Robert M.L. Winston

Birth of a Normal Girl after in Vitro Fertilization and Preimplantation Diagnostic Testing for Cystic Fibrosis

Abstract Background. Cystic fibrosis is a common, severe autosomal recessive disease caused in a majority of cases by a three-nucleotide deletion (ΔF508) 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 ΔF508 deletion. In vitro fertilization techniques were used to recover oocytes from each woman and fertilize them with her husbands 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 ...

BIOPSY OF HUMAN PREIMPLANTATION EMBRYOS AND SEXING BY DNA AMPLIFICATION

A single cell was removed, through a hole made in the zona pellucida, from each of 30 human embryos at the 6-10 cell cleavage stage three days after in-vitro fertilisation. A normal proportion of the embryos (37%) developed to the blastocyst stage by day six in culture and 6 hatched from the zona. Each male embryo was sexed from the DNA by amplification of a repeated sequence specific for the Y chromosome. In 15 embryos with the normal two pronuclei the sex was determined also by in-situ hybridisation with a Y specific probe or fluorescent chromosome staining to detect metaphase Y chromosomes; the results of Y specific amplification were confirmed. This approach may be valuable for couples at risk of transmitting X-linked disease.

Multicolour FISH detects frequent chromosomal mosaicism and chaotic division in normal preimplantation embryos from fertile patients

Abstract We have used multicolour fluorescent in situ hybridisation (FISH) with DNA probes for chromosomes X, Y and 1 to analyse spare untransferred cleavage-stage embryos after preimplantation diagnosis to avoid X-linked disease. In total, 93 morphologically normal embryos were available from seven patients (six of proven fertility) who had undergone fourteen in vitro fertilisation (IVF) cycles. The chromosome patterns observed were classified into four groups; normal, abnormal (non-mosaic), mosaic and chaotic (uncontrolled division). Approximately half of the embryos were normal for the chromosomes tested. Two embryos only were aneuploid (non-mosaic) throughout but, after excluding those showing chaotic division, 30% were considered to be chromosomal mosaics. Of these, a minority had arisen because of mitotic non-disjunction or chromosome loss or gain, whereas the majority were ploidy mosaics, with haploidy being the most common. The occurrence of chaotically dividing embryos was strongly patient-related, i.e. some patients had ‘chaotic’ embryos in repeated cycles, whereas other patients were completely free of this type of anomaly. ‘Chaotic’ embryos are unlikely to progress beyond implantation. These findings have important implications both for routine IVF and preimplantation genetic diagnosis.

Infertile couples with Robertsonian translocations : preimplantation genetic analysis of embryos reveals chaotic cleavage divisions

Abstract Preimplantation genetic diagnosis (PGD) may provide a feasible option for some Robertsonian translocation carriers who experience severe difficulty in achieving a normal pregnancy. We report on five PGD cycles for two such couples, 45,XY,der(13;14)(q10:q10) and 45,XX,der(13;21)(q10;q10), carried out by biopsy of two cells from day 3 post-insemination embryos generated by in vitro fertilisation. Locus-specific YAC probes for chromosomes 13, 14 and 21 were used to detect the chromosomes involved in the translocation using multicolour FISH. Three embryos transfers were carried out (two single embryo transfers and one double transfer) but no clinical pregnancies were established. In two cycles no embryos were transferred as all those biopsied were chromosomally abnormal. Combined results from both couples show 13% (6/45) of embryos analysed were normal for the translocation chromosomes and 87% (39/45) were chromosomally abnormal; these were categorised as 36% aneuploid or aneuploid mosaic and 51% chaotic where the chromosome constitution varied randomly from cell to cell. This suggests two factors may be acting to reduce fertility in these couples; the aneuploid segregation of the parental Robertsonian translocation and also a post-zygotic factor leading to uncontrolled chromosome distribution in early cleavage stages in an exceptionally high proportion of embryos.

Selection criteria for human embryo transfer: A comparison of pyruvate uptake and morphology

PurposePyruvate 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 or 3 and correlated the levels with implantation and pregnancy outcome.ResultsThe 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.ConclusionWe 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.

Anti-Apoptotic Action of Insulin-Like Growth Factor-I During Human Preimplantation Embryo Development

Abstract Insulin-like growth factor I (IGF-I) has been shown to increase the proportion of embryos forming blastocysts and the number of inner cell mass cells in human and other mammalian preimplantation embryos. Here we examined whether the increased cell number resulted from increased cell division or decreased cell death. Normally fertilized, Day 2 human embryos of good morphology were cultured to Day 6 in glucose-free Earles balanced salt solution supplemented with 1 mM glutamine, with (n = 42) and without (n = 45) 1.7 nM IGF-I. Apoptotic cells in Day 6 blastocysts were identified using terminal deoxynucleotidyl dUTP terminal transferase (TUNEL) labeling to detect DNA fragmentation and 4’-6-diamidino-2-phenylindole (DAPI) counterstain to evaluate nuclear morphology. The number of nuclei and extent of DNA and nuclear fragmentation was assessed using laser scanning confocal microscopy. IGF-I significantly increased the proportion of embryos developing to the blastocyst stage from 49% (control) to 74% (+IGF-I) (P < 0.05). IGF-I also significantly decreased the mean proportion of apoptotic nuclei from 16.3 ± 2.9% (–IGF-I) to 8.7 ± 1.4% (+IGF-I) (P < 0.05). The total number of cells remained similar between both groups (61.7 ± 4.6 with IGF-I; 54.5 ± 5.1 without IGF-I). The increased number of blastocysts combined with reduced cell death suggests that IGF-I is rescuing embryos in vitro which would otherwise arrest and acting as a survival factor during preimplantation human development.

