Laslo Nebel

Chromosome analysis of multipronuclear human oocytes fertilized in vitro

In an in vitro fertilization and embryo transfer program composed mainly of patients with tubal infertility, the incidence of fertilized oocytes with multiple pronuclei was 4.3%. An attempt was made to fix such oocytes with supernumerary pronuclei in order to assess the chromosome constitution of the gametes. Nine multipronuclear oocytes containing a total of 29 pronuclei were successfully fixed before the first cleavage division, and another oocyte containing 3 pronuclei was fixed before the second cleavage division. On analysis, chromosome counts could be obtained for 28 of the 29 total pronuclei, and 24 gave informative results. Nineteen pronuclei had a normal haploid chromosome count, 2 pronuclei contained one extra chromosome, and 3 pronuclei had one chromosome missing. The presence of a Y chromosome in six pronuclei identified their paternal origin; two of the six sperm pronuclei had a 22,Y,-E chromosome constitution. Only three pronuclei could be conclusively ascertained to be maternal in origin, yet two of these were aneuploid; one pronucleus had a 24,X,+D karyotype and the second had only 22 chromosomes. Multipronuclear oocytes present ideal material for analyzing the chromosome constitution of those human gametes which can undergo fertilization in vitro.

Chromosome Analysis of Human Oocytes and Embryos Fertilized in Vitro

The phenomenon of early embryonic loss in the human female in the presence or absence of an associated clinical diagnosis of pregnancy has never before been so clearly demonstrated as it is now observed in women undergoing treatment for infertility by in vitro fertilization (IVF) and embryo transfer (ET). In even the most successful IVF and ET treatment programs, only about 5-10% of the total embryos transferred back to the recipient patients will actually result in a live birth.’ This represents, at its maximum level for the population of women undergoing embryo transfer, an embryonic mortality of 90-95%. There are, of course, many factors acting simultaneously which together determine the success or failure of any given IVF treatment cycle and the fate of the resulting embryos. Factors related to the state of the recipients’ endometrial tissue,2 the ease with which the transfer is carried out,3 the number of embryos t ran~ferred,~ and the age of the patient’ are all equally important. However, even when all of these extraneous factors are at their optimum and most beneficial level, as is seen in those women who have undergone IVF and ET and have subsequently delivered a normal baby (TABLE I ) , there is still an embryo mortality rate of around 60%. Thus, with embryo mortality lying somewhere between 60% at its best and 95% at its worst in a highly successful IVF and ET program, we have attempted to determine to what extent chromosome abnormalities, specifically gamete aneuploidy, contribute to the lack of viability of the majority of human embryos produced in vitro.6 The material for our cytogenetic studies has come from two sources: (i) multipronuclear oocytes (MPOs), that is, those oocytes that were observed to have more than the usual two pronuclei 14-24 hours after insemination, and (ii) “spare embryos,” which remain after the maximum 4-6 with the most regular morphologic appearance have been transferred back to the patient.’ As yet we lack the facilities for storing such “spare embryos” by freezing and it has been our policy, like that of others,’ to allow

Influence of Insemination on the Implantation of Transferred Rat Blastocysts

Embryo transfer probably produces a lower incidence of implantation than the physiological incidence despite all other factors seeming similar. The only factor known to be present physiologically and absent in embryo transfer is the presence of sperm in the uterine cavity. Implantation and decidualization are often considered a modified form of inflammatory reaction. Semen contains factors which excite an inflammatory response. This project attempted to determine whether insemination would affect the implantation rate of transferred blastocysts in the rat. The figures showed a significantly increased implantation rate after insemination at day 4 of pseudopregnancy as compared to controls.

Selection of patients with habitual abortion for paternal leucocyte immunization

SummaryAfter potentiation of the immune response in habitual aborters 75–85% of subsequent pregnancies are claimed to result in healthy term infants. However, all publications to date have either been based on the authors concept of the immune processes involved or an attempt to demonstrate the efficacy of treatment either empirically or by matched trials. As immunization is coming into wider clinical use, it is necessary to determine which patients will benefit from this form of treatment. This paper presents our experience with paternal leucocyte immunization over the period 1985–1988. 207 patients were classified on a clinical basis and by immunological testing. 143 patients have been immunised, 129 pregnancies have occured in 108 patients. The vast majority of our patients have recurrent missed abortions. Only six women habitually aborted live fetuses. Two had subsequent live births. Secondary aborters seem to do well in subsequent pregnancies, whether immunized or not. The patient most likely to benefit from immunization is the Primary missed aborter who does not possess antipaternal antibody (APCA), but is induced to produce APCA by immunization. Using these criteria, 75% success rates are observed in the subsequent pregnancy. This success rate is irrespective of HLA antigen sharing or invitro mixed lymphocyte reactivity.

