Laser-assisted derivation of human embryonic stem cell lines from IVF embryos after preimplantation genetic diagnosis.

BACKGROUND: Human embryonic stem cells (hESCs) suitable for future transplantation therapy should preferably be developed in an animal-free system. Our objective was to develop a laser-based system for the isolation of the inner cell mass (ICM) that can develop into hESC lines, thereby circumventing immunosurgery that utilizes animal products. METHODS: Hatching was assisted by micromanipulation techniques through a laser-drilled orifice in the zona pellucida of 13 abnormal preimplantation genetic diagnosed blastocysts. ICMs were dissected from the trophectoderm by a laser beam and plated on feeders to derive hESC lines. RESULTS: eight ICMs were isolated from nine hatched blastocysts and gave rise to three hESC lines affected by myotonic dystrophy type 1, hemophilia A and a carrier of cystic fibrosis 405 + 1G > A mutation. Five blastocysts that collapsed during assisted hatching or ICM dissection were plated whole, giving rise to an additional line affected by fragile X. All cell lines expressed markers of pluripotent stem cells and differentiated in vitro and in vivo into the three germ layers. CONCLUSIONS: These hESC lines can serve as an important model of the genetic disorders that they carry. Laser-assisted isolation of the ICMs may be applied for the derivation of new hESC lines in a xeno-free system for future clinical applications.

Neural progenitors from human embryonic stem cells.

The derivation of neural progenitor cells from human embryonic stem (ES) cells is of value both in the study of early human neurogenesis and in the creation of an unlimited source of donor cells for neural transplantation therapy. Here we report the generation of enriched and expandable preparations of proliferating neural progenitors from human ES cells. The neural progenitors could differentiate in vitro into the three neural lineages--astrocytes, oligodendrocytes, and mature neurons. When human neural progenitors were transplanted into the ventricles of newborn mouse brains, they incorporated in large numbers into the host brain parenchyma, demonstrated widespread distribution, and differentiated into progeny of the three neural lineages. The transplanted cells migrated along established brain migratory tracks in the host brain and differentiated in a region-specific manner, indicating that they could respond to local cues and participate in the processes of host brain development. Our observations set the stage for future developments that may allow the use of human ES cells for the treatment of neurological disorders.

Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro.

We describe the derivation of pluripotent embryonic stem (ES) cells from human blastocysts. Two diploid ES cell lines have been cultivated in vitro for extended periods while maintaining expression of markers characteristic of pluripotent primate cells. Human ES cells express the transcription factor Oct-4, essential for development of pluripotential cells in the mouse. When grafted into SCID mice, both lines give rise to teratomas containing derivatives of all three embryonic germ layers. Both cell lines differentiate in vitro into extraembryonic and somatic cell lineages. Neural progenitor cells may be isolated from differentiating ES cell cultures and induced to form mature neurons. Embryonic stem cells provide a model to study early human embryology, an investigational tool for discovery of novel growth factors and medicines, and a potential source of cells for use in transplantation therapy.

New advances in sex preselection.

OBJECTIVE: To review the current developments in the field of preconceptual sex selection and to discuss the moral dilemmas that accompany the scientific progress. DESIGN: A survey of the major publications on sex preselection. RESULTS: Examination of current methods of preconceptual gender selection revealed that in vivo methods such as timing of intercourse, the use of ovulation induction medications, and artificial insemination do not appear to affect the sex ratio to a clinically significant degree. In vitro separation of X- and Y-bearing spermatozoa by gradient techniques have been reported to alter significantly the sex ratio at birth. However, these trials were noncontrolled, and molecular biological techniques could not validate that these methods indeed change the Y- to X-bearing spermatozoa ratio sufficiently for clinical use. Nevertheless recent scientific advances have made highly reliable preconceptual sex selection possible by using preimplantation diagnosis or sperm separation by flow cytometry combined with IVF. At present, these methods have been used to avoid sex-linked disorders. Both involve the invasive procedure of IVF and thus are held by most as inappropriate for nonmedical indications. However, improvement in flow cytometry output of sexed spermatozoa might provide in the near future sufficient sorted gametes for artificial insemination. This technique then will provide an available noninvasive method of sexing for social purposes. CONCLUSIONS: Reliable preconceptual sex selection is currently possible only by preimplantation diagnosis, or sperm separation by flow cytometry combined with IVF. Both methods involve invasive procedures and are at present exclusively used for medical indications. It may be that in the near future, an improvement in flow cytometry output of sexed spermatozoa will provide sufficient sorted gametes for artificial insemination. In such a case, the medical community will be forced to take a stand, whether this reliable noninvasive method of sexing will be allowed for social purposes.

