocyst biopsy for preimplantation diagnosis., Hum Reprod, Vol: 10, Pages: 1492-1496, ISSN: 0268-1161
Normally fertilized human embryos were biopsied at cleavage stages on the third day after in-vitro fertilization (IVF). One or two blastomeres at the 8-cell stage were removed and co-cultured with the biopsied embryos. Embryos and blastomeres were assessed daily for morphological development until day 6, when the number of cells were counted by labelling the nuclei. In all, 53% of the biopsied embryos (25 out of 47) reached the blastocyst stage between day 5 and 6 and the proportion was the same irrespective of the number of cells removed. There was no significant difference between biopsied embryos from which one or two blastomeres respectively had been removed with regard to total cell numbers at the blastocyst stage (56.2 +/- 3.0 and 64.7 +/- 5.5), number of trophectoderm (45.4 +/- 3.5 and 44.0 +/- 5.7) and inner cell mass cells (14.0 +/- 1.2 and 16.6 +/- 1.8). Overall, 72% of the isolated blastomers divided at least once over 3 days in culture and 50% divided more than once. The mean overall cell number after 3 days in culture was 3.7 +/- 0.48 per blastomere (range 1-8 cells) if one cell was removed and 6.9 +/- 1.0 if two cells were removed. If the undivided blastomeres are excluded, the mean cell number was 4.8 +/- 0.51 and 8.3 +/- 1.0 respectively. Over this period, 55% of the blastomeres cavitated. Of the blastomeres taken from embryos that developed to the blastocyst stage, 92% divided and 76% cavitated. In those from arrested embryos, 50% divided (P < 0.002) and 32% cavitated (P < 0.003).(ABSTRACT TRUNCATED AT 250 WORDS)
GEBER S, PARASCHOS T, ATKINSON G, et al., 1995, RESULTS OF IVF IN PATIENTS WITH ENDOMETRIOSIS - THE SEVERITY OF THE DISEASE DOES NOT AFFECT OUTCOME, OR THE INCIDENCE OF MISCARRIAGE, HUMAN REPRODUCTION, Vol: 10, Pages: 1507-1511, ISSN: 0268-1161
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- Citations: 83
Ray PF, Conaghan J, Winston RM, et al., 1995, Increased number of cells and metabolic activity in male human preimplantation embryos following in vitro fertilization., J Reprod Fertil, Vol: 104, Pages: 165-171, ISSN: 0022-4251
The number of cells and metabolic activity of male and female human preimplantation embryos were examined to determine whether male embryos are more advanced than female embryos following in vitro fertilization (IVF). The metabolic activity of embryos fertilized normally was assessed daily by non-invasive measurement of pyruvate and glucose uptake and lactate production between days 2 and 6 after insemination. On day 6, the numbers of nuclei from the trophectoderm and inner cell mass of blastocysts were counted by differential labelling and fluorescence microscopy. Nuclei were then recovered and the sex of the embryos identified using nested primers to amplify the amelogenin gene and pseudogene sequences on the X and Y chromosomes, respectively. Development of male and female embryos were then compared retrospectively. From 69 of 178 (39%) embryos that developed to the blastocyst stage, the sex of 57 was determined; 21 (37%) were male and 36 (63%) female. The number of cells in male embryos was significantly greater on day 2 (P < 0.005), and this difference was maintained up to the blastocyst stage (in both the trophectoderm and the inner cell mass), although differences were not always significant. Pyruvate uptake was significantly higher by male embryos between days 2 and 5 (P < 0.05). Glucose uptake and lactate production were significantly higher in male embryos on days 4-5 (P < 0.05); this difference was not significant on days 5-6. Extrapolation from differences in the number of cells indicates that female embryos are approximately 4.5 h delayed in their development from day 2 onwards compared with male embryos.(ABSTRACT TRUNCATED AT 250 WORDS)
Moohan JM, Winston RM, Lindsay KS, 1995, The variable effects of 2'-deoxyadenosine on human sperm motility and hyperactivation in vitro., Hum Reprod, Vol: 10, Pages: 1098-1103, ISSN: 0268-1161
The response of human sperm motility and hyperactivation to the stimulant 2'-deoxyadenosine (2'-DEA) was studied in vitro using computer-assisted sperm motion analysis. A total of 20 randomly selected individuals with normal sperm counts as defined by the World Health Organization were chosen and their migration-separated spermatozoa exposed to a range (0.1-10.0 mM) of concentrations of 2'-DEA. The straight line velocity (VSL) was increased above control values only at 0.1 mM, while the curvilinear velocity (VCL) and lateral head displacement (ALH) were increased significantly at all concentrations. Linearity of progression (LIN), on the other hand, declined with increasing concentration of 2'-DEA. These changes were related to a