Phillip E. Patton

Human Embryonic Stem Cells Derived by Somatic Cell Nuclear Transfer

Reprogramming somatic cells into pluripotent embryonic stem cells (ESCs) by somatic cell nuclear transfer (SCNT) has been envisioned as an approach for generating patient-matched nuclear transfer (NT)-ESCs for studies of disease mechanisms and for developing specific therapies. Past attempts to produce human NT-ESCs have failed secondary to early embryonic arrest of SCNT embryos. Here, we identified premature exit from meiosis in human oocytes and suboptimal activation as key factors that are responsible for these outcomes. Optimized SCNT approaches designed to circumvent these limitations allowed derivation of human NT-ESCs. When applied to premium quality human oocytes, NT-ESC lines were derived from as few as two oocytes. NT-ESCs displayed normal diploid karyotypes and inherited their nuclear genome exclusively from parental somatic cells. Gene expression and differentiation profiles in human NT-ESCs were similar to embryo-derived ESCs, suggesting efficient reprogramming of somatic cells to a pluripotent state.

Towards germline gene therapy of inherited mitochondrial diseases

Mutations in mitochondrial DNA (mtDNA) are associated with severe human diseases and are maternally inherited through the egg’s cytoplasm. Here we investigated the feasibility of mtDNA replacement in human oocytes by spindle transfer (ST; also called spindle–chromosomal complex transfer). Of 106 human oocytes donated for research, 65 were subjected to reciprocal ST and 33 served as controls. Fertilization rate in ST oocytes (73%) was similar to controls (75%); however, a significant portion of ST zygotes (52%) showed abnormal fertilization as determined by an irregular number of pronuclei. Among normally fertilized ST zygotes, blastocyst development (62%) and embryonic stem cell isolation (38%) rates were comparable to controls. All embryonic stem cell lines derived from ST zygotes had normal euploid karyotypes and contained exclusively donor mtDNA. The mtDNA can be efficiently replaced in human oocytes. Although some ST oocytes displayed abnormal fertilization, remaining embryos were capable of developing to blastocysts and producing embryonic stem cells similar to controls.

Vascular endothelial growth factor levels in serum and follicular fluid of patients undergoing in vitro fertilization

OBJECTIVE To define the relationship between serum and follicular fluid (FF) levels of vascular endothelial growth factor (VEGF), E2, and P in patients undergoing IVF; to quantify the effects of hCG on serum levels of VEGF during early pregnancy, and to report serial measurements of serum and ascites fluid levels of VEGF in a patient with severe ovarian hyperstimulation syndrome (OHSS). DESIGN Prospective observational study. SETTING University IVF program. PATIENTS(S) Women undergoing conventional IVF, receiving donated oocytes or spontaneously conceiving. One patient hospitalized with severe OHSS. MAIN OUTCOME MEASURE(S) Concentrations of VEGF, E2, and P in serum, FF, or peritoneal fluid. RESULT(S) At the time of egg retrieval, FF VEGF concentrations were positively correlated with serum and FF P concentrations and with patient age. At 11 to 14 days after ET, pregnant recipients of autologous fresh embryos had higher serum VEGF levels than both nonpregnant recipients of autologous fresh embryos and pregnant recipients of donor eggs. Elevated serum VEGF levels in a patient with severe OHSS coincided with the clinical onset and recurrence of symptoms. CONCLUSION(S) In patients undergoing IVF, FF VEGF levels at the time of egg retrieval correlated with the degree of follicular luteinization. There is a significant ovarian contribution to circulating VEGF levels during early gestation. Elevated serum VEGF levels may be a factor in the etiology of OHSS symptoms.

CA-125 levels in endometriosis

CA-125 is a cellular antigen detected in many patients with ovarian cancer, but it has also been detected in patients with endometriosis. Preoperative CA-125 levels were drawn from 113 patients before they underwent laparoscopy. Patients were categorized into diagnostic groups on the basis of pathologic findings. CA-125 levels in patients with evidence of intraabdominal adhesions were not statistically different from those in patients with normal pelvic anatomy. However, patients with advanced endometriosis had significantly elevated levels of CA-125 when compared with patients with normal pelvic anatomy (P less than 0.05). The clinical and immunologic implications of elevated CA-125 levels in patients with advanced endometriosis are discussed.

Intrauterine insemination outperforms intracervical insemination in a randomized, controlled study with frozen, donor semen

OBJECTIVE To assess the efficacy of intrauterine insemination (IUI) in a donor insemination program. DESIGN Prospective randomized clinical trial. SETTING Donor insemination program. PATIENTS, PARTICIPANTS Women undergoing insemination were randomly assigned to receive either IUI or intracervical insemination for a maximum of six cycles. INTERVENTIONS None. MAIN OUTCOME MEASURE(S) Cycle fecundity rates between the two routes were compared. RESULTS The monthly fecundity rate for intracervical insemination was 5.1% compared with 23% by IUI. By life table analysis, pregnancy rates for IUI were significantly higher than intracervical insemination (P = 0.02). CONCLUSIONS Intrauterine insemination with quarantined donor sperm is superior to intracervical insemination.

Percutaneous absorption of progesterone in postmenopausal women treated with transdermal estrogen.

