Blood lymphocyte chimerism associated with IVF and monochorionic dizygous twinning: case report.

We report on dizygotic (DZ) twins, conceived by IVF and ICSI with assisted hatching, who each had a mixture of 46,XX and 46,XY cells in blood lymphocytes. The female twin had mild genitalia abnormalities but further study revealed anatomically normal reproductive anatomy. Chromosome and fluorescence in situ hybridization studies of buccal, skin and ovarian tissue were normal, as were buccal tissue DNA studies. Fetal ultrasound and fetal membrane pathology were consistent with a monochorionic, diamniotic placenta (MCDAP). These twins thus have blood chimerism but are not chimeric in the other tissues studied. The mechanism for the chimerism could be due to either placental vascular anastamoses (after the development of the haematoblast stem cells) or due to an admixture of trophoblast cells during early blastocyst development. Such trophoblast cell admixtures would be restricted to the extraembryonic tissues so that general physical development in the fetus is normal and without somatic cell chimerism. This case in combination with others previously reported suggests that in IVF conceptions, the prevalence of blood chimerism associated with twinning, and the occurrence of DZ twinning associated with MCDAP, may be higher than previously thought.

Results on single cell PCR for Huntington's gene and WAVE product analysis for preimplantation genetic diagnosis.

Triple repeat base pair amplification is the basis for a number of prevalent genetic diseases such as Huntington's, Fragile X, Myotonic Dystrophy and others. We have chosen to investigate the use of PCR to amplify a portion of the Huntington's gene in single cells in order to develop a clinical test system for preimplantation genetic diagnosis (PGD). Amplification of CAG triple repeat sequences poses difficulties due to resistance of GC melting for amplification. Special PCR modifications are necessary to carry out the amplification of GC rich areas found in most triple base pair expansions. We have used a modified polymerase chain reaction (PCR) protocol to amplify the expanded repeat sequence of the Huntington's gene with satisfactory efficiency. Detection of the amplified expanded CAG repeats is shown to be possible using both agarose gel electrophoresis and high definition denaturing high pressure liquid (DHPLC) chromatography. The incidence of allele dropout (ADO) is documented.

Primer system for single cell detection of double mutation for Tay-Sachs disease.

PURPOSE: Nearly 100% of infantile Tay-Sachs disease is produced by two mutations occurring in the alpha chain of the lysosomal enzyme beta-N-acetylhexosaminidase (HEXA) in the Ashkenazi Jewish population. Although others have described primer systems used to amplify both sites simultaneously, few discuss the allele dropout problems inherent in this test. Our goal was to construct a more robust test enabling stronger signal generation for single cell preimplantation genetic diagnosis and to investigate the occurrence of allele dropout. METHODS: New nested primers were designed to optimize detection of both major Tay-Sachs mutations. Four hundred fifty-seven single cells, including normal cells and those carrying mutations of either the 4bp insertion exon 11 or splice-site intron 12 defects, were used to screen a new primer system. RESULTS: Based on PCR amplified product analysis, total efficiency of amplification was 85.3%, (390/457). The allele dropout rate for the 4bp insertion mutation in exon 11 and splice-site mutation in intron 12 was 4.8% and 5.8%, respectively. CONCLUSIONS: Multiple mutation detection and analysis within the Tay-Sachs disease gene (HEXA) is possible using single cells for clinical preimplantation genetic diagnosis. Alternative PCR primers and conditions offer various methods for developing systems compatible to specific program requirements.

Simultaneous single-cell detection of two mutations for cystic fibrosis.

PURPOSE: A single-cell diagnosis procedure using polymerase chain reaction (PCR) technology was developed to simultaneously detect two cystic fibrosis (CF) mutations (DF-508, W1282X). METHODS: The reported test procedures made use of specific cell lines (lymphoblasts, fibroblasts) of known CF mutation status to determine the efficiency of signal generation and prevalence of allele dropout (ADO) during amplification. RESULTS: Using cells carrying the DF-508 mutation, the PCR signal efficiency for the affected homozygous, normal homozygous, and carrier heterozygote cell populations were 91%, 81%, and 92%, respectively. The total combined PCR efficiency was 87.7% and the ADO rate was 5.7%. For W1282X carrier heterozygote cells, the PCR signal efficiency was 82.0% and the ADO rate was 8.7%. CONCLUSIONS: Methods have been developed to detect two common mutations simultaneously for CF in single-cell assays. The high signal efficiencies and low ADO rates obtained in these tests allow those embryos from couples wishing to avert the transmission of this serious genetic disease to their offspring to be screened by preimplantation genetic diagnosis.

A novel, simplified and cost effective protocol for superovulation and intrauterine insemination.