significant increase in the number of spermatozoa exhibiting hyperactive-like motion. There was, however, considerable intra-individual variability in the response to 2'-DEA. In some individuals VCL and ALH exhibited little or no response to 2'-DEA, whilst in others an increase above the control of 50-55% occurred. The maximum response for VCL and ALH occurred at 2.5 mM 2'-DEA. Individuals showed greater variability in the percentage of spermatozoa exhibiting hyperactivity in response to 2'-DEA, with increases ranging from 76 to 948% of the control value, although the maximum response was also most commonly seen at 2.5 mM 2'-DEA. The diversity of response to 2'-DEA emphasizes the importance of tailoring doses to the individual rather than employing one concentration for all. Further tests on a subgroup of the individuals examined the longevity of spermatozoa in response to 24 h of continued exposure to 2'-DEA.(ABSTRACT TRUNCATED AT 250 WORDS)
Chia CM, Winston RM, Handyside AH, 1995, EGF, TGF-alpha and EGFR expression in human preimplantation embryos., Development, Vol: 121, Pages: 299-307, ISSN: 0950-1991
Epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha) through their common receptor, epidermal growth factor receptor (EGFR) are known to enhance mitogenesis, development and implantation in several species. In the mouse, co-culture of grouped embryos in microdrops increases the cell number and proportion developing to the blastocyst stage. A similar effect is observed with culture of single embryos in medium supplemented with EGF or TGF-alpha highlighting their embryotrophic effects. To study the role of EGF, TGF-alpha and EGFR in early human development, two methods applicable for analysis of expression at the single embryo level have been employed. In the first method, reverse transcription-polymerase chain reaction has been used to examine the presence of transcripts. Following reverse transcription, strategically designed nested primers, optimised for specificity, were used for amplification from the cDNA equivalent of a single embryo. The products were then verified by restriction enzyme digestion and sequence analysis. In the second method, immunocytochemistry has been used to colocalise the expressed proteins. Individual embryos were paraffin embedded and serial sectioned, allowing adjacent sections to be examined with different antibodies and controls. Monoclonal TGF-alpha and polyclonal EGF and EGFR primary antibodies were used. Staining was performed by peroxidase-conjugated avidin-biotin immunocytochemistry with the appropriate controls. The combination of these two methods can potentially be used for simultaneous analysis of several growth factors and/or their receptors in the same human embryos. Transcripts for EGF, TGF-alpha and EGFR were detected in unfertilized oocytes and embryos between 8-cell and blastocyst stages on day 3 to 6 post-insemination.(ABSTRACT TRUNCATED AT 250 WORDS)
Dawson KJ, Conaghan J, Ostera GR, et al., 1995, Delaying transfer to the third day post-insemination, to select non-arrested embryos, increases development to the fetal heart stage., Hum Reprod, Vol: 10, Pages: 177-182, ISSN: 0268-1161
The purpose of this study was to determine whether delaying embryo transfer by 24 h, until day 3 post-insemination, allowed improved selection of non-arrested embryos for transfer. We have retrospectively analysed pregnancy rates in a large series of patients who had embryo transfer either on day 2 or on day 3 post-insemination over a 27 month period. From January 1990 to March 1992, 567 patients received embryo transfer on day 2, and 661 patients had transfer on day 3 post-insemination, but these transfers were not contemporary. Pregnancy rates were slightly higher in patients who had embryo transfer on day 3 (37%) than in those patients who had their embryos transferred on day 2 (35%), but this difference was not significant. The implantation rate, as measured by the proportion of embryos developing to the fetal heart stage, was significantly higher following transfer on day 3 (23%) than after transfer on day 2 (19%) (P < 0.05), suggesting that selection of viable embryos is improved on day 3. Furthermore, of the embryos which gave rise to a fetal sac, significantly fewer miscarried before the fetal heart stage (P < 0.05) following transfer on day 3 (6%) than after transfer on day 2 (12%). Delaying transfer until day 3 provides a further 24 h to observe embryo development. During this period 16% of embryos arrested or became developmentally retarded; thus waiting until day 3 allowed these embryos to be identified and avoided for consideration for transfer. Embryo transfer may be safely delayed until day 3, and this may help in selecting embryos most likely to implant and develop after transfer.