Mechanical isolation and in vitro growth of preantral and small antral human follicles.

OBJECTIVE To develop a procedure for isolating small human follicles and to determine their growth requirements. DESIGN Preantral and early antral follicles were isolated manually, allocated randomly to experimental groups, and cultured for a few weeks. SETTING Patients giving informed consent in hospitals. PATIENT(S) Women undergoing laparotomy or oophorectomy. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Follicular size, E2, histology. RESULT(S) Human FSH (at a dose of 1.5 U/mL) induced antral growth of follicles, and the addition of human LH (2.5 ng/mL) to human FSH stimulated growth and antral development. Histologic studies showed that most of the early antral follicles did not contain an oocyte and already had begun to undergo atresia before culturing. Levels of E2 increased in the incubation medium as the follicles increased in size, but those levels were significantly greater when the follicles contained oocytes. CONCLUSION(S) It is possible to grow small human follicles after they have been isolated manually. To develop successfully, they require a low concentration of human LH in addition to human FSH. The rate of atresia between the preantral and early antral stages in vivo is very high; therefore, it is worthwhile to develop techniques for isolating and culturing the follicles before the antral stages.

Dual fluorescent in situ hybridisation for simultaneous detection of X and Y chromosome-specific probes for the sexing of human preimplantation embryonic nuclei

SummaryDual fluorescent in situ hybridisation has been used for the simultaneous detection of X and Y chromosome-specific probes in single cleavage nuclei from disaggregated 4- to 7-cell human embryos. Based on the presence of a Y signal or 2 X signals in the absence of a Y, 89% of poor quality metaphases and 72% of interphase nuclei could be classified as male or female. With further refinements, this technique will offer a credible alternative to the polymerase chain reaction for the diagnosis of sex in human preimplantation embryos in families segregating for X-linked genetic disease.

Microsurgery of the Fallopian Tube: From Fantasy to Reality

The author presents 4 examples of experimental research being conducted in the techniques of microsurgery in animals. The surgically modified oviduct, capture of ova by the fimbriae, adhesion formation, and the pathophysiology of damaged tubes have been dealt with through microsurgery. Similar microsurgical techniques were applied clinically and the reported results are essentially raw data to be analyzed in the future. The operating microscope is used for all infertility surgery and it can be used over a range of magnifications. Other instruments are also mentioned, e.g., telescopic spectacles. The surgical approach is dealt with--routinely employed is the Pfannenstiel incision, a large incision. Tissues are handled as little as possible since peritoneal raw areas are an important precursor of later adhesion formation. Nonabsorbable sutures are best and nylon appears to cause the least reaction. It is helpful to leave hydrocortisone acetate in the peritoneal cavity to prevent adhesions. The success of tubal anastomosis is dependent on the method used and the patient selected. For example, of those with diathermy burns, 55% conceived. With diathermic coagulation, the pelvis remains relatively clean and the tubes adhesion-free. The authors, however, maintain that isthmic-isthmic anastomosis is best. The microscope is also used in cases of fimbrial damage; however, the results are poor. Results after repeat salpingostomy following previous surgery are even worse; of 59 patients, only 8.4% had term pregnancies. The technique is superior to those results obtained with implantation. While microsurgery is a tremendously useful technique, it would be better to develop better methods of preventive medicine. Furthermore, adequate training for microsurgery should come in the form of a fellowship at an appropriate institution.

Maintenance of the Inner Cell Mass in Human Blastocysts from Fragmented Embryos

Abstract The degree of fragmentation during early cleavage is universally used as an indicator of embryo quality during human in vitro fertilization treatment. Extensive fragmentation has been associated with reduced blastocyst formation and implantation. We examined the relationship between early fragmentation and subsequent allocation of cells to the trophectoderm and inner cell mass in the human blastocyst. We retrospectively analyzed data from 363 monospermic human embryos that exhibited varying degrees of fragmentation on Day 2. Embryos were cultured from Day 2 to Day 6 in Earle balanced salt solution with 1 mM glucose and human serum albumin. Rates of development and blastocyst formation were measured. The number of cells in the trophectoderm and inner cell mass and the incidence of apoptosis were assessed following differential labeling with polynucleotide-specific fluorochromes. Increasing fragmentation resulted in reduced blastocyst formation and lower blastocyst cell numbers. For minimal and moderate levels of fragmentation, the reduction in cell numbers was confined largely to the trophectoderm and a steady number of inner cell mass cells was maintained. However, with extensive fragmentation of more than 25%, cell numbers in both lineages were reduced in the few embryos that formed blastocysts. Apoptotic nuclei were present in both the trophectoderm and inner cell mass, with the lowest incidence in blastocysts that had developed from embryos with minor (5–10%) fragmentation. Paradoxically, higher levels of apoptosis were seen in embryos of excellent morphology, suggesting a possible role in regulation of cell number.