Hormonally active nontransformed human ovarian cell culture from oophorectomy specimens: methods of development and initial characterization.

We repeatedly established a nontransformed steroidogenically active human ovarian cell culture derived from oophorectomy specimens. The cells maintained steroidogenic activity for 3-5 passages (6-8 weeks) and responded to stimulation by insulin and gonadotropin. With pregnenolone as substrate, LH stimulated progesterone production up to 124% and FSH up to 121%. Insulin alone stimulated progesterone production up to 135%, in the presence of LH up to 191%, and in the presence of FSH up to 170%. With dehydroisoandrosterone (DHA) as substrate, insulin alone stimulated testosterone production up to 117%, and in the presence of LH (but not FSH) up to 125%. With androstenedione as substrate, insulin alone stimulated estradiol production up to 133%, FSH alone up to 188%, and LH with insulin up to 217%. With progesterone as substrate and in the presence of LH (but not FSH), 17-alpha-hydroxylase activity was stimulated up to 131%. With DHA as substrate and in the presence of LH, 3-beta-hydroxysteroid dehydrogenase (3-beta-HSD) activity was stimulated up to 139%. With androstenedione as substrate, insulin alone stimulated aromatase activity up to 202%, LH up to 208%, and FSH up to 251%. Under the same conditions, in the presence of LH and insulin, aromatase activity was stimulated up to 342%, and in the presence of FSH and insulin, up to 318%. With testosterone as substrate, insulin alone stimulated aromatase activity up to 122%. With testosterone as substrate, in the presence of LH and insulin, aromatase activity was stimulated up to 136%, and in the presence of FSH and insulin, up to 156%. Immunocytochemistry studies directly confirmed presence of aromatase and 3-beta-HSD in these cultured cells. We conclude that a steroidogenically active nontransformed long-term human ovarian cell culture can be repeatedly established from oophorectomy specimens, providing uninterrupted supply of cultured human ovarian cells for a variety of studies of ovarian physiology.

Egg production predicts a doubling of in vitro fertilization pregnancy rates even within defined age and ovarian reserve categories.

OBJECTIVE: To examine the age-independent association of ovarian response and IVF outcome in women with normal and abnormal ovarian reserve. DESIGN: Retrospective analysis. SETTING: Academic IVF center. PATIENT(S): Four thousand eight hundred sixty-two consecutive IVF cycles. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Outcome of IVF was analyzed as a function of ovarian response to controlled ovarian hyperstimulation and ovarian reserve. RESULT(S): The mean patient age was 36.2 +/- 4.5 years. Younger patients and patients with normal ovarian reserve were found to have better implantation and clinical pregnancy rates. Patients with normal ovarian reserve had a higher number of oocytes retrieved, mature oocytes, two-pronuclei embryos, and embryos transferred. A greater number of embryos were transferred for patients with higher ovarian response. Higher clinical pregnancy rates were seen in those patients who had more oocytes retrieved for all patients, regardless of age and ovarian reserve. In fact, clinical pregnancy rates more than doubled for specific patient groups. CONCLUSION(S): In an age-independent fashion, ovarian response is highly predictive of IVF outcome in women with normal and abnormal ovarian reserve. These findings highlight the importance of not solely relying on age when presenting and discussing IVF outcome data and are useful information when helping patients interpret their IVF cycle response.

Polymorphism in intron 2 of the fetal interleukin-1 receptor antagonist genotype influences midtrimester amniotic fluid concentrations of interleukin-1beta and interleukin-1 receptor antagonist and pregnancy outcome.