OBJECTIVE The objective of this study was to evaluate the serum levels of progesterone resulting from the application of a progesterone cream to the skin. STUDY DESIGN Six postmenopausal women were evaluated at a university clinic over a 4-week period. RESULTS Transdermal estradiol 0.05 mg was applied 2 days before the first application of progesterone (30 mg/d) and was continued throughout the study. Patches were changed twice a week. Progesterone cream was applied once a day for 2 weeks. On day 15 and for the next 2 weeks, the progesterone cream was applied twice daily (60 mg/d). Serum 17beta-estradiol and progesterone were measured at 9 different times over a 24-hour period on day 1 and at weekly intervals for the 4-week duration of the study. Serum 17beta-estradiol concentrations varied among women, with mean concentrations of 40 to 64 pg/mL observed. Consistency in 17beta-estradiol concentrations was found within individual persons throughout the study. Serum progesterone concentrations also varied among women, with mean concentrations ranging from 1.6 to 3.3 ng/mL. After 2 weeks of percutaneous dosing, progesterone concentrations were sustained for at least 8 hours and were consistent within a given person. An appropriate increase in progesterone concentration occurred after 4 weeks compared with 2 weeks of application. Individually, a 0.53 correlation, significant at P <.0001, was seen between the absorption of 17beta-estradiol and progesterone. CONCLUSION Significant increases in serum concentrations of progesterone were observed in all of the women studied. The percutaneous absorption of progesterone correlates strongly with the absorption of transdermal 17beta-estradiol. There is variance in absorption of progesterone just as with 17beta-estradiol, and the 2 measures are closely correlated. The percutaneous application of progesterone cream appears to be a safe and effective route of administration.

Anatomic uterine defects.

Congenital or acquired uterine defects remain important considerations in the investigation of recurrent pregnancy loss. When repeated first or second trimester losses, preterm delivery, or abnormal fetal presentations are documented, the suspicion of a structural uterine abnormality should be high. The diagnosis of uterine defects is no longer elusive. The combination of radiologic imaging techniques, hysteroscopy, and laparoscopy enables an accurate diagnosis in nearly every case. The optimal treatment for uterine malformations is still a matter of considerable controversy. Therefore, when a uterine defect is diagnosed, tough clinical decisions must be made. When alternate causes of pregnancy loss are excluded, pregnancy potential will depend primarily on the specific type of uterine anomaly that is detected. It is important to recognize that not all uterine defects are amenable to therapy, but in carefully selected patients, reparative surgery may be rewarding.

Microsurgical reconstruction of the proximal oviduct

Since the successful advent of in vitro fertilization and embryo transfer (IVF-ET), reproductive surgeons have been forced to compare pregnancy outcomes of surgical procedures for tubal infertility with those of IVF-ET. The current study reports pregnancy outcomes, with extended follow-up, in patients who had proximal oviductal obstruction. Twenty-seven patients who had proximal oviductal disease with limited distal disease were evaluated after microsurgical repair. The conception rate at the end of 3 years of observation was 69.3%. The live birth rate was 53.2% at 3 1/2 years. The results indicate that microsurgical reconstruction of proximal oviductal occlusion is a workable option for patients with infertility.

Metabolic changes during medical treatment of endometriosis: Nafarelin acetate versus danazol

In this double-blind study of changes in plasma lipid and lipoprotein concentrations during 6-month medical treatment of endometriosis, 53 patients were randomly assigned to one of four treatment schedules: danazol, 800 mg/day (n = 10); danazol, 600 mg/day (n = 8); intranasal nafarelin acetate, 800 micrograms/day (n = 10); or intranasal nafarelin acetate, 400 micrograms/day (n = 25). Plasma levels of triglycerides, cholesterol, and low-density lipoprotein, very low-density lipoprotein, and high-density lipoprotein cholesterol fractions were obtained before, during, and 1 month after treatment. High-density lipoprotein2 and high-density lipoprotein3 cholesterol concentrations were measured in selected patients. Body weight was also followed. The drugs were equally effective in achieving symptomatic relief and laparoscopically demonstrated resolution of endometriosis but differed significantly in their effects on lipid concentrations. Nafarelin acetate had no adverse effects on serum lipoprotein concentrations, whereas danazol significantly decreased high-density lipoprotein cholesterol (p less than 0.01), as well as the high-density lipoprotein2 subfraction (p less than 0.05), and increased low-density lipoprotein cholesterol (p less than 0.01). Danazol significantly increased body weight (p less than 0.01), whereas nafarelin did not.

The prevalence of autoantibodies in pregnant and nonpregnant women

The frequency of autoantibodies during pregnancy was studied. Sera from 136 women (84 pregnant and 52 nonpregnant) were tested for antibodies directed against nuclear antigens, smooth muscle antigens, gastric parietal cells, mitochondria, and striated muscle by indirect immunofluorescent microscopy. Rheumatoid factor was measured by a latex agglutination test, and thyroid microsomal and thyroglobulin antibodies were measured by a hemagglutination assay. Immunofluorescence assays were considered positive if antibodies were detected at a serum dilution of 1:20. All positive samples were retested at increasing dilutions until immunofluorescence or agglutination could not be detected. The control population consisted of 52 normal, healthy, ovulatory, and nonpregnant women. The study population consisted of 84 pregnant women, none of whom had a known autoimmune disorder. The mean ages of the two groups were 32 and 26 years, respectively. The frequency of autoantibody detection was not significantly different in comparing the study (46.2) and control groups (40.5). The results did not support the hypothesis that pregnancy has an effect on the induction or suppression of autoantibody production. Previous studies that suggested differences may have been influenced by the lack of a control group.