OBJECTIVE: To determine the efficacy and cost of a simplified superovulation regimen compared with traditional control ovarian hyperstimulation with gonadotropins (HMG). STUDY DESIGN: This was a retrospective study in a university referral center with 99 infertile couples undergoing 225 treatment cycles. The outcome was compared to outcomes of previously published studies. The simplified superovulation regimen included clomiphene citrate 100 mg on cycle days 5 through 9 and HMG 75 IU on cycle days 5, 7, 9, and 11, with estradiol and ultrasound monitoring on day 13. If adequate follicular maturity was documented, HCG 10,000 IU was administered, followed by intrauterine insemination 40 hours later. RESULTS: Fecundity rates were assessed by life table analysis. Average cycle fecundity was 8%, with a cumulative rate of 29% over 4 cycles, compared to 10% monthly fecundity with HMG/IUI and background rates of 1 to 3%. Costs averaged $662 per cycle compared to $1,854 with HMG/IUI. CONCLUSION: A simplified protocol of CC/HMG/ IUI is almost as effective as HMG/IUI and costs only one-third as much.

Gonadotropin-releasing hormone (GnRH) and GnRH receptor gene expression in human myometrium and leiomyomata and the direct action of GnRH analogs on myometrial smooth muscle cells and interaction with ovarian steroids in vitro.

The objective of the present study was to determine whether GnRH and GnRH receptor are expressed in myometrium and leiomyomata, and if GnRH analogs alone or in the presence of ovarian steroids can modulate the rate of DNA synthesis, proliferation, and transforming growth factor-beta 1 (TGF beta 1) production in myometrial smooth muscle cells in vitro. Reverse transcription-PCR revealed that leiomyomata, unaffected myometrium, and isolated myometrial smooth muscle cells express GnRH and GnRH receptor messenger ribonucleic acid. Furthermore, in a dose-dependent manner, GnRH agonist (leuprolide acetate) inhibited, but GnRH antagonist [D-pGlu1,D-Phe2,D-Trp3.6] (GnRH-Ant1) stimulated, the rate of [3H]thymidine incorporation into myometrial smooth muscle cells (P < 0.05), whereas GnRH-Ant2 (Ac-D-P-Cl-Phe1.2,D-Trp3,D-Arg6,D-Ala10) had no effect. 17 beta-Estradiol (E2) medroxyprogesterone acetate (MPA), and E2 plus MPA (1 micromol/L) stimulated the rate of DNA synthesis by smooth muscle cells (P < 0.05), which was inhibited by GnRH analogs used at 5 micromol/L (P < 0.05). GnRH analogs had no significant effect on myometrial smooth muscle cell proliferation, with the exception of GnRH-Ant1; however, they inhibited the stimulatory action of E2, MPA, and E2 plus MPA in a time-dependent manner (P < 0.05). These cells also synthesized and released approximately 1.32 +/- 0.02 ng/mL total (active plus latent) TGF beta 1, of which 0.73 +/- 0.02 ng/mL was in an active form. E2, MPA, E2 plus MPA, and GnRH analog treatments resulted in an increase in total TGF beta 1 production, whereas GnRH agonist and GnRH-Ant2, but not GnRH-An1, inhibited active TGF beta 1 (P < 0.05). GnRH analogs also inhibited the action of E2 plus MPA on total and active TGF beta 1 production, whereas GnRH-Ant1 further stimulated E2, MPA, or E2 plus MPA action on active TGF beta 1 production (P < 0.05). The data demonstrate for the first time that GnRH and GnRH receptor messenger ribonucleic acid are expressed in myometrium, leiomyomata, and myometrial smooth muscle cells. The local expression of GnRH and receptor along with the direct action of GnRH analogs on the smooth muscle cell DNA synthesis and TGF beta 1 production suggest an autocrine/paracrine role for GnRH in these tissues, a mechanism that may be involved in leiomyomata regression in women receiving GnRH agonist therapy.

Twelve-well culture plate for the efficient collection and culture of human oocytes and embryos.

OBJECTIVE: Our goal was to assess a 12-well oocyte collection and embryo culture plate for use in the IVF laboratory. DESIGN: This was a prospective nonrandomized study. SETTING: The setting was a university in vitro fertilization program. PATIENTS: Eighty-four consecutive infertility couples presenting for IVF were studied. MAIN OUTCOME MEASURES: The main outcome measure was pregnancy (delivered). RESULTS: A 34% delivered pregnancy rate per retrieval was attained using the 12-well collection and culture plate without the use of expensive culture media and special serum supplementation. CONCLUSIONS: The use of a 12-well plate for oocyte collection and embryo culture provides a simple, economical, efficient, and effective means of producing human embryos during in vitro fertilization. This system is capable of supporting high rates of ongoing and delivered pregnancies.