MASON HD, WILLIS DS, BEARD RW, et al., 1994, ESTRADIOL PRODUCTION BY GRANULOSA-CELLS OF NORMAL AND POLYCYSTIC OVARIES - RELATIONSHIP TO MENSTRUAL-CYCLE HISTORY AND CONCENTRATIONS OF GONADOTROPINS AND SEX STEROIDS IN FOLLICULAR-FLUID, JOURNAL OF CLINICAL ENDOCRINOLOGY & METABOLISM, Vol: 79, Pages: 1355-1360, ISSN: 0021-972X
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- Citations: 172
Harper JC, Robinson F, Duffy S, et al., 1994, Detection of fertilization in embryos with accelerated cleavage by fluorescent in-situ hybridization (FISH)., Hum Reprod, Vol: 9, Pages: 1733-1737, ISSN: 0268-1161
Embryos from a couple undergoing routine in-vitro fertilization for unexplained infertility had shown cleavage by day 1 in two consecutive cycles. The response of the woman to fertility drugs had been normal, and there were no known sperm abnormalities. In a subsequent cycle, accelerated cleavage occurred again and we used a modified method of spreading whole embryos and dual fluorescent in-situ hybridization (FISH) with directly labelled probes for chromosomes X and Y to determine if fertilization had occurred in these embryos. Nine oocytes were collected, five of which had cleaved when examined for pronuclei early on day 1 following late insemination. Pronuclei were observed in one of the remaining oocytes, but in this case, three were present. On day 2, all of the oocytes/embryos had cleaved and two were transferred to the patient. The remaining seven were spread for FISH analysis but nuclei were only obtained from five. In three, a Y signal was detected, indicating that fertilization had occurred. In all five embryos, a wide range of X chromosome signals were observed. These data suggest that the embryos had undergone abnormal fertilization and accelerated cleavage.
Harper JC, Coonen E, Ramaekers FC, et al., 1994, Identification of the sex of human preimplantation embryos in two hours using an improved spreading method and fluorescent in-situ hybridization (FISH) using directly labelled probes., Hum Reprod, Vol: 9, Pages: 721-724, ISSN: 0268-1161
Dual fluorescent in-situ hybridization (FISH) using X and Y chromosome specific probes has been used to identify the sex of human embryos for preimplantation diagnosis of X-linked disease. With a modified spreading method and directly labelled fluorescent DNA probes, we have examined the possibility of reducing the time of the FISH procedure from 7 to 2 h. A total of 17 normally fertilized human embryos were disaggregated and 98 intact blastomeres obtained. The spreading efficiency was 96% and FISH signals were obtained from 97% of nuclei. In all cases, sibling blastomeres from the same embryo were the same sex. Mosaicism was observed in some embryos. Five cells which lysed during the disaggregation process were spread to determine whether FISH was possible in these cells, but in all cases the morphology of the nuclei was poor and multiple signals were observed so that reliable diagnosis of sex was not possible. The data reported here confirm that by using an improved spreading method in combination with directly labelled DNA probes, we have increased the efficiency and reduced the time required for sexing embryos for preimplantation diagnosis of X-linked disease.