The use of vaginal tablets as a vehicle for steroid replacement in a donor oocyte program.

Nine women enrolled in the donor egg program received in a preparatory cycle 6 mg of E2 by vaginal tablets two times per day for the first 14 days, followed by a combined preparation of 6 mg of E2 and 50 mg of P as vaginal tablets, two times per day for another 14 days. Adequate serum levels of E2 and P and favorable sonographic endometrial thickness were achieved throughout the cycle in all patients. Eight endometrial biopsies taken on days 25 to 26 were independently assessed as being representative of day 25 +/- 2. One endometrial biopsy was out of phase. It is concluded that vaginal tablets of E2, followed by a combined preparation of vaginal tablets of E2 and P, is an effective method for establishing an appropriately developed endometrium in a donor oocyte program. Endometrial preparation in donor oocyte programs can be simplified by the use of these tablets.

Is the obstetric outcome of in vitro fertilized singleton gestations different from natural ones? A controlled study.

OBJECTIVE: To determine whether singleton IVF pregnancies carry adverse maternal or fetal outcome when compared with naturally conceived gestations. DESIGN: An analysis of the obstetric outcome of singleton IVF pregnancies in comparison with matched, naturally conceived singleton controls. SETTING: In vitro fertilization unit and obstetric service at a tertiary medical center. PATIENT(S): Two hundred sixty consecutive singleton IVF pregnancies and 260 naturally conceived singleton controls matched 1:1 for maternal age, parity, ethnic origin, and location and date of delivery. INTERVENTION(S): In vitro fertilization-ET. MAIN OUTCOME MEASURE(S): The rate of antenatal obstetric complications, nonvertex presentation, cesarean section, preterm labor, low birth weight, small and very small for gestational age, neonatal intensive care unit admissions, and perinatal mortality. RESULT(S): The rates of most antenatal complications were similar in both groups. Urinary tract infection was the only complication diagnosed significantly more frequently after IVF (7.3% versus 1.2%); however, the rates of severe urinary tract infection necessitating hospitalization were similar. The incidence of nonvertex presentation was also similar. The cesarean section rate was significantly higher among IVF patients (41.9% versus 15.5%). The rates of preterm labor, low birth weight, small and very small for gestational age, neonatal intensive care unit admissions, and perinatal mortality were comparable. CONCLUSION(S): When controlling for maternal age, parity, ethnic origin, and location and date of delivery, singleton IVF pregnancies do not carry an increased risk for prematurity, low birth weight, or maternal or fetal complications. Still, these pregnancies are associated with a high rate of cesarean sections.

Effects of hormone replacement therapy on weight, body composition, fat distribution, and food intake in early postmenopausal women: a prospective study.

OBJECTIVE: To evaluate the effects of hormone replacement therapy (HRT) on body weight and composition, fat distribution, and food intake in women entering the climacteric. DESIGN: Prospective clinical study. SETTING: Outpatient menopause clinic at a tertiary medical center. PARTICIPANTS: Sixty-three early postmenopausal women (44 to 54 years old) were prospectively studied for 1 year. They consisted of two groups: group A, 34 subjects who initiated continuous estrogen and progestin treatment (daily oral conjugated estrogen 0.625 mg and medroxyprogesterone acetate 2.5 mg), and group B, 29 women who refused hormonal therapy and served as controls. The age, menopausal status, initial anthropometric measurements (weight, body mass index [BMI], fat mass, and waist-to-hip girth ratio), and daily food intake (total caloric intake and food composition) were similar in both groups. INTERVENTIONS: Anthropometric measurements were performed before commencement of HRT use and after 12 months. MAIN OUTCOME MEASURES: Anthropometric measurements included BMI, waist-to-hip girth ratio, and body composition (the percentage of body fat and water) estimated by means of infrared interactance. Daily food intake was also recorded. RESULTS: The body weight and fat mass increased significantly in both the treatment (73.22 +/- 2.01 [mean +/- SE] to 75.57 +/- 1.12 kg) and the control group (71.45 +/- 3.11 to 73.51 +/- 1.23 kg). However, a significant shift from gynoid to android fat distribution was observed only in the control group (waist-to-hip ratio shifted from 0.80 +/- 0.01 to 0.85 +/- 0.01), whereas no significant change was observed in the treatment group (0.81 +/- 0.01 to 0.82 +/- 0.01). Caloric and macronutrient intake did not change in either group. CONCLUSIONS: These results indicate that continuous daily estrogen and progestin replacement therapy neither prevents nor increases early postmenopausal weight gain and fat accumulation. However, it does minimize the shift from gynoid to android fat distribution.