The Capacitation Rate of Rat Sperm in Vitro

In the rat, as in many rodents where semen is deposited directly into the uterus, it is believed that the principal site for capacitation is the oviduct. In vivo, there is a temporal correlation between the migration of fertilizing sperm into the ampulla and the appearance of oocyte-cumulus complexes there. It has been suggested (Gwatkin 77) that in some rodents cumulus cells play an important part in capacitation while in others (Hoppe & Whitten 74, Niwa & Chang 74) their importance was not shown.

Characterization of the progesterone receptor solubilized by micrococcal nuclease and DNase I digestion

In order to investigate the functional organization of the progesterone receptor in chromatin we characterized the physical-chemical properties of the receptor bound chromatin fragments released by micrococcal nuclease and DNase I digestion. The crude nuclear fraction was isolated from T 47 D cells, previously exposed to 0.1 microM [3H]ORG 2058. The parameters determined in low and high salt concentrated buffers were: sedimentation coefficients (S) on a sucrose gradient, Stokes radii (Rs) by gel filtration on a Sephadex G-200 column and the binding abilities to a DNA-cellulose column. The molecular weights (Mr) and frictional ratios (f/fo) were calculated from the S and Rs values. Micrococcal nuclease digestion solubilized a receptor form sedimenting as a single peak at 4.4 S with a Rs = 7.78 nm and an estimated Mr = 144,000. About 53% of the applied receptor bound to a DNA-cellulose column could be eluted by high salt concentrated buffer. 0.4 M KCl dissociated this receptor form into a smaller receptor sedimenting at 3.3 S with Rs = 5.53 nm and a calculated Mr = 76,000. A similar receptor form was extracted by 0.6 M KCl from the undigested crude nuclear fraction. DNase I digestion solubilized a receptor form sedimenting at 3.3 S with a Rs = 6.87 nm and a calculated Mr = 94,000. About 26% of the applied receptor bound to a DNA-cellulose column could be eluted by high salt concentrated buffer. Dissociation of this receptor form by 0.4 M KCl resulted in a receptor sedimenting at 2.8 S with a Rs = 6.53 nm and an estimated Mr = 76,000. These results suggest: The progesterone receptor in chromatin is associated with several molecules probably proteins which complexed it to DNA. Some of these molecules still associated with the progesterone receptor could be released by nucleases digestion. Micrococcal nuclease releases a larger portion of these molecules than those release by DNase I.

Comparison of Cornual Transfer via Laparotomy with Utero-Cervical Transfer of Cultured Preimplantation Rat Embryos

Most work on embryo transfer has used the cornual route via laparotomy for implantation. This method is time-consuming and costly in operating materials. Cervical transfer seems to offer a simpler route and the promise of time and cost saving. Technical difficulties, however, have prevented this method from becoming widely used. It was thought that mastery of this technique is essential to allow large-scale experiments in order to determine the optimal time for reimplantation, the optimal stage whether 8 cells, morulae, or blastocysts, and to compare culture media. All of these will have significant clinical applications. In this work Vickerys method of cervical transfer in mice was modified to include direct vision of the cervix and dilatation before implantation. With this modification equivalent results were found on cervical or cornual transfer, but a higher failure rate on cervical transfer. The results and implications are discussed.

The Influence of Endometrial Preparation Protocols on Embryo Implantation in Human Cryopreservation Treatment

In promoting successful implantation of embryos following in vitro fertilization (IVF) and embryo transfer, there is always a dilemma as to what is more important, uterine environment1 or qualities of the embryo itself.2 Pregnancies following IVF treatment have been described previously in spontaneous cycles, stimulated cycles, and in egg donation program, using exogenous substitutional hormone therapy. In this study in order to evaluate the importance of the uterine environment, three protocols for uterine preparation prior to embryo replacement were compared while transferring frozen thawed embryos.

Synchronization of Donor and Recipient Cycles in an Egg Donation Program: Fresh Versus Frozen/Thawed Embryo Transfers

A high pregnancy rate has been reported following egg donation in the range of 20%–40% per transfer procedure.1,2,3 The procedure of egg donation is used mainly for noncycling patients (ovarian failure). Besides preparing the endometrium in these patients by means of hormone replacement therapy4,5 one should also synchronize the date of the endometrium with the age of the donated embryos. Synchronization can be achieved either by transfer of fresh embryos1 or by freezing the embryos and replacing them on different timing.2 In the present study we investigated the effect of synchronization by cryopreservation versus fresh embryo transfers on the results of treatment of egg donation.