FISCH B, ROSE MP, ELDER MG, et al., 1994, EFFECTS OF ESTROGEN ON PROGESTERONE SYNTHESIS AND ARACHIDONIC-ACID METABOLISM IN HUMAN LUTEAL CELLS, CLINICAL ENDOCRINOLOGY, Vol: 40, Pages: 21-32, ISSN: 0300-0664
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- Citations: 11
Moohan JM, Winston RM, Lindsay KS, 1993, Variability of human sperm response to immediate and prolonged exposure to pentoxifylline., Hum Reprod, Vol: 8, Pages: 1696-1700, ISSN: 0268-1161
Pentoxifylline improves some motility characteristics of human spermatozoa, but the variability of response to this drug has not been clearly defined. We used computer-assisted sperm motion analysis to examine the in-vitro response of spermatozoa to pentoxifylline. Individuals (n = 31) with normal sperm counts were randomly selected and their spermatozoa exposed to different concentrations of pentoxifylline. Further tests on a subgroup of individuals examined the longevity of spermatozoa in response to this agent. Straight line velocity (VSL) was only improved at 0.1 mM and the major effect of the drug was on curvilinear velocity (VCL) and lateral head displacement (ALH). Prolonged exposure to pentoxifylline enhanced sperm motion only at 0.1 mM. Higher concentrations produced dose-dependent detrimental effects on all the motion characteristics. There was considerable inter-individual variability in both VCL and ALH response ranging from little or no detectable response to a 40% increase above control value. The maximum response was most commonly seen at a concentration of 2 mM pentoxifylline.
Conaghan J, Handyside AH, Winston RM, et al., 1993, Effects of pyruvate and glucose on the development of human preimplantation embryos in vitro., J Reprod Fertil, Vol: 99, Pages: 87-95, ISSN: 0022-4251
Although human embryos will develop in vitro for six days or more, little is known about the effects of the primary nutrients, pyruvate and glucose, on development. Because the nutrient requirements of embryos change throughout preimplantation development, the effects of altering substrate concentrations in the culture medium were examined, using 'surplus' human preimplantation embryos cultured from the two-four-cell stage to the blastocyst stage in medium containing various concentrations of pyruvate and glucose. Between the one-cell stage and the two-four-cell stage all of the embryos were exposed to 0.47 mmol pyruvate l-1 and 5.5 mmol glucose l-1. Pyruvate as sole substrate in the medium could support blastocyst development to an extent of 59% (10 of 17). Conversely, culture of embryos in pyruvate-free medium resulted in the developmental arrest of 84% (21 of 25) of embryos, and for the 16% (4 of 25) that did reach the blastocyst stage there was a significant decrease in metabolic activity on day 4-5, during the morula to blastocyst stage transition. Embryos could not use glucose to compensate for the lack of pyruvate in the medium. Pyruvate uptake was related to exogenous concentration and optimal development occurred at the highest concentration tested, 0.47 mmol l-1. Embryo development to the eight-cell stage was slightly enhanced 82% (14 of 17) versus 60% (24 of 40) when no glucose was added to the medium, and the resulting blastocysts had significantly more cells (99.1 +/- 13.5 versus 58.4 +/- 8.2; P < 0.02) than did embryos grown in the presence of 1 mmol glucose l-1.(ABSTRACT TRUNCATED AT 250 WORDS)
HARDY K, WINSTON RML, HANDYSIDE AH, 1993, BINUCLEATE BLASTOMERES IN PREIMPLANTATION HUMAN EMBRYOS IN-VITRO - FAILURE OF CYTOKINESIS DURING EARLY CLEAVAGE, JOURNAL OF REPRODUCTION AND FERTILITY, Vol: 98, Pages: 549-558, ISSN: 0022-4251
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- Citations: 130
Winston RM, Handyside AH, 1993, New challenges in human in vitro fertilization., Science, Vol: 260, Pages: 932-936, ISSN: 0036-8075
This review assesses some scientific and ethical problems with human in vitro fertilization. Improved selection of viable embryos, better culture conditions, and greater understanding of the uterine environment will increase success and prevent multiple pregnancy. Further advances will also improve oocyte cryopreservation, in vitro maturation of oocytes, knowledge of sperm function, and sperm microinjection. Preimplantation diagnosis will help avoid genetic diseases and increase understanding of embryonic defects and the viability of zygotes. The greatest ethical problem with all these developments seems to be delivery of these complex treatments when health-care resources are increasingly limited.