Comparison of magnetic resonance imaging and transvaginal ultrasonography with hysterosalpingography in the evaluation of women exposed to diethylstilbestrol.

OBJECTIVE: To compare the ability of magnetic resonance imaging (MRI) and transvaginal ultrasound (TV-US) with that of hysterosalpingography (HSG) in detecting uterine abnormalities caused by in utero diethylstilbestrol (DES) exposure. STUDY DESIGN: The study was a prospective MRI and TV-US blind comparison of DES-exposed and nonexposed subjects who had had previous HSG for infertility evaluation. RESULTS: MRI identified uterine constrictions in 60% of patients and T-shaped uteri in 25% of DES-exposed patients with HSG-confirmed abnormalities. TV-US did not identify uterine constrictions or T-shaped uteri in DES-exposed patients. CONCLUSION: HSG must still be considered the preferred method in evaluating DES-related uterine abnormalities. HSG-defined uterine abnormalities associated with in utero DES exposure were variably identified by MRI and not at all by TV-US.

POM-ZP3, a bipartite transcript derived from human ZP3 and a POM121 homologue.

Human POM-ZP3 is a novel bipartite RNA transcript that is derived from a gene homologous to rat POM121 (a nuclear pore membrane protein) and ZP3 (a sperm receptor ligand in the zona pellucida). The 5' region is 77% identical to the 5' end of the coding region of rat POM121 and appears to represent a partial duplication of a gene encoding a human homologue of this rodent gene. The 3' end of the POM-ZP3 transcript is 99% identical to ZP3 and appears to have arisen from a duplication of the last four exons (exons 5-8) of ZP3. Using Northern blots and RT-PCR, POM-ZP3 transcripts were detected in human ovaries, testes, spleen, thymus, lymphocytes, prostate, and intestines. The longest open reading frame encodes a conceptual protein of 210 amino acids, the first 76 of which are 83% identical to residues 241-315 of rat POM121. The next 125 amino acids are 98% identical to residues 239-363 of the 424-amino-acid human ZP3 protein. By fluorescence in situ hybridization, genomic fragments of ZP3 and a human homologue of POM121 were localized to chromosome 7q11.23. Taken together, these data suggest that partial duplications of human ZP3 and a POM121-like gene have resulted in a fusion transcript, POM-ZP3, that is expressed in multiple human tissues.

Successful implementation of a regional in vitro fertilization program.

In vitro fertilization and other assisted reproductive technologies are becoming widely accepted modalities of treatment for infertile couples. Like other modern technologies, in vitro fertilization requires specially trained physicians and scientists who need specially dedicated equipment and space. In addition, patients having the procedure must undergo timely laboratory tests and sonographic evaluations for monitoring follicular maturation. To make in vitro fertilization available to patients in north central Florida, the Division of Reproductive Endocrinology and Infertility of the Department of Obstetrics and Gynecology at the University of Florida College of Medicine in Gainesville established a system that utilizes local resources in three distant locations. Patients in an assisted reproductive technologies cycle are monitored at satellite locations but coordinated through the University of Florida in vitro fertilization team. Oocyte retrieval, laboratory handling of gametes and embryo transfer are performed at the Shands Teaching Hospital in Gainesville. Micromanipulation for severe male factor infertility and oocyte donation are available when needed. Preimplantation diagnosis for genetic conditions is under development. The ongoing pregnancy and delivery rate is 21% per cycle initiated from July 1, 1992, to July 3, 1993, 62% above the reported national pregnancy rate for 1991. This program may be a useful model for the development of collaborative efforts in delivering highly complex medical services to large populations.

The effect of insulin on aromatase activity in isolated human endometrial glands and stroma.

Hyperinsulinemic states have been associated with an increased incidence of estrogen-dependent endometrial neoplasia. To study the effect of insulin on the ability of endometrium to aromatize androgens to estrogens, late proliferative endometrium was obtained from normally cycling women at the time of indicated surgery, separated into component glands and stroma, and grown to confluence. Separated gland and stromal cultures were incubated in triplicate with increasing insulin concentrations and epidermal growth factor. Aromatase activity was assayed by the production of tritiated water from tritium-labeled androstenedione. The activity was noted to increase proportionally with increasing concentrations of insulin greater than 10 U/ml, and the effect was specific. These data suggest the following conclusions: (1) Insulin stimulates aromatase activity in both endometrial glands and stroma; (2) hyperinsulinemia may predispose to endometrial neoplasia by enhancing endogenous endometrial estrogen production.