OBJECTIVE: Preterm labor in experimental models is initiated by intra-amniotic interleukin-1beta (IL-1beta) and inhibited by interleukin-1 receptor antagonist (IL-1ra). The IL-1ra gene is polymorphic and the different alleles are associated with variations in IL-1beta and IL-1ra production. The relationship among the IL-1ra genotype of the fetus, concentrations of IL-1beta and IL-1ra in second-trimester amniotic fluid, and pregnancy outcome was determined. STUDY DESIGN: Amniotic fluids from 291 consecutive women with singleton pregnancies, obtained at 15 to 17 weeks' gestation, were tested for IL-1beta and IL-1ra concentrations by enzyme-linked immunosorbent assay. DNA from fetal cells was analyzed for a length polymorphism in intron 2 of the IL-1ra gene by polymerase chain reaction. Pregnancy outcomes were obtained after completion of testing. RESULTS: The distribution of fetal IL-1ra genotypes was similar to that found in other populations: 50.9% (148) were homozygous for allele 1 (IL1RN*1), 39.5% (115) were IL1RN*1/allele 2 (IL1RN*2) heterozygotes, 6.9% (20) were IL1RN*2 homozygotes, whereas 2.7% (8) had combinations of other alleles. Fetal possession of IL1RN*2 was associated with a greater than 50% increase in midtrimester intra-amniotic IL-1beta levels (P=.006) and a smaller increase in IL-1ra levels (P=.01) compared with fetuses who were IL1RN*1 homozygotes. Despite the low sample size, IL1RN*2 homozygosity, but not midtrimester intraamniotic levels of IL-1beta and IL-1ra, was related to an increased rate of preterm birth (P<.0001). In the 11 pregnancies that were subsequently terminated because of major malformations, there was a decreased frequency of IL1RN*1 homozygosity (P=.04). Birth weight was unrelated to IL-1ra genotype. CONCLUSION: Possession by the fetus of the IL1RN*2 allele is associated with enhanced intraamniotic IL-1beta production. Induction of an intra-amniotic proinflammatory immune response might be more likely to lead to preterm labor in fetuses carrying the IL1RN*2 allele.

Neonatal outcome of triplet versus twin and singleton pregnancies: a matched case control study.

OBJECTIVE: To determine the neonatal outcome of triplet gestations versus that of singletons and twins matched for gestational age. STUDY DESIGN: All live born triplet gestations delivered between 1 April 1993 and 31 March 2000 were compared to an age matched control group consisting of live born twins and singletons. The neonatal outcome of 116 sets of triplets was compared to that of 116 sets of twins and 116 singletons. RESULTS: During a 7-year period 116 sets of triplet pregnancies were reviewed. Of 116 sets of live born triplets (348 newborns), 70.67% triplets were born between 33- and 36-week gestation, 28.44% between 28 and 32 weeks and 0.86% less than 28 weeks. Triplets were smaller in weight than singletons but not twins. Apgar score, use of prenatal steroid and sex ratio were similar in the three groups. Incidence of respiratory distress syndrome (RDS), use of surfactant, infants requiring intubation, pneumothorax, patent ductus arteriosus, sepsis, intraventricular hemorrhage, periventricular leucomalacia, retinopathy of prematurity, necrotizing enterocolitis, gastroesophageal reflux and jaundice requiring phototherapy were not statistically different among the three groups. Incidence of major and minor congenital anomalies, percent neonatal intensive care unit (NICU) admissions, and mean duration of NICU stay were also similar. There was no influence of birth order on neonatal outcome of triplet pregnancy and outcome did not significantly change over 7 years of the study period. CONCLUSIONS: Triplets have a similar outcome to twins and singletons when matched for gestational age. Since outcome is dependent on gestational age, the closer the gestational age is to term the better is the outcome.

Preliminary experience with orthotopic and heterotopic transplantation of ovarian cortical strips.

The idea of fresh orthotopic autologous ovarian transplantation in humans is neither novel nor sophisticated; a New York surgeon reported on this technique as early as in 1906. It is the recent possibility of cryostorage of ovarian tissue and the development of new orthotopic and heterotopic techniques to autotransplant frozen-thawed ovarian cortical strips that brought a new dimension to the field. With the availability of more effective cryoprotectants, researchers were able to demonstrate that ovarian function and fertility could be restored after transplantation of cryopreserved ovarian tissue in large mammals. Experimental models of ovarian transplantation are discussed in the article by Shaw and Trounson; our focus will be on the clinical applications of ovarian transplantation and new developments in heterotopic transplantation. Patient selection and screening, details of the surgical techniques, and safety measures to avoid reseeding cancer cells via transplanted tissue will be discussed. Synchronization between the laboratory and the operating room will be detailed to provide guidance for clinicians who are contemplating ovarian transplantation with previously frozen ovarian tissue.