MASON HD, MARGARA R, WINSTON RML, et al., 1993, INSULIN-LIKE GROWTH FACTOR-I (IGF-I) INHIBITS PRODUCTION OF IGF-BINDING PROTEIN-1 WHILE STIMULATING ESTRADIOL SECRETION IN GRANULOSA-CELLS FROM NORMAL AND POLYCYSTIC HUMAN OVARIES, JOURNAL OF CLINICAL ENDOCRINOLOGY & METABOLISM, Vol: 76, Pages: 1275-1279, ISSN: 0021-972X
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- Citations: 54
BAKER G, JOUANNET P, VANSTEIRTEGHEM A, et al., 1993, TREATMENT OF SEVERE MALE-INFERTILITY WITH PURE FOLLICLE-STIMULATING-HORMONE (FSH) - THE NEED FOR A PROPERLY CONTROLLED MULTICENTER TRIAL, HUMAN REPRODUCTION, Vol: 8, Pages: 500-501, ISSN: 0268-1161
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- Citations: 9
Schrurs BM, Winston RM, Handyside AH, 1993, Preimplantation diagnosis of aneuploidy using fluorescent in-situ hybridization: evaluation using a chromosome 18-specific probe., Hum Reprod, Vol: 8, Pages: 296-301, ISSN: 0268-1161
Fluorescent detection of in-situ hybridization (FISH) with a chromosome 18-specific probe (P5041 B.5 D18Z1) has been used to assess the use of this method for preimplantation diagnosis of aneuploidy. Interphase nuclei (n = 802) have been analysed from 59 normally fertilized embryos developing in vitro at the normal rate between days 2 and 7 postinsemination. The efficiency of hybridization in control cells, as assessed by the proportion with two signals in normal female lymphocytes was 88.9% (n = 353) and with three signals in a trisomic (48,XXX+18) fibroblast cell line 74.0% (n = 290). Fifty-four of the human embryos were considered to be diploid on the basis that the majority of nuclei had two signals. Some nuclei in these embryos had one or no signal, especially on day 2, and tetraploid nuclei were also widespread. Among the remaining five embryos, one 5-cell embryo on day 2 had three hybridization signals in 4/5 nuclei and was trisomic for chromosome 18, one 4-cell embryo on day 2 had only one signal in 4/4 nuclei and was monosomic, and the three other embryos were aneuploid mosaics and/or had multi-nucleated blastomeres. Analysis of the incidence of interphase nuclei with more or less than the diploid number of hybridization signals indicates that more than a single nucleus will be necessary for accurate preimplantation diagnosis of aneuploidy.
Conaghan J, Hardy K, Handyside AH, et al., 1993, Selection criteria for human embryo transfer: a comparison of pyruvate uptake and morphology., J Assist Reprod Genet, Vol: 10, Pages: 21-30, ISSN: 1058-0468
PURPOSE: Pyruvate uptake is higher in human embryos developing to the blastocyst stage than those arresting at cleavage stages. To investigate whether pyruvate uptake provides an improved criterion for selecting embryos for transfer, we have measured uptakes by individual embryos noninvasively over 24-hr periods between the first day (day 1) postinsemination and embryo transfer on day 2 to 3 and correlated the levels with implantation and pregnancy outcome. RESULTS: The mean uptake was significantly lower for embryos that implanted than for those which failed to implant: 22.9 +/- 1.0 and 27.1 +/- 0.6 pmol/embryo/hr, respectively on day 2, and 22.4 +/- 1.5 and 26.9 +/- 0.8 pmol/embryo/hr, respectively, on day 3, but the wide range of uptakes by individual embryos was overlapping. CONCLUSION: We conclude that pyruvate uptake as the sole criterion for embryo selection cannot predict which embryos will implant after transfer. Assessment of embryos using morphological and developmental criteria, therefore, remains the most consistent, though inefficient, indicator of pregnancy potential.