Effects of low-dose estrogen oral contraceptives on weight, body composition, and fat distribution in young women.

OBJECTIVE: To determine prospectively whether the use of low-dose estrogen oral contraceptives (OC) is associated with changes in weight, body composition, or fat distribution. DESIGN: Anthropometric measurements were performed in 49 healthy young (16 to 21 years old) women before commencement of OC use (30 micrograms ethinyl estradiol [EF2] plus 75 micrograms gestodene) and after three and six treatment cycles. Thirty one age- and weight-matched women who were not using OC served as controls. SETTING: Outpatient gynecological clinic of Hadassah Medical Center, a tertiary level hospital, and the "Shilo" voluntary service for the prevention of unwanted pregnancy. MAIN OUTCOME MEASURES: Anthropometric measurements included body mass index (BMI), waist-to-hip girth ratio, and body composition (the percentage of body fat and water), estimated by mean of infrared interactance. RESULTS: In the group of OC users, baseline BMI, percent fat, percent water, and waist-to-hip girth ratio were 21.1 +/- 0.32 (kg/m2), 23.8% +/- 0.63%, 57.4% +/- 0.39%, and 0.73 +/- 0.01, respectively, and did not change significantly after six cycles (20.6 +/- 0.41 [kg/m2], 23.9% +/- 0.57%, 58.1% +/- 0.49%, and 0.72 +/- 0.03, respectively). These measurements were not significantly different when compared with the nonusers. Fifteen OC users (30.6%) gained weight (> 0.5 kg). Weight gain was due to a significant accumulation of fat (from 22.5% +/- 1.1% to 25.6% +/- 0.74%), whereas the percentage of body water remained stable. The waist-to-hip girth ratio also was not changed significantly. Similarly, 11 nonusers (35.4%) gained weight because of similar nonabdominal fat accumulation. Ten OC users (20.4%) lost weight (57 kg +/- 1.51 to 55.4 +/- 1.47 [mean +/- SEM]) and 6 nonusers (19.3%) also lost weight (59 kg +/- 1.42 to 57.3 +/- 1.92). In both groups the loss of weight was not associated with significant change in body composition. CONCLUSIONS: The use of low-dose OC (EE2 plus gestodene) was not associated with overall impact on weight, body composition, or fat distribution. However, when weight gain did occur during OC use, it was due to increase in body fat and not in volume of body water, and it was not associated with changes in fat distribution.

Hyperreactio luteinalis associated with non-immune hydrops fetalis--the role of pituitary hormones.

The level of pituitary hormones was measured in the serum and cysts' aspirate in a case of hyperreactio luteinalis (HL) complicating non-immune hydrops fetalis. The level of follicular stimulating hormone (FSH), prolactin and growth hormone (GH) was within normal range for pregnancy in both the serum and cysts' aspirate. The importance of increased luteinizing hormone (LH) level which was demonstrated in the cysts' fluid should further be determined.

HELLP syndrome--a syndrome of hemolysis, elevated liver enzymes and low platelet count--complicating preeclampsia-eclampsia.

The course of preeclamptic/eclamptic patients may be complicated by HELLP syndrome, a syndrome of intravascular hemolysis (H), elevated liver enzymes (EL) and low platelet count (LP). These patients typically present at early third trimester with epigastric or right upper quadrant pain, nausea and vomiting. They may present without the clinical signs of preeclampsia (hypertension and proteinuria or edema), thus an initial wrong nonobstetric diagnosis is not uncommon. The most frequent maternal complication is intravascular coagulopathy (30%). Placental abruption and acute renal failure are also common. Ten cases of maternal deaths were reported among 295 cases reviewed in the English language literature, while the perinatal mortality rate was 226/1000. The grave prognosis for mother and fetus warrants physician awareness in order to accomplish early diagnosis and proper management. This paper is a review of the literature in English.

Safety of surgical intervention during the second trimester of pregnancy. A case report.

Surgical and anesthetic interventions during pregnancy are not hazard free. While an increase in fetal resorption and the abortion rate during the first trimester is well documented, a second-trimester intervention is assumed to be relatively safe. A case of acute transient oligohydramnios, fetal growth arrest and limb reduction anomaly following second-trimester abdominal surgery is reported for the first time.

Maternal hemorrhagic complications following prophylactic low-dose aspirin and dipyridamole therapy.

A case is reported of a severe postpartum maternal hemorrhagic complication, which was related to prophylactic antithrombotic therapy with daily low-dose aspirin (75 mg) combined with dipyridamole (225 mg) for the prevention of preeclampsia. The postpartum course was complicated by recurrent episiotomy site hematomas of nonclotted blood and prolonged bleeding time. Transfusion of platelet concentrates was necessary to control the bleeding. This case report draws attention to maternal hemorrhagic complications which may be associated with prophylactic low-dose aspirin and dipyridamole in pregnancy.

Antenatal bleeding and fetal heart rate.

Objective of the paper was to determine the fetal heart rate (FHR) changes that occur in preterm fetuses whose mothers have suffered antepartum bleeding, versus uncomplicated controls. Over a 12-year span, 91 patients with significant antenatal bleeding (bleeding requiring inhospital observation) were examined and compared to 75 controls with uncomplicated normal pregnancies. None of the women were in labor and all were evaluated at 25-37 weeks' gestation. Excluded were: patients with any other complication (i.e., premature rupture of membranes, intrauterine growth retardation, diabetes, hypertension, collagen vascular disease, postuterine surgery, substance abuse and twins). Analysis of the FHR tracings included baseline heart rate, long-term FHR variability, and number and amplitude of FHR accelerations in 20-min segments. There was no difference in baseline heart rate in the preterm fetuses of pregnancies complicated by antepartum bleeding versus controls. However, the parameters associated with FHR reactivity (number of accelerations in 20 min, and amplitude of accelerations) were higher to a statistically significant degree in fetuses of pregnancies complicated by antenatal bleeding than in controls. Fetuses of mothers suffering antenatal bleeding exhibited significant higher rates of reactive FHR patterns at earlier gestational ages than did controls. In conclusion, there is a significant increase in FHR reactivity in pregnancies in which significant antenatal bleeding occurs, suggesting a probable acceleration in fetal central nervous system maturation in these fetuses.

Defective oocytes as a possible cause of infertility in a beta-thalassaemia major patient.

Impaired reproduction is common among patients afflicted with beta-thalassaemia major and is attributed mainly to the deposition of haemosiderin in the pituitary gland and ovaries. In-vitro fertilization (IVF)--embryo transfer cycles including ovum donation cycles are described for the first time in a patient with beta-thalassaemia major. The patient's oocytes were not fertilized by the husband's sperm in repeated IVF attempts. However, when donated oocytes were used, in two consecutive cycles, most were fertilized by the husband, yielding pregnancies after each embryo transfer. It is suggested that in this case of beta-thalassaemia major, impaired oocyte function contributed to infertility. However, a possible association between thalassaemia major and defective oocytes should be tested on a basis of additional IVF-embryo transfer cycles in these patients.

Recurrent gestational trophoblastic disease following in-vitro fertilization.

Recurrence of gestational trophoblastic disease (GTD) following two attempts at in-vitro fertilization (IVF)/embryo transfer is reported in a childless couple after 17 years of unsuccessful trials of ovulation induction. The diagnosis of bilateral tubal obstruction was finally established, indicating IVF treatment. After the first IVF/embryo transfer treatment, the woman developed GTD and was treated with methotrexate. After a second IVF attempt, GTD was again diagnosed. This time there was no response to methotrexate, thus necessitating second-line chemotherapy. Etoposide, methotrexate, actinomycin D, cyclophosphamide, oncovine was used, and after only four treatment cycles the beta-human chorionic gonadotrophin (HCG) dropped to < 5 mIU/ml. After 26 months of follow-up, the beta-HCG continues to be undetectable. Preimplantation evaluation and ovum donation are described as measures to minimize the risk for GTD recurrence in a future IVF/embryo transfer.

Intracytoplasmic sperm injection combined with preimplantation genetic diagnosis for the prevention of recurrent gestational trophoblastic disease.

A strategy for the prevention of repeated molar pregnancies by using intracytoplasmic sperm injection (ICSI) coupled with preimplantation genetic diagnosis (PGD) with fluorescence in-situ hybridization (FISH) was developed. In this approach, complete moles which arise from dispermic fertilization are avoided by the use of ICSI. ICSI is followed by preimplantation selection against the transfer of 46,XX embryos, thus preventing complete moles resulting from a fertilization of an inactive oocyte, by a haploid X-bearing spermatozoon which subsequently duplicates. Triploid partial moles which arise mainly from dispermic fertilization may also be prevented by ICSI. The preimplantation confirmation of diploidy by FISH guards against triploid partial moles which may result from mechanisms other than dispermic fertilization. The employment of this strategy in an attempt to prevent a repeated event of molar pregnancy in a patient with a history of two previous episodes of gestational trophoblastic disease is reported.