Tarín JJ, Conaghan J, Winston RM, et al., 1992, Human embryo biopsy on the 2nd day after insemination for preimplantation diagnosis: removal of a quarter of embryo retards cleavage., Fertil Steril, Vol: 58, Pages: 970-976, ISSN: 0015-0282
OBJECTIVE: To assess any reduction in viability and development in vitro after biopsy of a quarter of the cells of human embryos on day 2 after insemination. DESIGN: A prospective study in which normally fertilized surplus embryos of good morphology with two to eight cells approximately 48 hours after insemination were randomly allocated to a control or biopsied group, respectively. SETTING: In vitro fertilization (IVF) unit and laboratories of the Hammersmith Hospital, Institute of Obstetrics and Gynaecology, London University. PATIENTS, PARTICIPANTS: One hundred twenty-nine embryos from 28 infertile IVF patients. INTERVENTIONS: Follicular aspiration by ultrasound-guided transvaginal puncture and embryo biopsy by micromanipulative procedures. MAIN OUTCOME MEASURE(S): Pyruvate uptake and cell number at the blastocyst stage. RESULTS: Embryo biopsy did not have an adverse effect on either the proportion developing to the blastocyst stage (50% [32 of 64] and 47.7% [31 of 65] for the control and biopsied groups, respectively) or embryo viability, measured indirectly through pyruvate uptake. However, the proportion of embryos that reached the morula stage after day 4 (retarded embryos) was significantly higher (44%, 11 of 25 versus 8.7%, 2 of 23) in the biopsied group. The total number of cells (29.6 +/- 3.1 versus 62.4 +/- 4.7), numbers of inner cell mass (7.7 +/- 2.2 versus 24.5 +/- 1.4) and trophectoderm (24.0 +/- 5.2 versus 45.0 +/- 6.4) cells, and the inner cell mass:trophectoderm ratio (34.7 +/- 7.9 versus 59.5 +/- 11.7) were strikingly reduced at the blastocyst stage in the biopsied group. This reduction was greater in embryos that reached the morula stage after day 4. CONCLUSIONS: More investigation is needed to assess whether the detrimental effects observed were because of the biopsy method used in this study or to a high sensitivity of human embryos at early stages to manipulation in vitro.
Handyside AH, Lesko JG, Tarín JJ, et al., 1992, Birth of a normal girl after in vitro fertilization and preimplantation diagnostic testing for cystic fibrosis., N Engl J Med, Vol: 327, Pages: 905-909, ISSN: 0028-4793
BACKGROUND: Cystic fibrosis is a common, severe autosomal recessive disease caused in a majority of cases by a three-nucleotide deletion (delta F508) in the cystic fibrosis transmembrane regulator gene. Current methods of prenatal diagnosis involve chorionic-villus sampling or amniocentesis. In vitro fertilization and diagnosis during embryonic development before implantation would allow only unaffected embryos to be selected for transfer to the uterus, thereby avoiding the need to terminate a pregnancy. METHODS: Preimplantation diagnosis of cystic fibrosis was attempted in the cases of three couples, both members of which carried the delta F508 deletion. In vitro fertilization techniques were used to recover oocytes from each woman and fertilize them with her husband's sperm. Three days after insemination, embryos in the cleavage stage underwent biopsy and removal of one or two cells for DNA amplification and analysis. RESULTS: Only two oocytes from one woman were fertilized normally; DNA analysis of one of the embryos failed and cystic fibrosis was diagnosed in the other (i.e., it was homozygous for delta F508), so neither was transferred. The oocytes of each of the other two women produced noncarrier, carrier, and affected embryos. Both couples chose to have one noncarrier embryo and one carrier embryo transferred. One woman became pregnant and gave birth to a girl free of the deletion in both chromosomes. CONCLUSIONS: Preimplantation diagnosis of the delta F508 deletion causing cystic fibrosis is possible through in vitro fertilization, biopsy of a cleavage-stage embryo, and amplification of DNA from single embryonic cells. This approach should be equally applicable to other single-gene diseases in which the defect has been identified. Analysis of a series of pregnancies, however, will be required to assess the method adequately.
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